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Bispecific antibodies (bsAbs) are of significant importance to the development of novel antibody-based therapies, and heavy chain (Hc) heterodimers represent a major class of bispecific drug candidates. Current technologies for the generation of Hc heterodimers are suboptimal and often suffer from contamination by homodimers posing purification challenges. Here, we introduce a new technology based on biomimicry wherein the protein-protein interfaces of two different immunoglobulin (Ig) constant domain pairs are exchanged in part or fully to design new heterodimeric domains. The method can be applied across Igs to design Fc heterodimers and bsAbs. We investigated interfaces from human IgA CH3, IgD CH3, IgG1 CH3, IgM CH4, T-cell receptor (TCR) α/β, and TCR γ/δ constant domain pairs, and we found that they successfully drive human IgG1 CH3 or IgM CH4 heterodimerization to levels similar to or above those of reference methods. A comprehensive interface exchange between the TCR α/β constant domain pair and the IgG1 CH3 homodimer was evidenced by X-ray crystallography and used to engineer examples of bsAbs for cancer therapy. Parental antibody pairs were rapidly reformatted into scalable bsAbs that were free of homodimer traces by combining interface exchange, asymmetric Protein A binding, and the scFv × Fab format. In summary, we successfully built several new CH3- or CH4-based heterodimers that may prove useful for designing new bsAb-based therapeutics, and we anticipate that our approach could be broadly implemented across the Ig constant domain family. To our knowledge, CH4-based heterodimers have not been previously reported.

Neospora caninum is considered to be the main cause of bovine abortion in Europe and the USA, leading to considerable financial impact. Losses are caused directly by abortions or indirectly through breeding of calves with impaired viability. Due to the lack of effective chemotherapy against bovine neosporosis, there is a need to develop new anti-protozoal compounds, which would either eliminate the parasite or avoid its transmission. In order to identify compounds of interest, the in vitro activities of 41 di-cationic pentamidine derivatives were studied employing a transgenic N. caninum clone expressing beta-galactosidase as a reporter gene. The arylimidamide DB745, previously shown to be highly active against Leishmania donovani in vitro and in vivo, appeared as the most promising compound, with an IC50 of 80 nM in 3-day growth assays and severely affecting both host cell invasion as well as intracellular proliferation. TEM of intracellular tachyzoites identified distinct alterations related to the nucleolus and the nuclear and cellular membrane. Long-term growth assays showed that DB745 acted parasiticidal upon the Nc-Liv isolate, but not against the Nc-1 isolate of N. caninum. In vivo studies in N. caninum (Nc-1 isolate) infected mice showed that daily intraperitoneal application of DB745 for a period of 14 days resulted in a decreased number of clinically affected animals, and lower cerebral parasite burdens in DB745-treated mice compared to non-treated mice. These results illustrate the potential of dicationic arylimidamides for the treatment of N. caninum infections.

Neospora caninum and neosporosis 3. Host cell adhesion/invasion by N. caninum and related apicomplexan parasites 3.1. Initial adhesion is mediated by surface antigens (SAGs) 3.2. Apical attachment is mediated by microneme proteins (MICs) 3.3. Host cell entry is achieved by formation of a moving junction composed of microneme and rhoptry components 3.4. Formation of the parasitophorous vacuole membrane (PVM) is governed by rhoptry (ROPs) and dense granule (DG) proteins 3.5. Intracellular parasitism and parasite-host cell communication 4. Vaccines against Neospora caninum 4.1. Killed tachyzoite lysates / live-attenuated vaccines 4.2. Components of the host cell adhesion/invasion machinery of N. caninum and related apicomplexans represent potential vaccine targets 5. Where to go from here 6. Acknowledgements 7.References