Bispecific immunoglobulins (Igs) typically contain at least two distinct variable domains (Fv) that bind to two different target proteins. They are conceived to facilitate clinical development of biotherapeutic agents for diseases where improved clinical outcome is obtained or expected by combination therapy compared to treatment by single agents. Almost all existing formats are linear in their concept and differ widely in drug-like and manufacture-related properties. To overcome their major limitations, we designed cross-over dual variable Ig-like proteins (CODV-Ig). Their design is akin to the design of circularly closed repeat architectures. Indeed, initial results showed that the traditional approach of utilizing (G4S) x linkers for biotherapeutics design does not identify functional CODV-Igs. Therefore, we applied an unprecedented molecular modeling strategy for linker design that consistently results in CODV-Igs with excellent biochemical and biophysical properties. CODV architecture results in a circular self-contained structure functioning as a self-supporting truss that maintains the parental antibody affinities for both antigens without positional effects. The format is universally suitable for therapeutic applications targeting both circulating and membrane-localized proteins. Due to the full functionality of the Fc domains, serum half-life extension as well as antibody-or complement-dependent cytotoxicity may support biological efficiency of CODV-Igs. We show that judicious choice in combination of epitopes and paratope orientations of bispecific biotherapeutics is anticipated to be critical for clinical outcome. Uniting the major advantages of alternative bispecific biotherapeutics, CODV-Igs are applicable in a wide range of disease areas for fast-track multi-parametric drug optimization.

Various important biological pathways are modulated by TGFb isoforms; as such they are potential targets for therapeutic intervention. Fresolimumab, also known as GC1008, is a pan-TGFb neutralizing antibody that has been tested clinically for several indications including an ongoing trial for focal segmental glomerulosclerosis. The structure of the antigen-binding fragment of fresolimumab (GC1008 Fab) in complex with TGFb3 has been reported previously, but the structural capacity of fresolimumab to accommodate tight interactions with TGFb1 and TGFb2 was insufficiently understood. We report the crystal structure of the single-chain variable fragment of fresolimumab (GC1008 scFv) in complex with target TGFb1 to a resolution of 3.00 Å and the crystal structure of GC1008 Fab in complex with TGFb2 to 2.83 Å . The structures provide further insight into the details of TGFb recognition by fresolimumab, give a clear indication of the determinants of fresolimumab pan-specificity and provide potential starting points for the development of isoformspecific antibodies using a fresolimumab scaffold.

Proline cis-trans conformational isomerization is a mechanism that affects different types of protein functions and behaviors. Using analytical characterization, structural analysis, and molecular dynamics simulations, we studied the causes of an aberrant two-peak size-exclusion chromatography profile observed for a trispecific anti-HIV antibody. We found that proline isomerization in the tyrosine-proline-proline (YPP) motif in the heavy chain complementarity-determining region (CDR)3 domain of one of the antibody arms (10e8v4) was a component of this profile. The pH effect on the conformational equilibrium that led to these two populations was presumably caused by a histidine residue (H147) in the light chain that is in direct contact with the YPP motif. Finally, we demonstrated that, due to chemical equilibrium between the cis and trans proline conformers, the antigen-binding potency of the trispecific anti-HIV antibody was not significantly affected in spite of a potential structural clash of 10e8v4 YP trans P trans conformers with the membrane-proximal ectodomain region epitope in the GP41 antigen. Altogether, these results reveal at mechanistic and molecular levels the effect of proline isomerization in the CDR on the antibody binding and analytical profiles, and support further development of the trispecific anti-HIV antibody.

Most antibodies display very low brain exposure due to the blood-brain barrier (BBB) preventing their entry into brain parenchyma. Transferrin receptor (TfR) has been used previously to ferry antibodies to the brain by using different formats of bispecific constructs. Tetravalent bispecific tandem immunoglobulin Gs (IgGs) (TBTIs) containing two paratopes for both TfR and protofibrillar forms of amyloid-beta (Aβ) peptide were constructed and shown to display higher brain penetration than the parent anti-Aβ antibody. Additional structure-based mutations on the TfR paratopes further increased brain exposure, with maximal enhancement up to 13-fold in wild-type mice and an additional 4–5-fold in transgenic (Tg) mice harboring amyloid plaques, the main target of our amyloid antibody. Parenchymal target engagement of extracellular amyloid plaques was demonstrated using in vivo and ex vivo fluorescence imaging as well as histological methods. The best candidates were selected for a chronic study in an amyloid precursor protein (APP) Tg mouse model showing efficacy at reducing brain amyloid load at a lower dose than the corresponding monospecific antibody. TBTIs represent a promising format for enhancing IgG brain penetration using a symmetrical construct and keeping bivalency of the payload antibody.

A thermodynamic model was used in this study to predict the adhesion of Phanerochaete chrysosporium INA-12 as conidiospores or mycelium to various solid carriers. Theoretical predictions were closely reflected by experimental results. Amount of immobilized mycelium was higher for hydrophobic (polypropylene and polyurethane) than for hydrophilic carrier (stainless steel and grey). Lignin peroxidase production was stimulated in the same way. However, better results were obtained with polyurethane than with polypropylene and with grey than with stainless steel. These results were attributed to roughness effects of solid surfaces. Surface morphology characterization showed that the surface roughness parameter R(A) was higher for polyurethane and grey as compared to polypropylene and stainless steel, respectively. On the other hand, polyurethane is not simply rugous; it has an intraparticle porosity as well as a higher total surface area as compared to polypropylene.