A triple threat for HIV: The HIV virus continually evolves tricks to evade elimination by the host. Prevention and a cure will likely rely on broadly neutralizing antibodies that can recognize and conquer multiple viral strains or subtypes. Xu et al. engineered a single antibody molecule to recognize three highly conserved proteins needed for HIV infection (see the Perspective by Cohen and Corey). This “trispecific” antibody uses two sites (V1V2 and MPER) to bind HIV-infected cells, while the third site (CD4bs) recruits killer T lymphocytes that can eliminate the virus. When tested against >200 different HIV strains, trispecific antibodies were highly potent and broadly neutralized ∼99% of HIV viruses. This approach could potentially simplify HIV treatment regimens and improve therapy response. Science , this issue p. 85 ; see also p. 46

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.

[icon name="unlock"] OPEN-ACCESS [icon name="eye"] PEER-REVIEWED REVIEW ARTICLE Marta Amaral, Soraya Hölper, Christian Lange, Jennifer Jung, Hanno Sjuts, Sandra Weil,... This review covers in-depth analytics for the characterization of multispecific mAbs, focusing on mispairing analysis and functional characterization.

Recombinantly produced monoclonal antibodies (mabs) belong to the fastest growing class of biotherapeutics. In humans, antibodies are classified into five different classes: IgA, IgD, IgE, IgG and IgM. Most of the therapeutic mabs used in the clinic belong to the IgG class, albeit other antibody classes, e.g., IgM, have been evaluated in clinical stages. Antibodies are composed of heavy chains paired with a light chain. In IgM and IgA, an additional chain, the J-chain, is present. Two types of light chains exist in humans: the κ-light chain and the λ-light chain. The κ-light chain predominates in humans and is used in the vast majority of therapeutic IgG. The reason for the preference of the κ-light chain in humans is not known. Our study investigates whether light-chain selection influences the productivity of the clinically validated mabs adalimumab and trastuzumab. Both mabs were expressed as IgG and IgM with a κ- or a λ-light chain in HEK293 cells. Besides comparing the expression levels of the different mabs, we also evaluated whether the passage number of the cell line has an impact on product yield. In addition, the expressions of adalimumab, trastuzumab, an anti-CD38 and an anti-PD-L1-antibody were analyzed in HEK293 and CHO cells when both the κ- and λ-light chains are present. In summary, IgG outperformed IgM variants in expression efficacy, while light-chain selection had minimal impact on the overall expression levels. The yields of all mab variants were higher in fresh cells, despite cell cultures with a high cell passage number having higher cell densities and cell numbers at the time of harvest. The incorporation of a particular light chain occurred at similar rates in HEK293 and CHO cells.

Recombinantly produced monoclonal antibodies (mabs) belong to the fastest growing class of biotherapeutics. In humans, antibodies are classified into five different classes: IgA, IgD, IgE, IgG and IgM. Most of the therapeutic mabs used in the clinic belong to the IgG class, albeit other antibody-classes, e.g. IgM, have been evaluated in clinical stages. Antibodies are composed of heavy chains paired with a light chain. In IgM and IgA antibodies, an additional chain, the J-chain, is present. Two types of light chain exist in humans: The -light chain and the -light chain. The -light chain predominates in human and is used in the vast majority of therapeutically used IgGs. The reason for preference of the -light chain in human is not known. Our study investigates whether light chain selection influences productivity of the clinically validated mabs: adalimumab and trastuzumab. Both mabs were expressed as IgG and IgM with a  or a -light chain in HEK293 cells. Besides comparing the expression levels of the different mabs, we also evaluated, whether the passage number of the cell line has an impact on product yield. In addition, expression of adalimumab, trastuzumab, an anti-CD38 and an anti-PD-L1-antibody was analyzed in HEK293 and CHO cells when both, the κ- and λ-light chain are present. In summary, IgG outperformed IgM variants in expression efficacy while light chainselection had minimal impact on overall expression levels. Yields of all mab variants were higher in fresh cells, despite cell cultures with a high cell passage number having higher cell densities and cell numbers at the time of harvest. Incorporation of a particular light chain occurred at similar rates in HEK293 and CHO cells.