Search Results (2)

Reversible antibody self-association, while having major developability and therapeutic implications, is not fully understood or readily predictable and correctable. For a strongly self-associating humanized mAb variant, resulting in unacceptable viscosity, the monovalent affinity of self-interaction was measured in the low μM range, typical of many specific and biologically relevant protein–protein interactions. A face-to-face interaction model extending across both the heavy-chain (HC) and light-chain (LC) Complementary Determining Regions (CDRs) was apparent from biochemical and mutagenesis approaches as well as computational modeling. Light scattering experiments involving individual mAb, Fc, Fab, and Fab’2 domains revealed that Fabs self-interact to form dimers, while bivalent mAb/Fab’2 forms lead to significant oligomerization. Site-directed mutagenesis of aromatic residues identified by homology model patch analysis and self-docking dramatically affected self-association, demonstrating the utility of these predictive approaches, while revealing a highly specific and tunable nature of self-binding modulated by single point mutations. Mutagenesis at these same key HC/LC CDR positions that affect self-interaction also typically abolished target binding with notable exceptions, clearly demonstrating the difficulties yet possibility of correcting self-association through engineering. Clear correlations were also observed between different methods used to assess self-interaction, such as Dynamic Light Scattering (DLS) and Affinity-Capture Self-Interaction Nanoparticle Spectroscopy (AC-SINS). Our findings advance our understanding of therapeutic protein and antibody self-association and offer insights into its prediction, evaluation and corrective mitigation to aid therapeutic development. Full article

We report the novel crystal structure and characterization of symmetrical, homodimeric humanized heavy-chain-only antibodies or dimers (HC2s). HC2s were found to be significantly coexpressed and secreted along with mAbs from transient CHO HC/LC cotransfection, resulting in an unacceptable mAb developability attribute. Expression of full-length HC2s in the absence of LC followed by purification resulted in HC2s with high purity and thermal stability similar to conventional mAbs. The V_H and C_H1 portion of the heavy chain (or Fd) was also efficiently expressed and yielded a stable, covalent, and reducible dimer (Fd2). Mutagenesis of all heavy chain cysteines involved in disulfide bond formation revealed that Fd2 intermolecular disulfide formation was similar to Fabs and elucidated requirements for Fd2 folding and expression. For one HC2, we solved the crystal structure of the Fd2 domain to 2.9 Å, revealing a highly symmetrical homodimer that is structurally similar to Fabs and is mediated by conserved (C_H1) and variable (V_H) contacts with all CDRs positioned outward for target binding. Interfacial dimer contacts revealed by the crystal structure were mutated for two HC2s and were found to dramatically affect HC2 formation while maintaining mAb bioactivity, offering a potential means to modulate novel HC2 formation through engineering. These findings indicate that human heavy-chain dimers can be secreted efficiently in the absence of light chains, may show good physicochemical properties and stability, are structurally similar to Fabs, offer insights into their mechanism of formation, and may be amenable as a novel therapeutic modality. Full article