Manufacturability and functionality assessment of different formats of T-cell engaging bispecific antibodies

Abstract

T-cell-engaging bispecific antibodies (T-bsAbs) are promising immunotherapies for cancer treatment due to their capability of redirecting T-cells toward destroying tumor cells. Numerous T-bsAb formats have been developed, each with advantages and disadvantages in terms of developability, immunogenicity, effector functions, and pharmacokinetics. Here, we systematically compared T-bsAbs produced using eight different formats, evaluating the effect of molecular design of T-bsAbs on their manufacturability and functionality. These eight T-bsAb formats were constructed using antigen-binding fragments (Fabs) and single-chain variable fragments (scFvs) of antibodies linked to the crystallizable fragment (Fc) domain of immunoglobulin G. To ensure a fair comparison of growth and production data, we used recombinase-mediated cassette exchange technology to generate the T-bsAb-producing CHO cell lines. The produced T-bsAbs were assessed for their purification profile and recovery, binding capability, and biological activities. Our findings indicated that the manufacturability of bsAbs was adversely affected with increased number of scFv building blocks, while the functionality was affected by the combination of multiple factors, including the binding affinity and avidity of targeting moieties and the flexibility and geometry of formats. These results provide valuable insights into the impact of the format design on the optimal production and function of T-bsAbs.

RUBY® – a tetravalent (2+2) bispecific antibody format with excellent functionality and IgG-like stability, pharmacology and developability properties

“… 39 , 40 Indeed, a negative correlation between manufacturability and amount of scFv in a bsAb has been observed in previous studies. 41 Another feature that indicates good overall inherent format developability for RUBY bsAbs is the relatively low level of aggregation, measured after only protein A purification, observed from transient and stable cultures. This is beneficial for large-scale manufacturing and functional screening of several binder combinations where a “one-size-fits-all” automated approach is preferred to generate research material for hundreds of novel bsAbs as opposed to the need for multi-dimensional generation or optimization of bsAbs reported elsewhere.…”

“…This feature was engineered using high-avidity CEA binding domains and would not have been possible in a format with only one binding arm toward CEA. There are likely other applications for a 2 + 2 geometry as demonstrated by Loh et al 41 in a systematic study that included the evaluation of the effects of valency, avidity, and geometry on function. HER2×CD3 bsAbs were constructed in eight formats and results showed the highest cell killing for the tetravalent, symmetric, 2 + 2 format.…”