Engineering of stable bispecific antibodies targeting IL-17A and IL-23

Abstract

Bispecific antibodies (bsAbs) present an attractive opportunity to combine the additive and potentially synergistic effects exhibited by combinations of monoclonal antibodies (mAbs). Current challenges for engineering bsAbs include retention of the binding affinity of the parent mAb or antibody fragment, the ability to bind both targets simultaneously, and matching valency with biology. Other factors to consider include structural stability and expression of the recombinant molecule, both of which may have significant impact on its development as a therapeutic. Here, we incorporate selection of stable, potent single-chain variable fragments (scFvs) early in the engineering process to assemble bsAbs for therapeutic applications targeting the cytokines IL-17A/A and IL-23. Stable scFvs directed against human cytokines IL-23p19 and IL-17A/A were isolated from a human Fab phage display library via batch conversion of panning output from Fabs to scFvs. This strategy integrated a step for shuffling V regions during the conversion and permitted the rescue of scFv molecules in both the V(H)V(L) and the V(L)V(H) orientations. Stable scFvs were identified and assembled into several bispecific formats as fusions to the Fc domain of human IgG1. The engineered bsAbs are potent neutralizers of the biological activity of both cytokines (IC(50) < 1 nM), demonstrate the ability to bind both target ligands simultaneously and display stability and productivity advantageous for successful manufacture of a therapeutic molecule. Pharmacokinetic analysis of the bsAbs in mice revealed serum half-lives similar to human mAbs. Assembly of bispecific molecules using stable antibody fragments offers an alternative to reformatting mAbs and minimizes subsequent structure-related and manufacturing concerns.

Dual Inhibition of Interleukin-23 and Interleukin-17 Offers Superior Efficacy in Mouse Models of Autoimmunity

“…Although agents targeting both IL-17 and IL-23 have been reported, a complete understanding of the potential for improved efficacy with dual inhibition of IL-23 and IL-17 is lacking (Mabry et al, 2010). To investigate if greater efficacy could be achieved with dual blockade, we generated potent and selective inhibitors of IL-23 and IL-17A.…”

Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange

“…Since then, strategies to circumvent H-L chain mispairing [e.g., common L chain (7), speciesrestricted H-L chain pairing (8), crossover of CL and CH1 domains (9)] in combination with strategies to promote heterodimerization of the two H chains [e.g., "knobs-into-holes" (7), electrostatic steering (10), strand-exchange engineered domains (SEED) (11)] have increased homogeneity and yield of the desired end product (2). Alternatively, approaches using additional VH-VL pairs (12), single-chain (sc)Fv (13)(14)(15)(16), or domain antibody (dAb) (17,18) fragments for target binding allow incorporation of multiple antigen-combining sites in a single polypeptide chain (or single HL pair), which also increases product homogeneity and yield, although often at the expense of the physicochemical and/or pharmacokinetic properties of these agents (1-4).…”

Nanocell targeting using engineered bispecific antibodies

“…21,22,31 Previous studies have found that inclusion of a number of design modifications, including variations in CH3-scFv linker length and type, and also scFv stability engineering via addition of disulfide bonds, improved construct stability. [70][71][72][73][74][75] A concern with the Fc-linked dimeric design was whether the presence of 2 anti-LPS scFvs would result in EDV TM nanocell aggregation via binding of multiple nanocells rather than high avidity binding of a single nanocell. An alternative Fc-linked BsAb was engineered consisting of 2 EGFR targeting scFvs at the N-terminus (high avidity) and a single anti-LPS scFv at the C-terminus of the final product.…”