CDR-restricted engineering of native human scFvs creates highly stable and soluble bifunctional antibodies for subcutaneous delivery

Abstract

While myriad molecular formats for bispecific antibodies have been examined to date, the simplest structures are often based on the scFv. Issues with stability and manufacturability in scFv-based bispecific molecules, however, have been a significant hindrance to their development, particularly for high-concentration, stable formulations that allow subcutaneous delivery. Our aim was to generate a tetravalent bispecific molecule targeting two inflammatory mediators for synergistic immune modulation. We focused on an scFv-Fc-scFv format, with a flexible (A4T)3 linker coupling an additional scFv to the C-terminus of an scFv-Fc. While one of the lead scFvs isolated directly from a naïve library was well-behaved and sufficiently potent, the parental anti-CXCL13 scFv 3B4 required optimization for affinity, stability, and cynomolgus ortholog cross-reactivity. To achieve this, we eschewed framework-based stabilizing mutations in favor of complementarity-determining region (CDR) mutagenesis and re-selection for simultaneous improvements in both affinity and thermal stability. Phage-displayed 3B4 CDR-mutant libraries were used in an aggressive “hammer-hug” selection strategy that incorporated thermal challenge, functional, and biophysical screening. This approach identified leads with improved stability and >18-fold, and 4,100-fold higher affinity for both human and cynomolgus CXCL13, respectively. Improvements were exclusively mediated through only 4 mutations in VL-CDR3. Lead scFvs were reformatted into scFv-Fc-scFvs and their biophysical properties ranked. Our final candidate could be formulated in a standard biopharmaceutical platform buffer at 100 mg/ml with <2% high molecular weight species present after 7 weeks at 4 °C and viscosity <15 cP. This workflow has facilitated the identification of a truly manufacturable scFv-based bispecific therapeutic suitable for subcutaneous administration.

A Combination of Structural and Empirical Analyses Delineates the Key Contacts Mediating Stability and Affinity Increases in an Optimized Biotherapeutic Single-chain Fv (scFv)

“…As a result, the mechanisms that drive affinity improvements for antigen binding are still poorly understood. The development of the highly stable, pM potency, fully human, anti-CXCL13 scFv E10 presented a rare opportunity to study a molecule that had exhibited multi-parameter improvements via only four mutations in a single CDR loop (13). In the study presented here, we used both empirical analyses of point mutations and co-crystal structural analyses to investigate the mutations that enabled the generation of the affinity-and stability-optimized scFv E10.…”

“…This study shows that assumption to be flawed, as Arg 93 and Arg 94 in the V L -CDR3 of 3B4 are the only residues that deviate from the germline in this CDR, but the presence of Arg 93 is actually deleterious to CXCL13 binding function. Arg 93 and Arg 94 are somatic mutations that were most likely generated in the context of a different V H partner in the human B-cell background of the naive scFv library from which 3B4 was derived (13). In the original v-gene pairing, these two mutations could indeed have been beneficial for binding to an unknown antigen, but when isolated as part of 3B4, Arg 93 is a hindrance to high affinity binding of CXCL13.…”

“…The resulting proteins were buffer-exchanged into PBS using 40-kDa cut-off Zeba columns (Thermo Scientific) and quantified using a Micro BCA kit (Thermo Scientific). Thermal stability ELISAs and DSC analyses were performed as previously described (13).…”

“…2C). Conservation of these interactions provides structural rationale for the failure to retrieve any affinity-optimized mutants from the 3B4 V H -CDR3 mutagenesis libraries (13).…”

“…We recently described the successful concurrent affinity and stability optimization of an anti-CXCL13 scFv using CDR-restricted mutagenesis and affinity-and stability-driven selection by phage display (13). When reformatted into an scFv-Fc-scFv bispecific molecule, the lead scFv "E10" displayed an exemplary stability and solubility profile and could be formulated at 100 mg/ml in a standard biopharmaceutical platform buffer with the viscosity remaining well below the threshold of 20 centipoise required for subcutaneous administration.…”