Nature-inspired design and evolution of anti-amyloid antibodies

Abstract

3 The abbreviations used are: A␤, amyloid ␤; CDR, complementarity-determining region; scFv, single-chain variable fragment; V H , variable domain of heavy-chain; Fc, fragment crystallizable; HCDR3, heavy chain CDR3; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; IAPP, islet amyloid polypeptide; RCF, relative centrifugal force; APP, amyloid precursor protein; HFIP, hexafluoro-2-propanol; Ni-NTA, nickel-nitrilotriacetic acid; DMEM, Dulbecco's modified Eagle's medium; KLH, keyhole limpet hemocyanin; ssDNA, single-stranded DNA; RFU, relative fluorescence units. cro ARTICLE 8438 A␤ solubilization and fibril preparationA␤ peptides (biotin-A␤42 (AS-23523-05; Anaspec) and A␤42 (AS20276; Anaspec)) were initially dissolved in Nature-inspired anti-amyloid antibodies

Directed evolution of conformation‐specific antibodies for sensitive detection of polypeptide aggregates in therapeutic drug formulations

“…The conformational antibodies reported in this study against liraglutide fibrils and related conformational antibodies against glucagon fibrils (Stimple et al, 2019), which were produced using similar directed evolution methods, possess several unique molecular features relative to other conformational and conventional antibodies. First, the heavy chain CDR3s of our liraglutide and glucagon antibodies have unusually large numbers of aromatic amino acids (10.3 ± 1.4 Trp, Phe, and Tyr residues) relative to Aβ conformational (2.9 ± 1.9 aromatic residues) and nonconformational (2.6 ± 2.1 aromatic residues) antibodies (for a summary of these antibodies, see Julian et al, 2019) as well as human antibodies in general (3.3 ± 1.7 aromatic residues). Second, our liraglutide and glucagon conformational antibodies have heavy chain CDR3s that are much more negatively charged (−5.8 ± 0.4 net charge at pH 7.4) than those of Aβ conformational (−0.3 ± 1.6) and nonconformational (−1.4 ± 1.5) antibodies as well as human antibodies in general (−1.4 ± 1.3).…”

“…The antibodies were isolated through two stages of library sorting. In the first stage of sorting, a single‐chain variable fragment (scFv) library was generated by diversification of heavy chain CDR3 (HCDR3) of the 4D5 scFv (Julian et al, 2019; Stimple et al, 2019; Tiller et al, 2017). To avoid concerns related to selection bias towards positively charged residues (Rabia et al, 2018), the library was diversified at 15 sites in HCDR3, at a frequency of 2–6 amino acids per site, while omitting positively charged residues (and cysteine) from the library.…”

“…The diversified scFvs (theoretical library diversity of 3.6 × 10 7 ) were cloned in yeast (EBY100) via homologous recombination into a yeast surface display vector as C‐terminal fusions to the yeast mating protein Aga2. The yeast display vector was a closely related derivative of pCTCON2 containing a modified linker between Aga2 and the scFvs (Julian et al, 2019). Yeast harboring the plasmids were cultured, propagated, and recovered after sorting in low‐pH SDCAA medium (20 g dextrose, 5 g casamino acids, 6.7 g yeast nitrogen base without amino acids, 16.75 g sodium citrate, and 4 g anhydrous citric acid per liter) with shaking at 30°C.…”

Sensitive detection of glucagon aggregation using amyloid fibril‐specific antibodies

“…Antibodies (scFvs) specific for glucagon fibrils were isolated using mutagenesis (Tiller, Chowdhury et al, ), yeast surface display and magnetic sorting using Dynabeads (Dynabeads M‐280 Tosylactivated, 14203; Invitrogen, Carlsbad, CA) coated with immobilized fibrillar or soluble glucagon using methods related to those reported previously (Julian et al, ). Briefly, 8 × 10 7 beads were washed twice with 1 ml of PBS.…”

“…The antibodies were expressed transiently in adherent HEK 293T cells (CRL‐3216; ATCC, Manassas, VA) using Invitrogen Lipofectamine 2000 Transfection Reagent (11668019; Thermo Fisher Scientific), as described previously (Julian et al, ; Rabia, Zhang, Ludwig, Julian, & Tessier, ; Tiller, Li et al, ). Briefly, cells were cultured in ~15 ml of Dulbecco's Modified Eagle's Medium (DMEM) culture media (10569‐044; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (35010CV; Corning, Corning, NY) and 1% penicillin/streptomycin (15140122; Thermo Fisher Scientific) in 75 cm 2 cell culture flasks (0030711122; Eppendorf, Hamburg, Germany) until reaching ~80% confluence.…”