The anti-BACE1 affinity-matured 1A11 antibody (1A11AM) was used to generate bispecific antibodies. The engineering and expression of BBB00574 (1A11AM-BBB00515) and BBB00578 (1A11AM-BBB00533) were performed as previously described [16 (link),25 (link)]. Briefly, the DNA sequences encoding for the heavy chain composed of BBB00515 or BBB00533 fused to an Fc with knobs-into-holes (KiH) and ablated effector function mutations (human IgG1, L234A, L235A, P329G, T350V, T366L, K392L, T394W), the 1A11AM heavy chain with KiH and ablated effector function mutations (human IgG1, L234A, L235A, P329G, T350V, L351Y, F405A, Y407V), and the 1A11AM light chain (human kappa) were synthesized by Twist Bioscience and cloned in their pTwist CMV BetaGlobin WPRE Neo vector (Twist Bioscience). Expressions were performed using the Mirus CHOgro® High Yield Expression System (Mirus Bio) according to the manufacturer instructions. Briefly, ExpiCHO-S™ cells (Mirus Bio) were transfected with the nanobody-human Fc fusions and the 1A11AM heavy chain and light chain with a transfection ratio of 2:1:3 with TransIT-PRO Transfection Reagent (Mirus Bio) [25 (link)]. After an incubation time of 14 days, the antibodies were purified, first with AmMag™ Protein A Magnetic Beads (Genscript) and then over a CaptureSelect™ CH1-XL Pre-packed Column (ThermoFischer Scientific) according to the manufacturer’s instructions.
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