AX213 and AX132 PCSK9 antagonists and variants
An antagonist, antagonistic technology, applied in the direction of antibodies, antibody mimics/scaffolds, anti-enzyme immunoglobulins, etc.
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Embodiment 1
[0200] ABMAXIS PDL1 phage library panning against PCSK9 protein
[0201] A synthetic human Fab library was panned against human PCSK9. The antigenic protein PCSK9 was coated on Maxisorp well strips (Nunc-Immuno Modules) overnight at 4°C at a concentration of 1-10 μg / ml. For each library, prepare multiple antigen wells. Coated wells were blocked with 5% milk in PBS for 1-2 hours at room temperature. After washing with PBS, 100 μl of phage library solution / well (usually 1-5×10 12 , in 2% milk-PBS) were added to 4 parallel wells and incubated for the indicated length of time (usually 1-2 hours). After several washes with PBST and PBS, freshly prepared 2 Bound phage were eluted from the wells with 1.4% triethylamine in O (incubated for 10 minutes at room temperature), followed immediately by neutralization by the addition of 50 [mu]l 1M Tris-HCl, pH 6.8.
[0202] The eluted, enriched phage collection was further amplified by the following steps: First, TG1 cells were infected...
Embodiment 2
[0205] Fab ELISA Screening of PCSK9 Binders
[0206] Over 10,000 clones from the 3rd round of panning were picked by a MegaPix picking robot (Genetix) and inoculated into 384-well plates containing 60 μl 2YT / 2% glucose / carbenicillin and incubated at 30°C with shaking at 450 rpm overnight. Duplicate plates were prepared by transferring approximately 1-3 μl of the overnight culture from each well to a new plate containing 50 μl / well of 2YT / 0.1% glucose / carbenicillin. Replica plates were incubated for 6 hours at 30°C on a shaker, then 10 μl / well of IPTG was added to a final concentration of 1 mM. After overnight incubation at 22°C, soluble Fab was released in the IPTG-induced plates by adding lysozyme to each well.
[0207] To test the antigen-binding activity of soluble Fabs generated from the above experiments, antigen plates were prepared by coating overnight with 5 μg / ml human PCSK9 antigen. After blocking with 5% milk-PBS and washing with PBST, 15-20 μl of Fab samples wer...
Embodiment 3
[0218] Fab protein expression and purification from TG1 cells
[0219] A 50 ml overnight culture from a single clone was grown in 2YT / 2% glucose / carbenicillin 100 μg / ml in a 37°C shaker incubator. On the following day, each clone was inoculated with 750 mL to 1 L of 2YT / 0.1% glucose / 100 μg / mL carbenicillin by transferring 5-10 ml of the overnight culture. The culture was grown for approximately 3-4 hours at 30°C with shaking until the OD600 was about 1. IPTG was added to the cultures to a final concentration of 0.1-0.5 mM. Following overnight IPTG induction at 22°C, cell pellets were collected by centrifugation at 10,000 rpm for 10-15 minutes to proceed with periplasmic preparation.
[0220] Soluble Fab was extracted from the periplasm. Periplasmic preparation was performed as follows. The TG1 pellet was resuspended in 20 mL of pre-chilled PPB buffer (20% sucrose + 2 mM EDTA + 30 mM Tris, pH=8) and incubated on ice for 1 hour. The supernatant containing soluble Fab was co...
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