Affinity Maturation of a Humanized Rat Antibody for Anti-RAGE Therapy: Comprehensive Mutagenesis Reveals a High Level of Mutational Plasticity Both Inside and Outside the Complementarity-Determining Regions

Improving the pharmacokinetic properties of biologics by fusion to an anti-HSA shark VNAR domain

“…PCR products of approximately 400 bp from amplification using spiny dogfish library primers 61 were gel purified, digested with Sfi I and Not I followed by column-purification and ligated into the phagemid vector pWRIL-9 (derived from pWRIL-1). 62 Ligated DNA was transformed into electrocompetent TG1 cells (Lucigen,60502) resulting in a phagemid library of approximately 2 × 10 7 clones known as WySki2. The library was rescued with M13K07 helper phage at approximately a 1:20 ratio (cell:phage).…”

An Ultra-specific Avian Antibody to Phosphorylated Tau Protein Reveals a Unique Mechanism for Phosphoepitope Recognition

“…Phage Selections and Screening of Peptides-Selections were carried out using a solution-phase protocol with biotinylated peptides and streptavidin bead capture (43) in the presence of a 10-fold molar excess of cognate non-phosphopeptides and scrambled phosphopeptides as competitors (non-biotinylated). Selected bacterial clones were screened via direct binding ELISA as described previously (42,44).…”

Pharmacokinetics and lung distribution of a humanized anti-RAGE antibody in wild-type and RAGE-/-mice

“…Humanized anti-RAGE Ab (humanized from the rat IgG2b clone XT-M4) was generated at Pfizer (previously Wyeth Research, Cambridge, MA, USA). 16,25 Iodination was performed by the IODO-BEADS method according to manufacturer's instructions (Pierce, Rockford, IL, USA), using ~0.5 mg of test article re-formulated in phosphate buffer saline (PBS) and 5 mCi of 125-iodine (Perkin Elmer; Waltham, MA, USA). A dosing solution was prepared by mixing unlabeled test article, a trace amount of 125 I-labeled test article, and PBS to achieve the specific activity of ~38 μCi/mg (with <2% free iodine) and the final protein concentration of 1 mg/mL.…”