We present here, the design, implementation and validation of ALTHEA (from the Greek “to heal”) Gold Libraries?. ALTHEA Gold Libraries? are built with synthetic well-known and highly stable diversified human scaffolds and diverse human H3J fragments obtained from a pool of 200 donors assembled as scFvs. A universal VH scaffold is paired with two distinct VL scaffolds, providing two alternative antigen-binding site topographies to bind protein or peptide targets. ALTHEA Gold Libraries? are obtained by a proprietary process consisting of enrichment for thermostable and highly functional scFvs. Deep sequencing of over a million of scFv sequences indicates that all of them virtually unique. The potential of ALTHEA Gold Libraries? to generate specific scFvs is assessed with four targets, including a model protein (Hen Egg White Lysozyme) and three undisclosed therapeutic targets, two soluble proteins and a GPCR. In all the four targets diverse and specific antibodies are obtained. Also, we have developed and tested a Rapid Affinity Maturation (RAM) kit? for facile affinity maturation of selected scFvs or intermediary pools of scFvs obtained after a few rounds of selection.
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