, 2005), as well as the possible differences in the donor pools. Therefore, the performance characteristics of each library will differ, making it advantageous to have a variety of libraries available for selection. Although, fully human naïve Fab and scFv libraries have been made before
(Marks et al., 1991, Griffiths et al., 1994, Vaughan et al., 1996, de Haard et al., 1999, Glanville et al., 2009 and Lloyd et al., 2009), here we present the first direct comparison between the performances of the two formats. This comparison can be done because these two libraries were constructed Tofacitinib using similar donor sources, construction methods and vector backbones, limiting the variability between the libraries. Both XFab1 and XscFv2 were assessed for multiple qualification parameters, including percentage of open reading frame (%ORF), expression levels, V-gene family distribution, VH-CDR3 length, and germline occurrence. Our libraries have been used for selections against seven targets and the resulting clones analyzed to determine unique hit rate, V-gene usage, and affinity. These parameters have allowed us to validate and compare the libraries and demonstrate their utility as potential
sources for high affinity, functional therapeutic antibodies. The source RNA and cDNA used to amplify the V-genes Palbociclib was purchased from AllCells and Cureline. The E. coli strain TG1 (Lucigen) was used for all molecular cloning, phage production, and expression assays. Restriction endonucleases and T4-DNA ligase were purchased from New England Biolabs. KOD Hot Start DNA Polymerase and associated 10 × buffer, dNTP mix, Reverse transcriptase and MgSO4 (EMD Biosciences), were used for all PCR reactions. Some PCR reactions also included betaine (Sigma-Aldrich) and/or DMSO (Sigma-Aldrich). PCR primers were purchased from Elim Biosciences or IDT. ArrayScript™ Reverse Transcriptase
(Ambion) with Random primers (NEB) was used to make cDNA libraries from RNA samples. All media and solutions were purchased from Teknova. For the CHO cells expressing TIE2 and InsR used for screening, mammalian expression vectors encoding TIE2 and InsR were each transfected into CHO-K1 cells using a PEI transfection reagent (JetPEI®, Polyplus). Individual G-418-resistant clones were screened by FACS using commercially available antibodies to TIE2 or InsR. XFab1 used cDNA generated from 15 PBMC samples and 15 bone marrow samples. The variable regions were amplified from cDNA using primers designed based on sequences in V-Base to amplify each family of Vλ1–Vλ10, Vκ1–Vκ6, and VH1–VH6 individually with forward primers annealing to the V segment and reverse primers annealing in the Cλ or Cκ for Vλ and Vκ and in the VHJ region for VH (Table S1).