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. 2017 May 18;8(39):65152–65170. doi: 10.18632/oncotarget.17996

Figure 1. Antigens, post-selection analysis and characterization of scFv clones.

Figure 1

(A) Sequence alignment of v6 region of human CD44v6 and peptides used in this study. v6 1-21, v6 12-32 and v6 22-43 are overlapping peptides that span the entire sequence of v6. 14-mer peptides derived from human and rat CD44v6, respectively, were used for blocking and species specificity testing. (B) Phage-scFv ELISA with the population isolated after two selection rounds. Binding is specific to the v6-positive antigen, CD44v3-10, with very limited binding to the v6-negative protein, CD44v3-10Δv6, and no binding to three other proteins (hNRR1-Fc, hNRR1-CD4 and Fc-His-FLAG) included as controls. Bars and standard deviation are from two reads of each sample. Background signal from a negative control (no phage) is subtracted. (C) ELISA with polyclonal phage populations from affinity maturation selection rounds 3 (Rd3) and 4 (Rd4). Antigen concentrations used to select respective populations were between 10 - 0.1 pM as indicated. All populations have specific binding to CD44v3-10 (grey) with very limited reactivity to CD44v3-10Δv6 (black). A450 from background (no phage) has been subtracted. (D) Cluster of selected affinity-matured scFv clones. Seven clones identified after affinity maturation are included, the top candidates scFv-A11 and scFv-H12 (indicated in bold) belong to the same high affinity cluster. The scale bar indicates the number of substitutions per residue, i.e. a longer distance corresponds to larger differences between the sequences. The sequence similarity tree was constructed using the Geneious software v7.1.9 (Biomatters, Auckland, New Zealand). (E) Representative sensorgram for scFv-A11 binding to immobilized CD44v3-10. The curves represent 50, 25, 12.5 and 6.3 nM, respectively. Solid lines are fitted to a 1:1 Langmuir binding isotherm using the ProteOn Manager software 3.1.0.6. (F) Blocking and species specificity of scFv-A11. Both scFv-A11 alone (50 nM) and scFv-A11 pre-incubated with a 10-fold molar excess of rat 14-mer peptide bind to immobilized CD44v3-10 (top two curves). Pre-incubation with human 14-mer peptide blocked binding (lower curve). Injection of 14-mers alone did not give any signal (not shown).