Fig. 1 (abstract P-341).

(A) Schematic diagram of the pIT2-derived Anti-HER2 phagemid expression cassette. Trastuzumab scFv is cloned under the transcriptional control of lactose promoter (Lacpro), the sequence is in-frame with the gene encoding pIII phage coat protein (gIII); the vector contains a leader sequence (pelB) and a myc/His6tag. The cloning sites used are indicated in italics. (B) Flow Cytometry analysis of phage binding to HER2-positive cells (BT474, HCC1954) and HER2-negative cells (MCF-7, HCC1806). Cells were incubated with 1x1012 phage particles for 1 hour with Anti-HER2 phages (blue), non-relevant phages (red). Phage binding was detected by a mouse Anti-M13 antibody. Control staining shown in grey. Increase in fluorescence indicates binding of Anti-HER2 phage to cells. (C) Phagemid quantification by qPCR in each round of panning using primers specific to the trastuzumab scFv sequence. Phage DNA was isolated directly from 1/5 of the output phages and the number of phagemid copies was measured with the SYBR Green Master Mix I. Number of copies was estimated using a calibration with 108 – 102 Anti-HER2 phage particles. (D) Monoclonal phage-ELISA of 4 rounds of panning using the human HER2 protein. The graph shows an increase in percentage of clones with affinity to the human HER2 protein after each round of selection on BT474 (black bars), while for HCC1806 (grey bars) the percentage is always lower than 10%