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Additional file 4. Flow-chart of the participants’ selection.

Additional file 2. The specific plan of diagnosis and treatment. BP, blood pressure. PSA, prostate-specific antigen. TRUS, transrectal ultrasonography. MRI, magnetic resonance imaging.

Additional file 1. Abnormal appearances in patients’ MRI. The blue arrow points to the seminal vesicle cyst; the red arrow points to the stone in the right seminal vesicle. MRI, magnetic resonance imaging.

Additional file 3. Flowchart of the surgery. For each key step in the surgery, “positive or negative” findings together with further guidance of the next move are listed in the flowchart.

Additional file 6: Table S1. Efficacy of H5B14-Mediated RON Internalization in Comparison with Other Anti-RON mAbs.

Additional file 5: Figure S5. Effect of H5B14-based ADCs on mouse bodyweight. Female athymic nude mice (five mice per group) were injected with H5B14-MMAe or H5B14-DCM at 40, 60, 80, and 100â mg/kg in a single dose through the tail vein, respectively. Animals were monitored daily for activity, responsiveness, food consumption, and others. Individual mice were weighted every day to reach an average bodyweight for each group. All animals were sacrificed at the end of the study.

Additional file 3: Figure S3. Stability of H5B14-based ADCs in PBS. H5B14-MMAE and H5B14-DCM at 10 μg/ml were incubated with 1 ml PBS at room temperature for 28 days. Samples were collected at different time intervals and analyzed by HIC. Individual peaks with different numbers of MMAE or DCM conjugated to H5B14 were marked as P0 to P6. The average DAR combining P2, P4, and P6 for both ADCs were calculated accordingly [1–3].

Additional file 4: Figure S4. The concentration-dependent effect of H5B14-based ADCs on cell viability. A panel of fifteen cancer cell lines expressing variable levels of RON was used as the model. Cells at 8000 cells per well in a 96-well plate in triplicate were treated with different amounts of H5B14-MMAE (A) or H5B14-DCM (B) for 72â h. Cell viability was determined by the MTT assay. Zt/g4-MMAE or Zt/g4-DCM were used for comparison.

Additional file 2: Figure S2. Interaction of H5B14 with RONs from different species. Stable NIH3T3 cells expressing human, monkey, or mouse RON were incubated in duplicate with different amounts of H5B14 followed by goat anti-human IgG coupled with FITC. Immunofluorescent intensities from individual samples were determined by flow cytometric analysis. Results are shown as the percentages of H5B14 specific binding to RON. The binding affinity (IC50) was calculated using the GraphPad Prism 7 software.

Tables S1. Pathological and Biological Features of Primary PDAC Cell Lines from Patient-Derived Xenograft Tumors*. Table S2. Adverse Effects of H-Zt/g4-MMAE on blood leukocyte and erythrocytes in Cynomolgus monkey. Table S3. Effect of H-Zt/g4-MMAE in vivo on various enzymatic activities in blood samples collected from cynomolgus monkeys. (PDF 663 kb)

Figure S1. Immune reactivity of H-Zt/g4 to RON in multiple tissues from human and cynomolgus monkey. Figure S2. Effect of H-Zt/g4-MMAE on eradication of RON-expressing cells in PDAC xenograft tumors. Figure S3. Induction in vitro by H-Zt/g4-MMAE of death of PDAC stem-like cells. Figure S4. Effect of H-Zt/g4-MMAE on elimination of RON-expressing cells in PSC + 24/44/ESA cell-mediated xenografts. Figure S5A. Cell surface RON expression by a panel of primary PDX cell lines. Figure S5B. Induction of cell surface RON internalization by H-Zt/g4. Figure S5C. Effect of H-Zt/g4-MMAE on viability of primary PDAC cell lines derived from patient’ tumors. Figure S6. Histological examination of multiple organs and/or tissues from cynomolgus monkey treated with H-Zt/g4-MMAE. Figure S7. Histological examination of multiple organs and/or tissues from cynomolgus monkey treated with H-Zt/g4-MMAE. (PDF 2315 kb)

Additional file 1: Figure S1. Schematic representation of procedures for generation of mouse mAbs specific to the RON PSI domain. (A) Structure and sequence of the human RON PSI domain. A peptide containing 43 amino acids from Gly526 to Pro568 corresponding to the entire PSI domain was presented. (B) The synthetic peptide conjugated to KLH was used for mouse immunization. Individual hybridomas cell lines were obtained through classical methods for mouse mAb production. The mAbs specific to the RON PSI domain such as PCM5B14 were verified by ELISA, immunoprecipitation, and immunofluorescence analyses.