Supplementary Methods, SupplementaryTables 1-2, and Supplementary Figures 1-7 from MI130004, a Novel Antibody–Drug Conjugate Combining Trastuzumab with a Molecule of Marine Origin, Shows Outstanding <i>In Vivo</i> Activity against HER2-Expressing Tumors (2023)

&lt;p&gt;Description of synthetic method for PM120160 Supplementary Figure 1. Analysis of MI130004. A: Analytical size exclusion chromatography analysis performed on a Superdex-100 10/300 column running an isocratic method with PBS at 1 mL/min. B: HIC analysis of trastuzumab (dotted red line) and MI130004 (solid blue line) run as described under Materials and Methods. The dotted black line shows the ammonium sulfate gradient. C: UV-Vis chromatogram at 280 nm resulting from the elution of trastuzumab digested with IdeS and then reduced with DTT. D: UV-Vis chromatogram at 280 nm resulting from the elution of MI130004 digested with IdeS and then reduced with DTT. Supplementary Figure 2. Deconvoluted mass spectra of peaks common to trastuzumab and MI130004 in Suppl. Fig. 1. A: Analysis of peak eluted at 12.7 min. B: Analysis of peak eluted at 15.2 min. C: Analysis of peak eluted at 19.0 min. Supplementary Figure 3. Deconvoluted mass spectra of peaks exclusive for MI130004 in Suppl. Fig. 1. A: Analysis of peak eluted at 20.82 min. B: Analysis of peak eluted at 23.88 min. Supplementary Figure 4. Antiproliferative assay showing the in vitro potency of MI130004. The assay was performed as described under Materials and Methods. A: Effect on breast tumor cells: JIMT-1 (HER2 positive, solid circles), BT-474 (HER2 positive, solid squares), MDA-MB-231 (HER2 negative, hollow circles) and MCF7 (HER2 negative, hollow squares) cell lines. B: Effect on gastric tumor cells: HGC-27 (HER2 positive, solid circles) and Hs 746T (HER2 negative, hollow circles) cell lines. Supplementary Figure 5. Biological effects of PM050489 in tumor cell lines of bbreast and gastric origins. A: Fluorescence microscopy analysis of cells treated with 1% (v/v) DMSO ("control") or with 1 nM PM050489 in 1% DMSO. Cells were stained for α-tubulin (green), γ-tubulin (red) and nuclei (blue).. B: Effect of PM050489 in cell cycle as determined by flow cytometry. Figures denote the percentage of cells arrested in G2/M according to the area of the peaks pointed by the arrows. Supplementary Figure 6. Biological effects of MI130004 in tumor cell lines of breast and gastric origin. Fluorescence microscopy analysis of cells treated with MI130004 at 0.1 µg/mL. Cells were stained for α-tubulin (green), γ-tubulin (red) and nuclei (blue). Left hand column, control untreated cells. Right hand column, cells treated with MI130004. Supplementary Figure 7. Immunohistochemistry analysis of tumors from mice xenografts. Twenty-four hours after MI130004 first administration, HER2 levels were determined by immunohistochemistry, as described under Materials and Methods, in samples from MI130004-treated animals bearing breast JIMT-1, BT-474 (both HER2 positive), MDA-MB-231 (HER2 negative), gastric N87, Gastric-008 (bth HER2 positive), Hs 746T (HER2 negative) and ovarian SK-OV-3 and A2780cis (HER2 positive) tumor cells. Magnification is 20x. Supplementary Table 1: Supplementary Table 1. Mass assignments of species eluted in the UV chromatograms shown in Suppl. Fig. 1 Supplementary Table 2: Supplementary Table 2. Antiproliferative activity of PM050489 in HER2 positive and negative cell lines.&lt;/p&gt;