2.8. In vivo tumor xenograft studies in mice

All in vivo xenograft studies were approved by the IACUC of Tanabe Research Laboratories, USA, Inc. (San Diego, CA, USA) and performed according to the company’s Institutional Animal Care Guidelines. H1975 and H1373 cancer cell lines were implanted subcutaneously at 5 × 10⁶ cells/animal into the right flank of female Nu/Nu mice obtained from Charles River (Wilmington, MA, USA). Animals were randomized after the average tumor volume reached 200–300 mm³. Mice were given a single intravenous injection of ADC, nontargeting control ADC, or vehicle control at doses described in the Figs ​Figs1,1, ​,2,2, ​,3,3, ​,4.4. Body weight and tumor volume were measured 2–3 times per week over the entire duration of the studies. Tumor volume was calculated as follows: V(mm³) = 0.5236 × length (mm) × width² (mm). Tumor volumes ± SEM were plotted in prism 7 (GraphPad). Statistical significance was determined with a one‐way ANOVA with Tukey’s or Dunnett’s multiple comparison test dependent on whether groups were compared to a control group or not. When only two dose groups were compared, an unpaired two‐tailed t‐test was performed in prism 7.

In vitro potency and in vivo efficacy of TR1801‐ADC with lung cancer cell lines H1975 (60 000 cMet receptors/cell) and H1373 (97 000 cMet receptors/cell) with medium–low cMet expression. Five‐day CellTiter‐Glo® cytotoxicity assays were run as duplicates and repeated at least one time. Lung cancer xenografts in Nu/Nu mice were inoculated with 5 × 10⁶ cells/mouse, and mice were injected with test articles at an average tumor volume of 200–300 mm³. Tumor volume is plotted in mm³ ± SEM. (A) Lung cancer cell lines H1975 and H1373 were treated with TR1801‐ADC, nontargeting ADC secukinumab–SG3249, cMet‐vc‐MMAE (10‐point dilution series with a starting concentration of 100 nm), or free PBD toxin SG3199 (starting concentration of 10 nm). (B) Lung cancer xenografts H1975 and H1373 were treated with single intravenous doses of vehicle (1× PBS), TR1801‐ADC (1 and 0.5 mg·kg⁻¹), cMet‐vc‐MMAE (5 and 1 mg·kg⁻¹), and nontargeting ADC (1 mg·kg⁻¹) with eight animals per group. Statistics: one‐way ANOVA with Dunnett’s multiple comparison test. Shown is only the significance between cMet‐vc‐MMAE, TR1801‐ADC, and rituximab‐SSC‐SG3249 in comparison with control (*P < 0.05, **P < 0.01, ***P < 0.001).

Expression of cMet in patient samples of gastric, colon, biliary tract, and head and neck (H&N) cancers. IHC was performed with SP44 rabbit monoclonal cMet antibody on TMAs (80–100 cores per indication were analyzed). Intensity of cMet staining was scored on a scale from 0 to 300 (H‐score) and grouped in four levels of Met expression (none, low, medium, and high) and plotted as % of patient samples.

Preclinical assessment of TR1801‐ADC in 10 HuPrime® gastric cancer PDX models. Female BALB/c nude mice were treated with a single intravenous dose of vehicle control, TR1801‐ADC, or nontargeting ADC (secukinumab–SG3249) when subcutaneous tumors reached an average size of 200 mm³. Ex vivo 3D methylcellulose assays were performed on selected gastric PDX over a 7‐day period with TR1801‐ADC, free PBD toxin SG3199, and cisplatin. Nine‐point dilution series were prepared with starting concentrations of 50, 10, and 100 µm, respectively. Assay was run in triplicates with an n = 1. (A) Two representative gastric PDX models GA3121 and GA0152. Tumor growth of each group (n = 10) was monitored after a single intravenous administration of vehicle (1× PBS), TR1801‐ADC (1, 0.5, 0.25, and 0.125 mg·kg⁻¹), or nontargeting ADC (1 mg·kg⁻¹). Statistics: one‐way ANOVA with Dunnett’s multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.001). (B) Ex vivo 3D assay performed with GA3121 and GA0152 PDX models and treated with free PBD toxin (SG3199), TR1801‐ADC, or cisplatin. (C) Representative IHC staining with rabbit monoclonal cMet antibody (SP44) on tissue sections of gastric cancer PDX models GA3121 and GA0152. (D) Plot of 10 gastric cancer PDX models. Tumor growth inhibition (%) at different dose concentrations (1, 0.5, 0.25, and 0.125 mg·kg⁻¹) of TR1801‐ADC.