TF-positive patient-derived xenograft (PDX) models were performed in athymic nude mice to evaluate the efficacy of the ADCs in vivo. Study animals were implanted unilaterally on the left flank with tumor fragments. Animals were randomized and treated as indicated in the figures. Animals were removed from study and euthanized once tumor size reached 1,200 mm³ or skin ulceration was evident. In addition, the MTV curve for the treatment group in question was no longer shown once an animal was removed from study due to size TGI and statistical analyses were conducted in the same manner as for the CDX studies. The CR and PR response definitions were as follows for the PDX studies: a PR responder had a MTV 30% of MTV at day 1 for two consecutive measurements; a CR responder had an undetectable MTV for two consecutive measurement IHC analisys: Formalin-fixed paraffin-embedded (FFPE) tissues were sectioned at 4-m thickness and mounted onto positive-charged glass slides The tissue sections were stained with the anti-TF antibody HTF-1 ADC treatment started on day 1 after animals with a tumor size of approximately 190 mm³ The model dosed weekly at 25 mg/kg for 3 weeks.

TF-positive patient-derived xenograft (PDX) models were performed in athymic nude mice to evaluate the efficacy of the ADCs in vivo. Study animals were implanted unilaterally on the left flank with tumor fragments. Animals were randomized and treated as indicated in the figures. Animals were removed from study and euthanized once tumor size reached 1,200 mm³ or skin ulceration was evident. In addition, the MTV curve for the treatment group in question was no longer shown once an animal was removed from study due to size TGI and statistical analyses were conducted in the same manner as for the CDX studies. The CR and PR response definitions were as follows for the PDX studies: a PR responder had a MTV 30% of MTV at day 1 for two consecutive measurements; a CR responder had an undetectable MTV for two consecutive measurement IHC analisys: Formalin-fixed paraffin-embedded (FFPE) tissues were sectioned at 4-m thickness and mounted onto positive-charged glass slides The tissue sections were stained with the anti-TF antibody HTF-1 ADC treatment started on day 1 after animals with a tumor size of approximately 210 mm³ The model dosed weekly at 5 mg/kg for 2 weeks.

TF-positive patient-derived xenograft (PDX) models were performed in athymic nude mice to evaluate the efficacy of the ADCs in vivo. Study animals were implanted unilaterally on the left flank with tumor fragments. Animals were randomized and treated as indicated in the figures. Animals were removed from study and euthanized once tumor size reached 1,200 mm³ or skin ulceration was evident. In addition, the MTV curve for the treatment group in question was no longer shown once an animal was removed from study due to size TGI and statistical analyses were conducted in the same manner as for the CDX studies. The CR and PR response definitions were as follows for the PDX studies: a PR responder had a MTV 30% of MTV at day 1 for two consecutive measurements; a CR responder had an undetectable MTV for two consecutive measurement IHC analisys: Formalin-fixed paraffin-embedded (FFPE) tissues were sectioned at 4-m thickness and mounted onto positive-charged glass slides The tissue sections were stained with the anti-TF antibody HTF-1 ADC treatment started on day 1 after animals with a tumor size of approximately 140 mm³ The model dosed weekly at 4 mg/kg for 3 weeks.

Cell line-derived xenograft (CDX) models MDA-MB-231 epidermoid carcinoma and the HPAF-II pancreatic carcinoma cell lines were implanted subcutaneously in the flank of athymic nude mice Animals were removed from study and euthanized once tumor size reached 1200 mm³ or skin ulceration was evident ADC was dosed weekly at 2 mg/kg for 2 weeks.

Cell line-derived xenograft (CDX) models MDA-MB-231 epidermoid carcinoma and the HPAF-II pancreatic carcinoma cell lines were implanted subcutaneously in the flank of athymic nude mice Animals were removed from study and euthanized once tumor size reached 1200 mm³ or skin ulceration was evident ADC was dosed weekly at 4 mg/kg for 2 weeks.

Antibody-dependent cellular cytotoxicity (ADCC) A431 cells were plated on a microtiter plate The following day, the cells were incubated with a ten-point 1:3 dilution titration of anti-TF antibodies or the ADCs starting at 50 nM An ADCC effector-to-target cell ratio of 8:1 was added to each well and incubated for 6 h at 37°C Luciferase Assay Reagent was added to each well to measure luminescence on an Envision plate reader Antibody-dependent cellular cytotoxicity (ADCC) reporter luminescence was evaluated after a 6-hour incubation of the reporter Jurkat cell line with TF-positive A431 cells and a titration of anti-TF antibody or ADC The ADCC reporter luminescence EC50 values for each anti-TF antibody or ADC are listed.

To evaluate ADC cytotoxicity, cells were plated in 384-well plates Anti-TF antibodies conjugated to MC-vc-PAB-MMAE were serially diluted as shown Plates were incubated for 3 days, followed by lysis in CTG assay reagent For each ADC, the IC50 and its associated 95% confidence interval (95% CI) were calculated Titrations of the TF-specific ADCs were added to HPAF-II cells, with a 72-hour incubationThis treatment resulted in efficacious cell killing.

To evaluate ADC cytotoxicity, cells were plated in 384-well plates Anti-TF antibodies conjugated to MC-vc-PAB-MMAE were serially diluted as shown Plates were incubated for 3 days, followed by lysis in CTG assay reagent For each ADC, the IC50 and its associated 95% confidence interval (95% CI) were calculated Titrations of the TF-specific ADCs were added to 5-day old cultures of MDA-MB-231 cells, with a 72-hour incubationThis treatment resulted in efficacious cell killing.

To evaluate ADC cytotoxicity, cells were plated in 384-well plates Anti-TF antibodies conjugated to MC-vc-PAB-MMAE were serially diluted as shown Plates were incubated for 3 days, followed by lysis in CTG assay reagent For each ADC, the IC50 and its associated 95% confidence interval (95% CI) were calculated Titrations of the TF-specific ADCs were added to A431 cells, with a 72-hour incubationThis treatment resulted in efficacious cell killing.

To evaluate ADC cytotoxicity, cells were plated in 384-well plates Anti-TF antibodies conjugated to MC-vc-PAB-MMAE were serially diluted as shown Plates were incubated for 3 days, followed by lysis in CTG assay reagent For each ADC, the IC50 and its associated 95% confidence interval (95% CI) were calculated Titrations of the TF-specific ADCs were added to A431 cells, with a 4-hour incubation followed by removal of excess ADC and culture for another 68 hours This treatment resulted in efficacious cell killing.