In vivo therapeutic efficacy studies

All animal experiments were conducted in accordance with guidelines approved by Washington University School of Medicine's Research Animal Resource Center and Institutional Animal Care and Use Committee. Eight- to 10-week-old nu/nu female or male mice and 4 to 6-week-old Balb/c female or male mice were obtained from Charles River Laboratories. The therapies were initiated when the tumor volume reached ~200 mm³. Tumor volumes were measured twice a week by caliper measurements. Humane endpoints were defined as tumor volumes >1,500 mm³, bodyweight loss of >20%, tumor ulceration, and other clinical symptoms of acute toxicity. At the terminal stage, tumors were collected for the western blot analyses.

Mice bearing HER2⁺ NCIN87 xenografts were stratified into the following groups: control (saline), Panitumumab (Pan), T-DXd, no click (Panitumumab plus T-DXd) and click (Pan-TCO plus T-DXd-Tz) cohort. Pan-TCO or Pan was administered at 10 mg kg⁻¹ via tail vein. At 24 h post-injection of Pan-TCO, the mice received an intravenous injection of T-DXd or T-DXd-Tz (10 mg kg⁻¹). Mice in Pan or T-DXd cohorts received intravenous injection of Pan or T-DXd (10 mg kg⁻¹) alone.

Nu/nu female or male mice (Charles River Laboratories) were injected subcutaneously with 5 million NCIN87 cells, and/or 0.25 million A431 cells in a 100 μL cell suspension of a 1:1 (v/v) mixture of medium with reconstituted basement membrane (BD Matrigel, BD Biosciences). Mice-bearing NCIN87/A431 bilateral tumors were developed by subcutaneous injection of NCIN87 and A431 cancer cells on the bilateral dorsal flank regions. When the tumor volume reached the size of 200 mm³, mice were randomized them into six cohorts: control (saline), Pan (5 mg kg⁻¹), ADC T-DXd alone (5 mg kg⁻¹), no click (Pan plus T-DXd, both at 5 mg kg⁻¹), and click (Pan-TCO plus T-DXd-Tz, both at 5 mg kg⁻¹).

Nu/nu male and female mice bearing NCIN87 xenografts were treated with T-DXd monotherapy intravenously (5 mg kg⁻¹). Nearly one month after therapy, mice were stratified into responders versus non-responders, based on the tumor volume measurements, western blot analyses, and HER2-PET imaging. HER2-targeting immuno-PET was performed at 24 h post-intravenous injection of [⁶⁴Cu]Cu-NOTA-trastuzumab (50 μg, 200 μCi). The mice that initially did not respond to T-DXd therapy (i.e., non-responder) were treated intravenously with Pan-TCO (5 mg kg⁻¹) on day 1 and T-DXd-Tz on day 2 (5 mg kg⁻¹ as described above), while the responders continued to receive T-DXd therapy (5 mg kg⁻¹).

Nu/nu female mice were implanted subcutaneously with one million estrogen receptor-positive BT474-trastuzumab resistant cells. Drinking water of mice was supplemented with 0.67μg mL⁻¹ of β-estradiol (Sigma) from 1 week in advance of tumor inoculation and continued until mice were sacrificed. Fresh-estradiol supplemented water was provided twice a week. The T-DXd ADC therapy was initiated when the tumor volume reached 200 mm³. The tumor volumes were measured twice a week for a period of ~1 month. Mice were then stratified into responders versus non-responders, based on the tumor volume measurements. The responder mice were maintained on T-DXd (5 mg kg⁻¹) therapy and non-responder mice on the click therapy. In the click therapy cohort, the mice were injected with Per-TCO mAbs (5 mg kg⁻¹) on day 1. Following this, the mice were injected with T-DXd-Tz (5 mg kg⁻¹) on day 2 and tumor volumes were measured twice a week.

CT26-hHER2 cancer cells (0.25 million) were xenografted on the right shoulder of female BALB/c mice and treatments were initiated when the tumor volumes reached the size of 200–500 mm³. Then, mice were randomized into six cohorts: control (saline), Per (5 mg kg⁻¹), T-DXd (5 mg kg⁻¹), no click (Per plus T-DXd, 5 mg kg⁻¹), and click (Per-TCO plus T-DXd-Tz, 5 mg kg⁻¹). The mice that showed complete remission of the tumors after the therapy, were further rechallenged with 5 million of CT26-hHER2 cancer cells.