The in vivo efficacy of trastuzumab-Flexmab-SG3710 was investigated after single-dose injections (0.30 mg/kg) in female athymic mice bearing NCI-N87 HER2-positive subcutaneous xenografts. NCI-N87 cells (5 x10⁶) in 50% Matrigel were inoculated subcutaneously into 46 week-old female athymic nude mice. Five mice per group were dosed intravenously five days after their tumors reached a volume of 200 mm³.

The in vivo efficacy of trastuzumab-Flexmab-SG3710 was investigated after single-dose injections (0.6 mg/kg) in female athymic mice bearing NCI-N87 HER2-positive subcutaneous xenografts. NCI-N87 cells (5 x10⁶) in 50% Matrigel were inoculated subcutaneously into 46 week-old female athymic nude mice. Five mice per group were dosed intravenously five days after their tumors reached a volume of 200 mm³.

The in vivo efficacy of trastuzumab-Flexmab-SG3710 and trastuzumab-C239i-SG3249 ADCs was investigated after single-dose injections (1 mg/kg) in female athymic mice bearing NCI-N87 HER2-positive subcutaneous xenografts,and tumor growth was monitored for 85 days. NCI-N87 cells (5 x10⁶) in 50% Matrigel were inoculated subcutaneously into 46 week-old female athymic nude mice. Five mice per group were dosed intravenously five days after their tumors reached a volume of 200 mm³.

The in vivo efficacy of trastuzumab-C239i-SG3249 was investigated after single-dose injections (0.30 mg/kg) in female athymic mice bearing NCI-N87 HER2-positive subcutaneous xenografts. NCI-N87 cells (5 x10⁶) in 50% Matrigel were inoculated subcutaneously into 46 week-old female athymic nude mice. Five mice per group were dosed intravenously five days after their tumors reached a volume of 200 mm³.

The in vivo efficacy of trastuzumab-Flexmab-SG3710 and trastuzumab-C239i-SG3249 ADCs was investigated after single-dose injections (1 mg/kg) in female athymic mice bearing NCI-N87 HER2-positive subcutaneous xenografts,and tumor growth was monitored for 85 days. NCI-N87 cells (5 x10⁶) in 50% Matrigel were inoculated subcutaneously into 46 week-old female athymic nude mice. Five mice per group were dosed intravenously five days after their tumors reached a volume of 200 mm³.

Cells at 0.5 × 105/mL were cultured for 5 days in mAb or ADC or medium (in triplicate wells) in wells of flat-bottom 96-well plates. PrestoBlue Cell Viability reagent (Thermo Fisher Scientific) was added and incubated for 1.5 h. Fluorescence intensity from each well was detected using a FLUOstar OPTIMA microplate reader (BMG Labtech, Aylesbury, UK) using excitation filter of 560-10 and emission filter of 590-10, and gain at 1,500. Data were plotted using GraphPad Prism software, and sigmoid dose-response non-linear regression was carried out to determine IC50 values.

Cells at 0.5 × 105/mL were cultured for 5 days in mAb or ADC or medium (in triplicate wells) in wells of flat-bottom 96-well plates. PrestoBlue Cell Viability reagent (Thermo Fisher Scientific) was added and incubated for 1.5 h. Fluorescence intensity from each well was detected using a FLUOstar OPTIMA microplate reader (BMG Labtech, Aylesbury, UK) using excitation filter of 560-10 and emission filter of 590-10, and gain at 1,500. Data were plotted using GraphPad Prism software, and sigmoid dose-response non-linear regression was carried out to determine IC50 values.

Cells at 0.5 × 105/mL were cultured for 5 days in mAb or ADC or medium (in triplicate wells) in wells of flat-bottom 96-well plates. PrestoBlue Cell Viability reagent (Thermo Fisher Scientific) was added and incubated for 1.5 h. Fluorescence intensity from each well was detected using a FLUOstar OPTIMA microplate reader (BMG Labtech, Aylesbury, UK) using excitation filter of 560-10 and emission filter of 590-10, and gain at 1,500. Data were plotted using GraphPad Prism software, and sigmoid dose-response non-linear regression was carried out to determine IC50 values.

Cells at 0.5 × 105/mL were cultured for 5 days in mAb or ADC or medium (in triplicate wells) in wells of flat-bottom 96-well plates. PrestoBlue Cell Viability reagent (Thermo Fisher Scientific) was added and incubated for 1.5 h. Fluorescence intensity from each well was detected using a FLUOstar OPTIMA microplate reader (BMG Labtech, Aylesbury, UK) using excitation filter of 560-10 and emission filter of 590-10, and gain at 1,500. Data were plotted using GraphPad Prism software, and sigmoid dose-response non-linear regression was carried out to determine IC50 values.

Cells at 0.5 × 105/mL were cultured for 5 days in mAb or ADC or medium (in triplicate wells) in wells of flat-bottom 96-well plates. PrestoBlue Cell Viability reagent (Thermo Fisher Scientific) was added and incubated for 1.5 h. Fluorescence intensity from each well was detected using a FLUOstar OPTIMA microplate reader (BMG Labtech, Aylesbury, UK) using excitation filter of 560-10 and emission filter of 590-10, and gain at 1,500. Data were plotted using GraphPad Prism software, and sigmoid dose-response non-linear regression was carried out to determine IC50 values.

Cells at 0.5 × 105/mL were cultured for 5 days in mAb or ADC or medium (in triplicate wells) in wells of flat-bottom 96-well plates. PrestoBlue Cell Viability reagent (Thermo Fisher Scientific) was added and incubated for 1.5 h. Fluorescence intensity from each well was detected using a FLUOstar OPTIMA microplate reader (BMG Labtech, Aylesbury, UK) using excitation filter of 560-10 and emission filter of 590-10, and gain at 1,500. Data were plotted using GraphPad Prism software, and sigmoid dose-response non-linear regression was carried out to determine IC50 values.

A total of 1 × 104 T cell cancer cells or the normal human T cells was suspended in 200 ul of RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin in 96-well flat-bottom tissue culture-treated plates. The following drugs were resuspended in DMSO solution to make a 10 mM stock solution; exatecan mesylate (MedChemExpress HY-13631A), DM1 (MedChemExpress HY-19792), SN38 (MedChemExpress HY-13704), MMAE (MedChemExpress HY-15162), SG3199 (MedChemExpress HY-101161). The drugs were added to the target cells at the concentrations specified in the text for 5 days at 37 °C. For luciferase-expressing cells, cell viability was assayed using the ONE-Glo luciferase assay (E6110, Promega) according to the manufacturer's instructions. Luminescence data were collected using the Synergy H1 microplate reader and analysed with the Biotek Gen5 software. Viability was calculated as the ratio of the luminescence signal to the no antibody or control antibody condition: (antibody well luminescence)/ (no antibody or control antibody well luminescence).

A total of 1 × 104 T cell cancer cells or the normal human T cells was suspended in 200 ul of RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin in 96-well flat-bottom tissue culture-treated plates. The following drugs were resuspended in DMSO solution to make a 10 mM stock solution; exatecan mesylate (MedChemExpress HY-13631A), DM1 (MedChemExpress HY-19792), SN38 (MedChemExpress HY-13704), MMAE (MedChemExpress HY-15162), SG3199 (MedChemExpress HY-101161). The drugs were added to the target cells at the concentrations specified in the text for 5 days at 37 °C. For luciferase-expressing cells, cell viability was assayed using the ONE-Glo luciferase assay (E6110, Promega) according to the manufacturer's instructions. Luminescence data were collected using the Synergy H1 microplate reader and analysed with the Biotek Gen5 software. Viability was calculated as the ratio of the luminescence signal to the no antibody or control antibody condition: (antibody well luminescence)/ (no antibody or control antibody well luminescence).

EoL-1, EoL-1/hTIM-3 and Kasumi-3 cells were incubated with increasing concentrations of KK2845 or KM8047-SG3249 in RPMI1640 medium supplemented with 10 vol% (for EoL-1 and EoL-1/hTIM-3 cells) or 20 vol% (for Kasumi-3 cells) heat-inactivated fetal bovine serum, 20 vol% heat-inactivated human serum (Sigma), and a mixture of 100 U/mL penicillin and 100 ug/mL streptomycin at 37 °C for 4 days. After incubation, the cells were treated with Cell Counting Kit-8 (CCK-8, Dojindo Laboratories), and the absorbance was measured using a Multiskan Ascent device (Thermo Fisher Scientific, Ascent Software: version 2.6). Primary AML cells were incubated with increasing concentrations of KM8047-SG3249, KK2845, or CD33-ADC in IMDM medium (Thermo Fisher Scientific) supplemented with 20 vol% heat-inactivated human serum, a mixture of 100 U/mL penicillin and 100 ug/mL streptomycin, 25 ng/mL hIL-3 (Miltenyi Biotec), 25 ng/mL hSCF (Peprotech), 25 ng/mL hGM-CSF (R&D), and 25 ng/mL hG-CSF (R&D) at 37 °C for 4 days. After incubation, the cells were stained by anti-CD45 antibody-FITC (BD Pharmingen, HI30 clone), anti-CD34 antibody-APC (BD Bioscience, 8G12 clone), anti-CD38 antibody-BV421 (BD Bioscience, HIT2 clone) and 7-amino-actinomycin D (7-AAD, Bio-Rad Laboratories). The number of viable cells in the CD34+ fraction, CD34+CD38- fraction, or CD34- fraction in primary AML cells was counted by flow cytometry (FACSVerse, software program: BD FACSUITE-V1.0.5).

EoL-1, EoL-1/hTIM-3 and Kasumi-3 cells were incubated with increasing concentrations of KK2845 or KM8047-SG3249 in RPMI1640 medium supplemented with 10 vol% (for EoL-1 and EoL-1/hTIM-3 cells) or 20 vol% (for Kasumi-3 cells) heat-inactivated fetal bovine serum, 20 vol% heat-inactivated human serum (Sigma), and a mixture of 100 U/mL penicillin and 100 ug/mL streptomycin at 37 °C for 4 days. After incubation, the cells were treated with Cell Counting Kit-8 (CCK-8, Dojindo Laboratories), and the absorbance was measured using a Multiskan Ascent device (Thermo Fisher Scientific, Ascent Software: version 2.6). Primary AML cells were incubated with increasing concentrations of KM8047-SG3249, KK2845, or CD33-ADC in IMDM medium (Thermo Fisher Scientific) supplemented with 20 vol% heat-inactivated human serum, a mixture of 100 U/mL penicillin and 100 ug/mL streptomycin, 25 ng/mL hIL-3 (Miltenyi Biotec), 25 ng/mL hSCF (Peprotech), 25 ng/mL hGM-CSF (R&D), and 25 ng/mL hG-CSF (R&D) at 37 °C for 4 days. After incubation, the cells were stained by anti-CD45 antibody-FITC (BD Pharmingen, HI30 clone), anti-CD34 antibody-APC (BD Bioscience, 8G12 clone), anti-CD38 antibody-BV421 (BD Bioscience, HIT2 clone) and 7-amino-actinomycin D (7-AAD, Bio-Rad Laboratories). The number of viable cells in the CD34+ fraction, CD34+CD38- fraction, or CD34- fraction in primary AML cells was counted by flow cytometry (FACSVerse, software program: BD FACSUITE-V1.0.5).

EoL-1, EoL-1/hTIM-3 and Kasumi-3 cells were incubated with increasing concentrations of KK2845 or KM8047-SG3249 in RPMI1640 medium supplemented with 10 vol% (for EoL-1 and EoL-1/hTIM-3 cells) or 20 vol% (for Kasumi-3 cells) heat-inactivated fetal bovine serum, 20 vol% heat-inactivated human serum (Sigma), and a mixture of 100 U/mL penicillin and 100 ug/mL streptomycin at 37 °C for 4 days. After incubation, the cells were treated with Cell Counting Kit-8 (CCK-8, Dojindo Laboratories), and the absorbance was measured using a Multiskan Ascent device (Thermo Fisher Scientific, Ascent Software: version 2.6). Primary AML cells were incubated with increasing concentrations of KM8047-SG3249, KK2845, or CD33-ADC in IMDM medium (Thermo Fisher Scientific) supplemented with 20 vol% heat-inactivated human serum, a mixture of 100 U/mL penicillin and 100 ug/mL streptomycin, 25 ng/mL hIL-3 (Miltenyi Biotec), 25 ng/mL hSCF (Peprotech), 25 ng/mL hGM-CSF (R&D), and 25 ng/mL hG-CSF (R&D) at 37 °C for 4 days. After incubation, the cells were stained by anti-CD45 antibody-FITC (BD Pharmingen, HI30 clone), anti-CD34 antibody-APC (BD Bioscience, 8G12 clone), anti-CD38 antibody-BV421 (BD Bioscience, HIT2 clone) and 7-amino-actinomycin D (7-AAD, Bio-Rad Laboratories). The number of viable cells in the CD34+ fraction, CD34+CD38- fraction, or CD34- fraction in primary AML cells was counted by flow cytometry (FACSVerse, software program: BD FACSUITE-V1.0.5).

Primary human CD34+ cells were purchased from STEMCELL technologies (Vancouver, BC, Canada). The cells were obtained using Institutional Review Board (IRB)-approved consent forms and protocols. The steady-state human bone marrow CD34+ cells (3 donors, STEMCELL technologies) or purified mobilized CD34+ cells from two healthy rhesus macaques were cultured (2500 cells/well in 384-well plate) in serum-free StemSpan SFEM media (45 ul, STEMCELL Technologies) supplemented with 100 ng/ml each of Interleukin 6 (IL-6), fms-related tyrosine kinase 3 ligand (FLT3L), and thrombopoietin (TPO). Escalating doses of CD117-ADC as well as IgG isotype control-conjugated ADC were added (5 ul ADC added to 45 ul cells). After 6 days of culture, the number of viable CD34 + CD90+ cells were evaluated by flow cytometry using 7-Aminoactinomycin D (7-AAD as a viability marker, Biolegend, San Diego, CA, USA), CD34 BV785 (clone 561, BioLegend), and CD90 APC (Clone 5E10, BioLegend) detection antibodies. Cells were acquired on a BD Celesta Instrument and analyzed with FloJo. The EC50 and EC90 values were calculated with GraphPad Prism software.

Primary human CD34+ cells were purchased from STEMCELL technologies (Vancouver, BC, Canada). The cells were obtained using Institutional Review Board (IRB)-approved consent forms and protocols. The steady-state human bone marrow CD34+ cells (3 donors, STEMCELL technologies) or purified mobilized CD34+ cells from two healthy rhesus macaques were cultured (2500 cells/well in 384-well plate) in serum-free StemSpan SFEM media (45 ul, STEMCELL Technologies) supplemented with 100 ng/ml each of Interleukin 6 (IL-6), fms-related tyrosine kinase 3 ligand (FLT3L), and thrombopoietin (TPO). Escalating doses of CD117-ADC as well as IgG isotype control-conjugated ADC were added (5 ul ADC added to 45 ul cells). After 6 days of culture, the number of viable CD34 + CD90+ cells were evaluated by flow cytometry using 7-Aminoactinomycin D (7-AAD as a viability marker, Biolegend, San Diego, CA, USA), CD34 BV785 (clone 561, BioLegend), and CD90 APC (Clone 5E10, BioLegend) detection antibodies. Cells were acquired on a BD Celesta Instrument and analyzed with FloJo. The EC50 and EC90 values were calculated with GraphPad Prism software.