Stable and Potent Selenomab-Drug Conjugates Xiuling Li, Christopher G. Nelson, Rajesh R. Nair, Lori Hazlehurst, Tina Moroni, Pablo Martinez-Acedo, Alex R. Nanna, David Hymel, Terrence R. Burke, Christoph Rader  Cell Chemical Biology  Volume 24, Issue 4, Pages 433-442.e6 (April 2017) DOI: 10.1016/j.chembiol.2017.02.012 Copyright © 2017 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2017 24, 433-442. e6DOI: (10. 1016/j. chembiol Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Human Plasma Stability of Selenomab-Fluorescein Conjugates Anti-HER2 scFv-Fc-Sec/maleimide-fluorescein versus anti-HER2 scFv-Fc-Sec/iodoacetamide-fluorescein conjugate stability in human plasma at 37°C. (A) Fluorescent (top) and Coomassie-stained (bottom) SDS-PAGE gels are shown. Antibody and HSA bands are indicated by arrows. Molecular weights from a pre-stained protein ladder are shown on the left. (B) Summary of selenomab-fluorescein conjugate stability from three independent experiments (mean ± SD) with band intensities quantified by NIH ImageJ software. Note that 100% remaining maleimide conjugate refers to the intensity of the band at time point “0” in the left panel of (A), and 100% remaining iodoacetamide conjugate refers to the intensity of the band at time point “0” in the right panel of (A). (C) Scheme of the reaction between scFv-Fc-Sec and iodoacetamide-fluorescein (top) or maleimide-fluorescein (bottom). See also Figure S2. Cell Chemical Biology 2017 24, 433-442.e6DOI: (10.1016/j.chembiol.2017.02.012) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Drugs and In Vitro Activity of HER2-Targeting Selenomab-Drug Conjugates (A) Structural formulas of iodoacetamido-caproyl-MMAF derivatives CN27, CN28, and CN29. (B) Cytotoxicity of anti-HER2 scFv-Fc-Sec conjugated to CN27, CN28, and CN29 following incubation with HER2-high breast cancer cell lines SK-BR-3 and KPL-4, and HER2-low breast cancer cell line MCF-7 for 72 hr at 37°C (mean ± SD of triplicates). Anti-CD79B scFv-Fc-Sec/CN29 served as non-targeting control ADC. See also Figures S1, S2, and S4; Tables S1, S2, and S4. Cell Chemical Biology 2017 24, 433-442.e6DOI: (10.1016/j.chembiol.2017.02.012) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 In Vivo Activity of the HER2-Targeting Selenomab-Drug Conjugate Human breast cancer cell line KPL-4 was xenografted into the mammary fat pads of female CD-1 nude mice, grown to ∼100 mm3, randomized into five groups comprising five mice each, and treated with intravenous (tail vein) injections of the indicated ADCs and controls four times every 4 days at the indicated doses. Mean ± SD values are plotted; the p values (t test) compare the treatment groups with the vehicle-alone group. Cell Chemical Biology 2017 24, 433-442.e6DOI: (10.1016/j.chembiol.2017.02.012) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 In Vivo Activity of the CD138-Targeting Selenomab-Drug Conjugate Human multiple myeloma cell line U266-ffluc was injected intravenously (tail vein) into NSG mice. After 4 weeks, the mice were randomized into three groups comprising eight to nine mice each and treated with intravenous (tail vein) injections of 3 mg/kg of the ADC, the unconjugated antibody, and vehicle alone. (A) Human lambda light chain concentrations correlating with tumor burden were determined by ELISA at the indicated time points and plotted as mean ± SD values. The p value (t test) compares the ADC group with the unconjugated antibody group. (B) Kaplan-Meier survival curve. The p value (log-rank test) compares the ADC group with the unconjugated antibody group. See also Figure S3 and Table S3. Cell Chemical Biology 2017 24, 433-442.e6DOI: (10.1016/j.chembiol.2017.02.012) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 In Vitro Activity of the Second-Generation HER2-Targeting Selenomab-Drug Conjugate Cytotoxicity of anti-HER2 scFv-Fc-Sec/CN29, anti-HER2 scFv-Fc(Ser396Sec)/CN29, anti-HER2 scFv-Fc(Ser396Sec)/MMAF, and ado-trastuzumab emtansine following incubation with HER2-high breast cancer cell lines KPL-4 and MDA-MB-361/DYT2, HER2-low breast cancer cell line MCF-7, and HER2-negative breast cancer cell line MDA-MB-468 for 72 hr at 37°C (mean ± SD of triplicates). Anti-CD79B scFv-Fc(Ser396Sec)/CN29 served as non-targeting control ADC. See also Figures S5–S7 and Table S1. Cell Chemical Biology 2017 24, 433-442.e6DOI: (10.1016/j.chembiol.2017.02.012) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 In Vivo Activity and Pharmacokinetics of the Second-Generation HER2-Targeting Selenomab-Drug Conjugate (A) Human breast cancer cell line KPL-4 was xenografted into the mammary fat pads of female NSG mice, grown to ∼150 mm3, randomized into five groups comprising six mice each, and treated with intravenous (tail vein) injections of the indicated ADCs and controls four times every week at 5 mg/kg. Mean ± SD values are plotted. (B) Four female CD-1 mice were injected intravenously with 6 mg/kg anti-HER2 scFv-Fc(Ser396Sec)/CN29. The plasma concentrations of total antibody and intact ADC were quantified by ELISA at the indicated time points, using HER2 extracellular domain for capturing and either anti-His mAb (total antibody) or anti-MMAF mAb (intact ADC) for detection. Shown are mean ± SD values for each time point. See also Figures S5–S7 and Table S6. Cell Chemical Biology 2017 24, 433-442.e6DOI: (10.1016/j.chembiol.2017.02.012) Copyright © 2017 Elsevier Ltd Terms and Conditions

Scheme S1 Solid-phase Synthesis of CN27 Cell Chemical Biology 2017 24, 433-442.e6DOI: (10.1016/j.chembiol.2017.02.012) Copyright © 2017 Elsevier Ltd Terms and Conditions

Scheme S2 Solid-phase Synthesis of CN28 and CN29 Cell Chemical Biology 2017 24, 433-442.e6DOI: (10.1016/j.chembiol.2017.02.012) Copyright © 2017 Elsevier Ltd Terms and Conditions

Scheme S3 Solid-phase Synthesis of Iodoacetamido-caproyl-MMAF Cell Chemical Biology 2017 24, 433-442.e6DOI: (10.1016/j.chembiol.2017.02.012) Copyright © 2017 Elsevier Ltd Terms and Conditions