Stable and Potent Selenomab-Drug Conjugates

Abstract

Selenomabs are engineered monoclonal antibodies with one or more translationally incorporated selenocysteine residues. The unique reactivity of the selenol group of selenocysteine permits site-specific conjugation of drugs. Compared with other natural and unnatural amino acid and carbohydrate residues that have been used for the generation of site-specific antibody-drug conjugates, selenocysteine is particularly reactive, permitting fast, single-step, and efficient reactions under near physiological conditions. Using a tailored conjugation chemistry, we generated highly stable selenomab-drug conjugates and demonstrated their potency and selectivity in vitro and in vivo. These site-specific antibody-drug conjugates built on a selenocysteine interface revealed broad therapeutic utility in liquid and solid malignancy models.

Keywords: antibody-drug conjugates; cancer therapy; selenocysteine; site-specific conjugation.

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 3 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 (A), left and 100% remaining iodoacetamide conjugate refers to the intensity of the band at time point “0” in (A), right. (C) Scheme of the reaction between scFv-Fc-Sec and iodoacetamide-fluorescein (top) or maleimide-fluorescein (bottom). See also Figure S2.

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 h at 37° C (mean ± SD of triplicates). Anti-CD79B scFv-Fc-Sec/CN29 served as non-targeting control ADC. See also Figure S1, Figure S2, Figure S4, Table S1, Table S2, and Table S4.

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 5 groups comprising 5 mice each, and treated with i.v. (tail vein) injections of the indicated ADCs and controls four times every four days at the indicated doses. Mean ± SD values are plotted; the p values (t-test) compare the treatment groups to the vehicle alone group.

Figure 4. In vivo activity of the CD138-targeting selenomab-drug conjugate

Human multiple myeloma cell line U266-ffluc was i.v. (tail vein) injected into NSG mice. After 4 weeks, the mice were randomized into 3 groups comprising 8–9 mice each and treated with i.v. (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 to the unconjugated antibody group. (B) Kaplan-Meier survival curve. The p value (log-rank test) compares the ADC group to the unconjugated antibody group. See also Figure S3 and Table S3.

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 h at 37° C (mean ± SD of triplicates). Anti-CD79B scFv-Fc(Ser396Sec)/CN29 served as non-targeting control ADC. See also Figure S5, Figure S6, Figure S7, and Table S1.

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 5 groups comprising 6 mice each, and treated with i.v. (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 i.v. with 6 mg/kg of 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 ECD 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 Figure S5, Figure S6, Figure S7, and Table S6.