Exatecan Antibody Drug Conjugates Based on a Hydrophilic Polysarcosine Drug-Linker Platform

Abstract

We herein report the development and evaluation of a novel HER2-targeting antibody-drug conjugate (ADC) based on the topoisomerase I inhibitor payload exatecan, using our hydrophilic monodisperse polysarcosine (PSAR) drug-linker platform (PSARlink). In vitro and in vivo experiments were conducted in breast and gastric cancer models to characterize this original ADC and gain insight about the drug-linker structure-activity relationship. The inclusion of the PSAR hydrophobicity masking entity efficiently reduced the overall hydrophobicity of the conjugate and yielded an ADC sharing the same pharmacokinetic profile as the unconjugated antibody despite the high drug-load of the camptothecin-derived payload (drug-antibody ratio of 8). Tra-Exa-PSAR10 demonstrated strong anti-tumor activity at 1 mg/kg in an NCI-N87 xenograft model, outperforming the FDA-approved ADC DS-8201a (Enhertu), while being well tolerated in mice at a dose of 100 mg/kg. In vitro experiments showed that this exatecan-based ADC demonstrated higher bystander killing effect than DS-8201a and overcame resistance to T-DM1 (Kadcyla) in preclinical HER2+ breast and esophageal models, suggesting potential activity in heterogeneous and resistant tumors. In summary, the polysarcosine-based hydrophobicity masking approach allowsfor the generation of highly conjugated exatecan-based ADCs having excellent physicochemical properties, an improved pharmacokinetic profile, and potent in vivo anti-tumor activity.

Keywords: antibody–drug conjugates; camptothecin; deruxtecan; polysarcosine; topoisomerase I inhibitor.

Figure 1

Chemical structures of the antibody-drug conjugates (ADC) drug-linkers used in the present study. See Supplementary Materials for detailed chemical synthetic procedures and exact ADC structures.

Figure 2

Physicochemical characterization of ADCs. (A) Hydrophobic Interaction Chromatograms (HIC) of DAR8 ADCs. (B) Size Exclusion Chromatograms (SEC) of DAR8 ADCs. (C) Ex-vivo rat plasma stability studies, as assayed by immunocapture and reversed phase HPLC—mass spectrometry. Low immunocapture recovery of the heavy-chain of the Tra-deruxtecan conjugate prevented us to report stability data at day 7 for this ADC. (D) HER2 ELISA binding affinity profiles of ADCs.

Figure 3

In vitro evaluation of Tra-Exa-PSAR10. (A) Trastuzumab and ADC cell binding assayed by flow cytometry. (B) In vitro cytotoxicity of ADCs in breast and gastric HER2+ and HER2- (MCF-7) cancer cell lines after 6-day exposure to trastuzumab conjugates, as assayed by MTT assay, n = 3. (C) HER2 cell surface expression in breast and gastric tumor cell lines as assayed by flow cytometry.

Figure 4

In vivo evaluation of Tra-Exa-PSAR10. (A) ADC pharmacokinetic study in Sprague-Dawley rats after a single intravenous ADC dose of 3 mg/kg. Total ADC concentration was assayed by anti-human IgG ELISA. (B) Antitumor activity in HER2+ SCID/BT-474 breast cancer model following a single intravenous ADC dose of 10 mg/kg. (C) Antitumor activity in HER2+ SCID/NCI-N87 gastric cancer model following a single sub-curative intravenous ADC dose of 1 mg/kg. (D) Mice tolerability experiment following a single intraperitoneal ADC dose of 100 mg/kg. (**: p < 0.01, ***: p < 0.001).

Figure 5

Bystander killing effect of Tra-Exa-PSAR10 compared to T-DM1 and trastuzumab-deruxtecan (DS-8201a) in co-culture in vitro. (A) Passive cell membrane diffusion of Exatecan and DXd payloads as assayed by a Corning^® Gentest^TM PAMPA assay. (B) In vitro bystander killing effect of Tra-Exa-PSAR10 compared to Tra-deruxtecan in co-cultured SKBR-3 (HER2+) and A549 (HER2-) cells that were treated with 10 nM ADCs for 5 days. Cell number and ratio of HER2+ and HER2- cells were determined by flow cytometry. T-DM1 (Kadcyla^®) was used as a negative control. (C) Representative flow cytometry data presented for 0:1, 1:0 and 2:1 cell ratio, showing no impact of the ADCs on HER2- A549 cells (0:1), activity against HER2+ SKBR-3 cells (1:0) and a stronger bystander activity of Tra-Exa-PSAR10 compared to Tra-deruxtecan. ns: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001). n = 3.

Figure 6

In vitro cytotoxicity of Tra-Exa-PSAR10 in cells resistant to T-DM1. (A) Cytotoxicity assay of T-DM1 in MDA-MB-361 and OE-19 sensitive (S) and T-DM1-resistant (TR and TCR) cells showing an increase in the IC₅₀ values of resistant cells compared to parental. (B) Exposure of cells resistant to T-DM1 to Tra-Exa-PSAR10 showing comparable cytotoxicity in parental and T-DM1 resistant cells. (C) Relative resistance of TR and TCR cell lines to T-DM1 or Tra-Exa-PSAR10 represented as the IC₅₀ of resistant cell line over the parental cell line for each experiment. *: p < 0,05. S: sensitive cells, TR: T-DM1 resistant cells and TCR: T-DM1 resistant cells generated in the presence of ciclosporin A.