Trophoblast cell surface antigen 2 (TROP2) exhibits aberrant expression in pancreatic cancer, correlating with metastasis, advanced tumor stage, and poor prognosis in patients with pancreatic ductal adenocarcinoma. TROP2 has been recognized as a promising therapeutic target for antibody–drug conjugates (ADC), as evidenced by the approval of the anti-TROP2 ADC Trodelvy for the treatment of triple-negative breast cancer (TNBC). In this study, we report the generation of novel second-generation amanitin-based ADCs (ATAC) targeting TROP2, comprising the humanized RS7 antibody of Trodelvy (hRS7) and the highly potent payload amanitin. The specific in vitro binding, efficient antigen internalization, and high cytotoxicity of hRS7 ATACs with EC50 values in the picomolar range in TROP2-expressing cells constituted the foundation for preclinical in vivo evaluation. The hRS7 ATACs demonstrated a significant reduction in tumor growth in vivo in subcutaneous xenograft mouse models of pancreatic cancer and TNBC at well-tolerated doses. The antitumor efficacy correlated with the level of TROP2 expression on the tumors and the in vivo tumor uptake of the ATACs. The long half-life of 9.7 to 10.7 days of hRS7 ATACs without premature payload release in serum supported a high therapeutic index. Notably, the efficacy of the hRS7 ATACs was superior to that of Trodelvy with complete tumor eradication in both refractory pancreatic cancer and TNBC xenograft models. In summary, hRS7 ATACs represent a highly effective and well-tolerated targeted therapy, and our data support their development for pancreatic cancer and other TROP2-expressing tumors.

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©2024 The Authors; Published by the American Association for Cancer Research
2024
American Association for Cancer Research
This open access article is distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) license.
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Supplementary data

Supplementary Data

MS spectra of DAR determination. Western blotting of TROP2 expression on PDAC cell lines. Internalization of antibody in PDAC cell lines. In vitro cytotoxicity of ADCs MTD and efficacy studies. Spiderplot of animals treated with ADCs.

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Figure S1

Deconvoluted MS spectra for hRS7 ATAC 1 (A) and hRS7 ATAC 1 (B) conjugates. The drug-to-antibody (DAR) ratio was measured using intact LC-MS analysis.

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Figure S2

Western blotting of TROP2 expression on PANC-1, BxPC-3, Capan-1 and Capan-2 cells. hRS7-LALA-DC was used as primary antibody and rabbit anti-human-IgG-HRP (Abcam) as secondary antibody. Chemiluminescence signal was detected by WB imager (Azure C400 imaging). The different apparent size of TROP-2 in BxPC3 and Capan 2 cells is most likely due to differential glycosylation [44] and/or from different proteolytic cleavage of TROP2 [45]. In Capan-1 cells TROP2 is not detected, most likely due to proteolytical cleavage resulting in an isoform that cannot be detected by the hRS7 antibody [45], [46].

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Figure S3

Internalization of hRS7-LALA-DC antibody in PDAC cell lines. Flow cytometry gating was performed according to the PANC-1 cells that do not express TROP2.

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Figure S4

a. In vitro cytotoxicity of hRS7 ATAC 1 in tumor cells with medium (A; NCI-N87 cells) and low TROP-2 expression (B; MCF-7 cells) b. In vitro cytotoxicity of hRS7 ATAC 1 in PDAC cells upon transient acidosis or acid adaptation. BxPC-3 (TROP2+++) and Capan-1 (TROP2++) cells were incubated at normal or pH 6.6 medium for 24 hours, 7 days or 1 month and then treated for 96 hours with hRS7 ATAC 1. c. In vitro cytotoxicity of Trodelvy 1 in TNBC cells d. In vitro cytotoxicity of Trodelvy compared to ATAC 1 in TNBC cells.

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Figure S5

MTD of hRS7 ATAC 1 and hRS7 ATAC 2. The tolerability was assessed in 7-week-old tumor free female NMRI nude mice by single dose intravenous administration of hRS7 ATACs. At a dose of 10 mg/kg ATAC 1, one of three animals was found dead within 48 h after treatment and thus a dose of 8 mg/kg was considered to be MTD. ATAC2 was tested up to a maximum dose of 80 mg/kg – no higher doses were tested. Therefore, the MTD was considered to be ≥ 80 mg/kg.

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Figure S6

In vivo antitumor efficacy of individual animals treated with Trodelvy compared to hRS7 ATAC 1 in TNBC xenograft mouse model. Female NMRI nude mice bearing HCC70 subcutaneous tumors were treated either with PBS or the indicated doses of hRS7 ATAC 1 (cleavable linker) or Trodelvy. Efficacy was monitored for 76 days and evaluated by average tumor volume ±SEM, n = 10.

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Table S1

Dose of hRS7 ATAC 1 and ATAC 2 with indicated dose of α-amanitin used in BxPC-3 and Capan-1 dose-response efficacy studies.

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