TROP2-directed nanobody-drug conjugate elicited potent antitumor effect in pancreatic cancer

Abstract

Background: Pancreatic cancer is a highly aggressive malignancy with limited treatment options and a poor prognosis. Trophoblast cell surface antigen 2 (TROP2), a cell surface antigen overexpressed in the tumors of more than half of pancreatic cancer patients, has been identified as a potential target for antibody-drug conjugates (ADCs). Almost all reported TROP2-targeted ADCs are of the IgG type and have been poorly studied in pancreatic cancer. Here, we aimed to develop a novel nanobody-drug conjugate (NDC) targeting TROP2 for the treatment of pancreatic cancer.

Results: In this study, we developed a novel TROP2-targeted NDC, HuNb_TROP2-HSA-MMAE, for the treatment of TROP2-positive pancreatic cancer. HuNb_TROP2-HSA-MMAE is characterized by the use of nanobodies against TROP2 and human serum albumin (HSA) and has a drug-antibody ratio of 1. HuNb_TROP2-HSA-MMAE exhibited specific binding to TROP2 and was internalized into tumor cells with high endocytosis efficiency within 5 h, followed by intracellular translocation to lysosomes and release of MMAE to induce cell apoptosis in TROP2-positive pancreatic cancer cells through the caspase-3/9 pathway. In a xenograft model of pancreatic cancer, doses of 0.2 mg/kg and 1 mg/kg HuNb_TROP2-HSA-MMAE demonstrated significant antitumor effects, and a dose of 5 mg/kg even eradicated the tumor.

Conclusion: HuNb_TROP2-HSA-MMAE has desirable affinity, internalization efficiency and antitumor activity. It holds significant promise as a potential therapeutic option for the treatment of TROP2-positive pancreatic cancer.

Keywords: Antitumor effect; Mechanisms of action; Nanobody-drug conjugate; Pancreatic cancer; TROP2.

Fig. 1

Screening of anti-TROP2 nanobody. A Schematic illustration of the screening of TROP2-targeted nanobodies. B The affinity of TROP2 nanobody candidates for TROP2 protein was determined by ELISA. C Binding activity of TROP2 nanobody candidates to TROP2-expressing cells was assessed by flow cytometry. D Cells were incubated with nanobody candidates and the fluorescently labeled secondary antibodies, and the internalization of nanobodies was determined by measuring intracellular fluorescence intensity through flow cytometry

Fig. 2

Structure and binding affinity of HuNb_TROP2-HSA-MMAE. A Schematic structure of HuNb_TROP2-HSA-MMAE. B The SDS-PAGE analysis of HuNb_TROP2-HSA and HuNb_TROP2-HSA-MMAE with the addition of the reducing agent TCEP and/or the oxidizing agent CuSO₄. M represents marker. C The affinity of HuNb_TROP2-HSA and HuNb_TROP2-HSA-MMAE for TROP2 proteins was determined by ELISA. D Flow cytometry assessed the binding activities of HuNb_TROP2-HSA and HuNb_TROP2-HSA-MMAE to TROP2-positive pancreatic cancer cells in the presence and absence of 10% human serum

Fig. 3

Endocytosis and in vitro antitumor activity of HuNb_TROP2-HSA-MMAE. A The process of HuNb_TROP2-HSA-MMAE internalization into BxPC-3 cells. Blue: nuclei stained by Hoechst33342; green: HuNb_TROP2-HSA-MMAE labeled with Alexa Fluor™ 488. B HuNb_TROP2-HSA-MMAE underwent lysosomal degradation after internalization. Blue: nuclei; green: HuNb_TROP2-HSA-MMAE; red: lysosomes. C The expression of TROP2 on the surface of three human pancreatic cell lines was determined by flow cytometry and the cytotoxic effects of HuNb_TROP2-HSA-MMAE treatment for 2 or 3 days on these cancer cells were evaluated by MTT assay

Fig. 4

Cell apoptosis induced by HuNb_TROP2-HSA-MMAE. A The effect of HuNb_TROP2-HSA-MMAE on apoptosis of BxPC-3 and PK-59 cells was detected by Annexin V-FITC/PI staining. Annexin V⁺PI⁻ (Q4) represents early apoptotic cells and Annexin V⁺PI⁺ (Q2) represents late apoptotic cells. B A statistical analysis of three replicate experiments of (A). The proportion of apoptotic cells is the sum of Q2 and Q4. C Western Blot demonstrated the expressions of apoptosis-related proteins in pancreatic cancer cells after 72 h of treatment of HuNb_TROP2-HSA-MMAE

Fig. 5

In vivo distribution and metabolism of HuNb_TROP2-HSA-MMAE. A Living imaging after intravenous injection of 200 μg Cy7-HuNb_TROP2-HSA-MMAE for different times. B Fluorescence distribution in major organs at 24 h post-injection. C Quantitative analysis of the fluorescence intensity in (B)

Fig. 6

In vivo antitumor activity of HuNb_TROP2-HSA-MMAE. A Tumor volume during 4 weeks of treatment with different doses of HuNb_TROP2-HSA-MMAE. B Survival curves of mice receiving various treatments. C Each line represents the tumor volume for an individual mouse. D H&E staining and IHC for Ki-67, cleaved caspase-3 and cleaved PARP of tumor tissues