Targeting of colorectal cancer organoids with zoledronic acid conjugated to the anti-EGFR antibody cetuximab

Abstract

BackgroundAntibody-drug conjugates (ADC) are essential therapeutic options to treat solid and hematological cancers. The anti-epidermal growth factor-receptor (EGFR) antibody cetuximab (Cet) is used for the therapy of colorectal carcinoma (CRC). Anti-CRC Vδ2 cytolytic T lymphocytes can be elicited by the priming of tumor cells with the aminobisphosphonate zoledronic acid (ZA) and consequent presentation of isopentenyl pyrophosphates through butyrophilin (BTN) family members such as BTN3A1 and BTN2A1. A major drawback that impairs the targeting of ZA to CRC is the bone tropism of aminobisphosphonates.MethodsThe phosphoric group of ZA was linked to free amino groups of Cet in the presence of imidazole following the labeling of phosphoric groups of DNA to amino groups of proteins. The generation of Cet-ZA ADC was confirmed by matrix assisted laser desorption ionization mass spectrometry and inductively coupled plasma-mass spectrometry analysis. Thirteen CRC organoids were obtained with a chemically defined serum-free medium in Geltrex domes. Proliferation and activation of cytolytic activity against CRC organoids by Vδ2 T cells was detected with flow cytometry, crystal violet and cytotoxic probe assays and image analysis. Immunohistochemistry and quantification of BTN3A1 or BTN2A1 expression and the number of tumor infiltrating Vδ2 T cells in CRC were performed by automatic immunostaining, whole slide scanning and computerized analysis of digital pathology imaging.ResultsThe novel ADC Cet-ZA was generated with a drug antibody ratio of 4.3 and displayed a reactivity similar to the unconjugated antibody. More importantly, patient-derived CRC organoids, or CRC tumor cell suspensions, could trigger the expansion of Vδ2 T cells from peripheral blood and tumor infiltrating lymphocytes when primed with Cet-ZA. Furthermore, Cet-ZA triggered Vδ2 T cell-mediated killing of CRC organoids. The expression of BTN3A1 and BTN2A1 was detected not only in CRC organoids but also in CRC specimens, together with a considerable amount of tumor infiltrating Vδ2 T cells.ConclusionsThese findings are proof of concept that the Cet-ZA ADC can be used to target specifically CRC organoids and may suggest a new experimental approach to deliver aminobisphosphonates to EGFR+solid tumors.

Novel SPEA Superantigen Peptide Agonists and Peptide Agonist-TGFαL3 Conjugate. In Vitro Study of Their Growth-Inhibitory Effects for Targeted Cancer Immunotherapy

“…Studies reported that EGFR is overexpressed in cancer cells such as colorectal, breast, and non–small cell lung cancers [ 69 , 70 , 71 ]. Due to its overexpression on cancer cells, several investigations used the third loop (L3) of transforming growth factor-α (TGF-α) to conjugate with protein antibodies [ 72 , 73 , 74 , 75 ], or full superantigen SEB to exert the toxic effect of a drug in the vicinity of cancers to achieve targeted cancer therapy [ 76 ].…”

EGFR-Targeted Antibody–Drug Conjugate to Different Aminobisphosphonates: Direct and Indirect Antitumor Effects on Colorectal Carcinoma Cells

“…IBA, RIS, and ZA were purchased from Selleckchem (Houston, TX, USA), and Cet was obtained as a leftover of the therapeutic preparation used for patients suffering from CRC (the kind gift from the Pharmacy Unit of IRCCS Ospedale Policlinico San Martino, Genoa, Italy). Cet-IBA, Cet-RIS, and Cet-ZA ADCs were prepared by Nanovex Biotechnologies (Asturias, Spain), according to the procedure reported before for the Cet-ZA (Figure 1) [30]. The protein sample was purified via ultrafiltration (100 kDa cut off) to eliminate the presence of excipients.…”

“…Then, these incubation cells were lysed and subjected to Western blot (WB) analysis. WB was performed as described in detail [30]. Briefly, 30 µg/lane of total protein from 4 × 10 6 CRC cell lines Caco-2 or HCT-116 extracted with RIPA buffer were run on 8% PAGE gels (Express Plus, GenScript, Piscataway, NJ, USA) and blotted on PVDF membranes (GE-healthcare, Milan, Italy).…”

“…Recently, we reported the generation of the ADC composed of cetuximab covalently linked to zoledronate (Cet-ZA) [30]. This novel ADC can trigger the expansion and activation of Vδ2 T cells targeting CRC primary organoids [30].…”

“…Recently, we reported the generation of the ADC composed of cetuximab covalently linked to zoledronate (Cet-ZA) [30]. This novel ADC can trigger the expansion and activation of Vδ2 T cells targeting CRC primary organoids [30]. This effect was also detected when tumor-associated fibroblasts (TAF) from CRC mucosa were primed with Cet-ZA [31].…”