Development of recombinant immunotoxins targeting overexpressed cell surface biomarkers trophoblast cell-surface antigen 2 and mesothelin for cervical cancer treatment

Masuku, Sizalobuhle

Development of recombinant immunotoxins targeting overexpressed cell surface biomarkers trophoblast cell-surface antigen 2 and mesothelin for cervical cancer treatment

Thesis / Dissertation

2025

Publisher

University of Cape Town

Department

Department of Integrative Biomedical Sciences (IBMS)

Faculty

Faculty of Health Sciences

Abstract

Cervical cancer is a devastating disease that raises significant health concerns in Sub-Saharan Africa. In South Africa, cervical cancer is considered the second most common cause of cancer-related deaths, with high incidence rates. The disease is strongly linked to persistent infection by high-risk strains of the human papillomavirus (HPV). Despite the availability of HPV vaccines and early detection methods such as Pap smears and HPV testing, many women remain undiagnosed or only receive treatment in advanced stages of the disease in low- and middle-income countries (LMIC). The high costs associated with cervical cancer screening and the ineffectiveness of current conventional treatments, which often result in severe side effects, exacerbate this public health crisis. Addressing these challenges necessitates innovative therapeutic approaches, such as recombinant immunotoxins, which may offer targeted treatment options with reduced side effects compared to traditional therapies. In this study, we focused on developing a target-specific next-generation recombinant immunotoxin (rIT) to improve treatment outcomes. The rITs were targeting cell surface biomarkers, Trophoblast cell surface antigen 2 (TROP2) and mesothelin (MSLN), which are overexpressed in cervical cancer cells. Targeting TROP2 and MSLN presents a promising therapeutic approach. The study aimed to develop novel recombinant anti-TROP2 and anti-MSLN immunotoxin candidates based on truncated Pseudomonas exotoxin A (ETA) or its deimmunized version (dETA) with an R456T point mutation to compare their cytotoxicity activity towards cancer cells. Medical Biotechnology & Immunotherapy Research Unit Institute for Infectious Disease & Molecular Medicine South African Research Chair in Cancer Biotechnology ETA represents the wild type (wt) of Pseudomonas exotoxin A, a highly cytotoxic protein toxin produced by a bacterium that inhibits protein synthesis in host cells leading to cell death. dETA version of ETA being the modified form of the toxin generated using a supercomputing-based molecular dynamics simulation model to identify point mutations that minimize the risk of an immune response in patients while maintaining the cytotoxic activity of the wild type (wt) ETA. In this study, to generate anti-TROP2/MSLN-specific rIT, corresponding single-chain antibody fragment (αTROP2/MSLN (scFv)) genes were cloned into either a pMT ETA or dETA expression vectors to create pMT-TROP2/MSLN-ETA/dETA constructs. The constructs were expressed under osmotic stress in the presence of compatible solutes in Escherichia coli (BL21 DE3) cells. The rITs were extracted using sonication in a lysis buffer with protease inhibitors to recover soluble active proteins. Once expression was confirmed, purification was performed using two-step immobilized metal affinity chromatography (IMAC) and quantified. Purified proteins were characterized by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. The rITs were subsequently tested for their ability to bind to antigen-positive cervical cancer cell lines, and their cytotoxic effects were evaluated. The results of this study demonstrated that such difficult-to-express rITs can be successfully generated using the unique periplasmic stress expression protocol. This was illustrated by the SDS-PAGE results from the first IMAC purification showing full-length protein of anti- TROP2(scFv)-ETA, anti-TROP2(scFv)-dETA, anti-MSLN(scFv)-ETA, and anti-MSLN(scFv)-dETA at around 72 kDa. Despite initial success in purifying the MSLN rITs using IMAC, significant degradation of both anti-MSLN(scFv)-ETA and anti-MSLN(scFv)-dETA during the second purification limited their further characterisation. Future work needs to be conducted to reduce degradation. In contrast, the TROP2 rIT average protein yield from a 2L culture volume was calculated to be approximately 1.66 and 3.735 mg of anti-TROP2(scFv)-ETA and anti-TROP2(scFv)-dETA, respectively and these purified proteins demostrated excellent performance in downstream functionality assays. Both anti-TROP2(scFv)-ETA and anti-TROP2(scFv)-dETA showed strong antigen-dependent binding and internalisation, which were confirmed by confocal microscopy. Furthermore, cytotoxicity results demonstrated dose-dependent killing of target cervical cancer cells. The anti-TROP2(scFv)-ETA (wild type) showed greater cytotoxicity activity than the anti-TROP2(scFv)-dETA, with a two-fold difference in IC50 values, representing an improvement in the deimmunised version compared to previous studies. This study demonstrated the potential of recombinant immunotoxins (rITs) as a targeted strategy for treating cervical cancer in vitro, due to their specificity and cytotoxic activity against cancerous cells. However, the immunogenicity and therapeutic efficacy of the rITs described in this work still require validation through preclinical in vivo studies. Overall, the preliminary proof-of-concept experiments presented in this thesis provide a promising foundation for the development of rIT-based therapies aimed at selectively targeting and eliminating cervical cancer cells.

Keywords

Cervical cancer

Treatment

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