Conjugate vaccines targeting tumour vasculature: CLEC14A and ROBO4 as novel immunotherapeutic antigens

Download Statistics

Downloads

Downloads per month over past year

Kara, Muhammet Ali ORCID: https://orcid.org/0009-0002-0743-5392 (2025). Conjugate vaccines targeting tumour vasculature: CLEC14A and ROBO4 as novel immunotherapeutic antigens. University of Birmingham. Ph.D.

Preview

Kara2025PhD.pdf
Text - Accepted Version
Available under License All rights reserved.
Download (19MB) | Preview

Abstract

Tumour angiogenesis plays a fundamental role in the progression of solid tumours and their metastasis through the leaky vasculature, supplying nutrients and oxygen via these vessels. Targeting tumour endothelial markers (TEMs) such as CLEC14A and ROBO4 presents a promising immunotherapeutic strategy to disrupt angiogenesis and inhibit tumour growth. TEMs are distinct proteins predominantly or exclusively expressed on the endothelial cells that form tumour blood vessels, often showing limited or no expression in healthy vasculature, making them attractive targets. This thesis investigates the development of active vaccination approaches aimed at stimulating immune responses against CLEC14A and ROBO4, aiming to impair tumour vasculature and improve cancer treatment outcomes.
To achieve this, protein-based conjugate vaccine platforms were designed and optimised. This approach was chosen to address the inherent challenge of targeting self-antigens like CLEC14A and ROBO4, which typically have low immunogenicity and struggle to elicit robust immune responses due to insufficient T-cell support. Conjugate vaccines overcome this limitation by covalently linking these weak antigens to highly immunogenic carrier proteins, thereby recruiting essential T cell help and leading to a more robust and long-lasting immune response. Tetanus toxin fragment C (TTc) was selected as the carrier protein due to its well-established ability to elicit robust CD4+ T-cell responses and its safety profile in humans. Furthermore, widespread pre-existing T cell memory to tetanus toxoid (TT) in the general population ensures efficient and rapid recruitment of T cell help in vaccinated patients. Recombinant proteins were successfully expressed, purified, and characterised before being used in vivo immunisation studies.
The results demonstrate that vaccination against CLEC14A and ROBO4 induces a strong humoral immune response. Specifically, ROBO4-TTc-immunised mice exhibited significantly increased ROBO4-specific IgG1 antibody titres. Additionally, the ROBO4-TTc vaccine induces cellular immune responses.
A mouse model of triple-negative breast cancer (TNBC) was employed to evaluate the efficacy of the ROBO4-TTc vaccine in eliciting tumour-specific immune responses. TNBC is an aggressive and heterogeneous subtype of breast cancer that poses a significant clinical challenge due to the absence of the oestrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), and its limited immune response to immune checkpoint inhibitor treatment (ICT). This model closely mimics human TNBC, making it valuable for studying tumour biology and evaluating novel treatments. The efficacy of the ROBO4-TTc vaccine was evaluated by analysing tumour burden. This was complemented by a comprehensive assessment of immune activation, including antibody production, T and B cell responses, and immune cell infiltration into the tumour, using flow cytometry, enzyme-linked immunosorbent assay (ELISA), and histological techniques. Additionally, the mechanistic basis of ROBO4-targeted vaccination was investigated to determine its effects on tumour vasculature, endothelial cell function, and overall tumour microenvironment.
The ROBO4-TTc vaccine significantly inhibited tumour growth in the TNBC model (4T1). ROBO4-TTc-immunised mice exhibited reduced tumour vascularisation and increased immune cell infiltration, contributing to delayed tumour progression. This is likely due to vascular normalization, where the irregular, leaky tumour blood vessels become more structured and functional, improving immune cell access to the tumour microenvironment and reducing hypoxia-driven immunosuppression. These findings highlight the potential of active vaccination strategies targeting tumour endothelium as a well-characterised approach to cancer therapy. By leveraging immune-mediated destruction of tumour vasculature, this study provides a foundation for further exploration of endothelial-targeted immunotherapies in treating solid malignancies.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):