A novel combined phage and yeast display-based approach to discover binding domains for CAR T-cell therapies: Focus on EBV-associated malignancies

Barrena Díaz, Antonio Jesús (2021) A novel combined phage and yeast display-based approach to discover binding domains for CAR T-cell therapies: Focus on EBV-associated malignancies. PhD thesis, University of Glasgow.

Abstract

It is estimated that more than 90% of the adult population worldwide is infected with the Epstein-Barr virus (EBV). The infection is associated with certain lymphomas and carcinomas, causing around 1% of all cancers and these are challenging to treat. Cancer immunotherapy is an approach to help the patient’s body fight the disease by boosting antitumour immunity. A novel, cutting-edge immunotherapy treatment has been recently developed, among them the Chimeric Antigen Receptor T-cell therapy, also known as CAR T-cells.

CAR T-cells are modified T-cells designed to carry a gene coding for an artificial protein receptor (the CAR) able to recognise a specific tumour-associated antigen (TAA) and deliver a signal of T-cell activation by an HLA-independent interaction. The CAR can be designed in several ways by combining different domains obtained from existing proteins, including receptors expressed in T-cells and antibodies. The antigen-binding domain is the CAR domain able to interact with the TAA (typically on the target cell surface), triggering the activation singling cascade in the T-cells and thereby eliciting a T-cell response to the cancer cell. Single chain variable fragments (scFvs) are composite proteins derived from antibodies that have been commonly used as antigen-binding domains for CARs. Murine scFvs, formatted from existing murine antibodies obtained by mouse immunisation, have been used for the currently most successful CAR T-cell therapies, treating B-cell malignancies. However, mouse scFvs also increase the therapy's immunogenicity and have a negative outcome in efficacy and safety. Therefore, human scFvs are being used to develop new CAR T-cells therapies, and scFvs can be obtained from phage display libraries synthesised using B-cells from human donors.

In this research, a phage display library of human scFvs has been used to obtain scFvs to design CARs to treat EBV-associated malignancies. Three EBV TAAs, expressed in a wide range of EBV-associated malignancies, were selected to be targeted by the CARs: the human protein B7H4 and the viral proteins Latent membrane protein 1 (LMP1) and Latent membrane protein 2A (LMP2A). B7-H4 is a transmembrane immune checkpoint protein expressed on many cancer cells and tumour-associated macrophages. It acts to inhibit T-cell effector functions. LMP1 and LMP2A are viral proteins expressed on the plasma membrane of several EBV-associated malignancies, such as Nasopharyngeal carcinoma, Hodgkin lymphoma, or Diffuse large B-cell lymphoma.

A novel combined phage and yeast display-based approach was developed to screen the phage display library. This new approach uses a yeast display platform as a source of antigen. The yeast express the membrane antigen on their surface, and the phage display library is panned against them. Then, after one or several rounds of phage selection and for the final steps of the selection, the system is flipped, and the selected pool of scFvs is expressed in the yeast display system. In these final steps, the yeast are exposed to adherent mammalian cells expressing the membrane antigen. The scFvs, on the surface of the yeast, bind to the antigen on the surface of the mammalian cells. The strength of interaction yeastmammalian cells is boosted by avidity due to the high number of scFvs expressed on the surface of the yeast. This increases the chances of holding the binding yeast after several washes and washing off the non-binding yeast. This method helps to overcome phage display biases, limitations and, cost-effectively, allows the selection of scFvs. Using this new methodology, eleven anti-B7H4 scFvs were successfully isolated and one potential anti-LMP1 scFv. The anti-B7H4 scFvs show different strengths of interaction with B7H4, providing a diverse set of clones that can be used to explore the optimal affinity for an anti-B7H4 CAR T-cell therapy. Preliminary CAR constructs were designed using these novel isolated scFvs to perform functional characterisation. However, the expression of the CARs in a reporter T-cell line was challenging and their utility as CAR components remains to be determined. The next steps in this work will be to investigate the capacity of these scFvs to induce antigen-specific signalling in CAR format.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Supported by funding from Medical Research Scotland.
• Subjects: Q Science > QR Microbiology > QR180 Immunology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Life Sciences
• Supervisor's Name: Wilson, Professor Joanna B.
• Date of Award: 2021
• Embargo Date: 20 December 2023
• Depositing User: Theses Team
• Unique ID: glathesis:2021-82611
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 06 Jan 2022 11:17
• Last Modified: 27 Jan 2025 10:26
• Thesis DOI: 10.5525/gla.thesis.82611
• URI: https://theses.gla.ac.uk/id/eprint/82611