Towards Development of Antibodies Targeting Myeloid and Regulatory T Cells in Bladder Tumours

Abstract

Checkpoint inhibition is an attractive option for treating bladder cancer, the tenth most common type of cancer in the world. With currently approved drugs restricted to targeting the PD1/PD-L1 axis with limited response rates, finding alternative targets for manipulation of immune cells could improve the survival of bladder cancer patients. Recently, cell markers selectively expressed by immunosuppressive regulatory T cell and macrophage populations in bladder tumours were identified by single-cell RNAseq studies. The aim of this project was to develop single-chain variable fragments (scFv) as a basis for future antibodies, targeting two of these markers on macrophages (protein A) and Tregs (protein B). The phage display technology is used for selecting scFv binders against both of the proteins from an initial scFv sequence library. Three rounds of panning were carried out and the selected sequences were cloned and transfected into E. coli for the production of soluble scFvs. Enzyme-linked immunosorbent assay (ELISA) was then used for screening of the scFvs, with 95 and 404 being confirmed to bind target A and B, respectively. For protein A, flow cytometry and SPR were used to further evaluate binding. Thus, while SPR screening revealed 8 binders, screening for cell binding by flow cytometry against target A only revealed a tendency to bind in one scFv. In summary, the results indicate that it is feasible to produce scFVs against potential targets on immune cells in bladder tumours, although further evaluation and optimization is needed especially for target B.

Popular Abstract

Bladder cancer is one of the most common types of cancer in the world. While the survival rate is relatively high compared to for example lung or colon cancer, a high recurrence rate makes it the costliest one in economic terms. Also, around one in five patients diagnosed with bladder cancer have a high-risk variant and of those, 50% succumb due to the disease within 5 years. Recommended treatment for high-risk bladder cancer cases is chemotherapy and complete removal of the bladder, which have serious impact on the patient's quality of life. More efficient treatments are needed, and one strategy is to modulate the immune cell and thereby enable the immune system to eliminate cancer cells.
One of the immune system’s primary duties is tumour surveillance. That is, detecting and eliminating cells that grow uncontrollably, i.e. cancerous cells, before the tumour grows too large and can cause harm. However, tumours can adapt to evade immune surveillance, by for example modulating the immune cells within the tumour. For instance, the so-called macrophages are often altered in a way to promote the tumour. Additionally, so-called regulatory T-cells can prevent other immune cells from eliminating the tumour. Antibodies that bind to key immune cells can be used to stimulate an effective immune response. These antibodies can be developed to specifically hinder those cells that promote the tumours, or to stimulate those which can kill tumour cells. The specific activity is achieved by the antibodies binding to certain proteins on the cell surface.
The aim of this project was to develop antibodies that could target two different proteins on the surface of immune cells in bladder tumours. To this end, a technology called phage display was employed, where we can fish out molecules that specifically bind to the target proteins, and later use these binders in the format of antibodies. After several candidate molecules had been selected by phage display, they were screened using different techniques to assess their ability to bind the target proteins. Additionally, the genetic sequences of each molecule were outlined to check how many were actually different from each other.
In short, the results indicate that several molecules that can bind to the target proteins were identified. For one of the targets, an additional assay was performed to confirm binding to the target and a few candidate molecules showed positive results. Further, one of these candidate molecules seems to be capable of binding the target protein on the surface of cells, which would be important for potential use as a therapeutic. Next, further analyses of the candidate molecules is required, as is the optimization of the screening techniques. The identified molecules that bind the targets may be useful to further develop full antibodies.

• author: Martín Hinojosa, Francisco
• supervisor: Kristina Lundberg; Viktor Sincic; Sarah Richtmann; Magdalena Godzwon
• organization: Department of Immunotechnology; Educational programmes, LTH
• course: KIMM01 20231
• year: 2023
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• keywords: Bladder cancer, immune cell targets, antibody development, phage display
• language: English
• id: 9125600
• date added to LUP: 2023-07-10 09:59:20
• date last changed: 2023-07-10 09:59:20