Development of T-cell costimulatory antibodies for skin cancer immunotherapy

Despite advances in cancer treatment, current therapies are still not sufficiently effective in most patients with cancer. Immunostimulatory antibodies (ISAs), which bind to 'costimulatory receptors' on immune cells to activate these cells to attack the tumour, are a promising method for treating cancer in the future. However, more research is needed to determine whether ISAs can work as perfectly in human cancers as has been shown in mice, and to optimise ISAs so that they boost the human immune system's ability to destroy cancers without causing side effects elsewhere in the body. Some ISAs cause side effects by binding to receptors called Fc gamma receptors (FcgRs) found on cells throughout the body. Using a novel approach, we can improve the ability of ISAs to be directed to and retained in cancers, by creating 'bispecific' ISAs (bs-ISAs) which stick to molecules on tumour cells whilst targeting costimulatory receptors on immune cells within the tumour. As bs-ISAs do not require binding to FcgRs to work, they are likely to provide more effective treatment for cancer with fewer side effects.

The aim of this study is to test the effectiveness of different ISAs and bs-ISAs in laboratory experiments using two common human skin cancers (melanoma and cutaneous squamous cell cancer (cSCC)) to investigate whether ISAs can be improved so that they can be used to treat these cancers. This research is much needed because skin cancers are amongst the commonest human cancers, and melanomas and advanced cSCCs are often fatal, yet current immune therapies are effective in less than 20% of melanomas and advanced cSCCs.

I will investigate which costimulatory receptor target and format of ISA improves tumour immune responses most effectively. The Antibody and Vaccine group have generated anti-4-1BB, anti-CD27 and anti-DR3 ISAs in different versions (IgG1, IgG2 and IgG4 subclasses). Each of these ISAs will be tested in our 'tumour tissue slice culture' system, in which samples of melanomas and cSCCs from skin cancer surgery patients are thinly sliced and incubated, preserving the tumour and immune cells within each slice. In these experiments, the extent that immune cells called T-cells are activated to fight and kill tumour cells will be quantified for each ISA, and the effects on gene expression will be analysed to compare the mechanisms by which different ISAs work.

As each IgG subclass binds to different types of FcgRs, and as a type of FcgR called FcgRIIb seems particularly important for how some ISAs work, FcgRs will be characterised on various immune cell subsets in melanoma and cSCC. To investigate the relationship between FcgRs and clinical outcome in skin cancer, FcgRs will be characterised in melanomas and cSCCs with known clinical outcomes, including whether the cancer had spread to other parts of the body (metastasised) or led to death. I will also investigate whether/which FcgRs are needed for each ISA to enhance function of T-cells isolated from melanomas and cSCCs, and will examine mechanisms by which ISAs bound by FcgR reduce certain cancer-promoting immune cells called regulatory T-cells.

I will determine whether bs-ISAs which concurrently bind to costimulatory receptors (4-1BB, CD27 or DR3) on T-cells and a molecule called PD-L1 on tumour cells can improve the ability of ISAs to enhance immunity against the tumour. The different bs-ISAs will be tested to determine how well they activate costimulatory receptors and boost anti-tumour immunity in the tissue slice culture system. The effects of bs-ISAs will be compared to a combination of two separate antibody treatments (i.e. ISAs that only bind to costimulatory receptors and antibodies that bind to PD-L1) to determine whether bs-ISAs enhance tumour immunity more effectively.

The results of this study will guide the development of more effective ISAs and help determine whether ISAs/bs-ISAs can improve treatment for aggressive skin cancers and other cancers.

Checkpoint inhibitors are not effective for many patients with cancer. Immunostimulatory antibodies (ISAs) targeting T-cell costimulatory receptors (e.g. 4-1BB, CD27 and DR3) enhance anti-tumour immunity but research is needed to improve their effectiveness in human cancers. Some ISAs depend on Fc gamma receptor (FcgR)-mediated crosslinking for optimal activity, but this can result in off-tumour toxicity and depletion of activated T-cells. Bispecific ISAs (bs-ISAs) can target ISAs to tumours without requiring FcgR-mediated crosslinking. However, the optimal costimulatory receptor targets and ISA/bs-ISA formats for many human tumours have not been resolved.

I will examine the effects of ISAs/bs-ISAs on tumoral immune responses in human melanoma and cutaneous squamous cell carcinoma (cSCC), building on our experience of using these cancers as models for studying tumour immunity and immunotherapeutic antibodies. The effects of different ISAs (anti-4-1BB, anti-CD27 and anti-DR3) and isotypes of these ISAs (IgG1, IgG2 and IgG4) on tumoral immunity will be examined using ex vivo melanoma and cSCC tissue slice cultures. Flow cytometry will analyse T-cells from tumour tissue slices (for activation markers, cytotoxicity, proliferation, cytokine production), confocal microscopy will quantify tumour cell death and transcriptomic profiling will be conducted with targeted RNAseq. I will characterise FcgR expression in melanoma and cSCC and ascertain FcgR requirement for ISAs for enhancing tumoral T-cell function. Functional experiments and tumour tissue slice cultures will examine whether bs-ISAs targeting T-cell costimulatory receptors and PD-L1 provide superior co-stimulatory receptor activation and enhancement of tumoral T-cell responses compared to combination treatment with parental monospecific IgG1 antibodies and anti-PD-L1. The results of this study will evaluate whether ISAs/bs-ISAs can form a therapeutic approach for skin cancer and be informative for other cancers.