tocky illuminates mechanisms of immune checkpoint blockade

In 2017, after successfully developing the Tocky technology and the data transformation method for Tocky data, I began submitting our first Tocky papers. Around the same time, I launched a new cancer immunotherapy project that utilized the capabilities of Tocky technology.

A Pump Priming Collaborative Grant 2017

In 2016, I initiated discussions about a new cancer immunotherapy project with my collaborators at the Institute for Cancer Research, Prof Alan Melcher and Prof Kevin Harrington.

These discussions led to securing a pump-priming grant from the Cancer Research Centre of Excellence (CRCE, now known as the CRUK Convergence Science Centre). The grant, which was for a one-year project starting in September 2017, marked a significant milestone. It enabled us to commence a project focused on investigating the effects of immune checkpoint blockade on cancer-reactive T cells, utilizing tools such as Nr4a3-Tocky and Foxp3-Tocky.

A PhD studentship 2019

The next stage of our cancer research endeavor involved applying for a PhD studentship from the Convergence Science Centre to study immune checkpoint inhibitors, specifically PD-1 blockade. This application was successful, leading to the launch of a new PhD studentship project dedicated to investigating the mechanisms of immune checkpoint blockade using the Tocky tools.

The studentship project that recruited a new cross-institutional PhD student in 2019.

This project aimed to enhance the precision of cancer immunotherapy by exploring T cell regulation mechanisms in melanoma patients. With the establishment of Tocky technology, the project focused on two critical immune suppressors: PD-1 and regulatory T cells (Treg). It sought to reveal the mechanisms behind anti-PD-1 and anti-CTLA-4, which have become standard treatments in malignant melanoma and other cancers.

CRUK Programme Foundation Award 2021

In pursuit of substantial funding to fully launch my cancer immunotherapy project, I encountered several challenges. Despite these struggles, my persistent efforts culminated in a successful application for a CRUK Programme Foundation Award (PFA).

The pivotal interview for this award took place on March 10, 2020, just before the onset of the first lockdown in the UK.

The corner with toilet rolls in a supermarket near the interview venue on 10th March 2020.

I left the interview with a positive feeling, but I had to wait for the announcement of the results, which was unusually delayed by almost a year due to the pandemic.

First Collaboration Paper Published 2021

In the meantime, our first collaborative paper on cancer immunotherapy with the Institute for Cancer Research (ICR) was successfully published in 2021 (Bozhanova et al., 2022). This study explored the mechanism of an oncolytic virus therapy using Nr4a3-Tocky technology.

The first collaboration paper with Profs Melcher and Harrington at ICR published in 2021.

First Preprint Released in July 2022

Our first study on cancer immunotherapy, focusing on immune checkpoint blockade, was made available as a preprint in July 2022(Hassan et al., 2022). This was more delayed than anticipated due to the challenging periods during the pandemic and personal circumstances. Still, this was an important milestone for us.

This research explored the mechanisms of the anti-PD-L1 and anti-CTLA-4 antibodies using Nr4a3-Tocky technology. The findings led us to hypothesize and test the anti-OX40 antibody, which demonstrated significant additive effects on anti-cancer T-cell activities.

I am grateful to my great collaborators, Prof Alan Melcher and Prof Kevin Harrington at ICR, and Prof Taku Okazaki, who is a renowned expert in PD-1 and Lag-3 biology.

Hassan et al reveals the effects of PD1 blockade, CTLA-4 blockade, and anti-OX40 immunotherapy on T cell activities using Nr4a3 Tocky (Hassan et al., 2022).

The endeavor has just begun. We anticipate being able to discuss the full scale of our cancer immunotherapy project within this blog very soon.

References

2022

CD4 T cell dynamics shape the immune response to combination oncolytic herpes virus and BRAF inhibitor therapy for melanoma

Galabina Bozhanova , Jehanne Hassan , Lizzie Appleton , Victoria Jennings , Shane Foo , Martin McLaughlin , Charleen ML Chan Wah Hak , Emmanuel C Patin , Eva Crespo-Rodriguez , Gabby Baker , Edward Armstrong , Matthew Chiu , Hardev Pandha , Adel Samson , Victoria Roulstone , Joan Kyula , Richard Vile , Fiona Errington-Mais , Malin Pedersen , Kevin Harrington , Masahiro Ono, and Alan Melcher

Journal for ImmunoTherapy of Cancer, Sep 2022

Abstract

Background Combination herpes simplex virus (HSV) oncolytic virotherapy and BRAF inhibitors (BRAFi) represent promising immunogenic treatments for BRAF mutant melanoma, but an improved understanding of the immunobiology of combinations is needed to improve on the benefit of immune checkpoint inhibitors (ICI).Methods Using a BRAFV600E-driven murine melanoma model, we tested the immunogenicity of HSV/BRAFi in immunocompetent C57BL mice. In addition to standard FACS analysis, we used the ‘Timer of Cell Kinetics and Activity’ system, which can analyze the temporal dynamics of different T cell subsets. This immune data was used to inform the selection of ICI for triple combination therapy, the effects of which were then further characterized using transcriptomics.Results Adding BRAFi treatment to HSV improved anti-tumor effects in vivo but not in vitro. Immune characterization showed HSV or dual therapy led to fewer intratumoral Treg, although with a more activated phenotype, together with more effector CD8 +T cells. Tocky analysis further showed that HSV/BRAFi dual treatment reduced the Tocky signal (reflecting engagement with cognate antigen), in both Treg and conventional subsets of CD4+, but not in CD8 +cells. However, a higher percentage of Treg than of conventional CD4 +maintained frequent engagement with antigens on treatment, reflecting a predominance of suppressive over effector function within the CD4 +compartment. The only T cell subset which correlated with a reduction in tumor growth was within Tocky signal positive conventional CD4+, supporting their therapeutic role. Targeting CD25 high, antigen-engaged Treg with a depleting anti-CD25 ICI, achieved complete cures in 100% of mice with triple therapy. Transcriptomic analysis confirmed reduction in Foxp3 on addition of anti-CD25 to HSV/BRAFi, as well as increases in expression of genes reflecting interferon signaling and cytotoxic activity.Conclusions Combination HSV/BRAFi is an immunogenic therapy for BRAF mutant melanoma, but cannot fully control tumors. Dual therapy results in changes in T cell dynamics within tumors, with relatively maintained antigen signaling in Treg compared with conv CD4+. Antigen-engaged CD4 +effectors correlate with tumor growth control, and depletion of Treg by addition of an anti-CD25 ICI, releasing suppression of conventional CD4 +effectors by Treg, enhances survival and activates immune signaling within tumors.All data relevant to the study are included in the article or uploaded as online supplemental information. Not applicable.
Single-cell level temporal profiling of tumour-reactive T cells under immune checkpoint blockade

Jehanne Hassan , Elizabeth Appleton , Bahire Kalfaoglu , Malin Pedersen , José Almeida-Santos , Hisashi Kanemaru , Nobuko Irie , Shane Foo , Omnia Reda , Benjy J.Y. Tan , Il-mi Okazaki , Taku Okazaki , Yorifumi Satou , Kevin Harrington , Alan Melcher , and Masahiro Ono

bioRxiv, Sep 2022

Abstract Publication

The blockade of the immune checkpoints PD-1 and CTLA-4 enhances T cell response. However, it is largely unknown how antigen-reactive T cells regulate their checkpoint expression in vivo and whether and how the checkpoint blockade can change activation dynamics of tumour-reactive T cells. To address this, here we used Nr4a3-Timer-of-cell-kinetics-and-activity (Tocky), which allows analysis of temporal changes of activated T cells following TCR signalling in vivo. By analysing melanoma-bearing Nr4a3 Tocky mice, we elucidate hidden dynamics of tumour-reactive T cells in the steady-state. Checkpoint blockade depleted highly activated effector Treg, while promoting unique effector T cell populations, and thus differentially modulating activation of tumour-reactive T cell populations. Furthermore, multidimensional analysis and seamless analysis of Tocky and scRNA-seq revealed a full spectrum of T cell dynamics in response to tumour burden and treatment with checkpoint blockade. Lastly, we propose a rational design of combinatorial therapy to further enhance T cell activities.Competing Interest StatementThe authors have declared no competing interest.