Title: Combination immunotherapy for breast cancer: targeting cancer-associated fibroblasts to improving therapeutic vaccination
Funder: Cancer Research UK

Abstract: Background: Despite improvements in mortality rates, breast cancer remains a common cause of UK cancer deaths (11,500 deaths/year). Anti-PD1/PDL1 checkpoint immunotherapy has produced exciting results in many cancers; this is predicated on boosting a pre-existing (albeit ineffective) anti-tumour immune response. However, most breast cancer patients have ‘immune cold’ tumours, and around 80% fail to respond. Vaccines developed to generate immune responses against specific tumour antigens are producing promising clinical results, and have the potential to be effective in these patients. DNA vaccines are one of the most clinically advanced cancer vaccine modalities. The Southampton team have developed a number of therapeutic DNA vaccine approaches, which have undergone successful clinical testing. HER2 and MUC1 are two well-studied antigens in breast cancer, with expression in around 25-30% and 90% patients respectively). Successful vaccine-based (and other) immunotherapy requires trafficking of lymphocytes into tumours; several recent studies have described an ‘immune exclusion’ tumour phenotype, where myofibroblastic cancer-associated fibroblasts (CAF) shield tumours from T-cells; attempts to target CAF clinically have been unsuccessful. Using orthotopic models of CAF-rich breast cancer, we found that CAF suppress infiltration of CD8+ T-cells into tumours, resulting in ‘immune excluded’ tumours that respond poorly to immunotherapy. We identified NOX4 as a key regulator of CAF activation. Using a NOX1/4 inhibitor (GKT831; phase-2 tested as an anti-fibrotic), we successfully targeted CAF in vivo, inhibiting CAF accumulation, promoting intratumoral CD8+ T-cell infiltration and improving response to anti-cancer vaccination. We hypothesise that combining DNA vaccination with stromal targeting by NOX4 inhibition will improve therapeutic response in ‘immune-excluded’, CAF-rich breast cancers. Aims: To investigate the effect of combined DNA vaccination and stromal targeting through GKT831 CAF targeting in CAF-rich breast cancer. Methods: We will use established orthotopic murine models (4T1, Muc1-transfected 4T1, Her2-expressing D2F2/E2) to examine the effect of CAF on CD8+ and CD4+ T-cell phenotype and localisation within tumours (flow cytometry, immunochemistry, RNA-sequencing). Human breast cancer tissues (POSH cohort; triple-negative subset; n=558) will be analysed for spatial CAF/immune/tumour cell relationships (multicolour immunofluorescence, image analysis). We will test GKT831 as an immunotherapy adjunct combining DNA vaccines encoding Her2 and Muc1 antigenic epitopes, integrated into the clinically-tested Southampton vaccine design for optimal CD8+ and CD4+ T-cell induction, also tested in combination with anti-PD1. Standard immunological endpoints include tetramer analysis, flow cytometry, Elispot assays, multiplexed immunochemistry/immunofluorescence. Summary: If successful, this work will lead to a clinical trial combining DNA vaccination and GKT831 for aggressive, CAF-rich breast cancers.

Lay abstract: Background: Despite improvements in mortality rates, breast cancer remains a common cause of UK cancer deaths (11,500 deaths/year). Anti-PD1/PDL1 checkpoint immunotherapy has produced exciting results in many cancers; this is predicated on boosting a pre-existing (albeit ineffective) anti-tumour immune response. However, most breast cancer patients have ‘immune cold’ tumours, and around 80% fail to respond. Vaccines developed to generate immune responses against specific tumour antigens are producing promising clinical results, and have the potential to be effective in these patients. DNA vaccines are one of the most clinically advanced cancer vaccine modalities. The Southampton team have developed a number of therapeutic DNA vaccine approaches, which have undergone successful clinical testing. HER2 and MUC1 are two well-studied antigens in breast cancer, with expression in around 25-30% and 90% patients respectively). Successful vaccine-based (and other) immunotherapy requires trafficking of lymphocytes into tumours; several recent studies have described an ‘immune exclusion’ tumour phenotype, where myofibroblastic cancer-associated fibroblasts (CAF) shield tumours from T-cells; attempts to target CAF clinically have been unsuccessful. Using orthotopic models of CAF-rich breast cancer, we found that CAF suppress infiltration of CD8+ T-cells into tumours, resulting in ‘immune excluded’ tumours that respond poorly to immunotherapy. We identified NOX4 as a key regulator of CAF activation. Using a NOX1/4 inhibitor (GKT831; phase-2 tested as an anti-fibrotic), we successfully targeted CAF in vivo, inhibiting CAF accumulation, promoting intratumoral CD8+ T-cell infiltration and improving response to anti-cancer vaccination. We hypothesise that combining DNA vaccination with stromal targeting by NOX4 inhibition will improve therapeutic response in ‘immune-excluded’, CAF-rich breast cancers. Aims: To investigate the effect of combined DNA vaccination and stromal targeting through GKT831 CAF targeting in CAF-rich breast cancer. Methods: We will use established orthotopic murine models (4T1, Muc1-transfected 4T1, Her2-expressing D2F2/E2) to examine the effect of CAF on CD8+ and CD4+ T-cell phenotype and localisation within tumours (flow cytometry, immunochemistry, RNA-sequencing). Human breast cancer tissues (POSH cohort; triple-negative subset; n=558) will be analysed for spatial CAF/immune/tumour cell relationships (multicolour immunofluorescence, image analysis). We will test GKT831 as an immunotherapy adjunct combining DNA vaccines encoding Her2 and Muc1 antigenic epitopes, integrated into the clinically-tested Southampton vaccine design for optimal CD8+ and CD4+ T-cell induction, also tested in combination with anti-PD1. Standard immunological endpoints include tetramer analysis, flow cytometry, Elispot assays, multiplexed immunochemistry/immunofluorescence. Summary: If successful, this work will lead to a clinical trial combining DNA vaccination and GKT831 for aggressive, CAF-rich breast cancers.

Research activity codes: 5.1 PHARMACEUTICALS
Health categories: CANCER AND NEOPLASMS