Chisanga, Chrispin
(2024)
Inhibition of the p38 MAPK pathway as a strategy to overcome tumour immune suppression in CD1c+ dendritic cells.
PhD thesis, University of Nottingham.
Abstract
Introduction: Harnessing the potential of the immune system to destroy and eliminate cancer cells has been a long-term dream for many researchers in the field of cancer immunotherapy. The licencing of immune checkpoint inhibitors (ICI) and targeted therapies for malignancies such as malignant melanoma confirms the value of immune responses with important advances in survival being achieved despite some notable toxicity. Despite these major advances, there remains a keen interest in new treatments with improved efficacy and safety profiles and the exploration of adoptive immunotherapy with dendritic cells (DC) remains topical. Dendritic cells belong to a heterogeneous population of professional antigen presenting cells (APC) and reside in peripheral tissues and lymphoid organs. Previously, our group described loss of function of myeloid CD1c+ DC in patients with advanced melanoma and showed promising data to support the concept of restoring immune function by inhibiting mitogen activated protein kinase (MAPK) signalling pathways in these cells. Notably, inhibition of p38 MAPK (p38i) with small molecule research inhibitors was shown to increase the balance of IL-12:IL-10 secretion to favour the differentiation of Th1 responses. This thesis explored the impact of inhibiting p38 MAPK with candidate small molecules suitable for clinical use on the chemotaxis of CD1c+DC.
Methods: Health donor CD1c+ DC were isolated and cultured in the presence of highly selective clinical p38 inhibitors, AZD6703 and AZD7624, (AstraZeneca Ltd) and assayed for CCR7 and CD86 expression, IL-10 and IL-12p70 production. Experiments were designed to investigate how these compounds could be translated from the laboratory to a clinical manufacturing setting to align with the use of Miltenyi Biotech’s Prodigy platform for advanced cell manufacturer. RNA sequencing (RNASeq) was performed on mature dendritic cells (mDC) versus p38i+mDC to identify differentially expressed genes (DEG) that drive DC chemotaxis and motility. ViewRNA assay was also performed in partnership with AstraZeneca Ltd to understand the potential roles played by CD5, CD209 and Semaphorin 7A (Sema7A) in DC chemotaxis during reduced CCR7 expression.
Results: The two clinical p38i had comparable effect to the research inhibitor (BIRB0796 1µM) previously used but were 100-fold more efficacious as they were active at 10nM. As anticipated, inhibition of p38 MAPK with these compounds enhanced the secretion of IL-12 but inhibited IL-10 release by activated DC. Furthermore, p38i resulted in a marked increase in CD86 expression beyond that achieved with maturation alone. Importantly, treatment of mature DC with the clinical p38i led to increased chemotaxis to CCR7 ligand (CCL19) despite reduced display of CCR7 per se.
To explore the wider alterations in gene expression following inhibition of p38 MAPK, RNASeq analysis on CD1c+ DC was undertaken. Transcriptomics analysis revealed differentially expressed genes (DEG) which upon Kyoto encyclopaedia genes and genomes (KEGG) pathway analysis with WEB-based Gene Set Analysis Toolkit (WebGestalt) identified the chemokine signalling pathway as being crucial for driving DC migration and motility. Central to this pathway was the Wiskott-Aldrich syndrome protein (WASP). Importantly, the study showed concordance between cell culture results and transcriptomics data for CCR7 and CD86 expression as well as IL-10 and IL-12p70 production all of which served as important makers for Th1 polarisation. Surprisingly, no significant differences were recorded in the ViewRNA expression profiles of the target molecules.
To develop a process for future clinical manufacture of CD1c+ DC, we explored the scheduling of p38 MAPK inhibition. Importantly, it was possible to inhibit the p38 MAPK pathway in CD1c+ DC in the presence of other cells prior to their isolation and yet retain the functional impact of inhibiting p38 MAPK. However, in the presence of other cells a 100-fold increase in p38i was required to elicit functional efficacy comparable to MAPK inhibition of already isolated cells.
Conclusions: This study identified a suitable clinical inhibitor, AZD7624, that will now be used in an advanced cell therapy manufacturing for melanoma patients. Additionally, RNASeq analysis revealed that the chemokine signalling pathway was crucial for mediating DC migration and motility. The presence of WASP in this pathway has important implications for DC cytoskeleton rearrangement and immunological synapse formation with naïve T cells for potent adaptive immune responses. We will shortly initiate a first-in-man clinical trial (Innovate UK funded) of p38i CD1c+ DC plus plasmacytoid DC for patients with advanced malignant melanoma who have failed immune checkpoint blockade.
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