Antigen Specific Delivery of Chemokines by Activated T cells; Potential Strategy for Inducing Inflammation at the Tumour Site

Abstract

We have developed a new strategy to induce inflammation and other tumour destructive responses in a tumour specific manner. Inflammation can play an important role in tumour supression by stimulating an anti-tumour response. In this project we have harnessed the action of the angiostatic members of non-ELR CXC family chemokines (CXCL9 and CXCL10) with a view to directing lymphocyte chemotaxis to the tumour site. CXCL9 and CXCL10 recruit selective subsets of inflammatory cells by driving various cell types into the site of inflammation. They can also reduce angiogenesis in tumours. These chemokines are highly pleotropic, and in addition there are splice variants of the non-ELR CXC family receptor CXCR3 which may have contrasting effects. CXCR3A promotes cell survival and endothelial cell angiogenesis, while CXCR3B acts as a cellular inhibitor and is involved in angiostasis. Therefore, expression of these receptors is as important as expression of their ligands within the cancer in controlling the microenvironment and determining tumour behaviour. In order to target expression of CXCL9 and CXCL10, various constructs were created, with chemokines expressed under the control of the inducible promoters derived from the promoter of cytokine IL-2 gene, that is only expressed when T cells are activated. These constructs were transfected into primary T cells, and we showed that the promoter is activated when the T cell receptor is engaged and that production and activity of chemokine was restricted to activated T cells. The delivery method, by transfection of the constructs into tumour specific T cells, is designed to limit the production of the chemokine to the active tumour sites. The function and fidelity of the constructs were compared and optimized using T cell lines. We have determined the function and fidelity of the promoter and also confirmed the biological action of CXCL9 and CXCL10 in the construct. Quantitative real-time PCR experiments confirmed that expression of the construct results in production of the chemokine only when the T cell receptor is engaged. ELISAs were used to quantify, the amount of chemokine production. Chemotaxis assays showed the migration of cytotoxic lymphocyte populations and NK cells towards the supernatants of cells transfected with constructs. In preliminary experiments, tumour infiltrating lymphocytes from liver metastasis of colorectal adenocarcinomas were isolated and grown in culture. Transduction of the constructs into tumour specific cells will result in production and activity of the chemokine only in the presence of tumour antigen. Expression of CXCL9, CXCL10 and CXCR3A and CXCR3B in colorectal adenocarcinoma was validated by quantitative real time PCR in 50 fresh frozen liver metastasis samples from colorectal adenocarcinomas which showed strong correlation between the expression of two chemokines and also correlation between each chemokine and CXCR3B variant expression. This project forms a proof of concept, and would be a prelude for further work to demonstrate the in vivo efficacy of this approach.