Abstract
BackgroundCellular therapy is a promising therapeutic strategy for malignant diseases. The efficacy of this therapy can be limited by poor infiltration of the tumor by immune effector cells. In particular, NK cell infiltration is often reduced relative to T cells. A novel class of fusion proteins was designed to enhance the recruitment of specific leukocyte subsets based on their expression of a given chemokine receptor. The proteins are composed of an N-terminal chemokine head, the mucin domain taken from the membrane-anchored chemokine CX3CL1, and a C-terminal glycosylphosphatidylinositol (GPI) membrane anchor replacing the normal transmembrane domain allowing integration of the proteins into cell membranes when injected into a solid tumor. The mucin domain in conjunction with the chemokine head acts to specifically recruit leukocytes expressing the corresponding chemokine receptor.Methodology/Principal FindingsA fusion protein comprising a CXCL10 chemokine head (CXCL10-mucin-GPI) was used for proof of concept for this approach and expressed constitutively in Chinese Hamster Ovary cells. FPLC was used to purify proteins. The recombinant proteins efficiently integrated into cell membranes in a process dependent upon the GPI anchor and were able to activate the CXCR3 receptor on lymphocytes. Endothelial cells incubated with CXCL10-mucin-GPI efficiently recruited NK cells in vitro under conditions of physiologic flow, which was shown to be dependent on the presence of the mucin domain. Experiments conducted in vivo using established tumors in mice suggested a positive effect of CXCL10-mucin-GPI on the recruitment of NK cells.ConclusionsThe results suggest enhanced recruitment of NK cells by CXCL10-mucin-GPI. This class of fusion proteins represents a novel adjuvant in cellular immunotherapy. The underlying concept of a chemokine head fused to the mucin domain and a GPI anchor signal sequence may be expanded into a broader family of reagents that will allow targeted recruitment of cells in various settings.
Highlights
Cell-based immunotherapy harnesses the natural cytotoxic potential of immune cells to eliminate target cells in a highly specific manner
The underlying concept of a chemokine head fused to the mucin domain and a GPI anchor signal sequence may be expanded into a broader family of reagents that will allow targeted recruitment of cells in various settings
In addition to T lymphocytes, the activity of NK cells is desirable as they play a complementary role to CTL in the antitumor response by recognizing tumors which are resistant to T cell killing due to downregulation of MHC class I molecules [1,2,3]
Summary
Cell-based immunotherapy harnesses the natural cytotoxic potential of immune cells to eliminate target cells in a highly specific manner. A problem frequently encountered using cytotoxic lymphocytes as anti-tumor agents is insufficient infiltration of the tumor tissue, in particular evident for NK cells [4,5,6,7,8], which has been proposed as an explanation for the lack of efficacy of cellular tumor-therapy in many settings [9,10,11]. This has been linked to changes in the tumor vasculature leading to reduced expression of adhesion molecules on tumor endothelial cells, as well as reduced efficacy of proinflammatory cytokines in upregulating adhesion molecule expression [6,12,13,14,15]. The mucin domain in conjunction with the chemokine head acts to recruit leukocytes expressing the corresponding chemokine receptor
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