LEC/chTNT-3 fusion protein for the immunotherapy of experimental solid tumors.

Abstract

The human chemokine liver-expression chemokine (LEC) was originally found in an expressed sequence tag library, and later the LEC gene was located to chromosome 17q in the ML chemokine gene cluster. LEC has been shown to chemoattract monocytes, lymphocytes, and polymorphonuclear leukocytes (PMNs) by its binding to CCR1 and CCR8 chemokine receptors. Because of its potency as a chemoattractant for immune cells, LEC was used to genetically engineer a fusion protein with chTNT-3, a monoclonal antibody previously shown to target tumors by binding to DNA exposed in necrotic zones. Because the N-terminus of chemokines is important for their activity, the C-terminus of LEC was genetically linked to the chTNT-3 heavy chain variable region and, along with the light chain gene, cotransfected into NSO murine myeloma cells using the glutamine synthetase gene amplification system. The expressed LEC/chTNT-3 fusion protein was purified by tandem protein-A affinity and ion-exchange chromatography and chemotaxis and binding assays confirmed the bioactivity of the purified fusion protein. Pharmacokinetic and biodistribution studies in vivo showed that LEC/chTNT-3 had a biologic half-life of 3 hours and had good uptake in tumor (2.4% injected dose/g), which remained stable at 12 and 24 hours postinjection. Immunotherapy studies performed in three solid tumor models of the BALB/c mouse showed between 37% and 55% tumor reduction at 19 days post-implantation. Immunohistochemical studies using tumor sections obtained at different time points after the administration of control chTNT-3 and LEC/chTNT-3 showed heavy infiltration of CD4+ and CD8+ T cells, PMNs, B cells, and CD11c+CD11b+ dendritic cells in the LEC/chTNT-3 treated groups. The results of these studies demonstrate that this novel fusion protein has potent antitumor activity that is associated with the infiltration of different subpopulations of immune cells. The targeting of LEC to necrotic areas of tumors where the release of tumor antigens is prevalent may be a new approach for the immunotherapy of solid tumors.