Intratumoral virotherapy with 4-1BBL armed modified vaccinia Ankara eradicates solid tumors and promotes protective immune memory

Background Human cancers are extraordinarily heterogeneous in terms of tumor antigen expression, immune infiltration and composition. A common feature, however, is the host ' s inability to mount potent immune responses that prevent tumor growth effectively. Often, naturally primed CD8(+) T cells against solid tumors lack adequate stimulation and efficient tumor tissue penetration due to an immune hostile tumor microenvironment. Methods To address these shortcomings, we cloned tumor-associated antigens (TAA) and the immune-stimulatory ligand 4-1BBL into the genome of modified vaccinia Ankara (MVA) for intratumoral virotherapy. Results Local treatment with MVA-TAA-4-1BBL resulted in control of established tumors. Intratumoral injection of MVA localized mainly to the tumor with minimal leakage to the tumor-draining lymph node. In situ infection by MVA-TAA-4-1BBL triggered profound changes in the tumor microenvironment, including the induction of multiple proinflammatory molecules and immunogenic cell death. These changes led to the reactivation and expansion of antigen-experienced, tumor-specific cytotoxic CD8(+) T cells that were essential for the therapeutic antitumor effect. Strikingly, we report the induction of a systemic antitumor immune response including tumor antigen spread by local MVA-TAA-4-1BBL treatment which controlled tumor growth at distant, untreated lesions and protected against local and systemic tumor rechallenge. In all cases, 4-1BBL adjuvanted MVA was superior to MVA. Conclusion Intratumoral 4-1BBL-armed MVA immunotherapy induced a profound reactivation and expansion of potent tumor-specific CD8(+) T cells as well as favorable proinflammatory changes in the tumor microenvironment, leading to elimination of tumors and protective immunological memory

Intratumoral virotherapy with 4-1BBL armed modified vaccinia Ankara eradicates solid tumors and promotes protective immune memory

Background Human cancers are extraordinarily heterogeneous in terms of tumor antigen expression, immune infiltration and composition. A common feature, however, is the host ' s inability to mount potent immune responses that prevent tumor growth effectively. Often, naturally primed CD8(+) T cells against solid tumors lack adequate stimulation and efficient tumor tissue penetration due to an immune hostile tumor microenvironment. Methods To address these shortcomings, we cloned tumor-associated antigens (TAA) and the immune-stimulatory ligand 4-1BBL into the genome of modified vaccinia Ankara (MVA) for intratumoral virotherapy. Results Local treatment with MVA-TAA-4-1BBL resulted in control of established tumors. Intratumoral injection of MVA localized mainly to the tumor with minimal leakage to the tumor-draining lymph node. In situ infection by MVA-TAA-4-1BBL triggered profound changes in the tumor microenvironment, including the induction of multiple proinflammatory molecules and immunogenic cell death. These changes led to the reactivation and expansion of antigen-experienced, tumor-specific cytotoxic CD8(+) T cells that were essential for the therapeutic antitumor effect. Strikingly, we report the induction of a systemic antitumor immune response including tumor antigen spread by local MVA-TAA-4-1BBL treatment which controlled tumor growth at distant, untreated lesions and protected against local and systemic tumor rechallenge. In all cases, 4-1BBL adjuvanted MVA was superior to MVA. Conclusion Intratumoral 4-1BBL-armed MVA immunotherapy induced a profound reactivation and expansion of potent tumor-specific CD8(+) T cells as well as favorable proinflammatory changes in the tumor microenvironment, leading to elimination of tumors and protective immunological memory

A novel in vitro model of simulated twice a day iron supplementation for assessment of iron uptake and cellular damage

Hepcidin is the master regulator of iron homeostasis and its expression is mainly attributed to BMP6 protein which triggers the phosphorylation of SMAD1/5/8. Our previous results have shown that heparins strongly repress the BMP6/SMAD signaling resulting in a decrease of hepcidin expression. This finding suggested that not only heparin, but also endogenous heparan sulfates (HSs), ubiquitous heparin-like polysaccharides exposed on cells surface, may participate in the BMP6/SMAD pathway by facilitating the binding of BMP6 to its receptors. How BMP6 binds heparin is not clear, but different Heparin Binding Domains (HBDs) have been already characterized in BMP2 and BMP4 isoforms as lysine and arginine-rich regions.The aim of this work is to define the heparin/HS binding domains of BMP6 to clarify the mechanism of action of heparin in hepcidin inhibition and to verify if HSs are involved in hepcidin expression.BMP6 presents three basic regions rich in Arg and Lys, that are putative HBDs. The first is located at the unstructured N-terminus (HBD1), a second in central portion (HBD2) and the third at C-Terminus (HBD3). Synthetic 15-residue peptides representing the three domains were produced in fusion with biotin. They were used to track the energetics of binding of each HBD peptide to heparin adsorbed onto microcantilever biosensors. We found that N-terminus domain (HBD1) showed higher heparin affinity than the others and that it was dose dependent. Then we assayed their binding to heparan sulfates (HS) using monolayers of cells that express or not membrane HS. The results showed that also with this approach the N-terminus HBD peptide bound with higher affinity the surface of cells expressing HS, and that this binding was reduced in the presence of heparin. These findings prompted us to produce the recombinant mature form of human BMP6 in a prokaryotic system in order to study its heparin and HS binding activity in a completely folded protein. Human mature BMP6 with his-tag was cloned into the pASK-IBA43plus vector and expressed in E. Coli RosettaTM (DE3) host strain, as insoluble inclusion bodies. BMP6 was solubilized and then purified obtaining a highly pure denatured BMP6 on its monomeric form. Finally, we used an in vitro oxidative renaturation protocol to obtain the dimeric functional form of BMP6.We found that the produced dimeric BMP6 was able to bind heparin and that it binds more effectively to cells expressing HS. The biological activity of BMP6 seems to require the glycosylation, thus we cloned the mature human and mouse BMP6 in eucariotic vectors. We transfected the Hek293 with these plasmids and treated HepG2 cells with their supernatant. The transcription of hepcidin was induced by the BMP6 transfected Hek supernatant supporting the expression and secretion of BMP6. Mutagenesis of arginine in these prokaryotic and eukaryotic clones are in progress to identify the major HBD in the BMP6; the results will be present