During antiviral therapy, specific delivery of interferon‐α (IFNα) to infected cells may increase its antiviral efficacy, trigger a localized immune reaction, and reduce the side effects caused by systemic administration. Two T‐cell receptor‐like antibodies (TCR‐L) able to selectively bind hepatitis B virus (HBV)‐infected hepatocytes of chronic hepatitis B patients and recognize core (HBc18‐27) and surface (HBs183‐91) HBV epitopes associated with different human leukocyte antigen (HLA)‐A*02 alleles (A*02:01, A*02:02, A*02:07, A*02:11) were generated. Each antibody was genetically linked to two IFNα molecules to produce TCR‐L/IFNα fusion proteins. We demonstrate that the fusion proteins triggered an IFNα response preferentially on the hepatocytes presenting the correct HBV‐peptide HLA‐complex and that the mechanism of the targeted IFNα response was dependent on the specific binding of the fusion proteins to the HLA/HBV peptide complexes through the TCR‐like variable regions of the antibodies. Conclusion: TCR‐L antibodies can be used to target cytokines to HBV‐infected hepatocytes in vitro. Fusion of IFNα to TCR‐L decreased the intrinsic biological activity of IFNα but preserved the overall specificity of the protein for the cognate HBV peptide/HLA complexes. This induction of an effective IFNα response selectively in HBV‐infected cells might have a therapeutic advantage in comparison to the currently used native or pegylated IFNα. (HEPATOLOGY 2012;56:2027–2038)
Background PN-943, an oral gastrointestinal (GI) -restricted peptide antagonist of α4β7 integrin, is being developed for the treatment of inflammatory bowel disease (IBD). Blockade of α4β7 binding to mucosal addressin cell adhesion molecule-1 (MAdCAM1) is thought to treat IBD by preventing extravascular migration of blood T cells into the inflamed GI mucosa. Recent evidence suggests the involvement of other mechanisms. Saturation of circulating T-cell α4β7 by vedolizumab is not sufficient for optimal efficacy; and PTG-100, a PN-943 analogue, showed evidence of clinical and histological remission in ulcerative colitis patients at sub-saturating blood receptor occupancy (%RO). Here, we assessed the potential of a local GI-acting function of α4β7 by evaluating the ability of PN-943 to inhibit MAdCAM1-mediated CD4+ T-cell proliferation and cytokine production. Methods Primary CD4+ T cells were labelled fluorescently and incubated with plate-bound anti-CD3 alone or together with MAdCAM1 with or without inhibitors for three days. Phenotype, distribution of T-helper (Th) subsets and %RO analyses were conducted by flow cytometry. Supernatant cytokine levels were quantified by multiplex assays. Results MAdCAM1 combined with anti-CD3 markedly enhanced proliferation of CD4+ T cells compared with anti-CD3 alone (n = 7, 12–64%). Immune phenotyping revealed that proliferation occurred in both CD45RO- naïve and CD45RO+ memory T cells and was restricted to the β7+ population with successive cycles of proliferation showing increased β7 expression. Amongst the proliferated memory T cells, the percentage of the IFNγ-producing Th1 subset was higher than IL-17A-producing Th17 and IL-4-producing Th2 subsets. Multiplex profiling identified several cytokines, including but not limited to IL-6, IL-13, GM-CSF, and TNFα, whose release were promoted by MAdCAM1. PN-943 abolished MAdCAM1-mediated stimulation (n = 4, IC50 4.6 nM). The inhibitory effects were associated with lowered β7 expression indicative of internalisation by PN-943. Blockade was not observed with an inactive analogue indicating dependency on PN-943 binding to α4β7. Consistent with the requirement for a locally acting drug, 30 mg/kg oral dosing in mice demonstrated high exposure (n = 6, 39 nM) and occupancy of T-cell α4β7 (n = 6, 92%) in the GI compared within the blood. Conclusion The α4β7-MAdCAM1 interaction promoted β7+CD4+ T-cell proliferation and cytokine release, which may contribute to chronic inflammatory responses occurring in the diseased gut of IBD patients independent of T-cell trafficking. PN-943 inhibition of MAdCAM1-mediated signalling through α4β7 supports the potential therapeutic advantages for an oral GI-restricted approach, whereby PN-943 is delivered locally and directly blocks α4β7 function in the GI.
Human testisin, a serine protease, is highly expressed in ovarian cancer and premeiotic spermatocytes with relatively little expression in other normal tissues. We first showed that testisin was localized on the surface of cultured tumor cells as a glycosyl-phosphatidylinositol–linked protein. We next explored the biological function of testisin in malignant transformation through manipulation of testisin expression in cell culture model systems. Small interfering RNA–mediated knockdown of endogenous testisin mRNA and protein expression in tumor cell lines led to increased apoptosis and diminished growth in soft agar. Conversely, overexpression of testisin in an epithelial cell line induced colony formation in soft agar as well as s.c. tumor growth in severe combined immunodeficient mice. A catalytic domain mutant was unable to induce soft-agar growth indicating that testisin protease activity is required for transformation. Ectopic expression of testisin in a human ovarian cancer cell line without endogenous testisin expression, led to the formation of larger tumors in severe combined immunodeficient mice. Data presented here provide the first demonstration that testisin can promote cellular processes that drive malignant transformation. Our functional data coupled with the restricted normal tissue distribution of testisin and its overexpression in a majority of ovarian cancers validates this cell surface protein as a target for therapeutic intervention.
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