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The selective knock-down of cellular proteins has proven useful for in vivo studies of protein function and RNAi methods are readily available for this purpose. However, interfering directly at the protein level may have distinct advantages, with the intracellular targeting of antibodies (intrabodies) representing an attractive option, although not a general one. We demonstrate a novel, general strategy named suicide (or silencing) intrabody technology (SIT), based on the inducible degradation of intrabodies, which are equipped with proteasome-targeting sequences and thus converted into suicide intrabodies. We show that suicide intrabodies are able to redirect the target cellular proteins upon stimulus administration to the proteolytic machinery, thus resulting in selective protein knock-down. Remarkably, suicide intrabody acts in a catalytic fashion. SIT is a ligand-inducible strategy, potentially applicable to any protein of interest and does not require the engineering of cellular proteolytic enzymes. SIT represents a general approach to confer "neutralizing" properties to any intrabody, a valuable feature, given the present impossibility to select a priori intrinsically neutralizing antibodies. This knock-down strategy, together with available methods to isolate functional intrabodies, should allow the large-scale investigation of intracellular protein networks.

Allograft transplantation into sensitized recipients with antidonor antibodies results in accelerated antibody-mediated rejection (AMR), complement activation, and graft thrombosis. We have developed a membrane-localizing technology of wide applicability that enables therapeutic agents, including anticoagulants, to bind to cell surfaces and protect the donor endothelium. We describe here how this technology has been applied to thrombin inhibitors to generate a novel class of drugs termed thrombalexins (TLNs). Using a rat model of hyperacute rejection, we investigated the potential of one such inhibitor (thrombalexin-1 [TLN-1]) to prevent acute antibody-mediated thrombosis in the donor organ. TLN-1 alone was able to reduce intragraft thrombosis and significantly delay rejection. The results confirm a pivotal role for thrombin in AMR in vivo. This approach targets donor organs rather than the recipient and is intended to be directly translatable to clinical use.

CD46 is a complement regulator with important roles related to the immune response. CD46 functions as a pathogen receptor and is a potent costimulator for the induction of interferon-γ (IFN-γ)-secreting effector T helper type 1 (TH1) cells and their subsequent switch into interleukin 10 (IL-10)-producing regulatory T cells. Here we identified the Notch family member Jagged1 as a physiological ligand for CD46. Furthermore, we found that CD46 regulated the expression of Notch receptors and ligands during T cell activation and that disturbance of the CD46-Notch crosstalk impeded induction of IFN-γ and switching to IL-10. Notably, CD4+ T cells from CD46-deficient patients and patients with hypomorphic mutations in the gene encoding Jagged1 (Alagille syndrome) failed to mount appropriate T H 1 responses in vitro and in vivo, which suggested that CD46-Jagged1 crosstalk is responsible for the recurrent infections in subpopulations of these patients. © 2012 Nature America, Inc. All rights reserved.

Protein-protein interactions represent a major potential drug target for many human diseases, but these are unanimously considered undruggable with small chemical molecules. We have developed 3-SPLINT, a novel technology for the selection of antibodies that are intrinsically endowed with the ability to interfere with a given protein-protein interaction. The selection procedure exploits the recently described yeast SPLINT libraries of intrabodies, adapting them to a reverse-hybrid system, yielding the selection of recombinant antibodies that are able to disrupt a target protein-protein interaction in vivo. This class of antibodies should therefore perturb an individual protein-protein interaction, without perturbing the scaffolding function of the target protein in that complex, or other protein interactions of that same protein. We provide here a proof of concept of the technology, by the de novo selection of antibodies against two distinct interacting protein pairs: the GABARAP, which interact with the gamma2 subunit of GABA(A) receptor, and the p65 protein dimer, involved in the NF-kappaB-mediated signalling transduction pathway. Intrabodies selected against the latter were functionally validated in cells. Such antibodies, by interfering with the dimerization domain of p65, lead to an activation of the NF-kappaB-mediated transcriptional activity, which is normally inhibited by p65 knock-down RNAi. This provides a clear-cut demonstration that interfering with a protein interaction can be functionally very different from physically removing one of the interacting proteins. The 3-SPLINT approach provides a general and finer tool for the functional validation of selected protein interactions in protein networks, and is ideally applied to protein "hubs", displaying multiple distinct interactions. 3-SPLINT will therefore complement RNAi-based approaches, in the toolkit of target validation strategies, and is amenable to the systematic isolation of comprehensive sets of antibodies against most protein-protein interactions of a given protein network.

The tissue factor-thrombin pathway is known to mediate proliferation, adhesion, invasion and metastasis of many types of cancer cells. These effects are additional to the thromboembolic processes commonly occurring in cancer, particularly in patients on hormonal therapies. In prostate cancer, there is an overexpression of the thrombin receptor PAR1 correlating with tumor progression. Anticoagulants such as heparin and hirudin are known to inhibit metastasis of tumors and improve survival rates in prostate cancer patients with localized disease. However, anticoagulants given systemically have an associated risk of bleeding which is higher in cancer patients and can be compounded by an increased risk of thrombocytopenia. Anticoagulants which can be localized to a tumor lesion are therefore highly favorable. The aim of the following study was to determine the efficacy of membrane-tethered anticoagulants on thrombin mediated migration and proliferation of prostate cancer cells in vitro. The hirudin cytotopic analogs PTL004 and PTL006, previously designed for use with anti-complement therapeutics in transplantation, were assessed as inhibitors of proliferation and migration of prostate cancer cells, in comparison to non-tailed hirudin-derived peptide, a PAR-1 antagonist and hirudin itself. Methods: For proliferation assays, 2000 PC3 prostate cancer cells were seeded in 96 well plates in serum free culture medium. Cells were preincubated for 30 mins with anticoagulant inhibitors at concentrations from 1 µM to 0.001 µM, and then 0.5 nM thrombin was added. Proliferation was measured after 48 hrs using MTT. To measure effects on cell migration, 10,000 PC3 cells were seeded onto porous membrane inserts in 24 well plates in serum free medium and then incubated with the inhibitors at 1 µM for 30 mins followed by 0.5 nM thrombin. Migration of cells through the membrane was measured after a 24 hr period. Results: Thrombin increased proliferation of PC3 cells in serum-free medium with a 1.5 fold increase in cell number after 48h. The tailed derivative PTL004 inhibited proliferation with an IC50 of 1 µM, equivalent to hirudin and the PAR1 antagonist. Liposomes prepared with PTL004 on the surface were more active than the tailed preparation in free solution with an IC50 10-50 fold lower. However, the non-tailed hirudin-derived peptide, and the derivative PTL006, which is known to have a more rapid dissociation rate when used with other cell types, were not active under these assay conditions. In the migration assays, PTL004 was also most active among the derivatives we tested. Conclusions: The membrane localizing anti-coagulant PTL004 has comparable anti-migratory and anti-proliferative properties to hirudin, and liposomes bearing this compound are even more effective at inhibition of proliferation of PC3 cells. The results demonstrate the potential of a cytotopic approach using tailed anticoagulants in localized prostate cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 673. doi:10.1158/1538-7445.AM2011-673