Neutralisation of factor VIII inhibitors by anti-idiotypes isolated from phage-displayed libraries

SummaryFollowing replacement therapy with coagulation factor VIII (FVIII), up to 30 % of haemophilia A patients develop FVIII-specific inhibitory antibodies (FVIII inhibitors). Immune tolerance induction (ITI) is not always successful, resulting in a need for alternative treatments for FVIII inhibitor-positive patients. As tolerance induction in the course of ITI appears to involve the formation of anti-idiotypes specific for anti-FVIII antibodies, such anti-idiotypes might be used to restore haemostasis in haemophilia A patients with FVIII inhibitors. We isolated antiidiotypic antibody fragments (scFvs) binding to murine FVIII inhibitors 2-76 and 2-77 from phage-displayed libraries. FVIII inhibitor/anti-idiotype interactions were very specific as no cross-reactivity with other FVIII inhibitors or isotype controls was observed. ScFvs blocked binding of FVIII inhibitors to FVIII and neutralised their cognate inhibitors in vitro and a monoclonal mouse model. In addition, scFv JkH5 specific for FVIII inhibitor 2-76 stained 2-76-producing hybridoma cells. JkH5 residues R52 and Y226, located in complementary determining regions, were identified as crucial for the JkH5/2-76 interaction using JkH5 alanine mutants. SPR spectroscopy revealed that JkH5 interacts with FVIII inhibitor 2-76 with nanomolar affinity. Thus, FVIII inhibitorspecific, high-affinity anti-idiotypes can be isolated from phagedisplayed libraries and neutralise their respective inhibitors. Furthermore, we show that anti-idiotypic scFvs might be utilised to specifically target inhibitor-specific B cells. Hence, a pool of anti-idiotypes could enable the reestablishment of haemostasis in the presence of FVIII inhibitors in patients or even allow the depletion of inhibitors by targeting inhibitor-specific B cell populations.

Changes in 65-Kilodalton Glutamate Decarboxylase Antibody-Specific Antiidiotypic Antibody Levels Correlate with Changes in C-Peptide Levels and Progression to Islet Cell Autoimmunity

This article appears in The Journal of Clinical Endocrinology & Metabolism. 10.1210/jc.2010-0785

Antifungal and antitumor models of bioactive protective peptides

Peptides are remarkably reactive molecules produced by a great variety of species and able to display a number of functions in uni-and multicellular organisms as mediators, agonists and regulating substances. Some of them exert cytotoxic effects on cells other than those that produced them, and may have a role in controlling subpopulations and protecting certain species or cell types. Presently, we focus on antifungal and antitumor peptides and discuss a few models in which specific sequences and structures exerted direct inhibitory effects or stimulated a protective immune response. The killer peptide, deduced from an antiidiotypic antibody, with several antimicrobial activities and other Ig-derived peptides with cytotoxic activities including antitumor effects, are models studied in vitro and in vivo. Peptide 10 from gp43 of P. brasiliensis (P10) and the vaccine perspective against paracoccidioidomycosis is another topic illustrating the protective effect in vivo against a pathogenic fungus. The cationic antimicrobial peptides with antitumor activities are mostly reviewed here. Local treatment of murine melanoma by the peptide gomesin is another model studied at the Experimental Oncology Unit of UNIFESP.

Therapeutic Activity of an Engineered Synthetic Killer Antiidiotypic Antibody Fragment against Experimental Mucosal and Systemic Candidiasis

ABSTRACT Peptides derived from the sequence of a single-chain, recombinant, antiidiotypic antibody (IdAb; KT-scFv) acting as a functional internal image of a microbicidal, wide-spectrum yeast killer toxin (KT) were synthesized and studied for their antimicrobial activity by using the KT-susceptible Candida albicans as model organism. A decapeptide containing the first three amino acids (SAS) of the light chain CDR1 was selected and optimized by alanine replacement of a single residue. This peptide exerted a strong candidacidal activity in vitro, with a 50% inhibitory concentration of 0.056 μM, and was therefore designated killer peptide (KP). Its activity was neutralized by laminarin, a β1-3 glucan molecule, but not by pustulan, a β1-6 glucan molecule. KP also competed with the binding of a KT-like monoclonal IdAb to germinating cells of the fungus. In a rat model of vaginal candidiasis, local, postchallenge administration of KP was efficacious in rapidly abating infections caused by fluconazole-susceptible or -resistant C. albicans strains. In systemic infection of BALB/c or SCID mice preinfected intravenously with a lethal fungal load, KP caused a highly significant prolongation of the median survival time, with >80% of the animals still surviving after >60 days, whereas >90% of control mice died within 3 to 5 days. KP is therefore the first engineered peptide derived from a recombinant IdAb retaining KT microbicidal activity, probably through the interaction with the β-glucan KT receptor on target microbial cells.