RESUMO
Autoantigen-specific immunotherapy using peptides offers a more targeted approach to treat autoimmune diseases, but clinical implementation has been challenging. We previously showed that multivalent delivery of peptides as soluble antigen arrays (SAgAs) efficiently protects against spontaneous autoimmune diabetes in the non-obese diabetic (NOD) mouse model. Here, we compared the efficacy, safety, and mechanisms of action of SAgAs versus free peptides. SAgAs, but not their corresponding free peptides at equivalent doses, efficiently prevented the development of diabetes. SAgAs increased the frequency of regulatory T cells among peptide-specific T cells or induce their anergy/exhaustion or deletion, depending on the type of SAgA used (hydrolysable (hSAgA) and non-hydrolysable 'click' SAgA (cSAgA)) and duration of treatment, whereas their corresponding free peptides induced a more effector phenotype following delayed clonal expansion. Over time, the peptides induced an IgE-independent anaphylactic reaction, the incidence of which was significantly delayed when peptides were in SAgA form rather than in free form. Moreover, the N-terminal modification of peptides with aminooxy or alkyne linkers, which was needed for grafting onto hyaluronic acid to make hSAgA or cSAgA variants, respectively, influenced their stimulatory potency and safety, with alkyne-functionalized peptides being more potent and less anaphylactogenic than aminooxy-functionalized peptides. Immunologic anaphylaxis occurred in NOD mice in a dose-dependent manner but not in C57BL/6 or BALB/c mice; however, its incidence did not correlate with the level of anti-peptide antibodies. We provide evidence that SAgAs significantly improve the efficacy of peptides to induce tolerance and prevent autoimmune diabetes while at the same time reducing their anaphylactogenic potential.
Assuntos
Diabetes Mellitus Tipo 1 , Tolerância Imunológica , Camundongos Endogâmicos NOD , Peptídeos , Animais , Camundongos , Diabetes Mellitus Tipo 1/imunologia , Peptídeos/imunologia , Peptídeos/administração & dosagem , Feminino , Autoantígenos/imunologia , Linfócitos T Reguladores/imunologia , Imunoterapia/métodos , Anafilaxia/prevenção & controle , Anafilaxia/imunologia , Dessensibilização Imunológica/métodos , Dessensibilização Imunológica/efeitos adversosRESUMO
Fc fusions are a growing class of drugs comprising an antibody Fc domain covalently linked to a protein or peptide and can pose manufacturing challenges. In this study we evaluated three synthetic approaches to generate Fc fusions, using Fc-insulin as a model drug candidate. Engineered human IgG1 was digested with HRV3C to produce an Fc fragment with a C-terminal sortase tag (Fc-LPETGGH6). The synthesis of Fc-insulin2 from Fc-LPETGGH6 was evaluated with direct sortase-mediated ligation (SML) and two chemoenzymatic strategies. Direct SML was performed with triglycine-insulin, and chemoenzymatic strategies used to SML fuse either triglycine-azide or triglycine-DBCO prior to linking insulin with copper-catalyzed or strain-promoted azidealkyne cycloaddition. Reaction conditions were optimized by evaluating reagent concentrations, relative equivalents, temperature, and time. Direct SML provided the most effective reaction yields, converting 60-70% of Fc-LPETGGH6 to Fc-insulin2, whereas our optimized chemoenzymatic synthesis converted 30-40% of Fc-LPETGGH6 to Fc-insulin2. Here we show that SML is a practical and efficient method to synthesize Fc fusions and provide an optimized pathway for fusion drug synthesis.