Characterization of AT-02, a pan-amyloid-binding peptide fusion immunoglobulin with high binding potency, complement activation, and immune cell stimulation

Abstract

Abstract Background Cardiac amyloidosis is an ominous manifestation of systemic amyloidosis, notably in AL and ATTR-associated types, resulting in a restrictive hypertrophic cardiomyopathy with electrical conduction and atrioventricular strain dysfunction (1). Amyloid is not naturally cleared by the immune system due to the presence of extracellular matrix components in the deposits (2). However, recruiting and stimulating the innate immune system, notably phagocytic macrophages, is a promising strategy to effect amyloid removal. To facilitate this, we have generated AT-02, a humanized IgG1 incorporating the pan-amyloid-binding peptide, p5R (3), at the light chain C-terminus. This reagent binds diverse types of amyloid via the peptide interactions and retains immunomodulatory capacity. Purpose The goal of these studies was to characterize the bioactivity of AT-02 that underlies its potential therapeutic activity. Methods AT-02 was produced from a CHO pool using a perfusion cell culture production process, purified by Protein A, anion exchange, and cation exchange chromatographies, and characterized by mass spectrometry and SDS-capillary electrophoresis. AT-02 was biotinylated and used for immunohistochemical detection of both AL and ATTR amyloidosis in formalin fixed tissues (Figure 1A). Binding to heparin (as a surrogate for amyloid-associated hypersulfated heparan sulfate proteoglycans) and synthetic AL fibrils was studied using both ELISA and surface plasmon resonance assays (Figure 1B). Activation of complement following binding to amyloid substrates was determined using a C5b9 liberation assay in the presence of human plasma. The effect of AT-02 on amyloid fibril growth was determined using a fluorescence fibril elongation assay. In vivo clearance of amyloid and cell activation was demonstrated using a murine model of AL amyloidoma. Results AT-02 bound human cardiac amyloid in tissue sections with high specificity and intensity. The potency for heparin and synthetic amyloid fibrils, as well as human cardiac amyloid extracts, was sub-nanomolar in the ELISA assay with kinetics consistent with a high affinity antibody. In the presence of plasma, complement activation of amyloid-bound AT-02 was significantly higher as compared to controls, and the reagent inhibited synthetic amyloid fibril growth by ∼40%. Treatment of human amyloid with AT-02 expedited phagocytosis by murine macrophages in vivo and enhanced the intensity of immune response to the implanted amyloid. Conclusion Clearance of cardiac amyloid is a significant clinical unmet need for patients with systemic amyloidosis. The humanized IgG1-peptide fusion, AT-02, potently binds cardiac amyloid and may serve as an opsonin and induce macrophage-mediated phagocytosis and clearance of cardiac amyloid deposits.