Characterization of AT02 Bioactivity and an Assessment of Its Potential Use As an Amyloid-Clearing Therapeutic for AL Amyloidosis
Authors: Wall Jonathan S. , Kennel Stephen J. , Stuckey Alan , Richey Tina , Williams Angela D. , Balachandran Manasi , Hancock Trevor , Jackson Joseph W. , Wooliver D. Craig , Macy Sallie , Heidel R. Eric , Lands Ronald H. , Martin Emily B 
Published: December 2024

Background: Immunoglobulin light chain (AL) amyloidosis is a systemic protein folding disorder associated with a monoclonal plasma cell dyscrasia. Fibrillar aggregates of monoclonal free light chains can deposit in any organ or tissue, leading to architectural disruption and organ dysfunction. Amyloid is not naturally cleared by the immune system possibly due to the presence of extracellular matrix components in the deposits (PMID: 36541892). However, 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 peptide-fusion incorporating the pan amyloid-binding peptide, p5R (PMID: 23750281), at the light chain C-terminus. This reagent binds to AL amyloid via the peptide interactions and retains the immunomodulatory capacity of the IgG Fc domain. The goal of these studies was to characterize the binding and bioactivity of AT-02 with AL amyloid.

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 AL amyloid in formalin fixed tissues. 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. Activation of complement following binding to amyloid substrates was determined using a C5b9 liberation assay in the presence of human plasma. AT-02 labelled with the near infra-red fluorophore Dylight800 was administered to mice bearing subcutaneous human AL amyloid and the uptake monitored over ten (10) days using optical imaging.

Results: AT-02 bound cardiac AL amyloid in tissue sections with high specificity and intensity. The binding potency for synthetic rVl6WIL fibrils, as well as human AL amyloid extracts, was sub-nanomolar in the ELISA assay. Incubation of AT-02 with AL amyloid extract induced phagocytosis by activated human THP-1 cells via FcR-mediated uptake and induced complement activation in the presence of human plasma. Fluorophore labelled AT-02 co-localized with human AL amyloidomas in mice and remained bound to the amyloid for ten days post-injection. Pretreatment of human AL amyloid extract with AT-02 prior to subcutaneous injection enhanced phagocytosis and clearance of amyloid in the amyloidoma mouse model.

Conclusion: Clearance of AL amyloid from affected organs is a significant clinical unmet need for patients. Removing amyloid could positively impact organ function and reverse dysfunction, as seen in some patients where AL amyloid spontaneously regresses coincident with a complete hematologic remission. The humanized IgG1-peptide fusion, AT-02, potently binds AL amyloid and may serve as an opsonin to induce macrophage-mediated phagocytosis and clearance of tissue amyloid deposits.

Acknowledgments: This study is supported by a grant from Attralus Inc, CA, USA. AT-02 was provided by Attralus Inc.