NEOD001 Specifically Binds Aggregated Light Chain Infiltrates in Multiple Organs from Patients with AL Amyloidosis and Promotes Phagocytic Clearance of AL Aggregates in Vitro

Background: Light chain (AL) amyloidosis is the most common form of systemic amyloidosis and is characterized by the accumulation of aggregated, misfolded light chain protein in a variety of organs, resulting in serious organ damage and dysfunction. To date, no therapies have been approved to treat patients with AL amyloidosis, and current approaches do not directly target the underlying cause of organ dysfunction. NEOD001 is a conformation-specific antibody currently in a phase 3 clinical trial that specifically targets light chain amyloid aggregates and may promote the clearance of AL deposits by means of phagocytosis. In the present study, we compared the binding characteristics of NEOD001 to aggregates deposited in various AL organs using immunolabeling and binding assays. We also assessed the ability of the antibody to induce the clearance of AL amyloid by phagocytosis in vitro.

Methods: The murine form of NEOD001, the 2A4 antibody, was used in these studies to avoid the nonspecific detection of human immunoglobulin G in human tissue. Immunohistochemical and biochemical techniques were used to characterize 2A4 immunoreactivity to AL aggregates. A total of 15 organs (heart, kidney, spleen, and liver) derived from 10 AL patients were examined. Fluorescent and chromogenic immunohistochemistry (IHC) were performed on both fresh frozen and aldehyde-fixed samples. Thioflavin T (ThioT) labeling was used to identify amyloid in IHC-labeled tissue. To assess the binding of 2A4 to AL amyloidosis patient extracts and recombinant light chain, we used surface plasmon resonance and a newly developed, plate-based immunoassay specific to AL aggregates. Phagocytosis was assessed using a macrophage cell line cultured in the presence of aggregated light chain. All experiments included isotype-control antibodies.

Results: In fresh frozen sections, 2A4 demonstrated specific immunoreactivity to AL aggregates in patient samples, but not in samples from healthy subjects, for all organs examined. Although 2A4 immunolabeling largely colocalized with ThioT, additional 2A4+/ThioT-unlabeled deposits were present that likely represented amorphous light chain deposits. Immunostaining of deposits with 2A4 was almost completely attenuated in aldehyde-fixed samples, even after brief fixation (1 minute), and was minimally recovered with antigen-retrieval methods. The aggregated light chain immunoassay demonstrated 2A4 binding in all organ tissue extracts from patients with AL amyloidosis but not from healthy subjects. The specificity of 2A4 to aggregated versus monomeric light chain was confirmed by biochemical assays. 2A4 induced the rapid engagement of macrophages and the phagocytic clearance of light chain aggregate particles in vitro.

Conclusions: This study demonstrates that 2A4, the murine form of NEOD001, specifically binds to amyloid light chain and amorphous light chain aggregates in various organs of patients with AL amyloidosis and, in vitro, promotes the clearance of light chain aggregate particles by macrophage phagocytosis. NEOD001 may thus hold therapeutic potential for directly targeting the underlying cause of organ dysfunction in AL amyloidosis.