217 - The Peptide Fusion Immunoglobulin, AT-02, Exhibits Highly Potent Pan-Amyloid Reactivity And Immunomodulation

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Author Block: Jonathan Wall¹, Michael Klein², Spencer Guthrie², James S. Foster¹, Angela Williams¹, Tina Richey¹, Manasi Balachandran¹, Joseph Jackson¹, Trevor Hancock¹, Alan Stuckey¹, Emily Martin¹, Stephen Kennel¹. ¹University of Tennessee Graduate School of Medicine, Knoxville, TN; ²Attralus Inc, San Francisco, CA

Disclosure Block: E. Martin: None.

Background: Cardiac amyloidosis is an ominous manifestation of -systemic amyloidosis, notably in AL and ATTR-associated types, resulting in a restrictive hypertrophic cardiomyopathy with conduction and strain abnormalities. Amyloid is generally not cleared by the immune system. 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 amyoid-binding peptide, p5R, in the light chain. This reagent can bind 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 underlie 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 cardiac AL and ATTR amyoid in formalin fixed tissues. Binding to synthetic AL fibrils and heparin (as a surrogate for amyloid-associated hypersulfated heparan sulfate proteoglycans) 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. The effect of AT-02 on AL 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 agent exhibited potent, sub-nanomolar binding to synthetic amyloid fibrils, as well as human cardiac amyloid extracts, 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 amyloid fibril growth. Treatment of human amyloid with AT-02 expedited phagocytosis of the material 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, inhibits fibril growth and may serve as an effective opsonin to induce the macrophage-mediated phagocytosis and clearance of cardiac amyloid deposits.