Characterization and in vivo target engagement studies of a novel, preformed, amyloid-reactive peptope-antibody complex

324 Introduction: Systemic amyloidosis is a devastating, progressive, protein-deposition disease resulting from the formation of fibrils by normal serum protein components. More than 30 forms of amyloid-associated disease have been identified, with each defined by the precursor protein comprising the fibril. Clearance of tissue amyloid remains an important goal in the treatment of patients with systemic amyloidosis. The persistent accumulation of amyloid results in organ dysfunction and severe morbidity. Amyloid clearance may result in restoration of function and improved patient outcomes, and the prevailing mechanism for achieving this is via antibody (mAb) Fc-mediated phagocytosis of amyloid by macrophages. We have previously described a peptope construct comprising a pan-amyloid binding peptide and high-affinity epitope recognized by mAb 11-1F4 for use in a two-step pre-targeting protocol for amyloid opsonization. We now demonstrate that premixing mAb 11-1F4 with a bifunctional peptope, designated p105, yields a complex that can recognize multiple types of amyloid. We have shown that the p105-11-1F4 complex: 1) Forms a stable, functional complex; 2) Binds diverse amyloids; 3) Specifically targets amyloid in a murine model of AA amyloidosis, and; 4) Enhances the phagocytosis of amyloid in vitro. Objectives: The aim of this study was to characterize a novel peptope-mAb preformed complex and assess amyloid reactivity and biodistribution in preclinical studies. Methods: Peptope p105 was chemically synthesized (Genscript, NJ) and murine 11-1F4 was prepared by the National Cancer Institute. The complex was formed by incubation of peptope p105 with 11-1F4 at a molar ratio of 2.5:1. For certain assays, 11-1F4 was labeled with iodine-125 before addition to p105. After characterization of the complex, binding of p105-125I-11-1F4 to diverse amyloids was assessed using synthetic fibrils and human amyloid extracts in a pulldown assay. Reactivity with amyloid in vivo was evaluated using mice with inflammation-associated (AA) amyloidosis by small animal SPECT/CT imaging and microautoradiography. Amyloid free mice served as controls. Results: Incubation of p105 and 11-1F4 resulted in ~95% of the mAb complexed with peptope. In a pulldown assay, p105-125I-11-1F4 bound synthetic fibrils (84% bound vs. 2% for 11-1F4 alone) and human AL amyloid extracts consistent with the amyloid binding characteristics of the peptope. When injected into AA mice, p105-125I-11-1F4 was retained in the spleen (11.1 %injected dose/g tissue), liver (23.2 %ID/g) and pancreas (12 %ID/g) - sites of major AA amyloid deposition in this model. In amyloid-free mice, liver and splenic uptake was 0.4 and 1.6 %ID/g, respectively. When compared to 125I-11-1F4 mAb alone, the radiolabeled complex was shown to specifically co-localize with amyloid in these tissues by using microautoradiography, with no evidence of uptake in amyloid free organs. Conclusions: The p105-11-1F4 complex is a novel approach to developing a multi-amyloid targeting opsonizing reagent. The preformed peptope-mAb could be used as a single injection immunotherapeutic to effect resolution of diverse forms of tissue amyloid through cell-mediated uptake. Acknowledgment: This study was supported in part by the National Institute of Diabetes and Digestive and Kidney Diseases, DK110038.