C11-1F4 Binds and Promotes Clearance of Light Chain Fibrils By Phagocytosis

Introduction

Amyloid light-chain (AL) amyloidosis is a rare, progressive, systemic, life-threatening disorder, caused by deposition of amyloid fibrils in various organs resulting in organ failure and significant morbidity and mortality. AL amyloidosis results from a plasma cell dyscrasia (PCD) in which the plasma cells secrete light chains that misfold and aggregates in organs. Current standard of care in patients with AL amyloidosis is focused on arresting their PCD preventing formation of further amyloid deposits but does not target the existing fibrils. C11-1F4 was developed to specifically bind and eliminate fibrils from affected organs. It is hypothesized that C11-1F4 binds to fibrils resulting in the recruitment and activation of macrophages, neutrophils, and monocytes via the Fc portion, which in turn remove the fibrils through phagocytosis. In this study we tested the efficacy of C11-1F4 as a fibril opsonizing agent leading to the recruitment of immune cells which potentially contribute to amyloid removal.

Methods

Amyloid fibrils were generated from recombinant AL09 VL protein. Fibril formation was validated using ThT fluorescence analysis and Dynamic Light Scattering (DLS). For phagocytosis experiments, AL-09 fibrils were labeled using pHrodo™Red pH-sensitive dye which fluoresces bright red in acidic pH. THP-1 cells were differentiated to macrophage-like cells using 50 ng/ml PMA. THP-1 derived macrophages were treated with C11-1F4, IgG-1 isotype control (NIST), or NEOD-001 at various concentrations preincubated with 1uM of pHrodo™Red-labeled-AL-09 fibrils. NIST served as a negative isotype control. Phagocytosis of labeled AL-09 fibrils was captured using Incucyte cell imager.

To address whether C11-1F4 mediates phagocytosis by polymorphonuclear cells (PMN), whole blood was incubated with synthetic AL-09-labeled-fibrils (1 uM) and C11-1F4 at various concentrations. To confirm our observations from the whole blood assay, we also isolated PMN cells and performed the phagocytosis assay as outlined above. Phagocytosis of labeled AL-09 fibrils was captured using flow cytometry for the whole blood assay or by imaging with Incucyte for the isolated PMN cell assay.

Results and Conclusion

We developed microplate monolayer phagocytosis and solution phase flow cytometry assays that permitted the evaluation of the opsonizing ability of C11-1F4 which would result in enhanced phagocytosis by innate immune cells. Using pHrodo™Red-labeled AL09, we measured the uptake of fibrils in the presence of C11-1F4, NEOD-001, or NIST (isotype control). In vitro data with THP-1 derived macrophages shows that C11-1F4, at 10nM, enhances AL-09 fibril phagocytosis. Under similar conditions, NEOD-001 did not demonstrate any significant effect on AL-09 fibril phagocytosis and was similar to the IgG1 isotype control (Figure 1). Furthermore, in whole blood collected from healthy volunteers we showed that C11-1F4 binding to fibrils recruits PMN cells leading to phagocytosis of pHrodo™Red-labeled AL-09 fibrils (23% in the presence of C11-1F4 vs 3% in the presence of NIST antibody). At similar concentrations NEOD-001 showed no difference compared to isotype control. To confirm our observation from whole blood assay, we conducted the phagocytosis assay on isolated PMN cells. C11-1F4 significantly induces internalization of the pHrodo™Red-conjugated AL-09 fibrils by PMN cells compared to the isotype control antibody (Figure 2).

In conclusion, our studies highlight the role of C11-1F4 in fibril opsonization and recruitment of immune cells leading to fibril removal.

Esteghamat:Alexion/AstraZeneca Rare Disease Unit: Current Employment, Current equity holder in publicly-traded company. Ahlawat:Alexion/AstraZeneca Rare Disease Unit: Current Employment, Current equity holder in publicly-traded company. Raiker:Alexion/AstraZeneca Rare Disease Unit: Current Employment, Current equity holder in publicly-traded company. Batonick:Alexion/AstraZeneca Rare Disease Unit: Current Employment, Current equity holder in publicly-traded company. Quarta:Alexion, AstraZeneca Rare Disease: Current Employment. Usmani-Brown:Alexion/AstraZeneca Rare Disease Unit: Current Employment, Current equity holder in publicly-traded company.