(W1130-01-05) Fibronectin (FN) Binding Efficacy of PEGylated FUD (PEG-FUD) Peptide in Bleomycin Treated Animal Model for the Treatment of Pulmonary Fibrosis

Purpose: Idiopathic pulmonary fibrosis (IPF) is an irreversible and progressive chronic disease which causes scarring in the lungs due to which patients have difficulty in breathing as the lungs are unable to transport oxygen into the bloodstream effectively. Fibronectin (FN) is a blood and extracellular matrix (ECM) glycoprotein that plays an important role in fibrosis as it controls the deposition of collagen and other extracellular matrix molecules. It is an important ECM component found in fibrotic tissues and its deposition occurs before collagen deposition and is crucial to the evolution of fibrosis, thus a distinguishing hallmark of this disorder. FN deposition has been identified as a possible therapeutic target for the treatment of fibrosis. Functional Upstream Domain (FUD) is a 6-kD peptide which shows nanomolar binding affinity for the FN (at N-terminal 70-kD domain), but the delivery of biologics is often challenged by elimination barriers like proteolysis and organ clearance that limits in-vivo systemic exposure, therefore, to overcome these problems, alternative delivery strategies for FUD were considered to understand the peptide therapeutic potential. PEGylation of FUD was thought to be a viable delivery technique because it is a well-developed methodology for optimizing biologics delivery by altering their pharmacokinetic features. Our group has developed a PEGylated FUD/pUR4, a FN assembly inhibitor, using 20-kD PEG and evaluated its binding affinity and inhibitory potency. Using a high-affinity FN-binding peptide (FUD/pUR4) that inhibits FN deposition into extracellular matrix (ECM), we have shown the efficacy of a PEGylated FUD/pUR4 (PEG-FUD) to inhibit fibrosis by using a fluorescently labeled Cy5-PEG-FUD that demonstrates preferential targeting to the fibrotic lungs in the bleomycin-induced mouse model of pulmonary fibrosis.

Methods: FUD and its mutated version (mFUD, which does not inhibit FN fibrillogenesis) were recombinantly expressed in E. coli BL21 (DE3) using the pET-ELMER vector. The peptides were purified and analyzed for their therapeutic potential using FPLC, MALDI-MS, ITC, and competitive binding in-vitro assay. Both peptides were further PEGylated using 20kD methoxy-PEG and the concentration the peptides was determined using UV-Vis absorbance curve at 280 nm. For fluorescence imaging, peptides were labeled using sulfo-Cy5-NHS-ester to see its in-vivo/ex-vivo efficacy. For ex vivo assessment of peptide targeting, non-fixed IPF or normal human lung tissue slices were stained with Cy5-labeled PEG-FUD or PEG-mFUD and DAPI, followed by immunofluorescence imaging. For in vivo assessment of peptide targeting, pulmonary fibrosis was induced in 11±3-week-old C57Bl6/J mice via intratracheal delivery of bleomycin (1U/kg in 0.9 % normal saline, NS) or NS control. During different phases of the bleomycin-induced pulmonary fibrosis, mice were injected via subcutaneous dose (12.5 mg/kg) of 1.5% Cy5-PEG-FUD or Cy5-PEG-mFUD in addition to matching unlabeled peptide between the shoulder blades and imaged via in vivo imaging system (IVIS) 24 h post injection. Immediately following in vivo imaging, mouse vital organs were collected and imaged ex vivo via IVIS.

Results: PEGylated FUD and mFUD were successfully synthesized and purified using FPLC. The MALDI-MS analysis of PEG-FUD confirmed the N-terminal specific conjugation of 20kD PEG to the FUD. It was also observed that PEGylation of FUD peptide did not significantly compromise the FN binding affinity by using isothermal titration calorimetry (ITC). Staining of samples obtained from human IPF lungs revealed high levels of FN and collagen accumulation in fibroblastic foci, the leading edges of fibrotic development, confirming the role of these ECM markers in development of the disease. Tissue staining with Cy5-labeled PEG-FUD (Cy5-PEG-FUD) and the control (Cy5-PEG-mFUD), demonstrated that Cy5-PEG-FUD preferentially binds to the fibroblastic foci, while there was no significant targeting of the control to the human fibrotic tissues. These data suggest that PEG-FUD can detect and target the early development of pulmonary fibrosis. To demonstrate its potential to target the fibrotic lungs during various phases of fibrosis, we utilized the bleomycin-model of murine pulmonary fibrosis. Cy5-labeled peptides were SC administered to mice followed by quantification of fluorescent signal in vital organs 24 h post injection. Using IVIS, we found that Cy5-PEG-FUD preferentially targets the fibrotic lung in the pro-fibrotic phases of the model compared to controls (Cy5-PEG-mFUD in bleomycin treated mice and Cy5-PEG-FUD in NS controls) and that this compound is likely cleared through renal elimination.

Conclusion: In this study, we have concluded that: 1. FUD/mFUD were successfully synthesized and PEGylated using 20kD PEG without compromising with its FN binding affinity. 2. The PEG-FUD shows the FN binding affinity in the nanomolar range, and the in-vivo exposure was increased when administered SC with reasonable stability. 3. The ex-vivo studies in human IPF tissue samples shows specific targeting of Cy5-PEG-FUD to regions of nascent fibronectin fibrillogenesis, in contrast to the mutant control. 4. Cy5-PEG-FUD shows significant in-vivo FN binding capability in the bleomycin treated fibrotic murine lungs.

References: Lee et al., Multimodal imaging demonstrates enhanced tumor exposure of PEGylated FUD peptide in breast cancer, Journal of Controlled Release 2022, 350, 284.
Zbyszynski et al., Characterization of the PEGylated functional upstream domain peptide (PEG-FUD): a potent fibronectin assembly inhibitor with potential as an anti-fibrotic therapeutic, Pharm. Res. 2018, 35, 126.
Johansson et al., PEGylated pUR4/FUD peptide inhibitor of fibronectin fibrillogenesis decreases fibrosis in murine unilateral ureteral obstruction model of kidney disease, PLoS One 2018, 13, e0205360.
Zbyszynski et al., Probing the subcutaneous absorption of a PEGylated FUD peptide nanomedicine via in vivo fluorescence imaging, Nano Converg. 2019, 6, 22.

Acknowledgements: Fundings: DoD W81XWH-22-1-0692 (NS, GK), NIH R01HL146402 (NS), R01CA257837 (GK), P30CA014520, Mosher Family Philanthropic Gift, University of Wisconsin-Madison Department of Medicine Pilot Award (NS). Ethical Statement: Human tissue: De-identified human IPF tissue samples were obtained from thoracic surgical resection specimens from patient’s undergoing surgical biopsy or lung explant tissue from patient’s undergoing lung transplantation. Tissues were obtained by the University of Wisconsin-Madison Carbone Cancer Center Translational Science BioCore (Institutional Review Board approval # 2011-0840) and was released to the research team. Histologic assessment was performed to verify the presence of the usual interstitial pneumonia (UIP) architectural pattern for fibrotic tissue. Mouse studies: All animal experiments were approved by the University of Wisconsin-Madison’s Institutional Animal Care and Use Committee (Protocol # M005823). Conflict of interest statement: Authors declare no conflict of interest. Senior Authors: NS and GSK contributed equally to this work.

Fig. 1: FPLC chromatogram: Ion-exchange chromatography for the purification of PEG-FUD.

Fig 2: Ex-vivo imaging studies: Binding efficacy of Cy5-labeled PEG-FUD/mFUD on human OCT frozen tissue samples [IPF (a,b) and normal/non-IPF (c,d)].

Fig 3: In-vivo and ex-vivo imaging of Cy5-labeled PEG-FUD/mFUD peptides on bleomycin treated (d7) animal model.