HYpoxia and alternatively activated MAcrophaGEs as novel molecular imaging targets for early monitoring of therapy efficacy in Idiopathic Pulmonary Fibrosis – HYMAGE-IPF

1/ Specific inhibition of HIF1a, HSP90 or LRP1 will be studied in vitro in normoxia and hypoxia on myofibroblast differentiation and macrophage polarization on human lung epithelial/fibroblast cells and monocytes and in vivo in our animal models, 2/ Imaging of hypoxia, M2 macrophages and LRP1 will be performed in our animal models by PET or SPECT after injection i. v. of 18F-FMISO, 99mTc-Tilmanocept or LRP1 antagonist (RAP), radiolabeled with the SPECT isotope 111In and 3/ the impact of HIF1a, HSP90 or LRP1 inhibition and pirfenidone/nintedanib on hypoxia, M2 macrophage and LRP1 imaging will be evaluated in our animal models.

Our intermediate results demonstrated the anti-fibrosis effect of HSP90 inhibition by a blocking antibody in our pulmonary fibrosis model. The anti-HSP90 antibody reduces collagen accumulation in the lung in a dose-dependent manner.
In parallel, we have demonstrated and published the value of hypoxia imaging in PET via 18F-FMISO to detect and follow the progression of pulmonary fibrosis. This publication also demonstrates that hypoxia imaging can predict fibrosis progression and response to ninedanib, an anti-fibrosis therapy used in the clinic. These results also demonstrate the superiority of hypoxia imaging for the early detection of pulmonary fibrosis compared to CT and 18F-FDG PET imaging. Following this publication, we considered that the development of an imaging probe targeting CAIX would not bring any superiority to 18F-FMISO for hypoxia imaging in pulmonary fibrosis. In parallel, our results demonstrate the value of imaging type 2 macrophages via 99mTC-Tilmanocept targeting the CD206 receptor to detect pulmonary fibrosis progression and response to ninedanib. These results were confirmed by flow cytometry and scRNAseq study and are ready for submission to an international journal in 2022.
We have also developed 3 other imaging probes targeting HSP90 (via a modified antibody for SPECT imaging), Gp96 (via a modified antibody for SPECT imaging) and FAP via a modified antibody for SPECT imaging, a marker of myofibroblasts responsible for collagen synthesis. The Gp96 probe has just been produced and validated in vitro. The HSP90 probe has been validated in vitro and has been the subject of a first in vivo experiment in fibrous mice. The increase in lung accumulation of the probe targeting HSP90 was inconclusive in fibrous mice compared to control mice. Finally, the FAP probe was validated in vitro and successfully tested in vivo. Indeed, the accumulation of the FAP probe in the lung was more important in fibrous mice compared to control mice showing the increase of myofibroblasts.

In the next part of the project we wish to evaluate the inhibition of LRP1 via a blocking antibody that will serve as both a therapeutic agent and as a basis to form an imaging agent targeting LRP1.
We also wish to evaluate in vivo the efficacy of the probe targeting Gp96 for the detection of fibrosis in our animal models. Similarly, we wish to continue our investigations with the probe targeting HSP90 to test if we can detect the increase of HSP90 in vivo at different stages of fibrosis (earlier). Finally, we wish to study in detail the interactions between Gp96 and PAF.

Publications:
1. Bellaye, P.S., et al, Measurement of pulmonary hypoxia in idiopathic pulmonary fibrosis: a matter of control. Eur Respir J, 2022.
2. Tanguy, J., et al, [(18)F]FMISO PET/CT imaging of hypoxia as a non-invasive biomarker of disease progression and therapy efficacy in a preclinical model of pulmonary fibrosis: comparison with the [(18)F]FDG PET/CT approach. Eur J Nucl Med Mol Imaging, 2021. 48(10) : p. 3058-3074.

Extension lectures:
1. Plateforme d'Expertise Maladies Rares BFC, RareMène ta science, 1er RDV de rencontre associations/chercheurs, Développement d'agents d'imagerie médicale au service du diagnostic et du suivi de la fibrose pulmonaire. Tuesday, November 16, 2021.

Description of the disease and state of knowledge: Idiopathic pulmonary fibrosis (IPF) is a progressive disease of the lung of unknown aetiology with a median survival time of less than five years. Treatment options are limited with only two approved drugs, nintedanib and pirfenidone, which demonstrate efficacy to slow down fibrosis progression but are unable to reverse or stop the disease. Currently, tools to monitor the efficacy of these therapies are inexistent. IPF is characterized by a TGFß1-dependant proliferation of myofibroblasts responsible for the aberrant accumulation of extracellular-matrix (ECM) in the lungs. Chronic hypoxia of the lung is a significant clinical feature of patients with IPF but its exact role on the progression of the disease remains elusive. At the cellular level, hypoxia signalling is mainly mediated by the upregulation of HIF-1a which is involved in myofibroblast differentiation. Hypoxic area are found in the IPF lung and hypoxia exposure exacerbates lung fibrosis in animal models. Hypoxia promotes the alternative activation of macrophages into a so called M2 phenotype characterized by the expression of the CD206 receptor and the production of profibrotic mediators such as TGF-ß1. Secreted heat shock protein (HSP)-90 and low density lipoprotein-related protein receptor (LRP1) are involved in M2 polarisation in hypoxic condition. Our preliminary data demonstrate that 1/ imaging of hypoxia with the PET tracer 18F-FMISO correlates with lung fibrosis in preclinical models, 2/ imaging of M2 macrophages with the SPECT tracer 99mTc-Tilmanocept (targeting CD206) demonstrate infiltration of fibrotic lungs by these pro-fibrotic macrophages, 3/ M2 macrophages are detectable by 99mTc-Tilmnaocept SPECT imaging in area that appear “normal” of CT-scans of fibrotic lungs, 4/ LRP1 is upregulated in lung fibroblasts from IPF patients, 5/ secreted HSP90 interacts with LRP1 to activate pro-fibrotic signalling promoting IPF progression and 6/ inhibition of LRP1 abrogated pro-fibrotic signals mediated by secreted HSP90.

Objectives: 1/ to evaluate the therapeutic efficacy of the blocking of hypoxia signalling via inhibition of HIF1a, secreted HSP90 and LRP1 in experimental fibrosis. 2/ to develop and evaluate imaging probes targeting hypoxia and hypoxia signalling (M2 macrophages and LRP1) to detect/quantify lung fibrosis in preclinical models and 3/ to evaluate the efficacy of the developed probes to monitor the efficacy of existing anti-fibrotic therapies (nintedanib and pirfenidone) as well as novel anti-fibrotic therapies (anti-HIF1a, anti-LRP1 and anti-sHSP90) in preclinical models.

Description of the project methodology: 1/ Specific inhibition of HIF1a, secreted HSP90 or LRP1 will be investigated in vitro in normoxia and hypoxia on myofibroblasts differentiation and macrophage polarization on murine lung epithelial cells/fibroblasts and primary human monocytes and in vivo in our animal models, 2/ Hypoxia, M2 macrophage and LRP1 imaging will performed by PET or SPECT after i.v. injections of the 18F-FMISO, 99mTc-Tilmanocept or a LRP1 antagonist, RAP, radiolabelled with the SPECT isotope 111In in our animal models and 3/ the impact of HIF1a, secreted HSP90 or LRP1 inhibition and pirfenidone/nintedanib on Hypoxia, M2 macrophage and LRP1 imaging will be evaluated in our animal models.

Expected results and impacts: This project will allow supporting whether or not, hypoxia and its signalling through LRP1 and M2 macrophages polarisation could be surrogate imaging biomarkers for IPF. We believe that the development of innovative tools for the detection of specific biological pathways involved in pulmonary fibrosis will be a first step in the development of personalized therapies for IPF patients, which is currently unavailable. Our ambitious project will therefore promote the development of targeted therapies based on hypoxia inhibition which are inexistent for in IPF.