Community Research and Development Information Service - CORDIS

Periodic Report Summary 3 - MATHIAS (New Molecular-Functional Imaging Technologies and Therapeutic Strategies for Theranostic of Invasive Aspergillosis)

Project Context and Objectives:
The development of novel technologies to diagnose and treat lung infections caused by the opportunistic fungal pathogen Aspergillus fumigatus is the aim of this European research consortium. Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease of immunocompromised patients (typically neutropenic haematological malignancy and bone marrow transplant patients) caused by the ubiquitous environmental mould A. fumigatus. Diagnosis of this disease is extremely difficult since radiological signs in computed tomography scans are not specific for the disease, and so culture of the fungus from lung biopsies is needed for accurate diagnosis. Biopsy is an invasive procedure that can often not be performed in already sick patients, and so the MATHIAS consortium is using an Aspergillus-specific mouse monoclonal antibody (mJF5) developed by ISCA Diagnostics, in an antibody-guided imaging procedure known at immuno-PET/MR, which allows non-invasive detection of Aspergillus lung infections in vivo. This 5-year project has three scientific work packages.
The objective of WP1, led by ISCA is to generate antibody fragments (scFv, F(ab’) and F(ab’)2) and a humanised version of JF5 (hJF5). Once generated, the fragments and full-length mJF5 and hJF5 can be conjugated to a chelator and then radioactively labelled with 64Cu to allow pre-clinical imaging of IPA in a mouse model of disease.
WP2, led by EKUT, focuses on the preclinical evaluation of the candidate immunoconjugates in vitro, ex vivo and in vivo for their performance recognizing specifically and with high affinity A. fumigatus. Using fluorescence, fungal invasion and the immunoconjugate binding profile can be studied ex vivo and with invasive imaging techniques, providing highly resolved images of Aspergillosis progression. Using radiolabelling, enabling immune-PET/MR imaging, the potential of the radioimmunoconjugate for diagnosis of Aspergillosis is studied. The emphasis on specificity to this particular disease is of paramount importance in this study in order to efficiently prepare for clinical translation. Lastly, a hybrid diagnosis, therapy monitoring and therapeutic approach (“theranostic”) is conducted by capitalizing on the high affinity of JF5 for A. fumigatus to deliver therapeutic isotopes directly to the site of infection.
The overall goal of WP3 is the translation of the most suited radioimmunoconjugate from bench-to-bedside. This requires production of the radioimmunoconjugate, including chelator, antibody and radionucleides in accordance with Good Manufacturing Practice (GMP) procedures to ensure quality, reproducibility and suitability for human use. In parallel, the radioimmunoconjugate must be fully characterized not only in terms of stability and activity but also toxicity. Lastly, preparation of the clinical study is performed with appropriate registration and approval by constituted institutional ethics committee (IEC) and at the PEI as regulatory authority.
Project Results:
In WP1, we have successfully generated fragments of the Aspergillus-specific mouse monoclonal antibody JF5 (mJF5) by expression of recombinant scFv and F(ab’) in E. coli and have successfully developed a stable cGMP-grade Chinese Hamster Ovary (CHO) cell line expressing humanised JF5 antibody following grafting of the mouse JF5 complementarity determining regions (CDRs) into a human IgG1 framework. In addition to recombinant scFv and F(ab’) fragments, we have generated F(ab’) and F(ab’)2 fragments of mJF5 and hJF5 using papain and pepsin digestion of the full-length antibodies. The antibody fragments and full-length humanised antibody retain their specificity to Aspergillus, binding to active (invasive) growth phases of the pathogen only. Using the full-length mJF5 and hJF5 antibodies, we have explored a number of chelators and chemistries for conjugating 64Cu to the antibodies and WP2 has shown that the chelator NODAGA allows specific detection of invasive lung disease in vivo, with minimal uptake of the tracer by non-target organs such as the liver, and no accumulation in lungs infected by other diseases. We have shown that the hJF5-based tracer ([64Cu]NODAGA-hJF5) has superior PET/MR imaging capabilities compared to its mouse counterpart. In addition, we have characterised the epitope bound by JF5 as ß1,5-galactofuranose (Galf), an epitope not found in mammalian carbohydrates. In addition, improvements have been made in the early detection of the disease in vivo and in monitoring ongoing chemotherapy of Aspergillosis. This, coupled with the superior imaging capabilities of the [64Cu]NODAGA-hJF5 tracer, means it is an excellent candidate for clinical studies of radiological IPA detection in humans. In parallel, we have gained new insights in the progression and invasion of Aspergillosis in lungs with and without intact immune systems using fluorescence imaging.
Based on the work of WP3, a subcontractor generated a cGMP version of the humanised JF5 monoclonal antibody (hJF5). Functional assays with this antibody could proove long-term stability and immunoreactivity. Since the beginning of the project the purity of (R)-NODA-GA(tBu)3 has improved. Now, the production process allows a chemical purity of more than 99.5%. Additionally, production and purification methods for 64Cu have been further optimised, including the necessary steps to ensure GMP quality of the radio-isotope. Currently, toxicity tests of NODAGA-hJF5 (produced according to GMP guidelines) are performed by a subcontractor. So far, no adverse effects with the humanised JF5 antibody have been found in toxicity tests. At present a study protocol for a small scale clinical study phase I is developed, although the actual study will not be performed within the MATHIAS project.
Potential Impact:
Invasive pulmonary aspergillosis accounts for more than 200,000 human infections each year, with an associated mortality rate of 30-90% and attendant economic burden to healthcare systems. There is an urgent need to develop accurate, non-invasive methods for identifying IPA in the ever-expanding population of immune deficient patients at risk of acquiring the disease, to allow timely treatment with adequate antifungal drugs, ultimately improving survival rates. The MATHIAS consortium is using a non-invasive state-of-the-art procedure (immuno-PET/MR) to detect IPA by employing a humanised monoclonal antibody specific to the aspergillosis pathogen. Using this novel diagnostic approach, we are able to specifically identify active infection in the lung and to differentiate Aspergillus from other lung pathogens in animal models. The work breaks new ground in infectious disease diagnosis and is a paradigm shift in the way we diagnose this devastating disease in immunocompromised individuals such as haematological malignancy and bone marrow transplant patients. While the full potential of the approach will only be realized in a full clinical trial (outside of the scope of the funding plan), extensive preparations are already underway. The required human-use-ready immunotracer production is completed, and the current preparation of the documentation for the approval of a human clinical trial is ongoing. We expect to be able to start with fully fledged clinical trials shortly after expiration of the funding period, which would represent a major breakthrough in Aspergillosis care.
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