Periodic Reporting for period 2 - PhotoPHARMA (Light-induced synthesis of protein-drug conjugates for imaging and therapy)
Reporting period: 2023-08-01 to 2025-01-31
Protein-based drugs and related biological products (biologics) represent one of the fastest growing sectors in the pharmaceutical industry. The global biologics market has been projected to reach over 550 million EUR by 2026. Biologics include recombinant proteins, vaccines, monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs). In Nuclear Medicine, the diverse chemical and biophysical properties of mAbs and related immunoglobulin fragments are an attractive platform for building target-specific radiopharmaceuticals for use in diagnostic imaging with positron emission tomography (PET) or radioimmunotherapy (RIT).
Synthesis of radiolabelled mAbs (and other ADCs) requires chemical modification of the glycoprotein. Standard coupling chemistries rely almost exclusively on heat-induced reactions such as the post-translational modification of amino-acid side chains or the site-specific labelling of glycans by chemical or enzymatic methods. Although highly successful, these classic approaches suffer from several important limitations. Notably, existing methods are time consuming, difficult to automate, and usually involve multiple step syntheses that are incompatible with protein formulation buffers. Standard routes also require the isolation, characterisation, and storage of an intermediate new molecular entity (the conjugated mAb) which poses practical and economic problems during clinic translation. In addition, the conjugation strategy can have a profound and often detrimental impact on the biophysical properties of the protein-based construct. Alternative methods that offer simple, reproducible, and financially viable ways for making new protein-conjugates are essential.
The work outlined in this ERC Consolidator project (PhotoPHARMA) is designed to explore alternative radiochemical methods for creating labelled monoclonal antibodies for applications in diagnostic imaging and RIT. We aim to harness the potential of using light-induced photochemical reactions to create new types of bioconjugate bonds between our radiolabelled compounds and a series of cancer-specific mAbs. Fundamental aspects of photoradiosynthesis technology will be developed alongside new automated instrumentations to help facilitate future clinical translation. This proposal consists of 4 primary Objectives that will be completed in 5 years.
Objective 1. Explore the biochemical scope of light-induced reactions for bimolecular protein-ligation
Objective 2. Synthesis, characterisation and biological studies of photo(radio)labelled antibodies for use in diagnostic imaging and therapy
Objective 3. Develop automated technologies for photoradiochemical synthesis of 89Zr-antibodies
Objective 4. Establish the GLP-synthesis and Chemistry, Manufacturing and Controls (CMC) for future translation of photoradiolabelled 89Zr-antibodies to the clinic
The long-term goals are to facilitate the synthesis and clinical translation of protein-conjugates by introducing a radical new technology based on the fast, reliable and automated approach of using light-induced chemistry. In the short-term, PhotoPHARMA will advance on our recent discoveries and inventions (European and World Patent applications filed, October 2018/9) and will deliver key steps toward this goal by: i) exploring the reactivity and mechanisms of different photoactive groups for bimolecular protein-conjugation, ii) expanding our photochemical technology for the synthesis protein-conjugates labelled with metal-based radionuclides, cytotoxic drugs, and fluorophores, iii) developing an automated photo(radio)chemistry platform, and iv) overcoming crucial steps on the path toward the translation of photoradiolabelled 89Zr-mAbs for PET imaging in the clinic. Ultimately, we believe that our photochemical technologies will stimulate increased access to protein-conjugates – advancing fundamental science and clinical medicine.
Synthesis of radiolabelled mAbs (and other ADCs) requires chemical modification of the glycoprotein. Standard coupling chemistries rely almost exclusively on heat-induced reactions such as the post-translational modification of amino-acid side chains or the site-specific labelling of glycans by chemical or enzymatic methods. Although highly successful, these classic approaches suffer from several important limitations. Notably, existing methods are time consuming, difficult to automate, and usually involve multiple step syntheses that are incompatible with protein formulation buffers. Standard routes also require the isolation, characterisation, and storage of an intermediate new molecular entity (the conjugated mAb) which poses practical and economic problems during clinic translation. In addition, the conjugation strategy can have a profound and often detrimental impact on the biophysical properties of the protein-based construct. Alternative methods that offer simple, reproducible, and financially viable ways for making new protein-conjugates are essential.
The work outlined in this ERC Consolidator project (PhotoPHARMA) is designed to explore alternative radiochemical methods for creating labelled monoclonal antibodies for applications in diagnostic imaging and RIT. We aim to harness the potential of using light-induced photochemical reactions to create new types of bioconjugate bonds between our radiolabelled compounds and a series of cancer-specific mAbs. Fundamental aspects of photoradiosynthesis technology will be developed alongside new automated instrumentations to help facilitate future clinical translation. This proposal consists of 4 primary Objectives that will be completed in 5 years.
Objective 1. Explore the biochemical scope of light-induced reactions for bimolecular protein-ligation
Objective 2. Synthesis, characterisation and biological studies of photo(radio)labelled antibodies for use in diagnostic imaging and therapy
Objective 3. Develop automated technologies for photoradiochemical synthesis of 89Zr-antibodies
Objective 4. Establish the GLP-synthesis and Chemistry, Manufacturing and Controls (CMC) for future translation of photoradiolabelled 89Zr-antibodies to the clinic
The long-term goals are to facilitate the synthesis and clinical translation of protein-conjugates by introducing a radical new technology based on the fast, reliable and automated approach of using light-induced chemistry. In the short-term, PhotoPHARMA will advance on our recent discoveries and inventions (European and World Patent applications filed, October 2018/9) and will deliver key steps toward this goal by: i) exploring the reactivity and mechanisms of different photoactive groups for bimolecular protein-conjugation, ii) expanding our photochemical technology for the synthesis protein-conjugates labelled with metal-based radionuclides, cytotoxic drugs, and fluorophores, iii) developing an automated photo(radio)chemistry platform, and iv) overcoming crucial steps on the path toward the translation of photoradiolabelled 89Zr-mAbs for PET imaging in the clinic. Ultimately, we believe that our photochemical technologies will stimulate increased access to protein-conjugates – advancing fundamental science and clinical medicine.
During the first half of this PhotoPHARMA project we have made strong progress on the experimental goals. Specifically, after establishing our new team, we focused efforts on the synthesis and characterisation of different photoactivatable radiotracers. This work explored three main avenues: 1) development of new photoactive chelates for complexation of a range of different radionuclides; 2) exploration of the chemistry and mechanisms of photochemically activated conjugation handles to proteins; and 3) development of automated radiosynthesis tools to facilitate future clinical translation.
We have now reported experimental data on over a dozen photoactivatable compounds which facilitate complexation chemistry with a wide range of radionuclides ions including: 68Ga, 64Cu, 89Zr and 18F for positron emission tomography (PET) imaging, 111In for Auger therapy, 99mTc for single-photon emission computed tomography (SPECT) / -ray imaging, and the beta-emitting radiotherapeutic nuclides 177Lu, 188Re and 161Tb. Notably, the chemistry of these radionuclides is vastly different and required us to develop several new synthetic methods. For example, many of the photoactivatable groups that we have tested (see below) are unstable under the strongly reducing conditions which are an essential part of most 99mTc and 188Re-based radiolabelled methods. Here, we designed new chelates for stable complexation of Tc / Re ions in the 5+ oxidation state combined with photoreactive handles that are stable toward SnCl2 reduction (Jonas Genz; 2 manuscripts in preparation). Similarly, metal complexes of lanthanoid ions like 177Lu3+ and 161Tb3+ require different coordination environments. For this we turned to the chemistry of bispidine chelates which increase the overall complex stability and radiolabelling efficiency (177Lu: Cieslik et al. Chem. Eur. J., 2024; 161Tb Cieslik et al. manuscript in preparation; Klingler et al. manuscript in preparation; Nisli et al. manuscript in preparation). Other examples of new photoactive chelate development include (Guillou et al. Inorg. Chem. Frontiers, 2022; and Earley et al. Dalton Trans., 2022, which was selected by the editor as a ‘Hot Paper’).
We have also performed extensive fundamental photochemical and radiochemical studies exploring the light-induced reactivity of over 14 different functional groups. Experimental data from these studies have appeared in several publications (e.g. Fay et al. Chem. Eur. J., 2021; Guillou et al. Bioconjugate Chem., 2021; and detailed mechanistic work was reported in the article Earley et al. Charting the chemical and mechanistic scope of light-triggered protein ligation; JACS Au, 2022).
To facilitate a broader scope of utility, we have also expanded the chemistry beyond radiolabelled compounds of combining our photolabelling technology with fluorophores (see Guillou et al. J. Med. Chem., 2022).
In parallel to the fundamental chemistry, we have produced two new automated radiolabelling devices which use both batch-type photochemical reactors (Klingler et al. Automated light-induced synthesis of 89Zr-radiolabeled antibodies for immuno-positron emission tomography; Sci. Reports, 2022) and microfluidic technology (Earley et al. Molecules, 2021) for hands-free radiolabelling of proteins and antibodies. So far, it appears that batch-based reactors are more practical for implementing our new radiochemistry, but we will continue to explore flow-based options in the second half of PhotoPHARMA.
Other notable achievements which utilise the PhotoPHARMA chemistry, but which were not directly related to the initial objectives of the project include our recent development of supramolecular radiotracers (please see d’Orchymont et al., Chem. Sci., 2022, Commun. Chem., 2023; J. Am. Chem. Soc., 2023).
We have now reported experimental data on over a dozen photoactivatable compounds which facilitate complexation chemistry with a wide range of radionuclides ions including: 68Ga, 64Cu, 89Zr and 18F for positron emission tomography (PET) imaging, 111In for Auger therapy, 99mTc for single-photon emission computed tomography (SPECT) / -ray imaging, and the beta-emitting radiotherapeutic nuclides 177Lu, 188Re and 161Tb. Notably, the chemistry of these radionuclides is vastly different and required us to develop several new synthetic methods. For example, many of the photoactivatable groups that we have tested (see below) are unstable under the strongly reducing conditions which are an essential part of most 99mTc and 188Re-based radiolabelled methods. Here, we designed new chelates for stable complexation of Tc / Re ions in the 5+ oxidation state combined with photoreactive handles that are stable toward SnCl2 reduction (Jonas Genz; 2 manuscripts in preparation). Similarly, metal complexes of lanthanoid ions like 177Lu3+ and 161Tb3+ require different coordination environments. For this we turned to the chemistry of bispidine chelates which increase the overall complex stability and radiolabelling efficiency (177Lu: Cieslik et al. Chem. Eur. J., 2024; 161Tb Cieslik et al. manuscript in preparation; Klingler et al. manuscript in preparation; Nisli et al. manuscript in preparation). Other examples of new photoactive chelate development include (Guillou et al. Inorg. Chem. Frontiers, 2022; and Earley et al. Dalton Trans., 2022, which was selected by the editor as a ‘Hot Paper’).
We have also performed extensive fundamental photochemical and radiochemical studies exploring the light-induced reactivity of over 14 different functional groups. Experimental data from these studies have appeared in several publications (e.g. Fay et al. Chem. Eur. J., 2021; Guillou et al. Bioconjugate Chem., 2021; and detailed mechanistic work was reported in the article Earley et al. Charting the chemical and mechanistic scope of light-triggered protein ligation; JACS Au, 2022).
To facilitate a broader scope of utility, we have also expanded the chemistry beyond radiolabelled compounds of combining our photolabelling technology with fluorophores (see Guillou et al. J. Med. Chem., 2022).
In parallel to the fundamental chemistry, we have produced two new automated radiolabelling devices which use both batch-type photochemical reactors (Klingler et al. Automated light-induced synthesis of 89Zr-radiolabeled antibodies for immuno-positron emission tomography; Sci. Reports, 2022) and microfluidic technology (Earley et al. Molecules, 2021) for hands-free radiolabelling of proteins and antibodies. So far, it appears that batch-based reactors are more practical for implementing our new radiochemistry, but we will continue to explore flow-based options in the second half of PhotoPHARMA.
Other notable achievements which utilise the PhotoPHARMA chemistry, but which were not directly related to the initial objectives of the project include our recent development of supramolecular radiotracers (please see d’Orchymont et al., Chem. Sci., 2022, Commun. Chem., 2023; J. Am. Chem. Soc., 2023).
Photochemical reactions remain relatively unexplored in the fields of radiochemistry, bioconjugation and protein chemistry. Although we have made tremendous progress in exploring some of the reactive handles that allow for efficient labelling of mAbs, so far we have only looked at a small fraction of the chemistry that might be possible. In exploring this new type of chemical reactivity, we have been forced to develop methods that go beyond current state-of-the-art in terms of protein bioconjugation. Specifically, our reactions must be performed in water which presents a major departure from many classic organic-phase coupling chemistries. We focus exclusively on aqueous phase reactions and use this as a screening tool to select the most appropriate reagents and chelates. We identified 14 different functional groups that can be used to create new types of bioconjugate bonds to protein. With the exception of our work on the chemistry of aryl azide and tetrazole protein conjugates – which have been validated in biological models including animal imaging studies – biophysical properties of the other chemical groups remain to be explored. Our aryl azide technology facilitates lysine specific conjugation chemistry whereas recent results from our work on tetrazoles points toward selective reactivity with cysteine. These two groups allow us to perform chemoselective chemistry from which we are currently aiming to develop a unique site-specific photoradiolabelling approach (to date the reported photochemical reactions on protein are not necessarily site-specific so further work in this direction would be a major new advance).
The photochemistry allows us to label clinical grade antibodies in the presence of full formulation buffers (e.g. trastuzumab, onartuzumab, cetuximab, dinutuximab, etc). To the best of our knowledge, no other ‘standard’ chemical conjugation route is compatible with formulation buffers which makes our photoradiosynthesis approach unique. The importance of this observation is that we can also perform the radiolabelling and conjugation chemistry directly using fully automated radiosynthesis tools (custom build boxes like out ALISI system). This advance brings protein labelling chemistry closer in operation to the more established methods used for automated synthesis of radiolabelled peptides.
We expect that by the end of the PhotoPHARMA project, we will have explored a broader range of photoactive groups, and at the same time, to have developed robust methods / automated tools, that can be transferred to clinical radiopharmaceutical environments to help drive translation of radiolabelled antibodies in the clinic.
As a spin out project derived from our studies on photoconjugated proteins, we noted that the new covalent bonds that are formed with mAbs display vastly different chemistry to classic conjugation handles like amide, thiourea, and maleimido groups. We are currently exploring the reasons behind these biophysical differences, and we are using this information to develop new ways of designing radiotracers. Our ideas on this topic have recently been published (Berton et al. New tactics in the design of theranostic radiotracers; NPJ Imaging, 2024). This is important because photochemistry is one of the new tools that we are using to develop radiotracers that have improved dosimetry profiles which will facilitate the use of antibodies in radioimmunotherapy.
The photochemistry allows us to label clinical grade antibodies in the presence of full formulation buffers (e.g. trastuzumab, onartuzumab, cetuximab, dinutuximab, etc). To the best of our knowledge, no other ‘standard’ chemical conjugation route is compatible with formulation buffers which makes our photoradiosynthesis approach unique. The importance of this observation is that we can also perform the radiolabelling and conjugation chemistry directly using fully automated radiosynthesis tools (custom build boxes like out ALISI system). This advance brings protein labelling chemistry closer in operation to the more established methods used for automated synthesis of radiolabelled peptides.
We expect that by the end of the PhotoPHARMA project, we will have explored a broader range of photoactive groups, and at the same time, to have developed robust methods / automated tools, that can be transferred to clinical radiopharmaceutical environments to help drive translation of radiolabelled antibodies in the clinic.
As a spin out project derived from our studies on photoconjugated proteins, we noted that the new covalent bonds that are formed with mAbs display vastly different chemistry to classic conjugation handles like amide, thiourea, and maleimido groups. We are currently exploring the reasons behind these biophysical differences, and we are using this information to develop new ways of designing radiotracers. Our ideas on this topic have recently been published (Berton et al. New tactics in the design of theranostic radiotracers; NPJ Imaging, 2024). This is important because photochemistry is one of the new tools that we are using to develop radiotracers that have improved dosimetry profiles which will facilitate the use of antibodies in radioimmunotherapy.