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A training network for the chemical site-selective modification of proteins: Preparation of the next-generation of therapeutic chemically-defined protein conjugates

Periodic Reporting for period 2 - ProteinConjugates (A training network for the chemical site-selective modification of proteins: Preparation of the next-generation of therapeutic chemically-defined protein conjugates)

Reporting period: 2017-11-01 to 2019-10-31

According to the World Health Organization, Cancer was responsible for an estimated 9.6 million deaths in 2018, being the second leading cause of death globally. Thus, it is necessary to develop efficient treatments against cancer.
Recently, the conjugation of monoclonal antibodies directed against tumor marker proteins with highly potent cytotoxic drugs has emerged as a powerful strategy to fight cancer. In addition, chemical protein modification has become a key instrument in Chemical Biology and drug development, hence being of major importance for the development of more efficient ADCs.
With this in mind, ProteinConjugates was an inter- and multi-disciplinary network for the education of early young researchers who aimed to learn how to rationally design and construct the next generation of chemically-defined protein drug conjugates. The goal of the ProteinConjugates network was to develop new site-selective bioconjugation methods that explore natural proteinogenic amino acids and their use to build protein conjugates for the treatment of serious conditions such as cancer. Furthermore, the combined expertise of the beneficiaries and industrial partners in synthesis of complex molecules, molecular dynamics, supramolecular self-assembly, site-selective chemical protein modification, protein/antibody engineering, cancer therapeutics and drug development, allowed to develop exciting projects that led to new methods and pre-clinically validated conjugates. Within the course of the 10 ESR projects it was possible to develop several new methods for site-selective modification of proteins (disulfide bonds rebridging with pyridazinediones; disulfide bridging with boronic acids, cysteine modification with carbonylacrylic acid handles), to develop new antibodies (anti-MUC1 and anti-IL7 receptor) and to develop new nanoparticles and functionalized proteins. With these, new protein conjugates were built, leading to a promising anti T-ALL ADC, to an ADC against HER2+ cancer cells, two potential protein conjugates against colon carcinoma, fluorophore protein conjugates as imaging tools and a potent and selective therapy against glioblastoma. The later, resulted in Intellectual Property which led to the formation of a new spin-out company. These newly developed methodologies have and will contribute to the emergence of new potential therapeutic strategies against diseases that are a major burden for society. Moreover, exposure to the highly dynamic and multidisciplinary ProteinConjugates environment will contribute to the education of the next generation of leaders in the emerging field of protein biotherapeutics, a key area of research and drug development for Europe's competitiveness in the field.
During the first half of the ProteinConjugates ITN, several methods for new site selective modifications and new protein functionalization approaches were developed, namely:
- Three new methods of disulfide re-bridging using pyradizinediones (UCL);
- Site-selective installation of salicylhydroxamate handles on disulfide bonds (MPIP);
- Development of a boronic acid-based protein modification technique (FFUL);
- A new method that uses cysteine residues resulting from reduction of the interchain disulfides of the full-length IgG antibody, to create stable antibody-drug conjugates, using carbonylacrylic acid handles (IMM)
- A new method that uses iminoboronates as a multifunctional platform for the construction of functional homogeneous multivalent targeting drug conjugates (FFUL):
- Development of a new anti-MUC1-antibody with high affinity and selectivity for modified MUC1 glycopeptides (UniRioja);
- Development of a new antibody against IL7 receptor (IMM);
- Microfluidic setup for the generation of monodisperse protein nanoparticles that are susceptible to surface modification (UCAM);
- A new approach for functionalization of lectins (Institute Curie)
- A new strategy for functionalization of silaffins with polyamides at lysine residues (UniVie).
During the second half of the ITN, all these methods and newly developed proteins and antibodies were successfully used for the construction of new protein conjugates and new ADCs:
- The B12-MMAE , and ADC against T cell acute lymphoblastic leukemia, significantly promoted cell death in ALL cells and PDX samples (IMM);
- 2FBBA-Laminin conjugate which successfully delivered a fluorescent payload to HT29 cancer cells (FFUL);
- The two ADCs built with Trastuzumab, (Trastuzumab-coumarin and Trastuzumab- duocarmycin derivative) that retain their specificity against Her2+ cancer cells (UniRioja);
- A bombezin- bortezomib conjugate with shown internalization and release in HT29 cells (FFUL)
- The Quer-R5 conjugates that were shown to be taken up by HT29 cells into the nucleus (UniVie);
- The formulation of Piperlongumine for sustained intracranial release that proved to be effective against GBM (IMM).
All the results obtained during the course of the action where disseminated both by the ESRs and their supervisors, through participation in several national and international conferences and through seminars given at different institutions. Many of the results were also published in international, peer-reviewed publications. Furthermore, the achievements of the ITN were also disseminated through the website of the network and several activities for the civil society.
The work of the students of the ITN ProteinConjugates produced significant results both in terms of new methods for protein site-selective chemistry as well as their use to develop new drug-delivery strategies. The methods developed are robust and may be used by any laboratory to produce chemically-defined conjugates. This has potential economical impact since the routine construction of therapeutic proteins and antibodies with cargo chemically conjugated at precise sites remains a challenge. The methods our students have developed are highly selective and produce constructs that are stable in circulation, which allows the release of their cargo only at the site of disease. Thus, there is an enormous potential to translate these technologies to the production of current and future antibody-drug conjugates which is a major area of development within the pharmaceutical industry. Furthermore, the in vitro and in vivo results of the ADCs generated under this action suggest their further development in the clinical setting. Considering the need for safer and more efficient cancer treatments, the development of precision medicines such as ADCs can impact the life of patients. Thus, we believe that the methods and results obtained during the ITN ProteinConjugates will have an impact for the society.