Aim of the PhD Project:
- Develop new bioconjugate and chelator chemistry to incorporate metallic ions into antibodies.
- Study the distribution of the new chelator-antibody conjugates tagged with non-radioactive metals in vivo (tumour-bearing mice) using Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry (LA-ICP-MS).
- Radiolabel the new chelator-antibody conjugates with therapeutic radiometallic ions and undertake radiotherapeutic efficacy studies in cancer models.
Project Description / Background:
The overarching objective of this project is to develop new chemical technology to attach heavy metal tags to antibodies, and apply this technology to quantify/image the biodistribution of radioactive and non-radioactive antibody-based therapies in vivo in mouse models using radionuclide imaging methods. This will enable high resolution, sensitive and quantitative mapping of such therapies, and give insight into their therapeutic efficacy.
Imaging of receptor-targeted therapies
Receptor-targeted antibody therapies have had huge clinical impact in the treatment of cancer, and new discoveries will result in further antibody treatment options. Receptor-targeted radionuclide therapies based on beta-or alpha-emitting radiometals have been used effectively in cancer treatments. These molecular radiopharmaceuticals incorporate a radioactive metal bound via a chelatorattached to a peptide, protein or antibody, which targets cell-surface receptors of diseased cells.
Imaging the biodistribution of antibodies/radionuclide therapies in cells and in in vivo subjects has aided their preclinical and clinical development, and enabled clinicians to predict treatment efficacy and monitor patient response. However, state-of-the-art quantitative radionuclide in vivo tracking methods (e.g. positron emission technology, PET,or single photon emission computed tomography, SPECT) have relatively low spatial resolution (≥1 mm). On the other hand, fluorescence imaging methods, which can pinpoint sub-cellular localisation of antibodies, are not quantitative.
Mass spectrometry technologies
Mass cytometry uses IgG antibodies tagged with transition and lanthanide metal ions (referred to here as “heavy” metals) to provide information at the single cell level on protein expression and cell processes (1). Typically, these are single isotopes of non-endogenous metals – metal ions that are not used by organisms for normal physiological processes. Cells are first exposed to a panel of antibodies, with each antibody tagged with a single stable isotope of a heavy metal. Individual cells or populations of cells are then analysed by mass spectrometry (typically with inductively coupled plasma mass spectrometry, ICP-MS) to measure the metal content of cells, with metal content providing a measure of antibody accumulation. As mass spectrometry can quantitatively measure many heavy metals in a single sample (≥20), a cocktail of antibodies can provide a vast array of information on single cell processes.
LA-ICP-MS is a relatively new methodology. Laser ablation of a two-dimensional sample, for example a tissue slice, followed by ICP-MS analysis of ablated material, enables highly resolved mapping of elemental distribution.
Chemistry of IgG antibodies labelled with heavy metal tags
Stable metal-chelator complexes and stable chelator-antibody bioconjugates are required for studying the biodistribution of heavy metal tags in vivo. Dissociation of a metal ion from a chelator-antibody conjugate, or loss of the chelator from the antibody will compromise quantifiability of antibody distribution. Existing mass cytometry tagging technology uses:
- polymers of DOTA chelators complexed to rare earth lanthanide ions, and
- maleimide bioconjugate chemistry to attach the DOTA chelator to cysteine side-chains at the antibody “hinge” region (1).
DOTA complexes of lanthanide ions are known to be highly stable in vivo, however DOTA complexes of some transition metal ions (for example [Cu(DOTA)]2-) are known to dissociate in biological media. Maleimide-based bioconjugates exhibit in vivo instability, undergoing retro-Michael reactions and ultimately releasing their cargo or tags under in vivo conditions (2,3). Recent research has produced disubstituted maleimides (including dibromomaleimides and dithiophenylmaleimides) that react with two reduced thiol groups of antibodies, thus enabling concomitant attachment of tagging agents/cargo and re-bridging of pairs of cysteines at antibody hinge regions. The resulting antibody conjugate, containing a dithiomaleamic motif, is highly stable in biological media (3).
This project will develop new chelators/complexes containing dibromomaleimides for attachment to the breast cancer targeting antibody, trastuzumab. The new conjugates, containing either non-radioactive heavy metal tags or radioactive therapeutic isotopes, will be studied in vivo in breast cancer mouse models.
To test the feasibility of this chemical approach, we recently prepared a series of novel chelator-dibromomaleimide compounds that coordinate to transition metal ions, and stably attached these chelators/complexes to the monoclonal IgG antibody, trastuzumab, which targets HER2 overexpressed in some breast cancers. We have radiolabelled two of these new trastuzumab conjugates (containing chelators 1 and 2, blue inset in figure) with radioactive 89Zr4+, and used PET/CT imaging to track antibody conjugate distribution in healthy mice (e.g. PET/CT imaging in Figure).
- Bjornson, Z. B., Nolan, G. P., and Fantl, W. J. (2013) Single-cell mass cytometry for analysis of immune system functional states. Current Opinion in Immunology 25, 484-94.
- Morais, M., and Ma, M. T. (2018) Site-specific chelator-antibody conjugation for PET and SPECT imaging with radiometals. Drug Discovery Today: Technologies 30, 91-104.
- Nunes, J. P. M., Morais, M., Vassileva, V., Robinson, E., Rajkumar, V. S., Smith, M. E. B., Pedley, R. B., Caddick, S., Baker, J. R., and Chudasama, V. (2015) Functional native disulfide bridging enables delivery of a potent, stable and targeted antibody-drug conjugate (ADC). Chemical Communications 51, 10624-27