Vladimir Tolmachev's projects on radionuclide tumour targeting

Therapy of disseminated cancer can be improved by increasing treatment specificity with the use of molecular recognition of proteins that are aberrantly expressed in malignant cells. Antibodies, tyrosine kinase inhibitors and small interfering RNAs are just a few examples of novel specific therapeutics. However, the expression of a particular molecular target can vary from patient to patient and between lesions within the same patient. Therefore, molecular testing is becoming part of the paradigm of targeted therapy to choose drugs on an individual patient basis.

Radionuclide molecular imaging of tumour-associated targets has the clear advantages of being global, minimally-invasive and easily repeatable to follow changes in a target expression. Therefore, radionuclide molecular imaging might be used for patient stratification identifying patients who would most likely benefit from particular targeting therapy due to sufficient target expression. Thus, radionuclide molecular imaging may be a powerful and convenient tool to make treatment of disseminated cancer more personalised.

Predictive biomarkers identify only high probabilities of response to a targeting therapy. Some patients with positive predictive biomarkers will inevitably not respond. Targeted delivery of cytotoxic nuclides (e.g. beta- or alpha-emitters) may provide selective destruction of malignant cells sparing healthy tissues. The use of radionuclides offers advantage of cross-fire effect (when nuclides delivered to one cancer cell irradiate its malignant neighbours) and absence of multidrug resistance phaenomenon.

New type of targeting probes, scaffold proteins

Javad Garousi, Mohamed Altai, Hadis Honarvar, Maryam Oroujeni, Dan Sandberg, Jörgen Carlsson, Anna Orlova, Joachim Feldwisch, Fredrik Freijd, Vladimir Tolmachev
The use of robust protein scaffolds enables selection of high-affinity binders that are much smaller than antibodies. Our team pioneered in the use of scaffold protein for molecular imaging in vivo by radiolabelling of Affibody molecules. Affibody molecules are small (7 kDa) phage-display selected scaffold proteins, developed at Royal Institute of Technology (KTH), Stockholm. They can be selected for specific binding to a large variety of protein targets including tumour-associated antigens. Currently, anti-HER2 Affibody molecules are evaluated in clinical studies demonstrating exquisite sensitivity and specificity. In 2015, we reported successful use of another type of scaffold protein ADAPTs (5.2 kDa), for molecular imaging.

Our group focuses on evaluating the influence of different factors (format, labelling chemistry, off-target interactions) on tumour targeting using scaffold proteins. During 2016, we studied the influence of hexahistidine tags, N-terminal sequence, chemical nature of a radioactive labels and their position of targeting of ADAPTs. This helped us to appreciably improve the contrast of imaging using ADAPTs.

During 2016, we developed chemistry for labelling of Affibody molecules with positron-emitting nuclides 18F, 89Zr, 44Sc, 55Co, 64Cu and single-photon emitter 99mTc.

Personalising tyrosine kinase targeting

Javad Garousi, Mohamed Altai, Hadis Honarvar, Maryam Oroujeni, Anna Orlova, Fredrik Frejd, Vladimir Tolmachev
Trans-membrane receptor tyrosine kinases (RTKs) are overexpressed in many malignancies. RTK signalling triggers cell proliferation, the suppression of apoptosis, increased motility and the recruitment of neovasculature. Overexpressed RTKs are the molecular targets for an increasing number of anti-cancer drugs.

We focus on the use of radionuclide molecular imaging for personalising of tyrosine kinase treatment. The main targets are HER2, EGFR, HER3, IGF-1R, VEGFR2 and PDGFRb. Influence of target expression level in tumours and normal tissues, cellular processing of tracers in tumours and excretory organs, affinity of a tracer on imaging sensitivity is evaluated and used in rational molecular design of scaffold proteins-based probes for RTK imaging. During 2016, we investigated approaches enabling to improve imaging of HER2, EGFR, VEGFR2 and HER3.

Imaging of different molecular targets in tumour xenografts in mice
Imaging of expression of different molecular targets in tumour xenografts (left hind leg).
Images were obtained using radiolabelled Affibody molecules (see below). Images
are from (Rosik et al, Bioconjug Chem, 2014;25:82-92; Tolmachev, Eur J Nucl Med
2010; 613-22; Tolmachev, J Nucl Med 2012; 90-97;Tolmachev,J Nucl Med, 2014;
294-300; Orlova, Eur J Nucl Med , 2014;1450-1450; Honarvar, Int J Onc, 2015;46(2):513-20.

Affibody-based pre-targeting for radionuclide therapy of cancer

Hadis Honarvar, Mohamed Altai, Justin Velletta, Anna Orlova, Vladimir Tolmachev
High re-absorption of radiolabelled scaffold proteins in kidneys makes radionuclide therapy challenging. To avoid this issue, we started development of pre-targeting for Affibody-based therapy. Radionuclide pre-targeting is a two-step procedure for selective delivery of radionuclides to tumours. In this case, a primary Affibody-based targeting agent fitted with a recognition tag is injected first. After localization of the primary agent in a tumour and its clearance from blood and other non-targeted compartments, a radiolabelled secondary agent, which is specific to a recognition tag, is injected. The secondary agent is selected to have low re-absorption in kidneys.

Earlier, we have shown feasibility of the use of bioorthogonal chemistry and peptide nucleic acids (PNA) interactions as mechanisms for secondary recognition. In 2016, we have optimized labelling of PNA with therapeutic radionuclide 177Lu and studies influence such factors as dosing and timing on delivery of 177Lu to tumours. The data indicated high probability of improvement of survival using our method.