1. Towards clickable radio-immunoconjugates
as theranostic agents for TEM-1 targeting
Alice D’Onofrio1, L. Gano1, C. Oliveira1 , J. Fierle2, S. Dunn2, A. Rocha Paulo1
Instituto Tecnológico e Nuclear - IST Lisboa1, LAbCore Platform, Ludwig Cancer Research - Lausanne2
Introduction:
TEM-1 is a membrane receptor overexpressed in several solid tumors with a very low expression in normal tissues. TEM-1 is a tumor vascular markers essential for tumor progression, accessible from the blood circulation and less likely to undergo mutations and adaptations1.
Radio-immunocojugates combine the unique specificity of the antibody-antigen interaction with the capability of radioconjugates to provide both diagnostic and therapeutic tools. Theranostics are essential for personalized treatment, allowing the identification of the biomarkers expressed, a more accurate dosimetry and the monitoring of the therapy effectiveness.
Terbium is a particularly attractive radiometal in medical application because of the availability of four clinically relevant isotopes with identical chemical properties, which are excellent candidates for theranostic applications : 149Tb (α emitter), 152Tb (β+ emitter), 155Tb (γ emitter), and 161Tb (β- and Auger emitter)2.
Objectives and Results:
SYNTHESIS OF THE BIFUNCTIONAL CONJUGATES:
TEM-1 scFv-Fc for specific recognition of tumor cells.
Acridine Orange to enhance the effects of short-range Auger electrons.
Tetrazine moiety for in vivo click-reaction with the TCO on the TEM-1 scFv-Fc (better pharmacokinetic and lower exposure to radiation).
Bio-labile linker to help the DNA-targeting fragment reach the nucleus of tumor cells (enhancement of antitumor activity).
The conjugates will be labeled with 161Tb (which can be particularly useful for the eradication of small tumors and metastasis) and with other related medical radionuclides such as 111In and 177Lu.
PRE-CLINICAL EVALUATION OF A SMALL PANEL OF scFv-Fc:
A small panel of antibody fragments targeting TEM-1 receptor will be labelled first with 125I and then with 111In to evaluate their specific TEM-1 targeting ability and their stability to labelling conditions, in order to determine the best candidate for the design of our radio-immunoconjugate.
Selective uptake in TEM-1 positive and negative mu/hu cell lines.
Immunoreactive fraction determination through Lindmo assay.
Cellular internalization of labelled fragments.
Radiochemical stability in different media and temperature for the F1 and F2 antibody fragment radiolabelled with 125I (above 90% during 48h).
Schematic representation of a multifunctional clickable 161Tb radioimmunoconjugate for the DNA-targeting of tumor cells.
Cellular uptake assays showed higher uptake of F1-125I in TEM-1 positive cells, F2-125I showed non-specific lower uptake in TEM-1 positive and negative cells.
H-NMR spectra of the building block for the synthesis of the radio-immunoconjugates.
Future Perspectives:
Further in vitro and animal studies foreseen: bio-distribution and SPECT imaging in tumor-bearing mice, antitumor effects, radiotoxicity and mechanism of cell death, micro and nano-dosimetric studies.
The development of such conjugates and the investigation on innovative strategies to improve their pharmacological behaviour will provide new insights for the development of Tb-based radiopharmaceuticals on which few studies have been reported so far.
References:
[1] S. Dunn, G. Coukos et al., Oncotarget. 2014; 5: [2] C. Müller et al., J Nucl Med. 2012; 53:
This research project has been supported by a Marie Skłodowska-Curie Innovative Training Network Fellowship of the European Commission’s Horizon 2020 Program under contract number MEDICIS-PROMED.