Pretargeted PET imaging
Labeled antibodies and antibody fragments have been developed for PET imaging, but the target-to-background ratios have often been unsatisfactory. In pretargeted imaging a two-step approach is used to overcome the problems: in pretargeting step, an unlabelled immunoconjugate is administered and allowed to redistribute in the body until it is either bound to its specific tissue targets or cleared from the body. Immunoconjugate has also another binding site, for which a labeled small molecular compound (hapten, reporter, radiotracer) is then injected. In the second step ("click"), the radiotracer binds covalently to the immunoconjugate. Alternative to the covalent click reaction is to use a bispecific antibody, possibly with a hapten binding additional Fab fragment attached. This procedure provides better target-to-background ratios with shorter scan times, and in addition to immuno-PET, also has applications in radioimmunotherapy of cancer.
Pretargeting techniques are usually based on antibodies, antibody fragments, or affibodies targeted against tumour antigen, but any ligand/receptor systems could be used, including oligonucleotide aptamers, peptides, and nanoparticles. Also several techniques for the second step have been proposed. Good results have been obtained using affinity enhancement system (AES), where the labeled hapten has two (bivalent) or more binding sites for the immunoconjugate. Labelled hapten therefore binds very tightly to pretargeted (tumour) cells, while the non-bound hapten is excreted quickly from the body.
Reporters that can be labeled with wide range of isotopes have been developed. The same pretargeting system can thus be first researched and optimized using PET radionuclides with relatively short half-lives, and then labeled with isotopes that have favourable properties for radiotherapy, such as 90Y or 177Lu.
In vivo click reactions
Inverse-electron-demand Diels-Alder (IEDDA) reactions between 1,2,4,5-tetrazines and trans-cyclooctene (TCO) derivatives has been proven to be suitable for in vivo pretargeted imaging, and the small TCO compounds, even able to cross the blood-brain barrier, can be labelled with 18F (Billaud et al., 2017). It is possible to design the reaction both ways, attaching tetrazine to the antibody and labelling the TCO-derivative, or vice versa. 68Ga- and 18F-labelled tetrazines have been developed, and used to label TCO-modified mAbs in vivo (Devaraj et al., 2012; Evans et al., 2014; Keinänen et al., 2017a).
Specific binding can be studied by using TCO-modified mAb that is not specific to the target, and by using target-specific mAb that does not contain TCO (Devaraj et al., 2012).
In the pretargeting step, the free antibody or antibody fragment usually cannot be internalized into cells, and it therefore can only bind those targets (usually receptors, such as EGFR and HER2) that are on the cell surface. Receptor-endogenous ligand complex can be internalized, thus making fraction of receptors unavailable for in vivo visualization, while all receptors may be accountable in in vitro and ex vivo analysis, preventing direct comparison. When the administered antibody or antibody fragment binds its receptor target, that complex can also be internalized, making it unavailable for the reporter, unless it too can easily pass through the cell membrane. Most of the antibodies that have been successfully used for pretargeted imaging show high persistence on the cell surface even when bound to the antigen, or accumulate in the target tissue at exceptionally high concentrations (Keinänen et al., 2017a). Pretargeting approach is still feasible even if the antibody is internalized and the radiotracer is not (Sharkey et al., 2012; Houghton, 2016).
When enough time is given for the pretargeting step, the concentrations of the immunoconjugate in plasma is low, and concentration in tissue can be assumed to be constant during the relatively short duration of the second step, including the PET imaging. The radiolabelled reporter is usually given in large excess (high mass, low molar activity), compared to its target concentration, to maintain high reaction rate in vivo, and to block the remaining free immunoconjugate in the blood. In contrast, Keinänen et al (2017b) has shown that the higher the molar activity of the tetrazine radiotracer, the more efficient the in vivo reaction is, since high mass caused saturation in reaction sites. If the mass of reporter is high, we can assume that the reaction itself does not affect its concentration, which simplifies the second-order reaction kinetics into first-order equations, but on the other hand, possible saturation effect may reduce the radioactivity concentration in the target tissue and thus the image quality.
Quantitative analysis requires that input function is known, that is, the plasma concentration of the radiotracer as a function of time is measured during the second step. Simple Patlak and Logan plots can reveal whether radiotracer binding is irreversible or reversible during the PET scan (Keinänen et al., 2017b).
Due to the several confounding factors in the uptake process of the radiotracer, only semiquantitative analysis is usually done, for example SUV, tissue-to-reference tissue or tissue-to-blood ratio (Keinänen et al., 2017a).
Altai M, Membreno R, Cook B, Tolmachev V, Zeglis BM. Pretargeted imaging and therapy. J Nucl Med. 2017; 58(10): 1553-1559. doi: 10.2967/jnumed.117.189944.
Goldenberg DM, Chang C-H, Rossi EA, McBride WJ, Sharkey RM. Pretargeted molecular imaging and radioimmunotherapy. Theranostics 2012; 2(5): 523-540.
Goodwin DA, Meares CF, McCall MJ, McTigue M, Chaovapong W. Pre-targeted immunoscintigraphy of murine tumors with Indium-111-labeled bifunctional haptens. J Nucl Med. 1988; 29: 226-234.
Goodwin DA. Tumor pretargeting: almost the bottom line. J Nucl Med. 1995; 36(5): 876-879.
Goodwin DA, Meares CF. Pretargeted peptide imaging and therapy. Cancer Biother Radiopharm. 1999; 14(3): 145-152.
Houghton JL, Zeglis BM, Abdel-Atti D, Sawada R, Scholz WW, Lewis JS. Pretargeted immuno-PET of pancreatic cancer: overcoming circulating antigen and internalized antibody to reduce radiation doses. J Nucl Med. 2016; 57(3): 453-459. doi: 10.2967/jnumed.115.163824.
Kraeber-Bodéré F, Rousseau C, Bodet-Milin C, Frampas E, Faivre-Chauvet A, Rauscher A, Sharkey RM, Goldenberg DM, Chatal J-F, Barbet J. A pretargeting system for tumor PET imaging and radioimmunotherapy. Front Pharmacol. 2015; 6:54.
Liu G, Hnatowich DJ. A semiempirical model of tumor pretargeting. Bioconjug Chem. 2008; 19(11): 2095-2104.
Rossin R, Verkerk PR, van den Bosch SM, Vulders RC, Verel I, Lub J, Robillard MS. In vivo chemistry for pretargeted tumor imaging in live mice. Angew Chem Int Ed Engl. 2010; 49(19): 3375-3378.
Rossin R, Robillard MS. Pretargeted imaging using bioorthogonal chemistry in mice. Curr Opin Chem Biol. 2014; 21: 161-169.
Sharkey RM, Karacay H, McBride WJ, Rossi EA, Chang CH, Goldenberg DM. Bispecific antibody pretargeting of radionuclides for immuno single-photon emission computed tomography and immuno positron emission tomography molecular imaging: an update. Clin Cancer Res. 2007; 13(18 Pt 2): 5577s-5585s.
van de Watering FCJ, Rijpkema M, Robillard M, Oyen WJG, Boerman OC. Pretargeted imaging and radioimmunotherapy of cancer using antibodies and bioorthogonal chemistry. Front Med (Lausanne) 2014; 1:44.
van Duijnhoven SM, Rossin R, van den Bosch SM, Wheatcroft MP, Hudson PJ, Robillard MS. Diabody pretargeting with click chemistry in vivo. J Nucl Med. 2015; 56(9): 1422-1428.
Updated at: 2018-07-20
Created at: 2015-11-19
Written by: Vesa Oikonen