Strategies to improve cancer radioimmunotargeting

Sammanfattning: Radioimmunotherapy (RIT) and radioimmunolocalisation (RIL) are developing and promising technologies to diagnose and treat tumours by use of radiolabelled antibodies targeting tumour specific antigens. The major reason why RIL and RIT not are efficient enough, is the comparatively low accumulation of radiolabelled antibodies in the tumours. Irrespective of the antigen - antibody system used, the maximal tumour uptake in humans is often limited to below 0.1 % of the total injected dose, with significant radionuclide remaining in the blood pool and extravascular fluid.In the present thesis, the following putative improvement techniques for radioimmunotargeting have been evaluated in an experimental model using HeLa cell-xenografted nude mice: 1) Repetitive, simultaneous targeting of different antigens, 2) Removal of non-targeting antibodies using secondary antiidiotypic antibodies, 3) Preinjection of unlabelled antibody to remove shedded antigen and 4) Use of fractionated antibody administration. By use of multiple injections of mixtures of two different 131I-labelled monoclonal antibodies targeting placental alkaline phosphatase (H7) and cytokeratin 8 (TS1), respectively, a significant tumour growth inhibition compared to controls, was obtained. In the treated group, a negligible increase in tumour volume was seen compared to the control group, in which a 20-fold increase was observed. Quantitative determinations of volume densities of viable tumour cells, necrotic cells and connective tissue demonstrated no significant differences in the relative proportions between the groups, indicating that the irradiation caused decelerated growth. Using hybridoma technology, monoclonal antiidiotypic antibodies were generated against both TS1 and H7. The in vitro and in vivo effects of these antibodies, aH7 and aTSl, were investigated. Both these antiidiotypes were found to generate stable complexes with the radiolabelled idiotypic antibody, as revealed by gel-electrophoresis and autoradiography. Using biosensor technology (BIAcore, Pharmacia) the interactions were followed in real time and the association rate-, dissociation rate-, and affinity constants between the reactants were determined. In vivo, the antiidiotypes promoted a rapid dose dependent clearance of the 125I-labelled idiotypes with a decrease in total body radioactivity and concomitant dramatic increase in non-protein bound 125I excreted in the urine. The syngeneic monoclonal antiidiotypic antibody αTSl, was furthermore evaluated as a secondary clearing antibody at radioimmunolocalisation. Injection of αTSl in a molar ratio of 0.5-0.75:1 to TS1, 24 hours after the 125I-labelled TS1 improved the tumour to normal tissue ratio 2-3 fold. This was due to a decreased level of total body radioactivity as well as a slight decrease in tumour-radioactivity. A model describing the kinetics of the involved components, i.e. the antigen, the idiotype and the antiidiotype was presented. It is concluded that high affinity monoclonal antiidiotypes can be used as tools to regulate the levels of idiotypic antibodies in vivo. This strategy, combined with preinjection of non­labelled idiotypic antibodies, caused accumulated doses of 3 Gy to the tumour and 0.9 Gy to non tumour tissues as calculated for 125I-labelled antibodies (80 MBq/mg) by MIRD formalism based on repetitive quantitative radioimmunoscintigraphies. By approaching the maximal tolerated whole body radiation dose for mice (i.e. 6 Gy), it can be estimated that doses up to 20 Gy are possible to obtain following one single injection of labelled antibody. It was furthermore demonstrated that a single bolus injection of antibody is to be preferred, compared to exactly the same dose divided into three or ten fractions. Thus, not only the dose of radioactivity, but also the amount of antibody should be considered for fractionated RIT.In summary, the thesis demonstrates that several techniques can be used to improve radioimmunolocalisation and to approach the proposed 70 Gy required to sterilise tumours at radioimmunotherapy.