Development of radiolabeled monoclonal antibody constructs : capable of transporting high radiation dose into cancer cells

Abstract

The successful application of radioimmunotherapy for cancer has been limited by the availability of the small number of suitable isotopes, mostly emitting p-particle. Iodine- 131 is the most prevalent radionuclide for labeling monoclonal antibodies to high specific activity for therapeutic applications. Also yttrium-90 is a promising radiolabel because of its higher energy, longer ranged ß-particles and lack of γ-rays. The α-particle emitting radionuclides have a number of physical characteristics that make them attractive candidates for radioimmunotherapy: (a) high linear energy transfer; (b) short path lengths (50-80 μm); (c) limited ability of cells to repair damage to DNA; (d) cell kill via apoptotic mechanisms. The present study is focused on developing a radiolabeled monoclonal antibody construct that is capable of transporting a high radiation dose into cancer cells. Also the effects of iodination on the tyrosine residues and coupling a chelate on lysine residues to the immunoreactivity fraction of mAb have been studied. Direct iodine-131 radiolabeling methods yield decreased immunoreactivity of the antibody as a function of increased iodine incorporation. The amino acid sequences of a therapeutic IgG (HuM195), and in particular its complementary determining regions (CDR) were studied, in order to correlate the iodination of tyrosine residues in the antigen binding site with changes in immunoreactivity. The CDR contained an overabundance of tyrosines relative to an expected random distribution of amino acids. The immunoreactivity measured after varying levels of iodination fit to a theoretical curve generated based on the assumption that a single iodine incorporation anywhere on a tyrosine residue in a CDR destroys the immunoreactivity of the antibody. Using enzyme digestion, the distribution of iodine on different parts of the antibody was also studied. The iodinated residues were distributed non-uniformly throughout the IgG, with the heavy chain variable region tyrosines having a higher propensity for iodine incorporation than tyrosines in the other regions of the IgG. The data also suggest that high tyrosine fraction in the CDRs is a limiting factor for using direct radioiodination at the level required for the radioimmunotherapy. Conjugation of HuM195 to CHX-A-DTPA resulted in the attachment of up to 10 ligand molecules per antibody, and labeling efficiency with Bismuth-213 was typically over 90%. After injection into mice, there was no uptake or loss of bismuth to mouse tissues, that do not express antigen or to kidney, which has avidity for free, unbound bismuth. Toxicity of ²¹³Bi-CHX-A-DTPA was evaluated in normal mice with doses from 0.5 to 20 mCi/kg showing no toxicity, but at 70 mCi/kg significant toxicity was detected. Cell killing experiments with different specific activities of ²¹²Bi or ²¹³Bi labeled conjugate showed dose and specific activity dependent killing of HL60 cells. The results of this thesis indicate that bismuth-213 labeled HuM195 has high potency to specifically kill the target cells without remarkable toxicity to other tissues.