Labeling of monoclonal antibodies with diethylenetriaminepentaacetic acid-appended radioiodinated peptides containing D-amino acids

The optimal use of radioiodinated internalizing monoclonal antibodies (mAbs ) for radioimmunotherapy necessitates the development of practical methods for increasing the level of retention of I-131 in the tumor. Lysosomally tr apped ("residualizing") iodine radiolabels that have been previously design ed are based mostly on carbohydrate-tyramine adducts, but these methods hav e drawbacks of low overall yields and/or high levels of mAb aggregation. We have developed a method using thiol-reactive diethylenetriaminepentaacetic acid (DTPA)-peptide adducts wherein the peptides are assembled with one or more D-amino acids, including D-tyrosine. Two such substrates, R-Gly-D-Tyr -D-Lys[1-(p-thiocarbonylaminobenzyl)DTPA], referred to as IMP-R1, and [R-D- Ala-D-Tyr-D-Tyr-D-Lys](2)(CA-DTPA), referred to as IMP-R2, wherein R is 4-( N-maleimidomethyl)cydohexane-1-carbonyl, were synthesized by preparing func tional group-protected peptides on a solid phase, selectively derivatizing the lysine side chain with 1-(p-isothiocyanatobenzyl)DTPA or DTPA dianhydri de (CA-DTPA), deprotecting other functional groups, and finally derivatizin g the peptide's N-terminus so it contained a maleimide group. Radioiodinati ons of the peptides followed by conjugations to disulfide-reduced mAbs, car ried out as a one-vial procedure, resulted in 32-89% overall yields, at spe cific activities of 1.8-11.1 mCi/mg, with less than 2% aggregation. Two int ernalizing mAbs, LL2 (anti-CD 22 B-cell lymphoma mAb) and RS7 (an anti-aden ocarcinoma mAb which targets EGP-1 antigen), labeled with this procedure ex hibited a 2-3-fold better cellular retention in Ramos and Calu-S tumor cell lines, in vitro, respectively, compared to the same mAbs radioiodinated wi th the chloramine-T method. The rationale for the new approach, syntheses, radiochemistry and in vitro data are presented.