A FACILE, WATER-SOLUBLE METHOD FOR MODIFICATION OF PROTEINS WITH DOTA- USE OF ELEVATED-TEMPERATURE AND OPTIMIZED PH TO ACHIEVE HIGH SPECIFIC ACTIVITY AND HIGH CHELATE STABILITY IN RADIOLABELED IMMUNOCONJUGATES

We have developed a method for attachment of the macrocyclic chelating agent 1,4,7,10-tetraazacyclododecane N,N',N'':N'''-tetraacetic acid ( DOTA) to proteins by activation of a single carboxyl group with N-hydr oxysulfosuccinimide (sulfo-NHS). The sulfo-NHS active ester of DOTA wa s prepared in a single step using 1-ethyl-3-[3-(dimethylamino)propyl]c arbodiimide (EDC), and DOTA conjugates of cytochrome c and the anti-ca rcinoembryonic antigen chimeric monoclonal antibody cT84.66 were prepa red by adding the DOTA active ester reaction mixture to the proteins a t pH 8.5-9.0. Mass spectrometry of the cytochrome c conjugates showed that as the molar ratio of DOTA active ester to protein in the reactio n mixture was increased from 10:1 to 100:1, the average number of chel ators attached to the protein molecule increased from 2.64 to 8.79. Wh en DOTA active ester reacted with the antibody at a molar ratio of 100 :1, the conjugate averaged 3.8 chelates per antibody. Immunoreactivity of the antibody conjugate radiolabeled with In-111(III) and Y-90(III) remained quantitative. Variation of the DOTA:sulfo-NHS:EDC activation stoichiometry from 2:2:1 to 10:10:1 revealed that the kinetic stabili ty of the radioconjugates increased as the molar ratio of carbodiimide , relative to DOTA and sulfo-NHS, was decreased. Radiolabeling of the protein conjugates with In-111(III) and Y-90(III) proved to be sensiti ve to pH, buffer, and temperature effects. The optimum pH for the labe ling reaction was different for each protein and may be related to the isoelectric point of the protein. Radiometal incorporation at high sp ecific activity was accomplished in acetate and Tris buffers, but the presence of citrate inhibited the labeling reaction. Increasing the te mperature of the radiolabeling reaction from 25 to 43 degrees C greatl y increased both the efficiency of radiometal incorporation and the ki netic stability of the radioconjugates. Stability studies of the conju gates in human serum and in the presence of a 5000- to 250 000-fold ex cess of diethylenetriaminepentaacetic acid (DTPA) demonstrated that th e radiolabeled proteins are kinetically inert under physiological cond itions. In serum, the In-111(III)-labeled antibody showed a rate of ra diometal loss of approximately 0.08% per day. In the presence of exces s DTPA, both conjugates lost In-111(III) at a rate of about 0.3% per d ay. No loss of Y-90(III) from the conjugates was observed in serum, bu t in excess DTPA, both Y-90(III) labeled proteins showed a rate of rad iometal loss of approximately 0.2% per day. Therefore, kinetic analysi s of metal loss from a radiolabeled immunoconjugate in the presence of a vast excess of DTPA may provide a better indication of the in vivo stability of that immunoconjugate than serum stability studies.