Lysine reduces renal accumulation of radioactivity associated with injection of the [177Lu]alpha-[2-(4-aminophenyl) ethyl]-1,4,7,10-tetraaza-cyclodecane-1,4,7,10-tetraacetic acid-CC49 Fab radioimmunoconjugate.

The high uptake and prolonged renal retention of monoclonal antibody fragments that are conjugated with radiometal chelates precludes their routine clinical use due to high background counts, which may hinder detection of nearby lesions and/or cause renal radiotoxicity. We report on the potential use of Lys as a pharmacological agent to enhance renal excretion of the [177Lu]alpha-[2-(4-aminophenyl) ethyl]-1,4,7,10-tetraaza-cyclodecane-1,4,7,10-tetraacetic acid CC49 Fab ([177Lu]CC49 Fab) radioimmunoconjugate. The monoclonal antibody portion of this complex is directed toward the tumor-associated glycoprotein-72 antigen. Lys was administered to female BALB/c mice by i.p. injections. [177Lu]CC49 Fab bolus injections were given by the i.v. route. Results of our investigations showed that: (a) kidney radioactivity concentrations were inversely related to Lys dose. The optimal dose (50 mg/mouse) evoked a 3-fold reduction in kidney counts; (b) Lys was most effective when injected 15 min before, or at the same time as, [177Lu]CC49 Fab; (c) the renal effect was both rapid (3-fold decrease at 15 min after injection) and prolonged (4-fold decrease at 24 h after injection); (d) a single Lys dose decreased total body radioactivity by > 2.5-fold; (e) urine excretion of radioactivity was enhanced in Lys-treated mice. High pressure liquid chromatographic analyses using a GF-250 column showed that a large fraction of this urine radioactivity coeluted with a [177Lu]CC49 Fab injection standard. We conclude that Lys enhances the urinary excretion of radioactivity associated with [177Lu]CC49 Fab. These observations warrant further study with regard to the use of amino acids or their derivatives as pharmacological agents to enhance the urinary excretion of small-molecule radioimmunoconjugates.

Preparation, characterization, and in vivo biodistribution properties of synthetically cross-linked multivalent antitumor antibody fragments.

Two new antibody forms of the general structure F(ab')n (n = 3 or 4) were prepared and tested in vivo as part of an ongoing search for antibody candidates with improved biodistribution properties for cancer immunotargeting applications. The novel multivalent antibody forms, called F(ab')3-x (tribody, 150 kDa, x = cross-linker) and F(ab')4-x (tetrabody, 200 kDa), were constructed through chemical cross-linking of Fab' subunits derived from murine CC49 IgG, a monoclonal antibody which recognizes the tumor-associated antigen TAG-72. Two new chemical reagents (trismaleimide 1 and tetramaleimide 2) were synthesized for use in cross-linking cysteine sulfhydryl groups present on the hinge region of Fab'. Homogeneous Fab' was prepared by mild reduction of F(ab')2 followed by selective reoxidation of interchain disulfide bonds, leaving a single hinge-region cysteine sulfhydryl group available for modification. For biodistribution studies, the parent F(ab')2 fragment was first radiolabeled via lysine amine modification using the isothiocyanate derivative of the 105Rh(BA-2,3,2-tet)Cl2 complex. Both new fragment forms were shown to retain antigen binding ability in vitro using a solid-phase immunoassay. Although isolated yields for F(ab')3-x and F(ab')4-x were low (18 and 4%, respectively), sufficient quantities were prepared for preliminary biodistribution studies in Balb/c mice and, in the case of F(ab')3-x, for a 5-day biodistribution study in tumor-bearing nude mice. A large proportion of the 105Rh-labeled F(ab')4-x was found to accumulate in the liver, possibly indicating an upper size limit for the in vivo use of cross-linked fragments. The biodistribution behavior of 105Rh-labeled F(ab')3-x in both Balb/c and nude mice was intermediate between that of IgG and F(ab')2 for all organs studied. Kidney localization was reduced, while blood circulation time and tumor accumulation were slightly increased, for the trivalent species compared with F(ab')2. The unique biodistribution profile of F(ab')3-x suggests the possible use of this multivalent fragment for in vivo tumor targeting applications.