Biotin Reagents for Antibody Pretargeting. Synthesis, Radioiodination, and in Vitro Evaluation of Water Soluble, Biotinidase Resistant Biotin Derivatives

Abstract

As part of our development of antibody pretargeting for cancer therapy, an investigation has been conducted to examine the stability of water solubilized, radioiodinated biotin derivatives toward biotinidase degradation in mouse and human serum. Eight new biotin derivatives were synthesized to conduct the study. The biotin derivatives synthesized contained (1) the biotin moiety, (2) a water solubilizing linker moiety, (3) p-iodobenzoate or p-tri-n-butylstannylbenzoate moieties, and (4) in some of the compounds, N-methyl or alpha-methyl containing moieties were added to block biotinidase activity. The linker moiety, 4,7,10-trioxa-1,13-tridecanediamine, 5, was included in the biotin derivatives to improve their water solubility, and it also functioned as a 17 A spacer between the biotin and the benzoyl moieties. Four of the new biotin derivatives (12, 14, 22, and 23) contained a p-tri-n-butylstannylbenzoyl moiety as precursors which could be radioiodinated in the last synthetic step. The other four biotin derivatives (13, 15, 24, and 25) contained p-iodobenzoyl moieties and were used as HPLC reference standards. Initial studies involved radioiodination of 12 to yield [125I]13. Radioiodinated 13, which did not contain a moiety for blocking biotinidase activity, was found to be rapidly degraded in both mouse and human serum at 37 degrees C. Derivatives which were designed to be stable to biotinidase incorporated N-methyl and alpha-methyl moieties adjacent to the biotin carboxylate group. In one set of biotin derivatives (14 and 15), the N-methyl moiety was obtained by incorporating N,N-dimethyl-4,7,10-trioxa-1,13-tridecanediamine, 9, as a linker in the place of 5. In the second set of biotin derivatives (22 and 24), the N-methyl moiety was introduced by incorporating a sarcosine (N-methylglycine) moiety between biotin and 5. The radioiodinated N-methyl containing biotin derivatives [125I]15 and [125I]24 were found to be very stable to biotinidase degradation. An alpha-methyl group was obtained in a pair of biotin derivatives (23 and 25) by incorporating a 3-aminobutyric acid moiety between biotin and 5. The radioiodinated alpha-methyl containing derivative, [125I]25, was found to have an intermediate stability with regards to biotinidase degradation.

Endoprotease Profiling with Double-Tagged Peptide Substrates: A New Diagnostic Approach in Oncology

“…Finally, the biotin-tag was protected from serum biotinidase activity by incorporation of 2-aminobutyric acid (27,30 ) to avoid the use of iodacetamid, which inhibits not only biotinidase but also cysteine proteases (31 ).…”

Release of biotin from biotinylated proteins occurs enzymatically and nonenzymatically in human plasma

“…Reduced eYcacy of tumor imaging and therapy techniques that utilize unstable biotin labels has also been a concern of others [1,2,12,13,15]. In a previous study, we directly demonstrated that a variety of commercially available biotinylating agents produced biotin labels that are cleaved in the presence of plasma [4].…”

“…Wilbur et al [3] speculated that the loss of binding to (strept)avidin is the action of biotinidase cleavage of biotin from the Fab' and noted that other investigators had reported the need to develop metabolically stable biotin conjugates. Indeed, Wilbur et al published a series of papers describing modiWcations of the biotinamide bond to protect bond stability and retain aqueous solubility without aVecting binding to (strept)avidin [1,[12][13][14][15]. In many of these reports, biotinidase is inferred, although not directly shown, to catalyze the cleavage.…”

Target Visualization by MRI Using the Avidin/Biotin Amplification Route: Synthesis and Testing of a Biotin–Gd‐DOTA Monoamide Trimer

“…The focus of the studies was the investigation of antibody conjugates with recombinant streptavidin in tumor pretargeting protocols, a method for amplifying the amount of radioactivity delivered to cancer cells. [8] Many derivatives have been synthesized by Wilbur and coworkers containing two and three biotin moieties with varying distances between the carbon atoms of the biotin amide. [9] Only tris-biotinylated compounds completely polymerized in SAv solution and similar results were obtained in the surface-bound SAv cross-linking experiment.…”

“…[8,17] Since the relaxivity of the Gd 3 L2/Av adduct is more than two times higher than the relaxivity of free Gd 3 L2, the degradation of the adduct can be followed by measuring the proton relaxation rate (R 1obs ) of the solution. For this purpose, the R 1obs of solutions containing Gd 3 L2/Av (0.5 mm protein concentration) were measured after dilution 1:10 with human serum.…”