Influence of a peptide linker on biodistribution and metabolism of antibody-conjugated benzyl-EDTA. Comparison of enzymic digestion in vitro and in vivo

Abstract

Insight into the metabolism of radiolabeled antibodies is important for the design of better radioimaging and therapy agents. To test the effect of linkers that can be cleaved in vivo, we introduced Ala-Leu-Ala-Leu between the antibody Lym-1 and an 111In-labeled benzyl-EDTA. For comparison, we studied a conjugate without the linker. Digestion of the two conjugates in vitro showed that the one with Ala-Leu-Ala-Leu was cleaved rapidly by the liver protease cathepsin B1 (T1/2 approximately 6 h). After 100 h of digestion, reversed-phase HPLC product analysis of the Ala-Leu-Ala-Leu conjugate showed that 48% of the total radioactivity had the same retention time as (p-aminobenzyl)-EDTA(In), and 37% of the total radioactivity had the same retention time as [p-(Ala-Leu-amido)benzyl]-EDTA(In). After 97 h of digestion, the conjugate without the linker had 79% of the radioactivity activity still attached to the protein. We also tested the two conjugates in mice. Ala-Leu-Ala-Leu had only a moderate effect on the whole body and liver clearance in vivo. The excretion of the radioactivity was about 6% per day with the linker and about 3% per day without the linker. HPLC analysis of the urine of a single mouse showed products similar to the in vitro study; 54% of the excreted radioactivity had the same retention time as (p-aminobenzyl)-EDTA(In), while 10% had the retention time of [p-(Ala-Leu-amido)benzyl]-EDTA(In).

Preparation and evaluation of a new releasable PEGylated tumor necrosis factor-α (TNF-α) conjugate for therapeutic application

“…At higher concentrations (>5000 U mL 1 ); however, these conjugates exhibited stronger cytotoxicity, up to 60%, against VECs ( Figure 3A). At the same doses, both forms of PEGylated TNF- were more cytotoxic to B16 and L929 cells, a difference likely due to the presence of cathepsin B in tumor tissue [16]. Moreover, the cytotoxicity of rPEG-TNF- toward B16 (IC 50 (5368.06±46.28) IU mL 1 ; Figure 3B) and L929 (IC 50 (3263.18±32.27) IU mL 1 ; Figure 3C) cells, was about 5 and 9-fold higher than that of PEG-TNF- against B16 (IC 50 (30233.06±204.22) IU mL 1 ) and L929 (IC 50 (27356.81±274.08) IU mL 1 ) cells, respectively.…”

“…The general substrates of cathepsin B and L include dipeptide and tetrapeptide [14]. Tetrapeptide such as Gly-Phe-Leu-Gly and Ala-Leu-Ala-Leu were regarded as having distinct disadvantages, because of their relatively slow drug release and hydrophobic nature, which could cause these tetrapeptides to precipitate or aggregate [15][16][17]. Consequently, a dipeptide linker sensitive to cathepsin B such as the Val-Cit moiety has been widely utilized in releasable conjugates [18][19][20].…”

Understanding the in vivo fate of radioimmunoconjugates for nuclear imaging

“…[121][122][123][124] In a different approach, Studer et al introduced a tetrapeptide linker (Ala-Leu-Ala-Leu) between the antibody and the chelator to promote the specific cleavage of the 111 In-chelator complex in the liver. 125 However, this approach led to very limited-if any-improvements in the activity concentrations retained in the liver of mice. The authors suggest that the 111 In-chelate complexes were successfully cleaved from the antibody but were themselves subsequently (and unfortunately) trapped in the liver.…”

Protein– and Peptide–Drug Conjugates

“…These proteases have very low activities in blood due to endogenous inhibitors and unfavorably high pH of blood (Ciechanover, 2005). Early peptide linkers were tetrapeptides like, Gly-Phe-Leu-Gly (Koblinski, Ahram, & Sloane, 2000) and Ala-Leu-Ala-Leu (Studer, Kroger, DeNardo, Kukis, & Meares, 1992;Versluis, Rump, Rensen, Van Berkel, & Bijsterbosch, 1998), but, they release drug slowly and their hydrophobic nature leads to aggregation. On the other hand, dipeptide-based linkers Val-Cit and Phe-Lys are reasonably stable under physiological conditions and undergo rapid hydrolysis in the presence of lysosomal cathepsin B (Kirschke, Barrett, Rawlings, et al, 1995;Otto & Schirmeister, 1997).…”