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

Martin Studer, Linda A. Kroger, Sally J. DeNardo, David L. Kukis, Claude F. Meares

Research output: Contribution to journalArticle

27 Citations (Scopus)

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 ≈ 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).

Original languageEnglish (US)
Pages (from-to)424-429
Number of pages6
JournalBioconjugate Chemistry
Volume3
Issue number5
StatePublished - 1992

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alanyl-leucyl-alanyl-leucine
Ethylenediaminetetraacetic acid
Radioactivity
Metabolism
Antibodies
Peptides
Digestion
Liver
High Pressure Liquid Chromatography
Cathepsin B
Peptide Hydrolases
In Vitro Techniques
benzyl-EDTA
Urine
Proteins

ASJC Scopus subject areas

  • Chemistry(all)
  • Organic Chemistry
  • Clinical Biochemistry
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry

Cite this

Influence of a peptide linker on biodistribution and metabolism of antibody-conjugated benzyl-EDTA. Comparison of enzymatic digestion in vitro and in vivo. / Studer, Martin; Kroger, Linda A.; DeNardo, Sally J.; Kukis, David L.; Meares, Claude F.

In: Bioconjugate Chemistry, Vol. 3, No. 5, 1992, p. 424-429.

Research output: Contribution to journalArticle

Studer, Martin ; Kroger, Linda A. ; DeNardo, Sally J. ; Kukis, David L. ; Meares, Claude F. / Influence of a peptide linker on biodistribution and metabolism of antibody-conjugated benzyl-EDTA. Comparison of enzymatic digestion in vitro and in vivo. In: Bioconjugate Chemistry. 1992 ; Vol. 3, No. 5. pp. 424-429.
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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 ≈ 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).",
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N2 - 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 ≈ 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).

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