Sulfhydryl site-specific crosslinking and labeling of monoclonal antibodies by a fluorescent equilibrium transfer alkylation crosslink reagent

Abstract

The site-specific intramolecular cross-linking of sulfhydryls of monoclonal antibodies via a new class of "equilibrium transfer alkylation cross-link (ETAC) reagents" is described. Following complete or partial reduction of interchain disulfides with dithiothreitol (DTT), two murine IgG2a monoclonal antibodies, 225.28S and 5G6.4, were reacted with alpha,alpha-bis[(p-tolylsulfonyl)methyl]-m-aminoacetophenone (ETAC 1a) and a fluorescent conjugated derivative, sulforhodamine B m-(alpha,alpha-bis(p-tolysulfonylmethyl)acetyl)anilide derivative (ETAC 1b). Reducing SDS-polyacrylamide gel electrophoresis analysis of the products from 1b indicated the formation of S-ETAC-S interchain heavy and light chain cross-links (approximately 23-34% overall yield by video-camera densitometry) which do not undergo disulfide-thiol exchange with DTT at 100 degrees C. In contrast, no interchain cross-links were observed upon reaction of unreduced or reduced antibody wherein the thiols have been previously alkylated with iodoacetamide. These results indicated site-specific cross-linking of interchain sulfhydryls and places their distance within 3-4 A. Flow cytometry of the ETAC 1b 5G6.4 cross-linked product using 77 IP3 human ovarian carcinoma target cells showed positive binding and retention of immunoreactivity. The in vivo biodistributions of 131I-labeled intact 5G6.4 and 125I-labeled reduced 5G6.4 + ETAC 1a product in rats were essentially identical over a period of 24 h. The present study illustrates the potential applications of labelable ETAC reagents as thiol-specific probes for a wide variety of immunological studies.

Effect of chemical modification strategy on the characteristics of copper-67-Labeled immunoconjugates, Part I: Immunoreactivity

“…Arano et al (1994) reported mercaptoethanol reduction of the disulfide bonds of a monoclonal antibody against osteogenic sarcoma to generate 6 to 7 thiols per molecule. Using a procedure similar to that described herein, del Rosario et al (1990) reported the use of DTT at 37°C for approximately 5 h to cleave the hinge region disulfides of antibodies. Upon radioiodination, one of the half antibody preparations possessed immunoreactivity in the highest range typically obtained for routine radioiodination of this antibody.…”

Bioconjugation through Mesitylene Thiol Alkylation

“…Although bis-alkylating agents have been widely used for peptide cyclization and labelling, [22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] only a few examples of bis-alkylation of a previously reduced pair of Cys residues of an antibody using a bis-sulfone linker and bromomaleimides have been described in the literature. [39][40][41][42][43] Thus new chemical entities able to preserve the native structure of the antibodies after bioconjugation are needed.…”

Identification and insertion of 3-carbon bridges in protein disulfide bonds: a computational approach

“…Although we have shown that the 3-carbon bridge can be used to conjugate PEG efficiently in aqueous solution 10,13 , insertion of the bridge also offers the potential to enable the site-specific conjugation of pharmacological and imaging agents to the protein [14][15][16] .…”