Site-directed chemical modification and cross-linking of a monoclonal antibody using equilibrium transfer alkylating cross-link reagents

Bioconjug Chem. 1990 Jan-Feb;1(1):36-50. doi: 10.1021/bc00001a005.

Abstract

A new, more reactive group of protein cross-linkers in the class of equilibrium transfer alkylating cross-link (ETAC) reagents has been synthesized. These compounds include alpha,alpha-bis[(p-chlorophenyl)methyl]- and alpha,alpha-bis[(p-tolylsulfonyl)methyl]acetophenones substituted in the acetophenone ring with chloro, nitro, amino, and carboxyl groups and derivatives. Included are an 125I-labeled ETAC reagent and a 111In-labeled DTPA (diethylenetriaminepentaacetic acid) ETAC for site direction and biodistribution studies. These ETAC compounds were reacted with unreduced and partially reduced antibody under mild pH (pH 4-8) and room temperature conditions to give cross-linked structures. Examination of resultant cross-linked antibody via size-exclusion HPLC, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, and an enzyme linked immunosorbent assay revealed that (1) both interantibody as well as intraantibody cross-linking had occurred; (2) the level of inter- and intraantibody cross-linking varied with the substituent on the ETAC; (3) the stability of the cross-links on the reducing SDS gels varied with substituents on the ETAC; (4) little if any immunoreactivity was lost after reaction with one of the more effective ETAC cross-linking compounds; (5) the 125I-labeled ETAC sulfhydryl cross-linking in partially reduced antibody increased with pH whereas amine cross-linking with the unreduced antibody decreased with pH; (6) the optimum pH for sulfhydryl site direction was pH 5.0; (7) the 111In DTPA ETAC labeled antibody had a biodistribution in CD1 mice similar to that of the 111In bis cyclic anhydride DTPA labeled antibody.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Acetophenones*
  • Alkylating Agents
  • Animals
  • Antibodies, Monoclonal*
  • Cross-Linking Reagents* / chemical synthesis
  • Enzyme-Linked Immunosorbent Assay
  • Indicators and Reagents
  • Indium
  • Mice
  • Pentetic Acid
  • Protein Binding
  • Structure-Activity Relationship

Substances

  • Acetophenones
  • Alkylating Agents
  • Antibodies, Monoclonal
  • Cross-Linking Reagents
  • Indicators and Reagents
  • Indium
  • Pentetic Acid