Immunoconjugate design: a predictive approach for coupling of daunomycin to monoclonal antibodies

Abstract

There is increasing interest in the development of daunomycin-antibody immunoconjugates for the targeting of drug to specific cells or tissues. To this end, we have examined the factors influencing the synthesis of daunomycin-monoclonal antibody conjugates linked covalently by an acid-labile cis-aconitic spacer (which is considered to aid drug release from immunoconjugates in the lysosomes and thus enhance their cytotoxic potential). A rapid and efficient procedure for the purification of drug from contaminants and stabilizers was first developed; conditions for the optimal preparation of cis-aconityldaunomycin were established; products were analyzed and identified by TLC and HPLC. The coupling of cis-aconityldaunomycin to antibody was accomplished by activating the modified drug with a carbodiimide before addition to antibody. Several factors were identified which influenced the efficiency of the conjugation; in particular, the compositional features of the antibody which determine its electrophoretic charge characteristics were of profound effect. However, by appropriate choice and control of buffer pH during conjugation, it was possible to define conditions resulting in the controlled substitution of antibody with drug. The consequent effects upon the cell-binding activity of immunoconjugates were established and related to the extent of substitution. The procedures described enable appropriate reaction conditions to be selected for the linkage of daunomycin to antibody (at set drug/antibody molar ratios) and in good yield, based upon consideration of the compositional and charge properties of the antibody.

Reversible Covalent Chemistry in Drug Delivery

“…Antibodies are usually attached to conjugates through disulfide bonds either by attaching a thiol linker to a lysine residue in the antibody or by direct attachment to internal disulfide bonds naturally present within the antibody [29,30,31]. Thiol linkers attached to lysine residues often include N-succimidyl 3-(2-pyridyldithio)propionate [32] (SPDP) or the charge retaining 2-iminothiolane (Traut's reagent) [33].…”

Immunotargeting of daunomycin to localized and metastatic human colon adenocarcinoma in athymic mice

“…(b) The second conjugate, daunomycin (cis-aconitylated)-SF25Ab (oxidized, reacted with ethylenediamine and reduced), designated dan-CASF25Ab, was prepared as follows: cis-aconityldaunomycin (dan-CA) was synthesized according to Hudecz et al [ 13]. The coupling of dan-CA to SF25Ab was performed either directly using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide [13] or according to Dillman et al [7] by periodate oxidation of the antibody.…”

“…The coupling of dan-CA to SF25Ab was performed either directly using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide [13] or according to Dillman et al [7] by periodate oxidation of the antibody. The oxidized antibody was reacted with ethylenediamine and then reduced by sodium cyanoborohydride, in order to create additional amino groups.…”

“…Direct conjugation of the cis-aconitic-linked daunomycin to antibody [13,19,29] resulted in very low drug binding to antibody (2-3 mol drug/ml antibody). Therefore the variation described by Dillman et al, in which the antibody was first oxidized and then reacted with ethylenediamine [7], was preferred.…”

“…This type of conjugate has already been tested by others [6] and by us [1,15,28] in several experimental systems. In the second coupling procedure, daunomycin was attached to the antibody via a cis-aconityl spacer [13,22] and used in some of the more recent immunotargeting studies [7,29]. We have tested both conjugates against two colorectal carcinoma lines in vitro.…”