Preparation of Immunotoxin Conjugates via Disulfide Exchange Reactions

Preparation of Immunotoxin Conjugates via Disulfide Exchange Reactions 

N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) is by far the most popular heterobifunctional cross-linking agent used for immunotoxin conjugation (Chapter 5, Section 1.1). The activated NHS ester end of SPDP reacts with amine groups in one of the two proteins to form an amide linkage. The 2-pyridyldithiol group at the other end 

LC-SPDP (Chapter 5, Section 1.1) is an analog of SPDP containing a hexanoate spacer arm within its internal cross-bridge. The increased length of the extended crosslinker is important in some conjugations to avoid steric problems associated with closely-linked macromolecules. However, for the preparation of immunotoxins, no advantages were observed for LC-SPDP over SPDP (Singh et al., 1993). 

To activate the toxin, SPDP again can be used to modify the intact A—B component. After purification of the modified toxin from excess cross-linker, the SPDP—toxin is mixed with the thiolated antibody to effect the final conjugate (Fig. 319). 

For instance, if toxin A chain—antibody conjugates are to be prepared, the antibody can be similarly activated with SPDP, but in this case not treated with reductant. After removal of excess cross-linker, the activated antibody can be directly mixed with isolated A chain to create the conjugate (Fig. 320). This procedure makes use of the indigenous sulfhydryl residues produced during reductive separation of the A and B chains and therefore does not require cross-linker thiolation of one of the proteins. 

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