Immunotoxin SPDP conjugation

SMPT often is used in place of SPDP for the preparation of immunotoxin conjugates. The hindered disulfide of SMPT has distinct advantages in this regard. Thorpe et al. (1987) showed that SMPT conjugates had approximately twice the half-life in vivo as SPDP conjugates. Antibody-toxin conjugates prepared with SMPT possess a half-life in vivo of up to 22 hours, presumably due to the decreased susceptibility of the hindered disulfide toward reductive cleavage. 

SMPT, succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene, contains an NHS ester end and a pyridyl disulfide end similar to SPDP, but its hindered disulfide makes conjugates formed with this reagent more stable (Thorpe et al., 1987) (Chapter 5, Section 1.2). The reagent is especially useful in forming immunotoxin conjugates for in vivo administration (Chapter 21, Section 2.1). A water-soluble analog of this crosslinker containing an extended spacer arm is also commercially available as sulfo-LC-SMPT (Thermo Fisher). 

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). 

This multi-step crosslinking method employing SPDP on both molecules has been used to prepare a number of immunotoxin conjugates (Edwards et al., 1982 Thorpe et al., 1982 Colombatti et al., 1983 Wiels et al., 1984 Vogel, 1987 Reiter and Fishelson, 1989). While... 

Figure 21.7 An intact A-B subunit toxin molecule may be activated with 2-iminothiolane with good retention of cytotoxic activity. The thiolated toxin then may be conjugated with SPDP-activated antibody to generate the immunotoxin conjugate through a disulfide bond.

Figure 21.8 SMPT may be used to form immunotoxin conjugates by activation of the antibody component to form a thiol-reactive derivative. Reduction of an A-B toxin molecule with DTT can facilitate subsequent isolation of the A chain containing a free thiol. Mixing the A-chain containing a sulfhydryl group with the SMPT-activated antibody causes immunotoxin formation through disulfide bond linkage. The hindered disulfide of an SMPT crosslink has been found to survive in vivo for longer periods than conjugates formed with SPDP.

As in the case of MBS, discussed previously, SMPB was found to be more effective than aliphatic crosslinkers in producing immunotoxin conjugates with ricin that have high yields of cytotoxicity (Myers et al., 1989). This was attributed to the reagent s aromatic ring structure. A comparison with SPDP produced immunotoxin conjugates concluded that SMPB formed more stable complexes that survive in serum for longer periods (Martin and Papahadjopoulos, 1982). 

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... 

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