Immunotoxin cross-linkers

Myers, D.E., Uckun, F.M., Swaim, S.E., and Vallera, D.A. (1989) The effects of aromatic and aliphatic maleimde cross-linkers on and-CP5 ricin immunotoxins. J. Immunol. Meth. 121, 129-142. 

SMPT, succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene, contains an NHS ester end and a pyridyldisulfide end similar to those of SPDP, but its hindered disulfide makes conjugates formed with this reagent more stable (Thorpe etal., 1987) (Chapter 5, Section 1.2). The reagent is especially useful in forming immunotoxin conjugates for in vivo administration (Chapter 11, Section 2.1). A water-soluble analog of this cross-linker containing an extended spacer arm is also commercially available as Sulfo-LC-SMPT (Pierce). 

Figure 315 The basic design of an immunotoxin conjugate consists of an antibody targeting component cross-linked to a toxin molecule. The complexation typically includes a disulfide bond between the antibody portion and the cytotoxic component of the conjugate to allow release of the toxin intracellularly. In this illustration, an intact A—B toxin protein provides the requisite disulfide, but the linkage also may be designed into the cross-linker itself.

Although the use of these alternative disulfide generating agents has proven successful in some applications, pyridyldisulfide-containing cross-linkers, as discussed previously, by far are more common for producing immunotoxin conjugates. 

Preparation of Immunotoxin Conjugates via Amine- and Sulfhydryl-Reactive heterobifunctional Cross-linkers... 

To make effective immunotoxin conjugates using the following cross-linkers, it is necessary to cross-link intact A—B toxins to antibodies, not single-chain or A-chain toxins. Using intact two-subunit toxins allows the A chain to break free of the complex... 

However, since SMCC forms nonreversible thioether linkages with sulfhydryl groups, it only can be used in the preparation of immunotoxins if intact A-B toxins are employed in the conjugate. In such conjugates, the A chain still has the potential for reductive release from the B-chain subunit after cellular docking and internalization. Immunotoxins prepared with A-chain or single-subunit toxins will not display cytotoxicity if cross-linked with SMCC, since the cross-linker does not create cleavable disulfide bonds upon conjugation. 

SMCC has been used to prepare immunotoxins with cobra venom factor (Vogel, 1987) and was compared to other cross-linkers in the preparation of gelonin and PAP conjugates (Lambert et al., 1985). 

As in the case of MBS, discussed previously, SMPB was found to be more effective than aliphatic cross-linkers 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). 

The method for the preparation of immunotoxins with SMPB is identical to that used for MBS (above). Since the thioether bonds formed with sulfhydryl-containing molecules are noncleavable, A-chain isolates or single-chain toxin molecules cannot be conjugated with antibodies with retention of cytotoxicity. Only intact A—B toxin molecules may be used with this cross-linker, since the A chain still is capable of being reductively released from the complex. 

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