Antibody-toxin conjugates cross-linkers

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.

The conjugation process must leave the antigen binding sites on the antibody component free to interact with its intended target. Cross-linker modification or blockage of these binding sites by the attached toxin must be kept to a minimum. 

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. 

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... 

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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