Toxin molecules

The C-NMR spectrum of neosaxitoxin obtained from the feeding experiment showed an enhanced signal for C-4, which was split into a doublet by the spin-spin coupling with the neighboring (J=9.3 Hz) (Scheme 4). The result clearly indicate that the connectivity C-2 - N-2 of arginine was incorporated intact into the toxin molecule, supporting the pathway in Scheme 3. 

If we assume that those peculiarities of the toxin which cause their distribution are localized in a special group of the toxin molecules and the power of the organs and tissues to react with the toxin are localized in a special group of the protoplasm, we arrive at the basis of my side chain theory. The distributive groups of the toxin I call the haptophore group and the corresponding chemical organs of the protoplasm the receptor. . .. Toxic actions can only occur when receptors fitted to anchor the toxins are present. 

Figure 21.1 The basic design of an immunotoxin conjugate consists of an antibody-targeting component crosslinked 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 crosslinker itself.

A-chain immunotoxins, however, may not be quite as cytotoxic as conjugates formed from intact toxin molecules (Manske et al., 1989). In an alternative approach to A chain use, the intact toxin of two-subunit proteins is directly conjugated to a monoclonal without isolation of the A chain. Conjugation of an antibody with intact A-B chain toxins can be done without a cleavable linker, as long as the A chain can still separate from the B chain once it is internalized. Therefore, it is important to avoid intramolecular crosslinking during the conjugation process which can prevent release of the A-B complex. In addition, since the B chain... 

Figure 21.5 SPDP can be used to modify both an antibody and a toxin molecule for conjugation purposes. In this case, the antibody is thiolated to contain a sulfhydryl group by modification with SPDP followed by reduction with DTT. A toxin molecule is then activated with SPDP and reacted with the thiolated antibody to effect the final conjugate through a disulfide bond.

Figure 21.6 SPDP can be used to activate an antibody molecule through its available amine groups to form a sulfhydryl-reactive derivative. Toxin molecules containing disulfide-linked A-B chains may be reduced with DTT to isolate the A-chain component containing a free thiol. The SPDP-activated antibody is then mixed with the reduced A chain to effect the final conjugate by disulfide bond formation.

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.

Caution Toxins are highly toxic even in very low amounts. Handle all toxin molecules and their isolated subunits with extreme care. 

Caution toxin molecules are dangerously toxic even in small amounts. Use extreme care in handling. 

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.

Figure 21.12 SIAB can be used to activate toxin molecules for coupling with sulfhydryl-containing antibodies. In this case, the antibody molecule is thiolated using SATA and deprotected to reveal the free sulfhydryl. Reaction with the SIAB-activated toxin forms the final conjugate by thioether bond formation.

Figure 21.13 Sulfo-SMCC may be used to activate antibody molecules for coupling to thiolated toxin components. An intact A-B toxin molecule can be modified to contain sulfhydryls by treatment with 2-iminothiolane. Thiolation with this reagent retains the cytotoxic properties of the toxin while generating a sulfhydryl for conjugation. Reaction of the thiolated toxin with the maleimide-activated antibody creates the immunotoxin through thioether bond formation.

Figure 21.14 Activation of an intact A-B toxin molecule with MBS with subsequent conjugation with a reduced antibody fragment to produce an immunotoxin.

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 non-cleavable, A-chain isolates or single-chain toxin molecules can not be conjugated with antibodies with retention of cytotoxicity. Only intact A-B toxin molecules may be used with this crosslinker, since the A chain still is capable of being reductively released from the complex. 

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