Antibodies intact toxins

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 21.15 A periodate-oxidized dextran polymer may be reacted with both an antibody and an intact toxin component using reductive amination to form a multivalent immunotoxin complex.

Examination of genotoxicity of pharmaceuticals is required to assess the interaction of the drug with DNA. These studies are generally not applicable to immunotoxins. Unlike chemotherapeutics that cause cell death through DNA interaction, immunotoxins mediate cell death by preventing protein synthesis. However, immunotoxins use a linker to connect the toxin to the antibody that may need to be examined if it is an organic linker and has the ability to bind DNA (per ICH S6). The majority of immunotoxins use either a nonreducible thioether linker for intact toxins or a disulfide bond for A chains and ribosome-inactivating proteins and do not interact with DNA. 

For preparing whole ricin IT, the intact toxin is normally derivatized with a bifunctional cross-linking agent before being mixed with antibody prepared for cross-linking by partial reduction of disulphide bonds [183]. 

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.

Other investigators, however, have demonstrated that conjugations of antibody with intact, two-subunit toxins can be done using non-cleavable crosslinkers such as NHS ester-maleimide heterobifunctionals (Chapter 5, Section 1) (Myers et al., 1989). Presumably, the toxin is still able to release the A chain after the antibody has bound to the cell, since the conjugation process does not permanently attach the two toxin subunits together—only the toxin to the antibody. 

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 21.13 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. 

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). 

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... 

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