Immunotoxin cytotoxicity

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 21.7 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.

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 have 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 crosslinked with SMCC, since the crosslinker does not create cleavable disulfide bonds upon conjugation. 

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.

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). 

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. 

A second method of immunotoxin preparation by reductive amination involves the use a polysaccharide spacer. Soluble dextran may be oxidized with periodate to form a multifunctional crosslinking polymer. Reaction with antibodies and cytotoxic molecules in the presence of a reducing agent forms multivalent immunotoxin conjugates. The following sections discuss these options. 

Dell Arciprete, L., Colombatti, M., Rappuoli, R., and Tridente, G. (1988) A C terminus cysteine of diphtheria toxin B chain involved in immunotoxin cell penetration and cytotoxicity./. Immunol. 140, 2466-2471. 

Jansen, F.L., Blythman, H.E., Carriere, D., Casellas, P., Diaz, J., Gros, P., Hennequin, J.R., Paolucci, F., Pau, B., Poncelet, P., Richer, G., Salhi, S.L., Vidal, H., and Voisin, G.A. (1980) High specific cytotoxicity of antibody-toxin hybrid molecules (immunotoxins) for target cells. Immunol. Lett. 2, 97. 

Wawrzynczak, E. J. and Thorpe, P. E. (1988) Effect of chemical linkage upon the stability and cytotoxic activity of A chain immunotoxins, in Immunotoxins (Frankel, A. E., ed.), Kluwer Academic Publishers, Boston, pp. 239-251. 

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