Immunotoxin conjugation using

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... 

SMPT, succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene, contains an NHS ester end and a pyridyl disulfide end similar to SPDP, but its hindered disulfide makes conjugates formed with this reagent more stable (Thorpe et al., 1987) (Chapter 5, Section 1.2). The reagent is especially useful in forming immunotoxin conjugates for in vivo administration (Chapter 21, Section 2.1). A water-soluble analog of this crosslinker containing an extended spacer arm is also commercially available as sulfo-LC-SMPT (Thermo Fisher). 

SMPT or sulfo-LC-SMPT has been used to develop conjugates for in vivo delivery of siRNA to hepatocytes (Rozema et al., 2007), in preparing an anti-CD25-immunotoxin conjugate (Mielke et al., 2007), and in preparing conjugates for selective depletion of donor lymphocytes in stem cell transplantation (Solomon et al., 2005). 

PDPH has been used in the preparation of immunotoxin conjugates (Zara et al., 1991). It has also been used to create a unique conjugate of nerve growth factor (NGF) with an... 

Since immunotoxin conjugates are destined to be used in vivo, their preparation involves more critical consideration of crosslinking methods than most of the other conjugation protocols described in this book. The following sections discuss the problems associated with toxin conjugates and the main crosslinking methods for preparing them. 

This multi-step crosslinking method employing SPDP on both molecules has been used to prepare a number of immunotoxin conjugates (Edwards et al., 1982 Thorpe et al., 1982 Colombatti et al., 1983 Wiels et al., 1984 Vogel, 1987 Reiter and Fishelson, 1989). While... 

SMPT often is used in place of SPDP for the preparation of immunotoxin conjugates. The hindered disulfide of SMPT has distinct advantages in this regard. Thorpe et al. (1987) showed that SMPT conjugates had approximately twice the half-life in vivo as SPDP conjugates. Antibody-toxin conjugates prepared with SMPT possess a half-life in vivo of up to 22 hours, presumably due to the decreased susceptibility of the hindered disulfide toward reductive cleavage. 

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 21.10 Cystamine may be used to make immunotoxin conjugates by a disulfide interchange reaction. Modification of antibody molecules using an EDC-mediated reaction creates a sulfhydryl-reactive derivative. A-chain toxin subunits containing a free thiol can be coupled to the cystamine-modified antibody to form disulfide crosslinks.

Purification of the immunotoxin conjugate from unconjugated ricin can be done using a column of TSK3000 SW (Toya Soda, Japan) according to the method of Myers et al. (1989). 

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. 

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