Preparation of Immunotoxin Conjugates

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. 

It has become apparent that the method of crosslinking can dramatically affect the activity of an immunotoxin in vivo. This is true not only with regard to possible direct blocking by the crosslinker of the enzymatic active site which is responsible for inactivation of ribosomes, but the chemistry of conjugation also is an important factor in proper binding and entry of the 

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. 

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 

More elaborate methods of blocking or eliminating the B-chain galactose binding site also can be done to prevent nonspecific cytotoxicity. For instance, the crosslinking agent may have a 

Other investigators, however, have demonstrated that conjugations of antibody 

When using ricin A chains, it has been found that chemical deglycosylation of the subunit prevents its nonspecific binding to receptors for mannose on certain cells of the reticuloendothelial system (Vitetta and Thorpe, 1985 Ghetie et al., 1988, 1991). 

Regardless of their method of preparation, the required and ideal characteristics of immunotoxin conjugates can be summarized in the following points  

Lactose Modified with Cy amine at Its Reducing End via Reductive Amination 

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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... 

Preparation of Immunotoxin Conjugates via Disulfide Exchange Reactions... 

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. 

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 NGF with an antibody directed against the transferrin receptor OX-26, which could traverse the blood-brain barrier (Friden et al., 1993). Labeling of antibody molecules with PDPH at oxidized polysaccharide sites followed by reduction to free the sulfhydryl has been used to form a technetium-99m complex for radiopharmaceutical use (Ranadive et al., 1993) (Chapter 8, Section 2.5). 

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