Immunotoxin using

Chain A loses its toxicity when the B chain is removed. In ricin immunotoxins, the MAb replaces the B chain function and takes the responsibility for both the target cell specificity and cell entry of the A chain Ricin immunotoxins using the anti-CD5 antibody (a marker on T cells and some B cells) have been investigated in T and B cell lymphomas ricin immunotoxins using the anti-CD 19 antibody (a B lymphocyte marker) have been investigated in non-Hodgkin s lymphoma. 

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. 

Human parenteral toxicity for abrin is approximately 0.1-1 Jig/kg (Romano et al, 2007). However, based on clinical trials on abrin-immunotoxin use for cancer treatment, the human minimum lethal dose by intravenous injection was estimated to be >0.3 Jig/kg without occurrence of serious adverse effects (Gill, 1982). 

Wiley, R. G., Oeltmann, T. N. Lappi, D. A. (1991). Immunolesioning selective destruction of neurons using immunotoxin to rat NGF receptor. Brain Res. 562... 

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. 

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... 

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.

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