Toxin conjugates

Antibodies antibody fragments (e.g., collagen-specific drug-toxin conjugates)... 

SATA has been used to form conjugates with avidin or steptavidin with excellent retention of activity (Chapter 23, Section 3.1). It also has been used in the formation of a therapeutically useful toxin conjugate with recombinant CD4 (Ghetie et al., 1990), to study syntaxin proteins (Amessou et al., 2007), to prepare bispecific antibodies (Lindorfer et al., 2001), and to make a unique polylysine conjugate as a vehicle for drug delivery (Sakharov et al., 2001). 

Using this approach, EGF has been successfully conjugated by disulfide exchange to the A chain of diphtheria toxin (Shimisu et al., 1980). A cystaminyl derivative of insulin also could be conjugated to the A chain of diphtheria toxin by this method (Miskimins and Shimizu, 1979). Other references to disulfide exchange using cystamine include Oeltmann and Forbes (1981) and Bacha et al. (1983) who prepared antibody-toxin and peptide-toxin conjugates, respectively. 

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. 

The following methods are generalized to provide an overview of how SPDP can be used in these conjugation techniques. The appropriate optimization for a particular toxin conjugate should be done. 

React for 18 hours at room temperature to form the final conjugate. Isolation of the ideal 1 1 or 1 2 antibody-toxin conjugate can be done through gel filtration separation using a column of Sephacryl S-300 or the equivalent. 

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. 

Removal of nonspecific binding potential in the B chain must be done before using an A-B intact toxin conjugate in vivo. See step 5 of the MBS conjugation protocol discussed previous to this section. 

Blattler, W.A., Kuenzi, B.S., Lambert, J.M., and Senter, P.D. (1985b) New heterobifunctional protein cross-linking reagents and their use in the preparation of antibody-toxin conjugates. Photochem. Photobiol. 42, 231. 

Anti-tumour monoclonal antibodies can also be used to deliver toxins to tumour sites. Toxins conjugated to therapeutic antibodies include ricin, pokeweed toxin, Pseudomonas toxin and... 

Clinically, monoclonal antibodies are also proposed as drug delivery vehicles in certain tumors where specific tumor-associated antigens are expressed. In this context, investigators have found that by conjugating toxins such as the A chain polypeptide of the plant protein ricin or the bacterial toxin from Corynebacterium diphtheriae to monoclonal antibodies specific for certain tumor type, as few as one or two molecules of antibody-toxin conjugate can destroy a tumor cell in vitro. Some success has also been obtained in clinical trials with monoclonal antibody-toxin conjugates. 

D. M. Neville Jr., Monoclonal Antibody Mediated Drug Delivery and Antibody Toxin Conjugates , in Directed Drug Delivery-A Multidisciplinary Approach , Eds. R. T. Borchardt, A. J. Repta, V. J. Stella, Humana Press, Clifton, N. J., 1985, p. 211-230. 

Targeting Tumor Blood Vessels With a Toxin Conjugate... 

Therapeutic applications of antibody-toxin conjugates may be limited because (1) antibody-toxin conjugates distribute nonspecifically to organs such as the liver and cause severe toxicity, (2) the bacterial toxin is immunogenic to humans, (3) tumor-associated antigens are often found in normal tissue (although levels are low), and (4) premature release of toxin from the antibody conjugate leads to systemic toxicity. 

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