Antibody—toxin conjugates

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. 

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. 

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. 

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. 

The methods described below for the preparation of antibody-toxin conjugates containing A chains isolated from toxins or single-chain RIPs are generally applicable to the synthesis of conjugates using any type of antibody and all known RIPs of plant origin. 

The method described has been used to prepare antibody-toxin conjugates containing mouse and rat monoclonal antibodies of various IgG subtypes and polyclonal antibody from several animal species. Modifications of this method have also been used to prepare conjugates with antibodies of other classes. 

Antibody-toxin conjugates made with ricin A chain, abrin A chain, gelomn, and momordin can be stored for at least 4 yr at -70°C without detectable loss of activity. The bond between the antibody and the RIP breaks down very slowly at 4°C in PBSE but, provided that care is taken to ensure the sterility of the solution, conjugates can be stored under these conditions for up to one year with little deterioration in quality. 

Fig. 1. Gel permeation chromatography of an antibody-toxin conjugate reaction mixture. The reaction mixture obtained following the conjugation of a mouse monoclonal antibody (50 mg) and [l25I]-labeled abrin A chain was chromatographed on a column of Sephacryl S-200 (SF), dimensions 80 cm x 2.6 cm (id). Fractions eluting from the column were monitored spectrophotometrically at 280 nm (—) to measure total protein, and by gamma counting (—) to measure the A chain in its free or conjugated form. The hatched area indicates a typical pooled conjugate preparation.

Fig. 2. SDS-PAGE of an antibody-toxin conjugate preparation. (A) Antibody (starting material). (B) Abrin A chain conjugate (pooled fractions shown in Fig. 1). Samples were prepared under nonreducing conditions and run on a 2—27% gradient polyacrylamide gel.

Thorpe, P E and Ross, W. C J (1982) The preparation and cytotoxic properties of antibody-toxin conjugates. Immunol Rev 62, 119-158... 

Immunoaffinity Purification and Quantification of Antibody-Toxin Conjugates... 

Before using the column to purify antibody-toxin conjugate, apply a solution of the RIP at a concentration of 1 mg/mL to block any high affinity binding sites. Remove the RIP using the elution procedure (see Note 13)... 

Load the sample (1 mL) of the antibody-toxin conjugate preparation (at a concentration up to 1 mg/mL in running buffer) and elute with the running buffer at a flow rate of 0 5 mL/min... 

Dialyze the affinity-purified antibody-toxin conjugate into PBSA, filter-sterilize in a sterile hood, and store at 4°C or, freeze rapidly and store at-70°C (see Notes 15 and 16). 

Prepare dilutions of the antibody-toxin conjugate samples with casein buffer to give an approximate concentration of between 0 5 and 5 ng of conjugated RIP/ mL of buffer in each case. At least 300 pL of each dilution is required for step 7 (see Note 18)... 

Remove the casein buffer Add 100 pL of each diluted antibody-toxin conjugate sample (prepared in step 4) to the plates in triplicate... 

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