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Spring 1997Volume 4, Number 1 |
Immunotoxins offer the potential to selectively target cells of the immune system in order to promote the goal of tolerance to organ transplants. Conceptually, immunotoxins take advantage of the exquisite specificity of antibodies to selectively target drug delivery and the potency of toxins to kill the target cells. A variety of such agents have been developed for experimental use. Some immunotoxins and chimeric toxins have been designed to target cancer cells. Unfortunately, the toxicity of some of these agents has prevented their further clinical development. An additional problem has been the immunogenicity of the toxin. The ideal toxin molecule for use in an immunotoxin construct would have minimal toxicity and poor immunogenicity.
The targeting component of an immunotoxin is most frequently a monoclonal antibody reactive with the desired specificity but it may also be a receptor or ligand for a receptor on the surface of the targeted cell. Monoclonal antibodies can be used as whole molecules or as enzymatically cleaved fragments, which are less immunogenic but have shorter half-lives in vivo. Genetically-engineered fusion proteins containing immunoglobulin heavy chain constant regions confer a longer half-life, and humanized constructs can be engineered to eliminate almost all immunogenicity.
Toxins used in immunotoxin constructs are derived from bacteria, fungi, and plants, and most function by inhibiting protein synthesis. Toxins such as diphtheria toxin (DT) and Pseudomonas exotoxin (PE) prevent protein synthesis by an effect on elongation factor 2 (EF-2). They are synthesized as single chain proteins which are modified post-translationally into two-chain molecules joined by disulfide bonds. The interchain disulfide bond is necessary for the cytotoxic effect of the toxin. Toxins have separate functional domains committed to target cell binding, translocation across membranes, and inhibition of protein synthesis (see illustration below). In order to be effective, however, immunotoxin must be internalized and route to the appropriate intracellular compartment for translocation of their attached toxin into the cytosol. The targeting moiety and toxin are joined by a crosslinker which is stable extracellularly but labile intracellularly so that the toxin can function in the cytosol.
Figure 1. Construction of anti-T cell immunotoxin
The side effects of immunotoxins have included hepatotoxicity, hypoalbuminemia, vascular leak syndrome, and myalgias. Immunogenicity related to the toxin component has also limited their use. Anti-immunotoxin antibodies can reduce the effectiveness of immunotoxins by increasing their rate of clearance from the circulation or by blocking the functional domains of the antibody or the toxin. Preformed antibodies to diphtheria as a result of childhood immunization represent an additional obstacle to clinical application of diphtheria-based immunotoxins.
To accomplish T-cell killing, CD5 immunotoxins are more effective than CD2 immunotoxins, because anti-CD2 immunotoxins are preferentially routed to lysosomes where the A chain is degraded. CD3 immunotoxins have been associated with cytokine release related to stimulation of the T-cell receptor by the antibody. Selection of an anti-CD3 monoclonal antibody with a low affinity Fc receptor has minimized this problem, as has removal of the Fc receptor enzymatically or by constructing a fusion protein.
| The ability to design toxin and antibody genes capable of eliminating undesirable features while retaining potency of the immunotoxin has permitted continued improvement of immunotoxin constructs that may eventually have clinical applications to tolerance induction. |
The ability to design toxin and antibody genes capable of eliminating undesirable features while retaining potency of the immunotoxin has permitted continued improvement of immunotoxin constructs that may eventually have clinical applications to tolerance induction. For example, the wild-type diphtheria toxin has been modified by altering the B chain to produce the mutant CRM9. CRM9 has 1/300th of the toxicity of the wild-type diphtheria toxin but maintains its potency as an inhibitor of protein synthesis.
Drugs and toxins which have been coupled to monoclonal antibodies and used in experimental transplant models are listed in the table below. There are obviously a huge number of potential immunoconjugates which could be constructed with potential use as immunosuppressive agents for organ transplantation. Selection of the most appropriate toxin and targeting moiety for clinical use is the subject of ongoing studies.
| DRUGS/TOXINS CONJUGATED TO ANTIBODIES FOR USE IN TRANSPLANTATION |
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Preliminary work with an anti-CD3 epsilon monoclonal antibody coupled to CRM9 toxin (FN18-CRM9) has shown that this construct can efficiently kill T lymphocytes in both peripheral blood and lymph node of the rhesus monkey. The immunotoxin appears to be well tolerated with minimal systemic side effects. When given seven days before an MHC-mismatched renal allograft as the sole immunosuppressant therapy, long-term graft survival occurred in treated recipients, and (specific) tolerance to subsequent skin grafts was demonstrated. These promising results in a primate renal allograft model suggest the potential role of immunotoxins in human transplantation.
Stuart J. Knechtle, MD
Associate Professor of Surgery
Department of Surgery
University of Wisconsin
Madison, Wisconsin
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