We are all familiar with the terrible side effects of currently used therapies for cancer and viral diseases such as HIV. Radiation therapy for cancer severely lowers the quality of life for the treated patients, resulting in nausea, hair loss, and a severe drop in energy. Drug therapy, or chemotherapy, most often is also accompanied by these side effects. The problem is that chemotherapeutics and radiation therapy are poorly selective in which cells they attack. However, a better way to treat patients was envisioned by Paul Ehrlich in the early 1900s: By tethering therapeutic agents to antibodies, "magic bullets" could be produced that specifically bind and deliver the therapeutic agent to sick or malevolent cells, having little or no effect on healthy cells in the body. This works because antibodies are proteins that have exquisite binding selectivity, which can be produced to bind to only one targeted protein, while ignoring a multitude of other proteins they might come in contact with.
Immunotoxins are protein conjugates borne of this "magic bullet" concept. They are made by linking an antibody with the desired specificity (e.g., bind to cancer cells, bind to HIV-infected cells) to a highly toxic protein, termed a "toxin." The linker holding the antibody and the toxin together is designed to be stable outside of a cell (in the bloodstream), yet once inside a cell will cleave apart, freeing the toxin to do its dirty work. Immunotoxins have recently shown a great degree of promise in fighting cancer (specifically leukemias and lymphomas), autoimmune disease (such as arthritis), and are being brought to bear in the battle with AIDS.
The Antibody Moiety
Typically, antibodies are generated using monoclonal antibody technology, which can produce large quantities of a mouse antibody that has singular specificity for a protein to which it was designed to bind. Basically, the target protein (termed an antigen) is injected into a mouse, and when the mouse has developed a sufficient immune response to the antigen (including many protein-specific, antibody-producing B cells), its spleen cells (containing B cells) are harvested and fused to myeloma cells. Myeloma cells are an immortal cell line that will allow the fused cells to grow indefinitely and at a fast rate. These myeloma-B cell hybrid cells are called hybridoma cells, and can be selected for and tested to verify that they produce the desired antibody. The hybridoma clones that produce the antibody demonstrating the required specific binding activity can then be grown in large quantities, and the monoclonal antibodies can be harvested for use in an immunotoxin.
One of the problems with monoclonal antibodies is their mouse, or murine, origin. A human patient's own immune system will recognize the murine antibody as foreign, and will clear the antibody (and immunotoxin therapy) from the bloodstream quickly, greatly lessening the immunotoxin's effectiveness. To combat this, laboratories have engineered "humanized" antibodies where the part of the antibody that the human immune system identifies as of mouse origin, called the constant region of the antibody, is swapped out for a human constant region.
Another problem with monoclonal antibodies is their large size. Antibodies consist of four polypeptide chains and have a molecular mass of 150,000 daltons. Because of their size, they have a hard time penetrating solid cancer tumors where the blood supply is fairly restricted. As a solution to this size problem, scientists are making immunotoxins that utilize only the protein-binding part of the antibody, called the variable region. They do this by cleaving off this region with a protease (making an Fab fragment), or by cloning the variable region into bacteria and expressing as a single-chain antibody.
The Toxin
Protein toxins are among the most potent poisons known to man. Because of its incredible toxicity, ricin was used in probably the most famous political assassination in history. A minuscule amount of one toxin, ricin, was placed in the tip of an umbrella and used to assassinate Georgi Markov, a Bulgarian dissident. As little as 1 mg can kill an adult, and it has been extrapolated that as little as a single toxin molecule is enough to kill a cell. Toxins are produced in some bacteria and in low levels in common plants such as pokeweed and the castor bean plant. Bacterial toxins commonly used in immunotoxins include Diphtheria toxin (DT) and the toxin from Pseudomonas exotoxin (PE). Plant toxins utilized in immunotoxins include the A chain of ricin (RTA), and the ribosome inactivating proteins (RIPs) gelonin, pokeweed antiviral protein, and dodecandron. Toxins work by catalytically inactivating protein synthesis. Because it is an enzyme, one toxin molecule can work on many substrate molecules, having a devastating effect on the cell.
Immunotoxin Mechanism
The mechanism by which immunotoxins work to kill diseased cells in the body is quite simple. Using AIDS therapy as an example, let's say we have developed an immunotoxin to kill HIV-infected cells by raising antibodies that bind to GP120, a viral protein found on the outside of only HIV-infected cells. Once an AIDS patient has been treated, the immunotoxin floats around in the bloodstream until it binds to a GP120 molecule on the outside of an infected cell. Once bound, the GP120-immunotoxin complex gets pulled inside the cell by endocytosis, where it is either localized to an acidified endosome (if DT is the toxin), or the endoplasmic reticulum (ER) and trans-golgi apparatus in the cell. Inside these organelles, the linker holding the toxin to the antibody is cleaved. Usually the linker is made with an internal disulfide bond, so that it is stable in the oxidizing atmosphere outside the cell and cleaved by reduction in the reducing environment inside the cell.
Once freed from the antibody, the toxin now catalytically inactivates the protein synthesis machinery of the cell. The bacterial toxins perform this by inactivating the ribosome accessory protein elongation factor-2 (EF-2). The plant RIPs accomplish their task by cleaving a single adenine base from the ribosomal RNA so that it can no longer bind EF-2. Either way, the inactivation of protein synthesis leads to the death of the cell. For HIV this is important because it is the infected cells that are the virus-producing factories inside the AIDS patient.
Immunotoxin Web Site Links
Pictures of Toxin and Single-Chain Immunotoxin Structures
Seattle Genetics News - Announcing a phase I clinical trial of a single-chain immunotoxin for treating carcinomas
CD30-PAP - An immunotoxin that uses pokeweed antiviral protein conjugated to a CD30 receptor (instead of an antibody) to treat Hodgkin's disease
AIDS Treatment News - Announcing a new lymphoma immunotoxin study
Monoclonal Antibodies Facility - Core facility home page with simple cartoon showing how to make monoclonal antibodies
Bees — Latest Weapon in Cancer Fight - Use of a bee toxin in immunotoxin therapy for cancer (Pictures)
Coping with the Side Effects of Chemotherapy
Ricin - Everything about the plant toxin ricin
The Spying Game - Spy site that has ricin assassination story
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