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News News Recombinant Molecules May Mean Improved Toxin Therapies For years, and with varying success, researchers have tried to adapt natural plant and bacterial toxins to the treatment of cancer. Their potent cytotoxicity — a single toxin molecule penetrating a cell is enough to kill the cell — makes them appealing to researchers looking for better ways to kill cancer cells. Now, recombinant toxins, the latest refinement of this longstanding effort, are emerging from the laboratory and showing promise as a potential cancer treatment. "Toxins have several attractive features that make them useful in cancer treatment," said Ira Pastan, M.D., chief of NCI's Laboratory of Molecular Biology. "Toxins and conventional chemotherapeutic drugs have different mechanisms of action. Therefore, those cancer cells that are naturally resistant or acquire resistance to chemotherapeutic drugs will not be resistant to toxin-based therapies. Also, toxins kill non-dividing cells, which some conventional therapies do not." Although the basic strategy of linking toxins with molecules that preferentially bind to cancer cells has met with some success, the full potential of toxin-based therapies has not been achieved. Some researchers, like Pastan, are hopeful that recombinant toxins can better fulfill this potential.
DNA encoding these growth factors is fused to bacterial toxin genes. The fused DNA then produces a protein with both a cell-recognition and a cell-killing function. The engineered protein will selectively bind to and kill cancer cells with the appropriate growth factor receptor. One widely-studied molecule, TGFalpha-PE40 (TP40), provides a good example of why recombinant toxin therapy is thought to have a promising future. Pastan's lab has shown that TP40, a fusion of transforming growth factor-alpha and the cell-lethal portion of the Pseudomonas exotoxin, is extremely cytotoxic to cells that overproduce epidermal growth factor receptors, a characteristic of cancers of the lung, head and neck, breast, and bladder. TP40's antitumor effect was seen in studies of animals bearing
Targeted to Cancer Recombinant toxins, genetically engineered fusion molecules, are designed to kill cancer cells by taking advantage of a common characteristic of many cancers: the over-production of growth factor receptors. 1466
Dr. Ira Pastan
4 Dr. W. Marston Linehan
tumors that have more EGF receptors than normal cells.
Human Trials A phase I, multicenter study is currently under way to assess the safety of TP40 in patients with recurrent, superficial bladder cancer. In the treatment trial, TP40 is introduced directly into the bladder. "Superficial bladder cancer was chosen as a clinical target based on its high content of EGF receptors," explained W. Marston Linehan, M.D., chief of urologic oncology in NCI's Surgery Branch and collaborator with Pastan and Merck Research Laboratories in the TP40 trial. "Forty to 60% of superficial bladder cancer cases show an abundance of EGF receptors on the cell surface. "This is a very exciting and interesting new strategy for the treatment of cancer," said Linehan, who nevertheless remains cautious in his assessment of TP40's clinical performance to date. "Although well-tolerated, it is too early to determine whether this form of therapy will be effective," he said. Journal of the National Cancer Institute
News News Still,researchersanticipate that recombinant toxins will overcome the drawbacks associated with earlier "targeted toxins." The first targeted toxins, called immunotoxins, were made by chemically linking the toxin to monoclonal antibodies for cancer-specific antigens. Many immunotoxins produce selective killing of cancer cells in culture, but only a few have been able to cause substantial tumor regression in animals. Several human trials have been conducted using immunotoxins. Two recent trials using ricin, a plant toxin, have produced significant responses in patients with lymphoma. "So far," said Pastan, "the antibodies used for the preparation of immunotoxins to treat carcinomas or other solid tumors have been found to react with important normal human tissues and produce doselimiting toxicity without significant clinical responses. Producing immunotoxins by chemical coupling methods is also very expensive because it requires large amounts of antibody and toxin." The chemical conjugation techniques used to produce immunotoxins can also create problems. "The chemical modifications can change the antibody and affect its binding to antigen," said Pastan. And because chemical coupling produces a heterogeneous mixture of antibody-toxin molecules connected to each other in a variety of positions, the yield of the desired molecule is often poor. Recombinant fusion toxins, on the other hand, are homogeneous products which can be purified in large amounts.
Improved Antibodies Recombinant fusion toxins being developed exploit antibodies' cell-binding ability more efficiently. These molecules contain DNA sequences encoding the variable region of the antibody, the portion responsible for cell-binding. DNA sequences encoding the constant region of the antibody, which is not necessary Vol. 84, No. 19, October 7, 1992
for immunotoxic action, are omitted. The fusion molecules are thus much smaller and, consequently, more mobile, making them more active than their chemically linked counterparts. Until recently, the fact that antibody variableregionsnormally exist as two separate polypeptide chains had made it difficult to produce recombinants. Several tedious and time-consuming steps were required to clone the genes encoding the individual chains and then reassemble them into a functional antibody.
A strategy devised in Pastan's lab, however, has made it possible to rapidly clone the individual variable domain genes as a single-chain antibody fused to a toxin gene.
Single Chains Several of these "single-chain immunotoxins" have been constructed by Pastan's group, using either modified Pseudomonas exotoxin or diphtheria toxin genes. Recently, they reported the construction of a single-chain immunotoxin that was highly cytotoxic to cancer
Stat Bite
Breast Cancer Incidence, 1973-1989 In 1989, breast cancer incidence in women declined for the second straight year. The sharp rise from 1980-1987, mainly in small, early stage tumors, is thought to reflect increases in mammography screening. This earlier detection may result in lower mortality by the mid-1990s. However, screening may be stabilizing at lower levels than expected, reducing potential mortality benefits. Rate per 100,000 400
100
50
Under 50
1977
1981
1985
1989
Year of Diagnosis
f'itnion Cun
News News Recombinant Molecules May Mean Improved Toxin Therapies For years, and with varying success, researchers have tried to adapt natural plant and bacterial toxins to the treatment of cancer. Their potent cytotoxicity — a single toxin molecule penetrating a cell is enough to kill the cell — makes them appealing to researchers looking for better ways to kill cancer cells. Now, recombinant toxins, the latest refinement of this longstanding effort, are emerging from the laboratory and showing promise as a potential cancer treatment. "Toxins have several attractive features that make them useful in cancer treatment," said Ira Pastan, M.D., chief of NCI's Laboratory of Molecular Biology. "Toxins and conventional chemotherapeutic drugs have different mechanisms of action. Therefore, those cancer cells that are naturally resistant or acquire resistance to chemotherapeutic drugs will not be resistant to toxin-based therapies. Also, toxins kill non-dividing cells, which some conventional therapies do not." Although the basic strategy of linking toxins with molecules that preferentially bind to cancer cells has met with some success, the full potential of toxin-based therapies has not been achieved. Some researchers, like Pastan, are hopeful that recombinant toxins can better fulfill this potential.
DNA encoding these growth factors is fused to bacterial toxin genes. The fused DNA then produces a protein with both a cell-recognition and a cell-killing function. The engineered protein will selectively bind to and kill cancer cells with the appropriate growth factor receptor. One widely-studied molecule, TGFalpha-PE40 (TP40), provides a good example of why recombinant toxin therapy is thought to have a promising future. Pastan's lab has shown that TP40, a fusion of transforming growth factor-alpha and the cell-lethal portion of the Pseudomonas exotoxin, is extremely cytotoxic to cells that overproduce epidermal growth factor receptors, a characteristic of cancers of the lung, head and neck, breast, and bladder. TP40's antitumor effect was seen in studies of animals bearing
Targeted to Cancer Recombinant toxins, genetically engineered fusion molecules, are designed to kill cancer cells by taking advantage of a common characteristic of many cancers: the over-production of growth factor receptors. 1466
Dr. Ira Pastan
4 Dr. W. Marston Linehan
tumors that have more EGF receptors than normal cells.
Human Trials A phase I, multicenter study is currently under way to assess the safety of TP40 in patients with recurrent, superficial bladder cancer. In the treatment trial, TP40 is introduced directly into the bladder. "Superficial bladder cancer was chosen as a clinical target based on its high content of EGF receptors," explained W. Marston Linehan, M.D., chief of urologic oncology in NCI's Surgery Branch and collaborator with Pastan and Merck Research Laboratories in the TP40 trial. "Forty to 60% of superficial bladder cancer cases show an abundance of EGF receptors on the cell surface. "This is a very exciting and interesting new strategy for the treatment of cancer," said Linehan, who nevertheless remains cautious in his assessment of TP40's clinical performance to date. "Although well-tolerated, it is too early to determine whether this form of therapy will be effective," he said. Journal of the National Cancer Institute
News News Still,researchersanticipate that recombinant toxins will overcome the drawbacks associated with earlier "targeted toxins." The first targeted toxins, called immunotoxins, were made by chemically linking the toxin to monoclonal antibodies for cancer-specific antigens. Many immunotoxins produce selective killing of cancer cells in culture, but only a few have been able to cause substantial tumor regression in animals. Several human trials have been conducted using immunotoxins. Two recent trials using ricin, a plant toxin, have produced significant responses in patients with lymphoma. "So far," said Pastan, "the antibodies used for the preparation of immunotoxins to treat carcinomas or other solid tumors have been found to react with important normal human tissues and produce doselimiting toxicity without significant clinical responses. Producing immunotoxins by chemical coupling methods is also very expensive because it requires large amounts of antibody and toxin." The chemical conjugation techniques used to produce immunotoxins can also create problems. "The chemical modifications can change the antibody and affect its binding to antigen," said Pastan. And because chemical coupling produces a heterogeneous mixture of antibody-toxin molecules connected to each other in a variety of positions, the yield of the desired molecule is often poor. Recombinant fusion toxins, on the other hand, are homogeneous products which can be purified in large amounts.
Improved Antibodies Recombinant fusion toxins being developed exploit antibodies' cell-binding ability more efficiently. These molecules contain DNA sequences encoding the variable region of the antibody, the portion responsible for cell-binding. DNA sequences encoding the constant region of the antibody, which is not necessary Vol. 84, No. 19, October 7, 1992
for immunotoxic action, are omitted. The fusion molecules are thus much smaller and, consequently, more mobile, making them more active than their chemically linked counterparts. Until recently, the fact that antibody variableregionsnormally exist as two separate polypeptide chains had made it difficult to produce recombinants. Several tedious and time-consuming steps were required to clone the genes encoding the individual chains and then reassemble them into a functional antibody.
A strategy devised in Pastan's lab, however, has made it possible to rapidly clone the individual variable domain genes as a single-chain antibody fused to a toxin gene.
Single Chains Several of these "single-chain immunotoxins" have been constructed by Pastan's group, using either modified Pseudomonas exotoxin or diphtheria toxin genes. Recently, they reported the construction of a single-chain immunotoxin that was highly cytotoxic to cancer
Stat Bite
Breast Cancer Incidence, 1973-1989 In 1989, breast cancer incidence in women declined for the second straight year. The sharp rise from 1980-1987, mainly in small, early stage tumors, is thought to reflect increases in mammography screening. This earlier detection may result in lower mortality by the mid-1990s. However, screening may be stabilizing at lower levels than expected, reducing potential mortality benefits. Rate per 100,000 400
100
50
Under 50
1977
1981
1985
1989
Year of Diagnosis
f'itnion Cun
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