Heregulin-specific Diphtheria Toxin as a Cancer Therapy

Institution: Salk Institute for Biological Studies
Investigator(s): Gordon Louie, Ph.D. -
Award Cycle: 1998 (Cycle IV) Grant #: 4KB-0143 Award: $322,293
Award Type: New Investigator Awards
Research Priorities
Detection, Prognosis and Treatment>Innovative Treatment Modalities: search for a cure

Initial Award Abstract (1998)
Normally, the growth of human breast cells is closely controlled by steroid hormones, such as estrogen, and other growth-promoting proteins, such as heregulin. Cancerous cells that overproduce the HER2 and HER4 oncogene proteins have progressed to a diseased state where they are no longer responsive to anti-estrogen treatment (e.g., tamoxifen). Often, the unwanted growth of breast cancer cells is promoted by heregulin, a protein that interacts with and activates the HER2 and HER4 proteins on the cell surface. In some instances, the breast cancer cells themselves produce heregulin. Thus, neutralizing the growth-promoting activities of heregulin is one strategy for breast cancer therapy. Our goal is to design a toxin protein that will kill cells carrying heregulin.

Diphtheria toxin is known to kill certain types of cells carrying a specific receptor protein on their surface. Diphtheria toxin enters the cell via the receptor and arrests protein synthesis to cause cell death. Importantly, the normal diphtheria toxin receptor has strong similarity to the heregulin growth factors involved in stimulating breast cancer cells. Thus, diphtheria toxin will serve as the starting material in our design of a new toxin capable of recognizing and killing only cells that produce heregulin. We anticipate that this new toxin variant may be effective in several approaches to limit the overall growth of breast cancer. First, the targeted delivery of the toxin's killing activity to heregulin-producing cells will reduce circulating levels of heregulin that can cause the growth of breast cancer cells. Second, breast cancer cells that produce heregulin will be selectively killed. Third, binding of the toxin protein to heregulin will prevent it from activating the HER2 and HER4 proteins on cancerous cells.

Our approach is to custom design diphtheria toxin by using a three-dimensional picture produced by X-ray crystallography. This analysis shows in detail how atoms of the diphtheria toxin fit together with atoms of its normal cell surface receptor - like pieces of a jigsaw puzzle. This allows us to identify key atoms dictating this interaction. Then, we will construct variant diphtheria toxin molecules using biology techniques with the aim of targeting the toxin to heregulin-bearing cells. This appears feasible and will require only small changes on the toxin. The biologic activity of this designed toxin will be determined by using breast cancer cell lines that express heregulin.

Although only about 30% of patients appear to express the HER2 oncogene, these patients experience the least favorable clinical outlook. Our approach may provide an alternative to the current clinical development of antibody therapy to HER2-containing breast cancers.

Final Report (2000)
Note: The PI resigned the grant in October 2000 to pursue a career path in biotechnology.

The goal of this project is to design a cytotoxic protein using diphtheria toxin as a template that will recognize and bind to a cell that overproduces heregulin (HRG) precursor on the cell surface. HRG is an activating growth factor ligand for HER-4, whose overexpression is correlated with breast cancer cells. Given the recent success with the humanized monoclonal antibody (herceptin) against erb-B receptors as therapeutics against breast cancer, it is reasonable to hypothesize that the downregulation of the production of this ligand will lead to the inhibition of the growth of breast cancer cells. In 1997 we completed the crystal structure of diphtheria toxin (DT) complexed with an extracellular fragment of its receptor protein, a membrane-bound precursor of human heparin-binding EGF (HBEGF). HBEGF is structurally homologous to HRG, thus providing a point of departure for molecular design of diphtheria toxin in order to divert to a new target receptor, HRG.

A single molecule of diphtheria toxin of 535 amino-acid residues is sufficient to kill a cell. The killing action of DT involves three distinct steps. First, it binds to a receptor on the surface of sensitive cells and subsequent receptor-mediated endocytosis. Second, it translocates the catalytic domain of the toxin across the endosomal membrane and into the cytoplasm of the cell, a process induced by the acidic environment inside the endosome. Third, the catalytic transfer of an ADP-ribosyl group from NAD+ to elongation factor-2 by the toxin domain prevents protein synthesis in the cell and leads to cell death.

We have chosen selected number of positions at the toxin/receptor interface to introduce the new binding specificity. As a test, we attempted to direct the toxin to mouse. We have shown partial success for such systematic change in specificity, yet require extensive optimization of the assay methods to quantitatively measure the binding affinity. At the end of our continued pursuit of the goal of protein engineering the toxin molecule in order to modulate its target specificity from human HBEGF to heregulin, the ultimate test will be whether such engineered toxin will be of therapeutic value in the treatment of breast cancer. However, as of the termination date of the project, we have not convincingly demonstrated the feasibility of the recombination, screening, and assay methods. Further pursuit to target towards HRG, therefore, has been delayed for future study.