Innovative Treatment Modalities: Search for a Cure
Translational Research Collaboration – Pilot
Awards
Translational Research Collaboration – Full Research
Awards
Innovative Treatment and Models of Care Awards
New Investigator Awards
Postdoctoral Fellowship Awards
A priority issue of the BCRP is to fund research that develops and explores innovative breast cancer treatment modalities. Under this priority we awarded 12 grants in 1998. In most cases, the funded projects are a link between the basic science laboratory and the clinic. At this stage there is a need for refinement of the mechanisms of action of new treatments and validation of their potential before they are used in humans. These projects make strong use of both animal model systems of breast cancer and clinical samples. There is an increased awareness that more relevant model systems are needed to test potential treatments for breast cancer. This year our awards are clustered in three topic areas (i) metastasis and angiogenesis, (ii) immunotherapy and drug delivery approaches, and (iii) surgery, tumor markers, and drug efficacy.
Angiogenesis has recently emerged as a promising target for attacking breast cancer. The size, invasiveness, and spread (metastasis) of tumors can be limited by treating the local blood supply. The blood supply of tumors is poorly organized or ‘leaky’, which allows tumor access for new forms of drug delivery and targeting. Francis Markland, Jr. is exploring the delivery and formulation of a novel protein from snake venom that affects the adhesive properties of both endothelial and breast cancer cells. Similarly, Keith Laderoute has identified a new drug that disrupts the internal cytoskeleton of cells, and also targets both the blood vessels and breast cancer cells in tumors. John Park is attaching antibodies specific to the blood vessel endothelial cells onto special liposomes (small sacs composed of fat particles) that contain anti-angiogenesis proteins. It is expected that anti-angiogenesis compounds have a selective effect on tumors. The ability to target tumors would make these expensive compounds useful in much smaller doses and less toxic to healthy tissue. Finally, dietary agents could offer more long-term, preventative affects on tumor angiogenesis. Kent Erickson is investigating conjugated linoleic acid as a potential angiogenesis inhibitor to treat women at risk for developing metastasis following the diagnosis of breast cancer.
Immunotherapy and associated treatment strategies commonly focus on the breast cancer cell oncogene Her-2, which is present on about 30% of women diagnosed with breast cancer. Her-2 is a cell surface receptor that leads to unregulated cell growth. Women having tumors with the Her-2 marker experience a poorer clinical outcome. One possible approach to treatment is to use antibodies to Her-2 to directly target breast cancer cells. Daryl Drummond is attaching Her-2 antibodies to liposome particles to deliver chemotherapy drugs directly to the interior of breast cancer cells. In contrast, Malcolm Mitchell is trying to find portions of the Her-2 protein that will stimulate a cytotoxic T lymphocyte recognition of breast cancer cells. The idea is to generate a cell-mediated immune response, rather than just antibodies. Joseph Lustgarten will attach an immune-enhancing cytokine to Her-2 antibodies to ‘mark’ tumor cells for attack by the immune system. From a totally different perspective, Gordon Louie is using the diphtheria toxin x-ray crystal structure as a way to model a novel molecular interaction with a natural ligand for the Her oncogene family, called heregulin.
Several new innovative treatment awards are strongly in the clinical realm. Surgeons need more advanced molecular markers to use in diagnosing breast biopsies. Shanaz Dairkee will study the molecular pathology of breast samples to see if defects are present even when the visual histological appearance is normal. There is evidence to suggest that the stage of the menstrual cycle can effect the outcome of breast cancer surgery. Hillary Klonoff-Cohen, Helena Chang and Hungyi Shau will approach this issue by examining the differential effect of surgery during different phases of the menstrual cycle on disease outcome. The key strength here is collaborative association of oncologists and epidemiologists brought together in the Translational Research Collaboration (TRC) funding mechanism. Physicians and surgeons are confused and frustrated that, while so many breast cancer ‘markers’ are known, we are lacking in follow-up studies to see how effective they are in predicting clinical outcome and survival. Shelley Enger, Michael Press and Jon Greif are assembling a team (TRC Pilot) and developing a strategy for collecting and evaluating information on Her-2, p53 (a tumor suppressor gene) and Bax (which regulates apoptosis, or normal cell death) from a sample of patients, and analyzing this information with respect to the therapy provided and subsequent survival status. Finally, we know that only a portion of women actually respond to any given therapy, and this is most frustrating for those women considering chemotherapy. Silvia Formenti, Peter Danenberg and Franco Muggia will be studying whether they can identify tumor markers that will predict the response of a tumor to the drug, Paclitaxel, with or without radiation before actual treatment begins.
Tranlastional Research Collaboration – Pilort Award
Breast Tumor Markers and Response to Adjuvant Therapy
Shelley Enger, Ph.D.
Kaiser Foundation Research Institute- Department of Research and Evaluation
Michael F. Press, M.D., Ph.D.
University of Southern California- School of Medicine
Jon M. Greif, D.O.
Kaiser Permanente Medical Care Program
An estimated 184,000 women were diagnosed with breast cancer in the United States in 1996, with over 10% of these cases occurring in California. Many of these women will have undergone painful and risky treatments, which will have no benefit for some of them. Others who were deemed at low risk of having their tumor recur will not have undergone certain procedures that may have greatly benefited them. Although treatment decisions made by the physician and the patient are typically based on the extent of the woman’s disease, recent evidence suggests that specific factors related to the tumor ("tumor markers") may influence the extent to which a woman responds to her treatment. We propose to conduct a pilot study to lay the groundwork for a full study to determine whether women respond to breast cancer treatments differently depending on the status of certain tumor markers.
The specific aims of the project are as follows: 1) to develop a partnership between Kaiser Permanente Southern California (KPSC) and the University of Southern California (USC), representing three distinct research areas: epidemiology, laboratory research, and clinical research; 2) to gather preliminary data on breast cancer treatment, breast cancer recurrence, survival and the status of specific tumor markers among a group of breast cancer patients diagnosed from 1988 through 1994 at the KPSC San Diego Medical Center; and 3) to prepare an application for a full TRC Award to examine tumor markers that may influence a woman’s response to breast cancer treatments.
The proposed study will create the foundation for a larger study that can address fundamental questions regarding patient-treatment interactions, with the ultimate goals of improving survival among breast cancer patients and reducing the substantial discomforts and risks associated with breast cancer therapies among women who are unlikely to benefit from them.
Translational Research Collaborative – Full Research Awards
Biologic Determinants of Response to Paclitaxel & Radiation
Silvia C. Formenti, M.D.
University of Southern California- Radiation Oncology Department
Peter Danenberg, Ph.D
University of Southern California- Cancer Research Laboratory
Franco Muggia, M.D.
New York University- Kaplan Cancer Center (a subcontract through USC)
Since systemic therapy reduces the risk of dying of breast cancer, most breast cancer patients are prescribed adjuvant chemotherapy (after surgical removal of the tumor). Unfortunately, most patients derive no benefit from adjuvant chemotherapy. In fact, of 10 women treated by conventional adjuvant chemotherapy only one will benefit. The remaining nine derive no benefit, either because they would have fared as well in the absence of systemic treatment or because they will recur in spite of it. Patients and doctors share the frustration of not knowing how to identify that one patient before treatment.
We are proposing a clinical paradigm that allows us to monitor the effect of therapy on the original cancer, instead of removing the breast cancer first, and then giving therapy. Moreover, advances in molecular biology make it possible to study the original tumor cell characteristics (from pre-treatment biopsies), to correlate them with tumor response to a specific treatment.
We were successful in obtaining the support of the Breast Cancer Research Program for a pilot study that fostered collaboration among three institutions, the University of Southern California, Mayo Clinic-Jacksonville and New York University. Together we have generated preliminary data that demonstrates feasibility and safety of single agent paclitaxel in breast cancers measuring more than 2 cm in diameter (T2-T3). The pilot also generated biological information that justified the design of a second study that combines paclitaxel with radiation for women who present with locally advanced breast cancers.
Both studies are designed to enable us to measure the extent of residual cancer after treatment. As predicted, a large range of tumor response exists: in some patients the cancer cells completely disappear, while in others the cancer persists. Original biological features are studied from pre-treatment tumor biopsies and are correlated with the response found in the surgical specimen obtained after therapy. This method identifies specific markers in the original tumor that can predict for pathological response to paclitaxel (clinical study #1) or to paclitaxel and radiation (clinical study #2).
By joining the efforts of three separate institutions we plan to accrue over 36 months a total of 80 women for each study. Clinical study #1 will enables us to: a) measure chemosensitivity directly in the breast cancer, b) understand which molecular characteristics of the tumor may predict for response and, c) generate biological information on paclitaxel alone to better understand its impact when combined with radiation. Clinical study #2 is as important because it will generate data to: a) understand how to best combine paclitaxel and radiation, b) impact the treatment of breast cancer as well as tumors at other sites that are treated with this combination and, c) improve treatment for locally advanced tumors, a more common breast cancer presentation among underserved minority women.
We expect a more rational, patient-specific use of the available treatment modalities to increase survival from breast cancer.
Timing of Breast Cancer Surgery, Menstrual Cycle & Prognosis
Hillary S. Klonoff-Cohen, Ph.D.
University of California, San Diego- Department of Family and Preventive
Medicine
Helena Chang, M.D., Ph.D.
University of California, Los Angeles- Revlon/UCLA Breast Center
Hungyi Shau, Ph.D.
University of California, Los Angeles- Division of Oncology
Surgery is the most common treatment for early breast cancer. There may be a particular time during the menstrual cycle when breast cancer surgery is less successful and results in decreased survival. A multi-disciplinary research team consisting of an epidemiologist, reproductive hormone specialist, basic immunologist, and three breast cancer surgeons will evaluate breast cancer patients’ hormonal status to determine whether breast cancer surgery during a particular time of the menstrual cycle known as the follicular phase (i.e., occurring between menstruation and ovulation) will increase the chance that the tumor will re-occur. This three-year study will follow 400 White, African-American, Hispanic, and Asian or Pacific Islander premenopausal women who will undergo surgery for breast cancer at four different hospitals (University of California San Diego, University of California Los Angeles, Los Angeles County-University of Southern California, and USC/Kenneth Norris) between July 1998 and June 2001. Patients with other cancers, or those with a hysterectomy, will not be included in the study. A pathologist will classify the type of breast cancer and where it has spread. The medical and reproductive histories, as well as other important information will be obtained from a detailed telephone interview, medical records, and laboratory results. The phase of the menstrual cycle (i.e., early or late follicular or luteal) will be determined by measuring specific hormones in the urine (e.g., progesterone, estradiol, follicle stimulating hormone, and leutinizing hormone) on a daily basis, starting on the day of surgery, and continuing until the onset of the next menstrual cycle. Most factors that influence the long-term outcome of breast cancer are beyond the doctor's control. This study will work with the body's predictable biologic rhythms (referred to as chronotherapy), in order to search for a better way to treat breast cancer. If the timing of surgical treatment during a particular phase of the menstrual cycle plays a significant role in survival from premenopausal breast cancer, this could possibly extend and/or save a great number of women's lives. In fact, the greatest benefit for timing of surgery would be to those young women at highest risk of breast cancer recurrence. The ease of modifying the timing of breast cancer surgery in the clinical setting could be very rapid and inexpensive; hence, timing of surgery could serve as a potentially simple, but powerful therapeutic tool.
Innovative Treatment and Models of Care Awards (ITaMoCAs)
Molecular Mapping of Surgical Margins
Shanaz H. Dairkee, Ph.D.
California Pacific Medical Center- Geraldine Brush Cancer Research Institute
In a recent breakthrough in breast cancer biology, it has been discovered that the "normal" breast tissue that lies near a newly diagnosed tumor is not entirely "normal," but contains specific genetic changes previously known to be present only in tumor cells (Deng et al., Science 274:2057, 1996). This has led to the new concept that the breasts of women diagnosed with breast cancer may contain a "predisposing" field spanning a region of unknown dimensions that visually appears to be normal yet harbors specific genetic changes. It is thought that cells in this region become committed to further cellular changes, leading eventually to invasive cancer. If this predisposing field is relatively large and undefined, the patient is likely to be at an increased risk for multiple occurrences of malignancy.
In this application, we propose to determine the magnitude and define the physical limits of such a predisposing field within the breasts of individual patients. We will use molecular methods of analysis in conjunction with a unique tissue resource in which the spatial relationship of benign ducts to the tumor as well as other ducts is intact. Consequently, the entire affected region can be examined at the histological (tissue) and molecular level in a three-dimensional context.
Mechanistically, the loss of genetic material seen in "normal" tissue could represent the loss of genes that may contribute towards initiating events in cancer and early malignant progression. Identification of these genes, which would be based on the results of this work, has obvious basic and clinical relevance.
The overall translational impact of the proposed molecular pathology studies will be in defining effective margins of surgical excision for invasive and non-invasive breast lesions. From the standpoint of the best surgical intervention, being able to identify these normal-appearing "predisposing" regions may significantly reduce the risk of developing new tumors.
Reduced Breast Cancer Metastasis by Conjugated Linoleic Acid
Kent Erickson, Ph.D.
University of California, Davis- School of Medicine
The overall goal of this project is to determine how a specific type of dietary fat can reduce and potentially prevent the spread of breast cancer to other parts of the body, the process of metastasis. Despite advances in surgery and development of additional therapies for the treatment of tumors in the breast, most deaths are caused by the spread of the tumor. Since less than 10% of women have tumors that have completely spread to distant organs when their cancer is first detected, if agents were discovered to block metastasis, great advances could be made in prevention of the most devastating part of the disease.
Conjugated linoleic acid is a component found in some sources of dietary fat. It has been also found to be a potent chemoprotective agent against breast cancer in experimental animals. Although some dietary fats, such as those from plant oils, increased breast cancer while others, such as those from fish oils, reduced breast cancer growth, large changes in the amounts of dietary fat were necessary to achieve those alterations. In contrast, very low dietary concentrations of conjugated linoleic acid can significantly alter experimental breast cancer. Consequently, small alterations in dietary fat consumption, either through natural sources or as a supplement, has a great potential for reducing or preventing tumor metastasis.
In this project, we propose to investigate how conjugated linoleic acid reduces breast cancer by focusing on how it can alter metastasis. This step was selected because preliminary studies indicate that it may be altered by dietary fat. We believe this change may be due to reduction of prostaglandin E2, a fat-based mediator which has been shown to influence metastasis.
From this work, we hope to determine how dietary conjugated linoleic acid functions to reduce or prevent the spread of breast cancer. Although conjugated linoleic acid has not been tested in humans, these experiments in mice should provide a basis for future human studies to reduce the mortality of women diagnosed with the disease.
Novel Anti-vascular Agents for Breast Cancer Therapy
Keith R. Laderoute, Ph.D.
SRI International
Solid human tumors, such as breast cancers, cannot continue growing without a network of blood vessels derived from normal tissue by the process of angiogenesis to supply oxygen and nutrients and to remove waste products. A crisis in the development of a breast tumor occurs when it reaches a size that overwhelms the ability of its blood supply to support further growth. At this point, stressful internal microenvironments develop within the tumor. This localized stress can cause the rise of new and more aggressive cancer cells, and the generation of molecular signals necessary for the growth of new blood vessels to support further tumor growth. Thus, angiogenesis is essential for the survival and further progression of breast cancers to full malignancy. However, the new tumor-associated blood vessel networks have properties very different from blood vessels formed in normal tissues. The unique properties of this tumor-associated vascular lining makes it an attractive target for drug therapy. Our approach targets normal blood vessel endothelial cells within a tumor. Importantly, these cells have not undergone the profound genetic alterations and variability characteristic of tumor cells, and they might be expected to be susceptible to drug treatment.
Recent research has shown that tumor blood vessels can be targeted for disruption or collapse by drugs that disturb the formation of microtubules, structures that are found in all cells and are essential for growth, proliferation, and other vital processes. Vinblastine and paclitaxel (Taxol) are examples of anticancer drugs that damage microtubules in proliferating tumor and vascular cells, but both have toxic side effects. We have identified a novel anti-microtubule drug (BTO-956) that inhibits tumor cell proliferation and suppresses the growth of human breast and ovarian carcinoma implants in mice. BTO-956 has remarkably little normal tissue toxicity, even at very high orally administered doses. Preliminary studies show that our new compound also inhibits the proliferation of human blood vessel cells (i.e., endothelial cells) and antagonizes the formation of new blood vessels in a chicken embryo assay. Taken together, these findings indicate that our compound is both a safe and novel inhibitor of blood vessel growth for treating breast cancer. Our project will investigate the effectiveness of BTO-956 in inhibiting proliferating blood vessel cells using cell culture and animal models of angiogenesis and breast tumor development. In addition, we will explore new ways of formulating and delivering the drug to enhance its potency in breast carcinomas. Such pre-clinical information is essential for determining the value of BTO-956 or its derivatives as well-tolerated experimental drugs for breast cancer therapy, possibly in combination with other therapeutic agents.
Novel Anti-Angiogenic Therapy for Breast Cancer
Francis Markland Jr., Ph.D.
University of Southern California- School of Medicine
In order for a tumor mass to grow to a critical size, it is necessary for it to obtain a blood supply. The process of new blood vessel growth into the tumor is called angiogenesis. Blood vessels in tumor angiogenesis are disorganized ("leaky") compared to the normal vascular system in the body and provide an opportunity for targeted delivery of therapeutic compounds directly to the tumor mass. Contortrostatin (CN) is a protein we have isolated from the Southern copperhead snake venom. It is a potent inhibitor of breast cancer progression. CN binds to cell surface receptors, called integrins, on human breast cancer cells and inhibits growth and metastasis of a breast tumor in laboratory mice. In addition, CN blocks new blood vessel growth into the human mammary carcinoma. Recent studies have also shown that CN indirectly causes apoptosis (programmed cell death) of vascular endothelial cells during angiogenesis. Our goal is to characterize and quantitate the anti-angiogenic activity of CN, investigate the mechanism of its anti-angiogenic action on endothelial cells, and expand the clinical potential of this unique form of therapy.
In the present project we have four interrelated aims. First, we will quantitate the anti-angiogenic activity of CN in human breast cancer cells using immunodeficient mice. For this aim, we will grow small pieces of human skin on mice and inject human breast cancer cells into the skin flap. Using this model system we will be able to study the angiogenic process using immunohistochemistry in a context more closely related to human breast cancer. We will also determine the potential synergistic activity of combining a chemotherapeutic agent with CN therapy. In the second aim, we will examine the mechanism of CN activity towards specific integrin cell adhesion receptors using human endothelial cells. Our third approach is utilize tumor vessel "leakiness" in order to develop a liposome-based delivery system (lipid encapsulation of CN) for intravenous administration of CN to mice bearing experimental breast tumors. Our final aim is to chemically synthesize novel CN-like peptides that mimic the integrin-binding site. We will use these compounds in the liposome-based delivery system. To summarize, we will develop further information on CN mode of action, better means of delivery to sites of tumor growth, and new drug formulation based on the active site.
We believe that the major strengths of CN as a breast cancer therapeutic are its ability to inhibit both angiogenesis and to directly block tumor cell metastasis. We are focusing our work on developing this therapeutic using human cells and tissue whenever possible in order to maximize the potential translation of our findings to the therapy of human breast cancer.
Breast Cancer Immunotherapy Using CD4+ T Lymphocytes
Malcolm Mitchell, M.D.
University of California, San Diego- School of Medicine
Our interest is to develop and test novel strategies to mobilize and direct a person’s own immune system in combating breast cancer. In this approach we isolate immune cells from the patient and stimulate them with purified proteins or protein fragments from the cancer cells. The critical factors to be resolved are the type of lymphocyte to be used, the specific cancer antigen providing the most vigorous response, and the strategy for reintroducing the lymphocytes and supplementary cells back to the patient. We speculate that CD4+ T helper cells, after exposing the cells to fragments of the HER-2/neu oncogene protein, will cause measurable destruction of breast tumor cells. Furthermore, we believe that this will improve the survival for patients whose tumors carry HER-2/neu. The use of cytotoxic T lymphocytes (CTL) has been previously emphasized, but we predict that using treatment with T helper cells, will improve the outcome over CTL alone.
We will pursue three specific aims. First, we would like to determine the major protein fragments of HER-2/neu that effectively stimulate T helper cells. For this purpose, we will try to stimulate normal T cells from normal individuals or breast cancer patients who do not have high levels of HER-2/neu in their tumor, and thus have never encountered these fragments. We will first isolate "professional" antigen-presenting cells called "dendritic cells" from the blood. Two complementary strategies will be used to "load" the dendritic cells, so that they can stimulate the T helper cells. Then, we will try to immunize T helper cells with HER-2/neu protein fragments extending from the cell membrane and located furthest from the cell. This portion is least similar to normal growth factors and may generate a specific anti-tumor response with little or no toxicity to normal tissues. Our next aim will be to study whether T cells from patients with high levels of HER-2/neu in their tumors can be directly immunized against these protein fragments in the laboratory. Our goal is to modify the conditions to generate T helper cells to aid a cellular tumor-rejection response rather than antibody production. In our final aim we will investigate producing sufficient numbers of specific T helper cells in the laboratory for therapy of patients, with a goal to produce them within a clinically useful 4 to 6 week time frame.
If these experiments are successful, they will permit immunotherapy with CD4+ T cells to be used in the treatment of patients with high levels of HER-2/neu in their tumors. This would add cellular immunotherapy to what has heretofore been a chemotherapy-oriented approach to breast cancer. We hope to benefit this particular subset of breast cancer patients whose prognosis has been among the poorest of those with this disease.
Immunoliposomes for Cytotoxic Anti-Angiogenesis Therapy
John W. Park, M.D.
University of California, San Francisco- School of Medicine
Solid tumors, such as breast cancer, constantly require additional blood supply in order to grow and spread. Tumors use a process known as "tumor angiogenesis" to cause new blood vessels to form. Recently, a number of new treatments have been developed to suppress tumor angiogenesis, for example by inhibiting the growth of endothelial cells, the cells that line blood vessels. While promising, these treatments may not be the best way to target tumor angiogenesis. These treatments typically work by temporarily suppressing endothelial cell growth, but must be given frequently and over long time periods to continue to work. An alternative strategy is to target and kill the endothelial cells involved in tumor angiogenesis, thus doing rapid and irreversible damage to the tumor. Indeed, it appears that some types of chemotherapy already in use work in part by this mechanism, although chemotherapy also directly kills cancer cells (as well as many normal cells).
We propose to develop a novel strategy to target tumor endothelial cells using immunoliposomes (ILs), a combination of liposomes and antibodies. Liposomes are small, hollow particles of fat that can be used to carry large numbers of potent anti-cancer drugs. Antibodies, which can recognize and bind to cancer cells, can be physically linked to liposomes to produce immunoliposomes. We have previously developed anti-HER2 immunoliposomes (immunoliposomes that target the HER2 protein, which is associated with aggressive tumor growth), and have shown that these immunoliposomes efficiently deliver chemotherapy to tumors in animals, resulting in a treatment that is more effective as well as safer than conventional chemotherapy; this promising treatment is proceeding to clinical trials. In order to target tumor endothelial cells, we will construct immunoliposomes containing an antibody that recognizes a protein called Flk-1. Flk-1 plays a critical role in tumor angiogenesis, serving as the receptor for another protein called vascular endothelial growth factor (VEGF). Flk-1 is produced in overabundance by tumor endothelial cells, and can therefore be used as a target for immunoliposomes. We will prepare immunoliposomes that recognize Flk-1, load them with various chemotherapy drugs, and test them against target cells in the test tube and in relevant animal models.
This strategy is designed to kill tumor endothelial cells, while sparing other endothelial cells and normal tissues. This selectively killing of tumor endothelial cells is based on two separate principles. First, immunoliposomes will deliver chemotherapy to tumor endothelial cells, which have high amounts Flk-1, and not to normal cells. Second, chemotherapy itself kills dividing or growing cells; hence, tumor endothelial cells will be susceptible, but not most normal cells, including normal endothelial cells. A further advantage of this strategy is that it will allow us to test a battery of different chemotherapy drugs inside immunoliposomes. This will lead to a better understanding of the susceptibility of tumor endothelial cells to chemotherapy drugs, which has been hard to sort out because of the many effects of conventional chemotherapy without targeted delivery.
This proposal is highly innovative, as it seeks to develop immunoliposomes carrying chemotherapy as a targeted and lethal therapy against tumor angiogenesis, thus providing a novel strategy unlike existing approaches. It is multidisciplinary, drawing from lipid biochemistry, molecular biology, antibody engineering, cell biology, cancer biology, tumor immunology, pharmacology and pharmacokinetics, drug development, and clinical oncology. Finally, it is translational, as these studies may directly lead to a new and useful treatment for breast cancer. Such a treatment could be superior to existing therapies, and is likely to be substantially less toxic than conventional chemotherapy.
New Investigator Awards
Heregulin-Specific Diphtheria Toxin as a Cancer Therapy
Gordon Louie, Ph.D.
The Salk Institute for Biological Studies
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.
Antibody-IL-2 Fusion Protein for Breast Cancer Immunotherapy
Joseph Lustgarten, Ph.D.
Sidney Kimmel Cancer Center
Cancer immunotherapy relies on the ability of specialized white blood cells called cytolytic T lymphocytes (also called killer T cells) to eradicate tumor cells. These T cells recognize and destroy damaged or foreign cells that make proteins (antigens) that are not produced in an individual’s normal cells. However, killer T cells that specifically eliminate tumor cells are not frequently found in cancer patients. Our goal is to manipulate the immune system to produce killer T cells that specifically recognize and eliminate breast tumors. We will focus on three antigens that are found in breast tumors: Her-2/neu, Her-3, and Her4. Her-2/neu and Her-3 are expressed on 25-35% of all breast cancers and are associated with poor prognosis and low survival. It has been proposed that elimination of tumor cells expressing Her-family receptors could improve the effectiveness of drug treatment and prolong survival time.
We have recently generated a fusion protein that is a combination of an antibody and a cytokine (IL2 - a compound necessary for obtaining a vigorous immune response). This fusion protein is targeted to the Her-2/neu protein. We have observed that T cells in the presence of this fusion protein are able to kill target cells in a manner that is independent of the their normal route through the T cell receptor. It is our hypothesis that a treatment protocol using a combination of functional T cells from cancer patients with the anti-Her-2/neu fusion protein will improve the efficiency of immunotherapy for breast cancer. We are trying to recreate a pre-clinical mode that is analogous to the situation in breast cancer patients to test the efficacy of this strategy to control the growth of breast tumors. We will use a mouse model to test the ability of T cells from patients with breast cancer to eliminate the tumor from breast carcinomas cell lines expressing Her-2/neu. In addition, we will generate another fusion protein composed of the ligand for Her-3 and Her4. This work will assist in the development of a new strategy to reinforce the body's defenses against existing tumors.
Postdoctoral Fellowship Award
A New System for Breast Cancer Drug Delivery
Daryl Drummond, Ph.D.
California Pacific Medical Center- Geraldine Brush Cancer Research Institute
Drug delivery, specificity, and stability are critical issues in the application of new therapies to treat breast cancer. Our interest is in the development and application of lipid encapsulated (liposome-mediated) delivery of anti-cancer drugs. Liposome approaches have the advantage of improved circulation times in the blood, protection of the drug within the lipid particle, and avoiding general tissue penetration due to size considerations. Recent advances in liposome technology have led to the development of small liposomes coated with polyethylene glycol (PEG). These have a relatively long half-life in the circulation and a limited tissue distribution. Importantly, these PEG-coated liposomes are able to localize in tumors as a result of increased local retention and release. This is because in the tumors, the blood vessels are disorganized or 'leaky'. Nevertheless, there still remains a critical issue of targeting drug-containing liposomes directly to the cancer cells in a tumor mass. Such tumor targeting would increase drug accumulation specifically at or in the breast cancer cells, which would eliminate or reduce non-specific toxicities typically associated with chemotherapy. The focus of this project is to explore this aspect of the technology, so that an anti-tumor drug can not only be delivered to the cell, but also released inside the cell itself.
Our drug delivery project has two elements. First, the chemotherapeutic drug (doxorubicin) is enclosed in a liposome formulation that includes antibody fragments at the surface directed at the Her-2 growth factor receptor. Her-2 is found on about 30% of the more aggressive breast cancers, and the use of an antibody makes it an 'immunoliposome'. We believe this approach will effectively target the drug directly to the most cancerous cells in the tumor. However, the drug must still enter the cell to work. Fortunately, antibody-containing particles are efficiently internalized into cells and contained within internal structures called endosomes. Thus, the next critical issue in this project is to construct and test liposomes that become unstable in endosomes and release their drug upon exposure to the local acidic environment. This would result in an increased killing effect inside the cell, and an enhanced therapeutic effect. Our specific methodology will be to use acid-sensitive protecting groups and cross-linking reagents, which should allow us to prepare liposomes that are both stable at neutral pH (plasma conditions) and become destabilized in endosomes. We will ultimately test our approach in Her-2 tumor bearing mice after the issues of liposome formulation are resolved using cells in culture.
Researchers have been able to kill breast cancer cells in the laboratory with drugs for many decades, and novel therapies are continuously being developed. Our goal is to overcome the gap between our laboratory success and our clinical ability to deliver drugs directly to the cancer to make a meaningful difference to the patients' quality of life and survival.
