Members of the Cancer Immunobiology Center are involved in a broad range of significant research studies related to the body’s immune system and its interaction with cancer cells. They also study tumor dormancy, targeted therapy of cancer, and develop and test novel vaccines.
Following are several of the recent research projects in our laboratories.
Chimerization of an Anti-CD19 Antibody for the Therapy of Cancer
CD19 is a 95 kDa membrane glycoprotein that’s involved in signal transduction in B lymphocytes. CD19 appears on normal B cells early in ontogeny and is also expressed in virtually all malignant B cell lymphomas. The clinical utility of CD19 as an immunotherapeutic target was recently demonstrated by using transgenic mice expressing human CD19. Early studies performed in our Center demonstrated that HD37, an anti-CD19 antibody, can inhibit the growth of human B-cell tumor lines in vitro and in SCID mice by inducing cell cycle arrest. HD37 antibody can also overcome the resistance of tumor cells to chemotherapy. We have chimerized the murine HD37 monoclonal antibody (MAb), both divalent and tetravalent forms, and have evaluated them for their anti-tumor activity both in vivo and in vitro.
Tetravalent Monoclonal MAbs (TetraMabs)
We have demonstrated that monoclonal antibodies (MAbs) that have little or no signaling activity (i.e., anti-CD19, CD20, CD21, CD22, and anti- HER-2) can become potent anti-tumor therapies when they are converted into IgG-IgG homodimers. The homodimers exert anti-tumor activity by signaling G0/G1 arrest or apoptosis, depending on which cell surface molecule they bind. We have converted these into recombinant, chimeric TetraMAbs. The testing of these chimeric TetraMAbs for their anti-tumor activity both in vivo and in vitro is ongoing.
Increasing the Half-Life of IgG In Vivo
The anti-tumor efficacy of MAbs correlates with in vivo half-life, which is regulated by the interaction between the Fc portion of IgG and the neonatal Fc receptor (FcRn) expressed on endothelial cells. MAbs with longer half-lives should be more effective in treating cancer. We have genetically engineered chimeric IgGs to increase their serum half-life and hence improve their use as therapeutic agents. Preclinical studies of these "engineered" chimeric MAbs are ongoing.
Decreasing the Half-Life of IgG In Vivo
The therapeutic window of MAbs conjugated to either toxin or radioisotope (immunoconjugates) correlates to their serum persistence. Immunoconjugates with shorter half-lives have fewer side effects and increased therapeutic windows. These constructs can become more potent cancer therapies. We have genetically engineered chimeric IgG to decrease its serum half-life and have prepared immunoconjugates of these recombinant immunotoxins and tested them in mice xenografted with human tumors.
Modulation of Multidrug Resistance (MDR) by Inhibition of mTOR in Combination with MAb Therapy
We are studying the modulation of P-glycoprotein (P-gp) by targeting mTOR (the mammalian target of rapamycin), which is a critical effector in cell-signaling pathways that are commonly dysregulated in human cancers. Rapamycin, an mTOR inhibitor, is used currently under study in an attempt to overcome the multidrug resistance (MDR) induced by chemotherapy in patients with B-lymphoma. Recently three new MDR cell lines were generated by drug selection. The new MDR-cells stably express P-gp both in vitro and in vivo; their P-gp pump is active and can be blocked by verapamil and valspodar (PSC 833). These cells were induced drug-resistant by stepwise exposure to increasing concentrations of vincristine (VCR) but they became resistant to other drugs, too; therefore, a typical MDR phenotype was induced.
Targeting mTOR with rapamycin can block the expression and activity of P-gp and chemosensitize all three drug-selected, P-gp+ MDR cell lines. Moreover, the combination of mTOR inhibitor with UV3, an anti-tumor monoclonal antibody developed in Dr. Vitetta’s center that targets CD54 (ICAM-1) molecule that is over expressed in MDR B cell lymphoma and can act in synergy with mTOR inhibitors. The new MDR-cells are currently used to test the therapeutic efficacy and mechanism of action of Rapamycin +/- UV3 in SCID mice with MDR human lymphomas.
RiVax, a Recombinant Ricin Subunit Vaccine, Protects Mice against Ricin Delivered by Gavage or Aerosol
Ricin is a plant toxin that is classified by the CDC as a level B biothreat, due to it being easily obtainable in massive amounts in crude form as a byproduct of castor oil production. The estimated lethal dose of ricin in humans is 1-25 ug/kg, depending upon the route of exposure. Drs. Smallshaw and Vitetta have developed a recombinant ricin A chain vaccine (RiVax), which contains mutations in both known toxic sites, i.e., ribotoxicity and vascular leak-inducing ability, making it nontoxic at doses at least 800 times the immunogenic dose.
RiVax without adjuvant given intramuscularly protected mice against intraperitoneally administered ricin. Furthermore, the vaccine without alum was proven safe and immunogenic in human volunteers; immune serum from the volunteers was capable of neutralizing ricin in vitro and protected mice from a lethal intraperitoneally administered ricin challenge. Since ricin poisoning in humans is most likely to occur following accidental or intentional contamination of food, water, or air, we investigated whether RiVax administered intramuscularly would protect against ricin given by either route. They have determined the dose of ricin that’s lethal when delivered by aerosol or ingestion and shown that intramuscular vaccination protects mice in a dose-dependent manner against ricin delivered by either route.
RiVax also protects against aerosol-induced lung damage as determined by histology and lung function tests. The vaccine was also shown to work even better in all of the mouse challenge models with the addition of the adjuvant, aluminum hydroxide (‘alum’). Continuing studies have demonstrated that intradermal immunization enhances the immunogenicity of the vaccine in mice, making it more effective at low doses. A second human trial is under way using an alum formulation of the vaccine.
The Role of Regulatory T Cells (Tregs) in Tumor Dormancy
Cancer dormancy is a clinical state in which tumor cells are present in an individual but do not expand. Patients treated for various lymphomas, melanoma, or breast cancer can have remissions (dormancy) that last for many years followed by relapses. Relapses occur following a breach in dormancy when tumor cells re-acquire their ability to grow and seed sites in the periphery. Our lab developed the first well-defined model of dormancy: the B cell lymphoma (BCL1), a mouse model for prolymphocytic leukemia. Although there has been no clear demonstration of the role of cell-mediated immunity on the induction or maintenance of dormancy, examination of T cell subsets showed that CD8 T cells in collaboration with cytokines maintained dormancy after it was induced by anti-Idiotypic antibody.
Interestingly, CD4 T cells suppressed the activity of the CD8 T cells. We now know that CD4 cells contain a subset of immunosuppressive regulatory T cells (Tregs). Increased frequencies of Tregs in cancer patients have been reported. Therefore, it is important to determine whether Tregs are responsible for relapses of dormant tumors. Ongoing studies will determine whether Tregs can induce a breach in established dormancy and whether their depletion will promote anti-tumor responses and cure or prolong dormancy. Understanding the function of T regs in tumor development and dormancy should provide key insights into new strategies for prolonging remissions or curing patients with dormant cancers. Since primary and relapsed cancers occur most frequently in the elderly, insights into cancer dormancy will help design better therapies to prolong the life our aging population.
The Development of Peptoid-Based Vaccines
We are developing a new platform for identifying key B cell epitope mimetics in the form of synthetic peptides, called peptoids. They will determine whether peptoid-carrier conjugates can be used to safely generate antibodies against the native protein. Large libraries of peptoids, synthesized on beads, will be screened with monoclonal antibodies containing known to neutralize a broad panel of virus quasispecies. The peptoids that bind to these antibodies and that can be blocked by the native protein will be linked to conventional carrier proteins and injected with adjuvant to induce an immune response, which will then be evaluated for cross-reactivity with native antigen.
By systematically studying well-defined pairs of monoclonal antibodies and their corresponding protein/peptide antigens, such as anti-FLAG and FLAG antigen, each variable and parameter of the screening procedures will be tested and optimized for use with monoclonal antibodies. This is being done by a biomedical engineering student. This approach to vaccine development could be applied to any pathogen for which there exists neutralizing antibodies. We are currently applying this technology to vaccines for Hepatitis C Virus (HCV), West Nile Virus, and HIV.
Antibody-Conjugated Carbon Nanotubes (CNTs) for Selective Photothermal Ablation of Human Tumors
Along with our collaborator, Dr. Rockford Draper at UT Dallas, we are developing monoclonal antibody (MAb)-coupled CNTs (1/100,000 the diameter of a cell) to target and photothermally ablate cancer cells. The ultimate goal of this project is to develop a noninvasive method for selective ablation of primary tumors using the thermal energy generated by irradiation of cell-bound MAb-CNTs. The selectivity of heating is provided by the tumor-specific MAbs covalently attached to CNTs able to convert light or magnetic energy to thermal energy, and by local thermal energy generated by exposure of tumor site to external harmless 808 nm near infrared light, which is routinely used in various topic dermatological treatments.
This approach will create virtual “nanoheaters” that will selectively kill tumor cells, leaving unaffected the healthy tissue. In vitro studies have demonstrated a proof-of-concept of the specificity and efficacy of this approach. We hope to further develop and test these nanoparticles to treat primary tumors as an alternative to surgery.
Our Anti-CD54 Antibody (UV3) Is an Effective Anti-Tumor Agent
We have developed and tested an MAb (UV3). UV3 recognizes the cell adhesion molecule CD54 (ICAM-1), which is elevated on many human tumors. When human myeloma or lymphoma cell lines were grown in SCID mice, UV3 was highly effective at prolonging their survival. UV3 also slows the growth of human uveal melanoma, pancreatic cancer, non-small cell lung, breast, and prostate tumors in SCID mice. In addition, UV3 appears to be as effective as the chemotherapeutic agent, gemcitabine. A chimeric (partially human) version of UV3 has already been developed.
Dual Targeting of Angiogenesis and Tumor Cells in a Xenograft Model of Human Pancreatic Cancer
In collaboration with Dr. Rolf Brekken, Division of Surgical Oncology and Department of Pharmacology at UT Southwestern, we are interested in targeting both human pancreatic tumors cells and vasculature in mouse xenografts of human pancreatic, breast, and breast tumors. Our strategy is to combine an anti-angiogenic monoclonal antibody (MAb) (r84) [that recognizes vascular endothelial growth factor (VEGF) that is secreted by both human tumors and mouse stroma] with an anti-human tumor MAb that targets human CD54 MAb (UV3) and kills tumor cells by harnessing natural killer cells and macrophages.
Previous work has shown that each MAb when administered alone slows the growth of pancreatic tumor cells in SCID mice. In addition, UV3 kills pancreatic cancer cells as effectively as the current treatment for pancreatic cancer, gemcitabine, and that the combination of UV3 + gemicitabine have even better anti-tumor activity. They are working to optimize the use of these two MAbs alone and in combination with chemotherapy (gemcitabine) to inhibit the growth of both primary and metastatic pancreatic tumors in SCID mice.
Detection of Genetic Changes Associated with Cancer Progression
We have developed a highly sensitive test for detecting, enumerating, and characterizing carcinoma cells in the blood. The test can detect one such cell in 108 leukocytes. The circulating tumor cells (CTCs) can be immunophenotyped and analyzed genetically by FISH to follow genetic changes. There is no other way to accomplish this because repeated invasive procedures such as biopsies are unacceptable. The first major achievement using this technique is to challenge the current dogma involving HER-2 gene amplification. This genetic lesion in breast cancer indicates a very poor prognosis. However, there is an anti-HER-2 antibody called Herceptin that can reverse the unwanted effects of the amplification. The dogma is that amplification occurs early and if it is not present in the primary tumor then the patient will remain HER-2 negative.
We have found that a minimum of 30 percent of patients with progressive cancer acquire HER-2 gene amplification in their CTCs. Four of these patients had Herceptin added to their therapy and two had significant responses, one a complete response. Since targeted therapy is the wave of the future, it is critical to be able to detect genetic changes associated with cancer progression that call for a particular targeted treatment.
Mechanisms Underlying Cancer Dormancy in Humans
Cancer dormancy refers to a clinical phenomenon in which cancer recurs a very long time after removal of the primary tumor. It is seen commonly in breast and kidney carcinomas, melanoma, and non-Hodgkin's lymphoma. Circulating tumor cells (CTCs) have been isolated tumor cells in more than 35 percent of patients with breast cancer dormancy. These patients have a very low rate of recurrence. Unexpectedly, the CTCs have a very short half-life in the blood, about one to two hours. Therefore, they must be constantly replenished by replication of tumor cells, presumably in micrometastases. Hence, replication is precisely balanced by cell death.
Characterization of the intrinsic mechanisms that allow such individuals to control their cancer without the use of pharmacologic agents may lead to the discovery of new anti-tumor approaches. The results emphasize further that cancer is frequently a chronic disease that should be controlled rather then treated with large doses of cytotoxic agents in order to eradicate all tumor cells.