Chimerization of an anti-CD19 antibody for the Therapy of Cancer
CD19 is a 95 kDa membrane glycoprotein that is 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. Drs. Ellen Vitetta and Xiaoyun Liu and Immunology graduate student Lydia Wu have chimerized the murine HD37 monoclonal antibody (MAb) both divalent and tetravalent forms and are testing them for their anti-tumor activity both in vivo and in vitro.
Tetravalent Monoclonal MAbs (TetraMabs)
Drs. Ellen Vitetta and Maria-Ana Ghetie 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. Drs. Victor Ghetie, Laurentiu Pop and Xiaoyun Liu 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. Drs. Victor Ghetie, Laurentiu Pop and Xiaoyun Liu have genetically engineered chimeric IgG 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. Drs. Victor Ghetie, Laurentiu Pop and Xiaoyun Liu have genetically engineered chimeric IgG to decrease its serum half-life and are preparing immunoconjugates of these recombinant immunotoxins to test in mice xenografted with human tumors.
Modulation of Multidrug Resistance (MDR) by MAb therapy
Drs. Maria-Ana Ghetie, Iliodora Pop. Laurentiu Pop and Ellen Vitetta are studying MAb-mediated modulation of P-glycoprotein (P-gp) by translocation of other specific membrane markers of tumor cells (e.g. CD19, CD20, etc.) into low-density-insoluble membrane compartment (lipid rafts). Two MAbs (HD37 anti-CD19 and Rituximab anti-CD20) are currently under study, in an attempt to overcome the multidrug resistance induced by chemotherapy in patients with B-lymphoma. Recently three new MDR cell lines were obtained by Dr. Iliodora Pop following 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. These cells are currently used to test the therapeutic efficacy and mechanism of action of HD37 and Rituximab 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. Joan Smallshaw and Ellen 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 is 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 Immunology of the Gulf War Syndrome
In 1990 approximately 700,000 troops were sent to the Middle East for Operation Desert Storm; today as many 100,000 of them suffer from a collection of chronic symptoms termed Gulf War Syndrome (GWS). Soldiers suffering from GWS were subjected to numerous vaccinations given during a period of high stress as well as possible chemical exposures. To date, the possibility of immune dysregulation in these soldiers has not been thoroughly investigated. Dr. Vitetta and immunology graduate student Praveena Selvaduray plan to profile the immune system of mice that have been intensely vaccinated and/or exposed to battlefield chemicals in order to determine if immune dysregulation and brain damage occur.
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. Studies by Immunology graduate student Andrew Bitmansour 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 vaccine.
Hepatitis C Virus (HCV) currently infects 3% of the population, or 170 million people. Although 15% of these will clear the infection, the remaining 85% will develop chronic infection. There is no vaccine for HCV due mostly to virus mutation, which in time enables the virus to outrun the host immune system. Clearly, it is highly desirable to develop a vaccine to induce sterilizing immunity. Doing so will require identifying conserved epitopes on the virus common to all quasispecies which, when injected, induce the formation of protective antibodies against many viral strains. Drs. Vitetta and Kodadek along with Biomedical Engineering graduate student Allison Carroll 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 generate antibodies against the native protein. Housed on microarrays, these peptoids will be screened with sera containing known antibodies. The peptoids that bind to native-protein-reactive antibodies will be linked to conventional carrier proteins and injected in adjuvant to induce immunity. By systematically studying a well-defined system of a polyclonal antibody and a protein antigen, each variable and parameter will be tested and optimized.
Antibody-conjugated carbon nanotubes for selective photothermal ablation of human tumors.
Drs. Vitetta, Marches and Biomedical Engineering graduate student Pavitra Chakravarty along with their collaborator, Dr. Rockford Draper at UT Dallas are developing antibody-coupled carbon nanotubes (CNTs) to target cancer cells. CNTs have the potential to carry various agents across cell membranes, including genes, proteins and drugs. The strong absorbance of NIR light can be used for thermal ablation of the tumor cells while sparing normal tissue. We have prepared novel therapeutic agents that consist of CNTs (1/100,000 the diameter of a tumor cell) coupled to tumor-specific monoclonal antibodies (MAbs). The therapeutic potential of CNT-MAb conjugates relies on the ability of CNTs to absorb electromagnetic radiation (near-infrared, microwave, and radio frequency) and convert the energy to heat. Living tissue absorbs radiation poorly in areas of these spectral regions, which makes it possible to heat CNTs within living tissue. Dispersion of CNTs and attachment of tumor-specific antibodies will be achieved by methods that do not interfere with the critical optical and electronic properties. CNTs targeted to tumor cells can mediate the selective thermal ablation of the cells.
An Anti-CD54 Antibody (UV3) is an Effective Anti-Tumor Agent.
Dr. Vitetta and her former fellows and students have developed and tested a MAb (UV3) against human multiple myeloma cells. 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. Immunology graduate student, Kimberly Brooks has now demonstrated that UV3 also slows the growth of human uveal melanoma, pancreatic, 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 by Dr. Joan Smallshaw for use in clinical trials. The chimeric antibody will be further tested in patients in collaboration with Abiogen Pharma.
Detection of genetic changes associated with cancer progression
Dr. Jonathan Uhr and his group 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 which 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% 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 at 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. Dr. Jonathan Uhr’s method for detecting CTCs has isolated tumor cells in more than 35% of patients with breast cancer dormancy for the first time. These patients have a very low rate of recurrence. Unexpectedly, the CTCs have a very short half-life in the blood, about 1-2 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.
Clinical Trials
Currently, six clinical trials have been approved by the FDA and are in various stages of completion.
1. The use of an anti-CD25 immunotoxin (IT) to eliminate alloreactive T cells. Three clinical trials using anti-CD25 IT to eliminate alloreactive T cells have been initiated with 3 different investigators: Dr. Fisher in France, Dr. Brenner in Houston, and Dr. Barrett at the NIH. All three trials use an anti-CD25 IT to deplete alloreactive T cells prior to stem cell transplantation for leukemia. The objectives of the protocols were to determine whether depletion of alloreactive cells 1) depleted cells that were able to mediate graft vs. leukemia in vivo and 2) whether this led to more rapid immune recovery. The trials at the three sites are similar but are open to different types of patients and use different methods of in vitro allodepletion.
a. The trial in France has been completed and published. In summary, the trial was a success in that no severe GVHD was observed in 20 patients.
b. The trial at the NCI has been completed. Results of the trial showed a marked improvement over historical controls. The results of the study have been published.
c. The trial in Houston has accrued 20 patients. One patient had a Grade IV skin event that was either a drug reaction or GVHD. The results have been published but the trial is continuing.
2. ITs against CD19 and 22 (Combotox) are effective in killing Pre-B-ALL from pediatric patients. We have demonstrated that this strategy works ex vivo and have recently completed a Phase I multi-center clinical trial to determine whether this strategy is safe in relapsed patients. This study was highly successful with 3 complete responses and an overall response rate of 53%.
A Phase 2 study investigating a schedule of 6 doses per cycle rather than 3 has been approved and will start when funding has been obtained.
Because of the success in pediatric ALL, we have begun a trial in adult ALL. Four patients have been enrolled at UTSWMC, six at Albert Einstein in New York and two at MD Anderson in Houston.
3. A Pilot Clinical Trial of a Recombinant Ricin Vaccine in Normal Humans. We have generated a recombinant (r) RTA with two amino acid substitutions that disrupt its ribotoxic site (Y80A) and its VLS-inducing site (V76M). This mutant rRTA (named RiVax) was expressed and produced in E. coli and purified. When RiVax was injected intramuscularly (IM) into mice it protected them against a ricin challenge of 10 LD50s. Preclinical studies in both mice and rabbits demonstrated that Rivax was safe. Based on these results, we have now conducted a pilot clinical trial in humans under an investigational new drug application (IND) submitted to the FDA. In this study, 3 groups of 5 normal volunteers were injected three times at monthly intervals with 10, 33 or 100 mg of RiVax. The vaccine was safe and elicited ricin-neutralizing antibodies in one of five individuals in the low dose group, four of five in the intermediate dose group, and five of five in the high dose group.
A second clinical trial with RiVax with alum will start before the end of 2007. The objective is to determine whether the addition of alum results in higher serum concentration of anti-RTA antibody and/or longer lasting serum levels of antibody.
4. Depletion of regulatory T cells with an immunotoxin directed against CD25 (RFT5-dgA).
a. Melanoma: We are carrying out a study with Dr. Rosenberg at the NCI in patients with metastatic melanoma using RFT5-dgA. RFT5-dgA is being used to eliminate CD25+ T reg cells which protect tumors by preventing recognition of their tumor-associated antigens. Six patients have been treated and T regs were depleted by greater than 75%.
b. CTCL: A protocol is being written to conduct a Phase II trial with RFT5-dgA in patients CTCL with Dr. Simrit Parma at UTSWMC. The objective of the trial is to deplete both tumor cells and T regs. It will be submitted to the IRB by the end of October and the trial will be carried out at our institution.
