RNA-based Immunotherapy Targeting Antigens Unique To Brain Tumor Stem Cells
John H Sampson, Associate Professor
Surgeryduke University
Grant 1R01CA135272-01 from National Cancer Institute, IRG: ZRG1
Abstract: A subset of cells in glioblastoma multiforme (GBM) has been identified that enjoy a unique capacity to regenerate tumors. These brain tumor stem cells (BTSC) can be segregated by the neural stem cell marker, CD133, and are widely believed to be the cells responsible for resistance to conventional therapies. An effective means of specifically eliminating these cells may reduce the need for intensive and non-specific conventional therapy and lower the risk of tumor recurrence. EGFRvIII is a tumor-specific mutation found on BTSC. We have successfully targeted EGFRvIII using a peptide vaccine that allowed rapid translation to an ongoing Phase III trial. EGFRvIII expression is heterogeneous, however, and the recurrence of EGFRvIII-negative tumors suggests that BTSC can rely on other oncogenic pathways. While our data suggests that targeting tumor-specific mutations in BTSC may be important, few highly-conserved tumor-specific mutations like EGFRvIII will be identified and antigen defined vaccine approaches will ultimately be limited. Dendritic cells (DCs) loaded with amplified total tumor RNA is an innovative strategy to induce cellular and humoral antitumor immune responses. Although CD133(+) BTSC are a minority subpopulation of GBM that cannot be reliably isolated or propagated in sufficient quantities to serve as an antigen source for human vaccination protocols, we have been able to reproducibly amplify the RNA content from as few as 500 sorted CD133(+) tumor cells to generate RNA libraries sufficient for clinical scale DC-based vaccination. In order to focus the immunologic response on antigens preferentially or uniquely expressed within BTSC and limit the potential for autoimmune reactivity against shared antigens expressed in normal cells, we will evaluate approaches to enrich for antigens preferentially or uniquely expressed in BTSC by using full length cDNA affinity based substractive hybridization or an innovative strategy that leverages the ability of the DNA mismatch binding protein, MutS, to isolate cDNAs that contain tumor-specific mutations. These various preparations will be evaluated for differential toxicity and efficacy in an inbred transgenic murine malignant astrocytoma model, in which a subpopulation of CD133(+) tumor cells with BTSC qualities have been identified and CD8(+) and CD4(+) epitopes have been found. If efficacy is seen, the least toxic strategy will be translated into a Phase I study within the context of our existing clinical trial platform. Treatment for malignant primary brain tumors, which are the most common cause of death among children and account for more deaths in adults than melanoma, currently represents the most expensive medical therapy per quality-adjusted life-year saved currently provided in the United States. A subset of malignant primary brain tumor cells (BTSCs), called brain tumor stem cells, enjoy a unique capacity to regenerate tumors and to resist conventional therapies. In this proposal we will see if targeting antigens preferentially or uniquely expressed by BTSCs will enhance the efficacy and reduce toxicity of immunotherapy
Project start date: 2008-08-01
Project end date: 2013-05-31
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Grants awarded to John H Sampson
Effect On IL-2R Antibody On Regulatory T-cells In Patients With Malignant Gliomas
John H Sampson, Associate Professor
Surgeryduke University
2200 W. Main St.
durham, Nc 27705
Grant 1R21CA132891-01 from National Cancer Institute, IRG: ZRG1
Abstract: The immune system has the potential to eliminate altered neoplastic cells with incredible specificity. A consistent in-frame deletion in the extra-cellular domain of the epidermal growth factor receptor (EGFRvIII) represents a truly tumor-specific target amenable to immunotherapeutic attack. Our multi-institutional Phase II study demonstrated that vaccination with an EGFRvIII-specific peptide in patients with newly-diagnosed glioblastoma multiforme (GBM) induces potent T- and B-cell immunity, produces nearly complete radiographic responses in all patients with residual tumor, and universally eliminates EGFRvIII-expressing cells. Recurrent tumors, however, continue to express wild-type EGFR suggesting that the immune response is specific, but productive intra-molecular cross-priming against other potential tumor-associated antigens is incomplete. We believe that productive extension of such secondary immune responses is hindered by the presence of regulatory T-cells (TRegs). We have recently shown that TRegs are disproportionately represented within the peripheral blood and tumors of patients with GBM and serve to induce a state of profound, but reversible, immunosuppression. TRegs are characterized by constitutive expression of the high affinity interleukin (IL)-2 receptor (IL-2R1)(CD25) and are uniquely dependent on IL- 2R1 signaling for their function and survival. Using our spontaneous murine glioma model, we have demonstrated that treatment with an antibody that blocks IL-2R1 signaling functionally inactivates and eliminates TRegs without inducing autoimmune toxicity. Our pre-clinical studies have shown that these unarmed IL-2R1-specific antibodies when given in vivo to mice during recovery from lymphopenia induced by therapeutic temozolomide (TMZ) are capable of not only functionally inactivating TRegs, but also dramatically enhance vaccine-induced immune responses. Daclizumab, an existing, humanized, unarmed IL-2R1-specific antibody, functions identically to the antibody used for TReg inactivation studies in mice. We hypothesize that daclizumab therapy during the recovery from therapeutic TMZ-induced lymphopenia in patients with newly-diagnosed GBM will inhibit the functional recovery of TRegs, enhance immune responses against an EGFRvIII-targeted vaccine, and promote productive cross-priming without the induction of deleterious autoimmunity. Because NK cells also express CD25 and may be potent activators or inhibitors of innate and antigen-specific immune responses, the effect of daclizumab on NK cells will also be assessed
Project start date: 2008-01-04
Project end date: 2009-12-31
CMV-SPECIFIC ANTI-TUMOR IMMUNE RESPONSE IN ASTROCYTOMAS
John H Sampson, Associate Professor
Duke University 2200 W. Main St. Durham, Nc 27705
Grant 5P50CA108786-040003 from National Cancer Institute, IRG: ZCA1
Abstract: Malignant gliomas (MGs) are univerally fatal, and effective therapy is limited by collateral damage to normal tissue. Immunotherapy directed against tumor-specific antigens may allow neoplastic cells to be targeted more precisely, and our dendritic cell (DC)-based vaccinations targeting of a mutated tumor-specific epidermal growth factor receptor have produced immunologic and radiographic responses in patients with MGs. The discovery that MGs, but not surrounding normal brain, serve as a refuge for Cytomegalovirus (CMV) reactivation provides an unparalleled opportunity to subvert, as a tumor-specific antigen, the highly immunogenic CMV protein, pp65. Despite the numerous advantages of targeting CMV antigens in MGs with DC-based vaccines, a number of factors clearly limit ant/tumor immune responses in these patients. Innovative complementary strategies that eliminate CD25+ regulatory T cells or block cytotoxic 3; lymphocyte antigen-4-induced T cell tolerance may enhance such immune responses, but the indiscriminate application of these potent adjuvants carries the risk of inducing autoimmune encephalomyelitis. In order to understand the limitations and risks of targeting CMV antigens in MGs, we have developed a novel murine astrocytoma cell line that supports infection with murine CMV and is tumorigenic in syngeneic mice. Our preliminary murine studies demonstrate that these tumors in the brain can be targeted with RNA-loaded DCs. We have also shown that DCs from patients with MGs that are loaded with pp65mRNA, induce interferon-gamma, production from CD4+ and CDS+ T-cells in an antigen-specific manner and incite T-cells to kill malignant astrocytes infected with human CMV. Interestingly, we have also found that CMV-specific T-cells preferentially accumulate at the tumor site in patients with MGs. We believe that our murine model system and the complementary human studies proposed will allow selection and translation of the most effective strategies for targeting CMV-associated antigens in patients with MGs, without the induction of autoimmunity. In this project, we will use the murine model, in combination with in vitro human studies to evaluate the safety of, and to gain a better understanding of the mechanisms involved in the therapeutic targeting of CMV-associated proteins in malignant gliomas. The results will then be used to rationally design and conduct a clinical CMV-targeted clinical trial.
Keywords: astrocytoma, cytomegalovirus, neoplasm /cancer immunotherapy, neoplasm /cancer vaccine, therapy design /development, vaccine development, T lymphocyte, clinical trial phase I, clinical trial phase II, dendritic cell, human therapy evaluation, nonhuman therapy evaluation, vector vaccine, biotechnology, human subject, laboratory mouse, patient oriented research
Dendritic Cell Immunotherapy Of Malignant Gliomas
John H Sampson, Associate Professor
Duke University 2200 W. Main St. Durham, Nc 27705
Grant 5R01CA097222-05 from National Cancer Institute, IRG: ZRG1
Abstract: Despite aggressive surgical resections, high-dose radiation therapy, and toxic chemotherapy, the vast majority of patients with malignant brain tumors survive less than one year making conventional therapy for malignant brain tumors the most expensive therapy per quality-adjusted life-year saved currently provided. Moreover, the failure of these treatment modalities to be tumor-specific at the molecular level, results in inevitable damage to surrounding normal brain that incapacitates patients treated with these traditional modalities. The inherent specificity of immunologic recognition offers the prospect of targeting malignant cells more precisely. Several studies have documented the exceptional ability of dendritic cells (DCs) to activate the immune system and produce encouraging human antitumor responses. The epidermal growth factor mutation, EGFRvIII, found on the majority of malignant gliomas, represents a tumor-specific target for such an approach. Our preclinical results demonstrate that DCs loaded with a KLH conjugate of an EGFRvIII peptide induce potent humoral and cell-mediated immune responses. Although anti-EGFRvIII, DC-based immunotherapy will allow antigen-specific immune responses to be clearly monitored and potentially optimized, we believe that human antitumor responses will likely be enhanced and the spectrum and utility of this paradigm expanded by targeting additional antigens. However, existing techniques for identifying potential targets are labor intensive, do not systematically assess the entire neoplastic genome, and do not assess the potential risk of autoimmunity posed by targeting these antigens. Serial analysis of gene expression (SAGE) is a contemporary approach to gene expression analysis that allows rapid identification of genes that are over-expressed in neoplastic cells. SAGE databased mining and rapid expression screening has allowed our group to identify a large number of genes uniquely expressed in malignant gliomas that may function as specific tumor antigens. To select those with immunologic relevance and those that are unlikely to induce autoimmune reactivity, we have developed a unique system based on the loading of autologous DCs with genes or gene fragments. Using this technique, we have demonstrated that DCs loaded with tumor-specific RNAs can specifically activate autologous T cells without activating autoreactive T cells. The hypothesis to be tested in this project is that malignant gliomas can be selectively targeted for therapeutic immunotherapy without the induction of autoimmunity using DCs loaded with the tumor-specific EGFRvIII and other additional genes found by SAGE to be uniquely expressed by malignant gliomas.
Keywords: dendritic cell, epidermal growth factor, glioma, growth factor receptor, human therapy evaluation, neoplasm /cancer immunotherapy, receptor expression, antigen antibody reaction, antitumor antibody, autoimmunity, clinical trial phase I, colony stimulating factor, dosage, gene mutation, immunity, immunosuppression, interleukin 4, monocyte, tumor antigen, clinical research, human subject, leukapheresis, patient oriented research, serial analysis of gene expression
Project start date: 2002-09-30
Project end date: 2008-08-31
5R01CA097222-05 (2006): $300762
5R01CA097222-04 (2005): $308000
5R01CA097222-03 (2004): $305300
5R01CA097222-02 (2003): $305300
1R01CA097222-01 (2002): $299633
Intracerebral Infusion Of Radiolabeled Specific Antibody
John H Sampson, Associate Professor
Duke University 2200 W. Main St. Durham, Nc 27705
Grant 5R01CA097611-05 from National Cancer Institute, IRG: ZRG1
Abstract: Despite aggressive surgical resections, high-dose radiation therapy, and chemotherapy delivered at toxic doses, the vast majority of patients with malignant brain tumors survive less than one year making conventional therapy for malignant brain tumors the most expensive medical therapy per quality-adjusted life-year saved currently provided in the United States. Moreover, the failure of these treatment modalities to be tumor-specific at the molecular level, results in inevitable damage to surrounding normal brain that incapacitates patients treated with these traditional modalities. The inherent specificity of immunologic recognition offers the prospect of targeting malignant cells more precisely. Within our program, direct injection of 131-I-labeled, operationally-specific, monoclonal antibodies (MAbs) into brain tumor resection cavities delivers extremely high radiation doses to tumor cells around the resection cavity and has produced promising results in Phase II clinical trials. These MAbs diffuse only short distances beyond the cavity, however. Therefore, most of the radiation extending beyond the cavity is not specifically targeted to tumor cells and the radiation dose delivered beyond the cavity declines exponentially from the cavity interface. As a result tumor cells that are known to infiltrate the brain for significant distances beyond the cavity are sub-optimally treated and lethal tumors always recur within 2 cm of the radiated re section cavity. Continuous microinfusion is a promising technique that allows homogeneous delivery of even large molecular weight molecules at high concentrations throughout large areas of the brain. Although this technique may enhance the delivery of 131-I-labeled MAbs and other therapeutic agents to diffusely infiltrating malignant brain tumors and reduce recurrence rates, the parameters that govern this technique and its limitations have not been defined. One of the major goals of this proposal is to define these parameters. In addition, this proposal is designed to investigate whether targeted radiotherapy might be improved through the use of human chimeric MAbs with increased biostability and the use of high linear energy transfer radioisotopes, such as 211-At, with greater relative biological effectiveness.The hypothesis to be tested in this proposal is that continuous microinfusion will widely deliver operationally tumor-specific monoclonal antibodies conjugated to 131-I or the alpha-emitter 211-At such that they will be specific and potent therapeutic agents against malignant brain tumors with major reductions in toxicity to normal brain.
Keywords: antibody specificity, brain neoplasm, human therapy evaluation, injection /infusion, monoclonal antibody, neoplasm /cancer radioimmunotherapy, radiotracer, clinical trial phase I, dosage, drug screening /evaluation, neurotoxicology, nuclear medicine, tenascin, biotechnology, clinical research, human subject, patient oriented research
Project start date: 2002-09-30
Project end date: 2008-08-31
5R01CA097611-05 (2006): $300762
5R01CA097611-04 (2005): $308000
5R01CA097611-03 (2004): $308000
5R01CA097611-02 (2003): $308000
1R01CA097611-01 (2002): $308000
MENTORED PATIENT ORIENTED RESEARCH CAREER DEVELOPMENT AW
John H Sampson, Associate Professor
Duke University 2200 W. Main St. Durham, Nc 27705
Grant 5K23RR016065-05 from National Center For Research Resources, IRG: RIRG
Abstract: Adapted from s ) The brain is the most frequent site of crippling and incurable human disease, and malignant primary brain tumors alone are more common than Hodgkin s disease, and cause more deaths than cancer of the bladder or kidney, leukemia, or melanoma. Conventional therapy for malignant brain tumors is ineffective and incapacitating, and represents the most expensive medical therapy per quality- adjusted life-year saved currently provided in the U.S. At the investigators institution, direct injection of (131)I-labeled, operationally-specific, monoclonal antibodies (MAbs) into brain tumor resection cavities delivers extremely high radiation doses to tumor cells around the resection cavity and has produced promising results in Phase II clinical trials. However, these MAbs diffuse only short distances beyond the cavity. Therefore, most of the radiation extending beyond the cavity is not specifically targeted to tumor cells and the radiation dose delivered beyond the cavity declines exponentially from the cavity interface. As a result, tumor cells that are known to infiltrate the brain for significant distances beyond the cavity are subopitimally treated and lethal tumors always recur within 2cm of the radiated resection cavity. Continuous microinfusion is a promising technique that allows homogeneous delivery of even large molecular weight molecules at high concentrations throughout large areas of the brain. Although this technique may enhance the delivery of (131)I-labeled MAbs and other therapeutic agents to diffusely infiltrating malignant brain tumors and reduce recurrence rates, the parameters that govern this technique and its limitations have not been defined. One of the major goals of this proposal is to define these parameters. In addition, this proposal is designed to investigate whether targeted radiotherapy might be improved through the use of human chimeric MAbs with increased biostability and the use of high linear energy transfer radioisotopes, such as (211)At, with greater relative biological effectiveness. The hypothesis to be tested in this proposal is that continuous microinfusion will widely deliver operationally tumor-specific MAbs conjugated to (131)I or the alpha-emitter (211)At such that they will be specific and potent therapeutic agents against malignant brain tumors with major reductions in toxicity to normal brain over conventional whole brain radiotherapies.
Keywords: brain neoplasm, combination cancer therapy, human therapy evaluation, monoclonal antibody, neoplasm /cancer immunotherapy, neoplasm /cancer radiation therapy, chimeric protein, clinical trial phase I, cytotoxicity, drug delivery system, glioma, neoplasm /cancer relapse /recurrence, neoplastic cell, outcomes research, radiation dosage, tenascin, tumor antigen, tumor progression, clinical research, human subject, linear energy transfer, microinjection, radionuclide
Project start date: 2000-09-01
Project end date: 2005-08-31
5K23RR016065-05 (2004): $125496
5K23RR016065-04 (2003): $125496
5K23RR016065-02 (2001): $125253
1K23RR016065-01 (2000): $125172
RNA-based Immunotherapy Targeting Antigens Unique To Brain Tumor Stem Cells
John H Sampson, Associate Professor
Surgeryduke University
Grant 1R01CA135272-01 from National Cancer Institute, IRG: ZRG1
Abstract: A subset of cells in glioblastoma multiforme (GBM) has been identified that enjoy a unique capacity to regenerate tumors. These brain tumor stem cells (BTSC) can be segregated by the neural stem cell marker, CD133, and are widely believed to be the cells responsible for resistance to conventional therapies. An effective means of specifically eliminating these cells may reduce the need for intensive and non-specific conventional therapy and lower the risk of tumor recurrence. EGFRvIII is a tumor-specific mutation found on BTSC. We have successfully targeted EGFRvIII using a peptide vaccine that allowed rapid translation to an ongoing Phase III trial. EGFRvIII expression is heterogeneous, however, and the recurrence of EGFRvIII-negative tumors suggests that BTSC can rely on other oncogenic pathways. While our data suggests that targeting tumor-specific mutations in BTSC may be important, few highly-conserved tumor-specific mutations like EGFRvIII will be identified and antigen defined vaccine approaches will ultimately be limited. Dendritic cells (DCs) loaded with amplified total tumor RNA is an innovative strategy to induce cellular and humoral antitumor immune responses. Although CD133(+) BTSC are a minority subpopulation of GBM that cannot be reliably isolated or propagated in sufficient quantities to serve as an antigen source for human vaccination protocols, we have been able to reproducibly amplify the RNA content from as few as 500 sorted CD133(+) tumor cells to generate RNA libraries sufficient for clinical scale DC-based vaccination. In order to focus the immunologic response on antigens preferentially or uniquely expressed within BTSC and limit the potential for autoimmune reactivity against shared antigens expressed in normal cells, we will evaluate approaches to enrich for antigens preferentially or uniquely expressed in BTSC by using full length cDNA affinity based substractive hybridization or an innovative strategy that leverages the ability of the DNA mismatch binding protein, MutS, to isolate cDNAs that contain tumor-specific mutations. These various preparations will be evaluated for differential toxicity and efficacy in an inbred transgenic murine malignant astrocytoma model, in which a subpopulation of CD133(+) tumor cells with BTSC qualities have been identified and CD8(+) and CD4(+) epitopes have been found. If efficacy is seen, the least toxic strategy will be translated into a Phase I study within the context of our existing clinical trial platform. Treatment for malignant primary brain tumors, which are the most common cause of death among children and account for more deaths in adults than melanoma, currently represents the most expensive medical therapy per quality-adjusted life-year saved currently provided in the United States. A subset of malignant primary brain tumor cells (BTSCs), called brain tumor stem cells, enjoy a unique capacity to regenerate tumors and to resist conventional therapies. In this proposal we will see if targeting antigens preferentially or uniquely expressed by BTSCs will enhance the efficacy and reduce toxicity of immunotherapy
Project start date: 2008-08-01
Project end date: 2013-05-31
Related Publications
Stereotactic Body Radiotherapy for Lesions of The Spine and Paraspinal Regions. Int J Radiat Oncol Biol Phys. 2008 Nov 10. [Epub ahead of print] PMID: 19004569 CONVECTION-ENHANCED DELIVERY OF CINTREDEKIN BESUDOTOX (INTERLEUKIN-13-PE38QQR) FOLLOWED BY RADIATION THERAPY WITH AND WITHOUT TEMOZOLOMIDE IN NEWLY DIAGNOSED MALIGNANT GLIOMAS: PHASE 1 STUDY OF FINAL SAFETY RESULTS. Neurosurgery. 2008 Sep 15. [Epub ahead of print] PMID: 18827726 A novel inhibitor of signal transducers and activators of transcription 3 activation is efficacious against established central nervous system melanoma and inhibits regulatory T cells. Clin Cancer Res. 2008 Sep 15; 14( 18): 5759-68. PMID: 18794085 Detection of humoral response in patients with glioblastoma receiving EGFRvIII-KLH vaccines. J Immunol Methods. 2008 Nov 30; 339( 1): 74-81. Epub 2008 Sep 4. PMID: 18775433 Tumor-specific immunotherapy targeting the EGFRvIII mutation in patients with malignant glioma. Semin Immunol. 2008 Oct; 20( 5): 267-275. Epub 2008 Jun 9. PMID: 18539480 Combating immunosuppression in glioma. Future Oncol. 2008 Jun; 4( 3): 433-42. Review. PMID: 18518768 Immunotherapy against angiogenesis-associated targets: evidence and implications for the treatment of malignant glioma. Expert Rev Anticancer Ther. 2008 May; 8( 5): 717-32. Review. PMID: 18471045 Selective modification of antigen-specific T cells by RNA electroporation. Hum Gene Ther. 2008 May; 19( 5): 511-21. PMID: 18471037 Intracerebral infusion of an EGFR-targeted toxin in recurrent malignant brain tumors. Neuro Oncol. 2008 Jun; 10( 3): 320-9. Epub 2008 Apr 10. PMID: 18403491 Cryptococcal meningitis in patients with glioma: a report of two cases. J Neurooncol. 2008 Aug; 89( 1): 51-3. Epub 2008 Apr 9. PMID: 18398572 Immunotherapy of malignant brain tumors. Immunol Rev. 2008 Apr; 222: 70-100. PMID: 18363995 Cholesterol granuloma of the lateral ventricle. Case report. J Neurosurg. 2008 Feb; 108( 2): 357-60. PMID: 18240934 Convection-enhanced delivery of cintredekin besudotox (interleukin-13-PE38QQR) followed by radiation therapy with and without temozolomide in newly diagnosed malignant gliomas: phase 1 study of final safety results. Neurosurgery. 2007 Nov; 61( 5): 1031-7; discussion 1037-8. PMID: 18091279 Immunological responses in a patient with glioblastoma multiforme treated with sequential courses of temozolomide and immunotherapy: case study. Neuro Oncol. 2008 Feb; 10( 1): 98-103. Epub 2007 Dec 13. PMID: 18079360 Sensitive detection of human cytomegalovirus in tumors and peripheral blood of patients diagnosed with glioblastoma. Neuro Oncol. 2008 Feb; 10( 1): 10-8. Epub 2007 Oct 19. PMID: 17951512 EGFRvIII-targeted immunotoxin induces antitumor immunity that is inhibited in the absence of CD4+ and CD8+ T cells. Cancer Immunol Immunother. 2008 Jan; 57( 1): 115-21. Epub 2007 Jul 19. PMID: 17634939 
Clinical utility of a patient-specific algorithm for simulating intracerebral drug infusions. Neuro Oncol. 2007 Jul; 9( 3): 343-53. Epub 2007 Apr 13. PMID: 17435179 
Viruses in the treatment of malignant glioma. Expert Rev Neurother. 2007 Apr; 7( 4): 321-4. Review. No abstract available. PMID: 17425484 
Systemic CTLA-4 blockade ameliorates glioma-induced changes to the CD4+ T cell compartment without affecting regulatory T-cell function. Clin Cancer Res. 2007 Apr 1; 13( 7): 2158-67. PMID: 17404100 Direct intracerebral delivery of cintredekin besudotox (IL13-PE38QQR) in recurrent malignant glioma: a report by the Cintredekin Besudotox Intraparenchymal Study Group. J Clin Oncol. 2007 Mar 1; 25( 7): 837-44. PMID: 17327604