Vaccines

Vaccines

Vaccine Immunotherapy for Patients with Glioblastoma Multiforme

By
James Need, PhD and Al Musella, DPM
Last Updated 02/15/08
Approved by Paul Zeltzer, MD on 2/15/2008

Note: The authors of this paper are not M.D.s The contents of this paper are based on the listed references and are offered for educational and informational purposes only. This article is meant to bring attention to a timely treatment therapy concept that should be considered; it is not meant to be a complete literature review for either immunotherapy or vaccine therapy. Patients should refer to their health care provider for medical advice. Special acknowledgement and thanks are made to Meng-Yin Yang et al., 2006, and Ben A. Williams, 2007, for their recent reviews of this subject.

Background

As anyone familiar with glioblastoma multiforme (GBM) unfortunately knows too well, currently available treatments seldom provide anything more than temporary respite and tumor growth inevitably recurs. One area showing considerable promise is that using the patient’s immune system as an instrument for cancer therapy. An immune response directed against cells bearing tumor markers or antigens could provide a specific and effective mechanism for killing the unresectable, infiltrating residual tumor; thus halting the inevitable recurrence. While the theory is “elegant and persuasive”, a substantial clinical breakthrough has yet to be made and most clinical investigations have not yet translated into FDA-approved therapies for brain tumor patients (1).
Some of the factors limiting success thus far include the following (1, 2, 3, 4, and 5):

Different people have different tumors with different collections of antigens/proteins, so generic vaccines will not be effective (unless targeted against GBM-specific molecular pathways common to all patients) and patient-specific ones will be required.
A paucity of well-defined, tumor-specific antigens exists (most of the surface antigens for GBM cells are the same as those of “normal” host cells).
The human central nervous system (CNS) is immunologically quiescent.
- Absence of organized lymph tissue and traditional lymphatic drainage from the brain decrease the immunologic responsiveness of the CNS.
- The tight blood-brain barrier hinders the transport of many types of immunoreactive molecules and immune cells into the brain.
- High concentrations of immunoregulatory factors interfere with inflammatory responses within the CNS.
- Danger exists when stimulating inflammatory responses within the CNS.
- There is a lack of cellular major histocompatability complex (MHC) expression on normal cells within the CNS, which limits activation and migration of armed T cells.
- Ineffective antigen-processing events (6, 7) dampen both humoral and cellular immune responses.

Nevertheless, a great deal of promising immunotherapy-related research has been conducted over the past 10 years and continues today. The major areas of immune investigation have been focused on the following approaches: passive immunotherapy (introducing target (tumor)-specific monoclonal antibodies [mAb] into the patient) (1, 8, 9); radioactive antibody targeting (tagging a target-specific mAb with a radioisotope to deliver high-dose radiation to malignant cells) (1, 10-16); coupled targeted immunotoxins (with interleukin and other growth factors) (1, 17-25); adoptive immunotherapy (transferring the patient’s own immune cells [or other immune cells] that have been stimulated outside the body with tumor antigens) (1, 26, 27); lymphokine-activated killer cells (specific case of adoptive immunotherapy using “trained” peripheral blood lymphocytes to lyse natural killer-resistant tumor targets at the site of the tumor) (1, 28, 29); cytotoxic T lymphocytes (another specific case of adoptive immunotherapy using tumor cells to stimulate T cells and accelerate/activate the patient’s own cellular mediated resistance) (1, 30-35), and active immunotherapy (1, 36-38). This article will focus on active immunotherapy – the administration of the tumor antigenic material to induce a primary immune response, e.g. to “vaccinate” a patient against their own tumor.

Active Immunotherapy

Because most tumor antigens are poor stimulators of the immune system, active immunotherapy usually includes the use of adjuvants to enhance the immune response by prolonging the time of exposure to antigen and by increasing the activity of antigen presenting cells (APCs) which are essential in the activation of the body’s cellular mediated resistance process (1). The most common approaches include the use of dendritic cell (DC)-based, cytokine immunogene, and bacterial/viral tumor vaccine therapies.

Dendritic cell-based vaccines

DCs are the most effective APCs in the body as evidenced by their abilities to stimulate leukocyte reactions and to prime naïve T lymphocytes. This strategy involves exposure in the laboratory of the patient’s dendritic cells to their tumor antigen in combination with various stimulating factors, followed by an injection of these “primed” DCs into the patient to stimulate an internal immune response. Although soluble antigen is naturally released by tumor cells in later stages with the development of necrotic areas, such tumors are past the threshold when they can be checked by a patient’s natural immune reaction. This strategy accelerates the body’s natural process; hopefully inducing tumor destruction in the earliest stages of growth (1). In a 1999 Phase I trial, twelve (7 newly diagnosed, 5 recurrent) high-grade glioma patients received three separate vaccinations spaced two weeks apart in addition to standard of care which included external beam radiation therapy. Robust infiltration of T cells was detected in tumor specimens and median survival was 455 days (compared to 257 days for a control population (1, 2, and 39). Other Phase I DC-based clinical trials have been conducted (40-46) and shown promising results (median overall survival: 133 weeks; median overall survival 23.4 months; and 50% 2-year survival rate [41]), and in 2004, it was reported that the use of temodar after vaccination treatment improved outcomes relative to the vaccine alone (47). Proof of clinical benefit is still being established in Phase II clinical trials, which were started in 2002 (48).

Cytokine immunogene therapy

In 2000, a new strategy, the cytokine immunogene therapy, was reported (49). To prepare this type of vaccine, genes are transferred into a fibroblast cell line that causes the cells to produce cytokines, potent proteins known to stimulate the immune system. These cells are subsequently injected into the tumor bed, resulting in the development of an antitumor immune response. Glick et al. found that mice with a primary intracerebral glioma, melanoma, or breast cancer treated with this cytokine-secreting vaccine survived significantly longer than untreated mice. Additionally the vaccine was found to stimulate a systemic antitumor immune response, as shown by immunocytotoxic studies, histopathological examination, and delayed immune memory responses. Their results showed that immunogene therapy was a promising new targeted therapy for the treatment of intracerebral malignant tumors (49). In 2006, Glick et al. further demonstrated the efficacy of this potential therapy using intratumoral injection of interleukin-2-secreting cells as a treatment or protective vaccine in young mice. They demonstrated a significant prolongation of survival in animals harboring gliomas. Furthermore, long term survivors demonstrated immune memory, as evidenced by prolonged survival when rechallenged with tumor cells. Most impressive of all, 78% of long term survivors did not develop a tumor when rechallenged (50). While limited use of this therapy has been reported in humans (51, 52), studies continue. It is hoped that these findings and those like them will lead in the near future to more extensive clinical trials for the treatment of gliomas using this concept.

Bacterial and viral tumor vaccines

Live bacteria and/or viruses may also serve as a basis for active immunotherapies against brain tumors (1, 48) as their infections and the resulting damage can provide signals that attract APCs and initiate the cellular immune response (53). Studies have been conducted using Listeria monocytogenes (54-57), reovirus (2, 58), and reengineered polio virus (2, 59) in animals, and Salmonella typhimurium (1, 60), New Castle virus (1, 61), and herpes virus (2, 62) in humans. Four patients with advanced high-grade glioma, treated with the New Castle Disease Virus vaccine (MTH-68/H) had purported survival times of 5-9 years (61). More recently, Steiner et al., reported the results of a pilot clinical trial using patient tumor cell cultures infected with New Castle Disease Virus, followed by gamma irradiation. 39% had a 2 year survival rate compared to 11% in the controls (63). The use of a modified herpes virus was reported in Scotland in 2000. Four of nine patients were alive at the time of the report; 14-24 months after treatment (2, 64).

GBM-specific molecular pathway vaccines

One of the challenges mentioned earlier in using vaccines directed at tumors is that different people have different tumors with different collections of antigens/proteins; thus generic vaccines will not be effective. However, if a vaccine could be developed that was targeted to a metabolic pathway specific only to GBMs, that vaccine could be used “off the shelf” without modification for individual patients (2). Recently, a vaccine was developed that targets the epidermal growth factor, Variant III, which occurs in a high percentage of GBMs (but not all) but is rarely seen in anything other than GBM tumors. Patients received an initial set of 3 vaccinations at two-week intervals, standard temodar plus radiation, and monthly vaccines thereafter. Median time to tumor progression for 23 patients was 12.1 months, compared to a median time of 7.1 months for patients receiving the same treatment without vaccination. Median survival in the vaccinated patients had not been reached at the time of the report (65). A subsequent trial was completed in which the vaccine was given only to patients who were screened in advance for the mutant receptor before admission into the clinical trial. Median survival was 29 months, one of the best clinical outcomes thus far reported (Celdex Pharmaceuticals Report, June 2007).

Status

While these various strategies hold much promise, various challenges remain (1):

The use of corticosteroids, almost always a part of the standard care for brain tumor patients, is known to suppress the immune response. Therefore, its use must be considered when contemplating any of these immunotherapy strategies and is another reason that active immunotherapy should be used early, rather than late in a treatment protocol.
Due to the need for highly patient-selective treatments, the patient accrual on clinical trials is slower, the potential market for an approved product is lower, and the development times to FDA approval are prolonged.
There are significant manufacturing challenges facing the clinical development of such therapeutics.

When one considers the above it becomes easier to understand why it is generally accepted that simultaneous targeting of several components of the neoplastic process will provide the maximal chances of tumor control. The use of active immunotherapy, in combination with traditional surgery, radiation, chemotherapy, and molecularly targeted biological agents should prove to be synergistic and of low toxicity. The combined use of conventional treatments within the context of clinical trials of immunotherapy will allow for the evaluation of efficacy yet retain the ethical requirements for human investigation (1).

Vaccine Trials

This is a list of the vaccine trials we know about (all brain tumor types). If you know of any we missed - or if any of these closed, let us know!

Treatment Location Last Updated

Details A Phase II/III Randomized Study of CDX-110 with Radiation and Temozolomide in Patients with Newly Diagnosed Glioblastoma Multiforme San Francisco, CA 07/03/2008

Details A Phase l Trial of Surgical Resection With Gliadel Wafer Placement Followed by Vaccination With Dendritic Cells Pulsed With Tumor Lysate for Patients With Malignant Glioma Los Angeles, CA 03/02/2008

Details A Phase l Trial of Tumor Associated Antigen Pulsed Dendritic Cell Immunotherapy for Patients With Brain Stem Glioma and Glioblastoma Los Angeles, CA 03/02/2008

Details Chemotherapy, Radiation Therapy, and Vaccine Therapy With or Without Daclizumab in Treating Patients With Glioblastoma Multiforme That Has Been Removed by Surgery Durham, NC 03/02/2008

Details Evaluation of Recovery From Drug-Induced Lymphopenia Using Cytomegalovirus-Specific T-Cell Adoptive Transfer [ERaDICATe] Durham, NC 03/02/2008

Details Phase I Dose Escalation Study of Autologous Tumor Lysate-Pulsed Dendritic Cell Immunotherapy for Malignant Gliomas in Pediatric Patients Los Angeles, CA 03/02/2008

Details Phase I Study of Glioma-Associated Antigen (GAA) Peptide-Pulsed Dendritic Cell Vaccination in Malignant Glioma Patients Los Angeles, CA 03/02/2008

Details Phase I Study of Immunization With Autologous Tumor Lysate-Pulsed Dendritic Cells in Patients With Malignant Gliomas (Vaccine Therapy in Treating Patients With Malignant Glioma) Los Angeles, CA 03/02/2008

Details Phase I/II Study of GP96 Heat Shock Protein-Peptide Complex Vaccine in Patients With Recurrent or Progressive High-Grade Glioma San Francisco, CA 03/02/2008

Details Phase ll Trial of Tumor Lysate-Pulsed Dendritic Cell Immunotherapy for Patients With Atypical or Malignant, Primary or Metastatic Brain Tumors of the Central Nervous System Los Angeles, CA 03/02/2008

Details A phase I/II Evaluation of Vaccination with Type-1 Dendritic Cells Pulsed With Multiple Peptides In the Treatment of HLA-A2 Positive Patients With Recurrent Malignant Gliomas Pittsburgh, PA 03/02/2008

Details Phase I Study of Intratumoral and/or Resection Cavity Administration of Recombinant Measles Virus Encoding Human Carcinoembryonic Antigen in Patients With Recurrent Glioblastoma Multiforme Rochester, MN 03/02/2008

Details Phase I/II trial of Heat Shock Protein Peptide Complex-96 (HSPPC-96) Vaccine For Patients With Recurrent High Grade Glioma San Francisco, CA 10/03/2007

Details A Phase II Trial of Dendritic Cell Vaccine Pulsed with Tumor Lysate Followed by Temozolomide Upon Recurrence in Patients with Newly Diagnosed Glioblastoma Los Angeles, CA 08/18/2007

Details Gliadel and Dendritic Cell Vaccine for Malignant Glioma Los Angeles, CA 08/18/2007

Literature Cited:

1. Yang MY, Zetler PM, Prins RM, et al., “Immunotherapy for patients with malignant glioma: from theoretical principles to clinical applications.” Expert Rev. Neurotherapeutics. 2006; 6(10):1481-1494.

2. Williams BA, “Treatment Options for Glioblastoma and Other Gliomas”, 2007. http://virtualtrials.com/pdf/williams2007.pdf

3. Prins RM, Liau LM, “Immunology and immunotherapy in neurosurgical disease.” Neurosurgery. 2003; 53:144-152.

4. Walker PR, Calzascia T, Dietrich PV, “All in the head: obstacles for immune rejection of brain tumours.” Immunology. 2002; 107: 28-38.

5. Walker PR, Calzascia T, Schnuriger V, et al., The brain parenchema is permissive for full antitumor CTL effector function, even in the absence of CD4 T cells. J Immunol. 2000; 165:3128-3135.

6. Smits HA, van Beelen AJ, de Vos NM, et al., Activation of human macrophages by amyloid-B is attenuated by astrocytes. J Immunol. 2001; 166: 6869-6876.

7. Taniguchi Y, Ono K, Yoshida S, Tanaka R., “Antigen presenting capability of glial cells under glioma-harboring conditions and the effect of glioma-derived factors on antigen presentation. J Neuroimmunol. 2000; 111: 177-185.

8. Adams GP, Weiner JM, “Monoclonal antibody therapy of cancer.” Nat Biotechnol. 2005; 23: 1147-1157.

9. Sampson JH, Crotty LE, Lee S, et al., “Unarmed, tumor-specific monoclonal antibody effectively treats brain tumors.” Proc Natl Acad Sci. 2000; 97: 7503-7508.

10. Wikstrand CJ, Zalutsky MR, Bigner DD. “Radiolabeled antibodies for therapy for brain tumors.” In: Brain Tumor Immunotherapy, Human Press, NJ, USA, 2001: 205-209.

11. Riva P, Franceschi G, Frattarelli M, et al., “Loco-regional radioimmunotherapy of high-grade malignant gliomas using specific monoclonal antibodies labeled with 90Y: a Phase I study.” Clin Cancer Res. 1999; 5: S3275-S3280.

12. Reardon DA, Akabani G, Coleman RE, et al., “Phase II trial of murine (131)I-labeled antitenascin monoclonal antibody 81C6 administered into surgically created resection cavities of patients with newly diagnosed malignant gliomas.” J Clin Oncol. 2002; 20: 1389-1397.

13. Emrich JG, Brady LW, Quang TS, et al., “Radioiodinated (I-125) monoclonal antibody 425 in the treatment of high grade glioma patients: ten-year synopsis of a novel treatment. 2002; Am J Clin Oncol 25: 541-546.

14. Quang TS, Brady LW, “Radioimmunotherapy as a novel treatment regimen: 125I-labeled monoclonal antibody 425 in the treatment of high grade gliomas. Int J Radiat Oncol Biol Phys. 2004; 58: 972-975.

15. Paganelli G, Bartolomei M, Ferrari M, et al., “Pre-targeted locoregional radioimmunotherapy with 90Y-biotin in glioma patients: Phase I study and preliminary therapeutic results.” Cancer Biother Radiopharm. 2001; 16: 227-235.

16. Bartolomei M, Mazzetta C, Hadnkiewicz-Junal D, et al., “Combined treatment of glioblastoma patients with locoregional pre-targeted 90Y-biotin radioimmunotherapy and temozolomide.” Q J Nucl Med Mol Imaging. 2004; 48: 220-228.

17. Hall WA, “Targeted toxin therapy for malignant astrocytoma. Neurosurgery. 2000; 46: 544-551.

18. Rand RW, Kreitman RJ, Patronas N, et al., “Intratumorial administration of recombinant circularly permuted interleukin-4-Pseudomonas exotoxin in patients with high-grade glioma. Clin Cancer Res. 2000; 6: 2157-2165.

19. Kunwar S, “Convection-enhanced delivery of IL13PE38QQR for treatment of recurrent malignant glioma: presentation of interim findings from ongoing Phase I studies. Act Neurochir Suppl. 2003; 88: 105-111.

20. Parney IF, Kunwar S, McDermott M, et al., “Neuroradiographic changes following convection-enhanced delivery of the recombinant cytotoxin interleukin 13-PE38QQR for recurrent malignant glioma. J Neurosurg. 2005; 102: 267-275.

21. Sampson JH, Akabain G, Archer GE, et al., “Progress report of a Phase I study of the intracerebral microinfusion of a recombinant chimeric protein composed of transforming growth facto (TGF)-alpha and a mutated form of the Pseudomonas exotoxin termed PE-38 (TP-38) for the treatment of malignant brain tumors. J Neurooncol. 2003; 65: 27-35.

22. Husain SR, Joshi BH, Puri RK, “Interleukin-13 receptor as a unique target for anti-glioblastoma therapy. Int J Cancer. 2001; 92: 168-175.

23. Husain SR, Puri RK, “Interleukin-13 receptor-directed cytotoxin for malignant glioma therapy: from bench to bedside. J Neurooncol. 2003; 65: 37-48.

24. Kunwar S, Chang SM, Prados MD, et al., “Safety of intraparenchymal convection-enhanced delivery of cintredekin besudotoz in early-phase studies. Neurosurg Focus. 2006; 20: E15.

25. Shimamura T, Husain SR, Puri RK, “The IL-4 and IL-13 pseudomonas exotoxins: new hope for brain tumor therapy.” Neurosurg Focus. 2006; 20: E11.

26. Plautz GE, Mukai S, Cohen PA, Shu S, “Cross-presentation of tumor antigens to effector T cells is sufficient to mediate effective immunotherapy of established intracranial tumors. J Immunol. 2000; 165: 3656-3662.

27. Mitchell DA, Fecci PE, Sampson JH, “Adoptive immunotherapy for malignant glioma. Cancer J. 2003; 9: 157-166.

28. Dillman RO, Duma CM, Schiltz PM, et al., “Intracavity placement of autologous lymphokine-activated killer (LAK( cells after resection of recurrent glioblastoma. J Immunother. 2004; 27: 398-404.

29. Hayes RL, Arbit E, Odaimi M, et al., “Adoptive cellular immunotherapy for the treatment of malignant gliomas.” Crit Rev Oncol Hematol. 2001; 39: 31-42.

30. Kruse CA, Cepeda L, Owens B, et al., “Treatment of recurrent glioma with intracavity alloreactive cytotoxic T lymphocytes and interleukin-2. Cancer Immunol Immunother. 1997; 45: 77-87.

31. Merchant RE, Baldwin NG, Rice CD, Bear HD, “Adoptive immunotherapy of malignant glioma using tumor-sensitized T lymphocytes. Neurol Res. 1997; 19: 145-152.

32. Wood CW, Holladay FP, Turner T, et al., “A pilot study of autologous cancer cell vaccination and cellular immunotherapy using anti-CD3 stimulated lymphocytes in patients with recurrent grade III/IV astrocytoma. J Neurooncol. 2000; 48: 113-120.

33. Ishikawa E, Tsuboi K, Saijo K, et al., Autologous natural killer cell therapy for human recurrent malignant glioma. Anticancer Res. 2004; 24: 1861-1871.

34. Quattrocchi KB, Miller CH, Cush S, et al., “Pilot study of local autologous tumor infiltrating lymphocytes for the treatment of recurrent malignant gliomas. J Neurooncol. 1999; 45; 141-157.

35. Wang LX, Huang WX, Graor H, et al., “Adoptive immunotherapy of caner with polyclonal, 108-fold hyperexpanded, CD4+ and CD8+ T cells. J Transl Med. 2004; 2; 41.

36. Liau LM, Black KL, Prins RM, et al., “Treatment of intracranial gliomas with bone marrow-derived dendritic cells pulsed with tumor antigens.” J Neurosurg. 1999; 90; 1115-1124.

37. Okada H, Pollack IF, “Cytokine gene therapy for malignant glioma.” Exper Opin Biol Ther. 2004; 4; 1609-1620.

38. Glick RP, Lichtor T, Cohen EP, “Cytokine-based immune-gene therapy for brain tumors. In: Brain Tumor Immunotherapy, Humana Press, NJ, USA, 2001; 273-288.

39. Yu J. S., et al., “Vaccination of malignant glioma patients with peptide-pulsed dendritic cells elicits systemic cytotoxicity and intracranial T-cell infiltration.” Cancer Res. 2001; 61: 842-847.

40. Liau LM, Prins RM, Kiertscher SM, et al., “Dendritic cell vaccination in glioblastoma patients induces systemic and intracranial T-cell responses modulated by the local CNS tumor microenvironment.” Clin Cancer Res. 2005; 11: 5515-5525.

41. Yu JS, Liu G, Ying H, et al., “Vaccination with tumor lysate-pulsed dendritic cells elicits antigen-specific, cytotoxic T-cells in patients with malignant glioma.” Cancer Res. 2004; 64: 4973-4979.

42. Yu JS, Wheeler CJ, Zeltzer PM, et al., “Vaccination of malignant glioma patients with peptide-pulsed dendritic cells elicits systemic cytotoxicity and intracranial T-cell infiltration.” Cancer Res. 2001; 61: 842-847.

43. Yamanaka R, Abe T, Yajima N, et al., “Vaccination of recurrent glioma patients with tumour lysat-pulsed dendritic cells elicits immune responses: results of a clinical Phase I/II trial.” Br J Cancer. 2003; 89: 1172-1179.

44. Yamanaka R, Homma J, Yajima N, et al., “Clinical evaluation of dendritic cell vaccination for patients with recurrent glioma: results of a clinical Phase I/II Trial. Clin Cancer Res. 2005; 11: 4160-4167.

45. Kikuchi T, Akasaki Y, Irie M, et al., “Results of a Phase I clinical trial of vaccination of glioma patients with fusions of dendritic and glioma cells.” Cancer Immunol Immunother. 2001; 50: 337-344.

46. Kikuchi T, Akasaki Y, Abe T, et al., Vaccination of glioma patients with fusions of dendritic and glioma cells and recombinant human interleukin 12. J Immunother. 2004; 27: 452-459.

47. Wheeler, CJ et al., “Clinical responsiveness of glioblastoma multiforme to chemotherapy after vaccination.” Clin Cancer Res. 2004; 10: 5316-5326.

48. Chabalgoity JA, Dougan G, Mastroeni P, Aspinall RJ., “Live bacteria as the basis for immunotherapies against cancer. Expert Rev Vaccines. 2002; 1: 495-505.

49. Glick RP, Lichtor T, Cohen EP, “Cytokine immunogene therapy.” Neurosurg Focus. 2000; Dec 15; 9(6): e2.

50. Glick RP, Lichtor T, Lin H, et al., “Immunogene therapy as a treatment for malignant brain tumors in young mice.” J Neurosurg. 2006; 105: 65-77.

51. Okada H, Lieberman FS, Edington HD, et al., “Autologous glioma cell vaccine admixed with interleukin-4 gene transfected fibroblasts in the treatment of recurrent glioblastoma: preliminary observations in a patient with a favorable response to therapy. J Neuroonco. 2003; 64: 13-20.

52. Ren H, Boulikas T, Lundstrom K, et al., “Immunogene therapy of recurrent glioblastoma multiforme with a liposomally encapsulated replication-incompetent Semlik forest virus vector carrying the human interleukin-12 gene – a Phase I/II clinical protocol. J Neurooncol. 2003; 147-154.

53. Matzinger P, “The danger model: a renewed sense of self.” Science. 2002; 296: 301-305.

54. Barton GM, Medzhitov R., “Toll-like receptors and their ligands.” Curr Top Microbiol Immunol. 2002; 270: 81-92.

55. Ulevitch RJ., “Therapeutics targeting the innate immune system.” Nat Rev Immunol. 2004; 4: 512-520.

56. Liau LM, Jensen ER, Kremen TJ, et al., “Tumor immunity within the central nervous system stimulated by recombinant Listeria monocytogene vaccination. Cancer Res. 2002; 62: 2287-2293.

57. Calzascia T, Di Berardino-Besson W, Wilmotte R, et al., “Cutting edge: cross-presentation as a mechanism for efficient recruitment of tumor-specific CTL to the brain. J Immunol. 2003; 171: 2187-2191.

58. Wilcox ME, et al., “Reovirus as an oncolytic agent against experimental human malignant gliomas. J Nat Cancer Inst. 2001; 93: 903-912.

59. Gromeier, M, et al., “Intergeneric poliovirus recombinants for the treatment of malignant glioma. Proc Nat Acad Sci. 2000; 97: 6803-6808.

60. Rosenberg sA, Spiess PJ, Kleiner DE., “Antitumor effects in mice of the intravenous injection of attenuated Salmonella typhimurium. J Immunother. 2002; 25: 218-225.

61. Csatary LK, Gosztonyi G, Szeberenyi J, et al., “MTH-68/H oncolytic viral treatment in human high-grade gliomas. J Neurooncol. 2004; 67: 83-93.

62. Germano IM, et al., “Adenovirus/herpes simplex-thymidine kinase/ganciclovir complex: preliminary results of a phase I trial in patients with recurrent malignant gliomas. J Neurooncol. 2003; 65: 279-289.

63. Steiner HH, Bonsanto MM, Beckhove P, et al., “Antitumor vaccination of patients with glioblastoma multimform: a pilot study to assess feasibility, safety, and clinical benefit. J Clin Oncol. 2004; 22: 4272-4282.

64. Rampling R, et al., “Toxicity evaluation of replication-competent herpes simplex virus (ICP34.5 null mutant 1716) in patients with recurrent malignant glioma. Gene Ther. 2000; 7: 859-866.

65. Heimberger AB, et al., “An epidermal growth factor receptor variant III peptide vaccination appears promising in newly diagnosed GBM patients. 2006. Paper presented at American Association of Neurological Surgeons. .