Neuro-Oncology Neuro-Oncology 17:vii15 –vii25, 2015 doi:10.1093/neuonc/nov159

Vaccination strategies for neuro-oncology John H. Sampson and Duane A. Mitchell Preston Robert Tisch Brain Tumor Center at Duke, Duke Brain Tumor Immunotherapy Program, Division of Neurosurgery, Department of Surgery, Duke University Medical Center, Durham, North Carolina (J.H.S.); Preston A. Wells, Jr. Center for Brain Tumor Therapy, UF Brain Tumor Immunotherapy Program, Department of Neurosurgery, McKnight Brain Institute, University of Florida, Gainesville, Florida (D.A.M.) Corresponding Authors: [email protected] and [email protected]

Vaccination against cancer-associated antigens has long held the promise of inducting potent antitumor immunity, targeted cytotoxicity while sparing normal tissues, and long-lasting immunologic memory that can provide surveillance against tumor recurrence. Evaluation of vaccination strategies in preclinical brain tumor models has borne out the capacity for the immune system to effectively and safely eradicate established tumors within the central nervous system. Early phase clinical trials have established the feasibility, safety, and immunogenicity of several vaccine platforms, predominantly in patients with glioblastoma. Definitive demonstration of clinical benefit awaits further study, but initial results have been encouraging. With increased understanding of the stimulatory and regulatory pathways that govern immunologic responses and the enhanced capacity to identify novel antigenic targets using genomic interrogation of tumor cells, vaccination platforms for patients with malignant brain tumors are advancing with increasing personalized complexity and integration into combinatorial treatment paradigms. Keywords: cancer vaccines, glioblastoma, immunotherapy, tumor antigens.

Antigen Targets The underlying premise of all cancer vaccine strategies is that the induction of de novo antitumor immune responses or expansion of preexisting immunity against tumor antigens will elicit a cytotoxic response that is effective in mediating tumor regression. A first requisite therefore is an immunogenic formulation comprising one or more antigens that are expressed within malignant tumor cells. Antigens expressed within tumor cells can be classified very broadly into 2 general categories: (i) tumor-associated antigens (TAAs) and (ii) tumor-specific antigens (TSAs).1 – 3 TAAs are normal proteins that are overexpressed within tumor cells relative to normal tissues and may serve as immunogenic targets for the immune system. These antigens are most often lineage-differentiation antigens such as the melanoma-associated antigens (MAGE, CAGE),4,5 colorectal cancer antigens (carcinoembryonic antigen, [CEA]),6,7 and the hepatocellular carcinoma antigen, alpha-fetoprotein (AFP).8,9 Other overexpressed antigens with limited expression profiles in normal tissues such as HER2 and IL-13 receptor alpha (IL-13Ra) have also been identified as potential immunologic targets for therapeutic cancer vaccines.10 – 12 A concern in directing vaccination strategies against TAAs is the degree to which central or peripheral tolerance against these normal self-proteins is operative as an additional confounding factor

to the engendering of potent antitumor immunity, and the risk of autoimmunity directed at tissues with shared expression if potent immunologic responses are in fact elicited using immunotherapy.

Tumor-associated Antigens There have been several studies examining the expression of TAAs in glioblastoma (GBM) tumors that have collectively demonstrated the presence of several potential candidates for vaccine-directed immunotherapy. Many of these identified TAAs have been evaluated in early-stage therapeutic vaccine trials that have demonstrated safety and immunogenicity in patients with primary or recurrent GBM. The cancer/testis antigens (CTAs) and melanocyte differentiation antigens (MDAs) are broad classes of differentiation antigens that have been demonstrated to be variably expressed within GBM tumors. Syed et al.13 examined the expression of CTAs: LAGE-1, NY-ESO-1, MAGE-1, MAGE-3, MAGE-4, MAGE-10, CT-7, CT-10, HOM-MEL 40, BAGE, and SCP-1 and MDAs: tyrosinase, gp100, MELAN-A/MART-1, and TRP-2, in primary glioma tissues and glioma cell lines. Expression of at least one CTA at the RNA level was identified in 33% of gliomas, and 48% of tumors examined expressed at least one MDA. Freitas et al.14 recently conducted an extensive expression analysis of CTAs within GBM and

Received 23 June 2015; accepted 15 July 2015 # The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: [email protected].

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Sampson and Mitchell: Vaccination strategies for neuro-oncology

normal brain selected from 153 CTA genes. Using a combination of screening publically available gene expression databases and RT-PCR detection within normal brain as an exclusion for further consideration, these investigators identified 4 CTAs (ACTL8, CTCFL, OIP5, and XAGE3) as candidate CTAs uniquely expressed within GBM tumors. Other TAAs have also been advanced as candidates for vaccine therapy in malignant brain tumors such as survivin, telomerase, HER2neu, EphA2, and IL-13Ra.15 – 23 These targets are frequently upregulated in pediatric and adult gliomas as well as other cancers, and immunogenic epitopes have been identified within these antigens that can recognize and kill glial tumor cells in vitro. While most studies have examined the expression of TAAs in glioblastoma, studies of medulloblastoma, PNETs, and ependymomas have also confirmed the expression of CTAs, MDAs, and TAAs such as survivin and telomerase within these tumors.24 – 33 As with other TAAs, these antigens are largely, although not exclusively, restricted in their expression to the tumor microenvironment, and early phase studies of vaccination against these targets have shown safety, immunogenicity, and some evidence of efficacy. Further studies will be needed to determine whether a therapeutic window exists where vaccination against TAAs with shared low-level expression in normal tissues can elicit sufficient immunity to mediate a clinical benefit in the absence of intolerable autoimmune manifestations. Such demonstration would open the door to a large cohort of potential combinatorial antigenic targets within malignant gliomas and other tumors.

Viral Antigens Viral antigens represent a unique class of TAAs that are foreign to the host immune system and thus represent particularly attractive targets for immunotherapy due to their inherent immunogenicity. While tumor-associated viruses are not exclusively restricted to expression within tumor cells, viral gene expression is most often undetectable within normal tissues in patients harboring virus-associated cancers. Thus, the immunogenic viral antigens expressed in tumor cells may make excellent targets for tumor-directed immunotherapy. In the cases of virally induced cancers such as HPV-associated genitourinary cancers or EBV-associated lymphomas, immunotherapy targeting viral antigens has demonstrated a favorable safety profile and compelling early-stage clinical efficacy.34,35 Malignant brain tumors have not been shown to be virally induced, but recent reports from our laboratory and others have demonstrated frequent detection of low-level expression of human cytomegalovirus (CMV) genes within malignant gliomas.36,37 While the role of CMV in the biology of these tumors is a continued area of study,38 the potential for co-opting viral gene expression within tumor cells as targets for immunotherapy is particularly attractive given the history of safety and efficacy in targeting CMV with cellular immunotherapeutic modalities in immunocompromised patients such as those undergoing allogeneic bone marrow transplantation.39 – 41 Immunodominant CMV antigens such as immediate early 1 (IE1), phosphoprotein 65 (pp65), and glycoprotein B (gB) have been demonstrated to be expressed in GBM tumors by PCR, immunohistochemistry, in situ hybridization, and Western blot analysis in several independent studies, lending the possibility of

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targeting several immunogenic antigens expressed within these tumors.42 – 44

Tumor-specific Antigens In contrast to TAAs, TSAs are exquisitely expressed within tumor cells and do not have a shared expression within normal tissues. TSAs are potentially the ideal vaccine candidates for cancer immunotherapy due to their reduced likelihood for generating autoimmune manifestations if targeted effectively and for increased immunogenic potential due to comprising truly foreign antigens to the human immune system. TSAs most often arise in the context of tumor-specific mutations, which may be conserved across a subgroup of patients or arise as individual immunogenic alterations within a given patient. Conserved mutations that alter the protein-coding sequence within tumor cells can serve as targets for the development of cancer vaccine platforms such as the immunologic targeting of the epidermal growth factor receptor variant 3 (EGFRvIII) with a peptide conjugate vaccine (rindopepimut).45 The list of conserved mutations identified within malignant brain tumors is growing due to the rapid advances in the molecular characterization of adult and pediatric brain tumors.46 – 48 These mutations most often characterize a subpopulation of patients with a given histologic type of brain tumor, thus presenting some challenges in the clinical development of such focused targeting platforms due to limiting numbers of available patients who may derive benefit. Nonetheless, the immunologic targeting of conserved and immunogenic mutations such as EGFRvIII in GBM and IDH1 mutations in secondary GBMs and lower-grade gliomas represent prototypical genetic alterations that may serve as excellent candidates for immunotherapeutic development.49 – 53 An advantage to vaccination against defined tumor-specific mutations in comparison with other development of therapeutic modalities targeting mutated proteins in cancer is that the immune-mediated mechanism of killing tumor cells is agnostic to whether the mutated protein plays a critical role in tumor biology or to whether redundant pathways are operative within the tumor cell that drive tumor proliferation and survival. In addition to EGFRvIII in GBM, IDH1 in malignant gliomas, other conserved mutations and genomic alterations such as histone mutations in diffuse intrinsic pontine gliomas,54,55 BRAF mutations and fusions in low-grade gliomas,56,57 and C11orf95-RELA fusion protein in ependymomas58,59 constitute examples of a growing list of potential targets for the development of targeted immunotherapy.60 In addition to conserved mutations, there is a larger number of patient-specific mutations that arise within individuals and are unique to that patient’s tumor. Evidence that immunity against patient-specific antigens may be important for modulating tumor growth was recently highlighted in a study demonstrating that an increased number of immunogenic mutations, as assessed by tumor genome meta-analysis, was associated with increased survival in patients registered within The Cancer Genome Atlas (TCGA).61

Tumor Heterogeneity and Cancer Vaccine Development One of the challenges in the successful development of vaccine is addressing the heterogeneous gene expression patterns

Sampson and Mitchell: Vaccination strategies for neuro-oncology

inherent to malignant brain tumors that render immunologic coverage of all tumor cells difficult to achieve with defined antigen vaccines. An alternative approach has been to use unfractionated tumor antigens in the form of tumor lysates, eluted tumor peptides, or extracted and amplified total tumor RNA.62 – 64 These approaches have the advantage of allowing the natural antigen processing and presentation pathways operative in a patient’s immune system and the full complement of expressed antigens within a patient’s tumor to select the most appropriate antigens for immunologic recognition. While such approaches hold at least a theoretically increased risk of autoimmunity due to vaccination with a formulation containing both normal brain antigens as well as tumor antigens, preclinical, and clinical evaluation of such unfractionated vaccination strategies to date have been shown to be safe and capable of engendering antitumor immunity.

Clinical Trials EGFRvIII Vaccines (Rindopepimut) Epidermal growth factor receptor variant type III (EGFRvIII) is a deletion mutation that generates a novel extracellular tumorspecific epitope that is heterogeneously expressed in 30% – 35% of the primary glioblastoma (GBM) population. In a phase 2 multicenter trial from our laboratory, patients with newly diagnosed GBM were vaccinated with a 14 amino acid peptide from EGFRvIII encompassing the mutation site and conjugated to keyhole limpet hemocyanin (KLH) (rindopepimut) and given with GM-CSF. Rindopepimut was well tolerated, and immunized patients treated in the context of standard of care (SOC) temozolomide (TMZ) possessed an impressive overall survival (OS) of 26 months in comparison with 15 months in matched controls (hazard ratio [HR], 5.3, P ¼ .0013).53 Importantly, no patients possessed EGFRvIII-specific immune responses prior to vaccination, and the induction of these responses correlated with extended OS. Rindopepimut was also administered to patients with newly diagnosed GBM in the context of dose-intensified TMZ (OS of 23.6 months; HR, 0.23; P ¼ .019). Dose-intensified TMZ did not impair the induction of vaccine-specific immunity, and EGFRvIII-specific antibody titers and DTH responses were actually higher than those seen in patients treated with SOC TMZ.52 In patients with recurrent EGFRvIII-positive GBM, a randomized phase 2 study was undertaken in which patients received bevacizumab and rindopepimut or bevacizumab and KLH. While follow-up is ongoing, rindopepimut appears to prolong survival significantly in this context (rindopepimut median OS¼12.0 mo; 95% CI: 9.7 – vs KLH median OS¼8.8 mo; 95% CI: 6.8 – 11.4 mo; (HR¼0.47 [0.25 – 0.91]; P ¼ .0208). Furthermore, a relationship between vaccine-induced immunity and survival was again observed as the rapidity of EGFRvIII titer generation was associated with extended OS.65 Retrospective analysis demonstrates that rindopepimut eliminates nearly all EGFRvIII positive tumor cells, and this peptide vaccine is now being tested in an international phase 3 randomized trial (ACT IV) (that has completed accrual). However, recurrence after treatment is associated with the outgrowth of EGFRvIII-negative tumor cells.53 These data suggest a significant effect of the vaccine; however, it also serves as a

Neuro-Oncology

cautionary lesson regarding the risks of targeting heterogeneously expressed single antigens. We also performed a randomized placebo-controlled pilot trial in which patients with newly diagnosed GBM receiving SOC TMZ received a single infusion of daclizumab or saline at the time of first vaccination.66 Daclizumab is an antibody specific to the high affinity IL-2 receptor (IL-2Ra) that depletes and impairs immune suppressive regulatory T cells (TRegs), a cell population elevated in GBM and shown to inhibit immune function. Administration of daclizumab significantly reduced circulating TRegs and the frequency of TRegs inversely correlated with EGFRvIII antibody titers, suggesting that TReg depletion could serve to augment rindopepimut-induced humoral immunity.

SL-701 and Other Peptide Vaccines Additional consistent, somatic tumor-specific mutations such as EGFRvIII with high frequencies of expression within other specific tumor types (eg, the isositrate dehydrogenase mutation in lower-grade gliomas) have recently been revealed and may also serve as excellent targets.50 However, the number of consistent tumor-specific mutations is still quite low. Therefore, targeting TAAs that are highly expressed in tumor and negligibly expressed in normal brain with peptide vaccination has also been performed. A multipeptide vaccine composed of HLA-A2-restricted peptides from the TAAs interleukin-13 receptor alpha 2 (IL-13Ra2), survivin, and EphA2 was examined in a single arm trial of 26 pediatric patients with high-grade glioma (HGG).21 In a phase I trial with 23 adults diagnosed with high-risk low-grade glioma (LGG), HLA-A2-restricted peptides from the same 3 TAAs were used with the addition of a peptide from the TAA Wilms tumor 1 (WT1).67 In both trials, the vaccines were emulsified in Montanide-ISA, admixed with a tetanus peptide and given with intramuscular injections of poly-ICLC followed by booster vaccinations. In both trials, vaccination was well tolerated. Robust TAA-specific immune responses to one or more of the peptides were induced in the majority of patients, and some patients had extended progression-free survival (PFS) after treatment initiation and in some cases demonstrated radiographic tumor responses. The initial success of this platform has been parleyed into the optimized SL-701 vaccine from Stemline Therapeutics. SL-701 contains peptides targeting IL-13Ra2, survivin, and EphA2 and is currently in an accruing multicenter phase 1/2 study in adults with recurrent GBM (ClinicalTrials.gov: NCT02078648 https://clinicaltrials.gov/ct2/show/NCT02078648) with primary outcomes of safety, 12-month OS, and objective response rate. Importantly, if meaningful immune responses that impact antigen-positive tumor can be generated to more than one peptide, this may help prevent the outgrowth of antigennegative tumor cells. An early phase clinical trial in 25 patients with advanced malignant glioma also examined multipeptide vaccination with TAAs68 using a personalized approach in which peptides were selected for immunization based on patient cellular responses prior to immunization. Cellular or humoral responses were seen in more than half of the immunized patients after at least 6 vaccinations, and 5 partial radiographic responses were observed. A subsequent phase 1 trial in patients with recurrent or progressive GBM based on this approach demonstrated

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Sampson and Mitchell: Vaccination strategies for neuro-oncology

a similar PFS and prolonged OS, although the study is probably too small to be conclusive.69 In a nonrandomized phase 2 trial in 21 patients with recurrent GBM, WT1 was targeted with a single 9mer heteroclitic HLA-A2402-restricted peptide in Montanide ISA51 by weekly intradermal injection until progression.70 While the frequency of WT1 cytotoxic T lymphocytes did not increase after immunization, the 6-month PFS rate was 33.3% suggesting that WT1 immunization may possess activity. The cumulative results of these peptide vaccine trials indicate that peptide immunization is safe and may be associated with radiographic responses and prolonged survival. Larger trials validating these small, early phase trials need to be pursued, and future combinatorial approaches with other effective modalities may further these promising results.

Heat Shock Protein Vaccines (Alliance) Another immunization approach has leveraged heat shock proteins (HSP). HSPs are highly conserved, stress-induced molecular chaperones that bind peptides to assist in protein folding as well as transport. HSP-96 delivers peptides to antigenpresenting cells (APCs), and brain tumor-derived HSP-96 results in the presentation of HSP-96-chaperoned tumor antigen on class I and class II HLA and robust immunogenicity. In a phase 1 dose escalation trial, HSP-96 was purified from autologous tumor and pulsed onto patient APCs (HSPPC-96) and then administered as an autologous vaccine to patients with resectable recurrent GBM. This personalized and polyvalent vaccine approach resulted in 11 of 12 patients demonstrating peripheral immune responses to HSP-96 bound peptide with no severe adverse events.71 In the subsequent single-arm phase 2 trial, 41 treated patients with recurrent GBM demonstrated a 6-month OS of 90.2% (95% CI: 75.9 –96.8) with decreased OS observed in patients with reduced lymphocyte counts.72 A randomized phase 3 trial within the Alliance Consortium (ClinicalTrials.gov: NCT01814813 https://clinicaltrials.gov/ show/NCT01814813) is underway with a primary objective of determining whether there is an OS advantage of HSPPC-96 administered with bevacizumab, given concomitantly or at the point of progression when compared with bevacizumab given alone in patients with resected recurrent GBM.

DCVax and Other Tumor Lysate-pulsed Dendritic-cell Vaccines One of the major emphases of brain tumor immunotherapy has been direct vaccination with dendritic cells (DCs). While autologous cell vaccination is inherently complex and expensive, DC vaccines can engender robust immune responses. DCVax-L is a DC vaccine in which autologous DCs are pulsed with a lysate derived from the patient’s own resected tumor. A phase 3 trial of DCVax-L that was initiated in 2006 is currently enrolling patients with newly diagnosed GBM.(ClinicalTrials.gov: NCT00045968 https:// clinicaltrials.gov/ct2/show/NCT00045968). Patients in this randomized, placebo-controlled, multicenter trial receive DCVax-L or placebo at a 2:1 ratio, and crossover is permitted. The primary outcome is reported to be complete in September 2015 and will examine PFS between patients receiving vaccine or placebo with additional endpoints of performance status, immune response, and safety. Perhaps one of the most intriguing results

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using this platform was reported by Prins et al.73 in which a patient treated with one administration of DCs loaded with autologous tumor lysate mounted an impressive CD8+ T cell response to the pp65 epitope of CMV, a virus significantly associated with glioma. Tumor lysate-pulsed DC vaccines represent one of the most extensively pursued immunotherapeutic approaches.74 – 82 In the HGG-2006 phase 1/2 trial (EudraCT 2006– 002881 –20) 77 patients with newly diagnosed GBM received 4 vaccinations of DCs loaded with autologous whole tumor cell lysate followed by boosts of tumor lysate.75 The regimen was incorporated with SOC radiotherapy and TMZ. A median OS of 18.3 months was reported, which is superior to that reported from SOC alone.83 While it is difficult to draw conclusions from various early-phase clinical trials with differing methodologies, this immunotherapeutic platform was largely well tolerated. The results of the randomized phase 3 DCVax-L are being awaited with great interest.

ICT-107 and Other Peptide-pulsed Dendritic Cell Vaccines DCs pulsed with peptides, as opposed to autologous tumor lysate, have also been investigated. ICT-107 is a vaccine using autologous DCs pulsed with synthetic TAAs (AIM-2, MAGE-1, TRP-2, gp 100, HER-2, and IL-13Ra2). In an initial single arm, phase 1 study, 17 patients with newly diagnosed GBM treated with ICT-107 achieved an impressive OS rate of 55.6% (95%CI, 28.6–75.9) with a median OS of 38.4 months, and there were correlations with OS and immune responses to gp100 and HER2 antigens.84 ICT-107 underwent testing in a multicenter double-blinded phase 2b study sponsored by ImmunoCellular Therapeutics that is no longer recruiting (ClinicalTrials.gov: NCT01280552 https://clinicaltrials.gov/ct2/show/NCT01280552). Patients with newly diagnosed GBM were randomized at a 2:1 ratio to autologous DCs pulsed with TAAs or unpulsed autologous DCs and received a minimum of 4 intradermal vaccines. The primary endpoints were to compare OS and PFS between the 2 study arms. Phase 2 results demonstrated an apparent increase in PFS and OS in a predetermined subset of the population. Additional single-arm trials have examined DCs pulsed with acid-eluted tumor peptides,85 IL13Ra2-derived peptides,86 and DCs pulsed with other synthetic GAAs.87,88 In the trial by Okada et al.,87 the peptides were from Eph2A, IL13Ra2, YKL-40, and gp100 and were given along with injections of PolyICLC. Of the 22 patients with malignant glioma who were treated, 9 achieved a PFS of at least 12 months, and one patient with GBM demonstrated a sustained complete response.

Cytomegalovirus pp65 RNA-pulsed Dendritic Cells and Other RNA-pulsed Dendritic Cell Vaccines Work from our laboratory,89 and others90 has also examined vaccination with tumor-targeted mRNA-loaded DCs. In our recent publication in Nature, we reported a randomized, blinded phase 1/2 trial (ClinicalTrials.gov:NCT00639639 https:// clinicaltrials.gov/ct2/show/NCT00639639) in which 12 patients with newly diagnosed GBM were randomized to receive Cytomegalovirus pp65 mRNA-pulsed DC vaccination alone or DC vaccination with vaccine site preconditioned using tetanus toxoid (Td) or unpulsed DCs. Patients randomized to Td had

Sampson and Mitchell: Vaccination strategies for neuro-oncology

Table 1. Open studies from CT.Gov 20150528 NCT#

Study Name

Responsible Party

Primary Outcome

Study Plan

02287428

A Phase I Study of a Personalized NeoAntigen Cancer Vaccine With Radiotherapy Among MGMT Unmethylated, Newly Diagnosed Glioblastoma Patients

Catherine Wu, MD, Dana-Farber Cancer Institute

To evaluate safety and tolerability of administering NeoVax in participants with newly diagnosed glioblastoma

01902771

A Phase I Study of Dendritic Cell Vaccine Therapy With In Situ Maturation for Pediatric Brain Tumors

John Goldberg University of Miami

To demonstrate that dendritic cell vaccine loaded with tumor lysate is feasible and safe in patients diagnosed with brain cancer as children

00045968

A Phase 3 Clinical Trial Evaluating DCVaxw-L, Autologous Dendritic Cells Pulsed With Tumor Lysate Antigen For The Treatment Of GBM

Northwest Biotherapeutics

02010606

Phase I Trial of Vaccination With Autologous Dendritic Cells Pulsed With Lysate Derived From an Allogeneic Glioblastoma Stem-like Cell Line for Patients With Newly Diagnosed or Recurrent Glioblastoma Imiquimod/BTIC Lysate-Based Vaccine Immunotherapy for Diffuse Intrinsic Pontine Glioma in Children and Young Adults

Jethro Hu, Cedars-Sinai Medical Center

The primary objective of this study is to compare progression free survival from time of randomization between patients treated with DCVax-L and control patients We will assess possible vaccine-related adverse effects, including but not limited to cerebral edema, autoimmune reactions, and allergic reactions

Following standard radiation therapy, MGMT-unmethylated subjects will receive NeoVax using a dosing schedule that incorporates both priming and boost phases instead of TMZ DC vaccine manufactured and partially matured using our standard operating procedures, developed in collaboration with the HGG Immuno Group, loaded with tumor lysate, then administered through imiquimod treated skin DCVax-L(treatment cohort) or autologous PBMC (placebo cohort)

Masonic Cancer Center, University of Minnesota

Determined as Grade 3 or 4 toxicity observation after dosing with BTIC vaccination

Phase I Study of Safety and Immunogenicity of ADU-623, a Live-attenuated Listeria Monocytogenes Strain (DactA/ DinlB) Expressing the EGFRvIII-NY-ESO-1 Vaccine, in Patients With Treated and Recurrent WHO Grade 3/4 Astrocytomas

Providence Health & Services

The primary objective of this trial is to determine the maximum tolerated dose (MTD)(up to a dose of 1 ×10^9 cfu IV) and characterize the safety profile of ADU-623 vaccine in patients with recurrent WHO Grade 3/4 Astrocytomas

01400672

01967758

Dendritic cell vaccination, in addition to standard temozolomide chemotherapy with optional bevacizumab treatment

Following RT, Tumor Lysate Vaccine and Imiquimod topically at each site prior to and 24 h after vaccination. Vaccine will be produced by the University Of Minnesota Molecular and Cellular Therapeutics Facility using the established brain tumor initiating cell (BTIC) cell line GBM-6 as the antigen source. Additional 180 cGy fractions will be delivered in single doses in a novel effort to induce NKG2D ligand upregulation. The total radiation dose for each patient will be 5940 cGy A novel vaccine approach using a live-attenuated strain of Listeria monocytogenes expressing EGFRvIII and NY-ESO-1 antigens followed by a 3-day course of antibiotics

Continued

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Sampson and Mitchell: Vaccination strategies for neuro-oncology

Table 1. Continued NCT#

Study Name

Responsible Party

Primary Outcome

Study Plan

01814813

A Phase II Randomized Trial Comparing the Efficacy of Heat Shock Protein-Peptide Complex-96 (HSPPC-96) (NSC #725085, ALLIANCE IND # 15380) Vaccine Given With Bevacizumab vs Bevacizumab Alone in the Treatment of Surgically Resectable Recurrent GBM Recurrent Medulloblastoma and Primitive Neuroectodermal Tumor Adoptive T Cell Therapy During Recovery From Myeloablative Chemotherapy and Hematopoietic Stem Cell Transplantation (REMATCH)

Alliance for Clinical Trials in Oncology

This randomized phase II trial studies how well giving vaccine therapy with or without bevacizumab works in treating patients with recurrent glioblastoma multiforme that can be removed by surgery During Phase I, 9 patients will be treated with increasing doses of tumor-specific immune cells to determine the safety of this treatment. Phase I patients will also receive dendritic cell vaccines to help boost the function of these immune cells and maintain their growth.

The vaccine is called heat shock protein peptide complex 96 (HSPPC-96) with bevacizumab

01326104

University of Florida

02052648

A Phase I/II Study of the Combination of Indoximod and Temozolomide for Adult Patients With Temozolomide-Refractory Primary Malignant Brain Tumors

NewLink Genetics Corporation

02078648

Phase 1/2 Study of SL-701, a Subcutaneously Injected Multivalent Glioma-Associated Antigen Vaccine, in Adult Patients With Recurrent GBM A Pilot Study to Evaluate the Effects of Vaccinations With HLA-A2-Restricted Glioma Antigen-Peptides With Poly-ICLC for Children With Newly Diagnosed

Stemline Therapeutics, Inc

01130077

Ian F. Pollack, M.D., University of Pittsburgh

During Phase II, 35 patients will be treated with tumor-specific immune cells and dendritic cell vaccines to see what impact they have on the tumor In this study, investigators will conduct a phase I/II trial in recurrent (temozolomide resistant) glioma patients. The overall goal of this study is to provide a foundation for future studies with indoximod tested in newly diagnosed glioblastoma patients with radiation and temozolomide, or in combination with vaccine therapies The purpose of this study is to determine the safety and efficacy of SL-701 as a treatment for recurrent GBM The overall objective of this pilot study is to collect immunological and safety data following administration of

TTRNA-DCs and TTRNA-xALT with SOC therapy

Indoleamine 2, 3-dioxygenase (IDO) inhibitor – Indoximod is given in combination with TMZ or bevacizumab, or with TMZ and stereotactic radiation.

SL-701; Imiquimod Cream 5% and Leukine as adjuvants

HLA Restricted glioma antigen peptides plus Poly ICLC; GAAs were EphA2, interleukin-13 receptor alpha 2 (IL-13Ra2), and survivin, and their peptide epitopes were Continued

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Sampson and Mitchell: Vaccination strategies for neuro-oncology

Table 1. Continued NCT#

Study Name

Responsible Party

Primary Outcome vaccinations with HLA-A2. This data will be used to decide whether a larger study of clinical efficacy is warranted

Malignant Brain Stem Gliomas, Non-Brainstem High-Grade Gliomas, Recurrent Low-Grade Gliomas or Recurrent High Grade Gliomas 02146066

An Expanded Access Protocol for the Treatment of Glioblastoma Multiforme in Patients With Already Manufactured DCVaxw-L, Autologous Dendritic Cells Pulsed With Tumor Lysate Antigen Who Have Screen-Failed Protocol 020221

Northwest Biotherapeutics

00390299

Phase I Trial of a Measles Virus Derivative Producing CEA (MV-CEA) in Patients With Recurrent GBM

Mayo Clinic

01567202

A Triple-blind Randomized Clinical Study of Vaccination With Dendritic Cells Loaded With Glioma Stem-like Cells Associated Antigens Against Brain Glioblastoma Multiforme

Jinsong Wu, Huashan Hospital, Fudan University, Shanghai, China

01759810

Proteome-based Personalized Immunotherapy of Malignant Brain Tumors

NeuroVita Clinic, Moscow, Russian Federation

01782287

Proteome-based Personalized Immunotherapy of Brain Metastases From Lung Cancer

NeuroVita Clinic, Moscow, Russian Federation

The study is an open-label expanded access study for patients for whom vaccine was manufactured during the Northwest Biotherapeutics’ 020221 DCVax-L for GBM screening process, but who subsequently failed to meet specific enrollment criteria This phase I trial is studying the side effects and best dose of viral therapy in treating patients with recurrent GBM

This is a Phase II study in a single center to determine the efficacy of autologous dendritic cells (DCs) loaded with autogeneic glioma stem-like cells (A2B5+) administered as a vaccination in adults with glioblastoma multiforme (primary or secondary) Safety/Efficacy Study

Acute, progressing lethal neurooncological process can be transferred into chronic and non-lethal, the survival rates and life quality can be improved by of control of tumor cells (TCs) quantity and

Study Plan emulsified in Montanide-ISA-51with intramuscular polyinosinic-polycytidylic acid stabilized by lysine and carboxymethylcellulose Autologous Dendritic Cells Pulsed with Tumor Lysate Antigen

Carcinoembryonic antigen-expressing measles virus: Patients undergo stereotactic biopsy (to confirm the diagnosis) and placement of a catheter within the tumor, followed by MV-CEA administration into the tumor through the catheter Placebo vs DC vaccine with SOC therapy

Allogeneic haploidentical hematopoietic stem cells (HSCs), dendritic vaccine (DV) and cytotoxic lymphocytes (CTLs) is first line therapy. Second line therapy involves DV with recombinant proteins, CTLs and autologous HSC with modified proteome. Autologous HSCs are mobilized by G-CSF. Allogeneic haploidentical hematopoietic stem cells (HSCs), dendritic vaccine (DV) and cytotoxic lymphocytes (CTLs) is first line therapy. Second line therapy involves DV with recombinant proteins, CTLs and autologous HSC with modified proteome. Continued

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Sampson and Mitchell: Vaccination strategies for neuro-oncology

Table 1. Continued NCT#

01782274

Study Name

Proteome-based Personalized Immunotherapy of Brain Metastases From Breast Cancer

Responsible Party

NeuroVita Clinic, Moscow, Russian Federation

significantly greater bilateral migration of DCs to the draining lymph nodes, and the overall degree of lymph node DC uptake correlated with both PFS and OS (Cox proportional hazards model, PFS: HR ¼ 0.845, P ¼ .027; OS: HR ¼ 0.820, P ¼ .023). Remarkably, patients randomized to vaccine site preconditioning with Td possessed a median OS of .36 months. OS was significantly different in patients preconditioned with Td than the OS in patients randomized to unpulsed DCs (log rank P ¼ .013), and half of the patients in the Td arm survived almost 5 years or longer. Although a small clinical study, corroborating data in a murine model of DC immunotherapy demonstrated that Td acts as a novel adjuvant to enhance systemic DC migration through the chemokine CCL3 and may also improve clinical outcomes.

Whole Tumor Vaccines Whole tumor cell vaccines, in which tumor cells are rendered “safe” through formalin fixation91,92 or irradiation,93 – 95 have been examined for clinical efficacy in the context of brain tumors. The largest and most recent study on autologous formalin-fixed tumor vaccination (AFTV) examined the impact of 3 AFTVs 1 week apart in the context of radiotherapy in 22 resected patients with newly diagnosed GBM.92 The median OS was 19.8 months (95% CI: 13.8 – 31.3) with an actuarial 2-year survival rate of 40%. No severe adverse events were reported.

Human Umbilical Vein Endothelial Cell Vaccines Two clinical trials have also examined glutaraldehyde-fixed HUVEC vaccination in patients with recurrent GBM96 and recurrent malignant brain tumors97 on the premise that immune recognition of HUVEC antigen could induce an antiangiogenic response. In both trials, significant clinical responses were reported. Tanaka et al.96 reported that 17 patients with recurrent GBM treated with serial intradermal HUVEC immunizations in the context of radiotherapy and chemotherapy had a 12-month OS rate of 47.1% (95%CI, 25.5% – 69.7%). Okaji et al.97 reported that serial intradermal HUVEC immunizations

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Primary Outcome targeted regulation of effector functions of tumor stem cells (TSCs) Acute, progressing lethal neurooncological process can be transferred into chronic and non-lethal, the survival rates and life quality can be improved by of control of tumor cells (TCs) quantity and targeted regulation of effector functions of tumor stem cells (TSCs)

Study Plan Autologous HSCs are mobilized by G-CSF. Allogeneic haploidentical hematopoietic stem cells (HSCs), dendritic vaccine (DV) and cytotoxic lymphocytes (CTLs) is first line therapy. Second line therapy involves DV with recombinant proteins, CTLs and autologous HSC with modified proteome. Autologous HSCs are mobilized by G-CSF.

in 6 patients with recurrent malignant glioma resulted in 1 partial and 2 complete responses. While antiangiogenic immunotherapy is an intriguing proposition as an adjunct to direct tumor therapy, larger randomized studies will need to be performed. A list of ongoing vaccine trials for malignant brain tumors registered at clinicaltrials.gov is appended in Table 1.

Future Approaches and Considerations Vaccination strategies for malignant brain tumors have shown significant promise in preclinical studies and early-phase clinical evaluation, with a handful of pivotal phase 3 studies underway in patients with GBM. However, our current understanding of the mechanisms that influence the likely efficacy of such approaches has rapidly outpaced the clinical development pathway. We now know that complementary approaches such as immune checkpoint blockade, regulatory T cell depletion, and enhancement of DC migration are strategies that may synergize with tumor-specific vaccination and likely improve clinical outcomes. There are also significant advances being made in the development of novel adjuvants that can boost vaccine effectiveness through engagement of innate immune activation pathways. Thus, the future of vaccine development in neurooncology will likely yield a wave of combinatorial approaches that integrate the identification of patient-specific antigens98 with vaccination strategies and blockade of immunosuppressive pathways that attenuate the magnitude and duration of antitumor immunity in patients with brain tumors.

Conflict of interest statement. D.A.M. and J.H.S. have licensing agreements with Celldex Therapeutics, Inc. and Annias Immunotherapeutics, Inc. J.H.S. holds issued stock options in Celldex Therapeutics, Inc. J.H.S. serves on an advisory board for Annias Immunotherapeutics, Inc. and will receive stock options in Annias Immunotherapeutics, Inc. J.H.S. has served as a

Sampson and Mitchell: Vaccination strategies for neuro-oncology

consultant to Celldex Therapeutics, Inc. D.A.M. and J.H.S. have issued and pending patents related to vaccine technologies discussed in this review.

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