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PDF file - 277KB, Phenotypic analysis of CMV pp65 RNA-pulsed DCs.

PDF file - 69KB, Cytokine secretion analysis of CMV pp65-specific T cells co-cultured with primary GBM tumor cells.

supplementary figures and legends

Summary of adverse events with possible relation to study.

Ex vivo analysis of CMV pp65-specific CD8+ T cells in circulation before and after immunotherapy.

PDF file - 72KB, Phenotype of in vitro stimulated T cells.

Table S2 - Glioma cell lines and patient derived tumor samples used for study with the number of EGFRvIII receptors per cell, the number of EGFRwt receptors per cell and the proportion of cells expressing EGFRvIII in the given cell line or tumor sample.

Purpose: Despite aggressive conventional therapy, glioblastoma (GBM) remains uniformly lethal. Immunotherapy, in which the immune system is harnessed to specifically attack malignant cells, offers a treatment option with less toxicity. The expression of cytomegalovirus (CMV) antigens in GBM presents a unique opportunity to target these viral proteins for tumor immunotherapy. Although the presence of CMV within malignant gliomas has been confirmed by several laboratories, its relevance as an immunologic target in GBM has yet to be established. The objective of this study was to explore whether T cells stimulated by CMV pp65 RNA-transfected dendritic cells (DC) target and eliminate autologous GBM tumor cells in an antigen-specific manner.Experimental Design: T cells from patients with GBM were stimulated with autologous DCs pulsed with CMV pp65 RNA, and the function of the effector CMV pp65-specific T cells was measured.Results: In this study, we demonstrate the ability to elicit CMV pp65-specific immune responses in vitro using RNA-pulsed autologous DCs generated from patients with newly diagnosed GBM. Importantly, CMV pp65-specific T cells lyse autologous, primary GBM tumor cells in an antigen-specific manner. Moreover, T cells expanded in vitro using DCs pulsed with total tumor RNA demonstrated a 10- to 20-fold expansion of CMV pp65-specific T cells as assessed by tetramer analysis and recognition and killing of CMV pp65-expressing target cells.Conclusion: These data collectively demonstrate that CMV-specific T cells can effectively target glioblastoma tumor cells for immunologic killing and support the rationale for the development of CMV-directed immunotherapy in patients with GBM. Clin Cancer Res; 20(10); 2684–94. ©2014 AACR.

PDF file - 65KB, Analysis of maturation markers in dendritic cells pulsed with RNA and cultured in maturation cytokine cocktail.

Purpose: Conventional therapy for malignant glioma fails to specifically target tumor cells. In contrast, substantial evidence indicates that if appropriately redirected, T cells can precisely eradicate tumors. Here we report the rational development of a fully human bispecific antibody (hEGFRvIII-CD3 bi-scFv) that redirects human T cells to lyse malignant glioma expressing a tumor-specific mutation of the EGFR (EGFRvIII).Experimental Design: We generated a panel of bispecific single-chain variable fragments and optimized design through successive rounds of screening and refinement. We tested the ability of our lead construct to redirect naïve T cells and induce target cell–specific lysis. To test for efficacy, we evaluated tumor growth and survival in xenogeneic and syngeneic models of glioma. Tumor penetrance following intravenous drug administration was assessed in highly invasive, orthotopic glioma models.Results: A highly expressed bispecific antibody with specificity to CD3 and EGFRvIII was generated (hEGFRvIII-CD3 bi-scFv). Antibody-induced T-cell activation, secretion of proinflammatory cytokines, and proliferation was robust and occurred exclusively in the presence of target antigen. hEGFRvIII-CD3 bi-scFv was potent and target-specific, mediating significant lysis of multiple malignant glioma cell lines and patient-derived malignant glioma samples that heterogeneously express EGFRvIII. In both subcutaneous and orthotopic models, well-engrafted, patient-derived malignant glioma was effectively treated despite heterogeneity of EGFRvIII expression; intravenous hEGFRvIII-CD3 bi-scFv administration caused significant regression of tumor burden (P < 0.0001) and significantly extended survival (P < 0.0001). Similar efficacy was obtained in highly infiltrative, syngeneic glioma models, and intravenously administered hEGFRvIII-CD3 bi-scFv localized to these orthotopic tumors.Conclusions: We have developed a clinically translatable bispecific antibody that redirects human T cells to safely and effectively treat malignant glioma. On the basis of these results, we have developed a clinical study of hEGFRvIII-CD3 bi-scFv for patients with EGFRvIII-positive malignant glioma. Clin Cancer Res; 24(15); 3611–31. ©2018 AACR.

Median survival for glioblastoma (GBM) remains ex vivo analyses from this study found no change in the capacity of CMV pp65-specific T cells to gain multiple effector functions or polyfunctionality, which has been associated with superior antitumor efficacy. Previous studies have shown that dendritic cells (DC) could further enhance tumor-specific CD8+ T-cell polyfunctionality in vivo when administered as a vaccine. Therefore, we hypothesized that vaccination with CMV pp65 RNA-loaded DCs would enhance the frequency of polyfunctional CMV pp65-specific CD8+ T cells after ATCT. Here, we report prospective results of a pilot trial in which 22 patients with newly diagnosed GBM were initially enrolled, of which 17 patients were randomized to receive CMV pp65-specific T cells with CMV-DC vaccination (CMV-ATCT-DC) or saline (CMV-ATCT-saline). Patients who received CMV-ATCT-DC vaccination experienced a significant increase in the overall frequencies of IFNγ+, TNFα+, and CCL3+ polyfunctional, CMV-specific CD8+ T cells. These increases in polyfunctional CMV-specific CD8+ T cells correlated (R = 0.7371, P = 0.0369) with overall survival, although we cannot conclude this was causally related. Our data implicate polyfunctional T-cell responses as a potential biomarker for effective antitumor immunotherapy and support a formal assessment of this combination approach in a larger randomized study.Significance: A randomized pilot trial in patients with GBM implicates polyfunctional T-cell responses as a biomarker for effective antitumor immunotherapy. Cancer Res; 78(1); 256–64. ©2017 AACR.

Glioblastoma is an immunologically ‘cold’ malignancy where few tumor infiltrating lymphocytes (TILs) are present. We sought to overcome this by ‘seeding’ the tumor with TILs via administration of autologous lymphocytes (ALT). We hypothesized that prior administration of ALT will enhance subsequent immunomodulatory therapies (e.g. immune checkpoint blockade (ICB) or co-stimulation). To evaluate this, we initially generated a pro-infiltrative TIL phenotype. CD45.1 splenocytes (C57/Bl6 background) were co-cultured with either IL2 and Concanavalin A (Con A) or IL7 and Con A for 5 days. Flow cytometric analysis found that both conditions resulted in similar CD4:CD8 ratios (20:80%) but IL7 co-culture upregulated expression of VLA-4, a pro migratory integrin. We then compared the entry of TILs into tumor bearing CNS when co-cultured with either IL2 or IL7. 8–10-week-old C57/Bl6 mice (n=5-6 per group) were implanted orthotopically with 30,000 CT2AvIII cells (syngeneic glioma) which established for 14 days. Mice received (1) CD45.1 lymphocytes activated with IL-7 and Con A (single intravenous (IV) injection, 1 × 107 cells) or (2) CD45.1 lymphocytes activated with IL2 and serial Con-A stimulation. Mice were sacrificed 3- and 48-hours following ALT and brains analysed for CD45.1+CD3+ populations. Groups were compared using a Mann-Whitney U test. Mice in Group 1 demonstrated significantly enhanced entry of CD8+ effector and memory T cells into tumor bearing hemispheres at both time points following administration compared to Group 2 (p = 0.005, p = 0.0159 respectively). By 48 hours post ALT, IL7 co-culture resulted in an average 810.1% expansion of CD8 T cell numbers in tumor bearing CNS compared to counts at Day 0. We then evaluated if mice with a TIL enriched CNS might be more responsive to ICB or co-stimulatory therapy. 8–10-week-old C57/Bl6 mice (n=5-7 per group) were implanted with 30,000 CT2AvIII cells that established over 10 days. On day 10, mice received IV ALT of either IL7 T cells only, or IL-7 and treatment courses of IP ICB (anti-PD1, anti-CTLA 4 or anti-41BB). Mice given ICB following IL7 ALT demonstrated a median survival (aPD1: 27 days, aCTLA-4: 25 days) similar to mice given IL7 ALT alone (25 days). However, 4/7 mice who received IL7 ALT and anti-41BB were still alive 45 days post tumor implantation (p=0.0004). Taken together, we demonstrated that certain ex vivo autologous lymphocyte co-culture conditions can generate a T cell population which infiltrates tumor bearing CNS in significant numbers. Further, we found that seeding a tumor with TILs prior to stimulatory therapy yields long term survival in vivo. Future work will focus on optimizing ex vivo culturing approaches for ALT and determine whether therapeutic efficacy is due to expansion of resident TILs at the tumor site, or ongoing recruitment of endogenous T cells from the periphery. Citation Format: Kirit Singh, Kelly M. Hotchkiss, Eliese Moelker, Gary E. Archer, John H. Sampson, Mustafa Khasraw. Systemic autologous lymphocyte transfer can enhance tumor-infiltrating lymphocyte infiltration in glioblastoma and license co-stimulatory immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4061.

The application of adoptive T cell therapies, including those using chimeric antigen receptor (CAR)-modified T cells, to solid tumors requires combinatorial strategies to overcome immune suppression associated with the tumor microenvironment. Here we test whether the inflammatory nature of oncolytic viruses and their ability to remodel the tumor microenvironment may help to recruit and potentiate the functionality of CAR T cells. Contrary to our hypothesis, VSVmIFNβ infection is associated with attrition of murine EGFRvIII CAR T cells in a B16EGFRvIII model, despite inducing a robust proinflammatory shift in the chemokine profile. Mechanistically, type I interferon (IFN) expressed following infection promotes apoptosis, activation, and inhibitory receptor expression, and interferon-insensitive CAR T cells enable combinatorial therapy with VSVmIFNβ. Our study uncovers an unexpected mechanism of therapeutic interference, and prompts further investigation into the interaction between CAR T cells and oncolytic viruses to optimize combination therapy., Oncolytic viruses promote an inflammatory response and elicit anti-tumor immunity. Here the authors show, unexpectedly, that the oncolytic virus, VSVIFNβ, induces type I interferon responses that, when combined with chimeric antigen receptor (CAR) T therapy, lead to the attrition of both CAR T and conventional T cells, thus dampening their anti-tumor activity.

Purpose: Chimeric antigen receptor (CAR) T cells have shown promise against solid tumors, but their efficacy has been limited, due in part, to immunosuppression by CD4+FoxP3+ regulatory T cells (Tregs). Although lymphodepletion is commonly used to deplete Tregs, these regimens are nonspecific, toxic, and provide only a narrow window before Tregs repopulate hosts. Importantly, CARs have also been shown to inadvertently potentiate Tregs by providing a source of IL2 for Treg consumption. We explored whether disruption of the IL2 axis would confer efficacy against solid tumors without the need for lymphodepletion. Experimental Design: We developed second- (CD28z) and third- (CD28-4-1BBz) generation CARs targeting EGFRvIII. To eliminate secretion of IL2, 2 amino acid substitutions were introduced in the PYAP Lck–binding motif of the CD28 domain (ΔCD28). We evaluated CARs against B16 melanomas expressing EGFRvIII. Results: CD28z CARs failed to engraft in vivo. Although 4-1BB addition improved expansion, CD28-4-1BBz CARs required lymphodepletion to treat solid tumors. CARs deficient in Lck signaling, however, significantly retarded tumor growth without a need for lymphodepletion and this was dependent on inclusion of 4-1BB. To evaluate CAR vulnerability to Tregs, we lymphodepleted mice and transferred CARs alone or with purified Tregs. Cotransfer with Tregs abrogated the efficacy of CD28-4-1BBz CARs, whereas the efficacy of ΔCD28-4-1BBz CARs remained unperturbed. Conclusions: In the absence of lymphodepletion, CARs targeting solid tumors are hindered by Treg immunosuppression and poor persistence. Here, CARs were modified to circumvent Treg suppression and to simultaneously improve in vivo engraftment. Modified CARs treated solid tumors without a need for lymphodepletion.

Personalized cancer vaccines targeting neoantigens arising from somatic missense mutations are currently being evaluated for the treatment of various cancers due to their potential to elicit a multivalent, tumor-specific immune response. Several cancers express a low number of neoantigens; in these cases, ensuring the immunotherapeutic potential of each neoantigen-derived epitope (neoepitope) is crucial. In this study, we discovered that therapeutic vaccines targeting immunodominant major histocompatibility complex (MHC) I-restricted neoepitopes require a conjoined helper epitope in order to induce a cytotoxic, neoepitope-specific CD8+ T-cell response. Furthermore, we show that the universally immunogenic helper epitope P30 can fulfill this requisite helper function. Remarkably, conjoined P30 was able to unveil immune and antitumor responses to subdominant MHC I-restricted neoepitopes that were, otherwise, poorly immunogenic. Together, these data provide key insights into effective neoantigen vaccine design and demonstrate a translatable strategy using a universal helper epitope that can improve therapeutic responses to MHC I-restricted neoepitopes.

INTRODUCTION While primary GBM is largely heterogeneous and devoid of homogeneously expressed neoantigens, mutant IDH1 (R132H) is a uniformly expressed hallmark in >70% of low grade gliomas. As such, IDH1 mutations represent a potentially valuable vaccination target. METHODS Here, we report an update on the immunogenicity results of the mutant IDH1 peptide vaccine alone and in combination with temozolomide (TMZ). In the phase I RESIST clinical trial (NCT02193347), patients with recurrent and resectable IDH1 R132H mutant grade 2 glioma received peptide vaccinations composed of 500 µg of mutant IDH1 peptide and 150 µg of GM-CSF mixed 1:1 with Montanide adjuvant prior to surgical resection. Vaccines 1, 2, and 3 were given 15 (+/-) 3 days apart. 7-12 days after vaccine 3, patients underwent standard of care tumor (SOC) resection. After resection, patients with grade 2 gliomas were given up to 15 doses of peptide vaccine in combination with TMZ regimens while patients with transformed grade 3 gliomas were given up to 15 doses of peptide vaccine in combination with SOC radiation therapy + TMZ regimens. T cell responses against the mutant peptide were measured after vaccine 3 using IFN-γ ELISPOT and intracellular flow cytometry for IL-2, TNFα,and IFNγ. RESULTS 3/20 patients were taken off the study before completion of study related activities. 1/20 patients progressed before completion of all vaccines. Out of 134 total doses of vaccine delivered, only one dose generated a grade 2 or higher injection site reaction according to the CTCAE guidelines. Vaccination with the mutant peptide led to an overall increase in IFN-γ+ spot-forming splenocytes specific to the mutant peptide (p=0.0408). CONCLUSION Administering the mutant IDH1 peptide vaccine in patients with recurrent IDH-mutant gliomas was able to induce anti-IDH1 R132H immune responses in this initial phase I study.

Replication-competent retrovirus (RCR) is a safety concern for individuals treated with retroviral gene therapy. RCR detection assays are used to detect RCR in manufactured vector, transduced cell products infused into research subjects, and in the research subjects after treatment. In this study, we reviewed 286 control (n = 4) and transduced cell products (n = 282) screened for RCR in the National Gene Vector Biorepository. The transduced cell samples were submitted from 14 clinical trials. All vector products were previously shown to be negative for RCR prior to use in cell transduction. After transduction, all 282 transduced cell products were negative for RCR. In addition, 241 of the clinical trial participants were also screened for RCR by analyzing peripheral blood at least 1 month after infusion, all of which were also negative for evidence of RCR infection. The majority of vector products used in the clinical trials were generated in the PG13 packaging cell line. The findings suggest that screening of the retroviral vector product generated in PG13 cell line may be sufficient and that further screening of transduced cells does not provide added value. Keywords: retrovirus, safety testing, replicating virus, lentivirus

Median survival for glioblastoma (GBM) remains Significance: A randomized pilot trial in patients with GBM implicates polyfunctional T-cell responses as a biomarker for effective antitumor immunotherapy. Cancer Res; 78(1); 256–64. ©2017 AACR.

INTRODUCTION Vaccination with dendritic cells (DCs) fares poorly in primary and recurrent glioblastoma (GBM). Moreover, GBM vaccine trials are often underpowered due to limited sample size. METHODS To address these limitations, we conducted three sequential clinical trials utilizing Cytomegalovirus (CMV)-specific DC vaccines in patients with primary GBM. Autologous DCs were generated and electroporated with mRNA encoding for the CMV protein pp65. Serial vaccination was given throughout adjuvant temozolomide cycles, and 111Indium radiolabeling was implemented to assess migration efficiency of DC vaccines. Patients were followed for median overall survival (mOS) and OS. RESULTS Our initial study was the phase II ATTAC study (NCT00639639; total n=12) with 6 patients randomized to vaccine site preconditioning with tetanus-diphtheria (Td) toxoid. This led to an expanded cohort trial (ATTAC-GM; NCT00639639) of 11 patients receiving CMV DC vaccines containing granulocyte-macrophage colony-stimulating factor (GM-CSF). Follow-up data from ATTAC and ATTAC-GM revealed 5-year OS rates of 33.3% (mOS 38.3 months; CI95 17.5-undefined) and 36.4% (mOS 37.7 months; CI95 18.2-109.1), respectively. ATTAC additionally revealed a significant increase in DC migration to draining lymph nodes following Td preconditioning (P=0.049). Increased DC migration was associated with OS (Cox proportional hazards model, HR=0.820, P=0.023). Td-mediated increased migration has been recapitulated in our larger confirmatory trial ELEVATE (NCT02366728) of 43 patients randomized to preconditioning (Wilcoxon rank sum, Td n=24, unpulsed DC n=19; 24h, P=0.031 and 48h, P=0.0195). In ELEVATE, median follow-up of 42.2 months revealed significantly longer OS in patients randomized to Td (P=0.026). The 3-year OS for Td-treated patients in ELEVATE was 34% (CI95 19-63%) compared to 6% given unpulsed DCs (CI95 1-42%). CONCLUSION We report reproducibility of our findings across three sequential clinical trials using CMV pp65 DCs. Despite their small numbers, these successive trials demonstrate consistent survival outcomes, thus supporting the efficacy of CMV DC vaccine therapy in GBM.

INTRODUCTION Cancer vaccines involve the administration of tumor-associated antigens into the host to generate anti-tumor T cell responses. Glioblastoma (GBM) is a disease with poor prognosis. GBM has a limited number of immunotherapeutic targets due to low mutational load, and is also highly heterogeneous; targeting a single antigen leads to antigen escape and tumor growth. METHODS VMDK mice were subcutaneously implanted with 750,000 SMA560 cells and on days 1 and 8 post implantation, mice were treated with 15 nmol of the universal helper epitope, P30, conjoined to the MHCI-restricted neoepitopes Odc1MHCI-P30 or Topbp1MHCI-P30. Human CD27 (hCD27) transgenic mice were intracranially implanted with CT2A-Odc1, followed by anti-CD27 and 15 nmol of Odc1MHCI-P30. B16.OVA or B16.F10 tumor cells were intracranially implanted in hCD27 mice and received SIINFEKL-P30 or Trp2-P30 conjoined peptides. Tumor growth, survival, or IFNγ secretion of splenic or tumor-infiltrating cells was assessed. RESULTS Unlike Odc1MHCI mixed with P30, conjoined Odc1MHCI-P30 had equivalent immunogenicity and anti-tumor efficacy to that observed with native long Odc1 peptide. Native long peptide of Topbp1 did not elicit an antitumor response, yet Topbp1MHCI-P30 caused an increase in numbers of IFNγ-secreting splenocytes and a decrease in tumor growth and similar to that seen with Odc1MHCI-P30 . Anti-CD27 treatment significantly increased numbers of IFNγ secreting splenocytes in Odc1MHCI-P30 vaccinated hCD27 mice, and the use of anti-CD27 with Odc1MHCI-P30 achieved long-term survival in 90% of tumor bearing hCD27 mice. Anti-CD27 synergized with SIINFEKL-P30 and Trp2-P30 to significantly improve survival after administration of these peptides. CONCLUSIONS Our work shows that poorly immunogenic neoantigens can be conjoined to P30 and used to generate an anti-tumor response in mouse models of GBM, and anti-tumor responses of conjoined peptides can be enhanced with anti-CD27 treatment. Together, these data demonstrate the efficacy of neoantigen vaccines for the treatment of heterogeneous GBM.

INTRODUCTION Approximately 30% of glioblastomas harbor the tumor specific EGFRvIII mutation. hEGFRvIII-CD3 bi-scFv (Brain Bi-Specific T Cell Engager - BRiTE) is a novel bispecific antibody construct which can redirect a patient’s entire repertoire of T cells (TCR-agnostic) to specifically lyse EGFRvIII-positive tumor cells. Compared to CAR-T therapy, it offers a highly potent yet off-the-shelf approach for glioblastoma. BRiTE is now entering Phase I clinical trials (NCT04903795) and will be trialed as a monotherapy or with autologous T cell infusion. Migration of T cell binding macromolecules across the blood-brain barrier may be facilitated by activated T cells which adhere to the microvascular endothelium and enter the brain parenchyma. Concurrent administration of activated T cells could therefore enhance trafficking of BRiTE and other antibody-based macromolecules into the intracerebral compartment. HYPOTHESIS We hypothesize that treatment of EGFRvIII-positive WHO grade IV malignant glioma with BRiTE alone or with peripheral T-cell infusion is safe and can induce objective tumor shrinkage at tolerable doses. TRIAL DESIGN/OBJECTIVES A maximum of 18 patients with newly diagnosed or recurrent WHO grade IV malignant EGFRvIII+ glioma will be enrolled after undergoing standard of care treatment. The primary objective will be evaluating the safety of BRiTE alone and with autologous T cell infusion. Patients will receive a single BRiTE infusion, followed 14 days later by an infusion of activated autologous T cells and a second bolus BRiTE infusion. Dose escalation and de-escalation will be managed using a Bayesian optimal interval design. Secondary objectives will be to describe clinical benefit as determined by objective response rate per mRANO criteria, and to evaluate BRiTE pharmacokinetics in serum. CONCLUSION We describe a first-in-human trial of bispecific T cell engager therapy for glioblastoma. We also describe our novel approach for enhancing intracranial penetration of BRiTE using autologous T cell infusion.

Purpose: Patients with glioblastoma have less than 15-month median survival despite surgical resection, high-dose radiation, and chemotherapy with temozolomide. We previously demonstrated that targeting cytomegalovirus pp65 using dendritic cells (DC) can extend survival and, in a separate study, that dose-intensified temozolomide (DI-TMZ) and adjuvant granulocyte macrophage colony-stimulating factor (GM-CSF) potentiate tumor-specific immune responses in patients with glioblastoma. Here, we evaluated pp65-specific cellular responses following DI-TMZ with pp65-DCs and determined the effects on long-term progression-free survival (PFS) and overall survival (OS). Experimental Design: Following standard-of-care, 11 patients with newly diagnosed glioblastoma received DI-TMZ (100 mg/m2/d × 21 days per cycle) with at least three vaccines of pp65 lysosome–associated membrane glycoprotein mRNA-pulsed DCs admixed with GM-CSF on day 23 ± 1 of each cycle. Thereafter, monthly DI-TMZ cycles and pp65-DCs were continued if patients had not progressed. Results: Following DI-TMZ cycle 1 and three doses of pp65-DCs, pp65 cellular responses significantly increased. After DI-TMZ, both the proportion and proliferation of regulatory T cells (Tregs) increased and remained elevated with serial DI-TMZ cycles. Median PFS and OS were 25.3 months [95% confidence interval (CI), 11.0–∞] and 41.1 months (95% CI, 21.6–∞), exceeding survival using recursive partitioning analysis and matched historical controls. Four patients remained progression-free at 59 to 64 months from diagnosis. No known prognostic factors [age, Karnofsky performance status (KPS), IDH-1/2 mutation, and MGMT promoter methylation] predicted more favorable outcomes for the patients in this cohort. Conclusions: Despite increased Treg proportions following DI-TMZ, patients receiving pp65-DCs showed long-term PFS and OS, confirming prior studies targeting cytomegalovirus in glioblastoma. Clin Cancer Res; 23(8); 1898–909. ©2017 AACR.

BACKGROUND Low grade gliomas (LGGs) represent 10–15% of glial tumors in adults and while LGG patients have a better prognosis over high-grade gliomas, these cancers ultimately recur and transform to more aggressive tumors. Isocitrate dehydrogenase 1 (IDH1) is commonly mutated in LGG, and when mutated, it is the oncogenic driver by leading to the production of oncometabolite 2-hydroxyglutarate (2-HG). We developed a phase 1 study for recurrent resectable IDH1 mutant LGG utilizing a tumor-specific peptide vaccine targeting IDH1 mutant protein that spans the mutated region of IDHR132H (PEPIDH1M vaccine). METHODS We performed a phase 1, single-center, clinical trial in recurrent resectable IDH1 mutant WHO grade II glioma patients. Subjects received three PEPIDH1M vaccine q2wks and then proceeded to surgical resection. If subject’s tumor retained grade II status, then the subject proceeded with 12 cycles of daily TMZ (50 mg/m2 X 28 days) and PEPIDH1M vaccine (12 injections q4wks). If subject’s tumor transformed to grade III, then subject proceeded to radiation therapy (RT) with concurrent TMZ followed by 12 cycles of daily TMZ (50 mg/m2 X 28 days) and PEPIDH1M vaccine (12 injections q4wks). Primary endpoint was safety of PEPIDH1M vaccine in combination with adjuvant TMZ and/or XRT/TMZ and evaluable subjects needed to receive ≥6 PEPIDH1M vaccines. We assessed safety using CTCAE 4.03. RESULTS We enrolled 24 recurrent LGG subjects with mean age of 43.8 yrs (sd=11.4 yrs). Twenty subjects completed ≥6 PEPIDH1M vaccines. Most common related toxicity was grade 1 injection site reaction (N=20) and skin induration (n=17) with no grade 3–4 related toxicities. CONCLUSIONS PEPIDH1M vaccine in combination with surgical resection, daily TMZ and/or RT + TMZ with daily TMZ was safe and well tolerated in recurrent IDH1 mutant LGG. We are currently exploring secondary/correlative endpoints including immunogenicity of PEPIDH1M vaccine and magnetic resonance spectroscopy for 2-HG.

BACKGROUND Recurrent medulloblastoma and malignant glioma are lethal tumors that are virtually incurable. The cytomegalovirus (CMV) antigen pp65 is ubiquitously expressed on medulloblastoma and malignant glioma but not on healthy brain. We evaluated autologous CMV pp65 RNA-pulsed dendritic cell (DC) vaccines in children and young adults in a phase I trial. METHODS Circulating monocytes were harvested using leukapheresis, differentiated into DCs, matured, and pulsed with pp65 RNA using electroporation. DCs were packaged into vaccines (2x107DC/vaccine) and administered intradermally following tetanus-diphtheria toxoid site preconditioning every 2 weeks x3, then monthly. The primary objectives of the study were to establish the feasibility of generating at least 3 vaccines and safety. An exploratory objective was to evaluate the ability of vaccination to create and enhance patient pp65-specific T cell responses. RESULTS Eleven patients were enrolled with medulloblastoma (n=3) or glioblastoma (n=8). Ages ranged from 9–30 years old (mean 15.5y). Ten of 11 patients (91%) generated at least 3 vaccines (mean 6.2). Eight patients received at least 3 vaccines. To date, 4 patients have received all generated vaccines without progression, 4 patients have progressed, and 2 patients are still receiving vaccines. There have not been any severe adverse events probably or definitely related to vaccines. More mature data will be presented at ISPNO. CONCLUSIONS Leukapheresis and monocyte differentiation is a feasible strategy for generating adequate DCs for active immunization in children with malignant brain tumors. CMV pp65 RNA-pulsed DCs are well-tolerated and immunogenic. Efficacy endpoints will be evaluated in a subsequent phase II trial.

We recently reported the development of a fully-human, CD3-binding bispecific antibody for immunotherapy of malignant glioma. To translate this therapeutic (hEGFRvIII-CD3- bi-scFv) to clinical trials and to help further the translation of other similar CD3-binding therapeutics, some of which are associated with neurologic toxicities, we performed a good laboratory practice (GLP) toxicity study to assess for potential behavioral, chemical, hematologic, and pathologic toxicities including evaluation for experimental autoimmune encephalomyelitis (EAE). To perform this study, male and female C57/BL6 mice heterozygous for the human CD3 transgene (20/sex) were allocated to one of four designated groups. All animals were administered one dose level of hEGFRvIII-CD3 bi-scFv or vehicle control. Test groups were monitored for feed consumption, changes in body weight, and behavioral disturbances including signs of EAE. Urinalysis, hematologic, and clinical chemistry analysis were also performed. Vehicle and test chemical-treated groups were humanely euthanized 48 hours or 14 days following dose administration. Complete gross necropsy of all tissues was performed, and selected tissues plus all observed gross lesions were collected and evaluated for microscopic changes. This included hematoxylin-eosin histopathological evaluation and Fe-ECR staining for myelin sheath enumeration. There were no abnormal clinical observations or signs of EAE noted during the study. There were no statistical changes in food consumption, body weight gain, or final body weight among groups exposed to hEGFRvIII-CD3 bi-scFv compared to the control groups for the 2- and 14-day timepoints. There were statistical differences in some clinical chemistry, hematologic and urinalysis endpoints, primarily in the females at the 14-day timepoint (hematocrit, calcium, phosphorous, and total protein). No pathological findings related to hEGFRvIII-CD3 bi-scFv administration were observed. A number of gross and microscopic observations were noted but all were considered to be incidental background findings. The results of this study allow for further translation of this and other important CD3 modulating bispecific antibodies.

Patients undergoing current treatment for glioblastoma (GBM) still face a poor prognosis with a median survival of ~16 months. Current therapy is incapacitating and limited by non-specific toxicity to the surrounding brain. We have developed an immunotherapeutic approach that selectively targets GBM by redirecting the patients’ own macrophages present within the natural glioma milieu (representing up to 30% of the tumor volume) towards the tumor in an antigen-specific manner. This novel molecule, called bispecific activator of macrophages (BAM), bridges the macrophages with the tumor cells through the binding of CD64 and EGFRvIII, a tumor-specific antigen, respectively. We generated a stable fully human EGFRvIII-specific BAM Chinese hamster ovary cell line, and confirmed expression of BAM by western blot. Binding to CD64 and EGFRvIII was confirmed by flow cytometry on human CD64(+) (hCD64) transgenic murine macrophages and EGFRvIII(+) gliomas. Simultaneous BAM engagement of hCD64 on macrophages, and EGFRvIII led to the induction of macrophage tumoricidal activity measured by reactive oxygen species release. Furthermore, in vivo BAMadministration into glioma bearing mice significantly reduced tumor growth. In conclusion, we have developed a novel immunotherapeutic approach capable of harnessing the body’s own tumoricidal capacity by redirecting macrophages towards tumors via the administration of a bispecific activator of macrophages (BAM). While further studies are needed, this therapy offers promising results for the treatment of GBM.

Two of our previous clinical trials targeting Cytomegalovirus (CMV) protein pp65 in newly diagnosed glioblastoma (GBM) using pp65-specific dendritic cell (DC) vaccines have yielded long term survival rates greatly exceeding those predicted with standard of care. In the ATTAC trial (IND 12839), patients received sequential DC vaccination throughout monthly cycles of standard temozolomide (STD-TMZ) 200mg/m(2)/d for 5d and were randomized to one of two vaccine site preconditioning regimens. Significantly higher rates of DC migration (p=0.04) and survival (p=0.013) were observed in patients randomized to tetanus preconditioning, with half of the cohort living to nearly five years after diagnosis. In the ATTAC-GM trial, we treated a subsequent cohort with dose-intensified TMZ (DI-TMZ) 100mg/m(2)/d for 21d prior to and throughout vaccination with GM-CSF-containing DC vaccines and observed extraordinarily prolonged progression-free survival (PFS) and overall survival (OS) (median 25.3 and 41.1 months, respectively). In this trial, DI-TMZ-induced lymphopenia facilitated homeostatic expansion of pp65 responses after an initial DI-TMZ cycle but later ablated T cell responses when monthly cycles were reintroduced. The benefit of DI-TMZ thus warrants further study in a larger series of patients. Here we describe the initiation of a validation trial, “I-ATTAC: Improved Anti-Tumor Immunotherapy Targeted Against Cytomegalovirus in Patients with Newly-Diagnosed WHO Grade IV Unmethylated Glioma” (IND-16301). This prospective single arm Phase 2 trial of 48 patients will validate our findings that tetanus preconditioning and GM-CSF in conjunction with pp65-DCs extends OS in patients with newly diagnosed, unmethylated GBM. By withholding additional TMZ cycles in this patient population, we hypothesize that CMV immune responses will not be abrogated as previously observed, and that this greater expansion will translate into further prolonged survival (primary objective). A series of secondary objectives will be evaluated for the association between increased DC migration, systemic mediators of tetanus preconditioning, and T cell polyfunctionality with survival.

Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) is an effective immunotherapy for B-cell malignancies but has failed in some solid tumors clinically. Intracerebral tumors may pose challenges that are even more significant. In order to devise a treatment strategy for patients with glioblastoma (GBM), we evaluated CARs as a monotherapy in a murine model of GBM. CARs exhibited poor expansion and survival in circulation and failed to treat syngeneic and orthotopic gliomas. We hypothesized that CAR engraftment would benefit from host lymphodepletion prior to immunotherapy and that this might be achievable by using temozolomide (TMZ), which is standard treatment for these patients and has lymphopenia as its major side effect. We modelled standard of care temozolomide (TMZSD) and dose-intensified TMZ (TMZDI) in our murine model. Both regimens are clinically approved and provide similar efficacy. Only TMZDI pretreatment prompted dramatic CAR proliferation and enhanced persistence in circulation compared to treatment with CARs alone or TMZSD + CARs. Bioluminescent imaging revealed that TMZDI + CARs induced complete regression of 21-day established brain tumors, which correlated with CAR abundance in circulation. Accordingly, TMZDI + CARs significantly prolonged survival and led to long-term survivors. These findings are highly consequential, as it suggests that GBM patients may require TMZDI as first line chemotherapy prior to systemic CAR infusion to promote CAR engraftment and antitumor efficacy. On this basis, we have initiated a phase I trial in patients with newly diagnosed GBM incorporating TMZDI as a preconditioning regimen prior to CAR immunotherapy (NCT02664363).

The epidermal growth factor receptor variant III deletion mutation, EGFRvIII, results in a constitutively activated receptor with a novel, highly immunogenic extracelluar epitope. EGFRvIII is present in 25%–30% of glioblastomas1 but is not significantly expressed in healthy tissue. Expression of EGFRvIII correlates with increased tumorigenicity in mouse models.2 In glioblastoma, EGFRvIII has been associated with poor long-term survival, independent of other known significant prognostic factors, such as gross total resection (GTR).3–6 EGFRvIII expression is often heterogeneous in glioblastoma specimens, but EGFRvIII+ cells may influence neighboring EGFRvIII–tumor cells through cytokines and microvesicles, providing a proliferative signal even to nonexpressing cells.7–9 EGFRvIII is also frequently expressed in glioblastoma tumor stem cells.10,11 EGFRvIII and isocitrate dehydrogenase (IDH) 1/2 mutations, the latter associated with long-term survival, rarely coexist in the same patient.12,13 Rindopepimut vaccine consists of the unique 13 amino acid sequence created by the in-frame deletion of EGFRvIII, chemically conjugated to keyhole limpet hemocyanin (KLH) as described by Heimberger and colleagues.14 Rindopepimut is designed to generate a specific immune response against EGFRvIII+ tumor cells, an approach which may be particularly relevant for glioblastoma, where diffuse infiltration of tumor into healthy white matter presents a treatment challenge. Preclinical models have demonstrated that induction of humoral and cellular anti-EGFRvIII immune responses can be effective against EGFRvIII+ intracranial tumors.14 In 2 small single-arm phase II trials conducted at MD Anderson and Duke University (“ACTIVATE” and “ACT II”),6,15 rindopepimut was well tolerated in patients with resected, EGFRvIII+ glioblastoma with promising progression-free survival (PFS) and overall survival (OS) compared with a contemporary cohort of patients matched for major study eligibility. In addition, the vaccine elicited robust anti-EGFRvIII immune responses despite concurrent temozolomide chemotherapy, and EGFRvIII was routinely eliminated in posttreatment tumor samples obtained at recurrence. The current study (“ACT III”) was performed to confirm these results in a larger, multicenter trial.

Purpose: Conventional therapy for malignant glioma fails to specifically target tumor cells. In contrast, substantial evidence indicates that if appropriately redirected, T cells can precisely eradicate tumors. Here we report the rational development of a fully human bispecific antibody (hEGFRvIII-CD3 bi-scFv) that redirects human T cells to lyse malignant glioma expressing a tumor-specific mutation of the EGFR (EGFRvIII). Experimental Design: We generated a panel of bispecific single-chain variable fragments and optimized design through successive rounds of screening and refinement. We tested the ability of our lead construct to redirect naïve T cells and induce target cell–specific lysis. To test for efficacy, we evaluated tumor growth and survival in xenogeneic and syngeneic models of glioma. Tumor penetrance following intravenous drug administration was assessed in highly invasive, orthotopic glioma models. Results: A highly expressed bispecific antibody with specificity to CD3 and EGFRvIII was generated (hEGFRvIII-CD3 bi-scFv). Antibody-induced T-cell activation, secretion of proinflammatory cytokines, and proliferation was robust and occurred exclusively in the presence of target antigen. hEGFRvIII-CD3 bi-scFv was potent and target-specific, mediating significant lysis of multiple malignant glioma cell lines and patient-derived malignant glioma samples that heterogeneously express EGFRvIII. In both subcutaneous and orthotopic models, well-engrafted, patient-derived malignant glioma was effectively treated despite heterogeneity of EGFRvIII expression; intravenous hEGFRvIII-CD3 bi-scFv administration caused significant regression of tumor burden (P < 0.0001) and significantly extended survival (P < 0.0001). Similar efficacy was obtained in highly infiltrative, syngeneic glioma models, and intravenously administered hEGFRvIII-CD3 bi-scFv localized to these orthotopic tumors. Conclusions: We have developed a clinically translatable bispecific antibody that redirects human T cells to safely and effectively treat malignant glioma. On the basis of these results, we have developed a clinical study of hEGFRvIII-CD3 bi-scFv for patients with EGFRvIII-positive malignant glioma. Clin Cancer Res; 24(15); 3611–31. ©2018 AACR.

BACKGROUND Recurrent medulloblastoma and malignant glioma are virtually incurable. In children, these tumors are often disseminated or occur in deep midline structures that preclude surgery and are not amenable to highly inflammatory treatments. Active immunization is an attractive immunotherapeutic approach. We are conducting a phase I trial evaluating autologous CMV pp65 RNA-pulsed dendritic cell (DC) vaccines in children with recurrent malignant glioma or medulloblastoma. A primary objective is establishing the feasibility of using leukapheresis to harvest sufficient monocytes in order to generate at least 3 autologous DC vaccines. METHODS For children unable to receive two 16-guage IVs, a temporary pheresis catheter is placed in the jugular vein under anesthesia. Leukapheresis is performed with the goal of processing 3 total blood volumes. Circulating monocytes are collected and cultured for 7 days in media supplemented with GM-CSF and IL-4, then matured into dendritic cells and loaded with antigen. The target dose for a single vaccine is 2 x 107 dendritic cells. RESULTS To date, five patients (ages 9, 13, 16, 17, 30) have undergone leukapheresis. All patients tolerated leukapheresis without complications, with run times between 3 to 6 hours depending on achievable pheresis flow rate. Patients generated 2, 3, 7, 8, 8, and 8 vaccines. The medulloblastoma patient who generated 2 vaccines underwent a second leukapheresis that generated 8 vaccines. In this patient, although the initial leukapheresis collected a high number of monocytes, the conversion rate to mature DCs was low. Thusfar, there has not been a strong correlation between the number of monocytes collected and the eventual number of mature dendritic cells. CONCLUSIONS Leukapheresis is tolerable for younger children and appears to be a viable strategy for harvesting sufficient monocytes for creating DC vaccines. Conversion rates between harvested monocytes and the final number of mature dendritic cells vary.

Adoptive cell transfer (ACT) using ex vivo-expanded anti-tumor T cells such as tumor-infiltrated lymphocytes or genetically engineered T cells potently eradicates established tumors. However, these two approaches possess obvious limitations. Therefore, we established a novel methodology using total tumor RNA (ttRNA) to prime dendritic cells (DC) as a platform for the ex vivo generation of anti-tumor T cells. We evaluated the antigen-specific expansion and recognition of T cells generated by the ttRNA–DC–T platform, and directly modulated the differentiation status of these ex vivo-expanded T cells with a cytokine cocktail. Furthermore, we evaluated the persistence and in vivo anti-tumor efficacy of these T cells through murine xenograft and syngeneic tumor models. During ex vivo culture, IL-2 preferentially expanded CD4 subset, while IL-7 enabled homeostatic proliferation from the original precursors. T cells tended to lose CD62L during ex vivo culture using IL-2; however, IL-12 could maintain high levels of CD62L by increasing expression on effector T cells (Tem). In addition, we validated that OVA RNA–DC only selectively expanded T cells in an antigen-specific manner. A cytokine cocktail excluding the use of IL-2 greatly increased CD62Lhigh T cells which specifically recognized tumor cells, engrafted better in a xenograft model and exhibited superior anti-tumor activities in a syngeneic intracranial model. ACT using the ex vivo ttRNA–DC–T platform in conjunction with a cytokine cocktail generated potent CD62Lhigh anti-tumor T cells and imposes a novel T cell-based therapeutic with the potential to treat brain tumors and other cancers.

Background. Many cancers, including melanoma, exclusively express constitutive proteasomes (cPs) and are unable to express immunoproteasomes (iPs). In contrast, mature DCs used for immunotherapy exclusively express iPs. Since proteasomes generate peptides presented by HLA class I molecules, we hypothesized that mature melanoma antigen–loaded DCs engineered to process antigens through cPs would be superior inducers of antimelanoma immunity in vivo. Methods. Subjects with metastatic melanoma were vaccinated with mature DCs transfected with RNAs encoding melanoma antigens MART1, MAGE-3, gp100, and tyrosinase. These DCs were derived from monocytes that were untransfected (Arm A; n = 4), transfected with control siRNA (Arm B; n = 3), or transfected with siRNAs targeting the 3 inducible iP subunits (Arm C; n = 5). Results. Vaccination stimulated antigen-specific T cell responses in all subjects, which peaked after 3–4 vaccinations, but remained elevated in Arm C subjects. Also in Arm C, circulating melanoma cell levels (as detected by quantitative PCR) fell, and T cell lytic activity against autologous melanoma was induced. In HLA-A2+ subjects, CD8+ T cells that bound tetramers loaded with cP-derived melanoma antigenic peptides were found in the peripheral blood only in Arm C subjects. Of 2 subjects with active disease (both in Arm C), one had a partial clinical response, while the other, who exhibited diffuse dermal and soft tissue metastases, had a complete response. Conclusion. These results suggest that the efficacy of melanoma DC–based immunotherapy is enhanced when tumor antigen–loaded DCs used for vaccination express cPs. Trial registration. Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00672542","term_id":"NCT00672542"}}NCT00672542. Funding. Duke Clinical Research Institute/Duke Translational Medicine Institute, Duke Melanoma Consortium, and Duke University Department of Surgery.

Epidermal growth factor receptor variant III (EGFRvIII) is a tumor-specific mutation widely expressed in glioblastoma multiforme (GBM) and other neoplasms, but absent from normal tissues. Immunotherapeutic targeting of EGFRvIII could eliminate neoplastic cells more precisely but may be inhibited by concurrent myelosuppressive chemotherapy like temozolomide (TMZ), which produces a survival benefit in GBM. A phase II, multicenter trial was undertaken to assess the immunogenicity of an experimental EGFRvIII-targeted peptide vaccine in patients with GBM undergoing treatment with serial cycles of standard-dose (STD) (200 mg/m(2) per 5 days) or dose-intensified (DI) TMZ (100 mg/m(2) per 21 days). All patients receiving STD TMZ exhibited at least a transient grade 2 lymphopenia, whereas those receiving DI TMZ exhibited a sustained grade 3 lymphopenia (

Purpose Immunologic targeting of tumor-specific gene mutations may allow precise eradication of neoplastic cells without toxicity. Epidermal growth factor receptor variant III (EGFRvIII) is a constitutively activated and immunogenic mutation not expressed in normal tissues but widely expressed in glioblastoma multiforme (GBM) and other neoplasms. Patients and Methods A phase II, multicenter trial was undertaken to assess the immunogenicity of an EGFRvIII-targeted peptide vaccine and to estimate the progression-free survival (PFS) and overall survival (OS) of vaccinated patients with newly diagnosed EGFRvIII-expressing GBM with minimal residual disease. Intradermal vaccinations were given until toxicity or tumor progression was observed. Sample size was calculated to differentiate between PFS rates of 20% and 40% 6 months after vaccination. Results There were no symptomatic autoimmune reactions. The 6-month PFS rate after vaccination was 67% (95% CI, 40% to 83%) and after diagnosis was 94% (95% CI, 67% to 99%; n = 18). The median OS was 26.0 months (95% CI, 21.0 to 47.7 months). After adjustment for age and Karnofsky performance status, the OS of vaccinated patients was greater than that observed in a control group matched for eligibility criteria, prognostic factors, and temozolomide treatment (hazard ratio, 5.3; P = .0013; n = 17). The development of specific antibody (P = .025) or delayed-type hypersensitivity (P = .03) responses to EGFRvIII had a significant effect on OS. At recurrence, 82% (95% CI, 48% to 97%) of patients had lost EGFRvIII expression (P < .001). Conclusion EGFRvIII-targeted vaccination in patients with GBM warrants investigation in a phase III, randomized trial.

Object Convection-enhanced delivery (CED) is a novel intracerebral drug delivery technique with considerable promise for delivering therapeutic agents throughout the CNS. Despite this promise, Phase III clinical trials employing CED have failed to meet clinical end points. Although this may be due to inactive agents or a failure to rigorously validate drug targets, the authors have previously demonstrated that catheter positioning plays a major role in drug distribution using this technique. The purpose of the present work was to retrospectively analyze the expected drug distribution based on catheter positioning data available from the CED arm of the PRECISE trial. Methods Data on catheter positioning from all patients randomized to the CED arm of the PRECISE trial were available for analyses. BrainLAB iPlan Flow software was used to estimate the expected drug distribution. Results Only 49.8% of catheters met all positioning criteria. Still, catheter positioning score (hazard ratio 0.93, p = 0.043) and the number of optimally positioned catheters (hazard ratio 0.72, p = 0.038) had a significant effect on progression-free survival. Estimated coverage of relevant target volumes was low, however, with only 20.1% of the 2-cm penumbra surrounding the resection cavity covered on average. Although tumor location and resection cavity volume had no effect on coverage volume, estimations of drug delivery to relevant target volumes did correlate well with catheter score (p < 0.003), and optimally positioned catheters had larger coverage volumes (p < 0.002). Only overall survival (p = 0.006) was higher for investigators considered experienced after adjusting for patient age and Karnofsky Performance Scale score. Conclusions The potential efficacy of drugs delivered by CED may be severely constrained by ineffective delivery in many patients. Routine use of software algorithms and alternative catheter designs and infusion parameters may improve the efficacy of drugs delivered by CED.

Given the highly infiltrative growth pattern of malignant glioma and the lack of specificity associated with currently available treatment regimens, alternative strategies designed to eradicate cancer cells while limiting collateral toxicity in normal tissues remain a high priority. To this end, the development of specific immunotherapies against targeted neoplastic cells represents a promising approach. The epidermal growth factor receptor class III variant (EGFRvIII), a constitutively activated mutant of the wild-type tyrosine kinase, is present in a substantial proportion of malignant gliomas and other human cancers, yet completely absent from normal tissues. This receptor variant consists of an in-frame deletion, the translation of which produces an extracellular junction with a novel glycine residue, flanked by amino acid sequences that are not typically adjacent in the normal protein. In this review, both preclinical and early clinical development of a peptide vaccine directed against this portion of the EGFRvIII antigenic domain are recapitulated. Following vaccination, our group has demonstrated potent, redirected cellular and humoral immunity against cancer cells expressing the mutant receptor without significant toxicity. Additionally, the corresponding therapeutic outcomes observed in these studies lend credence to the potential role of peptide-based vaccination strategies among emerging antitumor immunotherapies in patients with malignant glioma.

Purpose: Activation of signal transducers and activators of transcription 3 (STAT3) has been identified as a central mediator of melanoma growth and metastasis. We hypothesized that WP1066, a novel STAT3 blockade agent, has marked antitumor activity, even against the melanoma metastasis to brain, a site typically refractory to therapies. Experimental Design: The antitumor activities and related mechanisms of WP1066 were investigated both in vitro on melanoma cell lines and in vivo on mice with subcutaneously syngeneic melanoma or with intracerebral melanoma tumors. Results: WP1066 achieved an IC50 of 1.6, 2.3, and 1.5 μmol/L against melanoma cell line A375, B16, and B16EGFRvIII, respectively. WP1066 suppressed the phosphorylation of Janus-activated kinase 2 and STAT3 (Tyr705) in these cells. Tumor growth in mice with subcutaneously established syngeneic melanoma was markedly inhibited by WP1066 compared with that in controls. Long-term survival (>78 days) was observed in 80% of mice with established intracerebral syngeneic melanoma treated with 40 mg/kg of WP1066 in contrast to control mice who survived for a median of 15 days. Although WP1066 did not induce immunologic memory or enhance humoral responses to EGFRvIII, this compound reduced the production of immunosuppressive cytokines and chemokines (transforming growth factor-β, RANTES, MCP-1, vascular endothelial growth factor), markedly inhibited natural and inducible Treg proliferation, and significantly increased cytotoxic immune responses of T cells. Conclusions: The antitumor cytotoxic effects of WP1066 and its ability to induce antitumor immune responses suggest that this compound has potential for the effective treatment of melanoma metastatic to brain.

Cytotoxic chemotherapy that induces lymphopenia is predicted to ablate the benefits of active antitumor immunization. Temozolomide is an effective chemo-therapeutic agent for patients with glioblastoma multiforme, but it induces significant lymphopenia. Although there is monthly fluctuation of the white blood cell count, specifically the CD4 and CD8 counts, there was no cumulative decline in the patient described in this case report. Depriving patients of this agent, in order to treat with immunotherapy, is controversial. Despite conventional dogma, we demonstrated that chemotherapy and immunotherapy can be delivered concurrently without negating the effects of immunotherapy. In fact, the temozolomide-induced lymphopenia may prove to be synergistic with a peptide vaccine secondary to inhibition of regulatory T cells or their delayed recovery.

Immunotoxins as anti-cancer therapeutics have several potential advantages over conventional agents including a high specificity, extraordinary potency, and a lack of an identified mechanism for resistance. It has been clearly demonstrated that Pseudomonas-based immunotoxins have a direct cytotoxic effect. However, delayed and often dramatic antitumor responses seen in human studies with targeted toxins led us to hypothesize that immunologic responses may be a secondary mechanism that enhances the therapeutic efficacy of these novel drugs.This hypothesis was tested in a murine system using an immunotoxin, MR1-1 [MR1-1(dsFv)-PE38KDEL], that targets a syngeneic murine homologue of the tumor-specific human epidermal growth factor mutation, EGFRvIII, expressed on a murine cell line.Intratumoral treatment with MR1-1 eliminated EGFRvIII-expressing tumors (P0.0001). The antitumor activity of MR1-1 was dependent on the expression of EGFRvIII on some, but not all tumors cells, and was significantly inhibited in the absence of CD4+ (P = 0.0193) and CD8+ (P = 0.0193) T cells. MR1-1 induced EGFRvIII-specific immunity (P0.0005) and produced long lasting immunity against tumors expressing EGFRvIII as well as EGFRvIII-negative tumors.These data suggest that immunotoxins may not be strictly dependent on direct cytotoxicity for their efficacy, but may also be potent inducers of antitumor immunity active even against cells that do not express the targeted antigen.

Purpose: Analyses of T-cell mRNA expression profiles in glioblastoma multiforme has not been previously reported but may help to define and characterize the immunosuppressed phenotype in patients with this type of cancer.Experimental Design: We did microarray studies that have shown significant and fundamental differences in the expression profiles of CD4+ and CD8+ T cells and immunosuppressive CD4+CD25+CD45RO+FoxP3+ regulatory T cells (Treg) from normal healthy volunteers compared with patients with newly diagnosed glioblastoma multiforme. For these investigations, we isolated total RNA from enriched CD4+ and CD8+ T cell or Treg cell populations from age-matched individuals and did microarray analyses.Results: ANOVA and principal components analysis show that the various T cell compartments exhibit consistently similar mRNA expression profiles among individuals within either healthy or brain tumor groups but reflect significant differences between these groups. Compared with healthy volunteers, CD4+ and CD8+ T cells from patients with glioblastoma multiforme display coordinate down-regulation of genes involved in T cell receptor ligation, activation, and intracellular signaling. In contrast, Tregs from patients with glioblastoma multiforme exhibit increased levels of transcripts involved in inhibiting host immunity.Conclusion: Our findings support the notion that key differences between expression profiles in T-cell populations from patients with glioblastoma multiforme results from differential expression of the immunologic transcriptome, such that a limited number of genes are principally important in producing the dysregulated T-cell phenotype.

PURPOSE: The toxicity and antitumor activity of regional intrathecal delivery of an oncolytic recombinant poliovirus, PVS-RIPO, was evaluated in rodent models of glioblastoma multiforme neoplastic meningitis. EXPERIMENTAL DESIGN: To evaluate for toxicity, PVS-RIPO was administered into the spinal cord of transgenic mice that express the human poliovirus receptor, CD155, and into the intrathecal space of athymic rats without tumor. To evaluate efficacy, two different doses of PVS-RIPO were administered intrathecally 3 days after athymic rats were inoculated intrathecally with an aggressive human glioblastoma multiforme xenograft. RESULTS: No clinical or histologic evidence of toxicity was found. In efficacy studies, median survival was increased by 174.47% from 8.5 days in the group treated with UV light-inactivated virus to 15 days in the rats treated with 1.0 x 10(7) plaque-forming units (pfu) of PVS-RIPO (P < 0.0001). A similar increase in median survival was seen in the group receiving 1.0 x 10(9) pfu PVS-RIPO (P < 0.0001); however, there was no statistically significant dose-response relationship (P = 0.345). In addition, 1 of 10 rats in lower-dose PVS-RIPO-treated group and 3 of 10 rats in higher-dose PVS-RIPO-treated group survived >60 days after tumor cell inoculation and had no evidence of residual tumor at autopsy. CONCLUSION: These results suggest that intrathecal treatment with PVS-RIPO may be useful for treatment of neoplastic meningitis in patients with glioblastoma multiforme and provides a rationale for clinical trials in this area.

Despite advancements in therapeutic regimens, the prognosis remains poor for patients with malignant gliomas. Specificity has been an elusive goal for current modalities, but immunotherapy has emerged as a potential means of designing more tumor-specific treatments. Dendritic cells (DC) are the specialized antigen presenting cells of the immune system and have served now as a platform for therapeutic immunizations against such cancers as lymphoma, multiple myeloma, melanoma, prostate cancer, renal cell carcinoma, non-small cell lung carcinoma, colon cancer, and even malignant gliomas. DC-based immunizations offer a number of advantages over traditional immunotherapeutic approaches to brain tumors, approaches that have proved promising despite concerns over central nervous system immune privilege and glioma-mediated immunosuppression. The future success of clinical trials will depend on the optimization and standardizing of procedures for DC generation, loading, and administration.

TP-38 is a recombinant chimeric targeted toxin composed of the EGFR binding ligand TGF-α and a genetically engineered form of the Pseudomonas exotoxin, PE-38. After in vitro and in vivo animal studies that showed specific activity and defined the maximum tolerated dose (MTD), we investigated this agent in a Phase I trial. The primary objective of this study was to define the MTD and dose limiting toxicity of TP-38 delivered by convection-enhanced delivery in patients with recurrent malignant brain tumors. Twenty patients were enrolled in the study and doses were escalated from 25ng/mL to 100 with a 40mL infusion volume delivered by two catheters. One patient developed Grade IV fatigue at the 100ng/mL dose, but the MTD has not been established. The overall median survival after TP-38 for all patients was 23 weeks whereas for those without radiographic evidence of residual disease at the time of therapy, the median survival was 31.9 weeks. Overall, 3 of 15 patients, with residual disease at the time of therapy, have demonstrated radiographic responses and one patient with a complete response and has survived greater than 83 weeks.

BACKGROUND: Dendritic cell (DC) vaccine efficacy is limited by suboptimal migration to vaccine site-draining lymph nodes (VDLNs). In mice, vaccine site conditioning with inflammatory cytokines or mature DCs increases DC trafficking and the induction of antigen-specific T cells. We assessed the impact of DC migration to VDLNs on clinical outcomes in patients with newly-diagnosed glioblastoma (GBM) by randomizing patients to one of two vaccine site conditioning strategies. METHODS: We compared effects of vaccine site conditioning with either unpulsed DCs or tetanus-diphtheria (Td) toxoid prior to a vaccine of 111Indium-labeled DCs and monitored subsequent migration to VDLNs using SPECT/CT imaging. Clinical responses following vaccine site conditioning before DC vaccination were evaluated with progression-free survival (PFS) and overall survival (OS). A preclinical model using DCs derived from fluorescent transgenic mice was employed to explore DC migration to VDLNs in the context of vaccine site conditioning. The capacity for increased DC migration to induce tumor-specific immune responses in mice was evaluated. RESULTS: In patients with GBM, migration of injected DCs to VDLNs via Td conditioning was significantly increased compared to controls (P = 0.04), and the extent of migration was strongly associated with clinical outcomes (HR = 0.845 P = 0.027 for PFS; HR = 0.820 P = 0.023 for OS; Cox proportional hazards model). Furthermore, patients randomized to Td demonstrated improved OS compared to controls (median OS 18.5 months vs >32.5 months, P = 0.013). In accordance with our clinical observations, our preclinical model showed that Td conditioning significantly increased DC migration systemically to VDLNs (P = 0.0001). Td conditioning resulted in a significant elevation in serum levels of the chemokine macrophage inflammatory protein-1 alpha (MIP-1α) (P = 0.028). In vitro chemotaxis assays revealed a selective increase in human and murine DCs migrating towards serum from Td-conditioned patients and mice compared to controls. Furthermore, this preferential migration was severely abrogated with MIP-1α blockade (P = 0.02). In mice, Td conditioning in MIP-1α knockout mice only induced baseline migration efficiency compared to controls (P = 0.003). Td conditioning in mice dramatically suppressed tumor growth and improved DC vaccines in a tumor antigen-specific manner (P = 0.0001). Moreover, Td-conditioned MIP-1α knockout mice demonstrated significantly increased tumor burden compared to that of normal mice receiving Td prior to DC vaccination (P < 0.05). CONCLUSIONS: Td conditioning increased DC migration and tumor responses in a preclinical murine model corroborating observations from our phase I/II clinical trial. Our studies demonstrate a relationship between dendritic cell trafficking and antitumor efficacy and highlight MIP-1α as an important chemokine for improving dendritic cell immunotherapy. SECONDARY CATEGORY: Clinical Neuro-Oncology.

Therapeutic vaccination of patients with cancer-targeting tumor-associated antigens is a promising strategy for the specific eradication of invasive malignancies with minimal toxicity to normal tissues. However, as increasingly potent modalities for stimulating immunologic responses are developed for clinical evaluation, the risk of inflammatory and autoimmune toxicities also may be exacerbated. In this report, we describe the induction of a severe (grade 3) immunologic reaction in a patient with newly diagnosed glioblastoma (GBM) receiving autologous RNA-pulsed dendritic cell (DC) vaccines admixed with GM-CSF and administered coordinately with cycles of dose-intensified temozolomide. Shortly after the eighth administration of the admixed intradermal vaccine, the patient experienced dizziness, flushing, conjunctivitis, headache, and the outbreak of a disseminated macular/papular rash and bilateral indurated injection sites. Immunologic workup of patient reactivity revealed sensitization to the GM-CSF component of the vaccine and the production of high levels of anti–GM-CSF autoantibodies during vaccination. Removal of GM-CSF from the DC vaccine allowed continued vaccination without incident. Despite the known lymphodepletive and immunosuppressive effects of temozolomide, these observations demonstrate the capacity for the generation of severe immunologic reactivity in patients with GBM receiving DC-based therapy during adjuvant dose-intensified temozolomide. Cancer Immunol Res; 3(4); 320–5. ©2014 AACR.

Purpose: Despite aggressive conventional therapy, glioblastoma (GBM) remains uniformly lethal. Immunotherapy, in which the immune system is harnessed to specifically attack malignant cells, offers a treatment option with less toxicity. The expression of cytomegalovirus (CMV) antigens in GBM presents a unique opportunity to target these viral proteins for tumor immunotherapy. Although the presence of CMV within malignant gliomas has been confirmed by several laboratories, its relevance as an immunologic target in GBM has yet to be established. The objective of this study was to explore whether T cells stimulated by CMV pp65 RNA-transfected dendritic cells (DC) target and eliminate autologous GBM tumor cells in an antigen-specific manner. Experimental Design: T cells from patients with GBM were stimulated with autologous DCs pulsed with CMV pp65 RNA, and the function of the effector CMV pp65-specific T cells was measured. Results: In this study, we demonstrate the ability to elicit CMV pp65-specific immune responses in vitro using RNA-pulsed autologous DCs generated from patients with newly diagnosed GBM. Importantly, CMV pp65-specific T cells lyse autologous, primary GBM tumor cells in an antigen-specific manner. Moreover, T cells expanded in vitro using DCs pulsed with total tumor RNA demonstrated a 10- to 20-fold expansion of CMV pp65-specific T cells as assessed by tetramer analysis and recognition and killing of CMV pp65-expressing target cells. Conclusion: These data collectively demonstrate that CMV-specific T cells can effectively target glioblastoma tumor cells for immunologic killing and support the rationale for the development of CMV-directed immunotherapy in patients with GBM. Clin Cancer Res; 20(10); 2684–94. ©2014 AACR.

Combinatorial variation of CDR3 of V(H) and V(L), followed by phage display, was used to select affinity mutants of the parental anti-epidermal growth factor receptor-vIII (EGFRvIII) scFv MR1. One mutant, MR1-1(scFv), had increased specific binding affinity for EGFRvIII. It was produced and radiolabeled, and its biodistribution was evaluated in human glioma-bearing athymic mice. MR1-1 targeted the same EGFRvIII epitope as MR1 with an approximately 15-fold higher affinity (K(d) = 1.5 x 10(-9) M) measured by surface resonance analysis. Labeling with (131)I or (125)I was performed, and the immunoreactive fraction of the labeled MR1-1(scFv) was 50% to 55%. After incubation at 37 degrees C for 4 days, the binding affinity was maintained at 60% of initial levels. The specificity of MR1-1 for EGFRvIII was demonstrated in vitro by flow cytometry and incubation of FITC-labeled scFv with the EGFRvIII-expressing U87MG. DeltaEGFR cell line or with the EGFRvIII-negative U87MG cell line in the presence or absence of competing unlabeled MR1-1(scFv). We also investigated the internalization and processing of MR1-1 compared with MR1; MR1-1 exhibited levels of both cell surface retention and internalization up to 5 times higher than those by MR1. In biodistribution studies performed in athymic mice bearing s.c. U87MG. DeltaEGFR tumor xenografts, animals received paired-label intratumoral infusions of (131)I-labeled MR1-1(scFv) and (125)I-labeled MR1(scFv). Our results showed an up to 244% +/- 77% increase in tumor uptake for MR1-1 compared with that for MR1. The improved tumor retention of MR1-1(scFv) combined with its rapid clearance from normal tissues also resulted in sustained higher tumor:normal organ ratios. These results suggest that the improved affinity of MR1-1 can significantly impact in vivo glioma-specific targeting and immunotherapy.