Tetanus toxoid and CCL3 improve dendritic cell vaccines in mice and glioblastoma patients.

After stimulation, dendritic cells (DCs) mature and migrate to draining lymph nodes to induce immune responses. As such, autologous DCs generated ex vivo have been pulsed with tumour antigens and injected back into patients as immunotherapy. While DC vaccines have shown limited promise in the treatment of patients with advanced cancers including glioblastoma, the factors dictating DC vaccine efficacy remain poorly understood. Here we show that pre-conditioning the vaccine site with a potent recall antigen such as tetanus/diphtheria (Td) toxoid can significantly improve the lymph node homing and efficacy of tumour-antigen-specific DCs. To assess the effect of vaccine site pre-conditioning in humans, we randomized patients with glioblastoma to pre-conditioning with either mature DCs or Td unilaterally before bilateral vaccination with DCs pulsed with Cytomegalovirus phosphoprotein 65 (pp65) RNA. We and other laboratories have shown that pp65 is expressed in more than 90% of glioblastoma specimens but not in surrounding normal brain, providing an unparalleled opportunity to subvert this viral protein as a tumour-specific target. Patients given Td had enhanced DC migration bilaterally and significantly improved survival. In mice, Td pre-conditioning also enhanced bilateral DC migration and suppressed tumour growth in a manner dependent on the chemokine CCL3. Our clinical studies and corroborating investigations in mice suggest that pre-conditioning with a potent recall antigen may represent a viable strategy to improve anti-tumour immunotherapy.

Systemic administration of a bispecific antibody targeting EGFRvIII successfully treats intracerebral glioma.

Bispecific antibodies (bscAbs), particularly those of the bispecific T-cell engager (BiTE) subclass, have been shown to effectively redirect T cells against cancer. Previous efforts to target antigens expressed in both tumors and normal tissues have produced significant toxicity, however. Moreover, like other large molecules, bscAbs may be restricted from entry into the "immunologically privileged" CNS. A tumor-specific mutation of the epidermal growth factor receptor, EGFRvIII, is a constitutively activated tyrosine kinase not found in normal tissues but frequently expressed in glioblastomas and many other neoplasms. Because it is localized solely to tumor tissue, EGFRvIII presents an ideal target for immunotherapy. Here we report the preclinical evaluation of an EGFRvIII-targeted BiTE, bscEGFRvIIIxCD3. Our results show that bscEGFRvIIIxCD3 activates T cells to mediate potent and antigen-specific lysis of EGFRvIII-expressing gliomas in vitro (P < 0.001) at exceedingly low concentrations (10 ng/mL) and effector-to-target ratios (2.5:1). Treatment with i.v. bscEGFRvIIIxCD3 yielded extended survival in mice with well-established intracerebral tumors (P < 0.05) and achieved durable complete cure at rates up to 75%. Antitumor efficacy was significantly abrogated on blockade of EGFRvIII binding, demonstrating the need for target antigen specificity both in vitro and in vivo. These results demonstrate that BiTEs can be used to elicit functional antitumor immunity in the CNS, and that peptide blockade of BiTE-mediated activity may greatly enhance the safety profile for antibody-redirected T-cell therapies. Finally, bscEGFRvIIIxCD3 represents a unique advancement in BiTE technology given its exquisite tumor specificity, which enables precise elimination of cancer without the risk of autoimmune toxicity.

A cytokine cocktail directly modulates the phenotype of DC-enriched anti-tumor T cells to convey potent anti-tumor activities in a murine model.

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.

Monoclonal antibody blockade of IL-2 receptor α during lymphopenia selectively depletes regulatory T cells in mice and humans.

Lymphodepletion augments adoptive cell transfer during antitumor immunotherapy, producing dramatic clinical responses in patients with malignant melanoma. We report that the lymphopenia induced by the chemotherapeutic agent temozolomide (TMZ) enhances vaccine-driven immune responses and significantly reduces malignant growth in an established model of murine tumorigenesis. Unexpectedly, despite the improved antitumor efficacy engendered by TMZ-induced lymphopenia, there was a treatment related increase in the frequency of immunosuppressive regulatory T cells (T(Regs); P = .0006). Monoclonal antibody (mAb)-mediated inhibition of the high-affinity IL-2 receptor α (IL-2Rα/CD25) during immunotherapy in normal mice depleted T(Regs) (73% reduction; P = .0154) but also abolished vaccine-induced immune responses. However, during lymphodepletion, IL-2Rα blockade decreased T(Regs) (93% reduction; P = .0001) without impairing effector T-cell responses, to augment therapeutic antitumor efficacy (66% reduction in tumor growth; P = .0024). Of clinical relevance, we also demonstrate that anti-IL-2Rα mAb administration during recovery from lymphodepletive TMZ in patients with glioblastoma reduced T(Reg) frequency (48% reduction; P = .0061) while permitting vaccine-stimulated antitumor effector cell expansion. To our knowledge, this is the first report of systemic antibody-mediated T(Reg) depletion during lymphopenia and the consequent synergistic enhancement of vaccine-driven cellular responses, as well as the first demonstration that anti-IL-2Rα mAbs function differentially in nonlymphopenic versus lymphopenic contexts.

A conjoined universal helper epitope can unveil antitumor effects of a neoantigen vaccine targeting an MHC class I-restricted neoepitope.

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.

GLP toxicology study of a fully-human T cell redirecting CD3:EGFRvIII binding immunotherapeutic bispecific antibody.

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.

A novel inhibitor of signal transducers and activators of transcription 3 activation is efficacious against established central nervous system melanoma and inhibits regulatory T cells.

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 IC(50) of 1.6, 2.3, and 1.5 mumol/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-beta, 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.

Preventing Lck Activation in CAR T Cells Confers Treg Resistance but Requires 4-1BB Signaling for Them to Persist and Treat Solid Tumors in Nonlymphodepleted Hosts.

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.

Immunological responses in a patient with glioblastoma multiforme treated with sequential courses of temozolomide and immunotherapy: case study.

Cytotoxic chemotherapy that induces lymphopenia is predicted to ablate the benefits of active antitumor immunization. Temozolomide is an effective chemotherapeutic 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.

Detection of humoral response in patients with glioblastoma receiving EGFRvIII-KLH vaccines.

The epidermal growth factor receptor variant III (EGFRvIII) is a consistent tumor-specific mutation that is widely expressed in glioblastoma multiforme (GBM) and other neoplasms. As such it represents a truly tumor-specific target for antitumor immunotherapy. Although endogenous humoral responses to EGFRvIII have been reported in patients with EGFRvIII-expressing breast cancer, it is not known whether de novo responses can be generated or endogenous responses enhanced with an EGFRvIII-specific vaccine. To assess this in clinical trials, we have developed and validated an immunoassay to measure and isolate anti-EGFRvIII and anti-KLH antibodies from the serum of patients vaccinated with an EGFRvIII-specific peptide (PEPvIII) conjugated to keyhole limpet hemocyanin (KLH). Using magnetic beads with immobilized antigen we captured and detected anti-EGFRvIII and anti-KLH antibodies in serum from patients before and after vaccinations. Using this assay, we found that significant levels of antibody for tumor-specific antigen EGFRvIII (>4 microg/mL) and KLH could be induced after vaccination with PEPvIII-KLH.

Systemic CTLA-4 blockade ameliorates glioma-induced changes to the CD4+ T cell compartment without affecting regulatory T-cell function.

PURPOSE: Patients with malignant glioma suffer global compromise of their cellular immunity, characterized by dramatic reductions in CD4(+) T cell numbers and function. We have previously shown that increased regulatory T cell (T(reg)) fractions in these patients explain T-cell functional deficits. Our murine glioma model recapitulates these findings. Here, we investigate the effects of systemic CTLA-4 blockade in this model. EXPERIMENTAL DESIGN: A monoclonal antibody (9H10) to CTLA-4 was employed against well-established glioma. Survival and risks for experimental allergic encephalomyelitis were assessed, as were CD4(+) T cell numbers and function in the peripheral blood, spleen, and cervical lymph nodes. The specific capacities for anti-CTLA-4 to modify the functions of regulatory versus CD4(+)CD25(-) responder T cells were evaluated. RESULTS: CTLA-4 blockade confers long-term survival in 80% of treated mice, without eliciting experimental allergic encephalomyelitis. Changes to the CD4 compartment were reversed, as anti-CTLA-4 reestablishes normal CD4 counts and abrogates increases in CD4(+)CD25(+)Foxp3(+)GITR(+) regulatory T cell fraction observed in tumor-bearing mice. CD4(+) T-cell proliferative capacity is restored and the cervical lymph node antitumor response is enhanced. Treatment benefits are bestowed exclusively on the CD4(+)CD25(-) T cell population and not T(regs), as CD4(+)CD25(-) T cells from treated mice show improved proliferative responses and resistance to T(reg)-mediated suppression, whereas T(regs) from the same mice remain anergic and exhibit no restriction of their suppressive capacity. CONCLUSIONS: CTLA-4 blockade is a rational means of reversing glioma-induced changes to the CD4 compartment and enhancing antitumor immunity. These benefits were attained through the conferment of resistance to T(reg)-mediated suppression, and not through direct effects on T(regs).

Increased regulatory T-cell fraction amidst a diminished CD4 compartment explains cellular immune defects in patients with malignant glioma.

Immunosuppression is frequently associated with malignancy and is particularly severe in patients with malignant glioma. Anergy and counterproductive shifts toward T(H)2 cytokine production are long-recognized T-cell defects in these patients whose etiology has remained elusive for >30 years. We show here that absolute counts of both CD4(+) T cells and CD4(+)CD25(+)FOXP3(+)CD45RO(+) T cells (T(regs)) are greatly diminished in patients with malignant glioma, but T(regs) frequently represent an increased fraction of the remaining CD4 compartment. This increased T(reg) fraction, despite reduced counts, correlates with and is sufficient to elicit the characteristic manifestations of impaired patient T-cell responsiveness in vitro. Furthermore, T(reg) removal eradicates T-cell proliferative defects and reverses T(H)2 cytokine shifts, allowing T cells from patients with malignant glioma to function in vitro at levels equivalent to those of normal, healthy controls. Such restored immune function may give license to physiologic antiglioma activity, as in vivo, T(reg) depletion proves permissive for spontaneous tumor rejection in a murine model of established intracranial glioma. These findings dramatically alter our understanding of depressed cellular immune function in patients with malignant glioma and advance a role for T(regs) in facilitating tumor immune evasion in the central nervous system.

Temozolomide lymphodepletion enhances CAR abundance and correlates with antitumor efficacy against established glioblastoma.

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).

A Rationally Designed Fully Human EGFRvIII:CD3-Targeted Bispecific Antibody Redirects Human T Cells to Treat Patient-derived Intracerebral Malignant Glioma.

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.

Dendritic Cells Enhance Polyfunctionality of Adoptively Transferred T Cells That Target Cytomegalovirus in Glioblastoma.

Median survival for glioblastoma (GBM) remains <15 months. Human cytomegalovirus (CMV) antigens have been identified in GBM but not normal brain, providing an unparalleled opportunity to subvert CMV antigens as tumor-specific immunotherapy targets. A recent trial in recurrent GBM patients demonstrated the potential clinical benefit of adoptive T-cell therapy (ATCT) of CMV phosphoprotein 65 (pp65)-specific T cells. However, 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.

Profiling of CD4+, CD8+, and CD4+CD25+CD45RO+FoxP3+ T cells in patients with malignant glioma reveals differential expression of the immunologic transcriptome compared with T cells from healthy volunteers.

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 (T(reg)) 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 T(reg) 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, T(regs) 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.

Targeted therapy for glioblastoma multiforme neoplastic meningitis with intrathecal delivery of an oncolytic recombinant poliovirus.

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.

Long-term Survival in Glioblastoma with Cytomegalovirus pp65-Targeted Vaccination.

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.

Treatment of neoplastic meningitis with intrathecal 9-nitro-camptothecin.

The topoisomerase I inhibitor, 9-nitro-camptothecin (9NC), is highly tumoricidal against glioblastoma multiforme (GBM) in vitro. However, systemic administration of 9NC has not shown the expected efficacy in clinical trials. This failure may be due to the rapid hydrolysis of 9NC in plasma from the active form to the inactive and myelosuppressive form in the presence of human albumin at physiologic pH. Concurrent treatment with anticonvulsants and dexamethasone, drugs indispensable for the supportive therapy of patients with GBM, has also been shown to decrease plasma concentrations of these drugs. Intrathecal drug delivery circumvents the blood-brain barrier and minimizes systemic toxicity. Intrathecal delivery of 9NC may also have the more specific advantage of significantly reducing the hydrolysis of 9NC that occurs after systemic delivery due to the more favorable pH and reduced albumin content in cerebrospinal fluid. The present study evaluated the toxicity and efficacy of intrathecal delivery of 9NC in an athymic rat model of neoplastic meningitis. Toxicity tests showed that 0.3 micromol (5000 microM), 0.03 micromol (500 microM), 0.003 micromol (50 microM), or 0.0003 micromol (5 microM) of 9NC administered intrathecally to the athymic rats caused no evidence of clinical or histological toxicity. Intrathecal administration of 0.3 micromol (5000 microM) of 9NC twice a week for three doses to athymic rats with neoplastic meningitis induced by the GBM cell line, U87MGDeltaEGFR, resulted in a 26% increase of median survival compared to the control group (p < 0.005). These results suggest that intrathecal treatment with 9NC may be useful for patients with GBM neoplastic meningitis.

Mutant epidermal growth factor receptor up-regulates molecular effectors of tumor invasion.

The gene most commonly altered in human glioblastomas is the epidermalgrowth factor receptor (EGFR). We profiled transcripts induced by mutantEGFR to better understand its role in tumor progression. The pattern found suggested enhanced tumor invasion. The highly induced genes included extracellular matrix components, metalloproteases, and a serine protease. We confirmed that mutant EGFR did make glioblastoma cells both more motile and invasive using in vitro assays. Furthermore, inhibitors of EGFR (OSI-774 and Tyrphostin AG1478) selectively down-regulated these molecular effectors in glioblastoma cells, eliminating enhanced invasion.