Xiuyu Cui

Systemic Anti-CD25 Monoclonal Antibody Administration Safely Enhances Immunity in Murine Glioma without Eliminating Regulatory T Cells

Purpose: Elevated proportions of regulatory T cells (Treg) are present in patients with a variety of cancers, including malignant glioma, yet recapitulative murine models are wanting. We therefore examined Tregs in mice bearing malignant glioma and evaluated anti-CD25 as an immunotherapeutic adjunct. Experimental Design: CD4+CD25+Foxp3+GITR+ Tregs were quantified in the peripheral blood, spleens, cervical lymph nodes, and bone marrow of mice bearing malignant glioma. The capacities for systemic anti-CD25 therapy to deplete Tregs, enhance lymphocyte function, and generate antiglioma CTL responses were assessed. Lastly, survival and experimental allergic encephalitis risks were evaluated when anti-CD25 was combined with a dendritic cell–based immunization targeting shared tumor and central nervous system antigens. Results: Similar to patients with malignant glioma, glioma-bearing mice show a CD4 lymphopenia. Additionally, CD4+CD25+Foxp3+GITR+ Tregs represent an increased fraction of the remaining peripheral blood CD4+ T cells, despite themselves being reduced in number. Similar trends are observed in cervical lymph node and spleen, but not in bone marrow. Systemic anti-CD25 administration hinders detection of CD25+ cells but fails to completely eliminate Tregs, reducing their number only moderately, yet eliminating their suppressive function. This elimination of Treg function permits enhanced lymphocyte proliferative and IFN-γ responses and up to 80% specific lysis of glioma cell targets in vitro. When combined with dendritic cell immunization, anti-CD25 elicits tumor rejection in 100% of challenged mice without precipitating experimental allergic encephalitis. Conclusions: Systemic anti-CD25 administration does not entirely eliminate Tregs but does prevent Treg function. This leads to safe enhancement of tumor immunity in a murine glioma model that recapitulates the tumor-induced changes to the CD4 and Treg compartments seen in patients with malignant glioma.

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.

Myeloablative Temozolomide Enhances CD8+ T-Cell Responses to Vaccine and Is Required for Efficacy against Brain Tumors in Mice

Temozolomide (TMZ) is an alkylating agent shown to prolong survival in patients with high grade glioma and is routinely used to treat melanoma brain metastases. A prominent side effect of TMZ is induction of profound lymphopenia, which some suggest may be incompatible with immunotherapy. Conversely, it has been proposed that recovery from chemotherapy-induced lymphopenia may actually be exploited to potentiate T-cell responses. Here, we report the first demonstration of TMZ as an immune host-conditioning regimen in an experimental model of brain tumor and examine its impact on antitumor efficacy of a well-characterized peptide vaccine. Our results show that high-dose, myeloablative (MA) TMZ resulted in markedly reduced CD4+, CD8+ T-cell and CD4+Foxp3+ TReg counts. Adoptive transfer of naïve CD8+ T cells and vaccination in this setting led to an approximately 70-fold expansion of antigen-specific CD8+ T cells over controls. Ex vivo analysis of effector functions revealed significantly enhanced levels of pro-inflammatory cytokine secretion from mice receiving MA TMZ when compared to those treated with a lower lymphodepletive, non-myeloablative (NMA) dose. Importantly, MA TMZ, but not NMA TMZ was uniquely associated with an elevation of endogenous IL-2 serum levels, which we also show was required for optimal T-cell expansion. Accordingly, in a murine model of established intracerebral tumor, vaccination-induced immunity in the setting of MA TMZ–but not lymphodepletive, NMA TMZ–led to significantly prolonged survival. Overall, these results may be used to leverage the side-effects of a clinically-approved chemotherapy and should be considered in future study design of immune-based treatments for brain tumors.

Selective modification of antigen-specific T cells by RNA electroporation.

It has been observed that the efficient transfection of T cells by RNA electroporation requires prior activation of T cells with mitogens or by anti-CD3 antibody stimulation. We hypothesized that this requirement for T cell activation could be leveraged to express marker genes within activated T cells responding to antigen-pulsed dendritic cells and allow for the selective enrichment and modification of antigen-specific T cells. Using electroporation of mRNA encoding green fluorescent protein as a marker gene, we demonstrate that RNA electroporation can efficiently allow for the separation of cytomegalovirus-specific CD8+ and CD4+ T cells from bulk culture responding to cytomegalovirus pp65 antigen-pulsed dendritic cells. Furthermore, we demonstrate that cytomegalovirus-specific T cells can be functionally modified by RNA transfection of the C-X-C chemokine receptor, CXCR2, to migrate efficiently toward a variety of CXCR2-specific chemokines in vitro and in vivo. These studies demonstrate the utility of RNA transfection as a simple method by which to purify and selectively modify the function of antigen-specific T cells for use in adoptive immunotherapy, and importantly provide evidence that transient expression of proteins by RNA transfection is an efficient means of modulating the in vivo function of activated T cells.

Novel role of hematopoietic stem cells in immunologic rejection of malignant gliomas.

Adoptive cellular therapy (ACT) after lymphodepletive conditioning can induce dramatic clinical responses, but this approach has been largely limited to melanoma due to a lack of reliable methods for expanding tumor-specific lymphocytes from the majority of other solid cancers. We have employed tumor RNA-pulsed dendritic cells (DCs) to reliably expand CD4+ and CD8+ tumor-reactive T lymphocytes for curative ACT in a highly-invasive, chemotherapy- and radiation-resistant malignant glioma model. Curative treatment of established intracranial tumors involved a synergistic interaction between myeloablative (MA) conditioning, adoptively transferred tumor-specific T cells, and tumor RNA-pulsed DC vaccines. Hematopoietic stem cells (HSCs), administered for salvage from MA conditioning, rapidly migrated to areas of intracranial tumor growth and facilitated the recruitment of tumor-specific lymphocytes through HSC-elaborated chemokines and enhanced immunologic rejection of intracranial tumors during ACT. Furthermore, HSC transplant under non-myeloablative (NMA) conditions also enhanced immunologic tumor rejection, indicating a novel role for the use of HSCs in the immunologic treatment of malignant gliomas and possibly other solid tumors.

T-Cell Exhaustion Signatures Vary with Tumor Type and Are Severe in Glioblastoma

Purpose: T-cell dysfunction is a hallmark of glioblastoma (GBM). Although anergy and tolerance have been well characterized, T-cell exhaustion remains relatively unexplored. Exhaustion, characterized in part by the upregulation of multiple immune checkpoints, is a known contributor to failures amid immune checkpoint blockade, a strategy that has lacked success thus far in GBM. This study is among the first to examine, and credential as bona fide, exhaustion among T cells infiltrating human and murine GBM. Experimental Design: Tumor-infiltrating and peripheral blood lymphocytes (TILs and PBLs) were isolated from patients with GBM. Levels of exhaustion-associated inhibitory receptors and poststimulation levels of the cytokines IFNγ, TNFα, and IL2 were assessed by flow cytometry. T-cell receptor Vβ chain expansion was also assessed in TILs and PBLs. Similar analysis was extended to TILs isolated from intracranial and subcutaneous immunocompetent murine models of glioma, breast, lung, and melanoma cancers. Results: Our data reveal that GBM elicits a particularly severe T-cell exhaustion signature among infiltrating T cells characterized by: (1) prominent upregulation of multiple immune checkpoints; (2) stereotyped T-cell transcriptional programs matching classical virus-induced exhaustion; and (3) notable T-cell hyporesponsiveness in tumor-specific T cells. Exhaustion signatures differ predictably with tumor identity, but remain stable across manipulated tumor locations. Conclusions: Distinct cancers possess similarly distinct mechanisms for exhausting T cells. The poor TIL function and severe exhaustion observed in GBM highlight the need to better understand this tumor-imposed mode of T-cell dysfunction in order to formulate effective immunotherapeutic strategies targeting GBM. Clin Cancer Res; 24(17); 4175–86. ©2018 AACR. See related commentary by Jackson and Lim, p. 4059

Sequestration of T cells in bone marrow in the setting of glioblastoma and other intracranial tumors

T cell dysfunction contributes to tumor immune escape in patients with cancer and is particularly severe amidst glioblastoma (GBM). Among other defects, T cell lymphopenia is characteristic, yet often attributed to treatment. We reveal that even treatment-naïve subjects and mice with GBM can harbor AIDS-level CD4 counts, as well as contracted, T cell–deficient lymphoid organs. Missing naïve T cells are instead found sequestered in large numbers in the bone marrow. This phenomenon characterizes not only GBM but a variety of other cancers, although only when tumors are introduced into the intracranial compartment. T cell sequestration is accompanied by tumor-imposed loss of S1P1 from the T cell surface and is reversible upon precluding S1P1 internalization. In murine models of GBM, hindering S1P1 internalization and reversing sequestration licenses T cell–activating therapies that were previously ineffective. Sequestration of T cells in bone marrow is therefore a tumor-adaptive mode of T cell dysfunction, whose reversal may constitute a promising immunotherapeutic adjunct.Patients with glioblastoma experience lymphopenia and sequestration of T cells in the bone marrow, which is recapitulated in mice with brain tumors, where the reversible nature of this effect is demonstrated by an approach that enables the efficacy of other immunotherapeutics.

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

Flow Cytometric Identification of Tumor-Infiltrating Lymphocytes from Glioblastoma

We describe an isolation method of tumor-infiltrating lymphocytes (TILs) from glioblastoma tumors for the purpose of analysis by flow cytometry. This protocol is unique from many others in that the use of a selective lymphocyte isolation procedure, such as a Ficoll or Percoll gradient, is not used. We find that staining of TILs and analysis by flow cytometry is not affected by the presence of heterogeneous populations, while other selective isolation procedures can significantly decrease lymphocyte yield from already rare populations.

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.