Abstract Despite new additions to the standard of care therapy for high grade primary malignant brain tumors, the prognosis for patients with this disease is still poor. A small contingent of clinical researchers are focusing their efforts on testing the safety, feasibility and efficacy of experimental active and passive immunotherapy approaches for gliomas and are primarily conducting Phase I and II clinical trials. Few trials have advanced to the Phase III arena. Here we provide an overview of the cellular therapies and vaccine trials currently open for patient accrual obtained from a search of http://www.clinicaltrials.gov. The search was refined with terms that would identify the Phase I, II and III immunotherapy trials open for adult glioma patient accrual in the United States. From the list, those that are currently open for patient accrual are discussed in this review. A variety of adoptive immunotherapy trials using ex vivo activated effector cell preparations, cell-based and non-cell-based vaccines, and several combination passive and active immunotherapy approaches are discussed.

Cancer cells derived from Glioblastoma multiforme possess membranous protrusions allowing these cells to infiltrate surrounding tissue, while resisting lymphocyte cytotoxicity. Microvilli and filopodia are supported by actin filaments cross-linked by fascin. Fascin-1 was genetically silenced within human U251 glioma cells; these knock-down glioma cells lost their microvilli/filopodia. The doubling time of these fascin-1 knock-down cells was doubled that of shRNA control U251 cells. Fascin-1 knock-down cells lost their transmigratory ability responding to interleukin-6 or insulin-like growth factor-1. Fascin-1 silenced U251 cells were more easily killed by cytolytic lymphocytes. Fascin-1 knock-down provides unique opportunities to augment glioma immunotherapy by simultaneously targeting several key glioma functions: like cell transmigration, cell division and resisting immune responses.

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on extracellular matrix proteins, for measuring the motility of fluorescently-labeled, non-adherent human or murine effector immune cells. This technique employs a stainless steel manifold and 10-well Teflon slide to focally deposit non-adherent T cells into wells prepared with either confluent tumor cell monolayers or extracellular matrix proteins. Light and/or multi-channel fluorescence microscopy is used to track the movement and behavior of the effector cells over time. Fluorescent dyes and/or viral vectors that code for fluorescent transgenes are used to differentially label the cell types for imaging. This method is distinct from similar-type in vitro assays that track horizontal or vertical migration/invasion utilizing slide chambers, agar or transwell plates. The assay allows detailed imaging data to be collected with different cell types distinguished by specific fluorescent markers; even specific subpopulations of cells (i.e., transduced/nontransduced) can be monitored. Surface intensity fluorescence plots are generated using specific fluorescence channels that correspond to the migrating cell type. This allows for better visualization of the non-adherent immune cell mobility at specific times. It is possible to gather evidence of other effector cell functions, such as cytotoxicity or transfer of viral vectors from effector to target cells, as well. Thus, the method allows researchers to microscopically document cell-to-cell interactions of differentially-labeled, non-adherent with adherent cells of various types. Such information may be especially relevant in the assessment of biologically-manipulated or activated immune cell types, where visual proof of functionality is desired with tumor target cells before their use for cancer therapy.

Background

Rasmussen encephalitis (RE) is a rare neuroinflammatory disease characterized by intractable seizures and progressive atrophy on one side of the cerebrum. Perivascular cuffing and clusters of T cells in the affected cortical hemisphere are indicative of an active cellular immune response.

Methods

Peripheral blood mononuclear cells (PBMCs) and brain-infiltrating lymphocytes (BILs) were isolated from 20 RE surgery specimens by standard methods, and CD3(+) T cell populations were analyzed by flow cytometry. Gamma delta T cell receptor spectratyping was carried out by nested PCR of reversed transcribed RNA extracted from RE brain tissue, followed by high resolution capillary electrophoresis. A MiSeq DNA sequencing platform was used to sequence the third complementarity determining region (CDR3) of δ1 chains.

Results

CD3(+) BILs from all of the RE brain specimens comprised both αβ and γδ T cells. The median αβ:γδ ratio was 1.9 (range 0.58-5.2) compared with a median ratio of 7.7 (range 2.7-40.8) in peripheral blood from the same patients. The αβ T cells isolated from brain tissue were predominantly CD8(+), and the majority of γδ T cells were CD4(-) CD8(-). Staining for the early activation marker CD69 showed that a fraction of the αβ and γδ T cells in the BILs were activated (median 42%; range 13-91%, and median 47%; range 14-99%, respectively). Spectratyping T cell receptor (TCR) Vδ1-3 chains from 14 of the RE brain tissue specimens indicated that the γδ T cell repertoire was relatively restricted. Sequencing δ1 chain PCR fragments revealed that the same prevalent CDR3 sequences were found in all of the brain specimens. These CDR3 sequences were also detected in brain tissue from 15 focal cortical dysplasia (FCD) cases.

Conclusion

Neuroinflammation in RE involves both activated αβ and γδ T cells. The presence of γδ T cells with identical TCR δ1 chain CDR3 sequences in all of the brain specimens examined suggests that a non-major histocompatibility complex (MHC)-restricted immune response to the same antigen(s) is involved in the etiology of RE. The presence of the same δ1 clones in CD brain implies the involvement of a common inflammatory pathway in both diseases.

Cancer cells derived from Glioblastoma multiforme possess membranous protrusions allowing these cells to infiltrate surrounding tissue, while resisting lymphocyte cytotoxicity. Microvilli and filopodia are supported by actin filaments cross-linked by fascin. Fascin-1 was genetically silenced within human U251 glioma cells; these knock-down glioma cells lost their microvilli/filopodia. The doubling time of these fascin-1 knock-down cells was doubled that of shRNA control U251 cells. Fascin-1 knock-down cells lost their transmigratory ability responding to interleukin-6 or insulin-like growth factor-1. Fascin-1 silenced U251 cells were more easily killed by cytolytic lymphocytes. Fascin-1 knock-down provides unique opportunities to augment glioma immunotherapy by simultaneously targeting several key glioma functions: like cell transmigration, cell division and resisting immune responses.

Purpose

Individual or combined strategies of cellular therapy with alloreactive CTLs (alloCTL) and gene therapy using retroviral replicating vectors (RRV) encoding a suicide prodrug activating gene were explored for the treatment of breast tumors metastatic to the brain.

Experimental design

AlloCTL, sensitized to the HLA of MDA-MB-231 breast cancer cells, were examined in vitro for antitumor functionality toward breast cancer targets. RRV encoding the yeast cytosine deaminase (CD) gene was tested in vivo for virus spread, ability to infect, and kill breast cancer targets when exposed to 5-fluorocytosine (5-FC). Individual and combination treatments were tested in subcutaneous and intracranial xenograft models with 231BR, a brain tropic variant.

Results

AlloCTL preparations were cytotoxic, proliferated, and produced IFN-γ when coincubated with target cells displaying relevant HLA. In vivo, intratumorally placed alloCTL trafficked through one established intracranial 231BR focus to another in contralateral brain and induced tumor cell apoptosis. RRV-CD efficiently spread in vivo, infected 231BR and induced their apoptosis upon 5-FC exposure. Subcutaneous tumor volumes were significantly reduced in alloCTL and/or gene therapy-treated groups compared to control groups. Mice with established intracranial 231BR tumors treated with combined alloCTL and RRV-CD had a median survival of 97.5 days compared with single modalities (50-83 days); all experimental treatment groups survived significantly longer than sham-treated groups (median survivals 31.5 or 40 days) and exhibited good safety/toxicity profiles.

Conclusion

The results indicate combining cellular and suicide gene therapies is a viable strategy for the treatment of established breast tumors in the brain.