Adoptive Immunotherapy Against Malignant Glioma Using Survivin-specific CTLs Expanded by W6/32 Antibody-mediated Artificial Antigen-presenting Cells

Malignant glioma patients usually undergo debulking surgery followed by radiation and aggressive rounds of chemotherapy. Despite the majority of the tumors being removed or killed, no lasting and effective antitumor immunity is generated and the patients die from the tumor cells escaping or resisting those therapies. These failures lead many to believe that an aggressive combination of standard therapies, along with other biologically based therapies, is needed to successfully treat this cancer. Although the central nervous system (CNS), and tumors that arise therein, reside in an “immunologically privileged” site [1], some glioma patients were successfully treated by immunotherapy [2-7]. Adjuvant immunotherapy modalities may be particularly useful for treating gliomas located next to critical brain control regions (brain stem or the thalamus) where surgery or radiation cannot be used, provided inflammation can be controlled. The advantage of generating an immune response toward the cancer cells is that the immunized T-cells can now seek and destroy the remaining tumor cells that resisted the therapies or were localized to sites that were inaccessible to the traditional treatments. Therefore, immunotherapy for glioma is an attractive alternative treatment option.


Introduction
Malignant glioma patients usually undergo debulking surgery followed by radiation and aggressive rounds of chemotherapy. Despite the majority of the tumors being removed or killed, no lasting and effective antitumor immunity is generated and the patients die from the tumor cells escaping or resisting those therapies. These failures lead many to believe that an aggressive combination of standard therapies, along with other biologically based therapies, is needed to successfully treat this cancer. Although the central nervous system (CNS), and tumors that arise therein, reside in an "immunologically privileged" site [1], some glioma patients were successfully treated by immunotherapy [2][3][4][5][6][7]. Adjuvant immunotherapy modalities may be particularly useful for treating gliomas located next to critical brain control regions (brain stem or the thalamus) where surgery or radiation cannot be used, provided inflammation can be controlled. The advantage of generating an immune response toward the cancer cells is that the immunized T-cells can now seek and destroy the remaining tumor cells that resisted the therapies or were localized to sites that were inaccessible to the traditional treatments. Therefore, immunotherapy for glioma is an attractive alternative treatment option.
Adoptive transfer of large numbers of tumor-specific cytotoxic T-lymphocytes (CTLs) is currently being developed to treat cancer by changing the relative balance between tumor load and the immune response [8,9]. CTLs recognize "processed" peptides that are derived from endogenous proteins and presented on the cell surface in association with major histocompatibility complex (MHC) class I molecules [10,11]. Peptides that bind to a MHC class I molecule have been shown to share common amino acid motifs, which are called major anchor motifs [11]. Hence, tumor-specific CTLs can recognize and select the antigenic peptides, then kill tumor cells in an antigenic peptide-specific fashion.
Survivin is a newly identified member of the inhibitor of apoptosis (IAP) gene family that has been implicated in suppression of apoptotic cell death and regulation of cell division [12,13]. Survivin expression has been found to be undetectable in normal adult tissues. However, it has been found to be abundantly expressed in fetal tissues and a wide variety of human malignancies [14]. The fact that survivin overexpression may provide a survival benefit for tumor cells and that its enhanced expression is almost completely restricted to malignant tissues makes survivin an interesting target for the development of immunotherapeutic strategies.

Materials and Methods
Cell line and cell culture T2 cells bear the HLA-A*0201 gene, but express a very low level of cell surface HLA-A2.1 molecules, and are unable to present endogenous antigens due to a deletion of most of the MHC class II region, including the transporter associated with antigen processing, and genes encoding immuno-proteasomal subunits. This cell line was maintained in Roswell Park Memorial Institute (RPMI)-1640 medium (Invitrogen, Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, USA) and 1% antibiotics (penicillin / streptomycin; 100 U/ml). Glioma cell lines U251 (HLA-A2 + , survivin + ) and A172 (HLA-A2 -, survivin + ) were cultured in our laboratory. These glioma cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM)-supplemented with 10% FBS and 1% antibiotics (penicillin / streptomycin; 100 U/ml). NOD/SCID mice, 6 to 8 weeks of age, were obtained from Vital River Laboratories (Beijing, China) and were maintained under a specific pathogen-free condition.

Synthetic peptide
Survivin-derived peptide pSurvivin 95-104 ELTLGEFLKL [17], and the control peptide HIV-Gag peptide SLYNTVATL (pHIV) [18] were used in this study; they were synthesized by standard solid-phase chemistry and characterized by mass spectrometry. The purity of the synthetic peptides was more than 95%, as indicated by analytical HPLC. Lyophilized peptides were dissolved in dimethylsulfoxide and stored at − 80ºC after dilution in phosphate-buffered saline (PBS).

HLA class I typing
HLA class I typing was performed with microcytotoxicity by Lambda antigen tray class I (One Lambda, Canoga Park, USA).

Preparation of HLA-A2-pSurvivin 95-104 tetramer
Synthesis of HLA-A2-pSurvivin 95-104 tetramer was carried out according to the protocol of Altman et al. [19]. Briefly, plasmids encoding HLA-A*0201 (heavy chain) molecules with a C-terminal biotinylation site and human β2m molecule were constructed by insertion of the target genes into pET28a. The heavy chain and β2m molecules were expressed in Escherichia coli, and purified from inclusion bodies, then refolded in the presence of excess pSurvivin 95-104 to form HLA-A2-pSurvivin 95-104 monomeric product. The folded product was then subjected to enzymatic biotinylation by BirA enzyme (Avidity, Denver, USA) at 25ºC for 12 h. This biotinylated HLA-A2-pSurvivin 95-104 monomer was used for the preparation of the HLA-A2-pSurvivin 95-104 tetramer, which was produced by mixing the purified biotinylated monomer with phycoerythrin (PE)-labeled streptavidin (Sigma, St. Louis, USA) at a molar ratio of 4:1. HLA-A2-pHIV tetramer was also prepared according to the protocol.

Tetramer staining
Tetramer staining was carried out as previously described [21,22]. In brief, 1 × 10 6 cells were incubated in 100 μl fluorescence activated cell sorter (FACS) staining buffer (PBS supplemented with 1% BSA and 0.05% NaN 3 ) with 20 μg/ml HLA-A2-peptide tetramer at 37ºC for 30 min. Cells were washed with PBS and subsequently incubated with FITC labeled anti-CD8 antibody (BD PharMingen, San Diego, USA) at 4ºC for 30 min. All cells were washed with PBS twice after being stained, and then they were fixed in 1% formaldehyde. Stained cells were analyzed with FACScalibur (Becton Dickinson, Heidelberg, Germany).

Inhibition of the cytotoxicity with HLA class I-specific monoclonal antibody
T2pSurvivin 95-104 and U251 target cells were incubated with anti-HLA class I monoclonal antibody (mAb) W6/32 (American Type Culture Collection [ATCC], http://www.atcc.org/) [18] and a control isotypic mAb of irrelevant specificity, immunoglobulin G2a (IgG2a; BD PharMingen) at a final concentration of 30 μg/ml for 40 min at 4°C before cytotoxicity assay. After incubation, the target cells were mixed with effector cells for the 51 Cr release assay.

Adoptive immunotherapy experiments
NOD/SCID mice were inoculated with 4 × 10 6 human glioma U251 cells in 0.5 ml PBS intravenously. Mice were divided into three groups (n = 10 each). For adoptive transfer experiments, mice were treated 3 times on days 5, 15, 25 after tumor inoculation with i.v. injection of induced pSurvivin 95-104 -specific CTLs (up to 3×10 6 cells per injection). Mice with injection of PBS or pHIV-specifc CTLs were served as the control groups. All mice received two injections of rhIL-2 (2 ×10 5 IU/mouse) i.p. on days 5, 15, 25. Tumor volumes were calculated using Vernier calipers at 5-day intervals after tumor cells inoculation according to the formula: d1 × (d2)2 ×0.5 (d1 = largest diameter, d2 = perpendicular diameter). The survival time of each group of mice was monitored and recorded on a regular basis. They were sacrificed when any single or combined tumor linear measurement exceeded 20 mm. Three independent experiments were performed.

Statistical analysis
All data in this study were analyzed using version SPSS 11.0 software (SPSS, Chicago, USA). P<0.05 was considered as statistically significant.

Survivin gene expression and HLA class I typing in glioma cell lines
The mRNA expression of the survivin gene was detected in U251 and A172 cell lines. As shown in Figure 1, the expression level of the survivin gene was high in U251 and A172 cell lines, whereas there was no expression in PBMC. The results of HLA class I typing detection showed that U251 was an HLA-A2 + cell line and A172 was an HLA-A2cell line.

Inhibition of cytotoxicity of pSurvivin 95-104 -specific CTLs by HLA class I specific mAb W6/32
To determine whether the CTLs expanded by W6/32 antibodymediated pSurvivin 95-104 -specific aAPCs could recognize the specific target cells in an HLA class I-restricted manner, anti-HLA class I mAb W6/32 was utilized to block the cytotoxicity of the expanded CTLs. The cytotoxic activity against the T2pSurvivin 95-104 and U251 was significantly eliminated by W6/32. As shown in Figure 5, W6/32     inhibited target cell lysis, whereas mouse IgG2a, used as an isotype control, showed no effect in T2pSurvivin 95-104 and U251 cells. These results suggested that the expanded CTLs lysed the specific targets in an HLA class I-restricted manner.

Adoptive transfer of the expanded pSurvivin 95-104 -specific CTLs can mediate effective therapy of human glioma in NOD/SCID mice
To evaluate the therapeutic effects of pSurvivin 95-104 -specific CTLs expanded by W6/32 antibody-mediated pSurvivin 95-104 -specific aAPCs, NOD/SCID mice bearing U251 cells were treated by adoptive transfer of the expanded CTLs. Tumor volumes of PBS group or pHIV-specific CTLs group expanded as determined by measuring every 5 days. In contrast, the rate of tumor growth in animals treated with pSurvivin 95-104 -specific CTLs was significantly decreased as compared with that in animals treated with PBS or pHIV-specific CTLs (P <0.05) ( Figure 6A). In addition, as shown in Figure 6B, none of treatment control mice survived longer than 40 days. However, treatment with the expanded pSurvivin 95-104 -specific CTLs was effective and cured 100% of the mice (> 60 days). These results demonstrated that adoptive transfer of the expanded pSurvivin 95-104 -specific CTLs can mediate effective therapy of human glioma in NOD/SCID mice.

Discussion
Malignant gliomas are the most common tumors in the central nervous system. When treated with conventional therapy such as surgery, radiation, or chemotherapy, the prognosis for patients with malignant glioma is poor [22]. Clearly, novel therapeutic strategies are necessary.
The adoptive transfer of tumor-specific CTLs provides a promising approach to the immunotherapy of cancer. Previous attempts to determine the impact of adoptive transfer in tumor immunotherapy have been limited by the difficulty of isolating T-cells of known antigen specificity. Melanoma and renal cell carcinoma were the first tumors in humans to be treated with adoptive immunotherapy [23]. Infusions of polyclonal T-cell populations isolated from the tumor (tumorinfiltrating lymphocytes) and nonspecifically expanded in vitro with high concentrations of IL-2 suggested the therapeutic potential of this approach in cancer patients [23]. The recent characterization of glioma-associated antigens recognized by human CTLs has opened new possibilities for the adoptive transfer of T-cells in glioma immunotherapy.
Rimoldi et al. [24] were the first to document that melanoma associated antigen-specific CTL lines could recognize HLA-matched glioma cells in vitro. Then, Chi et al. [25], Scarcella et al. [26], and Sahin et al. [7] reported that gp100 and MAGE-1 mRNA was expressed in glioma tumor cells and tumor tissue by RT-PCR, and HER-2 was found in brain tumor by immunohistochemical staining [28,29]. Prins et al. [30] validated melanoma-associated antigen gp100 and TRP-2 as immunotherapeutic targets in a murine glioma model. Liu et al. [31] reported on TRP-2 as a CTL target in malignant glioma,  which demonstrated that TRP-2 antigen can be naturally processed and recognized by TRP-2-specific CTLs. They also found that TRP-2-specific cytotoxic T-cell activity was detected when PBMCs were stimulated with autologous DCs pulsed with irradiated GBM tumor cells in vitro and in patents' PBMCs after DC-pulsed autologous tumor lysate vaccinations. Very importantly, IL-13 receptor α2 has been identified as a glioma-specific antigen [32], and a HLA-A2.1restricted CTL epitope (WLPFGFILI) might serve as an attractive component of peptide-based vaccines to treat glioma [33,34]. Survivin is a member of the inhibitors of apoptosis family and is overexpressed in different types of malignancies [35]. Cytotoxic T-cells recognizing survivin epitopes can be elicited in vitro and by vaccination in patients with leukemia, breast cancer, and melanoma. Our results indicate that pSurvivin 95-104 epitope could be used as tumor antigen targets for surrogate assays for antigen-specific CTLs or to develop antigenspecific active immunotherapy strategies for glioma patients.
There are several methods currently in use to activate and expand CD8 + T-cells to the numbers required for a trial of adoptive immunotherapy. One of the most widely used approaches has been based on the use of autologous antigen-loaded dendritic cells (DCs) as antigen presenting cells (APCs). However, the generation and maintenance of DCs is expensive and cumbersome. Furthermore, variability in DC number and the quality of the DCs are affected by both pretreatment of the patient and the patient's disease. Therefore, a number of different systems have been developed in which aAPC present antigen-HLA complexes in combination with the appropriate costimulatory molecules. The benefits of an aAPC system to expand T-cells include their consistency over time and over multiple donors, their "off-the-shelf" availability, and the lack of need for coculture of allogeneic or virally infected cells with the desired T-cells. Furthermore, the use of artificial APCs may be more attractive for clinical use because they are more amenable to manufacturing within the constraints of current Good Manufacturing Practices than are cellular APCs [36][37][38].
In the present study, we used W6/32 antibody-mediated pSurvivin 95-104 -specific aAPCs to induce specific CTL against malignant glioma from the PBLs of healthy donors. Following this procedure, we found that the expanded CTLs, derived from PBLs in an HLA-A2 + healthy donor, could kill the HLA-A2 + pSurvivin 95-104 + glioma cells when expanded with W6/32 antibody-mediated pSurvivin 95-104 -specific aAPCs in vitro. The specific killing activity of CTLs against specific target cells T2pSurvivin 95-104 and U251 was much more effective than that of any other control group. The results indicated the cytotoxicity of the CTLs expanded with W6/32 antibody-mediated pSurvivin 95-104specific aAPCs is antigen-specific, that is, against the target cells bearing the corresponding HLA-A2-pSurvivin 95-104 complexes. Intravenous injection of the expanded pSurvivin 95-104 -specific CTLs into nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mice harboring glioma cells resulted in glioma cells elimination, whereas transfer of control T-cells was ineffective.
Proper cell signaling during the expansion phase is also an essential component in producing effective, functioning CTLs following infusion. Excessive stimulation and improper or absent co-stimulation could result in deficient cytolytic activity, lack of persistence or apoptosis. June et al. found the beads coated with HLA class II tetramers in an indirect fashion, via an anti-Class II antibody covalently bound to the bead, were more efficient activators of antigenspecific CD4 + T-cells than beads directly and covalently coated with HLA monomers or tetramers [39]. Our results show the beads coated with HLA class I tetramers via antibody W6/32 covalently bound to the bead, were more efficient activators of antigen-specific CD8 + T-cells than beads directly and covalently coated with HLA monomers or tetramers (data not shown). In addition, in this study, we used a combination of anti-CD28, 4-1BBL and CD83 for the generation of a costimulatory signal. Recent studies have demonstrated that combining B7.1/CD28 stimulation and 4-1BB signaling was required for optimal induction of CD25 and bcl-XL expression in CD8 + T-cells, increased CTL activity and protective anti-tumor immunity [40][41][42]. In addition, CD83 molecule is important in: (a) priming naive CD8 + T-cells; (b) driving their antigen-specific expansion; and (c) supporting their long-term survival and function when coupled to aAPCs [43,44]. As we learn more about the in vivo response and the effects of differing amounts and types of co-stimulation on CTL function post-infusion, future generations of the aAPC might prove to be essential because the bead can easily adapt to the new stimulatory requirements by adding or removing different co-stimulatory complexes.
In summary, we have presented data showing that the use of aAPC represents the state-of-the-art in generation of antigen-specific CTLs for adoptive immunotherapy. Thus, The W6/32 antibody-mediated artificial antigen-presenting cells (aAPCs) made by coating HLA-A2/ pSurvivin 95-104 tetramer mediated by W6/32 antibody, anti-CD28 antibody, 4-1BBL and CD83 molecules to cell-sized latex beads could provide a useful tool for the reproducible expansion of peptidespecific CTLs in vitro and significantly advance the field of adoptive immunotherapy. The W6/32 antibody-mediated pSurvivin 95-104 - specific aAPCs could be used as a standardized, "off-the-shelf" reagent to enrich pSurvivin 95-104 -specific CTLs for the treatment of patients with malignant glioma.

Disclosures
The authors have no financial conflict of interest.