Antigenic profiling of glioma cells to generate allogeneic vaccines or dendritic cell-based therapeutics

Abstract

Purpose: Allogeneic glioma cell lines that are partially matched to the patient at class I human leukocyte antigen (HLA) loci and that display tumor-associated antigens (TAA) or antigenic precursors [tumor antigen precursor proteins (TAPP)] could be used for generating whole tumor cell vaccines or, alternatively, for extraction of TAA peptides to make autologous dendritic cell vaccines.

Experimental design: Twenty human glioma cell lines were characterized by molecular phenotyping and by flow cytometry for HLA class I antigen expression. Twelve of the 20 cell lines, as well as analyses of freshly resected glioma tissues, were further characterized for protein and/or mRNA expression of 16 tumor antigen precursor proteins or TAA.

Results: These 20 human glioma cell lines potentially cover 77%, 85%, and 78% of the U.S. Caucasian population at HLA-A, HLA-B, and HLA-C alleles, respectively. All cells exhibited multiple TAA expressions. Most glioma cells expressed antigen isolated from immunoselected melanoma-2 (Aim-2), B-cyclin, EphA2, GP100, beta1,6-N-acetylglucosaminyltransferase V (GnT-V), IL13Ralpha2, Her2/neu, hTert, Mage, Mart-1, Sart-1, and survivin. Real-time PCR technology showed that glioblastoma specimens expressed most of the TAA as well. Tumor-infiltrating lymphocytes and CD8(+) CTL killed T2 cells when loaded with specific HLA-A2(+) restricted TAA, or gliomas that were both HLA-A2(+) and also positive for specific TAA (Mart-1, GP100, Her2/neu, and tyrosinase) but not those cells negative for HLA-A2 and/or lacking the specific epitope.

Conclusions: These data provide proof-in-principle for the use of allogeneic, partially HLA patient-matched glioma cells for vaccine generation or for peptide pulsing with allogeneic glioma cell extracts of autologous patient dendritic cells to induce endogenous CTL in brain tumor patients.

Fig. 1

TAPP associated with U-251cells by immunofluorescence microscopy and by intracellular flow cytometry. A, immunofluorescence of U-251cells shows that the TAPP are present within their predicted intracellular locations. U-251gliomas were allowed to attach to coverslips overnight. The cells were fixed, permeabilized, and then stained with the specific antibodies. Top, left to right, Her2/neu, GnT-V, and survivin. Top middle, tyrosinase Mage-1andTrp-1 (which is as bright as the negative control; not shown). Bottom middle, EphA2, GP100, and IL13Rα2. Bottom, B-cyclin, Mart-1, and hTert. B, intracellular flow cytometry of TAPP within U-251glioma cells. U-251glioma cells (10⁶) were fixed and permeabilized. The permeabilized cells were incubated with the primary antibodies for 1h, washed, and subsequently incubated with a FITC-labeled secondary antibody directed against the primary antibody. Flow cytometric analysis of 10⁴ glioma cells is shown. The isotypic control (dark line) and the anti-TAPP staining (either a fine line or dashed line) are shown. a, Her2/neu; b, GnT-V and survivin; c, tyrosinase and Mage-1; d, Trp-1 and EphA2; e, GP100 and IL13Rα2; f, Mart-1; g, B-cyclin and hTert.

Fig. 2

Cytotoxicity assays done with HLA-A2–restricted CTL or TIL effectors and peptide-loaded T2 cells or glioma target cells. CTL (top and middle rows) were directed against Mart-1, GP100, EphA2, and Her/neu peptides and incubated with peptide-loaded T2 target cells (top row) or glioma target cells (middle row). TIL (bottom row) were incubated with either peptide-loaded T2 cells or the glioma target cells. The effector TIL 1374 cells are specific for tyrosinase (366-377; left) and TIL 771 cells are specific for GP100 (154-162; right) and incubated with the target glioma cells. The phenotypes of the glioma cells used are, for SNB19: HLA-A2+, Mart-1+, GP100+, EphA2+, Her2/neu+; U-251: HLA-A2+, Mart-1+, GP100+, EphA2+, Her2/neu+, tyro+; U-373: HLA-A2+, Mart-1+, GP100+, EphA2+, Her2/neu+, tyro−; A172:HLA-A2−, Mart-1+, GP100+, EphA2+;T98G: HLA-A2+, tyro+; LN229: HLA-A2−, tyro+.