Specific targeting of a naturally presented osteosarcoma antigen, papillomavirus binding factor peptide, using an artificial monoclonal antibody

Abstract

Osteosarcoma is a rare but highly malignant tumor occurring most frequently in adolescents. The prognosis of non-responders to chemotherapy is still poor, and new treatment modalities are needed. To develop peptide-based immunotherapy, we previously identified autologous cytotoxic T lymphocyte-defined osteosarcoma antigen papillomavirus binding factor (PBF) in the context of HLA-B55 and the cytotoxic T lymphocyte epitope (PBF A2.2) presented by HLA-A2. PBF and HLA class I are expressed in ∼90 and 70% of various sarcomas, respectively. However, the expression status of peptide PBF A2.2 presented by HLA-A2 on osteosarcoma cells has remained unknown because it is difficult to generate a specific probe that reacts with the HLA·peptide complex. For detection and qualification of the HLA-A*02:01·PBF A2.2 peptide complex on osteosarcoma cells, we tried to isolate a single chain variable fragment (scFv) antibody directed to the HLA-*A0201·PBF A2.2 complex using a naïve scFv phage display library. As a result, scFv clone D12 with high affinity (KD = 1.53 × 10(-9) M) was isolated. D12 could react with PBF A2.2 peptide-pulsed T2 cells and HLA-A2+PBF+ osteosarcoma cell lines and simultaneously demonstrated that the HLA·peptide complex was expressed on osteosarcoma cells. In conclusion, scFv clone D12 might be useful to select candidate patients for PBF A2.2 peptide-based immunotherapy and develop antibody-based immunotherapy.

Keywords: Antibody Engineering; Antigen Presentation; HLA-A2; HLA/Peptide Complex; Major Histocompatibility Complex (MHC); PBF; Phage Display; T Cell Receptor (TCR); scFv.

FIGURE 1.

Structure and sequence around the multicloning site of the phagemid vector pMARXL.

FIGURE 2.

Electrophoresis of amplified variable regions after primary PCR. Arrows indicate the adequate amplicons (around 350 bp) containing variable regions.

FIGURE 3.

Three scFv clones recognized HLA-A2·osteosarcoma antigen PBF-derived peptide complex. A, input/output ratios of phage particles. B, ELISA screening of the specific binders of 94 scFv clones reacting with biotinylated HLA-A*02:01·PBF A2.2 peptide complex. C, sequence analysis of complementarity-determining region 3 (CDR3) region of scFv clones.

FIGURE 4.

The scFv clones could react with peptide PBF A2.2 presented by HLA-A*02:01 on antigen-presenting cells with strong affinity. A, soluble fractions of D12 scFv-hIgG1 after purification with Protein G are visualized by SDS-PAGE. The reduced monomer (black arrows) and oxidized dimer of scFv-hIgG (red arrow) of fraction 1 are indicated. B, FACS analysis of scFv-hIgG1. T2 cells were pulsed with the indicated peptides and stained with each scFv-hIgG1 at a concentration of 10 μg/ml. BB7.2 was used to detect expression of HLA-A2 molecules. C and D, surface plasmon resonance analysis. Biotinylated HLA-A*02:01·PBF A2.2 peptide complex (C) or HLA-A*02:01·HIV-A2 peptide complex (D) was immobilized on the sensor tip as the target. Serially diluted D12 scFv-hIgG1 was used as the analyte as described under “Experimental Procedures.” E, ELISA screening of the reactivity of D12 scFv-hIgG against various biotinylated HLA-A*02:01·peptide complexes. RU, response units; EBV, Epstein-Barr virus; HTLV-1, human T-lymphotropic virus type I.

FIGURE 5.

scFv clone D12 could recognize PBF A2.2 peptide presented on the surface of sarcoma cells. A and B, FACS analysis of reactivity of D12 scFv-hIgG1 (1 mg/ml) and D12 scFv multimer (10 μg/ml) against T2 cells pulsed with the indicated peptides. %MFI increase is indicated. T2 without peptide was used as the negative control. A more than 20.0% MFI increase is indicated by underlining. C, FACS analysis of the reactivity of the D12 scFv multimer (3 μg/ml) with sarcoma cell lines and primary culture cells. Osteosarcoma cell line U2OS (A*02:01/A*3201, PBF+), OS2000 (A*2402, PBF+), KIKU (A*0206/A*2402, PBF+), HOS (A*02:11, PBF+), Saos-2 (A*02:01/A*24:02, PBF+), malignant fibrous histiocytoma cell line MFH2003 (A*2402, PBF−), and primary culture of osteosarcoma (Primary OS; A*02:01, PBF+) were used as target cells. A more than 20.0% MFI increase is indicated by underlining. The expression status of HLA-A2·PBF A2.2 peptide complex was graded as follows: strong (≥100% MFI increase), weak (≥20% but <100% MFI increase), and none (<20% MFI increase).

FIGURE 6.

Clone scFv D12 showed specificity similar to natural CTL recognizing HLA-A*02:01·PBF A2.2 peptide complex. A, tetramer staining of oligoclonal CTL line 5A9 directed to HLA-A*02:01·PBF A2.2 peptide before and after positive selection. B, CTL response against U2OS cells assessed by ELISA. Tetramer-positive cells after enrichment were used as the responder. C, inhibition effect of scFv-hIgG1 on CTL response. scFv D12-hIgG1 and irrelevant scFv D11-hIgG1 were used as blocking antibodies. The inhibition effect on CTL response was calculated as described under “Experimental Procedures.”