A TCR targeting the HLA-A*0201-restricted epitope of MAGE-A3 recognizes multiple epitopes of the MAGE-A antigen superfamily in several types of cancer

Abstract

Adoptive immunotherapy using TCR-engineered PBLs against melanocyte differentiation Ags mediates objective tumor regression but is associated with on-target toxicity. To avoid toxicity to normal tissues, we targeted cancer testis Ag (CTA) MAGE-A3, which is widely expressed in a range of epithelial malignancies but is not expressed in most normal tissues. To generate high-avidity TCRs against MAGE-A3, we employed a transgenic mouse model that expresses the human HLA-A*0201 molecule. Mice were immunized with two HLA-A*0201-restricted peptides of MAGE-A3: 112-120 (KVAELVHFL) or MAGE-A3: 271-279 (FLWGPRALV), and T cell clones were generated. MAGE-A3-specific TCR α- and β-chains were isolated and cloned into a retroviral vector. Expression of both TCRs in human PBLs demonstrated Ag-specific reactivity against a range of melanoma and nonmelanoma tumor cells. The TCR against MAGE-A3: 112-120 was selected for further development based on superior reactivity against tumor target cells. Interestingly, peptide epitopes from MAGE-A3 and MAGE-A12 (and to a lesser extent, peptides from MAGE-A2 and MAGE-A6) were recognized by PBLs engineered to express this TCR. To further improve TCR function, single amino acid variants of the CDR3 α-chain were generated. Substitution of alanine to threonine at position 118 of the α-chain in the CDR3 region of the TCR improved its functional avidity in CD4 and CD8 cells. On the basis of these results, a clinical trial is planned in which patients bearing a variety of tumor histologies will receive autologous PBLs that have been transduced with this optimized anti-MAGE-A3 TCR.

FIGURE 1.

A, Schematic illustration of the MSGV1-based retroviral vector encoding anti–MAGE-A3 TCR expression cassette. TCR α- and β-chains are linked with furin-spacer (SGSG)-P2A ribosomal skip peptide sequence. B and C, Flow cytometric analysis of MAGE-A3 TCR-transduced PBLs. B, Dot plots showing the FACS profile of PBLs stained with anti–human-CD8-PE and anti–mouse-TCR β-FITC Abs. C, Dot plots showing the FACS profile of PBLs stained with anti–human-CD8-FITC Ab and PE-conjugated MAGE-A3: 112–120 or MAGE-A3: 271–279 HLA-A*0201 tetramers.

FIGURE 2.

A and B, Recognition of peptide-pulsed T2 cells by the MAGE-A3 TCR-transduced PBLs. Human PBLs expressing TCR against MAGE-A3: 112–120 or MAGE-A3: 271–279 were cocultured for 16 h with T2 cells that were previously pulsed with different concentrations of the respective peptides. Coculture of PBLs expressing TCR against MAGE-A3: 112–120 or MAGE-A3: 271–279 with control T2 cells that were not pulsed with peptides produced background levels of IFN-γ. The concentration of IFN-γ secreted into the culture medium was measured by ELISA.

FIGURE 3.

Specific killing of tumor cell lines by MAGE-A3 TCR-transduced PBLs. TCR-transduced human PBLs were cocultured for 4 h with the indicated ⁵¹Cr-labeled target tumor cell lines. Specific lysis of tumor cells was measured at the given E:T ratio using the formula: ([specific release − spontaneous release]/[total release − spontaneous release]). Specific lysis of untransduced, MAGE-A3: 112–120- or MAGE-A3: 271–279-specific TCR-transduced human PBLs are plotted on the graph as indicated. A, Specific lysis of melanoma tumor cell lines 1300 melanoma (HLAA*0201⁺/MAGE-A3⁺), 526 melanoma (HLA-A*0201⁺/MAGE-A3⁺), and 938 melanoma (HLA-A*0201⁻/MAGE-A3⁺) by the TCR-engineered PBLs. B, Specific lysis of NSCLC cell lines H1299 (MAGE⁺/HLA-A2⁺) and H1299-A*0201 (MAGE⁺/HLA-A2⁻). C, The cytolytic activities of TCR-engineered CD8⁺ and CD4⁺ T cells were evaluated using purified lymphocytes.

FIGURE 4.

Single amino acid substitutions at the CDR3 region of the MAGE-A3 TCR α-chain.

FIGURE 5.

A, Flow cytometric analysis of wild-type and modified MAGE-A3 TCR-A118T-transduced PBLs. Dot blot showing the FACS profile of PBLs stained with anti–human-CD8-FITC Ab and PE-conjugated MAGE-A3: 112–120-HLA-A*0201 tetramer. B, Recognition of MAGE-A3: 112–120 peptide-pulsed T2 cells by the MAGE-A3 TCR-transduced PBLs. Human PBLs expressing MAGE-A3: 112–120 wild-type or A118T variant TCR were cocultured for 16 h with T2 cells that were previously pulsed with different concentrations of MAGE-A3: 112–120 peptide. The concentration of IFN-γ secreted into the culture medium was measured by ELISA, and values indicate the mean of duplicate samples. C, A representative assay showing GM-CSF release data from two donors after overnight coculture with tumor cell targets. The concentration of GM-CSF secreted into the culture medium was measured by ELISA, and values indicate the mean of duplicate samples. D, Proliferation of PBLs after coculture with H1299 or H1299-A2 cells measured by [³H]thymidine incorporation after 3 d in culture. Values indicate average counts of triplicate wells.

FIGURE 6.

Intracellular cytokine staining for IFN-γ and IL-2 of MAGE-A3 TCR-transduced PBLs after Ag-specific stimulation. Ten days after transduction with MAGE-A3: 112–120 wild-type or A118T, TCR-transduced PBLs were subjected to overnight coculture with H1299-A2 cells. Cells were then stained for the detection of intracellular IFN-γ and IL-2 and cell surface expression of CD3 molecule and analyzed by FACS. The transduced PBLs produced IFN-γ in an Ag-specific manner. The plots are gated on CD3⁺ lymphocytes, and the percentage on each quadrant is shown on the plot. A representative of three experiments is shown.

FIGURE 7.

Expression of degranulation marker CD107a on MAGE-A3 TCR-transduced PBLs after Ag exposure. Expression of CD107a on MAGE-A3: 112–120 wild-type or A118T TCR-transduced PBLs was detected by FACS analysis after coculture with H1299-A2 cells. The percentages of CD107a-positive cells are shown. Results from a representative of three experiments are presented.

FIGURE 8.

Recognition of other related MAGE peptides by the MAGE-A3: 112–120 TCR-transduced PBLs. PBLs expressing TCRs against MAGE-A3: 112–120 were cocultured for 16 h with T2 cells that were previously pulsed with different concentrations of respective peptides. The amount of IFN-γ secreted into the medium was measured by ELISA. A, Amino acid sequence alignment of related MAGE peptides. All of the peptides were 9-mers, and the amino acid variation from the MAGEA3 peptide is indicated in bold. B, Peptide recognition titration was measured as the IFN-γ production after coculture with peptide-pulsed T2 cells.