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. 2012:2012:586314.
doi: 10.1155/2012/586314. Epub 2012 Feb 12.

TCR gene transfer: MAGE-C2/HLA-A2 and MAGE-A3/HLA-DP4 epitopes as melanoma-specific immune targets

Trudy Straetemans  1 , Mandy van Brakel, Sabine van Steenbergen, Marieke Broertjes, Joost Drexhage, Joost Hegmans, Bart N Lambrecht, Cor Lamers, Pierre van Der Bruggen, Pierre G Coulie, Reno Debets
Affiliations

TCR gene transfer: MAGE-C2/HLA-A2 and MAGE-A3/HLA-DP4 epitopes as melanoma-specific immune targets

Trudy Straetemans et al. Clin Dev Immunol. 2012.

Abstract

Adoptive therapy with TCR gene-engineered T cells provides an attractive and feasible treatment option for cancer patients. Further development of TCR gene therapy requires the implementation of T-cell target epitopes that prevent "on-target" reactivity towards healthy tissues and at the same time direct a clinically effective response towards tumor tissues. Candidate epitopes that meet these criteria are MAGE-C2(336-344)/HLA-A2 (MC2/A2) and MAGE-A3(243-258)/HLA-DP4 (MA3/DP4). We molecularly characterized TCRαβ genes of an MC2/A2-specific CD8 and MA3/DP4-specific CD4 T-cell clone derived from melanoma patients who responded clinically to MAGE vaccination. We identified MC2/A2 and MA3/DP4-specific TCR-Vα3/Vβ28 and TCR-Vα38/Vβ2 chains and validated these TCRs in vitro upon gene transfer into primary human T cells. The MC2 and MA3 TCR were surface-expressed and mediated CD8 T-cell functions towards melanoma cell lines and CD4 T-cell functions towards dendritic cells, respectively. We intend to start testing these MAGE-specific TCRs in phase I clinical trial.

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Figures

Figure 1
Figure 1
Cloning and validation of MC2/A2 and MA3/DP4 TCRαβ genes. (a) Schematic representation of how TCR DNAs have been cloned, typed for TCR-V(D)J gene usage, and tested in T cells following gene transfer. (b) TCR-V(D)J and C classification of the TCRα and β chains expressed by EB81-CTL16 and R12-C9 according to http://www.imgt.org/. The arrow before the Jα6.01 indicates a frame shift preventing surface expression of this TCR-Vα2 chain. Sequence data for human TCR-Vα2, Vα3, and Vβ28 of EB81-CTL16-derived TCR genes are available from GenBank under accession nos. EU427373, EU427374, and EU427375, respectively; and sequence data for human TCR-Vα38 and Vβ22 of R12-C9-derived TCR genes are available from GenBank under accession nos. EU427376 and EU427377, respectively.
Figure 2
Figure 2
Primary human T cells transduced with TCR-Vα3/Vβ28 genes bind MC2/A2 pMHC. The MC2/A2 TCR T cells were labeled with PE-conjugated MC2336–344/A2 pentamers before flow cytometric analysis (solid lines). (a) T cells transduced either with TCR-Vα2Cα/Vβ28Cβ1 and Vα3Cα/Vβ28Cβ1 or control TCRαβ genes (Mock), and not sorted for MC2/A2 binding. (b) T cells transduced with TCR-Vα3Cα/Vβ28Cβ1 genes and FAC Sorted with MC2/A2 pentamer. Results are from a representative transduction out of 6 transductions of PBMC from 2 donors with similar results.
Figure 3
Figure 3
MC2/A2 TCR is functionally expressed by primary human T cells. (a) MC2/A2 TCR T cells lyse MC2/A2 positive target cells. TCR T cells were tested in a 6 h 51Cr-release assay. The following target cells were used: MC2/A2-positive EB81-MEL-2 melanoma cells (derived from the same patient from whom the MC2 TCR was derived), pretreated or not with IFNγ, and A2-positive BSM EBV-B cells, pulsed either with gp100 or MC2 peptide (both at 10 μM final). Mock T cells did not lyse MC2/A2-positive target cells (data not shown). Effector-to-target cell ratios are indicated on the x-axis and specific 51Cr-releases are indicated on the y-axis. (b) MC2/A2 TCR T cells produce cytokines upon coculture with MC2/A2-positive target cells. T-cell production of IFNγ and TNFα (in pg/mL) was measured by ELISA in supernatants harvested after an 18 h coculture between T cells and the target cells described in legend to Figure (a) No cytokines were produced by T cells only or Mock T cells cocultured with MC2-positive target cells (data not shown). Measurements were performed in triplicate and expressed as mean values corrected for medium values. Data shown are from representative experiments out of 4 experiments from 2 donors with similar results.
Figure 4
Figure 4
Surface expression of MA3/DP4 TCR on human primary T cells following gene transfer. Human primary T cells transduced with MA3/DP4 TCRαβ genes were stained with TCR-Vβ2 mAb (in which case nonstained MA3/DP4 TCR T cells served as a negative control since control TCRαβ genes also comprise the TCR-Vβ2 chain) (a) or MA3/DP4 tetramer (b) prior to analysis by flow cytometry. In (a), the following T cells were analyzed: parental CD4 T-cell clone R12-C9; TCR T cells, nondepleted (bulk) and TCR T cells depleted for either CD8 or CD4 T cells. These T-cell populations are not FAC sorted. In (b), TCR-transduced T cells, depleted for either CD8 or CD4 T cells nonsorted, or FAC sorted with MA3/DP4 tetramer, were analyzed. Results are from a representative transduction out of 4 transductions of PBMC from 2 donors with similar results.
Figure 5
Figure 5
MA3/DP4 TCR T cells specifically lyse MA3-transduced or peptide-loaded B cells, but not MA3-positive melanoma cells. (a) MA3/DP4 TCR T cells specifically lyse DP4-positive B cells transduced with MA3-encoding cDNA. Human T cells were tested in a 6 h 51Cr-release assay using EBV-MA3 target cells. The following effector T cells were used: CD4 T-cell clone R12-C9, MA3/DP4 TCR T cells, nondepleted T cells, MA3/DP4 TCR T cells depleted for CD8 T cells, or Mock T cells depleted for CD8 T cells. MA3-negative, DP4-positive B cells (BSM) were not recognized by MA3/DP4 TCR T cells (data not shown). (b) MA3/DP4 TCR T cells do not lyse MZ2-MEL43 melanoma cells, natively expressing MA3 and DP4. Effector T cells used were those described in legend to Figure (a). (c) MA3/DP4 TCR T cells do not lyse MZ2-MEL43 melanoma cells that are pretreated with IFNγ. Target cells were MZ2-MEL43 cells that were either pretreated with IFNγ or not, and effector T cells were MA3/DP4 TCR or Mock T cells. (d) MA3/DP4 TCR T cells lyse MZ2-MEL43 melanoma cells that are pulsed with MA3 peptide. Target cells were MZ2-MEL43 cells that were either pulsed with MA3 peptide or not, and effector T cells were MA3/DP4 TCR T cells. Measurements were performed in triplicate and expressed as mean values corrected for medium values. Data are from representative experiments out of 3 experiments with similar results.
Figure 6
Figure 6
MA3/DP4 TCR T cells specifically produce IFNγ and TNFα upon coculture with MA3-transduced or peptide-loaded B cells, but not MA3-positive melanoma cells. Cytokine production is determined in supernatants of T cells after an 18 h co-culture with (a) DP4-positive B cells transduced with Ii-MA3 cDNA (EBV-MA3) or (b) MZ2-MEL43 cells loaded with MA3 peptide or not. In (a), effector T cells were: the CD4 T-cell clone R12-C9; MA3/DP4 TCR or Mock T cells, either nondepleted or depleted for CD8 T cells. MA3-negative, DP4-positive B cells (such BSM) were not recognized by MA3/DP4 TCR T cells (data not shown). In (b), MA3/DP4 TCR or Mock T cells, non-depleted, were used as effector T cells. Supernatants were harvested and analyzed for IFNγ and TNFα by ELISA. Measurements were performed in triplicate and expressed as mean values corrected for medium values. Data are from representative experiments out of 3 experiments with similar results.
Figure 7
Figure 7
MA3/DP4 TCR CD4 T cells produce cytokines upon coculture with MA3 protein-loaded autologous dendritic cells. MA3/DP4 TCR, and Mock CD4 T cells were cultured with immature or mature autologous dendritic cells that were either loaded with 25 μg/mL MA3 protein or not. After 4 days, supernatants were harvested and analyzed for cytokine production by cytokine bead arrays. Cytokine production was not detected in case T cells were cultured without dendritic cells (data not shown). Measurements were performed in duplicate and expressed as mean values. Data are from a representative experiment out of 2 experiments with similar results.
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References

    1. Dudley ME, Wunderlich JR, Yang JC, et al. Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma. Journal of Clinical Oncology. 2005;23(10):2346–2357. - PMC - PubMed
    1. Yee C, Thompson JA, Byrd D, et al. Adoptive T cell therapy using antigen-specific CD8+ T cell clones for the treatment of patients with metastatic melanoma: in vivo persistence, migration, and antitumor effect of transferred T cells. Proceedings of the National Academy of Sciences of the United States of America. 2002;99(25):16168–16173. - PMC - PubMed
    1. Walter EA, Greenberg PD, Gilbert MJ, et al. Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor. The New England Journal of Medicine. 1995;333(16):1038–1044. - PubMed
    1. Savoldo B, Heslop HE, Rooney CM. The use of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus induced lymphoma in transplant recipients. Leukemia and Lymphoma. 2000;39(5-6):455–464. - PubMed
    1. Butler MO, Friedlander P, Milstein MI, et al. Establishment of antitumor memory in humans using in vitro-educated CD8+ T cells. Science Translational Medicine. 2011;3(80):p. 80ra34 . - PMC - PubMed

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