Peripheral blood lymphocytes genetically modified to express the self/tumor antigen MAGE-A3 induce antitumor immune responses in cancer patients by Raffaella Fontana, Marco Bregni, Arcadi Cipponi, Laura Raccosta, Cristina Rainelli, Daniela Maggioni, Francesca Lunghi, Fabio Ciceri, Sylvain Mukenge, Claudio Doglioni, Didier Colau, Pierre G. Coulie, Claudio Bordignon, Catia Traversari, and Vincenzo Russo Blood Volume 113(8):1651-1660 February 19, 2009 ©2009 by American Society of Hematology

Characterization of TK-specific immune responses. Characterization of TK-specific immune responses. (A) Ex vivo IFN-γ ELISPOT assay and (B) IFN-γ ELISA-based semiquantitative recall assays on multiple groups (105) of PBMCs collected before and after treatment. (C,D) HLA-I restriction of the anti-TK T cells in patients CIP-5 (C) and CIP-21 (D). Microcultures containing TK-specific effectors were challenged with Cos-7 cells transfected with available HLA-I alleles together with HSV-TK cDNAs. Supernatants were then assayed for IFN-γ release (*P < .01). Error bars represent the SD of experimental replicates. (E) Microcultures from the postfourth blood sample of patient CIP-25, a patient expressing the HLA-B7 allele, recognized autologous untransduced (UT)–GMLs pulsed with the TK279-288 epitope previously described.14 (F) Limiting dilution analysis estimating the frequency of anti-TK T cells in patients CIP-23 and CIP-25. Raffaella Fontana et al. Blood 2009;113:1651-1660 ©2009 by American Society of Hematology

Enumeration and characterization of circulating anti–MAGE-A3 T cells in patient CIP-19 and analysis of anti–MAGE-A3 T cells infiltrating the DTH. (A) Clinical evolution of patient CIP-19 and timing of treatments. Enumeration and characterization of circulating anti–MAGE-A3 T cells in patient CIP-19 and analysis of anti–MAGE-A3 T cells infiltrating the DTH. (A) Clinical evolution of patient CIP-19 and timing of treatments. (B) Anti–MAGE-A3 T-cell frequency and DTH reactivity after indicated treatments are shown. The frequency of total anti–MAGE-A3 T cells was measured by IFN-γ release assay performed on multiple cultures of PBMCs (105) stimulated and then tested against M3-GMLs and UT-GML. After 12 infusions, we detected a strong increase of circulating anti–MAGE-A3 T cells (1.73 × 10−5). This increase paralleled the development of a strong MAGE-A3–specific DTH reaction. (C) Antigen specificity of a selected CD4+ microculture. Microculture no. 8 was tested against MAGE-A3–transduced (M3) or untransduced (UT) autologous EBV in the presence or in the absence of anti-DR, anti-DP, and anti-NGFr (used as control antibody) mAbs. Anti–MAGE-A3 T cells were also tested against autologous EBV pulsed with the M3.DP*0401 peptide26 and against allogeneic MAGE-A3–transduced or untransduced EBV (BM21-EBV) sharing the HLA-DP*1001 allele. Microculture no. 8 specifically recognized the allogeneic HLA-DP*1001 EBV expressing MAGE-3 but not the M3.DP*0401 peptide. Error bars represent SD of experimental replicates. (D,E) MAGE-A3–specific long-term T-cell memory. (D) CD3+CD4+ T-cell clones from a punch biopsy of an anti–MAGE-A3 DTH performed 9 months after the 14th infusion, proliferated in the presence of MAGE-A3–transduced autologous EBV cells. (E) Upon in vitro expansion, clones no. 30 and no. 45 specifically released IFN-γ in response to MAGE-A3–expressing target cells. Raffaella Fontana et al. Blood 2009;113:1651-1660 ©2009 by American Society of Hematology

Enumeration and characterization of circulating anti–MAGE-A3 T cells in patient CIP-26 and analysis of anti–MAGE-A3 T cells infiltrating tumor lesions. Enumeration and characterization of circulating anti–MAGE-A3 T cells in patient CIP-26 and analysis of anti–MAGE-A3 T cells infiltrating tumor lesions. (A) Clinical evolution of patient CIP-26 and timing of treatments. PD indicates progressive disease; CR, complete response; and PR, partial response. (B) Frequency of circulating anti–MAGE-A3 T cells estimated before, during, and after treatment. The frequency of total anti–MAGE-A3 T cells was measured as described in Figure 2. P indicates after infusion; P30*, this blood sample was withdrawn in 2007, 1 year after the 30th vaccination. (C) HLA restriction of a representative MAGE-A3–specific CD8+ microculture. The microculture released IFN-γ against autologous M3-GMLs preincubated with 4E mAb (recognizing HLA-B and -C alleles) or with anti-NGFr mAb (control mAb). Release of IFN-γ was instead inhibited in the presence of W6/32 mAb (anti–HLA-I mAb). Error bars represent SD of experimental replicates. (D) Relationship between the increase of MAGE-A3–specific effectors and the development of MAGE-A3–specific DTH reaction. (E) Ex vivo IFN-γ release assay performed on anti–MAGE-A3 T cells from a regressing tumor lesion and a regional lymph node collected in 2005. T cells recognized autologous M3-GMLs but not mock-transduced lymphocytes (UT-GMLs). (F) Only CD8+ purified TILs specifically released IFN-γ when challenged with M3-GMLs. Raffaella Fontana et al. Blood 2009;113:1651-1660 ©2009 by American Society of Hematology

Immunohistochemical analysis of tumor lesions of patient CIP-26 collected during the treatment. Immunohistochemical analysis of tumor lesions of patient CIP-26 collected during the treatment. (A-G) Melanoma lesions collected during the treatment were stained with HC10 mAb (anti–HLA-B and -C), anti–β2-microglobulin mAb, W6/32 (anti–HLA-I) mAb and anti–HLA-A30 mAb. (A,B) The regressing tumor nodule collected in 2005 after 18 vaccinations, was almost completely negative for HC10 (A), but contained tumor areas stained with anti–β2-microglobulin mAb (B), W6/32 mAb (C) and with an anti–HLA-A30 mAb (D). A progressing tumor lesion collected in 2006, after 22 vaccinations, was not stained by mAb HC10 (E) and by anti–β2-microglobulin mAb (F). Some tumor areas from the progressing lesion were not stained by mAb W6/32 (G) and by an anti–HLA-A30 mAb (H). Objectives, ×200. Raffaella Fontana et al. Blood 2009;113:1651-1660 ©2009 by American Society of Hematology

Enumeration and characterization of circulating anti–MAGE-A3 T cells in patient CIP-5. Enumeration and characterization of circulating anti–MAGE-A3 T cells in patient CIP-5. (A) Clinical evolution of patient CIP-5 and timing of treatments; PD: progressive disease, NED: not evidence of disease. (B) Frequency of circulating anti–MAGE-A3 T cells estimated in patient CIP-5 during the treatment. The frequency of total anti–MAGE-A3 T cells was measured as described in Figure 2. The frequency of anti–MAGE-A3.A26 and anti–MAGE-A3.B44 T cells was measured by tetramer staining or IFN-γ release assay, performed on multiple cultures of PBMCs (105) stimulated with the peptide M3250-258. After 15 infusions, patient CIP-5 developed a strong increase of circulating anti–MAGE-A3 T cells (3.08 × 10−5) that paralleled a MAGE-A3–specific DTH reaction; P stands for after infusion. (C) HLA-I restriction of the selected CD8+ microculture no. 10. HLA-I restriction was characterized as described in Figure 1. Microculture no. 10 recognized HLA-A26, -B44 and Cw01-restricted MAGE-A3–derived epitopes and autologous tumor cells (CIP-5-mel). Autologous lymphocytes expressing MAGE-A3 (M3-GMLs) were used as positive control. Raffaella Fontana et al. Blood 2009;113:1651-1660 ©2009 by American Society of Hematology