Vaccination with mage-3A1 peptide-pulsed mature, monocyte-derived dendritic cells expands specific cytotoxic T cells and induces regression of some metastases in advanced stage IV melanoma

B Thurner, I Haendle, C Röder, D Dieckmann, P Keikavoussi, H Jonuleit, A Bender, C Maczek, D Schreiner, P von den Driesch, E B Bröcker, R M Steinman, A Enk, E Kämpgen, G Schuler, B Thurner, I Haendle, C Röder, D Dieckmann, P Keikavoussi, H Jonuleit, A Bender, C Maczek, D Schreiner, P von den Driesch, E B Bröcker, R M Steinman, A Enk, E Kämpgen, G Schuler

Abstract

Dendritic cells (DCs) are considered to be promising adjuvants for inducing immunity to cancer. We used mature, monocyte-derived DCs to elicit resistance to malignant melanoma. The DCs were pulsed with Mage-3A1 tumor peptide and a recall antigen, tetanus toxoid or tuberculin. 11 far advanced stage IV melanoma patients, who were progressive despite standard chemotherapy, received five DC vaccinations at 14-d intervals. The first three vaccinations were administered into the skin, 3 x 10(6) DCs each subcutaneously and intradermally, followed by two intravenous injections of 6 x 10(6) and 12 x 10(6) DCs, respectively. Only minor (less than or equal to grade II) side effects were observed. Immunity to the recall antigen was boosted. Significant expansions of Mage-3A1-specific CD8(+) cytotoxic T lymphocyte (CTL) precursors were induced in 8/11 patients. Curiously, these immune responses often declined after the intravenous vaccinations. Regressions of individual metastases (skin, lymph node, lung, and liver) were evident in 6/11 patients. Resolution of skin metastases in two of the patients was accompanied by erythema and CD8(+) T cell infiltration, whereas nonregressing lesions lacked CD8(+) T cells as well as Mage-3 mRNA expression. This study proves the principle that DC "vaccines" can frequently expand tumor-specific CTLs and elicit regressions even in advanced cancer and, in addition, provides evidence for an active CD8(+) CTL-tumor cell interaction in situ as well as escape by lack of tumor antigen expression.

Figures

Figure 2
Figure 2
Recall antigen–specific immunity (tuberculin in patient 10; TT in all others) as assayed by antigen-specific proliferation. The cpm values determined after therapy (14 d after vaccination 5) are shown as multiples of pretherapy cpm values. Absolute cpm (cpm with recall antigen minus cpm without antigen) after therapy was 68,917 in patient 02, 85,225 in patient 04, 16,759 in patient 05, 7,913 in patient 06, 16,367 in patient 07, 107,923 in patient 09, 22,790 in patient 10, 4,507 in patient 12, and 1,831 in patient 13 (SEM for all measurements was

Figure 1

Local reactions to DCs carrying…

Figure 1

Local reactions to DCs carrying Mage-3A1 peptide and TT at the intradermal and…

Figure 1
Local reactions to DCs carrying Mage-3A1 peptide and TT at the intradermal and subcutaneous vaccination sites in patient 09 (24 h after vaccination 2; top panel) and 02 (48 h after vaccination 3; bottom panel). Erythema at the 10 intradermal (left) and 2 subcutaneous (right) vaccination sites was followed by induration >10 mm in diameter (with secondary purpura in patient 02). These local reactions represent strong DTH reactions to DCs carrying TT, as such strong reactions did not occur in response to unpulsed DCs or DCs pulsed with Mage-3A1 peptide alone in DTH tests I–III (Table ; reactions not shown).

Figure 1

Local reactions to DCs carrying…

Figure 1

Local reactions to DCs carrying Mage-3A1 peptide and TT at the intradermal and…

Figure 1
Local reactions to DCs carrying Mage-3A1 peptide and TT at the intradermal and subcutaneous vaccination sites in patient 09 (24 h after vaccination 2; top panel) and 02 (48 h after vaccination 3; bottom panel). Erythema at the 10 intradermal (left) and 2 subcutaneous (right) vaccination sites was followed by induration >10 mm in diameter (with secondary purpura in patient 02). These local reactions represent strong DTH reactions to DCs carrying TT, as such strong reactions did not occur in response to unpulsed DCs or DCs pulsed with Mage-3A1 peptide alone in DTH tests I–III (Table ; reactions not shown).

Figure 3

Kinetic analysis of immunity to…

Figure 3

Kinetic analysis of immunity to recall antigens as assessed by TT-specific IFN-γ ELISPOT…

Figure 3
Kinetic analysis of immunity to recall antigens as assessed by TT-specific IFN-γ ELISPOT (SEM for all measurements was before vaccination 2, i.e., 14 d after vaccination 1), and finally after therapy. Time points at which vaccinations were not performed lack bars. Note the increase after the intracutaneous vaccinations and the decline upon the two vaccinations after intravenous ones. Patient 10, who received tuberculin-pulsed DCs, exhibited no significant change in the TT-specific IFN-γ ELISPOT as expected.

Figure 4

Mage-3A1 CTLp frequency analysis as…

Figure 4

Mage-3A1 CTLp frequency analysis as assessed by semiquantitative recall assay. The y-axis and…

Figure 4
Mage-3A1 CTLp frequency analysis as assessed by semiquantitative recall assay. The y-axis and the numbers above the bars indicate the percentage of positive wells found before vaccination 1, before vaccination 4 (14 d after vaccination 3), and after therapy (usually 14 d after vaccination 5).

Figure 5

Regression (arrows) of a globular…

Figure 5

Regression (arrows) of a globular (13 mm in diameter) lung metastasis in patient…

Figure 5
Regression (arrows) of a globular (13 mm in diameter) lung metastasis in patient 07 that was then no longer detectable in serial 6-mm-thick computed tomography scans.

Figure 5

Regression (arrows) of a globular…

Figure 5

Regression (arrows) of a globular (13 mm in diameter) lung metastasis in patient…

Figure 5
Regression (arrows) of a globular (13 mm in diameter) lung metastasis in patient 07 that was then no longer detectable in serial 6-mm-thick computed tomography scans.

Figure 6

Regressing subcutaneous metastases in patient…

Figure 6

Regressing subcutaneous metastases in patient 06 display a CD8 + lymphocytic infiltrate (alkaline…

Figure 6
Regressing subcutaneous metastases in patient 06 display a CD8+ lymphocytic infiltrate (alkaline phosphatase/antialkaline phosphatase immunohistochemical staining with anti-CD8 mAb) that surrounds (A) and infiltrates (B) the tumor. Areas of damaged (B, ★) and necrotic (C, ★) melanoma cells are obvious in the vicinity of the CD8+ T cell infiltrate. The metastasis expressed Mage-3, as demonstrated by RT-PCR (data not shown). Magnifications: A, 100; B, 250; C, 160.

Figure 6

Regressing subcutaneous metastases in patient…

Figure 6

Regressing subcutaneous metastases in patient 06 display a CD8 + lymphocytic infiltrate (alkaline…

Figure 6
Regressing subcutaneous metastases in patient 06 display a CD8+ lymphocytic infiltrate (alkaline phosphatase/antialkaline phosphatase immunohistochemical staining with anti-CD8 mAb) that surrounds (A) and infiltrates (B) the tumor. Areas of damaged (B, ★) and necrotic (C, ★) melanoma cells are obvious in the vicinity of the CD8+ T cell infiltrate. The metastasis expressed Mage-3, as demonstrated by RT-PCR (data not shown). Magnifications: A, 100; B, 250; C, 160.

Figure 6

Regressing subcutaneous metastases in patient…

Figure 6

Regressing subcutaneous metastases in patient 06 display a CD8 + lymphocytic infiltrate (alkaline…

Figure 6
Regressing subcutaneous metastases in patient 06 display a CD8+ lymphocytic infiltrate (alkaline phosphatase/antialkaline phosphatase immunohistochemical staining with anti-CD8 mAb) that surrounds (A) and infiltrates (B) the tumor. Areas of damaged (B, ★) and necrotic (C, ★) melanoma cells are obvious in the vicinity of the CD8+ T cell infiltrate. The metastasis expressed Mage-3, as demonstrated by RT-PCR (data not shown). Magnifications: A, 100; B, 250; C, 160.
All figures (10)
Figure 1
Figure 1
Local reactions to DCs carrying Mage-3A1 peptide and TT at the intradermal and subcutaneous vaccination sites in patient 09 (24 h after vaccination 2; top panel) and 02 (48 h after vaccination 3; bottom panel). Erythema at the 10 intradermal (left) and 2 subcutaneous (right) vaccination sites was followed by induration >10 mm in diameter (with secondary purpura in patient 02). These local reactions represent strong DTH reactions to DCs carrying TT, as such strong reactions did not occur in response to unpulsed DCs or DCs pulsed with Mage-3A1 peptide alone in DTH tests I–III (Table ; reactions not shown).
Figure 1
Figure 1
Local reactions to DCs carrying Mage-3A1 peptide and TT at the intradermal and subcutaneous vaccination sites in patient 09 (24 h after vaccination 2; top panel) and 02 (48 h after vaccination 3; bottom panel). Erythema at the 10 intradermal (left) and 2 subcutaneous (right) vaccination sites was followed by induration >10 mm in diameter (with secondary purpura in patient 02). These local reactions represent strong DTH reactions to DCs carrying TT, as such strong reactions did not occur in response to unpulsed DCs or DCs pulsed with Mage-3A1 peptide alone in DTH tests I–III (Table ; reactions not shown).
Figure 3
Figure 3
Kinetic analysis of immunity to recall antigens as assessed by TT-specific IFN-γ ELISPOT (SEM for all measurements was before vaccination 2, i.e., 14 d after vaccination 1), and finally after therapy. Time points at which vaccinations were not performed lack bars. Note the increase after the intracutaneous vaccinations and the decline upon the two vaccinations after intravenous ones. Patient 10, who received tuberculin-pulsed DCs, exhibited no significant change in the TT-specific IFN-γ ELISPOT as expected.
Figure 4
Figure 4
Mage-3A1 CTLp frequency analysis as assessed by semiquantitative recall assay. The y-axis and the numbers above the bars indicate the percentage of positive wells found before vaccination 1, before vaccination 4 (14 d after vaccination 3), and after therapy (usually 14 d after vaccination 5).
Figure 5
Figure 5
Regression (arrows) of a globular (13 mm in diameter) lung metastasis in patient 07 that was then no longer detectable in serial 6-mm-thick computed tomography scans.
Figure 5
Figure 5
Regression (arrows) of a globular (13 mm in diameter) lung metastasis in patient 07 that was then no longer detectable in serial 6-mm-thick computed tomography scans.
Figure 6
Figure 6
Regressing subcutaneous metastases in patient 06 display a CD8+ lymphocytic infiltrate (alkaline phosphatase/antialkaline phosphatase immunohistochemical staining with anti-CD8 mAb) that surrounds (A) and infiltrates (B) the tumor. Areas of damaged (B, ★) and necrotic (C, ★) melanoma cells are obvious in the vicinity of the CD8+ T cell infiltrate. The metastasis expressed Mage-3, as demonstrated by RT-PCR (data not shown). Magnifications: A, 100; B, 250; C, 160.
Figure 6
Figure 6
Regressing subcutaneous metastases in patient 06 display a CD8+ lymphocytic infiltrate (alkaline phosphatase/antialkaline phosphatase immunohistochemical staining with anti-CD8 mAb) that surrounds (A) and infiltrates (B) the tumor. Areas of damaged (B, ★) and necrotic (C, ★) melanoma cells are obvious in the vicinity of the CD8+ T cell infiltrate. The metastasis expressed Mage-3, as demonstrated by RT-PCR (data not shown). Magnifications: A, 100; B, 250; C, 160.
Figure 6
Figure 6
Regressing subcutaneous metastases in patient 06 display a CD8+ lymphocytic infiltrate (alkaline phosphatase/antialkaline phosphatase immunohistochemical staining with anti-CD8 mAb) that surrounds (A) and infiltrates (B) the tumor. Areas of damaged (B, ★) and necrotic (C, ★) melanoma cells are obvious in the vicinity of the CD8+ T cell infiltrate. The metastasis expressed Mage-3, as demonstrated by RT-PCR (data not shown). Magnifications: A, 100; B, 250; C, 160.

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