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A phase I clinical study of vaccination of melanoma patients with dendritic cells loaded with allogeneic apoptotic/necrotic melanoma cells. Analysis of toxicity and immune response to the vaccine and of IL-10 -1082 promoter genotype as predictor of disease progression.

von Euw EM, Barrio MM, Furman D, Levy EM, Bianchini M, Peguillet I, Lantz O, Vellice A, Kohan A, Chacón M, Yee C, Wainstok R, Mordoh J - J Transl Med (2008)

Bottom Line: The vaccine was well tolerated in all patients.The DTH score increased significantly in all patients after the first vaccination (Mann-Whitney Test, p < 0.05).We conclude that DC/Apo-Nec vaccine is safe, well tolerated and it may induce specific immunity against melanoma Ags.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro de Investigaciones Oncológicas FUCA, Cramer 1180, (1426) Buenos Aires, Argentina. evoneuw@leloir.org.ar

ABSTRACT

Background: Sixteen melanoma patients (1 stage IIC, 8 stage III, and 7 stage IV) were treated in a Phase I study with a vaccine (DC/Apo-Nec) composed of autologous dendritic cells (DCs) loaded with a mixture of apoptotic/necrotic allogeneic melanoma cell lines (Apo-Nec), to evaluate toxicity and immune responses. Also, IL-10 1082 genotype was analyzed in an effort to predict disease progression.

Methods: PBMC were obtained after leukapheresis and DCs were generated from monocytes cultured in the presence of GM-CSF and IL-4 in serum-free medium. Immature DCs were loaded with gamma-irradiated Apo-Nec cells and injected id without adjuvant. Cohorts of four patients were given four vaccines each with 5, 10, 15, or 20 x 106 DC/Apo-Nec cell per vaccine, two weeks apart. Immune responses were measured by ELISpot and tetramer analysis. Il-10 genotype was measured by PCR and corroborated by IL-10 production by stimulated PBMC.

Results: Immature DCs efficiently phagocytosed melanoma Apo-Nec cells and matured after phagocytosis as evidenced by increased expression of CD83, CD80, CD86, HLA class I and II, and 75.2 +/- 16% reduction in Dextran-FITC endocytosis. CCR7 was also up-regulated upon Apo-Nec uptake in DCs from all patients, and accordingly DC/Apo-Nec cells were able to migrate in vitro toward MIP-3 beta. The vaccine was well tolerated in all patients. The DTH score increased significantly in all patients after the first vaccination (Mann-Whitney Test, p < 0.05). The presence of CD8+T lymphocytes specific to gp100 and Melan A/MART-1 Ags was determined by ELISpot and tetramer analysis in five HLA-A*0201 patients before and after vaccination; one patient had stable elevated levels before and after vaccination; two increased their CD8 + levels, one had stable moderate and one had negligible levels. The analysis of IL-10 promoter -1082 polymorphism in the sixteen patients showed a positive correlation between AA genotype, accompanied by lower in vitro IL-10 production by stimulated PBMC, and faster melanoma progression after lymph nodes surgery (p = 0.04). With a mean follow-up of 49.5 months post-surgery, one stage IIC patient and 7/8 stage III patients remain NED but 7/7 stage IV patients have progressed.

Conclusion: We conclude that DC/Apo-Nec vaccine is safe, well tolerated and it may induce specific immunity against melanoma Ags. Patients with a low-producing IL-10 polymorphism appear to have a worst prognosis.

Trial registration: Clinicaltrials.gov (NHI) NCT00515983.

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Patient#1's lymphocytes in vitro proliferation in response to autologous tumor Ags presented by DCs. Pre and post vaccination Patient#1 PBMC were incubated either alone, with Apo-Nec#1 cells prepared from patient#1 tumor cells or with DC/Apo-Nec#1 cells; (H3) dThd incorporation was measured as described under Methods. Results represent mean ± SD cpm (counts per minute) of triplicates. Positive controls incubated with PHA incorporated more than 7 × 104 cpm (not shown).
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Figure 2: Patient#1's lymphocytes in vitro proliferation in response to autologous tumor Ags presented by DCs. Pre and post vaccination Patient#1 PBMC were incubated either alone, with Apo-Nec#1 cells prepared from patient#1 tumor cells or with DC/Apo-Nec#1 cells; (H3) dThd incorporation was measured as described under Methods. Results represent mean ± SD cpm (counts per minute) of triplicates. Positive controls incubated with PHA incorporated more than 7 × 104 cpm (not shown).

Mentions: In the case of patient #1, an autologous melanoma cell line was established, allowing us to analyze if lymphocytes were primed by DC/Apo-Nec vaccination (allogeneic cells) and recognized autologous Ags. We measured pre- and post-vaccination PBMC proliferation after a five-day coculture of mononuclear cells with Apo-Nec#1 cells (irradiated patient #1's tumor cells) or DC/Apo-Nec as described under Methods. As we had previously demonstrated, Apo-Nec cells alone were not able to stimulate PBMC by their own, probably due to the loss of HLA-class I molecules at the cell surface during the apoptotic process [21] (Figure 2). Instead, post-vaccination PBMC proliferated approximately 10-fold more than pre-vaccination lymphocytes in response to DC/Apo-Nec#1 cells, demonstrating that after vaccination with DC/Apo-Nec, lymphocyte clones recognizing Ags shared by the Apo-Nec mixture and the patient's tumor cells had expanded.


A phase I clinical study of vaccination of melanoma patients with dendritic cells loaded with allogeneic apoptotic/necrotic melanoma cells. Analysis of toxicity and immune response to the vaccine and of IL-10 -1082 promoter genotype as predictor of disease progression.

von Euw EM, Barrio MM, Furman D, Levy EM, Bianchini M, Peguillet I, Lantz O, Vellice A, Kohan A, Chacón M, Yee C, Wainstok R, Mordoh J - J Transl Med (2008)

Patient#1's lymphocytes in vitro proliferation in response to autologous tumor Ags presented by DCs. Pre and post vaccination Patient#1 PBMC were incubated either alone, with Apo-Nec#1 cells prepared from patient#1 tumor cells or with DC/Apo-Nec#1 cells; (H3) dThd incorporation was measured as described under Methods. Results represent mean ± SD cpm (counts per minute) of triplicates. Positive controls incubated with PHA incorporated more than 7 × 104 cpm (not shown).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2265680&req=5

Figure 2: Patient#1's lymphocytes in vitro proliferation in response to autologous tumor Ags presented by DCs. Pre and post vaccination Patient#1 PBMC were incubated either alone, with Apo-Nec#1 cells prepared from patient#1 tumor cells or with DC/Apo-Nec#1 cells; (H3) dThd incorporation was measured as described under Methods. Results represent mean ± SD cpm (counts per minute) of triplicates. Positive controls incubated with PHA incorporated more than 7 × 104 cpm (not shown).
Mentions: In the case of patient #1, an autologous melanoma cell line was established, allowing us to analyze if lymphocytes were primed by DC/Apo-Nec vaccination (allogeneic cells) and recognized autologous Ags. We measured pre- and post-vaccination PBMC proliferation after a five-day coculture of mononuclear cells with Apo-Nec#1 cells (irradiated patient #1's tumor cells) or DC/Apo-Nec as described under Methods. As we had previously demonstrated, Apo-Nec cells alone were not able to stimulate PBMC by their own, probably due to the loss of HLA-class I molecules at the cell surface during the apoptotic process [21] (Figure 2). Instead, post-vaccination PBMC proliferated approximately 10-fold more than pre-vaccination lymphocytes in response to DC/Apo-Nec#1 cells, demonstrating that after vaccination with DC/Apo-Nec, lymphocyte clones recognizing Ags shared by the Apo-Nec mixture and the patient's tumor cells had expanded.

Bottom Line: The vaccine was well tolerated in all patients.The DTH score increased significantly in all patients after the first vaccination (Mann-Whitney Test, p < 0.05).We conclude that DC/Apo-Nec vaccine is safe, well tolerated and it may induce specific immunity against melanoma Ags.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro de Investigaciones Oncológicas FUCA, Cramer 1180, (1426) Buenos Aires, Argentina. evoneuw@leloir.org.ar

ABSTRACT

Background: Sixteen melanoma patients (1 stage IIC, 8 stage III, and 7 stage IV) were treated in a Phase I study with a vaccine (DC/Apo-Nec) composed of autologous dendritic cells (DCs) loaded with a mixture of apoptotic/necrotic allogeneic melanoma cell lines (Apo-Nec), to evaluate toxicity and immune responses. Also, IL-10 1082 genotype was analyzed in an effort to predict disease progression.

Methods: PBMC were obtained after leukapheresis and DCs were generated from monocytes cultured in the presence of GM-CSF and IL-4 in serum-free medium. Immature DCs were loaded with gamma-irradiated Apo-Nec cells and injected id without adjuvant. Cohorts of four patients were given four vaccines each with 5, 10, 15, or 20 x 106 DC/Apo-Nec cell per vaccine, two weeks apart. Immune responses were measured by ELISpot and tetramer analysis. Il-10 genotype was measured by PCR and corroborated by IL-10 production by stimulated PBMC.

Results: Immature DCs efficiently phagocytosed melanoma Apo-Nec cells and matured after phagocytosis as evidenced by increased expression of CD83, CD80, CD86, HLA class I and II, and 75.2 +/- 16% reduction in Dextran-FITC endocytosis. CCR7 was also up-regulated upon Apo-Nec uptake in DCs from all patients, and accordingly DC/Apo-Nec cells were able to migrate in vitro toward MIP-3 beta. The vaccine was well tolerated in all patients. The DTH score increased significantly in all patients after the first vaccination (Mann-Whitney Test, p < 0.05). The presence of CD8+T lymphocytes specific to gp100 and Melan A/MART-1 Ags was determined by ELISpot and tetramer analysis in five HLA-A*0201 patients before and after vaccination; one patient had stable elevated levels before and after vaccination; two increased their CD8 + levels, one had stable moderate and one had negligible levels. The analysis of IL-10 promoter -1082 polymorphism in the sixteen patients showed a positive correlation between AA genotype, accompanied by lower in vitro IL-10 production by stimulated PBMC, and faster melanoma progression after lymph nodes surgery (p = 0.04). With a mean follow-up of 49.5 months post-surgery, one stage IIC patient and 7/8 stage III patients remain NED but 7/7 stage IV patients have progressed.

Conclusion: We conclude that DC/Apo-Nec vaccine is safe, well tolerated and it may induce specific immunity against melanoma Ags. Patients with a low-producing IL-10 polymorphism appear to have a worst prognosis.

Trial registration: Clinicaltrials.gov (NHI) NCT00515983.

Show MeSH
Related in: MedlinePlus