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- MATERIAL AND METHODS
Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that can be used for vaccination purposes, to induce a specific T-cell response in vivo against melanoma-associated antigens. We have shown that the sequential use of early-acting hematopoietic growth factors, stem cell factor, IL-3 and IL-6, followed by differentiation with IL-4 and granulocyte-macrophage colony-stimulating factor allows the in vitro generation of large numbers of immature DCs from CD34+ peripheral blood progenitor cells. Maturation to interdigitating DCs could specifically be induced within 24 hr by addition of TNF-α. Here, we report on a phase I clinical vaccination trial in melanoma patients using peptide-pulsed DCs. Fourteen HLA-A1+ or HLA-A2+ patients received at least 4 i.v. infusions of 5 × 106 to 5 × 107 DCs pulsed with a pool of peptides including either MAGE-1, MAGE-3 (HLA-A1) or Melan-A, gp100, tyrosinase (HLA-A2), depending on the HLA haplotype. A total of 83 vaccinations were performed. Clinical side effects were mild and consisted of low-grade fever (WHO grade I–II). Clinical and immunological responses consisted of anti-tumor responses in 2 patients, increased melanoma peptide-specific delayed-type hypersensitivity reactions in 4 patients, significant expansion of Melan-A- and gp100-specific cytotoxic T lymphocytes in the peripheral blood lymphocytes of 1 patient after vaccination and development of vitiligo in another HLA-A2+ patient. Our data indicate that the vaccination of peptide-pulsed DCs is capable of inducing clinical and systemic tumor-specific immune responses without provoking major side effects. Int. J. Cancer 86:385–392, 2000. © 2000 Wiley-Liss, Inc.
The cloning and characterization of tumor-associated antigens and tumor-associated antigen-derived peptides recognized by human cytotoxic T lymphocytes (CTLs) in a major histocompatibility complex (MHC) class I–restricted fashion has opened new possibilities for immunotherapeutic approaches to the treatment of human cancers, particularly malignant melanoma (van den Eynde and van der Bruggen, 1997). Encouraging results have been obtained in vivo with different vaccination strategies using antigenic peptides, plasmid DNA or recombinant viruses encoding tumor-associated antigens (Rosenberg et al.,1998a,b). The dominant role of professional antigen-presenting cells (APCs) in the induction of peptide-specific CTLs has been demonstrated in most of these approaches (Iwasaki et al.,1997). In addition, Toes et al. (1998) have shown that the in vivo CTL-tolerizing potential of some peptides can be converted to specific immunostimulation depending on the nature of the APCs.
Dendritic cells (DCs) are potent APCs and thus specifically involved in the initiation of antigen–specific immune responses (Banchereau and Steinman, 1998). Due to their potent co-stimulatory activity, they are well suited to activate T cells toward various antigens, such as tumor antigens.
The availability of large numbers of DCs, generated from hematopoietic progenitor cells or monocytes in vitro, has profoundly changed pre-clinical research as well as the clinical evaluation of these cells (Herbst et al.,1996, 1997). DCs are attractive for the in vivo induction and activation of antigens and peptide-specific T cells. Accordingly, appropriately pulsed or transfected DCs may be used for vaccination in the field of infectious diseases or tumor immunotherapy, to induce specific CTLs.
Previous studies on the vaccination of melanoma patients with peptide-pulsed DCs provided evidence of clinical and immunological anti-tumor responses (Nestle et al.,1998; Hu et al.,1996).
Here, we report on the vaccination of melanoma patients with peptide-pulsed DCs generated in vitro from CD34+ peripheral blood progenitor cells (PBPCs). We have demonstrated that the sequential use of early-acting hematopoietic growth factors, stem cell factor (SCF), IL-3 and IL-6 in vitro followed on day 8 by differentiation with IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) allows the generation of large numbers of immature DCs from CD34+ PBPCs (Herbst et al.,1996). Maturation to interdigitating DCs could specifically be induced within 24 hr by addition of TNF-α. Autologous DCs were pulsed with a cocktail of melanoma-associated peptides depending on the HLA haplotype of the patient. Since effective immunization with antigen-pulsed DCs injected i.v. has been demonstrated in studies on both humans and other species (Mayordomo et al.,1995; Hsu et al.,1996; Höltl et al.,1998), we injected the DCs i.v. after pulsing them with peptides. We report here on the results of 14 melanoma patients treated with this type of active immunotherapy.
- Top of page
- MATERIAL AND METHODS
We report a clinical phase I vaccination study in melanoma patients performed with peptide-pulsed DCs generated in vitro from CD34+ hematopoietic progenitor cells. The following conclusions emerge from this study, which included 83 vaccinations in 14 melanoma patients: (i) sufficient numbers of DCs can be generated from CD34+ PBPCs for a minimum of 4 vaccinations with 5 × 106 to 5 × 107 CD1a+ DCs; (ii) vaccination with peptide-pulsed DCs is possible without major side effects or signs of auto-immune disease; (iii) immunological responses consisted of peptide-specific DTH responses in 4 patients, a significant increase in circulating Melan-A- and gp100-reactive CD8+ CTLs after vaccination in 1 patient showing signs of clinical anti-tumor response and development of generalized vitiligo in 1 HLA-A2+ patient during vaccination; (iv) clinical anti-tumor responses were observed in 2 patients, including regression of s.c. metastasis.
Our study is a clinical trial with DCs derived from CD34+ hematopoietic progenitor cells. Previous studies were conducted with monocyte-derived DCs (Nestle et al.,1998; Höltl et al.,1998). We have demonstrated that the sequential use of early-acting hematopoietic growth factors, SCF, IL-3 and IL-6 followed on day 8 by differentiation with IL-4 and GM-CSF allows generation of large numbers of immature DCs that mature to interdigitating DCs within 24 hr of addition of TNF-α (Herbst et al.,1996). Comparative analysis of CD34+ PBPCs and monocyte-derived DCs revealed similar phenotypic and functional properties (Herbst et al.,1997). However, Mortarini et al. (1997) demonstrated that DCs derived from CD34+ progenitors are more efficient APCs for the activation of low-frequency, peptide-specific CTLs than DCs derived from monocytes.
In the present study, patients received peptide-pulsed DCs in 3 different cell-dose levels, ranging from 5 × 106 to 5 × 107 CD1a+ DCs. Clinical and immunological responses were observed independently of the dose level, thus supporting the view that relatively small numbers of DCs are effective at stimulating specific immune responses (Nestle et al.,1998; Hsu et al.,1996).
So far, 2 studies on vaccination with peptide-pulsed DCs have been published. Nestle et al. (1998) showed a specific DTH response in 11 of 15 patients but an objective response in 5 of 16 evaluable patients after vaccination with peptide- or tumor lysate–pulsed DCs. However, the tumor load in these selected patients was reported to be low, suggesting efficiency of this approach in melanoma patients with minimal residual disease, as achieved by cytoreductive therapy. Mukherji et al. (1995) vaccinated patients with MAGE-1 peptide-pulsed APCs and demonstrated accumulation of MAGE-1-specific CTLs at the vaccination site, though it required 3 in vitro restimulations to detect this response (Hu et al.,1996).
In the present study, 4 of 14 patients revealed a peptide-specific DTH response after vaccination. Interestingly, in 2 of these patients, positive DTH response correlated well with specific immune reponses. Patient 12, showing a positive DTH response against Melan-A-pulsed DCs after vaccination, developed generalized vitiligo after the 2nd vaccination. An association between high frequencies of Melan-A-specific CTLs and development of auto-immune vitiligo has been reported (Ogg et al.,1998). Vitiligo is a common progressive depigmentary skin disorder believed to be due to the auto-immune-mediated destruction of epidermal melanocytes. Patient 2 revealed a positive DTH response against Melan-A and gp100. In parallel, we detected a significant increase of circulating Melan-A- and gp100-reactive CTLs in PBLs after vaccination. Melan-A-specific CTLs are present ex vivo in melanoma-infiltrated lymph nodes (Romero et al.,1998) and circulating CD8+ T cells specific for Melan-A and tyrosinase in unvaccinated melanoma patients (Lee et al.,1999). However, antigen-specific unresponsiveness may explain why such cells are unable to control tumor growth (Lee et al.,1999).
The optimal mode of DC administration with respect to practical aspects and induction of specific immune responses remains unclear. We and others have shown that i.v. administration of in vitro generated human DCs delivers these APCs preferentially to the spleen and liver (Mackensen et al.,1999; Morse et al.,1999). Clinical anti-tumor responses after DC vaccination have been reported after i.v. injection (Hsu et al.,1996; Höltl et al.,1998) and after direct injection into lymph nodes (Nestle et al.,1998). Comparative analysis of clinical and immunological responses after different routes of DC vaccination are thus warranted.
In conclusion, our pilot study demonstrates that it is possible to significantly increase the number of circulating peptide-specific CTLs by vaccination with peptide-pulsed autologous DCs derived from hematopoietic progenitors and to mediate tumor regression in some patients with metastatic melanoma.