Melanoma: Mackensen A

Garbe C, Hauschild A, Volkenandt M, Schadendorf D, Stolz W, Reinhold U, Kortmann RD, Kettelhack C, Frerich B, Keilholz U, Dummer R, Sebastian G, Tilgen W, Schuler G, Mackensen A, Kaufmann R. · University Department of Dermatology, Tübingen, Germany. · Melanoma Res. · Pubmed #18337653 No free full text.

Abstract: Systemic medical treatment of melanoma is administered in the adjuvant and palliative setting. Adjuvant therapy may be considered in patients with primary melanoma with more than 1.5 mm tumor thickness and with regional node metastasis. Presently no indication for systemic adjuvant chemotherapy or for adjuvant therapy with nonspecific immune-stimulatory agents outside controlled studies is seen. Interferon-alpha is the first substance in the adjuvant therapy of melanoma, which has shown to present a significant advantage to the patients in some prospective randomized studies. Good arguments for using adjuvant interferon-alpha therapy in high-risk melanoma patients exist. Both high-dose and low-dose interferon-alpha show promise. The major indications for systemic chemotherapy and chemoimmunotherapy are inoperable recurrent tumors, inoperable regional metastases and distant metastases (stage IV). As treatment in such situations is primarily palliative, the effect of any regimen on the quality of life must be carefully weighed. As a first line treatment, single agent therapy is recommended, as polychemotherapy or biochemotherapy did not show significant advantages for prolongation of survival; hence they are more toxic. An urgent need for development of new treatment modalities is necessary and general principles of experimental immunotherapy are outlined.

Garbe C, Hauschild A, Volkenandt M, Schadendorf D, Stolz W, Reinhold U, Kortmann RD, Kettelhack C, Frerich B, Keilholz U, Dummer R, Sebastian G, Tilgen W, Schuler G, Mackensen A, Kaufmann R. · University Department of Dermatology, Tübingen, Germany. · Melanoma Res. · Pubmed #18227710 No free full text.

Abstract: The primary treatment of a melanoma is surgical excision. An excisional biopsy is preferred, and safety margins of 1 cm for tumor thickness up to 2 mm and 2 cm for higher tumor thickness should be applied either at primary excision or in a two-step procedure. When dealing with facial, acral or anogenital melanomas, micrographic control of the surgical margins may be preferable to allow reduced safety margins and conservation of tissue. The sentinel lymph node biopsy should be performed in patients whose primary melanoma is thicker than 1.0 mm and this operation should be performed in centers where both the operative and nuclear medicine teams are experienced. In clinically identified lymph node metastases, radical lymph node dissection is considered standard therapy. If distant metastases involve just one internal organ and operative removal is feasible, then surgery should be seen as therapy of choice. Radiation therapy for the primary treatment of melanoma is indicated only in those cases in which surgery is impossible or not reasonable. In regional lymph nodes, radiation therapy is usually recommended when excision is not complete (R1 resection) or if the nodes are inoperable. In distant metastases, radiation therapy is particularly indicated in bone metastases, brain metastases and soft tissue metastases.

Garbe C, Hauschild A, Volkenandt M, Schadendorf D, Stolz W, Kortmann RD, Kettelhack C, Frerich B, Keilholz U, Dummer R, Sebastian G, Tilgen W, Schuler G, Mackensen A, Kaufmann R. · Universitäts-Hautklinik Tübingen. · J Dtsch Dermatol Ges. · Pubmed #16638065 No free full text.

This publication has no abstract.

Meidenbauer N, Andreesen R, Mackensen A. · Department of Hematology/Oncology, University of Regensburg, Germany. · Biol Chem. · Pubmed #11405216 No free full text.

Abstract: The characterization of tumor-associated antigens recognized by human T lymphocytes in a major histocompatibility complex (MHC)-restricted fashion has opened new possibilities for immunotherapeutic approaches to the treatment of human cancers. Dendritic cells (DC) are professional antigen presenting cells that are well suited to activate T cells toward various antigens, such as tumor-associated antigens, due to their potent costimulatory activity. The availability of large numbers of DC, generated either from hematopoietic progenitor cells or monocytes in vitro or isolated from peripheral blood, has profoundly changed pre-clinical research as well as the clinical evaluation of these cells. Accordingly, appropriately pulsed or transfected DC may be used for vaccination in the field of infectious diseases or tumor immunotherapy to induce antigen-specific T cell responses. These observations led to pilot clinical trials of DC vaccination for patients with cancer in order to investigate the feasibility, safety, as well as the immunologic and clinical effects of this approach. Initial clinical studies of human DC vaccines are generating encouraging preliminary results demonstrating induction of tumor-specific immune responses and tumor regression. Nevertheless, much work is still needed to address several variables that are critical for optimizing this approach and to determine the role of DC-based vaccines in tumor immunotherapy.

Mackensen A, Meidenbauer N, Vogl S, Laumer M, Berger J, Andreesen R. · Department of Hematology/Oncology, University of Regensburg, Franz-Josef-Strauss-Allee 11, D-93042 Regensburg, Germany. · J Clin Oncol. · Pubmed #17075125 No free full text.

Abstract: PURPOSE: The adoptive transfer of in vitro generated tumor antigen-specific cytotoxic T lymphocytes (CTL) provides a promising approach to the immunotherapy of cancer. A phase I study was conducted to test the feasibility, safety, and survival of adoptively transferred Melan-A-specific CTL lines in melanoma patients. PATIENTS AND METHODS: Eleven HLA-A2+ patients with metastatic melanoma received at least three intravenous infusions of Melan-A-specific CTL at 2-week intervals. CTL were generated by four rounds of in vitro stimulation of purified CD8+ peripheral blood lymphocytes with autologous dendritic cells pulsed with an HLA-A2 binding Melan-A peptide. Each T-cell infusion was accompanied by a 6-day course of low-dose interleukin-2. RESULTS: A total of 52 T-cell infusions were administered, averaging 2.1 x 10(8) Melan-A-specific CTL per infusion. Clinical adverse effects were mild and consisted of chills and low-grade fever in seven of 11 patients. Clinical and immunologic responses revealed an antitumor response in three of 11 patients (one complete regression, one partial regression, one mixed response), an elevated frequency of circulating Melan-A tetramer+ T cells up to 2 weeks in all the patients with a maximal frequency of 2% of total CD8+ T cells, an increase in eosinophils to up to 50% in seven of 11 patients, and a selective loss of Melan-A expression in lymph node metastases in two evaluated patients after T-cell transfer. CONCLUSION: Our data indicate that the adoptive transfer of antigen-specific T cells in melanoma patients can induce clinical tumor-specific immune responses without major adverse effects.

Meidenbauer N, Marienhagen J, Laumer M, Vogl S, Heymann J, Andreesen R, Mackensen A. · Department of Hematology/Oncology, University of Regensburg, Franz-Josef-Strauss-Allee 11, D-93042 Regensburg, Germany. · J Immunol. · Pubmed #12574389 links to  free full text

Abstract: Adoptive T cell therapy has been successfully used for treatment of viral and malignant diseases. However, little is known about the fate and trafficking of transferred Ag-specific T cells. Using the tetramer (TM) technology which allows for detection and quantification of Ag-specific CTL, we assessed the frequency of circulating Melan-A-specific CTL in advanced melanoma patients during adoptive T cell therapy. Melan-A-specific CTL were generated from HLA-A2.1(+) patients by in vitro stimulation of CD8(+) T cells with dendritic cells pulsed with a mutated HLA-A2-binding Melan-A (ELAGIGILTV) peptide. Eight patients received three infusions of 0.25-11 x 10(8) Melan-A-specific CTL i.v. at 2-wk intervals along with low-dose IL-2. The transferred T cell product contained a mean of 42.1% Melan-A-TM(+) CTL. Before therapy, the frequencies of Melan-A-specific CTL in patients' circulating CD8(+) T cells ranged from 0.01 to 0.07%. Characterization of the TM frequencies before and at different time points after transfer revealed an increase of circulating Melan-A-specific CTL up to 2%, correlating well with the number of transferred CTL. An elevated frequency of TM(+) T cells was demonstrated up to 14 days after transfer, suggesting long-term survival and/or proliferation of transferred CTL. Combining TM analysis with a flow cytometry-based cytokine secretion assay, unimpaired production of IFN-gamma was demonstrated in vivo for at least 24 h after transfer. Indium-111 labeling of Melan-A-specific CTL demonstrated localization of transferred CTL to metastatic sites as early as 48 h after injection. Overall, the results suggest that in vitro-generated Melan-A-specific CTL survive intact in vivo for several weeks and localize preferentially to tumor.

Mackensen A, Dräger R, Schlesier M, Mertelsmann R, Lindemann A. · Department of Hematology/Oncology, Freiburg University Medical Center, Germany. · Cancer Immunol Immunother. · Pubmed #10881694 No free full text.

Abstract: Dendritic cells are professional antigen-presenting cells that can be generated in vitro either from monocytes or from CD34+ peripheral blood progenitor cells by using recombinant cytokines. These cells have potential implications for immunotherapeutic approaches in the treatment of cancer and other diseases. We have conducted a phase I study in melanoma patients using peptide-pulsed dendritic cells cultured in medium supplemented with 10% fetal calf serum (FCS) and a cocktail of cytokines. Peptide-pulsed dendritic cells were injected intravenously at 2-week intervals. Here we report on a case of type I hypersensitivity anaphylactic reaction after repetitive vaccination with autologous peptide-pulsed cells. Pre-vaccination and post-vaccination serum samples were evaluated for the presence of antibodies to FCS and bovine serum albumin (BSA). A retrospective study in 7 patients vaccinated with FCS-cultured dendritic cells demonstrated the presence of IgG and IgM antibodies to FCS and BSA after vaccination in 6 out of 7 patients. However, IgE antibodies were absent in all patients with the exception of the patient developing anaphylaxis. The patient's serum was demonstrated to contain a strong IgE response directed against BSA. In contrast, 2 patients vaccinated with dendritic cells cultured under serum-free conditions developed no antibodies to FCS and BSA after repetitive vaccination. We suggest that patients can be sensitized with an IgE response against BSA leading to anaphylactic reactions. On the basis of these data, dendritic cells cultured in autologous serum or under serum-free conditions are recommended for therapeutic applications in vivo.

Mackensen A, Herbst B, Chen JL, Köhler G, Noppen C, Herr W, Spagnoli GC, Cerundolo V, Lindemann A. · Department of Hematology/Oncology, Freiburg University Medical Center, Freiburg, Germany. · Int J Cancer. · Pubmed #10760827 No free full text.

Abstract: 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-alpha. 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 x 10(6) to 5 x 10(7) 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.

Mackensen A, Krause T, Blum U, Uhrmeister P, Mertelsmann R, Lindemann A. · Department of Hematology/Oncology, Freiburg University Medical Center, Germany. · Cancer Immunol Immunother. · Pubmed #10414465 No free full text.

Abstract: Dendritic cells (DC) are professional antigen-presenting cells that can be generated in vitro from CD34+ peripheral blood progenitor cells by recombinant cytokines. These cells have potential implications for immunotherapeutic approaches in the treatment of cancer and other diseases. Physiologically, immature DC in the periphery capture and process antigens, then mature to interdigitating DC and migrate to lymphoid organs, where they activate lymphocytes. However, it is not known if DC generated in vitro have the capacity to traffic in vivo to the lymphoid tissues, such as spleen and lymph nodes. We have investigated whether human radiolabeled DC differentiated in vitro migrate and localize to lymphoid tissues after intravenous and intralymphatic injection. The distribution and localization of the DC were evaluated in five patients with malignant melanoma using serial whole-body gamma camera imaging. Intravenously infused DC demonstrated transient lung uptake followed by localization in the spleen and liver for at least 7 days. DC injected into a lymphatic vessel at the dorsal foot were rapidly detected in the draining lymph nodes where they remained for more than 24 h. These data suggest that DC differentiated in vitro localize preferentially to lymphoid tissue, where they could induce specific immune responses.

Voelkl S, Moore TV, Rehli M, Nishimura MI, Mackensen A, Fischer K. · Department of Internal Medicine 5, Hematology/Oncology, University of Erlangen-Nuernberg, Krankenhausstrasse 12, Erlangen, Germany. · Cancer Immunol Immunother. · Pubmed #18836718 No free full text.

Abstract: The immune attack against malignant tumors require the concerted action of CD8+ cytotoxic T lymphocytes (CTL) as well as CD4+ T helper cells. The contribution of T cell receptor (TCR) alphabeta+ CD4- CD8- double-negative (DN) T cells to anti-tumor immune responses is widely unknown. In previous studies, we have demonstrated that DN T cells with a broad TCR repertoire are present in humans in the peripheral blood and the lymph nodes of healthy individuals. Here, we characterize a human DN T cell clone (T4H2) recognizing an HLA-A2-restricted melanoma-associated antigenic gp100-peptide isolated from the peripheral blood of a melanoma patient. Antigen recognition by the T4H2 DN clone resulted in specific secretion of IFN-gamma and TNF. Although lacking the CD8 molecule the gp100-specific DN T cell clone was able to confer antigen-specific cytotoxicity against gp100-loaded target cells as well as HLA-A2+ gp100 expressing melanoma cells. The cytotoxic capacity was found to be perforin/granzymeB-dependent. Together, these data indicate that functionally active antigen-specific DN T cells recognizing MHC class I-restricted tumor-associated antigen (TAA) may contribute to anti-tumor immunity in vivo.

Moore TV, Lyons GE, Brasic N, Roszkowski JJ, Voelkl S, Mackensen A, Kast WM, Le Poole IC, Nishimura MI. · Department of Surgery, The University of Chicago, Chicago, IL 60637, USA. · Cancer Immunol Immunother. · Pubmed #18836717 No free full text.

Abstract: Effective immunotherapy using T cell receptor (TCR) gene-modified T cells requires an understanding of the relationship between TCR affinity and functional avidity of T cells. In this study, we evaluate the relative affinity of two TCRs isolated from HLA-A2-restricted, gp100-reactive T cell clones with extremely high functional avidity. Furthermore, one of these T cell clones, was CD4- CD8- indicating that antigen recognition by this clone was CD8 independent. However, when these TCRs were expressed in CD8- Jurkat cells, the resulting Jurkat cells recognized gp100:209-217 peptide loaded T2 cells and had high functional avidity, but could not recognize HLA-A2+ melanoma cells expressing gp100. Tumor cell recognition by Jurkat cells expressing these TCRs could not be induced by exogenously loading the tumor cells with the native gp100:209-217 peptide. These results indicate that functional avidity of a T cell does not necessarily correlate with TCR affinity and CD8-independent antigen recognition by a T cell does not always mean its TCR will transfer CD8-independence to other effector cells. The implications of these findings are that T cells can modulate their functional avidity independent of the affinity of their TCRs.

Garbe C, Schadendorf D, Stolz W, Volkenandt M, Reinhold U, Kortmann RD, Kettelhack C, Frerich B, Keilholz U, Dummer R, Sebastian G, Tilgen W, Schuler G, Mackensen A, Kaufmann R, Hauschild A. · Sektion Dermatologische Onkologie, Universitäts-Hautklinik, Liebermeister Strasse 25, D-72076 Tübingen, Germany. · J Dtsch Dermatol Ges. · Pubmed #18801142 No free full text.

This publication has no abstract.

Durai M, Krueger C, Ye Z, Cheng L, Mackensen A, Oelke M, Schneck JP. · Department of Pathology, Johns Hopkins School of Medicine, Ross 664G, 720 Rutland Avenue, Baltimore, MD 21205, USA. · Cancer Immunol Immunother. · Pubmed #18563409 No free full text.

Abstract: Adoptive immunotherapy for treatment of cancers and infectious diseases is often hampered by a high degree of variability in the final T cell product and in the limited in vivo function and survival of ex vivo expanded antigen-specific cytotoxic T cells (CTL). This has stimulated interest in development of standardized artificial antigen presenting cells (aAPC) to reliably expand antigen specific CTL. However, for successful immunotherapy the aAPC ex vivo generated CTL must have anti-tumor activity in vivo. Here, we demonstrate that HLA-Ig based aAPC stimulated tumor-specific CTL from human peripheral blood T lymphocytes showed robust expansion and functional activity in a human/SCID mouse melanoma model. HLA-Ig based aAPC expanded CTL were detected in the peripheral blood up to 15 days after transfer. Non-invasive bioluminescence imaging of tumor bearing mice demonstrated antigen dependent localization of transferred CTL to the tumor site. Moreover, adoptive transfer of HLA-Ig based aAPC generated CTL inhibited the tumor growth both in prevention and treatment modes of therapy and was comparable to that achieved by dendritic cell expanded CTL. Thus, our data demonstrate potential therapeutic in vivo activity of HLA-Ig based aAPC expanded CTL to control tumor growth.

Garbe C, Hauschild A, Volkenandt M, Schadendorf D, Stolz W, Reinhold U, Kortmann RD, Kettelhack C, Frerich B, Keilholz U, Dummer R, Sebastian G, Tilgen W, Schuler G, Mackensen A, Kaufmann R. · University Department of Dermatology, Tübingen, Germany. · Melanoma Res. · Pubmed #17992123 No free full text.

Abstract: Melanoma is a malignant tumor that arises from melanocytic cells and primarily involves the skin. The most important exogenous etiological factor is exposure to ultraviolet irradiation. Diagnosis of melanoma is based primarily on its clinical features, and the A-B-C-D rule is useful in identifying pigmented lesions, which are suspicious for melanoma (Asymmetry, Border irregular, Color inhomogeneous and Diameter more than 5 mm). Dermoscopy is very helpful in clarifying the differential diagnosis of pigmented lesions. About 90% of melanomas are diagnosed as primary tumors without any evidence for metastasis. The tumor-specific 10-year survival for all such tumors is about 75-85%. The most important prognostic factors for primary melanoma without metastases are vertical tumor thickness (Breslow depth) as measured on the histological specimen, presence of histopathologically recognized ulceration, invasion level (Clark level) and identification of micrometastases in the regional lymph nodes via sentinel lymph node biopsy. The current tumor node metastasis classification for the staging of primary melanoma is based on these factors. Melanomas can metastasize either by the lymphatic or by the hematogenous route. About two-thirds of metastases are originally confined to the drainage area of regional lymph nodes. A regional metastasis can appear as satellite metastases up to 2 cm from the primary tumor, as intransit metastases in the skin between the site of the primary tumor and the first lymph node and as regional lymph node metastases. In the stage of regional metastasis, the differentiation between micrometastasis and macrometastasis and the number of lymph nodes involved are crucial. As soon as distant metastasis develops, prognosis depends on the site of the metastasis and on the lactate dehydrogenase levels in the blood. The frequency and extent of follow-up examinations is based on the initial tumor parameters. In thin primary melanomas up to 1-mm tumor thickness, clinical examinations at 6-month intervals are sufficient and in thicker primary melanomas, at 3-month intervals. Lymph node sonography as well as determination of the tumor marker protein S100beta are recommended. Additionally, in the stage of regional metastasis, whole body imaging should be performed every 6 months; in the stage of distant metastasis, surveillance has to be scheduled individually.

Grube M, Moritz S, Obermann EC, Rezvani K, Mackensen A, Andreesen R, Holler E. · Department of Hematology and Oncology, Institute of Pathology, University of Regensburg, Regensburg, Germany. · Clin Cancer Res. · Pubmed #17289902 links to  free full text

Abstract: PURPOSE: Survivin is a member of the inhibitors of apoptosis family and is overexpressed in different types of malignancies. Cytotoxic T cells recognizing survivin epitopes can be elicited in vitro and by vaccination in patients with leukemia, breast cancer, and melanoma. We did this study to investigate whether survivin-specific CD8+ T cells occur in patients with multiple myeloma. EXPERIMENTAL DESIGN: An HLA-A2.1-binding survivin peptide was used to detect peptide-specific T cells by a quantitative real-time PCR to measure antigen-specific IFN-gamma mRNA expression in 23 patients with myeloma and 21 healthy volunteers. T cells producing IFN-gamma in response to survivin were further analyzed for expression of CD45RA and CCR7 to determine phenotypic characterization. Additional immunohistochemical analyses of survivin antigen expression in bone marrow specimens of patients was done. RESULTS: T cells recognizing HLA-A2.1-binding survivin peptide were detected in 9 of 23 patients and in 1 of 21 healthy volunteers. Survivin-reactive T cells were identified as terminally differentiated effector T cells (CD8+, CD45RA+, and CCR7-). Positive survivin expression of myeloma cells in bone marrow specimens was shown in 7 of 11 patients. CONCLUSION: We provide, for the first time, evidence of T cell reactivity against survivin antigen in patients with multiple myeloma. Our data suggest the immunogenicity of survivin antigen in multiple myeloma and that immunotherapeutic strategies using survivin as a target antigen might be an option for patients with this disease.

Walton SM, Gerlinger M, de la Rosa O, Nuber N, Knights A, Gati A, Laumer M, Strauss L, Exner C, Schäfer N, Urosevic M, Dummer R, Tiercy JM, Mackensen A, Jaeger E, Lévy F, Knuth A, Jäger D, Zippelius A. · Medical Oncology, Department of Internal Medicine, University Hospital Zurich, Raemistrasse 100, CH-8091 Zurich, Switzerland. · J Immunol. · Pubmed #17114498 links to  free full text

Abstract: The melanocyte differentiation Ag RAB38/NY-MEL-1 was identified by serological expression cloning (SEREX) and is expressed in the vast majority of melanoma lesions. The immunogenicity of RAB38/NY-MEL-1 has been corroborated previously by the frequent occurrence of specific Ab responses in melanoma patients. To elucidate potential CD8 T cell responses, we applied in vitro sensitization with overlapping peptides spanning the RAB38/NY-MEL-1 protein sequence and the reverse immunology approach. The identified peptide RAB38/NY-MEL-1(50-58) exhibited a marked response in ELISPOT assays after in vitro sensitization of CD8 T cells from HLA-A *0201(+) melanoma patients. In vitro digestion assays using purified proteasomes provided evidence of natural processing of RAB38/NY-MEL-1(50-58) peptide. Accordingly, monoclonal RAB38/NY-MEL-1(50-58)-specific T cell populations were capable of specifically recognizing HLA-A2(+) melanoma cell lines expressing RAB38/NY-MEL-1. Applying fluorescent HLA-A2/RAB38/NY-MEL-1(50-58) multimeric constructs, we were able to document a spontaneously developed memory/effector CD8 T cell response against this peptide in a melanoma patient. To elucidate the Ag-processing pathway, we demonstrate that RAB38/NY-MEL-1(50-58) is produced efficiently by the standard proteasome and the immunoproteasome. In addition to the identification of a RAB38/NY-MEL-1-derived immunogenic CD8 T cell epitope, this study is instrumental for both the onset and monitoring of future RAB38/NY-MEL-1-based vaccination trials.

Blank C, Kuball J, Voelkl S, Wiendl H, Becker B, Walter B, Majdic O, Gajewski TF, Theobald M, Andreesen R, Mackensen A. · Department of Hematology and Oncology, University of Regensburg, Regensburg, Germany. · Int J Cancer. · Pubmed #16482562 No free full text.

Abstract: Human tumors frequently escape immune destruction, despite the presence of cytotoxic T cells (CTL) recognizing tumor-associated antigens (TAA). We have previously shown that programmed death ligand-1 (PD-L1), a recently identified ligand of the B7 superfamily, is expressed on murine tumors and can inhibit antitumor immune responses. To evaluate the clinical relevance of our animal model findings, we examined human tumors and tumor-specific T cells. We found PD-L1 to be constitutively expressed on human renal cell carcinoma (RCC) cell lines and upregulated on human melanoma cell lines upon exposure to interferon-gamma. Similarly, we found binding of anti-PD-L1 monoclonal antibody (mAb) on frozen sections from RCC and melanomas, but not on normal tissues. The corresponding inhibitory receptor of PD-L1, PD-1, revealed a higher expression on tumor-infiltrating lymphocytes than on peripheral blood lymphocytes (PBL) from melanoma patients upon specific antigen stimulation. Stimulation of PBL from healthy donors with peptide-loaded dendritic cells in the presence of anti-PD-L1 mAb altered neither the total T cell numbers after expansion, nor the percentage of peptide-specific CTL, when providing a T cell help by addition of cytokines. However, when stimulating TAA-specific CTL and T helper cells with Ag-pulsed dendritic cells in the absence of exogenous cytokines, PD-L1 blockade increased the cytokine production. Similar to the data achieved in the murine system, the blockade of PD-L1 on human tumors resulted in enhanced cytolytic activity of TAA-specific CTLs and cytokine production of TAA-specific T helper cells when interacting directly with the tumor. In summary, our data suggest that PD-L1/PD-1 interactions negatively regulate T cell effector functions predominantly in the absence of exogenous cytokine support, indicating an important role for this pathway in tumor evasion.

Gottfried E, Kunz-Schughart LA, Ebner S, Mueller-Klieser W, Hoves S, Andreesen R, Mackensen A, Kreutz M. · Department of Hematology and Oncology, Institute of Pathology, University of Regensburg, Franz-Josef Strauss Allee 11, 93042 Regensburg, Germany. · Blood. · Pubmed #16278308 links to  free full text

Abstract: The tumor milieu can influence dendritic cell (DC) differentiation. We analyzed DC differentiation in a 3-dimensional tumor model and propose a new mechanism of DC modulation by the tumor environment. Monocytes were cultured in the presence of IL-4 and GM-CSF within multicellular tumor spheroids (MCTSs) generated from different tumor cell lines. Monocytes invaded the MCTSs and differentiated into tumor-associated dendritic cells (TADCs). The antigen expression was altered on TADCs independent of the culture conditions (immature/mature DCs, Langerhans cells) and IL-12 secretion was reduced. Supernatants of MCTSs could partially transfer the suppressive effect. Conditioned media from urothelial carcinoma cell lines contained high levels of M-CSF and IL-6, both cytokines known to modulate DC differentiation. In contrast, melanoma and prostate carcinoma MCTS cocultures produced little M-CSF and IL-6, but high levels of lactic acid. Indeed, addition of lactic acid during DC differentiation in vitro induced a phenotype comparable with TADCs generated within melanoma and prostate carcinoma MCTSs. Blocking of lactic acid production in melanoma MCTS cocultures reverted the TADC phenotype to normal. We therefore conclude that tumor-derived lactic acid is an important factor modulating the DC phenotype in the tumor environment, which may critically contribute to tumor escape mechanisms.

Meidenbauer N, Zippelius A, Pittet MJ, Laumer M, Vogl S, Heymann J, Rehli M, Seliger B, Schwarz S, Le Gal FA, Dietrich PY, Andreesen R, Romero P, Mackensen A. · Department of Hematology/Oncology, University of Regensburg, Regensburg, Germany. · Cancer Res. · Pubmed #15342421 links to  free full text

Abstract: Tumor-reactive T cells play an important role in cancer immunosurveillance. Applying the multimer technology, we report here an unexpected high frequency of Melan-A-specific CTLs in a melanoma patient with progressive lymph node metastases, consisting of 18 and 12.8% of total peripheral blood and tumor-infiltrating CD8+ T cells, respectively. Melan-A-specific CTLs revealed a high cytolytic activity against allogeneic Melan-A-expressing target cells but failed to kill the autologous tumor cells. Loading of the tumor cells with Melan-A peptide reversed the resistance to killing, suggesting impaired function of the MHC class I antigen processing and presentation pathway. Mutations of the coding region of the HLA-A2 binding Melan-A26-35 peptide or down-regulation of the MHC class I heavy chain, the antigenic peptide TAP, and tapasin could be excluded. However, PCR and immunohistochemical analysis revealed a deficiency of the immunoproteasomes low molecular weight protein 2 and low molecular weight protein 7 in the primary tumor cells, which affects the quantity and quality of generated T-cell epitopes and might explain the resistance to killing. This is supported by our data, demonstrating that the resistance to killing can be partially reversed by pre-exposure of the tumor cells to IFN-gamma, which is known to induce the immunoproteasomes. Overall, this is the first report of an extremely high frequency of tumor-specific CTLs that exhibit competent T-cell-effector functions but fail to lyse the autologous tumor cells. Immunotherapeutic approaches should not only focus on the induction of a robust antitumor immune response, but should also have to target tumor immune escape mechanisms.

Zippelius A, Batard P, Rubio-Godoy V, Bioley G, Liénard D, Lejeune F, Rimoldi D, Guillaume P, Meidenbauer N, Mackensen A, Rufer N, Lubenow N, Speiser D, Cerottini JC, Romero P, Pittet MJ. · Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research and Multidisciplinary Oncology Center, University Hospital (Centre Hospitalier Universitaire Vaudois), Lausanne, Switzerland. · Cancer Res. · Pubmed #15087405 links to  free full text

Abstract: Although tumor-specific CD8 T-cell responses often develop in cancer patients, they rarely result in tumor eradication. We aimed at studying directly the functional efficacy of tumor-specific CD8 T cells at the site of immune attack. Tumor lesions in lymphoid and nonlymphoid tissues (metastatic lymph nodes and soft tissue/visceral metastases, respectively) were collected from stage III/IV melanoma patients and investigated for the presence and function of CD8 T cells specific for the tumor differentiation antigen Melan-A/MART-1. Comparative analysis was conducted with peripheral blood T cells. We provide evidence that in vivo-priming selects, within the available naive Melan-A/MART-1-specific CD8 T-cell repertoire, cells with high T-cell receptor avidity that can efficiently kill melanoma cells in vitro. In vivo, primed Melan-A/MART-1-specific CD8 T cells accumulate at high frequency in both lymphoid and nonlymphoid tumor lesions. Unexpectedly, however, whereas primed Melan-A/MART-1-specific CD8 T cells that circulate in the blood display robust inflammatory and cytotoxic functions, those that reside in tumor lesions (particularly in metastatic lymph nodes) are functionally tolerant. We show that both the lymph node and the tumor environments blunt T-cell effector functions and offer a rationale for the failure of tumor-specific responses to effectively counter tumor progression.

Oelke M, Maus MV, Didiano D, June CH, Mackensen A, Schneck JP. · Department of Pathology & Medicine, Johns Hopkins School of Medicine, Baltimore, Maryland, USA. · Nat Med. · Pubmed #12704385 No free full text.

Abstract: Adoptive immunotherapy holds promise as a treatment for cancer and infectious diseases, but its development has been impeded by the lack of reproducible methods for generating therapeutic numbers of antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs). As a result, there are only limited reports of expansion of antigen-specific CTLs to the levels required for clinical therapy. To address this issue, artificial antigen-presenting cells (aAPCs) were made by coupling a soluble human leukocyte antigen-immunoglobulin fusion protein (HLA-Ig) and CD28-specific antibody to beads. HLA-Ig-based aAPCs were used to induce and expand CTLs specific for cytomegalovirus (CMV) or melanoma. aAPC-induced cultures showed robust antigen-specific CTL expansion over successive rounds of stimulation, resulting in the generation of clinically relevant antigen-specific CTLs that recognized endogenous antigen-major histocompatibility complex complexes presented on melanoma cells. These studies show the value of HLA-Ig-based aAPCs for reproducible expansion of disease-specific CTLs for clinical approaches to adoptive immunotherapy.

Farkas B, Hantschel M, Magyarlaki M, Becker B, Scherer K, Landthaler M, Pfister K, Gehrmann M, Gross C, Mackensen A, Multhoff G. · Department of Dermatology, University Hospital Pecs, Pecs, Hungary. · Melanoma Res. · Pubmed #12690297 No free full text.

Abstract: Cell membrane localization of the 72 kDa heat shock protein 70 (Hsp70) has been found on different tumour cell lines, on biopsy material from solid tumours and metastases and on leukaemic blasts from acute myelogenous leukaemia patients, but not on the corresponding normal tissues, as determined by flow cytometry using the Hsp70-specific monoclonal antibody C92F3B1. In the present study Hsp70 membrane expression was studied on primary malignant melanomas, melanoma metastases, melanocytes, human skin fibroblasts and peripheral blood lymphocytes, together with expression of the melanoma-associated markers Mel-1, Mel-2 and Mel-5, major histocompatibility complex class I and the fibroblast-specific marker ASO2. As previously shown, fibroblasts and peripheral blood lymphocytes from healthy human volunteers were found to be negative for Hsp70 and for the melanoma-associated markers Mel-1, Mel-2 and Mel-5. Human melanocytes from healthy human donors were also negative for Hsp70, but were positive for Mel-1 and Mel-5. Independent of the Clark's level, all the malignant melanomas (n = 9) and metastases (n = 11) exhibited were positive for both Mel-1 and Mel-2. The primary melanomas could be divided into two groups according to their Hsp70 and Mel-5 expression pattern: those with an Hsp70-negative and a Mel-5-positive phenotype (-/+) (five out of nine), and those with an Hsp70-positive and a Mel-5-negative phenotype (+/-) (four out of nine). All the melanoma metastases (n = 11) had an Hsp70-positive, Mel-5-negative phenotype (+/-). These data provide the first hint that the marker combination Hsp70 positive/Mel-5 negative might be useful in estimating the metastatic potential of a melanoma. Investigations on changes in the marker combination Hsp70/Mel-5 during onset of melanoma disease and progression will clarify its potential as a prognostic risk factor.

Kurokawa T, Fischer K, Bertz H, Hoegerle S, Finke J, Mackensen A. · Department of Hematology/Oncology, Freiburg University Medical Center, Germany. · Int J Cancer. · Pubmed #12209588 No free full text.

Abstract: It has been shown that after allogeneic peripheral blood stem cell transplantation (PBSCT), donor T cells can induce potent graft-versus-tumor (GVT) effects in hematologic malignancies and possibly solid tumors such as renal cell carcinoma. Two patients (27 and 30 years old) with metastatic melanoma received allogeneic PBSCT from an HLA-identical sibling donor after reduced conditioning with fludarabine, carmustine and melphalan. One patient showed a delayed mixed response with complete regression of lymph node metastases but persistent liver metastasis at day +60 and +120, consistent with a GVT response. In order to generate donor-derived tumor-reactive cytotoxic T lymphocytes (CTLs), peripheral blood mononuclear cells were stimulated with donor dendritic cells (DCs) loaded with host tumor lysate. Using these culture conditions, a marked increase in CD8(+) CTLs was observed in both donors exhibiting a strong MHC class I-restricted cytotoxic activity against the host tumor without cross-reactivity against nonmalignant host cells. CDR3 spectratyping was used to analyze the complexity of T-cell subpopulations in both CTL lines. Results demonstrate that oligoclonal T cells are expanded in vitro, exhibiting a marked overexpression of TCRVbeta3 (donor 1) and TCRVbeta4/Vbeta11 (donor 2) subfamilies. Functional (ELISPOT assay) and phenotypic (CDR3 spectratyping, sequencing Vbeta transcripts) analysis of patients' T cells at different time points after transplantation demonstrated an expansion of alloreactive T cells with a limited TCR Vbeta pattern. The Vbeta3 cDNA clone, being predominant in the CTL line from donor 1, could not be identified in patient 1 peripheral blood lymphocytes after transplant. Altogether, our results provide the first evidence that GVT effects against melanoma can induce tumor regression and that oligoclonal donor-derived CTLs specific against host tumor cells can be generated in vitro that may be used for adoptive T-cell transfer after allogeneic transplantation.

Fischer K, Andreesen R, Mackensen A. · Department of Hematology/Oncology, University of Regensburg, Franz-Josef-Strauss-Allee 11, D-93042 Regensburg, Germany. · J Immunol Methods. · Pubmed #11730851 No free full text.

Abstract: The cytotoxic activity of T lymphocytes, natural killer and lymphokine-activated killer cells is usually tested by radioactive assays, which detect the release of cytoplasmic contents after plasma membrane disintegration of dying cells. In contrast to this indirect evaluation of cytotoxicity, we describe here an improved fluorescence assay that is based on the direct quantitative and qualitative flow cytometric analysis of cell damage at a single cell level. Target cells are stained with PKH-26, a lipophilic dye that stably integrates into the cell membrane and permits distinction between target and effector cells. After 3 h of in vitro incubation, costaining with AnnexinV-FITC (ann-FITC) and propidium iodide (PI) permitted discrimination between vital, early apoptotic and necrotic cells. Data analysis is performed first by gating on PKH-26-positive target cells followed by the analysis of ann-FITC- and PI-positive subpopulations. The percentage of cytotoxicity in the PKH-26-gated cell population is calculated by subtracting non-specific ann-FITC- or PI-positive target cells, measured in appropriate controls without effector cells. Membrane staining of target cells such as primary melanoma cells or leukemic blasts revealed high and stable loading of PKH-26 without altering the viability or the immunogenicity of the cells. Using in vitro-generated antigen-specific cytotoxic T lymphocytes (CTL), we could demonstrate that this flow cytometric assay is sensitive and correlates well with the standard 51Cr release assay. In conclusion, the improved fluorescence assay is a simple and highly reproducible procedure for evaluating the specific cytotoxicity of T cells.

Kurokawa T, Oelke M, Mackensen A. · Department of Hematology/Oncology, Freiburg University Medical Center, Freiburg, Germany. · Int J Cancer. · Pubmed #11275975 No free full text.

Abstract: Melanoma and renal cell carcinoma (RCC) are considered to be the most immunogenic tumors in humans. To generate conditions to induce primary T-cell responses against RCC and to allow further expansion of tumor-specific cytotoxic T lymphocytes (CTL) for adoptive transfer, peripheral blood mononuclear cells from RCC patients were stimulated with primary autologous tumor cells or monocyte-derived dendritic cells (DC) loaded with either tumor lysate (TU-LY) or apoptotic tumor cells (TU-AP). Whereas repetitive stimulation (4x) with tumor cells alone induced a predominant population of CD3(-) natural killer cells, 4 weeks of stimulation with tumor-loaded DC favored induction and expansion of CD4+ T cells (>80%). However, 2 weekly stimulation cycles with tumor-loaded DC followed by restimulation with autologous irradiated tumor cells alone were optimal for induction of tumor-specific CTL responses in vitro. Using these culture conditions a marked increase of CD4+ T cells was observed during the first 2 weeks of stimulation with tumor-loaded DC. Subsequent restimulation with autologous tumor cells alone gave rise to 500-fold expansion of CD8+ T cells. These CD8+ T cells were shown to exhibit strong major histocompatibility complex class I-restricted cytotoxic activity against the autologous tumor. Comparison of TU-LY and TU-AP as a source of tumor antigen for loading DC did not show any difference in stimulating tumor-specific CTL. Length pattern analysis of the complementary determining region 3 (CDR3) of the T-cell receptor Vbeta chain revealed expansion of oligoclonal CTL populations with outgrowth of 1 or 2 clones after prolonged stimulation with autologous tumor cells. Our study demonstrated an efficient method for generating tumor-specific CTL in vitro that may be used to identify tumor cell antigens or that can be expanded for adoptive T-cell transfer in tumor immunotherapy.