55-kd Tumor Necrosis Factor Receptor Is Expressed by Human Keratinocytes and Plays a Pivotal Role in Regulation of Human Keratinocyte ICAM-1 Expression

Tumor necrosis factor α (TNFα) is a potent modulator of human keratinocyte intercellular adhesion molecule-1 (ICAM-1) expression. TNFα is known to exert its biologic effects by binding to specific cell-surface receptors. Two distinct TNF binding molecules, the 55-kd and the 75-kd TNF receptor (TNFR) recently have been found to be expressed by human cells. These two receptor types are independently regulated and differ markedly in their intracellular regions, indicating functional dichotomy. In order to gain further insight into the mechanisms underlying ICAM-1 regulation in human keratinocytes, in the present study, the receptor molecules mediating TNFα induced ICAM-1 upregulation in human keratinocytes was defined. Human keratinocyte TNFR expression was assessed using monoclonal antibodies that specifically recognize the 55-kd or the 75-kd TNFR. Using FACS analysis, normal (HNK) as well as transformed (KB) human keratinocytes were found to react with anti-55-kd TNFR, but not anti-75-kd TNFR antibodies. These immunofluorescence data were confirmed by Northern blot analysis revealing clearly detectable amounts of mRNA specific for the 55-kd TNFR in KB cells. Incubation of human keratinocytes with anti-55-kd TNFR antibodies at 37°C for 24h increased ICAM-1 expression in a TNFα-like fashion. Moreover, the well known synergistic effect of IFNγ plus TNFα on keratinocyte ICAM-1 induction could be mimicked by stimulation of cells with IFNγ plus anti-55-kd TNFR antibodies. Synergistic ICAM-1 induction was not associated with increased expression of the 55-kd TNFR in IFNγ-stimulated human keratinocytes. These studies indicate that human keratinocytes express the 55-kd TNF receptor and that this surface molecule may play an important role in regulation of human keratinocyte ICAM-1 expression

The price of tumor control

Ipilimumab, a cytotoxic T-lymphocyte antigen-4 (CTLA-4) blocking antibody, has been approved for the treatment of metastatic melanoma and induces adverse events (AE) in up to 64% of patients. Treatment algorithms for the management of common ipilimumab-induced AEs have lead to a reduction of morbidity, e.g. due to bowel perforations. However, the spectrum of less common AEs is expanding as ipilimumab is increasingly applied. Stringent recognition and management of AEs will reduce drug-induced morbidity and costs, and thus, positively impact the cost-benefit ratio of the drug. To facilitate timely identification and adequate management data on rare AEs were analyzed at 19 skin cancer centers. Patient files (n = 752) were screened for rare ipilimumab-associated AEs. A total of 120 AEs, some of which were life-threatening or even fatal, were reported and summarized by organ system describing the most instructive cases in detail. Previously unreported AEs like drug rash with eosinophilia and systemic symptoms (DRESS), granulomatous inflammation of the central nervous system, and aseptic meningitis, were documented. Obstacles included patientś delay in reporting symptoms and the differentiation of steroid-induced from ipilimumab-induced AEs under steroid treatment. Importantly, response rate was high in this patient population with tumor regression in 30.9% and a tumor control rate of 61.8% in stage IV melanoma patients despite the fact that some patients received only two of four recommended ipilimumab infusions. This suggests that ipilimumab-induced antitumor responses can have an early onset and that severe autoimmune reactions may reflect overtreatment. The wide spectrum of ipilimumab-induced AEs demands doctor and patient awareness to reduce morbidity and treatment costs and true ipilimumab success is dictated by both objective tumor responses and controlling severe side effects

The price of tumor control: an analysis of rare side effects of anti-CTLA-4 therapy in metastatic melanoma from the ipilimumab network

Background: Ipilimumab, a cytotoxic T-lymphocyte antigen-4 (CTLA-4) blocking antibody, has been approved for the treatment of metastatic melanoma and induces adverse events (AE) in up to 64% of patients. Treatment algorithms for the management of common ipilimumab-induced AEs have lead to a reduction of morbidity, e.g. due to bowel perforations. However, the spectrum of less common AEs is expanding as ipilimumab is increasingly applied. Stringent recognition and management of AEs will reduce drug-induced morbidity and costs, and thus, positively impact the cost-benefit ratio of the drug. To facilitate timely identification and adequate management data on rare AEs were analyzed at 19 skin cancer centers. Methods and Findings: Patient files (n = 752) were screened for rare ipilimumab-associated AEs. A total of 120 AEs, some of which were life-threatening or even fatal, were reported and summarized by organ system describing the most instructive cases in detail. Previously unreported AEs like drug rash with eosinophilia and systemic symptoms (DRESS), granulomatous inflammation of the central nervous system, and aseptic meningitis, were documented. Obstacles included patientś delay in reporting symptoms and the differentiation of steroid-induced from ipilimumab-induced AEs under steroid treatment. Importantly, response rate was high in this patient population with tumor regression in 30.9% and a tumor control rate of 61.8% in stage IV melanoma patients despite the fact that some patients received only two of four recommended ipilimumab infusions. This suggests that ipilimumab-induced antitumor responses can have an early onset and that severe autoimmune reactions may reflect overtreatment. Conclusion: The wide spectrum of ipilimumab-induced AEs demands doctor and patient awareness to reduce morbidity and treatment costs and true ipilimumab success is dictated by both objective tumor responses and controlling severe side effects

Long-term therapy of interferon-alpha induced pulmonary arterial hypertension with different PDE-5 inhibitors: a case report

BACKGROUND: Interferon alpha2 is widely used in hepatitis and high-risk melanoma. Interferon-induced pulmonary arterial hypertension as a side effect is rare. CASE PRESENTATION: We describe a melanoma patient who developed severe pulmonary arterial hypertension 30 months after initiation of adjuvant interferon alpha2b therapy. Discontinuation of interferon did not improve pulmonary arterial hypertension. This patient could be treated successfully with phosphodiesterase-5 inhibitor therapy. CONCLUSION: This is only the 5th case of interferon-induced pulmonary arterial hypertension and the first documented case where pulmonary arterial hypertension was not reversible after termination of interferon alpha2 therapy. If interferon alpha2 treated patients develop respiratory symptoms, pulmonary arterial hypertension should be considered in the differential diagnosis. For these patients phosphodiesterase-5 inhibitors, e.g. sildenafil or vardenafil, could be an effective therapeutic approach

Proteome Serological Determination of Tumor-Associated Antigens in Melanoma

Proteome serology may complement expression library-based approaches as strategy utilizing the patients' immune responses for the identification pathogenesis factors and potential targets for therapy and markers for diagnosis. Melanoma is a relatively immunogenic tumor and antigens recognized by melanoma-specific T cells have been extensively studied. The specificities of antibody responses to this malignancy have been analyzed to some extent by molecular genetic but not proteomics approaches. We screened sera of 94 melanoma patients for anti-melanoma reactivity and detected seropositivity in two-thirds of the patients with 2–6 antigens per case detected by 1D and an average of 2.3 per case by 2D Western blot analysis. For identification, antigen spots in Western blots were aligned with proteins in 2-DE and analyzed by mass spectrometry. 18 antigens were identified, 17 of which for the first time for melanoma. One of these antigens, galectin-3, has been related to various oncogenic processes including metastasis formation and invasiveness. Similarly, enolase has been found deregulated in different cancers. With at least 2 of 18 identified proteins implicated in oncogenic processes, the work confirms the potential of proteome-based antigen discovery to identify pathologically relevant proteins

Antigen loss and possibilities for immunotherapy in malignant melanoma

Titelblatt, Inhaltsverzeichnis, Eidesstattliche Erklärung Einleitung Publikationen Diskussion, Abkürzungsverzeichnis LiteraturverzeichnisAntigenexpression ist für Immuntherapien, aber auch für die Immunhistochemie beim Melanom von elementarer Bedeutung. Es wurde hier mit dem monoklonalen Antikörper SM5-1 ein Reagenz generiert und charakterisiert, das die höchste Sensitivität aller bisher beschriebenen Melanomantikörper aufweist und somit gegenüber den Antikörpern gegen gp100, Tyrosinase oder MART-1 große Vorteile hat. Bei Antigen-Expressionsstudien mit großen Fallzahlen zeigten sich konkordante Antigenverluste der Melanom-Differenzierungsantigene von hoher statistischer Signifikanz, was Implikationen für antigenspezifische Immuntherapien hat. Diese konkordanten Verluste schlossen die Cancer Testis Antigene und mAk SM5-1 nicht mit ein. Das Immunoscreening einer Melanomzell cDNA library ergab für das von mAk SM5-1 erkannte Antigen eine Sequenzidentität für humanes Fibronektin mit zwei Isoformen, die die ED-A und die CS1 Regionen beinhalten. Hierbei liegt eine post-translationale Modifikation vor, da das von mAk SM5-1 erkannte Epitop nicht auf mAk SM5-1 negativen Zelllinien zu finden ist, diese aber die Standard Fibronektinform exprimieren. Somit wird diese Melanom-assoziierte Fibronektin Isoform von der Mehrzahl der Melanome exprimiert und kann, ähnlich wie die ED-A Region des Fibronektins möglicherweise als Immuntherapeutikum verwendet werden. Um Antigenverlustvarianten möglichst wenig Einfluss auf eine Immuntherapie zu verschaffen und um eine optimale T-Helfer Zell Aktivierung zu induzieren, wurden zwei klinische Studien einer Fusionsvakzine aus allogenen antigen- präsentierenden Zellen (B-Zellen bzw. DCs) und autologen Tumorzellen bei Patienten mit fortgeschrittenem Melanom durchgeführt. In beiden Studien zeigten sich Hinweise für durch Vakzine vermittelte Immunreaktionen, entweder inflammatorische Infiltrate (B-Zell Fusionsvakzine) oder Expansion von Tumorantigen-spezifischen CTLs und in-situ Verlust von Tumorantigenen und TAP-1 bei progredienten Metastasen (DC Fusionsvakzine). In beiden Studien wurden einige wenige Patienten beobachtet, die von der Vakzinierung profitierten, sei es durch objektive Tumorrückgänge oder aber durch teilweise lang anhaltende Stabilisierungen der Erkrankung. Bis auf die Entwicklung lokaler Vitiligo wurden keine Nebenwirkungen beobachtet. Gerade die Entwicklung einer Vitiligo unterstreicht den Ansatz der Vakzine, durch die Verwendung ganzer Tumorzellen in Kombination mit Ko-stimulation und T-Helfer Zell Aktivierung Effekte gegen eine Vielzahl von Antigenen zu induzieren, die nicht identifiziert werden müssen.Antigen expression is for immunotherapy, but also for immunohistochemistry of utmost importance. A monoclonal antibody, SM5-1 was generated and characterized and was shown to exhibit the highest sensitivity in comparison to the previously described melanoma antibodies. SM5-1 therefore has advantages compared to gp100, tyrosinase or MART-1 antibodies. Concordant antigen loss of melanoma differentiation antigens in large sample numbers has significant implications for antigen specific immunotherapy. These concordant losses did not include cancer testis antigens or mAb SM5-1. Immunoscreening of a melanoma cDNA library revealed sequence identity for human fibronectin with two isoforms which include the ED-A and the CS1 regions. This melanoma- associated fibronectin isoform could potentially be used for immunotherapy of melanoma. In order to decrease antigen loss variants and its potential negative effect for immunotherapeutic efforts, two clinical studies were conducted with a fusion cell vaccine, designed for enhanced T-helper cell activation, consisting of allogeneic antigen-presenting cells (B cells or dendritic cells) and autologous tumor cells in patients with advanced melanoma. In both studies signs of vaccine mediated immune reactions such as inflammatory infiltrates or expansion of tumor antigen-specific CTLs and in- situ loss of tumor antigens or TAP-1 in progressing metastases were observed. A few patients did benefit from this treatment, either by objective tumor response or by stabilization of the disease. Beside a few cases of vitiligo, no side effects were observed. The development of vitiligo underlines this vaccine approach of using whole tumor cells in combination with co-stimulation and T-helper cell activating effect in order to obtain effects against a multitude of antigens without the need of identifying them

The monoclonal antibody SM5-1 recognizes a fibronectin variant which is widely expressed in melanoma

Abstract Background Previously we have generated the monoclonal antibody SM5-1 by using a subtractive immunization protocol of human melanoma. This antibody exhibits a high sensitivity for primary melanomas of 99% (248/250 tested) and for metastatic melanoma of 96% (146/151 tested) in paraffin embedded sections. This reactivity is superior to the one obtained by HMB-45, anti-MelanA or anti-Tyrosinase and is comparable to anti-S100. However, as compared to anti-S100, the antibody SM5-1 is highly specific for melanocytic lesions since 40 different neoplasms were found to be negative for SM5-1 by immunohistochemistry. The antigen recognized by SM5-1 is unknown. Methods In order to characterize the antigen recognized by mAb SM5-1, a cDNA library was constructed from the metastatic human melanoma cell line SMMUpos in the Uni-ZAP lambda phage and screened by mAb SM5-1. The cDNA clones identified by this approach were then sequenced and subsequently analyzed. Results Sequence analysis of nine independent overlapping clones (length 3100–5600 bp) represent fibronectin cDNA including the ED-A, but not the ED-B region which are produced by alternative splicing. The 89aa splicing variant of the IIICS region was found in 8/9 clones and the 120aa splicing variant in 1/9 clones, both of which are included in the CS1 region of fibronectin being involved in melanoma cell adhesion and spreading. Conclusion The molecule recognized by SM5-1 is a melanoma associated FN variant expressed by virtually all primary and metastatic melanomas and may play an important role in melanoma formation and progression. This antibody is therefore not only of value in immunohistochemistry, but potentially also for diagnostic imaging and immunotherapy.</p