Heterogeneity in expression of human leukocyte antigens and melanoma-associated antigens in advanced melanoma

The study of tumor immunology has led to many innovative therapeutic strategies for the treatment of melanoma. The strategies are primarily dependent on melanoma-associated antigen peptide vaccination or T-cell-based therapy. These immunotherapies are totally reliant on proper copresentation of human leukocyte antigen class I molecules in sufficient quantity and the presence and availability of melanoma-associated antigenic peptides. Altered expression of either HLA class I molecules or melanoma antigens is known to occur. These defects lead to altered manufacture and copresentation of HLA class I molecules with melanoma-associated antigens to T-cells. Defects in any one combination can lead to loss of recognition of melanoma cells and their subsequent destruction by cytotoxic T-lymphocytes. Thus, these immunotherapy strategies can be thwarted by defects or heterogeneity of expression of human leukocyte antigen class I or of melanoma-associated antigens.     

Human melanoma-associated antigen expression on human neuroblastoma cells: Effects of differentiation inducers

Summary We have described two human melanoma-associated antigens (HMAA), recognized by the murine monoclonal antibodies LS62 and LS109. LS62 recognizes the neuroglandular antigen (NGA), which is overexpressed in neoplastic melanocytes as well as in several tissues of neuroectodermal origin. These antibodies were used to screen six neuroblastoma cell lines and one neuroepithelioma cell line. A melanoma cell line, G361, known to express the two antigens, was used as the positive control. Variable expression of the two antigens was detected in neuroblastoma cells. The surface expression of NGA and of the LS109 antigen was modulated in parallel with the morphological differentiation induced by retinoic acid, 5-bromodeoxyuridine, or cyclic AMP analog/activators. The modulation of the expression of the two HMAA was detected in G361 melanoma cells and in one of the neuroblastoma cell lines, SK-N-SH. These results suggest altered expression of both antigens during melanoma and neuroblastoma cell differentiation in culture.     

Demonstration and isolation of murine melanoma-associated antigenic surface proteins

Melanomas are antigenic and induce the formation of antibodies in both syngeneic and xenogeneic species. The nature of melanoma-associated antigens remains problematic, however. We found that xenogeneic (goat) antiserum to the mouse (C57BL/6) B16 melanoma, following appropriate absorptions with nonmelanoma cells, showed specificity for melanoma-associated surface antigens of B16 and one other murine melanoma. The antibody to B16 did not react with histocompatibility antigens, mouse-specific xenoantigens, viral antigens or melanin. The IgG fraction of the goat antibody was cross-linked covalently to protein A-Sepharose using dimethylsuberimidate. This immunoadsorbent was used to isolate shed antigens from cultures in which B16 cells had been grown and from detergent extracts of biosynthetically labeled (³H-leucine) B16 cells. The immune-affinity purified antigen preparation contained two major components of apparent molecular weight 60,000 and 50,000 daltons as assessed by SDS-polyacrylamide gel electrophoresis. Immunization of rabbits with immune-affinity purified B16 antigens induced antibodies which bound specifically to B16 cells.     

Differential Antitumor Effects of IgG and IgM Monoclonal Antibodies and Their Synthetic Complementarity-Determining Regions Directed to New Targets of B16F10-Nex2 Melanoma Cells

Malignant melanoma has increased incidence worldwide and causes most skin cancer-related deaths. A few cell surface antigens that can be targets of antitumor immunotherapy have been characterized in melanoma. This is an expanding field because of the ineffectiveness of conventional cancer therapy for the metastatic form of melanoma. In the present work, antimelanoma monoclonal antibodies (mAbs) were raised against B16F10 cells (subclone Nex4, grown in murine serum), with novel specificities and antitumor effects in vitro and in vivo. MAb A4 (IgG2ak) recognizes a surface antigen on B16F10-Nex2 cells identified as protocadherin β₁₃. It is cytotoxic in vitro and in vivo to B16F10-Nex2 cells as well as in vitro to human melanoma cell lines. MAb A4M (IgM) strongly reacted with nuclei of permeabilized murine tumor cells, recognizing histone 1. Although it is not cytotoxic in vitro, similarly with mAb A4, mAb A4M significantly reduced the number of lung nodules in mice challenged intravenously with B16F10-Nex2 cells. The V_H CDR3 peptide from mAb A4 and V_L CDR1 and CDR2 from mAb A4M showed significant cytotoxic activities in vitro, leading tumor cells to apoptosis. A cyclic peptide representing A4 CDR H3 competed with mAb A4 for binding to melanoma cells. MAb A4M CDRs L1 and L2 in addition to the antitumor effect also inhibited angiogenesis of human umbilical vein endothelial cells in vitro. As shown in the present work, mAbs A4 and A4M and selected CDR peptides are strong candidates to be developed as drugs for antitumor therapy for invasive melanoma.     

Demonstration of B700 cross-reactive antigens on human and other animal melanomas

B700 is a melanoma-associated antigen originally detected by immunologic and biochemical criteria; it is expressed by several murine melanomas but is not detectable on any normal murine cells, or on murine nonmelanoma neoplasms. We have used antibodies raised against purified B700 to study the presentation of B700 and B700 crossreactive molecules on the surfaces of melanoma cells of various species and origins. The antibodies are shown to bind to all the melanoma cells tested, including five different murine melanoma lines (S91, JB/RH, JB/MS, K1735, and B16), three different B16 sublines (F1, F10, and BL6), three human, one hamster, and two swine melanoma cell lines. These results suggest the candidacy of B700-like molecules as "pan-melanoma" antigens.     

Heterogeneity of primary and metastatic human malignant melanoma as detected with monoclonal mntibodies in cryostat sections of biopsies

Summary Monoclonal antibodies were generated against established melanoma cell lines and characterized by their reactivity with various sublines. The antibodies selected for their reaction with melanoma-associated antigens were tested on cryostat sections of melanoma tissue from various stages and on other tumors. The reactivity with normal tissues was also determined. Of 30 antibodies reacting with melanoma cell lines 11 did not react with melanoma biopsies. Of the remaining 19 antibodies nine displayed broad cross-reactivity with normal cells and structures and other benign or malignant tumor cells. Among the remaining antibodies five types were defined that detected antigens (nevocellular I, nevocellular II, neural, endothelial, basal cell) found on certain normal tissues and structures and on certain tumor phenotypes. Even though there seems to be a tendency for some antigens to be preferentially associated with certain stages of melanoma, it has not yet been possible to establish any clear-cut correlation between the expression of one of the differentiation antigens and a particular stage or malignancy potential of melanoma.     

Recombinant gamma-interferon induces changes in expression and shedding of antigens associated with normal human melanocytes, nevus cells, and primary and metastatic melanoma cells

Recombinant gamma-interferon (rIFN-gamma) induced or augmented the expression of HLA-DR class II antigens on melanocytes isolated from newborn foreskin, from congenital, common acquired, and dysplastic nevi, and from primary and metastatic melanoma. The stimulatory effect of rIFN-gamma on HLA-DR antigen expression was suppressed by the addition of the phorbol ester TPA or its analog PDBu to the culture medium. Whereas rIFN-gamma did not significantly alter the expression of melanoma-associated, non-class II antigens on melanoma cells, there was a marked decrease in the expression of antigens associated with nevus cells. In addition, rIFN-gamma stimulated shedding of antigens. Increased antigen shedding was most apparent for an intracytoplasmic melanoma-associated protein of 80kd, followed by the ganglioside GD2 and by an alkali labile ganglioside. The simultaneous stimulation of class II antigen expression and shedding of melanoma-associated antigens as well as suppression of nevus-associated antigen expression could play an important role in the host immune response to premalignant and malignant melanocytic lesions.     

A malignus melanoma immunterápiájának lehetoségei

Despite their well-documented immunogenicity, malignant melanomas belong to the most aggressive tumor types. A potential explanation for this is the suboptimal activation of tumor infiltrating T cells. In order to boost immune responses against tumors, a variety of treatment modalities have been tested in animal models and in clinical setting. Antigen-nonspecific approaches (e.g., IFN-alpha and IL-2), as well as active specific immunotherapeutical modalities based on the use of autologous or allogeneic tumor cell-save been investigated in clinical trials of melanoma. The identification of melanoma-associated antigens has opened new avenues in antigen-specific immunotherapy. A promising alternative for the delivery of different forms of melanoma antigens is the application of dendritic cells, the most potent antigen presenting cells capable of eliciting efficient T-cell response. Beside active immunotherapy, immune response against melanoma antigens could be increased through the adoptive transfer of tumor infiltrating lymphocytes or antigen specific T-cell clones. The most important conclusion that can be drawn from the results of published immunotherapy studies is that these modalities are able to induce durable complete tumor regressions,mostly with reasonable toxicity; however, generally only in a minority of patients. This points to the importance of appropriate patient selection, with regard to the expression of the targeted antigens and HLA molecules, as well as to the general immunocompetence of the patients. A crucial and still unsolved question is monitoring immune activation during treatment, although there are promising new tools that could prove useful in this respect. The presence of tumor-reactive CTL in the circulation or in the tumors does not guarantee an efficient immune response. It is important to assess if these T cells are in an activated and functional state. Finally, in several single target antigen-based clinical studies a therapy-induced immunoselection of antigen-negative clones, leading to disease progression, was observed. This could be overcome with the use of antigen cocktails or whole tumor approaches. A better understanding of the mechanisms of action of immunotherapeutical modalities may enhance the success rate of these strategies.     

Tumor antigens discovery: perspectives for cancer therapy.

The adoptive transfer of cytotoxic T lymphocytes (CTLs) derived from tumor-infiltrating lymphocytes (TIL) along with interleukin 2 (IL-2) into autologous patients with cancer resulted in the objective regression of tumor, indicating that these CTLs recognized cancer rejection antigens on tumor cells. To understand the molecular basis of T cell-mediated antitumor immunity, several groups started to search for such tumor antigens in melanoma as well as in other types of cancers. This led to the subject I will review in this article. A number of tumor antigens were isolated by the use of cDNA expression systems and biochemical approaches. These tumor antigens could be classified into several categories: tissue-specific differentiation antigens, tumor-specific shared antigens, and tumor-specific unique antigens. However, the majority of tumor antigens identified to date are nonmutated, self proteins. This raises important questions regarding the mechanism of antitumor activity and autoimmune disease. The identification of human tumor rejection antigens provides new opportunities for the development of therapeutic strategies against cancer. This review will summarize the current status and progress toward identifying human tumor antigens and their potential applications to cancer treatment.     

Humoral immune response to melanoma-associated membrane antigen and fetal brain antigen demonstrated by indirect membrane immunofluorescence. I

Summary A melanoma-associated membrane antigen and a fetal brain antigen were identified on the surface of a human melanoma cell line by indirect membrane immunofluorescence techniques. The target melanoma cells were grown in gamma globulin-depleted human serum. Sera from melanoma patients were used as the source of antimelanoma antibodies. To remove alloantibodies, the allogeneic sera were preabsorbed with cultured lymphoblastoid cells derived from the peripheral lymphocytes of the donor of the target cell line. To further define the antigen responsible for antibody activity, sequential absorption tests were performed with fetal brain cells, cultured sarcomas, and breast carcinomas. Some antibody activity was removed by fetal brain tissues. Further absorption with fetal brain or the cultured sarcoma or breast carcinoma did not remove additional activity. However, antibody activity was completely removed by either cultured or biopsy-derived melanoma cells. A serum autochthonous to the target cell line was also tested. The antibody titer of the serum was completely removed by absorption with either autochthonous biopsied tumor or an allogeneic melanoma cell line, but not with the normal tissues. Thus it appeared that sera from melanoma patients contained antibody to both a melanoma-associated membrane antigen and a fetal brain antigen.     

Lymphocytes cytotoxic to uveal and skin melanoma cells from peripheral blood of ocular melanoma patients

Summary To study antitumor immunity in patients with choroidal melanoma, T cells were generated from the peripheral blood of choroidal melanoma patients by mixed lymphocyte/tumor cell culture (MLTC). Because autologous tumors are generally unavailable, an allogeneic choroidal melanoma cell line, OCM-1, was used as the specific stimulus. Lymphocyte cultures from 27 patients were characterized by cell-surface phenotypes, patterns of reactivity towards cells of the melanocytic origin and T-cell-receptor gene usage. Antimelanoma reactivity was found in cell-sorter-purified CD4⁺ and CD8⁺ T cell subsets. To analyze this reactivity, sorter-purified CD4⁺ and CD8⁺ cells from a MLTC were cloned by limiting dilution in the presence of exogenous interleukin-2 and interleukin-4 as well as irradiated OCM-1. Under these conditions, CD4⁺ T cells did not proliferate, perhaps because of the absence of antigen-presenting cells. However, CD8⁺ grew vigorously and 29 cytolytic CD8⁺ T cell clones were isolated. On the basis of their pattern of lysis of OCM-1, a skin melanoma cell line M-7 and its autologous lymphoblastoid cell line LCL-7, the clones were categorized into three groups. Group 1, representing 52% of the clones, lysed all three target cells, and are alloreactive. However, since OCM-1 and M-7 did not share class I antigens, these clones recognized cross-reactive epitope(s) of the histocompatibility locus antigen (HLA) molecule. Group 2, constituting 28% of the clones, lysed both the ocular and skin melanoma cell lines but not LCL-7, and were apparently melanoma-specific. Unlike classical HLA-restricted cytolytic T lymphocytes, these T cells might mediate the lysis of melanoma cells via other ligands or a more degenerate type of HLA restriction. For the latter, the HLA-A2 and -A28 alleles would have to act interchangeably as the restriction element for shared melanoma-associated antigen(s). Group 3, representing only 10% of the T cell clones, was cytotoxic only to OCM-1, but not to M-7 or LCL-7. These clones may recognize antigens unique to ocular melanoma cells. Our data suggest that choroidal melanoma patients can recognize melanoma-associated antigens common to both ocular and cutaneous melanoma cells, and presumbly their autologous tumor. Thus, choroidal melanoma, like its skin counterpart, may be responsive to immunotherapeutic regimens such as active specific or adoptive cellular immunotherapy.This work is supported by National Institutes of Health research grants CA 36 233 and EY 9031, the Lucy Adams Memorial Fund and support from the Concern Foundation     

GD3-reactive antibodies can inhibit the lysis of autologous tumor cells by tumor-infiltrating lymphocytes

Summary GD3 is expressed in high concentrations on melanoma cells and may serve as a useful target antigen for mAb-mediated immunotherapy. Monoclonal antibodies (mAbs) against GD3 stimulate cell-mediated immune responses against tumor cells in vitro and this activity may contribute to antitumor effects in patients with melanoma treated with GD3-reactive mAbs. In the present study the effects of GD3-reactive mAbs on autologous tumor cell lysis by a human melanoma-derived tumor-infiltrating lymphocyte (TIL) population were examined. Unlike results reported for other GD3⁺ T cells isolated from melanoma patients, the tumor-specific lytic activity of the TIL line was inhibited by incubation with mAbs against GD3. Other melanoma-reactive mAbs, including those against GD2 and the high-molecular-weight melanoma-associated Ag, had no effect on the TIL lytic activity. Overall, these results indicate that mAbs against GD3 may have different effects on T cell/tumor cell interactions.     

T-cell recognition of melanoma-associated antigens

In this review, we summarize the significant progress that has been made in the identification of melanoma-associated antigens (MAA) recognized by cytotoxic T-lymphocytes (CTL). These antigens belong to three main groups: tumor-associated testis-specific antigens (e.g. , MAGE, BAGE, and GAGE); melanocyte differentiation antigens (e.g., tyrosinase, Melan-A/MART-1); and mutated or aberrantly expressed molecules (e.g, CDK4, MUM-1, beta-catenin). Although strong CTL activity may be induced ex vivo against most of these antigens, often in the presence of excess cytokines and antigen, a clear understanding of the functional status of CTL in vivo and their impact on tumor growth, is still lacking. Several mechanisms are described that potentially contribute to tumor cell evasion of the immune response, suggesting that any antitumor efficacy achieved by immune effectors may be offset by factors that result ultimately in tumor progression. Nevertheless, most of these MAA are currently being investigated as immunizing agents in clinical studies, the conflicting results of which are reviewed. Indeed, the therapeutic potential of MAA has still to be fully exploited and new strategies have to be found in order to achieve an effective and long-lasting in vivo immune control of melanoma growth and progression.     

Prognostic significance of expression of antigens on melanoma cells

Summary The expression of antigens on 33 human melanoma cells obtained directly from surgically excised tumours was investigated by means of antibody-dependent cell-mediated cytotoxic assays. Antisera used in the study were two antisera from human melanoma patients against different tumour-associated antigens on melanoma cells and antisera against carcinoembryonic antigen (CEA) and ₂ microglobulin (₂M). Considerable heterogeneity was observed in the expression of both melanoma-associated and non-melanoma antigens on melanoma cells from 33 different patients.Patients whose tumours were reactive with the melanoma-associated antiserum (CHI) had a significantly longer remission period to stage 2 melanoma. The period to development of stage 3 melanoma also appeared longer, but this was not statistically significant with the number of patients available for study. The expression of CEA, ₂M, and the tumour-associated antigen TIN was not significantly related to the recurrence-free interval. There appeared to be a reciprocal expression of the two melanoma-associated antigens, and patients with tumours expressing CHI but not TIN had a significantly longer recurrence-free interval than patients whose tumours had the opposite antigenic pattern.In the limited number of patients available for study the expression of the antigen CHI did not appear related to thickness of the primary tumour or to immune response of the patients to melanoma cells in leucocyte-dependent antibody and natural killer cell assays. Although the nature of the association between expression of this antigen and longer remission-free period is unknown these results suggest that the expression of certain melanoma antigens on the cell surface may be an important additional variable which has prognostic and therapeutic importance.     

In situ T cells in melanoma

During the past decade new insights have been gained into the role of T lymphocytes in the host's immune response to cancer in general and to melanoma in particular. Several melanoma-associated antigens (MAA) recognized by T cells have been characterized, and a number of HLA class I- and class II-restricted peptides have been identified. These antigens can be divided into three different groups: tumor-associated testis-specific antigens, melanocyte differentiation antigens, and mutated or aberrantly expressed antigens. These proteins give rise to several antigenic peptides. The number of known melanoma-associated peptides that can induce killing by cytotoxic T-lymphocytes (CTL) exceeds 30 and is still increasing. In line with these findings, clinical data indicate that the immune system is essential in the control of tumor growth. A brisk infiltration of lymphocytes is associated with a favorable prognosis, and complete or partial regression of primary melanoma occurs quite frequently. Furthermore, immunomodulatory therapies have accomplished complete or partial tumor regression in a number of patients. However, the immune response is in most cases inadequate to control tumor growth as tumor progression often occurs. Hence, the coexistence of a cellular immune response in melanoma lesions, demonstrated by the presence of clonally expanded T cells, remains a major paradox of tumor immunology. In the present paper we review current knowledge regarding tumor infiltrating lymphocytes (TIL) in melanoma and discuss possible mechanisms of escape from immune surveillance. Received: 20 March 1999 / Accepted: 3 March 1999     

Differential modulation by interferon γ of the sensitivity of human melanoma cells to cytolytic T cell clones that recognize differentiation or progression antigens

 Human melanoma is a highly immunogenic tumor capable of inducing a specific immune response. A number of melanoma-associated antigens have been characterized during the past several years and can be classified into two groups: differentiation antigens  –  present also in normal melanocytes  –  and tumor-specific antigens, which, with the exception of testis, are present only in tumor cells. In a previous publication [Kirkin A. F., Petersen T. R., Olsen A. C., Li L., thor Straten P., Zeuthen J. (1995) Cancer Immunol Immunother 41:71] we have described the production of clones of cytotoxic T lymphocytes (CTL) against the highly immunogenic human melanoma cell line FM3. Using these clones we have defined four previously unknown melanoma-associated antigens, which could be subdivided into differentiation and progression antigens. In the experiments reported in this paper, we have further compared CTL clones from different groups and shown that the sensitivity of melanoma cells to CTL that recognize differentiation or progression antigens is differentially modulated during tumor progression as well as by the lymphokines interferon γ (IFNγ) and interleukin-10 (IL-10). The interaction of CTL clones recognizing progression antigens was strongly increased after treatment of melanoma cells with IFNγ, while the recognition by CTL clones specific for differentiation antigens either was unchanged or significantly decreased. IL-10 treatment of melanoma cells induced up-regulation with respect to recognition by CTL clones specific for differentiation antigens without affecting the recognition of melanoma cells by CTL clones specific for progression antigens. Using cellular systems at different stages of tumor progression, we demonstrated that the progressed state of melanoma cells is associated with increased sensitivity to recognition by CTL clones detecting progression antigens, and with decreased sensitivity to CTL clones recognizing differentiation antigens. Mimicking tumor progression, treatment with IFN-γ induced apparent down-regulation of differentiation antigens. A hypothesis is suggested in which IFN-γ plays different roles in the immune response against poorly immunogenic and highly immunogenic melanoma cells, increasing the progression of poorly immunogenic tumor cells or promoting a strong immune response and regression of highly immunogenic melanoma cells. Received: 23 November 1995 / Accepted: 7 March 1996     

Evaluation of human monoclonal antibody (2-139-1) in cutaneous melanocytic neoplasms in fixed tissue sections.

Despite the growing list of xenogeneic monoclonal antibodies (MAb) that recognize malignant melanoma-associated antigens (MAA) in formalin-fixed, paraffin-embedded tissue, none has been able to detect epitopes found in malignant melanomas and not in melanocytic nevi. A human MAb, 2-139-1, that showed promise in this regard was evaluated against 85 melanocytic neoplasms, including malignant melanoma and histological simulators, particularly Spitz's nevus. MAb 2-139-1 stained 18 (53%) of 34 melanomas, eight (57%) of 14 dysplastic nevi, six (38%) of 16 Spitz's nevi, and three (14%) of 21 banal nevi, which included three small congenital nevi. We observed a significant increasing trend in reactivity (% positive cells x intensity) associated with the potential for malignancy (p for linear trend = 0.002). We conclude that human MAb 2-139-1 is applicable to the study of melanocytic neoplasms in routinely processed tissue. Although the ability of this MAb to separate benign from malignant cells is not absolute, our results suggest that the expression of the 2-139-1 epitope may be an early event in melanocytic tumor progression.