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PubMed1996

Binding and presentation of peptides derived from melanoma antigens MART-1 and glycoprotein-100 by HLA-A2 subtypes. Implications for peptide-based immunotherapy.

Cellular immune responses to melanoma-associated Ags are the focus of ongoing studies aimed at developing immunotherapies for treatment of malignant melanoma. Melanoma predominantly affects Caucasians, a population in whom expression of HLA-A2 is prevalent. Among HLA-A2 subtypes, HLA-A*0201 is widely expressed, and HLA-A*0201-restricted, tumor-reactive CTL responses are well studied. We have observed in a group of melanoma patients an unexpectedly high frequency (approximately 20%) of non-HLA-A*0201 subtypes (*0202, *0204, and *0205), and little is known regarding antimelanoma response profiles in patients expressing such subtypes. We analyzed non-HLA-A*0201 peptide response profiles using HLA-A*0201-restricted epitopes from melanoma Ags MART-1/Melan A and glycoprotein 100. Most of these peptides bound to the majority of subtypes tested with 50% inhibitory concentrations less than 500 nM. Recognition of cells pulsed with different peptides (MART-1(27-35), G9(154), and G9(280) Flu M1(58-66)) and expressing different subtype molecules by HLA-A*0201-restricted CTL was limited to only a subset of non-HLA-A*0201 molecules, and the peptide/subtype complexes recognized varied among the effector populations tested. CTL responses elicited from PBL of patients and healthy donors expressing subtypes HLA-A*0202 and HLA-A*0205 suggested significant differences among HLA-A2 subtype function in the context of melanoma Ag presentation. These observations imply the necessity of subtyping patients considered for peptide-based protocols and highlight the need for further study of melanoma-directed cellular responses among patients expressing non-HLA-A*0201 subtypes.

Alleles↗
PubMed1995

Induction of tumor-reactive CTL from peripheral blood and tumor-infiltrating lymphocytes of melanoma patients by in vitro stimulation with an immunodominant peptide of the human melanoma antigen MART-1.

MART-1 is an Ag expressed on melanomas and melanocytes, and is recognized by the majority of HLA-A2-restricted tumor-specific tumor-infiltrating lymphocytes (TIL) from melanoma patients. In the present study we have analyzed 10 potential 9-mer epitopes containing the HLA-A2.1 binding motifs for their ability to induce melanoma-specific T cell lines. Antimelanoma CTL could be generated only with MART-1(27-35) peptide, which has been previously shown to be recognized by a majority of HLA-A2-restricted TIL. Anti-MART-1(35-43)-specific CTL could also be induced, but these T cells did not recognize melanoma cells. MART-1(27-35)-specific CTL could be effectively generated from a total of 11 of 12 PBL and from 3 of 3 TIL derived from HLA-A2+ melanoma patients, as well as from 2 of 4 PBL from HLA-A2+ healthy donors by in vitro stimulation with autologous PBMC pulsed with the synthetic MART-1(27-35) peptide. These CTL lines specifically lysed and release cytokines (TNF-alpha, IFN-gamma, and GM-CSF) in response to T2 cells pulsed with MART-1(27-35), as well as to HLA-A2+ MART-1+ melanoma cells. CTL generated with MART-1(27-35) also lysed uncultured HLA-A2+ melanoma cells derived from tumor biopsies, indicating that this MART-1 epitope is likely to be expressed in association with HLA-A2 on the surface of tumor cells in vivo. CTL lines generated with MART-1(27-35) mediated 25- to 100-fold higher lytic activity than MART-1-reactive CTL grown from TIL in the presence of high dose IL-2. These results demonstrate that MART-1(27-35) peptide may represent an ideal candidate for Ag-specific immunotherapy in melanoma patients.

Amino Acid Sequence↗
PubMed1996

Melanocyte lysis by cytotoxic T lymphocytes recognizing the MART-1 melanoma antigen in HLA-A2 patients with Vogt-Koyanagi-Harada disease.

The MART-1/Melan-A melanoma antigen recognized by the majority of HLA-A2-restricted tumor-infiltrating lymphocytes is a self antigen expressed on melanocytes and the retina. We have investigated whether Vogt-Koyanagi-Harada (VKH) disease and sympathetic ophthalmia (SO), systemic inflammatory disorders affecting various organs containing melanocytes, are autoimmune diseases directed toward the MART-1 antigen. In two of three patients with VKH disease and one patient with SO, CD8(+) T cell clones (TCC) form intraocular fluid of HLA-A2(+) patients lysed T2 cells when pulsed with a HLA-A2-binding MART-1 peptide, but not a HLA-A2-binding pMel-17 or tyrosinase peptide, in a HLA-A2-restricted manner. In addition, Th, TCC recognizing a HLA-A2-binding MART-1 peptide were also established from peripheral blood mononuclear cells of a patient with VKH disease. In contrast, either CD4(+) TCC from these patients or CD8(+) TCC from the intraocular fluid of HLA-A2(+) patients with uveitis associated with Behcet's disease or HTLV-1 uveitis did not show this cytotoxicity. The results demonstrate that the MART-1 peptide-specific cytotoxic T lymphocytes lyse melanocytes in the eye of patients with VKH disease or SO, suggesting that these diseases are autoimmune diseases directed toward the MART-1 antigen in HLA-A2(+) patients.

Antigens, Neoplasm↗
PubMed1995

Characterization of the functional specificity of a cloned T-cell receptor heterodimer recognizing the MART-1 melanoma antigen.

T cells can play a central role in the immune response to cancer, with tumor-specific T-lymphocyte reactivity provided by the T-cell receptor (TCR) alpha and beta chain heterodimer. This study is the first report of the definitive identification and characterization of a functional tumor-associated, antigen-specific TCR by reconstitution in an alternate cell line. Jurkat T cells were transfected with the cDNAs encoding the full-length alpha and beta T-cell receptor chains from the HLA-A2 restricted, melanoma-reactive T-cell clone, clone 5. Expression of the transfected TCR was evaluated by immunofluorescence after down-modulation of the endogenous receptor with Jurkat T-cell receptor beta chain-specific mAb. Jurkat clone 5 TCR+ cells recognized MART-1 peptides presented by T2 cells in a pattern and sensitivity equivalent to native MART-1-reactive T-cells. Recognition of HLA-A2+ melanoma cell lines by the Jurkat clone 5 TCR+ cells, however, did not occur without the addition of exogenous MART-1 peptide. The cloning and expression of functional TCR genes which are capable of specifically recognizing MART-1 antigen provides reagents which could be used for the study of the mechanisms of T-cell/tumor antigen interactions and creates immortalized reagents which can facilitate studies requiring detection of the MART-1 antigen. The tumor reactivity provided by these genes could also have application in novel immunotherapeutic strategies for treating patients with melanoma, including redirection of tumor-infiltrating lymphocyte specificity and bone marrow stem cell therapy.

Amino Acid Sequence↗
PubMed1995

Generation of tumor-specific CTLs from melanoma patients by using peripheral blood stimulated with allogeneic melanoma tumor cell lines. Fine specificity and MART-1 melanoma antigen recognition.

PBLs were isolated from 13 patients with metastatic melanoma. Mixed lymphocyte tumor cell cultures (ML TCs) were established (15 times) by using irradiated HLA-matched (one class I locus) allogeneic melanoma tumor cell lines (13 times) or autologous melanoma tumor cell lines (two times) in medium containing 120 IU/ml IL-2 and 100 IU/ml IL-4. PBLs grew to levels that could be assessed for functional reactivity 9 of 15 times. In seven of nine cases, CD3+CD8+ CTLs grew from MLTCs that were tumor specific; five were restricted by HLA-A2 and two were restricted by HLA-A24. Four of the tumor-specific CTL lines lysed autologous fresh tumor cells. Tumor-specific CTLs from two of three patients had cytolytic activity identical with tumor-infiltrating lymphocytes (TIL) derived from tumor biopsies removed earlier and grown in high concentrations (6000 IU/ml) of IL-2. Three of the HLA-A2-restricted tumor-specific CTLs were shown to recognize 293 cells transfected with HLA-A2.1 cDNA and the gene encoding the melanoma Ag, MART-1. In addition, these CTLs recognized the T2 cell line pulsed exogenously with the peptide MART-1(27-35), which is the nine-amino acid immunodominant epitope of the MART-1 Ag recognized on melanoma tumor cells by nearly all HLA-A2-restricted TIL. Thus, we have demonstrated the ability to generate tumor-specific CTLs from PBLs that are similar in their reactivity to TIL. This technique obviates the need for autologous tumor tissue and suggests that PBLs contain sufficient CTL precursors for use in generating antitumor CTLs for cellular immunotherapy trials.

Amino Acid Sequence↗
PubMed1996

Suboptimal activation of melanoma infiltrating lymphocytes (TIL) due to low avidity of TCR/MHC-tumor peptide interactions.

Coculture of melanoma cells and T cell clones derived from tumor-infiltrating lymphocytes (TIL) generally results in lysis of the antigen-bearing tumor cells but to inefficient proliferation and IL-2 secretion by responder T cells. This suboptimal activation is classically explained by an inability of tumor cells to provide costimulatory signals. Here we analyzed the responses to synthetic peptides of HLA-A2.1-restricted CTL clones specific for melanoma antigens MART-1 and NA17-A. We showed that peptide concentrations ranging from 1 pM to 10 nM efficiently sensitized the peptide transporter-deficient T2 cells to lysis. T2 cells pulsed with melanoma peptides also induced TIL proliferation and detectable secretion of IL-2, IFN-gamma and GM-CSF, but only for peptide concentrations 10- to 10,000-fold higher than those required for lysis. Hence this suggests that partial triggering of TIL clones by melanoma cells could be due to expression of appropriate MHC-peptide complexes at subthreshold levels. In support of this, we showed that melanoma cells, unable to trigger IL-2 secretion, developed this ability when incubated with the appropriate peptide. These results indicate that the level of antigens expressed on melanoma tumors critically affects TIL activation status and thus, the efficiency of specific immune reactions mediated by these cells.

Antigens, Neoplasm↗
PubMed1995

Quantitative correlation between HLA class I allele expression and recognition of melanoma cells by antigen-specific cytotoxic T lymphocytes.

MHC class I antigen expression is necessary for CD8+ T-cell-mediated recognition of tumors. Recently, several mechanisms leading to loss or decreased expression of MHC antigens on the tumor cell surface have been described that may account for tumor escape from immune recognition. It is yet unknown whether tumor recognition by CTL occurs at a threshold amount of MHC molecules or correlates with the level of HLA-allele expression. In this study, a model was developed in which clones derived from the 624-MEL melanoma cell line and expressing varying amounts of HLA-A2 molecules were lysed in a standard 51Cr release assay by an HLA-A2-restricted CTL clone (A42) or a bulk culture of tumor-infiltrating lymphocytes. The A42 clone and the tumor-infiltrating lymphocyte culture were characterized previously as specifically recognizing the melanoma antigen MART-1(27-35) peptide. A marked heterogeneity in the susceptibility to lysis by A42 was observed in tumor clones and was not due to heterogeneous expression of MART-1 by the clones or loss of accessory molecules involved in the lymphocyte-target interaction. Lysis by A42 and by the tumor-infiltrating lymphocyte culture significantly correlated with the level of HLA-A2 expression, evaluated as mean channel number of fluorescence by flow cytometry (P < 0.001). Transfection of an HLA-A2-negative clone (624.28) with the HLA-A2.1 gene produced a panel of clones expressing different levels of HLA-A2, the lysis of which was highly correlated with the expression of HLA-A2 (P < 0.001). The addition of exogenous MART-1(27-35) peptide enhanced lysis of clones expressing intermediate amounts of HLA-A2 but did not affect clones with high expression. These data suggest that the number of HLA molecules present on the surface of tumor cells can quantitatively affect their lysis by CTL in situations with borderline amounts of peptide and/or MHC.

Alleles↗
PubMed1996

Identification of differentially expressed messenger RNAs in human melanocytes and melanoma cells.

The phenotype of malignant lesions is a reflection of genetic events altering the RNA and protein expression patterns of normal cells. We have investigated RNA expression patterns distinguishing normal melanocytes (FM 902), a primary melanoma cell line (WM 793), and its variant cell line (1205-LU), selected for metastatic phenotype in athymic mice. Using mRNA differential display, we identified 42 different cDNA PCR products with cell line-specific expression patterns. Direct sequence analysis matched approximately 50% of the cDNA PCR products with gene sequences accessible in DNA databases. Among the known genes, two functionally distinct groups were recognized: (a) genes encoding ribosomal and mitochondrial proteins that were predominantly up-regulated in the malignant cells; and (b) genes encoding modulators of the immune response. Among the immunomodulators, the T-cell antigen MART-1 and the protease inhibitor alpha2-macroglobulin were detected in the melanocyte cell line but not in the tumor cells. By contrast, mRNAs for the complement inhibitor CD59 and the cytokine IL-1beta were found to be overexpressed in the malignant melanoma cells. RNA slot blot hybridization on a larger panel of melanocyte and melanoma cell lines confirmed differential expression of 15 of 42 genes including MART-1, alpha2-macroglobulin, and CD59. This molecular screening approach identified also three partially characterized and three novel sequences with differential expression patterns in normal and malignant melanocytes.

Animals↗
PubMed1994

Identification of the immunodominant peptides of the MART-1 human melanoma antigen recognized by the majority of HLA-A2-restricted tumor infiltrating lymphocytes.

Four melanoma proteins, MART-1, gp100, tyrosinase, and tyrosinase-related protein-1 (gp75) were evaluated for recognition by HLA-A2-restricted melanoma-specific cytotoxic T lymphocytes (CTLs) derived from the tumor-infiltrating lymphocytes (TIL) of 10 different patients. 9 of 10 TIL recognized MART-1, 4 recognized gp100 (including 3 that also recognized MART-1), but none of the TIL recognized tyrosinase or gp75. Based on the known HLA-A2.1 peptide binding motifs, 23 peptides from MART-1 were synthesized in an attempt to identify the epitopes recognized by TIL. Three peptides were recognized by TIL when pulsed on T2 target cells. One of the 9-mer peptides, AAGIGILTV, was most effective in sensitizing the T2 cells for TIL lysis. This peptide was recognized by 9 of 10 HLA-A2-restricted melanoma-specific CTLs. Therefore, this peptide appears to be a very common immunogenic epitope for HLA-A2-restricted melanoma-specific TIL and may be useful for the development of immunotherapeutic strategies.

Amino Acid Sequence↗
PubMed1994

Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells infiltrating into tumor.

By cDNA expression cloning we have isolated a gene encoding a shared human melanoma antigen recognized by HLA-A2 restricted autologous and allogenic tumor-infiltrating lymphocytes (TILs) from patients with metastatic melanoma. By using both transient and stable expression systems, transfection of this gene into non-antigen-expressing HLA-A2+ cell lines resulted in recognition by the antigen-specific TILs. The sequence of this cDNA revealed a previously undescribed putative transmembrane protein whose expression was restricted to melanoma and melanocyte cell lines and human retina but no other fresh or cultured normal tissues tested or other tumor histologies. Thus, we have identified a gene encoding a melanocyte lineage-specific protein (MART-1; melanoma antigen recognized by T cells 1) that is a widely shared melanoma antigen recognized by the T lymphocytes of patients with established malignancy. Identification of this gene opens possibilities for the development of immunotherapies for patients with melanoma.

Amino Acid Sequence↗
PubMed1996

Antigen-specific tumor vaccines. Development and characterization of recombinant adenoviruses encoding MART1 or gp100 for cancer therapy.

The human melanoma tumor Ags, MART1 and gp100, are specifically recognized by HLA-A2-restricted CD8+ CTLs derived from melanoma patients and appear to be involved in tumor regression. In order to develop immunizing vectors for the treatment of patients with metastatic melanoma, replication-defective recombinant adenoviruses, Ad2CMV-MART1 and Ad2CMV-gp100, which encode these tumor Ags, have been generated. Infection of non-Ag expressing HLA-A2+ cell lines A375 and MDA-231 with the vectors resulted in recognition by Ag-specific CTLs as demonstrated by specific target cell lysis and release of cytokines, including IFN-gamma, TNF-alpha, and granulocyte-macrophage-CSF. Sodium butyrate and TNF-alpha can further augment adenovirus-mediated transgene expression and increase recognition by specific CTLs. Although adenovirus-infected cells expressed the E3/19K protein at detectable levels, significant reduction of surface MHC class I expression was observed in only 3 of 10 tumor cell lines infected with either Ad2CMV-MART1 or Ad2CMV-gp100. Because of the suspected homology between the human MART1 and gp100 genes and their murine counterparts, we immunized C57BL/6 mice with these recombinant adenoviruses and demonstrated that immunization with Ad2CMV-gp100 could protect mice from murine melanoma B16 challenge administered intradermally. Depletion of CD8+ but not CD4+ T cells in vivo from Ad2CMV-gp100-vaccinated mice eliminated the protective effect. The anti-gp100 T cells induced by Ad2CMV-gp100 vaccinated appeared to be responsible for the protection. Thus, these recombinant adenoviruses encoding tumor Ags may be useful as vaccines to induce specific T cell immunity for cancer therapy.

Adenoviridae↗
PubMed1995

A human melanoma cell line transduced with an interleukin-4 gene by a retroviral vector releases biologically active IL-4 and maintains the original tumor antigenic phenotype.

Experimental models of vaccination with tumor cells engineered to produce interleukin-4 (IL-4) have shown that the local release of this cytokine is associated with the development of antitumor immunity that may induce regression of established cancer. The aim of this study was to transduce a human melanoma cell line with the gene coding for human IL-4, and to analyze cytokine production, phenotypic characteristics, and antigen expression after transduction. A retroviral vector, constructed by inserting IL-4 cDNA into the LXSN vector, was used to infect the human melanoma cell line Me14932, known to express the MHC class I HLA-A2 and the melanoma-associated antigen Melan-A/MART-1, recognized by HLA-A2-restricted T-cells. The confluence of all G418-resistant cells (Me14932/IL-4) was then analyzed for proviral integration and IL-4 mRNA expression. Substantially stable IL-4 release was detected by ELISA in the supernatant of transduced cells, ranging from 1.6 to 4.6 ng/ml per 10(5) cells per 24 hr; such a cytokine displayed a specific biologic activity, as revealed by the stimulation of blast cell proliferation and the inhibition of lymphokine activated killer cell (LAK) induction by IL-2. After 200 Gy irradiation, IL-4 release remained detectable for 5 weeks, whereas cell proliferation ceased within 7 days. Morphology and immunophenotypic characteristics of the parental cell line (expression of MHC classes I and II, ICAM-1, LFA 3, melanoma-associated antigens, etc.) were retained by the IL-4 gene-transduced melanoma as assayed by microscopy and immunofluorescence; likewise, susceptibility to lysis by LAK cells as well as a T-cell clone recognizing the Melan-A/MART-1 antigen did not change. These results, together with the lack of replication-competent retrovirus, suggest that the Me14932/IL-4 cell line displays suitable characteristics for its use in the treatment of HLA-matched melanoma patients.

Antigens, Neoplasm↗
PubMed1996

Inverse relationship of melanocyte differentiation antigen expression in melanoma tissues and CD8+ cytotoxic-T-cell responses: evidence for immunoselection of antigen-loss variants in vivo.

Antigenic peptides derived from differentiation antigens of the melanocyte lineage were recently identified in human melanomas as targets for MHC-restricted cytotoxic T lymphocytes (CTL). CTL directed against peptides derived from the Melan A/MART-1, tyrosinase and gp100/Pmel17 antigens can be detected in melanoma patients and in healthy controls. The presence of defined antigenic peptides and corresponding precursor CTL in patients with metastatic melanoma opens perspectives for the development of antigen-specific tumor vaccines. In this study, we examined the expression of Melan A/MART-1, tyrosinase and gp100lPmel17 in fresh melanoma tissues of HLA-A2+ patients and the spontaneous CTL reactivity against antigenic peptides derived from these antigens. Our results demonstrate an inverse correlation of antigen expression and CTL response to Melan A/MART-1 and tyrosinase in patients with metastatic melanoma. In 2 patients with advanced disease, CTL responses against Melan A/MART-1 and tyrosinase were induced by intradermal immunization with synthetic nona- or deca-peptides derived from these antigens. Metastases increasing in size over time showed a loss of Melan A/MART-1 expression in the presence of CTL in one patient. The regression of a metastasis with persistent tyrosinase expression was observed in the other patient after the induction of CTL, reactive against tyrosinase. We conclude that CTL responses against melanocyte differentiation antigens may mediate regression of antigen-positive tumors and select for antigen-loss variants in vivo.

Amino Acid Sequence↗
PubMed1996

Serological analysis of Melan-A(MART-1), a melanocyte-specific protein homogeneously expressed in human melanomas.

Recent progress in the structural identification of human melanoma antigens recognized by autologous cytotoxic T cells has led to the recognition of a new melanocyte differentiation antigen, Melan-A(MART-1). To determine the properties of the Melan-A gene product, Melan-A recombinant protein was produced in Escherichia coli and used to generate mouse monoclonal antibodies (mAbs). Two prototype mAbs, A103 and A355, were selected for detailed study. Immunoblotting results with A103 showed a 20-22-kDa doublet In Melan-A mRNA positive melanoma cell lines and no reactivity with Melan-A mRNA-negative cell lines. A355, in addition to the 20-22-kDa doublet, recognized several other protein species in Melan-A mRNA-positive cell lines. Immunocytochemical assays on cultured melanoma cells showed specific and uniform cytoplasmic staining in Melan-A mRNA-positive cell lines. Immunohistochemical analysis of normal human tissues with both mAbs showed staining of adult melanocytes and no reactivity with the other normal tissues tested. Analysis of 21 melanoma specimens showed homogenous staining of tumor cell cytoplasm in 16 of 17 Melan-A mRNA-positive cases and no reactivity with the three Melan-A mRNA-negative cases.

Animals↗
PubMed1996

New treatment options for patients with melanoma: review of melanoma-derived T-cell epitope-based peptide vaccines.

Human melanoma represents the principal cause of death in patients with skin cancer in the United States and Europe. Tumour infiltrating lymphocytes recognizing melanoma have been used to identify the tumour antigens recognized by T-cells in the context of MHC class I or class II molecules. Such antigens include MAGE-1, MAGE-3, MART-1/Melan-A, gp100, tyrosinase, the tyrosinase-related antigen gp75, the antigen gp15 and the mutated CDK4 and beta-catenin gene-products. The identification of these T-cell epitopes provides us with novel reagents for the development of state-of-the-art treatments and for the (immuno-)monitoring of patients with melanoma. In order for treatments, including peptide-based vaccines, to be successful, several conceptual criteria must be met: (1) The patient's tumour must present the relevant epitope(s) integrated into the vaccine, (2) the tumour should express the appropriate restricting major histocompatibility complex (MHC) molecule(s) required for patient cytotoxic T lymphocyte (CTL) reactivity, and (3) the patient's T-cell repertoire should be able to react productively against the melanoma antigens present in the vaccine. Clinical trials implementing peptide-based vaccines or whole protein therapies have been initiated in the United States and Europe. We suggest that such treatments should include the careful monitoring of anti-tumour T-cell responses. This should include examination of melanoma antigen and MHC class I allele expression in the individual patient's tumour, assessment of the status of the peptide transporter molecules TAP1/TAP2 and evaluation of T-cell mediated immune responses reactive against peptides and autologous melanoma. Evaluation of clinical parameters (such as disease-free survival) in conjunction with an examination of immunological parameters may facilitate our understanding of the immune responses against T-cell antigens that are shared among melanoma and normal melanocytes, and may ultimately help to identify the most effective immunotherapy for patients with melanoma.

Amino Acid Sequence↗
PubMed1994

Identification of MART-1-specific T-cell receptors: T cells utilizing distinct T-cell receptor variable and joining regions recognize the same tumor epitope.

Tumor-specific cytotoxic T lymphocytes (CTLs) can mediate tumor regression in patients with metastatic melanoma and play a central role in the immune response to cancer. The recent identification of shared melanoma antigens has raised the possibility of a limited melanoma-specific T-cell receptor (TCR) repertoire, but subsequent studies have been controversial and difficult to interpret without knowing which tumor-associated antigens (TAAs) are being recognized by specific TCRs. However, the recent cloning of several melanoma TAAs now allows for the identification of the specifically recognized TAA and its epitope. We evaluated the TCR of two clonal CD8+ CTL lines, A42 and 1E2, from two HLA-A2+ patients with metastatic melanoma. Both CTL lines were MART-1 specific, and both demonstrate reactivity to the same epitope when presented in an HLA-A2.1 context. The TCR genes of the two clones were sequenced. All of the productively rearranged A42 TCR beta chain genes were V beta 7/D beta 2.1/J beta 2.7/C beta 2; the TCR alpha chain genes were V alpha 21/J alpha 42/C alpha. The 1E2 TCR beta chain genes were V beta 3/D beta 1.1/J beta 1.1/C beta 1, and TCR alpha chains were V alpha 25/J alpha 54/C alpha. This study is the first report of TCR sequences specific for a melanoma epitope. These TCR clones may be useful for the development of more effective immunotherapies and in studies of the mechanism of T-cell recognition of tumor antigen. They also provide direct evidence that the immune system can provide more than one TCR capable of recognizing a TAA epitope.

Antigens, Neoplasm↗
PubMed1995

Recognition of multiple epitopes in the human melanoma antigen gp100 by tumor-infiltrating T lymphocytes associated with in vivo tumor regression.

Four of ten HLA-A2-restricted melanoma specific CTL that were derived from tumor-infiltrating lymphocytes (TIL) and administered to patients recognized the gp100 melanoma Ag and nine of ten recognized the MART-1 Ag. Adoptive transfer of the four gp100-reactive CTL, but not the other TIL, resulted in tumor regression when infused into autologous patients along with IL-2. Tumor regression was thus correlated with the recognition of gp100 by the administered T cells (p = 0.0048). To identify the epitopes recognized by these four gp100-reactive CTL, 169 peptides containing HLA-A2.1 binding motifs were synthesized and screened for their recognition by TIL using cytotoxicity and IFN-gamma release assays. Five gp100 epitopes (two for TIL620, three for TIL660, one for TIL1143, and two for TIL1200) were recognized by CTL derived from different patients. Five of eight HLA-A2 binding melanoma epitopes (five gp100, one MART-1/Melan-A, two tyrosinase) had intermediate binding affinity to HLA-A2.1. These gp100 epitopes may be responsible for mediating tumor rejection in vivo and thus may be useful for the development of immunotherapies for patients with melanoma.

Amino Acid Sequence↗
PubMed1995

Peptide-specific CTL in tumor infiltrating lymphocytes from metastatic melanomas expressing MART-1/Melan-A, gp100 and Tyrosinase genes: a study in an unselected group of HLA-A2.1-positive patients.

Peptide specificity of cultured tumor-infiltrating lymphocytes (TIL) was systematically investigated in a group of HLA-A2.1+ metastatic melanoma patients consecutively referred to our department for surgical treatment. Seven samples from 6 patients were studied. All surgical specimens showed evidence of gp 100, MART-1/Melan-A and Tyrosinase gene expression as detectable by reverse PCR (rPCR). Cultured TIL from 2 patients displayed cytotoxic activity against autologous or HLA-matched EBV-transformed cells previously pulsed with MART-1/Melan-A27-35 peptide. In contrast, no CTL activity against gp100(280-288) or tyrosinase1-9 peptides could be observed. TIL were then repeatedly stimulated in vitro with the same peptides. After 6 restimulation courses at weekly intervals, specific recognition of gp100(280-288) and MART-1/Melan-A peptides was detectable in 3 and 5 TIL populations, respectively. In one case Tyrosinase1-9-specific CTL could be demonstrated. Two TIL populations from metastases resected from a melanoma patient at 6 months' distance showed a different peptide specificity pattern, and no specific CTL could be generated from simultaneously sampled peripheral blood mononuclear cells (PBMC). All peptide-specific CTL populations also displayed significant cytotoxic activity against HLA-A2.1 matched melanoma cell lines expressing the antigens under investigation. Our data indicate that CTL specific for MART-Melan-A27-35, gp100(280-288) or Tyrosinase1-9 peptides could be expanded with varying frequency from TIL derived from 4 out of 6 HLA-A2.1+ patients whose tumors expressed the genes encoding these tumor-associated antigens (TAA).

Adult↗