Published papers

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-127-35 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-127-35 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.

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-γ 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.

Cytotoxic T lymphocytes (CTL) reactive with human melanoma tumor cells occasionally display cross-reactivity with normal melanocytes. Previously, we identified the melanocyte lineage-specific antigen gp100 that is expressed by both melanoma cells and normal melanocytes, as a target antigen for tumor-infiltrating lymphocytes derived from a melanoma patient (TIL 1200). Here, we demonstrate that the oligoclonal HLA-A2.1-restricted TIL 1200 line is reactive with 2 distinct peptides derived from the gp100 protein. Apart from the peptide corresponding to gp100 amino acids 457-466, we identified the gp100 peptide 154-162 as a second epitope recognized by TIL 1200. A 100-fold lower concentration of this novel gp100 peptide was required for target-cell sensitization compared to peptide 457-466, indicating that the 154-162 peptide is the dominant gp100 epitope for TIL 1200. Together with the recently described gp100 280-288 epitope, 3 distinct CTL epitopes have now been identified in gp100, all presented in the context of HLA-A2.1. Therefore, gp100 is an attractive target antigen in the development of immune-therapeutic protocols against melanoma.

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-127-35, 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.

A number of Ags recognized by class I-restricted, melanoma-specific T cells have recently been identified. In this report we demonstrated that tumor-infiltrating lymphocytes (TIL) from melanoma patient 1413 recognize a tumor Ag, tyrosinase, in the context of HLA-A24. This Ag had previously been shown to be recognized by an HLA-A24-restricted TIL, TIL 888, as well as HLA- A2-restricted, melanoma-specific T cells isolated from two additional patients. The peptide epitope recognized by TIL 1413 was then identified through the use of sequential deletions of the tyrosinase cDNA, as well as through prediction of HLA-A24 binding peptides based on a previously identified motif. Two peptides, a 9-amino acid peptide (AFLPWHRLF) and an overlapping 10-amino acid peptide (AFLPWHRLFL) containing an additional leucine at the carboxyl terminus, were both recognized by TIL 1413. Anti- peptide-specific CTL could be induced by repeated stimulation of peripheral blood lymphocytes from melanoma patient 1413, and this CTL line specifically recognized both HLA-A24+ B cell lines pulsed with the peptide and HLA-A24+ tyrosinase+ melanoma cells. This peptide thus represents a reagent that may be used to generate melanoma-specific T cells for adoptive immunotherapy, as well as in peptide vaccines for HLA-A24+ melanoma patients.

Four often 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-γ 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 gpl 00, 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 devel opment of immunotherapies for patients with melanoma.

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) α and β 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 α and β 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 β 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.

Tumor-infiltrating lymphocytes (TILs) were grown from four distinct anatomic sites from a patient with metastatic melanoma. The metastatic sites included a tumor-involved lymph node, a subcutaneous lesion obtained from the chest wall, a portion of bowel, and adrenal gland. TILs grown from each anatomic site over the course of 20 days in the presence of 6,000 IU/ml recombinant interleukin-2 exhibited comparable growth rates. Between days 30 and 45, the TILs were a mixture of CD3+CD4+ and CD3+CD8+ lymphocytes expressing the αβ form of the T-cell receptor. TILs derived from each anatomic site specifically lysed autologous tumor obtained from all four anatomic sites. In fine specificity analysis, the TILs exhibited human leukocyte antigen (HLA-A2)-restricted lysis of fresh tumor targets and cultured melanoma cell lines. Each TIL recognized a product of the MART-1 gene, and specifically, the monomer peptide MART-127-35. Thus lymphocytes reactive with the MART-1 melanoma antigen appeared to be widely distributed in diverse metastases in this patient. This information, along with previous data on the reactivity of multiple patients to this antigen, attests to its dominance in the immune reactivity of humans to melanoma.

gplOO is a melanocyte lineage-specific antigen recognized by tumor-infiltrating lymphocytes whose adoptive transfer has been associated with tumor regression in patients with metastatic melanoma. The peripheral blood mononuclear cells of five melanoma patients were sensitized in vitro with synthetic peptides to elicit antigen-specific cytotoxic T lymphocyte (CTL) lines against four gplOO epitopes. These epitope-specific CTL lines were generated following weekly in vitro stimulation with the synthetic decamer G10476 (V-L-Y-R-Y-G-S-F-S-V) or the nonamers G9280 (Y-L-E-P-G-P-V-T-A), G9154 (K-T-W-G-Q-Y-W-Q-V), or G9209 (I-T-D-Q-V-P-F-S-V) pulsed onto autologous irradiated peripheral blood mononuclear cells. These lines grew as long as 4 months in culture in low-dose interleukin 2 (30 IU/ml) and exhibited antigen-specific, MHC class I-restricted lysis of peptide-pulsed T2 cells and HLA-A2+, gplOO+ established melanoma cell lines. G10476- and G9280-specific CTLs demonstrated specific release of granulocyte-macrophage-colony-stimnlating factor and tumor necrosis factor a in response to T2 cells pulsed with relevant peptide, as well as to gplOO+ melanoma cell lines. These results demonstrate that several peptides derived from the gplOO protein are presented on the surface of melanoma cells and are sufficiently immunogenic to generate, in vitro, potent CTLs capable of cytolysis and the secretion of cytokines. Therefore, for HLA-A2+ melanoma patients, these and possibly other gplOO peptides could represent good candidates for antigen-specific immunotherapy either singly or in a multivalent regimen.

New immunotherapies using cancer antigens recognized by T-cells have recently been developed and a variety of phase I clinical trals has been conducted in the US and Europe. An anti-tumor effect has already been observed in some protocols. However, many questions still remain to establish effective immunotherapies for cancer. Key points which were raised in the research on immunotherapies against melanoma are discussed in this article.

Adoptive transfer of cultured tumor infiltrating T-lymphocytes (TIL) with interleukin 2 (IL2) resulted in tumor regression in melanoma patients, indicating the important role of T-cells in mediating in vivo melanoma regression. TIL secrete cytokines and lyse tumor cells in vitro by recognizing autologous melanoma. Using cDNA expression cloning methods with these TIL, cDNAs encoding melanoma antigens including 5 melanosomal proteins (MART-1, gp100, tyrosinase, TRP1, and TRP2), p15 and β-catenin, were identified. Non-mutated and mutated T cell epitopes have also been identified. A variety of mechanisms to generate these T cell epitopes on melanoma has been identified including peptides derived from regular open reading frames (ORFs), an alternative ORF, introns, or sequences with a mutation. Some melanoma epitopes had relatively low major histocompatibility complex (MHC) binding affinities, suggesting that these were subdominant self epitopes. Several MART-1 and gp100 epitopes were recognized by TIL from many patients, suggesting that these were immunodominant common epitopes. By in vitro stimulation with these identified epitopes, melanoma reactive cytotoxic T lymphocytes (CTL) could be efficiently induced from peripheral blood lymphocytes (PBL) or TIL of melanoma patients. Since adoptive transfer of the TIL reacting to these melanoma antigens into autologous patients with IL2 resulted in tumor regression, these peptides may function as tumor rejection antigens. Tumor regression correlated with vitiligo development after IL2 based immunotherapies in melanoma indicating that these epitopes may also be present on the surface of melanocytes in vivo and that autoreactive T-cells may be involved in the tumor regression. A variety of Phase I clinical trials including immunization with the MART-1 and gp100 peptides, or recombinant viruses containing the MART-1 or gp100 gene, with or without administration of cytokines such as IL2, granulocyte macrophagecolony stimulating factor (GM-CSF), IL12, is being conducted in the Surgery Branch of the National Cancer Institute (NCI), and tumor regression has been observed in some patients.

The cloning of the genes encoding cancer antigens has opened new possibilities for the treatment of patients with cancer. In this study, immunodominant peptides from the gp100 melanoma-associated antigen were identified, and a synthetic peptide, designed to increase binding to HLA-A2 molecules, was used as a cancer vaccine to treat patients with metastatic melanoma. On the basis of immunologic assays, 91% of patients could be successfully immunized with this synthetic peptide, and 13 of 31 patients (42%) receiving the peptide vaccine plus IL-2 had objective cancer responses, and four additional patients had mixed or minor responses. Synthetic peptide vaccines based on the genes encoding cancer antigens hold promise for the development of novel cancer immunotherapies.

Clinical observations in the interleukin (IL) 2-based immunotherapies suggest that T cells play a central role in the rejection of melanoma. Using cDNA expression cloning, we have isolated genes encoding melanoma antigens recognized by tumor-infiltrating T lymphocytes. These antigens are categorized as (a) melanocyte-specific melanosomal proteins (MART-1/melan A, gp100, tyrosinase, TRP-1, and TRP-2), (b) tumor-specific mutated proteins (β-catenin), and (c) others (p15). A variety of mechanisms has been identified for the generation of T cell epitopes on tumor cells. Some of the HLA-A2 binding epitopes from the melanosomal antigens appear to be subdominant self-determinants with relatively low major histocompatibility complex binding affinity. The effectiveness of adoptive transfer into patients of cytotoxic T lymphocytes recognizing the melanosomal antigens, the significant correlation between vitiligo development and clinical response in patients receiving IL-2-based immunotherapies, and the sporadic tumor regressions observed in some patients following immunization with the MART-1 or gp100 peptides in incomplete Freund's adjuvant or recombinant viruses expressing the MART-1 antigen suggest that these epitopes may represent tumor rejection antigens. Phase I immunization trials using peptides or recombinant viruses containing genes encoding the melanosomal antigens MART- 1 or gp100, with or without co-administration of cytokines such as IL-2, IL-12, or granulocyte-macrophage colony-stimulating factor, are being conducted in the Surgery Branch of the National Cancer Institute. These studies may demonstrate the feasibility of using melanosomal proteins for the immunotherapy of patients with melanoma.

Melanoma is a relatively immunogenic cancer and a good model to evaluate the possibilities of immunotherapies. Recent progress of molecular biology and immunology has allowed us to understand immune responses to human cancer at the molecular level. Although antigens for CD4+ T cells have not well been identified, various melanoma antigens recognized by CD8+ T cells have been identified, including tissue specific proteins (melanosomal proteins), proteins expressed in testis and a variety of cancers (Cancer-Testis antigens) and tumor specific mutated proteins. The immunological features of these antigens have been characterized for development of new immunotherapies. Based on these findings, antigen specific immunotherapies have been developed. Some of the phase I clinical trials with the identified melanoma antigens demonstrated anti-tumor effects. Immunization with the modified gp100 epitope that had high MHC binding affinity, in conjunction with incomplete freund adjuvant and high-dose IL-2, resulted in a 42 % response rate (CR+PR). Immunization with dendritic cells pulsed with tumor lysates or epitope peptides resulted in a 31 % response rate in metastatic melanoma. These immunotherapies need to be improved through modifications and better understanding of tumor escape mechanisms.