Details of a Researcher - Kawakami, Yutaka

Papers - Kawakami, Yutaka

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

Cole D. J., Weil D. P., Shilyansky J., Custer M., Kawakami Y., Rosenberg S. A., Nishimura M. I.

Cancer Research 55 ( 4 ) 748 - 752 1995

ISSN 0008-5472

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.

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

Kawakami Yutaka, Eliyahu Siona, Jennings Christopher, Sakaguchi Kazuyasu, Kang Xiaoqiang, Southwood Scott, Robbins Paul F., Sette Alessandro, Appella Ettore, Rosenberg Steven A.

Journal of Immunology 154 ( 8 ) 3969 - 3974 1995

ISSN 0022-1767

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.

Identification of a tyrosinase epitope recognized by HLA-A24-restricted, tumor-infiltrating lymphocytes

Kang X., Kawakami Y., El-Gamil M., Wang R., Sakaguchi K., Yannelli J. R., Appella E., Rosenberg S. A., Robbins P. F.

Journal of Immunology 155 ( 3 ) 1343 - 1348 1995

ISSN 0022-1767

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.

Generation of tumor-specific CTLs from melanoma patients by using peripheral blood stimulated with allogeneic melanoma tumor cell lines

Stevens E. J., Jacknin L., Robbins P. F., Kawakami Y., El Gamil M., Rosenberg S. A., Yannelli J. R.

Journal of Immunology 154 ( 2 ) 762 - 771 1995

ISSN 0022-1767

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.

Identification of a novel peptide derived from the melanocyte-specific gp100 antigen as the dominant epitope recognized by an HLA-A2.1-restricted anti-melanoma CTL line

Bakker A. B H, Schreurs M. W J, Tafazzul G., De Boer A. J., Kawakami Y., Adema G. J., Figdor C. G.

International Journal of Cancer 62 ( 1 ) 97 - 102 1995

ISSN 0020-7136

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.

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

Kawakami Y., Eliyahu S., Jennings C., Sakaguchi K., Kang X., Southwood S., Robbins P. F., Sette A., Appella E., Rosenberg S. A.

Journal of Immunology 154 ( 8 ) 3961 - 3968 1995

ISSN 0022-1767

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.

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

Rivoltini L., Barracchini K. C., Viggiano V., Kawakami Y., Smith A., Mixon A., Restifo N. P., Topalian S. L., Simonis T. B., Rosenberg S. A., Marincola F. M.

Cancer Research 55 ( 14 ) 3149 - 3157 1995

ISSN 0008-5472

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.

Cloning of a new gene encoding an antigen recognized by melanoma-specific HLA-A24-restricted tumor-infiltrating lymphocytes

Robbins P. F., El-Gamil M., Li Y. F., Topalian S. L., Rivoltini L., Sakaguchi K., Appella E., Kawakami Y., Rosenberg S. A.

Journal of Immunology 154 ( 11 ) 5944 - 5950 1995

ISSN 0022-1767

The role of tumor-specific T cells in mediating the regression of metastatic melanoma has been suggested by the clinical response of patients to treatment with tumor infiltrating lymphocytes (TIL). A number of Ags recognized by class I-restricted melanoma-specific T cells have recently been isolated, raising the hope that this will lead to the development of improved therapies. In this study, we report the cloning of a tumor Ag recognized by T cells from melanoma patient 888. Previously, we reported that TIL 888, grown from the tumor of this patient, recognized tyrosinase in an HLA-A24- restricted fashion. This line, when infused into the autologous patient, resulted in complete regression of multiple metastases. Three years later, a second TIL line, TIL 1290, was isolated from a recurrent pelvic tumor. Infusion of a mixture of TIL 888 and TIL 1290 cell lines into the patient resulted in complete regression of a residual abdominal mass and the patient remains disease-free 2 yr later. The TIL 1290 cell line, which recognized melanoma in an HLA-A24-restricted manner, failed to recognize tyrosinase. TIL 1290 was then used to screen an 888 melanoma cDNA library, and air Ag was isolated that did not correspond to any found in sequence databases. This gene, termed p15, was found to be expressed in a variety of normal tissues, and a peptide epitope recognized by TIL 1290 was found to represent the product of an nonmutated gene. Screening of additional cDNA pools resulted in the isolation of a second clone which stimulated TIL 1290. This clone also appeared to represent a transcript of the p15 gene, indicating that this gene may encode the predominant Ag recognized by TIL 1290.

Identification of MART-1-specific T-cell receptors

Cole David J., Weil Daniel P., Shamamian Peter, Rivoltini Licia, Kawakami Yutaka, Topalian Suzanne, Jennings Christopher, Eliyahu Siona, Rosenberg Steven A., Nishimura Michael I.

Cancer Research 54 ( 20 ) 5265 - 5268 1994.10

ISSN 0008-5472

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 β chain genes were Vβ7/Dβ2.1/Jβ2.7/Cβ2; the TCR α chain genes were Vα21/Jα42/Cα. The 1E2 TCR β chain genes were Vβ3/Dβ1.1/Jβ1.1/Cβ1, and TCR α chains were Vα25/Jα54/Cα. 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.

Human CD4⁺ T cells specifically recognize a shared melanoma-associated antigen encoded by the tyrosinase gene

Topalian Suzanne L., Rivoltini Licia, Mancini Marie, Markus Nancy R., Robbins Paul F., Kawakami Yutaka, Rosenberg Steven A.

Proceedings of the National Academy of Sciences of the United States of America 91 ( 20 ) 9461 - 9465 1994.09

ISSN 0027-8424

Although commonly expressed human melanoma-associated antigens recognized by CD8⁺ cytolytic T cells have been described, little is known about CD4⁺ T-cell recognition of melanoma-associated antigens. Epstein-Barr virus- transformed B cells were used to present antigens derived from whole cell lysates of autologous and allogeneic melanomas for recognition by melanoma- specific CD4⁺ T-cell lines and clones cultured from tumor-infiltrating lymphocytes. HLA-DR-restricted antigens were detected in the lysates on the basis of specific release of cytokines from the responding T cells. Antigen sharing was demonstrated in the majority of melanomas tested, as well as in cultured normal melanocytes, but not in other normal tissues or nonmelanoma tumors. T-cell clones manifested a single recognition pattern, suggesting the presence of an immunodominant epitope. This epitope was identified as a product of the tyrosinase gene, which has also been shown to encode class I- restricted epitopes recognized by CD8⁺ T cells from melanoma patients. Identification of commonly expressed tumor-associated protein molecules containing epitopes presented by both class I and class II major histocompatibility molecules may provide optimal reagents for cancer immunization strategies.

Identification of a human melanoma antigen recognized by tumor-infiltrating lymphocytes associated with in vivo tumor rejection

Kawakami Yutaka, Eliyahu Siona, Delgado Cynthia H., Robbins Paul F., Sakaguchi Kazuyasu, Appella Ettore, Yannelli John R., Adema Gosse J., Miki Toru, Rosenberg Steven A.

Proceedings of the National Academy of Sciences of the United States of America 91 ( 14 ) 6458 - 6462 1994.07

ISSN 0027-8424

The cultured T-cell line TIL1200, established from the tumor-infiltrating lymphocytes (TILs) of a patient with advanced metastatic melanoma, recognized an antigen on most HLA-A2⁺ melanomas and on all HLA-A2⁺ cultured neonatal melanocytes in an HLA-A2 restricted manner but not on other types of tissues or cell lines tested. A cDNA encoding an antigen recognized by TIL1200 was isolated by screening an HLA-A2⁺ breast cancer cell line transfected with an expression cDNA library prepared from an HLA-A2⁺ melanoma cell line. The nucleotide and amino acid sequences of this cDNA were almost identical to the genes encoding glycoprotein gp100 or Pme117 previously registered in the GenBank. Expression of this gene was restricted to melanoma and melanocyte cell lines and retina but was not expressed on other fresh or cultured normal tissues or other types of tumor tested. The cell line transfected with this cDNA also expressed antigen recognized by the melanoma-specific antibody HMB45 that bound to gp100. A synthetic 10-amino acid peptide derived from gp100 was recognized by TIL1200 in the context of HLA-A2.1. Since the administration of TIL1200 plus interleukin 2 resulted in regression of metastatic cancer in the autologous patient, gp100 is a possible tumor rejection antigen and may be useful for the development of immunotherapies for patients with melanoma.

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

Kawakami Yutaka, Eliyahu Siona, Sakaguchi Kazuyasu, Robbins Paul F., Rivoltini Licia, Yannelli John R., Appella Ettore, Rosenberg Steven A.

Journal of Experimental Medicine 180 ( 1 ) 347 - 352 1994.07

ISSN 0022-1007

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.

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Recognition of tyrosinase by tumor-infiltrating lymphocytes from a patient responding to immunotherapy

Robbins Paul F., El-Gamil Mona, Kawakami Yutaka, Rosenberg Steven A.

Cancer Research 54 ( 12 ) 3124 - 3126 1994.06

ISSN 0008-5472

The observation that allogeneic melanoma cells matched for particular HLA class I alleles stimulate T-cells isolated from patients suggest that widely shared antigens exist on these tumors. A transient expression system was developed for screening a melanoma complementary DNA library using the highly transfectable human kidney cell line 293. Using this system, large numbers of complementary DNA clones can be rapidly screened for the expression of antigens which stimulate T-cells. Tumor-infiltrating lymphocytes from patient 888, which recognized melanoma in the context of HLA-A24, were used to screen a complementary DNA library made from the autologous melanoma. Our results demonstrate that these tumor-infiltrating lymphocytes recognize tyrosinase, a gene previously shown to be recognized by T-cells only in the context of HLA- A2. These data demonstrate that a single antigen can be recognized in the context of two different class 1 HLA alleles. In addition, this study suggests that recognition of tyrosinase by antigen-specific T-cells may be involved in tumor rejection.

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

Kawakami Yutaka, Eliyahu Siona, Delgado Cynthia H., Robbins Paul F., Rivoltini Licia, Topalian Suzanne L., Miki Toru, Rosenberg Steven A.

Proceedings of the National Academy of Sciences of the United States of America 91 ( 9 ) 3515 - 3519 1994.04

ISSN 0027-8424

By cDNA expression cloning we have isolated a gene encoding a shared human melanoma antigen recognized by HLA-A2 restricted autologous and allogeneic 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.

T-cell receptor usage by melanoma-specific clonal and highly oligoclonal tumor-infiltrating lymphocyte lines

Shilyansky Joel, Nishimura Michael I., Yannelli John R., Kawakami Yutaka, Jacknin Laura S., Charmley Patrick, Rosenberg Steven A.

Proceedings of the National Academy of Sciences of the United States of America 91 ( 7 ) 2829 - 2833 1994.03

ISSN 0027-8424

Tumor-infiltrating lymphocytes (TIL) obtained from human melanomas can specifically lyse autologous tumor in vitro and mediate tumor regression in vivo. To develop more effective therapeutic reagents and to further understand the T-cell response to tumors, the diversity of T-cell receptors (TCRs) involved in melanoma antigen recognition has been studied. The TCR variable (V) genes, joining (J) segments, and N diversity regions used by five clonal lines and one highly oligoclonal, melanoma-specific, CD8⁺ TIL line were examined utilizing PCR amplification with V gene subfamily-specific primers and anchor PCR. The TIL lysed multiple allogeneic melanomas expressing matched surface major histocompatibility complex class I molecules. TCR analysis confirmed the clonal nature of the TIL lines; however, the TCR repertoire was diverse. Even among the three HLA-A2 restricted TIL (TIL 1200, TIL F2-2, and TIL 5), no common V gene usage was found. Comparison of the third complementarity-determining regions of the TCRs from the HLA-A2 restricted TIL revealed no homology. Results presented here identify T-cell clonotypes that recognize epitopes on highly prevalent, shared melanoma tumor-associated antigens presented in the context of HLA- 855, HLA-A1, and HLA-A2. These T cells and the antigens they recognize represent important components for the design of new immunotherapies for patients with advanced melanoma.

Melanocyte lineage-specific antigen gp100 is recognized by melanoma-derived tumor-infiltrating lymphocytes

Bakker Alexander B H, Schreurs Marco W J, De Boer Annemiek J., Kawakami Yutaka, Rosenberg Steven A., Adema Gosse J., Figdor Carl G.

Journal of Experimental Medicine 179 ( 3 ) 1005 - 1009 1994.03

ISSN 0022-1007

We recently isolated a cDNA clone that encodes the melanocyte lineage-specific antigen glycoprotein (gp)100. Antibodies directed against gp100 are an important tool in the diagnosis of human melanoma. Since the gp100 antigen is highly expressed in melanocytic cells, we investigated whether this antigen might serve as a target for antimelanoma cytotoxic T lymphocytes (CTL). Here, we demonstrate that cytotoxic tumor-infiltrating lymphocytes (TIL) derived from a melanoma patient (TIL 1200) are directed against gp100. HLA-A2.1⁺ melanoma cells are lysed by TIL from this patient. In addition, murine double transfectants, expressing both HLA-A2.1 and gp100, are lysed by TIL 1200, whereas transfectants expressing only HLA-A2.1 are not susceptible to lysis. Furthermore, the HLA-A2.1⁺ melanoma cell line BLM, which lacks gp100 expression and is resistant to lysis, becomes susceptible after transfection of gp100 cDNA. Finally, HLA-A2.1⁺ normal melanocytes are lysed by TIL 1200. These data demonstrate that the melanocyte differentiation antigen gp100 can be recognized in the context of HLA-A2.1 by CTL from a melanoma patient. Gp100 may therefore constitute a useful target for specific immunotherapy against melanoma, provided that no unacceptable cytotoxicity towards normal tissue is observed.

Recognition of neuroectodermal tumors by melanoma-specific cytotoxic T lymphocytes

Shamamian Peter, Mancini Marie, Kawakami Yutaka, Restifo Nicholas P., Rosenberg Steven A., Topalian Suzanne L.

Cancer Immunology Immunotherapy 39 ( 2 ) 73 - 83 1994.03

ISSN 0340-7004

Melanomas from different patients have been shown to express shared tumor antigens, which can be recognized in the context of the appropriate MHC class 1 molecules by cytolytic T cells. To determine if T-cell-defined melanoma antigens are expressed on other tumors of neuroectodermal origin, four melanoma-specific cytotoxic T lymphocyte (CTL) cultures derived from tumor-infiltrating lymphocytes (TIL) were tested for lysis of a panel of 23 HLA-A2⁺ neuroectodermal tumor cell lines of various histologies, including retinoblastoma (1), neuroblastoma (8), neuroepithelioma (6), astrocytoma (2), neuroglioma (1), and Ewing's sarcoma (5). Low expression of MHC class I and/or ICAM-1 molecules was found on 22 of 23 neuroectodermal tumor lines, and could be enhanced by treatment with interferon γ (IFNγ). Following IFNγ treatment, three Ewing's sarcoma lines were lysed by at least one melanoma TIL culture, and levels of lysis were comparable to melanoma lysis by these TIL. Lysis could be inhibited by monoclonal antibodies directed against MHC class I molecules and against CD3, indicating specific immune recognition of tumor-associated antigens. None of the other neuroectodermal tumors tested were lysed by TIL, but they could be lysed by non-MHC-restricted lymphokine-activated killer cells. This demonstration of immunological cross-reactivity between melanomas and Ewing's sarcomas, two tumors of distinct histological types with a common embryonic origin, has implications for the developmental nature of these CTL-defined tumor antigens. It also raises the possibility that specific antitumor immunotherapies, such as vaccines, may be reactive against more than one form of cancer.

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T-cell receptor repertoire in tumor-infiltrating lymphocytes. Analysis of melanoma-specific long-term lines

Nishimura M. I., Kawakami Y., Charmley P., O'Neil B., Shilyansky J., Yannelli J. R., Rosenberg S. A., Hood L.

Journal of Immunotherapy 16 ( 2 ) 85 - 94 1994

ISSN 1053-8550

Cytotoxic T-lymphocytes (CTLs) can be isolated from human melanoma biopsies that specifically lyse autologous melanoma in vitro and can be effective therapeutic agents for patients with advanced disease. Recent evidence indicates that HLA-A2-restricted, melanoma-specific tumor- infiltrating lymphocytes (TILs) recognize melanomas obtained from different HLA-A2⁺ patients, suggesting the presence of one or more common melanoma antigens. Furthermore, T-cell receptor (TCR) repertoire analysis by other groups of TILs from fresh melanoma biopsies suggests that there is limited TCR V gene usage in TILs. One serious limitation in analyzing the TCR repertoire in fresh tumors has been the inability to correlate TCR usage with immune function. Therefore, the TCR repertoire was determined in long-term TIL cultures that specifically lysed autologous melanoma in vitro and in many cases mediated in vivo regression of metastatic cancer in patients with advanced disease. The TCR repertoire in cultured melanoma-specific TILs was diverse, with each TIL containing an average of 9.5 ± 5.7 of the 23 Vα and 11.2 ± 5.9 of the 23 Vβ subfamilies. Despite the large diversity observed, several Vα and Vβ genes (Vα1, Vα2, Vα22, Vβ13, Vβ14, and Vβ18) are very commonly found in melanoma-specific TILs. No statistically significant associations were observed between the presence of a TCR V gene subfamily in TILs and clinical response, HLA haplotype, or age of the culture. Even though the results in this study suggest that certain TCR V gene segments may be involved in immune responses to human melanoma, we were unable to demonstrate functionally that a particular T-cell clonotype recognizes melanoma tumor- associated antigens. Only the analysis of melanoma-specific CTL clones can determine which clonotypes are important in lysis of human melanoma.

Detection of shared MHC-restricted human melanoma antigens after vaccinia virus-mediated transduction of genes coding for HLA

O'Neil Bert H., Kawakami Yutaka, Restifo Nicholas P., Bennink Jack R., Yewdell Jonathan W., Rosenberg Steven A.

Journal of Immunology 151 ( 3 ) 1410 - 1418 1993.08

ISSN 0022-1767

To detect shared human melanoma Ag that are recognized by HLA-A2 restricted, melanoma-specific CTL derived from tumor infiltrating lymphocytes, we have developed a convenient method to insert and express foreign HLA genes capable of presenting Agon target cell lines. Seventeen melanoma cell lines and 11 nonmelanoma cell lines were infected with recombinant vaccinia virus containing the HLA-A2.1 gene. Infection by the vaccinia virus resulted in expression of functional HLA-A2 molecules on the cell surface of virtually 100% of infected cells within a 3.5-h period. The results showed that 11 of 17 (65%) naturally HLA-A2^- melanoma cell lines were specifically lysed by the HLA-A2-restricted, melanoma-specfic TIL after infection with the vaccinia-HLA-A2.1 virus. None of the nine human nonmelanoma cell lines tested (three colon cancer, four breast cancer, or two immortalized nontumor cell lines) or two murine melanoma cell lines were lysed by the HLA-A2-restricted TIL after vaccinia-HLA-A2.1 infection. Coinfection of the vaccinia virus containing the β2-microglobulin gene with the vaccinia-HLA-A2.1 virus increased the surface expression of HLA-A2 and subsequent lysis by melanoma-specific tumor infiltrating lymphocytes. With this new method we could extend previous findings demonstrating that shared melanoma Ag recognized by HLA-A2-restricted tumor infiltrating lymphocytes exist among melanoma cells from different patients regardless of HLA type. These Ag represent excellent candidates for the development of vaccines to induce T cell responses for the immunotherapy of patients with melanoma.

Identification of human cancers deficient in antigen processing

Restifo Nicholas P., Esquivel Fernando, Kawakami Yutaka, Yewdell Jonathan W., Mulé James J., Rosenberg Steven A., Bennink Jack R.

Journal of Experimental Medicine 177 ( 2 ) 265 - 272 1993.02

ISSN 0022-1007

Intracellular antigens must be processed before presentation to CD8⁺ T cells by major histocompatibility complex (MHC) class I molecules. Using a recombinant vaccinia virus (Vac) to transiently express the K^d molecule, we studied the antigen processing efficiency of 26 different human tumor lines. Three cell lines, all human small cell lung carcinoma, consistently failed to process endogenously synthesized proteins for presentation to K^d-restricted, Vac-specific T cells. Pulse-chase experiments showed that MHC class I molecules were not transported by these cell lines from the endoplasmic reticulum to the cell surface. This finding suggested that peptides were not available for binding to nascent MHC molecules in the endoplasmic reticulum. Northern blot analysis of these cells revealed low to nondetectable levels of mRNAs for MHC-encoded proteasome components LMP-7 and LMP-2, as well as the putative peptide transporters TAP-1 and TAP-2. Treatment of cells with interferon γ enhanced expression of these mRNAs and reversed the observed functional and biochemical deficits. Our findings suggest that downregulation of antigen processing may be one of the strategies used by tumors to escape immune surveillance. Potential therapeutic applications of these findings include enhancing antigen processing at the level of the transcription of MHC-encoded proteasome and transporter genes.

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