研究者詳細 - 河上　裕

論文 - 河上　裕

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The Intronic Region of an Incompletely Spliced gp100 Gene Transcript Encodes an Epitope Recognized by Melanoma-Reactive Tumor-Infiltrating Lymphocytes

Robbins Paul F., El-Gamil Mona, Li Yong F., Fitzgerald Ellen B., Kawakami Yutaka, Rosenberg Steven A.

Journal of Immunology 159 （ 1 ） 303 - 308 1997年

ISSN 0022-1767

Recent studies have characterized a number of the Ags that are recognized by melanoma-reactive T cells. Although the majority of tumor Ags appear to represent nonmutated gene products, a variety of epitopes have been shown to arise from either mutated or alternatively processed transcripts. Here, we report that the screening of a cDNA library with a HLA-A24-restricted melanoma-reactive T cell cloid derived from tumor infiltrating lymphocytes resulted in the isolation of a variant of the gp100 gene that had retained the entire fourth intron of this gene, termed gp100-in4. The gp100-in4 transcript could be detected by reverse transcriptase-PCR but could not be detected in Northern blots conducted with melanoma RNA, indicating that it represents a relatively rare transcript. Read-through of this transcript into the region corresponding to the fourth intron gave rise to an additional 35 amino acids not found in the normal gp100 glycoprotein, and a peptide within this region conforming to the HLA-A24 consensus motif (VYFFLPDHL) was shown to be recognized by the T cell cloid. The sequence of the intron was identical with that of a previously isolated genomic gp100 clone, and T cells that recognized the gp100-in4 gene product were found to recognize HLA-A24-matched allogeneic melanoma cell lines and melanocytes, demonstrating that this represents a nonmutated epitooe. These results further extend the types of Ags that can be recognized by melanoma-reactive T cells to aberrant transcripts of melanosomal genes.

Differential expression of MART-1 in primary and metastatic melanoma lesions

Kageshita Toshiro, Kawakami Yutaka, Hirai Shunji, Ono Tomomichi

Journal of Immunotherapy 20 （ 6 ） 460 - 465 1997年

ISSN 1053-8550

Twenty-eight primary and 29 metastatic melanoma lesions and 18 pigmented nevi lesions were analyzed by using the immunoperoxidase reaction with anti- MART-1 and anti-gp100 monoclonal antibodies (mAbs). The MART-1 was expressed in 28, 29, and 18, and gp100 was expressed in 27, 28, and eight of these lesions, respectively. Intensity and percentage of stained cells with anti- MART-1 mAb were stronger and higher than those with anti-gp100 mAb. MART-1 was expressed homogeneously in primary melanoma and pigmented nevi, whereas it was heterogeneously expressed in metastatic melanoma lesions. The level of expression of MART-1 in primary melanoma lesions did not correlate with any clinicopathologic parameters. These results suggest that anti-MART-1 mAb is a useful tool for immunohistochemical analysis of melanocytic lesions and also is useful for patient's selection and monitoring of antigen-loss variants in clinical trials with the MART-1-based immunotherapy.

Immunobiology of human melanoma antigens MART-1 and gp100 and their use for immuno-gene therapy

Kawakami Yutaka, Rosenberg Steven A.

International Reviews of Immunology 14 （ 2-3 ） 173 - 192 1997年

ISSN 0883-0185

Melanosomal proteins as melanoma-specific immune targets

Sakai C., Kawakami Y., Law L. W., Furumura M., Hearing V. J.

Melanoma Research 7 （ 2 ） 83 - 95 1997年

ISSN 0960-8931

Pigmentation of our skin, hair and eyes is essential for photoprotection, embryological development, detoxification end protective/cosmetic coloration. A number of proteins important to the production of melanin within melanosomes have now been identified including enzymatic and structural proteins encoded at the murine albino, brown, pinkeyed-dilution, MART1, siaty end silver loci. Interestingly, many of those melanosomal proteins (including epitopes derived from tyrosinase, TRP1/gp75, silver/gp100 end MART1/melan-A) function in vivo as targets of humoral and cellular autoimmune responses directed specifically against normal or transformed melanocytes. These findings have provided new impetus to research on immune responses to melanoma end, perhaps more importantly, examining why they are insufficient to provide protection against tumour growth end what type of immune therapy can be designed to correct that. The melanosome must now be considered beyond its function in pigmentation, end assumes the role of a valuable source for specific immune targets for malignant melenoma.

Identification of TRP-2 as a human tumor antigen recognized by cytotoxic T lymphocytes

Wang Rong Fu, Appella Ettore, Kawakami Yutaka, Kang Xiaoqiang, Rosenberg Steven A.

Journal of Experimental Medicine 184 （ 6 ） 2207 - 2216 1996年12月

ISSN 0022-1007

The infusion of TIL586 along with interleukin-2 into the autologous patient with metastatic melanoma resulted in the objective regression of tumor. A gene encoding a tumor antigen recognized by TIL586 was previously isolated and shown to encode gp75 or TRP-1. Here we report that TRP-2 was identified as a second tumor antigen recognized by a HLA-A31-restricted CTL clone derived from the TIL586 cell line. The peptide LLPGGRPYR epitope was subsequently identified from the coding region of TRP-2 based on studies of the recognition of truncated TRP-2 cDNAs and the HLA-A31 binding motif. This epitope peptide was capable of sensitizing target cells for lysis by a CTL clone at 1 nM peptide concentration. Although some modified peptides could be recognized by the CTL clone, none were found to be better recognized by T cells than the parental peptide. Like other melanoma differentiation antigens, TRP-2 was only expressed in melanoma, melanocytes, and retina, but not in other human tissues tested.

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Human tumor antigens recognized by T cells

Robbins Paul F., Kawakami Yutaka

Current Opinion in Immunology 8 （ 5 ） 628 - 636 1996年10月

ISSN 0952-7915

The ability of tumor-reactive T cells to mediate in vivo tumor regression has been demonstrated in murine tumor models and by the clinical responses to adoptive immunotherapy with tumor-infiltrating lymphocytes isolated from human melanomas. Investigations carried out in the past several years have resulted in the isolation of a number of the genes encoding antigens recognized by melanoma-reactive T cells. The ability of these products to serve as tumor regression antigens has now begun to be evaluated in clinical vaccine trials.

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Improved Induction of Melanoma-Reactive CTL with Peptides from the Melanoma Antigen gp100 Modified at HLA-A*0201-Binding Residues

Parkhurst Maria R., Salgaller Michael L., Southwood Scott, Robbins Paul F., Sette Alessandro, Rosenberg Steven A., Kawakami Yutaka

Journal of Immunology 157 （ 6 ） 2539 - 2548 1996年09月

ISSN 0022-1767

Recognition of the melanoma Ag gp100 by tumor-infiltrating lymphocytes (TIL) in vitro has been correlated with tumor regression in patients with metastatic melanoma treated with the adoptive transfer of TIL plus IL-2. Three common gp100 epitopes have been identified that are recognized in the context of HLA-A2 by TIL from different patients: G9₁₅₄ (KTWGQYWQV), G9₂₀₉ (ITDQVPFSV), and G9₂₈₀ (YLEPGPVTA). Upon stimulation with these peptides, melanoma-reactive CTL could be induced in vitro from PBL of some HLA-A2⁺ melanoma patients. However, numerous restimulations were required, and specific reactivity could not be generated in many patients. Therefore, to enhance the immunogenicity of gp100 peptides, amino acid substitutions were introduced into G9₁₅₄, G9₂₀₉, and G9₂₈₀ at HLA-A*0201-binding anchor positions, but not at TCR contact residues, to increase peptide class I MHC-binding affinity. Several modified gp100 peptides bound with greater affinity to HLA-A*0201 than unmodified peptides and were recognized by TIL specific for the natural epitopes. These peptides were used to sensitize PBL from HLA-A2⁺ melanoma patients in vitro using peptide-pulsed autologous PBMC as stimulators. After five weekly restimulations with either the native G9₂₀₉ or G9₂₈₀ peptide, melanoma-reactive CTL could only be induced from two of seven patients. However, amino acid substitutions in these peptides enabled the induction of melanoma-reactive CTL from all seven patients. These results suggest that modified gp100 peptides may be more immunogenic than the native epitopes, and may be useful in immunotherapy protocols for patients with melanoma.

T-cell recognition of self peptides as tumor rejection antigens

Kawakami Yutaka, Rosenberg Steven A.

Immunologic Research 15 （ 3 ） 179 - 190 1996年09月

ISSN 0257-277X

Human melanoma antigens and their epitopes recognized by T cells have recently been identified. HLA-A2 binding epitopes of melanoma antigens MART-1 and gp100 were characterized and suspected to be subdominant/cryptic self determinants. Together with other findings of tumor-specific mutated self peptides as tumor antigens recognized by T cells, the nature of the antitumor immune response to human melanoma has been revealed at a molecular level. These findings have implications not only for understanding of the immune response to self peptides in normal and pathologic conditions, but also for the development of immunotherapies for cancer and autoimmune diseases.

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Human melanoma antigens recognized by T lymphocytes

Kawakami Yutaka, Robbins Paul F., Rosenberg Steven A.

Keio Journal of Medicine 45 （ 2 ） 100 - 108 1996年06月

ISSN 0022-9717

Human melanoma antigens and their epitopes recognized by T cells have been identified using a variety of methods. These antigens are classified as 1) melanocyte specific melanosomal proteins (MART-1, gp100, tyrosinase and TRP-1), 2) proteins expressed in testis and a variety of cancers (MAGE-1, MAGE-3, BAGE and GAGE), 3) tumor specific mutated proteins (β-catenin, MUM-1 and CDK4), and 4) others (p15). Some of the HLA-A2 binding non-mutated melanoma epitopes contained non-dominant anchor amino acids and have relatively low HLA-A2 binding affinity, suggesting that these epitopes were likely to be subdominant or cryptic self determinants. The significant correlation observed between vitiligo development and IL2 based immunotherapy suggested that autoreactive T cells specific for these self peptides were involved in melanoma regression in vivo. In addition, since adoptive transfer into patients of CTL recognizing these epitopes resulted in tumor regression, these epitopes may be tumor rejection antigens. Melanoma reactive CTL were efficiently induced from PBL of patients by in vitro stimulation wilh PBMC pulsed with these melanoma epitopes and may be useful in adoptive transfer protocols for the treatment of patients with metastatic melanoma. An immunization trial using the MART-1 and gp100 peptides in conjunction with incomplete Freund's adjuvant is in progress. These identified antigens may be useful for the development of new immunotherapies for the treatment of melanoma patients as well as for understanding the mechanisms of anti-tumor immune responses and autoimmune disorders against melanocytes.

Utilization of an alternative open reading frame of a normal gene in generating a novel human cancer antigen

Wang Rong Fu, Parkhurst Maria R., Kawakami Yutaka, Robbins Paul F., Rosenberg Steven A.

Journal of Experimental Medicine 183 （ 3 ） 1137 - 1140 1996年03月

ISSN 0022-1007

Tumor infiltrating lymphocytes (TILs) derived from tumor-bearing patients recognize tumor-associated antigens presented by major histocompatibility complex (MHC) class I molecules The infusion of TIL586 along with interleukin (IL) 2 into an autologous patient with metastatic melanoma resulted in the objective regression of tumor. A gene encoding a tumor antigen recognized by TIL586 was recently isolated and shown to encode gp75. Here we report that an antigenic peptide, MSLQRQFLR, recognized by TIL586 was not derived from the normal gp75 protein. Instead, this nonamer peptide resulted from translation of an alternative open reading frame of the same gene. Thus, the gp75 gene encodes two completely different polypeptides, gp75 as an antigen recognized by immunoglobulin G antibodies in sera from a patient with cancer, and a 24-amino acid product as a tumor rejection antigen recognized by T cells. This represents the first demonstration that a human tumor rejection antigen can be generated from a normal cellular gene using an open reading frame other than that used to encode the normal protein. These findings revealed a novel mechanism for generating tumor antigens which may be useful as vaccines to induce tumor-specific cell-mediated immunity against cancer.

A mutated β-catenin gene encodes a melanoma-specific antigen recognized by tumor infiltrating lymphocytes

Robbins Paul F., El-Gamil Mona, Li Yong F., Kawakami Yutaka, Loftus Douglas, Appella Ettore, Rosenberg Steven A.

Journal of Experimental Medicine 183 （ 3 ） 1185 - 1192 1996年03月

ISSN 0022-1007

A number of antigens recognized by tumor-reactive T cells have recently been identified. The antigens identified in mouse model systems appear, with one exception, to represent thee products o mutated genes. In contrast, most of the antigens recognized by human tumor-reactive C cells reported to date appear to represent the products of non-mutated genes. Here we report the isolation o a cDNA clone encoding β-catenin, which was shown to be recognized by the tumor-infiltrating lymphocyte (TIL) 1290, a HLA-A24 restricted melanoma-specific CTL line from patient 888. Thee cDNA clone, which was isolated from thee autologous melanoma cDNA library, differed by a single base pair from the published β-catenin sequence, resulting in a change from a serine to a phenylalanine residue at position 37. Normal tissues from this patient did not express the altered sequence, nor did 12 allogeneic melanomas, indicating that this represented a unique mutation in this patient's melanoma. A peptide corresponding to the sequence between amino acids 29 and 37 of the mutant gene product was identified as the T cell epitope recognized by TIL 1290. The observation that HLA-A24 binding peptides contain an aromatic or hydrophobic residue at position 9 suggested that the change at position 37 may have generated a peptide (SYLDSGIHF) which was capable of binding to HLA-A24, and a competitive binding assay confirmed this hypothesis. The β-catenin protein has been shown previously to be involved in cell adhesion mediated through the cadherin family of cell surface adhesion molecules. The high frequency of mutations found in members of cellular adhesion complexes in a variety of cancers suggests that these molecules may play a role in development of the malignant phenotype.

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Loss of functional beta₂-microglobulin in metastatic melanomas from five patients receiving immunotherapy

Restifo Nicholas P., Marincola Francesco M., Kawakami Yutaka, Taubenberger Jeff, Yannelli John R., Rosenberg Steven A.

Journal of the National Cancer Institute 88 （ 2 ） 100 - 108 1996年01月

ISSN 0027-8874

Background: In a subset of patients with metastatic melanoma, T lymphocytes bearing the cell-surface marker CD8 (CD8⁺ T cells) can cause the regression of even large tumors. These antitumor CD8⁺ T cells recognize peptide antigens presented on the surface of tumor cells by major histocompatibility complex (MHC) class I molecules. The MHC class I molecule is a heterodimer composed of an integral membrane glycoprotein designated the α chain and a non-covalently associated, soluble protein called beta₂- microglobulin (β₂m). Loss of β₂m generally eliminates antigen recognition by antitumor CD8⁺ T cells. Purpose: We studied the loss of β₂m as a potential means of tumor escape from immune recognition in a cohort of patients receiving immunotherapy. Methods: We successfully grew 13 independent tumor cell cultures from tumor specimens obtained from 13 patients in a cohort of 411 consecutive patients undergoing immunotherapy for metastatic melanoma and for whom tumor specimens were available. These cell lines, as well as another melanoma cell line (called 1074mel) that had been derived from tumor obtained from a patient in a cytokine-gene therapy study, were characterized in vitro cytofluorometrically for MHC class I expression and by northern and western blot analyses for messenger RNA (mRNA) and protein expression, respectively, and ex vivo by immunohistochemistry. Results: After one melanoma cell line (107mel) was found not to express functional β₂m by cytofluorometric analysis, four (31%) of the 13 newly established melanoma cell lines were found to have an absolute lack of functional MHC class I expression. Northern blot analysis of RNA extracted from the five cell lines exhibiting no functional MHC class I expression showed that these cells contained normal levels of α-chain mRNA but variable levels of β₂m mRNA. In addition, no immunoreactive β₂m protein was detected by western blot analysis. When human β₂m was transiently expressed with the use of a recombinant vaccinia virus, cell-surface MHC class I expression was reconstituted and the ability of these five cell lines to present endogenous antigens was restored. Immunohistochemical staining of tumor sections revealed a lack of immunoreactive MHC class I in vivo, supporting the notion that the in vitro observations were not artifactual. Furthermore, archival tumor sections obtained from patients prior to immunotherapy were available from three patients and were found to be β₂m positive. This result was consistent with the hypothesis that loss of β₂m resulted from immunotherapy. Conclusions: These data suggest that the loss of β₂m may be a mechanism whereby tumor cells can acquire immunoresistance. This study represents the first characterization of a molecular route of escape of tumors from immune recognition in a cohort of patients being treated with immunotherapy.

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Antigen-specific tumor vaccines

Zhai Yifan, Yang James C., Kawakami Yutaka, Spiess Paul, Wadsworth Samuel C., Cardoza Lisa M., Couture Larry A., Smith Alan E., Rosenberg Steven A.

Journal of Immunology 156 （ 2 ） 700 - 710 1996年01月

ISSN 0022-1767

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-γ, TNF-α, and granulocyte-macrophage-CSF. Sodium butyrate and TNF-α 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 vaccination 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.

Analysis of expression of the melanoma-associated antigens MART-1 and gp100 in metastatic melanoma cell lines and in in situ lesions

Marincola Francesco M., Hijazi Yasmine M., Fetsch Patricia, Salgaller Michael L., Rivoltini Licia, Cormier Janice, Simonis Toni B., Duray Paul H., Herlyn Meenhard, Kawakami Yutaka, Rosenberg Steven A.

Journal of Immunotherapy 19 （ 3 ） 192 - 205 1996年

ISSN 1053-8550

MART-1 and gp100 melanoma associated antigens (MAA) are expressed by cells of the melanocytic lineage and are recognized by the majority of HLA- A2 restricted tumor-infiltrating lymphocytes. Heterogeneity of expression of MAA in tumor deposits may affect the natural history or response to therapy of patients with melanoma. In this study, we evaluated the expression of these MAA with a new monoclonal antibody (mAb) directed against MART-1 (M2- 7C10) and the commercially available HMB45 mAb directed against gp100. Expression was tested in vitro by intracellular fluorescence analysis and in vivo by immunophenotyping of tissue specimens. Nine melanoma cell lines and 25 tissue specimens from metastatic melanoma were analyzed. One cell line did not express MART-1 or gp100. The expression of both antigens was more heterogeneous and significantly reduced (p < 0.01) in melanoma cell lines compared with melanocytes, suggesting progressive loss of expression of MAA by neoplastic cells. None of the nonmelanoma cancer lines tested stained for MART-1 or gp100. Analysis of melanoma lesions by immunohistochemistry showed significant heterogeneity of expression of both MART-1 and gp100 MAA either as a percentage of cells expressing MAA or as intensity of expression. Ten of 25 frozen sections expressed MART-1 in <50% of the cells. In 6 of 25 lesions, immunoreactivity for MART-1 was totally absent. Fine needle aspiration of metastatic lesions seemed to yield information accurately about amount and heterogeneity of expression of MAA in tumor lesions in vivo. Heterogeneity of expression of MAA may be one of several mechanisms leading to tumor escape from immune recognition, and pretreatment evaluation of tumor lesion for expression of these antigens may help in selecting patients best suited to antigen-specific vaccine therapies.

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Identification of tumor-regression antigens in melanoma.

Kawakami Y., Robbins P. F., Wang R. F., Rosenberg S. A.

Important advances in oncology 3 - 21 1996年

ISSN 0883-5896

Recognition of Multiple Epitopes in the Human Melanoma Antigen gplOO by Peripheral Blood Lymphocytes Stimulated in Vitro with Synthetic Peptides

Salgaller Michael L., Afshar Alireza, Marincola Francesco M., Rivoltini Licia, Kawakami Yutaka, Rosenberg Steven A.

Cancer Research 55 （ 21 ） 4972 - 4979 1995年11月

ISSN 0008-5472

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.

Molecular characterization of defective antigen processing in human prostate cancer

Sanda Martin G., Restifo Nicholas P., Walsh Jonathan C., Kawakami Yutaka, Nelson William G., Pardoll Drew M., Simons Jonathan W.

Journal of the National Cancer Institute 87 （ 4 ） 280 - 285 1995年02月

ISSN 0027-8874

Background:Gene-modified tumor cell vaccines have shown efficacy in animal models of malignancy, including prostate cancer. Class I major histocompatibility complex (MHC) assembly and function in the cellular targets of such therapies is pivotal in determining the efficacy of specific cytokine-secreting tumor vaccines.Purpose To help guide development of genetically engineered vaccine therapy for human prostate cancer, potential immune resistance pathways were evaluated by analysis of class I MHC assembly in prostate cancer cells. Method: Class I MHC assembly in metastasis-derived human prostate cancer cell lines (LNCaP, PPC-1, DU-145, PC-3, and TSU) and a normal prostate-derived cell line (TP-2) were characterized by phenotypic, molecular, and functional assays. Assembled class I MHC and antigen was measured by flow cytometry; mRNA levels of assembly components (class I MHC heavy chain,; β2-microglobulin, and the antigen transporter gene product TAP-2) were determined; and antigen processing was measured with a chimeric reconstituted system using vaccinia vectors. Restoration of antigen processing was attempted by interferon gamma stimulation and by transfection with mouse class I MHC heavy-chain cDNA. Results: Assembled class I MHC was underexpressed in two (LNCaP and PPC-1) of five prostate cancer cell lines compared with normal prostate-derived controls. PPC-1 cells underexpressed TAP-2 mRNA despite abundant class I MHC and β2-microglobulin message. Induction of TAP-2 by interferon gamma indicated that coding sequences for TAP-2 message were present in PPC-1. Resistance to cytotoxic T lymphocytes (CTL) lysis showed a functional defect in antigen transport by PPC-1 cells; reversal of the molecular defect with interferon gamma led to restoration of functional antigen processing. In contrast, LNCaP cells had competent antigen transport but deficient class I MHC heavy-chain function despite abundant class I MHC RNA; though refractory to stimulation by interferon gamma, this defect responded to transfection of class I MHC heavy-chain cDNA. Conclusions: Metastatic prostate cancer cells can escape T-cell recognition via divergent mechanisms of defective class I MHC assembly. The specific under-expression of TAP-2 gene product in PPC-1 cells contrasts with prior studies of TAP gene underexpression in lung cancer (which concurrently underexpressed class I MHC heavy chain) and provides evidence for a regulatory pathway controlling TAP-2 gene expression in human cancers that may not affect class I MHC heavy-chain expression- Implications:In clinical application of gene therapy for prostate cancer, these findings provide a rationale for focusing on strategies that can circumvent sole reliance on class I MHC-mediated tumor cell recognition by CTL. [J Natl Cancer Inst 87: 280-285, 1995]

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Identification of a gene encoding a melanoma tumor antigen recognized by HLA-A31-restricted tumor-infiltrating lymphocytes

Wang Rong Fu, Robbins Paul F., Kawakami Yutaka, Kang Xiao Qiang, Rosenberg Steven A.

Journal of Experimental Medicine 181 （ 2 ） 799 - 804 1995年02月

ISSN 0022-1007

The availability of antitumor cytotoxic T lymphocytes which can be generated from either peripheral blood lymphocytes after stimulation in vitro or tumor infiltrating lymphocytes (TIL) has made it possible to identify a number of melanoma antigens presented by major histocompatibility complex class I molecules. The present and previous studies indicated that TIL586 recognized an antigen expressed on most melanoma and normal melanocytes in the context of the HLA-A31 molecule. We report here the cloning of a cDNA that directs the expression of the shared melanoma antigen recognized by this TIL. The DNA sequence analysis revealed that the cDNA was almost identical to the gene encoding tyrosinase-related protein 1 or glycoprotein gp75 which was originally identified by serum antibodies in a patient with melanoma. The gene was found to be expressed only in melanoma, normal melanocyte cell lines, and retina, but not in other normal tissues tested. The gp75 antigen presented by HLA-A31 may therefore constitute a useful immune target for specific treatment of patients with melanoma, since both antibody- and T cell mediated immune responses can be generated against this antigen.

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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

Rivoltini Licia, Kawakami Yutaka, Sakaguchi Kazuyasu, Southwood Scott, Sette Alessandro, Robbins Paul F., Marincola Francesco M., Salgaller Michael L., Yannelli John R., Appella Ettore, Rosenberg Steven A.

Journal of Immunology 154 （ 5 ） 2257 - 2265 1995年

ISSN 0022-1767

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-α, IFN-γ, 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.

Melanoma tumor-infiltrating lymphocytes derived from four distinct anatomic sites obtained from a single patient

Yannelli J. R., McConnell S., Parker L., Nishimura M., Robbins P., Yang J., El Gamil M., Kawakami Y.

Journal of Immunotherapy 18 （ 4 ） 263 - 271 1995年

ISSN 1053-8550

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-1_27-35. 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.

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