61 results on '"El-Gamil M"'
Search Results
2. Melanoma Cell Lines Contain a Proteasome-Sensitive, Nuclear Cytoskeleton-Associated Pool of beta-Catenin
- Author
-
BONVINI, P., primary, HWANG, S.-G., additional, EL-GAMIL, M., additional, ROBBINS, P., additional, NECKERS, L., additional, and TREPEL, J., additional
- Published
- 1999
- Full Text
- View/download PDF
3. The intronic region of an incompletely spliced gp100 gene transcript encodes an epitope recognized by melanoma-reactive tumor-infiltrating lymphocytes.
- Author
-
Robbins, P F, primary, El-Gamil, M, additional, Li, Y F, additional, Fitzgerald, E B, additional, Kawakami, Y, additional, and Rosenberg, S A, additional
- Published
- 1997
- Full Text
- View/download PDF
4. A mutated beta-catenin gene encodes a melanoma-specific antigen recognized by tumor infiltrating lymphocytes.
- Author
-
Robbins, P F, primary, El-Gamil, M, additional, Li, Y F, additional, Kawakami, Y, additional, Loftus, D, additional, Appella, E, additional, and Rosenberg, S A, additional
- Published
- 1996
- Full Text
- View/download PDF
5. Identification of a tyrosinase epitope recognized by HLA-A24-restricted, tumor-infiltrating lymphocytes.
- Author
-
Kang, X, primary, Kawakami, Y, additional, el-Gamil, M, additional, Wang, R, additional, Sakaguchi, K, additional, Yannelli, J R, additional, Appella, E, additional, Rosenberg, S A, additional, and Robbins, P F, additional
- Published
- 1995
- Full Text
- View/download PDF
6. Cloning of a new gene encoding an antigen recognized by melanoma-specific HLA-A24-restricted tumor-infiltrating lymphocytes.
- Author
-
Robbins, P F, primary, el-Gamil, M, additional, Li, Y F, additional, Topalian, S L, additional, Rivoltini, L, additional, Sakaguchi, K, additional, Appella, E, additional, Kawakami, Y, additional, and Rosenberg, S A, additional
- Published
- 1995
- Full Text
- View/download PDF
7. 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.
- Author
-
Stevens, E J, primary, Jacknin, L, additional, Robbins, P F, additional, Kawakami, Y, additional, el Gamil, M, additional, Rosenberg, S A, additional, and Yannelli, J R, additional
- Published
- 1995
- Full Text
- View/download PDF
8. Melanoma Cell Lines Contain a Proteasome-Sensitive, Nuclear Cytoskeleton-Associated Pool of β-Catenin.
- Author
-
BONVINI, P., HWANG, S.-G., EL-GAMIL, M., ROBBINS, P., NECKERS, L., and TREPEL, J.
- Published
- 1999
- Full Text
- View/download PDF
9. Melanoma Cell Lines Contain a Proteasome-Sensitive, Nuclear Cytoskeleton-Associated Pool of ?-Catenin
- Author
-
BONVINI, P., HWANG, S.-G., EL-GAMIL, M., ROBBINS, P., NECKERS, L., and TREPEL, J.
- Published
- 1999
- Full Text
- View/download PDF
10. Cloning of a new gene encoding an antigen recognized by melanoma-specific HLA-A24-restricted tumor-infiltrating lymphocytes
- Author
-
Robbins, P. F., El-Gamil, M., Li, Y. F., Topalian, S. L., Licia Rivoltini, Sakaguchi, K., Appella, E., Kawakami, Y., and Rosenberg, S. A.
- Subjects
Immunology ,Immunology and Allergy - Abstract
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 an 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.
11. Targeted cytokine production
- Author
-
Dm, Segal, Jh, Qian, Ja, Titus, Mb, Moreno, Andrew George, Cr, Jost, Kurucz I, el-Gamil M, and Jr, Wunderlich
- Subjects
Cytotoxicity, Immunologic ,Mice ,T-Lymphocytes ,Receptors, Antigen, T-Cell ,Animals ,Antibodies, Monoclonal ,Cytokines ,Mammary Neoplasms, Experimental ,Lymphocyte Activation ,Models, Biological ,Spleen - Abstract
It has been well established that bispecific antibodies containing anti-T-cell receptor MAbs crosslinked to anti-tumor MAbs induce T cells to lyse tumor cells, as measured in a 51Cr-release assay. Such lysis requires direct attachment between target and cytotoxic cells and most probably involves the exocytosis of cytolytic substances into the cell:cell interface. In addition, targeted T cells mediate a second activity, the secretion into the medium of factors that can block the growth of bound tumor cells and unbound bystander cells. In order to test how targeted effector cells mediate anti-tumor effects in vivo, we are currently developing a totally syngeneic murine system in which murine T cells are targeted against mouse mammary tumors. The system allows us to treat both primary tumors and tumor transplants, using a mammary-tumor-virus antigen as the entity that is specifically recognized on the tumor cells.
12. Evidence for the absence of I-E/C antigen expression on the cell surface in mice of the H-2b or H-2s haplotypes.
- Author
-
Ozato, K, primary, Lunney, J K, additional, El-Gamil, M, additional, and Sachs, D H, additional
- Published
- 1980
- Full Text
- View/download PDF
13. Binuclear copper(II), cobalt(II) and Nickel(II) complexes of N 1-ethyl-N 2-(pyridin-2-yl) hydrazine-1,2-bis(carbothioamide): Structural, spectral, pH-metric and biological studies
- Author
-
El-Gammal, O. A., Abu El-Reash, G. M., and El-Gamil, M. M.
- Subjects
- *
COPPER ions , *METAL complexes , *PYRIDINE , *HYDRAZINE , *MOLECULAR structure , *CHEMICAL stability , *DISSOCIATION (Chemistry) - Abstract
Binuclear Cu(II), Co(II) and Ni(II) complexes derived from N1-ethyl-N2-(pyridin-2-yl) hydrazine-1,2-bis(carbothioamide) (H2PET) have been prepared and characterized by elemental analysis, spectral (IR, UV–vis, EI mass, ESR and 1HNMR) and magnetic measurements. The isolated complexes assigned the general formula, [M(HPET)(H2O) n Cl]2·xH2O where M=Cu(II), Co(II) and Ni(II), n = 2, 1, 0 and x =0, 0.5 and 0, respectively. IR data revealed that the ligand behaves as monobasic tridentate through (C=N)py, (C–S) and new azomethine, (N = C)∗ groups in the Co(II) complex but in Cu(II) complex, the ligand coordinate via both (CS) groups, one of them in thiol form as well as the new azomethine group. In Ni(II) complex, H2PET acts as NSNS monobasic tetradente via (C=N)py, (C–S), (C=S) and the new azomethine, (N = C)∗ groups. An octahedral geometry is proposed for all complexes. pH- metric titration was carried out in 50% dioxane–water mixture at 298, 308 and 318 °K, respectively and the dissociation constant of the ligand as well as the stability constants of its complexes were evaluated. Also the kinetic and thermodynamic parameters for the different thermal decomposition steps of the complexes were determined by Coats–Redfern and Horowitz–Metzger methods. Moreover, the anti-oxidant, anti-hemolytic, and cytotoxic activities of the compounds have been tested. [Copyright &y& Elsevier]
- Published
- 2012
- Full Text
- View/download PDF
14. Unique Neoantigens Arise from Somatic Mutations in Patients with Gastrointestinal Cancers.
- Author
-
Parkhurst MR, Robbins PF, Tran E, Prickett TD, Gartner JJ, Jia L, Ivey G, Li YF, El-Gamil M, Lalani A, Crystal JS, Sachs A, Groh E, Ray S, Ngo LT, Kivitz S, Pasetto A, Yossef R, Lowery FJ, Goff SL, Lo W, Cafri G, Deniger DC, Malekzadeh P, Ahmadzadeh M, Wunderlich JR, Somerville RPT, and Rosenberg SA
- Subjects
- Biomarkers, Tumor, Gastrointestinal Neoplasms pathology, Humans, Lymphocyte Activation genetics, Lymphocyte Activation immunology, Lymphocytes, Tumor-Infiltrating immunology, Lymphocytes, Tumor-Infiltrating metabolism, Lymphocytes, Tumor-Infiltrating pathology, Receptors, Antigen, T-Cell metabolism, T-Lymphocyte Subsets immunology, T-Lymphocyte Subsets metabolism, T-Lymphocyte Subsets pathology, Antigens, Neoplasm genetics, Antigens, Neoplasm immunology, Disease Susceptibility, Gastrointestinal Neoplasms etiology, Gastrointestinal Neoplasms metabolism, Mutation
- Abstract
Immunotherapies can mediate regression of human tumors with high mutation rates, but responses are rarely observed in patients with common epithelial cancers. This raises the question of whether patients with these common cancers harbor T lymphocytes that recognize mutant proteins expressed by autologous tumors that may represent ideal targets for immunotherapy. Using high-throughput immunologic screening of mutant gene products identified via whole-exome sequencing, we identified neoantigen-reactive tumor-infiltrating lymphocytes (TIL) from 62 of 75 (83%) patients with common gastrointestinal cancers. In total, 124 neoantigen-reactive TIL populations were identified, and all but one of the neoantigenic determinants were unique. The results of in vitro T-cell recognition assays demonstrated that 1.6% of the gene products encoded by somatic nonsynonymous mutations were immunogenic. These findings demonstrate that the majority of common epithelial cancers elicit immune recognition and open possibilities for cell-based immunotherapies for patients bearing these cancers. SIGNIFICANCE: TILs cultured from 62 of 75 (83%) patients with gastrointestinal cancers recognized neoantigens encoded by 1.6% of somatic mutations expressed by autologous tumor cells, and 99% of the neoantigenic determinants appeared to be unique and not shared between patients. This article is highlighted in the In This Issue feature, p. 983 ., (©2019 American Association for Cancer Research.)
- Published
- 2019
- Full Text
- View/download PDF
15. Durable Complete Response from Metastatic Melanoma after Transfer of Autologous T Cells Recognizing 10 Mutated Tumor Antigens.
- Author
-
Prickett TD, Crystal JS, Cohen CJ, Pasetto A, Parkhurst MR, Gartner JJ, Yao X, Wang R, Gros A, Li YF, El-Gamil M, Trebska-McGowan K, Rosenberg SA, and Robbins PF
- Subjects
- Animals, Cell Line, Combined Modality Therapy, Epitopes, T-Lymphocyte genetics, Epitopes, T-Lymphocyte immunology, Exome, Humans, Male, Melanoma diagnosis, Middle Aged, T-Cell Antigen Receptor Specificity immunology, T-Lymphocytes metabolism, Tomography, X-Ray Computed, Treatment Outcome, Antigens, Neoplasm genetics, Antigens, Neoplasm immunology, Immunotherapy, Adoptive, Melanoma genetics, Melanoma immunology, Melanoma therapy, Mutation, T-Lymphocytes immunology
- Abstract
Immunotherapy treatment of patients with metastatic cancer has assumed a prominent role in the clinic. Durable complete response rates of 20% to 25% are achieved in patients with metastatic melanoma following adoptive cell transfer of T cells derived from metastatic lesions, responses that appear in some patients to be mediated by T cells that predominantly recognize mutated antigens. Here, we provide a detailed analysis of the reactivity of T cells administered to a patient with metastatic melanoma who exhibited a complete response for over 3 years after treatment. Over 4,000 nonsynonymous somatic mutations were identified by whole-exome sequence analysis of the patient's autologous normal and tumor cell DNA. Autologous B cells transfected with 720 mutated minigenes corresponding to the most highly expressed tumor cell transcripts were then analyzed for their ability to stimulate the administered T cells. Autologous tumor-infiltrating lymphocytes recognized 10 distinct mutated gene products, but not the corresponding wild-type products, each of which was recognized in the context of one of three different MHC class I restriction elements expressed by the patient. Detailed clonal analysis revealed that 9 of the top 20 most prevalent clones present in the infused T cells, comprising approximately 24% of the total cells, recognized mutated antigens. Thus, we have identified and enriched mutation-reactive T cells and suggest that such analyses may lead to the development of more effective therapies for the treatment of patients with metastatic cancer. Cancer Immunol Res; 4(8); 669-78. ©2016 AACR., (©2016 American Association for Cancer Research.)
- Published
- 2016
- Full Text
- View/download PDF
16. Isolation and Characterization of an HLA-DPB1*04: 01-restricted MAGE-A3 T-Cell Receptor for Cancer Immunotherapy.
- Author
-
Yao X, Lu YC, Parker LL, Li YF, El-Gamil M, Black MA, Xu H, Feldman SA, van der Bruggen P, Rosenberg SA, and Robbins PF
- Subjects
- Antigen Presentation, Antigens, Neoplasm immunology, CD4 Antigens metabolism, Cell Separation, Clone Cells, Cross Reactions, Genetic Therapy, HLA-DP beta-Chains metabolism, Humans, Melanoma immunology, Neoplasm Proteins immunology, Receptors, Antigen, T-Cell metabolism, T-Cell Antigen Receptor Specificity, T-Lymphocytes, Helper-Inducer transplantation, T-Lymphocytes, Regulatory transplantation, Vaccination, White People, Antigens, Neoplasm metabolism, Cancer Vaccines immunology, Immunotherapy, Adoptive methods, Melanoma therapy, Neoplasm Proteins metabolism, Receptors, Antigen, T-Cell genetics, T-Lymphocytes, Helper-Inducer immunology, T-Lymphocytes, Regulatory immunology
- Abstract
Long-term tumor regressions have been observed in patients following the adoptive transfer of autologous tumor-infiltrating lymphocytes or genetically modified T cells expressing MHC class I-restricted T-cell receptors (TCRs), but clinical trials have not evaluated responses to genetically modified T cells expressing antitumor MHC class II-restricted TCRs. As studies carried out in a murine tumor model system have demonstrated that the adoptive transfer of CD4 T cells could lead to the regression of established tumors, we plan to test the hypothesis that CD4 T cells can also induce tumor regressions in cancer patients. In this study, 2 MAGE-A3-specific TCRs were isolated from a regulatory T-cell clone (6F9) and an effector clone (R12C9), generated from the peripheral blood of 2 melanoma patients after MAGE-A3 vaccination. The results indicated that T cells transduced with 6F9 TCR mediated stronger effector functions than R12C9 TCR. The 6F9 TCR specifically recognized MAGE-A3 and the closely related MAGE-A6 gene product, but not other members of the MAGE-A family in the context of HLA-DPB1*04:01. To test the feasibility of a potential clinical trial using this TCR, a clinical-scale procedure was developed to obtain a large number of purified CD4 T cells transduced with 6F9 TCR. Because HLA-DPB1*04:01 is present in ∼60% of the Caucasian population and MAGE-A3 is frequently expressed in a variety of cancer types, this TCR immunotherapy could potentially be applicable for a significant portion of cancer patients.
- Published
- 2016
- Full Text
- View/download PDF
17. Isolation of neoantigen-specific T cells from tumor and peripheral lymphocytes.
- Author
-
Cohen CJ, Gartner JJ, Horovitz-Fried M, Shamalov K, Trebska-McGowan K, Bliskovsky VV, Parkhurst MR, Ankri C, Prickett TD, Crystal JS, Li YF, El-Gamil M, Rosenberg SA, and Robbins PF
- Subjects
- Adolescent, Adult, Algorithms, Amino Acid Sequence, Antigen-Antibody Reactions, Antigens, Neoplasm classification, Antigens, Neoplasm genetics, Cells, Cultured, DNA, Neoplasm genetics, DNA-Binding Proteins genetics, DNA-Binding Proteins immunology, Epitopes genetics, Epitopes immunology, Female, Genes, erbB-2, HLA-A1 Antigen chemistry, HLA-A1 Antigen immunology, HLA-A2 Antigen chemistry, HLA-A2 Antigen immunology, Humans, Interferon-gamma Release Tests, Male, Melanoma genetics, Middle Aged, Molecular Sequence Data, Mutation, Neoplasm Proteins genetics, Neoplasm Proteins immunology, Nuclear Proteins genetics, Nuclear Proteins immunology, Peptide Fragments immunology, Receptor, ErbB-2 immunology, TEA Domain Transcription Factors, Transcription Factors genetics, Transcription Factors immunology, Antigens, Neoplasm immunology, Exome, Lymphocytes, Tumor-Infiltrating immunology, Melanoma immunology, Melanoma secondary, RNA, Neoplasm genetics, T-Cell Antigen Receptor Specificity, T-Lymphocytes immunology
- Abstract
Adoptively transferred tumor-infiltrating T lymphocytes (TILs) that mediate complete regression of metastatic melanoma have been shown to recognize mutated epitopes expressed by autologous tumors. Here, in an attempt to develop a strategy for facilitating the isolation, expansion, and study of mutated antigen-specific T cells, we performed whole-exome sequencing on matched tumor and normal DNA isolated from 8 patients with metastatic melanoma. Candidate mutated epitopes were identified using a peptide-MHC-binding algorithm, and these epitopes were synthesized and used to generate panels of MHC tetramers that were evaluated for binding to tumor digests and cultured TILs used for the treatment of patients. This strategy resulted in the identification of 9 mutated epitopes from 5 of the 8 patients tested. Cells reactive with 8 of the 9 epitopes could be isolated from autologous peripheral blood, where they were detected at frequencies that were estimated to range between 0.4% and 0.002%. To the best of our knowledge, this represents the first demonstration of the successful isolation of mutation-reactive T cells from patients' peripheral blood prior to immune therapy, potentially providing the basis for designing personalized immunotherapies to treat patients with advanced cancer.
- Published
- 2015
- Full Text
- View/download PDF
18. A pilot trial using lymphocytes genetically engineered with an NY-ESO-1-reactive T-cell receptor: long-term follow-up and correlates with response.
- Author
-
Robbins PF, Kassim SH, Tran TL, Crystal JS, Morgan RA, Feldman SA, Yang JC, Dudley ME, Wunderlich JR, Sherry RM, Kammula US, Hughes MS, Restifo NP, Raffeld M, Lee CC, Li YF, El-Gamil M, and Rosenberg SA
- Subjects
- Adult, Aged, Antigens, Neoplasm genetics, Epitopes, T-Lymphocyte immunology, Female, Follow-Up Studies, HLA-A2 Antigen immunology, Humans, Immunotherapy, Adoptive, Male, Melanoma diagnosis, Melanoma genetics, Melanoma immunology, Melanoma metabolism, Melanoma mortality, Melanoma therapy, Membrane Proteins genetics, Middle Aged, Neoplasm Metastasis, Odds Ratio, Phenotype, Pilot Projects, Sarcoma, Synovial diagnosis, Sarcoma, Synovial genetics, Sarcoma, Synovial immunology, Sarcoma, Synovial metabolism, Sarcoma, Synovial mortality, Sarcoma, Synovial therapy, T-Lymphocyte Subsets immunology, T-Lymphocyte Subsets metabolism, Tomography, X-Ray Computed, Treatment Outcome, Young Adult, Antigens, Neoplasm immunology, Membrane Proteins immunology, Receptors, Antigen, T-Cell genetics, Receptors, Antigen, T-Cell metabolism, T-Lymphocytes immunology, T-Lymphocytes metabolism
- Abstract
Purpose: Although adoptive cell therapy can be highly effective for the treatment of patients with melanoma, the application of this approach to the treatment of other solid tumors has been limited. The observation that the cancer germline (CG) antigen NY-ESO-1 is expressed in 70% to 80% and in approximately 25% of patients with synovial cell sarcoma and melanoma, respectively, prompted us to perform this first-in-man clinical trial using the adoptive transfer of autologous peripheral blood mononuclear cells that were retrovirally transduced with an NY-ESO-1-reactive T-cell receptor (TCR) to heavily pretreated patients bearing these metastatic cancers., Experimental Design: HLA-*0201 patients with metastatic synovial cell sarcoma or melanoma refractory to standard treatments and whose cancers expressed NY-ESO-1 received autologous TCR-transduced T cells following a lymphodepleting preparative chemotherapy. Response rates using Response Evaluation Criteria in Solid Tumors (RECIST), as well as immunologic correlates of response, are presented in this report., Results: Eleven of 18 patients with NY-ESO-1(+) synovial cell sarcomas (61%) and 11 of 20 patients with NY-ESO-1(+) melanomas (55%) who received autologous T cells transduced with an NY-ESO-1-reactive TCR demonstrated objective clinical responses. The estimated overall 3- and 5-year survival rates for patients with synovial cell sarcoma were 38% and 14%, respectively, whereas the corresponding estimated survival rates for patients with melanoma were both 33%., Conclusions: The adoptive transfer of autologous T cells transduced with a retrovirus encoding a TCR against an HLA-A*0201 restricted NY-ESO-1 epitope can be an effective therapy for some patients bearing synovial cell sarcomas and melanomas that are refractory to other treatments., (©2014 American Association for Cancer Research.)
- Published
- 2015
- Full Text
- View/download PDF
19. Efficient identification of mutated cancer antigens recognized by T cells associated with durable tumor regressions.
- Author
-
Lu YC, Yao X, Crystal JS, Li YF, El-Gamil M, Gross C, Davis L, Dudley ME, Yang JC, Samuels Y, Rosenberg SA, and Robbins PF
- Subjects
- Alleles, Amino Acid Sequence, Animals, Antigens, Neoplasm chemistry, Autoantigens immunology, Cell Line, Disease Progression, Epitope Mapping, Epitopes, T-Lymphocyte chemistry, Epitopes, T-Lymphocyte immunology, Epitopes, T-Lymphocyte metabolism, Gene Library, Histocompatibility Antigens genetics, Histocompatibility Antigens immunology, Humans, Lymphocytes, Tumor-Infiltrating immunology, Lymphocytes, Tumor-Infiltrating metabolism, Neoplasms pathology, Peptides chemistry, Peptides immunology, Protein Binding immunology, Antigens, Neoplasm genetics, Antigens, Neoplasm immunology, Mutation, Neoplasms genetics, Neoplasms immunology, T-Lymphocytes immunology, T-Lymphocytes metabolism
- Abstract
Purpose: Cancer immunotherapy with adoptive transfer of tumor-infiltrating lymphocytes (TIL) represents an effective treatment for patients with metastatic melanoma, with the objective regressions in up to 72% of patients in three clinical trials. However, the antigen targets recognized by these effective TILs remain largely unclear., Experimental Design: Melanoma patients 2359 and 2591 both experienced durable complete regressions of metastases ongoing beyond five years following adoptive TIL transfer. Two conventional screening approaches were carried out to identify the antigens recognized by these clinically effective TILs. In addition, a novel approach was developed in this study to identify mutated T-cell antigens by screening a tandem minigene library, which comprised nonsynonymous mutation sequences identified by whole-exome sequencing of autologous tumors., Results: Screening of an autologous melanoma cDNA library using a conventional approach led to the identification of previously undescribed nonmutated targets recognized by TIL 2359 or TIL 2591. In contrast, screening of tandem minigene libraries encoding tumor-specific mutations resulted in the identification of mutated kinesin family member 2C (KIF2C) antigen as a target of TIL 2359, and mutated DNA polymerase alpha subunit B (POLA2) antigen as a target of TIL 2591. Both KIF2C and POLA2 have been found to play important roles in cell proliferation., Conclusions: These findings suggest that the minigene screening approach can facilitate the antigen repertoire analysis of tumor reactive T cells, and lead to the development of new adoptive cell therapies with purified T cells that recognize candidate-mutated antigens derived from genes essential for the carcinogenesis., (©2014 American Association for Cancer Research.)
- Published
- 2014
- Full Text
- View/download PDF
20. Mutated PPP1R3B is recognized by T cells used to treat a melanoma patient who experienced a durable complete tumor regression.
- Author
-
Lu YC, Yao X, Li YF, El-Gamil M, Dudley ME, Yang JC, Almeida JR, Douek DC, Samuels Y, Rosenberg SA, and Robbins PF
- Subjects
- Base Sequence, Clinical Trials as Topic, Flow Cytometry, Gene Knockdown Techniques, Gene Library, Humans, Lymphocyte Activation immunology, Male, Melanoma genetics, Molecular Sequence Data, Mutation, Phosphoprotein Phosphatases genetics, Protein Phosphatase 1 genetics, Reverse Transcriptase Polymerase Chain Reaction, Immunodominant Epitopes immunology, Immunotherapy, Adoptive methods, Lymphocytes, Tumor-Infiltrating immunology, Melanoma immunology, Phosphoprotein Phosphatases immunology, Protein Phosphatase 1 immunology
- Abstract
Adoptive cell therapy with tumor-infiltrating lymphocytes (TILs) represents an effective treatment for patients with metastatic melanoma. However, most of the Ag targets recognized by effective melanoma-reactive TILs remain elusive. In this study, patient 2369 experienced a complete response, including regressions of bulky liver tumor masses, ongoing beyond 7 y following adoptive TIL transfer. The screening of a cDNA library generated from the autologous melanoma cell line resulted in the isolation of a mutated protein phosphatase 1, regulatory (inhibitor) subunit 3B (PPP1R3B) gene product. The mutated PPP1R3B peptide represents the immunodominant epitope recognized by tumor-reactive T cells in TIL 2369. Five years following adoptive transfer, peripheral blood T lymphocytes obtained from patient 2369 recognized the mutated PPP1R3B epitope. These results demonstrate that adoptive T cell therapy targeting a tumor-specific Ag can mediate long-term survival for a patient with metastatic melanoma. This study also provides an impetus to develop personalized immunotherapy targeting tumor-specific, mutated Ags.
- Published
- 2013
- Full Text
- View/download PDF
21. Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells.
- Author
-
Robbins PF, Lu YC, El-Gamil M, Li YF, Gross C, Gartner J, Lin JC, Teer JK, Cliften P, Tycksen E, Samuels Y, and Rosenberg SA
- Subjects
- Adult, Antigens, Neoplasm immunology, Epitopes, T-Lymphocyte, Female, HLA-A Antigens metabolism, Humans, Male, Melanoma immunology, Middle Aged, Adoptive Transfer, Antigens, Neoplasm genetics, Exome, Lymphocytes, Tumor-Infiltrating immunology, Melanoma therapy, Mutation
- Abstract
Substantial regressions of metastatic lesions have been observed in up to 70% of patients with melanoma who received adoptively transferred autologous tumor-infiltrating lymphocytes (TILs) in phase 2 clinical trials. In addition, 40% of patients treated in a recent trial experienced complete regressions of all measurable lesions for at least 5 years following TIL treatment. To evaluate the potential association between the ability of TILs to mediate durable regressions and their ability to recognize potent antigens that presumably include mutated gene products, we developed a new screening approach involving mining whole-exome sequence data to identify mutated proteins expressed in patient tumors. We then synthesized and evaluated candidate mutated T cell epitopes that were identified using a major histocompatibility complex-binding algorithm for recognition by TILs. Using this approach, we identified mutated antigens expressed on autologous tumor cells that were recognized by three bulk TIL lines from three individuals with melanoma that were associated with objective tumor regressions following adoptive transfer. This simplified approach for identifying mutated antigens recognized by T cells avoids the need to generate and laboriously screen cDNA libraries from tumors and may represent a generally applicable method for identifying mutated antigens expressed in a variety of tumor types.
- Published
- 2013
- Full Text
- View/download PDF
22. Characterization of T-cell receptors directed against HLA-A*01-restricted and C*07-restricted epitopes of MAGE-A3 and MAGE-A12.
- Author
-
Zhu S, Van den Eynde BJ, Coulie PG, Li YF, El-Gamil M, Rosenberg SA, and Robbins PF
- Subjects
- Antigens, Neoplasm genetics, CD4-Positive T-Lymphocytes immunology, CD4-Positive T-Lymphocytes metabolism, CD8-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes metabolism, Cell Line, Tumor, Clone Cells, Cloning, Molecular, Cytotoxicity, Immunologic immunology, Epitopes, T-Lymphocyte chemistry, Humans, Melanoma immunology, Melanoma metabolism, Neoplasm Proteins genetics, Receptors, Antigen, T-Cell genetics, T-Lymphocytes immunology, T-Lymphocytes metabolism, Transduction, Genetic, Antigens, Neoplasm immunology, Epitopes, T-Lymphocyte immunology, HLA-A Antigens immunology, HLA-C Antigens immunology, Neoplasm Proteins immunology, Receptors, Antigen, T-Cell immunology
- Abstract
The ability of T cells that have been genetically engineered to express T-cell receptors (TCRs) directed against tumor antigens to mediate tumor regression has been demonstrated in several clinical trials. These TCRs have primarily targeted HLA-A*0201-restricted TCRs, as approximately 50% of whites, who represent the predominant population of patients who develop melanomas, expresses this HLA class I allele. These therapies could be extended to additional patients through the use of TCRs that target epitopes that are presented by additional class I alleles that are prevalent in this population such as HLA-C*07 and HLA-A*01, which are expressed by approximately 50% and 30% of the patient population respectively. Therefore, 2 TCRs that recognize an epitope of MAGE-A12 in the context of HLA-C*07 and 2 TCRs that recognize an epitope of MAGE-A3 in the context of HLA-A*01 were isolated from tumor-reactive T-cell clones and cloned in a recombinant retroviral expression vector. Comparative studies indicated that one of the 2 MAGE-A3-reactive TCRs and one of the 2 MAGE-A12-reactive TCRs were superior to the additional TCRs in conferring transduced peripheral blood mononuclear cells with the capacity to recognize a broad array of antigen and MHC-positive target cells. These results provide support for the use of these TCRs in cancer adoptive immunotherapy trials.
- Published
- 2012
- Full Text
- View/download PDF
23. Binuclear copper(II), cobalt(II) and nickel(II) complexes of N1-ethyl-N2-(pyridin-2-yl) hydrazine-1,2-bis(carbothioamide): structural, spectral, pH-metric and biological studies.
- Author
-
El-Gammal OA, Abu El-Reash GM, and El-Gamil MM
- Subjects
- Animals, Antineoplastic Agents pharmacology, Antioxidants pharmacology, Ascites pathology, Cobalt pharmacology, Copper pharmacology, Electron Spin Resonance Spectroscopy, Electrons, Erythrocytes drug effects, Free Radical Scavengers pharmacology, Hemolysis drug effects, Hydrogen-Ion Concentration drug effects, Kinetics, Magnetic Phenomena, Magnetic Resonance Spectroscopy, Mass Spectrometry, Mice, Models, Molecular, Molecular Conformation, Nickel pharmacology, Rats, Spectrophotometry, Infrared, Static Electricity, Thermogravimetry, Coordination Complexes chemistry, Coordination Complexes pharmacology, Hydrazines chemistry, Hydrazines pharmacology
- Abstract
Binuclear Cu(II), Co(II) and Ni(II) complexes derived from N(1)-ethyl-N(2)-(pyridin-2-yl) hydrazine-1,2-bis(carbothioamide) (H(2)PET) have been prepared and characterized by elemental analysis, spectral (IR, UV-vis, EI mass, ESR and (1)HNMR) and magnetic measurements. The isolated complexes assigned the general formula, [M(HPET)(H(2)O)(n)Cl](2)·xH(2)O where M=Cu(II), Co(II) and Ni(II), n=2, 1, 0 and x=0, 0.5 and 0, respectively. IR data revealed that the ligand behaves as monobasic tridentate through (CN)(py), (C-S) and new azomethine, (NC)(∗) groups in the Co(II) complex but in Cu(II) complex, the ligand coordinate via both (CS) groups, one of them in thiol form as well as the new azomethine group. In Ni(II) complex, H(2)PET acts as NSNS monobasic tetradente via (CN)(py), (C-S), (CS) and the new azomethine, (NC)(∗) groups. An octahedral geometry is proposed for all complexes. pH- metric titration was carried out in 50% dioxane-water mixture at 298, 308 and 318 °K, respectively and the dissociation constant of the ligand as well as the stability constants of its complexes were evaluated. Also the kinetic and thermodynamic parameters for the different thermal decomposition steps of the complexes were determined by Coats-Redfern and Horowitz-Metzger methods. Moreover, the anti-oxidant, anti-hemolytic, and cytotoxic activities of the compounds have been tested., (Copyright © 2012 Elsevier B.V. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
24. An antigenic peptide produced by reverse splicing and double asparagine deamidation.
- Author
-
Dalet A, Robbins PF, Stroobant V, Vigneron N, Li YF, El-Gamil M, Hanada K, Yang JC, Rosenberg SA, and Van den Eynde BJ
- Subjects
- Antibodies, Monoclonal, Chemical Fractionation, Chromatography, High Pressure Liquid, Endoplasmic Reticulum metabolism, Histocompatibility Antigens Class I metabolism, Humans, Lymphocytes, Tumor-Infiltrating metabolism, Melanoma metabolism, Monophenol Monooxygenase genetics, Peptides genetics, Peptides isolation & purification, Peptides metabolism, Protein Processing, Post-Translational genetics, Protein Processing, Post-Translational immunology, Protein Transport immunology, Antigen Presentation immunology, Histocompatibility Antigens Class I immunology, Melanoma immunology, Peptides immunology
- Abstract
A variety of unconventional translational and posttranslational mechanisms contribute to the production of antigenic peptides, thereby increasing the diversity of the peptide repertoire presented by MHC class I molecules. Here, we describe a class I-restricted peptide that combines several posttranslational modifications. It is derived from tyrosinase and recognized by tumor-infiltrating lymphocytes isolated from a melanoma patient. This unusual antigenic peptide is made of two noncontiguous tyrosinase fragments that are spliced together in the reverse order. In addition, it contains two aspartate residues that replace the asparagines encoded in the tyrosinase sequence. We confirmed that this peptide is naturally presented at the surface of melanoma cells, and we showed that its processing sequentially requires translation of tyrosinase into the endoplasmic reticulum and its retrotranslocation into the cytosol, where deglycosylation of the two asparagines by peptide-N-glycanase turns them into aspartates by deamidation. This process is followed by cleavage and splicing of the appropriate fragments by the standard proteasome and additional transport of the resulting peptide into the endoplasmic reticulum through the transporter associated with antigen processing (TAP).
- Published
- 2011
- Full Text
- View/download PDF
25. Irradiation enhances human T-cell function by upregulating CD70 expression on antigen-presenting cells in vitro.
- Author
-
Huang J, Wang QJ, Yang S, Li YF, El-Gamil M, Rosenberg SA, and Robbins PF
- Subjects
- Antigen-Presenting Cells immunology, Cell Proliferation, Cells, Cultured, Dendritic Cells immunology, Dendritic Cells radiation effects, Humans, Interferon-gamma metabolism, Interleukin-12 metabolism, Interleukin-23 metabolism, Leukocytes, Mononuclear immunology, Leukocytes, Mononuclear radiation effects, Lymphocyte Activation immunology, Radiation, Ionizing, T-Lymphocytes immunology, Up-Regulation, Antigen-Presenting Cells radiation effects, CD27 Ligand immunology, T-Lymphocytes radiation effects
- Abstract
In addition to the direct killing of tumor cells, radiation therapy can alter the balance of immune cells in vivo due to the differential radiosensitivity of different cell types. The addition of adjuvant radiation therapy before adoptive cell transfer therapy has been shown to enhance antitumor responses in both mouse models and clinical trials. This study examines the effects of in vitro irradiation on the phenotype and function of human antigen-presenting cells. The results indicated that irradiation upregulated CD70 expression on both B cells and mature dendritic cells (DCs). Expression of CD70 on mature DCs was enhanced in a dose-dependent manner, whereas under the same conditions, no significant upregulation of CD80, CD86, or CD40 was observed. The levels of expression of CD70 induced on mature DC by irradiation correlated highly with the ability of those cells to stimulate T-cell proliferation and interferon-γ production. Furthermore, significant reductions in T-cell proliferation and interferon-γ production were seen when CD70 expression on DCs was partially reduced using shRNA, as well as when DCs were incubated with a blocking anti-CD70 antibody. Radiation therapy may therefore enhance T-cell activation in vivo through the CD27 pathway by virtue of its ability to upregulate the expression of CD70 on antigen-presenting cells.
- Published
- 2011
- Full Text
- View/download PDF
26. Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1.
- Author
-
Robbins PF, Morgan RA, Feldman SA, Yang JC, Sherry RM, Dudley ME, Wunderlich JR, Nahvi AV, Helman LJ, Mackall CL, Kammula US, Hughes MS, Restifo NP, Raffeld M, Lee CC, Levy CL, Li YF, El-Gamil M, Schwarz SL, Laurencot C, and Rosenberg SA
- Subjects
- Adult, Cancer Vaccines immunology, Epigenesis, Genetic, Female, Genetic Engineering, Humans, Male, Melanoma immunology, Melanoma mortality, Melanoma secondary, Middle Aged, Neoplasm Metastasis, Neoplasm Staging, Prognosis, Risk Assessment, Sarcoma, Synovial immunology, Sarcoma, Synovial mortality, Sarcoma, Synovial secondary, Skin Neoplasms immunology, Skin Neoplasms mortality, Skin Neoplasms pathology, Survival Analysis, Treatment Outcome, Young Adult, Cancer Vaccines administration & dosage, Immunotherapy methods, Lymphocytes, Tumor-Infiltrating immunology, Melanoma therapy, Sarcoma, Synovial therapy, Skin Neoplasms therapy
- Abstract
Purpose: Adoptive immunotherapy using tumor-infiltrating lymphocytes represents an effective cancer treatment for patients with metastatic melanoma. The NY-ESO-1 cancer/testis antigen, which is expressed in 80% of patients with synovial cell sarcoma and approximately 25% of patients with melanoma and common epithelial tumors, represents an attractive target for immune-based therapies. The current trial was carried out to evaluate the ability of adoptively transferred autologous T cells transduced with a T-cell receptor (TCR) directed against NY-ESO-1 to mediate tumor regression in patients with metastatic melanoma and synovial cell sarcoma., Patients and Methods: A clinical trial was performed in patients with metastatic melanoma or metastatic synovial cell sarcoma refractory to all standard treatments. Patients with NY-ESO-1-positive tumors were treated with autologous TCR-transduced T cells plus 720,000 iU/kg of interleukin-2 to tolerance after preparative chemotherapy. Objective clinical responses were evaluated using Response Evaluation Criteria in Solid Tumors (RECIST)., Results: Objective clinical responses were observed in four of six patients with synovial cell sarcoma and five of 11 patients with melanoma bearing tumors expressing NY-ESO-1. Two of 11 patients with melanoma demonstrated complete regressions that persisted after 1 year. A partial response lasting 18 months was observed in one patient with synovial cell sarcoma., Conclusion: These observations indicate that TCR-based gene therapies directed against NY-ESO-1 represent a new and effective therapeutic approach for patients with melanoma and synovial cell sarcoma. To our knowledge, this represents the first demonstration of the successful treatment of a nonmelanoma tumor using TCR-transduced T cells.
- Published
- 2011
- Full Text
- View/download PDF
27. No correlation between clinical response to CTLA-4 blockade and presence of NY-ESO-1 antibody in patients with metastatic melanoma.
- Author
-
Goff SL, Robbins PF, El-Gamil M, and Rosenberg SA
- Subjects
- Antibodies, Blocking administration & dosage, Antibodies, Monoclonal administration & dosage, CTLA-4 Antigen, Clinical Trials as Topic, Enzyme-Linked Immunosorbent Assay, Humans, Immunoglobulins blood, Ipilimumab, Melanoma blood, Melanoma pathology, Membrane Glycoproteins administration & dosage, Neoplasm Metastasis, Peptide Fragments administration & dosage, gp100 Melanoma Antigen, Antibody Formation, Antigens, CD immunology, Antigens, Neoplasm immunology, Melanoma drug therapy, Melanoma immunology, Membrane Proteins immunology
- Published
- 2009
- Full Text
- View/download PDF
28. Recognition of NY-ESO-1+ tumor cells by engineered lymphocytes is enhanced by improved vector design and epigenetic modulation of tumor antigen expression.
- Author
-
Wargo JA, Robbins PF, Li Y, Zhao Y, El-Gamil M, Caragacianu D, Zheng Z, Hong JA, Downey S, Schrump DS, Rosenberg SA, and Morgan RA
- Subjects
- Cell Line, Tumor, Cloning, Molecular, Cysteine chemistry, Cytokines metabolism, Genetic Vectors, Humans, Immunotherapy methods, Lymphocytes metabolism, Models, Genetic, Neoplasms therapy, Peptides chemistry, Receptors, Antigen, T-Cell metabolism, T-Lymphocytes metabolism, Antigens, Neoplasm metabolism, Epigenesis, Genetic, Lymphocytes immunology
- Abstract
The therapeutic use of T cell receptor (TCR)-transduced peripheral blood lymphocytes (PBL) targeting tumor-associated antigens is emerging as a promising investigational treatment for patients with cancer. Initial response rates to therapy were low, suggesting the need to improve the function of TCR-transduced PBL. We constructed standard bicistronic retroviral vectors using an internal promoter or internal ribosomal entry site element as well as vectors incorporating coding sequences for 2A linker peptides between coding sequences for alpha and beta chains targeting the cancer-testis (CT) antigen, NY-ESO-1. Incorporation of coding sequences for 2A linker peptides in the bicistronic TCR expression cassette resulted in up to a fourfold increase in TCR expression and a significant improvement in effector function as measured by interferon-gamma release following co-culture with peptide-pulsed targets and NY-ESO-1+ tumors. We also sought to enhance reactivity of TCR-transduced PBL against tumor targets by modulation of tumor antigen expression on target cells. Induction of NY-ESO-1 expression on tumor targets using the demethylating agent 5-aza-2'-deoxycytidine (alone or in combination with the histone deacetylase inhibitor depsipeptide) resulted in enhanced interferon-gamma secretion by the TCR-transduced PBL on culture with treated targets. Taken together, these results indicate that design of TCR-based vectors incorporating 2A linker peptides improves TCR expression and effector function of transduced PBL. Furthermore, induction of CT antigen expression through treatment of tumor targets with chromatin-remodeling agents may augment TCR-based immunotherapy targeting these antigens. These results have relevance for TCR-based gene therapies targeting common epithelial malignancies.
- Published
- 2009
- Full Text
- View/download PDF
29. Single and dual amino acid substitutions in TCR CDRs can enhance antigen-specific T cell functions.
- Author
-
Robbins PF, Li YF, El-Gamil M, Zhao Y, Wargo JA, Zheng Z, Xu H, Morgan RA, Feldman SA, Johnson LA, Bennett AD, Dunn SM, Mahon TM, Jakobsen BK, and Rosenberg SA
- Subjects
- Antigens, Neoplasm genetics, Antigens, Neoplasm immunology, Cell Line, Tumor, Complementarity Determining Regions immunology, HLA-A Antigens genetics, HLA-A2 Antigen, Humans, MART-1 Antigen, Neoplasm Proteins genetics, Neoplasm Proteins immunology, Receptors, Antigen, T-Cell immunology, Amino Acid Substitution immunology, CD4-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes immunology, Complementarity Determining Regions genetics, HLA-A Antigens immunology, Receptors, Antigen, T-Cell genetics
- Abstract
Single and dual amino acid substitution variants were generated in the TCR CDRs of three TCRs that recognize tumor-associated Ags. Substitutions that enhance the reactivity of TCR gene-modified T cells to the cognate Ag complex were identified using a rapid RNA-based transfection system. The screening of a panel of variants of the 1G4 TCR, that recognizes a peptide corresponding to amino acid residues 157-165 of the human cancer testis Ag NY-ESO-1 (SLLMWITQC) in the context of the HLA-A*02 class I allele, resulted in the identification of single and dual CDR3alpha and CDR2beta amino acid substitutions that dramatically enhanced the specific recognition of NY-ESO-1(+)/HLA-A*02(+) tumor cell lines by TCR gene-modified CD4(+) T cells. Within this group of improved TCRs, a dual substitution in the 1G4 TCR CDR3alpha chain was identified that enhanced Ag-specific reactivity in gene-modified CD4(+) and CD8(+) T cells. Separate experiments on two distinct TCRs that recognize the MART-1 27-35 (AAGIGILTV) peptide/HLA-A*02 Ag complex characterized single amino acid substitutions in both TCRs that enhanced CD4(+) T cell Ag-specific reactivity. These results indicate that simple TCR substitution variants that enhance T cell function can be identified by rapid transfection and assay techniques, providing the means for generating potent Ag complex-specific TCR genes for use in the study of T cell interactions and in T cell adoptive immunotherapy.
- Published
- 2008
- Full Text
- View/download PDF
30. Enhanced antitumor activity of T cells engineered to express T-cell receptors with a second disulfide bond.
- Author
-
Cohen CJ, Li YF, El-Gamil M, Robbins PF, Rosenberg SA, and Morgan RA
- Subjects
- Cysteine genetics, Cysteine metabolism, Humans, Jurkat Cells, Leukocytes, Mononuclear immunology, Melanoma immunology, Protein Engineering, Receptors, Antigen, T-Cell biosynthesis, Receptors, Antigen, T-Cell genetics, T-Lymphocytes immunology, Cysteine immunology, Genetic Therapy methods, Immunotherapy, Adoptive methods, Leukocytes, Mononuclear physiology, Receptors, Antigen, T-Cell immunology, T-Lymphocytes physiology
- Abstract
Adoptive transfer of genetically T-cell receptor (TCR)-modified lymphocytes has been recently reported to cause objective cancer regression. However, a major limitation to this approach is the mispairing of the introduced chains with the endogenous TCR subunits, which leads to reduced TCR surface expression and, subsequently, to their lower biological activity. We here show that it is possible to improve TCR gene transfer by adding a single cysteine on each receptor chain to promote the formation of an additional interchain disulfide bond. We show that cysteine-modified receptors were more highly expressed on the surface of human lymphocytes compared with their wild-type counterparts and able to mediate higher levels of cytokine secretion and specific lysis when cocultured with specific tumor cell lines. Furthermore, cysteine-modified receptors retained their enhanced function in CD4(+) lymphocytes. We also show that this approach can be employed to enhance the function of humanized and native murine receptors in human cells. Preferential pairing of cysteine-modified receptor chains accounts for these observations, which could have significant implications for the improvement of TCR gene therapy.
- Published
- 2007
- Full Text
- View/download PDF
31. Modulation by IL-2 of CD70 and CD27 expression on CD8+ T cells: importance for the therapeutic effectiveness of cell transfer immunotherapy.
- Author
-
Huang J, Kerstann KW, Ahmadzadeh M, Li YF, El-Gamil M, Rosenberg SA, and Robbins PF
- Subjects
- Antigens, CD genetics, Antigens, CD metabolism, CD27 Ligand, CD8-Positive T-Lymphocytes cytology, CD8-Positive T-Lymphocytes metabolism, Cell Proliferation, Cells, Cultured, Clinical Trials as Topic, Humans, Interleukin-2 administration & dosage, Interleukin-2 metabolism, Lymphocyte Activation immunology, Lymphocytes, Tumor-Infiltrating immunology, Lymphocytes, Tumor-Infiltrating metabolism, Lymphocytes, Tumor-Infiltrating transplantation, Melanoma pathology, Membrane Proteins genetics, Membrane Proteins metabolism, Protein Binding immunology, Tumor Cells, Cultured, Tumor Necrosis Factor Receptor Superfamily, Member 7 genetics, Tumor Necrosis Factor Receptor Superfamily, Member 7 metabolism, Tumor Necrosis Factors genetics, Tumor Necrosis Factors metabolism, Up-Regulation immunology, Antigens, CD biosynthesis, CD8-Positive T-Lymphocytes immunology, Immunotherapy, Adoptive methods, Interleukin-2 physiology, Melanoma immunology, Melanoma therapy, Membrane Proteins biosynthesis, Tumor Necrosis Factor Receptor Superfamily, Member 7 biosynthesis, Tumor Necrosis Factors biosynthesis
- Abstract
Proper T cell function relies on the integration of signals delivered by Ag, cytokine, and costimulatory receptors. In this study, the interactions between IL-2, CD27, and its ligand CD70 and their effects on human T cell function were examined. Unstimulated CD8(+) T cells expressed relatively low levels of CD70 and high levels of CD27. Incubation in vitro with high doses of IL-2 (3,000 IU/ml) or administration of IL-2 in vivo resulted in substantial up-regulation of CD70 expression and the concomitant loss of cell surface CD27 expression on CD8(+) cells. Withdrawal of IL-2 from activated CD8(+) T cells that had been maintained in IL-2 resulted in a reversal of the expression of these two markers, whereas reciprocal changes were seen following treatment of PBMCs with IL-2. The proliferation observed in cells stimulated with IL-2 primarily occurred in a subset of the CD70(+)CD8(+) T cells that up-regulated IL-2 receptor expression but did not occur in CD70(-)CD8(+) T cells. Blocking CD70 resulted in a significant reduction of T cell proliferation induced by high-dose IL-2, indicating that the interaction of CD70 with CD27 played a direct role in T cell activation mediated by IL-2. Finally, studies conducted on tumor-infiltrating lymphocyte (TIL) samples that were administered to melanoma patients indicated that the size of the pool of CD27(+)CD8(+) T cells in bulk TILs was highly associated (p = 0.004) with the ability of these TILs to mediate tumor regression following adoptive transfer.
- Published
- 2006
- Full Text
- View/download PDF
32. Clonal persistence and evolution during a decade of recurrent melanoma.
- Author
-
Wang E, Voiculescu S, Le Poole IC, El-Gamil M, Li X, Sabatino M, Robbins PF, Nickoloff BJ, and Marincola FM
- Subjects
- Cell Line, Tumor, Clone Cells, Female, Humans, Karyotyping, Melanoma secondary, Mutation, Neoplasm Recurrence, Local pathology, Skin Neoplasms pathology, Melanoma genetics, Neoplasm Recurrence, Local genetics, Neoplastic Stem Cells, Skin Neoplasms genetics, beta Catenin genetics
- Abstract
A patient with metastatic cutaneous melanoma responsive to immunotherapy experienced several recurrences over a decade of observation. With each recurrence, biopsies were obtained and cell lines generated. A rare mutation of the beta-catenin gene and an unbalanced methylation of the androgen receptor were documented in all cell lines. Karyotyping and comparative genomic hybridization identified consistent genetic traits in spite of divergent phenotypes, suggesting that all the metastases were derived from the same primary tumor, although they were each probably not derived from the most recent previous metastasis in a sequential manner. Thus, metastatic melanoma recurs from a common progenitor cell and phenotypic changes occur around a central core of genetic stability. This observation may bear significance for the development of targeted anticancer therapies.
- Published
- 2006
- Full Text
- View/download PDF
33. Survival, persistence, and progressive differentiation of adoptively transferred tumor-reactive T cells associated with tumor regression.
- Author
-
Huang J, Khong HT, Dudley ME, El-Gamil M, Li YF, Rosenberg SA, and Robbins PF
- Subjects
- Adult, Antigens, CD analysis, Antigens, CD genetics, Antigens, Neoplasm, Cell Differentiation, Humans, Lymphocytes, Tumor-Infiltrating transplantation, Male, Neoplasm Proteins immunology, Phenotype, Receptors, Antigen, T-Cell, alpha-beta genetics, Immunotherapy, Adoptive, Lymphocytes, Tumor-Infiltrating immunology, Melanoma immunology, Melanoma therapy, Receptors, Antigen, T-Cell, alpha-beta chemistry
- Abstract
Objective clinical responses have been observed in approximately 50% of patients who received non-myeloablative chemotherapy prior to the adoptive transfer of autologous melanoma-reactive tumor-infiltrating lymphocytes (TILs). Recent studies carried out through the use of antibodies directed against T-cell-receptor beta chain variable region (TRBV) products, as well as by direct sequencing of the expressed TRBV gene products, indicated that clinical responses in this trial were associated with the level of persistence of adoptively transferred T cells. In an attempt to further characterize T cells that persist in vivo following adoptive transfer, five dominant T-cell clonotypes were identified in TIL 2035, an adoptively transferred TIL that was associated with the complete regression of multiple metastases. The most highly persistent clonotype, which expressed the BV1 TR gene product, recognized the MAGE-6 cancer/testis antigen in the context of HLA-A23. This clonotype was detected in peripheral blood for over 16 months following adoptive transfer, expressed relatively higher levels of the co-stimulatory markers CD28 and CD27, and possessed telomeres that were long relative to other clonotypes present in TIL 2035 that showed only short-term persistence. The long-term persistent BV1 clonotype appeared to differentiate more slowly toward an end-stage effector in vivo than short-term persistent clonotypes, as manifested by the downregulation of CD28, CD27, and CD45RO and upregulation of CD57 and CD45RA expression on these T cells. These results indicated that the differentiation stage and replicative history of individual TIL clonotypes might be associated with their ability to survive and to persist in vivo, and progressive differentiation of the persistent clonotypes occurred following adoptive transfer.
- Published
- 2005
- Full Text
- View/download PDF
34. Cutting edge: persistence of transferred lymphocyte clonotypes correlates with cancer regression in patients receiving cell transfer therapy.
- Author
-
Robbins PF, Dudley ME, Wunderlich J, El-Gamil M, Li YF, Zhou J, Huang J, Powell DJ Jr, and Rosenberg SA
- Subjects
- Cell Proliferation, Cell Survival genetics, Cell Survival immunology, Clone Cells, Gene Rearrangement, beta-Chain T-Cell Antigen Receptor, Humans, Immunoglobulin Variable Region administration & dosage, Immunoglobulin Variable Region analysis, Immunoglobulin Variable Region genetics, Lymphocyte Count, Lymphocytes, Tumor-Infiltrating immunology, Lymphocytes, Tumor-Infiltrating metabolism, Lymphocytes, Tumor-Infiltrating pathology, Melanoma secondary, Pilot Projects, Receptors, Antigen, T-Cell, alpha-beta administration & dosage, Receptors, Antigen, T-Cell, alpha-beta analysis, Receptors, Antigen, T-Cell, alpha-beta genetics, Immunotherapy, Adoptive methods, Lymphocyte Transfusion methods, Lymphocytes, Tumor-Infiltrating transplantation, Melanoma immunology, Melanoma therapy
- Abstract
The lack of persistence of transferred autologous mature lymphocytes in humans has been a major limitation to the application of effective cell transfer therapies. The results of a pilot clinical trial in 13 patients with metastatic melanoma suggested that conditioning with nonmyeloablative chemotherapy before adoptive transfer of activated tumor-reactive T cells enhances tumor regression and increases the overall rates of objective clinical responses. The present report examines the relationship between T cell persistence and tumor regression through analysis of the TCR beta-chain V region gene products expressed in samples obtained from 25 patients treated with this protocol. Sequence analysis demonstrated that there was a significant correlation between tumor regression and the degree of persistence in peripheral blood of adoptively transferred T cell clones, suggesting that inadequate T cell persistence may represent a major factor limiting responses to adoptive immunotherapy.
- Published
- 2004
- Full Text
- View/download PDF
35. T cells associated with tumor regression recognize frameshifted products of the CDKN2A tumor suppressor gene locus and a mutated HLA class I gene product.
- Author
-
Huang J, El-Gamil M, Dudley ME, Li YF, Rosenberg SA, and Robbins PF
- Subjects
- Adult, Amino Acid Sequence, Base Sequence, Cell Line, Tumor, Clone Cells, Cyclin-Dependent Kinase Inhibitor p16 genetics, Cyclin-Dependent Kinase Inhibitor p16 immunology, Epitopes, T-Lymphocyte genetics, Epitopes, T-Lymphocyte isolation & purification, Female, Genetic Markers, HLA-A Antigens genetics, HLA-A Antigens immunology, HLA-A11 Antigen, Humans, Immunotherapy, Adoptive, Lymphocytes, Tumor-Infiltrating metabolism, Lymphocytes, Tumor-Infiltrating transplantation, Melanoma, Experimental secondary, Molecular Sequence Data, Open Reading Frames immunology, T-Lymphocyte Subsets metabolism, T-Lymphocyte Subsets transplantation, Tumor Suppressor Protein p14ARF genetics, Tumor Suppressor Protein p14ARF isolation & purification, Cyclin-Dependent Kinase Inhibitor p16 metabolism, Frameshift Mutation immunology, HLA-A Antigens metabolism, Lymphocytes, Tumor-Infiltrating immunology, Melanoma, Experimental immunology, Melanoma, Experimental therapy, Remission Induction, T-Lymphocyte Subsets immunology
- Abstract
The dramatic tumor regression observed following adoptive T cell transfer in some patients has led to attempts to identify novel Ags to understand the nature of these responses. Nearly complete regression of multiple metastatic melanoma lesions was observed in patient 1913 following adoptive transfer of autologous tumor-infiltrating lymphocytes. The autologous 1913 melanoma cell line expressed a mutated HLA-A11 class I gene product that was recognized by the bulk tumor-infiltrating lymphocytes as well as a dominant T cell clone derived from this line. A second dominant T cell clone, T1D1, did not recognize the mutated HLA-A11 product, but recognized an allogeneic melanoma cell line that shared expression of HLA-A11 with the parental tumor cell line. Screening of an autologous melanoma cDNA library with clone T1D1 T cells in a cell line expressing the mutated HLA-A11 gene product resulted in the isolation of a p14ARF transcript containing a 2-bp deletion in exon 2. The T cell epitope recognized by T1D1, which was encoded within the frameshifted region of the deleted p14ARF transcript, was also generated from frameshifted p14ARF or p16INK4a transcripts that were isolated from two additional melanoma cell lines. The results of monitoring studies indicated that T cell clones reactive with the mutated HLA-A11 gene product and the mutated p14ARF product were highly represented in the peripheral blood of patient 1913 1 wk following adoptive transfer, indicating that they may have played a role in the nearly complete tumor regression that was observed following this treatment.
- Published
- 2004
- Full Text
- View/download PDF
36. Identification of a colorectal tumor-associated antigen (COA-1) recognized by CD4(+) T lymphocytes.
- Author
-
Maccalli C, Li YF, El-Gamil M, Rosenberg SA, and Robbins PF
- Subjects
- Amino Acid Sequence, Antigens, Neoplasm biosynthesis, Antigens, Neoplasm genetics, Base Sequence, Cell Line, Tumor, Colorectal Neoplasms genetics, Epitopes, T-Lymphocyte genetics, Epitopes, T-Lymphocyte immunology, Humans, Molecular Sequence Data, Antigens, Neoplasm immunology, CD4-Positive T-Lymphocytes immunology, Colorectal Neoplasms immunology
- Abstract
Only a limited number of target molecules have been shown to be recognized by colon tumor-reactive T cells, limiting the options for the development of immunotherapies for patients with colon cancer. The current studies were undertaken in an attempt to generate tumor-reactive T cells that could be used to identify and characterize novel colon tumor-associated antigens. Multiple CD4(+) T-cell clones isolated either from tumor-infiltrating lymphocytes or peripheral blood mononuclear cells that were sensitized in vitro with autologous tumor cells from a colon cancer patient, 1869, recognized autologous tumor cells in a class II HLA-DR-restricted manner. One of the peripheral blood mononuclear cell clones, clone C111, was used to screen pools of clones that were generated from an autologous colon tumor cell line cDNA library. A cDNA clone that was isolated encoded a protein that was termed colorectal tumor-associated antigen-1 (COA-1). This product was recognized in the context of the two autologous HLA-DRbeta1 alleles, HLA-DRbeta1*0402 and DRbeta1*1301. The nucleotide sequence of the COA-1 transcript was nearly identical to multiple expressed sequence tag sequences that encode variants of Socius, a protein that was found recently to bind to members of the Rnd family of GTPases. The COA-1 gene was expressed at relatively comparable levels in colorectal and melanoma tumor cells, EBV-infected B cells, normal B cells, and cultured fibroblast cell lines. However, the gene that was isolated from normal cell types contained a single nucleotide substitution, resulting in an amino acid change near the COOH terminus of the protein. Although the minimal epitope recognized by CD4(+) cells was encoded by sequences that were upstream from this substitution, C111 T cells did not appear to recognize the normal gene product. Therefore, this alteration may either affect the localization or the processing of this gene product, which may at least in part be responsible for the differential recognition of tumor and normal cells.
- Published
- 2003
37. Multiple HLA class II-restricted melanocyte differentiation antigens are recognized by tumor-infiltrating lymphocytes from a patient with melanoma.
- Author
-
Robbins PF, El-Gamil M, Li YF, Zeng G, Dudley M, and Rosenberg SA
- Subjects
- Alleles, Amino Acid Sequence, Animals, Antigens, Differentiation immunology, Antigens, Neoplasm immunology, CD4-Positive T-Lymphocytes immunology, CD4-Positive T-Lymphocytes metabolism, COS Cells, Clone Cells, Cytotoxicity Tests, Immunologic, Epitopes, T-Lymphocyte immunology, Epitopes, T-Lymphocyte metabolism, HLA Antigens immunology, HLA-DR Antigens genetics, HLA-DR Antigens immunology, HLA-DR Antigens metabolism, HLA-DRB1 Chains, HLA-DRB5 Chains, Histocompatibility Antigens Class II immunology, Humans, Isoantigens immunology, Isoantigens metabolism, Lymphocyte Activation immunology, Lymphocytes, Tumor-Infiltrating metabolism, Melanocytes metabolism, Melanoma metabolism, Membrane Glycoproteins immunology, Membrane Glycoproteins metabolism, Molecular Sequence Data, Neoplasm Proteins immunology, Neoplasm Proteins metabolism, Proteins immunology, Proteins metabolism, Tumor Cells, Cultured, gp100 Melanoma Antigen, Antigens, Differentiation metabolism, Antigens, Neoplasm metabolism, HLA Antigens metabolism, Histocompatibility Antigens Class II metabolism, Lymphocytes, Tumor-Infiltrating immunology, Melanocytes immunology, Melanoma immunology, Oxidoreductases
- Abstract
Dramatic clinical responses were observed in patient 888 following the adoptive transfer of autologous tumor-infiltrating lymphocytes (TIL). Previously, extensive analysis of the specificity of class I-restricted T cells from patient 888 TIL has revealed that these T cells recognize a mutated, as well as several nonmutated tumor Ags. Additional studies that were conducted on TIL from patient 888 indicated that they contained CD4-positive T cells that recognized the autologous tumor that had been induced to express HLA class II molecules. Tumor-reactive CD4-positive T cell clones were isolated from TIL and tested for their ability to react with Ags that are recognized by HLA class I-restricted, melanoma-reactive T cells. Using this approach, T cell clones were identified that recognized an epitope expressed in both the tyrosinase-related protein 1 and tyrosinase-related protein 2 Ags in the context of the HLA-DRbeta1*1502 class II gene product. Additional clones were found to recognize an epitope of gp100 in the context of the same HLA-DR restriction element. These observations provide an impetus to develop strategies directed toward generating HLA class II-restricted tumor-reactive T cells.
- Published
- 2002
- Full Text
- View/download PDF
38. Generation of NY-ESO-1-specific CD4+ and CD8+ T cells by a single peptide with dual MHC class I and class II specificities: a new strategy for vaccine design.
- Author
-
Zeng G, Li Y, El-Gamil M, Sidney J, Sette A, Wang RF, Rosenberg SA, and Robbins PF
- Subjects
- Amino Acid Sequence, Epitopes, T-Lymphocyte immunology, HLA-A Antigens immunology, HLA-DP beta-Chains, Humans, Melanoma blood, Melanoma immunology, Molecular Sequence Data, T-Lymphocytes, Cytotoxic immunology, Antigens, Neoplasm immunology, CD4-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes immunology, Cancer Vaccines immunology, HLA-A2 Antigen immunology, HLA-DP Antigens immunology, Membrane Proteins, Peptide Fragments immunology, Proteins immunology
- Abstract
The existence of overlapping CD8+ and CD4+ T-cell epitopes within certain tumor antigens provides an opportunity to test the hypothesis that relatively short peptides could be used to generate both CD8+ and CD4+ T cells against tumor. In this report, T-cell responses to a fragment of the tumor antigen NY-ESO-1 that contained an HLA-DP4-restricted helper T cell epitope as well as an HLA-A2-restricted cytotoxic T cell epitope were analyzed. One peptide, ESO:157-170 (SLLMWITQCFLPVF) was recognized by both NY-ESO-1-reactive CD8+ and CD4+ T-cell clones. Both CD4+ and CD8+ T cells were efficiently generated from the peripheral blood of multiple melanoma patients after in vitro stimulations using ESO:157-170. Dual-specific peptides containing both cytotoxic T-cell and helper T-cell epitopes may represent an attractive strategy of vaccine design aimed at generating tumor-reactive CD4+ and CD8+ T cells.
- Published
- 2002
39. Identification of BING-4 cancer antigen translated from an alternative open reading frame of a gene in the extended MHC class II region using lymphocytes from a patient with a durable complete regression following immunotherapy.
- Author
-
Rosenberg SA, Tong-On P, Li Y, Riley JP, El-Gamil M, Parkhurst MR, and Robbins PF
- Subjects
- Amino Acid Sequence, Antigens, Neoplasm biosynthesis, Cell Line, Clone Cells, Cloning, Molecular, Humans, Interferon-gamma biosynthesis, Melanoma pathology, Melanoma therapy, Molecular Sequence Data, Neoplasm Metastasis, Open Reading Frames, Peptides chemistry, Peptides immunology, Protein Biosynthesis, RNA, Messenger biosynthesis, Tumor Cells, Cultured, Antigens, Neoplasm genetics, Antigens, Neoplasm immunology, Genes, MHC Class II, Melanoma immunology, T-Lymphocytes, Cytotoxic immunology
- Abstract
Multiple human cancer Ags have been identified, although little is known concerning which would be most effectively used in cancer immunotherapy. To gain insight into the selection of appropriate Ags, the immunologic reactivity of a patient who had a durable complete regression of melanoma metastases was measured. PBMCs were directly cloned using the monoclonal anti-CD3 Ab OKT3 and IL-2 without any bias introduced by previous culture. A lymphocyte clone recognized a previously unknown shared melanoma Ag that was identified as the BING-4 protein encoded in a gene-rich region of the extended class II MHC. The HLA-A2-restricted BING-4 immunodominant peptide was translated from a 10-aa-long alternative open reading frame. In vitro sensitization against this peptide generated lymphocytes reactive against HLA-A2(+) melanomas. Real-time semiquantitative RT-PCR analysis revealed that 8 of 15 melanoma cell lines overexpressed BING-4, and this correlated with recognition by lymphocytes. Overexpression was not found in normal tissues or other tumor types. Thus, BING-4 represents another candidate Ag for possible use in the immunotherapy of patients with melanoma.
- Published
- 2002
- Full Text
- View/download PDF
40. Melanoma-Reactive CD8+ T cells recognize a novel tumor antigen expressed in a wide variety of tumor types.
- Author
-
Harada M, Li YF, El-Gamil M, Ohnmacht GA, Rosenberg SA, and Robbins PF
- Subjects
- Amino Acid Sequence, Base Sequence, Cells, Cultured, DNA-Binding Proteins, Epitopes immunology, Gene Expression, Humans, Lymphocyte Culture Test, Mixed, Molecular Sequence Data, Tumor Cells, Cultured, Antigens, Neoplasm immunology, CD8-Positive T-Lymphocytes immunology, HLA-A1 Antigen immunology, Interferon-gamma isolation & purification, Melanoma immunology, Nuclear Proteins genetics, Nuclear Proteins isolation & purification
- Abstract
An autologous melanoma cell line selected for loss of expression of the immunodominant MART-1 and gp100 antigens was initially used to carry out a mixed lymphocyte tumor culture (MLTC) in a patient who expressed the human leukocyte antigen (HLA)-AI and HLA-A2 class I major histocompatibility complex alleles. Ten clones identified from this MLTC seemed to recognize melanoma in an HLA-A1-restricted manner but failed to recognize a panel of previously described melanoma antigens. The screening of an autologous melanoma cDNA library with one HLA-Al-restricted melanoma-reactive T-cell clone resulted in the isolation of a cDNA clone called AIM-2 (antigen isolated from immunoselected melanoma-2). The AIM-2 transcript seemed to have retained an intronic sequence based on its alignment with genomic sequences as well as expressed sequence tags. This transcript was not readily detected after Northern blot analysis of melanoma mRNA, indicating that only low levels of this product may be expressed in tumor cells. Quantitative reverse transcriptase-polymerase chain reaction analysis, however, demonstrated a correlation between T-cell recognition and expression in HLA-A1-expressing tumor cell lines. A peptide that was encoded within a short open reading frame of 23 amino acids and conformed to the HLA-A1 binding motif RSDSGQQARY was found to represent the T-cell epitope. The AIM-2-reactive T-cell clone recognized a number of neuroectodermal tumors as well as breast, ovarian, and colon carcinomas that expressed HLA-A1, indicating that this represents a widely expressed tumor antigen. Thus, AIM-2 may represent a potential target for the development of vaccines in patients bearing tumors of a variety of histologies.
- Published
- 2001
- Full Text
- View/download PDF
41. Use of an in vitro immunoselected tumor line to identify shared melanoma antigens recognized by HLA-A*0201-restricted T cells.
- Author
-
Harada M, Li YF, El-Gamil M, Rosenberg SA, and Robbins PF
- Subjects
- Amino Acid Sequence, Antigen Presentation immunology, Antigens, Neoplasm genetics, Base Sequence, Cloning, Molecular, Crystallins, DNA, Complementary genetics, DNA, Complementary isolation & purification, DNA, Neoplasm genetics, DNA, Neoplasm isolation & purification, Epitope Mapping, Humans, Intramolecular Oxidoreductases genetics, Intramolecular Oxidoreductases immunology, Lymphocyte Culture Test, Mixed, MART-1 Antigen, Membrane Glycoproteins genetics, Membrane Glycoproteins immunology, Molecular Sequence Data, Neoplasm Proteins genetics, Neoplasm Proteins immunology, Protein Biosynthesis, Proteins genetics, Proteins immunology, T-Lymphocytes immunology, T-Lymphocytes, Cytotoxic immunology, Transfection, Tumor Cells, Cultured, gp100 Melanoma Antigen, Antigens, Neoplasm immunology, Epitopes, T-Lymphocyte immunology, HLA-A2 Antigen immunology, Melanoma immunology, Membrane Proteins
- Abstract
An immunoselected melanoma cell line that had lost expression of the dominant melanoma antigens MART-1 and gp100 was generated in an attempt to identify previously unknown tumor antigens. After repeated stimulation with the autologous immunoselected tumor line, a number of HLA-A*0201-restricted T-cell clones were established from the peripheral blood of a single melanoma patient. One T-cell clone (C-22) recognized 14 of 16 HLA-A2+ melanoma cell lines, as well as HLA-A2+ melanocytes but recognized neither HLA-A2+ fibroblasts nor autologous B cells. Screening of an autologous cDNA library resulted in the isolation of a transcript identical to an entry in the expressed sequence tag database. Northern blot analysis revealed that this gene was expressed in most melanoma cell lines and melanocytes but not in normal tissues. The peptide epitope (AMF-GREFCYA) recognized by clone C-22 was identified based on studies of the recognition of truncated cDNAs and the use of the consensus HLAA*0201 binding motif. A second T-cell clone (C-29) was found to recognize a new tyrosinase-related protein 2 epitope (455-463; YAIDLPVSV) in an HLA-A*0201-restricted manner. Together, these results provide additional targets that can be used for the development of immunotherapeutic protocols in HLA-A2+ melanoma patients and demonstrate the utility of immunoselected tumor lines for the identification of new melanoma antigens.
- Published
- 2001
42. Nuclear beta-catenin displays GSK-3beta- and APC-independent proteasome sensitivity in melanoma cells.
- Author
-
Bonvini P, Hwang SG, El-Gamil M, Robbins P, Kim JS, Trepel J, and Neckers L
- Subjects
- Adenomatous Polyposis Coli Protein, Cell Nucleus metabolism, Chromatin metabolism, Detergents chemistry, Glycogen Synthase Kinase 3, Glycogen Synthase Kinases, Humans, Melanoma, Multienzyme Complexes antagonists & inhibitors, Proteasome Endopeptidase Complex, Solubility, Subcellular Fractions metabolism, Tumor Cells, Cultured, beta Catenin, Calcium-Calmodulin-Dependent Protein Kinases metabolism, Cysteine Endopeptidases metabolism, Cytoskeletal Proteins metabolism, Multienzyme Complexes metabolism, Trans-Activators
- Abstract
Colon carcinoma and melanoma cells containing either a deletion of the adenomatous polyposis coli tumor suppressor protein (APC) or mutation of the site in beta-catenin phosphorylated by glycogen synthase kinase-3beta (GSK-3beta) display elevated levels of detergent-soluble beta-catenin due to insensitivity of the cytosolic protein to proteasome-dependent degradation. In this study, we have examined the effect of beta-catenin mutation (S37F) or APC loss on the proteasome sensitivity of additional subcellular beta-catenin pools in melanoma cells. In contrast to detergent-soluble beta-catenin, the detergent-insoluble protein remains proteasome-sensitive irrespective of S37F mutation or APC status. This insoluble component appears associated primarily with nuclear cytoskeletal elements. In addition, DNase I treatment solubilized a portion of detergent-insoluble beta-catenin, suggesting that this fraction also contains chromatin-associated protein, and correlating with a proteasome-sensitive elevation in beta-catenin-stimulated reporter activity. Since the detergent-insoluble nuclear component of beta-catenin displays GSK-3beta- and APC-independent proteasome sensitivity, distinct from the soluble nuclear and cytosolic pools of this protein, regulation of beta-catenin proteasome sensitivity and the contribution of this process to beta-catenin function may be more complex than previously appreciated.
- Published
- 2000
- Full Text
- View/download PDF
43. Stabilization of beta-catenin by genetic defects in melanoma cell lines.
- Author
-
Rubinfeld B, Robbins P, El-Gamil M, Albert I, Porfiri E, and Polakis P
- Subjects
- Adenomatous Polyposis Coli Protein, Animals, Cell Line, Cytoskeletal Proteins chemistry, Cytoskeletal Proteins metabolism, DNA-Binding Proteins metabolism, Humans, Lymphoid Enhancer-Binding Factor 1, Melanoma metabolism, Mice, Mutation, Point Mutation, RNA Splicing, RNA, Messenger genetics, RNA, Neoplasm genetics, Transcription Factors metabolism, Transfection, Tumor Cells, Cultured, Up-Regulation, beta Catenin, Cytoskeletal Proteins genetics, Gene Expression Regulation, Neoplastic, Genes, APC, Melanoma genetics, Trans-Activators
- Abstract
Signal transduction by beta-catenin involves its posttranslational stabilization and downstream coupling to the Lef and Tcf transcription factors. Abnormally high amounts of beta-catenin were detected in 7 of 26 human melanoma cell lines. Unusual messenger RNA splicing and missense mutations in the beta-catenin gene (CTNNB1) that result in stabilization of the protein were identified in six of the lines, and the adenomatous polyposis coli tumor suppressor protein (APC) was altered or missing in two others. In the APC-deficient cells, ectopic expression of wild-type APC eliminated the excess beta-catenin. Cells with stabilized beta-catenin contained a constitutive beta-catenin-Lef-1 complex. Thus, genetic defects that result in up-regulation of beta-catenin may play a role in melanoma progression.
- Published
- 1997
- Full Text
- View/download PDF
44. Identification of tyrosinase-related protein 2 as a tumor rejection antigen for the B16 melanoma.
- Author
-
Bloom MB, Perry-Lalley D, Robbins PF, Li Y, el-Gamil M, Rosenberg SA, and Yang JC
- Subjects
- Animals, Base Sequence, Female, Isomerases immunology, Melanoma, Experimental therapy, Mice, Mice, Inbred C57BL, Molecular Sequence Data, Mutagenesis, Site-Directed, Structure-Activity Relationship, T-Lymphocytes immunology, Tumor Cells, Cultured, Vaccination, Antigens, Neoplasm analysis, Intramolecular Oxidoreductases, Isomerases analysis, Melanoma, Experimental immunology
- Abstract
Recently, major advances have been made in the identification of antigens from human melanoma which are recognized by T cells. In spite of this, little is known about the optimal ways to use these antigens to treat patients with cancer. Progress in this area is likely to require accurate preclinical animal models, but the availability of such models has lagged behind developments in human tumor immunology. Whereas many of the identified human melanoma antigens are normal tissue differentiation proteins, analogous murine tumor antigens have not yet been identified. In this paper we identify a normal tissue differentiation antigen, tyrosinase-related protein 2 (TRP-2), expressed by the murine B16 melanoma which was found by screening a cDNA library from B16 with tumor-reactive cytotoxic T lymphocytes (CTL). A peptide conforming to the predicted MHC class I H2-Kb binding motif, TRP-2181-188, was identified as the major reactive epitope within TRP-2 recognized by these anti-B16 CTLs. By site-directed mutagenesis, it was shown that alteration of this epitope eliminated recognition of TRP-2. It was further demonstrated that a CTL line raised from splenocytes by repeated stimulation in vitro with this peptide could recognize B16 tumor and was therapeutic against 3-d-old established pulmonary metastases. The use of TRP-2 in a preclinical model of tumor immunotherapy may be helpful in suggesting optimal vaccination strategies for cancer therapy in patients.
- Published
- 1997
- Full Text
- View/download PDF
45. Melanoma tumor-infiltrating lymphocytes derived from four distinct anatomic sites obtained from a single patient: comparison of functional reactivity and melanoma antigen recognition.
- Author
-
Yannelli JR, McConnell S, Parker L, Nishimura M, Robbins P, Yang J, el Gamil M, and Kawakami Y
- Subjects
- Adrenal Gland Neoplasms pathology, Antigens, Neoplasm analysis, Axilla, Cell Division immunology, Cell Line, Colonic Neoplasms pathology, Cytotoxicity, Immunologic, Humans, Immunophenotyping, Lymph Nodes pathology, MART-1 Antigen, Neoplasm Proteins pharmacology, Thorax, Lymphocytes, Tumor-Infiltrating immunology, Lymphocytes, Tumor-Infiltrating pathology, Melanoma immunology, Melanoma pathology, Neoplasm Proteins immunology
- Abstract
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 alpha beta 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.
- Published
- 1995
- Full Text
- View/download PDF
46. Recognition of tyrosinase by tumor-infiltrating lymphocytes from a patient responding to immunotherapy.
- Author
-
Robbins PF, el-Gamil M, Kawakami Y, Stevens E, Yannelli JR, and Rosenberg SA
- Subjects
- Antigens, Neoplasm genetics, Antigens, Neoplasm immunology, DNA, Neoplasm genetics, HLA-A Antigens genetics, HLA-A24 Antigen, Humans, Melanoma immunology, Tumor Cells, Cultured, Immunotherapy, Adoptive, Lymphocytes, Tumor-Infiltrating, Melanoma enzymology, Melanoma therapy, Monophenol Monooxygenase genetics, Monophenol Monooxygenase immunology, T-Lymphocytes immunology
- Abstract
The observation that allogeneic melanoma cells matched for particular HLA class I alleles stimulate T-cells isolated from patients suggests 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 I HLA alleles. In addition, this study suggests that recognition of tyrosinase by antigen-specific T-cells may be involved in tumor rejection.
- Published
- 1994
47. Targeted cytokine production.
- Author
-
Segal DM, Qian JH, Titus JA, Moreno MB, George AJ, Jost CR, Kurucz I, el-Gamil M, and Wunderlich JR
- Subjects
- Animals, Lymphocyte Activation, Mice, Models, Biological, Spleen immunology, Antibodies, Monoclonal immunology, Cytokines biosynthesis, Cytotoxicity, Immunologic, Mammary Neoplasms, Experimental immunology, Receptors, Antigen, T-Cell immunology, T-Lymphocytes immunology
- Abstract
It has been well established that bispecific antibodies containing anti-T-cell receptor MAbs crosslinked to anti-tumor MAbs induce T cells to lyse tumor cells, as measured in a 51Cr-release assay. Such lysis requires direct attachment between target and cytotoxic cells and most probably involves the exocytosis of cytolytic substances into the cell:cell interface. In addition, targeted T cells mediate a second activity, the secretion into the medium of factors that can block the growth of bound tumor cells and unbound bystander cells. In order to test how targeted effector cells mediate anti-tumor effects in vivo, we are currently developing a totally syngeneic murine system in which murine T cells are targeted against mouse mammary tumors. The system allows us to treat both primary tumors and tumor transplants, using a mammary-tumor-virus antigen as the entity that is specifically recognized on the tumor cells.
- Published
- 1992
48. Expression of swine class II genes using recombinant retroviral vectors.
- Author
-
LeGuern C, Shafer GE, Alexander RC, Germana S, Gustafsson K, el-Gamil M, and Sachs DH
- Subjects
- Animals, Cell Line, Gene Expression, Genetic Vectors, Mice, Plasmids, Recombination, Genetic, Simian virus 40 genetics, Simplexvirus genetics, Swine, Transfection, Genes, MHC Class II
- Published
- 1991
49. Class II genes of miniature swine. II. Molecular identification and characterization of B (beta) genes from the SLAc haplotype.
- Author
-
Pratt K, Sachs DH, Germana S, el-Gamil M, Hirsch F, Gustafsson K, and LeGuern C
- Subjects
- Animals, Base Sequence, Chromosome Mapping, Cloning, Molecular, DNA genetics, DNA Probes, Exons, Molecular Sequence Data, Restriction Mapping, Histocompatibility Antigens Class II genetics, Major Histocompatibility Complex, Swine genetics
- Abstract
Genomic clones corresponding to class II beta genes of the SLAc haplotype of miniature swine have been isolated and characterized. These genes have been grouped into seven non-overlapping clusters on the basis of restriction mapping. Ordering of exons within each cluster was accomplished by hybridization of Southern blots of restriction fragments with exon-specific probes. The two clusters (clusters 2 and 3) encoding the DRB and DQB genes were identified on the basis of hybridization with locus-specific 3' untranslated cDNA probes. Cluster 4 contained exons of both DOB and DQB genes, the basis for which remains to be determined. The remaining four clusters (1, 5, 6, 7) were identified as containing DP, DR, and DO coding sequences, respectively, on the basis of sequence analysis. The porcine class II region appears very similar to that of man in number and nature of the class II genes identified and in the intron/exon organization of corresponding genes.
- Published
- 1990
- Full Text
- View/download PDF
50. Class II genes of miniature swine. I. Class II gene characterization by RFLP and by isolation from a genomic library.
- Author
-
Sachs DH, Germana S, el-Gamil M, Gustafsson K, Hirsch F, and Pratt K
- Subjects
- Animals, DNA genetics, Polymorphism, Restriction Fragment Length, Swine, Swine, Miniature immunology, Genes, MHC Class II, Histocompatibility Antigens genetics, Swine, Miniature genetics
- Abstract
Class II genes of miniature swine have been characterized by restriction fragment length polymorphism (RFLP) analysis and by analysis of a series of clones isolated from a lymphocyte genomic library. For RFLP analysis, DNA samples from three independent major histocompatibility complex homozygous lines and three intra-MHC recombinant lines were digested with a variety of restriction enzymes and analyzed in Southern blots using human cDNA probes for DP, DQ, DR, and DZ alpha genes, and DP, DQ, DR, and DO beta genes. One, or at most two, unique fragments were detected by hybridization with each of the human alpha probes tested. In contrast, multiple bands (five to six for most enzymes examined) were detected by each of the human beta probes tested, the majority of which were found to cross-react with at least three of these probes under conditions of moderate stringency. Genomic DNA from the SLAc haplotype was cloned into an EMBL-3 bacteriophage vector, and the corresponding genomic library was screened with each of these human cDNA probes. The class II genes thereby isolated from this library showed characteristics consistent with those anticipated from the RFLP analysis. Thus, unique alpha genes were obtained which showed no evidence of cross-hybridization, while beta genes showed extensive cross-hybridization and were frequently detected in the library by more than one human beta gene probe. These data are consistent with early evolutionary divergence of alpha genes, prior to mammalian speciation, and with continuing evolution of beta genes, with possible shared usage of these genes by different alpha loci. The data also imply that alpha genes can readily be assigned to loci homologous to their human counterparts, but that beta genes will require further mapping and/or sequence analysis to confirm assignments.
- Published
- 1988
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.