Peptide Tyrosinase (Asp371) – HLA-A*02:01 (YMDGTMSQV) is a human tyrosinase-derived (369-377) peptide by posttranslational conversion of the sequence YMNGTMSQV. Tyrosinase is an oxidase membrane-bound protein. Tyrosinase play a key role in the melanin synthesis pathway. Tyrosinase is presented on the surface of HLA-A*02:01 melanomas and also expressed in melanocytes. Tyrosinase has been still suggested to be a tumor antigen and might be implicated in improvement of immunotherapeutic strategies such as for efficient anticancer vaccine development.
Applications of Peptide Tyrosinase (Asp371) – HLA-A*02:01 (YMDGTMSQV)
Peptide Tyrosinase (Asp371) – HLA-A*02:01 (YMDGTMSQV) is used to stimulate specific cytotoxic T lymphocytes (CTL) in PBMCs and then to analyze CTL response especially the cytokine production by ELISPOT assay. Peptide Tyrosinase (Asp371) – HLA-A*02:01 (YMDGTMSQV) is also involved in experimental therapies of metastatic melanoma by allogeneic hematopoietic stem cell transplantation. In fact, cytotoxic T cells were generated from peripherical blood mononuclear cells (PBMCs) of HLA-A*02:01 healthy donors after being stimulated by injection of Asp371 antigen. This strategy raises issues which concern the graft versus tumor (GvT) effect and graft versus host disease (GvHD).
Many peptide epitopes for cytotoxic T lymphocytes (CTLs) have been identified from melanocytic differentiation proteins. Vaccine trials with these peptides have been limited mostly to those associated with HLA‐A2, and immune responses have been detected inconsistently. Cases of clinical regression have been observed after peptide vaccination in some trials, but melanoma regressions have not correlated well with T‐cell responses measured in peripheral blood lymphocytes (PBLs). We vaccinated stage IV melanoma patients with a mixture of gp100 and tyrosinase peptides restricted by HLA‐A1 (DAEKSDICTDEY), HLA‐A2 (YLEPGPVTA and YMDGTMSQV) and HLA‐A3 (ALLAVGATK) in an emulsion with GM‐CSF and Montanide ISA‐51 adjuvant. CTL responses were assessed in PBLs and in a lymph node draining a vaccine site (sentinel immunized node, SIN). We found CTL responses to vaccinating peptides in the SIN in 5/5 patients (100%). Equivalent assays detected peptide‐reactive CTLs in PBLs of 2 of these 5 patients (40%). CTLs expanded from the SIN lysed melanoma cells naturally expressing tyrosinase or gp100. We demonstrated immunogenicity for peptides restricted by HLA‐A1 and ‐A3 and for 1 HLA‐A2 restricted peptide, YMDGTMSQV. Immune monitoring of clinical trials by evaluation of PBLs alone may under‐estimate immunogenicity; evaluation of SIN provides a new and sensitive approach for defining responses to tumor vaccines and correlating these responses with clinical outcomes. This combination of an immunogenic vaccine strategy with a sensitive analysis of CTL responses demonstrates the potential for inducing and detecting anti‐tumor immune responses in the majority of melanoma patients.
2- Nolte A., Scheffold C., Slotty J., Huenefeld C., Schultze J. L., Grabbe S., Berdel W. E. and Kienast J. J. Immunother. 26(3):257-269 (2003)
To exploit alloreactive T-cell responses known as the graft-versus-tumor effect, allogeneic hematopoietic stem cell transplantation is being explored as experimental therapy in selected solid tumors, including metastatic melanoma. However, donor T-cell responses are often delayed and associated with severe graft-versus-host disease. Posttransplant adoptive immunotherapy using tumor-specific cytotoxic T lymphocytes (CTL) of donor origin might provide immediate graft-versus-tumor effects but not graft-versus-host disease. Therefore, the aim of the current study was to define in vitro conditions for the expansion of allogeneic major histocompatibility complex-matched CTLs targeting melanoma-associated antigens (MAA). The CTLs were generated from peripheral blood mononuclear cells (PBMCs) of HLA-A*0201+ healthy donors by repetitive stimulations with HLA-A*0201-restricted MAA-derived peptides. Melanoma reactivity, as determined by lysis of peptide-pulsed T2 cells and HLA-A2+/Ag+ melanoma cells, was detected using in vitro expanded CTL targeting MAA peptides AAGIGILTV(MT(27-35)), IMDQVPFSV(G(209-2M)), and YMDGTMSQV(T(368-376)). In contrast, FLWGPRALV(MAGE3(271)-(279)) and VLPDVFIRCV(GnT-V(nt38-67)) induced peptide-specific recognition of T2 target cells only, whereas ITDQVPFSV(G(209-217)), KTWGQYWQV(G9(154)), MLLAVLYCL(T(1-9)), and tumor lysate could not induce specific CTLs. Specific cytolytic activity was accompanied by interferon-gamma secretion. Peptide-pulsed dendritic cells were required only for the initial stimulation of CTLs and could be substituted by PBMCs during restimulations. The median expansion rate of CTL was five to six times, regardless of whether dendritic cells or PBMCs were used after the initial stimulation. The results delineate the conditions for effective ex vivo expansion of melanoma-specific CTLs from PBMCs of healthy donors to be used as an adjunct in allogeneic cell therapy of metastatic melanoma.
3- Lee P. et al. J Clin Oncol. 19(18):3836-3847 (2001)
PURPOSE: Forty-eight patients with high-risk re-sected stage III or IV melanoma were immunized with two tumor antigen epitope peptides derived from gp100(209-217)(210M) (IMDQVPSFV) and tyrosinase(368-376)(370D) (YMDGTMSQV) emulsified with incomplete Freund’s adjuvant (IFA). Patients received peptides/IFA with or without interleukin (IL)-12 30 ng/kg to evaluate the toxicities and immune responses in either arm with time to relapse and survival as secondary end points.
METHODS: Immunizations were administered every 2 weeks for 8 weeks, then every 4 weeks for 12 weeks, and then once 8 weeks later. A leukapheresis to obtain peripheral-blood mononuclear cells for immune analyses was done before and after vaccination. Skin testing with peptides and recall reagents was performed before and after vaccinations.
RESULTS: Local pain and granuloma formation, fever, and lethargy of grade 1 or 2 were observed. Transient vaccine-related grade 3-but no grade 4-toxicity was observed. Thirty-four of 40 patients developed a positive skin test response to the gp100 peptide but none to tyrosinase. Immune responses were measured by release of gamma-interferon in an enzyme-linked immunosorbent assay (ELISA) by effector cells in the presence of peptide-pulsed antigen-presenting cells or by an antigen-specific tetramer flow cytometry assay. Thirty-three of 38 patients demonstrated an immune response by ELISA after vaccination, as did 37 of 42 patients by tetramer assay. Twenty-four of 48 patients relapsed with a median follow-up of 20 months, and 10 patients in this high-risk group have died.
CONCLUSION: These data suggest a significant proportion of patients with resected melanoma mount an antigen-specific immune response against a peptide vaccine and indicate that IL-12 may increase the immune response and supporting further development of IL-12 as a vaccine adjuvant.
4- Chiritoiu G. N. et al. Electrophoresis. 37(11):1448-1460 (2016)
The degradation process of the antigens specific to MHC-I presentation depends mainly on the proteasomal proteases in the cytosol. However, since many antigens are glycoproteins, including tumor antigens or viruses envelope proteins, their glycosylation status could also affect their processing and presentation. Here, we investigate the processing of tyrosinase, a multiple glycosylated tumor antigen overexpressed in human malignant melanoma. By LC-MS/MS analysis of human tyrosinase expressed in a melanoma cell, we show that all seven sites of tyrosinase are at least partially N-glycosylated. Using human CD8+ T-cell clones specific for the tyrosinase epitope YMDGTMSQV (369-377), including an N-glycosylation site, we found that transfectants of single and triple N-glycosylation mutants are recognized by specific T cells. Importantly, single, triple, and the aglycosylated tyrosinase mutants lacking the epitope located N-glycosylation site (N371D) were able to trigger higher CD8+ T-cell activation. The LC/MS analysis showed significant increase of the amount of YMDGTMSQV peptide resulted from accelerated oligomerization and degradation of aglycosylated mutants. The generation of the antigenic peptide by the antigen processing machinery is therefore largely independent of tyrosinase N-glycosylation. However, while distal N-glycans had no effect on the epitope generation, the mutants lacking the N371 glycan generated the antigenic peptide more efficiently. We conclude that epitope located N-glycans limit the ability of human tyrosinase to provide HLA-A2-restricted antigen for recognition by specific CD8+ T cells.
5- Lewis J. J. et al. Int J Cancer. 87(3):391-398 (2000)
The lack of reproducible, quantitative assays for T-cell responses has been a limitation in the development of cancer vaccines to elicit T-cell immunity. We utilized the Elispot assay, which allows a quantitative and functional assessment of T cells directed against specific peptides after only brief in vitro incubations. CD8(+) T-cell reactivity was determined with an interferon (IFN)-gamma Elispot assay detecting T cells at the single cell level that secrete IFN-gamma. We studied both healthy individuals and patients with melanoma. Healthy HLA-A*0201-positive individuals showed a similar mean frequency of CD8(+) cells recognizing a tyrosinase peptide, YMDGTMSQV, when compared with melanoma patients prior to immunization. The frequencies of CD8(+) cells recognizing the tyrosinase peptide remained relatively constant over time in healthy individuals. Nine HLA-A*0201-positive patients with stage IV metastatic melanoma were immunized intradermally with the tyrosinase peptide together with the immune adjuvant QS-21 in a peptide dose escalation study with 3 patients per dose group. Two patients demonstrated a significant increase in the frequency of CD8(+) cells recognizing the tyrosinase peptide during the course of immunization, from approx. 1/16,000 CD8(+) T cells to approx. 1/4,000 in the first patient and from approx. 1/14,000 to approx. 1/2,000 in the second patient. These results demonstrate that modest expansion of peptide-specific CD8(+) T cells can be generated in vivo by immunization with peptide plus QS-21 in at least a subset of patients with melanoma.
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