Melanocyte Differentiation Antigens Research Articles

Pathological, histochemical, and immune-histochemical studies on some canine-skin neoplasm at Sharkia province, Egypt.

Cutaneous neoplastic disorders are often observed in small mammal pets, such as dogs, regardless of their gender. An important objective of this work was to give a full account of the clinical, pathological, and immune-histochemical features of several skin tumors in dogs. This study was a case series in the hospital clinic, Faculty of Veterinary Medicine, Zagazig University, Egypt. Twenty-five dogs (14 males and 11 females) were examined clinically during the period from March 2022 to October 2023. The skin swelling was collected from affected animals and then subjected to a detailed histopathological study to record the different gross and microscopic findings and confirm the diagnosis by immunohistochemistry. Skin neoplasia in dogs was exposed to various clinical signs, and the dogs' ages ranged between 3 and 11 years. Concerning tumor features, the majority of neoplasms were malignant (65.52%) more than benign (34.48%). The study revealed the presence of 29 cases of dogs showed neoplasia with different prevalence rates including squamous cell carcinoma (13.79%), mast cell tumor (6.89%), basal cell tumors (10.34%), histiocytoma (6.89%), trichoepithelioma (10.34%), transmissible venereal tumor (10.34%), trichilemmoma (3.44%), scalp paraganglioma (3.44%), pilomatricoma (10.34%), malignant melanomas (17.24%), and miscellaneous cases as fat necrosis (6.89%), in males and females dogs with different histopathological lesions and immunohistochemistry expressions for pan-cytokeratin (CK), melanocyte-differentiation antigens (S100 protein), and synaptophysin. Malignant melanomas (17.24%) are the extremely common cutaneous tumors diagnosed in this study. Meanwhile, benign tumors such as trichilemmoma, trichoepithelioma, pilomatricoma, and paraganglioma are less frequent in dogs.

Autoimmunity in immune checkpoint inhibitor-induced immune-related adverse events: A focus on autoimmune skin toxicity and pneumonitis.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy. However, their use is frequently associated with immune-related adverse events (irAEs) potentially affecting any organ. The mechanisms mediating such irAEs remain poorly understood and biomarkers to predict the development of irAEs are lacking. Growing evidence shows the importance of self-antigens in mediating irAEs during ICI therapy, in particular the well-described melanocyte differentiation antigens in melanoma patients. This review will focus on two novel classes of self-antigens involved in mediating autoimmune skin toxicity and pneumonitis in non-small cell lung cancer patients treated with immunotherapy. T cells specific for these self-antigens are thought to not only mediate irAEs but are thought to simultaneously mediate anti-tumor responses and are therefore associated with both autoimmune toxicity and response to ICI therapy. We further discuss emerging cellular and proteomic immune signatures of irAEs that may serve as biomarkers to help predict which patients are at higher risk of developing these irAEs. The determination of new tumor antigens involved in ICI therapy and the identification of related biomarkers brings us a step forward in the mechanistic understanding of ICIs and will help to monitor patients at higher risk of developing irAEs. Lastly, we discuss the current challenges in collecting research samples for the study of ICI-related mechanisms and in distinguishing between immune signatures of response and those of irAEs.

Abstract CT207: Phase I trial of vesicular stomatitis virus expressing human interferon beta and tyrosinase related protein 1 (VSV-IFNb-TYRP1) in metastatic ocular melanoma

Abstract Oncologic viruses (OV) can induce tumor death through both direct oncolysis and immune-mediated destruction. Our study investigates the use of a novel OV with a Vesicular Stomatitis Virus (VSV) vector modified to express interferon-beta (IFN-b) and Tyrosinase Related Protein 1 (TYRP1) (VSV-IFNb-TYRP1). VSV is an attractive vector since humans are not naturally infected with VSV; thus, pre-existing immunity is minimal. IFN-b leads to increased T-cell responses and tumor cell apoptosis, while TYRP1, a melanocyte differentiation antigen, increases immunogenicity of the therapy. We conducted a Phase 1 clinical trial with a 3+3 design in patients with metastatic uveal melanoma. VSV-IFNb-TYRP1 was injected into a liver metastasis under image guidance, then administered on the same day as a single intravenous (IV) infusion. IV doses started at dose level (DL) 1 1 × 1010 TCID50, then escalated to DL2 3 × 10 10, DL3 1 × 1011, and DL4 3 × 1011. The dose delivered intratumorally (IT) varied based on the IV dose. The primary endpoints were safety and maximum tolerated dose (MTD). Efficacy was a secondary endpoint. Correlative studies focused on understanding viral pharmacokinetics (PK) and immunological responses induced by VSV-IFNb-TYRP1 therapy. Twelve patients with previously treated metastatic uveal melanoma were enrolled. Median follow up was 19.1 months. Four DLs were evaluated with one patient at DL4 experiencing dose limiting toxicities (DLTs), including decreased platelet count (grade 3), increased aspartate aminotransferase (AST), and cytokine release syndrome (CRS). Non-DLTs at DL4 were fatigue, fever, CRS, hematological toxicities (decreased platelets and lymphocytes), and AST elevation. Four patients had stable disease (SD) and 8 had progressive disease (PD) as their best response. ELIspot data show that three of the four patients with response to TYRP1 also had a response to gp100, suggesting possible epitope spreading. These four patients received immune checkpoint inhibitor treatment as the next line of therapy, which led to clinical benefit for two patients. Our study evaluated VSV-IFNb-TYRP1 administered via IT and IV routes in a previously treated population of metastatic uveal melanoma patients and found no major safety events. Although there were no clear objective responses to VSV-IFNb-TYRP1, dose-dependent immunogenicity to melanoma antigens was seen. While OV response alone is not sufficient for clinical benefit, the evidence of epitope spreading is encouraging. Future studies of VSV-IFNb-TYRP1 will evaluate combinations with other therapies. Citation Format: Katherine E. Smith, Adam Weisbrod, Carrie Strand, Jacob Allerd, Jose S. Pulido, Alysha Newsom, Lianwen Zhang, Nandakumar Packiriswamy, Heather Montane, Lisa Kottschade, Anastasios Dimou, Yiyi Yan, Markovic Svetomir, Kah Whye Peng, Richard G. Vile, Matthew S. Block, Roxana S. Dronca. Phase I trial of vesicular stomatitis virus expressing human interferon beta and tyrosinase related protein 1 (VSV-IFNb-TYRP1) in metastatic ocular melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT207.

Viral vector-mediated reprogramming of the fibroblastic tumor stroma sustains curative melanoma treatment

The tumor microenvironment (TME) is a complex amalgam of tumor cells, immune cells, endothelial cells and fibroblastic stromal cells (FSC). Cancer-associated fibroblasts are generally seen as tumor-promoting entity. However, it is conceivable that particular FSC populations within the TME contribute to immune-mediated tumor control. Here, we show that intratumoral treatment of mice with a recombinant lymphocytic choriomeningitis virus-based vaccine vector expressing a melanocyte differentiation antigen resulted in T cell-dependent long-term control of melanomas. Using single-cell RNA-seq analysis, we demonstrate that viral vector-mediated transduction reprogrammed and activated a Cxcl13-expressing FSC subset that show a pronounced immunostimulatory signature and increased expression of the inflammatory cytokine IL-33. Ablation of Il33 gene expression in Cxcl13-Cre-positive FSCs reduces the functionality of intratumoral T cells and unleashes tumor growth. Thus, reprogramming of FSCs by a self-antigen-expressing viral vector in the TME is critical for curative melanoma treatment by locally sustaining the activity of tumor-specific T cells.

Heterologous Prime Boost Vaccination Induces Protective Melanoma-Specific CD8+ T Cell Responses

Cancer vaccination aims at inducing an adaptive immune response against tumor-derived antigens. In this study, we utilize recombinant human adenovirus serotype 5 (rAd5) and recombinant lymphocytic choriomeningitis virus (rLCMV)-based vectors expressing the melanocyte differentiation antigen gp100. In contrast to single or homologous vaccination, a heterologous prime boost vaccination starting with a rAd5-gp100 prime immunization followed by a rLCMV-gp100 boost injection induces a high magnitude of polyfunctional gp100-specific CD8+ T cells. Our data indicate that an optimal T cell induction is dependent on the order and interval of the vaccinations. A prophylactic prime boost vaccination with rAd5- and rLCMV-gp100 protects mice from a B16.F10 melanoma challenge. In the therapeutic setting, combination of the vaccination with low-dose cyclophosphamide showed a synergistic effect and significantly delayed tumor growth. Our findings suggest that heterologous viral vector prime boost immunizations can mediate tumor control in a mouse melanoma model.

Abstract PR16: Specific activation of the G protein-coupled estrogen receptor inhibits melanoma and other cancers and potentiates immune and targeted therapies

Abstract Modern immune and targeted therapies appear more effective against advanced melanoma in women than in men. Melanoma incidence is also lower in women. Similar sex differences are observed in other tumors that are not classically considered sex hormone responsive. Here, we show that much of the protective effect likely results from estrogen signaling through the G protein-coupled estrogen receptor (GPER) on tumor cells. Selective GPER activation in primary male and female melanocytes, and in melanoma cells, induced long-term changes that maintained a more differentiated cell state as defined by increased expression of well-established melanocyte differentiation antigens, increased pigment production, decreased proliferative capacity, and decreased expression of the oncodriver and stem cell marker c-Myc. GPER signaling also rendered melanoma cells more vulnerable to immunotherapy. Systemic delivery of a small-molecule, selective GPER agonist was well tolerated, and cooperated with PD-1 directed immune checkpoint blockade in melanoma-bearing mice to dramatically extend survival, with up to half of mice clearing their tumor. Complete responses were associated with immune memory that protected against rechallenge with naive tumor cells. Specific GPER activation in the BRaf-driven murine melanoma model (YUMM1.7) also delayed resistance to targeted BRaf inhibition in vivo. Similar antitumor responses were observed in genetically defined KRas-driven syngeneic pancreatic ductal carcinoma (PDAC) models. The GPER agonist used has favorable pharmacokinetics and oral bioavailability in mice and higher vertebrates and appears to have a large therapeutic window. Together, these data suggest that GPER may be a useful, pharmacologically accessible target for melanoma and other common cancers. This abstract is also being presented as Poster B15. Citation Format: Chris Natale, Jinyang Li, Jun Zhang, Cristina Aquirre, Ankit Dahal, Tzvete Dentchev, John Seykora, Ben Stanger, Todd Ridky. Specific activation of the G protein-coupled estrogen receptor inhibits melanoma and other cancers and potentiates immune and targeted therapies [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR16.

Syngeneic B16-F1 cells are more efficient than allogeneic Cloudman cells as antigen source in DC-based vaccination in the B16-F1 murine melanoma model

A major obstacle to obtaining relevant results in cancer vaccination has been the lack of identification of immunogenic antigens. Dendritic cell (DC)-based cancer vaccines used preventively may afford protection against tumor inoculation, but the effect of antigen choice on anti-tumor protection is not clear. When using irradiated syngeneic tumor cells to load DCs, tumor self-antigens are provided, including tumor-associated antigens (TAAs) and neoantigens generated by tumor mutations. On the other hand, allogeneic tumor cells could only supply shared TAAs. To assess the advantages of each source in protective vaccination, we analyzed in C57BL/6 mice the effect of loading DCs with irradiated syngeneic B16-F1 or allogeneic Cloudman melanoma cells; both cell lines were characterized by whole exome sequencing and RNAseq. Tumor cell components from the two irradiated cell lines were efficiently internalized by DCs, and transported to MHC-class II positive tubulovesicular compartments (MIICs). DCs loaded with allogeneic irradiated Cloudman cells (DC-ApoNecALLO) induced a partially effective anti-melanoma protection, although Cloudman and B16-F1 cells share the expression of melanocyte differentiation antigens (MDAs), cancer-testis antigens (CTAs) and other TAAs. DCs loaded with syngeneic B16-F1 cells (DC-ApoNecSYN) established a more potent and long-lasting protection and induced a humoral anti-B16F1 response, thus suggesting that neoepitopes are needed for inducing long-lasting protection.

Abstract 5010: Potentiating immune checkpoint blockade therapeutic efficacy using a small molecule activator of integrin cell adhesion receptors

Abstract Background: Immune checkpoint blockade (ICB) has therapeutic benefit in several human cancers, but in many patients, ICB - induced T cells do not infiltrate tumors, preventing clinical benefit. Intratumoral T cell accumulation requires firm adhesion mediated by the integrins very late antigen-4 (VLA-4) and lymphocyte function-associated antigen-1 (LFA-1) on activated T cells. The integrin class of cell adhesion receptors also play critical roles in multiple phases of tumor immune responses. Blocking the LFA-1 interaction with its ligand Intercellular adhesion molecule 1 (ICAM-1) abrogates CD8+ tumor infiltration after anti-CTLA4 therapy. Here, we evaluated the effect of an integrin activator, 7HP349, on promoting intratumoral T cell accumulation to potentiate CTLA-4 and PD-L1 checkpoint blockade anti-tumor activity. Experimental Procedure: To evaluate the effect of 7HP349 in promoting ICB therapeutic activity, we combined 7HP349 with anti-CTLA-4 or anti-PD-L1 antibodies in preclinical murine models for melanoma and colon carcinoma. Results: In cell adhesion assays, 7HP349 increased by >100-fold the number of VLA-4 or LFA-1 expressing T cells that bind to vascular cell adhesion molecule 1 (VCAM-1) and ICAM-1, respectively. 7HP349 facilitated chemo-attraction of T cells across matrices of VCAM-1 or ICAM-1, induced by the chemokine SDF-1α. In a PDL-1 negative B16/BL6 melanoma model, 7HP349 was dosed twice weekly at 1 mg/Kg intratumorally either alone as a single agent compared to vehicle control (13% vs 0%, p<0.01) or in combination with anti-CTLA-4 (44% vs 21%, p<0.01). After intraperitoneal administration, 7HP349 increased the complete response in combinations with anti-CTLA-4 (77% vs 27%, p<0.01). In addition, 7HP349 increased median survival in mice with CT-26 colon cancer when dosed either as a single agent versus control (21 vs 15 days, P<0.0012) or in combination with anti-PD-L1 (24 vs 21 days, P<0.02). 7HP349 may also increase the effectiveness of anti-CD137 without increasing liver toxicity. Levels of CD8 effector T cells (Teff) detected in tumor were significantly higher in mice treated with anti-CTLA-4 and 7HP349 than anti-CTLA-4 and vehicle. Some of these Teff showed specificity to p15E, an endogenous retroviral epitope expressed on B16. Mice treated with anti-CTLA-4 and 7HP349 showed increased depigmentation (vitiligo) suggesting immunity to melanocyte differentiation antigens. Conclusion: 7HP349 described here represents a first-in-concept agent that positively regulate VLA-4 and LFA-1 function. Activation of integrin cell adhesion molecules with 7HP349 is a promising approach to enhance the anti-cancer activity of checkpoint blockade therapy with antibodies against CTLA-4 and PD-(L)1. Citation Format: Yared Hailemichael, Peter Vanderslice, Robert V. Market, Ronald J. Biediger, Darren G. Woodside, Upendra K. Marathi, Willem W. Overwijk. Potentiating immune checkpoint blockade therapeutic efficacy using a small molecule activator of integrin cell adhesion receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5010.

Systematic assessment of LCMV based vaccine vectors expressing melanocyte differentiation antigens in human in vitro assays and in mouse melanoma models.

e14299 Background: Antibodies blocking the immune checkpoint pathways represent an important milestone in the treatment of patients with metastatic melanoma. However, individuals presenting cold tumors do usually not show a clinical benefit. New cancer vaccine approaches are needed to cover this gap. Methods: A novel replication attenuated vaccine vector based on lymphocytic choriomeningitis virus expressing full length melanocyte differentiation antigens is evaluated in mouse melanoma models and in PBMCs and T cell cultures from melanoma patients. Results: Here, we demonstrate that intratumoral but not intravenous injection of a recombinant propagating LCMV vector expressing the melanoma-associated antigen TRP2 leads to T cell-dependent eradication of established s.c. melanoma. Importantly, intratumoral vaccination shows an abscopal effect on distant lung metastasis and protects from a rechallenge with melanoma. Confocal microscopy and flow cytometry reveal that intratumoral injection of rLCMV vectors reprograms the tumor microenvironment resulting in sustained T cell fitness. In addition, we demonstrate that rLCMV vectors can efficiently transduce human antigen presenting cells. Moreover, in vitro data confirm that rLCMV efficiently induces T cells against various melanoma-associated antigens in PBMCs from melanoma patients. Conclusions: Preclinical assessment of propagating rLCMV vectors shows unique features of this cancer vaccine resulting in a profound and multistep activation of the cancer immunity cycle resulting in eradication of established melanomas in the B16F10 mouse model after one single immunization. Positive proof of principle experiments using PBMCs from melanoma patients suggest a rapid evaluation in clinical trials.

Antibodies as biomarker candidates for response and survival to checkpoint inhibitors in melanoma patients

BackgroundLong-term survival of stage IV melanoma patients has improved significantly with the development of immune checkpoint inhibitors (CIs). Reliable biomarkers to predict response and clinical outcome are needed.MethodsWe investigated the role of melanoma-associated antibodies as predictive markers for CI therapy in two independent cohorts. In cohort 1, a prospective study, we measured specific antibodies before treatment, after one week and after six to nine weeks of treatment. Cohort 2 consisted of serum samples prior to CI therapy initiation. ELISA assays were performed to quantify specific IgG directed against melanocyte differentiation antigens tyrosinase-related proteins 1 and 2 (TRP1/TYRP1 and TRP2/TYRP2), glycoprotein 100 (gp100), MelanA/MART1, and the cancer-testis antigen NY-ESO-1. Response was defined as either complete or partial remission on CT scan according to RECIST 1.1.ResultsIn cohort 1, baseline levels of these antibodies were higher in the responder group, although statistical significance was only reached for NY-ESO-1 (p = 0.007). In cohort 2, significantly higher antibody baseline levels for MelanA/MART1 (p = 0.003) and gp100 (p = 0.029) were found. After pooling the results from both cohorts, higher levels of MelanA/MART1 (p = 0.013), TRP1/TYRP1 (p = 0.048), TRP2/TYRP2 (p = 0.047) and NY-ESO-1 (p = 0.005) specific antibodies at baseline were independently associated with response.ConclusionsMelanoma-associated antibodies may be candidate biomarkers for response and survival in metastatic melanoma patients being treated with CIs. These markers may be used to complement patient assessment, in combination with PD-L1 status, tumor-infiltrating lymphocytes and tumor mutational burden, with the aim to predict outcome of CI treatment in patients with metastatic melanoma.Trial registrationEthikkommission Ostschweiz, EKOS 16/079 https://ongoingprojects.swissethics.ch/runningProjects_list.php?q=%28BASECID~contains~2016-00998%29&orderby=dBASECID.

Tyrosinase and nestin immunohistochemical expression in melanocytic nevi as a histopathologic pattern to trace melanocyte differentiation and nevogenesis.

While histological analysis represents a powerful tool for the classification of melanocytic lesions as benign or malignant, a clear-cut distinction between a nevus and a melanoma is sometimes a challenging step of the diagnostic process. The immunohistochemical detection of tyrosinase, cardinal melanogenic enzyme during melanocytic maturation, has often been helpful in formulating a differential diagnosis due to the peculiar staining pattern in nevocytes compared with melanoma cells. Tyrosinase distribution in nevi appears to overlap with the cytoarchitectural changes observable within these lesions, that result in epidermal or superficial dermal nevocytes being larger and strongly expressing melanocytic differentiation antigens, such as tyrosinase, compared with deeper dermal nevus cells. Our study aimed to evaluate the immunohistochemical expression pattern of tyrosinase in different histological types of acquired dysplastic melanocytic nevi, including junctional, compound, and intradermal nevi. Moreover, to estimate whether in nevocytes the expression of tyrosinase was associated with their differentiation state, we investigated the expression of two recognized markers of pluripotency, CD34 and nestin. In all examined nevi, our analysis revealed a remarkable immunoreactivity for tyrosinase in junctional and superficial dermal nevocytes and a decreasing gradient of staining in dermal nevocytes, up to become negative in deeper dermis. Meanwhile, junctional and dermal nevocytes were lacking in CD34 protein. Furthermore, nestin immunostaining showed an opposite distribution compared with tyrosinase, leading us to look into the tyrosinase/nestin expression pattern in melanocytic nevus as a tool to better understand the final stages of differentiation of melanocyte precursors toward their ultimate anatomical site into the epidermis.

Oncogenic BRAFV600E Governs Regulatory T-cell Recruitment during Melanoma Tumorigenesis.

Regulatory T cells (Treg) are critical mediators of immunosuppression in established tumors, although little is known about their role in restraining immunosurveillance during tumorigenesis. Here, we employ an inducible autochthonous model of melanoma to investigate the earliest Treg and CD8 effector T-cell responses during oncogene-driven tumorigenesis. Induction of oncogenic BRAFV600E and loss of Pten in melanocytes led to localized accumulation of FoxP3+ Tregs, but not CD8 T cells, within 1 week of detectable increases in melanocyte differentiation antigen expression. Melanoma tumorigenesis elicited early expansion of shared tumor/self-antigen-specific, thymically derived Tregs in draining lymph nodes, and induced their subsequent recruitment to sites of tumorigenesis in the skin. Lymph node egress of tumor-activated Tregs was required for their C-C chemokine receptor 4 (Ccr4)-dependent homing to nascent tumor sites. Notably, BRAFV600E signaling controlled expression of Ccr4-cognate chemokines and governed recruitment of Tregs to tumor-induced skin sites. BRAFV600E expression alone in melanocytes resulted in nevus formation and associated Treg recruitment, indicating that BRAFV600E signaling is sufficient to recruit Tregs. Treg depletion liberated immunosurveillance, evidenced by CD8 T-cell responses against the tumor/self-antigen gp100, which was concurrent with the formation of microscopic neoplasia. These studies establish a novel role for BRAFV600E as a tumor cell-intrinsic mediator of immune evasion and underscore the critical early role of Treg-mediated suppression during autochthonous tumorigenesis.Significance: This work provides new insights into the mechanisms by which oncogenic pathways impact immune regulation in the nascent tumor microenvironment. Cancer Res; 78(17); 5038-49. ©2018 AACR.

In Vitro TyRP-1 Knockdown Based on siRNA Carried by Liquid Crystalline Nanodispersions: an Alternative Approach for Topical Treatment of Vitiligo.

Vitiligo is a skin disease characterized by depigmentation and the presence of white patches that are associated with the loss of melanocytes. The most common explanation for the cause of this condition is that it is an autoimmune condition. TyRP-1 is involved in melanin pigment synthesis but can also function as a melanocyte differentiation antigen. This protein plays a role in the autoimmune destruction of melanocytes, which results in the depigmentation, characteristic of this disease. In this study, we evaluated liquid crystalline nanodispersions as non-viral vectors to deliver siRNA-TyRP-1 as an alternative for topical treatment of vitiligo. Liquid crystalline nanodispersions were obtained and characterized with respect to their physical-chemical parameters including size, PdI and zeta potential, as well as Small Angle X-ray Scattering and complexing to siRNA. The effects of the liquid crystalline nanodispersions on the cellular viability, cell uptake and levels of the knockdown target TyRP-1 were evaluated in melan-A cells after 24h of treatment. The liquid crystalline nanodispersions demonstrated adequate physical-chemical parameters including nanometer size and a PdI below 0.38. These systems promoted a high rate of cell uptake and an impressive TyRP-1 target knockdown (> 80%) associated with suitable loading of TyRp-1 siRNA. We demonstrated that the liquid crystalline nanodispersions showed promising alternative for the topical treatment of vitiligo due to their physical parameters and ability in knockdown the target protein involved with autoimmune destruction of melanocytes.

Activation of G protein-coupled estrogen receptor signaling inhibits melanoma and improves response to immune checkpoint blockade.

Female sex and history of prior pregnancies are associated with favorable melanoma outcomes. Here, we show that much of the melanoma protective effect likely results from estrogen signaling through the G protein-coupled estrogen receptor (GPER) on melanocytes. Selective GPER activation in primary melanocytes and melanoma cells induced long-term changes that maintained a more differentiated cell state as defined by increased expression of well-established melanocyte differentiation antigens, increased pigment production, decreased proliferative capacity, and decreased expression of the oncodriver and stem cell marker c-Myc. GPER signaling also rendered melanoma cells more vulnerable to immunotherapy. Systemically delivered GPER agonist was well tolerated, and cooperated with immune checkpoint blockade in melanoma-bearing mice to dramatically extend survival, with up to half of mice clearing their tumor. Complete responses were associated with immune memory that protected against tumor rechallenge. GPER may be a useful, pharmacologically accessible target for melanoma.

SLC45A2: A Melanoma Antigen with High Tumor Selectivity and Reduced Potential for Autoimmune Toxicity.

Cytotoxic T lymphocyte (CTL)-based immunotherapies have had remarkable success at generating objective clinical responses in patients with advanced metastatic melanoma. Although the melanocyte differentiation antigens (MDA) MART-1, PMEL, and tyrosinase were among the first melanoma tumor-associated antigens identified and targeted with immunotherapy, expression within normal melanocytes of the eye and inner ear can elicit serious autoimmune side effects, thus limiting their clinical potential as CTL targets. Using a tandem mass spectrometry (MS) approach to analyze the immunopeptidomes of 55 melanoma patient-derived cell lines, we identified a number of shared HLA class I-bound peptides derived from the melanocyte-specific transporter protein SLC45A2. Antigen-specific CTLs generated against HLA-A*0201- and HLA-A*2402-restricted SLC45A2 peptides effectively killed a majority of HLA-matched cutaneous, uveal, and mucosal melanoma cell lines tested (18/25). CTLs specific for SLC45A2 showed significantly reduced recognition of HLA-matched primary melanocytes that were, conversely, robustly killed by MART1- and PMEL-specific T cells. Transcriptome analysis revealed that SLC45A2 mRNA expression in normal melanocytes was less than 2% that of other MDAs, therefore providing a more favorable melanoma-to-melanocyte expression ratio. Expression of SLC45A2 and CTL sensitivity could be further upregulated in BRAF(V600E)-mutant melanoma cells upon treatment with BRAF or MEK inhibitors, similarly to other MDAs. Taken together, our study demonstrates the feasibility of using tandem MS as a means of discovering shared immunogenic tumor-associated epitopes and identifies SLC45A2 as a promising immunotherapeutic target for melanoma with high tumor selectivity and reduced potential for autoimmune toxicity. Cancer Immunol Res; 5(8); 618-29. ©2017 AACR.

Abstract 5596: Intratumoral administration of a TLR9-adjuvanted nanoparticle cancer vaccine stimulates more effective immunity in both injected and un-injected tumor sites compared to subcutaneous administration

Abstract Therapeutic success of a cancer vaccine requires substantial expansion of vaccine-primed CTL and efficient differentiation to polyfunctional T cells capable of migrating from the site of vaccination to metastatic sites. We have evaluated the tumor itself as a potential site of vaccination, compared to vaccination at subcutaneous sites distant from the tumor. For vaccination, we have developed a highly efficient nanoparticle platform comprised of multiple copies of a CpG-rich oligonucleotide (a TLR9 agonist) and long peptide tumor antigens, both covalently linked to the sucrose polymer Ficoll. Multiple peptide antigens where studied, including ovalbumin and a long peptide comprising epitopes from three melanocyte differentiation antigens. In all of these models, intratumoral vaccination was superior to subcutaneous vaccination for elimination of both injected tumors and, more importantly, distant site skin and lung metastases. Intratumoral vaccination substantially increased the magnitude of the systemic CTL response measured in the blood and in the spleen and enhanced capacity cell homing to distant site tumors. A greater fraction of tumor-antigen specific CD8+ T cells generated in the tumor were polyfunctional, expressing both IFN-γ and TNF-α. They were also less exhausted as measured by decreased overall PD-1 levels, increased T bet and the increased number of PD-1+ cells that retained cytotoxicity and proliferative capacity. Intratumoral vaccination also induced spread of the CD8+ T cell response to tumor antigens not included in the vaccine. These results demonstrate that direct intratumoral immunization with peptide antigens combined with a strong TLR9-activating adjuvant significantly increases the magnitude and quality of vaccine-primed CTL and enhances control of metastatic disease. Note: This abstract was not presented at the meeting. Citation Format: Edwina Naik, Chi Ying, Robert Milley, Carlo Calacsan, Stewart Chipman, Thomas Tüting, Robert Coffman, Cristiana Guiducci. Intratumoral administration of a TLR9-adjuvanted nanoparticle cancer vaccine stimulates more effective immunity in both injected and un-injected tumor sites compared to subcutaneous administration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5596. doi:10.1158/1538-7445.AM2017-5596

Identification of Novel HLA-A*0201-Restricted CTL Epitopes in Chinese Vitiligo Patients.

Generalized vitiligo is an autoimmune disease characterized by melanocyte loss, which results in patchy depigmentation of skin and hair. Recent studies suggested the key role of CD8+T lymphocytes for mediating immune response in vitiligo through melanocyte differentiation antigens, including tyrosinase, gp100 and MelanA/Mart-1. However, the specific epitopes of these auto-antigens are still unknown. In our study, we predicted the possible HLA-A*0201-restricted nonapeptides overlaying the full-length amino acid sequences of these three known antigens and investigated the lymphocytes reactivity to these nonapeptides by Elispot assay. In addition, we evaluated the abilities of these nonapeptides to activate CD8+T cells. We screened out 5 possible epitopes originated from tyrosinase and gp100, numbered P28, P41, P112, P118 and P119. Among these 5 epitopes, notably, P28 and P119 played the dominant role in activating CTLs, with a significant increase in proliferation rate and Interferon-γ (IFN-γ) production of CD8+T cells. Nevertheless, antigen-specific T cell reactivity was not detected in MelanA/Mart-1 peptides. Our studies identified two novel epitopes originated from proteins of gp100 and tyrosinase, which may have implications for the development of immunotherapies for vitiligo.

Adoptive transfer of induced pluripotent stem cell-derived regenerated T cells mediates effective regression of large tumors and establishes immunological memory

Abstract Current approaches to adoptive T cell therapy are limited by the difficulty of obtaining sufficient numbers of T cells against targeted antigens with effective in vivo characteristics. Theoretically, this limitation can be overcome by using induced pluripotent stem cells (iPSCs) that could provide an unlimited source of autologous T cells. However, therapeutic efficacy of iPSC-derived regenerated T cells remains to be elucidated. To this end, we established iPSCs from Pmel-1 T cell receptor (TCR) transgenic CD8+ T cells specific for the gp100 melanocyte differentiation antigen as a preclinical model of evaluating antitumor reactivity of regenerated antigen-specific T cells from iPSCs (Pmel-1 iPSCs). Pmel-1 iPSCs retained the same rearranged configuration as the original Pmel-1 CD8+ T cells, and differentiated into CD4+CD8+ double positive (DP) T cells that expressed CD3 and the desired TCR in vitro. Stimulation of iPSC-derived DP T cells with the cognate antigen in the presence of IL-7 and IL-15 followed by expansion with IL-2, IL-7 and IL-15 generated large numbers of less-differentiated CD8+ single positive T cells with antigen-specific potent cytokine production and cytolytic capacity. Furthermore, adoptively transferred iPSC-derived CD8+ T cells escaped immune rejection, mediated effective regression of large tumors, improved survival, and established antigen-specific immunological memory. These findings support the translational potential of iPSCs to provide an unlimited number of phenotypically defined, functional and expandable autologous antigen-specific T cells harboring characteristics necessary for in vivo effectiveness.

Protein Expression Analysis of Melanocyte Differentiation Antigen TRP-2

Melanocyte differentiation antigens, such as gp100, tyrosinase, and Melan-A and their corresponding antibodies HMB45, T311, and A103, are major diagnostic tools in surgical pathology. Little is known about tyrosinase-related protein 2 (TRP-2, or dopachrome tautomerase/DCT) another melanocyte differentiation antigen, which is an enzymatic component of melanogenesis. We identified a commercial reagent to TRP-2, monoclonal antibody (mAb) C-9 and undertook a comprehensive analysis to assess its specificity and usefulness for surgical pathology. Subsequently, we analyzed panels of normal tissues and tumors. We show that TRP-2 is regularly expressed in melanocytes of the normal skin. In cutaneous nevi, TRP-2 is present in junctional as well as in dermal nevocytes. In malignant tumors, C-9 reactivity is restricted to melanocytic and related lesions and present in 84% and 58% of primary and metastatic melanomas, respectively. Ten primary melanomas of the anorectal mucosa were all positive. Like the other melanocyte differentiation antigens, TRP-2 was absent in 6 desmoplastic melanomas. Also, only 2 of 9 angiomyolipomas were TRP-2 positive. We conclude that mAb C-9 is a valuable reagent for the analysis of TRP-2 expression in archival surgical pathology material. The expression pattern of TRP-2 in melanocytic and related lesions appears to parallel other melanocyte differentiation antigens, although the overall incidence is lower than other antigens, such as Melan-A or gp100.

Abstract B035: Self-tumor-antigen-specific CD8 T cells were suppressed by regulatory T cells

Abstract Background: Naturally occurring regulatory T (Treg) cells play an essential role for immunological unresponsiveness to self-tumor-antigens. Yet, how they stably suppress self-tumor-antigen-specific T cells remains unclear. In particular, it is unknown whether Treg cell-mediated suppression for a limited period has a critical long-lasting effect on cell fate and antigen reactivity of self-tumor-antigen-specific T cells. Experimental procedures: To demonstrate Treg-cell effect on proliferation, cytokine production, and cell fate of self-tumor-antigen-specific CD8+ T cells, CD8+ T cells from peripheral blood mononuclear cells (PBMCs) of healthy individuals were stimulated with a melanocyte differentiation antigen Melan-A/ MART-1, expressed by normal melanocytes and some melanoma cells, with/without FoxP3+CD25+CD4+ Treg cells. Melan-A/ MART-1-specific CD8+ T-cell induction and their phenotypes were investigated. Results: Melan-A/ MART-1-specific CD8+ T-cell induction was reduced by the presence of Treg cells. Self-tumor-antigen-specific CD8+ T cells co-cultured with Treg cells underwent one cell division and stopped further proliferation due to the lack of co-stimulation from antigen-presenting cells through Treg-cell suppression. They harbored low-affinity T-cell receptors and an anergic phenotype (i.e., hypo-proliferative and cytokine hypo-producing upon antigen re-stimulation) defined by expression of CCR7 and co-inhibitory molecules such as CTLA-4. Melan-A/ MART-1-specific CD8+ T cells with this anergic phenotype were detected ex vivo in the periphery and contributed to the tolerance of self-tumor-antigens. Conclusions: Treg-cell-mediated induction of anergy in self- antigen tumor-specific CD8+ T cells inhibits their activation in cancer patients after immunotherapy, and neo-antigens associated with tumor-specific gene mutations may become a main target of CD8+ T cells inducing actual tumor regression. Citation Format: Yuka Maeda, Hiroyoshi Nishikawa, Shimon Sakaguchi. Self-tumor-antigen-specific CD8 T cells were suppressed by regulatory T cells. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B035.