Clinical and pathological characterization of tebentafusp-associated skin toxicity: A cohort study with 33 patients.

BACKGROUND: Tebentafusp is a novel treatment for patients with metastatic uveal melanoma and often causes cutaneous side effects. OBJECTIVES: The aim of this study was to better characterize these heterogenous cutaneous side effects. METHODS: This prospective cohort study evaluated all patients from a tertiary hospital center who were treated with tebentafusp between January 2019 and June 2023 clinically and assessed skin biopsies histologically. RESULTS: In total, 33 patients were analyzed. Skin toxicity was observed in 78.8% of patients and was classified into 5 clinical categories: (1) symmetrical erythematous patches (83.8%), (2) hemorrhagic macules (11.8%), (3) urticarial lesions (7.4%), (4) bullous lesions (1.5%), and (5) skin (8.5%) and hair depigmentation (11.4%). Histopathologic features were focal lymphocytic interface dermatitis with epidermal infiltration of CD8-positive lymphocytes. Patients with skin reactions had a significantly longer median overall survival compared to patients without any cutaneous events (34 versus 4 months, P < .001). LIMITATION: Monocentric study with a limited number of patients. CONCLUSION: Tebentafusp frequently induces cutaneous reactions. Pathogenesis is likely due to binding of tebentafusp to stimulated melanocytes in the skin, followed by infiltration and activation of lymphocytes. Development of treatment-induced skin reactions may be associated with survival benefits.

ER-aminopeptidase 1 determines the processing and presentation of an immunotherapy-relevant melanoma epitope.

Dissecting the different steps of the processing and presentation of tumor-associated antigens is a key aspect of immunotherapies enabling to tackle the immune response evasion attempts of cancer cells. The immunodominant glycoprotein gp100209-217 epitope, which is liberated from the melanoma differentiation antigen gp100PMEL17 , is part of immunotherapy trials. By analyzing different human melanoma cell lines, we here demonstrate that a pool of N-terminal extended peptides sharing the common minimal epitope is generated by melanoma proteasome subtypes. In vitro and in cellulo experiments indicate that ER-resident aminopeptidase 1 (ERAP1)-but not ERAP2-defines the processing of this peptide pool thereby modulating the T-cell recognition of melanoma cells. By combining the outcomes of our studies and others, we can sketch the complex processing and endogenous presentation pathway of the gp100209-217 -containing epitope/peptides, which are produced by proteasomes and are translocated to the vesicular compartment through different pathways, where the precursor peptides that reach the endoplasmic reticulum are further processed by ERAP1. The latter step enhances the activation of epitope-specific T lymphocytes, which might be a target to improve the efficiency of anti-melanoma immunotherapy.

Adoptive T-Cell Transfer to Treat Lymphangioleiomyomatosis.

Patients with lymphangioleiomyomatosis (LAM) develop pulmonary cysts associated with neoplastic, smooth muscle-like cells that feature neuroendocrine cell markers. The disease preferentially affects premenopausal women. Existing therapeutics do not cure LAM. As gp100 is a diagnostic marker expressed by LAM lesions, we proposed to target this immunogenic glycoprotein using TCR transgenic T cells. To reproduce the genetic mutations underlying LAM, we cultured Tsc2-/- kidney tumor cells from aged Tsc2 heterozygous mice and generated a stable gp100-expressing cell line by lentiviral transduction. T cells were isolated from major histocompatibility complex-matched TCR transgenic pmel-1 mice to measure cytotoxicity in vitro, and 80% cytotoxicity was observed within 48 hours. Antigen-specific cytotoxicity was likewise observed using pmel-1 TCR-transduced mouse T cells, suggesting that transgenic T cells may likewise be useful to treat LAM in vivo. On intravenous injection, slow-growing gp100+ LAM-like cells formed lung nodules that were readily detectable in severe combined immunodeficient/beige mice. Adoptive transfer of gp100-reactive but not wild-type T cells into mice significantly shrunk established lung tumors, even in the absence of anti-PD-1 therapy. These results demonstrate the treatment potential of adoptively transferred T cells to eliminate pulmonary lesions in LAM.

Syngeneic murine model for prostate cancer using RM1 cells transfected with gp100.

BACKGROUND: Prostate cancer (PC) is the most commonly diagnosed solid tumor in men. A major challenge in PC immunotherapy is the lack of an animal model that resembles human adenocarcinoma and allows for manipulation or monitoring of the immune response. Mouse models are needed for preclinical testing of new immunotherapies, whether used alone or in combination with established drugs, and to develop companion biomarkers that can be validated in clinical trials. METHODS: To develop a syngeneic prostate adenocarcinoma model with a well-defined tumor antigen, murine RM1 PC cells were transfected with the endogenous mouse melanoma protein, gp100 (RM1-gp100). Gp100 was attractive because it is a self-protein and it instantly allowed us to use the large trove of immune research tools developed for melanoma research. A dendritic cell (DC) vaccine was used as model immunotherapy to demonstrate that adoptive immunotherapy against gp100 decreases the growth of RM1-gp100 but not RM1. RESULTS: Expressing gp100 did not change the growth of RM1 cell in vitro or in vivo. The DCs pulsed with RM1-gp100 could be used to stimulate Pmel-1 lymphocyte proliferation and activation. Pmel-1 lymphocytes could be adoptively transferred into C57Bl/6 mice that were treated with DCs pulsed with RM1-gp100. The resulting Pmel-1 lymphocytes were monitored to assess the primary cellular immune response and memory response. CONCLUSION: We describe a murine model for prostate adenocarcinoma with a well-characterized antigen that can be used in an immunologically intact mice to monitor the temporal CD8+ lymphocyte-mediated antitumor immunity.

Effective oral delivery of gp100 plasmid vaccine against metastatic melanoma through multi-faceted blending-by-blending nanogels.

Plasmid DNA gp100 is able to act as an available vaccine against metastatic melanoma, but its administration is usually limited to parenteral route. Since oral delivery of plasmid DNA is intervened by various physical obstacles, here we constructed a nanogel (Alg-Tat-gp100) with multi-faceted functions through blending-by-blending method. Due to the cooperation of alginate and Tat peptide, Alg-Tat-gp100 demonstrated the significant improvement of stability in the stomach, mucus penetration ability in intestine, and transport across mucus layer and MDCK cells. Moreover, the bone marrow-derived cells were activated with an enhanced co-stimulatory molecule expression. Following immunization using Alg-Tat-gp100 nanogels in C57BL/6 mice, the secretion of IFN-γ and the activation of cytotoxic T cells were significantly improved. Benefiting from those cases, the B16F10 tumor inhibition rate achieved 42.5% by this oral DNA vaccine, suggesting that this multi-faceted nanogel prepared by simple blending-by-blending method may provide a new strategy for oral DNA vaccine delivery.

A Molecular Switch Abrogates Glycoprotein 100 (gp100) T-cell Receptor (TCR) Targeting of a Human Melanoma Antigen.

Human CD8(+) cytotoxic T lymphocytes can mediate tumor regression in melanoma through the specific recognition of HLA-restricted peptides. Because of the relatively weak affinity of most anti-cancer T-cell receptors (TCRs), there is growing emphasis on immunizing melanoma patients with altered peptide ligands in order to induce strong anti-tumor immunity capable of breaking tolerance toward these self-antigens. However, previous studies have shown that these immunogenic designer peptides are not always effective. The melanocyte differentiation protein, glycoprotein 100 (gp100), encodes a naturally processed epitope that is an attractive target for melanoma immunotherapies, in particular peptide-based vaccines. Previous studies have shown that substitutions at peptide residue Glu(3) have a broad negative impact on polyclonal T-cell responses. Here, we describe the first atomic structure of a natural cognate TCR in complex with this gp100 epitope and highlight the relatively high affinity of the interaction. Alanine scan mutagenesis performed across the gp100(280-288) peptide showed that Glu(3) was critically important for TCR binding. Unexpectedly, structural analysis demonstrated that the Glu(3) → Ala substitution resulted in a molecular switch that was transmitted to adjacent residues, abrogating TCR binding and T-cell recognition. These findings help to clarify the mechanism of T-cell recognition of gp100 during melanoma responses and could direct the development of altered peptides for vaccination.

Tebentafusp Induces a T-Cell-Driven Rash in Melanocyte-Bearing Skin as an Adverse Event Consistent with the Mechanism of Action.

Tebentafusp is a gp100xCD3-bispecific ImmTAC designed to redirect polyclonal T cells against cells presenting the melanocyte lineage-specific antigen gp100 on HLA-A∗02:01. Skin-related adverse events, predominantly rash, are frequent and occur within a few hours after initial infusions; yet, the mechanisms are unknown. In this study, we analyzed clinical data from the randomized phase 3 trial (NCT03070392) of tebentafusp (n = 252) versus investigator's choice (n = 126). Translational analyses were performed on paired on-treatment skin samples from 19 patients collected in the phase 1 trial (NCT01211262). Our analyses showed that rash is a clinical manifestation of tebentafusp-induced recruitment of T cells to cutaneous melanocytes. Development of rash depended on baseline expression levels of gp100 and other melanin pathway genes in the skin. On treatment, melanocyte number was reduced, and expression of melanocytic genes decreased, whereas gene expression related to immunity and cytokine signaling increased. When adjusted for baseline prognostic features, patients with rash within the first week of tebentafusp treatment had the same overall survival as patients without a rash in the phase 3 randomized trial IMCgp100-202 (hazard ratio = 0.84, 95% confidence interval = 0.53-1.32). In summary, skin rash is an off-tumor, on-target effect of tebentafusp against gp100+ melanocytes, in line with the mechanism of action.

Tebentafusp: a first-in-class treatment for metastatic uveal melanoma.

Tebentafusp is a first-in-class immunotherapy agent that comprises an engineered T-cell receptor targeting a gp100 epitope presented by human leukocyte antigen-A*02:01 cells, fused to an anti-CD3 single-chain variable fragment. Tebentafusp is both the first bispecific T-cell engager to show efficacy in the treatment of advanced solid cancer and the first anti-cancer treatment to demonstrate an overall survival benefit in patients with uveal melanoma (UM). This review article will focus on the clinical development of tebentafusp, the mechanism of action and resultant evolution of the management of advanced UM.

Liposomal celecoxib combined with dendritic cell therapy enhances antitumor efficacy in melanoma.

Cancer vaccine efficacy is limited by the immunosuppressive nature of the tumor microenvironment created by inflammation, immune inhibitory factors, and regulatory T cells (Tregs). Inspired by the role of cyclooxygenase-2 (COX-2) in inflammation in the tumor site, we proposed that normalization of the tumor microenvironment by celecoxib as a COX-2 inhibitor might improve the efficacy of Dendritic Cell (DC) therapy in a melanoma model. In the present study, liposomal celecoxib (Lip-CLX) was combined with ex vivo generated DC vaccines pulsed with gp100 peptide (in liposomal and non-liposomal forms) for prophylactic and therapeutic evaluation in the B16F10 melanoma model. Tumor site analysis by flow cytometry demonstrated that intravenous administration of Lip-CLX at a dose of 1 mg/kg in four doses effectively normalized the tumor microenvironment by reducing Tregs and IL-10 production. Furthermore, in combination with DC vaccination (DC + Lip-peptide+Lip-CLX), it significantly increased tumor-infiltrating CD4+ and CD8+ T cells and secretion of IFN-γ. This combinatorial strategy produced an effective prophylactic and therapeutic antitumor response, which reduced tumor growth and prolonged the overall survival. In conclusion, our findings suggest that the liposomal celecoxib targets the inhibitory mechanisms of the tumor microenvironment and broadens the impact of DC therapy to improve the outcome of immunotherapy in solid tumors.

Correlation Studies between S100 Protein Level and Soluble MIA or Tissue MelanA and gp100 (HMB45) Expression in Cutaneous Melanoma.

(1) Background: Cutaneous melanoma (CM) originates from melanocytes and causes 90% of skin cancer deaths; therefore, the comparison of different soluble and tissue markers could be valuable in the detection of melanoma progression and therapy monitoring. The present study is focused on the potential correlations between soluble S100B and MIA protein levels in different melanoma stages or with tissue expression of S100, gp100 (HMB45), and MelanA biomarkers. (2) Methods: Soluble S100B and MIA levels were evaluated by means of immunoassay methods in blood samples from 176 patients with CM, while tissue expressions of S100, MelanA, and gp100 (HMB45) were detected by means of immunohistochemistry in 76 melanomas. (3) Results: Soluble S100B correlated with MIA in stages III (r = 0.677, p < 0.001) and IV (r = 0.662, p < 0.001) but not in stages I and II; however, 22.22% and 31.98% of stage I and II patients, respectively, had high values for at least one of the two soluble markers. S100 tissue expression correlated with both MelanA (r = 0.610, p < 0.001) and HMB45 (r = 0.476, p < 0.01), while HMB45 and MelanA also significantly positively correlated (r = 0.623, p < 0.001). (4) Conclusions: Blood levels of S100B and MIA corroborated with melanoma tissue markers expression could help to improve the stratification process for patients with a high risk of tumor progression.

Tebentafusp in the Treatment of Metastatic Uveal Melanoma: Patient Selection and Special Considerations.

Uveal melanoma (UM) is a rare type of melanoma with distinct features from cutaneous melanoma, low response rates to immune checkpoint inhibition, and poor survival rates. Tebentafusp, a bispecific antibody engaging T cells with gp 100 on HLA-A*02:01, was recently approved by the FDA as the first drug of its class and the first treatment approved by the FDA to treat UM. In this review, we summarize the preclinical and clinical data on tebentafusp for UM. We additionally discuss patient selection and the relevant challenges. For the literature search, PubMed search and relevant articles presented at international conferences were used.

Evaluating Anti-tumor Immune Responses of Protein Nanoparticle-Based Cancer Vaccines.

Cancer vaccines displaying tumor-associated antigens (TAAs) train the immune system for enhanced tumor recognition and elimination. Nanoparticle-based cancer vaccines are ingested and processed by dendritic cells, which subsequently activate antigen-specific cytotoxic T cells, allowing them to identify and eliminate tumor cells displaying these TAAs. Here, we describe the procedures to conjugate TAA and adjuvant to a model protein nanoparticle platform (E2), followed by assessment of vaccine performance. Utilizing a syngeneic tumor model, the efficacy of in vivo immunization was determined by cytotoxic T lymphocyte assays and IFN-γ ELISpot ex vivo assays to measure tumor cell lysis and TAA-specific activation, respectively. In vivo tumor challenge directly allows evaluation of anti-tumor response and survival over time.

Tebentafusp for the treatment of HLA-A*02:01-positive adult patients with unresectable or metastatic uveal melanoma.

INTRODUCTION: Metastatic uveal melanoma is associated with poor prognosis and few treatment options. Tebentafusp recently became the first FDA-approved agent for metastatic uveal melanoma. AREAS COVERED: In this review, we describe the mechanism of action of tebentafusp as well as preclinical data showing high tumor specificity of the drug. We also review promising early-phase trials in which tebentafusp demonstrated activity in metastatic uveal melanoma patients with an acceptable toxicity profile that included cytokine-mediated, dermatologic-related, and liver-related adverse events. Finally, we summarize findings from a pivotal phase III randomized trial in which tebentafusp demonstrated significant improvement in overall survival in comparison with investigator choice therapy. EXPERT OPINION: Tebentafusp has transformed the treatment paradigm for metastatic uveal melanoma and should be the preferred frontline agent for most HLA-A*0201 positive patients. However, patients with rapidly progressing disease or high tumor benefit may not derive the same benefit. Areas of future study should focus on its role in the adjuvant setting as well as strategies to improve the efficacy of tebentafusp in the metastatic setting.

Designing a novel multiepitope peptide vaccine against melanoma using immunoinformatics approach.

Malignant melanoma is the most aggressive and life-threaten skin cancer. Nowadays, the prevention and treatment of melanoma are challenging areas for researchers and physicians. Therefore, we implemented an in silico-based approach to design a multi-epitope peptide vaccine for melanoma. This approach consists of immunoinformatics, molecular docking, and dynamic stimulation assessments to identify potent targets. Three most immunogenic melanoma proteins; NEYSO-1, gp-100, and MART-1were considered to predict immunodominant B and T cell epitopes. The prioritized epitopes had significant potential to induce strong humoral and cellular immunity and INF-γ responses without the possibility of allergenicity. To enhance the immunogenic properties of the vaccine, we used adjuvants HBHA, the helper epitope of PADRE, and three segments of the helper epitope from TTFrC. To design the final vaccine construct, appropriate linkers are used to join immunogenicscreened-epitopes and also the adjuvants. The physicochemical and immunological properties of the vaccine were evaluated.The designed-vaccine construct was docked to TLR4 to visualize the complex affinity and then conformational dynamics simulation was used to analyze time-dependent interaction behavior. In silico cloning demonstrated that the vaccine can be efficiently expressed in E.coli. Therefore, the designed vaccine might have the ability to induce humoral and cellular immune responses against melanoma cancer antigens. This vaccine has a high-quality structure and suitable characteristics such as high stability, solubility, and a high potential for expression in a prokaryotic system. However, these results need the experimental study to ensure the immunogenicity and safety profile of the melanoma candidate vaccine construct.

Tumor rejection properties of gp100209-specific T cells correlate with T cell receptor binding affinity towards the wild type rather than anchor-modified antigen.

Although there are exceptions and outliers, T cell functional responses generally correlate with the affinity of a TCR for a peptide/MHC complex. In one recently described outlier case, the most promising clinical candidate in a series of TCRs specific for the gp100209 melanoma antigen bound with the weakest solution affinity and produced the least amount of cytokine in vitro. Hypotheses for this outlier behavior included unusual cytokine expression patterns arising from an atypical TCR binding geometry. Studying this instance in more detail, we found here that outlier behavior is attributable not to unusual cytokine patterns or TCR binding, but the use of a position 2 anchor-modified peptide variant in in vitro experiments instead of the wild type antigen that is present in vivo. Although the anchor-modified variant has been widely used in basic and clinical immunology as a surrogate for the wild type peptide, prior work has shown that TCRs can clearly distinguish between the two. We show that when this differential recognition is accounted for, the functional properties of gp100209-specific TCRs track with their affinity towards the peptide/MHC complex. Beyond demonstrating the correlates with T cell function for a clinically relevant TCR, our results provide important considerations for selection of TCRs for immunotherapy and the use of modified peptides in immunology.

Comparison of two routes of administration of a cationic liposome formulation for a prophylactic DC vaccination in a murine melanoma model.

Dendritic cell (DC) vaccination can be achieved via straight loading of vaccine into DCs ex vivo or administration to DCs in vivo. However, there is no certain consensus on which approach is preferable, and each strategy has its advantages and disadvantages, which affect the efficacy and safety of vaccines. It will also be more complicated when a vaccine delivery system is included. In this study, the efficacy of ex vivo pulsed DC-based vaccine compared with in vivo subcutaneous administration of a cationic liposomes (CLs) formulation containing gp100 antigen (gp100-CLs) was evaluated in a murine melanoma model. In combination with an anti-PD-1 antibody, the ex vivo approach of gp100-CLs yielded a significant (P < 0.01) increase in the number of antigen-specific tumors infiltrated lymphocytes (TILs) with a significant upregulation of IFN-γ (P < 0.0001) and PD-1 (P < 0.0001) expression level. They also dampened the function of immunosuppressive regulatory T cells (Tregs) via significant downregulation of IL-10 and TGF-ß (P < 0.0001) expression level compared to in vivo approach in the tumor microenvironment (TME). Furthermore, prophylactic immunization with gp100-CLs pulsed DCs ex vivo delayed tumor growth and induced the survival benefit over in vivo immunization. Collectively, the ex vivo DC-based vaccination pulsed with gp100 encapsulated in liposomes synergizes with anti-PD-1 antibody and represents a preferable approach against melanoma.

Expression of Potential Targets for Cell-Based Therapies on Melanoma Cells.

Tumor antigen-specific redirection of cytotoxic T cells (CTLs) or natural killer (NK) cells including chimeric antigen receptor (CAR-) and T cell receptor (TCR-) cell therapy is currently being evaluated in different tumor entities including melanoma. Expression of melanoma-specific antigen recognized by the respective CAR or TCR directly or presented by HLA molecules is an indispensable prerequisite for this innovative therapy. In this study, we investigated in 168 FFPE tumor specimens of patients with stage I-IV melanoma the protein expression of HER2, TRP2, ABCB5, gp100, p53, and GD2 by immunohistochemistry (IHC). These results were correlated with clinical parameters. Membrane expression of HER2 and GD2 was also investigated in ten melanoma cell lines by flow cytometry for which corresponding tumors were analyzed by IHC. Our results demonstrated that gp100 was the most frequently overexpressed protein (61%), followed by TRP2 (50%), GD2 (38%), p53 (37%), ABCB5 (17%), and HER2 (3%). TRP2 expression was higher in primary tumors compared to metastases (p = 0.005). Accordingly, TRP2 and ABCB5 expression was significantly associated with lower tumor thickness of the primary (p = 0.013 and p = 0.025). There was no association between protein expression levels and survival in advanced melanoma patients. Flow cytometric analysis revealed abundant surface expression of GD2 and HER2 in all melanoma cell lines. The discordant HER2 expression in situ and in vitro suggests a tissue culture associated induction. In summary, our data support the use of gp100 and GD2 as a potential target for developing engineered TCR- or CAR-cell therapies, respectively, against melanoma.

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.

Liposomal gp100 vaccine combined with CpG ODN sensitizes established B16F10 melanoma tumors to anti PD-1 therapy.

OBJECTIVES: Program death 1 (PD-1)/ program death-ligand 1 (PD-L1) pathways, as the main inhibitory checkpoints, induce immunosuppression in the tumor microenvironment (TME). Despite the importance of inhibitor checkpoint receptor (ICR) blockers, their outcomes have been limited by the low immune response rate and induced acquired resistance. Pre-existing tumor-specific T cells is related to the improvement of their therapeutic efficacy. In the present study, we show that the combination of liposomal gp100 nanovaccine with anti PD-1 monoclonal antibody (mAb) potentiates the therapeutic effect in the melanoma model. MATERIALS AND METHODS: In this study, we first decorate the cationic liposome with gp10025-33 self-antigen and then characterize it. Mice bearing B16F10 melanoma tumors were vaccinated with different formulations of gp100 peptide (free or liposomal form) with or without CpG ODN adjuvant in combination with anti PD-1 mAb. RESULTS: Therapeutic combination of liposomal nanovaccine and CpG with anti PD-1 mAb, demonstrated the increased number of tumor infiltrated lymphocytes (TILs) in TME with the highest IFN-γ production and cytotoxic activity, which led to remarkable tumor regression. CONCLUSION: Our results demonstrated the synergism between Lip-peptide+CpG nanovaccine and anti PD-1 regime, which improved the therapeutic efficacy of PD-1 checkpoint blocker in melanoma mice models.

Vaccination with dendritic cells pulsed ex vivo with gp100 peptide-decorated liposomes enhances the efficacy of anti PD-1 therapy in a mouse model of melanoma.

BACKGROUND: Targeting antigens to dendritic cells (DCs) via nanoparticles is a powerful strategy which improves the efficacy of ex vivo antigen-pulsed DC vaccines. METHODS: In this study, liposomes were first decorated with gp10025-33 self-antigen and then characterized. Then, DCs were pulsed ex vivo with liposomal gp100 and injected subcutaneously in mice bearing B16F10 established melanoma tumors in combination with anti-PD-1 therapy. RESULTS: Treatment with liposomal pulsed DC vaccine elicited the strongest anticancer immunity and enhanced intratumoral immune responses based on infiltration of gp100-specific CD4+ and CD8+ T cells to the tumor leading to significant tumor growth regression and prolonged survival rate. Treatment with liposomal pulsed DC vaccine also markedly enhanced specific cytotoxic T lymphocytes (CTL) responses with a significant higher titer of IFN-γ in the spleen. Moreover, a significant increase of PD-1 expressing CD8+ tumor infiltrating lymphocytes (TILs) was detected in tumors. CONCLUSION: Our results demonstrate an optimum dose of liposomal gp100 significantly increases the efficacy of anti-PD-1 therapy in mice and might be an effective strategy to overcome resistance to anti-PD-1 therapy.