Engineering Cancer Antigen-Specific T Cells to Overcome the Immunosuppressive Effects of TGF-ß.

Adoptive T cell therapy with T cells expressing affinity-enhanced TCRs has shown promising results in phase 1/2 clinical trials for solid and hematological tumors. However, depth and durability of responses to adoptive T cell therapy can suffer from an inhibitory tumor microenvironment. A common immune-suppressive agent is TGF-ß, which is secreted by tumor cells and cells recruited to the tumor. We investigated whether human T cells could be engineered to be resistant to inhibition by TGF-ß. Truncating the intracellular signaling domain from TGF-ß receptor (TGFßR) II produces a dominant-negative receptor (dnTGFßRII) that dimerizes with endogenous TGFßRI to form a receptor that can bind TGF-ß but cannot signal. We previously generated specific peptide enhanced affinity receptor TCRs recognizing the HLA-A*02-restricted peptides New York esophageal squamous cell carcinoma 1 (NY-ESO-1)157-165/l-Ag family member-1A (TCR: GSK3377794, formerly NY-ESO-1c259) and melanoma Ag gene A10254-262 (TCR: ADP-A2M10, formerly melanoma Ag gene A10c796). In this article, we show that exogenous TGF-ß inhibited in vitro proliferation and effector functions of human T cells expressing these first-generation high-affinity TCRs, whereas inhibition was reduced or abolished in the case of second-generation TCRs coexpressed with dnTGFßRII (e.g., GSK3845097). TGF-ß isoforms and a panel of TGF-ß-associated genes are overexpressed in a range of cancer indications in which NY-ESO-1 is commonly expressed, particularly in synovial sarcoma. As an example, immunohistochemistry/RNAscope identified TGF-ß-positive cells close to T cells in tumor nests and stroma, which had low frequencies of cells expressing IFN-γ in a non-small cell lung cancer setting. Coexpression of dnTGFßRII may therefore improve the efficacy of TCR-transduced T cells.

Targeting cancer testis antigens in synovial sarcoma.

Synovial sarcoma (SS) is a rare cancer that disproportionately affects children and young adults. Cancer testis antigens (CTAs) are proteins that are expressed early in embryonic development, but generally not expressed in normal tissue. They are aberrantly expressed in many different cancer types and are an attractive therapeutic target for immunotherapies. CTAs are expressed at high levels in SS. This high level of CTA expression makes SS an ideal cancer for treatment strategies aimed at harnessing the immune system to recognize aberrant CTA expression and fight against the cancer. Pivotal clinical trials are now underway, with the potential to dramatically alter the landscape of SS management and treatment from current standards of care. In this review, we describe the rationale for targeting CTAs in SS with a focus on NY-ESO-1 and MAGE-A4, the current state of vaccine and T-cell receptor-based therapies, and consider emerging opportunities for future development.

Correlating SS18-SSX immunohistochemistry (IHC) with SS18 fluorescent in situ hybridization (FISH) in synovial sarcomas: a study of 36 cases.

The recently introduced, highly sensitive and specific SS18-SSX immunohistochemistry (IHC) is an attractive alternative to SS18 fluorescence in situ hybridization (FISH) testing in synovial sarcoma (SS). However, little is known about how SS18-SSX IHC correlates with SS18 FISH. We correlated the SS18 FISH results of SS from 36 patients with SS18-SSX IHC. Twenty-six tumours had a classic break-apart FISH pattern (1 fused, 1 red and 1 green signal) and all stained positive for the IHC. Ten had an atypical (non-classic) FISH pattern of which 5 stained positive for the IHC. Four of these (including two with novel atypical SS18 FISH patterns) were confirmed to harbour the SS18-SSX fusion on targeted RNA sequencing, while one had classic features of a biphasic SS. The remaining 5 tumours stained negative for the IHC. One had a TPM3-NTRK1 fusion, and one had no fusion, while the remaining three had insufficient tissue/RNA for sequencing. The sensitivity of the IHC was 91% (after excluding the 2 cases with confirmed absence of SS18-SSX fusion). Twenty histologic mimics of SS also stained negative for the IHC (100% specificity). Our study shows that the SS18-SSX IHC is more specific than SS18 FISH in diagnosing SS, especially in cases with atypical FISH patterns. It correlates well with RNA sequencing result and has the potential to replace SS18 FISH testing. A positive IHC result supports the diagnosis of SS, while a tumour with atypical FISH pattern and negative IHC result should undergo further molecular testing.

IL-15 mediated expansion of rare durable memory T cells following adoptive cellular therapy.

BACKGROUND: Synovial sarcoma (SS) and myxoid/round cell liposarcoma (MRCL) are ideal solid tumors for the development of adoptive cellular therapy (ACT) targeting NY-ESO-1, as a high frequency of tumors homogeneously express this cancer-testes antigen. Data from early phase clinical trials have shown antitumor activity after the adoptive transfer of NY-ESO-1-specific T cells. In these studies, persistence of NY-ESO-1 specific T cells is highly correlated with response to ACT, but patients often continue to have detectable transferred cells in their peripheral blood following progression. METHOD: We performed a phase I clinical trial evaluating the safety of NY-ESO-1-specific endogenous T cells (ETC) following cyclophosphamide conditioning. Peripheral blood mononuclear cells (PBMCs) from treated patients were evaluated by flow cytometry and gene expression analysis as well as through ex vivo culture assays with and without IL-15. RESULTS: Four patients were treated in a cohort using ETC targeting NY-ESO-1 following cyclophosphamide conditioning. Treatment was well tolerated without significant toxicity, but all patients ultimately had disease progression. In two of four patients, we obtained post-treatment tumor tissue and in both, NY-ESO-1 antigen was retained despite clear detectable persisting NY-ESO-1-specific T cells in the peripheral blood. Despite a memory phenotype, these persisting cells lacked markers of proliferation or activation. However, in ex vivo culture assays, they could be induced to proliferate and kill tumor using IL-15. These results were also seen in PBMCs from two patients who received gene-engineered T-cell receptor-based products at other centers. CONCLUSIONS: ETC targeting NY-ESO-1 with single-agent cyclophosphamide alone conditioning was well tolerated in patients with SS and those with MRCL. IL-15 can induce proliferation and activity in persisting NY-ESO-1-specific T cells even in patients with disease progression following ACT. These results support future work evaluating whether IL-15 could be incorporated into ACT trials post-infusion or at the time of progression.

HLA Genotyping in Synovial Sarcoma: Identifying HLA-A*02 and Its Association with Clinical Outcome.

PURPOSE: To determine if a targeted exome panel utilizing matched normal DNA can accurately detect germline and somatic HLA genes in patients with synovial sarcoma (SS) and whether select HLA-A*02 genotypes are prognostic or predictive of outcome in metastatic SS. EXPERIMENTAL DESIGN: Patients with metastatic SS consented to HLA typing by a Clinical Laboratory Improvement Amendments (CLIA)-certified test to determine eligibility for a clinical trial of NY-ESO-1-specific engineered T cells restricted to carriers of HLA-A*02:01, -A*02:05, or -A*02:06 (HLA-A*02 eligible). HLA genotype was determined from Memorial Sloan Kettering Integrated Molecular Profiling of Actionable Cancer Targets (MSK-IMPACT), where feasible, and somatic loss of heterozygosity (LOH) in HLA alleles was identified. Overall survival (OS) was estimated and stratified by HLA-A*02 eligibility. RESULTS: A total of 23 patients had HLA genotyping by a CLIA-certified lab and MSK-IMPACT. Ninety percent (108/110) of the sequenced alleles were concordant between IMPACT and the outside lab. LOH of HLA genes was detected in three tumors, one had loss of HLA-A*02:01. In total, 66 patients were screened for T-cell therapy and 20 (30%) were HLA-A*02 eligible on outside testing. Univariate analysis of OS from the time of metastasis found HLA-A*02 eligibility was marginally associated with shorter OS [HR = 1.95; 95% confidence interval (CI), 0.995-3.813; P = 0.052]. On multivariate analysis, older age and larger tumor size, but not HLA-A*02 eligibility, were significantly associated with decreased OS. HLA-A*02 eligibility did not impact OS after chemotherapy or pazopanib in the metastatic setting. CONCLUSIONS: Targeted gene panels like MSK-IMPACT may accurately report HLA type and identify loss of somatic HLA alleles. In a multivariable model, HLA-A*02 eligibility was not significantly associated with OS in patients with metastatic SS.

Tumor mutation burden and checkpoint immunotherapy markers in primary and metastatic synovial sarcoma.

Synovial sarcoma (SS) is a soft-tissue malignancy that most often affects patients aged between 15 and 40 years, and the prognosis for patients with metastatic disease is generally poor. This study was performed to evaluate checkpoint blockade immunotherapy markers in SS, including tumor mutational burden (TMB), DNA mismatch repair (MMR) status, and PDL-1 (programmed cell death ligand 1), PD1 (programmed cell death 1), and CD8 expression by normal-tumor paired whole-exome sequencing (WES) and immunohistochemistry (IHC). Outcomes evaluated included event-free and overall survival. Twenty one (21) FISH (Fluorescence In Situ Hybridization)-confirmed SS cases (11 F, 10 M) were studied, with age ranging from 8 to 89 years at diagnosis and follow-up ranging from 1 to 16 years. TMB (n = 16) ranged from 0.83 to 212/Mb (median, 1.7). Only one case showed a high TMB of 212/Mb and missense variants of MMR genes in the primary tumor, while the other 15 cases had a low TMB of less than 5/Mb. IHC was performed on all 21 tumor samples for PD-L1, PD1, CD8, and MMR proteins. PD-L1 membranous staining was detected in 3 of 21 cases (14.3%), ranging from 1 to 5% for tumor proportion score and 1-10 for combined positive score. PD1 was detected in 15 of 21 cases (71.4%), ranging from 1 to 25/HPF (high power field) (median, 2). CD8 stain was seen in all cases, ranging from 2 to 60/HPF (median, 5). PD1 staining results correlated with CD8 staining results (P < 0.0001). No correlation of TMB or IHC markers was found with survival. No fixed pattern of TMB or IHCs between primary and metastatic tumors was observed; there was no correlation between TMB or IHCs and age, location, or diagnosis subtype. All of the cases tested showed retained expression of MMR proteins. The results show that for SS, a tumor with strong driver translocation, most cases have a low TMB, but occasionally a high TMB may be present, as observed in 1 of the 16 (6.25%) cases. The demonstration of a subgroup of SS cases with high TMB might explain the 10% response rate to checkpoint immunotherapy observed in clinical trials in patients with SS.

Histiocyte predominant myocarditis resulting from the addition of interferon gamma to cyclophosphamide-based lymphodepletion for adoptive cellular therapy.

BACKGROUND: Adoptive cellular therapy (ACT) is a promising treatment for synovial sarcoma (SS) with reported response rates of over 50%. However, more work is needed to obtain deeper and more durable responses. SS has a 'cold' tumor immune microenvironment with low levels of major histocompatibility complex (MHC) expression and few T-cell infiltrates, which could represent a barrier toward successful treatment with ACT. We previously demonstrated that both MHC expression and T-cell infiltration can be increased using systemic interferon gamma (IFN-γ), which could improve the efficacy of ACT for SS. CASE PRESENTATION: We launched a phase I trial incorporating four weekly doses of IFN-γ in an ACT regimen of high-dose cyclophosphamide (HD Cy), NY-ESO-1-specific T cells, and postinfusion low-dose interleukin (IL)-2. Two patients were treated. While one patient had significant tumor regression and resultant clinical benefit, the other patient suffered a fatal histiocytic myocarditis. Therefore, this cohort was terminated for safety concerns. CONCLUSION: We describe a new and serious toxicity of immunotherapy from IFN-γ combined with HD Cy-based lymphodepletion and low-dose IL-2. While IFN-γ should not be used concurrently with HD Cy or with low dose IL-2, IFN-γ may still be important in sensitizing SS for ACT. Future studies should avoid using IFN-γ during the immediate period before/after cell infusion. TRIAL REGISTRATION NUMBERS: NCT04177021, NCT01957709, and NCT03063632.

Guillain-Barre syndrome observed with adoptive transfer of lymphocytes genetically engineered with an NY-ESO-1 reactive T-cell receptor.

BACKGROUND: Adoptive transfer of autologous T-lymphocytes transduced with a high affinity NY-ESO-1-reactive T-cell receptor (NY-ESO-1c259 T-cells) has emerged as a promising therapeutic strategy for patients with refractory synovial sarcoma. Secondary autoimmune T-cell mediated toxicities can occur long after initial adoptive T-cell transfer. We report on the first two cases of the development and management of Guillain-Barre syndrome in synovial sarcoma patients who received NY-ESO-1c259 T-cells. CASE PRESENTATION: A 47 year-old woman and 39 year-old woman with refractory metastatic SS were treated with fludarabine-cyclophosphamide lymphodepletion followed by adoptive transfer of NY-ESO-1c259 T-cells. On day 42 after adoptive T-cell therapy, patient one presented to the emergency room with a one-week history of numbness, paresthesia, and heaviness to both legs progressing to difficulty walking on the day of presentation. Although MRI brain and lumbar puncture were negative, electromyography (EMG) and nerve conduction studies (NCS) of the lower extremities and right arm performed revealed an abnormal study suggestive of a very mild, distal, motor, axonal polyneuropathy. Patient two presented on day 113 with bilateral foot numbness, left foot drop, unsteady gait, and pain in the left thigh, which progressed over two week to bilateral leg weakness, inability to walk, and numbness bilaterally in the hands, legs, and feet. Both patients received intravenous immunoglobulin (IVIG) 0.4 g/kg/day for 5 days for possible acute inflammatory demyelinating polyneuropathy (AIDP) likely related to NY-ESO-1 targeting T-cell therapy. After 3 and 5 doses, respectively, of IVIG, the patients reported improvement in symptoms and strength, and were later transferred to an inpatient rehabilitation facility to continue gaining strength. At patient one's neurology follow-up on day 95, she reported only mild left lower extremity (LLE) weakness and was gradually successfully regaining independence in motor function. At patient two's 9-month follow-up, the patient had regained normal function and independence. CONCLUSIONS: Given the expanding applications of immunotherapy in cancer management, clinicians should stay vigilant against the potential development of unusual but life-threatening immune-mediated toxicities.

Systemic and local immunity following adoptive transfer of NY-ESO-1 SPEAR T cells in synovial sarcoma.

BACKGROUND: Gene-modified autologous T cells expressing NY-ESO-1c259, an affinity-enhanced T-cell receptor (TCR) reactive against the NY-ESO-1-specific HLA-A*02-restricted peptide SLLMWITQC (NY-ESO-1 SPEAR T-cells; GSK 794), have demonstrated clinical activity in patients with advanced synovial sarcoma (SS). The factors contributing to gene-modified T-cell expansion and the changes within the tumor microenvironment (TME) following T-cell infusion remain unclear. These studies address the immunological mechanisms of response and resistance in patients with SS treated with NY-ESO-1 SPEAR T-cells. METHODS: Four cohorts were included to evaluate antigen expression and preconditioning on efficacy. Clinical responses were assessed by RECIST v1.1. Engineered T-cell persistence was determined by qPCR. Serum cytokines were evaluated by immunoassay. Transcriptomic analyses and immunohistochemistry were performed on tumor biopsies from patients before and after T-cell infusion. Gene-modified T-cells were detected within the TME via an RNAish assay. RESULTS: Responses across cohorts were affected by preconditioning and intra-tumoral NY-ESO-1 expression. Of the 42 patients reported (data cut-off 4June2018), 1 patient had a complete response, 14 patients had partial responses, 24 patients had stable disease, and 3 patients had progressive disease. The magnitude of gene-modified T-cell expansion shortly after infusion was associated with response in patients with high intra-tumoral NY-ESO-1 expression. Patients receiving a fludarabine-containing conditioning regimen experienced increases in serum IL-7 and IL-15. Prior to infusion, the TME exhibited minimal leukocyte infiltration; CD163+ tumor-associated macrophages (TAMs) were the dominant population. Modest increases in intra-tumoral leukocytes (≤5%) were observed in a subset of subjects at approximately 8 weeks. Beyond 8 weeks post infusion, the TME was minimally infiltrated with a TAM-dominant leukocyte infiltrate. Tumor-associated antigens and antigen presentation did not significantly change within the tumor post-T-cell infusion. Finally, NY-ESO-1 SPEAR T cells trafficked to the TME and maintained cytotoxicity in a subset of patients. CONCLUSIONS: Our studies elucidate some factors that underpin response and resistance to NY-ESO-1 SPEAR T-cell therapy. From these data, we conclude that a lymphodepletion regimen containing high doses of fludarabine and cyclophosphamide is necessary for SPEAR T-cell persistence and efficacy. Furthermore, these data demonstrate that non-T-cell inflamed tumors, which are resistant to PD-1/PD-L1 inhibitors, can be treated with adoptive T-cell based immunotherapy. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01343043 , Registered 27 April 2011.

Systemic Interferon-γ Increases MHC Class I Expression and T-cell Infiltration in Cold Tumors: Results of a Phase 0 Clinical Trial.

Interferon-γ (IFNγ) has been studied as a cancer treatment with limited evidence of clinical benefit. However, it could play a role in cancer immunotherapy combination treatments. Despite high expression of immunogenic cancer-testis antigens, synovial sarcoma (SS) and myxoid/round cell liposarcoma (MRCL) have a cold tumor microenvironment (TME), with few infiltrating T cells and low expression of major histocompatibility complex class I (MHC-I). We hypothesized that IFNγ treatment could drive inflammation in a cold TME, facilitating further immunotherapy. We conducted a phase 0 clinical trial treating 8 SS or MRCL patients with weekly systemic IFNγ. We performed pre- and posttreatment biopsies. IFNγ changed the SS and MRCL TME, inducing tumor-surface MHC-I expression and significant T-cell infiltration (P < 0.05). Gene-expression analysis suggested increased tumor antigen presentation and less exhausted phenotypes of the tumor-infiltrating T cells. Newly emergent antigen-specific humoral and/or T-cell responses were found in 3 of 7 evaluable patients. However, increased expression of PD-L1 was observed on tumor-infiltrating myeloid cells and in some cases tumor cells. These findings suggest that systemic IFNγ used to convert SS and MRCL into "hot" tumors will work in concert with anti-PD-1 therapy to provide patient benefit.

Prognostic impact of the tumor immune microenvironment in synovial sarcoma.

The association between the immune status within the tumor microenvironment and prognosis in synovial sarcoma is not well understood. We aimed to investigate the tumor immune microenvironment and analyze its prognostic impact for patients with synovial sarcoma. A total of 36 primary patients who were treated in our institution were retrospectively evaluated. Infiltration of lymphocytes (CD4+, CD8+, and FOXP3+), CD163+ macrophages, and expression of human leukocyte antigen (HLA) class I and programmed death ligand 1 (PD-L1) were evaluated by immunohistochemistry. Moreover, we investigated PD-L1 and programmed death ligand 2 (PD-L2) mRNA expression in 19 of the 36 cases, using real-time PCR. The Kaplan-Meier method was used to estimate overall survival and progression-free survival. Infiltration of lymphocytes and macrophages varied among the patients. Furthermore, the expression of HLA class I was negative or downregulated in 11 specimens. No PD-L1 expression was observed using immunohistochemistry. Moreover, although PD-L1 mRNA expression was observed in 18 of 19 specimens, the expression level was low. A higher infiltration of CD8+ or FOXP3+ lymphocytes in patients was associated with a favorable overall survival. In addition, a higher infiltration of CD163+ macrophages indicated a significantly worse overall and progression-free survival. Infiltration of CD4+ lymphocytes, HLA class I, PD-L1, and PD-L2 expression were not associated with patient prognosis. This represents the first report investigating the tumor immune microenvironment as a prognostic factor in synovial sarcoma, indicating that CD163+ macrophages are associated with tumor progression. Our results underscore the clinical significance of the tumor immune microenvironment in synovial sarcoma.

Immune profiles of desmoplastic small round cell tumor and synovial sarcoma suggest different immunotherapeutic susceptibility upfront compared to relapse specimens.

BACKGROUND: Desmoplastic small round cell tumor (DSRCT) and synovial sarcoma are rare tumors with dismal outcomes requiring new therapeutic strategies. Immunotherapies have shown promise in several cancer types, but have not been evaluated in DSRCT and synovial sarcoma. Because the immune microenvironment can provide indications of the inflammatory nature of tumors, immunohistochemical staining is able to assess the tumor immune infiltrates in both tumor types. PROCEDURE: Using tissue microarrays of DSRCT and synovial sarcoma tumor samples, we detected tumoral HLA-A/B/C, beta-2-microglobulin(B2M), and PD-L1 expression, and quantified tumor-infiltrating lymphocytes expressing CD4, CD8, CD56, CD45RO, or FOXP3 by immunohistochemistry. We used staining intensity on a scale of 0-3 and percentage of tumor stained to determine HLA, B2M, and PD-L1 scores. We calculated the cytotoxic T lymphocyte (CTL) target score as HLA score × B2M score/100. RESULTS: In diagnostic samples, we found high HLA and CTL target scores and low PD-L1 expression with decreased scores in recurrence for both tumor types. We found an increase in CD56+ natural killer cells in DSRCT samples from diagnosis to recurrence. CONCLUSIONS: We found similar immunostimulatory profiles in DSRCT and synovial sarcoma. Our findings suggest that DSRCT and synovial sarcoma may be amenable to immunotherapies, albeit there was significant heterogeneity. Interestingly, HLA and CTL target scores decreased at recurrence, possibly reflecting immunoevasion. Our findings suggest both tumor types may be amendable to CTL-based therapies at diagnosis but less so at relapse. Our results support further investigation into the prognostic and predictive value of these findings in a larger dataset.

Antitumor Activity Associated with Prolonged Persistence of Adoptively Transferred NY-ESO-1 c259T Cells in Synovial Sarcoma.

We evaluated the safety and activity of autologous T cells expressing NY-ESO-1c259, an affinity-enhanced T-cell receptor (TCR) recognizing an HLA-A2-restricted NY-ESO-1/LAGE1a-derived peptide, in patients with metastatic synovial sarcoma (NY-ESO-1c259T cells). Confirmed antitumor responses occurred in 50% of patients (6/12) and were characterized by tumor shrinkage over several months. Circulating NY-ESO-1c259T cells were present postinfusion in all patients and persisted for at least 6 months in all responders. Most of the infused NY-ESO-1c259T cells exhibited an effector memory phenotype following ex vivo expansion, but the persisting pools comprised largely central memory and stem-cell memory subsets, which remained polyfunctional and showed no evidence of T-cell exhaustion despite persistent tumor burdens. Next-generation sequencing of endogenous TCRs in CD8+ NY-ESO-1c259T cells revealed clonal diversity without contraction over time. These data suggest that regenerative pools of NY-ESO-1c259T cells produced a continuing supply of effector cells to mediate sustained, clinically meaningful antitumor effects.Significance: Metastatic synovial sarcoma is incurable with standard therapy. We employed engineered T cells targeting NY-ESO-1, and the data suggest that robust, self-regenerating pools of CD8+ NY-ESO-1c259T cells produce a continuing supply of effector cells over several months that mediate clinically meaningful antitumor effects despite prolonged exposure to antigen. Cancer Discov; 8(8); 944-57. ©2018 AACR.See related commentary by Keung and Tawbi, p. 914This article is highlighted in the In This Issue feature, p. 899.

Primary Subcutaneous Synovial Sarcoma: First Reported Subcutaneous Case Showing TLE1 Immunoreactivity.

Synovial sarcoma (SS) accounts for 5%-10% of all soft tissue sarcomas. It is a well-defined soft tissue neoplasm with biphasic and monophasic histologic subtypes and unknown histogenesis. It usually occurs in the extremities, especially the thigh-knee region of young adults. Recurrences are frequent and distant metastasis developed in approximately half of the patients. SSs are characterized by a recurrent nonrandom chromosomal translocation, t(X; 18) (p11; q11), which is considered the primary genetic event in more than 90% of cases. Only 4 cases of cutaneous and subcutaneous SSs have been published in the literature so far. We report a case of primary subcutaneous SS in the forearm of a young woman and discuss the histopathologic differential diagnosis with other similar neoplasms. This is the first reported case of primary cutaneous SS showing immunoreactivity for TLE1 in the nuclei of neoplastic cells, supporting the use of this marker for diagnosis of this rare cutaneous neoplasm.

Human Leukocyte Antigen-A Allele Distribution in Nasopharyngeal Carcinoma Patients Showing Anti-Melanoma-Associated Antigen A or Synovial Sarcoma X-2 T Cell Response in Blood.

BACKGROUND: Development of innovative immunotherapy is imperative to improve the poor survival of the nasopharyngeal carcinoma (NPC) patients. In this study, we evaluated the T cell response to melanoma-associated antigen (MAGE)-A1, MAGE-A3, or synovial sarcoma X-2 (SSX-2) in the peripheral blood of treatment-naive NPC patients. The relationship of responses among the three proteins and the human leukocyte antigen (HLA)-A types were analyzed to provide evidence of designing novel therapy. METHODS: Sixty-one NPC patients admitted into the Tumor Hospital affiliated to the Xinjiang Medical University between March 2015 and July 2016 were enrolled. Mononuclear cells were isolated from the peripheral blood before any treatment. HLA-A alleles were typed with Sanger sequence-based typing technique. The T cell response to the MAGE-A1, MAGE-A3, or SSX-2 was evaluated with the Enzyme-Linked ImmunoSpot assay. Mann-Whitney U-test was used to compare the T cell responses from different groups. Spearman's rank correlation was used to analyze the relationship of T cell responses. RESULTS: HLA-A*02:01, A*02:07, and A*24:02 were the three most frequent alleles (18.9%, 12.3%, and 11.5%, respectively) among the 22 detected alleles. 31.1%, 19.7%, and 16.4% of the patients displayed MAGE-A1, MAGE-A3, or SSX-2-specific T cell response, respectively. The magnitudes of response to the three proteins were 32.5, 38.0, and 28.7 SFC/106 peripheral blood mononuclear cells, respectively. The T cell response against the three proteins correlated with each other to different extent. The percentage of A*02:01 and A*24:02 carriers were significantly higher in patients responding to any of the three proteins compared to the nonresponders. CONCLUSION: MAGE-A1, MAGE-A3, or SSX-2-specific T cell responses were detectable in a subgroup of NPC patients, the frequency and magnitude of which were correlated.

The potential of the CMB305 vaccine regimen to target NY-ESO-1 and improve outcomes for synovial sarcoma and myxoid/round cell liposarcoma patients.

INTRODUCTION: Synovial Sarcoma (SS) and Myxoid Round Cell Liposarcoma (MRCL) are devastating sarcoma subtypes with few treatment options and poor outcomes in the advanced setting. However, both these diseases may be ideal for novel immunotherapies targeting the cancer-testis antigen, NY-ESO-1. AREAS COVERED: In this review, we discuss the novel NY-ESO-1 targeted vaccine regimen, CMB305. This regimen uses a unique integration-deficient, dendritic-cell targeting lentiviral vector from the ZVex® platform, LV305, in order to prime NY-ESO-1 specific T cells. LV305 has single agent activity, and, in one case, caused a durable partial response in a refractory SS patient. CMB305 also includes a boost from a NY-ESO-1 protein vaccine given along with a potent toll-like-4 receptor agonist, glycopyranosyl lipid A. CMB305 induces NY-ESO-1 specific T cell responses in both SS and MRC patients and these patients had excellent overall survival (OS) outcomes in the initial phase I study. EXPERT COMMENTARY: CMB305 is a therapeutic vaccine regimen targeting NY-ESO-1 based on the lentiviral vaccine vector, LV305. Phase I studies have proven this vaccine is active immunologically. Data suggesting this vaccine may improve OS for SS and MRCL patients is exciting but early, and on-going work is testing the impact of CMB305 on patient outcomes.

First-in-Human Treatment With a Dendritic Cell-targeting Lentiviral Vector-expressing NY-ESO-1, LV305, Induces Deep, Durable Response in Refractory Metastatic Synovial Sarcoma Patient.

Effective induction of antitumor T cells is a pivotal goal of cancer immunotherapy. To this end, lentiviral vectors (LV) are uniquely poised to directly prime CD8 T-cell responses via transduction of dendritic cells in vivo and have shown promise as active cancer therapeutics in preclinical tumor models. However, until now, significant barriers related to production and regulation have prevented their widespread use in the clinic. We developed LV305, a dendritic cell-targeting, integration-deficient, replication incompetent LV from the ZVex platform, encoding the full-length cancer-testis antigen NY-ESO-1. LV305 is currently being evaluated in phase 1 and 2 trials in metastatic recurrent cancer patients with NY-ESO-1 positive solid tumors as a single agent and in combination with anti-PD-L1. Here we report on the first patient treated with LV305, a young woman with metastatic, recurrent, therapy-refractive NY-ESO-1 synovial sarcoma. The patient developed a robust NY-ESO-1-specific CD4 and CD8 T-cell response after 3 intradermal injections with LV305, and subsequently over 85% disease regression that is continuing for >2.5 years posttherapy. No adverse events >grade 2 occurred. This case demonstrates that LV305 can be safely administered and has the potential to induce a significant clinical benefit and immunologic response in a patient with advanced stage cancer.

Infiltration of CD8 T Cells and Expression of PD-1 and PD-L1 in Synovial Sarcoma.

Tumors expressing programmed death ligand 1 (PD-L1) interact with the corresponding negative-signal generating immune receptor on the surface of CD8 T cells, PD-1, thereby suppressing antitumor activity. Therapeutics blocking this interaction have shown promise in various cancers by restoring functional antitumor T-cell activity. We explored the degree of PD-L1, PD-1, and CD8 expression in a retrospective analysis of 29 clinical synovial sarcoma samples. Quantitative immunohistochemistry and multiplex immunofluorescence were used to determine relative quantification of CD8+ and PD-1+ T cells and PD-L1 expression within the intratumor area and the interface between the tumor and the surrounding nontumor tissue (i.e., invasive margin), and colocalization of these factors, respectively. PD-L1, PD-1, and CD8 cell densities in the tumor-invasive margins were significantly higher in the metastatic tumors than the primary tumors (P < 0.01), and PD-L1, PD-1, and CD8 cell densities were all significantly positively correlated with one other (P < 0.0001). PD-1 cell density in the tumor-invasive margin was significantly associated with worse progression-free survival. Multiplex immunofluorescence demonstrated coexpression of PD-1 and CD8 on lymphocytes within the invasive margin, as well as relative proximity between PD-1+ CD8 cells and PD-L1+ tumor cells. Our results provide a preclinical rationale for screening of patients with synovial sarcoma for the colocalization of CD8, PD-1, and PD-L1, which may be a marker for response to PD-1 blockade therapy. Cancer Immunol Res; 5(2); 118-26. ©2016 AACR.

Cancer-testis antigen expression in synovial sarcoma: NY-ESO-1, PRAME, MAGEA4, and MAGEA1.

Synovial sarcoma (SS) is regarded as a relatively chemosensitive sarcoma, but the prognosis of advanced SSs remains poor. Here we identified highly expressed cancer-testis antigens that could be promising immunotherapy targets for SS, using a previously conducted cDNA microarray, and we assessed the clinicopathological or prognostic relationships of these antigens in SS. We compared the gene expression profiles of 11 SSs with those of 3 normal adipose tissues. Among the up-regulated cancer-testis antigens, we analyzed PRAME, MAGEA1, and MAGEA4 and another cancer-testis antigen (NY-ESO-1) together, by immunohistochemistry and real-time polymerase chain reaction in 108 SSs. Immunohistochemically, NY-ESO-1, PRAME, MAGEA4, and MAGEA1 were positive in 66 (61%), 93 (86%), 89 (82%), and 16 (15%) of 108 SSs, respectively, and 104 (96%) of 108 SSs showed the immunohistochemical expression of at least 1 of NY-ESO-1, PRAME, and MAGEA4. Moreover, the high expression of at least 1 of these 3 antigens was observed in 83% of the SSs. High expression of NY-ESO-1 and MAGEA4 was significantly correlated with the presence of necrosis and advanced clinical stage. The immunohistochemical expression of these cancer-testis antigens was not correlated with prognosis, but the coexpression of NY-ESO-1, PRAME, and MAGEA4 was significantly associated with adverse prognosis. The real-time polymerase chain reaction results were closely related to the immunohistochemical results: NY-ESO-1 (P = .0019), PRAME (P = .039), MAGEA4 (P = .0149), and MAGEA1 (P = .0766). These data support the potential utility of NY-ESO-1, PRAME, and MAGEA4 as immunotherapy targets and ancillary prognostic parameters, suggesting the possible benefit of the combined use of these cancer-testis antigens as an SS immunotherapy target.

Current status of engineered T-cell therapy for synovial sarcoma.

Synovial sarcoma is a rare soft tissue sarcoma characterized by a t(X;18) translocation, which results in a SYT-SSX gene fusion. In the metastatic setting, chemotherapy has limited, durable efficacy prompting the necessity for new therapeutic modalities. One emerging new strategy involves T-cell-directed therapy such as tumor-infiltrating lymphocytes or the development of T cells that are genetically engineered to express a T-cell receptor against a cancer testis antigen. Of these approaches, engineered T cells that recognize NY-ESO-1 are the furthest along in development. Completed and on-going clinical trials have shown promise and there are efforts to continue to optimize the current approach.