Antibody Responses to NY-ESO-1 in Primary Breast Cancer Identify a Subtype Target for Immunotherapy

The highly immunogenic human tumor antigen NY-ESO-1 (ESO) is a target of choice for anti-cancer immune therapy. In this study, we assessed spontaneous antibody (Ab) responses to ESO in a large cohort of patients with primary breast cancer (BC) and addressed the correlation between the presence of anti-ESO Ab, the expression of ESO in the tumors and their characteristics. We found detectable Ab responses to ESO in 1% of the patients. Tumors from patients with circulating Ab to ESO exhibited common characteristics, being mainly hormone receptor (HR)− invasive ductal carcinomas of high grade, including both HER2− and HER2+ tumors. In line with these results, we detected ESO expression in 20% of primary HR− BC, including both ESO Ab+ and Ab− patients, but not in HR+ BC. Interestingly, whereas expression levels in ESO+ BC were not significantly different between ESO Ab+ and Ab− patients, the former had, in average, significantly higher numbers of tumor-infiltrated lymph nodes, indicating that lymph node invasion may be required for the development of spontaneous anti-tumor immune responses. Thus, the presence of ESO Ab identifies a tumor subtype of HR− (HER2− or HER2+) primary BC with frequent ESO expression and, together with the assessment of antigen expression in the tumor, may be instrumental for the selection of patients for whom ESO-based immunotherapy may complement standard therapy

NYESO-1/LAGE-1s and PRAME Are Targets for Antigen Specific T Cells in Chondrosarcoma following Treatment with 5-Aza-2-Deoxycitabine

Chondrosarcoma has no proven systemic option in the metastatic setting. The development of a non-cross-resistant strategy, such as cellular immunotherapy using antigen-specific T cells would be highly desirable. NY-ESO-1 and PRAME are members of the Cancer Testis Antigen (CTA) family that have been identified as promising targets for T cell therapy. LAGE-1 is a cancer testis antigen 90% homologous to NY-ESO-1, sharing the 157-165 A*0201 NY-ESO-1 epitope with its transcript variant, LAGE-1s. A number of CTA's have been induced using 5-Aza-2-Deoxycitabine (5-Aza-dC) in other cancers. We sought to evaluate the feasibility of targeting chondrosarcoma tumors using NY-ESO-1/LAGE-1s and PRAME specific T cells using 5-Aza-dC to induce antigen expression.We used 11 flash frozen tumors from the University of Washington tumor bank to test for the expression of NY-ESO-1, PRAME, LAGE-1s and LAGE-1L in chondrosarcoma tumors. Using four chondrosarcoma cell lines we tested the expression of these CTA's with and without 5-Aza-dC treatments. Finally, using NY-ESO-1/LAGE-1s and PRAME specific effectors that we generated from sarcoma patients, we evaluated the ability of these T cells to lyse A*0201 expressing chondrosarcoma cell lines in vitro both with and without 5-Aza-dC treatment.A minority (36%) of chondrosarcoma tumors expressed either NY-ESO-1 or LAGE-1s at >10% of our reference value and none expressed PRAME at that level. However, in all four of the chondrosarcoma cell lines tested, NY-ESO-1 and PRAME expression could be induced following treatment with 5-Aza-dC including in cell lines where expression was absent or barely detectable. Furthermore, NY-ESO-1/LAGE-1s and PRAME specific CD8+ effector T cells were able to specifically recognize and lyse A*0201 expressing chondrosarcoma cell lines following 5-Aza-dC treatment.These data suggest that adoptive immunotherapy in combination with 5-Aza-dC may be a potential strategy to treat unresectable or metastatic chondrosarcoma patients where no proven systemic therapies exist

Successful treatment of metastatic melanoma by adoptive transfer of blood-derived polyclonal tumor-specific CD4+ and CD8+ T cells in combination with low-dose interferon-alpha

A phase I/II study was conducted to test the feasibility and safety of the adoptive transfer of tumor-reactive T cells and daily injections of interferon-alpha (IFNα) in metastatic melanoma patients with progressive disease. Autologous melanoma cell lines were established to generate tumor-specific T cells by autologous mixed lymphocyte tumor cell cultures using peripheral blood lymphocytes. Ten patients were treated with on average 259 (range 38–474) million T cells per infusion to a maximum of six infusions, and clinical response was evaluated according to the response evaluation criteria in solid tumors (RECIST). Five patients showed clinical benefit from this treatment, including one complete regression, one partial response, and three patients with stable disease. No treatment-related serious adverse events were observed, except for the appearance of necrotic-like fingertips in one patient. An IFNα-related transient leucopenia was detected in 6 patients, including all responders. One responding patient displayed vitiligo. The infused T-cell batches consisted of tumor-reactive polyclonal CD8+ and/or CD4+ T cells. Clinical reactivity correlated with the functional properties of the infused tumor-specific T cells, including their in vitro expansion rate and the secretion of mainly Th1 cytokines as opposed to Th2 cytokines. Our study shows that relatively low doses of T cells and low-dose IFNα can lead to successful treatment of metastatic melanoma and reveals a number of parameters potentially associated with this success

Identification of novel helper epitopes of MAGE-A4 tumour antigen: useful tool for the propagation of Th1 cells

MAGE-A4 has been considered as an attractive cancer-testis (CT) antigen for tumour immunotherapy. It has been well accepted that T-helper type 1 (Th1) cell-dominant immunity is critical for the successful induction of antitumour immunity in a tumour-bearing host. The adoptive Th1 cell therapy has been shown to be an attractive strategy for inducing tumour eradication in mouse systems. However, Th1-cell therapy using human tumour-specific Th1 cells, which were expanded from peripheral blood mononuclear cells (PBMCs) in a clinically useful protocol, has never been performed. Here, we first identified MAGE-A4-derived promiscuous helper epitope, peptide (MAGE-A4 280–299), bound to both HLA-DPB1*0501 and DRB1*1403. Using the peptide, we established a suitable protocol for the propagation of MAGE-A4-specific Th1 cells in vitro. Culture of CD4+ T cells with IFN-γ-treated PBMC-derived adherent cells in the presence of helper epitope peptide resulted in a great expansion of MAGE-A4-reactive Th cells producing IFN-γ , but not IL-4. Moreover, it was shown that ligation of MAGE-A4-reactive Th1 cells with the cognate peptide caused the production of IFN-γ and IL-2. Thus, our identified MAGE-A4 helper epitope peptide will become a good tool for the propagation of tumour-specific Th1 cells applicable to adoptive immunotherapy of human cancer

Expansion and Characterization of Human Melanoma Tumor-Infiltrating Lymphocytes (TILs)

Various immunotherapeutic strategies for cancer are aimed at augmenting the T cell response against tumor cells. Adoptive cell therapy (ACT), where T cells are manipulated ex vivo and subsequently re-infused in an autologous manner, has been performed using T cells from various sources. Some of the highest clinical response rates for metastatic melanoma have been reported in trials using tumor-infiltrating lymphocytes (TILs). These protocols still have room for improvement and furthermore are currently only performed at a limited number of institutions. The goal of this work was to develop TILs as a therapeutic product at our institution.TILs from 40 melanoma tissue specimens were expanded and characterized. Under optimized culture conditions, 72% of specimens yielded rapidly proliferating TILs as defined as at least one culture reaching ≥3×10(7) TILs within 4 weeks. Flow cytometric analyses showed that cultures were predominantly CD3+ T cells, with highly variable CD4+:CD8+ T cell ratios. In total, 148 independent bulk TIL cultures were assayed for tumor reactivity. Thirty-four percent (50/148) exhibited tumor reactivity based on IFN-γ production and/or cytotoxic activity. Thirteen percent (19/148) showed specific cytotoxic activity but not IFN-γ production and only 1% (2/148) showed specific IFN-γ production but not cytotoxic activity. Further expansion of TILs using a 14-day "rapid expansion protocol" (REP) is required to induce a 500- to 2000-fold expansion of TILs in order to generate sufficient numbers of cells for current ACT protocols. Thirty-eight consecutive test REPs were performed with an average 1865-fold expansion (+/- 1034-fold) after 14 days.TILs generally expanded efficiently and tumor reactivity could be detected in vitro. These preclinical data from melanoma TILs lay the groundwork for clinical trials of ACT

Ex Vivo Expansion of Human CD8+ T Cells Using Autologous CD4+ T Cell Help

Background: Using in vivo mouse models, the mechanisms of CD4+ T cell help have been intensively investigated. However, a mechanistic analysis of human CD4+ T cell help is largely lacking. Our goal was to elucidate the mechanisms of human CD4+ T cell help of CD8+ T cell proliferation using a novel in vitro model. Methods/Principal Findings: We developed a genetically engineered novel human cell-based artificial APC, aAPC/mOKT3, which expresses a membranous form of the anti-CD3 monoclonal antibody OKT3 as well as other immune accessory molecules. Without requiring the addition of allogeneic feeder cells, aAPC/mOKT3 enabled the expansion of both peripheral and tumor-infiltrating T cells, regardless of HLA-restriction. Stimulation with aAPC/mOKT3 did not expand Foxp3+ regulatory T cells, and expanded tumor infiltrating lymphocytes predominantly secreted Th1-type cytokines, interferon-γ and IL-2. In this aAPC-based system, the presence of autologous CD4+ T cells was associated with significantly improved CD8+ T cell expansion in vitro. The CD4+ T cell derived cytokines IL-2 and IL-21 were necessary but not sufficient for this effect. However, CD4+ T cell help of CD8+ T cell proliferation was partially recapitulated by both adding IL-2/IL-21 and by upregulation of IL-21 receptor on CD8+ T cells. Conclusions: We have developed an in vitro model that advances our understanding of the immunobiology of human CD4+ T cell help of CD8+ T cells. Our data suggests that human CD4+ T cell help can be leveraged to expand CD8+ T cells in vitro

Identification and manipulation of tumor associated macrophages in human cancers

Evading immune destruction and tumor promoting inflammation are important hallmarks in the development of cancer. Macrophages are present in most human tumors and are often associated with bad prognosis. Tumor associated macrophages come in many functional flavors ranging from what is known as classically activated macrophages (M1) associated with acute inflammation and T-cell immunity to immune suppressive macrophages (M2) associated with the promotion of tumor growth. The role of these functionally different myeloid cells is extensively studied in mice tumor models but dissimilarities in markers and receptors make the direct translation to human cancer difficult. This review focuses on recent reports discriminating the type of infiltrating macrophages in human tumors and the environmental cues present that steer their differentiation. Finally, immunotherapeutic approaches to interfere in this process are discussed