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Purpose Peripheral blood–derived antigen-specific cytotoxic T cells (CTLs) provide a readily available source of effector cells that can be administered with minimal toxicity in an outpatient setting. In metastatic melanoma, this approach results in measurable albeit modest clinical responses in patients resistant to conventional therapy. We reasoned that concurrent cytotoxic T-cell lymphocyte antigen-4 (CTLA-4) checkpoint blockade might enhance the antitumor activity of adoptively transferred CTLs. Patients and Methods Autologous MART1-specific CTLs were generated by priming with peptide-pulsed dendritic cells in the presence of interleukin-21 and enriched by peptide-major histocompatibility complex multimer-guided cell sorting. This expeditiously yielded polyclonal CTL lines uniformly expressing markers associated with an enhanced survival potential. In this first-in-human strategy, 10 patients with stage IV melanoma received the MART1-specific CTLs followed by a standard course of anti–CTLA-4 (ipilimumab). Results The toxicity profile of the combined treatment was comparable to that of ipilimumab monotherapy. Evaluation of best responses at 12 weeks yielded two continuous complete remissions, one partial response (PR) using RECIST criteria (two PRs using immune-related response criteria), and three instances of stable disease. Infused CTLs persisted with frequencies up to 2.9% of CD8+ T cells for as long as the patients were monitored (up to 40 weeks). In patients who experienced complete remissions, PRs, or stable disease, the persisting CTLs acquired phenotypic and functional characteristics of long-lived memory cells. Moreover, these patients also developed responses to nontargeted tumor antigens (epitope spreading). Conclusion We demonstrate that combining antigen-specific CTLs with CTLA-4 blockade is safe and produces durable clinical responses, likely reflecting both enhanced activity of transferred cells and improved recruitment of new responses, highlighting the promise of this strategy.

BACKGROUND: Myxoid/round cell liposarcoma (MRCL) is the second most common liposarcoma subtype, accounting for >33% of liposarcomas and approximately 10% of all soft tissue sarcomas. Although MRCL is a chemosensitive subtype, patients with metastatic disease have a poor outcome. NY-ESO-1 is a cancer-testis antigen (also known as cancer germ cell antigen) that has been successfully targeted in vaccine trials and in adoptive T-cell therapy trials for the treatment of several solid tumors. METHODS: The authors investigated the feasibility of targeting NY-ESO-1 in patients with MRCL by evaluating the prevalence of NY-ESO-1 expression in tumors using immunohistochemistry and quantitative reverse transcriptase-polymerase chain reaction analysis. NY-ESO-1–specific tumor recognition by NY-ESO-1–specific T-cells also was analyzed using a chromium release assay. RESULTS: A search of the University of Washington Sarcoma Tissue Bank identified paraffin-embedded tumor samples from 25 patients with MRCL. NY-ESO-1 expression was observed in every MRCL tumor assessed (100%); in 18 tumors (72%), staining was homogenous. In all but 2 tumors, staining was sufficiently robust (2þ) that such patients would be eligible for clinical trials of NY-ESO-1–directed therapy. By using NY-ESO-1 specific, CD8-positive T-cells, the in vitro sensitivity of myxoid liposarcoma cell lines to antigen-specific lysis was demonstrated. CONCLUSIONS: The current results establish NY-ESO-1 as an important target antigen for the treatment of patients with MRCL. Cancer 2012;118:4564-70. V C 2012 American Cancer Society.

Adoptively transferred tumor-specific T cells offer the potential for non–cross-resistant therapy and long-term immunoprotection. Strategies to enhance in vivo persistence of transferred T cells can lead to improved antitumor efficacy. However, the extrinsic (patient conditioning) and intrinsic (effector cell) factors contributing to long-term in vivo persistence are not well-defined. As a means to enhance persistence of infused T cells in vivo and limit toxicity, 11 patients with refractory, progressive metastatic melanoma received cyclophosphamide alone as conditioning before the infusion of peripheral blood mononuclear cell-derived, antigen-specific, CD8 + cytotoxic T-lymphocyte (CTL) clones followed by low-dose or high-dose IL-2. No life-threatening toxicities occurred with low-dose IL-2. Five of 10 evaluable patients had stable disease at 8 wk, and 1 of 11 had a complete remission that continued for longer than 3 y. On-target autoimmune events with the early appearance of skin rashes were observed in patients with stable disease or complete remission at 4 wk or longer. In vivo tracking revealed that the conditioning regimen provided a favorable milieu that enabled CTL proliferation early after transfer and localization to nonvascular compartments, such as skin and lymph nodes. CTL clones, on infusion, were characterized by an effector memory phenotype, and CTL that persisted long term acquired phenotypic and/or functional qualities of central memory type CTLs in vivo. The use of a T-cell product composed of a clonal population of antigen-specific CTLs afforded the opportunity to demonstrate phenotypic and/or functional conversion to a central memory type with the potential for sustained clinical benefit.

Background 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. Methods 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. Results 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. Conclusion 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.

Purpose: Merkel cell polyomavirus (MCPyV) is prevalent in the general population, integrates into most Merkel cell carcinomas (MCC), and encodes oncoproteins required for MCC tumor growth. We sought to characterize T-cell responses directed against viral proteins that drive this cancer as a step toward immunotherapy. Experimental Design: Intracellular cytokine cytometry, IFN-γ enzyme-linked immunospot (ELISPOT) assay, and a novel HLA-A*2402–restricted MCPyV tetramer were used to identify and characterize T-cell responses against MCPyV oncoproteins in tumors and blood of MCC patients and control subjects. Results: We isolated virus-reactive CD8 or CD4 T cells from MCPyV-positive MCC tumors (2 of 6) but not from virus-negative tumors (0 of 4). MCPyV-specific T-cell responses were also detected in the blood of MCC patients (14 of 27) and control subjects (5 of 13). These T cells recognized a broad range of peptides derived from capsid proteins (2 epitopes) and oncoproteins (24 epitopes). HLA-A*2402–restricted MCPyV oncoprotein processing and presentation by mammalian cells led to CD8-mediated cytotoxicity. Virus-specific CD8 T cells were markedly enriched among tumor infiltrating lymphocytes as compared with blood, implying intact T-cell trafficking into the tumor. Although tetramer-positive CD8 T cells were detected in the blood of 2 of 5 HLA-matched MCC patients, these cells failed to produce IFN-γ when challenged ex vivo with peptide. Conclusions: Our findings suggest that MCC tumors often develop despite the presence of T cells specific for MCPyV T-Ag oncoproteins. The identified epitopes may be candidates for peptide-specific vaccines and tumor- or virus-specific adoptive immunotherapies to overcome immune evasion mechanisms in MCC patients. Clin Cancer Res; 17(21); 6671–80. ©2011 AACR.

Myxoid/round cell liposarcoma (MRCL) is the second most common liposarcoma subtype, accounting for33% of liposarcomas and approximately 10% of all soft tissue sarcomas. Although MRCL is a chemosensitive subtype, patients with metastatic disease have a poor outcome. NY-ESO-1 is a cancer-testis antigen (also known as cancer germ cell antigen) that has been successfully targeted in vaccine trials and in adoptive T-cell therapy trials for the treatment of several solid tumors.The authors investigated the feasibility of targeting NY-ESO-1 in patients with MRCL by evaluating the prevalence of NY-ESO-1 expression in tumors using immunohistochemistry and quantitative reverse transcriptase-polymerase chain reaction analysis. NY-ESO-1-specific tumor recognition by NY-ESO-1-specific T-cells also was analyzed using a chromium release assay.A search of the University of Washington Sarcoma Tissue Bank identified paraffin-embedded tumor samples from 25 patients with MRCL. NY-ESO-1 expression was observed in every MRCL tumor assessed (100%); in 18 tumors (72%), staining was homogenous. In all but 2 tumors, staining was sufficiently robust (2+) that such patients would be eligible for clinical trials of NY-ESO-1-directed therapy. By using NY-ESO-1 specific, CD8-positive T-cells, the in vitro sensitivity of myxoid liposarcoma cell lines to antigen-specific lysis was demonstrated.The current results establish NY-ESO-1 as an important target antigen for the treatment of patients with MRCL.

Persistent expression of Merkel cell polyomavirus (MCPyV) T antigen oncoproteins is required for the growth of most Merkel cell carcinomas (MCCs). MCPyV oncoproteins are thus attractive tumor antigens for evaluating the feasibility of antigen-specific adoptive T-cell therapy in MCC. Importantly, unlike mammalian tumor associated antigens that have some degree of expression within normal tissues, MCPyV oncoprotein expression is restricted to MCCs. We have recently identified several dozen MCC-associated viral peptides recognized by T cells. However, a major challenge for T cell therapy is that MCCs often downregulate HLA-I expression (51% of 114 tumors). We report that a single dose of either radiotherapy (2-8 Gy) or intralesional interferon reverses HLA-I downregulation in MCCs and can be used as an adjuvant in combination with virus-specific T cells for a multi-modality approach to target MCC. The feasibility and efficacy of this combined treatment has been explored in a 68 year-old man. He initially presented with a primary MCC on the hip that expressed low levels of HLA-I, high levels of MCPyV oncoprotein, and contained MCPyV-specific CD8 T cells. Based on rising antibody titers to MCPyV T-antigen, he was later found to have peri-pancreatic metastases. Blood-derived virus-specific T cells failed to respond to peptide challenge and expressed high levels of T cell exhaustion markers PD-1 and Tim-3. We used a novel approach to generate functional, polyclonal virus-specific therapeutic T cells. Lymphocytes from blood were cultured with peptide-loaded autologous dendritic cells, IL-2, IL-7, and IL-21, then sorted using an HLA-I/peptide tetramer and further expanded. 86% of these polyclonal CD8+ T cells were virus-specific and killed appropriate target cells. 24h after injecting interferon-beta into a pancreatic metastasis, the patient received 10^10 T cells/m2. Four weeks later, a single 8 Gy dose of radiation was given to the tumor area and was followed by a second T cell infusion 24h later. Treatment was well tolerated. After 2 treatments, all 3 metastatic lesions had markedly responded (>60% shrinkage by RECIST). No new distant disease has been detected at 456 days after the first metastasis (far beyond a median of 200 days for historical stage-matched controls). At >140 days after initiation of therapy, each of the following parameters remained markedly increased (relative to pre-treatment baseline) among virus-specific T cells in the blood: frequency, proliferation (Ki-67) and antigen-induced interferon-gamma production. TCR sequence analysis was used to track the prevalence and tumor infiltration of infused T cell clonotypes. After treatment, an increased fraction of virus-specific T cells was observed in the metastasis compared to a pre-treatment biopsy of the primary tumor. The use of polyclonal T cells may have been important because we observed that only a subset of the infused clones effectively infiltrated the sampled metastasis. This study represents a first-in-man treatment of a carcinoma using T cells targeting an oncogenic viral antigen and benefitted from the ability to accurately track adoptively transferred T cells specific for a non-self tumor antigen. Citation Format: Olga Afanasiev, Aude Chapuis, Jayasri Iyer, Kotaro Nagase, Kelly Paulson, Aaron Seo, Ivy Lai, Ilana Roberts, Erik Farrar, Joo Ha Hwang, Upendra Parvathaneni, David Koelle, Cassian Yee, Paul Nghiem. Merkel cell carcinoma therapy with viral oncoprotein-specific T cells in combination with immunostimulatory adjuvants. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr IA17.

For advanced, refractory malignancies, adoptive T cell therapy represents an increasingly attractive modality with the potential for significant anti-tumor efficacy, minimal toxicity and longterm immunoprotection. This approach involves the ex vivo isolation and expansion of a patient's tumor-reactive T cells. Such T cells are rare, and difficult to isolate. Iterative cycles of in vitro re-stimulation, limiting dilution cloning and screening of hundreds to thousands of T cell colonies are required to select for a tumor-reactive population of T cells. Although remarkable responses have been achieved, the time and resource intensive nature of this process has limit the use of adoptive T cell therapy to a handful of centers with specialized facilities and personnel. A closed-system clinical-grade cell sorter that provides a high-speed, sterile and multi-parametric system for isolating low frequency antigen specific T-cells in a cost-effective, easy-to-use and high performance manner would be desirable. A personal-sized cell sorter was developed which uses classical Fluorescence Activated Cell Sorter (FACS) laser excitation and fluorescence emission for interrogation and identification of cells; however, the laser excitation and detection regions, sorting actuator, sample reservoirs, and all of the fluidic channels are contained in a single, closed, disposable, low cost cartridge. The heart of the sorting cartridge is the MEMS microchip which is the world's fastest valve which can open and close in about 15-20 usec allowing a sort rate of more than 100 million cells/hour Cell Sorting of Human Antigen-specific CD8 T cells We determined the lowest threshold limit for MEMS-based antigen-specific T cell single color sorting using fluorescent-conjugated peptide-MHC multimers using a panel of well-characterized tumor-associated antigen (TAA)-specific T cell clones established in our lab were titrated o autologous PBMCs. We demonstrate discrete recognition of the antigen-specific population and the ability to routinely achieve a sort purity of > 85%, yield of > 90% and viability of > 95% starting from a T cell frequency of < 0.5%, with retention of viability, cytolytic, cytokine-producing and proliferative functions. We present data demonstrating routine feasibiligy in achieving enrichment of rare antigen-specific human CD8 and CD4 T cell populations to purity of > 85% with preserved functionality, enhanced viability over conventional stream in air sorters (where cells are subject to 10-50 fold greater shear forces), and expansion capacity after sort to >2000 fold reducing time to treatment for patients from 84 to 24 days. The use of a completely self-contained, disposable cartridge system provides substantial cost savings and allows for multiple samples to be run by a single operator. Development of multicolor and tandem sorting to facilitate enrichment of large volume samples is being pursued. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-137. doi:1538-7445.AM2012-LB-137

Merkel cell carcinoma (MCC) represents a highly aggressive neuroendocrine skin malignancy with 46% 5 year disease-associated mortality making it 3x as lethal as melanoma. Merkel cell polyomavirus (MCPyV), a recently discovered viral agent related to SV40, is clonally integrated into ∼80% of MCC tumors. MCPyV produces T Antigen (T-Ag) oncoproteins that are persistently expressed, necessary for survival and proliferation of most MCCs, representing an optimal target for immunotherapy. An HLA A*2402-restricted T-Ag epitope recognized by autologous CD8+ T-cells was identified in a patient with MCC. The presence of a small endogenous population (0.1% of CD8+ T-cells) of MCC-specific CD8+ T-cells failed to prevent new metastases, and responses may have been impaired by MHC class I down-regulation observed in the patient's tumor. As adoptively transferred antigen-specific T cells (CTL) have demonstrated clinical benefit in other tumor settings, we set out to examine the safety, in vivo persistence and anti-tumor effect of CTL targeting MCC. Autologous polyclonal antigen-specific CD8+ T cells targeting the HLA A*2402-restricted epitope of the MCPyV T-Ag oncoprotein were generated for this patient. Ex-vivo cultures were supplemented with the γc-chain cytokine IL-21, which has been demonstrated to promote less terminal differentiation of cultured cells as a means to increase CTL in vivo survival. To induce tumor MHC class I up-regulation with minimal confounding therapeutic effect, the patient received a single intra-lesional injection of 3.3 x 106 IU IFNβ 1B to 1 of 3 measurable metastatic lesions 24h prior to a first infusion of 1010/m2 of polyclonal multimer sorted MCPyV-specific CTL. Four weeks later, a single 8Gy dose of radiation to detectable disease was followed 24h later by a second T cell infusion. Both infusions were followed by low-dose IL-2 (500,000 IU/m2 s.c. x 14 days). The patient experienced no treatment-associated side effects. Four weeks after the first infusion, the metastatic lesion infused with IFNβ 1B regressed by 40% whereas the non-infused lesions increased by an average of 32%. Eight weeks after the second infusion, all lesions had regressed by an average of 62% with complete disappearance of the metastatic lesion previously infused with IFNβ 1B, and no development of new systemic lesions. Infused CTL peaked at 8% of total CD8+ T cells 7 days after infusions and persisted at ≥1% for at least 6 weeks. Infused CTL remained polyfunctional (secretion of IFNγ, TNFα and IL-2 in response to cognate antigen) over the observed period. Overall, this first-in-human trial suggests infusion of IL-21 derived polyclonal CTL targeting MCPyV is safe, and combined with up-regulation of tumor MHC class I, promotes in vivo tumor regression. Plans are underway to extend this investigational treatment to MCC patients with a broader array of MHC-restricted viral epitopes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-430. doi:1538-7445.AM2012-LB-430

Adoptive transfer of melanoma-reactive CD8+ T Cells (CTL) has demonstrated antitumor activity that may be enhanced by prolonging survival of transferred T cells in vivo. CTLA4 is up-regulated on activated T cells and when engaged to its B7-ligand, delivers inhibitory signals that impede TCR signaling, IL-2 transcription, and T cell proliferation. Human anti-CTLA4 monotherapy benefits about 25% of patients (pts), but most pts likely lack a sufficiently high frequency of pre-existing melanoma-reactive T cells susceptible to CTLA4 blockade and fail to respond. We propose a combined biologic strategy involving the adoptive transfer of melanoma-reactive CTL with concurrent administration of anti-CTLA4 as a means of enhancing the in vivo CTL survival and anti-tumor effect. Autologous polyclonal CTL were generated by multimer sorting and expanding melanoma-specific CTL lines to increase the probability of infusing cells derived from central memory (Tcm), as such cells were shown to exhibit prolonged persistence and superior replicative potential in murine and non-human primate models. Ex-vivo cultures were supplemented with the γc-chain cytokine Interleukin-21 (IL-21), which has been demonstrated to promote effector CTL helper-independence and to arrest differentiation at a Tcm stage. To date, 6 pts with bulky, therapy-resistant progressive melanoma were infused with 1010/m2 polyclonal IL-21-derived CTL targeting MART1. The infusions were preceded by intravenous cyclophosphamide 300mg/m2 and followed by low-dose subcutaneous IL-2 (250,000 IU/m2 BID) x 14 days. Anti CTLA4 (ipilimumab) 3mg/kg was started on the day after the CTL infusion. Side effects included fevers and chills starting 1-2 hours post-CTL infusion and moderately erythematous maculo-papular skin rashes in all 6 patients. No unexpected or serious (grade ≥3) toxicity was observed. Persistence of the infused CTL to frequencies >2x baseline levels was documented at 6 weeks (5/5 evaluable pts) and 12 weeks (4/4 pts). 3/5 and 2/3 evaluable pts experienced stable disease by RECIST 1.1 criteria at 6 and 12 weeks respectively. One patient had a delayed response with 25% reduction in tumor mass at 12 weeks after having failed previous ipilimumab monotherapy and prior CTL therapy without CTLA4 blockade. Tracking of infused multimer+ CTL in vivo revealed acquisition of both phenotypic (CD27, CD28, CD127, CD62L and CCR7) and functional (secretion of IL-2 in response to cognate antigen) characteristics of Tcm within 12 weeks. Our preliminary results suggest polyclonal IL-21-stimulated CTL targeting melanoma in combination with Ipilmumab is safe and promotes persistent anti-tumor responses such that CTL are poised for enhanced therapeutic activity in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-136. doi:1538-7445.AM2012-LB-136

Background Adoptive T cell therapy represents an attractive modality for the treatment of patients with cancer. Peripheral blood mononuclear cells have been used as a source of antigen specific T cells but the very low frequency of T cells recognizing commonly expressed antigens such as NY-ESO-1 limit the applicability of this approach to other solid tumors. To overcome this, we tested a strategy combining IL-21 modulation during in vitro stimulation with first-in-class use of tetramer-guided cell sorting to generate NY-ESO-1 specific cytotoxic T lymphocytes (CTL). Methods CTL generation was evaluated in 6 patients with NY-ESO-1 positive sarcomas, under clinical manufacturing conditions and characterized for phenotypic and functional properties. Results Following in vitro stimulation, T cells stained with NY-ESO-1 tetramer were enriched from frequencies as low as 0.4% to >90% after single pass through a clinical grade sorter. NY-ESO-1 specific T cells were generated from all 6 patients. The final products expanded on average 1200-fold to a total of 36 billion cells, were oligoclonal and contained 67-97% CD8+, tetramer+ T cells with a memory phenotype that recognized endogenous NY-ESO-1. Conclusion This study represents the first series using tetramer-guided cell sorting to generate T cells for adoptive therapy. This approach, when used to target more broadly expressed tumor antigens such as WT-1 and additional Cancer-Testis antigens will enhance the scope and feasibility of adoptive T cell therapy. Electronic supplementary material The online version of this article (doi:10.1186/s40425-014-0036-y) contains supplementary material, which is available to authorized users.