Adoptive Cell Transfer Immunotherapy Research Articles

CTLA-4 Blockade Plus Adoptive T-Cell Transfer Promotes Optimal Melanoma Immunity in Mice

Immunotherapeutic approaches to the treatment of advanced melanoma have relied on strategies that augment the responsiveness of endogenous tumor-specific T-cell populations [eg, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) blockade-mediated checkpoint inhibition] or introduce exogenously prepared tumor-specific T-cell populations [eg, adoptive cell transfer (ACT)]. Although both approaches have shown considerable promise, response rates to these therapies remain suboptimal. We hypothesized that a combinatorial approach to immunotherapy using both CTLA-4 blockade and nonlymphodepletional ACT could offer additive therapeutic benefit. C57BL/6 mice were inoculated with syngeneic B16F10 melanoma tumors transfected to express low levels of the lymphocytic choriomeningitis virus peptide GP33 (B16GP33), and treated with no immunotherapy, CTLA-4 blockade, ACT, or combination immunotherapy of CTLA-4 blockade with ACT. Combination immunotherapy resulted in optimal control of B16GP33 melanoma tumors. Combination immunotherapy promoted a stronger local immune response reflected by enhanced tumor-infiltrating lymphocyte populations, and a stronger systemic immune responses reflected by more potent tumor antigen-specific T-cell activity in splenocytes. In addition, whereas both CTLA-4 blockade and combination immunotherapy were able to promote long-term immunity against B16GP33 tumors, only combination immunotherapy was capable of promoting immunity against parental B16F10 tumors as well. Our findings suggest that a combinatorial approach using CTLA-4 blockade with nonlymphodepletional ACT may promote additive endogenous and exogenous T-cell activities that enable greater therapeutic efficacy in the treatment of melanoma.

Serum CEACAM1 Elevation Correlates with Melanoma Progression and Failure to Respond to Adoptive Cell Transfer Immunotherapy.

Malignant melanoma is a devastating disease whose incidences are continuously rising. The recently approved antimelanoma therapies carry new hope for metastatic patients for the first time in decades. However, the clinical management of melanoma is severely hampered by the absence of effective screening tools. The expression of the CEACAM1 adhesion molecule on melanoma cells is a strong predictor of poor prognosis. Interestingly, a melanoma-secreted form of CEACAM1 (sCEACAM1) has recently emerged as a potential tumor biomarker. Here we add novel evidences supporting the prognostic role of serum CEACAM1 by using a mice xenograft model of human melanoma and showing a correlation between serum CEACAM1 and tumor burden. Moreover, we demonstrate that serum CEACAM1 is elevated over time in progressive melanoma patients who fail to respond to immunotherapy as opposed to responders and stable disease patients, thus proving a correlation between sCEACAM1, response to treatment, and clinical deterioration.

Immune escape mechanisms associated with tumor recurrence after adoptive cell transfer immunotherapy.

3054 Background: Adoptive cell transfer immunotherapy (ACT) with tumor infiltrating lymphocytes (TILs) results in unprecedented rates of durable clinical responses but some patients initially respo...

Inhibition of CSF-1 receptor improves the antitumor efficacy of adoptive cell transfer immunotherapy.

Colony stimulating factor 1 (CSF-1) recruits tumor-infiltrating myeloid cells (TIM) that suppress tumor immunity, including M2 macrophages and myeloid-derived suppressor cells (MDSC). The CSF-1 receptor (CSF-1R) is a tyrosine kinase that is targetable by small molecule inhibitors such as PLX3397. In this study, we used a syngeneic mouse model of BRAF(V600E)-driven melanoma to evaluate the ability of PLX3397 to improve the efficacy of adoptive cell therapy (ACT). In this model, we found that combined treatment produced superior antitumor responses compared with single treatments. In mice receiving the combined treatment, a dramatic reduction of TIMs and a skewing of MHCII(low) to MHCII(hi) macrophages were observed. Furthermore, mice receiving the combined treatment exhibited an increase in tumor-infiltrating lymphocytes (TIL) and T cells, as revealed by real-time imaging in vivo. In support of these observations, TILs from these mice released higher levels of IFN-γ. In conclusion, CSF-1R blockade with PLX3397 improved the efficacy of ACT immunotherapy by inhibiting the intratumoral accumulation of immunosuppressive macrophages.

Expression of New York esophageal squamous cell carcinoma-1 in primary and metastatic melanoma

New York esophageal squamous cell carcinoma-1 (NY-ESO-1), a cancer testis antigen, is an ideal target for adoptive cell transfer immunotherapy. Evidence from several clinical trials in melanoma and other malignancies shows the potential value of targeting the NY-ESO-1 antigen in immune-based therapy of metastatic tumors. However, the incidence of NY-ESO-1 expression in metastatic melanoma is unknown, and thus, it is unclear how many patients might benefit from this therapy. In this study, we analyzed NY-ESO-1 expression in 222 melanoma specimens, including 16 primary and 206 metastatic tumors. Our results support previous findings showing higher expression of NY-ESO-1 in metastatic (58/206; 28.2%) versus primary (0/16) tumors. In addition, our results show that the epithelioid subtype of melanoma has the highest incidence of NY-ESO-1 expression. These findings provide evidence of the value of this specific adoptive cell transfer therapy for the treatment of metastatic melanoma.

The who's who of T‐cell differentiation: Human memory T‐cell subsets

Following antigen encounter and subsequent resolution of the immune response, a single naïve T cell is able to generate multiple subsets of memory T cells with different phenotypic and functional properties and gene expression profiles. Single-cell technologies, first and foremost flow cytometry, have revealed the complex heterogeneity of the memory T-cell compartment and its organization into subsets. However, a consensus has still to be reached, both at the semantic (nomenclature) and phenotypic level, regarding the identification of these subsets. Here, we review recent developments in the characterization of the heterogeneity of the memory T-cell compartment, and propose a unified classification of both human and nonhuman primate T cells on the basis of phenotypic traits and in vivo properties. Given that vaccine studies and adoptive cell transfer immunotherapy protocols are influenced by these recent findings, it is important to use uniform methods for identifying and discussing functionally distinct subsets of T cells.

A Sea Change in Melanoma

As this issue of Clinical Cancer Research goes to press, Vical, Inc., a pharmaceutical company developing an intralesional gene therapy for melanoma, Allovectin-7, announced its decision to discontinue development following negative results in a phase III trial (1). This story highlights the sea change that has occurred in therapy for metastatic melanoma. The phase III pivotal trial, which launched in 2006 (NCT00395070), compared Allovectin-7 to standard therapy—dacarbazine or temozolomide (2). The pivotal trial followed a phase II study in which an 11.8% response rate was observed, with durability for responding patients, yet a median time to progression of 1.6 months (3). These results, which were at that time sufficient to launch a pivotal trial in an era defined by negative phase III trials, contrast sharply with the 48% response rate for vemurafenib and 52% for dabrafenib in pivotal trials, with progression-free survivals of 5.3 and 5.1 months, respectively (4). Allovectin-7 represented a novel therapy with modest activity that might have received U.S. Food and Drug Administration approval a decade ago on the basis of durable responses alone, but in the wake of new drug approvals, expectations about therapy for melanoma have changed dramatically. We can only hope for such a sea change in other solid tumors. This edition of CCR Focus highlights the progress in our molecular understanding of melanoma, discusses the new agents that have raised the bar on efficacy, and reviews the challenges facing the field in combining the new strategies. With Guest Editors Keith Flaherty and Patricia LoRusso, the articles in this CCR Focus section discuss new molecular targets in melanoma, targeted therapy, the new immune checkpoint inhibitors, and adoptive cell transfer immunotherapy. As with every edition of CCR Focus, we hope to inform those interested but not expert in the field and to challenge those working in the field.Susan E. BatesDeputy Editor, CCR FocusNational Cancer Institute

Improving the in vivo Persistence, Distribution and Function of Cytotoxic T Lymphocytes by Inhibiting the Tumor Immunosuppressive Microenvironment

Adoptive cell transfer immunotherapy of malignant tumors has the problem of symbiosis between effector cells and tumor cells, a short in vivo residence time, and a poor killing efficiency of effector cells. Thus, releasing effector cells from the cancer immunosuppressive microenvironment and improving their effective time and functional status in vivo would seem to be ideal strategies for facilitating immunotherapy. Low-dose cyclophosphamide administration can effectively break immunotolerance by inhibiting regulatory T cells. In the present study, in order to verify whether the persistence, distribution and function of effector cells can be improved by inhibiting immunosuppressive microenvironment, low-dose cyclophosphamide was previously intraperitoneally injected into melanoma-bearing C57BL/6 mice, thereafter, CFSE-labeled cytotoxic T lymphocytes were transfused intravenously, and their effective time, distributive pattern, and killing efficiency in different groups were observed by measuring the fluorescence intensity and cell cycle of cytotoxic T lymphocytes distributed in various organs, in comparison with tumor growth. We found down-regulating Tregs in vivo can simultaneously reduce the levels of interleukin-10 and transforming growth factor-β. Migration and distribution of cytotoxic T lymphocytes in vivo was found to vary with time. Inhibition of immunotolerance can significantly improve the persistence, distribution, and function of cytotoxic T lymphocytes. Correspondingly, significantly higher secretion of perforin, granzyme B, IL-2, and IFN-γ in tumor tissues with decreased tumor growth was seen in the cyclophosphamide injection group than in the control group. Our study may provide useful information on the cyclophosphamide-mediated mechanism for facilitating tumor immunotherapy by inhibiting the immunosuppressive tumor microenvironment.

Abstract 3969: Blocking colony stimulating factor 1 receptor (CSF1R) improves anti-tumor effects of adoptive T cell transfer therapy.

Abstract Tumors can escape immune responses by creating an inflammatory milieu that supports and recruits tumor-infiltrating myeloid cells (TIMs) with immune suppressive functions, such as myeloid derived suppressor cells (MDSC) and M2-polarized macrophages. Therapies that block recruitment of suppressor TIMs have the potential to enhance adoptive cell transfer (ACT) of T cell based-immunotherapies. We originally established a BRAFV600E mutant murine melanoma cell line (SM1) for which ACT of melanoma-targeted T cells induced anti-tumor responses, but not complete eradication in vivo. By gene expression profiling and single-nucleotide polymorphism (SNP) arrays, we found that, out of 108 cytokines produced by SM1, colony stimulating factor 1 (CSF1) was highly expressed. CSF1 induces proliferation of immune suppressors Gr-1(+) CD11b(+) MDSC and F4/80(+) CD11b(+) macrophages. In order to block the recruitment of TIMs, we tested a potent CSF1R inhibitor, PLX3397. Cultured primary murine T cells exposed to PLX3397 with a broad range of concentrations (0.1 to 50 μM) showed no evidence of cytotoxicity by MTS assay. We then tested the combination of PLX3397 and ACT in vivo using two models. In the first one, SM1 cells stably expressing the chicken ovalbumin (OVA) antigen were implanted in C57BL/6 mice. Daily oral gavage with PLX3397 (50mg/kg) combined with ACT of OVA-specific TCR transgenic cells (OT-1) demonstrated superior effects of the combined treatment (tumor size on day 14_vehicle: 1611.2±22.5 mm2, PLX3397: 1028.0±24.0 mm2, OT-1: 1112.5±35.7 mm2, combined PLX3397+OT-1: 347.3±15.5 mm2, p<0.001). We have observed a dramatic reduction of the number of macrophages in the tumor tissue (vehicle: 10.3±1.6%, PLX3397: 1.35±0.5%, OT-1: 12.0±0.6%, combined: 2.33±0.3%, p<0.001) and a skewing of type M2 to type M1 macrophages. In vivo real-time T cell tracking by luciferase bioluminescence imaging demonstrated an increased number of tumor infiltrating lymphocytes (TILs) in the combined therapy group (photon counts/pixel on day 5 post ACT: OT-1: 4424.5±412, combined: 9176±1780, p<0.001). The second in vivo model was based on pmel-1 transgenic T cells, which targeted the endogenous melanoma antigen (gp100) expressed by SM1. The combined treatment of PLX3397 and ACT of pmel-1 also resulted in significant tumor shrinkage, reduction of M2 macrophages, and increased number of TILs. TILs were also collected and analyzed for function by intracellular staining of IFNγ. TILs from the combined treatment group released significantly higher levels of IFNγ (ACT: 48.2±8.7%, combined: 78.8±3.5%, p<0.001). In conclusion, our data derived from two independent in vivo models demonstrated dramatic potentiation of anti-tumor effects with the combination of CSF1R blockade and ACT immunotherapy, supporting the rationale for further testing in patients with metastatic melanoma. Citation Format: Stephen Mok, Christopher Tsui, Jingying Xu, Begonya Comin-Anduix, Thinle Chodon, Antoni Ribas, Richard C. Koya. Blocking colony stimulating factor 1 receptor (CSF1R) improves anti-tumor effects of adoptive T cell transfer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3969. doi:10.1158/1538-7445.AM2013-3969

Multifunctional T-cell Analyses to Study Response and Progression in Adoptive Cell Transfer Immunotherapy

Adoptive cell transfer (ACT) of genetically engineered T cells expressing cancer-specific T-cell receptors (TCR) is a promising cancer treatment. Here, we investigate the in vivo functional activity and dynamics of the transferred cells by analyzing samples from 3 representative patients with melanoma enrolled in a clinical trial of ACT with TCR transgenic T cells targeted against the melanosomal antigen MART-1. The analyses included evaluating 19 secreted proteins from individual cells from phenotypically defined T-cell subpopulations, as well as the enumeration of T cells with TCR antigen specificity for 36 melanoma antigens. These analyses revealed the coordinated functional dynamics of the adoptively transferred, as well as endogenous, T cells, and the importance of highly functional T cells in dominating the antitumor immune response. This study highlights the need to develop approaches to maintaining antitumor T-cell functionality with the aim of increasing the long-term efficacy of TCR-engineered ACT immunotherapy. A longitudinal functional study of adoptively transferred TCR–engineered lymphocytes yielded revealing snapshots for understanding the changes of antitumor responses over time in ACT immunotherapy of patients with advanced melanoma.

Abstract A37: CTLA-4 blockade improves the balance of CD8+ effector and CD4+ regulatory T cells following adoptive cell transfer melanoma immunotherapy.

Abstract Background: Clinical trials have shown efficacy for CTLA-4 blockade and adoptive cell transfer (ACT) immunotherapy in the treatment of melanoma. We hypothesized that the addition of CTLA-4 blockade to a non-lymphodepleting regimen of ACT could enhance the control of melanoma tumor growth in a murine model. Methods: A B16F10 melanoma cell line was stably transfected to express low levels of the lymphocytic choriomeningitis virus (LCMV) peptide GP33 (B16GP33). Immunocompetent Ly5.2+/C57BL/6 mice were inoculated with subcutaneous flank injections of 106 B16GP33 cells on day 0. GP33-specific CD8+ T cells were harvested from syngeneic Ly5.1+/C57BL/6 donor mice 8 days after LCMV infection. Mice received no treatment, CTLA-4 blockade (intraperitoneal injections of 200 μg anti-CTLA-4 mAb on days 2, 5, and 8), ACT (intravenous injections of 5x104 GP33-specific CD8+ T cells on day 1), or both. Tumor growth was measured every three days. Subsets of mice were euthanized between days 9 and 18 for TIL and splenocyte analyses. Mice exhibiting minimal B16GP33 tumor growth > 30 days after immunotherapy were challenged with contralateral flank injections of 106 B16F10 and B16GP33 cells. Results: Combination CTLA-4 blockade and ACT immunotherapy resulted in significantly greater inhibition of B16GP33 tumor growth compared to CTLA-4 blockade or ACT alone. TIL analysis identified stronger CD8+ T cell infiltration in tumors treated with combination immunotherapy. This augmented TIL response was due to the induction of endogenous populations of CD8+ T cells, as there were no differences in Ly5.1+/CD8+ T cell infiltration between mice treated with combination immunotherapy versus ACT alone. Significantly fewer CD4+CD25+FoxP3+ regulatory T cells were observed within TIL populations after combination immunotherapy. Splenocyte response to in vitro stimulation with GP33 peptide was stronger after combination immunotherapy compared to single agent immunotherapy. A subset of mice treated with CTLA-4 blockade alone and combination immunotherapy showed minimal B16GP33 tumor growth after prolonged observation. Both groups of mice showed immunity against B16GP33 challenge tumors, but only mice treated with combination immunotherapy exhibited immunity against B16F10 challenge tumors as well. Discussion: The combination of CTLA-4 blockade with non-lymphodepletion ACT may represent a novel and effective form of melanoma immunotherapy. Our findings suggest that the additive benefit of CTLA-4 may be due to the enhanced recruitment of endogenous melanoma-directed effector T cell responses and the induction of smaller populations of regulatory T cells. Moreover, the promotion of a durable and generalized protection from recurrent tumors suggests that this combination may have synergistic immunological effects against melanoma. Citation Format: Lucy Wentworth, David A. Mahvi, Justin V. Meyers, Samantha Durbin, Clifford S. Cho. CTLA-4 blockade improves the balance of CD8+ effector and CD4+ regulatory T cells following adoptive cell transfer melanoma immunotherapy. [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 A37.

CXCR1 as a novel target for directing reactive T cells toward melanoma: implications for adoptive cell transfer immunotherapy.

Adoptive cell transfer therapy with reactive T cells is one of the most promising immunotherapeutic modalities for metastatic melanoma patients. Homing of the transferred T cells to all tumor sites in sufficient numbers is of great importance. Here, we seek to exploit endogenous chemotactic signals in order to manipulate and enhance the directional trafficking of transferred T cells toward melanoma. Chemokine profiling of 15 melanoma cultures shows that CXCL1 and CXCL8 are abundantly expressed and secreted from melanoma cultures. However, the complimentary analysis on 40 melanoma patient-derived tumor-infiltrating lymphocytes (TIL) proves that the corresponding chemokine receptors are either not expressed (CXCR2) or expressed at low levels (CXCR1). Using the in vitro transwell system, we demonstrate that TIL cells preferentially migrate toward melanoma and that endogenously expressing CXCR1 TIL cells are significantly enriched among the migrating lymphocytes. The role of the chemokines CXCL1 and CXCL8 is demonstrated by partial abrogation of this enrichment with anti-CXCL1 and anti-CXCL8 neutralizing antibodies. The role of the chemokine receptor CXCR1 is validated by the enhanced migration of CXCR1-engineered TIL cells toward melanoma or recombinant CXCL8. Cytotoxicity and IFNγ secretion activity are unaltered by CXCR1 expression profile. Taken together, these results mark CXCR1 as a candidate for genetic manipulations to enhance trafficking of adoptively transferred T cells. This approach is complimentary and potentially synergistic with other genetic strategies designed to enhance anti-tumor potency.

Ex vivo enrichment of circulating anti-tumor T cells from both cutaneous and ocular melanoma patients: clinical implications for adoptive cell transfer therapy

Tumor-infiltrating lymphocytes (TILs) have been successfully used for adoptive cell transfer (ACT) immunotherapy; however, due to their scarce availability, this therapy is possible for a limited fraction of cutaneous melanoma patients. We assessed whether an effective protocol for ex vivo T-cell expansion from peripheral blood mononuclear cells (PBMCs), suitable for ACT of both cutaneous and ocular melanoma patients, could be identified. PBMCs from both cutaneous and ocular melanoma patients were stimulated in vitro with autologous, irradiated melanoma cells (mixed lymphocyte tumor cell culture; MLTCs) in the presence of IL-2 and IL-15 followed by the rapid expansion protocol (REP). The functional activity of these T lymphocytes was characterized and compared with that of TILs. In addition, the immune infiltration in vivo of ocular melanoma lesions was analyzed. An efficient in vitro MLTC expansion of melanoma reactive T cells was achieved from all PBMC’s samples obtained in 7 cutaneous and ocular metastatic melanoma patients. Large numbers of melanoma-specific T cells could be obtained when the REP protocol was applied to these MLTCs. Most MLTCs were enriched in non-terminally differentiated TEM cells homogeneously expressing co-stimulatory molecules (e.g., NKG2D, CD28, CD134, CD137). A similar pattern of anti-tumor activity, in association with a more variable expression of co-stimulatory molecules, was detected on short-term in vitro cultured TILs isolated from the same patients. In these ocular melanoma patients, we observed an immune infiltrate with suppressive characteristics and a low rate of ex vivo growing TILs (28.5% of our cases). Our MLTC protocol overcomes this limitation, allowing the isolation of T lymphocytes with effector functions even in these patients. Thus, anti-tumor circulating PBMC-derived T cells could be efficiently isolated from melanoma patients by our novel ex vivo enrichment protocol. This protocol appears suitable for ACT studies of cutaneous and ocular melanoma patients.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-011-1179-z) contains supplementary material, which is available to authorized users.

Determinants of Successful CD8+ T-Cell Adoptive Immunotherapy for Large Established Tumors in Mice

Adoptive cell transfer (ACT) of tumor infiltrating or genetically engineered T cells can cause durable responses in patients with metastatic cancer. Multiple clinically modifiable parameters can comprise this therapy, including cell dose and phenotype, in vivo antigen restimulation, and common gamma-chain (γ(c)) cytokine support. However, the relative contributions of each these individual components to the magnitude of the antitumor response have yet to be quantified. To systematically and quantitatively appraise each of these variables, we employed the Pmel-1 mouse model treating large, established B16 melanoma tumors. In addition to cell dose and magnitude of in vivo antigen restimulation, we also evaluated the relative efficacy of central memory (T(CM)), effector memory (T(EM)), and stem cell memory (T(SCM)) subsets on the strength of tumor regression as well as the dose and type of clinically available γ(c) cytokines, including IL-2, IL-7, IL-15, and IL-21. We found that cell dose, T-cell differentiation status, and viral vaccine titer each were correlated strongly and significantly with the magnitude of tumor regression. Surprisingly, although the total number of IL-2 doses was correlated with tumor regression, no significant benefit to prolonged (≥6 doses) administration was observed. Moreover, the specific type and dose of γ(c) cytokine only moderately correlated with response. Collectively, these findings elucidate some of the key determinants of successful ACT immunotherapy for the treatment of cancer in mice and further show that γ(c) cytokines offer a similar ability to effectively drive antitumor T-cell function in vivo.

Cell transfer immunotherapy for metastatic solid cancer—what clinicians need to know

Cancer immunotherapy using the adoptive transfer of autologous tumor-infiltrating lymphocytes results in objective cancer regression in 49-72% of patients with metastatic melanoma. In a pilot trial combining cell transfer with a maximum lymphodepleting regimen, complete durable responses were seen in 40% of patients, with complete responses ongoing beyond 3 to 7 years. Current approaches to cell transfer therapy using autologous cells genetically engineered to express conventional or chimeric T-cell receptors have mediated cancer regression in patients with metastatic melanoma, synovial sarcoma, neuroblastoma and refractory lymphoma. Adoptive cell transfer immunotherapy is a rapidly developing new approach to the therapy of metastatic cancer in humans. This Review will emphasize the current available applications of cell transfer immunotherapy for patients with cancer.

Abstract SY03-04: Novel imaging approaches for immunotherapy and targeted therapy of melanoma in animal models and humans

Abstract Molecular imaging may allow gaining a better understanding of the pharmacodynamic (PD) effects of immunotherapy strategies and novel targeted therapies for cancer. Two general approaches for imaging can be used, the administration of radiolabeled small molecule tracers that specifically accumulate in cells modulated by the therapy or the genetic labeling of immune cells followed by the administration of a detection probe. The use of targeted agents that block the MAPK pathway is providing unprecedented response rates in melanoma with the BRAF V600 mutation. The positron emitting tomography (PET) probe [18F]fluorodeoxy-glucose ([18F]FDG) can noninvasively image an effect of the therapy in inhibiting glucose metabolism, and effects on cell replication can be studied with the PET probe 3′-deoxy-3′-[18F]fluorothymidine ([18F]FLT). In vitro studies in a panel of BRAF V600E mutant melanoma cell lines showed differential effects of the BRAF inhibitor PLX4032 with most cell lines being highly sensitive and a minority being resistant. Oncogenic analysis did not discern between these cell lines. Despite evidence of blocking the MAPK pathway by detecting cell cycle arrest both by flow cytometry or the incorporation of FLT in nearly all cell lines, some cell lines failed to lose viability when exposed to the BRAF inhibitor. The best way to discern between poorly responsive and highly responsive cells was the differential uptake of 2DG. In the clinic, PLX4032 has resulted in response rates up to 80% in patients with BRAF V600+ metastatic melanoma, which are preceded by dramatic decrease in [18F]FDG uptake in PET scans. Areas of progression due to acquired resistance to PLX4032 are visualized by reactivation of [18F]FDG uptake in PET scans. Therapy with anti-CTLA4 antibodies results in durable objective responses in approximately 10% of patients with metastatic melanoma, and the anti-CTLA4 antibody ipilimumab has demonstrated improvement in overall survival in a randomized clinical trial. A key issue is the location where lymphocyte proliferation occurs after the release of the CTLA4 cell cycle inhibitory checkpoint in lymphocytes. Immune monitoring assays had failed to detect a large lymphocytic expansion in blood, while tumors of patients responding to this therapy had markedly increased lymphocytic intratumoral infiltrates. We analyzed changes in glucose metabolism using the PET probe [18F]FDG, and cell replication with the PET probe [18F]FLT, after the administration of the CTLA4-blocking antibody tremelimuamb. In these studies, molecular imaging with [18F]FLT allowed mapping and noninvasive imaging of cell proliferation in the spleen, the largest secondary lymphoid organ, after CTLA4 blockade in patients with metastatic melanoma. A better understanding of the distribution and tumor targeting of lymphocytes may allow advancing adoptive cell transfer (ACT) immunotherapy for cancer. We have used T cell receptor (TCR) engineering together with molecular imaging to study the antitumor activity in an animal model of TCR-engineered ACT therapy. Splenocytes were activated ex vivo and genetically modified with a retroviral vector expressing a TCR for tyrosinase and either luciferase for bioluminescence imaging (BLI) or sr39tk for positron emitting tomography (PET) imaging. Genetic labeling with the BLI and PET reporter genes allowed visualization of the distribution and antigen-specific tumor homing of TCR transgenic T cells, with trafficking correlated with antitumor efficacy. After an initial brief stage of systemic distribution, TCR redirected and genetically labeled T cells demonstrated an early pattern of specific distribution to antigen-matched tumors and locoregional lymph nodes, followed by a more promiscuous distribution one week later with additional accumulation in antigen-mismatched tumors. Therefore, the molecular imaging reporter gene labeling approach allowed the non-invasive imaging of the in vivo dynamics of the TCR transgenic cells. The approach of TCR engineering can result in responses in patients with advanced cancer. In an ongoing clinical trial we have treated patients with metastatic melanoma with the ACT of lymphocytes genetically modified to express a high affinity TCR for the melanosomal antigen MART-1. This approach has led to high initial antitumor responses followed by tumor progression once the T cells lost their immunological potency in vivo. PET imaging with [18F]FDG demonstrated profound effects on tumor lesions beyond the changes in size criteria provided by CT scans, allowing the study of T cell responses to cancer. In conclusion, the use of molecular imaging in preclinical models and in the clinic allows longitudinal analysis of biological processes without altering their course, thereby providing important information to advance immunotherapy and targeted therapy strategies for cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY03-04. doi:10.1158/1538-7445.AM2011-SY03-04

CpG Oligodeoxynucleotides Enhance the Efficacy of Adoptive Cell Transfer Using Tumor Infiltrating Lymphocytes by Modifying the Th1 Polarization and Local Infiltration of Th17 Cells

Adoptive cell transfer immunotherapy using tumor infiltrating lymphocytes (TILs) was an important therapeutic strategy against tumors. But the efficacy remains limited and development of new strategies is urgent. Recent evidence suggested that CpG-ODNs might be a potent candidate for tumor immunotherapy. Here we firstly reported that CpG-ODNs could significantly enhance the antitumor efficacy of adoptively transferred TILs in vivo accompanied by enhanced activity capacity and proliferation of CD8+ T cells and CD8+ T cells, as well as a Th1 polarization immune response. Most importantly, we found that CpG-ODNs could significantly elevate the infiltration of Th17 cells in tumor mass, which contributed to anti-tumor efficacy of TILs in vivo. Our findings suggested that CpG ODNs could enhance the anti-tumor efficacy of adoptively transferred TILs through modifying Th1 polarization and local infiltration of Th17 cells, which might provide a clue for developing a new strategy for ACT based on TILs.

Depletion of CD4+CD25high regulatory T cells from tumor infiltrating lymphocytes predominantly induces Th1 type immune response in vivo which inhibits tumor growth in adoptive immunotherapy

Adoptive cell transfer (ACT) immunotherapy administered with prior host immunosuppression significantly improved the anti-tumor efficacy in a murine model. However, bulk transfer of lymphocytes containing suppressor lymphocyte subsets, including regulatory T cells to mice bearing late-stage tumors impaired this anti-tumor effect. In this study, we investigated the enhanced anti-tumor efficacy by adoptive transfer of Treg-depleted autologous tumor infiltrating lymphocytes in advanced murine breast cancer. We found that, compared to bulk cell transfer, Treg-depleted cell transfer enhanced the activation and proliferation of both CD4+ and CD8+ T cells. Most importantly, the immune response deviated towards the Th1 response reflected by increased IFN-γand reduced IL-4 secretion in both CD4+ and CD8+ T cells and an enhanced granzyme B release of CTL. Furthermore, the elicited Th1 response subsequently resulted in delayed tumor growth and prolonged mice survival as well as reduced lung metastasis in tumor-bearing nude mice. These results strongly indicated that Treg-depleted autologous cell transfer greatly enhanced Th1 type immune response, consequently leading to delayed tumor growth and reduced tumor burden. Therefore, ACT immunotherapy based on ex vivo selection of tumor-reactive lymphocytes resulted in enhanced anti-tumor immunity and provides important implications for further human studies.

Human TSLP directly enhances expansion of CD8+ T cells

Human thymic stromal lymphopoietin (TSLP) promotes CD4(+) T-cell proliferation both directly and indirectly through dendritic cell (DC) activation. Although human TSLP-activated DCs induce CD8(+) T-cell proliferation, it is not clear whether TSLP acts directly on CD8(+) T cells. In this study, we show that human CD8(+) T cells activated by T-cell receptor stimulation expressed TSLP receptor (TSLPR), and that TSLP directly enhanced proliferation of activated CD8(+) T cells. Although non-stimulated human CD8(+) T cells from peripheral blood did not express TSLPR, CD8(+) T cells activated by anti-CD3 plus anti-CD28 did express TSLPR. After T-cell receptor stimulation, TSLP directly enhanced the expansion of activated CD8(+) T cells. Interestingly, using monocyte-derived DCs pulsed with a cytomegalovirus (CMV)-specific pp65 peptide, we found that although interleukin-2 allowed expansion of both CMV-specific and non-specific CD8(+) T cells, TSLP induced expansion of only CMV-specific CD8(+) T cells. These results suggest that human TSLP directly enhances expansion of CD8(+) T cells and that the direct and indirect action of TSLP on expansion of target antigen-specific CD8(+) T cells may be beneficial to adoptive cell transfer immunotherapy.

In vitro methods for generating highly purified EBV associated tumor antigen-specific T cells by using solid phase T cell selection system for immunotherapy

Adoptive cell transfer immunotherapy has been utilized to treat EBV related human malignancies including post-transplant lymphoproliferative diseases, Hodgkin's lymphoma and nasopharyngeal carcinoma. However, there are limited options available for tumor antigen-specific T cell purification. Here we describe a novel solid phase T cell selection system, in which monocytes or EBV transformed B-lymphocytes are immobilized on solid support for antigen-specific T cell purification. We hypothesize and prove that antigen-specific T cells recognize their cognate antigens and bind to them faster than non-antigen specific T cells. Therefore antigen-specific T cells can be concentrated on the surface after removing the non-adherent cells by washing. The optimal selection time for both EBV-specific T cells and LMP2-specific T cells is studied. Our data demonstrate that the frequency of antigen-specific T cells can be increased by > 20-fold after selection. Moreover, activated antigen-specific T cells proliferate more rapidly than non-specific T cells, further increasing the frequency and purity of antigen-specific T cells. This new T cell selection system is superior to traditional repeated stimulation methods in generating tumor antigen-specific T cells. We are able to generate large quantities of highly purified T cells of subdominant antigens LMP2 within 2 weeks after T cell activation for adoptive cell transfer immunotherapy with this simple, rapid and inexpensive T cell selection system.