Adoptive T-cell Transfer Therapy Research Articles

A phase I study of GPC3 targeted CAR-T cell therapy in advanced GPC3-expressing hepatocellular carcinoma (HCC).

TPS624 Background: The mortality of hepatocellular carcinoma (HCC) is increasing worldwide, but outcome of systemic treatments in advanced HCC is suboptimal. Adoptive T-cell transfer therapy represents a promising field that exploits the ability of T-cells to recognize and eliminate their target. Targeting the tumor-associated antigen glypican- 3 (GPC3) through chimeric antigen receptors (CAR) engineered T cells is a mechanistically rational novel treatment for advanced HCC. This study aims to determine the dose and early signals of GPC3 targeted (CAR)-T cells in advanced GPC3 expressing HCC (NCT05003895). Methods: This phase I first in human dose escalation trial will study the safety and feasibility of CAR (hYP7)- T cells in advanced HCC patients expressing GPC3. Eligibility criteria includes advanced HCC, not candidates for curative interventions, progressed on first line systemic treatment, tumor GPC3 positivity of ³ 25% by IHC, Child-Pugh Class A, ³1 measurable lesion, ECOG 0 or 1, adequate organ and marrow function. ParticipantsÕ T cells collected through leukapheresis will be transduced with a lentivirus encoding the CAR construct to generate CAR expressing T cells. Patients receive a conditioning chemotherapy regimen of cyclophosphamide and fludarabine prior to the infusion of the GPC3 directed CAR-T cells (Table). The trial has a 4-level modified Fibonacci dose escalation with a minimum of 3 patients at each level and a 28-day interval between the first three patients. Response will be assessed by imaging every two months during the first year. Patients undergo close monitoring with safety assessments during the first year and are followed for life. The primary objective is to determine the MTD, DLT, safety and feasibility of anti-GPC3 CAR expressing T- cells in patients with GPC3-expressing advanced HCC. Secondary objectives include best overall response and overall survival. Exploratory objectives are multiple and include studies to evaluate the persistence and peak levels of anti-GPC3 CAR-T cells after infusion. Recruitment began in December 2021 and the two patients at dose level -1 has been treated; the planned sample size is 38 patients. Table 1 Dose Level Anti-GPC3 CAR-T Cyclophosphamide (mg/m2) Fludarabine (mg/m2) Level -1 0.3x106 CAR-T per kg bw 200 30 Level 1 0.3x106 CAR-T per kg bw 300 30 Level 2 1x106 CAR-T per kg bw 300 30 Level 3 3x106 CAR-T per kg bw 300 30. Clinical trial information: NCT05003895 .

Paraspeckle Promotes Hepatocellular Carcinoma Immune Escape by Sequestering IFNGR1 mRNA.

Background & AimsHepatocellular carcinoma (HCC) is the most common type of hepatic malignancies, with poor prognosis and low survival rate. Paraspeckles, which are unique subnuclear structures, are recently found to be involved in the development of various tumors, including HCC, and are related to induction in chemoresistance of HCC. This study aimed to investigate the possibility of paraspeckle in HCC cells participating in immune escape and its underlying mechanism in vitro and in vivo.MethodsExpression of NEAT1_2, the framework of paraspeckle, in HCC cells and tissues was detected by qRT-PCR and RNA-FISH. mRNAs interacted with NEAT1_2 were pull-downed and sequenced in C-terminal S1-aptamer-tagged NEAT1_2 endogenously expressed HCC cells constructed using CRISPR-CAS9 knock-in technology. The effects of paraspeckle on HCC sensitivity to T-cell-mediated cytolysis were detected by T-cell mediated tumor cell killing assay. The roles of NEAT1_2 or NONO on IFNGR1 expression and IFN-γ signaling by applying gene function loss analysis in HCC cells were detected by qRT-PCR, RNA immunoprecipitation, Western blotting, and ELISA. The role of paraspeckle during adoptive T-cell transfer therapy for HCC in vivo was performed with a subcutaneous xenograft mouse.ResultsParaspeckle in HCC cells is negatively related to T-cell-mediated cytolysis. Destruction of paraspeckle in HCC cells by knockdown of NEAT1_2 or NONO significantly improved the sensibility of resistant HCC cells to T-cell killing effects. Furthermore, IFNGR1 mRNA, which is sequestered by NEAT1_2 and NONO, is abundant in paraspeckle of T-cell killing-resistant HCC cells. Incapable IFN-γ-IFNGR1 signaling accounts for paraspeckle mediated-adoptive T-cell therapy resistance. Moreover, NEAT1_2 expression negatively correlates with IFNGR1 expression in clinical HCC tissues.ConclusionsParaspeckle in HCC cells helps tumor cells escape from immunosurveillance through sequestering IFNGR1 mRNA to inhibiting IFN-γ-IFNGR1 signaling, thereby avoiding T-cell killing effects. Collectively, our results hint that NEAT1_2 highly expressed HCC patient is more resistant to T-cell therapy in clinic, and NEAT1_2 may be potential target for HCC immunotherapy.

Abstract 4407: Leveraging spatial profiling combined with bulk RNA sequencing to study patient-specific immunotherapeutic mechanisms

Abstract Introduction: Mechanisms of resistance to adoptive T-cell transfer therapy (ACT) in clinical samples have been identified using bulk tumor sequencing, gene expression profiling, and immunohistochemistry. Furthermore, these datatypes are independently used to predict response to immunotherapy. However, this is often done using relative values quantified across clinical cohorts, which is not always feasible in single sample case analyses. Samples and Methods: A patient (INY3) presented with a synovial sarcoma lesion (INY3-P) and had an objective response (OR) to ACT targeting NY-ESO-1. A lung metastasis (INY3-M) progressed 10 months post-treatment, and she had a second OR to ACT plus ipilimumab. Seven months later, the primary lesion recurred (INY3-R). RNAseq was performed on snap-frozen biopsies at each timepoint. Two formalin-fixed, paraffin-embedded slides from each timepoint were assessed by either the Nanostring GeoMx Digital Spatial Profiling (DSP) 60-plex protein or 84-plex RNA panel. Regions-of-interest (ROIs) were selected for those positive for NY-ESO-1, pan-CK, CD45, or combinations of these proteins. We evaluated the correlation of DSP targets across ROIs within each sample, correlation of matched protein/RNA DSP targets across timepoints, the impact of DSP heterogeneity on bulk tumor RNAseq, and DSP patterns in ACT-resistant samples. Results: The Spearman correlation across ROIs ranged from -0.57-0.84 (median -0.14) in DSP protein and -0.59-0.75 (median -0.11) in DSP RNA expression. The variance in correlation was greatest in INY3-M (0.18 protein; 0.11 RNA), compared to INY3-P (0.08 protein; 0.13 RNA) and INY3-R (0.07 protein; 0.02 RNA), suggesting greater differences in ROIs selected in INY3-M. This was confirmed by lowest correlation between DSP RNA expression with bulk RNAseq in INY3-M (0.43-0.76 across ROIs; INY3-P, 0.31-0.61; INY3-R, 0.67-0.79). The median correlation between matched protein/RNA targets (N=31) in each ROI was 0.41 (-0.17-0.90), establishing some differences in RNA and protein expression. This was confirmed by overall lower correlation of DSP protein expression with bulk RNA expression (0.32-0.65 across ROIs; median 0.61). There were three patterns observed in ACT-resistant samples: increased CTNNB1 (INY3-M); lower expression of T cell proteins, as well as B2M, HLA-DRB, CXCL9, and PSMB10 (INY3-M, INY3-R); and increased expression of T cell surface proteins, and antigen presentation machinery (INY3-M, INY3-R). Conclusions: Bulk tumor RNAseq analysis is complicated by tumor heterogeneity, which can be overcome by DSP. Collectively, our results support complementary use of these technologies to better study individual samples, without requiring comparison to clinical cohorts, as well as address the complexities of immunotherapeutic mechanisms, identifying cell type-specific signals that may be lost using bulk RNAseq. Citation Format: Katie M. Campbell, Theodore S. Nowicki, Antoni Ribas. Leveraging spatial profiling combined with bulk RNA sequencing to study patient-specific immunotherapeutic mechanisms [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4407.

Abstract 3155: Interferon-gamma-induced melanoma plasticity and response to PD-1 blockade therapy

Abstract Melanoma is a cancer of melanocytes, which develop from neural crest cells during embryogenesis. One of the ways melanoma can resist targeted therapy or adoptive T cell transfer therapy (ACT) is by dedifferentiation, whereby the tumor becomes less melanocytic and more neural crest-like. ACT-induced dedifferentiation and therapy resistance was a result of tumor necrosis factor (TNF) from antitumor T cells (Landsberg, Nature 2012), which can serve as a positive control for inflammatory cytokine-induced melanoma plasticity. To study whether immune checkpoint blockade induces melanoma dedifferentiation, we analyzed RNA-seq data from paired biopsies of 68 patients before and during anti-PD-1 therapy. Surprisingly, the tumors of patients with complete or partial response dedifferentiate following therapy (paired p= 0.00015), but those of stable or progressive disease do not (paired p= 0.31 and 0.32, respectively). As the presence of interferon-gamma signatures in biopsies is best correlated with response to anti-PD-1 therapy, we hypothesized that interferon-gamma mediates dedifferentiation. We dedifferentiated four human melanoma cell lines by in vitro exposure to long-term (three to five weeks) interferon-gamma and compared it to three days of TNF as positive control. Although TNF and long-term interferon-gamma induced similar phenotypic changes based on flow cytometry, ATAC-seq showed that they each drive different global chromatin remodeling. Motif enrichment analyses revealed STAT6, TFAP2C, and SOX proteins are key transcription factors involved in interferon-gamma-induced dedifferentiation. Inferred protein activity analysis of the matching RNA-seq data showed that these regulators, as well as IRF3 and HMGA1, alter in activity selectively with dedifferentiation. In conclusion, dedifferentiation may be a marker of positive response to anti-PD-1 therapy, mediated by chronic exposure to T cells producing interferon-gamma. Citation Format: Yeon Joo Kim, Katherine M. Sheu, Jennifer Tsoi, Gabriel Abril-Rodriguez, Catherine Grasso, Alexander Hoffmann, Stephen T. Smale, Thomas G. Graeber, Cristina Puig-Saus, Antoni Ribas. Interferon-gamma-induced melanoma plasticity and response to PD-1 blockade therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3155.

Synergistic effect of adoptive immunotherapy and docetaxel inhibits tumor growth in a mouse model

Adoptive T cell transfer therapy (ACT) has emerged as a promising approach to cancer immunotherapy; however, the efficacy of ACT is limited by the T-cell suppressive activity of myeloid-derived suppressor cells (MDSCs), which accumulate in the tumor microenvironment after ACT. We sought to determine whether the efficacy of ACT could be enhanced by co-treatment with docetaxel, a taxane chemotherapy agent that has been shown previously to inhibit MDSC function. Using a mouse tumor model, we demonstrated that ACT and docetaxel synergistically inhibit the growth either of engrafted CT26 colon cancer or 4T1 mammary carcinoma cells. While ACT mediated an increase in the recruitment of MDSCs to the site of the tumor, docetaxel reversed this increase. Furthermore, ex vivo cultures of tumor-associated MDSCs suppressed the cytotoxic activity of tumor-specific T cells, and this suppressive activity was abolished by docetaxel treatment. These results suggest that docetaxel inhibits both the tumor recruitment and T cell suppressive activity of MDSCs. Inhibitors of iNOS and arginase partially inhibited ex vivo MDSC activity, and combined inhibition of iNOS and arginase had a similar effect as docetaxel, which supports the possibility that docetaxel may function by inhibiting ACT-associated activation of these pathways. Furthermore, docetaxel mediated inhibition of the T cell suppressive activity of MDSCs from human blood, which supports the potential clinical applicability of these findings. On the basis of these findings, docetaxel treatment may represent an effective therapeutic approach for reversing immunosuppression by MDSCs subsequent to ACT-based therapy.

Fueling Cancer Immunotherapy With Common Gamma Chain Cytokines.

Adoptive T cell transfer therapy (ACT) using tumor infiltrating lymphocytes or lymphocytes redirected with antigen receptors (CAR or TCR) has revolutionized the field of cancer immunotherapy. Although CAR T cell therapy mediates robust responses in patients with hematological malignancies, this approach has been less effective for treating patients with solid tumors. Additionally, toxicities post T cell infusion highlight the need for safer ACT protocols. Current protocols traditionally expand T lymphocytes isolated from patient tumors or from peripheral blood to large magnitudes in the presence of high dose IL-2 prior to infusion. Unfortunately, this expansion protocol differentiates T cells to a full effector or terminal phenotype in vitro, consequently reducing their long-term survival and antitumor effectiveness in vivo. Post-infusion, T cells face further obstacles limiting their persistence and function within the suppressive tumor microenvironment. Therapeutic manipulation of T cells with common γ chain cytokines, which are critical growth factors for T cells, may be the key to bypass such immunological hurdles. Herein, we discuss the primary functions of the common γ chain cytokines impacting T cell survival and memory and then elaborate on how these distinct cytokines have been used to augment T cell-based cancer immunotherapy.

An overview of cancer immunotherapeutic strategies.

Artificially boosting body's immune response is one of the most exciting, effective and promising advancements in the treatment of cancers. Cancer immunotherapeutics consist of variety of treatment approaches such as cytokine therapy, adoptive T-cell transfer therapy, and antibodies that stimulate innate and adoptive immune responses. In addition to this, development of HPV vaccine has paved way toward the development of other cancer vaccines. Checkpoint blockade inhibitors, for example, anti-programmed cell death protein 1 and anti-cytotoxic T-lymphocyte-associated antigen-4, chimeric antigen receptor T-cell therapy and monoclonal antibodies are emerging as other major breakthroughs that are highly effective against cancer. This review addresses the current status of immunotherapeutic strategies against cancer and provides baseline data for future research.

In Vitro Priming of Adoptively Transferred T Cells with a RORγ Agonist Confers Durable Memory and Stemness In Vivo.

Adoptive T-cell transfer therapy is an FDA- approved treatment for leukemia that relies on the ex vivo expansion and reinfusion of a patient's immune cells, which can be engineered with a chimeric antigen receptor (CAR) for more efficient tumor recognition. Type 17 T cells, controlled transcriptionally by RORγ, have been reported to mediate potent antitumor effects superior to those observed with conventionally expanded T cells. Here, we demonstrate that addition of a synthetic, small-molecule RORγ agonist during ex vivo expansion potentiates the antitumor activity of human Th17 and Tc17 cells redirected with a CAR. Likewise, ex vivo use of this agonist bolstered the antitumor properties of murine tumor-specific CD4+ and CD8+ T cells. Expansion in the presence of the RORγ agonist enhanced IL17A production without compromising IFNγ secretion in vitroIn vivo, cytokine neutralization studies revealed that IFNγ and IL17A were required to regress murine melanoma tumors. The enhanced antitumor effect of RORγ agonist treatment was associated with recovery of more donor T cells in the tumor and spleen; these cells produced elevated levels of cytokines months after infusion and expressed markers of long-lived stem and central memory cells such as Tcf7 and CD62L. Conversely, untreated cells mainly exhibited effector phenotypes in the tumor. Cured mice previously treated with agonist-primed T cells were protected from tumor rechallenge. Collectively, our work reveals that in vitro treatment with a RORγ agonist generates potent antitumor Type 17 effector cells that persist as long-lived memory cells in vivoSignificance: RORγ agonists can be used in vitro during T-cell expansion to enhance the efficacy of adoptive cell therapy (e.g., CAR-T) and to provide long-term protection against tumors.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/14/3888/F1.large.jpg Cancer Res; 78(14); 3888-98. ©2018 AACR.

Abstract 1537: Anti-CD19 ARTEMIS™ T cells prevent excessive inflammatory cytokine release, including IL-6, in a co-culture model of CRS

Abstract We designed the ARTEMISTM T-cell signaling platform to be a safer ACT therapy by relying on endogenous T-cell signaling factors. We show evidence that the ARTEMISTM receptor disentangles efficacy from CRS and holds the potential to be a clinically safer therapy by preventing ACT-triggered CRS. Despite the remarkable curative potential of anti-CD19-CAR T cells the therapy continues to trigger a potentially life threatening cytokine release syndrome (CRS) that is characterized by excessive concentrations of inflammatory cytokines. We reasoned that by redesigning the T-cell activating antigen receptor to require signaling through the endogenous CD3 complex the T-cell could better regulate its cellular responses and thus reduce the risk of CRS when used as an adoptive T-cell transfer (ACT) therapy. Through protein engineering, we developed the ARTEMIS™ signaling platform which when coupled with Eureka's human anti-CD19 antibody, ET190L1, and expressed on primary T-cells, results in a significant reduction of cytokine release during antigen specific T-cell activation. When CAR vs. ARTEMIS™ comparisons were tested in vitro, ET190L1-ARTEMISTM T-cells and ET190L1-CD28z-CAR T-cells specifically lysed multiple CD19+ leukemia and lymphoma cell lines with similar potencies. However, during the 16 hour in vitro killing assays, ARTEMIS™ T-cells secreted demonstrably less IL-2, IFN-γ, GM-CSF, and TNFα as compared to CAR T-cells. ARTEMISTM T-cells also accumulated less PD-1, LAG3, and TIM3 on their surface during culturing and following in vitro killing, indicating a diminished propensity for exhaustion compared to corresponding CAR T-cells. The ability of the anti-IL6 antibody tocilizumab to mitigate toxicity in patients that develop severe CRS in the clinic suggests that limiting IL-6 expression could improve the safety of adoptive T-cell therapies. However, T-cells are not efficient expressors of IL-6 and the vast majority of elevated IL-6 is produced by monocytes in response to activated CAR T-cells. To assay the effects of T-cell cytotoxicity on upregulation of cytokine expression within monocytes as a model for CRS, we co-cultured T-cells with CD19-positive ALL tumor cells on one side of a transwell and monocytes on the other side. After tumor cell lysis, RNA expression levels were measured from both T-cells and monocytes. Using ET190L1-ARTEMIS™ T-cells to kill NALM-6 tumor cells resulted in dramatically reduced levels of IL-6 and IL-5 mRNA upregulation within monocytes compared to that observed with CAR transduced T-cells. When tested in vivo against CD19+ xenografts mouse models, intravenous administration of ET190L1-ARTEMISTM T-cells caused efficient tumor regression. In agreement with the in vitro data, mice treated with ARTEMISTM T-cells released significantly lower levels of cytokines in their blood at 24 hours post dosing than mice treated with CAR T-cells. Citation Format: Hong Liu, Lucas H. Horan, Stephan A. Grupp, David M. Barrett, Cheng Liu. Anti-CD19 ARTEMIS™ T cells prevent excessive inflammatory cytokine release, including IL-6, in a co-culture model of CRS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1537.

NKG2D receptor activation makes CD8+ T cells resistant to the suppressive effects of TGF-β

Abstract Natural killer group 2, member D (NKG2D) is a stimulatory receptor majorly present on the surface of natural killer and CD8+ T cells. We and others have shown that NKG2D receptor enhances the function, persistence and proliferation of CD8+ T cells. On the other hand, a major hurdle in the field of cancer vaccine and adoptive T cell transfer therapy (ACT) is diminished effectiveness and persistence of CD8+ T cells due to immuno-suppressive factors. TGF-β is a highly prevalent immuno-suppressive factor that negatively regulates the function and persistence of CD8+ T cells. We hypothesized that NKG2D activation opposes the suppressive effects of TGF-β on CD8+ T cells, and enhances their anti-tumor activity in tumor microenvironment. Here, we show that activating the NKG2D receptor makes human CD8+ T cells resistant to TGF-β’s suppressive effects on the activation, proliferation and granzyme B expression. Further, we found that NKG2D knock-out mouse CD8+ T cells are more susceptible to these TGF-β mediated suppressive effects in vitro. Interestingly, under influence of TGF-β, NKG2D KO CD8+ T cells’ ability to secrete multiple cytokines in vivo was significantly less than their WT counterparts. Our next objective is to determine the molecular mechanisms by which NKG2D makes CD8+ T cells resistant to TGF-β. For the proximal mechanism, we will determine NKG2D’s immediate signaling pathway, namely PI3K or Grb2-SOS-Ras, mediating resistance to TGF-β. Moreover, our preliminary data shows that NKG2D activation increases the levels of regulator of G protein signaling 3 (RGS3), inhibitor of TGF-β signaling, in CD8+ T cells. We will investigate if up-regulation of RGS3 is a distal mechanism underlying NKG2D mediated resistance to TGF-β in CD8+ T cells.

VSV immunotherapy to improve treatment outcomes in a mouse model of metastatic melanoma

Abstract Vesicular Stomatitis Virus (VSV) engineered to express tumor-associated antigens (TAA) is a promising systemic therapy against widely disseminated malignancy. Using a mouse model of metastatic melanoma, we have demonstrated that VSV immunotherapy is a rational addition to current treatment strategies. Combining VSV immunotherapy with adoptive T-cell transfer and localized radiation therapy enhances the overall survival and control of disease in mice challenged with disseminated melanoma. Furthermore, our previous data have shown that the use of stereotactic hypofractionated radiotherapy provides control of subcutaneous localized disease while systemically delivered VSV immunotherapies contribute to the control of metastatic disease. From these experiments we have demonstrated that this therapy is mediated by multiple immune subsets including CD4+, CD8+, and NK1.1+ populations. Thus, we hypothesized that the additional combination of the above clinically relevant therapies with adoptive T-cell transfer and immune checkpoint inhibitors would result in distinct and quantifiable improvements in immunological tumor control. Forthcoming data will characterize the expression dynamics of the inhibitory immune receptors PD-1 and TIM3 on effector and naïve populations (as defined by CD44 and CD62L expression) in response to each of the above therapies in order to design rationale treatment strategies against metastatic melanoma.

TCR γ4δ1-Engineered αβT Cells Exhibit Effective Antitumor Activity

T cell engineering with T cell receptors (TCRs) specific for tumors plays an important role in adoptive T-cell transfer (ATC) therapy for cancer. Here, we present a novel strategy to redirect peripheral blood-derived αβT cells against tumors via TCRγ4δ1 gene transduction. The broad-spectrum anti-tumor activity of TCRδ1 cells in innate immunity is dependent on CDR3δ1. TCRγ4δ1-engineered αβT cells were prepared by lentiviral transduction and characterized by analyzing in vitro and in vivo cytotoxicity to tumors, ability of proliferation and cytokine production, and their potential role in autoimmunity. Results show TCRγ4δ1 genes were transduced to approximately 36% of polyclonal αβT cells. TCRγ4δ1-engineered αβT cells exhibited an effective in-vitro TCRγδ-dependent cytotoxicity against various tumor cells via the perforin-granzyme pathway. They also showed a strong proliferative capacity and robust cytokine production. TCRγ4δ1-engineered αβT cells neither expressed mixed TCR dimers nor bound/killed normal cells in vitro. More importantly, adoptive transfer of TCRγ4δ1-engineered αβT cells into nude mice bearing a human HepG2 cell line significantly suppressed tumor growth. Our results demonstrate a novel role for TCRγ4δ1 in gene therapy and ATC for cancer.

Identification of a Novel Immunodominant HLA-B*07: 02-restricted Adenoviral Peptide Epitope and Its Potential in Adoptive Transfer Immunotherapy.

Adenovirus infections of immunocompromised patients, particularly following allogeneic hematopoietic stem cell transplantation, are associated with morbidity and mortality. Immunotherapy by adoptive transfer of hexon-specific and penton-specific T cells has been successfully applied, but many approaches are impeded by the low number of HLA class I-restricted adenoviral peptide epitopes described to date. We use a novel method to identify naturally presented adenoviral peptide epitopes from infected human cells, ectopically expressing defined HLA, using peptide elution and liquid chromatography-mass spectrometry analysis. We show that the previously described HLA-A*01:01-restricted peptide epitope LTDLGQNLLY from hexon protein is naturally presented, and demonstrate the functionality of LTDLGQNLLY-specific T cells. We further identify a novel immunodominant HLA-B*07:02-restricted peptide epitope VPATGRTLVL from protein 13.6 K, and demonstrate the high proliferative, cytotoxic, and IFN-γ-producing capacity of peptide-specific T cells. Lastly, LTDLGQNLLY-specific T cells can be detected ex vivo following adoptive transfer therapy, and LTDLGQNLLY-specific and VPATGRTLVL-specific T cells have memory phenotypes ex vivo. Given their proliferative and cytotoxic capacity, such epitope-specific T cells are promising candidates for adoptive T-cell transfer therapy of adenovirus infection.

Studying the antagonistic relationship between NKG2D and TGF-β in CD8+ T cells (IRM14P.449)

Abstract One of the major hurdles in cancer vaccine and adoptive T cell transfer therapy (ACT) development is diminished effectiveness and persistence of T lymphocytes due to immuno-suppressive factors. TGF-β is highly prevalent immuno-suppressive factor secreted by tumor which negatively regulates persistence and functionality of CD8+ T cells. Natural killer group 2, member D (NKG2D) is a stimulatory receptor majorly expressed on the surface of NK and CD8+ T cells. Others including our lab have shown that NKG2D activation on CD8+ T cells enhances their function, proliferation and persistence. Accordingly, we hypothesized that NKG2D signaling reverses the inhibitory effects of TGF-β on CD8+ T cells. We found that in vitro NKG2D activation prevented TGF-β mediated inhibition of proliferation and granzyme B expression in human CD8+ T cells. Interestingly, we also found that TGF-β down regulates the surface expression of NKG2D in human and mouse CD8+ T cells. In the presented research we will test two alternative hypotheses to elucidate molecular mechanism underlying NKG2D and TGF-β’s antagonistic effects. NKG2D opposes TGF-β’s suppressive effects in CD8+ T cells by i) repressing levels of transcription factor T-bet ii) increasing levels of inhibitor of TGF-β signaling-regulator of G-protein signaling 3 (RGS3). The findings from this research will form basis to develop a therapeutic strategy to reverse TGF-β’s immunosuppressive effects in vivo that results in better anti-tumor immunity.

MiR-23a blockade enhances adoptive T cell transfer therapy by preserving immune-competence in the tumor microenvironment

In adoptive T cell transfer therapy (ACT), the antitumor efficacy of cytotoxic CD8+ T lymphocytes (CTLs) has been limited by tumor-induced immunosuppression. We have demonstrated that miR-23a blockade in tumor-specific CTLs conferred resilience to TGFβ-mediated immunosuppression, resulting in superior tumor control. Our studies highlight miR-23a in tumor-specific CTLs as a clinically relevant target to enhance ACT.

Tumor-infiltrating lymphocyte therapy for melanoma: rationale and issues for further clinical development.

Cancer immunotherapy has become an important area for the future development of cancer therapy; this includes T-cell-based therapies that involve adoptive transfer of autologous T cells derived from the tumors or peripheral blood of cancer patients, vaccines, oncolytic virus therapy, and immunomodulatory antibodies and ligands. Here, we summarize the current approaches and clinical data in the field of adoptive T-cell transfer therapy using tumor-infiltrating lymphocytes (TILs) for metastatic melanoma. We also discuss current knowledge on the mechanism of transferred TILs in mediating tumor regression and the growing need for and recent advances in the identification of predictive biomarkers to better select patients for TIL therapy. The current technical limitations of current TIL expansion methods for out-scaling are discussed as well as how these are being addressed in order to further "industrialize" this form of cell therapy. Lastly, how TIL adoptive transfer can be incorporated into the current melanoma treatment continuum, especially as combination therapy with other immunomodulators and targeted drugs, is discussed.

Phase I Clinical Trial of Fibronectin CH296-Stimulated T Cell Therapy in Patients with Advanced Cancer

BackgroundPrevious studies have demonstrated that less-differentiated T cells are ideal for adoptive T cell transfer therapy (ACT) and that fibronectin CH296 (FN-CH296) together with anti-CD3 resulted in cultured cells that contain higher amounts of less-differentiated T cells. In this phase I clinical trial, we build on these prior results by assessing the safety and efficacy of FN-CH296 stimulated T cell therapy in patients with advanced cancer.MethodsPatients underwent fibronectin CH296-stimulated T cell therapy up to six times every two weeks and the safety and antitumor activity of the ACT were assessed. In order to determine immune function, whole blood cytokine levels and the number of peripheral regulatory T cells were analyzed prior to ACT and during the follow up.ResultsTransferred cells contained numerous less-differentiated T cells greatly represented by CD27+CD45RA+ or CD28+CD45RA+ cell, which accounted for approximately 65% and 70% of the total, respectively. No ACT related severe or unexpected toxicities were observed. The response rate among patients was 22.2% and the disease control rate was 66.7%.ConclusionsThe results obtained in this phase I trial, indicate that FN-CH296 stimulated T cell therapy was very well tolerated with a level of efficacy that is quite promising. We also surmise that expanding T cell using CH296 is a method that can be applied to other T- cell-based therapies.Trial RegistrationUMIN UMIN000001835

Adoptive T-cell transfer therapy and oncogene-targeted therapy for melanoma: the search for synergy.

The clinical strengths of immunotherapy and small-molecule inhibitors targeting the mitogen-activated protein kinase (MAPK) pathway appear to be largely complementary for the treatment of advanced melanoma. In current practice, most patients with BRAF V600 mutant melanomas will see both modalities. Several in vitro and in vivo studies suggest that combining immunotherapy with MAPK inhibition may have synergistic effects. First, mouse models show that adoptive cell therapy (ACT) can be enhanced by vaccination. Rapid tumor destruction by vemurafenib could provide a vaccine-like stimulus to adoptively transferred T cells. Second, both in mice and in early clinical trials, melanoma metastases treated with MAPK inhibitors seem to display increased T-cell infiltrates. Third, MAPK inhibition upregulates the expression of some melanoma antigens and, therefore, may enhance T-cell recognition of vemurafenib-treated melanomas. Fourth, vemurafenib may sensitize tumor cells to immune destruction. Finally, some investigators have found that an optimal antitumor effect from MAPK inhibition is dependent on an intact host immune response. Currently, the Surgery Branch of the National Cancer Institute has initiated a phase II trial combining the BRAF inhibitor vemurafenib with ACT using tumor-infiltrating lymphocytes in patients with BRAF-mutant tumors to investigate the safety and efficacy of this combination. The proposed mechanisms for synergy between these two modalities can be complex, and their optimal combination may require testing a variety of sequences and schedules.

Ex vivo characterization of γδ T-cell repertoire in patients after adoptive transfer of Vγ9Vδ2 T cells expressing the interleukin-2 receptor β-chain and the common γ-chain

Adoptive immunotherapy is emerging as a potent anti-tumor treatment modality; Vγ9Vδ2 T cells may represent appropriate agents for such cancer immunotherapy. To improve the currently limited success of Vγ9Vδ2 T-cell-based immunotherapy, we examined the in vivo dynamics of these adoptively-transferred cells and hypothesized that interleukin (IL)-15 is the potential factor for Vγ9δ2 T cell in vivo survival. We conducted a clinical trial of adoptive Vγ9Vδ2 T-cell transfer therapy in six colorectal cancer patients who received pulmonary metastasectomy. Patients' peripheral blood mononuclear cells were stimulated with zoledronate (5 μmol/L) and IL-2 (1000 IU/mL) for 14 d. Harvested cells, mostly Vγ9Vδ2 T cells, were given intravenously weekly without additional IL-2 eight times in total. The frequency, phenotype and common γ-chain cytokine receptor expression of Vγ9Vδ2 T cells in peripheral blood was monitored by flow cytometry at each time point during treatment and 4 and 12 weeks after the last administration. Adoptively transferred Vγ9Vδ2 T cells expanded well without exogenous IL-2 administration or lymphodepleting preconditioning. They maintained effector functions in terms of interferon-γ secretion and prompt release of cytotoxic granules in response to PMA/ionomycin or isopentenyl pyrophosphate-positive cells. Because they are IL-2Rα(-)IL-7Rα(-)IL-15Rα(-)IL-2Rβ(+)γc(+), it is likely that IL-2 or IL-15 is required for their maintenance. The persistence of large numbers of functionally active adoptively transferred Vγ9Vδ2 T cells in the absence of exogenous IL-2 implies that an endogenous factor, such as IL-15 transpresentation, is adequate to support these cells in vivo.

New immune therapy targets tumor-associated environment: from bone marrow to tumor site

New immune therapy targets tumor-associated environment: from bone marrow to tumor site