T-cell Surface Research Articles

Engagement of distinct epitopes on CD43 induces different co-stimulatory pathways in human T cells.

SummaryCo‐receptors, being either co‐stimulatory or co‐inhibitory, play a pivotal role in T‐cell immunity. Several studies have indicated that CD43, one of the abundant T‐cell surface glycoproteins, acts not only as a potent co‐receptor but also as a negative regulator for T‐cell activation. Here we demonstrate that co‐stimulation of human peripheral blood (PB) T cells through two distinct CD43 epitopes recognized by monoclonal antibodies (mAb) CD43‐6E5 (T6E5‐act) and CD43‐10G7 (T10G7‐act) potently induced T‐cell proliferation. However, T‐cell co‐stimulation through two CD43 epitopes differentially regulated activation of nuclear factor of activated T cells (NFAT) and nuclear factor‐κB (NF‐κB) transcription factors, T‐cell cytokine production and effector function. T6E5‐act produced high levels of interleukin‐22 (IL‐22) and interferon‐γ (IFN‐γ) similar to T cells activated via CD28 (TCD 28‐act), whereas T10G7‐act produced low levels of inflammatory cytokines but higher levels of regulatory cytokines transforming growth factor‐β (TGF‐β) and interleukin‐35 (IL‐35). Compared with T6E5‐act or to TCD 28‐act, T10G7‐act performed poorly in response to re‐stimulation and further acquired a T‐cell suppressive function. T10G7‐act did not directly inhibit proliferation of responder T cells, but formed stable heterotypic clusters with dendritic cells (DC) via CD2 to constrain activation of responder T cells. Together, our data demonstrate that CD43 is a unique and polarizing regulator of T‐cell function.

Abstract 2307: Improved CAR safety by a non-lethal switch to control CAR activity at the T-cell surface membrane

Abstract Adoptive immunotherapy using engineered T-cells has emerged as a powerful approach to treat cancer. The potential of this approach relies on the ability to redirect the specificity of T cells through genetic engineering. Novel specificities in T cells have been typically implemented through the genetic transfer of the so-called chimeric antigen receptors (CARs). CARs are synthetic receptors that associated an extracellular targeting moiety with one or more intracytoplasmic signaling domain derived from lymphocyte activation receptors. Present CAR architectures are designed to combine all relevant domains within a single polypeptide, thereby; they combines advantages of MHC unrestricted target recognition to the potent native effector mechanisms of the T cell. Although adoptive transfer of CAR T cells is proven to be an effective strategy to cancer therapy, potential adverse effects such as Cytokines Release Syndrome (CRS) and/or the risk of on-target off-tumor targeting are still a major concern. To date only suicide mechanisms that can eradicate the engineered T-Cell “at will” or mRNA CAR transfection approaches have been proposed to addresses this safety issue. Here, we describe the development of a small molecule based switch-on technology to control the surface presentation of a chimeric antigen receptor in T-cells. By grafting protein domains that can interact upon addition of a small molecule drug in the hinge domain of the CAR architecture, we are able to turn a T-cell endowed with an engineered CAR from an off-state to an on-state, in term surface presentation as well as for its specific cytolytic properties. This system offers the advantage of a “transient CAR T-cell” for safety while letting open the possibility of multiple cytotoxicity cycles using a small molecule drug. This non-lethal control system of CAR engineered T-cells represents an important advance for the safety of this technology. Citation Format: Alexandre Juillerat, Alan Marechal, Jean-Marie Filhol, Philippe Duchateau, Laurent Poirot. Improved CAR safety by a non-lethal switch to control CAR activity at the T-cell surface membrane. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2307.

Cancer immunotherapy by immuno-checkpoint blockade

As cancer immunotherapies utilizing anti-tumor T-cell responses, immuno-checkpoint blockade and adoptive T-cell immunotherapy have recently achieved durable responses even in advanced cancer patients with metastases. Administration of antibodies on the T-cell surface, CTLA-4 and PD-1 (or PD-1 ligand PD-L1), resulted in tumor regression of not only melanoma and renal cell cancer which were known to be relatively sensitive to immunotherapy, but also various malignancies including lung, bladder, ovarian, gastric, and head and neck cancers, as well as hematological malignancies such as Hodgkin and B-cell malignant lymphomas. These findings have changed the status of immunotherapy in the development of cancer treatments. Currently, development of combinations employing cancer immunotherapy with immuno-checkpoint blockade, as well as personalized cancer immunotherapy based on the evaluation of pretreatment immune status, are in progress.

Multiparameter mass cytometry of T cell development and selection

Abstract T cell development and selection in the thymus play key roles in shaping the adaptive immune system and maintaining self-tolerance. Previous studies characterizing thymic development have largely relied on genetic perturbations and subsequent cell sorting that invariably perturb specific developmental compartments. Using mass-cytometry, allow us to capture the entire developmental progression of T cells, from the double-negative (DN) stages, through double-positive (DP) and the CD4 and CD8 branches, without perturbation. With 42 channels simultaneously measured, we collected a mass cytometry dataset profiling the mouse thymus with T-cell surface markers and transcription factors, chosen based on their broad functionality in T cell development. Using the newly developed “Wishbone” algorithm, we recovered the known stages in T cell development with high accuracy and developmental resolution. This allowed us to place DN, DP, CD4+ and CD8+ cells from a single thymus along a unified bifurcating trajectory. Our data allows for precise ordering of multiple events along the trajectory using un-sorted cells from mice that were not perturbed genetically. To characterize the selection process along the development trajectory we used Nur77-GFP mice in which GFP is induced by TCR signaling that occurs during positive and negative selection. By following GFP strength together with cell trajectory we were able to recognize and characterize the behavior of proteins that participate in selection and that are involved in the transition from the DP to SP stage. This approach enables us for the first time to place and follow coordinated changes in multiple markers that take part in T cell development and selection in the thymus using single snapshots

αβ T-cell receptor bias in disease and therapy (Review).

The diversity and specificity of T cell receptors (TCR), the characteristics of T-cell surface marker, are central to the adaptive immunity. TCR variability is required for successful immunization coverage because this structural foundation is indispensable for the valid identification of short antigen peptides (derived from degraded antigens) that are presented by major histocompatibility molecules on the surfaces of antigen-presenting cells. Despite the vast T-cell repertoire, biased αβ TCR has become a common theme in immunology. To date, numerous examples of TCR bias have been observed in various diseases. Immunotherapy strategies that are based on αβ T cell responses are also emerged as a prominent component of clinical treatment. In the present review, we briefly summarize the current knowledge regarding basic structural information and the molecular mechanisms underlying TCR diversity. Moreover, we outline the role of TCR repertoire bias in some diseases, and its application for therapeutic interventions, as these play significant roles in disease progression, even with patients with a good prognosis.

CD5 expression is regulated during human T-cell activation by alternative polyadenylation, PTBP1, and miR-204.

T lymphocytes stimulated through their antigen receptor (TCR) preferentially express mRNA isoforms with shorter 3´ untranslated regions (3´-UTRs) derived from alternative pre-mRNA cleavage and polyadenylation (APA). However, the physiological relevance of APA programs remains poorly understood. CD5 is a T-cell surface glycoprotein that negatively regulates TCR signaling from the onset of T-cell activation. CD5 plays a pivotal role in mediating outcomes of cell survival or apoptosis, and may prevent both autoimmunity and cancer. In human primary T lymphocytes and Jurkat cells we found three distinct mRNA isoforms encoding CD5, each derived from distinct poly(A) signals (PASs). Upon T-cell activation, there is an overall increase in CD5 mRNAs with a specific increase in the relative expression of the shorter isoforms. 3´-UTRs derived from these shorter isoforms confer higher reporter expression in activated T cells relative to the longer isoform. We further show that polypyrimidine tract binding protein (PTB/PTBP1) directly binds to the proximal PAS and PTB siRNA depletion causes a decrease in mRNA derived from this PAS, suggesting an effect on stability or poly(A) site selection to circumvent targeting of the longer CD5 mRNA isoform by miR-204. These mechanisms fine-tune CD5 expression levels and thus ultimately T-cell responses.

T-Cell Surface Antigens and sCD30 as Biomarkers of the Risk of Rejection in Solid Organ Transplantation.

T-cell activation is a characteristic of organ rejection. T cells, located in the draining lymph nodes of the transplant recipient, are faced with non-self-molecules presented by antigen presenting cells and become activated. Activated T cells are characterized by up-regulated surface antigens, such as costimulatory molecules, adhesion molecules, chemokine receptors, and major histocompatibility complex class II molecules. Surface antigen expression can be followed by flow cytometry using monoclonal antibodies in either cell function assays using donor-specific or nonspecific stimulation of isolated cells or whole blood and without stimulation on circulating lymphocytes. Molecules such as CD30 can be proteolytically cleaved off the surface of activated cells in vivo, and the determination of the soluble protein (sCD30) in serum or plasma is performed by immunoassays. As promising biomarkers for rejection and long-term transplant outcome, CD28 (costimulatory receptor for CD80 and CD86), CD154 (CD40 ligand), and sCD30 (tumor necrosis factor receptor superfamily, member 8) have been identified. Whereas cell function assays are time-consuming laboratory-developed tests which are difficult to standardize, commercial assays are frequently available for soluble proteins. Therefore, more data from clinical trials have been published for sCD30 compared with the surface antigens on activated T cells. This short review summarizes the association between selected surface antigens and immunosuppression, and rejection in solid organ transplantation.

Characterization of a double-CRD-mutated Gal-8 recombinant protein that retains co-stimulatory activity on antigen-specific T-cell response.

Galectins (Gals) constitute a family of mammalian lectins with affinity for β-galactosides, characterized by the presence of conserved CRDs (carbohydrate-recognition domains). We have found previously that Gal-8, from the tandem-repeat group with two linked CRDs, exerts two separate actions on CD4(+)T-cells: antigen-independent proliferation and, at lower concentration, antigen-specific co-stimulation. Whereas proliferation can be ascribed to the pro-inflammatory role of Gal-8, the co-stimulatory activity of borderline T-cell-specific responses allows the proposal of Gal-8 as an adjuvant in vaccination. To study the relevance of glycan-lectin interaction to these T-cell activities, we generated a double-mutated protein (Gal-8mut) by replacing canonical arginine residues on each CRD, so as to abolish sugar-binding capacity. As expected, Gal-8mut was unable to bind to lactosyl-Sepharose, confirming that lactose recognition was precluded; however, preservation of lectin activity was still evident since Gal-8mut displayed haemoagglutinatory effects and binding capacity to the T-cell surface. To search for glycan affinity, a glycan microarray analysis was conducted which revealed that Gal-8mut lost most low- and intermediate-, but retained high-, affinity interactions, mainly to polylactosamines and blood group antigens. These findings were supported further by molecular modelling. Regarding biological activity, Gal-8mut was unable to induce T-cell proliferation, but efficiently co-stimulated antigen-specific responses, bothin vitroandin vivo.Therefore Gal-8mut represents a useful tool to dissect the specificities of lectin-glycan interactions underlying distinctive Gal-8 activities on T-cell biology. Moreover, given its distinguishing properties, Gal-8mut could be used to enhance borderline immune responses without the non-specific pro-inflammatory activity or other potential adverse effects.

Preclinical Advances in Combined-Modality Cancer Immunotherapy With Radiation Therapy

Cancer immunotherapy as a field seeks to exploit the pathways by which tumors and their microenvironment escape detection and elimination by the host immune system. Cancer cells have demonstrated a variety of ways of escaping immune detection, including decreased presentation of tumor-specific antigens, release of immunosuppressive cytokines, and activation of T-cell inhibitory pathways. Interventions against the latter mechanism have demonstrated the greatest potential of cancer immunotherapy to date. The cytotoxic T-lymphocyte antigen 4 (CTLA4) and programmed cell death 1 receptor (PD-1) are T-cell surface receptors that, when activated by their respective ligands, serve to suppress T-cell functions by separate pathways. Anti-CTLA4 (ipilimumab) and anti-PD1 (nivolumab) have been developed as immune checkpoint inhibitors and approved by the US Food and Drug Administration for clinical use in the treatment of advanced melanoma (ipilimumab and nivolumab) and metastatic squamous non-small cell lung cancer (nivolumab). The finding that dual immune checkpoint blockade in patients with metastatic melanoma prolongs overall survival and induces greater responses over monotherapy advances the promise of immunotherapy as a treatment modality (1). Radiation therapy has long been used as a local treatment modality in cancer care. The cytotoxic effect of radiation results in release of tumor-associated antigens and damage-associated molecular patterns that can produce an immunogenic response, which in some cases can produce an abscopal effect. The earliest report of an abscopal effect, defined as an “effect of radiation that is manifested at a distance from the volume of tissue which is directly irradiated,” listed in PubMed.gov was authored by Law and Mole in 1961 (2). The earliest evidence that we are aware of that abscopal effects involved immune responses was by Mikuriya et al (3), who found that large doses of ionizing radiation could be leveraged into a systemic antitumor response. The initial report by Postow

Abstract B101: Engineered PD-1 variants as immunotherapies for cancer

Abstract Immunotherapies represent the next generation of treatments for cancer. The recent clinical successes of immunotherapies have highlighted the potential to achieve stunning efficacy against otherwise intractable cancers. One such clinically validated immunotherapy target is the T-cell surface receptor PD-1 (programmed cell death protein 1), which inhibits T-cell cytotoxicity upon ligation with PD-L1 (programmed death-ligand 1) on a target cell. Antibody therapies targeting PD-1/PD-L1 can be strikingly effective cancer treatments, as demonstrated in the clinic by the recently approved pembrolizumab and nivolumab. However, antibody therapies have significant drawbacks as agents against this pathway. Due to their large size (~150 kDa), antibodies have limited tissue penetrance. Furthermore, due to the presence of the prophagocytic Fc domain, antibodies bound to PD-1 or PD-L1 on the T-cell cause counterproductive lymphocyte depletion. We hypothesized that a small engineered protein capable of blocking PD-1:PD-L1 signaling could avoid these issues and function as a superior immunotherapy. To accomplish this goal, we used yeast display-based directed evolution to affinity-mature a soluble form of the PD-1 ectodomain to increase its affinity for human PD-L1. After several rounds of selection from two generations of mutant libraries, we identified a high-affinity PD-1 variant. By surface plasmon resonance, high-affinity PD-1 binds to human PD-L1 with 110 pM affinity and a half-life of binding of >40 minutes, a dramatic improvement from wild-type PD-1. Direct in vivo binding assays demonstrate that this small protein penetrates solid tissues much more effectively than an anti-PD-L1 monoclonal antibody. Unlike anti-PD-L1 antibodies, high-affinity PD-1 does not lead to the depletion of effector T-cells. Consistent with these advantages, in syngeneic CT26 tumor models, high affinity PD-1 was effective in treating both small (~50 mm3) and large tumors (>150 mm3), whereas the activity of anti-PD-L1 antibodies was completely abrogated against large tumors. Furthermore, radiolabeling of high-affinity PD-1 enabled us to utilize our reagent as a non-invasive tracer for PET imaging. Taken together, our data demonstrates the benefits of using small, non-antibody therapeutics as cancer immunotherapy and immune diagnostic agents. Citation Format: Sydney R. Gordon, Roy Maute, Aaron Mayer, Melissa McCracken, Arutselvan Natarajan, Nan Guo, Richard Kimura, Jonathan M. Tsai, Aashish Manglik, Andrew Kruse, Sanjiv Gambhir, Irving L. Weissman, Aaron M. Ring. Engineered PD-1 variants as immunotherapies for cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B101.

IL-25/IL-33–responsive TH2 cells characterize nasal polyps with a default TH17 signature in nasal mucosa

BackgroundChronic rhinosinusitis with nasal polyposis (CRSwNP) in Western countries is characterized by eosinophilia, IgE production, and TH2 cytokine expression. Type 2 innate lymphoid cells from polyps produce IL-5 and IL-13 in response to IL-25 and IL-33, although the relevance of this axis to local mucosal T-cell responses is unknown.ObjectiveWe sought to investigate the role of the IL-25/IL-33 axis in local mucosal T-cell responses in patients with CRSwNP.MethodsPolyp tissue and blood were obtained from patients undergoing nasal polypectomy. Control nasal biopsy specimens and blood were obtained from healthy volunteers. Tissue was cultured in a short-term explant model. T-cell surface phenotype/intracellular cytokines were assessed by means of flow cytometry. T-cell receptor variable β-chain analysis was performed with the immunoSEQ assay. Microarrays were performed for gene expression analysis.ResultsIL-25 receptor (IL-17RB)–expressing TH2 effector cells were identified in nasal polyp tissue but not the healthy nasal mucosa or periphery. IL-17RB+CD4+ polyp–derived TH2 cells coexpressed ST2 (IL-33 receptor) and responded to IL-25 and IL-33 with enhanced IL-5 and IL-13 production. Within IL-17RB+CD4+ T cells, several identical T-cell receptor variable β-chain complementarity-determining region 3 sequences were identified in different subjects, suggesting clonal expansion driven by a common antigen. Abundant IL-17–producing T cells were observed in both healthy nasal mucosal and polyp populations, with TH17-related genes the most overexpressed compared with peripheral blood T cells.ConclusionIL-25 and IL-33 can interact locally with IL-17RB+ST2+ polyp T cells to augment TH2 responses in patients with CRSwNP. A local TH17 response might be important in healthy nasal mucosal immune homeostasis.

Safety, Maximum Tolerated Dose and Immunogenicity of CMV-MVA-Triplex in Healthy Volunteers with or without Prior Immunity to CMV and Vaccinia

In the era of preemptive antiviral therapy, cytomegalovirus (CMV) still remains the cause of major health complications, profound defects in immune reconstitution, and significant morbidity in post-transplant recovery of immune-compromised HCT recipients. Substituting antivirals with a vaccine that harnesses the native immune response to CMV may improve outcomes for HCT recipients.Modified Vaccinia Ankara (MVA), a vaccinia virus being investigated as a smallpox vaccine by the Defense Department, is a safe and robust delivery system to treat or prevent a wide range of diseases including HIV and cancer. Replication-defective MVA is safe, well tolerated and strongly immunogenic when given to HCT recipients or AIDS patients. We developed a multiple-antigen recombinant MVA with genes encoding 3 immunodominant CMV proteins: UL83 (pp65), UL123 (IE1-exon4), and UL122 (IE2-exon5) (CMV-MVA-Triplex), and established its pre-clinical safety and immunogenicity using humanized HLA transgenic mouse models and human PBMC from CMV-seropositive healthy volunteers and HCT recipients.Defining safety, persistence of the virus, maximum tolerated dose and immunogenicity of CMV-MVA-Triplex in healthy volunteers is a critical first step in its clinical development for HCT recipients as required by the FDA. In a Phase I trial (NCT01941056), these endpoints were evaluated in 24 healthy volunteers (age: 18-60), with or without prior immunity to CMV and vaccinia. Three escalating dose levels (DL) were administered intramuscularly (DL1=10xE7; DL2=5x10E7; DL3=5x10E8 pfu/dose) in 8 subjects/DL, with a booster injection 28 days later, and follow up for 1 year.As of July 2015, all 24 planned volunteers were enrolled, vaccinated and followed for at least 4 months. Vaccinations at all DL were well-tolerated, with only a few expected injection reactions and no SAE or dose limiting toxicities. Immunogenicity of the vaccine was evaluated by measuring the levels of the CD137 T-cell surface marker representing functional activation of PBL harvested from vaccinees and stimulated 24 hours with full-length pp65, IE-1 and IE2 overlapping peptide libraries, or direct measurement of CMV-specific T-cells using HLA multimers. CMV-MVA-Triplex induced robust expansion of pp65-, IE1- and IE2-specific CD8 and CD4 T-cells in vaccinated CMV-seropositives, at each DL (Cf. plot showing geometric mean with upper lower/limits of the pp65 T-cell levels for DL2 cohort). HLA multimers identified CMV-specific T-cells whose expansion closely followed CD137-activated CMV-specific T-cells in vaccinees with common HLA alleles which have a corresponding known CMV-CTL epitope (data to be presented).A statistical analysis performed using generalized estimated equations indicated that the post-vaccination levels of pp65-, IE1- or IE2-specific CD8 and CD4 T-cells were significantly increased, with p-values ranging from 3x10-5 to 0.025. For example, the pre-/post-vaccination median pp65-reactive CD4+ CD137+ T-cells rose from 1.3 to 4.4 cells/µL (p=3x10-5); and pp65-reactive CD8+ CD137+ T-cells rose from a median of 0.22 to 3.1 cells/µL (p=0.003). Importantly, robust immunity was detected in CMV-seronegatives (as shown in the plot for UPN 14 and 18), as well as in subjects who had received smallpox vaccinations. Elevated frequencies of CMV-specific CD4 and CD8 T cells for all 3 antigens plateaued after day 56, but in most cases remained elevated up to one year post-vaccination (data to be presented). Circulating MVA vector in blood was assessed by real-time PCR post-injection and showed only minimal residual vector DNA [10-30 gc/mL] in just 2 vaccinees in the DL3 cohort that disappeared within 3 months. These results provide evidence that CMV-seropositive HCT recipients, whether they receive stem cell product from CMV-seropositive or -seronegative donors could respond to CMV-MVA-Triplex by generating protective CMV-specific immunity.CMV-MVA-Triplex is the first vaccine against CMV that uses a recombinant MVA incorporating multiple CMV antigens, developed for HCT recipients, who are at risk for CMV reactivation. The safety and marked immunogenicity of CMV-MVA-Triplex in this Phase I trial, warrant testing of the vaccine in the HCT setting. A Phase 2 multicenter, placebo-controlled trial to assess CMV-MVA-Triplex in CMV seropositive recipients, receiving HCT from matched related or unrelated donors will start in Fall 2015. [Display omitted] [Display omitted] DisclosuresDiamond:Helocyte, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Co-Expression of NUP98-HOXD13 and Mutant IDH2 Triggers an Early T-Cell Precursor-like Leukemia in Mice

Background and hypothesis: Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy, comprising of B lineage ALL and T-cell lineage ALL (about 85% and 15%, respectively). In most cases, treatment regimens for ALL patients have proved efficient and prognosis is usually encouraging. However, a recently described subgroup of T-cell ALL patients have been found to have a poor prognosis when treated with conventional therapy. These higher risk T-cell leukemias have a cell of origin thought to resemble an early T-cell precursor (ETP), which retains the capability to differentiate into both myeloid or T-lineage cells, and are therefore termed ETP ALL. ETP ALL is characterized by lack of or low expression of typical T-cell surface markers, aberrant expression of myeloid and hematopoietic stem cell (HSC) surface markers and a gene expression signature resembling that of murine ETP cells. Recently, ETP ALL patients were found to have a unique mutational landscape that includes acquired mutations in genes typically associated with myeloid differentiation. The ambiguous lineage attributes of ETP ALL may explain the ineffectiveness of current therapies, stressing the need for establishing pre-clinical animal models. Here we show that co-expression of Nup98-Hoxd13 (NHD13) fusion gene and a mutant IDH2 gene (IDH2 R140Q) triggers an ETP-like leukemia in mice. Expression of NHD13 has been reported in MDS and AML patients, and NHD13 mice have been previously shown to develop MDS, AML, and T-cell ALL. Mutations in IDH1/2 genes occur in several cancers, including AML; the most common IDH mutation in AML patients is the IDH2 R140Q. However, expression of mutant IDH1/2 in mice has been reported to induce an expansion of the HSC compartment but was not sufficient for establishment of leukemia. Targeted sequencing of NHD13 mice has identified recurrent mutations in IDH1, suggesting that these two aberrations may collaborate to cause leukemia.Study Design and Methods: IDH2 R140Q mice were crossed with NHD13 mice and the progeny were monitored for survival. Classification of leukemic samples was done using flow cytometry and PCR analysis of TCRβ gene rearrangements. Global expression and mutational patterns of IDH2/NHD13 leukemias were analyzed using gene chip arrays and whole exome sequencing (WES).Results and conclusions: Transgenic IDH2/NHD13 mice showed decreased survival compared to all control groups. Detailed analysis of surface marker expression revealed that most double Tg cases (10/11 tested) developed leukemias resembling ETP (cKit+CD44+CD25-) or double negative (DN) 2 cells (cKit+CD44+CD25+). PCR analysis of the TCRβ locus revealed that unlike NHD13 T-cell leukemias, IDH2/NHD13 leukemias do not have clonal VDJ rearrangements. However, most of IDH2/NHD13 leukemias have clonal or oligoclonal DJ rearrangements of the TCRβ locus, suggesting that these leukemias emerged during an early stage in T cell development. Expression analysis revealed that the IDH2/NHD13 leukemia expression signature is enriched in genes that are upregulated in ETP or DN2 cells. In addition, we found that the IDH2/NHD13 leukemia expression signature is also enriched in genes uniquely expressed in ETP ALL patients. Finally, WES showed correspondence between genes mutated in the mouse ETP-like leukemia and genes recurrently mutated in ETP ALL patients. These results indicate that IDH2/NHD13 leukemias resemble ETP ALL in terms of gene expression, immunophenotype, and cooperative mutations.Relevance and importance: The poor prognosis of ETP ALL may be related to incomplete effectiveness of treatment strategies, emphasizing the need for novel therapies. IDH2/NHD13 mice can serve as an excellent pre-clinical model to study ETP ALL and to test potential treatments. DisclosuresAplan:NIH Office of Technology Transfer: Patents & Royalties.

Results from a Phase 1/2, Open-Label, Dose-Finding Study of Pralatrexate and Oral Bexarotene in Patients with Relapsed/Refractory Cutaneous T-Cell Lymphoma

Background: Cutaneous T-cell Lymphomas (CTCL) include several variants of extra-nodal non-Hodgkin's lymphomas characterized by their skin lesions and T-cell surface markers. The most common, mycosis fungoides (MF), often has an indolent course but can transform and rapidly disseminate. While skin-directed therapies are effective for early-stage MF, patients with refractory, transformed or late-stage disease require systemic therapy.Methods: Adult patients with CTCL including MF, transformed MF (T-MF), Sézary syndrome (SS), primary cutaneous anaplastic large cell lymphoma (ALCL), ≥ 1 prior systemic therapy, and an Eastern Cooperative Oncology Group performance status (ECOG PS) ≤ 2 were eligible and signed consent. FOL was administered weekly via intravenous push for 3 of 4 weeks; BEX was self-administered orally with food. The standard 3 + 3 dose-escalation design was used to determine the maximum tolerated dose (MTD), with cohort expansion for dose-limiting toxicities (DLTs). The MTD was the highest dose with < 33% DLT incidence in any cohort. DLTs included the following in Cycle 1: ≥ Grade 3 neutropenia (or G-CSF administered), thrombocytopenia, treatment-related non-hematologic toxicity, hyperlipidemia, or hypothyroidism, and treatment-related adverse events (AEs) causing BEX dose omission for ≥10/28 days or FOL dose omission/reduction.Results: In Cohort 1 (15 mg/m2 FOL + 150 mg/m2 BEX), 0/3 patients had DLTs. In Cohort 2a (15 mg/m2 FOL + 300 mg/m2 BEX), 2/3 patients had DLTs: Grade 3 neutropenia and Grade 2 hypotension (n = 1) and Grade 4 neutropenia and thrombocytopenia (n = 1). Therefore, the combination MTD was identified as 15 mg/m2 + 150 mg/m2. An additional 28 patients were enrolled at the MTD for a total of 34 patients (53% male, 47% female) in the study; all have discontinued treatment. The median age was 66 (range 39-85) years, and the median number of prior therapies was 4 (range 2-14). Histology included MF (53%), T-MF (32%), SS (12%), and ALCL (3%). ECOG PS was 0 (68%), 1 (18%), or 2 (15%). Patients received a median of 6 (range 1-33) cycles of therapy.All patients reported ≥ 1 AE. The most common (>20%) FOL-related AEs were mucositis or mucosal inflammation (68%), fatigue (41%), neutropenia and nausea (32% each), and anemia (24%). The most common (>20%) BEX-related AEs were hypertriglyceridemia (56%), fatigue (44%), neutropenia (32%), nausea (26%), and uncorrected hypothyroidism (24%). Grade 3 and 4 AEs were reported for 79% and 9% of patients, respectively: neutropenia (29% and 6%), hypertriglyceridemia (29% and 0%), and stomatitis (21% and 0%) and one Grade 5 AE of respiratory failure. Serious adverse events (SAEs) were reported for 35% of all patients; SAEs in > 1 patient were neutropenia and hypotension (6% each). Dose omissions and reductions for AEs were required for 74% and 21% of patients, respectively. AEs led to discontinuation for 32% of all patients, most commonly stomatitis (9%) and anemia, fatigue, and neutropenia (6% each).The overall Objective Response Rate was 61% (20/33 evaluable patients); 18/30 [60%] in Cohort 1 and 2/3 [67%] in Cohort 2. Four [12%] patients had a complete response, 16 [48%] partial response, 11 [33%] stable disease, and 2 [6%] progressive disease. Duration of Response ranged from 0-29+ months; median Response Duration was not reached, as 14 patients (3 CRs, 11 PRs) remained in response. Median PFS at the MTD was 12.8 (range 0.5-29.9+) months.Table 1Response by CTCL SubtypeObjective Response RatesCTCL SubtypeOverallCRPRAll Evaluable, n=3320 (61%)416ALCL, n = 11 (100%)10MF, n = 1912 (63%)210T-MF, n = 105 (50%)14SS, n = 32 (67%)02Conclusions: The combination of FOL + BEX was well tolerated and efficacious in patients with various CTCL subtypes, as measured by response rates (61% including patients with durable CRs) in this dose-finding study. DisclosuresDuvic:Celgene: Membership on an entity's Board of Directors or advisory committees; Therakos: Research Funding, Speakers Bureau; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kyowa Hakko Kirin, Co: Membership on an entity's Board of Directors or advisory committees, Research Funding; Array Biopharma: Consultancy; Allos (spectrum): Research Funding; Rhizen Pharma: Research Funding; Spatz Foundation: Research Funding; Oncoceutics: Research Funding; Innate Pharma: Research Funding; Tetralogics SHAPE: Research Funding; Cell Medica Ltd: Consultancy; Soligenics: Research Funding; Eisai: Research Funding; MiRagen Therapeutics: Consultancy; Huya Bioscience Int'l: Consultancy. Bhat:Spectrum Pharmaceuticals, Inc: Employment.

Developing an Immunotherapy Strategy for the Effective Treatment of Oral, Head and Neck Squamous Cell Carcinoma

The recent understanding of the role of tumor-infiltrating lymphocytes in different solid cancers suggests that only patients with evidence of an immune response will have durable responses to therapeutic intervention. Therefore, for more patients to achieve clinical benefit, it is necessary to find effective approaches to prime de novo antitumor immunity. This is particularly true for oral, head and neck squamous cell carcinomas (OHNSCCs), which are known for their immune-suppressive character and for which clinical outcome has not appreciably improved during the past 4 decades. The authors' group is exploring an immunotherapy strategy for the effective treatment of OHNSCC, which uses antibodies that modulate T-cell surface proteins with the use of next-generation vaccines and radiation therapy as an antigen source to prime T-cell response and induce immune-mediated rejection of tumors. The purpose of this review is to describe recent advances in immuno-oncology and the authors' developing therapeutic strategies.

Systemic expression of inflammatory mediators in patients with chronic rhinosinusitis and nasal polyps with and without Aspirin Exacerbated Respiratory Disease

BackgroundSystemic reactions are related to the pathogenesis of Aspirin Exacerbated Respiratory Disease (AERD). With this work we wanted to study the changes in the systemic levels of inflammatory mediators in both baseline and after oral aspirin challenge in patients with and without AERD. MethodsPatients with nasal polyposis and asthma with AERD (n=20) and without (n=18) were orally challenged with aspirin in a single-blind placebo controlled study. Serum samples and urine were collected before and 6h after placebo and aspirin oral challenges. Serum levels of inflammatory mediators were assayed by using the Luminex technology and ELISA. The concentrations of 9-alpha, 11-beta prostaglandin F2, and leukotriene E4 (uLTE4) were measured in urine samples by ELISA. The expression of T-cell surface markers was analyzed in peripheral blood mononuclear cells isolated before and after the challenges. ResultsAERD patients showed significantly higher baseline levels of s-IL-5R-alpha, uLTE4 and percentage of CD4+CD25+CD127pos and CD4+CD45RA−CD45RO+ but decreased levels of TGF-β1 and number of CD4+CD25+CD127neg cells. Aspirin challenge induced the release of uLTE4, IL-6 and increased the number of CD4+CD45RA−CD45RO+ memory T-cells only in AERD patients but failed to reduce the levels of sCD40L as observed in non-AERD subjects. Further, IL-8 and sIL-5R-alpha levels directly correlated with the PD20ASA and the effects of aspirin on IL-6 and number of memory T-cells was more pronounced in subjects showing more strong reaction (bronchial and nasal). ConclusionsAERD patients have a differential baseline inflammatory pattern that supports the role inflammation as underlying mechanism of the disease. Systemic response to oral aspirin challenge was related to an increase in serum IL-6 and the number of circulating memory T-cells in AERD patients.

Prosthetic Antigen Receptors.

Chimeric antigen receptors (CARs) have shown great promise for the immunological treatment of cancer. Nevertheless, the need to genetically engineer a patient's T-cells has presented significant production and safety challenges. To address these issues, we have demonstrated that chemically self-assembled nanorings (CSANs) displaying single chain antibodies can bind to both the CD3 ε subunit of the T-cell-receptor/CD3 complex and the CD22 antigen on malignant B cells such as B-leukemias or lymphomas. We demonstrate that the multivalent and bispecific format allows the antiCD3/antiCD22 CSANs to stably bind to T-cell surfaces for greater than 4 days, while being easily disassembled on the cell membrane by treatment with the nontoxic FDA approved drug, trimethoprim. In the presence of CD22+ Raji cells, T-cells modified with antiCD3/antiCD22 CSANs were shown to selectively up-regulate the production of interleukin-2 (IL-2) and interferon-γ (IFN-γ) and to initiate cytotoxicity. Taken together, our results demonstrate that antiCD3/antiCD22 bispecific CSANs offer a potential alternative to CARs, as prosthetic antigen receptors.

Abstract 4636: Investigation of exome variants associated with overall survival in ovarian cancer

Abstract Many germline genetic variants have been found to be associated with susceptibility to epithelial ovarian cancer (EOC), but currently few associations have been identified for EOC outcomes such as overall survival. In the absence of evidence for common germline markers, it is possible that rare variants not captured in GWAS arrays may be associated with overall survival. We hypothesize that rare variants, either individually or combined across a gene, may be associated with overall survival in EOC. We utilized data from two genotyping projects of the Ovarian Cancer Association Consortium (OCAC) that used commercial exome-based arrays. The primary sample, genotyped on the Affymetrix Axiom Exome Array at 227,892 standard and custom variants, consisted of 14 independent EOC studies (4293 cases; 2257 deaths). The secondary sample, genotyped on the Illumina Infinium HumanExome BeadChip at 114,620 variants, consisted of six additional EOC studies (1744 cases; 1027 deaths). Both sets were analyzed using Cox proportional hazards regression to determine the association of each variant with overall survival time. Meta-analysis was conducted across samples at 73,203 overlapping variants. Gene-level tests of over 18,000 genes were also conducted using burden tests and Sequencing Kernel Association Tests (SKAT) adapted for Cox proportional hazards models. Gene-level tests were conducted separately for the primary and secondary cases and then were meta-analyzed. Models were adjusted for age, site, and three principle components. Six individual variants had meta-analysis p-values &amp;lt; 1.0E-4, five of which were rare (MAF &amp;lt; 0.003). Common variant rs8170 (MAF = 0.2) in BABAM1 encoding BRISC and BRCA1 A complex member 1 - previously reported to be associated with EOC risk_showed evidence of association with overall survival (Pmeta = 1.5E-5) and consistent estimates of effect size across both samples (HR1 = 1.18, HR2 = 1.19). In imputed genotype data from over 18,000 subjects from OCAC genotyped on the COGS array, this association reaches genome-wide significance (HR = 1.15, P = 4.7E-9). Five genes had gene-level meta-analysis p-values &amp;lt; 1.0E-4, including top ranked ATG2B (p = 1.1E-6), an autophagy gene important for apoptosis; zinc finger protein PEG3 (p = 1.8E-5), playing a role in autophagy and down-regulated in EOC; helicase-like transcription factor HLTF (p = 1.9E-5), involved in post-replication DNA repair; mitochondrial fission regulator 1-like gene FAM54B (p = 3.1E-5); and T-cell surface glycoprotein CD1E (p = 8.8E-5). With over 6000 EOC cases in the primary and secondary samples, this is the largest genome-wide assessment of rare variant genetic associations with EOC overall survival. Variant rs8170 in BABAM1, a necessary component of the BRCA1 complex, is associated with overall survival. Single variant and gene-level analyses provide complementary approaches, suggesting additional candidates that warrant follow-up study. Citation Format: Stacey J. Winham, Brooke L. Fridley, Melissa C. Larson, Zachary Fogarty, Andrew Berchuck, Yian Ann Chen, Hui-Yi Lin, Georgia Chenevix-Trench, Jenny Permuth-Wey, Thomas A. Sellers, Ailith Pirie, Ellen L. Goode, Ovarian Cancer Association Consortium. Investigation of exome variants associated with overall survival in ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4636. doi:10.1158/1538-7445.AM2015-4636

Recombinant Leishmania major lipophosphoglycan 3 activates human T-lymphocytes via TLR2-independent pathway

Leishmaniasis is one of the most common infectious diseases transmitted by an obligate intracellular genus Leishmania. As there is no efficient vaccination strategy for leishmaniasis, new immunostimulatory components may enhance protective immune responses against this parasite. Lipophosphoglycan 3 (LPG3) is an essential protein required for LPG assembling. In this study, the ability of recombinant LPG3 (rLPG) and its fragments to activate isolated healthy human T-cells and cytokine secretion was evaluated in vitro. The results showed that rLPG3 and its N-terminal fragment (rNT-LPG3) enhanced expression of CD69 on the surface of T-cells and promoted differentiation of CD4+ T-lymphocytes toward a T-helper 1 (TH1) phenotype, in part, through up-regulation of interferon (IFN)-γ expression in a TLR2-independent manner. These results indicated the protective effects of LPG3 (particularly NT-LPG3 fragment) as a potent immunostimulatory component of leishmania in vaccination against leishmaniasis. Further investigations in in vivo assays are clearly warranted.

Genetic Engineering of T Cells to Target HERV-K, an Ancient Retrovirus on Melanoma.

The human endogenous retrovirus (HERV-K) envelope (env) protein is a tumor-associated antigen (TAA) expressed on melanoma but not normal cells. This study was designed to engineer a chimeric antigen receptor (CAR) on T-cell surface, such that they target tumors in advanced stages of melanoma. Expression of HERV-K protein was analyzed in 220 melanoma samples (with various stages of disease) and 139 normal organ donor tissues using immunohistochemical (IHC) analysis. HERV-K env-specific CAR derived from mouse monoclonal antibody was introduced into T cells using the transposon-based Sleeping Beauty (SB) system. HERV-K env-specific CAR(+) T cells were expanded ex vivo on activating and propagating cells (AaPC) and characterized for CAR expression and specificity. This includes evaluating the HERV-K-specific CAR(+) T cells for their ability to kill A375-SM metastasized tumors in a mouse xenograft model. We detected HERV-K env protein on melanoma but not in normal tissues. After electroporation of T cells and selection on HERV-K(+) AaPC, more than 95% of genetically modified T cells expressed the CAR with an effector memory phenotype and lysed HERV-K env(+) tumor targets in an antigen-specific manner. Even though there is apparent shedding of this TAA from tumor cells that can be recognized by HERV-K env-specific CAR(+) T cells, we observed a significant antitumor effect. Adoptive cellular immunotherapy with HERV-K env-specific CAR(+) T cells represents a clinically appealing treatment strategy for advanced-stage melanoma and provides an approach for targeting this TAA on other solid tumors.