Costimulatory Molecule OX40 Research Articles

Comparison of CD30L and OX40L Reveals CD30L as a Promising Therapeutic Target in Atopic Dermatitis.

Blocking IL-13 is highly efficacious in patients with Th2-biased atopic dermatitis (AD), and recent clinical data have highlighted that targeting the T cell costimulatory molecules OX40 and OX40L (TNFSF4) holds promise for future treatment of AD. We asked whether targeting another T cell costimulatory molecule, CD30L (TNFSF8), might also be a possible treatment option in AD. Single-cell RNA-seq data from human AD skin lesions was analyzed to identify pathogenic IL-13- or IL-22-producing T cells and assess expression of CD30 and its ligand in comparison to OX40 and its ligand. Additionally, a murine model of AD with repetitive exposure to house dust mite allergen was used to compare neutralizing antibodies against CD30L with those against IL-13 or OX40L. Analysis of several scRNA-seq datasets from skin lesions of AD patients showed that transcripts for CD30 or CD30L were found expressed with OX40 or OX40L in the primary T cell populations that also expressed mRNA for IL13 and/or IL22. Suggesting that this could be therapeutically relevant, mice treated prophylactically with a blocking CD30L antibody were protected from developing maximal allergen-induced AD features, including epidermal and dermal thickening, immune cell infiltration, and expression of AD-related genes, similar to mice treated with a blocking IL-13 antibody. Moreover, therapeutic neutralization of CD30L in mice with experimental AD also reduced all of the pathological skin lesion features to a comparable extent as blocking OX40L. These data suggest that targeting the CD30-CD30L axis might hold promise as a future therapeutic intervention in human AD, similar to targeting the OX40-OX40L axis.

Soluble levels of 4-1BB (CD137) and OX40 (CD134) are associated with cancer progression in gastric adenocarcinoma.

Previous studies have demonstrated that soluble forms of T-cell costimulatory molecules 4-1BB (s4-1BB) and OX40 (sOX40) interact with immune cells and may constitute a mechanism of immune evasion by tumors in various cancers. The role of the soluble forms of 4-1BB and OX40 in GC remains unclear. We aimed to examine the association between serum levels of s4-1BB and sOX40 and tumor progression in patients with GC. Between 2017 and 2018, a cross-sectional study was performed with serum samples of 83 GC patients and 20 healthy controls. Patients with stage IV metastatic gastric cancer had significantly higher levels of soluble OX40 in comparison with stage III patients with lymph nodes metastasis (p = 0.0003) and stages I and II patients (p = 0.005), whereas the opposite was found for soluble 4-1BB levels, with lower levels being found in advanced stage III (p = 0.003) compared with initial stages I/II. The sOX40 and s4-1BB-mediated T cell interactions may be involved in antitumor immune responses in GC, possibly favoring tumor escape and progression. Serum levels of sOX40 and s4-1BB are associated with staging in GC and may constitute biomarkers for prognosis, as well as potential targets for immunotherapy.

Inborn errors of immunity underlying defective T-cell memory.

T-cell memory is a complex process not well understood involving specific steps, pathways and different T-cell subpopulations. Inborn errors of immunity (IEIs) represent unique models to decipher some of these requirements in humans. More than 500 different IEIs have been reported to date, and recently a subgroup of monogenic disorders characterized by memory T-cell defects has emerged, providing novel insights into the pathways of T-cell memory generation and maintenance, although this new knowledge is mostly restricted to peripheral blood T-cell memory populations. This review draws up an inventory of the main and recent IEIs associated with T-cell memory defects and their mice models, with a particular focus on the nuclear factor kappa B (NF-κB) signalling pathway, including the scaffold protein capping protein regulator and myosin 1 linker 2 (CARMIL2) and the T-cell co-stimulatory molecules CD28 and OX-40. Besides NF-κB, IKZF1 (IKAROS), a key transcription factor of haematopoiesis and STAT3-dependent interleukin-6 signals involving the transcription factor ZNF341 also appear to be important for the generation of T cell memory. Somatic reversion mosaicism in memory T cells is documented for several gene defects supporting the critical role of these factors in the development of memory T cells with a potential clinical benefit. Systematic examination of T-cell memory subsets could be helpful in the diagnosis of IEIs.

Costimulatory Molecules OX40 and OX40L Upregulation in Oral Squamous Cell Carcinoma: A Blood-Based Study.

This research aimed to determine OX40 and OX40L mRNA expression in blood samples of naive oral squamous cell carcinoma (OSCC) patients in different histological grades and clinical stages. The in silico analysis was performed using the STRING database for functional association and a better understanding of the interactions of OX40 and its ligand with other proteins. In this study, we recruited 141 newly diagnosed patients of OSCC. Levels of OX40 and OX40L mRNA expression were explored using real-time quantitative polymerase chain reaction. An in silico tool was also utilized to evaluate the OX40/OX40L interactome. The results showed higher OX40 expressional levels in the late stage (23-fold) compared with the early stage (8.5-fold) (p = < 0.001). A similar trend was seen in OX40L mRNA expression, revealing a fold change of 5.8 in the early stage in comparison to 9.9-fold change in the late stage (p = < 0.001). Overexpression of OX40 and OX40L was found in different histological grades (p = 0.005 and p = < 0.001, respectively). Overexpression of OX40 and OX40L was detected in habits such as smoking and paan intake, whereas statistically significant upregulation was observed in the cheek, lip, and alveolus tumors. However, there was no substantial difference in OX40 and OX40L expression based on age or gender. The functional interactions, that is, interactomes of OX40 and OX40L with other proteins have been determined by in silico analysis. Based on current study findings, despite OX40 and OX40L upregulation in newly diagnosed OSCC patients, it is speculated that the physiological function of these molecules is altered due to immune system exhaustion.

A novel coculture system for assessing respiratory sensitizing potential by IL-4 in T cells.

Although several in vitro assays that predict the sensitizing potential of chemicals have been developed, none can distinguish between chemical respiratory and skin sensitizers. Recently, we established a new three-dimensional dendritic cell (DC) coculture system consisting of a human airway epithelial cell line, immature DCs derived from human peripheral monocytes, and a human lung fibroblast cell line. In this coculture system, compared to skin sensitizers, respiratory sensitizers showed enhanced mRNA expression in DCs of the key costimulatory molecule OX40 ligand (OX40L), which is important for T helper 2 (Th2) cell differentiation. Herein, we established a new two-step DC/T cell coculture system by adding peripheral allogeneic naïve CD4+ T cells to the DCs stimulated in the DC coculture system. In this DC/T cell coculture system, model respiratory sensitizers, but not skin sensitizers, enhanced mRNA expression of the predominant Th2 marker interleukin-4 (IL-4). To improve the versatility, in place of peripheral monocytes, monocyte-derived proliferating cells called CD14-ML were used in the DC coculture system. As in peripheral monocytes, enhanced mRNA expression of OX40L was induced in CD14-ML by respiratory sensitizers compared to skin sensitizers. When these cell lines were applied to the DC/T cell coculture system with peripheral allogeneic naïve CD4+ T cells, respiratory sensitizers but not skin sensitizers enhanced the mRNA expression of IL-4. Thus, this DC/T cell coculture system may be useful for discriminating between respiratory and skin sensitizers by differential mRNA upregulation of IL-4 in T cells.

Combined Treatment With H1 and H4 Receptor Antagonists Improves Th2 Inflammatory Responses in the Nasal Mucosa of Allergic Rhinitis Rats.

Histamine H1 receptor (H1R) antagonists are the first-line drugs for the treatment of allergic rhinitis (AR) at present. Emerging evidence supports an important role of histamine H4 receptor (H4R) in allergic diseases. However, information regarding the effects of combined treatment with H1 and H4 receptor antagonists in AR is limited. We aimed to assess the effects of combined treatment with H1R and H4R antagonists on Th2 inflammatory responses in the nasal mucosa of AR rats. Sprague Dawley rats were sensitized with ovalbumin and treated with H1R antagonist desloratadine or/and H4R antagonist JNJ7777120. Western blotting was used to assay the phenotypic markers of mature dendritic cells in the nasal mucosa, including major histocompatibility complex class II (MHC-II) and co-stimulatory molecules CD80, CD86 and OX40 ligand (OX40L). Th2 inflammatory cytokines including interleukin-4, 5 and 13 in nasal lavage fluids were determined by using enzyme-linked immunoassay. The treatment with desloratadine alone down-regulated the CD86 expression, and decreased the production of Th2 cytokines, but had no impact on the expression of MHC-II, CD80 and OX40L. The administration of NJ7777120 alone reduced the levels of CD86, OX40L and Th2 cytokines, whereas MHC-II and CD80 expression was unaffected. The combination of desloratadine and JNJ7777120 showed more significant synergistic therapeutic effects than monotherapy. H4R antagonist acted synergistically with H1R antagonist to reduce Th2 inflammatory responses by down-regulating CD86 and OX40L expression in the nasal mucosa of AR rats. The combination with H1R and H4R antagonists might be a new strategy for AR treatment.

CD137 agonist-based combination immunotherapy enhances activated, effector memory T cells and prolongs survival in pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma(PDAC) is resistant to the PD-1/PD-L1 blockade therapy. Previously, the combination of PD-1 blockade and vaccine therapy was shown to have a modest antitumor activity in murine models of PDAC. We used a murine syngeneic model of metastatic PDAC to identify, among multiple T cell modulators tested, which therapeutic agents in combination with the GVAX cancer vaccine and an anti-PD-1 antagonist antibody(αPD-1) are able to improve the survival. We found that an anti-CD137 agonist antibody(αCD137) most significantly improved survival in the mouse PDAC model. Moreover, αPD-1 and αCD137 together in combination with vaccine therapy more significantly increased the expression of costimulatory molecules CD137 and OX40 on CD4+PD-1+ and CD8+PD-1+ T cells comparing to αPD-1 or αCD137, respectively, suggesting that T cell activation within PDACs were enhanced by a synergy of αCD137 and αPD-1. On another hand, αCD137 treatment led to an increase in effector memory T cells independent of αPD-1. Although αCD137 does not increase the cytotoxic effector T cell function, the addition of αCD137 to GVAX+αPD-1 increased expression of IFNγ in EOMES+exhausted tumor-infiltrating T cells. Taken together, this preclinical study established the mechanism of targeting CD137 to enhance effector memory and activated T cells in PDAC. Immunohistochemistry analysis of resected human PDACs following the neo-adjuvant GVAX treatment showed increased levels of CD8+ T cells in those with high levels of CD137 expression, supporting an ongoing clinical trial of testing CD137 as a potential target in treating PDACs that are inflamed with T cells by vaccine therapy.

THU126 - Co-stimulatory molecules OX40 and CD30 mediates the regeneration of the liver epithelium in a mouse primary scletosing cholangitis model

THU126 - Co-stimulatory molecules OX40 and CD30 mediates the regeneration of the liver epithelium in a mouse primary scletosing cholangitis model

Profiles of expression of costimulatory molecules OX40 and OX40l in PBMCs and levels of soluble OX40/OX40l in plasma in chronic hepatitis B patients and health subjects

Profiles of expression of costimulatory molecules OX40 and OX40l in PBMCs and levels of soluble OX40/OX40l in plasma in chronic hepatitis B patients and health subjects

Targeting MerTK Enhances Adaptive Immune Responses After Radiation Therapy

The role of MerTK, a member of the Tyro3-Axl-MerTK family of receptor tyrosine kinase, in the immune response to radiation therapy (RT) is unclear. We investigated immune-mediated tumor control after RT in murine models of colorectal and pancreatic adenocarcinoma using MerTK wild-type and knock-out hosts and whether inhibition of MerTK signaling with warfarin could replicate MerTK knock-out phenotypes. Wild-type and MerTK-/- BALB/c mice were grafted in the flanks with CT26 tumors and treated with computed tomography guided RT. The role of macrophages and CD8 T cells in the response to radiation were demonstrated with cell depletion studies. The role of MerTK in priming immune responses after RT alone and with agonist antibodies to the T cell costimulatory molecule OX40 was evaluated in a Panc02-SIY model antigen system. The effect of warfarin therapy on the in-field and abscopal response to RT was demonstrated in murine models of colorectal adenocarcinoma. The association between warfarin and progression-free survival for patients treated with SABR for early-stage non-small cell lung cancer was evaluated in a multi-institutional retrospective study. MerTK-/- hosts had better tumor control after RT compared with wild-type mice in a macrophage and CD8 T cell-dependent manner. MerTK-/- mice showed increased counts of tumor antigen-specific CD8 T cells in the peripheral blood after tumor-directed RT alone and in combination with agonist anti-OX40. Warfarin therapy phenocopied MerTK-/- for single-flank tumors treated with RT and improved abscopal responses for RT combined with anti-CTLA4. Patients on warfarin therapy when treated with SABR for non-small cell lung cancer had higher progression-free survival rates compared with non-warfarin users. MerTK inhibits adaptive immune responses after SABR. Because warfarin inhibits MerTK signaling and phenocopies genetic deletion of MerTK in mice, warfarin therapy may have beneficial effects in combination with SABR and immune therapy in patients with cancer.

New pathways in immune stimulation: targeting OX40

Immune checkpoint blockers (ICB) reinvigorate the immune system by removing the molecular brakes responsible for the scarce activity of immune phenotypes against malignant cells. After having proven their remarkable role as monotherapy, combinations of anti-Programmed cell death 1 (PD-1)/Programmed death-ligand 1 (PD-L1) agents with cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4) antibodies, chemotherapy and/or anti-angiogenic compounds provide unprecedented results and durable responses across a variety of tumour types. Nevertheless, the main drawbacks of ICB are represented by primary and acquired resistance, translating into disease progression, as well as by immune-related toxicities. In this sense, novel strategies to foster the immune system through its direct stimulation are being tested in order to provide additional clinical improvements in patients with cancer. In this scenario, the co-stimulatory molecule OX40 (CD134) belongs to the next generation of immune therapeutic targets. Preliminary results of early clinical trials evaluating OX40 stimulation by means of different agents are encouraging. Here we review the rationale of OX40 targeting, highlighting the combination of OX40-directed therapies with different anticancer agents as a potential strategy to foster the immune system against malignant phenotypes.

From the Editor’s desk…: June 2019

From the Editor’s desk…: June 2019

OX40 stimulation and PD-L1 blockade synergistically augment HBV-specific CD4 T cells in patients with HBeAg-negative infection

Current antiviral therapies lack the potential to eliminate persistent hepatitis B virus (HBV) infection. HBV-specific T cells are crucial for HBV control and have recently been shown to be protective in patients following discontinuation of antiviral therapy. Thus, T cell-based approaches may greatly improve the therapeutic landscape of HBV infection. We aimed to augment HBV-specific CD4 T cells from chronically infected patients by targeting different immunological pathways. Expression of various co-stimulatory and inhibitory receptors on HBV- and influenza-specific CD4 T cells was analyzed directly ex vivo by MHC class II-tetramers. Patients infected with HBV genotype D were screened for CD4 T cell responses by IFN-γ ELISpot and intracellular cytokine staining following stimulation with overlapping peptides (OLPs) spanning the HBV-polyprotein. Stimulation with recombinant IL-7, an agonistic OX40-antibody or blockade of PD-L1 was performed in antigen-specific in vitro cultures. Cytokine secretion and expression of transcription factors were analyzed by flow cytometry. Responses targeting influenza, Epstein-Barr virus and tetanus toxoid served as controls. Tetramer-staining revealed that the IL-7 receptor-alpha (CD127), OX40 and PD-1 constitute possible therapeutic targets as they were all strongly expressed on HBV-specific CD4 T cells ex vivo. The HBV-specific CD4 T cell responses identified by OLP screening targeted predominantly the HBV-polymerase and core proteins. Combined OX40 stimulation and PD-L1 blockade significantly augmented IFN-γ and IL-21 producing HBV-specific CD4 T cells in vitro, suggesting active T helper type 1 cell and follicular T helper cell programs. Indeed, transcription factors T-bet and Bcl6 were strongly expressed in cytokine-producing cells. Combined OX40 stimulation and PD-L1 blockade augmented secretion of the helper T cell signature cytokines IFN-γ and IL-21, suggesting that immunotherapeutic approaches can improve HBV-specific CD4 T cell responses. CD4 T cells are important in controlling viral infections but are impaired in the context of chronic hepatitis B virus (HBV) infection. Therapeutic approaches to cure chronic HBV infection are highly likely to require an immune-stimulatory component. This study demonstrates that HBV-specific CD4 T cells can be functionally augmented by combined stimulation of the co-stimulatory molecule OX40 and blockade of the inhibitory PD-1 pathway.

Late-Stage Tumor Regression after PD-L1 Blockade Plus a Concurrent OX40 Agonist.

The protective capability of tumor antigen-specific T cells is regulated by costimulatory and inhibitory signals. Current approaches in cancer immunotherapy seek to restore the function of unresponsive T cells by blocking inhibitory pathways. In contrast, providing exogenous costimulatory signals to T cells also enhances antitumor functionality. By combining these two clinical approaches, we demonstrate the synergy of targeting PD-L1 together with the costimulatory molecule OX40, to enhance antitumor immunity. Concurrently blocking PD-L1 and providing a costimulatory agonist to OX40 increased the presence and functionality of tumor antigen-specific CD8+ T cells with simultaneous enhancement of T-helper type 1 (Th1)-skewed CD4+ T cells. This shift was functionally supported by increased glucose metabolism of antigen-specific CD8+ T cells and the acquisition of granzyme B by regulatory T cells. Together, this mechanism promoted tumor regression of late-stage tumors beyond that achieved by either blockade as monotherapy. These findings indicate that targeting both T-cell intrinsic (OX40) and extrinsic (PD-L1) regulatory molecules increases the bioenergetic potential of T cells, thereby expanding functional and tumor antigen-specific T cells.

Intraperitoneal neutrophils activated by KRAS-induced ovarian cancer exert antitumor effects by modulating adaptive immunity.

Increased neutrophil counts are a hallmark of a poor prognosis for cancer. We previously reported that KRAS promoted tumorigenesis and increased neutrophil counts in a mouse peritoneal cancer model. In the current study, we evaluated the role of increased neutrophils in cancer progression, as well as their influence on the intraperitoneal microenvironment. A mouse peritoneal cancer model was established using the KRAS-transduced mouse ovarian cancer cell line, ID8-KRAS. Neutrophil function was assessed by neutrophil depletion in ID8-KRAS mice. Neutrophil depletion markedly accelerated tumor formation; this was accompanied by an increase in interleukin-6 concentrations in ascites. Neutrophil depletion significantly decreased the amount of local and systemic CD8+ T cells, while increasing the amount of local CD4+ T cells, accompanied by an increased amount of monocytic myeloid-derived suppressor cells (M-MDSCs) and regulatory T cells (Tregs) (P<0.05). The roles of peritoneal neutrophils (PENs) in CD8+ T cell activation were assessed in vitro. PENs of ID8-KRAS mice had a strong potential to enhance T cell proliferation with a higher expression of the T cell costimulatory molecules OX40 ligand (OX40L) and 4-1BB ligand (4-1BBL), as compared with peripheral blood neutrophils (PBNs). These findings suggest that neutrophils recruited into the KRAS-induced tumor microenvironment (TME) have antitumor properties with the potential to modulate the numbers of M-MDSCs and Tregs and activate CD8+ T cells through T cell costimulatory molecules.

Synergy of TLR4 agonist GSK1795091, an innate immune activator, with agonistic antibody against co-stimulatory immune checkpoint molecule OX40 in cancer immunotherapy.

12055Background: GSK1795091 (aka, GSK’091) is a synthetic glycolipid toll-like receptor 4 (TLR4) agonist. TLR4 is a Pattern Recognition Receptor for host defense against bacterial infection, expres...

Characterisation of chicken OX40 and OX40L

The Tumour Necrosis Factor superfamilies of receptors and ligands play a crucial role in the regulation of effective immune responses against pathogens and malignant cells. In chickens, only few members have been identified. Here, we characterise the chicken homologues for mammalian costimulatory molecules OX40 and OX40L, which are involved in sustaining T cell responses. Both genes were identified by virtue of their genomic localisation close to highly conserved genes and their structural relationship to their mammalian homologues. Following cloning and expression of soluble and cell-associated chicken OX40 and OX40L, we confirmed their mutual interaction via ELISA and flow cytometric analyses. In addition, we showed the application of soluble OX40-Fc in staining of chicken cells. Whereas non-activated cells did not express OX40L, activation by IL-2 and IL-12 resulted in upregulation of OX40L on αβ and γδ T cell populations. Our results demonstrate the existence of the costimulatory OX40-OX40L system in the chicken and provide the basis for further investigations of chicken T cell responses.

Abstract 4598: Triggering of OX40 on T cells by a novel monoclonal antibody elicits robust antitumor immunity&amp;lt;!–EndFragment–&amp;gt;

Abstract T cell costimulation is an attractive strategy for cancer treatment in addition to check point inhibitors. Costimulatory molecule OX40 is a member of the TNF receptor superfamily that is transiently expressed on activated T cells. Activation of OX40 leads to enhanced T cell proliferation and cytokine secretion and in turn results in better anti-tumor efficacy as shown by multiple mouse syngeneic tumor models. PF-04518600 (PF-8600) is a fully human IgG2 agonist antibody to human OX40 with high affinity and specificity. We used in vitro and in vivo methods to evaluate the co-stimulatory functions of PF-8600. PF-8600 demonstrated better agonist activity in a luciferase reporter cell line expressing OX40 and NFκB as compared to a human IgG1 antibody. In human primary T cells, PF-8600 dose dependently increased T cell proliferation and cytokine secretion in vitro. Furthermore in a mouse line expressing human OX40, PF-8600 in a mouse IgG1 framework increased the proliferation of OT1 cells and inhibited EG7 tumor growth as compared to isotype control antibody, further confirming the mechanism of action as an OX40 agonist. As regulatory T cells are a major inhibitory population in the tumor microenvironment, their specific depletion in the tumor is highly desirable. In an in vitro assay using monocyte derived macrophages as effector cells, PF-8600 mediated the depletion of OX40 expressing cells similar to a human IgG1 antibody, and this activity is dependent on the binding to hFcγRIIA. In human FcγR knock-in mice, anti-OX40-hIgG1 showed increased Treg reduction in the tumor comparing to anti-OX40-hIgG2, but less T cell proliferation and activation in the periphery. In combination with an anti-PD-L1 antibody, both anti-OX40-hIgG1 and hIgG2 showed similar enhanced anti-tumor activity in the MC38 colon carcinoma model as comparing to anti-PD-L1 alone. Both in vitro and in vivo results demonstrated PF-8600 enhanced T cells functions and inhibited tumor growth in a mouse syngeneic tumor model. The activity of the hIgG2 antibody is similar to that of an hIgG1 in both phagocytosis assays and hFcγR KI in vivo assays. Based on the mechanism of action and robust anti-tumor efficacy in preclinical models, PF-8600 is currently in clinical development in a broad spectrum of malignancies. Citation Format: Hua Long, Ann White, Jie Wei, Brittany Jiang, Reid Feldman, Danielle Pappas, Aymen Al-Shamkhani, John Lin. Triggering of OX40 on T cells by a novel monoclonal antibody elicits robust antitumor immunity&amp;lt;!–EndFragment–&amp;gt; [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4598. doi:10.1158/1538-7445.AM2017-4598

Abstract 5676: Functional pairing of immunomodulatory targets in anaplastic thyroid cancer

Abstract Thyroid cancer is the most common type of endocrine malignancy that has an escalating global frequency. Although most well differentiated thyroid cancers (WDTC) are manageable and respond to current therapeutic modalities, undifferentiated anaplastic thyroid cancers (ATC) exhibit a dramatically different clinical behavior and poor prognosis. With the recent development of immunotherapies, targeted, well-defined treatment plans can demonstrate promising treatment outcomes in ATC patients. Precise immunological targets in ATCs with potential clinical relevance are unknown. Major progress has been made in last 5 years toward development of immune checkpoint inhibitors using anti-CTLA-4 and anti-PD-1/PD-L1 antibodies for cancer treatment which has made immunotherapies one of the mainstream treatment choices. Few additional members of the immunoglobulin superfamily of receptors, like LAG3, TIM3 and VISTA have recently been identified as potential checkpoint targets. Interestingly some of these molecules including TIM3 and PD-L1 promote tumor progression and immune escape. Identification of specific immunotherapeutic targets requirs a better understanding of the immune microenvironment in ATC. To this end we evaluated the expression of prominent co-stimulatory and co-inhibitory cell surface molecules by RT-PCR in three thyroid cancer cell lines - TPC-1 (papillary), CGTH-W-1 (follicular) and 8505C (anaplastic). We observed that many co-inhibitory molecules were upregulated in all three tumor cell lines. CTLA4, interestingly, had the highest expression in 8505C. Additionally we observed differential expression of BTLA, LAIR1, TIM3 and VISTA between TPC-1 and 8505C. LAG3, PD-1 and PD-L1 were also upregulated in 8505C compared to TPC-1. Similar pattern was observed with the expression of co-stimulatory molecules, CD40L and GITR . GITR has been shown to have a tumor suppressor function in multiple myeloma. Another co-stimulatory molecule OX40, which has shown promise in tumor recession when targeted, was upregulated in all three cell lines and 8505C showed the highest expression. Our findings suggest that the aggressive and less immunogenic phenotype of ATC might be attributed to the differential expression of these molecules. Targeting these immunomodulatory molecules in ATC warrants a better understanding of the crosstalk between them and it might provide an efficient means for the disease management. Citation Format: Sanjukta Chakraborty, Rachana R. Maniyar, Neha Y. Tuli, Ghada Ben Rahoma, Cameron Budenz, Sarnath Singh, Jan Geliebter, Raj Tiwari. Functional pairing of immunomodulatory targets in anaplastic thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5676. doi:10.1158/1538-7445.AM2017-5676

Abstract 1682: Correlation of immune co-stimulatory molecule OX40 and outcome in trastuzumab treated HER2-positive breast cancer patients in the NCCTG-N9831

Abstract Background: Trastuzumab (H), a monoclonal antibody against HER2, has revolutionized the treatment for HER2-positive breast cancer. Besides inhibiting downstream signaling of HER2, several studies showed that H also exerts its anti-tumor activity via immune-related mechanisms. While H is quite effective in preventing recurrence, significant numbers of patients still develop recurrence despite adjuvant H-based chemotherapy. In this study, we analyzed immune-related genes that were associated with poor outcome in N9831. Methods: NanoStringTM technology was used to quantify mRNA of immune-related genes in baseline samples from 1,280 patients in N9831. In N9831, patients in arm A were treated with chemotherapy alone (AC-T), arm B received chemotherapy followed by sequential H (AC-T-H), and arm C received H concurrently with chemotherapy (AC-TH). Cox proportional hazard ratio (HR) was used to determine the association of each immune-related gene with recurrence free survival (RFS). Different immune subset signatures, including CD45, CD8, cytotoxic-cells, and T-cells were analyzed using algorithms developed by NanoString. Results: With the median follow up of 10.6 years, we identified a total 77 genes that were associated with improved outcome in arm C. Among these 77 genes, there were 20 tumor necrosis factor (TNF)-related genes. Of those, only OX40 (TNFRSF4) and its ligand TNFSF4 have interaction p &amp;lt; 0.10. Interestingly, we found uneven distribution of OX40 expression in the N9831 specimens. Approximately, 9.5% of HER2-positive breast cancer expressed OX40 at distinctly low level. Low expression of OX40 was significantly associated with HR positivity (OX40 low 61% vs. OX40 high 51%, p 0.003) and larger tumor size but not patients’ age, tumor grade, and lymph node status. Low expression of OX40 was significantly associated with low mTIL-CD45, CD8, cytotoxic-cells, and T-cells immune signature scores. Tumors with low OX40 expression had significantly lower levels of CTLA4 (p 9.11e-71) and PD-L1 expressions (p 2.49e-92). Low expression of OX40 is associated with poorer outcome among patients treated with H in both sequential (HR 0.88, 95% CI 0.79-0.98, p 0.022) and concurrent arms (HR 0.86, 95%CI 0.76-0.98, p 0.027) but not in chemotherapy only arm (HR 0.9, 95%CI 0.90-1.09, p 0.8). Similar findings were observed with its ligand TNFSF4 in arm A (HR 1.02, 95%CI 0.92-1.15, p 0.68), arm B (HR 0.87, 95%CI 0.76-0.99, p 0.04), and arm C (HR 0.81, 95%CI 0.68-0.95, p 0.01). Conclusion: Our study suggests that pre-existing expression of OX40 and its ligand TNFSF4 are prognostic and may also be predictive of adjuvant H benefit. Patients with distinctly low level of OX40 had poor outcome despite adjuvant H-based adjuvant chemotherapy. Our study provides a rationale to further evaluate the strategy to increase immune activation to improve outcome in this group of patients. Citation Format: Saranya Chumsri, Daniel J. Serie, Afshin Mashadi-Hossein, Sarah Warren, Alvaro Moreno-Aspitia, Geraldo Colon-Otero, Keith L. Knutson, Edith A. Perez, E. Aubrey Thompson. Correlation of immune co-stimulatory molecule OX40 and outcome in trastuzumab treated HER2-positive breast cancer patients in the NCCTG-N9831 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1682. doi:10.1158/1538-7445.AM2017-1682