anti-OX40 Antibody Research Articles

Soluble OX40L favors tumor rejection in CT26 colon carcinoma model

OX40 receptor-expressing regulatory T cells (Tregs) populate tumors and suppress a variety of immune cells, posing a major obstacle for cancer immunotherapy. Different ways to functionally inactivate Tregs by triggering OX40 receptor have been suggested, including anti-OX40 antibodies and Fc:OX40L fusion proteins. To investigate whether the soluble extracellular domain of OX40L (OX40Lexo) is sufficient to enhance antitumor immune response, we generated an OX40Lexo-expressing CT26 colon carcinoma cell line and studied its tumorigenicity in immunocompetent BALB/c and T cell deficient nu/nu mice.We found that soluble OX40L expressed in CT26 colon carcinoma favors the induction of an antitumor response which is not limited just to cells co-expressing EGFP as an antigenic determinant, but also eliminates CT26 cells expressing another fluorescent protein, KillerRed. Tumor rejection required the presence of T lymphocytes, as indicated by the unhampered tumor growth in nu/nu mice. Subsequent re-challenge of tumor-free BALB/c mice with CT26 EGFP cells resulted in no tumor growth, which is indicative of the formation of immunological memory. Adoptive transfer of splenocytes from mice that successfully rejected CT26 OX40Lexo EGFP tumors to naïve mice conferred 100% resistance to subsequent challenge with the CT26 EGFP tumor.

Intratumoral Injection of TLR4 Agonist (G100) Leads to Tumor Regression of A20 Lymphoma and Induces Abscopal Responses

Background: Toll-like receptors (TLRs) are components of the innate immune system that recognize pathogen-associated molecular patterns present on bacterial, fungal, or viral pathogens. Glucopyranosyl lipid A (GLA) is a synthetic TLR4 agonist that activates dendritic cells (DC) and enhances antigen-specific Th1 cell responses.Scientific question: Can an oil-in-water emulsion of GLA (G100) induce an anti-tumor effect due to modulation of the tumor microenvironment?Results: In our A20 murine lymphoma tumor model, intratumoral (i.t) administration of G100 resulted in tumor regression in approximately 50% of the mice in a dose dependent manner with an optimal dose of 10-20 μg/mouse. Surviving mice remained tumor-free at three months post G100 treatment and were resistant to a secondary tumor challenge with A20 cells, but not with CT26 colon cancer cells or 4T1 breast cancer cells. The effect was CD8+ T cell mediated as CD8+ depletion resulted in a significant loss of the effect. To examine the potential systemic effect of G100 i.t. treatment, Balb/c mice were implanted bilaterally with A20 tumors. Injection of G100 into the ipsilateral tumor three times a week resulted in an abscopal effect on the contralaterally implanted, untreated tumor, as evidenced by reduced tumor growth. Furthermore, the potential synergy between G100 and immunomodulatory antibodies was investigated: mice with bilateral A20 tumors received intratumoral G100 alone, systemic anti-PD-1 mAb alone, or a combination of both agents. Mice receiving combination therapy showed best overall survival, as measured by regression or reduced growth of both treated and untreated tumors. In addition, injection of low doses of anti-CTLA4 and anti-OX40 mAbs together with G100, directly into the tumor, was able to cure mice with established disease.Significance: Immunomodulatory antibodies are currently under clinical development for cancer treatment. We show here that combining these antibodies with a TLR4 agonist is sufficient to trigger a systemic anti-tumor response able to eradicate tumor at a distant site. This anti-tumor immune response was long lasting, specific and required CD8+ T cells as the effect was lost in CD8+ T cell depleted mice.Impact: We recently published positive results of intra-tumoral CpG (TLR9 agonist) in combination with immunomodulatory antibodies (Marabelle, Levy, et al. JCI, 2013). Both anti-CTLA4 and anti-PD1 antibodies have been FDA approved, and anti-PD-L1 and anti-OX40 antibodies are currently in advanced clinical trials. Our results suggest that G100 alone or in combination with immunomodulatory mAbs may be a promising new treatment for patients with injectable sites of lymphoma. Clinical studies investigating intratumoral G100 are underway. DisclosuresOff Label Use: G100- a TLR4 agonist. Lu:Immune Design: Employment. Hewitt:Immune Design: Employment. Hsu:Immune Design: Employment. Meulen:Immune Design: Employment. Levy:Immune Design: Research Funding; Dynavax: Research Funding.

OX40 expression enhances the prognostic significance of CD8 positive lymphocyte infiltration in colorectal cancer.

OX40 is a TNF receptor family member expressed by activated T cells. Its triggering by OX40 ligand promotes lymphocyte survival and memory generation. Anti-OX40 agonistic monoclonal antibodies (mAb) are currently being tested in cancer immunotherapy. We explored the prognostic significance of tumor infiltration by OX40+ cells in a large colorectal cancer (CRC) collective. OX40 gene expression was analyzed in 50 freshly excised CRC and corresponding healthy mucosa by qRT-PCR. A tissue microarray including 657 clinically annotated CRC specimens was stained with anti-OX40, -CD8 and -FOXP3 mAbs by standard immunohistochemistry. The CRC cohort was randomly split into training and validation sets. Correlations between CRC infiltration by OX40+ cells alone, or in combination with CD8+ or FOXP3+ cells, and clinical-pathological data and overall survival were comparatively evaluated. OX40 gene expression in CRC significantly correlated with FOXP3 and CD8 gene expression. High CRC infiltration by OX40+ cells was significantly associated with favorable prognosis in training and validation sets in univariate, but not multivariate, Cox regression analysis. CRC with OX40(high)/CD8(high) infiltration were characterized by significantly prolonged overall survival, as compared to tumors with OX40(low)/CD8(high), OX40(high)/CD8(low) or OX40(low)/CD8(low) infiltration in both uni- and multivariate analysis. In contrast, prognostic significance of OX40+ and FOXP3+ cell infiltration was not enhanced by a combined evaluation. Irrespective of TNM stage, CRC with OX40(high)/CD8(high) density infiltrates showed an overall survival similar to that of all stage I CRC included in the study. OX40(high)/CD8(high) density tumor infiltration represents an independent, favorable, prognostic marker in CRC with an overall survival similar to stage I cancers.

Abstract LB-229: Agonistic antibodies to costimulatory molecules, OX40 and GITR, significantly enhance the antitumor efficacy of Listeria monocytogenes (Lm-LLO)-based immunotherapy

Abstract Combinational anti-cancer immune therapies that target multiple tumor-mediated suppressive mechanisms or enhance effector immunity are emerging as promising strategies for cancer treatment. Recently, we demonstrated that the combination of Listeria monocytogenes (Lm-LLO)-based immunotherapy (ADXS11-001) and anti-PD-1 antibody results in improved anti-tumor therapeutic and immune efficacy. Interestingly, regardless of the presence of an antigen or treatment with anti-PD-1 antibody, treatment with the Lm-LLO alone significantly inhibited the suppressive arm of the immune system, including regulatory T cells and myeloid-derived suppressor cells. We therefore hypothesized that combination of ADXS11-001 with agonistic antibodies against co-stimulatory molecules will lead to further improvement of immune and therapeutic efficacy as a result of the combined down-regulation of the suppressive arm and the enhancement of the effector arm. Here we demonstrate that combining Lm-LLO-based immunotherapy with anti-OX40 or anti-GITR antibodies lead to significant inhibition of tumor growth and prolonged survival of animals. The complete regression of established TC-1 tumors occurs in 40% and 60% of animals treated with ADXS11-001 in combination with anti-OX40 and anti-GITR antibodies, respectively. We show that this therapeutic potency enhancement is due to a significant increase in antigen-specific immune responses along with ADXS11-001-mediated inhibition of suppressor cells. Thus, we believe that simultaneous stimulation of T cells while inhibiting the suppressor cells, specifically using Lm-LLO-based immunotherapy combination with agonistic anti-co-stimulatory molecule antibody is a feasible and translatable approach that can lead to overall enhancement of the efficacy of an anti-tumor immunotherapy. Citation Format: Mikayel Mkrtichyan, Rajeev Shrimali, Shamim Ahmad, Rasha Abu Eid, Zuzana Berrong, Anu Wallecha, Robert Petit, Samir N. Khleif. Agonistic antibodies to costimulatory molecules, OX40 and GITR, significantly enhance the antitumor efficacy of Listeria monocytogenes (Lm-LLO)-based immunotherapy. [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 LB-229. doi:10.1158/1538-7445.AM2015-LB-229

Abstract DDT01-02: Unleashing anti-tumor immunity through anti-OX40 monotherapy and in combination with anti-PD-L1

Abstract Cancer immunotherapy has the potential to induce durable responses through the generation of tumor-specific T cell memory. In addition to cognate antigen recognition, T cells require costimulation via stimulatory receptors for optimal memory T cell development and the establishment of anti-tumor immunity. OX40 is a costimulatory molecule that is expressed on activated effector and regulatory T cells. Agonistic antibodies against OX40 are predicted to counteract the immunosuppressive tumor microenvironment and promote T cell-dependent, anti-tumor immunity via two mechanisms: (1) binding to OX40 on antigen-experienced effector T cells increases their proliferation and cytokine production and (2) binding to OX40 on activated regulatory T cell inhibits their suppressive function, which in turn enhances the T cell based immune response. MOXR0916 is a humanized monoclonal antibody that binds to the human OX40 costimulatory receptor in an agonistic fashion. Binding of MOXR0916 to OX40 on activated human T cells in vitro resulted in the costimulation of effector T cell proliferation and IFN-g production, whereas binding to regulatory T cells inhibited their suppressive function. Due to sequence divergence between human and mouse OX40, a murine agonist antibody was employed to assess anti-tumor efficacy and T cell dynamics in murine syngeneic tumor models. In preclinical tumor models, anti-mouse OX40 administration induced a spectrum of anti-tumor activity, including durable responses. Animals with a complete response to anti-OX40 treatment were resistant to tumor rechallenge, indicating generation of tumor-specific memory. Tumor regression in anti-OX40 sensitive tumor models was associated with increased intratumoral expression of IFN-g, granzymes, and perforin at baseline, suggesting that anti-OX40 treatment augments latent immune responses to the tumor. Administration of anti-OX40 in several tumor models results in an increase in intratumoral CD8+ T cells and a decrease in regulatory T cells. Both anti-tumor efficacy and pharmacodynamic modulation of effector and regulatory T cells required antibody crosslinking. Similarly, anti-human OX40 MOXR0916 required antibody crosslinking to costimulate effector T cells. Treatment of agonistic anti-OX40 antibodies was capable of enhancing tumor immunity despite the activity of multiple inhibitory pathways including the PD-1- PD-L1 axis. In preclinical tumor models, the combination of anti-OX40 and a blocking antibody to the inhibitory receptor PD-L1 led to increased tumor growth inhibition and increased number of complete responses compared to either agent alone. This combination activity was associated with an increase in the expression of IFN-g associated genes and activated CD8+ T cells in the tumor. Together, these studies demonstrate that anti-OX40, both as a single agent and in combination with anti-PD-L1 can induce robust immune responses in animal tumor models. Citation Format: Jeong M. Kim. Unleashing anti-tumor immunity through anti-OX40 monotherapy and in combination with anti-PD-L1. [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 DDT01-02. doi:10.1158/1538-7445.AM2015-DDT01-02

Characteristics of immunosuppressive regulatory T cells in cutaneous squamous cell carcinomas and role in metastasis

BackgroundNon-melanoma skin cancer is the most common cancer worldwide, and cutaneous squamous cell carcinomas (SCCs) account for substantial morbidity and mortality because of their potential for metastasis. SCCs are surrounded by an immune cell infiltrate containing regulatory T cells (Tregs). The aim of this study was to characterise Tregs in SCCs and investigate whether increased Treg numbers in primary skin SCCs are associated with subsequent metastasis. MethodsLymphocytes were extracted from freshly excised skin SCC tumours and corresponding peripheral blood and normal skin. Flow cytometry was used for T-cell analysis and cell sorting. Tritiated thymidine based lymphocyte proliferation assays and interferon γ (IFNγ) ELISPOT assays were used to assess peritumoral lymphocyte function in vitro. Immunohistochemistry was performed on primary cutaneous SCC sections from tumours that subsequently metastasised and from those that did not after 5-year follow-up. FindingsIncreased frequencies of CD3+CD4+CD25hiCD127loFOXP3+ Tregs were found in SCCs (21·5% of CD4+ immune infiltrate, n=60 tumours) compared with corresponding peripheral blood (5·4%) and normal skin (7·6%). SCC Tregs expressed significantly higher levels of the co-stimulatory molecules OX40 (37·2% of FOXP3+ cell population, n=10 tumours) and 4-1BB (12·6%, n=9) than peritumoral non-regulatory T cells and Tregs from peripheral blood and normal skin (p=0·0005). The inhibitory receptor CTLA4 and the transcription factor Helios were expressed at high levels in peritumoral Tregs. SCC Tregs significantly suppressed phytohaemagglutinin-stimulated peritumoral CD4+ T-cell proliferation (p=0·005, n=10), peritumoral CD8+ T-cell proliferation (p=0·015, n=9), and IFNγ secretion by CD4+ effector T cells (p=0·026, n=10). Increased in-vitro proliferation of phytohaemagglutinin-stimulated peritumoral CD4+ T cells was shown after the addition of anti-OX40 antibodies (p=0·0078, n=9 tumours) and anti-4-1BB antibodies (p=0·0039, n=9). Immunohistochemistry showed fewer CD8+ T cells in SCCs that metastasised (n=29) than in non-metastatic SCCs (n=26) (28·5% of immune infiltrate vs 44·6%%, p<0·0001) and more FOX3+ Tregs (28·5% of immune infiltrate vs 49·3%, p<0·0001). InterpretationOur study shows that immunosuppressive Tregs are present in the immune infiltrate of cutaneous SCCs, and contribute to ineffective anti-tumour immune responses, thereby permitting SCC development and promoting metastasis. FundingWellcome Trust, National Institute for Health Research.

Three Steps to Breaking Immune Tolerance: A Microparticle Approach

Lymphoma immunotherapy can result in durable immune and clinical responses. Nevertheless, the clinical impact of such therapy remains suboptimal and there is still a need to apply our growing understanding of cancer immunity to the design and testing of rational combination approaches to lymphoma immunotherapy. This includes consideration of the three steps necessary to generate an antitumor response: (1) providing tumor antigens to dendritic cells (DCs) in a manner that enhances their ability to process and present antigens to T cells; (2) enhancing T cell activation; and (3) overcoming immunosuppression so that activated anti-lymphoma T cells can proceed unrestrained.In situ immunization generates systemic immune responses through local injection of agents into the tumor, thus providing DCs with the patient’s own tumor antigens. We evaluated a three-step approach to in situ immunization against lymphoma using Doxorubicin, anti-CTLA-4 and anti-OX40: (1) Doxorubicin (Dox) to induce the expression of “eat-me” signals by dying tumor cells, facilitating their phagocytosis by DCs; (2) anti-OX40 antibody to augment OX40-mediated stimulation of T cells; (3) anti-CTLA-4 antibody to block immunosuppression imposed by CTLA-4 on T cells. While Dox is highly effective as a systemic anti-lymphoma agent and has been reported to induce immunogenic cell death, intratumoral injection of soluble Dox is not clinically feasible due to its strong vesicant effect. Poly(lactide-co-glycolide) (PLGA) is an FDA-approved polymer that is used in biodegradable surgical sutures and microparticles (MPs) with sustained-release properties. Thus, PLGA MPs loaded with Dox (Dox MPs) represent a clinically translatable approach for delivering Dox, as its slow release would decrease likelihood of vesication. In addition, PLGA MPs at an optimal size of 1-µm enhance antigen-specific immune responses by activating the NALP3 inflammasome in DCs.While both tumor cells and DCs are exposed to Dox released by MPs in the tumor, we found in vitro that Dox MPs (1-µm) are less cytotoxic to DCs than to A20 B-lymphoma cells and do not require internalization for their cytotoxic activity. Dox MPs significantly enhanced phagocytosis of A20 by DCs as compared to soluble Dox. In vivo, we used the two-tumor mouse model to assess immune responses. This allowed us to monitor the local effect (on the tumor injected with MP) and the systemic effect (on the distant tumor that was not injected with MP) of therapy. Dox MPs injected intratumorally do not induce vesication even at doses as high as 100 µg Dox. Using a low dose of Dox MPs (2 µg Dox) and of antibody to limit systemic toxicity, we found that three-step therapy induced CD4- and CD8- T cell-dependent systemic immune responses that enhanced T cell infiltration into distant tumors. This led to their eradication and significantly improved survival as compared to antibody-only therapy (87% of mice treated with all three components became tumor-free). Moreover, all three components were required for maximum efficacy (Figure 1). These results demonstrate that systemic antitumor immune responses can be generated locally by three-step therapy. The potential value of this approach to immunotherapy is not limited to lymphoma and merits further evaluation in lymphoma and other cancers. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Anti-tumor efficacy and biomarker evaluation of agonistic anti-OX40 antibodies in preclinical models

Meeting abstracts OX40, a TNFRSF member, is a co-stimulatory molecule expressed on antigen experienced effector T (Teff) and regulatory T (Treg) cells, including infiltrating cells in mouse and human tumors. Activation of OX40 by agonistic antibodies is hypothesized to promote anti-tumor immunity

Anti-tumor efficacy and biomarker evaluation of agonistic anti-OX40 antibodies in preclinical models

Meeting abstracts OX40, a TNFRSF member, is a co-stimulatory molecule expressed on antigen experienced effector T (Teff) and regulatory T (Treg) cells, including infiltrating cells in mouse and human tumors. Activation of OX40 by agonistic antibodies is hypothesized to promote anti-tumor immunity

424 Nonclinical safety assessment of a humanized anti-OX40 agonist antibody, MOXR0916

424 Nonclinical safety assessment of a humanized anti-OX40 agonist antibody, MOXR0916

209 Pre-clinical and translational pharmacology, pharmacokinetics and pharmacodynamics for a humanized anti-OX40 antibody MOXR0916, a T-cell agonist in the treatment of solid tumors

209 Pre-clinical and translational pharmacology, pharmacokinetics and pharmacodynamics for a humanized anti-OX40 antibody MOXR0916, a T-cell agonist in the treatment of solid tumors

Small-molecule modulators of the OX40-OX40 ligand co-stimulatory protein-protein interaction.

The OX40-OX40L protein-protein interaction (PPI) is an important cell-surface signalling co-stimulatory regulator within the TNFR superfamily (TNFRSF) and a promising therapeutic target for immunomodulation. PPIs are difficult to modulate using small-molecules. Here, we describe the identification of a small-molecule OX40 modulator and confirm its partial agonist character. Cell-free screening assays were developed and used to identify OX40-OX40L inhibitors. Modified versions of this assay were used to elucidate the binding partner and the binding nature of active compounds. OX40-transfected sensor cells with NF-κB reporters were constructed and used to confirm and characterize activity and specificity. Immunomodulatory activity and partial agonist nature were further confirmed by ex vivo T-cell polarization assays. Several compounds that concentration-dependently affected OX40-OX40L were identified. Cell assays indicated that they were partial agonists with low micromolar potency and adequate selectivity. Under polarizing conditions based on TGF-β, the most promising compound mimicked the effect of an agonistic anti-OX40 antibody in suppressing regulatory T-cell generation and diverting CD4(+) CD62L(+) Foxp3(-) cells to TH 9 phenotype in vitro. We identified, to our knowledge, the first small-molecule compounds able to interfere with OX40-OX40L binding and, more importantly, to act as partial agonists of OX40. This is particularly interesting, as small-molecule agonism or activation of PPIs is considered unusually challenging and there are only few known examples. These results provide proof-of-principle evidence for the feasibility of small-molecule modulation of the OX40-OX40L interaction and for the existence of partial agonists for TNFRSF-PPIs.

PD-1 Blockade and OX40 Triggering Synergistically Protects against Tumor Growth in a Murine Model of Ovarian Cancer

The co-inhibitory receptor Programmed Death-1 (PD-1) curtails immune responses and prevent autoimmunity, however, tumors exploit this pathway to escape from immune destruction. The co-stimulatory receptor OX40 is upregulated on T cells following activation and increases their clonal expansion, survival and cytokine production when engaged. Although antagonistic anti-PD-1 or agonistic anti-OX40 antibodies can promote the rejection of several murine tumors, some poorly immunogenic tumors were refractory to this treatment. In the present study, we evaluated the antitumor effects and mechanisms of combinatorial PD-1 blockade and OX40 triggering in a murine ID8 ovarian cancer model. Although individual anti-PD-1 or OX40 mAb treatment was ineffective in tumor protection against 10-day established ID8 tumor, combined anti-PD-1/OX40 mAb treatment markedly inhibited tumor outgrowth with 60% of mice tumor free 90 days after tumor inoculation. Tumor protection was associated with a systemic immune response with memory and antigen specificity and required CD4+ cells and CD8+ T cells. The anti-PD-1/OX40 mAb treatment increased CD4+ and CD8+ cells and decreased immunosuppressive CD4+FoxP3+ regulatory T (Treg) cells and CD11b+Gr-1+ myeloid suppressor cells (MDSC), giving rise to significantly higher ratios of both effector CD4+ and CD8+ cells to Treg and MDSC in peritoneal cavity; Quantitative RT-PCR data further demonstrated the induction of a local immunostimulatory milieu by anti-PD-1/OX40 mAb treatment. The splenic CD8+ T cells from combined mAb treated mice produced high levels of IFN-γ upon tumor antigen stimulation and exhibited antigen-specific cytolytic activity. To our knowledge, this is the first study testing the antitumor effects of combined anti-PD-1/OX40 mAb in a murine ovarian cancer model, and our results provide a rationale for clinical trials evaluating ovarian cancer immunotherapy using this combination of mAb.

PLGA-nanoparticle mediated delivery of anti-OX40 monoclonal antibody enhances anti-tumor cytotoxic T cell responses

OX40 (CD134) is a tumor necrosis factor (TNF) receptor expressed mainly on activated T cells and transmits a potent costimulatory signal once engaged. Agonistic anti-OX40 monoclonal antibody (mAb) enhances tumor immune response leading to therapeutic effects in mouse tumor models. However, when tested in phase I clinical trials it did not show objective clinical activity in cancer patients. In this study, we examined the feasibility of nanoparticle (NP)-mediated delivery of anti-OX40 mAb to efficiently induce cytotoxic T lymphocyte (CTL) responses. The biodegradable poly(dl-lactide-co-glycolide) nanoparticle (PLGA-NP) carrying anti-OX40 mAb, anti-OX40-PLGA-NP, was prepared by double emulsion method and showed an average diameter of 86nm with a loading efficiency of 25%. We found that anti-OX40-PLGA-NP induced CTL proliferation and tumor antigen-specific cytotoxicity as well as cytokine production more strongly than free anti-OX40 mAb. These results suggest that PLGA-based nanoparticle formulation may provide efficient delivery system of anti-OX40 mAb for cancer immunotherapy.

Immune combinational therapy targeting OX40 and IDO synergistically enhances efficacy of a cancer vaccine

Enhancing an effector immune response by cancer vaccines has not been clinically successful so far. Although the necessary immune response may be elicited using vaccine-based therapies, this has not been sufficient for a positive clinical outcome; since most cancers can escape immune surveillance via multiple immune suppressive mechanisms induced in the tumor environment. A relatively recent strategy to increase the therapeutic efficacy of tumor vaccination is to combine it with alternative immunotherapeutic approaches. Here we tested if simultaneous targeting of the effector arm of the immune system with an agonist anti-OX40 mAb together with targeting of suppressive mechanisms in the tumor, via inhibition of indoleamine-(2,3)-dioxygenase (IDO), could enhance the anti-tumor activity of vaccine in mouse models of cancer. The OX40 molecule is a co-stimulatory receptor expressed on T-cells that can lead to proliferation and enhancement of T-cell effector function when triggered with an agonistic antibody. However, the tumor secretes a number of suppressive signals as a protective mechanism against its destruction, one of which is IDO. We demonstrate that treatment of TC-1 tumor bearing mice with an anti-OX40 agonist antibody in combination with vaccine leads to the enhancement of antigen-specific T cell responses. The dose of 1 mg/kg anti-OX40 antibody stimulated the effector arm of T cells, which ultimately led to a significant increase in the CD8+/regulatory T-cell (Treg) ratio within tumors. Further, this combination of vaccine and anti-OX40 antibody treatment led to a significant inhibition of tumor growth and to a prolonged survival of tumor bearing mice compared to control, and resulted in complete tumor regression in 20% of treated mice. We found, however, that this anti-tumor activity could be further enhanced through the combination of vaccine, anti-OX40 and 1-methyltryptophan (1-MT), an IDO activity inhibitor. IDO has been shown to be secreted by tumor cells, suppressive dendritic cells and macrophages in tumor environment, and is known to be responsible for suppressing effector cells and inducing Tregs. Our data demonstrate that vaccine/anti-OX40 antibody/1-MT combination leads to a more profound inhibition of tumor growth in mice and complete regression of established tumors in 60% of treated mice. In conclusion, we provide evidence that simultaneous targeting of the effector arm of the immune system, via anti-OX40 antibody, and suppressive mechanisms within the tumor, via 1-MT, in combination with cancer vaccine has a synergistic effect for tumor eradication and is a promising strategy that could potentially enhance the overall efficacy of cancer treatment in patients.

Natural OX40L expressed on human T cell leukemia virus type-I-immortalized T cell lines interferes with infection of activated peripheral blood mononuclear cells by CCR5-utilizing human immunodeficiency virus

BackgroundOX40 ligand (OX40L) co-stimulates and differentiates T cells via ligation of OX40 that is transiently induced on T cells upon activation, resulting in prolonged T cell survival and enhanced cytokine production by T cells. This view has led to the targeting of OX40 as a strategy to boost antigen specific T cells in the context of vaccination. In addition, the ligation of OX40 has also been shown to inhibit infection by CCR5-utilizing (R5) but not CXCR4-utilizing (X4) human immunodeficiency virus type-1 (HIV-1) via enhancement of production of CCR5-binding β-chemokines. It was reasoned that human T cell leukemia virus type-I (HTLV-1) immortalized T cell lines that express high levels of OX40L could serve as an unique source of physiologically functional OX40L. The fact that HTLV-1+ T cell lines simultaneously also express high levels of OX40 suggested a potential limitation.ResultsResults of our studies showed that HTLV-1+ T cell lines bound exogenous OX40 but not OX40L, indicating that HTLV-1+ T cell lines express an active form of OX40L but an inactive form of OX40. Anti-OX40 non-blocking monoclonal antibody (mAb), but not blocking mAb, stained HTLV-1+ T cell lines, suggesting that the OX40 might be saturated with endogenous OX40L. Functionality of the OX40L was confirmed by the fact that a paraformaldehyde (PFA)-fixed HTLV-1+ T cell lines inhibited the infection of autologous activated peripheral blood mononuclear cells (PBMCs) with R5 HIV-1 which was reversed by either anti-OX40L blocking mAb or a mixture of neutralizing mAbs against CCR5-binding β-chemokines.ConclusionsAltogether, these results demonstrated that autologous T cell lines immortalized by HTLV-1 can be utilized as a conventional source of physiologically functional OX40L.

FDC Like Cell Line HK With IL-2, IL-4 and OX40 Ligand Supports The Growth Of ATLL Cells

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive neoplasm derived from CD4+ T-cells with HTLV-I infection, and its mechanisms of tumorigenesis still remain to be elucidated. The fact that tumor cells rarely proliferate in vitro is one of the most important problems to be solved. The establishment of cell line from ATLL patient samples has been difficult even in the presence of interleukins. Previously we established one cell line (HU-ATTAK) from acute or lymphoma types of 10 ATLL cases which did not proliferate in the presence of IL-2 and/or IL-4. HU-ATTAK is critically dependent on IL-2 and human umbilical cord vein endothelial cells (HUVEC) as feeder cells. In HU-ATTAK, adding anti-OX40 ligand antibody into the culture system completely inhibited its proliferation. So, OX40 ligand as well as L-2 and/or IL-4 is suggested be necessary for the proliferation of ATLL cells, and feeder cells may also confer the favorable environment. As the substitute of HUVEC, follicular dendritic cell like cell line HK which expresses OX40 ligand was used by introducing human OX40 ligand cDNA (HK-OX40L). When 9 ATLL patient samples were co-cultured with HK-OX40L in the presence of L-2 and/or IL-4, two ATLL cells proliferate vigorously only in the presence of both IL-2 and IL-4 simultaneously. These cell lines were confirmed to be derived from original tumor cells by array CGH analysis, and continued the growth for more than a year. Depletion of IL-2 and IL-4 made these cell lines stop growing within 6 days even on HK-OX40L. In the presence of IL-2 and IL-4, the conditions such as HK alone without OX40 ligand or OX40 ligand alone without HK made the cell lines growing for three months at most. In the presence of IL-2 and IL-4 without HK-OX40L, these cell lines vigorously proliferated for more than three months but finally stopped growing. These data suggested that for the growth of these two cell lines, the cell division is dependent on IL-2 and IL-4, and the maintenance of immortalization is dependent on OX40 ligand and HK cells. This culture analysis would provide important factors for cell growth of ATLL which will explore new targets for ATLL treatment.*HK cells are kindly provided by Dr. Young S Choi at Ochsner Cancer Center, New Orleans. Disclosures:No relevant conflicts of interest to declare.

Retinoic acid prevents mesenteric lymph node dendritic cells from inducing IL-13-producing inflammatory Th2 cells.

The vitamin A (VA) metabolite retinoic acid (RA) affects the properties of T cells and dendritic cells (DCs). In VA-deficient mice, we observed that mesenteric lymph node (MLN)-DCs induce a distinct inflammatory T helper type 2 (Th2)-cell subset that particularly produces high levels of interleukin (IL)-13 and tumor necrosis factor-α (TNF-α). This subset expressed homing receptors for skin and inflammatory sites, and was mainly induced by B220(-)CD8α(-)CD11b(+)CD103(-) MLN-DCs in an IL-6- and OX40 ligand-dependent manner, whereas RA inhibited this induction. The corresponding MLN-DC subset of VA-sufficient mice induced a similar T-cell subset in the presence of RA receptor antagonists. IL-6 induced this subset differentiation from naive CD4(+) T cells upon activation with antibodies against CD3 and CD28. Transforming growth factor-β inhibited this induction, and reciprocally enhanced Th17 induction. Treatment with an agonistic anti-OX40 antibody and normal MLN-DCs enhanced the induction of general inflammatory Th2 cells. In VA-deficient mice, proximal colon epithelial cells produced TNF-α that may have enhanced OX40 ligand expression in MLN-DCs. The repeated oral administrations of a T cell-dependent antigen primed VA-deficient mice for IL-13-dependent strong immunoglobulin G1 (IgG1) responses and IgE responses that caused skin allergy. These results suggest that RA inhibits allergic responses to oral antigens by preventing MLN-DCs from inducing IL-13-producing inflammatory Th2 cells.

OX40-OX40L Interaction Promotes Proliferation and Activation of Lymphocytes via NFATc1 in ApoE-Deficient Mice

BackgroundOur previous studies have shown that OX40-OX40L interaction regulates the expression of nuclear factor of activated T cells c1(NFATc1) in ApoE−/− mice during atherogenesis. The aim of this study was to investigate whether OX40-OX40L interaction promotes Th cell activation via NFATc1 in ApoE−/− mice.Methods and ResultsThe lymphocytes isolated from spleen of ApoE−/− mice were cultured with anti-CD3 mAb in the presence or absence of anti-OX40 or anti-OX40L antibodies. The expression of NFATc1 mRNA and protein in isolated lymphocytes were measured by real time PCR (RT-PCR) and flow cytometry (FCM), respectively. The proliferation of lymphocytes was analyzed by MTT method,and the expression of IL-2, IL-4 and IFN-γ in the cultured cells and supernatant were measured by RT-PCR and enzyme-linked immunosorbent assary (ELISA), respectively. After stimulating OX40-OX40L signal pathway, the expression of NFATc1 and the proliferation of leukocytes were significantly increased. Anti-OX40L suppressed the expression of NFATc1 in lymphocytes of ApoE−/− mice. Anti-OX40L or the NFATc1 inhibitor (CsA) markedly suppressed the cell proliferation induced by anti-OX40. Moreover, the expression of IL-2 and IFN-γ was increased in lymphocytes induced by OX40-OX40L interaction. Blocking OX40-OX40L interaction or NFATc1 down-regulated the expression of IL-2 and IFN-γ, but didn’t alter the expression of IL-4 in supernatants.ConclusionThese results suggest that OX40-OX40L interaction promotes the proliferation and activation of lymphocytes through NFATc1.

Biomarkers for Immunostimulatory Monoclonal Antibodies in Combination Strategies for Melanoma and Other Tumor Types

Modulation of the immune system by targeting coinhibitory and costimulatory receptors has become a promising new approach of immunotherapy for cancer. The recent approval of the CTLA-4-blocking antibody ipilimumab for the treatment of melanoma was a watershed event, opening up a new era in the field of immunotherapy. Ipilimumab was the first treatment to ever show enhanced overall survival (OS) for patients with stage IV melanoma. However, measuring response rates using standard Response Evaluation Criteria in Solid Tumors (RECIST) or modified World Health Organization criteria or progression-free survival does not accurately capture the potential for clinical benefit for ipilimumab-treated patients. As immunotherapy approaches are translated into more tumor types, it is important to study biomarkers, which may be more predictive of OS to identify the patients most likely to have clinical benefit. Ipilimumab is the first-in-class of a series of immunomodulating antibodies that are in clinical development. Anti-PD1 (nivolumab and MK-3475), anti-PD-L1 (BMS-936 559, RG7446, and MEDI4736), anti-CD137 (urelumab), anti-OX40, anti-GITR, and anti-CD40 monoclonal antibodies are just some of the agents that are being actively investigated in clinical trials, each having the potential for combination with the ipilimumab to enhance its effectiveness. Development of rational combinations of immunomodulatory antibodies with small-molecule pathway inhibitor therapies such as vemurafenib makes the discovery of predictive biomarkers even more important. Identifying reliable biomarkers is a necessary step in personalizing the treatment of each patient's cancer through a baseline assessment of tumor gene expression and/or immune profile to optimize therapy for the best chance of therapeutic success.