anti-OX40 Agonist Antibody Research Articles

Reprogramming macrophage by targeting VEGF and CD40 potentiates OX40 immunotherapy

The low clinical response rate of checkpoint blockades, such as PD-1 and CTLA-4, highlighted the requirements of agonistic antibodies to boost optimal T cell responses. OX40, a co-stimulatory receptor on the T cells, plays a crucial role in promoting T cell survival and differentiation. However, the clinical efficacy of anti-OX40 agonistic antibodies was unimpressive. To explore the mechanism underlying the action of anti-OX40 agonists to improve the anti-tumor efficacy, we analyzed the dynamic changes of tumor-infiltrating immune cells at different days post-treatments using single-cell RNA-sequencing (scRNA-seq). In this study, we found that tumor-infiltrating regulatory T (Treg) cells were reduced after two rounds of anti-OX40 treatment, but the increase of infiltration and activation of CD8+ effector T cells, as well as M1 polarization in the tumor were only observed after three rounds of treatments. Moreover, our group first analyzed the antitumor effect of anti-OX40 treatments on regulating the macrophages and discovered the dynamic changes of vascular endothelial growth factor (VEGF) and CD40 signaling pathways on macrophages, indicating their possibility to being potential combination targets to improve the anti-OX40 agonists efficacy. The combination of VEGFR inhibitors or anti-CD40 agonist antibody with anti-OX40 agonists exhibited more remarkable inhibition of tumor growth. Therefore, the mechanism-driven combination of anti-OX40 agonists with VEGFR inhibitors or anti-CD40 agonists represented promising strategies.

Loss of the OX40 Target in Lymphoma Patients after Combining an Anti-OX40 Agonist Antibody with in Situ Vaccination

INTRODUCTION:Previous clinical trials have shown that in situ vaccination using the combination of low-dose radiotherapy and intratumoral injection of CpG, a Toll-like receptor 9 (TLR9) agonist, can elicit T cell immune responses in patients with low grade lymphoma and regression at both the treated and non-treated distal tumor sites in patients with low-grade lymphoma (Brody et al., JCO 2010, Frank et al., Cancer Discovery 2018, Hammerich et al., Nat Med 2019). OX40 is a T cell activating co-receptor, belonging to the TNFR superfamily. Ligation of this receptor by an agonist anti-OX40 antibody leads to enhanced T cell activation of effector CD4 T cells while inhibiting Treg function. In preclinical models, addition of an anti-OX40 antibody to intratumoral CpG cured established tumors (Sagiv-Barfi I, et al. Sci Transl Med. 2018). Intratumoral injection of CpG up-regulated OX40 on CD4 T cells in the microenvironment enhancing the binding of agonistic anti-OX40 antibody that triggered a robust immune response. These results provided the rationale for a clinical trial of in situ vaccination (NCT03410901) using a human IgG1 agonistic anti-OX40 antibody (BMS-986178). STUDY:Fourteen patients with low-grade lymphoma received low-dose (2Gy x 2) radiotherapy to a single tumor site followed by 5 weekly intratumoral injections of 2 mg CpG-ODN (SD-101, TriSalus Life Sciences) and 3.75 mg anti-OX40 antibody into the same site. 40mg of anti-OX40 was administered intravenously starting on day 2 and continuing every 4 weeks for a total of 6 infusions. Therapy was well tolerated with no dose-limiting toxicities. Response to therapy was assessed using the revised Lugano criteria (Cheson et al., JCO 2014) based on CT scans at 12, 24, 48, 72, and 96 weeks. Fine needle aspirates (FNA) were performed at both the injected and a non-injected site outside the radiation field prior to, and at 1 and 6 weeks after initiation of treatment. Cells were analyzed by flow cytometry and droplet-based single-cell RNA sequencing to determine population dynamics, response to therapy as well as antibody binding and target occupancy by the anti-OX40 antibody. RESULTS:Of the 14 patients enrolled, 1 patient attained a partial response (PR), while 9 had stable disease (SD) and 4 had disease progression (PD) as their best overall response. Investigation into the binding of the treatment antibody and target occupancy by flow cytometry showed saturation of all the OX40 sites on CD4 T cells by week 6 at both the injected and non-injected tumors. However, further investigation also showed there was significantly less OX40 on the cell surface after treatment, suggesting a loss of the antibody target (Figure 1). This loss was reproduced by in vitro assays where activated blood CD4 T cells exhibited a loss of cell surface OX40 when cultured with BMS-986178. Additionally, Quigley et al ( Clin Cancer Res 2019) previously showed that an OX40 receptor occupancy of greater than 40% led to a loss of target in mice and in blood T cells of solid tumor patients. We further observed that the loss of cell surface OX40 occurred in all CD4 T subsets, including effector CD4 T cells and T Regulatory cells (Tregs). This result contrasted with our in vivo and in vitro findings in successful therapeutic mouse models where we did not observe this loss of cell surface OX40 on effector CD4 T cells after treatment with a mouse-specific anti-OX40 agonist antibody. C ONCLUSION: Our observations suggest that treatment with an agonistic anti-OX40 antibody induced a loss of OX40 on the cell surface of effector CD4 T cells in the tumor microenvironment. Fewer molecules of OX40 receptor may have constrained the efficacy of subsequent anti-OX40 infusions and may explain why the clinical responses observed in this study were lower than observed in our past clinical trials of in situ vaccination.

Structural Basis of a Novel Agonistic Anti-OX40 Antibody.

Agonistic antibodies targeting co-stimulating receptor OX40 on T cells are considered as important as (or complementary to) the immune checkpoint blockers in cancer treatment. However, none of these agonistic antibodies have reached the late stage of clinical development partially due to the lack of intrinsic potency with the correlation between binding epitope and activity of the antibody not well understood. Here, we identified a novel anti-OX40 agonistic antibody DF004, which stimulated the proliferation of human CD4+ T cells in vitro and inhibited tumor growth in a mouse model. Our crystallography structural studies showed that DF004 binds to the CRD2 region of OX40 while RG7888, an OX40 agonist antibody developed by Roche, binds to CRD3 of OX40 to the diametrically opposite position of DF004. This suggests that the agonistic activities of the antibodies are not necessarily epitope dependent. As their agonistic activities critically depend on clustering or cross-linking, our structural modeling indicates that the agonistic activity requires the optimal positioning of three Fc receptor/antibody/OX40 complexes on the cell membrane to facilitate the formation of one intracellular hexameric TRAF complex for downstream signal transduction, which is relatively inefficient. This may explain the lack of sufficient potency of these OX40 antibodies in a therapeutic setting and sheds light on the development of cross-linking-independent agonistic antibodies.

Antibody-mediated delivery of a viral MHC-I epitope into the cytosol of target tumor cells repurposes virus-specific CD8+ T cells for cancer immunotherapy

BackgroundRedirecting pre-existing virus-specific cytotoxic CD8+ T lymphocytes (CTLs) to tumors by simulating a viral infection of the tumor cells has great potential for cancer immunotherapy. However, this strategy is limited by lack of amenable method for viral antigen delivery into the cytosol of target tumors. Here, we addressed the limit by developing a CD8+T cell epitope-delivering antibody, termed a TEDbody, which was engineered to deliver a viral MHC-I epitope peptide into the cytosol of target tumor cells by fusion with a tumor-specific cytosol-penetrating antibody.MethodsTo direct human cytomegalovirus (CMV)-specific CTLs against tumors, we designed a series of TEDbodies carrying various CMV pp65 antigen-derived peptides. CMV-specific CTLs from blood of CMV-seropositive healthy donors were expanded for use in in vitro and in vivo experiments. Comprehensive cellular assays were performed to determine the presentation mechanism of TEDbody-mediated CMV peptide-MHC-I complex (CMV-pMHCI) on the surface of target tumor cells and the recognition and lysis by CMV-specific CTLs. In vivo CMV-pMHCI presentation and antitumor efficacy of TEDbody were evaluated in immunodeficient mice bearing human tumors.ResultsTEDbody delivered the fused epitope peptides into target tumor cells to be intracellularly processed and surface displayed in the form of CMV-pMHCI, leading to disguise target tumor cells as virally infected cells for recognition and lysis by CMV-specific CTLs. When systemically injected into tumor-bearing immunodeficient mice, TEDbody efficiently marked tumor cells with CMV-pMHCI to augment the proliferation and cytotoxic property of tumor-infiltrated CMV-specific CTLs, resulting in significant inhibition of the in vivo tumor growth by redirecting adoptively transferred CMV-specific CTLs. Further, combination of TEDbody with anti-OX40 agonistic antibody substantially enhanced the in vivo antitumor activity.ConclusionOur study offers an effective technology for MHC-I antigen cytosolic delivery. TEDbody may thus have utility as a therapeutic cancer vaccine to redirect pre-existing anti-viral CTLs arising from previously exposed viral infections to attack tumors.

Inactivated Cowpea Mosaic Virus in Combination with OX40 Agonist Primes Potent Antitumor Immunity in a Bilateral Melanoma Mouse Model.

Viral immunotherapies are being recognized in cancer treatment, with several currently approved or undergoing clinical testing. While contemporary approaches have focused on oncolytic viral therapies, our efforts center on the development of plant virus-based cancer immunotherapies. In a previous work, we demonstrated the potent efficacy of the cowpea mosaic virus (CPMV), a plant virus that does not replicate in animals, applied as an in situ vaccine. CPMV is an immunostimulatory drug candidate, and intratumoral administration remodels the tumor microenvironment leading to activation of local and systemic antitumor immunity. Efficacy has been demonstrated in multiple tumor mouse models and canine cancer patients. As wild-type CPMV is infectious toward various legumes and because shedding of infectious virus from patients may be an agricultural concern, we developed UV-inactivated CPMV (termed inCPMV) which is not infectious toward plants. We report that as a monotherapy, wild-type CPMV outperforms inCPMV in mouse models of dermal melanoma or disseminated colon cancer. Efficacy of inCPMV is less than that of CPMV and similar to that of RNA-free CPMV. Immunological investigation using knockout mice shows that inCPMV does not signal through TLR7 (toll-like receptor); structure-function studies indicate that the RNA is highly cross-linked and therefore unable to activate TLR7. Wild-type CPMV signals through TLR2, -4, and -7, whereas inCPMV more closely resembles RNA-free CPMV which signals through TLR2 and -4 only. The structural features of inCPMV explain the increased potency of wild-type CPMV through the triple pronged TLR activation. Strikingly, when inCPMV is used in combination with an anti-OX40 agonist antibody (administered systemically), exceptional efficacy was demonstrated in a bilateral B16F10 dermal melanoma model. Combination therapy, with in situ vaccination applied only into the primary tumor, controlled the progression of the secondary, untreated tumors, with 10 out of 14 animals surviving for at least 100 days post tumor challenge without development of recurrence or metastatic disease. This study highlights the potential of inCPMV as an in situ vaccine candidate and demonstrates the power of combined immunotherapy approaches. Strategic immunocombination therapies are the formula for success, and the combination of in situ vaccination strategies along with therapeutic antibodies targeting the cancer immunity cycle is a particularly powerful approach.

Radiation Augments the Local Anti-Tumor Effect of In Situ Vaccine With CpG-Oligodeoxynucleotides and Anti-OX40 in Immunologically Cold Tumor Models.

IntroductionCombining CpG oligodeoxynucleotides with anti-OX40 agonist antibody (CpG+OX40) is able to generate an effective in situ vaccine in some tumor models, including the A20 lymphoma model. Immunologically “cold” tumors, which are typically less responsive to immunotherapy, are characterized by few tumor infiltrating lymphocytes (TILs), low mutation burden, and limited neoantigen expression. Radiation therapy (RT) can change the tumor microenvironment (TME) of an immunologically “cold” tumor. This study investigated the effect of combining RT with the in situ vaccine CpG+OX40 in immunologically “cold” tumor models.MethodsMice bearing flank tumors (A20 lymphoma, B78 melanoma or 4T1 breast cancer) were treated with combinations of local RT, CpG, and/or OX40, and response to treatment was monitored. Flow cytometry and quantitative polymerase chain reaction (qPCR) experiments were conducted to study differences in the TME, secondary lymphoid organs, and immune activation after treatment.ResultsAn in situ vaccine regimen of CpG+OX40, which was effective in the A20 model, did not significantly improve tumor response or survival in the “cold” B78 and 4T1 models, as tested here. In both models, treatment with RT prior to CpG+OX40 enabled a local response to this in situ vaccine, significantly improving the anti-tumor response and survival compared to RT alone or CpG+OX40 alone. RT increased OX40 expression on tumor infiltrating CD4+ non-regulatory T cells. RT+CpG+OX40 increased the ratio of tumor-infiltrating effector T cells to T regulatory cells and significantly increased CD4+ and CD8+ T cell activation in the tumor draining lymph node (TDLN) and spleen.ConclusionRT significantly improves the local anti-tumor effect of the in situ vaccine CpG+OX40 in immunologically “cold”, solid, murine tumor models where RT or CpG+OX40 alone fail to stimulate tumor regression.

Advances in Anti-Cancer Immunotherapy: Car-T Cell, Checkpoint Inhibitors, Dendritic Cell Vaccines, and Oncolytic Viruses, and Emerging Cellular and Molecular Targets.

Unlike traditional cancer therapies, such as surgery, radiation and chemotherapy that are typically non-specific, cancer immunotherapy harnesses the high specificity of a patient’s own immune system to selectively kill cancer cells. The immune system is the body’s main cancer surveillance system, but cancers may evade destruction thanks to various immune-suppressing mechanisms. We therefore need to deploy various immunotherapy-based strategies to help bolster the anti-tumour immune responses. These include engineering T cells to express chimeric antigen receptors (CARs) to specifically recognise tumour neoantigens, inactivating immune checkpoints, oncolytic viruses and dendritic cell (DC) vaccines, which have all shown clinical benefit in certain cancers. However, treatment efficacy remains poor due to drug-induced adverse events and immunosuppressive tendencies of the tumour microenvironment. Recent preclinical studies have unveiled novel therapies such as anti-cathepsin antibodies, galectin-1 blockade and anti-OX40 agonistic antibodies, which may be utilised as adjuvant therapies to modulate the tumour microenvironment and permit more ferocious anti-tumour immune response.

Development and characterization of a novel anti-OX40 antibody for potent immune activation.

With the great success of anti-CTLA-4 and anti-PD-1 therapeutics in cancer immunotherapy, tumor necrosis factor receptor superfamily members have been recognized as ideal targets to provide co-stimulatory signals in combination with immune checkpoint blocking antibodies. Among these is OX40 (CD134), a co-stimulatory molecule expressed by activated immune cells. Recently, several anti-OX40 agonistic monoclonal antibodies, pogalizumab as the most advanced, have entered early phase clinical trials. Using ayeast platform and multiple screening methods, we identified a fully human anti-OX40 antibody (IBI101) with distinct modes of action. Unlike pogalizumab, IBI101 partially blocks the binding of OX40 to its ligand OX40L and exhibits both FcγR-dependent and independent agonistic activities in NF-κB luciferase reporter assays. IBI101 also promotes T cell activation and proliferation in vitro. These unique properties partially explain the more potent anti-tumor activity of IBI101 than that of pogalizumab in humanized NOG mice bearing LoVo tumors. In addition, IBI101 shows efficacious anti-tumor activity in mice when administrated alone or in combination with anti-PD-1 antibodies. In human OX40 knock-in mice bearing MC38 colon carcinoma, IBI101 treatment induces tumor antigen-specific CD8+ T-cell responses, decreases immunosuppressive regulatory T cells in tumor, and enhances the immune response to PD-1 inhibition. Preclinical studies of IBI101 in non-human primates demonstrate typical pharmacokinetic characteristics of an IgG antibody and no drug-related toxicity. Collectively, IBI101 has desirable preclinical attributes which support its clinical development for cancer treatment.

Anti-OX40 Antibody Directly Enhances The Function of Tumor-Reactive CD8+ T Cells and Synergizes with PI3Kβ Inhibition in PTEN Loss Melanoma.

OX40 agonist-based combinations are emerging as a novel avenue to improve the effectiveness of cancer immunotherapy. To better guide its clinical development, we characterized the role of the OX40 pathway in tumor-reactive immune cells. We also evaluated combining OX40 agonists with targeted therapy to combat resistance to cancer immunotherapy.Experimental Design: We utilized patient-derived tumor-infiltrating lymphocytes (TILs) and multiple preclinical models to determine the direct effect of anti-OX40 agonistic antibodies on tumor-reactive CD8+ T cells. We also evaluated the antitumor activity of an anti-OX40 antibody plus PI3Kβ inhibition in a transgenic murine melanoma model (Braf mutant, PTEN null), which spontaneously develops immunotherapy-resistant melanomas. We observed elevated expression of OX40 in tumor-reactive CD8+ TILs upon encountering tumors; activation of OX40 signaling enhanced their cytotoxic function. OX40 agonist antibody improved the antitumor activity of CD8+ T cells and the generation of tumor-specific T-cell memory in vivo. Furthermore, combining anti-OX40 with GSK2636771, a PI3Kβ-selective inhibitor, delayed tumor growth and extended the survival of mice with PTEN-null melanomas. This combination treatment did not increase the number of TILs, but it instead significantly enhanced proliferation of CD8+ TILs and elevated the serum levels of CCL4, CXCL10, and IFNγ, which are mainly produced by memory and/or effector T cells. These results highlight a critical role of OX40 activation in potentiating the effector function of tumor-reactive CD8+ T cells and suggest further evaluation of OX40 agonist-based combinations in patients with immune-resistant tumors.

Cancer combinatorial immunotherapy using anti-OX40 agonist and anti-PD-L1 antagonist: a patent evaluation of US2018256711A1

ABSTRACTIntroduction: OX40 is checkpoint inhibitor in cancer that coordinates the downregulation of the proliferation of antigen-specific lymphocytes. There is a great need to discover and develop new therapies focused on inhibiting the action of OX40 and consequently improving the immune response in the various types of cancer. Authors of patent US2018256711A1 propose a method to eradicate cancer that utilizes anti-OX40 agonist antibody in combination with anti-PD-L1 antagonist antibody.Areas covered: Patent US2018256711A1 describes a method of cancer combinatorial treatment consisting of the utilization of a pharmaceutical cocktail containing anti-OX40 and an anti-PD-L1 antibody.Expert opinion: The results of the clinical trials only support trials regarding the tolerability of combinatorial therapy, even when the objectives of determining the safety and pharmacokinetics of the treatment are proposed.

Abstract LB-110: Anti-OX40 agonistic antibody enhances ex vivo CD8+ TIL expansion with increased T-cell effector function

Abstract Adoptive T cell therapy (ACT) using tumor infiltrating lymphocytes (TILs) has demonstrated efficacy in metastatic melanoma patients, with ~50% objective responses. Amongst parameters recognized as important for the efficacy of ACT is a high proportion of CD8+ T cells in the infusion product. OX40 (CD134) belongs to the tumor necrosis factor receptor super family and is mainly expressed by activated T lymphocytes. Activation of OX40 signaling promotes proliferation and survival of T-cells via the NF-kB pathway. Agonistic anti-OX40 antibodies have been developed as immunotherapy drugs for the treatment of cancer, one of which was recently shown to increase reactivity of tumor antigen-specific CD8+ T cells in patient's peripheral blood. In this work, we studied the expression of the OX40 receptor on TILs and investigated the impact of an anti-OX40 agonistic antibody on the ex vivo expansion and effector function of TILs. We found that OX40 was enriched in the CD4+ T cell subset of TILs. Its expression was up-regulated in response to anti-CD3 stimulation during the rapid expansion protocol (REP) of TIL production. An anti-OX40 monoclonal antibody was obtained from commercial sources and characterized in vitro. It was shown to bind OX40 specifically and to mediate NF-κB activation in a dose-dependent manner upon clustering. Consistent with its agonistic activity in the reporter assay, the anti-OX40 antibody also heightened IFN-γ production of OKT3-stimulated TILs, suggesting improved T cell effector function (N=6; p=0.03) and supporting its use during TIL expansion. When TILs were propagated by REP in the presence of anti-OX40, the frequency of CD8+ T cells increased significantly in the final product relative to control (N=21, p=0.001). The effect was dose-dependent. Interestingly, cell surface expression of the OX40 receptor was specifically down-regulated in the responding anti-OX40-treated TILs, suggesting a link between regulation of the OX40 signaling axis and the antibody-mediated effect on the TIL subsets. The TCR Vβ repertoire of TILs expanded with anti-OX40 was not changed significantly (N=3). However, expression of T cell surface markers was altered by the antibody, which skewed the T cell phenotype towards a differentiated and activated state. Taken together, maintenance of T cell diversity and differentiation state of the T cells suggest that anti-OX40 acted by selectively promoting the proliferation of CD8+ T cells. When assessed functionally in an IFNγ assay, the potency of the TILs produced in the presence of anti-OX40 was shown to be comparable to that of control TILs. In conclusion, our work shows that activation of OX40 signaling using an agonistic antibody could significantly improve expansion of CD8+ TILs. Implications on in vivo anti-tumor activity remain to be assessed. Citation Format: Krit Ritthipichai, Marcus Machin, Maria Fardis, Cecile Chartier. Anti-OX40 agonistic antibody enhances ex vivo CD8+ TIL expansion with increased T-cell effector function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-110.

Abstract 4559: KY-B602, a humanized anti-human OX40 antibody, enhances CD4+T cell activation and inhibits tumor growth in a syngeneic model

Abstract OX40, a member of the tumor necrosis factor (TNF) receptor superfamily, is mainly expressed on activated T cells. As a secondary costimulatory molecule, activated OX40 can increase effector T-cell survival, proliferation, and memory. Anti-OX40 monoclonal antibodies have shown anti-tumor efficacy. Recent preclinical studies further support the potential synergistic effects of agonist OX40 immunotherapy in combination with cancer vaccination or in sequential co-administration with PD1 antibody to revert PD1 resistance. Several anti-OX40 agonistic monoclonal antibodies are currently tested in early phase cancer clinical trials either in monotherapy or in combination with other immune modulators. KY-B602 is a novel humanized IgG1 anti-OX40 antibody under pre-clinical development. It was generated from hybridoma and humanized by CDR grafting and structure simulation. KY-B602 specifically binds to the extracellular domain of human OX40 with high affinity (KD = 5.1 nM, determined by Biacore). FACS study showed KY-B602 also binds to both human and cynomolgus monkey OX40 over-expressed on 293T cells (EC50 =4.6nM, EC50=6.1nM, respectively). Another, in HT1080 cell reporter assay, KY-B602 significantly increased IL-8 secretion through OX40 pathway. In addition, KY-B602 dose-dependently augmented IL-2 and IFN-gamma secretion from CD4+T cells, that were pre-stimulated with IL-2 and PHA. However, in FACS competition assay, KY-B602 did not affect OX40L binding to OX40, suggesting that KY-B602 binds to a different epitope with that of OX40L. Moreover, KY-B602 inhibits in vivo tumor growth in MC38 syngeneic model. In summary, the current data of KY-B602 support its potency for cancer therapy. Citation Format: feng hao, feng he, mingxuan ning, zhaoshuai bai, jinying ning. KY-B602, a humanized anti-human OX40 antibody, enhances CD4+T cell activation and inhibits tumor growth in a syngeneic model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4559.

Advances in novel anti-OX40 cancer immunotherapy

OX40 (CD134) is a member of the tumor necrosis factor receptors and mainly expressed on the surface of active lymphocytes. Being an important pair of co-stimulatory factors in the process of immune response, OX40 and its ligand OX40L play an important role in inducing anti-tumor immune response. They regulate the immunosuppressive effect in tumor microenvironment and enhance the killing activities of effector cells. Pre-clinical animal studies have shown that agonists for OX40, whether they are used alone or in combination with other treatments, have stronger anti-tumor effects. Several anti-OX40 agonistic monoclonal antibodies (McAb) are currently tested in early phase cancer clinical trials. This review focuses on the advances in anti-OX40 mAbs-based therapy in terms of molecular and biological properties, mechanism of action, monotherapy and combined therapy. Key words: OX40; Immune checkpoint; Tumor; Immunotherapy

In Situ Vaccination with a TLR9 Agonist and Anti-OX40 Antibody Leads to Tumor Regression and Induces Abscopal Responses in Murine Lymphoma

Background:Toll-like receptors (TLRs) are components of the innate immune system that recognize pathogen-associated molecular patterns on bacterial, fungal, or viral pathogens. Intratumoral (IT) injection of unmethylated CG-enriched oligodeoxynucleotide (CpG), a TLR9 agonist, results in local tumor eradication but on its own is not able to induce a systemic anti-tumor immune response. OX40 is a potent costimulatory receptor that can potentiate the action of conventional T cells leading to their proliferation, effector function and survival, but can also inhibit or kill T regulatory cells by ADCC. In previous preclinical studies systemic injection of OX40 agonists increased antitumor immunity and improved survival.Scientific question:Does local injection of both CpG and low doses of an anti-OX40 agonistic antibody trigger a systemic anti-tumor immune response?Results:We implanted the A20 lymphoma tumor bilaterally on opposite sides of the abdomen. After tumors were established we administered microgram quantities of CpG and anti-OX40 antibody into the tumor on one side and monitored both the injected and the uninjected tumor sites. This treatment resulted in both a local and an abscopal effect on the contralateral, untreated tumor. In addition, the animals were protected from a second challenge with A20 cells. This anti-tumor effect was T cell dependent, since depletion of either CD4+ or CD8+ T cells abrogated the therapeutic effect. There was no evidence of toxicity or autoimmunity in the treated animals.To examine the potential of this maneuver to treat spontaneous, non-transplanted cancers we chose the mouse mammary tumor model- FVB/N-Tg(MMTV-PyVT)634Mul/J. These animals all develop invasive breast cancer tumors in all of their mammary glands by 12 weeks of age. Injections of CpG and anti-OX40 antibody into the first arising tumor not only prevented its growth but significantly reduced the incidence and outgrowth of subsequent tumors at un-injected susceptible mammary glands and reduced the number of lung metastases.Significance:TLR9 agonists and anti-OX40 antibodies are currently under clinical development for cancer treatment. We show here that combining anti-OX40 antibody with a TLR9 agonist at a single established tumor is sufficient to trigger a systemic anti-tumor response able to eradicate tumor at distant sites in both transplantable and spontaneously occurring oncogene-driven murine tumors. This anti-tumor effect was long lasting, specific and required T cells.Impact:Our current results suggest that CpG and anti-OX40 are sufficient to induce fully protective and curative anti-tumor immune responses, even in spontaneously arising cancer. Anti-OX40 and CpG are both currently in phase-I clinical trials as single agents. Our results provide the rationale for testing these agents clinically in combination as described here, injected locally in low doses in order to induce therapeutic anti-lymphoma immunity. DisclosuresLevy:Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding.

Abstract 3204: INCAGN01949: an anti-OX40 agonist antibody with the potential to enhance tumor-specific T-cell responsiveness, while selectively depleting intratumoral regulatory T cells

Abstract OX40 (CD134, TNFRSF4) is a T cell co-stimulatory receptor that potentiates T cell receptor (TCR) signaling during CD4+ and CD8+ T cell priming, effector cell differentiation and memory T cell recall responses. In preclinical mouse tumor models, surrogate anti-OX40 agonist antibodies have shown remarkable single agent anti-tumor efficacy, as well as the ability to combine effectively with other immunomodulatory antibodies and immune education strategies, such as therapeutic cancer vaccines. Agonistic antibodies targeting OX40 are predicted to counteract the immunosuppressive tumor microenvironment and promote tumor-specific T cell immunity via two primary mechanisms: 1) binding and activating OX40 signaling in tumor-specific effector and memory T cells, thereby enhancing their responsiveness to tumor-associated antigens, and 2) co-engaging Fcγ receptors expressed by tumor-associated effector cells, and facilitating the selective depletion of intratumoral regulatory T cells. INCAGN01949 is a novel fully human IgG1 monoclonal antibody identified using the Retrocyte Display™ platform that is being developed for the treatment of advanced malignancies. INCAGN01949 recognizes human and cynomolgus monkey OX40 with comparable binding affinity. INCAGN01949 has been optimized to potently mediate receptor forward signaling under conditions of suboptimal TCR stimulation, leading to features like enhanced production of TNFα and IFNγ, and concomitant suppression of IL-10. INCAGN01949 achieves this functionality through OX40 clustering and downstream activation of the NFκB pathway in T cells, which is sustained across a broad range of antibody concentrations. Consistent with mouse preclinical tumor models, OX40 was found to be selectively overexpressed by intratumoral regulatory T cells from a variety of primary human tumor samples. Commensurate with its human IgG1 Fc region, INCAGN01949 can effectively co-engage activating Fcγ receptors on immune effector cells, including natural killer cells and macrophages. Therefore INCAGN01949 has the potential to mediate selective effector cell activity toward intratumoral populations of regulatory T cells. The biophysical and functional attributes of INCAGN01949 make it suited for clinical development, both as a single agent and in combination with other immunomodulatory antibodies or immune education strategies. Citation Format: Ana Maria Gonzalez, Mariana L. Manrique, Ekaterina Breous, David Savitsky, Jeremy Waight, Randi Gombos, Yuqi Liu, Shiwen Lin, Taha Merghoub, Daniel Hirschhorn-Cymerman, Gerd Ritter, Jedd Wolchok, Peggy Scherle, Gregory Hollis, Reid Huber, Marc Van Dijk, Robert Stein, Nicholas S. Wilson. INCAGN01949: an anti-OX40 agonist antibody with the potential to enhance tumor-specific T-cell responsiveness, while selectively depleting intratumoral regulatory T cells. [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 3204.

Abstract CT027: A phase I, open-label study of GSK3174998 administered alone and in combination with pembrolizumab in patients (pts) with selected advanced solid tumors (ENGAGE-1)

Abstract Background: OX40 (CD134) belongs to the tumor necrosis factor receptor (TNFR) family, and transduces a costimulatory signal during T-cell activation. In the periphery, OX40 expression is limited to recently activated CD4+ and CD8+ cells. In tumors, infiltrating T lymphocytes are enriched for OX40, where it augments T-cell activation, proliferation and survival. OX40 is also expressed on tumor infiltrating regulatory T cells (Tregs). Intratumoral Treg depletion is critical for the in vivo antitumor activity of immune checkpoint antibodies. OX40 can also be found on natural killer (NK) cells, mediating antibody-dependent cellular cytotoxicity. Together, these mechanisms make stimulation of OX40 with agonistic agents an attractive target for a novel anti-cancer immunotherapy. GSK3174998 is a humanized IgG1 anti-OX40 agonistic monoclonal antibody identified through collaboration with MD Anderson Cancer Center and is currently in phase I development. Methods: The primary objective of this open-label non-randomized multicenter phase I study is to evaluate the safety of GSK3174998, as monotherapy in escalating doses every 3 weeks IV for up to 48 weeks (Part 1) or in combination with 200 mg pembrolizumab administered every 3 weeks IV for up to 2 years (Part 2). Secondary objectives include assessment of antitumor activity, pharmacokinetics, pharmacodynamics and immunogenicity. Adverse events will be monitored using NCI CTCAE v. 4.0. Radiographic imaging will be obtained every 12 weeks to assess clinical response defined by immune-related RECIST. Exploratory objectives include examination of changes in genomic DNA, RNA, protein, measures of immune function in tumor, and the association of these with antitumor activity. The study will enroll up to approximately 180 pts; eligible pts will have measurable disease with documentation of metastatic non-small cell lung cancer, squamous cell carcinoma of the head and neck, renal cell carcinoma, melanoma, bladder, soft tissue sarcoma, triple-negative breast cancer, and colorectal carcinoma displaying microsatellite instability that has progressed after standard therapy, or for which standard therapy is ineffective, intolerable, or inappropriate. A pre-treatment biopsy is requested of all pts. Matched pre-treatment and on-treatment biopsies will be performed in expansion cohorts to allow investigation of candidate biomarkers which may predict clinical response. As of Feb 1, 2016, monotherapy cohorts 1 and 2 have completed without DLT and enrollment to Cohort 3 is ongoing. This study (NCT02528357) is funded by GlaxoSmithKline. Citation Format: Jeffrey Infante, Christoph M. Ahlers, F Stephen Hodi, Sopie Postel-Vinay, Jan HM Schellens, John V. Heymach, Karen Autio, Mary Barnette, Herbert Struemper, Maura Watmuff, Elaine M. Paul, David R. Kaufman, Jeffrey S. Weber, Axel Hoos. A phase I, open-label study of GSK3174998 administered alone and in combination with pembrolizumab in patients (pts) with selected advanced solid tumors (ENGAGE-1). [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 CT027.

Abstract 4864: Engaging the immune system with GSK3174998, a potent anti-OX40 agonist antibody

Abstract Introduction: GSK3174998, a humanized IgG1 agonistic anti-OX40 monoclonal antibody (mAb) identified in collaboration between GSK and MDACC is currently in Phase I clinical development. Critical for the development of more effective cancer immunotherapy are agents that stimulate effector T cells (Teff) and inhibit the immunosuppressive function of regulatory T cells (Treg) that typically infiltrate tumors. OX40 is a tumor necrosis factor receptor superfamily member expressed on the surface of activated CD4+ and CD8+ T cells. OX40 agonism stimulates both immune effector and memory functions and attenuation of Tregs. Therefore, OX40 agonistic mAbs are ideal candidates to potentially increase the efficacy of immune-checkpoint blocking antibodies, like anti-PD1 (aPD1). Methods: GSK3174998 has suitable cross-reactivity to cynomolgus monkey OX40 to inform directly on toxicology, pharmacokinetic and pharmacodynamic (PD) preclinical endpoints. However, to understand the antitumor efficacy of OX40 agonism in vivo, studies were performed using a surrogate mAb to murine OX40 (OX86) alone or in combination with a surrogate aPD1 antibody in A20 lymphoma and CT26 colon carcinoma syngeneic tumor models. Intratumoral (i.e. tumor infiltrating T cells) and peripheral (blood) PD biomarkers, including T cell intracellular and surface protein expression, cytokine production and gene regulation were analyzed. Results: GSK3174998 was well tolerated in monkeys up to 100 mg/kg. In vitro T-cell activation of OX40 with GSK3174998 resulted in enhanced CD4+ and CD8+ effector T-cell proliferation, both in plate-bound as well as soluble PBMC assays. Suppression of Treg differentiation was observed with GSK3174998 as compared with an Fc-disabled mAb, which did not demonstrate these effects. In vitro GSK3174998 induced Th1 cytokine production (IFN and TNFá) and this was further enhanced by the addition of pembrolizumab. In vitro OX86 demonstrated similar characteristics to GSK3174998. In vivo OX86 induced a significant dose-dependent, durable anti-tumor response as monotherapy, which was significantly enhanced when combined with an aPD1 checkpoint inhibitor. Preclinical efficacy correlated with PD changes in several immunological markers including T-cell proliferation and activation. In silico and IHC analysis of expression of OX40 and PDL1 in human tumors was utilized to prioritize cancers most likely to respond to monotherapy and combination therapy for the first-time-in-human (FTIH) clinical study. Conclusions: GSK3174998 is a potent anti-OX40 agonist that engages the immune system via several T-cell-mediated pathways and may further enhance the antitumor activity observed with PD1 inhibition. Preclinical studies provide a strong rationale to support the ongoing FTIH Phase I study of GSK3174998 administered alone and in combination with pembrolizumab to patients with selected advanced solid tumors. Citation Format: Carlo Toniatti, Niranjan Yanamandra, Kui Voo, Amin Al-Shami, Laura Bover, Peter Morley, Sara Brett, Tim Lofton, Jennifer Greer, Ningping Feng, Ignacio Ivan Wistuba, Sabyasachi Bhattacharya, Christopher Hopson, David Kilian, Heather Jackson, Paul Bojczuk, Mili Mandal, Junping Jing, Kevin French, Roopa Srinivasan, Axel Hoos. Engaging the immune system with GSK3174998, a potent anti-OX40 agonist antibody. [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 4864.

Abstract 551: In situ vaccination with TLR9 agonist and anti-Ox40 antibody is sufficient to induce abscopal responses even in mice with spontaneous oncogene-driven tumors

Abstract Background: Toll-like receptors (TLRs) are components of the innate immune system that recognize pathogen-associated molecular patterns on bacterial, fungal, or viral pathogens. Intratumoral (IT) injection of unmethylated CG-enriched oligodeoxynucleotides (CpG), a TLR9 agonist, results in local tumor eradication but on its own is not able to induce a systemic anti-tumor immune response. OX40 is a potent costimulatory receptor that can potentiate the action of conventional T cells leading to their proliferation, effector function and survival, but can also inhibit or kill T regulatory cells by ADCC. In preclinical studies OX40 agonists increased antitumor immunity and improve tumor-free survival. Scientific question: Does local injection of a CpG with anti-OX40 agonistic antibody trigger a systemic anti-tumor immune response? Results: Using transplantable syngeneic tumor models, we implanted the tumor bilaterally in opposite sides of the abdomen. After tumors were established we administered CpG and anti-OX40 antibody into the tumor on one side and monitored both the injected and the uninjected sites. Mice bearing the A20 lymphoma tumors were cured and were protected from a second challenge with A20 cells. To examine the potential to treat spontaneous, non-transplanted cancers we chose the mouse mammary gland tumor model- FVB/N-Tg(MMTV-PyVT)634Mul/J. Injections of CpG and anti-OX40 antibody to the first arising tumor significantly reduced the incidence and outgrowth of subsequent tumors at un-injected susceptible mammary glands and the number of lung metastases. This anti tumor effect was T cell dependent, since depletion of either CD4+ or CD8+ T cells abrogated the therapeutic effect of in situ vaccination. Significance: TLR9 agonists and anti-OX40 antibodies are currently under clinical development for cancer treatment. We show here that combining anti-OX40 antibody with a TLR9 agonist at a single established tumor is sufficient to trigger a systemic anti-tumor response able to eradicate tumor at distant sites in both transplantable and spontaneously occurring oncogene-driven murine tumors. This anti-tumor effect was long lasting, specific and required T cells. Impact: We recently published positive results showing that the combination of CpG with antibodies against CTLA4 and OX40 induced a therapeutic immune response against transplantable syngeneic tumors (Marabelle, Levy, et al. JCI, 2013). Our current results suggest that CpG and anti-OX40 are sufficient to induce fully protective and curative anti tumor immune responses, even in spontaneously arising cancer. Anti-OX40 and CpG are both currently in phase-I clinical trials as single agents. Our results provide the rationale for testing these agents in combination in the form of locally injected inducers of therapeutic anti-cancer immunity. Citation Format: Idit Sagiv-Barfi, Debra K. Czerwinski, Ronald Levy. In situ vaccination with TLR9 agonist and anti-Ox40 antibody is sufficient to induce abscopal responses even in mice with spontaneous oncogene-driven tumors. [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 551.

Optimizing Timing of Immunotherapy Improves Control of Tumors by Hypofractionated Radiation Therapy.

The anecdotal reports of promising results seen with immunotherapy and radiation in advanced malignancies have prompted several trials combining immunotherapy and radiation. However, the ideal timing of immunotherapy with radiation has not been clarified. Tumor bearing mice were treated with 20Gy radiation delivered only to the tumor combined with either anti-CTLA4 antibody or anti-OX40 agonist antibody. Immunotherapy was delivered at a single timepoint around radiation. Surprisingly, the optimal timing of these therapies varied. Anti-CTLA4 was most effective when given prior to radiation therapy, in part due to regulatory T cell depletion. Administration of anti-OX40 agonist antibody was optimal when delivered one day following radiation during the post-radiation window of increased antigen presentation. Combination treatment of anti-CTLA4, radiation, and anti-OX40 using the ideal timing in a transplanted spontaneous mammary tumor model demonstrated tumor cures. These data demonstrate that the combination of immunotherapy and radiation results in improved therapeutic efficacy, and that the ideal timing of administration with radiation is dependent on the mechanism of action of the immunotherapy utilized.

ENGAGE-1: A first in human study of the OX40 agonist GSK3174998 alone and in combination with pembrolizumab in patients with advanced solid tumors.

TPS3107Background: OX40 (CD134) is a potent costimulatory tumor necrosis factor receptor expressed on activated CD4+ and CD8+ T cells. OX40 agonism promotes T-cell division and survival, resulting in stimulation of both immune effector and memory functions, while also blocking the suppressive function of regulatory T cells. This holds potential to overcome immune resistance and enhance immune mediated anti-tumor activity with OX40 agonism, particularly in combination with checkpoint inhibition. GSK3174998 is a humanized IgG1 anti-OX40 agonistic monoclonal antibody identified through collaboration with MD Anderson Cancer Center and is currently in phase I development. Methods: This is an open-label, non-randomized, multicenter study of GSK3174998 administered alone and in combination with pembrolizumab in patients (pts) with selected advanced or recurrent solid tumors: non-small cell lung cancer, squamous cell carcinoma of the head and neck, renal cell carcinoma, melanoma, bladder, soft tissue sarcoma, tri...