Agonistic anti-CD40 Antibody Research Articles

Murine mesothelioma induces locally-proliferating IL-10+TNF-α+CD206−CX3CR1+ M3 macrophages that can be selectively depleted by chemotherapy or immunotherapy

ABSTRACTWe used a murine model to monitor changes to myeloid cell subsets, i.e., myeloid-derived suppressor cells (MDSCs), M1 macrophages that secrete pro-inflammatory cytokines and express CD40 and CD80 and suppressive M2 macrophages that secrete anti-inflammatory cytokines and express CD206 and CX3CR1, during mesothelioma progression and during chemotherapy or immunotherapy-induced tumor regression. In vitro studies showed that mesothelioma-conditioned media generated CD206−CX3CR1+MCP-1+TGF-β+ macrophages that induced T cell proliferation but prevented T cell IFNγ production. In vivo studies showed that co-inoculation of macrophages with mesothelioma cells led to faster tumor growth, and depleting macrophages using anti-F4/80 antibody induced tumor regression. Flow cytometry revealed increasing levels of different suppressive myeloid cells in lymphoid organs: MDSCs dominated bone marrow (BM) and spleens, M2 macrophages dominated tumor-draining lymph nodes (DLN) and a mixed IL-10+TNF-α+CD206−CX3CR1+ M1/M2 (M3) macrophage subset dominated the mesothelioma microenvironment. Ki67 staining and cell cycle analysis showed that tumor-associated M1 and M3, but not M2, macrophages were proliferating in situ, with M1 cells arrested in the G1 phase while M3 cells progressed to mitosis. Immunohistochemistry showed that M1 and M3 cells were co-located supporting the hypothesis that M1 cells transition to M3 cells during proliferation. Gemcitabine reduced tumor-associated M3 and MDSCs, but not M2 macrophages, the latter likely contributing to the tumor outgrowth seen following treatment cessation. In contrast, IL-2/agonist anti-CD40 antibody therapy reduced M3 cells and polarized macrophages into M1 cells coinciding with tumor regression. These data show that myeloid cells, particularly M3 cells, represent a therapeutic target for the generation of antitumor immunity.

Local convection-enhanced delivery of an anti-CD40 agonistic monoclonal antibody induces antitumor effects in mouse glioma models.

Glioblastoma is one of the most malignant brain tumors in adults and has a dismal prognosis. In a previous report, we reported that CD40, a TNF-R-related cell surface receptor, and its ligand CD40L were associated with glioma outcomes. Here we attempted to activate CD40 signaling in the tumor and determine if it exerted therapeutic efficacy. CD40 expression was examined in 3 mouse glioma cell lines (GL261, NSCL61, and bRiTs-G3) and 5 human glioma cell lines (U87, U251, U373, T98, and A172). NSCL61 and bRiTs-G3, as glioma stem cells, also expressed the glioma stem cell markers MELK and CD44. In vitro, we demonstrated direct antitumor effects of an anti-CD40 agonistic monoclonal antibody (FGK45) against the cell lines. The efficacy of FGK45 was examined by local convection-enhanced delivery of the monoclonal antibody against each glioma model. CD40 was expressed in all mouse and human cell lines tested and was found at the cell membrane of each of the 3 mouse cell lines. FGK45 administration induced significant, direct antitumor effects in vitro. The local delivery of FGK45 significantly prolonged survival compared with controls in the NSCL61 and bRiTs-G3 models, but the effect was not significant in the GL261 model. Increases in apoptosis and CD4(+) and CD8(+) T cell infiltration were observed in the bRiTs-G3 model after FGK45 treatment. Local delivery of FGK45 significantly prolonged survival in glioma stem cell models. Thus, local delivery of this monoclonal antibody is promising for immunotherapy against gliomas.

Abstract A042: A predictive nomogram to determine CD137 upregulation on NK cells in patients receiving monoclonal antibody therapy

Abstract Monoclonal antibody (mAb) therapy, including rituximab (anti-CD20), trastuzumab (anti-HER2) and cetuximab/panitumumab (anti-EGFR) has changed the natural history of patients with B cell lymphomas, breast cancer, and colorectal and head and neck cancers, respectively. Despite their promise, response rates are suboptimal at less than 25%, highlighting the need to enhance mAb activity. Natural killer (NK) cells are important effector cells mediating antibody dependent cell-mediated cytotoxicity (ADCC), a primary antitumor mechanism of action of mAbs. Combining a tumor-targeting mAb with a second antibody that activates NK cells can improve the therapeutic effects of mAbs against tumors. We have previously validated this approach using an agonistic mAb against the co-stimulatory molecule CD137 (4-1BB), a member of the tumor necrosis factor receptor superfamily that is expressed by activated T cells and NK cells. Anti-CD137 augmented ADCC in vitro and demonstrated synergistic anti-tumor activity in multiple in vivo tumor models, including lymphoma, breast, colon, and head and neck cancers each uniquely to the targeting mAb (rituximab, trastuzumab, and cetuximab). Agonistic anti-CD137 antibodies are currently in early-phase clinical trials. In order to enhance clinical success, we sought to develop a nomogram for predicting the amplitude of CD137 upregulation following therapy with rituximab, trastuzumab, or cetuximab. This nomogram is of high importance as it has the opportunity to demonstrate the potential clinical value of anti-CD137 in combination antibody therapy by providing a biomarker of putative clinical benefit allowing selection and individualization of this therapeutic approach. On the basis of data from ongoing clinical trials of patients with lymphoma, breast, colorectal, and head and neck cancers receiving mAb therapy, we are building a nomogram that is based on patient tumor and immune characteristics identified from peripheral blood samples collected pre-and post-mAb therapy. CD137 expression on NK cells and other immune characteristics were assessed by mass cytometry time of flight (CyTOF) analysis. The model is being subjected to bootstrap internal validation, and its predictive accuracy and discriminative ability are being measured by concordance index (C-index) and risk group stratification. Given the cost of drug development and clinical trials, the nomogram will prove highly useful for drug discovery focused on identifying synergy through CD137 immunomodulation, an innovative and paradigm changing approach to improving the survival of patients with all histologies of cancer. Citation Format: Amani Makkouk, Cariad Chester, Serena Chang, Vandana Sundaram, Manisha Desai, Inna Sayfer, Holden T. Maecker, Holbrook E. Kohrt. A predictive nomogram to determine CD137 upregulation on NK cells in patients receiving monoclonal antibody therapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A042.

Dendritic cells require NIK for CD40-dependent cross-priming of CD8+ T cells

Dendritic cells (DCs) link innate and adaptive immunity and use a host of innate immune and inflammatory receptors to respond to pathogens and inflammatory stimuli. Although DC maturation via canonical NF-κB signaling is critical for many of these functions, the role of noncanonical NF-κB signaling via the serine/threonine kinase NIK (NF-κB-inducing kinase) remains unclear. Because NIK-deficient mice lack secondary lymphoid organs, we generated transgenic mice with targeted NIK deletion in CD11c(+) cells. Although these mice exhibited normal lymphoid organs, they were defective in cross-priming naive CD8(+) T cells following vaccination, even in the presence of anti-CD40 or polyinosinic:polycytidylic acid to induce DC maturation. This impairment reflected two intrinsic defects observed in splenic CD8(+) DCs in vitro, namely antigen cross-presentation to CD8(+) T cells and secretion of IL-12p40, a cytokine known to promote cross-priming in vivo. In contrast, antigen presentation to CD4(+) T cells was not affected. These findings reveal that NIK, and thus probably the noncanonical NF-κB pathway, is critical to allow DCs to acquire the capacity to cross-present antigen and prime CD8 T cells after exposure to licensing stimuli, such as an agonistic anti-CD40 antibody or Toll-like receptor 3 ligand.

Activation of innate and adaptive immunity as an effective combined strategy for cancer immunotherapy

Immunotherapeutic approaches can demonstrate some antitumor benefit, but their efficacy is limited when they are used as a single modality. We asked if a combinatory approach activating both innate and adaptive immunity would improve cancer immunotherapy. We have previously shown that an agonistic anti-CD40 monoclonal antibody (anti-CD40) in combination with a toll-like receptor 9 agonist, CpG, can activate macrophages in mice, leading to tumor cell killing. Separately, we have shown that a direct intratumoral injection of an immunocytokine (IC) consisting of anti-GD2 antibody linked to interleukin-2 can activate NK and T cells, resulting in antitumor effects. We hypothesize that activation of macrophages (with anti-CD40/CpG) and NK cells (with IC) will increase tumor destruction and presentation of tumor antigens, leading to T cell activation, which in turn could be further augmented by anti-CTLA-4 antibody, resulting in tumor eradication and prevention of tumor recurrence. Using the mouse GD2+ B78 melanoma model, we show that anti-CD40/CpG and IC/anti-CTLA-4 synergistically induced regression of established subcutaneous tumors, resulting in the cure of 50% of mice and development of immunological memory against B78 as well as wild type B16 tumors. While the antitumor effect of anti-CD40/CpG was T cell independent, the antitumor effect of IC/anti-CTLA-4 required T cells. Anti-CD40/CpG treatment led to upregulation of T cell activation markers in draining lymph nodes. Finally, the combined treatment with anti-CD40/CpG and IC/anti-CTLA-4 was effective against B16 lung metastases. We suggest that a combination of anti-CD40/CpG and IC/anti-CTLA-4 should be tested as a clinically relevant novel treatment strategy.

Generation of a soluble recombinant trimeric form of bovine CD40L and its potential use as a vaccine adjuvant in cows

Vaccination is the most cost-effective way to control infectious diseases in cattle. However, many infectious diseases leading to severe economical losses worldwide still remain for which a really effective and safe vaccine is not available. These diseases are most often due to intracellular pathogens such as bacteria or viruses, which are, by their localization, protected from antibiotics and/or CD4+ T cell-dependent humoral responses. We therefore postulated that strategies leading to induction of not only CD4+ T cell responses but also CD8+ cytotoxic T lymphocyte (CTL) responses against infected cells should be privileged in the development of new vaccines against problematic intracellular pathogens in bovines. CD40 signaling in antigen-presenting cells may lead to the induction of robust CD4-independent CTL responses and several studies, especially in mice, have used CD40 stimulation to promote CD8+ T cell-mediated immunity. For example, we have recently shown that immunization of mice with heat-killed Staphylococcus aureus (HKSA) and agonistic anti-CD40 monoclonal antibodies elicits strong CTL responses capable of protecting mice from subsequent staphylococcal mastitis. Unfortunately, there is at present no tool available to efficiently stimulate CD40 in cattle. In this study, we therefore first produced a soluble recombinant trimeric form of the natural bovine CD40 ligand (sboCD40LT). We then observed that sboCD40LT was able to potently stimulate bovine cells in vitro. Finally, we provide evidence that immunization of cows with sboCD40LT combined with HKSA was able to significantly increase the number of both HKSA-specific CD4+ and CD8+ T cells in the draining lymph nodes. In conclusion, we suggest that this new molecular tool could help in the development of vaccine strategies against bovine diseases caused by intracellular pathogens.

Synergy between CD40 and MyD88 Does Not Influence Host Survival to Salmonella Infection.

Previous studies using purified toll-like receptor (TLR) ligands plus agonistic anti-CD40 antibodies showed that TLRs and CD40 can act synergistically on dendritic cells (DCs) to optimize T cell activation and Th1 differentiation. However, a synergistic effect of TLRs and CD40 during bacterial infection is not known. Here, we show that mice lacking the TLR adaptor MyD88 alone, or lacking both MyD88 and CD40 [double knockout (DKO) mice], are compromised in survival to Salmonella infection but have intact recruitment of neutrophils and inflammatory monocytes as well as unaltered abundance of DC subsets and DC activation in infected tissues. In contrast to infected wildtype and CD40−/− mice, both MyD88−/− mice and DKO mice lack detectable serum IFN-γ and have elevated IL-10. A synergistic effect of TLRs and CD40 was revealed in co-culture experiments where OT-II T cell proliferation was compromised when DKO DCs were pulsed with OVA protein and OVA323–339 peptide, but not with heat-killed Salmonella expressing OVA (HKSOVA), relative to MyD88−/− DCs. By contrast, MyD88−/− or DKO DCs pulsed with any of the antigens had a similar ability to induce IFN-γ that was lower than WT or CD40−/− DCs. DKO DCs pulsed with HKSOVA, but not with OVA or OVA323–339, had increased IL-10 relative to MyD88−/− DCs. Finally, HKSOVA-pulsed MyD88−/− and DKO DCs had similar and low induction of NFκB-dependent and -independent genes upon co-culture with OT-II cells. Overall, our data revealed that synergistic effects of CD40 and MyD88 do not influence host survival to Salmonella infection or serum levels of IFN-γ or IL-10. However, synergistic effects of MyD88 and CD40 may be apparent on some (IL-10 production) but not all (OT-II proliferation and IFN-γ production) DC functions and depend on the complexity of the antigen. Indeed, synergistic effects observed using purified ligands and well-defined antigens may not necessarily apply when complex antigens, such as live bacteria, challenge the immune system.

Vaccine-induced but not tumor-derived Interleukin-10 dictates the efficacy of Interleukin-10 blockade in therapeutic vaccination

ABSTRACTBlocking antibodies against immunosuppressive molecules have shown promising results in cancer patients. However, there are not enough data to define those conditions dictating treatment efficacy. In this scenario, IL-10 is a cytokine with controversial effects on tumor growth. Thus, our aim was to characterize in which setting IL-10 blockade may potentiate the beneficial effects of a therapeutic vaccine In the IL-10-expressing B16-OVA and TC-1 P3 (A15) tumor models, therapeutic vaccination with tumor antigens plus the TLR7 ligand Imiquimod increased IL-10 production. Although blockade of IL-10 signal with anti-IL-10R antibodies did not inhibit tumor growth, when combined with vaccination it enhanced tumor rejection, associated with stronger innate and adaptive immune responses. Interestingly, a similar enhancement on immune responses was observed after simultaneous vaccination and IL-10 blockade in naive mice. However, when using vaccines containing as adjuvants the TLR3 ligand poly(I:C) or anti-CD40 agonistic antibodies, despite tumor IL-10 expression, anti-IL-10R antibodies did not provide any beneficial effect on tumor growth and antitumor immune responses. Of note, as opposed to Imiquimod, vaccination with this type of adjuvants did not induce IL-10 and correlated with a lack of in vitro IL-10 production by dendritic cells (DC). Finally, in B16-OVA-bearing mice, blockade of IL-10 during therapeutic vaccination with a multiple adjuvant combination (MAC) with potent immunostimulatory properties but still inducing IL-10 led to superior antitumor immunity and complete tumor rejection. These results suggest that for therapeutic antitumor vaccination, blockade of vaccine-induced IL-10 is more relevant than tumor-associated IL-10.

Abstract 2473: KIR3DL2 is a novel target for antibody-therapy of T cell lymphomas

Abstract Intro: The killer immunoglobulin-like receptor, three domains, long cytoplasmic tail 2 (KIR3DL2) is a transmembrane glycoprotein expressed by natural killer (NK) cells and subsets of T cells. On NK cells, KIRD3L2 modulates NK cell activation by transducing inhibitory signals upon ligation by human leukocyte antigen (HLA) class I molecules. Interestingly, KIR3DL2 is also expressed on several subtypes of T cell lymphomas/leukemias, such as Sézary Syndrome and transformed Mycosis Fungoides. This expression pattern provides a promising target for antibody-based therapy. Recently, antibody-based therapies have been shown to synergize with co-stimulatory immunomodulation via agonistic immunotherapeutic antibodies, such as anti-CD137. Methods: We evaluated the efficacy of an anti-KIR3DL2 monoclonal antibody, IPH4102 (Innate Pharma S.A.), in inducing effector cell cytotoxicity of KIR3DL2+ tumors. In vitro experiments utilized primary samples and three KIR3DL2+ cell lines: HUT78, a Sézary cell line with constitutive KIR3DL2 expression; a RAJI-KIR3DL2 high-expression sub clone with ∼28,000 KIR3DL2 antigen sites per cell; and a RAJI-KIR3DL2 low-expression sub clone with ∼ 6,000 antigens sites per cell. All antibody-mediated cell cytotoxicity (ADCC) experiments used 7-AAD and Annexin-V to identify apoptotic cells. Results: In a CD107a /IFN-γ assay, NK cells co-cultured with IPH4102-coated HUT78 cells demonstrated increased activation profiles relative to NK cells co-cultured with untreated HUT78 cells. Activated NK cells displayed elevated expression of CD137, OX40, and CD69 and produced more CD107a and IFN-γ. In allo-ADCC assays using HUT78 cells at varying target to effector ratios, IPH4102 treatment induced more target apoptosis than treatment with positive control anti-CD52 alemtuzumab. In additional allo-ADCC assays, RAJI-KIR3DL2 high-expression tumors demonstrated more apoptosis than RAJI-KIR3DL2 low-expression tumors. The IPH4102-mediated cytotoxicity could be enhanced by the concurrent administration of an agonistic anti-CD137 monoclonal antibody. In ADCC assays of primary Sézary cells with autologous NK cells, we confirmed the upregulation of KIR3DL2 and observed enhanced NK cytotoxicity following tumor treatment with IPH4102. Again, the combination of anti-KIR3DL2 and anti-CD137 therapy proved synergistic leading to increased tumor eradication relative to either therapy alone. Conclusion: Our findings support a role for anti-KIR3DL2 monoclonal antibodies in the treatment of KIR3DL2+ tumors. IPH4102 effectively increased ADCC of KIR3DL2+ tumors by NK cells. The addition of agonistic anti-CD137 antibodies will further augment the anti-tumor efficacy of this therapeutic strategy. Citation Format: Cariad Chester, Sean Lim, Anne Marie-Cardine, Naren Rajasekaran, Hélène Sicard, Youn Kim, Holbrook Kohrt. KIR3DL2 is a novel target for antibody-therapy of T cell lymphomas. [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 2473. doi:10.1158/1538-7445.AM2015-2473

Abstract 289: The combination of CD40 agonism and PD-L1 blockade enhances anti-tumor immunity in a mouse syngeneic orthotopic pancreatic tumor model

Abstract Infiltration of immunosuppressive myeloid cells into tumors correlates with poorer prognosis in many cancer types. The immunosuppressive effects of these cells help to create a microenvironment that may inhibit responses to T cell-directed immunotherapies. Anti-CD40 agonistic antibodies (αCD40) promote myeloid cell maturation, license cross-presentation of antigens to cytotoxic T cells, and enhance direct macrophage tumoricidal activity. We tested the hypothesis that αCD40 can improve responses to PD-L1 blockade via enhancing anti-tumor immunity in a syngeneic orthotopic mouse model of pancreatic cancer (Pan02). Whilst PD-L1 blockade alone had limited effect on tumor growth, we demonstrated that αCD40 drove antigen presenting cell maturation and systemic immune activation, inhibiting tumor growth and improving overall survival. αCD40 also induced upregulation of PD-L1 both systemically and within the tumor microenvironment. Furthermore, the combination of αCD40 and PD-L1 blockade led to enhanced tumor growth inhibition and improved anti-tumor immunity compared to either monotherapy alone. These data provide further support for the potential benefits of re-educating tumor myeloid cells. Citation Format: Nadia Luheshi, Jane Coates-Ulrichsen, James Harper, Gareth Davies, James Legg, Robert Wilkinson. The combination of CD40 agonism and PD-L1 blockade enhances anti-tumor immunity in a mouse syngeneic orthotopic pancreatic tumor model. [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 289. doi:10.1158/1538-7445.AM2015-289

Abstract SY21-04: Starting an immune response against pancreatic cancer: Getting past the stroma

Abstract Pancreatic ductal adenocarcinoma (PDA) is a highly aggressive and lethal cancer which is poorly responsive to standard therapies. Although the PDA tumor microenvironment is considered especially immunosuppressive, recent data mostly from genetically engineered and other mouse models of the disease suggest that novel immunotherapeutic approaches hold promise. Here, both laboratory and clinical efforts to target the CD40 pathway for immunotherapy in PDA will be described. Findings suggest that CD40 agonists can mediate both T-cell-dependent and T-cell-independent immune mechanisms of tumor regression in mice and patients. T-cell-independent mechanisms are associated with macrophage activation and the destruction of PDA tumor stroma, but these effects are transient. T cell-dependent mechanisms can be elicited with novel chemotherapy combinations and further extended by checkpoint blockade (with anti-PD-1 and/or anti-CTLA4). In the absence of anti-CD40 agonist antibody, neither chemotherapy nor checkpoint blockade can achieve meaningful tumor regressions nor long-term remissions, but inclusion of anti-CD40 can establish immune memory and cures in murine models. Overall, immune modulation of the PDA tumor microenvironment represents a useful approach in cancer immunotherapy. Citation Format: Robert H. Vonderheide. Starting an immune response against pancreatic cancer: Getting past the stroma. [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 SY21-04. doi:10.1158/1538-7445.AM2015-SY21-04

Abstract 2472: SEA-CD40, a sugar engineered non-fucosylated anti-CD40 antibody with improved immune activating capabilities

Abstract SEA-CD40 is a non-fucosylated, humanized IgG1 monoclonal antibody directed against human CD40, a co-stimulatory receptor of the TNF receptor superfamily. SEA-CD40 is derived from dacetuzumab, a humanized IgG1 previously developed and studied for B-lineage malignancies. Glycosylation of the antibody Fc is essential for Fc receptor-mediated activity and non-fucosylated antibodies show improved efficacy, particularly via increased binding to low affinity FcγRIIIa. Enhanced functionality of SEA-CD40 was determined through FcγRIIIa binding affinity, antibody-dependent cellular cytotoxicity (ADCC) activity, activation of the immune response, and induction of antigen-specific T-cells. While SEA-CD40 and the parent antibody dacetuzumab bind to CD40 with similar affinity, the non-fucosylated SEA-CD40 binds equally well to the low (158F) and high (158V) affinity versions of FcγRIIIa with higher affinity than dacetuzumab. The consequence of enhanced SEA-CD40/FcγRIIIa binding is potent ADCC activity against a CD40+ lymphoma B cell line and improved agonistic signaling to antigen presenting cells (APCs). SEA-CD40 treatment of human PBMCs elicits a robust immune response as measured by increased cytokine production and up-regulation of maturation markers on APCs with maintained activity at antibody concentrations as low as 10 ng/ml. The immune stimulatory properties of SEA-CD40 were observed in vivo as increased activity in xenograft and syngenic tumor models as well as induction of cytokine production in cynomolgus monkeys. Both in-vitro and in-vivo activity of SEA-CD40 was significantly greater than with dacetuzumab. The increased functionality of SEA-CD40 occurs through the non-fucosylated Fc domain as a F(ab’)2 version loses the ability to induce ADCC, stimulate cytokines, or up-regulate APC activation markers. SEA-CD40 induction of antigen specific T-cells was assessed using human peripheral blood mononuclear cells (PBMCs) exposed to influenza antigen. In the presence of SEA-CD40 influenza specific T-cells mount a robust antigen-specific response characterized by tetramer staining and elevated production of IFNγ. SEA-CD40 activity on PBMCs from donors with melanoma, pancreatic, or breast cancer was assessed and tumor antigen specific T-cell proliferation and IFNγ production was observed. SEA-CD40 is a non-fucosylated agonistic anti-CD40 antibody that shows enhanced binding to FcγRIIIa resulting in amplified cytokine production, co-stimulatory molecule up regulation, and ultimately stimulation of antigen specific T-cell responses to viral and tumor antigens. Citation Format: Shyra J. Gardai, Angela Epp, Germein Linares, Lori Westendorf, May Sutherland, Haley Neff-LaFord, Stanford L. Peng, Che-Leung Law. SEA-CD40, a sugar engineered non-fucosylated anti-CD40 antibody with improved immune activating capabilities. [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 2472. doi:10.1158/1538-7445.AM2015-2472

Boosting Cancer Immunotherapy with Anti-CD137 Antibody Therapy.

In the past 5 years, immunomodulatory antibodies have revolutionized cancer immunotherapy. CD137, a member of the tumor necrosis factor receptor superfamily, represents a promising target for enhancing antitumor immune responses. CD137 helps regulate the activation of many immune cells, including CD4(+) T cells, CD8(+) T cells, dendritic cells, and natural killer cells. Recent studies indicate that the antitumor efficacy of therapeutic tumor-targeting antibodies can be augmented by the addition of agonistic antibodies targeting CD137. As ligation of CD137 provides a costimulatory signal in multiple immune cell subsets, combination therapy of CD137 antibody with therapeutic antibodies and/or vaccination has the potential to improve cancer treatment. Recently, clinical trials of combination therapies with agonistic anti-CD137 mAbs have been launched. In this review, we discuss the recent advances and clinical promise of agonistic anti-CD137 monoclonal antibody therapy.

Controlled release of a model vaccine by nanoporous ceramic microneedle arrays

Current vaccination technology can advance from the use of novel ceramic nanoporous microneedle arrays (npMNA), where the material serves as a storage reservoir for vaccines. Moreover, npMNA will enhance vaccine efficacy by more precisely reaching skin dendritic cells, the kickstarters of T and B cell immunity. In the present study we assessed the efficacy of vaccination using npMNAs by in vivo application of OVA257-264 peptides mixed with agonistic anti-CD40 antibodies as adjuvant. The induction of OVA-specific CD8+ T cells via npMNA was comparable with the frequency induced via intradermal injection using needle-syringe. However, only when expanding the vaccination area by using two npMNAs the frequencies of induced IFN-γ-specific effector CD8+ T cells were comparable with those induced via needle-syringe injection. Analysis of vaccine release from npMNA in a human ex vivo skin explant model revealed that OVA257-264 peptides were indeed delivered intradermal, and release also increased by prolonging the npMNA application time on the human skin. Together, our studies demonstrate the potential of npMNA for vaccine delivery in human skin and in vivo induction of CD8+ effector T cell responses.

Functional expression of CD137 (4-1BB) on T helper follicular cells

CD137 (4-1BB) is a surface protein initially discovered to mark activated T lymphocytes. However, its broader expression pattern also encompasses activated NK cells, B cells and myeloid cells, including mature dendritic cells. In this study, we have immunostained for CD137 on paraffin-embedded lymphoid tissues including tonsils, lymph nodes, ectopic tertiary lymphoid tissue in Hashimoto thyroiditis and cancer. Surprisingly, immunostaining mainly decorated intrafollicular lymphocytes in the tissues analyzed, with only scattered staining in interfollicular areas. Moreover, pathologic lymphoid follicles in follicular lymphoma and tertiary lymphoid tissue associated with non-small cell lung cancer showed a similar pattern of immunostaining. Multispectral fluorescence cytometry demonstrated that CD137 expression was restricted to CD4+ CXCR5+ follicular T helper lymphocytes (TFH cells) in tonsils and lymph nodes. Short-term culture of lymph node cell suspensions in the presence of either an agonistic anti-CD137 monoclonal antibody (mAb) or CD137-ligand stimulated the functional upregulation of TFH cells in 3 out of 6 cases, as indicated by CD40L surface expression and cytokine production. As a consequence, immunostimulatory monoclonal antibodies targeting CD137 (such as urelumab and PF-05082566) should be expected to primarily act on this lymphocyte subset, thus modifying ongoing humoral immune responses in patients with autoimmune disease and cancer.

Systemic Agonistic Anti-CD40 Treatment of Tumor-Bearing Mice Modulates Hepatic Myeloid-Suppressive Cells and Causes Immune-Mediated Liver Damage.

Immune-stimulatory mAbs are currently being evaluated as antitumor agents. Although overall toxicity from these agents appears to be moderate, liver toxicities have been reported and are not completely understood. We studied the effect of systemic CD40 antibody treatment on myeloid cells in the spleen and liver. Naïve and tumor-bearing mice were treated systemically with agonistic anti-CD40 antibody. Immune cell subsets in the liver and spleen, serum transaminases, and liver histologies were analyzed after antibody administration. Nox2(-/-), Cd40(-/-), and bone marrow chimeric mice were used to study the mechanism by which agonistic anti-CD40 mediates its effects in vivo. Suppressor function of murine and human tumor-induced myeloid-derived suppressor cells (MDSC) was studied upon CD40 ligation. Agonistic CD40 antibody caused liver damage within 24 hours after injection in two unrelated tumor models and mice strains. Using bone marrow chimeras, we demonstrate that CD40 antibody-induced hepatitis in tumor-bearing mice was dependent on the presence of CD40-expressing hematopoietic cells. Agonistic CD40 ligation-dependent liver damage was induced by the generation of reactive oxygen species. Furthermore, agonistic CD40 antibody resulted in increased CD80-positive and CD40-positive liver CD11b(+)Gr-1(+) immature myeloid cells. CD40 ligation on tumor-induced murine and human CD14(+)HLA-DR(low) peripheral blood mononuclear cells from patients with cancer reduced their immune suppressor function. Collectively, agonistic CD40 antibody treatment activated tumor-induced myeloid cells, caused myeloid-dependent hepatotoxicity, and ameliorated the suppressor function of murine and human MDSC. Collectively, our data suggest that CD40 may mature immunosuppressive myeloid cells and thereby cause liver damage in mice with an accumulation of tumor-induced hepatic MDSC.

Combination of anti-CD73 and agonistic anti-CD137 antibody therapy leads to tumor regression (TUM2P.1030)

Abstract Agonist antibodies (Ab) directed against costimulatory molecule CD137 (4-1BB) on the surface of antigen-primed T-lymphocytes are currently in various stages of pre-clinical and clinical development, though therapeutic benefit has been limited as single agents. Here, we examined the inhibitory role of CD73 on the antitumor efficacy of agonistic anti-CD137 Ab therapy. CD73-deificient mice treated with anti-CD137 Ab completely rejected CD73-nonexpressing B16-SIY melanoma growth when compared with wild-type mice, highlighting the importance of an inhibitory role for host-derived CD73. To explore the therapeutic relevance of these observations, we combined anti-CD137 and anti-CD73 Ab to treat mice with established B16-SIY melanoma. This combination therapy induced tumor regression associated with enhanced antitumor CD8+ T cell responses and reduced CD4+Foxp3+ regulatory T cells number and function. In contrast, neither anti-41BB nor anti-CD73 monotherapy is effective to control the growth of established melanoma. Our study demonstrates that CD73 blockade has critical implications for effective clinical targeting of CD137 and possibly other costimulatory molecules.

Abstract O.27: The Role Of CD40L And CD40 In The Pathogenesis Of Kawasaki Disease

Kawasaki Disease (KD) is characterized by acute, systemic inflammation followed by a persistent immune response localized to the coronary arteries. Lactobacillus Casei Cell Wall Extract (LCWE) contains among its active ingredients a superantigen (SAg) that induces coronary arteritis in mice. LCWE induces massive immune activation followed by apoptosis or anergy. Previous work has shown that co-stimulatory signals can rescue a subset of SAg-reactive T-cells from apoptosis. CD40-CD40L (ligand) are members of the TNF superfamily which play a role in T-cell co-stimulation. Moreover, genome wide association studies have implicated CD40/CD40L as candidate genes in the pathogenesis of KD. We hypothesize that co-stimulation via the CD40 pathway regulates survival of SAg-reactive T-cells during disease development. Using thymidine proliferation assay and flow cytometry we show that activation of CD40 signaling via agonistic anti-CD40 antibody or recombinant soluble CD40L (rsCD40L) upregulates CD86 (ligand responsible for CD28 signaling) expression, reduces active-caspase-3 expression, indicating decreased cell death and promotes lymphocyte proliferation, Additionally, using CD28 deficient mice we show that lymphocytes do not proliferate in response to SAg plus CD40 stimulation indicating that CD28 is the intermediary that mediates the proliferative effects of CD40 activation. Furthermore, platelets, which are markedly elevated in KD, produce approximately 90% of the soluble CD40L in the circulation. We show that activated platelets (CD62P+) express CD40L and promotes cell survival as evidenced by enhanced lymphocyte proliferation. Likewise, the absence of CD40L negates the proliferative effects of platelet mediated proliferation. Lastly, to determine the role of CD40 stimulation on in vivo development of KD, LCWE was injected into B6 and CD40L deficient mice. We show that inflammation in the coronary arteries does not develop in the absence of CD40L. Therefore, CD40L plays a critical role in immune activation via upregulation of CD86 on antigen presenting cells leading to enhanced co-stimulation and T-cell survival.

Mo1760 Pharmacology of the Anti-CD40 Agonist Acute Colitis Model

Originally described by Uhlig et. al., the anti-CD40 agonist acute model of gut inflammation is a useful tool for interrogating myeloid-driven gut inflammation. Previous studies have shown that this model is sensitive to intervention with anti-TNFα, anti-IFNγ and anti-IL12/ 23p40. Here we present data that extend previous findings in this model and demonstrate an anti-CD40 dose dependent severity of disease pathogenesis as well as dose response curves for treatment with anti-TNFα and anti-IL12/23p40. In addition, we have identified both circulating and fecal biomarkers of disease. Methods: RAG2 deficient mice on the C57BL/6 background were injected i.p. with a single dose of anti-CD40 agonist antibody (FGK45) on day 0 and monitored daily for body weight loss until day 7. Prophylactic treatment with either anti-TNFα or anti-IL12/23p40 was performed using i.p. injections on day -1 and day 2 prior to sacrifice at day 7. Spleens were collected and weights recorded, plasma and feces were also collected for drug exposure levels and biomarker analysis. The colon was removed, flushed with PBS and buffered formalin prior to histological sectioning and scoring. Results: Mice treated with anti-CD40 (single dose 1.25-20mpk) exhibited a dose-dependent increase in splenomegaly, colonic inflammation and liver pathology with an induction ED50 of 2.5mpk. In addition, plasma levels of CXCL-10, MCP-1, MIG and other inflammatory mediators were also increased dose-dependently with CXCL-10 levels highly correlating with histology scores (r2 = 0.85). Fecal calprotectin (S100A8-S100A9 dimer), a biomarker routinely used in the clinic, also increased dose-dependently and correlated well with histologic scores (r2 = 0.72 and 0.68 respectively). As significant liver necrosis was observed at doses of anti-CD40 above 5mpk, this dose was selected for intervention studies. Prophylactic treatment with either anti-IL12/23p40 or anti-TNFα attenuated colonic pathology in a dose-dependent manner at day 7 of disease with an EC50 of 100 μg/mL for anti-p40. Therapeutic intervention (i.e. a single injection given at day 3 of disease) with anti-TNFα also significantly reduced colon histopathology scores while antiIL-12/23p40 only showed therapeutic significance at the highest dose (EC50 of 7 and 1.3 μg/mL respectively). Reduced colonic inflammation in all cases was accompanied by a reduction in circulating CXCL-10 levels. In summary, we have shown that the anti-CD40 acute model of gut inflammation is a useful drug discovery model for interrogating myeloid mechanisms involved in colitis and have identified circulating CXCL-10 and fecal calprotectin as biomarkers related to both pathogenesis and intervention in this model. We have also generated dose-efficacy relationships for clinically validated therapies such as anti-TNF and anti-IL12/23p40.

Synergy of anti-CD40, CpG and MPL in activation of mouse macrophages

Activation of macrophages is a prerequisite for their antitumor effects. Several reagents, including agonistic anti-CD40 monoclonal antibody (anti-CD40), CpG oligodeoxynucleotides (CpG) and monophosphoryl lipid A (MPL), can stimulate activation of macrophages. Our previous studies showed synergy between anti-CD40 and CpG and between anti-CD40 and MPL in macrophage activation and antitumor efficacy in mice. In the present study, we asked whether there was synergy among these three reagents. The activation of adherent peritoneal exudate cells (PEC) obtained from mice injected with anti-CD40 and then treated with CpG and/or MPL in vitro was determined by their ability to suppress proliferation of tumor cells and to produce various cytokines and chemokines in vitro. Cell sorting and histology followed by functional testing showed that macrophages were the main cell population in PEC activated by CD40 ligation in vivo. A combination of anti-CD40, CpG or MPL activated PEC to suppress proliferation of B16 cells and produce nitric oxide far greater than the single reagents or any of the double combinations of these reagents. In addition, the combination of all three reagents activated PEC to secrete IL-12, IFN-γ and MCP-1 to a greater degree than any single reagent or any two combined reagents. These results demonstrate that macrophages can be synergistically activated by anti-CD40, CpG and MPL, suggesting that this novel combined approach might be further investigated as potential cancer therapy.