Macrophage Tumoricidal Activity Research Articles

Abstract LB-194: First-in-human Phase I study of the CD40 agonist mAb CDX-1140 and in combination with CDX-301 (rhFLT3L) in patients with advanced cancers: Interim results

Abstract CDX-1140 is an agonist CD40 mAb intended to promote priming of CD8+ effector T cells through activation of dendritic cells (DC), enhance macrophage tumoricidal activity, and mediate direct killing of CD40-expressing tumor cells. CDX-1140 is a human IgG2 mAb with Fc receptor independent activity and is synergistic with sCD40L, suggesting a potential to enhance CD40L-mediated immune activation. CDX-1140 has a broad dose-dependent activity that may achieve maximal agonist activity at doses that provide significant tumor penetration with minimal toxicities from systemic CD40 activation. CDX-301 (rhFLT3L) is a DC growth factor that markedly increases CD141+ DCs, including in the tumor microenvironment (TME). CD141+ DCs are critical for initiating anti-tumor immune responses and are often depleted within the TME. Preclinical studies have shown that treatment with CDX-301 synergizes with CD40 signaling to enhance anti-tumor immune responses, suggesting that combining CDX-1140 and CDX-301 could result in enhanced clinical benefit. CDX1140-01(NCT03329950) is a Phase I dose-escalation study of CDX-1140 as monotherapy or in combination with CDX-301 evaluating safety, PK, PD, and preliminary clinical activity in patients (pts) with refractory solid tumors and non-Hodgkin lymphoma. CDX-1140 is given iv q 4 weeks at doses from 0.01 to 3 mg/kg utilizing a 3+3 design (first 2 cohorts are single-pts). In combination cohorts (solid tumors only), CDX-301 at 75 μg/kg daily x 5 is administered sc prior to cycles 1 and 2 of escalating doses of CDX-1140 starting at 0.09 mg/kg. Tumor-specific expansion cohorts are planned to further explore the activity of monotherapy or combination treatment. To date, 18 pts across 10 tumor types have been dosed in CDX-1140 monotherapy cohorts with doses ranging from 0.01 to 0.72 mg/kg and without identification of an MTD. There has been one DLT (0.18 mg/kg): grade 3 pneumonitis and hypoxia. One pt (0.72 mg/kg) experienced grade 3 fatigue requiring hospitalization 48 hrs after the first infusion. There have been 3 additional grade 3 treatment-related adverse events: dyspnea (0.18 mg/kg), nausea (0.36 mg/kg), and vomiting (0.36 mg/kg). The first combination cohort (n=5) has been completed. In monotherapy and combination cohorts there have been no significant drug-related changes in liver function tests to date. CDX-1140 PK is quantifiable at doses ≥ 0.09 mg/kg and exposure appears dose proportional. Expected dose-dependent PD effects have shown activation of peripheral blood lymphocytes and increases in pro-inflammatory cytokines and chemokines. Combination with CDX-301 has shown enhanced cytokine responses. The data suggest that CDX-1140 has the expected biologic and safety profile predicted from preclinical studies and may achieve dose levels optimal for systemic exposure. The addition of CDX-301 may enhance the activity of CDX-1140. Citation Format: Rachel E. Sanborn, Nashat Y. Gabrail, Nina Bhardwaj, Michael S. Gordon, Mark O'Hara, Danny Khalil, Thomas Hawthorne, Richard Gedrich, Laura Vitale, Mark Rogalski, Tianshu Li, Tracey Rawls, Tibor Keler, Michael Yellin. First-in-human Phase I study of the CD40 agonist mAb CDX-1140 and in combination with CDX-301 (rhFLT3L) in patients with advanced cancers: Interim results [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-194.

Both Type I and Type II Interferons Can Activate Antitumor M1 Macrophages When Combined With TLR Stimulation.

Triggering or enhancing antitumor activity of tumor-associated macrophages is an attractive strategy for cancer treatment. We have previously shown that the cytokine interferon-γ (IFN-γ), a type II IFN, could synergize with toll-like receptor (TLR) agonists for induction of antitumor M1 macrophages. However, the toxicity of IFN-γ limits its clinical use. Here, we investigated whether the less toxic type I IFNs, IFN-α, and IFN-β, could potentially replace IFN-γ for induction of antitumor M1 macrophages. We measured in vitro the ability of type I and II IFNs to synergize with TLR agonists for transcription of inducible nitric oxide synthase (iNOS) mRNA and secretion of nitric oxide (NO) by mouse bone marrow-derived macrophages (BMDMs). An in vitro growth inhibition assay was used to measure both cytotoxic and cytostatic activity of activated macrophages against Lewis lung carcinoma (LLC) cancer cells. We found that both type I and II IFNs could synergize with TLR agonists in inducing macrophage-mediated inhibition of cancer cell growth, which was dependent on NO. The ability of high dose lipopolysaccharide (LPS) to induce tumoricidal activity in macrophages in the absence of IFN-γ was shown to depend on induction of autocrine type I IFNs. Antitumor M1 macrophages could also be generated in the absence of IFN-γ by a combination of two TLR ligands when using the TLR3 agonist poly(I:C) which induces autocrine type I IFNs. Finally, we show that encapsulation of poly(I:C) into nanoparticles improved its potency to induce M1 macrophages up to 100-fold. This study reveals the potential of type I IFNs for activation of antitumor macrophages and indicates new avenues for cancer immunotherapy based on type I IFN signaling, including combination of TLR agonists.

Killed Propionibacterium acnes enhances immunogenicity and tumor growth control of a dendritic-tumor cell hybrid vaccine in a murine melanoma model.

Hybrid vaccines have been investigated in clinical and experimental studies once expresses total antigens of a tumor cell combined with the ability of a dendritic cell (DC) to stimulate immune responses. However, the response triggered by these vaccines is often weak, requiring the use of adjuvants to increase vaccine immunogenicity. Killed Propionibacterium acnes (P. acnes) exerts immunomodulatory effects by increasing the phagocytic and tumoricidal activities of macrophages, promoting DC maturation, inducing pro-inflammatory cytokines production and increasing the humoral response to different antigens. Here, we evaluated the effect of P. acnes on a specific antitumor immune response elicited by a hybrid vaccine in a mouse melanoma model. Hybrid vaccine associated with P. acnes increased the absolute number of memory T cells, the IFN-γ secretion by these cells and the IgG-specific titers to B16F10 antigens, polarizing the immune response to a T helper 1 pattern. Furthermore, the addition of P. acnes to a hybrid vaccine increased the cytotoxic activity of splenocytes toward B16F10 in vitro and avoided late tumor progression in a pulmonary colonization model. These results revealed the adjuvant effect of a killed P. acnes suspension, as it improved specific humoral and cellular immune responses elicited by DC-tumor cell hybrid vaccines.

Abstract 3800: Inhibition of Polycomb Repressive Complex 2 EZH2 lysine methyltransferase improves tumoricidal activity of macrophages towards mesothelioma cells

Abstract Rationale: Clinical evidence indicates that tumor infiltration by tumor associated macrophages (TAMs) correlates with poor prognosis in malignant mesothelioma (MM). By attenuating the immune response, TAMs indeed promote survival of MM cells. TAMs share properties with alternative macrophages (M2) that are activated by anti-inflammatory (e.g. IL-10) or Th2-associated (i.e. IL-4, IL-13) cytokines. Objectives: We hypothesized that macrophage activation is mediated by a transcriptional program tightly regulated by epigenetic modifications. We focused on the Polycomb Repressive Complex 2 (PRC2) EZH2 lysine methyltransferase responsible for trimethylation of histone H3 at lysine 27 (H3K27me3). We investigated the effect of a selective EZH2 inhibitor (EPZ005687) on tumoricidal activity of primary human monocyte-derived macrophages. Results: Our data show that inhibition of EZH2 reduces CD206 expression and phagocytic activity of macrophages conditioned in MM pleural effusions (PE-macrophages). Moreover, EZH2 inhibition enhances the cytotoxic potential of PE-macrophages towards MM cells. Inhibition of EZH2 also increases the ability of macrophages to support proliferation of autologous T-cells. Conclusion: Induction of cytotoxic activity of PE-macrophages via PRC2 EZH2 lysine methyltransferase could be of therapeutic value in MM. Citation Format: Malik Hamaidia, Clotilde Hoyos, Luc L. Willems. Inhibition of Polycomb Repressive Complex 2 EZH2 lysine methyltransferase improves tumoricidal activity of macrophages towards mesothelioma cells [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 3800.

Cabazitaxel regimens inhibit the growth of prostate cancer cells and enhances the anti‐tumor properties of PEDF with various efficacy and toxicity

Taxanes chemotherapies represent the major therapeutic alternative for symptomatic mCRPC. While docetaxel is the most commonly prescribed Taxane for mCRPC; cabazitaxel has been approved for patients unresponsive to docetaxel. Still mCRPC remains incurable and patients often experience severe side effects. Recently, the FIRSTANA trial first demonstrated the absence of superiority in overall survival between cabazitaxel and docetaxel in mCRPC patients. Inversely, different toxicity were reported suggesting that cabazitaxel may provide a first line treatment option for some patients urging for a deeper characterization of cabazitaxel mechanisms of action as well as a re-evaluation of cabazitaxel conventional dose and schedule. In this study, our goal was therefore to evaluate the anti-tumor efficacy of various cabazitaxel regimens delivered as monotherapy or in combination with PEDF, a known anti-angiogenic and anti-neoplastic agent. CRPC cells undergoing Taxane treatment were evaluated for cell proliferation, migration and death, and apoptosis using crystal violet staining, chemotaxis, cell cycle, and TUNEL assays. In vitro data were corroborated in CL1 CRPC xenografts where mice received intermittent or metronomic low-doses cabazitaxel ± PEDF. We found that cabazitaxel inhibits the proliferation of CRPC cells with a higher efficacy than docetaxel in vitro. As expected, high-doses of Taxanes blocked the cells in mitosis. Surprisingly, low-doses of cabazitaxel induced more cell death than docetaxel mainly through apoptosis. In vivo, intermittent cabazitaxel lead to disease stabilization when combined with PEDF. Unexpectedly, low-doses of cabazitaxel delayed tumor growth with severe toxicity for some of the doses tested. Other results showed that PEDF and low-doses of cabazitaxel combination inhibited the migration of tumor cell and increased the tumoricidal activity of macrophages toward prostate tumor cells. Our findings highlight the great promise of cabazitaxel drug and predict a possible move of cabazitaxel forward within the therapeutic sequence of prostate cancer.

Control of polarization and tumoricidal activity of macrophages by multicellular spheroid formation

Immune cell-based therapy is a promising approach for cancer immunotherapy. Macrophages can be used for this purpose if their tumoricidal activity and viability are properly controlled. In the present study, we aimed to enhance these properties of macrophages by constructing uniformly sized multicellular spheroids. Mouse macrophage-like J774.1 cells were selected as model macrophages, and poly(N-isopropylacrylamide)-coated polydimethylsiloxane-based microwell plates with an approximate diameter of 750μm were used to prepare J774.1 spheroids. J774.1 spheroids were successfully generated, and the viability of cells in the spheroids was over 95%. J774.1 spheroids showed higher mRNA expression of induced nitric oxide synthase, a marker of M1-type activated macrophages, than monolayered J774.1 cells. The production of reactive oxygen species was also high in J774.1 spheroids, suggesting the existence of hypoxic regions in the spheroids. J774.1 spheroids released more tumor necrosis factor-α than monolayered cells upon stimulation with lipopolysaccharide. Moreover, J774.1 spheroids in the upper compartment of the Transwell system more efficiently inhibited the proliferation of mouse adenocarcinoma colon 26 cells in its lower compartment than monolayered J774.1 cells did. These results indicate that spheroid formation can be used to increase the tumoricidal activity of macrophages for use in cell-based cancer immunotherapy.

Metabolism and TAM functions-it takes two to tango.

From the evidence on clinical studies and experimental mouse models we now know that tumor-associated macrophages (TAMs) sustain tumor development in many different ways. They play a role in angiogenesis, tumor cell invasion, and metastasis formation. Additionally, TAMs interfere with natural killer and T-cell antitumoral activities, producing an immune-suppressive environment that protects tumor cell growth. This indicates that the tumoricidal activity of macrophages within the tumor microenviroment is lost due to an imbalance of the regulatory mechanisms underpinning these cells' function. Since metabolism is emerging as a major modulator of macrophage function, metabolic changes in response to signals coming from cancer or other immune cells might promote this imbalance, enhancing the tumorigenic activities of TAMs. In this review we describe the novel, most recent findings on how metabolism shapes TAM functions or conversely, how TAMs influence the activity of other cells through metabolic mechanisms. The complete elucidation of the metabolic switches between pro- and antitumoral properties of macrophages, now still in its infancy, is destined to provide scientists with new instruments not only to understand but also to combat cancer.

Toll-Like Receptor Ligands and Interferon-γ Synergize for Induction of Antitumor M1 Macrophages.

Tumor-associated macrophages may either promote or suppress tumor growth depending on their activation status. Interferon-γ (IFN-γ) has been identified as a key factor for inducing tumoricidal M1 phenotype in macrophages. However, it remains unclear whether IFN-γ is sufficient or if additional stimuli are required. Here, we tested IFN-γ and a panel of toll-like receptor (TLR) agonists for the ability to activate murine macrophages toward a tumoricidal M1 phenotype. The following TLR ligands were used: TLR1/TLR2 agonist Pam3CSK4, TLR2/TLR6 agonist lipotechoic acid, TLR3 agonist poly(I:C), TLR4 agonist lipopolysaccharide (LPS), TLR5 agonist flagellin, TLR7 agonist CL264, and TLR9 agonist CpG. We used an in vitro growth inhibition assay to measure both cytotoxic and cytostatic activity of mouse macrophages against Lewis lung carcinoma (LLC) and MOPC315 plasmacytoma tumor cells. Production of nitric oxide (NO) and cytokines by activated macrophages was quantified. We found that IFN-γ alone was not able to render macrophages tumoricidal. Similarly, macrophage activation with single TLR agonists was inefficient. In sharp contrast, IFN-γ was shown to synergize with TLR agonists for induction of macrophage tumoricidal activity and production of both NO and pro-inflammatory cytokines (TNF-α, IL-12p40, and IL-12p70). Furthermore, IFN-γ was shown to suppress macrophage IL-10 secretion induced by TLR agonists. NO production was necessary for macrophage tumoricidal activity. We conclude that two signals from the microenvironment are required for optimal induction of antitumor M1 macrophage phenotype. Combination treatment with IFN-γ and TLR agonists may offer new avenues for macrophage-based cancer immunotherapy.

Abstract 250: Methionine restriction increases macrophage tumoricidal activity and significantly inhibits prostate cancer growth

Abstract Background: Our previous work showed a significant reduction of tumor growth, macrophage infiltration, circulating IGF-1, and mTOR activation with low protein diet in a patient derived xenograft model of prostate cancer. The evolutionarily conserved, nutrient sensing, mTOR pathway plays a central role in both development of advanced stage prostate cancer and immune responsiveness. This study presents novel data on the impact of dietary protein modification on the function of the host immune system in response to prostate cancer and immunotherapy. Methods: Our In vitro studies utilized bone marrow or tumor derived (RP-B6 Myc) macrophages. In vivo studies utilized the recently characterized RP-B6 Myc model. Mice were fed ad libitum control or methionine restricted diets for four weeks prior to S.C. implantation with ~1mm2 tumor pieces. Treatment of survivin peptide vaccine (1mg/ml S.C. 1 X week) and anti-PD-1 (20mg/kg I.P. 2 X week) began at ~50mm2 tumor size. Tumor volumes were blindly recorded 2 X week. End point analyses include: tumor weight, flow cytometric analysis, proteomic profiler analyses, and microbiome analyses from each diet and treatment group. Results: We show here that while methionine restriction (MR) has little impact on our RP-B6 Myc prostate cancer cell line, it does yield a significant alteration in both the polarization and function of M1 and M2 macrophages. In the in vitro MR conditions, we observed significantly enhanced polarization of M1 macrophages and reduced polarization of M2 macrophages. Functional analysis revealed increased tumoricidal activity of both M1, ‘antitumor’, and M2, ‘pro-tumor,’ macrophages suggesting a flip in M2 function from tumor-promoting to tumoricidal. Further analysis of the released cytokines in MR media conditions yielded significant increase of antitumor cytokines & chemokines, such as IL-12, IL-27, CXCL9, CXCL10, CXCL11, CCL2, CCL4, and TNF-alpha, a double-edged sword which in our system correlates with decreased cancer cell viability upon co-culture with amino acid restricted M1 and M2 macrophages. Our preliminary results show dietary MR yielding a significant inhibition of prostate cancer growth in the RP-B6-Myc model and our ongoing study with survivin peptide vaccine and anti-PD-1 immunotherapies will be available to present in the conference. Conclusions: Our data suggest that restricting methionine is sufficient to alter both the polarization and tumoricidal function of macrophages. Our preliminarily results show that restricted dietary methionine is able to inhibit prostate cancer growth, modify the host immune response and better inhibit prostate cancer growth alone or in combination with immunotherapy. These results provide a strong basis to consider diet restriction as a means to limit cancer growth targeting tumor immune system and potentially to enhance cancer patient responses to immunotherapy. Citation Format: Ashley R. Orillion, Sreenivasulu Chintala, Remi Adelaiye-Ogala, Li Shen, Nur Damayanti, May Elbanna, Sreevani Arisa, Bennett Elzey, Chinghai Kao, Luigi Fontana, Roberto Pili. Methionine restriction increases macrophage tumoricidal activity and significantly inhibits prostate cancer growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 250. doi:10.1158/1538-7445.AM2017-250

Abstract 5661: Targeting CD47 expression on macrophages regulates immunometabolism enhancing tumoricidal activity against cancer cells

Abstract Macrophage mediated elimination of cancer cells is critical for control of tumor growth. Cancer cells evade immune recognition by expressing receptors that send inhibitory signals to prevent macrophage mediated cytotoxicity. Clinical studies show that overexpression of the cell surface receptor CD47 is a poor prognostic factor in cancer, allowing tumors to bypass immunosurveillance and activation pro-growth pathways. Prior studies have described that CD47 cell surface expression on cancer cells inhibits phagocytic activity through engagement of its counter receptor SIRPα on macrophages. However, the role of CD47 expression on macrophage function is not known. Here, we show that CD47 is expressed on primary mouse macrophages, RAW 264.7 and the U937 cell lines. Moreover, knockdown of CD47 on macrophages enhanced the tumoricidal capacity against melanoma and breast cancer cells as measured by cell impedance and LDH release. Blockade of CD47 enhanced the macrophage-mediated cytotoxicity against B16 melanoma and 4T1 cells when compared to untreated effector/target pairs. Blockade of CD47 on differentiated U937 human monocytes also enhanced tumoricidal activity against the triple-negative breast cancer cell line MDA-MB-231 by over 50% when compared to untreated co-cultures. This indicates that expression of CD47 on macrophages may play a role in polarization and macrophage tumoricidal capacity against cancer cells beyond the known interactions with SIRPα. Our previous studies show that blockade of CD47 preserves T cell viability under stress by increasing glycolytic flux. Activation of M1 cytotoxic macrophages requires a shift in metabolism from oxidative phosphorylation to glycolysis. Our data shows that blockade of CD47 on RAW 264.7 macrophages increases glycolytic flux when compared to untreated controls as measured by extracellular acidification rate (ECAR) using a Seahorse© XF analyzer. The increase in glycolysis was associated with a significant elevation of glucose uptake by macrophages treated with anti-sense CD47 morpholino, indicating that targeting this receptor regulates glucose metabolism to improve macrophage tumoricidal activity. Interestingly, blockade of CD47 in the 4T1 breast cancer cell line reduced ECAR and reduced glucose uptake indicating that targeting CD47 reverses Warburg metabolism in cancer cells. Overall, these results show a new role of CD47 expression that extends beyond suppressing macrophage mediated killing through engagement of SIRPα. Therefore, anti-CD47 immunotherapy may regulate immunometabolism to improve clinical outcomes of cancer patients. Citation Format: Ashley A. Smith, Adam S. Wilson, David R. Soto-Pantoja. Targeting CD47 expression on macrophages regulates immunometabolism enhancing tumoricidal activity against cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5661. doi:10.1158/1538-7445.AM2017-5661

PEDF increases the tumoricidal activity of macrophages towards prostate cancer cells in vitro.

BackgroundAlthough inflammation and prostate cancer (PCa) have been linked, the molecular interactions between macrophages and PCa cells are poorly explored. Pigment Epithelium-Derived Factor (PEDF) is an anti-angiogenic and anti-tumor factor. We previously showed that PEDF induces macrophages recruitment in vitro, correlates with macrophages density in human prostate, and stimulates macrophages polarization towards the classically activated pathway. Here, we demonstrate that PEDF modulates the interaction between macrophages and PCa cells through a bidirectional signalling leading to tumor cell apoptosis and phagocytosis.MethodsRAW 264.7 and THP-1 cells, and BMDMs were grown in vitro as mono- or co-cultures with PC3 or CL1 tumor cells. The effects of PEDF and its derived P18 peptide were measured on macrophages differentiation, migration, and superoxide production, and tumor cell apoptosis and phagocytosis. PEDF receptors (ATP5B, PNPLA2, and LRP6) and CD47 mRNA and protein expression were quantified in macrophages and tumor cells by quantitative RT-PCR, western blot, immunofluorescence and flow cytometry.ResultsWe found that PEDF induced the migration of macrophages towards tumor 3D spheroids and 2D cultures. In co-culture, PEDF increased PCa cells phagocytosis through an indirect apoptosis-dependent mechanism. Moreover, PEDF stimulated the production of superoxide by macrophages. Conditioned media from macrophages exposed to PEDF induced tumor cells apoptosis in contrast to control conditioned media suggesting that ROS may be involved in tumor cells apoptosis. ATP5B and PNPLA2 PEDF receptors on macrophages and CD47 on tumor cells were respectively up- and down-regulated by PEDF. As PEDF, blocking CD47 induced phagocytosis. Inhibiting ATP5B reduced phagocytosis. Inversely, PNPLA2 inhibition blocks differentiation but maintains phagocytosis. CD47-induced phagocytosis was partially reverted by ATP5B inhibition suggesting a complementary action. Similar effects were observed with P18 PEDF-derived peptide.ConclusionsThese data established that modulating the molecular interactions between macrophages and PCa cells using PEDF may be a promising strategy for PCa treatment.

A CD47xCD19 Bispecific Antibody That Remodels the Tumor Microenvironment for Improved Killing and Provokes a Memory Immune Response to Cancer B Cells

Up-regulation of CD47 in hematological and solid cancers correlates with poor clinical prognosis. CD47 interaction with SIRPα provides a ‘don’t eat me' signal that allows healthy cells to limit elimination by immune cells, in particular macrophages. Although tumor-associated macrophages (TAMs) are often considered pro-tumorigenic, several studies report a high phagocytic potential and tumoricidal function in the presence of therapeutic antibodies (Ab). Therefore, targeting the CD47-SIRPα pathway in the tumor microenvironment is an attractive approach to maximize the tumor killing potential of TAMs to boost tumor destruction. However, clinical development of monoclonal Abs to CD47 is likely to be hindered by the ubiquitous expression of CD47 leading to rapid drug elimination and toxicity including anemia. To address these concerns, we have created NI-1701, a bispecific Ab that drives efficacious binding only to CD19+B cells by pairing a high affinity anti-CD19 targeting arm to an anti-CD47 arm of optimized affinity..In addition to in vitro data demonstrating that the bispecific Ab, NI-1701, effectively kills CD19+ human tumor B cells through ADCP (antibody-dependent cellular phagocytosis) and antibody-dependent cell-mediated cytotoxicity (ADCC), we have observed significant tumor killing in vivo, as either a monotherapy or in a combination approach. NI-1701 controlled sub-cutaneously implanted Raji cell tumor growth in NOD/SCID mice in a manner dependent on the co-ligation of both CD19 and CD47. Examination of the excised tumors revealed that NI-1701 reshaped the tumor microenvironment by enhancing the tumoricidal activity of macrophages (i.e., more macrophages engulfing tumor cells), by promoting an antitumor M1-like phenotype, and reducing the proportion of CD11b+Gr1+myeloid-derived suppressor cells (MDSCs). Extending these findings to a disseminated in vivo model, NI-1701 eliminated tumor cells from the peripheral blood, bone marrow and liver in mice transplanted either with the B-Acute Lymphocytic Leukemia (B-ALL) cell line NALM-6 or with primary cells from B-ALL patients. Furthermore, NI-1701 also abrogated tumor growth more efficiently than the BTK inhibitor ibrutinib in a Diffuse Large B-Cell Lymphoma (DLBCL) patient-derived xenograft (PDX) mouse model.As combination therapies are gaining traction as successful treatment strategies in the clinic, we next tested the effect of blocking CD47 biology in combination with clinically validated molecules. Interestingly, in NOD/SCID mice implanted with Raji cells, NI-1701 was shown to be more efficacious at controlling tumor cell growth than Rituximab. A combination of NI-1701 and Rituximab was shown to act synergistically at controlling tumor growth and leading to tumor regression in some mice. Finally, in a syngeneic re-challenge model, using bispecific reagents targeting CD47 blockade to the A20 murine B-cell lymphoma, we observed the induction of a durable and protective anti-tumor response when combined with a single administration of cyclophosphamide.Importantly, in vitro safety studies demonstrate a favorable binding profile of NI-1701 to B cells compared with erythrocytes, no evidence of platelet activation or aggregation and no haemagglutination at and above anticipated therapeutic concentrations. Single and multiple dose studies in non-human primates demonstrated favorable elimination kinetics and no effects on hematological parameters (e.g., red blood cell and platelet counts) up to 100mg/kg, the highest dose tested.Taken together, we describe a novel bispecific approach that balances a safe yet effective blockade of CD47 with a high selectivity for a B cell associated antigen resulting in impressive tumor cell killing in a range of preclinical models. The effects on both the reshaping of the tumor microenvironment and the induction of long term tumor immunity provide further evidence that manipulation of myeloid lineage cells (e.g., macrophages and dendritic cells) is a promising approach for the next frontier in immune-oncology treatment strategies. NI-1701 is in preclinical enabling studies in preparation for a Phase I clinical study in patients with CD19+ B cell malignancies, planned for early 2017. DisclosuresFerlin:Novimmune S.A.: Employment, Equity Ownership. Chauchet:Novimmune S.A.: Employment. Buatois:Novimmune S.A.: Employment. Salgado-Pires:Novimmune S.A.: Employment. Shang:Novimmune S.A.: Employment. Dheilly:Novimmune S.A.: Employment. Masternak:Novimmune S.A.: Employment. Johnson:Novimmune S.A.: Employment. DiPersio:Incyte Corporation: Research Funding. Kosco-Vilbois:Novimmune S.A.: Employment. Fischer:Novimmune S.A.: Employment.

Tumoricidal Activation of Macrophages using Jatropha curcas Leaf Extract: As a Proxy for the Treatment of Cancer

Abstract Background: Jatropha curcas (J. curcas) is a plant rich in methanolic. Macrophages can be activated to become tumoricidal through interactions with immunomodulators. The aim of this study was to investigate the effect of J. curcas leaf extracts on the phagocytic activity of macrophages. Methods: Peritoneal-derived macrophages from male BALB/c mice were obtained using the method of Colligan et al. with some modifications. Latex was added as an antigen to cultures of BALB/c mice macrophages treated with two different concentrations of J. curcas extract and the cultures were observed every 30 minutes for 2 hours. Results: Macrophage latex vacuoles were observed after treatment with 15 μg/mL J. curcas leaf extract, with a mean latex vacuole count of 7.2 vacuoles/cell and a phagocytic index (PI) of 369. Macrophage treatment with 250 μg/mL J. curcas leaf extract produced a mean latex vacuole count of 8.1 vacuoles/cell and a PI of 785. Conclusion: Treatment with J. curcas leaf extracts increased the phagocytic activity of mice macrophages.

Jacalin-Activated Macrophages Exhibit an Antitumor Phenotype.

Tumor-associated macrophages (TAMs) have an ambiguous and complex role in the carcinogenic process, since these cells can be polarized into different phenotypes (proinflammatory, antitumor cells or anti-inflammatory, protumor cells) by the tumor microenvironment. Given that the interactions between tumor cells and TAMs involve several players, a better understanding of the function and regulation of TAMs is crucial to interfere with their differentiation in attempts to skew TAM polarization into cells with a proinflammatory antitumor phenotype. In this study, we investigated the modulation of macrophage tumoricidal activities by the lectin jacalin. Jacalin bound to macrophage surface and induced the expression and/or release of mainly proinflammatory cytokines via NF-κB signaling, as well as increased iNOS mRNA expression, suggesting that the lectin polarizes macrophages toward the antitumor phenotype. Therefore, tumoricidal activities of jacalin-stimulated macrophages were evaluated. High rates of tumor cell (human colon, HT-29, and breast, MCF-7, cells) apoptosis were observed upon incubation with supernatants from jacalin-stimulated macrophages. Taken together, these results indicate that jacalin, by exerting a proinflammatory activity, can direct macrophages to an antitumor phenotype. Deep knowledge of the regulation of TAM functions is essential for the development of innovative anticancer strategies.

Lenalidomide Enhances MOR202 Dependent Macrophage-Mediated Effector Functions Via the Vitamin D Pathway

Multiple myeloma (MM) is considered a chronic and incurable disease due to its highly complex and heterogeneous molecular abnormalities and the support from myeloma microenvironment factors. Macrophages are an abundant component of the stromal cell compartment and are believed to support proliferation, survival, and drug resistance of MM cells. Conversely, macrophages are key immune effector cells for the therapeutic effect of monoclonal antibodies and can directly eliminate tumor cells. However, myeloma-associated macrophages (MAMs) regularly fail to exert direct effector functions. Given their abundance in MM, an attractive therapeutic approach would be to stimulate their tumoricidal activity in order to promote antitumor immunity. Lenalidomide, an immunomodulatory agent that enhances antibody dependent cell mediated cytotoxicity (ADCC), has the potential to synergize with MOR202, an anti-CD38 monoclonal IgG1 antibody currently in phase I/IIa for the treatment of MM. Furthermore, vitamin D plays a key role in regulating effector functions of human macrophages. This is closely linked to the expression of the vitamin D-1-hydroxylase CYP27B1, which catalyzes the conversion of 25-hydroxy-vitamin D (25D) to the bioactive form 1,25-di-hydroxy-vitamin D (1,25D). We have previously shown, that vitamin D promotes tumoricidal activity of macrophages and improves the efficacy of rituximab-dependent cytotoxicity (Bruns et al., Sci. Transl. Med., 2015; Bittenbring et al., JCO, 2014). Therefore, we hypothesized that the combination of MOR202 with lenalidomide and MOR202 with 1,25D would enhance the MOR202- dependent macrophage-mediated effector functions against myeloma cells.Here we report that MAMs exhibit an altered vitamin D metabolism with a reduced expression of the vitamin D receptor (VDR) and CYP27B1, while the expression of CYP24A1, which catabolizes 1,25D, is increased. As a consequence MAMs cannot convert 25D into bioactive 1,25D. Given the importance of the vitamin D pathway for antibody mediated cytotoxicity, we screened several drugs for their ability to restore the vitamin D pathway in human macrophages. We found, by RNA-sequencing, that lenalidomide treatment modulates the phenotype of monocyte-derived macrophages and isolated MAMs, and that lenalidomide significantly increases the expression of the VDR, CYP27B1 and CAMP (cathelicidin antimicrobial peptide) in these macrophages. Furthermore, we demonstrate that isolated MAMs regularly fail to eliminate primary myeloma cells, and that the lack of effector functions can be overcome by treatment with lenalidomide and vitamin D. Moreover, we show that MOR202-dependent elimination of myeloma cells is enhanced by pre-treatment of isolated MAMs with lenalidomide and supplementation of vitamin D.In summary, these data show that the bioactive form of vitamin D (1,25D) is essential for the effector functions of MAMs and that the therapeutic activation of the vitamin D pathway by lenalidomide may restore their tumoricidal effector mechanisms. Furthermore, these findings highlight that stimulating the tumoricidal activity of MAMs, an abundant component of the stromal cell compartment, with lenalidomide and 1,25D is an attractive therapeutic approach in combination with the anti-CD38 monoclonal IgG1 antibody MOR202. DisclosuresBruns:Morphosys: Research Funding; Celgene: Research Funding.

SIRPαFc, a CD47-Blocking Cancer Immunotherapeutic, Sensitizes Malignant B Cells to Macrophage-Mediated Destruction

Macrophages commonly infiltrate tumor microenvironments and can phagocytose and destroy malignant cells. Cancer cells, however, can inhibit the tumoricidal activity of macrophages by expressing CD47 on their surface. CD47 delivers an anti-phagocytic ("do not eat") signal by binding signal-regulatory protein α (SIRPα) on the surface of macrophages. There is strong evidence that many liquid and solid tumors exploit the CD47-SIRPα pathway to escape macrophage-mediated destruction. Blockade of this inhibitory axis using a soluble SIRPα-Fc fusion protein (SIRPαFc) has emerged as a promising strategy to neutralize the suppressive effects of CD47 and promote the eradication of tumor cells. Here we have examined the effect of SIRPαFc on malignant human B cells in vitro and in vivo.We first assessed the binding of SIRPαFc to a panel of established cell lines and primary cells from patients with diffuse large B cell lymphoma, Burkitt's lymphoma, multiple myeloma and acute lymphoblastic leukemia. SIRPαFc exhibited strong, dose-dependent binding to all tumor cells, with an average effective half-maximal concentration of approximately 150 nM. Next, the ability of SIRPαFc to promote macrophage-mediated phagocytosis of human tumor cells was examined using confocal microscopy. In cultures left untreated or treated with a control Fc fragment, macrophages exhibited a low level of phagocytosis, consistent with CD47-mediated suppression. Blockade of CD47 on the target cells using SIRPαFc dramatically increased macrophage phagocytosis of tumor cells. The majority of established cell lines and all primary human tumors were sensitized to macrophage-mediated destruction, including both peripheral blood- and bone marrow-derived primary tumor samples. Finally, we assessed the in vivo activity of SIRPαFc in CD20hi (Raji) and CD20low (Namalwa) B lymphoma xenograft models. SIRPαFc treatment significantly reduced Raji growth and increased host mouse survival (time to euthanasia), and completely ablated the growth of Namalwa tumors, the latter being insensitive to rituximab therapy.In conclusion, SIRPαFc demonstrated in vitro activity against a broad range of human B cell tumors and was highly effective at controlling the growth of aggressive B lymphoma xenografts in mice, including a CD20low tumor that was non-responsive to rituximab. These data support the evaluation of SIRPαFc in patients with B cell malignancies. DisclosuresNielsen Viller:Trillium Therapeutics Inc.: Employment. Vareki:Trillium Therapeutics Inc.: Research Funding. Dodge:Trillium Therapeutics Inc.: Employment. Chen:Trillium Therapeutics Inc.: Employment. Lee:Trillium Therapeutics Inc.: Employment. Chai:Trillium Therapeutics Inc.: Employment. Pang:Trillium Therapeutics Inc.: Employment. Wong:Trillium Therapeutics Inc.: Employment. Trudel:Novartis: Honoraria; Oncoethix: Research Funding; BMS: Honoraria; Trillium Therapeutics Inc.: Research Funding; Celgene: Equity Ownership, Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau. Figueredo:Trillium Therapeutics Inc.: Research Funding. Pampillo:Trillium Therapeutics Inc.: Research Funding. Koropatnick:Trillium Therapeutics Inc.: Research Funding. Petrova:Trillium Therapeutics Inc.: Employment. Uger:Trillium Therapeutics Inc.: Employment.

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

Vitamin D lends a helping hand

Vitamin D promotes tumoricidal activity of macrophages and improves the efficacy of antibody-dependent cellular cytotoxicity.

Vitamin D-dependent induction of cathelicidin in human macrophages results in cytotoxicity against high-grade B cell lymphoma.

Infiltration by macrophages represents a characteristic morphological hallmark in high-grade lymphatic malignancies such as Burkitt's lymphoma (BL). Although macrophages can, in principle, target neoplastic cells and mediate antibody-dependent cellular cytotoxicity (ADCC), tumor-associated macrophages (TAMs) regularly fail to exert direct cytotoxic functions. The underlying mechanisms responsible for this observation remain unclear. We demonstrate that inflammatory M1 macrophages kill proliferating high-grade B cell lymphoma cells by releasing the antimicrobial peptide cathelicidin in a vitamin D-dependent fashion. We show that cathelicidin directly induces cell death by targeting mitochondria of BL cells. In contrast, anti-inflammatory M2 macrophages and M2-like TAMs in BL exhibit an altered vitamin D metabolism, resulting in a reduced production of cathelicidin and consequently in inability to lyse BL cells. However, treatment of M2 macrophages with the bioactive form of vitamin D, 1,25D3, or a vitamin D receptor agonist effectively induces cathelicidin production and triggers tumoricidal activity against BL cells. Furthermore, rituximab-mediated cytotoxicity of vitamin D-treated M2 macrophages is cathelicidin-dependent. Finally, vitamin D treatment of 25-hydroxyvitamin D (25D)-deficient volunteers in vivo or primary TAMs in vitro improves rituximab-mediated ADCC against B cell lymphoma cells. These data indicate that activation of the vitamin D signaling pathway activates antitumor activity of TAMs and improves the efficacy of ADCC.

Interferon regulatory factor (IRF)-1 is a master regulator of the cross talk between macrophages and L929 fibrosarcoma cells for nitric oxide dependent tumoricidal activity.

Macrophage tumoricidal activity relies, mainly, on the release of Tumor Necrosis Factor alpha (TNFα) and/or on reactive oxygen or nitrogen intermediates. In the present work, we investigated the cytotoxic activity of resident peritoneal macrophages against L929 fibrosarcoma cell line in vitro and in vivo. Resident macrophages lysed L929 cells in a mechanism independent of TNFα and cell-to-cell contact. The cytotoxic activity was largely dependent on nitric oxide (NO) release since treatment with L-NAME (NOS inhibitor) inhibited L929 cells killing. Macrophages from mice with targeted deletion of inducible NO synthase (iNOS) together with L929 cells produced less NO and displayed lower, but still significant, tumoricidal activity. Notably, NO production and tumor lysis were abolished in co-cultures with macrophages deficient in Interferon Regulatory Factor, IRF-1. Importantly, the in vitro findings were reproduced in vivo as IRF-1 deficient animals inoculated i.p with L929 cells were extremely susceptible to tumor growth and their macrophages did not produce NO, while WT mice killed L929 tumor cells and their macrophages produced high levels of NO. Our results indicate that IRF-1 is a master regulator of bi-directional interaction between macrophages and tumor cells. Overall, IRF-1 was essential for NO production by co-cultures and macrophage tumoricidal activity in vitro as well as for the control of tumor growth in vivo.