Murine Colon Carcinoma Model Research Articles

Intensive therapy with gastropodan hemocyanins increases their antitumor properties in murine model of colon carcinoma

Various natural compounds have been tested as anticancer therapeutics in clinical trials. Most promising direction for antitumor therapy is the use of substances which enhance the immune system response stimulating tumor-specific lymphocytes.Hemocyanins are large extracellular oxygen transport glycoproteins isolated from different arthropod and mollusk species which exhibit strong anticancer properties. Immunized in mammals they trigger Th1 immune response that promotes unspecific stimulation and adjuvant activity in experimental therapeutic vaccines for cancer and antibody development.In the present study we used two hemocyanins – one isolated from marine snail Rapana thomasiana (RtH) and another one, from the terrestrial snail Helix pomatia (HpH) which have been investigated by using different administration schedules (intensive and mild) in murine model of colon carcinoma.The treatment with RtH and HpH generated high levels of antitumor IgG antibodies, antibody-producing plasma cells and tumor-specific CTLs, stimulated secretion of proinflammatory cytokines, suppressed the manifestation of carcinoma symptoms as tumor growth and size, and prolonged the life span of treated mice.Our results showed a significant anti-cancer effect of RtH and HpH hemocyanins on a murine model of colon carcinoma with promising potential for immunotherapy in various schemes of administration based on cross-reactive tumor-associated epitopes.

Radiation-induced change of PD-1/PD-L1 immune checkpoint in mouse colon cancer models

Abstract Background Radiotherapy (RT), a major part of anti-cancer treatment, directly kills tumor cells, and subsequent anti-tumor immune responses are up-regulated. However, immunologic impacts of RT on PD-1/PD-L1 immune checkpoint activity has not been much investigated. This study evaluated RT-induced alterations of the PD-1/PD-L1 checkpoint molecules based on murine colon carcinoma models. Methods CT26 colon carcinoma cell line was subcutaneously inoculated on the right hind leg of BALB/c mice. Based on tumor growth curves after irradiation of 15 Gy x 1 fx or 5 Gy x 3 fx, mouse tumors were surgically resected on 4 different time points: “Pre-RT”, non-irradiated status just prior to initiation of RT; “Early”, the early phase of RT response; “Nadir”, representing minimal tumor volume; and “Regrowth”, with regrown tumors after RT. PD-L1 expression on tumor cells, PD-1 expression on tumor-infiltrating CD4+ and CD8+ T cells, and proportions of tumor-infiltrating CD4+ and CD8+ T cell populations were estimated using flow cytometry analysis. Results PD-L1 expression on mouse tumor cells surged within a few days after completion of RT, followed by abrupt decreases on the “Nadir” and “Regrowth” phases (P Conclusions This study verified radiation-induced immunologic shift and dynamic change of the PD-1/PD-L1 checkpoint activity and tumor-infiltrating lymphocytes. The change was maximal at the early phase of RT response, which highlights the need of concurrent combinatory strategy of PD-L1 blockade and RT. Legal entity responsible for the study The author. Funding National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Science, ICT & Future Planning) (NRF-2019R1C1C1007994). Disclosure The author has declared no conflicts of interest.

Abstract 508: Combined MEK and PD-1 inhibition in acute myeloid leukemia: Effect on cell proliferation and phenotype

Abstract Although most patients with acute myeloid leukemia (AML) respond to standard of care chemotherapy, the majority of patients will relapse, and the 5-year overall survival rate persists at 25%. The high rates of relapse and poor survival have contributed to increased interest in the use of other therapies, including immunotherapies, in hematological malignancies in order to achieve more durable responses. In particular, immune checkpoint blockade, such as anti-PD-1, has elicited efficacious responses in Hodgkin’s lymphoma. In addition, trametinib, a small molecule MEK inhibitor, was shown to have activity in AML patients with a RAS-mutation in a phase 1/2 trial, but demonstrated the need for rational combinational therapies to produce longer lasting responses. Interestingly, in a murine model of colon carcinoma harboring a KRAS mutation, the combination of these two agents resulted in a more durable tumor response compared to either agent alone. We hypothesized that in AML, combined MEK and PD-1 inhibition would result in direct inhibition of blast proliferation while simultaneously promoting proliferation and activation of T cells with a mature/memory phenotype. Bone marrow and peripheral blood samples isolated from patients with AML were cultured in our laboratory for 5 days with CD3 stimulation to induce T-cell activation. Cultures were treated with trametinib, anti-PD-1, or both. Additional samples were cultured with interferon-gamma (IFN-γ) to induce PD-L1 expression. Mouse spleen cells from a genetically engineered mouse model of AML (FLT3-ITD/TET2+/-) were cultured under the same conditions for 3 days. Samples were evaluated by flow cytometry to measure treatment effects on T-cell and blast proliferation and phenotype. In the patient samples, trametinib consistently reduced PD-L1 expression in a dose-dependent manner on the myeloid cell population and was further able to attenuate PD-L1 expression induced by IFN-γ. A mean decrease of 36.6% was seen in PD-L1 expression after treatment with trametinib at 100 nm (p=0.0177). In most samples, trametinib decreased blast proliferation and increased blast cell death. Additionally, in a NRAS-mutant sample with high PD-L1 expression, the combination therapy best restored T cell proliferation and decreased the naive T cell population resulting in increased effector and memory T cells. We observed similar changes in our mouse model of AML. In conclusion, IFN-γ increased PD-L1 expression on cancer cells suggesting a possible mechanism of immunosuppression inhibiting autologous immune responses, and the increased PD-L1 expression could be at least partially reversed by trametinib. Our study suggests that combined MEK and PD-1 inhibition may result in greater therapeutic efficacy over each agent alone and provide a rationale for administering this combination to patients with AML. Citation Format: Kaycee B. Moshofsky, Hyun J. Cho, Yoko Kosaka, Matthew T. Newman, Jeffrey W. Tyner, Evan F. Lind. Combined MEK and PD-1 inhibition in acute myeloid leukemia: Effect on cell proliferation and phenotype [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 508.

Modulated electro-hyperthermia-enhanced liposomal drug uptake by cancer cells.

PurposeModulated electro-hyperthermia (mEHT) stands to be a significant technological advancement in the hyperthermia field, utilizing autofocusing electromagnetic power on the cell membrane to create massive apoptosis. Since mEHT possesses the unique ability to excite cell membranes, we hypothesized that mEHT could enhance the uptake of liposomal drugs by enhancing phagocytic activity.Materials and methodsWater bath control and mEHT were used to compare the enhancement of liposome-encapsulated doxorubicin (Lipodox®) uptake by cancer cells. Cancer cells were made visible by doxorubicin fluorescence to investigate drug uptake. Viable cell yield was determined via the Trypan Blue exclusion method. Various substrates were used to investigate the mechanism of drug-uptake enhancement. The murine colon carcinoma model, CT26, was used to confirm the tissue infiltration of Lipodox® and its therapeutic effect.ResultsmEHT treatment showed a significant enhancement of Lipodox® uptake of doxorubicin fluorescence compared with 37°C or 42°C water bath treatment. Tumor tissue sections also confirmed that mEHT treatment achieved the highest doxorubicin concentration in vivo (1.44±0.32 µg/g in mEHT group and 0.79±0.32 µg/g in 42°C water bath). Wortmannin was used to inhibit the macropinocytosis effect and 70 kDa dextran-FITC served as uptake substance. The uptake of dextran-FITC by cancer cells significantly increased after mEHT treatment whereas such enhancement was significantly inhibited by wortmannin.ConclusionThe result showed mEHT-induced particle-uptake through macropinocytosis. mEHT-enhanced uptake of Lipodox® may amplify the therapeutic effect of liposomal drugs. This novel finding warrants further clinical investigation.

Genetically Encoding Albumin Binding into Chemotherapeutic-loaded Polypeptide Nanoparticles Enhances Their Antitumor Efficacy.

We report the development of drug-encapsulating nanoparticles that bind endogenous albumin upon intravenous injection and evaluate their in vivo performance in a murine as well as canine animal model. The gene encoding a protein-G derived albumin binding domain (ABD) was fused to that of a chimeric polypeptide (CP), and the ABD-CP fusion was recombinantly synthesized by bacterial expression of the gene. Doxorubicin (DOX) was conjugated to the C-terminus of the ABD-CP fusion, and conjugation of multiple copies of the drug to one end of the ABD-CP triggered its self-assembly into ∼100 nm diameter spherical micelles. ABD-decorated micelles exhibited submicromolar binding affinity for albumin and also preserved their spherical morphology in the presence of albumin. In a murine model, albumin-binding micelles exhibited dose-independent pharmacokinetics, whereas naked micelles exhibited dose-dependent pharmacokinetics. In addition, in a canine model, albumin binding micelles resulted in a 3-fold increase in plasma half-life and 6-fold increase in plasma exposure as defined by the area under the curve (AUC) of the drug, compared with naked micelles. Furthermore, in a murine colon carcinoma model, albumin-binding nanoparticles demonstrated lower uptake by the reticuloendothelial system (RES) system organs, the liver and spleen, that are the main target organs of toxicity for nanoparticulate delivery systems and higher uptake by the tumor than naked micelles. The increased uptake by s.c. C26 colon carcinoma tumors in mice translated to a wider therapeutic window of doses ranging from 20 to 60 mg equivalent of DOX per kg body weight (mg DOX equiv·kg-1 BW) for albumin-binding ABD-CP-DOX micelles, as compared to naked micelles that were only effective at their maximum tolerated dose of 40 mg DOX equiv·kg-1 BW.

Association of IL-36γ with tertiary lymphoid structures and inflammatory immune infiltrates in human colorectal cancer.

IL-1 family cytokines play a dual role in the gut, with different family members contributing either protective or pathogenic effects. IL-36γ is an IL-1 family cytokine involved in polarizing type-1 immune responses. However, its function in the gut, including in colorectal cancer pathogenesis, is not well appreciated. In a murine model of colon carcinoma, IL-36γ controls tertiary lymphoid structure formation and promotes a type-1 immune response concurrently with a decrease in expression of immune checkpoint molecules in the tumor microenvironment. Here, we demonstrate that IL-36γ plays a similar role in driving a pro-inflammatory phenotype in human colorectal cancer. We analyzed a cohort of 33 primary colorectal carcinoma tumors using imaging, flow cytometry, and transcriptomics to determine the pattern and role of IL-36γ expression in this disease. In the colorectal tumor microenvironment, we observed IL-36γ to be predominantly expressed by M1 macrophages and cells of the vasculature, including smooth muscle cells and high endothelial venules. This pattern of IL-36γ expression is associated with a CD4+ central memory T cell infiltrate and an increased density of B cells in tertiary lymphoid structures, as well as with markers of fibrosis. Conversely, expression of the antagonist to IL-36 signaling, IL-1F5, was associated with intratumoral expression of checkpoint molecules, including PD-1, PD-L1, and CTLA4, which can suppress the immune response. These data support a role for IL-36γ in the physiologic immune response to colorectal cancer by sustaining inflammation within the tumor microenvironment.

Abstract 1740: Epigenetic reprogramming of the tumor microenvironment by entinostat increases tumor sensitivity to multivalent immunotherapy combinations with an IL-15 superagonist plus vaccine or immune checkpoint blockade

Abstract The clinical promise of cancer immunotherapy relies on the immune system recognizing and eliminating tumor cells identified as non-self. However, the tumor microenvironment (TME) can greatly impede a tumor targeted immune response via increasing immunosuppressive cells or hampering T and NK cell maturation, recruitment, and function through numerous pathways, including upregulation of immune checkpoints such as PD-L1. Hence, there is an unmet clinical need to develop effective therapeutic strategies that can reprogram the TME to restore tumor immune recognition and reverse immune evasion. We recently demonstrated that Entinostat, a class I histone deacetylase (HDAC) inhibitor, reverses tumor immune escape to T cell-mediated lysis. We hypothesize that the immune-mediated tumor elimination promoted by the IL-15/IL-15Rα superagonist ALT803 in combination with PD-L1 checkpoint blockade or a therapeutic adenoviral vaccine targeting CEA (Ad-CEA) will be augmented by the epigenetic reprograming of the TME induced by Entinostat. In preclinical studies, ALT803 has been shown to exhibit potent antitumor activity in multiple murine models of cancer through the expansion of NK and CD8+ T cells with high effector function. Here, we demonstrate that Entinostat increased extracellular expression of immune-relevant proteins on murine colon and breast carcinoma cells in vitro. Additionally, the frequency of T cells with an activated phenotype were increased in non-tumor bearing mice. In the MC38-CEA murine model of colon carcinoma, the addition of Entinostat augmented the antitumor activity promoted by ALT803 plus Ad-CEA resulting in increased survival. Furthermore, in the 4T1 murine model of triple-negative breast cancer, the combination of Entinostat with ALT803 and a monoclonal antibody targeting PD-L1 significantly reduced primary tumor weight relative to ALT803 plus anti-PD-L1 therapy and resulted in a significant reduction of the number of 4T1 tumor-forming cells in the lung. The immune mechanism associated with antitumor efficacy of these multivalent therapies was examined in both the periphery and TME. Overall, these studies provide a rationale for combining Entinostat with multivalent immunotherapy combinations, including cytokines, antibodies targeting PD-L1, and therapeutic cancer vaccines. Citation Format: Kristin C. Hicks, Karin M. Knudson, Frank R. Jones, Peter Ordentlich, Shahrooz Rabizadeh, Hing C. Wong, James W. Hodge, Jeffrey Schlom, Sofia R. Gameiro. Epigenetic reprogramming of the tumor microenvironment by entinostat increases tumor sensitivity to multivalent immunotherapy combinations with an IL-15 superagonist plus vaccine or immune checkpoint blockade [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 1740.

Abstract 1683: A comparative evaluation of immunotherapy responses in murine colon carcinoma

Abstract With the drive in oncology to develop molecules targeting the immune system, there is a need for reliable and well-characterized preclinical models. To this end, the impact of checkpoint inhibitors, such as anti-PD-1 and anti-PD-L1, has been extensively investigated preclinically, and the landscape of available models varies greatly in response to these molecules. With several recent checkpoint inhibitor-based drug approvals, the focus of immuno-oncology drug development has shifted to immunometabolism targets and costimulatory molecules, particularly with combination strategies. Comparative preclinical tumor model validation with respect to these targets is generally lacking; however, the same level of granularity as was afforded anti-PD-1 and anti-PD-L1 would be needed with these molecules to allow confidence in designing preclinical proof of concept studies for future clinical development. To better understand the utility of these agents in preclinical development, we examined the response of two murine colon carcinoma models, CT26 and MC38, against a panel of costimulatory molecules and epacadostat, an IDO inhibitor, using in vivo tumor growth delay studies. Our data demonstrate that CT26 tumors are sensitive to the costimulatory molecules, anti-GITR, anti-CD137, and anti-OX40, resulting in 6/10, 7/10, and 2/10 complete responses (CRs), respectively. In contrast, the response of MC38 tumors to these agents was modest with no CRs and times to evaluation size (TES) extending only 1.4 to 6.9 days beyond control. Epacadostat treatment did not impact growth of CT26 or MC38, with TES's extended by 4.4 or 2.3 days, respectively, compared to control. Interestingly, addition of epacadostat did not improve the response of anti-PD-1 in either model. To determine whether the changes in response could be related to differences in immune infiltration of these tumors, immunophenotyping from untreated CT26 and MC38 tumors was evaluated. We found that immune cell populations also highlighted potential differences in these models. CT26 tumors were well infiltrated with a mean of 25.2% CD45+ cells, with 54.8% of CD3+ cells being CD8+ T cells. Comparatively, MC38 tumors were weakly infiltrated with CD45+ cells (5.3%) with 35.9% of CD3+ cells being CD8+ T cells. Macrophage and MSDC infiltrates, as a percent of CD11b+ cells, in CT26 tumors were generally double that found in MC38 tumors. Our results indicate that CT26 is more sensitive to the costimulatory molecules tested than MC38. Further, epacadostat as monotherapy or in combination with anti-PD-1 did not elicit clinically meaningful tumor growth delay in either model. Stark differences were also seen in overall lymphocyte infiltrate and composition between CT26 and MC38. The differences in baseline immune infiltrate and efficacy allow us to determine which models and immunotherapies are best suited for rational drug combination approaches. Citation Format: Sheri R. Barnes, Sumithra Urs, Mary Anne Meade, David Draper, Hillary Evens, Daniel Saims, Scott Wise, Maryland Rosenfeld Franklin. A comparative evaluation of immunotherapy responses in murine colon carcinoma [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 1683.

Abstract LB-149: Targeting shared hotspot cancer mutations with a Listeria monocytogenes immunotherapy induce potent anti-tumor immunity

Abstract Introduction: Virtually all tumors contain somatic mutations that can result in novel antigenic sequences that may be targeted by the host cellular immune response. Some of these mutations occur in preferential regions of specific genes commonly referred to as hotspot mutations. Hotspot mutations are commonly shared by cancer patients both within and across multiple tumor types. These hotspot mutations often confer loss or gain of function contributing to oncogenesis, which makes them promising therapeutic targets. One such mutation commonly found in several human tumor types is an aspartic acid substitution for glycine at position 12 (G12D) in KRAS. This same mutation occurs in the CT26 murine colorectal tumor model. To determine if expression of the KRAS G12D sequence in a bacterial immunotherapy vector can control tumor growth in the CT26 murine model, the Advaxis Listeria monocytogenes (Lm)-based platform was engineered to express a 21-amino acid KRAS sequence peptide containing the G12D mutation (Lm-Hot KRAS_G12D). In addition, we evaluated control of tumor growth using an ADXS-HOT construct (ADXS-503) that expresses multiple shared human hotspot and tumor-associated antigens, including the G12D KRAS. The ADXS-HOT clinical program is comprised of several Lm-based immunotherapies designed to target multiple shared hotspot and tumor-associated antigens commonly found in specific cancer types. In this study, we demonstrate control of tumor growth in a mouse model by targeting a commonly shared hotspot mutation using an Lm-based immunotherapy. Results: We show that the Lm-HOT KRAS_G12D therapy significantly delayed tumor growth and improved long-term survival in the murine CT26 colon carcinoma model. This response was associated with an increase in the frequency of tumor infiltrating antigen-specific CD8 T cells and γδ T cells within the tumor microenvironment and a decrease in the frequency of intratumoral regulatory T cells (Tregs). Furthermore, tumor-specific CD8 T cells displayed lower expression of exhaustion markers as well as increased functionality upon restimulation. Interestingly, our proprietary ADXS-503 (a clinical ADXS-HOT construct) which includes KRAS G12D as one of its multiple targets, was also capable of significantly suppressing tumor growth in the CT26 tumor model. Conclusion: These results suggest that our ADXS-HOT platform is a promising approach to target shared hotspot mutations. That ADXS Lm constructs targeting a single hotspot mutation can significantly control tumor growth whether it is in a single or multi-target construct. These data describe an exciting translatable discovery with the potential for broad utility across multiple tumor types and patients who share common hotspot mutations. Citation Format: Daniel Villarreal, Brandon Coder, Susan Armington, Andrew L'Huillier, Cristina Mottershead, Elena Filippova, Nithya Thambi, Kim Ramos, David Balli, Robert Petit, Michael Princiotta. Targeting shared hotspot cancer mutations with a Listeria monocytogenes immunotherapy induce potent anti-tumor immunity [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-149.

The ceramide structure of sulfatide-analogues influences the functional activity of type II NKT cells

Abstract The immune system plays a dual role in tumor elimination. Tumor antigens activate cytotoxic cells that kill tumor cells, but the autologous origin of tumors also activates regulatory cells supporting tumor escape. The CD1d-restricted sulfatide–reactive type II NKT cells have a regulatory role by increasing the development of lung metastasis after sulfatide injection in a murine model of colon carcinoma. The identification of compounds able to control the activation of type II NKT cells is essential. Sulfatide (C24:1), a lipid enriched in many tumors, is composed of a galactose head with a sulfate group β-linked to a ceramide moiety. The structure of the ceramide influences the TCR/lipid-CD1d complex interaction and determines the reactivity to the lipid. We synthesized diverse sulfatide-analogues varying in the number of double bonds and hydroxyl groups in the ceramide. Using analogue-loaded-CD1d tetramers, we identified analogue-specific cells in the lung. These cells were also sulfatide-reactive but distinct from α-GalCer-specific type I NKT cells. In vitro, analogues exhibiting a phytosphingosine chain activated both type II and type I NKT cell hybridomas. All analogues induced CD1d-dependent IL-13 production by lung cells but in differing amounts. In vivo, analogues had variable effects on lung metastasis. Interestingly, the analogue C24:2, recognized only by the type II NKT hybridoma, induced significantly more IL-13 production by lung cells than sulfatide while significantly reducing the development of lung metastasis. The distinct effects of sulfatide and C24:2 on the specific activation of type II NKT cells and on tumor elimination could make C24:2 a valuable addition to the armamentarium of anti-tumor immunotherapies.

M7824, a novel bifunctional anti-PD-L1/TGFβ Trap fusion protein, promotes anti-tumor efficacy as monotherapy and in combination with vaccine

ABSTRACTTumors evade host immune surveillance through multiple mechanisms, including the generation of a tumor microenvironment that suppresses immune effector function. Secretion of TGFβ and upregulation of immune checkpoint programmed cell death ligand-1 (PD-L1) are two main contributors to immune evasion and tumor progression. Here, we examined the efficacy of a first-in-class bifunctional checkpoint inhibitor, the fusion protein M7824, comprising the extracellular domain of human TGFβRII (TGFβ Trap) linked to the C-terminus of human anti-PD-L1 heavy chain (αPD-L1). We demonstrate that M7824 reduces plasma TGFβ1, binds to PD-L1 in the tumor, and decreases TGFβ-induced signaling in the tumor microenvironment in mice. In murine breast and colon carcinoma models, M7824 decreased tumor burden and increased overall survival as compared to targeting TGFβ alone. M7824 treatment promoted CD8+ T cell and NK cell activation, and both of these immune populations were required for optimal M7824-mediated tumor control. M7824 was superior to TGFβ- or αPD-L1-targeted therapies when in combination with a therapeutic cancer vaccine. These findings demonstrate the value of using M7824 to simultaneously target TGFβ and PD-L1/PD-1 immunosuppressive pathways to promote anti-tumor responses and efficacy. The studies also support the potential clinical use of M7824 as a monotherapy or in combination with other immunotherapies, such as therapeutic cancer vaccines, including for patients who have progressed on αPD-L1/αPD-1 checkpoint blockade therapies.

Fibromodulin deficiency reduces collagen structural network but not glycosaminoglycan content in a syngeneic model of colon carcinoma.

Tumor barrier function in carcinoma represents a major challenge to treatment and is therefore an attractive target for increasing drug delivery. Variables related to tumor barrier include aberrant blood vessels, high interstitial fluid pressure, and the composition and structure of the extracellular matrix. One of the proteins associated with dense extracellular matrices is fibromodulin, a collagen fibrillogenesis modulator expressed in tumor stroma but scarce in normal loose connective tissues. Here, we investigated the effects of fibromodulin on stroma ECM in a syngeneic murine colon carcinoma model. We show that fibromodulin deficiency decreased collagen fibril thickness but glycosaminoglycan content and composition were unchanged. Furthermore, vascular density, pericyte coverage and macrophage amount were unaffected. Fibromodulin can therefore be a unique effector of dense collagen matrix assembly in tumor stroma and, without affecting other major matrix components or the cellular composition, can function as a main agent in tumor barrier function.

Abstract 594: Dual targeting of TGFb and PD-L1 promotes potent anti-tumor efficacy in multiple murine models of solid carcinomas

Abstract Tumors evade host immune surveillance through multiple mechanisms, including promoting a tumor microenvironment able to effectively suppress immune effector functions. Secretion of cytokines such as TGFb and upregulation of immune checkpoint molecules are two main contributors to immune evasion and tumor progression. TGFb is an important immunosuppressive cytokine with pleiotropic actions in cancer, including promotion of epithelial to mesenchymal transition and metastasis. It promotes tumor immune evasion by impairing T and NK cell maturation, recruitment, and function. Tumors also inhibit antitumor immune responses through tumor cell-immune cell interaction, in which the negative regulatory checkpoint PD-L1/PD-1 interaction plays a major role. Therapies that independently target TGFb signaling or PD-L1/PD-1 interaction demonstrate limited clinical efficacy. Anti-PD-L1/TGFbTRAP (M7824) is a novel immunotherapeutic agent designed to simultaneously block the PD-L1 and TGFb immunosuppressive pathways. It is comprised of the extracellular domain of human TGFbRII (TGFbTRAP) linked to the C-terminus of human anti-PD-L1 heavy chain (anti-PD-L1). M7824 has shown potent anti-tumor activities in preclinical models1 and some evidence of clinical efficacy in a phase 1 study2. Here, we demonstrate that anti-PD-L1/ TGFbTRAP neutralizes murine TGFb-induced signaling and binds to murine PD-L1 in multiple murine breast and colon carcinoma models. In non-tumor bearing mice, anti-PD-L1/TGFbTRAP increases the number of CD8+ T cells in the lymph nodes, and elicits splenic CD8+ T and NK cells with a more active phenotype. Initial examination of peripheral immune subsets in tumor-bearing mice indicates that anti-PD-L1/TGFbTRAP induces CD8+ T cells and NK cells with a more active, less exhausted phenotype. Furthermore, dual targeting of PD-L1 and TGFb increases tumor expression of MHC-I, MHC-II, and PD-L1, suggesting additional immune effects in the tumor microenvironment. Importantly, anti-PD-L1/TGFbTRAP displays potent anti-tumor efficacy against various murine models of breast and colorectal carcinomas. This response is durable, preventing relapses up to 7 months. Anti-PD-L1/TGFbTRAP also decreases spontaneous metastasis in a murine model of triple-negative breast cancer. Taken together, these findings support the preclinical proof of concept for dual targeting of TGFb and PD-L1/PD-1 pathways with anti-PD-L1/ TGFbTRAP, its mode of action, and its potential clinical use as a monotherapy or in combination with other immunotherapies or standards of care. 1 Lan Y et al, Preclinical evaluation of M7824 (MSB0011359C), a novel bifunctional fusion protein targeting the PD-L1 and TGFb pathways. Keystone Symposium, Jan 2017. 2 Strauss J et al, Phase I trial of M7824 (MSB0011359C), a bifunctional fusion protein targeting PD-L1 and TGFb, in advanced solid tumors. Keystone Symposium, Jan 2017. Citation Format: Karin M. Knudson, Sofia R. Gameiro, Kin-Ming Lo, Jeffrey Schlom. Dual targeting of TGFb and PD-L1 promotes potent anti-tumor efficacy in multiple murine models of solid carcinomas [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 594. doi:10.1158/1538-7445.AM2017-594

Abstract 2612: Anti-Tigit induces T cell mediated anti-tumor immune response and combines with immune checkpoint inhibitors to enhance strong and long term anti-tumor immunity

Abstract TIGIT (T cell immunoreceptor with Ig and ITIM domains) has been recently described as an inhibitory receptor which blocks CD8 T cell-mediated anti-tumor immune responses. We have generated an anti-mouse TIGIT antibody (313R12) to evaluate drug efficacy and mechanism of action in pre-clinical tumor models. Anti-TIGIT as a single agent promoted an anti-tumor immune response in multiple syngeneic mouse tumor models. Anti-TIGIT enhanced tumor specific T cell responses, particularly of the Th1 type and reduced Th2 type responses and also increased the function of cytotoxic T cells. Furthermore, anti-TIGIT displayed combination activity with immune checkpoint inhibitors anti-PD1 and anti-PDL1 in inhibiting tumor growth, promoting complete tumor rejection and significantly increasing mouse survival in the murine CT26 colon carcinoma model as compared to controls and single agents alone. Mice “cured” with anti-TIGIT/anti-PDL1 or anti-TIGIT/anti-PD1 combination treatments did not form tumors upon subsequent re-challenges with increasing number of CT26 tumor cells, suggesting the existence of immunologic memory. IL2 and tumor-specific IFN-γ production by splenic T cells were increased in mice who responded to combination treatment compared to controls. Additionally, both effector and memory CD8+ T cell frequencies were increased within the total CD8+ T cell population in responding mice. We also demonstrated a systemic increase in tumor-specific CD8 T cells after anti-TIGIT/anti-PDL1 combination treatment compared to controls. Therefore, these results suggest that co-targeting of TIGIT and PD1 or PDL1 may be an effective and durable cancer therapy by increasing T cell-mediated anti-tumor immune responses and promoting long-term immunological memory. Citation Format: Minu K. Srivastava, Rui Yun, Erin Mayes, Janice Yu, Hyun-Bae Jie, Fumiko Axelrod, Ming-Hong Xie, Jorge Monteon, Andrew Lam, May Ji, Yuwang Liu, John Lewicki, Tim Hoey, Austin Gurney, Angie Inkyung Park. Anti-Tigit induces T cell mediated anti-tumor immune response and combines with immune checkpoint inhibitors to enhance strong and long term anti-tumor immunity [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 2612. doi:10.1158/1538-7445.AM2017-2612

Abstract 2619: Combination of ECP1014 and anti-PD-L1 reduces tumor growth in the CT26 murine colon carcinoma model of a cold tumor

Abstract Objective: The objective of this study was to evaluate the effect of ECP1014, a new selective COX2 inhibitor, in combination with a PDL1 inhibitor on tumor growth in a CT26 murine colon carcinoma model. Colon cancer has been described as a “cold tumor” and antiPDL1monotherapy has shown limited or no benefit. In addition, many colon cancer cell lines produce high levels of PGE2, which may in turn dampen the immune system. The hypothesis was that combining ECP1014, which we have shown potently decreases PGE2 in CT26, with a checkpoint inhibitor would increase immune response to the tumor, turning it “hot” and producing superior tumor control versus either agent alone. Methods: Balb/C mice were implanted with CT26 cells and randomly assigned to 7 treatment groups; vehicle (G1), 10mg/kg rat IgG2b control (G2), 10mg/kg antiPDL1 (G3), 10mg/kg ECP1014 (G4), 10mg/kg ECP1014+10mg/kg rat IgG2b (G5), 10mg/kg ECP1014+10mg/kg antiPDL1 (G6), 1mg/kg ECP1014+10mg/kg antiPDL1(G7). ECP1014 was given daily by oral gavage; rat IgG2b and antiPDL1 (clone 10F.9G2) were administered IP. Treatment was started on day 3 post implant and mice were followed for 16 days. Tumor measurements were taken every 2 to 3 days, and a nonparametric approach was taken to assess the pairwise differences among groups using day 16 tumor volumes. In addition, a responder analysis was used. Tumor infiltrating lymphocytes (TIL) were also assessed by flow cytometry on day 16. Results: All animals developed tumors. G4 to 7 demonstrated statistically significantly (p<0.05) slowing of tumor growth when compared to vehicle (G1). In particular, G7 produced an 87% suppression of tumor growth compared to vehicle. Three animals in G7 showed regression of tumor growth and one additional animal had no detectable growth until day 16. G6 produced 65% suppression of tumor growth compared to G1. G2 to 5 produced 30, 54, 73 and 50% suppression of tumor growth respectively when compared to G1. G7 had the highest number of responders 75% vs. 62.5% for G6 and G4, with all other groups having < 25% responders. TIL analysis from G4, G6 and G7 also displayed the highest levels of CD8+ Tcells (6.3%, 6.8%, and 6.7% respectively, versus 3.8% for antiPDL1 and 2.5% for vehicle groups). Summary: ECP1014 alone was superior to antiPDL1 in slowing tumor growth. However, the combination of ECP1014+antiPDL1showed the greatest tumor response and growth suppression and markedly increased CD8+ Tcells infiltrating into the tumor vs. single agent anti-PDL1 treatment. Citation Format: Bobby W. Sandage, John J. Talley, Eduardo J. Martinez, Maryland R. Franklin, Mary Anne Meade, Dan Saims, Matt Thayer, Scott Wise, David Draper, Wilber Leopold. Combination of ECP1014 and anti-PD-L1 reduces tumor growth in the CT26 murine colon carcinoma model of a cold tumor [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 2619. doi:10.1158/1538-7445.AM2017-2619

Abstract 5624: Characterization of proliferation in multiple T-cell subsets in the CT26 murine colon carcinoma model by multi-color flow cytometry

Abstract The efficacy of immune-modulating anti-cancer therapeutic antibodies that have been FDA-approved in recent years, such as anti-CTLA-4, anti-PD-1 and anti-PD-L1, has altered the paradigm of cancer treatment. Subsequently, the growing interest in the development of new single agent and combination therapies with immune-modulatory effects has generated a need for more powerful immunophenotyping techniques capable of in-depth cell characterization and proliferation assessment. To this end, using the CT26 syngeneic murine colorectal cancer model we have developed an 8 color flow cytometry antibody panel that focuses on the identification of lymphocyte subsets and analysis of proliferation within them utilizing the high-throughput-capable 4-laser, 14-color Attune NxT Flow Cytometer (Thermo Fisher Scientific). Splenocytes from tumor-naïve and CT26 tumor-bearing C57BL/6 mice were stained with CFSE (carboxyfluorescein succinimidyl ester), an amine-reactive fluorescent dye which enables generational tracking for up to seven rounds of cell division, allowing for proliferation to be quantified. Splenocytes from these mice were then stimulated with anti-CD3, anti-CD28, and IL-2. After stimulation for four days, cells were stained with antibodies against CD45, CD3, CD19, CD49b, CD335, CD4, CD8, and FoxP3. We were able to identify differential proliferative capacity between naïve and tumor-bearing mice in CD4+ T-cell, CD8+ T-cell, regulatory T-cell, and B-cell populations. Finally, we show that the accuracy of analysis is enhanced by the use of fluorescence minus-one (FMO) controls to identify those markers that generate dim signals, as well as a viability dye used to exclude dead cells from analysis. Identification of potentially responsive immune compartments, as well as characterizing proliferation, will facilitate identification and development of potential combination therapies by expanding the depth of our ability to provide mechanistic descriptions of drug function and efficacy. Following this proof-of-concept work, we will also present data from tumor-bearing mice treated with immune checkpoint inhibitors in order to identify potential differences in proliferation after treatment with immune-modulating agents. Citation Format: Matt Thayer, Alden Wong, David Draper, Dan Saims, Scott C. Wise. Characterization of proliferation in multiple T-cell subsets in the CT26 murine colon carcinoma model by multi-color flow cytometry [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 5624. doi:10.1158/1538-7445.AM2017-5624

TLR9 agonist lefitolimod to improve antitumor effect of checkpoint inhibitors in vivo.

e14625 Background: TLR9 agonists are potent activators of the immune system via induction of cellular and humoral responses. Preclinical and ongoing clinical studies support the use of TLR9 agonists for immunotherapeutic approaches. Lefitolimod/MGN1703 is a covalently-closed dumbbell-like immune surveillance reactivator (ISR) with a broad immunomodulatory potential. Due to promising data from a phase 2 trial (IMPACT) as maintenance therapy after first-line chemotherapy in mCRC patients lefitolimod is recently evaluated in a phase 3 trial in mCRC patients (IMPALA). Furthermore, lefitolimod is currently investigated in a phase 2 trial in SCLC patients (IMPULSE) and in a phase 1 / 2 trial in HIV patients (TEACH). Methods: It was shown that lefitolimod reduces tumor growth in several murine tumor models. Since the mode-of-action of lefitolimod starts upstream of the initiation points of checkpoint inhibitors like anti-CTLA-4 or anti-PD-1/anti-PD-L1 a combinatory approach may result in an enhanced anti-tumor effect. Therefore, two syngeneic murine models were used – a colon carcinoma CT26 and a lymphoma A20 model – for evaluation of the anti-tumor effect in vivo. Results: In the CT26 model treatment with anti-PD-L1 (i.p.) had no effect on the tumor growth, whereas addition of lefitolimod (s.c.) to anti-PD-L1 led to an anti-tumor effect (tumor growth inhibition, TGI 48%) which consequently resulted in prolonged survival of the mice. This combinatory effect was even more pronounced in the A20 model where treatment with anti-PD-1 (i.p.) alone had a moderate anti-tumor effect which was vastly increased by the combination (TGI – anti-PD-1: 46%, anti-PD-1/lefitolimod 99%). Conclusions: In conclusion, we showed that lefitolimod, a member of dSLIM® family of TLR9 agonists and an ISR, can enhance the limited anti-tumor effects of checkpoint inhibitors in pilot studies in murine colon carcinoma and lymphoma tumor models in vivo. These data show the promising potential for the combination with checkpoint inhibitors. Notably, a clinical trial in cooperation with MD Anderson evaluating the benefit of lefitolimod in combination with ipilimumab is currently ongoing.

Syndecan-1 deficiency promotes tumor growth in a murine model of colitis-induced colon carcinoma.

Syndecan-1 (Sdc1) is an important member of the cell surface heparan sulfate proteoglycan family, highly expressed by epithelial cells in adult organisms. Sdc1 is involved in the regulation of cell migration, cell-cell and cell-matrix interactions, growth-factor, chemokine and integrin activity, and implicated in inflammatory responses and tumorigenesis. Gastrointestinal tract represents an important anatomic site where loss of Sdc1 expression was reported both in inflammation and malignancy. However, the biological significance of Sdc1 in chronic colitis-associated tumorigenesis has not been elucidated. To the best of our knowledge, this study is the first to test the effects of Sdc1 loss on colorectal tumor development in inflammation-driven colon tumorigenesis. Utilizing a mouse model of colitis-related colon carcinoma induced by the carcinogen azoxymethane (AOM), followed by the inflammatory agent dextran sodium sulfate (DSS), we found that Sdc1 deficiency results in increased susceptibility to colitis-associated tumorigenesis. Importantly, colitis-associated tumors developed in Sdc1-defficient mice were characterized by increased local production of IL-6, activation of STAT3, as well as induction of several STAT3 target genes that act as important effectors of colonic tumorigenesis. Altogether, our results highlight a previously unknown effect of Sdc1 loss in progression of inflammation-associated cancer and suggest that decreased levels of Sdc1 may serve as an indicator of colon carcinoma progression in the setting of chronic inflammation.

Combination of TLR9 agonist lefitolimod/MGN1703 with checkpoint inhibitors for cancer immunotherapy.

634 Background: TLR9 agonists are potent activators of the immune system via induction of cellular and humoral responses. Preclinical and ongoing clinical studies support the use of TLR9 agonists for immunotherapeutic approaches. Lefitolimod/MGN1703 is a covalently-closed dumbbell-like immune surveillance reactivator (ISR) with a broad immunomodulatory potential. After promising data from a phase II trial (IMPACT) as maintenance therapy after first-line induction chemotherapy in patients with metastatic colorectal cancer lefitolimod is recently evaluated in a phase III trial in mCRC patients (IMPALA). Methods: It was previously shown that lefitolimod can reduce tumor growth in several murine tumor models. Since the mode-of-action of lefitolimod starts upstream of the initiation points of checkpoint inhibitors aCTLA-4 or aPD-1/aPD-L1 a combinatory approach may result in an enhanced anti-tumor effect. Therefore, we employed two syngeneic murine tumor models: One with s.c. inoculation of CT26 cells and another with s.c. A20 cell challenge. Multiple doses of lefitolimod were injected s.c. or i.tu. Results: In the colon carcinoma CT26 model i.p. injection of aPD-L1 had no effect on the tumor growth, whereas peritumoral injection of lefitolimod led to a slowed tumor growth. The tumor growth was further inhibited by the combination (tumor growth inhibition, TGI - aPD-L1: no, lefitolimod: 28%, combination: 48%) resulting in prolonged survival of the mice. This combinatory effect was even more pronounced in the lymphoma A20 model: Injections of anti-PD-1 (i.p.) or lefitolimod (i.tu.) alone had a moderate anti-tumor effect which was vastly increased by the combination (TGI - aPD-1: 46%, lefitolimod: 50%, combination: 99%). Conclusions: In conclusion, we showed that lefitolimod, a member of dSLIM family of TLR9 agonists and an ISR, can enhance the limited anti-tumor effects of checkpoint inhibitors in pilot studies in murine colon carcinoma and lymphoma tumor models in vivo. These data show the promising potential for the combination with checkpoint inhibitors. In fact, a clinical trial in cooperation with MD Anderson evaluating the benefit of lefitolimod in combination with ipilimumab is currently ongoing.

Abstract 3242: In depth myeloid cell characterization in the murine syngeneic CT26 colon carcinoma model by 10 color flow cytometry

Abstract The efficacy of immune-modulating anti-cancer therapeutic antibodies that have been FDA-approved in recent years, such as anti-CTLA-4 and anti-PD-1, has driven growing interest in methods that provide a mechanistic understanding of drug function. Development of new mono- and combination therapies with immune-modulatory effects requires more powerful immunophenotyping techniques capable of in depth cell characterization. To this end, using the CT26 murine syngeneic colorectal cancer model we have developed a 10 color flow cytometry antibody panel that focuses on the identification of tumor-infiltrating immune cell subsets derived from myeloid lineage precursors utilizing the high-throughput-capable 4-laser, 14-color Attune NxT Flow Cytometer with autosampler. The panel includes a combination of antibodies against CD45, CD3, CD19, CD49b, CD335, CD11b, CD11c, Ly-6G, Ly-6C, F4/80, and CD115. By excluding cells of lymphoid lineage, we show that this panel facilitates analysis of myeloid derived cells including natural killer (NK) cells, macrophages, neutrophils, dendritic cells (DCs), and monocytic or granulocytic myeloid-derived suppressor cell (mMDSCs and gMDSCs) subsets in tumor and peripheral blood. In addition, this combination of antibodies allows for a more complete analysis of MDSCs which can differentially express several disease-relevant myeloid specific markers including Ly-6G, Ly-6C, F4/80, CD11c, and CD115. In the tumor, the Ly-6G-high population demonstrates differential expression of Ly-6C, 21% Ly-6C-high (granulocytes) and 77% Ly-6C-low (mMDSCs). The majority of the granulocytic population was identified as gMDSCs and the remainder as neutrophils based on CD115 expression. Macrophages constitute 27% of Ly-6G-low cells. In blood, 98% of the Ly-6G-high population was also Ly-6C-high, and this population is predominantly neutrophils. No macrophages (Ly6G-low and F4/80+) were identified in the peripheral blood. These data confirm the expected distribution of myeloid lineages in the tissue types investigated. Finally, we show that the accuracy of analysis is enhanced by the use of fluorescence minus-one (FMO) controls to identify those markers that generate dim signals, as well as a viability dye used to exclude dead cells from analysis. Identification of additional potentially responsive immune compartments will facilitate identification and development of potential combination therapies otherwise overlooked by looking primarily at T-cells. This panel allows for a significant expansion of our ability to provide a complex description of the myeloid subset. Citation Format: Matt Thayer, David Draper, Daniel Saims, Maryland Rosenfeld-Franklin, Scott C. Wise. In depth myeloid cell characterization in the murine syngeneic CT26 colon carcinoma model by 10 color flow cytometry. [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 3242.