Abstract The Stimulator of Interferon Genes (STING) protein is a key mediator of innate immunity that plays a central role in the immune response to invading pathogens (bacterial, viral) and transformed cells. A next generation human STING agonist, JNJ-‘6196 was developed that cures mice of their tumors in preclinical models when administered by the IV route. JNJ-‘6196 was rationally selected to have a weaker binding affinity and fast off rate but functionally is a strong cytokine inducer and an efficient activator of human dendritic cells. JNJ-‘6196 exhibits a unique cytokine induction profile in human PBMCs compared to other cyclic dinucleotides (CDNs) that are not curative by the IV route in mice with higher levels of pro-inflammatory cytokines that mediate antitumor activity and lower levels of those that promote suppressive M2 macrophages. In preclinical models of cancer in mice, JNJ-‘6196 eliminates bilateral tumors when administered IV, and demonstrates activity over a wide therapeutic range. Cured mice are immune to further re-challenge due to the expansion and persistence of tumor specific CD8+ T-cells following JNJ-‘6196 administration. Moreover, JNJ-‘6196 increased the effectiveness of checkpoint inhibitors, turning a PD-1 resistant model into a responsive model. Although it is a very potent inducer of antitumor cytokines in mouse and cyno, it is tolerated at similar dose levels as other CDNs that are not systemically active. The functional properties that confer systemic activity were investigated by comparing gene signatures of JNJ-‘6196 to another CDN that was not curative when administered by the IV route. Differences in the intensity of cytokine gene induction were likely responsible for systemic activity rather than genes that were selectively induced by this IV-active compound. The pharmacologic mode of action of JNJ-‘6196 was investigated and found to be Cmax driven based on efficacy and cytokine readouts. JNJ-‘6196 creates an immune inflamed microenvironment in tumors and could expand the population of patients that respond to immunotherapy. The ability to administer JNJ-'6196 systemically and the potential to synergize with other immunotherapeutics could create unique combination modalities and differentiate this compound from other STING agonists. Citation Format: Szeman Ruby Chan, Gilles Bignan, Emily Pierson, Sally Mahady, Hayley Ta, Wim Schepens, Jan Willem Thuring, Heng Keang Lim, Monicah Otieno, Thomas Wilde, Monica Singer, Nancy Bogdan, Shefali Patel, Leo Luistro, Liam Campion, Melissa Smith, Diana Wiley, Kathryn Packman, Michael Allegrezza, Caitlin Morgan, Jocelyn Sendecki, Glenn Van Aller, Daniel Krosky, Peter Connolly, James Edwards, Kim Staquet, Stuart L. Emanuel. JNJ-‘6196: A next generation STING agonist with potent preclinical activity by the IV route [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5567A.

Abstract CD24 is a heavily glycosylated glycosylphosphatidylinositol- anchored protein that is overexpressed in many different tumor types, including colon cancer (>80 %), and has been shown to correlate with shortened patient survival. CD24 plays a role in regulating cancer cell proliferation and tumor microenvironment interactions. The mechanism by which CD24 regulates cell survival and proliferation is not very well understood. What is better known is that CD24 modulates cancer cell adhesion to the vasculature wall and cancer cell-platelet thrombi formation by its binding to P-selectin expressed on activated platelets and endothelial cells. CD24 was also reported to be a functional marker for liver, colon and pancreatic cancer stem cells. All of these functions may contribute to tumor growth and metastasis. The aim of this study was to investigate the function of CD24 as a target for colon cancer therapy. In order to confirm that CD24 expressing cells acquire oncogenic properties, we transfected the CD24 gene into the CD24− colon cancer line SW480. We confirmed that CD24 overexpression induces SW480 tumor growth in vivo. In order to understand the mechanism by which CD24 promotes tumorigenesis, we found that CD24 overexpression activates several oncogenic pathways. Previous publications have shown that CD24 increases expression of p-Raf, p-ERK, and p-JNK in SW480CD24+ cells. Here, using a reporter assay, we show that CD24 expression activates not only ERK and JNK but also the Wnt pathway. It was also shown in the past that treatment of HT-29 colon cancer cells with the antiproliferative anti-CD24 mAb SWA11 caused a decrease in hypoxia and VEGF pathways. We show here that CD24 induced VEGF as well as FGF-2, IL-10, and MMP2 expression and activation of the hypoxia pathway. Finally, we have tested the tumor inhibitory effect of two different anti-CD24 antibodies, SWA11 (mouse IgG2a) and ALB9 (mouse IgG1). Results suggest that both antibodies bind specifically to the CD24 protein core as shown by competition assays with CD24 Fc and binding assays to the deglycosylated CD24. We show that SWA11 significantly inhibited both HT-29 and SW480CD24+ tumor growth in vivo while ALB9 inhibited SW480CD24+ tumor growth only. SWA11 didn't show any neutralizing activity in vitro in inhibiting cell proliferation or CD24 expression but showed effector function activity that may account for its mechanism of action in vivo. ALB9 mechanism of action was not explored in this study, but as it is a mouse IgG1 antibody, ADCC activity is not likely to be part of its mechanism of action. Together, these results provide support for the hypothesis that CD24 has oncogenic properties and that CD24-expressing tumor can be inhibited with antibody immunotherapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A190. Citation Format: Luciana F. Macedo, Elizabeth Kaiser, Haiyan Jiang, Hillary Millar, Diana Wiley, Adam Cotty, Fred Kaplan, Barry Morse, Jill M. Carton, Michael F. Naso, Randall Brezski, Allison Oberholtze, E. Christine Pietsch, Li Yingzhe, Debbie Marshall, Linda A. Snyder. Colon tumor cells expressing CD24 have oncogenic properties and are inhibited by monoclonal antibody immunotherapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A190.

CD24 is a heavily glycosylated glycosylphosphatidylinositol-anchored protein that is over-expressed in many different tumor types including lung cancer (NSCLC and SCLC), and has been shown to correlate with disease progression. Around 70% of the primary NSCLC was shown to overexpress CD24 and these patients tended to have higher risk of disease progression. CD24 plays a role in regulating cancer cell proliferation and tumor microenvironment interactions. The mechanism by which CD24 regulates cell survival and proliferation is not very well understood. It is better known that CD24 modulates cancer cell adhesion to the vasculature wall and cancer cell-platelet thrombi formation by its binding to P-selectin expressed on activated platelets and endothelial cells. The aim of this study was to investigate CD24 as a target for NSCLC therapy. In order to assess CD24 as a target for NSCLC, we have investigated CD24 expression at the mRNA level in NSCLC tissues. CD24 mRNA was shown to be expressed both in squamous cell carcinoma, and in KRas and EGFR mutant adenocarcinoma tumors (123/168 samples). In order to investigate if CD24 has a critical role in cancer cell functions, we performed knock-down experiments using siRNA and several different NSCLC lines. CD24 siRNA treatment significantly affected NSCLC cancer cell line survival in vitro, by causing cell viability inhibition of 38 to 92% (depending on the cell line tested and the siRNA used), as measured by MTS assay. CD24 knockdown also caused inhibition of H358 colony formation in a soft agar anchorage-independent growth assay. Moreover, CD24 knock-down caused a 4-13 fold increase in apoptosis, as measured by propidium iodide staining. To further test the impact of CD24 knock-down on cell viability, we developed an in vitro 3D model system that is more reflective of the tumor microenvironment, containing basement membrane extract and lung cancer associated fibroblasts (CAFs) in co-culture with H358 lung tumor cells. CD24 knock-down significantly decreased cell viability and inhibited spheroid formation by H358 lung cancer cells grown alone or with CAFs. The migration of H358 cells in the scratch-wound assay was also significantly inhibited by CD24 knock down. Finally, using the H358 xenograft tumor model, we tested the effect of an anti-mouse CD24 antibody (SWA11). Treatment was initiated on the same day of cell implantation and repeated twice a week for 2 weeks. SWA11 significantly inhibited tumor formation. Together, these results demonstrate that CD24 expression affects both the viability and motility of lung cancer cell lines and is a potential target for NSCLC treatment. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C189. Citation Format: Luciana F. Macedo, Elizabeth Kaiser, Haiyan Jiang, Hillary Millar, E. Christine Pietsch, Fred Kaplan, Diana Wiley, Linda A. Snyder, Debbie Marshall. CD24 plays an important role on NSCLC cell functions relevant to tumor growth. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C189.

Abstract Integrins are cell-surface adhesion proteins that interact with various components of the extracellular matrix and play an important role in intracellular signaling, cell adhesion, migration, and proliferation. The expression of αV integrins has been associated with poor clinical outcome in patients with non-small cell lung cancer (NSCLC). Intetumumab (CNTO 95) is a novel, fully human monoclonal antibody against all members of the αV integrin family including αVβ1, αVβ3, αVβ5, αVβ6, and αVβ8. Intetumumab inhibits cell adhesion, migration, proliferation, and induces apoptosis in tumor and endothelial cells in vitro. Intetumumab inhibits tumor growth, metastasis, and angiogenesis in vivo in rat xenograft models. Here, we describe experiments to understand the mechanism of action of intetumumab in NSCLC models in vitro and in vivo. The focal adhesion complex and αV integrins play crucial roles in adhesion, motility, and migration in both normal and cancer cells. NSCLC cells were cultured on a vitronectin matrix to stimulate signaling through the αV integrin pathway and then treated with intetumumab (10 µg/ml). Intetumumab treatment resulted in the reduction of phosphorylated FAK (Y397) and Paxillin (Y31) compared to controls up through 48 hours. FAK has been shown to participate in downstream processes such as cell cycle progression by modulating ERK1/2, AKT and the cyclin-dependent kinase inhibitor p27KIP1. Intetumumab treatment of NSCLC cell lines in vitro resulted in the reduction of phosphorylated ERK1/2 and AKT and the subsequent upregulation of p27KIP1. Furthermore, intetumumab treatment activated the intrinsic pathway of apoptosis as evidenced by the cleavage of caspase 9 and PARP as well as increased Bim expression. In vivo administration of intetumumab (10 mg/kg, i.p., 3x weekly) alone or in combination with docetaxel (up to 20 mg/kg, i.p., weekly for 3 weeks) significantly inhibited the growth of established A549 human NSCLC tumor xenografts in nude rats as a monotherapy (p<0.030) or in combination with docetaxel (p<0.039). Moreover, in three separate experiments, combination treatment resulted in complete tumor regressions in 76% (25/33) of animals showing superior efficacy of the combination versus either agent alone (p=0.0138). In conclusion, the results presented here demonstrate the impact of intetumumab on the cellular and molecular mechanisms mediating cell survival through the αV integrin signaling pathway resulting in disruption of focal adhesion complexes, inhibition of cell cycle progression at the G1 phase and induction of the cell intrinsic pathway of apoptosis. Taken together these results demonstrate enhanced anti-tumor efficacy in an established NSCLC preclinical model and suggest the potential for clinical evaluation of intetumumab in NSCLC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2334.

Abstract CCL2 (C-C chemokine ligand 2; also known as MCP-1) is a pleiotropic chemokine overexpressed by many types of tumors. CCL2 is believed to promote tumor growth by increasing macrophage infiltration, angiogenesis, tumor proliferation/survival, and metastasis. The purpose of these studies was to assess potential mechanisms of action associated with the efficacy of neutralizing CCL2 in human pancreatic xenograft models. Cell lines included BxPC-3, PANC-1 and AsPC-1 human pancreatic carcinoma, all of which are p53mut/krasmut, but each differentially expresses CCL2 and its receptor, CCR2. The three tumor cell lines were implanted subcutaneously in immunocompromised mice. When tumors reached ∼50-100 mm3, a cocktail of neutralizing antibodies to human CCL2, mouse MCP-1 and mouse MCP-5 (termed CCL2 blockade) was administered i.p. at 10 mg/kg each, twice a week for the study duration, either alone or in combination with gemcitabine i.p. at 120 mg/kg q3dx4. CCL2 blockade alone significantly inhibited primary tumor growth for BxPC-3 (66-78% tumor growth inhibition (TGI); P<0.001) or prolonged survival for PANC-1 (by seven days; P<0.004). CCL2 in combination with gemcitabine significantly inhibited AsPC-1 tumor growth compared to either therapy alone (55% TGI; P<0.034). By IHC analysis, macrophage infiltration in AsPC-1 tumors was reduced significantly by CCL2 blockade (P=0.049). In vitro studies were conducted to further understand the mechanism of action. Recombinant huCCL2 had no effect on pancreatic tumor cell proliferation in vitro, suggesting that the in vivo anti-tumor effect by CCL2 blockade may be through CCL2 neutralization within the host stroma. Co-culture studies to mimic the tumor-stroma interaction were conducted, using pancreatic tumor cells and normal human lung fibroblasts (NHLF). Secretion of CCL2, as well as IL-5, IL-6, IL-6R, IL-8 and GRO, was significantly enhanced 2-10-fold during co-culture of BxPC3 with NHLF (P<0.001), while only CCL2 was enhanced 2-fold during PANC-1/NHLF co-culture (P<0.001). Furthermore, cell-cell contact was not required for cytokine induction for both BxPC-3 and PANC-1 with NHLF. Tumor cell conditioned medium (CM) stimulated NHLF to produce cytokines, not vice versa, suggesting that a factor(s) in tumor CM is responsible for cytokine induction. Neutralizing antibodies to either CCL2 or IL-6 did not abolish induction of other cytokines in the BxPC-3/NHLF model. The inducers of chemokines/cytokines as well as in vitro migration and in vivo gene profiling studies are underway to better understand the impact of CCL2 blockade on pancreatic tumor growth. These results demonstrate the significant effect of CCL2 blockade on pancreatic tumor growth in vivo, and suggest that the tumor cell/fibroblast interaction may be an important source of CCL2 at the tumor site. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 555.

Tumor histopathology is an important method to understand the mechanism of action of anti‐neoplastic therapies and immunotherapeutics in animal models. An experimental metastasis model was established with the MDA‐MB‐231 human breast adenocarcinoma cell line in SCID beige mice. Compared to subcutaneous and orthotopic models, this model allows evaluation of multiple lesions at earlier time points. Mice were injected i.v. with MDA‐MB‐231 cells and sacrificed either 14 or 21 days post tumor cell injection. Lungs were processed for histology and stained with H&E. Multiple focal lesions were observed and these lesions had a random distribution in the lung and were variable in size. The lesions increased in size and number over time. These observations were confirmed by a rank order analysis on disease severity, and morphometry on size of the lesions. Morphometry also revealed a significant increase in proliferating cells within lung lesions at Day 21 compared to Day 14. Numerous infiltrating macrophages could also be identified within the lesions. No evidence of angiogenesis within the lesions was observed in this time course. It is hypothesized that the lesions studied have not yet crossed the angiogenic switch and therefore no new blood vessel formation was observed. Lesions can be further characterized by additional histologic methods.