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.

Glucagon-like peptide-2 (GLP-2) is a member of the glucagon multigene family that is produced by intestinal enteroendocrine cells in response to food intake. GLP-2 stimulates growth of the intestinal epithelium, enhances its barrier functions, and increases nutrient uptake. Therefore, a GLP-2 agonist may be efficacious in human diseases characterized by malabsorption or injury to the gastrointestinal epithelium. MIMETIBODY™ refers to a proprietary scaffold developed to extend the half-life of rapidly cleared peptides. It consists of a peptide linked to a scaffold that contains sequence elements from a human immunoglobulin G including those that allow recycling through the FcRn. The GLP-2 sequence was engineered into the MIMETIBODY™ scaffold. The primary state of both GLP-2 and the GLP-2 MIMETIBODY™ in DPBS was a noncovalently associated dimer indicative of self-interaction. The increased heterogeneity and the decreased lot-to-lot reproducibility caused by the self-interaction of therapeutic proteins are a challenge to drug development. A similar protein, GLP-1 MIMETIBODY™, contains the related GLP-1 peptide and does not form a dimer under similar conditions. Therefore, to minimize or abrogate dimerization, several variants were made by substituting GLP-2 amino acids with the corresponding amino acids from GLP-1. Molecular weight and secondary structure analyses reveal that substituting leucine for glutamine at position 17 (L17Q) reduces dimerization and α-helix content yet retains bioactivity.