1. Figure 3. Kaplan-Meier analysis demonstrates a significant survival advantage in animals treated with the anti-ITGA6 antibody J8H compared to controls. Animals were followed for up to 8 weeks and were sacrificed if an imminent fracture in any lesion was detected by radiologist, as dictated by the protocol. Figure 5. A) PC3B1a conditioned media prepared in the presence of J8H antibody engagement induces associated Beta-galactosidase activity (SA-B-Gal) when incubated with human fibroblast foreskin (HFF) B). Incubation of PC3B1a cells with J8H antibody induces expression of the matricellular protein CCN1 (CYR61). CCN1 concentration was determined by ELISA assay, and the concentration was calculated based on standard curve. Figure 1. A. Working Model of Laminin Receptor Cross-talk. A6 integrin resides on the tumor cell surface and is post- translationally modified by a serine protease, uPA. uPA cleaves the integrin at specific arginine residues to facilitate migration and adhesion release from laminin substrates. J8H antibody blocks ITGA6 cleavage and prevents migration, but does not inhibit the adhesion function of the receptor. B. Detection of Laminin binding Integrins in prostate tumor cells. Western Blot Analysis shows the full length A3 (150kD, detected by AB1920), and the Alpha 6 Integrin (full-length 150kD, and N-terminal fragment 75kD, detected by AA6A). Model Terry H. Landowski, Jaime Gard, Erika Pond, Gerald D. Pond, Raymond B. Nagle, Christopher P. Geffre, and Anne E. Cress Non-cytotoxic targeting of Integrin A6 stimulates curative-type bone metastasis lesions in a xenograft model Introduction Conclusions Study design J8H antibody prolongs survival in a mouse model J8H treatment reduces bone tumor progression Figure 4. Radiographic analyses demonstrate a reduction in both the frequency of progressive lesions, and the rate of progression in animals treated with J8H antibody. A) All individual lesions (n=69) were scored for progression by the radiologist without knowledge of the treatment groups. B) Specific growth rate is measured using microCT analysis of the three-dimensional volume of the tumor over time. Figure 6. Radiographic analysis and histopathology demonstrate a higher frequency of sclerotic lesions in animals treated with the J8H antibody. Three different micro CT views were used for analysis of each lesion. B) Matched histological sections obtained at the end of the study of the same lesion and tumor mass identified (B, black dotted circle) abutting the epiphyseal plate. Increased magnification of the lesion indicated a sclerotic type lesion, encircled by vessels and new bone formation (Panel B, right image, arrow). C) The frequency of lytic vs sclerotic lesions were scored using MicroCT images from untreated (n=29, solid bars) or J8H-treated (n=40, hatched bars) animals. J8H treatment induces sclerotic lesions Laminin binding integrin receptors are key mediators of epithelial cell migration and tumor metastasis. Recent studies have demonstrated a role for the alpha 6 integrin (ITGA6/CD49f) in maintaining stem cell compartments within normal bone marrow, and in residency of tumors metastatic to bone. In this study, we tested a function-blocking antibody specific for ITGA6, called J8H, to determine if pre-existing cancer lesions in bone could be slowed and/or animal survival improved. Human prostate tumors were established by intracardiac injection into male SCID mice and treatment with J8H (4mg/kg) antibody was initiated after one week. Tumor progression was monitored by microCT imaging of skeletal lesions. Animals that received weekly injections of the anti-ITGA6 antibody showed radiographic progression in only 40% of osseous tumors (femur or tibia), compared to control animals, where 80% of the lesions (femur or tibia) showed progression at 5 weeks. Kaplan-Meier survival analysis demonstrated a significant survival advantage for J8H-treated animals. Unexpectedly, CT image analysis revealed an increased proportion of bone lesions displaying a sclerotic rim of new bone formation encapsulating the arrested lytic lesions in animals that received the anti-ITGA6 antibody treatment. Histopathology of the sclerotic lesions demonstrated well-circumscribed tumor within bone, surrounded by fibrosis. These data suggest that systemic targeting of the ITGA6- dependent function of established tumors in bone may offer a non- cytotoxic approach to arrest the osteolytic progression of metastatic prostate cancer, thereby providing a new therapeutic strategy for advanced disease. J8H engagement of ITGA6 induces HFF senescence Figure 2. PC3B1a bone metastases were established in SCID mice using intracardiac (I.C.) injection. Treatment with 4 mg/kg anti- ITGA6 antibody (J8H) was initiated 1 week after cell injection, and animals were imaged weekly. A) Schematic illustrating the animal protocol; B) Representative micro CT scans of control vs. J8H- treated animals are shown before injection (Wk 0) or weekly beginning with week 3, which is the first point that radiographic lesions could be detected. The ROI used for lesion volume analysis is shown (white dotted circle). Animals were removed from the study (X) when imminent fracture was indicated by the radiologist (*). n=9 n=13 B. A. J8H A2BII The anti-ITGA6 monoclonal antibody, J8H, blocks the cleavage of ITGA6 and prevents tumor cell migration in vitro and in vivo. J8H treatment of animals with pre-existing bone metastases reduces osteolytic tumor progression and increases overall survival. Bone lesions in animals treated with the J8H monoclonal antibody show an increased frequency of sclerotic type lesions compared to untreated animals. Sclerotic lesions are associated with fewer pathologic fractures and less bone pain in patients with prostate cancer metastases. Ex vivo assays suggest that extracellular engagement of the ITGA6 receptor on prostate tumor cells may alter the vicious cycle of bone destruction by altering the tumor microenvironment. Inhibition of integrin function may represent a new, non-cytotoxic treatment for metastatic bone disease. Supported by RO1 CA159406 (AEC), and P01 CA017094 (THL, RBN). Shared services supported by the University of Arizona Cancer Center Support Grant, NIH P30 CA023074.