WO2016033489A2 - Compositions and methods for reducing interaction between receptor for advanced glycation end products (rage) and its ligands - Google Patents

Description

COMPOSITIONS AND METHODS FOR REDUCING INTERACTION BETWEEN RECEPTOR FOR ADVANCED GLYCATION END PRODUCTS (RAGE) AND

ITS LIGANDS

CROSS- REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62/043,352 filed August 28, 2014, which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

This invention was partially funded by the faculty start-up packages of Dr. Vetter at Florida Atlantic University and Dr. Leclerc at North Dakota State University. The start-up package of Dr. Leclerc also included a State EPSCoR New Faculty Award (#FAR0016296). The student performing part of the experiments was partially funded by an Infra-structure Improvement Program-Doctoral Dissertation Assistantship (IIP-DDA), North Dakota EPSCoR grant NSF#0814442.

BACKGROUND OF THE INVENTION

1. Field of Invention

Provided herein are methods, compositions, and uses relating to antibody inhibitors of the Receptor for Advanced Glycation End Products (RAGE) protein. More particularly, the disclosure concerns RAGE targeting antibodies for the detection and treatment of various cancers and other RAGE mediated diseases.

2. Description of Related Art

The receptor for advanced glycation end products (RAGE) is a pattern recognition receptor and a multi-ligand receptor of the immunoglobulin superfamily. Under physiological conditions, RAGE is expressed at low levels by many cell types, e.g., endothelial and smooth muscle cells, macrophages and lymphocytes and most tissues. However, in pathophysiological conditions, such as diabetes, atherosclerosis, rheumatoid arthritis, inflammatory bowel disease or other chronic inflammation, sepsis and neurodegenerative disorders, RAGE expression is increased drastically in different tissues, such as in the vasculature, in the hematopoietic compartment, or in the central nervous system.

RAGE binds several different classes of molecules leading to various cellular responses, including cytokine secretion and cell migration. RAGE includes one extracellular domain, one small transmembrane domain and one cytosoiic domain.

The extracellular domain is capable of binding to structurally and functionally diverse ligands, including advanced glycation end products (AGE), S100/calgranulin family of proteins, transthyretin, high mobility group box-1 chromosomal protein 1 (HMGB-1 ), amyloid β peptide, mac-1, phophatidyl serine, and C3A. Following ligand binding, the cytosoiic domain functions in signal transduction through cellular activation of several kinases and transcription factors including mitogen activated protein kinases (MAPKs), nuclear factor kappa-light-chain-enhancer of activated B cells (NF- Β), AKT, and Ras resulting in oxidative stress and sustained inflammation associated with numerous pathologies. In addition to the membrane bound RAGE, there also exists a soluble form of RAGE secreted into the blood stream

AGEs are formed by non-enzymatic glycosylation of endogenous or dietary proteins by reducing sugars. AGE-RAGE interaction has been shown to activate AKT, NF-kB, Ras, and MAPK signaling pathways and to contribute to various diseases such as those stated above.

The contribution of AGE-RAGE mediated signaling in complications of diabetes has been studied in detail. Accumulation of AGEs in the blood of people with diabetes leads to AGE-RAGE mediated activation of pro-inflammatory signals implicated in diabetic vasculopathy. Tissue accumulation of RAGE ligands can lead to enhanced RAGE expression to amplify and sustain overall signal.

Rage signaling has also been implicated in several cancers such as melanoma and pancreatic cancer. Melanoma is a type of skin cancer that develops in the skin's pigment-producing cells, called melanocytes. These cells make melanin, which is responsible for the color in skin, eyes and hair. The incidence and mortality rates of melanoma have been increasing over the last few decades. The American Cancer Society (ACS) estimates that the lifetime risk of developing melanoma is approximately 1 in 50 for Caucasians, 1 in 1 ,000 for African- Americans, and 1 in 200 for Hispanics. Overall, melanoma is the sixth most common cancer in men and the seventh most common cancer in women (ACS). The National Cancer Institute estimates there will be 76,100 cases of melanoma in the United States and 9,710 deaths from melanoma in 2014 alone.

Pancreatic cancer is the fourth leading cause of cancer-related death in the USA. While the 5-year survival is only 5%, this has been shown to increase with early surgical intervention, however, the disease is notoriously difficult to diagnose in its early stages.

Pancreatic cancers can arise from both the exocrine and endocrine portions of the pancreas. Of pancreatic tumors, almost all develop from the exocrine portion of the pancreas, including the ductal epithelium, acinar cells, connective tissue, and lymphatic tissue. Approximately 75% of all pancreatic carcinomas occur within the head or neck of the pancreas, 15-20% occur in the body of the pancreas, and 5-10% occur in the tail of the pancreas.

AGEs can upregulate RAGE expression in human vascular endothelial cells via NF-kB dependent signaling. Moreover, high glucose consumption by cancer cells leads to accumulation of AGE products within the tumor microenvironment, which can upregulate RAGE expression in the tumors. The AGE/RAGE axis has also been shown to enhance melanoma cell proliferation and migration, which suggests it is active contribution in promoting melanoma growth. RAGE has been detected at high levels in a subset of metastatic tumor samples, both at the transcription and protein levels, suggesting that RAGE might contribute to tumor development only in certain melanoma tumors.

Furthermore, RAGE is expressed by pancreatic cancer cells and is associated with their increased proliferation and high metastatic potential. RAGE promotes constitutive activation of NFkB and phosphorylation and mitochondrial translocation of STAT3 leading to chronic inflammation and resistance to apoptosis. RAGE is also known to regulate autophagy in pancreatic cancers, which facilitates tumor growth. Silencing of RAGE through, for example siRNA leads to enhanced apoptosis and decreased autophagy in pancreatic cancer cells. Such silencing also leads to reduced growth of tumors as shown when these cells are injected into mice. Silencing of RAGE also reduces levels of autophagy markers that may be elevated in pancreatic cancer. Additionally, soluble RAGE is a negative regulator of RAGE function and its levels are inversely correlated with pancreatic cancer etiology. Taken as a whole, RAGE appears to be an important regulator of inflammatory, stress and survival pathways that contribute to resistance to chemotherapy, enhanced proliferation and the high metastatic potential of melanoma and pancreatic cancer.

Inhibition of binding of ligands to RAGE could be an effective therapeutic strategy against diseases associated with and ligand-RAGE mediated signaling.

Accordingly, there is a need for compositions effective in inhibiting binding of ligands to RAGE. The present invention addresses that need.

SUMMARY OF THE INVENTION

The present disclosure provides for Anti-RAGE antibodies for use as a therapeutic agent, diagnostic agent and pharmaceutical compositions. The disclosure further provides for methods of treating RAGE-mediated disease. The antibodies described herein are generally, but not limited to monoclonal antibodies that specifically bind the V domain, C1 domain, C2 domain, or in combination.

The antibodies described herein specific for RAGE can reduce or prevent triggering of the RAGE inflammation cascade, and can reduce or slow disease progression. Moreover, some embodiments of the disclosure show highly specific binding to the V-domain, a known ligand binding domain, where binding to said domain does not induce RAGE signaling, thus minimizing any side effect causes by RAGE binding.

In other embodiments, the use of anti-RAGE antibodies lowers the effective amount of pharmaceutical or cytotoxic agent required to treat a disease state. It is also possible to conjugate the anti-RAGE antibodies with an effector molecule for targeted delivery to diseased cells.

Some embodiments of the present disclosure include, but are not limited to antibodies or antigen-binding regions comprising a heavy chain variable region having at least 80%, at least 90% or at least 95% amino acid identity to SEQ ID NO: 1-8 and a light chain variable region having at least 80%, at least 90% or at least 95% amino acid identity to SEQ ID NO: 9-16.

In other embodiments, antibodies or antigen-binding regions of the present disclosure contain a heavy chain variable region comprising a heavy chain variable region CDR1 , CDR2, and CDR3 having at least 80%, at least 90% or at least 95% amino acid identity to a heavy chain variable region CDR1 , CDR2, and CDR3 of SEQ ID NO: 1-8, and a light chain variable region comprises a light chain variable region CDR1 , CDR2, and CDR3 having at least 80%, at least 90% or at least 95% amino acid identity to a light chain variable region CDR1 , CDR2, and CDR3 of SEQ

ID NO: 9-16.

In further embodiments, the antibodies disclosed herein contain a heavy chain variable region CDR1, CDR2, and CDR3 having at least 80%, at least 90% or at least 95% amino acid sequence identity, or are identical to the heavy chain variable region CDR1, CDR2, and CDR3 of SEQ ID NO: 1 and the light chain variable region CDR1 , CDR2, and CDR3 having at least 80%, at least 90% or at least 95% sequence identity, or are identical to the light chain variable region CDR1 , CDR2, and CDR3 of SEQ ID NO: 9.

In other embodiments, antibodies disclosed herein contain a heavy chain variable region comprising a heavy chain variable region CDR1, CDR2, and CDR3 and a light chain variable region comprising a light chain variable region CDR1 , CDR2, and CDR3, the heavy chain variable region CDR1, CDR2, and CDR3 and the light chain variable region CDR1 , CDR2, and CDR3 may comprise any CDR of SEQ

ID NO: 1-16.

Furthermore, the antibodies described herein may be a fragment of an antibody, such as the Fv or Fab fragment.

In other embodiments, the antibody can be a monoclonal antibody and of any isotype including IgG, IgM, IgD, IgE or IgA, but preferably IgG. The antibodies of present disclosure preferably reduce binding of a RAGE ligand to RAGE and specifically binds to the V, C1, C2 or C1C2 domain of RAGE, or in combination. Rage ligands include S100 family of proteins, AGE-s and HMGB1. Antibody binding to RAGE occurs both in vivo and in vitro.

In further embodiments, there is provided a method for treating RAGE- mediated diseases. Such disease include, but are not limited to diabetes, cancers such as melanoma and pancreatic cancer, Alzheimer's disease, multiple sclerosis and chronic inflammation associated with these diseases. Such methods for treating a subject with a condition mediated by binding of a RAGE ligand to RAGE comprising administering an effective amount of the antibodies described herein. In some embodiments, the method includes the use of an antibody formulated into a pharmaceutically acceptable composition.

In some embodiments, the treated disease is melanoma or pancreatic cancer that may further comprise administering to a subject a pharmaceutical agent effective in treating said melanoma or pancreatic cancer. In still further embodiments, the anti-RAGE antibodies are administered, or co-administered to a subject with a pharmaceutical or cytotoxic agent. The pharmaceutical or cytotoxic agent is preferably an alkylating agent, and more preferably is dacarbazine.

The present disclosure also provides for methods of detecting RAGE in a biological sample, comprising contacting said biological sample with any one of the antibodies described herein. The biological samples can be obtained using any method known to a skilled worker, including, but not limited to urine, blood, serum, plasma, circulating cells saliva, ascites, circulating tumor cells, cells that are not tissue associated, tissues, biopsies, fine needle aspiration samples, surgically resected tumor tissue, or histological preparations.

In other embodiments, the anti-RAGE antibodies contain a detectable moiety. Such detectable moiety may include a radio label, a fluorescent label, an epitope tag, biotin, a chromophore label, a chemiluminescence label, or an enzyme. The anti- RAGE antibodies may further be used in an immunoassay such as a western blot assay, ELISA assay or immunohistochemical assay to detect RAGE In biological samples.

BRIEF DESCRIPTION OF THE DRAWINGS

The preferred embodiments of the invention will be described in conjunction with the appended drawings provided to illustrate and not to the limit the invention, where like designations denote like elements, and in which:

Figure 1 illustrates A: growth of human melanoma xenograft tumors. Xenograft tumors were grown from WM115-MOCK (black triangles) and WM- 115RAGE cells (black spheres). The size of the RAGE tumors were significantly larger than those of the MOCK tumor, after day 10. Mean ± SEM are shown; *p<0.05; **p<.0.01 ; ***p<0.001. B. Microscopic images from WM115-MOCK (a, b) and WM115-RAGE tumors (c, d) stained with either eosin and hematoxylin (a, c) or using IgG 2A11 and peroxidase-conjugated anti-mouse secondary antibody (b, d). Scale bar: 50 μΜ.

Figure 2 illustrates A: protein levels of S100B, S100A2, S100A4, S100A6, and S100A10 in tumors established from WM115-MOCK and WM115-RAGE cells. A representative Western blot is shown. B: Level of Akt and p-Akt, Erk and p-Erk and JNK and p-JNK in tumors established from WM115-MOCK and WM115-RAGE cells. Representative Western blots are shown. Mean ± SD are shown; *p<0.05; **p<0.01 ; ***p<0.001. The blots were performed on at least three different tumors. The density of each band was normalized to that of β-actin. The corresponding densitometric analysis is shown above each blot.

Figure 3 illustrates A: the binding of IgG 2A11 in the presence of the ligand S100B. IgG 2A11 was kept at 100 nM in this experiment. B, C and D illustrate the effect of anti-RAGE antibody treatment on melanoma xenograft growth. B Mice bearing WM115-RAGE tumors of about 80 mm³ diameters were treated with IgG 2a11 (filled spheres) or saline solution (filled triangles). Treatment with IgG 2A11 resulted in growth delay of about 8 days (vertical lines) as defines by the difference of days needed for the IgG treated tumors to reach a tumor size of 500 mm³, compares to the control tumors. ^*p<0.05; ^**p<0.005; ^***p<0.001. C. Tumor volume of mice bearing WM115-MOCK tumors of about 80 mm3 diameters during the treatment with either control IgG (filled squares) or lgG2A11 (filled triangles). D. Body weight the mice bearing the melanoma xenograft during treatment with PBS (filled triangles) or the anti-RAGE antibody (filled spheres).

Figure 4 illustrates A: In vivo imaging of tumors by Cy5.5-conjugated lgG2A11 in the whole animal bearing WM115-RAGE (up) or WM115-MOCK (bottom) animals. B. Fluorescence images of the tumor sections. RAGE tumor sections originating from mice treated with PBS only (saline) were used as negative controls (a, b). Tumor sections originating from WM115-MOCK cells (c, d). Tumor sections originating from WM115-RAGE cells (e, f). The tumor sections were imaged in bright field (a, c, e) or infrared fluorescence mode (b, d, f), Scale bar: 100 micrometers . C. Time dependent accumulation of Cy5.5 conjugated IgG 2A11 at the tumor site. From left to right, Cy5.5-lgG2A11 injected in mice bearing WM1 15-RAGE tumors (light grey bars), Cy5.5-lgG2A11 injected in mice bearing WM115-MOCK tumors (black bars) and Cy5.5-labeled control mouse antibody injected into mice bearing WM115- RAGE tumors (dark grey bars). D. Biodistribution of Cy5.5 conjugated IgG 2A1 1 in different organs of the mouse. Mice implanted with WM115-RAGE cells and injected with Cy5.5 conjugated 2A11 antibody (light grey bars). Mice implanted with WM115- MOCK cells and injected with Cy5.5 conjugated 2A11 (black bars). Mice implanted with WM115-RAGE cells and injected with Cy5.5 conjugated control IgG (dark grey bars).

Figure 5 illustrates A: tumor volumes of WM115-RAGE xenografts at different time points of treatment with DTIC (dacarbazine) alone: 12.5mg/kg (filled squares); 25mg/kg (filled triangles); 50 mg/kg (filled spheres). B. Tumor volumes of WM115- RAGE xenograft tumors at different time points of treatment with DTIC alone: 12.5mg/kg (filled triangles); or in combination with IgG 2A11 (12.5mg/kg DTIC and 0.5 mg/mouse every 5 days, filled squares). C. Tumor volumes of WM115-RAGE xenograft tumors at different time points of treatment with DTIC alone (25mg/kg, filled squares); 50mg/kg (filled triangles) or DTIC (25mg/kg) in combination with IgG 2A11 (filled squares).

Figure 6 illustrates blocking the interaction of RAGE with its ligands (AGEs, HMGB1 or S100P) by neutralizing antibodies could result in reduction of autophagy, increase of apoptosis and decrease in desmoplasia-associated fibroblast activation and in reduction of pancreatic cancer tumor and increased sensitivity towards chemotherapeutic agents.

Figure 7 illustrates RAGE expression in tumors. RAGE (depicted as in Figure 6) is expressed in most cell types forming a tumor: pancreatic cancer cells, myofibroblasts (activated fibroblasts), T cells, endothelial cells and myeloid derived suppressor cells (MSDC). RAGE ligands (AGEs, HMGB1 or S100P) are also found in high levels in pancreatic tumor stroma and act as inflammatory mediators.

Figure 8 illustrates a binding sensograms of monoclonal IgG 2A11 (A) and 2B6 (B) to their respective RAGE domains by SPR. The kinetic parameters k_on and k_0ff were extracted from the global fits and were as follows: IgG 2A11 : k_on = 1.6 x 10⁵ M^-1s^-1; k_off≈ 6.05 x10^-5 s^{-1 ;} IgG 2B6: k_on = 1.74 x 105 M^-1s^-1; k_off≈ 5.9 x 10^-5 s ^-1.

Figure 9 illustrates binding of IgG 2A11 to the surface of RAGE expressing

HEK-293 cells. Nuclei were stained with DAPI.

Figure 10 illustrates spheroids of BxPC-3 (top) and PANC-1 (bottom) stained with anti-RAGE polyclonal antibodies. The nuclei were stained with DAPI. Figure 11 illustrates antibodies produced from the hybridomas and the Hek 293 or CHO cells are similar in terms of binding affinity to RAGE. 2A11 antibody was titrated to cell surface RAGE by flow cytometry. Applicant used Hek 293 cells that overexpress RAGE as a cell line. IgG 2A11 produced from CHO cells binds to cell surface RAGE with a K_D = 2.1 nM (Left panel, full line). IgG 2A11 produced from hybridomas binds to cell surface RAGE with a similar affinity K_D = 1.8 nM (Right panel, full line). On the left panel, the dashed line represents the binding of IgG 2A11 to non-transfected Hek 293 cells. These cells express very little amount of RAGE on their surface.

Figure 12 illustrates a therapeutic antibody that can block RAGE activation by its ligands in cells. Applicant tested if the antibody could suppress the proliferation of pancreatic cancer cells (PANC-1 ) after stimulation with RAGE ligands. (Left panel) Applicant stimulated the proliferation of the cells with 3 concentrations of S100P (1 nM, 10 nM and 100 nM). S100P has been described as a ligand of RAGE in pancreatic cancer cells. Treatment of the PANC-1 cells with IgG 2A11 alone (25ug/ml) slightly reduced the proliferation of the cells, as compared to the treatment of the cells with saline only (CTRL column). Co-treatment of the cells with S100P (100 nM) and IgG 2A11 (25ug/ml) significantly reduced the increased of proliferation observed with S100P. IgG 2A11 could inhibit the interaction between RAGE and its ligands in cells. ^* p<0.05. Experiments were performed in triplicate. (Right panel) IgG 2A11 was tested to suppress RAGE activation by another RAGE ligand: advanced glycation end products (AGE). AGES are glycated proteins that activate RAGE. AGEs have been suggested to contribute to macro- and microvasculature in diabetic patients. They have also been suggested to play important functions in cancer. Applicant showed that the proliferation of PANC-1 cells can be significantly increased when cells are treated with AGEs (1mg/ml). AGEs here have been produced by incubating BSA with 500 mM ribose. Here again, treatment of the cells with IgG 2A11 alone did not significantly change the proliferation of the cells. However, co-treatment of the cells with AGEs (1mg/ml) and IgG 2A11 (25ug/ml) resulted in a significant reduction of the AGE-induced proliferation. Alamar Blue (AB) was used to evaluate the changes in cell proliferation. AB changes its fluorescence properties following changes in reducing agents produced by the proliferative cells. Figure 13 illustrates that IgG 2A11 can suppress AGE-induced proliferation in both PANC-1 and BxPC3 cells. AGE is ribose BSA (1mg/ml). IgG 2A11 (25ug/ml). RAP is a peptide derived from S100P that plays the role of RAGE inhibitor.

Figure 14 illustrates that IgG 2A11 (25ug/ml) can suppress AGE- induced reactive oxygen species (ROS) production and NF-kB activation in PANC-1 cells. (AGE = ribose-BSA; 1mg/ml) * p<0.05.

Figure 15 illustrates the effect of 2A11 on AGE induced proliferation observed in PANC-1 cells could be reproduced in MIAPaCa2 cells. IgG 2A1 1 : 25ug/ml; AGE = ribose-BSA; I mg/ml, ^* p<0.05.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Provided herein are methods and compositions relating to inhibitors of RAGE mediated signaling. For example, Applicant discloses antibodies targeting RAGE, methods of producing antibodies targeting RAGE, and methods for treating diseases as well as for research and diagnostic uses. In some embodiments, the compositions and methods herein provide therapies relating to inhibiting RAGE signaling. Some embodiments provide an isolated antibody that targets RAGE. The compositions and methods provided herein find further use in treating various RAGE mediated cancers such as pancreatic cancer or melanomas.

Applicant has developed antibodies that inhibits activation of the receptor for advanced glycation end products (RAGE). The antibodies specifically bind the RAGE receptor at the V domain, C1 domain or C2 domain or a combination thereof to block activation of RAGE by its ligands (Figure 6). In some embodiments, the antibodies specifically bind to the V domain. The V domain can bind multiple structurally and functionally diverse ligands to trigger signal transduction by RAGE's cytosolic domain, and result in sustained inflammation that is associated with, but not limited to diabetes, diabetic nephropathy, cancers including melanomas and pancreatic cancer, Alzheimer's disease, multiple sclerosis, many chronic inflammatory diseases, including rheumatoid and psoriatic arthritis and inflammatory bowel disease, amyloidoses, cardiovascular diseases and sepsis. As a result, the anti-RAGE antibodies have potential to treat a wide variety of diseases, and in some cases might reduce or slow disease progression. In some embodiments, the antibodies delay tumor growth in a xenograft melanoma model.

Known ligands for RAGE receptors include advanced glycation end products (AGE), S100 family of proteins (S100A1-S100A16, S100B, S100P and S100Z), transthyretin, HMGB-1, amyloid β peptide, mac-1, phophatidyl serine, and C3A. Many of these ligands bind to the V domain of Rage. Therefore, antibodies that inhibit ligand binding to the V domain can reduce RAGE-ligand mediated signaling. Moreover, antibodies that bind to the C1 or C2 domain can also inhibit RAGE mediated signaling. In some embodiments, antibodies that specifically bind to RAGE can be an isolated antibody comprising a heavy chain variable region having at least 80%, at least 90% or at least 95% amino acid identity to SEQ ID NO: 1 , SEQ ID NO: 2. SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 and a light chain variable region having at least 80%, at least 90% or at least 95% amino acid identity to SEQ ID NO: 9 , SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15 OR SEQ ID NO: 16.

In other embodiments, antibodies of the present disclosure contain a heavy chain variable region comprising a heavy chain variable region CDR1 , CDR2, and CDR3 having at least 80%, at least 90% or at least 95% amino acid identity to a heavy chain variable region CDR1 , CDR2, and CDR3 of SEQ ID NO: 1 , SEQ ID NO: 2. SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8; and said light chain variable region comprises a light chain variable region CDR1 , CDR2, and CDR3 having at least 80%, at least 90% or at least 95% amino acid identity to a light chain variable region CDR1 , CDR2, and CDR3 of SEQ

ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 OR SEQ ID NO: 16.

In further embodiments, the antibodies disclosed herein contain a heavy chain variable region CDR1, CDR2, and CDR3 have at least 80%, at least 90% or at least 95% amino acid sequence identity to a heavy chain variable region CDR1 , CDR2, and CDR3 of SEQ ID NO: 1 and a light chain variable region CDR1, CDR2, and CDR3 have at least 80%, at least 90% or at least 95% sequence identity to a light chain variable region CDR1 , CDR2, and CDR3 of SEQ ID NO: 9. In still further embodiments, the antibodies disclosed herein contain a heavy chain variable region CDR1, CDR2, and CDR3 that are identical to the heavy chain variable region CDR1, CDR2, and CDR3 of SEQ ID NO: 1 and a light chain variable region CDR1, CDR2, and CDR3 that are identical to the light chain variable region CDR1 , CDR2, and CDR3 of SEQ ID NO: 9.

In further embodiments, the antibodies may contain a mixture of any CDR's listed in SEQ ID No: 1-16. By way of example, an antibody containing the heavy chain CDR's corresponding to SEQ ID NO: 1 and the light chain CDR's corresponding to SEQ ID NO: 9 may have one or more of the CDR's replaced by a heavy chain CDR from SEQ ID NO: 3 and a light chain CDR from SEQ ID NO: 10, respectively. As another example, an antibody having a heavy chain CDR1 , CRD2 and CDR3 from SEQ ID NO: 1 may have the CDR1 replaced with the CDR1 of SEQ

ID NO: 2. An antibody created in this way will generally contain at least one original CDR, and may have 1, 2, 3, 4 or 5 CDR's replaced with another CDR listed in Appendix A. However, in some embodiments, all six CDR's may be replaced with another CDR of choice. Methods for replacing CDR's are well known in the art.

In still further embodiments, antibodies contain heavy and light chain variable regions identical to SEQ ID NO: 1 and SEQ ID NO: 9. In other embodiments, the antibody is a fragment such as the Fab portion.

As used herein, RAGE particularly refers to human RAGE. Unless otherwise stated the term "RAGE" also encompasses RAGE molecules isolated or obtained from other, different from human, species, such as rodents, like mice or rats; or bovine RAGE molecules.

As used herein, the term "antibody" includes monoclonal antibodies (for e.g., full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity) and may also include certain antibody fragments (as described in greater detail herein).

An "antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as V_H) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as V_L) and a light chain constant region. The light chain constant region is comprised of one domain, C_L. The V_H and V_L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each V_H and V_L is composed of three CDRs (for a total of 6 CDR's) and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

An antibody that "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide is one that binds to that particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

The term "binding affinity" is herein used as a measure of the strength of a non-covalent interaction between two molecules, e.g. an antibody, or fragment thereof, and an antigen. The term "binding affinity" is used to describe monovalent interactions (intrinsic activity). Binding affinity between two molecules, e.g. an antibody, or fragment thereof, and an antigen, through a monovalent interaction may be quantified by determination of the dissociation constant (K_D). In turn, KD can be determined by measurement of the kinetics of complex formation and dissociation, e.g. by the Surface Plasmon Resonance method. The rate constants corresponding to the association and the dissociation of a monovalent complex are referred to as the association rate constant k_a (or k_on) and dissociation rate constant k_d (or k_off), respectively. K_D is related to k_a and k_d through the equation K_D=k_d/k_a.

Typically, the K_D for the antibody with respect to the target will be 2-fold, preferably 5-fold, more preferably 10-fold less than K_D with respect to the other, non- target molecule such as unrelated material or accompanying material in the environment or control. More preferably, the K_D will be 50-fold less, such as 100-fold less, or 200-fold less; even more preferably 500-fold less, such as 1 , 000-fold less, or 10,000-fold less. The value of this dissociation constant can be determined directly by well- known methods, and can be computed even for complex mixtures by methods such as those, for example, set forth in Caceci et al. (Byte 9:340-362, 1984). For example, the K_D may be established using a double-filter nitrocellulose filter binding assay such as that disclosed by Wong & Lohman (Proc. Natl. Acad. Sci. USA 90, 5428- 5432, 1993). Other standard assays to evaluate the binding ability of ligands such as antibodies towards targets are known in the art including, for example, ELISAs, Western blots, radioimmuno assays, and flow cytometry analysis. The binding kinetics and binding affinity of the antibody also can be assessed by standard assays known in the art, such as Surface Plasmon Resonance.

The term "surface plasmon resonance", as used herein, refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

A competitive binding assay can be conducted in which the binding of the antibody to the target is compared to the binding of the target by another ligand of that target, such as another antibody. The concentration at which 50% inhibition occurs is known as the Ki. Under ideal conditions, the Ki is equivalent to K_D. The Ki value will never be less than the K_D, SO measurement of Ki can conveniently be substituted to provide an upper limit for K_D.

The terms "protein" and "polypeptide" refer to compounds comprising amino acids joined via peptide bonds and are used interchangeably. A "protein" or "polypeptide" encoded by a gene is not limited to the amino acid sequence encoded by the gene, but includes post-translational modifications of the protein.

Where the term "amino acid sequence" is recited herein refers to an amino acid sequence of a protein molecule, "amino acid sequence" and like terms, such as "polypeptide" or "protein" are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule. Furthermore, an "amino acid sequence" can be deduced from the nucleic acid sequence encoding the protein.

As used herein, the term "treating" includes reducing or alleviating at least one adverse effect, sign, or symptom of a disease or disorder through introducing in any way a therapeutic composition of the present technology into or onto the body of a subject. "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.

As used herein, "therapeutically effective amount" refers to an amount of a therapeutic agent sufficient to bring about a beneficial or desired clinical effect. Said dose can be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or administration route. However, the precise determination of what would be considered an effective dose may be based on factors individual to each patient, including, but not limited to, the patient's age, size, type or extent of disease, stage of the disease, route of administration, the type or extent of supplemental therapy used, ongoing disease process, and type of treatment desired (e.g., aggressive vs. conventional treatment).

As used herein, the term "pharmaceutical composition" refers to the combination of an active agent with, as desired, a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vitro, in vivo, or ex vivo.

As used herein, the terms "pharmaceutically acceptable" or

"pharmacologically acceptable" refer to compositions that do not substantially produce adverse reactions, e.g., toxic, allergic, or immunological reactions, when administered to a subject.

The term "domain" when used in reference to a polypeptide refers to a subsection of the polypeptide which possesses a unique structural and/or functional characteristic; typically, this characteristic is similar across diverse polypeptides. The subsection typically comprises contiguous amino acids, although it may also comprise amino acids which act in concert or which are in close proximity due to folding or other configurations. Examples of a protein domain include the transmembrane domains, and the glycosylation sites.

As used herein, the term "isolated" in the context of a peptide, polypeptide, fusion protein, antibody or antigen-binding antibody fragment refers to a peptide, polypeptide, fusion protein, antibody or antigen-binding antibody fragment which is substantially free of cellular material or contaminating proteins from the cell or tissue source from which it is derived or obtained, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material or contaminating protein" includes preparations of a peptide, polypeptide, fusion protein, antibody or antigen-binding antibody fragment in which the peptide, polypeptide, fusion protein, antibody or antigen- binding antibody fragment is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, a peptide, polypeptide, fusion protein, antibody or antigen-binding antibody fragment that is substantially free of cellular material or contaminating protein includes preparations of a peptide, polypeptide, fusion protein, antibody or antigen-binding antibody fragment having less than about 30%, about 20%, about 10%, or about 5% (by dry weight) of other protein. When the peptide, polypeptide, fusion protein, antibody or antigen-binding antibody fragment is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, about 10%, or about 5% of the volume of the protein preparation. When the peptide, polypeptide, fusion protein, antibody or antigen-binding antibody fragment is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the peptide, polypeptide, fusion protein, antibody or antigen-binding antibody fragment. Accordingly, such preparations of a peptide, polypeptide, fusion protein, antibody or antigen-binding antibody fragment have less than about 30%, about 20%, about 10%, about 5% (by dry weight) of chemical precursors or compounds other than the peptide, polypeptide, fusion protein, antibody.

The term "contacting" refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the cellular or molecular level, for example, to bring about a physiological reaction, a chemical reaction, or a physical change, e.g., in a solution, in a reaction mixture, in vitro, or in vivo.

The term "about" can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent. For integer ranges, the term "about" can include one or two integers greater than and/or less than a recited integer at each end of the range. Unless indicated otherwise herein, the term "about" is intended to include values, e.g., weight percentages, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment. The term about can also modify the end-points of a recited range as discuss above in this paragraph.

As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements.

"Polynucleotide," synonymously referred to as "nucleic acid molecule," "nucleotides" or "nucleic acids," refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotides" include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double- stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications may be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also embraces relatively short nucleic acid chains, often referred to as oligonucleotides. A "vector" as used herein is a replicon, such as plasmid, phage, cosmid, or virus in which another nucleic acid segment may be operably inserted so as to bring about the replication or expression of the segment.

The terms "express" and "produce" are used synonymously herein, and refer to the biosynthesis of a gene product. These terms encompass the transcription of a gene into RNA. These terms also encompass translation of RNA into one or more polypeptides, and further encompass all naturally occurring post-transcriptional and post-translational modifications. The expression or production of an antibody or antigen-binding fragment thereof may be within the cytoplasm of the cell, or into the extracellular milieu such as the growth medium of a cell culture.

Anti-RAGE antibodies and methods of production

In certain embodiments, an antibody or a fragment thereof that binds to at least a portion of the RAGE protein and inhibits RAGE signaling are contemplated. The antibody may be selected from the group consisting of a chimeric antibody, an affinity matured antibody, a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, or an antigen-binding antibody fragment or a natural or synthetic ligand. Preferably, the anti-RAGE antibody is a monoclonal antibody.

Thus, polyclonal or monoclonal antibodies, antibody fragments, binding domains and CDRs (including engineered forms of any of the foregoing) may be created that are specific to RAGE protein, one or more of its respective epitopes, or conjugates of any of the foregoing, whether such antigens or epitopes are isolated from natural sources or are synthetic derivatives or variants of the natural compounds.

The term "epitope" or "antigenic determinant" includes any polypeptide determinant capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody. In certain embodiments, an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. Animals may be inoculated with an antigen, such as a full-length RAGE proteins, or RAGE protein domains such as the V (amino acids 29-129), C1 (amino acids 130-234) or C2 domains (amino acids 235-336), or a combination thereof in order to produce antibodies specific for RAGE protein (SEQ ID NO: 17). Optionally, an antigen is bound or conjugated to another molecule to enhance the immune response. As used herein, a conjugate is any peptide, polypeptide, protein, or non- proteinaceous substance bound to an antigen that is used to elicit an immune response in an animal. Antibodies produced in an animal in response to antigen inoculation comprise a variety of non-identical molecules (polyclonal antibodies) made from a variety of individual antibody producing B lymphocytes. A polyclonal antibody is a mixed population of antibody species, each of which may recognize a different epitope on the same antigen. Given the correct conditions for polyclonal antibody production in an animal, most of the antibodies in the animal's serum will recognize the collective epitopes on the antigenic compound to which the animal has been immunized. This specificity is further enhanced by affinity purification to select only those antibodies that recognize the antigen or epitope of interest.

A monoclonal antibody is a single species of antibody wherein every antibody molecule recognizes the same epitope because all antibody producing cells are derived from a single B-lymphocyte cell line. The methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. In some embodiments, rodents such as mice and rats are used in generating monoclonal antibodies. In some embodiments, rabbit, sheep, or frog cells are used in generating monoclonal antibodies. The use of rats is well known and may provide certain advantages. Mice (e.g., BALB/c mice) are routinely used and generally give a high percentage of stable fusions.

Hybridoma technology involves the fusion of a single B lymphocyte from a mouse previously immunized with a RAGE antigen with an immortal myeloma cell (usually mouse myeloma). This technology provides a method to propagate a single antibody- producing cell for an indefinite number of generations, such that unlimited quantities of structurally identical antibodies having the same antigen or epitope specificity (monoclonal antibodies) may be produced.

Plasma B cells may be isolated from freshly prepare peripheral blood mononuclear cells of immunized animals and further selected for RAGE binding cells. After enrichment of antibody producing B cells, total RNA may be isolated and cDNA synthesized. DNA sequences of full length antibody or variable regions from both heavy chains and light chains may be amplified, constructed into a phage display expression vector, and transformed into E. coli. RAGE specific binding full length antibody or Fab fragments may be selected through multiple rounds enrichment panning and then sequenced. Selected RAGE binding candidates may be expressed as full length IgG, for example, in mouse and mouse/human chimeric forms using a mammalian expression vector system in human embryonic kidney (HEK293) cells (Invitrogen) and purified using a protein G resin with a fast protein liquid chromatography (FPLC) separation unit, or alternatively as described in Example 1 below.

In one embodiment, the antibody is a chimeric antibody, for example, an antibody comprising antigen binding sequences from a non-human donor grafted to a heterologous non-human, human, or humanized sequence (e.g., framework and/or constant domain sequences). Methods have been developed to replace light and heavy chain constant domains of the monoclonal antibody with analogous domains of human origin, leaving the variable regions of the foreign antibody intact. Alternatively, "fully human" monoclonal antibodies are produced in transgenic mice from human antibody genes. Methods have also been developed to convert variable domains of monoclonal antibodies to more human form by recombinantly constructing antibody variable domains having both rodent, for example, mouse, and human amino acid sequences. In "humanized" monoclonal antibodies, only the hypervariable CDR is derived from mouse monoclonal antibodies, and the framework and constant regions are derived from human amino acid sequences. It is thought that replacing amino acid sequences in the antibody that are characteristic of rodents with amino acid sequences found in the corresponding position of human antibodies will reduce the likelihood of adverse immune reaction during therapeutic use. A hybridoma or other cell producing an antibody may also be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced by the hybridoma.

Methods for producing polyclonal antibodies in various animal species, as well as for producing monoclonal antibodies of various types, including humanized, chimeric, and fully human, are well known in the art and highly predictable. Antibodies may be produced from any animal source, including birds and mammals. Preferably, the antibodies are human, bovine, murine (e.g., mouse and rat), rabbit, goat, guinea pig, camel, horse, chicken or chimeric antibodies thereof.

It is fully expected that antibodies to RAGE will have the ability to neutralize or counteract the effects of RAGE regardless of the animal species, monoclonal cell line, or other source of the antibody, both in vivo and in vitro. Certain animal species may be less preferable for generating therapeutic antibodies because they may be more likely to cause allergic response due to activation of the complement system through the "Fc" portion of the antibody. However, whole antibodies may be enzymatically digested into "Fc" (complement binding) fragment, and into antibody fragments having the binding domain or CDR. Removal of the Fc portion reduces the likelihood that the antigen antibody fragment will elicit an undesirable immunological response, and thus, antibodies without Fc may be preferential for prophylactic or therapeutic treatments. As described above, antibodies may also be constructed so as to be chimeric or partially or fully human, so as to reduce or eliminate the adverse immunological consequences resulting from administering to an animal an antibody that has been produced in, or has sequences from, other species.

Also contemplated herein are antibodies with substitutional variations, and more specifically, antibodies with substitutional variations in the variable heavy and light chain regions, and even more specifically, antibodies with substitutional variations in heavy and light chain CDR's. Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, substitutions may be non-conservative such that a function or activity of the polypeptide is affected. Non- conservative changes typically involve substituting a residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa.

As used herein, "sequence identity" between two polypeptide sequences indicates the percentage of amino acids that are identical between the sequences. "Sequence similarity" indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions. Preferred polypeptide sequences of the invention have a sequence identity in the CDR regions of at least 60%, more preferably, at least 70% or 80%, still more preferably at least 90% and most preferably at least 95%. Preferred antibodies also have a sequence similarity in the CDR regions of at least 80%, more preferably 90% and most preferably 95%. Preferred polypeptide sequences of the invention have a sequence identity in the variable regions of at least 60%, more preferably, at least 70% or 80%, still more preferably at least 90% and most preferably at least 95%. Preferred antibodies also have a sequence similarity in the variable regions of at least 80%, more preferably 90% and most preferably 95%.

In addition, newer technology permits the development of and screening for human antibodies from human combinatorial antibody libraries. The recombinant antibody library may be from a subject immunized with RAGE, or a portion of RAGE. For example, bacteriophage antibody expression technology allows specific antibodies to be produced in the absence of animal immunization, as described in U.S. Pat. No. 6,946,546, which is incorporated herein by reference. Other antibody libraries can be screened to identify an antibody having the desired binding specificity. Methods for such screening of recombinant antibody libraries are well known in the art and include methods described in, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT Publication No. WO 92/18619 and Dower et al. PCT Publication No. WO 91/17271.

As a further example, in phage display methods, functional antibody domains are displayed on the surface of phage particles, which carry the polynucleotide sequences encoding them. In a particular, such phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present disclosure include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9- 18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994).

In another approach the antibodies of the present invention can also be generated using yeast display methods known in the art. In yeast display methods, genetic methods are used to tether antibody domains to the yeast cell wall and display them on the surface of yeast. In particular, such yeast can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine).

In one aspect, the disclosure pertains to an isolated antibody, or an antigen- binding portion thereof, that binds human RAGE. Particularly, the antibody is a neutralizing antibody. In various embodiments, the antibody is a recombinant antibody or otherwise synthesized in vitro or a monoclonal antibody.

The affinity of the described antibodies, or antigen-binding fragments, may be determined by a variety of methods know in the art, such as Surface Plasmon Resonance or ELISA-based methods. To select antibodies of the invention having a particular neutralizing activity for RAGE, such as those with a particular IC₅₀, standard methods known in the art for assessing the inhibition of RAGE activity may be used.

Antibodies with the desired properties can be generated and purified using any suitable method. In some embodiments, the expressed polypeptides are secreted into and recovered from the periplasm of the host cells. Protein recovery typically involves disrupting the microorganism, generally by such means as osmotic shock, sonication or lysis. Once cells are disrupted, cell debris or whole cells may be removed by centrifugation or filtration. The proteins may be further purified, for example, by affinity resin chromatography.

Alternatively, proteins can be transported into the culture media and Isolated therein. Cells may be removed from the culture and the culture supernatant being filtered and concentrated for further purification of the proteins produced. The expressed polypeptides can be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assay. In other embodiments, blood/serum from an immunized subject may be collected and the antibodies therein isolated by interaction with a solid or semi-solid substrate bound antigen and the interacting antibodies recovered through elution with an appropriate solvent.

Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; herein incorporated by reference) can be adapted to produce specific single chain antibodies as desired. Single-chain Fv antibody fragments comprise the V_H and V_L domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the V_H and V_L domains that enables the single-chain Fv antibody fragments to form the desired structure for antigen binding.

In other embodiments, Grafting of CDR regions may be accomplished to produce an antibody of choice. CDR Grafting is performed by replacing one or more CDRs of an acceptor antibody (e.g., a human antibody or other antibody comprising desired framework residues) with CDRs of a donor antibody (e.g., a non-human antibody). Acceptor antibodies may be selected based on similarity of framework residues between a candidate acceptor antibody and a donor antibody. For example, according to one approach, human framework regions are identified as having substantial sequence homology to each framework region of the relevant non-human antibody, and CDRs of the non-human antibody are grafted onto the composite of the different human framework regions. A related method also useful for preparation of antibodies of the invention is described in U.S. Patent Application Publication No. 2003/0040606. It is contemplated that any of the CDR regions disclosed in SEQ ID No: 1-16 may be grafted to form a functional antibody.

CDR's of the present disclosure can also be utilized in small antibody mimetics, which comprise two CDR regions and a framework region. Chimeric or humanized antibodies of the present disclosure can be prepared based on the sequence of a murine antibody described herein. DNA encoding the heavy and light chain immunoglobulins can be obtained from the murine hybridoma of interest and engineered to contain non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to create a chimeric antibody, the murine variable regions can be linked to human constant regions using methods known in the art (see e.g., U.S. Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody, the murine CDR regions can be inserted into a human framework using methods known in the art.

Additional contemplated antibody variants include glycosylation isoforms that result in improved functional properties. For example, modification of the Fc portion by glycosylation can result in altered effector functions, e.g., increased binding to Fc gamma receptors and improved ADCC and/or increased or decreased C1q binding and Complement Mediated Cytotoxicity.

In certain embodiments provided herein, it is desirable to use an antibody fragment. These fragments can contain the full-length variable domain or a portion thereof such as those antibodies described herein. Various techniques are known for the production of antibody fragments. Traditionally, these fragments are derived via proteolytic digestion of intact antibodies (for example Morimoto et al., 1993, Journal of Biochemical and Biophysical Methods 24:107-117 and Brennan et al., 1985, Science, 229:81). For example, papain digestion of antibodies produces two identical antigen-binding fragments, called Fab fragments, each with a single antigen-binding site, and a residual Fc fragment. Pepsin treatment yields an F(ab')₂ fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

However, these fragments can be typically produced directly by recombinant host cells as described above. Thus Fab, Fv, and scFv antibody fragments can all be expressed in and secreted from E. coli or other host cells, thus allowing the production of large amounts of these fragments. Alternatively, such antibody fragments can be isolated from the antibody phage libraries discussed above. The antibody fragment can also be linear antibodies as described in U.S. Pat. No. 5,641 ,870, for example, and can be monospecific or bispecific. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. Fv is the minimum antibody fragment which contains a complete antigen- recognition and antigen-binding site. This region consists of a dimer of one heavy- chain and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the V_H-V_L dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1 ) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. F(ab')₂ antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known to the skilled artisan.

The disclosure herein also contemplates modifying an antibody to increase its serum half-life. This can be achieved, for example, by incorporating a salvage receptor binding epitope into the antibody fragment by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag that is then fused to the antibody fragment at either end or in the middle (e.g., by DNA or peptide synthesis).

The present disclosure further provides for the use of recombinant DNA constructs comprising one or more of the nucleotide sequences of the present invention. The recombinant constructs are used in connection with a vector, such as a plasmid or viral vector, into which a DNA molecule encoding an antibody for use in the invention is inserted.

The vectors can be expression vectors. Recombinant expression vectors containing a sequence encoding a polypeptide of interest are thus contemplated as within the scope of this disclosure. The expression vector may contain one or more additional sequences such as but not limited to regulatory sequences (e.g., promoter, enhancer), a selection marker, and a polyadenylation signal. Vectors for transforming a wide variety of host cells are well known and include, but are not limited to, plasmids, phagemids, cosmids, baculoviruses, bacmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), as well as other bacterial, yeast and viral vectors.

Recombinant expression vectors within the scope of the description include synthetic, genomic, or cDNA-derived nucleic acid fragments that encode at least one recombinant protein which may be operably linked to suitable regulatory elements. Such regulatory elements may include a transcriptional promoter, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation. Expression vectors, especially mammalian expression vectors, may also include one or more nontranscribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, other 5' or 3' flanking nontranscribed sequences, 5' or 3' nontranslated sequences (such as necessary ribosome binding sites), a polyadenylation site, splice donor and acceptor sites, or transcriptional termination sequences. An origin of replication that confers the ability to replicate in a host may also be incorporated.

In some embodiments, the antibody or antigen-binding fragment coding sequence is placed under control of a constitutive promoter, such as the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin, human myosin, human hemoglobin, human muscle creatine, and others. In addition, many viral promoters function constitutively in eukaryotic cells and are suitable for use with the described embodiments. Such viral promoters include without limitation, Cytomegalovirus (CMV) immediate early promoter, the early and late promoters of SV40, the Mouse Mammary Tumor Virus (MMTV) promoter, the long terminal repeats (LTRs) of Maloney leukemia virus, Human Immunodeficiency Virus (HIV), Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV), and other retroviruses, and the thymidine kinase promoter of Herpes Simplex Virus. In one embodiment, the antibody or antigen- binding fragment thereof coding sequence is placed under control of an inducible promoter such as the metallothionein promoter, tetracycline-inducible promoter, doxycycline-inducible promoter, promoters that contain one or more interferon- stimulated response elements (ISRE) such as protein kinase R 2',5'-oligoadenylate synthetases, Mx genes, ADAR1 , and the like. Vectors described herein may contain one or more Internal Ribosome Entry Site(s) (IRES). Inclusion of an IRES sequence into fusion vectors may be beneficial for enhancing expression of some proteins. In some embodiments the vector system will include one or more polyadenylation sites (e.g., SV40), which may be upstream or downstream of any of the aforementioned nucleic acid sequences. Vector components may be contiguously linked, or arranged in a manner that provides optimal spacing for expressing the gene products (i.e., by the introduction of "spacer" nucleotides between the ORFs), or positioned in another way. Regulatory elements, such as the IRES motif, may also be arranged to provide optimal spacing for expression.

The vectors may comprise selection markers, which are well known in the art. Selection markers include positive and negative selection markers, for example, antibiotic resistance genes (e.g., neomycin resistance gene, a hygromycin resistance gene, a kanamycin resistance gene, a tetracycline resistance gene, a penicillin resistance gene), glutamate synthase genes, HSV-TK, HSV-TK derivatives for ganciclovir selection, or bacterial purine nucleoside phosphorylase gene for 6- methylpurine selection (Gadi et al., 7 Gene Ther. 1738-1743 (2000)). A nucleic acid sequence encoding a selection marker or the cloning site may be- upstream or downstream of a nucleic acid sequence encoding a polypeptide of interest or cloning site.

The vectors described herein may be used to transform various cells with the genes encoding the described antibodies or antigen-binding fragments. For example, the vectors may be used to generate antibody or antigen-binding fragment- producing cells. Thus, another aspect features host cells transformed with vectors comprising a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof that specifically binds RAGE, such as the antibodies or antigen- binding fragments described and exemplified herein.

Numerous techniques are known in the art for the introduction of foreign genes into cells and may be used to construct the recombinant cells for purposes of carrying out the described methods, in accordance with the various embodiments described and exemplified herein. The technique used should provide for the stable transfer of the heterologous gene sequence to the host cell, such that the heterologous gene sequence is heritable and expressible by the cell progeny, and so that the necessary development and physiological functions of the recipient cells are not disrupted. Techniques which may be used include but are not limited to chromosome transfer (e.g., cell fusion, chromosome mediated gene transfer, micro cell mediated gene transfer), physical methods (e.g., transfection, spheroplast fusion, microinjection, electroporation, liposome carrier), viral vector transfer (e.g., recombinant DNA viruses, recombinant RNA viruses) and the like. Calcium phosphate precipitation and polyethylene glycol (PEG)-induced fusion of bacterial protoplasts with mammalian cells may also be used to transform cells.

Cells suitable for use in the expression of the antibodies or antigen-binding fragments described herein are preferably eukaryotic cells, more preferably cells of plant, rodent, or human origin, for example but not limited to NSO, CHO, CHOK1 , perC.6, Tk-ts13, BHK, HEK293 cells, COS-7, T98G, CV-1/EBNA, L cells, C127, 3T3, HeLa, NS1 , Sp2/0 myeloma cells, and BHK cell lines, among others. In addition, expression of antibodies may be accomplished using hybridoma cells. Methods for producing hybridomas are well established in the art.

Cells transformed with expression vectors described herein may be selected or screened for recombinant expression of the antibodies or antigen-binding fragments described herein. Recombinant-positive cells are expanded and screened for subclones exhibiting a desired phenotype, such as high level expression, enhanced growth properties, or the ability to yield proteins with desired biochemical characteristics, for example, due to protein modification or altered post-translational modifications. These phenotypes may be due to inherent properties of a given subclone or to mutation. Mutations may be effected through the use of chemicals, UV-wavelength light, radiation, viruses, insertional mutagens, inhibition of DNA mismatch repair, or a combination of such methods.

In some embodiments, the entire heavy and light chain variable regions (such as SEQ ID NO: 20-21) can be inserted into a suitable vector such as gWIZ or similar vectors. These vectors can then be transiently transfected into, for example, Hek293 or CHO cells to produce functional antibodies. This approach can beneficial as hybridomas can slow production of antibodies over time. Antibodies made by this method are the functional equivalent of antibodies produced by hybridomas (see Figure 1 1 ). In other embodiments, only a portion of the antibody is cloned into a suitable expression vector. Such portions may include the variable heavy and light chain regions (such as SEQ ID NO: 22-35). A suitable vector may also contain an Fc region (such as the Fc region of 2A11 , or another Fc region), while the variable regions of choice are added by known cloning techniques to produce a full length antibody. In other instances, only the variable heavy and light chain regions are cloned into an expression vector to produce Fv or Fab fragments.

In addition to Eukaryotic cells, bacterial systems can be employed to produce antibodies of the present disclosure. Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desirable, to provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.

Bacterial vectors may be, for example, bacteriophage-, plasmid- or phagemid- based. These vectors can contain a selectable marker and bacterial origin of replication derived from commercially available plasmids typically containing elements of the well-known cloning vector pBR322 (ATCC 37017). Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is de-repressed/induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the protein being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of antibodies or to screen peptide libraries, for example, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Diagnostic and therapeutic uses of anti-RAGE antibodies

The antibodies of the present disclosure have in vitro and in vivo diagnostic and therapeutic utilities. For example, these molecules can be administered to cells in culture, e.g. in vitro or in vivo, or in a subject, e.g., in vivo, to treat, prevent or diagnose a variety of RAGE-mediated disorders.

The methods are particularly suitable for treating, preventing or diagnosing RAGE-mediated disorders such as, but not limited to diabetes, Alzheimer's disease, various cancers such as melanoma and pancreatic cancer, multiple sclerosis or another disease associated with chronic inflammation such as acne vulgaris, asthma, autoimmune diseases, autoinflammatory disease, Celiac disease, chronic prostatitis, glomerulonephritis, hypersensitivities, inflammatory bowel diseases, pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, sarcoidosis, vasculitis and interstitial cystitis.

The disclosure also provides methods for decreasing or suppressing RAGE- ligand induced signaling response by administering a composition comprising a therapeutically efficient amount of the antibodies of the as disclosed herein to a biological sample or subject.

Accordingly, in some embodiments, the antibodies of the present disclosure, or fragments thereof, can be used to detect RAGE (e.g., in a biological sample, such as serum or plasma), using a conventional immunoassays, such as an enzyme linked immunosorbent assays (ELISA), a radioimmunoassay (RIA) or tissue immunohistochemistry. The present disclosure also provides a method for detecting RAGE in a biological sample comprising contacting a biological sample with an antibody, or antibody fragments thereof, of the disclosure and detecting either the antibody (or antibody portion) bound to RAGE or unbound antibody (or antibody portion), to thereby detect RAGE in the biological sample.

In further embodiments, the described methods involve determining whether a subject is afflicted by a RAGE mediated disease by determining the amount of RAGE present in a biological sample derived from the subject; and comparing the observed amount of RAGE with the amount of RAGE in a control, or reference, sample, wherein a difference between the amount of RAGE in the sample derived from the subject and the amount of RAGE in the control, or reference, sample is an indication that the subject is afflicted with a RAGE-mediated disease. In another embodiment the amount of RAGE observed in a biological sample obtained from a subject may be compared to levels of RAGE known to be associated with certain forms of diseases. In some embodiments the amount of RAGE in the sample derived from the subject is assessed by contacting the sample with an antibody, or an antigen-binding fragment thereof, that specifically binds RAGE, such as the antibodies described herein. Preferred antibodies or antigen-binding regions for use as a diagnostic or therapeutic compound comprise isolated antibodies that specifically bind to RAGE comprising a heavy chain variable region comprising a heavy chain variable region CDR1 , CDR2, and CDR3 having at least 80%, at least 90% or at least 95% amino acid identity to a heavy chain variable region CDR1 , CDR2, and CDR3 of SEQ ID NO: 1 , SEQ ID NO: 2. SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8; and said light chain variable region comprises a light chain variable region CDR1 , CDR2, and CDR3 having at least 80%, at least 90% or at least 95% amino acid identity to a light chain variable region CDR1, CDR2, and CDR3 of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 OR SEQ

ID NO: 16.

In further embodiments, the antibodies disclosed herein contain a heavy chain variable region CDR1, CDR2, and CDR3 have at least 80%, at least 90% or at least 95% amino acid sequence identity to a heavy chain variable region CDR1 , CDR2, and CDR3 of SEQ ID NO: 1 and a light chain variable region CDR1, CDR2, and CDR3 have at least 80%, at least 90% or at least 95% amino acid sequence identity to a light chain variable region CDR1 , CDR2, and CDR3 SEQ ID NO: 9.

In still further embodiments, the antibodies disclosed herein contain a heavy chain variable region CDR1, CDR2, and CDR3 that are identical to the heavy chain variable region CDR1, CDR2, and CDR3 of SEQ ID NO: 1 and a light chain variable region CDR1, CDR2, and CDR3 that are identical to the light chain variable region CDR1 , CDR2, and CDR3 of SEQ ID NO: 9.

The term "subject" refers to human and non-human animals, including all vertebrates, e.g., mammals and non-mammals, such as non-human primates, mice, rabbits, sheep, dogs, cats, horses, cows, chickens, amphibians, and reptiles. In many embodiments of the described methods, the subject is a human. The term "sample" as used herein refers to a collection of similar fluids, cells, or tissues (e.g., surgically resected tumor tissue, biopsies, including fine needle aspiration), isolated from a subject, as well as fluids, cells, or tissues present within a subject. In some embodiments the sample is a biological fluid. Biological fluids are typically liquids at physiological temperatures and may include naturally occurring fluids present in, withdrawn from, expressed or otherwise extracted from a subject or biological source. Certain biological fluids derive from particular tissues, organs or localized regions and certain other biological fluids may be more globally or systemically situated in a subject or biological source. Examples of biological fluids include blood, serum and serosal fluids, plasma, lymph, urine, saliva, cystic fluid, tear drops, feces, sputum, mucosal secretions of the secretory tissues and organs, vaginal secretions, ascites, ascites fluids such as those associated with non-solid tumors, circulating tumor cells, cells that are not tissue associated, fluids of the pleural, pericardial, peritoneal, abdominal and other body cavities, fluids collected by bronchial lavage and the like. Biological fluids may also include liquid solutions contacted with a subject or biological source, for example, cell and organ culture medium including cell or organ conditioned medium, lavage fluids and the like. The term "sample," as used herein, encompasses materials removed from a subject or materials present in a subject including tissues, biopsies, fine needle aspiration samples, surgically resected tumor tissue, or histological preparations

The described antibodies and antigen-binding fragments may be used in a variety of assays to detect RAGE in a biological sample. Some suitable assays include, but should not be considered limited to, western blot analysis, radioimmunoassay, surface plasmon resonance, immunofluorimetry, immunoprecipitation, equilibrium dialysis, immunodiffusion, electro- chemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence- activated cell sorting (FACS) or ELISA assay.

ELISA assays are widely used method for the detection of specific antigens in a biological sample. It involves the immobilization of an antibody (primary antibody) to a solid support surface such as plastic microplates, and detecting a specific antigen via binding to the immobilized antibody, followed by the addition of a secondary antibody or antibodies, the latter usually being conjugated to enzymes such as alkaline phosphatase or horseradish peroxidase in order to facilitate detection. Addition of a chemical substrate of the enzyme results in the development of a colored reaction product, which indicates the presence of the antigen of interest in the sample.

Hence, according to a preferred embodiment, the immune affinity procedure may be an ELISA immunoassay selected from the group consisting of direct enzyme-linked immunosorbent assays, indirect enzyme-linked immunosorbent assays, direct sandwich enzyme-linked immunosorbent assays, indirect sandwich enzyme-linked immunosorbent assays, and competitive enzyme-linked immunosorbent assays.

In one embodiment, detection is effected through capture ELISA. Capture ELISA (also known as "sandwich" ELISA) is a sensitive assay to quantify picogram to microgram quantities of substances (such as hormones, cell signaling chemicals, infectious disease antigens and cytokines.). This type of ELISA is particularly sought after when the substance to be analyzed may be too dilute to bind to the microtiter plate (such as a protein in a cell culture supernatant) or does not bind well to plastics (such as a small organic molecule). Optimal dilutions for the capture antibodies, samples, controls, and detecting antibodies as well as incubation times are determined empirically and may require extensive titration. Ideally, one would use an enzyme-labeled detection antibody. However, if the detection antibody is unlabeled, the secondary antibody should not cross-react with either the coating antibody or the sample. Optimally, the appropriate negative and positive controls should also be included.

The capture or coating antibody to be used should be diluted in carbonate- bicarbonate buffer or PBS. Capture antibodies are typically plated at 0.2 to 10 μg/ml.

It is preferable to use affinity purified antibodies or at a minimum use an IgG fraction. Generally samples are diluted in PBS (the more sensitive the assay, the less sample is required).

The antibodies may be labeled directly or indirectly by a detectable moiety. As used herein, the term "detectable moiety" refers to any atom, molecule or a portion thereof, the presence, absence or level of which may be monitored directly or indirectly. One example includes radioactive isotopes. Other examples include (i) enzymes which can catalyze color or light emitting (luminescence) reactions and (ii) fluorophores. The detection of the detectable moiety can be direct provided that the detectable moiety is itself detectable (i.e. can be directly visualized or measured), such as, for example, in the case of fluorophores. Alternatively, the detection of the detectable moiety can be indirect. In the latter case, a second moiety that reacts with the detectable moiety, itself being directly detectable is preferably employed. The detectable moiety may be inherent to the antibody. For example, the constant region of an antibody can serve as an indirect detectable moiety to which a secondary antibody having a direct detectable moiety can specifically bind.

Thus, secondary antibodies are a particularly suitable means for the detection of the primary antibody in the method of the invention. This secondary antibody may be itself conjugated to a detectable moiety. One of the ways in which an antibody in accordance with the present invention can be detectably labeled is by linking the same to an enzyme. The enzyme, in turn, when exposed to an appropriate substrate, will react with the substrate in such a manner as to allow its detection, for example by producing a chemical or visual signal which can be detected by the user.

Non-limiting examples of detectable moieties include enzymes that can be conjugated to an antibody or antigen-binding antibody fragment of the disclosure such as β-lactamases, β-galactosidases, phosphatases, peroxidases, reductases, esterases, hydrolases, isomerases and proteases, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; non-limiting examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, green fluorescent protein, red fluorescent protein, dansyl chloride or phycoerythrin; a non-limiting example of a luminescent material includes luminol. Non-limiting examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include ¹²⁵l, ¹³¹l, ¹¹¹ln, ^99mTc, or ⁹⁰Y.

The solid support surface to which the first antibody is bound may be any water-insoluble, water-insuspendible, solid support. Examples of suitable solid supports include, but are not limited to, are large beads, e.g., of polystyrene, filter paper, slides, chips, test tubes, and microtiter plates. The first antibody may be bound to the solid support surface as described above. For example, the antibody may be bound to the surface through a biotin-streptavidin interaction, or through the interaction with an amine reactive maleimide anhydride.

The solid support surface mentioned above can include polymers, such as polystyrene, agarose, Sepharose (a crosslinked, beaded-form of agarose), cellulose, glass beads and magnetizable particles of cellulose or other polymers. The solid- support can be in the form of large or small beads or particles, tubes, plates, slides, chips or other forms. As a solid support surface, use is preferably made of a test tube, or a microtiter plate the inner walls of which are coated with a first antibody.

In another preferred embodiment, microfluidic devices, which may also be referred to as "lab-on-a-chip" systems, biomedical micro-electro-mechanical systems (bioMEMs), or multicomponent integrated systems, can be used for detecting an antigen of interest. Such systems miniaturize and compartmentalize processes that allow for detection of antigens of interest, and other processes.

Array-based assays and bead-based assays can be used with microfluidic devices. For example, an antibody can be coupled to beads and the binding reaction between the coated beads and antigen of interest can be performed in a microfluidic device. Multiplexing, or detecting more than one antigen of interest at once, can also be performed using a microfluidic device. Different compartments can comprise different antibody populations for different antigens of interest, where each population has a different target antigen.

In another embodiment, microarrays are used to detect antigens of interest. Microarrays are typically small, high throughput chips generally made of a solid support structure, typically glass slides, nitrocellulose, or microtiter plates. Generally, antibodies to the antigen of interest are bound to the solid support surface. Detection of the captured antigen can be accomplished as discussed above for ELISA detection, or through any method known to a person of ordinary skill in the art.

Additionally, antibodies may be further used in immunohistochemical (IHC) staining assays to differentiate between normal cells and those cells with a RAGE- medlated disorder. IHC may be carried out according to well-known techniques. For example, see, e.g., Antibodies: A Laboratory Manual, Chapter 10, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988). Briefly, paraffin-embedded tissue (e.g., tumor tissue) can be prepared for immunohistochemical staining by deparaffinizing tissue sections with xylene followed by ethanol; hydrating in water then PBS; unmasking antigen by heating slide in sodium citrate buffer; incubating sections in hydrogen peroxide; blocking in blocking solution; incubating slide in primary antibody and secondary antibody; and finally detecting using ABC avidin/biotin or other labels according to manufacturer's instructions.

For example, the detection of foci of such detectably labeled antibodies might be indicative of tumor development. In one embodiment, this analysis is done by removing samples of tissue or blood and incubating such samples in the presence of the detectably labeled antibodies as described herein. In a preferred embodiment, this technique is done in a non-invasive manner through the use of magnetic imaging, fluorography, etc. Such a diagnostic test may be employed in monitoring the success of treatment of diseases, where presence or absence of RAGE-positive (or over-expressing) cells is a relevant indicator. The disclosure also contemplates the use of an anti-RAGE antibody, as described herein for diagnostics in an ex vivo setting.

As described herein, Applicant has identified a battery of monoclonal antibodies that selectively bind to RAGE and may be used in a diagnostic applications. Binding sensograms performed on these anti-RAGE antibodies using Surface Plasmon Resonance, ELISA and flow cytometry show nanomolar affinities toward RAGE. For instance, the binding affinities of a subset of these monoclonal anti-RAGE antibodies designated IgG 2A11 , 2D3 and 2B6 were k_on = 1.6 x 10⁵ M^-1s- ¹; k_off ≈ 6.05 x10^-5 s^-1 and k_on = 1.74 x 105 M^-1s^-1; k_off ≈ 5.9 x 10^-5 s^-1 respectively (Figure 8).

Additionally, these antibodies were able to bind human RAGE displayed on a cell surface of mammalian cells (HEK-93 cells) as shown by immunofluorescence microscopy (Figure 9). Moreover, the same antibodies were capable of specifically binding to RAGE on the surface of spheroids from PANC-1 and BxPC-3 cancer cells (Figure 10). Spheroids are microscale, spherical cell aggregates that grow free of foreign material. Spheroids are commonly employed in 3D cell culture systems that more closely mimic a cells natural in vivo microenvironment as compared to conventional single layer cell culture systems. Hence, antibodies of the present disclosure can play an important role in not only treating RAGE-mediated diseases, but also in the identification of RAGE-mediated diseases cells, or cells showing an overexpression of RAGE.

A further aspect of the disclosure relates to a method for treating or preventing a disorder in a subject, the method comprising administering to a subject a therapeutic amount of any one, or a combination of antibodies as described herein.

An embodiment of the disclosure thus relates to a method for treatment of an RAGE-mediated disease or disorder. Such diseases or disorders include but are not limited to diabetes, cancer (e.g. melanoma, pancreatic cancer), multiple sclerosis and inflammation associated with such diseases.

A method of treatment may aim at curing a disease or disorder, but in relation to some diseases including immunological and inflammatory diseases such as a chronic disease or disorder, relief of one or more symptoms is also considered a treatment, which may be a significant improvement for the subject even if only a partial relief of symptoms is obtained or the effect is only temporary or partial.

In a further embodiments, the disclosure relates to an antibody, an isolated antibody or antibody composition as described herein, for treatment of a disease or disorder. In further embodiments, the antibody, isolated antibody or antibody composition is for treatment of one or more of the diseases and disorders described herein above in relation to a method of treatment.

Other embodiments of the disclosure relate to the use of an antibody, an isolated antibody or antibody composition as described herein, for the preparation of a medicament for treatment of a disease or disorder, wherein the disease or disorder may be as described herein in relation to a method of treatment. Such antibodies include their corresponding Fab fragments since Fabs can have distinct pharmacokinetic properties when compared to full length antibodies and have been shown to present enhanced tumor penetration in certain instances.

Preferred antibodies or antigen-binding regions of the disclosure for use as a therapeutic compound contain a heavy chain variable region comprising a heavy chain variable region CDR1, CDR2, and CDR3 having at least 80%, at least 90% or at least 95% amino acid identity to a heavy chain variable region CDR1 , CDR2, and CDR3 of SEQ ID NO: 1, SEQ ID NO: 2. SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8; and said light chain variable region comprises a light chain variable region CDR1 , CDR2, and CDR3 having at least 80%, at least 90% or at least 95% amino acid identity to a light chain variable region CDR1 , CDR2, and CDR3 of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 OR SEQ ID NO: 16.

In further embodiments, the antibodies disclosed herein contain a heavy chain variable region CDR1 , CDR2, and CDR3 have at least 80%, at least 90% or at least 95% amino acid sequence identity, or are identical to a heavy chain variable region CDR1 , CDR2, and CDR3 of SEQ ID NO: 1 and a light chain variable region CDR1 , CDR2, and CDR3 have at least 80%, at least 90% or at least 95% sequence identity, or are identical to a light chain variable region CDR1, CDR2, and CDR3 SEQ ID NO: 9.

As a cell-surface receptor, the extracellular domain of RAGE is an appropriate target for therapeutic antibodies. Antibodies offer an advantage over small molecules because they are more specific towards their targets. Anti-RAGE antibodies have been previously used by Applicant, in cell-based assays and in animal experiments, to demonstrate the involvement of RAGE in different models of human diseases, in particular pancreatic cancer and melanomas.

Pancreatic cancer is characterized by high resistance to both chemotherapy and radiotherapy due to the presence of an abundant reactive stroma, poor vascularization and high interstitial tumor fluid pressure. RAGE has been shown to promote both pancreatic cancer tumor growth and chemoresistance through increased autophagy triggered by the engagement of RAGE by its ligands (AGEs, S100P and HMGB1) that are abundantly secreted into the tumor stroma. Antibodies described herein against RAGE can block RAGE/ligand (S100P, and AGEs) interaction and reduce both pancreatic cancer cell growth and desmoplasia in pancreatic cancer tumors. Accordingly, RAGE blockage with anti-RAGE antibodies can reduce pancreatic cancer cell growth and can also reduce the secretion of extracellular matrix (ECM) proteins by activated fibroblasts.

Applicant has shown that antibodies described herein can suppress the proliferation of pancreatic cancer cells (PANC-1 , MIA PaCa2 cells, and BxPc3 cells) after stimulation with RAGE ligands (Figures 12-13). For example, treatment of the PANC-1 cells with antibodies reduced the proliferation of the cells, as compared to the treatment of the cells with saline only. However, co-treatment of the cells with S100P and antibody significantly reduced the increased of proliferation observed with the addition of S100P (Figure 11). The antibodies could also suppress RAGE activation by another RAGE ligand: advanced glycation end products (AGE). AGES are glycated proteins that activate RAGEs. Antibodies can also suppress AGE- induced reactive oxygen species (ROS) production and NF-kB activation in PANC-1 cells and MIA PaCa2 cells (Figures 14-15).

Applicants have also investigated RAGE-mediated signaling in melanoma cancers where at least the RAGE receptor and consequently the RAGE-ligand interaction is up-regulated. Melanoma can include lentigo maligna, lentigo maligna melanoma, superficial spreading melanoma, acral lentiginous melanoma, mucosal melanoma, nodular melanoma, polypoid melanoma, desmoplastic melanoma, amelanotic melanoma and soft-tissue melanoma. Applicants have shown that the antibodies described herein reduced the growth rate of melanoma tumors in mice on average about 1.7 fold after 27 days of treatment with said antibody (Figure 3). Moreover, labeled antibodies to RAGE showed a 4-fold higher fluorescence signal than in mice having RAGE mediated melanoma tumors as compared to mice strains having no tumors (Figure 4). This suggests that anti-RAGE antibodies are effective in treating RAGE-mediated tumors in melanoma.

Applicant has also shown that antibodies described herein can specifically bind the V domain of RAGE to inhibit ligand binding and subsequent downstream signaling. Many of the RAGE ligands have been shown to bind RAGE in the V domain, especially the S100 family of ligands. More specifically, Applicant has found the monoclonal antibody 2A11 will inhibit RAGE/ligand interaction in cells. The cellular proliferation of human pancreatic PANC-1 cancer cells, following treatment with RAGE ligand (S100P and AGEs), in the presence or absence of the IgG 2A11 anti-RAGE antibody was tested. These experiments indicate the anti-RAGE antibody is able to suppress the increase in proliferation triggered by S100P or AGEs (Figures 12-15). This result suggests that S100P, AGEs and lgG2A11 compete for a similar epitope of RAGE.

RAGE has been found up-regulated not only in cancer cells, but also in other cell-types constituting the tumor micro-environment such as endothelial cells, fibroblasts and myofibroblasts, T cell and myeloid derived suppressor cells (Figure 7). Among these cells, tumor associated myofibroblasts have been shown to be responsible for the desmoplastic reaction characteristic of pancreatic carcinomas. Desmoplasia is characterized by a dense network of ECM components such as collagen I, III or IV, hyaluronan, fibronectin, that hinders the delivery of chemotherapeutic agents to the cancer cells in the tumor core. The use of anti- RAGE antibodies can alleviate this problem.

In further embodiments, the present disclosure features bispecific or multispecific molecules comprising an anti-RAGE antibody as described herein. A bispecific or multispecific antibody of the disclosure can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The antibodies of the disclosure may in fact be derivatized or linked to more than one other functional molecule to generate multi-specific molecules that bind to more than two different binding sites and/or target molecules; such multi-specific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule of the invention, an antibody of the disclosure can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, such that a bispecific molecule results.

In some embodiments, the antibodies may also be linked to a pharmaceutical or cytotoxic agent. Suitable pharmaceutical or cytotoxic agent include, but are not limited to cytotoxic compounds for use as effector molecules in compounds according to the disclosure include cytostatic compounds. Particular compounds include for example, alkylating agents, such as nitrogen mustards (e.g. chlorambucil, melphalan, mechlorethamine, cyclophosphamide, or uracil mustard) and derivatives thereof, triethylenephosphoramide, triethylenethiophosphoramide, busulphan, or cisplatin, antimetabolites, such as methotrexate, fluorouracil and floxuridine, cytarabine, mercaptopurine, thioguanine, fluoroacetic acid or fluorocitric acid; antibiotics, such as bleomycins (e.g. bleomycin sulphate), doxorubicin, daunorubicin, mitomycins (e.g. mitomycin C), actinomycins (e.g. dactinomycin) or plicamycin, mitotic inhibitors, such as etoposide, vincristine or vinblastine; ureas, such as hydroxyurea; hydrazines, such as procarbazine; or imidazoles, such as dacarbazine; calicheamicin, esperamicin or taxol, combrestatins, dolastatins, epothilones, staurosporin, maytansinoids, spongistatins, rhizoxin, halichondrins, roridins, hemiasterlins, cytochalasin B, gramicidin D, ethidium bromide, emetine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. In a specific embodiment, the pharmaceutical agent is dacarbazine.

Other molecules include proteins, peptides and enzymes. Enzymes of interest include, but are not limited to, proteolytic enzymes, hydrolases, lyases, isomerases, transferases. Proteins, polypeptides and peptides of interest include, but are not limited to, immunoglobulins, toxins such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin, a protein such as insulin, tumour necrosis factor, a- interferon, β-interferon, nerve growth factor, platelet derived growth factor or tissue plasminogen activator, a thrombotic agent or an anti-angiogenic agent, e.g. angiostatin or endostatin, or, a biological response modifier such as a lymphokine, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), nerve growth factor (NGF) or other growth factor and immunoglobulins.

Still further molecules may include chelated radionuclides such as ¹¹ ln and ⁹⁰Y, Lu¹⁷⁷, Bismuth²¹³, Californium²⁵², Iridium¹⁹² and Tungsten¹⁸⁸/Rhenium¹⁸⁸; or drugs such as but not limited to, alkylphosphocholines, topoisomerase I inhibitors, taxoids and suramin.

In other examples, bi-functional crosslinkers can be used to conjugate the pharmaceutical or cytotoxic agent of choice to the antibody via the primary amino groups of the antibody. An example of a drug/antibody conjugate is a hyaluronidase conjugated antibody. Hyaluronidase can be targeted by the anti-RAGE antibody to the site of the tumor and degrade hyaluronic acid at the tumor site, which could help the penetration of pharmaceutical or cytotoxic agents to the core of the tumor. The targeted delivery of pharmaceutical agents or cytotoxic drugs to the tumor site could reduce the side effects of non-targeted drugs.

In some embodiments, the antibodies are administered to a patient in need of treatment, or to a biological sample from said patient with an antibody, or combination of antibodies of the present disclosure with a pharmaceutical or cytotoxic compound such as those disclosed above. In some embodiments, the cytotoxic agent is an alkylating agent. In a specific embodiment, the alkylating agent is dacarbazine. Applicants have found that co-treatment of melanoma tumors, or subjects having melanoma tumors with anti-RAGE antibodies and dacarbazine resulted in a 1.7 fold decrease in tumor size when compared to treatment with only dacarbazine. Surprisingly, the effective amount of dacarbazine was reduced by 50% (50 mg/kg vs. 25 mg/kg) in the presence of anti-RAGE antibodies to produce the same effect (Figure 5). The reduction of the dosage of dacarbazine has the advantage of minimizing any general side effects.

In addition to therapeutic use, the antibodies described herein may be used in other applications such as research, for example, of the role of RAGE in various cancers such as, but not limited to pancreatic cancer using various immunoassays or other assays described herein. In more specific embodiments, the research may concern the ability of RAGE activation in fibroblasts present in a tumor microenvironment to trigger the secretion of ECM components contributing to desmoplasia. Anti-RAGE antibodies could reduce the tumor-associated desmoplasia by blocking RAGE activation in these activated fibroblasts.

Pancreatic cancer tumors are rich in myofibroblasts (activated fibroblasts) that actively secrete multiple components of the extracellular matrix (ECM), resulting in a dense stroma. This ECM protein network hinders the delivery of drugs and contributes to mechanical chemoresistance. Fibroblasts have been shown to express RAGE and to contribute to sustained inflammation in the tumor microenvironment (Figure 7). In addition, fibroblasts communicate with other cell types present in the tumor, by secreting chemokines and growth factors resulting in activation of these cells and sustained tumor growth. Accordingly, RAGE-mediated signaling in such systems presents and important area of research.

In some embodiments, pancreatic cancer cells (PANC-1, BxPC-3 or MIAPaCa-2 cells) can be co-cultured with fibroblast cells to generate 3D spheroids. Primary fibroblast or immortalized fibroblasts can be used. The growth conditions for generating the co-culture of spheroids with fibroblasts can be optimized by adjusting, for example, the cell culture media, nature of the support for growing the spheroids, number of seeded cells, incubation time or ratio of the two cell types according to various protocols that are known in the art. For example, fibroblast activity in the 3D spheroids can be examined following RAGE blockage by anti-RAGE antibodies. Levels of several ECM proteins (Collagen

I, III, IV, hyluronan, fibronectin, laminin and osteonectin), chemokines (IL-8 and MCP-1) in the co-cultured spheroids treated or non-treated with the anti-RAGE antibodies described herein can be analyzed. The spheroids co-cultured with the fibroblasts can also be "fixed" with formaldehyde, or another agent and prepared for immunohistochemistry analysis using appropriate antibodies as is known in the art. Activated fibroblasts are converted into myofibroblasts in the co-culture, and their presence in the spheroids can be ascertained by assaying for alpha smooth muscle actin (a-SMA), a characteristic of fibroblast activation. Additionally, comparative analysis of the levels of RAGE and its ligands, AGE, HMGB1 and S100P in antibody treated and non-treated co-cultured spheroids by immunohistochemistry may be achieved.

Pharmaceutical compositions containing anti-RAGE antibodies

The antibodies for use in the present disclosure can be formulated according to known methods to prepare pharmaceutically useful compositions, wherein an antibody for use in the invention (including any functional fragment thereof) is combined in a mixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation are described, for example, in Remington's Pharmaceutical Sciences (18th ed., Alfonso R. Gennaro, Ed., Easton, Pa.: Mack Pub. Co., 1990). In order to form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of one or more of the antibodies for use in the present invention, together with a suitable amount of carrier vehicle. Preferred antibodies or antigen-binding regions are provided herein.

Preparations may be suitably formulated to give controlled-release of the active compound. Controlled-release preparations may be achieved through the use of polymers to complex or absorb anti-RAGE antibody. The controlled delivery may be exercised by selecting appropriate macromolecules (for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinyl-acetate, methylcellulose, carboxymethylcellulose, or protamine, sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release. Another possible method to control the duration of action by controlled release preparations is to incorporate anti-RAGE antibody into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively, instead of incorporating these agents into polymeric particles, it is possible to entrap these materials in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatine-microcapsules and poly(methylmethacylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.

The pharmaceutical composition described herein may be in the form of a liposome encapsulating at least an anti-RAGE antibody molecule, such as one described herein is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids that exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers while in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like.

The anti-RAGE containing compounds described herein can be used to prepare therapeutic pharmaceutical compositions, for example, by combining the compounds with a pharmaceutically acceptable diluent, excipient, or carrier. The compounds may be added to a carrier in the form of a salt or solvate. For example, in cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, α-ketoglutarate, and β-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, halide, sulfate, nitrate, bicarbonate, and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid to provide a physiologically acceptable ionic compound. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example, calcium) salts of carboxylic acids can also be prepared by analogous methods.

The anti-RAGE containing compounds of the formulas described herein can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient, in a variety of forms. The forms can be specifically adapted to a chosen route of administration, e.g., oral or parenteral administration, by intravenous, intramuscular, topical or subcutaneous routes.

The anti-RAGE containing compounds described herein may be systemically administered in combination with a pharmaceutically acceptable vehicle, such as an inert diluent or an assimilable edible carrier. For oral administration, compounds can be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the food of a patient's diet. Compounds may also be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations typically contain at least 0.1% of active compound. The percentage of the compositions and preparations can vary and may conveniently be from about 0.5% to about 60%, about 1% to about 25%, or about 2% to about 10%, of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions can be such that an effective dosage level can be obtained.

The tablets, troches, pills, capsules, and the like may also contain one or more of the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; and a lubricant such as magnesium stearate. A sweetening agent such as sucrose, fructose, lactose or aspartame; or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring, may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propyl parabens as preservatives, a dye and flavoring such as cherry or orange flavor. Any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.

In addition, the active compound may be incorporated into sustained-release preparations and devices.

The active compound may be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can be prepared in glycerol, liquid polyethylene glycols, triacetin, or mixtures thereof, or in a pharmaceutically acceptable oil. Under ordinary conditions of storage and use, preparations may contain a preservative to prevent the growth of microorganisms.

Pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions, dispersions, or sterile powders comprising the active ingredient adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and/or antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by agents delaying absorption, for example, aluminum monostearate and/or gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, optionally followed by filter sterilization.

In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation can include vacuum drying and freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the solution.

For topical administration, anti-RAGE containing compounds may be applied in pure form, e.g., when they are liquids. However, it will generally be desirable to administer the active agent to the skin as a composition or formulation, for example, in combination with a dermatologically acceptable carrier, which may be a solid, a liquid, a gel, or the like.

Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina, and the like. Useful liquid carriers include water, dimethyl sulfoxide (DMSO), alcohols, glycols, or water-alcohol/glycol blends, in which a compound can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using a pump-type or aerosol sprayer.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses, or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

Examples of dermatological compositions for delivering active agents to the skin are known to the art; for example, see U.S. Patent Nos. 4,992,478 (Geria), 4,820,508 (Wortzman), 4,608,392 (Jacquet et al.), and 4,559,157 (Smith et al.). Such dermatological compositions can be used in combinations with the compounds described herein where an ingredient of such compositions can optionally be replaced by a compound described herein, or a compound described herein can be added to the composition

Useful dosages of the anti-RAGE containing compounds described herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Patent No. 4,938,949 (Borch et al.). The amount of a compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular compound or salt selected but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will be ultimately at the discretion of an attendant physician or clinician.

The compound can be conveniently administered in a unit dosage form, for example, containing 5 to 1000 mg/m², conveniently 10 to 750 mg/m², most conveniently, 50 to 500 mg/m² of active ingredient per unit dosage form. The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub- doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.

The disclosure provides therapeutic methods of treating cancer in a mammal, which involve administering to a mammal having cancer an effective amount of anti- RAGE compound or composition described herein. A mammal includes a primate, human, rodent, canine, feline, bovine, ovine, equine, swine, caprine, bovine and the like. Cancer refers to any various type of malignant neoplasm, for example, colon cancer, breast cancer, melanoma and leukemia, and in general is characterized by an undesirable cellular proliferation, e.g., unregulated growth, lack of differentiation, local tissue invasion, and metastasis.

The ability of an anti-RAGE compound of the invention to treat cancer may be determined by using assays well known to the art. For example, the design of treatment protocols, toxicity evaluation, data analysis, quantification of tumor cell kill, and the biological significance of the use of transplantable tumor screens are known.

Provided herein are also kits for detecting RAGE in a biological sample. These kits include one or more of the RAGE-specific antibodies described herein, or an antigen-binding fragment thereof, and instructions for use of the kit. In some embodiments the antibody, or antigen-binding fragment, provided in the described kits may be one or more of the antibodies described herein. The provided antibody, or antigen-binding fragment, may be in solution; lyophilized; affixed to a substrate, carrier, or plate; or detectably labeled.

The described kits may also include additional components useful for performing the methods described herein. By way of example, the kits may comprise means for obtaining a sample from a subject, a control or reference sample, e.g., a sample from a subject having slowly progressing cancer and/or a subject not having cancer, one or more sample compartments, and/or instructional material which describes performance of a method of the invention and tissue specific controls or standards.

The means for determining the level of RAGE can further include, for example, buffers or other reagents for use in an assay for determining the level of RAGE. The instructions can be, for example, printed instructions for performing the assay and/or instructions for evaluating the level of expression of RAGE.

The described kits may also include means for isolating a sample from a subject. These means can comprise one or more items of equipment or reagents that can be used to obtain a fluid or tissue from a subject. The means for obtaining a sample from a subject may also comprise means for isolating blood components, such as serum, from a blood sample. Preferably, the kit is designed for use with a human subject.

The following examples are provided to supplement the prior disclosure and to provide a better understanding of the subject matter described herein. These examples should not be considered to limit the described subject matter. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be apparent to persons skilled in the art and are to be included within, and can be made without departing from, the true scope of the invention.

Example 1. Novel antibodies specifically bind to the Receptor for Advanced Glycation End products.

New monoclonal antibodies were generated against ligand specific domains of RAGE that inhibit RAGE activation by its ligands. These antibodies may be used to reduce ligand-RAGE interaction, and in turn, reduce activation of downstream pathways to provide a novel therapeutic strategy for the treatment of RAGE dependent pathologies. In certain embodiments, these antibodies may be used as research or diagnostic tools.

The generated panel of monoclonal antibodies was characterized in vitro on recombinant RAGE domains. Subsequently, the monoclonal antibodies were used in cell-based assays to determine their potential mechanisms of action and finally in an animal model to observe their potential diagnostic and therapeutic properties. A series of monoclonal antibodies (IgGs) that specifically bind to the various domains on RAGE was generated using hybridoma technology. The eight hybridomas producing IgG antibodies that gave the strongest signals in ELISA against sRAGE were selected for further characterization for specificity against the isolated recombinant RAGE domains (V, C1 , C2, C1C2 and VC1C2). Four clones showed specificity towards the V domain of RAGE and three clones showed specificity towards the C2 domain. The clone 6B12 did npt react against a single domain but was reactive against VC1C2, suggesting binding to a conformational epitope. (Table 1 ), as described previously in Development of New Antibody Based Theranostic Agents Targeting the Receptor for Advanced Glycation End-Product (RAGE), which is incorporated by reference in its entirety.

All antibodies bind to sRAGE and VC1C2 with nanomolar affinity (Table 2 and

Sequences of variable domains of IgG antibodies 2A11 (2148), 3D1 (2148), 5H5 (2160), 2B6 (2168), 2H9 (2171 ), 2A12 (2201), 6B8 (2204), and 6B12 (2206) were recently obtained and are provided in Appendix A, attached hereto as part of this application.

Using FACS, it was shown that the antibodies bind to RAGE on the cell surface of HEK-293 cells at a nanomolar affinity except for 6B12, which binds at a submicromolar affinity.

Immunochemistry was used to visualize binding of IgGs to RAGE on cell surfaces. All tested IgGs except for 6B12 show distinct binding to RAGE on cell surfaces. The immunochemistry image of 6B12 suggests non-specific binding of IgG 6B12 to RAGE on the surface of cells.

Competition experiments were used to examine the ability of the antibodies to compete for RAGE ligand binding sites on recombinant RAGE domains. RAGE ligands specific for their extracellular domains were selected to compete against the generated antibodies. S100B, which binds to the V domain of RAGE, was used to evaluate antibodies that bind specifically to the V domain (2A11, 3D11 and 5H5). At a constant antibody concentration of 50 nM, S100B was shown to compete with IgG 2A11, 3D1 , and 5H5 for binding to RAGE.

The inhibitory activity of one antibody, lgG2A11 was investigated in vivo by monitoring tumor size in a mouse model.

The effect of lgG2A11 was evaluated in vivo in severe combined immune- deficiency (SCID) mice with human melanoma tumor xenografts formed by injection with WM115-RAGE or WM115-MOCK cells. The inhibitory activity of IgG 2A11 on tumor growth was investigated by treating mice with several doses of the antibody. The mouse tumors treated with phosphate buffered saline (PBS) continued to grow aggressively, reaching a size of approximately 1 ,200 mm³ by day 30. In contrast, treatment with IgG 2A11 antibody resulted in a significant reduction in tumor size (780 mm³).

IgG 2A11 was evaluated for the ability to reduce Akt and ERK1/2 kinase activities in vivo. Western blot analysis was performed on tumor lysate of PBS and

IgG 2A11 treated groups. Two out of three PBS treated tumors showed higher levels of phosphorylated Akt as compared to IgG 2A11 treated groups. There was no significant difference in the activity of ERK1/2 kinase in PBS and IgG 2A11 treated mice.

In recent work by the Applicant, WM115-RAGE cells were used to study the effect of RAGE overexpression on the cellular proliferation and invasion of these cells. Cell migration was significantly enhanced in RAGE overexpressing cells, at the expense of cell proliferation, which was significantly reduced. Comparison of the organization of the actin filaments in WM115-RAGE and WM115-MOCK further suggested that the RAGE overexpressing WM115 cells exhibited mesenchymal-like morphologies compared to the control cells. Based on these results, it was proposed that RAGE overexpression in the WM115 cells resulted in a metastatic-like switch of the WM115 cells, where cells switched from a proliferative to a migrative phenotype.

To further understand the role of RAGE in melanoma tumor progression, the transfected WM115 cells were implanted in SCID mice and the growth of resulting tumor xenografts was compared, and the levels of RAGE and its S100 protein ligands in the tumors were analyzed. Materials and Methods

Reagents and antibodies

All reagents were of biochemical grade. The antibodies used for Western blot were purchased from the following providers. Anti-β-actin (#4970), Akt (#4691 ), phospho-Akt (Ser473, #4060S), SAPK/JNK (#9258), phospho-SAPK/JNK (#4668, Thr183/T yr185), p44/42 (#4695) and phospho-p44/42 (#9101 , Thr202/Tyr204) were all from Cell signaling (Danvers, MA). Anti-S100B (#Z0311) was from DakoCytomation (Denmark), anti- S100A10 (#ab52272) was from Abeam (Cambridge, MA). Rabbit sera directed against, S100A2, S100A4 and S100A6 were a generous gift from Prof. Heizmann (Children's Hospital, Zurich, Switzerland). The non-specific murine IgG antibodies were from Innovative Research, Novi, Ml. Horseradish peroxidase secondary antibodies specific for the species of the primary antibodies were from Jakson ImmunoResearch laboratories (West Grove, PA).

The IgG 2A11 producing hybridoma cell line was generated by the Hybridoma Core Facility of the University of Gainesville (FL) and IgG 2A11 was purified from hybridoma supernatants using a single step chromatography on protein-G sepharose column (GE Healthcare). The purity and integrity of the purified antibody were determined by Coomassie Blue stained SDS PAGE and was estimated to be more than 95%.

Cell-lines

The generation and characterization of the WM115-MOCK and WM115- RAGE cell lines has been described in a previous publication (Meghnani et al. RAGE overexpression confers a metastatic phenotype to the WM115 human primary melanoma cell line. Biochimica et Biophysica Acta: Molecular Basis of Disease. 2014, 1842: pp1017-1027). The stably transfected cells were maintained in Opti- MEM (Invitrogen) containing 4% FBS (Invitrogen), 1% penicillin/streptomycin and in the presence of 1 mg/ml G418 (WM115-RAGE cells) or 0.5 mg/ml G418 (WM115- MOCK cells).

Animal studies

All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at NDSU and in compliance with the NIH's Principles of Laboratory Animal Care. Five to six week old female SCID mice (20-25 g) (Charles River Laboratories, Wilmington, MA) were used in all mouse studies.

To compare the growth rate of tumors established from WM115-MOCK and WM115-RAGE cells, two groups of mice (n=8) were implanted subcutaneously with either WM115-MOCK or WM115-RAGE cells (1 x 10⁶ in 50 μL) under anesthesia. The growth of the tumors was measured every three days with a digital caliper and the tumor volumes were determined using the following equation: 0.52 x L x W², where W is the width and L is the length of the tumor. To determine the effect of anti-RAGE antibody treatment on tumor growth, 40 SCID mice were implanted either with WM115-MOCK cells (n=16) or WM115-RAGE cells (n=24) as described above. When the tumor sizes reached 80 mm³, the mice implanted with WM115-RAGE cells were divided into 3 groups: Mice in group A (n=8) were treated with PBS; mice in group B (n=8) were treated with non-specific murine antibodies; mice in group C (n=8) were treated with IgG 2A11 in PBS (0.5 mg/mouse, in 100 μL) every 5 days, for 30 days. The mice implanted with WM115- MOCK cells were also separated into two groups: mice in group D (n=8) were treated with murine control antibodies; mice in group E (n=8) were treated with IgG 2A11 in PBS (0.5 mg/mouse) every 5 days, for 30 days. The antibody was administered via intraperitoneal injection.

To determine the effect of dacarbazine on the growth of the melanoma tumor xenografts, 32 mice were implanted with WM115-RAGE cells as described above. When the tumor sizes reached 80 mm³, the mice were randomly assigned to 5 groups. Mice in groups F, G and H (n=8) were treated with dacarbazine at dosages of 50mg/kg, 25mg/kg and 12.5 mg/kg, respectively. Mice in group H (n=4) were treated with dacarbazine (25 mg/kg) and IgG 2A11 (0.5 mg in 100 μL of PBS per mouse every 5 days), and mice in group I (n=4) were treated with dacarbazine (12.5 mg/kg) and IgG 2A11 (0.5 mg in 100 μL of PBS per mouse every 5 days). The antibody and dacarbazine were administered through intraperitoneal injection.

To image the tumors in mice, Applicant used the anti-RAGE lgG2A11 antibody labeled with the infra-red fluorescence dye Cy5.5 (Hsiao et al. , 2006). A non-specific murine control antibody was used as negative control. Briefly, 16 mice were implanted with wither WM115-RAGE cells (Groups J, K and L, n=4/group) or WM115-MOCK cells (Group M; n=4). When the tumor volumes reached 80 mm³, the mice implanted with WM115-RAGE cells were separated into three groups. Mice in groups J, K and L were injected in the tail vein with the Cy5.5 labeled RAGE antibody (100μg in 125μL), the Cy5.5-labeled mouse control IgG (100μg in125μL) and PBS respectively. Mice in group M (n=4) were injected with Cy5.5 labeled RAGE antibody (100μg in 125μL). At 0, 4, 24, and 48 h after injection, the mice were anaesthetized and subjected to Near Infra-Red Fluorescence (NIRF) imaging using a Kodak FX Pro imager (Carestream Health Incorporation, Rochester, NY). The image acquisition time was set at 1 min. The images were analyzed using the Kodak Digital Science 1D software (Carestream Health Incorporation, Rochester, NY). The average fluorescence intensities at the region of interest (ROI) were corrected by subtracting the background fluorescence at the adjacent skin.

Biodistribution of IgG 2A11 in mice was studied using the mice used for the imaging studies (Groups J-M). At 48 h after antibody injection, the animals (n = 3 from each group of animals) were sacrificed and the tumors and organs (liver, spleen, kidneys, heart, brain, bladder and stomach) were excised, washed with PBS, and subjected to imaging. The average fluorescence intensities were determined by selecting the whole organs as the region of interests (ROI). The image acquisition time was set at 1 min.

In all the studies, the health of the animals was observed daily, the tumor size and the body weight of animals were recorded every three days. At the end of the treatment or if the tumor size exceeded 1200 mm³, the mice were euthanized and the tumor were excised. These tumors were either immediately prepared for immunohistochemistry or snap frozen in liquid nitrogen for RT-PCR and Western blot analysis.

Western Blots

Protein extracts were prepared from tumors using the PARIS kit (Ambion Life Sciences, USA) according to the manufacturer's instructions. The protein content was determined with the Pierce BCA protein assay kit (Pierce/Thermo Scientific, Rockford, IL). The proteins (40μg to 100μg) were separated on either 10% or 15% SDS gels that were electro-blotted onto nitrocellulose membranes. Depending of the manufacturer's recommendations, the blot was blocked (2-12h) with 4% BSA/TBS at room temperature (RT) or 4°C and incubated with the corresponding primary antibody diluted in 1% BSA/TBS/0.1% tween. The blots were then incubated with the HRP conjugated antibodies diluted in 1% BSA/TBS/0.1 % tween for 1h at RT, and developed using a chemoluminescent substrate (ECL Western Blotting Susbstrate, Pierce/Thermoscientific). The X-ray films were scanned (200 dpi) and the intensities of the bands of the films were analyzed using ImageJ (NIH).

Immunohistochemistry

The excised tumors were embedded in Tissue-Tek OCT solution (Sakura Finetek USA Inc., Torrance, CA), frozen and sliced into 7 pm-thick sections. The sections were either stained with hematoxylin-eosin or treated for immunohistochemistry. For this purpose, the tumor sections were treated with aqueous H₂0₂ (3% v/v) to neutralize the endogenous peroxidase activity, blocked by 10% (v/v) normal goat serum to avoid non-specific binding, and stained with IgG 2A11. Horseradish peroxidase-conjugated goat anti-mouse antibody was then used as secondary antibody and the peroxidase activity was detected using 3,3'- diaminobenzidine (DAB) substrate (Vector Laboratories, Burlingame, CA).

Real-Time PCR (RT-PCR)

Total RNAs were extracted using a commercial kit (Ambion, Invitrogen) according to the manufacturer's instructions. The quality of the RNAs was assessed by absorbance spectroscopy and by agarose gel electrophoresis. The RNAs were reverse transcribed into cDNA using the Reverse Transcription System from Promega (Madison, Wl).

RT-PCR was run with 10 ng cDNA per well on a Stratagene Mx3000p thermocycler using the Brilliant II SYBER Green QPCR Master mix (Stratagene) in 25ml volume sample. The genes of β-actin and glyceraldyde-3-phosphate dehydrogenase (GAPDH) were used as housekeeping genes. The primers used to detect the transcripts of RAGE, S100B, S100A2, S100A4, S100A6, S100A10 and actin are listed in a previous publication (Leclerc, Heizmann, 2009b). The other primers were as follows: GAPDH_Forward: GAAGGTGAAGGTCGGAGTC; GAPDH_Reverse: GAAGATGGTGATGGGATTTC. The following RT-PCR program was used: 10 min at 95 °C followed by 40 cycles of 30 sec at 95°C, 30 sec at 58 °C, 30 sec at 72 °C. A melting curve was recorded at the end of the cycles to evaluate the quality of the amplified products. The Ct value was obtained with the integrated software of the Stratagene Mx3000p thermocycler.

The Ct values for each gene were calculated and initially normalized for b - actin and GAPDH, respectively using the formula ACt = Ctgene-Ct_actin or GAPDH- Because we obtained similar ACt values with both housekeeping genes (β -actin and GAPDH), we decided to normalize all the Ct values for β -actin. To compare the gene transcription levels between RAGE and MOCK transfected cells, we calculated the A(ACt) using the formula A(ACt) = ΔCt_MOCK - ΔCt_RAGE- For each gene, the fold of change (F_gene) of gene expression was then calculated using F = 2^Δ(Δ2Ct). The experiments were run in triplicate using cDNAs obtained from three independent RNA preparations. The standard deviations were calculated from the folds of changes in gene expression.

ELISA

Protein extracts were prepared from tumors as described in the Western blot section. RAGE protein levels in the tumor extracts were determined using the Quantikine human RAGE Immunoassay kit (R&D Systems) according to the manufacturer's procedure.

The blood of mice bearing MOCK and RAGE tumors and treated for 30 days with non-specific murine antibodies (groups B and D) were collected and the plasma prepared. S100B levels in the tumor extracts and in the plasma of the mice were determined with the help of a calibration curve obtained from a sandwich ELISA. For the sandwich ELISA, Applicant used a polyclonal anti-S100B antibody (DakoCytomation, Denmark) to capture S100B. A calibration curve was generated using recombinant human S100B (0.01 nM to 150 nM), which was purified according to standard procedures (Smith et al. , 1996). To detect bound S100B, we used a second anti-S100B antibody (MAB1820, R&D Systems). The detection was performed using an alkaline phosphatase-conjugated (AP) secondary antibody and para-nitrophenyl phosphate as AP substrate. The tumor extracts and plasma samples from two independent experiments were run in duplicate.

Statistical analysis

Data are presented as means ± standard deviations (SD) or standard errors of the mean (SEM), as indicated in the figure legends. For the densitometric analysis of the Western blots and the tumor growth curve analysis, the statistical analysis was performed using one-way ANOVA and the Bonferroni Post-Hoc test. For the in vivo imaging and antibody biodistribution studies, the statistical analysis was performed by student's t-test. The p-value of less than 0.05 was considered as statistically significant. Results and Discussion

RAGE overexpressing WM115 cells generate tumors faster than MOCK control cells when implanted in mice. This study was designed to investigate the role of RAGE in the progression and development of melanoma tumors by evaluating how melanoma cells stably transfected with RAGE proliferated and formed tumors in vivo using a xenograft melanoma mouse model.

The effect of RAGE overexpression on the in vitro proliferation and migration of the WM115 cells has been recently studied by Applicant. It was shown that although the WM115 cells overexpressing RAGE presented a significant increase in their abilities to migrate in response to a gradient of serum, they exhibited a moderate decrease in their proliferative capabilities, when grown as monolayers.

Interestingly, it was also shown that the WM115-RAGE cells could form larger colonies when grown in anchorage-free conditions. These results as well as a careful morphologic observation of the transfected cells led suggested that RAGE could trigger a metastatic-like switch in the WM115 melanoma cell line (Meghnani, Vetter, 2014). Based on these in vitro results, it was decided to next investigate how RAGE overexpression would influence the formation of tumors, in a xenograft melanoma model.

The WM115-MOCK and WM115-RAGE cells were subcutaneously implanted in the flanks of SCID mice and the growth of the xenograft tumors was followed every three days (Figure 1 ). For clarity, throughout the rest of the manuscript, the tumors generated from the WM115-MOCK and WM115-RAGE tumors are named MOCK or RAGE tumors respectively. The RAGE tumors grew faster than the MOCK controls. After 28 days, the mean volume of the RAGE tumors was 2-fold larger than that of the MOCK tumors (V = 60 ± 13 mm³ vs. 27 ± 14 mm³), demonstrating a positive correlation between the level of RAGE and the growth of these tumors. RAGE tumors had a higher level of RAGE than the MOCK tumors, as shown by immunohistochemistry (Figure 1 ), and by ELISA (Table 4). Interestingly, we observed that RAGE was 194-fold more abundant in RAGE tumors than in MOCK tumors, whereas it was only 94-fold more abundant in the RAGE transfected cells than in the MOCK transfected cells, suggesting the up-regulation of RAGE in the tumors (Table 4).

Table 4: Protein levels of RAGE in MOCK and RAGE overexpressing cells and tumors, expressed in pg RAGE per mg of total protein present in the cell or tumor extract. The ratio of RAGE in the WM115-RAGE cells over that in the WM115-MOCK cells is indicated

S100 protein RAGE ligands are up-regulated in RAGE overexpressing tumors.

The observation that RAGE was up-regulated in the tumors led to a search for RAGE ligands that could be present in the tumor microenvironment and influence the growth of the tumors through RAGE. In recent years, the tumor microenvironment has emerged as a key contributor to cancer growth and development. It is a complex milieu that includes many cell types (immune cells, fibroblasts or endothelial cells) that communicate with the cancer cells and influence their growth.

The primary focus of this study was on the S100 protein ligands of RAGE because many of this protein's family members have been shown to modulate melanoma tumor growth and to bind to RAGE.

Among the S100 proteins, S100B has been well studied in melanoma. S100B is secreted by melanoma cells and is used as a prognostic marker in stage IV melanoma patients. In these patients, high serum levels of S100B correlate with poor prognosis. S100B levels were discovered to be higher in RAGE tumors than in MOCK tumors, both at the transcript (15-fold increase, Table 5) and at the protein levels (2.7-fold, Figure 2). A significantly higher level of S100B in the plasma of mice bearing RAGE tumors (25.1 ± 3.6 nM) than MOCK tumors (11.5 ± 1.9 nM) was also detected. The increased serum level of S100B in the mice bearing the RAGE tumors suggests that the RAGE tumors exhibited higher tumorigenic potentials than the MOCK tumors. This study also suggests for the first time, that RAGE directly influences the level of serum S100B in melanoma and strongly supports a role of RAGE in melanoma progression.

Among the other S100 proteins investigated, transcript levels of S100A2, S100A4, S100A6 and S100A10 were significantly increased in the RAGE tumors compared to the MOCK tumors by 2.1 fold, 4.6 fold, 2.8 fold and 6.1 fold, respectively (Table 5). This observation was confirmed at the protein levels of S100A2, S100A4, S100A6 and S100A10, which were increased 1.77 fold, 1.4 fold, 1.3 fold and 3.2 fold respectively (Table 5).

The observation that S100A2 levels are higher in RAGE tumors compared to MOCK tumors appears to be contradictory to previous results that S100A2 was suggested to play a role of tumor suppressor in melanoma. However, contradictory reports on the role of S100A2 as tumor suppressor or inducer have also been reported within the same type of tumor such as head and neck squamous cell carcinoma and non-small cell lung cancers, suggesting complex functions of S100A2 in each type of tumor.

Among the other S100 proteins, S100A4, S100A6 and S100A10 have all been positively correlated with cancer progression, although their exact roles in melanoma are not well understood. For example, S100A4 was found to be secreted in the tumor milieu and to be essential for metastatic colonization. S100A6 and S100A10 have both been found to play important roles in maintaining a tumorigenic microenvironment by promoting the growth of tumor-associated fibroblasts or by promoting the degradation of the extracellular matrix and by attracting tumor- promoting macrophages. The observation that S100B, S100A2, S100A4, S100A6 and S100A10 are up- regulated in RAGE tumors strongly suggests that RAGE controls the up-regulation of these S100 proteins in tumors. A higher level of RAGE in tumors than in cells was also observed (Table 4), suggesting an up-regulation of the receptor itself in the tumor. Similar positive feedback loops have been previously reported for RAGE and other ligands.

Akt and Erk signaling pathways are up-regulated in tumors established from WM115-RAGE. This group previously showed that overexpression of RAGE in WM115 cells correlated with a decrease in the levels of the activated form of ERK1/2 with no change in the levels of activated Akt. It was suggested that the decrease in ERK1/2 activity correlated with the decrease in cell proliferation observed in the WM115-RAGE cells compared to the control cells, The levels of the activated forms of Akt and Erk1/2 in the extracts of RAGE and MOCK tumors were analyzed and it was discovered that the levels of p-Akt and p-Erk1/2 were both significantly higher in RAGE than in MOCK tumors, with a 1.9-fold and 2.1 -fold increase in p-Akt and p- Erk, respectively (Figure 2). Analysis of the levels of non-phosphorylated and phosphorylated JNK did not reveal changes in activation of this kinase (Figure 2). Akt and Erk belong to two signaling pathways that are important for melanoma cell proliferation and melanoma tumor growth and that are activated in melanoma tumors, suggesting that RAGE influences melanoma cell proliferation and tumor growth.

Tumor growth can be reduced with a monoclonal antibody targeting RAGE. Because RAGE tumors were found to grow faster than the MOCK tumors and many S100 protein RAGE ligands were up-regulated in the RAGE tumors, whether the growth rate of the RAGE tumors could be reduced by using anti-RAGE antibodies was considered. The monoclonal anti-RAGE antibody IgG 2A11 , which competes with S100B for binding to RAGE V domain, was evaluated. RAGE tumors were found to grew slower when the mice were treated with IgG 2A11 (Figure 3A). After 27 days of treatment, the volume of the IgG 2A11 -treated tumors (753 ± 130 mm³) was in average 1.7-fold smaller than the saline treated ones (1292 ± 88 mm³). From the growth curves, treatment with IgG 2A11 resulted in an estimated growth delay of about 8 days (Figure 3A). The MOCK tumors did not respond to the anti-RAGE antibody treatment, as seen in Figure 3B. Whether the IgG treatment was associated with some toxicity that would have been manifested itself by severe body weight losses was also investigate. Treated mice did not lose weight during the duration of the treatment (Figure 3C).

Imaging and biodistnbution studies were performed to confirm that IgG 2A11 targets the tumors when injected into the mice. lgG2A11 was conjugated with the Cy5.5 fluorescent label and the labeled antibody injected intravenously into the animals carrying either the RAGE or MOCK tumors. Strong fluorescence signals in tumor areas of the mice carrying either type of tumor were observed (Figure 4A). The fluorescence signal was 4-fold higher in the RAGE tumors than in MOCK tumors, indicative of a larger accumulation of the antibody in the RAGE tumors (Figure 4C). The targeting of IgG 2A11 at the tumor site was also confirmed when tumor sections were imaged (Figure 4B). Time course experiments demonstrated that IgG 2A11 was delivered to the tumor site after 4h (Figure 4C), and the accumulation continued for 24h and 48h. To ascertain the specificity of tumor targeting with IgG 2A11, accumulation of IgG 2A11at the tumor site was compared to that of a non-specific murine antibody; a larger (3.3-fold) accumulation of IgG 2A11 at the tumor site than the non-relevant antibody (Figure 4C). The biodistnbution of

IgG 2A11 was also studied and showed that the antibody accumulated mainly at the tumor site but was also found in the liver, kidneys and at lowest levels in the hearts and brain of the mice (Figure 4D).

These data suggest that IgG 2A11 targets RAGE at the site of the tumors. However, the 4-fold increase in fluorescence signal between the MOCK and RAGE tumors does not reflect the 194-fold difference in RAGE level observed between the two types of tumors and suggest that within the RAGE tumors, only a fraction of RAGE expressed by the cells could be targeted by the anti-RAGE antibody. This could be due to an abundant cytoplasmic expression of RAGE in the WM115 melanoma cells, as previously reported in other melanoma cell lines. The accumulation of the antibody in different organs of the mice reflects the general biodistnbution mechanisms of antibodies in the body. For example, antibodies can easily diffuse through the sinusoidal clefts of the liver and spleen, as a result from the physical properties of the blood capillaries in these organs lgG2A11 interacts specifically with the V domain of RAGE and can compete with S100B for binding to RAGE. S100B, S100A2 and S100A6 can all interact with RAGE V domain in vitro. S100A4 has also been shown to interact with RAGE although the exact domain of interaction is not known. A direct interaction between S100A10 and RAGE has not been demonstrated yet. It may be that within the RAGE tumor, lgG2A11 blocks the interaction between RAGE and the S100 proteins, resulting in reduced RAGE signaling. This could explain the effect of lgG2A11 in reducing melanoma tumor growth. The observation that the treatment with the anti- RAGE antibody had no effect on the growth of the MOCK tumors could be the result of both low levels of RAGE on the surface of the WM115-MOCK cells as well as of reduced levels of S100 proteins in the MOCK tumors. This result could also be explained by differences in conformations of RAGE on the surface of the WM115- MOCK and WM115-RAGE cells. Indeed, recent studies have suggested large variations in oligomeric forms of RAGE both in vitro and in melanoma cells. These distinct oligomers could exhibit slightly distinct binding domains for their ligands.

Anti-RAGE antibody combined with dacarbazine is more effective in reducing tumor growth than dacarbazine alone. Despite the recent approval of two new anti-melanoma drugs targeting the mutant BRAF (Vemurafenib) or the CTLA-4 antigen (Ipilimumab), dacarbazine remains widely used as cytotoxic agent to treat patients suffering from metastatic melanoma. Dacarbazine is an alkylating agent that exerts its tumor activities either by methylating DNA, resulting in cell death by both apoptosis and necrosis. Patients treated with dacarbazine experience very low five years survival rates and the duration of the response is often brief. In order to improve the efficacy of dacarbazine, combination therapies have been designed and have shown promising results in pre-clinical studies and clinical trials. Whether an anti-RAGE antibody could increase the anti-tumor efficacy of dacarbazine (DTIC) in a combination treatment was investigated by treating mice bearing melanoma xenografts with dacarbazine at three different dosages (50 mg/kg, 25 mg/kg or 12.5 mg/kg every 5 days) with or without a fixed dosage of anti-RAGE antibody (0.5 mg/mouse every 5 days) (Figure 5A, B). After 36 days of treatment, the mean tumor size (58.4 ± 3.3 mm³) of the animals treated with DTIC (12.5 mg/kg) in combination with IgG 2A1 1 was 1.7-fold smaller than the volume of the animals treated with DITC alone (100.29 ± 17 mm³) (Figure 5B). These data suggest the anti-RAGE antibody enhanced the effect of the alkylating drug DTIC. The combination of DTIC (25mg/kg) with IgG 2A11 was more powerful in reducing tumor growth than DTIC alone at the standard concentration (50mg/kg) (Figure 5 C).

Cancer stem cell versus phenotypic switching model

Two main models have been proposed to explain the mechanisms of melanoma tumor progression and formation of metastases: the cancer stem cell (CSC) model; and the phenotypic switching model. The relevance of each model has been evaluated and recent studies argue against the CSC model, due to large phenotypic variations in melanoma.

The phenotype switching model proposes that cells within melanoma tumors can switch back-and-forth between states of proliferation and invasion, depending on signals received from the tumor micro-environment. In support with this model, several studies have suggested that many tumorigenic markers could reversibly be turned on and off by signaling elements present in the micro-environment of melanoma tumors.

The data appear to be in agreement with the phenotype switching model.

Indeed, RAGE can turn on a metastatic-like switch in WM115 cells grown in in vitro conditions. On the other hand, the in vivo data suggest that the WM115-RAGE cells turned back to a proliferative phenotype in the complex tumor microenvironment.

The data demonstrate that RAGE expression controls the levels of selected S100 protein in melanoma tumors and that an anti-RAGE antibody could reduce tumor growth rate in a xenograft mouse model of melanoma tumor. The data also show that IgG 2A11 significantly enhanced the anti-tumor effect of the alkylating drug dacarbazine, possibly opening a way to new therapeutic approaches to treat metastatic melanoma.

While the disclosure is susceptible to various modifications and alternative forms, specific exemplary embodiments of the present invention have been shown by way of example in the drawings and have been described in detail. It should be understood, however, that there is no intent to limit the disclosure to the particular embodiments disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the scope of the disclosure as defined by the appended claims.

Claims

CLAIMS What is claimed is:

1. An isolated antibody that specifically binds to RAGE comprising a heavy chain variable region CDR1 , CDR2, and CDR3 having at least 90% amino acid identity to a heavy chain variable region CDR1 , CDR2, and CDR3 of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8; and a light chain variable region CDR1, CDR2, and CDR3 having at least 90% amino acid identity to a light chain variable region CDR1 , CDR2, and CDR3 of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 OR SEQ ID NO: 16.

2. The antibody of claim 1, wherein said heavy chain variable region CDR1 , CDR2, and CDR3 have at least a 90% amino acid sequence identity to said heavy chain variable region CDR1, CDR2, and CDR3 of SEQ ID NO: 1 and said light chain variable region CDR1, CDR2, and CDR3 have at least a 90% sequence identity to said light chain variable region CDR1, CDR2, and CDR3 of SEQ ID NO: 9.

3. The antibody of claim 2, wherein said heavy chain variable region CDR1 , CDR2, and CDR3 are identical to said heavy chain variable region CDR1, CDR2, and CDR3 of SEQ ID NO: 1 and said light chain variable region CDR1, CDR2, and CDR3 are identical to said light chain variable region CDR1 , CDR2, and CDR3 of SEQ ID NO: 9.

4. The antibody of claim 1 , wherein said antibody is a monoclonal antibody.

5. The antibody of claim 1 , wherein said antibody is an IgG antibody.

6. The antibody of claim 1 , wherein said antibody reduces binding of a RAGE ligand to RAGE.

7. The antibody of claim 1, wherein said antibody specifically binds to V, C1 or C2 domain of RAGE, or in combination.

8. A method of treating a subject with a condition mediated by binding of a RAGE ligand to RAGE comprising administering an effective amount of said antibody of claim 1 to said subject.

9. The method of claim 8, wherein said condition mediated by binding of said RAGE ligand to RAGE is diabetes, a cancer, multiple sclerosis or another disease associated with chronic inflammation.

10. The method of claim 9, wherein said cancer is a melanoma or pancreatic cancer.

11. The method of claim 8, wherein said antibody is formulated into a pharmaceutically acceptable composition.

12. The method of claim 10, further comprising administering to said subject a pharmaceutical agent effective in treating said melanoma or pancreatic cancer.

13. The method of claim 12, wherein said pharmaceutical agent is dacarbazine.

14. A method for detecting RAGE in a biological sample, comprising contacting said biological sample with said antibody of claim 1.

15. The method of claim 14, wherein said biological sample is derived from blood, plasma, serum, saliva, urine, ascites, circulating cells, circulating tumor cells, cells that are not tissue associated, tissues, biopsies, fine needle aspiration samples, surgically resected tumor tissue, or histological preparations.

16. The method of claim 14, wherein said antibody contains a detectably moiety, said detectable moiety being selected from the group consisting of a radio label, a fluorescent label, an epitope tag, biotin, a chromophore label, a chemiluminescence label, or an enzyme.

17. The method of claim 14, wherein said method for detecting RAGE is a diagnostic assay comprising an immunoassay.

18. The method of claim 17 wherein said immunoassay is a western blot assay, ELISA assay or immunohistochemical assay.

19. The antibody of claim 1 wherein the heavy chain variable region CDR1, CDR2, and CDR3 and the light chain variable region CDR1, CDR2, and CDR3 comprise any CDR of SEQ ID NO: 1 -16.