Monoclonal antibodies against hmgb1

This application is a continuation of U.S. application Ser. No. 10/938,992, filed Sep. 10, 2004, which claims the benefit of U.S. Provisional Application No. 60/502,568, filed Sep. 11, 2003.

The entire teachings of the above applications are incorporated herein by reference.

Inflammation is often induced by proinflammatory cytokines, such as tumor necrosis factor (TNF), interleukin (IL)-1α, IL-1β, IL-6, macrophage migration inhibitory factor (MIF), and other compounds. These proinflammatory cytokines are produced by several different cell types, most importantly immune cells (for example, monocytes, macrophages and neutrophils), but also non-immune cells such as fibroblasts, osteoblasts, smooth muscle cells, epithelial cells, and neurons. These proinflammatory cytokines contribute to various disorders during the early stages of an inflammatory cytokine cascade.

The early proinflammatory cytokines (e.g., TNF, IL-1, etc.) mediate inflammation, and induce the late release of high mobility group box 1 (HMGB 1; also known as HMG-1 and HMG1), a protein that accumulates in serum and mediates delayed lethality and further induction of early proinflammatory cytokines. HMGB1 was first identified as the founding member of a family of DNA-binding proteins, termed high mobility group box (HMGB) proteins, which are critical for DNA structure and stability. It was identified as a ubiquitously expressed nuclear protein that binds double-stranded DNA without sequence specificity. The HMGB1 molecule has three domains: two DNA binding motifs termed HMGB A and HMGB B boxes, and an acidic carboxyl terminus. The two HMGB boxes are highly conserved 80 amino acid, L-shaped domains. HMG boxes are also expressed in other transcription factors including the RNA polymerase I transcription factor human upstream-binding factor and lymphoid-specific factor.

Recent evidence has implicated HMG1 as a cytokine mediator of delayed lethality in endotoxemia (Andersson, U., et al., J. Exp. Med. 192(4):565-570 (2000)). That work demonstrated that bacterial endotoxin (lipopolysaccharide (LPS)) activates monocytes/macrophages to release HMG1 as a late response to activation, resulting in elevated serum HMG1 levels that are toxic. Antibodies against HMG1 prevent lethality of endotoxin even when antibody administration is delayed until after the early cytokine response. Like other proinflammatory cytokines, HMG1 is a potent activator of monocytes. Intratracheal application of HMG1 causes acute lung injury, and anti-HMG1 antibodies protect against endotoxin-induced lung edema (Abraham, E., et al., J. Immunol. 165:2950-2954 (2000)). Serum HMG1 levels are elevated in critically ill patients with sepsis or hemorrhagic shock, and levels are significantly higher in non-survivors as compared to survivors.

HMG1 has also been implicated as a ligand for RAGE, a multi-ligand receptor of the immunoglobulin superfamily. RAGE is expressed on endothelial cells, smooth muscle cells, monocytes, and nerves, and ligand interaction transduces signals through MAP kinase, P21 ras, and NF-kB. The delayed kinetics of HMG1 appearance during endotoxemia makes it a potentially good therapeutic target, but little is known about the molecular basis of HMG1 signaling and toxicity.

Therefore, given the importance of HMGB proteins in mediating inflammation, it would be useful to identify antibodies that bind HMGB for diagnostic and therapeutic purposes.

In various embodiments, the present invention is drawn to antibodies or antigen-binding fragments thereof that bind to a vertebrate high mobility group box (HMGB) polypeptide, methods of detecting and/or identifying an agent that binds to an HMGB polypeptide, methods of treating a condition in a subject characterized by activation of an inflammatory cytokine cascade and methods of detecting an HMGB polypeptide in a sample.

In one embodiment, the invention is an antibody or antigen-binding fragment thereof that specifically binds to a vertebrate HMGB A box but does not specifically bind to non-A box epitopes of HMGB, wherein the antibody or antigen-binding fragment inhibits release of a proinflammatory cytokine from a vertebrate cell treated with an HMGB protein.

In certain embodiments, the invention is an antibody produced by murine hybridoma 6E6 HMGB1 mAb, murine hybridoma 6H9 HMGB 1 mAb, murine hybridoma 2G7 HMGB1 mAb, murine hybridoma 2E11 HMGB1 mAb, or murine hybridoma 10D4 HMGB1 mAb. In other embodiments, the invention is an antibody or antigen-binding fragment thereof, wherein the binding of the antibody or antigen-binding fragment to a vertebrate HMGB polypeptide can be inhibited by 6E6 HMGB1 mAb, 6H9 HMGB1 mAb, 2G7 HMGB1 mAb, 2E11 HMGB1 mAb and/or 10D4 HMGB1 mAb. In still other embodiments, the invention is an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment has the epitopic specificity of 6E6 HMGB1 mAb, 6H9 HMGB1 mAb, 2G7 HMGB1 mAb, 2E11 HMGB1 mAb and/or 10D4 HMGB1 mAb.

In certain embodiments, the invention is an antibody or antigen-binding fragment that binds to a peptide consisting of amino acid residues 46 to 63 of SEQ ID NO:1, amino acid residues 61 to 78 of SEQ ID NO:1 and/or amino acid residues 151 to 168 of SEQ ID NO: 1. In one embodiment, the invention is an antibody or antigen-binding fragment, wherein the binding of the antibody or antigen-binding fragment to a peptide consisting of amino acid residues 46 to 63 of SEQ ID NO: 1, can be inhibited by 2G7 HMGB1 mAb. In another embodiment, the invention is an antibody or antigen-binding fragment, wherein the binding of the antibody or antigen-binding fragment to a peptide consisting of amino acid residues 61 to 78 of SEQ ID NO: 1, can be inhibited by 6E6 HMGB1 mAb and/or 6H9 HMGB1 mAb. In yet another embodiment, the invention is an antibody or antigen-binding fragment, wherein the binding of the antibody or antigen-binding fragment to a peptide consisting of amino acid residues 151 to 168 of SEQ ID NO:1, can be inhibited by 2E11 HMGB1 mAb.

In certain embodiments, the invention is an antibody or antigen-binding fragment that comprises the light chain CDRs (CDR1, CDR2 and CDR3) and the heavy chain CDRs (CDR1, CDR2 and CDR3) of an antibody selected from the group consisting of 6E6 HMGB1 mAb, 6H9 HMGB1 mAb, 2G7 HMGB1 mAb, 10D4 HMGB1 mAb and 2E11 HMGB1 mAb.

In other embodiments, the invention is murine hybridoma 6E6 HMGB1 mAb, murine hybridoma 6H9 HMGB1 mAb, murine hybridoma 2G7 HMGB1 mAb, murine hybridoma 2E11 HMGB1 mAb or murine hybridoma 10D4 HMGB1 mAb.

In another embodiment, the invention is an isolated cell that produces an antibody or antigen-binding fragment that specifically binds to a vertebrate HMGB A box but does not specifically bind to non-A box epitopes of HMGB. In other embodiments, the invention is an isolated cell that produces 6E6 HMGB1 mAb, 6H9 HMGB1 mAb, 2G7 HMGB1 mAb, 2E11 HMGB1 mAb or 10D4 HMGB1 mAb. In still other embodiments, the invention is an isolated cell that produces an antibody or antigen-binding fragment thereof, wherein the binding of the antibody or antigen-binding fragment to a vertebrate HMGB polypeptide can be inhibited by 6E6 HMGB1 mAb, 6H9 HMGB1 mAb, 2G7 HMGB1 mAb, 2E11 HMGB1 mAb and/or 10D4 HMGB1 mAb. In still other embodiments, the invention is an isolated cell that produces an antibody or antigen-binding fragment that has the epitopic specificity of 6E6 HMGB1 mAb, 6H9 HMGB1 mAb, 2G7 HMGB1 mAb, 2E11 HMGB1 mAb and/or 10D4 HMGB1 mAb.

In other embodiments, the invention is a composition that comprises an antibody or antigen-binding fragment of the invention and a pharmaceutically-acceptable excipient.

In another embodiment, the invention is a method of detecting and/or identifying an agent that binds to a vertebrate HMGB polypeptide comprising combining an antibody or antigen-binding fragment of the invention, a test agent and a composition comprising a vertebrate HMGB polypeptide. In the method, the formation of a complex between the antibody or antigen-binding fragment and the HMGB polypeptide is detected or measured and a decrease in complex formation, as compared to a suitable control, indicates that the test agent binds to the HMGB polypeptide.

In another embodiment, the invention is a method of treating a condition in a subject characterized by activation of an inflammatory cytokine cascade comprising administering to the subject an antibody or antigen-binding fragment of the invention.

In certain embodiments, the condition is sepsis, arthritis or lupus.