SBIR-STTR Award

RA is one of the most common chronic autoimmune disorders that can lead to complete joint destruction and severe disability if untreated. There is no cure for RA and up to 50% of RA patients do not respond to anti-TNF therapies as circulating Th-17/IL-17 levels are highly elevated subsequent to TNF blockade. For this subset of RA patients, disruption of a novel pathway that impairs the synergy between TNF (M1 macrophages) and IL-17 (Th-17 cells) cascades may resolve the critical barrier in RA treatment. Hence objective of this project is to develop a therapeutic human TLR5 antibody (Ab) for RA patients whose disease is driven by the cross-talk between the effector macrophages and T cells. We documented that ligation of TLR5 to its natural ligand expressed in the joints, transforms RA peripheral blood (PB) cells into proinflammatory M1 macrophages which produce high levels of TNF, IL-1 and IL-6. In addition, IL-6 produced from TLR5 driven M1 macrophages can differentiate the naïve T cells into inflammatory RA TH-17 cells that secrete IL-17A, IL-17F, IL-22, IL-24, IL-26, CCL20 and GM-CSF. In mice, systemic and local injection of a TLR5 agonist exacerbates joint swelling; conversely anti-TLR5 Ab treatment alleviates collagen induced arthritis (CIA) joint inflammation. To investigate the role of TLR5 Ab as a potential treatment for RA, we have partnered with scientists at Abwiz Inc. Using a TLR5 antigen, a human fragment antigen-binding (Fab) phage display library was screened for TLR5 high affinity binders. Up to 40 positive clones were sequenced and 10 selected TLR5 Fab clones were expressed, purified and assessed by ELISA for TLR5 binding and cross reactivity. Ten Fab clones were examined by the Shahrara lab for TLR5 neutralization capacity in human and murine cells and one was selected based on its superior blocking capacity. The overall goal of this project is to develop an anti-TLR5 Ab for RA therapy. In Phase I, our approach is to enhance the affinity of anti-TLR5 Ab in order to reach a Kd value that is within the range of commercially available Abs. Candidates provided through site directed mutagenesis using phage-display Ab library, will be tested for their TLR5 blocking affinity in human cells. Subsequently, the most promising candidates will be tested for their ability to abrogate RA synovial fluid from promoting inflammatory response in humanized RA mouse model. The long term goal of this project is to generate a safe and completely novel TLR5 Ab for RA patients that do not respond to the current therapies.

Public Health Relevance Statement:
A receptor has been identified in rheumatoid arthritis (RA) patients, which can strongly promote joint inflammation and bone erosion. We show strong evidence that blocking the function of this receptor potently alleviates the inflammatory response and bone loss in RA and preclinical models. The goal of this proposal is to engineer a highly specific antibody against the identified receptor so that it can be used as a potential therapy in RA patients.

Project Terms:
Achievement; Affect; Affinity; Agonist; alternative treatment; Anti-Inflammatory Agents; Anti-Tumor Necrosis Factor Therapy; Antibodies; antibody libraries; Antibody Therapy; antigen binding; Antigens; Antirheumatic Agents; arthritis therapy; Attenuated; Autoimmune Diseases; Bacteriophages; base; Binding; Blood Cells; bone; bone erosion; bone loss; CCL20 gene; Cell Differentiation process; Cell Line; Cells; cellular transduction; Chronic; Collagen Arthritis; commercialization; Complementarity Determining Regions; cross reactivity; Data; Dependovirus; Development; disability; Disease; Drug Kinetics; Effectiveness; Engineering; Enzyme-Linked Immunosorbent Assay; Epitopes; Fab Immunoglobulins; Flagellin; Funding; Goals; Granulocyte-Macrophage Colony-Stimulating Factor; Human; humanized mouse; Impairment; in vivo; Inflammation; Inflammatory; Inflammatory Response; Injections; Interleukin-1; Interleukin-1 beta; Interleukin-17; interleukin-22; Interleukin-6; Investigational New Drug Application; joint destruction; joint inflammation; Joints; Lead; Libraries; Ligands; Ligation; Light; macrophage; Maximum Tolerated Dose; Methotrexate; Molecular Conformation; monocyte; mouse model; multidisciplinary; Mus; Mutate; Myelogenous; nonhuman primate; novel; Pathway interactions; Patients; peripheral blood; Phage Display; Phase; pre-clinical; Pre-Clinical Model; Process; receptor; receptor function; Recombinants; Reporter; Rheumatoid Arthritis; Rodent; Role; safety study; Scientist; Site-Directed Mutagenesis; synergism; Synovial Fluid; T-Lymphocyte; Testing; Therapeutic; TLR5 gene; TNF gene; Toxicology

RA is one of the most common chronic autoimmune disorders that can lead to complete joint destruction and severe disability if untreated. There is no cure for RA and up to 50% of RA patients do not respond to anti-TNF therapies as circulating Th-17/IL-17 levels are highly elevated subsequent to TNF blockade. For this subset of RA patients, disruption of a novel pathway that impairs the synergy between TNF (M1 macrophages) and IL-17 (Th-17 cells) cascades may resolve the critical barrier in RA treatment. Hence objective of this project is to develop a therapeutic human TLR5 antibody (Ab) for RA patients whose disease is driven by the cross-talk between the effector macrophages and T cells.We documented that ligation of TLR5 to its natural ligand expressed in the joints, transforms RA peripheral blood (PB) cells into proinflammatory M1 macrophages which produce high levels of TNF, IL-1 and IL-6. In addition, IL-6 produced from TLR5 driven M1 macrophages can differentiate the naïve T cells into inflammatory RA TH-17 cells that secrete IL-17A, IL-17F, IL-22, IL-24, IL-26, CCL20 and GM-CSF. In mice, systemic and local injection of a TLR5 agonist exacerbates joint swelling; conversely anti-TLR5 Ab treatment alleviates collagen induced arthritis (CIA) joint inflammation.To investigate the role of TLR5 Ab as a potential treatment for RA, we have partnered with scientists at Abwiz Inc. Using a TLR5 antigen, a human fragment antigen-binding (Fab) phage display library was screened for TLR5 high affinity binders. Up to 40 positive clones were sequenced and 10 selected TLR5 Fab clones were expressed, purified and assessed by ELISA for TLR5 binding and cross reactivity. Ten Fab clones were examined by the Shahrara lab for TLR5 neutralization capacity in human and murine cells and one was selected based on its superior blocking capacity.The overall goal of this project is to develop an anti-TLR5 Ab for RA therapy. In Phase I, our approach is to enhance the affinity of anti-TLR5 Ab in order to reach a Kd value that is within the range of commercially available Abs. Candidates provided through site directed mutagenesis using phage-display Ab library, will be tested for their TLR5 blocking affinity in human cells. Subsequently, the most promising candidates will be tested for their ability to abrogate RA synovial fluid from promoting inflammatory response in humanized RA mouse model. The long term goal of this project is to generate a safe and completely novel TLR5 Ab for RA patients that do not respond to the current therapies. Public Health Relevance Statement A receptor has been identified in rheumatoid arthritis (RA) patients, which can strongly promote joint inflammation and bone erosion. We show strong evidence that blocking the function of this receptor potently alleviates the inflammatory response and bone loss in RA and preclinical models. The goal of this proposal is to engineer a highly specific antibody against the identified receptor so that it can be used as a potential therapy in RA patients.