Anti-αvβ8 integrin antibody for use in the treatment of renal diseases

Abstract

Methods and compositions for treating renal diseases, such as chronic kidney disease (CKD), are provided, comprising an antibody or antigen-binding fragment thereof that specifically and selectively binds to human αvβ8 integrin, which, as described, has been found to be highly expressed on renal cells and renal tissue, particularly diseased or fibrotic renal tissue. The disclosed anti-αvβ8 integrin antibodies bind to human αvβ8 integrin in the kidney and block the activation of TGF-β from its latent form in renal tissue. In the disclosed methods, the anti-αvβ8 antibodies reduce, attenuate, or abrogate renal fibrosis associated with the activity of αvβ8 integrin and TGF-β in renal tissue. The disclosed antibodies and methods effectively treat fibrosis associated with renal diseases, particularly renal diseases such as CKD, in individuals in need thereof.

Description

Background Art

[0001] Kidney diseases generally refer to a pathological condition in which an individual's kidneys are damaged and cannot properly perform the function of filtering waste products and excess water from the blood or assisting in blood pressure control. The kidneys have functions of regulating blood pressure, producing vitamin D, and releasing hormones that control the production of red blood cells. Kidney damage can potentially accumulate waste products in the body and may also cause or increase the risk of other health problems such as heart disease, heart attack, or stroke in an individual. The main risk factors for kidney diseases include diabetes, hypertension, and a family history of kidney failure. Kidney diseases can include acute kidney injury (AKI) that is sudden and sometimes accompanied by a temporary loss of kidney function, and chronic kidney disease (CKD) that refers to a pathological condition causing a decline in kidney function over a long period. CKD progresses over the years and can lead to end-stage renal disease (ESRD).

[0002] According to the 2015 kidney disease statistics of the American Kidney Fund, kidney disease is the ninth leading cause of death in the United States. Approximately 10% - 14% of the general adult population in the United States has CKD, which is more common in women. However, men with CKD are 50% more likely than women for the pathological condition of CKD to progress to kidney failure or ESRD. Furthermore, certain racial and ethnic groups have a higher risk of kidney failure than other groups. For example, the prevalence of ESRD is approximately 3.7 times higher in African Americans, approximately 1.4 times higher in Native Americans, and approximately 1.5 times higher in Asian Americans compared to white people. The likelihood of having ESRD in Hispanic Americans is nearly 1.5 times that of non-Hispanic Americans.

[0003] The progression of kidney disease often leads to the complete destruction of the functional tissue of the kidney, and ultimately, the affected individual may require lifelong dialysis or receive a kidney allograft. The progression of kidney disease is characterized by fibrosis, which contributes to the destruction of glomeruli (glomerulosclerosis) and tubules (tubulointerstitial fibrosis). Important factors in the progression of renal fibrosis are the cytokine TGF-β, its interacting molecules and receptors, and its downstream cell signaling cascades, which activate the pathophysiological cellular mechanisms that result in renal fibrosis and decreased kidney function. The expression of TGF-β and the excretion of TGF-β in the kidney correlate with a decrease in glomerular filtration rate (GFR) and protein leakage (proteinuria). The progression of kidney disease often leads to the complete destruction of the functional tissue of the kidney, and the affected individual may require lifelong dialysis or receive a kidney allograft. At the molecular level, the binding and interaction between TGF-β, which is involved in many pathological conditions including neoplastic diseases and inflammation, and its receptors and signaling mechanisms are still not well understood.

[0004] For the treatment of kidney disease, there is a great need for tissue- and disease-specific modulators of TGF-β activity that do not cause unwanted off-target effects and do not adversely affect other physiological contributions of this cytokine to normal cell function. The methods and reagents described herein provide specific target antibodies as therapeutic components that have the effect of directly blocking, neutralizing, regulating, and / or inhibiting important integrin targets that have been found to play important roles in the pathological conditions of renal cells and tissues, as well as in the mechanisms of kidney disease, and in the beneficial treatment of kidney disease.

Summary of the Invention

Means for Solving the Problems

[0005] As described below, the present disclosure features a method of treating a subject, particularly a mammalian subject, more particularly a human subject, having a kidney disease, particularly chronic kidney disease (CKD) and / or its symptoms, using an antibody or antigen-binding fragment thereof that specifically binds to αvβ8 integrin, i.e., an anti-αvβ8 integrin antibody. Based on the findings described herein, high-level expression of αvβ8 integrin on renal cells and renal tissue, particularly on diseased renal cells and renal tissue in a subject having a kidney disease such as CKD, enables an anti-αvβ8 integrin antibody or antigen-binding fragment thereof to specifically target αvβ8 integrin expressed on the diseased renal tissue of a subject suffering from a kidney disease such as CKD. The anti-αvβ8 integrin antibody described does not cross-react with other integrin receptor isoforms such as αvβ1, αvβ3, αvβ5, or αvβ6. In embodiments, the anti-αvβ8 integrin antibody is an isolated and purified antibody. In certain embodiments, the anti-αvβ8 integrin antibody is a humanized antibody. In another specific embodiment, the anti-αvβ8 integrin antibody is humanized and affinity-optimized to have good structural, binding, and / or functional properties such as specificity, affinity, and / or stability.

[0006] The anti-αvβ8 integrin antibodies and methods described herein were developed based on the discovery that renal cells and renal tissue, particularly renal epithelial tissue, express high levels of αvβ8 integrin, which is the latent receptor for the TGF-β cytokine (LAP-TGF-β). In particular, increased levels of αvβ8 integrin expressed in renal epithelium were found to be well correlated with fibrosis of renal tissue and / or the severity of fibrosis and fibrotic diseases in human subjects and animal models with renal diseases, as demonstrated by the experimental embodiments described herein. In particular, glomerular and tubular cells in the epithelial tissue of the kidney from individuals with renal diseases such as diabetic nephropathy (DN), as compared to normal renal cells and renal tissue, showed high levels of αvβ8 integrin expression, particularly in the podocytes and tubules (such as proximal and distal cortical tubules) of the affected kidney, as exemplified herein. In addition to diabetic nephropathy, other non-limiting renal diseases in which fibrosis of renal tissue results in damage or dysfunction that reduces the activity and function of the kidney include chronic kidney disease (CKD), acute kidney disease, hypertension-related renal disease, hyperglycemia-related renal disease, renal fibrosis, inflammation-related renal disease, end-stage renal disease (ESRD), autoimmune-related renal fibrosis (e.g., lupus nephritis), and fibrosis after kidney transplantation, among others.

[0007] For the treatment of kidney diseases and CKD, αvβ8 integrin that binds to latent TGF-β has been shown to be a very effective and useful target for reducing and attenuating fibrosis and tissue damage characteristic of kidney diseases such as CKD by the implementation of methods comprising the antibody reagents described herein. As described and exemplified herein, αvβ8 integrin is involved in the high-level expression of αvβ8 integrin in renal cells and renal tissues, particularly renal epithelial cells and epithelial tissues, such as glomerular podocytes and tubular cells of the kidney, and the fibrosis of renal tissues of subjects with kidney diseases, and considering the regulation of the activity of TGF-β that increases its damaging effect, it plays a direct and significant role in renal fibrosis that has not been previously recognized. The anti-αvβ8 integrin antibodies used in the methods described herein can specifically and selectively regulate the activities of both αvβ8 integrin and its TGF-β ligand in renal cells and renal tissues, thereby resulting in the reduction, inhibition, attenuation, decrease, and / or prevention of fibrosis in renal tissues caused by the binding of αvβ8 integrin to its ligand, latent TGF-β, and then enabling the activation / release of active TGF-β and liberating the harmful effects of active TGF-β in causing fibrosis of renal tissues.

[0008] One way TGF-β can be activated in renal tissue is by binding to the αvβ8 integrin expressed on the membrane of renal cells as the latent-associated peptide (LAP) TGF-β. As shown herein, in the renal tissue of subjects with renal disease and fibrosis, it was found that the expression of αvβ8 integrin was significantly increased. When TGF-β activation persists in the kidney over a long period of time, fibrosis of the renal tissue develops. Targeting αvβ8 integrin with the anti-αvβ8 integrin antibodies described herein, particularly in renal tissue expressing high levels of αvβ8 integrin, is an effective treatment for renal diseases such as CKD, while it has been determined that many potential systemic effects of non-selectively targeting and suppressing TGF-β in other non-renal tissues of the body are avoided. The UUO model is a model of fibrosis, and as shown by the anti-αvβ8 integrin antibodies described herein, inhibition of fibrosis and TGF-β activation results in a reduction and / or prevention of CKD progression. As described herein, high-affinity anti-αvβ8 integrin antibodies that specifically bind to αvβ8 integrin highly expressed in diseased and / or fibrotic renal tissue also selectively reduce, inhibit, attenuate, decrease, neutralize, and / or inhibit or prevent the interaction between αvβ8 integrin and latent TGF-β in the membranes of renal cells and renal tissue. Other tissues and organs that do not express αvβ8 integrin are not affected. Specific binding of the anti-αvβ8 antibody to αvβ8 integrin expressed in renal cells thereby blocks the activation of TGF-β in diseased and / or fibrotic kidneys, such as in CKD, and treats the associated fibrosis, with minimal or no significant adverse effects on the activity of TGF-β in non-renal cells and tissues.

[0009] Advantageously, the anti-αvβ8 integrin antibody specifically reduces the effect of the interaction between αvβ8 integrin and latent TGF-β on fibrosis of renal tissue and the onset and progression of kidney diseases, while avoiding much of the contribution of TGF-β activity to normal cell function. In embodiments, the methods described herein further provide a therapeutic benefit for protecting functional renal epithelium in individuals having a kidney disease involving fibrosis, such as CKD and other kidney diseases, e.g., diabetic nephropathy (DN)-associated kidney diseases, acute kidney diseases, hypertension-associated kidney diseases, hyperglycemia-associated kidney diseases, renal fibrosis, inflammation-associated kidney diseases, end-stage renal disease (ESRD), autoimmune-associated renal fibrosis (e.g., lupus nephritis), and fibrosis after kidney transplantation.

[0010] In one aspect, there is provided a method of treating renal fibrosis in a subject having a kidney disease, the method comprising administering to the subject an effective amount of an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof.

[0011] In another aspect, there is provided a method of reducing or attenuating renal fibrosis in a subject having a kidney disease, the method comprising administering to the subject in need thereof an effective amount of an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof, thereby reducing or attenuating fibrosis in the kidney.

[0012] In another aspect, there is provided a method of inhibiting the activity of αvβ8 integrin associated with renal fibrosis, the method comprising administering to the subject in need thereof an effective amount of an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof, blocking the binding of αvβ8 integrin to latent TGF-β, thereby inhibiting the activity of αvβ8 integrin associated with renal fibrosis. In one embodiment of the method, the subject has a kidney disease.

[0013] In yet another aspect, there is provided a method of treating renal fibrosis by blocking the activation of TGF-β from its latent form in renal cells and renal tissue, the method comprising administering to a subject in need thereof an effective amount of an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof, wherein the αvβ8 integrin binds to the latent form of TGF-β and blocks the production of active TGF-β, thereby treating renal fibrosis. In one embodiment of the method, the subject has a renal disease.

[0014] A method of treating renal injury characterized by an increase in plasma creatinine and / or urinary protein excretion levels, the method comprising administering to a subject in need thereof an effective amount of an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof, wherein administration of the anti-αvβ8 integrin antibody or an antigen-binding fragment thereof suppresses plasma creatinine and / or urinary protein excretion levels in the subject, thereby treating renal injury.

[0015] In one embodiment, the anti-αvβ8 integrin antibody or an antigen-binding fragment thereof reduces αvβ8-mediated TGF-β activation in the renal tissue of the subject.

[0016] In embodiments of any of the aspects of the methods described herein, the renal disease is selected from diabetic nephropathy (DN), chronic kidney disease (CKD), acute kidney disease, hypertension-related renal disease, hyperglycemia-related renal disease, renal fibrosis, inflammation-related renal disease, end-stage renal disease (ESRD), autoimmune-related renal fibrosis (e.g., lupus nephritis), and fibrosis after renal transplantation. In certain embodiments, the renal disease is CKD. In certain embodiments, the renal disease is diabetic nephropathy.

[0017] In one embodiment of any of the aspects of the methods described herein, the anti-αvβ8 integrin antibody or an antigen-binding fragment thereof binds to αvβ8 integrin expressed in renal cells and / or renal tissue and blocks the activation of TGF-β from its latent form.

[0018] In another aspect described herein, a method for detecting renal fibrosis in renal tissue is provided, which comprises contacting the renal tissue with an effectively labeled anti-αvβ8 integrin antibody or an antigen-binding fragment thereof, and detecting the binding of the anti-αvβ8 integrin antibody to αvβ8 integrin in the renal tissue.

[0019] In one embodiment of any of the aspects of the methods described herein, the anti-αvβ8 integrin antibody or an antigen-binding fragment thereof that specifically binds to αvβ8 integrin is (a) a heavy chain variable region complementarity determining region 1 (CDR1) comprising the amino acid sequence RYWMS (SEQ ID NO: 1) ; (b) a heavy chain variable region complementarity determining region 2 (CDR2) comprising the amino acid sequence EINPDSSTINYTSSL (SEQ ID NO: 2) ; and (c) a heavy chain variable region complementarity determining region 3 (CDR3) CDR3 comprising the amino acid sequence LITTEDY (SEQ ID NO: 3) ; and (d) a light chain variable region CDR1 comprising the amino acid sequence KASQDINSYLS (SEQ ID NO: 4) ; (e) a light chain variable region CDR2 comprising the amino acid sequence YANRLVD (SEQ ID NO: 5) ; and (f) a light chain variable region CDR3 comprising the amino acid sequence LQYDEFPYT (SEQ ID NO: 6) comprises.

[0020] In another embodiment of any of the aspects of the methods described herein, the anti-αvβ8 integrin antibody or an antigen-binding fragment thereof that specifically binds to αvβ8 integrin has a heavy chain variable region (V H ) amino acid sequence EVQLVESGGGLVQPGGSLRLSCAVSGFVFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYTSSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAILITTEDYWGQGTTVTVSS (SEQ ID NO: 7); and Light chain variable region (V L ) amino acid sequence DIQLTQSPSSLSASVGDRVTITCKASQDINSYLSWFQQKPGKAPKSLIYYANRLVDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPYTFGGGTKVEIK (SEQ ID NO: 8) comprising.

[0021] In one embodiment of any of the aspects of the methods described herein, an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof that specifically binds to αvβ8 integrin is (a) Amino acid sequence RSWIS (SEQ ID NO: 9) comprising heavy chain variable region CDR1; (b) Amino acid sequence EINPDSSTINYTSSL (SEQ ID NO: 2) comprising heavy chain variable region CDR2; and (c) Amino acid sequence LITTEDY (SEQ ID NO: 3) comprising heavy chain variable region CDR3; and (d) Amino acid sequence KASQDINKYLS (SEQ ID NO: 10) comprising light chain variable region CDR1; (e) Amino acid sequence YANRLVD (SEQ ID NO: 5) comprising light chain variable region CDR2; and (f) Amino acid sequence LQYDVFPYT (SEQ ID NO: 11) comprising light chain variable region CDR3 comprising.

[0022] In another embodiment of any aspect of the methods described herein, an anti-αvβ8 integrin antibody (referred to herein as "B5-15") that specifically binds to αvβ8 integrin, or an antigen-binding fragment thereof, has a heavy chain variable region (V H ) amino acid sequence EVQLVESGGGLVQPGGSLRLSCAVSGFVFSRSWISWVRQAPGKGLEWIGEINPDSSTINYTSSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAILITTEDYWGQGTTVTVSS (SEQ ID NO: 12); and a light chain variable region (V L ) amino acid sequence DIQLTQSPSSLSASVGDRVTITCKASQDINKYLSWFQQKPGKAPKSLIYYANRLVDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDVFPYTFGGGTKVEIK (SEQ ID NO: 13) and comprises

[0023] In an embodiment of any aspect of the treatment methods described herein, an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof that specifically binds to αvβ8 integrin is administered to a subject as an adjuvant therapeutic agent or in combination with a treatment for a renal disease. In an embodiment, an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof that specifically binds to αvβ8 integrin is administered to a subject before, simultaneously with, or after the administration of an adjuvant therapeutic agent or treatment.

[0024] In one embodiment of any aspect of the treatment methods described herein, an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof binds to αvβ8 integrin in fibrotic renal cells and renal tissue with increased expression, and reduces or inhibits fibrosis associated with increased expression of αvβ8 integrin in podocytes and interstitial tubular cells of the renal tissue of a subject having a renal disease such as CKD.

[0025] In another aspect described herein, an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof, wherein (a) amino acid sequence RYWMS (SEQ ID NO: 1) Heavy chain variable region CDR1 containing: (b) Amino acid sequence EINPDSSTINYTSSL (SEQ ID NO: 2) Heavy chain variable region CDR2; and (c) Amino acid sequence LITTEDY (SEQ ID NO: 3) Heavy chain variable region CDR3; and (d) Amino acid sequence KASQDINSYLS (SEQ ID NO: 4) Light chain variable region CDR1 containing: (e) Amino acid sequence YANRLVD (SEQ ID NO: 5) Light chain variable region CDR2; and (f) Amino acid sequence LQYDEFPYT (SEQ ID NO: 6) Light chain variable region CDR3 An antibody or antigen-binding fragment thereof containing the above is provided.

[0026] In another aspect described herein, an anti-αvβ8 integrin antibody or antigen-binding fragment thereof, Heavy chain variable region (V H ) amino acid sequence EVQLVESGGGLVQPGGSLRLSCAVSGFVFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYTSSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAILITTEDYWGQGTTVTVSS (SEQ ID NO: 7); and Light chain variable region (V L ) amino acid sequence DIQLTQSPSSLSASVGDRVTITCKASQDINSYLSWFQQKPGKAPKSLIYYANRLVDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPYTFGGGTKVEIK (SEQ ID NO: 8) An antibody or antigen-binding fragment thereof containing the above is provided.

[0027] In one embodiment, the antibody or antigen-binding fragment thereof binds to αvβ8 integrin, the expression of which is increased in fibrotic renal cells and renal tissues of a subject having a renal disease such as CKD. In one embodiment, the anti-αvβ8 integrin antibody or antigen-binding fragment thereof specifically binds to αvβ8 integrin, the expression of which is increased in fibrotic renal cells and renal tissues, blocks the binding of latent TGF-β to αvβ8 integrin, thereby suppressing the activity of αvβ8 integrin associated with renal fibrosis. In one embodiment, the anti-αvβ8 integrin antibody or antigen-binding fragment thereof attenuates or suppresses fibrosis associated with increased expression of αvβ8 integrin in podocytes and interstitial tubular cells of renal tissues of a subject having a renal disease.

[0028] In another aspect described herein, an anti-αvβ8 integrin antibody or antigen-binding fragment thereof, (a) a heavy chain variable region CDR1 comprising the amino acid sequence RSWIS (SEQ ID NO: 9) ; (b) a heavy chain variable region CDR2 comprising the amino acid sequence EINPDSSTINYTSSL (SEQ ID NO: 2) ; and (c) a heavy chain variable region CDR3 comprising the amino acid sequence LITTEDY (SEQ ID NO: 3) ; and (d) a light chain variable region CDR1 comprising the amino acid sequence KASQDINKYLS (SEQ ID NO: 10) ; (e) a light chain variable region CDR2 comprising the amino acid sequence YANRLVD (SEQ ID NO: 5) ; and (f) a light chain variable region CDR3 comprising the amino acid sequence LQYDVFPYT (SEQ ID NO: 11) is provided. An antibody or antigen-binding fragment thereof comprising the same is provided.

[0029] In another aspect described herein, an anti-αvβ8 integrin antibody or antigen-binding fragment thereof, wherein the heavy chain variable region (VH ) Amino acid sequence EVQLVESGGGLVQPGGSLRLSCAVSGFVFSRSWISWVRQAPGKGLEWIGEINPDSSTINYTSSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAILITTEDYWGQGTTVTVSS (SEQ ID NO: 12) and Light chain variable region (V L ) Amino acid sequence DIQLTQSPSSLSASVGDRVTITCKASQDINKYLSWFQQKPGKAPKSLIYYANRLVDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDVFPYTFGGGTKVEIK (SEQ ID NO: 13) An antibody or an antigen-binding fragment thereof containing the same is provided.

[0030] In one embodiment, the above antibody or its antigen-binding fragment binds to αvβ8 integrin whose expression is increased in fibrotic renal cells and renal tissues of a subject having a kidney disease such as CKD. In one embodiment, the anti-αvβ8 integrin antibody or its antigen-binding fragment specifically binds to αvβ8 integrin whose expression is increased in fibrotic renal cells and renal tissues, blocks the binding of latent TGF-β to αvβ8 integrin, thereby suppressing the activity of αvβ8 integrin associated with renal fibrosis. In one embodiment, the anti-αvβ8 integrin antibody or its antigen-binding fragment attenuates or suppresses fibrosis associated with the increased expression of αvβ8 integrin in podocytes and interstitial tubular cells of renal tissues of a subject having a kidney disease.

[0031] In one embodiment of any of the above aspects, the anti-αvβ8 integrin antibody or its antigen-binding fragment is of the IgG class. In a specific embodiment, the antibody or its antigen-binding fragment is of the IgG1 isotype.

[0032] In another aspect, an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof that competes with any of the anti-αvβ8 integrin antibodies or antigen-binding fragments thereof described in the above aspects for binding to αvβ8 integrin is provided. In one embodiment, the anti-αvβ8 integrin antibody or antigen-binding fragment thereof is an IgG antibody. In one embodiment, the anti-αvβ8 integrin antibody or antigen-binding fragment thereof is an IgG1 antibody.

[0033] In another aspect, a polynucleotide encoding the anti-αvβ8 integrin antibody or antigen-binding fragment thereof described herein is provided. In one embodiment, the polynucleotide sequence encoding the V H region of the antibody comprises the following nucleic acid sequence

Chemical formula

Chemical formula

[0034] In another aspect, an expression vector comprising the above polynucleotide is provided. In an embodiment, the expression vector is a prokaryotic, eukaryotic, or mammalian expression vector.

[0035] In another aspect, a cell comprising the above expression vector is provided. In an embodiment, the cell is a prokaryotic, eukaryotic, or mammalian host cell.

[0036] In another aspect, a pharmaceutical composition comprising the above anti-αvβ8 integrin antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier, excipient, or diluent is provided.

[0037] In another aspect, a pharmaceutical composition comprising the above polynucleotide and a pharmaceutically acceptable carrier, excipient, or diluent is provided.

[0038] In another aspect, there is provided a kit comprising an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof described herein, or a pharmaceutical composition comprising an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof.

[0039] Other features and advantages of the present disclosure will become apparent from the detailed description and the claims.

[0040] Definitions Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide one of ordinary skill in the art with many general definitions of terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below unless otherwise specified.

[0041] The term "agent" refers to a protein, polypeptide, peptide (or fragment or portion thereof), nucleic acid molecule, small compound, drug, or pharmaceutical. An agent can be an antagonist and can block or inhibit the activity of another molecule such as a cognate ligand.

[0042] As used in this disclosure, the term "antibody" refers to an immunoglobulin or fragment, portion or derivative thereof, and includes any polypeptide containing an antigen-binding site, whether produced in vitro or in vivo. This term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, variant, and grafted antibodies. Unless specifically modified by the term "intact" as in "intact antibody", for the purposes of this disclosure, the term "antibody" includes antibody fragments (or portions) such as Fab, F(ab’)2, Fv, scFv, Fd, dAb, etc., and other antibody fragments (or portions) that retain the antigen-binding function or epitope-binding function, i.e., the ability to specifically bind to a polypeptide. Typically, such fragments (or portions) contain an antigen-binding domain.

[0043] By way of example, an immunoglobulin (antibody) contains a tetrameric structural unit. Each tetramer contains two identical pairs of polypeptide chains, each pair having one "light" (L) chain (about 25 kD) and one "heavy" (H) chain (about 50 - 70 kD). The amino (N) terminus of each polypeptide chain defines a variable (V) region of about 100 - 110 or more amino acids that is primarily involved in antigen recognition and binding. The variable light chain region (V L ) and variable heavy chain region (V H ) refer to the variable regions of the light and heavy chains of an immunoglobulin molecule (antibody), respectively. The variable region or "V region" refers to the antibody variable region that results from the genetic rearrangement of the heavy and light chain V region genes during B cell differentiation, i.e., framework 1 (F1), CDR1, framework 2 (F2), CDR2, framework 3 (F3), CDR3, and framework 4 (F4).

[0044] The V H and V L regions of an immunoglobulin (antibody) molecule are the V H and V LIt contains three Complementary Determining Regions (CDRs), which are three hypervariable regions located within the framework region. CDRs are mainly involved in binding to the epitope of the antigen. The V of the antibody H and V LThe CDRs of a domain typically are numbered sequentially starting from the N-terminus of the variable region segment and are called CDR1, CDR2, and CDR3. The amino acid sequences of the framework regions of the different heavy and light antibody chains are relatively conserved within a species. The framework regions (FW1–FW4) of the V regions of the heavy and light chains of the antibody components provide the structural arrangement and alignment of the CDRs in three-dimensional space. The characterization (and numbering) of the amino acid sequences of the CDRs and framework regions in an antibody molecule can be determined, for example, as reported by Kabat, Chothia, the international ImMunoGeneTics database (IMGT), and AbM (e.g., Chothia & Lesk, 1987, J. Mol. Biol., 196:901–917; Chothia et al., 1989, Nature, 342:877–883; Chothia et al., 1992, J. Mol. Biol., 227:799–817; Al-Lazikani et al., 1997, J. Mol. Biol., 273(4):927–948). The definition of the antigen-binding site is reported in Ruiz et al., 2000, Nucleic Acids Res., 28:219–221 and Lefranc, 2001, Nucleic Acids Res., 29(1):207–209; MacCallum et al., 1996, J. Mol. Biol., 262:732–745; Martin et al, 1989, Proc. Natl Acad. Sci. USA, 86:9268–9272; Martin, et al, 1991, Methods Enzymol., 203:121–153; Pedersen et al, 1992, Immunomethods, 1:126–136; and Rees et al, 1996, In: Sternberg M.J.E. (ed.), Protein Structure Prediction. Oxford University Press, Oxford, England, pp. 141–172.

[0045] A "chimeric antibody" is an antibody molecule in which the constant region of an antibody molecule of a different or altered class and / or species, or a fragment thereof, of the antigen-binding site (variable region, CDR, or fragment thereof), or a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug, etc.) that confers new properties or effector functions to the chimeric antibody, is linked to the constant region or a fragment thereof, or the constant region or a fragment thereof has been modified, substituted, or exchanged. Alternatively, a chimeric antibody may contain a variable region or a fragment thereof that has been modified, substituted, or exchanged with a variable region having a different or altered antigen specificity (e.g., one or more CDRs and framework regions from different species).

[0046] The terms "antigen-binding portion", "antigen-binding domain", "antigen-binding fragment", "binding fragment", or "binding portion" refer to the portion of an antibody molecule that contains the amino acids involved in the specific binding of the antibody to an antigen. When the antigen is large, the antigen-binding domain may bind only to a portion of the antigen. The portion of the antigen molecule that is responsible for the specific interaction with the antigen-binding domain is referred to as the "binding site", "epitope", or "antigenic determinant". In certain embodiments, the antigen-binding domain comprises the variable region of the antibody light chain (V L ) and the variable region of the antibody heavy chain (V H ), but not necessarily both. For example, the so-called Fd antibody fragment is a V HIt consists only of domains but still retains some of the antigen-binding function of an intact antibody. The binding site of an antibody produced against a given antigen, i.e., the epitope, can be determined using methods known in the art. For example, a competition assay (e.g., an enzyme-linked immunosorbent assay (ELISA)) can be performed using an antibody having a known epitope. If a test antibody competes for binding to a given antigen, that antibody is likely to share at least a portion of the same epitope. Epitopes can also be localized using domain swapping or site-directed mutagenesis of the antigen. That is, each region or amino acid of the antigen can be "swapped" or replaced with an amino acid or component known not to interact with the test antibody. If substitution of a given region or amino acid results in a decrease in the binding of the test antibody to the substituted antigen compared to its binding to the unsubstituted antigen, that region or amino acid is likely to be an epitope, within the epitope, or at least part of the epitope.

[0047] Binding fragments (or portions) of an antibody are produced by recombinant DNA techniques or by enzymatic or chemical cleavage of an intact antibody. Examples of binding fragments or portions include Fab, Fab’, F(ab’)2, Fv, and single-chain antibodies. Antibodies other than "bispecific" or "bifunctional" antibodies are understood to have identical binding sites. Digestion of an antibody with the enzyme papain yields two identical antigen-binding fragments, also known as "Fab" fragments, and an "Fc" fragment that has no antigen-binding activity but has the ability to crystallize. Digestion of an antibody with the enzyme pepsin yields an F(ab’)2 fragment that contains two antigen-binding sites with the two arms of the antibody molecule still linked. The F(ab’)2 fragment has the ability to cross-link antigens. "Fv", as used herein, refers to the smallest fragment of an antibody that retains both the antigen recognition site and the antigen-binding site. "Fab", as used herein, refers to a fragment of an antibody that includes the constant domain of the light chain and the CHI domain of the heavy chain.

[0048] The term "mAb" refers to a monoclonal antibody. The antibodies disclosed herein include, but are not limited to, all native antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab’, single-chain V region fragments (scFv), fusion polypeptides, and non-conventional antibodies.

[0049] The term "humanized antibody" refers to an antibody derived from a non-human (e.g., mouse, rat or rabbit) immunoglobulin that has been engineered to contain minimal non-human (e.g., mouse, rat or rabbit) sequences. Typically, a humanized antibody is a human immunoglobulin in which residues from hypervariable complementarity-determining regions (CDRs) are replaced by residues from CDRs of a non-human species (e.g., mouse, rat, rabbit, or hamster) having the desired specificity, affinity, and / or capacity (Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536). Thus, the framework regions of a humanized antibody are substantially those of a human immunoglobulin. In some cases, the Fv framework region (FW) residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species having the desired specificity, affinity, and / or capacity.

[0050] Humanized antibodies can be further modified by substituting additional residues in the Fv framework region and / or in substituted non-human residues to refine and optimize antibody specificity, affinity, and / or ability. Generally, a humanized antibody will substantially include at least one, typically two or three, variable domains that contain all or substantially all of the CDR regions corresponding to a non-human immunoglobulin, while all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. A humanized antibody may also include at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to make humanized antibodies are described in U.S. Patent Nos. 5,225,539 or 5,639,641.

[0051] "Fragment" means a portion of a polypeptide or nucleic acid molecule. This "fragment" or "portion" preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full-length reference nucleic acid molecule or polypeptide. In certain embodiments, a fragment or portion of a polypeptide may contain 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 300 amino acids. In embodiments, the fragment or portion retains the full or at least partial activity and / or function of the polypeptide or nucleic acid molecule as a whole.

[0052] "Detecting" refers to identifying the presence, absence, or amount of an analyte to be detected. In various embodiments, the analyte is a polypeptide or nucleic acid biomarker.

[0053] As used herein in connection with antibodies, "competing" generally means that a first antibody or an antigen-binding fragment thereof competes for binding with a second antibody or an antigen-binding fragment thereof, where the binding of the first antibody (to its cognate antigen-binding site or epitope, e.g., on integrin αvβ8) in the presence of the second antibody is detectably decreased compared to the binding of the first antibody in the absence of the second antibody. Alternatively, in some cases, the binding of the second antibody to its cognate antigen-binding site or epitope is also detectably decreased in the presence of the first antibody. However, this is not always the case. Thus, a first antibody can inhibit the binding of a second antibody to its cognate antigen-binding site or epitope without the second antibody inhibiting the binding of the first antibody to its cognate antigen-binding site or epitope. However, antibodies are said to "cross-compete" with each other for binding to their respective binding sites or epitopes if each antibody inhibits the binding of the other antibody to its cognate epitope or ligand to a detectable extent, whether to the same extent, a greater extent, or a lesser extent. As used herein, both competing antibodies and cross-competing antibodies are contemplated. Regardless of the mechanism by which antibody competition or cross-competition occurs, such as steric hindrance, conformational changes, or binding to a common binding site, epitope, or fragment thereof, both competing antibodies and / or cross-competing antibodies are included herein and may be useful in the disclosed methods.

[0054] As used herein in connection with the treatments described herein, the term "ameliorate" refers to decreasing, reducing, alleviating, suppressing, attenuating, arresting, halting, preventing, blocking, neutralizing, or stabilizing the development or progression of a disease or condition such as fibrosis (renal fibrosis) in renal cells and / or renal tissue.

[0055] "Integrin", as referred to herein, is a cell surface glycoprotein that is a major receptor used by mammalian cells to bind to the extracellular matrix and mediate cell-cell and cell-extracellular matrix interactions. They are heterodimers (having α and β subunits non-covalently bound to each other) and function as transmembrane linkers between the extracellular matrix and the actin cytoskeleton of the cell. Integrin proteins do not function as passive adhesives but rather as dynamic molecules that mediate the transmission of information across the cell membrane in both directions. Integrin-mediated adhesion can be regulated in response to signals by clustering and conformational changes induced at the cytoplasmic tail of integrin, which functions as a signaling molecule that activates various intracellular signaling pathways when activated by ligand binding. Furthermore, integrin signaling controls cell survival, cell cycle progression, and differentiation. Regulation of integrin-mediated adhesion structures is important for many forms of cell migration. Integrins are also involved in the development of a variety of acquired and genetic diseases.

[0056] The integrin family of proteins has several members, some of which have a broad tissue distribution. There are approximately 24 integrins in vertebrates, and a single cell may express multiple different types of integrin receptors on its surface. The human integrin β8 subunit encoded by the ITGB8 gene has ligands including fibronectin and TGF-β1 and TGF-β2 isoforms. αVβ8 integrin (a heterodimer containing an alpha V (αV) subunit bound to a beta 8 (β8) subunit as further described below) is expressed on the cell surface in combination with MT1 matrix metalloproteinase (MMP), interacts with latent TGF-β in the cell matrix, and mediates its activation. MT1 protease cleaves latent TGF-β to release the mature active TGF-β polypeptide. Reactive oxygen species, other proteases, inflammation, and pH changes have also been demonstrated to be involved in the release of active TGFβ.

[0057] "αvβ8" means an "αvβ8 integrin receptor", "αvβ8 integrin", or "integrin αvβ8" polypeptide, or a fragment thereof, that has at least about 85% or more amino acid sequence identity to the human αvβ8 integrin amino acid sequence provided by NCBI Reference Sequence: NM_002214.2 and has the αvβ8 activity and / or function described below. Similar to other integrin beta (β) subunits, human αvβ8 contains an N-terminal signal peptide, a large extracellular domain containing four cysteine-rich repeats, a transmembrane domain, and a short C-terminal cytoplasmic domain. αvβ8 has a molecular weight of approximately 95 kD, consistent with substantial glycosylation of the predicted 81 kD β8 gene product. (M. Moyle et al., 1991, J. Biol. Chem., 266:19650-19658). Northern blot analysis revealed that human αvβ8 is expressed as an approximately 8.5 kilobase (kb) mRNA in osteosarcoma cell lines. When expressed in mammalian cells, the β8 integrin subunit binds to the alpha V (αV) subunit to form a cell surface αVβ8 integrin complex. In certain embodiments, the polypeptide is human αvβ8 integrin. As used herein, the term "αvβ8" is synonymous with "αvβ8 integrin receptor", "αvβ8 integrin", and "integrin αvβ8". The name "itgb8" typically refers to the human gene sequence of the β8 subunit.

[0058] The human β8 integrin (ITGB8, integrin beta 8, integrin β8, β8, and terms used interchangeably) protein sequence can be found at Uniprot accession number P26012 or NCBI Reference Sequence: NM_002214.2 and is as follows:

Chemical formula

[0059] The itgb8 polynucleotide coding sequence of human β8 integrin is shown below (8787bp itgb8 mRNA nucleic acid sequence). The polynucleotide sequence of human β8 integrin can be found in the accession number: NCBI reference sequence: NM_002214.2. Polynucleotides or fragments thereof having at least about 85% or more nucleotide sequence identity to the itgb8 polynucleotide sequence encoding the human β8 integrin polypeptide are encompassed by the present disclosure.

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

[0060] The alpha V integrin (α-V, ITGAV) subunit (also called alpha V and αv) binds to any of the β-1 (ITGB1), β-3 (ITGB3), β-5 (ITGB5), β-6 (ITGB6), and β-8 (ITGB8) subunits to form a heterodimer of the alpha (αV) and beta (β1-8) subunits. The alpha subunit is composed of a heavy chain (integrin α-V heavy chain) and a light chain (integrin α-V light chain) linked by disulfide bonds. In certain embodiments, the αv integrin subunit binds to the β8 integrin subunit to form the αvβ8 integrin. Human α-V (ITAV), which can bind to β-8 (ITGB8) as described above, contains 1040 amino acids and can be found in the Uniprot (UniProtKB) accession number P06756 as follows:

Chemical Structure

Chemical Structure

Chem.

[0061] The polynucleotide coding sequence encoding human alpha V (ITGAV) is shown below (3147 nucleotide base pairs). The polynucleotide sequence of human alpha v integrin (CCDS 2292.1) can be found at accession number: NCBI reference sequence: NM_002210.4. A polynucleotide having at least about 85% or more nucleotide sequence identity to the alpha v (ITGAV) integrin polynucleotide sequence encoding the human ITGAV integrin polypeptide or a fragment thereof is encompassed by the present disclosure.

Chem.

Chem.

[0062] The humanized anti-alpha v beta 8 integrin antibody designated herein as "MEDI-hu37E1B5" is useful in the compositions and methods of the present disclosure. The MEDI-hu37E1B5 antibody has the heavy and light chain variable region amino acid sequences shown below. This antibody specifically and selectively binds to the alpha v beta 8 integrin protein that is expressed in renal cells and renal tissue, and more particularly, is highly expressed in diseased renal cells and renal tissue such as fibrotic renal tissue of a subject having a renal disease such as chronic kidney disease (CKD). In one embodiment, an anti-alpha v beta 8 integrin antibody or an antigen-binding fragment thereof having at least about 85% or more or at least 85% or more amino acid sequence identity to the amino acid sequences of the heavy chain variable region (V H ), (116 amino acid residues), and the light chain variable region (V L ), (107 amino acid residues) of the MEDI-hu37E1B5 alpha v beta 8 integrin antibody is encompassed by the present disclosure. Amino acid sequence of the V H of the MEDI-hu37E1B5 anti-alpha v beta 8 integrin antibody:

Chem.

Chemical formula

[0063] The above V of MEDI-hu37E1B5 antibody H and V L In the sequences, underline the three CDR regions (defined by Kabat). More specifically, the amino acid sequences of the three CDRs of the heavy chain variable region (V H ) of the MEDI-hu37E1B5 antibody are as follows: V H CDR1: RYWMS (SEQ ID NO: 1) V H CDR2: EINPDSSTINYTSSL (SEQ ID NO: 2) V H CDR3: LITTEDY (SEQ ID NO: 3)

[0064] The amino acid sequences of the three CDRs of the light chain variable region (V L ) of the MEDI-hu37E1B5 antibody are as follows: V L CDR1: KASQDINSYLS (SEQ ID NO: 4) V L CDR2: YANRLVD (SEQ ID NO: 5) V L CDR3: LQYDEFPYT (SEQ ID NO: 6)

[0065] The V H and V L regions of the MEDI-hu37E1B5 anti-αvβ8 integrin antibody or its antigen-binding fragment identified above, and the polynucleotide sequences encoding them are also included in the present disclosure. In one embodiment, having at least about 85% or more nucleotide sequence identity with the MEDI-hu37E1B5 nucleotide sequence, the above V of the MEDI-hu37E1B5 anti-αvβ8 integrin antibody or its antigen-binding fragment Hand V L Polynucleotide sequences encoding the region are also encompassed by the present disclosure.

[0066] Another anti-αvβ8 integrin antibody, referred to herein as "B5-15", is particularly useful in the compositions and methods of the present disclosure. The B5-15 anti-αvβ8 integrin antibody is a humanized and affinity-optimized antibody (IgG1 isotype) that specifically binds to αvβ8 integrin and has the heavy and light chain variable region amino acid sequences shown below. The humanized B5-15 antibody is derived from the above-described MEDI-hu37E1B5 antibody, which is the "parent" of the B5-15 antibody described herein, and has optimized affinity. This B5-15 antibody binds very specifically and selectively to αvβ8 integrin, particularly to αvβ8 integrin expressed in renal cells and renal tissues, and more particularly to αvβ8 integrin protein highly expressed in diseased renal cells and renal tissues such as fibrotic renal tissues of subjects with renal diseases such as chronic kidney disease (CKD). In one embodiment, the heavy chain variable region (V L ) of the B5-15 αvβ8 integrin antibody shown below, (116 amino acid residues), and the light chain variable region (V L ) of the B5-15 αvβ8 integrin antibody shown below, (107 amino acid residues), anti-αvβ8 integrin antibodies or antigen-binding fragments thereof having at least about 85% or more, or at least 85% or more amino acid sequence identity are encompassed by the present disclosure. The V H amino acid sequence of the humanized and optimized B5-15 anti-αvβ8 integrin antibody:

Chemical formula

Chemical formula

[0067] The above V H and V LUnderline the amino acid sequences of the three CDR regions (defined by Kabat) in the array. More specifically, the amino acid sequences of the three CDRs of the heavy chain variable region (V H ) of the B5-15 antibody are as follows: V H CDR1: RSWIS (SEQ ID NO: 9) V H CDR2: EINPDSSTINYTSSL (SEQ ID NO: 2) V H CDR3: LITTEDY (SEQ ID NO: 3)

[0068] The amino acid sequences of the three CDRs of the light chain variable region (V L ) of the B5-15 humanized and affinity-optimized antibody are as follows: V L CDR1: KASQDINKYLS (SEQ ID NO: 10) V L CDR2: YANRLVD (SEQ ID NO: 5) V L CDR3: LQYDVFPYT (SEQ ID NO: 11)

[0069] The polynucleotide sequences encoding the V H and V L regions of the above B5-15 anti-αvβ8 integrin antibody or its antigen-binding fragment are also included in the present disclosure. The polynucleotide sequence encoding the V H region of the optimized B5-15 anti-αvβ8 integrin antibody is presented below:

Chemical Structure

[0070] The polynucleotide sequence encoding the V L (kappa) region of the optimized B5-15 anti-αvβ8 integrin antibody is presented below:

Chemical Structure

[0071] In one embodiment, the V of the above B5-15 anti-αvβ8 integrin antibody or antigen-binding fragment thereof having at least about 85% or more nucleotide sequence identity with the B5-15 nucleotide sequence H and V L region encoding polynucleotide sequences are also encompassed by the present disclosure.

[0072] The cytokine transforming growth factor beta (β) (TGF-β) is a multifunctional regulator that regulates cell proliferation, differentiation, apoptosis, adhesion and migration of various cell types. TGF-β induces the production of extracellular matrix (ECM) proteins and of almost all cell types, such as activated T and B cells, hematopoietic cells, macrophages, dendritic cells, which produce TGF-β and / or are sensitive to its effects. (S. Dennler et al., 2002, J. Leukoc. Biol., 71: 731-740). TGF-β is a member of a diverse superfamily that includes more than 30 related members in mammals, namely, three TGF-β isoforms, four activins, and more than 20 bone morphogenetic proteins (BMPs). The three mammalian isoforms of TGF-β (TGF-β1, TGF-β2 and TGF-β3) share 70-82% homology at the amino acid level and have qualitatively similar activities in different systems. The active form of TGF-β is a dimer stabilized by hydrophobic interactions and is most often further strengthened by inter-subunit disulfide bridges. The TGF-β1 isoform is the most abundantly present isoform in renal cells.

[0073] The mechanism by which TGF-β initiates intracellular signal transduction at the cell membrane is generally well understood. (See, for example, I. Loeffler and G. Wolf, 2013, Nephrol. Dial. Transplant, 29: i37-i45). The intracellular mediators of TGF-β signaling are called Smads and act downstream of the type I TGF-β receptor (TβR-1) and are classified into three classes. Receptor-regulated Smads (R-Smads), such as Smad1, Smad2, Smad3, Smad5, and Smad8, are directly phosphorylated and activated by TβR-1, which is a transmembrane receptor serine / threonine kinase, and form hetero-oligomeric complexes with a common mediator Smad (Co-Smad), such as Smad4, which is the second class of Smad. These Smad complexes translocate to the nucleus where they interact with site-specific DNA transcription factors and are involved in the regulation of target genes. Smad2 and Smad3 respond to signaling by the TGF-β subfamily. The third identified class of Smads includes inhibitory Smads, Smad6 and Smad7, which can antagonize the activity of receptor-regulated Smads by physically interacting with the activated TβR-1 receptor and preventing the docking and phosphorylation of R-Smads. (Ibid.). TGF-β can also directly activate other signaling cascades, including MAPK pathways such as Ras, Raf, Erk, JNK, and p38, in addition to Smad-mediated transcription, by virtue of its pleiotropic effects. Furthermore, TGF-β can activate the phosphatidylinositol-3-kinase (PI-3K) cascade by phosphorylation of its effector Akt, as well as Rho-like GTPases including RhoA, Rac, and cdc42. (Ibid.).

[0074] TGF-β is synthesized by many renal cell types and exerts its biological (and pathophysiological) effects via the signal transduction pathways described above. TGF-β is upregulated in kidney diseases, inducing renal cells to produce extracellular matrix proteins, leading to glomerulosclerosis and tubulointerstitial (TI) fibrosis, which is a damaging process characterized by progressive and detrimental connective tissue deposition on the renal parenchyma and resulting in the deterioration of renal function. Various types of renal cells are subject to various pathophysiological changes induced by the activity of TGF-β, resulting in apoptosis, tissue hypertrophy, and abnormal podocyte foot processes, ultimately causing renal dysfunction. (Ibid.).

[0075] As used herein, the terms "determine," "evaluate," "assess," "assay," "measure," and "detect," as well as "identify," refer to both quantitative and qualitative determinations, and thus the term "determine" is used herein synonymously with "assay," "measure," etc. When a quantitative determination is intended, the phrase "determine the amount, level, or concentration of" an analyte, substance, protein, etc. is used. When a qualitative and / or quantitative determination is intended, the phrase "determine the level of" an analyte or "detect" an analyte is used.

[0076] "Disease" means any condition or disorder that impairs, interferes with, or dysregulates the normal function of cells, tissues, or organs. Kidney diseases and kidney-related diseases referred to herein include, by way of non-limiting example, diabetic nephropathy (DN), chronic kidney disease (CKD), acute kidney disease, hypertension-related kidney disease, hyperglycemia-related kidney disease, renal fibrosis, inflammation-related kidney disease, end-stage renal disease (ESRD), autoimmune-related renal fibrosis (e.g., lupus nephritis), and fibrosis after kidney transplantation, among others. Such diseases, conditions, lesions, and / or their symptoms related to the kidney may be acute or chronic in a subject and are not intended to be limiting.

[0077] Generally speaking, "fibrosis" is the formation of excessive connective tissue in an organ or tissue that can occur as a result of a reactive (e.g., response to injury; disease) or repair process. Fibrosis can occur in a reactive, benign, or pathological state. In response to injury, fibrosis can be called scarring. Renal scarring leads to progressive loss of kidney function and ultimately end-stage renal disease and the need for dialysis or kidney transplantation.

[0078] Renal fibrosis is an inevitable result of the excessive accumulation of extracellular matrix that occurs in virtually every type of chronic kidney disease. The etiology of renal fibrosis is a progressive process that ultimately leads to a devastating disorder requiring end-stage renal disease / renal failure, dialysis, or kidney transplantation. Generally, renal fibrosis represents the failure of the wound healing process in renal tissue following chronic, persistent injury or damage. Several cellular pathways, including the activation of mesangial cells and fibroblasts, and epithelial-mesenchymal transition (EMT) of tubular epithelial cells, have been identified as the major ways in which matrix-producing cells are generated in the diseased state. (See, e.g., Y. Liu, 2006, Kidney Int., 69(2):213-217).

[0079] Among the many fibrogenic factors that regulate the renal fibrosis process, TGF-β plays a central role. Defective matrix degradation can contribute to tissue scarring, but the exact role and mechanism of matrix-degrading enzymes in the injured kidney are complex and not well understood. Intervening in the activity of endogenous antifibrotic factors can provide a strategy for antagonizing the fibrogenic action of the TGF-β / Smad signaling pathway.

[0080] A "podocyte" is a highly differentiated epithelial cell in the Bowman's capsule of the kidney that wraps around the capillaries of the glomerulus. It is the foot processes or the processes of podocytes that wrap around the capillaries and form the filtration slits (or slit diaphragms) through which blood and blood components are filtered. The Bowman's capsule filters the blood, retaining larger molecules (e.g., proteins), while filtering smaller molecules (e.g., water, salts, sugars) as the first step in urine formation. Together with the endothelial cells of the glomerular capillary loops and the glomerular basement membrane, podocytes form the filtration barrier. The kidney podocytes and mesangial cells support the structure and function of the glomerulus.

[0081] The "glomerulus" in the kidney is a network or cluster of capillaries called the tuft, which is located inside the cup-like sac (glomerular capsule) at the end of each renal tubule (nephron) and is involved in the filtration of blood. The composition of the glomerular capillary wall determines what and how much is filtered into the glomerular capsule. The capillary wall consists of an endothelial layer with relatively large pores through which solutes, plasma proteins, and fluid can pass but blood cells cannot, a basement membrane layer fused to the endothelial layer that prevents the filtration of plasma proteins from the blood, and an epithelial layer consisting of podocytes that adhere to the basement membrane by foot processes. The fluid passes through the filtration slits formed by the podocytes. The thin diaphragm between the slits functions as the final filtration barrier before the fluid enters the glomerular space.

[0082] The terms "isolated," "purified," or "biologically pure" refer to a substance that contains varying degrees of the components that are normally associated with it in its natural state. "Isolating" indicates a certain degree of separation from its original source or environment. "Purifying" indicates a higher degree of separation than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials so that no impurities substantially affect the protein's biological properties or cause other adverse consequences. That is, a nucleic acid or peptide, as used herein, is purified when it is produced by recombinant DNA techniques and substantially free of cellular material, viral contaminants, or culture medium, or when chemically synthesized and substantially free of chemical precursors or other chemicals. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis, column chromatography, high performance liquid chromatography (HPLC), mass spectrometry, and the like. The term "purified" can mean that a nucleic acid or protein yields substantially one band in an electrophoretic gel. For example, for a protein that can undergo modifications such as phosphorylation or glycosylation, different modifications may give rise to different isolated proteins that can be purified separately.

[0083] "Isolated polynucleotide" means a nucleic acid (e.g., DNA) that does not contain the genes adjacent to its gene in the naturally occurring genome of the organism from which the nucleic acid molecule is derived. Thus, the term includes, for example, recombinant DNA incorporated into a vector such as an expression vector, recombinant DNA incorporated into an autonomously replicating plasmid or virus, or recombinant DNA incorporated into the genomic DNA of a prokaryote or eukaryote, or recombinant DNA present as a separate molecule independent of other sequences (e.g., cDNA or genomic or cDNA fragments produced by PCR or restriction endonuclease digestion). Further, the term includes RNA molecules transcribed from a DNA molecule, and recombinant DNA that is part of a hybrid gene encoding one or more additional polypeptide sequences.

[0084] "Isolated polypeptide" means a polypeptide or molecule of the present disclosure, such as an isolated anti-αvβ8 integrin antibody or an antigen-binding fragment thereof, that has been separated from the components with which it is naturally associated or from components present during isolation or purification. Such a polypeptide or molecule is substantially free of other elements present in its natural environment. For example, an isolated protein is substantially free of cellular material or other proteins from the cell or tissue source from which it is derived. Typically, a polypeptide or molecule is isolated when it contains at least 60% by weight less of the proteins and naturally occurring organic molecules to which it is naturally bound. Preferably, the preparation is at least 75% by weight, more preferably at least 90% by weight, most preferably at least 99% by weight, of the polypeptide of the present disclosure. The isolated polypeptides of the present disclosure can be obtained, for example, by extraction from natural sources, by expression of recombinant nucleic acids encoding such polypeptides, or by chemically synthesizing the protein. The term "isolated" also refers to a preparation in which the isolated protein is pure enough to be administered as a pharmaceutical composition, or at least 70-80% (w / w) pure, more preferably at least 80-90% (w / w) pure, even more preferably 90-95% pure; most preferably at least 95%, 96%, 97%, 98%, 99% or 100% (w / w) pure. Purity can be measured by any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, HPLC analysis, and / or mass spectrometry.

[0085] The term "dosage" refers to the measured amount, quantity, or concentration of a therapeutic agent, such as a small molecule or a biological, a drug, a pharmaceutical, a compound, etc., that is administered (without limiting the route of administration) to a subject or patient in need of the agent for, for example, a therapeutic or therapeutic benefit.

[0086] "Increase" means a positive change, for example, an increase of at least 10%, 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 1000%, or more.

[0087] "Decrease" means a negative change, for example, a decrease of at least 10%, 25%, 50%, 75%, or 100%.

[0088] "Reference" or "control" means a basis for comparison, such as a placebo, although not limited thereto. In one embodiment, the reference level is the level, expression, or activity of a biomarker in a biological sample obtained from non-diseased tissue.

[0089] "Reference sequence" is a defined sequence used as a basis for sequence comparison. The reference sequence may be, for example, a segment of a full-length cDNA or gene sequence, or a subset or entirety of a defined sequence such as a full cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence is generally at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acid molecules, the length of the reference nucleic acid sequence is generally at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, even more preferably about 100 nucleotides or about 300 nucleotides, or any integer before, after, or between them.

[0090] "Responsive" in relation to treatment means being susceptible to the effects of treatment.

[0091] "Specifically binds" or "selectively binds" refers to an agent (e.g., an antibody) that recognizes and binds to a molecule (e.g., a polypeptide, an antigen, a ligand), but substantially does not recognize or bind to other molecules in a sample such as a biological sample. For example, two molecules that specifically bind to each other (e.g., an antibody and its ligand) form a relatively stable complex under physiological conditions. Specific binding is characterized by high affinity and low to moderate capacity, and is usually distinguished from non-specific binding which has low affinity and moderate to high capacity.

[0092] "Biological sample" or "sample" means any liquid, cell or tissue obtained from a subject. In some embodiments, the biological sample is blood, serum, plasma, cerebrospinal fluid, bronchoalveolar lavage fluid, sputum, tears, saliva, urine, semen, feces, etc. A cell or tissue sample such as a kidney sample can be further processed in a suitable buffer to generate a homogenate or suspension in which the cells and intracellular components of the cells and tissues are provided.

[0093] "Subject" means a mammal including, but not limited to, a human such as a human patient, human subject, human individual, non-human primate, or a non-human mammal such as a bovine, equine, canine, ovine or feline. In one embodiment, the subject is a human. In one embodiment, the subject is a human patient having or at risk of having a renal condition or disease such as CKD and / or its symptoms, or being treated therefor. The terms "subject", "individual" and "patient" may be used interchangeably herein.

[0094] The ranges provided herein are to be understood as shorthand notations for all values within the range including the first and last recited values. For example, the range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0095] "Pharmaceutical composition" or "formulation" refers to a composition (physiologically acceptable composition) suitable for pharmaceutical use in a subject such as an animal or mammal including a human. The pharmaceutical composition includes a therapeutically or prophylactically effective amount of the anti-αvβ8 integrin antibody or antigen-binding fragment thereof described herein and a pharmaceutically acceptable excipient, carrier, vehicle, or diluent. In one embodiment, the pharmaceutical composition includes the active ingredient (anti-αvβ8 integrin antibody or antigen-binding portion or fragment thereof), the inert ingredients that make up the carrier, and any product directly or indirectly resulting from the combination, complexation, or aggregation of any two or more components, or from the separation of one or more of the components, or from any other various reactions or interactions of one or more of the components. In one embodiment, the pharmaceutical composition optionally includes another biologically active agent, compound, drug, or medicine. Thus, the pharmaceutical compositions of the present disclosure include any composition made by mixing an anti-αvβ8 integrin antibody or antigen-binding portion or fragment thereof with a pharmaceutically acceptable excipient, carrier, vehicle, or diluent.

[0096] "Pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, buffers, etc., such as phosphate buffered saline, optionally another biologically active agent, an aqueous (e.g., 5%) solution of dextrose, and an emulsion (e.g., oil / water or water / oil emulsion). Non-limiting examples of excipients include adjuvants, binders, fillers, diluents, disintegrants, emulsifiers, wetting agents, lubricants, glidants, sweeteners, flavorants, and colorants. Suitable pharmaceutical carriers, excipients, vehicles, and diluents can be found in Remington’s Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co., Easton, 1995 (or updated versions of this reference)). The pharmaceutical carrier suitable for inclusion in a composition or formulation typically depends on the intended method of administration of the active agent, e.g., an anti-αvβ8 integrin antibody or an antigen-binding portion or fragment thereof described herein. Exemplary methods of administration include enteral (e.g., oral) or parenteral (e.g., subcutaneous, intramuscular, intravenous, or intraperitoneal injection, intravenous infusion, or topical, transdermal, or transmucosal administration).

[0097] "Pharmaceutically acceptable salt" refers to a salt that can be formulated into a compound for pharmaceutical use, including, but not limited to, metal salts (e.g., sodium, potassium, magnesium, calcium, etc.), and salts of ammonia or organic phosphoric acids.

[0098] "Pharmaceutically acceptable", "physiologically acceptable", or "pharmacologically acceptable" refers to a substance that is not biologically, physiologically, or otherwise harmful, i.e., the substance can be administered to an individual without causing any undesirable biological effects or interacting in a harmful manner with any of the components of the composition in which it is contained or with any components present in the body or within the body of the individual.

[0099] "Physiological state" refers to the state within the body of an animal or mammal such as a human. Examples of physiological states include, but are not limited to, body temperature and the aqueous environment such as physiological ionic strength, pH, and enzymes. Physiological state also encompasses the state within the body of a particular subject that is different from the "normal" state that exists in most subjects, such as normal human body temperature (about 37°C) or normal human blood pH (about 7.4).

[0100] As used herein, the terms "treat", "treating", "treatment", etc. refer to reducing, alleviating, decreasing, mitigating, suppressing, neutralizing, or improving a disorder and / or associated symptoms. It will be understood that treating a disorder or condition does not necessarily require completely eliminating or alleviating the disorder, condition, or associated symptoms, although this is not excluded. "Treatment" may refer to prophylactic treatment, therapeutic treatment, or diagnostic treatment. In certain embodiments, "treatment" refers to administering a compound or composition to a subject for therapeutic, prophylactic, or diagnostic purposes.

[0101] Treatment or treating according to the described methods includes administering an anti-αvβ8 integrin antibody described herein. In one embodiment, the anti-αvβ8 integrin antibody is administered parenterally, e.g., intravenously or subcutaneously, to a subject in need thereof. As will be understood by those skilled in the art, intravenous administration generally refers to providing or delivering an active ingredient, therapeutic agent, substance, agent, drug, or antibody, such as an anti-αvβ8 integrin antibody, to a vein or blood vessel of a subject and delivering the active ingredient into the subject's systemic circulation. Intravenous administration can include, for example, intravenous injection or infusion into a vein or blood vessel by means of a syringe and needle or catheter. Intravenous injection or infusion may involve the use of a plastic tube and infusion bag (e.g., an infusion set) such that the active ingredient is delivered into the infusion bag through a tube and then into the subject through a catheter and / or port placed within the subject's body at a flow rate determined conventionally and practically by a physician. Intravenous injection or infusion may be performed by the use of a pump or via a drip.

[0102] "Preventive treatment" (such as preventive or protective treatment) is a treatment administered to a subject who does not exhibit symptoms of a disease, or exhibits only initial symptoms of a disease, or is at risk of having a disease, for the purpose of reducing, decreasing, alleviating, or eliminating the risk of developing a disease, lesion, or condition, or the risk of developing a more severe or critical form of a disease, lesion, or condition. It is contemplated that the anti-αvβ8 integrin antibodies or antigen-binding fragments thereof, or compositions thereof, described herein can be administered as a preventive or protective treatment to reduce the likelihood that a subject will develop a renal disease, lesion, or condition, or to minimize the severity of a renal disease, lesion, or condition if it develops in the subject.

[0103] "Therapeutic" treatment is a treatment administered to a subject who exhibits symptoms or signs of a disease or lesion for the purpose of reducing, alleviating, or eliminating the symptoms or signs. The symptoms or signs of a disease or lesion may be, but are not limited to, biochemical, behavioral, cellular, phenotypic, genotypic, histological, functional, physical, subjective, or objective. In one embodiment, the anti-αvβ8 integrin antibodies of the present disclosure can be administered as a therapeutic treatment.

[0104] As used herein, a therapeutic agent that "prevents" a disorder or condition is a biological agent, compound, or pharmaceutical substance that reduces the occurrence of a disorder or condition in a treated sample compared to an untreated control or reference sample, or delays the onset or reduces the severity of one or more symptoms of a disorder or condition compared to an untreated reference or control sample, in a statistical sample. In one embodiment, the anti-αvβ8 integrin antibodies of the present disclosure are preventive therapeutic agents in the methods described herein.

[0105] The term "effective amount" refers to an amount of administration sufficient to produce a desired result (e.g., reduction, alleviation, elimination, or improvement of symptoms) in relation to the health condition, lesion, or disease of interest, or for diagnostic purposes. The desired result may include subjective or objective improvement in the subject to whom the dose or dosage is administered. "Therapeutically effective amount" refers to the amount of a drug effective to produce the intended beneficial effect on health. It will be understood that the specific dosage level and frequency for a particular patient may depend on a variety of factors including the activity of the specific compound used; the bioavailability, metabolic stability, excretion rate, and duration of action of that compound; the method and time of administration of that compound; the age, weight, general health, sex, and diet of the patient; and the severity of the particular medical condition of the patient.

[0106] The terms "protein," "peptide," and "polypeptide" refer to amino acid chains regardless of length or post-translational modifications (e.g., glycosylation or phosphorylation). Thus, these terms may be used interchangeably herein to refer to polymers of amino acid residues. These terms also apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding natural amino acids. Thus, the term "polypeptide" includes full-length naturally occurring proteins; as well as recombinant or synthetically produced polypeptides corresponding to full-length naturally occurring proteins or to specific domains or portions of naturally occurring proteins. This term also includes mature proteins to which an amino-terminal methionine has been added to facilitate expression in prokaryotic cells. Polypeptides can be synthesized chemically, can be synthesized by recombinant DNA methods, or can be purified from naturally expressing tissue according to standard biochemical purification methods. A "functional polypeptide" has one or more biological functions or activities of a given protein or polypeptide, such as, for example, an anti-αvβ8 integrin antibody. A functional polypeptide may contain a primary amino acid sequence that has been modified from what is considered to be the standard sequence of an anti-αvβ8 integrin antibody. Preferably, such modifications are conservative amino acid substitutions that do not change the normal function or activity of the protein, nor cause a substantial change. A polypeptide fragment, portion, or segment refers to a continuous stretch of amino acid residues of at least about 6 contiguous amino acids from a particular sequence, more typically at least about 10-12 contiguous amino acids.

[0107] Nucleic acid molecules (polynucleotides) encoding polypeptides such as the anti-αvβ8 integrin antibodies of the present disclosure include nucleic acid molecules encoding the polypeptides of the present disclosure, for example, nucleic acid molecules encoding anti-αvβ8 integrin antibodies or antigen-binding fragments thereof. Such nucleic acid molecules need not be 100% identical to the endogenous nucleic acid sequence, but will typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence will typically be able to hybridize to at least one strand of a double-stranded nucleic acid molecule. A polynucleotide having "substantial identity" to an endogenous sequence will typically be able to hybridize to at least one strand of a double-stranded nucleic acid molecule. "Hybridize" means to pair between complementary polynucleotide sequences (e.g., genes) or portions thereof under various stringency conditions to form a double-stranded molecule. (See, e.g., Wahl, G.M. and S.L.Berger, 1987, Methods Enzymol., 152:399; Kimmel, A.R., 1987, Methods Enzymol., 152:507).

[0108] As a non-limiting example, stringent salt concentrations are typically less than about 750 mM NaCl and less than about 75 mM trisodium citrate, preferably less than about 500 mM NaCl and less than about 50 mM trisodium citrate, more preferably less than about 250 mM NaCl and less than about 25 mM trisodium citrate. Low stringency hybridization can be obtained, for example, in the absence of organic solvents such as formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, more preferably at least about 50% formamide. Stringent temperature conditions typically include a temperature of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Various additional parameters such as hybridization time, the concentration of detergents such as sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA are well known to those of skill in the art. Various levels of stringency are achieved by combining these various conditions as needed. In certain embodiments, hybridization is performed at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another specific embodiment, hybridization is performed at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA. In another specific embodiment, hybridization is performed at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml salmon sperm DNA. Useful variations of these conditions will be readily apparent to those of skill in the art.

[0109] In most applications, the washing step following hybridization also varies by stringency. Washing stringency conditions can be defined by salt concentration and temperature. As noted above, washing stringency can be increased by decreasing the salt concentration or increasing the temperature. For example, a stringent salt concentration for a washing step is less than about 30 mM NaCl and less than about 3 mM trisodium citrate, particularly less than about 15 mM NaCl and less than about 1.5 mM trisodium citrate. Stringent temperature conditions for a washing step typically include a temperature of at least about 25°C, or at least about 42°C, or at least about 68°C. In certain embodiments, the washing step is performed at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In another specific embodiment, the washing step is performed at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In another specific embodiment, the washing step is performed at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations of these conditions will be readily apparent to those of skill in the art. Hybridization techniques are well known to those of skill in the art and are described, for example, in Benton and Davis (Science, 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA, 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0110] The terms "identical" or "identity" percent in the context of two or more nucleic acids or polypeptides refer to two or more sequences or subsequences that are identical or have a defined percentage of identical nucleotide or amino acid residues when compared and aligned for maximum correspondence (introducing gaps if necessary) without considering conservative amino acid substitutions as part of sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain an alignment of amino acid or nucleotide sequences are known in the art (see, for example, Karlin et al., 1990, Proc. Natl. Acad. Sci., 87:2264-2268 (modified in Karlin et al., 1993, Proc. Natl. Acad. Sci., 90:5873-5877 and incorporated into the NBLAST and XBLAST programs (Altschul et al., 1991, Nucleic Acids Res., 25:3389-3402))). In certain embodiments, Gapped BLAST can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. BLAST-2, WU-BLAST-2 (Altschul et al., 1996, Methods in Enzymology, 266:460-480), ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or Megalign (DNASTAR).

[0111] "Substantially identical" means a polypeptide or nucleic acid molecule that exhibits at least 50% identity with a reference amino acid sequence or nucleic acid sequence. Such sequences can have at least 60%, or at least 80% or 85%, or at least 90%, 95%, or 99% identity at the amino acid level or nucleic acid with the sequence used for comparison.

[0112] Sequence identity is typically measured using sequence analysis software (e.g., the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or the PILEUP / PRETTYBOX programs). Such software aligns identical or similar sequences by assigning a degree of homology for various substitutions, deletions, and / or other changes. Conservative substitutions typically include substitutions within the following amino acid groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method of determining the degree of identity, the BLAST program can be used, and the probability score between e -3 ~e -100 suggests closely related sequences.

[0113] Mammalian anti-αvβ8 integrin antibodies (particularly, anti-human αvβ8 integrin antibodies), or immunologically functional allelic variants or isoforms thereof, can be useful in the described methods, as can other variants or isoforms, including antibody fragments, having the binding and blocking activities of anti-αvβ8 integrin antibodies. An "allelic variation" in the context of a polynucleotide or gene is an alternative form (allele) of a gene that exists in two or more forms in a population. At the polypeptide level, an "allelic variant" generally differs from another by only one or, at most, a few amino acid substitutions. A "species variation" of a polynucleotide or polypeptide is one that naturally exists with diversity between different species.

[0114] In this disclosure, terms such as "comprises," "comprising," "contains," and "has" may have the meanings ascribed to them in the United States Patent Law, may mean "includes," "including," etc., and "consisting essentially of" or "consists essentially" likewise have the meanings ascribed to them in the United States Patent Law. The terms are open-ended and allow for the presence of elements beyond those recited, provided that the basic or novel characteristics of the recited elements are not changed by the presence of elements beyond those recited, except for prior art embodiments.

[0115] Unless otherwise specified or apparent from the context, the term "or" as used herein is understood to be inclusive when used in this specification. Unless otherwise specified or apparent from the context, the terms "a," "an," and "the" as used herein are understood to be singular or plural when used in this specification.

[0116] Unless otherwise specified or apparent from the context, the term "about" as used herein is understood to be within the normal tolerance in the art, e.g., within two standard deviations of the mean value. The term "about" is understood to refer to within 5%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the recited value. Unless clear from the context, all numerical values provided herein are modified by the term about.

[0117] Any of the compositions or methods provided herein may be combined with one or more of any of the other compositions and methods provided herein.

Brief Description of the Drawings

[0118]

Fig. 1A-1B

Fig. 2A-2D

Fig. 3A-3B

Fig. 4

Fig. 5

Fig. 6

Chem.

Chem.

Fig. 7A-7C

Fig. 8A-8E

Fig. 9A-9J

Fig. 10A-10B

Fig. 11

Mode for Carrying Out the Invention

[0119] The present disclosure generally features antibodies, compositions, and methods for treating kidney diseases, such as diabetic nephropathy (DN), chronic kidney disease (CKD), acute kidney disease, hypertension-related kidney disease, hyperglycemia-related kidney disease, kidney fibrosis, inflammation-related kidney disease, end-stage renal disease (ESRD), autoimmune-related kidney fibrosis (e.g., lupus nephritis), and fibrosis after kidney transplantation, in an individual in need thereof, as described herein. In particular, the antibodies, compositions, and methods are directed to treating kidney fibrosis associated with kidney diseases such as CKD.

[0120] The present disclosure is directed to a method of treatment for ameliorating, reducing, inhibiting, decreasing, or alleviating fibrosis of renal tissue in a subject having a renal disease such as CKD. Generally, fibrosis refers to the formation of excessive fibrous connective tissue (scar tissue) in organs such as the kidney, which causes thickening and scarring of the renal connective tissue. As described above, the method involves administration of an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof that specifically binds to αvβ8 integrin, which has been found to be highly expressed in diseased renal cells and renal tissue, particularly renal epithelial cells and renal epithelial tissue of subjects having a renal disease such as CKD. The anti-αvβ8 integrin antibody or an antigen-binding fragment thereof selectively binds to αvβ8 integrin on fibrotic renal cells and renal tissue, thereby blocking, neutralizing, or inhibiting the interaction of latent TGF-β (LAP-TGF-β) on the renal cell surface with renal-expressed αvβ8 integrin. Anti-αvβ8 integrin antibody binding interferes with the αvβ8 integrin / LAP TGF-β interaction, which in turn blocks or prevents activation of TGF-β at the renal cell surface, such that active TGF-β is not produced and thus cannot exert its cellular effects associated with renal fibrosis in renal tissue of a subject such as a human or non-human subject. The method provides a therapeutic benefit, particularly in the treatment of renal diseases, by reducing, decreasing, inhibiting, or lessening the detrimental fibrosis induced by active TGF-β in, for example, renal diseases such as CKD.

[0121] Although not wishing to be bound by a particular theory, an anti-αvβ8 integrin antibody or antigen-binding fragment thereof reduces the local activation of TGF-β in renal cells and renal tissue where αvβ8 integrin is highly expressed, for example, by directly binding to the αvβ8 integrin receptor of LAP TGF-β. Binding of an anti-αvβ8 integrin antibody to αvβ8 integrin, which blocks TGF-β activation from its latent form, may also reduce or prevent the recruitment of proteases that cleave latent TGF-β and release mature active TGF-β peptides. This can occur by the anti-αvβ8 integrin antibody inhibiting the binding of αvβ8 integrin on the renal cell surface to latent TGF-β associated with the extracellular matrix, thereby inhibiting subsequent activation of TGF-β as described below.

[0122] Transforming growth factor beta (β), (TGF-β) and its interaction with αvβ8 integrin In cells, the TGF-β cytokine is synthesized and secreted into the extracellular matrix as an inactive precursor that complexes with the "latency-associated peptide (LAP)" and the "latent TGFβ-binding protein (LTBP)". The latent form of TGF-β must be activated to bind to its receptor, such as αvβ8 integrin, and must have biological function (J.J. Worthington et al., 2011a, Trends Biochem. Sci., 36, 47-54). LAP is cleaved from active TGF-β but remains non-covalently attached in a structure that prevents TGF-β from binding to its receptor. TGF-β activators include various proteases and cell surface molecules that alter the latent complex to allow active TGF-β to bind to its receptor. Putative TGF-β activators include, but are not limited to, proteases that degrade LAP, thrombospondin 1, reactive oxygen species (ROS), and integrins. Therefore, activation of the latent complex is essential for the regulation of TGF-β function, and TGF-β activators are the rate-limiting step in the conversion of latent TGF-β to active TGF-β. As an example, in human CKD kidneys (n = 4), the amount of latent TGF-β is more than 53-fold that of active TGF-β.

[0123] Fibrosis is an important driver of the progression of chronic kidney disease (CKD) in human patients and correlates with renal dysfunction and kidney damage. TGF-β is involved in the development of renal fibrosis in CKD. Renal TGF-β is upregulated in human fibrotic CKD compared to control kidneys (D.S. Goumenos et al., 2002, Nephrol. Dial. Transplant., 17:2145-2152). Urinary TGF-β has been shown to correlate with kidney damage (albuminuria) in type 2 diabetes. (Marwood et al., 2002, Exp. Biol. Med., 227(11):943-956).

[0124] The αv-integrin transmembrane receptor, e.g., αvβ8, plays an important role in the regulation of extracellular matrix physiology and the activation of TGF-β. Briefly, αv-integrin mediates the activation of latent TGF-β. In particular, αvβ8 binds to the RGD (arginine-glycine-aspartic acid) motif of the TGF-β binding latency-associated peptide (LAP), thereby regulating the levels of free and active TGF-β in tissues (Mu, D. et al., 2002, J. Cell Biol., 157(3):493-507; Araya, J., 2006, Am. J. Pathol., 169(2):405-415).

[0125] Unlike the activities of other αvβ integrins, αvβ8 integrin is constitutively active and mediates the activation of LAP TGF-β (by the release of the active TGF-β cytokine after the binding of LAP TGF-β to αvβ8 integrin) by cleavage with MMP-14 protease rather than by immobilization to cytoplasmic actin (without traction effect). αvβ8 integrin expression is abundant in renal tissue, and the gene encoding αvβ8 integrin is highly expressed in renal tissue compared to other tissues such as, for example, pancreas, liver, gallbladder, salivary gland, esophagus, stomach, intestine, lung, heart, or bladder as exemplified below.

[0126] As described herein, both in vitro and in vivo studies have shown that high levels of expression of αvβ8 integrin in renal epithelial cells directly correlate with high levels of renal tissue fibrosis resulting from the activation of TGF-β. High levels of TGF-β activity induce and increase damage to renal cells and renal tissue, cause fibrosis, and thus increase the severity of renal diseases such as CKD. The anti-αvβ8 integrin antibodies described herein specifically bind to αvβ8 integrin expressed on renal cells, block and / or reduce the binding of αvβ8 integrin to latent TGF-β (LAP TGF-β), and inhibit the release of the active form of TGF-β, thereby inhibiting the destructive activity of TGF-β and the resulting fibrosis in renal cells and renal tissue. The action of this specific anti-αvβ8 integrin antibody serves as a treatment for the damage to renal cells and renal tissue caused by TGF-β activity, for example, by inhibiting the intracellular signaling cascade of TGF-β. According to the methods disclosed and exemplified herein, by providing an antibody that specifically binds to αvβ8 integrin in the kidney and blocking αvβ8 integrin activity, the activation of TGF-β localized in the kidney is greatly reduced, thereby specifically reducing the damage to renal cells and renal tissue, i.e., renal fibrosis, in the affected kidney.

[0127] The use of an anti-αvβ8 integrin antibody that specifically targets the αvβ8 integrin receptor on renal cells, as described herein, arises from the finding that αvβ8 integrin is preferentially expressed in the kidneys of normal subjects and that the expression of αvβ8 integrin is significantly increased and localized in renal epithelial cells (e.g., podocytes and interstitial tubules) of the kidneys of subjects with fibrotic kidney diseases, such as human patients with CKD. As noted above, the present disclosure provides the surprising finding that the αvβ8 protein is highly upregulated in the kidneys of human patients with CKD. Furthermore, the activation of TGF-β by the specific binding of αvβ8 integrin to the latent active form of TGF-β in the kidney is the direct cause of destructive fibrosis in renal tissue. These findings are in contrast to the prior art finding that transgenic animals that have αvβ8 integrin mainly present in renal mesangial cells but do not express mesangial cell αvβ8 integrin possess active TGF-β that causes endothelial cell apoptosis (S. Khan et al., 2011, Am. J. Pathology, 178(2):609 - 620). In contrast, as described and exemplified herein, the method involves inhibiting and blocking the interaction of αvβ8 integrin with LAP-TGF-β and binding to LAP-TGF-β such that active TGF-β is not released at the renal cell membrane and cannot cause fibrosis (and / or further damage) in renal cells and renal tissue of subjects suffering from renal diseases such as CKD.

[0128] Specific antibody against αvβ8 integrin The present disclosure encompasses the development and use of antibodies that target, specifically bind to, and bind to αvβ8 integrin, particularly αvβ8 integrin expressed in renal cells and renal tissue, as well as fibrotic renal cells and renal tissue. These antibodies or antigen-binding fragments thereof are highly beneficial in methods of treating renal fibrosis in renal diseases of a subject in need thereof, particularly renal diseases such as chronic kidney disease (CKD). In embodiments, the subject may be associated with injury or damage to renal cells and renal tissue and have a condition that causes fibrosis of renal tissue as described herein, or an acute, chronic, or end-stage renal disease. Treatments of subjects having renal fibrosis and renal diseases associated with fibrosis, regardless of the etiology, using the antibodies, compositions, and methods described herein provide significant medical and clinical benefits to subjects in need thereof, particularly patients suffering from renal diseases such as CKD or DN. In one embodiment, the anti-αvβ8 integrin antibody is a humanized antibody.

[0129] In one embodiment, the anti-αvβ8 integrin antibody is a humanized antibody called the "MEDI-hu37E1B5" antibody described above that specifically targets and binds to human αvβ8 integrin. In certain embodiments, the MEDI-hu37E1B5 antibody specifically targets and binds to human αvβ8 integrin that is expressed in the kidney and highly expressed in fibrotic kidneys. In one embodiment, the MEDI-hu37E1B5 antibody does not cross-react with antibodies to other integrins.

[0130] In another embodiment, the anti-αvβ8 integrin antibody is a humanized and affinity-optimized antibody called the above-mentioned "B5-15" anti-αvβ8 integrin antibody, which specifically targets human αvβ8 integrin and exhibits high-affinity binding to human αvβ8 integrin. The optimized B5-15 antibody is of the IgG1 subtype, specifically and selectively binds to human αvβ8 integrin, and exhibits functional activity by blocking or inhibiting the binding interaction or association between human αvβ8 integrin and latent TGF-β, and thus blocks or inhibits the activation of TGF-β by the release of active TGF-β from its latent form. As shown herein (Figure 4), B5-15 has a good profile with respect to binding to αvβ8 integrin as compared to the CDR-grafted MEDI-hu37E1B5 anti-αvβ8 integrin antibody described in Example 1.

[0131] The B5-15 antibody blocks the binding of αvβ8 integrin to LAP-TGF-β, blocks the activation of TGF-β and intracellular signaling by TGF-β, and as a result, protects renal cells and renal tissue from the damaging effects of active TGF-β peptides that can induce and exacerbate fibrosis. Without wishing to be bound by theory, the B5-15 antibody allosterically modifies αvβ8 integrin, reducing its affinity for the latent TGF-β (LAP) binding domain, thereby preventing the activation of TGF-β from its latent form and preventing the release of active TGF-β peptides. Thus, the antibody induces a structural change in αvβ8 integrin, such that αvβ8 can no longer bind to latent TGF-β and cannot promote its activation (WO 2015 / 195835 pamphlet). Until the present disclosure, the binding characteristics and functional activity of anti-αvβ8 integrin antibodies, such as the B5-15 antibody, in renal fibrosis were unknown.

[0132] In an embodiment, the anti-αvβ8 integrin antibodies disclosed herein specifically bind to αvβ8 integrin receptors that are upregulated in diseased, damaged, and / or fibrotic kidney tissue, such as that found in individuals with kidney disease, e.g., CKD or DN, in kidney cells and kidney tissue. Compositions comprising these antibodies, and their use in methods of treating kidney disease and nephropathy, particularly kidney disease associated with fibrosis, are encompassed by the present disclosure. The antibodies described bind only to human αvβ8 integrin and do not cross-react with other integrins.

[0133] The antibodies described also selectively target and specifically bind to the αvβ8 integrin receptor of latent TGF-β, without directly targeting the cytokine itself, and are thus advantageous in providing a safer therapy for treating kidney disease, particularly kidney disease associated with fibrosis. Furthermore, inhibition, blockade, or neutralization of the activity of the TGF-β1 isoform is particularly advantageous since this TGF-β isoform is generally thought to account for the majority of the disease-related activity of TGF-β. The presence of the TGF-β1 isoform in the kidney is likely to result in the involvement of the active form of TGF-β1 in kidney fibrosis and kidney disease.

[0134] Directly targeting TGF-β can be one way to inhibit or prevent lesions caused by TGF-β activity, but the general neutralization and / or chronic inhibition of TGF-β action resulting from directly targeting cytokines can have significant side effects in the treated individual, considering the involvement of TGF-β in the regulation of diverse cellular functions and pathways. Accordingly, methods of using the anti-αvβ8 integrin antibodies provided herein to block, inhibit, neutralize, and thus effectively prevent αvβ8 integrin / LAP TGF-β interactions in renal cells and renal tissue, without impairing in vivo TGF-β activation in other cells, tissues, and organs, or for other physiological purposes, provide useful therapeutic tools and methods for treating renal diseases such as CKD or DN and fibrosis. Advantageously, the specificity of the anti-αvβ8 antibodies described herein for renal cells and renal tissue expressing high levels of αvβ8 integrin reduces adverse effects such as autoimmune responses, rapidly developing atherosclerosis, and the development of cancer. Adverse effects are seen with pan-TGF-β inhibition, and thus specifically targeting αvβ8 integrin, which affects TGF-β activation, is likely to reduce adverse events.

[0135] As another advantage, the anti-αvβ8 integrin antibodies described herein do not cross the blood-brain barrier (BBB) and thus cannot bind to αvβ8 integrin expressed on brain cells and tissues.

[0136] The anti-αvβ8 antibodies described herein specifically block the binding of TGF-β to latent form of αvβ8 integrin expressed on renal epithelial cells, and thus block fibrosis caused by the release of active TGF-β in renal tissue, which plays a central role in glomerular and tubulointerstitial lesions of kidney diseases that induce changes in glomerular filtration barrier, glomerulosclerosis and fibrosis, as well as tubular degeneration leading to persistent renal dysfunction. Therefore, the present method does not target TGF-β itself, but specifically binds to a target receptor, namely αvβ8 integrin, which is highly expressed on the surface of damaged kidneys and / or renal cells of individuals with kidney diseases, thereby treating kidney diseases, such as chronic kidney diseases or diabetic nephropathy characterized by harmful renal tissue fibrosis, including the use of specific anti-αvβ8 integrin antibodies. Targeting and binding of αvβ8 integrin by the specific anti-αvβ8 integrin antibodies provided herein inactivates TGF-β cytokine activity, a major cause of renal tissue fibrosis and further damage to renal tissue in kidney diseases, and effectively prevents it.

[0137] The anti-αvβ8 integrin antibodies described herein specifically bind to one or more regions of the αvβ8 integrin receptor protein containing the antigen-binding site or epitope. In one embodiment, the epitope of αvβ8 integrin bound by anti-αvβ8 integrin antibodies such as MEDI-hu37E1B5 antibody or B5-15 antibody was mapped to a region approximately 28 Å (angstrom) away from the αvβ8 integrin and the LAP-TGF-β binding site. (S. Minagawa et al., 2014, Sci. Transl. Med., 6(241):241re79. Doi:10.1126:scitranslmed.3008074).

[0138] In one embodiment, for binding to αvβ8 integrin, as described herein, the following three light chain CDRs: V L CDR1: KASQDINSYLS (SEQ ID NO: 4), V L CDR2: YANRLVD (SEQ ID NO: 5), and V LA light chain variable region comprising CDR3: LQYDEFPYT (SEQ ID NO: 6), and the following three heavy chain CDRs: V H CDR1: RYWMS (SEQ ID NO: 1), V H CDR2: EINPDSSTINYTSSL (SEQ ID NO: 2), and V H An antibody that competes with an antibody having a heavy chain variable region comprising CDR3: LITTEDY (SEQ ID NO: 3) is contemplated. The antibody can be monoclonal, chimeric, humanized, etc., and can be of isotype IgG1, IgG2, IgG2a, IgG3 or IgG4. In certain embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is a human, humanized or chimeric antibody.

[0139] In another embodiment, for binding to αvβ8 integrin, as described herein, the following three light chain CDRs: V L CDR1: KASQDINKYLS (SEQ ID NO: 10), V L CDR2: YANRLVD (SEQ ID NO: 5), and V L A light chain variable region comprising CDR3: LQYDVFPYT (SEQ ID NO: 11), and the following three heavy chain CDRs: V H CDR1: RSWIS (SEQ ID NO: 9), V H CDR2: EINPDSSTINYTSSL (SEQ ID NO: 2), and V H An antibody that competes with an antibody having a heavy chain variable region comprising CDR3: LITTEDY (SEQ ID NO: 3) is contemplated. The antibody can be monoclonal, chimeric, humanized, etc., and can be of isotype IgG1, IgG2, IgG2a, IgG3 or IgG4. In certain embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is a human, humanized or chimeric antibody.

[0140] Also provided are an isolated polynucleotide encoding the described anti-αvβ8 integrin antibody or antigen-binding fragment thereof, a prokaryotic, eukaryotic, or mammalian vector suitable for encoding and expressing the described anti-αvβ8 integrin antibody or antigen-binding fragment thereof, and a host cell (prokaryotic, eukaryotic, or mammalian).

[0141] In other aspects, antibodies useful in the described methods and compositions include immunoglobulins, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two different αvβ8 integrin epitope-binding fragments, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, single-chain Fv (scFv), single-chain antibodies, single-domain antibodies, domain antibodies, Fab fragments, F(ab’)2 fragments, antibody fragments that exhibit a desired biological activity (e.g., antigen-binding portions), disulfide-bonded Fv (dsFv), intracellularly expressed antibodies, and antigen or epitope-binding fragments of any of the foregoing. In particular, suitable antibodies include immunoglobulin molecules and immunologically and functionally active fragments of immunoglobulin molecules such as molecules containing at least one antigen-binding site.

[0142] Anti-αvβ8 integrin antibodies include monoclonal human, humanized or chimeric anti-αvβ8 antibodies. The anti-αvβ8 antibodies used in the compositions and methods described herein can be naked antibodies, immune complexes or fusion proteins. In certain embodiments, the anti-αvβ8 antibody is a human, humanized or chimeric antibody of the IgG isotype, particularly the IgG1, IgG2, IgG3 or IgG4 human isotype, or any IgG1, IgG2, IgG3 or IgG4 allele found in the human population. Antibodies of the human IgG class have advantageous functional properties such as a long half-life in serum and the ability to mediate various effector functions (Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc., Chapter 1 (1995)). Human IgG class antibodies are further classified into the following subclasses: IgG1, IgG2, IgG3 and IgG4. In one embodiment, the anti-αvβ8 integrin antibody is of the human IgG1 subclass or isotype. The human IgG1 subclass has high ADCC activity and CDC activity in humans (Clark, Chemical Immunology, 65, 88 (1997)). In one embodiment, the anti-αvβ8 integrin antibody is a humanized antibody containing human framework regions and CDRs from a parental antibody such as the MEDI-hu37E1B5 antibody. In another embodiment, the anti-αvβ8 integrin antibody contains an amino acid sequence optimized to improve one or more antibody properties including specificity, function, stability, half-life / lifetime to the antigen, etc.

[0143] Therapeutic methods including administration of anti-αvβ8 integrin antibodies The methods described provide treatment for kidney diseases, particularly fibrotic kidney diseases, particularly chronic kidney disease (CKD) in which kidney function declines over a period of time with normal kidney function (estimated glomerular filtration rate (GFR) ≥ 90 mL / min / 1.73 m 2Stage 1, characterized by kidney damage with proteinuria (≥ 3 months) and persistence (≥ 3 months); regardless of the presence or absence of persistent (≥ 3 months) proteinuria, mild kidney function loss (estimated GFR 60 - 89 mL / min / 1.73m 2 Stage 2, characterized by kidney damage with mild to moderate kidney function loss (estimated GFR 30 - 59 mL / min / 1.73m 2 Stage 3, characterized by mild to severe kidney function loss (estimated GFR 30 - 59 mL / min / 1.73m 2 Stage 4, characterized by severe kidney function loss (estimated GFR 15 - 29 mL / min / 1.73m 2 Stage 5, characterized by kidney failure requiring dialysis or transplantation for survival. CKD at stage 5 is also known as ESRD (estimated GFR < 15 mL / min / 1.73m

[0144] The methods described herein, including administration of an anti-αvβ8 integrin antibody or antigen-binding fragment thereof, are also useful for the treatment of kidney diseases and / or renal fibrosis associated with damage or injury to renal cells and renal tissue caused by, for example, diabetic nephropathy (DN), chronic kidney disease (CKD), acute kidney disease, hypertension-related kidney disease, hyperglycemia-related kidney disease, renal fibrosis, inflammation-related kidney disease, end-stage renal disease (ESRD), autoimmune-related renal fibrosis (e.g., lupus nephritis), and fibrosis after kidney transplantation. General and local tissue inflammation in the kidney contributes to the pathophysiology and progression of diabetic nephropathy. The pathologies of hyperglycemia and hypertension typically associated with diabetic nephropathy can further lead to glomerular hypertension, mechanical stress on renal cells and renal tissue, podocyte injury and detachment, glomerular inflammation, and renal tubular inflammation, all of which result in fibrosis (scarring) in the kidney, particularly in the renal glomeruli and tubules.

[0145] Combination therapy In another embodiment, one or more of the anti-αvβ8 integrin antibodies may be administered in combination with another drug, medicine, or therapeutic agent or compound such as would be provided to a patient having a renal disease or CKD. As is often seen, individuals having a renal disease or CKD also have hypertension. Medicines and drugs that lower blood pressure serve to maintain blood pressure within a target range and help delay or halt further kidney damage. Common blood pressure medications include, but are not limited to, acetylcholinesterase (ACE) inhibitors, angiotensin II receptor blockers (ARBs), beta blockers, calcium channel blockers, direct renin inhibitors, diuretics, and vasodilators. Medicines and drugs administered to treat symptoms and complications of CKD include, but are not limited to, erythropoietin (EPO), (recombinant human erythropoietin, rhEPO), electrolyte imbalance correctives, diuretics, ACE inhibitors and ARBs, and iron therapy and vitamin D.

[0146] In combination therapies, one or more anti-αvβ8 integrin antibodies may optionally be included in the same pharmaceutical composition as other drugs or medicines. Alternatively, the anti-αvβ8 integrin antibody may be a separate pharmaceutical composition and may be administered simultaneously or at a different time point from one or more other drugs or medicines. The anti-αvβ8 integrin antibodies described herein, or pharmaceutical compositions containing the anti-αvβ8 integrin antibodies, are suitable for administration before, simultaneously with, or after the administration of other drugs or medicines, or pharmaceutical compositions containing such drugs or medicines. In certain examples, the administration of one or more anti-αvβ8 integrin antibodies to a subject overlaps with the administration time of another or companion drug or medicine provided separately or in a separate composition.

[0147] Pharmaceutical Compositions and Formulations The present disclosure encompasses the use of pharmaceutical compositions and formulations comprising one or more of the described anti-αvβ8 integrin antibodies, as well as one or more pharmaceutically acceptable excipients, carriers, and / or diluents. In certain embodiments, the composition may include one or more other biologically active agents (e.g., protease inhibitors).

[0148] Non-limiting examples of excipients, carriers, and diluents include vehicles, liquids, buffers, tonicity agents, additives, stabilizers, preservatives, solubilizers, surfactants, emulsifiers, wetting agents, adjuvants, and the like. The composition can contain liquids (e.g., water, ethanol); dilutions of various buffer components (e.g., Tris HCl, phosphate, acetate buffer, citrate buffer), pH and ionic strength; surfactants and solubilizers (e.g., polysorbate 20, polysorbate 80); antioxidants (e.g., methionine, ascorbic acid, sodium metabisulfite); preservatives (e.g., Thimerosol, benzyl alcohol, m-cresol); and bulking substances (e.g., lactose, mannitol, sucrose). The use of excipients, diluents, and carriers in the formulation of pharmaceutical compositions is known in the art. See, e.g., Remington’s Pharmaceutical Sciences, 18th Edition, pages 1435-1712, Mack Publishing Co. (Easton, Pennsylvania (1990)). This reference is hereby incorporated by reference in its entirety into this specification.

[0149] As non-limiting examples, carriers can include diluents, vehicles, and adjuvants, as well as implant carriers, and inert and non-toxic solid or liquid fillers and encapsulating materials that do not react with the active ingredient. Non-limiting examples of carriers include phosphate buffered saline, physiological saline, water, and emulsions (e.g., oil / water emulsions). The carrier can be a solvent or dispersion medium that contains, for example, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, and mixtures thereof.

[0150] Formulations comprising one or more anti-αvβ8 integrin antibodies for parenteral administration can be prepared, for example, as liquid solutions or suspensions, as solid forms suitable for solubilization or suspension in a liquid medium prior to injection, or as emulsions. Sterile injectable solutions and suspensions can be formulated according to techniques known in the art using suitable diluents, carriers, solvents (e.g., buffered aqueous solutions, Ringer's solution, isotonic sodium chloride solution), dispersing agents, wetting agents, emulsifying agents, suspending agents, etc. Sterile non-volatile oils, fatty esters, polyols and / or other inert components can also be used. Furthermore, formulations for parenteral administration can include aqueous sterile injectable solutions, which can contain antioxidants, buffers, bacteriostatic agents, solutes that render the formulation isotonic with the blood of the intended subject, and aqueous and non-aqueous sterile suspensions that can contain suspending agents and thickening agents. Injectable solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0151] Embodiments include sterile pharmaceutical formulations of anti-αvβ8 integrin antibodies useful as therapeutic agents for kidney diseases. Such formulations inhibit the binding of ligands to αVβ8 integrin, and thereby will effectively treat, for example, conditions in which tissue αVβ8 is abnormally increased. The anti-αvβ8 integrin antibody can have an appropriate affinity to potently inhibit αVβ8 integrin activity and can have an appropriate duration of action that allows for infrequent administration in humans. The prolonged duration of action allows for an infrequent and more convenient dosing schedule by alternative parenteral routes such as subcutaneous or intramuscular injection.

[0152] Sterile formulations can be made, for example, by filtration through a sterile filtration membrane before or after lyophilization and reconstitution of the antibody. The antibody is usually stored in lyophilized form or in solution. Therapeutic antibody compositions are generally placed in containers having a sterile access port, e.g., an intravenous solution bag or vial having an adapter that allows for retrieval of the formulation, such as a stopper pierceable by a subcutaneous injection needle.

[0153] For therapeutic use, for example, in the treatment of kidney diseases, particularly kidney fibrosis, the anti-αvβ8 integrin antibody or antigen-binding fragment thereof can be administered at a dosage according to the patient's needs, the patient's physical health and characteristics, and the severity of the condition being treated, such as the stage of CKD. For example, the dosage can be determined empirically taking into account the type and stage of the kidney disease and / or fibrosis diagnosed in a particular patient. In the context of the present compositions and methods, the dosage administered to a patient should be sufficient to provide a beneficial therapeutic response to the patient over a given period. The size of the dosage is also determined by the antibody dosage in combination with another therapeutic agent in a particular patient and / or the presence, nature, and extent of any harmful side effects associated with the administration of the dosage. Determination of the appropriate dosage for a particular patient and situation is within the scope of the physician's skill. Generally, treatment is initiated using a lower, less than optimal dosage of the therapeutic agent. Thereafter, the dosage is increased incrementally until efficacy, such as optimal efficacy, is achieved. For convenience or as needed, the total daily dosage may be administered in divided doses throughout the day. Treatment with the determined or optimal dosage can be continued for a short period (e.g., several hours or days) or for a longer period (e.g., several days, weeks, months, years).

[0154] Detection method In some embodiments, the anti-αvβ8 integrin antibody is used for detection, for example, for imaging or for determining the presence of αvβ8 integrin in vivo, ex vivo, or in vitro. In such embodiments, the antibody is labeled directly or indirectly with a detectable moiety. Accordingly, in some embodiments, a method (in vivo, ex vivo, or in vitro) is provided for determining the presence of αvβ8 integrin in a biological sample obtained from a subject, the method comprising contacting the biological sample with a labeled anti-αvβ8 integrin antibody described herein, detecting the presence of the labeled antibody bound to αvβ8 integrin, and thereby determining the presence of αvβ8 integrin in the sample. Such methods can be used for diagnosing kidney diseases, or kidney-related conditions such as kidney fibrosis, inflammation, or CKD.

[0155] In one embodiment, the antibody binds to an "effector" moiety or molecule, which can be, but is not limited to, a labeled moiety such as a radiolabel or a fluorescent label, or a therapeutic moiety or molecule. In one embodiment, effector moieties or molecules can include, but are not limited to, anti-cancer agents, toxins, cytotoxic agents, radiopharmaceuticals, cytokines, a second antibody, or enzymes. In another embodiment, the activity of the therapeutic moiety or molecule is regulated by binding to the antibody. In another embodiment, the antibody is linked to an enzyme that converts a prodrug into a cytotoxic agent.

[0156] An immune complex comprising an antibody or an antigen-binding fragment thereof can be used to target an effector moiety or molecule to cells expressing αvβ8 integrin on their surface, particularly diseased renal cells and renal tissue, such as CKD renal cells and renal tissue. Non-limiting examples of cytotoxic agents that can be effector molecules include radioisotopes, ricin, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracenedione, actinomycin D, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, steroids, glucocorticoids, and other chemotherapeutic agents. Detectable markers can include, but are not limited to, radioisotopes, fluorescent compounds, bioluminescent or chemiluminescent compounds, metal chelating agents, or enzymes.

[0157] In one embodiment, an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof, either alone (not complexed) or complexed with a detectable label or an effector moiety, such as an adjuvant therapeutic agent such as a suitable therapeutic drug or therapeutic agent for renal disease or CKD, is used as a therapeutic agent that reduces, inhibits, attenuates, decreases, blocks, or inhibits TGF-β activation in the kidney of an individual in need thereof, particularly in a diseased kidney or a CKD kidney.

[0158] "Detectable label or moiety" can be a diagnostic agent or component detectable by physical or chemical means, such as spectroscopic, radiological, photochemical, biochemical, immunochemical means, etc. As an example, detectable labels can include radioactive labels (e.g., 111 In, 99 mTc, 131 I, 67 Ga), and other FDA-approved imaging agents. Further labels can include 32 P, fluorescent dyes, high electron density reagents, enzymes, biotin, digoxigenin, or haptens and proteins or other molecules that can be made detectable, for example, by incorporating a radioactive label into a targeting agent. For example, methods for binding nucleic acids or nanocarriers known in the art to a label can be used by employing the methods described in Hermanson, Bioconiugate Techniques 1996, Academic Press, Inc., San Diego.

[0159] A "labeled" or "tagged" antibody or agent is one that is covalently attached by a linker or chemical bond, or non-covalently attached by ionic, van der Waals, electrostatic, or hydrogen bonds, to a label that enables the detection of the presence of the antibody, its antigen-binding fragment, or agent by detecting the label attached to the antibody or agent. Techniques for conjugating detectable therapeutic agents to antibodies are known and practiced by those skilled in the art, for example, as described in Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (Eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, Controlled Drug Delivery (2nd Ed.), Robinson et al. (Eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, Monoclonal Antibodies’84: Biological And Clinical Applications, Pinchera et al. (Eds.), pp. 475-506 (1985); and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62: 119-58 (1982).

[0160] Method of Administration In addition to the dosing regimens described herein, an anti-αvβ8 integrin antibody or antigen-binding fragment thereof, or a pharmaceutical composition or formulation comprising an anti-αvβ8 integrin antibody or antigen-binding fragment thereof, can be administered to a subject in a manner and via a route suitable for administering and / or delivering a biological drug such as a protein or antibody. Generally, suitable methods of biological delivery or administration include parenteral administration methods or routes. Such delivery methods include, but are not limited to, subcutaneous (SC) delivery, subcutaneous injection or infusion, such as intravenous (IV) delivery, for example, intravenous infusion or injection or bolus injection. Other delivery and administration methods or regimens include, but are not limited to, intra-articular, intra-arterial, intraperitoneal, intramuscular, intradermal, rectal, transdermal, or intrathecal. In certain embodiments, the anti-αvβ8 integrin antibody is provided to the subject by intravenous administration, for example, by IV infusion or bolus IV injection. In another specific embodiment, the anti-αvβ8 integrin antibody is provided to the subject by subcutaneous injection, such as a single subcutaneous injection.

[0161] The anti-αvβ8 integrin antibody can be administered in a long-term dosing regimen. The antibody may be administered for a period or a predetermined period, followed by a period during which it is not administered. The dosing regimen or cycle can also be repeated. In some embodiments, treatment (e.g., administration of an anti-αvβ8 integrin antibody) includes administration of a first dose, followed by administration of a second dose and / or one or more subsequent maintenance doses, for a period, for example, including several days. Subsequent or maintenance doses can be administered at regular intervals, for example, weekly, bi-weekly, tri-weekly, or more, for example, at four-week, five-week, six-week, seven-week, eight-week, nine-week, ten-, eleven-week, twelve-week intervals, or at longer intervals such as monthly or yearly intervals, after the first dose, second dose, or subsequent dose.

[0162] If feasible, it is also contemplated that anti-αvβ8 integrin antibodies can be administered by direct delivery, such as by infusion or injection, to the disease site or in the vicinity thereof. Injection into or in the vicinity of the kidney or renal tissue may be useful. It is also contemplated that anti-αvβ8 integrin antibodies can be administered by implantation of a depot that releases the antibody at the target site of action, such as renal tissue. Alternative methods of administration or delivery of anti-αvβ8 integrin antibodies can include inhalation (e.g., using an inhaler or aerosol spray), intranasal delivery, or transdermal delivery (e.g., via a patch on the skin). Further, administration can be effected by an osmotic pump (e.g., an Alzet pump) or a minipump (e.g., an Alzet mini-osmotic pump), which allows for controlled continuous and / or sustained release delivery of the anti-αvβ8 integrin antibody, or a pharmaceutical composition thereof, over a predetermined period. The osmotic pump or minipump can also be implanted subcutaneously at or in the vicinity of the kidney or renal tissue as the target site.

[0163] Kit Also provided is a kit for the treatment of kidney diseases associated with fibrosis, such as kidney diseases such as CKD or DN. In one embodiment, the kit includes a composition containing an effective amount of an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof, such as a therapeutic composition. In one embodiment, the anti-αvβ8 integrin antibody or an antigen-binding fragment thereof is in unit dosage form.

[0164] In some embodiments, the kit includes a sterile container containing an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof, for example, in aqueous or lyophilized form. If the antibody is in lyophilized form, the kit may include a container containing a suitable diluent, excipient, or vehicle for mixing with the dried antibody to prepare a solution containing the antibody suitable for administration, for example, intravenous administration. Such containers may be ampoules, bottles, vials, tubes, bags, pouches, blister packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding the agent, for example, in aqueous or dry form. The container can be placed in a box for protection against damage or breakage. One or more syringes for antibody dilution and / or administration can be included in the kit.

[0165] The kit may further provide instructions for administering the anti-αvβ8 integrin antibody, or a composition containing the antibody, to a subject having a renal disease, a fibrotic renal disease, such as CKD or DN. The instructions generally include information about the use of the antibody or composition for the treatment of renal disease, fibrotic renal disease, such as CKD or DN. In other embodiments, the instructions include one or more of the following: a description of the therapeutic antibody; a dosing schedule and administration for the treatment of renal disease, fibrotic renal disease, such as CKD or DN, or their symptoms; dosing information; cautions; warnings; indications; contraindications; overdose information; side effects; animal pharmacology; clinical trials; and / or references. The instructions may be printed directly on the container (if present), on a label affixed to the container, or on another sheet, pamphlet, card, or folder provided within the kit or with the container within the kit.

[0166] Unless otherwise indicated, the present disclosure encompasses the prior art in molecular biology (including any recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the skill of those in the art. Such techniques are fully described in documents such as "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology", "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of polynucleotides (polynucleotides) encoding anti-αvβ8 integrin antibodies or antigen-binding portions or fragments thereof, and / or anti-αvβ8 integrin antibodies or antigen-binding portions or fragments thereof (polypeptides), and thus can be considered in making and practicing the present invention.

[0167] The following examples are set forth to provide those skilled in the art with a complete disclosure and description of how to make and use the therapeutic methods of the present invention and are not intended to limit the scope of what the inventors regard as their invention.

Example

[0168] Example 1 Anti-αvβ8 integrin antibody Several anti-αvβ8 integrin antibodies are encompassed by the present disclosure and are used according to the methods, compositions, and products described herein and / or as reference or control antibodies. Specifically, a chimeric anti-αvβ8 integrin antibody herein referred to as "Chi-37E1B5" was obtained from The Regents of University of California (UCSF). A second anti-αvβ8 integrin antibody herein referred to as "hu37E1B5" was generated at MedImmune using the humanized sequences reported in the published PCT International Application WO 2013 / 026004 pamphlet (UCSF). When the hu37E1B5 antibody was evaluated in an affinity binding assay, it was found to have a very low binding affinity for the αvβ8 integrin protein, as shown in FIG. 1A. Accordingly, a third humanized anti-αvβ8 integrin antibody herein referred to as "MEDI-hu37E1B5" was generated using CDR grafting techniques known and practiced in the art. The CDRs used to generate the humanized MEDI-hu37E1B5 anti-αvβ8 integrin antibody were obtained from the above-described Chi-37E1B5 antibody. The MEDI-hu37E1B5 antibody showed a binding affinity for the αvβ8 integrin protein similar to that of the Chi-37E1B5 antibody, as shown in FIG. 1B. The V H and V L regions and the amino acid sequences of the CDRs are shown in FIG. 6. Surprisingly, the binding affinity was retained upon humanization of the Chi-37E1B5 antibody. In contrast, the UCSF humanized antibody ("hu37E1B5") showed a very low binding affinity upon humanization from Chi-37E1B5.

[0169] In addition, to obtain an anti-αvβ8 integrin antibody with improved binding affinity for αvβ8 integrin, a fourth optimized anti-αvβ8 integrin antibody, referred to herein as "B5-15", was generated from the parental MEDI-hu37E1B5 antibody using affinity maturation techniques known and used in the art. The resulting B5-15 anti-αvβ8 integrin antibody (also referred to as "optimized" or "affinity-optimized" B5-15) showed an improved binding profile to the αvβ8 integrin protein compared to the MEDI-hu37E1B5 anti-αvβ8 integrin antibody, as shown in FIG. 4. The V H and V L regions and the amino acid sequences of the CDRs are also shown in FIG. 6.

[0170] Humanization of the chimeric Chi-37E1B5 antibody by CDR grafting Using the CDR grafting method known and practiced in the art, the mouse / human chimeric 37E1B5 (Chi-37E1B5) antibody was humanized to generate the humanized MEDI-hu37E1B5 anti-αvβ8 integrin antibody. For humanization, the closest individual human germline framework (FW) having the same standard class was selected to mimic the antibody folding structure. Mouse FW residues important for back mutations were identified, genes were synthesized, IgG was converted and generated by transient transfection using 293 cells, and the resulting antibody was screened for binding to αvβ8 integrin. This process generated a fully humanized light chain clone and a hybrid human germline FW with four important mouse residues. The humanized MEDI-hu37E1B5 anti-αvβ8 integrin antibody obtained from the above method was surprisingly shown to retain the full binding activity of the original chimeric 37E1B5 (Chi-37E1B5) antibody (Figure 1B). As observed in Figure 1B, both the humanized MEDI-hu37E1B5 antibody and the Chi-37E1B5 antibody had increased binding to αvβ8 integrin compared to the hu37E1B5 antibody (this sequence is reported in WO 2013 / 026004 pamphlet as described above).

[0171] Site-saturation mutagenesis and affinity maturation result in the production of affinity-optimized humanized B5-15 anti-αvβ8 integrin antibody Furthermore, site-saturation mutagenesis was performed on the humanized MEDI-hu37E1B5 antibody, and the Cys94 residue (which has been shown to be a defect in the antibody structure as it is associated with potential fragmentation / peptide cleavage of the antibody backbone) was removed by first converting the residue to each of the other 19 amino acids. All of the resulting mutant antibodies were screened for binding to αvβ8 integrin by ELISA analysis. Depending on the residue at position 94, the binding affinity decreased. The best αvβ8 integrin-binding variants obtained from this procedure were designated MEDI-hu37E1B5-C94I and MEDI-hu37E1B5-C94G. The MEDI-hu37E1B5-C94I mutant antibody had an approximately 3-fold decrease in αvβ8 binding affinity. The humanized MEDI-hu37E1B5-C94I antibody was selected for further analysis and affinity optimization.

[0172] Affinity maturation of humanized MEDI-hu37E1B5-C94I having an N-glycosylation site was performed using economical mutagenesis, a method recognized in the art. Briefly, saturation point mutations for each CDR position of Medi-hu37E1B5-C94I were first generated. The mutations covered all six CDRs of the V H and V L regions of the antibody. A total of 6528 individual clones were screened for binding to αvβ8 integrin (redundancy >4×). From these, the following 10 primary hits were identified: 3 were in V H -CDR1; 2 were in V H -CDR3; 1 was in V L -CDR1; and 4 were in V L -CDR3. All of the hits showed a 2- to 5-fold improvement in binding to αvβ8 integrin.

[0173] Figures 2A-2C show the MEDI-hu37E1B5 C94I anti-αvβ8 integrin antibody, and representative anti-αvβ8 integrin antibody "hits" (referred to as "P1" or "P2" hits) identified in the screening analysis, e.g., the V H CDR1 hit (Figure 2A), VH CDR3 hits (Figure 2B) and V L Graphs showing the binding affinity analysis of V hits (Figure 2C) are shown. For example, to designate an antibody clone hit, "P" represents a given multi-well plate, and the number following P represents the well number in the plate.

[0174] Figure 2D shows the V of representative primary clone anti-αvβ8 integrin antibody hits named "P2-23", "P2-33", "P2-25", "P1-21", "P1-35", "P1-42", "P2-16", "P2-19", "P2-36", and "P2-14" obtained from the screening of affinity matured anti-αvβ8 integrin antibody clones. H and V L region amino acid sequence alignments. The framework (FW1-FW4) regions and CDRs (CDR1-CDR3) in the V of the clone are designated above the sequence. The differences in amino acid residues in the CDR regions are indicated by double underlines. H and V L region.

[0175] Next, a combinatorial library of the 10 most beneficial point mutation combinations was generated in a combinatorial fashion. 4608 clones were screened for binding to αvβ8 integrin. 88 clones were selected for confirmation. Six hits were identified from the combinatorial evaluation as showing further improvement in binding to αvβ8 integrin compared to the best primary hit, P2-23. The αvβ8 integrin binding data from the combinatorial library screening are shown in Figures 3A and 3B. A humanized and affinity-optimized antibody designated B5-15 ("optimized B5-15" or "affinity-optimized B5-15") expressed in CHO (G22) cells was selected as the final optimized antibody based on its binding affinity for αvβ8 integrin higher than that of MEDI-hu37E1B5 (Figure 4) and its in vitro potency in the TMLC luciferase assay higher than that of Chi-37E1B5 (Figure 5).

[0176] TGF-β activation bioassay In this technical field, the TMLC luciferase bioassay is used to measure TGF-β activation by integrins such as αvβ8 integrin. This bioassay is based on the mink lung cell line TMLC stably transfected with a plasminogen activator inhibitor-1 (PAI-1) promoter fused to luciferase, as described, for example, in M. Abe et al., 1994, Anal. Biochem., 216(2):276-284; L.A. Randall et al., 1993, J. Immunol. Methods, 164(1):61-67; M.A. van Waarde et al., 1997, Anal. Biochem., 247(1):45-51); and I. Tesseur et al., 2006, BMC Cell Biology, 7:15 (https: / / doi.org / 10.1186 / 1471-2121-7-15).

[0177] A part of the plasminogen activator inhibitor-1 (PAI-1) promoter linked to a luciferase reporter was stably transfected, and TGF-β activation was measured using transformed mink lung epithelial cells (TMLC) (cells provided by Daniel Rifkin, New York University) cultured as previously described (M. Abe et al., 1994, Anal. Biochem., 216(2):276-284). In 96-well plates, HeLa-B8 cells (1.5×104 cells / well) were co-cultured overnight with TMLC (1.5×104 cells / well) in DMEM high glucose (Life Technologies / Thermo Fisher) supplemented with 10% FBS and 10 U / ml penicillin G, 10 μg / mL streptomycin G sulfate (with or without the test antibody). After 16 hours, the supernatant was removed, the cells were lysed in 100 μL of cell lysis buffer (Promega), and luciferase activity was measured by transferring 80 μL of the lysate to a white-wall clear-bottom 96-well plate and mixing with 80 μL of substrate using a luciferase assay system (Promega). Samples were immediately read with a luminometer and expressed as relative luciferase units (RLU) or percent of maximum response, determined using TMLC alone as the assay baseline or 0% control and TMLC co-cultured with HeLa-B8 cells as the maximum or 100% response.

[0178] Generation of the HeLa-B8 cell line The HeLa-B8 cells are a derivative of the HeLa cell line (ECACC). Briefly, confluent HeLa cells were maintained in MEM (Life Technologies / Thermo Fisher) supplemented with 10% FBS, 1% non-essential amino acids (Life Technologies / Thermo Fisher), and 10 U / ml penicillin G (Life Technologies / Thermo Fisher), 10 μg / mL streptomycin G sulfate. Before use, the cells were detached using Accutase and resuspended at 1×106 cells / mL in PBS. LIVE / DEAD Fixable Aqua Dead Cell Stain (Life Technologies / Thermo Fisher, 1:1000) was added to the cells on ice for 20 minutes. The cells were pelleted and washed in cold flow cytometry staining buffer (eBioscience). Recombinant 37E1B5-mIgG1 or isotype-mIgG1 (100 μg / ml of 1×106 cells / ml) was added to the cells and incubated on ice for 30 minutes. The cells were pelleted, washed, and secondary anti-mouse Alexa-647 (Jackson ImmunoResearch, 1:200) was added to the cells and incubated on ice for 30 minutes. The cells were pelleted, washed, and resuspended at 10×106 cells / ml in HeLa cell medium containing 1% FBS. Cells were sorted using a BD FACSAria III cell sorter (BD Biosciences) with the Chi-37E1B5 antibody. Subsequently, the highly αvβ8+ sorted cells were cultured and expanded in complete HeLa cell medium and stored for future use. The cells remained positive for high αvβ8 expression for at least 1 month of culture.

[0179] Characterization of Humanized Affinity-Optimized B5-15 Anti-αvβ8 Integrin Antibody The light chain (L) variable region (V L , κ) amino acid (aa) sequence of the humanized and optimized B5-15 antibody polypeptide has 107 amino acid residues as follows: B5-15V L (kappa (κ)) DIQLTQSPSSLSASVGDRVTITCKASQDINKYLSWFQQKPGKAPKSLIYYANRLVDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDVFPYTFGGGTKVEIK (107aa) (SEQ ID NO: 13)

[0180] The variable region (V H ) of the heavy chain (H) of the B5-15 antibody polypeptide has 116 amino acid residues as follows: B5-15 V H EVQLVESGGLVQPGGSLRLSCAVSGFVFSRSWISWVRQAPGKGLEWIGEINPDSSTINYTSSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAILITTEDYWGQGTTVTVSS (116aa) (SEQ ID NO: 12)

[0181] In particular, the variable region (V L ) of the light chain (L) of the B5-15 antibody contains three CDRs with the following amino acid sequences: V L CDR1: KASQDINKYLS (SEQ ID NO: 10) V L CDR2: YANRLVD (SEQ ID NO: 5) V L CDR3: LQYDVFPYT (SEQ ID NO: 11)

[0182] The variable region (V H ) of the heavy chain (L) of the B5-15 antibody contains three CDRs with the following amino acid sequences: V H CDR1: RSWIS (SEQ ID NO: 9) V H CDR2: EINPDSSTINYTSSL (SEQ ID NO: 2) V H CDR3: LITTEDY (SEQ ID NO: 3)

[0183] The V of Chi-37E1B5, hu37E1B5, MEDI-hu37E1B5 and the B5-15 anti-αvβ8 integrin antibodyH and V L A comparison of the amino acid sequences of the regions is shown in FIG. 6.

[0184] Example 2 Immunohistochemical (IHC) detection method of αvβ8 integrin expression in formalin-fixed paraffin-embedded (FFPE) human tissues using anti-αvβ8 integrin antibody IHC method To prepare formalin-fixed paraffin-embedded (FFPE) tissue sections, slides on which human tissue samples were fixed were taken out from the storage location. The slides were appropriately labeled and loaded into an automatic staining device XL rack. The stained slides were deparaffinized and rehydrated with tap water.

[0185] The slide rack was transferred to an autoclave containing Dako antigen retrieval solution (Dako S1699). Heat-mediated antigen retrieval was performed under pressure (SP DDCP_5024) for 2 minutes with the following modifications: After antigen retrieval, the autoclave was cooled and depressurized, the autoclave was placed in running tap water, the lid was removed, the autoclave was cooled for 5 minutes, then its contents were rinsed with running tap water, the slides were taken out and rinsed with running water for 5 minutes. The slides were blocked with peroxidase (3% hydrogen peroxide in methanol) for 10 minutes.

[0186] Immunohistochemical staining was performed as follows: · Draw a pap pen at an appropriate position on the slide (to draw a hydrophobic barrier on the tissue); · Load the slides into a Dako automatic staining device; · Wash once (×1) with standard Dulbecco's PBS (PBST) containing 0.1% Tween20; · Incubate the slides in 2.5% horse serum (from the ImmPRESS kit) for 20 minutes; · Blowing step; Incubate with Calico antibody CAL16 (purified rabbit recombinant anti-αvβ8 integrin antibody) 1.0 μg / ml (diluted with PBST), Dako rabbit immunoglobulin isotype control 1.0 μg / ml or Vector Ki67 (diluted 1:200 as an experimental control) for 60 minutes; · Rinse once (×1) with PBST; · Incubate in the labeled polymer, Vector ImmPRESS™ HRP, donkey anti-rabbit IgG (peroxidase) polymer detection kit (Catalog No. MP-7401) for 30 minutes; · Wash once with PBST; · Incubate in PBST for 5 minutes; · Wash once with PBST; switch to hazardous waste × 1; · Incubate in DAB+ substrate / chromagen (Dako, K3468) for 5 minutes; · Wash once with pure water (automated stainer does this automatically); · Remove the slide from the Dako automated stainer; · Using Program 9, counterstain with Gill hematoxylin I, dehydrate, and cover the slide with a coverslip; and · Remove the slide from the Leica CV5030 coverslipper, and dry / set.

[0187]

Table 1

[0188] Alternatively, the steps of "Program No. 9" can be performed manually. To do this, the slide can be placed in the described reagent for the described time. Either an automated program or manual execution of the steps may be used.

[0189] Example 3 αvβ8 integrin is preferentially expressed in the kidney The IHC staining analysis described in Example 2 was performed on a number of tissue samples to determine the expression and distribution of avβ8 integrin in human tissues. Table 2 below shows the results of the IHC analysis.

[0190]

Table 2

[0191] As can be seen in Table 2, renal tissue expresses high levels of αvβ8 integrin. Furthermore, αvβ8 integrin was found to be present at high density in human renal tissue compared to 33 other human tissue types, namely, heart, lung, spleen, lymph node, thymus, tonsil, liver, gallbladder, pancreas, cerebellum and cerebrum of the brain, thyroid, adrenal gland, parotid gland, skin, skeletal muscle, stomach, ileum, colon, ovary, fallopian tube, myometrium of the uterus, endometrium, cervix of the uterus, ectocervix, breast, placenta, prostate, testis, seminal vesicle, bladder and ureter. In Figure 7A (left side), strong staining of αvβ8 integrin was observed in human renal tissue, particularly in the podocytes and epithelial cells of the renal tubules. In contrast, staining was found to be weak, inconsistent, or absent in the other tissue types examined.

[0192] In the IHC staining analysis shown in Figure 7A, the CAL16 clone anti-αvβ8 integrin rabbit monoclonal antibody (purified rabbit recombinant anti-αvβ8 integrin antibody from Calico Biolabs Inc (Pleasanton, CA)) was used. This commercially available antibody was optimized and validated for binding to αvβ8 integrin expressed in both human and mouse tissues.

[0193] Example 4 Avβ8 expression increases in renal tissue of patients with chronic kidney disease (CKD) The expression of αvβ8 integrin was evaluated in human renal tissue samples taken from patients with diabetic nephropathy (DN) and individuals with normal renal tissue as "healthy" controls. DN renal tissue samples were obtained from the Addenbrooke’s Biobank and the MedImmune (Gaithersburg) Biobank. Normal kidney samples were obtained from the MedImmune (Cambridge) tissue bank. More specifically, samples from 9 patients with diabetic nephropathy chronic kidney disease, DN-CKD, were obtained by needle biopsy. Samples from 4 healthy "normal" individuals were used as controls. In the normal samples, the presence of some areas of mild chronic inflammation was evident but did not affect the study design or results (Figure 7A, right side).

[0194] As described in Example 3, the antibody used in the IHC staining experiment was the CAL16 clone anti-αvβ8 integrin rabbit monoclonal antibody (purified rabbit recombinant antibody) from Calico Biolabs Inc (Pleasanton, CA). After staining, the slides were reviewed by experienced senior pathologists.

[0195] The results of this IHC staining analysis were as follows: In healthy individuals, glomeruli showed positive staining with the anti-αvβ8 integrin antibody compared to isotype-matched control antibody staining; anti-αvβ8 integrin antibody staining was generally weak (3 / 4 samples), but one sample (1 / 4) showed strong staining in podocytes (podocyte pattern). In renal tubules, weak multifocal staining was observed from the cortex of the tubules, membranes, and from the basal to the apical side. (Figure 7A, right side). Tubule staining was not seen in the collecting ducts. The overall staining pattern in healthy human kidneys was mostly in glomeruli similar to healthy transgenic mice, but staining of αvβ8 integrin by IHC was observed in the tubule structure in both CKD patients and UUO transgenic mice.

[0196] In patients with DN-CKD, the degree of αvβ8 staining in glomeruli varied and was associated with the degree of glomerular injury. Loss of podocytes in the affected tissue correlated with less staining. Due to loss of podocytes in the affected kidneys, the staining intensity varied. Tubule staining in DN-CKD kidney tissue varied from weak staining to strong staining of the cytoplasm and membranes mainly in the areas of inflammation / fibrosis. An overall increase in the expression of αvβ8 integrin was observed in DN-CKD kidneys as demonstrated by the staining pattern of the anti-αvβ8 integrin antibody. The overexpression was seen substantially in renal tubules. (Figure 7B). Based on anti-αvβ8 integrin antibody staining, the changes in the expression of αvβ8 integrin in DN-CKD kidney tissue appeared to appropriately approximate the αvβ8 integrin expression in the kidneys of mouse models showing tubulointerstitial inflammation and fibrosis.

[0197] Figure 7C shows photomicrographs of renal tissue cells obtained from human patients with kidney disease. The renal tissue cells were stained with an anti-αvβ8 integrin antibody and analyzed by IHC. The IHC staining results showed that the αvβ8 integrin protein was upregulated in renal cells and renal tissues of human patients with diabetic nephropathy (DN) compared to normal renal cells and renal tissues (Figure 7C, upper panel). In particular, in renal tissue samples obtained from DN and CKD patients, overexpression of αvβ8 integrin was substantially observed in the tubules (Figure 7C, lower panel). The glomeruli of the kidneys of DN patients showed a decrease in αvβ8 integrin staining, probably as a result of podocyte loss due to renal tissue fibrosis and damage. The unstained areas in renal tissue samples from patients with stage 2 and stage 3 DN are fibrous matrices that replaced functional nephrons, as indicated by an asterisk ( * ) in Figure 7C. This result emphasizes the importance of targeting αvβ8 integrin to protect functional epithelium. In the IHC staining analysis shown in Figure 7C, the CAL16 clone anti-αvβ8 integrin rabbit monoclonal antibody (purified rabbit recombinant anti-αvβ8 integrin antibody from Calico Biolabs Inc. (Pleasanton, CA)) was used. This commercially available antibody was optimized and validated for binding to αvβ8 integrin expressed in both human and mouse tissues.

[0198] Example 5 The itgb8 gene is upregulated in the kidneys of individuals with CKD and shows high expression compared to other β integrins Transcriptomic analysis provided evidence that ITGB8 (encoding β8 integrin) is more highly expressed in the kidneys of human CKD patients compared to the kidneys of healthy human subjects. In these analyses, relative β-integrin family mRNA expression was measured in human CKD kidney homogenates. Briefly, one punch of renal biopsy (2 mm puncher) was homogenized in RLT lysis buffer using TissueLyserII. RNA from the lysate was isolated using an RNAeasy Mini kit column. RNA concentration was measured with Nanodrop and adjusted, and qPCR analysis was performed using the TaqMan RNA-to-Ct 1-step kit and probes specific for all integrins (including hprt-1 as a housekeeping gene). Figure 8A shows a bar graph depicting the relative expression levels of mRNAs encoding different isoforms of β-integrin in the kidneys from human patients with CKD. As seen in Figure 8A, β8 integrin mRNA expression was dominant over the expression of other β-integrins (i.e., β1, β3, β5, and β6) in the kidneys of CKD patients.

[0199] In another experiment, the transcriptome profiles of 157 patients with different degrees of CKD were analyzed and compared with those of living donors (LD). Diabetic neuropathy (DN) was recognized in 12 out of 157 cases. The glomerular and tubulointerstitial compartments were separated, and whole-genome gene expression analysis was performed as described by S. Martini et al. (2014, J. Am. Soc. Nephrol., 25(11):2559-2572). In this analysis, first, the expression of itgb8 mRNA in the renal glomerular compartment was evaluated in relation to nephrin (encoded by the NPHS1 gene). Nephrin is a podocyte protein necessary for the proper function of the kidney filtration barrier, which consists of fenestrated endothelial cells, the glomerular basement membrane, and the podocytes of epithelial cells. Mutations in NPHS1 are associated with congenital nephrotic syndrome. NPHS1 expression is an indicator of the number of podocytes. In CKD, the number of podocytes decreases, so NPHS1 expression decreases. Figure 8B shows that itgb8 mRNA expression is positively correlated with NPHS1, a podocyte marker gene, supporting the expression of this gene in renal podocytes. To better evaluate itgb8 expression in the glomerular cortex considering podocyte loss, itgb8 expression was normalized by NPHS1. Therefore, to understand the expression changes in podocytes under conditions of podocyte loss such as in chronic kidney disease, the data were normalized for the expression of nephrin (encoded by the NPHS1 gene). Figure 8C shows a box plot graph indicating that itgb8 mRNA expression was higher in the tubulointerstitial (TI) of DN patient kidney samples compared to its expression in living donors (LD) as healthy controls. Figure 8D shows a dot plot graph indicating that itgb8 mRNA expression is strongly correlated with the overall TGF-β activation score in TI of patients with CKD, supporting the role of αvβ8 integrin in TGF-β activation in CKD.

[0200] In another cohort, the tubulointerstitium (Tub) and glomerulus (Glom) of kidney samples obtained from 20 human patients with DN were profiled by whole-genome transcriptional profiling using RNAseq, compared to the TI and glomerulus of kidney samples obtained from 19 LD patients. The results showed increased itgb8 mRNA expression in the tubulointerstitium of DN patients (represented as "Tub-DN" in the graph) compared to living donors (LD) (Figure 8E). The finding of high itgb8 mRNA levels in the tubulointerstitium of patients with kidney disease, namely diabetic nephropathy, correlates with the pathology of kidney injury and fibrosis in these kidney disease patients.

[0201] The main findings of these analyses were as follows: itgb8 mRNA expression was increased in glomeruli from DN patient samples after normalization to the podocyte marker gene nephrin (NPHS1). itgb8 mRNA expression was increased in the tubulointerstitium (TI) of DN patients. In the TI, itgb8 mRNA expression was positively correlated with the putative TGF-β activation score, which is consistent with the proposed role of αvβ8 integrin in the control of TGF-β activation in fibrotic diseases. Similar results were also found by analysis of the mRNA expression of WT1, another podocyte marker gene (data not shown). These findings support the discovery that αvβ8 integrin expression correlates with fibrosis in human CKD kidneys, and that fibrosis in CKD is associated with the activation of TGF-β, which plays an important role in causing and exacerbating renal fibrosis.

[0202] Example 6 In vivo efficacy of anti-αvβ8 integrin antibody Using a mouse model of fibrosis, the in vivo efficacy of an anti-αvβ8 integrin antibody in the treatment of renal fibrosis was tested. This model involved performing a procedure called unilateral ureteral obstruction (UUO) (ligation of one side of the ureter) on the animals. In this model, sham or UUO treatment was performed on humanized αvβ8 transgenic (Tg) male mice for 5 and 8 days after injury. The Tg mice were generated by mating mice with a knocked-out αvβ8 gene (αvβ8 KO mice) with human αvβ8 BAC transgenic mice. The generation of Tg mice expressing the human ITGB8 gene is described, for example, in S. Minagawa et al., 2014, Sci. Transl. Med., 6(241):241ra79 (doi:10.1126 / scitranslmed.3008074). The humanized αvβ8 transgenic mice expressed human αvβ8 integrin mainly in renal glomeruli in a pattern similar to that observed in healthy humans. Induction of fibrosis by ureteral ligation (UUO) increased αvβ8 integrin expression in renal tubules, similar to what is observed in human CKD.

[0203] The test agent used was B5-15 of the above-described IgG1 humanized and sequence-optimized anti-αvβ8 integrin antibody. The control antibody was an isotype-matched IgG antibody.

[0204] Protocol for the UUO Tg mouse model test: Model: 91 humanized αvβ8 transgenic (Tg) male mice were subjected to sham or unilateral ureteral obstruction (UUO) treatment; 5 or 8 days after injury. The animals in these groups were administered the respective antibodies every other day (EOD), i.e., on days -1, 1, 3, 5, and 7. The sham-treated animals were administered vehicle on days 0, 2, 4, and 6.

[0205] Age of the mice at the start of the test: 92 - 121 days old.

[0206] Test agent / compound: Anti-αvβ8 integrin antibody (Chi-37E1B5 monoclonal antibody), B5-15 sequence-optimized anti-αvβ8 integrin antibody), IgG isotype control and / or vehicle (PBS) were administered to the groups at the doses and frequencies shown in Table 3 below.

[0207]

Table 3

[0208] Test endpoints: Morphology: Body weight (initial, final, Δ); Kidney weight (obstructed and contralateral) and index; and Tibia length.

[0209] Renal cortex mRNA expression by Luminex: · Connective tissue growth factor (CTGF) · α-smooth muscle actin (ACTA2) · Fibronectin 1 (FN1) · Collagen 1a1 (Col1a1) · Collagen 3a1 (Col3a1)

[0210] Renal cortex hydroxyproline content Histological reading: Picrosirius red (PRS) and αvβ8 staining. The results of determining renal fibrosis and its degree by IHC staining of renal tissues of humanized αvβ8 transgenic mice using an anti-αvβ8 integrin antibody are shown in Figures 9A-9D. The microscopic photographs of IHC staining with the anti-αvβ8 integrin antibody shown in Figures 9A and 9B indicate that αvβ8 integrin was expressed mainly in the glomeruli of the kidneys, similar to what is typically observed in healthy human kidneys in humanized αvβ8 transgenic mice. It was shown that induction of fibrosis by UUO treatment increased αvβ8 expression in renal tubules, similar to what is typically observed in the kidneys of humans with CKD (Figures 9C and 9D). Figures 9E-9H show the results obtained from the in vivo test by UUO treatment described above and outlined in Table 3.

[0211] As shown in Figure 9E, the anti-αvβ8 integrin antibodies Chi-37E1B5 (labeled with the parental Avb8 Ab) and B5-15 (labeled with the lead Avb8 Ab) attenuated the increase in collagen 1a1 mRNA expression induced by UUO on day 8 after UUO surgery, compared to the UUO control. As shown in Figure 9F, the anti-αvβ8 integrin antibodies Chi-37E1B5 (labeled with the parental Avb8 Ab) and B5-15 (labeled with the lead Avb8 Ab) attenuated the increase in Col3a1 expression induced by UUO on day 8 after UUO surgery, compared to the UUO control. As shown in Figure 9G, UUO increased the expression of fibronectin 1 (Fn1) mRNA in the obstructed renal cortex on day 8 after UUO surgery, compared to the sham control. The anti-αvβ8 integrin antibodies Chi-37E1B5 (labeled with the parental Avb8 Ab) and antibody B5-15 (labeled with the lead Avb8 Ab) attenuated the increase in Fn-1 expression induced by UUO during the 8-day injury period, compared to the UUO control. As shown in Figure 9H, the anti-αvβ8 integrin antibody B5-15 (labeled with the lead Avb8 Ab) attenuated the increase in α-smooth muscle actin (α-SMA) mRNA expression induced by UUO on day 8 after UUO surgery, compared to the UUO control. The Chi-37E1B5 antibody (labeled with the parental Avb8 Ab) did not reduce the increase in α-SMA induced by UUO. Since the presence of α-SMA+ cells is detrimental to normal renal function, the reduction of α-SMA is important because these cells are contractile and not only act directly on fibrotic remodeling but also produce highly synthesized inflammatory and fibrotic mediators. As shown in Figure 9I, the anti-αvβ8 integrin antibodies Chi-37E1B5 (labeled with the parental Avb8 Ab) and B5-15 (labeled with the lead Avb8 Ab) attenuated the increase in connective tissue growth factor (CTGF) mRNA expression induced by UUO on day 8 after UUO surgery, compared to the UUO control. As shown in Figure 9J, UUO increased the percentage of hydroxyproline (OH-P) in the obstructed renal cortex on day 8 after UUO surgery.The Chi-37E1B5 antibody (labeled with parental Avb8 Ab) and the B5-15 antibody (labeled with lead Avb8 Ab) attenuated the increase in OH-P% induced by UUO during the 8-day injury period compared to the control. The reading of renal cortical hydroxyproline serves as a measure of the actual fibrosis content / fibrosis of the tissue.

[0212] In summary, on day 8 after UUO surgery, Chi-37E1B5 and B5-15 attenuated the UUO-induced increases in Col1a1, Col3a1, FN-1, and CTGF mRNA expression, as well as hydroxyproline content%. Furthermore, on day 8 after UUO surgery, B5-15 attenuated the increase in α-SMA mRNA expression induced by UUO.

[0213] The two anti-αvβ8 integrin antibodies, Chi-37E1B5 and B5-15, used in this example were administered at the maximum dose to mice in the UUO model. The purpose of this study was to demonstrate whether antibodies against αvβ8 integrin could effectively reduce TGF-β-induced fibrosis caused by binding to αvβ8 integrin. The inventors expected that there would be a difference in the reduction of TGF-β-induced fibrosis at lower doses (i.e., EC 50 ) of any of these anti-αvβ8 integrin antibodies. That is, the inventors expected that a greater reduction in TGF-β-induced fibrosis would be seen in the UUO model from treatment with B5-15 than with Chi-37E1B5 at equivalent doses. This is mainly due to the fact that B5-15 has a higher binding affinity for αvβ8 integrin than Chi-37E1B5 (see FIGS. 1B and 4), and B5-15 has higher in vitro potency than Chi-37E1B5 (see FIG. 5). Treatment with B5-15 is more advantageous than with Chi-37E1B5 because it allows for a lower frequency or lower dose of administration to patients, resulting in fewer adverse events, if any, and leading to greater patient compliance.

[0214] As discussed above, the αvβ8 integrin target receptor is preferentially and highly expressed in diseased / fibrotic kidney tissue, is bound in kidney tissue by an anti-αvβ8 integrin antibody, which interferes with the binding interaction of latent TGF-β to αvβ8 integrin. The anti-αvβ8 integrin antibodies disclosed herein are particularly advantageous and beneficial for the treatment of fibrotic kidney disease in subjects having a kidney disease, because the use of antibodies against αvβ8 integrin that bind latent TGF-β prevents and avoids the targeting of systemic TGF-β, and thus avoids potentially serious problems associated with systemic inhibition of TGF-β in other tissues in a subject being treated.

[0215] Example 7 Ex vivo assay using the B5-15 anti-αvβ8 integrin antibody To evaluate the binding and involvement of the anti-αvβ8 integrin antibody (B5-15) with the TGF-β target αvβ8 integrin receptor, the activation of the downstream TGF-β signaling pathway in kidney lysates was evaluated by measuring total phosphorylated kidney SMAD2 / 3. Briefly, kidney samples from the animals used in the assay described in Example 5 were homogenized in a specific lysis buffer (1× diluted with distilled water + 10 μl / ml protease and phosphatase inhibitors) using a TissueLyser II, the protein content was measured using the bicinchoninic acid (BCA) assay known and used by those skilled in the art, and the protein concentration was normalized for all samples. The total phosphorylated form of the SMAD2 / 3 protein (phospho-SMAD2(Ser465 / 467) / SMAD3(Ser423 / 425)) was analyzed by ELISA according to the manufacturer's protocol. As noted above, members of the Smad family of signaling molecules are components of an intracellular pathway that transmits the TGF-β signal from the cell surface to the nucleus.

[0216] The results of the experiment showed that the B5-15 anti-αvβ8 integrin antibody decreased the downstream TGF-β signaling pathway in the kidneys of transgenic mice with the human αvβ8-encoding gene (「humanized αvβ8 transgenic mice」) that had undergone unilateral ureteral obstruction (UUO) for 5 days. In Figures 10(a) and 10(b), * =≤0.05, and **** =≤0.0001. In Figures 10A and 10B, 「Sham + NIP228 (IgG isotype control), n = 6; 「UUO + NIP228 (IgG isotype control)」, n = 8; and 「UUO + B5-15 (anti-αvβ8 integrin antibody)」, n = 8.

[0217] As can be seen in Figures 10A and 10B, UUO surgery in humanized αvβ8 mice resulted in a 5.7-fold increase in TGF-β-dependent SMAD2 / 3 phosphorylation compared to the Sham-treated group. Interestingly, the anti-αvβ8 integrin antibody (B5-15) significantly decreased SMAD2 / 3 activation by 1.6-fold compared to treatment with the isotype control. The total levels of SMAD2 / 3 increased in all UUO groups compared to Sham-treated animals.

[0218] Example 8 Treatment of a tri-culture cell line with the B5-15 anti-αvβ8 integrin antibody To evaluate the effect of B5-15 (anti-αvβ8 integrin antibody) on a model of human glomerulosclerosis (described in Waters et al., 2017, J Pathol, 243(3):390-400), the inventors treated a tri-culture cell line (glomerular endothelial cells, podocytes, and mesangial cells that form a vascular network) with 10 ng / ml of TGF-β or 25 ng / ml of CTGF that induces fibrosis. An increase in the number of nodules reflects the progression of fibrosis. Treatment with 15 μg / ml of B5-15 significantly decreased the number of nodules compared to treatment with 15 μg / ml of the isotype control (NIP228) (see Figure 11).

[0219] 3D tri-culture formation In tri-culture, human podocytes (Celprogen, CA, USA), glomerular endothelial cells (GEC) and mesangial cells (MC) (both GEC and MC from ScienCell Research Laboratories, CA, USA) were suspended at 4°C in rat tail type I collagen (1.5 mg / ml; Corning, MA, USA), human plasma fibronectin (90 μg / ml; Merck Millipore, MA, USA), 1.5 mg / ml NaHCO3, 25 mM HEPES and M199 medium (10×; Sigma, MO, USA). The gel was pH adjusted to pH 7.4 with 0.1 M HCl (Fisher Scientific, UK). This cell / gel suspension was pipetted into 48-well plates (Corning Incorporated, NY, USA) at a volume of 320 μl per well. Renal glomerular cells were used at a ratio of 16:3:1 (GEC:POD:MC), i.e., 330,000 - 340,000 GEC, 50,000 - 70,000 POD, and 20,000 - 24,000 MC per 320 μl. The cell / gel suspension was polymerized at 37°C for 20 minutes, after which 500 μl of medium was pipetted onto the gel. The tri-culture medium consisted of RPMI 1640 (by GibcoTM, Thermo Fisher, UK), 2% FBS, 1% penicillin / streptomycin, 1% insulin, apo-transferrin, sodium selenite (in ITS mixture) and 1% ECGS (all supplements from ScienCell Research Laboratories, CA, USA). The cultures were maintained for 24 hours. Cells were used for experiments between p2 - p6.

[0220] Stimulation assays with TGF-β, NIP228, anti-αvβ8 antibody and CTGF For stimulation, 10 ng / ml of TGF-β (R&D Systems (Bio-Techne Ltd), MN, USA), 15 μg / ml of NIP228, 15 μg / ml of anti-αvβ8 integrin antibody, and 25 ng / ml of CTGF (Invitrogen, CA, USA) were added, either alone or in combination, to the medium placed on the culture gel for 24-hour incubation. The control treatment was medium alone.

[0221] The inventors demonstrate that treatment with an anti-αvβ8 integrin antibody can inhibit the progression of fibrosis caused by TGF-β activation.

[0222] Other embodiments Embodiments of the present invention are further described in the following sections: [Item 1] A method for treating renal fibrosis in a subject having a renal disease, comprising administering to the subject an effective amount of an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof, thereby treating renal fibrosis. [Item 2] A method for reducing or attenuating renal fibrosis in a subject having a renal disease, comprising administering to the subject in need thereof an effective amount of an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof, thereby reducing or attenuating fibrosis in the kidney. [Item 3] A method for suppressing the activity of αvβ8 integrin associated with renal fibrosis, comprising administering to the subject in need thereof an effective amount of an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof, thereby suppressing the activity of αvβ8 integrin associated with renal fibrosis. [Item 4] A method for treating renal fibrosis by blocking the activation of TGF-β from its latent form in renal cells and renal tissues, comprising administering to the subject in need thereof an effective amount of an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof, thereby treating the renal fibrosis. [Item 5] A method for treating kidney injury characterized by an increase in plasma creatinine and / or urinary protein excretion levels, comprising administering to a subject in need thereof an effective amount of an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof, wherein said administration of the anti-αvβ8 integrin antibody or an antigen-binding fragment thereof suppresses plasma creatinine and / or urinary protein excretion levels in said subject, thereby treating kidney injury. [Item 6] The method according to any one of Items 3 to 5 above, wherein the subject has a kidney disease. [Item 7] The method according to any one of Items 1 to 6 above, wherein the kidney disease is selected from diabetic nephropathy (DN), chronic kidney disease (CKD), acute kidney disease, hypertension-related kidney disease, hyperglycemia-related kidney disease, renal fibrosis, inflammation-related kidney disease, end-stage renal disease (ESRD), autoimmunity-related renal fibrosis (e.g., lupus nephritis), and fibrosis after kidney transplantation. [Item 8] The method according to Item 7 above, wherein the kidney disease is CKD. [Item 9] The method according to any one of Items 1 to 8 above, wherein the antibody or an antigen-binding fragment thereof binds to αvβ8 integrin expressed in renal cells and / or renal tissue, and blocks the activation of TGF-β from its latent form in said renal cells and / or renal tissue. [Item 10] A method for detecting renal fibrosis in renal tissue, comprising contacting the renal tissue with an effective amount of a detectably labeled anti-αvβ8 integrin antibody or an antigen-binding fragment thereof, and detecting the binding of the anti-αvβ8 integrin antibody to αvβ8 integrin in said renal tissue. [Item 11] The anti-αvβ8 integrin antibody or an antigen-binding fragment thereof (a) A heavy chain variable region complementarity-determining region 1 (CDR1) comprising the amino acid sequence RYWMS ; (b) A heavy chain variable region complementarity-determining region 2 (CDR2) CDR2 comprising the amino acid sequence EINPDSSTINYTSSL ; and (c) Amino acid sequence LITTEDY Heavy chain variable region complementarity-determining region 3 (CDR3) CDR3 containing; and (d) Amino acid sequence KASQDINSYLS Light chain variable region CDR1 containing; (e) Amino acid sequence YANRLVD Light chain variable region CDR2 containing; and (f) Amino acid sequence LQYDEFPYT Light chain variable region CDR3 containing The method according to any one of items 1 to 10 above, containing [Item 12] The anti-αvβ8 integrin antibody or its antigen-binding fragment has a heavy chain variable region (V H ) Amino acid sequence EVQLVESGGGLVQPGGSLRLSCAVSGFVFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYTSSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAILITTEDYWGQGTTVTVSS; and Light chain variable region (V L ) Amino acid sequence DIQLTQSPSSLSASVGDRVTITCKASQDINSYLSWFQQKPGKAPKSLIYYANRLVDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPYTFGGGTKVEIK The method according to item 11 above, containing [Item 13] The anti-αvβ8 integrin antibody or its antigen-binding fragment is (a) Amino acid sequence RSWIS Heavy chain variable region CDR1 containing; (b) Amino acid sequence EINPDSSTINYTSSL Heavy chain variable region CDR2 containing; and (c) Amino acid sequence LITTEDY a heavy chain variable region CDR3 comprising; and (d) an amino acid sequence KASQDINKYLS a light chain variable region CDR1 comprising; (e) an amino acid sequence YANRLVD a light chain variable region CDR2 comprising; and (f) an amino acid sequence LQYDVFPYT a light chain variable region CDR3 comprising The method according to any one of items 1 to 10 above, comprising. [Item 14] The anti-αvβ8 integrin antibody or antigen-binding fragment thereof has a heavy chain variable region (V H ) amino acid sequence EVQLVESGGGLVQPGGSLRLSCAVSGFVFSRSWISWVRQAPGKGLEWIGEINPDSSTINYTSSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAILITTEDYWGQGTTVTVSS and a light chain variable region (V L ) amino acid sequence DIQLTQSPSSLSASVGDRVTITCKASQDINKYLSWFQQKPGKAPKSLIYYANRLVDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDVFPYTFGGGTKVEIK The method according to item 13 above, comprising. [Item 15] The antibody or antigen-binding fragment thereof reduces or suppresses fibrosis associated with increased expression of αvβ8 integrin in podocytes and interstitial tubular cells of renal tissue of the subject having a renal disease. The method according to any one of items 1 to 14 above. [Item 16] The antibody or antigen-binding fragment thereof is administered to the subject as an adjuvant therapeutic agent for renal disease or in combination with treatment. The method according to any one of items 1 to 9 or 11 to 15 above. [Item 17] The method according to item 16 above, wherein the antibody or antigen-binding fragment thereof is administered to the subject before, simultaneously with, or after the administration of the adjuvant therapeutic agent or treatment. [Item 18] (a) Amino acid sequence RYWMS Heavy chain variable region CDR1 containing: (b) Amino acid sequence EINPDSSTINYTSSL Heavy chain variable region CDR2; and (c) Amino acid sequence LITTEDY Heavy chain variable region CDR3; and (d) Amino acid sequence KASQDINSYLS Light chain variable region CDR1 containing: (e) Amino acid sequence YANRLVD Light chain variable region CDR2; and (f) Amino acid sequence LQYDEFPYT Light chain variable region CDR3 An anti-αvβ8 integrin antibody or antigen-binding fragment thereof containing: [Item 19] Heavy chain variable region (V H ) Amino acid sequence EVQLVESGGGLVQPGGSLRLSCAVSGFVFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYTSSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAILITTEDYWGQGTTVTVSS; and Light chain variable region (V L ) Amino acid sequence DIQLTQSPSSLSASVGDRVTITCKASQDINSYLSWFQQKPGKAPKSLIYYANRLVDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPYTFGGGTKVEIK An anti-αvβ8 integrin antibody or antigen-binding fragment thereof according to item 18 above containing: [Item 20] (a) Amino acid sequence RSWIS Heavy chain variable region CDR1 containing; (b) Amino acid sequence EINPDSSTINYTSSL Heavy chain variable region CDR2 containing; (c) Amino acid sequence LITTEDY Heavy chain variable region CDR3 containing; and (d) Amino acid sequence KASQDINKYLS Light chain variable region CDR1 containing; (e) Amino acid sequence YANRLVD Light chain variable region CDR2 containing; and (f) Amino acid sequence LQYDVFPYT Light chain variable region CDR3 containing An anti-αvβ8 integrin antibody or an antigen-binding fragment thereof containing. [Item 21] Heavy chain variable region (V H ) Amino acid sequence EVQLVESGGGLVQPGGSLRLSCAVSGFVFSRSWISWVRQAPGKGLEWIGEINPDSSTINYTSSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAILITTEDYWGQGTTVTVSS and Light chain variable region (V L ) Amino acid sequence DIQLTQSPSSLSASVGDRVTITCKASQDINKYLSWFQQKPGKAPKSLIYYANRLVDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDVFPYTFGGGTKVEIK The anti-αvβ8 integrin antibody or an antigen-binding fragment thereof according to Item 20 above, containing. [Item 22] An anti-αvβ8 integrin antibody or an antigen-binding fragment thereof that competes with the antibody or an antigen-binding fragment thereof according to any one of Items 18 to 21 above for binding to αvβ8 integrin. [Item 23] An antibody or antigen-binding fragment thereof according to any one of items 18 to 22 above for use in a method of treating renal fibrosis, which specifically binds to αvβ8 integrin and thereby treats renal fibrosis. [Item 24] An antibody or antigen-binding fragment thereof according to item 23 above, which specifically binds to αvβ8 integrin expressed in fibrotic renal cells and renal tissue, blocks the binding of latent TGF-β to αvβ8 integrin, and thereby inhibits the activity of αvβ8 integrin associated with renal fibrosis to treat renal diseases. [Item 25] A polynucleotide encoding the antibody or antigen-binding fragment thereof according to item 18 or item 19 above. [Item 26] A polynucleotide encoding the antibody or antigen-binding fragment thereof according to item 20 or item 21 above. [Item 27] The sequence encoding the V H region contains the nucleic acid sequence TIFF0007706458000024.tif29160, and the sequence encoding the V L region contains the nucleic acid sequence TIFF0007706458000025.tif29160. The polynucleotide according to item 26 above. [Item 28] An expression vector containing the polynucleotide according to any one of items 25 to 27 above. [Item 29] The expression vector according to item 28 above, which is an expression vector of prokaryote, eukaryote, or mammal. [Item 30] A cell containing the expression vector according to item 28 or 29 above. [Item 31] The cell according to item 30 above, which is a host cell of prokaryote, eukaryote, or mammal. [Item 32] A pharmaceutical composition containing the antibody or antigen-binding fragment thereof according to any one of items 18 to 24 above and a pharmaceutically acceptable carrier, excipient, or diluent. [Item 33] A pharmaceutical composition comprising the polynucleotide according to any one of items 25 to 27 above and a pharmaceutically acceptable carrier, excipient or diluent. [Item 34] A kit comprising an antibody or an antigen-binding fragment thereof that specifically binds to αvβ8 integrin according to any one of items 18 to 24 above, or a pharmaceutical composition comprising the antibody or the antigen-binding fragment thereof. From the foregoing description, it will be apparent that variations and modifications may be made to the present invention described herein to adapt it to various uses and conditions. Such embodiments are also within the scope of the following claims.

[0223] The citation of a list of elements in any definition of a variable herein includes the definition of the variable as any single element or combination (or sub-combination) of the recited elements. The detailed description of embodiments herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0224] All patents and publications described herein are incorporated herein by reference to the same extent as if each individual patent and publication were specifically and individually indicated to be incorporated by reference.

Claims

1. (a) A heavy chain variable region CDR1 comprising the amino acid sequence RSWIS ; (b) A heavy chain variable region CDR2 comprising the amino acid sequence EINPDSSSTINYTSSSL ; (c) A heavy chain variable region CDR3 comprising the amino acid sequence LITTEDY ; and (d) A light chain variable region CDR1 comprising the amino acid sequence KASQDINKYLS ; (e) A light chain variable region CDR2 comprising the amino acid sequence YANRLVD ; and (f) A light chain variable region CDR3 comprising the amino acid sequence LQYDVFPYT An anti-αvβ8 integrin antibody or an antigen-binding fragment thereof comprising the same.

2. EVQLVESGGGLVQPGGSLRLSCAVSGFVFSRSWISWVRQAPGKGLEWIGEINPDSSSTINYTSSSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAILITTEDYWGQGTTVTVSS and Heavy chain variable region (V Ｈ ) amino acid sequence DIQLTQSPSSLSASVGDRTITCKASQDINKYLSWFQQKPGKAPKSLIYYANRLVDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDVFPYTFGGGTKVEIK Light chain variable region (V Ｌ ) amino acid sequence An anti-αvβ8 integrin antibody or an antigen-binding fragment thereof according to Claim 1, comprising the same.

3. A pharmaceutical composition for treating renal fibrosis in a subject having a renal disease, comprising the antibody or an antigen-binding fragment thereof according to Claim 1 or 2, and a pharmaceutically acceptable carrier, excipient or diluent.

4. A pharmaceutical composition for treating kidney injury characterized by an increase in plasma creatinine and / or urinary protein excretion levels in a subject, comprising the antibody or an antigen-binding fragment thereof according to Claim 1 or 2, and a pharmaceutically acceptable carrier, excipient or diluent, wherein the subject may have a renal disease.

5. (i) The renal disease is selected from diabetic nephropathy (DN), chronic kidney disease (CKD), acute kidney disease, hypertension-related renal disease, hyperglycemia-related renal disease, renal fibrosis, inflammation-related renal disease, end-stage renal disease (ESRD), autoimmune-related renal fibrosis (e.g., lupus nephritis), and fibrosis after kidney transplantation; and / or (ii) The antibody or an antigen-binding fragment thereof binds to αvβ8 integrin expressed in renal cells and / or renal tissue, and blocks the activation of TGF-β from its latent form in the renal cells and / or renal tissue. The pharmaceutical composition according to Claim 3 or 4.

6. ​ The pharmaceutical composition according to any one of claims 3 to 5, which reduces or suppresses fibrosis associated with increased expression of ανβ8 integrin in podocytes and interstitial tubular cells of the renal tissue of the subject having a kidney disease.

7. The pharmaceutical composition according to any one of claims 3 to 6, which is administered to the subject as an adjuvant therapeutic agent for kidney disease or in combination with a treatment.

8. A polynucleotide encoding the antibody or an antigen-binding fragment thereof according to claim 1 or claim 2.

9. V Ｈ The array encoding the region is a nucleic acid sequence 【Chemical 1】 comprising V Ｌ The array encoding the region is a nucleic acid sequence 【Chemical 2】 The polynucleotide according to claim 8, comprising

10. An expression vector comprising the polynucleotide according to claim 8 or 9.

11. A cell comprising the expression vector according to claim 10.

12. A pharmaceutical composition comprising the polynucleotide according to claim 8 or 9 and a pharmaceutically acceptable carrier, excipient or diluent.

13. A kit comprising the anti-ανβ8 integrin antibody or an antigen-binding fragment thereof according to claim 1 or claim 2, or a pharmaceutical composition comprising the antibody or an antigen-binding fragment thereof.

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