ANTI-avB8 INTEGRIN ANTIBODIES FOR USE IN TREATING KIDNEY DISEASE
20230112035 · 2023-04-13
Inventors
- David James BAKER (Cambridge, GB)
- Stephanie Claire HEASMAN (Cambridge, GB)
- Maria Marcela HERRERA (Cambridge, GB)
- Elena LIARTE MARIN (Cambridge, GB)
- Carol Patricia MORENO-QUINN (Cambridge, GB)
- Lynne Anne MURRAY (Cambridge, GB)
- Ping Tsui (Gaithersburg, MD)
- Yanli WU (Gaithersburg, MD, US)
Cpc classification
C07K2317/76
CHEMISTRY; METALLURGY
G01N2333/70546
PHYSICS
C07K2317/92
CHEMISTRY; METALLURGY
C07K2317/24
CHEMISTRY; METALLURGY
International classification
Abstract
Provided are methods and compositions for treating kidney disease, such as chronic kidney disease (CKD), in which the methods and compositions comprise antibodies or an antigen binding fragment thereof that specifically and selectively bind to human αvβ8 integrin, which was discovered, as described, to be highly expressed on kidney cells and tissue, and, in particular, diseased or fibrotic kidney 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 kidney tissue. The anti-αvβ8 antibodies in the disclosed methods reduce, attenuate, or abrogate kidney fibrosis, which is associated with the activities of αvβ8 integrin and TGF-β in kidney tissue. The disclosed antibodies and methods effectively treat kidney disease, in particular, fibrosis associated with kidney disease, such as CKD, in individuals in need thereof.
Claims
1. A method of treating kidney fibrosis in a subject having kidney disease, the method comprising administering to the subject an effective amount of an anti-αvβ8 integrin antibody or an antigen binding fragment thereof, thereby treating kidney fibrosis.
2. A method of reducing or attenuating kidney fibrosis in a subject having kidney disease, 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, thereby reducing or attenuating fibrosis in the kidney.
3. A method of abrogating the activity of αvβ8 integrin associated with kidney fibrosis, 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, thereby abrogating the activity of αvβ8 integrin associated with kidney fibrosis.
4. A method of treating kidney fibrosis by blocking the activation of TGF-β from its latent form in kidney cells and 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, thereby treating the kidney fibrosis.
5. A method of treating kidney damage 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 said administration of the anti-αvβ8 integrin antibody or an antigen binding fragment thereof abrogates the plasma creatinine and/or urinary protein excretion levels in the subject, thereby treating kidney damage.
6. The method of any one of claims 3-5, wherein the subject has kidney disease.
7. The method of any one of claims 1-6, wherein the kidney disease is selected from diabetic nephropathy (DN), chronic kidney disease (CKD), acute kidney disease, hypertension-associated kidney disease, hyperglycemia-associated kidney disease, renal fibrosis, inflammation-associated kidney disease, end stage renal disease (ESRD), autoimmune-associated kidney fibrosis (for example, lupus nephritis) and fibrosis post-kidney transplant.
8. The method of claim 7, wherein the kidney disease is CKD.
9. The method of any one of claims 1-8, wherein the antibody or an antigen binding fragment thereof binds to αvβ8 integrin expressed on kidney cells and/or tissue and blocks the activation of TGF-β from its latent form in the kidney cell and/or tissue.
10. A method of detecting kidney fibrosis in kidney tissue, the method comprising contacting kidney tissue with an effective amount of a detectably labeled anti-αvβ8 integrin antibody or an antigen binding fragment thereof, thereby detecting the binding of the anti-αvβ8 integrin antibody to αvβ8 integrin in the kidney tissue.
11. The method of any one of claims 1-10, wherein the anti-αvβ8 integrin antibody, or an antigen-binding fragment thereof, comprises: (a) a heavy chain variable region complementarity determining region 1 (CDR1) comprising the amino acid sequence: TABLE-US-00062 RYWMS; (b) a heavy chain variable region complementarity determining region 2 (CDR2) CDR2 comprising the amino acid sequence: TABLE-US-00063 EINPDSSTINYTSSL; and (c) a heavy chain variable region complementarity determining region 3 (CDR3) CDR3 comprising the amino acid sequence: TABLE-US-00064 LITTEDY; and (d) a light chain variable region CDR1 comprising the amino acid sequence: TABLE-US-00065 KASQDINSYLS; (e) a light chain variable region CDR2 comprising the amino acid sequence: TABLE-US-00066 YANRLVD; and (f) a light chain variable region CDR3 comprising the amino acid sequence: TABLE-US-00067 LQYDEFPYT.
12. The method of claim 11, wherein the anti-αvβ8 integrin antibody, or an antigen-binding fragment thereof, comprises a heavy chain variable region (V.sub.H) amino acid sequence: TABLE-US-00068 EVQLVESGGGLVQPGGSLRLSCAVSGFVFSRYWMSWVRQAPGKGLEWIGE INPDSSTINYTSSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAILI TTEDYWGQGTTVTVSS; and a light chain variable region (V.sub.L) amino acid sequence: TABLE-US-00069 DIQLTQSPSSLSASVGDRVTITCKASQDINSYLSWFQQKPGKAPKSLIYY ANRLVDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPYTFGG GTKVEIK.
13. The method of any one of claims 1-10, wherein the anti-αvβ8 integrin antibody, or an antigen-binding fragment thereof, comprises: (a) a heavy chain variable region CDR1 comprising the amino acid sequence: TABLE-US-00070 RSWIS; (b) a heavy chain variable region CDR2 comprising the amino acid sequence: TABLE-US-00071 EINPDSSTINYTSSL; and (c) a heavy chain variable region CDR3 comprising the amino acid sequence: TABLE-US-00072 LITTEDY; and (d) a light chain variable region CDR1 comprising the amino acid sequence: TABLE-US-00073 KASQDINKYLS; (e) a light chain variable region CDR2 comprising the amino acid sequence: TABLE-US-00074 YANRLVD; and (f) a light chain variable region CDR3 comprising the amino acid sequence: TABLE-US-00075 LQYDVFPYT.
14. The method of claim 13, wherein the anti-αvβ8 integrin antibody, or an antigen-binding fragment thereof, comprises a heavy chain variable region (V.sub.H) amino acid sequence: TABLE-US-00076 EVQLVESGGGLVQPGGSLRLSCAVSGFVFSRSWISWVRQAPGKGLEWIGE INPDSSTINYTSSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAILI TTEDYWGQGTTVTVSS and a light chain variable region (V.sub.L) amino acid sequence: TABLE-US-00077 DIQLTQSPSSLSASVGDRVTITCKASQDINKYLSWFQQKPGKAPKSLIYY ANRLVDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDVFPYTFGG GTKVEIK.
15. The method of any one of claims 1-14, wherein the antibody, or an antigen binding fragment thereof, attenuates or abrogates fibrosis associated with increased expression of αvβ8 integrin in podocytes and interstitial tubule cells in kidney tissue of the subject with kidney disease.
16. The method of any one of claims 1-9, or 11-15, wherein the antibody or an antigen binding fragment thereof, is administered to the subject in combination with an adjunct therapeutic agent or treatment for kidney disease.
17. The method of claim 16, wherein the antibody or an antigen binding fragment thereof, is administered to the subject prior to, at the same time as, or after the administration of the adjunct therapeutic agent or treatment.
18. An anti-αvβ8 integrin antibody, or an antigen binding fragment thereof, comprising: (a) a heavy chain variable region CDR1 comprising the amino acid sequence: TABLE-US-00078 RYWMS; (b) a heavy chain variable region CDR2 comprising the amino acid sequence: TABLE-US-00079 EINPDSSTINYTSSL; and (c) a heavy chain variable region CDR3 comprising the amino acid sequence: TABLE-US-00080 LITTEDY; and (d) a light chain variable region CDR1 comprising the amino acid sequence: TABLE-US-00081 KASQDINSYLS; (e) a light chain variable region CDR2 comprising the amino acid sequence: TABLE-US-00082 YANRLVD; (f) a light chain variable region CDR3 comprising the amino acid sequence: TABLE-US-00083 LQYDEFPYT.
19. The anti-αvβ8 integrin antibody or an antigen binding fragment thereof of claim 18, comprising a heavy chain variable region (V.sub.H) amino acid sequence: TABLE-US-00084 EVQLVESGGGLVQPGGSLRLSCAVSGFVFSRYWMSWVRQAPGKGLEWIGE INPDSSTINYTSSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAILI TTEDYWGQGTTVTVSS; and a light chain variable region (V.sub.L) amino acid sequence: TABLE-US-00085 DIQLTQSPSSLSASVGDRVTITCKASQDINSYLSWFQQKPGKAPKSLIYY ANRLVDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPYTFGG GTKVEIK.
20. An anti-αvβ8 integrin antibody or an antigen binding fragment thereof, comprising: (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 EINPDSSTINYTSSL; (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 a light chain variable region CDR3 comprising the amino acid sequence LQYDVFPYT.
21. The anti-αvβ8 integrin antibody or an antigen binding fragment thereof of claim 20, comprising a heavy chain variable region (V.sub.H) amino acid sequence: TABLE-US-00086 EVQLVESGGGLVQPGGSLRLSCAVSGFVFSRSWISWVRQAPGKGLEWIGE INPDSSTINYTSSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAILI TTEDYWGQGTTVTVSS and a light chain variable region (V.sub.L) amino acid sequence: TABLE-US-00087 DIQLTQSPSSLSASVGDRVTITCKASQDINKYLSWFQQKPGKAPKSLIYY ANRLVDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDVFPYTFGG GTKVEIK.
22. An anti-αvβ8 integrin antibody or an antigen binding fragment thereof that competes for binding to αvβ8 integrin with the antibody or an antigen binding fragment thereof of any one of claims 18-21.
23. The antibody or an antigen binding fragment thereof of any one of claims 18-22, for use in a method of treating kidney fibrosis, wherein said antibody or an antigen binding fragment thereof specifically binds to αvβ8 integrin, thereby treating kidney fibrosis.
24. The antibody or an antigen-binding fragment thereof of claim 23, wherein said antibody or an antigen-binding fragment thereof specifically binds to αvβ8 integrin expressed on fibrotic kidney cells and tissue and blocks binding of αvβ8 integrin to latent TGF-β, thereby abrogating the activity of αvβ8 integrin associated with kidney fibrosis to treat kidney disease.
25. A polynucleotide encoding the antibody or an antigen binding fragment thereof of claim 18 or claim 19.
26. A polynucleotide encoding the antibody or an antigen binding fragment thereof of claim 20 or claim 21.
27. The polynucleotide of claim 26, wherein the V.sub.H region coding sequence comprises nucleic acid sequence: TABLE-US-00088 gaggtgcagctggtggaaagcggcggaggactggtgcagcctggcggcag cctgagactgagctgcgccgtgtccggcttcgtgttcagccggagctgga tcagctgggtccgccaggccccagggaagggcctggaatggatcggcgag atcaaccccgacagcagcaccatcaactacaccagcagcctgaaggaccg gttcaccatcagccgggacaacgccaagaacagcctgtacctgcagatga acagcctgcgggccgaggacaccgccgtgtactactgcgccatcctcatc accaccgaggactactggggccagggcaccaccgtgaccgtgtcctct; and the V.sub.L region coding sequence comprises nucleic acid sequence: TABLE-US-00089 gacatccagctgacccagagccccagcagcctgagcgccagcgtgggcga cagagtgaccatcacatgcaaggccagccaggacatcaacaagtacctga gctggttccagcagaagcccggcaaggcccccaagagcctgatctactac gccaaccggctggtggacggcgtgcccagcagattttctggcagcggcag cggcaccgacttcaccctgaccatcagcagcctgcagcccgaggacttcg ccacctactactgcctgcagtacgacgtgttcccctacaccttcggcgga ggcaccaaggtggaaatcaag.
28. An expression vector which comprises the polynucleotide of any one of claims 25-27.
29. The expression vector of claim 28, which is a prokaryotic, eukaryotic, or mammalian expression vector.
30. A cell comprising the expression vector of claim 28 or claim 29.
31. The cell of claim 30, which is a prokaryotic, a eukaryotic, or a mammalian host cell.
32. A pharmaceutical composition comprising the antibody or an antigen-binding fragment thereof of any one of claims 18-24, and a pharmaceutically acceptable carrier, excipient, or diluent.
33. A pharmaceutical composition comprising the polynucleotide of any one of claims 25-27, and a pharmaceutically acceptable carrier, excipient, or diluent.
34. A kit comprising the antibody or an antigen binding fragment thereof that specifically binds to αvβ8 integrin of any one of claims 18-24, or a pharmaceutical composition comprising the antibody or the antigen binding fragment thereof.
Description
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0148]
[0149]
[0150]
[0151]
[0152]
[0153]
[0154] As shown in
TABLE-US-00049 (SEQ ID NO: 20) EVQLVESGGGLVQPGGSLNLSCAVSGFVFSRYWMSWVRQAPGKGLEWIGEI NPDSSTINYTSSLKDKFIISRDNAKNTLYLQMNKVRSEDTALYYCACLITT EDYWGQGTSVTVSS.
The nucleotide sequence of the V.sub.H region of the Chi-37E1B5 antibody is as follows:
TABLE-US-00050 (SEQ ID NO: 21) gaagtgcagctggtggagtctggaggtggcctggtgcagcctggaggatcc ctgaacctctcctgtgcagtctcaggattcgtttttagtagatactggatg agttgggtccggcaggctccagggaaagggctagaatggattggagaaatt aatccagatagcagtacgataaactatacgtcatctctaaaggataaattc atcatctccagagacaacgccaaaaatacgttgtacctgcaaatgaacaaa gtgagatctgaggacacagccctttattactgtgcatgtcttattactacg gaggactactggggtcaaggaacctcagtcaccgtctcctca.
The amino acid sequence of the V.sub.L (kappa) region of the Chi-37E1B5 anti-αvβ8 integrin antibody is as follows:
TABLE-US-00051 (SEQ ID NO: 22) EIVLTQSPSSMYASLGERVTIPCKASQDINSYLSWFQQKPGKSPKTLIYYA NRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGGGT KLEIK.
The nucleotide sequence of the V.sub.L (kappa) region of the Chi-37E1B5 antibody is as follows:
TABLE-US-00052 (SEQ ID NO: 23) gaaattgtgctgactcagtctccatcttccatgtatgcatctctaggagag agagtcactatcccttgcaaggcgagtcaggacattaatagctatttaagc tggttccagcagaaaccagggaaatctcctaagaccctgatctattatgca aacagattggtagatggggtcccatcaaggttcagtggcagtggatctggg caagattattctctcaccatcagcagcctggagtatgaagatatgggaatt tattattgtctacagtatgatgagtttccgtacacgttcggaggaggcacc aagctggaaatcaaa.
Also in
TABLE-US-00053 (SEQ ID NO: 24) EVQLVESGGGLVQPGGSLRLSCAVSGFVFSRYWMSWVRQAPGKGLEWIGEI NPDSSTINYTSSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASLITT EDYWGQGTTVTVSS.
The amino acid sequence of the V.sub.L (kappa) region of the UCSF hu37E1B5 antibody is as follows:
TABLE-US-00054 (SEQ ID NO: 25) EIVLTQSPSSLSLSPGERVTITCKASQDINSYLSWYQQKPGKAPKLLIYYA NRLVDGVPARFSGSGSGQDYTLTISSLEPEDFAVYYCLQYDEFPYTFGGGT KLEIK.
[0155]
[0156]
[0157]
[0158] The IHC staining photomicrographs shown in
[0159] As shown in
[0160] As shown in
[0161] As shown in
[0162] As shown in
[0163] As shown in
[0164] As shown in
[0165]
[0166]
DETAILED DESCRIPTION
[0167] The present disclosure generally features antibodies, compositions and methods for treating kidney disease, e.g., diabetic nephropathy (DN), chronic kidney disease (CKD), acute kidney disease, hypertension-associated kidney disease, hyperglycemia-associated kidney disease, renal fibrosis, inflammation-associated kidney disease, end stage renal disease (ESRD), autoimmune-associated kidney fibrosis (for example, lupus nephritis) and fibrosis post-kidney transplant, and the like, in an individual in need as described herein. In particular, the antibodies, compositions and methods are directed to treating kidney fibrosis, which is associated with kidney disease, such as CKD.
[0168] The present disclosure is directed to a treatment method for ameliorating, attenuating, abrogating, reducing, or alleviating fibrosis in kidney tissue in a subject having kidney disease, such as CKD. In general, fibrosis refers to the formation of excess fibrous connective tissue (scar tissue) in an organ such as the kidney, which causes thickening and scarring of the kidney connective tissue. As noted supra, the methods involve the administration of an anti-αvβ8 integrin antibody, or an antigen binding fragment thereof, which specifically binds to αvβ8 integrin found to be highly expressed on diseased kidney cells and in kidney tissue, particularly, kidney epithelial cells and tissue in subjects having kidney disease, such as CKD. The anti-αvβ8 integrin antibodies, or an antigen binding fragment thereof, selectively bind to αvβ8 integrin on fibrotic kidney cells and tissues, thereby blocking, neutralizing, or inhibiting the interaction of the kidney-expressed αvβ8 integrin with the latent form of TGF-β (LAP-TGF-β) at the kidney cell surface. The anti-αvβ8 integrin antibody binding interferes with the αvβ8 integrin/LAP TGF-β interaction, which, in turn, blocks or prevents the activation of TGF-β at the kidney cell surface, so that active TGF-β is not produced and thus cannot exert its cellular effects associated with kidney fibrosis in the kidney tissue of a subject, such as a human or a non-human subject. The methods provide therapeutic benefit, particularly in the treatment of kidney disease, for example, by reducing, attenuating, abrogating, or decreasing the damaging fibrosis induced by active TGF-β in kidney disease, e.g., CKD.
[0169] Without wishing to be bound by a particular theory, the anti-αvβ8 integrin antibody, or an antigen-binding fragment thereof, reduces local TGF-β activation in kidney cells and tissue where αvβ8 integrin is highly expressed, for example, by directly binding to the αvβ8 integrin receptor of LAP TGF-β. The binding of an anti-αvβ8 integrin antibody to αvβ8 integrin, which blocks the activation of TGF-β from its latent form, may also reduce or prevent recruitment of the protease that cleaves latent TGF-β and releases the mature, active TGF-β peptide. This could occur by the anti-αvβ8 integrin antibody inhibiting the binding of αvβ8 integrin on the kidney cell surface to latent TGF-β associated with the cell matrix, thereby inhibiting the subsequent activation of TGF-β as described infra.
Transforming Growth Factor Beta (n), (TGF-β) and its Interaction with αvβ8 Integrin
[0170] In cells, the TGF-β cytokine is synthesized and secreted to the extracellular matrix as an inactive precursor that is complexed to a “latency-associated peptide (LAP)” and a “latent TGFβ binding protein (LTBP).” The latent form of TGF-β must be activated in order to bind to its receptor, e.g., αvβ8 integrin, and have biological function (J. J. Worthington et al., 2011a, Trends Biochem. Sci., 36:47-54). The LAP is cleaved from the active TGF-β, but remains non-covalently attached in a conformation that prevents TGF-β from engaging its receptor. Activators of TGF-β include a variety of proteases and cell surface molecules that alter the latent complex allowing active TGF-β to engage its receptor. Putative TGF-β activators include, without limitation, proteases that degrade LAP, thrombospondin-1, reactive oxygen species (ROS) and integrins. Activation of the latent complex is thus essential for the regulation of TGF-β function, and TGF-β activators are the rate-limiting step in the conversion of latent to active TGF-β. By way of example, in human CKD kidneys (n=4), the amount of latent TGF-β is 53-fold higher than that of active TGF-β.
[0171] Fibrosis is an important driver of chronic kidney disease (CKD) progression in human patients and correlates with renal dysfunction and damage. TGF-β is involved in the development of renal fibrosis in CKD. Renal TGF-β is upregulated in human fibrotic CKD versus control kidney (D. S. Goumenos et al., 2002, Nephrol. Dial. Transplant., 17:2145-2152). Urinary TGF-β was shown to correlate with renal damage (albuminuria) in Type 2 diabetes. (Marwood et al., 2002, Exp. Biol. Med., 227(11):943-956).
[0172] The αv-integrin transmembrane receptors, e.g., αvβ8, are important players in the regulation of extracellular matrix physiology and in the activation of TGF-β. Briefly, αv-integrins mediate 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).
[0173] Unlike the activity of other αvβ integrins, αvβ8 integrin is constitutively active, and the activation of LAP TGF-β (by release of active TGF-β cytokine after binding of LAP TGF-β to αvβ8 integrin) is mediated by cleavage by the MMP-14 protease, rather than by anchoring to cytoplasmic actin (no traction effect). αvβ8 integrin expression is enriched in kidney tissue, and the gene encoding αvβ8 integrin is highly expressed in kidney tissue compared with other tissues, such as, for example, pancreas, liver, gallbladder, salivary gland, esophagus, stomach, intestine, lung, heart, or bladder, as exemplified infra.
[0174] As described herein, both in vitro and in vivo studies have demonstrated that high levels of expression of αvβ8 integrin on kidney epithelial cells directly correlated with high levels of kidney tissue fibrosis resulting from the activation of TGF-β. High levels of TGF-β activity induce and increase damage to renal (kidney) cells and tissue, causing fibrosis, and thus seriously exacerbate kidney disease, such as CKD. The anti-αvβ8 integrin antibodies described herein specifically bind to αvβ8 integrin expressed by kidney cells and inhibit TGF-β's destructive activity and consequent fibrosis in kidney cells and tissue by blocking and/or reducing αvβ8 integrin's binding to latent TGF-β (LAP TGF-β) and inhibiting release of the active form of TGF-β. This action of the specific anti-αvβ8 integrin antibodies serves as a treatment against kidney cell and tissue destruction resulting from TGF-β activity, e.g., by inhibiting TGF-β's intracellular signaling cascade. In accordance with the methods disclosed and exemplified herein, blocking αvβ8 integrin activity by providing antibodies that specifically bind αvβ8 integrin in the kidney significantly reduce activation of TGF-β localized in kidney and thereby specifically reducing kidney cell and tissue damage, namely, kidney fibrosis, in diseased kidneys.
[0175] The use of anti-αvβ8 integrin antibodies that specifically target the αvβ8 integrin receptor on kidney cells stemmed from the discoveries, as described herein, that αvβ8 integrin is preferentially expressed in the kidney in normal subjects and that the expression of αvβ8 integrin is significantly increased and localized in kidney epithelial cells (e.g., podocytes and interstitial tubules) in the kidneys of subjects with fibrotic kidney disease, e.g., human patients with CKD. As noted supra, the present disclosure provides surprising findings that the αvβ8 protein is highly up-regulated in kidneys of human patients with CKD. Moreover, the activation of TGF-β by the specific binding of αvβ8 integrin to the latent active form of TGF-β in the kidney is a direct cause of destructive fibrosis in kidney tissue. The present discoveries are contradictory to a prior finding in the art that αvβ8 integrin is found primarily in mesangial cells of the kidney and that transgenic animals which did not express mesangial cell αvβ8 integrin nevertheless harbored active TGF-β that caused endothelial cell apoptosis (S. Khan et al., 2011, Am. J. Pathology, 178(2):609-620). By contrast and as described and exemplified herein, the present methods involve the inhibition and blockage of αvβ8 integrin's interaction with and binding to LAP-TGF-β, such that active TGF-β is not released at the kidney cell membrane and is not able to cause fibrosis (and/or further damage) to kidney cells and tissue in subjects afflicted with kidney disease such as CKD.
Antibodies Specifically Directed Against αvβ8 Integrin
[0176] The present disclosure encompasses the development and use of antibodies that are directed against and specifically target and bind to αvβ8 integrin, particularly, to αvβ8 integrin expressed in kidney cells and tissue and in fibrotic kidney cells and tissue. These antibodies, or antigen binding fragments thereof, are of great benefit in methods of treating kidney disease, particularly, kidney fibrosis in kidney disease, such as chronic kidney disease (CKD), in a subject in need of treatment. In embodiments, the subject may have a condition that is associated with damage or injury to kidney cells and tissue and that causes fibrosis of kidney tissue as described herein, or an acute, chronic, or end stage kidney disease. Treatment of subjects having kidney fibrosis and kidney disease involving fibrosis, regardless of the etiology, using the antibodies, compositions and methods described herein provides an important medical and clinical benefit to subjects in need, especially patients afflicted with kidney disease, such as CKD or DN. In an embodiment, the anti-αvβ8 integrin antibody is a humanized antibody.
[0177] In one embodiment, the anti-αvβ8 integrin antibody is a humanized antibody, referred to as “MEDI-hu37E1B5” antibody as described supra, which specifically targets and binds to human αvβ8 integrin. In a particular embodiment, the MEDI-hu37E1B5 antibody specifically targets and binds to human αvβ8 integrin that is expressed in the kidney and that is highly expressed in fibrotic kidney. In an embodiment, the MEDI-hu37E1B5 antibody does not cross-react with antibodies against other integrins.
[0178] In another embodiment, the anti-αvβ8 integrin antibody is a humanized and affinity optimized antibody, referred to as “B5-15” anti-αvβ8 integrin antibody as described supra, which specifically targets and demonstrates high affinity binding to the human αvβ8 integrin. The optimized B5-15 antibody is of the IgG1 subtype, demonstrates specific and selective binding to human αvβ8 integrin and exhibits functional activity by blocking or inhibiting the binding interaction or association between human αvβ8 integrin with TGF-β latent form, thus blocking or inhibiting the activation of TGF-β by release of active TGF-β from its latent form. As demonstrated herein (
[0179] The B5-15 antibody blocks the binding of αvβ8 integrin to LAP-TGF-β and blocks the activation of TGF-β and intracellular signaling by TGF-β, thus protecting kidney cells and tissue from the damaging effects of the active TGF-β peptide, which can induce and exacerbate fibrosis. Without wishing to be bound by theory, the B5-15 antibody allosterically modifies the αvβ8 integrin and reduces its affinity for the latent TGF-β (LAP) binding domain, which prevents the activation of TGF-β from its latent form so that no active TGF-β peptide is released. Thus, the antibody induces a conformational change in αvβ8 integrin, such that αvβ8 can no longer bind to latent TGF-β to facilitate its activation (WO 2015/195835). Until the present disclosure, the binding properties and functional activities of anti-αvβ8 integrin antibodies, such as the B5-15 antibody, in renal (kidney) fibrosis were unknown.
[0180] In embodiments, the anti-αvβ8 integrin antibodies disclosed herein specifically bind to the αvβ8 integrin receptor that has elevated expression on kidney cells and tissue, particularly diseased, damaged, and/or fibrotic kidney tissue such as is found in individuals with kidney disease, e.g., CKD or DN. Compositions comprising these antibodies and their use in methods of treating kidney disease and nephropathy, particularly, kidney disease involving fibrosis, are encompassed by the present disclosure. The described antibodies bind only to human αvβ8 integrin and do not cross-react with any other integrins.
[0181] The described antibodies are also advantageous because they selectively target and specifically bind to the αvβ8 integrin receptor for latent TGF-β and do not directly target the cytokine itself, thus providing a safer therapeutic approach for treating kidney disease, particularly, kidney disease involving fibrosis. In addition, the inhibition, blocking, or neutralization of the activity of the TGF-β1 isoform is especially advantageous, as this TGF-β isoform is generally considered to account for the majority of the disease-related activity of TGF-β. The prevalence of the TGF-β1 isoform in kidney is likely to result in the involvement of the active form of TGF-β1 in kidney fibrosis and kidney disease.
[0182] While targeting TGF-β directly may be one approach for inhibiting or preventing pathologies caused by TGF-β activity, a general neutralization and/or chronic inhibition of the actions of TGF-β resulting from directly targeting the cytokine could have grave side effects in the treated individual, given the involvement of TGF-β in modulating diverse cellular functions and pathways. Thus, the approach of using anti-αvβ8 integrin antibodies as provided herein to block, inhibit, neutralize, and thus effectively prevent, the αvβ8 integrin/LAP TGF-β interaction on kidney cells and tissue without compromising in vivo TGF-β activation in other cells, tissues and organs, or for other physiological purposes, provides a valuable therapeutic tool and method for treating kidney disease and fibrosis, such as CKD or DN. Advantageously, the specificity of the anti-αvβ8 antibodies described herein for kidney cells and tissue expressing high levels of the αvβ8 integrin decreases adverse effects, such as autoimmune responses, rapid-onset atherosclerosis and carcinoma development. Adverse effects have been seen with pan-TGF-β inhibition, therefore specifically targeting αvβ8 integrin to affect TGF-β activation is likely to result in reduced adverse events.
[0183] As another advantage, the anti-αvβ8 integrin antibodies described herein do not cross the blood-brain-barrier (BBB) and thus cannot result in binding to αvβ8 integrin expressed on cells and tissue of the brain.
[0184] The described anti-αvβ8 antibodies specifically block the binding of kidney epithelial cell-expressed αvβ8 integrin to the latent form of TGF-β and thus block fibrosis caused by the release in kidney tissue of active TGF-β, which has been found to play a central role in the glomerular and tubule-interstitial pathobiology of renal disease that induce alterations of glomerular filtration barrier, glomerulosclerosis and fibrosis, as well as the degeneration of tubules leading to permanent renal dysfunction. Accordingly, the present methods involve the use of specific anti-αvβ8 integrin antibodies to treat kidney disease, such as chronic kidney disease or diabetic nephropathy characterized by deleterious kidney tissue fibrosis, by specifically binding to a target receptor, i.e., αvβ8 integrin, that is highly expressed on the surface of kidney cells in individuals having damaged kidneys and/or kidney disease, rather than targeting TGF-β itself. The targeting and binding of αvβ8 integrin by the specific anti-αvβ8 integrin antibodies provided herein abrogates and effectively prevents TGF-β cytokine activity that is a major culprit in causing kidney tissue fibrosis and further damage to kidney tissue in kidney disease.
[0185] The anti-αvβ8 integrin antibodies described herein specifically bind to one or more regions of the αvβ8 integrin receptor protein that contain antigen binding sites or epitopes. In an embodiment, the epitope of αvβ8 integrin bound by the anti-αvβ8 integrin antibody, such as the MEDI-hu37E1B5 antibody or the B5-15 antibody was mapped to a region approximately 28A (Angstroms) away from the αvβ8 integrin and LAP-TGF-β binding site. (S. Minagawa et al., 2014, Sci. Transl. Med., 6(241): 241re79. Doi:10.1126/scitranslmed.3008074).
[0186] In an embodiment, an antibody that competes for binding to αvβ8 integrin with an antibody having a light chain variable region comprising the following three light chain CDRs: V.sub.L CDR1: KASQDINSYLS (SEQ ID NO: 4); V.sub.L CDR2: YANRLVD (SEQ ID NO: 5); and V.sub.L CDR3: LQYDEFPYT (SEQ ID NO: 6); and a heavy chain variable region comprising the following three heavy chain CDRs: V.sub.H CDR1: RYWMS (SEQ ID NO: 1); V.sub.H CDR2: EINPDSSTINYTSSL (SEQ ID NO: 2); and V.sub.H CDR3: LITTEDY (SEQ ID NO: 3) as described herein is contemplated. The antibody can be monoclonal, chimeric, humanized, etc., and can be of isotype IgG1, IgG2, IgG2a, IgG3 or IgG4. In a particular embodiment, the antibody is an IgG1 antibody. In some embodiments, the antibody is a human, humanized, or chimeric antibody.
[0187] In another embodiment, an antibody that competes for binding to αvβ8 integrin with an antibody having a light chain variable region comprising the following three light chain CDRs: V.sub.L CDR1: KASQDINKYLS (SEQ ID NO: 10); V.sub.L CDR2: YANRLVD (SEQ ID NO: 5); and V.sub.L CDR3: LQYDVFPYT (SEQ ID NO: 11); and a heavy chain variable region comprising the following three heavy chain CDRs: V.sub.H CDR1: RSWIS (SEQ ID NO: 9); V.sub.H CDR2: EINPDSSTINYTSSL (SEQ ID NO: 2); and V.sub.H CDR3: LITTEDY (SEQ ID NO: 3) as described herein is contemplated. The antibody can be monoclonal, chimeric, humanized, etc., and can be of isotype IgG1, IgG2, IgG2a, IgG3 or IgG4. In a particular embodiment, the antibody is an IgG1 antibody. In some embodiments, the antibody is a human, humanized, or chimeric antibody.
[0188] Also provided is an isolated polynucleotide encoding the described anti-αvβ8 integrin antibody or an antigen binding fragment thereof; a prokaryotic, eukaryotic, or mammalian vector or vectors; and host cells, (prokaryotic, eukaryotic, or mammalian), suitable for encoding and expressing the anti-αvβ8 integrin antibody or an antigen binding fragment thereof as described.
[0189] In other aspects, antibodies useful in the described methods and compositions include immunoglobulins, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies formed from at least two different αvβ8 integrin epitope binding fragments (e.g., bispecific antibodies), human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, single-chain Fvs (scFv), single-chain antibodies, single domain antibodies, domain antibodies, Fab fragments, F(ab′)2 fragments, antibody fragments that exhibit the desired biological activity (e.g., the antigen binding portion), disulfide-linked Fvs (dsFv), intrabodies, and antigen or epitope-binding fragments of any of the above. In particular, suitable antibodies include immunoglobulin molecules and immunologically and functionally active fragments of immunoglobulin molecules, e.g., molecules that contain at least one antigen-binding site.
[0190] Anti-αvβ8 integrin antibodies encompass monoclonal human, humanized or chimeric anti-αvβ8 integrin antibodies. Anti-αvβ8 integrin antibodies used in compositions and methods described herein can be naked antibodies, immunoconjugates, or fusion proteins. In certain embodiments, an anti-αvβ8 integrin antibody is a human, humanized, or chimeric antibody of the IgG isotype, particularly an 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 characteristics, 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)). The human IgG class antibody is further classified into the following subclasses: IgG1, IgG2, IgG3 and IgG4. In an 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 an embodiment, the anti-αvβ8 integrin antibody is a humanized antibody containing human framework regions and CDRs from a parent antibody, such as the MEDI-hu37E1B5 antibody. In another embodiment, the anti-αvβ8 integrin antibody comprises an optimized amino acid sequence to improve one or more antibody properties, including specificity for antigen, function, stability, half-life/longevity and the like.
Treatment Methods Involving Administration of an Anti-αvβ8 Integrin Antibody
[0191] The described methods provide treatment of kidney disease, especially fibrotic kidney disease, and, in particular, chronic kidney disease (CKD) in which kidney function is reduced over a period of time and extensive fibrosis of kidney tissue typically occurs and is exacerbated over time. In general, the five stages of CKD are: Stage 1, characterized by kidney damage with normal kidney function (estimated glomerular filtration rate (GFR)≥90 mL/min per 1.73 m.sup.2) and persistent (≥3 months) proteinuria; Stage 2, characterized by kidney damage with mild loss of kidney function (estimated GFR 60-89 mL/min per 1.73 m.sup.2) with or without persistent (≥3 months) proteinuria; Stage 3, characterized by mild-to-severe loss of kidney function (estimated GFR 30-59 mL/min per 1.73 m.sup.2); Stage 4, characterized by severe loss of kidney function (estimated GFR 15-29 mL/min per 1.73 m.sup.2); and Stage 5, characterized by kidney failure requiring dialysis or transplant for survival. Stage 5 CKD is also known as ESRD (estimated GFR<15 mL/min per 1.73 m.sup.2). Glomerular filtration rate (GFR), measured in milliliters per minute (mL/min), refers to the rate at which the kidneys filter wastes and extra fluids from the blood.
[0192] The described methods involving administration of an anti-αvβ8 integrin antibody or an antigen-binding fragment thereof are also useful for treating kidney disease and/or fibrosis associated with damage or injury to kidney cells and tissue, as caused, for example, by diabetic nephropathy (DN), chronic kidney disease (CKD), acute kidney disease, hypertension-associated kidney disease, hyperglycemia-associated kidney disease, renal fibrosis, inflammation-associated kidney disease, end stage renal disease (ESRD), autoimmune-associated kidney fibrosis (for example, lupus nephritis) and fibrosis post-kidney transplant, and the like. General and localized tissue inflammation in the kidney contributes to the pathophysiology and progression of diabetic nephropathy. The conditions of hyperglycemia and hypertension that typically accompany diabetic nephropathy, can further lead to glomerular hypertension and mechanical stress on kidney cells and tissue, podocyte injury and detachment, inflammation of the glomerulus and inflammation of the kidney tubules, all of which results in fibrosis (scarring) in the kidney, and more particularly, in the kidney glomerulus and tubules.
Combination Treatments
[0193] In another embodiment, one or more of the anti-αvβ8 integrin antibodies may be administered in conjunction with another drug, medication, or therapeutic agent or compound, such as would be provided to a patient having kidney disease or CKD. As is frequently the case, individuals who have kidney disease or CKD also have high blood pressure. Medicines and drugs that lower blood pressure help to maintain blood pressure in a target range and delay or stop further kidney damage. Common blood pressure medications include, without limitation, acetylcholine esterase (ACE) inhibitors, angiotensin II receptor blockers (ARBs), beta blockers, calcium channel blockers, direct renin inhibitors, diuretics and vasodilators. Medications and drugs that are administered to treat the symptoms and complications of CKD include, without limitation, erythropoietin (EPO), (recombinant human erythropoietin, rhEPO), electrolyte imbalance correcting medicines, diuretics, ACE inhibitors and ARBs, as well as iron therapy and vitamin D.
[0194] In co-therapy, one or more anti-αvβ8 integrin antibodies may be optionally included in the same pharmaceutical composition as the other drug or medication. Alternatively, an anti-αvβ8 integrin antibody may be in a separate pharmaceutical composition and may be administered at the same time or at a different time from one or more other drugs or medications. An anti-αvβ8 integrin antibody as described herein, or a pharmaceutical composition comprising the anti-αvβ8 integrin antibody is suitable for administration prior to, simultaneously with, or following the administration of another drug or medication, or a pharmaceutical composition comprising the drug or medication. In certain instances, the administration of one or more of the anti-αvβ8 integrin antibodies to a subject overlaps with the time of administration of another or companion drug or medication provided separately or in a separate composition.
Pharmaceutical Compositions and Formulations
[0195] The present disclosure encompasses the use of pharmaceutical compositions and formulations comprising one or more of the described anti-αvβ8 integrin antibodies and one or more pharmaceutically acceptable excipients, carriers and/or diluents. In certain embodiments, the compositions may comprise one or more other biologically active agents (e.g., inhibitors of proteases).
[0196] Non-limiting examples of excipients, carriers and diluents include vehicles, liquids, buffers, isotonicity agents, additives, stabilizers, preservatives, solubilizers, surfactants, emulsifiers, wetting agents, adjuvants, etc. The compositions can contain liquids (e.g., water, ethanol); diluents of various buffer content (e.g., Tris-HCl, phosphate, acetate buffers, citrate buffers), pH and ionic strength; detergents and solubilizing agents (e.g., Polysorbate 20, Polysorbate 80); anti-oxidants (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, Pa. (1990)), which is incorporated herein by reference in its entirety.
[0197] By way of nonlimiting example, carriers can include diluents, vehicles and adjuvants, as well as implant carriers, and inert, non-toxic solid or liquid fillers and encapsulating materials that do not react with the active ingredient(s). Non-limiting examples of carriers include phosphate buffered saline, physiological saline, water, and emulsions (e.g., oil/water emulsions). A carrier can be a solvent or dispersing medium containing, e.g., ethanol, a polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), a vegetable oil, and mixtures thereof.
[0198] Formulations comprising one or more of the 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 solution, Ringer's solution, isotonic sodium chloride solution), dispersing agents, wetting agents, emulsifying agents, suspending agents, and the like. Sterile fixed oils, fatty esters, polyols and/or other inactive ingredients can also be used. In addition, formulations for parenteral administration can include aqueous sterile injectable solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended subject and aqueous and nonaqueous sterile suspensions, which can contain suspending agents and thickening agents. Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.
[0199] Embodiments include sterile pharmaceutical formulations of anti-αvβ8 integrin antibodies that are useful as treatments for kidney diseases. Such formulations would inhibit the binding of ligands to the αvβ8 integrin, thereby effectively treating pathological conditions where, for example, tissue αvβ8 integrin is abnormally elevated. Anti-αvβ8 integrin antibodies may possess adequate affinity to potently inhibit αvβ8 integrin activity, and may have an adequate duration of action to allow for infrequent dosing in humans. A prolonged duration of action will allow for less frequent and more convenient dosing schedules by alternate parenteral routes such as subcutaneous or intramuscular injection.
[0200] Sterile formulations can be created, for example, by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution of the antibody. The antibody ordinarily will be stored in lyophilized form or in solution. Therapeutic antibody compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having an adapter that allows retrieval of the formulation, such as a stopper pierceable by a hypodermic injection needle.
[0201] For therapeutic use, e.g., in the treatment of kidney disease, especially, kidney fibrosis, an anti-αvβ8 integrin antibody or an antigen binding fragment thereof, may be administered at a dose depending upon the requirements of the patient, the physical health and characteristics of the patient and the severity of the condition, e.g., the stage of CKD, being treated. For example, dosages can be empirically determined considering the type and stage of kidney disease and/or fibrosis diagnosed in a particular patient. The dose administered to a patient, in the context of the present compositions and methods should be sufficient to result in a beneficial therapeutic response in the patient over a given period of time. The size of the dose also will be determined by the existence, nature, and extent of any adverse side effects that accompany the administration of the antibody dose and/or the dose in combination with another therapeutic agent in a particular patient. The determination of the proper dose for a particular patient and situation is within the skill of a medical practitioner. In general, treatment is initiated using smaller doses, which are less than the optimum dose of the therapeutic. Thereafter, the dose is increased by small increments until effectiveness, such as optimum effectiveness, is achieved. For convenience and if desired, the total daily dosage may be divided and administered in portions during the day. Treatment with a determined or optimum dose may be continued for a short time period (e.g., hours or days), or over a longer time period (e.g., days, weeks, months, years).
Detection Methods
[0202] In some embodiments, the anti-αvβ8 integrin antibody is used for detection, for example, for imaging or to determine 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, methods are provided for determining the presence of αvβ8 integrin in a biological sample obtained from a subject (in vitro, ex vivo, or in vivo), which involves contacting the biological sample with a labeled anti-αvβ8 integrin antibody as described herein and detecting the presence of the labeled antibody bound to αvβ8 integrin, thereby determining the presence of αvβ8 integrin in the sample. Such methods may be used to diagnose kidney disease or a kidney-related condition such as kidney fibrosis, inflammation, or CKD.
[0203] In one embodiment, the antibody is conjugated to an “effector” moiety or molecule, which can be, without limitation, labeling moieties, such as radioactive labels or fluorescent labels, or a therapeutic moiety or molecule. In an embodiment, an effector moiety or molecule may include, but is not limited to, an anti-tumor drug, a toxin, a cytotoxic agent, a radioactive agent, a cytokine, a second antibody, or an enzyme. In another embodiment, the activity of the therapeutic moiety or molecule is modulated by virtue of its being conjugated to the antibody. In another embodiment, the antibody is linked to an enzyme that converts a prodrug into a cytotoxic agent.
[0204] An immunoconjugate comprising the antibody or an antigen binding fragment thereof can be used to target an effector moiety or molecule to a cell that expresses αvβ8 integrin on its surface, particularly diseased kidney cells and tissue, e.g., CKD kidney cells and tissue. Nonlimiting examples of cytotoxic agents that can be effector molecules include radioisotopes, ricin, doxorubicin, daunorubicin, taxol, ethiduim bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracin dione, actinomycin D, diphteria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, steroids, glucocorticoids and other chemotherapeutic agents. Detectable markers include, without limitation, radioisotopes, fluorescent compounds, bioluminescent or chemiluminescent compounds, metal chelators, or enzymes.
[0205] In an embodiment, an anti-αvβ8 integrin antibody or an antigen binding fragment thereof is used as a therapeutic agent to reduce, abrogate, attenuate, decrease, block, or inhibit TGF-β activation in the kidneys, particularly, diseased or CKD kidneys, of an individual in need, either by itself (unconjugated), or conjugated to a detectable label or an effector moiety, such as an adjunct therapeutic treatment agent, such as a suitable treatment or therapeutic for kidney disease or CKD.
[0206] A “detectable label or moiety” may be a diagnostic agent or component that is detectable by a physical or chemical means, e.g., spectroscopic, radiological, photochemical, biochemical, immunochemical means, and the like. By way of example, detectable labels include radiolabels (e.g., .sup.111In, .sup.99mTc, .sup.131I, .sup.67Ga) as well as other FDA-approved imaging agents. Additional labels may include .sup.32P, fluorescent dyes, electron-dense reagents, enzymes, biotin, digoxigenin, or haptens and proteins or other molecules that can be made detectable, for example, by incorporating a radiolabel into the targeting agent. Any method known in the art for conjugating a nucleic acid or a nanocarrier to the label can be used, such as by using methods as described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.
[0207] A “labeled” or “tagged” antibody or agent is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonding, to a label that allows the detection of the presence of the antibody, an antigen binding fragment thereof, or agent by detecting the label that is bound to the antibody or agent. Techniques for conjugating detectable and therapeutic agents to antibodies are known and practiced by those in the art, for example, as described in Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (Eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery” in 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” in 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).
Modes of Administration
[0208] In addition to the administration regimens described herein, an anti-αvβ8 integrin antibody or an antigen binding fragment thereof, or pharmaceutical compositions or formulations comprising an anti-αvβ8 integrin antibody or an antigen binding fragment thereof, can be administered to subjects by modes and routes that are suitable for administering and/or delivering a biologic drug, such as a protein or antibody, to a subject. In general, suitable biological delivery or administration methods embrace parenteral administration modes or routes. Such delivery methods include, without limitation, subcutaneous (SC) delivery, subcutaneous injection or infusion, intravenous (IV) delivery, e.g., intravenous infusion or injection or IV push. Other delivery and administration modes or regimens may include, without limitation, intra-articular, intra-arterial, intraperitoneal, intramuscular, intradermal, rectal, transdermal or intrathecal. In particular embodiments, the anti-αvβ8 integrin antibody is provided to a subject by intravenous administration, e.g., IV infusion or a bolus IV injection. In another particular embodiment, the anti-αvβ8 integrin antibody is provided to a subject by subcutaneous injection, such as a single subcutaneous injection.
[0209] An anti-αvβ8 integrin antibody can be administered in a chronic treatment regimen. The antibody can be administered for a period of time or a predetermined period of time followed by a period of no treatment. A dosing regimen or cycle can also be repeated. In some embodiments, the treatment (e.g., administration of the anti-αvβ8 integrin antibody) involves the administration of a first dose, followed by a second dose and/or one or more subsequent maintenance doses, e.g., for a time period comprising multiple days. Subsequent or maintenance doses may be administered at periodic intervals, e.g., weekly intervals, such as 1 week, 2 weeks, 3 weeks, or longer, e.g., 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, or at monthly intervals, or longer intervals, such as years, following the initial, second, or subsequent doses.
[0210] It is also contemplated that the anti-αvβ8 integrin antibody can be administered by direct delivery, e.g., infusion or injection, at or near a site of disease, as practicable. Injection in or near the kidney or kidney tissue may be useful. It is also contemplated that the anti-αvβ8 integrin antibody can be administered by implantation of a depot, which releases the antibody at the target site of action, such as in kidney tissue. Alternative modes of administration or delivery of the anti-αvβ8 integrin antibody may include inhalation (e.g., inhaler or aerosol spray), intranasal delivery, or transdermal delivery (e.g., by means of a patch on the skin). In addition, administration may be by osmotic pump (e.g., an Alzet pump) or mini-pump (e.g., an Alzet mini-osmotic pump), allowing for controlled, continuous and/or slow-release delivery of the anti-αvβ8 integrin antibody, or a pharmaceutical composition thereof, over a pre-determined period. The osmotic pump or mini-pump can also be implanted subcutaneously at or near the kidney or kidney tissue as the target site.
Kits
[0211] Also provided are kits for the treatment of kidney disease, such as kidney disease involving fibrosis, e.g., CKD or DN. In an embodiment, the kit includes a composition, e.g., a therapeutic composition, containing an effective amount of an anti-αvβ8 integrin antibody, or an antigen binding fragment thereof. In an embodiment, the anti-αvβ8 integrin antibody, or an antigen binding fragment thereof, is in unit dosage form.
[0212] In some embodiments, the kit comprises a sterile container which comprises the anti-αvβ8 integrin antibody, or an antigen binding fragment thereof, e.g., in aqueous or lyophilized form. If the antibody is in a lyophilized form, the kit may include a container with an appropriate diluent, excipient, or vehicle for admixing with the dried antibody to prepare a solution containing the antibody, suitable for administration, e.g., intravenous administration. The containers can 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 medicaments, e.g., in aqueous or dried form. The containers can be in boxes for protection from damage or breakage. One or more syringes for antibody dilution and/or for administration may be included in the kit.
[0213] The kit may further provide instructions for administering the anti-αvβ8 integrin antibody, or a composition containing the antibody, to a subject having kidney disease, fibrotic kidney disease, e.g., CKD or DN. The instructions will generally include information about the use of the antibody or the composition for the treatment of kidney disease, fibrotic kidney disease, e.g., CKD or DN. In other embodiments, the instructions include one or more of the following: description of the therapeutic antibody; dosage schedule and administration for treatment of kidney disease, fibrotic kidney disease, e.g., CKD or DN, or symptoms thereof; dosage information; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), on a label applied to the container, or on a separate sheet, pamphlet, card, or folder supplied in the kit or with the container in the kit.
[0214] The present disclosure encompasses, unless otherwise indicated, conventional techniques of molecular biology (including any recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, 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 a polynucleotide encoding an anti-αvβ8 integrin antibody, or an antigen binding portion or fragment thereof polynucleotides, and/or anti-αvβ8 integrin antibody, or an antigen binding portion or fragment thereof polypeptides as described herein, and, as such, may be considered in making and practicing the invention.
[0215] The following examples are set forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.
EXAMPLES
Example 1
Anti-αvβ8 Integrin Antibodies
[0216] Several anti-αvβ8 integrin antibodies are embraced by the present disclosure and used in accordance with the methods, composition and products described herein, and/or as reference or control antibodies. Specifically, a chimeric anti-αvβ8 integrin antibody, called “Chi-37E1B5” herein, was in-licensed from The Regents of the University of California (UCSF). A second anti-αvβ8 integrin antibody, called “hu37E1B5” herein, was produced at MedImmune using a humanized sequence that was reported in published International PCT Application WO 2013/026004 (UCSF). When the hu37E1B5 antibody was evaluated in affinity binding studies, it was found to have very poor binding affinity for the αvβ8 integrin protein, as shown in
[0217] In addition, to obtain an anti-αvβ8 integrin antibody with improved binding affinity for αvβ8 integrin, a fourth, optimized, anti-αvβ8 integrin antibody, called “B5-15” herein, was generated from the MEDI-hu37E1B5 antibody as a parental antibody using affinity maturation techniques known and used in the art. The resulting B5-15 anti-αvβ8 integrin antibody (also called “optimized” or “affinity optimized” B5-15) exhibited an improved binding profile for αvβ8 integrin protein compared with that of the MEDI-hu37E1B5 anti-αvβ8 integrin antibody as shown in
Humanization of the Chimeric Chi-37E1B5 Antibody by CDR Grafting
[0218] CDR grafting methods as known and practiced in the art were employed to humanize the mouse/human chimeric 37E1B5 (Chi-37E1B5) antibody and to produce the humanized MEDI-hu37E1B5 anti-αvβ8 integrin antibody. For humanization, the closest individual human germline framework (FW) with the same canonical class was selected to mimic the antibody folding structure. Critical murine FW residues for back mutations were identified, genes were synthesized, IgG was converted and produced by transient transfection using 293 cells, and the resulting antibodies were screened for binding to αvβ8 integrin. This process produced a fully humanized light chain clone, and a hybrid human germline FWs with 4 key mouse residues. The humanized MEDI-hu37E1B5 anti-αvβ8 integrin antibody resulting from the above methods was demonstrated to surprisingly retain the full binding activity of the original chimeric 37E1B5 (Chi-37E1B5) antibody (
Site-Saturation Mutagenesis and Affinity Maturation Leading to the Production of the Affinity Optimized, Humanized B5-15 Anti-αvβ8 Integrin Antibody
[0219] In addition, site-saturation mutagenesis was performed on the humanized MEDI-hu37E1B5 antibody to remove a Cys 94 residue (which has been shown to be a liability in antibody structure as it is associated with potential fragmentation/peptide cleavage of the antibody backbone) by first converting the residue to all of the other 19 amino acids. All of the resulting mutant antibodies were screened for binding to αvβ8 integrin via ELISA analysis. Depending on the residue at position 94, the binding affinity was reduced. The best αvβ8 integrin-binding mutants obtained from this procedure were called MEDI-hu37E1B5-C94I and MEDI-hu37E1B5-C94G. The MEDI-hu37E1B5-C94I mutant antibody had a roughly 3-fold reduction in αvβ8 binding affinity. The humanized MEDI-hu37E1B5-C94I antibody was selected for further analysis and affinity optimization.
[0220] Affinity maturation of the humanized MEDI-hu37E1B5-C94I, with the N-glycosylation site, was performed using parsimonious mutagenesis, an art-recognized method. Briefly, saturation point mutations to each CDR position of Medi-hu37E1B5-C94I were first generated. The mutations covered all 6 CDRs of the antibody V.sub.H and V.sub.L regions. A total of 6528 individual clones were screened (>4× redundancy) for binding to αvβ8 integrin. From these, 10 primary hits were identified: 3 were in V.sub.H-CDR1; 2 were in V.sub.H-CDR3; 1 was in V.sub.L-CDR1; and 4 were in V.sub.L-CDR3. All of the hits showed 2-5-fold improvement in binding to αvβ8 integrin.
[0221]
[0222]
[0223] A combination library of the 10 most beneficial point mutations was then created in a combinatorial fashion. 4608 clones were screened for binding to αvβ8 integrin. 88 clones were selected for confirmation. 6 hits were identified from the combinatorial evaluation as showing additive improvement in binding to αvβ8 integrin compared with the best primary hit, P2-23. αvβ8 integrin binding data from the combination library screening are shown in
TGF-β Activation Bioassay
[0224] The TMLC luciferase bioassay is used in the art to measure TGF-β activation via integrins, such as αvβ8 integrin. The bioassay is based on a mink lung cell line, TMLC, that is 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).
[0225] TGF-β activation was measured using transformed mink lung epithelial cells (TMLC) stably transfected with a portion of the plasminogen activated inhibitor 1 (PAI-1) promoter linked to a luciferase reporter (cells provided by Daniel Rifkin, New York University) and cultured as described previously (M. Abe et al., 1994, Anal. Biochem., 216(2):276-284). HeLa-B8 cells (1.5×104 cells/well) were co-cultured with TMLCs (1.5×104 cells/well) in a 96-well plate overnight 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 test antibody. After 16 hours, supernatants were removed and cells were lysed in 100 μL of cell lysis buffer (Promega) and luciferase activity determined using the luciferase assay system (Promega) by transferring 80 μL of lysate and mixing with 80 μL of substrate in a white walled clear bottom 96-well plate. Samples were read immediately on a luminometer and shown as either relative luciferase units (RLU) or percent maximal response, determined by using TMLCs alone as the baseline or 0% control and TMLCs co-cultured with HeLa-B8 cells as maximal or 100% response in the assay.
Generation of the HeLa-B8 Cell Line
[0226] HeLa-B8 cells is a derivative of the HeLa cell line (ECACC). Briefly, confluent HeLa cells 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 and prior to use, cells were removed using accutase and resuspended in PBS at 1×106 cells/mL. LIVE/DEAD fixable aqua dead cell stain (Life Technologies/Thermo Fisher, 1:1000) was added to the cells on ice for 20 minutes. 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. Cells were pelleted and washed and a secondary anti-mouse-Alexa-647 (Jackson ImmunoResearch, 1:200) added to the cells and incubated on ice for 30 minutes. Cells were pelleted and washed and resuspended at 10×106 cells/ml in HeLa cell medium containing 1% FBS. Cells were sorted on a BD FACSAria III cell sorter (BD Biosciences) using Chi-37E1B5 antibody. High αvβ8+ sorted cells were then cultured in complete HeLa cell medium, expanded and banked for future use. Cells remained positive for high αvβ8 expression for at least 1 month of culture.
Characteristics of Humanized, Affinity Optimized B5-15 Anti-αvβ8 Integrin Antibody
[0227] The light chain (L) variable region (V.sub.L, κ) amino acid (aa) sequence of the humanized and optimized B5-15 antibody polypeptide has 107 amino acid residues as follows:
TABLE-US-00055 B5-15 V.sub.L (kappa (κ)) (SEQ ID NO: 13) DIQLTQSPSSLSASVGDRVTITCKASQDINKYLSWFQQKPGKAPKSLIYYA NRLVDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDVFPYTFGGGT KVEIK (107 aa)
[0228] The heavy chain (H) variable region (V.sub.H) amino acid sequence of the B5-15 antibody polypeptide has 116 amino acid residues as follows:
TABLE-US-00056 B5-15 V.sub.H (SEQ ID NO: 12) EVQLVESGGGLVQPGGSLRLSCAVSGFVFSRSWISWVRQAPGKGLEWIGEI NPDSSTINYTSSLKDRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAILITT EDYWGQGTTVTVSS (116 aa)
[0229] In particular, the light chain (L) variable region (V.sub.L) of the B5-15 antibody includes three CDRs having the amino acid sequences as follows:
TABLE-US-00057 V.sub.L CDR1: (SEQ ID NO: 10) KASQDINKYLS V.sub.L CDR2: (SEQ ID NO: 5) YANRLVD V.sub.L CDR3: (SEQ ID NO: 11) LQYDVFPYT
The heavy chain (H) variable region (V.sub.H) of the B5-15 antibody includes three CDRs having the amino acid sequences as follows:
TABLE-US-00058 V.sub.H CDR1: (SEQ ID NO: 9) RSWIS V.sub.H CDR2: (SEQ ID NO: 2) EINPDSSTINYTSSL V.sub.H CDR3: (SEQ ID NO: 3) LITTEDY
[0230] A comparison of the amino acid sequences of the V.sub.H and V.sub.L regions of Chi-37E1B5, hu37E1B5, MEDI-hu37E1B5 and B5-15 anti-αvβ8 integrin antibodies is presented in
Example 2
[0231] Immunohistochemistry (IHC) Detection Method for αvβ8 Integrin Expression in Formalin-Fixed Paraffin-Embedded (FFPE) Human Tissue Using an Anti-αvβ8 Integrin Antibody
IHC Method
[0232] To prepare formalin-fixed paraffin-embedded (FFPE) tissue sections, slides onto which human tissue samples were affixed were removed from storage. The slides were appropriately labeled and loaded into Autostainer XL rack(s). The stained slides were dewaxed and rehydrated in tap water.
[0233] The slide racks were transferred to a pressure cooker containing Dako Antigen Retrieval Solution (Dako S1699). Heat Mediated Antigen Retrieval was performed for 2 minutes at pressure (SP DDCP_5024) with the following alterations: following antigen retrieval, the pressure cooker was allowed to cool and de-pressurize; the pressure cooker was placed in running tap water and the lid was removed; the pressure cooker was cooled for 5 minutes and its contents were then flushed with running tap water; the slides were removed and rinsed in running tap water for 5 minutes. Slides were blocked with peroxidase (3% Hydrogen Peroxide in methanol) for 10 minutes.
[0234] Immunohistochemistry was performed as follows: [0235] PAP pen slides at appropriate locations (for drawing hydrophobic barriers on tissue); [0236] Load slides onto Dako Autostainer; [0237] Rinse in standard Dulbecco's PBS with 0.1% Tween 20 (PBST)×1 (one time); [0238] Incubate slides in 2.5% Horse Serum (from ImmPRESS kit) 20 minutes; [0239] Blow step; Incubate in either Calico antibodies CAL16 (a purified rabbit recombinant anti-αvβ8 integrin antibody) @ 1.0 ug/ml diluted in PBST, Dako rabbit immunoglobulin isotype control @ 1.0 ug/ml or Vector Ki67 at a 1:200 dilution as experiment control for 60 minutes; [0240] Rinse in PBST×1; [0241] Incubate in labeled polymer, Vector ImmPRESS™ HRP, horse anti-Rabbit IgG (Peroxidase) Polymer Detection Kit, (Catalog No. MP-7401) for 30 minutes; [0242] Rinse in PBST×1; [0243] Incubate in PBST for 5 minutes; [0244] Rinse in PBST×1; Switch to hazardous waste ×1; [0245] Incubate in DAB+ substrate/chromagen (Dako, K3468) for 5 minutes; [0246] Rinse in Pure water (automatically done by Autostainer) ×1; [0247] Unload slides from Dako Autostainer; [0248] Counterstain with Gill I Haematoxylin, dehydrate and coverslip slides using program 9; and [0249] Unload slides from Leica CV5030 Coverslipper and allow to dry/set.
TABLE-US-00059 TABLE 1 COUNTERSTAIN AND DEHYDRATION PROGRAM NO: 9 TIME STEP STATION REAGENT [M:S] EXACT 1 WASH 5 RUNNING TAP WATER 3:00 NO 2 11 HAEMATOXYLIN GILL I 0:25 YES 3 WASH 4 RUNNING TAP WATER 4:00 NO 4 12 1% ACID ALCOHOL 0:02 YES 5 WASH 2 RUNNING TAP WATER 5:00 NO 6 13 95% IMS 1:30 NO 7 14 IMS 1:00 NO 8 15 IMS 1:30 NO 9 16 IMS 1:00 NO 10 17 XYLENE 2:00 NO 11 18 XYLENE 2:00 NO 12 EXIT XYLENE /:/ /
[0250] Alternatively, one can perform the steps in “Program No: 9” manually. To do this, one can place the slides in the stated reagents for the time stated. One could use either the automated program or perform the steps manually.
Example 3
[0251] αvβ8 Integrin is Preferentially Expressed in Kidney
[0252] IHC staining analysis as described in Example 2 was carried out on numerous tissue samples to determine αvβ8 integrin expression and distribution in human tissues. Table 2 below presents the results of the IHC analysis.
TABLE-US-00060 TABLE 2 Number of samples that express αvβ8 Tissue N integrin Location Heart 3 0 Lung 3 0 Kidney 3 3 Glomerular and tubular Spleen 3 0 Lymph node 3 0 Thymus 3 2 Epithelial cells surrounding Hassall's corpuscles Tonsil 3 2 Trabecula stratified squamous epithelium Liver 3 0 Gall bladder 3 1 Weak cytoplasmic staining columnar epithelium Pancreas 3 1 Small area of stroma positive expression Brain 3 1 Weak homogeneous parenchyma cerebellum Brain 3 2 Weak homogeneous parenchyma cerebellum Thyroid 3 0 Adrenal 3 2 Cortex cells - rare interstitial αv8β integrin staining detected Capillaries Parotid 3 0 Skin 3 0 Skeletal 3 0 muscle Stomach 3 0 Ileum 3 2 Adventitia nerve cells Colon 3 0 Ovary 3 1 ? nerve cells Fallopian tube 3 1 Rare weak columnar epithelium Uterus 3 0 myometrium Endometrium 3 1 Endometrial gland epithelium Endocervix 3 1 Weak stratified squamous Exocervix 3 2 Epithelial membrane. Columnar/ stratified squamous Breast 3 0 Placenta 3 3 Weak scanty trophoblast, rare membrane Prostate 3 1 Moderate glandular epithelium Testis 3 2 Weak cytoplasmic primary spermatocytes Seminal 3 2 Glandular/vesicle epithelium vesicles Bladder 3 0 Ureter 3 0
[0253] As observed in Table 2, kidney tissue expresses a high level of αvβ8 integrin. In addition, αvβ8 integrin was found to be highly enriched in human kidney tissue compared with 33 other human tissue types, namely, heart, lung, spleen, lymph node, thymus, tonsil, liver, gallbladder, pancreas, brain cerebellum and cerebrum, thyroid, adrenal, parotid, skin, skeletal muscle, stomach, ileum, colon, ovary, fallopian tube, uterus myometrium, endometrium, endocervix, exocervix, breast, placenta, prostate, testis, seminal vesicle, bladder and ureter). In
[0254] For the IHC staining analyses presented in
Example 4
[0255] Avβ8 Expression is Increased in the Kidney Tissue of Patients with Chronic Kidney Disease (CKD)
[0256] The expression of αvβ8 integrin was evaluated in human kidney tissue samples taken from patients with diabetic nephropathy (DN) and from individuals with normal kidney tissue as “healthy” controls. DN kidney tissue samples were obtained from Addenbrooke's Biobank and MedImmune (Gaithersburg) Biobank. Normal kidney samples were obtained from MedImmune (Cambridge) tissue bank. More specifically, samples from 9 patients having 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, some areas of mild chronic inflammation were evident, but did not impact the study design or results (
[0257] As described in Example 3, the antibody used in the IHC staining experiments was CAL16 clone anti-αvβ8 integrin rabbit monoclonal antibody (purified rabbit recombinant antibody) from Calico Biolabs Inc. (Pleasanton, Calif.). After staining, the slides were reviewed by an experienced senior pathologist.
[0258] The results from this IHC staining analysis were as follows: In the healthy individuals, the glomeruli showed positive staining with the anti-αvβ8 integrin antibody compared with isotype-matched control antibody staining; the anti-αvβ8 integrin antibody staining was generally light (¾ samples), although one sample (¼) showed strong staining in podocytes (podocyte pattern). In kidney tubules, light multifocal staining was observed in cortical tubules, membrane, basal to apical. (
[0259] In the patients having DN-CKD, the extent of αvβ8 staining in the glomeruli was variable and was in relation to the degree of glomerular damage. The loss of podocytes in the diseased tissue correlated with less staining. Because of podocyte loss in diseased kidney, the staining intensity was variable. Staining of tubules in DN-CKD kidney tissue varied from light staining to strong staining of cytoplasm and membrane, mostly in areas of inflammation/fibrosis. An overall increased expression of αvβ8 integrin was observed in DN-CKD kidneys as evidenced by the staining pattern of the anti-αvβ8 integrin antibody. The overexpression was essentially seen in kidney tubules. (
[0260]
Example 5
[0261] Itgb8 Gene is Upregulated in Kidneys from Individuals with CKD and has Elevated Expression Compared with Other β Integrins
[0262] Transcriptomics analyses provided evidence that kidneys of human CKD patients had higher expression of ITGB8 (which encodes for (38 integrin) compared with the kidneys of healthy human subjects. In these analyses, the relative (3 integrin family mRNA expression was measured in human CKD kidney homogenates. In brief, 1 punch (2 mm puncher) of kidney biopsies was homogenized in RLT lysis buffer using a TissueLyserII. RNA from the lysates was isolated with RNAeasy Mini kit columns. RNA concentration was measured with a Nanodrop and concentrations were adjusted to perform qPCR analyses using the TaqMan RNA to Ct 1-step Kit and specific probes for all the integrins, with the hprt-1 included as a housekeeping gene.
[0263] In other experiments, the transcriptomic profiles of 157 patients having different degrees of CKD were analyzed and compared with those of living donors (LD). Twelve (12) of the 157 patients had diabetic neuropathy (DN). Glomerular and tubulo-interstitital compartments were separated and whole genome gene expression analysis was performed as described by S. Martini et al. (2014, 1 Am. Soc. Nephrol., 25(11):2559-2572). In this analysis, the expression of itgb8 mRNA was first assessed in the renal glomerular compartment in relation to nephrin (encoded by NPHS1 gene). Nephrin is a podocyte protein necessary for the proper functioning of the renal 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 podocyte number. In CKD, as podocyte numbers decrease, there is a reduction in NPHS1 expression.
[0264] In a separate cohort, the tubulo-interstitium (Tub) and glomerulus (Glom) of kidney samples obtained from 20 human patients with DN compared with the TI and glomerulus of kidney samples obtained from 19 LD patients were profiled by whole genome transcriptional profiling using RNAseq. The results showed that itgb8 mRNA expression increased in the tubulo-interstitium of DN patients (designated as “Tub-DN” in the graph) versus that in living donors (LD), (
[0265] The key findings of these analyses were as follows: itgb8 mRNA expression was elevated in the glomeruli from DN patient samples after normalization to nephrin (NPHS1), a podocyte marker gene. itgb8 mRNA expression was elevated in the tubule-interstitium (TI) of DN patients. In the TI, itgb8 mRNA expression was positively correlated with a putative TGF-β activation score, consistent with a proposed role of αvβ8 integrin in controlling TGF-β activation in fibrotic diseases. Similar results were found following the analysis of mRNA expression of WT1, another podocyte marker gene (data not shown). These findings support the discovery that in human CKD kidney, αvβ8 integrin expression correlates with fibrosis in CKD, which is associated with the activation of TGF-β, an important player in causing and exacerbating kidney fibrosis.
Example 6
In Vivo Efficacy of Anti-αvβ8 Integrin Antibodies
[0266] A mouse model of fibrosis induction was used to study the in vivo efficacy of the anti-αvβ8 integrin antibody in treating fibrosis in the kidney. This model involved performing a procedure called unilateral ureteral occlusion (UUO), (unilateral ligation of the ureter), on the animals. For the model, male, humanized αvβ8 transgenic (Tg) mice underwent a sham or a UUO procedure involving five (5) and eight (8) day duration of injury. The Tg mice were produced by crossing a mouse in which the αvβ8 gene was knocked out (αvβ8 KO mouse) with a human αvβ8 BAC transgenic mouse. The generation of Tg mice expressing 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 the kidney glomerulus, in a pattern similar to that observed in healthy humans. The induction of fibrosis following ureteral ligation (UUO) increased αvβ8 integrin expression in kidney tubules, similar to what is observed in human CKD.
[0267] The test agent used was B5-15, the IgG1 humanized and sequence optimized anti-αvβ8 integrin antibody as described supra. The control antibody was an isotype-matched IgG antibody.
[0268] Protocol for the UUO Tg Mouse Model Study:
[0269] Model: 91 male humanized αvβ8 transgenic (Tg) mice underwent sham or a unilateral ureteral occlusion (UUO) procedure; 5- or 8-day duration of injury. The animals in the groups were dosed with respective antibody treatment every other day (EOD) on Days −1, 1, 3, 5 and 7. The sham-treated animals were administered vehicle on Days 0, 2, 4 and 6.
[0270] Mice Age at Study Inception: 92-121 days old.
[0271] Test Agents/Compounds: 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 at doses, frequencies, and to groups as displayed in Table 3 below.
TABLE-US-00061 TABLE 3 Compound and Surgical Administration Preparation N/group Interval Dose and Interval (Animal #s) Strain (Day (D)) Route (Day (D)) Condition n = 6 Tg IgG Control 10 mg/kg Once at D 0 Sham (3428-3433) D −1, D 1, D 3, D 5, D 7 i.p. Surgery Vehicle N/A D 0, D 2, D 4, D 6 i.p. n = 10 Tg IgG Control 10 mg/kg Once at D 0 Permanent (3434-3443) D −1, D 1, D 3 i.p. UUO Vehicle N/A D 0, D 2, D 4, D 6 i.p. n = 10 Tg Chi-37E1B5 10 mg/kg Once at D 0 Permanent (3454-3463) D −1, D 1, D 3, D 5, D7 i.p. UUO Vehicle N/A D 0, D 2, D 4, D 6 i.p. n = 10 Tg B5-15 10 mg/kg Once at D 0 Permanent (3464-3473) D −1, D 1, D 3, D 5, D 7 i.p. UUO Vehicle N/A D 0, D 2, D 4, D 6 i.p.
[0272] Study Endpoints: Morphology: Body (initial, final, A); Kidney weight (obstructed and contralateral) and index; and Tibia length.
[0273] Renal Cortical mRNA Expression via Luminex: [0274] Connective Tissue Growth Factor (CTGF) [0275] α-Smooth Muscle Actin (ACTA2) [0276] Fibronectin-1 (FN1) [0277] Collagen 1a1 (Col1a1) [0278] Collagen 3a1 (Col3a1)
[0279] Renal Cortical Hydroxyproline Content
[0280] Histology readout: Picrosirius Red (PRS) and αvβ8 staining.
The results of IHC staining of kidney tissue of humanized αvβ8 transgenic mice with anti-αvβ8 integrin antibodies to determine kidney fibrosis and the extent thereof are shown in
[0281] As shown in
[0282] In summary, at 8-days post-UUO surgery, Chi-37E1B5 and B5-15 attenuated UUO-induced increases in Col1a1, Col3a1, FN-1 and CTGF mRNA expression and % hydroxyproline content. Additionally, at 8-days post-UUO surgery, B5-15 attenuated a UUO-induced increase in α-SMA mRNA expression.
[0283] The two anti-αvβ8 integrin antibodies used in this Example, Chi-37E1B5 and B5-15, were administered to the mice in the UUO model at maximal dose. The purpose of this study was to demonstrate whether an antibody against αvβ8 integrin could effectively reduce TGF-β-induced fibrosis caused by association with the αvβ8 integrin. We would expect to see a difference in the reduction of TGF-β-induced fibrosis at lower doses (i.e. EC.sub.50) of either of these anti-αvβ8 integrin antibodies. That is, we would expect to see a greater reduction in TGF-β-induced fibrosis in the UUO model from treatment with B5-15 than with Chi-37E1B5 at an equivalent dose. This is primarily because B5-15 demonstrates a greater binding affinity for the αvβ8 integrin than Chi-37E1B5 (see
[0284] As discussed supra, the αvβ8 integrin target receptor is preferentially and highly expressed in diseased/fibrotic kidney tissue and is bound in kidney tissue by the anti-αvβ8 integrin antibody, which interferes with the binding interaction of αvβ8 integrin to latent TGF-β. The anti-αvβ8 integrin antibodies as disclosed herein are particularly advantageous and beneficial for treating fibrotic kidney disease in subjects having kidney disease because use of an antibody directed against the αvβ8 integrin, which binds latent TGF-β, obviates and avoids the targeting of systemic TGF-β, and thus avoids potentially serious problems that could accompany a systemic inhibition of TGF-β in other tissues in the subject undergoing treatment.
Example 7
Ex Vivo Studies Using the B5-15 Anti-αvβ8 Integrin Antibody
[0285] To evaluate the binding and engagement of the anti-αvβ8 integrin antibody (B5-15) with the target αvβ8 integrin receptor of TGF-β, the activation of the downstream TGF-β signaling pathway was assessed in kidney lysates by measuring total and phosphorylated kidney SMAD2/3. In brief, kidney samples from the animals used in the study described in Example 5 were homogenized in a specific lysis buffer (1× diluted in distilled water+10 μl/ml of protease and phosphatase inhibitor) using a TissueLyser II; protein content was measured using a bicinchoninic acid (BCA) assay as known to and used by those skilled in the art; and protein concentration was normalized for all samples. The total and phosphorylated forms of SMAD2/3 protein (phospho-SMAD2 (Ser465/467)/SMAD3 (Ser423/425)) were analyzed by ELISA following the manufacture's protocol. As noted supra, members of the Smad family of signal transduction molecules are components of the intracellular pathway that transmits TGF-β signals from the cell surface into the nucleus.
[0286] The results of the experiments showed that the B5-15 anti-αvβ8 integrin antibody reduced the downstream TGF-β signaling pathway in the kidneys of transgenic mice carrying the human αvβ8-encoding gene (“humanized αvβ8 transgenic mice”) that had undergone unilateral ureteral occlusion (UUO) of 5 days' duration. In
[0287] As observed in
Example 8
Treatment of a Tri-Culture Cell System Using B5-15, an Anti-αvβ8 Integrin Antibody
[0288] To evaluate the effect of B5-15 (an anti-αvβ8 integrin antibody) on a model of human glomerulosclerosis (described in Waters et al., 2017, J Pathol, 243(3):390-400), we treated the tri-culture cell system (where glomerular endothelial cells, podocytes, and mesangial cells form a vascular network) with 10 ng/ml TGF-β or 25 ng/ml CTGF to induce fibrosis. An increase in nodule number is reflective of progression of fibrosis. Treatment with 15 μg/ml of B5-15 significantly reduced nodule number in comparison to treatment with 15 μg/ml of an isotype control (NIP228), see
3D Tri-Culture Formation
[0289] In tri-culture human podocytes (Celprogen, CA, USA), glomerular endothelial cells (GECs) and mesangial cells (MCs) (both GECs and MCs from ScienCell Research Laboratories, CA, USA) were suspended within rat tail type 1 collagen (1.5 mg/ml; Corning, Mass., USA), human plasma fibronectin (90 μg/ml; Merck Millipore, Mass., USA), 1.5 mg/ml NaHCO.sub.3, 25 Mm HEPES and M199 medium (10×; Sigma, Mo., USA) at 4° C. Gel was pH adjusted with 0.1M HCl (Fisher Scientific, UK) to pH 7.4. The cell/gel suspension was pipetted into 48 well plates (Corning Incorporated, NY, USA) in a volume of 320 μl per well, respectively. Renal glomerular cells were used at a ratio of 16:3:1 (GECs:PODs:MCs), 330,000-340,000 GECs, 50,000-70,000 PODs and 20,000-24,000 MCs per 320 μl. Cell/gel suspension was polymerised at 37° C. for 20 minutes, after which 500 μl of media was pipetted on top of the gel. Tri-culture media was composed of RMPI 1640 (Gibco™ by Thermo Fisher, UK), 2% FBS, 1% penicillin/streptomycin, 1% insulin, Apo-transferrin, sodium selenite (in ITS mix) and 1% ECGS (supplements all from ScienCell Research Laboratories, CA, USA). Cultures were maintained for 24 hrs. Cells were used in experiments between p2-p6.
Stimulation Assays with TGF-β, NIP228, an Anti-αvβ8 Antibody and CTGF
[0290] For stimulation 10 ng/ml TGF-β (R&D Systems (Bio-Techne Ltd), MN, USA), 15 μg/ml NIP228, 15 μg/ml an anti-αvβ8 integrin antibody and 25 ng/ml CTGF (Invitrogen, CA, USA) alone or in combination, were added to media placed on top of culture gels for 24-hour incubation. Control treatment was media alone.
[0291] We demonstrate that treatment with an anti-αvβ8 integrin antibody can inhibit the progression of fibrosis caused by TGF-β activation.
OTHER EMBODIMENTS
[0292] From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
[0293] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
[0294] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.