WO2007146160A2 - Methods for the treatment of renal disease using anti-pdgf-dd antibodies - Google Patents

Abstract

The invention described herein relates to antibodies directed to platelet derived growth factor-DD (PDGF-DD) and uses of such antibodies. The antibodies of the invention find use as diagnostics, in the treatment of renal fibrosis, and in the treatment of renal disease, renal dysfunction and/or renal injury.

Description

METHODS FOR THE TREATMENT OF RENAL DISEASE, RENAL FIBROSIS AND OTHER RENAL INJURIES USING ANTI-PDGF-DD ANTIBODIES

RELATED APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Patent

Application Serial No. 60/811,834, filed June 7, 2007 which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The invention described herein relates to antibodies directed to platelet derived growth factor-DD (PDGF-DD) and uses of such antibodies. The antibodies of the invention find use as diagnostics, and in the treatment of renal disease, renal fibrosis, renal dysfunction and/or renal injury.

BACKGROUND OF THE INVENTION

[0003] Diabetic nephropathy underlies up to 35% of end stage renal disease cases, and glomerulonephritides, in particular the most common type IgA nephropathy, account for another 20% of cases in most Western countries. Both diabetic nephropathy and the majority of progressive glomerulonephritides are histologically characterized by glomerular mesangial cell proliferation and/or matrix accumulation. In addition all of these diseases progress to renal failure via secondary tubulointerstitial damage and fibrosis. Treatments targeting both of these processes would therefore be of major clinical relevance.

SUMMARY OF THE INVENTION

[0004] The invention described herein relates to antibodies directed to platelet derived growth factor-DD (PDGF-DD) and uses of such antibodies. The antibodies of the invention find use as diagnostics, and in the treatment of renal disease, renal fibrosis, renal dysfunction and/or renal injury.

[0005] Accordingly, one embodiment of the invention is the use of fully human anti-PDGF-DD antibodies, and anti-PDGF-DD antibody preparations with desirable properties from a therapeutic perspective, to treat, prevent or delay the progression of renal fibrosis in a subject. Another embodiment of the invention is the use of fully human anti- PDGF-DD antibodies, and anti-PDGF-DD antibody preparations with desirable properties from a therapeutic perspective, to treat, prevent or delay the progression of renal failure in a subject. Yet another embodiment of the invention is the use of MIy human anti-PDGF-DD antibodies, and anti-PDGF-DD antibody preparations with desirable properties from a therapeutic perspective, to reduce proteinuria in a subject.

[0006] Preferably, the antibodies have a heavy chain amino acid having a sequence selected from the group consisting of SEQ ID NOS: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42,

46, 50, 54, 58, 62, 66, 70, and 74, and further have a light chain amino acid having a sequence selected from the group consisting of SEQ ID NOS: 4, 8, 12, 16, 20, 24, 28, 32,

36, 40, 44, 48, 52, 56, 60, 64, 68, and 72.

[0007] In a preferred embodiment, the anti-PDGF-DD antibody has the following light chain and heavy chain sequences:

[0008] Nucleotide sequence encoding the variable region of the heavy chain: caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggata caccttcacc agttatgata tcaactgggt gcgacaggcc 120 actggacaag ggcttgagtg gatgggatgg ataaacccta atagtggtaa cacagactat 180 gcacagaagt tccagggcag agtcaccatg accagggaca cctccataag cacagcctac 240 atggagctga gcagcctgag atctgaggac acggccatat attattgtgt gagaggcttt 300 ggatacagct ataattacga ctactattac ggtatggacg tctggggcca agggaccacg 360 gtcaccgtct cctcagt (SEQ ID NO : 1 ) 377

[0009] Amino acid sequence encoding the variable region of the heavy chain:

Gin VaI GIn Leu VaI GIn Ser GIy Ala GIu VaI Ly s Ly s Pro GIy Ala

1 5 10 15

Ser VaI Lys VaI Ser Cys Lys Ala Ser GIy Tyr Thr Phe Thr Ser Tyr

20 25 30

Asp lie Asn Trp VaI Arg GIn Ala Thr GIy GIn GIy Leu GIu Trp Met

35 40 45

GIy Trp lie Asn Pro Asn Ser GIy Asn Thr Asp Tyr Ala GIn Lys Phe

50 55 60

GIn GIy Arg VaI Thr Met Thr Arg Asp Thr Ser lie Ser Thr Ala Tyr

65 70 75 80

Met GIu Leu Ser Ser Leu Arg Ser GIu Asp Thr Ala lie Tyr Tyr Cys

85 90 95

VaI Arg GIy Phe GIy Tyr Ser Tyr Asn Tyr Asp Tyr Tyr Tyr GIy Met

100 105 110

Asp VaI Trp GIy GIn GIy Thr Thr VaI Thr VaI Ser Ser (SEQ ID NO: 2) 115 120 125

[0010] Nucleotide sequence encoding the variable region ofthe light chain: gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagt agtagttact tagcctggta ccagcagaag 120 cctggccagg ctcccaggct cctcatctat gctacatcca gcagggccac tggcatccca 180 gaσaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240 cctgaagatt ttgcagtgta ttactgtcag cagtatggta gttcaccgtg cagtttfcggc 300 caggggacca agctggaaat caagc (SEQ ID NO:3) 325 [0011] Amino acid sequence encoding the variable region of the light chain:

GIu He VaI Leu Thr GIn Ser Pro GIy Thr Leu Ser Leu Ser Pro GIy

1 5 10 15

GIu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser VaI Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr GIn GIn Lys Pro GIy GIn Ala Pro Arg Leu Leu

35 40 45 lie Tyr Ala Thr Ser Ser Arg Ala Thr GIy lie Pro Asp Arg Phe Ser

50 55 60

GIy Ser GIy Ser GIy Thr Asp Phe Thr Leu Thr He Ser Arg Leu GIu

65 70 75 80

Pro GIu Asp Phe Ala VaI Tyr Tyr Cys GIn GIn Tyr GIy Ser Ser Pro

85 90 95

Cys Ser Phe GIy GIn GIy Thr Lys Leu GIu He Lys (SEQ ID NO: 4) 100 105

[0012] It will be appreciated that embodiments of the invention are not limited to any particular anti-PDGF-DD antibody, or any specific form of an antibody. For example, the anti-PDGF-DD antibody may be a full length antibody (e.g. having an intact human Fc region) or an antibody fragment {e.g. a Fab, Fab' or F(ab')2>. In addition, the antibody may be manufactured from a hybridoma that secretes the antibody, or from a recombinantly produced cell that has been transformed or transfected with a gene or genes encoding the antibody.

[0013] In one embodiment, the anti-PDGF-DD antibody forms a pharmaceutical composition comprising an effective amount of the antibody, or a fragment thereof, in association with a pharmaceutically acceptable carrier or diluent. In an alternative embodiment, an anti-PDGF-DD antibody is linked to a radioisotope or a toxin. In another embodiment, the anti-PDGF-DD antibody or fragment thereof is conjugated to a therapeutic agent. The therapeutic agent can be a toxin or a radioisotope.

[0014] Preferably, the anti-PDGF-DD antibodies of the invention are used for the treatment of diseases, such as, for example, nephritis, progressive renal diseases, and related diseases, such as mesangial proliferative nephritis, mesangial proliferative glomerulonephritis, mesangiocapillary glomerulonephritis, systemic lupus erythematosus, glomerular nephritis, renal interstitial fibrosis, renal failure, and diabetic nephropathy. [0015] In yet another embodiment, the invention includes a method for inhibiting cell proliferation associated with, or caused by, renal failure, renal disease, renal injury and/or other renal dysfunction, by contacting cells expressing PDGF-DD with an effective amount of an anti-PDGF-DD antibody or a fragment thereof and incubating the cells and antibody, wherein the incubation results in inhibited proliferation of cells. In one embodiment, the cell proliferation is mesangial cell proliferation. Further, the mesangial cells can be human mesangial cells. In addition, the method can be performed in vivo. [0016] In one embodiment, the invention includes a method for diagnosing a condition associated with the expression of PDGF-DD, e.g., associated with, or caused by, renal failure, renal disease, renal injury and/or other renal dysfunction, in a cell by contacting the cell with an anti-PDGF-DD antibody, and detecting the presence of PDGF- DD. Preferred conditions include, without limitation, mesangial proliferative nephritis, mesangial proliferative glomerulonephritis, mesangiocapillary glomerulonephritis, systemic lupus erythematosus, glomerular nephritis, renal failure, and diabetic nephropathy. [0017] In still another embodiment, the invention includes an assay kit for the detection of PDGF-DD in mammalian tissues or cells to screen for renal failure, renal disease, renal injury, other renal dysfunction and related diseases in humans, including but not limited to, mesangial proliferative nephritis, mesangial proliferative glomerulonephritis, mesangiocapillary glomerulonephritis, systemic lupus erythematosus, glomerular nephritis, renal failure, and diabetic nephropathy. The kit includes an antibody that binds to PDGF- DD and a means for indicating the reaction of the antibody with PDGF-DD, if present. Preferably the antibody is a monoclonal antibody. In one embodiment, the antibody that binds PDGF-DD is labeled. In another embodiment the antibody is an unlabeled first antibody and the means for indicating the reaction is a labeled antiimmunoglobulin antibody. Preferably, the antibody is labeled with a marker selected from the group consisting of: a fluorochrome, an enzyme, a radionuclide and a radiopaque material. [0018] Yet another embodiment is the use of an anti-PDGF-DD antibody in the preparation of a medicament for the treatment of renal failure, renal disease, renal injury, other renal dysfunction and related diseases. In one embodiment, the disease is selected from the group comprising nephritis, progressive renal diseases, and related diseases, such as mesangial proliferative nephritis, mesangial proliferative glomerulonephritis, mesangiocapillary glomerulonephritis, systemic lupus erythematosus, glomerular nephritis, renal interstitial fibrosis, renal failure, and diabetic nephropathy.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a graph depicting PDGF-D induced proliferation of rat renal fibroblasts to a similar degree as PDGF-B, and that the addition of CR002 specifically inhibited the effect of PDGF-D, but not that of PDGF-B. Incorporation of 5-Bromo- 2'deoxyuridine in growth arrested rat fibroblasts NRK-49F after stimulation with PDGF-BB and -DD (10 ng/ml and 100 ng/ml, respectively) with or without the anti-PDGF-D fully human monoclonal antibody CR002 (1 μg/ml). Data are means ± SEM of four independent experiments. * p<0.05 versus unstimulated control; ^§ p<0.05 versus PDGF-D stimulated cells.

[0020] Figure 2 is a graph depicting that the development of proteinuria was transiently decreased by CR002. Proteinuria was significantly ameliorated by the treatment at days 49, 56 and 77. Arrows indicate the time points of CR002 administration. The insert shows the concentration of circulating CR002 on days 42, 49 and 56 and the grey area represents the approximate duration of effective PDGF-D inhibition. *p<0.05 versus IgG group.

[0021] Figures 3A-3C are a series of photographs and a graph depicting that tubulointerstitial damage was significantly ameliorated by CR002. PAS stained sections from IgG treated (A) or CR002 treated (B) animals. Inhibition of PDGF-D improved the tubulointerstitial damage score (C). Data are mean ± SEM. *p<0.05 versus IgG group.

Magnification 10Ox.

[0022] Figures 4A-4J are a series of photographs depicting the sinus red staining

(A, B) and specific immunohistochemistry for collagen type I (C, D) and III (E, F), vimentin (G, H) and α-smooth muscle actin (I, J) in IgG control animals (images A, C, E, G,

I) or CR002 treated animals (B, D, F, H, J). Magnification 10Ox.

[0023] Figures 5A-5J are a series of graphs depicting the quantitative assessment of glomerular and tubulointerstitial damage on day 100. Panels show Sirius red staining (A, B) and immunohistochemistry for collagen type I (C, D) and type III (E, F), vimentin (G, H) and α-smooth muscle actin (I, J). Graphs showing the glomerular compartment are the upper ones (A, C, E, G, I), those showing tubulointerstitial compartment are below (B, D, F,

H, J). The Y-axis gives the relative area [%] of tissue staining positively with Sirius red or the various immunostains. Data are means ± SEM. *p<0.05 versus IgG group.

DETAILED DESCRIPTION

[0024] The invention described herein relates to methods for effectively treating, preventing, delaying the progression of, or otherwise ameliorating a renal disease, renal fibrosis, renal dysfunction and/or renal injury. In one particular embodiment, the invention includes administering a therapeutically effective amount of anti-PDGF-DD antibodies as a treatment for renal disease, renal fibrosis, renal dysfunction, renal injury and related diseases. In preferred embodiments, the antibodies are fully human antibodies against the dimer PDGF-DD.

[0025] Diabetic nephropathy underlies up to 35% of end stage renal disease cases, and glomerulonephritides, in particular the most common type IgA nephropathy, account for another 20% of cases in most Western countries. Both diabetic nephropathy and the majority of progressive glomerulonephritides are histologically characterized by glomerular mesangial cell proliferation and/or matrix accumulation (Klahr S, et al., N Engl J Med, vol. 318:1657-1666 (1988); Striker LJ, et al., Lab Invest, vol. 64:446-456 (1991)). In addition all of these diseases progress to renal failure via secondary tubulointerstitial damage and fibrosis. Treatments targeting both of these processes would therefore be of major clinical relevance.

[0026] The PDGF system consists of four PDGF chains, PDGF-A to -D, that are secreted as homo- or heterodimers and bind to dimeric PDGF receptors composed of α- and/or β-chains. Whereas PDGF-A and -C bind to the α-chain only, PDGF-B is a ligand for all receptor types and PDGF-DD binds predominantly to the PDGF ββ-receptor (Li X, et al., Nat Cell Biol, vol. 2:302-309 (2000); Bergsten et al., Nat Cell Biol vol. 3:512-516 (2001); LaRochelle WJ, et al., Nat Cell Biol, vol. 3:517-521 (2001)). All four PDGF isoforms, as well as both receptor chains are expressed in the kidney, albeit in distinct spatial arrangements (Floege and Ostendorf, Kidney Int vol. 59:1592-1593 (2001); Changsirikulchai et al., Kidney Int vol. 62:2043-2054 (2002); and Eitner et al., J Am Soc Nephrol, vol. 13:910-917 (2002)).

[0027] The role of both PDGF-B and -D chain in mediating mesangioproliferative changes in glomerular disease is now well established (Bergsten et al., Nat Cell Biol vol. 3:512-516 (2001); LaRochelle WJ, et al., Nat Cell Biol, vol. 3:517-521 (2001); Johnson et al., J Am Soc Nephrol vol. 4:119-128 (1993); Floege and Johnson, Miner Electrolyte Metab vol. 21:271-282 (1995); Ostendorf T, et al., J Am Soc Nephrol, vol. 12:909-918 (2001); Ostendorf T, et al., J Am Soc Nephrol, vol.14:2237-2247 (2003); Hudkins et al., J Am Soc Nephrol, vol. 15:286-298 (2004)). With respect to mediating fϊbrotic damage, some evidence implicates the actions of PDGF-B (Tang et al., Am J Pathol vol 148:1169-1180 (1996)), but increasing evidence also implicates PDGF-D in various organs: a) its renal interstitial expression increases in obstructive uropathy in both humans and mice (Taneda et al., J Am Soc Nephrol vol 14:2544-2555 (2003)), b) it is strongly up-regulated in an in vitro model of hepatic fibrogenesis (Breitkopf et al., Cytokine vol 31:349-357 (2005)), c) it is expressed by synovial fibroblasts and macrophages of patients with rheumatoid arthritis and osteoarthritis (Pohlers et al. Arthritis Rheum vol 54:788-794 (2006)), and most importantly, d) in transgenic mice overexpressing PDGF-D in the heart pronounced cardiac fibrosis developed (Ponten et al. Circ Res 97:1036-1045 (2005)). Finally, the receptor for both PDGF-B and -D, i.e. PDGF receptor-ββ, is also upregulated in the fϊbrotic renal interstitium (Taneda et al., J Am Soc Nephrol vol 14:2544-2555 (2003); Kliem et al. Kidney Int 49:666-678 (1996)).

[0028] It has been shown that specific antagonism of PDGF-D using a fully human monoclonal antibody (CR002) in a progressive model of mesangioproliferative glomerulonephritis, i.e., anti-Thy 1.1 nephritis in rats, potently reduced the early glomerular damage. Despite cessation of the treatment on day 17 after disease induction, it also partially prevented the subsequent development of tubulointerstitial damage (Ostendorf T, et al., J Am Soc Nephrol, vol. 17(4): 1054-62 (2006)). The Examples provided herein were designed to evaluate whether such treatment is also effective if initiated after the acute antibody-mediated phase of glomerular damage has subsided and first tubulointerstitial damage is already established. Treatment was therefore confined to days 17, 28 and 35 after disease induction.

[0029] Arresting or regressing kidney scarring is of major clinical relevance. Platelet

Derived Growth Factor D (PDGF-D) is widely expressed in fibrotic kidneys. Administration of the PDGF-D neutralizing, fully human monoclonal antibody CR002 in the acute phase of progressive anti-Thy 1.1 glomerulonephritis reduced glomerular and secondary tubulointerstitial damage. Using the latter model, the effects of CR002 (n=15) versus irrelevant control IgG (n=17) administered on days 17, 28 and 35 after disease induction, i.e. after acute glomerular damage had subsided has been evaluated as shown in the Examples provided herein. In vitro, CR002 potently reduced the PDGF-D- but not the PDGF-B- induced proliferation of rat renal fibroblasts. Following the first CR002 injection on day 17, exposure to therapeutic levels was maintained until day 49. Proteinuria in the CR002 treated group was transiently reduced between days 49 and 77 (-19 to -23% in comparison to the control group; p<0.05), but the antiprotemuric effect was not maintained until day 100. On day 100 CR002 treatment significantly reduced the number of rats that had doubled their serum creatinine (CR002: 40% vs. controls: 71%; p<0.05). Compared to controls, the CR002 treated group, on day 100, significantly lowered glomerular expression of vimentin and collagens (Sirius red and types I and III collagen) as well as tubulointerstitial damage scores, interstitial fibrosis (Sirius red staining and interstitial type I, type III collagen), vimentin and cortical PDGF-D mRNA levels. Thus, the Examples provided herein demonstrate that PDGF-D antagonism, even after the phase of acute glomerular damage, exerts beneficial effects on the course of tubulointerstitial damage, i.e., the final common pathway of most renal diseases.

[0030] Previous studies demonstrated that administration of the CR002 antibody against PDGF-D on days 3, 10 and 17 after induction of anti-Thy 1.1 nephritis not only ameliorated glomerular damage but also the secondary interstitial fibrosis (Ostendorf T, et al., J Am Soc Nephrol, vol. 17(4): 1054-62 (2006)). Since PDGF-D is overexpressed in both rodent and human fibrotic renal interstitium (Changsirikulchai et al., Kidney Int vol. 62:2043-2054 (2002); Taneda et al., J Am Soc Nephrol vol 14:2544-2555 (2003)), the studies described herein were designed to determine whether PDGF-D antagonism would still be beneficial if initiated after the acute phase of mesangioproliferative nephritis. Since the phase of increased glomerular mesangial cell proliferation in the anti-Thy 1.1 nephritis model subsides at around day 10 after disease induction, CR002 was administered on days 17, 28 and 35. Due to concerns regarding the immunogenicity of the fully human antibody in rats no longer treatment duration was attempted.

[0031] In vitro studies were used to verify that PDGF-D acts as a potent mitogenic stimulus in rat interstitial fibroblasts and that this can be reversed by adding CR002 (Figure 1). The findings demonstrated herein extend data showing that PDGF-D induces proliferation of rat vascular fibroblasts, primary mouse cardiac fibroblasts and human synovial fibroblasts (Pohlers et al. Arthritis Rheum vol 54:788-794 (2006); Ponten et al. Circ Res 97:1036-1045 (2005); Chen et al. Biochem Biophys Res Coramun vol 329:976- 983 (2005)).

[0032] The present study determined that treatment with CR002 on days 17, 28 and

35 transiently decreased proteinuria, slowed progression of renal failure and ameliorated glomerular as well as tubulointerstitial fibrotic changes at day 100. Most effects of CR002 persisted 65 days after the last treatment and approximately 50 days after circulating CR002 concentrations fell below the therapeutic level. In some embodiments, continuous antagonism of PDGF-D from day 17 to 100 is employed.

[0033] The studies described herein are the first to specifically inhibit one of the two ligands of the PDGF β-receptor, i.e. PDGF-B and -D, in a model of renal tubulointerstitial damage. In the case of PDGF-B, no specific antagonism has been tested so far. However, it has been shown that PDGF-B, like PDGF-D, is overexpressed in fibrotic renal interstitium and that the prolonged administration of high doses of PDGF-B can induce renal interstitial fibrosis (Ostendorf T, etal., J Am Soc Nephrol, vol. 12:909-918 (2001); Tang et al., Am J Pathol vol 148: 1169-1180 (1996); Kliem et al. Kidney Int 49:666-678 (1996) and Lassila et al. IJ Am Soc Nephrol vol 16:363-373 (2005)). Without intending to be bound by theory, differences in the pro-fibrotic activity of PDGF-B and -D might be due to the fact that only PDGF-B can activate the PDGF-αα receptor, which is very widely expressed in fibrotic renal tissue (Floege et al. J Am Soc Nephrol vol 9:211-223 (1998); Floege et al. Kidney Int vol 51:1140-1150 (1997)). However, heart-specific overexpression of PDGF-D in mice caused fatal cardiac fibrosis, whereas PDGF-B led to a non-lethal phenotype with only focal fibrosis (Ponten et al. Circ Res 97:1036-1045 (2005)). Also, in mice hepatic overexpression of PDGF-D caused a more profound glomerulopathy than overexpression of PDGF-B (Hudkins et al., J Am Soc Nephrol, vol. 15:286-298 (2004)). At least in mesangial cells, it has been shown that both PDGF-isoforms activated the cells almost exclusively via the β receptor despite the presence of both PDGF a- and β-receptors on these cells (van Roeyen, et al. Kidney Int vol 69: 1393-1402 (2006)). Collectively these observations identify PDGF-D as a more potent agonist of the PDGF β-receptor, whereas the role of the PDGF α-receptor in vivo is less clear.

[0034] Several studies have investigated the effects of imatinib (STI-571), a receptor tyrosin kinase blocker, in models of renal disease. Imatinib is widely used in cancer therapy as a blocker of the c-abl kinase, but also blocks signal transduction of the PDGF-receptor tryrosine kinase (Roskoski R, Jr. Biochem Biophys Res Commun 309:709-717, 2003). Imatinib retarded the development of experimental diabetic nephropathy (Lassila et al. IJ Am Soc Nephrol vol 16:363-373 (2005)), ameliorated experimental chronic allograft nephropathy (Savikko J, Taskinen E, Von Willebrand E. Transplantation 75:1147-1153, 2003) and the course of renal fibrosis after unilateral ureter ligation (Wang S, Wilkes MC, Leof EB, et al. Faseb J 19:1-11, 2005). It also was heart- and renoprotective in a model of hypertensive end-organ damage (Schellings MW, Baumann M, van Leeuwen RE, et al. Hypertension 47:467-474, 2006) and attenuated pulmonary and liver fibrosis in different models (Abdollahi A, Li M, Ping G, et al. J Exp Med 201 :925-935, 2005; Neef M, Ledermann M, Saegesser H, et al. J Hepatol 44:167-175, 2006). However, whether this beneficial effect of imatinib was indeed mediated via reduction of PDGF signaling remains uncertain, since Wang et al (Faseb J 19:1-11, 2005) demonstrated that renal fibroblasts express c-abl kinase, that the major pro-fibrotic cytokine TGF-β acts via c-abl kinase, and that imatinib can interfere with this process. More relevant in the context of the present study is the observation that specific inhibition of PDGF-B chain by antisense mRNA in rat model of liver fibrosis significantly attenuated the overexpression of type I collagen and α- smooth muscle actin (Borkham-Kamphorst E, Stoll D, Gressner AM, et al. A Biochem Biophys Res Commun 321:413-423, 2004).

[0035] De novo expression of vimentin and α-SMA and the tubular epithelial loss of

E-cadherin expression are markers of epithelial-mesenchymal transition, i.e. a phenotypic change of tubular epithelial cells to (myo-) fibroblast-like cells. EMT is believed to play an important role in kidney fibrosis (Kalluri R, Neilson EG. J Clin Invest 112:1776-1784, 2003; Thiery JP, Sleeman JP. Nat Rev MoI Cell Biol 7: 131-142, 2006). Late PDGF-D antagonism had inconsistent effects on EMT, since it did not influence the loss of E- cadherin, but downregulated the tubular de novo vimentin expression (Fig.4 G-J). Thus late PDGF-D inhibition did not interfere with the process of EMT as potently as the early inhibition (Ostendorf T, et al., J Am Soc Nephrol, vol. 17(4): 1054-62 (2006)). [0036] PDGF-D antagonism in the studies presented herein did not affect the glomerular or tubulointerstitial monocyte/macrophage influx on day 100. It remains unclear, whether PDGF-D has a direct chemotactic role for macrophages similar to that of PDGF-B (Siegbahπ A, Hammacher A, Westermark B, et al. J Clin Invest 85:916-920, 1990; Krettek A, Ostergren-Lunden G, Fager G, et al. Atherosclerosis 156:267-275, 2001). Previous data as well as those of Uutela et al. (Ostendorf T, et al., J Am Soc Nephrol, vol. 17(4):1054-62 (2006); Uutela M, Wirzenius M, Paavonen K, et al. Blood 104:3198-3204, 2004) suggest that PDGF-D is capable of recruiting monocytes/macrophages into the kidney and skin, respectively. However, the PDGF receptor tyrosin kinase inhibitor imatinib also had inconsistent effects in this respect, since it reduced macrophage infiltration in diabetic nephropathy (Lassila et al. IJ Am Soc Nephrol vol 16:363-373 (2005)) but not in rat renal interstitial fibrosis (Wang S, Wilkes MC, Leof EB, et al. Faseb J 19: 1-11, 2005). [0037] CR002 did not affect the renal cortical mRNA levels of PDGF-B and

PDGFR-β on day 100 but led to a persistent decrease of PDGF-D mRNA. Whereas an autocrine, self-stimulatory loop is well recognized in the case of PDGF-BB (Bhandari B, Grandaliano G, Abboud HE. Biochem J 297 (Pt 2):385-388, 1994), the data presented herein suggest the existence of a similar auto-regulatory loop for PDGF-D in vivo. [0038] Thus, the methods provided herein are the first to use specific antagonism of

PDGF-D as a therapeutically effective treatment in the phase of active, immune-mediated glomerular injury, but also in later phases where progression of renal disease has become independent of the initiating condition. PDGF-D antagonism thereby targets two clinically important processes and provides therapeutic efficacy to patients in various phases of progressive glomerular disease.

Sequence Listing

[0039] The heavy chain and light chain variable region nucleotide and amino acid sequences of representative human anti-PDGF-DD antibodies are provided in the sequence listing, the contents of which are summarized in Table 1 below.

Table 1

Definitions

[0040] Unless otherwise defined, scientific and technical terms used in connection with the invention described herein shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (2001)), which is incorporated herein by reference. The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. [0041] As utilized in accordance with the embodiments provided herein, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0042] Mesangial cells are cells found within the glomerular lobules of mammalian kidney where they serve as structural supports, may regulate blood flow, are phagocytic and may act as accessory cells, presenting antigen in immune responses.

[0043] Mesangial proliferative nephritis is glomerulonephritis with an increase in glomerular mesangial cells or matrix, or mesangial deposits.

[0044] Mesangial proliferative glomerulonephritis is an inflammation of the kidney glomerulus (blood filtering portion of the kidney) due to the abnormal deposition of IgM antibody in the mesangium layer of the glomerular capillary.

[0045] Mesangiocapillary glomerulonephritis is a kidney disorder which results in kidney dysfunction. Inflammation of the glomeruli result from an abnormal immune response and the deposition of antibodies within the kidney (glomerulus). Symptoms include cloudy urine (pyuria), decreased urine output, swelling and hypertension. The disorder often results in end-stage renal disease.

[0046] The mesangium is the central part of the glomerulus between capillaries.

Mesangial cells are phagocytic and for the most part separated from capillary lumina by endothelial cells. Extraglomerular mesangium are mesangial cells that fill the triangular space between the macula densa and the afferent and efferent arterioles of the juxtaglomerular apparatus.

[0047] Glomerulonephritis is a variety of nephritis which is characterized by inflammation of the capillary loops in the glomeruli of the kidney. It occurs in acute, subacute and chronic forms and may be secondary to infection or autoimmune disease.

[0048] The term "PDGF-DD "includes PDGF-DD in its full length and mature form, along with its variants, and fragments thereof. Accordingly, PDGF-DD can include, but is not limited to, variants CG52053-01, CGS2053-02, CG52053-03, CG52053-04,

CG52053-05, CG52053-06, and CG52053-07. (CuraGen, New Haven, CT). More information can be found in PCT Publication WO 01/25433 filed October 7, 1999.

[0049] The term "isolated polynucleotide" as used herein shall mean a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the "isolated polynucleotide" (1) is not associated with all or a portion of a polynucleotide in which the "isolated polynucleotide" is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence.

[0050] The term "isolated protein" referred to herein means a protein of cDNA, recombinant RNA, or synthetic origin or some combination thereof, which by virtue of its origin, or source of derivation, the "isolated protein" (1) is not associated with proteins found in nature, (2) is free of other proteins from the same source, e.g. free of murine proteins, (3) is expressed by a cell from a different species, or (4) does not occur in nature. [0051] The term "polypeptide" is used herein as a generic term to refer to native protein, fragments, or analogs of a polypeptide sequence. Hence, native protein, fragments, and analogs are species of the polypeptide genus. Preferred polypeptides in accordance with the invention comprise the human heavy chain immunoglobulin molecules and the human kappa light chain immunoglobulin molecules, as well as antibody molecules formed by combinations comprising the heavy chain immunoglobulin molecules with light chain immunoglobulin molecules, such as the kappa light chain immunoglobulin molecules, and vice versa, as well as fragments and analogs thereof.

[0052] The term "naturally occurring" as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory or otherwise is naturally occurring.

[0053] The term "operably linked" as used herein refers to positions of components so described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0054] The term "control sequence" as used herein refers to polynucleotide sequences which are necessary to effect the expression and processing of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term "control sequences" is intended to include, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. [0055] The term "polynucleotide" as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.

[0056] The term "oligonucleotide" referred to herein includes naturally occurring, and modified nucleotides linked together by naturally occurring, and non-naturally occurring oligonucleotide linkages. Oligonucleotides are a polynucleotide subset generally comprising a length of 200 bases or fewer. Preferably oligonucleotides are 10 to 60 bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides are usually single stranded, e.g. for probes; although oligonucleotides may be double stranded, e.g. for use in the construction of a gene mutant. Oligonucleotides of the invention can be either sense or antisense oligonucleotides. [0057] The term "naturally occurring nucleotides" referred to herein includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" referred to herein includes nucleotides with modified or substituted sugar groups and the like. The term "oligonucleotide linkages" referred to herein includes oligonucleotides linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoroamidate, and the like. See e.g., LaPlanche et al. Nucl. Acids Res. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984); Stein et al. Nucl. Acids Res. 16:3209 (1988); Zon et al. Anti-Cancer Drug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: A Practical Approach, pp. 87- 108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec et al. U.S. Patent No. 5,151,510; Uhlmann and Peyman Chemical Reviews 90:543 (1990), the disclosures of which are hereby incorporated by reference. An oligonucleotide can include a label for detection, if desired.

[0058] The term "selectively hybridize" referred to herein means to detectably and specifically bind. Polynucleotides, oligonucleotides and fragments thereof in accordance with the invention selectively hybridize to nucleic acid strands under hybridization and wash conditions that minimize appreciable amounts of detectable binding to nonspecific nucleic acids. High stringency conditions can be used to achieve selective hybridization conditions as known in the art and discussed herein. Generally, the nucleic acid sequence homology between the polynucleotides, oligonucleotides, and fragments of the invention and a nucleic acid sequence of interest will be at least 80%, and more typically with preferably increasing homologies of at least 85%, 90%, 95%, 99%, and 100%. Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less are preferred with 2 or less being more preferred. Alternatively and preferably, two protein sequences (or polypeptide sequences derived from them of at least 30 amino acids in length) are homologous, as this term is used herein, if they have an alignment score of at more than 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater. See M.O. Dayhoff, in Atlas of Protein Sequence and Structure, Vol. 5, 101-110 and Supplement 2 to Vol. 5, 1-10 (National Biomedical Research Foundation 1972). The two sequences or parts thereof are more preferably homologous if their amino acids are greater than or equal to 50% identical when optimally aligned using the ALIGN program. The term "corresponds to" is used herein to mean that a polynucleotide sequence is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence. In contradistinction, the term "complementary to" is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. For illustration, the nucleotide sequence "TATAC" corresponds to a reference sequence "TATAC" and is complementary to a "GTATA".

[0059] The following terms are used to describe the sequence relationships between two or more polynucleotide or amino acid sequences: "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity," and "substantial identity". A "reference sequence" is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing or may comprise a complete cDNA or gene sequence. Generally, a reference sequence is at least 18 nucleotides or 6 amino acids in length, frequently at least 24 nucleotides or 8 amino acids in length, and often at least 48 nucleotides or 16 amino acids in length. Since two polynucleotides or amino acid sequences may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide or amino acid sequence) that is similar between the two molecules, and (2) may further comprise a sequence that is divergent between the two polynucleotides or amino acid sequences, sequence comparisons between two (or more) molecules are typically performed by comparing sequences of the two molecules over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window," as used herein, refers to a conceptual segment of at least 18 contiguous nucleotide positions or 6 amino acids wherein a polynucleotide sequence or amino acid sequence may be compared to a reference sequence of at least 18 contiguous nucleotides or 6 amino acid sequences and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions, deletions, substitutions, and the like (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoI. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 (1988), by computerized implementations of these algorithms (GAP₅ BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, (Genetics Computer Group, 575 Science Dr., Madison, Wis.), Geneworks, or Mac Vector software packages), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected.

[0060] The term "sequence identity" means that two polynucleotide or amino acid sequences are identical (i.e., on a nucleotide-by-nucleotide or residue-by-residue basis) over the comparison window. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) or residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms "substantial identity" as used herein denotes a characteristic of a polynucleotide or amino acid sequence, wherein the polynucleotide or amino acid comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 18 nucleotide (6 amino acid) positions, frequently over a window of at least 24-48 nucleotide (8-16 amino acid) positions, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the comparison window. The reference sequence may be a subset of a larger sequence.

[0061] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology - A Synthesis (2d ed., Golub, E.S. and Gren, D.R. eds., Sinauer Associates, Sunderland, Mass. 1991), which is incorporated herein by reference. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the invention described herein. Examples of unconventional amino acids include: 4-hydroxyproline, γ -carboxyglutamate, ε-N,N,N-trirnethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4- hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

[0062] Similarly, unless specified otherwise, the left-hand end of single-stranded polynucleotide sequences is the 5' end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5' direction. The direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA and which are 5' to the 5' end of the RNA transcript are referred to as "upstream sequences"; sequence regions on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the RNA transcript are referred to as "downstream sequences".

[0063] As applied to polypeptides, the term "substantial identity" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity, and most preferably at least 99 percent sequence identity. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic-aspartic, and asparagine-glutamine.

[0064] As discussed herein, minor variations in the amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the invention described herein, providing that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% of the original sequence. In particular, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into families: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3) non-polar=alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. More preferred families are: serine and threonine are aliphatic-hydroxy family; asparagine and glutamine are an amide-containing family; alanine, valine, leucine and isoleucine are an aliphatic family; and phenylalanine, tryptophan, and tyrosine are an aromatic family. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding or properties of the resulting molecule, especially if the replacement does not involve an amino acid within a framework site. Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those of ordinary skill in the art. Preferred amino- and carboxy-termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and/or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function. Methods to identify protein sequences that fold into a known three-dimensional structure are known. Bowie et ah, Science 253:164 (1991). Thus, the foregoing examples demonstrate that those of skill in the art can recognize sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with the invention.

[0065] Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (4) confer or modify other physicochemical or functional properties of such analogs. Analogs can include various muteins of a sequence other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally occurring sequence (preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, ed., W. H. Freeman and Company, New York 1984); Introduction to Protein Structure (Branden, C. and Tooze, J. eds., Garland Publishing, New York, N. Y. 1991); and Thornton et al, Nature 354:105 (1991), which are each incorporated herein by reference.

[0066] The term "polypeptide fragment" as used herein refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally occurring sequence deduced, for example, from a full-length cDNA sequence. Fragments typically are at least 5, 6, 8 or 10 amino acids long, preferably at least 14 amino acids long, more preferably at least 20 amino acids long, usually at least 50 amino acids long, and even more preferably at least 70 amino acids long. The term "analog" as used herein refers to polypeptides which are comprised of a segment of at least 25 amino acids that has substantial identity to a portion of a deduced amino acid sequence and which has at least one of the following properties: (1) specific binding to a PDGF-DD dimer, under suitable binding conditions, (2) ability to block appropriate PDGF-DD binding, or (3) ability to inhibit PDGF-DD expressing cell growth in vitro or in vivo. Typically, polypeptide analogs comprise a conservative amino acid substitution (or addition or deletion) with respect to the naturally occurring sequence. Analogs typically are at least 20 amino acids long, preferably at least 50 amino acids long or longer, and can often be as long as a full-length naturally occurring polypeptide.

[0067] Peptide analogs are commonly used in the pharmaceutical industry as non- peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed "peptide mimetics" or "peptidomimetics." Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger, TINS p.392 (1985); and Evans et al., J. Med. Chem. 30:1229 (1987), which are incorporated herein by reference. Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biochemical property or pharmacological activity), such as human antibody, but have one or more peptide linkages optionally replaced by a linkage selected from the group consisting of: -CH₂NH--, --CH₂S- -, --CH₂-CH₂-, -CH=CH-(CiS and trans), -COCH₂-, --CH(OH)CH₂-, and -CH₂SO-, by methods well known in the art. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L- lysine) may be used to generate more stable peptides. In addition, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein by reference); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide. [0068] "Antibody" or "antibody peptide(s)" refer to an intact antibody, or a binding fragment thereof that competes with the intact antibody for specific binding. Binding fragments are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab', F(ab')₂, Fv, and single- chain antibodies. An antibody other than a "bispecific" or "bifunctional" antibody is understood to have each of its binding sites identical. An antibody substantially inhibits adhesion of a receptor to a counterreceptor when an excess of antibody reduces the quantity of receptor bound to counterreceptor by at least about 20%, 40%, 60% or 80%, and more usually greater than about 85% (as measured in an in vitro competitive binding assay). [0069] The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. An antibody is said to specifically bind an antigen when the dissociation constant is <1 μM, preferably < 100 nM and most preferably ≤ 10 nM. [0070] The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.

[0071] "Active" or "activity" for the purposes herein refers to form(s) of PDGF-DD polypeptide which retain a biological and/or an immunological activity of native or naturally occurring PDGF-DD polypeptides, wherein "biological" activity refers to a biological function (either inhibitory or stimulatory) caused by a native or naturally occurring PDGF-DD polypeptide other than the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally occurring PDGF- DD polypeptide and an "immunological" activity refers to the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally occurring PDGF-DD polypeptide.

[0072] "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.

[0073] "Mammal" refers to any animal classified as a mammal, including humans, other primates, such as monkeys, chimpanzees and gorillas, domestic and farm animals, and zoo, sports, laboratory, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, rodents, etc. For purposes of treatment, the mammal is preferably human. [0074] "Carriers" as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0075] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment yields an "F(ab')2" fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

[0076] "Fv" is the minimum antibody fragment that contains a complete antigen- recognition and binding site of the antibody. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen- binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen- binding specificity to the antibody. However, for example, even a single variable domain (e.g., the VH or VL portion of the Fv dimer or half of an Fv comprising only three CDRs specific for an antigen) may have the ability to recognize and bind antigen, although, possibly, at a lower affinity than the entire binding site.

[0077] A Fab fragment also contains the constant domain of the light chain and the first constant domain (CHl) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHl domain including one or more cysteines from the antibody hinge region. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. [0078] "Solid phase" means a non-aqueous matrix to which the antibodies described herein can adhere. Examples of solid phases encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid phases can comprise the well of an assay plate; in others it is a purification column (e.g., an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Patent No.4,275,149.

[0079] The term "liposome" is used herein to denote a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as a PDGF-DD polypeptide or antibody thereto) to a mammal. The components of the liposomes are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.

[0080] The term "small molecule" is used herein to describe a molecule with a molecular weight below about 500 Daltons.

[0081] As used herein, the terms "label" or "labeled" refers to incorporation of a detectable marker, e.g., by incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). In certain situations, the label or marker can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0082] The term "pharmaceutical agent or drug" as used herein refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient. Other chemistry terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)), incorporated herein by reference). [0083] As used herein, "substantially pure" means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, more preferably more than about 85%, 90%, 95%, and 99%. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. [0084] The term "patient" includes human and veterinary subjects.

Anti-PDGF-DD antibodies

[0085] Antibodies, or parts, fragments, mimetics, or derivatives thereof, may be any type of antibody or part which recognizes a PDGF-DD dimer. In certain embodiments, it is preferred that the antibody, or part thereof, can neutralize PDGF-DD. In additional embodiments, it is preferred that the antibody, or part thereof, can reduce the symptoms associated with PDGF-DD and kidney disease and/or renal fibrosis, renal failure, renal injury or other renal dysfunction, including but not limited to inflammation, fluid retention, tissue swelling, pain, puffmess, high blood pressure, brain swelling, visual disturbances, low urine volume, and urine containing blood. According to one embodiment, the antibody can be anti-PDGF-DD mAb 6.4 (also referred to herein as CR002), for example. Further examples of such antibodies can be found in U.S. Patent No. 7,135,174; United States Patent Application Publication No. US 2004-0141969 Al; and PCT Application Publication No. WO 2007/059234.

Antibody Structure

[0086] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as rau, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2d ed. Raven Press, N. Y. (1989)) (incorporated by reference in its entirety for all purposes). The variable regions of each light/heavy chain pair form the antibody binding site. Thus, an intact antibody has two binding sites. Except in bifunctional or bispecifϊc antibodies, the two binding sites are the same.

[0087] The chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminal to C- terminal, both light and heavy chains comprise the domains FRl, CDRl, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & LβskJ. MoI. Biol. 196:901-917 (1987); Chothia et al. Nature 342:878-883 (1989).

[0088] A bispecifϊc or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bi specific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 19: 315-321 (1990), Kostelny et al., J. Immunol. 148:1547-1553 (1992). Production of bispecifϊc antibodies can be a relatively labor intensive process compared with production of conventional antibodies and yields and degree of purity are generally lower for bispecifϊc antibodies. Bispecifϊc antibodies do not exist in the form of fragments having a single binding site (e.g., Fab, Fab', and Fv).

[0089] It will be appreciated that such bifunctional or bispecifϊc antibodies are contemplated and encompassed by the invention.

Human Antibodies and Humanization of Antibodies

[0090] Embodiments of the invention described herein also contemplate and encompass human antibodies. For treatment of a human, human antibodies avoid certain of the problems associated with antibodies that possess murine or rat variable and/or constant regions. The presence of such murine or rat derived proteins can lead to the rapid clearance of the antibodies or can lead to the generation of an immune response against the antibody by a patient. In order to avoid the utilization of murine or rat derived antibodies, it has been postulated that one can develop humanized antibodies or generate fully human antibodies through the introduction of human antibody function into a rodent so that the rodent would produce fully human antibodies.

Human Antibodies

[0091] One method for generating fully human antibodies is through the use of

XenoMouse® strains of mice that have been engineered to contain human heavy chain and light chain genes within their genome. For example, a XenoMouse® mouse containing 245 kb and 190 kb-sized germline configuration fragments of the human heavy chain locus and kappa light chain locus is described in Green et al., Nature Genetics 7:13-21 (1994). The work of Green et al. was extended to the introduction of greater than approximately 80% of the human antibody repertoire through utilization of megabase-sized, germline configuration YAC fragments of the human heavy chain loci and kappa light chain loci, respectively. See Mendez et al., Nature Genetics 15:146-56 (1997) and U.S. Patent Application Serial No. 08/759,620, filed December 3, 1996, the disclosures of which are hereby incorporated by reference. Further, XenoMouse® mice have been generated that contain the entire lambda light chain locus (U.S. Patent Application Serial No. 60/334,508, filed November 30, 2001). And, XenoMouse® mice have been generated that produce multiple isotypes {see, e.g., WO 00/76310). XenoMouse® strains are available from Abgenix, Inc. (Fremont, CA).

[0092] The production of XenoMouse® mice is further discussed and delineated in

U.S. Patent Application Serial Nos. 07/466,008, filed January 12, 1990, 07/610,515, filed November 8, 1990, 07/919,297, filed July 24, 1992, 07/922,649, filed July 30, 1992, filed 08/031,801, filed March 15,1993, 08/112,848, filed August 27, 1993, 08/234,145, filed April 28, 1994, 08/376,279, filed January 20, 1995, 08/430,938, April 27, 1995, 08/464,584, filed June 5, 1995, 08/464,582, filed June 5, 1995, 08/463,191, filed June 5, 1995, 08/462,837, filed June 5, 1995, 08/486,853, filed June 5, 1995, 08/486,857, filed June 5, 1995, 08/486,859, filed June 5, 1995, 08/462,513, filed June 5, 1995, 08/724,752, filed October 2, 1996, and 08/759,620, filed December 3, 1996 and U.S. Patent Nos. 6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598 and Japanese Patent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2. See also Mendez et al. Nature Genetics 15:146-156 (1997) and Green and Jakobovits J. Exp. Med., 188:483-495 (1998). See also European Patent No., EP 463,151 Bl, grant published June 12, 1996, International Patent Application No., WO 94/02602, published February 3, 1994, International Patent Application No., WO 96/34096, published October 31, 1996, WO 98/24893, published June 11, 1998, WO 00/76310, published December 21, 2000. The disclosures of each of the above-cited patents, applications, and references are hereby incorporated by reference in their entirety. [0093] In an alternative approach, others, including GenPharm International, Inc., have utilized a "minilocus" approach. In the minilocus approach, an exogenous Ig locus is mimicked through the inclusion of pieces (individual genes) from the Ig locus. Thus, one or more V_H genes, one or more D_H genes, one or more J_H genes, a mu constant region, and a second constant region (preferably a gamma constant region) are formed into a construct for insertion into an animal. This approach is described in U.S. Patent No. 5,545,807 to Surani et al and U.S. Patent Nos. 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,877,397, 5,874,299, and 6,255,458 each to Lonberg and Kay, U.S. Patent No. 5,591,669 and 6,023.010 to Krimpenfort and Berns, U.S. Patent Nos. 5,612,205, 5,721,367, and 5,789,215 to Berns et al, and U.S. Patent No. 5,643,763 to Choi and Dunn, and GenPharm International U.S. Patent Application Serial Nos. 07/574,748, filed August 29, 1990, 07/575,962, filed August 31, 1990, 07/810,279, filed December 17, 1991, 07/853,408, filed March 18, 1992, 07/904,068, filed June 23, 1992, 07/990,860, filed December 16, 1992, 08/053,131, filed April 26, 1993, 08/096,762, filed July 22, 1993, 08/155,301, filed November 18, 1993, 08/161,739, filed December 3, 1993, 08/165,699, filed December 10, 1993, 08/209,741, filed March 9, 1994, the disclosures of which are hereby incorporated by reference. See also European Patent No. 0 546 073 Bl, International Patent Application Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884 and U.S. Patent No. 5,981,175, the disclosures of which are hereby incorporated by reference in their entirety. See further Taylor et al, 1992, Chen et al , 1993, Tuaillon et al., 1993, Choi et al, 1993, Lonberg et al, (1994), Taylor et al., (1994), and Tuaillon et al, (1995), Fishwild et al, (1996), the disclosures of which are hereby incorporated by reference in their entirety.

[0094] The inventors of Surani et al, cited above and assigned to the Medical

Research Counsel (the "MRC"), produced a transgenic mouse possessing an Ig locus through use of the minilocus approach. The inventors on the GenPharm International work, cited above, Lonberg and Kay, following the lead of the present inventors, proposed inactivation of the endogenous mouse Ig locus coupled with substantial duplication of the Surani et al. work.

[0095] An advantage of the minilocus approach is the rapidity with which constructs including portions of the Ig locus can be generated and introduced into animals. Commensurately, however, a significant disadvantage of the minilocus approach is that, in theory, insufficient diversity is introduced through the inclusion of small numbers of V, D, and J genes. Indeed, the published work appears to support this concern. B-cell development and antibody production of animals produced through use of the minilocus . approach appear stunted. Therefore, research surrounding the invention described herein has consistently been directed towards the introduction of large portions of the Ig locus in order to achieve greater diversity and in an effort to reconstitute the immune repertoire of the animals.

[0096] Kirin has also demonstrated the generation of human antibodies from mice in which, through microcell fusion, large pieces of chromosomes, or entire chromosomes, have been introduced. See European Patent Application Nos.: 773 288 and 843 961, the disclosures of which are hereby incorporated by reference.

[0097] Lidak Pharmaceuticals (now Xenorex) has also demonstrated the generation of human antibodies in SCID mice modified by injection of non-malignant mature peripheral leukocytes from a human donor. The modified mice exhibit an immune response characteristic of the human donor upon stimulation with an immunogen, which consists of the production of human antibodies. See U.S. Patent Nos. 5,476,996 and 5,698,767, the disclosures of which are herein incorporated by reference.

[0098] Human anti-mouse antibody (HAMA) responses have led the industry to prepare chimeric or otherwise humanized antibodies. While chimeric antibodies have a human constant region and a murine variable region, it is expected that certain human anti- chimeric antibody (HACA) responses will be observed, particularly in chronic or multi-dose utilizations of the antibody. Thus, it would be desirable to provide fully human antibodies against PDGF-DD in order to vitiate concerns and/or effects of HAMA or HACA response.

Humanization and Display Technologies

[0099] As discussed above in connection with human antibody generation, there are advantages to producing antibodies with Teduced immunogenicity. To a degree, this can be accomplished in connection with techniques of humanization and display techniques using appropriate libraries. It will be appreciated that murine antibodies or antibodies from other species can be humanized or primatized using techniques well known in the art. See e.g., Winter and Harris, Immunol Today 14:43-46 (1993) and Wright et al., Crit, Reviews in Immunol. 12:125-168 (1992). The antibody of interest may be engineered by recombinant DNA techniques to substitute the CHl, CH2, CH3, hinge domains, and/or the framework domain with the corresponding human sequence {see WO 92/02190 and U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,792, 5,714,350, and 5,777,085). Also, the use of Ig cDNA for construction of chimeric immunoglobulin genes is known in the art (Liu et al., P.N.A.S. 84:3439 (1987) and J. Immunol 139:3521 (1987)). mRNA is isolated from a hybridoma or other cell producing the antibody and used to produce cDNA. The cDNA of interest may be amplified by the polymerase chain reaction using specific primers (U.S. Pat. Nos. 4,683,195 and 4,683,202). Alternatively, a library is made and screened to isolate the sequence of interest. The DNA sequence encoding the variable region of the antibody is then fused to human constant region sequences. The sequences of human constant regions genes may be found in Kabat et al., "Sequences of Proteins of Immunological Interest," N.I.H. publication no. 91-3242 (1991). Human C region genes are readily available from known clones. The choice of isotype will be guided by the desired effector functions, such as complement fixation, or activity in antibody-dependent cellular cytotoxicity. Preferred isotypes are IgGl, IgG3 and IgG4. Either of the human light chain constant regions, kappa or lambda, may be used. The chimeric, humanized antibody is then expressed by conventional methods.

[00100] Antibody fragments, such as Fv, F(ab').sub.2 and Fab may be prepared by cleavage of the intact protein, e.g., by protease or chemical cleavage. Alternatively, a truncated gene is designed. For example, a chimeric gene encoding a portion of the F(ab')₂ fragment would include DNA sequences encoding the CHl domain and hinge region of the H chain, followed by a translational stop codon to yield the truncated molecule. [00101] Consensus sequences of heavy and light J regions may be used to design oligonucleotides for use as primers to introduce useful restriction sites into the J region for subsequent linkage of V region segments to human C region segments. C region cDNA can be modified by site directed mutagenesis to place a restriction site at the analogous position in the human sequence.

[00102] Expression vectors include plasmids, retroviruses, YACs, EBV derived episomes, and the like. A convenient vector is one that encodes a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any VH or VL sequence can be easily inserted and expressed. In such vectors, splicing usually occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C region, and also at the splice regions that occur within the human CH exons. Polyadenylatϊon and transcription termination occur at native chromosomal sites downstream of the coding regions. The resulting chimeric antibody may be joined to any strong promoter, including retroviral LTRs, e.g., SV-40 early promoter, (Okayama et al, MoI. Cell. Bio. 3:280 (1983)), Rous sarcoma virus LTR (Gorman et al, P.N.A.S. 19:6111 (1982)), and moloney murine leukemia virus LTR (Grosschedl et al., Cell 41:885 (1985)). Also, as will be appreciated, native Ig promoters and the like may be used. [00103] Further, human antibodies or antibodies from other species can be generated through display-type technologies, including, without limitation, phage display, retroviral display, ribosomal display, and other techniques, using techniques well known in the art and the resulting molecules can be subjected to additional maturation, such as affinity maturation, as such techniques are well known in the art. Wright and Harris, supra., Hanes and Plucthau, PNAS USA 94:4937-4942 (1997) (ribosomal display), Parmley and Smith, Gene 73:305-318 (1988) (phage display), Scott, TIBS 17:241-245 (1992), Cwirla etal, PNAS USA 87:6378-6382 (1990), Russel et al.,Nucl. Acids Res. 21:1081-1085 (1993), Hoganboom et al., Immunol. Reviews 130:43-68 (1992), Chiswell and McCafferty, TIBTECH 10:80-84 (1992), and U.S. Patent No. 5,733,743. If display technologies are utilized to produce antibodies that are not human, such antibodies can be humanized as described above.

[00104] Using these techniques, antibodies can be generated to PDGF-DD expressing cells, PDGF-DD itself, forms of PDGF-DD, epitopes or peptides thereof, and expression libraries thereto (see e.g. U.S. Patent No. 5,703,057) which can thereafter be screened as described above for the activities described above.

Preparation of Antibodies

[00105] Through use of XenoMouse® technology, fully human monoclonal antibodies specific for the dimer form of PDGF-D were produced. Essentially, XenoMouse™ lines of mice were immunized with PDGF-DD; or fragments thereof, lymphatic cells (such as B-cells) were recovered from the mice that express antibodies, recovered cells were fused with a myeloid-type cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines were screened and selected to identify hybridoma cell lines that produced antibodies specific to PDGF-DD. Further, a characterization of the antibodies produced by such cell lines is described herein, including nucleotide and amino acid sequence analyses of the heavy and light chains of such antibodies. [00106] In preferred embodiments the antibody is selected from neutralizing anti-

PDGF-DD mAbs 1.6, 1.9, 1.18, 1.19, 1.22, 1.29, 1.33, 1.40.1, 1.45, 1.46, 1.51, 1.59, and 6.4 described herein. See PCT publication WO 03/057,857, dated July 17, 2003, which is hereby expressly incorporated by reference in its entirety. Of course, the disclosed methods are not limited to use of any particular anti-PDGF-DD monoclonal antibody, but rather encompass the use of any such antibody.

[00107] Alternatively, instead of being fused to myeloma cells to generate hybridomas, the recovered cells, isolated from immunized XenoMouse™ lines of mice, can be screened further for reactivity against the initial antigen, preferably PDGF-DD protein. Such screening includes ELISA with PDGF-DD-His protein, a competition assay with known antibodies that bind the antigen of interest, and in vitro binding to transiently transfected CHO cells expressing full length PDGF-DD. Single B cells secreting antibodies of interest are then isolated using a PDGF-DD-specifϊc hemolytic plaque assay (Babcook et al, Proc. Natl. Acad. ScL USA, 93:7843-7848 (1996)). Cells targeted for lysis are preferably sheep red blood cells (SRBCs) coated with the PDGF-DD antigen. In the presence of a B cell culture secreting the immunoglobulin of interest and complement, the formation of a plaque indicates specific PDGF-DD-mediated lysis of the target cells. The single antigen-specific plasma cell in the center of the plaque can be isolated and the DNA that encodes the antibody can then be isolated from the single plasma cell. Using reverse- transcriptase PCR, the DNA encoding the variable region of the antibody secreted can be specifically cloned. Such cloned DNA can then be further inserted into a suitable expression vector, preferably a vector cassette such as a pcDNA, more preferably such a pcDNA vector containing the constant domains of immunoglobulin heavy and light chain. The generated vector can then be transfected into host cells, preferably CHO cells, and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. The isolation of multiple single plasma cells that produce antibodies specific to PDGF-DD is described herein. Further, the genetic material that encodes the specificity of the anti- PDGF-DD antibody is isolated, introduced into a suitable expression vector which is then transfected into host cells.

[00108] In general, it was found that antibodies produced by the above-mentioned cell lines possessed fully human IgG2 heavy chains with human kappa light chains. The antibodies had high affinities, typically possessing Kd's of from about 10^"6 through about 10^"11 M, when measured by either solid phase and solution phase. These mAbs can be stratified into groups or "bins" based on antigen binding competition studies. See PCT publication WO 03/048,731, dated June 12, 2003, which is hereby expressly incorporated by reference, for a description of this process.

[00109] Regarding the importance of affinity to therapeutic utility of anti-PDGF-DD antibodies, it will be understood that one can generate anti-PDGF-DD antibodies, for example, combinatorially, and assess such antibodies for binding affinity. One approach that can be utilized is to take the heavy chain cDNA from an antibody, prepared as described above and found to have good affinity to PDGF-DD, and combine it with the light chain cDNA from a second antibody, prepared as described above and also found to have good affinity to PDGF-DD, to produce a third antibody. The affinities of the resulting third antibodies can be measured as described herein and those with desirable dissociation constants are isolated and characterized. Alternatively, the light chain of any of the antibodies described above can be used as a tool to aid in the generation of a heavy chain that when paired with the light chain will exhibit a high affinity for PDGF-DD, or vice versa. These heavy chain variable regions in this library could be isolated from naϊve animals, isolated from hyperimmune animals, generated artificially from libraries containing variable heavy chain sequences that differ in the CDR regions, or generated by any other methods that produce diversity within the CDR regions of any heavy chain variable region gene (such as random or directed mutagenesis). These CDR regions, and in particular CDR3, may be a significantly different length or sequence identity from the heavy chain initially paired with the original antibody. The resulting library could then be screened for high affinity binding to PDGF-DD to generate a therapeutically relevant antibody molecule with similar properties as the original antibody (high affinity and neutralization). A similar process using the heavy chain or the heavy chain variable region can be used to generate a therapeutically relevant antibody molecule with a unique light chain variable region. Furthermore, the novel heavy chain variable region, or light chain variable region, can then be used in a similar fashion as described above to identify a novel light chain variable region, or heavy chain variable region, that allows the generation of a novel antibody molecule.

[00110] Another combinatorial approach that can be utilized is to perform mutagenesis on germ line heavy and/or light chains that are demonstrated to be utilized in the antibodies in accordance with the invention described herein, particularly in the complementarity determining regions (CDRs). The affinities of the resulting antibodies can be measured as described herein and those with desirable dissociation constants isolated and characterized. Upon selection of a preferred binder, the sequence or sequences encoding the same may be used to generate recombinant antibodies as described above. Appropriate methods of performing mutagenesis on an oligonucleotide are known to those skilled in the art and include chemical mutagenesis, for example, with sodium bisulfite, enzymatic misincorporation, and exposure to radiation. It is understood that the invention described herein encompasses antibodies with substantial identity, as defined herein, to the antibodies explicitly set forth herein, whether produced by mutagenesis or by any other means. Further, antibodies with conservative or non-conservative amino acid substitutions, as defined herein, made in the antibodies explicitly set forth herein, are included in embodiments of the invention described herein.

[00111] Another combinatorial approach that can be used is to express the CDR regions, and in particular CDR3, of the antibodies described above in the context of framework regions derived from other variable region genes. For example, CDRl, CDR2, and CDR3 of the heavy chain of one anti-PDGF-DD antibody could be expressed in the context of the framework regions of other heavy chain variable genes. Similarly, CDRl , CDR2, and CDR3 of the light chain of an anti-PDGF-DD antibody could be expressed in the context of the framework regions of other light chain variable genes. In addition, the germline sequences of these CDR regions could be expressed in the context of other heavy or light chain variable region genes. The resulting antibodies can be assayed for specificity and affinity and may allow the generation of a novel antibody molecule. [00112] As will be appreciated, antibodies prepared in accordance with the invention described herein can be expressed in cell lines other than hybridoma cell lines. Sequences encoding particular antibodies can be used for transformation of a suitable mammalian host cell. Transformation can be by any known method for introducing polynucleotides into a host cell, including, for example packaging the polynucleotide in a virus (or into a viral vector) and transducing a host cell with the virus (or vector) or by transfection procedures known in the art, as exemplified by U.S. Patent Nos.: 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which patents are hereby incorporated herein by reference). The transformation procedure used depends upon the host to be transformed. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.

[00113] Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and a number of other cell lines. Cell lines of particular preference are selected through determining which cell lines have high expression levels and produce antibodies with constitutive PDGF-DD binding properties.

Additional Criteria for Antibody Therapeutics

[00114] As discussed herein, the function of the PDGF-DD antibody appears important to at least a portion of its mode of operation. By function, is meant, by way of example, the activity of the anti-PDGF-DD antibody in response to PDGF-DD. Accordingly, in certain respects, it may be desirable in connection with the generation of antibodies as therapeutic candidates against PDGF-DD that the antibodies may be made capable of effector function, including complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). There are a number of isotypes of antibodies that are capable of the same, including, without limitation, the following: murine IgM, murine IgG2a, murine IgG2b, murine IgG3, human IgM, human IgGl , and human IgG3. It will be appreciated that antibodies that are generated need not initially possess such an isotype but, rather, the antibody as generated can possess any isotype and the antibody can be isotype switched thereafter using conventional techniques that are well known in the art. Such techniques include the use of direct recombinant techniques (see, e.g., U.S. Patent No. 4,816,397 and U.S. Patent No. 6,331,415), cell-cell fusion techniques (see, e.g., U.S. Patent Nos. 5,916,771 and 6,207,418), among others.

[00115] In the cell-cell fusion technique, a myeloma or other cell line is prepared that possesses a heavy chain with any desired isotype and another myeloma or other cell line is prepared that possesses the light chain. Such cells can, thereafter, he fused and a cell line expressing an intact antibody can be isolated.

[00116] By way of example, the anti-PDGF-DD antibodies discussed herein are human anti-PDGF-DD IgG2 and IgG4 antibodies. If such antibody possessed desired binding to the PDGF-DD molecule, it could be readily isotype switched to generate a human IgM, human IgGl , or human IgG3, IgAl or IgGA2 isotypes, while still possessing the same variable region (which defines the antibody's specificity and some of its affinity). Such molecule would then be capable of fixing complement and participating in CDC. [00117] Accordingly, as antibody candidates are generated that meet desired

"structural" attributes as discussed above, they can generally be provided with at least certain of the desired "functional" attributes through isotype switching.

Epitope Mapping

Immunoblot Analysis

[00118] The binding of the antibodies described herein to PDGF-DD can be examined by a number of methods. For example, PDGF-DD may be subjected to SDS- PAGE and analyzed by irnmunoblotting. The SDS-PAGE may be performed either in the absence or presence of a reduction agent. Such chemical modifications may result in the methylation of cysteine residues. Accordingly, it is possible to determine whether the PDGF-DD antibodies described herein bind to a linear epitope on PDGF-DD.

Surface-enhanced laser desorption/ionization

[00119] Epitope mapping of the epitope for the PDGF-DD antibodies described herein can also be performed using SELDI. SELDI ProteinChip® arrays are used to define sites of protein-protein interaction. Antigens are specifically captured on antibodies covalently immobilized onto the Protein Chip array surface by an initial incubation and wash. The bound antigens can be detected by a laser-induced desorption process and analyzed directly to determine their mass. Such fragments of the antigen that bind are designated as the "epitope" of a protein.

[00120] The SELDI process enables individual components within complex molecular compositions to be detected directly and mapped quantitatively relative to other components in a rapid, highly-sensitive and scalable manner. SELDI utilizes a diverse array of surface chemistries to capture and present large numbers of individual protein molecules for detection by a laser-induced desorption process. The success of the SELDI process is defined in part by the miniaturization and integration of multiple functions, each dependent on different technologies, on a surface ("chip"). SELDl BioChips and other types of SELDI probes are surfaces "enhanced" such that they become active participants in the capture, purification (separation), presentation, detection, and characterization of individual target molecules (e.g., proteins) or population of molecules to be evaluated. [00121] A single SELDI protein BioChip, loaded with only the original sample, can be read thousands of times. The SELDI protein BioChips from LumiCyte hold as many as 10,000 addressable protein docking locations per 1 square centimeter. Each location may reveal the presence of dozens of individual proteins. When the protein composition information from each location is compared and unique information sets combined, the resulting composition map reveals an image with sets of features that are used collectively to define specific patterns or molecular "fingerprints." Different fingerprints may be associated with various stages of health, the onset of disease, or the regression of disease associated with the administration of appropriate therapeutics.

[00122] The SELDI process may be described in further detail in four parts. Initially, one or more proteins of interest are captured or "docked" on the ProteinChip Array, directly from the original source material, without sample preparation and without sample labeling. In a second step, the "signal-to-noise" ratio is enhanced by reducing the chemical and biomolecular "noise." Such "noise" is reduced through selective retention of target on the chip by washing away undesired materials. Further, one or more of the target protein(s) that are captured are read by a rapid, sensitive, laser-induced process (SELDI) that provides direct information about the target (molecular weight). Lastly, the target protein at any one or more locations within the array may be characterized in situ by performing one or more on-the-chip binding or modification reactions to characterize protein structure and function.

Phage Display

[00123] The epitope for the PDGF-DD antibodies described herein can be determined by exposing the ProteinChip Array to a combinatorial library of random peptide 12-mer displayed on Filamentous phage (New England Biolabs).

[00124] Phage display describes a selection technique in which a peptide is expressed as a fusion with a coat protein of a bacteriophage, resulting in display of the fused protein on the surface of the virion. Panning is carried out by incubation of a library of phage displayed peptide with a plate or tube coated with the target, washing away the unbound phage, and eluting the specifically bound phage. The eluted phage is then amplified and taken through additional binding and amplification cycles to enrich the pool in favor of binding sequences. After three or four rounds, individual clones binding are further tested for binding by phage ELISA assays performed on antibody-coated wells and characterized by specific DNA sequencing of positive clones.

[00125] After multiple rounds of such panning against the PDGF-DD antibodies described herein, the bound phage may be eluted and subjected to further studies for the identification and characterization of the bound peptide.

Design and Generation of Other Therapeutics

[00126] Moreover, based on the activity of the antibodies that are produced and characterized herein with respect to PDGF-DD, the design of other therapeutic modalities beyond antibody moieties is facilitated. Such modalities include, without limitation, advanced antibody therapeutics, such as bispecific antibodies, immunotoxins, and radiolabeled therapeutics, generation of peptide therapeutics, gene therapies, particularly intrabodies, antisense therapeutics, and small molecules.

[00127] In connection with the generation of advanced antibody therapeutics, where complement fixation is a desirable attribute, it may be possible to sidestep the dependence on complement for cell killing through the use of bispecifics, immunotoxins, or radiolabels, for example.

[00128] For example, in connection with bispecific antibodies, bispecific antibodies can be generated that comprise (i) two antibodies one with a specificity to PDGF-DD and another to a second molecule that are conjugated together, (ii) a single antibody that has one chain specific to PDGF-DD and a second chain specific to a second molecule, or (iii) a single chain antibody that has specificity to PDGF-DD and the other molecule. Such bispecific antibodies can be generated using techniques that are well known for example, in connection with (i) and (ii) see, e.g., Fanger et al., Immunol Methods 4:72-81 (1994) and Wright and Harris, supra and in connection with (iii) see, e.g., Traunecker et al, Int. J. Cancer (Suppl.) 7:51-52 (1992). In each case, the second specificity can be made to the heavy chain activation receptors, including, without limitation, CDl 6 or CD64 {see, e.g., Deo et al, 18: 127 (1997)) or CD89 (see, e.g., Valerius et al, Blood 90:4485-4492 (1997)). Bispecific antibodies prepared in accordance with the foregoing would be likely to kill cells expressing PDGF-DD, and particularly those cells in which the PDGF-DD antibodies described herein are effective.

[00129] With respect to immunotoxins, antibodies can be modified to act as immunotoxins utilizing techniques that are well known in the art. See, e.g., Vitetta, Immunol Today 14:252 (1993). See also U.S. Patent No. 5,194,594. In connection with the preparation of radiolabeled antibodies, such modified antibodies can also be readily prepared utilizing techniques that are well known in the art. See, e.g., Junghans et al., in Cancer Chemotherapy and Biotherapy 655-686 (2d ed., Chafher and Longo, eds., Lippincott Raven (1996)). See also U.S. Patent Nos.: 4,681,581, 4,735,210, 5,101,827, 5,102,990 (RE 35,500), 5,648,471, and 5,697,902. Each of immunotoxins and radiolabeled molecules would be likely to kill cells expressing PDGF-DD, and particularly those cells in which the antibodies described herein are effective.

[00130] In connection with the generation of therapeutic peptides, through the utilization of structural information related to PDGF-DD and antibodies thereto, such as the antibodies described herein (as discussed below in connection with small molecules) or screening of peptide libraries, therapeutic peptides can be generated that are directed against PDGF-DD. Design and screening of peptide therapeutics is discussed in connection with Houghten et al., Biotechniques 13:412-421 (1992), Houghten, PNAS USA 82:5131-5135 (1985), Pinalla et al., Biotechniques 13:901-905 (1992), Blake and Litzi-Davis, BioConjugate Chem. 3:510-513 (1992). Immunotoxins and radiolabeled molecules can also be prepared, and in a similar manner, in connection with peptidic moieties as discussed above in connection with antibodies.

[00131] Assuming that the PDGF-DD molecule (or a form, such as a splice variant or alternate form) is functionally active in a disease process, it will also be possible to design gene and antisense therapeutics thereto through conventional techniques. Such modalities can be utilized for modulating the function of PDGF-DD. In connection therewith the antibodies, as described herein, facilitate design and use of functional assays related thereto. A design and strategy for antisense therapeutics is discussed in detail in International Patent Application No. WO 94/29444. Design and strategies for gene therapy are well known. However, in particular, the use of gene therapeutic techniques involving intrabodies could prove to be particularly advantageous. See, e.g., Chen et al., Human Gene Therapy 5:595- 601 (1994) and Marasco, Gene Therapy 4:11-15 (1997). General design of and considerations related to gene therapeutics is also discussed in International Patent Application No.: WO 97/38137.

[00132] Small molecule therapeutics can also be envisioned. Drugs can be designed to modulate the activity of PDGF-DD, as described herein. Knowledge gleaned from the structure of the PDGF-DD molecule and its interactions with other molecules, as described herein, such as the antibodies described herein, and others can be utilized to rationally design additional therapeutic modalities. In this regard, rational drug design techniques such as X-ray crystallography, computer-aided (or assisted) molecular modeling (CAMM), quantitative or qualitative structure-activity relationship (QSAR), and similar technologies can be utilized to focus drug discovery efforts. Rational design allows prediction of protein or synthetic structures which can interact with the molecule or specific forms thereof which can be used to modify or modulate the activity of PDGF-DD. Such structures can be synthesized chemically or expressed in biological systems. This approach has been _v reviewed in Capsey et al., Genetically Engineered Human Therapeutic Drugs (Stockton Press, NY (1988)). Further, combinatorial libraries can be designed and synthesized and used in screening programs, such as high throughput screening efforts.

Therapeutic Administration and Formulations

[00133] The anti-PDGF-DD compounds including, but not limited to, antibodies and fragments thereof are suitable for incorporation into pharmaceuticals that treat organisms in need of a compound that modulates PDGF-DD. These pharmacologically active compounds can be processed in accordance with conventional methods of galenic pharmacy to produce medicinal agents for administration to organisms, e.g., animals and mammals including humans. In certain embodiments, the active ingredients can be incorporated into a pharmaceutical product with or without modification. Additional embodiments include the manufacture of pharmaceuticals or therapeutic agents that deliver the pharmacologically active compounds, described herein, by several routes. For example, and not by way of limitation, DNA, RNA, and viral vectors having sequence encoding the antibodies or fragments thereof can be used in certain embodiments. Additionally, nucleic acids encoding antibodies or fragments thereof can be administered alone or in combination with other active ingredients.

[00134] It will be appreciated that administration of therapeutic entities described herein can be administered in admixture with suitable carriers, excipients, stabilizers, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. Pharmaceutically acceptable carriers include organic or inorganic carrier substances suitable for parenteral, enteral (for example, oral) or topical application that do not deleteriously react with the pharmacologically active ingredients of this invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc. Additional carriers, excipients, and stabilizers include buffers such as TRIS HCl, phosphate, citrate, acetate and other organic acid salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) peptides such as polyarginine, proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidinone; amino acids such as glycine, glutamic acid, asparric acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium and/or nonionic surfactants such as TWEEN, PLURONICS or polyethyleneglycol. Many more suitable vehicles are described in Remmington's Pharmaceutical Sciences, 15th Edition, Easton:Mack Publishing Company, pages 1405-1412 and 1461-1487(1975) and The National Formulary XTV, 14th Edition, Washington, American Pharmaceutical Association (1975), herein incorporated by reference.

[00135] The route of antibody administration can be in accord with known methods, including, for example, but are not limited to, topical, transdermal, parenteral, gastrointestinal, transbronchial, and transalveolar. Parenteral routes of administration include, but are not limited to, electrical or direct injection or infusion such as direct injection into a central venous line, intravenous, intracerebral, intramuscular, intraperitoneal, intradermal, intraarterial, intrathecal, or intralesional routes. The antibody is preferably administered continuously by infusion, by bolus injection, or by sustained release systems as noted below. In a preferred embodiment the administration route can be subcutaneous injection. In an alternative embodiment, the antibodies are administered via the renal artery. Gastrointestinal routes of administration include, but are not limited to, ingestion and rectal. Transbronchial and transalveolar routes of administration include, but are not limited to, inhalation, either via the mouth or intranasally. [00136] When used for in vivo administration, the antibody formulation may be sterile. This can be readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution. The antibody ordinarily will be stored in lyophilized form or in solution. In addition, the therapeutic composition can be pyrogen-free and in a parenterally acceptable solution having due regard for pH, isotoniciry, and stability. Therapeutic antibody compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[00137] Sterile compositions for injection can be formulated according to conventional pharmaceutical practice as described in Remington 's Pharmaceutical Sciences (18^th ed., Mack Publishing Company, Easton, PA (1990)). The pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, for example, lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, antioxidants, coloring, flavoring and/or aromatic substances and the like that do not deleteriously react with the active compounds. For example, dissolution or suspension of the active compound in a vehicle such as water or naturally occurring vegetable oil like sesame, peanut, or cottonseed oil or a synthetic fatty vehicle like ethyl oleate or the like may be desired.

[00138] Suitable compositions having the pharmacologically active compounds of this invention that are suitable for parenteral administration include, but are not limited to, pharmaceutically acceptable sterile isotonic solutions. Such solutions include, but are not limited to, saline and phosphate buffered saline for injection into a central venous line, intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous injection. [00139] Compositions having the pharmacologically active compounds of this invention that are suitable for gastrointestinal administration include, but not limited to, pharmaceutically acceptable powders, pills or liquids for ingestion and suppositories for rectal administration.

[00140] Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) as described by Langer e/ α/., J. BiomedMater. Res., 15:167-277 (1981) and Langer, Chetn. Tech., 12:98- 105 (1982) or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al, Biopolymers, 22:547-556 (1983)), non-degradable ethylene-vinyl acetate (Langer et al, supra), degradable lactic acid-glycolic acid copolymers such as the LUPRON Depot™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid (EP 133,988).

[00141] While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated proteins remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for protein stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

[00142] Sustained-release compositions also include liposomally entrapped antibodies of the invention. Liposomes containing such antibodies are prepared by methods known per se: U.S. Pat. No. DE 3,218,121 ; Epstein et al, Proc. Natl. Acad. ScL USA, 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. ScL USA, 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; 142,641; Japanese patent application 83- 118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.

[00143] An effective amount of antibody to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the patient. The dosage of the antibody will be determined by the attending physician taking into consideration various factors known to modify the action of drugs including severity and type of disease, body weight, sex, diet, time and route of administration, other medications and other relevant clinical factors. Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. Typically, the clinician will administer antibody until a dosage is reached that achieves the desired effect. Therapeutically effective dosages may be determined by either in vitro or in vivo methods. The progress of this therapy is easily monitored by conventional assays or by the assays described herein. [00144] Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, ED50 (the dose therapeutically effective in 50% of the population). The data obtained from treating the rat model of nephritis or an alternative model may be used in formulating a range of dosage for use with other organisms, including humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with no toxicity. The dosage varies within this range depending upon type of evectin, hybrid, binding partner, or fragment thereof, the dosage form employed, sensitivity of the organism, and the route of administration.

[00145J Normal dosage concentrations of various antibodies or fragments thereof can vary from approximately 0.1 to 100 mg/kg. Desirable dosage concentrations include, for example, 0.1mg/kg, 0.2mg/kg, 0.3mg/kg, 0.4mg/kg, 0.5mg/kg, 0.6mg/kg, 0.7mg/kg, 0.8mg/kg, 0.9mg/kg, 1.0mg/kg, 1.5mg/kg, 2.0mg/kg, 2.5mg/kg, 3.0mg/kg, 3.5mg/kg, 4.0mg/kg, 4.5mg/kg, 5.0mg/kg, 5.5mg/kg, 6.0mg/kg, 6.5mg/kg, 7.0mg/kg, 7.5mg/kg, 8.0mg/kg, 8.5mg/kg, 9.0mg/kg, 10mg/kg, 15mg/kg, 20mg/kg, 25mg/kg, 30mg/kg, 35mg/kg, 40mg/kg, 45mg/kg, 50mg/kg, 55mg/kg, 60mg/kg, 65mg/kg, 70mg/kg, 75mg/kg, 80mg/kg, 85mg/kg, 90mg/kg, 95mg/kg, and lOOmg/kg or more. One preferred dosage is 1 to 10mg/kg.

[00146] In some embodiments, the dose of antibodies or fragments thereof produces a tissue or blood concentration or both from approximately 0.1 μM to 50OmM, preferably about 1 to 800μM, and more preferably greater than about lOμM to about 500μM. Preferable doses are, for example, the amount required to achieve a tissue or blood concentration or both of lOμM, 15μM, 20μM, 25μM, 30μM, 35μM, 40μM, 45μM, 50μM, 55μM, 60μM, 65μM, 70μM, 75μM, 80μM, 85μM, 90μM, 95μM, lOOμM, HOμM, 12OuM₅ 130μM, 140μM, 145μM, 15OuM₃ 160μM, 170μM, 180μM, 190μM, 200μM, 220μM, 240μM, 250μM, 260μM, 280μM, 300μM, 320μM, 340μM, 360μM, 380μM, 400μM, 420μM, 440μM, 460μM, 48OuM, and 500μM. In alternative embodiments, doses that produce a tissue concentration of greater than 800μM are can be used. A constant infusion of the antibodies, hybrids, binding partners, or fragments thereof can also be provided so as to maintain a stable concentration in the tissues as measured by blood levels. [00147] ^' Dosage and administration can be adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Embodiments herein include both short acting and long acting pharmaceutical compositions. Accordingly, embodiments include schedules where pharmaceutical compositions are administered approximately every 1, 2, 3, 4, 5, or 6 days, every week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, or once every 8 weeks. Depending on half-life and clearance rate of the particular formulation, the pharmaceutical compositions described herein can be administered about once, twice, three, four, five, six, seven, eight, nine, and ten or more times per day.

[00148] Additional therapeutics may be administered in combination with, before, or after administration of the anti-PDGF-DD antibodies. These therapeutics may be used to treat symptoms of the disease or may decrease the side effects of the anti-PDGF-DD antibodies. They may also be used to enhance the activity of the anti-PDGF-DD antibodies. Any type of therapeutic may be used including, but not limited to, for example, antibiotics, diuretics, anesthetics, analgesics, anti-inflammatories, and insulin. Examples of agents that are typically used to treat glomerulonephritis and may be used in combination with the antibodies include prednisone, cyclophosphamide, chlorambucil, and blood thinning agents, such as, for example, warfarin, dipyradamole, and aspirin.

Diagnostic Use

[00149] PDGF-DD has been found to be expressed at low levels in normal kidney but its expression is increased dramatically in postischemic kidney (Ichimura T, Bonventre JV, Bailly V, Wei H, Hession CA, Gate RL, Sanicola M., J. Biol. Chem. 273(7):4135-42 (1998)). As immunohistochemical staining with anti-PDGF-DD antibody shows positive staining of renal, kidney, prostate and ovarian carcinomas (see below), PDGF-DD overexpression relative to normal tissues can serve as a diagnostic marker of such diseases. [00150] Accordingly, embodiments of the invention are also useful for assays, particularly in vitro diagnostic assays, for example, for use in determining the level of PDGF-DD in patient samples. Such assays may be useful for diagnosing diseases associated with over expression of PDGF-DD. In some embodiments, the disease is nephritis. The patient samples can be, for example, bodily fluids, preferably blood, more preferably blood serum, synovial fluid, tissue lysates, and extracts prepared from diseased tissues. Other embodiments of the invention are useful for diagnosing and staging nephritis and diseases related to mesangial proliferation. Monitoring the level of PDGF-DD may be used as a surrogate measure of patient response to treatment and as a method of monitoring the severity of the disease in a patient. Elevated levels of PDGF-DD compared to levels of other soluble markers would indicate the presence of postischemic kidney. The concentration of the PDGF-DD antigen present in patient samples can be determined using a method that specifically determines the amount of the antigen that is present. Such a method includes an ELISA method in which, for example, antibodies of the invention may be conveniently immobilized on an insoluble matrix, such as a polymer matrix. Alternatively, immunohistochemistry staining assays using anti-PDGF-DD antibodies may be used to determine levels of PDGF-DD in a sample. Using a population of samples that provides statistically significant results for each stage of progression or therapy, a range of concentrations of the antigen that may be considered characteristic of each stage of disease can be designated.

[00151] In one embodiment, a sample of blood is taken from the subject and the concentration of the PDGF-DD antigen present in the sample is determined to evaluate the stage of the disease in a subject under study, or to characterize the response of the subject to a course of therapy. The concentration so obtained is used to identify in which range of concentrations the value falls. The range so identified correlates with a stage of disease progression or a stage of therapy identified in the various populations of diagnosed subjects, thereby providing a stage in the subject under study.

[00152] Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA (Thomas, Proc. Natl. Acad. ScL USA, 77:5201-5205 (1980)), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-KNTA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay can be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected. [00153] For example, antibodies, including antibody fragments, can be used to qualitatively or quantitatively detect the expression of PDGF-DD proteins. As noted above, the antibody preferably is equipped with a detectable, e.g., fluorescent label, and binding can be monitored by light microscopy, flow cytometry, fluorimetry, or other techniques known in the art. These techniques are particularly suitable if the amplified gene encodes a cell surface protein, e.g., a growth factor. Such binding assays are performed as known in the art.

[00154] In situ detection of antibody binding to the PDGF-DD protein can be performed, for example, by immunofluorescence or immunoelectron microscopy. For this purpose, a tissue specimen is removed from the patient, and a labeled antibody is applied to it, preferably by overlaying the antibody on a biological sample. This procedure also allows for determining the distribution of the marker gene product in the tissue examined. It will be apparent for those skilled in the art that a wide variety of histological methods are readily available for in situ detection.

[00155] One of the most sensitive and most flexible quantitative methods for quantitating differential gene expression is RT-PCR, which can be used to compare mRNA levels in different sample populations, in normal and tumor tissues, with or without drug treatment, to characterize patterns of gene expression, to discriminate between closely related mRNAs, and to analyze RNA structure.

[00156] The first step is the isolation of mRNA from a target sample. The starting material is typically total RNA isolated from a disease tissue and corresponding normal tissues, respectively. Thus, mRNA can be extracted, for example, from frozen or archived paraffin-embedded and fixed {e.g. formalin-fixed) samples of diseased tissue for comparison with normal tissue of the same type. Methods for mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel etal., Current Protocols of Molecular Biology, John Wiley and Sons (1997). Methods for RNA extraction from paraffin embedded tissues are disclosed, for example, in Rupp and Locker, Lab Invest., 56:A67 (1987), and De Andres et al., BioTechniques, 18:42044 (1995). In particular, RNA isolation can be performed using purification kit, buffer set and protease from commercial manufacturers, such as Qiagen, according to the manufacturer's instructions. For example, total RNA from cells in culture can be isolated using Qiagen RNeasy mini-columns. Total RNA from tissue samples can be isolated using RNA Stat-60 (Tel-Test).

[00157] As RNA cannot serve as a template for PCR, the first step in differential gene expression analysis by RT-PCR is the reverse transcription of the RNA template into cDNA, followed by its exponential amplification in a PCR reaction. The two most commonly used reverse transcriptases are avilo myeloblastosis virus reverse transcriptase (AMV-RT) and Moloney murine leukemia virus reverse transcriptase (MMLV-RT). The reverse transcription step is typically primed using specific primers, random hexarners, or oligo-dT primers, depending on the circumstances and the goal of expression profiling. For example, extracted RNA can be reverse-transcribed using a GeneAmp RNA PCR kit (Perkin Elmer, CA, USA), following the manufacturer's instructions. The derived cDNA can then be used as a template in the subsequent PCR reaction.

[00158] Although the PCR step can use a variety of thermostable DNA-dependent

DNA polymerases, it typically employs the Taq DNA polymerase, which has a 5'-3' nuclease activity but lacks a 3'-5' endonuclease activity. Thus, TaqMan PCR typically utilizes the 5 '-nuclease activity of Taq or Tth polymerase to hydrolyze a hybridization probe bound to its target amplicon, but any enzyme with equivalent 5' nuclease activity can be used. Two oligonucleotide primers are used to generate an amplicon typical of a PCR reaction. A third oligonucleotide, or probe, is designed to detect nucleotide sequence located between the two PCR primers. The probe is non-extendible by Taq DNA polymerase enzyme, and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe. During the amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner. The resultant probe fragments disassociate in solution, and signal from the released reporter dye is free from the quenching effect of the second fluorophore. One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.

{00159] TaqMan RT-PCR can be performed using commercially available equipments, such as, for example, ABI PRIZM 7700TM Sequence Detection SystemTM (Perkin-Elmer-Applied Biosystems, Foster City, CA, USA), or Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany). In a preferred embodiment, the 5' nuclease procedure is run on a real-time quantitative PCR device such as the ABI PRIZM 7700TM Sequence Detection SystemTM. The system consists of a thermocycler, laser, charge-coupled device (CCD), camera and computer. The system amplifies samples in a 96-well format on a thermocycler. During amplification, laser-induced fluorescent signal is collected in real-time through fiber optics cables for all 96 wells, and detected at the CCD. The system includes software for running the instrument and for analyzing the data. [00160] 5'-Nuclease assay data are initially expressed as Ct, or the threshold cycle.

As discussed above, fluorescence values are recorded during every cycle and represent the amount of product amplified to that point in the amplification reaction. The point when the fluorescent signal is first recorded as statistically significant is the threshold cycle (Ct). The ΔCt values are used as quantitative measurement of the relative number of starting copies of a particular target sequence in a nucleic acid sample when comparing the expression of RNA in a cell from a diseased tissue with that from a normal cell.

[00161] To minimize errors and the effect of sample-to-sample variation, RT-PCR is usually performed using an internal standard. The ideal internal standard is expressed at a constant level among different tissues, and is unaffected by the experimental treatment. RNAs most frequently used to normalize patterns of gene expression are mRNAs for the housekeeping genes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and β-actin. [00162] Differential gene expression can also be identified, or confirmed using the microarray technique. In this method, nucleotide sequences of interest are plated, or arrayed, on a microchip substrate. The arrayed sequences are then hybridized with specific DNA probes from cells or tissues of interest.

[00163] In a specific embodiment of the microarray technique, PCR amplified inserts of cDNA clones are applied to a substrate in a dense array. Preferably at least 10,000 nucleotide sequences are applied to the substrate. The microarrayed genes, immobilized on the microchip at 10,000 elements each, are suitable for hybridization under stringent conditions. Fluorescently labeled cDNA probes may be generated through incorporation of fluorescent nucleotides by reverse transcription of RNA extracted from tissues of interest. Labeled cDNA probes applied to the chip selectively hybridize to each spot of DNA on the array. After stringent washing to remove non-specifically bound probes, the chip is scanned by confocal laser microscopy. Quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance. With dual color fluorescence, separately labeled cDNA probes generated from two sources of RNA are hybridized pairwise to the array. The relative abundance of the transcripts from the two sources corresponding to each specified gene is thus determined simultaneously. The miniaturized scale of the hybridization affords a convenient and rapid evaluation of the expression pattern for large numbers of genes. Such methods have been shown to have the sensitivity required to detect rare transcripts, which are expressed at a few copies per cell, and to reproducibly detect at least approximately two-fold differences in the expression levels (Schena et al, Proc. Natl. Acad. ScL USA, 93(2O)L 106-49). The methodology of hybridization of nucleic acids and microarray technology is well known in the art.

[00164] Selected embodiments of the antibodies and methods are illustrated in the

Examples below:

EXAMPLES

[00165] The following examples, including the experiments conducted and results achieved are provided for illustrative purposes only and are not to be construed as limiting upon the embodiments of the invention described herein.

Example 1 Materials and Methods

[00166] Fully Human PDGF-D Monoclonal Antibody CR002: Generation and specificity of the fully human PDGF-DD mAb CR002 was described previously in Ostendorf T, et al., J Am Soc Nephrol, vol.14:2237-2247 (2003), the contents of which are hereby incorporated by reference in their entirety.

[00167] Cell Culture Experiments: To examine the autogenic effect of PDGF-D on rat renal interstitial fibroblasts, NRK-49F cells (German National Resource Center for Biological Material (DSMZ), Braunschweig, Germany) were seeded in 96-well plates (Nalge Nunc, Naperville, IL), grown to subconfluency and growth-arrested for 72 hrs in serum free medium. Cells were then stimulated for 24 hours with recombinant human PDGF-DD (100 ng/ml; produced as described previously LaRochelle WJ, et al., Nat Cell Biol, vol. 3:517-521 (2001), the contents of which are hereby ) or recombinant human PDGF-BB (10 ng/ml; Sigma-Aldrich, Deisenhofen, Germany) with or without the addition of neutralizing PDGF-D antibody CR002 (1 μg/ml). DNA synthesis was determined by a 5- bromo-2'-deoxyuridine (BrdU) incorporation assay according to the manufacturer's instructions (Roche, Mannheim, Germany). The experiments were performed in quadruplicates.

[00168] Experimental Design: All animal experiments were approved by the local review boards. Animals were held in rooms with constant temperature and humidity, 12h/12h light cycles, and had ad libitum access to drinking water (ozone-treated and acidified). Daily fluid was measured, and the different treatment groups were pair-fed (normal chow diet).

[00169] Progressive mesangioproliferative glomerulonephritis was induced in 32 male Wistar rats weighing 200 g (Charles River, Sulzfeld, Germany) by unilateral nephrectomy followed by injection of 1 mg/kg monoclonal anti-Thy 1.1 antibody (clone OX.-1; European Collection of Animal Cell Cultures, Salisbury, England) as described previously (Ostendorf T, et al., J Am Soc Nephrol, vol. 17(4):1054-62 (2006)). On day 3 rats were randomized according to proteinuria into 2 groups: the first group received control IgG₂ (5mg/kg body weight; n=17) and the second was treated with fully human anti-PDGF- D monoclonal antibody CR002 (5mg/kg body weight; n=15). The antibodies were dissolved in 20 mM Tris-HCl/100 mM NaCl, pH 7.4 and administered by intraperitoneal injections on days 17, 28 and 35 after disease induction. Longer treatment was not attempted given concerns about the potential immunogenicity of the neutralizing human antibody in rats. Non-nephritic control groups with a uni-nephrectomy were not included in the present study, as it was shown that CR002 treatment over 3 weeks has no significant effects under these conditions (Ostendorf T, et al., J Am Soc Nephrol, vol. 17(4): 1054-62 (2006)). No rat treated with CR002 died during the experiment. In animals that received control IgG two rats had to be sacrificed due to rapid progression of the disease on day 24 and 95 after disease induction (the second was included into the study).

[00170] Venous blood samples (drawn from a tail vein or the inferior vena cava at sacrifice) and 24 hour urine collections were performed on days 3, 35, 42, 49, 56, 77, 90 and 100. Blood pressure was measured by tail cuff phlethysmography on days 2, 35, 56, 73 and 99. Following sacrifice, serum samples as well as renal tissues for histological evaluation were collected. The remaining cortical tissue of each rat was used to isolate RNA. [00171] Renal Morphology: Tissue for light microscopy and immunoperoxidase staining was fixed in methyl Carnoy's solution as well as in formalin and embedded in paraffin. Four μm sections were stained with the PAS reagent and counterstained with hematoxylin. In PAS-stained sections of day 100 biopsies, the percentage of glomeruli exhibiting focal or global glomerulosclerosis was determined as described in Floege J, et al., Kidney Int, vol. 51:230-243 (1997), the contents of which are hereby incorporated by reference in their entirety. Tubulointerstitial injury on day 100 was graded on a scale of 0 to 4 as described previously in Ostendorf T, et al., JAm Soc Nephrol, vol. 12:909-918 (2001) , the contents of which are hereby incorporated by reference in their entirety. [00172] For the evaluation of total collagen content, renal tissues were stained with

Sirius-red and evaluated by computer based morphometry (Soft Imaging System GmbH, Mϋnster, Germany). The percentage of positively stained area in each tissue was calculated separately for glomeruli and in 20 interstitial fields with each field having an area of 0.37 mm². In all analyses the investigator was unaware of the origin of the slides. [00173] Immunoperoxidase Staining: Four μm sections of methyl Carnoy's fixed biopsy tissue were processed as previously described (Floege J, et al., Kidney Int, vol. 51:230-243 (1997)). Primary antibodies included a murine monoclonal antibody (clone 1 A4) to α-smooth muscle actin; a murine monoclonal IgG antibody (clone EDl) to monocytes, macrophages and dendritic cells; affinity purified polyclonal goat antibodies against human type I and type III collagen (Southern Biotechnology, Birmingham, Alabama, USA); a murine monoclonal antibody against porcine vimentin (clone V9, DAKO, Glostrup, Denmark); a murine monoclonal IgG against human E-cadherin (Dako Cytomation, Glostrup, Denmark) plus appropriate negative controls as described previously (Burg M, etal., Lab Invest, vol. 76:505-516 (1997)).

[00174] The stains for cortical type I and type III collagen, α-smooth-muscle actin, infiltrating monocytes/macrophages (EDl), vimentin, E-cadherin were evaluated by computer based morphometry as described for Sirius red (see above). [00175] Cortical RNA Extraction and Analyses: Total RNA was extracted from renal cortex using the RNeasy Mini Kit (Qiagen, Hilden, Germany). cDNA syntheses and realtime-quantitative PCRs were performed as previously described (Ostendorf T, et al., J Am Soc Nephrol, vol. 17(4): 1054-62 (2006)). Sequences of primers and probes used in this study are listed in Table 2. mRNA for each sample was normalized to GAPDH.

Table 2

Primers and probes

Forward Primer ' Reverse Primer Taqman Probe

TAPror 5'-ACAAGATGGTGAAGGTCGGTG-S' 5'-AGAAGGCAGCCCTGGTAACC-S' 5'-CGGATTTGGCCGTATCGGACGC-S'

^(JΆ^Ϊ-UΆ (SEQ ID NO: 83) (SEQ ID NO: 84) (SEQ ID NO: 85)

Type I 5'-GAAGGCAACAGTCGATTCACC-S' 5'-GACTGTCTTGCCCCAAGTTCC-S' 5'-ACAGCACGCTTGTGGATGGCTGC-S'

Collagen (SEQ ID NO: 86) (SEQ ID NO: 87) (SEQ ID NO: 88)

Type m 5'-GGGATGCAACTACCTTGGTCA-S' 5'-TCCCGAGTCGCAGACACATA-S' 5'-TGACATGGTTCTGGCTTCCAGACATCTCTAG-S'

Collagen (SEQ ID NO: 89) (SEQ ID NO: 90) (SEQ ID NO: 91)

5'-GCAAGACGCGTACAGAGGTG-S' 5'-GAAGTTGGCATTGGTGCGA-S' 5'-TCCAGATCTCGCGGAACCTCATCG-S'

(SEQ ID NO: 92) (SEQ ID NO: 93) (SEQ ED NO: 94)

« _{pnrp π} 5'-ATCGGGACACTTTTGCGACT-S' 5'-GTGCCTGTCACCCGAATGTT-S' 5'-TTGCGCAATGCCAACCTCAGGAG-S'

(SEQ ID NO: 95) (SEQ ID NO: 96) (SEQ ID NO: 97)

PDGFR- 5'-AATGACCACGGCGATGAGA-S' 5'-TCTTCCAGTGTTTCCAGCAGC-S' 5'-CATCAACGTTACTGTGATCGAAAATGGCTATG-S' β (SEQ ID NO: 98) (SEQ ID NO: 99) (SEQ ID NO: 100)

GAPDH - glyceraldehyde-3-phosphate dehydrogenase; PDGF - platelet-derived growth factor; PDGFR - receptor for PDGF

[00176] Miscellaneous Measurements: Urinary albumin levels were determined by an enzyme-linked immunosorbent assay (Nephrat, Exocell, Philadelphia, PA). Serum creatinine and urine protein and creatinine levels were determined by an autoanalyzer. All measurements were performed in duplicate. Blood pressure measurements were performed by the tail cuff method, using a programmed sphygmomanometer (Softron Co., Tokyo, Japan).

[00177] Circulating levels of anti-PDGF-DD (CR002) mAb were measured by

ELISA as described previously (Ostendorf T, et al., J Am Soc Nephrol, vol. 17(4): 1054-62 (2006)). To assess for potential immunogenicity of CR002, i.e. formation of rat anti-human IgG, an ELISA assay was used as previously described (Ostendorf T, et al., J Am Soc Nephrol, vol. 17(4): 1054-62 (2006)).

[00178] Statistical Analysis: All values are expressed as means ± standard error of the mean. To compare the mean values between the anti-PDGF-D and IgG group unpaired /-tests was employed. For multiple groups comparison in the in vitro experiments one-way ANOVA with least square difference post-hoc test was used. For comparison of categorical variables Pearson Chi-Quadrat test was used (one-tailed). Statistical significance was defined as p < 0.05.

Example 2 In Vitro Effects of PDGF-D on Renal Fibroblasts

[00179] PDGF-D is a mitogen for renal fibroblasts in vitro. Figure 1 shows that

PDGF-D induced proliferation of rat renal fibroblasts to a similar degree as PDGF-B. Addition of CR002 specifically inhibited the effect of PDGF-D, but not that of PDGF-B.

Example 3 Anti-PDGF-DD Antibodies in Nephritis Model

[00180] Serum levels were obtained after injection of CR002 into nephritic rats.

Seven days after the last i.p. injection (i.e. on day 42) the mean concentration of circulating CR002 was 4.00 ± 0.45 μg/ml and no rat had to be excluded due to insufficient serum concentrations of CR002. Compared to day 42, serum CR002 concentrations decreased two and eight fold on days 49 and 56, respectively (Figure 2) and 100 fold on day 100 (0.035 ± 0.003 μg/ml).

[00181] No production of rat anti-human IgG that could have neutralized the effect of

CR002 was detected in sera obtained on days 49 and 100 (data not shown).

Example 4 PDGF-D Antagonism in Treatment of Proteinuria and Progressive Renal Failure

[00182] PDGF-D antagonism transiently reduces proteinuria and retards progressive

Tenal failure. CR002 treatment led to significant reductions in proteinuria as compared to the group receiving irrelevant IgG, on days 49, 56 and 77 (Figure 2). At later time points differences were no longer significant and considerable variability in the extent of proteinuria was noted. Albuminuria on day 100 also did not differ between the two groups (84±5 mg/d in IgG treated rats versus 77±6 in CR002 treated rats; not significant). [00183] Serum creatinine concentrations and creatinine clearances did not differ significantly throughout the study, although the means were constantly lower (creatinine) or higher (creatinine clearance) in the CR002 treated group versus the IgG group (Table 3). To account for the large variability in renal failure progression between different rats, the number of rats that doubled their serum-creatinine in the course of the experiment (i.e. > 92 μmol/1 at day 100) was calculated. This analysis showed that 6 of 15 rats receiving CR002 versus 12 of 17 rats receiving IgG doubled their serum creatinine (p < 0.05). [00184] Since both groups received pair-feeding, body weight was similar in both groups (Table 3). Measured daily water intake was also not different between the two groups at various time points throughout the study (data not shown). [00185] Systolic and diastolic blood pressure were constantly elevated between days

36 and 100 but did not differ significantly throughout the study (day 100: systolic pressure 161±5 mmHg vs. 153±7 mmHg in IgG and CR002 treated rats, respectively; diastolic pressure 124±5 mmHg vs. 122±7 mmHg in IgG and CR002 treated rats, respectively). Table 3

Body weight and parameters of kidney function in nephritic rats treated with irrelevant IgG (IgG, n=17) and CR002 (anti-PDGFD Ab, n=15).

Day after Disease lnductior ϊ

Parameter Group 3 35 42 49 56 77 90 100

Body Weight IgG 201+4 347±9 366±9 377±9 389±10 429±10 445±12 426±12

(g) CR002 205+6 352±8 365±9 382+9 395±9 437+10 456+12 436+12

Serum Creatinine IgG 46±2 52±3 54±3 57±3 60±4 74±6 85±10 127±21

'Jl

OO (μmol/l) CR002 45±1 55+4 51±3 53+4 62±4 69±6 71+7 100±10

Creatinine Clearance IgG 0.38±0.02 0.42±0.03 0.39±0.03 0.30±0.03 0.32±0.3 0.28±0.03 0.25±0.03 0.20±0.02 (ml/min/100g) CR002 0.37±0.02 0.41+0.04 0.41+0.02 0.34±0.03 0.29±0.02 0.30±0.03 0.30±0.03 0.24+0.02

All differences between CR002 and IgG treated group not significant.

Example 5 PDGF-D Antagonism in Treatment of Tubulointerstitial Damage

[00186] PDGF-D antagonism reduces overall tubulointerstitial damage on day 100.

Widespread glomerular and tubulointerstitial damage with considerable glomerulosclerosis and tubulointerstitial fibrosis developed in both groups. However, as compared to rats receiving irrelevant IgG, the CR002 treated group exhibited significantly lower tubulointerstitial damage scores (Figure 3). The development of focal segmental glomerulosclerosis was also reduced in CR002 treated rats, but the difference was not significant (66±4 % vs. 57±4 % of glomeruli in IgG and CR002 treated animals, respectively).

Example 6

Effect of PDGF-D Antagonism on Glomerular Mesagnial Cell Activation and Matrix

Accumulation

[00187] PDGF-D antagonism reduces glomerular mesangial cell activation and matrix accumulation on day 100. The glomerular de novo expression of α-smooth muscle actin (α-SMAj and interstitial types of collagen (i.e. types I and III) as well as over- expression of vimentin is characteristic of a myofibroblast-like phenotype acquisition of mesangial cells. As shown in Figure 5, Sinus red staining as well as imrnunostaining for types I and III collagen and vimentin were significantly reduced in CR002 treated rats as compared to rats receiving irrelevant IgG by 33, 49, 35%, and 20%, respectively. Glomerular α- smooth muscle actin expression was reduced by PDGF-D antagonism by 39% (Figure 3). Glomerular monocyte/macrophage infiltration was not affected by CR002 treatment on day 100 (data not shown).

Example 7

Effect of PDGF-D Antagonism on Tubulointerstitial Matrix Accumulation and on Cortical PDGF-D. PDGF-B and PDGFR-β Expression

[00188] PDGF-D antagonism reduces tubulointerstitial matrix accumulation on day

100. Tubulointerstitial fibrosis was assessed by a global marker (i.e. Sirius red) and by the expression of specific matrix molecules. Figures 4 and 5 show that CR002 treatment as compared to irrelevant IgG significantly reduced the tubulointerstitial Sirius red positive area (-20%) as well as the renal cortical areas covered by type I collagen (-28%), type III collagen (-32%), and vimentin (-41%). Renal cortical mRNA levels of type I and III collagen were not different between the two groups (Table 4). Expression of α-smooth muscle actin decreased by 33% but failed to reach statistical significance. Again, monocyte/macrophage infiltration on day 100 was not affected by CR002 treatment (data not shown). There was no difference in E-cadherin between the groups (O.85±O.11 % vs. 0.70±0.09 % of stained area in IgG and CR002 treated animals, respectively). [00189] PDGF-D antagonism downregulates cortical PDGF-D but not -B or PDGFR- β mRNA on day 100. Treatment with CR002 showed no effect on cortical mRNA levels of PDGF-B and PDGFR-β, whereas PDGF-D levels were significantly downregulated on day 100 (Table 4).

Table 4: Relative renal cortical mRNA expression of types I and HI collagen, FDGF-B, -D, and PDGFR-β in nephritic rats treated with irrelevant IgG (IgG, n=17) or CR002 (anti- PDGF-D Ab, n=l 5).

Relative mRNA IgG CR002 expression

Type I Collagen 1.18 ± 0.18 1.27 ± 0.20

Type πi Collagen 1.15 ± 0.17 1.01 ± 0.15

PDGF-B 7.7/ ± 0.12 0.91 ± 0.07

PDGF-D 1.09 ± 0.11 0.77 ± 0.08 *

PDGFR-β 1.10 ± 0.14 1.01 ± 0.10

PDGF - platelet-derived growth factor; PDGFR - receptor for PDGF *p<0.05 CR002 vs. IgG group Example 8 Analyzing the risk for developing, the diagnosis of. and staging of a renal disease with

ELISA

[00190] Serum levels of PDGF-DD from patients afflicted with, or suspected of being afflicted with, a renal disease are analyzed. The concentration of PDGF-DD is assessed using a quantitative sandwich ELISA with 2 fully human mAbs raised against PDGF-DD. An elevated PDGF-DD level in the patient, as compared to normal patients, is indicative of the presence of a renal disease. For example, it is found that PDGF-DD levels are elevated four to seven fold in the sera of nephritis patients compared to normal patients. These differences in the level of PDGF-DD can accordingly help form diagnostics and help practitioners track staging of renal diseases such as nephritis and related diseases.

Example 9 Safety. Pharmacokinetic and Pharmacodynamics of Anti-PDGF-DD Antibodies in Healthy

Human Subjects

[00191] Studies were performed to examine the safety, pharmacokinetics, and pharmacodynamics of CR002 in a Phase I clinical trial. Volunteers were dosed with 0.3, 1, 3, 10, 30 mg/kg of CR002 or placebo. Six volunteers were in each cohort receiving CR002. Two volunteers received placebo at each of the dose levels. Treatment was administered as a single 300 ml IV infusion over 2 hours.

[00192] Safety: The decision to dose escalate was made based on having no more than one grade 3 or any grade 4 drug-related toxicity for one month after dosing. Subjects were evaluated for a total of 3 months following each dose. CR002 was well tolerated at all dose levels. There were no dose dependent adverse events (AEs), and no dose limiting toxicities were observed at any dose. There were no drug related serious adverse events (SAEs).

[00193] Pharmacokinetic Properties: CR002 was measured in human serum using an ELISA method in which samples were added to PDGF-DD coated microtiter plates. Bound CR002 was detected with murine anti-human-IgG2-HRP conjugate, followed by colorimetric detection with substrate, TMB. The assay was validated within a calibration range of 37.5-1600 ng/mL. Serum concentrations from a 2 hour infusion of CR002 (mean of 6 patients +/- SEM) were calculated. The sampling extended out to 90 days from date of infusion due to the long terminal phase half-life of CR002 in human serum. Mean pharmacokinetic parameters Cmax, AUC and half-life demonstrated linear exposure across all dose levels (n=6).

[00194] Determination ofCR002 Half-Life: PK parameters for determining the half- life of CR002 were calculated from plasma concentration data. CR002 exposure was found to be linear from 0.3 to 30 mg/kg. The terminal phase half-life was not dependent on dosing, and ranged from 483 hours in the 0.3 mg/kg group to 820 hours in the 10 mg/kg group. Stated another way, CR002 has a long half-life, with a T ¹A ranging from 17.4 to 25.6 days. [00195] Pharmacodynamic Analysis of PDGF-DD: Studies were designed to determine the level of "free" platelet derived growth factor-D (PDGF-DD; PDGF-DD not bound to CR002) and total PDGF-DD (free and CR002-bound PDGF-DD) in normal subjects before and after a single bolus treatment of CR002.

[00196] Subjects were divided into cohorts. In each cohort six subjects were treated with a single dose of CR002 (0.3, 1.0, 3, 10, 30 mg/kg) and two subjects were treated with placebo (PBS). Serum was sampled before drug injection at day 0 and then at days 2, 7, 21, 30, 45 and 90 after treatment. Each serum sample was then evaluated for PDGF-DD levels using an Enzyme Linked Immunosorbent Assay (ELISA) employing CR002 as a capture antibody and a second ELISA employing the anti-PDGF-DD monoclonal antibody (mAb), 1.38. The CR002 -based ELISA detects only free PDGF-DD; whereas the 1.38 mAb-based ELISA can detect both free PDGF-DD as well as PDGF-DD bound to CR002. [00197] PDGF-DD levels in human serum indicated that measurable PDGF-DD levels are present in healthy volunteers, but free PDGF-DD levels decline below detectable levels in the presence of CR002. This decrease was sustained in approximate duration of serum plasma levels of CR002. Since serum free PDGF-DD was below detection level after treatment with CR002, this provides proof of principle that CR002 binds PDGF-DD in human serum.

[00198] The various methods and techniques described above provide a number of ways to carry out numerous embodiments. Of course, it is to be understood that not necessarily all objectives or advantages described may be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods may be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as may be taught or suggested herein. [00199] Furthermore, the skilled artisan will recognize the interchangeability of various features from different embodiments. Similarly, the various features and steps discussed above, as well as other known equivalents for each such feature or step, can be mixed and matched by one of ordinary skill in this art to perform methods in accordance with principles described herein.

[00200] Although the methods described herein have been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that these methods extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and obvious modifications and equivalents thereof. Accordingly, the methods described herein are not intended to be limited by the specific disclosures of preferred embodiments herein, but instead by reference to claims attached hereto.

Claims

WHAT IS CLAIMED IS:

1. A method of treating, preventing or delaying the progression of renal fibrosis in a subject comprising administering to said subject a therapeutically effective dose of an antibody, or binding fragment thereof, that binds to platelet derived growth factor-DD (PDGF-DD).

2. The method of claim 1 , wherein said subject is a human.

3. The method of claim 1, wherein said antibody is a fully human monoclonal antibody.

4. The method of claim 1, wherein said antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

5. The method of claim 1 , wherein said administration is via subcutaneous injection.

6. The method of claim 1, wherein said administration is via intramuscular injection.

7. The method of claim 1 , wherein said administration is via intravenous infusion.

8. The method of claim 1 , wherein said antibody is administered after the phase of acute glomerular damage in said subject.

9. A method of treating, preventing or delaying the progression of renal failure in a subject comprising administering to said subject a therapeutically effective dose of an antibody, or binding fragment thereof, that binds to platelet derived growth factor-DD (PDGF-DD).

10. The method of claim 9, wherein said subject is a human.

11. The method of claim 9, wherein said antibody is a fully human monoclonal antibody.

12. The method of claim 9, wherein said antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

13. The method of claim 9, wherein said administration is via subcutaneous injection.

14. The method of claim 9, wherein said administration is via intramuscular injection.

15. The method of claim 9, wherein said administration is via intravenous infusion.

16. The method of claim 9, wherein said antibody is administered after the phase of acute glomerular damage in said subject.

17. A method of reducing proteinuria in a subject comprising administering to said subject a therapeutically effective dose of an antibody, or binding fragment thereof, that binds to platelet derived growth factor-DD (PDGF-DD).

18. The method of claim 17, wherein said subject is a human.

19. The method of claim 17, wherein said antibody is a fully human monoclonal antibody.

20. The method of claim 17, wherein said antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

21. The method of claim 17, wherein said administration is via subcutaneous injection.

22. The method of claim 17, wherein said administration is via intramuscular injection.

23. The method of claim 17, wherein said administration is via intravenous infusion.