WO2007118084A2 - Compositions and methods for treating polycystic kidney disease - Google Patents

Abstract

Disclosed herein are methods and compositions for the treatment of polycystic kidney disease or other diseases that are associated with alteration, either directly or indirectly, by tumor necrosis factor-alpha (TNF-α) of receptor-calcium channel complexes or calcium regulation such that cyst formation is stimulated.

Description

COMPOSITIONS AND METHODS FOR TREATING POLYCYSTIC

KIDNEY DISEASE

FIELD OF INVENTION

[0001] The present invention generally relates to methods and compositions for the treatment of autosomal dominant polycystic kidney disease (ADPKD) or other cyst-forming diseases. Specifically, the compositions and methods of the invention may be used to inhibit the formation of cysts associated with altered calcium regulation. BACKGROUND OF INVENTION

[0002] Autosomal dominant polycystic kidney disease (ADPKD) is a common hereditary disorder characterized by cystic dilation of the kidney tubules. As the disease advances, cysts enlarge and eventually become isolated from their nephrons. The resultant decrease in kidney function often necessitates hemodialysis or organ transplantation. The majority of ADPKD cases (85-90%) are caused by mutations within the PKDl gene, which encodes a 460 kDa G protein-coupled receptor known as polycystin-1 (PCl) ^1>2. Mutations in a second gene, PKD2, abrogate the function of the Ca²⁺-permeable cation channel protein, polycystin-2 (PC2) and account for 10-15% of all ADPKD cases ^8>9>19>20. PCI and PC2 form a complex through interaction of their cytoplasmic domain and reside, among other locations, the primary cilia at the apical surface of kidney epithelia. The primary cilium is an organelle that extends from the basal body and comprises organized arrays of microtubules and their associated proteins. Recent studies have implicated the primary cilia in a variety of physiological functions and human diseases, such as PKD. It is hypothesized that defects in cilia and polycycstin- mediated calcium signaling in response to fluid flow result in altered epithelial cell polarity, cell- cell contact and cell cycle control, leading to disruption of epithelial integrity and cystogenesis. [0003] A particularly important question regarding the development of ADPKD is how recessive loss of function mutations in PKDl and PKD2 could cause cyst growth in both human and mice heterozygous for these mutations. Analysis of somatic mutations in isolated human cysts led to the hypothesis that cystogenesis in ADPKD results from clonal expansion of epithelial cells harboring "second hit" mutations affecting the functional copy of the polycystin gene, including mostly somatic loss of heterozygosity and in some cases point mutations that may alter gene expression or function. However, for this hypothesis to be the sole explanation for the autosomal dominant nature of the disease, one must assume that the PKDl locus is exceptionally mutable, given the estimated 1 in 1000 frequency of ADPKD in the human population and 1 in 5 frequency of cyst development in PKD2-/+ mice. Other studies indicated that polycystin is expressed in most cyst lining cells and argued that somatic mutations may not be required for at least the adult form of ADPKD. A main caveat of the "two hit" hypothesis is the difficulty to directly demonstrate the causal relationship between somatic mutations and the disease phenotype, and it has been pointed out that the high frequency of mutations observed in cyst lining cells may be due to loss of proper growth control, similar to the high-level genetic instablility observed in cancer cells (Harris PC and Watson ML). Alternative to evoking genetic changes, it is possible that some epigenetic factors, probably those embedded in the cellular networks involving polycystins, is the culprit in the phenotypic expression of ADPKD. In short, the cause(s) of ADPKD are not currently fully understood, and as a consequence, devising treatment for the disease has not been fully successful. No cures are currently known. SUMMARY OF INVENTION

[0004] Provided herein is a method of inhibiting cyst formation, which may be by inhibiting TNF-α signaling. The TNF-α signaling may be selected from the group consisting of: TNF-α, Rab8, TNF-α receptor, fibrocystin, FIP2, and TNF-α receptor-associated proteins. Inhibiting TNF-α signaling may maintain polycystin function in a cell, which may be a pancreatic cell, kidney cell, or liver cell. TNF-α signaling may be inhibited in vitro or in vivo. Maintaining polycystin function may inhibit cystogenesis and may also localize PC2 to the plasma membrane.

[0005] Also provided herein is a method of inhibiting the disruption of calcium influx into a cell, which may be by inhibiting TNF-α signaling and maintaining polycystin function in a cell. FIP2 interaction with PC2 may also be inhibited. Inhibiting FIP2 interaction with PC2 may localize PC2 to the plasma membrane of a cell.

[0006] Further provided herein is a method of treating a haploinsufficiency- associated disease, which may be by inhibiting TNF-α signaling and maintaining polycystin function in a cell. The haploinsufficiency-associated disease may comprise autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, neurofibromatosis, neuroblastoma, or MODY diabetes. TNF-α signaling may be inhibited by an antibody to TNF-α, Rab8, TNF-α receptor, fibrocystin, FIP2, or a TNF-α receptor- associated protein. TNF-α may also be inhibited by etanercept, infliximab, D2E7, CDP 571, CDP 870, onercept, OR1384, tenidap, rapamycin, or leflunomide.

[0007] Also provided herein is a composition, which may be an antibody or an antigen-binding fragment thereof. The antibody may be a monoclonal, IgG, or IgM antibody. The antibody may also be a dimmer, trimer, or multimer. The antibody may be human, humanized, or part-human. The antibody may also be chimeric or recombinant. The antibody may also be operatively attached to at least a first therapeutic or diagnostic agent. The antibody may also be operatively attached to at least a first and a second therapeutic agent. The antigen- binding fragment may be a scFv, Fv, Fab', Fab, diabody or F(ab')₂ fragment. The antibody or antigen-binding fragment thereof may bind substantially to TNF-α, Rab8, TNF-α receptor, fibrocystin, FIP2, or a TNF-α receptor- associated protein. The binding may maintain polycystin function and inhibit cystogenesis.

[0008] Further provided herein is a pharmaceutical composition, which may comprise a biologically effective amount of at least a first immunoconjugate. The immunoconjugate may comprise an antibody or antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof may bind substantially to TNF-α, Rab8, TNF-α receptor, fibrocystin, FIP2 or a TNF-α receptor-associated protein. The binding may inhibit cystogenesis and maintain polycystin function in a cell.

[0009] Also provided herein is a compound, which may comprise an antibody or an antigen-binding portion thereof. The antibody or antigen-binding portion thereof may bind substantially to TNF-α, Rab8, TNF-α receptor, fibrocystin, FIP2 or a TNF-α receptor- associated protein. The antibody or antigen-binding portion thereof may be attached to a therapeutic agent. The therapeutic agent may inhibit TNF-α signaling and may also maintain polycystin function in a cell.

BRIEF DESCRIPTION OF DRAWINGS

[00010] This application contains at least one photograph executed in color.

Copies of this patent application publication with color photographs will be provided by the Office upon request and payment of the necessary fee. The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[00011] FIG. IA-E. PC2 interaction with FIP2 in yeast and mammalian cells is shown. A, Deletion construct Gal4-AD-FIP2, shown schematically, was used to determine the interaction domains in FIP2 and PC2 with yeast two-hybrid assays. The strengths of interactions are indicated as either positive interaction (+) or lacking interaction (-); and grey boxes represent coiled-coil regions. B, Deletion construct Gal4-BD-PC2 was used to determine the interaction domains in FIP2 and PC2 with yeast two-hybrid assays. The strengths of interactions are indicated as either positive interaction (+) or lacking interaction (-); and grey boxes represent coiled-coil regions. C, PC2 co-immunoprecipitated with FIP2 from lysates of 293T cells co- transfected with Myc-tagged PC2 and TJ- or Flag-tagged FIP2 with PC2 antibody (96525), or respective anti-tag antibodies. The PC2-FIP2 complex can be co-immunoprecipitated reciprocally. D, Western blot analyses, on the left-hand side, revealed an increase in the protein

levels of FIP2 but not PC2 upon TNF-α stimulation. E, Endogenous PC2 co-

immunoprecipitated with FIP2 with anti-PC2 or FIP2 antibody. TNF-α induction resulted in an

increase in the amount of PC2 that was pulled down by FIP2.

[00012] FIG. 2A-M. TNF-α signaling disrupts PC2 localization in IMCD cells.

A-D, Double immunofluorescence staining of endogenous PC2 (green) and acetylated tubulin

(ac-tub, red) before (A, A') or after (B, B') treatment with TNF-α, observed with confocal

microscopy. TNF-α stimulation results in the loss of PC2 localization to the plasma membrane

and primary cilia and enrichment of PC2 within the perinuclear regions (B, B'), overlapping with a Golgi marker (C). A' AND B' show enlarged cilia images in the boxed regions in A and B,

respectively. The loss of proper localization of PC2 was not due to cell death, as the TNF-α-

treated cells showed normal morphology and were negative for TUNEL staining (D). E-K, Transfection of siRNA against FIP2 into IMCD cells inhibited FIP2 expression and prevented

mislocalization of PC2, caused by TNF-α. E, Transfected IMCD cells with 4 different siRNA

against FIP2 showed diminished FIP2 expression (siRNA#08 had the strongest effect). F,G, Western blot to determine the FIP2 levels in IMCD cells which were transfected with siRNA#05

(F) and siRNA#08 (G) and then treated with TNF-α for 16 hours showed diminished FIP2

expression. H-K, Immunofluorescence staining of IMCD cells transfected with siRNA#05 (H,I)

or siRNA#08 (J,K) against FIP2 before (H, J) or after (I, K) treatment with TNF-α showed that

siRNA prevented the mislocalization of PC2 (green) (within the perinuclear regions). L,M, Immunofluorescence staining of PC2 (green) and transfected Flag-tagged FIP2 (red), showed a loss of PC2 localization to the plasma membrane in cells transfected with FIP2. L, Flag-FIP2- transfected cell, demonstrating colocalization of PC2 and FIP2 in the perinuclear region, and M, an untransfected cell with normal PC2 localization.

[00013] FIG. 3. TNF-α modulates PC2 complex formation with Rab8 and PC 1.

A, Co-immunoprecipitation of PC2 and Rab8 in cells stimulated with TNF-α for various lengths

of time, showing that the interaction was induced by TNF-α. B,C, Reciprocal co-

immunoprecipitation of PCl and PC2. After TNF-α induction, PCl failed to be co-

immunoprecipitated by a PC2 antibody (B), and PC2 by a PCl antibody (C), even though the same amounts of PCl and PC2 were immunoprecipitated with their respective antibodies in all time points. D,E, Merged double immunofluorescence staining of endogenous PCl (green) and

acetylated tubulin (ac-tub, red) before (D) or after (E) treatment with TNF-α, observed with confocal microscopy. The images shown are 2D projections of all the confocal sections. D' and E' show enlarged cilia images in the boxed regions in d and e, respectively.

[00014] FIG. 4. TNF-α inhibits fluid flow-induced cytosolic Ca²⁺ mobilization in

IMCD cells. A, Control IMCD cells without the addition of TNF-α display their normal Ca²⁺-

response to fluid flow stimulation. B, Treatment with TNF-α for 16 h blocks the response of

IMCD cells to flow stimulation. C, TNF-α does not inhibit the ATP-induced increase in

cytosolic calcium in IMCD cells. When challenged with lμM ATP, both control (no treatment)

and TNF-α-treated cells (treated for either 4 or 16 hours) show an increase in cytosolic calcium.

Fifty cells were randomly selected within a cell population, and changes in their cytosolic calcium levels were averaged and plotted in the graphs.

[0010] FIG. 5. Treatment of cultured kidneys from E15.5 embryos with TNF-α

for 5 days, in concentrations ranging from 3.125 ng/mL to 25 ng/mL, resulted in the formation of numerous cysts in wild- type kidneys and enhanced cyst formation in Pkdl+/- and Pkd2+/- kidneys. A, Treatment of wild type cultured kidneys with 3.125 ng/mL, 6.25 ng/mL, 12.5

ng/mL, or 25 ng/mL of TNF-α triggered cyst formation. Each vertical pair of kidneys represents

left and right kidneys from the same embryo. B, C, Treatment with 25 ng/mL or 12.5 ng/mL

TNF-α triggered cyst formation in wild-type littermates and their respective (b) Pkdl+/-

littermates or (c) Pkd2+/- littermates. D, Enlarged images of the kidneys from the kidney organ

cultures with or without TNF-α addition. Cyst-lining cells resulting from TNF-α treatment

exhibited the expected flat morphology, as opposed to the cuboidal shape characteristic of normal renal tubule lining cells.

[0011] FIG. 6 Quantification of TNF-α levels in ADPKD human cysts by

ELISA indicated that the TNF-α concentration in small cysts reached the ng/mL range and that there was an inverse relationship between cyst size and TNF-α concentration. A, The data

represent 20 cysts from 10 ADPKD patients. The horizontal axis indicates cyst volumes; the left

vertical axis indicates TNF-α concentration; and the right vertical axis indicates total TNF-α

amount in each cyst. Levels of FIP2 increased in ADPKD cells. B, The amount of FIP2 protein

as well as TNF-α receptor (TNFR) was compared between cultured normal human kidney cells

(NHK) and ADPKD human cyst lining cells (PKD) and there was a 2.5-fold increase of FIP2 in the PKD cells as well as a 4-fold increase of TNFR. Consistent with this, levels of TNFR exhibited an increase of 35% in TNF-α-treated IMCD cells and an increase of 3.5-fold in Pkdl-/-

mutant mouse cells, as compared to wild-type cells. C, Immunoblot analysis of TNF-α receptor

(TNFR) protein levels, comparing wild-type cells with or without TNF-α treatment and wild- type and Pkdl-/- cells.

[0012] FIG. 7. Double negative feedback loops constitute an epigenetic switch controlling cystogenesis in ADPKD. A, Schematic diagram depicting the network harboring two

double negative feedback loops connecting TNF-α signaling and cystogenesis. +: positive

effects; -: negative effects. In red: stochastic factors that could trigger the feedback loops. B-D, Simulation of ADPKD disease progression using functional Petri nets. Cyst formation was allowed to occur only when polycystin level falls below 0.4 (1 being the maximum). Below this threshold, cyst formation is a stochastic process with a probability of occurring that increases linearly as PclPc2 level decreases; hence, cyst formation is continuous if PclPc2 level falls to zero. Cysts decay was set to occur at a constant rate. Renal injury was set to occur with a probability of approximately 1% every 4 years. In the wild type (B), expression of polycystin proteins is high enough to ensure that functional polycystin level remains well above the threshold. In individuals harboring heterozygous polycystin gene mutations (C), a stochastic dip in functional polycystin levels, due to transcriptional fluctuation, or a spike in TNF-α level due

renal injury, triggered the positive feedback loop, causing permanent disease onset. If feedback was inhibited in the heterozygous case (D), cysts could appear transiently due to dips in polycystin level, but uncontrolled cyst formation never occurred.

DETAILED DESCRIPTION

[0013] In an attempt to identify the components of the signaling pathway involving polycystins, we performed a yeast two-hybrid screen of a human fetal kidney cDNA library, using bait containing the entire C-terminal intracellular domain of human PC2. This

screen identified FIP2, a coiled coil-containing, TNF-α-induced protein, as a potential PC2

binding partner. To test the specificity of this interaction and to map the PC2 interacting domain within FIP2, we made a series of deletion constructs of FIP2 (FIG. IA) and tested their interaction with PC2 in the yeast two-hybrid system. As shown in FIG. IA, of the three coiled- coil domains within FIP2, only the constructs containing the 2nd coiled-coil domain interacted with the PC2 C-terminal domain as efficiently as the full-length FIP2. Deletion constructs containing either the N-terminal domain or the C-terminal domain of FIP2 did not interact with PC2, further indicating that the PC2-binding domain is located within the second coiled-coil region of FIP2 (amino acid 143-260). Co-transformation of yeast with the full-length FIP2 and a series of PC2 C-terminal deletion constructs showed that the FIP2-binding domain is located around the coiled-coil domain of PC2 between amino acid 741-802 (FIG. IB). To gain biochemical evidence that PC2 associates with FIP2 in mammalian cells, Iy sates prepared from IMCD cells co-transfected with full-length myc-tagged PC2 and flag-tagged or T7-tagged FIP2 were immunoprecipitated with anti-flag, anti-T7, anti-Myc or an anti-PC2 antibody (FIG. 1C). We found that PC2 co-immunoprecipitated with FIP2 but not when the control anti-IgG antibody was used for immunoprecipitation.

[0014] TNF-α is a pro-inflammatory cytokine with a number of important

biologic functions ^26~29 . TNF-α mRNA and proteins are markedly increased after hypertensive

stress ³⁴ and renal injury ^35~37 . It has been reported that in the cystic kidneys of cpk/cpk mice

TNF-α level was increased progressively with age . TNF-α was also found to be present in the

cyst fluid in the ADPKD patients ³⁸ . These facts, together with the finding that PC2 binds a

TNF-α-induced protein, suggest that TNF-α may influence the expression or function of PC2.

Since TNF-α can induce the expression of a number of genes, such as FIP2 ⁴⁰ , we first tested

whether it also alters the expression of PC2. Western blot analyses of FIP2 and PC2 expression

after TNF-α induction revealed that TNF-α increased the expression of FIP2 by 3-4 fold, but not

that of PC2 at any of the time points investigated (FIG. ID). Immunoprecipitation using anti- FIP2 or anti-PC2 antibody could co-precipitate endogenous PC2 or FIP2, respectively, in IMCD

cells induced with TNF-α (FIG. IE). The amount of PC2 in the FIP2 immunoprecipitate

increased as FIP2 level rose during the TNF-α treatment (FIG. IE).

[0015] Since TNF-α did not affect the level of PC2 expression, we tested whether

localization of PC2 was altered in TNF-α-treated cells. Immunofluorescence staining using anti-

PC2 antibody in the untreated IMCD cells showed that the endogenous PC2 localized to the plasma membrane, especially enriched in the primary cilia in IMCD cells (FIG. 2A,A'), as

demonstrated in previous work ²¹. However, in cells treated with TNF-α for 16 hours, we

observed a striking difference in PC2 localization: PC2 no longer localized to the plasma membrane but was strongly enriched in perinuclear regions, overlapping with a Golgi marker (FIG. 2C), and the cilia were completely devoid of PC2 (FIG. 2B,B'). The loss of proper localization of PC2 was not due to cell death, as the TNF- α- treated cells showed normal

morphology and were negative for TUNEL staining (FIG. 2D). To test if this observed effect was due to elevated FIP2 expression, we over-expressed Flag-tagged FIP2 in IMCD cells by transient transfection and we transfected IMCD cells with siRNA against FIP2.

[0016] IMCD cells were transiently transfected with full length FIP2. PC2 localization to the plasma membrane was diminished in the FIP2 transfected cells but not in the surrounding non-transfected cells, and PC2 colocalizes with FIP2 in perinuclear structures (FIG. 2L, M). FIP2 siRNA reduced FIP2 expression by 95% (FIG. 2E-G) and restored normal PC2

localization in TNF-α-treated cells (FIG. 2H-K). These results suggest that induction of FIP2

downstream of TNF-α signaling disrupts PC2 localization to its normal site of function.

[0017] The lack of PC2 cilia localization and accumulation of PC2 in the

perinuclear region in TNF-α-treated cells raised the possibility that PC2 was prevented from

effectively trafficking through the exocytic pathway or was mis- sorted. In support of this possibility, FIP2, also known as Optineurin, was previously found to be a binding partner for the GTP-bound form of Rab8, a GTPase localized in the recycling endosomes and important for basal-lateral protein targeting . FIP2 interacts with Rab8 through the N-terminal -200 amino acid and was shown to link the Huntingtin protein and myosin VI to Rab8 . To test if FIP2 plays a role in promoting complex formation between PC2 and Rab8, an antibody specific to Rab8 was

used in immunoprecipitation experiments. Without TNF-α stimulation, PC2 did not co-

immunoprecipiate with Rab8, even though Rab8 protein level was unaffected by TNF-α

treatment. In cells treated with TNF-α for 4 or 16 hours, PC2 was clearly present in Rab8

immunoprecipitate (FIG. 3A). This result suggests that TNF-α treatment induces the formation

of a PC2-FIP2-Rab8 complex, which hijacks PC2 from the apical membrane and accumulates it in the perinuclear structures, likely to be the recycling endosomes where Rab8 was previously shown to reside . The Huntingtin protein, another binding partner for FIP2, was also shown to be sequestered, upon over-expression of FIP2, to perinuclear particulate structures that contained Rab8 in HT 1080 cells .

[0018] The function of PC2 requires formation of a mechanosensitive receptor- channel complex with PCl, which transduces mechanical signals such as fluid flow sheer stress into a chemical signal important for epithelial tissue homeostasis ¹⁵. The C-terminal cytoplasmic domain of PC2 that binds to PCl overlaps with the domain that binds FIP2. In cells not treated

with TNF- α, PCl and PC2 can be co-immunoprecipiated with the antibody against either protein

(FIG. 3B, C). After TNF-α induction, however, this interaction was no longer detectable, even

though the expression levels of PCl and PC2 remained the same as in the untreated cells. Furthermore, immunofluorescence staining of PCl with the antibody 96521 revealed that PCl

remained on the primary cilia after TNF-α treatment, although we noticed subtle but

reproducible changes: whereas in the untreated cells PCl localizes along the entire length of the

cilia, in the TNF-α treated cells, PCl was often absent at either end of the cilia (FIG.

3D,D',E,E'). The subtle alteration in PCl localization could result from the absence of its normal binding partner, PC2, on cilia membrane.

[0019] It was recently shown that the co-distribution of PCl and PC2 in the primary cilium mediates Ca²⁺ influx in response to physiological fluid flow over the apical

surface . Because TNF-α stimulation disrupts the PC2-PC1 complex and PC2 localization to the

cilia, TNF-α should also abrogate fluid flow-induced Ca²⁺ influx. To test this prediction, we

performed flow assay in IMCD cells after 16 hours TNF-α stimulation. We found that TNF-α

stimulation completely abolished the flow-induced Ca²⁺ response, whereas a normal flow- induced Ca²⁺ response was seen in the control IMCD cells (FIG. 4A-B). This effect was

specific, as TNF-α did not affect the Ca²⁺ rise in response to ATP (FIG. 4C).

[0020] The above results suggest a pathway through which TNF-α could down-

regulate polycystin function and thereby stimulate cyst growth. This pathway could be an important contributing factor in the onset of ADPKD. To test this hypothesis, a mouse embryonic kidney organ culture system was used to determine if TNF-α can induce cyst formation in kidneys from wild-type, PKDl+/- and PKD2+/- mice. Previously, it was found that cAMP, an activator of in vitro cyst formation, induced cyst formation in cultured mouse E 13.5 kidneys. The culture conditions were modified using E15.5-E17.5 kidneys such that no cyst formation was observed in the absence of TNF-α. However, treatment for five days with concentrations as low as 6.25 ng/ml TNF-α resulted in the formation of numerous cysts in wild- type kidneys (FIG. 5A), and enhanced cyst formation in Pkdl+/- and Pkd2+/- kidneys (FIG. 5B,C). Cyst-lining cells resulting from TNF-α treatment exhibited the expected flat morphology, as opposed to the cuboidal shape characteristic of normal renal tubule cells (FIG. 5D). These results provide direct evidence that TNF-α promotes kidney cyst formation.

[0021] Furthermore, the presence of TNF-α in cyst fluids from ADPKD patients has been reported in a previous study, which measured 10-73 pg/ml TNF-α in cyst fluids from ADPKD patients. The effective concentrations that we have observed with kidney organ cultures were in the ng/ml range. We measured the concentration of TNF-α in cyst fluids obtained from 10 ADPKD patients by using ELISA. The TNF-α concentration in small cysts reached the ng/ml range, with the highest concentration reaching 3.8 ng/ml. There was an inverse relationship between cyst size and TNF-α concentration (FIG. 6A), suggesting that TNF- α might be diluted in the lumens of large cysts. Importantly, the total amount of TNF-α appeared to increase with larger cyst size (FIG. 6A), suggesting that TNF-α might be continuously accumulated as cysts grow. To determine if FIP2 levels are increased in ADPKD cells, the amount of FIP2 protein was compared between cultured normal human kidney cells (NHK) and ADPKD human cyst lining cells (PKD). As shown in FIG. 6B, top panel, there was a 2.5-fold increase of FIP2 in the PKD cells. The levels of TNF-α receptor (TNFR) also exhibited an increase (about 4-fold) in the cyst lining cells (PKD) (FIG. 6B, middle panel). Consistent with

this, in comparing wild-type IMCD cells, with or without TNF-α treatment, or wild-type and

pkdl-/- mutant kidney epithelial cell lines, TNFR levels were increased by 35% in TNF-α-treated IMCD cells and were increased 3.5-fold in Pkdl-/- mutant mouse cells, as compared to wild-type

cells (FIG. 6C). This result indicates that exposure to TNF-α or loss of polycystin function

could stimulate expression of TNFR, and this change could render the epithelia even more

sensitive to TNF-α.

[0022] A network scheme emerging from above results consists of two nested

double negative feedback loops connecting TNF-α signaling, polycystin function and

cystogenesis (FIG. 7A). Double negative feedback loops are observed in many regulatory networks that give rise to bistable system behavior in such processes as cell cycle transition and cell fate determination . Positive feedback loops and double negative loops have the potential to turn transient and minute signals to stable end states. We hypothesize that such regulatory system could also function as an "epigenetic switch" controlling the stable transition from the healthy state to the disease state in response to spontaneous perturbation of the normal function.

In the case of ADPKD, it is conceivable that an acute increase in TNF-α level could result from

hypertensive stress ³⁴ and renal injury ^35~37 , and could activate the feedback loops and precipitate full-scale cystogenesis. There are indeed clinical observations linking hypertension and renal injury to the progression of PKD . Stochastic triggers of the epigenetic switch could also result from fluctuations in gene expression. There is increasing evidence that the initiation and termination of gene transcription are probabilistic, giving rise to high noise in gene expression . The degree of expression fluctuation is increased with reduced gene copy number . In cells with only a single copy of functional PKDl or 2 gene, where the steady-state level of gene expression is on average only 50% of the normal, the probability of the gene product level stochastically dipping below certain functional threshold would be much higher than in normal cells. Consequently, the high probability of triggering the epigenetic switch due to sub-threshold level of polycystin function would result in sudden transition to and perpetuation of a stable disease state. A similar mechanism has been proposed for at least another autosomal dominant disease - maturity-onset diabetes of the young.

[0023] We used a network model based on functional Petri nets [Doi, 2004;

Nagasaki, 2004] to simulate the induction of cystogenesis through the TNF-α-mediated

epigenetic switch. Our modeling approach has adopted fuzzy logic methods from control systems engineering to modeling reaction kinetics [Kecman, 2001; Ross, 2004; Tsoukalas, 1997]. Reactions are connected as in a Petri net and occur at every time step. This model computes time courses for protein concentrations and gene expression levels from the network specification. A feature of this model is that reactions in the network may have a probability assigned, which allows stochastic processes to be represented (more details of the model can be found in Bosl, 2006. PCl and PC2 are modeled as a functional unit, the level of which is affected by gene dosage of PKDl and PKD2, as well as proper targeting of PC2 to the cilia, which is inhibited by FIP2. There are two sources of random fluctuations in the model: stochastic gene expression and renal injury, which occurs infrequently but is sufficient to generate a spike in TNF-α production. In the wild-type population, where two copies of the gene

are present to direct each polycystin transcription, the fluctuation in functional PC1-PC2 level is less in magnitude than in the heterozygous situation and is always above the functional threshold required for preventing cyst initiation (FIG. 7B). However, in the heterozygous population, where the level of expression is in general sufficient to suppress cyst formation, random fluctuations may cause the feedback loops to be switched on and cyst growth continues unabated (FIG. 7C). Significantly, if the feedback loops are disabled, full-scale cystogenesis is prevented even with stochastic sub-threshold dips in polycystin level (FIG. 7D). It is important to state that the involvement of an epigenetic switch for the onset of ADPKD does not rule out disease induction due to loss of heterozygosity or other genetic modifications. In fact, genetic changes could be particularly important for the stabilization of the disease state and further enhance the rate of cyst growth and renal degeneration. Unraveling the epigenetic control pathways that potentially function as a "second hit" in ADPKD may provide hopeful targets, such as

components of the TNF-α signaling pathway, for therapeutic intervention of disease initiation

and progression.

[0024] In particular, inhibitors and antagonists of TNF-α may be used to prevent

or treat ADPKD. Inhibitors and antagonists of TNF-α reduce or inhibit the action of TNF-α.

These agents include fusion proteins, such as etanercept (Enbrel®, Amgen and Wyeth); monoclonal antibodies, such as infliximab (Remicade®, Centocor), D2E7 (Humira®, Abbott), and CDP 571 (Celltech); binding proteins, such as onercept (Serono); and antibody fragments, such as CDP 870 (Celltech and Pfizer). Other TNF-alpha antagonists and inhibitors include OR1384, tenidap (5-chloro-2,3-dihydro-3-(hydroxy-2-thienylmethylene)-2-oxo-lH- indole-1- carboxamide), rapamycin (sirolimus or Rapamune®), and leflunomide (Arava®). [0025] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

EXAMPLES

[0026] Example 1. Cell culture

[0027] IMCD (ATCC catalog no. CRL-2123) cells were cultured in Dulbecco' s modified Eagle's medium/F12 medium supplemented with 10% (v/v) fetal bovine serum (Invitrogen). The final concentration for TNF-α induction is 200 ng/ml for all the experiments. . Mouse embryonic kidney (MEK) cells isolated from E15.5 wild-type and Pkdl mutant mice (Pkdl-/-) were cultured at 33°C as described previously (1). Cells were cultured to 30%-50% confluence and transiently transfected with Fugene 6 transfection reagent (Roche, Indianapolis, IN) following the manufacturer's protocol. Cells were harvested for further analysis 48 hours after transfection.

[0028] Example 2. Embryonic Kidney Organ Culture [0029] Embryonic kidneys were dissected in PBS (with calcium and magnesium) plus penicillin-streptomycin-glutamine (GiBCO, Grand Island, NY) from embryos of wild type CDl mice at E15.5, E16.5 or E17.5 and of C57BL/6 Pkdl+Λ or C57BL/6 Pkd2+Λ mice or their wild type counterparts at E15.5. The dissected kidneys were cultured at 37°C in DMEM/F12 containing 2 mM L-glutamine, 10 mM Hepes, 5 μg/ml insulin, 5μg/ml transferrin, 2.8 mM selenium, 25 ng/ml prostaglandin El, 32 pg/ml T3 and 250 U/ml penicillin- streptomycin. The kidneys were cultured with or without TNF-α at different concentrations for 48 hours and then 50 μM 8-bromo-cyclic AMP (Sigma, St. Louis, MO) was added for 5 days. The cultured kidneys were then fixed with 4% paraformaldehyde (PFA) in PBS for 6 hours. Washed with PBS twice for 5 minutes each and transferred to 70% EtOH for short-term storage at room temperature or for more extended storage at 4°C. The fixed kidney samples were subsequently processed for Hematoxylin and Eosin staining following common histology protocol.

[0030] Example 3. TUNEL Assay

[0031] Cells treated with or without TNF-α were fixed with 4% PFA. TUNEL assay was performed using a fluorescent apoptosis-detection system (R&D systems, Minneapolis, MN). Fixed cells were treated with TACS-nuclease for the positive control.

[0032] Example 4. Immunoprecipitation and Western Blot Analysis

[0033] Immunoprecipitation and Western blotting were performed on whole-cell lysates as previously described (REFS). The antibodies used for Western blotting included rabbit polyclonal anti-FIP2 (2), rabbit anti-PC2 polyclonal antibody 96525 (1), rabbit anti-PCl polyclonal antibody 96521, rabbit anti-Rab8 polyclonal antibody and rabbit anti-TNFR-I polyclonal antibody. Secondary antibodies used include: goat-anti-rabbit IgG-fluorescein isothiocyanate (FITC Molecular Probes, Eugene, OR), goat-anti-mouse IgG-Texas Red, (1:500 dilution; Molecular Probes). For western blotting, goat-anti-rabbit Ig-horseradish peroxidase (HRP or goat- anti-mouse IgG-HRP, 1:10,000 dilution; Amersham Pharmacia Biotech) were used as secondary antibodies.

[0034] Example 5. RNAi to Inhibit FIP2 Expression

[0035] The oligo sequences used for FIP2 RNAi (Dharmacon, Chicago, IL) are listed below: #05 Sense sequence: 5'-GCUAUGAAAGGGCGAUUUGUU (SEQ ID NO: 1); #05 Antisense sequence: 5'-PCAAAUCGCCCUUUCAUAGCUU (SEQ ID NO:2); #06 Sense sequence: 5'-UGAGCUGCCUGACUGAGAAUU (SEQ ID NO:3); #06 Antisense sequence: 5'- PUUCUCAGUCAGGCAGCUCAUU (SEQ ID NO:4); #07 Sense sequence: 5'- GAAAUGCAGUGCCGACACGUU (SEQ ID NO:5); #07 Antisense sequence: 5'- PCGUGUCGGCACUGCAUUUCUU (SEQ ID NO:6); #08 Sense sequence: 5'- CCAUGAAGCUAAAUAAUCAUU (SEQ ID NO:7); #08 Antisense sequence: 5'- PUGAUUAUUUAGCUUCAUGGUU (SEQ ID NO:8).

[0036] The transfection was performed using the DharmaFECT siRNA transfection reagent (Dharmacon, Chicago, IL).

[0037] Example 6. Immunofluorescence Microscopy

[0038] Immunofluorescence was carried out as previously described (1). Primary antibodies were used at the following dilutions: FIP2 (1:100), PCl (1:500), PC2 (1:500), and flag (1:500). Secondary antibodies used included goat-anti-rabbit IgG-fluorescein isothiocyanate (Molecular Probes, Eugene, OR) and goat-anti-mouse IgG-Texas Red (1:500 dilution; Molecular Probes). Images were captured on an inverted microscope (Axiovert 200M, Carl Zeiss, inc.) equipped with a spinning disc confocal head (Yogogawa), Argon-Krypton laser system (Prairie Technologies, Inc.), and ORCA-ER CCD camera (Hamamatsu). Images were acquired using the Metamorph software (Molecular Devices) and 3D image reconstruction was performed using the Volocity (Improvision, Inc.) software.

[0039] Example 7. Primary Cultures of Human Kidney Cells

[0040] Primary cultures of ADPKD and normal human kidney (NHK) cells were generated with the assistance of the PKD Biomaterials Research Core laboratory at the University of Kansas Medical Center (KUMC). Normal regions of human kidneys, confirmed by histological examination, were collected from nephrectomy specimens removed for the treatment of renal carcinomas. ADPKD kidneys were obtained from hospitals participating in the Polycystic Kidney Research Retrieval Program with the assistance of the PKD Foundation (Kansas City, MO). The kidneys were packaged within ice and shipped to the laboratory overnight. The protocol for the use of discarded human tissues complies with federal regulations and was approved by the Institutional Review Board at KUMC.

[0041] Primary cell cultures were prepared as described (3). Cells are propagated in DMEM/F12 supplemented with 5% FBS, 5 μg/ml insulin, 5 μg/ml transferrin and 5 ng/ml sodium selenite (ITS) and 100 IU/ml penicillin G and 0.1 mg/ml streptomycin. Primary cultures of ADPKD and NHK cells appear epithelial (3-5) and stain with Arachis hypogaea and Dolichos biflorus lectins that bind the collecting ducts and distal tubules (6).

[0042] Example 8. Measurement of TNF- α Concentrations in Human

ADPKD Cyst Fluid

[0043] Cyst fluids were collected from 10 ADPKD kidneys maintained at 4° C.

The fluid was cleared by centrifugation and aliquots were frozen at -20° C. The concentrations and total amounts of TNF-α in individual cyst fluids were measured using the DuoSet ELISA Development kit for human TNF-α/TNFSFIA (R&D Systems, Minneapolis, MN). [0044] Example 9. Ca²⁺ Microfluorimetry

[0045] The experimental setup for flow was as previously described (7). Briefly, cells were incubated for 30 min with the Ca²⁺ sensitive probe Fura2-AM (5 uM) at 37°C. Cells were then washed three times to remove excess Fura2-AM and placed in a perfusion chamber with a thickness of 0.0254 cm and width of 1.0 cm (GlycoTech). The chamber was positioned under a Nikon Diaphot microscope equipped with a CCD camera using IPLab software for Macintosh. Paired Fura images were captured every 5 s at excitation wavelengths of 340 nm and 380 nm. After equilibration in the microscopy media for at least 10 min, the primary cilia of these cells were stimulated at a fluid shear stress of 0.75 dyne cm^"2. The Ca²⁺ level relative to the baseline value was calculated radiometric ally using R_min and R₁^_x values of 0.3 and 6.0, respectively. In some experiments, cells were incubated with TNF-α (200 ng/ml) for 4 or 16 hours before flow activation. In other experiments, cells were incubated with antibodies against PC2 either extracellular or intracellular domain at a dilution of 1:50 for at least 45 min. Cells were then washed at least three times with phosphate-buffered saline.

[0046] Example 10. Data analysis and statistics

[0047] Cytosolic free Ca²⁺ concentrations were calculated using the formula = Ka

((R - Rm_1n) / (Rmax - R)) (Fmax at 380 nm / F_min at 380 nm) where Ka denotes apparent dissociation constant of Fura-2 indicator (145 nM), R is the ratio of 510 nm emission intensity when excited at 340 nm to 510 emission intensity when excited at 380 nm, R_min and R_max are ratios at zero and saturating (10 mM) Ca²⁺ concentrations, respectively, and F_max and F_1111n are the fluorescence intensity excited at 380 nm at zero and saturating free Ca²⁺ concentrations, respectively. At the end of the Fura-2 experiments, cells were incubated with calcium-free solution containing 140 mM potassium, 2 mM EGTA and 10 M ionomycin (at pH 8.6 to optimize the ionomycin effect) for about 5 min. This allowed the minimum signal ratio and values of R_1111n and F_n^_x at 380 nm to be obtained. Excess Ca²⁺ was supplied to the cell by adding 10 mM CaCl₂ to determine the maximum signal ratio. After the 340-nm and 380-nm signals were stable (roughly 3 min), the values of R_max and F_min at 380 nm were obtained. All of the fluorescence measurements were then corrected for autofluorescence. All values for statistical significance represent means (s.e.), and each data set was verified to be normally distributed before analysis. Comparisons were carried out between means using paired Student's t-test. For all comparisons, power analyses were carried out to enable reliable conclusions, and coefficient variances were below 10%. All comparisons with negative results had statistical powers of 0.8, and the statistical significance implies P < 0.05.

[0048] Example 11. Modeling

[0049] The signaling network for TNF-α represented in figures 7b-d was modeled using an approach that adopts intelligent hybrid systems methods from control systems engineering for modeling reaction kinetics (8). Reactions are embedded in a network structure. The resulting model is similar to functional Petri nets (9, 10). Computer codes are available upon request. This model computes the relationship between protein concentrations and gene expression levels over a time course. Reactions are executed in a time stepping loop, with time steps of one week. On each time step, the outcome is determined by computing the current rate based on substrate, activator and inhibitor levels. Protein concentrations and gene expression levels are all normalized to a scale ranging from 0 to 1. The balance between production and decay is more important than the precise value of these rates since this balance determines the equilibrium level of each protein. Furthermore, the strength of activation or inhibition of a reaction is defined in qualitative terms (very low, low, medium, etc). Reaction rates were first assigned by relative values. Then, they were adjusted such that the pathway behavior matched observations. This is essentially manual parameter-fitting to the data.

[0050] Two production reactions are defined for PCl and PC2, representing two genes that produce the respective proteins. In the mutant case, one of the PCl genes is silenced, so the production rate is reduced by 50%. The decay reaction for each of these proteins is constant, at a rate that is 25% of the maximum production rate. To introduce stochastic changes in gene expression, each gene expression "reaction" is set to occur on only 70% of the time steps, chosen randomly. The relative production rates for each PCl and PC2 reaction are 0.4 and the decay rates are 0.1. If only production and decay reactions occurred for PCl and PC2, these would have mean equilibrium levels on a 0 to 1 scale of approximately 0.74 in the case of one gene and 0.83 with 2 genes. This follows from basic kinetics and with production rates determined by the rate times the mean expression probability of 0.7.

[0051] The polycystin complex is produced by interaction between PCl and PC2 proteins, and decays at a constant rate, set again at 1/4 of the maximum production rate. The polycystin complex level fluctuates as a result of random PCl and PC2 expression. Note that in the wild type the stochastic variation is much smaller than in the mutant. This is due to offsetting variation in the two separate reactions that represent gene expression and protein production. Polycystin complex formation is inhibited by FIP2. Thus, as FIP2 levels increase, the reaction for polycystin complex production is inhibited by FIP2 and the decay reaction is activated by FIP2.

[0052] Cyst growth occurs when polycystin levels drop below an arbitrarily set level of 0.4. In the wild type, polycystin fluctuations are always above this level, so cyst growth never occurs. Below 0.4, cyst growth occurs stochastically, with the chance of cyst growth rising linearly as polycystin levels fall. Cyst growth causes TNF-α production, then TNFR and FIP2 production increase, resulting in suppression of polycystin complex formation. Renal injury is also defined as a randomly occurring reaction whose only product is TNF-α. This is modeled as a sudden event that is distinguished from TNF-α increases caused by cyst growth by the sharp rise in concentration that is not preceded by an increase in cyst growth. Rather, cyst growth follows. Renal injury is defined as having a relatively small chance of occurrence - 0.02% in any given week. In the simulations, renal injury events occur in approximately every other lifespan of 80 years and are not always sufficiently large to engage the positive feedback loop and cause PKD to set in. When injury occurs during a time when polycystin levels are particularly low, such as when cysts have been sporadically forming, the threshold is usually reached and disease occurs.

REFERENCES

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Claims

CLAIMSWhat is claimed is:

1. A method of inhibiting cyst formation comprising inhibiting TNF-α signalling such that

polycystin function is maintained in a cell.

2. The method of claim 1, wherein the TNF-α signalling is selected from the group

consisting of TNF-α, Rab8, TNF-α receptor, fibrocystin, FIP2 and TNF-α receptor-

associated proteins.

3. The method of claim 2, wherein the TNF-α signalling comprises fibrocystin.

4. The method of claim 2, wherein the TNF-α signalling comprises FIP2.

5. The method of claim 2, wherein the TNF-α signalling comprises TNF-α.

6. The method of claim 1, wherein the cell comprises a pancreatic cell, a kidney cell or a liver cell.

7. The method of claim 6, wherein the cell comprises a pancreatic cell.

8. The method of claim 6, wherein the cell comprises a kidney cell.

9. The method of claim 6, wherein the cell comprises a liver cell.

10. The method of claim 1, wherein the TNF-α signalling is inhibited in vivo.

11. The method of claim 1, wherein the TNF-α signalling is inhibited in vitro.

12. The method of claim 1, wherein polycystin function is maintained such that cystogenesis is inhibited.

13. The method of claim 1, wherein polycystin function is maintained such that PC2 remains localized to the plasma membrane of the cell.

14. A method of inhibiting the disruption of calcium influx into a cell comprising inhibiting

TNF-α signalling such that polycystin function is maintained in a cell.

15. A method of inhibiting the disruption of calcium influx into a cell comprising inhibiting FIP2 interaction with PC2 such that PC2 remains localized to the plasma membrane of a cell.

16. A method of treating a haploinsufficiency-associated disease comprising inhibiting TNF-

α signalling such that polycystin function is maintained in a cell.

17. The method of claim 16, wherein the haploinsufficiency-associated disease comprises autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, neurofibromatosis, neuroblastoma or MODY diabetes.

18. The method of claim 16, wherein TNF-α signalling is inhibited by an antibody to TNF-α,

Rab8, TNF-α receptor, fibrocystin, FIP2 or a TNF-α receptor- associated protein.

19. The method of claim 16, wherein TNF-α signalling is inhibited by etanercept, infliximab,

D2E7, CDP 571, CDP 870, onercept, OR1384, tenidap, rapamycin, or leflunomide.

20. A composition comprising an antibody, or antigen-binding fragment thereof, that binds

substantially to TNF-α, Rab8, TNF-α receptor, fibrocystin, FIP2 or a TNF-α receptor-

associated protein such that polycystin function is maintained in a cell and cystogenesis is inhibited.

21. The composition of claim 19, wherein the antibody is a monoclonal antibody or an antigen-binding fragment thereof.

22. The composition of claim 19, wherein the antibody is an IgG antibody or an IgM antibody.

23. The composition of claim 19, wherein the antigen-binding fragment is an scFv, Fv, Fab', Fab, diabody, linear antibody or F(ab')₂ antigen-binding fragment of an antibody.

24. The composition of claim 19, wherein the antibody is a dimer, trimer or multimer of the antibody or antigen-binding fragments thereof.

25. The composition of claim 19, wherein the antibody is a human, humanized or part-human antibody or antigen-binding fragment thereof.

26. The composition of claim 19, wherein the antibody is a chimeric antibody.

27. The composition of claim 19, wherein the antibody is a recombinant antibody.

28. The composition of claim 19, wherein the antibody is operatively attached to at least a first therapeutic or diagnostic agent.

29. The composition of claim 27, wherein the antibody is operatively attached to at least a first and a second therapeutic agent.

30. A pharmaceutical composition comprising a biologically effective amount of at least a first immunoconjugate comprising an antibody, or antigen-binding fragment thereof, that

binds substantially to TNF-α, Rab8, TNF-α receptor, fibrocystin, FIP2 or a TNF-α

receptor- associated protein such that cystogenesis is inhibited and polycystin function is maintained in a cell.

31. A compound comprising an antibody or an antigen-binding portion thereof operatively attached to a therapeutic agent, wherein the antibody, or antigen-binding portion thereof,

binds substantially to TNF-α, Rab8, TNF-α receptor, fibrocystin, FIP2 or a TNF-α

receptor- associated protein, such that the therapeutic agent inhibits TNF-α signalling and

polycystin function is maintained in a cell.