WO2015021291A2 - Antibodies to acetylated cyclophilin and use thereof - Google Patents

Abstract

Disclosed is an isolated antibody or binding fragment thereof which bind specifically to an acetylated epitope of Cyclophilin A, kits containing the antibody or binding fragment, and methods of their use.

Description

ANTIBODIES TO ACETYLATED CYCLOPHILIN A AND USE THEROF

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

No. 61/863,709, filed August 8, 2013, the disclosure of which is incorporated herein by reference in its entirety.

[0002] This invention was made with government support under grant number

HL49192 awarded by the National Institutes of Health. The government has certain rights in the invention.

TECHNOLOGICAL FIELD

[0003] Described herein are isolated antibodies or binding fragments thereof which bind specifically to an acetylated epitope of Cyclophilin A, kits containing the antibodies or binding fragments, and methods of their use.

BACKGROUND

[0004] Cyclophilin A ("CyPA") is a ubiquitously expressed protein which possesses peptidyl-propyl cis-trans isomerase ("PPIase") activity as well as non-enzymatic scaffold function (Marks, "Cellular Functions of Immunophilins," Physiol. Rev. 76:631-649 (1996); Handschumacher et al., "Cyclophilin: A Specific Cytosolic Binding Protein for Cyclosporin A," Science 226:544-547 (1984)). It plays an important role in various cell functions, including protein folding, intracellular trafficking, signal transduction, and transcription regulation (Satoh et al., "Circulating Smooth Muscle Progenitor Cells: Novel Players in Plaque Stability,"

Cardiovasc. Res. 77:445-447 (2008)). Vascular smooth muscle cells ("VSMC") secrete CyPA in response to oxidative stress, which is regulated by a Rho-dependent vesicular secretion pathway (Suzuki et al, "Cyclophilin A is Secreted by a Vesicular Pathway in Vascular Smooth Muscle Cells," Circ. Res. 98:811-817 (2006); Jin et al, "Cyclophilin A is a Secreted Growth Factor Induced by Oxidative Stress," Circ. Res. 87:789-796 (2000)). Secreted CyPA is a

proinflammatory mediator which can stimulate VSMC growth via ER activation (Jin et al., "Cyclophilin A is a Secreted Growth Factor Induced by Oxidative Stress," Circ. Res. 87:789-796 (2000)), cause endothelial cell ("EC") dysfunction by inducing adhesion molecules expression (Jin et al., "Cyclophilin A is a Proinflammatory Cytokine that Activates Endothelial Cells," Arterioscler. Thromb. Vase. Biol. 24: 1186-1191 (2004)), activate monocytes, and recruit inflammatory cells (Satoh et al., "Cyclophilin A Mediates Vascular Remodeling by Promoting Inflammation and Vascular Smooth Muscle Cell Proliferation," Circulation 117:3088-3098 (2008); Yuan et al., "Pro-Inflammatory Activities Induced by CyPA-Emmprin Interaction in Monocytes," Atherosclerosis 213(2):415-421 (2010)). Furthermore, CyPA deficiency prevents Angiotensin II ("AngII")-induced abdominal aortic aneurysm formation in ApoE-/- mice by regulating reactive oxygen species ("ROS") production and matrix metalloproteinase 2

("MMP2") activation in VSMC (Satoh et al, "Cyclophilin A Enhances Vascular Oxidative Stress and the Development of Angiotensin II-Induced Aortic Aneurysms," Nat. Med. 15:649- 656 (2009)).

[0005] Oxidative stress regulates many cell signaling pathways by affecting post- translational modification of target proteins (Shao et al., "Redox Modification of Cell Signaling in the Cardiovascular System," J. Mol. Cell Cardiol. 52:550-558 (2012)), which in turn affects protein function, stability, and degradation. For example, acetylation of lysine residues is a reversible post-translational process that neutralizes the amino acid's positive charge altering enzymatic activity, protein-protein interactions, and DNA binding (Wang et al., "Acetylation and Nuclear Receptor Action," J. Steroid Biochem. Mol. Biol. 123:91-100 (2011); Xiong et al, "Mechanistic Insights into the Regulation of Metabolic Enzymes by Acetylation," J. Cell Biol. 198: 155-164 (2012); de la Vega et al, "A Redox-Regulated Sumo/Acetylation Switch of hipk2 Controls the Survival Threshold to Oxidative Stress," Mol. Cell. 46:472-483 (2012)).

[0006] CyPA can be post-translationally modified by phosphorylation (Pan et al.,

"Cyclophilin A is Required for CXCR4-Mediated Nuclear Export of Heterogeneous Nuclear Ribonucleoprotein A2, Activation and Nuclear Translocation of ERKl/2, and Chemotactic Cell Migration," J. Biol. Chem. 283:623-637 (2008)) or acetylation (Massignan et al., "Proteomic Analysis of Spinal Cord of Presymptomatic Amyotrophic Lateral Sclerosis g93a Sodl Mouse," Biochem. Biophys. Res. Commun. 353:719-725 (2007); Lammers et al, "Acetylation Regulates Cyclophilin A Catalysis, Immunosuppression and HIV Isomerization," Nat. Chem. Biol. 6:331- 337 (2010); Kim et al., "Substrate and Functional Diversity of Lysine Acetylation Revealed by a Proteomics Survey," Mol. Cell. 23:607-618 (2006)). Proteomic analyses of spinal cord extracts from amyotrophic lateral sclerosis G93A SOD1 mice revealed that acetylated CyPA ("Ac- CyPA") is associated with oxidative stress. Synthetic Ac-CyPA at lysine ("AcK-CyPA") 125 affects CyPA's enzymatic PPIase activity, its ability to bind to cyclosporin A ("CsA"), and calcineurin, as well as affect HIV-1 incorporation into a cell (Lammers et al., "Acetylation Regulates Cyclophilin A Catalysis, Immunosuppression and HIV Isomerization," Nat. Chem. Biol. 6:331-337 (2010)). Furthermore, acetylated and methylated CyPA were secreted from irradiated breast cancer cells, suggesting that post-translational modification is required for CyPA secretion (Chevalier et al., "Accumulation of Cyclophilin A Iso forms in Conditioned Medium of Irradiated Breast Cancer Cells," Proteomics 12: 1756-1766 (2012)). Although many studies have indicated that post-translational modification of CyPA is important in signal transduction and cell development, its role specifically in vascular pathology has remained unclear.

[0007] Aspects illustrated herein are directed to overcoming deficiencies in the art pertaining to cardiovascular disease.

SUMMARY

[0008] According to aspects illustrated herein, there is provided an isolated antibody or binding fragment thereof which binds specifically to acetylated Cyclophilin A (Ac-CyPA).

[0009] According to other aspects illustrated herein, there is provided a kit for detecting

Ac-CyPA. The kit includes an antibody or binding fragment according to aspects illustrated herein having a label bound to the antibody or binding fragment and one or more buffer solutions or reagent solutions to detect the label.

[0010] According to further aspects illustrated herein, there is provided a method of detecting Ac-CyPA in a biological sample. This method involves contacting the biological sample with an antibody or binding fragment according to aspects illustrated herein having a label bound to the antibody or binding fragment and detecting specific binding that occurs between the biological sample and the antibody or binding fragment to identify Ac-CyPA in the biological sample.

[0011] According to additional aspects illustrated herein, there is provided a method of screening an individual for cardiovascular disease. This method involves providing a biological sample from an individual, contacting the biological sample with an antibody or binding fragment according to aspects illustrated herein having a label bound to the antibody or binding fragment, and detecting specific binding that occurs between the biological sample and the antibody or binding fragment to identify Ac-CyPA in the biological sample, where the presence of Ac-CyPA in the biological sample indicates the individual has cardiovascular disease.

[0012] According to other aspects illustrated herein, there is provided a method of treating an individual for symptoms associated with cardiovascular disease. This method involves contacting an individual with an antibody or binding fragment according to aspects illustrated herein under conditions effective for the antibody or binding fragment to specifically bind to Ac-CyPA, where the binding treats the individual for symptoms associated with cardiovascular disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIGs. 1A-1F demonstrate that Angll induces acetylation of CyPA in VSMC. In

FIGs. 1 A and IB, protein acetylation was detected by Western blot from total cell lysates (RASMC-TCL) stimulated with Angll (300 nM). CyPA and GAPDH were used as internal loading controls. In FIGs. 1C and ID, Angll or Trichostatin A (TSA, 1 μΜ for 8 hour)- stimulated RASMC-TCL were immunoprecipitated with anti-CyPA antibody and immune complexes were immunob lotted with anti-acetyl lysine (AcK) or CyPA antibody. In FIGs. IE and IF, RASMC were pretreated with ROS scavenger Tiron (5 mM) or N-acetylcysteine (NAC, 10 mM) for 30 minutes and stimulated with Angll for 16 hours followed by immunoprecipitation and Western blot. Data represent three independent experiments and are shown as mean±SEM (*p<0.05 relative to t=0, #p<0.05 vs Angll alone).

[0014] FIGs. 2A-2C demonstrate that CyPA lysine residues K82 and K125 are targets for

Angll-induced acetylation. In the graph of FIG. 2 A, GFP fluorescence was determined by flow cytometry as a measure for transduction efficiency in Ppia-I- MASMC transduced with lentiviral particles expressing FlagCyPA or vector alone. Non-transduced cells were used as control. In FIGs. 2B and 2C, Ppia-I- MASMC were transduced with FlagCyPA with or without K/R substitutions and stimulated with 1 μΜ Angll for 24 hours. Cell lysates were

immunoprecipitated with anti-Flag antibody and immunoblotted with Anti-AcK or Flag. GFP and Flag expression in total cell lysates (TCL) were analyzed by Western analysis. Experiments were performed three independent times and are shown as mean±SEM (*p<0.05 versus untreated WT, # p<0.05 versus WT-Angll).

[0015] FIGs. 3A-3H demonstrate that Angll induces acetylated-CyPA secretion in

VSMC. In FIGs. 3A-3C, Angll-induced secretion of proteins in conditioned-medium (CM) as measured by Western blot using anti-AcK or CyPA antibodies. In FIGs. 3D and 3E, Angll- induced AcK-CyPA in CM was measured by immunoprecipitation and immunoblotting. In FIGs. 3F-3H, ROS scavenger Tiron (5 mM) or N-acetylcysteine (NAC, 10 mM) 30 minutes pretreated RASMC were stimulated with Angll for 16 hours, followed by immunoprecipitation and immunoblotting. The results are normalized to the fluorescence intensity at the 0 time point or vehicle treated cells, which was set to 1.0. Data represent three experiments and are shown as mean ± SEM (*p<0.05 versus 0 time point or vehicle, # p<0.05 versus Angll).

[0016] FIGs. 4A-4B demonstrate that acetylation is necessary for Angll-induced CyPA secretion. In FIG. 4A, CM from Angll (1 μΜ for 24 hours) stimulated Ppia-I- MASMC- transduced with lentiviral particles expressing FlagCyPA with or without K/R substitutions was immunoprecipitated with anti-Flag antibody and immunoblotted with anti-AcK or Flag. FIG. 4B shows the ratio of secreted CyPA (or KtoR mutants) to total-CyPA in the total cell lysate (TCL) (or KtoR mutant), which was normalized to the ratio for WT CyPA, which was set to 100.

Experiments were performed three independent times and data are shown as ±SEM (*p<0.05 versus WT).

[0017] FIGs. 5A-5G relate to ERK1/2 activity measured in RASMC-stimulated with CM titrated to assure equal amounts of extracellular CyPA (Vector, WT, or K/R substitutions) for 10 minutes (FIGs. 5 A and 5B) or 50 nM rhCyPA or AcK-rhCyPA for 10 minutes (FIG. 5C and 5D). HAT buffer contains no CyPA protein; rhCyPA is present in HAT buffer lacking p300 acetyltransferase; and AcK-rhCyPA is present in buffer containing CyPA, p300, and acetyl-CoA (see also FIG. 10). In FIG. 5E, ROS production was measured by flow cytometry in RASMC stimulated with CM for 4 hours. In FIGs. 5F and 5G, representative gelatin zymography showed MMP2 activity in CM from Ppia-I- MASMC transduced with lentiviral particles. All experiments were repeated three different times and are shown as mean ± SEM (*p<0.05 versus corresponding control, #p<0.05 versus WT-Angll).

[0018] FIGs. 6A-6J show that acetylated extracellular CyPA enhances adhesion molecules expression and EC-monocytes adhesion. Human umbilical vein endothelial cells ("HUVEC") were stimulated with CM from Ppia-I- MASMC transduced with lentiviral particles (WT or K/R mutant) or 50 nM rhCyPA or AcK-rhCyPA. After 6 hour incubation, VCAM-1 and ICAM-1 expression were measured by Western blot (FIGs. 6A-6F) or U937 monocytes were added to HUVEC and the adherent monocytes were counted in five different optical fields for each well (FIGs. 6G-6J). Quantified data show fold increase of monocyte adherence to EC. Data are shown as mean ± SEM of values from three independent experiments (*p<0.05 versus corresponding control, #p<0.05 versus WT or rhCyPA).

[0019] FIGs. 7A-D provide further evidence that Angll induces acetylation of CyPA. In

FIGs. 7A and 7B, Angll-induced CyPA acetylation was measured in Flag-CyPA over expressed mouse aortic smooth muscle cells (MASMC- FlagCyPA) using immunoprecipitation and immunoblotting method. The results are normalized to the fluorescence intensity at the 0 time point, which was set to 1.0. In FIGs. 7C and 7D, TCL from WT or CyPA knockout mouse aortic smooth muscle cell (Ppia-I- MASMC) were analyzed by western blot to detect 17 kDa protein acetylation. All experiments are performed three independent times and data are shown as mean ± SEM. (*p<0.05 versus untreated cells).

[0020] FIGs. 8A-8E demonstrate Rho kinase inhibitor and AcK-CyPA secretion. In FIG.

8A, RASMC were pretreated with Rho kinase inhibitor Y-27632 (30 μΜ) for 30 minutes and stimulated with Angll (300 nM) for 24 hours. CM were immunoprecipitated with anti-CyPA antibody and immunoblotted with anti-AcK or CyPA, respectively. FIGs. 8B and 8C show the quantitative analysis of AcK-CyPA using Image J (NIH). Data are representative of three independent experiments and values are mean ± SEM. (*p<0.05 versus vehicle, # p<0.05 versus Angll). In FIG. 8D, equal amounts of total cell lysates from RASMC treated as described were immunoprecipitated with anti-CyPA and the complexes Western blotted for CyPA and acetylation. Y27632, in the presence or absence of Angll did not affect CyPA expression. FIG. 8E shows quantitation of ratio of AcK-CyPA to CyPA in the presence of Y27632.

[0021] FIG. 9 shows confirmation of the total amount of Flag-CyPA in mixtures of CM, which is used for pERKl/2, ROS production and MMP2 activity experiments. CyPA in the CM from Ppia-/- MASMC transduced with WT or mutant lentiviral particles was determined from quantitation of the Western blot reactivity (FIGs. 4A-4B) and the volumes were normalized using conditioned medium from Ppia-/- MASMC. Aliquots of the mixtures used in zymography and ROS experiments and pErk activation were subjected to Western blot and immunoblotted with anti-Flag antibody.

[0022] FIG. 10 provides in vitro acetylation confirmation. rhCyPA was acetylated in vitro in HAT buffer containing acetyl CoA and p300 acetyltransferase. In vitro acetylated rhCyPA (AcK-rhCyPA) and native rhCyPA (in HAT buffer lacking p300) were subjected to Western analysis to detect acetylation using anti-AcK or CyPA antibody.

[0023] FIGs. 11 A- 1 IB illustrate mechanistic details for Angll. The schematic model in

FIG. 11 A shows the role of Angll-induced acetylated CyPA in secretion and regulation of vascular cells activation. In FIG. 1 IB, Angiotensin II (Angll)-induced oxidative stress regulates lysine acetylase (KAT) and/or deacetylase (KDAC) activity, which alters the cellular equilibrium of the two enzyme activities. CyPA is one of the substrates whose acetylation is affected by the alteration of this equilibrium. Acyl-CyPA is preferentially secreted and is a more potent agonist than CyPA as measured by ER l/2 and MMP2 activation.

[0024] FIGs. 12A-12B demonstrate that AcK-CypA K82 antibody is specific for acetylated CypA. In FIG. 12A, affinity purified (AcK peptide followed by non-AcK peptide to remove non-acetyl-specific Abs) bleeds 5+6 and bleeds 7 through 9 were titrated against

TrichlorostatinA (an HDAC inhibitor that causes an increase in acetylation of CyPA) treated

RASMC by Western blot. Total serum from bleed 8 (143 B8 crude) is a control to show effectiveness of affinity purification strategy. a-CyPA (ENZO) is a control lane probed with a commercially available, validated (not AcK specific) CyPA antibody. For each lane the designation is AcK-CyPA Ab concentration (1 or 0.5 μg/ml). The second designation (1/lOK or

1/25K) is the dilution of the secondary goat anti rabbit-HRP secondary antibody. In FIG. 12B, upper panel, affinity purified bleeds 5+6 and bleeds 7 through 9 were reacted against TSA treated (AcK-CyPA) or control treated (-) RASMC extracts by Western blot to determine the specificity of AcK-K82 Ab for AcK-CyPA. AcK-K82 detected AcK-CyPA in only the TSA treated extracts. a-CyPA (ENZO) is a control lane probed with a commercially available, validated (not AcK specific) CyPA antibody. The reactivity with proteins of greater molecular mass likely represents the large isoform of cyclophilin A, the multi-domain enzyme cyclophilin- 40. In FIG. 12B, lower panel, a Western blot stripped and reprobed with CyPA (ENZO) antibody demonstrates equal CyPA mass in each lane.

[0025] FIGs. 13 A- 13B relate to secreted AcK-CyPA. In FIG 13 A, acetylated CyPA is detected in endothelial and smooth muscle cells. Equal amounts of total cell lysates from the indicated cell lines were analyzed by Western blot for total CyPA and acetylated CyPA (AcK- CyPA). PAC1— Rat pulmonary artery smooth muscle (SM) cell; RASMC-Rat aortic SM;

RPMEC-Rat pulmonary endothelial cell. FIG. 13B is a graph showing quantitation of relative abundance of AcK-CyPA to total CyPA.

[0026] FIG. 14 illustrates that acetylated CypA is secreted from endothelial cells following hypoxia. RPMEC were subject to 6 or 24 hours of hypoxia (1% 0₂) or normoxia. Total cell lysates and culture supematants were Western blotted for total (CyPA) and acetylated (AcK) CyPA.

[0027] FIG. 15 shows acetylated CyPA in lungs of a rat model of Pulmonary Artery

Hypertension. Lung sections from healthy rats and those treated with monocrotaline to induce pulmonary artery hypertension were stained with K82 AcK-CyPA antibody and reactivity detected by immunofluorescence (Alexa-fluor). Control IgG was used in place of primary AcK- CypA antibody to show specificity of staining for AcK-CypA in endothelial cells of vessels in the lung. Sections were counterstained with DAPI to delineate nuclei.

[0028] FIG. 16 shows AcK-CyPA expression in human lungs. Human lung tissue from control (panel A) and a PAH patient (panels B, C) was stained by immunofluorescence for AcK- CyPA. Sections were counterstained with DAPI to delineate nuclei. Bright red circles showed autofluorescence from red blood cells in the vessel.

DETAILED DESCRIPTION

[0029] According to aspects illustrated herein, there is provided an isolated antibody or binding fragment thereof which binds specifically to acetylated Cyclophilin A.

[0030] Cyclophilin A is a ubiquitously expressed protein which possesses peptidyl- propyl cis-trans isomerase (PPIase) activity as well as non-enzymatic scaffold function (Marks, "Cellular Functions of Immunophilins," Physiol. Rev. 76:631-649 (1996) and Handschumacher et al, "Cyclophilin: A Specific Cytosolic Binding Protein for Cyclosporin A," Science 226:544- 547 (1984), the disclosures of which are incorporated herein by reference in their entirety). CyPA plays an important role in various cell functions including protein folding, intracellular trafficking, signal transduction, and transcription regulation.

[0031] Cyclophilin A is a proinflammatory mediator involved in oxidative stress related cardiovascular diseases. It is secreted from vascular smooth muscle cells in response to reactive oxygen species in a highly regulated manner. Extracellular CyPA activates vascular smooth muscle cells and endothelial cells promoting inflammation, cell growth, and cell death.

[0032] Human Cyclophilin A has an amino acid sequence as described in GenBank

Accession No. NM 021130, the disclosure of which is incorporated herein by reference in its entirety. Specifically, the amino acid sequence of human CyPA is SEQ ID NO: l, as follows:

MVNPTVFFDI AVDGEPLGRV SFELFADKVP KTAENFRALS TGEKGFGYKG

SCFHRIIPGF MCQGGDFTRH NGTGGKS IYG EKFEDENFIL KHTGPGILSM

ANAGPNTNGS QFFICTAKTE WLDGKHVVFG KVKEGMNIVE AMERFGSRNG

KTSKKITIAD CGQLE

[0033] The nucleotide sequence that encodes human CyPA is SEQ ID NO:2, as follows: gaacgtggta taaaaggggc gggaggccag gctcgtgccg ttttgcagac

gccaccgccg aggaaaaccg tgtactatta gccatggtca accccaccgt

gttcttcgac attgccgtcg acggcgagcc cttgggccgc gtctcctttg

agctgtttgc agacaaggtc ccaaagacag cagaaaattt tcgtgctctg

agcactggag agaaaggatt tggttataag ggttcctgct ttcacagaat

tattccaggg tttatgtgtc agggtggtga cttcacacgc cataatggca

ctggtggcaa gtccatctat ggggagaaat ttgaagatga gaacttcatc

ctaaagcata cgggtcctgg catcttgtcc atggcaaatg ctggacccaa

cacaaatggt tcccagtttt tcatctgcac tgccaagact gagtggttgg

atggcaagca tgtggtgttt ggcaaagtga aagaaggcat gaatattgtg

gaggccatgg agcgctttgg gtccaggaat ggcaagacca gcaagaagat

caccattgct gactgtggac aactcgaata agtttgactt gtgttttatc

ttaaccacca gatcattcct tctgtagctc aggagagcac ccctccaccc

catttgctcg cagtatccta gaatctttgt gctctcgctg cagttccctt

tgggttccat gttttccttg ttccctccca tgcctagctg gattgcagag

ttaagtttat gattatgaaa ataacaattg tcctcgtttg

agttaagagt gttgatgtag gctttatttt aagcagtaat gggttacttc tgaaacatca cttgtttgct taattctaca cagtacttag atttttttta ctttccagtc ccaggaagtg tcaatgtttg ttgagtggaa tattgaaaat

gtaggcagca actgggcatg gtggctcact gtctgtaatg tattacctga

ggcagaagac cacctgaggg taggagtcaa gatcagcctg ggcaacatag

tgagacgctg tctctacaaa aaataattag cctggcctgg tggtgcatgc

ctagtcctag ctgatctgga ggctgacgtg ggaggattgc ttgagcctag

agtgagctat tatcatgcca ctgtacagcc tgggtgttca cagatcttgt

gtctcaaagg taggcagagg caggaaaagc aaggagccag aattaagagg

ttgggtcagt ctgcagtgag ttcatgcatt tagaggtgtt cttcaagatg

actaatgtca aaaattgaga catctgttgc ggtttttttt tttttttttt

cccctggaat gcagtggcgt gatctcagct cactgcagcc tccgcctcct

gggttcaagt gattctagtg cctcagcctc ctgagtagct gggataatgg

gcgtgtgcca ccatgcccag ctaatttttg tatttttagt atagatgggg

tttcatcatt ttgaccaggc tggtctcaaa ctcttgacct cagctgatgc

gcctgccttg gcctcccaaa ctgctgagat tacagatgtg agccaccgca

ccctacctca ttttctgtaa caaagctaag cttgaacact gttgatgttc

ttgagggaag catattgggc tttaggctgt aggtcaagtt tatacatctt

aattatggtg gaattcctat gtagagtcta aaaagccagg tacttggtgc

tacagtcagt ctccctgcag agggttaagg cgcagactac ctgcagtgag

gaggtactgc ttgtagcata tagagcctct ccctagcttt ggttatggag

gctttgaggt tttgcaaacc tgaccaattt aagccataag atctggtcaa

agggataccc ttcccactaa ggacttggtt tctcaggaaa ttatatgtac

agtgcttgct ggcagttaga tgtcaggaca atctaagctg agaaaacccc

ttctctgccc accttaacag acctctaggg ttcttaaccc agcaatcaag

tttgcctatc ctagaggtgg cggatttgat catttggtgt gttgggcaat

ttttgtttta ctgtctggtt ccttctgcgt gaattaccac caccaccact

tgtgcatctc agtcttgtgt gttgtctggt tacgtattcc ctgggtgata

ccattcaatg tcttaatgta cttgtggctc agacctgagt gcaaggtgga

aaacatcttt tcatta

[0034] Mouse Cyclophilin A has an amino acid sequence as described in GenBank

Accession No. DQ985731 , the disclosure of which is incorporated herein by reference in its entirety. Specifically, the amino acid sequence of mouse CyPA is SEQ ID NO:3, as follows: MVNPTVFFDI TADDEPLGRV SFELFADKVP KTAENFRALS TGEKGFGYKG

SSFHRIIPGF MCQGGDFTRH NGTGGRS IYG EKFEDENFIL KHTGPGILSM

ANAGPNTNGS QFFICTAKTE WLDGKHVVFG KVKEGMNIVE AMERFGSRNG

KTSKKITISD CGQL

[0035] The nucleotide sequence that encodes mouse CyPA is SEQ ID NO:4, as follows: atggtcaacc ccaccgtgtt cttcgacatc acggccgatg acgagccctt

gggccgcgtc tccttcgagc tgtttgcaga caaagttcca aagacagcag

aaaactttcg agctctgagc actggagaga aaggatttgg ctataagggt

tcctcctttc acagaattat tccaggattc atgtgccagg gtggtgactt

tacacgccat aatggcactg gcggcaggtc catctacgga gagaaatttg

aggatgagaa cttcatccta aagcatacag gtcctggcat cttgtccatg

gcaaatgctg gaccaaacac aaacggttcc cagtttttta tctgcactgc

caagactgaa tggctggatg gcaagcatgt ggtctttggg aaggtgaaag

aaggcatgaa tattgtggaa gccatggagc gttttgggtc caggaatggc

aagaccagca agaagatcac catttccgac tgtggacagc tctaa

[0036] Rat Cyclophilin A has an amino acid sequence as described in GenBank

Accession No. BC091153, the disclosure of which is incorporated herein by reference in its entirety. Specifically, the amino acid sequence of rat CyPA is SEQ ID NO:5, as follows:

MVNPTVFFDI TADGEPLGRV CFELFADKVP KTAENFRALS TGEKGFGYKG SSFHRIIPGF MCQGGDFTRH NGTGGKS IYG EKFEDENFIL KHTGPGILSM ANAGPNTNGS QFFICTAKTE WLDGKHVVFG KVKEGMS IVE AMERFGSRNG KTSKKITISD CGQL

[0037] The nucleotide sequence that encodes rat CyPA is SEQ ID NO:6, as follows: tcgccgcttg ctgcagacat ggtcaacccc accgtgttct tcgacatcac

ggctgatggc gagcccttgg gtcgcgtctg cttcgagctg tttgcagaca

aagttccaaa gacagcagaa aactttcgtg ctctgagcac tggggagaaa

ggatttggct ataagggttc ctcctttcac agaattattc caggattcat

gtgccagggt ggtgacttca cacgccataa tggcactggt ggcaagtcca tctacggaga gaaatttgag gatgagaact tcatcctgaa gcatacaggt cctggcatct tgtccatggc aaatgctgga ccaaacacaa atggttccca

gttttttatc tgcactgcca agactgagtg gctggatggc aagcatgtgg

tctttgggaa ggtgaaagaa ggcatgagca ttgtggaagc catggagcgt

tttgggtcca ggaatggcaa gaccagcaag aagatcacca tctccgactg

tggacaactc taatttcttt gacttgcggg cattttaccc atcaaaccat

tccttctgta gctcaggaga gcacccccac cccatctgct cgcaataccc

tgtaatctct gctctcactg aagttctttg ggttccatat tttcctcatt

ccccttcaag tctagcagga ttgcaaagtt aagtttatga ttatgaataa

cL cL cL C t cL cL cL t Cf cL C[ cL cL cL cL cL cL cL cL cL cL cL cL cL cL cL cL cL cL cL cL cL cL cL cL cL cL cL cL

[0038] According to one embodiment, Ac-CyPA comprises an acetylated lysine residue

(AcK-CyPA). The acetylated lysine residue is, according to one embodiment, the lysine residue in position 82. For example, the acetylated lysine residue is position 82 of SEQ ID NO: 1 , SEQ ID NO:3, or SEQ ID NO:5. According to another embodiment, the lysine residue is in position 125. For example, the acetylated lysine residue is in position 125 of SEQ ID NO: l, SEQ ID NO:3, or SEQ ID NO:5.

[0039] In one embodiment, AcK-CyPA comprises an amino acid sequence of SEQ ID

NO:7, as follows:

SIYGE [K-ac] FEDENFI .

[0040] In another embodiment, AcK-CyPA comprises an amino acid sequence of SEQ

ID NO:8, as follows:

TEWLDG [K-ac] FEDENFI .

[0041] In one embodiment, the isolated antibody or binding fragment thereof is a monoclonal antibody or binding fragment of a monoclonal antibody.

[0042] In another embodiment, the isolated antibody or binding fragment thereof is a polyclonal antibody or binding fragment of a polyclonal antibody.

[0043] The term "antibody" as used herein is any specific binding substance having a binding domain with the required specificity. Thus, this term covers antibody fragments, derivatives, functional equivalents, and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or synthetic, monoclonal, or polyclonal. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are also included, as described in more detail infra.

[0044] As used herein, the phrase "binds specifically to" means that the antibody or binding fragment thereof binds to the stated target {e.g., an acetylated epitope of Cyclophilin A) and not to other targets {e.g., non-acetylated Cyclophilin A, non-acetylated epitopes of

Cyclophilin A, or acetylated or non-acetylated proteins other than Cyclophilin A). According to one embodiment, the isolated antibody or binding fragment thereof is a monospecific, polyclonal antibody, with monospecificity to an acetylated epitope of Cyclophilin A. However, as described infra, the antibody or binding fragment thereof may also include monospecific, monoclonal antibodies. Isolated antibodies or binding fragments thereof which bind specifically to an acetylated epitope of Cyclophilin A are distinguishable from antibodies that bind to acetylated residues non-specifically without regard to any particular peptide or any particular acetylated residue or epitope.

[0045] Procedures for raising polyclonal antibodies are well known. Typically, such antibodies can be raised by administering the antigen {e.g., acetylated Cyclophilin A, or a peptide or peptide fragment comprising SEQ ID NO:7 or SEQ ID NO:8) subcutaneously to rabbits, mice, rats, or chickens which have first been bled to obtain pre-immune serum. The antigens can be injected as tolerated. Each injected material can contain adjuvants and the selected antigen {e.g., in substantially pure or isolated form). Suitable adjuvants include, without limitation, Freund's complete or incomplete mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, and potentially useful human adjuvants such as bacille Calmette-Guerin and Carynebacterium parvum. The subject mammals are then bled one to two weeks after the first injection and periodically boosted with the same antigen {e.g., three times every six weeks). A sample of serum is then collected one to two weeks after each boost. Polyclonal antibodies can be recovered from the serum by affinity chromatography using the corresponding antigen to capture the antibody. This and other procedures for raising polyclonal antibodies are disclosed in Harlow & Lane, editors, Antibodies: A Laboratory Manual (1988), the disclosure of which is incorporated herein by reference in its entirety.

[0046] Monoclonal antibody production can also be carried out by techniques that are well known in the art. Basically, the process involves first obtaining immune cells

(lymphocytes) from the spleen of a mammal {e.g. , mouse) that has been previously immunized with the antigen of interest {e.g., acetylated Cyclophilin A, or a peptide or peptide fragment comprising SEQ ID NO: 7 or SEQ ID NO: 8) either in vivo or in vitro. The antibody-secreting lymphocytes are then fused with myeloma cells or transformed cells, which are capable of replicating indefinitely in cell culture. The resulting fused cells, or hybridomas, are immortal, immunoglobulin-secreting cell lines that can be cultured in vitro. Upon culturing the

hybridomas, the resulting colonies can be screened for the production of desired monoclonal antibodies. Colonies producing such antibodies are cloned and grown either in vivo or in vitro to produce large quantities of antibody. A description of the theoretical basis and practical methodology of fusing such cells is set forth in Kohler and Milstein, "Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity," Nature 256:495 (1975), the disclosure of which is incorporated herein by reference in its entirety.

[0047] Mammalian lymphocytes are immunized by in vivo immunization of the animal

{e.g., a mouse, rat, rabbit, or human). Such immunizations are repeated as necessary at intervals of up to several weeks to obtain a sufficient titer of antibodies. Following the last antigen boost, the animals are sacrificed and spleen cells removed.

[0048] Fusion with mammalian myeloma cells or other fusion partners capable of replicating indefinitely in cell culture is effected by standard and well-known techniques, for example, by using polyethylene glycol ("PEG") or other fusing agents {see Milstein and Kohler,

"Derivation of Specific Antibody-producing Tissue Culture and Tumor Lines by Cell Fusion,"

Eur. J. Immunol. 6:511 (1976), the disclosure of which is incorporated herein by reference in its entirety). This immortal cell line, which is, e.g., murine, but may also be derived from cells of other mammalian species, including but not limited to rats and humans, is selected to be deficient in enzymes necessary for the utilization of certain nutrients, to be capable of rapid growth, and to have good fusion capability. Many such cell lines are known to those skilled in the art, and others are regularly described. Human hybridomas can be prepared using the EBV-hybridoma technique for monoclonal antibodies {see Cole et al, in Monoclonal Antibodies and Cancer

Therapy, Alan R. Liss, Inc., pp. 77-96 (1985), the disclosure of which is incorporated herein by reference in its entirety). Human antibodies may be used and can be obtained by using human hybridomas (Cote et al., "Generation of Human Monoclonal Antibodies Reactive with Cellular

Antigens," Proc. Natl. Acad. Sci. USA 80:2026- 2030 (1983), the disclosure of which is incorporated herein by reference in its entirety) or by transforming human B cells with EBV virus in vitro (Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.

77-96 (1985), the disclosure of which is incorporated herein by reference in its entirety). In addition, monoclonal antibodies can be produced in germ-free animals {see PCT/US90/02545, the disclosure of which is incorporated herein by reference in its entirety).

[0049] In addition, techniques developed for the production of chimeric antibodies

(Morrison et al, "Chimeric Human Antibody Molecules: Mouse Antigen-binding Domains with

Human Constant Region Domains," Proc. Natl. Acad. Sci. USA 81 :6851-6855 (1984); Neuberger et al, "Recombinant Antibodies Possessing Novel Effector Functions," Nature 312:604-608 (1984); Takeda et al., "Construction of Chimaeric Processed Immunoglobulin Genes Containing Mouse Variable and Human Constant Region Sequences," Nature 314:452-454 (1985), the disclosures of which are incorporated herein by reference in their entirety) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region (see e.g., U.S. Patent No. 4,816,567 to Cabilly et al, and U.S. Patent No. 4,816,397 to Boss et al., the disclosures of which are incorporated herein by reference in their entirety).

[0050] Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g., by a peptide linker) but unable to associate with each other to form an antigen binding site: antigen binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (see PCT Publication No. WO 94/13804, the disclosure of which is incorporated herein by reference in its entirety).

[0051] Alternatively, techniques described for the production of single chain antibodies

(see e.g., U.S. Patent No. 4,946,778 to Ladner et al; Bird et al, "Single-chain Antigen-binding Proteins," Science 242:423-426 (1988); Huston et al, "Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-dogoxin Single-chain Fv Analogue Produced in Escherichia coli," Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); Ward et al, "Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted from

Escherichia coli," Nature 334:544-546 (1989), the disclosures of which are incorporated herein by reference in their entirety) can be adapted to produce single chain antibodies against modified bases. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.

[0052] In addition to whole antibodies, binding portions (or fragments) of such antibodies are also contemplated. Such binding portions include Fab fragments, F(ab')₂ fragments, Fv fragments, single-chain antibodies, a V_H domain, or a V_L domain. These antibody fragments can be made by conventional procedures, such as proteolytic fragmentation procedures, as described in Goding, Monoclonal Antibodies: Principles and Practice, pp. 98-118 (N.Y. Academic Press, 1983), the disclosure of which is incorporated herein by reference in its entirety. [0053] Antibodies may be isolated by standard techniques known in the art, such as immunoaffmity chromatography, centrifugation, precipitation, etc. The antibodies (or fragments or variants thereof) are e.g., prepared in a substantially purified form {i.e., at least about 85% pure, or e.g., at least about 90% pure, or e.g., at least about 95% to 99% pure). Immunoaffinity ligands for total IgG include, but are not limited to, Protein A, Protein G, or cells of

Staphylococcus aureus. Epitope specific antibodies are subsequently purified by adsorption on and elution from the specific epitope immobilized to a solid support. For the purposes of antibodies recognizing specifically modified residues {e.g., acetylation), a post-depletion step is performed. Antibodies of undesired affinity {i.e., those recognizing the non-modified epitope) are removed by adsorption against the non-modified epitope. Non-binding antibodies are highly enriched for affinity against the modified residue.

[0054] As a further alternative, "synthetic antibodies" can be generated using

recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage. This term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, where the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.

[0055] To isolate DNA encoding an antibody, for example, DNA is extracted from an antibody expressing phage. Such extraction techniques are well known in the art and are described, for example, in Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, New York (1989); Ausubel et al, "Short Protocols in Molecular Biology," New York: Wiley (1999), the disclosures of which are incorporated herein by reference in their entirety.

[0056] Another form of antibody includes a nucleic acid sequence which encodes the antibody and which is operably linked to promoter/regulatory sequences which can directly express the antibody in vivo. For a discussion of this technology, see, e.g., Cohen, Science 259: 1691-1692 (1993); Fynan et al. Proc. Natl. Acad. Sci. 90: 11478-11482 (1993); and Wolff et al. Biotechniques 11 :474-485 (1991), the disclosures of which are incorporated herein by reference in their entirety), which describe the use of naked DNA as an antibody/vaccine. For example, a plasmid containing suitable promoter/regulatory sequences operably linked to a DNA sequence encoding an antibody may be directly administered to a patient using the technology described in the aforementioned references.

[0057] Alternatively, the promoter/enhancer sequence operably linked to DNA encoding the antibody may be contained within a vector, which vector is administered to a subject. The vector may be a viral vector which is suitable as a delivery vehicle for delivery of the DNA encoding the antibody to the subject, or the vector may be a non- viral vector which is suitable for the same purpose.

[0058] According to one embodiment, the antibody or binding fragment of the present invention further comprises a label bound to the antibody or binding fragment. Suitable labels are selected from a fluorescent label, a biologically-active enzyme label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label, and a chromophore label.

[0059] An antibody bound to a label is useful for diagnostic use, such as for detecting the presence or absence of Ac-CyPA, as described in more detail infra.

[0060] Examples of labels useful for diagnostic imaging in accordance with the present invention are radiolabels such as ¹³¹I, ^mIn, ¹²³I, "mTc, ³²P, ¹²⁵1, ³H, ¹⁴C, and ¹⁸⁸Rh, fluorescent labels such as fluorescein and rhodamine, nuclear magnetic resonance active labels, positron emitting isotopes detectable by a positron emission tomography ("PET") scanner,

chemiluminescers such as luciferin, and enzymatic markers such as peroxidase or phosphatase. Short-range radiation emitters, such as isotopes detectable by short-range detector probes, such as a transrectal probe, can also be employed. The antibody or antibody fragment can be labeled with such reagents using techniques known in the art. For example, Wensel et al,

Radioimmunoimaging and Radioimmunotherapy, Elsevier, New York (1983), the disclosure of which is incorporated herein by reference in its entirety, teach techniques relating to the radiolabeling of antibodies, as does Colcher et al., "Use of Monoclonal Antibodies as

Radiopharmaceuticals for the Localization of Human Carcinoma Xenografts in Athymic Mice," Meth. Enzymol. 121 : 802-816 (1986), the disclosure of which is incorporated herein by reference in its entirety.

[0061] A radiolabeled antibody or antibody fragment of the present invention can be used for in vitro diagnostic tests. The specific activity of a tagged antibody, or binding portion thereof, depends upon the half-life, the isotopic purity of the radioactive label, and how the label is incorporated into the antibody. Table 1 lists several commonly-used isotopes, their specific activities, and half-lives. In immunoassay tests, the higher the specific activity, in general, the better the sensitivity. TABLE 1

[0062] Procedures for labeling antibodies with the radioactive isotopes listed in Table 1 are generally known in the art. Tritium labeling procedures are described in U.S. Patent No. 4,302,438, to Zech, the disclosure of which is incorporated herein by reference in its entirety. Iodinating, tritium labeling, and 35 S labeling procedures especially adapted for murine monoclonal antibodies are described by Goding, Monoclonal Antibodies: Principles and

Practice, pp. 124-126 (N.Y. Academic Press, 1983) and the references cited therein, the disclosure of which is incorporated herein by reference in its entirety. Other procedures for iodinating antibodies, or binding portions thereof, are described by Hunter et al, Nature 144:945

(1962); David et al, Biochemistry 13: 1014-1021 (1974); and U.S. Patent Nos. 3,867,517 and

4,376,110, the disclosures of which are incorporated herein by reference in their entirety.

Radiolabeling elements which are useful in imaging include ¹²³I, ¹³¹I, ^{1 U}In, and ^99mTc, for example. Procedures for iodinating antibodies are described by Greenwood et al, Biochem. J.

89: 114-123 (1963); Marchalonis, Biochem. J. 113:299-305 (1969); and Morrison et al,

Immunochemistry 8:289-297 (1971), the disclosures of which are incorporated herein by reference in their entirety. Procedures for ^99mTc-labeling are described by Rhodes et al., in

Burchiel et al. (eds.), Tumor Imaging: The Radioimmunochemical Detection of Cancer, New

York: Masson 111-123 (1982), and the references cited therein, the disclosures of which are incorporated herein by reference in their entirety. Procedures suitable for ^luIn-labeling antibodies are described by Hnatowich et al, J. Immul. Methods 65: 147-157 (1983); Hnatowich et al, J. Applied Radiation 35:554-557 (1984); and Buckley et al, F.E.B.S. 166:202-204 (1984), the disclosures of which are incorporated herein by reference in their entirety.

[0063] A radiolabeled antibody is useful for administration to a patient, as described in more detail infra, because it may be detected or "imaged" in vivo using known techniques such as radionuclear scanning using e.g., a gamma camera or emission tomography. See e.g.,

Bradwell et al, "Developments in Antibody Imaging", Monoclonal Antibodies for Cancer

Detection and Therapy, Baldwin et al, (eds.), pp. 65-85 (Academic Press 1985), the disclosure of which is incorporated herein by reference in its entirety. Alternatively, a positron emission transaxial tomography scanner, such as designated Pet VI located at Brookhaven National

11 18 15 13

Laboratory, can be used where the radiolabel emits positrons (e.g., C, F, O, and N).

[0064] Fluorophore and chromophore labeled antibodies can be prepared from standard moieties known in the art. Since antibodies and other proteins absorb light having wavelengths of up to about 310 nm, the fluorescent moieties should be selected to have substantial absorption at wavelengths above 310 nm or e.g., above about 400 nm. A variety of suitable fiuorescers and chromophores are described by Stryer, Science 162:526 (1968) and Brand et al, Annual Review of Biochemistry 41 :843-868 (1972), the disclosures of which are incorporated herein by reference in their entirety. The antibodies can be labeled with fluorescent chromophore groups by conventional procedures such as those disclosed in U.S. Patent Nos. 3,940,475; 4,289,747; and 4,376,110; the disclosures of which are incorporated herein by reference in their entirety.

[0065] One group of fiuorescers having a number of the desirable properties described supra are the xanthene dyes, which include the fluoresceins derived from

3,6-dihydroxy-9-henylxanthhydrol and resamines and rhodamines derived from

3,6-diamino-9-phenylxanthydrol and lissanime rhodamine B. The rhodamine and fluorescein derivatives of 9-o-carboxyphenylxanthhydrol have a 9-o-carboxyphenyl group. Fluorescein compounds having reactive coupling groups such as amino and isothiocyanate groups such as fluorescein isothiocyanate and fiuorescamine are readily available. Another group of fluorescent compounds are the naphthylamines, having an amino group in the a- or β position.

[0066] Antibodies can be labeled with fluorochromes or chromophores by the procedures described by Goding, Monoclonal Antibodies: Principles and Practice, pp. 208-249 (N.Y.

Academic Press, 1983), the disclosure of which is incorporated herein by reference in its entirety. The antibodies can be labeled with an indicating group containing the NMR-active ¹⁹F atom, or a plurality of such atoms inasmuch as (i) substantially all of the naturally abundant fluorine atoms are the ¹⁹F isotope and, thus, substantially all fluorine-containing compounds are NMR-active; (ii) many chemically active polyfiuorinated compounds such as trifiuoracetic anhydride are commercially available at relatively low cost, and (iii) many fiuorinated compounds have been found medically acceptable for use in humans such as the perfluorinated polyethers utilized to carry oxygen as hemoglobin replacements. After permitting such time for incubation, a whole body NMR determination is carried out using an apparatus such as one of those described by Pykett, Scientific American 246:78-88 (1982), the disclosure of which is incorporated herein by reference in its entirety.

[0067] Conditions effective to bind an antibody to a protein are known or can be determined by persons of ordinary skill in the art. For example, an appropriate ionic balance in the sample can assist the antibody in effectively binding to the protein {e.g., AcK-CyPA). The H of a sample can be controlled by addition of suitable buffers such as sodium phosphate, which will maintain the pH at approximately 7.0. Salts, such as sodium chloride may also be added to the buffer and/or the sample.

[0068] According to other aspects illustrated herein, there is provided a kit for detecting

Ac-CyPA. The kit includes an antibody or binding fragment according to aspects illustrated herein, where the antibody or binding fragment is bound to a label. The kit also includes one or more buffer solutions or reagent solutions to detect the label.

[0069] Suitable labels and their detection are described supra.

[0070] In the kit of the present invention, the one or more buffer solutions or reagent solutions to detect the label are suitable for carrying out detection of binding between a biological sample and the antibody or binding fragment using an assay selected from, e.g. , a

Western blot, immunoassay, ELISA assay, flow cytometry, radiography, or

immunoscintography.

[0071] In one embodiment, the kit further includes a secondary antibody or binding fragment that binds specifically to the antibody or binding fragment. Thus, according to the embodiment, the kit includes both primary and secondary antibodies. The primary antibodies include the antibody or binding fragment thereof the present invention which binds specifically to Ac-CyPA. The secondary antibody is an antibody that binds to primary antibodies or antibody fragments. Secondary antibodies are typically labeled with probes that make them useful for detection, purification, or cell sorting applications. Secondary antibodies may be polyclonal or monoclonal, and are available with specificity for whole Ig molecules or antibody fragments such as the Fc or Fab regions. Specific secondary antibodies are chosen to work in specific laboratory applications. Frequently, any of several secondary antibodies perform adequately in a particular application. Secondary antibodies may be selected according to the source of the primary antibody, the class of the primary antibody {e.g. , IgG or IgM), and the kind of label employed.

[0072] A kit that includes primary and secondary antibodies can be useful in carrying out a variety of biochemical assays including, without limitation, ELISA, Western blot,

immunostaining, immunohistochemistry, and immunocytochemistry.

[0073] A variety of configurations and formats are possible for each type of

immunoassay. The capture antibody (primary antibody), for example, can be attached to a variety of different solid phases to enable the washing away of unreacted assay reagents during the course of the assay. These include: microwells, coated test tubes, coated magnetic particles, wands or sticks, and membranes (nitrocellulose and others). [0074] The capture antibody (primary antibody) can be attached by passive adsorption, covalent coupling, or by using a solid phase pre-coated with a secondary binder such as protein A, protein G, a secondary antibody specific for the primary antibody, avidin, or an antibody specific for a particular ligand (i.e. : biotin, dinitrophenol, fluorescein, and others). In the case of avidin or any of the ligand specific antibodies, it is necessary to covalently attach the ligand to the capture antibody.

[0075] An Ac-CyPA specific antibody can be either directly labeled by covalent coupling or a labeled secondary antibody that is specific for the corresponding primary antibody and can be used without the need to chemically modify the primary antibody. A labeled secondary binder such as avidin or a labeled antibody specific for a particular ligand (i.e., dinitrophenol, fluorescein, and others) can also be employed. In the case of avidin or any of the ligand specific antibodies, it is necessary to covalently attach the corresponding ligand to the primary antibody.

[0076] In another embodiment, a suitable assay system may include a quantity of the capture antibody sufficient to optimize the detection of Ac-CyPA. In addition, the concentration of detector antibody, the particular anti-goat conjugate and its concentration, the formulation of the reagent diluent buffer, the formulation of the non-specific binding (NSB) reagent, and the type of microwell can all be optimized to yield the lowest background and highest signal-to- noise ratio. Furthermore, the reagent configuration (i.e., 10 x concentrates of detector reagent, enzyme conjugate reagent and NSB reagent, with a separate reagent diluent buffer) can be designed to maximize kit stability and shelf life.

[0077] A small amount of bovine gamma globulin can be added to a reagent diluent buffer used to prepare working solutions of detector antibody and enzyme conjugate. As a result, the background signal may be significantly reduced.

[0078] The kit can also include instructions for employing the kit components and the use of any other reagent not included in the kit. In one embodiment, the kit includes instructions for using the antibody or binding fragment thereof that specifically binds Ac-CyPA and necessary buffer solutions for carrying out methods described herein. Instructions may include variations that can be implemented.

[0079] According to other aspects illustrated herein, there is provided a method of detecting Ac-CyPA in a biological sample. This method involves contacting the biological sample with an antibody or binding fragment as illustrated herein having a label bound to the antibody or binding fragment and detecting specific binding that occurs between the biological sample and the antibody or binding fragment to identify Ac-CyPA in the biological sample. [0080] Detection of binding between the biological sample and the antibody or binding fragment is carried out using an assay selected from the group consisting of a Western blot, immunoassay, ELISA assay, flow cytometry, radiography, and immunoscintography.

[0081] In carrying out this method, the antibody or binding fragment thereof may be administered to a subject. Suitable subjects include a mammal, a human, a mouse, a rat, a rabbit, a sheep, a goat, a horse, a cow, a pig, or any other suitable animal. Administering may be carried out orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intravesical instillation, by intracavitary, intravesical instillation, intraocularly, intra-arterially, intralesionally, or by application to mucous membrane.

[0082] According to one embodiment, when administered to a subject, detection of specific binding is carried out using an in vivo detection method, which may include, without limitation, diagnostic imaging, ultrasound, tomography, magnetic resonance, elastography, and radionuclear scanning.

[0083] The detection method may be carried out to diagnose a subject for cardiovascular disease. Alternatively, or in addition, the detection method may be carried out for prognostic analysis of cardiovascular disease in a subject.

[0084] As used herein, cardiovascular disease includes, without limitation, abdominal aortic aneurysm, atherosclerosis, cardiac hypertrophy, vascular remodeling, pulmonary arterial hypertension, systemic hypertension, stroke, vascular dementia, peripheral vascular disease, wound healing, and myocardial infarction.

[0085] According to other aspects illustrated herein, there is provided a method of screening an individual for cardiovascular disease. This method involves providing a biological sample from an individual, contacting the biological sample with an antibody or binding fragment as illustrated herein having a label bound to the antibody or binding fragment, and detecting specific binding that occurs between the biological sample and the antibody or binding fragment to identify Ac-CyPA in the biological sample. The presence of Ac-CyPA in the biological sample indicates the individual has cardiovascular disease.

[0086] According to other aspects illustrated herein, there is provided a method of treating an individual for symptoms associated with cardiovascular disease. This method involves contacting an individual with an antibody or binding fragment according to the present invention under conditions effective for the antibody or binding fragment to specifically bind to Ac-CyPA, where said binding treats the individual for symptoms associated with cardiovascular disease.

[0087] In carrying out the treatment method, it may be desirable to administer to the individual a pharmaceutical composition comprising a carrier and one or more antibodies or binding fragments thereof as illustrated herein. Thus, the present invention also relates to pharmaceutical compositions comprising one or more antibodies or binding fragments thereof as illustrated herein. A suitable pharmaceutical composition may contain two or more antibodies or binding fragments where all antibodies or binding fragments recognize the same epitope.

Alternatively, the pharmaceutical composition may contain an antibody or binding fragment mixture where one or more antibodies or binding fragments recognize more than one epitope. For example, the mixture may contain one or more antibodies that bind specifically to Lys82 of AcK-CyPA in combination with any other antibody that binds to Lysl25 of AcK-CyPA, as described supra. The pharmaceutical composition further contains a pharmaceutically acceptable carrier or other pharmaceutically acceptable components as described infra.

[0088] A pharmaceutical composition containing an antibody as illustrated herein can be administered to a subject having, or at risk of having, cardiovascular disease. Various delivery systems are known and can be used to administer an antibody to an individual for treatment purposes. Methods of administering include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The antibody can be administered, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings {e.g., oral mucosa, rectal and intestinal mucosa, and the like) and can be administered together with other biologically active agents. Administration can be systemic or local.

[0089] A pharmaceutical composition may include one or more pharmaceutical carriers

{e.g., sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like). Water is a common carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, e.g., for injectable solutions. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like. The composition, if desired, can also contain amounts {e.g., minor amounts) of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington 's Pharmaceutical

Sciences by E. W. Martin, the disclosure of which is incorporated herein by reference in its entirety. Such compositions will contain a therapeutically effective amount of the antibody, typically in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulations correspond to the mode of administration.

[0090] Effective doses of the pharmaceutical compositions for the treatment of cardiovascular disease may vary depending upon many different factors, including mode of administration, target site, physiological state of the patient, other medications administered, and whether treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage may be administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and, e.g. , until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

EXAMPLES

[0091] Further illustration of the disclosure is made by reference to the following examples.

Example 1 ~ Acetylation of CyPA is Required for Secretion and Vascular Cell

Activation

Materials and Methods

Materials

[0092] Human Angll and Rho kinase inhibitor Y-27632 were purchased from

Calbiochem. Trichostatin A and Acetyl-CoA were purchased from Sigma- Aldrich.

Recombinant human Cyclophilin A and rabbit anti-Cyclophilin A were purchased from Enzo Life Science. Recombinant p300 HAT domain and anti-GAPDH antibody were from EMD Millipore. 2',7'-dic-hlorodihydrofluorescein diacetate (H₂DCFDA) was from Invitrogen. Protein A- and Protein G-agarose were from Roche. Mouse anti-FlagM2 antibody was from Agilent Technologies. Rabbit anti-acetyl lysine (#9441) and phospho-ER l/2 were from Cell Signaling Technology. Mouse anti-ERK2, goat anti-VCAMl, mouse anti-ICAMl, and mouse anti-CyPA were purchased from Santa Cruz Biotechnology. Cell Isolation and Culture

[0093] Animal experiments were performed using protocols approved by the Institutional

Animal Care and Use Committee at the University of Rochester published by the United States National Institutes of Health. Animals (8-12 weeks old) were anesthetized with single intraperitoneal injection of ketamine (130 mg/kg) and xylazine (8.8 mg/kg). The depth of surgical anesthesia was determined by toe pinch and euthanasia was by exsanguination following perfusion. Aortic smooth muscle cells from rats ("RASMC") or mice ("MASMC") from WT, Ppia-I-, or overexpressed Flag-CyPA in smooth muscle cell were isolated by enzymatic digestion and maintained in Dulbecco's modified Eagle's medium ("DMEM") containing 10% fetal bovine serum ("FBS") as described previously (Satoh et al, "Cyclophilin A Mediates Vascular Remodeling by Promoting Inflammation and Vascular Smooth Muscle Cell

Proliferation," Circulation 117:3088-3098 (2008), the disclosure of which is incorporated herein by reference in its entirety). RASMC at passages 6-12 or MASMC at passage 4-6 at 70% to 80% confluence were growth arrested by incubation in DMEM containing 0.3% FBS for 24 hours and stimulated with Angll for the indicated times. HUVEC were isolated as previously described (Kim et al., "P62 Binding to Protein Kinase C Zeta Regulates Tumor Necrosis Factor Alpha-Induced Apoptotic Pathway in Endothelial Cells," Arterioscler. Thromb. Vase. Biol. 32:2974-2980 (2012), the disclosure of which is incorporated herein by reference in its entirety) and seeded onto 0.2%> gelatin pre-coated dishes maintained in Medium 200 (Cascade Biologic) with low serum growth supplement (LSGS; Invitrogen), 5% FBS, 100 μg/ml streptomycin, and 100 IU/ml penicillin. U937 monocytes were maintained in RPMI-1460 medium containing 10% FBS, 100 ug/ml streptomycin and 100 IU/ml penicillin. HEK293 cell lines were maintained in 10%) FBS, 100 μg/ml streptomycin, and 100 IU/ml penicillin. All cells were cultured at 37°C in a humidified atmosphere of 95% air and 5% C0₂.

Plasmid

[0094] Mutants of CyPA (lysine to arginine) were generated by site directed mutagenesis according to the manufacturer's protocol (QuikChange Site-Directed Mutagenesis; Agilent Technologies). The primers used were: 44R F: 5 ^'- GCA CTG GGG AGA GAG GAT TTG GCT ATA AG - 3 ^' (SEQ ID NO: 15); K44R R: 5 ^" - CTT ATA GCC AAA TCC TCT CTC CCC AGT GC - 3 ^' (SEQ ID NO: 16); K82R F: 5 ^" - CCA TCT ACG GAG AGA GAT TTG AGG ATG AGA AC - 3 ^' (SEQ ID

NO: 17);

K82R R: 5 ^" - GTT CTC ATC CTC AAA TCT CTC TCC GTA GAT GG - 3 ^' (SEQ ID NO: 18); K125R F: 5 ^" - GTG GCT TGG CAG GCA TGT GGT CTT TGG - 3 ^' (SEQ ID NO: 19); and K125R R: 5 ^" - CCA AAG ACC AC A TGC CTG CCA TCC AGC CAC - 3 ^' (SEQ ID NO:20); DNA sequences were verified at the University of Rochester Genomics Research Center.

Construction of Recombinant Lentivirus and VSMC Transduction

[0095] pLV-CMV-IRES-GFP is an HIV-1 based lentiviral expression vector that allows simultaneous expression of CyPA cDNA (and mutants thereof) from the CMV promoter and EGFP by means of an IRES element. Infectious viral particles were generated by co-transfection of the transgene, with plasmids expressing viral gag/pol genes (psPAX2) and VSV-G coat protein (pMD2.G) into HEK293T cells using Fugene6 (Promega). 48 hours post-transfection, viral containing supernatant were collected, filtered through 0.45 μιη cellulose acetate filters, and stored in aliquots at -80°C. For transduction, VSMC were plated at subconfluence in 6 well culture plates and spin-oculated (1500 x g for 1.5 hours at room temperature) with virus in the presence of polybrene (8 μ^ιηΐ). Cells were incubated for 36 hours and the media changed to serum free DMEM 1 hour before Angll stimulation.

Preparation of Conditioned Medium (CM)

[0096] Conditioned medium ("CM") was prepared as described previously (Suzuki et al,

"Cyclophilin A is Secreted by a Vesicular Pathway in Vascular Smooth Muscle Cells," Circ. Res. 98:811-817 (2006), the disclosure of which is incorporated herein by reference in its entirety). Briefly, VSMC were growth arrested with DMEM containing 0.3% FBS for 24 hours and the medium was changed to serum free DMEM 1 hour before the experiment. The culture medium was collected and concentrated 100-fold using Amicon Ultra Centrifuge filter-3K. Measurement of Reactive Oxygen Species (ROS) by Flow Cytometry

[0097] Growth arrested VSMC were collected by trypsinization and incubated with 2 mM 2',7'-dichiorodihydrofluorescein diacetate (H₂DCFDA, Invitrogen) in 3% FBS/PBS at 37°C for 30 minutes. Cells were centrifuged, washed, and incubated with conditioned medium or rhCyPA at 37°C. After, 4 hours of incubation, cells were centrifuged, washed, and resuspended in 3% FBS/PBS, followed by flow cytometry measurement (Accuri® C6). The data were analyzed using Flow Jo software (TreeStar Inc.).

Gelatin Zymography for MMP2 Activity

[0098] Gelatin zymography for the detection of MMP2 activity in conditioned medium was performed as follows. Samples were mixed in loading buffer (125 mM Tris pH6.8, 5% SDS, 20% glycerol, 0.03% bromophenol blue) and incubated at room temperature for 5 minutes. Samples were resolved through 8% non-denaturing PAGE gels containing 0.1% gelatin in Tris/Glycine/SDS running buffer. Following electrophoresis, gels were immersed in renaturation buffer (2.5% Triton X-100 in 50 mM Tris pH 7.5) for 1 hour at room temperature. Gels were then immersed in digestion buffer (50 m Tris pH7.5, 4 mM CaCl₂, 200 mM NaCl, 0.02% Brij35) for 48 hours at 37°C. MMP2 activation was detected as clear areas on a blue background by staining in 0.1% Coomassie Blue R250 (in 40% methanol/10% acetic acid) and destaining in 40% methanol/10% acetic acid).

In vitro Acetylation Assay

[0099] Reactions (20 μΐ) containing rhCyPA 50 iiM, 1.2 mM acetyl-CoA, and 1 μg p300 protein in HAT buffer (50 mM Tris HO, pH 7.5, 150 mM NaCl, 1 mM PMSF, 1 mM DTT, 10 mM sodium Butyrate, and 10% glycerol) were incubated at 30°C for 45 minutes and reaction was stopped by incubation on ice.

Immunoprecipitation and Western Blotting

[00100] VSMC were lysed in NP-40 buffer (1 % P-40, 50 mM Tris HCl; pH 7.5, 150 mM NaCl, 10 mM sodium fluoride, 1 mM PMSF, 2 mM EDTA, 0.1% SDS, 0.5% sodium deoxycholate, and 1 : 1000 protease inhibitor cocktail (Sigma)) and protein concentrations were determined by Bradford protein assay (Bio-Rad). Lysates containing equal amounts of soluble proteins were incubated with antibody overnight at 4°C. Antibody complexes were collected by incubation with protein A agarose for 2 hours at 4°C. Precipitates were washed 3 times in lysis buffer and then resuspended in SDS-PAGE sample buffer. Samples were separated by SDS- PAGE and analyzed by Western blot. Protein reactivity was detected using ECL (GE Healthcare).

Endothelial to Monocytes Adhesion Assay

[00101] HUVEC cultured in 35 mm dish were growth arrested for 2 hours with serum free medium and stimulated with conditioned medium or 50 nM rhCyPA for 6 hours. Then, medium was removed and U937 monocytes (1 x 10⁴ cells / 2 ml RPMI) were added and incubated for 30 minutes at 37°C. Unbound cells were removed by washing 3 times with PBS. Adherent cells were counted in 5 randomly selected optical fields in each well. Phase-contrast

microphotographs of cells were obtained using an inverted fluorescent microscope (1X50, Olympus) with 20x lens.

Statistical Analysis

[00102] Data are means ± SEM of at least three independent experiments. The significance between samples was determined by Student's t-test for two group comparisons or ANOVA for more than two groups using Graphpad Prism software. p<0.05 was considered statistically significant.

Results

Angiotensin II Stimulates CyPA Acetylation in VSMC

[00103] To investigate Angll induced acetylation of proteins in VSMC, RASMC were treated with Angll and acetylation was analyzed using an acetyl lysine antibody (AcK), which detects general lysine acetylation. It was observed that Angll increased acetylation of numerous proteins in RASMC. The time course of acetylation for different proteins varied, as

demonstrated in FIGs. 1A and IB. In particular, a protein of molecular weight 17 kDa is the most heavily acetylated and exhibited a bi-phasic pattern with peak lysine acetylation at 1-2 hours and 24 hours. Proteins of mass 20-25 kDa lysine acetylation peaked between 4-8 hours, and 25-37 kDa molecular weight exhibited a bi-phasic pattern with peak lysine acetylation at 1-2 hours and 24 hours (FIG. IB). Given that CyPA has a molecular weight of 17 kDa, and that CyPA molecular weight on Western blot (WB) was coincident with the acetylated proteins, it is very plausible that CyPA is one of the acetylated proteins.

[00104] To prove that CyPA is acetylated, total cell lysates from Angll or HDAC inhibitor

TrichostatinA (TSA)-stimulated RASMC were immunoprecipitated with a CyPA antibody and probed for AcK (FIGs. 1C and ID). Angll-induced CyPA acetylation occurred in a time- dependent manner with a peak at 16-24 hours. The time course of the acetylation of CyPA (FIG. ID) differed from that observed in the acetylation of TCL, suggesting that the other proteins of MW 17 kDa are also included in the highly acetylated band. Based on this time course, CyPA acetylation at 24 hours was studied, which was the peak. Similarly, it was observed that TSA induced CyPA acetylation in VSMC. Similar to the RASMC, Angll increased acetylation of exogenous Flag-CyPA in MASMC over-expressing CyPA (MASMC-FlagCyPA) with a peak at 16-24 hours (FIGs. 7A and 7B) further suggesting that CyPA is acetylated in VSMC in response to Angll. In addition, when wild type (WT -MASMC) and CyPA (gene: peptidyl-prolyl isomerase) knockout mouse aortic smooth muscle cells (Ppia-/- MASMC) were compared, Angll-induced acetylation of the 17 kDa protein band detected in WT -MASMC (arrow) was not observed in Ppia-/- MASMC, further providing evidence that CyPA is acetylated in VSMC (FIGs. 7C and 7D). To understand the role of ROS in CyPA acetylation, the ROS scavengers Tiron and N-acetylcysteine were used and then acetylation of Cyclophilin A was assessed. As shown in FIGs. IE and IF, ROS inhibition decreased Angll-induced CyPA acetylation in VSMC.

Lysine Residues K82 and K125 Regulate Angll-induced CyPA

Acetylation

[00105] To determine lysine residues important for Angll-induced acetylation, UniProt and PHOSIDA were used to predict potential acetylated residues. There are fourteen lysine residues in CyPA, of which 5 were potential acetylation targets (K28, K44, K82, K125, and K131). Of these 5, K44 is conserved in all species (Masse et al., "Cloning and Characterisation of the Immunophilin X-Cypa in Xenopus laevis," Gene Expr. Patterns 5:51-60 (2004), the disclosure of which is incorporated herein by reference in its entirety), K82 is located on the surface of CyPA and is involved in calcineurin and CsA-cyclophilin A complex binding (Ivery, "A Proposed Molecular Model for the Interaction of Calcineurin with the Cyclosporin A- Cyclophilin A Complex," Bioorg. Med. Chem. 7: 1389-1402 (1999); Mikol et al, "X-Ray Structure of a Cyclophilin B/Cyclosporin Complex: Comparison with Cyclophilin A and

Delineation of its Calcineurin-Binding Domain," Proc. Natl. Acad. Sci. U.S.A. 91 :5183-5186 (1994), the disclosures of which are incorporated herein by reference in their entirety) and K125, also located at the surface, is involved in CsA binding and PPlase regulation (Lammers et al., "Acetylation Regulates Cyclophilin A Catalysis, Immunosuppression and HIV Isomerization," Nat. Chem. Biol. 6:331-337 (2010); Etzkorn et al, "Cyclophilin Residues that Affect

Noncompetitive Inhibition of the Protein Serine Phosphatase Activity of Calcineurin by the Cyclophilin. Cyclosporin A Complex," Biochemistry 33:2380-2388 (1994), the disclosures of which are incorporated herein by reference in their entirety). Therefore, K44, K82, and K125 were mutated to arginine (K44R, K82R, and K125R or K82/125R double mutant) and their ability to be acetylated in response to Angll was tested. Lentiviral transduction was used to express the mutants in MASMC, because plasmid transfection efficiency in MASMC was too low. The HIV-based lentiviral expression vector pLV-CMV-IRES-GFP allows simultaneous expression of Flag-CyPA cDNA (and mutants thereof) from the CMV promoter and EGFP from an IRES element. GFP fluorescence measured by flow cytometry indicated 80±0.3% and 86±0.7% transduction efficiency in vector and WT Flag-CyPA transduced cells, respectively (FIG. 2A). To determine the acetylation of WT and mutant Flag-CyPA, Ppia-/- MASMC were infected with virus followed by Angll stimulation. Immunoprecipitation results showed that WT-CyPA was acetylated strongly in response to Angll (FIG. 2B, upper panel, and FIG. 2C). The K44R mutant was acetylated in response to Angll to similar levels as WT-CyPA while acetylation of the K82R and K125R mutants was decreased by approximately 50% compared to wild type ("WT"). Angll-induced acetylation of the K82/125R double mutant was completely inhibited compared with WT-CyPA. Importantly, GFP expression levels were the same in all viral infections and expression levels of WT and all CyPA mutants were equivalent, as demonstrated by Flag reactivity in total cell lysates, suggesting the mutations do not affect protein stability (FIG. 2B, lower panel). Thus, K82 and K125 are important residues for Angll- induced CyPA acetylation in VSMC.

Angll Induces Acetylated-CyPA Secretion in VSMC

[00106] CyPA is a secreted protein involved in oxidative stress conditions (Satoh et al,

"Circulating Smooth Muscle Progenitor Cells: Novel Players in Plaque Stability," Cardiovasc. Res. 77:445-447 (2008), the disclosure of which is incorporated herein by reference in its entirety). To determine whether AcK-CyPA can be secreted, conditioned medium from Angll- treated RASMC was analyzed by Western blot. Angll induced secretion of several acetylated proteins with molecular masses between 17 kDa and 50 kDa in a time-dependent manner (FIGs. 3A and 3B). When the acetylation blot was reprobed with anti-CyPA antibody, the

immunoreactivity was coincident with the 17 kDa acetylated protein (FIG. 3 A, lower panel), suggesting that acetylated-CyPA (AcK-CyPA) was one of the proteins secreted into the conditioned medium in a time-dependent manner with a peak at 16 and 24 hours (FIG. 3C). There was a strong similarity in the time course of Angll-stimulated extracellular CyPA and extracellular AcK-CyPA (FIGs. 3A-C), as well as intracellular CyPA-AcK (FIGs. 1C and ID) at 16 and 24 hours. Secretion of AcK-CyPA was confirmed by immunoprecipitation and immunoblot analysis. Angll induced secretion of AcK-CyPA and total CyPA in a time- dependent manner with a peak at 24 hours (FIGs. 3D and 3E). Moreover, ROS scavengers inhibited AcK-CyPA secretion, suggesting that Angll-induced AcK-CyPA secretion is ROS dependent (FIGs. 3F-3H). It was previously shown that secretion of CyPA was dependent on Rho activity (Suzuki et al., "Cyclophilin A is Secreted by a Vesicular Pathway in Vascular Smooth Muscle Cells," Circ. Res. 98:811-817 (2006), the disclosure of which is incorporated herein by reference in its entirety). To determine whether secretion of AcK-CyPA depends on the Rho-pathway, RASMC were pretreated with the Rho kinase inhibitor, Y27632, followed by stimulation with Angll for 24 hours. AcK-CyPA secretion was dramatically inhibited while intracellular CyPA expression levels were unaffected by Y27632 (FIG. 8D).

Acetylation is Required for CyPA Secretion in VSMC

[00107] To further understand the role of CyPA acetylation on its secretion, AcK-CyPA was measured in conditioned medium from lentivirally transduced Ppia-/- MASMC (FIG. 4A). An equal volume of conditioned medium was used from each sample and immunoprecipitation analysis was performed using anti-Flag antibody. These data demonstrated that Angll induced secretion of acetylated WT-FlagCyPA. Secretion of K82R and K125R CyPA mutants was reduced compared to WT. Secretion of the CyPA K82/125R double mutant was barely detectable. In contrast, secretion of the K44R mutant, which showed a high level of intracellular AcK, did not differ from secretion of WT-FlagCyPA. Similar results were shown for the total level of secreted Flag-CyPA in the conditioned medium. The total intracellular level of CyPA and mutants, as measured by FLAG reactivity in the total cell lysate (TCL), was unaffected by acetylation status (FIG. 4A, lower panel) suggesting that acetylation of CyPA is required for its secretion but not its stability. It is evident that by determining the ratio of secreted Flag-CyPA to intracellular FLAG-CyPA (FIG. 4B) the acetylation status of CyPA affects its secretion.

Acetylation Promotes Extracellular CyPA Induced VSMC Activation

[00108] To assess whether AcK-CyPA can affect VSMC activation, RASMC were stimulated with CM prepared from WT and mutants transduced Ppia-/- MASMC. An equal amount of CyPA (the amount of CyPA was determined from quantitation of Western blot reactivity from FIG. 4A) was used in each assay and volumes were normalized with conditioned medium from Angll-treated Ppia-/- MASMC. The Western blot in FIG. 9 shows total CyPA from a series of normalizations of conditioned medium from the mutants relative to the WT CyPA. Each aliquot contains an equivalent amount of CyPA, except for the double mutant in which undetectable amounts of CyPA were secreted into the original conditioned medium. In so doing, differences between mutants can be interpreted as being the result of their altered acetylation status and not simply as the effect of different amounts of CyPA added in each assay. ER 1/2 activation measured by pER l/2 increased significantly after stimulation with conditioned medium from WT-CyPA and the K44R mutant (FIG. 5A and 5B). However, conditioned medium from the K82R and K125R mutants displayed decreased pERKl/2 activation while the double mutant, K82/125R, exhibited dramatically reduced ER l/2 activation. To support the role of AcK-CyPA in VSMC activation, recombinant CyPA

(rhCyPA) was acetylated in vitro using recombinant histone acetyltransferase p300 and acetyl - CoA. Western blot analysis demonstrated the presence of acetylated-CyPA (AcK-rhCyPA) (FIG. 10). RASMC treated with AcK-rhCyPA exhibited greater pERKl/2 activation compared with native rhCyPA (FIGs. 5C and 5D).

[00109] Further, intracellular ROS production in RASMC was increased following stimulation with conditioned medium from WT-CyPA expressing cells compared to conditioned medium from vector expressing cells. The conditioned medium prepared from the K82/125R expressing cells did not significantly stimulate ROS production compared to vector expressing cells (FIG. 5E).

[00110] It was previously demonstrated that Angll-induced MMP2 activation was inhibited in Ppia-I- MASMC (Satoh et al., "Cyclophilin A Enhances Vascular Oxidative Stress and the Development of Angiotensin II-Induced Aortic Aneurysms," Nat. Med. 15:649-656 (2009), the disclosure of which is incorporated herein by reference in its entirety). To understand if AcK-CyPA is important in regulating MMP2 activity, gelatin zymography of conditioned medium prepared from Angll-stimulated WT and mutant CyPA expressing cells was performed. As described above, an approximately equal amount of CyPA was used in each assay, and volumes were normalized with conditioned medium from Angll treated Ppia-I- cells. Zymography results indicated that pro-MMP9 activity was unaffected by CyPA or its mutants. However, WT-CyPA increased MMP2 activity relative to the vector control (FIGs. 5F and 5G). Expression of the K82R and K125R mutants resulted in reduced MMP2 activation while the K82/125R double mutant significantly inhibited MMP2 activity over WT-CyPA. Together, these data support the hypothesis that acetylation of CyPA increases its potential for VSMC activation. Acetylated-CyPA Increases Adhesion Molecule Expression and Monocyte Adhesion to Endothelial Cells

[00111] Extracellular CyPA can cause endothelial cell (EC) dysfunction by increasing adhesion molecules (VCAM-1 and ICAM-1) expression (Jin et al., "Cyclophilin A is a

Proinflammatory Cytokine that Activates Endothelial Cells," Arterioscler. Thromb. Vase. Biol. 24: 1186-1191 (2004), the disclosure of which is incorporated herein by reference in its entirety). To understand the role of AcK-CyPA in EC dysfunction, VCAM-1 and ICAM-1 expression were measured from EC treated with conditioned medium prepared from Ppia-I- MASMC expressing WT or K82/125R CyPA or rhCyPA (native and acetylated form). VCAM-1 and ICAM-1 expression were significantly (p<0.01) decreased in EC treated with conditioned medium from K82/125R expressing cells (FIGs. 6A-6C). In contrast, AcK-rhCyPA increased VCAM-1 and ICAM-1 expressions compared to rhCyPA (FIGs. 6D-6F). However, total CyPA expression was not changed in any of the experiments (see lower panel of each immunoblot figure).

[00112] Additionally, monocyte adhesion in response to conditioned medium or rhCyPA was measured. EC treated with K82/125R-CM exhibited less monocyte adhesion compared to cells treated with WT-conditioned medium (FIGs. 6G and 6H). In contrast, AcK-rhCyPA treated EC showed augmented monocyte adhesion compared to rhCyPA (FIGs. 61 and 6J).

Discussion

[00113] This study shows that acetylation of CyPA is required for its secretion, and acetylated extracellular CyPA is functionally more active than the non-acetylated form. Site directed mutagenesis identified K82 and K125 as the predominant CyPA residues acetylated in response to Angll and required for CyPA secretion. AcK-CyPA stimulated significantly greater activation of ER l/2 and MMP2, as well as ROS production in VSMC than non AcK-CyPA as shown by two methods: (i) use of conditioned medium containing secreted CyPA comparing WT-CyPA versus K82/125R-CyPA and (ii) acetylated recombinant CyPA. Moreover, AcK- CyPA increased adhesion molecules expression (VCAM-1 and ICAM-1) in EC, which promoted monocyte adhesion.

[00114] Lysine acetylation plays roles in various cardiovascular diseases (Bush et al,

"Protein Acetylation in the Cardiorenal Axis: The Promise of Histone Deacetylase Inhibitors," Circ. Res. 106:272-284 (2010); Lu et al., "The Emerging Characterization of Lysine Residue Deacetylation on the Modulation of Mitochondrial Function and Cardiovascular Biology," Circ. Res. 105:830-841 (2009); the disclosures of which are incorporated herein by reference in their entirety). Previous research demonstrates that lysine acetylation of CyPA at K125 was important for its functions of immunity and viral infection (Lammers et al., "Acetylation Regulates Cyclophilin A Catalysis, Immunosuppression and HIV Isomerization," Nat. Chem. Biol. 6:331- 337 (2010), the disclosure of which is incorporated herein by reference in its entirety). Because it is an important regulator in many cardiovascular diseases (Satoh et al., "Oxidative Stress and Vascular Smooth Muscle Cell Growth: A Mechanistic Linkage by Cyclophilin A," Antiox.

Redox. Signal. 12:675-682 (2010), the disclosure of which is incorporated herein by reference in its entirety), a global screening of lysine acetylated proteins using Angll as the agonist was performed. Moreover, Angll plays multiple roles in VSMC functions in which increased ROS production is one of the important mechanisms for its signaling regulation (Clempus et al., "Reactive Oxygen Species Signaling in Vascular Smooth Muscle Cells," Cardiovasc. Res.

71 :216-225 (2006), the disclosure of which is incorporated herein by reference in its entirety). It was demonstrated that Angll increased acetylation of many VSMC proteins in a biphasic manner with peaks at early and late time points following stimulation. Further analysis of these bands indicated that CyPA was one of the highly acetylated proteins.

[00115] Acetylation is a reversible process in which alterations in lysine acetyltransferase

("HAT") and lysine deacetylase ("HDAC") activity mediates cellular protein acetylation levels

(FIG. 1 IB). (The more established terms HAT and HDAC are used, but there is an increasing realization that the more recent terminology of KAT/KDAC, which reflects

acetylation/deacetylation of non-histone proteins would also be appropriate here.) ROS regulates

HAT/HDAC balance by either decreasing (Ito et al., "Oxidative Stress Reduces Histone

Deacetylase 2 Activity and Enhances IL-8 Gene Expression: Role of Tyrosine Nitration,"

Biochem. Biophys. Res. Commun. 315:240-245 (2004), the disclosure of which is incorporated herein by reference in its entirety) or increasing (Pang et al., "GIT1 Mediates HDAC5 Activation by Angiotensin II in Vascular Smooth Muscle Cells," Arterioscler. Thromb. Vase. Biol. 28:892-

898 (2008), the disclosure of which is incorporated herein by reference in its entirety) endogenous HDAC activity. Angll stimulates a rapid and sustained increase in ROS generated by VSMC (Griendling et al, "Angiotensin II Stimulates NADH and NADPH Oxidase Activation in Cultured Vascular Smooth Muscle Cells," Circ. Res. 74: 1141-1148 (1994), the disclosure of which is incorporated herein by reference in its entirety), but also directly increases HAT {e.g., p300/CBP) activation in VSMCs (Sahar et al, "Cooperation of SRC-1 and p300 with NF- kappaB and CREB in Angiotensin II-Induced IL-6 Expression in Vascular Smooth Muscle

Cells," Arterioscler. Thromb. Vase. Biol. 27: 1528-1534 (2007), the disclosure of which is incorporated herein by reference in its entirety). The data using Angll mirrored that in which

Trichostatin A was used, a Class I and Class II HDAC inhibitor suggesting that one of these

HDACs is responsible for maintaining the appropriate level of CyPA acetylation. Large-scale proteomic analyses of the cellular acetylome suggest the presence of multiple deacetylases with both nuclear and cytoplasmic activities (Lundby et al., "Proteomic Analysis of Lysine

Acetylation Sites in Rat Tissues Reveals Organ Specificity and Subcellular Patterns," Cell Rep. 2:419-431 (2012), the disclosure of which is incorporated herein by reference in its entirety).

[00116] Many studies have reported that CyPA is a major target of redox regulation

(Satoh et al, "Vascular-Derived Reactive Oxygen Species for Homeostasis and Diseases," Nitric Oxide: Biology and Chemistry / Official Journal of the Nitric Oxide Society (2011), the disclosure of which is incorporated herein by reference in its entirety). CyPA is acetylated in spinal cord tissue from amyotrophic lateral sclerosis G93A SODl mice in which oxidative stress is highly induced (Massignan et al., "Proteomic Analysis of Spinal Cord of Presymptomatic Amyotrophic Lateral Sclerosis g93a Sodl Mouse," Biochem. Biophys. Res. Commun. 353:719- 725 (2007), the disclosure of which is incorporated herein by reference in its entirety). It was shown that CyPA acetylation was increased in a time-dependent manner with a peak at later time points. Since Angll produces a sustained increase in ROS in VSMC, it was concluded that the time dependent increase in CyPA acetylation was largely due to ongoing ROS production.

Inhibition of ROS decreased CyPA acetylation, providing the evidence that ROS is involved in CyPA acetylation regulation.

[00117] Uniprot and PHOSIDA predict 5 predominant lysine residues in CyPA that can be acetylated. Proteomic analysis revealed that CyPA can be acetylated at many lysine residues, although K82 and K125 are the primary targets in immune cells and human cancer cells

(Lammers et al., "Acetylation Regulates Cyclophilin A Catalysis, Immunosuppression and HIV

Isomerization," Nat. Chem. Biol. 6:331-337 (2010); Kim et al., "Substrate and Functional

Diversity of Lysine Acetylation Revealed by a Proteomics Survey," Mol. Cell. 23:607-618

(2006); Masse et al., "Cloning and Characterisation of the Immunophilin X-Cypa m Xenopus laevis," Gene Expr. Patterns 5:51-60 (2004); Etzkorn et al, "Cyclophilin Residues that Affect

Noncompetitive Inhibition of the Protein Serine Phosphatase Activity of Calcineurin by the

Cyclophilin. Cyclosporin A Complex," Biochemistry 33:2380-2388 (1994); Lundby et al,

"Proteomic Analysis of Lysine Acetylation Sites in Rat Tissues Reveals Organ Specificity and

Subcellular Patterns," Cell Rep. 2:419-431 (2012); the disclosures of which are incorporated herein by reference in their entirety). These are located on solvent accessible surfaces of CyPA

(Lammers et al., "Acetylation Regulates Cyclophilin A Catalysis, Immunosuppression and HIV

Isomerization," Nat. Chem. Biol. 6:331-337 (2010); Mikol et al, "X-Ray Structure of a

Cyclophilin B/Cyclosporin Complex: Comparison with Cyclophilin A and Delineation of its

Calcineurin-Binding Domain," Proc. Natl. Acad. Sci. U.S.A. 91 :5183-5186 (1994); the disclosures of which are incorporated herein by reference in their entirety). Mutagenesis studies demonstrate that K82 and K125 are the target lysine residues involved in Angll-induced CyPA acetylation in VSMC. Importantly, it is also shown that total CyPA expression was not changed by mutation of K82 or K 125, suggesting that acetylation does not affect the steady state level of intracellular CyPA. Given the location of K82 and K125 on the surface of CyPA, these residues could be potential pharmacological targets.

[00118] CyPA is secreted in many pathological conditions such as oxidative stress, inflammation, cardiovascular disease (Jin et al., "Cyclophilin A is a Secreted Growth Factor Induced by Oxidative Stress," Circ. Res. 87:789-796 (2000); Nishioku et al, "Cyclophilin A Secreted from Fibroblast-Like Synoviocytes is Involved in the Induction of cdl47 Expression in Macrophages of Mice with Collagen-Induced Arthritis," J. Inflamm. (Lond.) 9:44 (2012); the disclosures of which are incorporated herein by reference in their entirety). In particular, it is highly secreted from VSMC in response to Angll and oxidative stress (Satoh et al, "Cyclophilin A Enhances Vascular Oxidative Stress and the Development of Angiotensin II-Induced Aortic Aneurysms," Nat. Med. 15:649-656 (2009), the disclosure of which is incorporated herein by reference in its entirety). CyPA is among the most abundant intracellular proteins, including 0.1- 0.6% of the total cytosolic proteins (Ryffel et al., "Distribution of the Cyclosporine Binding Protein Cyclophilin in Human Tissues," Immunology 72:399-404 (1991), the disclosure of which is incorporated herein by reference in its entirety). While it is likely that the majority of cytosolic CyPA remains in the cell, post-translational modification of CyPA, such as acetylation, can promote its secretion. For example, secretion of some acetylated proteins, such as hsp90a or sterol, is controlled by acetylation/deacetylation cycle (Tiwari et al., "An

Acetylation/Deacetylation Cycle Controls the Export of Sterols and Steroids from S. cerevisiae " EMBO J. 26:5109-5119 (2007); Yang et al, "Role of Acetylation and Extracellular Location of Heat Shock Protein 90alpha in Tumor Cell Invasion," Cancer Res. 68:4833-4842 (2008); the disclosures of which are incorporated herein by reference in their entirety). The results herein demonstrate that Angll induced secretion of AcK-CyPA as evident by immunoprecipitation of CyPA from conditioned medium following Angll stimulation of VSMC. Moreover, ROS inhibition attenuated intracellular CyPA acetylation resulting in decreased AcK-CyPA secretion, further suggesting that acetylation of CyPA controls its secretion. Furthermore, it was shown that AcK-CyPA secretion was mediated via a Rho-dependent pathway, which was previously described as the mechanism for CyPA secretion (Suzuki et al, "Cyclophilin A is Secreted by a Vesicular Pathway in Vascular Smooth Muscle Cells," Circ. Res. 98:811-817 (2006), the disclosure of which is incorporated herein by reference in its entirety).

[00119] Increasing evidence demonstrates that extracellular CyPA is an important agonist for many cell types. The present study shows that extracellular AcK-CyPA is more functionally active than native CyPA. Specifically, it was shown that AcK-CyPA was secreted in response to ROS and its acetylation was integrally involved in activating ER 1/2 and ROS formation. In addition, synthetic acetylated rhCyPA dramatically increased ERK1/2 activation as well as ROS production. The mechanism by which extracellular CyPA activates cell-signaling cascades is still unclear, although recent publications suggest at least three mechanisms. First there may be increased binding and/or activation of the extracellular CyPA receptor, since CyPA acetylation alters its surface electrostatic charges (Lammers et al., "Acetylation Regulates Cyclophilin A Catalysis, Immunosuppression and HIV Isomerization," Nat. Chem. Biol. 6:331-337 (2010), the disclosure of which is incorporated herein by reference in its entirety). As previous studies have shown that CyPA binds to CD 147 (EMMPRIN), and regulates downstream signaling in immune cells (Yurchenko et al, "Cyclophilin-cdl47 Interactions: A New Target for Anti-Inflammatory Therapeutics," Clin. Exp. Immunol. 160(3):305-317 (2010), the disclosure of which is incorporated herein by reference in its entirety), it will be important to determine the effect of acetylation on CyPA binding to EMMPRIN. Second, a recent paper demonstrated that acetylation markedly inhibited CyPA catalysis of cis to trans isomerization, suggesting a plausible mechanism for altered biological activity (Lammers et al., "Acetylation Regulates Cyclophilin A Catalysis, Immunosuppression and HIV Isomerization," Nat. Chem. Biol. 6:331- 337 (2010), the disclosure of which is incorporated herein by reference in its entirety). The loss of enzyme activity might stabilize interactions of acetylated CyPA with receptors or other signal mediators. Third, because AcK-CyPA is more stable in acidic conditions (Lammers et al., "Acetylation Regulates Cyclophilin A Catalysis, Immunosuppression and HIV Isomerization," Nat. Chem. Biol. 6:331-337 (2010), the disclosure of which is incorporated herein by reference in its entirety), the microenvironment of cell culture and pathologic conditions may make AcK- CyPA a more potent ligand.

[00120] A previous study reported that CyPA regulates Angll-induced MMP2 activation in VSMC and inflammatory cell accumulation (Satoh et al., "Cyclophilin A Enhances Vascular

Oxidative Stress and the Development of Angiotensin Il-Induced Aortic Aneurysms," Nat. Med.

15:649-656 (2009), the disclosure of which is incorporated herein by reference in its entirety).

MMP2 is secreted as proMMP2, which interacts with the cell surface receptor (MT1-MMP and

TIMP2 complex), and subsequently undergoes cleavage and results in its activation (Haas et al,

"Extracellular Matrix -Driven Matrix Metalloproteinase Production in Endothelial Cells:

Implications for Angiogenesis," Trends Cardiovasc. Med. 9:70-77 (1999), the disclosure of which is incorporated herein by reference in its entirety). In this study, it is demonstrated that the acetylated form of CyPA is integral to promoting MMP2 activity. When approximately the same amount of CyPA from WT-conditioned medium was used versus K82R-conditioned medium or K125R-conditioned medium, WT-conditioned medium showed significant MMP2 activation, whereas K82R, K125R, and double mutant showed activation to a lesser extent. However there was no observation of a dramatic difference in pro-MMP2 or pro-MMP9 level in CyPA transduced cells. It is possible that extracellular AcK-CyPA has increased affinity to bind with proMMP2, and serves as a chaperone for interaction with the cell surface receptor for MMP2.

[00121] Increased adhesion molecule expression in EC is one of the mechanisms involved in inflammatory conditions. CyPA has a proinflammatory effect on EC by inducing mitogen- activated protein kinases and adhesion molecules expression (Jin et al., "Cyclophilin A is a Proinflammatory Cytokine that Activates Endothelial Cells," Arterioscler. Thromb. Vase. Biol. 24: 1186-1191 (2004), the disclosure of which is incorporated herein by reference in its entirety). Here, it was demonstrated that extracellular AcK-CyPA enhances EC adhesion molecules (VCAM-1 and ICAM-1) expression, which promotes adhesion of inflammatory cells. It is possible that AcK-CyPA has more affinity to bind with its receptor in EC to regulate adhesion molecules expression.

[00122] In summary, it was demonstrated that lysine residues K82 and K125 are targets for Angll-induced CyPA acetylation. Acetylation of CyPA is required for its secretion and secreted AcK-CyPA plays an important role in its signaling by regulating VSMC and EC activation (FIG. 11 A). This study provides rationale for designing a more targeted approach to cardiovascular diseases treatment.

Example 2 - Generation of Antibodies Specific to Acetylated CyPA

[00123] Acetylated and non-modified peptides corresponding to the sequence of

Cyclophilin A surrounding K82 and K125 were synthesized by standard chemistries using a commercial company (21^st Century Biochemicals). Each modified peptide was synthesized with a hydrophobic spacer arm (Ahx-; aminohexanoic acid) at either its amino- or carboxy- terminal residue to permit conjugation to an immune carrier (KLH; keyhole limpet hemocyanin). The sequence of the modified peptides is as follows:

CypA K125 NT: C-Ahx-AKTEWLDG[K-ac]HVVFGKV(SEQ ID NO:9)-amide; CypA K125 CT: Acetyl-TEWLDG[K-ac]HVVFGKV(SEQ ID NO: 10)-Ahx-C-amide; CypA K82 NT: C-Ahx-SIYGE[K-ac]FEDENFI(SEQ ID NO: l l)-amide; and CypA K82 CT: Acetyl-SIYGE[K-ac]FEDENFI(SEQ ID NO: 12)-Ahx-C-amide. [00124] The sequence of the non-modified peptides (for affinity purification protocols) are as follows:

CyPA K125 NP: C-Ahx-AKTEWLDGKHVVFGKV(SEQ ID NO: 13)-amide and CyPA K82 NP: C-Ahx-SIYGEKFEDENFI(SEQ ID NO: 14)-amide.

[00125] Sequence of peptides was verified by Collision-induced dissociation and Tandem

Mass Spec (CID MS/MS).

[00126] HPLC purified peptides, conjugated to KLH, were injected into rabbits and bleeds were acquired at standard intervals. Rabbits were initially immunized with 0.4 mg acetylated peptide in Complete Freunds Adjuvant (CFA). At days 14, 28, 42, 62, 83, 114, and 152, rabbits were reinjected (boosted) with a further 0.2 mg acetylated peptide per rabbit in Incomplete Freunds adjuvant. Serum was collected at days 38, 52, 72, 124, 131, 159, 166, and 172 for Western blot assay of specific reactivity to acetylated CypA. Bleeds 5+6 (days 124 and 131) and bleeds 7-9 (days 159, 166, 172) were pooled and affinity purified. Complete exsanguination occurred on day 187.

Example 3 - Validation of Monospecific, Polyclonal Antibodies Against AcK-CyPA

[00127] FIGs. 12A-12B demonstrate that AcK-CyPA K82 antibody is specific for acetylated CypA. In FIG. 12A, affinity purified (AcK peptide followed by non-AcK peptide to remove non-acetyl-specific Abs) bleeds 5+6 and bleeds 7 through 9 were titrated against TrichlorostatinA (an HDAC inhibitor that causes an increase in acetylation of CyPA) treated RASMC by Western blot. Total serum from bleed 8 (143 B8 crude) is a control to show effectiveness of affinity purification strategy. a-CyPA (ENZO) is a control lane probed with a commercially available, validated (not AcK specific) CypA antibody. For each lane the designation is Ack-CyPA Ab concentration (1 or 0.5 μg/ml). The second designation (1/10 K or 1/25 K) is the dilution of the secondary goat anti rabbit-HRP secondary antibody. In FIG. 12B, upper panel, affinity purified bleeds 5+6 and bleeds 7 through 9 were reacted against TSA treated (AcK-CypA) or control treated (-) RASMC extracts by Western blot to determine the specificity of AcK-K82 Ab for Ack-CyPA. Ack-K82 detected Ack-CyPA in only the TSA treated extracts. a-CyPA (ENZO) is a control lane probed with a commercially available, validated (not AcK specific) CyPA antibody. The reactivity with proteins of greater molecular mass likely represents the large isoform of cyclophilin A, the multi-domain enzyme cyclophilin- 40. In FIG. 12B, lower panel, a Western blot stripped and reprobed with CyPA (ENZO) antibody demonstrates equal CyPA mass in each lane. These data indicate that the antibody has specificity for the detection of acetylated CyPA by Western blot. [00128] In FIG 13 A, acetylated CypA is detected in endothelial and smooth muscle cells.

Equal amounts of total cell lysates from the indicated cell lines were analyzed by Western blot for total CyPA and Acetylated CyPA (AcK-CyPA). PAC1— Rat pulmonary artery smooth muscle (SM) cell; RASMC-Rat aortic SM; RPMEC-Rat pulmonary endothelial cell. FIG. 13B is a graph showing quantitation of relative abundance of AcK-CyPA to total CyPA.

[00129] FIG. 14 illustrates that acetylated CypA is secreted from endothelial cells following hypoxia. RPMEC were subject to 6 or 24 hours of hypoxia (1% 0₂) or normoxia. Total cell lysates and culture supematants were Western blotted for total (CyPA) and acetylated (Ack) CyPA.

[00130] FIG. 15 shows acetylated CyPA in lungs of a rat model of Pulmonary Artery

Hypertension. Lung sections from healthy rats and those treated with monocrotaline to induce pulmonary artery hypertension were stained with K82 AcK-CyPA antibody and reactivity detected by immunofluorescence (Alexa-fluor). Control IgG was used in place of primary Ack- CyPA antibody to show specificity of staining for Ack-CyPA in endothelial cells of vessels in the lung. Sections were counterstained with DAPI to delineate nuclei. These data indicate that the antibody has specificity for the detection of acetylated CyPA by immunofluorescence in formalin fixed, paraffin embedded rat tissues.

[00131] FIG. 16 shows AcK-CyPA expression in human lungs. Human lung tissue from control (panel A) and a PAH patient (panels B, C) was stained by immunofluorescence for AcK- CyPA. Sections were counterstained with DAPI to delineate nuclei. Bright red circles showed autofluorescence from red blood cells in the vessel. These data indicate that the antibody has specificity for the detection of human acetylated CypA by immunofluorescence in formalin fixed, paraffin embedded tissues.

[00132] It will be appreciated that variants of the above-disclosed and other features and functions, or alternatives thereof, may be combined into many other different systems or applications. Various presently unforeseen or unanticipated alternatives, modifications, variations, or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims.

Claims

WHAT IS CLAIMED:

1. An isolated antibody or binding fragment thereof which binds specifically to an acetylated epitope of Cyclophilin A ("Ac-CyPA").

2. The antibody or binding fragment according to claim 1 , wherein the acetylated epitope comprises an acetylated lysine residue.

3. The antibody or binding fragment according to claim 2, wherein the lysine residue is in position 82 of Cyclophilin A.

4. The antibody or binding fragment according to claim 3, wherein the acetylated epitope comprises SEQ ID NO:7.

5. The antibody or binding fragment according to claim 2, wherein the lysine residue is in position 125 of Cyclophilin A.

6. The antibody or binding fragment according to claim 5, wherein the acetylated epitope comprises SEQ ID NO:8.

7. The antibody or binding fragment according to claim 1 , wherein the antibody is a monoclonal antibody.

8. The antibody or binding fragment according to claim 1 , wherein the antibody is a polyclonal antibody.

9. The antibody or binding fragment according to claim 1 further comprising: a label bound to the antibody or binding fragment.

10. The antibody or binding fragment according to claim 9, wherein the label is selected from the group consisting of a fluorescent label, a biologically-active enzyme label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label, and a chromophore label.

11. An antibody binding fragment according to claim 1.

12. The antibody binding fragment according to claim 11 , wherein the binding fragment comprises a Fab fragment, Fv fragment, single-chain antibody, a V_H domain, or a V_L domain.

13. A kit for detecting Ac-CyPA, said kit comprising:

an antibody or binding fragment according to claim 9 and

one or more buffer solutions or reagent solutions to detect the label.

14. The kit according to claim 13, wherein the label is selected from the group consisting of a fluorescent label, a biologically-active enzyme label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label, and a chromophore label.

15. The kit according to claim 13 further comprising:

a secondary antibody or binding fragment that binds specifically to the antibody or binding fragment.

16. A method of detecting Ac-CyPA in a biological sample, said method comprising:

contacting the biological sample with an antibody or binding fragment according to claim 9; and

detecting specific binding that occurs between the biological sample and the antibody or binding fragment to identify Ac-CyPA in the biological sample.

17. The method according to claim 16, wherein the acetylated epitope comprises an acetylated lysine residue.

18. The method according to claim 17, wherein the lysine residue is in position 82 of Cyclophilin A.

19. The method according to claim 18, wherein the acetylated epitope comprises SEQ ID NO:7.

20. The method according to claim 17, wherein the lysine residue is in position 125 of Cyclophilin A.

21. The method according to claim 20, wherein the acetylated epitope comprises SEQ ID NO:8.

22. The method according to claim 16, wherein the antibody is a monoclonal antibody.

23. The method according to claim 16, wherein the antibody is a polyclonal antibody.

24. The method according to claim 16, wherein the label is selected from the group consisting of a fluorescent label, a biologically-active enzyme label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label, and a chromophore label.

25. The method according to claim 16, wherein said contacting is carried out with the binding fragment.

26. The method according to claim 25, wherein the binding fragment comprises a Fab fragment, Fv fragment, single-chain antibody, a V_H domain, or a V_L domain.

27. The method according to claim 16, wherein said detecting is carried out using an assay selected from the group consisting of a Western blot, immunoassay, ELISA assay, flow cytometry, radiography, and immunoscintography.

28. The method according to claim 16, wherein the antibody or a binding fragment thereof is administered to a subject and said detecting is carried out in vivo.

29. The method according to claim 28, wherein said detecting is carried out using detection procedures selected from the group consisting of diagnostic imaging, ultrasound, tomography, magnetic resonance, elastography, and radionuclear scanning.

30. The method according to claim 28, wherein the subject is selected from a human, mouse, or rat.

31. The method according to claim 30, wherein said method is carried out to diagnose a subject for cardiovascular disease.

32. The method according to claim 30, wherein said method is carried out for prognostic analysis of cardiovascular disease in a subject.

33. A method of screening an individual for cardiovascular disease, said method comprising:

providing a biological sample from an individual;

contacting the biological sample with an antibody or binding fragment according to claim 9; and

detecting specific binding that occurs between the biological sample and the antibody or binding fragment to identify Ac-CyPA in the biological sample, wherein the presence of Ac-CyPA in the biological sample indicates the individual has cardiovascular disease.

34. The method according to claim 33, wherein the acetylated epitope comprises an acetylated lysine residue.

35. The method according to claim 34, wherein the lysine residue is in position 82 of Cyclophilin A.

36. The method according to claim 35, wherein the acetylated epitope comprises SEQ ID NO:7.

37. The method according to claim 34, wherein the lysine residue is in position 125 of Cyclophilin A.

38. The method according to claim 37, wherein the acetylated epitope comprises SEQ ID NO:8.

39. The method according to claim 33, wherein the antibody is a monoclonal antibody.

40. The method according to claim 33, wherein the antibody is a polyclonal antibody.

41. The method according to claim 33, wherein the label is selected from the group consisting of a fluorescent label, a biologically-active enzyme label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label, and a chromophore label.

42. The method according to claim 33, wherein said contacting is carried out with the binding fragment.

43. The method according to claim 42, wherein the binding fragment comprises a Fab fragment, Fv fragment, single-chain antibody, a V_H domain, or a V_L domain.

44. The method according to claim 33, wherein said detecting is carried out using an assay selected from the group consisting of a Western blot, immunoassay, ELISA assay, flow cytometry, radiography, and immunoscintography.

45. The method according to claim 33, wherein said detecting is carried out using detection procedures selected from the group consisting of diagnostic imaging, ultrasound, tomography, magnetic resonance, elastography, and radionuclear scanning.

46. The method according to claim 33, wherein the individual is human.

47. A method of treating an individual for symptoms associated with cardiovascular disease, said method comprising:

contacting an individual with an antibody or binding fragment according to claim 1 under conditions effective for the antibody or binding fragment to specifically bind to an acetylated epitope of Cyclophilin A, wherein said binding treats the individual for symptoms associated with cardiovascular disease.