Chymase compositions, antibodies, diagnostics, and therapeutic methods related thereto

Abstract

The disclosure relates to chymase, antibodies to chymase, and diagnostic and therapeutic methods relates thereto. It has been discovered that above certain circulating levels of chymase, a patient has a high likelihood of arteriovenous fistula nonmaturation. Compositions and methods of detecting and measuring chymase levels are disclosure herein. In certain embodiments, the disclosure relates to methods of determining the effectiveness of creating an arteriovenous fistula in a subject diagnosed with chronic kidney disease.

Claims

1. A method comprising: a) assaying a sample for a plasma chymase level, wherein the sample is obtained from a subject diagnosed with chronic kidney disease; and b) correlating an elevated level of plasma chymase with a likelihood of developing arteriovenous fistula nonmaturation, wherein the elevated level of plasma chymase is greater than 210 ng/mL, wherein the assaying comprises measuring the plasma chymase level with an anti-chymase antibody, and wherein the anti-chymase antibody binds an epitope comprising PSKFCGG (SEQ ID NO:1).

2. The method of claim 1, further comprising the step of c) reporting the plasma chymase level to the subject or a medical professional or representative thereof.

3. An anti-chymase antibody that binds to an epitope comprising PSKFCGG (SEQ ID NO:1).

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Non-CKD vein (a, b) with no intimal hyperplasia and minimal chymase activity (green); CKD vein with intimal hyperplasia (c) and abundant chymase (d), co-localized with α-smooth muscle actin (α-SMA).

(2) FIG. 2: Serial vein segments from the cephalic vein of a pre-dialysis CKD (a,c,e) and an ESRD (b,d,f) patient. Immunofluorescence (a,b) shows abundant chymase (pink) expression in the thickened vessel intima and media (separated by elastic lamina, blue) of both the CKD and ESRD patient. Immunohistochemistry shows localization of TGF-β (c,d) (brown) and IL-6 (e,f) (brown) primarily within the intima and media, in a similar distribution as chymase.

(3) FIG. 3: Plasma chymase concentration by patient cohort. Note bimodal distribution of plasma chymase in CKD patients. Elevated plasma chymase is shown with corresponding mast cell chymase expression in veins of same patients with IH. ESRD patients have a different plasma chymase concentration, suggesting that creatinine clearance may influence plasma chymase.

DETAILED DISCUSSION

(4) Chymase is suspected of playing a causative role in the formation of venous IH via stimulation of two primary contributors to IH formation: angiotensin II (Ang II) and transforming growth-factor beta 1 (TGF-β1). Chymase causes Ang II formation, which has been shown to increase the expression of pro-TGF-β and MMP-9. In addition to this indirect effect, chymase also converts the latent form of TGF-β to its active form. These direct and indirect effects of chymase may contribute to IH and fibrosis. The role of chymase in IH formation is supported by the ability of chymase inhibition to attenuate intimal proliferation in canine models of balloon injury, vein grafting, and AV fistula (AVF) creation and AV graft (AVG) insertion.

(5) Uremia is a state of chronic inflammation and oxidative stress. Chymase is believed to play a role in venous IH formation and is highly expressed in the veins of CKD and ESRD patients with IH. Data suggest that mast cell chymase expression may be reflected by an elevated plasma chymase concentration.

(6) Increased Plasma Chymase Concentration and Mast Cell Chymase Expression in Venous Neointimal Lesions of CKD and ESRD Patients

(7) Progressive IH restricts AVF blood flow, preventing adequate dialysis and increasing the likelihood of AVF thrombosis. Important contributors to IH development such as chymase are present in veins of CKD and ESRD patients. Plasma chymase is increased as much as a 33-fold in CKD patients. The finding that chymase inhibition attenuates IH in animal models, and findings herein that chymase levels are elevated in venous IH lesions of CKD and ESRD patients suggest that chymase inhibition may be useful therapeutically. It also points to the utility of using plasma chymase as a biomarker to predict AVF nonmaturation.

(8) Studies of mast cell chymase suggest that it is localized to the adventitial layer of blood vessels and that chymase inhibition limits IH development in animal models of vascular injury. In CKD patients it was found that chymase expression is diffuse, with greater chymase immunoreactivity in the vessel intima and media. TGF-β is a well-recognized promoter of VSMC proliferation, and induces fibrosis and ECM production in blood vessels. Studies herein suggest that TGF-β co-localizes with chymase in the veins of subjects with IH, and is most prominent in the intima and media layers of blood vessels. Although it is not intended that embodiments, of the disclosure be limited by any particular mechanism, one possibility for this co-localization is that chymase increases the expression of TGF-β indirectly via promotion of Ang II formation and directly by activating pro-TGF-β1. Another possibility for the chymase/TGF-β/IL-6 co-localization in these diseased vessels could be the finding that mast cells store and release all of these mediators into the vascular interstitium. Chymase and TGF-β in vessels with IH, in addition to IL-6, indicate an inflammatory state and suggest their involvement in IH progression.

(9) Studies herein suggest that plasma chymase levels were increased in CKD patients between 4 and 33-fold. Interestingly, chymase distribution was bimodal, yet there were no significant differences in co-morbidities between the groups except BMI. Circulating chymase levels may be a predictor of extant intimal hyperplasia in patients with CKD. Studies herein suggest significantly greater plasma chymase levels in CKD patients than previous studies on subjects with mastocytosis and aortic aneurysm.

(10) Although there are several endogenous inhibitors of chymase, such as alpha-1 antitrypsin, circulating chymase exists in a protected state in complex with alpha-2 macroglobulin such that it is capable of converting angiotensin I to angiotensin II in the circulation. Further, angiotensin receptor blockers (ARB's) but not ACE inhibitors have been shown to attenuate IH after vascular injury. It is therefore contemplated that elevated chymase levels could underlie the therapeutic advantage of ARB's versus ACE inhibitors in this setting.

(11) Antibodies

(12) Within certain embodiments, the disclosure contemplates antibodies with an epitope to a polypeptide of PSKFCGG (SEQ ID NO: 1) or EIVTSNGPSKFCGG (SEQ ID NO: 2). The disclosure should not be construed as being limited solely to one type of antibody. The antibodies can be produced by immunizing an animal such as, but not limited to, a horse, rabbit, guinea pig, hamster, rat, or a mouse, with a protein, or a portion thereof, or by immunizing an animal using a protein comprising at least a portion of the corresponding polypeptide. One skilled in the art would appreciate, based upon the disclosure provided herein, smaller fragments of these proteins can also be used to produce antibodies that specifically bind the polypeptide. Antibody production may be enhanced by the addition of an adjuvant including, but not limited to, alum. The antibodies may be conjugated to a marker, such as a fluorescent dye or protein, e.g, green fluorescent protein. Sections of the antibodies may be detected by secondary antibodies containing markers.

(13) Certain embodiments of the disclosure encompass polyclonal, monoclonal, synthetic antibodies, and the like. Moreover, the antibody can be used to detect and or measure the amount of protein present in a biological sample using well-known methods such as, but not limited to, Western blotting, radio immunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA). The antibody can also be used to immunoprecipitate and/or immuno-affinity purify their cognate antigen using methods well-known in the art.

(14) It is also contemplated the antibodies may be contained in a pharmaceutical composition for uses that any chymase inhibitor could be used for, such as being administered to a subject for the treatment or prevention of thickening of the walls of arteries or vessels, renal disease, heart disease, or heart attack. Thus, by administering the antibody to a cell or to the tissue of an animal, or to the animal itself, the activity of chymase would therefore be inhibited.

(15) In another embodiment, a monoclonal antibody is obtained from the non-human animal, and then modified, e.g., humanized, deimmunized, chimeric, or produced using recombinant DNA techniques known in the art. A variety of approaches for making chimeric antibodies have been described. See, e.g., U.S. Pat. No. 4,816,567 and U.S. Pat. No. 4,816,397. Humanized antibodies may also be produced, for example, using transgenic mice that express human heavy and light chain genes, but are incapable of expressing the endogenous mouse immunoglobulin heavy and light chain genes. Winter describes an exemplary CDR-grafting method that may be used to prepare the humanized antibodies described herein (U.S. Pat. No. 5,225,539). All of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR, or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody to a predetermined antigen.

(16) Humanized antibodies or fragments thereof can be generated by replacing sequences of the Fv variable domain that are not directly involved in antigen binding with equivalent sequences from human Fv variable domains. Exemplary methods for generating humanized antibodies or fragments thereof are provided by U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No. 5,693,762; U.S. Pat. No. 5,859,205; and U.S. Pat. No. 6,407,213. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable domains from at least one of a heavy or light chain. Such nucleic acids may be obtained from a hybridoma producing an antibody against a specific target, as described above, as well as from other sources. The recombinant DNA encoding the humanized antibody molecule can then be cloned into an appropriate expression vector.

(17) In certain embodiments, a humanized antibody is optimized by the introduction of conservative substitutions, consensus sequence substitutions, germline substitutions and/or backmutations. An antibody or fragment thereof may also be modified by specific deletion of human T cell epitopes or “deimmunization” by the methods disclosed in U.S. Pat. No. 7,125,689 and U.S. Pat. No. 7,264,806. Briefly, the heavy and light chain variable domains of an antibody can be analyzed for peptides that bind to MHC Class II; these peptides represent potential T-cell epitopes. For detection of potential T-cell epitopes, a computer modeling approach termed “peptide threading” can be applied, and in addition a database of human MHC class II binding peptides can be searched for motifs present in the VH and VL sequences. These motifs bind to any of the 18 major MHC class II DR allotypes and thus constitute potential T cell epitopes. Potential T-cell epitopes detected can be eliminated by substituting small numbers of amino acid residues in the variable domains or preferably, by single amino acid substitutions. Typically, conservative substitutions are made. Often, but not exclusively, an amino acid common to a position in human germline antibody sequences may be used. The V BASE directory provides a comprehensive directory of human immunoglobulin variable region sequences. These sequences can be used as a source of human sequence, e.g., for framework regions and CDRs. Consensus human framework regions can also be used, e.g., as described in U.S. Pat. No. 6,300,064.

EXPERIMENTAL

(18) CKD Subjects

(19) Stage 4 and 5 CKD patients were identified between Jul. 14, 2008 and Jul. 31, 2010 who had no prior AV access creation (including central venous catheters), were six or more months from initiating hemodialysis, and were suitable candidates for AVF based on upper extremity venous mapping at the Emory Dialysis Access Center of Atlanta, Emory University Hospital and Emory Midtown Hospital. Subjects were enrolled as part of a study to evaluate the cardiovascular effects of AVF creation. A baseline visit occurred prior to AVF creation, at which time a blood sample, echocardiogram, and six-minute walk test were obtained. Additionally, demographic data, clinical history and medication use were obtained via direct patient interview. Study subjects underwent AVF creation within two days-3 weeks following the baseline visit. The Institutional Review Board (IRB) of Emory University Medical Center approved the study protocol and informed consent was obtained from each patient prior to study enrollment.

(20) ESRD Subjects

(21) Adult ESRD patients receiving outpatient hemodialysis at Emory University dialysis units between Mar. 24, 2009 and Jun. 25, 2010 were identified who were suitable candidates for AVF creation based on upper extremity venous mapping at the Emory Dialysis Access Center of Atlanta, Emory University Hospital and Emory Midtown Hospital. Subjects were enrolled as part of a pilot study to evaluate the impact of vitamin D on AVF maturation, and all subjects included in this analysis were using a central venous catheter at the time of study enrollment. The average time on dialysis was 11.7 months (±19.8 months). A baseline visit occurred prior to AVF creation, and demographic and clinical data were collected via direct patient interview, at which time a blood sample was obtained. Study subjects underwent AVF creation within 1-3 weeks following the baseline visit. The Institutional Review Board (IRB) of Emory University Medical Center approved the study protocol and informed consent was obtained from each patient prior to study enrollment.

(22) Tissue Collection

(23) Remnants of surgically excised cephalic and basilic veins were collected from 11 CKD and 12 ESRD subjects at the time of AVF creation at Emory University and Emory Midtown Hospitals between Jul. 15, 2008-Jul. 30, 2010, by four vascular surgeons.

(24) A peripheral vein from a non-CKD patient was used as control. Remnant vein segments, which are normally discarded, were harvested from the cephalic or basilic vein to be used as the AVF conduit by the surgeon. It was not possible to obtain vein segments from all study patients because of the limited length of the vessel in some cases. The vein remnants were carefully placed directly into 10% normal buffered formalin without use of forceps. The specimens were transferred to 70% ethanol 24 h after initial placement in formalin. Within 1 week, venous segments were processed and embedded in paraffin blocks. Hematoxylin and eosin stain (H and E) was used to show cellularity and general morphological characteristics.

(25) Tissue Immunohistochemistry

(26) Paraffin-embedded vein segments were sectioned into 3.0-μm-thick serial sections. After heat-mediated antigen retrieval, slides were immunohistochemically processed in a DAKO Automated Immunostainer (DAKO Corp.) using a labeled streptavidin-biotin method for IL-6 (rabbit polycolonal antibody to IL-6 manufactured by Abcam) and TGF-β1 (rabbit polyclonal IgG to TGF-β1 manufactured by Santa Cruz). After processing, slides were coverslipped with a Leica CV5000 Coverslipper (Leica Microsystems, Inc.). Each staining batch contained positive and negative slides; normal colon tissue was the positive control tissue for IL-6, and normal tonsil tissue was the positive control for TGF-β. The negative and positive control slides were treated identically to the patients' slides except that antibody diluents were used rather than primary antibody on the negative slides.

(27) Tissue Immunofluorescence and Confocal Laser Scanning Microscopy

(28) Human veins were immersion-fixed in 4% paraformaldehyde and stored in 70% ethanol until paraffin embedding and sectioning. Sections (5 μm) were mounted on slides, deparaffinized in xylene and rehydrated in ethanol. Sections were blocked with 5% goat serum in 1×PBS for 1 h at room temperature. Primary antibodies (final concentration): ACE rabbit monoclonal (Sigma, 1:250); Chymase (our custom synthesized antibody using services of affinity bioreagents, 1:750) were combined in an appropriate volume of 5% goat serum in 1×PBS, and applied to sections by overnight incubation at 4° C. The sections were incubated with ALEXA® FLOUR® 488 or ALEXA® FLOUR® 647 goat anti-rabbit, to visualize the specific stains. All secondary antibodies were from Molecular Probes. 4′ 6-diamidino-2-phenylindole (DAPI) (Vector) was used to visualize nuclear DNA. Image Acquisition was performed on a Zeiss LSM510 META confocal laser scanning microscope (Zeiss, Thornwood, N.Y.) with a LSM510 system cooled CCD camera and software Zeiss ZEN 2008 (Zeiss, Thornwood, N.Y.). In showing chymase staining (micrographs in FIG. 1), chymase was pseudocolored in green and α-SMA in red. In micrographs 2 and 3, chymase was pseudocolored in pink and ACE rendered in green.

(29) Plasma Chymase ELISA Assay

(30) Plasma from normal, CKD and ESRD subjects were diluted in 50 mM of sodium carbonate buffer, pH 9.6 (Sigma, St. Louis, Mo., USA) and plated in COSTAR® 96 well flat bottom Medical-grade polystyrene plates (Corning Life Sciences, Massachusetts, USA) overnight at 4° C. The next day, the samples were washed in PBS containing 1% bovine serum albumin (BSA) and 0.05% TWEEN® 20 (Sigma) and blocked for 2 h with PBS containing 3% goat serum (Sigma) at 37° C. After three washes with PBS containing 1% BSA and 0.05% TWEEN® 20, primary polyclonal antibody specific for chymase (our custom synthesized antibody using services of Affinity Bioreagents) was diluted 1:1000 in PBS containing 3% goat serum and applied to the plate followed by a 37° C. incubation for 1 h. After three washes with PBS containing 1% BSA and 0.05% TWEEN® 20, bound antibody was detected with a secondary horseradish peroxidase (HRP)-conjugated antibody (Sigma) and incubated for 1 h at 37° C. The plate was washed three times with PBS containing 1% BSA and 0.05% TWEEN® 20 and developed with Ultra TMB (PIERCE® Biotechnology, IL, USA). The reaction was stopped using 2 M sulfuric acid (Sigma), and its OD was read at 490 nm by the microplate reader (ELX® 808; Bio-Tek instruments). Standard curve for chymase was prepared by measuring optical absorption from known concentrations.

(31) Human Chymase amino acid 40-53 custom peptide sequence: EIVTSNGPSKFCGG (SEQ ID NO: 2) was used to create antibodies for human chymase after antibody peptide sequence: MAYLEIVTSNGPSKF (SEQ ID NO: 3) failed to produce antibodies that were useful for the detection assay. In the successful EIVTSNGPSKFCGG (SEQ ID NO: 2), PSKF is unique for human chymase.

(32) Clinical data for 29 CKD and 13 ESRD subjects were available for the analysis. Venous tissue was available for analysis on 11 CKD subjects and 12 ESRD subjects. Overall, the average patient age was 58 years, 67% of patients were Black and 67% male. Mean SBP was 140, 93% had hypertension, 43% had diabetes, 59% had a history of tobacco use, and 17% had a history of myocardial infarction (MI) (Table 1). CKD patients were older, less likely to be Black, with lower systolic and diastolic blood pressure compared with ESRD patients. There was no difference between patient cohorts in gender, BMI, prevalence of hypertension, diabetes, smoking history, or history of MI.

(33) TABLE-US-00001 TABLE 1 Clinical Characteristics of Study Subjects Control CKD ESRD Characteristics (n = 5) (n = 29) (n = 13) p-value* Age  26 ± 12.9 61.2 ± 12.2 51.4 ± 7.9 0.004 Black 0 (0) 15 (51.7) 13 (100)   0.002 Female   2 (40.0) 11 (37.9) 3 (23.1) 0.485 BMI 22.6 ± 2.3  31.1 ± 8.2  29.6 ± 6.1 0.508 SBP 113 ± 6.0  135 ± 18.8 .sup. 154 ± 27.8 0.042 DBP  71 ± 7.3 .sup. 72 ± 11.9  86 ± 18 0.025 Hypertension 0 (0) 28 (96.6) 11 (84.6)  0.222 Diabetes 0 (0) 12 (41.4) 6 (46.2) 1 Smoking 0 (0) 17 (58.6) 8 (61.5) 1 History of MI 0 (0)  4 (13.8) 3 (23.1) 0.657 Results are % or standard deviation. *p-value compares CKD vs. ESRD patients

(34) Pre-existing IH varied. 10 of 11 (91%) CKD vein segments exhibited IH and luminal stenosis prior to surgical AVF creation, demonstrating that IH exists in CKD patients prior to AVF creation, and cannot be attributed solely to the effects of flow and pressure alterations resulting from AVF creation. 9 of 12 (75%) ESRD vein segments had IH. Abundant chymase was present in vessels with IH, and was diffuse, within the intima and adventitia, and co-localizing in the vessel media with α-smooth muscle actin (α-SMA), reflecting myofibroblast formation (FIG. 1). The same finding was observed in vein samples from ESRD subjects. By contrast, the non-CKD control subject lacked IH, with little evidence of mast cell chymase and no evidence of α-SMA within the vein sample.

(35) The distribution of vascular chymase was then compared with two factors which induce IH and are associated with AVF stenosis: transforming growth factor-β (TGF-β) and interleukin-6 (IL-6) (FIG. 2). TGF-β expression was greater in vessels with IH compared to those without IH and was found to be greater in vessel intima and media layers, indicating that TGF-β production is greater in diseased vessels with IH. Veins with IH expressed more IL-6, which was more pronounced in the venous intimal and medial layers. The intensity of TGF-β and IL-6 expression was similar in CKD and ESRD veins with IH.

(36) After finding abundant mast cell chymase in the veins of CKD and ESRD subjects preparing for AVF creation, the relationship between mast cell chymase and plasma chymase concentrations was examined (FIG. 3). The non-CKD control subjects had undetectable plasma chymase concentrations (<8 ng/ml), which corresponded with the virtual lack of mast cell chymase. CKD subjects demonstrated a bimodal distribution of plasma chymase, with plasma chymase concentrations that were as much as 6-fold greater than the lowest group, and abundant mast cell chymase expression. Finally, ESRD subjects had a different plasma chymase distribution than CKD subjects, suggesting that reduced creatinine clearance may be responsible for elevated plasma chymase levels in CKD subjects. Dialysis had an effect on plasma chymase levels, however, it is unlikely that this was due to direct removal of chymase from the bloodstream by dialysis, because chymase exists as a 200 kDA complex with alpha-2 macroglobulin in plasma. This led us to speculate that plasma markers of systemic inflammation may be elevated among CKD patients with the greatest plasma chymase concentrations. However, no association between plasma c-reactive protein and plasma chymase concentrations was found (data not shown). Moreover, the plasma chymase bi-modal distribution was not associated with statin use. Interleukin-6 (IL-6), a local marker of inflammation, was present in vascular IH lesions which express high levels of chymase (FIG. 2).

(37) In a group of CKD patients (n=25) in whom AVF suitability for dialysis within 6 months of AVF creation was determined clinically (ability to cannulate the AVF with 2 needles with dialysis blood flow >300 ml/min for at least 6 dialysis sessions in one month and within 6 months of AVF creation), plasma chymase concentration was examined as a predictor of AVF outcomes. The risk of experiencing AVF nonmaturation for patients with plasma chymase >210 ng/ml is 10.3 times the risk of experiencing AVF nonmaturation for patients with chymase <210 ng/ml. (Table 2). Notably, the bimodal distribution (FIG. 3) suggests that 150 ng/ml may provide the cutpoint for predicting AVF maturation, but this preliminary data suggests otherwise.

(38) TABLE-US-00002 TABLE 2 Odds of AVF nonmaturation by chymase concentration Dichotomized Chymase Continuous Outcome Estimated odds Relative p- Chymase Variable ratio (CI)* Risk** (CI) value*** p-value**** AVF >=150 vs <150 (referent) 0.124 mature 0.263  3.14 0.341 (0 = no,  (0.0046, 3.308) (0.407, 24.3)  referent) >=210 vs <210 (referent) 0.0534 10.3 0.012 (0.00086, 0.753) (1.38, 76.8) >=215 vs <215 (referent) 0.0204 16 0.002 (0.00026, 0.379)  (2.25, 113.6)