WO2004029238A1 - Human tissue kallikrein-specific immunoglobulin, immunologic formulation comprising the immunoglobulin and related diagnostic kit for the diagnosis of cerebral ischemia - Google Patents

Abstract

The present invention relates to an immunoglobulin specific for human tissue kallikrein as a whole or for a portion thereof, the immunologic formulation comprising this immunoglobulin and the related diagnostic kit for the diagnosis and monitoring of cerebral ischemia.

Description

HUMAN TISSUE KALLIKREIN-SPECIFIC IMMUNOGLOBULIN, IMMUNOLOGIC FORMULATION COMPRISING THE IMMUNOGLOBULIN AND RELATED DIAGNOSTIC KIT FOR THE DIAGNOSIS OF CEREBRAL ISCHEMIA

Field of the invention The present invention relates to an immunoglobulin specific for human tissue kallikrein (hereinafter referred to as "TK"), the immunologic formulation comprising the immunoglobulin and the related diagnostic kit for the diagnosis and monitoring of cerebral ischemia. State of the art TK is an enzyme, present in circulating blood as well as in other fluids and tissues of the body, that generates kinins, biological end-products with potent vasodilator activity. TK can be uncoupled or form a complex with its natural inhibitors, such as kallistatin. The TK-kinins system is present in the brain where it participates in the central regulation of cardiovascular function (Madeddu P. et al., Hypertension 1996; 28:980-989; Emanueli C. et al., Br. J. Pharmacol. 1999; 126:1769-1776). TK- generated kinins act as potent vasodilators in cerebral vasculature (Toda N., Am. J. Physiol., 1997; 222:H267-H274). The TK-kinin system is activated by ischemia and contributes to re-establish tissue perfusion by promoting neovascularization (Emanueli C. et al., Circulation. 2001 ; 103: 125-132).

Stroke, a cerebral accident caused by acute interruption of blood flow to the brain, is one of the leading causes of mortality and infirmity. Stroke usually occurs in patients affected by atherosclerotic obstructions of carotid arteries as a consequence of plaque rupture with subsequent thrombosis and/or embolism to cerebral vessels. Cerebrovascular insufficiency represents a major clinical problem especially in elderly. In epidemiological studies, as many as 10% of men and 7% of women older than 65 years were found to have greater than 50% carotid artery stenosis (hereinafter referred to as "CAS"). As lifespan progressively increases, cerebroVascular insufficiency is gaining relevance as a major social and economic burden in developed countries. Evidence from large-scale trials indicates that patients with high-grade CAS are stroke-prone. Revascularization, i.e. reopening of obstructed artery, is usually recommended in this category, in consideration of the favourable overall benefit- to-risk ratio. In contrast, treatment options for patients with angiographic evidence of mild to moderate-grade CAS remain matter of debate, because peri-operative risk overwhelms the advantages of revascularization. Consequently, low-risk patients are generally lumped together and dismissed without attention to the deleterious consequences that chronic hypo-perfusion might produce to the brain, including progressive cognitive deterioration and dementia. Further, prevalence of moderate-grade CAS is such a high that an excess of acute ischemic strokes is expected to derive from not recognising functionally relevant vascular obstructions. Unfortunately, angiography, i.e. the radiological method commonly used to visualise the degree of arterial obstruction, is not suitable for the assessment of cerebral blood flow. Further, the high cost of recently-introduced diagnostic procedures, that allow an indirect estimation of cerebral perfusion, precludes their use for general screening of the population at risk of stroke.

It follows that identification of new biomarkers of cerebral ischemia may lead to significant advances in the diagnosis, monitoring, and therapy of the disease, particularly in patients apparently at low risk, who represent the majority among those with cerebrovascular obstruction. SUMMARY

Now the Applicant has found that circulating TK represents a biomarker of cerebral ischemia and that measurement of this biomarker in peripheral blood allows revealing the presence of hemodynamically relevant cerebral insufficiency in subjects affected by carotid artery obstruction or stenosis. In fact, the Applicant has found that TK levels are increased in patients suffering cerebrovascular insufficiency due to atherosclerotic plaque obstruction of carotid arteries. Moreover, the increase in circulating TK levels is proportional to the obstruction grade. Therefore, measurement of TK concentration in serum or plasma derived from a single peripheral blood sample can elucidate the current status and the evolution of ischemic disease. Further, evaluation of biomarker levels represents an easy and economic way for identification of the patients in whom carotid artery revascularization and/or reinforcement of preventive measures and medical treatment is recommendable.

Subject of the present invention is therefore an immunoglobulin specific for TK, either uncoupled or bound to an inhibitor of TK, or specific for a portion of TK; for example, the immunoglobulin produced by immunisation of a host animal with a suitable immunogen comprising TK or a portion thereof.

The process for the preparation of the above immunoglobulin is a further subject of the present invention.

In addition, subjects of the invention are: the immunologic formulation comprising an immunoglobulin specific for TK and a detection agent, and a diagnostic kit for the diagnosis of cerebral ischemia comprising the above said immunologic formulation.

Further subject of the invention is a method for measuring TK levels in a biologic sample, comprising the following steps: formation of an immune complex between an immunoglobulin, specific for the whole TK or a TK fragment, and TK in a biological sample to be analysed, and between the above immunoglobulin and TK in a reference standard in which TK concentration is known; detection of the immune complex coming from step 1 ); comparison of the results obtained in the biological sample to be analysed with those obtained for the reference standard.

Finally, subject of the invention is a method for the diagnosis of cerebral ischemia, comprising the following steps: measurement of TK in a biological sample by reaction with an immunoglobulin specific for the whole TK, uncoupled or bound to a TK inhibitor, or specific for a portion of TK; assessment of the severity of cerebral ischemia, where increased TK levels in the biologic sample with respect to the average value in the normal population are directly proportional to the degree of carotid artery obstruction and therefore correlated to the severity of cerebrovascular ischemia.

The characteristics and the advantages of the present invention will be illustrated in details in the following description. BRIEF DESCRIPTION OF FIGURES

Figure 1 : Dispersion graph of serum TK levels (expressed in pg/ml) in patients classified according to the degree of carotid artery obstruction as indicated by angiography. Shaded area delimited by dotted lines represents normal distribution (mean±2 standard deviation) of TK in healthy population..

Figure 2: Histogram graph of the data illustrated in Figure 1 , showing the percent of patients with TK levels above the upper limits of normal distributions. Patients were classified according to the risk of stroke calculated on clinical and angiographic criteria: low/moderate risk patients are shown in the left side and high risk patients on the right side;

Figure 3: Line graph indicating the changes in TK values (expressed in pg/ml) in before and over the first 3 days after surgical revascularization of carotid artery stenosis by endo-arteriectomy (CEA). Figures 4: Panel A shows TK levels (expressed in pg/ml) of single patients before and at 3 months from CEA of unilateral CAS.

Panel B reproduces the results of panel A in form of histograms indicating mean TK values±standard error of the mean. The symbol "*" is used to denote that mean post-operative levels were significantly lower as compared with mean basal levels; Panel C shows TK values (expressed in pg/ml) of single patients before and at 3 months from CEA of bilateral CAS;

Panel D reproduces the results of panel C in form of histograms indicating mean TK values±standard error of the mean.

Figure 5: Line graph shows TK levels (expressed in pg/ml) of 2 patients suffering peri-operative ischemic complications. Values before and after CEA are indicated as "Basal" and "CEA", respectively.

Figure 6: Line graph shows TK levels (expressed in pg/ml) of single patients before and at 12 months from CEA. The symbol "*" is used to denote that mean post-operative levels were significantly lower as compared with mean basal levels. DETAILED DESCRIPTION OF THE INVENTION The Applicant has found that the enzyme TK is an effective and selective biomarker, suitable for the diagnosis of cerebral ischemia. Therefore, the Applicant has developed a diagnostic kit and a method based on this kit that opens new opportunities for the diagnosis of cerebral ischemia and for monitoring the evolution of the disease. Moreover, measurement of TK levels performed by means of the present kit simplifies the screening of patients. In fact, the test represents a simple and rapid way to identify, among patients affected by cerebral ischemia, those requiring carotid revascularization and/or reinforcement of preventive measures and medical treatment. In particular, the present diagnostic kit makes possible to measure TK concentration in a single sample to verify the divergence from the average value in healthy population and thereby allows to gain indirect insights of cerebral perfusion. Further, sequential measurements of TK concentration over time by means of the present method makes possible to detect changes of TK levels and thereby allows to monitor the evolution of cerebrovascular disease, whereby decreasing levels would indicate an improving condition either spontaneous or induced by therapy, while increasing levels indicate a deteriorating condition. Further, in patients with bilateral carotid obstruction, persistence of elevated TK levels after unilateral revascularization would suggest surgical reopening of contralateral carotid artery not involved in the first surgical operation.

Moreover, measurement of TK levels by means of the present method allows a simple and rapid estimation of the risk/benefit ratio of performing revascularization of obstructed carotid artery, whereby increasing elevation of TK levels suggests the immediate need of interventions aimed at reestablishing brain perfusion, while normal TK levels suggest a less aggressive approach.

According to the invention, TK measurement in a biological sample is performed by an immunological method using a suitable antibody specific for TK. In particular, the present method for evaluation of TK concentration in a biological sample comprises the following steps: formation of an immune complex between an immunoglobulin, specific for the whole TK or a TK fragment, and TK in a biological sample to be analysed, and between the above immunoglobulin and TK in a reference standard in which TK concentration is known; detection of the immune complex coming from step 1 ); comparison of the results obtained in the biological sample to be analysed with )

those obtained for the reference standard.

For "biological sample" is intended a blood or blood-derived sample or a sample of other biological fluid harvested from the subject under examination and intended to be processed out of the human body. In the present invention, "TK" is intended as tissue kallikrein uncoupled or bound in form of a complex with a kallikrein inhibitor, such as kallistatin. The suitable TK-specific antibody is prepared according to the methodology commonly used by experts in the field, for instance by immunisation of a host animal with a suitable immunogen, for instance human TK obtained from urine or other biological liquids and purified with conventional purification methods. As an alternative, recombinant TK obtained with genetic engineering and molecular biology techniques commonly used by experts in the field may be used for immunisation. TK fragments may also be used in order to produce a suitable TK- specific immunoglobulin. As an example, an immunogen conjugate comprising a synthetic peptide, whose aminoacid sequence encompasses an epitope of TK, coupled to a conventional immunogenic carrier molecule could be used. The serum of the immunised animal is utilised to obtain the above immunoglobulin following purification performed according to purification methods commonly used by experts in the field. Sheep, goat, rabbit, donkey, horse, rat, mouse, monkey, and similar can be used for immunisation as host animals.

Suitable antibody reagents to be used for TK detection according to the invention can be labelled, e.g. by conjugation with an enzyme, or immobilised, e.g. coated onto a microtiter plate, bound to plastic or magnetic beads or particles, and can comprise whole immunoglobulins, e.g. IgG, or fragments, e.g., Fab, Fab', and F(ab').sub.2 fragments.

TK-specific immunoglobulin, prepared as indicated above, can be used to prepare an immunologic formulation comprising the immunoglobulin itself and a detection agent, wherein with the expression "detection agent" is intended any agent suitable to detect the immune antigen/antibody complex and to quantify antigen concentration. According to the invention, detection of the antigen/antibody complex in the biological sample can be performed through a system based on colour or fluorescence development, where the intensity of colour or fluorescence, as measured by methods known to the experts in the field, is proportional to TK concentration in the sample. According to the invention, the detection agent may be selected for example from the group consisting of biotin-avidin-peroxidase, biotin-avidin-alkaline phosphatase, fluorescein, and other molecules or supramolecular complexes commonly used for this purpose and well known to any person skilled in the field.

According to a preferred embodiment of the invention, measurement of TK levels in a biological sample is performed by sandwich ELISA immunoassay; the assay is carried out in a 96-well plate, where the immunoglobulin specific for TK (either uncoupled or forming a complex with its inhibitor) or specific for a TK fragment reacts with the TK present in the biological sample and the complex produced in such a way is recognised by a secondary antibody linked to a detection agent comprising one of the formulation described above. As evident to any skilled person in the field, alternative methods based on the use of the specific immunoglobulin subject of the invention may be suitable for determining TK, TK fragments or TK-inhibitor complex in samples such as blood, blood-derivates, or other biological fluids. As example, immuno-enzymatic and radio-immunologic techniques may be considered suitable. In addition, physical or chemical methodologies based on molecular weight or electric charge or the combination of both may be suitably used as known by the skilled worker in the field. Consequently, chromatographic methods used either alone or in combination with mass spectrophotometry may be used to determine TK. The diagnostic kit according to the invention comprises at least one antibody specific for uncoupled TK, TK-inhibitor complex, or a TK portion as described above and a detection agent. Moreover, the kit may comprise one or more buffers aimed at maintaining optimal chemical conditions for the reaction, suitable standards with known TK concentration and other components commonly used in the field of the invention and known by any skilled person in the field. The following examples are provided to give a non-limiting illustration of the invention. EXAMPLE 1 Purification of TK from human urine Human TK was purified from pooled human urine with affinity chromatographic methods similar to a method previously described (Shimamoto K., Chao, J., Margolius H. S. (1980) J. Clin. Endocrinology and Metabolism. 51 : 840-848). Approximately 12 I urine were collected and concentrated daily with a hollow fiber ultrafiltration apparatus (Amicon Corp., Lexington, MA) to approximately 300 ml and frozen at -20° C. The concentrates from approximately 200 I urine were thawed, pooled, and filtered through three layers of gauze upon one layer of Nitex (pore size 100 Dm; Tetko, Elmsford, NY). The filtrate was centrifuged at 5000 rounds per min for 30 min. A precipitate was fractionated from the supernatant with 35-80% ammonium sulphate, resolved, and dialysed. This solution was subjected to DE-52 cellulose column chromatography. Approximately 5% of the Tos-Arg-Ome esterase activity flowed through the column. The remaining was subsequently eluted with a linear gradient of 0.1-0.5 M NaCI and 0.01 M sodium phosphate buffer, pH 6.5. Tos-Arg-OMe esterase-containing fractions were pooled and dialysed against 0.2 M NaCI and 0.1 M sodium phosphate, pH 6.5, and applied to an aprotinin-agarose affinity column (Affi-Gel^® 10, Bio-Rad Laboratories, Richmond, CA). The column was washed with the dialysis buffer until protein could not be detected in the effluent. Absorbed Tos-Arg-Ome esterase was then eluted with 0.1 M acetate buffer, pH 3.5. Five-ml fractions were collected into tubes containing 1 ml 0.5 M Tris-HCI, pH 8.5. The eluted Tos-Arg-OMe esterase from the affinity column was further purified by Sephacryl^® S-200 column chromatography. The purity of TK was determined by polyacrylamide gel electrophoresis. The purification factor from the crude urine concentrates to purified enzyme was 2114-fold. Both the Tos-Arg-OMe esterase activity and the kinin-generating activity of this purified protein were measured. Kinin-generating activity was measured using dog or bovine low molecular weight kininogen as substrate, with subsequent RIA of the product kinin using the method described in Shimamoto K, et al. J Lab Clin Med. 1978,91 :721-728. Preparation and purification of polyclonal antisera Female New Zealand white rabbits were immunised with 100 mg of an emulsion of TK purified from human urine as described in example 1 and Freund's complete adjuvant. This procedure was repeated twice to boost the immune response except that 50 mg of an emulsion of TK was used. In order to amplify immune response, the shoot was repeated one or more times at 1 month from the first injection. In this case, 50 mg of the TK-containing emulsion was used. After 1-2 months from the last immunogen injection, the blood of immunised rabbit is sequentially obtained from the ear vein under anaesthesia. The blood is collected in glass tubes and immediately centrifuged (1500 g/min, 15 min) to separate the serum that is then stored at -20°C until analysis.

A sample of 15-20 ml of the antiserum raised in rabbits to human urinary TK is loaded onto a Sepharose^® column (Sigma Chemical Co.) previously equilibrated with 0.05 M H₃P0₄, pH 8.0 The column is washed with 0.05 M H₃P0₄, pH 8.0 until the absorbance reaches the base line.

The column is eluted with 0.1 M glycine/HCI buffer, pH 3.0 (34 drops/min, 100 drops/tube), and the eluent neutralised with 0.5 M Tris-base, pH 9.0 immediately (or otherwise appropriate amount of Tris-base is added to collecting tubes before the elution). The fractions containing IgG with absorbance at 280 nm greater than 0.15 are pooled.

Preparation of biotin-conjugated human TK- IgG

The method is based on that described by Guesdon, J. L. et al. in J. Hischem Cytochem. 1997, 27: 1131-11139. The solution containing purified rabbit anti- human TK IgG is dialysed against 0.1 M sodium bicarbonate buffer, pH 9.5, at 4°C for 24 hours and added to 10 ml freshly prepared 0.1 M biotinyl-N hydroxysuccinimide ester (dissolved in dimethyl formamide). The reaction is carried out at room temperature for 1 hour and the mixture is dialysed against PBS. An equal volume of double-distilled glycerol is added, and the biotin-labelled anti-human TK IgG is stored at -80°C. Human TK-specific immunoassav

Microtiter plates (96-well, Corning) are coated with non-labelled antihuman TK IgG (2 mg/ml, 100 μl per well) overnight at 4°C. The plates are then blocked with 200 ml PBS (10 mmol/l sodium phosphate, pH 7.4, 150 mmol/l NaCI) containing 1% bovine serum ml albumin at 37°C for 1 hour. The plates are washed three times with PBS containing 0.1% Tween^® 20 (washing solution). Purified human TK standard (0.04 to 2.5 ng/ml) and serum samples are added to individual wells in a total volume of 100 Dl PBS containing 0.05% Tween-20 and 0.5% gelatin (dilution buffer). The plates are incubated at 37°C for 90 min. After incubation, the plates are washed three times with the washing solution. One hundred Dl of 1 Dg/ml biotin-labelled anti-human TK IgG diluted in the dilution buffer is added to each well. The reaction is carried out at 37°C for 1 hour. After incubation, excess labelled IgG is washed off three times with the washing solution. One hundred Dl of 1 Dg/ml peroxidase-avidin diluted in the dilution buffer is added to each well, and the plates are incubated at 37°C for 1 hour. After incubation, the plates are washed five times with the washing solution and once with PBS. The colour reaction is performed by adding 100 D I freshly prepared substrate solution [(0.03% 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and 0.03% H₂0₂ in 0.1 M citrate buffer, pH 4.31] to each well and incubating at room temperature for 30 min. The plates are read at 414 nm on a Titertek Multiskan ELISA reader (Flow Laboratories). A calibrator standard curve is constructed by plotting the concentrations of the purified TK used in the assay against the readings obtained at 414 nm. As an alternative, standard and sample reading may be performed at 405 nm. The amount of TK in the serum samples is determined for each test sample by comparison with the calibrator standard curve. CLINICAL TESTS Serum TK levels were measured in a consecutive series of patients admitted to Medical University of Sassari. Excluded from the study were patients with major cardiac, renal, hepatic, or cancerous disease or infection. Age-matched healthy subjects were used as control subjects. All recruited subjects were Caucasian. Risk factors such as smoking, hypertension, diabetes, and hypercholesterolemia were also assessed. Severity of the disease was classified on clinical grounds and on the basis of Doppler PW evaluation and carotid angiography. Angiograms were reviewed by two skilled neuro-radiologists and were used to determine severity of carotid obstruction that was then graded according to NASCET (North American Symptomatic Carotid Endarterectomy Trial Collaborators. N Engl J Med. 1991 ; 325: 445-453) and collateral flow pattern was established according to the classification proposed by Ozgur HT et al. (Am J Neuroradiol. 2001; 22:928-936). Blood samples for TK measurements were obtained between 8 and 9am from an indwelling intravenous catheter inserted into the left brachial vein of patients, who had remained in recumbent position since the night before. In subsets of patients, measurements were repeated during the first days or 3 and 12 months after revascularization. Sera obtained from blood samples by conventional procedure was stored at -20°C until assay. TK concentration in sera was measured in duplicate with the ELISA described above by a researcher who was blind to subject identification. Results were expressed as mean±SEM (Standard Error of the Mean). Multivariate statistical analysis was performed to identify possible influence of aggregate risk factors on the parameters under study, and then univariate analysis was used to check the impact of each single risk factor. The SigmaStat statistical package (Jandel Co) was used for all analyses. Results Sixty-seven patients (53 men and 14 women; age, 42 to 85 years; mean age, 70 years) and 45 control subjects (33 men and 12 women; age, 40 to 82 years; mean age, 66 years) were studied. Among patients, twenty-nine (43.2%) were asymptomatic, 19 (28.4%) had experienced one or more episodes of transient ischemic attack (TIA), and the remaining 19 (28.4%) had suffered stroke. Angiographic evidence of unilateral carotid obstruction was obtained in 41 (61 %) while bilateral carotid lesions were detected in the remainders (39%). The examined patients were further stratified according to the severity of obstruction as following (in the case of bilateral lesions, the more stenotic side was considered for grading):

- Moderate-grade, 50-69% CAS (n=10, 14.9%); - High-grade, 70-99% CAS (n=40, 59.7%);

- Carotid Occlusion (CAO, n=17, 25.4%).

Collateral flow pattern was determinable in 28 angiograms. Accordingly, patients were classified as stage 1 (n=2), 2 (n=6), 3 (n=16), and 4 (n=4), with the type 1 being the least compromised and type 4 the most. By Pulsed Doppler ultrasonography, we documented plaque instability in 39 (58%). Instability recognised by Doppler was confirmed by gross examination in all patients who underwent reconstructive surgery. Risk factors (age over 65, gender, smoke, hypercholesterolemia, diabetes, hypertension, plaque instability) were similarly represented among patients classified according to severity of obstruction. Asymptomatic peripheral obstructive lesions were present in 12 patients. Serum Levels of TK and carotid artery obstruction grade. Inter-assay variability was less than 10% and inter-assay variability less than 5%. TK averaged 1164±148 pg/ml (ranging from 199 to 5894 pg/ml, PO.01 vs. healthy controls) and in 48 (72%) exceeded the upper confidence limits of normal distribution (375±33 pg/ml, ranging from 160 to 658 pg/ml). TK levels increasingly augmented as a function of obstruction grading (Figure 1 ), from 595±36 in 50-69% CAS (P<0.05), 1175±169 in 70-99% CAS (PO.01 ), and 1534±426 pg/ml in CAO (P<0.01). Accordingly, the number of patients exceeding the upper limits of normal distribution was 5 (50%) of 10 in 50-69% CAS, 30 (75%) of 40 in 70-99% CAS, and 13 (76%) of 17 in CAO. The highest levels were associated with previous ischemic stroke (1376±352 pg/ml) or TIA (1225±314 pg/ml), corresponding to a figure of 74% above upper limits of normalcy. However, the biomarker was increased also in 20 (69%) of 29 asymptomatic patients (averaging 1015±174 pg/ml). Furthermore, tK levels were positively correlated with collateral angiographic score (r=0.61 , P<0.001 ). No interaction was detected between tK levels and gender, single or associated risk factors, hematological tests, or plaque instability.

The results reported above indicate that circulating TK levels increases in direct correlation with carotid artery obstruction grade and are independent from associated risk factors. Consequently, TK measurement in sera obtained from peripheral blood represents a useful screening test in the population at risk for stroke.

Biomarker Levels and Risk of Stroke.

Patients were stratified according to provisional risk of stroke as following:

1 ) High-risk, encompassing symptomatic 70-99% CAS or CAO;

2) Moderate-risk, encompassing asymptomatic 50-79% CAS or symptomatic 50- 69% CAS.

Among 50 patients of the high-risk subgroup (Figure 2, right), TK levels were high in 74%. Among the 17 patients of the moderate-risk subgroup (Figure 2, left), TK level was increased in 65%. All high-TK patients, classified at moderate-risk were asymptomatic, except one who had experienced ischemic transitory attack (TIA). Eight had unilateral and the remainder 3 bilateral CAS. The degree of accuracy for TK levels in distinguishing patients with different degree of risk was 64%. Based on angiographic criteria, revascularization was recommendable in 74% of this series of patients. However, when taking into account of tK levels, the figure reached 91 %.

The above results indicate the utility of TK as a biomarker of cerebral ischemia. Further, measurement of TK may have special utility for recognising patients with moderate CAS, either symptomatic or not, who may need reinforcement of current therapy or operative revascularization. The issue is clinically important since therapeutic decision in this category of patients remains controversial, as documented in clinical trials such as NASCET (North American Symptomatic Carotid Endarterectomy Trial Collaborators. N Engl J Med. 1991 ; 325: 445-453), ACAS (Asymptomatic Carotid Atherosclerosis Study. JAMA. 1995; 273:1421- 1428) and VACS (Veterans Affairs Cooperative Study. N Engl J Med. 1993; 328:221-227).

One major limitation of NASCET, ACAS, and VACS consists of that these clinical trials heavily rely on angiography for calculation of risk grading. Unfortunately, angiography has a modest informative power with regard to cerebral hemodynamics. Therefore, a number of subjects with high-degree CAS are currently exposed to the risks of revascularization without assessing if brain perfusion is preserved by an efficient collateralization. On the other hand, moderate-degree CAS are usually dismissed, without the necessary attention to individual hemodynamic status. Given the prevalence of this category of patients in the general population, a significant number could eventually benefit from reperfusion.

Newly developed techniques, such as SPECT, are now available for assessing cerebrovascular perfusion. However, screening of low-risk population for identification of patients with significant cerebrovascular insufficiency is currently precluded by both the high cost of the procedure and the long waiting lists. By contrast, measurement of serum TK as a biomarker of cerebral ischemia is easy, rapid, and affordable. Moreover, it requires minimal laboratory work-up and is more acceptable and safe to the patient.

Circulating TK assay may also be useful for reinforcing any available preventive measures aimed at delaying the progression of the disease, including the development of cognitive impairment and dementia, and at ameliorating the quality of life.

Assay of Serum TK and Clinical Follow up

Thirty-five patients (23 with unilateral and 12 with bilateral CAS) underwent revascularization by carotid endoarteriectomy (CEA). The procedure was successful in 33 patients. In the remainders, post-operative complications occurred, consisting 1 ipsilateral stroke and 1 TIA.

TK was re-examined consecutively during the first 3 days or at 3 and months from CEA. As shown in Figure 3, improving brain perfusion by CEA results in significant reduction of the circulating TK. This effect starts by the first day and reaches half of preoperative values by 3 days after surgery.

In 17 vascular patients, the biomarker was measured again 3 months after surgery. On the same occasion, restenosis was excluded by ultrasound examination. TK levels were reduced from a pre-operative value of 862±147 to 416±56 pg/ml (P<0.01). As shown in Figure 4A and 4B, the decrease in TK level was particularly evident in patients who underwent CEA for unilateral CAS. Patients with bilateral CAS are usually revascularized at 2 different occasions. These patients not always experienced a decrease of TK levels following the first CEA (Figure 4C and D). However, the decrease in TK circulating levels following the second CEA was universal. Thus, persistently elevated TK levels, indicative of residual brain ischemia, underline the opportunity of revascularization of the artery initially left untouched. Serum TK levels increased following CEA in the 2 patients that experienced peri- operative ischemic complications (Figure 5). Finally, in 18 patients, tK was measured 1 year after CEA. On the same occasion, restenosis was excluded by angiography. As shown in Figure 6, tK declined from 1410±352 to 482±76 pg/ml (P<0.01) and the percent of patients with biomarker levels above upper limits of normal distribution dropped from 87 to 27%, regardless they showed or not neurological symptoms prior to revascularization. To exclude the possibility that early post-operative changes were due to tissue injury, the biomarker was assayed in 10 patients who underwent different surgical procedures of the neck. We found that tK levels remained unchanged before (430±23 pg/ml) and after 3 (415±12 pg/ml) or 7 days (439±18 pg/ml) from surgery (P=N.S.).

The current results indicate that assaying serum TK concentration provides useful information on the course of cerebrovascular insufficiency after revascularization, inasmuch successful reperfusion is always associated with a drop in circulating TK levels; while peri-operative complications are associated with increases in TK levels.

It will be understood that, similar to other types of accepted disease diagnostic and monitoring methods, the present biomarker will not be useful on every patient diagnosed with cerebral ischemia to make therapeutic decisions. Rather, the physician will use TK values in combination with other diagnostic values and clinical observations to diagnose the onset of cerebral ischemia, and further to develop a course of treatment and therapy for each individual patient. It is also contemplated that monitoring TK levels will provide a means for monitoring the progress of a course of therapy for an individual patient. In conclusion, circulating tK is elevated in patients suffering atherosclerosis- induced vascular disease and, being directly implicated in the reparative response, it provides insight into the disease process itself. Therefore, measurement of tK represents a simple and accurate way for assessing the severity of carotid disease and making the point on the necessity of treatment reinforcement in symptomatic or asymptomatic patients.

Claims

1. An immunoglobulin specific for human tissue kallikrein, uncoupled or bound to a tissue kallikrein inhibitor, or specific for a portion of the tissue kallikrein molecule.

2. An immunoglobulin specific for human tissue kallikrein, uncoupled or bound to a tissue kallikrein inhibitor, or specific for a portion of the tissue kallikrein molecule, obtainable by immunising a host animal with a suitable immunogen comprising tissue kallikrein or a tissue kallikrein fragment.

3. The immunoglobulin according to claim 2, wherein the said immunogen is selected from the group consisting of an immunogen conjugate comprising a synthetically prepared peptide having an aminoacidic sequence that includes an epitope of tissue kallikrein, purified tissue kallikrein from urine or from other biological fluids, and tissue kallikrein produced by genetic engineering and molecular biology methods.

4. The immunoglobulin according to claim 1 , wherein the said immunoglobulin is selected from the group consisting of IgG and IgG fragments, Fab, Fab' and

F(ab').sub.2.

5. The immunoglobulin according to claim 2, wherein the said immunoglobulin is selected from the group consisting of IgG and IgG fragments, Fab, Fab' and F(ab').sub.2.

6. A process for the preparation of immunoglobulin specific for human tissue kallikrein or for human tissue kallikrein fragments comprising the immunisation of a host animal with a suitable immunogen comprising tissue kallikrein or tissue kallikrein fragments.

7. An immunologic formulation comprising an immunoglobulin specific for tissue kallikrein, or a fragment of said immunoglobulin, and a detection agent.

8. The immunologic formulation according to claim 7, wherein the said detection agent is selected from the group consisting of biotin-avidin-peroxidase complex, biotin-avidin-alkaline phosphatase, and fluorescein.

9. A diagnostic kit for the diagnosis of cerebral ischemia comprising an immunological formulation comprising an immunoglobulin specific for tissue kallikrein or a fragment of said immunoglobulin, and a detection agent.

10. The diagnostic kit according to claim 9, wherein the detection agent is selected from the group consisting of biotin-avidin-peroxidase complex, biotin-avidin- alkaline phosphatase, and fluorescein.

11. The diagnostic kit according to claim 9, additionally comprising a control standard.

12. The diagnostic kit according to claim 9, additionally comprising one or more buffers to be used for dilution, washing and maintenance of the optimal pH.

13. A method for measuring the tissue kallikrein amount in a biologic sample, comprising the following steps: formation of an immune complex between an immunoglobulin, specific for the whole tissue kallikrein or a tissue kallikrein fragment, and tissue kallikrein in a biological sample to be analysed, and between the above immunoglobulin and tissue kallikrein in a reference standard in which tissue kallikrein concentration is known; detection of the immune complex coming from step 1 ); comparison of the results obtained in the biological sample to be analysed with those obtained for the reference standard.

14. The method according to claim 13, wherein the step 2) is performed by using a immunoenzymatic or radioimmunologic system.

15. The method according to claim 13, wherein the step 2) is performed by using a detection agent based on a biochemical enzyme-substrate reaction leading to colour production or fluorescence.

16. A method for the diagnosis of cerebral ischemia comprising the following steps: measurement of tissue kallikrein levels in a biological sample by reaction with an immunoglobulin specific for tissue kallikrein, uncoupled or bound to a tissue kallikrein inhibitor, or specific for a portion of the tissue kallikrein molecule, evaluation of the severity of cerebral ischemia, where the increase in tissue kallikrein levels in the biological sample in comparison with the tissue kallikrein average in healthy population is directly proportional to carotid artery obstruction grade and to cerebrovascular insufficiency.

17. The method according to claim 16, wherein step a) consists of a series of measurements on biological samples collected over time and wherein in the evaluation of step b) over time increases in tissue kallikrein values indicates a deteriorating condition, while decreases in tissue kallikrein levels indicate an improving condition.