WO2004005934A2

WO2004005934A2 - Toxicity markers

Info

Publication number: WO2004005934A2
Application number: PCT/GB2003/002893
Authority: WO
Inventors: Lasantha Ranasinghe-Bandara
Original assignee: Oxford Glycosciences (Uk) Ltd
Priority date: 2002-07-04
Filing date: 2003-07-04
Publication date: 2004-01-15
Also published as: AU2003281287A8; AU2003281287A1; WO2004005934A3

Abstract

The present invention relates to the use of the following polypeptides: T-kininnogen, alpha-1-macroglobulin, apolipoprotein E, clusterin, complement C3, complement C4, fibrinogen-alpha-chain, fibrinogen-beta-chain, inter-alpha-inhibitor H4P heavy chain and plasma retinol binding protein, in the screening or diagnosis of kidney toxicity. Specifically, their use in screening for kidney toxicity effects in drug development is contemplated.

Description

TOXICITY MARKERS

1. INTRODUCTION

The present invention relates to the use of the following polypeptides: T-kininnogen, alpha-1- macroglobulin, apolipoprotein E, clusterin, complement C3, complement C4, fibrinogen-alpha-chain, fibrinogen-beta-chain, inter-alpha-inhibitor H4P heavy chain and plasma retinol binding protein, in the screening or diagnosis of kidney toxicity. Specifically, their use in screening for kidney toxicity effects in drug development is contemplated.

2. BACKGROUND OF THE INVENTION

The mammalian kidney is an extremely complex organ, both anatomically and functionally, and plays an important role in the control and regulation of homeostasis. Thus, the kidney has a key role in the regulation of extracellular fluid volume and electrolyte composition. It is also the site of hormone synthesis and production of certain vasoactive prostaglandins and kinins that influence systemic metabolic function (Brenner B M and Rector Jr F C, Eds (1991) The Kidney, 4^th edition, 1-2, Saunders, Philadelphia & Seldin D W & Giebisch G, Eds (1985) The Kidney: Physiology and Pathophysiology, 1-2, Raven Press, New York, 1-36).

The kidney can be divided into two major anatomical areas - the cortex and the medulla. The functional anatomy of the kidney is based on the nephron structure, which has three separate elements, the vasculature, the glomeruli and the tubular component. All nephrons have their major vascular components and glomeruli in the cortex. The proximal convoluted tubules (pars convoluta/S 1 & S2 regions) are located in the cortex, with the straight portions of the proximal tubules (pars recta /S3 region) extending into the outer medulla. For a more detailed description, see Kinter and Short, 1993, in Toxicology of the Kidney, 2^nd edn, Eds. J B Hok & R S Goldstein, Raven Press, New York.

Since the cortex forms the major part of the kidney it receives most of the blood supply, about 80% of total renal blood flow. Thus when a foreign chemical, e.g. a drug, enters the blood stream, a high percentage will be delivered to the cortex and, hence, have a greater chance of altering cortical function. The renal medulla receives a much lower blood supply and hence the delivery of foreign chemicals to it is lower than in the cortex. However, as the chemical passes down the nephron, the countercurrent mechanism present in this region may lead to the chemical becoming concentrated in the medulla and in the papilla to a concentration many times that in the plasma. This has been reported for example for paracetamol (Duggin and Mudge, 1976, J. Pharmacol. Exp. Ther; 199, 1-9).

Maintenance of renal function requires the delivery of large quantities of oxygen and metabolic substrates to the kidney. Thus the kidney, especially the pars recta of the proximal tubule, is particularly susceptible to agents that produce cellular anoxia - for instance a decrease in blood pressure or blood volume, as in a shock or haemorrhage. Once a chemical has been delivered to the kidney and concentrated within renal tubular cells, the chemical may act directly on key enzymes, such as those involved in energy metabolism, or may itself undergo metabolism to either generate a chemically reactive moiety or become detoxified.

A combination of the ability of chemicals to concentrate in certain regions of the nephron and the heterogeneity of chemical metabolising enzymes along the nephron are contributory factors to site- specific renal injury.

Thus, a toxic insult to the kidney, may affect some or all of its functions. A toxic insult may also change over time, for example beginning with the formation of acidic vascular inclusions and transitioning to collagen fibre deposition over time.

Given the high degree of variability in its causes and classifications, there currently is no specific measure of kidney toxicity. The following list outlines currently validated measures of kidney homeostasis: Nonintrusive assays, such as, serum creatinine and blood urea nitrogen (BUN) levels; creatinine clearance rates; urine creatinine and protein levels; radioisotope metabolic labelling or soft tissue imaging, including, sonography, magnetic resonance imaging and computed tomography. The non- intrusive assays show poor correlation with kidney histopathology and generally provide no prospective measure of how the kidney will further change over time. The only reliable way of identifying structural alteration to the kidney is invasive using histological examination in experimental animals. In humans, renal biopsy is sometimes used to aid diagnosis. In addition, the intrusive assays require time-consuming and costly interpretation by expert pathologists.

Due to the costly and time consuming nature of existing, often ambiguous, tests it would be highly desirable to measure a substance or substances in samples of blood or kidney that would lead to a positive diagnosis of specific kidney toxicity or that would help to exclude kidney toxicity from a differential diagnosis.

In the development of new pharmaceutical compositions, kidney toxicity is a major cause of compound attrition and is not always efficiently predicted by routine toxicity studies in animals using standard parameters for assessment of kidney damage. Traditionally, toxicologists have defined the preliminary risks of a new compound to human safety using animal studies, as recommended by the

International Conference on Harmonization (ICH, www.mcclurenet.com/ICHsafety.html), together with histopathological and biochemical techniques (Evans, G O & Davies, D T, 1996, In Animal Clinical Chemistry, Evans G O ed., p 1-19, Taylor and Francis). Some tissue and plasma specific biomarkers of kidney toxicity are provided in WO 02/054081. In order to change the current approach to safety assessment in animals and humans, both in the laboratory and the clinic we need to improve our understanding of kidney toxicity and improve the predictability of preclinical testing. Therefore, there is a need to identify more sensitive and predicitive kidney toxicity markers that can be used to screen canditate compounds for their ability to induce specific kidney toxicity. Such new markers will enable kidney toxicity to be detected at lower drug doses than is possible using conventional methods, such as histopathology and clinical chemistry. The implications for drug development are that, at early stages of the drug discovery process, candidate compounds could be ranked according to their toxic effects. This would inevitably contribute to significant financial savings in the latter stages of the development process. Kidney toxicity was investigated using models of chemically induced kidney toxicity. Specifically, three categories of kidney toxicity have been defined; renal tubular necrosis, glomerular injury and papilla injury.

'T-kinninogen' is part of a family of rat kinnogens which all show differing biological properties. Rats express a HMW (high molecular weight) and a LMW (low molecular weight) kinninogen, produced by alternative splicing of the same gene. In addition, they express a 'LMW-like' T-kininogens. LMW T- kinninogen precursor has at least three known variants entered in the Genbank database under the following accession numbers: LMW T-kininogen-1 205085; T kininogen-I 125520 and T kininnogen-II 125521. LMW T-Kininogen (I/IT) is an acute phase protein and possibly a precursor of T-kinin (although it appears not to be released by tissue or plasma kalikrein) and may possibly act as a cysteine protease inhibitor.

The present invention provides novel protein markers of kidney toxicity. 3. SUMMARY OF THE INVENTION

The present invention provides T-kinninogen and/or inter-alpha-inhibitor H4P heavy chain as novel markers of kidney toxicity. The invention also provides the use of T-kinninogen and/or inter-alpha- inhibitor H4P heavy chain optionally in combination with one or more of the following polypeptides, alpha-1-macroglobulin, apolipoprotein E, clusterin, complement C3, complement C4, fibrinogen-alpha- chain, fibrinogen-beta-chain, inter-alpha-inhibitor H4P heavy chain or plasma retinol binding protein polypeptides for the diagnosis and/or screening of kidney toxicity. The present invention provides methods for screening and diagnosis of kidney toxicity and in particular, the screening of environmental factors, such as drug compounds e.g. for their ability to induce kidney toxicity.

The invention provides methods for screening or diagnosis of kidney toxicity in a target cell, tissue or mammal which comprises the step of detecting and/or quantifying in a test sample obtained from said target T-kinninogen and/or inter-alpha-inhibitor H4P heavy chain optionally in combination with one or more of the following polypeptides: alpha-1-macroglobulin, apolipoprotein E, clusterin, complement C3, complement C4, fibrinogen-alpha-chain, fibrinogen-beta-chain, or plasma retinol binding protein polypeptides as defined herein. The invention also provides kits that may be used in the above recited methods.

The methods and kits of the invention may also find use in monitoring the effectiveness of treatment for kidney toxicity, for selecting participants in clinical trials, for identifying patients most likely to respond to a particular therapeutic treatment and for screening and development of drugs. 4.0 DETAILED DESCRIPTION OF THE INVENTION The present invention provides the use of T-kinninogen and/or inter-alpha-inhibitor as markers of kidney toxicity. It further provides the use of T-kinninogen and/or inter-alpha-inhibitor optionally in combination with one or more of the following polypeptides: H4P heavy chain alpha- 1-macroglobulin, apolipoprotein E, clusterin, complement C3, complement C4, fibrinogen-alpha-chain, fibrinogen-beta- chain, and plasma retinol binding protein polypeptides for the screening or diagnosis of kidney toxicity. 4.1 Diagnosis of Kidney Toxicity

The present invention provides a method for screening or diagnosis of kidney toxicity in a target cell, tissue or mammal which comprises the step of detecting and/or quantifying in a test sample obtained from said target T-kinninogen and/or inter-alpha-inhibitor H4P heavy chain optionally in combination with one or more of the following polypeptides: alpha- 1-macroglobulin, apolipoprotein E, clusterin, complement C3, complement C4, fibrinogen-alpha-chain, fibrinogen-beta-chain, or plasma retinol binding protein polypeptides.

In accordance with the present invention, a test sample e.g. tissue, blood, serum, plasma or urine obtained from a target is used for diagnosis of kidney toxicity. In one embodiment of the invention, an increased abundance of T-kinninogen and /or inter-alpha-inhibitor H4P heavy chain optionally in combination with one or more of the following polypeptides: complement C3, complement C4, fibrinogen-alpha chain, fibrinogen-beta chain in a first sample or sample set compared to a second sample or sample set or a previously determined reference range indicates the presence of kidney toxicity. Preferred, is T-kinninogen polypeptides.

In another embodiment of the invention, an increased abundance of T-kinninogen and /or inter- alpha-inhibitor H4P heavy chain optionally in combination with a decreased abundance of one or more of the following polypeptides: alpha- 1-macroglobulin, apolipoprotein E, clusterin, fibrinogen-alpha chain, fibrinogen-beta chain or plasma retinol binding protein (or any combination of them) in a first sample or sample set compared to a second sample or sample set or a previously determined reference range indicates the presence of kidney toxicity. In yet another embodiment, the relative abundance of one or more polypeptides for use in the invention (or any combination of them) in a first sample or sample set relative to a second sample or sample set or a previously determined reference range indicates the degree or severity of kidney toxicity.

In any of the aforesaid methods, detection of one or more of the polypeptides for use in the invention as described herein may optionally be combined with detection of one or more additional biomarkers for kidney toxicity.

Preferably, kidney toxicity is detected in a mammal, more preferably in a rodent, e.g. a rat. Any suitable method in the art can be employed to measure the level/expression of polypeptides for use in the invention, including but not limited to, two dimensional electrophoresis, kinase assays or immunoassays to detect and/or visualize the polypeptides for use in the invention (e.g. competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, immunocytochemistry, fluorescent immunoassays and protein A immunoassays. Alternatively, an assay for polypeptide activity may be used to measure polypeptide expression. A preferred method of detection is by immunoassay. In a further embodiment, an increased abundance of mRNA encoding one or more polypeptides for use in the invention in a first sample or sample set relative to a second sample or sample set or previously determined reference range indicates the presence of kidney toxicity. Any suitable hybridization assay can be used to detect polypeptide expression by detecting and or visualizing mRNA encoding the polypeptide for use in the invention (e.g. Northern assays, dot blots, in situ hybridization, etc.).

In a specific embodiment, the polypeptides for use in the invention are detected and/ or quantified using one or more capture reagents that specifically binds to a polypeptide for use in the invention.

As used herein "caputure reagent" refers to an agent that specifically recognises and binds a polypeptide for use in the invention e.g. an antibody. Such capture reagents are useful in the preparation of a kit for screening or diagnosis of kidney toxicity, as described herein.

Thus, in another embodiment of the invention, one or more labelled capture reagents, e.g. antibodies, derivatives and analogs thereof, which specifically bind to a polypeptide for use in the invention can be used for diagnostic purposes to detect, diagnose, or monitor kidney toxicity.

Preferred capture reagents bind specifically to polypeptides for use in the present invention so that they can be used to detect, purify and/or inhibit the activity of said polypeptides. Preferably, the capture reagent is an antibody specific for a polypeptide for use in the invention. More preferably, an antibody specific for a polypeptide for use in the invention preferentially binds to said polypeptide rather than to other isoforms of the same protein. In a preferred embodiment, said antibody binds to said polypeptide with at least 2-fold greater affinity, more preferably at least 5 -fold greater affinity, still more preferably at least 10-fold greater affinity, than to other isoforms of the same protein.

4.1.1 Assessing the effects of an environmental factor on kidney toxicity.

The methods of the invention can be used to evaluate drug candidates for their ability to induce kidney toxicity. This aspect of the invention is particularly useful in drug development in the pharmaceutical sector. Environmental factors which may be tested according to the invention include any physical, chemical or biological factors which have the potential to induce unwanted kidney toxicity or relieve kidney toxicity effects. The environmental factor is preferably an exogenous compound, for example a candidate drug for use in medicine.

In one embodiment, environmental factors are tested for their ability to increase levels of T- kinninogen and/or inter-alpha-inhibitor H4P heavy chain optionally in combination with one or more of the following polypeptides: complement C3, complement C4, fibrinogen-alpha chain, fibrinogen-beta chain or in a subject treated with said environmental factor compared to said levels found in subjects not treated with the environmental factor (i.e. control subjects or subjects free from kidney toxicity), which indicates the ability of the environmental factor to induce specific kidney toxicity effects. Preferably the levels of one or more of the following protein isoforms: LMW-TKPI-1, LMW-TKPI-2, LMW-TKPI-3, LMW-TKPI-4, LMW-TKPI-5, LMW-TKPI-6, C3-PI-1, C3-PI-2, C4-PI-1, C4-PI-2, FA-PI-1, FB-PI-1, H4P-1, H4P-2 or H4P-3, as detailed in Table I, are assessed. Preferred protein isoforms are LMW-TKPI- 1, LMW-TKPI-2, LMW-TKPI-3, LMW-TKPI-4, LMW-TKPI-5, LMW-TKPI-6, C3-PI-1, C3-PI-2, H4P- 1, H4P-2 or H4P-3. Highly preferred protein isoforms are LMW-TKPI-1, LMW-TKPI-2, LMW-TKPI-3, LMW-TKPI-4, LMW-TKPI-5, or LMW-TKPI-6. In another embodiment, environmental factors are tested for their ability to increase levels of T- kinninogen and or inter-alpha-inhibitor H4P heavy chain optionally in combination with decreases in levels of one or more of the following polypeptides: alpha- 1-macroglobulin, apolipoprotein E, clusterin, fibrinogen-alpha chain, fibrinogen-beta chain or plasma retinol binding protein, in a subject treated with said environmental factor compared to said levels found in subjects not treated with the environmental factor (i.e. control subjects or subjects free from kidney toxicity), which indicates the ability of the environmental factor to induce specific kidney toxicity. Preferably the levels of one or more of the following protein isoforms: A1M-PI-1, A1M-PI-2, APOE-PI-1, CLUS-PI-1, FA-PI-2, FB-PI-1 or PRBP- PI-1, as detailed in Table I, are assessed. Preferred protein isoforms are A1M-PI-1, A1M-PI-2 or PRBP- PI-1. In another embodiment, environmental factors are tested for their ability to restore levels of one or more polypeptides defined above in a subject having kidney toxicity to levels found in subjects free from kidney toxicity, or to preserve levels of one or more polypeptides of said polypeptides at or near non-kidney toxicity values. Preferably the levels of one or more protein isoforms, as listed in Table I, are assessed. In another embodiment, the present invention is used to screen candidates for a clinical study to identify, individuals having kidney toxicity, such individuals can then be either excluded from or included in the study or can be placed in a separate cohort for treatment or analysis. If desired, the candidates can concurrently be screened to identify individuals with elevated blood urea nitrogen or creatine; procedures for these screens are well known in the art. In additional aspects, the invention provides a method for predicting the ability of an environmental factor to cause kidney toxicity comprising: a) exposing a target cell, tissue or mammal to the environmental factor; b) obtaining a test sample from the target; c) detecting and/or quantifying in the sample one or more of the polypeptides defined above; and d) comparing the abundance of one or more of said polypeptides in the test sample with the abundance of said polypeptide in a control sample, or with a previously determined reference range for said polypeptide in targets free from kidney toxicity, or with the abundance at least one standard polypeptide in the test sample.

The invention also provides a method for predicting the ability of an environmental factor to cause kidney toxicity comprising: a) providing a plurality of identical spatial arrays of capture reagents comprising at least one capture reagent that specifically binds to one or more of the polypeptides defined above; b) exposing a target cell, tissue or mammal to the environmental factor; c) extracting and isolating a polypeptide containing mixture from said exposed kidney target of step b); d) extracting and isolating a control polypeptide containing mixture from an equivalent kidney cell, tissue or mammal not exposed to said environmental factor of step c); e) exposing a spatial array as defined in a) to the mixture extracted in d), and detecting the binding of polypeptides to the capture reagents; f) exposing a spatial array as defined in a) to the mixture extracted in c) and detecting the binding of polypeptides to the capture reagents; g) comparing the first and second binding patterns detected in step e) and f) to identify any change in said test pattern from the control pattern, indicative of the toxic effect of the environmental factor.

Those skilled in the art will appreciate that the use of a control target may not always be necessary once a standard reference for comparison has been established.

In a specific embodiment the test sample is obtained within 21 days, e.g. 7 days, of exposure to the environmental factor. Preferably the test sample is obtained within 12h, 24h or 48h of exposure to the environmental factor.

The skilled person will appreciate that one or more of the polypeptides defined above can then be detected and/or quantified, in the test sample, at any convenient time point. Preferably the test sample is stabilised to prevent degradation of one or more of said polypeptides present in said test sample.

In a preferred embodiment, environmental factors that modulate the abundance (i.e. upregulate or downregulate) the expression, activity or both the expression and activity of a polypeptide for use in the invention, as described herein, are identified in a mammalian subject e.g. an animal model. Examples of suitable animals include, but are not limited to, mice, rats, rabbits, monkeys, guinea pigs, dogs and cats. A preferred animal model is the rodent, e.g. a rat. In accordance with this embodiment, the environmental factor or a control agent is administered (e.g. orally, rectally or parenterally such as intraperitoneally or intravenously) to a suitable animal and the effect on the expression, activity or both expression and activity of the polypeptide is determined. Changes in the expression of a polypeptide can be assessed by any suitable method described above, e.g. immunoassay. 4.1.2 Kits of the invention

The invention also provides diagnostic kits for use in the methods of the invention, comprising one or more capture reagents specific for a polypeptide for use in the invention. In addition, such a kit may optionally comprise one or more of the following: (1) instructions for using the capture reagent of the invention for screening or diagnosis of kidney toxicity or any combination of these applications; (2) a labelled binding partner to the capture reagent; (3) a solid or semi-solid phase (such as a reagent strip) upon which the capture reagent of the invention is immobilized; and (4) a label or insert indicating regulatory approval for diagnostic use for kidney toxicity or any combination thereof. If no labelled binding partner to the capture reagent is provided, the capture reagent of the invention itself can be labelled with a detectable marker.

Hence in one aspect, the capture reagent is labelled, for example with a radioactive label (such as

P, S or I), a fluorescent label (such as fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde or fluorescamine), chemiluminescent or enzymatic label to enable detection of an interaction with an capture reagent. Alternatively, the capture reagent can be biotinylated using techniques well known to those of skill in the art (e.g. biotinylation kit, Pierce Chemicals; Rockford, IL). The ability of the capture reagent to interact directly or indirectly can be determined by methods known to those of skill in the art. For example, the interaction between an capture reagent of the invention and a polypeptide for use in the invention can be determined by flow cytometry, a scintillation assay, immunoprecipitation or western blot analysis. Another aspect of the invention provides for the use of a plurality of capture reagents in the preparation of a diagnostic array for clinical screening or diagnosis of kidney toxicity. Preferably the capture reagent are antibodies. The capture reagents can be selected for use as diagnostic markers of specific kidney toxicity indications, such as markers of renal tubular necrosis, glomerular injury or papilla injury. Preferably the array will contain markers that are diagnostic for all three indications of kidney toxicity. Preferably, the capture reagent is first immobilized, by, for example, contacting it with an immobilized antibody which specifically recognizes and binds it, or by contacting a purified preparation of capture reagent with a surface designed to bind proteins. The capture reagent may be partially or completely purified (e.g. partially or completely free of other polypeptides). The invention also provides a kit comprising a nucleic acid probe capable of hybridizing to RNA encoding a polypeptide for use in the invention. In a specific embodiment, a kit comprises in one or more containers a pair of primers (e.g. each in the size range of 6-30 nucleotides, more preferably 10-30 nucleotides and still more preferably 10-20 nucleotides) that under appropriate reaction conditions can prime amplification of at least a portion of a nucleic acid encoding a polypeptide for use in the invention, such as by polymerase chain reaction (see, e.g. Innis et al., 1990, PCR Protocols, Academic Press, Inc., San Diego, CA), ligase chain reaction (see EP 320,308) use of Q replicase, cyclic probe reaction, or other methods known in the art.

A kit can optionally further comprise a predetermined amount of an isolated polypeptide or a nucleic acid molecule as defined above, e.g. for use as a standard or control. Another aspect of the invention provides for the use of a plurality of capture reagents in the preparation of a diagnostic array for clinical screening or diagnosis of kidney toxicity. Hence, kits are also provided wherein the capture reagent is optionally provided within a spatial array of additional capture reagents that specifically bind to one or more other known biomarkers of kidney toxicity, e.g. those described in WO02/054081. Kits are also provided which may optionally be combined with one or more additional capture reagents polypeptides of known biomarkers for kidney toxicity.

The capture reagents can be selected for use in diagnostics of specific kidney toxicity indications, such as renal tubular necrosis, glomerular injury or papilla injury. Preferably the kits will contain capture reagents that can be used in diagnostics for renal tubular necrosis. More preferably, all three kidney toxicity indications are diagnosed. 4.2 Definitions

"Kidney Toxicity" refers to and includes the physiological manifestation or derangement in kidney function, and/or other organ or cellular function and/or any condition that comes about from the interaction of the kidney with an 'environmental factor'. Kidney toxicity includes but is not limited to any aspect of destruction to kidney cells, e.g. nephron cell metabolic pathway modulation and conditions for example, such as but not limited to, glomerular / proximal tubular nephritis, glomerular / papillary necrosis, acute renal failure, chronic renal failure, and end-stage renal disease. Environmental factors that may induce kidney toxicity include, but are not limited to, xenobiotics, chemicals, viruses and other biological agents. In the context of the present invention the test samples are tissue or body fluids, for example, blood, serum, plasma or urine. Preferably the body fluid sample is a plasma or urine sample and more preferably the body fluid is urine. "Plasma" refers to the supernatant fluid produced by inhibition of clotting (for example, by citrate or EDTA) and centrifugal sedimentation of a blood sample.

In the context of the present invention, the polypeptide mixture that is isolated from the target is determined by the type of kidney target. Wherein, if the target is isolated kidney cells or tissue, then the preferred isolated polypeptide mixture for use in the method can be for example, the supernatant. If the target is the kidney organ in a mammalian subject, the isolated polypeptide mixture is preferably a body fluid, such as but not limited to blood, serum, plasma or urine. 4.2.1 Polypeptides for use in the invention Polypeptides for use in the invention include: T-kinninogen and or inter-alpha-inhibitor H4P heavy chain optionally in combination with one or more of the following polypeptides: alpha-1- macroglobulin, apolipoprotein E, clusterin, complement C3, complement C4, fibrinogen-alpha-chain, fibrinogen-beta-chain, or plasma retinol binding protein polypeptides.

For all aspects of the invention, reference to said polypeptides encompasses any mammalian polypeptide, such as but not limited, to the rat polypeptides as provided in Figure 1. The skilled person will appreciate that, for example, the rat polypeptides: a) comprise or consist an amino acid sequence selected from SEQ ED NOs: 1 to 12; b) are derivatives having one or more amino acid substitutions, deletions or insertions relative to an amino acid sequence given in SEQ ID NOs: 1 to 12, provided that such a derivative exhibits the activity a polypeptide with an amino acid sequence selected from SEQ ID NOs: 1 to 12 provided in a); or c) are fragments of a) or b) above, which are at least ten amino acids long.

A polypeptide within the scope of a), may consist of the particular amino acid sequence given in Figure 1 or may have an additional N-terminal and/or an additional C-terminal amino acid sequence relative to the sequence given in Figure 1. Additional N-terminal or C-terminal sequences may be provided for various reasons. Techniques for providing such additional sequences are well known in the art.

Additional sequences may be provided in order to alter the characteristics of a particular polypeptide. This can be useful in improving expression or regulation of expression in particular expression systems. For example, an additional sequence may provide some protection against proteolytic cleavage. This has been done for the hormone Somatostatin by fusing it at its N-terminus to part of the β galactosidase enzyme (Itakwa etα/., Science 198: 105-63 (1977)).

Additional sequences can also be useful in altering the properties of a polypeptide to aid in identification or purification. For example, a fusion protein may be provided in which a polypeptide is linked to a moiety capable of being isolated by affinity chromatography. The moiety may be an antigen or an epitope and the affinity column may comprise immobilised antibodies or immobilised antibody fragments which bind to said antigen or epitope (desirably with a high degree of specificity). The fusion protein can usually be eluted from the column by addition of an appropriate buffer.

Additional N-terminal or C-terminal sequences may, however, be present simply as a result of a particular technique used to obtain a polypeptide for use in the present invention and need not provide any particular advantageous characteristic to the polypeptide for use in the present invention. Such polypeptides are within the scope of the present invention.

Whatever additional N-terminal or C-terminal sequence is present, it is preferred that the resultant polypeptide should exhibit the immunological activity of the polypeptide having the amino acid sequence shown in Figure 1. It will be appreciated by the person skilled in the art that these polypeptides are variants of the polypeptides defined above, provided that such variants exhibit the activity of a polypeptide having the amino acid sequence shown in Figure 1.

Alterations in the amino acid sequence of a protein can occur which do not affect the function of a protein. These include amino acid deletions, insertions and substitutions and can result from alternative splicing and/or the presence of multiple translation start sites and stop sites. Polymorphisms may arise as a result of the infidelity of the translation process. Thus changes in amino acid sequence may be tolerated which do not affect the protein's function.

The skilled person will appreciate that various changes can often be made to the amino acid sequence of a polypeptide which has a particular activity to produce variants (sometimes known as "muteins") having at least a proportion of said activity, and preferably having a substantial proportion of said activity. Such variants of the polypeptides described in a) above are within the scope of the present invention and are discussed in greater detail below. They include allelic and non-allelic variants.

An example of a variant for use in the present invention is a polypeptide relative to one of the amino acid sequences shown in Figure 1, apart from the substitution of one or more amino acids with one or more other amino acids. The skilled person is aware that various amino acids have similar properties. One or more such amino acids of a substance can often be substituted by one or more other such amino acids without eliminating a desired activity of that substance.

Thus, the amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids having aliphatic side chains). Of these possible substitutions, it is preferred that glycine and alanine are used to substitute for one another (since they have relatively short side chains) and that valine, leucine and isoleucine are used to substitute for one another (since they have larger aliphatic side chains which are hydrophobic).

Other amino acids which can often be substituted for one another include:

- phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); - lysine, arginine and histidine (amino acids having basic side chains);

- aspartate and glutamate (amino acids having acidic side chains);

- asparagine and glutamine (amino acids having amide side chains); and

- cysteine and methionine (amino acids having sulphur-containing side chains).

Substitutions of this nature are often referred to as "conservative" or "semi-conservative" amino acid substitutions.

Amino acid deletions or insertions may also be made relative to the amino acid sequence given in a) above. Thus, for example, amino acids which do not have a substantial effect on the activity of the polypeptide, or at least which do not eliminate such activity, may be deleted. Such deletions can be advantageous since the overall length and the molecular weight of a polypeptide can be reduced whilst still retaining activity. This can enable the amount of polypeptide required for a particular purpose to be reduced - for example, dosage levels can be reduced.

Amino acid insertions relative to the sequence given in a) above can also be made. This may be done to alter the properties of a polypeptide for use in the present invention (e.g. to assist in identification, purification or expression, as explained above in relation to fusion proteins). Amino acid changes relative to the sequence given in a) above can be made using any suitable technique e.g. by using site-directed mutagenesis (Hutchinson et al, 1978, J. Biol. Chem. 253:6551).

It should be appreciated that amino acid substitutions or insertions within the scope of the present invention can be made using naturally occurring or non-naturally occurring amino acids. Whether or not natural or synthetic amino acids are used, it is preferred that only L- amino acids are present. Whatever amino acid changes are made (whether by means of substitution, insertion or deletion), preferred polypeptides for use in the present invention have at least 50% sequence identity with a polypeptide as defined in a) above, more preferably the degree of sequence identity is at least 75%. Yet more preferably the degree of sequence identity is at least 80% or at least 85%. Sequence identities of at least 90% or at least 95% are most preferred. The percent identity of two amino acid sequences or of two nucleic acid sequences is determined by aligning the sequences for optimal comparison purposes (e.g. gaps can be introduced in the first sequence for best alignment with the sequence) and comparing the amino acid residues or nucleotides at corresponding positions. The "best alignment" is an alignment of two sequences which results in the highest percent identity. The percent identity is determined by the number of identical amino acid residues or nucleotides in the sequences being compared (i.e. % identity = # of identical positions/total # of positions x 100). The determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. The NBLAST and XBLAST programs of Altschul, et al, (1990) J. Mol. Biol. 215:403-410 have incorporated such an algorithm. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilised as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilising BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g. XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov. Another example of a mathematical algorithm utilised for the comparison of sequences is the algorithm of Myers & Miller, CABIOS (1989). The ALIGN program (version 2.0) which is part of the CGC sequence alignment software package has incorporated such an algorithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis & Robotti (1994) Comput. Appl Bioscl, 10 :3-5; and FASTA described in Pearson & Lipman (1988) Proc. Natl. Acad. Sci. §5:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search.

Where high degrees of sequence identity are present there will be relatively few differences in amino acid sequence. Thus for example there may be less than 20, less than 10, or even less than 5 differences.

As discussed supra, it is often advantageous to reduce the length of a polypeptide, provided that the resultant reduced length polypeptide still has a desired activity or can give rise to useful antibodies. Feature c), therefore, covers fragments of the amino acid sequences shown in Figure 1 or, of a polypeptide within the scope of b) above.

The skilled person can determine whether or not a particular fragment has activity using the techniques disclosed above. Preferred fragments are at least 10 amino acids long. They may be at least 20, at least 50 or at least 100 amino acids long.

If it is necessary to prepare polypeptides for use in the invention for, e.g. as controls, in the present invention, the skilled person will appreciate that for the preparation of polypeptides, the preferred approach will be based on recombinant DNA techniques. 4.2.2 Isoforms As is well known in the art, a given protein may be expressed as variants that differ in their amino acid composition (e.g. as a result of alternative mRNA or premRNA processing, e.g. alternative splicing or limited proteolysis) or as a result of differential post-translational modification (e.g. glycosylation, phosphorylation, acylation), or both, so that proteins of identical amino acid sequence can differ in their pi, MW, or both. It follows that differential presence of a protein isoform (PI) does not require differential expression of the gene encoding the protein in question. These protein isoforms can be characterised by two-dimensional electrophoresis and further sequenced by mass spectrometry as described in the Examples infra.

Six specific T-kinninogen protein isoforms, herein designated as LMW-TKPIs for use in the methods of the invention are defined in Table I. LMW-TKPI-1 (accession 205085/125520) and LMW- TKPI-2, LMW-TKPI-4 and LMW-TKPI-5 (accession 205085/1225520/125521). In addition to LMW-TKPIs, specific protein isoforms for alpha- 1-macroglobulin (AlM-PIs) apolipoprotein E (APOE-PIs), clusterin (CLUS-PIs), complement C3 (C3-PIs), complement C4 (C4-PIs), fibrinogen-alpha-chain (FA-PIs), fibrinogen-beta-chain (FB-PIs), inter-alpha-inhibitor H4P heavy chain (H4P-PIs) and plasma retinol binding (PRBP-PIs) of the invention were also identified. Details for all the protein isoforms are provided in Table I.

Preferred polypeptides for use in the invention are polypeptides within the scope of a). Most preferably, the polypeptide is a polypeptide isoform of the invention, specifically one or more of the protein isoforms listed in Table I. As those of skill in the art will readily appreciate, the apparent MW and pl of a given protein isoform will vary to some extent depending on the precise protocol used for its identification e.g. as described in the Example infra). As used herein, the terms "MW" and "pi" are defined, respectively, to mean the apparent molecular weight and the apparent isoelectric point of a protein isoform as measured in exact accordance with the Reference Protocol identified in the Example provided below. When the Reference Protocol is followed and when samples are run in duplicate or a higher number of replicates, variation in the measured mean pi of an protein isoform is typically less than 3% and variation in the measured mean MW of protein isoform is typically less than 5%. Where the skilled artisan wishes to deviate from the Reference Protocol, calibration experiments should be performed to compare the MW and pi for each protein isoform as detected (a) by the Reference Protocol and (b) by the deviant protocol. In one aspect of the invention, two dimensional electrophoresis is used to analyse a test sample from a target, preferably a mammal, in order to detect or quantify one or more polypeptides for use in the invention, preferably, one or more protein isoforms of the invention selected from the protein isoforms listed in Table I.

In a preferred embodiment, the signal associated with one or more polypeptides for use in the invention in the test sample of a subject (e.g. a subject suspected of having or known to have kidney toxicity is normalised with reference to one or more Expression Reference Features (ERFs), (i.e. a feature whose abundance is substantially invariant, in the population of subjects being examined) detected in the same 2D gel. As will be apparent to one of ordinary skill in the art, such ERFs may readily be determined by comparing different samples using the methods, described in the Example infra. In a further embodiment, a test sample is analyzed for quantitative detection of one or more polypeptides for use in the invention and one or more previously known biomarkers of kidney toxicity (e.g. histology, soft tissue imaging). In accordance with this embodiment, the abundance of one or more polypeptides for use in the invention and known biomarkers relative to a control or reference range is an indicator of kidney toxicity. 4.2.3 Nucleic acid molecules for use in the invention The present invention also provides a method for screening or diagnosis of kidney toxicity in a target cell, tissue or mammal which comprises the step of detecting and/or quantifying in a test sample obtained from said target one or more nucleic acid molecules which comprises or consists of the DNA sequence that encodes a polypeptide for use in the invention or its RNA equivalent. The nucleic acid molecules are also useful for the expression of polypeptides for use in the invention e.g. as standards and/or controls. They can be inserted into vectors and cloned to provide large amounts of DNA or RNA for further study. Suitable vectors may be introduced into host cells to enable the expression of polypeptides for use in the present invention using techniques known to the person skilled in the art. Unless the context indicates otherwise, nucleic acid molecules may have one or more of the following characteristics:

1) they may be DNA or RNA;

2) they may be single or double stranded;

3) they may be provided in recombinant form i.e. covalently linked to a 5' and/or a 3' flanking sequence to provide a molecule which does not occur in nature; 4) they may be provided without 5' and/or 3' flanking sequences which normally occur in nature;

5) they may be provided in substantially pure form. Thus they may be provided in a form which is substantially free from contaminating proteins and/or from other nucleic acids; and

6) they may be provided with introns or without introns (e.g. as cDNA).

7) they may hybridise to a nucleic acid molecule encoding a polypeptide for use in the invention, such hybridising molecules being at least 10 nucleotides in length and preferably are at least 25 or at least 50 nucleotides in length.

It is preferred if sequences l)-7) show substantial identity with nucleic acid molecules encoding a polypeptide of the invention e.g. have at least 75%, at least 80%, at least 85% or at least 90% or 95% sequence identity. 4.2.4 Antibodies for use in the invention

The polypeptides for use in the invention, their fragments, isoforms or other derivatives, or analogues thereof, may be used as immunogens to generate antibodies which immunospecifically bind such an immunogen. Antibodies include, but are not limited to polyclonal, monoclonal, bispecific, humanized or chimeric antibodies, single chain antibodies, Fab fragments and F(ab') fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above or complementarity determining regions (CDRs).

The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. molecules that contain an antigen binding site that specifically binds an antigen. Immunoglobulin molecules can be of any class (e.g. IgG, IgE, IgM, IgD and IgA) or subclass of immunoglobulin molecule.

In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art, e.g. ELISA (enzyme-linked immunosorbent assay). For example, to select antibodies, which recognize a specific domain of a polypeptide for use in the invention, one may assay generated hybridomas for a product, which binds to a polypeptide fragment containing such domain. For selection of an antibody that specifically binds a first polypeptide homblogue but which does not specifically bind to (or binds less avidly to) a second polypeptide homologue, one can select on the basis of positive binding to the first polypeptide homologue and a lack of binding to (or reduced binding to) the second polypeptide homologue.

For preparation of monoclonal antibodies (mAbs) directed toward a polypeptide for use in the invention or fragment or analogue thereof, any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used. For example, the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al, 1983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole et al, 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAbs may be cultivated in vitro or in vivo. Monoclonal antibodies can also be produced in germ-free animals utilizing known technology.

The monoclonal antibodies include but are not limited to human monoclonal antibodies and chimeric monoclonal antibodies (e.g. human-mouse chimeras). A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a human immunoglobulin constant region and a variable region derived from a murine mAb. (See, e.g. Cabilly et al, U.S. 4,816,567; and Boss et al, U.S. 4,816,397. Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non- human species and a framework region from a human immunoglobulin molecule, see, e.g. U.S. 5,585,089.

Chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in WO 87/02671; EP 184,187; EP 171,496; EP 173,494; WO 86/01533; U.S. 4,816,567; EP 125,023; Better et al, 1988, Science 240:1041-1043; Liu et al, 1987, Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al, 1987, J. Immunol. 139:3521-3526; Sun et al, 1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al, 1987, Cane. Res. 47:999-1005; Wood et al, 1985, Nature 314:446-449; and Shaw et al, 1988, J. Natl. Cancer List. 80: 1553-1559; Morrison, 1985, Science 229: 1202-1207; Oi et al, 1986, Bio/Techniques 4:214; U.S. 5,225,539; Jones et al, 1986, Nature 321:552-525; Verhoeyan et al, (1988) Science 239:1534; and Beidler et al, 1988, J. Immunol. 141:4053-4060. Completely human antibodies can be produced using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chain genes, but which can express human heavy and light chain genes. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g. all or a portion of a polypeptide for use in the invention. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harboured by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g. U.S. 5,625,126; U.S. 5,633,425; U.S. 5,569,825; U.S. 5,661,016; and U.S. 5,545,806. In addition, companies such as Abgenix, Inc. (Freemont, CA) and Genpharm (San Jose, CA) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

Completely human antibodies, which recognize a selected epitope, can be generated using a technique referred to as "guided selection." In this approach a selected non-human monoclonal antibody, e.g. a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al, (1994) Bio/technology 12:899-903).

The antibodies for use in the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular, such phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g. human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g. using labelled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Phage display methods that can be used to make the antibodies for use in the present invention include those disclosed in Brinkman et al, J. Immunol. Methods 182:41-50 (1995); Ames et al, J. Immunol. Methods 184:177- 186 (1995); Kettleborough et al, Eur. J. Immunol. 24:952-958 (1994); Persic et al, Gene 187 9-18 (1997); Burton et al, Advances in Immunology 57:191-280 (1994); WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.

As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g. as described in detail below. For example, techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in WO 92/22324; Mullinax et al, BioTechniques 12(6):864-869 (1992); and Sawai et al, AJRI 34:26-34 (1995); and Better et al, Science 240: 1041-1043 (1988). Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. 4,946,778 and 5,258,498; Huston et al, Methods in Enzymology 203:46-88 (1991); Shu et al, PNAS 90:7995-7999 (1993); and Skerra et al, Science 240:1038-1040 (1988). The invention further provides for the use of bispecific antibodies, which can be made by methods known in the art. Traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Milstein et al, 1983, Nature 305:537-539). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829 and in Traunecker et al, 1991, EMBO J. 10:3655-3659.

According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.

In a preferred embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details for generating bispecific antibodies see, for example, Suresh et al, Methods in Enzy ology, 1986, 121:210.

The invention provides functionally active fragments, derivatives or analogues of the anti-polypeptide immunoglobulin molecules. Functionally active means that the fragment, derivative or analogue is able to elicit anti-anti-idiotype antibodies (i.e. tertiary antibodies) that recognize the same antigen that is recognized by the antibody from which the fragment, derivative or analogue is derived. Specifically, in a preferred embodiment the antigenicity of the idiotype of the immunoglobulin molecule may be enhanced by deletion of framework and CDR sequences that are C-terminal to the CDR sequence that specifically recognizes the antigen. To determine which CDR sequences bind the antigen, synthetic peptides containing the CDR sequences can be used in binding assays with the antigen by any binding assay method known in the art. In native IgG antibodies the variable heavy and light chains each contribute three CDR regions which are responsible for binding the antigen. Each region is typically 7-20 amino acids long, e.g. 15-17 amino acids, and its sequence defines the specificity and affinity of that CDR for the antigen. There is increasing evidence that these regions of CDR-peptides are capable of autonomous specific binding to antigens, see for example Steinbergs, J. et al, 1996, Hum. Antibodies Hybridomasi 7: 106-112; William, W. et al, 1991, J. Biol. Chem., 266: 5182-5190; Saragovi, H. et al, 1991, 253: 792-795; Welling, W. et al, 1991, J Chromatogr., 548: 235-242; and Levi, M. et al, 1993, Proc. Natl. Acad. Sci. USA., 90: 4374- 4378; and hence they have been proposed as new generation antibody fragments with reduced immunogenicity and as anti-viral molecules (see Sivolapenko, G. et al, 1995, Lancet 346: 1662-1666; and Rossenu, S. et al, 1997, J. Prot. Chem., 16, 499-503). CDRs can be produced by recombinant means or can be chemically synthesized. CDRs can be chemically synthesized according to known CDR sequences using either standard protein synthesis or using a combinatorial synthesis approach.

The present invention provides antibody fragments such as, but not limited to, F(ab')2 fragments and Fab fragments. Antibody fragments which recognize specific epitopes may be generated by known techniques. F(ab')2 fragments consist of the variable region, the light chain constant region and the CHI domain of the heavy chain and are generated by pepsin digestion of the antibody molecule. Fab fragments are generated by reducing the disulfide bridges of the F(ab')2 fragments. Heavy chain and light chain dimmers of the antibodies for use in the invention, or any minimal fragment thereof such as Fvs or single chain antibodies (SCAs) (e.g. as described in U.S. 4,946,778; Bird, 1988, Science 242:423-42; Huston et al, 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al, 1989, Nature 334:544- 54), or any other molecule with the same specificity as the antibody for use in the invention are also encompassed. 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. Techniques for the assembly of functional Fv fragments in E. coli may be used (Skerra et al, 1988, Science 242: 1038-1041). In other embodiments, the invention provides fusion proteins of immunoglobulins (or functionally active fragments thereof), for example in which the immunoglobulin is fused via a covalent bond (e.g. a peptide bond), at either the N-terminus or the C-terminus to an amino acid sequence of another protein (or portion thereof, preferably at least 10, 20 or 50 amino acid portion of the protein) that is not the immunoglobulin. Preferably the immunoglobulin, or fragment thereof, is covalently linked to the other protein at the N-terminus of the constant domain. As stated above, such fusion proteins may facilitate purification, increase half -life in vivo, and enhance the delivery of an antigen across an epithelial barrier to the immune system. The immunoglobulins of the invention include analogues and derivatives that are either modified, i.e. by the covalent attachment of any type of molecule as long as such covalent attachment that does not impair immunospecific binding. For example, but not by way of limitation, the derivatives and analogues of the immunoglobulins include those that have been further modified, e.g. by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, etc. Additionally, the analogue or derivative may contain one or more non-classical amino acids. The foregoing antibodies can be used in methods known in the art relating to the localization and activity of the polypeptides for use in the invention, e.g. for imaging or radioimaging these proteins, measuring levels thereof in appropriate physiological samples, in diagnostic methods, etc. and for radiotherapy.

Antibodies can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression, and are preferably produced by recombinant expression technique.

Recombinant expression of antibodies, or fragments, derivatives or analogues thereof, requires construction of a nucleic acid that encodes the antibody. If the nucleotide sequence of the antibody is known, a nucleic acid encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g. as described in Kutmeier et al, 1994, BioTechniques 17:242), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding antibody, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

Alternatively, the nucleic acid encoding the antibody may be obtained by cloning the antibody. If a clone containing the nucleic acid encoding the particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the antibody may be obtained from a suitable source (e.g. an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the antibody) by PCR amplification using synthetic primers hybridisable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence. If an antibody molecule that specifically recognizes a particular antigen is not available (or a source for a cDNA library for cloning a nucleic acid encoding such an antibody), antibodies specific for a particular antigen may be generated by any method known in the art, for example, by immunizing an animal, such as a rabbit, to generate polyclonal antibodies or, more preferably, by generating monoclonal antibodies. Alternatively, a clone encoding at least the Fab portion of the antibody may be obtained by screening Fab expression libraries (e.g. as described in Huse et al., 1989, Science 246: 1275-1281) for clones of Fab fragments that bind the specific antigen or by screening antibody libraries (See, e.g. Clackson et al, 1991, Nature 352:624; Hane et al, 1997 Proc. Natl. Acad. Sci. USA 94:4937).

Once a nucleic acid encoding at least the variable domain of the antibody molecule is obtained, it may be introduced into a vector containing the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g. WO 86/05807; WO 89/01036; and U.S. 5,122,464). Vectors containing the complete light or heavy chain for co-expression with the nucleic acid to allow the expression of a complete antibody molecule are also available. Then, the nucleic acid encoding the antibody can be used to introduce the nucleotide substitution(s) or deletion(s) necessary to substitute (or delete) the one or more variable region cysteine residues participating in an intrachain disulfide bond with an amino acid residue that does not contain a sulfhydryl group. Such modifications can be carried out by any method known in the art for the introduction of specific mutations or deletions in a nucleotide sequence, for example, but not limited to, chemical mutagenesis, in vitro site directed mutagenesis (Hutchinson et al, 1978, J. Biol. Chem. 253:6551), PCR based methods, etc.

In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al, 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al, 1984, Nature 312:604-608; Takeda et al, 1985, Nature 314:452-454) by splicing 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. As described supra, 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 niAb and a human antibody constant region, e.g. humanized antibodies. Once a nucleic acid encoding an antibody molecule has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing the protein by expressing nucleic acid containing the antibody molecule sequences are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing an antibody molecule coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al, (1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) and Ausubel et al, (eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY). The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody for use in the invention.

The host cells used to express a recombinant antibody for use in the invention may be either bacterial cells such as Escherichia coli, or, preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule. In particular, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al, 198, Gene 45:101; Cockett et al, 1990, Bio/Technology 8:2).

A variety of host-expression vector systems may be utilized to express an antibody. Such host- expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express the antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g. E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g. Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g. baculovirus) containing the antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g. cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g. Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g. COS, CHO, BHK, 293, 3T3 cells) harbouring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g. metallothionein promoter) or from mammalian viruses (e.g. the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al, 1983, EMBO J. 2:1791), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety. In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). In mammalian host cells, a number of viral-based expression systems (e.g. an adenovirus expression system) may be utilized.

As discussed above, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g. glycosylation) and processing (e.g. cleavage) of protein products may be important for the function of the protein. For long-term, high-yield production of recombinant antibodies, stable expression is preferred.

For example, cells lines that stably express an antibody of interest can be produced by transfecting the cells with an expression vector comprising the nucleotide sequence of the antibody and the nucleotide sequence of a selectable (e.g. neomycin or hygromycin), and selecting for expression of the selectable marker. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.

The expression levels of the antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al, 1983, Mol. Cell. Biol. 3:257).

The host cell may be co-transfected with two expression vectors, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers, which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

Once the antibody molecule has been recombinantly expressed, it may be purified by any method known in the art for purification of an antibody molecule, for example, by chromatography (e.g. ion exchange chromatography, affinity chromatography such as with protein A or specific antigen, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purif icati on of proteins . Alternatively, any fusion protein may be readily purified by utilizing an antibody specific for the fusion protein being expressed. For example, a system described by Janknecht et al, allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino- terminal tag consisting of six histidine residues. The tag serves as a matrix-binding domain for the fusion protein. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni²⁺ nitriloacetic acid-agarose columns and histidine-tagged proteins are selectively eluted with imidazole-containing buffers. In a preferred embodiment, antibodies for use in the invention or fragments thereof are conjugated to a diagnostic moiety. The antibodies can be used for diagnosis, clinical screening or prognosis. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive nuclides, positron emitting metals (for use in positron emission tomography), and nonradioactive paramagnetic metal ions. See generally U.S. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin and biotin; suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin, and aequorin; and suitable

125 131 il l 99 radioactive nuclides include I, I, In and Tc.

Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. 4,676,980.

Preferred features of each aspect of the invention are as for each of the other aspects mutatis mutandis.

The invention will now be described with reference to the following example, which should not in any way be construed as limiting the scope of the present invention. The example refers to the figures in which:

Figure la: shows the amino acid sequence of LMW T-kinninogen- 1, accession number 205085,

SEQ ID NO: 1, in the Genbank database.

Figure lb: shows the amino acid sequence of T-kinninogen-I KNT-1, accession number

125520, SEQ ED NO: 2, in the Genbank database. Figure lc: shows the amino acid sequence of T-kinninogen-II/ KNT-2, accession number

125521, SEQ ID NO: 3, in the Genbank database.

Figure Id: shows the amino acid sequence of alpha-1-macroglobulin, accession number 205384,

SEQ ED NO: 4, in the Genbank database.

Figure le: shows the amino acid sequence of apolipoprotein E, accession number P02650, SEQ ID NO: 5, in the Swissprot database.

Figure If: shows the amino acid sequence of clusterin, accession number P05371, SEQ ED NO:

6, in the Swissprot database.

Figure lg: shows the amino acid sequence of complement C3, accession number P01026, SEQ

ED NO: 7, in the Swissprot database. Figure lh: shows the amino acid sequence of complement C4, accession number P01029, SEQ

ED NO: 8, in the Swissprot database. Figure li: shows the amino acid sequence of fibrinogen-alpha-chain, accession number P06399 SEQ ED NO: 9, in the Swissprot database.

Figure lj: shows the amino acid sequence of fibrinogen-beta-chain, accession number P14480, SEQ ED NO: 10, in the Swissprot database. Figure Ik: shows the amino acid sequence of inter-alpha-inhibitor H4P heavy chain, accession number 7441758, SEQ ED NO: 11, in the Genbank database.

Figure 11: shows the amino acid sequence of plasma retinol binding protein, accession number P04916, SEQ ED NO: 12, in the Swissprot database. The genbank database is held by the National Institute of Health (NEH) available at www.ncbi.nlm.nih.gov/ and the Swissprot database is available at www.ca.expasy.org/.

Figure 2: is representative of an image obtained from 2-dimensional electrophoresis of rat plasma, which has been annotated to show the twelve landmark features, designated FI to F7; F9 to FI 1 and F13 to F14 defined in Table II. , _

Figure 3: shows the dosage (A and C) and temporal (B and D) response profiles of LMW TKPI- 5 and LMW TKPI-6 respectively to D-serine treatment. Graphs A and C show that for both protein isoforms, induction of protein expression occurs at dosages of 250 mg and 750 mg of D- serine, as compared to the control (no treatment) and L-serine treatment (negative control). Graphs B and D show that the induction in LMW-T kininnogen occurs at 24h post exposure to 750 mg D-serine and that levels remain elevated for up to 3 weeks after treatment.

5.0 EXAMPLE: IDENTIFICATION OF POLYPEPTIDES DIFFERENTIALLY EXPRESSED IN THE PLASMA OF SUBJECTS HAVING KIDNEY TOXICITY, IN PARTICULAR, RENAL TUBULAR NECROSIS

5.1 Introduction Compounds that reliably produce injury to the proximal tubule, glomerulus or papilla of the kidney in a rat model were used to illustrate the role of specific polypeptides as a marker of kidney toxicity. 5.1.1 Models of Chemically Induced Renal Tubular Necrosis

A number of chemicals and drugs have been shown to cause selective toxicity to the proximal tubule in rats and in some cases humans. Chemically-induced renal tubular necrosis is associated with cisplatin, aminoglycosidases, such as gentamycin, tobramycin, netilmicin, amikacin, kanamycin, streptomycin and neomycin C, β-lactam antibiotics, 4-aminophenol, mercuric chloride, haloalkenes and D-serine. Three models of chemically-induced renal tubular necrosis were used. For selective necrosis to the proximal tubule of the kidney, in particular the pars recta region, the following chemically diverse drugs were used: 4-aminophenol, D-serine and cisplatin. 5.1.2 Models of Chemically induced Glomerular Injury

A number of compounds have been shown to cause selective toxicity to the glomerulus leading to changes in glomerular morphology and/ or function in both experimental animals and humans. Many of the drugs produce their effect by an extra-renal, often immunologically mediated mechanism. Glomerular injury in the form of immune complex glomerulonephritis is thought to arise when drugs or other chemicals interfere with the immune sysytem, resulting in antibody formation. Examples in this area would be mercuric chloride, gold, penicillamine and hydrocarbon solvents. Drugs such as cyclosporin, gentamicin and amphotericin B can also alter functional properties of the glomerulus without producing any structural changes. Two compounds that cause epithelial cell damage to the glomerulus as models of nephrotic syndrome are puromycin aminonucleoside and adriamycin. 5.1.3 Models of Chemically Induced Papilla Injury

Renal papillary necrosis (RPN) is a leading cause of chronic renal failure throughout the world (Kincaid Smith P (1978) Kidney Int. 13, 1-3 & Sabatini S (1984) Fundam. Appl. Toxicol. 4, 909-921). RPN is most commonly clinically seen in association with obstructive nephropathy, diabetes mellitus and sickle cell disease. Papillary necrosis is also a hallmark of analgesic abuse. Chemically induced RPN has been associated with analgesics, nonsteroidal anti-inflammatory drugs, other therapeutic agents such as, cyclophosphamide, dapsone, radiocontrast media and chemicals such as 2 bromoethanamine (2-BEA), and jet-fuel (Bach P H and Bridges J W (1985) CRC Crit. Rev. Toxicol. 15, 217-329 and 331-441 & Bach P H and Hardy T L (1985) Kidney Int. 28, 605-613). 5.2 Identification of polypeptides differentially expressed in renal tubular necrosis

5.2.1 4-aminophenol

4-aminophenol is a known nephrotoxic metbolite of the analgesic drug paracetamol. The kidney injury induced with paracetamol, however, shows strain specificity with the Fischer 344 rat being more sensitive than the Alderley-Park Wistar rat (Newton et al, 1983, Toxicol. Appl. Pharmacol; 69, 291-306).

Groups of five male Fischer-344 rats were treated with 4-aminophenol at the following dose levels: 20, 50 or 80 mg/kg/day. A group of five male Alderly Park Wistar rats were used as a negative control and were treated with a dose of 80 mg/kg/day. The dosage was administered as a single dose by intraperitoneal injection. Plasma samples from treated rats were collected for proteome analysis at 4, 8 and 24 hours. A further plasma sample was collected after 21 days and a kidney cortex tissue sample was also taken and prepared for histologic examination according to standard tissue preparation protocols.

The clinical parameters blood urea nitrogen and blood creatine were measured. Histologic examination revealed evidence of renal tubular necrosis at doses of 50 and 80 mg/kg/day with tissue regeneration observed by day 21. There was a positive correlation between the histopathological and clinical chemistry data.

5.2.2 D-serine

D-serine is the unnatural D-enantiomer of the amino acid L-serine. Its' toxic effects can be induced within 24h following a single intraperitoneal or intravenous injection (Wachstein and Beson, 1964, Am. J. Pathol; 44, 383-393 & Kaltenbach et al, 1982, Exp. Mol. Pathol; 37, 225-234). The natural L-serine is not nephrotoxic and was used as a control.

Groups of five male Alderly Park Wistar rats were treated with D-serine at the following dose levels 75, 250 and 750 mg/kg/day. The control group were treated with L-serine at a dose of 750 mg/kg/day. The dosage was administered as a single dose by intraperitoneal injection. Both, plasma and kidney samples were collected, and clinical and histopathological parameters were assessed, as described above. Once again, there was a positive correlation between the histopathological and clinical chemistry data.

5.2.3 Cisplatin

Cisplatin is a platinum antitumour drug and as such is widely used for the treatment of a range of cancers. Nephrotoxicity is frequently a side effect that limits it's dosage. It's inactive isomer transplatin was used as a negative (non-nephrotoxic) control.

Groups of five male Alderly Park Wistar rats were treated with cisplatin at the following dose levels 1, 2 and 6mg kg. The control group were treated with transplatin at a dose of 6mg/kg. The dosage was administered as a single dose by intraperitoneal injection. Plasma samples from treated rats were collected for proteome analysis at 24, 48 and 72 hours. A further plasma sample was collected at 21 days and a kidney cortex tissue sample was also taken and prepared for histologic examination according to standard tissue preparation protocols. Evidence of kidney toxicity was observed at doses of 2 and 6mg/kg cisplatin whereas no toxicity was observed with transplatin at doses of 6mg/kg. 5.3 Materials and Methods

Proteins in plasma samples, taken from subjects having kidney toxicity (treated group) and from subjects free from kidney toxicity (control group), were separated by isoelectric focusing followed by SDS-PAGE and analyzed. The procedure set forth below is hereby designated the "Reference Protocol". 5.3.1 Sample Preparation

Approximately 2ml of fresh venous blood was collected in pre-labelled EDTA collection tubes, (yields 0.8-lml plasma). The sample was mixed thoroughly and gently. The samples were then centrifuged, as soon as possible, after collection for 10 in exactly at 1500xg at 4°C. This results in the separation of the blood into two layers. The top layer (the plasma layer) was drawn off and added to another prelabelled tube containing protease inhibitor solution (Sigma P2714) (150μl protease inhibitor solution /ml plasma). The contents were then mixed by gentle vortexing. The samples were then snap frozen and stored at -70°C. A protein assay (Pierce BCA Cat # 23225) was performed on each sample as received. Prior to protein separation, each plasma sample was processed for selective depletion of certain proteins, in order to enhance and simplify protein separation and facilitate analysis by removing proteins that may interfere with or limit analysis of proteins of interest, see WO 99/63351.

Removal of albumin, haptoglobin, transferrin and immunoglobin G (IgG) from plasma ("plasma depletion") was achieved by an affinity chromatography purification step in which the sample was passed through a series of "Hi-Trap" columns containing immobilized antibodies for selective removal of albumin, haptoglobin and transferrin, and protein G for selective removal of immunoglobin G. Two affinity columns in a tandem assembly were prepared by coupling antibodies to protein G-sepharose contained in Hi-Trap columns (Protein G-Sepharose Hi-Trap columns (1 ml) Pharmacia Cat. No. 17- 0404-01). This was done by circulating the following solutions sequentially through the columns: (1) Dulbecco's Phosphate Buffered Saline (Gibco BRL Cat. No. 14190-094); (2) concentrated antibody solution; (3) 200 mM sodium carbonate buffer, pH 8.35; (4) cross-linking solution (200 mM sodium carbonate buffer, pH 8.35, 20 mM dimethylpimelimidate); and (5) 500 mM ethanolamine, 500 mM NaCl. A third (un-derivatised) protein G Hi-Trap column was then attached to the lower end of the tandem column assembly.

The chromatographic procedure was automated using an Akta Fast Protein Liquid Chromatography (FPLC) System such that a series of up to seven runs could be performed sequentially. The samples were passed through the series of 3 Hi-Trap columns in which the affinity chromatography media selectively bind the above proteins thereby removing them from the sample. Fractions (typically 3 ml per tube) were collected of unbound material ("Flowthrough fractions") that eluted through the column during column loading and washing stages and of bound proteins ("Bound/Eluted fractions") that were eluted by step elution with Lmmunopure Gentle Ag/Ab Elution Buffer (Pierce Cat. No. 21013). The eluate containing unbound material was collected in fractions which were pooled and desalted/concentrated by centrifugal ultrafiltration. The sample was recovered in 2D Sample Buffer (see below) containing a cocktail of protease inhibitors (Sigma P2714) and stored at -70°C to await further analysis by 2D PAGE.

An aliquot of the stored sample containing 300 microg of protein was prepared for 2D analysis by adding Resolytes 3.5-10 (BDH 44338 2x) to 2% (v/v), as well as a trace of Bromophenol Blue and further 2D Sample Buffer in a final volume of 370 microl. 2D Sample Buffer: 8M urea (BDH 452043w )

2M thiourea (Fluka 88810) 4% CHAPS (Sigma C3023) 65mM dithiotheitol (DTT) This mixture was vortexed, and centrifuged at 13000 rpm for 5 min at 15°C, and the supernatant was analyzed by isoelectric focusing. 5.3.2 Isoelectric Focusing

Isoelectric focusing (EEF), was performed using the Immobiline™ DryStrip Kit (Pharmacia BioTech), following the procedure described in the manufacturer's instructions, see Instructions for Immobiline™ DryStrip Kit, Pharmacia, # 18-1038-63, Edition AB. Immobilized pH Gradient (EPG) strips (18cm, pH 3-10 non-linear strips; Pharmacia Cat. # 17-1235-01) were rehydrated overnight at 20°C in a solution of 8M urea, 2% (w/v) CHAPS, lOmM DTT, 2% (v/v) Resolytes 3.5-10, as described in the

Immobiline DryStrip Users Manual. For EEF, 50ml of supernatant (prepared as above) was loaded onto a strip, with the cup-loading units being placed at the basic end of the strip. The loaded gels were then covered with mineral oil (Pharmacia 17-3335-01) and a voltage was immediately applied to the strips according to the following profile, using a Pharmacia EPS3500XL power supply (Cat 19-3500-01): Initial voltage = 300V for 2 h

Linear Ramp from 300V to 3500V over 3h Hold at 3500V for 19h For all stages of the process, the current limit was set to 10mA for 12 gels, and the wattage limit to 5W. The temperature was held at 20°C throughout the run.

5.3.3 Gel Equilibration and SDS-PAGE

After the final 19hr step, the strips were immediately removed and immersed for 10 min at 20°C in a first solution of the following composition: 6M urea; 2% (w/v) DTT; 2% (w/v) SDS; 30% (v/v) glycerol (Fluka 49767); 0.05M Tris/HCl, pH 6.8 (Sigma Cat T-1503). The strips were removed from the first solution and immersed for 10 min at 20°C in a second solution of the following composition: 6M urea; 2% (w/v) iodoacetamide (Sigma 1-6125); 2% (w/v) SDS; 30% (v/v) glycerol; 0.05M Tris HCl, pH 6.8. After removal from the second solution, the strips were loaded onto supported gels for SDS-PAGE according to Hochstrasser et al, 1988, Analytical Biochemistry 173: 412-423, with modifications as specified below. 5.3.4 Preparation of supported gels

The gels were cast between two glass plates of the following dimensions: 23cm wide x 24cm long (back plate); 23cm wide x 24cm long with a 2cm deep notch in the central 19cm (front plate). To promote covalent attachment of SDS-PAGE gels, the back plate was treated with a 0.4% solution of g-methacryl-oxypropyltrimethoxysilane in ethanol (BindSilane™; Pharmacia Cat. # 17-1330-01). The front plate was treated with a 2% solution of dimethyldichlorosilane dissolved in octamethyl cyclo- octasilane (RepelSilane™ Pharmacia Cat. # 17-1332-01) to reduce adhesion of the gel. Excess reagent was removed by washing with water, and the plates were allowed to dry. At this stage, both as identification for the gel, and as a marker to identify the coated face of the plate, an adhesive bar-code was attached to the back plate in a position such that it would not come into contact with the gel matrix. The dried plates were assembled into a casting box with a capacity of 13 gel sandwiches. The top and bottom plates of each sandwich were spaced by means of 1mm thick spacers, 2.5 cm wide. The sandwiches were interleaved with acetate sheets to facilitate separation of the sandwiches after gel polymerization. Casting was then carried out according to Hochstrasser et al, op. cit.

A 9-16% linear polyacrylamide gradient was cast, extending up to a point 2cm below the level of the notch in the front plate, using the Angelique gradient casting system (Large Scale Biology). Stock solutions were as follows. Acrylamide (40% in water) was from Serva (Cat. # 10677). The cross-linking agent was PDA (BioRad 161-0202), at a concentration of 2.6% (w/w) of the total starting monomer content. The gel buffer was 0.375M Tris/HCl, pH 8.8. The polymerization catalyst was 0.05% (v/v) TEMED (BioRad 161-0801), and the initiator was 0.1% (w/v) APS (BioRad 161-0700). No SDS was included in the gel and no stacking gel was used. The cast gels were allowed to polymerize at 20°C overnight, and then stored at 4°C in sealed polyethylene bags with 6ml of gel buffer, and were used within 4 weeks.

5.3.5 SDS-PAGE

A solution of 0.5% (w/v) agarose (Fluka Cat 05075) was prepared in running buffer (0.025M Tris, 0.198M glycine (Fluka 50050), 1% (w/v) SDS, supplemented by a trace of bromophenol blue). The agarose suspension was heated to 70°C with stirring, until the agarose had dissolved. The top of the supported 2nd D gel was filled with the agarose solution, and the equilibrated strip was placed into the agarose, and tapped gently with a palette knife until the gel was intimately in contact with the 2nd D gel. The gels were placed in the 2nd D running tank, as described by Amess et al, 1995, Electrophoresis 16: 1255-1267. The tank was filled with running buffer (as above) until the level of the buffer was just higher than the top of the region of the 2nd D gels which contained polyacrylamide, so as to achieve efficient cooling of the active gel area. Running buffer was added to the top buffer compartments formed by the gels, and then voltage was applied immediately to the gels using a Consort E-833 power supply. For lh, the gels were run at 20mA/gel. The wattage limit was set to 150W for a tank containing 6 gels, and the voltage limit was set to 600V. After lh, the gels were then run at 40mA/gel, with the same voltage and wattage limits as before, until the bromophenol blue line was 0.5cm from the bottom of the gel. The temperature of the buffer was held at 16°C throughout the run. Gels were not run in duplicate.

5.3.6 Staining

Upon completion of the electrophoresis run, the gels were immediately removed from the tank for fixation. The top plate of the gel cassette was carefully removed, leaving the gel bonded to the bottom plate. The bottom plate with its attached gel was then placed into a staining apparatus, which can accommodate 12 gels. The gels were completely immersed in fixative solution of 40% (v/v) ethanol (BDH 28719), 10% (v/v) acetic acid (BDH 100016X), 50% (v/v) water (MilliQ-Millipore), which was continuously circulated over the gels. After an overnight incubation, the fixative was drained from the tank, and the gels were primed by immersion in 7.5% (v/v) acetic acid, 0.05% (w/v) SDS, 92.5% (v/v) water for 30 min. The priming solution was then drained, and the gels were stained by complete immersion for 4 h in a staining solution of pyridinium, 4-[2-[4-(dipentylamino)-2-trifluoromethylphenyl] ethenyl]-l-(sulfobutyl)-, inner salt, prepared by diluting a stock solution of this dye (2mg/ml in DMSO) in 7.5% (v/v) aqueous acetic acid to give a final concentration of 1.2 mg/1; the staining solution was vacuum filtered through a 0.4μm filter (Duropore) before use. 5.3.7 Imaging of the gel

A computer-readable output was produced by imaging the fluorescently stained gels with a scanner (Oxford Glycosciences, Oxford, UK). This scanner has a gel carrier with four integral fluorescent markers (Designated Ml, M2, M3, M4) that are used to correct the image geometry and are a quality control feature to confirm that the scanning has been performed correctly. For scanning, the gels were removed from the stain, rinsed with water and allowed to air dry briefly, and imaged on the preferred scanner. After imaging, the gels were sealed in polyethylene bags containing a small volume of staining solution, and then stored at 4°C. 5.3.8 Digital Analysis of the Data

The data were processed as described in WO 98/23950 and as set forth more particularly below. The output from the scanner was first processed using the MELANEE7 II 2D PAGE analysis program (Release 2.2, 1997, BioRad Laboratories, Hercules, California, Cat. # 170-7566) to autodetect the registration points, Ml, M2, M3 and M4; to autocrop the images (i.e. to eliminate signals originating from areas of the scanned image lying outside the boundaries of the gel, e.g. the reference frame); to filter out artifacts due to dust; to detect and quantify features; and to create image files in GIF format. Features were detected using the following parameters:

Smooths = 2

Laplacian threshold = 1

Partials threshold = 2

Saturation = 25

Peakedness = 100

Minimum Perimeter = 8 5.3.9 Assignment of pi and MW Values

Landmark identification was used to determine the pi and MW of features detected in the images. Twelve landmark features, designated FI to F7; F9 to FI 1 and F13 to F14, were identified in a standard plasma image. These landmark features are shown in Figure 2 and were assigned the pi and/or MW values identified in Table II.

Table II. Landmark Features Used in this Study

As many of these landmarks as possible were identified in each gel image of the dataset. Each feature in the study gels was then assigned a pi value by linear interpolation or extrapolation (using the MELANEE7-II software) to the two nearest landmarks, and was assigned a MW value by linear interpolation or extrapolation (using the MELANIE7-II software) to the two nearest landmarks. 5.3.10 Matching With Primary Master Image

Images were edited to remove gross artifacts such as dust, to reject images which had gross abnormalities such as smearing of protein features, or were of too low a loading or overall image intensity to allow identification of more than the most intense features, or were of too poor a resolution to allow accurate detection of features. Images were then compared by pairing with one common image from the whole sample set. This common image, the "primary master image", was selected on the basis of protein load (maximum load consistent with maximum feature detection), a well resolved myoglobin region, (myoglobin was used as an internal standard), and general image quality. Additionally, the primary master image was chosen to be an image which appeared to be generally representative of all those to be included in the analysis. (This process by which a primary master gel was judged to be representative of the study gels was rechecked by the method described below and in the event that the primary master gel was seen to be unrepresentative, it was rejected and the process repeated until a representative primary master gel was found.)

Each of the remaining study gel images was individually matched to the primary master image such that common protein features were paired between the primary master image and each individual study gel image as described below.

5.3.11 Cross-matching Between Samples

To facilitate statistical analysis of large numbers of samples for purposes of identifying features that are differentially expressed, the geometry of each study gel was adjusted for maximum alignment between its pattern of protein features, and that of the primary master, as follows. Each of the study gel images was individually transformed into the geometry of the primary master image using a multi-resolution warping procedure. This procedure corrects the image geometry for the distortions brought about by small changes in the physical parameters of the electrophoresis separation process from one sample to another. The observed changes are such that the distortions found are not simple geometric distortions, but rather a smooth flow, with variations at both local and global scale. The fundamental principle in multi-resolution modelling is that smooth signals may be modelled as an evolution through 'scale space', in which details at successively finer scales are added to a low resolution approximation to obtain the high resolution signal. This type of model is applied to the flow field of vectors (defined at each pixel position on the reference image) and allows flows of arbitrary smoothness to be modelled with relatively few degrees of freedom. Each image is first reduced to a stack, or pyramid, of images derived from the initial image, but smoothed and reduced in resolution by a factor of 2 in each direction at every level (Gaussian pyramid) and a corresponding difference image is also computed at each level, representing the difference between the smoothed image and its progenitor (Laplacian pyramid). Thus the Laplacian images represent the details in the image at different scales.

To estimate the distortion between any 2 given images, a calculation was performed at level 7 in the pyramid (i.e. after 7 successive reductions in resolution). The Laplacian images were segmented into a grid of 16x16 pixels, with 50% overlap between adjacent grid positions in both directions, and the cross correlation between corresponding grid squares on the reference and the test images was computed. The distortion displacement was then given by the location of the maximum in the correlation matrix. After all displacements had been calculated at a particular level, they were interpolated to the next level in the pyramid, applied to the test image, and then further corrections to the displacements were calculated at the next scale.

The warping process brought about good alignment between the common features in the primary master image, and the images for the other samples. The MELANIE7 II 2D PAGE analysis program was used to calculate and record approximately 500-700 matched feature pairs between the primary master and each of the other images. The accuracy of this program was significantly enhanced by the alignment of the images in the manner described above. To improve accuracy still further, all pairings were finally examined by eye in the MelView interactive editing program and residual recognizably incorrect pairings were removed. Where the number of such recognizably incorrect pairings exceeded the overall reproducibility of the Preferred Technology (as measured by repeat analysis of the same biological sample) the gel selected to be the primary master gel was judged to be insufficiently representative of the study gels to serve as a primary master gel. In that case, the gel chosen as the primary master gel was rejected, and different gel was selected as the primary master gel, and the process was repeated.

All the images were then added together to create a composite master image, and the positions and shapes of all the gel features of all the component images were super-imposed onto this composite master as described below. Once all the initial pairs had been computed, corrected and saved, a second pass was performed whereby the original (unwarped) images were transformed a second time to the geometry of the primary master, this time using a flow field computed by smooth interpolation of the multiple tie-points defined by the centroids of the paired gel features. A composite master image was thus generated by initialising the primary master image with its feature descriptors. As each image was transformed into the primary master geometry, it was digitally summed pixel by pixel into the composite master image, and the features that had not been paired by the procedure outlined above were likewise added to the composite master image description, with their centroids adjusted to the master geometry using the flow field correction. The final stage of processing was applied to the composite master image and its feature descriptors, which now represent all the features from all the images in the study transformed to a common geometry. The features were grouped together into linked sets or "clusters", according to the degree of overlap between them. Each cluster was then given a unique identifying index, the molecular cluster index (MCI). An MCI identifies a set of matched features on different images. Thus an MCI represents a protein or proteins eluting at equivalent positions in the 2D separation in different samples.

5.3.12 Construction of Profiles

After matching all component gels in the study to the final composite master image, the intensity of each feature was measured and stored. The end result of this analysis was the generation of a digital profile which contained, for each identified feature: 1) a unique identification code relative to corresponding feature within the composite master image (MCI), 2) the x, y coordinates of the features within the gel, 3) the isoelectric point (pi) of the features, 4) the apparent molecular weight (MW) of the features, 5) the signal value, 6) the standard deviation for each of the preceding measurements, and 7) a method of linking the MCI of each feature to the master gel to which this feature was matched. By virtue of a Laboratory Information Management System (LIMS), this MCI profile was traceable to the actual stored gel from which it was generated, so that proteins identified by computer analysis of gel profile databases could be retrieved. The LIMS also permitted the profile to be traced back to an original sample or patient.

5.3.13 Statistical Analysis of the Profiles The MCI selection strategy was based the following statistical approach:

(a) the use of the fold change. A fold change representing the ratio of the average normalized protein abundances of the MCI, was calculated for each MCI between the set of samples taken from subjects having kidney toxicity and the set of subjects free from kidney toxicity (control). Statistical comparisons were made as follows: between treated and control groups at the 80mg/kg/day dose level for 4-aminophenol treated subjects, at the 750 mg/kg/day dose level for D-serine treated subjects and at the 6mg/kg dose level for cisplatin treated subjects. The data from the all the time points were used to generate statistical comparisons. MCIs that were significantly altered (p < 0.05) were selected.

(b) qualitative presence or absence. Only features that had a high feature presence were examined. Features were further qualitatively selected based on their dose response and temporal response profiles.

Thus selected, these MCIs are then considered to be selected features.

5.3.14 Recovery and analysis of selected proteins

Proteins in selected features were robotically excised and processed to generate tryptic digest peptides. Tryptic peptides were analyzed by mass spectrometry using a PerSeptive Biosystems Voyager- DETM STR Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) mass spectrometer, and selected tryptic peptides were analyzed by tandem mass spectrometry (kidney toxicity /kidney toxicity) using a Micromass Quadrupole Time-of -Flight (Q-TOF) mass spectrometer (Micromass, Altrincham, U.K.) equipped with a nanoflowTM electrospray Z-spray source. For partial amino acid sequencing and identification of KTPIs uninterpreted tandem mass spectra of tryptic peptides were searched using the SEQUEST search program (Eng et al, 1994, J. Am. Soc. Mass Spectrom. 5:976-989), version v.C.1. Criteria for database identification included: the cleavage specificity of trypsin; the detection of a suite of a, b and y ions in peptides returned from the database. The database searched was database constructed of protein entries in the non-redundant database held by the National Centre for Biotechnology Information (NCBI) which is accessible at www.ncbi.nlm.nih.gov/. Following identification of proteins through spectral-spectral correlation using the SEQUEST program, masses detected in MALDI-TOF mass spectra were assigned to tryptic digest peptides within the proteins identified. In cases where no amino acid sequences could be identified through searching with uninterpreted kidney toxicity /kidney toxicity spectra of tryptic digest peptides using the SEQUEST program, tandem mass spectra of the peptides were interpreted manually, using methods known in the art. (In the case of interpretation of low-energy fragmentation mass spectra of peptide ions see Gaskell et al, 1992, Rapid Commun. Mass Spectrom. 6:658-662). The method described in WO02/021139 was also used to interpret mass spectra. 5.4 Results

The following polypeptides were identified in selected features as described above: LMW T- kinninogen, alpha-1-macroglobulin, apolipoprotein E, clusterin, complement C3, complement C4, fibrinogen-alpha-chain, fibrinogen-beta-chain, inter-alpha-inhibitor H4P heavy chain and plasma retinol binding protein. Their specific polypeptide Protein Isoforms (Pis) are described by pi, MW and partial amino acid sequence data and is provided in Table I.

Table III details the differential expression of the polypeptide protein isoforms identified in subjects having kidney toxicity induced by treatment with 4-aminophenol, D-serine or cisplatin. Specifically, protein isoforms of LMW T-kinninogen (I/II) (LMW-TKPIs), alpha- 1-macroglobulin (A1M- PIs) apolipoprotein E (APOE-PIs), clusterin (CLUS-PIs), complement C3 (C3-PIs), complement C4 (C4- PIs), fibrinogen-alpha-chain (FA-PIs), fibrinogen-beta-chain (FB-PIs), inter-alpha-inhibitor H4P heavy chain (H4P-PIs) and plasma retinol binding (PRBP-PIs).

Tablelll. Differential Expression of polypeptides identified in subjects with kidney toxicity, in articular, renal tubular necrosis

The following specific isoforms LMW-TKPI-1, LMW-TKPI-2, LMW-TKPI-3, LMW-TKPI-4, LMW-TKPI-5, LMW-TKPI-6, C3-PI-1, C3-PI-2, C4-PI-1, C4-PI-2, FA-PI-1, FB-PI-1, H4P-1, H4P-2 or H4P-3, were all increased in subjects with kidney toxicity, in particular renal tubular necrosis, as compared with subjects free from kidney toxicity. Preferred protein isoforms are LMW-TKPI-1, LMW- TKPI-2, LMW-TKPI-3, LMW-TKPI-4, LMW-TKPI-5, LMW-TKPI-6, C3-PI-1, C3-PI-2, H4P-1, H4P-2 or H4P-3. Highly preferred protein isoforms are LMW-TKPI-1, LMW-TKPI-2, LMW-TKPI-3, LMW- TKPI-4, LMW-TKPI-5, or LMW-TKPI-6. Whereas the following specific isoforms AIM-PI- 1, A1M-PI-2, APOE-PI-1, CLUS-PI-1, FA-PI- 2, FB-PI-1 or PRBP-PI-1 were all decreased in subjects with kidney toxicity, in particular renal tubular necrosis, as compared with subjects free from kidney toxicity. Preferred protein isoforms are A1M-PI-1, A1M-PI-2 or PRBP-PI-1.

The present invention is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the invention. Functionally equivalent methods and apparatus within the scope of the invention, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications and variations are intended to fall within the scope of the appended claims.

Claims

1. A method for screening or diagnosis of kidney toxicity in a target cell, tissue or mammal which comprises the step of detecting and/or quantifying in a test sample obtained from said target T-kinninogen and/or inter-alpha-inhibitor H4P heavy chain optionally in combination with one or more of the following polypeptides: alpha-1-macroglobulin, apolipoprotein E, clusterin, complement C3, complement C4, fibrinogen-alpha-chain, fibrinogen-beta-chain, or plasma retinol binding protein polypeptides.

2. The method according to claim 1 wherein the abundance of said polypeptides in the test sample is compared with the abundance of said polypeptide in a sample from one or more targets free from kidney toxicity, or with a previously determined reference range for said polypeptides in targets free from kidney toxicity, or with the abundance of at least one standard polypeptide in the test sample.

3. A method for predicting the ability of an environmental factor to cause kidney toxicity comprising: a) exposing a target cell, tissue or mammal to the environmental factor; b) obtaining a test sample from the target; c) detecting and/or quantifying in the sample T-kinninogen and/or inter-alpha-inhibitor H4P heavy chain optionally in combination with one or more of the following polypeptides: alpha- 1-macroglobulin, apolipoprotein E, clusterin, complement C3, complement C4, fibrinogen-alpha-chain, fibrinogen-beta- chain, and plasma retinol binding protein, as defined in claim 1; and d) comparing the abundance of the polypeptides detected in c) in the test sample with the abundance of said polypeptides in a control sample, or with a previously determined reference range for said polypeptides in targets free from kidney toxicity, or with the abundance at least one standard polypeptide in the test sample.

4. The method according to claim 3 wherein the control sample is obtained from a target which has not been exposed to the environmental factor.

5. The method according to claim 3 or 4 wherein the environmental factor is a drug compound.

6. The method according to claims 3, 4, or 5 wherein the test sample is obtained within 12h, 24h or

48h of exposure to the environmental factor.

7. The method according to any one of the preceding claims wherein said polypeptides are selected from one or more of the protein isoforms listed in Table I.

8. The method according to any one of the preceding claims wherein the target is a rodent.

9. The method of claim 8 wherein the rodent is a rat.

10. The method according to any one of the preceding claims wherein the test sample is selected from tissue, blood, serum, plasma or urine.

11. The method according to any one of the preceding claims wherein one or more of the polypeptides as defined in claim 1 are detected and/or quantified by 2D gel electrophoresis.

12. The method according to any one of the preceding claims wherein one or more of the polypeptides as defined in claim 1, are detected and/ or quantified using one or more capture reagents that specifically bind to said polypeptides.

13. The method according to claim 12 wherein the capture reagents are antibodies that specifically bind to polypeptides as defined in claim 1.

14. The method according to claim 12 or 13 wherein the capture reagents are immobilised on a solid or semi-solid support.

15. The method according to claims 12, 13 or 14 wherein the capture reagents are provided within a spatial array of one or more additional capture reagents that specifically bind to other known biomarkers of kidney toxicity.

16. The use of one or more capture reagents, that specifically bind to a polypeptide as defined in claim 1, in the preparation of a kit for screening or diagnosis of kidney toxicity.

17. A kit for use in the screening or diagnosis of kidney toxicity in a target, comprising one or more capture reagents, that specifically bind to a polypeptide as defined in claim 1, immobilised on a solid or semi- solid support and instructions for use.

18. A kit according to claim 17 wherein the solid support also has immobilised one or more capture reagents that specifically bind to other known biomarkers of kidney toxicity.

19. A method or kit, according to any of the preceding claims, wherein the kidney toxicity is renal tubular necrosis.