WO2007066131A2

WO2007066131A2 - A method of detecting skeletal muscle damage

Info

Publication number: WO2007066131A2
Application number: PCT/GB2006/004598
Authority: WO
Inventors: Julie Christine Barnes; Paul Michael Bradley
Original assignee: Biowisdom Ltd.
Priority date: 2005-12-08
Filing date: 2006-12-08
Publication date: 2007-06-14
Also published as: GB0525048D0; WO2007066131A3

Abstract

The present invention relates to a method of detecting skeletal muscle damage and to the use of certain proteins and fragments thereof as biological markers (commonly known as 'biomarkers') for such damage. The present invention has particular reference to the detection of muscle toxicity in mammals, particularly humans.

Description

A method of detecting skeletal muscle damage

The present invention relates to a method of detecting skeletal muscle damage and to the use of certain proteins and fragments thereof as biological markers (commonly known as "biomarkers") for such damage. The present invention has particular reference to the detection of muscle toxicity in mammals, particularly humans.

Muscle toxicity is an undesirable side-effect of the administration of some medicinal or veterinary products to some human or animal patients. Whether or not a given product causes toxicity depends upon factors such as the properties of the product itself and on the susceptibility of the patient to such toxicity. Some patients may be genetically predisposed to produce an adverse toxic reaction to certain products.

A toxic or other insult to mammalian tissue may provoke a variety of different cellular responses that are characteristic of stress to the tissue. The nature of such responses may depend upon the severity or duration of the insult, but may ultimately result in damage to the tissue giving rise to symptoms that in some cases may be chronic.

Accordingly it is desirable to test new products for such toxicity. In particular it is desirable to test new products for muscle toxicity and to be able to diagnose skeletal muscle damage in patients.

Biomarkers are commonly used to measure the progress of a disease or other condition. Biomarkers can range from imaging readouts to proteins that can be measured specifically in accessible body fluids such as blood, serum and urine. The identification of protein biomarkers is popular because of ease and cost of measurement, and there are a number of well-established protein biomarkers available such, for example, as prostate specific antigen (PSA) for prostate cancer. Certain troponin isoforms have been proposed as biomarkers for drug-induced cardiac muscle injury. Myosin heavy polypeptides have also been proposed for use as biomarkers for cardiac muscle toxicity. Developing biomarkers for a specific disease or pathology is challenging because any useful biomarker should be specific for that condition, sensitive enough to detect early forms of the disease or pathology, measurable in body fluids that can be readily sampled (often several times in any one subject) and should have a strong signal-to-noise ratio.

Biomarkers for skeletal muscle damage should be specific for skeletal muscle damage and should not normally be associated with damage to other tissue types, particularly heart muscle, liver or kidney tissue which are known to be susceptible to toxic insult.

An object of the present invention is to provide biomarkers for detecting or monitoring skeletal muscle damage in mammals, particularly humans or non-human animals (e.g. experimental animals such as mice, rats and the like). A particular object of the present invention is to provide biomarkers for detecting early stage muscle stress.

Another object of the invention is to provide improved biomarkers for skeletal muscle damage that meet the requirements of specificity, sensitivity and measurability.

A different object of the present invention is to provide a method for detecting skeletal muscle damage in mammals, particularly humans or non-human animals.

Yet another object of the present invention is to provide a method for diagnosing muscle toxicity in mammals, particularly humans or non-human animals.

Yet another object of the present invention comprehends the use of known proteins and splice variants or fragments thereof as biomarkers for skeletal muscle damage. Yet another object of the present invention is to provide a method for investigating the potential skeletal muscle toxicity of a medicinal or veterinary product for

administration to the mammalian body, particularly in humans or non-human animals. According to one aspect of the present invention therefore there is provided the use of a protein, or a splice variant or fragment of said protein, as a biomarker for muscle damage in a mammal, wherein said protein is selected from mitogen- activated protein kinase 12 (MAPK12), rho GTPase activating protein 26 (ARHGAP26), lactoperoxidase (LPO), acrosin (ACR), cathepsin E (CTSE),

four and a half LIM domains 3 (FHL3),

kelch repeat and BTB (POZ) domain containing 10 (KBTBDlO),

Fanconi anemia complementation group A (FANCA) and myosin binding protein H (MYBPH).

According to another aspect of the present invention there is provided a method of detecting skeletal muscle damage, said method comprising assaying a sample of body fluid obtained from a mammal for one or more proteins, or splice variants or fragments of said proteins, as biomarkers for such skeletal muscle damage, which one or more proteins are selected from mitogen-activated protein kinase 12 (MAPK12),

rho GTPase activating protein 26 (ARHGAP26), lactoperoxidase (LPO), acrosin (ACR), cathepsin E (CTSE), four and a half LIM domains 3 (FHL3),

kelch repeat and BTB (POZ) domain containing 10 (KBTBDlO),

Said mammal may comprise a human or a non-human animal, preferably a human.

Said method may be conducted entirely ex vivo. Said body fluid may comprise blood, plasma, serum or urine. Preferably said body fluid is serum or plasma obtained from a blood sample.

The above-mentioned proteins that may be used as biomarkers for skeletal muscle damage according to the present invention are expressed in skeletal muscle tissue, but are normally absent, or expressed to a lesser extent, under normal physiological conditions (i.e. in the absence of disease) in whole blood or other tissues, including particularly heart, liver and kidney tissues. It will be appreciated that all proteins expressed in skeletal muscle have potential use as biomarkers for muscle damage or toxicity. Over three thousand different mRNAs are expressed in human skeletal muscle, but many of them are also normally co-expressed in other tissues that are known to be susceptible to drug- induced toxicity and are therefore not specific for skeletal muscle damage. For instance, many such mRNAs are expressed in heart, liver and kidney tissues, which are known to be particularly prone to toxic insult. Further, proteins that are normally resident in blood would have limited use as biomarkers as their presence in a detection medium under normal physiological conditions could mask any elevation following tissue damage, resulting in a poor signal-to-noise ratio.

The above-mentioned proteins are also associated with muscle-specific functions or cellular stress. Typical muscle-specific functions include myoblast differentiation, myoblast cell fate determination, muscle development, muscle contraction, sarcomere alignment, myoblast fusion, actin filament based movement, muscle cell differentiation, somatic muscle development, myogenesis, neuromuscular junction development striated and muscle contraction.

Further, the above-mentioned proteins are located within the cytoplasmic or soluble fraction of the cell or contain a signal sequence that targets the protein for secretion.

Soluble proteins are more likely to be released from the cell upon lysis than those that are membrane bound. Those that can be secreted from the cell prior to lysis might be expected to show even more sensitivity.

Preferably the biomarkers are selected from the human forms of the above- mentioned proteins. In some embodiments said one or more protein biomarkers may comprise mitogen- activated protein kinase 12 (MAPK12). Preferably, said MAPK12 protein comprises the amino acid sequence of SEQ ID NO. 1.

In some embodiments said one or more protein biomarkers may comprise rho GTPase activating protein 26 (ARHGAP26). Preferably, said ARHGAP26 protein comprises the amino acid sequence of SEQ ID NO. 2. Alternatively, said ARHGAP26 protein may comprise the isoform amino acid sequence of SEQ ID NO. 10 which is a fragment SEQ ID NO. 2. In some embodiments said one or more protein biomarkers may comprise lactoperoxidase (LPO). Preferably, said LPO protein comprises the amino acid sequence ofSEQ ID NO. 3. In some embodiments said one or more protein biomarkers may comprise acrosin

(ACR). Preferably, said ACR protein comprises the amino acid sequence of SEQ ID NO. 4.

In some embodiments said one or more protein biomarkers may comprise cathepsin E (CTSE). Preferably, said CTSE protein comprises the amino acid sequence of SEQ IDNO. 5.

In some embodiments said one or more protein biomarkers may comprise four and a half LIM domains 3 (FHL3). Preferably, said FHL3 protein comprises the amino acid sequence of SEQ ID NO. 6.

In some embodiments said one or more protein biomarkers may comprise kelch repeat and BTB (POZ) domain containing 10 (KBTBDlO). Preferably, said

KBTBDlO protein comprises the amino acid sequence of SEQ ID NO. 7.

In some embodiments said one or more protein biomarkers may comprise

Fanconi anemia complementation group A (FANCA). Preferably, said FANCA protein comprises the amino acid sequence of SEQ ID NO. 8. In some embodiments said one or more protein biomarkers may comprise myosin binding protein H (MYBPH). Preferably, said MYBPH protein comprises the amino acid sequence of SEQ ID NO. 9.

Each of the above-mentioned proteins may exist in a number of different respective variants in which one or more amino acids are deleted, substituted or inserted. The present invention comprehends the use of any of such variants which cross-react immunogenically.

Accordingly, in some embodiments, said one or more protein biomarkers may be selected from poly- or oligo-peptides comprising or consisting essentially of: (i) a polypeptide of any one of SEQ ID NOS. 1 to 9;

(ii) a polypeptide having at least 80% identity to any one of the polypeptides of SEQ ID NOS. 1 to 9;

(iii) a polypeptide of any one of SEQ ID NOS. 1 to 9 having one or a few amino acid deletions, substitutions or insertions; or

(iv) fragments of at least five contiguous amino acids of said polypeptides (i), (ii) or (iii), which fragments are capable of binding to antibodies that bind specifically to said respective polypeptides. By "identity" is meant herein the extent to which two polypeptides are invariant.

Where the two polypeptides are non-identical, then they should be aligned for maximal correspondence in accordance with a computer algorithm known in the art for such purpose. For instance, two polypeptide sequences may be compared using the BLAST 2 program [13]. Two popular multiple sequence alignment algorithms for polypeptides are ClustalW [14] and T-Coffee [15].

Preferably said polypeptide (ii) has at least 90%, and more preferably at least 95%, identity with any one of polypeptides of SEQ ID NOS. 1 to 10. For instance, said polypeptide (ii) may have at least 96%, 97%, 98% or 99% identity with said any one of polypeptides of SEQ ID NOS. 1 to 10.

Preferably, said fragments comprise at least ten, and more preferably at least fifteen, contiguous amino acids of (i), (ii) or (iii). By "fragments" of said proteins is meant polypeptides that comprise fewer amino acids than the corresponding full-length protein, including splice-variants. Antibodies cross-reacting with said variants also have specificity for the corresponding full-length proteins. Said fragments and variants may therefore share one or more epitopes with the full-length protein and would not normally comprehend portions of said full-length proteins that are not distinctive or characteristic of the proteins such, for example, as some transmembrane portions that are highly conserved amongst many membrane-bound proteins. The amino acid sequence of SEQ ID NO. 10 is an exemplary fragment of the amino acid sequence of SEQ ID NO. 2. In some embodiments, said protein or proteins may comprise mitogen-activated protein kinase 12 or rho GTPase activating protein 26, since these proteins are associated with one or more early stage stress functions and may therefore constitute biomarkers with high sensitivity, especially during the early stages of a response to a toxic or other insult to the tissue. Early stage stress functions include mitosis, cell proliferation, cell growth, hyperplasia, intracellular signalling cascade and signal transduction pathway.

By targeting early stage stress, one can identify biological markers of early and perhaps reversible toxicity-induced stress.

In some embodiments, said protein or proteins may be selected from Fanconi anemia, complementation Group A and lactoperoxidase, since these proteins are associated with one or more intermediate phase stress functions. Intermediate phase stress functions include DNA Repair, response to stress, oxidative stress response, cell ageing, JAK-STAT cascade, double-strand break repair and oxidation. The use of lactoperoxidase may be especially advantageous, since this protein possesses a predicted signal peptide and is therefore probably secreted.

Further, in some embodiments, said protein or proteins may be selected from acrosin, cathepsin E, mitogen-activated protein kinase 12, since these proteins are associated with one or or more late phase stress functions. Late phase stress functions include peptidolysis, proteolysis, endocytosis, digestion, apoptosis, ATP-dependent proteolysis, inflammatory response, cell death, response to wounding, cell cycle arrest, necrosis and inflammation. Like lactoperoxidase, acrosin possesses a predicted signal peptide and may therefore be secreted, making it especially suitable for use as a biomarker.

Said protein biomarkers may be qualitatively or quantitatively assayed using any suitable method known to those skilled in the art such, for example, as an enzyme-linked immunosorbent assays (ELISA) or Western blotting, both of which make use of antibodies to the protein biomarkers. Preferably, sandwich ELISA may be used. Such antibodies may be monoclonal or polyclonal antibodies, and methods of obtaining such antibodies are also well-known in the art. In a particular aspect of the present invention, said sample of body fluid may be obtained from said patient following administration of a medicinal product to said patient. The method of the present invention may therefore be used to investigate the toxicology of said medicinal product. Said assay may be carried out on a serum sample, whole blood or plasma obtained from a blood sample.

In some embodiments, a series of samples taken periodically from said patient may be assayed to monitor the toxicity of a medicinal or veterinary product over time. In some embodiments, said sample or samples may be assayed for only one of the above-mentioned proteins.

Alternatively, said sample or samples may be tested for a plurality of said proteins. Assaying a series of samples obtained over time for a panel of biomarkers may be advantageous where one biomarker is expressed at an earlier stage during the progression of a toxic response than another biomarker. The results of such assays may therefore be used to indicate the extent of progression of said toxic response.

Assaying for two or more biomarkers may also serve to reduce the risk of misdiagnosis.

Accordingly, in yet another aspect of the present invention there is provided a method of diagnosing muscle toxicity in a mammalian patient which comprises obtaining a sample of body fluid from said patient and assaying said sample for at least one protein, or a splice variant or fragment of said protein, as a biomarker for muscle toxicity, said at least one protein being selected from mitogen-activated protein kinase 12 (MAPK 12), rho GTPase activating protein 26 (ARHGAP26), lactoperoxidase (LPO), acrosin (ACR), cathepsin E (CTSE), four and a half LIM domains 3 (FHL3),

kelch repeat and BTB (POZ) domain containing 10 (KBTBDlO),

Fanconi anemia complementation group A (FANCA) and myosin binding protein H

(MYBPH).

As described above, said method may comprise assaying said sample for two or more of said proteins. In yet another aspect of the present invention there is provided a method for investigating the toxicology of a candidate medicinal or veterinary product in mammalian patients, which method comprises administering said candidate product to one or more patients, obtaining a sample of body fluid from the or each patient and assaying said sample for at least one protein, or a splice variant or fragment of said protein, said at least one protein being selected from mitogen-activated protein kinase 12 (MAPK 12), rho GTPase activating protein 26 (ARHGAP26), lactoperoxidase (LPO), acrosin (ACR), cathepsin E (CTSE), four and a half LIM domains 3 (FHL3),

kelch repeat and BTB (POZ) domain containing 10 (KBTBDlO),

Said method may comprise assaying said sample for two or more of said proteins.

Said method may further comprise periodically obtaining samples from the or each patient to provide a series of samples over time and assaying each of said samples for one or more of said protein biomarkers. Preferably, said candidate medicinal or veterinary product is not insulin or medication presently prescribed for diabetes.

Preferably, skeletal muscle damage does not refer to age-related changes in skeletal muscle, or to changes in patients with diabetes, in particular type 2 diabetes, or to changes in skeletal muscle associated with cancer.

Following is a description by way of example only of embodiments of the present invention. Example 1

MAPK 12 is selected as a protein biomarker for skeletal muscle damage in accordance with the present invention. Mitogen-activated protein kinase 12 (MAPK12) is located in skeletal muscle [I]. It is also known that MAPK12 is a cytoplasmic protein that is involved in myoblast differentiation, cell cycle arrest, signal transduction, muscle development and the cell cycle process [2]. MAPK 12 may be a biomarker for early and late stage skeletal muscle stress.

A candidate drug X is administered daily in a prescribed dosage amount to a plurality, e.g. twenty to one hundred, of healthy human volunteers or experimental non- human animals.

Preparation of plasma samples

Periodically a blood sample is taken from each volunteer. Li this example, the samples are taken daily at a predetermined time, but in other embodiments, the samples may be taken more or less frequently. The blood samples are collected into an anticoagulant solution, e.g. 3.8% trisodium citrate in the proportion of 9 volumes of blood to 1 volume of anticoagulant solution. The two components are gently mixed and centrifuged at 3,000 rpm for 10 minutes. The supernatant (plasma) is carefully removed without disturbing the pellet of red cells.

The following protocol assumes that the biomarkers of muscle damage will reach sufficient concentrations through tissue leakage to allow quantitation by sandwich ELISA without additional sample preparation. Should concentration of the plasma sample be necessary prior to analysis, this can be achieved by a variety of methods, such as vacuum evaporation, ultrafiltration or TCA precipitation.

Sandwich ELISA Protocol

A 96 well microtitre plate is pre-coated with 50 μl of a 10 μg/ml solution of unlabelled antibody and incubated at 37°C for 1 hour or overnight at 4°C. Anti-MAPK12 antibodies are available commercially, for example from Abgent [3]. The plate is washed twice in phosphate buffered saline (PBS) to remove unbound antibody then incubated for 1 hour at 37°C in blocking buffer, a solution of PBS containing 1% bovine serum albumin (BSA), to saturate any non-specific binding sites.

An antigen standard is serially diluted in blocking buffer, to prepare a standard curve comprising at least five points in the range of 50-150% of the expected concentration of antigen in plasma. Samples are diluted, if required, in blocking buffer and a negative control sample (a human plasma that tests negative for the antigen under consideration) is treated in the same way as the samples. Diluted samples and standards are added at each concentration in at least duplicate (-50 μl per well) and incubated for 1 hour at 37⁰C. After washing four times in PBS, 50 μl of biotin-labelled antibody, diluted in blocking buffer according to the manufacturer's recommendations, is added to each well and incubated for 1 hour at 37⁰C. The PBS washing step is repeated before adding 50 μl of horseradish peroxidase (HRP)-streptavidin (diluted in blocking buffer according to manufacturer's recommendations) and further incubating at 37°C for 60 minutes.

After repeating the washing step, 200 μl of substrate is added to each well. A suitable substrate is ABTS (2,2'-azino-di-(3-ethylbenz-thiazoline sulfonic acid)). The plate is incubated at room temperature until the colour has developed sufficiently, typically between 2 and 20 minutes. The absorbance at 414 nm is measured using an ELISA plate reader, blanking against the negative control sample. The concentration of antigen present in the sample is determined by selecting one or more sample concentrations that fall within the linear portion of the standard curve, and correcting for the dilution performed prior to analysis. The presence of MAPK 12 in any plasma sample is indicative of skeletal muscle damage, possibly resulting from toxicity produced by the administration of drug X.

Example 2

ARHGAP26 is selected as a protein biomarker for skeletal muscle damage in accordance with the present invention. Rho GTPase activating protein 26 (ARHGAP26) is known to be located in skeletal muscle [4] and to be involved in actin filament biogenesis [2]. Example 1 is repeated using goat anti-ARHGAP26 polyclonal antibodies which are commercially available from, for example, IMGENEX [5]. Example 3

LPO is selected as a protein biomarker for skeletal muscle damage in accordance with the present invention. Lactoperoxidase (LPO) is known to be located in skeletal muscle [1] and to be involved in the response to oxidative stress [2]. The precursor form of the protein possesses a potential signal peptide and is therefore likely to be secreted [6]. Example 1 is repeated using sheep anti-bovine LPO polyclonal antibodies, which are commercially available, for example, from Research Diagnostics, Inc. [7]. This antibody had been raised against the bovine LPO orthologue; the literature suggests that human salivary peroxidase and bovine lactoperoxidase are cross-reactive [8].

Example 4

ACR is selected as a protein biomarker for skeletal muscle damage in accordance with the present invention. Acrosin (ACR) is known to be located in skeletal muscle [1] and to be involved in proteolysis [2]. The precursor form of the protein possesses a potential signal peptide and is therefore likely to be secreted [6]. Example 1 is repeated using, for example, two from a panel of anti-acrosin monoclonal antibodies that can be purchased from Biosonda [9]. Example 5

CTSE is selected as a protein biomarker for skeletal muscle damage in accordance with the present invention. Cathepsin E (CTSE) is known to be located in skeletal muscle [1] and to be involved in digestion and proteolysis [2]. Example 1 is repeated using an anti-cathepsin E antibody, for example goat anti-human cathepsin E available from R&D Systems [10].

Example 6

FHL3 is selected as a protein biomarker for skeletal muscle damage in accordance with the present invention. Four and a half LLVl domains 3 (FHL3) is known to be located in skeletal muscle [1] and to be involved in muscle development [2]. Example 1 is repeated using a suitable antibody, e.g. chicken anti-FHL3 polyclonal antibody, which is available from Abeam [H].

Example 7

FANCA is selected as a protein biomarker for skeletal muscle damage in accordance with the present invention. Fanconi anemia complementation group A (FANCA) is known to be located in skeletal muscle [1] and to be involved in DNA repair [2]. Example 1 is repeated using an appropriate antibody, for example rabbit anti-human FANCA antibody, which can be obtained from Abeam [H]. Example 8

MYBPH is selected as a protein biomarker for skeletal muscle damage in accordance with the present invention. Myosin binding protein H (MYBPH) is known to be located in skeletal muscle [1] and to be involved in muscle development [2]. Example 1 is repeated using an appropriate anti-MYBPH antibody; generation of a site-directed polyclonal antibody to MYBPH is described in the literature [12].

Example 9

KBTBDlO is selected as a protein biomarker for skeletal muscle damage in accordance with the present invention. Kelch repeat and BTB (POZ) domain containing 10 (BCBTBD 10) is known to be located in skeletal muscle [4] and to be involved in striated muscle contraction [2]. Example 1 is repeated using a polyclonal anti-human KBTBDlO antibody; such a product does not currently exist commercially, so antibodies should be raised against human KBTBD 10 protein.

Example 10

Example 1 is repeated using a panel of protein biomarkers, said panel including at least one protein that is associated with one or more early stage stress functions as described above, at least one that is associated with one or more intermediate phase stress functions and at least one that is associated with one or or more late stage functions, e.g. Rho GTPase activating protein 26 (early), Fanconi anemia complementation group A (intermediate) and Cathepsin E (late). This combination of biomarkers is used to monitor progress of damage to the muscle.

References

The contents of the following references are all incorporated severally herein by reference.

I. Haverty et al, Nucleic Acids Res. 2002 Jan l;30(l):214-7

2. Maglott et al., Nucleic Acids Res. 2005 Jan l;33(Database issue):D54-8

3. MAPK12 antibodies: http://www.abgent.com/

4. Wheeler et al., Nucleic Acids Res. 2005 Jan 1 ;33(Database issue):D39-45

5. ARHGAP26 antibodies: http://www.imgenex.com/

6. Bairoch et al., Nucleic Acids Res. 2005 Jan 1 ;33(Database issue):Dl 54-9

7. LPO antibodies: http://www.researchd.com/

8. Mansson-Rahemtulla et al., J Dent Res. 1990 Dec;69(12): 1839-46

9. ACR antibodies: http://www.biosonda.com

10. CTSE antibodies: http://www.rndsystems.com/

II. FHL3 and FANCA antibodies: http://www.abcam.com/

12. Alyonycheva et al., Circ Res. 1997 May; 80(5): 665 -72

13. Tatiana et al., FEMS Microbiol Lett. 174:247-250

14. Thompson et al., Nucleic Acids Res. 1994 Nov 11 ;22(22):4673-80

15. Notredame et al., J MoI Biol. 2000 Sep 8;302(l):205-17

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<211> 712

<212> PRT

<213> Homo sapiens

<400> 3

Met Arg VaI Leu Leu His Leu Pro Ala Leu Leu Ala Ser Leu lie Leu

1 5 10 15

Leu GIn Ala Ala Ala Ser Thr Thr Arg Ala GIn Thr Thr Arg Thr Ser

20 25 30

Ala lie Ser Asp Thr VaI Ser GIn Ala Lys VaI GIn VaI Asn Lys Ala

35 40 45

Phe Leu Asp Ser Arg Thr Arg Leu Lys Thr Ala Met Ser Ser GIu Thr 50 55 60

Pro Thr Ser Arg GIn Leu Ser GIu Tyr Leu Lys His Ala Lys GIy Arg 65 70 75 80

Thr Arg Thr Ala lie Arg Asn GIy GIn VaI Trp GIu GIu Ser Leu Lys

85 90 95

Arg Leu Arg GIn Lys Ala Ser Leu Thr Asn VaI Thr Asp Pro Ser Leu

100 105 110

Asp Leu Thr Ser Leu Ser Leu GIu VaI GIy Cys GIy Ala Pro Ala Pro

115 120 125

VaI VaI Arg Cys Asp Pro Cys Ser Pro Tyr Arg Thr lie Thr GIy Asp 130 135 140

Cys Asn Asn Arg Arg Lys Pro Ala Leu GIy Ala Ala Asn Arg Ala Leu 145 150 155 160

Ala Arg Trp Leu Pro Ala GIu Tyr GIu Asp GIy Leu Ser Leu Pro Phe

165 170 175

GIy Trp Thr Pro GIy Lys Thr Arg Asn GIy Phe Pro Leu Pro Leu Ala

180 185 190 Arg GIu VaI Ser Asn Lys lie VaI GIy Tyr Leu Asn GIu GIu GIy VaI 195 200 205

Leu Asp GIn Asn Arg Ser Leu Leu Phe Met GIn Trp GIy GIn lie VaI 210 215 220

Asp His Asp Leu Asp Phe Ala Pro Asp Thr GIu Leu GIy Ser Ser GIu 225 230 235 240

Tyr Ser Lys Ala GIn Cys Asp GIu Tyr Cys lie GIn GIy Asp Asn Cys

245 250 255

Phe Pro lie Met Phe Pro Pro Asn Asp Pro Lys Ala GIy Thr Gin GIy

260 265 270

Lys Cys Met Pro Phe Phe Arg Ala GIy Phe VaI Cys Pro Thr Pro Pro

275 280 285

Tyr Lys Ser Leu Ala Arg GIu GIn lie Asn Ala Leu Thr Ser Phe Leu 290 295 300

Asp Ala Ser Phe VaI Tyr Ser Ser GIu Pro Ser Leu Ala Ser Arg Leu 305 310 315 320

Arg Asn Leu Ser Ser Pro Leu GIy Leu Met Ala VaI Asn GIn GIu VaI

325 330 335

Ser Asp His GIy Leu Pro Tyr Leu Pro Tyr Asp Ser Lys Lys Pro Ser

340 345 350

Pro Cys GIu Phe lie Asn Thr Thr Ala Arg VaI Pro Cys Phe Leu Ala

355 360 365

GIy Asp Ser Arg Ala Ser GIu His lie Leu Leu Ala Thr Ser His Thr 370 375 380

Leu Phe Leu Arg GIu His Asn Arg Leu Ala Arg GIu Leu Lys Arg Leu 385 390 395 400

Asn Pro GIn Trp Asp GIy GIu Lys Leu Tyr GIn GIu Ala Arg Lys lie

405 410 415

Leu GIy Ala Phe VaI GIn lie lie Thr Phe Arg Asp Tyr Leu Pro lie

420 425 430

Leu Leu GIy Asp His Met GIn Lys Trp lie Pro Pro Tyr GIn GIy Tyr

435 440 445

Ser GIu Ser VaI Asp Pro Arg lie Ser Asn VaI Phe Thr Phe Ala Phe 450 455 460

Arg Phe GIy His Leu GIu VaI Pro Ser Ser Met Phe Arg Leu Asp GIu 465 470 475 480

Asn Tyr GIn Pro Trp GIy Pro GIu Pro GIu Leu Pro Leu His Thr Leu 485 490 495

Phe Phe Asn Thr Trp Arg Met VaI Lys Asp GIy GIy lie Asp Pro Leu

500 505 510

VaI Arg GIy Leu Leu Ala Lys Lys Ser Lys Leu Met Lys GIn Asn Lys

515 520 525

Met Met Thr GIy GIu Leu Arg Asn Lys Leu Phe GIn Pro Thr His Arg 530 535 540

lie His GIy Phe Asp Leu Ala Ala lie Asn Thr GIn Arg Cys Arg Asp 545 550 555 560

His GIy GIn Pro GIy Tyr Asn Ser Trp Arg Ala Phe Cys Asp Leu Ser

565 570 575

Gin Pro Gin Thr Leu GIu GIu Leu Asn Thr VaI Leu Lys Ser Lys Met

580 585 ^' 590

Leu Ala Lys Lys Leu Leu GIy Leu Tyr GIy Thr Pro Asp Asn lie Asp

595 600 605

lie Trp lie GIy Ala lie Ala GIu Pro Leu VaI GIu Arg GIy Arg VaI 610 615 620

GIy Pro Leu Leu Ala Cys Leu Leu GIy Lys GIn Phe GIn GIn lie Arg 625 630 635 640

Asp GIy Asp Arg Phe Trp Trp GIu Asn Pro GIy VaI Phe Thr Asn GIu

645 650 655

GIn Lys Asp Ser Leu GIn Lys Met Ser Phe Ser Arg Leu VaI Cys Asp

660 665 670

Asn Thr Arg lie Thr Lys VaI Pro Arg Asp Pro Phe Trp Ala Asn Ser

675 680 685

Tyr Pro Tyr Asp Phe VaI Asp Cys Ser Ala lie Asp Lys Leu Asp Leu 690 695 700

Ser Pro Trp Ala Ser VaI Lys Asn

705 710

<210> 4

<211> 421

<212> PRT

<213> Homo sapiens

<400> 4

Met VaI GIu Met Leu Pro Thr Ala lie Leu Leu VaI Leu Ala VaI Ser

1 5 10 15

VaI VaI Ala Lys Asp Asn Ala Thr Cys Asp GIy Pro Cys GIy Leu Arg

20 25 30

Phe Arg GIn Asn Pro GIn GIy GIy VaI Arg lie VaI GIy GIy Lys Ala 35 40 45

Ala Gin His GIy Ala Trp Pro Trp Met VaI Ser Leu GIn lie Phe Thr 50 55 60

Tyr Asn Ser His Arg Tyr His Thr Cys GIy GIy Ser Leu Leu Asn Ser 65 70 75 80

Arg Trp VaI Leu Thr Ala Ala His Cys Phe VaI GIy Lys Asn Asn VaI

85 90 95

His Asp Trp Arg Leu VaI Phe GIy Ala Lys GIu lie Thr Tyr GIy Asn

100 105 110

Asn Lys Pro VaI Lys Ala Pro Leu GIn GIu Arg Tyr VaI GIu Lys He

115 120 125

He He His GIu Lys Tyr Asn Ser Ala Thr GIu GIy Asn Asp He Ala 130 135 140

Leu VaI GIu He Thr Pro Pro He Ser Cys GIy Arg Phe He GIy Pro 145 150 155 160

GIy Cys Leu Pro His Phe Lys Ala GIy Leu Pro Arg Gly Ser GIn Ser

165 170 175

Cys Trp VaI Ala GIy Trp GIy Tyr He GIu GIu Lys Ala Pro Arg Pro

180 185 190

Ser Ser He Leu Met GIu Ala Arg VaI Asp Leu He Asp Leu Asp Leu

195 200 205

Cys Asn Ser Thr GIn Trp Tyr Asn GIy Arg VaI GIn Pro Thr Asn VaI 210 215 220

Cys Ala Gly Tyr Pro VaI GIy Lys He Asp Thr Cys GIn Gly Asp Ser 225 230 235 240

Gly Gly Pro Leu Met Cys Lys Asp Ser Lys GIu Ser Ala Tyr VaI VaI

245 250 255

VaI Gly He Thr Ser Trp Gly VaI Gly Cys Ala Arg Ala Lys Arg Pro

260 265 270

Gly He Tyr Thr Ala Thr Trp Pro Tyr Leu Asn Trp He Ala Ser Lys

275 280 285

He Gly Ser Asn Ala Leu Arg Met He GIn Ser Ala Thr Pro Pro Pro 290 295 300

Pro Thr Thr Arg Pro Pro Pro He Arg Pro Pro Phe Ser His Pro He 305 310 315 320

Ser Ala His Leu Pro Trp Tyr Phe Gin Pro Pro Pro Arg Pro Leu Pro

325 330 335 Pro Arg Pro Pro Ala Ala GIn Pro Pro Pro Pro Pro Ser Pro Pro Pro 340 345 350

Pro Pro Pro Pro Pro Ala Ser Pro Leu Pro Pro Pro Pro Pro Pro Pro

355 360 365

Pro Pro Thr Pro Ser Ser Thr Thr Lys Leu Pro GIn GIy Leu Ser Phe 370 375 380

Ala Lys Arg Leu GIn GIn Leu lie GIu VaI Leu Lys GIy Lys Thr Tyr 385 390 395 400

Ser Asp GIy Lys Asn His Tyr Asp Met GIu Thr Thr GIu Leu Pro GIu

405 410 415

Leu Thr Ser Thr Ser

420

<210> 5

<211> 401

<212> PRT

<213> Homo sapiens

<400> 5

Met Lys Thr Leu Leu Leu Leu Leu Leu VaI Leu Leu GIu Leu GIy GIu

1 5 10 15

Ala Gin GIy Ser Leu His Arg VaI Pro Leu Arg Arg His Pro Ser Leu

20 25 30

Lys Lys Lys Leu Arg Ala Arg Ser Gin Leu Ser GIu Phe Trp Lys Ser

35 40 45

His Asn Leu Asp Met lie Gin Phe Thr GIu Ser Cys Ser Met Asp GIn 50 55 60

Ser Ala Lys GIu Pro Leu He Asn Tyr Leu Asp Met GIu Tyr Phe GIy 65 70 75 80

Thr He Ser He GIy Ser Pro Pro GIn Asn Phe Thr VaI He Phe Asp

85 90 95

Thr GIy Ser Ser Asn Leu Trp VaI Pro Ser VaI Tyr Cys Thr Ser Pro

100 105 110

Ala Cys Lys Thr His Ser Arg Phe GIn Pro Ser Gin Ser Ser Thr Tyr

115 120 125

Ser GIn Pro GIy GIn Ser Phe Ser He GIn Tyr GIy Thr GIy Ser Leu 130 135 140

Ser GIy He He GIy Ala Asp Gin VaI Ser Ala Phe Ala Thr GIn VaI 145 150 155 160

GIu GIy Leu Thr VaI VaI GIy GIn GIn Phe GIy GIu Ser VaI Thr GIu

165 170 175 Pro GIy GIn Thr Phe VaI Asp Ala GIu Phe Asp GIy lie Leu GIy Leu 180 185 190

GIy Tyr Pro Ser Leu Ala VaI GIy GIy VaI Thr Pro VaI Phe Asp Asn

195 200 205

Met Met Ala GIn Asn Leu VaI Asp Leu Pro Met Phe Ser VaI Tyr Met 210 215 220

Ser Ser Asn Pro GIu GIy GIy Ala GIy Ser GIu Leu He Phe GIy GIy 225 230 235 240

Tyr Asp His Ser His Phe Ser GIy Ser Leu Asn Trp VaI Pro VaI Thr

245 250 255

Lys GIn Ala Tyr Trp GIn He Ala Leu Asp Asn He GIn VaI GIy GIy

260 265 270

Thr VaI Met Phe Cys Ser GIu GIy Cys GIn Ala He VaI Asp Thr GIy

275 280 285

Thr Ser Leu He Thr GIy Pro Ser Asp Lys He Lys GIn Leu GIn Asn 290 295 300

Ala He GIy Ala Ala Pro VaI Asp GIy GIu Tyr Ala VaI GIu Cys Ala 305 310 315 320

Asn Leu Asn VaI Met Pro Asp VaI Thr Phe Thr He Asn GIy VaI Pro

325 330 335

Tyr Thr Leu Ser Pro Thr Ala Tyr Thr Leu Leu Asp Phe VaI Asp GIy

340 345 350

Met GIn Phe Cys Ser Ser GIy Phe GIn GIy Leu Asp He His Pro Pro

355 360 365

Ala GIy Pro Leu Trp He Leu GIy Asp VaI Phe He Arg GIn Phe Tyr 370 375 380

Ser VaI Phe Asp Arg GIy Asn Asn Arg VaI GIy Leu Ala Pro Ala VaI 385 390 395 400

Pro

<210> 6

<211> 280

<212> PRT

<213> Homo sapiens

<400> 6

Met Ser GIu Ser Phe Asp Cys Ala Lys Cys Asn GIu Ser Leu Tyr GIy

1 5 10 15

Arg Lys Tyr He GIn Thr Asp Ser GIy Pro Tyr Cys VaI Pro Cys Tyr

20 25 30 Asp Asn Thr Phe Ala Asn Thr Cys Ala GIu Cys GIn GIn Leu lie GIy 35 40 45

His Asp Ser Arg GIu Leu Phe Tyr GIu Asp Arg His Phe His GIu GIy 50 55 60

Cys Phe Arg Cys Cys Arg Cys GIn Arg Ser Leu Ala Asp GIu Pro Phe 65 70 75 80

Thr Arg GIn Asp Ser Glμ Leu Leu Cys Asn Asp Cys Tyr Cys Ser Ala

85 90 95

Phe Ser Ser GIn Cys Ser Ala Cys GIy GIu Thr VaI Met Pro GIy Ser

100 105 110

Arg Lys Leu GIu Tyr GIy GIy GIn Thr Trp His GIu His Cys Phe Leu

115 120 125

Cys lie GIy Cys GIu GIn Pro Leu GIy Ser Arg Pro Phe VaI Pro Asp 130 135 140

Lys GIy Ala His Tyr Cys VaI Pro Cys Tyr GIu Asn Asn Phe Ala Pro 145 150 155 160

Arg Cys Ala Arg Cys Thr Lys Thr Leu Thr GIn GIy GIy Leu Thr Tyr

165 170 175

Arg Asp Leu Pro Trp His Pro Lys Cys Leu VaI Cys Thr GIy Cys GIn

180 185 190

Thr Pro Leu Ala GIy GIn GIn Phe Thr Ser Arg Asp GIu Asp Pro Tyr

195 200 205

Cys VaI Ala Cys Phe GIy GIu Leu Phe Ala Pro Lys Cys Ser Ser Cys 210 215 220

Lys Arg Pro lie VaI GIy Leu GIy GIy GIy Lys Tyr VaI Ser Phe GIu 225 230 235 240

Asp Arg His Trp His His Asn Cys Phe Thr Cys Asp Arg Cys Ser Asn

245 250 255

Ser Leu VaI GIy GIn GIy Phe VaI Pro Asp GIy Asp GIn VaI Leu Cys -——— 260 265 270

GIn GIy Cys Ser GIn Ala GIy Pro

275 280

<210> 7

<211> 606

<212> PRT

<213> Homo sapiens

<400> 7

Met Asp Ser GIn Arg GIu Leu Ala GIu GIu Leu Arg Leu Tyr GIn Ser

1 5 10 15 Thr Leu Leu GIn Asp GIy Leu Lys Asp Leu Leu Asp GIu Lys Lys Phe 20 25 30 lie Asp Cys Thr Leu Lys Ala GIy Asp Lys Ser Leu Pro Cys His Arg

35 40 45

Leu lie Leu Ser Ala Cys Ser Pro Tyr Phe Arg GIu Tyr Phe Leu Ser 50 55 60

GIu lie Asp GIu Ala Lys Lys Lys GIu VaI VaI Leu Asp Asn VaI Asp 65 70 75 80

Pro Ala lie Leu Asp Leu lie lie Lys Tyr Leu Tyr Ser Ala Ser lie

85 90 95

Asp Leu Asn Asp GIy Asn VaI Gin Asp lie Phe Ala Leu Ala Ser Arg

100 105 110

Phe GIn He Pro Ser VaI Phe Thr VaI Cys VaI Ser Tyr Leu GIn Lys

115 120 125

Arg Leu Ala Pro GIy Asn Cys Leu Ala He Leu Arg Leu GIy Leu Leu 130 135 140

Leu Asp Cys Pro Arg Leu Ala He Ser Ala Arg GIu Phe VaI Ser Asp 145 150 155 160

Arg Phe VaI GIn He Cys Lys GIu GIu Asp Phe Met GIn Leu Ser Pro

165 170 175

GIn GIu Leu He Ser VaI He Ser Asn Asp Ser Leu Asn VaI GIu Lys

180 185 190

GIu GIu Ala VaI Phe GIu Ala VaI Met Lys Trp VaI Arg Thr Asp Lys

195 200 205

GIu Asn Arg VaI Lys Asn Leu Ser GIu VaI Phe Asp Cys He Arg Phe 210 215 220

Arg Leu Met Thr GIu Lys Tyr Phe Lys Asp His VaI GIu Lys Asp Asp 225 230 235 240

He He Lys Ser Asn Pro Asp Leu GIn Lys Lys He Lys VaI Leu Lys

245 250 255

Asp Ala Phe Ala GIy Lys Leu Pro GIu Pro Ser Lys Asn Ala Ala Lys

260 265 270

Thr GIy Ala GIy GIu VaI Asn GIy Asp VaI GIy Asp GIu Asp Leu Leu

275 280 285

Pro GIy Tyr Leu Asn Asp He Pro Arg His GIy Met Phe VaI Lys Asp 290 295 300

Leu He Leu Leu VaI Asn Asp Thr Ala Ala VaI Ala Tyr Asp Pro Thr 305 310 315 320 GIu Asn GIu Cys Tyr Leu Thr Ala Leu Ala GIu GIn lie Pro Arg Asn 325 330 335

His Ser Ser lie VaI Thr GIn GIn Asn GIn He Tyr VaI VaI GIy GIy

340 345 350

Leu Tyr VaI Asp GIu GIu Asn Lys Asp GIn Pro Leu GIn Ser Tyr Phe

355 360 365

Phe GIn Leu Asp Ser He Ala Ser GIu Trp VaI GIy Leu Pro Pro Leu 370 375 380

Pro Ser Ala Arg Cys Leu Phe GIy Leu GIy GIu VaI Asp Asp Lys He 385 390 395 400

Tyr VaI VaI Ala GIy Lys Asp Leu Gin Thr GIu Ala Ser Leu Asp Ser

405 410 415

VaI Leu Cys Tyr Asp Pro VaI Ala Ala Lys Trp Asn GIu VaI Lys Lys

420 425 430

Leu Pro He Lys VaI Tyr GIy His Asn VaI He Ser His Lys GIy Met

435 440 445

He Tyr Cys Leu GIy GIy Lys Thr Asp Asp Lys Lys Cys Thr Asn Arg 450 455 460

VaI Phe He Phe Asn Pro Lys Lys GIy Asp Trp Lys Asp Leu Ala Pro 465 470 475 480

Met Lys He Pro Arg Ser Met Phe GIy VaI Ala VaI His Lys GIy Lys

485 490 495

He VaI He Ala GIy GIy VaI Thr GIu Asp GIy Leu Ser Ala Ser VaI

500 505 510

GIu Ala Phe Asp Leu Thr Thr Asn Lys Trp Asp VaI Met Thr GIu Phe

515 520 525

Pro GIn GIu Arg Ser Ser He Ser Leu VaI Ser Leu Ala GIy Ser Leu 530 535 540

Tyr Ala He GIy GIy Phe Ala Met He GIn Leu GIu Ser Lys GIu Phe 545 550 555 560

Ala Pro Thr GIu VaI Asn Asp He Trp Lys Tyr GIu Asp Asp Lys Lys

565 570 575

GIu Trp Ala GIy Met Leu Lys GIu He Arg Tyr Ala Ser GIy Ala Ser

580 585 590

Cys Leu Ala Thr Arg Leu Asn Leu Phe Lys Leu Ser Lys Leu

595 600 605

<210> 8 <211> 1455

<212> PRT

<213> Homo sapiens

<400> 8

Met Ser Asp Ser Trp VaI Pro Asn Ser Ala Ser GIy GIn Asp Pro GIy

1 5 10 15

GIy Arg Arg Arg Ala Trp Ala GIu Leu Leu Ala GIy Arg VaI Lys Arg

20 25 30

GIu Lys Tyr Asn Pro GIu Arg Ala GIn Lys Leu Lys GIu Ser Ala VaI

35 40 45

Arg Leu Leu Arg Ser His Gin Asp Leu Asn Ala Leu Leu Leu GIu VaI 50 55 60

GIu GIy Pro Leu Cys Lys Lys Leu Ser Leu Ser Lys VaI lie Asp Cys 65 70 75 80

Asp Ser Ser GIu Ala Tyr Ala Asn His Ser Ser Ser Phe lie GIy Ser

85 90 95

Ala Leu GIn Asp GIn Ala Ser Arg Leu GIy VaI Pro VaI GIy lie Leu

100 105 110

Ser Ala GIy Met VaI Ala Ser Ser VaI GIy GIn lie Cys Thr Ala Pro

115 120 125

Ala GIu Thr Ser His Pro VaI Leu Leu Thr VaI GIu Gin Arg Lys Lys 130 135 140

Leu Ser Ser Leu Leu GIu Phe Ala GIn Tyr Leu Leu Ala His Ser Met 145 150 155 160

Phe Ser Arg Leu Ser Phe Cys GIn GIu Leu Trp Lys lie Gin Ser Ser

165 170 175

Leu Leu Leu GIu Ala VaI Trp His Leu His VaI GIn GIy lie VaI Ser

180 185 190

Leu GIn GIu Leu Leu GIu Ser His Pro Asp Met His Ala VaI GIy Ser

195 200 205

Trp Leu Phe Arg Asn Leu Cys Cys Leu Cys GIu GIn Met GIu Ala Ser 210 215 220

Cys GIn His Ala Asp VaI Ala Arg Ala Met Leu Ser Asp Phe VaI GIn 225 230 235 240

Met Phe VaI Leu Arg GIy Phe GIn Lys Asn Ser Asp Leu Arg Arg Thr

245 250 255

VaI GIu Pro GIu Lys Met Pro GIn VaI Thr VaI Asp VaI Leu GIn Arg

260 265 270

Met Leu He Phe Ala Leu Asp Ala Leu Ala Ala GIy VaI Gin GIu GIu 275 280 285

Ser Ser Thr His Lys lie VaI Arg Cys Trp Phe GIy VaI Phe Ser GIy 290 295 300

His Thr Leu GIy Ser VaI lie Ser Thr Asp Pro Leu Lys Arg Phe Phe 305 310 315 320

Ser His Thr Leu Thr GIn lie Leu Thr His Ser Pro VaI Leu Lys Ala

325 330 335

Ser Asp Ala VaI GIn Met GIn Arg GIu Trp Ser Phe Ala Arg Thr His

340 345 350

Pro Leu Leu Thr Ser Leu Tyr Arg Arg Leu Phe VaI Met Leu Ser Ala

355 360 365

GIu GIu Leu VaI GIy His Leu GIn GIu VaI Leu GIu Thr Gin GIu VaI 370 375 380

His Trp Gin Arg VaI Leu Ser Phe VaI Ser Ala Leu VaI VaI Cys Phe 385 390 395 400

Pro GIu Ala GIn GIn Leu Leu GIu Asp Trp VaI Ala Arg Leu Met Ala

405 410 415

GIn Ala Phe GIu Ser Cys GIn Leu Asp Ser Met VaI Thr Ala Phe Leu

420 425 430

VaI VaI Arg GIn Ala Ala Leu GIu GIy Pro Ser Ala Phe Leu Ser Tyr

435 440 445

Ala Asp Trp Phe Lys Ala Ser Phe GIy Ser Thr Arg GIy Tyr His GIy 450 455 460

Cys Ser Lys Lys Ala Leu VaI Phe Leu Phe Thr Phe Leu Ser GIu Leu 465 470 475 480

VaI Pro Phe GIu Ser Pro Arg Tyr Leu Gin VaI His lie Leu His Pro

485 490 495

Pro Leu VaI Pro Ser Lys Tyr Arg Ser Leu Leu Thr Asp Tyr lie Ser

500 505 510

Leu Ala Lys Thr Arg Leu Ala Asp Leu Lys VaI Ser lie GIu Asn Met

515 520 525

GIy Leu Tyr GIu Asp Leu Ser Ser Ala GIy Asp lie Thr GIu Pro His 530 535 540

Ser Gin Ala Leu GIn Asp VaI GIu Lys Ala lie Met VaI Phe GIu His 545 550 555 560

Thr Gly Asn lie Pro VaI Thr VaI Met GIu Ala Ser lie Phe Arg Arg

565 570 575 Pro Tyr Tyr VaI Ser His Phe Leu Pro Ala Leu Leu Thr Pro Arg VaI 580 585 590

Leu Pro Lys VaI Pro Asp Ser Arg VaI Ala Phe lie GIu Ser Leu Lys

595 600 605

Arg Ala Asp Lys lie Pro Pro Ser Leu Tyr Ser Thr Tyr Cys GIn Ala 610 615 620

Cys Ser Ala Ala GIu GIu Lys Pro GIu Asp Ala Ala Leu GIy VaI Arg 625 630 635 640

Ala GIu Pro Asn Ser Ala GIu GIu Pro Leu GIy Gin Leu Thr Ala Ala

645 650 655

Leu GIy GIu Leu Arg Ala Ser Met Thr Asp Pro Ser GIn Arg Asp VaI

660 665 670 lie Ser Ala GIn VaI Ala VaI lie Ser GIu Arg Leu Arg Ala VaI Leu

675 680 685

GIy His Asn GIu Asp Asp Ser Ser VaI GIu lie Ser Lys lie GIn Leu 690 695 700

Ser lie Asn Thr Pro Arg Leu GIu Pro Arg GIu His Met Ala VaI Asp 705 710 715 720

Leu Leu Leu Thr Ser Phe Cys GIn Asn Leu Met Ala Ala Ser Ser VaI

725 730 735

Ala Pro Pro GIu Arg GIn GIy Pro Trp Ala Ala Leu Phe VaI Arg Thr

740 745 750

Met Cys GIy Arg VaI Leu Pro Ala VaI Leu Thr Arg Leu Cys GIn Leu

755 760 765

Leu Arg His GIn GIy Pro Ser Leu Ser Ala Pro His VaI Leu GIy Leu 770 775 780

Ala Ala Leu Ala VaI His Leu GIy GIu Ser Arg—Ser Ala Leu Pro GIu 785 790 795 800

VaI Asp VaI GIy Pro Pro Ala Pro GIy Ala GIy Leu Pro VaI Pro Ala

805 810 815

Leu Phe Asp Ser Leu Leu Thr Cys Arg Thr Arg Asp Ser Leu Phe Phe

820 825 830

Cys Leu Lys Phe Cys Thr Ala Ala lie Ser Tyr Ser Leu Cys Lys Phe

835 840 845

Ser Ser GIn Ser Arg Asp Thr Leu Cys Ser Cys Leu Ser Pro GIy Leu 850 855 860

lie Lys Lys Phe GIn Phe Leu Met Phe Arg Leu Phe Ser GIu Ala Arg 865 870 875 880 Gin Pro Leu Ser GIu GIu Asp VaI Ala Ser Leu Ser Trp Arg Pro Leu 885 890 895

His Leu Pro Ser Ala Asp Trp Gin Arg Ala Ala Leu Ser Leu Trp Thr

900 905 910

His Arg Thr Phe Arg GIu VaI Leu Lys GIu GIu Asp VaI His Leu Thr

915 920 925

Tyr GIn Asp Trp Leu His Leu GIu Leu GIu lie GIn Pro GIu Ala Asp 930 935 940

Ala Leu Ser Asp Thr GIu Arg GIn Asp Phe His GIn Trp Ala lie His 945 950 955 960

GIu His Phe Leu Pro GIu Ser Ser Ala Ser GIy GIy Cys Asp GIy Asp

965 970 975

Leu GIn Ala Ala Cys Thr lie Leu VaI Asn Ala Leu Met Asp Phe His

980 985 990

GIn Ser Ser Arg Ser Tyr Asp His Ser GIu Asn Ser Asp Leu VaI Phe

995 1000 . 1005

GIy GIy Arg Thr GIy Asn GIu Asp lie lie Ser Arg Leu Gin GIu 1010 1015 1020

Met VaI Ala Asp Leu GIu Leu GIn GIn Asp Leu lie VaI Pro Leu 1025 1030 1035

GIy His Thr Pro Ser GIn GIu His Phe Leu Phe GIu lie Phe Arg 1040 1045 1050

Arg Arg Leu GIn Ala Leu Thr Ser GIy Trp Ser VaI Ala Ala Ser 1055 1060 1065

Leu GIn Arg GIn Arg GIu Leu Leu Met Tyr Lys Arg lie Leu Leu 1070 1075 1080

Arg Leu Pro Ser Ser VaI Leu Cys GIy Ser Ser Phe GIn Ala GIu 1085 1090 1095

GIn Pro lie Thr Ala Arg Cys GIu GIn Phe Phe His Leu VaI Asn 1100 1105 1110

Ser GIu Met Arg Asn Phe Cys Ser His GIy GIy Ala Leu Thr GIn 1115 1120 1125

Asp lie Thr Ala His Phe Phe Arg GIy Leu Leu Asn Ala Cys Leu 1130 1135 1140

Arg Ser Arg Asp Pro Ser Leu Met VaI Asp Phe lie Leu Ala Lys 1145 1150 1155

Cys Gin Thr Lys Cys Pro Leu He Leu Thr Ser Ala Leu VaI Trp 1160 1165 1170

Trp Pro Ser Leu GIu Pro VaI Leu Leu Cys Arg Trp Arg Arg His 1175 1180 1185

Cys GIn Ser Pro Leu Pro Arg GIu Leu GIn Lys Leu GIn GIu GIy 1190 1195 1200

Arg GIn Phe Ala Ser Asp Phe Leu Ser Pro GIu Ala Ala Ser Pro 1205 1210 1215

Ala Pro Asn Pro Asp Trp Leu Ser Ala Ala Ala Leu His Phe Ala 1220 1225 1230

lie GIn GIn VaI Arg GIu GIu Asn lie Arg Lys GIn Leu Lys Lys 1235 1240 1245

Leu Asp Cys GIu Arg GIu GIu Leu Leu VaI Phe Leu Phe Phe Phe 1250 1255 1260

Ser Leu Met GIy Leu Leu Ser Ser His Leu Thr Ser Asn Ser Thr 1265 1270 1275

Thr Asp Leu Pro Lys Ala Phe His VaI Cys Ala Ala lie Leu GIu 1280 1285 1290

Cys Leu GIu Lys Arg Lys lie Ser Trp Leu Ala Leu Phe GIn Leu 1295 1300 1305

Thr GIu Ser Asp Leu Arg Leu GIy Arg Leu Leu Leu Arg VaI Ala 1310 1315 1320

Pro Asp GIn His Thr Arg Leu Leu Pro Phe Ala Phe Tyr Ser Leu 1325 1330 1335

Leu Ser Tyr Phe His GIu Asp Ala Ala lie Arg GIu GIu Ala Phe 1340 1345 1350

Leu His VaI Ala VaI Asp Met Tyr Leu Lys Leu VaI GIn Leu Phe 1355 1360 1365

VaI Ala GIy Asp Thr Ser Thr VaI Ser Pro Pro Ala GIy Arg Ser 1370 1375 1380

Leu GIu Leu Lys GIy Gin GIy Asn Pro VaI GIu Leu lie Thr Lys 1385 1390 1395

Ala Arg Leu Phe Leu Leu Gin Leu lie Pro Arg Cys Pro Lys Lys 1400 1405 1410

Ser Phe Ser His VaI Ala GIu Leu Leu Ala Asp Arg GIy Asp Cys 1415 1420 1425

Asp Pro GIu VaI Ser Ala Ala Leu GIn Ser Arg GIn GIn Ala Ala 1430 1435 1440 Pro Asp Ala Asp Leu Ser GIn GIu Pro His Leu Phe 1445 1450 1455

<210> 9

<211> 477

<212> PRT

<213> Homo sapiens

<400> 9

Met Met GIu Lys Asn Thr Ser GIu GIy Pro Ala Cys Ser Pro GIu GIu

1 5 10 15

Thr Ala Ser GIu Ser Ala Lys VaI Pro Thr Ala GIu Pro Pro GIy GIu

20 25 30

VaI Ala VaI Ser GIu Ser Thr Arg GIu GIu GIn VaI Pro Lys Pro His

35 40 45

GIy Pro Ala Pro GIn Ala Pro Thr Ala Ser Thr Ala Thr Lys Pro Ala 50 55 60

Pro Pro Ser GIu Asp VaI Pro Ser Ala Pro Leu Leu Leu Thr Leu Asp 65 70 75 80

Asp VaI Ser Ser Ser Ser VaI Thr VaI Ser Trp GIu Pro Pro GIu Arg

85 90 95

Leu GIy Arg Leu GIy Leu GIn GIy Tyr VaI Leu GIu Leu Cys Arg GIu

100 105 110

GIy Ala Ser GIu Trp VaI Pro VaI Ser Ala Arg Pro Met Met VaI Thr

115 120 125

Gin GIn Thr VaI Arg Asn Leu Ala Leu GIy Asp Lys Phe Leu Leu Arg 130 135 140

VaI Ser Ala VaI Ser Ser Ala GIy Ala GIy Pro Pro Ala Met Leu Asp 145 150 155 160

GIn Pro He His lie Arg GIu Asn He GIu Ala Pro Lys He Arg VaI

165 170 175

Pro Arg His Leu Arg GIn Thr Tyr He Arg GIn VaI GIy GIu Thr VaI

180 185 190

Asn Leu GIn He Pro Phe Gin GIy Lys Pro Lys Pro GIn Ala Thr Trp

195 200 205

Thr His Asn GIy His Ala Leu Asp Ser Gin Arg VaI Ser Met Arg Thr 210 215 220

GIy Asp GIn Asp Ser He Leu Phe He Arg Ser Ala GIn Arg Ser Asp 225 230 235 240

Ser GIy Arg Tyr GIu Leu Thr VaI Arg VaI GIu Asp Leu GIu Ala Lys

245 250 255 Ala VaI lie Asp lie Leu VaI lie GIu Lys Pro GIy Pro Pro Ser Ser 260 265 270 lie Arg Leu Leu Asp VaI Trp GIy Cys Asn Ala Ala Leu GIn Trp Thr

275 280 285

Pro Pro GIn Asp Thr GIy Asn Thr GIu Leu Leu GIy Tyr Met VaI GIn 290 295 300

Lys Ala Asp Lys Lys Thr GIy GIn Trp Phe Thr VaI Leu GIu Arg Tyr 305 310 315 320

His Pro Thr Thr Cys Thr lie Ser Asp Leu lie lie GIy Asn Ser Tyr

325 330 335

Ser Phe Arg VaI Phe Ser GIu Asn Leu Cys GIy Leu Ser Thr Ser Ala

340 345 350

Thr VaI Thr Lys GIu Leu Ala His He GIn Lys Ala Asp He Ala Ala

355 360 365

Lys Pro Lys GIy Phe He GIu Arg Asp Phe Ser GIu Ala Pro Ser Phe 370 375 380

Thr GIn Pro Leu Ala Asp His Thr Ser Thr Pro GIy Tyr Ser Thr GIn 385 390 395 400

Leu Phe Cys Ser VaI Arg Ala Ser Pro Lys Pro Lys He He Trp Met

405 410 415

Lys Asn Lys Met GIu He GIn GIy Asn Pro Lys Tyr Arg Ala Leu Ser

420 425 430

GIu Gin GIy VaI Cys Thr Leu GIu He Arg Lys Pro Ser Pro Phe Asp

435 440 445

Ser GIy VaI Tyr Thr Cys Lys Ala He Asn VaI Leu GIy GIu Ala Ser 450 455 460

VaI Asp Cys Arg Leu GIu VaI Lys Ala Ser Ala Ala His

465 470 475

<210> 10

<211> 759

<212> PRT

<213> Homo sapiens

<400> 10

Met GIy Leu Pro Ala Leu GIu Phe Ser Asp Cys Cys Leu Asp Ser Pro

1 5 10 15

His Phe Arg GIu Thr Leu Lys Ser His GIu Ala GIu Leu Asp Lys Thr

20 25 30

Asn Lys Phe He Lys GIu Leu He Lys Asp GIy Lys Ser Leu He Ser

35 40 45 Ala Leu Lys Asn Leu Ser Ser Ala Lys Arg Lys phe Ala Asp Ser Leu 50 55 60

Asn GIu Phe Lys Phe GIn Cys lie GIy Asp Ala GIu Thr Asp Asp GIu 65 70 75 80

Met Cys lie Ala Arg Ser Leu Gin GIu Phe Ala Thr VaI Leu Arg Asn

85 90 95

Leu GIu Asp GIu Arg lie Arg Met lie GIu Asn Ala Ser GIu VaI Leu

100 105 110 lie Thr Pro Leu GIu Lys Phe Arg Lys GIu GIn lie GIy Ala Ala Lys

115 120 125

GIu Ala Lys Lys Lys Tyr Asp Lys GIu Thr GIu Lys Tyr Cys GIy lie 130 135 140

Leu GIu Lys His Leu Asn Leu Ser Ser Lys Lys Lys GIu Ser GIn Leu 145 150 155 160

GIn GIu Ala Asp Ser Gin VaI Asp Leu VaI Arg GIn His Phe Tyr GIu

165 170 175

VaI Ser Leu GIu Tyr VaI Phe Lys VaI GIn GIu VaI GIn GIu Arg Lys

180 185 190

Met Phe GIu Phe VaI GIu Pro Leu Leu Ala Phe Leu GIn GIy Leu Phe

195 200 205

Thr Phe Tyr His His GIy Tyr GIu Leu Ala Lys Asp Phe GIy Asp Phe 210 215 220

Lys Thr Gin Leu Thr lie Ser lie GIn Asn Thr Arg Asn Arg Phe GIu 225 230 235 240

GIy Thr Arg Ser GIu VaI GIu Ser Leu Met Lys Lys Met Lys GIu Asn

245 250 255

Pro Leu GIu His Lys Thr lie Ser Pro Tyr Thr Met GIu GIy Tyr Leu

260 265 270

Tyr VaI GIn GIu Lys Arg His Phe GIy Thr Ser Trp VaI Lys His Tyr

275 280 285

Cys Thr Tyr GIn Arg Asp Ser Lys GIn lie Thr Met VaI Pro Phe Asp 290 295 300

GIn Lys Ser GIy GIy Lys GIy GIy GIu Asp GIu Ser VaI lie Leu Lys 305 310 315 320

Ser Cys Thr Arg Arg Lys Thr Asp Ser lie GIu Lys Arg Phe Cys Phe

325 330 335

Asp VaI GIu Ala VaI Asp Arg Pro GIy VaI lie Thr Met GIn Ala Leu 340 345 350

Ser GIu GIy Asp Arg Arg Leu Trp Met GIu Ala Met Asp GIy Arg GIu

355 360 365

Pro VaI Tyr Asn Ser Asn Lys Asp Ser GIn Ser GIu GIy Thr Ala Gin 370 375 380

Leu Asp Ser lie GIy Phe Ser lie He Arg Lys Cys He His Ala VaI 385 390 395 400

GIu Thr Arg GIy He Asn GIu GIn GIy Leu Tyr Arg He VaI GIy VaI

405 410 415

Asn Ser Arg VaI GIn Lys Leu Leu Ser VaI Leu Met Asp Pro Lys Thr

420 425 430

Ala Ser GIu Thr GIu Thr Asp He Cys Ala GIu Trp GIu He Lys Thr

435 440 445

He Thr Ser Ala Leu Lys Thr Tyr Leu Arg Met Leu Pro GIy Pro Leu 450 455 460

Met Met Tyr Gin Phe GIn Arg Ser Phe He Lys Ala Ala Lys Leu GIu 465 470 475 480

Asn GIn GIu Ser Arg VaI Ser GIu He His Ser Leu VaI His Arg Leu

485 490 495

Pro GIu Lys Asn Arg Gin Met Leu GIn Leu Leu Met Asn His Leu Ala

500 505 510

Asn VaI Ala Asn Asn His Lys GIn Asn Leu Met Thr VaI Ala Asn Leu

515 520 525

GIy VaI VaI Phe GIy Pro Thr Leu Leu Arg Pro GIn GIu GIu Thr VaI 530 535 540

Ala Ala He Met Asp He Lys Phe GIn Asn He VaI He GIu He Leu 545 550 555 560

He GIu Asn His GIu Lys He Phe Asn Thr VaI Pro Asp Met Pro Leu

565 570 575

Thr Asn Ala GIn Leu His Leu Ser Arg Lys Lys Ser Ser Asp Ser Lys

580 585 590

Pro Pro Ser Cys Ser GIu Arg Pro Leu Thr Leu Phe His Thr VaI GIn

595 600 605

Ser Thr GIu Lys GIn GIu GIn Arg Asn Ser He He Asn Ser Ser Leu 610 615 620

GIu Ser VaI Ser Ser Asn Pro Asn Ser He Leu Asn Ser Ser Ser Ser 625 630 635 640 Leu GIn Pro Asn Met Asn Ser Ser Asp Pro Asp Leu Ala VaI VaI Lys 645 650 655

Pro Thr Arg Pro Asn Ser Leu Pro Pro Asn Pro Ser Pro Thr Ser Pro

660 665 670

Leu Ser Pro Ser Trp Pro Met Phe Ser Ala Pro Ser Ser Pro Met Pro

675 680 685

Thr Ser Ser Thr Ser Ser Asp Ser Ser Pro VaI Ser Thr Pro Phe Arg 690 695 700

Lys Ala Lys Ala Leu Tyr Ala Cys Lys Ala GIu His Asp Ser GIu Leu 705 710 715 720

Ser Phe Thr Ala GIy Thr VaI Phe Asp Asn VaI His Pro Ser GIn GIu

725 730 735

Pro GIy Trp Leu GIu GIy Thr Leu Asn GIy Lys Thr GIy Leu lie Pro

740 745 750

GIu Asn Tyr VaI GIu Phe Leu

755

Claims

1. A method of detecting skeletal muscle damage, said method comprising assaying a sample of body fluid obtained from a mammal for one or more proteins, or splice variants or fragments of said proteins, as biomarkers for skeletal muscle damage, which proteins are selected from mitogen-activated protein kinase 12, rho GTPase activating protein 26, lactoperoxidase, acrosin, cathepsin E, four and a half LIM domains 3,

kelch repeat and BTB (POZ) domain containing 10, Fanconi anemia complementation group A and myosin binding protein H.

2. A method as claimed in claim 1, wherein said sample is taken from said mammal following administration of a medicinal product to said mammal.

3. A method as claimed in claim 1 or claim 2, characterised by testing a series of samples taken periodically from said mammal.

4. A method as claimed in claim 1, claim 2 or claim 3, characterised by testing said sample or samples for only one of said proteins.

5. A method as claimed in claim 1, claim 2 or claim 3, characterised by testing said sample or samples for a plurality of said proteins.

6. A method as claimed in claim 5, characterised in that one of said biomarkers is expressed at an earlier stage of muscle damage than another of said biomarkers.

7. A method as claimed in claim 6, characterised in that at least one of the proteins is associated with one or more early or intermediate stage stress functions and at least one is associated with one or more intermediate or late phase stress functions.

8. A method as claimed in any preceding claim, wherein said body fluid is plasma, serum or urine.

9. A method as claimed in any preceding claim, wherein said one or more protein biomarkers are selected from poly- or oligopeptides comprising or consisting essentially of:

(i) a polypeptide of any one of SEQ ID NOS. 1 to 10; (ii) a polypeptide having at least 80% identity to any one of polypeptides of SEQ ID NOS. 1 to 10;

(iii) a polypeptide of any one of SEQ ID NOS. 1 to 10 having one or a few amino acid deletions, substitutions or insertions; or

(iv) fragments of at least five contiguous amino acids of (i), (ii) or (iii), which fragments are capable of binding to antibodies that bind specifically to said respective polypeptides.

10. A method as claimed in claim 9, wherein said fragments comprise at least ten contiguous amino acids of (i), (ii) or (iii).

11. A method of diagnosing muscle toxicity in a mammal which comprises obtaining a sample of body fluid from said mammal and assaying said sample for at least one protein, or a splice variant or fragment of said protein, wherein said at least one protein is selected from mitogen-activated protein kinase 12, rho GTPase activating protein 26, lactoperoxidase, acrosin, cathepsin E, four and a half LIM domains 3, kelch repeat and BTB (POZ) domain containing 10, Fanconi anemia complementation group A and myosin binding protein H.

12. A method as claimed in claim 11, further comprising assaying said sample for two or more of said proteins.

13. A method as claimed in claim 12, characterised in that one of said biomarkers is expressed at an earlier stage during the progression of a toxic response than another of said biomarkers.

14. A method as claimed in claim 13, characterised in that at least one of the proteins is associated with one or more early or intermediate stage stress functions and at least one is associated with one or more intermediate or late phase stress functions.

15. A method for investigating the toxicology of a candidate medicinal or veterinary product in mammals, which method comprises administering said candidate product to one or more mammals, obtaining a sample of body fluid from the or each mammal and assaying said sample for at least one protein, or a splice variant or fragment of said protein, wherein said at least one protein is selected from mitogen-activated protein kinase 12, rho GTPase activating protein 26, lactoperoxidase, acrosin, cathepsin E, four and a half LIM domains 3, kelch repeat and BTB (POZ) domain containing 10, Fanconi anemia complementation group A and myosin binding protein H.

16. A method as claimed in claim 15, further comprising assaying said sample for two or more of said proteins.

17. A method as claimed in claim 16, characterised in that one of said biomarkers is expressed at an earlier stage during the progression of a toxic response than another of said biomarkers.

18. A method as claimed in claim 17, characterised in that at least one of the proteins is associated with one or more early or intermediate stage stress functions and at least one is associated with one or more intermediate or late phase stress functions.

19. A method as claimed in claim 16, claim 17 or claim 18, further comprising periodically obtaining samples from the or each mammal to provide a series of samples over time and assaying each of said samples for one or more of said proteins.

20. A method of detecting skeletal muscle damage substantially as hereinbefore described in the Examples.

21. A method of diagnosing skeletal muscle toxicity in a mammal substantially as hereinbefore described in Examples 1 to 9.

22. A method for investigating the skeletal muscle toxicity of a candidate medicinal or veterinary product in mammalian patients substantially as hereinbefore described in Example 10.