TW201923346A - Methods and use for detecting and inhibiting cell-free mg53 - Google Patents

Abstract

The present invention provides a method for detecting an MG53 related disease or predicting a risk of an MG53 related disease, comprising the following steps: a) obtaining a test sample; and b) detecting cell-free MG53 in the test sample. The present invention also provides a method for reducing or inhibiting activity of cell-free MG53 in a subject, comprising administrating an effective amount of MG53 inhibitor to a subject in need thereof. Also provided are an MG53 antibody, a nucleic acid encoding the antibody, a clone or expression vector including the nucleic acid, a host cell including the clone or expression vector, and a pharmaceutical composition including above.

Description

Method and use for detecting or inhibiting extracellular free MG53

The present application relates to the field of biomedicine, and in particular, the present application relates to a method for detecting extracellular free MG53, or detecting related diseases or predicting the risk of MG53 related diseases, and a method for reducing or inhibiting extracellular free MG53 in an individual. Active method, and composition for said method.

Mitsugumin 53 (MG53), also known as TRIM72, is a member of the Tripartite motif-containing Proteins (TRIM) family. MG53 consists of the N-terminal TRIM domain and the C-terminal SPRY domain. The TRIM domain consists of the Ring, B-box, and coiled-coil domains (see Chuanxi Cai et al., The Journal of Biological Chemistry , Vol. 284 (5), 3314-3322 (2009)). MG53 is mainly expressed in striated muscle, and plays an important role in maintaining the homeostasis of skeletal muscle and heart, and even the entire body. It was previously discovered that MG53 has a cell repair function and a cardioprotective function (see, for example, Chuanxi Cai et al., Nature Cell Biology , Vol. 11, 56-64 (2009); CN101797375B). In addition, further research found that MG53 is also one of the important protective molecules in ischemic preconditioning (IPC) and ischemic postconditioning (PostC). The two ends of the MG53 molecule can be associated with Caveolin-3 and p85-PI3K kinases combine with each other to form a complex, and activate the downstream reperfusion injury salvage kinase (RISK) pathway, thereby achieving myocardial cell and heart protection (see Chun-Mei Cao et al., Circulation 121, 2565-2574, (2010)).

Previous studies on MG53 also found that MG53 has E3 ubiquitin ligase activity and is involved in regulating the occurrence and development of insulin resistance and metabolic syndrome. For example, it has been reported in two documents that the Ring domain of the N-terminal TRIM domain of MG53 has E3 ubiquitin ligase activity and can interact with insulin receptor (IR) and insulin receptor substrate-1 , IRS1), and mediate their ubiquitination and degradation of the proteasome pathway, thereby inhibiting the insulin message transmission pathway, leading to obesity, diabetes, hypertension, dyslipidemia and other metabolic classes with insulin resistance as the core link of pathogenesis The occurrence and development of diseases (see, for example, R. Song et al., Nature 494, 375-379, (2013); JSYi et al., Nature communications 4, 2354 (2013)).

However, it is generally believed in the prior art that MG53 exerts activities related to regulating the occurrence and development of insulin resistance and metabolic syndrome only in cells.

The present application relates to a method for detecting or predicting the risk of MG53-related diseases and a method for reducing or inhibiting the activity of extracellular free MG53 in an individual. In addition, the present application also relates to an MG53 antibody or antigen-binding fragment thereof, a nucleic acid encoding the antibody or antigen-binding fragment thereof, including a clone or expression vector of the nucleic acid, and a host cell including the clone or expression vector. , And a pharmaceutical composition including each of the above products. Moreover, the present application also relates to the use of a detection reagent for detecting extracellular free MG53 in a preparation kit and the use of an MG53 inhibitor in the manufacture of a medicament for reducing or inhibiting extracellular free MG53 in an individual.

In one aspect, the present application relates to a method for detecting or predicting the risk of MG53-related diseases, including the steps of: a. Obtaining a sample to be tested; and b. Detecting extracellular freeness in the sample to be tested MG53.

In some embodiments, the test sample is a body fluid. In some embodiments, the test sample is selected from the group consisting of whole blood, plasma, serum, interstitial fluid, urine, or sweat. In some embodiments, the test sample is selected from whole blood, plasma, or serum. In certain embodiments, the test sample is substantially free of cells.

In some embodiments, the step b comprises contacting the MG53 detection reagent with the test sample. In certain embodiments, the MG53 detection reagent is an MG53 antibody or an antigen-binding fragment thereof, an MG53 ligand, or an MG53-binding fragment thereof. In certain embodiments, the MG53 detection reagent is a small molecule compound that can bind to MG53. In certain embodiments, the MG53 detection reagent has a detectable label. In certain embodiments, the detectable label is luminescent, magnetic, radioactive, or enzymatic. In some embodiments, the step b includes performing radioimmunoassay, western blotting method, adjacent connection technology, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescence method, immunohistochemistry Assay, immunospot test or slot blot detection.

In some embodiments, the method described in this application further comprises: c. Comparing the detection value of MG53 obtained in step b with a control value. In certain embodiments, the control value is from a control sample. In some embodiments, the control sample and the test sample are from the same or different individuals. In some embodiments, the control sample and the test sample are from the same individual, but the control sample is collected earlier or later than the test sample for a period of time. In certain embodiments, the individual has received treatment or a change in health status over the period of time. In certain embodiments, the control value is obtained from a healthy individual or an individual suffering from an MG53-related disease. In certain embodiments, the individual is a human or non-human mammal.

In certain embodiments, the MG53-related disease is a metabolic syndrome. In certain embodiments, the MG53-related disease is a disease related to glucose metabolism or lipid metabolism. In certain embodiments, the glucose or lipid metabolism related disease is selected from the group consisting of insulin resistance, diabetic cerebrovascular disease, diabetic ocular complications, diabetic neuropathy, diabetic foot, hyperinsulinemia, hypercholesterolemia , Hyperglycemia, hyperlipidemia, hypertension and obesity.

In another aspect, the present application relates to a method for reducing or inhibiting the activity of extracellular free MG53 in an individual, which comprises administering to an individual in need thereof an effective amount of an MG53 inhibitor.

In certain embodiments, the MG53 inhibitor specifically binds MG53.

In certain embodiments, the MG53 inhibitor is an MG53 antibody or an antigen-binding fragment thereof, an MG53 ligand or an MG53-binding fragment thereof, or a non-functional protein fragment of MG53. In certain embodiments, the MG53 antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions, the three heavy chain complementarity determining regions are contained in SEQ ID NO: 1, 9, 17, 25, or 33. Shown in the sequence. In certain embodiments, the MG53 antibody or antigen-binding fragment thereof further comprises three light chain complementarity determining regions, the three light chain complementarity determining regions are contained in SEQ ID NO: 2, 10, 18, 26, or 34 Shown in the sequence. In certain embodiments, the MG53 antibody or antigen-binding fragment thereof comprises a heavy chain comprising the following sequence or a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence homology to it Sequence: SEQ ID NO: 1, 3, 9, 11, 17, 19, 25, 27, 33 or 35. In certain embodiments, the MG53 antibody or antigen-binding fragment thereof comprises a light chain comprising the following sequence or a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence homology to it Sequence: SEQ ID NO: 2, 4, 10, 12, 18, 20, 26, 28, 34 or 36.

In certain embodiments, the MG53 inhibitor is a small molecule compound. In certain embodiments, the MG53 inhibitor is a compound having Formula Ia or Ib or a pharmaceutically acceptable salt thereof,

Where L is a chemical bond or an optionally substituted C ₁ -C ₁₂ alkylene group; M is a chemical bond or an optionally substituted C ₆ -C ₁₂ aromatic subunit or a 5-12 membered heterocyclic subunit; X is a chemical bond , Or optionally substituted -CH = N-NH-C (O)-, -CH2-O-, -O-CH ₂ -C (O) -NH-, -OC (O)-, or -OC (O ) -CH = CH-; Y is optionally substituted C ₆ -C ₁₂ aryl or 5-12 membered heterocyclic group; Q is optionally substituted C ₁ -C ₁₂ alkylene or C ₂ -C ₁₂ sub Alkenyl; T is an optionally substituted C ₆ -C ₁₂ aryl or 5-12 membered heterocyclic group; each R _{1 is} independently hydrogen, nitro, halogen, hydroxyl, cyano, or optionally substituted C ₁ -C ₁₂ alkyl or C ₁ -C ₁₂ alkoxy; n is any positive integer from 1-5.

In certain embodiments, L is a chemical bond or a C ₁ -C ₃ alkylene group. In certain embodiments, M is a chemical bond, or an optionally substituted phenylene or furanyl. In certain embodiments, Y is an optionally substituted phenyl, furyl, quinolinyl, or benzodioxoyl. In certain embodiments, Q is an optionally substituted C ₁ -C ₃ alkylene or C ₂ -C ₃ alkenylene. In certain embodiments, T is optionally substituted phenyl or 1,3,4-thiadiazolyl.

In certain embodiments, the MG53 inhibitor is a compound having Formula II or a pharmaceutically acceptable salt thereof,

Wherein A is hydrogen, or optionally substituted C ₆ -C ₁₂ aryl or 5-12 membered heterocyclic group; G is -C (O) -OR ₃ , wherein R ₃ is optionally substituted C ₁ -C ₁₂ alkyl; J is an optionally substituted C ₆ -C ₁₂ aryl; or G and J together with the carbon atom to which they are attached Wherein R _{4 is} each independently hydrogen, nitro, halogen, hydroxy, cyano, or optionally substituted C ₁ -C ₁₂ alkyl or C ₁ -C ₁₂ alkoxy, and n is any An integer; B is a chemical bond, or optionally substituted C ₆ -C ₁₂ aromatic subunit or 5-12 membered heterocyclic subunit; D is a chemical bond, or optionally substituted -OR _5- , wherein R _{5 is} selected from optional Substituted C ₁ -C ₁₂ alkylene; E is optionally substituted C ₆ -C ₁₂ aryl or 5-12 membered heterocyclyl.

In certain embodiments, A is optionally substituted naphthyl, phenyl, or thienyl. In certain embodiments, G is -C (O) -OR ₃ , wherein R ₃ is optionally substituted C ₁ -C ₃ alkyl; J is selected from optionally substituted phenyl; or G and J Together with its connected carbon atoms Where R ₄ is hydrogen. In certain embodiments, B is a chemical bond, or optionally substituted phenylene or furanyl. In certain embodiments, D is a chemical bond, or optionally substituted -O-CH _2- . In certain embodiments, E is an optionally substituted phenyl.

In certain embodiments, the MG53 inhibitor is a compound having Formula III or a pharmaceutically acceptable salt thereof,

Formula III
Wherein A is hydrogen, or optionally substituted C ₆ -C ₁₂ aryl or 5-12 membered heterocyclic group; Y is each independently an optionally substituted C ₆ -C ₁₂ aryl or 5-12 membered heterocyclic group R ₇ is hydrogen, nitro, halogen, hydroxy, cyano, or optionally substituted C ₁ -C ₁₂ alkyl or C ₁ -C ₁₂ alkoxy.

In certain embodiments, A is optionally substituted phenyl. In certain embodiments, Y is optionally substituted phenyl. In certain embodiments, R ₇ is hydrogen, nitro, halogen, hydroxy, or cyano.

In certain embodiments, the "optionally substituted" mentioned in any one of the above embodiments refers to being unsubstituted or substituted with one or more substituents selected from the group consisting of nitro, halogen , Hydroxy, cyano, C ₁ -C ₁₂ alkyl, C ₁ -C ₁₂ alkoxy, benzamidine, or -C (O) -OR ₆ , wherein the C ₁ -C ₁₂ alkyl, C ₁ -C ₁₂ alkoxy or benzamidine may be further substituted with nitro, halogen, hydroxyl, cyano, C ₁ -C ₃ alkyl or C ₁ -C ₃ alkoxy, where R ₆ is hydrogen Or C ₁ -C ₃ alkyl.

In certain embodiments, the MG53 inhibitor is a compound or a pharmaceutically acceptable salt thereof.

In certain embodiments, the methods described herein can be used to alleviate, treat, or prevent MG53-related diseases or symptoms thereof. In certain embodiments, the MG53-related disease is a metabolic syndrome. In certain embodiments, the MG53-related disease is a disease related to glucose metabolism or lipid metabolism. In certain embodiments, the glucose or lipid metabolism related disease is selected from the group consisting of insulin resistance, diabetic cerebrovascular disease, diabetic ocular complications, diabetic neuropathy, diabetic foot, hyperinsulinemia, hypercholesterolemia , Hypertension, and obesity.

In certain embodiments, diabetic cerebrovascular disease includes cerebral arteriosclerosis, ischemic cerebrovascular disease, cerebral hemorrhage, cerebral atrophy, and cerebral infarction.

In certain embodiments, diabetic ocular complications include diabetic retinopathy, diabetic cataract, diabetes-related uveitis, and blindness.

In certain embodiments, the diabetic neuropathy includes diabetic peripheral neuropathy.

In another aspect, the present application relates to an MG53 antibody or an antigen-binding fragment thereof, the MG53 antibody or an antigen-binding fragment thereof includes three heavy chain complementarity determining regions, and the three heavy chain complementarity determining regions are contained in SEQ ID NO: 1 , 9, 17, 25, or 33. In certain embodiments, the MG53 antibody or antigen-binding fragment thereof further comprises three light chain complementarity determining regions, the three light chain complementarity determining regions are contained in SEQ ID NO: 2, 10, 18, 26, or 34 Shown in the sequence. In certain embodiments, the MG53 antibody or antigen-binding fragment thereof comprises a heavy chain, wherein the heavy chain has an amino acid sequence as follows or has at least 80%, 85%, 90%, 95%, or 99% sequence thereof Homologous sequence: SEQ ID NO: 1, 3, 9, 11, 17, 19, 25, 27, 33 or 35. In certain embodiments, the MG53 antibody or antigen-binding fragment thereof further comprises a light chain, wherein the light chain has the following amino acid sequence or has at least 80%, 85%, 90%, 95% thereof Or 99% sequence homology: SEQ ID NO: 2, 4, 10, 12, 18, 20, 26, 28, 34 or 36.

In another aspect, the present application relates to an antibody or antigen-binding fragment that competitively binds MG53 with the antibody or antigen-binding fragment described in any of the above embodiments.

In another aspect, the present application relates to an isolated nucleic acid that encodes the antibody or antigen-binding fragment described in any one of the above embodiments. In certain embodiments, the isolated nucleic acid includes the following nucleotide sequence or a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence homology to it: SEQ ID NOs: 5, 6 , 7, 8, 13, 14, 15, 16, 21, 22, 23, 24, 29, 30, 31, 32, 37, 38, 39, or 40.

In another aspect, the present application relates to a clone or an expression vector, which includes the isolated nucleic acid described in any one of the above embodiments.

In another aspect, the present application relates to a host cell, which includes a colony or an expression vector described in any one of the above embodiments.

In another aspect, the present application relates to a pharmaceutical composition comprising the antibody or antigen-binding fragment described in any one of the above embodiments, the colony or expression vector described in any one of the above embodiments, or any one of the above embodiments. Cells as described, and pharmaceutically acceptable excipients.

In another aspect, the present application relates to the use of a detection reagent for detecting extracellular free MG53 in a preparation kit for detecting MG53-related diseases, predicting the risk or progression of MG53-related diseases, and identifying potential MG53 Inhibitors, evaluation of the therapeutic effect on MG53-related diseases or detection of the activity of extracellular free MG53. In some embodiments, the detection reagent for detecting extracellular free MG53 may be the MG53 detection reagent described in any one of the above embodiments. In some embodiments, the MG53-related disease may be the MG53-related disease described in any one of the above embodiments.

In another aspect, the application relates to the use of an MG53 inhibitor in the manufacture of a medicament for reducing or inhibiting extracellular free MG53 in an individual. In certain embodiments, the MG53 inhibitor may be an MG53 inhibitor described in any of the above embodiments. In certain embodiments, the methods can be used to alleviate, treat or prevent MG53-related diseases or symptoms thereof. In some embodiments, the MG53-related disease may be the MG53-related disease described in any one of the above embodiments.

Although this application will disclose various aspects and embodiments below, it is obvious that persons with ordinary knowledge in the technical field to which this invention pertains can make various equivalent changes and modifications without departing from the spirit and scope of the subject matter of this application. The various aspects and embodiments disclosed in this application are for illustration purposes only, and are not intended to limit this application. The actual scope of protection of this application is based on the scope of patent application. Unless otherwise indicated, all technical and scientific terms used in this application have meanings commonly understood by those having ordinary knowledge in the technical field to which the present invention belongs. All references, patents, and patent applications cited in this application are incorporated herein by reference in their entirety.

Detection MG53 Related diseases or predictions MG53 Risk-Related Approaches

In one aspect, the present application relates to a method for detecting or predicting the risk of MG53-related diseases, comprising the steps of: a. Obtaining a sample to be tested; and b. Detecting extracellular free MG53 in the sample to be tested .

MG53

The term "MG53" as used in this application refers to naturally occurring wild-type MG53 and MG53 mutants expressed in animals.

The MG53 protein exists in many animals, including, but not limited to, mammals such as humans, gorillas, monkeys, pigs, cattle, dogs, rabbits, rats, and mice. MG53 protein is a multifunctional protein, and its structure is shown in Figure 1. The full-length MG53 protein of each species is slightly different in length, but usually has about 477 amino acids, consisting of the N-terminal TRIM domain and the C-terminal SPRY domain. The TRIM domain consists of Ring, B-box, and coiled- Coil domain (RBCC). MG53 protein is one of the important components of membrane repair, and plays an important role in the pre- and post-adaptation protection of ischemia-reperfusion injury. At the same time, the high expression of MG53 protein can also cause insulin resistance and metabolic syndrome. The structure, function, and interaction with other proteins of MG53 have been reported in the art in detail (see, for example, Chuanxi Cai et al., Journal of Biological Chemistry , 284 (5), 3314-3322, (2009); Xianhua Wang et al., Circulation Research 107, 76-83, (2010); Eun Young Park et al., Proteins , 790-795 (2009)).

In certain embodiments, the amino acid sequence of wild-type MG53 is as shown in SEQ ID NO: 41, 42, 43, 44, 45, 46, 47, or 61, corresponding to human, rhesus / cynomolgus monkey, respectively , Rat, mouse, pig, dog, rabbit and marmoset wild-type MG53 protein. In certain embodiments, the term "MG53" as used herein refers to MG53 derived from an individual being tested or treated.

Test sample

The term “sample to be tested” used in the present application refers to a sample obtained from an individual or from an in vitro or ex vivo sample of the individual, including a person with ordinary knowledge in the technical field to which the present invention perceives contains or may contain extracellular Any material from an individual that is free of MG53. In some embodiments, the test sample is a blood sample. In some embodiments, the test sample is a body fluid. In some embodiments, the test sample is selected from the group consisting of whole blood, plasma, serum, interstitial fluid, urine, or sweat. In some embodiments, the test sample is selected from whole blood, plasma, or serum. In some embodiments, the test sample may further include other substances, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

In some embodiments, the sample to be tested is substantially free of cells, such as cardiomyocytes, striated muscle cells, or blood cells. The term "substantially free" used in this application means that it does not contain more than a specific amount, or does not contain more than the amount remaining in its field through the use of general impurity removal techniques, or does not contain substantially affecting the function of other substances The amount of specific substance. For example, substantially free of cells in the sample may mean that the sample does not contain more than a specific amount of cells (eg, not more than 1000, 800, 500, 200, 100, 50, 20 per ml) , 10, 5, or 1 cell); or refers to cells that do not contain the number of cells normally remaining after being separated by conventional separation means (eg, centrifuging the supernatant). Samples can be obtained using any method known in the art. For example, a blood sample can be obtained using a vacuum or atmospheric lancet. In some embodiments, the collected blood sample may be further processed, such as by centrifugation, prior to performing the test.

MG53 Detection reagent

In certain embodiments, the MG53 detection reagent is an MG53 antibody or an antigen-binding fragment thereof, or an MG53 ligand or an MG53-binding fragment thereof. Any of the MG53 antibodies or antigen-binding fragments thereof, or MG53 ligands or MG53-binding fragments thereof, or a combination thereof described in this application can be used as the MG53 detection reagent.

In certain embodiments, the MG53 detection reagent is a small molecule compound that can bind to MG53. Any MG53-binding small molecule compound described in this application, or a combination thereof can be used as the MG53 detection reagent.

In certain embodiments, the MG53 detection reagent further has a detectable label. In certain embodiments, the detectable label is luminescent, magnetic, radioactive, or enzymatic. Examples of detectable labels include fluorescent labels, enzyme labels, radioisotope labels, chemiluminescent labels, electroluminescent labels, metal particle labels, and hapten labels.

Examples of fluorescent labels include 5- (6) -carboxyfluorescein, 5- or 6-carboxyfluorescein, 6- (luciferin) -5- (6) -carboxamidohexanoic acid, iso- Thiocyanate fluorescein, rhodamine, tetramethyl rhodamine, dyes (such as Cy2, Cy3, and Cy5), optionally substituted coumarins (eg, AMCA, PerCP), phycobiliproteins (eg, R- Phycoerythrin (RPE) and allophycoerythrin (APC), Texas red, Princeton red, green fluorescent protein (GFP) and their analogs, conjugates of R-phycoerythrin and allophycoerythrin, and inorganic Fluorescent labels (such as particles based on semiconductor materials (such as particles coated with CdSe nanocrystals)).

Examples of enzyme labels include horseradish peroxidase (HRP), alkaline phosphatase (ALP or AP), β-galactosidase (GAL), glucose-6-phosphate dehydrogenase, β-N-acetamidine Glucosidase, β-glucuronidase, invertase, xanthine oxidase, firefly luciferase and glucose oxidase (GO).

Examples of common substrates for horseradish peroxidase include 3,3'-diaminobenzidine (DAB), nickel-enhanced diaminobenzidine, 3-amino-9-ethyloxazole (AEC), benzidinediamine Hydrochloride (BDHC), Hanker-Yates reagent (HYR), indophenol blue (IB), tetramethylbenzidine (TMB), 4-chloro-1-naphthol (CN), α-naphthol paronine (Α-NP), o-anisidine (OD), 5-bromo-4-chloro-3-indole phosphate (BCIP), nitrotetrazolium blue (NBT), 2- (p-iodophenyl) ) -3-p-nitrophenyl-l-5-phenyltetrazolium chloride (INT), tetranitrotetrazole blue (TNBT), and 5-bromo-4-chloro-3-indole Phenol-β-D-galactoside / iron-potassium ferricyanide (BCIG / FF).

Examples of common substrates for alkaline phosphatase include naphthol-AS-B 1-phosphate / fast red TR (NABP / FR), naphthol-AS-MX-phosphate / fast red TR (NAMP / FR), naphthalene Phenol-AS-B1-Phosphate / -Fast Red TR (NABP / FR), Naphthol-AS-MX-Phosphate / Fast Red TR (NAMP / FR), Naphthol-AS-B1-Phosphate / New Fuchsin (NABP / NF), bromochloroindole phosphate / nitrotetrazole blue (BCIP / NBT), and 5-bromo-4-chloro-3-indolyl-bd-galactopyranoside (BCIG) .

Examples of radioisotope labels include isotopes of iodine, cobalt, selenium, thallium, carbon, sulfur, and phosphorus.

Examples of chemiluminescent labels include luminox, isoluminox, acridinium ester, 1,2-dioxolane, and pyridopyridazine.

Examples of the electroluminescent label include a ruthenium derivative.

Examples of metal particle markers include gold particles and coated gold particles, which can be converted through silver staining.

Examples of hapten labels include DNP, luciferin isothiocyanate (FITC), biotin, and digoxin.

In certain embodiments, exemplary detectable labels have one or more of the following structures.

The detectable label can be through covalent binding, affinity binding, embedding, malocclusion, combined, mixed or added, etc. is connected with the detection reagent MG53.

Detection method

Step b. Detection of extracellular free MG53 in the test sample can be performed using methods commonly used in the art. In certain embodiments, these methods include, but are not limited to, radioimmunoassay, western blotting, ortho-linking, immunofluorescence, enzyme immunoassay, immunoprecipitation assay, chemiluminescence, immunohistochemistry Assay, immunospot test or immunoslit test.

Those of ordinary skill in the art are aware of the general techniques and other variations of techniques used in performing the various assays described above, and can also use the methods described above alone or with isonuclear magnetic resonance (NMR), matrix-assisted laser desorption ionization time of flight (MALDI- TOF), liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), etc. are used in combination or alternatively.

In one embodiment, the enzyme immunoassay is a sandwich enzyme immunoassay that uses antibodies that specifically bind MG53 to detect extracellular free MG53. An exemplary sandwich enzyme immunoassay includes the following steps: a. Linking an antibody that specifically binds to extracellular free MG53 with a solid phase carrier to form a solid phase antibody, and washing to remove unbound antibody and impurities; b. Adding to be tested The specimen is contacted with the solid-phase antibody for a certain period of time to form a solid-phase antibody-antigen complex, and washed to remove other unbound materials; c. Add an enzyme-labeled antibody capable of binding to extracellular free MG53, and make it contact with The solid-phase antibody-antigen complex is contacted for a certain period of time to fully bind the solid-phase antibody-antigen complex, and the unbound enzyme-labeled antibody is washed to remove the unbound enzyme; d. The enzyme-labeled substrate is added, and the enzyme is made under appropriate conditions. The catalytic matrix is labeled and the amount of extracellular free MG53 is determined based on the degree of matrix response.

data processing

The detection method described in this application may further include a data processing step. In some embodiments, the method described in this application further comprises: c. Comparing the detection value of MG53 obtained in step b with a control value.

In certain embodiments, the control value is from a control sample. In some embodiments, the control sample and the test sample are from different individuals. For example, a control sample may be from a healthy individual or an individual identified as having an MG53-related disease, and the test sample may be from an individual having or suspected of having an MG53-related disease.

In some embodiments, the control sample and the test sample are from the same individual, but the control sample is collected earlier or later than the test sample for a period of time. In certain embodiments, the individual has received treatment or a change in state of health over the period of time. In other embodiments, the control sample and the test sample are collected from different parts of the same individual.

In certain embodiments, the control value is obtained from a healthy individual or an individual identified as having an MG53-related disease. In certain embodiments, the control value is expressed as the concentration of MG53.

In some embodiments, these control values may be negative control values, which are equal to or lower than the value indicating that the individual from which the test sample is derived does not have an MG53-related disease or has a lower risk of having an MG53-related disease. In some embodiments, a negative control value can be formulated based on the average value of MG53 test values obtained from a certain number of healthy individuals, for example, the average value, 80%, 90%, 110%, 120 of the average value %, 150% or 200% were used as negative control values.

In other embodiments, these control values may be positive control values, which are equal to or higher than the value indicating that the individual from which the test sample is derived has a higher risk of MG53-related disease or MG53-related disease. In some embodiments, a positive control value may be formulated based on an average value of MG53 detection values obtained from a certain number of individuals identified to have MG53-related diseases, for example, such average values, 80% of the average values, 90%, 110%, 120%, 150% or 200% were used as positive control values.

individual

The term "individual" as used in this application refers to humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals. "Individual" can also be a livestock animal (for example, a cow, pig, sheep, chicken, rabbit, or horse), or a rodent (for example, a rat or mouse), or a primate (for example, a gorilla or monkey ), Or domestic animals (for example, dogs or cats). "Individuals" can be male or female, or they can be of different ages. Human "individuals" can be Caucasians, Africans, Asians, Semites, or other races, or hybrids of different races. A human "individual" can be old, adult, adolescent, child, or infant.

In certain embodiments, the individual is a human or non-human mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig or hamster.

MG53 Related diseases

The term "MG53-related diseases" used in the present application refers to diseases related to the activity of MG53, such as the occurrence, onset, and development of such diseases. Those with ordinary knowledge in the technical field to which the present invention belongs can determine whether a certain disease is related to MG53 through conventional technical means. For example, the amount of MG53 in an individual can be reduced and the disease can be assessed in the individual by over-expressing the MG53 protein, or by removing MG53, or by techniques such as antisense nucleic acid technology, CRISPR / Cas9 technology, and Trim21 vector proteasome degradation Condition to determine whether the disease is associated with MG53.

In certain embodiments, the MG53-related disease is a metabolic syndrome. In certain embodiments, the MG53-related disease is a disease related to glucose metabolism or lipid metabolism. In certain embodiments, the glucose or lipid metabolism related disease is selected from the group consisting of insulin resistance, diabetic cerebrovascular disease, diabetic ocular complications, diabetic neuropathy, diabetic foot, hyperinsulinemia, hypercholesterolemia , Hyperglycemia, hyperlipidemia, hypertension and obesity.

The term "metabolic syndrome" used in the present application refers to a disorder characterized by a cluster of lipid and non-lipid cardiovascular risk factors caused by metabolic disorders. In certain embodiments, the metabolic syndrome is identified by the presence of any three of the following risk factors: waist circumference greater than 102 cm in men or waist greater than 88 cm in women; serum triglycerides of at least 150 mg / dL; men Medium to high density lipoprotein cholesterol (HDL-C) is less than 40 mg / dL or less than 50 mg / dL in women; blood pressure of at least 130/85 mmHg; and fasting blood glucose of at least 110 mg / dL. These risk factors can be easily measured in clinical practice, for example, according to the Executive Summary of the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III), JAMA, 2001, 285: 2486-2497, the entire contents of which are incorporated herein by reference.

Purpose of the test method

The method for detecting MG53-related diseases or predicting the risk of MG53-related diseases described herein can be used for a variety of purposes.

In certain embodiments, the detection method can be used to detect whether an individual has an MG53-related disease or to predict his risk of having an MG53-related disease. For example, as described above, the amount of extracellular free MG53 in an individual is equal to or lower than the negative control value, indicating that the individual does not have MG53-related disease or has a lower risk of having MG53-related disease; the amount of extracellular free MG53 in the individual A value equal to or higher than the positive control indicates that the individual is at higher risk for or with MG53-related disease.

In certain embodiments, the detection method can be used to predict the progression of MG53-related diseases in an individual. For example, by determining changes in the amount and / or activity of extracellular free MG53 in an individual compared to values of the same individual over a period of time, the condition (eg, improvement or worsening) of MG53-related disease in the individual is predicted. In certain embodiments, a decrease in the amount and / or activity of extracellular free MG53 in an individual compared to the values of the same individual over a period of time indicates an improvement in the MG53-related disease in that individual; a value from the same individual over time In contrast, an increase in the amount and / or activity of extracellular free MG53 in an individual indicates that the MG53-related disease in that individual has become worse.

In certain embodiments, the detection method can be used to identify potential extracellular free MG53 inhibitors. In certain embodiments, the use generally comprises contacting the compound or reagent to be tested with extracellular free MG53, followed by detection of extracellular free MG53. In certain embodiments, a test compound or agent capable of reducing the content of extracellular free MG53 by at least 10% can be considered an extracellular free MG53 inhibitor. In certain embodiments, the amount of extracellular free MG53 can be reduced by at least 20%, 40%, 50%, 80%, 90%, 95% or more. These identified extracellular free MG53 inhibitors can be used to treat, prevent, or alleviate MG53-related diseases. The compounds or reagents to be tested include, for example, small molecule organic and inorganic compounds (for example, molecules obtained from synthetic chemical libraries and natural product libraries), antibodies or antigen-binding fragments thereof, ligands or binding fragments thereof, or MG53 No functional protein fragments.

In certain embodiments, the detection method can be used to determine whether an individual will respond to an extracellular free MG53 inhibitor. After exposure to the extracellular free MG53 inhibitor, the presence and amount of extracellular free MG53 in the test sample can indicate whether the individual from which the test sample is derived will respond to the extracellular free MG53 inhibitor. For example, a decrease in the content and / or activity of extracellular free MG53 in the test sample by at least 10% indicates that individuals from the source of the test sample may respond to inhibitors of extracellular free MG53. In certain embodiments, the amount of extracellular free MG53 in the test sample is reduced by at least 20%, 40%, 50%, 80%, 90%, 95% or more compared to a control sample.

In certain embodiments, the detection method can be used to evaluate the therapeutic effect on MG53-related diseases. In these embodiments, the control sample and the test sample are from the same individual, but the control sample is collected a time earlier than the test sample, and the individual is treated for a period of time. In certain embodiments, a decrease in the amount of extracellular free MG53 in a test sample by at least 10% compared to a control sample indicates that the individual has responded to the currently received treatment. In certain embodiments, the amount and / or activity of extracellular free MG53 in the test sample is reduced by at least 20%, 40%, 50%, 80%, 90%, 95% or more compared to a control sample. Depending on the results of the response assessment, the treatment regimen for the individual can be maintained or changed.

In some embodiments, the detection method can be used to assess the change in the content of extracellular free MG53 or a mutant thereof after treatment with MG53 or a mutant thereof for a related disease. In these embodiments, the control sample and the test sample are from the same individual, but the control sample was collected a time earlier than the test sample, and the individual received MG53 or a mutant protein for a period of time Treatment results in changes in the content and / or activity of extracellular free MG53 or its mutant protein. Based on the results of the assessment, the individual's treatment regimen can be maintained or changed.

Reduces or inhibits extracellular release in individuals MG53 Active method

In another aspect, the present application relates to a method for reducing or inhibiting the activity of extracellular free MG53 in an individual, which comprises administering to an individual in need thereof an effective amount of an MG53 inhibitor.

MG53 Inhibitor

MG53 inhibitors can be small molecule organic or inorganic compounds (for example, molecules obtained from synthetic chemical libraries and natural product libraries), antibodies or antigen-binding fragments thereof, ligands or MG53-binding fragments thereof, or non-functioning MG53 Protein fragments. In certain embodiments, the MG53 inhibitor is capable of reducing extracellular free MG53 activity by at least 5%, 10%, 20%, 40%, 50%, 80%, 90%, 95% or more.

In the present application, when "activity" is used together with increase or decrease, it refers to a detected functional activity, which can be expressed as a change in the content of extracellular free MG53, or a constant content but a change in functional activity. In some embodiments, the activity of the MG53 is associated with a metabolic syndrome. In some embodiments, the activity of the MG53 is related to sugar metabolism or lipid metabolism. In some embodiments, the activity of the MG53 is associated with insulin resistance, hyperinsulinemia, hypercholesterolemia, hyperglycemia, hyperlipidemia, hypertension, or obesity. In some embodiments, the activity of MG53 refers to the binding activity to the insulin receptor and the insulin receptor matrix.

In certain embodiments, the MG53 inhibitor specifically binds MG53.

The term "specific binding" as used in this application when used to describe an MG53 inhibitor means that the MG53 inhibitor preferably recognizes MG53 in a complex mixture, and the binding constant of the inhibitor to MG53 is its binding to other non-MG53 At least 2 times the binding constant of the specific binding protein. In certain embodiments, the equilibrium dissociation constant of the MG53 inhibitor and MG53 is less than or equal to 10 ^-6 or 10 ^-7 M. In certain embodiments, the equilibrium dissociation constant of the MG53 inhibitor and MG53 is less than or equal to 10 ^-7 M or 10 ^-8 M.

In certain embodiments, the MG53 inhibitor is an MG53 antibody or an antigen-binding fragment thereof, an MG53 ligand or an MG53-binding fragment thereof, or a non-functional protein fragment of MG53. MG53 antibodies or antigen-binding fragments thereof, MG53 ligands or MG53-binding fragments thereof, or non-functional protein fragments of MG53 that can be used in this application include, but are not limited to, any of the MG53 antibodies or antigen-binding fragments thereof described in this application, MG53 ligand or MG53 binding fragment thereof, or non-functional protein fragment of MG53, or a combination thereof.

In certain embodiments, the MG53 inhibitor is a small molecule compound that can bind to MG53. Small molecule compounds that bind to MG53 that can be used in this application include, but are not limited to, any small molecule compound that binds to MG53 described in this application, or a combination thereof.

Uses of reduction or suppression methods

In certain embodiments, the methods of reducing or inhibiting described herein can be used to alleviate, treat or prevent MG53-related diseases or symptoms thereof, said methods of reducing or inhibiting comprising administering to a subject a therapeutically effective amount of an MG53 inhibitor. See above for the definition of MG53 related diseases.

"Relieving," "treating," or "preventing" a disease or symptom includes preventing or reducing a disease or discomfort, reducing the rate at which a disease or discomfort occurs or developing, and reducing the development of a disease or discomfort The risk of conditions, preventing or delaying the development of symptoms associated with a disease or condition, reducing or eliminating symptoms associated with a disease or condition, completely or partially reversing a disease or condition, curing a disease or condition Discomfort, or a combination of the above.

The term "therapeutically effective amount" used in the present application refers to an amount of a drug that can alleviate or eliminate a disease or symptom of an individual, or can prevent or prevent the disease or symptom from occurring. A therapeutically effective amount may be the amount of a drug that relieves one or more diseases or symptoms of an individual to a certain degree; those drugs that partially or completely return to one or more physiological or biochemical parameters related to the cause of the disease or symptom to normal An amount; and / or an amount of a drug that reduces the likelihood of a disease or symptom occurring.

The therapeutically effective dose of the MG53 inhibitor provided in this application depends on a variety of factors known in the art, such as weight, age, past medical history, currently undergoing treatment, subject's health and strength of drug interactions, allergies (Allergy), hypersensitivity and side effects, as well as the route of administration and the extent of disease development. Those skilled in the art (eg, a doctor or veterinarian) can reduce or increase the dose accordingly based on these or other conditions or requirements.

In certain embodiments, the MG53 inhibitor provided herein can be administered at a therapeutically effective dose of about 0.01 mg / kg to about 100 mg / kg (e.g., about 0.01 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg , About 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, or about 100 mg / kg). In certain embodiments, the MG53 inhibitor is administered at a dose of about 50 mg / kg or less. In certain embodiments, the administered dose is 10 mg / kg or less, 5 mg / kg or less, 1 mg / kg or less, 0.5 mg / kg or less, or 0.1 mg / kg or less . A particular dose can be divided into multiple intervals, such as once a day, two or more times a day, two or more times a month, once a week, once every two weeks, once every three weeks, once a month, or Once every two or more months. In certain embodiments, the dosage administered may vary with the course of treatment. For example, in certain embodiments, the initial administered dose may be higher than the subsequent administered dose. In certain embodiments, the administered dose is adjusted during the course of treatment based on the response of the subject to be administered.

The dosing regimen can be adjusted to achieve an optimal response (eg, a therapeutic response). For example, a single dose may be administered or divided into multiple divided doses over a period of time.

The MG53 inhibitor disclosed in the present application can be administered by a method of administration known in the art, such as injection (eg, subcutaneous injection, intraperitoneal injection, intravenous injection, including intravenous drip, intramuscular injection or intradermal injection). ) Or non-injectable (eg, oral, nasal, sublingual, rectal or topical). The MG53 inhibitor disclosed in the present application may be administered in the form of a pharmaceutical composition described in the present application.

In certain embodiments, the MG53 inhibitors disclosed in this application can be combined with other agents or therapies to treat related diseases or conditions. In some embodiments, the MG53 inhibitor is administered concurrently or sequentially with other agents or therapies. In some embodiments, the other agent or therapy is for the treatment of insulin resistance, diabetic cerebrovascular disease, diabetic ocular complications, diabetic neuropathy, diabetic foot, hyperinsulinemia, hypercholesterolemia, high An agent or therapy for one or more of blood glucose, hyperlipidemia, hypertension, and obesity. In some embodiments, the other agent is a hypoglycemic agent, for example, a PPAR agonist, a dipeptidyl peptidase (IV) inhibitor, a GLP-1 analog, insulin or an insulin analog, insulin secretagogue, SGLT2 Inhibitors, human dextrin analogs, biguanides, alpha-glucosidase inhibitors, or combinations thereof.

MG53 Antibodies and antigen-binding fragments thereof

Another aspect of the present application also provides an MG53 antibody or an antigen-binding fragment thereof.

In certain embodiments, the MG53 antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions, the three heavy chain complementarity determining regions are contained in SEQ ID NO: 1, 9, 17, 25, or 33. Shown in the sequence. In certain embodiments, the MG53 antibody or antigen-binding fragment thereof further comprises three light chain complementarity determining regions, the three light chain complementarity determining regions are contained in SEQ ID NO: 2, 10, 18, 26, or 34 Shown in the sequence. In certain embodiments, the MG53 antibody or antigen-binding fragment thereof includes three heavy chain complementarity determining regions and three light chain complementarity determining regions, and the six complementarity determining regions are included in the following group: SEQ ID NO: 1 / 2, 9/10, 17/18, 25/26, or 33/34.

In certain embodiments, the MG53 antibody or antigen-binding fragment thereof comprises a heavy chain, and the heavy chain variable region of the MG53 antibody or antigen-binding fragment thereof has the following amino acid sequence or has at least 80%, 85% thereof , 90%, 95%, or 99% sequence homology: the sequence shown in SEQ ID NO: 1, 9, 17, 25, or 33.

In certain embodiments, the heavy chain of the MG53 antibody or antigen-binding fragment thereof has the following amino acid sequence or a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence homology to it: SEQ ID NO: 3, 11, 19, 27 or 35.

In certain embodiments, the MG53 antibody or antigen-binding fragment thereof further comprises a light chain, and the light chain variable region of the MG53 antibody or antigen-binding fragment thereof has the following amino acid sequence or has at least 80%, 85% %, 90%, 95% or 99% sequence homology sequence: the sequence shown in SEQ ID NO: 2, 10, 18, 26 or 34.

In certain embodiments, the MG53 antibody or antigen-binding fragment thereof, wherein the light chain has an amino acid sequence as follows or has at least 80%, 85%, 90%, 95%, or 99% sequence homology to it Sequence: SEQ ID NO: 4, 12, 20, 28 or 36

Another aspect of the present application also provides a competitive antibody or an antigen-binding fragment thereof that competitively binds MG53 with the above-mentioned antibody or antigen-binding fragment.

The term "competitive binding" as used in this application refers to the ability of an antibody or antigen-binding fragment thereof to specifically inhibit the interaction between the antigen to which it is directed and another antibody molecule (eg, MG53 and other MG53 antibodies). In certain embodiments, a competitive antibody or antigen-binding fragment that blocks the binding of two antigen-antibody molecules can inhibit the interaction between the two antigen-antibody molecules by at least 50%. In certain embodiments, such an inhibitory effect can be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

The term "antibody" as used in this application includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, or bispecific (bivalent) antibody that can bind to a specific antigen. A natural intact antibody contains two heavy chains and two light chains. Each heavy chain consists of a variable region and first, second, and third constant regions; each light chain consists of a variable region and a constant region. Mammalian heavy chains can be divided into α, δ, ε, γ, and μ subtypes, and mammalian light chains can be divided into λ or κ subtypes. The antibody is of "Y" type, and the neck of the "Y" type structure is composed of the second and third constant regions of two heavy chains, which are bound through disulfide bonds. Each arm of the "Y" structure includes a variable region and a first constant region of a heavy chain, and a variable region and a constant region of a light chain associated with it. The first constant region of the heavy chain is connected to the second constant region through the hinge region. The variable regions of the light and heavy chains determine the specificity of antigen binding. The variable region of each chain contains three hypervariable regions, called complementarity determining regions (CDRs). The CDRs of the light chain (L) include LCDR1, LCDR2, and LCDR3, and the CDRs of the heavy chain (H) include HCDR1, HCDR2, and HCDR3. . The CDR boundaries of the antibodies and antigen-binding fragments disclosed in this application can be named or identified by Kabat, Chothia, or Al-Lazikani nomenclature. For details, see Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273 (4), 927 (1997); Chothia, C. et al., J Mol Biol. Dec 5; 186 (3): 651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196,901 (1987); Chothia, C. et al., Nature. Dec 21-28; 342 (6252): 877-83 (1989) and Kabat EA et al., National Institutes of Health, Bethesda, Md. . (1991). Among them, the three CDRs are separated by a continuous portion called a framework region (FR). The framework region is more highly retained than the CDR and forms a structure that supports the variable region. The constant regions of the heavy and light chains have nothing to do with the specificity of antigen binding, but have multiple effector functions. Antibodies can be divided into several classes based on the amino acid sequence of the constant region of the heavy chain. Depending on whether they contain α, δ, ε, γ, and μ heavy chains, antibodies can be divided into five major classifications or isomers: IgA, IgD, IgE, IgG, and IgM. Several major antibody classifications can also be divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (Α2 heavy chain) and so on.

The term "antigen-binding fragment" as used in this application refers to an antibody fragment formed from an antibody fragment that contains an antibody portion of one or more CDRs but does not have a complete antibody structure. Examples of antigen-binding fragments include, but are not limited to, Fab fragments, Fab 'fragments, F (ab') ₂ fragments, Fv fragments, single-chain antibody molecules (scFv), scFv dimers, and camelized single domain antibodies domain antibody) and nanobodies. The antigen-binding fragment can bind the same antigen as the parent antibody.

The "Fab" fragment of an antibody refers to the portion of the antibody that is composed of a light chain (including a variable region and a constant region), a variable region of a heavy chain, and a first constant region that are bound by a disulfide bond.

A "Fab '" fragment refers to a Fab fragment that contains a portion of the hinge region.

The "F (ab ') ₂ " fragment refers to a dimer of Fab'.

The "Fv" fragment of an antibody consists of a light chain variable region and a heavy chain variable region.

A "single-chain antibody molecule" or "scFv" refers to an engineered antibody in which the light chain variable region and the heavy chain variable region are directly connected or connected through a peptide chain. For a detailed introduction, see, for example, Huston JS, etc., Proc Natl Acad Sci USA, 85: 5879 (1988).

"ScFv dimer" refers to a polymer formed from two scFv.

"Camelized single domain antibody" (also known as "heavy chain antibody" or "HCAb (Heavy-chain-only antibodies)") refers to the two heavy chain variable regions without Light chain antibodies (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231 (1-2): 25-38 (1999); Muyldermans S., J Biotechnol. Jun; 74 (4): 277- 302 (2001); WO94 / 04678; WO94 / 25591; US Patent No. 6,005,079). Heavy chain antibodies were originally derived from the camel family (camel, dromedary, and llama). Although the light chain is missing, camelized antibodies have the full function of antigen binding (see Hamers-Casterman C. et al., Nature. 363 (6428): 446-8 (1993); Nguyen VK. Et al., "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation, "Immunogenetics. 54 (1): 39-47 (2002); Nguyen VK. Et al., Immunology. 109 (1): 93-101 (2003)), the entire contents of which are incorporated by reference Into this application.

A "nano antibody" is composed of a heavy chain variable region derived from a heavy chain antibody and two constant regions, CH2 and CH3.

In some embodiments, the antibody described herein is a fully human antibody, a humanized antibody, a chimeric antibody, a mouse antibody, or a rabbit antibody. In certain embodiments, the antibody described herein is a polyclonal antibody, a monoclonal antibody or a recombinant antibody. In certain embodiments, the antibodies described herein are monospecific antibodies, bispecific antibodies, or multispecific antibodies. In certain embodiments, the antibodies described herein can be further labeled. In certain embodiments, the antibody or antigen-binding fragment thereof is a fully human monoclonal antibody, which is optionally produced by a transgenic rat, eg, endogenous rat immunoglobulin gene expression is deactivated Transgenic rats carrying a recombinant human immunoglobulin locus with a deletion of the J locus and a C-kappa mutation can also be produced from engineered cells (eg, CHO cell expression).

The term "fully human" as used in this application when applied to an antibody or antigen-binding fragment means that the amino acid sequence of the antibody or antigen-binding fragment corresponds to that produced by a human or human immune cell, or from, for example, use The gene of the human antibody library is transgenic into the amino acid sequence of non-human derived antibodies such as non-human animals, or other sequences encoding human-derived antibodies.

The term "humanized" as used in this application, when used in antibodies or antigen-binding fragments, refers to CDRs derived from non-human animals, FR regions derived from humans, and constant regions derived from humans (when applicable) ) Antibodies or antigen-binding fragments. Because humanized antibodies or antigen-binding fragments are less immunogenic, they can be used as human therapeutics in certain embodiments. In certain embodiments, the non-human animal is a mammal (eg, a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster). In some embodiments, the humanized antibody or antigen-binding fragment consists essentially of human-derived sequences, except that the CDR sequences are of non-human origin.

The term "chimeric" as used in this application when used in an antibody or antigen-binding fragment means having a portion of the heavy and / or light chain derived from one species, and the rest of the heavy and / or light chain Antibodies or antigen-binding fragments from different species. In some embodiments, a chimeric antibody may include a constant region derived from a human and a variable region derived from a non-human animal, such as a mouse or rabbit.

In some embodiments, the MG53 antibody and the antigen-binding fragment thereof described herein can be ≤10 ^-6 M (for example, ≤5x10 ^-7 M, ≤2x10 ^-7 M, ≤10 ^-7 M, ≤5x10 ^-8 M, ≤2x10 ^-8 The binding affinity of M specifically binds to MG53. The binding affinity can be expressed by K _D value, which is the ratio of the dissociation rate to the binding rate when the binding of the antigen and the antigen-binding molecule reaches equilibrium (koff / kon) calculated. The antigen-binding affinity (eg, K _D ) can be detected by a suitable method known in the art, including a plasma resonance binding method using an instrument (eg, Biacore), for example, see Murphy, M. et al., Current protocols in protein science, Chapter 19, unit 19.14, 2006.

In certain embodiments, the antibodies and antigen-binding fragments thereof described herein range from 0.2 nM-2000 nM (eg, 1 nM-1500 nM, 5 nM-1000 nM, 10 nM-900 nM, 20 nM-800 nM, or 50 nM-800 nM) IC ₅₀ inhibits the binding of MG53 to its ligand.

The term "% homology" as used in this application means that for amino acid sequences, the candidate amino acid sequence is compared with the comparative amino acid sequence, and a gap is introduced when necessary to make the same The number of amino acids is the highest, and the percentage of the same amino acid between the two amino acid sequences is calculated on the basis of this; for the nucleic acid sequence, the candidate nucleic acid sequence is compared with the comparative nucleic acid sequence, and if necessary, The interval is introduced to maximize the number of identical nucleotides, and the percentage of identical nucleotides between two nucleic acid sequences is calculated on this basis. Alignment can be performed in a variety of ways known in the art to determine the percentage of homology. For example, sequence alignment can be performed using public tools such as BLASTp (National Center for Biotechnology Information (NCBI): http://blast.ncbi.nlm.nih.gov/Blast.cgi, see also, Altschul SF, etc. Human, J. Mol. Biol., 215: 403–410 (1990); Stephen F. et al., Nucleic Acids Res., 25: 3389–3402 (1997)), ClustalW2 (European Bioinformatics Institute website: http: //www.ebi.ac.uk/Tools/msa/clustalw2/, see, Higgins DG et al., Methods in Enzymology, 266: 383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, England), 23 (21): 2947-8 (2007)) and TCoffee (Swiss Institute of Bioinformatics, see Poirot O. et al., Nucleic Acids Res., 31 (13): 3503-6 (2003); Notredame C. et al., J. Mol. Boil., 302 (1): 205-17 (2000)). When using software to perform sequence alignment, the preset parameters provided by the software can be used, and the parameters can be appropriately adjusted according to the needs of the alignment, which are all within the knowledge of those with ordinary knowledge in the technical field to which the present invention belongs.

In some embodiments, the MG53 antibodies or antigen-binding fragments thereof described herein further include those MG53 antibodies or Its antigen-binding fragment.

The term "reserved substitution" of amino acid residues as used herein refers to substitutions between amino acids of similar nature, such as substitutions between polar amino acids (e.g., between glutamine and asparagine) Replacements), replacements between hydrophobic amino acids (eg, replacements between leucine, isoleucine, methionine, and valine), and between amino acids with the same charge Replacement (for example, arginine, lysine and histidine, or between glutamic acid and aspartic acid).

In certain embodiments, the antibodies or antigen-binding fragments thereof described herein further include a conjugate.

In certain embodiments, the conjugate can be a detectable label, a pharmacokinetic modification moiety, a purification moiety, or a cytotoxic moiety. A variety of conjugates can be bound to the antibodies or antigen-binding fragments thereof (see, for example, Conjugate Vaccines, Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. (eds.), Carger Press, New York ( 1989)). In certain embodiments, the MG53 antibody or antigen-binding fragment disclosed herein can be engineered to contain one or more conjugate binding sites, and these sites can be used to bind one or more conjugates. For example, such a site may contain one or more reactive amino acid residues (eg, cysteine residues and histidine residues) to assist covalent attachment to the conjugate. In certain embodiments, the MG53 antibody or antigen-binding fragment thereof may be linked indirectly to the conjugate or through another conjugate. For example, the MG53 antibody or antigen-binding fragment thereof may be linked to biotin and then indirectly bound to a second conjugate (eg, avidin), which is linked to biotin. In certain embodiments, the conjugate can be a detectable label, for example, a fluorescent label (eg, luciferin, rhodamine, rhododendron, phycoerythrin, or Texas red), an enzyme-matrix Labels (e.g. horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, sugar oxidase or β-D-galactosidase), radioisotopes (e.g. ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ³⁵ S, ³ H, ¹¹¹ In, ¹¹² In, ¹⁴ C, ⁶⁴ Cu, ⁶⁷ Cu, ⁸⁶ Y, ⁸⁸ Y, ⁹⁰ Y, ¹⁷⁷ Lu, ²¹¹ At, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹² Bi, and ³² P or other lanthanides), chromophore moiety, digoxin, biotin / avidin, DNA molecule or gold. In certain embodiments, the conjugate can be PEG, which helps extend the half-life of the antibody. In certain embodiments, the conjugate can be a purified portion (eg, magnetic beads). In certain embodiments, the conjugate can be a cytotoxic moiety, for example, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, scutellan, mitomycin, etoposide, Teniposide, vincristine, vincristine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithromycin, actinomycin D, 1- Dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and their analogs, antimetabolites (for example, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbaba), alkylating agents (e.g., nitrogen mustard, cetipine benzoate mustard, melphalan, carmustine (BSNU), and lomo Sting (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cisplatin), anthracycline antibiotics (for example, daunorubicin and doxorubicin ), Antibiotics (for example, dactinomycin, bleomycin, mithromycin, and anthromycin (AMC)), and antimitotic agents (for example, vincristine and vinblastine ).

MG53 Ligands and MG53 Combine fragments

An aspect of the present application provides an MG53 ligand and an MG53 binding fragment thereof. In the present application, MG53 ligands and MG53 binding fragments thereof can be used as MG53 detection reagents or MG53 inhibitors. Without wishing to be bound by theory, the presence or concentration of extracellular free MG53 can be detected by detecting the binding of MG53 ligand or its MG53 binding fragment to extracellular free MG53; it can also be performed by administering MG53 ligand or its MG53 binding to an individual The fragment thus competitively binds MG53, thereby blocking or reducing the binding of MG53 to the original reactive ligand / receptor in vivo.

In some embodiments, the MG53 ligand is an insulin receptor or a variant thereof, such as a human insulin receptor as shown in SEQ ID NO: 58. In some embodiments, the MG53 ligand is an extracellular region of an insulin receptor or a variant thereof.

MG53 Non-functional protein fragment

One aspect of the application provides a non-functional protein fragment of MG53. The "non-functional protein fragment of MG53" described in the present application may be any protein fragment lacking one or more physiological functions compared to natural MG53. In certain embodiments, non-functional protein fragments of MG53 are capable of competitively binding to MG53 ligands / receptors with native MG53. In certain embodiments, the activity of non-functional protein fragments of MG53 is reduced by at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared to native MG53. In certain embodiments, the reduced activity described above is an activity associated with a MG53-related disease. In certain embodiments, the aforementioned reduced activity is the activity of MG53 to activate AKT phosphorylation.

In certain embodiments, the non-functional protein fragment of MG53 is a MG53 mutant. The term "MG53 mutant" or "MG53 protein mutant" used in the present application refers to a MG53 protein variant in which the amino acid sequence of the natural wild-type MG53 protein has been modified. Such modifications include, but are not limited to, deletion, addition, and / or substitution of one or more amino acids. In certain embodiments, the MG53 mutant in the present application has, based on the amino acid sequence of the wild-type MG53, at least one serine deletion in the coiled-coil-SPRY domain of the wild-type MG53 and / Or mutated to any other non-serine or threonine amino acid.

In certain embodiments, the MG53 mutant is based on the amino acid sequence of wild-type MG53, and at least one serine is mutated to a non-polar amine in the coiled-coil-SPRY region of the wild-type MG53. Acid, the non-polar amino acid is selected from the group consisting of glycine, alanine, leucine, isoleucine, valine, proline, phenylalanine, methionine, and tryptophan . Preferably, in some embodiments, the non-polar amino acid is alanine. In some embodiments, the MG53 mutant is based on the amino acid sequence of wild-type MG53, and at least one serine is mutated to deseramide in the coiled-coil-SPRY region of the wild-type MG53. Polar amino acids other than acids and threonines. In certain embodiments, the polar amino acid is selected from the group consisting of glutamine, cysteine, asparagine, tyrosine, aspartic acid, glutamine, lysine, Arginine and Histidine. Preferably, in some embodiments, the polar amino acid is cysteine.

In certain embodiments, the MG53 mutant has an amino acid sequence as set forth in any one of SEQ ID NOs: 48-57. In certain embodiments, the amino acid sequence of the MG53 mutant has at least 70%, at least 75%, at least 80%, at least 80%, and at least the amino acid sequence shown in any one of SEQ ID NOs: 48-57. 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence homology.

For other contents of the MG53 mutant, please refer to Chinese Patent Application 201610847346.4, the entire contents of which are incorporated herein by reference.

MG53 Antibodies or antigen-binding fragments thereof, MG53 Ligand or MG53 Combine fragments, or MG53 For preparing non-functional protein fragments

MG53 antibodies or antigen-binding fragments thereof, MG53 ligands or MG53-binding fragments thereof, or non-functional protein fragments of MG53 in the present application can be prepared by conventional methods in the art. For example, they can be prepared by chemical synthesis methods or by gene Engineering method.

Chemical synthesis methods mainly include two methods of solid phase synthesis and liquid phase synthesis. Solid-phase polypeptide synthesis methods include, for example, the Merrifield solid-phase synthesis method, which has been described in detail in the literature Merrifield, J. Am. Chem. Soc. 85: 2149-2154, M. Bodanszky et al., Peptide Synthesis, John Wiley & Sons , Second Edition, 1976 ", and J. Meienhofer," Hormonal Proteins and Peptides ", Vol. 2, p. 46, Academic Press (New York), 1983, the entire contents of which are incorporated herein by reference. Merrifield Solid Phase The synthesis method mainly includes the following steps. According to the amino acid sequence of the target polypeptide, the protected carboxy-terminal amino acid is first connected to the resin; the resin is washed after the connection; and the protective group on the α-amino group of the carboxy-terminal amino acid is removed (for example, , Tert-butoxycarbonyl), when removing this protective group, it must be ensured that the connection between the amino acid and the resin is not broken; then the penultimate carboxy-terminal protected amino acid is coupled to the obtained resin When carrying out this coupling, an amido bond is formed between the free carboxyl group of the second amino acid and the amino group of the first amino acid connected to the resin; according to the amino acid connection order of the target polypeptide, In turn The aforementioned reaction process is repeated until all amino acids are connected to the resin; finally, the protected peptide is cut from the resin and the protective group is removed to obtain the target polypeptide. The polypeptide of the present application can also be prepared by liquid phase synthesis For example, it can be prepared by standard solution peptide synthesis method, which has been described in detail in the literature E. Schroder and K. Kubke, The Peptides, Vol. 1, Academic Press (New York), 1965, the entire contents of which are incorporated by reference and The liquid phase synthesis method mainly includes the stepwise coupling of an amino acid or a peptide fragment by a chemical or enzymatic method that forms an amidine bond.

Genetic engineering methods can be used to produce MG53 antibodies or antigen-binding fragments thereof, MG53 ligands or MG53-binding fragments thereof, or non-functional protein fragments of MG53 through cell culture and expression. In this method, a host cell is transformed with a clone or an expression vector carrying a target gene encoding a target protein to be expressed, and the transformed host cell is cultured in a nutrient medium, which is modified to be suitable for inducing startup For detailed descriptions of this method, please refer to molecular colony edited by Sambrook et al. (Experiment Manual, Cold Spring Harbor, 1989).

The host cells used to produce the antibody or the antigen-binding fragment thereof, the MG53 ligand or the MG53-binding fragment thereof, or the non-functional protein fragment of MG53 in the present application can be cultured in various culture media. Commercially available media such as Ham's F10, Minimum Basic Medium (MEM), RPMI-1640 and Dulbecco's Modified Eagle's Medium (DMEM) produced by Sigma can be used to culture the host cells. In addition, any of Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102: 255 (1980), US Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87/00195; or the medium mentioned in US Patent Application RE. 30,985 can be used as a medium for the host cell. These media can be supplemented with necessary hormones and / or other growth factors (for example, insulin, transferrin or epidermal growth factor), salts (for example, sodium chloride, calcium chloride, magnesium chloride or phosphate), buffers ( For example, HEPES), nucleotides (eg, adenylic acid and thymine), antibiotics (eg, gentamicin), trace elements (final concentrations are usually in the micromolar range and inorganic compounds used to provide nutrition, such as sulfuric acid Ferrous, copper sulfate, zinc sulfate or manganese chloride), and glucose or equivalent energy sources. The medium may also contain any other necessary additives at appropriate concentrations known in the art. Those skilled in the art to which the present invention pertains are familiar with how to select conditions for culturing host cells, such as temperature and pH.

When the above-mentioned target protein is expressed using a genetic engineering technique, the target protein may be generated in a cell, a cell wall membrane space, or directly secreted into a culture medium. If the antibody is produced intracellularly, the host cell is first lysed, and then the lysed fragment or particle debris of the host cell is removed, for example, by centrifugation or ultrasound. If the antibody is produced in the parietal space, the target protein can be isolated using, for example, the method described in the literature Carter et al., Bio / Technology 10: 163-167 (1992). Briefly, cells were lysed in the presence of sodium acetate (pH = 3.5), EDTA and benzylsulfonium fluoride (PMSF) for more than 30 minutes, followed by centrifugation to remove cell debris. If the antibody is secreted into the culture medium, the supernatant of the expression system is usually first concentrated using a commercially available protein concentration filter (such as Amicon or Pellicon ultrafiltration unit from Millipore). Protease inhibitors (eg, PMSF) can be added in any of the foregoing steps to inhibit protein degradation, and antibiotics to prevent the growth of accidental contaminants.

The products prepared from the cells can be purified by purification methods such as hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity Chromatographic analysis, of which affinity chromatography is a better purification technique. After any preliminary purification steps, low pH hydrophobic interaction chromatography can be used to treat mixtures containing the product of interest and impurities, using a washing buffer at a pH of about 2.5-4.5, preferably at a low salt concentration ( For example, from about 0 to 0.25 M salt concentration).

Isolated nucleic acid

In another aspect, the application relates to an isolated nucleic acid comprising an antibody or an antigen-binding fragment encoding any one of the embodiments described above. In certain embodiments, the isolated nucleic acid includes the following nucleotide sequence or a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence homology to it: SEQ ID NOs: 5, 6 , 7, 8, 13, 14, 15, 16, 21, 22, 23, 24, 29, 30, 31, 32, 37, 38, 39, or 40.

The term "isolated" as used in this application refers to a substance (eg, a polypeptide or a nucleic acid) that is separate from or present in an environment in which it normally exists in nature.

"Isolated" matter has been artificially altered from its natural state. If some "isolated" substance or component appears in nature, it has been altered or removed from its original state, or both. For example, a naturally occurring polynucleotide or polypeptide in a living animal is not isolated, but if these polynucleotides or polypeptides are sufficiently separated from substances that coexist in their natural state and exist in a sufficiently pure state, they can Think of it as "separated." In certain embodiments, the purity of the antibody and antigen-binding fragment is at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, and the purity can be determined by electrophoresis methods (eg, SDS-PAGE, etc. Electrofocusing or capillary electrophoresis), or chromatographic analysis (for example, ion-exchange chromatography or reversed-phase HPLC).

The term "nucleic acid" or "polynucleotide" as used herein refers to a ribonucleic acid (RNA), a deoxyribonucleic acid (DNA), or a RNA-deoxyribonucleic acid mixture (eg, a DNA-RNA hybrid). The nucleic acid or polynucleotide may be single- or double-stranded DNA or RNA, or a DNA-RNA hybrid. Nucleic acids or polynucleotides can be linear or circular.

As used herein, the term "encoded" or "encoded for" means capable of being transcribed into mRNA and / or translated into a peptide or protein.

In some embodiments, a polynucleotide comprising a polynucleotide may be in a modified form. Such modifications include base modifications (for example, bromouridine), ribose modifications (for example, cytarabine and 2 ', 3'-dideoxyribose), and internucleotide bond modifications (for example, phosphorothioate) Esters, dithiophosphates, phosphoroselenoate, phosphoroseleneoate, phosphoranilothioate, phosphanilide, and phosphoramidate).

The isolated nucleic acids described herein also include nucleic acids that have been replaced according to the degeneracy of the genetic code. The term "degenerateness of the genetic code" used in this application refers to a phenomenon in which the same amino acid has two or more codons. For example, proline has 4 synonymous codons, namely CCU, CCC, CCA, CCG. It is known in the art that due to the degeneracy of the nucleic acid genetic code, nucleic acids at certain positions in a known nucleic acid sequence can be replaced without changing the encoded amino acid sequence. Those with ordinary knowledge in the technical field to which the present invention pertains can easily perform the replacement of the degeneracy of the genetic code, for example, a site-directed mutation technique through bases. Different organisms have different preferences for different codons. In order to express the antibody or antigen-binding fragment of the present application in a selected biological cell, the codons preferred by the biological cell can be selected to obtain the corresponding coding gene. The antibody or antigen-binding fragment of the present application is obtained by recombinant expression.

Clone or expression vector

In another aspect, the present application relates to a clone or an expression vector, which includes the isolated nucleic acid described in any one of the above embodiments.

The term "vector" as used in this application refers to a vehicle that can be operatively inserted into a polynucleotide encoding a protein and obtain expression of the protein. Vectors can be used to transform, transduce, or transfect host cells so that the genetic material elements they carry are expressed within the host cells. For example, vectors include: plastids, phages, cosmids, artificial chromosomes (eg, yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC)), phages (eg, lambda phage or M13 phage), and animal viruses. Exemplary animal viruses that can be used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (eg, herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, Papillary polyoma vacuole virus (for example, SV40). Vectors can contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication initiation site. A vector may also include components that assist it in entering cells, including, but not limited to, viral particles, liposomes, or protein shells.

Host cell

In yet another aspect, the present application relates to a host cell, which includes a clone or an expression vector described in any one of the above embodiments.

The term "host cell" as used herein refers to a cell that has been introduced or is capable of introducing a nucleic acid sequence ("selected gene") encoding one or more antibodies, MG53 ligands, or nonfunctional MG53 fragments described herein, and It further expresses or is capable of expressing a selected gene of interest. The term includes the progeny of the parent cell, whether or not it is the same in morphology or genetic composition as the parent, as long as the selected gene is present.

A vector comprising a polynucleotide encoding a selected gene can be introduced into a host cell for selection or gene expression. The host cells suitable for colonizing or expressing the DNA in the vector in this application are prokaryotic cells or eukaryotic cells. Prokaryotic cells suitable for use in this application include Eubacteria and Archaea. Among them, Eubacteria include Gram-negative bacteria, Gram-positive bacteria, and actinomycetes. Exemplary Eubacteria include, for example, Escherichia coli, Irwinia, Klebsiella, Proteus, Salmonella, Serratia, Shigella, Pseudomonas, etc.

In addition to prokaryotic cells, eukaryotic microorganisms (for example, filamentous fungi or yeast) can also be used as vectors for host cell colonization or expression of selected genes. Saccharomyces cerevisiae, or baker's yeast, are the most commonly used lower eukaryotic host microorganisms. However, many other genera, species, and strains are more commonly used and applicable in this application, such as Schizosaccharomyces pombe; Kluyveromyces hosts (e.g., Kluyveromyces lactis, Kluyveromyces fragilis (ATCC 12,424 ), Kluyveromyces bulgaricus (ATCC 16,045), Kluyveromyces welchii (ATCC 24,178), Kluyveromyces kluyveri (ATCC 56,500), Kluyveromyces drosophila (ATCC 36,906), Kluyveromyces thermotolerance and Max Kluyveromyces); Yarrowia lipolytica (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesei (EP 244,234); Streptomyces; Western Schwann yeast (eg , Western Schwann yeast); and filamentous fungi (for example, Neurospora, Penicillium, Curvularia and Aspergillus).

The host cells provided in this application may be mammalian host cells, and the cultivation of mammalian host cells has become a routine operation. Examples of usable mammalian host cells are SV40 transformed monkey kidney cell CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or subcultured 293 cell suspensions, see Graham et al., J Gen Virol. 36:59 (1977)); young hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, see Urlaub et al., Proc. Natl. Acad. Sci. USA 77 : 4216 (1980)); mouse testicular support cells (TM4, see Mather, Biol. Reprod. 23: 243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO- 76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse breast tumors (MMT 060562, ATCC CCL51); TRI cells (see Mather et al., Annals NY Acad. Sci. 383: 44-68 ( 1982)); MRC 5 cells; FS4 cells; and human liver cancer cell lines (Hep G2).

Small molecule compounds

One aspect of the application provides small molecule compounds that bind to MG53. In the present application, a small molecule compound bound to MG53 can be used as an MG53 detection reagent or an MG53 inhibitor.

In certain embodiments, the small molecule compound bound to MG53 is a compound having Formula Ia or Ib or a pharmaceutically acceptable salt thereof,

Where L is a chemical bond or an optionally substituted C ₁ -C ₁₂ alkylene group; M is a chemical bond or an optionally substituted C ₆ -C ₁₂ aromatic subunit or a 5-12 membered heterocyclic subunit; X is a chemical bond , Or optionally substituted -CH = N-NH-C (O)-, -CH ₂ -O-, -O-CH ₂ -C (O) -NH-, -OC (O)-, or -OC ( O) -CH = CH-; Y is optionally substituted C ₆ -C ₁₂ aryl or 5-12 membered heterocyclic group; Q is optionally substituted C ₁ -C ₁₂ alkylene or C ₂ -C ₁₂ Alkenylene; T is an optionally substituted C ₆ -C ₁₂ aryl or 5-12 membered heterocyclic group; each R _{1 is} independently hydrogen, nitro, halogen, hydroxyl, cyano, or optionally substituted C _1- C ₁₂ alkyl or C ₁ -C ₁₂ alkoxy; n is any positive integer from 1-5.

In certain embodiments, the small molecule compound bound to MG53 is a compound having Formula II or a pharmaceutically acceptable salt thereof,

Wherein A is hydrogen, or optionally substituted C ₆ -C ₁₂ aryl or 5-12 membered heterocyclic group; G is -C (O) -OR ₃ , wherein R ₃ is optionally substituted C ₁ -C ₁₂ alkyl; J is an optionally substituted C ₆ -C ₁₂ aryl; or G and J together with the carbon atom to which they are attached Wherein R _{4 is} each independently hydrogen, nitro, halogen, hydroxy, cyano, or optionally substituted C ₁ -C ₁₂ alkyl or C ₁ -C ₁₂ alkoxy, and n is any An integer; B is a chemical bond, or optionally substituted C ₆ -C ₁₂ aromatic subunit or 5-12 membered heterocyclic subunit; D is a chemical bond, or optionally substituted -OR _5- , wherein R _{5 is} selected from optional Substituted C ₁ -C ₁₂ alkylene; E is optionally substituted C ₆ -C ₁₂ aryl or 5-12 membered heterocyclyl.

In certain embodiments, the small molecule compound bound to MG53 is a compound having Formula III or a pharmaceutically acceptable salt thereof,

Formula III
Wherein A is hydrogen, or optionally substituted C ₆ -C ₁₂ aryl or 5-12 membered heterocyclic group; Y is each independently an optionally substituted C ₆ -C ₁₂ aryl or 5-12 membered heterocyclic group R ₇ is hydrogen, nitro, halogen, hydroxy, cyano, or optionally substituted C ₁ -C ₁₂ alkyl or C ₁ -C ₁₂ alkoxy.

For the sake of brevity, the various features of the application described in the context of a single embodiment may also be provided in a separate manner or in any suitable sub-combination.

The term "substituted" as used in this application, when referring to a chemical group, means that one or more hydrogen atoms of the chemical group are removed and substituted with a substituent.

The term "substituent" as used in this application has its ordinary meaning as is known in the art and refers to a chemical moiety covalently attached to or, where appropriate, fused to the parent group.

The term "optionally substituted" as used in this application means that a chemical group may have no substituents (ie, unsubstituted) or may have one or more substituents (ie, substituted). It should be understood that substitution at a given atom is limited by the number of valences. In certain embodiments, illustrative examples of the substituents mentioned in any of the above embodiments are: nitro, halogen, hydroxyl, cyano, C ₁ -C ₁₂ alkyl, C ₁ -C ₁₂ alkoxy , Benzamidine, or -C (O) -OR ₆ , wherein the C ₁ -C ₁₂ alkyl, C ₁ -C ₁₂ alkoxy, or benzamidine may be further nitro, halogen, Hydroxyl, cyano, C ₁ -C ₃ alkyl or C ₁ -C ₃ alkoxy, wherein R ₆ is hydrogen or C ₁ -C ₃ alkyl.

The term “C _n -C _m ” used in the present application means a range of the number of carbon atoms, where n and m are integers and the range of the number of carbon atoms includes end points (ie, n and m) and each integer point therebetween. For example, C ₁ -C ₆ represents a range of 1 to 6 carbon atoms, including 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, and 6 carbon atoms.

The term "alkyl" as used in this application, whether used as part of other terms or alone, refers to a saturated hydrocarbyl group, which may be straight or branched. The term "C _n -C _m alkyl" refers to an alkyl group having n to m carbon atoms. In certain embodiments, the alkyl group contains 1 to 12, 1 to 8, 1 to 6, 1 to 4, 1 to 3, or 1 to 2 carbon atoms. Examples of alkyl groups include, but are not limited to, chemical groups such as methyl, ethyl, n -propyl, isopropyl, n -butyl, tert-butyl, isobutyl, sec-butyl, 2-methyl 1-butyl, n -pentyl, 3-pentyl, n -hexyl, 1,2,2-trimethylpropyl and the like.

The term "alkenyl" as used in this application, whether used as part of other terms or alone, refers to an unsaturated hydrocarbyl group, which may be straight or branched, with at least one carbon-carbon double bond. In certain embodiments, an alkenyl group contains 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms. In certain embodiments, the alkenyl group can also have 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1 carbon-carbon double bond. Examples of alkenyl groups include, but are not limited to, chemical groups such as vinyl, n -propenyl, isopropenyl, n -butenyl, sec-butenyl, and the like.

The term "alkylene" as used in this application, whether used as part of other terms or alone, refers to a divalent saturated hydrocarbon moiety, which may be straight or branched, and which connects two other molecules of the molecule section. The term "C _n -C _m alkylene" refers to an alkylene group having n to m carbon atoms. In certain embodiments, the alkylene group contains 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, or 1 to 3 carbon atoms. Examples of alkylene groups include, but are not limited to, chemical groups such as methylene, ethylene, 1-methyl-methylene, propylene, butylene, and the like.

The term "alkenylene" as used in this application, whether used as part of other terms or alone, refers to a divalent unsaturated hydrocarbyl moiety, which may be straight or branched, having at least one carbon-carbon bicarbonate Bond, and it connects two other parts of the molecule. The term "C _n -C _m alkenylene" refers to an alkenylene having n to m carbon atoms. In certain embodiments, the alkenylene group contains 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms. In certain embodiments, the alkenylene group can also have 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1 carbon-carbon double bond.

The term "aryl" or "aromatic" as used in this application, whether used as part of other terms or alone, refers to a single- or multiple-, having alternating double and single bonds between the carbon atoms forming the ring Carbocyclic ring group. The term "C _n -C _m aryl" means an aryl group having n to m carbon atoms form a ring. In certain embodiments, the aryl ring system has 5 to 10, 5 to 8 or 5 to 6 carbon atoms in one or more rings. In certain embodiments, the aryl ring system has 2 or more rings fused together. Examples of aryl groups include, but are not limited to, chemical groups such as phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like. In certain embodiments, phenyl includes a fused phenyl, such as a benzodioxocene group.

The term "aromatic subunit" as used in this application, whether used as part of other terms or alone, refers to a divalent aromatic ring or ring system that connects two other parts of a molecule, that is, the two parts described above Different ring positions are bonded. When the aromatic ring of the aromatic subunit is a monocyclic ring system, the above two parts are bonded to two different ring positions of the same ring. When the aromatic ring of the aromatic subunit is a polycyclic ring system, the above two parts may be bonded to two different ring positions of the same ring or different rings. The term "C _n -C _m aryl subunit" refers to an aryl subunit having n to m ring carbon atoms. The aromatic subunit may be substituted or unsubstituted. An unsubstituted aromatic subunit has no substituents other than the two parts of the molecule to which it is attached. A substituted aromatic subunit has other substituents in addition to the two parts of the molecule to which it is attached.

The term "alkoxy" used in this application, whether used as part of other terms or alone, refers to a group represented by the formula "-O-alkyl". The term "C _n -C _m alkyl group" means the alkyl portion of an alkoxy group having n to m carbon atoms. In certain embodiments, the alkyl moiety has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Examples of alkoxy groups include, but are not limited to, chemical groups such as methoxy, ethoxy, propoxy (eg, n -propoxy and isopropoxy), t -butoxy, and the like.

The term "n-membered" as used in this application, where n is an integer, is commonly used with ring systems to describe the number of atoms forming a ring in the ring system. For example, pyridinyl is an example of a 6-membered heterocycloalkyl ring, pyrazolyl is an example of a 5-membered heteroaryl ring, pyridyl is an example of a 6-membered heteroaryl ring, and 1,2,3 , 4-Tetrahydro-naphthalene is an example of a 10-membered aryl group.

The term "heteroaryl" as used in this application refers to an aryl group in which at least one ring atom in the aromatic ring is a heteroatom and the remaining ring atoms are carbon atoms. The term "n-m-membered heteroaryl" refers to a heteroaryl group having n to m ring-forming members. Examples of heteroatoms include, but are not limited to, oxygen, sulfur, nitrogen, phosphorus, and the like. In certain embodiments, a heteroaryl group may have 5 to 10, 5 to 8 or 5 to 6 ring-forming members. In certain embodiments, the heteroaryl is a 5- or 6-membered heteroaryl. Examples of heteroaryl include, but are not limited to, furyl, thienyl, pyridyl, quinolinyl, pyrrolyl, N-lower alkylpyrrolyl, pyridyl-N-oxide, pyrimidinyl, pyrazinyl, imidazolyl , Indolyl and so on.

A 5-membered heteroaryl is a heteroaryl having a ring having 5 ring atoms, in which one or more (eg, 1, 2 or 3) ring atoms may be independently selected from N, O, P and S. Examples of 5-membered heteroaryl include, but are not limited to, thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-trisyl Oxazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazole, 1,3,4-oxadiazolyl and the like.

A 6-membered heteroaryl is a heteroaryl having a ring having 6 ring atoms, in which one or more (eg, 1, 2 or 3) ring atoms may be independently selected from nitrogen, oxygen, sulfur, and phosphorus. Examples of 6-membered heteroaryl include, but are not limited to, pyridyl, pyrazinyl, pyrimidinyl, triazinyl, pyridazinyl, and the like.

The term "heteroaryl subunit" as used in this application, whether used as part of another term or used separately, refers to a divalent heteroaryl group that connects two other parts of a molecule, that is, the two parts described above differ from the ring in two Ring position bonding. When the aromatic ring of the heteroaryl subunit is a monocyclic ring system, the above two parts are bonded to two different ring positions of the same ring. When the aromatic ring of the heteroaryl subunit is a polycyclic ring system, the above two parts may be bonded to two different ring positions of the same ring or different rings. Heteroarylene may be substituted or unsubstituted. An unsubstituted heteroaryl subunit has no substituents other than the two parts of the molecule to which it is attached. A substituted heteroaryl subunit has other substituents in addition to the two parts of the molecule to which it is attached.

The term "heterocycloalkyl" as used herein refers to a cycloalkyl group in which at least one atom in the ring system is a heteroatom and the remaining ring atoms are carbon atoms. The term "n-m-membered heterocycloalkyl" refers to a heterocycloalkyl group having n to m ring-forming members. In addition, the ring may also have one or more double bonds, but not a fully conjugated system. In certain embodiments, the heterocycloalkyl is a saturated heterocycloalkyl. Examples of heteroatoms include, but are not limited to, oxygen, sulfur, nitrogen, phosphorus, and the like. In certain embodiments, heterocycloalkyl has 3 to 8, 3 to 6, or 4 to 6 carbon atoms forming a ring. Examples of heterocycloalkyl include, but are not limited to, azetidine, aziridine, pyrrolidinyl, pyrimidinyl, pyrazinyl, morpholinyl, thiomorpholine homopyrazine, and the like.

The term "heterocycloalkylene" as used in this application, whether used as part of another term or used separately, refers to a divalent heterocycloalkyl group that connects two other parts of a molecule, that is, the two parts described above and the ring are in two Bonding at different ring positions. The term "C _nm heterocycloalkylene" refers to a heterocycloalkylene group having n to m carbon atoms. In certain embodiments, the heterocycloalkylene group contains 3 to 8, 3 to 6, or 4 to 6 ring-forming carbon atoms.

The term "heterocyclyl" as used in this application includes heteroaryl and heterocycloalkyl.

The term "heterocyclic subunit" as used in this application includes heteroaryl and heterocycloalkylene.

The term "halogen" as used in this application refers to an atom selected from fluorine, chlorine, bromine and iodine.

The term "cyano" used in this application refers to a group represented by the formula "-CN".

The term "hydroxy" used in the present application refers to a group represented by the formula "-OH".

The term "nitro" used in the present application refers to a group represented by the formula "-NO ₂ ".

The term "benzylamino" as used in this application refers to the formula "-NH-C (= O) -phenyl".

The term "compound" as used in this application is intended to include all stereoisomers (eg, enantiomers and diastereomers), geometric isomers, tautomers, and isotopes of the structures shown. Unless otherwise specified, a compound identified in this application by name or structure as one particular tautomeric form is intended to include other tautomeric forms.

The compounds described herein may be asymmetric (eg, having one or more stereocenters). Unless otherwise indicated, all stereoisomers, such as enantiomers and diastereomers, are intended to be included. Compounds of the present application containing asymmetrically substituted carbon atoms can be isolated in optically activated or racemic form. Methods of preparing optically activated forms from starting materials that are not optically active are well known in the art, such as by isolating racemic mixtures or by stereoselective synthesis. Various geometric isomers such as olefins and carbon-carbon double bonds can also exist in the compounds described in this application, and all these stable isomers have been considered in this application. The present application describes cis and trans geometric isomers of compounds and they can be separated as a mixture of isomers or as individual isomers.

In certain embodiments, the compounds described herein have a ( R ) -configuration. In certain embodiments, the compounds described herein have a ( S ) -configuration.

Isolation can be performed by a racemic mixture of compounds using any of a variety of methods known in the art. An exemplary method includes fractional crystallization using an optically isolated acid, which is an optically active salt-forming organic acid. Suitable isolation reagents for the fractional recrystallization process are, for example, optically active acids (e.g., tartaric acid, diethyl tartaric acid, dibenzotartaric acid, mandelic acid, malic acid, D and L forms of lactic acid) or various Optically active camphor sulfonic acid. Other isolation reagents suitable for fractional crystallization methods include N-methylbenzylamine, 2-phenylglycine, norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine, 1,2 -A stereoisomeric pure form of diaminocyclohexane and the like.

The isolation of the racemic mixture can also be carried out by elution on a column packed with an optically active separation reagent such as dinitrobenzylidenephenylglycine. Suitable elution solvent compositions can be determined by those having ordinary skill in the art to which this invention belongs.

The compounds of the present application also include tautomeric forms. Tautomeric forms are caused by the exchange of a single bond with an adjacent double bond accompanied by the migration of a proton. Tautomeric forms include tautomers of protons in an isomeric protonated state with the same experimental formula and total charge. Examples of proton tautomers include keto-enol pairs, amidine-imine pairs, lactam-imide pairs, enamine-imines pairs, and cyclic forms in which protons can occupy heterocyclic rings Two or more positions in the system, such as 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole . Tautomeric forms can be balanced or sterically locked into one form by suitable substitution.

The compounds of the present application may also include all isotopes of atoms present in the intermediate or final compound. Isotopes include those atoms that have the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium, deuterium, and tritium.

Method for obtaining small molecule compounds

In certain embodiments, the small molecule compounds of the present application can be obtained through organic synthesis. The compounds of the present application, including their salts, esters, hydrates or solvates, can be prepared using any conventional organic synthesis technique and can be synthesized according to a number of possible synthetic routes.

The reaction for preparing the compound of the present application can be performed in a suitable solvent, and a person having ordinary knowledge in the field of organic synthesis can easily select a solvent. Suitable solvents are capable of substantially not reacting with the starting materials (reactants), intermediates, or products at the temperature at which the reaction proceeds (for example, a temperature range from the freezing temperature of the solvent to the boiling temperature of the solvent). A given reaction may be performed in one solvent or a mixture of more than one solvent. According to a specific reaction step, those having ordinary knowledge in the technical field to which the present invention pertains can select an appropriate solvent for a specific reaction step.

The preparation of the compounds of the present application may involve the protection and deprotection of various chemical groups. Those with ordinary knowledge in the technical field to which the present invention belongs can easily determine whether protection and deprotection are required and select a suitable protecting group. For protecting group chemistry, see, for example, T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd Ed., Wiley & Sons, Inc., New York (1999), the entire contents of which are incorporated herein by reference.

The reaction can be monitored according to any suitable method known in the art. For example, spectroscopic methods such as nuclear magnetic resonance spectroscopy (such as ¹ H or ¹³ C), infrared spectroscopy, spectrophotometry (such as UV-visible), mass spectrometry, or chromatographic methods such as high performance liquid chromatography ( HPLC), liquid chromatography-mass spectrometry (LCMS) or thin layer chromatography (TLC) were used to monitor the formation of the product. Those with ordinary knowledge in the technical field to which the present invention pertains can purify compounds using a variety of methods, including high performance liquid chromatography (HPLC) (for example, see "Preparative LC-MS Purification: Improved Compound Specific Method Optimization" Karl F. Blom, Brian Glass, Richard Sparks, Andrew P. Combs J. Combi. Chem. 2004, 6 (6), 874-883, the entire contents of which are incorporated herein by reference) and normal phase silica gel column chromatography.

In certain embodiments, the small molecule compounds of the present application are commercially available. In certain embodiments, the small molecule compounds of the present application may be a commercially available compound library, such as the SPECS compound library in the Netherlands.

Pharmaceutical composition

In another aspect, the present application relates to a pharmaceutical composition comprising an active substance (for example, an antibody or antigen-binding fragment described in any one of the above embodiments, an MG53 ligand or a non-functional fragment of MG53, The colony strain or expression vector, or the cell or any one of the small molecule compounds described in any one of the above embodiments), and a pharmaceutically acceptable excipient.

These pharmaceutical compositions can be prepared by methods known in the pharmaceutical art. In certain embodiments, an active substance of the present application can be mixed with a pharmaceutically acceptable carrier for the preparation of a pharmaceutical composition.

The term "pharmaceutically acceptable" as used in this application means within the scope of sound medical judgment that it is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, with reasonableness The benefit / risk ratio of those compounds, materials, compositions and / or dosage forms. In certain embodiments, a pharmaceutically acceptable compound, material, composition, and / or dosage form is approved by a regulatory agency (such as the U.S. Food and Drug Administration, China Food and Drug Administration, or European Medicines Agency) or is listed in generally recognized Those in the Pharmacopoeia of the United States (such as the United States Pharmacopoeia, the Chinese Pharmacopoeia, or the European Pharmacopoeia) for animals, and more particularly for humans.

Pharmaceutically acceptable excipients that can be used in the pharmaceutical composition of the present application include, but are not limited to, for example, a pharmaceutically acceptable liquid, gel, or solid carrier, an aqueous medium (eg, sodium chloride Injection, Ringer's solution injection, isotonic glucose injection, sterile water injection, or glucose and lactate Ringer injection), non-aqueous media (for example, plant-derived nonvolatile oil, cottonseed oil , Corn oil, sesame oil, or peanut oil), antimicrobial substances, isotonic substances (such as sodium chloride or glucose), buffers (such as phosphate or citrate buffers), antioxidants (such as sodium bisulfate), Anesthetics (such as procaine hydrochloride), suspending / dispersing agents (such as sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, or polyvinylpyrrolidone), chelating agents (such as EDTA (ethylenediaminetetraacetic acid) Or EGTA (ethylene glycol bis (2-aminoethyl ether) tetraacetic acid)), emulsifiers (such as polysorbate 80 (Tween-80)), diluents, adjuvants, adjuvants, or nontoxic auxiliary substances , Others in this field Known components, or multiple combinations of the above. Suitable ingredients may include, for example, fillers, binders, disintegrating agents, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants or emulsifiers.

In certain embodiments, the pharmaceutical composition is an oral formulation. Oral preparations include, but are not limited to, capsules, capsules, pills, tablets, lozenges (bases for taste, usually sucrose and gum arabic or astragalus gum), powders, granules, or water or non-aqueous solutions or Suspensions, or water-in-oil or oil-in-water emulsions, or elixirs or syrups, or candy lozenges (suitable for inert bases such as gelatin and glycerin, or sucrose or gum arabic) and / or mouthwashes and Its analog.

Oral solid preparations (such as capsules, tablets, pills, dragees, powders, granules, etc.) include the active substance and one or more pharmaceutically acceptable excipients, such as sodium citrate or dicalcium phosphate, And / or the following: (1) fillers or supplements such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders such as carboxymethyl cellulose, alginates, Gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar, calcium carbonate, potato or cassava starch, alginic acid, and some silicic acid Salts and / or sodium carbonate; (5) retarder solutions, such as paraffin; (6) accelerated absorption agents, such as quaternary ammonium compounds; (7) lubricants, such as acetol and glyceryl monostearate; (8) ) Absorbents, such as kaolin and bentonite; (9) glidants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and (10 )Colorant.

Liquid preparations for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to active substances, liquid dosage forms can also contain commonly used inert diluents, such as water or other solvents, solubilizers and emulsifiers such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzene ( (A) Benzyl acid, propylene glycol, 1,3-butanediol, oils (in particular, cottonseed oil, peanut oil, corn oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acids Sorbitol esters, and mixtures of two or more of the above. In addition to inert diluents, adjuvants such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, pigments, perfumes and preservatives can also be added to oral liquid preparations.

In some embodiments, the pharmaceutical composition is an injectable preparation. Injectable preparations include sterile aqueous solutions or dispersions, suspensions or emulsifiers. In all cases, the injectable preparation should be sterile and should be liquid to facilitate injection. It should be stable under the conditions of manufacture and storage and should be resistant to contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof and / or vegetable oils. The injectable preparation should maintain proper fluidity. The proper fluidity can be maintained in various ways, for example, by using a coating such as lecithin, using a surfactant, and the like. Antimicrobial contamination can be achieved by adding various antibacterial and antifungal agents (for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.).

In some embodiments, the pharmaceutical composition is an oral spray formulation or a nasal spray formulation. The spray formulation includes, but is not limited to, an aqueous aerosol, a non-aqueous suspension, a liposome formulation, or a solid particle formulation. Aqueous aerosols are formulated by incorporating an aqueous solution or suspension of the agent with conventional pharmaceutically acceptable excipients and stabilizers. The carrier and stabilizer vary depending on the needs of the particular compound, but they generally include non-ionic surfactants (Tweens, or polyethylene glycols), oleic acid, lecithin, and amino acids such as glycine Amino acids, buffers, salts, sugars or sugar alcohols. Aerosols are usually prepared from isotonic solutions and can be delivered through a nebulizer.

In some embodiments, the pharmaceutical composition can be used in combination with one or more other drugs. In certain embodiments, the pharmaceutical composition comprises at least one other drug. In some embodiments, the other drugs are cardiovascular drugs, drugs for treating kidney diseases, drugs for cell repair, and the like.

In certain embodiments, the pharmaceutical composition can be delivered to a subject by an appropriate route, including, but not limited to, an oral route, an injection route (such as intravenous, intramuscular, subcutaneous, intradermal, intracardiac) Injection, intrathecal injection, intrapleural injection, intraperitoneal injection, etc.), mucosal route (such as intranasal, intraoral, etc.), sublingual route, rectal route, transdermal route, intraocular route, lung way. In certain embodiments, the pharmaceutical composition can be administered by an injection route.

Kit

In another aspect, the present application relates to a kit for detecting extracellular free MG53.

In certain embodiments, the kit includes an MG53 detection reagent (eg, the MG53 detection reagent described herein). In some embodiments, the kit further includes one or more other ingredients, such as a MG53 standard solution, a diluent, a washing solution, a stop solution, and a developer. The kit described in this application can be used in the method for detecting extracellular free MG53 described in this application.

The reagents in the kit can be placed in any kind of container, so that each component in the reagent can be stably stored without being adsorbed or changed by the material of the container. For example, a sealed glass ampoule may contain a lyophilized matrix / molecule and / or buffer, which is packaged under a neutral, non-reactive gas (eg, nitrogen). The ampoule may be composed of any suitable material, such as glass, organic polymers (for example, polycarbonate or polystyrene, etc.), ceramics, metals, or any other material commonly used to store reagents. Other examples of suitable containers include simple bottles, which can be made of substances similar to ampoules, and envelopes, which can consist of a metal foil liner (for example, aluminum or an alloy). Other containers include test tubes, vials, flasks, bottles, syringes, etc. The container may have a sterile inlet, such as a bottle with a stopper that can be punctured with a hypodermic needle. Other containers may have two compartments, which are separated by a membrane that is easily removed, and the ingredients can be mixed after the membrane is removed. Removable films can be glass, plastic, rubber, and the like.

In certain embodiments, the kit further comprises an enzyme-linked immunoassay disk and a disk sealing membrane.

In some embodiments, the kit can also be provided with the instructions. The instructions may be printed on paper or other materials, and / or provided in the form of electronically readable media such as magnetic disks, CD-ROMs, DVD-ROMs, hard disks, video tapes, optical disks, audio tapes, and the like. The detailed instructions may not be physically associated with the kit; instead, the user may be directed to a website designated by the manufacturer or distributor of the kit, or provided by e-mail. A method for detecting extracellular free MG53 is described in the specification.

Pharmaceutical use

In another aspect, the application relates to the use of an MG53 inhibitor in the manufacture of a medicament for reducing or inhibiting extracellular free MG53 in an individual. In certain embodiments, the MG53 inhibitor may be any of the MG53 inhibitors described above. In certain embodiments, the methods can be used to alleviate, treat or prevent MG53-related diseases or symptoms thereof. In some embodiments, the MG53-related disease may be any of the MG53-related diseases described above. In some embodiments, the medicament is a pharmaceutical composition described herein.

In another aspect, the present application relates to the use of a MG53 detection reagent in the preparation of a kit for detecting extracellular free MG53. In certain embodiments, the kit can be used to detect MG53-related diseases, predict the risk or progression of MG53-related diseases, identify potential MG53 inhibitors, evaluate the therapeutic effect on MG53-related diseases, or detect the activity of free MG53 . In some embodiments, the MG53 detection reagent is provided in the form of a pharmaceutical composition as described herein.

Examples

The following examples are provided to explain the application. It is not intended to be limiting in any way.

Examples 1 : MG53 Preparation and performance of antibodies

MG53 Preparation of antibodies

The total RNA extracted from mice was used for reverse transcription to synthesize cDNA. Oligo-dT was used as a primer, and reverse transcriptase was used for reverse transcription reaction to obtain a cDNA library. Using the synthetic cDNA library as a template, mouse MG53 (NP_001073401.1) encoding nucleic acid was obtained by PCR using specific primers. The PCR procedure is as follows: denaturation, 98 degrees Celsius for 3 minutes; cycling process 98 degrees Celsius for 30 seconds, 65 degrees Celsius for 30 seconds, 72 degrees Celsius for 90 seconds, 30 cycles; then after 72 degrees Celsius for 5 minutes extension, save at 4 degrees Celsius. The primer sequence is as follows: upstream primer: 5'-ataggtaccg ccaccatgtc ggctgcaccc ggcct-3 '(SEQ ID NO: 59); downstream primer: 5'-atactcgagc ggcctgttcc tgctccggcc-3' (SEQ ID NO: 60); carrying KpnI and XhoI Restriction sites.

KpnI and XhoI were used to double-digest the PCR product. At the same time, empty vector plastid pcDNA4 / TO / myc-HisB was treated with the same double-digestion. The gel was recovered and the PCR product was ligated into the vector using T4 ligase to form the plastid. pcDNA4-mMG53-myc. Sequencing verifies that the sequence is correct.

Expression plasmid pcDNA4-mMG53-myc was transiently transfected into HEK293 cells. After 2 days, the supernatants harvested from the transiently transfected cell cultures were used for protein purification. The protein was purified and quantified using SDS-PAGE for immunization.

Three rabbits were immunized with 100 μg of MG53 protein by intraperitoneal injection. After three to five immunizations, blood was collected from the rabbits to collect serum and the antibody titer was determined by ELISA test. The rabbit with the highest titer was selected.

B lymphocytes were isolated from the spleen of the highest titer rabbit. Cell fusion was performed on the isolated B lymphocytes and myeloma cells (at a ratio of 1: 1). The specific experimental steps for fusion are as follows: Wash and resuspend the cell mixture with 5-10 mL of ECF solution. ECF solution was added to adjust the concentration to 2 × 10 ⁶ cells / mL. Immediately after the cells are electrofused, the cell suspension in the fusion chamber is transferred into a sterile tube containing a larger volume of culture medium. After incubation at 37 ° C for more than 24 hours, the cell suspension was mixed and pipetted into a 96-well plate (0.5 x 10 ⁶ cells / plate). Cells were cultured at 37 ° C and 5% CO ₂ . When the colonies were large enough, 100 μL of the supernatant was transferred from a 96-well plate for antibody screening tests.

ELISA was used to detect the binding of the fusion tumor supernatant to the MG53 protein. Briefly, a 1 μg / mL human MG53 protein was coated at 4 ° C overnight. After blocking and washing, the fusion tumor supernatant was diluted at different times, transferred to the coating pan and incubated for 1 hour at room temperature. The coated plate was then washed and then incubated with goat anti-rabbit IgG (H + L) HRP secondary antibody (Goat Anti-Rabbit HRP (IgG H & L) (ab6721) | Abcam) for 45 minutes. After washing, TMB matrix was added and the reaction was stopped with 2M HCl. The absorbance at 450 nm was read using a microdisk analyzer (Molecular Device). According to the results, five single-cell fusion tumor cells with the best binding properties and suitable for detection were selected. The binding data of the five single-cell fusion tumors are shown in the following table.

Among them, the positive standard value (commercially available MG53 antibody) was 0.456; the negative standard value (irrelevant antibody) was 0.083.

Five fusion tumor cell lines were reselected. Briefly, for each fused tumor cell line, cells were counted and diluted to 5 cells / well, 1 cell / well, and 0.5 cells / well in the selection medium. 200 μL / well was plated into a 96-well plate, one plate was 5 cells / well, one plate was 1 cell / well, and four plates were 0.5 cells / well. All plates were placed 37 degrees Celsius, 5% CO _2. Incubate until all cell lines can be tested by ELISA.

Subsequently, genetic sequencing was performed on 5 individual fusion tumor cells, and the results showed that the MG53 antibodies were antibody # 6 (the heavy chain nucleotide sequence: as shown in SEQ ID NO: 7, and the light chain nucleotide sequence: as shown in SEQ ID NO: 8), antibody # 110 (heavy chain nucleotide sequence: as shown in SEQ ID NO: 15, light chain nucleotide sequence: as shown in SEQ ID NO: 16), antibody # 84 (heavy chain Nucleotide sequence: as shown in SEQ ID NO: 23, light chain nucleotide sequence: as shown in SEQ ID NO: 24), antibody # 9 (heavy chain nucleotide sequence: as shown in SEQ ID NO: 31 Light chain nucleotide sequence: as shown in SEQ ID NO: 32), antibody # 43 (heavy chain nucleotide sequence: as shown in SEQ ID NO: 39, light chain nucleotide sequence: as shown in SEQ ID NO: 40).

MG53 Performance of antibody binding

Surface plasmon resonance (SPR) was used to verify the ability of MG53 antibodies to bind to MG53. Recombinant human MG53 protein (rhMG53) was dissolved in 10 mM sodium acetate buffer (pH = 4.5), and then rhMG53 was immobilized on a CM5 wafer with a primary amino group using Amine Coupling kit (GE Healthcare), and 1M amino group was used. Ethanol blocks other active sites. The control fluid chamber was activated and blocked in the absence of MG53. Fix the fixed level at 500 Biacore reaction units (RU) and gradually increase the concentration of MG53 antibody # 84 flowing through the surface of the wafer (amino acid heavy chain sequence: as shown in SEQ ID NO: 19, light chain sequence: as shown in SEQ ID NO: 20 (concentrations from 1.95 nM to 1000 nM), and the data results are shown in Figure 2. From the above experimental results, it can be seen that the MG53 antibody of the present application can bind to rhMG53 with high affinity.

Surface plasmon resonance (SPR) was used to verify the ability of MG53 antibodies to block the binding of MG53 to the extracellular domain of insulin receptors. The recombinant insulin receptor extracellular region (IR) was dissolved in 10 mM sodium acetate buffer (pH = 4.5), and the IR was then immobilized on the CM5 chip through the primary amine group using the Amine Coupling kit (GE Healthcare), 1M aminoethanol was used to block other active sites. The control fluid chamber was activated and blocked in the absence of IR. Fix the fixed level at 500 Biacore reaction units (RU), and gradually increase the concentration of the reactants flowing through the wafer surface (in the experiment shown in Figure 3a, rhMG53 was used with concentrations ranging from 0.49 nM to 500 nM; in the figure In the experiment shown in 3b, a mixture of rhMG53 and MG53 antibody # 84 was used, where the concentration of MG53 antibody was fixed at 500 nM and the concentration of rhMG53 was from 0 nM to 2000 nM).

The above two binding experiments are in 25 ° C, PBS-P (20 mM phosphate buffer, 2.7 mM NaCl, 137 mM KCl, 0.05% (volume ratio) surfactant P-20, pH = 7.4) buffer get on. Regeneration was performed with 10 mM NaOH (pH = 10) after each sample injection. Testing was performed using a Biacore T200 instrument and data analysis was performed using its software.

Examples 2 : Extracellular free MG53 Functional research

The procedure of the western dot method used in Example 2 is as follows:

(1) Glue making: Wash and dry the glass plate. Assemble the vertical electrophoresis instrument and perfuse 8% separation gel and 5% concentrated gel in order.

(2) Preparation of electrophoresis buffer: use Tris and glycine to prepare a 5 × stock solution of the electrophoresis buffer, dilute to 1 ×, and add 600 mL to the vertical electrophoresis tank.

(3) Loading: The prepared protein sample is cooled to room temperature, thawed and centrifuged to mix. Load the protein standard from left to right. For protein samples, if there is no sample in the well, add an equal amount of 1 × Loading buffer was added for balance, leaving no empty lanes to prevent bands from skewing.

(4) Running gel: Connect the electrophoresis device to the power supply, apply a voltage of 7 V / cm, and increase the voltage to 11 V / cm after the bromophenol blue front edge enters the separation gel and the protein standard isolates a clear band. Continue electrophoresis until bromophenol blue reaches the bottom of the gel.

(5) Preparation of transfection buffer solution: Tris and glycine were used to prepare a 20 × transfection buffer mother solution, diluted to 1 ×, and added with 20% methanol for mixing.

(6) Transfer film: transfer the film with a wet electric trough, and place the sponge, double-layer filter paper, gel, PVDF film, double-layer filter paper, and sponge on the black plastic plate in the order of "sandwich", and carefully clamp it Then put it into the electric rotator, (note that there should be no air bubbles between the PVDF membrane and the gel, otherwise it will affect the effect of the transfer membrane) Add 1000 mL of transfer membrane buffer solution, place the device on ice, connect the power supply, constant current 275 mA Transfer film for 2 hours.

(7) Blocking: Dilute 10 × TBS solution to 1 ×, add 0.1% Tween-20 to make 1 × TBST solution, and then add 5% (w / v) skim milk powder as blocking buffer. After the transfer is complete, carefully remove the PVDF membrane and place it in blocking buffer. Slowly shake for 1 hour at room temperature on a shaker.

(8) Incubation of primary antibody: Preparation of primary antibody solution. Add 1 ‰ primary antibody to TBST solution containing 5% (w / v) BSA. After the membrane was blocked, wash with TBST three times for 5 min each time, add the corresponding primary antibody solution, and incubate overnight at 4 ° C with a slow shake in a shaker.

(9) Incubate the secondary antibody: prepare a secondary antibody solution. The secondary antibody is diluted with a ratio of 1: 2000 in TBST containing 5% skimmed milk powder to make the secondary antibody solution. Note that the secondary antibody needs to correspond to the primary antibody. Resistance or rabbit resistance. The membrane that had been incubated overnight with the primary antibody was taken out, washed three times with TBST for 5 min each, and then the secondary antibody solution was added, and incubated at room temperature for 1 hour with slow shaking on a shaker.

(10) Chemiluminescence detection: Remove the PVDF membrane incubated with the secondary antibody, wash it three times with TBST for 5 min each time, and immerse the membrane in TBST for later use. The chemiluminescence reaction substrates A and B were prepared and mixed according to a volume ratio of 1: 1, and the operation was protected from light, and then added to a PVDF membrane for 5 minutes, and the chemiluminescence detection bands were performed using ChemiDocXRS from BIO-RAD.

(11) Re-incubate the primary antibody: wash the membrane twice with TBST for 5 min each time. Add elution buffer and wash the membrane at 50 ° C, 50 rpm for 30 min. Wash the membrane 6 times with TBST for 5 min each. After the antibody is eluted, the corresponding protein can be detected by incubating other primary and secondary antibodies. Chemiluminescence detection strip blot images were analyzed with ImageJ software for gray value.

Detection of extracellular freeness using antibodies of the present application MG53

Blood was collected from the tail vein of 14-week-old WT mice and MG53 ^-/- (the MG53 gene was removed by gene knockout) mice, and the serum was centrifuged. Using 0.5 μL of serum for sample loading, using MG53 antibody # 84 as the primary antibody and HRP anti-rabbit antibody as the secondary antibody, the Western blot method was used for detection. The test results are shown in FIG. 4. From the experimental results, it can be seen that using the MG53 antibody of the present application can clearly distinguish between samples that express MG53 and samples that do not express MG53.

Blood was collected from the tail vein of 14-week-old WT mice and TG mice (overexpression of the MG53 gene by gene technology), and the serum was collected by centrifugation. Using 0.5 μL of serum for sample loading, MG53 antibody # 84 was used as the primary antibody, and HRP anti-rabbit antibody was used as the secondary antibody. From the experimental results, it can be known that the experiment using the MG53 antibody of the present application can clearly distinguish serum MG53 with different concentrations.

MG53 antibody # 84 was used to detect rmMG53-myc (myc-tagged recombinant mouse MG53 protein expressed by HEK293 cells) and rhMG53 (purified recombinant human MG53 protein expressed by HEK293 cells) using the same western blot method as described above. As shown in Figure 6. It can be known from the experimental results that the MG53 antibody of the present application can be used to detect extracellular free MG53 of human and mouse origin.

Extracellular free MG53 Functional verification

Serum (T2D) from 37 patients with type 2 diabetes and serum from 21 normal persons (control) were obtained. Using the same western blot method as described above, the above serum samples were tested, and the test results are shown in FIG. 7. From the experimental results, it can be seen that the serum MG53 content in patients with type 2 diabetes is statistically significant (using the t-test test), so that whether or not they have diabetes and have Risk of diabetes.

Serum from 18 mice on a high-fat diet (using Cat. # D12492 feed, purchased from ResearchDiets Inc., 60% of which is derived from fat and fed for 35 weeks) and 15 mice on a normal diet. The same Western blot method as described above was used to test the above serum samples, and the test results are shown in FIG. 8. It can be known from the experimental results that a high-fat diet can induce an increase in serum MG53, so that the risk of suffering from fat metabolism diseases can be determined according to the content of serum MG53.

Obtain sera from 6 12-week-old ZDF rats (Zucker diabetic fatty rat (purchased from Vital River Laboratories (Beijing, China), Cat # 123) and 6 normal SD rats, and use ELISA kit (Cusbio) to detect serum The content of MG53 in the blood glucose was measured with a blood glucose meter (Roche), the blood insulin content was measured with an ELISA kit (Millipore), and the body weight was measured. The data are shown in Figure 9. From the experimental results, it can be seen that the content of serum MG53 is linearly related to body weight, blood glucose and blood insulin content, so that the risk and progression of diabetes can be judged based on the content of serum MG53, and the response to treatment.

C57 (purchased from Viton Lee Hua) mice, 8-10 weeks old, were selected and fasted overnight, followed by injection of recombinant human MG53 protein (rhMG53) or BSA at a dose of 6 mg / kg in the tail vein, followed by 1 minute after 10 minutes. A dose of U / kg is given intraperitoneally or without insulin. After another 10 minutes, the mice were sacrificed and the corresponding tissue material was obtained. The western blot method was used to detect the phosphorylation content of akt, an important molecule in the insulin signaling pathway, in each group of skeletal muscle, liver, fat, and heart tissues, and then to detect insulin sensitivity in several tissues. The source of each antibody is as follows: Anti-phospho Ser473 Akt (p-AktS473), CST Cat # 4060; Anti-total Akt (t-Akt), CST Cat # 9272; Anti-GAPDH, Bioeasy Technology, Cat # BE0023. Statistical analysis was performed using One-way anova. ** indicates P <0.01. The test results are shown in Figure 10. It can be known from the experimental results that serum MG53 can reduce insulin sensitivity, and thus can improve insulin sensitivity by reducing the concentration or activity of serum MG53.

High glucose and high insulin-induced perfusion from rodent hearts MG53 freed

Adult SD rats (250 to 300 g) or mice (20 to 30 g) were anesthetized with pentobarbital (70 mg / kg body weight, intraperitoneally). Rat or mouse hearts were excised and perfused on a Langendorff device with Krebs-Henseleit buffer at a constant pressure of 55 mm Hg (NaCl 118 mM, KCl 4.7 mM, CaCl ₂ 2.5 mM, MgSO ₄ • 7H ₂ O 1.2 mM, KH ₂ PO ₄ 1.2 mM and glucose 11.1 mM). The buffer is continuously supplied with 95% O ₂ /5% CO ₂ (pH 7.4) and is heated by a hot bath / circulation pump. The temperature of the heart is continuously monitored and maintained at 37 ± 0.5 ° C. Collect the perfusate from different time periods. For EDTA or BFA treatment, the perfusate solution included 0.75 mM EDTA-Na (no Ca ²⁺ in the perfusate solution) or 30M BFA. The collected perfusate samples were centrifuged for 15 minutes at 3,000 rpm using an Amicon Ultra-15 10K filter centrifuge (Millipore, Cat # UFC801096) and repeated 3 times. The concentrated perfusate sample was used for subsequent analysis.

After Langendorff perfusion was performed on the hearts of isolated rodents with physiological saline, we performed western blotting by accumulating the outflow perfusate accumulated in the rat heart over a 30-minute period and in the mouse heart over a 60-minute period. Point analysis showed the release of a large amount of MG53 (Figure 32). Strikingly, the metabolic challenge with high glucose (25 mM) increased rat myocardial MG53 release three-fold in the first 30 minutes after stimulation (Figure 32A). Elevated MG53 release continued into the second 30-minute period (30–60 minutes) of the glucose challenge, and increased in a glucose concentration-dependent manner (Figures 32A and 32B). In sharp contrast to this, L-glucose, which cannot be utilized by the organism, cannot alter the release of MG53 from the heart muscle (Figure 32C). To mimic the conditions of metabolic syndrome and T2D, we investigated the effects of glucose and insulin co-stimulation. Stimulation of insulin with basal concentration of glucose (11.1 mM) increased release of myocardial MG53 in rat hearts perfused in a time-dependent and concentration-dependent manner (Figures 32D and 32E). The coexistence of high glucose and high insulin stimulated MG53 to be released at a rate of ~ 4 times the baseline (Figure 32C). In addition, we used MG53 over-expression (mg53 TG) and knock ^- out (mg53 ^-/- ) mouse models to demonstrate that the combined stimulation of sugar and insulin stimulated the release of MG53 from mg53 TG hearts compared to wild-type (wt) mice The increase was at least two-fold, and no release of MG53 was detected in the perfusate of the mg53 ^-/- heart (Figures 32G and 32H). This confirms the released protein detected by Western blotting. As shown in Figures 35A and 35B, the release of MG53 caused by high glucose plus insulin does not cause myocardial lactate dehydrogenase (LDH) or creatine kinase (CK) release in perfused rat hearts (indicating cell membrane integrity) Loss), which means that metabolically regulated release of MG53 is not due to myocardial damage.

High glucose and high insulin cause MG53 Release and in vivo MG53 freed

Adult male C57 mice (20 to 30 g) were anesthetized with pentobarbital (70 mg / kg, intraperitoneally). Soleus muscle was isolated from the mice and modified at room temperature (22 ^o C) with a modified Krebs-Henseleit solution (NaCl 118 mM, KCl 4.7 mM, CaCl ₂ 2.5 mM, MgSO ₄ • 7H ₂ O 1.2 mM, KH ₂ PO ₄ 1.2 mM, glucose 5 mM) continuous perfusion at a rate of 1.0 mL / min in the presence of 95% O ₂ /5% CO ₂ gas mixture. Ten minutes after acclimatizing to perfusion, the soleus muscle was treated with modified Krebs-Henseleit solution buffer with or without high glucose (25 mM) combined with high insulin (10 U / L) for 2 hours. The perfusate samples were analyzed by Western blot method after being concentrated by ultrafiltration.

In addition to the heart muscle, the combined treatment of high glucose (25 mM) and high insulin (10 U / L) also effectively caused the release of MG53 from skeletal muscle (soleus muscle) perfused from wild-type mice in vitro (Figure 33A). The occurrence of glucose-induced release of MG53 from skeletal muscle did not cause any increase in LDH or CK concentrations in the perfusate (Figures 35C and 35D). This result indicates that metabolic stimulation can promote the release of MG53 from skeletal muscle and myocardium under physiological conditions. Since skeletal muscle accounts for approximately 40% of the body's non-fat weight, we hypothesized that high glucose-induced MG53 release would be sufficient to increase circulating MG53 content in the body. Hyperglycemia (glucose, 2 g / kg, intraperitoneal injection, repeated three times at 40 minute intervals) increased serum MG53 in wild-type mice (Figure 33B).

Oral delivery of glucose in healthy people

Healthy volunteers fasted overnight and then delivered glucose (75 g) orally twice at 30-minute intervals. Blood samples were taken before glucose treatment and 30 and 90 minutes after the second dose. Blood samples were centrifuged at 3,000 rpm, and serum was collected for Western blotting.

In healthy humans, oral administration of glucose significantly increased serum MG53 levels (Figure 33C), while increasing blood glucose levels without causing changes in serum LDH concentrations.

Regulates secretory pathways to mediate metabolic induction MG53 freed

Using pharmacological and genetic methods, we subsequently attempted to explain the underlying mechanisms of metabolically regulated release of MG53. First, in order to distinguish between the regulation of MG53 secretion and passive leakage, we evaluated the effect of a protein transport inhibitor Brefeldin A (BFA). In HEK293 cells expressing MG53-myc, BFA inhibited the release of MG53 induced by high glucose in a dose-dependent manner (Figure 33D). BFA (30 μM) can also block glucose-induced MG53 release in isolated rat perfused hearts (Figure 33E), suggesting that glucose-induced MG53 release is mediated by the classical secretory pathway. For most of the regulation of secretion, an increase in cytoplasmic Ca ²⁺ concentration is a prerequisite for the fusion of secretory vesicle membranes with cell membranes and the release of vesicle contents through exocytosis. Buffering extracellular Ca2 ⁺ ions with EDTA (0.75 mM) actually blocked metabolically regulated MG53 secretion to a large extent (Figure 33F). In syt7 ^-/- mice, high glucose and insulin stimulated MG53 release in the perfused heart and in vivo was blocked (Figures 33G and 33H), suggesting that Syt7 is required for metabolically regulated MG53 secretion. It is worth noting that neither EDTA treatment nor Syt7 deletion altered basal MG53 release in perfused rodent hearts (Figures 33F and 33G) or in vivo (Figure 33H), suggesting that MG53 release may be attributed to atypical Secretion or leakage. Collecting these results together, we believe that, like many cytokines, metabolically-induced MG53 release is mediated by Ca ²⁺ ions and SNARE binding protein-dependent secretion mechanisms.

MG53 Heart-specific overexpression is sufficient to cause systemic insulin resistance and metabolic syndrome

In order to further study the possible causal relationship between chronic elevation of MG53 in blood and the occurrence of metabolic diseases, we constructed cardiac-specific MG53 gene-transgenic mice (mg53 h-TG). Myocardial MG53 secretion continues to provide circulating MG53, while MG53 expression in skeletal muscle is not disturbed. Figure 34A shows that the basal serum MG53 content in young adult mg53 h-TG mice at 14 weeks of age is significantly higher than that in wild-type mice in the littermates.

Fasting weight was measured for wild-type and mg53 h-TG mice at 3 to 38 weeks of age, and glucose tolerance test (GTT) and insulin resistance were performed on wild-type and mg53 h-TG mice at 14 and 30 weeks of age, respectively. Tested (ITT). Briefly, for GTT, mice were fasted overnight (16 hours) and then injected intraperitoneally with D-glucose (2 g / kg, Sigma-Aldrich). For ITT, mice were fed at random and challenged with bovine insulin (0.75 units / kg, Sigma-Aldrich, intraperitoneal injection) collected from the tail vein before and at different time points after injection (as shown in Figures 34C and 34D). Blood and measure blood sugar.

Young mg53 h-TG mice showed moderate obesity (Figure 34B), glucose intolerance, and insulin intolerance (Figures 34C and 34D). At 30 weeks of age, the heart-specific overexpression of MG53 is sufficient to cause a comprehensive metabolic syndrome manifested by hyperglycemia, hyperinsulinemia, and dyslipidemia (Figure 34E-34G), abdominal fat accumulation, white fat, brown fat, and Fat-to-fat ratio increased (Figures 34H-34M), and fatty liver and pancreatic hyperplasia (Figures 34N and 34O). With the development of systemic insulin resistance, mg53 h-TG mice showed severe obesity, glucose intolerance, and insulin intolerance at this time point (Figures 34B-34D). At the same time, the daily energy expenditure of mg53 h-TG mice was significantly lower than that of the wild type control (Figure 34Q-34S), but their daily food intake, core body temperature or physical activity did not change (Figure 34Q-34S). At later time points, mg53 h-TG mice also developed diabetic complications such as diabetic cardiomyopathy. Therefore, chronic elevation of circulating MG53 firstly impairs the insulin response throughout the body, and secondly leads to systemic metabolic syndromes that cause obesity, diabetes and various cardiovascular complications.

Examples 3 ： Study on the effect of antibody therapy

Twenty 8- to 10-week-old 20 db / db mice (diabetic model mice purchased from Jackson Laboratory (Bar Harbor, ME) Cat # 000642) were selected, of which 10 mice were used as a control group and injected with IgG only (Sigma, I5381); the other 10 were treated as a treatment group and injected with MG53 antibody # 84. Five db / + mice of the same age were ordered at the same time. db / + mice were used only for final sampling and were not injected.

After the mice arrived, they were stable for 1 week, and then the body weight and blood glucose of the mice were measured, and the status was checked. On the day of the experiment, animals were not fasted, and body weight and blood glucose were measured at 8 am. Then group them, paying attention to strictly matching weight and blood sugar. Inject according to grouping. Subsequently, the injection volume was 0.24 ml / head / time at 1 mg / head / time, and the injection was performed once at 9 o'clock. Mice were then fasted and blood glucose was measured at 15:00 and 19:00 on the day of the experiment. Then, the body weight was measured at 9 o'clock every day, blood glucose was measured at 9 and 15 o'clock, and the measurement was continued for 10 days.

The insulin resistance test (ITT) was performed on the 8th day after injection. The experimental conditions are described in the literature by Song et al., Central role of E3 biquitin ligase MG53 in insulin resistance and metabolic disorders, Nature, 494, 375-381, 2013 .

The test results are shown in Figure 11. From the experimental results, we can see that MG53 antibody can effectively reduce blood sugar and improve insulin sensitivity, so it can be used to treat diabetes.

Examples 4 : MG53 Study of small molecule inhibitors

All small molecule compounds used in Example 4 were purchased from the SPECS compound library in the Netherlands, and the MG53 protein was synthesized and prepared by ORIGENE.

Computer Virtual Screening

First, perform homology simulation on MG53 protein, select the protein primary structure, that is, the known protein structure with higher amino acid sequence homology and resolved structural resolution as the reference structure, and then use Cavity 2.0 to bind the small molecule pocket For prediction, select a pocket with a relatively large volume, a large dissociation constant (pKD), and an amphiphilic pocket as the target pocket.

The principle of screening is to screen through different angles and different methods. Each screening method gives points according to the combination of small molecule compounds and proteins, and each time selects a higher score. Finally, the intersection of the screening results of different methods is taken. The specific operation is as follows: first, fix each group in the protein pocket, use small molecule compounds to freely dock, and select 20,000 compounds with a high score; then let the small molecule compounds and groups can be freely rotated and docked, and select Four thousand compounds with high scores; then manual screening using pymol software, the screening criteria are the following five: a. The molecular weight of the compound is greater than 300 Da, the molecular weight is too small, the specific reaction will be weak; b. The compound and the pocket There must be at least three hydrogen bonding interactions between the groups in the group, which is to increase the interaction between the compound and the protein; c. The compound must have a share of more than 80% of the protein pocket, which is also to reduce the non- Specificity; d. The compound cannot be a polypeptide, this is to avoid non-specific binding of the polypeptide in the body; e. The compound does not contain metal atoms. In the end, 140 small molecules were obtained.

Cell screening

The next 140 molecules were screened at the cell level. In order to conduct a large number of screening experiments, a special cell screening system was constructed. First construct GFP fluorescently labeled IRS1 plastids (IRS1-GFP) and Myc-labeled MG53 plastids (MG53-myc). At the same time, these two plastids are transfected into cells to make them highly expressed. Under normal circumstances, MG53 will use its E3 ligase activity to mediate the ubiquitination degradation of IRS1, causing its fluorescence intensity to be significantly reduced compared to the control group, thereby disrupting the cell insulin signaling pathway. When these two plastids are cotransformed into a cell, a small molecule compound is added. If the small molecule compound has an inhibitory effect on the MG53 protein activity, it will prevent it from performing its normal function in the cell. At this time, the fluorescence intensity of IRS1 will be With a certain recovery, small molecule compounds with inhibitory effects on MG53 were screened. In order to select a cell with better controllability and easy handling, a comparative experiment was performed in HEK293T and HEK293A cells. That is, MG53-Myc and IRS1-GFP were cotransfected in these two cells, respectively. The screening results are shown in Figure 12. It was found that in HEK293T cells, MG53 can significantly reduce the fluorescence intensity of IRS1. In order to increase the operating window for subsequent screening experiments, HEK293T cells were selected for subsequent experiments.

After the screening system was established, 140 small molecule compounds were screened in HEK293T cells. First, HEK293T cells were cultured in a 10 mL cell culture dish. The medium was changed every two days, and cells with good growth health were selected. When the cells covered the entire cell culture dish, 80% of MG53-Myc and IRS1-GFP were co-transformed. Co-transfection of pcDNA4 empty vector and IRS1-GFP in the control group. Cells were cultured for 24 hours after transfection. The control group could see a strong expression of GFP under a fluorescent microscope, while the fluorescence intensity of the experimental group was reduced by more than 50%. Next, the cells were individually digested with 0.125% trypsin, and the cells were collected by low-speed centrifugation. After the cells were mixed, they were spread on enzyme-linked immunoassay plates in 96-well plates and continued to culture. After 24 hours, the cells were exchanged and the small molecules to be tested were added. The culture was continued for 24 hours. Finally, the cells were washed 3 times with PBS, and the fluorescence signal was detected at 485nm / 528nm. Compounds were screened at concentrations of 100 μM, 10 μM, and 1 μM, respectively. A total of 48 candidate compound molecules were screened.

SPR filter

The 48 candidate compounds were screened by SPR experiments. According to theoretical calculations, the theoretical reaction value of the binding between MG53 protein and small molecule compounds is 5, so one of the screening criteria is that the reaction value is greater than or equal to 5. We selected two concentrations, 50 μM and 100 μM for screening, and the control group was a PBS solution. As shown in Figure 13, among the 48 small-molecule compounds, a total of 15 molecules have a reaction value of not less than 5 (including 10, 14, 16, 16, 17, 25, 68, 13, and 16 No. 28 and No. 28 relatively high response; No. 1, No. 3, No. 26, No. 28, No. 56 and No. 10 relatively low response), these compounds as candidate molecules for the next screening.

Next, the kinetic characteristics of the binding of these 15 candidate molecules to MG53 were explored through concentration-dependent SPR experiments. The screening criteria included a dissociation constant of less than 50 μM, not non-specific binding, and high molecular purity. Finally, four candidate molecules were screened (see Figure 14 for numbers 1, 10, 26, and 16). The dissociation constants of these 4 molecules are all less than 50 μM (see the corresponding S-shaped curve on the right, the dissociation constant is the concentration of the compound when the reaction value reaches half of the maximum reaction value), and the minimum reaction concentration is very small, reaching the nanomole level. This means that the higher the specificity of the compound's binding to the MG53 protein, the better the drugability. The corresponding kinetic curves are also "S" type curves, indicating that the four compound molecules have better kinetic characteristics. The other 11 compounds have insufficient molecular purity (detected by small molecule mass spectrometry), or the dissociation constant is too large (the dissociation constant is above 50 μM), or they show poor kinetic characteristics (kinetic curves A straight line or not an S-shaped curve indicates that the molecule has a strong non-specific binding to the MG53 protein), and so on. Among all these four candidate molecules, it is worth mentioning that molecule No. 26 has a minimum reaction concentration of only 1.2 nM and a dissociation constant of 100 nM, indicating that this molecule has a very good specific binding to the MG53 protein, and its The response curve of SPR is "slow up, slow down" type, which means that if it is used for drug administration, it will show many advantages such as low dosage, long efficacy, stable efficacy, and long interval of administration. , So there is a great possibility of medicine.

Degradation experiment verification

Next, the intracellular activities of the four candidate molecules were further verified. HEK293T cells in good culture were divided into 6 groups, which were transfected with IRS1 and MG53-myc. The 4 groups of cells transfected with IRS1 and MG53-myc were treated with compounds, and the concentration of the added compounds was 50 μM. The compounds were extracted for total protein after 24 hours and subjected to SDS-PAGE gel vertical electrophoresis experiments. The expressions of IRS1 and MG53 proteins were detected during exposure, and GAPDH was used as an internal control. We found that these four compound molecules have a certain degree of recovery effect on MG53-mediated ubiquitination of IRS1 (see Figure 15), which indicates that these compound molecules have the function of MG53 to perform its E3 ligase function in cells. Has a certain degree of inhibition.

The structures of the 4 compounds screened are as follows:

no

Figure 1: Schematic diagram of the molecular structure of wild-type MG53.

Figure 2: SPR experiment results used to test the affinity of MG53 antibody to antigen protein, K _D = 17.7 nM for this antibody.

Figure 3: a. SPR experiment results for detecting the affinity of MG53 and the extracellular region of insulin receptor, K _D = 8.0 nM for MG53 and the extracellular region of insulin receptor; b. For detecting MG53 antibody blocking MG53 and insulin A graph of the results of SPR experiments for receptor extracellular domain binding. The Ki of the antibody was 265 nM.

Figure 4: Using the antibody of the present application as a primary antibody to detect the MG53 content in the serum of MG53 ^-/- mice (MG53 knockout) and wild-type mice by Western blotting. The upper picture is a western dot map, and the lower figure is a histogram drawn based on the values calculated by the western dot map. Among them, ** indicates that P <0.01, which is significantly different, using t-test statistical test.

Figure 5: Using the antibody of the present application as a primary antibody to detect the MG53 content in the serum of TG mice (MG53 gene overexpression) and wild-type mice by Western blot method. The upper picture is a western dot map, and the lower figure is a histogram drawn based on the values calculated by the western dot map. Among them, ** indicates that P <0.01, which is significantly different, using t-test statistical test.

Figure 6: Detection of recombinant mouse MG53 and recombinant human MG53 by Western blot method using the antibody of the present application as a primary antibody.

Figure 7: Using the antibody of the present application as a primary antibody to detect the content of MG53 in the serum of type 2 diabetic patients and normal people by Western blot method. The upper picture is the western blotting diagram of the protein, and the lower figure is a histogram drawn based on the values calculated by the western blotting diagram. Among them, ** indicates that P <0.01, which is significantly different, using t-test statistical test.

Figure 8: Using the antibody of the present application as a primary antibody to detect the content of MG53 in the serum of high-fat diet mice and normal diet mice by Western blot method. The upper picture is a western dot map, and the lower figure is a histogram drawn based on the values calculated by the western dot map. Among them, ** indicates that P <0.01, which is significantly different, using t-test statistical test.

Figure 9: a. Serum MG53 content in fasted normal rats and fasted ZDF rats; b. Relationship between serum MG53 concentration and body weight in fasted normal rats and fasted ZDF rats; c. Fasting normal Relationship between serum MG53 concentration and blood glucose content in rats and fasted ZDF rats; d. Relationship between serum MG53 concentration and serum insulin content in fasted normal rats and fasted ZDF rats. Among them, ** indicates that P <0.01, which is significantly different, using t-test statistical test.

Figure 10: Detection of GAPDH (control), Akt, and insulin-induced phosphorylation in different tissues (skeletal muscle, liver, visceral fat, and heart) in mice with or without insulin and subsequent injections of BSA or rhMG53 The concentration of Akt. The upper picture is a western dot map, and the lower figure is a histogram drawn based on the values calculated by the western dot map. Among them, ** means P <0.01, which is significantly different, using One-way anova statistical test.

Figure 11: a. Serum MG53 levels in normal mice and diabetes model mice; b. Blood glucose levels in the control group (administered with IgG) and the treatment group (administered with MG53 antibody); c. In the insulin resistance test, The blood glucose levels of the control group (administered IgG) and the treatment group (administered MG53 antibody). Among them, ** means P <0.01, which has a significant difference, and * means P <0.05, which has a significant difference, using t-test statistical test.

Figure 12: Co-transfection of IRS1 plastids (IRS1-GFP) and Myc-labeled MG53 plastids (MG53-myc) in HEK293T cells and HEK293A cells, respectively, to detect the fluorescence intensity of GFP, where ** indicates P <0.01 Significant difference, * means P <0.05, significant difference, using t-test statistical test.

Figure 13: Response values of SPR experiments used to screen different candidate molecules.

Figure 14: a. Response value of SPR experiment of compound 1; b. Response value of SPR experiment of compound 10; c. Response value of SPR experiment of compound 26; d. Response value of SPR experiment of compound 16.

Figure 15: IRS1 plastids (IRS1-GFP) and Myc-labeled MG53 plastids (MG53-myc) were co-transfected in HEK293T, and compounds 1, 10, 26, and 16 were added, respectively, and the fluorescence intensity of GFP was subsequently detected. *** indicates P <0.001, which is significantly different, using One-way anova statistical test.

Figure 16: Shows the heavy chain amino acid sequence of SEQ ID NO: 3 and its encoding nucleic acid sequence of SEQ ID NO: 7 for antibody # 6. Among them, the shaded sequence corresponds to the heavy chain variable region of antibody # 6.

Figure 17: Shows the light chain amino acid sequence of SEQ ID NO: 4 and its encoding nucleic acid sequence of SEQ ID NO: 8 for antibody # 6. Among them, the shaded sequence corresponds to the light chain variable region of antibody # 6.

Figure 18: Shows the heavy chain amino acid sequence of SEQ ID NO: 11 and its encoding nucleic acid sequence of SEQ ID NO: 15 for antibody # 110. The shaded sequence corresponds to the heavy chain variable region of antibody # 110.

Figure 19: Shows the light chain amino acid sequence of SEQ ID NO: 12 and its encoding nucleic acid sequence of SEQ ID NO: 16 for antibody # 110. Among them, the shaded sequence corresponds to the light chain variable region of antibody # 110.

Figure 20: Shows the heavy chain amino acid sequence of SEQ ID NO: 19 and its encoding nucleic acid sequence of SEQ ID NO: 23 for antibody # 84. Among them, the shaded sequence corresponds to the heavy chain variable region of antibody # 84.

Figure 21: shows the light chain amino acid sequence of SEQ ID NO: 20 and its encoding nucleic acid sequence of SEQ ID NO: 24 for antibody # 84. Among them, the shaded sequence corresponds to the light chain variable region of antibody # 84.

Figure 22: Shows the heavy chain amino acid sequence of SEQ ID NO: 27 and its encoding nucleic acid sequence of SEQ ID NO: 31 for antibody # 9. Among them, the shaded sequence corresponds to the heavy chain variable region of antibody # 9.

Figure 23: Shows the light chain amino acid sequence of SEQ ID NO: 28 and its encoding nucleic acid sequence of SEQ ID NO: 32 for antibody # 9. Among them, the shaded sequence corresponds to the light chain variable region of antibody # 9.

Figure 24: Shows the heavy chain amino acid sequence of SEQ ID NO: 35 and its encoding nucleic acid sequence of SEQ ID NO: 39 for antibody # 43. Among them, the shaded sequence corresponds to the heavy chain variable region of antibody # 43.

Figure 25: Shows the light chain amino acid sequence of SEQ ID NO: 36 and its encoding nucleic acid sequence of SEQ ID NO: 40 for antibody # 43. Among them, the shaded sequence corresponds to the light chain variable region of antibody # 43.

Figure 26: Amino acid sequence of human wild type MG53 SEQ ID NO: 41, amino acid sequence of monkey wild type MG53 SEQ ID NO: 42, amino acid sequence of rat wild type MG53 SEQ ID NO: 43 and The amino acid sequence of mouse wild-type MG53 is SEQ ID NO: 44.

Figure 27: shows the amino acid sequence of pig wild-type MG53 SEQ ID NO: 45, the amino acid sequence of dog wild-type MG53 SEQ ID NO: 46, the amino acid sequence of rabbit wild-type MG53 SEQ ID NO: 47, and 狨The amino acid sequence of monkey wild type MG53 is SEQ ID NO: 61.

Figure 28: shows the sequence of some MG53 mutants (SEQ ID NOs: 48-50).

Figure 29: shows the sequence of some MG53 mutants (SEQ ID NOs: 51-53).

Figure 30: shows the sequence of some MG53 mutants (SEQ ID NOs: 54-57).

Figure 31: shows the sequence of the human insulin receptor (SEQ ID NO: 58).

Figure 32: Shows high glucose and high insulin-induced release of MG53 in rodent hearts perfused in vitro. Figure 32 A shows a representative western blotting plot (top block) and average data (bottom block), which indicates that high glucose increases perfusate in a time-dependent manner in isolated rat perfused heart MG53 (n = 12 hearts). Figure 32B shows a representative western dot map (upper block) and average data (lower block), which indicates that high glucose increases perfusion fluid in a concentration-dependent manner in isolated rat perfused heart MG53 content (n = 10 hearts). Figure 32C shows the perfusion with high concentrations of D-glucose (25 mM) or L-glucose (13.9 mM L-glucose in the presence of 11.1 mM basal D-glucose) (n = 8 / group). MG53 in perfusate in the heart of in vivo rats; data were obtained under basal conditions (0 minutes, 11.1 mM D-glucose) and after 30 minutes of processing. Figure 32D was stimulated with insulin, other conditions were consistent with Figure 32A (n = 11). Figure 32E was stimulated with insulin, other conditions were consistent with Figure 32A (n = 6). Figure 32F shows metabolic stimulation (n = 15) with high glucose (25 mM) combined with high insulin (10 units / liter) (glucose + insulin) in rat hearts perfused in vitro. Figures 32G and 32H show that MG53 in heart perfusion fluid of MG53 transgenic mice is more abundant than wild-type control mice, both in the basal state and the metabolically stimulating state (glucose + insulin, 25 mM glucose combined with 10 units Per liter of insulin) (Fig. 32G), whereas MG53 could not be detected in mg53 ^-/- mouse heart perfusate (Fig. 32H) (n = 8 / group). In all histograms, the data has been normalized to the corresponding non-specific bars (NS) obtained through bright green staining, and expressed as the mean ± standard deviation (* P <0.05, ** P <0.01); AE, G, and H perform one-way ANOVA, and F performs t-test. The control of each histogram is the first lane of the respective gel.

Figure 33: Shows high glucose and high insulin-induced MG53 secretion in vivo and MG53 secretion in vivo. FIG. 33A shows a representative western dot map (upper block) and average data (lower block), which represents the presence or absence of high concentrations of glucose (25 mM) and insulin (10 units / liter) (glucose) + Insulin) treatment (n = 12), MG53 content in perfusate of mouse soleus muscle. Figure 33B shows a representative Western blot map and average data showing that hyperglycemia (glucose, 2 g / kg, intraperitoneal injection, repeated three times at 40-minute intervals) increased serum MG53 (n = 8). Figure 33C shows a representative Western blot map and average data showing serum MG53 from healthy humans before and after oral glucose administration (75 g, twice at 30-minute intervals) (n = 8). Figure 33D shows that BFA dose-dependently inhibits MG53 release in C2C12 cells. Cells were infected with Ad-b-gal or Ad-MG53 and incubated with high glucose (25 mM) for 3 hours (n = 8). Figure 33E shows BFA (30 mM) blocking high glucose (25 mM) combined with high insulin (10 units / liter) (glucose + insulin) -induced increase in MG53 in perfusate in rat perfused heart (n = 10). Figure 33F shows the increase of MG53 in perfusate in rat perfused heart induced by EDTA-Na (0.75 mM) blocking metabolic stress (glucose + insulin, 25 mM glucose combined with 10 units / liter of insulin) (n = 10). Figure 33G shows that metabolic stimulation (glucose + insulin, 25 millimoles of glucose combined with 10 units / liter of insulin) increased MG53 release in wild-type mice, but not in the heart of syt7 ^-/- mice (n = 10). Figure 33H shows that hyperglycemia (glucose, 2 g / kg, intraperitoneal injection, repeated three times at 40-minute intervals) increased serum MG53 in wild-type mice, but not syt7 ^-/- mice (n = 10). In all the lower blocks, the data have been normalized to the corresponding non-specific bars (NS) obtained by bright green staining, and expressed as mean ± standard deviation (* P ＜ 0.05, ** P ＜ 0.01) A performs t-test and BH performs one-way ANOVA.

Figure 34: Shows that cardiac-specific MG53 overexpression leads to systemic insulin resistance and metabolic syndrome. Figure 34A shows representative Western blotting plots and average data showing serum MG53 content (wild type) in 14-week-old wild-type and MG53 heart-specific overexpressing genetically transgenic mice (mg53 h-TG). n = 12, mg53 h-TG n = 14). Figure 34B shows fasting weights of wild-type and mg53 h-TG mice at 3 to 38 weeks of age (wild-type n = 28, mg53 h-TG n = 22). Figure 34C shows a glucose tolerance test in wild-type and mg53 h-TG mice of a specified age (14-week-old, wild-type and mg53 h-TG are both n = 9; 30-week-old, wild-type n = 7, mg53 h-TG n = 6). Figure 34D shows insulin resistance tests in wild-type and mg53 h-TG mice of the specified age (14 weeks of age, wild-type n = 9, mg53 h-TG n = 10; 30-week-old, wild-type n = 8, mg53 h-TG n = 7). Figure 34E shows blood glucose at 30 weeks of age (wild-type n = 11, mg53 h-TG n = 13). Figure 34F shows fasting serum insulin (wild type n = 11, mg53 h-TG n = 13) at 30 weeks of age. Figure 34G shows fasting serum triglyceride content at 30 weeks of age (wild-type n = 11, mg53 h-TG n = 13). Figure 34H shows photographs taken during necropsy of 30-week-old wild-type and mg53 h-TG mice showing fat deposition in the abdominal cavity (wild-type n = 11, mg53 h-TG n = 14). Figure 34I shows the subcutaneous and visceral fat weights of wild-type and mg53 h-TG mice at 30 weeks of age (wild-type n = 11, mg53 h-TG n = 14). Figure 34J shows the brown fat weight of wild-type and mg53 h-TG mice at 30 weeks of age (wild-type n = 11, mg53 h-TG n = 14). Figure 34K shows the body non-fat weight and fat weight of wild-type and mg53 h-TG mice at 30 weeks of age (wild-type n = 11, mg53 h-TG n = 14). Figure 34L shows the body non-fat weight and fat weight as a percentage of body weight of wild-type and mg53 h-TG mice at 30 weeks of age (wild-type n = 11, mg53 h-TG n = 14). Figure 34M shows white and brown fat hematoxylin and eosin staining of wild-type and mg53 h-TG mice at 30 weeks of age (wild-type n = 11, mg53 h-TG n = 14). Figure 34N shows hematoxylin and eosin staining of 30-week-old livers of wild-type and mg53 h-TG mice (wild-type n = 11, mg53 h-TG n = 14). Figure 34O shows hematoxylin and eosin staining of the pancreas of wild-type and mg53 h-TG mice at 30 weeks of age (wild-type n = 11, mg53 h-TG n = 14). Figure 34P shows hematoxylin and eosin staining of skeletal muscle of wild-type and mg53 h-TG mice at 30 weeks of age (wild-type n = 11, mg53 h-TG n = 14). Ruler size: white fat, 50 μm; brown fat, 25 μm, liver, 100 μm; and pancreas, 100 μm. Figure 34Q shows oxygen consumption (n = 12) in wild-type and mg53 h-TG mice at 30 weeks of age. Figure 34R shows CO ₂ production (n = 12) of 30-week-old wild-type and mg53 h-TG mice. Figure 34S shows the daily energy expenditure of wild-type and mg53 h-TG mice at 30 weeks of age (n = 12). Data are mean ± standard deviation. Relative to wild type, * P <0.05, ** P <0.01; t-test.

Figure 35: Shows the release of MG53 from striated muscle by high glucose and high insulin. Figure 35A shows lactate dehydrogenase (LDH, n = 10) in perfusate from mouse hearts stimulated for 30 minutes with or without high glucose (25 mM) and high insulin (10 units / liter). Perfusion fluid. Figure 35A shows creatine kinase (CK, n-8) from perfusate from mouse hearts stimulated with or without high glucose (25 mM) and high insulin (10 units / liter) for 30 minutes. Figure 35C shows the LDH content (n = 10) of perfusate from skeletal muscle (soleus muscle) of mice treated with or without high glucose (25 mM) and high insulin (10 units / liter). Figure 35D shows the CK content (n = 10) of perfusate from skeletal muscle (soleus muscle) of mice treated with or without high glucose (25 mM) and high insulin (10 units / liter). Data are mean ± standard deviation.

Sequence Listing

<110> Peking University

<120> Method and use for detecting or inhibiting extracellular free MG53

<130> 066467-8002CN01

<140> 107138745
<141> 2018-11-01

<160> 61

<170> PatentIn version 3.5

<210> 1
<211> 136
<212> PRT
<213> Rabbit

<400> 1

Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly
1 5 10 15


Val Gln Cys Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro
20 25 30


Gly Thr Pro Leu Thr Leu Thr Cys Thr Ala Ser Gly Ile Asp Leu Ser
35 40 45


Arg Asn Asp Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
50 55 60


Trp Ile Gly Tyr Val Asp Phe Glu Asp Asp Arg Tyr Tyr Ala Ser Trp
65 70 75 80


Val Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp
85 90 95


Leu Lys Met Thr Ser Leu Thr Ser Ala Asp Thr Ala Thr Tyr Tyr Cys
100 105 110


Val Arg Gly Asn Pro Gly Ala Thr Arg Gly Gly Ile Ile Trp Gly Pro
115 120 125


Gly Thr Leu Val Thr Val Ser Ser
130 135


<210> 2
<211> 134
<212> PRT
<213> Rabbit

<400> 2

Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15


Leu Pro Gly Ala Thr Phe Ala Ile Val Val Thr Gln Thr Pro Ser Ser
20 25 30


Lys Ser Val Pro Val Gly Gly Thr Val Thr Ile Ser Cys Gln Ser Ser
35 40 45


Gln Ser Val Tyr Ser Asn Asn Arg Leu Ala Trp Tyr Gln Gln Lys Pro
50 55 60


Gly Gln Pro Pro Lys Leu Leu Ile Tyr Tyr Ala Ser Ile Leu Ala Ser
65 70 75 80


Gly Val Ser Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr
85 90 95


Leu Thr Ile Ser Asp Val Val Cys Asp Asp Ala Ala Thr Tyr Tyr Cys
100 105 110


Ala Gly Tyr Lys Tyr Arg Asp Ser Asp Asn Thr Ala Phe Gly Gly Gly
115 120 125


Thr Glu Val Val Val Lys
130


<210> 3
<211> 459
<212> PRT
<213> Rabbit

<400> 3

Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly
1 5 10 15


Val Gln Cys Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro
20 25 30


Gly Thr Pro Leu Thr Leu Thr Cys Thr Ala Ser Gly Ile Asp Leu Ser
35 40 45


Arg Asn Asp Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
50 55 60


Trp Ile Gly Tyr Val Asp Phe Glu Asp Asp Arg Tyr Tyr Ala Ser Trp
65 70 75 80


Val Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp
85 90 95


Leu Lys Met Thr Ser Leu Thr Ser Ala Asp Thr Ala Thr Tyr Tyr Cys
100 105 110


Val Arg Gly Asn Pro Gly Ala Thr Arg Gly Gly Ile Ile Trp Gly Pro
115 120 125


Gly Thr Leu Val Thr Val Ser Ser Gly Gln Pro Lys Ala Pro Ser Val
130 135 140


Phe Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr Val Thr
145 150 155 160


Leu Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr Val Thr
165 170 175


Trp Asn Ser Gly Thr Leu Thr Asn Gly Val Arg Thr Phe Pro Ser Val
180 185 190


Arg Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Ser Val Thr
195 200 205


Ser Ser Ser Gln Pro Val Thr Cys Asn Val Ala His Pro Ala Thr Asn
210 215 220


Thr Lys Val Asp Lys Thr Val Ala Pro Ser Thr Cys Ser Lys Pro Thr
225 230 235 240


Cys Pro Pro Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro
245 250 255


Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
260 265 270


Cys Val Val Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe Thr
275 280 285


Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Leu Arg
290 295 300


Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile
305 310 315 320


Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val His
325 330 335


Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Arg
340 345 350


Gly Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg Glu
355 360 365


Glu Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly Phe
370 375 380


Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala Glu
385 390 395 400


Asp Asn Tyr Lys Thr Thr Pro Ala Val Leu Asp Ser Asp Gly Ser Tyr
405 410 415


Phe Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg Gly
420 425 430


Asp Val Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
435 440 445


Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys
450 455


<210> 4
<211> 238
<212> PRT
<213> Rabbit

<400> 4

Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15


Leu Pro Gly Ala Thr Phe Ala Ile Val Val Thr Gln Thr Pro Ser Ser
20 25 30


Lys Ser Val Pro Val Gly Gly Thr Val Thr Ile Ser Cys Gln Ser Ser
35 40 45


Gln Ser Val Tyr Ser Asn Asn Arg Leu Ala Trp Tyr Gln Gln Lys Pro
50 55 60


Gly Gln Pro Pro Lys Leu Leu Ile Tyr Tyr Ala Ser Ile Leu Ala Ser
65 70 75 80


Gly Val Ser Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr
85 90 95


Leu Thr Ile Ser Asp Val Val Cys Asp Asp Ala Ala Thr Tyr Tyr Cys
100 105 110


Ala Gly Tyr Lys Tyr Arg Asp Ser Asp Asn Thr Ala Phe Gly Gly Gly
115 120 125


Thr Glu Val Val Val Lys Gly Asp Pro Val Ala Pro Thr Val Leu Ile
130 135 140


Phe Pro Pro Ala Ala Asp Gln Val Ala Thr Gly Thr Val Thr Ile Val
145 150 155 160


Cys Val Ala Asn Lys Tyr Phe Pro Asp Val Thr Val Thr Trp Glu Val
165 170 175


Asp Gly Thr Thr Gln Thr Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln
180 185 190


Asn Ser Ala Asp Cys Thr Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr
195 200 205


Ser Thr Gln Tyr Asn Ser His Lys Glu Tyr Thr Cys Lys Val Thr Gln
210 215 220


Gly Thr Thr Ser Val Val Gln Ser Phe Asn Arg Gly Asp Cys
225 230 235


<210> 5
<211> 408
<212> DNA
<213> Rabbit

<400> 5
atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60

tcgctggagg agtccggggg tcgcctggtc acgcctggga cacccctgac actcacctgc 120

acagcctctg gaatcgacct cagtaggaat gatataaatt gggtccgcca ggctccaggg 180

aaggggctgg aatggatcgg atacgttgat tttgaggatg acagatacta cgcgagctgg 240

gtgaaaggcc gcttcaccat ctccaaaacc tcgtcgacca cggtggatct gaaaatgacc 300

agtctgacaa gcgcggacac ggccacatat tactgtgtca gaggcaatcc tggcgctact 360

cgtggtggaa tcatctgggg cccaggcacc ctggtcaccg tctcctcg 408


<210> 6
<211> 402
<212> DNA
<213> Rabbit

<400> 6
atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60

acatttgcca tcgtggtgac ccagactcca tcttccaagt ctgtccctgt gggaggcaca 120

gtcaccatca gttgccagtc cagtcagagt gtttatagta acaatcgctt agcctggtat 180

cagcagaaac cagggcagcc tcccaagctc ctgatctatt atgcatccat tctggcatct 240

ggggtctcat cgcggttcaa aggcagtgga tctgggacac agttcactct caccatcagc 300

gatgtggtgt gtgacgatgc tgccacttac tactgtgcag gatataaata tcgtgacagt 360

gataatactg ctttcggcgg agggaccgag gtggtggtca aa 402


<210> 7
<211> 1380
<212> DNA
<213> Rabbit

<400> 7
atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60

tcgctggagg agtccggggg tcgcctggtc acgcctggga cacccctgac actcacctgc 120

acagcctctg gaatcgacct cagtaggaat gatataaatt gggtccgcca ggctccaggg 180

aaggggctgg aatggatcgg atacgttgat tttgaggatg acagatacta cgcgagctgg 240

gtgaaaggcc gcttcaccat ctccaaaacc tcgtcgacca cggtggatct gaaaatgacc 300

agtctgacaa gcgcggacac ggccacatat tactgtgtca gaggcaatcc tggcgctact 360

cgtggtggaa tcatctgggg cccaggcacc ctggtcaccg tctcctcggg gcaacctaag 420

gctccatcag tcttcccact ggccccctgc tgcggggaca cacccagctc cacggtgacc 480

ctgggctgcc tggtcaaagg gtacctcccg gagccagtga ccgtgacctg gaactcgggc 540

accctcacca atggggtacg caccttcccg tccgtccggc agtcctcagg cctctactcg 600

ctgagcagcg tggtgagcgt gacctcaagc agccagcccg tcacctgcaa cgtggcccac 660

ccagccacca acaccaaagt ggacaagacc gttgcgccct cgacatgcag caagcccacg 720

tgcccacccc ctgaactcct ggggggaccg tctgtcttca tcttcccccc aaaacccaag 780

gacaccctca tgatctcacg cacccccgag gtcacatgcg tggtggtgga cgtgagccag 840

gatgaccccg aggtgcagtt cacatggtac ataaacaacg agcaggtgcg caccgcccgg 900

ccgccgctac gggagcagca gttcaacagc acgatccgcg tggtcagcac cctccccatc 960

gcgcaccagg actggctgag gggcaaggag ttcaagtgca aagtccacaa caaggcactc 1020

ccggccccca tcgagaaaac catctccaaa gccagagggc agcccctgga gccgaaggtc 1080

tacaccatgg gccctccccg ggaggagctg agcagcaggt cggtcagcct gacctgcatg 1140

atcaacggct tctacccttc cgacatctcg gtggagtggg agaagaacgg gaaggcagag 1200

gacaactaca agaccacgcc ggccgtgctg gacagcgacg gctcctactt cctctacagc 1260

aagctctcag tgcccacgag tgagtggcag cggggcgacg tcttcacctg ctccgtgatg 1320

cacgaggcct tgcacaacca ctacacgcag aagtccatct cccgctctcc gggtaaatga 1380


<210> 8
<211> 717
<212> DNA
<213> Rabbit

<400> 8
atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60

acatttgcca tcgtggtgac ccagactcca tcttccaagt ctgtccctgt gggaggcaca 120

gtcaccatca gttgccagtc cagtcagagt gtttatagta acaatcgctt agcctggtat 180

cagcagaaac cagggcagcc tcccaagctc ctgatctatt atgcatccat tctggcatct 240

ggggtctcat cgcggttcaa aggcagtgga tctgggacac agttcactct caccatcagc 300

gatgtggtgt gtgacgatgc tgccacttac tactgtgcag gatataaata tcgtgacagt 360

gataatactg ctttcggcgg agggaccgag gtggtggtca aaggtgatcc agttgcacct 420

actgtcctca tcttcccacc agctgctgat caggtggcaa ctggaacagt caccatcgtg 480

tgtgtggcga ataaatactt tcccgatgtc accgtcacct gggaggtgga tggcaccacc 540

caaacaactg gcatcgagaa cagtaaaaca ccgcagaatt ctgcagattg tacctacaac 600

ctcagcagca ctctgacact gaccagcaca cagtacaaca gccacaaaga gtacacctgc 660

aaggtgaccc agggcacgac ctcagtcgtc cagagcttca ataggggtga ctgttag 717


<210> 9
<211> 136
<212> PRT
<213> Rabbit

<400> 9

Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly
1 5 10 15


Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro
20 25 30


Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser
35 40 45


Arg Asn Ala Met Ser Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu
50 55 60


Trp Ile Gly Ser Ile Asn Asn Leu Gly Ser Ala Tyr Tyr Ala Ser Trp
65 70 75 80


Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp
85 90 95


Leu Lys Met Thr Ser Leu Thr Ile Glu Asp Thr Ala Thr Tyr Phe Cys
100 105 110


Ala Arg Gly Ser Gly Gly Thr Ser Asn Ser Tyr Asn Ile Trp Gly Pro
115 120 125


Gly Thr Leu Val Thr Val Ser Ser
130 135


<210> 10
<211> 134
<212> PRT
<213> Rabbit

<400> 10

Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15


Leu Pro Gly Ala Thr Phe Ala Ile Val Met Thr Gln Thr Pro Ser Ser
20 25 30


Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser
35 40 45


Gln Thr Ile Tyr Asn Asn Asn Gln Leu Ser Trp Phe Gln Gln Lys Pro
50 55 60


Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Thr Leu Asp Ser
65 70 75 80


Gly Val Ser Ser Arg Phe Lys Gly Ser Gly Ser Gly Ala Gln Phe Thr
85 90 95


Leu Thr Ile Ser Asp Val Val Cys Asp Asp Ala Ala Thr Tyr Tyr Cys
100 105 110


Ala Gly Tyr Lys Ser Ser Ser Thr Asp Gly Ser Ala Phe Gly Gly Gly
115 120 125


Thr Glu Val Val Val Lys
130


<210> 11
<211> 459
<212> PRT
<213> Rabbit

<400> 11

Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly
1 5 10 15


Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro
20 25 30


Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser
35 40 45


Arg Asn Ala Met Ser Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu
50 55 60


Trp Ile Gly Ser Ile Asn Asn Leu Gly Ser Ala Tyr Tyr Ala Ser Trp
65 70 75 80


Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp
85 90 95


Leu Lys Met Thr Ser Leu Thr Ile Glu Asp Thr Ala Thr Tyr Phe Cys
100 105 110


Ala Arg Gly Ser Gly Gly Thr Ser Asn Ser Tyr Asn Ile Trp Gly Pro
115 120 125


Gly Thr Leu Val Thr Val Ser Ser Gly Gln Pro Lys Ala Pro Ser Val
130 135 140


Phe Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr Val Thr
145 150 155 160


Leu Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr Val Thr
165 170 175


Trp Asn Ser Gly Thr Leu Thr Asn Gly Val Arg Thr Phe Pro Ser Val
180 185 190


Arg Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Ser Val Thr
195 200 205


Ser Ser Ser Gln Pro Val Thr Cys Asn Val Ala His Pro Ala Thr Asn
210 215 220


Thr Lys Val Asp Lys Thr Val Ala Pro Ser Thr Cys Ser Lys Pro Thr
225 230 235 240


Cys Pro Pro Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro
245 250 255


Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
260 265 270


Cys Val Val Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe Thr
275 280 285


Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Leu Arg
290 295 300


Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile
305 310 315 320


Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val His
325 330 335


Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Arg
340 345 350


Gly Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg Glu
355 360 365


Glu Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly Phe
370 375 380


Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala Glu
385 390 395 400


Asp Asn Tyr Lys Thr Thr Pro Ala Val Leu Asp Ser Asp Gly Ser Tyr
405 410 415


Phe Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg Gly
420 425 430


Asp Val Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
435 440 445


Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys
450 455


<210> 12
<211> 238
<212> PRT
<213> Rabbit

<400> 12

Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15


Leu Pro Gly Ala Thr Phe Ala Ile Val Met Thr Gln Thr Pro Ser Ser
20 25 30


Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser
35 40 45


Gln Thr Ile Tyr Asn Asn Asn Gln Leu Ser Trp Phe Gln Gln Lys Pro
50 55 60


Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Thr Leu Asp Ser
65 70 75 80


Gly Val Ser Ser Arg Phe Lys Gly Ser Gly Ser Gly Ala Gln Phe Thr
85 90 95


Leu Thr Ile Ser Asp Val Val Cys Asp Asp Ala Ala Thr Tyr Tyr Cys
100 105 110


Ala Gly Tyr Lys Ser Ser Ser Thr Asp Gly Ser Ala Phe Gly Gly Gly
115 120 125


Thr Glu Val Val Val Lys Gly Asp Pro Val Ala Pro Thr Val Leu Ile
130 135 140


Phe Pro Pro Ala Ala Asp Gln Val Ala Thr Gly Thr Val Thr Ile Val
145 150 155 160


Cys Val Ala Asn Lys Tyr Phe Pro Asp Val Thr Val Thr Trp Glu Val
165 170 175


Asp Gly Thr Thr Gln Thr Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln
180 185 190


Asn Ser Ala Asp Cys Thr Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr
195 200 205


Ser Thr Gln Tyr Asn Ser His Lys Glu Tyr Thr Cys Lys Val Thr Gln
210 215 220


Gly Thr Thr Ser Val Val Gln Ser Phe Asn Arg Gly Asp Cys
225 230 235


<210> 13
<211> 408
<212> DNA
<213> Rabbit

<400> 13
atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60

tccgtggagg agtccggggg tcgcctggtc acgcctggga cacccctgac actcacctgc 120

accgtctctg gattctccct cagtcgcaat gcaatgagct gggtccgcca ggctccaggg 180

gaggggctgg aatggatcgg aagtattaat aatcttggta gcgcatacta cgcgagctgg 240

gcaaaaggcc gattcaccat ctccaaaacc tcgtcgacca cggtggatct gaaaatgacc 300

agtctgacaa tcgaggacac ggccacctat ttctgtgcca gaggtagtgg tggtactagc 360

aattcatata acatctgggg cccaggcacc ctggtcaccg tctcctcg 408


<210> 14
<211> 402
<212> DNA
<213> Rabbit

<400> 14
atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60

acatttgcca tcgtgatgac ccagactcca tcctccgtgt ctgcagctgt gggaggcaca 120

gtcaccatca attgccaggc cagtcaaact atttataata acaaccagtt atcgtggttt 180

cagcagaaac cagggcagcc tcccaagctc ctgatctatc tggcatccac tctggattct 240

ggggtctcat cgcggttcaa aggcagtgga tctggggcac agttcactct caccatcagc 300

gatgtggtgt gtgacgatgc tgccacttac tactgtgcag gatataagtc tagtagcact 360

gatggtagtg ctttcggcgg agggaccgag gtggtggtca aa 402


<210> 15
<211> 1380
<212> DNA
<213> Rabbit

<400> 15
atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60

tccgtggagg agtccggggg tcgcctggtc acgcctggga cacccctgac actcacctgc 120

accgtctctg gattctccct cagtcgcaat gcaatgagct gggtccgcca ggctccaggg 180

gaggggctgg aatggatcgg aagtattaat aatcttggta gcgcatacta cgcgagctgg 240

gcaaaaggcc gattcaccat ctccaaaacc tcgtcgacca cggtggatct gaaaatgacc 300

agtctgacaa tcgaggacac ggccacctat ttctgtgcca gaggtagtgg tggtactagc 360

aattcatata acatctgggg cccaggcacc ctggtcaccg tctcctcggg gcaacctaag 420

gctccatcag tcttcccact ggccccctgc tgcggggaca cacccagctc cacggtgacc 480

ctgggctgcc tggtcaaagg gtacctcccg gagccagtga ccgtgacctg gaactcgggc 540

accctcacca atggggtacg caccttcccg tccgtccggc agtcctcagg cctctactcg 600

ctgagcagcg tggtgagcgt gacctcaagc agccagcccg tcacctgcaa cgtggcccac 660

ccagccacca acaccaaagt ggacaagacc gttgcgccct cgacatgcag caagcccacg 720

tgcccacccc ctgaactcct ggggggaccg tctgtcttca tcttcccccc aaaacccaag 780

gacaccctca tgatctcacg cacccccgag gtcacatgcg tggtggtgga cgtgagccag 840

gatgaccccg aggtgcagtt cacatggtac ataaacaacg agcaggtgcg caccgcccgg 900

ccgccgctac gggagcagca gttcaacagc acgatccgcg tggtcagcac cctccccatc 960

gcgcaccagg actggctgag gggcaaggag ttcaagtgca aagtccacaa caaggcactc 1020

ccggccccca tcgagaaaac catctccaaa gccagagggc agcccctgga gccgaaggtc 1080

tacaccatgg gccctccccg ggaggagctg agcagcaggt cggtcagcct gacctgcatg 1140

atcaacggct tctacccttc cgacatctcg gtggagtggg agaagaacgg gaaggcagag 1200

gacaactaca agaccacgcc ggccgtgctg gacagcgacg gctcctactt cctctacagc 1260

aagctctcag tgcccacgag tgagtggcag cggggcgacg tcttcacctg ctccgtgatg 1320

cacgaggcct tgcacaacca ctacacgcag aagtccatct cccgctctcc gggtaaatga 1380


<210> 16
<211> 717
<212> DNA
<213> Rabbit

<400> 16
atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60

acatttgcca tcgtgatgac ccagactcca tcctccgtgt ctgcagctgt gggaggcaca 120

gtcaccatca attgccaggc cagtcaaact atttataata acaaccagtt atcgtggttt 180

cagcagaaac cagggcagcc tcccaagctc ctgatctatc tggcatccac tctggattct 240

ggggtctcat cgcggttcaa aggcagtgga tctggggcac agttcactct caccatcagc 300

gatgtggtgt gtgacgatgc tgccacttac tactgtgcag gatataagtc tagtagcact 360

gatggtagtg ctttcggcgg agggaccgag gtggtggtca aaggtgatcc agttgcacct 420

actgtcctca tcttcccacc agctgctgat caggtggcaa ctggaacagt caccatcgtg 480

tgtgtggcga ataaatactt tcccgatgtc accgtcacct gggaggtgga tggcaccacc 540

caaacaactg gcatcgagaa cagtaaaaca ccgcagaatt ctgcagattg tacctacaac 600

ctcagcagca ctctgacact gaccagcaca cagtacaaca gccacaaaga gtacacctgc 660

aaggtgaccc agggcacgac ctcagtcgtc cagagcttca ataggggtga ctgttag 717


<210> 17
<211> 134
<212> PRT
<213> Rabbit

<400> 17

Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly
1 5 10 15


Val Gln Cys Leu Ser Val Glu Glu Ser Gly Gly Gly Leu Val Gln Pro
20 25 30


Gly Gly Ser Leu Lys Val Thr Cys Thr Ala Ser Gly Phe Asp Leu Ser
35 40 45


Arg Tyr Glu Met Ser Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu
50 55 60


Trp Ile Gly Ile Ile Arg Thr Ser Gly Asn Thr Tyr Tyr Ala Ser Trp
65 70 75 80


Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Ala Asp
85 90 95


Leu Lys Met Thr Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys
100 105 110


Val Arg Gly Gly Pro Gly Ser Gly Ala Ala Asn Trp Gly Pro Gly Thr
115 120 125


Leu Val Thr Val Ser Ser
130


<210> 18
<211> 132
<212> PRT
<213> Rabbit

<400> 18

Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15


Leu Pro Gly Ala Thr Phe Ala Gln Val Leu Thr Gln Thr Pro Ser Ser
20 25 30


Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Ser Cys Gln Ser Ser
35 40 45


Gln Ser Val Tyr Asn Asn Asn Tyr Leu Ser Trp Tyr Gln Gln Lys Pro
50 55 60


Gly Gln Pro Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Thr Leu Pro Ser
65 70 75 80


Gly Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr
85 90 95


Leu Thr Ile Ser Asp Leu Glu Cys Asp Asp Ala Ala Thr Tyr Tyr Cys
100 105 110


Ala Gly Ala Tyr Ser Val Asn Ile Asp Ala Phe Gly Gly Gly Thr Glu
115 120 125


Val Val Val Arg
130


<210> 19
<211> 457
<212> PRT
<213> Rabbit

<400> 19

Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly
1 5 10 15


Val Gln Cys Leu Ser Val Glu Glu Ser Gly Gly Gly Leu Val Gln Pro
20 25 30


Gly Gly Ser Leu Lys Val Thr Cys Thr Ala Ser Gly Phe Asp Leu Ser
35 40 45


Arg Tyr Glu Met Ser Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu
50 55 60


Trp Ile Gly Ile Ile Arg Thr Ser Gly Asn Thr Tyr Tyr Ala Ser Trp
65 70 75 80


Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Ala Asp
85 90 95


Leu Lys Met Thr Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys
100 105 110


Val Arg Gly Gly Pro Gly Ser Gly Ala Ala Asn Trp Gly Pro Gly Thr
115 120 125


Leu Val Thr Val Ser Ser Gly Gln Pro Lys Ala Pro Ser Val Phe Pro
130 135 140


Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr Val Thr Leu Gly
145 150 155 160


Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr Val Thr Trp Asn
165 170 175


Ser Gly Thr Leu Thr Asn Gly Val Arg Thr Phe Pro Ser Val Arg Gln
180 185 190


Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Ser Val Thr Ser Ser
195 200 205


Ser Gln Pro Val Thr Cys Asn Val Ala His Pro Ala Thr Asn Thr Lys
210 215 220


Val Asp Lys Thr Val Ala Pro Ser Thr Cys Ser Lys Pro Thr Cys Pro
225 230 235 240


Pro Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys
245 250 255


Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
260 265 270


Val Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe Thr Trp Tyr
275 280 285


Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Leu Arg Glu Gln
290 295 300


Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile Ala His
305 310 315 320


Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val His Asn Lys
325 330 335


Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Arg Gly Gln
340 345 350


Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg Glu Glu Leu
355 360 365


Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly Phe Tyr Pro
370 375 380


Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala Glu Asp Asn
385 390 395 400


Tyr Lys Thr Thr Pro Ala Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu
405 410 415


Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg Gly Asp Val
420 425 430


Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
435 440 445


Lys Ser Ile Ser Arg Ser Pro Gly Lys
450 455


<210> 20
<211> 236
<212> PRT
<213> Rabbit

<400> 20

Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15


Leu Pro Gly Ala Thr Phe Ala Gln Val Leu Thr Gln Thr Pro Ser Ser
20 25 30


Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Ser Cys Gln Ser Ser
35 40 45


Gln Ser Val Tyr Asn Asn Asn Tyr Leu Ser Trp Tyr Gln Gln Lys Pro
50 55 60


Gly Gln Pro Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Thr Leu Pro Ser
65 70 75 80


Gly Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr
85 90 95


Leu Thr Ile Ser Asp Leu Glu Cys Asp Asp Ala Ala Thr Tyr Tyr Cys
100 105 110


Ala Gly Ala Tyr Ser Val Asn Ile Asp Ala Phe Gly Gly Gly Thr Glu
115 120 125


Val Val Val Arg Gly Asp Pro Val Ala Pro Thr Val Leu Ile Phe Pro
130 135 140


Pro Ala Ala Asp Gln Val Ala Thr Gly Thr Val Thr Ile Val Cys Val
145 150 155 160


Ala Asn Lys Tyr Phe Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly
165 170 175


Thr Thr Gln Thr Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser
180 185 190


Ala Asp Cys Thr Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr
195 200 205


Gln Tyr Asn Ser His Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr
210 215 220


Thr Ser Val Val Gln Ser Phe Asn Arg Gly Asp Cys
225 230 235


<210> 21
<211> 402
<212> DNA
<213> Rabbit

<400> 21
atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtctg 60

tcggtggagg agtccggggg aggcctggtc cagcctgggg gatccctgaa agtcacctgc 120

acagcctctg gattcgacct cagtagatat gaaatgagct gggtccgcca ggctccaggg 180

gaggggctgg aatggatcgg aatcattcgt actagtggta acacatatta cgcgagctgg 240

gcgaaaggcc gattcaccat ctccaaaacc tcgtcgacca cggcggatct gaaaatgacc 300

agtctgacaa ccgaggacac ggccacctat ttctgtgtca gaggtggtcc tggtagtggt 360

gctgctaatt ggggcccagg caccctggtc accgtctcct ca 402


<210> 22
<211> 396
<212> DNA
<213> Rabbit

<400> 22
atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60

acatttgccc aagtgctgac ccagactcca tcctccgtgt ctgcagctgt gggaggcaca 120

gtcaccatca gttgccagtc cagtcagagt gtttataata acaactactt atcctggtat 180

cagcagaaac cagggcagcc tcccaagctc ctgatctatt atacatccac tctgccatct 240

ggggtcccat cgcggttcaa aggcagtgga tctgggacac agttcactct caccatcagc 300

gacctggagt gtgacgatgc tgccacttac tactgtgcag gcgcttatag cgttaatatt 360

gatgctttcg gcggagggac cgaggtggtg gtcaga 396


<210> 23
<211> 1374
<212> DNA
<213> Rabbit

<400> 23
atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtctg 60

tcggtggagg agtccggggg aggcctggtc cagcctgggg gatccctgaa agtcacctgc 120

acagcctctg gattcgacct cagtagatat gaaatgagct gggtccgcca ggctccaggg 180

gaggggctgg aatggatcgg aatcattcgt actagtggta acacatatta cgcgagctgg 240

gcgaaaggcc gattcaccat ctccaaaacc tcgtcgacca cggcggatct gaaaatgacc 300

agtctgacaa ccgaggacac ggccacctat ttctgtgtca gaggtggtcc tggtagtggt 360

gctgctaatt ggggcccagg caccctggtc accgtctcct cagggcaacc taaggctcca 420

tcagtcttcc cactggcccc ctgctgcggg gacacaccca gctccacggt gaccctgggc 480

tgcctggtca aagggtacct cccggagcca gtgaccgtga cctggaactc gggcaccctc 540

accaatgggg tacgcacctt cccgtccgtc cggcagtcct caggcctcta ctcgctgagc 600

agcgtggtga gcgtgacctc aagcagccag cccgtcacct gcaacgtggc ccacccagcc 660

accaacacca aagtggacaa gaccgttgcg ccctcgacat gcagcaagcc cacgtgccca 720

ccccctgaac tcctgggggg accgtctgtc ttcatcttcc ccccaaaacc caaggacacc 780

ctcatgatct cacgcacccc cgaggtcaca tgcgtggtgg tggacgtgag ccaggatgac 840

cccgaggtgc agttcacatg gtacataaac aacgagcagg tgcgcaccgc ccggccgccg 900

ctacgggagc agcagttcaa cagcacgatc cgcgtggtca gcaccctccc catcgcgcac 960

caggactggc tgaggggcaa ggagttcaag tgcaaagtcc acaacaaggc actcccggcc 1020

cccatcgaga aaaccatctc caaagccaga gggcagcccc tggagccgaa ggtctacacc 1080

atgggccctc cccgggagga gctgagcagc aggtcggtca gcctgacctg catgatcaac 1140

ggcttctacc cttccgacat ctcggtggag tgggagaaga acgggaaggc agaggacaac 1200

tacaagacca cgccggccgt gctggacagc gacggctcct acttcctcta cagcaagctc 1260

tcagtgccca cgagtgagtg gcagcggggc gacgtcttca cctgctccgt gatgcacgag 1320

gccttgcaca accactacac gcagaagtcc atctcccgct ctccgggtaa atga 1374


<210> 24
<211> 711
<212> DNA
<213> Rabbit

<400> 24
atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60

acatttgccc aagtgctgac ccagactcca tcctccgtgt ctgcagctgt gggaggcaca 120

gtcaccatca gttgccagtc cagtcagagt gtttataata acaactactt atcctggtat 180

cagcagaaac cagggcagcc tcccaagctc ctgatctatt atacatccac tctgccatct 240

ggggtcccat cgcggttcaa aggcagtgga tctgggacac agttcactct caccatcagc 300

gacctggagt gtgacgatgc tgccacttac tactgtgcag gcgcttatag cgttaatatt 360

gatgctttcg gcggagggac cgaggtggtg gtcagaggtg atccagttgc acctactgtc 420

ctcatcttcc caccagctgc tgatcaggtg gcaactggaa cagtcaccat cgtgtgtgtg 480

gcgaataaat actttcccga tgtcaccgtc acctgggagg tggatggcac cacccaaaca 540

actggcatcg agaacagtaa aacaccgcag aattctgcag attgtaccta caacctcagc 600

agcactctga cactgaccag cacacagtac aacagccaca aagagtacac ctgcaaggtg 660

acccagggca cgacctcagt cgtccagagc ttcaataggg gtgactgtta g 711


<210> 25
<211> 139
<212> PRT
<213> Rabbit

<400> 25

Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly
1 5 10 15


Val Gln Cys Gln Ser Ala Glu Glu Ser Gly Gly Arg Leu Val Thr Pro
20 25 30


Gly Thr Pro Leu Thr Leu Thr Cys Ala Val Ser Gly Ile Ser Leu Ser
35 40 45


Ser Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
50 55 60


Trp Ile Gly Val Ile Tyr Ala Ser Gly Gly Thr Tyr Tyr Ala Ser Trp
65 70 75 80


Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp
85 90 95


Leu Lys Met Thr Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys
100 105 110


Ala Arg Leu Ser Tyr Tyr Thr Tyr Gly His Ala Tyr Leu Lys Asp Ile
115 120 125


Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser
130 135


<210> 26
<211> 135
<212> PRT
<213> Rabbit

<400> 26

Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15


Leu Pro Gly Ala Thr Phe Ala Gln Val Leu Thr Gln Thr Ala Ser Pro
20 25 30


Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ser Ser
35 40 45


Gln Ser Leu Tyr Asn His Asn Ser Leu Ala Trp Phe Gln Gln Lys Ala
50 55 60


Gly Gln Pro Pro Lys Arg Leu Ile Tyr Ser Ala Ser Thr Leu Ala Ser
65 70 75 80


Gly Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr
85 90 95


Leu Thr Ile Ser Asp Val Gln Cys Ala Asp Ala Ala Thr Tyr Tyr Cys
100 105 110


Gln Gly Ser Tyr Asp Cys Ser Ser Ala Asp Cys Thr Ala Phe Gly Gly
115 120 125


Gly Thr Glu Val Val Val Lys
130 135


<210> 27
<211> 462
<212> PRT
<213> Rabbit

<400> 27

Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly
1 5 10 15


Val Gln Cys Gln Ser Ala Glu Glu Ser Gly Gly Arg Leu Val Thr Pro
20 25 30


Gly Thr Pro Leu Thr Leu Thr Cys Ala Val Ser Gly Ile Ser Leu Ser
35 40 45


Ser Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
50 55 60


Trp Ile Gly Val Ile Tyr Ala Ser Gly Gly Thr Tyr Tyr Ala Ser Trp
65 70 75 80


Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp
85 90 95


Leu Lys Met Thr Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys
100 105 110


Ala Arg Leu Ser Tyr Tyr Thr Tyr Gly His Ala Tyr Leu Lys Asp Ile
115 120 125


Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Gly Gln Pro Lys Ala
130 135 140


Pro Ser Val Phe Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser
145 150 155 160


Thr Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val
165 170 175


Thr Val Thr Trp Asn Ser Gly Thr Leu Thr Asn Gly Val Arg Thr Phe
180 185 190


Pro Ser Val Arg Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
195 200 205


Ser Val Thr Ser Ser Ser Gln Pro Val Thr Cys Asn Val Ala His Pro
210 215 220


Ala Thr Asn Thr Lys Val Asp Lys Thr Val Ala Pro Ser Thr Cys Ser
225 230 235 240


Lys Pro Thr Cys Pro Pro Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
245 250 255


Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
260 265 270


Glu Val Thr Cys Val Val Val Asp Val Ser Gln Asp Asp Pro Glu Val
275 280 285


Gln Phe Thr Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro
290 295 300


Pro Leu Arg Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr
305 310 315 320


Leu Pro Ile Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys
325 330 335


Lys Val His Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
340 345 350


Lys Ala Arg Gly Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro
355 360 365


Pro Arg Glu Glu Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile
370 375 380


Asn Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly
385 390 395 400


Lys Ala Glu Asp Asn Tyr Lys Thr Thr Pro Ala Val Leu Asp Ser Asp
405 410 415


Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu Trp
420 425 430


Gln Arg Gly Asp Val Phe Thr Cys Ser Val Met His Glu Ala Leu His
435 440 445


Asn His Tyr Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys
450 455 460


<210> 28
<211> 239
<212> PRT
<213> Rabbit

<400> 28

Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15


Leu Pro Gly Ala Thr Phe Ala Gln Val Leu Thr Gln Thr Ala Ser Pro
20 25 30


Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ser Ser
35 40 45


Gln Ser Leu Tyr Asn His Asn Ser Leu Ala Trp Phe Gln Gln Lys Ala
50 55 60


Gly Gln Pro Pro Lys Arg Leu Ile Tyr Ser Ala Ser Thr Leu Ala Ser
65 70 75 80


Gly Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr
85 90 95


Leu Thr Ile Ser Asp Val Gln Cys Ala Asp Ala Ala Thr Tyr Tyr Cys
100 105 110


Gln Gly Ser Tyr Asp Cys Ser Ser Ala Asp Cys Thr Ala Phe Gly Gly
115 120 125


Gly Thr Glu Val Val Val Lys Gly Asp Pro Val Ala Pro Thr Val Leu
130 135 140


Ile Phe Pro Pro Ala Ala Asp Gln Val Ala Thr Gly Thr Val Thr Ile
145 150 155 160


Val Cys Val Ala Asn Lys Tyr Phe Pro Asp Val Thr Val Thr Trp Glu
165 170 175


Val Asp Gly Thr Thr Gln Thr Thr Gly Ile Glu Asn Ser Lys Thr Pro
180 185 190


Gln Asn Ser Ala Asp Cys Thr Tyr Asn Leu Ser Ser Thr Leu Thr Leu
195 200 205


Thr Ser Thr Gln Tyr Asn Ser His Lys Glu Tyr Thr Cys Lys Val Thr
210 215 220


Gln Gly Thr Thr Ser Val Val Gln Ser Phe Asn Arg Gly Asp Cys
225 230 235


<210> 29
<211> 417
<212> DNA
<213> Rabbit

<400> 29
atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60

tcggcggagg agtccggggg tcgcctggtc acgcctggga cacccctgac actcacctgc 120

gcggtctctg gaatatccct cagtagctat gcaatgaact gggtccgcca ggctccaggg 180

aaggggctgg aatggatcgg agtcatttat gctagtggtg gcacatacta cgcgagctgg 240

gcgaaaggcc gattcaccat ctccaaaacc tcgtcgacca cggtggatct gaaaatgacc 300

agtctgacaa ccgaggacac ggccacctat ttctgtgcaa gattgagtta ctatacttat 360

ggccatgctt atcttaagga catctggggc ccaggcaccc tggtcaccgt ctcctca 417


<210> 30
<211> 405
<212> DNA
<213> Rabbit

<400> 30
atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60

acatttgccc aagtgctgac ccagactgca tcgcccgtgt ctgcagctgt gggaggcaca 120

gtcaccatca agtgccagtc cagtcagagt ctttataatc acaactcctt agcctggttt 180

cagcagaaag cagggcagcc tcccaagcgc ctgatctatt ctgcatccac tctggcatct 240

ggggtcccat cgcggttcaa aggcagtgga tctgggacac agttcactct caccatcagc 300

gacgtgcagt gtgccgatgc tgccacttac tactgtcaag gcagttatga ttgtagtagt 360

gctgattgta ctgctttcgg cggagggacc gaggtggtgg tcaaa 405


<210> 31
<211> 1389
<212> DNA
<213> Rabbit

<400> 31
atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60

tcggcggagg agtccggggg tcgcctggtc acgcctggga cacccctgac actcacctgc 120

gcggtctctg gaatatccct cagtagctat gcaatgaact gggtccgcca ggctccaggg 180

aaggggctgg aatggatcgg agtcatttat gctagtggtg gcacatacta cgcgagctgg 240

gcgaaaggcc gattcaccat ctccaaaacc tcgtcgacca cggtggatct gaaaatgacc 300

agtctgacaa ccgaggacac ggccacctat ttctgtgcaa gattgagtta ctatacttat 360

ggccatgctt atcttaagga catctggggc ccaggcaccc tggtcaccgt ctcctcaggg 420

caacctaagg ctccatcagt cttcccactg gccccctgct gcggggacac acccagctcc 480

acggtgaccc tgggctgcct ggtcaaaggg tacctcccgg agccagtgac cgtgacctgg 540

aactcgggca ccctcaccaa tggggtacgc accttcccgt ccgtccggca gtcctcaggc 600

ctctactcgc tgagcagcgt ggtgagcgtg acctcaagca gccagcccgt cacctgcaac 660

gtggcccacc cagccaccaa caccaaagtg gacaagaccg ttgcgccctc gacatgcagc 720

aagcccacgt gcccaccccc tgaactcctg gggggaccgt ctgtcttcat cttcccccca 780

aaacccaagg acaccctcat gatctcacgc acccccgagg tcacatgcgt ggtggtggac 840

gtgagccagg atgaccccga ggtgcagttc acatggtaca taaacaacga gcaggtgcgc 900

accgcccggc cgccgctacg ggagcagcag ttcaacagca cgatccgcgt ggtcagcacc 960

ctccccatcg cgcaccagga ctggctgagg ggcaaggagt tcaagtgcaa agtccacaac 1020

aaggcactcc cggcccccat cgagaaaacc atctccaaag ccagagggca gcccctggag 1080

ccgaaggtct acaccatggg ccctccccgg gaggagctga gcagcaggtc ggtcagcctg 1140

acctgcatga tcaacggctt ctacccttcc gacatctcgg tggagtggga gaagaacggg 1200

aaggcagagg acaactacaa gaccacgccg gccgtgctgg acagcgacgg ctcctacttc 1260

ctctacagca agctctcagt gcccacgagt gagtggcagc ggggcgacgt cttcacctgc 1320

tccgtgatgc acgaggcctt gcacaaccac tacacgcaga agtccatctc ccgctctccg 1380

ggtaaatga 1389


<210> 32
<211> 720
<212> DNA
<213> Rabbit

<400> 32
atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60

acatttgccc aagtgctgac ccagactgca tcgcccgtgt ctgcagctgt gggaggcaca 120

gtcaccatca agtgccagtc cagtcagagt ctttataatc acaactcctt agcctggttt 180

cagcagaaag cagggcagcc tcccaagcgc ctgatctatt ctgcatccac tctggcatct 240

ggggtcccat cgcggttcaa aggcagtgga tctgggacac agttcactct caccatcagc 300

gacgtgcagt gtgccgatgc tgccacttac tactgtcaag gcagttatga ttgtagtagt 360

gctgattgta ctgctttcgg cggagggacc gaggtggtgg tcaaaggtga tccagttgca 420

cctactgtcc tcatcttccc accagctgct gatcaggtgg caactggaac agtcaccatc 480

gtgtgtgtgg cgaataaata ctttcccgat gtcaccgtca cctgggaggt ggatggcacc 540

acccaaacaa ctggcatcga gaacagtaaa acaccgcaga attctgcaga ttgtacctac 600

aacctcagca gcactctgac actgaccagc acacagtaca acagccacaa agagtacacc 660

tgcaaggtga cccagggcac gacctcagtc gtccagagct tcaatagggg tgactgttag 720


<210> 33
<211> 133
<212> PRT
<213> Rabbit

<400> 33

Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly
1 5 10 15


Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro
20 25 30


Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Leu Ser Leu Ser
35 40 45


Ser Tyr Ala Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
50 55 60


Trp Ile Gly Ile Ile Tyr Ala Ser Gly Lys Thr Tyr Tyr Ala Ser Trp
65 70 75 80


Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu
85 90 95


Lys Val Thr Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala
100 105 110


Arg Asp Arg Asp Tyr Tyr Gly Met Asp Pro Trp Gly Pro Gly Thr Leu
115 120 125


Val Thr Val Ser Ser
130


<210> 34
<211> 135
<212> PRT
<213> Rabbit

<400> 34

Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15


Leu Pro Gly Ala Arg Cys Ala Asp Ile Val Met Thr Gln Thr Pro Ala
20 25 30


Ser Val Glu Ala Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala
35 40 45


Ser Gln Ser Ile Ser Asp Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly
50 55 60


Gln Arg Pro Lys Leu Leu Ile Tyr Glu Ala Ser Lys Leu Ala Ser Gly
65 70 75 80


Val Pro Ser Arg Phe Ser Gly Ser Gly Tyr Gly Thr Glu Phe Thr Leu
85 90 95


Thr Ile Ser Asp Leu Glu Cys Ala Ala Ala Ala Ile Tyr Tyr Cys Gln
100 105 110


Thr Thr Tyr Gly Ser Ser Gly Ala Gly Tyr Tyr Asn Val Phe Gly Gly
115 120 125


Gly Thr Glu Val Val Val Lys
130 135


<210> 35
<211> 456
<212> PRT
<213> Rabbit

<400> 35

Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly
1 5 10 15


Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro
20 25 30


Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Leu Ser Leu Ser
35 40 45


Ser Tyr Ala Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
50 55 60


Trp Ile Gly Ile Ile Tyr Ala Ser Gly Lys Thr Tyr Tyr Ala Ser Trp
65 70 75 80


Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu
85 90 95


Lys Val Thr Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala
100 105 110


Arg Asp Arg Asp Tyr Tyr Gly Met Asp Pro Trp Gly Pro Gly Thr Leu
115 120 125


Val Thr Val Ser Ser Gly Gln Pro Lys Ala Pro Ser Val Phe Pro Leu
130 135 140


Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr Val Thr Leu Gly Cys
145 150 155 160


Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr Val Thr Trp Asn Ser
165 170 175


Gly Thr Leu Thr Asn Gly Val Arg Thr Phe Pro Ser Val Arg Gln Ser
180 185 190


Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Ser Val Thr Ser Ser Ser
195 200 205


Gln Pro Val Thr Cys Asn Val Ala His Pro Ala Thr Asn Thr Lys Val
210 215 220


Asp Lys Thr Val Ala Pro Ser Thr Cys Ser Lys Pro Thr Cys Pro Pro
225 230 235 240


Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro
245 250 255


Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
260 265 270


Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe Thr Trp Tyr Ile
275 280 285


Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Leu Arg Glu Gln Gln
290 295 300


Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile Ala His Gln
305 310 315 320


Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val His Asn Lys Ala
325 330 335


Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Arg Gly Gln Pro
340 345 350


Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg Glu Glu Leu Ser
355 360 365


Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly Phe Tyr Pro Ser
370 375 380


Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala Glu Asp Asn Tyr
385 390 395 400


Lys Thr Thr Pro Ala Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr
405 410 415


Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg Gly Asp Val Phe
420 425 430


Thr Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
435 440 445


Ser Ile Ser Arg Ser Pro Gly Lys
450 455


<210> 36
<211> 239
<212> PRT
<213> Rabbit

<400> 36

Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15


Leu Pro Gly Ala Arg Cys Ala Asp Ile Val Met Thr Gln Thr Pro Ala
20 25 30


Ser Val Glu Ala Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala
35 40 45


Ser Gln Ser Ile Ser Asp Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly
50 55 60


Gln Arg Pro Lys Leu Leu Ile Tyr Glu Ala Ser Lys Leu Ala Ser Gly
65 70 75 80


Val Pro Ser Arg Phe Ser Gly Ser Gly Tyr Gly Thr Glu Phe Thr Leu
85 90 95


Thr Ile Ser Asp Leu Glu Cys Ala Ala Ala Ala Ile Tyr Tyr Cys Gln
100 105 110


Thr Thr Tyr Gly Ser Ser Gly Ala Gly Tyr Tyr Asn Val Phe Gly Gly
115 120 125


Gly Thr Glu Val Val Val Lys Gly Asp Pro Val Ala Pro Thr Val Leu
130 135 140


Ile Phe Pro Pro Ala Ala Asp Gln Val Ala Thr Gly Thr Val Thr Ile
145 150 155 160


Val Cys Val Ala Asn Lys Tyr Phe Pro Asp Val Thr Val Thr Trp Glu
165 170 175


Val Asp Gly Thr Thr Gln Thr Thr Gly Ile Glu Asn Ser Lys Thr Pro
180 185 190


Gln Asn Ser Ala Asp Cys Thr Tyr Asn Leu Ser Ser Thr Leu Thr Leu
195 200 205


Thr Ser Thr Gln Tyr Asn Ser His Lys Glu Tyr Thr Cys Lys Val Thr
210 215 220


Gln Gly Thr Thr Ser Val Val Gln Ser Phe Asn Arg Gly Asp Cys
225 230 235


<210> 37
<211> 399
<212> DNA
<213> Rabbit

<400> 37
atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60

tcggtggagg agtccggggg tcgcctggtc acgcctggga cacccctgac actcacctgc 120

acagtctctg gattgtccct cagtagctat gcagtgagct gggtccgcca ggctccaggg 180

aaggggctgg aatggatcgg aatcatttat gctagtggta aaacatacta cgcgagctgg 240

gcgaaaggcc gattcaccat ctccaaaacc tcgaccacgg tggatctgaa ggtgaccagt 300

ctgacaaccg aggacacggc cacctatttc tgtgccagag accgagatta ttacggcatg 360

gacccctggg gcccagggac cctcgtcacc gtctcttca 399


<210> 38
<211> 405
<212> DNA
<213> Rabbit

<400> 38
atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60

agatgtgctg acattgtgat gacccagact ccagcctccg tggaggcagc tgtgggaggc 120

acagtcacca tcaagtgcca ggccagtcag agcattagtg actacttatc ctggtatcag 180

cagaaaccag ggcagcgtcc caagctcctg atctatgaag catccaagct ggcctctggg 240

gtcccgtcgc ggttcagcgg cagtggatat gggacagagt tcactctcac catcagcgac 300

ctggagtgtg ccgctgctgc catttactac tgtcaaacca cttatggtag tagtggtgct 360

ggttattata atgtttttgg cggagggacc gaggtggtgg tcaaa 405


<210> 39
<211> 1371
<212> DNA
<213> Rabbit

<400> 39
atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60

tcggtggagg agtccggggg tcgcctggtc acgcctggga cacccctgac actcacctgc 120

acagtctctg gattgtccct cagtagctat gcagtgagct gggtccgcca ggctccaggg 180

aaggggctgg aatggatcgg aatcatttat gctagtggta aaacatacta cgcgagctgg 240

gcgaaaggcc gattcaccat ctccaaaacc tcgaccacgg tggatctgaa ggtgaccagt 300

ctgacaaccg aggacacggc cacctatttc tgtgccagag accgagatta ttacggcatg 360

gacccctggg gcccagggac cctcgtcacc gtctcttcag ggcaacctaa ggctccatca 420

gtcttcccac tggccccctg ctgcggggac acacccagct ccacggtgac cctgggctgc 480

ctggtcaaag ggtacctccc ggagccagtg accgtgacct ggaactcggg caccctcacc 540

aatggggtac gcaccttccc gtccgtccgg cagtcctcag gcctctactc gctgagcagc 600

gtggtgagcg tgacctcaag cagccagccc gtcacctgca acgtggccca cccagccacc 660

aacaccaaag tggacaagac cgttgcgccc tcgacatgca gcaagcccac gtgcccaccc 720

cctgaactcc tggggggacc gtctgtcttc atcttccccc caaaacccaa ggacaccctc 780

atgatctcac gcacccccga ggtcacatgc gtggtggtgg acgtgagcca ggatgacccc 840

gaggtgcagt tcacatggta cataaacaac gagcaggtgc gcaccgcccg gccgccgcta 900

cgggagcagc agttcaacag cacgatccgc gtggtcagca ccctccccat cgcgcaccag 960

gactggctga ggggcaagga gttcaagtgc aaagtccaca acaaggcact cccggccccc 1020

atcgagaaaa ccatctccaa agccagaggg cagcccctgg agccgaaggt ctacaccatg 1080

ggccctcccc gggaggagct gagcagcagg tcggtcagcc tgacctgcat gatcaacggc 1140

ttctaccctt ccgacatctc ggtggagtgg gagaagaacg ggaaggcaga ggacaactac 1200

aagaccacgc cggccgtgct ggacagcgac ggctcctact tcctctacag caagctctca 1260

gtgcccacga gtgagtggca gcggggcgac gtcttcacct gctccgtgat gcacgaggcc 1320

ttgcacaacc actacacgca gaagtccatc tcccgctctc cgggtaaatg a 1371


<210> 40
<211> 720
<212> DNA
<213> Rabbit

<400> 40
atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60

agatgtgctg acattgtgat gacccagact ccagcctccg tggaggcagc tgtgggaggc 120

acagtcacca tcaagtgcca ggccagtcag agcattagtg actacttatc ctggtatcag 180

cagaaaccag ggcagcgtcc caagctcctg atctatgaag catccaagct ggcctctggg 240

gtcccgtcgc ggttcagcgg cagtggatat gggacagagt tcactctcac catcagcgac 300

ctggagtgtg ccgctgctgc catttactac tgtcaaacca cttatggtag tagtggtgct 360

ggttattata atgtttttgg cggagggacc gaggtggtgg tcaaaggtga tccagttgca 420

cctactgtcc tcatcttccc accagctgct gatcaggtgg caactggaac agtcaccatc 480

gtgtgtgtgg cgaataaata ctttcccgat gtcaccgtca cctgggaggt ggatggcacc 540

acccaaacaa ctggcatcga gaacagtaaa acaccgcaga attctgcaga ttgtacctac 600

aacctcagca gcactctgac actgaccagc acacagtaca acagccacaa agagtacacc 660

tgcaaggtga cccagggcac gacctcagtc gtccagagct tcaatagggg tgactgttag 720


<210> 41
<211> 477
<212> PRT
<213> Homo sapiens

<400> 41

Met Ser Ala Ala Pro Gly Leu Leu His Gln Glu Leu Ser Cys Pro Leu
1 5 10 15


Cys Leu Gln Leu Phe Asp Ala Pro Val Thr Ala Glu Cys Gly His Ser
20 25 30


Phe Cys Arg Ala Cys Leu Gly Arg Val Ala Gly Glu Pro Ala Ala Asp
35 40 45


Gly Thr Val Leu Cys Pro Cys Cys Gln Ala Pro Thr Arg Pro Gln Ala
50 55 60


Leu Ser Thr Asn Leu Gln Leu Ala Arg Leu Val Glu Gly Leu Ala Gln
65 70 75 80


Val Pro Gln Gly His Cys Glu Glu His Leu Asp Pro Leu Ser Ile Tyr
85 90 95


Cys Glu Gln Asp Arg Ala Leu Val Cys Gly Val Cys Ala Ser Leu Gly
100 105 110


Ser His Arg Gly His Arg Leu Leu Pro Ala Ala Glu Ala His Ala Arg
115 120 125


Leu Lys Thr Gln Leu Pro Gln Gln Lys Leu Gln Leu Gln Glu Ala Cys
130 135 140


Met Arg Lys Glu Lys Ser Val Ala Val Leu Glu His Gln Leu Val Glu
145 150 155 160


Val Glu Glu Thr Val Arg Gln Phe Arg Gly Ala Val Gly Glu Gln Leu
165 170 175


Gly Lys Met Arg Val Phe Leu Ala Ala Leu Glu Gly Ser Leu Asp Arg
180 185 190


Glu Ala Glu Arg Val Arg Gly Glu Ala Gly Val Ala Leu Arg Arg Glu
195 200 205


Leu Gly Ser Leu Asn Ser Tyr Leu Glu Gln Leu Arg Gln Met Glu Lys
210 215 220


Val Leu Glu Glu Val Ala Asp Lys Pro Gln Thr Glu Phe Leu Met Lys
225 230 235 240


Tyr Cys Leu Val Thr Ser Arg Leu Gln Lys Ile Leu Ala Glu Ser Pro
245 250 255


Pro Pro Ala Arg Leu Asp Ile Gln Leu Pro Ile Ile Ser Asp Asp Phe
260 265 270


Lys Phe Gln Val Trp Arg Lys Met Phe Arg Ala Leu Met Pro Ala Leu
275 280 285


Glu Glu Leu Thr Phe Asp Pro Ser Ser Ala His Pro Ser Leu Val Val
290 295 300


Ser Ser Ser Gly Arg Arg Val Glu Cys Ser Glu Gln Lys Ala Pro Pro
305 310 315 320


Ala Gly Glu Asp Pro Arg Gln Phe Asp Lys Ala Val Ala Val Val Ala
325 330 335


His Gln Gln Leu Ser Glu Gly Glu His Tyr Trp Glu Val Asp Val Gly
340 345 350


Asp Lys Pro Arg Trp Ala Leu Gly Val Ile Ala Ala Glu Ala Pro Arg
355 360 365


Arg Gly Arg Leu His Ala Val Pro Ser Gln Gly Leu Trp Leu Leu Gly
370 375 380


Leu Arg Glu Gly Lys Ile Leu Glu Ala His Val Glu Ala Lys Glu Pro
385 390 395 400


Arg Ala Leu Arg Ser Pro Glu Arg Arg Pro Thr Arg Ile Gly Leu Tyr
405 410 415


Leu Ser Phe Gly Asp Gly Val Leu Ser Phe Tyr Asp Ala Ser Asp Ala
420 425 430


Asp Ala Leu Val Pro Leu Phe Ala Phe His Glu Arg Leu Pro Arg Pro
435 440 445


Val Tyr Pro Phe Phe Asp Val Cys Trp His Asp Lys Gly Lys Asn Ala
450 455 460


Gln Pro Leu Leu Leu Val Gly Pro Glu Gly Ala Glu Ala
465 470 475


<210> 42
<211> 477
<212> PRT
<213> Rhesus / Crab-eating monkey

<400> 42

Met Ser Ala Ala Pro Gly Leu Leu His Gln Glu Leu Ser Cys Pro Leu
1 5 10 15


Cys Leu Gln Leu Phe Asp Ala Pro Val Thr Ala Glu Cys Gly His Ser
20 25 30


Phe Cys Arg Ala Cys Leu Gly Arg Val Ala Gly Glu Pro Ala Ala Asp
35 40 45


Gly Thr Val Leu Cys Pro Cys Cys Gln Ala Pro Thr Arg Pro Gln Ala
50 55 60


Leu Ser Thr Asn Leu Gln Leu Ala Arg Leu Val Glu Gly Leu Ala Gln
65 70 75 80


Val Pro Gln Gly His Cys Glu Glu His Leu Asp Pro Leu Ser Ile Tyr
85 90 95


Cys Glu Gln Asp Arg Ala Leu Val Cys Gly Val Cys Ala Ser Leu Gly
100 105 110


Ser His Arg Gly His Arg Leu Leu Pro Ala Ala Glu Ala His Ala Arg
115 120 125


Leu Lys Thr Gln Leu Pro Gln Gln Lys Leu Gln Leu Gln Glu Ala Cys
130 135 140


Met Arg Lys Glu Lys Ser Val Ala Val Leu Glu His Gln Leu Val Glu
145 150 155 160


Val Glu Glu Thr Val Arg Gln Phe Arg Gly Ala Val Gly Glu Gln Leu
165 170 175


Gly Lys Met Arg Val Phe Leu Ala Ala Leu Glu Gly Ser Leu Asp Arg
180 185 190


Glu Ala Glu Arg Val Arg Gly Glu Ala Gly Val Ala Leu Arg Arg Glu
195 200 205


Leu Gly Ser Leu Asn Ser Tyr Leu Glu Gln Leu Arg Gln Met Glu Lys
210 215 220


Val Leu Glu Glu Val Ala Asp Lys Pro Gln Thr Glu Phe Leu Met Lys
225 230 235 240


Tyr Cys Leu Val Thr Ser Arg Leu Gln Lys Ile Leu Ala Glu Ser Pro
245 250 255


Pro Pro Ala Arg Leu Asp Ile Gln Leu Pro Ile Ile Ser Asp Asp Phe
260 265 270


Lys Phe Gln Val Trp Arg Lys Met Phe Arg Ala Leu Met Pro Ala Leu
275 280 285


Glu Glu Leu Thr Phe Asp Pro Ser Ser Ala His Pro Ser Leu Val Val
290 295 300


Ser Ser Ser Gly Arg Arg Val Glu Cys Ser Glu Gln Lys Ala Pro Pro
305 310 315 320


Ala Gly Glu Asp Pro Arg Gln Phe Asp Lys Ala Val Ala Val Val Ala
325 330 335


His Gln Gln Leu Ser Glu Gly Glu His Tyr Trp Glu Val Glu Val Gly
340 345 350


Asp Lys Pro Arg Trp Ala Leu Gly Val Ile Ala Ala Glu Gly Pro Arg
355 360 365


Arg Gly Arg Leu His Ala Val Pro Ser Gln Gly Leu Trp Leu Leu Gly
370 375 380


Leu Arg Glu Gly Lys Ile Leu Glu Ala His Val Glu Ala Lys Glu Pro
385 390 395 400


Arg Ala Leu Arg Ser Pro Glu Arg Arg Pro Thr Arg Ile Gly Leu Tyr
405 410 415


Leu Ser Phe Gly Asp Gly Val Leu Ser Phe Tyr Asp Ala Ser Asp Ala
420 425 430


Asp Ala Leu Val Pro Leu Phe Ala Phe His Glu Arg Leu Pro Gly Pro
435 440 445


Val Tyr Pro Phe Phe Asp Val Cys Trp His Asp Lys Gly Lys Asn Ser
450 455 460


Gln Pro Leu Leu Leu Val Gly Ser Glu Gly Ala Glu Ala
465 470 475


<210> 43
<211> 477
<212> PRT
<213> Rat

<400> 43

Met Ser Thr Ala Pro Gly Leu Leu Arg Gln Glu Leu Ser Cys Pro Leu
1 5 10 15


Cys Leu Gln Leu Phe Asp Ala Pro Val Thr Ala Glu Cys Gly His Ser
20 25 30


Phe Cys Arg Ala Cys Leu Ile Arg Val Ala Gly Glu Pro Ala Asp Asp
35 40 45


Gly Thr Val Ala Cys Pro Cys Cys Gln Ala Ser Thr Arg Pro Gln Ala
50 55 60


Leu Ser Thr Asn Leu Gln Leu Ala Arg Leu Val Glu Gly Leu Ala Gln
65 70 75 80


Val Pro Gln Gly His Cys Glu Glu His Leu Asp Pro Leu Ser Ile Tyr
85 90 95


Cys Glu Gln Asp Arg Thr Leu Val Cys Gly Val Cys Ala Ser Leu Gly
100 105 110


Ser His Arg Gly His Arg Leu Leu Pro Ala Ala Glu Ala His Ala Arg
115 120 125


Leu Lys Thr Gln Leu Pro Gln Gln Lys Ala Gln Leu Gln Glu Ala Cys
130 135 140


Met Arg Lys Glu Lys Ser Val Ala Val Leu Glu His Gln Leu Val Glu
145 150 155 160


Val Glu Glu Thr Val Arg Gln Phe Arg Gly Ala Val Gly Glu Gln Leu
165 170 175


Gly Lys Met Arg Met Phe Leu Ala Ala Leu Glu Ser Ser Leu Asp Arg
180 185 190


Glu Ala Glu Arg Val Arg Gly Glu Ala Gly Val Ala Leu Arg Arg Glu
195 200 205


Leu Ser Ser Leu Asn Ser Tyr Leu Glu Gln Leu Arg Gln Met Glu Lys
210 215 220


Val Leu Glu Glu Val Ala Asp Lys Pro Gln Thr Glu Phe Leu Met Lys
225 230 235 240


Phe Cys Leu Val Thr Ser Arg Leu Gln Lys Ile Leu Ser Glu Ser Pro
245 250 255


Pro Pro Ala Arg Leu Asp Ile Gln Leu Pro Val Ile Ser Asp Asp Phe
260 265 270


Lys Phe Gln Val Trp Lys Lys Met Phe Arg Ala Leu Met Pro Glu Leu
275 280 285


Glu Glu Leu Thr Phe Asp Pro Ser Ser Ala His Pro Ser Leu Val Val
290 295 300


Ser Ala Ser Gly Arg Arg Val Glu Cys Ser Glu Gln Lys Ala Pro Pro
305 310 315 320


Ala Gly Glu Asp Thr Cys Gln Phe Asp Lys Thr Val Ala Val Val Ala
325 330 335


Lys Gln Leu Leu Ser Gln Gly Glu His Tyr Trp Glu Val Glu Val Gly
340 345 350


Asp Lys Pro Arg Trp Ala Leu Gly Val Met Ala Ala Asp Ala Ser Arg
355 360 365


Arg Gly Arg Leu His Ala Val Pro Ser Gln Gly Leu Trp Leu Leu Gly
370 375 380


Leu Arg Asp Gly Lys Ile Leu Glu Ala His Val Glu Ala Lys Glu Pro
385 390 395 400


Arg Ala Leu Arg Thr Pro Glu Arg Pro Pro Ala Arg Ile Gly Leu Tyr
405 410 415


Leu Ser Phe Ala Asp Gly Val Leu Thr Phe Tyr Asp Ala Ser Asn Thr
420 425 430


Asp Ala Leu Thr Pro Leu Phe Ser Phe His Glu Arg Leu Pro Gly Pro
435 440 445


Val Tyr Pro Met Phe Asp Val Cys Trp His Asp Lys Gly Lys Asn Ser
450 455 460


Gln Pro Leu Leu Leu Val Gly Pro Asp Ser Glu Gln Ala
465 470 475


<210> 44
<211> 477
<212> PRT
<213> mouse

<400> 44

Met Ser Ala Ala Pro Gly Leu Leu Arg Gln Glu Leu Ser Cys Pro Leu
1 5 10 15


Cys Leu Gln Leu Phe Asp Ala Pro Val Thr Ala Glu Cys Gly His Ser
20 25 30


Phe Cys Arg Ala Cys Leu Ile Arg Val Ala Gly Glu Pro Ala Ala Asp
35 40 45


Gly Thr Val Ala Cys Pro Cys Cys Gln Ala Pro Thr Arg Pro Gln Ala
50 55 60


Leu Ser Thr Asn Leu Gln Leu Ser Arg Leu Val Glu Gly Leu Ala Gln
65 70 75 80


Val Pro Gln Gly His Cys Glu Glu His Leu Asp Pro Leu Ser Ile Tyr
85 90 95


Cys Glu Gln Asp Arg Thr Leu Val Cys Gly Val Cys Ala Ser Leu Gly
100 105 110


Ser His Arg Gly His Arg Leu Leu Pro Ala Ala Glu Ala Gln Ala Arg
115 120 125


Leu Lys Thr Gln Leu Pro Gln Gln Lys Met Gln Leu Gln Glu Ala Cys
130 135 140


Met Arg Lys Glu Lys Thr Val Ala Val Leu Glu His Gln Leu Val Glu
145 150 155 160


Val Glu Glu Thr Val Arg Gln Phe Arg Gly Ala Val Gly Glu Gln Leu
165 170 175


Gly Lys Met Arg Met Phe Leu Ala Ala Leu Glu Ser Ser Leu Asp Arg
180 185 190


Glu Ala Glu Arg Val Arg Gly Asp Ala Gly Val Ala Leu Arg Arg Glu
195 200 205


Leu Ser Ser Leu Asn Ser Tyr Leu Glu Gln Leu Arg Gln Met Glu Lys
210 215 220


Val Leu Glu Glu Val Ala Asp Lys Pro Gln Thr Glu Phe Leu Met Lys
225 230 235 240


Phe Cys Leu Val Thr Ser Arg Leu Gln Lys Ile Leu Ser Glu Ser Pro
245 250 255


Pro Pro Ala Arg Leu Asp Ile Gln Leu Pro Val Ile Ser Asp Asp Phe
260 265 270


Lys Phe Gln Val Trp Lys Lys Met Phe Arg Ala Leu Met Pro Ala Leu
275 280 285


Glu Glu Leu Thr Phe Asp Pro Ser Ser Ala His Pro Ser Leu Val Val
290 295 300


Ser Ser Ser Gly Arg Arg Val Glu Cys Ser Asp Gln Lys Ala Pro Pro
305 310 315 320


Ala Gly Glu Asp Thr Arg Gln Phe Asp Lys Ala Val Ala Val Val Ala
325 330 335


Gln Gln Leu Leu Ser Gln Gly Glu His Tyr Trp Glu Val Glu Val Gly
340 345 350


Asp Lys Pro Arg Trp Ala Leu Gly Val Met Ala Ala Asp Ala Ser Arg
355 360 365


Arg Gly Arg Leu His Ala Val Pro Ser Gln Gly Leu Trp Leu Leu Gly
370 375 380


Leu Arg Asp Gly Lys Ile Leu Glu Ala His Val Glu Ala Lys Glu Pro
385 390 395 400


Arg Ala Leu Arg Thr Pro Glu Arg Pro Pro Ala Arg Ile Gly Leu Tyr
405 410 415


Leu Ser Phe Ala Asp Gly Val Leu Ala Phe Tyr Asp Ala Ser Asn Pro
420 425 430


Asp Val Leu Thr Pro Ile Phe Ser Phe His Glu Arg Leu Pro Gly Pro
435 440 445


Val Tyr Pro Ile Phe Asp Val Cys Trp His Asp Lys Gly Lys Asn Ala
450 455 460


Gln Pro Leu Leu Leu Val Gly Pro Glu Gln Glu Gln Ala
465 470 475


<210> 45
<211> 478
<212> PRT
<213> Pig

<400> 45

Met Ser Ala Ala Pro Gly Leu Leu His Gln Glu Leu Ser Cys Pro Leu
1 5 10 15


Cys Leu Gln Leu Phe Asp Ala Pro Val Thr Ala Glu Cys Gly His Ser
20 25 30


Phe Cys Arg Ala Cys Leu Gly Arg Val Ala Gly Glu Pro Ala Ala Asp
35 40 45


Gly Thr Val Leu Cys Pro Ser Cys Gln Ala Pro Thr Arg Pro Gln Ala
50 55 60


Leu Ser Thr Asn Gln Gln Leu Ala Arg Leu Val Glu Gly Leu Ala Gln
65 70 75 80


Val Pro Gln Gly His Cys Glu Glu His Leu Asp Pro Leu Ser Ile Tyr
85 90 95


Cys Glu Gln Asp Arg Val Leu Val Cys Gly Val Cys Ala Ser Leu Gly
100 105 110


Ser His Arg Gly His Arg Leu Leu Pro Ala Ala Glu Ala His Ala Arg
115 120 125


Leu Lys Thr Gln Leu Pro Gln Gln Lys Leu Gln Leu Gln Glu Ala Cys
130 135 140


Met Arg Lys Glu Lys Ser Val Gly Val Leu Glu Gln Gln Leu Val Glu
145 150 155 160


Val Glu Glu Thr Val Arg Gln Phe Arg Gly Ala Val Gly Glu Gln Leu
165 170 175


Gly Lys Met Arg Leu Phe Leu Ala Ala Leu Glu Gly Ser Leu Asp Arg
180 185 190


Glu Ala Glu Arg Val Arg Gly Glu Ala Gly Val Ala Leu Arg Arg Glu
195 200 205


Leu Gly Ser Leu Lys Ser Tyr Leu Glu Gln Leu Arg Gln Met Glu Lys
210 215 220


Val Leu Glu Glu Val Ala Asp Lys Pro Gln Thr Glu Phe Leu Met Lys
225 230 235 240


Tyr Cys Leu Val Thr Ser Arg Leu Gln Lys Ile Leu Ala Glu Ser Pro
245 250 255


Pro Pro Ala Arg Leu Asp Ile Gln Leu Pro Val Ile Ser Asp Asp Phe
260 265 270


Lys Phe Gln Val Trp Arg Lys Met Phe Arg Ala Leu Met Pro Ala Met
275 280 285


Gln Glu Leu Thr Phe Asp Pro Ser Thr Ala His Pro Ser Leu Val Leu
290 295 300


Ser Ala Ser Gly Arg Arg Val Glu Cys Ser Glu Gln Lys Ala Pro Pro
305 310 315 320


Ala Gly Glu Asp Pro Arg Gln Phe Asp Lys Ala Val Ala Val Val Thr
325 330 335


His Gln Leu Leu Ser Glu Gly Glu His Tyr Trp Glu Val Glu Val Gly
340 345 350


Asp Lys Pro Arg Trp Ala Leu Gly Val Ile Gly Ala Gln Ala Gly Arg
355 360 365


Arg Gly Arg Leu His Ala Val Pro Ser Gln Gly Leu Trp Leu Leu Gly
370 375 380


Leu Arg Glu Gly Lys Ile Leu Glu Ala His Val Glu Ala Lys Glu Pro
385 390 395 400


Arg Ala Leu Arg Thr Pro Glu Arg Arg Pro Ser Arg Ile Gly Ile Tyr
405 410 415


Leu Ser Phe Ala Asp Gly Val Leu Ser Phe Tyr Asp Ala Ser Asp Ala
420 425 430


Asp Ala Leu Glu Leu Leu Phe Ala Phe His Glu Arg Leu Pro Gly Pro
435 440 445


Val Tyr Pro Phe Phe Asp Val Cys Trp His Asp Lys Gly Lys Asn Ala
450 455 460


Gln Pro Leu Leu Leu Val Gly Pro Asp Ser Gly Gly Glu Ala
465 470 475


<210> 46
<211> 477
<212> PRT
<213> Dog

<400> 46

Met Ser Ala Ala Pro Gly Leu Leu His Gln Glu Leu Ser Cys Pro Leu
1 5 10 15


Cys Leu Gln Leu Phe Asp Ala Pro Val Thr Ala Glu Cys Gly His Ser
20 25 30


Phe Cys Arg Ala Cys Leu Ser Arg Val Ala Gly Glu Pro Ala Ala Asp
35 40 45


Gly Thr Val Pro Cys Pro Cys Cys Gln Ala Leu Thr Arg Pro Gln Ala
50 55 60


Leu Ser Thr Asn Gln Gln Leu Ala Arg Leu Val Glu Gly Leu Ala Gln
65 70 75 80


Val Pro Gln Gly His Cys Glu Glu His Leu Asp Pro Leu Ser Ile Tyr
85 90 95


Cys Glu Gln Asp Arg Ala Leu Val Cys Gly Val Cys Ala Ser Leu Gly
100 105 110


Ser His Arg Gly His Arg Leu Leu Pro Ala Ala Glu Ala His Ala Arg
115 120 125


Leu Lys Thr Gln Leu Pro Gln Gln Lys Leu Gln Leu Gln Glu Ala Cys
130 135 140


Met Arg Lys Glu Lys Ser Val Ala Leu Leu Glu His Gln Leu Met Glu
145 150 155 160


Val Glu Glu Met Val Arg Gln Phe Arg Gly Ala Val Gly Glu Gln Leu
165 170 175


Gly Lys Met Arg Val Phe Leu Ala Ala Leu Glu Gly Ser Leu Asp Arg
180 185 190


Glu Ala Glu Arg Val Arg Gly Glu Ala Gly Val Ala Leu Arg Arg Glu
195 200 205


Leu Gly Ser Leu Asn Ser Tyr Leu Glu Gln Leu Arg Gln Met Glu Lys
210 215 220


Val Leu Glu Glu Val Ala Asp Lys Pro Gln Thr Glu Phe Leu Met Lys
225 230 235 240


Tyr Cys Leu Val Thr Ser Arg Leu Gln Lys Ile Leu Ala Glu Ser Pro
245 250 255


Pro Pro Ala Arg Leu Asp Ile Gln Leu Pro Val Ile Ser Asp Asp Phe
260 265 270


Lys Phe Gln Val Trp Arg Lys Met Phe Arg Ala Leu Met Pro Val Thr
275 280 285


Lys Glu Leu Thr Phe Asp Pro Ser Ser Ala His Pro Ser Leu Val Leu
290 295 300


Ser Pro Ser Gly Arg Arg Val Glu Cys Ser Asp Gln Lys Ala Pro Pro
305 310 315 320


Ala Gly Glu Asp Pro Cys Gln Phe Asp Lys Ala Val Ala Val Val Ala
325 330 335


Gln Gln Val Leu Ser Asp Gly Glu His Tyr Trp Glu Val Gln Val Gly
340 345 350


Glu Lys Pro Arg Trp Ala Leu Gly Val Ile Ala Ala Gln Ala Ser Arg
355 360 365


Arg Gly Arg Leu His Ala Val Pro Ser Gln Gly Leu Trp Leu Leu Gly
370 375 380


Leu Arg Asp Gly Lys Ile Leu Glu Ala His Val Glu Ala Lys Glu Pro
385 390 395 400


Arg Ala Leu Arg Thr Pro Glu Arg Arg Pro Thr Arg Ile Gly Ile Tyr
405 410 415


Leu Ser Phe Gly Asp Gly Val Leu Ser Phe Tyr Asp Ala Ser Asp Pro
420 425 430


Asp Ala Leu Glu Leu Leu Phe Ala Phe His Glu Arg Leu Pro Gly Pro
435 440 445


Val Tyr Pro Phe Phe Asp Val Cys Trp His Asp Lys Gly Lys Asn Ala
450 455 460


Gln Pro Leu Leu Leu Val Gly Pro Asp Gly Glu Glu Ala
465 470 475


<210> 47
<211> 477
<212> PRT
<213> Rabbit

<400> 47

Met Ser Ala Ala Pro Gly Leu Leu His Gln Glu Leu Ser Cys Pro Leu
1 5 10 15


Cys Leu Gln Leu Phe Asp Ala Pro Val Thr Ala Glu Cys Gly His Ser
20 25 30


Phe Cys Arg Ala Cys Leu Ser Arg Val Ala Gly Glu Pro Ala Ala Asp
35 40 45


Gly Thr Val Asn Cys Pro Cys Cys Gln Ala Pro Thr Arg Pro Gln Ala
50 55 60


Leu Ser Thr Asn Leu Gln Leu Ala Arg Leu Val Glu Gly Leu Ala Gln
65 70 75 80


Val Pro Gln Gly His Cys Glu Glu His Leu Asp Pro Leu Ser Ile Tyr
85 90 95


Cys Glu Gln Asp Arg Val Leu Val Cys Gly Val Cys Ala Ser Leu Gly
100 105 110


Ser His Arg Gly His Arg Leu Leu Pro Ala Ala Glu Ala His Ser Arg
115 120 125


Leu Lys Thr Gln Leu Pro Gln Gln Lys Leu Gln Leu Gln Glu Ala Ser
130 135 140


Met Arg Lys Glu Lys Ser Val Ala Val Leu Glu His Gln Leu Thr Glu
145 150 155 160


Val Glu Glu Thr Val Arg Gln Phe Arg Gly Ala Val Gly Glu Gln Leu
165 170 175


Gly Lys Met Arg Val Phe Leu Ala Ala Leu Glu Gly Ser Leu Asp Arg
180 185 190


Glu Ala Glu Arg Val Arg Ser Glu Ala Gly Val Ala Leu Arg Arg Glu
195 200 205


Leu Gly Gly Leu His Ser Tyr Leu Glu Gln Leu Arg Gln Met Glu Lys
210 215 220


Val Leu Glu Glu Val Ala Asp Lys Pro Gln Thr Glu Phe Leu Met Lys
225 230 235 240


Tyr Cys Leu Val Thr Ser Arg Leu Gln Lys Ile Leu Ala Glu Ser Pro
245 250 255


Pro Pro Ala Arg Leu Asp Ile Gln Leu Pro Ile Ile Ser Asp Asp Phe
260 265 270


Lys Phe Gln Val Trp Arg Lys Met Phe Arg Ala Leu Met Pro Ala Leu
275 280 285


Glu Glu Leu Thr Phe Asp Pro Ser Ser Ala His Pro Ser Leu Val Val
290 295 300


Ser Pro Thr Gly Arg Arg Val Glu Cys Ser Glu Gln Lys Ala Pro Pro
305 310 315 320


Ala Gly Asp Asp Ala Arg Gln Phe Asp Lys Ala Val Ala Val Val Ala
325 330 335


Gln Gln Leu Leu Ser Asp Gly Glu His Tyr Trp Glu Val Glu Val Gly
340 345 350


Asp Lys Pro Arg Trp Ala Leu Gly Val Met Ala Ser Glu Ala Ser Arg
355 360 365


Arg Gly Arg Leu His Ala Val Pro Ser Gln Gly Leu Trp Leu Leu Gly
370 375 380


Leu Arg Asp Gly Lys Thr Leu Glu Ala His Val Glu Ala Lys Glu Pro
385 390 395 400


Arg Ala Leu Arg Thr Pro Glu Arg Arg Pro Thr Arg Leu Gly Leu Tyr
405 410 415


Leu Ser Phe Gly Asp Gly Val Leu Ala Phe Tyr Asp Ala Ser Asp Ala
420 425 430


Asp Ala Leu Glu Leu Leu Phe Ala Phe Arg Glu Arg Leu Pro Gly Pro
435 440 445


Val Tyr Pro Phe Phe Asp Val Cys Trp His Asp Lys Gly Lys Asn Ala
450 455 460


Gln Pro Leu Leu Leu Val Gly Pro Asp Gly Gln Glu Ala
465 470 475


<210> 48
<211> 477
<212> PRT
<213> Artificial sequence

<400> 48

Met Ser Ala Ala Pro Gly Leu Leu His Gln Glu Leu Ser Cys Pro Leu
1 5 10 15


Cys Leu Gln Leu Phe Asp Ala Pro Val Thr Ala Glu Cys Gly His Ser
20 25 30


Phe Cys Arg Ala Cys Leu Gly Arg Val Ala Gly Glu Pro Ala Ala Asp
35 40 45


Gly Thr Val Leu Cys Pro Cys Cys Gln Ala Pro Thr Arg Pro Gln Ala
50 55 60


Leu Ser Thr Asn Leu Gln Leu Ala Arg Leu Val Glu Gly Leu Ala Gln
65 70 75 80


Val Pro Gln Gly His Cys Glu Glu His Leu Asp Pro Leu Ser Ile Tyr
85 90 95


Cys Glu Gln Asp Arg Ala Leu Val Cys Gly Val Cys Ala Ser Leu Gly
100 105 110


Ser His Arg Gly His Arg Leu Leu Pro Ala Ala Glu Ala His Ala Arg
115 120 125


Leu Lys Thr Gln Leu Pro Gln Gln Lys Leu Gln Leu Gln Glu Ala Cys
130 135 140


Met Arg Lys Glu Lys Ser Val Ala Val Leu Glu His Gln Leu Val Glu
145 150 155 160


Val Glu Glu Thr Val Arg Gln Phe Arg Gly Ala Val Gly Glu Gln Leu
165 170 175


Gly Lys Met Arg Val Phe Leu Ala Ala Leu Glu Gly Ser Leu Asp Arg
180 185 190


Glu Ala Glu Arg Val Arg Gly Glu Ala Gly Val Ala Leu Arg Arg Glu
195 200 205


Leu Gly Ser Leu Asn Ser Tyr Leu Glu Gln Leu Arg Gln Met Glu Lys
210 215 220


Val Leu Glu Glu Val Ala Asp Lys Pro Gln Thr Glu Phe Leu Met Lys
225 230 235 240


Tyr Cys Leu Val Thr Ser Arg Leu Gln Lys Ile Leu Ala Glu Ala Pro
245 250 255


Pro Pro Ala Arg Leu Asp Ile Gln Leu Pro Ile Ile Ser Asp Asp Phe
260 265 270


Lys Phe Gln Val Trp Arg Lys Met Phe Arg Ala Leu Met Pro Ala Leu
275 280 285


Glu Glu Leu Thr Phe Asp Pro Ser Ser Ala His Pro Ser Leu Val Val
290 295 300


Ser Ser Ser Gly Arg Arg Val Glu Cys Ser Glu Gln Lys Ala Pro Pro
305 310 315 320


Ala Gly Glu Asp Pro Arg Gln Phe Asp Lys Ala Val Ala Val Val Ala
325 330 335


His Gln Gln Leu Ser Glu Gly Glu His Tyr Trp Glu Val Asp Val Gly
340 345 350


Asp Lys Pro Arg Trp Ala Leu Gly Val Ile Ala Ala Glu Ala Pro Arg
355 360 365


Arg Gly Arg Leu His Ala Val Pro Ser Gln Gly Leu Trp Leu Leu Gly
370 375 380


Leu Arg Glu Gly Lys Ile Leu Glu Ala His Val Glu Ala Lys Glu Pro
385 390 395 400


Arg Ala Leu Arg Ser Pro Glu Arg Arg Pro Thr Arg Ile Gly Leu Tyr
405 410 415


Leu Ser Phe Gly Asp Gly Val Leu Ser Phe Tyr Asp Ala Ser Asp Ala
420 425 430


Asp Ala Leu Val Pro Leu Phe Ala Phe His Glu Arg Leu Pro Arg Pro
435 440 445


Val Tyr Pro Phe Phe Asp Val Cys Trp His Asp Lys Gly Lys Asn Ala
450 455 460


Gln Pro Leu Leu Leu Val Gly Pro Glu Gly Ala Glu Ala
465 470 475


<210> 49
<211> 477
<212> PRT
<213> Artificial sequence

<400> 49

Met Ser Ala Ala Pro Gly Leu Leu Arg Gln Glu Leu Ser Cys Pro Leu
1 5 10 15


Cys Leu Gln Leu Phe Asp Ala Pro Val Thr Ala Glu Cys Gly His Ser
20 25 30


Phe Cys Arg Ala Cys Leu Ile Arg Val Ala Gly Glu Pro Ala Ala Asp
35 40 45


Gly Thr Val Ala Cys Pro Cys Cys Gln Ala Pro Thr Arg Pro Gln Ala
50 55 60


Leu Ser Thr Asn Leu Gln Leu Ser Arg Leu Val Glu Gly Leu Ala Gln
65 70 75 80


Val Pro Gln Gly His Cys Glu Glu His Leu Asp Pro Leu Ser Ile Tyr
85 90 95


Cys Glu Gln Asp Arg Thr Leu Val Cys Gly Val Cys Ala Ser Leu Gly
100 105 110


Ser His Arg Gly His Arg Leu Leu Pro Ala Ala Glu Ala Gln Ala Arg
115 120 125


Leu Lys Thr Gln Leu Pro Gln Gln Lys Met Gln Leu Gln Glu Ala Cys
130 135 140


Met Arg Lys Glu Lys Thr Val Ala Val Leu Glu His Gln Leu Val Glu
145 150 155 160


Val Glu Glu Thr Val Arg Gln Phe Arg Gly Ala Val Gly Glu Gln Leu
165 170 175


Gly Lys Met Arg Met Phe Leu Ala Ala Leu Glu Ser Ser Leu Asp Arg
180 185 190


Glu Ala Glu Arg Val Arg Gly Asp Ala Gly Val Ala Leu Arg Arg Glu
195 200 205


Leu Ser Ser Leu Asn Ser Tyr Leu Glu Gln Leu Arg Gln Met Glu Lys
210 215 220


Val Leu Glu Glu Val Ala Asp Lys Pro Gln Thr Glu Phe Leu Met Lys
225 230 235 240


Phe Cys Leu Val Thr Ser Arg Leu Gln Lys Ile Leu Ser Glu Ala Pro
245 250 255


Pro Pro Ala Arg Leu Asp Ile Gln Leu Pro Val Ile Ser Asp Asp Phe
260 265 270


Lys Phe Gln Val Trp Lys Lys Met Phe Arg Ala Leu Met Pro Ala Leu
275 280 285


Glu Glu Leu Thr Phe Asp Pro Ser Ser Ala His Pro Ser Leu Val Val
290 295 300


Ser Ser Ser Gly Arg Arg Val Glu Cys Ser Asp Gln Lys Ala Pro Pro
305 310 315 320


Ala Gly Glu Asp Thr Arg Gln Phe Asp Lys Ala Val Ala Val Val Ala
325 330 335


Gln Gln Leu Leu Ser Gln Gly Glu His Tyr Trp Glu Val Glu Val Gly
340 345 350


Asp Lys Pro Arg Trp Ala Leu Gly Val Met Ala Ala Asp Ala Ser Arg
355 360 365


Arg Gly Arg Leu His Ala Val Pro Ser Gln Gly Leu Trp Leu Leu Gly
370 375 380


Leu Arg Asp Gly Lys Ile Leu Glu Ala His Val Glu Ala Lys Glu Pro
385 390 395 400


Arg Ala Leu Arg Thr Pro Glu Arg Pro Pro Ala Arg Ile Gly Leu Tyr
405 410 415


Leu Ser Phe Ala Asp Gly Val Leu Ala Phe Tyr Asp Ala Ser Asn Pro
420 425 430


Asp Val Leu Thr Pro Ile Phe Ser Phe His Glu Arg Leu Pro Gly Pro
435 440 445


Val Tyr Pro Ile Phe Asp Val Cys Trp His Asp Lys Gly Lys Asn Ala
450 455 460


Gln Pro Leu Leu Leu Val Gly Pro Glu Gln Glu Gln Ala
465 470 475


<210> 50
<211> 477
<212> PRT
<213> Artificial sequence

<400> 50

Met Ser Thr Ala Pro Gly Leu Leu Arg Gln Glu Leu Ser Cys Pro Leu
1 5 10 15


Cys Leu Gln Leu Phe Asp Ala Pro Val Thr Ala Glu Cys Gly His Ser
20 25 30


Phe Cys Arg Ala Cys Leu Ile Arg Val Ala Gly Glu Pro Ala Asp Asp
35 40 45


Gly Thr Val Ala Cys Pro Cys Cys Gln Ala Ser Thr Arg Pro Gln Ala
50 55 60


Leu Ser Thr Asn Leu Gln Leu Ala Arg Leu Val Glu Gly Leu Ala Gln
65 70 75 80


Val Pro Gln Gly His Cys Glu Glu His Leu Asp Pro Leu Ser Ile Tyr
85 90 95


Cys Glu Gln Asp Arg Thr Leu Val Cys Gly Val Cys Ala Ser Leu Gly
100 105 110


Ser His Arg Gly His Arg Leu Leu Pro Ala Ala Glu Ala His Ala Arg
115 120 125


Leu Lys Thr Gln Leu Pro Gln Gln Lys Ala Gln Leu Gln Glu Ala Cys
130 135 140


Met Arg Lys Glu Lys Ser Val Ala Val Leu Glu His Gln Leu Val Glu
145 150 155 160


Val Glu Glu Thr Val Arg Gln Phe Arg Gly Ala Val Gly Glu Gln Leu
165 170 175


Gly Lys Met Arg Met Phe Leu Ala Ala Leu Glu Ser Ser Leu Asp Arg
180 185 190


Glu Ala Glu Arg Val Arg Gly Glu Ala Gly Val Ala Leu Arg Arg Glu
195 200 205


Leu Ser Ser Leu Asn Ser Tyr Leu Glu Gln Leu Arg Gln Met Glu Lys
210 215 220


Val Leu Glu Glu Val Ala Asp Lys Pro Gln Thr Glu Phe Leu Met Lys
225 230 235 240


Phe Cys Leu Val Thr Ser Arg Leu Gln Lys Ile Leu Ser Glu Ala Pro
245 250 255


Pro Pro Ala Arg Leu Asp Ile Gln Leu Pro Val Ile Ser Asp Asp Phe
260 265 270


Lys Phe Gln Val Trp Lys Lys Met Phe Arg Ala Leu Met Pro Glu Leu
275 280 285


Glu Glu Leu Thr Phe Asp Pro Ser Ser Ala His Pro Ser Leu Val Val
290 295 300


Ser Ala Ser Gly Arg Arg Val Glu Cys Ser Glu Gln Lys Ala Pro Pro
305 310 315 320


Ala Gly Glu Asp Thr Cys Gln Phe Asp Lys Thr Val Ala Val Val Ala
325 330 335


Lys Gln Leu Leu Ser Gln Gly Glu His Tyr Trp Glu Val Glu Val Gly
340 345 350


Asp Lys Pro Arg Trp Ala Leu Gly Val Met Ala Ala Asp Ala Ser Arg
355 360 365


Arg Gly Arg Leu His Ala Val Pro Ser Gln Gly Leu Trp Leu Leu Gly
370 375 380


Leu Arg Asp Gly Lys Ile Leu Glu Ala His Val Glu Ala Lys Glu Pro
385 390 395 400


Arg Ala Leu Arg Thr Pro Glu Arg Pro Pro Ala Arg Ile Gly Leu Tyr
405 410 415


Leu Ser Phe Ala Asp Gly Val Leu Thr Phe Tyr Asp Ala Ser Asn Thr
420 425 430


Asp Ala Leu Thr Pro Leu Phe Ser Phe His Glu Arg Leu Pro Gly Pro
435 440 445


Val Tyr Pro Met Phe Asp Val Cys Trp His Asp Lys Gly Lys Asn Ser
450 455 460


Gln Pro Leu Leu Leu Val Gly Pro Asp Ser Glu Gln Ala
465 470 475


<210> 51
<211> 477
<212> PRT
<213> Artificial sequence

<400> 51

Met Ser Ala Ala Pro Gly Leu Leu His Gln Glu Leu Ser Cys Pro Leu
1 5 10 15


Cys Leu Gln Leu Phe Asp Ala Pro Val Thr Ala Glu Cys Gly His Ser
20 25 30


Phe Cys Arg Ala Cys Leu Gly Arg Val Ala Gly Glu Pro Ala Ala Asp
35 40 45


Gly Thr Val Leu Cys Pro Cys Cys Gln Ala Pro Thr Arg Pro Gln Ala
50 55 60


Leu Ser Thr Asn Leu Gln Leu Ala Arg Leu Val Glu Gly Leu Ala Gln
65 70 75 80


Val Pro Gln Gly His Cys Glu Glu His Leu Asp Pro Leu Ser Ile Tyr
85 90 95


Cys Glu Gln Asp Arg Ala Leu Val Cys Gly Val Cys Ala Ser Leu Gly
100 105 110


Ser His Arg Gly His Arg Leu Leu Pro Ala Ala Glu Ala His Ala Arg
115 120 125


Leu Lys Thr Gln Leu Pro Gln Gln Lys Leu Gln Leu Gln Glu Ala Cys
130 135 140


Met Arg Lys Glu Lys Ser Val Ala Val Leu Glu His Gln Leu Val Glu
145 150 155 160


Val Glu Glu Thr Val Arg Gln Phe Arg Gly Ala Val Gly Glu Gln Leu
165 170 175


Gly Lys Met Arg Val Phe Leu Ala Ala Leu Glu Gly Ser Leu Asp Arg
180 185 190


Glu Ala Glu Arg Val Arg Gly Glu Ala Gly Val Ala Leu Arg Arg Glu
195 200 205


Leu Gly Ser Leu Asn Ser Tyr Leu Glu Gln Leu Arg Gln Met Glu Lys
210 215 220


Val Leu Glu Glu Val Ala Asp Lys Pro Gln Thr Glu Phe Leu Met Lys
225 230 235 240


Tyr Cys Leu Val Thr Ser Arg Leu Gln Lys Ile Leu Ala Glu Ala Pro
245 250 255


Pro Pro Ala Arg Leu Asp Ile Gln Leu Pro Ile Ile Ser Asp Asp Phe
260 265 270


Lys Phe Gln Val Trp Arg Lys Met Phe Arg Ala Leu Met Pro Ala Leu
275 280 285


Glu Glu Leu Thr Phe Asp Pro Ser Ser Ala His Pro Ser Leu Val Val
290 295 300


Ser Ser Ser Gly Arg Arg Val Glu Cys Ser Glu Gln Lys Ala Pro Pro
305 310 315 320


Ala Gly Glu Asp Pro Arg Gln Phe Asp Lys Ala Val Ala Val Val Ala
325 330 335


His Gln Gln Leu Ser Glu Gly Glu His Tyr Trp Glu Val Glu Val Gly
340 345 350


Asp Lys Pro Arg Trp Ala Leu Gly Val Ile Ala Ala Glu Gly Pro Arg
355 360 365


Arg Gly Arg Leu His Ala Val Pro Ser Gln Gly Leu Trp Leu Leu Gly
370 375 380


Leu Arg Glu Gly Lys Ile Leu Glu Ala His Val Glu Ala Lys Glu Pro
385 390 395 400


Arg Ala Leu Arg Ser Pro Glu Arg Arg Pro Thr Arg Ile Gly Leu Tyr
405 410 415


Leu Ser Phe Gly Asp Gly Val Leu Ser Phe Tyr Asp Ala Ser Asp Ala
420 425 430


Asp Ala Leu Val Pro Leu Phe Ala Phe His Glu Arg Leu Pro Gly Pro
435 440 445


Val Tyr Pro Phe Phe Asp Val Cys Trp His Asp Lys Gly Lys Asn Ser
450 455 460


Gln Pro Leu Leu Leu Val Gly Ser Glu Gly Ala Glu Ala
465 470 475


<210> 52
<211> 478
<212> PRT
<213> Artificial sequence

<400> 52

Met Ser Ala Ala Pro Gly Leu Leu His Gln Glu Leu Ser Cys Pro Leu
1 5 10 15


Cys Leu Gln Leu Phe Asp Ala Pro Val Thr Ala Glu Cys Gly His Ser
20 25 30


Phe Cys Arg Ala Cys Leu Gly Arg Val Ala Gly Glu Pro Ala Ala Asp
35 40 45


Gly Thr Val Leu Cys Pro Ser Cys Gln Ala Pro Thr Arg Pro Gln Ala
50 55 60


Leu Ser Thr Asn Gln Gln Leu Ala Arg Leu Val Glu Gly Leu Ala Gln
65 70 75 80


Val Pro Gln Gly His Cys Glu Glu His Leu Asp Pro Leu Ser Ile Tyr
85 90 95


Cys Glu Gln Asp Arg Val Leu Val Cys Gly Val Cys Ala Ser Leu Gly
100 105 110


Ser His Arg Gly His Arg Leu Leu Pro Ala Ala Glu Ala His Ala Arg
115 120 125


Leu Lys Thr Gln Leu Pro Gln Gln Lys Leu Gln Leu Gln Glu Ala Cys
130 135 140


Met Arg Lys Glu Lys Ser Val Gly Val Leu Glu Gln Gln Leu Val Glu
145 150 155 160


Val Glu Glu Thr Val Arg Gln Phe Arg Gly Ala Val Gly Glu Gln Leu
165 170 175


Gly Lys Met Arg Leu Phe Leu Ala Ala Leu Glu Gly Ser Leu Asp Arg
180 185 190


Glu Ala Glu Arg Val Arg Gly Glu Ala Gly Val Ala Leu Arg Arg Glu
195 200 205


Leu Gly Ser Leu Lys Ser Tyr Leu Glu Gln Leu Arg Gln Met Glu Lys
210 215 220


Val Leu Glu Glu Val Ala Asp Lys Pro Gln Thr Glu Phe Leu Met Lys
225 230 235 240


Tyr Cys Leu Val Thr Ser Arg Leu Gln Lys Ile Leu Ala Glu Ala Pro
245 250 255


Pro Pro Ala Arg Leu Asp Ile Gln Leu Pro Val Ile Ser Asp Asp Phe
260 265 270


Lys Phe Gln Val Trp Arg Lys Met Phe Arg Ala Leu Met Pro Ala Met
275 280 285


Gln Glu Leu Thr Phe Asp Pro Ser Thr Ala His Pro Ser Leu Val Leu
290 295 300


Ser Ala Ser Gly Arg Arg Val Glu Cys Ser Glu Gln Lys Ala Pro Pro
305 310 315 320


Ala Gly Glu Asp Pro Arg Gln Phe Asp Lys Ala Val Ala Val Val Thr
325 330 335


His Gln Leu Leu Ser Glu Gly Glu His Tyr Trp Glu Val Glu Val Gly
340 345 350


Asp Lys Pro Arg Trp Ala Leu Gly Val Ile Gly Ala Gln Ala Gly Arg
355 360 365


Arg Gly Arg Leu His Ala Val Pro Ser Gln Gly Leu Trp Leu Leu Gly
370 375 380


Leu Arg Glu Gly Lys Ile Leu Glu Ala His Val Glu Ala Lys Glu Pro
385 390 395 400


Arg Ala Leu Arg Thr Pro Glu Arg Arg Pro Ser Arg Ile Gly Ile Tyr
405 410 415


Leu Ser Phe Ala Asp Gly Val Leu Ser Phe Tyr Asp Ala Ser Asp Ala
420 425 430


Asp Ala Leu Glu Leu Leu Phe Ala Phe His Glu Arg Leu Pro Gly Pro
435 440 445


Val Tyr Pro Phe Phe Asp Val Cys Trp His Asp Lys Gly Lys Asn Ala
450 455 460


Gln Pro Leu Leu Leu Val Gly Pro Asp Ser Gly Gly Glu Ala
465 470 475


<210> 53
<211> 477
<212> PRT
<213> Artificial sequence

<400> 53

Met Ser Ala Ala Pro Gly Leu Leu His Gln Glu Leu Ser Cys Pro Leu
1 5 10 15


Cys Leu Gln Leu Phe Asp Ala Pro Val Thr Ala Glu Cys Gly His Ser
20 25 30


Phe Cys Arg Ala Cys Leu Ser Arg Val Ala Gly Glu Pro Ala Ala Asp
35 40 45


Gly Thr Val Pro Cys Pro Cys Cys Gln Ala Leu Thr Arg Pro Gln Ala
50 55 60


Leu Ser Thr Asn Gln Gln Leu Ala Arg Leu Val Glu Gly Leu Ala Gln
65 70 75 80


Val Pro Gln Gly His Cys Glu Glu His Leu Asp Pro Leu Ser Ile Tyr
85 90 95


Cys Glu Gln Asp Arg Ala Leu Val Cys Gly Val Cys Ala Ser Leu Gly
100 105 110


Ser His Arg Gly His Arg Leu Leu Pro Ala Ala Glu Ala His Ala Arg
115 120 125


Leu Lys Thr Gln Leu Pro Gln Gln Lys Leu Gln Leu Gln Glu Ala Cys
130 135 140


Met Arg Lys Glu Lys Ser Val Ala Leu Leu Glu His Gln Leu Met Glu
145 150 155 160


Val Glu Glu Met Val Arg Gln Phe Arg Gly Ala Val Gly Glu Gln Leu
165 170 175


Gly Lys Met Arg Val Phe Leu Ala Ala Leu Glu Gly Ser Leu Asp Arg
180 185 190


Glu Ala Glu Arg Val Arg Gly Glu Ala Gly Val Ala Leu Arg Arg Glu
195 200 205


Leu Gly Ser Leu Asn Ser Tyr Leu Glu Gln Leu Arg Gln Met Glu Lys
210 215 220


Val Leu Glu Glu Val Ala Asp Lys Pro Gln Thr Glu Phe Leu Met Lys
225 230 235 240


Tyr Cys Leu Val Thr Ser Arg Leu Gln Lys Ile Leu Ala Glu Ala Pro
245 250 255


Pro Pro Ala Arg Leu Asp Ile Gln Leu Pro Val Ile Ser Asp Asp Phe
260 265 270


Lys Phe Gln Val Trp Arg Lys Met Phe Arg Ala Leu Met Pro Val Thr
275 280 285


Lys Glu Leu Thr Phe Asp Pro Ser Ser Ala His Pro Ser Leu Val Leu
290 295 300


Ser Pro Ser Gly Arg Arg Val Glu Cys Ser Asp Gln Lys Ala Pro Pro
305 310 315 320


Ala Gly Glu Asp Pro Cys Gln Phe Asp Lys Ala Val Ala Val Val Ala
325 330 335


Gln Gln Val Leu Ser Asp Gly Glu His Tyr Trp Glu Val Gln Val Gly
340 345 350


Glu Lys Pro Arg Trp Ala Leu Gly Val Ile Ala Ala Gln Ala Ser Arg
355 360 365


Arg Gly Arg Leu His Ala Val Pro Ser Gln Gly Leu Trp Leu Leu Gly
370 375 380


Leu Arg Asp Gly Lys Ile Leu Glu Ala His Val Glu Ala Lys Glu Pro
385 390 395 400


Arg Ala Leu Arg Thr Pro Glu Arg Arg Pro Thr Arg Ile Gly Ile Tyr
405 410 415


Leu Ser Phe Gly Asp Gly Val Leu Ser Phe Tyr Asp Ala Ser Asp Pro
420 425 430


Asp Ala Leu Glu Leu Leu Phe Ala Phe His Glu Arg Leu Pro Gly Pro
435 440 445


Val Tyr Pro Phe Phe Asp Val Cys Trp His Asp Lys Gly Lys Asn Ala
450 455 460


Gln Pro Leu Leu Leu Val Gly Pro Asp Gly Glu Glu Ala
465 470 475


<210> 54
<211> 477
<212> PRT
<213> Artificial sequence

<400> 54

Met Ser Ala Ala Pro Gly Leu Leu His Gln Glu Leu Ser Cys Pro Leu
1 5 10 15


Cys Leu Gln Leu Phe Asp Ala Pro Val Thr Ala Glu Cys Gly His Ser
20 25 30


Phe Cys Arg Ala Cys Leu Gly Arg Val Ala Gly Glu Pro Ala Ala Asp
35 40 45


Gly Thr Val Leu Cys Pro Cys Cys Gln Ala Pro Thr Arg Pro Gln Ala
50 55 60


Leu Ser Thr Asn Leu Gln Leu Ala Arg Leu Val Glu Gly Leu Ala Gln
65 70 75 80


Val Pro Gln Gly His Cys Glu Glu His Leu Asp Pro Leu Ser Ile Tyr
85 90 95


Cys Glu Gln Asp Arg Ala Leu Val Cys Gly Val Cys Ala Ser Leu Gly
100 105 110


Ser His Arg Gly His Arg Leu Leu Pro Ala Ala Glu Ala His Ala Arg
115 120 125


Leu Lys Thr Gln Leu Pro Gln Gln Lys Leu Gln Leu Gln Glu Ala Cys
130 135 140


Met Arg Lys Glu Lys Ser Val Ala Val Leu Glu His Gln Leu Val Glu
145 150 155 160


Val Glu Glu Thr Val Arg Gln Phe Arg Gly Ala Val Gly Glu Gln Leu
165 170 175


Gly Lys Met Arg Val Phe Leu Ala Ala Leu Glu Gly Ser Leu Asp Cys
180 185 190


Glu Ala Glu Arg Val Arg Gly Glu Ala Gly Val Ala Leu Arg Arg Glu
195 200 205


Leu Gly Ser Leu Asn Ser Tyr Leu Glu Gln Leu Arg Gln Met Glu Lys
210 215 220


Val Leu Glu Glu Val Ala Asp Lys Pro Gln Thr Glu Phe Leu Met Lys
225 230 235 240


Tyr Cys Leu Val Thr Ser Arg Leu Gln Lys Ile Leu Ala Glu Ala Pro
245 250 255


Pro Pro Ala Arg Leu Asp Ile Gln Leu Pro Ile Ile Ser Asp Asp Phe
260 265 270


Lys Phe Gln Val Trp Arg Lys Met Phe Arg Ala Leu Met Pro Ala Leu
275 280 285


Glu Glu Leu Thr Phe Asp Pro Ser Ser Ala His Pro Ser Leu Val Val
290 295 300


Ser Ser Ser Gly Arg Arg Val Glu Cys Ser Glu Gln Lys Ala Pro Pro
305 310 315 320


Ala Gly Glu Asp Pro Arg Gln Phe Asp Lys Ala Val Ala Val Val Ala
325 330 335


His Gln Gln Leu Ser Glu Gly Glu His Tyr Trp Glu Val Asp Val Gly
340 345 350


Asp Lys Pro Arg Trp Ala Leu Gly Val Ile Ala Ala Glu Ala Pro Arg
355 360 365


Arg Gly Arg Leu His Ala Val Pro Ser Gln Gly Leu Trp Leu Leu Gly
370 375 380


Leu Arg Glu Gly Lys Ile Leu Glu Ala His Val Glu Ala Lys Glu Pro
385 390 395 400


Arg Ala Leu Arg Ser Pro Glu Arg Arg Pro Thr Arg Ile Gly Leu Tyr
405 410 415


Leu Ser Phe Gly Asp Gly Val Leu Ser Phe Tyr Asp Ala Ser Asp Ala
420 425 430


Asp Ala Leu Val Pro Leu Phe Ala Phe His Glu Arg Leu Pro Arg Pro
435 440 445


Val Tyr Pro Phe Phe Asp Val Cys Trp His Asp Lys Gly Lys Asn Ala
450 455 460


Gln Pro Leu Leu Leu Val Gly Pro Glu Gly Ala Glu Ala
465 470 475


<210> 55
<211> 333
<212> PRT
<213> Artificial sequence

<400> 55

Met Arg Lys Glu Lys Ser Val Ala Val Leu Glu His Gln Leu Val Glu
1 5 10 15


Val Glu Glu Thr Val Arg Gln Phe Arg Gly Ala Val Gly Glu Gln Leu
20 25 30


Gly Lys Met Arg Val Phe Leu Ala Ala Leu Glu Gly Ser Leu Asp Arg
35 40 45


Glu Ala Glu Arg Val Arg Gly Glu Ala Gly Val Ala Leu Arg Arg Glu
50 55 60


Leu Gly Ser Leu Asn Ser Tyr Leu Glu Gln Leu Arg Gln Met Glu Lys
65 70 75 80


Val Leu Glu Glu Val Ala Asp Lys Pro Gln Thr Glu Phe Leu Met Lys
85 90 95


Tyr Cys Leu Val Thr Ser Arg Leu Gln Lys Ile Leu Ala Glu Ala Pro
100 105 110


Pro Pro Ala Arg Leu Asp Ile Gln Leu Pro Ile Ile Ser Asp Asp Phe
115 120 125


Lys Phe Gln Val Trp Arg Lys Met Phe Arg Ala Leu Met Pro Ala Leu
130 135 140


Glu Glu Leu Thr Phe Asp Pro Ser Ser Ala His Pro Ser Leu Val Val
145 150 155 160


Ser Ser Ser Gly Arg Arg Val Glu Cys Ser Glu Gln Lys Ala Pro Pro
165 170 175


Ala Gly Glu Asp Pro Arg Gln Phe Asp Lys Ala Val Ala Val Val Ala
180 185 190


His Gln Gln Leu Ser Glu Gly Glu His Tyr Trp Glu Val Asp Val Gly
195 200 205


Asp Lys Pro Arg Trp Ala Leu Gly Val Ile Ala Ala Glu Ala Pro Arg
210 215 220


Arg Gly Arg Leu His Ala Val Pro Ser Gln Gly Leu Trp Leu Leu Gly
225 230 235 240


Leu Arg Glu Gly Lys Ile Leu Glu Ala His Val Glu Ala Lys Glu Pro
245 250 255


Arg Ala Leu Arg Ser Pro Glu Arg Arg Pro Thr Arg Ile Gly Leu Tyr
260 265 270


Leu Ser Phe Gly Asp Gly Val Leu Ser Phe Tyr Asp Ala Ser Asp Ala
275 280 285


Asp Ala Leu Val Pro Leu Phe Ala Phe His Glu Arg Leu Pro Arg Pro
290 295 300


Val Tyr Pro Phe Phe Asp Val Cys Trp His Asp Lys Gly Lys Asn Ala
305 310 315 320


Gln Pro Leu Leu Leu Val Gly Pro Glu Gly Ala Glu Ala
325 330


<210> 56
<211> 333
<212> PRT
<213> Artificial sequence

<400> 56

Met Arg Lys Glu Lys Ser Val Ala Val Leu Glu His Gln Leu Val Glu
1 5 10 15


Val Glu Glu Thr Val Arg Gln Phe Arg Gly Ala Val Gly Glu Gln Leu
20 25 30


Gly Lys Met Arg Val Phe Leu Ala Ala Leu Glu Gly Ser Leu Asp Arg
35 40 45


Glu Ala Glu Arg Val Arg Gly Glu Ala Gly Val Ala Leu Arg Arg Glu
50 55 60


Leu Gly Ser Leu Asn Ser Tyr Leu Glu Gln Leu Arg Gln Met Glu Lys
65 70 75 80


Val Leu Glu Glu Val Ala Asp Lys Pro Gln Thr Glu Phe Leu Met Lys
85 90 95


Tyr Cys Leu Val Thr Ser Arg Leu Gln Lys Ile Leu Ala Glu Ala Pro
100 105 110


Pro Pro Ala Arg Leu Asp Ile Gln Leu Pro Ile Ile Ser Asp Asp Phe
115 120 125


Lys Phe Gln Val Trp Arg Lys Met Phe Arg Ala Leu Met Pro Ala Leu
130 135 140


Glu Glu Leu Thr Phe Asp Pro Ser Ser Ala His Pro Ser Leu Val Val
145 150 155 160


Ser Ser Ser Gly Arg Arg Val Glu Cys Ser Gly Gln Lys Ala Pro Pro
165 170 175


Ala Gly Glu Asp Pro Arg Gln Phe Asp Lys Ala Val Ala Val Val Ala
180 185 190


His Gln Gln Leu Ser Glu Gly Glu His Tyr Trp Glu Val Asp Val Gly
195 200 205


Asp Lys Pro Arg Trp Ala Leu Gly Val Ile Ala Ala Glu Ala Pro Arg
210 215 220


Arg Gly Arg Leu His Ala Val Pro Ser Gln Gly Leu Trp Leu Leu Gly
225 230 235 240


Leu Arg Glu Gly Lys Ile Leu Glu Ala His Val Glu Ala Lys Glu Pro
245 250 255


Arg Ala Leu Arg Ser Pro Glu Arg Arg Pro Thr Arg Ile Gly Leu Tyr
260 265 270


Leu Ser Phe Gly Asp Gly Val Leu Ser Phe Tyr Asp Ala Ser Asp Ala
275 280 285


Asp Ala Leu Val Pro Leu Phe Ala Phe His Glu Arg Leu Pro Arg Pro
290 295 300


Val Tyr Pro Phe Phe Asp Val Cys Trp His Asp Lys Gly Lys Asn Ala
305 310 315 320


Gln Pro Leu Leu Leu Val Gly Pro Glu Gly Ala Glu Ala
325 330


<210> 57
<211> 269
<212> PRT
<213> Artificial sequence

<400> 57

Met Ser Ala Ala Pro Gly Leu Leu His Gln Glu Leu Ser Cys Pro Leu
1 5 10 15


Cys Leu Gln Leu Phe Asp Ala Pro Val Thr Ala Glu Cys Gly His Ser
20 25 30


Phe Cys Arg Ala Cys Leu Gly Arg Val Ala Gly Glu Pro Ala Ala Asp
35 40 45


Gly Thr Val Leu Cys Pro Cys Cys Gln Ala Pro Thr Arg Pro Gln Ala
50 55 60


Leu Ser Thr Asn Leu Gln Leu Ala Arg Leu Val Glu Gly Leu Ala Gln
65 70 75 80


Val Pro Gln Gly His Cys Glu Glu His Leu Asp Pro Leu Ser Ile Tyr
85 90 95


Cys Glu Gln Asp Arg Ala Leu Val Cys Gly Val Cys Ala Ser Leu Gly
100 105 110


Ser His Arg Gly His Arg Leu Leu Pro Ala Ala Glu Ala His Ala Arg
115 120 125


Leu Lys Thr Gln Leu Pro Gln Gln Lys Leu Gln Leu Gln Glu Ala Cys
130 135 140


Met Arg Lys Glu Lys Ser Val Ala Val Leu Glu His Gln Leu Val Glu
145 150 155 160


Val Glu Glu Thr Val Arg Gln Phe Arg Gly Ala Val Gly Glu Gln Leu
165 170 175


Gly Lys Met Arg Val Phe Leu Ala Ala Leu Glu Gly Ser Leu Asp Arg
180 185 190


Glu Ala Glu Arg Val Arg Gly Glu Ala Gly Val Ala Leu Arg Arg Glu
195 200 205


Leu Gly Ser Leu Asn Ser Tyr Leu Glu Gln Leu Arg Gln Met Glu Lys
210 215 220


Val Leu Glu Glu Val Ala Asp Lys Pro Gln Thr Glu Phe Leu Met Lys
225 230 235 240


Tyr Cys Leu Val Thr Ser Arg Leu Gln Lys Ile Leu Ala Glu Ala Pro
245 250 255


Pro Pro Ala Arg Leu Asp Ile Gln Leu Pro Ile Ile Ser
260 265


<210> 58
<211> 1382
<212> PRT
<213> Homo sapiens

<400> 58

Met Gly Thr Gly Gly Arg Arg Gly Ala Ala Ala Ala Pro Leu Leu Val
1 5 10 15


Ala Val Ala Ala Leu Leu Leu Gly Ala Ala Gly His Leu Tyr Pro Gly
20 25 30


Glu Val Cys Pro Gly Met Asp Ile Arg Asn Asn Leu Thr Arg Leu His
35 40 45


Glu Leu Glu Asn Cys Ser Val Ile Glu Gly His Leu Gln Ile Leu Leu
50 55 60


Met Phe Lys Thr Arg Pro Glu Asp Phe Arg Asp Leu Ser Phe Pro Lys
65 70 75 80


Leu Ile Met Ile Thr Asp Tyr Leu Leu Leu Phe Arg Val Tyr Gly Leu
85 90 95


Glu Ser Leu Lys Asp Leu Phe Pro Asn Leu Thr Val Ile Arg Gly Ser
100 105 110


Arg Leu Phe Phe Asn Tyr Ala Leu Val Ile Phe Glu Met Val His Leu
115 120 125


Lys Glu Leu Gly Leu Tyr Asn Leu Met Asn Ile Thr Arg Gly Ser Val
130 135 140


Arg Ile Glu Lys Asn Asn Glu Leu Cys Tyr Leu Ala Thr Ile Asp Trp
145 150 155 160


Ser Arg Ile Leu Asp Ser Val Glu Asp Asn Tyr Ile Val Leu Asn Lys
165 170 175


Asp Asp Asn Glu Glu Cys Gly Asp Ile Cys Pro Gly Thr Ala Lys Gly
180 185 190


Lys Thr Asn Cys Pro Ala Thr Val Ile Asn Gly Gln Phe Val Glu Arg
195 200 205


Cys Trp Thr His Ser His Cys Gln Lys Val Cys Pro Thr Ile Cys Lys
210 215 220


Ser His Gly Cys Thr Ala Glu Gly Leu Cys Cys His Ser Glu Cys Leu
225 230 235 240


Gly Asn Cys Ser Gln Pro Asp Asp Pro Thr Lys Cys Val Ala Cys Arg
245 250 255


Asn Phe Tyr Leu Asp Gly Arg Cys Val Glu Thr Cys Pro Pro Pro Tyr
260 265 270


Tyr His Phe Gln Asp Trp Arg Cys Val Asn Phe Ser Phe Cys Gln Asp
275 280 285


Leu His His Lys Cys Lys Asn Ser Arg Arg Gln Gly Cys His Gln Tyr
290 295 300


Val Ile His Asn Asn Lys Cys Ile Pro Glu Cys Pro Ser Gly Tyr Thr
305 310 315 320


Met Asn Ser Ser Asn Leu Leu Cys Thr Pro Cys Leu Gly Pro Cys Pro
325 330 335


Lys Val Cys His Leu Leu Glu Gly Glu Lys Thr Ile Asp Ser Val Thr
340 345 350


Ser Ala Gln Glu Leu Arg Gly Cys Thr Val Ile Asn Gly Ser Leu Ile
355 360 365


Ile Asn Ile Arg Gly Gly Asn Asn Leu Ala Ala Glu Leu Glu Ala Asn
370 375 380


Leu Gly Leu Ile Glu Glu Ile Ser Gly Tyr Leu Lys Ile Arg Arg Ser
385 390 395 400


Tyr Ala Leu Val Ser Leu Ser Phe Phe Arg Lys Leu Arg Leu Ile Arg
405 410 415


Gly Glu Thr Leu Glu Ile Gly Asn Tyr Ser Phe Tyr Ala Leu Asp Asn
420 425 430


Gln Asn Leu Arg Gln Leu Trp Asp Trp Ser Lys His Asn Leu Thr Ile
435 440 445


Thr Gln Gly Lys Leu Phe Phe His Tyr Asn Pro Lys Leu Cys Leu Ser
450 455 460


Glu Ile His Lys Met Glu Glu Val Ser Gly Thr Lys Gly Arg Gln Glu
465 470 475 480


Arg Asn Asp Ile Ala Leu Lys Thr Asn Gly Asp Gln Ala Ser Cys Glu
485 490 495


Asn Glu Leu Leu Lys Phe Ser Tyr Ile Arg Thr Ser Phe Asp Lys Ile
500 505 510


Leu Leu Arg Trp Glu Pro Tyr Trp Pro Pro Asp Phe Arg Asp Leu Leu
515 520 525


Gly Phe Met Leu Phe Tyr Lys Glu Ala Pro Tyr Gln Asn Val Thr Glu
530 535 540


Phe Asp Gly Gln Asp Ala Cys Gly Ser Asn Ser Trp Thr Val Val Asp
545 550 555 560


Ile Asp Pro Pro Leu Arg Ser Asn Asp Pro Lys Ser Gln Asn His Pro
565 570 575


Gly Trp Leu Met Arg Gly Leu Lys Pro Trp Thr Gln Tyr Ala Ile Phe
580 585 590


Val Lys Thr Leu Val Thr Phe Ser Asp Glu Arg Arg Thr Tyr Gly Ala
595 600 605


Lys Ser Asp Ile Ile Tyr Val Gln Thr Asp Ala Thr Asn Pro Ser Val
610 615 620


Pro Leu Asp Pro Ile Ser Val Ser Asn Ser Ser Ser Gln Ile Ile Leu
625 630 635 640


Lys Trp Lys Pro Pro Ser Asp Pro Asn Gly Asn Ile Thr His Tyr Leu
645 650 655


Val Phe Trp Glu Arg Gln Ala Glu Asp Ser Glu Leu Phe Glu Leu Asp
660 665 670


Tyr Cys Leu Lys Gly Leu Lys Leu Pro Ser Arg Thr Trp Ser Pro Pro
675 680 685


Phe Glu Ser Glu Asp Ser Gln Lys His Asn Gln Ser Glu Tyr Glu Asp
690 695 700


Ser Ala Gly Glu Cys Cys Ser Cys Pro Lys Thr Asp Ser Gln Ile Leu
705 710 715 720


Lys Glu Leu Glu Glu Ser Ser Phe Arg Lys Thr Phe Glu Asp Tyr Leu
725 730 735


His Asn Val Val Phe Val Pro Arg Lys Thr Ser Ser Gly Thr Gly Ala
740 745 750


Glu Asp Pro Arg Pro Ser Arg Lys Arg Arg Ser Leu Gly Asp Val Gly
755 760 765


Asn Val Thr Val Ala Val Pro Thr Val Ala Ala Phe Pro Asn Thr Ser
770 775 780


Ser Thr Ser Val Pro Thr Ser Pro Glu Glu His Arg Pro Phe Glu Lys
785 790 795 800


Val Val Asn Lys Glu Ser Leu Val Ile Ser Gly Leu Arg His Phe Thr
805 810 815


Gly Tyr Arg Ile Glu Leu Gln Ala Cys Asn Gln Asp Thr Pro Glu Glu
820 825 830


Arg Cys Ser Val Ala Ala Tyr Val Ser Ala Arg Thr Met Pro Glu Ala
835 840 845


Lys Ala Asp Asp Ile Val Gly Pro Val Thr His Glu Ile Phe Glu Asn
850 855 860


Asn Val Val His Leu Met Trp Gln Glu Pro Lys Glu Pro Asn Gly Leu
865 870 875 880


Ile Val Leu Tyr Glu Val Ser Tyr Arg Arg Tyr Gly Asp Glu Glu Leu
885 890 895


His Leu Cys Val Ser Arg Lys His Phe Ala Leu Glu Arg Gly Cys Arg
900 905 910


Leu Arg Gly Leu Ser Pro Gly Asn Tyr Ser Val Arg Ile Arg Ala Thr
915 920 925


Ser Leu Ala Gly Asn Gly Ser Trp Thr Glu Pro Thr Tyr Phe Tyr Val
930 935 940


Thr Asp Tyr Leu Asp Val Pro Ser Asn Ile Ala Lys Ile Ile Ile Gly
945 950 955 960


Pro Leu Ile Phe Val Phe Leu Phe Ser Val Val Ile Gly Ser Ile Tyr
965 970 975


Leu Phe Leu Arg Lys Arg Gln Pro Asp Gly Pro Leu Gly Pro Leu Tyr
980 985 990


Ala Ser Ser Asn Pro Glu Tyr Leu Ser Ala Ser Asp Val Phe Pro Cys
995 1000 1005


Ser Val Tyr Val Pro Asp Glu Trp Glu Val Ser Arg Glu Lys Ile
1010 1015 1020


Thr Leu Leu Arg Glu Leu Gly Gln Gly Ser Phe Gly Met Val Tyr
1025 1030 1035


Glu Gly Asn Ala Arg Asp Ile Ile Lys Gly Glu Ala Glu Thr Arg
1040 1045 1050


Val Ala Val Lys Thr Val Asn Glu Ser Ala Ser Leu Arg Glu Arg
1055 1060 1065


Ile Glu Phe Leu Asn Glu Ala Ser Val Met Lys Gly Phe Thr Cys
1070 1075 1080


His His Val Val Arg Leu Leu Gly Val Val Ser Lys Gly Gln Pro
1085 1090 1095


Thr Leu Val Val Met Glu Leu Met Ala His Gly Asp Leu Lys Ser
1100 1105 1110


Tyr Leu Arg Ser Leu Arg Pro Glu Ala Glu Asn Asn Pro Gly Arg
1115 1120 1125


Pro Pro Pro Thr Leu Gln Glu Met Ile Gln Met Ala Ala Glu Ile
1130 1135 1140


Ala Asp Gly Met Ala Tyr Leu Asn Ala Lys Lys Phe Val His Arg
1145 1150 1155


Asp Leu Ala Ala Arg Asn Cys Met Val Ala His Asp Phe Thr Val
1160 1165 1170


Lys Ile Gly Asp Phe Gly Met Thr Arg Asp Ile Tyr Glu Thr Asp
1175 1180 1185


Tyr Tyr Arg Lys Gly Gly Lys Gly Leu Leu Pro Val Arg Trp Met
1190 1195 1200


Ala Pro Glu Ser Leu Lys Asp Gly Val Phe Thr Thr Ser Ser Asp
1205 1210 1215


Met Trp Ser Phe Gly Val Val Leu Trp Glu Ile Thr Ser Leu Ala
1220 1225 1230


Glu Gln Pro Tyr Gln Gly Leu Ser Asn Glu Gln Val Leu Lys Phe
1235 1240 1245


Val Met Asp Gly Gly Tyr Leu Asp Gln Pro Asp Asn Cys Pro Glu
1250 1255 1260


Arg Val Thr Asp Leu Met Arg Met Cys Trp Gln Phe Asn Pro Lys
1265 1270 1275


Met Arg Pro Thr Phe Leu Glu Ile Val Asn Leu Leu Lys Asp Asp
1280 1285 1290


Leu His Pro Ser Phe Pro Glu Val Ser Phe Phe His Ser Glu Glu
1295 1300 1305


Asn Lys Ala Pro Glu Ser Glu Glu Leu Glu Met Glu Phe Glu Asp
1310 1315 1320


Met Glu Asn Val Pro Leu Asp Arg Ser Ser His Cys Gln Arg Glu
1325 1330 1335


Glu Ala Gly Gly Arg Asp Gly Gly Ser Ser Leu Gly Phe Lys Arg
1340 1345 1350


Ser Tyr Glu Glu His Ile Pro Tyr Thr His Met Asn Gly Gly Lys
1355 1360 1365


Lys Asn Gly Arg Ile Leu Thr Leu Pro Arg Ser Asn Pro Ser
1370 1375 1380


<210> 59
<211> 35
<212> DNA
<213> Artificial sequence

<400> 59
ataggtaccg ccaccatgtc ggctgcaccc ggcct 35


<210> 60
<211> 30
<212> DNA
<213> Artificial sequence

<400> 60
atactcgagc ggcctgttcc tgctccggcc 30


<210> 61
<211> 609
<212> PRT
<213> Marmoset


<220>
<221> misc_feature
<222> (53) .. (53)
<223> Xaa can be any naturally occurring amino acid

<220>
<221> misc_feature
<222> (66) .. (71)
<223> Xaa can be any naturally occurring amino acid

<220>
<221> misc_feature
<222> (480) .. (480)
<223> Xaa can be any naturally occurring amino acid

<400> 61

Met Ser Ala Ala Pro Gly Leu Leu His Gln Glu Leu Ser Cys Pro Leu
1 5 10 15


Cys Leu Gln Leu Phe Asp Ala Pro Val Thr Ala Glu Cys Gly His Ser
20 25 30


Phe Cys His Ile Cys Leu Arg Arg Met Lys Thr Glu Pro Ala Ala Asp
35 40 45


Asp Thr Val Pro Xaa Thr Ser Cys Gln Ala Asn Lys Pro Thr Asn Leu
50 55 60


Lys Xaa Xaa Xaa Xaa Xaa Xaa Gly Ala Arg Trp Pro Pro Gly Ser Thr
65 70 75 80


Ala Arg Glu His Leu Asp Pro Leu Ser Ile Tyr Cys Glu Gln Asp Arg
85 90 95


Ala Leu Val Cys Gly Val Cys Ala Ser Leu Gly Ser His Arg Gly His
100 105 110


Arg Leu Leu Pro Ala Ala Glu Ala His Ala Arg Ser Arg Asp Arg Glu
115 120 125


Met Gly Gly Arg Ile Pro Gly Ala Gly Leu Met Gly Gly Ala Glu Pro
130 135 140


Gly Gly Ala Arg Thr Gly Gln Arg Pro Lys Trp Tyr Ser Cys Thr Arg
145 150 155 160


Trp Ala Gly Arg Thr Gly Ser Pro Arg Gly Ala Gly Gly Gly Ala Ala
165 170 175


Phe Gly Arg Glu Pro Gly Gly Arg Gly Gly Ala Val Leu Met Ser Ser
180 185 190


Ala Glu Ala His Ala Pro Leu Lys Leu Arg Asn Leu Gly Arg Pro Asp
195 200 205


Gly Leu Gln Ala Gly Leu Thr Gly Arg Glu Ala Gly Trp Pro Gly Arg
210 215 220


Pro Phe Ala Thr Gly Glu Gly Leu Ala Gly Gly Leu Arg Glu Asp Pro
225 230 235 240


Arg Arg Glu Pro Glu Gly Gly Gly Gly Arg Pro Glu Gly Ser Gly Arg Asp
245 250 255


Pro Ile Pro Gly Ser Leu Thr Gln Leu Pro Gln Gln Lys Leu Gln Leu
260 265 270


Gln Glu Ala Cys Met Arg Lys Glu Lys Ser Val Ala Val Leu Glu His
275 280 285


Gln Leu Val Glu Val Glu Glu Thr Val Arg Gln Phe Arg Ala Ala Val
290 295 300


Gly Asp Gln Leu Gly Lys Met Arg Leu Phe Leu Ala Ala Leu Glu Gly
305 310 315 320


Ser Leu Asp Arg Glu Ala Glu Arg Val Arg Gly Glu Ala Gly Val Ala
325 330 335


Leu Arg Arg Glu Leu Gly Ser Leu Asn Ser Tyr Leu Glu Gln Leu Arg
340 345 350


Gln Met Glu Lys Val Leu Glu Glu Val Thr Asp Lys Pro Gln Thr Glu
355 360 365


Phe Leu Met Lys Tyr Cys Leu Val Thr Ser Arg Leu Gln Lys Ile Leu
370 375 380


Ala Glu Ser Pro Pro Pro Ala Arg Leu Asp Ile Gln Leu Pro Ile Ile
385 390 395 400


Ser Asp Asp Phe Lys Phe Gln Val Trp Arg Lys Met Phe Arg Ala Leu
405 410 415


Met Pro Ala Leu Glu Glu Leu Thr Phe Asp Pro Ser Ser Ala His Pro
420 425 430


Ser Leu Val Val Ser Ser Ser Gly Arg Arg Val Glu Cys Ser Glu Gln
435 440 445


Lys Ala Pro Pro Ala Gly Glu Asp Pro Arg Gln Phe Asp Lys Ala Val
450 455 460


Ala Val Val Ala His Gln Arg Leu Ser Glu Gly Glu His Tyr Trp Xaa
465 470 475 480


Val Glu Val Gly Asp Lys Pro Arg Trp Ala Leu Gly Val Ile Ser Ala
485 490 495


Glu Ala Pro Arg Arg Gly Arg Leu His Pro Val Pro Ser Gln Gly Leu
500 505 510


Trp Leu Leu Gly Leu Arg Glu Ser Lys Ile Leu Glu Ala His Val Glu
515 520 525


Ala Lys Glu Pro Arg Ala Leu Arg Ser Pro Glu Arg Arg Pro Thr Arg
530 535 540


Ile Gly Leu Tyr Met Ser Phe Ser Asp Gly Val Leu Ser Phe Tyr Asp
545 550 555 560


Ala Ser Asp Ala Asp Ala Leu Val Leu Leu Phe Ala Phe His Glu Arg
565 570 575


Leu Pro Gly Pro Val Tyr Pro Phe Phe Asp Val Cys Trp His Asp Lys
580 585 590


Gly Lys Asn Ala Gln Pro Leu Leu Leu Leu Gly Pro Glu Gly Ala Glu
595 600 605


Ala

Claims (59)

A method for detecting or predicting the risk of MG53-related diseases, including the following steps: a. Obtain a sample to be tested; and b. Detection of extracellular free MG53 in the test sample. The method according to claim 1, wherein the test sample is selected from the group consisting of whole blood, plasma, serum, and interstitial fluid. The method according to claim 1, wherein the test sample is substantially free of cells. The method according to claim 1, wherein the step b comprises contacting the MG53 detection reagent with the sample to be tested. The method according to claim 4, wherein the MG53 detection reagent is an MG53 antibody or an antigen-binding fragment thereof, an MG53 ligand or an MG53-binding fragment thereof. The method according to claim 4, wherein the MG53 detection reagent is a small molecule compound capable of binding to MG53. The method according to claim 4, wherein the MG53 detection reagent has a detectable label. The method according to claim 7, wherein the detectable label has a luminescent, magnetic, radioactive or enzymatic activity. The method according to claim 1, wherein said step b includes performing radioimmunoassay, western blotting method, adjacent connection technology, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescence method, Immunohistochemical assays, immunospot assays, or slot blot assays. The method according to claim 1, further comprising: c. Compare the detection value of MG53 obtained in step b with the control value. The method of claim 10, wherein the control value is from a control sample. The method according to claim 11, wherein the control sample and the test sample are from the same or different individuals. The method according to claim 11, wherein the control sample and the test sample are from the same individual, but the collection time of the control sample is earlier or later than the test sample. The method of claim 13, wherein the individual has received treatment or a change in health status during the period of time. The method according to claim 10, wherein the control value is obtained from a healthy individual or an individual suffering from an MG53-related disease. The method of any of claims 12-15, wherein the individual is a human or a non-human mammal. The method according to claim 1, wherein the MG53-related disease is a metabolic syndrome. The method according to claim 1, wherein the MG53-related disease is a disease related to glucose metabolism or lipid metabolism. The method according to claim 18, wherein the disease related to glucose metabolism or lipid metabolism is selected from the group consisting of insulin resistance, diabetic cerebrovascular disease, diabetic ocular complications, diabetic neuropathy, diabetic foot, hyperinsulinemia, high Cholesterolemia, hyperglycemia, hyperlipidemia, hypertension, and obesity. A method for reducing or inhibiting the activity of extracellular free MG53 in an individual, comprising administering to the individual in need thereof an effective amount of an MG53 inhibitor. The method according to claim 20, wherein the MG53 inhibitor specifically binds to MG53. The method according to claim 20, wherein the MG53 inhibitor is an MG53 antibody or an antigen-binding fragment thereof, an MG53 ligand or an MG53-binding fragment thereof, or a non-functional protein fragment of MG53. The method according to claim 22, wherein the MG53 antibody or antigen-binding fragment thereof comprises a heavy chain including the following sequence or having the same sequence as at least 80%, 85%, 90%, 95%, or 99% Derived sequence: SEQ ID NO: 1, 3, 9, 11, 17, 19, 25, 27, 33 or 35. The method according to claim 22, wherein the MG53 antibody or antigen-binding fragment thereof comprises a light chain comprising the following sequence or having the same sequence as at least 80%, 85%, 90%, 95%, or 99% Derived sequence: SEQ ID NO: 2, 4, 10, 12, 18, 20, 26, 28, 34 or 36. The method according to claim 20, wherein the MG53 inhibitor is a small molecule compound. The method according to claim 20, wherein the MG53 inhibitor is a compound having Formula Ia or Ib or a pharmaceutically acceptable salt thereof, Where L is a chemical bond or optionally substituted C ₁ -C ₁₂ alkylene; M is a chemical bond or optionally substituted C ₆ -C ₁₂ aromatic subunit or 5-12 membered heterocyclic subunit; X is a chemical bond , Or optionally substituted -CH = N-NH-C (O)-, -CH ₂ -O-, -O-CH ₂ -C (O) -NH-, -OC (O)-, or -OC ( O) -CH = CH-; Y is optionally substituted C ₆ -C ₁₂ aryl or 5-12 membered heterocyclic group; Q is optionally substituted C ₁ -C ₁₂ alkylene or C ₂ -C ₁₂ Alkenylene; T is optionally substituted C ₆ -C ₁₂ aryl or 5-12 membered heterocyclic group; R _{1 is} each independently hydrogen, nitro, halogen, hydroxy, cyano, or optionally substituted C _1- C ₁₂ alkyl or C ₁ -C ₁₂ alkoxy; n is any positive integer from 1-5. The method according to item 26 request, wherein the L is a bond or C ₁ -C ₃ alkylene group. The method according to claim 26, wherein the M is a chemical bond, or an optionally substituted phenylene or furanyl. The method according to claim 26, wherein the Y is an optionally substituted phenyl, furyl, quinolinyl, or benzodioxoyl. The method according to claim 26, wherein the Q is an optionally substituted C ₁ -C ₃ alkylene or C ₂ -C ₃ alkenylene. The method according to claim 26, wherein the T is an optionally substituted phenyl or 1,3,4-thiadiazolyl. The method according to claim 20, wherein the MG53 inhibitor is a compound having Formula II or a pharmaceutically acceptable salt thereof, Wherein A is hydrogen, or optionally substituted C ₆ -C ₁₂ aryl or 5-12 membered heterocyclic group; G is -C (O) -OR ₃ , wherein R ₃ is optionally substituted C ₁ -C ₁₂ alkyl; J is an optionally substituted C ₆ -C ₁₂ aryl; or G and J together with the carbon atom to which they are attached Wherein R _{4 is} each independently hydrogen, nitro, halogen, hydroxy, cyano, or optionally substituted C ₁ -C ₁₂ alkyl or C ₁ -C ₁₂ alkoxy, and n is any An integer; B is a chemical bond, or optionally substituted C ₆ -C ₁₂ aromatic subunit or 5-12 membered heterocyclic subunit; D is a chemical bond, or optionally substituted -OR _5- , wherein R _{5 is} selected from optional Substituted C ₁ -C ₁₂ alkylene; E is optionally substituted C ₆ -C ₁₂ aryl or 5-12 membered heterocyclyl. The method according to claim 32, wherein said A is optionally substituted naphthyl, phenyl or thienyl. The method according to item 32 request, wherein said G is -C (O) -OR _3, wherein R ₃ is optionally substituted C ₁ -C ₃ alkyl; J is selected from optionally substituted phenyl; or G and J together with the carbon atoms to which they are attached Where R ₄ is hydrogen. The method according to claim 32, wherein the B is a chemical bond, or an optionally substituted phenylene or furanyl group. The method according to claim 32, wherein the D is a chemical bond, or optionally substituted -O-CH _2- . The method according to claim 32, wherein the E is an optionally substituted phenyl. The method according to claim 20, wherein the MG53 inhibitor is a compound having Formula III or a pharmaceutically acceptable salt thereof, Formula III wherein A is hydrogen, or optionally substituted C ₆ -C ₁₂ aryl or 5-12 membered heterocyclic group; Y is each independently an optionally substituted C ₆ -C ₁₂ aryl or 5-12 membered Heterocyclyl; R ₇ is hydrogen, nitro, halogen, hydroxy, cyano, or optionally substituted C ₁ -C ₁₂ alkyl or C ₁ -C ₁₂ alkoxy. The method according to claim 38, wherein A is optionally substituted phenyl. The method according to claim 38, wherein Y is an optionally substituted phenyl. The method according to claim 38, wherein R ₇ is hydrogen, nitro, halogen, hydroxyl or cyano. The method according to any one of claims 26-41, wherein the optionally substituted refers to being unsubstituted or substituted with one or more substituents selected from the group consisting of nitro, halogen, hydroxyl , Cyano, C ₁ -C ₁₂ alkyl, C ₁ -C ₁₂ alkoxy, benzamidine, or -C (O) -OR ₆ , wherein the C ₁ -C ₁₂ alkyl, C ₁ -C ₁₂ alkoxy or benzamidine may be further substituted with nitro, halogen, hydroxy, cyano, C ₁ -C ₃ alkyl or C ₁ -C ₃ alkoxy, where R ₆ is hydrogen or C _1- C ₃ alkyl. The method according to claim 20, wherein the MG53 inhibitor is a compound or a pharmaceutically acceptable salt thereof . The method according to claim 20, which is used for relieving, treating or preventing MG53-related diseases or symptoms thereof. The method according to claim 44, wherein the MG53-related disease is a metabolic syndrome. The method according to claim 44, wherein the MG53-related disease is a disease related to glucose metabolism or lipid metabolism. The method according to claim 46, wherein the disease related to glucose metabolism or lipid metabolism is selected from the group consisting of insulin resistance, diabetic cerebrovascular disease, diabetic ocular complications, diabetic neuropathy, diabetic foot, hyperinsulinemia, high Cholesterolemia, hypertension, and obesity. An MG53 antibody or an antigen-binding fragment thereof includes three heavy chain complementarity determining regions contained in the sequence shown in SEQ ID NO: 1, 9, 17, 25, or 33. The MG53 antibody or antigen-binding fragment thereof according to claim 48, comprising three light chain complementarity determining regions contained in SEQ ID NO: 2, 10, 18, 26, or 34 Shown in the sequence. The MG53 antibody or antigen-binding fragment thereof according to claim 48, comprising a heavy chain, wherein the heavy chain has the following amino acid sequence or has at least 80%, 85%, 90%, 95%, or 99% sequence therewith Homologous sequence: SEQ ID NO: 1, 3, 9, 11, 17, 19, 25, 27, 33 or 35. The MG53 antibody or antigen-binding fragment thereof according to claim 50, further comprising a light chain, wherein the light chain has an amino acid sequence as follows or has at least 80%, 85%, 90%, 95%, or 99% thereof Sequences of sequence homology: SEQ ID NO: 2, 4, 10, 12, 18, 20, 26, 28, 34 or 36. An antibody or antigen-binding fragment that competitively binds MG53 with the antibody or antigen-binding fragment according to any one of claims 48-51. An isolated nucleic acid encoding the antibody or antigen-binding fragment according to any one of claims 48-52. The nucleic acid according to claim 53, comprising the following nucleotide sequence or a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence homology to it: SEQ ID NO: 5, 6, 7 , 8, 13, 14, 15, 16, 21, 22, 23, 24, 29, 30, 31, 32, 37, 38, 39, or 40. A clone or an expression vector comprising the isolated nucleic acid according to claim 53 or 54. A host cell comprising the colony or expression vector of claim 55. A pharmaceutical composition comprising the antibody or antigen-binding fragment according to any one of claims 48 to 52, the colony or expression vector according to claim 55, or the cell according to claim 56, and a pharmaceutically acceptable Accepted excipients. Use of a detection reagent for detecting extracellular free MG53 in a preparation kit for detecting MG53-related diseases, predicting the risk or progression of MG53-related diseases, identifying potential MG53 inhibitors, and evaluating MG53-related The therapeutic effect of the disease or detection of the activity of extracellular free MG53. Use of an MG53 inhibitor in the manufacture of a medicament for reducing or inhibiting extracellular free MG53 in an individual.