HK1171767A - Marker protein for type 2 diabetes - Google Patents

Abstract

The present invention provides a marker protein for the early detection of type II diabetes, antibodies directed to the marker protein and their use in a diagnostic method for type II diabetes and in drug development.

Description

Marker protein for type 2 diabetes

The present invention provides diagnostic marker proteins for the early detection of type II diabetes, antibodies directed against said marker proteins and their use in diagnostic methods and drug development for type II diabetes.

Type 2 diabetes (Type 2 diabetes), non-insulin dependent diabetes mellitus (NIDDM), is a disorder characterized by hyperglycemia in the context of insulin resistance and relative insulin deficiency. It is estimated that 2360 million people (7.8% of the population) in the United states have diabetes and 1790 million people are diagnosed, 90% of which are type 2. As the prevalence has doubled between 1990 and 2005, CDC (Centers for Disease Control and Prevention) has characterized this increase as epidemic.

Thus, there is a need for diagnostic markers and methods that allow for the early detection of type II diabetes.

In a first object, the present invention relates to a method for diagnosing type II diabetes or for determining a predisposition of an individual to develop type II diabetes, comprising the steps of:

measuring the polypeptide level of OLFM4 in a tissue sample of the individual, wherein a decreased level of OLFM4 polypeptide in the sample of the individual compared to the level of OLFM4 polypeptide representative of a healthy population is indicative of type II diabetes or a predisposition to develop type II diabetes (predisposition).

In a preferred embodiment, the tissue is blood, preferably plasma.

In a second object, the present invention provides a method of identifying a compound for treating type II diabetes, comprising the steps of:

a) administering the compound to a non-human animal having type II diabetes,

b) measuring the level of OLFM4 polypeptide in the tissue sample of the non-human animal of step a), wherein an altered level of OLFM4 polypeptide in the tissue sample of the non-human animal of step a) compared to the level of OLFM4 polypeptide in the tissue sample of a non-human animal having type II diabetes that has not been administered a compound is indicative of a compound for treating type II diabetes.

In a preferred embodiment, the tissue is blood, preferably plasma.

In another preferred embodiment, the non-human animal is a rodent, preferably a mouse or a rat, more preferably a DIO mouse, an ob/ob mouse or a ZDF rat.

In a third object, the present invention relates to the use of an OLFM4 polypeptide for diagnosing type II diabetes or for determining a predisposition of an individual to develop type II diabetes.

In a preferred embodiment, the OLFM4 polypeptide is a human OLFM4 polypeptide. The amino acid sequence of human OLFM4 is disclosed in seq.

In a fourth object, the present invention provides the use of an antibody that specifically binds to an OLFM4 polypeptide for diagnosing type II diabetes or for determining a predisposition of an individual to develop type II diabetes.

In a preferred embodiment, the antibody binds to a human OLFM4 polypeptide.

In a fifth object, the present invention relates to a kit for diagnosing type II diabetes or determining a predisposition to develop type II diabetes in an individual, comprising:

a) an antibody specific for an OLFM4 polypeptide, preferably an antibody of the invention,

b) labeled antibody (labeled antibody) that binds to OLFM4 captured by the antibody of a), or a labeled antibody that binds to the antibody of a), and

c) reagents for performing diagnostic assays.

In a preferred embodiment, the antibody specific for OLFM4 polypeptide binds to human OLFM4 polypeptide.

The methods of the invention are useful for monitoring the response of type II diabetes therapy in patients undergoing diabetes therapy by measuring the level of OLFM4 polypeptide in tissue samples (preferably blood samples) of these patients. Patients showing altered levels of OLFM4 polypeptide in tissue samples during treatment are responsive to the diabetes therapy compared to the level of OLFM4 polypeptide at the start of the therapy.

In another object, the invention relates to monoclonal antibodies directed against the human OLFM4 polypeptide.

In a preferred embodiment, the antibody is an antibody comprising a V of an antibody obtainable from a hybridoma cell line selected from the group consisting of_HCDR1-CDR3 of Domain: OLFM42/3(DSM ACC3012), OLFM41/46(DSM ACC3011), OLFM 42/1 (DSM ACC3013), OLFM 42/14 (DSM ACC3014), OLFM 42/28 (DSM ACC3015) and OLFM41/23 (DSMAC 3010), and V of an antibody obtainable from a hybridoma cell line selected from the group consisting of_LCDR1-CDR3 of Domain: OLFM42/3(DSM ACC3012), OLFM41/46(DSM ACC3011), OLFM 42/1 (DSMACC3013), OLFM 42/14 (DSM ACC3014), OLFM 42/28 (DSM ACC3015) and OLFM41/23(DSM ACC 3010).

In another preferred embodiment, the antibody is a chimeric antibody comprising a V of an antibody obtainable from a hybridoma cell line selected from the group consisting of_HDomains and V_LDomain (b): OLFM42/3(DSM ACC3012), OLFM41/46(DSM ACC3011), OLFM 42/1 (DSM ACC3013), OLFM 42/14 (DSM ACC3014), OLFM 42/28 (DSM ACC3015) and OLFM41/23(DSM ACC 3010).

In another preferred embodiment, the antibody is produced by a hybridoma cell line selected from the group consisting of: OLFM42/3(DSM ACC3012), OLFM41/46(DSM ACC3011), OLFM 42/1 (DSM ACC3013), OLFM 42/14 (DSM ACC3014), OLFM 42/28 (DSM ACC3015) and OLFM41/23(DSM ACC 3010).

Methods for detecting and/or measuring polypeptides in biological samples are well known in the art and include, but are not limited to, western blots, ELISAs or RIAs, or various proteomic techniques. Monoclonal or polyclonal antibodies or peptide fragments thereof recognizing OLFM4 polypeptide/fragments thereof may be generated (e.g. by immunizing rabbits with purified proteins) for the purpose of detecting the polypeptide or peptide fragments, or known antibodies recognizing the polypeptide or peptide fragments may be used. For example, antibodies capable of binding to denatured proteins (e.g., polyclonal antibodies) can be used to detect OLFM4 polypeptide/fragments thereof in western blots. One example of a method for measuring the label is ELISA. This type of protein quantification is based on an antibody capable of capturing a specific antigen, and a second antibody capable of detecting the captured antigen. Methods for making and using antibodies and the above-mentioned assays are described in Harlow, e, and Lane, d.antibodies: laboratory Manual (antibodies: A laboratory Manual) (1988), Cold Spring harbor laboratory Press (Cold Spring harbor laboratory Press).

In another object, the present invention provides a method of detecting pancreatic β -cells in a tissue sample comprising:

a) providing a pancreatic tissue sample from an individual or non-human animal,

b) detecting OLFM4 positive cells in the tissue sample of a), wherein the OLFM4 positive cells are β -cells.

In a preferred embodiment, the OLFM4 positive cells are detected by an antibody specific for OLFM4, preferably an antibody of the invention.

Methods for detecting β -cells in tissue samples of human subjects or non-human animals can be used to assess the effect of type II diabetes therapy on pancreatic physiology/histology. For example, in the development of compounds for the treatment of type II diabetes, the methods of detecting β -cells of the invention can be used to assess whether the compound has an effect on the physiology/histology of the pancreas, i.e., whether the compound is able to reverse some of the effects of type II diabetes on the pancreas in an animal model of type II diabetes.

In another object, the present invention provides a kit for detecting β -cells in a pancreatic tissue sample, comprising:

a) an antibody specific for an OLFM4 polypeptide, preferably an antibody of the invention,

b) a labeled antibody binding to the antibody of a) or a labeled antibody specific for an OLFM4 polypeptide, and

c) reagents for performing immunohistochemical assays.

Synonyms for polypeptide Olfactomedin 4(OLFM4) are hGC-1 and GW 112.

The term "polypeptide" as used herein refers to a polymer of amino acids, not to a specific length. Thus, peptides, oligopeptides and peptide fragments are encompassed by the definition of polypeptide.

The term "compound" is used herein in the context of a "test compound" or "drug candidate compound" as described in the assays pertaining to the invention. Thus, these compounds include organic or inorganic compounds, obtained synthetically or obtained from natural sources. These compounds include inorganic or organic compounds such as polynucleotide, lipid or hormone analogs, which are characterized by relatively low molecular weights. Other biopolymer organic test compounds include: peptides comprising from about 2 to about 40 amino acids and larger polypeptides comprising from about 40 to about 500 amino acids, such as antibodies or antibody conjugates.

The term "antibody" encompasses various forms of antibody structures, including, but not limited to, intact antibodies and antibody fragments. The antibody of the present invention is preferably a humanized antibody, a chimeric antibody or a further genetically engineered antibody, as long as it retains the characteristic properties described in the present invention.

An "antibody fragment" includes a portion of a full-length antibody, preferably its variable domain, or at least its antigen-binding site. Examples of antibody fragments include diabodies (diabodies), single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. scFv antibodies are, for example, the antibodies described in Houston, J.S., Methodsin Enzymol (methods in enzymology) 203(1991) 46-96). In addition, antibody fragments include single chain polypeptides having the properties of a VH domain (i.e. capable of assembly with a VL domain), or a VL domain which binds ANG-2 (i.e. capable of assembly with a VH domain), which assembly forms a functional antigen binding site and thereby provides said properties.

The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules of single amino acid composition.

The term "chimeric antibody" refers to an antibody comprising a variable region, i.e., a binding region, from one source or species and at least a portion of a constant region obtained from a different source or species, which is typically prepared by recombinant DNA techniques. Chimeric antibodies comprising murine variable regions and human constant regions are preferred. Other preferred forms of "chimeric antibodies" encompassed by the invention are those in which the constant region from the original antibody has been modified or altered to produce the properties of the invention, particularly with respect to C1q binding and/or Fc receptor (FcR) binding. Such chimeric antibodies are also referred to as "type-switch antibodies". Chimeric antibodies are the product of expressed immunoglobulin genes comprising a DNA segment encoding an immunoglobulin variable region and a DNA segment encoding an immunoglobulin constant region. Methods for producing chimeric antibodies involve conventional recombinant DNA, and gene transfection techniques are well known in the art. See, e.g., Morrison, S.L., et al, Proc. Natl. Acad. Sci. USA (Proc. Natl. Acad. Sci. USA) 81(1984) 6851-6855; U.S. patent nos. 5,202,238 and 5,204,244.

The term "human antibody" as used herein is intended to include antibodies having variable and constant regions obtained from human germline immunoglobulin sequences. Human antibodies are well known in the state of the art (van Dijk, m.a., and van de Winkel, j.g., curr. opin. chem.biol. (current chemical biology perspective) 5(2001) 368-. Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable of producing a complete repertoire or selection of human antibodies upon immunization, without endogenous immunoglobulin production. Transfer of human germline immunoglobulin gene arrays in such germline mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al, Proc. Natl. Acad. Sci. USA (Proc. Natl. Acad. Sci. USA) 90(1993) 2551-2555; Jakobovits, A., et al, Nature (Nature) 362(1993) 255-258; Bruggemann, M., et al, Yeast Immunol. (annual immunology) 7(1993) 33-40). Human antibodies can also be generated in phage display libraries (Hoogenboom, H.R., and Winter, G., J.mol.biol. (J.Mol.Biol.) (J.Biol.) -227 (1992)) 381-. The techniques of Cole et al and Boerner et al are also available for the preparation of human Monoclonal Antibodies (Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, p.77 (1985); and Boerner, P.et al, J.Immunol.147 (1991) 86-95). As already mentioned in the chimeric and humanized antibodies according to the invention, the term "human antibody" as used herein also includes antibodies which have been modified in the constant region to produce the properties described in the present invention, in particular with respect to C1q binding and/or FcR binding, for example by "type switching", i.e. by altering or mutating the Fc part (e.g. from IgG1 to IgG4 and/or IgG1/IgG4 mutations).

The term "epitope" includes any polypeptide determinant capable of specifically binding to an antibody. Epitope determinants include groups of chemically active surface molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and may, in certain embodiments, have specific three-dimensional structural characteristics, and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody.

"variable Domain" (light chain variable Domain (V)_L) Heavy chain variable domain (V)_H) As used herein) means each of the pair of light and heavy chain domains that are directly involved in binding of the antibody to the antigen. The variable light and heavy chain domains have the same general structure, and each domain includes four Framework (FR) regions, the sequences of which are universally conserved and connected by three "hypervariable regions" (or complementarity determining regions, CDRs). The framework regions adopt a β -sheet conformation and the CDRs may form loops connecting the β -sheet structures. The CDRs in each chain retain their three-dimensional structure through the framework regions and form together with the CDRs from the other chain an antigen binding site. The antibody heavy and light chain CDR3 regions play a particularly important role in the binding specificity/affinity of the antibodies according to the invention and thus provide another object of the invention.

The term "antigen-binding portion of an antibody" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The antigen-binding portion of an antibody includes amino acid residues from a "complementarity determining region" or "CDR". "framework" or "FR" regions are those variable domain regions other than the hypervariable region residues defined herein. Thus, the light and heavy chain variable domains of an antibody comprise, from N-terminus to C-terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR 4. In particular, the CDR3 of the heavy chain is the region that contributes primarily to antigen binding and defines the properties of the antibody. CDR and FR regions are determined according to the standard definition of Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition Public Health Service, National Institutes of Health, Besserda, MD (1991) and/or those residues from the "hypervariable loop".

Monoclonal antibodies can be prepared using hybridoma methods such as those described by Kohler and Milstein, Nature (Nature) 256: 495 (1975). In the hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to induce lymphocytes that produce, or are capable of producing, antibodies that specifically bind to the immunizing agent.

Any of a number of well-known protocols for fusing lymphocytes and immortalized Cell lines can be used for the purpose of producing Monoclonal Antibodies of the invention (see, e.g., G.Galfre et al (1977) Nature (Nature) 266: 55052; Gefter et al viral Cell Genet. (Somatic genetics), cited above; Lerner, Yale J.biol. Med. (J.Med. Yale., cited above; Kenneth, Monoclonal Antibodies, cited above). In addition, the skilled worker will appreciate that there are many variations of such methods, which would also be useful. Typically, the immortalized cell line (e.g., a myeloma cell line) is obtained from the same mammalian species as the lymphocytes. For example, murine hybridomas can be prepared by fusing lymphocytes from mice immunized with an immunogenic formulation of the invention with an immortal mouse cell line. A preferred immortalized cell line is a mouse myeloma cell line that is sensitive to medium containing hypoxanthine, aminopterin and thymidine ("HAT medium"). Any of a number of myeloma cell lines, such as the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines, may be used as fusion partners according to standard techniques. These myeloma lines are available from ATCC. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG"). The fusion-producing hybridoma cells are then selected using HAT medium, which kills unfused and non-productive fused myeloma cells (unfused splenocytes die after a few days because they are not transformed). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for bound antibody, e.g., using a standard ELISA assay.

Antibodies can be produced using recombinant methods and compositions (e.g., as described in U.S. Pat. No. 4,816,567). In one embodiment, an isolated nucleic acid encoding an anti-OLFM 4 antibody described herein is provided. Such nucleic acids may encode V comprising an antibody_LAnd/or a V comprising an antibody_HE.g., the light chain and/or heavy chain of an antibody. In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In another embodiment, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) vectors comprising nucleic acids encoding V comprising antibodies_LAnd V comprising an antibody_HOr (2) comprises a V encoding an antibody-containing antibody_LAnd a first vector comprising a nucleic acid encoding an antibody-containing V_HA second vector for a nucleic acid of the amino acid sequence of (a). In one embodiment, the host cell is eukaryotic, such as a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, there is provided a method of making an anti-TMEM 27 antibody, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody, as provided above, and optionally recovering the antibody from the host cell (or host cell culture medium).

For recombinant production of the antibodies of the invention, nucleic acids encoding the antibodies, e.g., as described above, are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of an antibody).

Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, especially when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. nos. 5,648,237, 5,789,199, and 5,840,523. (see also Charlton, Methods in molecular Biology, vol.248 (B.K.C.Lo, eds., Humana Press, Totowa, NJ, 2003), pp.245-254, describing the expression of antibody fragments in E.coli (E.coli.). After expression, the antibody can be isolated from the bacterial cell paste as a soluble fraction and can be further purified.

Methods for cloning antibody genes from monoclonal antibody-producing hybridoma cells are known to those skilled in the art. For example, the variable heavy and light chain domains (V)_HAnd V_L) The genetic information of (a) can be amplified from hybridoma cells by immunoglobulin-specific primers using the Polymerase Chain Reaction (PCR) (Methods Mol med.2004; 94: 447-58). Encoding variable heavy and light chain domains (V)_HAnd V_L) The nucleic acid of (a) may then be cloned in an appropriate vector for expression in a host cell.

Brief description of the drawings:

FIG. 1A: OLFM4 polypeptide was detected in 10 human plasma samples by ELISA using antibody pairs OLFM4-1/23 and OLFM4-2/3,

FIG. 1B: OLFM4 polypeptide was detected in 10 human plasma samples by ELISA using antibody pairs OLFM4-2/1 and OLFM4-2/28,

FIG. 1C: OLFM4 polypeptide was detected in 10 human plasma samples by ELISA using antibody pairs OLFM4-2/28 and OLFM4-2/14,

FIG. 2A: immunoprecipitation (IP) using monoclonal antibody OLFM4-2/1 with 10 human plasma samples,

FIG. 2B: immunoprecipitation (IP) using monoclonal antibody OLFM4-2/3 with 10 human plasma samples,

FIG. 2C: immunoprecipitation (IP) using monoclonal antibody OLFM4-2/28 with 10 human plasma samples,

FIG. 2D: immunoprecipitation (IP) using monoclonal antibody OLFM4-2/14 with 10 human plasma samples,

FIG. 3A: detecting OLFM4 polypeptide in plasma samples of human subjects selected from the group consisting of: healthy controls, Impaired Fasting Glucose (IFG), Impaired Glucose Tolerance (IGT), impaired fasting glucose + impaired glucose tolerance (IFG + IGT), type 1 diabetes mellitus patients (T1D) and type 2 diabetes mellitus patients (T2D),

FIG. 3B: detecting OLFM4 polypeptide in plasma samples of human subjects selected from the group consisting of the following by ELISA using antibody pairs OLFM 42/28 and OLFM 42/14: healthy controls, Impaired Fasting Glucose (IFG), Impaired Glucose Tolerance (IGT), and impaired fasting glucose + impaired glucose tolerance (IFG + IGT), type 1 diabetes mellitus patients (T1D) and type 2 diabetes mellitus patients (T2D),

FIG. 4A: immunohistochemical (IHC) staining of a human tissue array using monoclonal antibody hOLFM 41/46,

FIGS. 4B and C: human islets were stained with monoclonal antibody hOLFM 41/46 (OLFM 4: green, glucagon: red, DAPI: blue).

Examples

Monoclonal anti-human OLFM4 antibody of the present invention

The following five mouse hybridoma cell lines producing monoclonal antibodies against human OLFM4 have been deposited at DSMZ- (german collection of microorganisms and cell cultures (deutsche samlun von Mikroorganismen und Zellkulturen GmbH)) on behalf of hoffmann-larch Ltd at 10/7 th 2009 and received the following deposit numbers:

OLMF4-1/23 ＝DSM ACC3010

OLMF4-1/46 ＝DSM ACC3011

OLMF4-2/3 ＝DSMACC3012

OLMF4-2/1 ＝DSM ACC3013

OLMF4-2/14 ＝DSM ACC3014

OLMF4-2/28 ＝DSM ACC3015

generation of mouse monoclonal antibodies against human OLFM4 (mouse OLFM4 mAbs)

The amino acid sequence of the recombinant human OLFM4 fusion polypeptide used to produce monoclonal antibodies is given below:

GSGGSVSQLFSNFTGSVDDRGTCQCSVSLPDTTFPVDRVERLEFTAHVLSQKFEKE

LSKVREYVQLISVYEKKLLNLTVRIDIMEKDTISYTELDFELIKVEVKEMEKLVIQL

KESFGGSSEIVDQLEVEIRNMTLLVEKLETLDKNNVLAIRREIVALKTKLKECEASK

DQNTPVVHPPPTPGSCGHGGVVNISKPSVVQLNWRGFSYLYGAWGRDYSPQHPN

KGLYVAPLNTDGRLLEYYRLYNTLDDLLLYINARELRTYGQGSGTAVYNNNMYV

NMYNTGNIARVNLTTNTIAVTQTLPNAAYNNRFSYAVAWQDIDFAVDENGLWVI

YSTEASTGNMVISKLNDTTLQVLNTWYTKQYKPSASNAFMVCGVLYATRTMNTR

TEEIFYYDTNTGKEGKLDIVMHKMQEKVQSINYNPFDQLYVYNDGYLL-

NYDLSVLQKPQHHHHHH(Seq.Id.No.2)

mice were immunized with His-tag-coupled recombinant OLFM 45 μ g/injection generated in insect cells. Immunization was carried out on days 0, 13 and 28 in ImmunEasy adjuvant (ALHY-DROGEL 2% + CPG-ODN) in a volume ip of 20. mu.l. Animals were evaluated for immune response to recombinant OLFM4 by ELISA by collecting blood from day 41. Superboost (superbost) (5. mu.g recombinant OLFM4 iv in PBS) 2 selected animals on day 56, splenocytes were fused with PAI-cells after 2 days. Hybridoma screening and clonal evaluation were performed by ELISA against recombinant OLFM 4.

ELISA specificity verification

Three pairs of mAbs of the invention were used in ELISA:

	coating-mAbs	detection-mAb
			1	OLFM4-1/23	OLFM 4-2/3-biotin
2	OLFM4-2/1	OLFM 4-2/28-biotin
			3	OLFM4-2/28	OLFM 4-2/14-biotin

ELISA-results of 10 control human plasma

The results of the assay are given in fig. 1A-1C:

hu 1-Hu 10: control human serum (blood donor human plasma)

Positive control (OLFM 4): INS-1hOLFM4 WT F11

Negative control (med): culture medium

Testing 10 human plasma samples using three different ELISA assays gave very similar results. This verifies the specificity of the assay. These samples were further used to qualitatively verify these ELISA results by Immunoprecipitation (IP).

Qualitative confirmation of ELISAs by IP (immunoprecipitation (IP) -Final selected mAb pairs blood donor human plasma and INS-1hOLFM4 WT F11/Medium)

Human plasma samples used in previous ELISAs were used for Immunoprecipitation (IP) to assess whether similar results were obtained by different techniques (see fig. 2A-D). There was a perfect match between ELISA and IP results. This is very important as it is a qualitative confirmation of the results using two different techniques.

The following samples and antibodies were used in the IP experiments:

sample preparation:

MWM: labeling with molecular weight; the control was the same as in the ELISA assay.

Antibody:

FIG. 2A: OLFM4-2/1

FIG. 2B: OLFM4-2/3

FIG. 2C: OLFM4-2/28

FIG. 2D: OLFM4-2/14

The results of the IP assay are given in FIGS. 2A-D.

Positive samples (#1-5 and #7-10) in ELISA were also positive in IP. Samples (#6) that were negative in ELISA were also negative in IP. INS-1 OLFM4 supernatant and medium were used as positive and negative controls, respectively. This is a qualitative confirmation of the ELISA results by IP.

Results of ELISA on human cross-sectional cohort (cross sectional cohort): OLFM4 was significantly reduced in pre-diabetes (pre-diabetic) and diabetic patients (Bratislava cohort).

FIG. 3A +3B

Human plasma queue

Subject screening

Approximately 200 subjects with a metabolic risk of T2D from an outpatient clinic registry who meet the following inclusion criteria:

sex: male sex

Age: 40-55 years old

BMI：25-32kg/m2

HbAlC≤7.0％

An oral glucose tolerance test (75g) was performed. Exclusion criteria included previously known alterations in glucose metabolism, use of drugs known to alter insulin secretion or action, and the presence of liver or endocrine disorders. Before blood samples were collected, height and weight were assessed using standard protocols. Body Mass Index (BMI) is calculated as weight (kilograms) divided by height (meters) squared. Whole blood samples (20ml) from the antecubital vein were collected into EDTA tubes after 10-12 hours overnight fast and after 2 hours glucose load. To obtain a suitable fasting state, the participants are provided with precise instructions regarding the type of food and the time of their last intake. Plasma separated by centrifugation was stored in 1ml aliquots (10 ×) at-80 ℃ prior to analysis.

Patients who signed informed consent and met eligibility criteria were enrolled in the study. The Ethical committee (The Ethical committee) of The Institute for experimental Endocrinology (Institute of experimental Endocrinology) of The scholvacco Academy of Sciences approved The protocol of The study.

Diagnosis of

Subjects were treated according to ADA guidelines 2005(Diabetes Care.(diabetes Care) 1 month 2005; 28 supplement 1: s37-42) into 4 different groups:

healthy control: fasting Plasma Glucose (FPG) < 5.6mmol/l and Normal Glucose Tolerance (NGT) < 7.8 mmol/l.

Impaired Fasting Glucose (IFG): IFG is defined as FPG value between ≥ 5.6 and < 6.9mmol/l and Normal Glucose Tolerance (NGT) < 7.8mmol/l 2 hours after challenge.

Impaired Glucose Tolerance (IGT): IGT is defined as the glucose concentration between ≥ 7.8 and < 11.1mmol/l 2 h after loading.

Impaired fasting glucose and glucose tolerance (IFG + IGT): IGT + IFG is defined as a FPG value between 5.6 and < 6.9mmol/l and an NGT value 2 hours after the challenge between 7.8 and < 11.1 mmol/l).

In addition, a group of 8 type 1 diabetic patients and a group of 11 type 2 patients from an outpatient clinic registry were also selected. Patients with dyslipidemia are treated with hypolipidemic agents (hypolipidemics), such as statins or fibrates.

Summary of diabetic subjects

Average anthropometric and laboratory characterization of subjects with type 1 diabetes (T1-DM), type 2 diabetes (T2-DM), Impaired Glucose Tolerance (IGT), Impaired Fasting Glucose (IFG), Impaired Glucose Tolerance (IGT), and IFG + IGT

The following OLFM4 monoclonal antibody pairs were used in the ELISA assay:

FIG. 3A: antibody: 2/1-2/28

FIG. 3B: antibody: 2/28-2/14

Cross-cohort ELISA results showed significantly lower levels of OLFM4 in pre-diabetic patients (IFG + IGT, IFG and IGT) than in healthy control patients (fig. 3A and 3B). OLFM4 levels were also lower in patients with T2 DM. Interestingly, OLFM4 levels were higher in T1DM patients, although not significant (Dunnett's modified ANOVA). Patients in both T2DM and T1DM groups were undergoing treatment.

Given the significant reduction in OLFM4 in untreated pre-diabetic patients, we believe that OLFM4 may be useful as a marker for early T2D pathogenesis.

OLFM4 as a marker for pancreatic beta-cells

Immunohistochemical (IHC) staining of a Human Tissue Array (Human Tissue Array) using monoclonal antibody hOLFM 41/46. Reliable (robust) results showed no specific staining in any of the tissues tested, whereas a very strong and specific signal was detected in the beta-cells of human islets (human pancreatic sections). Note that pancreatic sections show negativity in tissue microarrays, as only exocrine tissue and no islet structure is present in the pancreatic spots of such tissue microarrays.

FIG. 4A: human tissue array stained with monoclonal antibody hOLFM 41/46

FIGS. 4B and C: human islets stained with monoclonal antibody hOLFM 41/46 (OLFM 4: green, glucagon: red, DAPI: blue).

Materials and methods

ELISA protocol

Coating:

coating-mAb: 5. mu.g/ml in PBS, 100. mu.l/well

→ in a humid cabinet overnight at 4 ℃

Wash PBS-Tween → 2 ×

And (3) sealing:

b-buffer solution

200. mu.l/well

→ at 37 ℃ for 1h

Wash PBS-Tween → 2 ×

Sample and detection-mAb:

biotinylated detection-mAb, 1 μ g/ml in B-buffer: 25 μ l/well

Sample (human plasma): dilution in B-buffer, starting from undiluted plasma, 1: 2 to 1: 128(8 concentrations) 30. mu.l/well

Add the first 25. mu.l of detection-mAb to the plate, followed by 30. mu.l of sample

→ in a humid cabinet at 4 ℃ overnight on a shaker

Wash PBS-Tween → 4 ×

Conjugate:

PIERCE streptavidin (Streptavidine) -HRPO (No 21126), 1. mu.g/ml, in B-buffer

50 μ l/well

→ at room temperature for 1h

Wash PBS-Tween → 4 ×

Substrate：

3, 3 ', 5, 5' -Tetramethylbenzidine (TMB), 100. mu.l/well

After 5min, the decoction is taken with 0.5M H₂SO₄Reaction was stopped at 100. mu.l/well

Reading at 450nm

Cell culture

Doxycycline (doxycline) induced the rat insulinoma INS-1hOLFM4 WT and INS-1hOLFM4-His stable cell lines (wild type (hOLFM4 WT) and His-tagged (hOLFM4-His) human OLFM4 forms, respectively) were cultured as previously described (Wang et al 2001). Both INS-1 cell lines were cultured in RPMI 1640+ GlutaMAX-1 medium (Invitrogen, Calsbad, Calif.) containing 10mM Hepes (pH 7.4), 1mM sodium pyruvate, 50. mu.M 2-mercaptoethanol, 10% heat-inactivated Fetal Bovine Serum (FBS), penicillin and streptomycin. Fifty μ G/ml G418 sulfate (Promega, Madison, Wis.) and 50 μ G/ml zeocin (Invitrogen) were added for growth selection. Overexpression of hOLFM4 WT and hOLFM4-His was induced by 500ng/ml doxycycline (Dox) (Sigma) for 96 hours. Cells were grown in a humidified incubator at 37 ℃ and 5% CO2 (subscript 2).

Immunoprecipitation (IP) and immunoblotting (Western blot, WB)

60-90% confluent cells were cultured in 10cm dishes for 96 hours in the presence or absence of 500ng/ml doxycycline. The supernatant (cell culture medium) was harvested under sterile conditions, centrifuged at 2000rpm for 10 minutes, and stored at 4 ℃. Cells were washed twice in 1 × PBS and lysed with 1mL lysis buffer. After 5 minutes, the cells were collected in a 1.5mL Eppendorf tube and centrifuged at full speed for 5 minutes. The supernatant (whole cell extract) was collected, aliquoted, snap frozen in liquid nitrogen and stored at-80 ℃. For IP, 3mL of supernatant (cell culture medium) was mixed with 1. mu.g of each mAb and incubated for 48 hours at 4 ℃ on an orbital shaker. Twenty-five μ L protein a sepharose CL-4B diluted 50% in 1X PBS-tween (0.05%) was added to each reaction and incubated for 1 hour at RT on an orbital shaker. The tube was spun down to obtain a pellet and the pellet was washed 2 times with 1X PBS-tween (0.05%) and 1 time with 1X PBS. Thirty-five μ L of 1X LDS-SB/10% β -ME was added to each pellet and the sample was vortexed vigorously (which word: vortexed. Immunoblotting, using enhanced chemiluminescence (Pierce, Rockford, IL, USA) detection, was performed as described previously (WangH, J Biol Chem (journal of biochemistry) 2001).

Immunohistochemistry (IHC)

Formalin Fixed Paraffin Embedded (FFPE) sections were used to assemble slides. The samples were dehydrated sequentially by soaking the slides in xylene (x2), 100% EtOH, 95% EtOH, 80% EtOH, 70% EtOH, and 1XPBS (3 minutes each). Antigen retrieval was performed by soaking slides in 1X citrate buffer and boiling them in a microwave oven (at 850 watts) for 3 minutes. After rinsing the slides twice with water, the cells were permeabilized (permeilized) for 10 min at RT with 100 μ L of 0.2% Triton in 1X PBS. After 3 washes with 1 × PBS, blocking was performed with 2% BSA in 1 × PBS at RT for 30' to 1 h. Washed three more times with 1XPBS, followed by primary Ab incubation (1-2 hours at 37 ℃ or O/N at 4 ℃). After three more washes with 1 × PBS, the secondary Ab was incubated with RT for 1h in the dark. Three more washes were performed and DAPI staining was performed (5-10 min at RT in the dark). Three final washes and assemble the coverslip.

FDA standard human tissue microarrays (T8234700, Biochain) were stained with mouse anti-OLFM 4 monoclonal antibody, followed by Alexa 488-conjugated donkey anti-mouse and Alexa 555 donkey anti-rabbit secondary antibody (Invitrogene).

Human pancreatic sections obtained by Asterand were co-stained with both mouse anti-OLFM 4 monoclonal antibody and rabbit anti-glucagon polyclonal antibody, followed by staining with Alexa 488-conjugated donkey anti-mouse and Alexa 555 donkey anti-rabbit secondary antibody (Invitrogene).

While there have been shown and described what are at present considered to be the preferred embodiments of the invention, it is to be distinctly understood that the invention is not limited thereto but may be otherwise embodied and carried out within the scope of the appended claims.

Claims (24)

1. A method for diagnosing type II diabetes or determining a predisposition of an individual to develop type II diabetes, comprising the steps of:

measuring the level of OLFM4 polypeptide in a tissue sample of the subject, wherein a decreased level of OLFM4 polypeptide in the sample of the subject as compared to the level of OLFM4 polypeptide representative of a healthy population is indicative of type II diabetes or a predisposition to develop type II diabetes.

2. The method of claim 1, wherein the tissue is blood, preferably plasma.

3. A method of identifying a compound for treating type II diabetes comprising the steps of:

c) administering the compound to a non-human animal having type II diabetes,

d) measuring the level of OLFM4 polypeptide in the tissue sample of the non-human animal of step a), wherein an altered level of OLFM4 polypeptide in the tissue sample of the non-human animal of step a) compared to the level of OLFM4 polypeptide in the tissue sample of a non-human animal having type II diabetes that has not been administered a compound is indicative of a compound for treating type II diabetes.

4. The method of claim 3, wherein the tissue is blood, preferably plasma.

5. The method of claim 3 or 4, wherein the non-human animal is a rodent, preferably a mouse or a rat.

6. The method of claim 5, wherein the rodent is a ZDF rat or an ob/ob mouse.

Use of an OLFM4 polypeptide for diagnosing type II diabetes or for determining a predisposition of an individual to develop type II diabetes.

8. The use of claim 7, wherein said OLFM4 polypeptide is a human OLFM4 polypeptide.

9. Use of an antibody that specifically binds to an OLFM4 polypeptide for diagnosing type II diabetes or for determining a predisposition of an individual to develop type II diabetes.

10. The use of claim 9, wherein the antibody binds to a human OLFM4 polypeptide.

11. A kit for diagnosing type II diabetes or determining a predisposition for developing type II diabetes in an individual, comprising:

d) an antibody specific for an OLFM4 polypeptide, preferably an antibody according to claims 13-16,

e) a labeled antibody that binds to a) the antibody, or a labeled antibody that binds to a) the captured OLFM4 polypeptide, and

f) reagents for performing diagnostic assays.

12. The kit of claim 11, wherein said antibody specific for an OLFM4 polypeptide binds to a human OLFM4 polypeptide.

13. A monoclonal antibody directed against a human OLFM4 polypeptide.

14. The antibody of claim 13, wherein the antibody comprises a V of an antibody obtainable from a hybridoma cell line selected from the group consisting of_HCDR1-CDR3 of Domain: OLFM42/3(DSM ACC3012), OLFM41/46(DSM ACC3011), OLFM 42/1 (DSMACC3013), OLFM 42/14 (DSM ACC3014), OLFM 42/28 (DSM ACC3015) and OLFM41/23(DSM ACC3010), and V of an antibody obtainable from a hybridoma cell line selected from the group consisting of_LCDR1-CDR3 of Domain: OLFM42/3 (DSMACC3012), OLFM41/46(DSM ACC3011), OLFM 42/1 (DSM ACC3013), OLFM 42/14 (DSM ACC3014), OLFM 42/28 (DSM ACC3015) and OLFM41/23(DSM ACC 3010).

15. The antibody of claim 13 or 14, wherein the antibody comprises a V of an antibody obtainable from a hybridoma cell line selected from the group consisting of_HDomains and V_LDomain (b): OLFM42/3(DSM ACC3012), OLFM41/46(DSM ACC3011), OLFM 42/1 (DSMACC3013), OLFM 42/14 (DSM ACC3014), OLFM 42/28 (DSM ACC3015) and OLFM41/23(DSM ACC 3010).

16. The antibody of claims 13-15, wherein the antibody is produced by a hybridoma cell line selected from the group consisting of: OLFM42/3(DSM ACC3012), OLFM41/46 (DSMACC3011), OLFM 42/1 (DSM ACC3013), OLFM 42/14 (DSM ACC3014), OLFM 42/28 (DSM ACC3015) and OLFM41/23(DSM ACC 3010).

17. A hybridoma cell line selected from the group consisting of: OLFM42/3 (DSMACC3012), OLFM41/46(DSM ACC3011), OLFM 42/1 (DSM ACC3013), OLFM 42/14 (DSM ACC3014), OLFM 42/28 (DSM ACC3015) and OLFM41/23(DSM ACC 3010).

18. A nucleic acid sequence comprising a V encoding an antibody obtainable from a hybridoma cell line selected from the group consisting of_HSequence of the domains: OLFM42/3(DSM ACC3012), OLFM41/46(DSM ACC3011), OLFM 42/1 (DSM ACC3013), OLFM 42/14 (DSMACC3014), OLFM 42/28 (DSM ACC3015) and OLFM41/23(DSM ACC 3010).

19. A nucleic acid sequence comprising a V encoding an antibody obtainable from a hybridoma cell line selected from the group consisting of_LSequence of the domains: OLFM42/3(DSM ACC3012), OLFM41/46(DSM ACC3011), OLFM 42/1 (DSM ACC3013), OLFM 42/14 (DSMACC3014), OLFM 42/28 (DSM ACC3015) and OLFM41/23(DSM ACC 3010).

Use of an OLFM4 polypeptide as a marker for pancreatic β -cells.

21. A method of detecting pancreatic β -cells in a tissue sample, comprising:

c) providing a pancreatic tissue sample from an individual or non-human animal,

d) detecting OLFM4 positive cells in the tissue sample of a), wherein the OLFM4 positive cells are β -cells.

22. The method of claim 21, wherein the OLFM4 positive cells are detected by an antibody specific for OLFM4, preferably an antibody of claims 13-16.

23. A kit for detecting β -cells in a pancreatic tissue sample comprising:

a) an antibody specific for an OLFM4 polypeptide, preferably an antibody according to claims 13-16,

b) a labeled antibody that binds to the antibody of a), and

c) reagents for performing immunohistochemical assays.

24. The methods and antibodies substantially as hereinbefore described, especially with reference to the foregoing examples.