The present invention relates to an in vitro method aiding in the assessment of type II diabetes (T2D). It discloses the use of the protein TMEM 27 as a marker of T2D. Furthermore, it especially relates to a method for assessing T2D from a sample, derived from an individual by measuring the protein TMEM27 in said sample in vitro. The invention further relates to antibodies against human TMEM27 protein, methods for their production, and uses thereof.

Background of the Invention

Type 2 diabetes (T2D, also known as non-insulin dependent diabetes mellitus (NIDDM)) is a disorder that is characterized by high blood glucose in the context of insulin resistance and relative insulin deficiency. There are an estimated 23.6 million people in the U.S. (7.8% of the population) with diabetes with 17.9 million being diagnosed, 90% of whom are type 2. With prevalence rates doubling between 1990 and 2005, CDC (Centers for Disease Control and Prevention) has characterized the increase as an epidemic.

Thus, it is an object of the present invention to find diagnostic markers and develop sensitive in vitro methods allowing an early detection of type II diabetes in a body fluid sample obtained from an individual.

In order to be of clinical utility, a new diagnostic marker as a single marker should be comparable to other markers known in the art, or better. Or, a new marker should lead to an improvement in diagnostic sensitivity or specificity either if used alone or if used in combination with one or more other markers, respectively.

Whole blood, serum or plasma are the most widely used sources of sample in clinical routine. The identification of an early T2D marker that would aid in the reliable T2D detection or provide early prognostic information could lead to a method that would greatly aid in the diagnosis and in the management of this disease. Therefore, an urgent clinical need exists to improve the in vitro assessment of T2D. It is especially important to improve the early diagnosis of T2D, since for patients diagnosed in early stages of T2D have later on less complications as compared to those patients diagnosed at a more progressed stage of disease. It was the object of the present invention to investigate whether a biochemical marker can be identified which may be used in assessing T2D in vitro. In particular, the inventors of the present invention investigated whether a biochemical marker could be identified for the assessment of T2D in a body fluid sample. Summary of the invention

It has now been found that the use of protein TMEM27 can at least partially overcome some of the problems of the methods available for assessment of T2D presently known.

It has further been found, that monoclonal antibodies according to the invention show benefits in the in vitro assessment of type II diabetes (T2D) in a human subject.

Surprisingly it was found in the present invention that an increased concentration of human transmembrane protein 27 (TMEM27) in a serum, plasma, or whole blood sample compared to a concentration of protein TMEM27 representative for a reference sample is indicative for diagnosis of type II diabetes (T2D) and thereby will also allow to identify the majority of those patients at risk to develop a T2D.

Disclosed herein is an in vitro method for assessing T2D comprising determining in a serum, plasma, or whole blood sample the concentration of human protein TMEM27 by an immunological detection method and using the determined result, particularly the concentration determined, in the assessment of T2D.

The invention also relates to an in vitro method for assessing type II diabetes (T2D) in a human subject, comprising a) determining the concentration of human transmembrane protein 27 (TMEM27) in a serum, plasma, or whole blood sample by an immunoassay procedure, and b) comparing the concentration of protein TMEM27 determined in step (a) with a reference concentration of protein

TMEM27, wherein a concentration of protein TMEM27 above a reference concentration is indicative for the presence of T2D.

In a further embodiment the present invention relates to a use of the human protein TMEM27 in the in vitro assessment of T2D in a serum, plasma, or whole blood sample, wherein a concentration of protein TMEM27 above a reference concentration for protein TMEM27 is indicative for the presence of T2D. In a further embodiment the present invention relates to the use of a monoclonal antibody that specifically binds to human protein TMEM27 in the in vitro assessment of T2D in a serum, plasma, or whole blood sample, wherein a concentration of protein TMEM27 above a reference concentration for protein TMEM27 is indicative for the presence of T2D.

In a further embodiment the present invention relates to a monoclonal antibody directed to human protein TMEM27.

In a further embodiment the present invention relates to the hybridoma cell line MAK<H-TMEM27>M-2.3.3 which was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) on February 1, 2011 and received the deposit number DSM ACC3116 or hybridoma cell line MAK<H-TMEM27>M- 2.1.34 which was deposited with the DSMZ on February 1, 2011 and received the deposit number DSM ACC3115, respectively.

In a further embodiment the present invention relates to a nucleic acid molecule comprising a sequence encoding the V _H domain of the monoclonal antibody obtained from hybridoma cell line MAK<H-TMEM27>M-2.3.3 or hybridoma cell line MAK<H-TMEM27>M-2.1.34, respectively.

In a further embodiment the present invention relates to a nucleic acid molecule comprising a sequence encoding the V _L domain of the monoclonal antibody obtained from hybridoma cell line MAK<H-TMEM27>M-2.3.3 or hybridoma cell line MAK<H-TMEM27>M-2.1.34, respectively.

In a further embodiment the present invention relates to a nucleic acid molecule comprising a sequence encoding the monoclonal antibody produced by hybridoma cell line MAK<H-TMEM27>M-2.3.3 which was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) on February 1, 2011 and received the deposit number DSM ACC3116 or hybridoma cell line MAK<H- TMEM27>M-2.1.34 which was deposited with the DSMZ on February 1, 2011 and received the deposit number DSM ACC3115, repsectively.

In a further embodiment the present invention relates to a vector comprising a nucleic acid sequence of the monoclonal antibody obtained from hybridoma cell line MAK<H-TMEM27>M-2.3.3 or hybridoma cell line MAK<H-TMEM27>M- 2.1.34, respectively. In a further embodiment the present invention relates to a host cell comprising a vector comprising a nucleic acid sequence of the monoclonal antibody obtained from hybridoma cell line MAK<H-TMEM27>M-2.3.3 or hybridoma cell line MAK<H-TMEM27>M-2.1.34, respectively. Also provided is a kit for performing the in vitro method for assessing T2D according to the present invention comprising the reagents required to specifically determine the concentration of human protein TMEM27.

Detailed Description of the Invention

The inventors of the present invention have surprisingly been able to demonstrate that the marker protein transmembrane protein 27 (TMEM27) is useful in the assessment of T2D.

The method of the present invention is suitable for the assessment of T2D. In more detail the method is suitable for the assessment of the presence or absence of T2D. Increased concentrations of human protein TMEM27 in a serum, plasma, or whole blood sample as compared to normal controls have been found to be indicative for T2D.

In an embodiment the present invention relates to an in vitro method for assessing type II diabetes (T2D) in a human subject, comprising a) determining the concentration of human transmembrane protein 27 (TMEM27) in a serum, plasma, or whole blood sample by an immunoassay procedure, and b) comparing the concentration of protein TMEM27 determined in step (a) with a reference concentration of protein TMEM27, wherein a concentration of protein TMEM27 above a reference concentration is indicative for the presence of T2D.

In a further embodiment the present invention relates to an in vitro method for assessing the presence or absence of type II diabetes (T2D) in a human subject, comprising a) determining the concentration of human TMEM27 in a serum, plasma, or whole blood sample by an immunoassay procedure, and b) comparing the concentration of protein TMEM27 determined in step (a) with a reference concentration of protein TMEM27, wherein a concentration of protein TMEM27 above a reference concentration is indicative for the presence of T2D.

In a further embodiment the concentration of TMEM27 is determined in the in vitro method according to the present invention with an monoclonal antibody produced by hybridoma cell line MAK<H-TMEM27>M-2.3.3 (DSM ACC3116) or hybridoma cell line MAK<H-TMEM27>M-2.1.34 (DSM ACC3115), respectively.

Definitions

The practicing of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Sambrook, et al., Molecular Cloning: A Laboratory Manual, second edition (1989); Gait, M.J., (ed.), Oligonucleotide Synthesis (1984); Freshney, R.I., (ed.), Animal Cell Culture (1987); Methods in Enzymology (Academic Press, Inc.); Ausubel,

F.M:, et al., (eds.), Current Protocols in Molecular Biology (1987) and periodic updates; Mullis, et al., (eds.), PCR: The Polymerase Chain Reaction (1994).

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton, et al., Dictionary of Microbiology and Molecular

Biology, 2nd ed., J. Wiley & Sons, New York, N.Y. (1994); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure, 4th ed., John Wiley & Sons, New York, N.Y. (1992); Lewin, B., Genes V, published by Oxford University Press (1994), ISBN 0-19-854287 9; Kendrew, J., et al., (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd. (1994), ISBN 0-632-02182-9; and Meyers, R.A., (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc. (1995), ISBN 1-56081-569 8) provide one skilled in the art with a general guide to many of the terms used in the present application. All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.

As used herein, each of the following terms has the meaning associated with it in this section.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an antibody" means one antibody or more than one antibody. The term "at least" is used to indicate that optionally one or more further objects may be present. By way of example, an array comprising at least two discrete areas may optionally comprise two or more than one discrete test areas.

The expression "one or more" denotes 1 to 50, preferably 1 to 20 also preferred 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 15.1 The term "marker" or "biochemical marker" as used herein refers to a molecule to be used as a target for analyzing an individual's test sample. In one embodiment examples of such molecular targets are proteins or polypeptides. Proteins or polypeptides used as a marker in the present invention are contemplated to include naturally occurring variants of said protein as well as fragments of said protein or said variant, in particular, immunologically detectable fragments. Immunologically detectable fragments preferably comprise at least 6, 7, 8, 10, 12, 15 or 20 contiguous amino acids of said marker polypeptide. One of skill in the art would recognize that proteins which are released by cells or present in the extracellular matrix may be damaged, e.g., during inflammation, and could become degraded or cleaved into such fragments. Certain markers are synthesized in an inactive form, which may be subsequently activated by proteolysis. As the skilled artisan will appreciate, proteins or fragments thereof may also be present as part of a complex. Such complex also may be used as a marker in the sense of the present invention. In addition, or in the alternative a marker polypeptide or a variant thereof may carry a post-translational modification. Preferred posttranslational modifications are glycosylation, acylation, or phosphorylation. "Detection" includes any means of detecting, including direct and indirect detection. The term "detection" is used in the broadest sense to include both qualitative and quantitative measurements of an analyte, herein measurements of an analyte such as an marker protein. In one aspect, a detection method as described herein is used to identify the mere presence of an analyte of interest in a sample. In another aspect, the method can be used to quantify an amount of analyte in a sample.

The term "assessing type 2 diabetes" or "assessing T2D" is used to indicate that the method according to the present invention will alone or together with other markers or variables, e.g., aid the physician to establish or confirm the absence or presence of T2D.

A "marker for T2D" in the sense of the present invention is a marker that, as single marker, or if combined with another marker of T2D, adds relevant information in the assessment of T2D to the diagnostic question under investigation. The information is considered relevant or of additive value if at a given specificity the sensitivity, or if at a given sensitivity the specificity, respectively, for the assessment of T2D can be improved by including said marker into a marker panel (marker combination) already comprising the marker TMEM27. Preferably the improvement in sensitivity or specificity, respectively, is statistically significant at a level of significance of p = 0.05, 0.02, 0.01 or lower.

The term "sample" or "test sample" as used herein refers to a biological sample obtained from an individual for the purpose of evaluation in vitro. In the methods of the present invention, the sample or patient sample may comprise in an embodiment of the present invention any body fluid. Preferred samples are body fluids, such as serum, plasma, or whole blood, with serum or plasma being most preferred. In one embodiment, the sample is a clinical sample. In another embodiment, the sample is used in a diagnostic assay. In an embodiment, a sample is obtained from a subject or patient prior to type 2 diabetes (T2B) therapy. In an embodiment, a sample is obtained from a subject or patient under T2B therapy.

The term„reference sample" as used herein refers to a biological sample provided from a reference group of apparently healthy individuals for the purpose of evaluation in vitro. The term„reference concentration" as used herein refers to a value established in a reference group of apparently healthy individuals. It is known to a person skilled in the art that the measurement results of step (a) according to the method(s) of the present invention will be compared to a reference concentration. Such reference concentration can be determined using a negative reference sample, a positive reference sample or a mixed reference sample comprising one or more than one of these types of controls. A negative reference sample preferably will comprise a sample from apparently healthy individuals with no diagnosis of T2D. A positive reference sample preferably will comprise a sample from a subject with the diagnosis of T2D.

The expression "comparing the concentration ... , wherein a concentration of ... above a reference concentration" as established in a control sample is merely used to further illustrate what is obvious to the skilled artisan anyway. The control sample may be an internal or an external control sample. In one embodiment an internal control sample is used, i.e. the marker level(s) is(are) assessed in the test sample as well as in one or more other sample(s) taken from the same subject to determine if there are any changes in the level(s) of said marker(s). In another embodiment an external control sample is used. For an external control sample the presence or amount of a marker in a sample derived from the individual is compared to its presence or amount in an individual known to suffer from, or known to be at risk of, a given condition; or an individual known to be free of a given condition, i.e., "normal individual". For example, a marker level in a patient sample can be compared to a level known to be associated with a specific course of T2D. Usually the sample's marker level is directly or indirectly correlated with a diagnosis and the marker level is e.g. used to determine whether an individual is at risk for T2D. Alternatively, the sample's marker level can e.g. be compared to a marker level known to be associated with a response to therapy in T2D patients, the diagnosis of T2D, the guidance for selecting an appropriate drug to T2D, in judging the risk of disease progression, or in the follow-up of T2D patients. Depending on the intended diagnostic use an appropriate control sample is chosen and a control or reference value for the marker established therein. It will be appreciated by the skilled artisan that such control sample in one embodiment is obtained from a reference population that is age-matched and free of confounding diseases. As also clear to the skilled artisan, the absolute marker values established in a control sample will be dependent on the assay used. Preferably samples from 100 well- characterized individuals from the appropriate reference population are used to establish a control (reference) value. Also preferred the reference population may be chosen to consist of 20, 30, 50, 200, 500 or 1000 individuals. Healthy individuals represent a preferred reference population for establishing a control value.

The term "measurement",„measuring" or„determining" preferably comprises a qualitative, semi-quanitative or a quantitative measurement of protein TMEM27 in a sample. In a preferred embodiment the measurement is a semi-quantitative measurement, i.e. it is determined whether the concentration of protein TMEM27 is above or below a cut-off value. As the skilled artisan will appreciate, in a Yes- presence) or No- (absence) assay, the assay sensitivity is usually set to match the cut-off value. A cut-off value can for example be determined from the testing of a group of healthy individuals. A value above the cut-off value can for example be indicative for the presence of T2D. A value below the cut-off value can for example be indicative for the absence of T2D.

In a further preferred embodiment the measurement of protein TMEM27 is a quantitative measurement. In further embodiments the concentration of protein

TMEM27 is correlated to an underlying diagnostic question. As the skilled artisan will appreciate, any such assessment is made in vitro. The sample (test sample) is discarded afterwards. The sample is solely used for the in vitro diagnostic method of the invention and the material of the sample is not transferred back into the patient's body. Typically, the sample is a liquid sample, e.g., serum, plasma, or whole blood.

As indicated above a sample comprising T2D in certain settings might be used as a positive control and preferably assayed in parallel with the sample to be investigated. In such setting a positive result for the marker protein TMEM27 in the positive control sample indicates that the testing procedure has worked on the technical level.

Transmembrane protein 27 (TMEM27, collectrin or NX- 17) was first described in the kidney as angiotensin-converting enzyme 2 (ACE2) homologue involved in amino acid transport (Zhang, et al., 2001; Danilczyk, et al., 2006). The amino acid sequence of the human TMEM27 glycoprotein is shown in SEQ ID NO: l (222 AA) and has an apparent signal peptide and a transmembrane domain. Human TMEM27 has 47.8% identity with non-catalytic extracellular, transmembrane, and cytosolic domains of ACE2; however, unlike ACE and ACE2, TMEM27 lacks active dipeptidyl carboxypeptidase catalytic domains. There is no consensus on the biological function of TMEM27. While one study established a potential positive role of TMEM27 in glucose-stimulated insulin exocytosis (Fukui, et al., 2005), another study linked TMEM27 to β-cell proliferation (Akpinar, et al., 2005). A more recent study confirmed the TMEM27 is mainly produced in β-cells (Altirriba, et al., 2010), where it regulates pancreatic β-cell mass and insulin secretion. TMEM27 is inactivated at the plasma membrane by proteolytic cleavage and shedding. It plays a role in controlling insulin exocytosis by regulating formation of the SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) complex in pancreatic B-cells. This finding renders TMEM27 to be good β-cell mass marker. Recent studies however find TMEM27 also cleaved by kidney tubular cells, ruling out a function as a β-cell mass biomarker. As obvious to the skilled artisan, the present invention shall not be construed to be limited to the full-length protein TMEM27 of SEQ ID NO: l . Physiological or artificial fragments of protein TMEM27, secondary modifications of protein TMEM27, as well as allelic variants of protein TMEM27 are also encompassed by the present invention. Variants of a polypeptide are encoded by the same gene, but may differ in their isoelectric point (=PI) or molecular weight (=MW), or both e.g., as a result of alternative mRNA or pre-mRNA processing. The amino acid sequence of a variant is to 95% or more identical to the corresponding marker sequence. Artificial fragments preferably encompass a peptide produced synthetically or by recombinant techniques, which at least comprises one epitope of diagnostic interest consisting of at least 6, 7, 8, 9 or 10 contiguous amino acids as derived from the sequence disclosed in SEQ ID NO: l . Such fragment may advantageously be used for generation of antibodies or as a standard in an immunoassay.

The term "polypeptide" as used herein, refers to a polymer of amino acids, and not to a specific length. Thus, peptides, oligopeptides and proteins are included within the definition of polypeptide.

By "correlate" or "correlating" is meant comparing, in any way, the performance or results of a first analysis or protocol with the performance or results of a second analysis or protocol. For example, one may use the results of a first analysis or protocol in carrying out a second protocols or one may use the results of a first analysis or protocol to determine whether a second analysis or protocol should be performed.

The terms "anti-TMEM27 antibody" and "an antibody that binds to protein TMEM27" refer to an antibody that is capable of binding protein TMEM27 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting protein TMEM27. In one embodiment, the extent of binding of an anti-TMEM27 antibody to an unrelated, non- TMEM27 protein is less than about 10% of the binding of the antibody to protein TMEM27 as measured, e.g., by a radioimmunoassay (RIA) or BIAcore™ technology. In certain embodiments, an antibody that binds to protein TMEM27 has a dissociation constant (Kd) of < ΙμΜ,

< 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g. 10 ^"8 M or less, e.g. from 10 ^"8 M to 10 ^"13 M, e.g., from 10 ^"9 M to 10 ^"13 M). In certain embodiments, an anti-TMEM27 antibody binds to an epitope of protein TMEM27 that is conserved among protein TMEM27 from different species. The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. "Antibody fragment" comprises a portion of an intact antibody, preferably comprising the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab') ₂ and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen- binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab') ₂ fragment that has two antigen-combining sites and is still capable of cross-linking antigen. "Single-chain Fv" (scFv) antibodies are, e.g. described in Houston, J.S., Methods in Enzymol. 203 (1991)

46-96). In addition, antibody fragments comprise single chain polypeptides having the characteristics of a V _H domain, namely being able to assemble together with a V _L domain, or of a V _L domain, namely being able to assemble together with a V _H domain to a functional antigen binding site and thereby providing the antigen binding property of full length antibodies.

The term "monoclonal antibody" (MAb) as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site.

Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler, et al., Nature 256 (1975) 495, or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson, et al., Nature 352 (1991) 624-628 and Marks, et al., J. Mol. Biol. 222 (1991) 581-597, for example.

An antibody that "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide is one that binds to that particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

In an embodiment the antibody according to the invention shows no binding to a linear epitope of human protein TMEM27. Epitope binding is investigated by using synthetic peptides of human protein TMEM27 from position 1-222 of

SEQ ID NO: 1 (see example 7). In an embodiment the antibody according to the present invention binds to a conformative epitope of human protein TMEM27.

The term "epitope" means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris, "Epitope Mapping Protocols", in Methods in Molecular Biology, vol. 66, Humana Press, Totowa, NJ (1996). Preferably an antibody according to the invention binds specifically to native but not to denatured TMEM27.

Alternatively, for example, a method can be used in which in a first step epitope overlapping of two antibodies binding to the same target antigen is determined with the help of a competitive test system. For this purpose, for example with the help of an enzyme immunoassay, there is tested the extent to which the antibody in question competes with the known antibody for the binding to an immobilized target antigen, e.g. an antibody produced by the cell lines according to the invention. For this purpose in a sandwich type ELISA a first specific antibody (e.g.

MAb-<TMEM27>-2.1.34) or a fragment thereof is immobilized and incubated with the antigen protein TMEM27 and an amount of a labeled second specific antibody (e.g. MAb-<TMEM27>-2.3.3) is added. By addition of an antibody in question (e.g. MAb TMEM27 8/9) can easily be ascertained the extent to which the antibody in question can displace the known antibody (e.g. labeled second specific antibody) from the binding. If there is a displacement of more than 20%, preferably of more than 30%), at the same concentration or a displacement of more than 70%, preferably of more than 80%>, at higher concentrations, preferably in the case of 10 ³ -10 ⁵ -fold excess of the antibody in question, referred to the known antibody, then epitope overlapping is present and both antibodies bind to the same or an overlapping part of the same epitope. If there is a displacement of less than 10%, preferabley of less than 5% at the same concentration referred to the known antibody (e.g. second specific antibody), then no epitope overlapping is present and both antibodies bind to different or non overlapping epitopes. In a control the addition of unlabeled antibody (e.g. MAb-<TMEM27>-2.3.3) leads to a signal reduction, as the unlabeld antibody (e.g. MAb-<TMEM27>-2.3.3) competes with the labeled second specific antibody (e.g. MAb-<TMEM27>-2.3.3) in binding to the same epitope (data shown in example 8).

The "variable region" (variable region of a light chain (V _L ), variable region of a heavy chain (V _H )) as used herein denotes each of the pair of light and heavy chains which is involved directly in binding the antibody to the antigen. The domains of variable light and heavy chains have the same general structure and each domain comprises four framework (FR) regions whose sequences are widely conserved, 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 structure. The CDRs in each chain are held in their three-dimensional structure by the framework regions and form together with the CDRs from the other chain the 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 therefore provide a further object of the invention.

The terms "hypervariable region" or "antigen-binding portion of an antibody" when used herein refer to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region comprises amino acid residues from the "complementarity determining regions" or "CDRs".

"Framework" or "FR" regions are those variable domain regions other than the hypervariable region residues as herein defined. Therefore, the light and heavy variable chains of an antibody comprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Especially, CDR3 of the heavy chain is the region which contributes most to antigen binding and defines the antibody.

CDR and FR regions are determined according to the standard definition of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) and/or those residues from a "hypervariable loop". The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

The antibodies may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. Purification is performed in order to eliminate other cellular components or other contaminants, e.g. other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. See Ausubel, F.M, et al., (eds.), Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987). Secernated or secreted antibodies may be present in the cell culture medium and can be purified from the cell culture medium by conventional precipitation, ion exchange, affinity chromatography, or the like.

An "isolated" antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than

95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). For review of methods for assessment of antibody purity, see e.g., Flatman, et al., J. Chromatogr. B 848 (2007) 79-87.

The monoclonal antibodies are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. DNA and RNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures. The hybridoma cells can serve as a source of such DNA and RNA. Once isolated, the DNA may be inserted into expression vectors, which are then transfected into host cells such as F£EK 293 cells, CHO cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of recombinant monoclonal antibodies in the host cells. An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

"Isolated nucleic acid encoding an anti-TMEM27 antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

The term "nucleic acid molecule", as used herein, is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded. Preferably a nucleic acid molecule is DNA.

Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are colinear, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.

The term "diagnosis" is used herein to refer to the identification of a molecular or pathological state, disease or condition or to refer to identification of a patient who may benefit from a particular treatment regimen. The term "prognosis" is used herein to refer to the prediction of the likelihood of clinical benefit from a therapy. The term "prediction" is used herein to refer to the likelihood that a patient will respond either favorably or unfavorably to a particular therapy. In one embodiment, the prediction relates to the extent of those responses. In one embodiment, the prediction relates to whether the probability that a patient will survive or improve following treatment, for example treatment with a particular therapeutic agent, and for a certain period of time without disease recurrence. The predictive methods of the invention can be used clinically to make treatment decisions by choosing the most appropriate treatment modalities for any particular patient. The predictive methods of the present invention are valuable tools in predicting if a patient is likely to respond favorably to a treatment regimen, such as a given therapeutic regimen, including for example, administration of a given therapeutic agent or combination, surgical intervention, steroid treatment, etc., or whether long-term survival of the patient, following a therapeutic regimen is likely. The term

"selecting" and "selection" is used herein to refer to a choice from a number of alternatives. As an example a "selection" is the process to choose one drug, from two or more available drugs available for treatment of a disease.

As used herein, the term "Immunoassay" (IA) means a specific binding assay in which an analyte is detected by use of at least one antibody as a specific binding partner or agent. Immunoassay includes, but is not limited to, radioimmunoassay (RIA), fluoroluminescence assay (FLA), chemiluminescence assay (CLA), electrochemiluminescence assay (ECLA), and enzyme linked immunosorbant assay (ELISA). ELISA methods are described, for example, in WO 2001/36972. The term "detection agent" refers to an agent that binds to an analyte and is detectably labeled. Examples of detection agents include, but are not limited to, an antibody, antibody fragment, soluble receptor, receptor fragment, and the like. Detection of a detection agent is either possible directly, i.e. via a label directly linked to the agent or indirectly via a labeled second binding partner, such as a further antibody or receptor that specifically binds the detection agent.

The term "label" as used herein refers to any substance that is capable of producing a signal via direct or indirect detection.

For direct detection the labeling group or label suitable for use in the present invention can be selected from any known detectable marker groups, but are not limited to, chromogens, fluorescent, chemiluminescent groups (e.g. acridinium esters or dioxetanes), electrochemiluminescent compounds, catalysts, enzymes, enzymatic substrates, dyes, fluorescent dyes (e.g. fluorescein, coumarin, rhodamine, oxazine, resorufin, cyanine and derivatives thereof), colloidal metallic and nonmetallic particles, and organic polymer latex particles. Other examples of labeling groups are luminescent metal complexes, such as ruthenium or europium complexes, enzymes, e.g. as used for ELISA, and radioisotopes.

Indirect detection systems comprise, for example, that the detection reagent, e.g. the detection antibody, is labeled with a first partner of a bioaffine binding pair. Examples of suitable binding pairs are hapten or antigen/antibody, biotin or biotin analogues such as aminobiotin, iminobiotin or desthiobiotin/avidin or streptavidin, sugar/lectin, nucleic acid or nucleic acid analogue/complementary nucleic acid, and receptor/ligand, e.g. steroid hormone receptor/steroid hormone. Preferred first binding pair members comprise hapten, antigen and hormone. Especially preferred are haptens like digoxin and biotin and analogues thereof. The second partner of such binding pair, e.g. an antibody, streptavidin, etc., usually is labeled to allow for direct detection, e.g. by the labels as mentioned above.

It would appear that in the prior art the presence or level of the protein TMEM27 in a serum, plasma, or whole blood is not known to have a diagnostic utility in the assessment of type 2 diabetes (T2D).

The inventors of the present invention have now found and could establish that an increased concentration for human protein TMEM27 as determined from a serum, plasma, or whole blood sample derived from an individual is indicative for T2D.

Protein TMEM27, particularly soluble forms of protein TMEM27, are determined in vitro in appropriate samples. In an embodiment the sample is a liquid sample.

Preferred typical samples are body fluids, such as serum, plasma, or whole blood. Preferably, the sample is derived from a human subject, e.g. a T2D patient or a person in risk of T2D or a person suspected of having T2D. In a further preferred embodiment the sample is selected from the group consisting of serum, plasma, or whole blood. Also preferred protein TMEM27 is determined in a serum or plasma sample.

In a further embodiment the protein TMEM27 is the human protein TMEM27. The amino acid sequence of human protein TMEM27 is shown in SEQ ID NO: l . The values for protein TMEM27 as determined in a control group or a control population are for example used to establish a cut-off value or a reference range. A value above such cut-off value or out-side the reference range at its higher end is considered as elevated. In an embodiment a fixed cut-off value is established. Such cut-off value is chosen to match the diagnostic question of interest. In an embodiment the cut-off is set to result in a specificity of 90%, also preferred the cut-off is set to result in a specificity of 95%, or also preferred the cut-off is set to result in a specificity of 98%). In an embodiment the cut-off is set to result in a sensitivity of 90%, also preferred the cut-off is set to result in a sensitivity of 95%, or also preferred the cut-off is set to result in a sensitivity of 98%.

In one embodiment values for protein TMEM27 as determined in a control group or a control population are used to establish a reference range. In a preferred embodiment an TMEM27 concentration is considered as elevated if the value determined is above the 90%-percentile of the reference range. In further preferred embodiments an TMEM27 concentration is considered as elevated if the value determined is above the 95%-percentile, the 96%-percentile, the 97%-percentile or the 97.5%-percentile of the reference range.

In one embodiment the control sample will be an internal control sample. In this embodiment serial samples are obtained from the individual under investigation and the marker levels are compared. This may for example be useful in assessing the efficacy of therapy.

The method according to the present invention is based on a liquid sample which is obtained from an individual and on the in vitro determination of protein TMEM27 in such sample. An "individual", "subject" or "patient" as used herein refers to a single human or non-human organism. Thus, the methods and compositions described herein are applicable to both human and veterinary disease. Preferably the individual, subject, or patient is a human being.

The method according to the present invention is based on a liquid sample which is obtained from an individual and on the in vitro determination of protein TMEM27 in such sample. In the methods of the present invention, the sample or patient sample may comprise in an embodiment of the present invention any body fluid. Preferred samples are body fluids, such as serum, plasma, or whole blood, with serum or plasma being most preferred. Preferably the marker protein TMEM27 is specifically determined in vitro from a liquid sample by use of a specific binding agent.

In a preferred embodiment according to the present invention, the concentration of protein TMEM27 is determined. In an embodiment, the concentration of marker protein TMEM27 is specifically determined in vitro from a serum, plasma, or whole blood sample by use of a specific binding agent.

A specific binding agent is, e.g., a receptor for the protein TMEM27, a lectin binding to protein TMEM27, or an antibody to protein TMEM27, peptidebodies to protein TMEM27, bispecific dual binders or bispecific antibody formats. A specific binding agent has at least an affinity of 10 ⁷ 1/mol for its corresponding target molecule. The specific binding agent preferably has an affinity of 10 ⁸ 1/mol or also preferred of 10 ⁹ 1/mol for its target molecule.

As the skilled artisan will appreciate the term specific is used to indicate that other biomolecules present in the sample do not significantly bind to the binding agent specific for the marker protein TMEM27. Preferably, the level of binding to a biomolecule other than the target molecule results in a binding affinity which is at most only 10% or less, only 5% or less only 2% or less or only 1% or less of the affinity to the target molecule, respectively. A preferred specific binding agent will fulfill both the above minimum criteria for affinity as well as for specificity. A specific binding agent preferably is an antibody reactive with the protein

TMEM27. The term antibody refers to a polyclonal antibody, a monoclonal antibody, antigen binding fragments of such antibodies, single chain antibodies as well as to genetic constructs comprising the binding domain of an antibody.

Any antibody fragment retaining the above criteria of a specific binding agent can be used. Antibodies are generated by state of the art procedures, e.g., as described in Tijssen (Tijssen, P., Practice and Theory of Enzyme Immunoassays, 11, Elsevier Science Publishers B.V., Amsterdam, the whole book, especially pages 43-78). In addition, the skilled artisan is well aware of methods based on immunosorbents that can be used for the specific isolation of antibodies. By these means the quality of polyclonal antibodies and hence their performance in immunoassays can be enhanced (Tijssen, P., supra, pages 108-115).

For the achievements as disclosed in the present invention antibodies may be used. However, clearly monoclonal antibodies or polyclonal antibodies from different species, e.g., rabbits, sheep, goats, rats, hamster or guinea pigs can be used. Since monoclonal antibodies can be produced in any amount required with constant properties, they represent ideal tools in development of an assay for clinical routine.

Alternative strategies to generate antibodies may be used. Such strategies comprise amongst others the use of synthetic peptides, representing an epitope of protein TMEM27 for immunization, DNA immunization, also known as DNA vaccination, or the use of purified recombinant protein TMEM27. In an embodiment DNA immunization is used to generate the antibody according to the present invention.

The present invention also relates in an embodiment to the use of an antibody directed against protein TMEM27 in a method according to the present invention.

The generation and the use of monoclonal antibodies to the marker protein TMEM27 and their use in a method according to the present invention, respectively, represent yet other preferred embodiments.

As the skilled artisan will appreciate now, that protein TMEM27 has been identified as a marker which is useful in the assessment of T2D. Various immunodiagnostic procedures may be used to reach a result comparable to the achievements of the present invention. For example, alternative strategies to generate antibodies may be used. Such strategies comprise amongst others the use of synthetic or recombinant peptides, representing a clinically relevant epitope of protein TMEM27 for immunization. Alternatively, DNA immunization also known as DNA vaccination may be used. In a preferred embodiment DNA immunization is used for the generation of antibodies against protein TMEM27. In a further preferred embodiment the DNA expression plasmid, containing the cDNA fragment encoding for the extracellular residues 1-125 of human TMEM27 (see Example 1 and Figures 1, 2 and 3), is used for DNA immunization.

For determination the sample obtained from an individual is incubated in vitro with the specific binding agent for protein TMEM27 under conditions appropriate for formation of a binding agent TMEM27 complex. Such conditions need not be specified, since the skilled artisan without any inventive effort can easily identify such appropriate incubation conditions. The amount of binding agent TMEM27 complex is determined and used in the assessment of T2D. As the skilled artisan will appreciate there are numerous methods to determine the amount of the specific binding agent TMEM27 complex all described in detail in relevant textbooks (cf, e.g., Tijssen, P., supra, or Diamandis, E.P., and Christopoulos, T.K. (eds.), Immunoassay, Academic Press, Boston (1996)).

Immunoassays are well known to the skilled artisan. Methods for carrying out such assays as well as practical applications and procedures are summarized in related textbooks. Examples of related textbooks are Tijssen, P., Preparation of enzyme- antibody or other enzyme-macromolecule conjugates, In: Practice and Theory of Enzyme Immunoassays, pp. 221-278, Burdon, R.H. and v. Knippenberg, P.H., (eds.), Elsevier, Amsterdam (1990), and various volumes of Colowick, S.P., and Caplan, N.O., (eds.), Methods in Enzymology, Academic Press, dealing with immunological detection methods, especially volumes 70, 73, 74, 84, 92 and 121.

In a further embodiment protein TMEM27 is detected in an immunoassay procedure. Such immunoassay procedure uses two monoclonal antibodies or fragments thereof that specifically bind to human TMEM27.

In a further embodiment protein TMEM27 is detected in an enzyme-linked immunoassay (ELIS A).

In an embodiment protein TMEM27 is detected in a sandwich assay (sandwich- type assay format). In such assay, a first specific binding agent is used to capture protein TMEM27 on the one side and a second specific binding agent, which is labeled to be directly or indirectly detectable, is used on the other side. The first specific binding agent used at one side of such sandwich is bound or capable of binding to a solid phase (often referred to as capture antibody), whereas the second specific binding agent used at the other side of such sandwich is labeled in such a manner that direct or indirect detection is facilitated (so-called detection antibody). The specific binding agents used in a sandwich-type assay format may be antibodies specifically directed against protein TMEM27. On the one hand, the detection may be carried out by using different capturing and labelled antibodies, i.e. antibodies which recognize different epitopes on the TMEM27 polypeptide. On the other hand, a sandwich-type assay may also be carried out with a capture and labelling antibody which is directed against the same epitope of protein TMEM27. In this embodiment, only di- and multimeric forms of protein TMEM27 may be detected. In an embodiment an antibody to protein TMEM27 is used in a qualitative (TMEM27 present or absent) or quantitative (amount of TMEM27 is determined) immunoassay.

In a further embodiment protein TMEM27 is detected in a competitive assay. Such assays, competitive assay or sandwich assay, respectively, may be performed in an embodiment without washing steps (homogeneous immunoassay) or with washing steps (heterogeneous immunoassay).

The concentration of the protein TMEM27 in test samples may be determined in vitro using a specific ELISA, as already described above. Using this assay format, the inventors have shown that samples from patients already diagnosed as having T2D by classical methods can be distinguished from samples from apparently healthy individuals. Results are shown in the example section of this application.

The inventors of the present invention surprisingly are able to detect protein TMEM27 in a serum, plasma, or whole blood sample. Even more surprising they are able to demonstrate that the presence of protein TMEM27 in such serum, plasma, or whole blood sample obtained from an individual can be correlated to T2D. No tissue and no biopsy sample is required to make use of the marker TMEM27 in the assessment of T2D. Measuring the serum, plasma, or whole blood level of protein TMEM27 is considered very advantageous in the field of T2D.

In a preferred embodiment the method according to the present invention is practiced with serum as liquid sample material. In a further preferred embodiment the method according to the present invention is practiced with plasma as liquid sample material. In a further preferred embodiment the method according to the present invention is practiced with whole blood as liquid sample material.

In a further preferred embodiment, the present invention relates to use of protein TMEM27 as a marker molecule in the in vitro assessment of T2D from a serum, plasma, or whole blood sample obtained from an individual.

The ideal scenario for diagnosis would be a situation wherein a single event or process would cause the respective disease as, e.g., in infectious diseases. In all other cases correct diagnosis can be very difficult, especially when the etiology of the disease is not fully understood. As the skilled artisan will appreciate, no biochemical marker is diagnostic with 100% specificity and at the same time 100% sensitivity for a given multifactorial disease, as for example for T2D. Rather, biochemical markers are used to assess with a certain likelihood or predictive value an underlying diagnostic question, e.g., the presence, absence, or the severity of a disease. Therefore in routine clinical diagnosis, generally various clinical symptoms and biological markers are considered together in the assessment of an underlying disease. The skilled artisan is fully familiar with the mathematical/statistical methods that routinely are used to calculate a relative risk or likelihood for the diagnostic question to be assessed. In routine clinical practice various clinical symptoms and biological markers are generally considered together by a physician in the diagnosis, treatment, and management of the underlying disease.

In the assessment of T2D the marker protein TMEM27 will be of advantage in one or more of the following aspects: assessment; screening; diagnostic aid; monitoring of the response to therapy. In an embodiment the measurement of protein TMEM27 is a quantitative measurement. In further embodiments the concentration of protein TMEM27 is correlated to an underlying diagnostic question like e.g. presence or absence of the disease, disease progression, or response to therapy. In an embodiment, the marker protein TMEM27 may be used for T2D monitoring as well as for T2D screening purposes.

It will be appreciated that a particular and unique benefit of the invention is the ease of prognosis which may be performed requiring only a simple body fluid sample. In the methods of the present invention, the test sample or patient sample preferably is a sample selected from the group consisting of serum, plasma and whole blood, with serum or plasma being most preferred.

Preferred areas of diagnostic relevance in assessing an individual suspected or known to have T2D are judging the risk of disease progression, guidance for selecting an appropriate drug, monitoring of response to therapy, and the follow-up of T2D patients.

Screening (assessment whether individuals are at risk for developing T2D or have T2D): Screening is defined as the systematic application of a test to identify individuals e.g. at risk individuals, for indicators of a disease, e.g., the presence of T2D. Preferably the screening population is composed of individuals known to be at higher than average risk of T2D. For example, a screening population for T2D is composed of individuals known to be at higher than average risk of T2D, like individuals having unhealthy eating habits (being overweight), having a sedentary lifestyle, having a family history or genetic predisposition of T2D, being over the age of 45, having high blood pressure, having prediabetes or have developed gestational diabetes. Screening in the sense of the present invention relates to the unbiased assessment of individuals regarding their risk for developing T2D. Whereas such screening may in theory be performed on any sample, in clinical practice such screening option will usually be given to individuals somehow at risk for development of T2D. As discussed above, such individuals may clinically be asymptomatic, i.e., they have no signs or symptoms of T2D. In one embodiment screening for T2D will be given to individuals at risk of developing T2D.

The present invention provides a novel blood plasma protein signature that can distinguish healthy from type-2 diabetic (T2D) human patients. The abundance of protein TMEM27 showed a significant change in concentration with upcoming

T2D and therefore represents a candidate novel diagnostic marker for T2D, in particular for early detection of T2D diabetes patients.

In an embodiment the diagnostic method according to the present invention is used for screening purposes. I.e., it is used to assess subjects without a prior diagnosis of T2D by a) determining the concentration of human protein TMEM27 in a serum, plasma, or whole blood sample in vitro, and b) comparing the concentration of protein TMEM27 determined in step (a) with a reference concentration of protein TMEM27, wherein a concentration of protein TMEM27 above the reference concentration is indicative for the presence of T2D. In an embodiment, a body fluid sample such as blood, serum, or plasma is used as a sample in the screening for T2D, with serum or plama being most preferred.

In the assessment of T2D the protein TMEM27 will aid the physician to assess the presence or absence of T2D in an individual suspected to have T2D.

In an embodiment the present invention relates to an in vitro method for assessing the presence or absence of type II diabetes (T2D) in a human subject, comprising a) determining the concentration of human protein TMEM27 in a serum, plasma, or whole blood sample, and b) comparing the concentration of protein TMEM27 determined in step (a) with a reference concentration of protein TMEM27, wherein a concentration of protein TMEM27 above the reference concentration is indicative for the presence of T2D. In a further preferred embodiment the sample is selected from the group consisting of serum, plasma and whole blood. In a further preferred embodiment the sample is serum or plasma.

In an embodiment the present invention relates to an in vitro method for assessing the presence or absence of type II diabetes (T2D) in a human subject, comprising a) determining the concentration of protein TMEM27 in a serum, plasma, or whole blood sample, b) comparing the concentration of protein TMEM27 determined in step (a) with a reference concentration of protein TMEM27, and c) assessing the presence or absence of T2D based on the comparison of step (b), wherein a concentration of protein TMEM27 above the reference concentration is indicative for the presence of T2D. . In a further preferred embodiment the sample is selected from the group consisting of serum, plasma and whole blood. In a further preferred embodiment the sample is serum or plasma.

In an embodiment the in vitro method according to the present invention is characterized in that the assessment of the protein TMEM27 takes place for classifying a patient according to be at risk for developing T2D.

In an embodiment the in vitro method according to the present invention is characterized in that the assessment takes place for classifying a patient according to be at risk to have T2D for clinical decisions, particularly further treatment by means of medications for the treatment or therapy of T2D.

In an embodiment the present invention relates to an in vitro method for assessing whether an individual is at risk for developing T2D comprising the steps of a) determining the concentration of human protein TMEM27 in a serum, plasma, or whole blood sample, and b) of assessing said individual's risk for developing T2D by comparing the concentration of protein TMEM27 determined in step (a) with a reference concentration of protein TMEM27, wherein a concentration of protein TMEM27 above a reference concentration is indicative for an individual to be at risk for developing T2D. In a preferred embodiment the sample is selected from the group consisting of serum, plasma and whole blood. In a further embodiment the concentration of TMEM27 is determined in the in vitro method according to the present invention with an monoclonal antibody produced by hybridoma cell line MAK<H-TMEM27>M-2.3.3 (DSM ACC3116) or MAK<H-TMEM27>M-2.1.34 (DSM ACC3115), respectively. In a further embodiment the concentration of TMEM27 is determined in the in vitro method according to the present invention with a fragment of monoclonal antibody produced by hybridoma cell line MAK<H-TMEM27>M-2.3.3 (DSM ACC3116) or MAK<H-TMEM27>M-2.1.34 (DSM ACC3115), respectively. Said fragment is in a preferred embodiment selected from the group consisting of Fab, Fab' and F(ab') ₂ of the antibody produced by hybridoma cell line MAK<H-TMEM27>M-2.3.3 or hybridoma cell line MAK<H-TMEM27>M-2.1.34, respectively, with Fab or Fab' being most presferred.

Diagnostic aid

Markers may either aid the differential diagnosis of diseases in a particular organ, help to distinguish between different histological types of a disease, or to establish baseline marker values before surgery.

Today, an important method used in the detection of T2D is the in vitro measurement of the glucose concentration in blood. However, the diagnosis of T2D is often in a late state of the disease. Use of the marker TMEM27 may aid in the early detection of T2D. A concentration of protein TMEM27 above a reference concentration for protein TMEM27 is directly correlated to the diagnosis of T2D.

Protein TMEM27 as a single marker is expected to have superior specificity for T2D as compared to other markers. Therefore protein TMEM27 is likely to be used as a diagnostic aid, especially by establishing a baseline value before therapy. The present invention thus also relates to the use of protein TMEM27 for establishing a baseline value before T2D therapy.

Assessing the risk of disease progression

The progression of T2D disease may be evaluated by in vitro monitoring of the concentration of protein TMEM27 in test samples. In an embodiment the present invention relates to an in vitro method for assessing a T2D-patient's risk for disease progression, comprising the steps of a) determining the concentration of human protein TMEM27 in a serum, plasma, or whole blood sample, b) comparing the concentration of protein TMEM27 determined in step (a) with a reference concentration of protein TMEM27, and of establishing said individuals' s risk for disease progression by comparing the concentration determined in step (a) to the concentration of this marker to its reference value. In a preferred embodiment the sample is selected from the group consisting of serum, plasma and whole blood.

At present it is very difficult to assess or to even predict with a reasonable likelihood whether a patient diagnosed with T2D has a more or less stable status or whether the disease will progress and the patient's health status as result is likely to worsen. Severity and progression of T2D is usually established by assessing the glucose concentration in a blood sample. Guidance in selecting an appropriate T2D therapy

In an embodiment the present invention relates to an in vitro method, aiding in the selection of an appropriate T2D-therapy, comprising the steps of a) determining the concentration of human protein TMEM27 in a serum, plasma, or whole blood sample, b) comparing the concentration of protein TMEM27 determined in step (a) with a reference concentration of protein TMEM27, and of selecting an appropriate therapy by comparing the concentration determined in step (a) to the concentration of this marker to its reference value. In a preferred embodiment the sample is selected from the group consisting of serum, plasma and whole blood. It is expected that the marker TMEM27 will be of help in aiding the physician to select the most appropriate treatment regimen from the various treatment regimens at hand in the area of T2D. In a further preferred embodiment therefore relates to the use of the marker TMEM27 in selecting a treatment regimen for a patient suffering from T2D.

Monitor a patient's response to therapy The progression of T2D disease may be evaluated by in vitro monitoring of the concentration of protein TMEM27 in test samples.

When used in patient monitoring the diagnostic method according to the present invention may help to assess efficacy of treatment of T2D and T2D progression in the follow-up of patients. In an embodiment the present invention relates to an in vitro method for monitoring a patient's response to T2D-therapy, comprising the steps of a) determining the concentration of human protein TMEM27 in a serum, plasma, or whole blood sample, b) comparing the concentration of protein TMEM27 determined in step (a) with a reference concentration of protein TMEM27, and of monitoring a patient's response to T2D therapy by comparing the concentration determined in step (a) to the concentration of this marker to its reference value. In a preferred embodiment the sample is selected from the group consisting of serum, plasma and whole blood. Alternatively the above method for motoring a patient's response to therapy can be practiced by establishing the pre- and post-therapeutic marker level for protein TMEM27 serum, plasma, or whole blood sample and by comparing the pre- and the post-therapeutic marker level.

The marker TMEM27 appears to be appropriate to monitor a patient's response to therapy. The present invention thus also relates to the use of marker TMEM27 in monitoring a patient's response to therapy. In that diagnostic area the marker TMEM27 can also be used for establishing a baseline value before therapy and to determine TMEM27 at one time-point or several time-points after therapy. In the follow-up of T2D patients a decreased level of TMEM27 is a positive indicator for an effective treatment of T2D.

Kit

The present invention also provides a kit for performing the in vitro method according to the present invention comprising the reagents required to specifically determine the concentration of human protein TMEM27. In a further embodiment the present invention provides a kit for performing the in vitro method according to the present invention comprising a first antibody specific for human protein TMEM27, a second antibody specific for human protein TMEM27.

In a further embodiment the present invention provides a kit for performing the in vitro method according to the present invention comprising a first antibody specific for human protein TMEM27, a second antibody specific for human protein TMEM27 and reagents required to specifically determine the concentration of protein TMEM27. In a further embodiment said first antibody is produced by the hybridoma cell line MAK<H-TMEM27>M-2.3.3 and the second antibody is produced by the hybridoma cell line MAK<H-TMEM27>M-2.1.34. In a further embodiment an antibody fragment selected from the group consisting of Fab, Fab', and F(ab')2 of said first and/or said second antibody is provided in said kit.

In a further embodiment the present invention relates to a diagnostic device for carrying out the in vitro method for assessing T2D according to the present invention. A "diagnostic device" as used herein refers to an in vitro diagnostic medical device (IVD) if it is a reagent, calibrator, control material, kit, specimen receptacle, software, instrument, apparatus, equipment or system, whether used alone or in combination with other diagnostic goods for in vitro use. It must be intended by the manufacturer to be used in vitro for the examination of samples or specimens derived from the human body, solely or principally for the purpose of giving information about a concentration of a marker, physiological or pathological state, a congenital abnormality or to determine safety and compatibility with a potential recipient, or to monitor therapeutic measures. Use

In an embodiment the present invention relates to the use of the human protein TMEM27 in the assessment of T2D. Preferably human protein TMEM27 is used in the assessment of the presence or absence of T2D. In a further embodiment the present invention relates to the use of the human protein TMEM27 in the in vitro assessment of T2D in a serum, plasma, or whole blood sample, wherein a concentration of protein TMEM27 above a reference concentration for protein TMEM27 in a serum, plasma, or whole blood sample is indicative for the presence of T2D. In a further embodiment the present invention relates to the use of the human protein TMEM27 in the in vitro assessment of T2D in a serum, plasma, or whole blood sample by an immunological method, wherein a concentration of protein TMEM27 is determined using the antibody according to the present invention, and wherein a concentration above a reference concentration for protein TMEM27 in a serum, plasma, or whole blood sample is indicative for the presence of T2D.

Preferably the antibody or a fragment thereof used in the in vitro assessment of T2D is produced by hybridoma cell line MAK<H-TMEM27>M-2.3.3 (DSM ACC3116) or hybridoma cell line MAK<H-TMEM27>M-2.1.34 (DSM ACC3115), respectively. Also preferably antibodies or fragments thereof produced by hybridoma cell line MAK<H-TMEM27>M-2.3.3 (DSM ACC3116) and hybridoma cell line MAK<H-TMEM27>M-2.1.34 (DSM ACC3115) are used in the in vitro assessment of T2D in a sandwich type immunoassay.

Antibody binding protein TMEM27

One aspect of the present invention are antibodies that specifically bind to human protein TMEM27, for example as means for preforming the method as reported herein.

In an embodiment the present invention comprises an antibody that binds to protein TMEM27. In a preferred embodiment said antibody specifically binds to protein TMEM27. In an embodiment the antibody according to the present invention specifically binds human protein TMEM27. In an embodiment the antibody of the current invention is further characterized in that it has been produced by immunizing suitable animals with DNA expressing the human TMEM27 polypeptide, also known to the skilled artisan as DNA immunization of suitable animals. In a preferred embodiment the antibody directed to protein TMEM27 is a monoclonal antibody.

There is a high protein sequence homology of human protein TMEM27 to the protein sequence of murine TMEM27 or the protein sequence of TMEM27 from other primates, so that the antibodies of the present invention are useful in preclinical studies, e.g. for T2D assessment, screening, diagnostic aid, prognosis, monitoring of the response to therapy of mice or primates.

The invention further provides hybridoma cell lines which produce monoclonal antibodies according to the invention. Preferred hybridoma cell lines according to the invention were deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Germany. The antibodies obtained from said cell lines are preferred embodiments of the invention.

A further embodiment of the invention is the antibody binding to human TMEM27, characterized in that it is produced by hybridoma cell line MAK<H-TMEM27>M- 2.3.3, which was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) on February 1, 2011 and received the deposit number DSM ACC3116, or the antibody binding to human TMEM27, characterized in that it is produced by hybridoma cell line MAK<H-TMEM27>M-2.1.34 which was deposited with the DSMZ on February 1, 2011 and received the deposit number DSM ACC3115, respectively.

The sequence of the polynucleotides contained in this deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are controlling in the event of any conflict with the sequences described in the sequence protocol.

It has been found that the antibodies as reported herein bind to a novel epitope. Characterization of the epitope recognized by the monoclonal antibody according to the present invention leads to the assumption that the antibody recognizes a native discontinuous epitope (conformational epitope) or various domains of protein TMEM27 (see Example 7). It has been shown by the inventors in competition experiments that the antibody of the present invention generated by DNA immunization has a higher binding affinity as compared to the monoclonal antibody TMEM27 8/9 (DSMZ ACC2995) generated by immunization with native human protein TMEM27. This substantiates the method of DNA immunization used for generation of antibodies according to the present invention that specifically bind TMEM27. In an embodiment the antibody of the present invention binds to a conformational epitope of human protein TMEM27. In an embodiment the antibody of the present invention binds to a native discontinous epitope of human protein TMEM27. In an embodiment the antibody according to the present invention binds to a native discontinous epitope of human protein TMEM27. In an embodiment the monoclonal antibody of the present invention binds to a native human protein TMEM27 but not to a denatured human protein TMEM27.

The skilled artisan is aware of methods, like cross blocking experiments or competition experiments (as exemplary shown in example 8), to check whether his antibody binds or binds not to the same conformational epitope of human protein

TMEM27 as the antibodies disclosed in this patent application.

In a further embodiment the present invention relates to an antibody binding to the same or an overlapping epitope as bound by the monoclonal antibody produced by hybridoma cell line DSM ACC3116 (MAK<H-TMEM27>M-2.3.3 (DSM ACC3116) or hybridoma cell line MAK<H-TMEM27>M-2.1.34 (DSM ACC3115), respectively.

In an embodiment the antibody binds to at least one amino acid in human protein TMEM27 within position 31-45 (SEQ ID NO: 6), 59-69 (SEQ ID NO: 7), 91-105 (SEQ ID NO: 8), 111-123 (SEQ ID NO: 9), 127-141 (SEQ ID NO: 10), or 171-181 (SEQ ID NO: 11).

In an embodiment antibody binds to at least one amino acid in human protein TMEM27 within each of positions 31-45 (SEQ ID NO: 6), 59-69 (SEQ ID NO: 7), 91-105 (SEQ ID NO: 8), 111-123 (SEQ ID NO: 9), 127-141 (SEQ ID NO: 10), and 171-181 (SEQ ID NO: 11). In an embodiment monoclonal antibody 2.3.3 produced by MAK<H-TMEM27>M-

2.3.3 (DSM ACC3116) binds to at least one amino acid in human protein TMEM27 within each of positions 31-45 (SEQ ID NO: 6), 59-69 (SEQ ID NO: 7), 91-105 (SEQ ID NO: 8), 111-123 (SEQ ID NO: 9), 127-141 (SEQ ID NO: 10), and 171-181 (SEQ ID NO: 11). In an embodiment the antibody binds to at least one amino acid in human protein

TMEM27 within position 93-107 (SEQ ID NO: 12). In an embodiment monoclonal antibody 2.1.34 produced by hybridoma cell line MAK<H-TMEM27>M-2.1.34 binds to at least one amino acid in human protein TMEM27 within position 93-107 (SEQ ID NO: 12).

The monoclonal antibody according to the present invention is preferably produced by hybridoma cell line MAK<H-TMEM27>M-2.3.3 (DSM ACC3116) or hybridoma cell line MAK<H-TMEM27>M-2.1.34 (DSM ACC3115), respectively. In more detail, hybridoma cell line MAK<H-TMEM27>M-2.3.3 is producing the monoclonal antibody 2.3.3, and hybridoma cell line MAK<H-TMEM27>M-2.1.34 is producing the monoclonal antibody 2.1.34. The CDR sequences can be determined according to the standard definition of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). CDRs on each chain are separated by framework amino acids.

The monoclonal antibody according to the invention is preferably characterized in that said monoclonal antibody comprises CDR3 of the V _H domain of the antibody obtained from hybridoma cell line MAK<H-TMEM27>M-2.3.3 (DSM ACC3116) or CDR3 of the V _H domain of the antibody obtained from hybridoma cell line MAK<H-TMEM27>M-2.1.34 (DSM ACC3115), respectively, and CDR3 of the V _L domain of the antibody obtained from hybridoma cell line MAK<H- TMEM27>M-2.3.3 (DSM ACC3116) or CDR3 of the V _L domain of the antibody obtained from hybridoma cell line MAK<H-TMEM27>M-2.1.34 (DSM ACC3115), respectively.

The monoclonal antibody according to the invention is preferably characterized in that said monoclonal antibody comprises CDR1 to CDR3 of the V _H domain of the antibody obtained from hybridoma cell line MAK<H-TMEM27>M-2.3.3 (DSM

ACC3116) or CDR1 to CDR3 of the V _H domain of the antibody obtained from hybridoma cell line MAK<H-TMEM27>M-2.1.34 (DSM ACC3115), respectively, and CDR1 to CDR3 of the V _L domain of the antibody obtained from hybridoma cell line MAK<H-TMEM27>M-2.3.3 (DSM ACC3116) or CDR1 to CDR3 of the V _L domain of the antibody obtained from hybridoma cell line MAK<H-

TMEM27>M-2.1.34 (DSM ACC3115), respectively.

The monoclonal antibody according to the invention is preferably characterized in comprising both, the V _H domain and the V _L domain of the antibody obtained from hybridoma cell line MAK<H-TMEM27>M-2.3.3 (DSM ACC3116) or the V _H domain and the V _L domain of the antibody obtained from hybridoma cell line MAK<H-TMEM27>M-2.1.34 (DSM ACC3115), respectively.

In an embodiment monoclonal antibody specifically binds to protein TMEM27 with a kd value of 5 x 10 ^"4 1/s or less. In an embodiment monoclonal antibody specifically binds to protein TMEM27 with a kd value of 7 x 10 ^"4 1/s or less. In an embodiment monoclonal antibody specifically binds to protein TMEM27 with a kd value of 3 x 10 ^"5 1/s or less.

In an embodiment monoclonal antibody specifically binds to protein TMEM27 with a t/2 diss value of 10 min or more. In an embodiment monoclonal antibody specifically binds to protein TMEM27 with t/2 diss value of 15 min or more. In an embodiment monoclonal antibody specifically binds to protein TMEM27 with t/2 diss value of 350 min or more.

In one embodiment the kd value and/or the t/2 diss value is determined at 25 °C.

In an embodiment the invention further comprises a fragment selected from the group consisting of Fab, Fab' and F(ab') ₂ of the antibody produced by hybridoma cell line MAK<H-TMEM27>M-2.3.3. In an embodiment the invention further comprises a fragment selected from the group consisting of Fab, Fab' and F(ab') ₂ of the antibody produced by hybridoma cell line MAK<H-TMEM27>M-2.1.34. In an embodiment a fragment selected from the group consisting of Fab, Fab' and F(ab') ₂ derived from MAK<H-TMEM27>M-2.3.3 or MAK<H-TMEM27>M-2.1.34, respectively, is used in a method according to the present invention, preferably in an immunoassay procedure.

In an embodiment the invention further comprises a fragment selected from the group consisting of Fab and Fab' of the antibody produced by hybridoma cell line MAK<H-TMEM27>M-2.3.3. In an embodiment the invention further comprises a fragment selected from the group consisting of Fab and Fab' of the antibody produced by hybridoma cell line MAK<H-TMEM27>M-2.1.34. The fragment selected from the group consisting of Fab and Fab' derived from MAK<H- TMEM27>M-2.3.3 or MAK<H-TMEM27>M-2.1.34, respectively, is used in an embodiment in a method according to the present invention, preferably in an immunoassay procedure.

In an embodiment the invention relates to a nucleic acid molecule comprising a sequence encoding the V _H domain of the antibody obtained from hybridoma cell line MAK<H-TMEM27>M-2.3.3 or hybridoma cell line MAK<H-TMEM27>M- 2.1.34, respectively. In an embodiment the invention relates to a nucleic acid molecule comprising a sequence encoding the V _L domain of the antibody obtained from hybridoma cell line MAK<H-TMEM27>M-2.3.3 or hybridoma cell line MAK<H-TMEM27>M-2.1.34, respectively.

The invention further comprises a nucleic acid molecule encoding an antibody chain, a variable chain or a CDR domain thereof according to the invention. Encoded polypeptides are capable of assembling together with a respective other antibody chain to result in an antibody molecule against protein TMEM27 according to the invention.

The invention further relates to a nucleic acid molecule comprising a sequence encoding the antibody produced by hybridoma cell line MAK<H-TMEM27>M- 2.3.3 which was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) on February 1, 2011 and received the deposit number DSM ACC3116 or hybridoma cell line MAK<H-TMEM27>M-2.1.34 which was deposited with the DSMZ on February 1, 2011 and received the deposit number DSM ACC3115, respectively.

The invention further provides prokaryotic and eukaryotic expression vectors containing said nucleic acids, and host cells containing such vectors for the recombinant production of such an antibody.

The invention further comprises a host cell comprising a vector according to the invention. In a preferred embodiment said host cell is a prokaryotic or eukaryotic host cell.

The invention further comprises a method for the production of a recombinant antibody according to the invention, characterized by expressing a nucleic acid according to the invention in a prokaryotic or eukaryotic host cell and recovering said antibody from said cell. The invention further comprises the antibody obtained by such a recombinant method.

Monoclonal antibodies according to the invention show benefits in the in vitro assessment of type II diabetes (T2D) in a subject. Antibody Deposition

The preferred hybridoma cell lines according to the invention, MAK<H- TMEM27>M-2.3.3 (producing monoclonal antibody MAb-<TMEM27>-M-2.3.3; also denoted as antibody 2-3) and MAK<H-TMEM27>M-2.1.34 (producing monoclonal antibody MAb-<TMEM27>-M-2.1.34; also denoted as antibody 2-34) were deposited, under the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure, with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Germany:

The antibodies obtained from said cell lines are preferred embodiments of the invention.

The following examples, references and sequence listings are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

Description of the Sequences

SEQ ID NO: 1 human protein TMEM27 (Swiss Prot Accession No.

Q9HBJ8).

SEQ ID NO: 2 DNA sequence of expression construct TMEM27(125)-His8. SEQ ID NO: 3 Deduced protein sequence of expression construct

TMEM27(125)-His8.

SEQ ID NO: 4 DNA sequence of expression construct TMEM27(125)- sCD40L.

SEQ ID NO: 5 Deduced protein sequence of expression construct

TMEM27(125)-sCD40L.

SEQ ID NO: 6 human protein TMEM27(31-45).

SEQ ID NO: 7 human protein TMEM27(59-69).

SEQ ID NO: 8 human protein TMEM27(91-105). SEQ ID NO: 9 human protein TMEM27(111-123).

SEQ ID NO: 10 human protein TMEM27(127-141).

SEQ ID NO: 11 human protein TMEM27(171-181).

SEQ ID NO: 12 human protein TMEM27(93-107). Description of the Figures

Figure 1: Vector map of pMl-MT.

Figure 2: DNA sequence (SEQ ID NO: 2) and deduced protein sequence

(SEQ ID NO: 3) of expression construct TMEM27(125)-His8. Expression construct TMEM27(125)-His8 used for expression and selection of the monoclonal antibodies according to the present invention (see Example 1).

Figure 3: DNA sequence (SEQ ID NO: 4) and deduced protein sequence

(SEQ ID NO: 5) of expression construct TMEM27Q25)- sCD40L). Expression construct TMEM27(125)-sCD40L used for DNA immunization (see Example 1, 2).

Figure 4: Reducing SDS-PAGE of TMEM27(125)-His8 following Ni-NTA purification: Both upper and lower protein band feature the same N-terminal sequence and may be products of different N-linked glycosylation at residues 76 and 93. The protein marker used was Novex® Sharp Protein Standard (Invitrogen).

Figure 5: Graphical representation of TMEM27 serum levels in ng/ml provided from Diabetes (Diabetes mellitus type II, T2D) patients and Normal (apparently healthy, reference sample) persons.

Figure 6: Binding Late [BL] / Stability Late [SL] Plot visualizing the

Kinetic Screening approach using recombinant TMEM 27 as antigen. Trendlines denote the theoretical antigen complex stabilities, which is expected according to a 1 : 1 Langmuir interaction with kd = lOE-01 1/s, kd = 10E-02 1/s, kd = 10E-03 1/s, kd = 10E-04 1/s, kd = 10E-05 1/s. The trendline t l/2diss lOmin indicates an antigen complex half life of 10 min according to the formula tl/2 diss = ln(2) / ( 60 * kd). Each dot stands for a tested <TMEM-27>M primary culture. Only primary cultures were selected with highest Binding Late and Stability Late values, which populate the corridor between tl/2 diss 10 min (trendline) and kd = 10E-05 1/s. The star indicate the primary monoclonal antibody culture 2-3 (deposited as DSM ACC3116) and the triangle indicate the primary monoclonal antibody culture 2-34 (deposited as DSM ACC3115), which were selected.

Figure 7: Clinical Samples - in vitro measurement of protein TMEM27 in ng/ml in serum samples provided from 15 Normal (apparently healthy, reference sample) and 15 Diabetes (T2D) donors.

Figure 8: Competitive binding analysis of MAb TMEM27 8/9 (DSM

ACC2995). X-axis: recombinant TMEM27 [pg/ml]; Y-axis: counts measured on multiplex platform IMPACT.

Figure 9: Western blot of linear epitope mapping (dot blot analysis) of

MAb-<TMEM27>-M-2.3.3 produced by hybridoma cell line MAK<H-TMEM27>M-2.3.3.

Figure 10: Western blot of linear epitope mapping (dot blot analysis) of

MAb-<TMEM27>-M-2.1.34 produced by hybridoma cell line MAK<H-TMEM27>M-2.1.34.

Example 1:

Cloning and transient gene expression of TMEM27(125)

The cDNA fragment encoding for the extracellular residues 1-125 of TMEM27 (Swiss Prot accession no. Q9HBJ8, SEQ ID NO: 1) was PCR amplified from template IRATp970E0577D (imaGenes GmbH, Berlin). The PCR primers featured a 5' Sal I-site and a 3' Not I-site for cloning into derivatives of plasmid pMl-MT (Roche Applied Science, Indianapolis) as shown in Figure 1.

For generation of the expression construct TMEM27(125)-His ₈ to be used for transient gene expression, the TMEM27(125) coding region was fused to a biotinylation / His ₈ -purification tag, as shown in Figure 2. TMEM27(125)-His8 DNA expression contruct DNA sequence has SEQ ID NO: 2 and the deduced protein sequence SEQ ID NO: 3.

For generation of the fusion construct TMEM27(125)-sCD40L to be used for DNA immunization of mice, the TMEM27(125) coding region was fused to a cDNA fragment encoding the soluble form of mouse CD40L, i.e. residues 112-260 of Swiss Prot accession no. P27548 as displayed in Figure 3, showing DNA sequence (SEQ ID NO: 4) and deduced protein sequence (SEQ ID NO: 5). sCD40L was previously reported to enhance the immune response (Wei Li, Immunology 115 (2005) 215-222: Synergistic antibody induction by antigen-CD40 ligand fusion protein as improved immunogen).

For transient gene expression in human embryonic kidney (F£EK)293 cells, the Freestyle™ 293-F expression system (Invitrogen Corp., Carlsbad) was used in combination with X-tremeGE E Ro-539 transfection reagent (Roche Applied

Science, Indianapolis). Three days post-transfection, the supernatant was cleared by centrifugation, supplied with DNase I from bovine pancreas and complete, EDTA-free Protease Inhibitor Cocktail Tablets (both: Roche Applied Science, Mannheim) and the pH adjusted to pH 7.5. The TMEM27(125)-His ₈ protein product was purified by standard Ni-NTA affinity chromatography using Ni-NTA

Superflow (QIAGEN GmbH, Hilden). In general, 1.2 L supernatant yielded 1.6 mg protein of reasonable purity as shown in Figure 4. The expected cleavage of the N-terminal signal sequence was demonstrated by protein sequencing: Both protein bands 1 and 2 exhibited the same N-terminal sequence (MLWLLFFLVTAIHA) [ ELCQPGAENAFKVRL.

For DNA immunization of mice, the expression plasmid was purified by a EndoFree Plasmid kit (QIAGEN GmbH, Hilden).

Example 2:

Production and screening for monoclonal antibodies against native TMEM27 Mice immunizations

Female NMRI mice, 8-12 weeks old, were injected intradermally with 30 μΐ DNA solution containing 50 μg of TMEM27(125)-sCD40L plasmid DNA into the base of the tail. The injection was followed by in vivo electroporation. Two plate electrodes were placed on the surface of the skin by squeezing the dermis blister developed by the injection. Electric pulses (two 450V pulses of 100 μβεϋ duration followed by four 115V pulses of 10 msec duration) were generated by a commercially available square wave pulse generator. The time interval between the pulses was 0.125 sec. After three weeks four further immunizations were performed at 3-week intervals. Ten days after the last immunization blood was taken and the antibody titer was determined in the serum of the immunized mice. Selected mice were given an intravenous booster injection of 50 μg of recombinant TMEM27 dissolved in PBS three days before fusion. Hybridoma production

Spleen cells of the immunized mice were fused with myeloma cells following the procedure of Galfre and Milstein, Meth. Enzymol. 73 (1981) 3-46. In this process lxlO ⁸ spleen cells of the immunized mouse were mixed with 2xl0 ⁷ myeloma cells (P3X63-Ag8-653, ATCC CRL1580) and centrifuged. The cells were then washed once in RPMI 1640 medium w/o FCS and again centrifuged at 400 g. The supernatant was discarded, the cell sediment was gently loosened by tapping, 1 ml PEG was added to this within one minute and mixed with the cells by gently swirling in a 37°C. warm water bath. Subsequently 5 ml RPMI 1640 medium w/o FCS was added dropwise within 5 min and mixed in a 37°C warm water bath by continuous swirling. After the addition of 25 ml RPMI 1640 medium w/o FCS the cells were centrifuged for 10 min at 400 g. The cell pellet was taken up in RPMI 1640 medium, 5% FCS and inoculated into azaserine-hypoxanthine selection medium (5.7 μΜ azaserine, 100 μΜ hypoxanthine, 2 mM glutamine, 1 mM sodium pyruvate, 50 μΜ 2-mercaptoethanol and 100 μΜ non-essential amino acids in

RPMI 1640 supplemented with 5% FCS). Mouse recombinant interleukin 6 (50U/ml) was added to the medium as a growth factor. After 10 days the primary cultures were tested for the synthesis of TMEM27-specific antibodies. TMEM27-specific hybridoma primary cultures were cloned in microtitre plates by means of fluorescence activated cell sorting (FACS).

Determination of the specificity of the produced antibodies

MAb production in hybridoma culture supernatants was assayed by indirect enzyme-linked immunosorbent assay (ELISA). Microtiter plates coated with sheep anti-rabbit Fey polyclonal antibody (Microcoat, Bernried, Germany) were incubated with anti-AviTag rabbit raw serum (Roche, Germany) diluted 1 :2000 in incubation buffer (phosphate buffered saline pH 7.3, 0.5% BycoC) for lh at room temperature. After washing with washing buffer (0.9% NaCl solution, 0.05% Tween 20 ^® ) the microtiter plates were incubated for lh at room temperature with lOOng/ml purified rec. TMEM27 diluted in incubation buffer. The microtiter plates were washed again and hybridoma supernatants were added to the coated well and incubated for lh at room temperature. After washing the bound MAbs were detected by a lh incubation with goat anti-mouse IgG peroxidase conjugate (Calbiochem, Germany) diluted 1 :5000 in incubation buffer followed by substrate reaction with ABTS solution (Roche, Germany) after further washing step. The color change was measured in an ELISA reader at 405/490 nm after 20-30 min. Production of sample IgG

Selected hybridoma clones were adapted to serum free medium (HyClone ADCF- MAb; Thermo Fisher) supplemented with 0.1% Nutridoma CS (Roche, Germany) and cultivated in 175cm ² tissue culture flask to a density of lxlO ⁵ cells/ml. 2xl0 ⁷ cells obtained from the preculture were resuspended in 10 ml of fresh medium and inoculated into the cell compartment of a CELLine classic 1000 bioreactor (Integra Biosciences, Germany). 500ml of fresh medium were added to the medium compartment and the cells were incubated for 6 to 7 days in C0 ₂ incubator. After the initial incubation medium change within the medium compartment and harvesting of 5 ml hybridoma suspension from the cell compartment were performed twice a week. The harvested cell suspension was centrifuged at 400 g and the cell free supernatant collected for subsequent IgG purification.

Example 3:

Characterization of <TMEM27>-specific monoclonal antibodies Kinetic Screening of hybridoma primary cultures

The BIAcore™ A100 system under the control of the Software V. l . l was prepared as follows: A BIAcore™ CM5 sensor (series S) was mounted into the system and was hydrodynamically addressed according to the manufacturer's recommendations. A polyclonal rabbit IgG antibody (<IgGFCyM>R, Jackson ImmunoResearch Laboratories Inc., USA) at 30 μg/ml was immobilized at 10,000

RU on spots 1, 2, 4 and 5 in the flow cells 1, 2, 3 and 4 via EDC/NHS chemistry according to the manufacturer's instructions using 10 mM sodium acetate buffer pH 4.5 as pre-concentration buffer. The sensor surface was finally blocked with ethanolamine. Hybridoma culture supernatants from different immunization campaigns were processed as outlined below.

The spots 2 and 4 of the sensor chip were used as a reference (1-2, 5-4). In order to capture antibody on the sensor surface hybridoma culture supernatants were diluted 1 :5 with running buffer HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05 % P20, BIAcore™) and were injected at 30 μΐ/min for 1 min.

Subsequently, the 150 nM of the respective 30 kDa recombinant TMEM27-GS- Avi-GS-Hiss protein was injected at 30 μΐ/min for 2 min association time. The dissociation phase was monitored for 5 min. Finally the surface was regenerated with a 3 min injection of 10 mM Glycine-HCl pH 1.7 at 30 μΐ/min.

For the selection of primary hybridomas the following procedure was used: A Binding Late (BL) reference point was set shortly before the antigen's injection end. A Stability Late (SL) reference point was set shortly before the end of the complex dissociation phase. The BL and SL data were graphically visualized (Figure 6). Hybridoma supernatants with highest BL and SL values and being located in the corridor between the indicator lines ti/2diss = 10 min and kd = 10e-05 1/s were positively selected. The data of the quantification of the kinetic screening approach are shown in

Table 1 and visualized in Figure 6.

Table 1:

Date in Table 1 are from left to right: Antibody No.: Primary Culture number; Antigen: recombinant, 30 kDa TMEM-27; CL: Capture Level in Response Units, describing the amount of antibody, which is selectively captured on the <M- IgGFCy>R coated sensor surface from the crude primary culture supernatant; BL: Binding Late in Response Units, describing the signal height at the end of the antigen association phase; SL: Stability Late in Response Units, describing the remaining signal height after 5 min of dissociation; kd (1/s): dissociation rate according to a Langmuir linear fit; tl/2 diss: Calculated antigen complex half life in minutes according to the formula tl/2 diss [min] = ln(2) / (60 * kd); MR: Molar Ratio, calculated upon the formula 150 kDa / 30 kDa * ( BL / CL); n.d. = not determined.

The kinetic signatures of the primary cultures were quantified using the BIAcore™ A100 evaluation software V.1.1.1. Antibody Capture Level, Binding Late, Stability Late were determined and the dissociation rate (kd), antigen complex half time (tl/2diss) and Molar Ratio (MR) were calculated. Primary cultures were selected according to highest possible Binding Late and Stability Late values, tm diss > 10 min and a Molar Ratio, indicating functionality of the antibody. Negative MR values iondicate no functionality, MR values > 2.5 indicate overstoichiometric binding.

Selected primary antibody cultures with suitable values were: 1-4, 1-12, 2-3 (deposited as DSM ACC3116), 2-32, 2-34 (deposited as DSM ACC3115), 2-66 and 2-72.

Crossblocking Experiment to identify suitable sandwich forming antibody pairs

The BIAcore™ A100 system under the control of the Software V. l . l was prepared like follows: A BIAcore™ CM5 sensor (series S) was mounted into the system and was hydrodynamically addressed according to the manufacturer's recommendations. A polyclonal rabbit IgG antibody (<IgGFCyM>R, Jackson ImmunoResearch Laboratories Inc., USA) at 30 μg/ml was immobilized at 10,000 RU on spots 1, 2, 4 and 5 in the flow cells 1, 2, 3 and 4 via EDC/NHS chemistry according to the manufacturer's instructions using 10 mM sodium acetate buffer pH 4.5 as pre-concentration buffer. The sensor surface was finally blocked with ethanolamine.

Hybridoma clone culture supernatants were processed as outlined below.

The spots 2 and 4 of the sensor chip were used as a reference (spot 1-2, spot 5-4). In order to capture antibody on the sensor surface hybridoma clone culture supernatant of the respective antibody producing cell line was diluted 1 :2 with running buffer HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05 % P20, BIAcore™) and was injected at 30 μΐ/min for 1 min. 3 μΜ of an IgG fragment (IgGl, IgG2a, IgG2b, IgG3) containing blocking solution was injected at 30 μΐ/min for 5 min. 150 nM recombinant TMEM27-GS-Avi-GS-His ₈ protein was injected at 30 μΐ/min for 3 min association time. Hybridoma clone culture supernatant of the respective antibody producing cell line was diluted 1 :2 and was injected at 30 μΐ/ min for 5 min.

Finally the surface was regenerated with a 3 min injection of 10 mM Glycine-HCl pH 1.7 at 30 μΐ/min.

The data was evaluated using the BIAcore™ evaluation software V.1.1 (Antibody Extension Package) according to the recommendations of the supplier.

All Clone Culture Supernatants 1-4, 1-12, 2-3 (deposited as DSM ACC3116), 2-32, 2-34 (deposited as DSM ACC3115), 2-66 and 2-72 were reciprocatively tested to determine the optimal epitope accessibility.

In Table 2 the expected epitope accessibility in % as it is calculated by the BIAcore™ A100 antibody extension package evaluation software is shown. Depending on the primary antibody/secondary antibody injection, the antibody pair 2-34/2-3 and 2-3/2-34 generates a sandwich with 26% and 54% accessibility. The noise of the measurement is +/- 8 %. Table 2: Expected epitope accessibility in %

The monoclonal antibody producing clones MAK<H-TMEM27>M-2.3.3 (Deposit No. DSM ACC3116) producing monoclonal antibody MAb-<TMEM27>-M-2.3.3 (also denoted as antibody 2-3) and MAK<H-TMEM27>M-2.1.34 (Deposit No. DSM ACC3115) producing monoclonal antibody MAb-<TMEM27>-M-2.1.34 (also denoted as antibody 2-34) have been deposited at the DSMZ.

Example 4:

Production of fragments and conjugates Purification of cell culture supernatants

The cell culture supernatant was purified using affinity chromatography on protein A coated to sepharose particles. The supernatant was loaded on a pre-equilibrated column (50 mM TRIS, 150 mM NaCl, pH 8.0) and washed by 3 volumes of the equilibration buffer. The bound IgG fraction was eluted accordingly to the M-IgG subclass:

- the M-IgGl (antribody clone 2-34) was eluted using 0.2 M citrate buffer, pH 4.5

- the M-IgG2b (antibody clone 2-3) was eluted using 0.2 M glycine buffer, pH 3.0. The fractions were collected in 0.1 M TRIS, pH 8.0 buffer in order to increase immediately the pH value of the eluate. The IgG fractions were pooled und dialysed against a 50 M phosphate, 150 NaCl, pH 7.8 buffer and stored at -80°C.

Preparation of biotin conjugated F(ab')2 fragment of the antibody 2-34

F(ab') ₂ fragment: The full length therapeutic antibody of the class G (IgG) in 100 mM sodium citrate buffer, pH 3.7 was incubated with pepsin (1 to 15 μg pepsin per mg IgG). The fragmentation was analyzed by analytical gel permeation chromatography and stopped after 90 minutes by adjusting the pH value to 6.5 by the addition of potassium phosphate.

Purification: The fragmentation mixture was dialysed against 10 mM sodium phosphate buffer with 10 mM sodium chloride, pH 5.5, the solution was applied to an SP-sepharose chromatography column, the isolated fractions eluted in a salt gradient were analyzed individually by analytical gel filtration. The pool containing the antibody F(ab') ₂ fragments was applied to an affinity matrix with immobilized polyclonal antibodies against mouse Fcg to eliminate trace amounts of Fcg fragments, the flow through was pooled and analyzed to a residual Fcg content. The affinity purification procedure was repeated at least three times until the residual Fcg concentration fell below 0.5 ppm. The product was concentrated to about 10 mg/ml and finally applied to a gel filtration column (Superdex 200).

Preparation of biotin conjugated Fab' fragment of the antibody 2-34 The purified F(ab') ₂ fragment was incubated with 5 mM cysteamine for 1 hour, the reduction to a Fab' fragment was monitored by analytical gel permeation chromatography. The raw product was applied to a gel filtration column (Superdex 200) and the pooled Fab' fractions were immediately conjugated with MEA activated biotin labels (stoichiometry 1 : 10, 1 hour). The final analytical characterization was performed by ESI-MS in order to confirm the conjugation site and yield, respectively.

Preparation of digoxigenin conjugated IgG (antibody clone 2-3)

Conjugation: the purified IgG was conjugated using NHS activated digoxigenin label at pH value of 8.2-8.4. The reaction stoichiometry was 1 :5 (IgG:label), the reaction was stopped by addition of 1 M lysine solution after 1 hour and the raw conjugate were purified on a gel filtration column (Superdex200). Example 5:

Assay development for detection of TMEM27 in human samples

Roche Professional Diagnostics (Roche Diagnostics GmbH) has developed a multiplex platform called IMPACT (Immunological Multi-Parameter Chip Technology), which is based on a small polystyrene chip manufactured by highly tested and validated procedures (Hornauer, et al., BlOspectrum, Special Proteomics 10 (2004) 564-565 and Hornauer, et al., Laborwelt 4 (2004) 38-39). During chip manufacturing, the chip surface is coated with a streptavidin layer, onto which biotinylated antibodies, proteins, or peptides are spotted in vertical rows for the duplicate analysis of samples. Each chip contains up to 10 markers, and each marker is arrayed on the chip as a vertical row of 10-12 spots; a minimum of 5 spots is required for determination of the mean level of a specific analyte in a sample. During the assay, the arrayed markers are probed with a small volume (40μ1) of diluted sample and with a digoxigenylated secondary monoclonal antibody. The secondary antibody is then detected by the addition of an anti-digoxigenin antibody conjugated to a fluorescent latex label. This label enables highly sensitive detection of < 10 individual binding events in a single spot, down to the fM concentration. After a final incubation, chips are transferred to a detection unit where a charge-coupled device camera creates an image that is converted to signal intensities, and fluorescence intensity of the array features is quantified by image analysis.

This technology platform was used to develop a TMEM27 immunoassay. A highly sensitive immunoassay (LDL = 0.67 pg/ml) was developed. For that purpose MAb-<TMEM27>-M-2.1.34 was used as a biotinylated Fab' fragment and spotted onto the streptavidin coated surface of the chip. Patient samples were diluted 1 :2 and each chip was probed with 40 μΐ of a diluted patient sample. After a washing step each chip was probed with 40 μΐ of a digoxigenylated MAb-<TMEM27>-M- 2.3.3. A serial dilution of recombinantly expressed TMEM27 (see Example 1) was used as a standard. Example 6:

Detection of TMEM27 in human samples

A set of 30 human plasma samples was used to assess the presence of protein TMEM27 in human plasma in vitro. These samples comprised 15 samples derived from type 2-diabetes patients (T2D) and 15 reference samples from apparently healthy individuals. Protein TMEM27 was detectable in the majority of the samples. Apparently, increased levels of protein TMEM27 are observed in T2D samples (data shown in Figure 5 and Figure 7).

Example 7:

Epitope characterization of TMEM27 antibodies

To determine, whether the TMEM27 antibodies MAb-<TMEM27>-M-2.3.3 or MAb-<TMEM27>-M-2.1.34 detect a conformation epitope or a linear epitope, a dot-blot analytic western blot was performed.

During this linear epitope mapping test (dot blot analysis) 105 screening peptides (15'mer peptides, each shifted by 2 amino acids) of human protein TMEM27 from position 1-222 of SEQ ID NO: 1 were spotted to a nitrocellulose membrane. The 105 peptides spotted on said screening membrane were incubated with the antibody of interest at a concentration of 1 μg/mL.

It has been found that MAb-<TMEM27>-M-2.3.3 produced by hybndoma cell line MAK<H-TMEM27>M-2.3.3 binds to the following peptides (Figure 9):

TMEM27(31-45) IRT ALGDKAYAWDTN (SEQ ID NO : 6),

TMEM27(59-69) RKVPNREATEI (SEQ ID NO: 7),

TMEM27(91-105) SKNHTLP AVE VQ S AI (SEQ ID NO: 8),

TMEM27(111-123) RINNAFFLNDQTL (SEQ ID NO: 9), TMEM27(127-141) KIPSTLAPPMDPSVP (SEQ ID NO: 10),

TMEM27(171-181) RKNKEP SEVDD (SEQ ID NO: 1 1).

It has been found that MAb-<TMEM27>-M-2.1.34 produced by hybridoma cell line MAK<H-TMEM27>M-2.1.34 binds to (Figure 10):

TMEM27(93-107) NHTLP AVE VQ S AIRM (SEQ ID NO: 12). Example 8:

Competition characterization of TMEM27 antibodies

The immunoassay described in example 5 was used to assess the ability of monoclonal antibody TMEM27 8/9 produced by hybridoma cell line DSMZ ACC2995 to compete for binding on recombinant and native human TMEM27, respectively. For this purpose a serial dilution of recombinant protein TMEM27 (production of recombinant protein TMEM27 shown in example 1) and a native sample containing a high level of human protein TMEM27 was analyzed. The assay was run under standard conditions on multiplex platform called IMPACT as described in example 5 in the presence of 4-fold and 10-fold excess of monoclonal antibody TMEM27 8/9 and, as a positive control, in the presence of 5 μg/ml (equal amount) of unlabeled MAb-<TMEM27>-M-2.3.3, respectively. As can be seen from Figure 8 (Competitive binding of MAb TMEM27 8/9) addition of 5 μg/ml of unlabeled MAb-<TMEM27>-M-2.3.3 reduced the signal for TMEM27 on recombinant protein TMEM27 as well as in native samples (Table 3), significantly. In contrast, the addition of a 4- and 10-fold excess of monoclonal antibody TMEM27 8/9 led only to a minor decrease in signal for recombinant protein and had no effect on the native detection. Obviously, MAb-<TMEM27>- M-2.3.3 and MAb-<TMEM27>-M-2.1.34 detect epitopes which are different from the one of monoclonal antibody TMEM27 8/9 and/or have a better binding affinity to protein TMEM27.

Table 3: Competition in native sample containing a high level of native protein

TMEM27

cts. = counts measured