NZ621727B2 - B7-h6 therapeutically active monoclonal antibody against b7-h6 polypeptide - Google Patents

Abstract

Disclosed is an antibody which specifically binds to an epitope formed by a portion of the extracellular domain of the B7-H6 polypeptide, said portion having an amino acid sequence of GDHQEAFRPGAIVSPWRLKSGDASLRLPGIQLEEAGEYRCEVVVTPLKAQGTVQLEVV.

Description

WO 37727 B7—H6 therapeutically active monoclonal antibody against B7-H6 polypeptide The present invention is concerned with diagnostic methods and means. Specifically, it relates to an antibody which specifically binds to a portion of the extracellular domain of the B7-H6 polypeptide. Moreover, said antibody is provided for use in the treatment or diagnosis of cancer or inflammatory disease. Furthermore, provided are a method for diagnosing cancer in a sample of a subject suspected to suffer from cancer or an inflammatory disease. Further, the present invention concerns a device and a kit for diagnosing cancer or inflammation.

BACKGROUND Until today, cancer is one of the leading causes of deaths in the United States, even though ss has been made in reducing incidence and ity rates and improving survival (see Jemal et al. 2010, CA Cancer J Clin. Sep-Oct 60(5):277—300). Further progress can be accelerated by improving stic methods and means due to the fact that cancer development is often ated with the lack of specific recognition of tumor cells by the immune system.

Targeted cancer therapy comprises medication which interferes with specific targeted molecules (e.g., monoclonal or polyclonal antibodies) to directly block the growth of cancer cell. Thus, targeted cancer therapy may be more effective than traditional therapeutic approaches (e. g., resection, radiation, chemotherapy) and may be less harmful to normal cells. Monoclonal antibodies (mAb) can be designed to specifically bind to an extracellular domain or to a cell surface target of the target cell to stimulate the immune system of the patient. Monoclonal antibodies can also be created for numerous serious diseases (e.g., inflammatory diseases or different types of cancers). Thus, monoclonal antibodies may provide reliable and efficient eutic and diagnostic methods and means to e.g., detect early developmental stages of these diseases or to offer therapeutic ches. l killer cells (NK cells) constitute a major component of the innate immune system shaping the atory and adaptive immune response (see Vivier et a1. 2008, Nat. lmmuno. 510) and g a crucial role in the rejection of transformed and virally infected cells (see Smyth et al. 2002, Nat. Rev. Cancer 2:850-861; Lanier 2005, Annu. Rev. Immunol. 23 74). NK cells survey target cells for expression of major histocompatibility complex (MHC) class I (see Parham 2005, Nat. Rev.

Immunol. 5:201 —204) which protects the target cell from NK cell activation and from NK cell attack. Target cells which lack MHC class I are directly killed by NK cells due to the induction of apoptosis (programmed cell death). The discovery ofNK- activating receptors (e.g., the natural cytotoxicity receptor (NCR) family like NKp30) revealed that also activation signals are necessary for the activation ofNK cells and tumor cell lysis (see Pende et al. 1999, Cancer Res. 62:6178—6186; Moretta et al. 2001, Annu. Rev. Immunol. 19: 197-223).

Recently, it could be shown that the human NKp30 directly cts with the B7 family member B7-H6 whose expression on tumor cells induces NKp3 O-dependent cell activation and cytotoxity (see Brandt et a1. 2009, J. Exp. Med. : 1495— 1503; US 2011/0081346). Hereby, the extracellular domain ofNKp30 directly interacts with the extracellular domain of B7-H6 which is ively expressed on the surface of several tumor cell lines (see Brandt et a1. 2009, J. Exp. Med. 206(7): 1495—1503).

SUMMARY OF THE INVENTION Accordingly, the present ion provides an antibody which specifically binds to an epitope formed by a portion of the extracellular domain of the B7-H6 polypeptide, wherein said portion having an amino acid sequence as shown in SEQ ID NO: 22.

Preferably, said antibody comprises complementarity ining regions (CDRS) as shown in SEQ ID NOs: 5, 7, 9, 15, 17, and 19.

Preferably, said antibody is a onal antibody.

Preferably, said sequence represents an IgV-like domain.

In a preferred embodiment of the antibody of the invention, said antibody comprises complementarity determining s (CDRS) as shown in SEQ ID NOs: 5, 7, 9, 15, 17, and 19. Nucleic acid sequences of the above mentioned CDRS were annotated ,va op2375 p2-3a p19a speci,2 according to the lMGT—ONTOLOGY (see Giudicelli and Lefranc 1999, Bioinformatics 15: 1047—1054).

In a preferred embodiment of the antibody of the invention, said antibody is a monoclonal antibody. More ably, said antibody is the antibody deposited under accession number DSM ACC 3117 at the DSMZ, Braunschweig, Germany under the Budapest treaty on February 02, 2011.

Preferably, an antibody of the invention for use in the treatment or diagnosis of cancer. Preferably, the cancer is T cell lymphoma, myeloid leukemia, colon carcinoma, B cell lymphoma, melanoma, or al carcinoma.

Preferably, an antibody of the invention for use in the treatment or diagnosis of inflammatory disease. Preferably, the inflammatory disease is a viral infection. ably, a method for diagnosing cancer in a sample of a subject suspected to suffer from cancer comprising: a) contacting the sample with the antibody of the invention under conditions which allow for binding of said antibody to its epitope on the B7-H6 polypeptide; and b) determining binding of the antibody to the said epitope, whereby cancer is diagnosed.

In a red embodiment of the method of the ion, the cancer is T cell lymphoma, myeloid leukemia, colon carcinoma, B cell lymphoma, melanoma, or cervical carcinoma.

Preferably, a method for diagnosing an inflammatory disease in a sample of a subject suspected to suffer from an inflammatory e comprising: i 30 a) ting the sample with the antibody of the invention under ions i which allow for binding of said antibody to its e on the B7-H6 i polypeptide; and b) determining g of the antibody to the said epitope, whereby the inflammatory disease is diagnosed.

In a preferred embodiment of the method of the invention, said sample is a tissue or body fluid sample. /03/15,va op2375 p2-3a pl9a speci,3 -3a- Preferably, a device for diagnosing cancer or an inflammatory disease in a sample comprising: a) an analyzing unit comprising the antibody of the invention; and b) a detector which detects g of the antibody in the analyzing unit to its epitope on the B7-H6 polypeptide.

In a preferred embodiment of the device of the invention, said sample is a tissue or body fluid sample.

A kit for diagnosing cancer or an inflammatory disease comprising the dy of the invention and, preferably, an agent for detection of binding of said antibody to its epitope on the B7-H6 ptide. /03/15,va op2375 p2-3a pl9a speci,a FIGURES Figure 1 shows the nucleic acid and amino acid sequences of the B7—H6-Ig-fusionprotein.

Italic c acid and amino acid sequences indicate enzymatic restriction sites. Nucleic acid and amino acid sequences of the extracellular domain of human B7-H6 are underlined bo 1d, whereby said sequences of Fcm are dotted underlined.

Figure 2 shows the amino acid sequence of the extracellular domain of the human B7-H6 polypeptide and indicates the IgV—like domain and the ke domain.

Figure 3 shows that the anti-B7-H6 clone 1.18 reacts with B7-H6 using an enzyme-linked immunoabsorbant assay (ELISA).

Figure 4a depicts that the anti-B7—H6 clone 1.18 binds to B7—H6 on ectants (BA/F3— B7—H6) using fluorescence—activated cell sorting (FACS). Figure 4b shows that the anti— B7-H6 clone 1.18 binds to B7—H6 on cell lines (haematopoictic and solid tumor origin), but not to healthy peripheral blood mononuclear cells (PBMCS).

Figure 5 shows that a portion of the IgV domain of B7—H6 is involved in binding of anti- B7-H6 clone 1.18.

Figure 6 depicts that cell surface expression of B7-H6 determined by fluorescence- activated cell g (FACS) and mRNA expression in the different cell lines. Figure 6a shows the expression of B7-H6 in tumor cell lines of hematopoietic origin. Figure 6b shows the expression of B7-H6 in tumor cell lines of solid tumor origin.

Figure 7 shows that the anti-B7-H6 mAb 1.18 detects B7-H6 on cytospins n sections) of BA/F—3-B7—H6 transfectants.

Figure 8 shows that y natural killer (NK) cells degranulate upon co—culture with BA/F3-B7-H6 transfectants.

DETAILED DESCRIPTION OF THE INVENTION The present ion relates to an antibody which ically binds to an epitope formed by a portion of the extracellular domain of the B7—H6 polypeptide, said portion having an PCT/EP2()12/067637 amino acid sequence as shown in SEQ ID NO: 22. Preferably, said sequence ents an IgV-like domain.

The term ody” refers to all types of dies which specifically bind to an epitope comprised in a portion of the extracellular domain of the B7-H6 polypeptide. Epitopes as referred. to herein are, preferably, defined by stretches of 7 to 15, preferably 8 to 11 contiguous amino acids in length. However, an epitope in accordance with the present ion can also be formed by a ceitain dimensional structure and such structural epitopes are also envisaged herein. Specific binding in this context means that the antibody of the invention essentially binds to the epitope without significant cross—reactivity (i.e. binding) to other epitopes either on the B7-H6 polypeptide or other polypeptides. Specific binding can be ined by techniques well known in the art. Preferably, the antibody binds specifically to the said epitope. The aforementioned epitope shall be located in a portion of the extracellular domain of the B7-H6 polypeptide. ably, the B7-H6 polypeptide has an amino acid sequence as shown in SEQ ID NO: 2 and the said extracellular domain corresponds to amino acids 58 to 300 of said sequence (see also Figures 1 and 2). It will be understood that the B7-H6 polypeptide may also be represented by a variant ce of SEQ ID NO: 2 which differs therefrom by substitution, addition and/or deletion of one or more amino acids. Such t sequences may be orthologous amino acid sequences from other species as well as paralogous or other homologous ces of the aforementioned specific B7—H6. Preferably, such variant ces are at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identical over the entire length or at least 50% of SEQ ID NO: 2 with the said sequence. The term “sequence ty” as used herein refers to a relationship between two or more polypeptide sequence, namely a reference sequence and a given sequence to be compared with the reference sequence. Sequence identity can be ined by comparing the given ce to the reference sequence after the ces have been d optimally to produce the highest degree of sequence similarity which can be determined by the match between strings of such sequences. Said alignment can be performed by a skilled artisan without further ado. 3O Accordingly, sequence identity provides information on the total number of said matches.

Sequence identity can be, preferably, calculated using publicly available computer programs which are known by a skilled artisan, e.g., BLAST and PASTA. Other sequence variants envisaged in accordance with the present invention are those which are encoded by nucleic acid molecules capable of hybridizing under stringent hybridization conditions to the nucleic acid ce encoding B7—H6 shown in SEQ ID NO: 1. Preferably, the B7- H6 polypeptide is encoded by the nucleic acid sequence shown in SEQ ID NO: 1.

Stringent hybridization conditions referred to in accordance wit the present invention are equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in l X SSC, 0.1 % SDS at 50°C or 65°C, wherein nucleic acid molecule probe comprising at least 100, more preferably at least 150, even more ably at least 200, most ably at least 250 consecutive nucleotides of SEQ ID N02] or its reverse complement is used. It will be understood that the first and the last amino acid of the extracellular domain in such sequence ts may differ from the positions indicated for SEQ ID NO: 2, above. However, the extracellular domain will start and end at positions corresponding to the said positions. Such corresponding positions can be determined by sequence is tools by the skilled artisan without further ado.

Preferably, an antibody as referred to in accordance with the present invention encompasses a monoclonal antibody, a single chain antibody, a chimeric antibody or any fragment or derivative of such antibodies having the above ned binding properties.

Such fragments and derivatives comprised by the term dy as used herein encompass a synthetic dy, an Fab, F(ab)2 Fv or scFV fragment, or a chemically modified derivative of any of these antibodies. Chemical modifications envisaged preferably by the present invention include those which aim to couple the antibody to a detectable marker as specified elsewhere in this specification. Antibodies or fragments thereof, in l, can be obtained by using s which are described, e.g., in Harlow and Lane odies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988.

Advantageously, the antibody of the present invention specifically binds to B7-H6 with a high affinity. In the studies underlying the present invention it has been found that compared to other anti-B7—H6 antibodies described or suggested in the prior art (Brandt 2009, J. Exp. Med. 206(7): 1495-1503 and US 2011/0081346), the antibody is particularly useful in in vivo applications such as FACS sorting and cell culture as well as in Vitro applications including immunohistochemistry on, e.g., frozen tissue sections. Thanks to the present invention, cancer diagnosis based on the determination of B7-H6 will improve. er, therapeutic approaches which aim to target anti—tumor drugs to B7-H6 positive 3O cells are le.

In a preferred embodiment of the antibody of the present ion, said antibody comprises complementarity determining regions (CDRs) as shown in SEQ ID NOS: 5, 7, 9, 15, 17, and 19. Nucleic acid sequences of the above mentioned CDRs were annotated according to the IMGT-ONTOLOGY (see Giudicclli and Lefranc 1999, Bioinformatics :1047—1054).

The term “complementarity determining region” or “CDR” as used herein refers to variable domains of an antibody which are responsible for the specificity in antigen binding. An antigen, usually, comprises three CDRs (CDRl, CDR2 and CDR3). These CDRs are arranged in a non-consecutive manner. Since the antigen recognizing portions of the antibody are typically composed of two variable domains on a heavy and a light chain, six CDRs come into contact with the antigen upon binding. The CDRs can be transferred from one dy species to another by tional molecular biology techniques such as CDR grafting (see Ewert 2004, s 34(2): 184—199; Benny KC. L0 in Antibody ering — Methods in Molecular Biology 2004, Volume 248, 11, 135—159, DOI 10.1385/1—59259—666—5zl35).

It will be understood from the above that in another preferred embodiment, the antibody of the present invention is a monoclonal antibody.

Preferably, such a monoclonal antibody can be prepared by applying an immunogenic polypeptide having the portion of the ellular domain as characterized above to a mammal, preferably a mouse. More preferably, the immunogenic polypeptide is conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). Depending on the host species, various adjuvants can be used to increase the immunological response. Such adjuvants encompass, ably, Freund’s adjuvant, mineral gels, e.g., aluminum hydroxide, and surface active substances, e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Monoclonal antibodies according to the invention can be subsequently ed using the well known hybridoma technique, the human B cell hybridoma technique, and the EBV hybridoma que. Further details on the preparation of an dy of the invention are described in the anying Examples below.

In a more red embodiment of the antibody of the t invention, the antibody is 3O the antibody or the antibody produced by the corresponding hybridoma cell clone as deposited under accession number DSM ACC 3117 at the “DSMZ — Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH”, 38124 Braunschweig, GERMANY on February 2, 2011 according to the Budapest Treaty by “Deutsches orschungszentrum”, Heidelberg, GERMANY.

The entioned anti—B7-H6 mAb shall comprise at least one heavy chain and at least one light chain. Preferably, anti—B7—H6 mAb has an amino acid sequence of the heavy chain (lGHV/lGHD/lGHJ) as shown in SEQ ID NO: 3, whereby the secreted form (IGHV/IGHD/IGHJ/IGHGl) is shown in SEQ ID NO: 11 and the membrane bound form (IGHV/IGHD/IGHJ/IGHGl) is shown in SEQ ID NO: 12. The nucleic acid ces of fragments 1-4 of the heavy chain are shown in SEQ ID NO: 4, 6, 8, and 10 and the nucleic acid sequences of CDRs 1-3 of the heavy chain are shown in SEQ ID NO: 5, 7, and 9.

Further, said antibody has an amino acid sequence of the light chain (IGLV/IGLJ) as shown in SEQ ID NO: 13, y the ce of IGLV/IGLJ/IGLC is shown in SEQ ID NO: 21. The nucleic acid sequences of fragments 1-4 of the light chain are shown in SEQ ID NO: 14, 16, 18, and 20 and the nucleic acid sequences of CDRs 1-3 of the light chain are shown in SEQ ID NO: 15, 17, and 19. It will be understood that the anti-B7-H6 mAb may also be represented by variant sequences of the aforementioned SEQ ID NOS: 3— 21 which differ therefrom by substitution, addition and/or deletion of one or more amino acids. Such variant ces may be orthologous amino acid sequences from other species as well as paralogous or other homologous sequences of the aforementioned specific anti—B7—H6 mAb. Preferably, such variant sequences are at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identical over the entire length or at least 50% of SEQ ID NOS: 3-21 with said sequences. The term ce identity has been defined elsewhere in this description and applies mutatis mutandis.

The present invention further relates to an antibody of the invention for use in the treatment or diagnosis of .

The term "treatment" as used herein encompasses the amelioration of a disease referred to herein or its ms as well as curing the disease, i.e. the reestablishment of the healthy condition in a subject with respect to the disease or its symptoms. Amelioration as referred to herein refers to a significant ement of the health condition with respect to the disease or a symptom of the disease. Such a significant improvement is, preferably, clinically apparent in, e.g._, staging or grading systems applied in order to igate a subject. As will be understood by those d in the art, treatment as used herein is 3O usually not intended to be correct for all (i.e. 100%) of the subjects under a given treatment. The term, however, requires that a statistically significant portion of subjects can be treated (eg. a cohort in a cohort study). Whether a n is statistically significant can be determined without further ado by the person skilled in the art using vaiious well known tic evaluation tools, e.g., determination of nce intervals, p-value determination, Student's t—test, Mann— Whitney test etc.. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. 2()12/()67637 Preferably, the antibody of the invention for use in treating cancer is either coupled to a cytotoxic agent or an anti-tumor agent or is capable of recruiting such agents suitable for treating cancer. The term “agent” as used herein refers to an element, nd, or other molecular entity (e.g., a pharmaceutical compound, a therapeutic compound, or a pharmacologic compound). Such an agent can be natural, synthetic or a combination thereof. The term “therapeutic agent” as used herein refers to an agent that either alone or in combination with another agent exhibits a eutic or cial effect on a cell or a tissue. Preferably, a eutic agent in accordance with the present invention shall comprise drugs, toxins, immunomoduiators, chelators, boron compounds, photoactive agents or dyes, and radioisotopes. Techniques for coupling therapeutic agents to polypeptides such as to antibodies are well—known by the skilled artisan (e.g., Amen et a1. 1985 "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer y (Reisfeld et al. eds, Alan R. Liss, Inc, 1985)). The term “cytotoxic agen ” as used herein refers to an agent that has a cytotoxic or cytostatic effect on a cell, thereby depleting or inhibiting the growth of, respectively, cells within a cell population. Preferably, cytotoxic agents in accordance with the present invention shall comprise anti-tubulin agents (e.g., dolastatins, Vinca alkaloids, podophyllatoxins, taxanes, in derivatives, cryptophysins, maytansinoids, and combretastatins), DNA minor groove binding agents, DNA replication inhibitors, ting agents (e.g., platinum complexes), anthracyclines, antibiotics, lates, antimetabolites, herapy sensitizers, duocarmycins, ides, ated pyrimidines, ionophores, lexitropsins, nitro soureas, platinols, pre-forming compounds, purine tabolites, puromyeins, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, Vinca alkaloids, or the like. The term “anti-tumor agent” as used herein refers to an agent that has a cytotoxic or malign effect on cancer cells, thereby arresting the growth of, respectively, cancer cells within a tumor resulting, preferably, in cell death. Preferably, the antibody of the invention binds to a target cell (c.g., a cancer cell) and specific effector cells expressing receptors for said antibody (e.g., natural killer cells, monocytes, granulocytes) which results in target cell death. In r preferred embodiment of the 3O invention the antibody of the invention is coupled to a cytotoxic agent or an anti-tumor agent Via a linker. Preferably, a linker in accordance with the present invention shall comprise linker that are cieavabie under intracellular conditions (e.g., a peptide linker cleavable by an intracellular se, dipeptide linker, disulflde linker, and hydrolysable linker which are e.g., hydrolysable at a pH of less than 5.5). However, the antibody of the invention may also be used for treating cancer due to its blocking and binding ties on B7-H6 as a modulator of ing cascades involved in cancer.

PCT/EP2()12/067637 The term "diagnosis" as used herein means the assessment whether a subject s from a disease referred to herein, or not. As will be tood by those skilled in the art, such an assessment is usually not intended to be correct for all (i.e. 100%) of the subjects to be identified. The term, however, requires that a statistically significant portion of subjects can be fied (e.g., a cohort in a cohort study). Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools referred to elsewhere herein. Diagnosis according to the present invention es applications of the method in monitoring, confirmation, and sub-classification of the nt disease. Moreover, the establishment of a diagnosis as used herein also includes establishing a prognosis for a subject. Such a prognosis is a predictive indicator for the further development of the disease in a future time window, i.e. the predictive window. Thus, a diagnosis as used herein, preferably, encompasses a prediction ofwhether a subject will improve with respect to the disease or diseases ms in the future or whether the disease or symptoms will become worse.

Accordingly, the antibody of the invention can be also applied for risk stratification approaches and, thus, for determining the amount of intensive care and alization which will be required for an individual subject suffering from a disease referred to . ably, the antibody of the invention for use in diagnosis is either coupled to a detection agent or is capable of recruiting such an agent. A detection agent as used herein encompasses a radioactive e (e.g., radioactive isotopes of Iodide Technetium), fluorescent or chemoluminescent agents (e.g., FITC, rhodamin), an enzyme which is capable of generating a detectable signal by converting a substrate (e.g., horseradish peroxidase, firefly luciferase, or beta galactosidase), a fluorescent protein (e.g., green—, blue- or red— fluorescent protein). Suitable detection agents are well known in the art. Also preferably, the antibody to be applied in the method of the present invention can be coupled to an agent that is capable of attracting a ion agent. Such an agent may be biotin. In such a case an avidin— or streptavidin coupled detection agent can be used which upon binding of the biotin of the bound antibody will serve as a detectable marker.

Suitable detectable markers in such a case are those referred to above, more preferably, an enzyme shall be used as a detectable marker in such a case. rmore, a secondary dy may be used for detection of the first antibody, i.e. the antibody to be applied in the method of the present invention which is bound to the B7-H6 polypeptide of the sample. Such a secondary antibody shall be coupled to a detectable marker as describe above. Thus, in the latter case, the secondary antibody will upon g to the first antibody generate a detectable signal and thereby enables the detection of the bound first antibody. The principle of detection of bound antibodies with a secondary antibody is well known in the art and routinely applied, e.g., for ining dy binding on tissue 2012/067637 sections. Dependent on the type of detectable , different detection methods can be d using a reader system for the signal generated by the detectable marker. Such systems include automatic signal reader device, such as an ELISA or RIA reader, but also microscopic device for manual or automatic detection of the detectable signal. Moreover, the reader system may determine additional ation of the sample, e. g., a microscopic system may display the cells of a tissue section optically or an automated signal reader may determine further biomarkers comprised by the sample in addition.

The term “cancer” as used herein refers to any ant neoplasm. The malignant neoplasm refers to diseases ing from the undesired growth, the invasion, and under certain conditions metastasis of impaired cells in an organism. The cells giving rise to cancer are genetically impaired and have usually lost their ability to control cell division, cell migration behavior, differentiation status and/or cell death machinery. Most cancers form a tumor but some hematopoietic cancers, such as ia, do not. The cancer in accordance with the present invention shall comprise cancer cells expressing a B7—H6 polypeptide as specified elsewhere . Preferred types of cancer are selected from the group ting of: T cell ma, myeloid leukemia, colon carcinoma, B cell lymphoma, melanoma, or cervical carcinoma. Symptoms and staging systems for the different cancers are well known in the art and described in standard text books of pathology. Cancer as used herein encompasses any stage, grade, morphological feature, invasiveness, aggressiveness or malignancy of the cancer or the tissue or organ affected thereby.

The present invention further relates to an antibody of the invention for use in the treatment or diagnosis of an inflammatory disease.

Preferably, the antibody of the ion for use in treating an inflammatory disease is either coupled to an anti-inflammation agent or is capable of rec1uiting such an agent as specified ere herein. However, the antibody of the invention may also be used for an inflammatory disease due to its blocking and binding properties on B7—H6 as a modulator of signaling cascades involved in inflammation.

Preferably, the antibody of the invention for use in diagnosis is either coupled to a detection agent or is capable of recruiting such an agent as specified elsewhere herein.

The term “inflammatory disease” as used herein refers to a tissue response involving atory cytokines and inflammatory cell rates in response to an injury or destruction of tissue. The inflammatory disease in ance with the present invention shall comprise a viral infection, and bacterial infection. In addition, autoimmune diseases such as diabetes, multiple sclerosis and inflammatory bowl disease are included.

If follows from the above that the present ion also relates to a method for diagnosing cancer in a sample of a subject suspected to suffer from cancer comprising: a) contacting the sample with the antibody of the invention under conditions which allow for binding of said antibody to its epitope on the B7-H6 polypeptide; and b) ining binding of the antibody to the said epitope, whereby cancer is sed.

The term “diagnosing” as used herein means the assessment whether a subject suffers from a disease referred to herein, or not. As will be understood by those skilled in the art, such an assessment is usually not intended to be correct for all (i.e. 100%) of the subjects to be identified. The term, however, requires that a tically significant portion of subjects can be identified (e.g., a cohort in a cohort . Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools referred to elsewhere herein. sis according to the t invention includes applications of the method in monitoring, confirmation, and sub-classification of the relevant disease. Moreover, the establishment of a diagnosis as used herein also includes establishing a prognosis for a subject. Such a prognosis is a predictive indicator for the further development of the disease in a future time window, i.e. the tive window. Thus, a sis as used herein, preferably, encompasses a prediction of whether a subject will improve with t to the disease or diseases symptoms in the future or whether the disease or symptoms will become worse.

Accordingly, the antibody of the invention can be also applied for risk stratification approaches and, thus, for determining the amount of ive care and hospitalization which will be required for an individual subject suffering from a disease referred to .

The aforementioned method for diagnosing cancer in a sample of the subject also, preferably, encompasses the step of recommending an ancer therapy for a subject based on the diagnostic result obtained by the method. The term “recommending” as used herein refers to making a recommendation for an anti-cancer therapy or excluding (i.e. not recommending) a certain anti cancer therapy for a subject. Such a endation shall serve optionally together with other information, e.g., ation from histopathological investigations, as a basis for a clinician to apply a certain anti—cancer therapy for an individual subject, or not. Based on the diagnosis of the present invention, i.e. the 2012/067637 diagnosis of cancer or no cancer, a endation for an anti-cancer therapy will be made. It will be understood that only in cases where the diagnosis of cancer has been established by the method of the t invention, the recommendation for the anti-cancer y shall be made. In cases where no cancer is established as diagnosis based on the method of the present invention, the recommendation would be to refrain from an anti- cancer therapy. As set forth above, further ation from the subject from which the sample originates can be used as well for improving the recommendation. In an aspect, a combined anti—cancer therapy, e.g., with different anti tumor drugs, can be recommended if the method of the present invention identifies cancer cells but if further cancer cells which are not fied by the method of the present invention are detected in the investigated cancer, e. g., by histopathological analyses.

The term “sample” refers to a sample of separated cells or to a sample from a tissue or an organ. Tissue or organ samples may be obtained from any tissue or organ by, e.g., biopsy.

Separated cells may be obtained from the body fluids, such as lymph, blood, plasma, serum, liquor and other, or from the tissues or organs by ting techniques such as centrifugation or cell sorting. Preferably, the sample is a tissue or body fluid sample which expresses or produces the polypeptides referred to herein. The sample can be obtained from the subject by routine techniques which are well known to the person skilled in the art, e.g., open biopsy including tion of tissue or cellular material from a subject. For those areas which cannot be easily d via an open biopsy, a surgery and, preferably, minimal invasive surgery can be performed.

The term ct" as used herein relates to animals, preferably mammals, and, more preferably, humans. The method of the present invention shall be applied for ts suspected to suffer from cancer. A subject suspect to suffer from cancer is either a subject exhibiting clinically apparent symptoms of the cancer or is a subject having an increased predisposition for cancer. In the context of large scale diagnostic screening trials, a subject suspected to suffer from cancer can be even a healthy subject, i.e., a subject who does not show symptoms of the disease nor a subject having a predisposition therefor.

The terms “contacting” and “contacting the sample” as used herein refer to bringing the antibody and the sample into physical contact thereby allowing specific binding of the antibody to the epitope on the B7-H6 polypeptide if comprised by the sample. It will be tood that ting as meant herein is carried out for a time and under conditions sufficient for allowing the antibody to bind ically to the B7—I-I6 polypeptide.

Depending on the nature of the sample, pre-treatment steps might be ary in order to release the B7—H6 polypeptide or to de-mask the epitope in the B7—H6 polypeptide so that the antibody has access and can specifically bind thereto. Moreover, dependent on the kind of sample, the handling might be different. For example, a tissue sample which shall be analyzed for the presence or absence of a B7-H6 polypeptide is, preferably, homogenized and the proteins comprised by the tissue are isolated and separated, e.g., by SDS PAGE or other n separation methods known by a skilled artisan. The separated ns are analyzed for the presence or e of the B7-H6 polypeptide by immunological methods such as Westem Blot using the antibody defined herein above. These s also include incubation steps which allow specific binding of the antibody to the B7-H6 polypeptide. In order to increase the specificity washing steps are to be carried out. How to carry out such measures is well known to the person skilled in the art. If a tissue section is used as a sample (ie. a tissue section sample), it will be understood that it is envisaged to analyze not only the presence or absence of the B7-H6 polypeptide but also the cellular or sub cellular zation thereof. Accordingly, the tissue shall be kept intact and may be also stained by histoehemical staining techniques prior or after dy binding. Suitable techniques which allow for immunostaining of tissue sections are well known to the person skilled in the art. Dependent on whether the tissue section sample has been embedded in an embedding medium, such as paraffin, removal of said embedding medium might be necessary. The relevant ques are also well known in the art.

The term “determining” as used herein refers to the detection of the antibody which is specifically bound to the B7-H6 polypeptide comprised by the sample, if any. Detection methods for antibodies which are specifically bound to an antigen are also well known in the art. Preferably, the antibody to be applied in the method of the present invention itself can be coupled to a detectable marker such as a radioactive isotope (e.g., radioactive isotopes of Iodide Technetium), fluorescent or chemoluminescent agents (e.g., FITC, rhodamin), an enzyme which is capable of generating a detectable signal by converting a substrate (e.g., horseradish peroxidase, firefly luciferase, or beta osidase), a fluorescent protein (c.g., green-, blue- or red- fluorescent protein). Suitable detectable markers are well known in the art. Also ably, the antibody to be applied in the method of the present invention can be coupled to an agent that is capable of attracting a ion agent. Such an agent may be . In such a case an avidin— or streptavidin coupled detection agent can be used which upon binding of the biotin of the bound dy will serve as a detectable marker. Suitable detectable markers in such a case are those ed to above, more preferably, an enzyme shall be used as a able marker in such a case. Furthermore, a secondary dy may be used for detection of the first antibody, i.e. the antibody to be d in the method of the present invention which is bound to the B7-H6 polypeptide of the sample. Such a secondary antibody shall be coupled to a detectable marker as describe above. Thus, in the latter case, the secondary antibody will upon binding to the first antibody generate a detectable signal and thereby enables the detection of the bound first antibody. The principle of detection of bound antibodies with a secondary antibody is well known in the art and routinely applied, e.g., for ining dy binding on tissue sections. Dependent on the type of detectable marker, different detection methods can be applied using a reader system for the signal generated by the detectable marker. Such systems include automatic signal reader device, such as an ELISA or RIA reader, but also microscopic device for manual or automatic detection of the detectable signal. Moreover, the reader system may determine onal ation of the sample, e.g., a microscopic system may display the cells of a tissue section optically or an automated signal reader may determine further biomarkers comprised by the sample in addition.

In a preferred embodiment of the method of the present invention, the cancer is T cell lymphoma, myeloid ia, colon carcinoma, B cell lymphoma, melanoma, or cervical carcinoma.

The present invention also provides a method for diagnosing an atory disease in a sample of a subject suspected to suffer from an inflammatory disease comprising: a) contacting the sample with the antibody of invention under conditions which allow for binding of said antibody to its epitope on the B7-H6 ptide; and b) determining binding of the antibody to the said epitope, y the inflammatory disease is diagnosed.

Explanations of the terms made in connection with the method for diagnosing cancer or other embodiments elsewhere herein apply mutatis mutandis for the terms in connection with the aforementioned method except as ied otherwise herein below.

The term ct" as used herein relates to animals, preferably mammals, and, more preferably, humans. The method of the present invention shall be applied for ts 3O suspected to suffer from an inflammatory disease. A subject suspect to suffer from an inflammatory disease is either a subject exhibiting clinically apparent symptoms of the an inflammatory disease or is a t having an sed predisposition for an inflammatory disease. In the context of large scale diagnostic screening trials, a subject suspected to suffer from an inflammatory disease can be even a healthy subject, i.e. a subject who does not show symptoms of the disease nor a subject having a predisposition therefor.

WO 37727 As sed elsewhere herein, the inflammatory e referred to above is, preferably, a viral infection.

The invention also relates to a device for diagnosing cancer or an inflammatory disease in a sample comprising: a) an ing unit comprising the antibody ofthe invention; and b) a detector which s binding of the antibody in the ing unit to its cpitope on the B7—H6 polypeptide.

The term “device” as used herein relates to a system comprising at least the aforementioned analyzing unit and the evaluation unit operatively linked to each other.

How to link the units of the device in an operating manner will depend on the type of units included into the device. For example, where units for automatic analysis of a sample are applied, the data obtained by said automatically operating analyzing unit can be processed by, e.g., a computer program in order to obtain the desired results by the evaluation unit.

Preferably, the units are comprised by a single device in such a case. The analyzing unit may comprise the antibody in lized form on a solid support. Such an ing unit is particular useful for liquid samples. The sample to be investigated with the device of the present invention is preferably a tissue sample and, more preferably, a tissue section sample. Thus, in another aspect, the antibody may be comprised in a detection solution which will be applied to tissue samples such as tissue section by the analyzing unit. The detection solution can be stored in the analyzing unit or a separate vial, even outside the device. The tion unit, preferably a computer or data processing device, comprises implemented rules, i.e. an algorithm, for ting the binding determined by the analyzing unit whereby the binding is evaluated into icant or non-significant binding based on the signal type, strength and, in the case of tissue s, position of the signal with respect to the tissue. For samples which are evaluated to show non-significant binding the diagnosis “no cancer” will be established. If significant binding is obtained as result of the evaluation, the diagnosis cancer shall be established.

Preferably, the device in its evaluation unit also comprises an implemented expert system with an algorithm that is adapted for making recommendations based on the established diagnosis for a suitable therapy or treatment as set forth elsewhere herein in more .

In a preferred embodiment of the device of the present invention, said sample is a tissue or body fluid sample.

Finally, the present invention relates to a kit for diagnosing cancer or an atory disease comprising the antibody of the invention and, preferably, an agent for ion of binding of said antibody to its epitope on the B7-H6 ptide.

The term “kit” as used herein refers to a collection of the aforementioned antibody and ctions provided in a ready—to-use manner for diagnosing cancer in a sample. The antibody and the instructions are, preferably, provided in a single container. Preferably, the kit also comprises further components which are necessary for carrying out the diagnosis.

Such components may be auxiliary agents which are ed for the detection of the antibody binding, agents for eating the sample to be analyzed or calibration standards.

All nces cited in this specification are herewith incorporated by reference with respect to their entire sure content and the disclosure content specifically mentioned in this specification.

EXAMPLES The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

Example 1: Method for Immunization to obtain anti-B7-H6 monoclonal Antibody (mAb) 1.18: Six weeks old BALB/c mice were immunized with 100 ug of a B7—H6-Ig-fusionprotein consisting of the extracellular domain ofB7—H6 fused to an IgGl- Fe domain (B7-H6—lg—FP) shown in Fig. l in te Freud’s Adjuvant ed so at four different sites. Three weeks later, 100 pg lg-FP was injected i.p. in PBS. After three weeks, BA/F3 (pro—B cells)-B7-H6 transfectants (2 x 107 cells) in PBS were injected i.p.. Two months later, 100 ug B7-H6-lg-FP in PBS was applied i.p.. After three weeks, 3O injection with BA/F3—B7-H6 ectants (2 x 107 cells) in PBS i.p. was performed and five days later spleen cells were fused with Ag8 mouse myeloma cells. 910 hybridoma were screened by flow cytometry for binding of produced immunoglobulins to BA/F3-B7- H6 cells. Additionally, 480 clones were screened by ELISA for binding to the B7-H6-Ig- FP. Anti-B7-H6 clone 1.18 was selected for further studies, because it stained BA/F3-B7- H6 transfectants and not control vector transduced BA/F3 cells at high levels and it bound to cell lines expressing B7-H6 endogenously at high levels.

PCT/EPZOIZ/(l67637 Example 2: Binding of 7—H6 mAb 1.18 to B7—H6-Ig-FP by ELISA and to BA/F3-B7-H6 transfected cells by flow cytometry: For ELISA: B7-H6-Ig-FP (3 ug/rnl) was immobilized on ELISA plates and incubated with the indicated concentrations of anti-B7-H6 mAb 1.18 and developed with HRP- ated mAbs.

For flow cytometry: BA/F3 or BA/F3—B7—H6 transfectants were stained with anti—B7-H6 mAb 1.18 (2 ng/ml), the isotype controls, NKp30-FP and a control PP and PE-conjugated secondary mAbs.

The data depict binding of anti—B7-H6 mAb 1.18 to B7—H6—Ig-FP by ELISA and to BA/F3— B7-H6 transfected cells by flow cytometry.

Example 3: Binding of anti—B7-H6 1.18 mAb involves the IgV domain of B7-H6: The following constructs based on the pcDNA3.l with ODS-leader peptide and a C-terminal HA—tag ng for the following portions of B7-H6 were prepared: B7—H6_l (amino acids ) B7—H6_2 (amino acids 83-454) B7-H6_3 (amino acids 141-454) B7—H6_4 (amino acids 190-454) 5 (amino acids 239-454) Resulting plasmids were transiently ected in HEK cells and uently stained with the anti-B7—H6 1.18 mAb as described in Example 2. As can be seen in Figure 5, the anti-B7-H6 1.18 mAb bound to B7—H6_l (amino acids 24-454) and B7-H6_2 (amino acids 83-454), but not to B7-H6_3 (amino acids 4) ting that amino acids 83—141 of B7—H6 (GDHQEAFRPGAIVSPWRLKSGDASLRLPGIQLEEAGEYRCEVVVTPLKAQGT VQLEVV, as shown in SEQ ID NO: 22 and Figures 1 and 2) are involved in the binding of anti-B7-H6 mAb 1.18. All proteins of truncated B7-H6 were expressed and were 3O detectable by western blotting using the A-tag mAb.

Example 4: Binding of anti-B7-H6 mAb 1.18 to cell lines of different origin: Cell lines of different origin were stained with anti-B7-H6 mAb 1.18 and analyzed by flow cytometry as described in Example 2. The data reveal binding of anti-B7-H6 mAb 1.18 to cell lines of different origin.

Example 5: Quantitative real-time PCR to ine B7—H6 mRNA expression: RNA was isolated from tumor cell lines using the RNeasy Mini kit (Qiagen), contaminating PCT/EP2()12/067637 DNA was removed using TURBO DNase (Ambion) and the RNA was reverse transcribed using the ProtoScript M—MuLV First Strand cDNA synthesis kit (NEB). Quantitative real- time PCR was performed using the SYBR Green 1 Master and ycler480 ).

Specific primers for B7—H6 (GACCTGGAGCCATTGTGTCT as shown in SEQ ID NO: 23 and AAGCTGGACTGTTCCCTGTG as shown in SEQ ID NO: 24) and the housekeeping gene GAPDH (GCAAATTCCATGGCACCGT as shown in SEQ ID NO: 25 and TCGCCCCACTTGATTTTGG as shown in SEQ ID NO: 26) were used in order to calculate the B7—H6 mRNA expression level relative to GAPDH. The data depict that cell lines of different origin that are stained with anti-B7—H6 mAb 1.18 express mRNA of B7— H6 in different s.

Example 6: histochemical staining of B7-H6 on cytospins 0f Ba/F3—B7—H6 transfectants. Acetone-fixed cytospins of a 1:1 e of Ba/F3 and Ba/F3-B7-H6 cells were stained using the Dual Envision+ System—HRP (Dako). After ng nous peroxidase activity, cytospins were blocked with 10% goat serum and 0.1 mg/ml human IgG. The cytospins were incubated with 5 ug/ml anti-B7-H6 mAb 1.18 or a mouse IgGl isotype control (clone 11711, R&D) in Dako antibody diluent, washed and incubated with Dako Peroxidase labeled polymer conjugated to goat anti—mouse and goat abbit immunoglobulins. After incubation with 3,3’—diaminobenzidine (DAB) substrate solution, cell nuclei were counterstained with Hematoxylin and mounted cyotospins were analyzed by light microscopy. The data reveal that anti—B7—H6 mAb 1.18 stains B7-H6 Ba/F3-B7— H6 transfectants on cytospins.

Example 7: Degranulation of primary NK cells after co—culture with BA/F3 cells transduced with B7—H6. Primary NK cells expanded with IL-2 for 14 days were cultured in media, with BA/F3, BA/F3—B7-H6 (ligand for NKp30) or BA/F3-MlCA (ligand for the activating receptor NKG2D) cells in the presence of a PE—conjugated anti-CD107 mAb for 5h. Degranulation ofNK cells was determined as percentages of CD 107—positive NK cells after co-culture by flow cytometry. Error bars depict mean+/— SD of triplicate cultures. The data reveal that BA/F3—B7—H6 cells induce ulation of primary NK cells. —19a- Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of rs or steps but not the exclusion of any other r or step or group of integers or steps.

The reference to any prior art in this specification is not and should not be taken as an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge in New Zealand. /03/15,va op2375 p2-3a pl9a speci,a

Claims (8)

1. An antibody which specifically binds to an epitope formed by a portion of the extracellular domain of the B7-H6 polypeptide, wherein said portion having an amino acid sequence as shown in SEQ ID NO: 22.

2. The antibody according to claim 1, wherein said antibody comprises mentarity determining regions (CDRs) as shown in SEQ ID NOS: 5, 7, 9, 15, 17, and 19.

3. The antibody according to claim 1 or claim 2, wherein said antibody is a monoclonal antibody.

4. The antibody according to any one of claim 1 to claim 3, n said antibody is the antibody deposited under ion number DSM ACC 3117 at the DSMZ, Braunschweig, Germany under the Budapest treaty on February 02, 2011.

5. An antibody ing to any one of claim 1 to claim 4 for use in the treatment or diagnosis of cancer.

6. The antibody according to claim 4, wherein the cancer is any one selected from the following group: T cell lymphoma; myeloid leukemia; colon carcinoma; B cell lymphoma; melanoma; and cervical carcinoma.

7. An antibody according to any one of claim 1 to claim 4, for use in the treatment or diagnosis of inflammatory e.

8. The antibody according to claim 7, wherein the inflammatory disease is a Viral infection. 15,va op23 75 claims,20