Monoclonal antibodies against growth differentiation factor 15 (GDF-15) and their use for treating cancer cachexia and cancer

Abstract

To provide monoclonal anti-human-GDF-15 antibodies.SOLUTION: The antibodies include chimeric antibodies and humanized antibodies. The invention also relates to monoclonal anti-human-GDF-15 antibodies including murine antibodies, chimeric antibodies and humanized antibodies for use in methods for the treatment of cancer cachexia and also for the treatment of cancer. The invention also provides pharmaceutical compositions, kits, methods and uses and cell lines capable of producing the monoclonal antibodies of the invention.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THE PRESENT APPLICATION The present invention relates to monoclonal anti-human GDF-15 antibodies, pharmaceutical compositions, kits, methods and uses, as well as cell lines capable of producing the monoclonal antibodies described herein. The present invention further relates to antibodies against human GDF-15 capable of inhibiting cancer growth, treating cancer-induced weight loss and cancer cachexia. [Background technology]

[0002] background To date, many cancers remain areas of unmet medical need and, therefore, means to treat cancer more effectively and in a broader range of cancers are needed.

[0003] In addition to the suffering caused by the cancer itself, many patients suffer from cancer cachexia, a cancer-induced medical condition that is typically accompanied by weight loss and loss of skeletal muscle mass. Cancer cachexia accounts for more than 20 percent of all cancer-related deaths (Murphy KT and Lynch GS: Update on emerging drugs for cancer cachexia. Expert Opin Emerg Drugs. 2009 Dec;14(4):619-32).

[0004] Therefore, there is a need for a treatment regimen that targets both of these medical conditions to improve the treatment and prognosis of cancers that lead to cancer cachexia.To date, most of the emerging drugs for the treatment of cancer cachexia are drugs that target cachexia but not cancer itself (see Murphy KT and Lynch GS: Update on emerging drugs for cancer cachexia. Expert Opin Emerg Drugs. 2009 Dec;14(4):619-32).Very few drugs are effective against both cancer and cancer cachexia, and therefore complex treatment regimens that combine anti-cancer drugs and anti-cancer cachexia drugs are often required.Therefore, there is an unmet medical need for drugs that can be used to effectively treat both cancer and cancer cachexia in a wide range of cancers.

[0005] Many types of cancer are known to express growth factors, including factors such as VEGF, PDGF, TGF-β and GDF-15.

[0006] GDF-15, growth differentiation factor 15, is a branched member of the TGF-β superfamily. It is a protein that is expressed intracellularly as a precursor, subsequently processed, and finally secreted from the cell into the environment. Both the active and fully processed (mature) form and the precursor of GDF-15 can be found extracellularly. The precursor is covalently bound to the extracellular matrix via its COOH-terminal amino acid sequence (Bauskin AR et al., Cancer Research 2005) and is therefore present outside the cell. The active and fully processed (mature) form of GDF-15 is soluble and is found in the serum of the blood. Therefore, the processed form of GDF-15 can potentially act on any target cell in the body that is connected to the blood circulation, if the potential target cell expresses a receptor for the soluble GDF-15 ligand.

[0007] During pregnancy, GDF-15 is found in the placenta under physiological conditions. However, many malignant cancers, especially aggressive brain cancer, melanoma, lung cancer, gastrointestinal tumors, colon cancer, pancreatic cancer, prostate cancer and breast cancer (Mimeault M and Batra SK, J. Cell Physiol 2010), show increased levels of GDF-15 in tumors and serum. Similarly, correlations have been described between high expression of GDF-15 and chemotherapy resistance (Huang CY et al., Clin. Cancer Res. 2009) and between high expression of GDF-15 and poor prognosis (Brown DA et al., Clin. Cancer Res. 2009), respectively.

[0008] GDF-15 is expressed in gliomas of various WHO grades as assessed by immunohistochemistry (Roth et al., Clin. Cancer Res. 2010). Furthermore, Roth et al. stably expressed DNA constructs expressing short hairpin RNAs targeting endogenous GDF-15 or control constructs in SMA560 glioma cells. Using these pre-established stable cell lines, they observed that tumor formation in mice bearing GDF-15 knockdown SMA560 cells was delayed compared to mice bearing the control construct.

[0009] Patent application PCT / EP2013 / 070127 relates to monoclonal anti-GDF-15 antibodies, in particular antibodies produced in the hybridoma cell line B1-23 deposited under the Budapest Treaty at the German Collection of Microorganisms and Cell Cultures (DSMZ) under deposit number DSM ACC3142. PCT / EP2013 / 070127 also relates to uses of the anti-GDF-15 antibodies.

[0010] Patent applications WO 2005 / 099746 and WO 2009 / 021293 relate to an anti-human GDF-15 antibody (Mab26) capable of antagonizing the effect of human GDF-15 on tumor-induced weight loss in mice in vivo: in these documents, immune-compromised mice were administered human tumor cells (prostate cancer cells DU145) transfected with a plasmid overexpressing human GDF-15. Tumor cells carrying a plasmid lacking the GDF-15 sequence served as a negative control. These mice expressing xenografted GDF-15 presented with tumor-induced weight loss (clinical term: cachexia) and anorexia. A single intraperitoneal administration of 1 mg of Mab26 from WO 2005 / 099746 resulted in a complete reversal of tumor-induced weight loss. WO 2005 / 099746 and WO 2009 / 021293 do not disclose the effect of anti-human GDF-15 antibodies on tumor growth. Moreover, these documents are silent as to whether anti-human GDF-15 antibodies could result in an increase in the body weight of treated mice compared to their body weight before the onset of cachexia.

[0011] Similarly, Johnen H et al. (Nature Medicine, 2007) reported the effect of anti-human GDF-15 monoclonal antibodies on cancer-induced anorexia and weight loss, but no effect of anti-human GDF-15 antibodies on the size of tumors formed by cancer was observed even when the antibodies were administered at a high dosage of 1 mg, and therefore the antibodies did not inhibit cancer growth.

[0012] Thereby, to date, there remains a need in the art for means to effectively treat cancer and cancer cachexia, as well as means to treat cancer and cancer cachexia in a broader range of cancers.

[0013] Therefore, the object of the present invention is to obtain a means which can be used not only to effectively treat cancer cachexia, but also to effectively treat cancer, as well as a means which can be used not only to effectively treat cancer cachexia, but also to effectively treat cancer in a wider range of cancers.

[0014] In an effort to find means to achieve these goals, the inventors surprisingly found that monoclonal antibodies against human GDF-15 can be used not only to treat cancer cachexia, but also to treat cancer in human xenograft tumors in mice.

[0015] In addition, the antibody against human GDF-15 according to the present invention has an equilibrium dissociation constant of about 790 pM for recombinant GDF-15 even without additional affinity maturation, which is a higher affinity than most known therapeutic antibodies.

[0016] Therefore, the antibody against human GDF-15 according to the present invention has superior properties compared to antibodies known in the art, and is particularly useful for inhibiting cancer growth and cancer cachexia.Therefore, the antibody of the present invention is useful for treating cancer and for treating cancer cachexia.Therefore, the present invention has been completed. Summary of the Invention

[0017] BRIEF DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention solves the above mentioned objectives by providing monoclonal antibodies, pharmaceutical compositions, kits, uses and cell lines capable of producing the monoclonal antibodies described herein.

[0018] In particular, the inventors have surprisingly shown that the monoclonal antibodies against human GDF-15 and antigen-binding portions thereof according to the present invention are capable of inhibiting cancer cachexia and / or cancer growth, which is unexpected since these monoclonal antibodies against GDF-15 previously known in the art (WO 2005 / 099746, WO 2009 / 021293 and Johnen H et al., Nature Medicine, 2007) were only known to cause reversal of cancer-induced weight loss (i.e., reversal of secondary symptoms induced by GDF-15 expressed by cancer), but they have been shown to fail to inhibit cancer growth.

[0019] By showing that the monoclonal antibodies against human GDF-15 according to the present invention can be used to treat cancer-induced weight loss and / or cancer cachexia and to treat cancer, the inventors also surprisingly show that human GDF-15 protein can be targeted by the antibodies of the present invention in such a way that cancer growth is inhibited and cancer-induced weight loss and cancer cachexia are treated. It is expected that the same mechanism of cancer growth inhibition and treatment of cancer-induced weight loss and cancer cachexia is applicable to many cancers that overexpress human GDF-15, including those listed below.

[0020] The monoclonal antibody and its antigen-binding portion according to the invention are derived from the murine anti-GDF-15 antibody, mAb-B1-23, described in PCT / EP2013 / 070127 and deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) under the Budapest Treaty under deposit number DSM ACC3142. The anti-human GDF-15 mAb-B1-23 antibody according to the invention can be generated by replacing the constant domains of the murine antibody mAb-B1-23 with the constant domains of a human IgG1 antibody.

[0021] Surprisingly, it has been observed that the chimeric and humanized B1-23 antibodies according to the invention do not exhibit a tendency to aggregate. These antibody properties according to the invention improve the bioavailability of these antibodies and are advantageous for the clinical formulation of these antibodies.

[0022] Thus, the present invention relates to a monoclonal antibody or antigen-binding portion thereof capable of binding to human GDF-15, wherein the heavy chain variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO:5 or an amino acid sequence at least 90% identical thereto, and the light chain variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO:7 or an amino acid sequence at least 85% identical thereto, and wherein the heavy chain constant domain comprises the amino acid sequence of SEQ ID NO:29 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95% identical thereto, and wherein the light chain constant domain comprises the amino acid sequence of SEQ ID NO:32 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95% identical thereto.

[0023] The present invention also relates to a monoclonal antibody or antigen-binding portion thereof capable of binding to human GDF-15 for use in a method for treating cancer cachexia in a mammal, wherein the heavy chain variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO:5 or an amino acid sequence at least 90% identical thereto, and the light chain variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO:7 or an amino acid sequence at least 85% identical thereto. The method comprises administering the antibody or antigen-binding portion thereof to the mammal. In addition, the present invention relates to corresponding methods for treatment.

[0024] Furthermore, the present invention also relates to a monoclonal antibody or antigen-binding portion thereof capable of binding to human GDF-15, wherein the binding is to a conformational or discontinuous epitope on human GDF-15 consisting of the amino acid sequences of SEQ ID NO:25 and SEQ ID NO:26, wherein the constant domain of the heavy chain comprises the amino acid sequence of SEQ ID NO:29 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95% identical thereto, and wherein the constant domain of the light chain comprises the amino acid sequence of SEQ ID NO:32 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95% identical thereto.

[0025] Furthermore, the present invention also relates to a monoclonal antibody or antigen-binding portion thereof capable of binding to human GDF-15 for use in a method for treating cancer cachexia in a mammal, wherein the binding is to a conformational or discontinuous epitope on human GDF-15 consisting of the amino acid sequences of SEQ ID NO: 25 and SEQ ID NO: 26. The method comprises administering the antibody or antigen-binding portion thereof to the mammal. Additionally, the present invention relates to corresponding methods for treatment.

[0026] The present invention also relates to a pharmaceutical composition comprising an antibody or antigen-binding portion thereof according to the invention. The present invention also relates to an antibody or an antigen-binding portion thereof according to the invention for use in medicine.

[0027] Furthermore, the present invention relates to an antibody or antigen-binding portion thereof or a pharmaceutical composition according to the invention for use in a method for treating cancer in a mammal, the method comprising administering to said mammal the antibody or antigen-binding portion thereof or the pharmaceutical composition.

[0028] Furthermore, the present invention relates to an antibody or antigen-binding portion thereof or a pharmaceutical composition according to the invention for use in a method for treating cancer cachexia in a mammal, the method comprising administering to said mammal the antibody or antigen-binding portion thereof or the pharmaceutical composition.

[0029] Additionally, the present invention relates to a kit comprising a pharmaceutical composition according to the present invention. The present invention also relates to an expression vector comprising a nucleotide sequence encoding an antibody or antigen-binding portion thereof according to the invention.

[0030] Furthermore, the present invention relates to a cell line capable of producing the antibody or antigen-binding portion thereof according to the invention. Thus, by providing monoclonal antibodies against human GDF-15, the present invention provides a means for the treatment of cancer cachexia and a cancer growth inhibitor that fulfills the above-defined need in the art. [Brief description of the drawings]

[0031] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1: NKG2D expression on NK cells after treatment with or without GDF-15. Cell surface expression of NKG2D was determined on NK cells after treatment with the indicated cytokines in the presence or absence of anti-GDF-15 antibody mAb B1-23. The figure displays the specific fluorescence intensity determined by flow cytometry and quantified against a nonspecific control antibody.

[0032] [Diagram 2] Figure 2: Akt phosphorylation in the ovarian cancer cell line SK-OV-3. To quantify the Western blots for the ovarian cancer cell line SK-OV-3, the ratio of phosphorylated Akt to total Akt was calculated and normalized to the untreated control.

[0033] [Diagram 3] Figure 3: JNK1 / 2 phosphorylation in immune cells. To quantitate western blots, the ratio of phosphorylated JNK1 / 2 to total JNK was calculated and normalized to untreated controls.

[0034] [Figure 4]Figure 4: Antitumor effect of murine B1-23 in vivo. Balb / cnu / nu nude mice were used in a xenograft setting with the melanoma cell line UACC-257. Tumor size in the cohort of animals treated with B1-23 (open squares) was significantly reduced compared to the PBS control group (filled circles). Significance was defined as p<0.05 as assessed by the Wilcoxon log-rank test.

[0035] [Diagram 5] Figure 5: Treatment of cancer cachexia with anti-GDF-15 antibodies. The figure shows a comparison of the average body weight of all treated Balb / cnu / nu nude mice inoculated with UACC-257 cells. The change in body weight is expressed as a percentage compared to the starting weight on day 0 over a period of 38 days.

[0036] [Figure 6] Figure 6: Coomassie staining of antibodies used in study no. 140123. [Figure 7] Figure 7: Improved solubility of chimeric and humanized antibodies at physiological pH. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] Detailed Description of the Invention definition Unless specifically defined below, terms used herein are to be understood according to their ordinary meanings as known to those of ordinary skill in the art.

[0038] The term "antibody" as used herein means any functional antibody capable of specifically binding to an antigen of interest, as generally reviewed in Chapter 7 of Paul, WE (Ed.): Fundamental Immunology 2nd Ed. Raven Press, Ltd., New York 1989, which is incorporated herein by reference. Without any limitation, the term "antibody" encompasses antibodies from any suitable source species, including mammals such as chicken, mouse, goat, non-human primates and humans. Preferably, the antibody is a humanized antibody.

[0039] The antibody is preferably a monoclonal antibody, which may be prepared by methods well known in the art. The term "antibody" includes antibodies of the following isotypes: IgG-1, -2, -3 or -4, IgE, IgA, IgM or IgD. The term "antibody" includes monomeric antibodies (such as IgD, IgE, IgG) or oligomeric antibodies (such as IgA or IgM). The term "antibody" also includes - without limitation - isolated antibodies and modified antibodies, e.g. genetically engineered antibodies such as chimeric antibodies.

[0040] The nomenclature of the domains of an antibody follows the terminology known in the art. Each monomer of an antibody contains two heavy chains and two light chains, as is generally known in the art. Of these, each heavy and light chain contains a variable domain (V for heavy chains) that is important for antigen binding. H , and V for light chains. L The heavy and light chain variable domains comprise (from N-terminus to C-terminus) the regions FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 (FR is a framework region; CDR is a complementarity determining region, also known as a hypervariable region).

[0041] Identification and assignment of the above-mentioned antibody regions within the antibody sequence may generally be performed according to Kabat et al. (Sequences of proteins of immunological interest, US Dept. of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, Md. 1983) or Chothia et al. (Conformations of immunoglobulin hypervariable regions. Nature. 1989 Dec 21-28;342(6252):877-83), or by using the IMGT / V-QUEST software described in Giudicelli et al. (IMGT / V-QUEST, an integrated software program for immunoglobulin and T cell receptor VJ and VDJ rearrangement analysis. Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W435-40), which are incorporated herein by reference. Preferably, the above-indicated antibody regions are identified and assigned by using the IMGT / V-QUEST software.

[0042] A "monoclonal antibody" is an antibody from an essentially homogeneous population of antibodies in which the antibodies are substantially identical in their sequence (i.e., identical except for minor fragments containing naturally occurring sequence modifications, such as amino acid modifications at their N- and C-termini). Unlike polyclonal antibodies, which contain a mixture of different antibodies directed against multiple epitopes, monoclonal antibodies are directed against the same epitope and are therefore highly specific.

[0043] The term "monoclonal antibody" includes, but is not limited to, antibodies obtained from a monoclonal cell population derived from a single cell clone, such as antibodies produced by the hybridoma method described by Koehler and Milstein (Nature, 1975 Aug 7;256(5517):495-7) or Harlow and Lane ("Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1988). Monoclonal antibodies may also be obtained by other suitable methods, including phage display technology, such as that described by Clackson et al. (Nature. 1991 Aug 15;352(6336):624-8) or Marks et al. (J Mol Biol. 1991 Dec 5;222(3):581-97).

[0044] The monoclonal antibody may be an antibody that has been optimized by methods known in the art for antigen-binding properties such as reduced Kd value, optimized association and dissociation rates, etc. For example, the Kd value may be optimized by display methods, including phage display, resulting in affinity-matured monoclonal antibodies. The term "monoclonal antibody" is not limited to antibody sequences derived from a particular species or from only a single species. Thus, the meaning of the term "monoclonal antibody" includes chimeric monoclonal antibodies, such as humanized monoclonal antibodies.

[0045] A "humanized antibody" is an antibody that contains human sequences and a small portion of non-human sequences that confer binding specificity to an antigen of interest (e.g., human GDF-15). Typically, humanized antibodies are generated by replacing hypervariable region sequences from a human acceptor antibody with hypervariable region sequences from a non-human donor antibody (e.g., mouse, rabbit, rat donor antibody) that binds to the antigen of interest (e.g., human GDF-15). In some cases, the framework region sequences of the acceptor antibody may also be replaced by the corresponding sequences of the donor antibody.

[0046] In addition to sequences derived from the donor and acceptor antibodies, a "humanized antibody" may or may not contain other (additional or alternative) residues or sequences. Such other residues or sequences can serve to further improve antibody properties, such as binding properties (e.g., reducing Kd values) and / or immunogenic properties (e.g., reducing antigenicity in humans). Non-limiting examples for methods of generating humanized antibodies are known in the art, e.g., from Riechmann et al. (Nature. 1988 Mar 24;332(6162):323-7) or Jones et al. (Nature. 1986 May 29-Jun 4;321(6069):522-5).

[0047] The term "human antibody" refers to an antibody containing human variable and constant domain sequences. This definition encompasses antibodies with human sequences with single amino acid substitutions or modifications that may serve to further improve antibody properties such as binding characteristics (e.g., decreasing Kd values) and / or immunogenic properties (e.g., decreasing antigenicity in humans). The term "human antibody" excludes humanized antibodies in which a portion of non-human sequence confers binding specificity for an antigen of interest.

[0048] As used herein, an "antigen-binding portion" of an antibody refers to a portion of an antibody that retains the antibody's ability to specifically bind to an antigen (e.g., GDF-15), i.e., the "antigen-binding portion" can compete with the antibody for specific binding to the antigen. An "antigen-binding portion" may contain one or more fragments of an antibody. It may be produced by any suitable method known in the art, including, but not limited to, recombinant DNA methods and preparation by chemical or enzymatic fragmentation of an antibody. An antigen-binding portion may be a Fab fragment, a F(ab') fragment, a F(ab')2 fragment, a single chain antibody (scFv), a single domain antibody, a bispecific antibody, or any other portion or portions of an antibody that allows it to retain binding to an antigen.

[0049] An "antibody" (e.g., a monoclonal antibody) or an "antigen-binding portion" may be derivatized or linked to a different molecule. For example, molecules that may be linked to an antibody are other proteins (e.g., other antibodies), molecular labels (e.g., fluorescent, luminescent, colored or radioactive molecules), pharmaceuticals and / or toxins. An antibody or antigen-binding portion may be linked directly (e.g., in the form of a fusion between two proteins) or via a linker molecule (e.g., any suitable type of chemical linker known in the art).

[0050] As used herein, the term "binding" or "binding" refers to specifically binding to an antigen of interest (e.g., human GDF-15). Preferably, the Kd value is less than 100 nM, more preferably less than 50 nM, even more preferably less than 10 nM, even more preferably less than 5 nM, and most preferably less than 2 nM.

[0051] As used herein, the term "epitope" refers to a small portion of an antigen that forms a binding site for an antibody. In the context of the present invention, binding or competitive binding of antibodies or antigen-binding portions thereof to an antigen of interest (e.g., human GDF-15) is measured by using surface plasmon resonance measurements as a reference standard assay, as described below.

[0052] The term “K D " or "K D The "K value" refers to the equilibrium dissociation constant as known in the art. In the context of the present invention, these terms relate to the equilibrium dissociation constant of an antibody with respect to a particular antigen of interest (e.g. human GDF-15). The equilibrium dissociation constant is a measure of the tendency of a complex to reversibly dissociate into its components (e.g. antigen and antibody). For antibodies according to the present invention, the K D Values ​​(such as for the antigen human GDF-15) are generally determined by using surface plasmon resonance measurements, as described below.

[0053] The term "cancer growth" as used herein refers to any measurable growth of cancer. For cancers that form solid tumors, "cancer growth" refers to the measurable increase in tumor volume over time. If a cancer forms only a single tumor, "cancer growth" refers only to the increase in the volume of the single tumor. If a cancer forms multiple tumors, such as metastases, "cancer growth" refers to the increase in the volume of all measurable tumors. For solid tumors, tumor volume can be measured by any method known in the art, including magnetic resonance imaging and computed tomography (CT scan).

[0054] For leukemias characterized by the presence of hematologic cancerous cells in the blood, "cancer growth" refers to a measurable increase in the number of cancer cells per blood volume. To make such a measurement, cancer cells can be identified from blood samples by measuring cell morphology or using any method known in the art, including, for example, staining with specific antibodies for tumor cell marker proteins, such as tumor marker cell surface proteins, and the cancer cells can be counted.

[0055] As used herein, the term "inhibit cancer growth" refers to measurable inhibition of cancer growth in patients treated with the antibody. Preferably, the inhibition is statistically significant. Inhibition of cancer growth may be assessed by comparing the cancer growth in a group of patients treated according to the present invention with a control group of untreated patients, or by comparing a group of patients treated according to the present invention in addition to standard cancer treatments in the art with a control group of patients who only received standard cancer treatments in the art.

[0056] Such studies to assess inhibition of cancer growth are designed according to accepted standards for clinical trials, e.g., double-blind, randomized trials with sufficient statistical power. The term "inhibiting cancer growth" includes inhibition where cancer growth is partially inhibited, inhibition where cancer growth (i.e., where cancer growth in the patient is delayed compared to a control group of patients), inhibition where cancer growth is completely inhibited (i.e., where cancer growth in the patient is halted), and inhibition where cancer growth is reversed (i.e., the cancer shrinks).

[0057] As used herein, an "isolated antibody" is an antibody that has been identified and separated from the majority (by weight) of components from its source environment, e.g., from components of a hybridoma cell culture or a different cell culture used for its production (e.g., producer cells such as CHO cells that recombinantly express the antibody). The separation is performed so as to sufficiently remove components that may otherwise interfere with the suitability of the antibody for the desired use (e.g., therapeutic use of an anti-human GDF-15 antibody according to the invention).

[0058] Methods of preparing isolated antibodies are known in the art and include Protein A chromatography, anion exchange chromatography, cation exchange chromatography, viral retention filtration and ultrafiltration. Preferably, the isolated antibody preparation is at least 70% (w / w) pure, more preferably at least 80% (w / w), even more preferably at least 90% (w / w), even more preferably at least 95% (w / w), and most preferably at least 99% (w / w) pure, as measured by using the Lowry protein assay.

[0059] As used herein, a "bispecific antibody" is a bivalent small antigen-binding antibody moiety that comprises a heavy chain variable domain linked to a light chain variable domain on the same polypeptide chain, linked by a peptide linker that is too short to allow pairing between the two domains on the same chain. This leads to pairing with a complementary domain on another chain and assembly of a dimeric molecule with two antigen-binding sites. Bispecific antibodies may be bivalent and monospecific (such as a bispecific antibody with two antigen-binding sites for human GDF-15) or bivalent and bispecific (for example, a bispecific antibody with two antigen-binding sites, one for human GDF-15 and the other for a different antigen). A detailed description of bispecific antibodies can be found in Holliger P et al. ("Diabodies": small bivalent and bispecific antibody fragments." Proc Natl Acad Sci US A. 1993 Jul 15;90(14):6444-8).

[0060] As used herein, a "single domain antibody" (also referred to as "Nanobody®") is an antibody fragment consisting of a single monomeric variable antibody domain. Structures and methods for producing single domain antibodies are known from the art, e.g. from Holt LJ et al. ("Domain antibodies: proteins for therapy." Trends Biotechnol. 2003 Nov;21(11):484-90), Saerens D et al. ("Single-domain antibodies as building blocks for novel therapeutics." Curr Opin Pharmacol. 2008 Oct;8(5):600-8. Epub 2008 Aug 22), and Arbabi Ghahroudi M et al. ("Selection and identification of single domain antibody fragments from camel heavy-chain antibodies." FEBS Lett. 1997 Sep 15;414(3):521-6).

[0061] As used herein, the term "higher" means that the value (e.g., GDF-15 level) in a patient sample is higher than the value in a corresponding control sample or group of control samples. Preferably, the difference is statistically significant.

[0062] As used herein, the term "elevated GDF-15 levels" means that a human patient has a higher level of GDF-15 in serum compared to the median level of GDF-15 in blood serum of healthy human control individuals as a reference prior to administration of an antibody or antigen-binding portion thereof or pharmaceutical composition according to the invention.

[0063] The preferred median standard for GDF-15 levels in blood serum of healthy human control individuals is <0.8ng / ml. The expected range is between 0.2ng / ml and 1.2ng / ml in healthy human controls (see Tanno T et al.: "Growth differentiation factor 15 in erythroid health and disease." Curr Opin Hematol. 2010 May; 17(3): 184-190). Preferably, the level is 1.2-fold higher, more preferably 1.5-fold higher, even more preferably 2-fold higher, and most preferably 5-fold higher.

[0064] The term "prior to administration" as used herein refers to the period immediately prior to administration of an antibody, fragment thereof, or pharmaceutical composition according to the invention. Preferably, the term "prior to administration" refers to a period of 30 days immediately prior to administration; most preferably, a period of 1 week immediately prior to administration.

[0065] As used herein, the terms "significant," "significantly," and the like refer to a statistically significant difference between values. The terms "cancer" and "cancer cell" are used herein according to their common meaning in the art (see, e.g., Weinberg R. et al.: The Biology of Cancer. Garland Science: New York 2006. 850p).

[0066] The term "cancer-induced weight loss" is used herein according to its general meaning in the art.Cancer-induced weight loss is frequently seen as an adverse event in individuals with cancer (see, for example, Fearon K. et al.: Definition and classification of cancer cachexia: an international consensus. Lancet Oncol. 2011 May; 12(5):489-95.; Tisdale MJ.: Mechanisms of cancer cachexia. Physiol Rev. 2009 Apr; 89(2):381-410).The term "cancer-induced weight loss" refers to weight loss induced by cancer.

[0067] In addition to cancer-induced weight loss, additional weight loss, for example weight loss induced by cancer treatment such as surgery, chemotherapy and radiation therapy, may also occur in individuals with cancer. It is understood that the meaning of the term "cancer-induced weight loss" does not include this additional weight loss. However, this does not exclude the possibility that the antibodies of the present invention, in addition to their effect on cancer-induced weight loss and cancer growth, may have a beneficial effect on such additional weight loss, for example by reversing or partially reversing such additional weight loss, or by preventing or partially preventing such additional weight loss.

[0068] Body weight can be easily measured by weighing, and body weight is typically expressed in units of mass, such as kg.

[0069] The term "cancer cachexia" is used herein according to its typical meaning in the art (see, for example, Fearon K. et al.: Definition and classification of cancer cachexia: an international consensus. Lancet Oncol. 2011 May; 12(5):489-95.; Tisdale MJ.: Mechanisms of cancer cachexia. Physiol Rev. 2009 Apr;89(2):381-410.). The most common symptom of cancer cachexia is cancer-induced weight loss. Thus, according to one definition, cancer cachexia is characterized by a continuing loss of skeletal muscle mass (with or without fat mass loss) that is not fully reversed by conventional nutritional intake.

[0070] In human patients, cancer cachexia is characterized by weight loss of more than 5% over the past 6 months or by a weight loss of less than 20 g / m 2 It may be defined by a body mass index below 18.5 and any degree of continued weight loss greater than 2%, or by sarcopenia (i.e., degenerative loss of muscle mass) and ongoing weight loss greater than 2% (see Fearon K. et al.: Definition and classification of cancer cachexia: an international consensus. Lancet Oncol. 2011 May; 12(5):489-95.). A further symptom of cancer cachexia may be the depletion of adipose tissue.

[0071] With respect to cancer cachexia, a "treatment" according to the present invention may be a treatment to prevent and / or a treatment to inhibit or reverse cancer cachexia. Typically, a treatment to prevent cancer cachexia is a treatment administered prophylactically at a cancer disease stage where cancer cachexia has not yet developed. A treatment to inhibit cancer cachexia is a treatment administered at a cancer disease stage where substantial cancer cachexia has developed, in order to inhibit further progression of cancer cachexia.

[0072] Treatments for reversing cancer cachexia are typically initiated at a stage of cancer where significant cancer cachexia has developed, and are treatments that reverse cancer cachexia. The effect of the treatment can be a partial effect, i.e. partial prevention, partial inhibition or partial reversal of cancer cachexia, or a complete effect, i.e. complete prevention, complete inhibition or complete reversal of cancer cachexia. Preferably, according to the present invention, the effect of the treatment is complete prevention, complete inhibition or complete reversal of cancer cachexia. More preferably, the effect of the treatment according to the present invention is complete prevention or complete reversal of cancer cachexia.

[0073] The term "completely" as used herein in connection with the treatment of cancer cachexia according to the present invention means that in the case of treatment for prevention, cancer cachexia does not develop in the treated individual during and / or after treatment. In the case of treatment for inhibiting cancer cachexia, the term "completely" means that further progression of cancer cachexia does not occur in the treated individual during and / or after treatment. In the case of treatment for reversing cancer cachexia, the term "completely" means that cancer cachexia is completely reversed during or after treatment, such that cancer cachexia is not present in the treated individual.

[0074] With respect to these effects of the treatment according to the present invention, the term "absence of cancer cachexia" means that cancer cachexia is not detectable by using standard methods for measurement and diagnosis known in the art. Similarly, the term "absence of further progression of cancer cachexia" means that further progression of cancer cachexia is not detectable by using standard methods for measurement and diagnosis known in the art. Methods known in the art and referenced herein are described, for example, in Fearon KC.: Cancer cachexia: developing multimodal therapy for a multidimensional problem. Eur J Cancer. 2008 May;44(8):1124-32;Fearon K. et al.: Definition and classification of cancer cachexia: an international consensus. Lancet Oncol. 2011 May;12(5):489-95.; or Tisdale MJ.: Mechanisms of cancer cachexia. Physiol Rev. 2009 Apr;89(2):381-410.

[0075] In addition to completely preventing or reversing cancer cachexia, the methods of treatment and products for use in these methods according to the present invention may increase the body weight of the treated mammal compared to its body weight before the onset of cancer cachexia. As used herein, the term "before the onset of cancer cachexia" refers to a point in the course of cancer disease after which cancer cachexia becomes measurable by methods known in the art, such as those referenced above.

[0076] Preferably, the above-defined efficacy of the treatment of cancer cachexia according to the present invention will be statistically significant when assessed against a suitable control group, whilst individual treated patients will not exhibit severe cachexia. According to the present invention, each occurrence of the term "comprises" may optionally be replaced with the term "consisting of."

[0077] Methods and Techniques Generally, unless otherwise defined herein, the methods used in the present invention (e.g., cloning methods or antibody-related methods) are performed according to procedures known in the art, such as those described, for example, in Sambrook et al. ("Molecular Cloning: A Laboratory Manual," 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1989), Ausubel et al. ("Current Protocols in Molecular Biology," Greene Publishing Associates and Wiley Interscience; New York 1992) and Harlow and Lane ("Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1988); these documents are incorporated herein by reference.

[0078] Molecular weight is measured by methods known in the art, such as mass spectrometry, and is expressed in Daltons (Da) or kilodaltons (kDa). The binding of monoclonal anti-human GDF-15 antibodies according to the invention is generally assessed by employing surface plasmon resonance measurements using a Bio-Rad® ProteOn® XPR36 system and a Bio-Rad® GLC sensor chip, as described for anti-human GDF-15 mAb-B1-23 in Example 1.

[0079] Sequence alignment of sequences according to the present invention is carried out by using the BLAST algorithm (see Altschul et al. (1990) "Basic local alignment search tool." Journal of Molecular Biology 215. p. 403-410; Altschul et al.: (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402). Preferably, the following parameters are used: maximum target sequence 10; word size 3; BLOSUM 62 matrix; gap cost: presence 11, extension 1; conditional composition score matrix adjustment. Thus, when used in relation to sequences, terms such as "identity" or "identical" refer to the identity value obtained by using the BLAST algorithm.

[0080] The monoclonal antibodies according to the invention may be produced by any method known in the art, including but not limited to the methods mentioned in Siegel DL ("Recombinant monoclonal antibody technology." Transfus Clin Biol. 2002 Jan;9(1):15-22). In a preferred embodiment, the antibodies according to the invention are produced by the hybridoma cell line B1-23, deposited under the Budapest Treaty at the German Collection of Microorganisms and Cell Cultures (DSMZ) under the deposit number DSM ACC3142. The deposit was filed on September 29, 2011.

[0081] Cell proliferation may be measured by any suitable method known in the art, including but not limited to, light microscopy, metabolic assays such as those measuring mitochondrial redox potential (e.g., the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay; resazurin staining, also known as the Alamar Blue® assay, staining for known endogenous proliferation biomarkers (e.g., Ki-67), and methods measuring cellular DNA synthesis (e.g., BrdU and [ 3 H-thymidine incorporation assay).

[0082] Immunosuppression can be measured by any suitable method known in the art, including (but not limited to) immune cell growth, cytokine secretion, intracellular cytokine staining by flow cytometry, cytokine measurement by qRT-PCR, redirected target cell lysis, further cytotoxicity or degranulation assay, downregulation of immune cell receptor activation (such as NKG2D), upregulation of inhibitory immune cell receptor, immunological synapse formation, immune cell infiltration. In order to apply the term immunosuppression, the effect must be measurable in at least one of these or any other suitable assay. The lack of effect in a particular test does not imply the general absence of immunosuppression.

[0083] Human GDF-15 levels can be measured by any method known in the art, including, but not limited to, measuring GDF-15 mRNA levels by methods including, but not limited to, quantitative real-time PCR (qRT-PCR) of human GDF-15 mRNA using primers specific for human GDF-15, in situ hybridization of mRNA with a probe specific for human GDF-15, mRNA deep sequence method; and measuring GDF-15 protein levels by methods including, but not limited to, mass spectrometry for proteins or peptides derived from human GDF-15, western blotting using an antibody specific for human GDF-15, a strip test using an antibody specific for human GDF-15, or immunocytochemistry using an antibody specific for human GDF-15.

[0084] For such methods using antibodies specific for human GDF-15, the anti-human GDF-15 antibodies of the invention are preferred, most preferably the antibodies of the invention produced by the hybridoma cell line B1-23 deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) under deposit number DSM ACC3142.

[0085] Aspects of the invention As described above, the inventors show that human GDF-15 protein can be targeted by antibodies according to the present invention in such a manner that not only can cancer cachexia and cancer-induced weight loss be treated, but cancer growth is also inhibited.

[0086] In view of the present invention, it is clear that the anti-GDF-15 antibodies known from WO 2005 / 099746, WO 2009 / 021293 and Johnen H et al., Nature Medicine, 2007 only inhibit one of the effects of human GDF-15 (i.e. cancer-induced weight loss), but are unable to inhibit other effects of human GDF-15, such as those related to cancer growth. One possible explanation for this failure, in view of the present invention, is the following: the antibodies known from the above documents only interfere with the transport of human GDF-15 across the blood-brain barrier (by forming large complexes that cannot be transported across the blood-brain barrier), but are unable to bind to human GDF-15 in such a way that they generally cannot interact with its receptors (e.g. receptors present on cells outside the brain). Furthermore, unlike the antibodies of the present invention, the anti-GDF-15 antibodies known from WO 2005 / 099746, WO 2009 / 021293 and Johnen H et al., Nature Medicine, 2007 did not lead to a detectable increase in the body weight of a mammal compared to its body weight before the onset of cancer cachexia.

[0087] Thereby, the efficacy of antibodies for use according to the present invention is unexpected in view of the art. The following properties of the antibodies of the invention are expected to contribute to their ability to more completely inhibit the effects of human GDF-15, including treating cachexia and inhibiting cancer growth.

[0088] Broad binding specificity for forms of human GDF-15 The antibodies of the invention are capable of binding to mature recombinant human GDF-15 (represented by SEQ ID NO:8) and therefore to active, fully processed (mature) human GDF-15. In addition, by performing staining experiments with the mAb-B1-23 antibody according to the present invention on human cells, the inventors show that the mAb-B1-23 antibody according to the present invention is capable of binding to human GDF-15 precursor on human cells.

[0089] Thus, it is expected that the binding and effect of antibodies according to the invention, particularly inhibition of cancer growth, is not limited to an effect on any particular form of human GDF-15.

[0090] Regarding the effects of human GDF-15 on cancer cachexia, these effects may be exerted on a subset of forms of human GDF-15, such as the soluble form of human GDF-15 that can cross the blood-brain barrier. As illustrated in the examples of the present invention, all of the tested anti-GDF-15 antibodies according to the present invention may be used to treat cancer-induced cachexia. Thus, the antibodies according to the present invention may interfere with the forms of human GDF-15 that are responsible for cancer cachexia.

[0091] High binding affinity The antibodies and antigen-binding portions thereof according to the invention have high binding affinity, as demonstrated by the mAb-B1-23 antibody according to the invention, which has an equilibrium dissociation constant of about 790 pM for recombinant human GDF-15. Notably, such affinity value is superior to most existing therapeutic antibodies, for example the therapeutic antibody rituximab, which has an equilibrium dissociation constant of about 8 nM.

[0092] High binding affinity will ensure that the antibody against human GDF-15 according to the present invention will stably bind to human GDF-15, so that the effects of human GDF-15, including the effects on cancer growth, are effectively inhibited.Similarly, the stable binding of the antibody according to the present invention is expected to ensure that forms of human GDF-15 that cause cancer cachexia cannot carry out their pathological functions.This may be due, for example, to antibody-dependent capture of these forms of human GDF-15 from the sites in the brain where they can act.Such binding and capture may occur, for example, at the site of cancer, or the antibody according to the present invention may interfere with the transport of human GDF-15 across the blood-brain barrier.

[0093] Binding to discontinuous or conformational epitopes The antibodies and antigen-binding portions thereof according to the invention bind to discontinuous or conformational epitopes, as demonstrated below for the mAb-B1-23 antibody according to the invention.

[0094] The binding of the antibody and its antigen-binding portion according to the present invention to a discontinuous or conformational GDF-15 epitope can help maintain human GDF-15 in a specific conformation. This conformational specificity is advantageous for maintaining GDF-15 in a form that cannot be released from tumors or cross the blood-brain barrier to cause cancer cachexia at the site of its action in the brain. In addition, such binding to a discontinuous or conformational GDF-15 epitope may contribute to the effective inhibition of the effects of human GDF-15, including the effects on cancer growth, by, for example, maintaining GDF-15 in a conformation that cannot functionally interfere with its receptor. Thus, the present invention relates to the following aspects:

[0095] A) Antibodies, vectors and cell lines Specifically, the present invention relates to a monoclonal antibody or antigen-binding portion thereof capable of binding to human GDF-15, wherein the heavy chain variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO:5 or an amino acid sequence at least 90% identical thereto, and the light chain variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO:7 or an amino acid sequence at least 85% identical thereto, wherein the heavy chain constant domain comprises the amino acid sequence of SEQ ID NO:29 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95% identical thereto, and wherein the light chain constant domain comprises the amino acid sequence of SEQ ID NO:32 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95% identical thereto.

[0096] In a preferred aspect of this embodiment, the constant domain of the heavy chain comprises the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence at least 98%, preferably at least 99% identical thereto, and the constant domain of the light chain comprises the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence at least 98%, preferably at least 99% identical thereto.

[0097] In another preferred aspect of this embodiment, the constant domain of the heavy chain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 29, and the constant domain of the light chain comprises an amino acid sequence of SEQ ID NO: 32 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% identical thereto.

[0098] In another preferred aspect of this embodiment, the constant domain of the heavy chain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 29, and the constant domain of the light chain comprises an amino acid sequence of SEQ ID NO: 32 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% identical thereto.

[0099] In another preferred aspect of this embodiment, the constant domain of the heavy chain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 29, and the constant domain of the light chain comprises an amino acid sequence of SEQ ID NO: 32 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% identical thereto.

[0100] In another preferred aspect of this embodiment, the constant domain of the heavy chain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 29, and the constant domain of the light chain comprises an amino acid sequence of SEQ ID NO: 32 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% identical thereto.

[0101] In another preferred aspect of this embodiment, the constant domain of the heavy chain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 29, and the constant domain of the light chain comprises an amino acid sequence of SEQ ID NO: 32 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% identical thereto.

[0102] In another preferred aspect of this embodiment, the constant domain of the heavy chain comprises an amino acid sequence of SEQ ID NO:29 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% identical thereto, and the constant domain of the light chain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:32.

[0103] In another preferred aspect of this embodiment, the constant domain of the heavy chain comprises an amino acid sequence of SEQ ID NO:29 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% identical thereto, and the constant domain of the light chain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:32.

[0104] In another preferred aspect of this embodiment, the constant domain of the heavy chain comprises an amino acid sequence of SEQ ID NO:29 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% identical thereto, and the constant domain of the light chain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:32.

[0105] In another preferred aspect of this embodiment, the heavy chain constant domain comprises an amino acid sequence of SEQ ID NO:29 or at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% identical thereto, and the light chain constant domain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:32.

[0106] In another preferred aspect of this embodiment, the constant domain of the heavy chain comprises an amino acid sequence of SEQ ID NO:29 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% identical thereto, and the constant domain of the light chain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:32.

[0107] In this embodiment, most preferably, the constant domain of the heavy chain comprises the amino acid sequence of SEQ ID NO:29 and the constant domain of the light chain comprises the amino acid sequence of SEQ ID NO:32.

[0108] In an alternative embodiment, the invention relates to a monoclonal antibody or antigen-binding portion thereof capable of binding to human GDF-15, wherein the heavy chain variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO:5 or an amino acid sequence that differs by no more than one amino acid from the amino acid sequence of SEQ ID NO:5, and the light chain variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO:7 or an amino acid sequence that differs by no more than one amino acid from the amino acid sequence of SEQ ID NO:7, wherein the heavy chain constant domain comprises the amino acid sequence of SEQ ID NO:29 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95% identical thereto, and wherein the light chain constant domain comprises the amino acid sequence of SEQ ID NO:32 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95% identical thereto.

[0109] In a preferred aspect of this embodiment, the constant domain of the heavy chain comprises the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence at least 98%, preferably at least 99% identical thereto, and the constant domain of the light chain comprises the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence at least 98%, preferably at least 99% identical thereto.

[0110] In another preferred aspect of this embodiment, the constant domain of the heavy chain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 29, and the constant domain of the light chain comprises an amino acid sequence of SEQ ID NO: 32 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% identical thereto.

[0111] In another preferred aspect of this embodiment, the constant domain of the heavy chain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 29, and the constant domain of the light chain comprises an amino acid sequence of SEQ ID NO: 32 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% identical thereto.

[0112] In another preferred aspect of this embodiment, the constant domain of the heavy chain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 29, and the constant domain of the light chain comprises an amino acid sequence of SEQ ID NO: 32 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% identical thereto.

[0113] In another preferred aspect of this embodiment, the constant domain of the heavy chain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 29, and the constant domain of the light chain comprises an amino acid sequence of SEQ ID NO: 32 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% identical thereto.

[0114] In another preferred aspect of this embodiment, the constant domain of the heavy chain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 29, and the constant domain of the light chain comprises an amino acid sequence of SEQ ID NO: 32 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% identical thereto.

[0115] In another preferred aspect of this embodiment, the constant domain of the heavy chain comprises an amino acid sequence of SEQ ID NO:29 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% identical thereto, and the constant domain of the light chain comprises an amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO:32.

[0116] In another preferred aspect of this embodiment, the constant domain of the heavy chain comprises an amino acid sequence of SEQ ID NO:29 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% identical thereto, and the constant domain of the light chain comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:32.

[0117] In another preferred aspect of this embodiment, the constant domain of the heavy chain comprises an amino acid sequence of SEQ ID NO:29 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% identical thereto, and the constant domain of the light chain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:32.

[0118] In another preferred aspect of this embodiment, the constant domain of the heavy chain comprises an amino acid sequence of SEQ ID NO:29 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% identical thereto, and the constant domain of the light chain comprises an amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:32.

[0119] In another preferred aspect of this embodiment, the constant domain of the heavy chain comprises an amino acid sequence of SEQ ID NO:29 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% identical thereto, and the constant domain of the light chain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:32.

[0120] In this embodiment, most preferably, the constant domain of the heavy chain comprises the amino acid sequence of SEQ ID NO:29 and the constant domain of the light chain comprises the amino acid sequence of SEQ ID NO:32.

[0121] Further provided is a monoclonal antibody or antigen-binding portion thereof capable of binding to human GDF-15, wherein the heavy chain variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO:5 or an amino acid sequence at least 90% identical thereto, and the light chain variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO:7 or an amino acid sequence at least 85% identical thereto.

[0122] Preferably, the constant domain of the heavy chain of this monoclonal antibody or antigen-binding portion thereof comprises the amino acid sequence of SEQ ID NO:29 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95% identical thereto, and the constant domain of the light chain of this monoclonal antibody or antigen-binding portion thereof comprises the amino acid sequence of SEQ ID NO:32 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95% identical thereto.

[0123] More preferably, the constant domain of the heavy chain comprises the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence at least 98%, preferably at least 99% identical thereto, and the constant domain of the light chain comprises the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence at least 98%, preferably at least 99% identical thereto. Even more preferably, the constant domain of the heavy chain comprises the amino acid sequence of SEQ ID NO: 29 and the constant domain of the light chain comprises the amino acid sequence of SEQ ID NO: 32.

[0124] Further provided is a monoclonal antibody or antigen-binding portion thereof capable of binding to human GDF-15, wherein the heavy chain variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO:5 or an amino acid sequence that differs by no more than one amino acid from the amino acid sequence of SEQ ID NO:5, and the light chain variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO:7 or an amino acid sequence that differs by no more than one amino acid from the amino acid sequence of SEQ ID NO:7.

[0125] Preferably, the constant domain of the heavy chain of this monoclonal antibody or antigen-binding portion thereof comprises the amino acid sequence of SEQ ID NO:29 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95% identical thereto, and the constant domain of the light chain of this monoclonal antibody or antigen-binding portion thereof comprises the amino acid sequence of SEQ ID NO:32 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95% identical thereto.

[0126] More preferably, the constant domain of the heavy chain comprises the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence at least 98%, preferably at least 99% identical thereto, and the constant domain of the light chain comprises the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence at least 98%, preferably at least 99% identical thereto. Even more preferably, the constant domain of the heavy chain comprises the amino acid sequence of SEQ ID NO: 29 and the constant domain of the light chain comprises the amino acid sequence of SEQ ID NO: 32.

[0127] In a second embodiment according to the above aspect, the heavy chain variable domain of the monoclonal antibody or antigen-binding portion thereof comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO:5, or alternatively the light chain variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO:7.

[0128] In a third embodiment according to the above aspects, the heavy chain variable domain of the monoclonal antibody or antigen-binding portion thereof comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO:5, and the light chain variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO:7.

[0129] In a fourth embodiment according to the above aspects, the heavy chain variable domain of the monoclonal antibody or antigen-binding portion thereof comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO:3 and a CDR2 region comprising the amino acid sequence of SEQ ID NO:4, and the light chain variable domain of the monoclonal antibody or antigen-binding portion thereof comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO:6 and a CDR2 region comprising the amino acid sequence ser-ala-ser.

[0130] In a fifth embodiment according to the above aspects the antibody is a humanized antibody. Preferably all of the variable domains of the humanized antibody are humanized variable domains.

[0131] In a further embodiment according to the above embodiment, the heavy chain variable domain of the monoclonal antibody or antigen-binding portion thereof comprises the amino acid sequence of SEQ ID NO:28, or an amino acid sequence at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably at least 99% identical thereto, and the light chain variable domain of the monoclonal antibody or antigen-binding portion thereof comprises the amino acid sequence of SEQ ID NO:31, or an amino acid sequence at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably at least 99% identical thereto. In a most preferred aspect of this embodiment, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:28, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:31.

[0132] In a further preferred embodiment according to the above, the heavy chain of the monoclonal antibody or antigen-binding portion thereof comprises the amino acid sequence of SEQ ID NO:27, and the light chain of the monoclonal antibody or antigen-binding portion thereof comprises the amino acid sequence of SEQ ID NO:30.

[0133] In another preferred embodiment according to the above embodiment, the heavy chain variable domain of the monoclonal antibody or antigen-binding portion thereof comprises the amino acid sequence of SEQ ID NO: 34, or an amino acid sequence at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, even more preferably at least 99% identical thereto, and the light chain variable domain of the monoclonal antibody or antigen-binding portion thereof comprises the amino acid sequence of SEQ ID NO: 37, or an amino acid sequence at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, even more preferably at least 99% identical thereto. In a most preferred aspect of this embodiment according to the above embodiment, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 34, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 37.

[0134] In yet another embodiment according to the above first to third embodiments, the heavy chain variable domain comprises a region including FR1, CDR1, FR2, CDR2 and FR3 regions and comprising the amino acid sequence of SEQ ID NO: 1 or a sequence 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain variable domain comprises a region including FR1, CDR1, FR2, CDR2 and FR3 regions and comprising the amino acid sequence of SEQ ID NO: 2 or a sequence 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0135] In a preferred embodiment according to the above first to third aspects, the heavy chain variable domain comprises a region including FR1, CDR1, FR2, CDR2 and FR3 regions, said region comprising the amino acid sequence of SEQ ID NO: 1 or a sequence 95% identical thereto, and the light chain variable domain comprises a region including FR1, CDR1, FR2, CDR2 and FR3 regions, said region comprising the amino acid sequence of SEQ ID NO: 2 or a sequence 95% identical thereto.

[0136] In a more preferred embodiment according to the above first to third embodiments, the heavy chain variable domain comprises a region including FR1, CDR1, FR2, CDR2 and FR3 regions and comprising the amino acid sequence of SEQ ID NO: 1 or a sequence 98% identical thereto, and the light chain variable domain comprises a region including FR1, CDR1, FR2, CDR2 and FR3 regions and comprising the amino acid sequence of SEQ ID NO: 2 or a sequence 98% identical thereto.

[0137] In an even more preferred embodiment according to the above first to third embodiments, the heavy chain variable domain comprises a region comprising FR1, CDR1, FR2, CDR2 and FR3 regions, said region comprising the amino acid sequence of SEQ ID NO:1, and the light chain variable domain comprises a region comprising FR1, CDR1, FR2, CDR2 and FR3 regions, said region comprising the amino acid sequence of SEQ ID NO:2.

[0138] Further provided is a monoclonal antibody or antigen-binding portion thereof capable of binding to human GDF-15, wherein the heavy chain variable domain comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 3 and a CDR2 region comprising the amino acid sequence of SEQ ID NO: 4, and the light chain variable domain comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 6 and a CDR2 region comprising the amino acid sequence of SEQ ID NO: 7. In a preferred aspect of this embodiment, the antibody may have a CDR3 sequence as defined in any of the embodiments of the invention above.

[0139] In another embodiment, a monoclonal antibody, or antigen-binding portion thereof, capable of binding to human GDF-15 is provided, wherein the antibody, or antigen-binding portion thereof, is capable of inhibiting cancer growth in a mammal, preferably a human patient.

[0140] In another embodiment according to the above embodiment, the invention relates to an antigen-binding moiety capable of binding to human GDF-15, wherein the antigen-binding moiety is a single domain antibody (also referred to as a "Nanobody®"). In one aspect of this embodiment, the single domain antibody comprises the CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively. In one aspect of this embodiment, the single domain antibody comprises the CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively. In a preferred aspect of this embodiment, the single domain antibody is a humanized antibody.

[0141] Preferably, an antibody or antigen-binding portion thereof of the invention capable of binding to human GDF-15 has an equilibrium dissociation constant for human GDF-15 of 100 nM or less, less than 20 nM, preferably less than 10 nM, more preferably less than 5 nM and most preferably between 0.1 nM and 2 nM.

[0142] In another embodiment of the invention, the antibody or antigen-binding portion thereof capable of binding to human GDF-15 binds to the same human GDF-15 epitope as the antibody against human GDF-15 obtained from cell line B1-23 deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) under accession number DSM ACC3142. As described herein, the antibody that binds to human GDF-15 according to the invention is assessed by surface plasmon resonance measurements as a reference standard method, according to the procedure described in Example 1. Binding to the same epitope on human GDF-15 can likewise be assessed by competitive binding experiments of surface plasmon resonance between the antibody against human GDF-15 obtained from cell line B1-23 and an antibody predicted to bind to the same human GDF-15 epitope as the antibody against human GDF-15 obtained from cell line B1-23.

[0143] In a highly preferred embodiment, the antibody is a monoclonal antibody capable of binding to human GDF-15, or an antigen-binding portion thereof, obtained from cell line B1-23 deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) under accession number DSM ACC3142.

[0144] In a preferred embodiment, the antibody or antigen-binding portion thereof capable of binding to human GDF-15 according to the invention is a humanized monoclonal antibody or antigen-binding portion thereof. For any non-human antibody sequence (i.e., donor antibody sequence) according to the invention, a humanized monoclonal anti-human GDF-15 antibody or antigen-binding portion thereof of the invention can be generated according to techniques known in the art as described above.

[0145] In a highly preferred embodiment, the monoclonal antibody or antigen-binding portion thereof capable of binding to human GDF-15 is a humanized antibody or antigen-binding portion thereof derived from a monoclonal antibody capable of binding to human GDF-15 obtained from cell line B1-23 deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) under accession number DSM ACC3142. In a non-limiting aspect of this embodiment, the heavy chain variable domain of the humanized antibody or antigen-binding portion thereof comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO:5, and the light chain variable domain of the humanized antibody or antigen-binding portion thereof comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO:7.

[0146] In a further non-limiting aspect of this embodiment, the heavy chain variable domain of the humanized antibody, or antigen-binding portion thereof, comprises or further comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO:3 and a CDR2 region comprising the amino acid sequence of SEQ ID NO:4, and the light chain variable domain of the humanized antibody, or antigen-binding portion thereof, comprises or further comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO:6 and a CDR2 region comprising the amino acid sequence of SEQ ID NO:7.

[0147] Further provided is a monoclonal antibody or antigen-binding portion thereof capable of binding to human GDF-15, wherein the binding is to a conformational or discontinuous epitope on human GDF-15 consisting of the amino acid sequences of SEQ ID NO: 25 and SEQ ID NO: 26. In a preferred aspect of this embodiment, the antibody or antigen-binding portion thereof is an antibody or antigen-binding portion thereof defined in any one of the above embodiments.

[0148] In another embodiment of the invention according to the above embodiment, the antibody or antigen-binding portion thereof capable of binding to human GDF-15 is a bispecific antibody. In one aspect of this embodiment, the bispecific antibody is bivalent and monospecific, having two identical antigen-binding sites for human GDF-15. In a second alternative aspect of this embodiment, the bispecific antibody is bivalent and bispecific, with one antigen-binding site being a binding site for human GDF-15 and the other antigen-binding site being a binding site for a different antigen.

[0149] Non-limiting examples of different antigens according to the second aspect of this embodiment include i) cell surface antigens that are co-expressed with GDF-15 at high levels in the same cancer (e.g., at higher levels compared to a control sample obtained from a non-cancerous part of tissue that is the primary site tissue of the cancer from the same patient), and ii) cell surface antigens on cells of the immune system that are known to be useful antigens for recruiting cells of the immune system to tumors.

[0150] In yet another embodiment of the invention according to the above embodiment, the antibody or antigen-binding portion thereof capable of binding to human GDF-15 is linked to a drug. In a non-limiting aspect of this embodiment, the drug can be a known anti-cancer drug and / or an immune stimulating molecule.

[0151] Known anticancer drugs include alkylating agents such as cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, and ifosfamide; antimetabolites such as azathioprine and mercaptopurine; alkaloids such as vinca alkaloids (e.g., vincristine, vinblastine, vinorelbine, and vindesine), taxanes (e.g., paclitaxel, docetaxel), etoposide, and teniposide; topoisomerase inhibitors such as camptothecins (e.g., irinotecan and topotecan); cytotoxic antibiotics such as actinomycin, anthracyclines, doxorubicin, daunorubicin, valrubicin, idarubicin, epirubicin, bleomycin, plicamycin, and mitomycin; and radioisotopes.

[0152] Linking the antibodies of the invention, or antigen-binding portions thereof, to anti-cancer drugs is expected to result in more potent inhibition of cancer tumor growth compared to the antibody without the anti-cancer drug, because the resulting conjugate will accumulate at the tumor site due to the presence of GDF-15 in the tumor, leading to accumulation of the anti-cancer drug at the site of the tumor and enhanced effect of the anti-cancer drug on the tumor.

[0153] In a further embodiment according to the above embodiment, the antibody or antigen-binding portion thereof capable of binding to human GDF-15 is modified by an amino acid tag. Non-limiting examples of such tags include polyhistidine (His) tag, FLAG tag, hemagglutinin (HA) tag, glycoprotein D (gD) tag and c-myc tag. Tags may be used for various purposes. For example, they may be used to aid in the purification of the antibody or antigen-binding portion thereof capable of binding to human GDF-15, or may be used for the detection of the antibody or antigen-binding portion thereof (e.g., when used in diagnostic assays). Preferably, such tags are present at the C-terminus or N-terminus of the antibody or antigen-binding portion thereof capable of binding to human GDF-15.

[0154] In a preferred embodiment according to the above, the antibody or antigen-binding portion thereof capable of binding to human GDF-15 is capable of inhibiting cancer growth in a mammal, preferably a human patient.

[0155] In another preferred embodiment according to the above, the human GDF-15 is recombinant human GDF-15 having the amino acid sequence set forth in SEQ ID NO:8.

[0156] In yet another preferred embodiment according to the above, the binding of the antibody or antigen-binding portion thereof capable of binding to human GDF-15 is to a conformational or discontinuous epitope on human GDF-15.

[0157] Preferably, the monoclonal antibody or antigen-binding portion thereof of the invention capable of binding to human GDF-15 is an isolated antibody.

[0158] In a preferred embodiment of the above antibody or antigen-binding portion thereof according to the present invention, the antibody has a size of more than 100 kDa, preferably more than 110 kDa, more preferably more than 120 kDa, even more preferably more than 130 kDa, and most preferably more than 140 kDa. Preferably, the antibody is a full-length antibody, more preferably a full-length IgG antibody.

[0159] The present invention also relates to an expression vector comprising a nucleotide sequence encoding an antibody or an antigen-binding portion thereof as defined above. Furthermore, the present invention also provides cell lines capable of producing the antibodies or antigen-binding portions thereof according to the present invention.

[0160] In one embodiment, the cell line can be derived from any cell line known in the art and suitable for the production of antibodies or antigen-binding portions thereof. In a preferred embodiment, the cell line is cell line B1-23 deposited at the German Collection of Microbial Cell Cultures (DSMZ) under accession number DSM ACC3142. In another preferred embodiment, the cell line contains an expression vector according to the invention as defined above.

[0161] B) Pharmaceutical Compositions In a further aspect, the present invention relates to a pharmaceutical composition comprising any of the antibodies or antigen-binding portions thereof defined above. Pharmaceutical compositions according to the invention are prepared in accordance with known standards for the preparation of pharmaceutical compositions containing antibodies and portions thereof.

[0162] For example, the compositions are prepared in a manner such that they may be appropriately stored and administered, such as by using pharma- ceutically acceptable components, such as carriers, excipients, or stabilizers. Such pharma- ceutically acceptable components are non-toxic in the amounts used when the pharmaceutical composition is administered to a patient. The pharma- ceutically acceptable components added to a pharmaceutical composition may depend on the particular intended use and route of administration of the pharmaceutical composition.

[0163] Generally, pharma- ceutically acceptable components used in connection with the present invention are used in accordance with the knowledge available in the art, e.g. from Remington's Pharmaceutical Sciences, Ed. AR Gennaro, 20th edition, 2000, Williams & Wilkins, PA, USA.

[0164] C) Methods of Treatment and Products for Use in These Methods The present invention further relates to a method for treating cancer cachexia in a mammal, the method comprising administering to said mammal an antibody or antigen-binding portion thereof as defined above or a pharmaceutical composition as defined above. Instead, the present invention relates to an antibody or antigen-binding portion thereof as defined above or a pharmaceutical composition as defined above for use in these methods. In highly preferred aspects of these embodiments, the mammal is a human patient.

[0165] The present invention further relates to methods for treating cancer in a mammal, comprising administering to said mammal an antibody or antigen-binding portion thereof as defined above or a pharmaceutical composition as defined above. Instead, the present invention relates to an antibody or antigen-binding portion thereof as defined above or a pharmaceutical composition as defined above for use in these methods. In highly preferred aspects of these embodiments, the mammal is a human patient.

[0166] In view of the present invention, it becomes apparent that anti-GDF-15 antibodies known from WO 2005 / 099746, WO 2009 / 021293 and Johnen H et al., Nature Medicine, 2007 only inhibit cancer-induced weight loss, but fail to inhibit other effects of human GDF-15, such as those associated with cancer growth. The present invention relates to several surprising advantages over the effects observed in the art.

[0167] Among other things, one major benefit of the present invention is that the anti-GDF-15 antibodies disclosed herein can be used to more effectively treat cancer-induced weight loss and / or cancer cachexia. For example, treatment with an antibody according to the invention may completely prevent cancer cachexia (when administered prophylactically) or may completely reverse cancer cachexia (when administered after the onset of cancer cachexia).

[0168] Moreover, the antibodies according to the invention may increase the body weight of the treated mammal even during prophylactic treatment for the prevention of cachexia. Similarly, in the course of therapeutic treatment initiated after the onset of cancer cachexia, it is expected that the antibodies according to the invention may not only reverse the loss of body weight, but also increase the body weight of the treated mammal compared to its body weight before the onset of cancer cachexia.

[0169] This unexpected effect of the antibody according to the invention may be beneficial in various clinical situations. For example, the administration of many pharmacologic active ingredients against cancer (e.g. various chemotherapeutic drugs) may result in a loss of body weight in mammals, including human patients. Such additional loss of body weight may be countered by the weight gain resulting from the administration of the antibody according to the invention.

[0170] Thus, the use of the antibodies according to the invention may be particularly advantageous and safe for combination regimens with additional chemotherapeutic agents.Similarly, the use of the antibodies according to the invention may be particularly advantageous for mammals, such as human patients, who were already underweight before the onset of cancer and / or before the onset of cancer cachexia.Underweight patients include, for example, cachectic patients, e.g., those with a body mass index of 18 kg / m 2 The patient may be less than

[0171] Moreover, unexpectedly, according to the present invention, the antibodies are not only effective in treating cancer cachexia, but are also effective in treating cancer. Thus, the methods of treatment and products for use of the antibodies according to the invention are expected to be particularly beneficial in the treatment of cancer patient sub-groups suffering from cancer-induced weight loss and / or cancer cachexia, respectively.

[0172] However, the effects according to the invention are also expected to be advantageous for the treatment of a complete patient group of cancers mentioned herein: by using antibodies according to the invention that are effective against both cancer itself and cancer-induced weight loss and / or cancer cachexia, the treatment of cancer and cancer cachexia may be simplified by using the same treatment for all cancer patients, regardless of whether they suffer from cancer-induced weight loss and / or cancer cachexia or not. This is why it is expected that due to the dual effect of the antibodies against cancer and cancer cachexia, these antibodies will make unnecessary the need for additional drugs for the treatment of cancer cachexia.

[0173] Similarly, due to the dual effect of the antibodies according to the invention, the need to diagnose cancer-induced weight loss and / or cancer cachexia may also be eliminated, and it is therefore expected that the overall costs of treatment and diagnosis will be reduced.

[0174] Thus, in a preferred embodiment of the above methods or of the antibodies, antigen-binding portions thereof or pharmaceutical compositions for use in these methods according to the present invention, the method for treating cancer cachexia is a method for completely preventing or completely reversing cancer cachexia. In a more preferred embodiment of the method or of the antibodies, antigen-binding portions thereof or pharmaceutical compositions for use in the method, the method for treating cancer cachexia is a method for completely preventing cancer cachexia. In an alternative more preferred embodiment of the method or of the antibodies, antigen-binding portions thereof or pharmaceutical compositions for use in the method, the method for treating cancer cachexia is a method for completely reversing cancer cachexia.

[0175] In a preferred embodiment of the above methods or of the antibody, antigen-binding portion thereof or pharmaceutical composition for use in these methods according to the invention, i) cancer, and ii) Cancer cachexia Only mammals suffering from both are treated in the method.

[0176] In a preferred embodiment of the above methods or of the antibodies, antigen-binding portions thereof or pharmaceutical compositions for use in these methods according to the invention, the method increases the body weight of the mammal compared to its body weight before the onset of cancer cachexia. Preferably, the increase in body weight of the mammal is at least 1.5%, preferably at least 2.5%, more preferably at least 5%, compared to its body weight before the onset of cancer cachexia.

[0177] In a preferred embodiment of the above methods or the antibodies, antigen-binding portions thereof or pharmaceutical compositions for use in these methods according to the invention, the method is a method for treating not only cancer but also cancer cachexia in the same mammal.

[0178] In a preferred embodiment of the above methods or of the antibodies, antigen-binding portions thereof or pharmaceutical compositions for use in these methods according to the invention, the antibodies have a size greater than 100 kDa, preferably greater than 110 kDa, more preferably greater than 120 kDa, even more preferably greater than 130 kDa, and most preferably greater than 140 kDa. Preferably, the antibodies are full-length antibodies, more preferably full-length IgG antibodies.

[0179] In a further preferred embodiment of the above methods or the antibody, antigen-binding portion thereof or pharmaceutical composition for use in these methods according to the invention, the antibody has an Fc portion capable of binding to an Fc receptor.

[0180] In a preferred embodiment of the above methods or of the antibodies, antigen-binding portions thereof or pharmaceutical compositions for use in these methods according to the invention, the mammalian cancer cells endogenously express GDF-15 and / or the mammalian cancer cells stimulate endogenous expression of GDF-15 in mammalian non-cancerous cells.

[0181] In a preferred embodiment of the above methods or the antibodies, antigen-binding portions thereof or pharmaceutical compositions for use in these methods according to the invention, the mammalian cancer cells are characterized in that they endogenously express GDF-15.

[0182] In a preferred embodiment of the above methods or the antibodies, antigen-binding portions thereof or pharmaceutical compositions for use in these methods according to the invention, the mammal is a human patient.

[0183] In a preferred embodiment of the above methods or of the antibody, antigen-binding portion thereof or pharmaceutical composition for use in these methods according to the invention, the human GDF-15 is recombinant human GDF-15 having the amino acid sequence set forth in SEQ ID NO:8.

[0184] In a preferred embodiment of the above methods or antibodies, antigen-binding portions thereof or pharmaceutical compositions for use in these methods, the human patient has elevated serum GDF-15 levels prior to administration. In the subgroup of patients with elevated serum GDF-15 levels, the treatment method according to the invention is expected to be particularly effective in inhibiting cancer growth. In the most preferred aspect of this embodiment, the GDF-15 level is the GDF-15 protein level measured using an antibody according to the invention obtained from the hybridoma cell line B1-23 deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) under the accession number DSM ACC3142, preferably measured by immunochemistry.

[0185] In another embodiment of the above methods or the antibody, antigen-binding portion thereof, or pharmaceutical composition for use in these methods, the antibody or antigen-binding portion thereof is the only component used in the method that is pharma- ceutical active against cancer.

[0186] In alternative embodiments of the above methods or the antibodies, antigen-binding portions thereof or pharmaceutical compositions for use in these methods, the antibodies or antigen-binding portions thereof are used in combination with one or more additional components pharmaceutical active against cancer. In one aspect of this embodiment, the one or more additional components pharmaceutical active against cancer are known anti-cancer agents and / or immune stimulating molecules as defined above.

[0187] Thus, the anticancer drug may be selected from, for example, alkylating agents such as cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil and ifosfamide; antimetabolites such as azathioprine and mercaptopurine; alkaloids such as vinca alkaloids (e.g., vincristine, vinblastine, vinorelbine and vindesine), taxanes (e.g., paclitaxel, docetaxel), etoposide and teniposide; topoisomerase inhibitors such as camptothecins (e.g., irinotecan and topotecan); cytotoxic antibiotics such as actinomycin, anthracyclines, doxorubicin, daunorubicin, valrubicin, idarubicin, epirubicin, bleomycin, plicamycin and mitomycin; and radioisotopes.

[0188] Due to the enhancing effect of the antibodies according to the invention on the body weight of mammals, including human patients, these combinations of antibodies or antigen-binding portions thereof with pharmacologic active ingredients against cancer are expected to be particularly safe, since they offset the additional weight loss that may result from the administration of pharmacologic active ingredients against cancer.

[0189] In preferred embodiments of the above methods or antibodies, antigen-binding portions thereof, or pharmaceutical compositions for use in these methods, the cancer is selected from the group consisting of brain cancer including glioma, cancer of the nervous system, melanoma, lung cancer, lip and oral cavity cancer, liver cancer, leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, bladder cancer, cervical cancer, uterine cancer, testicular cancer, thyroid cancer, kidney cancer, gallbladder cancer, multiple myeloma, nasopharyngeal cancer, laryngeal cancer, pharynx cancer, tumors of the digestive tract including esophageal cancer, gastric cancer and colorectal cancer, pancreatic cancer, prostate cancer, ovarian cancer and breast cancer, preferably melanoma, prostate cancer, breast cancer, brain cancer including glioma, colorectal cancer, gastric cancer, esophageal cancer and ovarian cancer, and most preferably melanoma.

[0190] In one embodiment, the cancer is selected from the above group, further including endometrial cancer, such as endometrial cancer, breast cancer, including subtypes of breast cancer, especially triple negative breast cancer and bladder cancer, such as urothelial cell carcinoma.

[0191] In another preferred embodiment of the above methods or antibodies, antigen-binding portions thereof or pharmaceutical compositions for use in these methods, the one or more tumor(s) formed by the cancer have higher human GDF-15 levels before administration, preferably 1.2-fold higher, more preferably 1.5-fold higher, even more preferably 2-fold higher, and most preferably 5-fold higher, compared to a control sample obtained from a non-cancerous part of the tissue that is the primary site of the cancer of the same patient. In the subgroup of patients having higher GDF-15 levels in the one or more tumor(s) formed by the cancer compared to the above control sample, the treatment method according to the present invention is expected to be particularly effective in inhibiting cancer growth.

[0192] In highly preferred embodiments of the above methods or antibodies, antigen-binding portions thereof, or pharmaceutical compositions for use in these methods, the method for treating cancer comprises inhibiting cancer growth. In a preferred aspect of this embodiment, cancer growth is halted. In a more preferred aspect, the cancer is reduced.

[0193] In a preferred embodiment of the above methods or antibodies, antigen-binding portions thereof or pharmaceutical compositions for use in these methods, the method for treating cancer comprises inducing the killing of cancer cells by NK cells and CD8+ T cells in human patients. Due to their ability to interfere with GDF-15-mediated downregulation of the known immunological surveillance regulator NKG2D, the antibodies or antigen-binding portions thereof according to the present invention are expected to restore immunological surveillance and induce the killing of cancer cells by NK cells and CD8+ T cells, in addition to the effect of the antibodies or antigen-binding portions thereof independent of the immune system.

[0194] D) Kit The present invention also provides a kit comprising the pharmaceutical composition defined above. In one embodiment the kit is for use in a method according to the invention as defined above. In a further aspect, the present invention also provides a diagnostic kit comprising any of the antibodies or antigen-binding portions thereof according to the present invention.

[0195] In one embodiment, the diagnostic kit may be used to detect whether a tumor or tumors formed by a cancer patient have higher levels of human GDF-15 compared to a control sample obtained from a non-cancerous part of tissue that is the primary site tissue of the cancer in the same patient. In another embodiment, the diagnostic kit may be used to detect whether a human cancer patient has elevated GDF-15 levels in the serum.

[0196] E) Sequence The amino acid sequences referred to in this application are as follows (ordered from N-terminus to C-terminus; represented by the single letter amino acid code): SEQ ID NO:1 (region of the heavy chain variable domain including FR1, CDR1, FR2, CDR2 and FR3 regions from the polypeptide sequence of monoclonal anti-human GDF-15 mAb-B1-23): QVKLQQSGPGILQSSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDDKRYNPTLKSRLTISKDPSRNQVFLKITSVDTADTATYYC

[0197] SEQ ID NO:2 (region of the light chain variable domain including FR1, CDR1, FR2, CDR2 and FR3 regions from the polypeptide sequence of monoclonal anti-human GDF-15 mAb-B1-23): DIVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWFLQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFC SEQ ID NO:3 (heavy chain CDR1 region peptide sequence of monoclonal anti-human GDF-15 mAb-B1-23): GFSLSTSGMG

[0198] SEQ ID NO:4 (heavy chain CDR2 region peptide sequence of monoclonal anti-human GDF-15 mAb-B1-23): IYWDDDK SEQ ID NO:5 (heavy chain CDR3 region peptide sequence of monoclonal anti-human GDF-15 mAb-B1-23): ARSSYGAMDY SEQ ID NO:6 (light chain CDR1 region peptide sequence of monoclonal anti-human GDF-15 mAb-B1-23): QNVGTN

[0199] Light chain CDR2 region peptide sequence of monoclonal anti-human GDF-15 mAb-B1-23: SAS SEQ ID NO:7 (light chain CDR3 region peptide sequence of monoclonal anti-human GDF-15 mAb-B1-23): QQYNNFPYT SEQ ID NO:8 (recombinant mature human GDF-15 protein): GSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI

[0200] SEQ ID NO:9 (human GDF-15 precursor protein): MPGQELRTVNGSQMLLVLLVLSWLPHGALSLAEASRASFPGPSELHSEDSRFRELRKRYEDLLTRLRANQSWEDSNTDLVPAPAVRILTPEVRLGSGGHLHLRISRAALPEGLPEASRLHRALFRLSPTASRSWDVTRPLRRQLSLARPQAPA LHLRLSPPSQSDQLLAESSSARPQLELHLRPQAARGRRRARARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI

[0201] SEQ ID NO:10 (human GDF-15 precursor protein + N- and C-terminal GSGS linkers): GSGSGSGMPGQELRTVNGSQMLLVLLVLSWLPHGGALSLAEASRASFPGPSELHSEDSRFRELRKRYEDLLTRLRANQSWEDSNTDLVPAPAVRILTPEVRLGSGGHLHLRISRAALPEGLPEASRLHRALFRLSPTASRSWDVTRPLRRQLSLARPQAPA LHLRLSPPSQSDQLLAESSSARPQLELHLRPQAARGRRRARARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCIGSGSGSG

[0202] SEQ ID NO: 11 (Flag peptide): DYKDDDDKGG SEQ ID NO: 12 (HA peptide): YPYDVPDYAG SEQ ID NO: 13 (human GDF-15 derived peptide): ELHLRPQAARGRR SEQ ID NO: 14 (human GDF-15 derived peptide): LHLRPQAARGRRR SEQ ID NO: 15 (human GDF-15 derived peptide): HLRPQAARGRRRA

[0203] SEQ ID NO: 16 (human GDF-15 derived peptide): LRPQAARGRRRAR SEQ ID NO: 17 (human GDF-15 derived peptide): RPQAARGRRRARA SEQ ID NO: 18 (human GDF-15 derived peptide): PQAARGRRRARAR SEQ ID NO: 19 (human GDF-15 derived peptide): QAARGRRRARARN SEQ ID NO: 20 (human GDF-15 derived peptide): MHAQIKTSLHRLK

[0204] SEQ ID NO: 25 (GDF-15 peptide containing a portion of the GDF-15 epitope that binds to B1-23): EVQVTMCIGACPSQFR SEQ ID NO:26 (GDF-15 peptide containing a portion of the GDF-15 epitope that binds to B1-23): TDTGVSLQTYDDLLAKDCHCI The nucleic acid sequences referred to in this application are as follows (5' to 3' order; represented according to the standard nucleic acid code):

[0205] SEQ ID NO:21 (DNA nucleotide sequence encoding the amino acid sequence defined in SEQ ID NO:1): CAAGTGAAGCTGCAGCAGTCAGGCCCTGGGATATTGCAGTCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGTACTTCTGGTATGGGTTGAGCTGGATTCGTCAGCCTTCAGGAAAGGGTCTGGAGT GGCTGGCACACATTTACTGGGATGATGACAAGCGCTATAACCCAACCCTGAAGAGCCGGCTCACAATCTCCAAGGATCCCTCCAGAAACCAGGTATTCCTCAAGATCACCAGTGTGGACACTGCAGATACTGCCACATACTACTGT

[0206] SEQ ID NO:22 (DNA nucleotide sequence encoding the amino acid sequence defined in SEQ ID NO:2): GACATTGTGCTCACCCAGTCTCCAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTAATGTGGCCTGGTTTCTACAGAAACCAGGGCAATCTCCT AAAGCACTTATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAACGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGT

[0207] SEQ ID NO:23 (DNA nucleotide sequence encoding the amino acid sequence defined in SEQ ID NO:5): GCTCGAAGTTCCTACGGGGCAATGGACTAC SEQ ID NO:24 (DNA nucleotide sequence encoding the amino acid sequence defined in SEQ ID NO:7): CAGCAATATAACAACTTTCCGTACACG

[0208] SEQ ID NO: 27 (amino acid sequence of the heavy chain of the H1L5 humanized B1-23 anti-GDF-15 antibody): QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYNPTLKSRLTITKDPSKNQVVLTMTNMDPPVDTATYYCARSSYGAMDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0209] SEQ ID NO: 28 (amino acid sequence of the heavy chain variable domain of the H1L5 humanized B1-23 anti-GDF-15 antibody): QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKGLEWLAHIYWDDDKRYNPTLKSRLTITKDPSKNQVVLTMTTNMDPVDTATYYCARSSYGAMDYWGQGTLVTVSS SEQ ID NO: 29 (amino acid sequence of the heavy chain constant domain of the H1L5 humanized B1-23 anti-GDF-15 antibody): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0210] SEQ ID NO: 30 (amino acid sequence of the light chain of the H1L5 humanized B1-23 anti-GDF-15 antibody): DIVLTQSPSFLSASVGDRVTITCKASQNVGTNVAWFQQKPGKSPKALIYSASYRYSGVPDRFTGSGSGTEFTLTISSLQPEDFAAYFCQQYNNFPYTFGGGTKLE IKRAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:31 (amino acid sequence of the light chain variable domain of the H1L5 humanized B1-23 anti-GDF-15 antibody): DIVLTQSPSFLSASVGDRVTITCKASQNVGTNVAWFQQKPGKSPKALIYSASYRYSGVPDRFTGSGSGTEFTLTISSLQPEDFAAYFCQQYNNFPYTFGGGTKLEIKR

[0211] SEQ ID NO: 32 (amino acid sequence of the light chain constant domain of the H1L5 humanized B1-23 anti-GDF-15 antibody): APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 33 (amino acid sequence of the heavy chain of the chimeric B1-23 anti-GDF-15 antibody): QVKLQQSGPGILQSSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDDKRYNPTLKSRLTISKDPSRNQVFLKITSVDTADTATYYCARSSYGAMDYWGQGT SVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0212] SEQ ID NO: 34 (amino acid sequence of the heavy chain variable domain of chimeric B1-23 anti-GDF-15 antibody): QVKLQQSGPGILQSSQTLSLLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDDKRYNPTLKSRLTISKDPSRNQVFLKITSVDTADTATYYCARSSYGAMDYWGQGTSVTVSS SEQ ID NO: 35 (amino acid sequence of the heavy chain constant domain of chimeric B1-23 anti-GDF-15 antibody): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0213] SEQ ID NO: 36 (amino acid sequence of the light chain of the chimeric B1-23 anti-GDF-15 antibody): DIVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWFLQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNNFPYTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0214] SEQ ID NO: 37 (amino acid sequence of the light chain variable domain of chimeric B1-23 anti-GDF-15 antibody): DIVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWFLQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNNFPYTFGGGTKLEIKRTVA SEQ ID NO: 38 (amino acid sequence of the light chain constant domain of chimeric B1-23 anti-GDF-15 antibody): APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0215] F) Example The invention is illustrated by the following non-limiting examples. Example 1 Generation and characterization of murine GDF-15 antibody B1-23 and generation of chimeric and humanized antibodies

[0216] Antibody B1-23 was generated in GDF-15 knockout mice. Recombinant human GDF-15 (SEQ ID NO: 8) was used as the immunogen. The hybridoma cell line B1-23 producing mAb-B1-23 was deposited in accordance with the Budapest Treaty at the German Collection of Microbial and Cell Cultures (DSMZ) under the accession number DSM ACC3142.

[0217] B1-23 was identified as an IgG2a (kappa chain) isotype by a commercially available test strip system. Surface plasmon resonance measurements were used to determine the dissociation constant (Kd) as follows: Binding of the monoclonal anti-human GDF-15 antibody, anti-human GDF-15 mAb-B1-23, was measured employing surface plasmon resonance measurements using a Bio-Rad® ProteOn™ XPR36 system and a Bio-Rad® GLC sensor chip.

[0218] To fabricate the biosensor, recombinant mature human GDF-15 protein was immobilized on flow cells 1 and 2. On one flow cell, recombinant GDF-15 from baculovirus-transfected insect cells (HighFive insect cells) was used, and on the other flow cell, another recombinant protein from expression in E. coli was used. The GLC sensor chip was activated using Sulfo-NHS (N-hydroxysulfosuccinimide) and EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) (Bio-Rad® ProteOn™ Amine Coupling Kit) according to the manufacturer's recommendations, and approximately 600 RU (1Ru = 1 pgmm) of GDF-15 was then applied to the sensor surface. -2 Proteins were loaded to a density of 1000 ng / ml.

[0219] Unreacted binding groups were then quenched by perfusion with 1 M ethanolamine pH 8.5, and the biosensor was equilibrated by perfusing the chip with running buffer (10 M HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.005% Tween® 20, pH 7.4, referred to as HBS150). As controls, two flow cells were used, one empty with no protein binding and one coupled to a non-physiological protein partner (human interleukin-5), which was immobilized using the same coupling chemistry and the same coupling density. For interaction measurements, anti-human GDF-15 mAb-B1-23 was dissolved in HBS150 and used as analyte in six different concentrations (concentrations: 0.4, 0.8, 3, 12, 49 and 98 nM).

[0220] Analytes were perfused over the biosensor using a one-shot rate setting to avoid intermittent regeneration, and all measurements were performed at 25°C, using a flow rate of 100 μl / min. To address the bulk face effect, nonspecific binding to the sensor matrix was removed by subtracting the SPR data of an empty flow cell (flow cell 3) from all other SPR data. The resulting sensograms were analyzed using the software Proteon Manager version 3.0. For the analysis of the binding kinetics, a 1:1 Langmuir-type interaction was assumed. For the association rate constant, 5.4 ± 0.06 × 10 5 / Ms(k on ) and the dissociation rate constant, 4.3 ± 0.03 × 10 -4 / s(k off ) values ​​could be determined (values ​​are for the interaction of anti-human GDF-15 mAb-B1-23 with insect cell-expressed GDF-15).

[0221] The equilibrium dissociation constant is given by the formula K D =k off / k on The affinity values ​​for the interaction of E. coli-expressed GDF-15 and anti-human GDF-15 mAb-B1-23 differed by less than two-fold, and the rate constants for insect cell and E. coli-derived GDF-15 were biased by approximately 45%, thus within the precision of the SPR measurements and likely not reflecting real affinity differences. Under the conditions used, anti-human GDF-15 mAb-B1-23 showed no binding to human interleukin-5, thus confirming the interaction data and the specificity of anti-human GDF-15 mAb-B1-23.

[0222] The amino acid sequence of recombinant human GDF-15 (expressed in Baculovirus transfected insect cells) is: GSARNGDHCP LGPGRCCRLH TVRASLEDLG WADWVLSPRE VQVTMCIGAC PSQFRAANMH AQIKTSLHRL KPDTVPAPCC VPASYNPMVL IQKTDTGVSL QTYDDLLAKD CHCI (SEQ ID NO:8) Thus, using surface plasmon resonance measurements, a dissociation constant (Kd) of 790 pM was determined. In comparison: the therapeutically used antibody Rituximab has a significantly lower affinity (Kd=8 nM).

[0223] From the mouse anti-human GDF-15 mAb-B1-23, a chimeric anti-human GDF-15 mAb-B1-23 antibody according to the invention was generated by replacing the constant domains of the mouse antibody with the constant domains of a human IgG1 antibody (Trastuzumab scaffold). The amino acid sequence of the heavy chain of this chimeric antibody is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain of this chimeric antibody is shown in SEQ ID NO: 36.

[0224] From the chimeric anti-human GDF-15 mAb-B1-23, the humanized anti-human GDF-15 mAb-B1-23 antibody according to the invention was developed by humanizing the variable domains of the chimeric antibody, i.e. by replacing the framework regions of the chimeric antibody with human sequences. The amino acid sequence of the heavy chain of this humanized antibody is shown in SEQ ID NO: 27, and the amino acid sequence of the light chain of this humanized antibody is shown in SEQ ID NO: 30. This antibody is also referred to as H1L5 anti-GDF-15 antibody or humanized B1-23-H1L5 antibody or H1L5 antibody.

[0225] To generate the chimeric anti-human GDF-15 mAb-B1-23 antibody and the humanized B1-23-H1L5 antibody shown above, the cDNA encoding the antibody sequence was optimized and the gene was synthesized. The gene sequence was then cloned into a cloning / expression vector system. Plasmid DNA with low endotoxin levels was synthesized from these vectors.

[0226] The plasmid DNA was then transiently transfected into CHO cells followed by analysis and quantification of antibody expression using a Protein A biosensor. The cDNA of candidate cultures for antibody expression was sequenced. The resulting monoclonal antibodies were analyzed (see Examples 7-9).

[0227] Example 2 Antagonism of GDF-15-mediated effects by mAB B1-23 a) The NKG2D (natural killer group 2D) receptor expressed on NK cells and CD8+ T cells is known to play an important role in immunosurveillance against tumors. Transformed and virus-infected cells express ligands that bind to the NKG2D receptor and activate the cytotoxic effector functions of the described immune cells. Thus, transformed cells can be detected and eliminated by the immune system. After treating immune cells with either recombinant human GDF-15 or tumor cell-secreted GDF-15 for 72 hours in vitro, the expression level of NKG2D on the cell surface of lymphocytes was downregulated (Figure 1).

[0228] After 72 hours of incubation, immune cells were stained with the following FACS antibodies: anti-CD3, anti-CD56, anti-NKG2D. Using this antibody combination, the experiments focused on NK cells and their NKG2D surface expression. Low NKG2D levels on immune cells resulted in impaired tumor / target cell lysis. GDF-15-mediated downregulation of NKG2D was prevented by mAb B1-23.

[0229] Therefore, it is concluded that human GDF-15 downregulates the expression of NKG2D on the cell surface of lymphocytes, thereby downregulating the immunosurveillance against tumors.By binding to human GDF-15, the antibody of the present invention can prevent the GDF-15-mediated downregulation of NKG2D, restore the immunosurveillance, and induce the killing of cancer cells by NK cells and CD8+ T cells.Given that the CDR region of mAb B1-23 antibody corresponds to the CDR region of chimeric and humanized antibodies, it is expected that the functional properties, including the binding properties, of these antibodies are similar.

[0230] b) Treatment of ovarian cancer cell line SK-OV-3 with recombinant GDF-15 resulted in phosphorylation of AKT. AKT is a molecule that is part of the PI3K pathway and contributes to cell activation and growth. In this experiment, SK-OV-3 cells were treated with 10 ng / ml recombinant GDF-15 for 10 min at 37°C and 5% CO2. Pre-incubation of 2 μg mAb-B1-23 with 10 ng / ml GDF-15 at 37°C blocked GDF-15-mediated AKT phosphorylation (Figure 2), indicating the neutralizing effect of mAb-B1-23.

[0231] c) Treatment of immune cells with recombinant GDF-15 resulted in phosphorylation of JNK, a kinase that is activated by either cytokines or stress. Antagonism of 10 ng / ml GDF-15 with 2 μg mAb-B1-23 (preincubation for 5 min at 37° C.) blocked GDF-15-mediated JNK1 / 2 phosphorylation (FIG. 3).

[0232] Example 3 Inhibition of cancer cell growth using mAb B1-23 The data generated by B1-23 showed the anti-growth effect of the antibody on cancer cells in vitro. The most potent anti-growth effect was observed using the prostate cancer cell line LnCaP, which produces a lot of GDF-15. The metabolic assay (Alamar Blue® assay) showed a 30% reduction in growth after 72 hours in the presence of mAb-B1-23 compared to the control group where no antibody was applied. This effect demonstrates a significant reduction in the cell division rate after blocking GDF-15, since the cytotoxic effect of the antibody was excluded in a different assay.

[0233] Example 4 :mAb B1-23 inhibits tumor growth in vivo The following in vivo tests were performed. To evaluate the antitumor effect of B1-23 in vivo, Balb / c nu / nu Nude mice were used in a xenograft setting of the melanoma cell line UACC-257. Mice were treated with either antibody B1-23 or PBS. Each treatment cohort contained 10 Balb / c mice. nu / nu Nude mice were included.

[0234] Prior to injection, UACC-257 melanoma cells were grown in complete medium to remove all contaminants. Cells were harvested when they reached 70-80% confluence in cell culture flasks. Cells were then washed with PBS and counted. 1 × 10 7 Viable cells were suspended in PBS.

[0235] The first injection / treatment was performed on 6-week-old Balb / c nu / nu Nude mice were administered. The inoculation area of ​​the mice was cleaned with ethanol. UACC 257 cells were mixed and drawn into a syringe without a needle to avoid negative pressure on the tumor cells. 1 × 10 7 A cell suspension containing cells was injected subcutaneously (sc) into the lower flank of mice.

[0236] Intraperitoneal (ip) injections of either B1-23 (25 mg / kg body weight) or an equal volume of PBS were administered twice weekly starting immediately after tumor cell inoculation (designated day 1). Tumors were allowed to grow for 48 days. Tumor diameters were measured with calipers and expressed in mm 3 The tumor volume at 0.05 mm was calculated by the following formula. Volume = (Width) 2 ×Length / 2 The results from the study are shown in FIG.

[0237] As demonstrated in the figure, tumor size in the B1-23 treated animal cohort was significantly reduced compared to the PBS control group. Given that the CDR regions of the mAb B1-23 antibody correspond to the CDR regions of the chimeric and humanized antibodies, the functional properties, including binding characteristics, of these antibodies are expected to be similar.

[0238] Example 5 mAb B1-23 recognizes conformational or discontinuous epitopes on human GDF-15 Epitope mapping: Monoclonal mouse antibody GDF-15 against a 13-mer linear peptide derived from GDF-15 Antigen: GDF-15: [ka] (322 amino acids with linker) (SEQ ID NO: 10)

[0239] The protein sequence was translated into a 13-mer peptide with a one amino acid shift, and the C- and N-termini were extended with neutral GSGS linkers to avoid truncated peptides (bold). Control peptide: Flag: DYKDDDDKGG (SEQ ID NO: 13), 78 spots; HA: YPYDVPDYAG (SEQ ID NO: 14), 78 spots (each array copy) Peptide Chip Identifier: 000264_01 (10 / 90, Ala2Asp linker)

[0240] Dyeing conditions: Standard buffer: PBS, pH7.4+0.05%Tween(R) 20 Blocking buffer: Rockland Blocking Buffer MB-070 Incubation buffer: Standard buffer and 10% Rockland blocking buffer MB-070 Primary sample: Monoclonal mouse antibody GDF-15 (1 μg / μl): stained at a dilution of 1:100 in incubation buffer at 4° C. for 16 hours with slight shaking at 500 rpm Secondary antibody: Goat anti-mouse IgG (H+L) IRDye680, diluted 1:5000, stained for 30 min at room temperature (RT) in incubation buffer Control antibodies: monoclonal anti-HA(12CA5)-LL-Atto 680 (1:1000), monoclonal anti-FLAG(M2)-FluoProbes752 (1:1000); staining for 1 h at RT in incubation buffer.

[0241] scanner: Odyssey® Imaging System, LI-COR Biosciences Settings: Offset: 1mm; Resolution: 21μm. Red / Green Intensity: 7 / 7

[0242] result: After pre-swelling in standard buffer for 30 min and blocking buffer for 30 min, peptide arrays with 10-, 12- and 15-mer B7H3-derived linear peptides were incubated with secondary goat anti-mouse IgG(H+L)IRDye680 antibody at 1:5000 dilution for 1 h at room temperature to analyze background interactions of the secondary antibodies. PEPperCHIP® was washed 2×1 min with standard buffer, rinsed with distilled water and dried in airflow. Readout was performed with an Odyssey® Imaging System at a resolution of 21 μm and a green / red intensity of 7 / 7: a weak interaction was observed between the arginine-rich peptides known as frequent binders (ELHLRPQAARGRR (SEQ ID NO: 15), LHLRPQAARGRRR (SEQ ID NO: 16), HLRPQAARGRRRA (SEQ ID NO: 17), LRPQAARGRRRAR (SEQ ID NO: 18), RPQAARGRRRARA (SEQ ID NO: 19), PQAARGRRRARAR (SEQ ID NO: 20) and QAARGRRRARARN (SEQ ID NO: 21)) and the basic peptide MHAQIKTSLHRLK (SEQ ID NO: 22) due to ionic interactions with the charged antibody dye.

[0243] After 10 min pre-swelling in standard buffer, the peptide microarrays were incubated overnight at 4° C. with monoclonal mouse antibody GDF-15 at a dilution of 1:100. They were then washed repeatedly with standard buffer (2×1 min) followed by incubation with secondary antibody at a dilution of 1:5000 for 30 min at room temperature. After 2×10 s washes with standard buffer and a short rinse with distilled water, the PEPperCHIP® was dried in an air stream. Readouts were performed using an Odyssey® Imaging System at a resolution of 21 μm and a green / red intensity of 7 / 7 before and after staining with anti-HA and anti-FLAG (M2) antibodies for the control peptides.

[0244] None of the linear 13-mer peptides derived from GDF-15 were shown to interact with the monoclonal mouse antibody GDF-15, even at over-controlled intensities, however staining of the Flag and HA control peptides framing the array produced good and uniform spot intensities.

[0245] overview: Epitope mapping of the monoclonal mouse GDF-15 antibody against GDF-15 did not reveal any linear epitopes with antigen-derived 13-mer peptides. This finding suggests that the monoclonal mouse antibody GDF-15 is highly likely to recognize a partial low affinity conformational epitope or a discontinuous epitope. Due to the apparent lack of any GDF-15 signal above background staining with the secondary antibody alone, quantification of spot intensity with the PepSlide® analyzer and subsequent peptide annotation was omitted.

[0246] Example 6 Structural identification of peptide ligand epitopes by mass spectrometric epitope excision and epitope extraction The epitope of recombinant human GDF-15 that binds to antibody B1-23 was identified by epitope excision and epitope extraction methods (Suckau et al. Proc Natl Acad Sci US A. 1990 December; 87(24): 9848-9852.; R.Stefanescu et al., Eur.J.Mass Spectrom. 13, 69-75 (2007)). For preparation of the antibody column, antibody B1-23 was added to NHS-activated 6-aminohexanoic acid bound to Sepharose. The Sepharose-bound antibody B1-23 was then loaded onto a 0.8 ml microcolumn and washed with blocking and washing buffers.

[0247] Epitope extraction experiments: Recombinant human GDF-15 was digested with trypsin for 2 hours at 37°C (in solution) to obtain different peptides depending on the trypsin cleavage site in the protein. After complete digestion, the peptides were loaded onto an affinity column containing immobilized antibody B1-23. Unbound and potentially bound peptides of GDF-15 were used for mass spectrometry. Identification of the peptides by mass spectrometry was not possible. This further indicated that the binding region of GDF-15 in the immune complex B1-23 contains discontinuous or conformational epitopes. In the case of continuous linear epitopes, the digested peptide should bind to its interaction partner unless a trypsin cleavage site is present in the epitope peptide. Discontinuous or conformational epitopes could be confirmed by the epitope excision method described below.

[0248] Epitope excision experiments: The immobilized antibody B1-23 on the affinity column was then incubated with recombinant GDF-15 for 2 hours. The immune complex formed on the affinity column was then incubated with trypsin at 37°C for 2 hours. Cleavage resulted in different peptides derived from recombinant GDF-15. The immobilized antibody itself is proteolytically stable. The peptides obtained from the digested GDF-15 protein, masked by the antibody and therefore protected from proteolytic cleavage, were eluted under acidic conditions (TFA, pH 2), collected and identified by mass spectrometry.

[0249] The epitope excision method using MS / MS identification yielded the following peptides: [Table 1]

[0250] The portion of human GDF-15 that binds to antibody B1-23 contains a discontinuous or conformational epitope. Mass spectrometry identified two peptides in the GDF-15 protein that are involved in the formation of immune complexes. These peptides are confined to positions 40-55 (EVQVTMCIGACPSQFR) and 94-114 (TDTGVSLQTYDDLLAKDCHCI) of the GDF-15 amino acid sequence. Thus, these two peptides contain the epitope of the GDF-15 protein that binds to antibody B1-23.

[0251] However, since the CDR regions of the mAb B1-23 antibody correspond to the CDR regions of the chimeric and humanized antibodies, the binding properties of these antibodies are expected to be similar.

[0252] Example 7 Treatment of cancer-induced weight loss with anti-GDF-15 antibodies In Basic Animal Research No. 140123, 10 Balb / cnu / nu mice per treatment group were administered at 10 × 10 6 UACC-257 cells were subcutaneously implanted with Matrigel at a 1:1 volume ratio (100 μl cells + 100 μl Matrigel). Animals were treated on the same day with the respective antibodies as indicated below:

[0253] [Table 2]

[0254] [Table 3]

[0255] The dacarbazine group (Group 1) served as a positive control for the reference group / Tumor Growth Arrest (a cytostatic drug for the treatment of malignant melanoma in humans). The PBS group (Group 2) served as the growth control / vehicle control group since all substances used in the other groups were administered in PBS.

[0256] The murine B1-23 lead candidate antibody group (group 3) served as a reference group for comparison with the chimeric B1-23 antibody and humanized B1-23 H1L5 (groups 4 and 5). Group 4 is a group of chimerized B1-23 lead candidate antibodies containing the murine variable and constant domains of a human IgG1 antibody (trastuzumab backbone). Group 5 is the group of H1L5 humanized B1-23 lead candidate antibodies that contain a humanized framework within the murine variable and constant domains of a human IgG1 antibody (Trastuzumab backbone).

[0257] Group 6 is a group of B12 isotype antibodies. In this isotype control group, the antibody B12 (Lot. No.: ID3195) was produced by the company Evitria AG. Since B12 binds to HIV antigens, it should not bind to antigens in nude mice or to antigens of human tumors. B12 was chosen as a very suitable isotype control because its immunoglobulin backbone is also composed of the human IgG1 antibody trastuzumab, and is therefore almost identical to the chimeric B1-23 and H1L5 humanized B1-23 antibodies, except for their variable regions.

[0258] The study was performed in a double-blind fashion for treatment with antibody and treatment with PBS. In groups 1, 2 and 6 that did not receive anti-GDF-15 antibodies, a weight loss of more than 10% was observed (i.e., a relative weight loss of less than 90% compared to day 0). In contrast, a weight gain was observed in groups that did not receive treatment with anti-GDF-15 antibodies B1-23, chimeric B1-23 and humanized B1-23-H1L5, respectively (Figure 5). Thus, surprisingly, treatment with all tested anti-GDF-15 antibodies completely prevented cancer-induced weight loss in mice, an effect that was significant for all anti-GDF-15 antibody-treated groups (2-way ANOVA; p<0.05).

[0259] It is also noteworthy that the mice in the group that did not receive treatment with anti-GDF-15 antibodies exhibited weight loss of more than 10%. In humans, weight loss of as little as 5% over a 6-month period is considered indicative of cancer cachexia (Fearon K. et al.: Definition and classification of cancer cachexia: an international consensus. Lancet Oncol. 2011 May; 12(5):489-95.). Given that the greater weight loss of the mice, even more than 10%, was observed in this study, it is expected that the mice in this study that did not receive treatment with anti-GDF-15 antibodies exhibited not only weight loss, but also cancer cachexia. This effect is completely prevented by the tested anti-GDF-15 antibodies. It is therefore expected that the anti-GDF-15 antibodies according to the present invention are capable of treating both cancer-induced weight loss and cancer cachexia.

[0260] Of note, the degree of weight loss did not correlate with the respective tumor size (r2=10-6). A correlation between tumor size and weight loss would not be expected if the prevention of weight loss was merely a secondary effect resulting from the inhibition of cancer growth and smaller tumor size. Thus, the absence of such a correlation indicates that the use of anti-GDF-15 antibodies according to the invention results in two unrelated treatment effects: -Inhibition of cancer growth, and - Prevention of weight loss as an additional effect that is unrelated to inhibition of cancer growth and is expected to reflect prevention of cancer cachexia. Despite their mechanistic independence, it was observed that these effects could occur simultaneously in the same animal.

[0261] In addition to assessing the average body weight of the mice, the food consumption of the mice was evaluated by pairwise comparison of the study groups (Table 1). Of note, the food consumption of the mice in the anti-GDF-15 antibody groups (B1-23, chimeric B1-23, and humanized B1-23-H1L5) was significantly higher than the food consumption of the mice in the group that did not receive the anti-GDF-15 antibody.

[0262] [Table 4]

[0263] Table 1: Comparative evaluation of food consumption between the various treatment groups. For the measured time intervals (days 17-20, 20-24, 24-27, 27-31, 31-34), the mean of food consumption per mouse and day was calculated for each respective group. Values ​​are shown together with their standard deviations.

[0264] The quality of the humanized anti-GDF-15 antibody B1-23-H1L5 used in this study was tested by using gel electrophoresis and Coomassie staining of the antibody (see FIG. 6). Of note, the bands of the humanized anti-GDF-15 antibody B1-23-H1L5 were sharp and clear, whereas the bands of the mouse B1-23 anti-GDF-15 antibody and the B12 control antibody appeared less sharp and of higher molecular weight. This suggests that the humanized anti-GDF-15 antibody B1-23-H1L5 is not prone to aggregation, and that the significant aggregation could be shifted to higher values ​​from the molecular weight of the other antibodies.

[0265] In addition, by using colorimetric analysis, it was confirmed that all anti-GDF-15 antibodies used in this study bound to GDF-15 in a concentration-dependent manner. Colorimetric ELISA experiments were performed to determine the binding of B1-23 antibody variants to GDF-15. B12 antibody served as an isotype antibody that did not bind to human GDF-15. Therefore, Maxisorp 96-well plates (Nunc) were coated with hrGDF-15 (25ng protein per well, 50μl volume) overnight at 4℃. The next day, the plates were washed to remove unbound protein (3 times with 150μl PBS 0.05% Tween®) and non-specific binding sites were blocked with 150μl PBS 1% BSA for 2 hours at room temperature.

[0266] Plates were washed again and different variants of B1-23 test antibody were applied (50 μl volume). To investigate the specificity of antibody binding, endpoint dilutions were performed starting from 333 ng / ml with serial dilutions of 1:3. As background control, PBS 1% BSA was applied. After 1 hour of binding at room temperature, wells were washed as above. As secondary antibody, HRP-conjugated anti-human IgG (Life technologies, 1:5000) was applied for 1 hour at room temperature. Wells were washed as above to remove unbound secondary antibody.

[0267] For detection, 50 μl of peroxide substrate (TMB 1:100 in 0.1 M sodium acetate pH 6) was added, followed by 10 min incubation and 50 μl of stop solution (2N H2SO4). As negative controls, wells without GDF-15 coating and wells without secondary antibody were included. For analysis, the optical density at 450 nm was quantified using an ELISA reader (Tecan Sunrise) and the corresponding Magellan software. In comparison with the B12 antibody, it was observed that the humanized H1L5 antibody, the chimeric B1-23 antibody and the mouse B1-23 antibody exhibited clear concentration-dependent binding to GDF-15.

[0268] Example 8 Determination of Kd value of anti-GDF-15 antibody The Kd values ​​of different anti-GDF-15 antibodies were compared using Surface Acoustic Wave (SAW) gold chip biosensor technology (SAW Instruments GmbH, Schwertberger Str. 16, D-53177 Bonn, Germany).

[0269] [Table 5]

[0270] The mouse antibody (B1-23) as well as the chimeric B1-23 antibody were present in purified form. The H1L5 humanized B1-23 antibody was serum-free CHO cell culture supernatant. The Kd value of mouse B1-23 deviates by 35-fold from the Kd value determined by Biacore® analysis (surface plasmon resonance).

[0271] This deviation - apart from the differences in the measurement methods - can be explained by the reduced availability of free mouse B1-23 antibody, since it was found that this antibody, as a mouse antibody, can form aggregates in that form. Therefore, the solution of mouse B1-23 antibody was stabilized by adding 0.2% BSA. Therefore, the availability of mouse B1-25 antibody could be reduced by the binding of the antibody to albumin, which could explain the differences in the affinity values ​​obtained by the various measurement methods. Compared to the mouse B1-23 antibody, the H1L5 humanized B1-23 antibody surprisingly showed no tendency to aggregate (see also Example 9 below).

[0272] In this assay, the chimeric B1-23 anti-GDF-15 antibody and the H1L5 humanized B1-23 antibody exhibited affinities for human GDF-15 that were approximately 2-fold and 5-fold higher than the affinity of the murine B1-23 anti-GDF-15 antibody, respectively. Thus, the chimeric B1-23 anti-GDF-15 antibody and the H1L5 humanized B1-23 antibody are high affinity antibodies.

[0273] Example 9 Aggregation studies of anti-GDF-15 antibodies To test the aggregation properties of anti-GDF-15 antibodies, antibody samples were shaken in microcentrifuge tubes at room temperature for 48 hours, and the tubes were then visually analyzed for agglutinated antibody properties.

[0274] In comparison with the murine B1-23 antibody, it was surprisingly observed that the H1L5 humanized B1-23 antibody showed no tendency to aggregate, even when the antibody was present only in phosphate-buffered saline (PBS) and in the absence of a stabilizing protein such as BSA.

[0275] Moreover, in freeze / thaw and dilution experiments, it was observed that the H1L5 humanized B1-23 antibody did not aggregate during any of the dilution steps or freeze / thaw cycles. In addition, the following experiment was performed (see Example 7).

[0276] 5 mg of antibodies (B1-23, Chimera B1-23 = "ChimB1-23", H1L5) were loaded onto a Proteus® Protein A column, eluted and collected with TRIS buffer at physiological pH. After elution, the quality of the purified antibodies was assessed by Coomassie Brilliant Blue gel analysis. The concentration of the eluted antibodies was measured photometrically as well as by Bradford assay (Roti-Quant, Carl Roth, Karlsruhe, Germany). All three antibody solutions (B1-23, ChimB1-23, H1L5) showed similar concentrations and were adjusted to 0.5 mg / ml. The antibodies were then concentrated 10-fold via spin columns (Centricon, MWCO 30). After this step, turbidity indicated the presence of a precipitate in the B1-23 containing sample, whereas ChimB1-23 and H1L5 showed no signs of aggregation. All concentrated eluates were then centrifuged at 1300 rpm for 5 min to precipitate antibody aggregates, and the remaining amount of soluble antibody was finally determined from the supernatant via a Bradford assay.

[0277] These properties of the antibody are expected to be advantageous for clinical formulation of the antibody.

[0278] G) Industrial Applicability The antibodies, antigen-binding portions thereof, pharmaceutical compositions and kits according to the invention can be industrially manufactured and sold as products for the claimed methods and uses (e.g. for treating cancer cachexia and cancer) in accordance with known standards for the manufacture of pharmaceuticals, thereby making the invention industrially applicable.

[0279] Preferred Aspects 1. A monoclonal antibody or antigen-binding portion thereof capable of binding to human GDF-15, wherein the heavy chain variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence at least 90% identical thereto, and the light chain variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence at least 85% identical thereto, and wherein the heavy chain constant domain comprises the amino acid sequence of SEQ ID NO:29, or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95% identical thereto, and the light chain constant domain comprises the amino acid sequence of SEQ ID NO:32, or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95% identical thereto.

[0280] 2. A monoclonal antibody or antigen-binding portion thereof capable of binding to human GDF-15, wherein the binding is to a conformational or discontinuous epitope on human GDF-15 consisting of the amino acid sequences of SEQ ID NO:25 and SEQ ID NO:26, wherein the constant domain of the heavy chain comprises the amino acid sequence of SEQ ID NO:29 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95% identical thereto, and wherein the constant domain of the light chain comprises the amino acid sequence of SEQ ID NO:32 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95% identical thereto.

[0281] 3. The antibody or antigen-binding portion thereof according to item 1 or 2, wherein the constant domain of the heavy chain comprises the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence at least 98%, preferably at least 99% identical thereto, and the constant domain of the light chain comprises the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence at least 98%, preferably at least 99% identical thereto. 4. The antibody or antigen-binding portion thereof according to any one of items 1 to 3, wherein the heavy chain constant domain comprises the amino acid sequence of SEQ ID NO: 29 and the light chain constant domain comprises the amino acid sequence of SEQ ID NO: 32.

[0282] 5. The antibody or antigen-binding portion thereof according to any one of items 1 to 4, wherein the antibody is a humanized antibody. 6. The antibody or antigen-binding portion thereof according to item 5, wherein all of the variable domains of the antibody are humanized variable domains. 7. The antibody or antigen-binding portion thereof according to any one of items 1 to 6, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably at least 99% identical thereto, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 31 or an amino acid sequence at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably at least 99% identical thereto.

[0283] 8. The antibody or antigen-binding portion thereof according to any one of items 1 to 7, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 28 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 31. 9. The antibody or antigen-binding portion thereof according to any one of items 1 to 8, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 27 and the light chain comprises the amino acid sequence of SEQ ID NO: 30.

[0284] 10. The antibody or antigen-binding portion thereof according to any one of items 1 to 4, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 34, or an amino acid sequence at least 75%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, even more preferably at least 99% identical thereto, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 37, or an amino acid sequence at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, even more preferably at least 99% identical thereto.

[0285] 11. The antibody or antigen-binding portion thereof according to item 10, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 34 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 37. 12. The antibody or antigen-binding portion thereof according to any one of items 1 to 11, wherein the heavy chain variable domain comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 3 and a CDR2 region comprising the amino acid sequence of SEQ ID NO: 4, and the light chain variable domain comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 6 and a CDR2 region comprising the amino acid sequence ser-ala-ser.

[0286] 13. The antibody or antigen-binding portion thereof according to any one of items 2 to 12, wherein the heavy chain variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence at least 90% identical thereto, and the light chain variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence at least 85% identical thereto. 14. The antibody or antigen-binding portion thereof according to any one of items 1 and 3 to 12, wherein the binding is to a conformational or discontinuous epitope on human GDF-15 consisting of the amino acid sequences of SEQ ID NO: 25 and SEQ ID NO: 26.

[0287] 15. The antibody or antigen-binding portion thereof according to any one of items 1 to 14, wherein the antibody has a size greater than 100 kDa, preferably greater than 110 kDa, more preferably greater than 120 kDa, even more preferably greater than 130 kDa, and most preferably greater than 140 kDa. 16. The antibody or antigen-binding portion thereof according to item 15, wherein the antibody is a full-length antibody. 17. The antibody or antigen-binding portion thereof according to item 16, wherein the antibody is a full-length IgG antibody, preferably a full-length IgG1 antibody.

[0288] 18. The antibody or antigen-binding portion thereof according to any one of items 1 to 17, wherein the antibody has an Fc portion capable of binding to an Fc receptor. 19. The antibody or antigen-binding portion thereof according to any one of items 1 to 18, wherein the human GDF-15 is recombinant human GDF-15 having the amino acid sequence set forth in SEQ ID NO:8. 20. An antibody or antigen-binding portion thereof according to any one of items 1 to 19 for use in medicine.

[0289] 21. An antibody or antigen-binding portion thereof according to any one of items 1 to 19 for use in a method for treating cancer cachexia in a mammal. 22. An antibody or antigen-binding portion thereof according to any one of items 1 to 19 for use in a method for treating cancer in a mammal. 23. The antibody or antigen-binding portion thereof according to any one of items 21 to 22, for use according to any one of items 21 to 22, wherein the method is a method for treating not only cancer but also cancer cachexia in the same mammal.

[0290] 24. The antibody or antigen-binding portion thereof according to any one of items 21 to 23, for use according to any one of items 21 to 23, wherein the mammal is a human patient. 25. The antibody or antigen-binding portion thereof according to items 21 or 23 to 24, for use according to items 21 or 23 to 24, wherein the method for treating cancer cachexia is a method for completely preventing or completely reversing cancer cachexia. 26. The antibody or antigen-binding portion thereof according to item 25, for use according to item 25, wherein the method for treating cancer cachexia is a method for preventing cancer cachexia altogether.

[0291] 27. The antibody or antigen-binding portion thereof according to item 25, for use according to item 25, wherein the method for treating cancer cachexia is a method for completely reversing cancer cachexia. 28. An antibody or an antigen-binding portion thereof according to any one of items 21 to 27 for use according to any one of items 21 to 27, wherein the method comprises: i) cancer, and ii) Cancer cachexia Only mammals suffering from both are treated. 29. The antibody or antigen-binding portion thereof according to any one of items 21 or 23 to 28, for use according to any one of items 21 or 23 to 28, wherein the method increases the body weight of the mammal compared to its body weight before the onset of cancer cachexia.

[0292] 30. The antibody or antigen-binding portion thereof according to item 29, for use according to item 29, wherein the increase in body weight of the mammal is at least 1.5%, preferably at least 2.5%, more preferably at least 5%, compared to its body weight before the onset of cancer cachexia. 31. The antibody or antigen-binding portion thereof according to any one of items 21 to 30, for use according to any one of items 21 to 30, wherein the mammalian cancer cells endogenously express GDF-15 and / or the mammalian cancer cells stimulate endogenous expression of GDF-15 in mammalian non-cancerous cells.

[0293] 32. The antibody or antigen-binding portion thereof according to any one of items 21 to 31, for use according to any one of items 21 to 31, wherein mammalian cancer cells endogenously express GDF-15. 33. The antibody or antigen-binding portion thereof according to any one of items 22 to 32, for use according to any one of items 22 to 32, wherein the method for treating cancer is a method comprising inhibiting cancer growth. 34. The antibody or antigen-binding portion thereof according to any one of items 22 to 33, for use according to any one of items 22 to 33, wherein the method for treating cancer comprises inducing the killing of cancer cells by NK cells and CD8+ T cells in a human patient.

[0294] 35. The antibody or antigen-binding portion thereof according to any one of items 21 to 34, for use according to any one of items 21 to 34, wherein the human patient has elevated levels of GDF-15 in serum prior to administration. 36. The antibody or antigen-binding portion thereof according to any one of items 21 to 35, for use according to any one of items 21 to 35, wherein the antibody or antigen-binding portion thereof is A) is the only component used in the method that is pharmacologic active against cancer, or B) In combination with one or more further ingredients that are pharma- ceutical active against cancer.

[0295] 37. The antibody or antigen-binding portion thereof according to any one of items 21 to 36, for use according to any one of items 21 to 36, wherein the cancer is selected from the group consisting of brain cancer including glioma, cancer of the nervous system, melanoma, lung cancer, lip and oral cavity cancer, liver cancer, leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, bladder cancer, cervical cancer, uterine cancer, testicular cancer, thyroid cancer, kidney cancer, gallbladder cancer, multiple myeloma, nasopharyngeal cancer, larynx cancer, pharynx cancer, tumors of the digestive tract including esophageal cancer, gastric cancer and colorectal cancer, pancreatic cancer, prostate cancer, ovarian cancer and breast cancer, preferably from the group consisting of melanoma, prostate cancer, breast cancer, brain cancer including glioma, colorectal cancer, gastric cancer, esophageal cancer and ovarian cancer, and most preferably melanoma.

[0296] 38. The antibody or antigen-binding portion thereof according to any one of items 21 to 37, for use according to any one of items 21 to 37, wherein prior to administration, the tumor or tumors formed by the cancer have higher levels of human GDF-15, preferably 1.2-fold higher, more preferably 1.5-fold higher, even more preferably 2-fold higher and most preferably 5-fold higher, compared to a control sample from the same patient obtained from a non-cancerous part of the tissue that is the primary site tissue of the cancer.

[0297] 39. The antibody or antigen-binding portion thereof according to item 38, for use according to item 38, wherein the antibody or antigen-binding portion thereof is combined with one or more further components pharmacologiciZably active against cancer, and the one or more further components pharmacologiciZably active against cancer are selected from the group consisting of alkylating agents; antimetabolites; alkaloids, taxanes; topoisomerase inhibitors; cytotoxic antibiotics; and radioisotopes.

[0298] 40. The antibody or antigen-binding portion thereof according to item 39, for use according to item 39, wherein the one or more further components pharma- ceutical active against cancer are selected from the group consisting of cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil and ifosfamide; azathioprine and mercaptopurine; vincristine, vinblastine, vinorelbine and vindesine, paclitaxel, docetaxel, etoposide and teniposide; irinotecan and topotecan; actinomycin, anthracyclines, doxorubicin, daunorubicin, valrubicin, idarubicin, epirubicin, bleomycin, plicamycin and mitomycin.

[0299] 41. A kit comprising the antibody or antigen-binding portion thereof described in any one of items 1 to 19. 42. The kit according to item 41, for use according to any one of items 21 to 40. 43. An expression vector comprising a nucleotide sequence encoding the antibody or antigen-binding portion thereof according to any one of items 1 to 19. 44. A cell line capable of producing the antibody or antigen-binding portion thereof according to any one of items 1 to 19.

[0300] 45. A monoclonal antibody, or antigen-binding portion thereof, capable of binding to human GDF-15, for use in a method for treating cancer cachexia in a mammal, wherein the heavy chain variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence at least 90% identical thereto, and the light chain variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence at least 85% identical thereto.

[0301] 46. ​​A monoclonal antibody or antigen-binding portion thereof capable of binding to human GDF-15, for use in a method for treating cancer cachexia in a mammal, wherein the binding is to a conformational or discontinuous epitope on human GDF-15 consisting of the amino acid sequences of SEQ ID NO:25 and SEQ ID NO:26. 47. The antibody or antigen-binding portion thereof according to item 45 or 46, for use according to item 45 or 46, wherein the method for treating cancer cachexia is a method for completely preventing cancer cachexia or completely reversing it.

[0302] 48. The antibody or antigen-binding portion thereof according to item 47, for use according to item 47, wherein the method for treating cancer cachexia is a method for preventing cancer cachexia altogether. 49. The antibody or antigen-binding portion thereof according to item 47, for use according to item 47, wherein the method for treating cancer cachexia is a method for completely reversing cancer cachexia. 50. An antibody or antigen-binding portion thereof according to any one of items 45 to 49, for use according to any one of items 45 to 49, wherein in the method iii) cancer, and iv) Cancer cachexia Only mammals suffering from both are treated.

[0303] 51. The antibody or antigen-binding portion thereof according to any one of items 49 to 50, for use according to any one of items 49 to 50, wherein the method increases the body weight of the mammal compared to its body weight before the onset of cancer cachexia. 52. The antibody or antigen-binding portion thereof according to item 51, for use according to item 51, wherein the mammal's body weight gain is at least 1.5%, preferably at least 2.5%, more preferably at least 5%, compared to its body weight before the onset of cancer cachexia.

[0304] 53. The antibody or antigen-binding portion thereof according to any one of items 45 to 52, for use according to any one of items 45 to 52, wherein the method is a method for treating not only cancer but also cancer cachexia in the same mammal. 54. The antibody or antigen-binding portion thereof according to any one of items 45 to 53, for use according to any one of items 45 to 53, wherein the antibody has a size greater than 100 kDa, preferably greater than 110 kDa, more preferably greater than 120 kDa, even more preferably greater than 130 kDa, and most preferably greater than 140 kDa.

[0305] 55. The antibody or antigen-binding portion thereof according to item 54, for use according to item 54, wherein the antibody is a full-length antibody. 56. The antibody or antigen-binding portion thereof according to item 55, for use according to item 55, wherein the antibody is a full-length IgG antibody. 57. An antibody or antigen-binding portion thereof according to any one of items 45 to 56, for use according to any one of items 45 to 56, wherein the antibody has an Fc portion capable of binding to an Fc receptor.

[0306] 58. The antibody or antigen-binding portion thereof according to any one of items 45 to 57, for use according to any one of items 45 to 57, wherein the mammalian cancer cells endogenously express GDF-15 and / or the mammalian cancer cells stimulate endogenous expression of GDF-15 in mammalian non-cancerous cells. 59. The antibody or antigen-binding portion thereof according to any one of items 45 to 58, for use according to any one of items 45 to 58, wherein mammalian cancer cells endogenously express GDF-15. 60. The antibody or antigen-binding portion thereof according to any one of items 45 to 59, for use according to any one of items 45 to 59, wherein the mammal is a human patient.

[0307] 61. The antibody or antigen-binding portion thereof according to any one of items 45 to 60, for use according to any one of items 45 to 60, wherein the human GDF-15 is recombinant human GDF-15 having the amino acid sequence represented by SEQ ID NO: 8. 62. The antibody or antigen-binding portion thereof according to any one of items 46 to 61, for use according to any one of items 46 to 61, wherein the heavy chain variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence at least 90% identical thereto, and the light chain variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence at least 85% identical thereto.

[0308] 63. The antibody or antigen-binding portion thereof according to any one of items 45 and 47 to 61, for use according to any one of items 45 and 47 to 61, wherein the binding is to a conformational or discontinuous epitope on human GDF-15 consisting of the amino acid sequences of SEQ ID NO: 25 and SEQ ID NO: 26. 64. The antibody or antigen-binding portion thereof according to any one of items 53 to 63, for use according to any one of items 53 to 63, wherein the method for treating cancer is a method comprising inhibiting cancer growth.

[0309] 65. The antibody or antigen-binding portion thereof according to any one of items 53 to 64, for use according to any one of items 53 to 64, wherein the method for treating cancer comprises inducing the killing of cancer cells by NK cells and CD8+ T cells in a human patient. 66. The antibody or antigen-binding portion thereof according to any one of items 45 to 65, for use according to any one of items 45 to 65, wherein the heavy chain variable domain comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 3 and a CDR2 region comprising the amino acid sequence of SEQ ID NO: 4, and the light chain variable domain comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 6 and a CDR2 region comprising the amino acid sequence ser-ala-ser.

[0310] 67. The antibody or antigen-binding portion thereof according to any one of items 45 to 66, for use according to any one of items 45 to 66, in which the constant domain of the heavy chain comprises the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95% identical thereto, and the constant domain of the light chain comprises the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence at least 85%, preferably at least 90%, more preferably at least 95% identical thereto.

[0311] 68. The antibody or antigen-binding portion thereof according to any one of items 45 to 67, for use according to any one of items 45 to 67, wherein the constant domain of the heavy chain comprises the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence that is at least 98%, preferably at least 99%, identical thereto, and the constant domain of the light chain comprises the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence that is at least 98%, preferably at least 99%, identical thereto.

[0312] 69. The antibody or antigen-binding portion thereof according to any one of items 45 to 68, for use according to any one of items 45 to 68, wherein the constant domain of the heavy chain comprises the amino acid sequence of SEQ ID NO: 29 and the constant domain of the light chain comprises the amino acid sequence of SEQ ID NO: 32. 70. The antibody or antigen-binding portion thereof according to any one of items 45 to 69, for use according to any one of items 45 to 69, wherein the antibody is a humanized antibody and all of the variable domains of the antibody are humanized variable domains.

[0313] 71. The antibody or antigen-binding portion thereof according to any one of items 45 to 70, for use according to any one of items 45 to 70, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably at least 99% identical thereto, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 31 or an amino acid sequence at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably at least 99% identical thereto.

[0314] 72. The antibody or antigen-binding portion thereof according to any one of items 45 to 71, for use according to any one of items 45 to 71, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 28 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 31. 73. The antibody or antigen-binding portion thereof according to any one of items 45 to 72, for use according to any one of items 45 to 72, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 27 and the light chain comprises the amino acid sequence of SEQ ID NO: 30.

[0315] 74. The antibody or antigen-binding portion thereof according to any one of items 45 to 69, for use according to any one of items 45 to 69, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence at least 75%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, even more preferably at least 99% identical thereto, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 37 or an amino acid sequence at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, even more preferably at least 99% identical thereto.

[0316] 75. The antibody or antigen-binding portion thereof according to item 74, for use according to item 74, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 34 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 37. 76. The antibody or antigen-binding portion thereof according to any one of items 45 to 66, for use according to any one of items 45 to 66, wherein the antibody is An antibody or antigen-binding portion thereof against human GDF-15 obtained from cell line B1-23 deposited at the German Collection of Microorganisms and Cell Cultures (DMSZ) under accession number DSM ACC3142.

[0317] 77. The antibody or antigen-binding portion thereof according to any one of items 45 to 75, for use according to any one of items 45 to 75, wherein the heavy chain variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO: 5, or the light chain variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO: 7. 78. The antibody or antigen-binding portion thereof according to any one of items 45 to 75 and 77, for use according to any one of items 45 to 75 and 77, wherein the heavy chain variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO: 5, and the light chain variable domain comprises a CDR3 region comprising the amino acid sequence of SEQ ID NO: 7.

[0318] 79. The antibody or antigen-binding portion thereof according to any one of items 45 to 69 and 77 to 78, for use according to any one of items 45 to 69 and 77 to 78, wherein the heavy chain variable domain comprises a region comprising the FR1, CDR1, FR2, CDR2 and FR3 regions and comprises the amino acid sequence of SEQ ID NO: 1 or a sequence with 95% identity thereto, and the light chain variable domain comprises a region comprising the FR1, CDR1, FR2, CDR2 and FR3 regions and comprises the amino acid sequence of SEQ ID NO: 2 or a sequence with 95% identity thereto.

[0319] 80. The antibody or antigen-binding portion thereof according to any one of items 45 to 69 and 77 to 79, for use according to any one of items 45 to 69 and 77 to 79, wherein the heavy chain variable domain comprises a region comprising the FR1, CDR1, FR2, CDR2 and FR3 regions and comprises the amino acid sequence of SEQ ID NO: 1 or a sequence with 98% identity thereto, and the light chain variable domain comprises a region comprising the FR1, CDR1, FR2, CDR2 and FR3 regions and comprises the amino acid sequence of SEQ ID NO: 2 or a sequence with 98% identity thereto.

[0320] 81. The antibody or antigen-binding portion thereof according to any one of items 45 to 80, for use according to any one of items 45 to 80, wherein the antibody or antigen-binding portion thereof has an equilibrium dissociation constant for human GDF-15 that is equal to or less than 20 nM, preferably less than 10 nM, more preferably less than 5 nM and most preferably between 0.1 nM and 2 nM. 82. The antibody or antigen-binding portion thereof according to any one of items 45 to 75 and 77 to 81, for use according to any one of items 45 to 75 and 77 to 81, wherein the antibody or antigen-binding portion thereof binds to the same epitope of human GDF-15 as the antibody against human GDF-15 obtained from the cell line B1-23 deposited at the German Collection of Microorganisms and Cell Cultures (DMSZ) under the accession number DSM ACC3142.

[0321] 83. The antibody or antigen-binding portion thereof according to any one of items 45 to 82, for use according to any one of items 45 to 82, wherein the human patient has elevated levels of GDF-15 in serum before administration. 84. The antibody or antigen-binding portion thereof according to any one of items 45 to 83, for use according to any one of items 45 to 83, wherein the antibody or antigen-binding portion thereof is A) is the only ingredient pharmacologic active against cancer used in the method, or B) In combination with one or more further ingredients that are pharma- ceutical active against cancer.

[0322] 85. The antibody or antigen-binding portion thereof according to any one of items 45 to 84, for use according to any one of items 45 to 84, wherein the cancer is selected from the group consisting of brain cancer including glioma, cancer of the nervous system, melanoma, lung cancer, lip and oral cavity cancer, liver cancer, leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, bladder cancer, cervical cancer, uterine cancer, testicular cancer, thyroid cancer, kidney cancer, gallbladder cancer, multiple myeloma, nasopharyngeal cancer, larynx cancer, pharynx cancer, tumors of the digestive tract including esophageal cancer, gastric cancer and colorectal cancer, pancreatic cancer, prostate cancer, ovarian cancer and breast cancer, preferably from the group consisting of melanoma, prostate cancer, breast cancer, brain cancer including glioma, colorectal cancer, gastric cancer, esophageal cancer and ovarian cancer, and most preferably melanoma.

[0323] 86. The antibody or antigen-binding portion thereof according to any one of items 45 to 85, for use according to any one of items 45 to 85, wherein prior to administration, the tumor or tumors formed by the cancer have higher levels of human GDF-15, preferably 1.2-fold higher, more preferably 1.5-fold higher, even more preferably 2-fold higher and most preferably 5-fold higher, compared to a control sample of the same patient obtained from a non-cancerous part of the tissue that is the primary site tissue of the cancer.

[0324] 87. The antibody or antigen-binding portion thereof according to item 84, for use according to item 84, wherein the antibody or antigen-binding portion thereof is combined with one or more further components pharmacologiciZably active against cancer, and the one or more further components pharmacologiciZably active against cancer are selected from the group consisting of alkylating agents; antimetabolites; alkaloids, taxanes; topoisomerase inhibitors; cytotoxic antibiotics; and radioisotopes.

[0325] 88. The antibody or antigen-binding portion thereof according to item 87 for use according to item 87, wherein the one or more further components pharma- ceutical active against cancer are selected from the group consisting of cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil and ifosfamide; azathioprine and mercaptopurine; vincristine, vinblastine, vinorelbine and vindesine, paclitaxel, docetaxel, etoposide and teniposide; irinotecan and topotecan; actinomycin, anthracyclines, doxorubicin, daunorubicin, valrubicin, idarubicin, epirubicin, bleomycin, plicamycin and mitomycin.

[0326] References [Table 6]

[0327] [Table 7]

[0328] [Table 8]

[0329] [Table 9]

Claims

1. A monoclonal antibody or antigen-binding portion thereof capable of binding to human GDF-15, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence at least 99% identical thereto, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 31 or an amino acid sequence at least 99% identical thereto; and the constant domain of the heavy chain comprises the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence at least 90% identical thereto, and the constant domain of the light chain comprises the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence at least 90% identical thereto; The antibody or antigen-binding portion thereof has an equilibrium dissociation constant for human GDF-15 that is less than 5 nM.

2. The antibody or antigen-binding portion thereof of claim 1, capable of inhibiting cancer growth in a mammal.

3. the constant domain of the heavy chain comprises the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence at least 95% identical thereto, and the constant domain of the light chain comprises the amino acid sequence of SEQ ID NO: 32, or an amino acid sequence at least 95% identical thereto; or the constant domain of the heavy chain comprises the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence at least 98% identical thereto, and the constant domain of the light chain comprises the amino acid sequence of SEQ ID NO: 32, or an amino acid sequence at least 98% identical thereto; or the constant domain of the heavy chain comprises the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence at least 99% identical thereto, and the constant domain of the light chain comprises the amino acid sequence of SEQ ID NO: 32, or an amino acid sequence at least 99% identical thereto; or The constant domain of the heavy chain comprises the amino acid sequence of SEQ ID NO: 29, and the constant domain of the light chain comprises the amino acid sequence of SEQ ID NO: 32; 3. An antibody or antigen-binding portion thereof according to claim 1 or 2.

4. An antibody or antigen-binding portion thereof according to any one of claims 1 to 3, wherein the antibody is a humanized antibody or all of the variable domains of the antibody are humanized variable domains.

5. the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:28 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:31; and / or the heavy chain comprises the amino acid sequence of SEQ ID NO: 27, and the light chain comprises the amino acid sequence of SEQ ID NO: 30; An antibody or antigen-binding portion thereof according to any one of claims 1 to 4.

6. the heavy chain variable domain comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO:3, a CDR2 region comprising the amino acid sequence of SEQ ID NO:4, and a CDR3 region comprising the amino acid sequence of SEQ ID NO:5; and 6. The antibody or antigen-binding portion thereof according to any one of claims 1 to 5, wherein the light chain variable domain comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 6, a CDR2 region comprising the amino acid sequence of ser-ala-ser, and a CDR3 region comprising the amino acid sequence of SEQ ID NO:

7.

7. The antibody or antigen-binding portion thereof according to any one of claims 1 to 6, wherein the binding is to a conformational or discontinuous epitope on human GDF-15 consisting of the amino acid sequences of SEQ ID NO: 25 and SEQ ID NO:

26.

8. An antibody or antigen-binding portion thereof according to any one of claims 1 to 7 for use in a method comprising: the method is for treating cancer cachexia and / or cancer in a mammal; and / or the method is for both treating cancer and treating cancer cachexia in the same mammal; and / or The antibody or antigen-binding portion thereof, wherein the mammal is a human patient.

9. A kit comprising an antibody or antigen-binding portion thereof according to any one of claims 1 to 7.

10. An expression vector comprising a nucleotide sequence encoding the antibody or antigen-binding portion thereof according to any one of claims 1 to 7.

11. A cell line capable of producing the antibody or antigen-binding portion thereof according to any one of claims 1 to 7.

12. A monoclonal antibody or antigen-binding portion thereof capable of binding to human GDF-15, the antibody or antigen-binding portion thereof has an equilibrium dissociation constant for human GDF-15 that is less than 5 nM; (i) the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence at least 99% identical thereto, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence at least 99% identical thereto; and / or (ii) the binding is to a conformational or discontinuous epitope on human GDF-15 consisting of the amino acid sequences of SEQ ID NO:25 and SEQ ID NO:26, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:28 or an amino acid sequence at least 99% identical thereto, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:31 or an amino acid sequence at least 99% identical thereto; The antibody or antigen-binding portion thereof for use in a method for treating cancer cachexia in a mammal.

13. The antibody or antigen-binding portion thereof of claim 12, capable of inhibiting cancer growth in a mammal.

14. In the method, i) cancer, and ii) Cancer cachexia and / or the method is for both treating cancer and treating cancer cachexia in the same mammal; and / or The mammal is a human patient.

14. An antibody or antigen-binding portion thereof according to claim 12 or 13.

15. The antibody or antigen-binding portion thereof according to any one of claims 12 to 14, wherein the human GDF-15 is recombinant human GDF-15 having the amino acid sequence represented by SEQ ID NO:

8.

16. the heavy chain variable domain comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO:3, a CDR2 region comprising the amino acid sequence of SEQ ID NO:4, and a CDR3 region comprising the amino acid sequence of SEQ ID NO:5; and 16. The antibody or antigen-binding portion thereof according to any one of claims 12 to 15, wherein the light chain variable domain comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 6, a CDR2 region comprising the amino acid sequence ser-ala-ser, and a CDR3 region comprising the amino acid sequence of SEQ ID NO:

7.

17. the constant domain of the heavy chain comprises the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence at least 90% or at least 95% identical thereto, and the constant domain of the light chain comprises the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence at least 90% or at least 95% identical thereto; or the constant domain of the heavy chain comprises the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence at least 98% identical thereto, and the constant domain of the light chain comprises the amino acid sequence of SEQ ID NO: 32, or an amino acid sequence at least 98% identical thereto; or the constant domain of the heavy chain comprises the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence at least 99% identical thereto, and the constant domain of the light chain comprises the amino acid sequence of SEQ ID NO: 32, or an amino acid sequence at least 99% identical thereto; or The constant domain of the heavy chain comprises the amino acid sequence of SEQ ID NO: 29, and the constant domain of the light chain comprises the amino acid sequence of SEQ ID NO: 32; An antibody or antigen-binding portion thereof according to any one of claims 12 to 16.

18. 18. The antibody or antigen-binding portion thereof according to any one of claims 12 to 17, wherein the antibody is a humanized antibody and all of the variable domains of the antibody are humanized variable domains.

19. 19. The antibody or antigen-binding portion thereof according to any one of claims 12 to 18, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 28 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:

31.

20. 20. The antibody or antigen-binding portion thereof according to any one of claims 12 to 19, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 27 and the light chain comprises the amino acid sequence of SEQ ID NO: 30.

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