Therapeutic inhibitors of GDF15 signaling

Abstract

The present invention relates to antibodies that bind to and inhibit the activity of glial cell line-derived neurotrophic factor family receptor alpha-like (GFRAL) protein. The present invention also relates to the GDF15-GFRAL signaling pathway as a therapeutic target for cachectic conditions and conditions involving reduced food intake and loss of muscle and fat mass.

Description

[Technical field]

[0001] The present invention relates to antibodies that bind to and inhibit the activity of glial cell line-derived neurotrophic factor family receptor alpha-like (GFRAL) protein. The present invention also relates to the GDF15-GFRAL signaling pathway as a therapeutic target for cachectic conditions and conditions involving reduced food intake and loss of muscle and fat mass. [Background technology]

[0002] A cell surface receptor known as "glial cell line-derived neurotrophic factor family receptor alpha-like" is encoded by the GFRAL gene, which is located on human chromosome 6p12.1 and has nine exons that code for a sequence of 394 amino acids.

[0003] In the developing mouse brain, Gfral mRNA levels in the cerebral cortex and hippocampus have been shown to reach a maximum at birth and then decrease, suggesting that it may be involved in neuroprotection and brain development [1]. In adult mice, Gfral transcripts are found primarily in the central nervous system. Its expression has been found to be restricted to certain neurons in the brainstem, particularly the occipital cortex (AP) and the nucleus tractus solitariusu (NTS). Subsequently, Gfral expression was also detected at low levels in the testis and adipose tissue [2, 3, 4, 5].

[0004] The ligand for GFRAL has been identified as the hormone growth and differentiation factor 15 (GDF15) [2, 3, 4, 5, 6]. Upon interaction with GDF15, the GDF15-GFRAL complex interacts with the tyrosine protein kinase receptor RET and induces cell signaling through activation of the MAPK, AKT, and PLC-γ signaling pathways. This signaling has been shown to have downstream effects in regulating food intake, energy expenditure, and body weight [2, 3, 4, 5]. The active signaling complex is an assembly of six polypeptides, including a homodimer of GDF15-GFRAL-RET.

[0005] GDF15 is a distant member of the TGF-β family. It is a stress-inducible cytokine that can be upregulated by tissue injury [7], ionizing radiation [8], hypoxia [9], inflammation

[10] , chemical toxins [42, 43], and a variety of disease states, including cancer

[11] , rheumatoid arthritis

[12] , chronic renal failure

[13] , cardiovascular disease

[14] , obesity, and diabetes

[15] . It is also induced by tissue-damaging toxins such as chemotherapeutic agents [16, 17]. GDF15 is a stress-responsive cytokine that reduces appetite / food intake, as demonstrated in mouse

[18] and non-primate models [4]. Circulating levels of GDF15 have been reported to be elevated in a wide range of conditions, including many disease states [42, 44]. GDF15 is thought to represent a “sentinel” hormone response to cellular stress, and its protective actions include limiting systemic exposure to recently ingested toxins (e.g., through vomiting) and promoting the avoidance of future exposure to agents that previously led to cellular stress, through the induction of conditioned aversion

[42] .

[0006] High levels of GDF15 are associated with cachexia in some cancer patients. The presence of cancer in the body may increase the levels of GDF15 and promote cancer cachexia. Cachexia is a multifactorial disease characterized by weight loss due to loss of skeletal muscle and adipose tissue, imbalance in metabolic regulation, and reduced food intake

[19] . Cachexia is estimated to affect up to 74% of patients with many types of cancer worldwide, with the highest incidence in head and neck, pancreatic, gastric, and liver cancers

[20] . Cancer cachexia not only negatively impacts the quality of life of cancer patients [21, 22], but also reduces the efficacy of anticancer chemotherapy [23, 24] and increases its toxicity [25, 26, 27], leading to increased cancer-related mortality [27, 28, 29], and increased expenditure of medical resources. A wide range of diseases other than cancer are also associated with cachexia, including pulmonary and cardiac conditions (e.g., congestive heart failure, chronic obstructive pulmonary disease), as well as chronic kidney disease, acquired immune deficiency syndrome (AIDS), and advanced stages of cystic fibrosis, multiple sclerosis, motor neuron disease, Parkinson's disease, dementia, tuberculosis, multiple system atrophy, mercury poisoning, Crohn's disease, rheumatoid arthritis, and celiac disease.

[0007] In cachexia animal models, administration of monoclonal antibodies against GDF15 led to increased food intake, improved motor function and energy expenditure, and could reverse weight loss and restore skeletal muscle [30, 31]. Blocking the GDF15-GFRAL pathway reversed weight loss caused by cancer cachexia and extended survival

[30] . An anti-GFRAL antagonist antibody that inhibits the RET signaling complex in brainstem neurons was reported to reverse excessive lipid oxidation and prevent cancer cachexia when injected into tumor-bearing mice

[32] . In 2019, a clinical trial was initiated in the treatment of cancer anorexia / cachexia syndrome using the anti-GFRAL monoclonal antibody NGM120 (NCT04068896).

[0008] Gfral - / -("knockout") mice appear normal weight on a chow diet, similar to wild-type mice, and become obese on a high-fat diet. Some reports indicate that Gfral / GFRAL knockout mice become slightly heavier than wild-type mice on a high-fat diet. Wild-type mice treated with GDF15 show a significant reduction in food intake and a sustained loss of body weight compared to vehicle-treated mice, whereas Gfral - / - Mice are resistant to the effects of GDF15 [3, 4], indicating the suppression of weight gain by GDF15-GFRAL signaling. Moreover, overexpression of GDF15 has been shown to protect mice from the development of obesity and improve glucose tolerance on a high-fat diet [11, 33]. In mice or rats fed a chow or high-fat diet, GDF15 administration reduces food intake and body weight. Spontaneously obese cynomolgus monkeys exhibit reduced food intake after 4 weeks of exposure to recombinant HAS-GDF15, resulting in a significant loss of body weight [2, 3, 4, 5, 6]. In a rat model, serum GDF15 levels were positively correlated with tumor volume and negatively correlated with food intake, indicating that GDF15 induces anorexia through nausea and vomiting [34, 35].

[0009] There are also reports that the GDF15-GFRAL pathway is highly associated with hyperemesis gravidarum

[36] . Thus, the signal transduction induced by GDF15-GFRAL-RET complex regulates food intake and body weight, and its activity plays an important role in conditions such as cancer cachexia.By inhibiting the activity of GDF15-GFRAL-RET complex, for example by inhibiting the formation of this complex on the cell surface, antagonists provide a potential route to the treatment of these and other conditions.The activation of the hypothalamic-pituitary-adrenal (HPA) axis by exogenous and endogenous GDF15 is described by Non-Patent Document 1, the contents of which are incorporated herein by reference. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Cimino et al., PNAS 118(27)2021 Summary of the Invention [Means for solving the problem]

[0011] The present invention provides antibodies against human GFRAL. The present inventors have generated and selected antibodies against GFRAL protein, which inhibit the activity of GFRAL with high potency and antagonize its downstream signaling. These antibodies show high affinity binding to GFRAL on the surface of GFRAL-expressing cells and inhibit GDF15-induced intracellular signaling in GFRAL-expressing cells. Binding of the antibodies to the GFRAL extracellular domain may inhibit the formation of the GDF15-GFRAL-RET complex, for example, by inhibiting the association of RET with GDF15-GFRAL. The present inventors have selected antibodies that show high affinity and high potency in assays with human GFRAL, combined with cross-reactivity against non-human GFRAL, for example mouse GFRAL.

[0012] Exemplary antibodies of the invention are designated QUEL-0101, QUEL-0201 and QUEL-0301. Additionally, related antibodies of the invention are designated QUEL-0102, QUEL-0103, QUEL-0104, QUEL-0105, QUEL-0302, QUEL-0303 and QUEL-0304. The present invention extends to anti-GFRAL binding molecules incorporating the antigen-binding sequence of QUEL-0101, QUEL-0201 or QUEL-0301, or any of QUEL-0102, QUEL-0103, QUEL-0104, QUEL-0105, QUEL-0302, QUEL-0303 or QUEL-0304, including their complementarity determining regions (CDRs), such as heavy chain CDRs (HCDRs) HCDR1, HCDR2 and / or HCDR3 and / or light chain CDRs (LCDRs) LCDR1, LCDR2 and / or LCDR3, and optionally the heavy and / or light chain variable (VH and / or VL) domains, and variants thereof. The binding molecule may comprise HCDR1, HCDR2 and / or HCDR3 of QUEL-0101, QUEL-0102, QUEL-0103, QUEL-0104, QUEL-0105, QUEL-0201, QUEL-0301, QUEL-0302, QUEL-0302, QUEL-0303 or QUEL-0304 in a polypeptide scaffold and / or may comprise LCDR1, LCDR2 and / or LCDR3 of QUEL-0101, QUEL-0102, QUEL-0103, QUEL-0104, QUEL-0105, QUEL-0201, QUEL-0301, QUEL-0302, QUEL-0303 or QUEL-0304 in a polypeptide scaffold. For example, the binding molecule comprises the HCDR3 of QUEL-0201, optionally the set of HCDRs of QUEL-0201, and optionally the HCDRs and LCDRs of QUEL-0201.

[0013] An embodiment of the invention includes an antibody comprising a VH domain and a VL domain, wherein the VH domain comprises a set of HCDRs, HCDR1, HCDR2 and HCDR3, and the VL domain comprises a set of LCDRs, LCDR1, LCDR2 and LCDR3, (i) the set of HCDRs is the QUEL-0101 set of HCDRs defined as HCDR1 being SEQ ID NO: 3, HCDR2 being SEQ ID NO: 4, and HCDR3 being SEQ ID NO: 5; (ii) the set of HCDRs is the QUEL-0201 set of HCDRs defined as HCDR1 being SEQ ID NO: 13, HCDR2 being SEQ ID NO: 14, and HCDR3 being SEQ ID NO: 15; or (iii) The set of HCDRs is the QUEL-0301 set of HCDRs defined as HCDR1 is SEQ ID NO: 23, HCDR2 is SEQ ID NO: 24, and HCDR3 is SEQ ID NO: 25.

[0014] Further embodiments of the invention include antibodies comprising a VH domain and a VL domain, wherein the VH domain comprises a set of HCDRs HCDR1, HCDR2 and HCDR3, and the VL domain comprises a set of LCDRs LCDR1, LCDR2 and LCDR3, wherein: (iv) the set of HCDRs is the QUEL-0102 set of HCDRs defined as HCDR1 is SEQ ID NO: 99, HCDR2 is SEQ ID NO: 100, and HCDR3 is SEQ ID NO: 101; (v) the set of HCDRs is the QUEL-0103 set of HCDRs defined as HCDR1 is SEQ ID NO: 107, HCDR2 is SEQ ID NO: 108, and HCDR3 is SEQ ID NO: 5; (vi) the set of HCDRs is the QUEL-0104 set of HCDRs defined as HCDR1 being SEQ ID NO: 107, HCDR2 being SEQ ID NO: 108, and HCDR3 being SEQ ID NO: 113; or (vii) The set of HCDRs is the QUEL-0105 set of HCDRs defined as HCDR1 is SEQ ID NO: 118, HCDR2 is SEQ ID NO: 108, and HCDR3 is SEQ ID NO: 119.

[0015] Further embodiments of the invention include antibodies comprising a VH domain and a VL domain, wherein the VH domain comprises a set of HCDRs HCDR1, HCDR2 and HCDR3, and the VL domain comprises a set of LCDRs LCDR1, LCDR2 and LCDR3, wherein: (viii) the set of HCDRs is the QUEL-0302 set of HCDRs defined as HCDR1 is SEQ ID NO: 23, HCDR2 is SEQ ID NO: 124, and HCDR3 is SEQ ID NO: 125; (ix) the set of HCDRs is the QUEL-0303 set of HCDRs defined as HCDR1 being SEQ ID NO: 133, HCDR2 being SEQ ID NO: 134, and HCDR3 being SEQ ID NO: 135; or (x) The set of HCDRs is the QUEL-0304 set of HCDRs defined as HCDR1 is SEQ ID NO: 133, HCDR2 is SEQ ID NO: 142, and HCDR3 is SEQ ID NO: 143.

[0016] The antibody of the invention may comprise a VH domain and a VL domain, The VH domain comprises a set of HCDRs, HCDR1, HCDR2 and HCDR3, and The VL domain comprises a set of LCDRs, LCDR1, LCDR2 and LCDR3, HCDR1 is SEQ ID NO:3, SEQ ID NO:99, SEQ ID NO:107 or SEQ ID NO:118; HCDR2 is SEQ ID NO:4, SEQ ID NO:100, or SEQ ID NO:108; HCDR3 is SEQ ID NO:5, SEQ ID NO:101, SEQ ID NO:113 or SEQ ID NO:119, and optionally LCDR1 is SEQ ID NO:8 LCDR2 is SEQ ID NO: 9; LCDR3 is SEQ ID NO:10 or SEQ ID NO:104.

[0017] The antibody of the invention may comprise a VH domain and a VL domain, the VH domain comprises the QUEL-0101 set of HCDRs: HCDR1 SEQ ID NO:3, HCDR2 SEQ ID NO:4 and HCDR3 SEQ ID NO:5; and The VL domain comprises the QUEL-0101 set of LCDRs: LCDR1 SEQ ID NO:8, LCDR2 SEQ ID NO:9 and LCDR3 SEQ ID NO:10.

[0018] In another embodiment, the antibody comprises a VH domain and a VL domain: the VH domain comprises the QUEL-0102 set of HCDRs: HCDR1 SEQ ID NO: 99, HCDR2 SEQ ID NO: 100 and HCDR3 SEQ ID NO: 101; The VL domain comprises the QUEL-0102 set of LCDRs: LCDR1 SEQ ID NO:8, LCDR2 SEQ ID NO:9 and LCDR3 SEQ ID NO:104.

[0019] In another embodiment, the antibody comprises a VH domain and a VL domain: the VH domain comprises the QUEL-0103 set of HCDRs: HCDR1 SEQ ID NO: 107, HCDR2 SEQ ID NO: 108 and HCDR3 SEQ ID NO: 5; The VL domain comprises the QUEL-0103 set of LCDRs: LCDR1 SEQ ID NO:8, LCDR2 SEQ ID NO:9 and LCDR3 SEQ ID NO:104.

[0020] In another embodiment, the antibody comprises a VH domain and a VL domain: the VH domain comprises the QUEL-0104 set of HCDRs: HCDR1 SEQ ID NO: 107, HCDR2 SEQ ID NO: 108 and HCDR3 SEQ ID NO: 113; The VL domain comprises the QUEL-0104 set of LCDRs: LCDR1 SEQ ID NO:8, LCDR2 SEQ ID NO:9 and LCDR3 SEQ ID NO:104.

[0021] In another embodiment, the antibody comprises a VH domain and a VL domain: the VH domain comprises the QUEL-0105 set of HCDRs: HCDR1 SEQ ID NO: 118, HCDR2 SEQ ID NO: 108 and HCDR3 SEQ ID NO: 119; The VL domain comprises the QUEL-0105 set of LCDRs: LCDR1 SEQ ID NO:8, LCDR2 SEQ ID NO:9 and LCDR3 SEQ ID NO:104. The antibody of the present invention may comprise the QUEL-0101 VH domain SEQ ID NO:2 or a variant thereof in which there are one or more amino acid modifications, and / or the QUEL-0101 VL domain SEQ ID NO:7 or a variant thereof in which there are one or more amino acid modifications. The amino acid modifications may be amino acid substitutions, deletions or insertions. Such modifications may optionally be in the variable domain framework outside the CDRs. For example, there may be one or two amino acid modifications in the VH and / or VL domain framework. The modifications may be conservative substitutions and / or represent germline lining of framework residues. Germline lining of an amino acid residue refers to restoring the amino acid residue to the residue present at that position encoded by the germline gene segment from which the variable domain was generated. Corresponding germline gene segments can be identified by comparing the variable domain sequences to human v, d and j (for VH) or human v and j (for VL) gene segments and identifying the closest match. Variants of the QUEL-0101 VH domain include VH domains having one or more modifications present in the QUEL-0102, QUEL-0103, QUEL-0104 or QUEL-0105 VH domains, e.g., VH domains present in one or more frameworks of these VH domains. The antibodies of the invention optionally comprise QUEL-0102 VH domain SEQ ID NO:98, QUEL-0103 VH domain SEQ ID NO:106, QUEL-0104 VH domain SEQ ID NO:112 or QUEL-0105 VH domain SEQ ID NO:117.

[0022] The VH domain is derived from the heavy chain v gene segment IGHV3-30 (e.g., IGHV3-30 *18) and the d gene segment and the heavy chain j gene segment, for example, IGHJ6 (e.g., IGHJ6 * 02) may be encoded by the nucleotide sequence produced by recombination with. Thus, it may contain a VH domain framework (the "IGHV3-30 IGHJ6 framework") produced by recombination of IGHV3-30 and IGHJ6. Optionally, the VH domain has been produced by recombination of IGHV3-30, IGHD3-10 and IGHJ6.

[0023] The antibody may contain a VH domain having at least 90% amino acid sequence identity, optionally at least 95%, at least 98% or at least 99% sequence identity with SEQ ID NO: 2 of the QUEL-0101 VH domain. The VH domain may contain or consist of SEQ ID NO: 2. The VH domain may contain or consist of the VH domain encoded by SEQ ID NO: 1 expressed in mammalian cells, such as CHO.

[0024] The VL domain may be encoded by the nucleotide sequence produced by recombination of the light chain v gene segment IGKV1-27 (e.g., IGKV1-27 * 01) and the light chain j gene segment, for example, a κj segment such as IGKJ4 (e.g., IGKJ4 * 01). Thus, it may contain a VL domain framework (the "IGKV1-27 IGKJ4 framework") produced by recombination of IGKV1-27 and IGKJ4.

[0025] The antibody may comprise a VL domain having at least 90% amino acid sequence identity, optionally at least 95%, at least 98% or at least 99% sequence identity to the QUEL-0101 VH domain SEQ ID NO: 7. The VL domain may comprise or consist of SEQ ID NO: 7. The VL domain may comprise or consist of a VL domain encoded by SEQ ID NO: 6 expressed in a mammalian cell, e.g. CHO.

[0026] An anti-GFRAL antibody of the invention may comprise the QUEL-0101 VH domain SEQ ID NO:2 and the QUEL-0101 VL domain SEQ ID NO:7.

[0027] In another embodiment, the QUEL-0102 VH domain comprises SEQ ID NO:98 and the QUEL-0102 VL domain comprises SEQ ID NO:103.

[0028] In another embodiment, the VH domain comprises SEQ ID NO:106 and the VH domain comprises SEQ ID NO:110.

[0029] In another embodiment, the VH domain comprises SEQ ID NO:112 and the VL domain comprises SEQ ID NO:115.

[0030] In another embodiment, the VH domain comprises SEQ ID NO:117 and SEQ ID NO:121.

[0031] The antibody of the invention may comprise a VH domain and a VL domain, The VH domain comprises the QUEL-0201 set of heavy chain complementarity determining regions (HCDRs) HCDR1 SEQ ID NO: 13, HCDR2 SEQ ID NO: 14 and HCDR3 SEQ ID NO: 15; and The VL domain comprises the QUEL-0201 set of light chain complementarity determining regions (LCDRs) LCDR1 SEQ ID NO:18, LCDR2 SEQ ID NO:19 and LCDR3 SEQ ID NO:20.

[0032] The antibody of the present invention may comprise QUEL-0201 VH domain SEQ ID NO: 12 or a variant thereof with one or more amino acid modifications, and / or QUEL-0201 VL domain SEQ ID NO: 17 with one or more amino acid modifications. The amino acid modifications may be amino acid substitutions, deletions or insertions. Such modifications may be in the variable domain framework outside the CDRs in some cases. For example, there may be one or two amino acid modifications in the VH and / or VL domain framework. The modifications may be conservative substitutions and / or represent germline lining of framework residues.

[0033] The VH domain is derived from the heavy chain v gene segment IGHV1-3 (e.g., IGHV1-3 * 01) and a d gene segment and a heavy chain j gene segment, e.g., IGHJ6 (e.g., IGHJ6 * 02). Thus, it may comprise a VH domain framework produced by recombination of IGHV1-3 and IGHJ6 ("IGHV1-3 IGHJ6 framework"). Optionally, the VH domain has been produced by recombination of IGHV1-3, IGHD5-18 and IGHJ6.

[0034] The antibody may comprise a VH domain having at least 90% amino acid sequence identity, optionally at least 95%, at least 98% or at least 99% sequence identity to the QUEL-0201 VH domain SEQ ID NO: 12. The VH domain may comprise or consist of SEQ ID NO: 12. The VH domain may comprise or consist of a VH domain encoded by SEQ ID NO: 11 expressed in a mammalian cell, e.g. CHO.

[0035] The VL domain is derived from the light chain v gene segment IGLV1-40 (e.g., IGLV1-40 *01) and a light chain j gene segment, e.g., IGLJ3 (e.g., IGLJ3 * 02)と(IGLV1-40)と(IGLV3)と(IGLV4)と(IGLV5)と(IGLV6)と(IGLV7)と(IGLV8)と(IGLV9)と(IGLV10)と(IGLV11) and (IGLV12)))))).

[0036] The antibody of the invention may comprise the QUEL-0201 VL domain SEQ ID NO: 17, or a variant thereof having one or more amino acid modifications. The antibody may comprise a VL domain having at least 90% amino acid sequence identity, optionally at least 95%, at least 98% or at least 99% sequence identity to the QUEL-0201 VH domain SEQ ID NO: 17. The VL domain may comprise or consist of SEQ ID NO: 17. The VL domain may comprise or consist of a VL domain encoded by SEQ ID NO: 16 expressed in a mammalian cell, e.g., CHO.

[0037] An anti-GFRAL antibody of the invention may comprise the QUEL-0201 VH domain SEQ ID NO:12 and the QUEL-0201 VL domain SEQ ID NO:17.

[0038] The antibody of the invention may comprise a VH domain and a VL domain, The VH domain comprises a set of HCDRs, HCDR1, HCDR2 and HCDR3, and The VL domain comprises a set of LCDRs, LCDR1, LCDR2 and LCDR3, HCDR1 is SEQ ID NO: 23 or SEQ ID NO: 133; HCDR2 is SEQ ID NO:24, SEQ ID NO:124, SEQ ID NO:134 or SEQ ID NO:142; HCDR3 is SEQ ID NO:25, SEQ ID NO:125, SEQ ID NO:135 or SEQ ID NO:143, and optionally LCDR1 is SEQ ID NO:28, SEQ ID NO:128 or SEQ ID NO:138; LCDR2 is SEQ ID NO: 29 or SEQ ID NO: 129; LCDR3 is SEQ ID NO:30, SEQ ID NO:130, SEQ ID NO:139 or SEQ ID NO:146.

[0039] The antibody of the invention may comprise a VH domain and a VL domain, The VH domain comprises the QUEL-0301 set of heavy chain complementarity determining regions (HCDRs) HCDR1 SEQ ID NO:23, HCDR2 SEQ ID NO:24 and HCDR3 SEQ ID NO:25; and The VL domain comprises the QUEL-0301 set of light chain complementarity determining regions (LCDRs) LCDR1 SEQ ID NO:28, LCDR2 SEQ ID NO:29 and LCDR3 SEQ ID NO:30.

[0040] In another embodiment, the antibody comprises a VH domain and a VL domain, wherein the VH domain comprises the QUEL-0302 set of HCDRs: HCDR1 SEQ ID NO:23, HCDR2 SEQ ID NO:124 and HCDR3 SEQ ID NO:125; and The VL domain comprises the QUEL-0302 set of LCDRs: LCDR1 SEQ ID NO:128, LCDR2 SEQ ID NO:129 and LCDR3 SEQ ID NO:130.

[0041] In another embodiment, the antibody comprises a VH domain and a VL domain, wherein the VH domain comprises the QUEL-0303 set of HCDRs: HCDR1 SEQ ID NO: 133, HCDR2 SEQ ID NO: 134, and HCDR3 SEQ ID NO: 135; and The VL domain comprises the QUEL-0303 set of LCDRs: LCDR1 SEQ ID NO:138, LCDR2 SEQ ID NO:29 and LCDR3 SEQ ID NO:139.

[0042] In another embodiment, the antibody comprises a VH domain and a VL domain, wherein the VH domain comprises the QUEL-0304 set of HCDRs: HCDR1 SEQ ID NO: 133, HCDR2 SEQ ID NO: 142 and HCDR3 SEQ ID NO: 143; and The VL domain comprises the QUEL-0304 set of LCDRs: LCDR1 SEQ ID NO:138, LCDR2 SEQ ID NO:29 and LCDR3 SEQ ID NO:146.

[0043] The antibody of the present invention may comprise the QUEL-0301 VH domain SEQ ID NO:22 or a variant thereof with one or more amino acid modifications, and / or the QUEL-0301 VL domain SEQ ID NO:27 with one or more amino acid modifications. The amino acid modifications may be amino acid substitutions, deletions or insertions. Such modifications may optionally be in the variable domain framework outside the CDRs. For example, there may be one or two amino acid modifications in the VH and / or VL domain framework. The modifications may be conservative substitutions and / or may represent germline lining of framework residues. Variants of the QUEL-0301 VH domain include VH domains with one or more modifications present in the QUEL-0302, QUEL-0303 or QUEL-0304 VH domain, for example modifications present in one or more frameworks of these VH domains. An antibody of the invention optionally comprises QUEL-0302 VH domain SEQ ID NO: 123, QUEL-0303 VH domain SEQ ID NO: 132 or QUEL-0304 VH domain SEQ ID NO: 141.

[0044] The VH domain is derived from the heavy chain v gene segment IGHV3-7 (e.g., IGHV3-7 * 01) and a d gene segment and a heavy chain j gene segment, e.g., IGHJ4 (e.g., IGHJ4 *02). Thus, it may comprise a VH domain framework produced by recombination of IGHV3-7 and IGHJ4 ("IGHV3-7 IGHJ4 framework"). Optionally, the VH domain has been produced by recombination of IGHV3-7, IGHD1-7 and IGHJ4. Alternatively, the VH domain has been produced by recombination of IGHV3-7, IGHD1-20 and IGHJ4.

[0045] The antibody may comprise a VH domain having at least 90% amino acid sequence identity, optionally at least 95%, at least 98% or at least 99% sequence identity to the QUEL-0301 VH domain SEQ ID NO: 22. The VH domain may comprise or consist of SEQ ID NO: 22. The VH domain may comprise or consist of a VH domain encoded by SEQ ID NO: 21 expressed in a mammalian cell, e.g. CHO.

[0046] The VL domain is derived from the light chain v gene segment IGLV1-44 (e.g., IGLV1-44 * 01) or IGLV1-47 (e.g., IGLV1-47 * 01) and a light chain j gene segment, e.g., IGLJ3 (e.g., IGLJ3 * The light chain v gene segment is encoded by a nucleotide sequence produced by recombination with a lambda j segment such as IGLV1-44 or IGLV1-47. In one embodiment, the light chain v gene segment is IGLV1-44. In another embodiment, the light chain v gene segment is IGLV1-47. Thus, it may comprise a VL domain framework produced by recombination of IGLV1-44 with IGLJ3 (the "IGLV1-44 IGLJ3 framework"). Alternatively, it may comprise a VL domain framework produced by recombination of IGLV1-47 with IGLJ3 (the "IGLV1-47 IGLJ3 framework").

[0047] The antibody may comprise a VL domain having at least 90% amino acid sequence identity, optionally at least 95%, at least 98% or at least 99% sequence identity to the QUEL-0301 VL domain SEQ ID NO: 27. The VL domain may comprise or consist of SEQ ID NO: 27. The VL domain may comprise or consist of a VL domain encoded by SEQ ID NO: 26 expressed in a mammalian cell, e.g., CHO.

[0048] An anti-GFRAL antibody of the invention may comprise the QUEL-0301 VH domain SEQ ID NO:22 and the QUEL-0301 VL domain SEQ ID NO:27.

[0049] In another embodiment, the VH domain comprises SEQ ID NO:123 and the VL domain comprises SEQ ID NO:127.

[0050] In another embodiment, the QUEL-0303 VH domain comprises SEQ ID NO:132 and the QUEL-0303 VL domain comprises SEQ ID NO:137.

[0051] In another embodiment, the VH domain comprises SEQ ID NO:141 and the VL domain comprises SEQ ID NO:145.

[0052] The anti-GFRAL antibody according to the present invention may compete with QUEL-0101, QUEL-0201 or QUEL-0301 for binding to human GFRAL. For example, it may compete with QUEL-0201 IgG comprising QUEL-0201 VH domain SEQ ID NO: 12 and QUEL-0201 VL domain SEQ ID NO: 17. Alternatively, it may compete with QUEL-0101 IgG comprising QUEL-0101 VH domain SEQ ID NO: 2 and QUEL-0101 VL domain SEQ ID NO: 7, and / or it may compete with QUEL-0301 IgG comprising QUEL-0301 VH domain SEQ ID NO: 22 and QUEL-0301 VL domain SEQ ID NO: 27. The ability of a binding molecule to compete with a reference molecule for binding to GFRAL may be determined in vitro, for example by surface plasmon resonance (SPR) in a sandwich assay.

[0053] An anti-GFRAL antibody according to the invention may be one that does not compete with GDF15 for binding to GFRAL. The ability of a binding molecule to compete with GDF15 for binding to GFRAL may also be determined by an SPR sandwich assay.

[0054] High affinity binding to GFRAL is advantageous. Anti-GFRAL antibodies preferably have an affinity (K) of 1 nanomolar (nM) or greater (i.e., 1 nM or less), given the limited expression of GFRAL protein. D ) binds to human GFRAL. Affinity can be determined by SPR, for example, as described in Example 2. K D can be 0.5 nM (500 picomolar, pM) or less, 400 pM or less, 300 pM or less, 200 pM or less, or 100 pM or less. D can be 50 pM or less, for example 10 pM or less. D can be in the range of about 100 pM, for example, 50 pM to 200 pM. D can be 5 pM or less, 4 pM or less, 3 pM or less, 2 pM or less, or 1 pM or less. Dcan be about 1 pM (e.g., 0.5 pM to 2 pM). In some embodiments, K D may be at least 0.1 pM. D may be at least 0.5 pM. D may be at least 1 pM, for example in the range of 1 pM to 1 nM.

[0055] Anti-GFRAL antibodies preferably bind to non-human GFRAL (e.g., mouse GFRAL, rat GFRAL and / or cynomolgus GFRAL) in addition to human GFRAL. Optimally, the antibodies are cross-reactive (affinity / potency within 10-fold) with mouse and cynomolgus GFRAL. Human GFRAL (Figure 1) and mouse GFRAL (Figure 2) share 70% amino acid sequence identity, while human and cynomolgus amino acid sequence identity is 94%. The degree of cross-reactivity of an antibody is determined by the K D Preferably, the antibody has an affinity for mouse GFRAL that is within 100-fold, within 10-fold, within 5-fold, or more preferably within 2-fold of its affinity for human GFRAL.

[0056] The anti-GFRAL antibody has an affinity (K D ) The affinity for mouse GFRALL is optionally 100 pM or less.

[0057] The anti-GFRAL antibodies described herein are inhibitors of GDF15 signal transduction. Specifically, they inhibit GDF15 signal transduction via GFRAL by binding to the GFRAL extracellular domain and inhibiting the formation of cell surface GDF15-GFRAL-RET signal transduction complex. The potency of inhibition by anti-GFRAL antagonist antibodies can be determined in an in vitro assay, such as ERK phosphorylation assay. This is a cell-based assay that determines the ability of a candidate antagonist molecule to inhibit RET signal transduction caused by the addition of GDF15. The potency can be quantified as IC50 in the assay. Preferably, the antibody inhibits GFRAL activation with a potency (IC50) of 15 nM or more (i.e., 15 nM or less) in ERK phosphorylation assay. Preferably, the IC50 is 10 nM or less, for example 5 nM or less.

[0058] Nucleic acids encoding the antibodies described herein are also provided, as are cells containing said nucleic acids. An in vitro host cell can contain the nucleic acid, which is optionally integrated into the cellular (e.g., genomic) DNA of the host cell or transiently transfected (e.g., plasmid DNA).

[0059] Anti-GFRAL antagonist antibodies are suitable for medical use. GFRAL-targeted antagonist antibodies that inhibit the signaling of hormone GDF15 through the GDF15-GFRAL-RET pathway represent potential therapeutic agents for conditions such as cachexia (e.g., cancer cachexia) and hyperemesis gravidarum. Inhibition of GDF15:GFRAL interaction by antagonist anti-GFRAL antibodies, as exemplified herein, inhibits the effect of GDF15 on food intake and body weight in vivo. Anti-GFRAL antibodies or nucleic acids encoding same can be administered to patients (e.g., for patients with cancer-related cachexia or anorexia) to increase food intake. Anti-GFRAL antibodies or nucleic acids encoding same can be used as adjunctive therapeutic agents in combination with other anti-cancer interventions, such as surgery, radiation therapy and / or administration of anti-neoplastic drugs. Herein, we show that antagonist anti-GFRAL antibodies can inhibit the production of corticosterone induced by GDF15 in mice. Corticosterone is a steroid hormone of the glucocorticoid class in mice. The human equivalent of corticosterone is cortisol. Plasma cortisol levels are elevated in response to various stressors. Cortisol suppresses the immune system, promotes protein catabolism, modulates lipolysis differentially in different adipose tissue sites, and promotes gluconeogenesis. Glucocorticoids are essential for life. However, prolonged excess of circulating levels of glucocorticoids can have deleterious effects, such as promoting muscle wasting. Cachexia is a condition characterized by a loss of lean and fat mass. In several conditions associated with cachexia, there is evidence for chronic activation of the hypothalamic pituitary adrenal (HPA) axis [37, 38, 39]. Given the known effects of glucocorticoids, it is likely that excessive activation of the HPA axis contributes to the loss of lean mass in conditions characterized by cachexia. The studies we present here suggest that blocking GDF15 signaling at its receptor GFRAL inhibits pathological activation of the HPA axis by elevated levels of GDF15, thereby reducing the detrimental effects of chronically elevated glucocorticoids on lean mass, including skeletal muscle.

[0060] Thus, a further aspect of the present invention relates to the treatment of conditions in which elevated levels of GDF15 promote the excessive activation of the hypothalamic pituitary adrenal axis (HPA), which leads to the harmful effects of pathologically elevated levels of circulating cortisol in patients, wherein the treatment comprises administering to the patient an inhibitor of GDF15 signaling. The inhibitor of GDF15 signaling can reduce circulating levels of cortisol (e.g., as measured in plasma or by assessing the excretion of free cortisol in urine).

[0061] These and other aspects and embodiments of the invention, including methods of producing antibodies, pharmaceutical compositions, and methods of treating patients, are described in more detail below. [Brief description of the drawings]

[0062] [Figure 1] Figure 3 shows the amino acid sequence of human GFRAL SEQ ID NO: 31, NCBI NP_997293.2, which contains 394 amino acids, with residues 1-18 representing the signal peptide. The boxed asparagine residues are N-linked glycosylation (GlcNAc) sites at N23, N50, N62, N67, N103 and N116. GFRAL contains two GDNF / GAS1 domains, formed by boxed residues 131-210 and residues 220-316, respectively. The region believed to be necessary for interaction with GDF15 is residues 149-228, shown in italics. The transmembrane domain spans residues 352-371, shown in bold. Polymorphic variants at residue 33, underlined, are present in the human population, with residues R occurring in approximately 62% of cases and C occurring in approximately 38% of cases according to the 1000 Genomes Project. Residue P386 in the cytoplasmic tail is also variable and may be S. [Diagram 2] Figure 3 shows the amino acid sequence of mouse GFRAL SEQ ID NO: 32, NCBI NP_995316.2. Annotation of the sequence structure uses the same convention as Figure 1. [Diagram 3] Shown is ΔF% plotted against log human GDF15 molar concentration in the ERK phosphorylation assay. [Figure 4] Fluorescence change of antagonist titration in an ERK phosphorylation assay with 4 nM (0.05 μg / ml) GDF15 is shown. [Diagram 5] Sensorgrams from SPR analysis of antibodies binding to human GFRAL: (A) QUEL-0101, (B) QUEL-0201, (C) QUEL-0301. [Figure 6] Sensorgrams from SPR analysis of antibodies bound to mouse GFRAL: (A) QUEL-0101, (B) QUEL-0201, and (C) QUEL-0301. [Figure 7] 7 shows the ability of anti-GFRAL antibodies to inhibit GDF15-induced loss of (A) food intake and (B) body weight. Data are from left to right for the following treatment groups: isotype control; low dose QUEL-0301; low dose QUEL-0201; high dose QUEL-0301; high dose QUEL-0201. [Figure 8] Data from Mouse Study 15 (MS15) on the validation of the GFRAL blocking antibody QUEL-0201 in mice. Percent change in (AA) food intake and (BB) body weight 24 hours after administration of control vehicle (left bar) or human recombinant GDF15 (right bar). Data are presented as mean ± SEM, n=6-8 per group. **p<0.01, ***p<0.001, ****p<0.0001 by ANOVA. [Figure 9] Data from Mouse Study 3 (MS3): Serum corticosterone levels in QUEL-0201- and IgG control-treated mice with (right bars) or without (left bars) 0.1 mg / kg human recombinant GDF15. Data are presented as mean ± SEM, n = 6-8 per group. **p<0.01, ***p<0.001, ****p<0.0001 by ANOVA. [Figure 10]MS3 data: Human GDF15 serum concentrations in QUEL-0201 and IgG groups treated with human recombinant GDF15. Data are presented as mean ± SEM, n=6-8 per group. **p<0.01, ***p<0.001, ****p<0.0001 by one-tailed Student's t-test. [Figure 11A] Figure 11A-D shows data from Example 8 in the following muscles: (A) tibialis anterior; (B) gastrocnemius; (C) extensor digitorum longus; (D) soleus. Relative fold change (normalized to β-2 microglobulin) in mRNA expression levels of Mafbx, Murf1, and Foxo1 in QUEL-0201- or IgG control-treated mice receiving 0.1 mg / kg human recombinant GDF15 (right bar, filled squares) or sham injection (left bar, open circles). Data are presented as mean ± SEM, n=4-5 per group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA and multiple comparisons. [Figure 11B] Figure 11A-D shows data from Example 8 in the following muscles: (A) tibialis anterior; (B) gastrocnemius; (C) extensor digitorum longus; (D) soleus. Relative fold change (normalized to β-2 microglobulin) in mRNA expression levels of Mafbx, Murf1, and Foxo1 in QUEL-0201- or IgG control-treated mice receiving 0.1 mg / kg human recombinant GDF15 (right bar, filled squares) or sham injection (left bar, open circles). Data are presented as mean ± SEM, n=4-5 per group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA and multiple comparisons. [Figure 11C]Figure 11A-D shows data from Example 8 in the following muscles: (A) tibialis anterior; (B) gastrocnemius; (C) extensor digitorum longus; (D) soleus. Relative fold change (normalized to β-2 microglobulin) in mRNA expression levels of Mafbx, Murf1, and Foxo1 in QUEL-0201- or IgG control-treated mice receiving 0.1 mg / kg human recombinant GDF15 (right bar, filled squares) or sham injection (left bar, open circles). Data are presented as mean ± SEM, n=4-5 per group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA and multiple comparisons. [Figure 11D] Figure 11A-D shows data from Example 8 in the following muscles: (A) tibialis anterior; (B) gastrocnemius; (C) extensor digitorum longus; (D) soleus. Relative fold change (normalized to β-2 microglobulin) in mRNA expression levels of Mafbx, Murf1, and Foxo1 in QUEL-0201- or IgG control-treated mice receiving 0.1 mg / kg human recombinant GDF15 (right bar, filled squares) or sham injection (left bar, open circles). Data are presented as mean ± SEM, n=4-5 per group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA and multiple comparisons. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0063] The role of GDF15-GFRAL-RET signaling in HPA axis activation Activation of the hypothalamic-pituitary-adrenal (HPA) axis, which leads to increased circulating levels of glucocorticoids, occurs in response to a wide range of stressful stimuli. Glucocorticoid hormones (mainly cortisol in humans) act on inflammation, metabolism, and blood vessels to help the organism survive life-threatening challenges

[40] . In response to infection, proinflammatory cytokines such as TNFα / β, IL-1, and IL-6 activate this axis

[41] .

[0064] In studies of cellular responses to chemical toxins, GDF15 has frequently been identified as one of the most highly upregulated genes [42, 43]. GDF15 is ubiquitously produced in the body, and circulating concentrations rapidly increase upon exposure to a wide range of stressors [42, 44]. Cisplatin is known to increase circulating levels of both corticosterone (the rodent equivalent of cortisol) [45, 46] and GDF15 [35, 35, 47]. Endoplasmic reticulum (ER) stress is mechanistically distinct from genotoxicity, and its action of increasing GDF15 expression and secretion is well established [48, 49]. Actions attributed to GDF15-GFRAL-RET signaling have been primarily focused on the control of food intake, anorexia, cachexia, vomiting, and conditioned aversion, as discussed in the Background section herein. These various actions are consistent with a role for GDF15 in signaling the presence of chemical threats to the organism, which may be mitigated by reducing the rate of exposure to or excretion of ingested toxins and facilitating their future avoidance.

[0065] As reported herein, we found that antagonism of GFRAL with anti-GFRAL antibodies (i) inhibited the effects of GDF15 on food intake and body weight in vivo, and (ii) at the same doses inhibited the increase in circulating glucocorticoid levels in response to GDF15. Thus, we show that inhibiting GFRAL inhibits neuroendocrine responses, including the effect of GDF15 to increase glucocorticoid levels. Anti-GFRAL counters the GDF15-induced increase in circulating glucocorticoids.

[0066] Inhibitors of GDF15 signaling Inhibitors of GDF15 signal transduction include molecules that inhibit the activation of GDF15-GFRAL-RET signal transduction complex. Inhibitors can inhibit the formation of this complex (e.g., by inhibiting the binding of GDF15 to GFRAL or inhibiting the binding of GDF15-GFRAL to RET) and / or its functional activity (e.g., by biasing the receptor to a less active conformation). Inhibition of GDF15 signal transduction is optionally measured in an in vitro assay, such as the ERK phosphorylation assay described herein. Other inhibitors of GDF15 signal transduction can reduce the level of GDF15, GFRAL or RET, for example, by downregulating its expression or increasing its degradation. Inhibitors can downregulate GFRAL or RET by reducing its presence on the cell surface.

[0067] Inhibitors can optionally bind to GDF15, GFRAL or RET or the nucleic acid that encodes them.Inhibitors can optionally be small molecules, nucleic acids (e.g., inhibitory RNA), or binder polypeptides.Binder polypeptides are polypeptide molecules that have the ability to specifically bind and inhibit their target antigens (e.g., GDF15, GFRAL or RET).

[0068] Many classes of binder polypeptides are known in the art, including traditional IgG antibodies and other binding proteins based on immunoglobulin domains. Molecules that are not bound to immunoglobulins are also known, and binding loops can be engineered into other polypeptide scaffolds, such as fibronectin.

[0069] Preferably, the binder polypeptide of the invention comprises an immunoglobulin domain in which the binding site for the antigen is formed by a loop region of the immunoglobulin domain. A preferred embodiment of the binder polypeptide is an antibody. Examples of anti-GFRAL antibodies are described in detail herein.

[0070] Other GDF15 signaling inhibitors, including, for example, anti-GFRAL antibodies and anti-GDF15 antibodies, are disclosed in WO 2017 / 189724, WO 2017 / 172260, WO 2017 / 152105, WO 2017 / 147742, WO 2017 / 121865, WO 2017 / 055613, WO 2016 / 0 49470, WO 2015 / 144855, WO 2014 / 100689, WO 2014 / 049087, WO 2013 / 023557, WO 2013 / 012648, WO 2011 / 070177, WO 2005 / 099746, and WO 2005 / 072112.

[0071] therapeutic use Aspects of the invention include: Methods of treating patients with inhibitors of GDF15 signaling, an inhibitor of GDF15 signaling for use in treating a patient; and The use of an inhibitor of GDF15 signaling for the manufacture of a medicament for treating a patient is included.

[0072] The inhibitors of GDF15 signaling described herein, such as anti-GFRAL antibodies, can be used to treat any medical condition associated with excessive activation of the GDF15-GFRAL pathway. Examples of such conditions are described herein and below. (i) Cachexia (loss of appetite and loss of body weight, including fat and lean mass, including skeletal muscle), due to a wide range of conditions including cancer, heart failure, kidney failure, chronic and chronic lung disease, and rare genetic disorders such as mitochondrial myopathies and thalassemias. (ii) Neuroendocrine dysfunction due to high levels of GDF15 in all of the conditions listed in (i) above (iii) Nausea and vomiting as a result of drugs that rapidly increase GDF15, such as cancer chemotherapy (iv) Hyperemesis gravidarum Includes.

[0073] Inhibitors of GDF15 signaling in GFRAL can be used to treat any condition in which levels of GDF15 are acutely or chronically elevated.

[0074] Acute elevations of GDF15 occur in response to stimuli such as cytotoxic chemotherapy, acute exposure to ionizing radiation, or early pregnancy and are known to be associated with nausea, vomiting, and reduced physical activity, as well as activation of the hypothalamic pituitary adrenal axis. Blockade of GDF15 signaling in the GFRAL would be predicted to reduce all of these phenomena.

[0075] Chronically elevated GDF15 occurs in a wide range of conditions, including cancer, chronic heart failure, chronic respiratory disease, chronic renal failure, and various rare diseases such as thalassemia and thalassemia mitochondrial myopathy. In all of these conditions, elevated GDF15 is associated with chronic decreased appetite and food intake, decreased fat mass, and decreased muscle mass, all of which are collectively referred to as cachexia. In at least some forms of cachexia, there is evidence of chronic overactivation of the HPA axis. Given the known effects of cortisol on muscle protein breakdown, it is at least plausible that preventing overactivation of the HPA axis would have a beneficial effect on muscle mass loss.

[0076] Treatment may include prophylactic treatment, where the therapeutic composition is administered prior to the appearance of the pathology to be treated, to prevent or at least ameliorate the effects of the condition. Treatment may also be performed after the appearance of the condition, for example, treatment may be prescribed after the diagnosis of the condition. The above conditions may be side effects of other medical interventions, such as treatment with cytotoxic agents (e.g., anti-neoplastic agents used in the treatment of cancer) or radiation therapy.

[0077] Treatment may be directed toward suppressing elevated cortisol in patients who may have elevated circulating levels of cortisol, detectable in the blood or urine by sampling at different times of the day (e.g., by measuring 24-hour urinary free cortisol

[50] ).

[0078] Patients may have elevated physiological levels of GDF15 detectable in the blood. Human serum levels of GDF15 have been reported to be 150-1150 pg / mL in one study

[51] . Methods for measuring GDF15 from blood samples have been described, for example ELISA or sandwich immunoassays such as the Elecsys® GDF-15 immunoassay for the in vitro quantitative determination of GDF-15 in human serum and plasma.

[0079] The patient to be treated may be a cancer patient. As is clear from the published literature in the art, many types of cancer are associated with an increase in GDF15, promoting cancer cachexia. The cancer may be a solid tumor (possibly metastatic) or a blood cancer. The cancer may be bladder cancer, brain cancer, breast cancer, colorectal cancer, head and neck cancer, kidney cancer, lung cancer (e.g., non-small cell lung cancer), lymphoma (e.g., non-Hodgkin's lymphoma), melanoma, mesothelioma, neuroblastoma, esophageal cancer, oral or oropharyngeal cancer, gastrointestinal cancer (e.g., gastric cancer), pancreatic cancer, prostate cancer, testicular cancer, thyroid cancer or uterine cancer.

[0080] Alternatively, treatment may include treating cachexia in patients with pulmonary and / or cardiac conditions (e.g., congestive heart failure, chronic obstructive pulmonary disease), chronic kidney disease, acquired immune deficiency syndrome (AIDS), cystic fibrosis, multiple sclerosis, motor neuron disease, Parkinson's disease, dementia, tuberculosis, multiple system atrophy, mercury poisoning, Crohn's disease, rheumatoid arthritis, or celiac disease.

[0081] As a result of treatment, the condition may be alleviated, and an increase in food intake and / or a stabilization or increase in the patient's weight may be observed. For example, a patient may be treated for cancer-related cachexia, and treatment results in weight gain or reduced weight loss.

[0082] A patient treated according to the present invention may be a patient who has been exposed or will be exposed to a cytotoxic chemical. A patient treated according to the present invention may be a patient who has been exposed or will be exposed to ionizing radiation.

[0083] The cytotoxic agent may be an anti-neoplastic agent, examples of which are provided below and elsewhere herein. The treatment may include combination therapy in which the composition of the present invention is administered to a patient who is also undergoing treatment with an anti-neoplastic agent, such as cancer chemotherapy. The composition of the present invention may be administered before or after the anti-neoplastic agent, or may be administered simultaneously. Although generally administered in separate formulations, there is the possibility of a single pharmaceutical product containing both agents, if both agents can be formulated together. The treatment according to the present invention may enhance the effect of chemotherapy. The treatment may extend the patient's survival time. It is also expected to significantly improve the patient's quality of life.

[0084] The patient to be treated may be one who has undergone or will undergo treatment with one or more of the following cancer chemotherapeutic agents: Alkylating agents (e.g., bifunctional alkylating agents such as cyclophosphamide, mechlorethamine, chlorambucil, melphalan, or monofunctional alkylating agents such as dacarbazine, nitrosoureas, temozolomide); Anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, barbicin); Cytoskeletal disrupting agents (e.g., taxanes such as paclitaxel, docetaxel, Abraxane, and Taxotere) Epothilones (e.g., epothilone A, B, C, D, E, or F) Histone deacetylase inhibitors (e.g., vorinostat, romidepsin) Topoisomerase I inhibitors (e.g., irinotecan, topotecan, tafluposide) Topoisomerase II inhibitors (e.g., etoposide, teniposide, tafluposide) Kinase inhibitors (e.g., bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, vismodegib) Nucleotide analogues and precursor analogues (e.g., azacitidine, azathioprine, capecitabine, cytarabine, doxifluridine, fluorouracil, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, thioguanine) Peptide antibiotics (e.g., bleomycin, actinomycin) Platinum-based drugs (e.g., carboplatin, cisplatin, oxaliplatin) Retinoids (e.g., alitretinoin, bexarotene, tretinoin) Vinca alkaloids and derivatives (e.g., vinblastine, vincristine, vindesine, vinorelbine).

[0085] antibody The antibody according to the present invention is an immunoglobulin or a molecule containing an immunoglobulin domain, natural or partially or wholly synthetically produced. The antibody may be an IgG, IgM, IgA, IgD, or IgE molecule, or an antigen-specific antibody fragment thereof (including but not limited to Fab, F(ab')2, Fv, disulfide-linked Fv, scFv, single domain antibodies, closed conformation multispecific antibodies, disulfide-linked scfv, diabodies), derived from any species that naturally produces antibodies or produced by recombinant DNA technology; isolated from serum, B cells, hybridomas, transfectomas, yeast, or bacteria. The antibody may be humanized using conventional techniques. The term antibody encompasses any polypeptide or protein that contains an antibody antigen-binding site. The antigen-binding site (paratope) is the portion of an antibody that binds to and is complementary to the epitope of its target antigen, e.g., GFRAL.

[0086] The term "epitope" refers to the region of an antigen to which an antibody binds. Epitopes are defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have residues that are directly involved in the affinity of the interaction. Epitopes can also be conformational, i.e., composed of non-linear amino acids. In certain embodiments, epitopes can include antigenic determinants that are chemically active molecular surface groups such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, can have specific three-dimensional structural characteristics, and / or specific charge characteristics.

[0087] An antigen-binding site is a polypeptide or domain that contains one or more CDRs of an antibody and can bind to an antigen. For example, the polypeptide contains CDR3 (e.g., HCDR3). For example, the polypeptide contains CDR1 and 2 (e.g., HCDR1 and 2) or CDR1-3 (e.g., HCDR1-3) of the variable domain of an antibody.

[0088] The antigen-binding site of an antibody is provided by one or more antibody variable domains. In one example, the antibody binding site is provided by a single variable domain, such as a heavy chain variable domain (VH domain) or a light chain variable domain (VL domain). In another example, the binding site comprises a VH / VL pair, or two or more such pairs. Thus, the antigen-binding site of an antibody may comprise a VH and a VL.

[0089] An antibody can be a whole immunoglobulin, including the constant region, or it can be an antibody fragment. An antibody fragment is a portion of an intact antibody, including, for example, the antigen-binding region and / or the variable region of the intact antibody. Examples of antibody fragments include: (i) a Fab fragment, i.e., a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bond at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of a VH or VL domain; and (vi) an isolated complementarity determining region (CDR) that retains specific antigen-binding functionality; Examples include:

[0090] A further example of an antibody is the H2 antibody, which comprises a dimer of heavy chains (5'-VH-(optional hinge)-CH2-CH3-3') and lacks light chains.

[0091] Single chain antibodies (e.g., scFv) are commonly used fragments. Multispecific antibodies are formed from antibody fragments. The antibodies of the present invention may utilize any such format as desired.

[0092] Optionally, the binder polypeptide, or its antibody immunoglobulin domain, can be fused or conjugated to additional polypeptide sequences and / or to labels, tags, toxins, or other molecules.The binder polypeptide can be fused or conjugated to one or more different antigen-binding regions to provide a molecule that can bind to a second antigen in addition to GFRAL.For example, the antibody of the present invention can be a multispecific antibody, such as a bispecific antibody, that comprises (i) an antibody antigen-binding site for GFRAL, and (ii) an additional antigen-binding site that recognizes another antigen (optionally the antibody antigen-binding site described herein).

[0093] An antibody usually comprises an antibody VH and / or VL domain. The isolated VH and VL domains of an antibody are also part of the present invention. An antibody variable domain is a part of an antibody light chain and heavy chain, which comprises the amino acid sequences of the complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and framework regions (FRs). Thus, in each of the VH and VL domains, there are CDRs and FRs. The VH domain comprises a set of HCDRs, and the VL domain comprises a set of LCDRs. VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain. Each of the VH and VL typically comprises three CDRs and four FRs, arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. According to the method used in the present invention, the amino acid positions assigned to CDRs and FRs are defined according to the IMGT nomenclature.

[0094] The antibody may comprise an antibody VH domain comprising VH CDR1, CDR2 and CDR3 and a framework. The antibody may alternatively or additionally comprise an antibody VL domain comprising VL CDR1, CDR2 and CDR3 and a framework. Examples of antibody VH and VL domains and CDRs according to the invention are as listed in Table A. All VH and VL sequences, CDR sequences, sets of CDRs, and sets of HCDRs and sets of LCDRs disclosed herein are aspects and embodiments of the invention. As described herein, a "set of CDRs" comprises CDR1, CDR2 and CDR3. Thus, a set of HCDRs refers to HCDR1, HCDR2 and HCDR3, and a set of LCDRs refers to LCDR1, LCDR2 and LCDR3. Unless otherwise stated, a "set of CDRs" comprises HCDRs and LCDRs.

[0095] As described in more detail in the Examples, the inventors have isolated and characterized antibodies of particular interest, designated QUEL-0101, QUEL-0201 and QUEL-0301. Subsequently, structural variants of QUEL-0101 and QUEL-0301, respectively, namely QUEL-0102, QUEL-0103, QUEL-0104, QUEL-0105, QUEL-0302, QUEL-0303 and QUEL-0304, have been identified. In various embodiments of the present invention, unless the context dictates otherwise, the anti-GFRAL antibody may optionally be selected from QUEL-0101, QUEL-0102, QUEL-0103, QUEL-0104, QUEL-0105, QUEL-0201, QUEL-0301, QUEL-0302, QUEL-0303 and QUEL-0304. Optionally, selected from QUEL-0101, QUEL-0201 and QUEL-0301.

[0096] The present invention includes anti-GFRAL antibodies having the VH and / or VL domain sequences of these antibodies shown in the accompanying sequence listing, as well as antibodies comprising the HCDRs and / or LCDRs of these antibodies, and optionally the entire heavy chain amino acid sequences and / or the entire light chain amino acid sequences.

[0097] Where the VH or VL domain of an antibody comprises one or more residues in the framework regions that differ from the germline gene segment from which it was derived by recombination, the non-germline residues may be retained or mutated to different residues, e.g., the non-germline residues may be reverted to germline residues. The corresponding germline gene segment can be identified as the gene segment with which the sequences of the variable domains are most closely aligned, and the germline gene segments corresponding to the VH and VL domains of QUEL-0101, QUEL-0201 and QUEL-0301, respectively, and their corresponding related antibodies are set forth in Table G herein.

[0098] The antibody according to the invention may comprise one or more CDRs described herein, such as CDR3, and may optionally also comprise CDR1 and CDR2 to form a set of CDRs. The CDR or set of CDRs may be any of the CDRs or set of CDRs of QUEL-0101, QUEL-0102, QUEL-0103, QUEL-0104, QUEL-0105, QUEL-0201, QUEL-0301, QUEL-0302, QUEL-0303 and QUEL-0304.

[0099] The present invention provides an antibody that comprises the HCDR1, HCDR2, and / or HCDR3 of any of the antibodies QUEL-0101, QUEL-0102, QUEL-0103, QUEL-0104, QUEL-0105, QUEL-0201, QUEL-0301, QUEL-0302, QUEL-0303, and QUEL-0304, and / or the LCDR1, LCDR2, and / or LCDR3, such as a set of CDRs, of any of these antibodies. The antibody may comprise a set of VH CDRs of one of these antibodies. Optionally, the antibody may also comprise a set of VL CDRs of one of these antibodies, and the VL CDRs may be derived from the same or different antibody as the VH CDRs.

[0100] Also provided by the invention are VH domains which comprise a set of disclosed HCDRs, and / or VL domains which comprise a set of disclosed LCDRs.

[0101] As discussed further below, although either the VH or VL domain alone can be used to bind to an antigen, typically the VH domain is paired with the VL domain to generate the antigen-binding site of the antibody. The VH domain of QUEL-0201 can be paired with the VL domain of QUEL-0201, thus forming an antigen-binding site of the antibody that includes both the VH and VL domains of QUEL-0201. Similar embodiments are provided for other VH and VL domains disclosed herein. In other embodiments, the QUEL-0201 VH is paired with a different VL domain, e.g., a λ VL domain, e.g., the QUEL-0301 VL domain. Conversely, the QUEL-0301 VH can be paired with a different VL domain, e.g., a λ VL domain, e.g., the QUEL-0201 VL domain. Promiscuous pairing of light chains is well established in the art. For example: The QUEL-0101 VH domain can be paired with any of the VL domains of QUEL-0101, QUEL-0102, QUEL-0103, QUEL-0104 and QUEL-0105. The QUEL-0102 VH domain can be paired with any of the VL domains of QUEL-0101, QUEL-0102, QUEL-0103, QUEL-0104 and QUEL-0105. The QUEL-0103 VH domain can be paired with any of the VL domains of QUEL-0101, QUEL-0102, QUEL-0103, QUEL-0104 and QUEL-0105. The QUEL-0104 VH domain can be paired with any of the VL domains of QUEL-0101, QUEL-0102, QUEL-0103, QUEL-0104 and QUEL-0105. The QUEL-0105 VH domain can be paired with any of the VL domains of QUEL-0101, QUEL-0102, QUEL-0103, QUEL-0104 and QUEL-0105. The QUEL-0301 VH domain can be paired with any of the VL domains of QUEL-0301, QUEL-0302, QUEL-0303 and QUEL-0304. The VH domain of QUEL-0302 can be paired with the VL domain of any of QUEL-0301, QUEL-0302, QUEL-0303 and QUEL-0304. The QUEL-0303 VH domain can be paired with any of the VL domains of QUEL-0301, QUEL-0302, QUEL-0303 and QUEL-0304. The QUEL-0304 VH domain can be paired with any of the VL domains of QUEL-0301, QUEL-0302, QUEL-0303 and QUEL-0304.

[0102] An antibody may comprise one or more CDRs, e.g., a set of CDRs, within an antibody framework. The framework regions may be of human germline gene segment sequences. Thus, an antibody may be a human antibody with a VH domain that comprises a set of HCDRs in a human germline framework. Usually, an antibody may also have a VL domain that comprises a set of LCDRs, e.g., in a human germline framework. An antibody "gene segment", e.g., a VH gene segment, a D gene segment, or a JH gene segment, refers to an oligonucleotide having a nucleic acid sequence from which that portion of an antibody is derived, e.g., a VH gene segment is an oligonucleotide that comprises a nucleic acid sequence corresponding to a portion of FR1 to CDR3 of a polypeptide VH domain. Human V, D, and J gene segments are recombined to produce a VH domain, and human V and J segments are recombined to produce a VL domain. A D domain or D region refers to the diversity domain or region of an antibody chain. A J domain or J region refers to the joining domain or region of an antibody chain. Although somatic hypermutation may result in antibody VH or VL domains with framework regions that do not exactly match or align with the corresponding gene segments, sequence alignment can be used to identify the closest gene segments and thus which particular combination of gene segments a particular VH or VL domain is derived from. When aligning an antibody sequence with a gene segment, the amino acid sequence of the antibody is aligned with the amino acid sequence encoded by the gene segment, or the nucleotide sequence of the antibody is directly aligned with the nucleotide sequence of the gene segment.

[0103] The antibodies of the invention can be human antibodies or chimeric antibodies comprising human variable regions and non-human (e.g., murine) constant regions. Antibodies of the invention, for example, have human variable regions and, optionally, also have human constant regions.

[0104] Thus, the antibody optionally comprises a constant region or a portion thereof, such as the constant region or a portion thereof of a human antibody. For example, the VL domain may be attached at its C-terminus to an antibody light chain kappa or lambda constant domain. Similarly, the antibody VH domain may be attached at its C-terminus to all or a portion of an immunoglobulin heavy chain constant region (e.g., CH1 domain or Fc region) from any antibody isotype, such as IgG, IgA, IgE, and IgM, and any of the isotype subclasses, such as IgG1 or IgG4. A preferred example is IgG4PE, e.g., SEQ ID NO: 60. Further examples of human heavy chain constant region sequences are shown in Table C.

[0105] In a preferred embodiment, the anti-GFRAL antibody is (i) a heavy chain comprising the QUEL-0201 VH domain SEQ ID NO: 12 and a human IgG4 heavy chain constant region (e.g., IgG4PE SEQ ID NO: 60); and (ii) a light chain comprising the QUEL-0201 VL domain SEQ ID NO:17 and a human lambda constant region (e.g., having a sequence as shown in Table C); and QUEL-0201 IgG, which contains

[0106] In another preferred embodiment, the anti-GFRAL antibody is (i) a heavy chain comprising the QUEL-0301 VH domain SEQ ID NO:22 and a human IgG4 heavy chain constant region (e.g., IgG4PE SEQ ID NO:60); and (ii) a light chain comprising the QUEL-0301 VL domain SEQ ID NO:17 and a light chain consisting of a human lambda constant region (e.g., having a sequence as shown in Table C). and QUEL-0301 IgG, which contains

[0107] In another preferred embodiment, the anti-GFRAL antibody is (i) a heavy chain comprising the QUEL-0101 VH domain SEQ ID NO:2 and a human IgG4 heavy chain constant region (e.g., IgG4PE SEQ ID NO:60); and (ii) a light chain comprising the QUEL-0101 VL domain SEQ ID NO:7 and a human kappa constant region (e.g., having a sequence as shown in Table C). and QUEL-0101 IgG, which contains

[0108] The constant region of the antibody of the present invention may alternatively be a non-human constant region. For example, when the antibody is produced in a transgenic animal (examples of which are described elsewhere herein), a chimeric antibody is produced that contains a human variable region and a non-human (host animal) constant region. Some transgenic animals produce fully human antibodies. Others have been engineered to produce antibodies that contain a chimeric heavy chain and a fully human light chain. When the antibody contains one or more non-human constant regions, replacing them with human constant regions reduces their immunogenicity, and such replacement may be performed to produce antibodies that are more suitable for administration to humans as therapeutic compositions.

[0109] Digestion of an antibody with the enzyme papain produces two identical antigen-binding fragments, also known as "Fab" fragments, and an "Fc" fragment that has no antigen-binding activity but has the ability to crystallize. As used herein, "Fab" refers to a fragment of an antibody that contains one constant domain and one variable domain of each of the heavy and light chains. The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. "Fc fragment" refers to the carboxy-terminal portions of both H chains that are held together by disulfides. The effector functions of an antibody are determined by the sequence in the Fc region, the region that is also recognized by the Fc receptor (FcR) found on certain types of cells. Digestion of an antibody with the enzyme pepsin produces an F(ab')2 fragment, in which the two arms of the antibody molecule remain attached and contain two antigen-binding sites. The F(ab')2 fragment has the ability to cross-link antigens.

[0110] "Fv" as used herein refers to the minimum fragment of an antibody that retains both antigen recognition and binding sites. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain that are tightly, non-covalently or covalently associated. In this conformation, the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Taken together, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half an Fv, which contains only three antigen-specific CDRs) has the ability to recognize and bind antigen, although with a lower affinity than the entire binding site.

[0111] The antibodies disclosed herein are modified to increase or decrease serum half-life. In one embodiment, one or more of the following mutations are introduced to increase the antibody's biological half-life: T252L, T254S, or T256F. Biological half-life can also be determined by the CH mutation in the heavy chain constant region, as described in U.S. Patent No. 5,869,046 and U.S. Patent No. 6,121,022, the modifications of which are incorporated herein by reference. 1 The CH domain or CL region of the Fc region of IgG 2 In another embodiment, the Fc hinge region of the antibody or antigen-binding fragment of the invention is mutated to decrease the biological half-life of the antibody or fragment. One or more amino acid mutations are added to the CH of the Fc-hinge fragment. 2 -CH 3The antibody or fragment is introduced into the domain interface region, such that the antibody or fragment has impaired Staphylococcus aureus protein A (SpA) binding compared to native Fc-hinge domain SpA binding. Other methods of increasing serum half-life are known to those skilled in the art. Thus, in one embodiment, the antibody or fragment is PEGylated. In another embodiment, the antibody or fragment is fused to an albumin binding domain, such as an albumin-binding single domain antibody (dAb). In another embodiment, the antibody or fragment is PASylated (i.e., genetic fusion of a polypeptide sequence composed of PAS (XL-Protein GmbH), which forms an uncharged random coil structure with a large hydrodynamic volume). In another embodiment, the antibody or fragment is XTENylated / rPEGylated (i.e., genetic fusion of an inexact repeat peptide sequence (Amunix, Versartis) to a therapeutic peptide). In another embodiment, the antibody or fragment is ELPylated (i.e., genetic fusion to an ELP repeat sequence (PhaseBio)). These various half-life extending fusions are described in more detail in Strohl, BioDrugs (2015) 29:215-239, e.g., in Tables 2 and 6, which fusions are incorporated herein by reference.

[0112] Antibody constant region As mentioned above, antibodies are provided in various isotypes and with different constant regions. The Fc region of an antibody is recognized by Fc receptors and determines the ability of the antibody to mediate cellular effector functions, including antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP). These cellular effector functions involve recruiting Fc receptor-bearing cells to the site of target cells and killing the cells bound to the antibody.

[0113] In the context of the present invention, it is desirable to avoid cell effector functions such as ADCC, ADCP, and / or CDC. Thus, the antibodies according to the present invention may lack Fc effector functions, for example, the antibodies according to the present invention may have an Fc region that does not mediate ADCC, ADCP, and / or CDC, or they may lack an Fc region or lack the entire antibody constant region. The antibodies may have an effector-null constant region.

[0114] An antibody may have a heavy chain constant region that binds to one or more types of Fc receptor but does not induce cellular effector function, i.e., does not mediate ADCC, CDC, or ADCP activity. Such a constant region may not be capable of binding to a particular Fc receptor involved in triggering ADCC, CDC, or ADCP activity.

[0115] The antibody may have a heavy chain constant region that does not bind to Fcγ receptors, for example, the constant region may contain an "E" mutation, such as a Leu235Glu mutation (i.e., the wild type leucine residue is mutated to a glutamic acid residue), referred to as IgG4-E. Another optional mutation in the heavy chain constant region is Ser228Pro (a "P" mutation), which increases stability by reducing Fab arm exchange. The heavy chain constant region may be an IgG4 that contains both the Leu235Glu and Ser228Pro mutations. This "IgG4-PE" heavy chain constant region is effector null. An alternative effector null human constant region is a disabled IgG1.

[0116] IgG4PE is a preferred antibody isotype of the present invention. The binder polypeptide may be an IgG4PE antibody comprising the sequence of the IgG4PE constant region shown in Table C.

[0117] The antibody constant region is engineered to increase its half-life in vivo. Examples include the "YTE" mutation and other half-life extension mutations (Dall'Acqua, Kiener, and Wu, JBC 281(33):23514-23524, 2006, and WO02 / 060919, which are incorporated herein by reference). The triple mutation YTE is a substitution of three amino acids in the CH2 domain of IgG, which provide a tyrosine at residue 252, a threonine at residue 254, and a glutamic acid at residue 256, numbered according to the EU index of Kabat. As described in the referenced publications, the YTE modification increases the half-life of the antibody compared to the half-life of the corresponding antibody with a human CH2 wild-type domain. To increase in vivo shelf life, the antibodies of the invention can comprise an antibody constant region (e.g., an IgG constant region, e.g., an IgG CH2 domain) with one or more mutations that increase the half-life of the antibody compared to a corresponding wild-type human constant region (e.g., an IgG, e.g., an IgG CH2 domain). Half-life can be determined by standard methods, such as those described in WO02 / 060919.

[0118] Further examples of constant regions are shown in Table C.

[0119] Binding to GFRAL The primary structures of human and mouse GFRAL are shown in Figures 1 and 2, respectively.

[0120] A GDF15 signaling inhibitor, such as an anti-GFRAL antibody, may recognize an epitope within the extracellular domain of GFRAL. It may bind within the sequence of residues 19-351, as shown in Figure 1. For example, the epitope may include the N-terminal region of GFRAL, including residues upstream of the first GDNF / GAS1 domain.

[0121] The residues of GFRAL that bind to the antibody (i.e., the precise structural epitope) can be determined by structural analysis of the antibody:antigen complex, e.g., cryo-electron microscopy or X-ray crystallography. Binding residues include residues that form salt bridges, hydrophobic interactions or hydrogen bonds with the antibody through side chains or the polypeptide backbone. Epitopes may further include carbohydrate moieties on glycosylated antigens.

[0122] Anti-GFRAL antibodies may recognize epitopes of GFRAL that are the same as or overlap with the epitopes recognized by QUEL-0101, QUEL-0201, or QUEL-0301. Anti-GFRAL antibodies may, for example, bind to epitopes that include one or more, and possibly all, of the residues bound by QUEL-0101, QUEL-0201, or QUEL-0301. Recognition of these epitopes is associated with antagonist activity, i.e., inhibition of GDF15-GFRAL-RET signaling, and thus are valuable epitopes to target.

[0123] Competition between antibodies or other inhibitors can also be determined.For example, an anti-GFRAL binder can compete with an antibody (e.g., IgG or scFv) that contains the VH and VL domains of QUEL-0101, QUEL-0201 or QUEL-0301, or with an IgG that contains complete heavy and light chains.For example, it can compete with QUEL-0201 IgG.

[0124] Competition between binder polypeptides or other agents indicates that they have epitopes in the same region of GFRAL, e.g., they may both bind to the same domain with overlapping binding footprints, which can be confirmed by other techniques, e.g., structural analysis of the anti-GFRAL molecule bound to GFRAL, which can be accomplished, for example, using cryo-electron microscopy or X-ray crystallography as described above.

[0125] Competition is sometimes determined by a sandwich assay that evaluates the ability of two binders to bind to the GFRAL extracellular domain simultaneously in solution. In this assay, a first binder (e.g., anti-GFRAL antibody QUEL-0101, QUEL-0201, or QUEL-0301 IgG) is bound to a solid support, and a GFRAL antigen is added in solution, allowing the antibody-antigen (or other binder-antigen) complex to form. A test antibody or other anti-GFRAL binding molecule is then added in solution. If binding of the test molecule is detected, it indicates that it does not compete with the first binder (e.g., reference (QUEL) antibody). If binding of the test molecule is not detected, it indicates that it does not compete with the first binder (e.g., reference antibody) for binding to GFRAL. The assay can be performed using SPR, in which the first binder is bound to the surface of a biosensor chip. In the case of IgG, binding is generally via the Fc region, for example, using a chip coated with anti-Fc antibody. See Example 4 for further details of the SPR sandwich assay.

[0126] In some cases, the anti-GFRAL binder does not inhibit binding of GDF15 to GFRAL, which can be determined in a sandwich assay using the above principles, in which the binder is bound to a solid support, GFRAL antigen in solution is added, and then GDF15 binding or absence of binding is determined.

[0127] Affinity for binding The affinity of a binder (e.g., an anti-GFRAL antibody) for GFRAL can be quantified in terms of the equilibrium dissociation constant KD, which is the ratio Ka / Kd of the association or on-rate (Ka) and dissociation or off-rate (kd) of the binding interaction. The KD, Ka and Kd of antigen binding can be measured using surface plasmon resonance (SPR). Examples of SPR procedures and conditions are shown in Examples 2 and 3. Affinity (KD) is a measure of the strength of the interaction between the antibody and the antigen. The association rate (ka) indicates the rate at which the antigen is recognized. The dissociation rate (ka) is a measure of the stability of the binding. Taken together, kinetic data provide valuable information that can provide insight into biological activity, pharmacokinetics, and dosing regimens.

[0128] SPR involves coating or immobilizing (directly or indirectly) an anti-GFRAL binder onto a biosensor chip, exposing the binder to an antigen in a buffer at a range of concentrations, detecting binding, and determining the equilibrium dissociation constant, K D For IgG antibodies, binding to the chip can be conveniently performed via Fc capture on an anti-Fc coated chip (a chip with anti-human Fc antibodies on the chip surface, e.g., chemically immobilized on the chip surface). Binding data can be fitted to a 1:1 model using standard algorithms that can be specific to the instrument used. A variety of SPR instruments are known, such as Biacore™, ProteOn XPR36™ (Bio-Rad®), and KinExA® (Sapidyne Instruments, Inc.). Briefly, SPR can be performed at 25° C. by capturing binder at a concentration of 1 μg / ml on the chip for 60 seconds (e.g., about 80-140 RU can be captured), injecting soluble GFRAL (analyte) at 30 μL / min for 120 seconds (association time), and monitoring dissociation for 1200 seconds. The analyte may be injected in a dilution series (e.g., 100, 25, 6.25, 1.56, 0.39, 0.098, 0.024, 0 nM concentrations). A suitable running buffer is 1 mM CaCl 2The binder polypeptide sensorgrams are generated and the data are fitted to a 1:1 interaction model. D and optionally other kinetic data are calculated. The isolated and purified GFRAL extracellular domain can be conveniently used in assays and is a suitable analyte for SPR (see Example 2).

[0129] Species cross-reactivity for GFRAL binding To allow for therapeutic use or testing of anti-GFRAL antibodies in non-human animals, the antibodies must be cross-reactive with the corresponding antigen in the species of interest.

[0130] Preferably, the antibodies of the invention bind to mouse GFRAL, rat GFRAL and / or cynomolgus GFRAL, in addition to human GFRAL.

[0131] The degree of interspecies cross-reactivity of anti-GFRAL antibody or other anti-GFRAL binding molecule is expressed as the fold difference in affinity to one kind of antigen compared to the antigen of another kind, for example, the fold difference in affinity to human antigen versus mouse antigen.Affinity can be quantified as KD, which refers to the equilibrium dissociation constant of the binding of antigen to antigen-binding molecule.KD can be determined by SPR, as described elsewhere herein.

[0132] Certain cross-reactive binding molecules may have a fold difference in binding affinity between the human and non-human antigens of 100-fold or less, 50-fold or less, 30-fold or less, 25-fold or less, 20-fold or less, 15-fold or less, 10-fold or less, 5-fold or less, or 2-fold or less. In other words, the K that binds to the extracellular domain of a human antigen may be less than 100-fold, 50-fold or less, 30-fold or less, 25-fold or less, 20-fold or less, 15-fold or less, 10-fold or less, 5-fold or less, or 2-fold or less. D is the K for binding to the extracellular domain of a non-human antigen D It may be within 100 times, 50 times, 30 times, 25 times, 20 times, 15 times, 10 times, 5 times, or 2 times of the above.

[0133] Preferably, the binding affinity between the human and non-human antigens is within a 10-fold range, more preferably 5-fold or 2-fold. D is determined, for example, by SPR and is the K D It may be up to 10 times (preferably up to 5 times or up to 2 times) greater than or up to 10 times (preferably up to 5 times or up to 2 times) lower than.

[0134] In addition, the K D If the threshold is met, for example, the K D and K binding to non-human (e.g., mouse) antigens. D A binding molecule can be considered to be species cross-reactive when K is less than or equal to 10 mM, preferably less than or equal to 5 mM, and more preferably less than or equal to 1 mM. D may be 100 nM or less, 50 nM or less, 25 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less.

[0135] The binding molecule may have a measurable ability to inhibit GDF15 signaling in cell-based assays using GFRAL from multiple species (e.g., one or more of human and mouse, rat and cynomolgus monkey GFRAL, or all of them).It may show dose-dependent inhibition of GDF15-induced GFRAL signaling activity in assays described herein using human and non-human (e.g., mouse, rat or cynomolgus monkey) GFRAL, for example, ERK phosphorylation assay.

[0136] Although species cross-reactivity for different species antigen binding may be favorable, the selectivity of binder for GFRAL is still desirable to avoid undesirable side effects.Therefore, it is preferred that GFRAL is the only antigen that binds to the antigen-binding site of binder polypeptide in the body.Notwithstanding this, binder polypeptide can be optionally engineered to include additional binding sites, and the antibody that includes antibody constant region can, for example, optionally bind to one or more Fc receptors.

[0137] Inhibition of the GDF15-GFRAL-RET signaling complex The functional activity of inhibitors of GDF15 signaling, such as anti-GFRAL antibodies, can be tested in vitro. A suitable assay is the ERK phosphorylation assay. In this assay, cells co-expressing GFRAL and RET at the cell surface are incubated with GDF15, resulting in the formation of a GDF15-GFRAL-RET signaling complex and downstream signaling, including the phosphorylation of ERK. ERK phosphorylation can be quantified in lysed cells by detecting changes in fluorescence, for example, using HTRF. In the absence of inhibitor, the addition of GDF15 in a dilution series generates a sigmoidal curve (Figure 3). The effect of adding an inhibitor in this assay can be quantified by its effect on the change in fluorescence detected by HTRF. A person skilled in the art will include appropriate controls and calibrate the assay to determine the appropriate concentration of the reagent. For example, to determine the concentration of GDF15 sufficient to give a maximal response in the assay in the absence of GDF15 inhibitor, GFRAL / RET expressing cells can be exposed to a range of concentrations of GDF15 and the inhibition at each concentration can be determined before selecting the appropriate concentration. For example, the inventors found that a concentration of 4 nM produced a maximal response in the present experiments, and therefore selected 4 nM as the GDF15 concentration to use in this assay. Further details are provided in Example 1.

[0138] The inhibitor of GDF15 signal transduction can show dose-dependent inhibition of GDF15-induced GFRAL signal transduction activity, for example, in the ERK phosphorylation assay using human and / or non-human (e.g., mouse, rat or cynomolgus monkey) GFRAL.The ERK phosphorylation assay can be carried out using various concentrations of inhibitor to generate a dose-response curve from which IC50 value can be calculated.Therefore, the inhibitor according to the present invention can be identified by dose-dependent inhibition in such enzyme assay.

[0139] The potency of an inhibitor can be quantified as IC50.

[0140] For example, an anti-GFRAL antibody may have an IC50 of 15 nM or less in such an assay. The potency of a binder polypeptide, such as an anti-GFRAL antibody, may be compared to one or more anti-GFRAL antibodies described herein for reference. For example, an antibody comprising the VH and VL domains of QUEL-0101, QUEL-0201 or QUEL-0301 may be used as a reference antibody. The reference antibody is provided as an IgG. An inhibitor of GDF15 signaling, such as an anti-GFRAL antibody according to the present invention, may have an IC50 within 50% or within 10% of the IC50 of QUEL-0101, QUEL-0201 or QUEL-0301 IgG, or may have an IC50 lower than the IC50 of said reference antibody. "Within x% of" means that the IC50 of the binder polypeptide under test is x% or less than the IC50 of the reference antibody.

[0141] The anti-GFRAL antibody may have an IC50 in an ERK phosphorylation assay of 10 nM or less, and optionally 5 nM or less.

[0142] Production and modification of binder polypeptides Methods for identifying and producing binder polypeptides, including antibodies, are well known in the art.

[0143] For example, antibodies are generated using laboratory animals, such as mice, including transgenic mice (e.g., Kymouse®, Velocimouse®, Omnimouse®, Xenomouse®, HuMab Mouse®, or MeMo Mouse®), rats (e.g., Omnirat®), camelids, sharks, rabbits, chickens, or other non-human animals, that have been immunized with GFRAL or its encoding nucleic acid, and then optionally humanized in the constant and / or variable regions to produce human or humanized antibodies. In one example, display technologies such as yeast, phage, or ribosome display are used, as will be apparent to one of skill in the art. Standard affinity maturation, e.g., using display technologies, is performed as a further step after isolation of antibodies derived from the transgenic animals, phage display libraries, or other libraries. Representative examples of suitable techniques are described in US20120093818 (Amgen, Inc.), the entire contents of which are incorporated herein by reference, e.g., in the methods set out in paragraphs

[0309] to

[0346] .

[0144] There are many reasons why it may be desirable to create variants of a binder, including optimizing the polypeptide sequence for large-scale manufacturing, facilitating purification, enhancing stability, or improving suitability for inclusion in a desired pharmaceutical formulation. For example, protein engineering work is performed at one or more targeted residues of the antibody sequence, such as substituting one amino acid with an alternative amino acid (possibly generating variants containing all naturally occurring amino acids at this position, with possible exceptions of Cys and Met), and monitoring the effect on function and expression to determine the best substitution. It may not be desirable to substitute a residue with Cys or Met, or to introduce these residues into the sequence, since doing so may cause problems in manufacturing, for example, due to the formation of new intramolecular or intermolecular cysteine-cysteine ​​bonds. Once a lead candidate has been selected and optimized for manufacturing and clinical development, it will generally be desirable to change its antigen-binding properties as little as possible, or at least retain the affinity and potency of the parent molecule. However, variants can also be generated to modulate important antibody characteristics, such as affinity, cross-reactivity, or neutralization potency.

[0145] The antibody may comprise any set of H and / or L CDRs of the disclosed antibodies, including one or more amino acid mutations in the disclosed set of H and / or L CDRs. The mutations may be amino acid substitutions, deletions or insertions. Thus, for example, there may be one or more amino acid substitutions in the disclosed set of H and / or L CDRs. For example, there may be up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 mutations, e.g., substitutions, in a set of H and / or L CDRs. For example, there may be up to 6, 5, 4, 3 or 2 mutations, e.g., substitutions, in HCDR3 and / or up to 6, 5, 4, 3 or 2 mutations, e.g., substitutions, in LCDR3. The antibody may comprise a set of HCDRs, LCDRs, or a set of the six (H and L) CDRs set forth for any QUEL antibody herein, or may comprise that set of CDRs containing one or two conservative substitutions.

[0146] One or more amino acid mutations are optionally introduced into the framework regions of the antibody VH or VL domains disclosed herein. For example, one or more residues that differ from the corresponding human germline segment sequence are reverted to the germline. Human germline gene segment sequences corresponding to the VH and VL domains of exemplary anti-GFRAL antibodies are shown in Table G.

[0147] The antibody may comprise a VH domain having at least 60, 70, 80, 85, 90, 95, 98 or 99% amino acid sequence identity with the VH domain of any of the antibodies shown in the attached sequence listing, and / or a VL domain having at least 60, 70, 80, 85, 90, 95, 98 or 99% amino acid sequence identity with the VL domain of any of those antibodies. Algorithms that can be used to calculate the percent identity of two amino acid sequences include, for example, BLAST, FASTA, or the Smith-Waterman algorithm, for example using default parameters. Particular variants may contain one or more amino acid sequence alterations (addition, deletion, substitution and / or insertion of amino acid residues).

[0148] The modifications are made to one or more framework regions and / or one or more CDRs. The variants are optionally provided by CDR mutagenesis. The modifications do not usually result in loss of function, so that the antibody comprising the amino acid sequence modified in this way can retain the ability to bind to human GFRAL and / or mouse GFRAL. The antibody can retain the same quantitative binding ability as the antibody that is not modified, for example, as measured by the assay described herein. The antibody comprising the amino acid sequence modified in this way can have improved ability to bind to and / or inhibit human and / or mouse GFRAL.

[0149] The alterations may include replacing one or more amino acid residues with non-naturally occurring or non-standard amino acids, modifying one or more amino acid residues to non-naturally occurring or non-standard forms, or inserting one or more non-naturally occurring or non-standard amino acids into the sequence. Examples of the number and location of alterations in the sequences of the invention are described elsewhere in the present invention. Naturally occurring amino acids include the 20 "standard" L-amino acids, identified by the standard one-letter code as G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C, K, R, H, D, E. Non-standard amino acids include any other residue that may be incorporated into a polypeptide backbone or result from the modification of an existing amino acid residue. Non-standard amino acids may be naturally occurring or non-naturally occurring.

[0150] The term "variant" as used herein refers to a peptide or nucleic acid that differs from a parent polypeptide or nucleic acid by the deletion, substitution or addition of one or more amino acids or nucleic acids, but nevertheless retains one or more specific functions or biological activities of the parent molecule. Amino acid substitutions include alterations in which an amino acid is replaced with a different naturally occurring amino acid residue. Such substitutions are classified as "conservative", in which an amino acid residue contained in a polypeptide is replaced with another naturally occurring amino acid of similar characteristics, either in terms of polarity, side chain functionality or size. Such conservative substitutions are well known in the art. Substitutions encompassed by the present invention may also be "non-conservative", in which an amino acid residue present in a peptide is replaced with an amino acid having different properties, such as a naturally occurring amino acid from a different group (e.g., replacing a charged or hydrophobic amino acid with alanine), or alternatively, a naturally occurring amino acid is replaced with a non-conventional amino acid. In some embodiments, the amino acid substitutions are conservative. Also, when used with respect to a polynucleotide or polypeptide, a polynucleotide or polypeptide encompassed within the term variant refers to a polynucleotide or polypeptide that may differ in primary, secondary, or tertiary structure compared to a reference polynucleotide or polypeptide, respectively (e.g., compared to a wild-type polynucleotide or polypeptide).

[0151] In some embodiments, "synthetic variants," "recombinant variants," or "chemically modified" polynucleotide or polypeptide variants isolated or produced using methods well known in the art can be used. "Modified variants" may include conservative or non-conservative amino acid changes as described below. Polynucleotide changes can result in substitutions, additions, deletions, fusions, and truncations of amino acids in a polypeptide encoded by a reference sequence. Some embodiments use insertion variants, deletion variants, or substitution variants with amino acid substitutions, including insertions and substitutions of amino acids and other molecules that do not normally occur in the peptide sequence on which the variant is based, such as, but not limited to, the insertion of ornithine, which does not normally occur in human proteins. The term "conservative substitution," when describing a polypeptide, refers to a change in the amino acid composition of a polypeptide that does not substantially alter the activity of the polypeptide. For example, a conservative substitution refers to the substitution of an amino acid residue for a different amino acid residue with similar chemical properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.). Conservative amino acid substitutions include leucine with isoleucine or valine, aspartic acid with glutamic acid, or threonine with serine. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following six groups each contain amino acids that are conservative substitutions for each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine, (I), leucine, (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W). (See also Creighton, Proteins, WH Freeman and Company (1984), which is incorporated by reference in its entirety.) In some embodiments, individual substitutions, deletions or additions that change, add or delete a single amino acid or a small percentage of amino acids can also be considered "conservative substitutions" if the change does not reduce the activity of the peptide.Insertions or deletions typically range from about 1 to 5 amino acids. The selection of conservative amino acids is based on the position of the amino acid to be substituted in the peptide, for example, whether the amino acid is on the exterior of the peptide and exposed to the solvent, or on the interior and not exposed to the solvent.

[0152] The amino acid to be substituted for the existing amino acid can be selected based on the position of the existing amino acid, including exposure to solvent (i.e., whether the amino acid is exposed to solvent or is present on the outer surface of the peptide or polypeptide, compared to the amino acid that is not exposed to solvent and is located inside).The selection of such conservative amino acid substitutions is well known in the art, for example, as disclosed in Dordo et al., J. MoI Biol, 1999, 217, 721-739, and Taylor et al., J. Theor. Biol. 119(1986); 205-218, and S. French and B. Robson, J. Mol. Evol. 19(1983) 171. Thus, conservative amino acid substitutions suitable for the external amino acids of a protein or peptide (i.e., amino acids exposed to the solvent) can be selected, for example and without limitation, the following substitutions are used: substitution of Y with F, substitution of T with S or K, substitution of P with A, substitution of E with D or Q, substitution of N with D or G, substitution of R with K, substitution of G with N or A, substitution of T with S or K, substitution of D with N or E, substitution of I with L or V, substitution of F with Y, substitution of S with T or A, substitution of R with K, substitution of G with N or A, substitution of K with R, substitution of A with S, K or P.

[0153] In alternative embodiments, conservative amino acid substitutions that are appropriate for amino acids in the interior of a protein or peptide can also be selected for inclusion, for example, conservative substitutions appropriate for amino acids that are in the interior of a protein or peptide (i.e., amino acids not exposed to solvent) can be used, for example, but not limited to, the following conservative substitutions can be used: Y is substituted with F, T is substituted with A or S, I is substituted with L or V, W is substituted with Y, M is substituted with L, N is substituted with D, G is substituted with A, T is substituted with A or S, D is substituted with N, I is substituted with L or V, F is substituted with Y or L, S is substituted with A or T, and A is substituted with S, G, T or V. In some embodiments, non-conservative amino acid substitutions are also encompassed within the term variant.

[0154] The present invention includes methods of making antibodies containing VH and / or VL domain variants of the antibody VH and / or VL domains shown in Table A. Such antibodies include (i) the parent antibody VH domain is the VH domain of QUEL-0101, QUEL-0201 or QUEL-0301, or a VH domain comprising the heavy chain complementarity determining regions of any of these antibodies; providing an antibody VH domain which is an amino acid sequence variant of a parent antibody VH domain by adding, deleting, substituting or inserting one or more amino acids into the amino acid sequence of the parent antibody VH domain; (ii) optionally combining the VH domain thus provided with a VL domain to provide a VH / VL combination; (iii) testing the VH domains or VH / VL domain combinations thus provided to identify antibodies having one or more desired characteristics; The composition can be prepared by a method comprising the steps of:

[0155] The VH domain may be the VH domain of QUEL-0201.

[0156] Desired characteristics include binding to human and / or non-human GFRAL. Antibodies with similar or higher affinity to human and / or mouse GFRAL compared to the parent antibody are identified. Other desired characteristics include inhibition of GDF15 signaling assays described herein, such as ERK phosphorylation assays. Identification of antibodies with desired characteristics may include identification of antibodies with functional attributes described herein, such as their affinity, cross-reactivity, specificity, or neutralization potency, any of which can be determined by the assays described herein.

[0157] When a VL domain is included in the method, the VL domain may be the VL domain of any of QUEL-0101, QUEL-0201 or QUEL-0301, or may be a variant provided by addition, deletion, substitution or insertion of one or more amino acids into the amino acid sequence of the parent VL domain, the parent VL domain being the VL domain of any of QUEL-0101, QUEL-0201 or QUEL-0301, or a VL domain comprising the light chain complementarity determining region of any of those antibodies. The VL domain may be the VL domain of the same antibody as the VH domain. The VL domain may be the VL domain of QUEL-0201.

[0158] The method of generating a variant antibody may optionally include making a copy of the antibody or VH / VL domain combination. The method may further include expressing the resulting antibody. Nucleotide sequences corresponding to the desired antibody VH and / or VL domains may optionally be made in one or more expression vectors. Suitable methods of expression, including recombinant expression in a host cell, are described in detail herein.

[0159] Encoding nucleic acid and method of production An isolated nucleic acid encoding the antibody according to the present invention is provided. The nucleic acid may be DNA and / or RNA. Genomic DNA, cDNA, mRNA or other RNA, or any combination thereof, of synthetic origin, may encode the antibody.

[0160] The present invention provides constructs in the form of plasmids, vectors, transcription or expression cassettes that contain at least one polynucleotide as described above.Exemplary nucleotide sequences are included in the sequence listing.Reference to the nucleotide sequences described herein includes DNA molecules with the specified sequences, and includes RNA molecules with the specified sequences with U substituted for T, unless the context otherwise requires.

[0161] The present invention also provides a recombinant host cell comprising one or more nucleic acids encoding the antibody. The method of producing the encoded antibody may include, for example, expression from the nucleic acid by culturing a recombinant host cell containing the nucleic acid. The antibody may then be obtained, isolated and / or purified using any suitable technique, and then used as required. The production method may include formulating the product into a composition comprising at least one additional component, such as a pharma- ceutically acceptable excipient.

[0162] Systems for cloning and expression of polypeptides in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, plant cells, filamentous fungi, yeast and baculovirus systems, as well as transgenic plants and animals.

[0163] The expression of antibodies and antibody fragments in prokaryotic cells is well established in the art. A common bacterial host is Escherichia coli. Expression in eukaryotic cells in culture is also available to those skilled in the art as a production option. Mammalian cell lines available in the art for the expression of heterologous polypeptides include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, YB2 / 0 rat myeloma cells, human embryonic kidney cells (e.g., HEK293), human embryonic retina cells and many other cells.

[0164] The vector may contain appropriate control sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as necessary. The nucleic acid encoding the antibody is introduced into the host cell. The nucleic acid of the invention may be integrated into the genome (e.g., chromosome) of the host cell. Integration may be promoted by including sequences that promote recombination with the genome, according to standard techniques. The nucleic acid may be introduced into eukaryotic cells by a variety of methods, including calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, such as vaccinia or, for insect cells, baculovirus. Introduction of nucleic acids into host cells, particularly eukaryotic cells, may use virus or plasmid-based systems. The plasmid system may be maintained episomally or integrated into the host cell or artificial chromosomes. Integration may be by either random or targeted integration of one or more copies at single or multiple loci. For bacterial cells, suitable techniques include calcium chloride transformation, electroporation, and transfection using bacteriophage. Following introduction, the nucleic acid is expressed, for example by culturing the host cells under conditions for expression of the gene, and then the binder polypeptide, e.g., antibody, may optionally be isolated or purified.

[0165] Formulation and Administration According to the present invention, a composition is provided that comprises an inhibitor of GDF15 signal transduction, for example, an anti-GFRAL antibody.The composition is also provided in a form that includes a nucleic acid that codes for an inhibitor of GDF15 signal transduction, which is a binder polypeptide, for example, an antibody.Such a composition can be provided for use in the treatment of the human or animal body by therapy, including any of the exemplary medical treatments described herein.The composition can further comprise one or more pharma-ceutically acceptable excipients in addition to the active ingredient (inhibitor or coding nucleic acid).

[0166] The binder polypeptides according to the present invention, and their encoding nucleic acid molecules, will usually be provided in isolated form. The VH and / or VL domains, and nucleic acids, are provided purified from their natural environment or from the environment in which they are produced. Isolated binder polypeptides and isolated nucleic acids will be free or substantially free of materials with which they are naturally associated, such as other polypeptides or nucleic acids with which they are found in vivo, or the environment in which they are produced (if such production is by in vitro recombinant DNA techniques) (e.g., cell culture). Optionally, an isolated binder polypeptide or nucleic acid (1) is free of at least some other proteins with which it is normally found, (2) is essentially free of other proteins from the same source, e.g., the same species, (3) is expressed by cells from a different species, (4) is separated from at least about 50 percent of the polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated, (5) is operably associated (by covalent or non-covalent interactions) with polypeptides with which it is not naturally associated, or (6) is not naturally occurring.

[0167] Binder polypeptides or their coding nucleic acids are formulated with diluents or adjuvants, and even isolated for practical purposes. For example, they are mixed with carriers if used to coat microtiter plates for use in immunoassays, and mixed with pharma- ceutically acceptable carriers or diluents if used in therapy. As described elsewhere in the present invention, other active ingredients can also be included in the therapeutic product. Binder polypeptides are either naturally glycosylated in vivo or by heterologous eukaryotic systems such as CHO cells, or non-glycosylated (e.g., when produced by expression in prokaryotic cells). The present invention encompasses antibodies with modified glycosylation patterns.

[0168] Typically, an isolated product will comprise at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample. The binder polypeptide may be substantially free of proteins or polypeptides or other contaminants found in its natural or produced environment that would interfere with its therapeutic, diagnostic, prophylactic, research or other uses.

[0169] The present invention provides therapeutic compositions comprising binder polypeptides as described herein. Therapeutic compositions comprising nucleic acids encoding such binder polypeptides are also provided. The encoding nucleic acids are described in more detail elsewhere in the present invention, and include DNA and RNA, such as mRNA. In the therapeutic methods described herein, the use of nucleic acids encoding binder polypeptides, and / or the use of cells containing such nucleic acids, can be used as an alternative (or in addition) to compositions comprising the binder polypeptides themselves. Cells containing nucleic acids encoding binder polypeptides, optionally with the nucleic acid stably integrated into the genome, are thus pharmaceuticals for therapeutic use in patients. Nucleic acids encoding binder polypeptides are introduced into human cells derived from the intended patient and modified ex vivo. Administration of cells containing encoding nucleic acids to a patient provides a reservoir of cells capable of expressing the binder polypeptide, which may provide longer-term therapeutic benefits compared to administration of isolated nucleic acids or isolated binder polypeptides. Nucleic acids can also be administered directly to a patient for gene therapy. Thus, nucleic acids encoding binder polypeptides are provided for use in gene therapy which involves introducing the encoding nucleic acid into the cells of a patient in vivo, such that the nucleic acid is expressed in the patient's cells to provide a therapeutic effect, examples of which are disclosed herein.

[0170] The composition may contain suitable carriers, excipients, and other agents that are incorporated into the formulation to improve transfer, delivery, tolerability, etc. Numerous suitable formulations can be found in formularies known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as LIPOFECTINT™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci Technol 52:238-311. The composition may include antibodies or nucleic acids in combination with medical injection buffers and / or adjuvants.

[0171] The binder polypeptides, or their encoding nucleic acids, can be formulated for the desired route of administration to the patient, for example, in an injectable liquid, optionally an aqueous solution. The composition can optionally be formulated for intravenous or subcutaneous injection.

[0172] A variety of delivery systems are known and can be used to administer the pharmaceutical composition of the present invention. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local. The antigen-binding molecule is preferably administered by subcutaneous injection. Administration can also be self-administration by the patient, for example, self-injection.

[0173] Pharmaceutical compositions can also be delivered in vesicles, in particular liposomes (see Langer (1990) Science 249:1527-1533; Treat et al. (1989), in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327; see generally, ibid.).

[0174] In certain circumstances, pharmaceutical compositions can be delivered in controlled release systems. In one embodiment, pumps are used (see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials are used; see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974). In yet another embodiment, controlled release systems are placed near the target of the composition, thus requiring only a fraction of the systemic dose (see, for example, Medical Applications of Controlled Release, Goodson, supra, vol. 2, pp. 115-138, 1984).

[0175] The injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions, and the like. These injectable preparations can be prepared by known methods. For example, the injectable preparations can be prepared by dissolving, suspending or emulsifying, for example, the above-described antibody or its salt in a sterile aqueous or oily medium that is routinely used for injections. Aqueous media for injection include, for example, saline, isotonic solutions containing glucose, and other auxiliary agents, which are used in combination with suitable solubilizing agents such as alcohol (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], and the like. As oily media, for example, sesame oil, soybean oil, and the like, which are used in combination with solubilizing agents such as benzyl benzoate, benzyl alcohol, and the like, are used. The injections thus prepared can be filled into suitable ampoules. The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. It is envisioned that the treatment is not limited to use in a clinic. Thus, subcutaneous injection using a needleless device is also advantageous. For subcutaneous delivery, a pen delivery device is easily adapted to deliver the pharmaceutical composition of the present invention. Such a pen delivery device may be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge is easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Conversely, a disposable pen delivery device becomes pre-filled with the pharmaceutical composition held in a reservoir in the device. Once the reservoir is empty of the pharmaceutical composition, the entire device is discarded. Numerous reusable pen and autoinjector delivery devices are adapted to subcutaneous delivery of the pharmaceutical composition of the present invention.Examples include AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burgdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPENT™, OPTIPEN PRO™, OPTIPEN IV ... Examples of disposable pen-type delivery devices that are applied to subcutaneous delivery of the pharmaceutical composition of the present invention include, but are not limited to, SOLOSTAR pen (Sanofi-Aventis), FLEXPEN (Novo Nordisk), and KWIKPEN (Eli Lilly).

[0176] Advantageously, the above-described oral or parenteral pharmaceutical compositions are prepared in dosage forms with unit doses suitable for the dosage of the active ingredient. Such dosage forms of unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antibody contained is generally about 5 to about 500 mg per dosage form of unit dose; particularly in the form of injection, the antibody is contained in about 5 to about 100 mg, and for other dosage forms, it is contained in about 10 to about 250 mg.

[0177] The binder polypeptide, nucleic acid, or composition comprising it is contained in a medical container, such as a vial, a syringe, an IV container, or an injection device.In one example, the binder polypeptide, nucleic acid, or composition is in vitro and can be in a sterile container.In one example, a kit is provided that includes the binder polypeptide, packaging, and instructions for use in the treatment method described herein.

[0178] One aspect of the present invention is a composition comprising the binder polypeptide or nucleic acid of the present invention and one or more pharma- ceutically acceptable excipients, examples of which are listed above. "Pharmaceutically acceptable" refers to being approved or expected to be approved by the regulatory agencies of the United States Federal or State Governments for use in animals, including humans, or being listed in the United States Pharmacopeia or other generally recognized pharmacopoeias. A pharma-ceutically acceptable carrier, excipient, or adjuvant can be administered to a patient together with a binder polypeptide, for example, any antibody or polypeptide molecule described herein, and is non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the drug, without impairing its pharmacological activity.

[0179] In some embodiments, the binder polypeptide will be the only active ingredient in the composition according to the invention. Thus, the composition may consist of an antibody, or the composition may consist of a binder polypeptide together with one or more pharma- ceutically acceptable excipients. However, the composition according to the invention may optionally include one or more additional active ingredients. Other therapeutic agents that may be desirable to administer with the binder polypeptide or nucleic acid according to the invention include other therapeutic agents for cancer, examples of which are described herein. Any such agent or combination of agents may be administered in combination with the binder polypeptide or nucleic acid according to the invention, or provided in a composition with the binder polypeptide or nucleic acid according to the invention, whether as a combined preparation or as a separate preparation. The binder polypeptide or nucleic acid according to the invention may be administered separately and sequentially, or simultaneously and optionally as a combined preparation with another therapeutic agent, such as those described herein.

[0180] The compositions can be administered separately or simultaneously. Separate administration refers to two compositions administered at different times, for example at least 10, 20, 30, or 10-60 minutes apart, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 hours apart. The compositions can also be administered 24 hours apart, or even longer apart. Alternatively, two or more compositions can be administered simultaneously, for example less than 10 minutes or less than 5 minutes apart. Compositions administered simultaneously are, in some embodiments, administered as a mixture with or without similar or different time release mechanisms for each component.

[0181] Binder polypeptides and their encoding nucleic acids can be used as therapeutic agents. The patient herein is generally a mammal, typically a human. Binder polypeptides or nucleic acids can be administered to a mammal, for example, by any of the administration routes described herein. In a preferred embodiment, binder polypeptides are administered by subcutaneous injection.

[0182] Administration is usually in a "therapeutically effective amount", which is an amount sufficient to show benefit to the patient and to produce the desired effect for which it is administered. The exact amount will depend on the purpose of the treatment and will be ascertainable by those skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding). The prescription of treatment, e.g., the amount administered, etc., is within the responsibility of general practitioners and other medical doctors and may depend on the severity of the symptoms and / or progression of the disease being treated. The therapeutically effective amount or appropriate dose of the binder polypeptide or nucleic acid can be determined by comparing its in vitro and in vivo activity in animal models. Methods for extrapolating effective dosages to humans in mice and other test animals are known.

[0183] In the treatment methods described herein, one or more doses are administered. In some cases, a single administration may be effective to achieve long-term benefits. Thus, the method may include a single administration of binder polypeptide, its encoding nucleic acid, or composition. Alternatively, multiple doses are administered, usually consecutively, and separated by a period of several days, weeks, or months. For example, administration may be every two weeks, three weeks, or four weeks. In some cases, the binder polypeptide is administered to the patient once a month, or less frequently, for example, every two or three months.

[0184] As used herein, the term "treat", "treating" or "amelioration" refers to a therapeutic treatment that aims to reverse, alleviate, improve, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder. A treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, a treatment is "effective" if the progression of a disease is reduced or stopped. That is, "treatment" includes not only the improvement of symptoms or markers, but also the cessation or at least slowing of the progression or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms(s), whether detectable or undetectable, reduction in the extent of disease, stabilization of disease state (i.e., not worsening), delay or slowing of disease progression, improvement or palliation of disease state, remission (whether partial or complete remission), and / or reduction in mortality. The term "treatment" of a disease also includes providing relief from disease symptoms or side effects (including palliative treatment). With respect to effective treatment, complete cure is not intended. The method may also include cure in certain embodiments. In the context of the present invention, treatment may be preventive treatment.

[0185] Long half-life is a desirable feature of the binder polypeptide of the present invention. Prolonged half-life leads to less frequent administration, and fewer injections are required to maintain a therapeutically effective concentration of the molecule in the bloodstream. The in vivo half-life of the antigen-binding molecule of the present invention in humans may be 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days or more. The in vivo half-life of the antigen-binding molecule in non-human primates, such as cynomolgus monkeys, may be 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days or more.

[0186] Administration of the binder polypeptide at regular intervals of one week, two weeks, three weeks, four weeks, or one month is provided.

[0187] item The following numbered items represent embodiments of the present invention and are a part of this specification. 1. An antibody that binds to human GFRAL, comprising an antibody heavy chain variable (VH) domain and an antibody light chain variable (VL) domain, the VH domain comprises the QUEL-0201 set of heavy chain complementarity determining regions (HCDRs) HCDR1 SEQ ID NO: 13, HCDR2 SEQ ID NO: 14 and HCDR3 SEQ ID NO: 15; and An antibody whose VL domain comprises the QUEL-0201 set of light chain complementarity determining regions (LCDRs) LCDR1 SEQ ID NO: 18, LCDR2 SEQ ID NO: 19 and LCDR3 SEQ ID NO: 20. 2. An antibody that binds to human GFRAL, A VH domain having at least 90% amino acid sequence identity to the QUEL-0201 VH domain SEQ ID NO: 12, and QUEL-0201 VL domain A VL domain having at least 90% amino acid sequence identity to SEQ ID NO: 17 An antibody comprising: 3. An antibody that binds to human GFRAL, A VH domain encoded by a nucleotide sequence produced by recombination of gene segments IGHV1-3 (e.g., IGHV1-3*01) and IGHJ6 (e.g., IGHJ6*02); and VL domains encoded by nucleotide sequences produced by recombination of gene segments IGLV1-40 (e.g., IGLV1-40*01) and IGLJ3 (e.g., IGLJ3*02). An antibody comprising: 4. The antibody of item 3, wherein the VH domain is encoded by a nucleotide sequence produced by recombination of gene segments IGHV1-3 (e.g., IGHV1-3*01), IGHD5-18 (e.g., IGHD5-18*01) and IGHJ6 (e.g., IGHJ6*02). 5. An antibody that competes with QUEL-0201 IgG for binding to human GFRAL comprising the QUEL-0201 VH domain SEQ ID NO:12 and the QUEL-0201 VL domain SEQ ID NO:17. 6. The antibody according to any one of items 1 to 5, which binds to human GFRAL with an affinity of 1 nM or greater as determined by surface plasmon resonance. 7. The antibody described in item 6, which binds to human GFRAL with an affinity of 100 pM or greater as determined by surface plasmon resonance. 8. The antibody described in item 6, which binds to human GFRAL with an affinity in the range of 50 pM to 200 pM. 9. The antibody according to any of items 1 to 8, which cross-reacts with mouse GFRAL and has affinity for mouse GFRAL within 10-fold of its affinity for human GFRAL. 10. The antibody according to any of items 1 to 9, which inhibits GFRAL with a potency of 15 nM or greater, as determined as IC50 in an in vitro assay of ERK phosphorylation in response to GDF15. 11. The antibody described in item 10, having a titer of 10 nM or more. 12. QUEL-0201 VH domain A VH domain having at least 95% amino acid sequence identity to SEQ ID NO: 12. QUEL-0201 VL domain A VL domain having at least 95% amino acid sequence identity to SEQ ID NO: 17 12. The antibody according to any one of items 1 to 11, comprising: 13. A VH domain having at least 98% amino acid sequence identity to the QUEL-0201 VH domain SEQ ID NO: 12, and QUEL-0201 VL domain A VL domain having at least 98% amino acid sequence identity to SEQ ID NO: 17 4. The antibody of any of the preceding items, comprising: 14. The QUEL-0201 VH domain SEQ ID NO:12, optionally with one or two amino acid modifications; and QUEL-0201 VL domain SEQ ID NO: 17, optionally including one or two amino acid modifications 14. The antibody according to item 13, 15. The QUEL-0201 VH domain SEQ ID NO: 12, comprising the QUEL-0201 set of HCDRs: HCDR1 SEQ ID NO: 13, HCDR2 SEQ ID NO: 14 and HCDR3 SEQ ID NO: 15, and optionally comprising one or two amino acid modifications in the VH domain framework; and QUEL-0201 VL domain SEQ ID NO: 17, comprising the QUEL-0201 set of LCDRs: LCDR1 SEQ ID NO: 18, LCDR2 SEQ ID NO: 19 and LCDR3 SEQ ID NO: 20, optionally including one or two amino acid modifications in the VL domain framework. 15. The antibody according to item 14, comprising: 16. The antibody of item 14 or 15, wherein one or two amino acid modifications are conservative substitutions. 17. The antibody of any of the preceding items, comprising the QUEL-0201 VH domain SEQ ID NO:12 and the QUEL-0201 VL domain SEQ ID NO:17. 18. An antibody that binds to human GFRAL, comprising an antibody heavy chain variable (VH) domain and an antibody light chain variable (VL) domain, the VH domain comprises a set of heavy chain complementarity determining regions (HCDRs), HCDR1 being SEQ ID NO:23 or SEQ ID NO:133, HCDR2 being SEQ ID NO:24, SEQ ID NO:124, SEQ ID NO:134 or SEQ ID NO:142, and HCDR3 being SEQ ID NO:25, SEQ ID NO:125, SEQ ID NO:135 or SEQ ID NO:143, and An antibody wherein the VL domain comprises a set of light chain complementarity determining regions (LCDRs), wherein LCDR1 is SEQ ID NO:28, SEQ ID NO:128 or SEQ ID NO:138, LCDR2 is SEQ ID NO:29 or SEQ ID NO:129, and LCDR3 is SEQ ID NO:30, SEQ ID NO:130, SEQ ID NO:139 or SEQ ID NO:146. 19. An antibody that binds to human GFRAL, comprising an antibody heavy chain variable (VH) domain and an antibody light chain variable (VL) domain, A VH domain comprising the QUEL-0301 set of heavy chain complementarity determining regions (HCDRs) HCDR1 SEQ ID NO: 23, HCDR2 SEQ ID NO: 24 and HCDR3 SEQ ID NO: 25; and A VL domain comprising the QUEL-0301 set of light chain complementarity determining regions (LCDRs) LCDR1 SEQ ID NO: 28, LCDR2 SEQ ID NO: 29 and LCDR3 SEQ ID NO: 30. An antibody comprising: 20. An antibody that binds to human GFRAL, comprising a VH domain and a VL domain, a VH domain comprising the QUEL-0302 set of HCDRs: HCDR1 SEQ ID NO:23, HCDR2 SEQ ID NO:124 and HCDR3 SEQ ID NO:125; and A VL domain comprising the QUEL-0302 set of LCDRs: LCDR1 SEQ ID NO: 128, LCDR2 SEQ ID NO: 129 and LCDR3 SEQ ID NO: 130. An antibody comprising: 21. An antibody that binds to human GFRAL, comprising a VH domain and a VL domain, wherein the VH domain comprises the QUEL-0303 set of HCDRs: HCDR1 SEQ ID NO: 133, HCDR2 SEQ ID NO: 134, and HCDR3 SEQ ID NO: 135; and A VL domain comprising the QUEL-0303 set of LCDRs: LCDR1 SEQ ID NO: 138, LCDR2 SEQ ID NO: 29 and LCDR3 SEQ ID NO: 139 An antibody comprising: 22. An antibody that binds to human GFRAL, comprising a VH domain and a VL domain, a VH domain comprising the QUEL-0304 set of HCDRs: HCDR1 SEQ ID NO: 133, HCDR2 SEQ ID NO: 142 and HCDR3 SEQ ID NO: 143; and A VL domain comprising the QUEL-0304 set of LCDRs: LCDR1 SEQ ID NO: 138, LCDR2 SEQ ID NO: 29 and LCDR3 SEQ ID NO: 146 An antibody comprising: 23. An antibody that binds to human GFRAL, A VH domain having at least 90% amino acid sequence identity to QUEL-0301 VH domain SEQ ID NO:22, and QUEL-0301 VL domain A VL domain having at least 90% amino acid sequence identity to SEQ ID NO:27 An antibody comprising: 24. An antibody that binds to human GFRAL, A VH domain encoded by a nucleotide sequence produced by recombination of gene segments IGHV3-7 (e.g., IGHV3-7*01) and IGHJ4 (e.g., IGHJ4*02); and A VL domain encoded by a nucleotide sequence produced by recombination of gene segments IGLV1-44 (e.g., IGLV1-44*01) and IGLJ3 (e.g., IGLJ3*02). An antibody comprising: 25. The antibody of item 24, wherein the VH domain is encoded by a nucleotide sequence produced by recombination of gene segments IGHV3-7 (e.g., IGHV3-7*01), IGHD1-7 (e.g., IGHD1-7*01) and IGHJ4 (e.g., IGHJ4*02). 26. An antibody that binds to human GFRAL, A VH domain encoded by a nucleotide sequence produced by recombination of gene segments IGHV3-7 (e.g., IGHV3-7*01) and IGHJ4 (e.g., IGHJ4*02); and A VL domain encoded by a nucleotide sequence produced by recombination of gene segments IGLV1-47 (e.g., IGLV1-47*01) and IGLJ3 (e.g., IGLJ3*02). An antibody comprising: 27. The antibody of item 26, wherein the VH domain is encoded by a nucleotide sequence produced by recombination of gene segments IGHV3-7 (e.g., IGHV3-7*01), IGHD1-20 (e.g., IGHD1-20*01) and IGHJ4 (e.g., IGHJ4*02). 28. An antibody that competes for binding to human GFRAL with QUEL-0301 IgG comprising QUEL-0301 VH domain SEQ ID NO:22 and QUEL-0301 VL domain SEQ ID NO:27. 29. The antibody according to any of items 18 to 28, which binds to human GFRAL with an affinity of 1 nM or greater as determined by surface plasmon resonance. 30. The antibody described in item 29, which binds to human GFRAL with an affinity of 1 pM or greater as determined by surface plasmon resonance. 31. The antibody described in item 29, which binds to human GFRAL with an affinity in the range of 0.5 pM to 2 pM. 32. The antibody according to any one of items 18 to 31, which cross-reacts with mouse GFRAL and has an affinity for mouse GFRAL of 10 nM or more as determined by surface plasmon resonance. 33. The antibody according to any of items 18 to 32, which inhibits GFRAL with a potency of 15 nM or greater, the potency being determined as IC50 in an in vitro assay of ERK phosphorylation in response to GDF15. 34. The antibody described in item 33, having a titer of 10 nM or more. 35. A VH domain having at least 95% amino acid sequence identity to the QUEL-0301 VH domain SEQ ID NO:22, and QUEL-0301 VL domain A VL domain having at least 95% amino acid sequence identity to SEQ ID NO:27 35. The antibody according to any one of items 18 to 34, comprising: 36. A VH domain having at least 98% amino acid sequence identity to the QUEL-0301 VH domain SEQ ID NO: 22, and QUEL-0301 VL domain A VL domain having at least 98% amino acid sequence identity to SEQ ID NO:27 4. The antibody of any of the preceding items, comprising: 37. The QUEL-0301 VH domain SEQ ID NO:22, optionally comprising one or two amino acid modifications; and QUEL-0301 VL domain SEQ ID NO:27, optionally with one or two amino acid modifications. 37. The antibody according to Item 36, comprising: 38. The QUEL-0301 VH domain SEQ ID NO:22, comprising the QUEL-0301 set of HCDRs: HCDR1 SEQ ID NO:23, HCDR2 SEQ ID NO:24 and HCDR3 SEQ ID NO:25, optionally with one or two amino acid modifications in the VH domain framework; and QUEL-0301 VL domain SEQ ID NO:27, comprising the QUEL-0301 set of LCDRs: LCDR1 SEQ ID NO:28, LCDR2 SEQ ID NO:29 and LCDR3 SEQ ID NO:30, optionally with one or two amino acid modifications in the VL domain framework. 38. The antibody according to Item 37, comprising: 39. The antibody of item 37 or 38, wherein one or two amino acid modifications are conservative substitutions. 40. The antibody of any of items 18 to 39, comprising the QUEL-0301 VH domain SEQ ID NO: 22 and the QUEL-0301 VL domain SEQ ID NO: 27. 41. The antibody of any of items 18, 20, and 23 to 28, comprising QUEL-0302 VH domain SEQ ID NO: 123 and QUEL-0302 VL domain SEQ ID NO: 127. 42. The antibody of any of items 18, 21, and 23 to 28, comprising the QUEL-0303 VH domain SEQ ID NO: 132 and the QUEL-0303 VL domain SEQ ID NO: 137. 43. The antibody of any of items 18 and 22 to 28, comprising QUEL-0304 VH domain SEQ ID NO: 141 and QUEL-0304 VL domain SEQ ID NO: 145. 44. An antibody that binds to human GFRAL, comprising an antibody heavy chain variable (VH) domain and an antibody light chain variable (VL) domain, comprising a set of HCDRs, HCDR1, HCDR2 and HCDR3; HCDR1 is SEQ ID NO:3, SEQ ID NO:99, SEQ ID NO:107 or SEQ ID NO:118; HCDR2 is SEQ ID NO:4, SEQ ID NO:100 or SEQ ID NO:108; a VH domain, the HCDR3 of which is SEQ ID NO:5, SEQ ID NO:101, SEQ ID NO:113 or SEQ ID NO:119, and comprising a set of LCDRs: LCDR1, LCDR2 and LCDR3; LCDR1 is SEQ ID NO:8, LCDR2 is SEQ ID NO:9, and The VL domain, wherein LCDR3 is SEQ ID NO: 10 or SEQ ID NO: 104. An antibody comprising: 45. An antibody that binds to human GFRAL, comprising an antibody heavy chain variable (VH) domain and an antibody light chain variable (VL) domain, the VH domain comprises the QUEL-0101 set of heavy chain complementarity determining regions (HCDRs) HCDR1 SEQ ID NO:3, HCDR2 SEQ ID NO:4 and HCDR3 SEQ ID NO:5; and An antibody whose VL domain comprises the QUEL-0101 set of light chain complementarity determining regions (LCDRs) LCDR1 SEQ ID NO:8, LCDR2 SEQ ID NO:9 and LCDR3 SEQ ID NO:10. 46. ​​An antibody that binds to human GFRAL, comprising a VH domain and a VL domain, the VH domain comprises the QUEL-0102 set of HCDRs: HCDR1 SEQ ID NO: 99, HCDR2 SEQ ID NO: 100 and HCDR3 SEQ ID NO: 101; and An antibody wherein the VL domain comprises the QUEL-0102 set of LCDRs: LCDR1 SEQ ID NO:8, LCDR2 SEQ ID NO:9 and LCDR3 SEQ ID NO:104. 47. An antibody that binds to human GFRAL, comprising a VH domain and a VL domain, the VH domain comprises the QUEL-0103 set of HCDRs: HCDR1 SEQ ID NO: 107, HCDR2 SEQ ID NO: 108 and HCDR3 SEQ ID NO: 5; and An antibody wherein the VL domain comprises the QUEL-0103 set of LCDRs: LCDR1 SEQ ID NO:8, LCDR2 SEQ ID NO:9 and LCDR3 SEQ ID NO:104. 48. An antibody that binds to human GFRAL, comprising a VH domain and a VL domain, the VH domain comprises the QUEL-0104 set of HCDRs: HCDR1 SEQ ID NO: 107, HCDR2 SEQ ID NO: 108 and HCDR3 SEQ ID NO: 113; and An antibody wherein the VL domain comprises the QUEL-0104 set of LCDRs: LCDR1 SEQ ID NO:8, LCDR2 SEQ ID NO:9 and LCDR3 SEQ ID NO:104. 49. An antibody that binds to human GFRAL, comprising a VH domain and a VL domain, the VH domain comprises the QUEL-0105 set of HCDRs: HCDR1 SEQ ID NO: 118, HCDR2 SEQ ID NO: 108 and HCDR3 SEQ ID NO: 119; and An antibody wherein the VL domain comprises the QUEL-0105 set of LCDRs: LCDR1 SEQ ID NO:8, LCDR2 SEQ ID NO:9 and LCDR3 SEQ ID NO:104. 50. An antibody that binds to human GFRAL, A VH domain having at least 90% amino acid sequence identity to the QUEL-0101 VH domain SEQ ID NO:2, and QUEL-0101 VL domain A VL domain having at least 90% amino acid sequence identity to SEQ ID NO:7 An antibody comprising: 51. An antibody that binds to human GFRAL, A VH domain encoded by a nucleotide sequence produced by recombination of gene segments IGHV3-30 (e.g., IGHV3-30*18) and IGHJ6 (e.g., IGHJ6*02); and VL domains encoded by nucleotide sequences produced by recombination of gene segments IGKV1-27 (e.g., IGKV1-27*01) and IGKJ4 (e.g., IGKJ4*01). An antibody comprising: 52. The antibody of item 51, wherein the VH domain is encoded by a nucleotide sequence produced by recombination of gene segments IGHV3-30*18 (e.g., IGHV3-30), IGHD3-10 (e.g., IGHD3-10*01) and IGHJ6 (e.g., IGHJ6*02). 53. An antibody that competes for binding to human GFRAL with QUEL-0101 IgG comprising the QUEL-0101 VH domain sequence number 2 and the QUEL-0101 VL domain sequence number 7. 54. The antibody according to any of items 44 to 53, which binds to human GFRAL with an affinity of 1 nM or greater as determined by surface plasmon resonance. 55. The antibody described in item 54, which binds to human GFRAL with an affinity of 200 pM or more as determined by surface plasmon resonance. 56. The antibody of item 54, which binds to human GFRAL with an affinity in the range of 100 pM to 400 pM. 57. The antibody according to any of items 44 to 56, which cross-reacts with mouse GFRAL and has an affinity for mouse GFRAL within 10-fold of its affinity for human GFRAL. 58. The antibody according to any of items 44 to 57, which inhibits GFRAL with a potency of 15 nM or more, the potency being determined as IC50 in an in vitro assay of ERK phosphorylation in response to GDF15. 59. The antibody described in item 58, having a titer of 10 nM or more. 60. The antibody described in item 59, having a titer of 5 nM or more. 61. A VH domain having at least 95% amino acid sequence identity to the QUEL-0101 VH domain SEQ ID NO:2, and QUEL-0101 VL domain A VL domain having at least 95% amino acid sequence identity to SEQ ID NO:7 61. The antibody according to any one of items 44 to 60, comprising: 62. A VH domain having at least 98% amino acid sequence identity to the QUEL-0101 VH domain SEQ ID NO:2, and QUEL-0101 VL domain A VL domain having at least 98% amino acid sequence identity to SEQ ID NO:7 62. The antibody according to any one of items 44 to 61, comprising: 63. The QUEL-0101 VH domain SEQ ID NO:2, optionally with one or two amino acid modifications; and QUEL-0101 VL domain SEQ ID NO:7, optionally with one or two amino acid modifications 63. The antibody of item 62, comprising: 64. QUEL-0101 VH domain SEQ ID NO:2, comprising the QUEL-0101 set of HCDRs: HCDR1 SEQ ID NO:3, HCDR2 SEQ ID NO:4 and HCDR3 SEQ ID NO:5, optionally with one or two amino acid modifications in the VH domain framework; and QUEL-0101 VL domain SEQ ID NO:7, comprising the QUEL-0101 set of LCDRs: LCDR1 SEQ ID NO:8, LCDR2 SEQ ID NO:9, and LCDR3 SEQ ID NO:10, optionally comprising one or two amino acid modifications in the VL domain framework. 64. The antibody according to Item 63, comprising: 65. The antibody of item 63 or 64, wherein one or two amino acid modifications are conservative substitutions. 66. The antibody according to any of items 44 to 65, comprising the QUEL-0101 VH domain SEQ ID NO: 2 and the QUEL-0101 VL domain SEQ ID NO: 7. 67. The antibody according to any one of items 44, 46, and 50 to 53, comprising the QUEL-0102 VH domain SEQ ID NO: 98 and the QUEL-0102 VL domain SEQ ID NO: 103. 68. The antibody of any of items 44, 47, and 50 to 53, comprising QUEL-0103 VH domain SEQ ID NO: 106 and QUEL-0103 VH domain SEQ ID NO: 110. 69. The antibody of any of items 44, 48, and 50 to 53, comprising QUEL-0104 VH domain SEQ ID NO: 112 and QUEL-0104 VL domain SEQ ID NO: 115. 70. The antibody of any of items 44 and 49 to 53, comprising QUEL-0105 VH domain SEQ ID NO: 117 and QUEL-0105 VH domain SEQ ID NO: 121. 71. The antibody of any of the preceding items, comprising a heavy chain Fc region. 72. The antibody described in item 71, which is a human IgG. 73. The antibody according to item 72, which is human IgG4. 74. The antibody described in item 73, comprising a heavy chain constant region sequence number 60. 75. A nucleic acid encoding an antibody according to any of the preceding items. 76. An in vitro host cell comprising the nucleic acid according to item 75. 77. A composition comprising the antibody according to any of items 1 to 74 or the nucleic acid according to item 75, formulated together with a pharma- ceutically acceptable excipient. 78. The composition according to item 77, formulated for intravenous or subcutaneous injection. 79. A method for treating a medical condition associated with the GDF15-GFRAL pathway in a patient, comprising administering to the patient a composition described in item 77 or 78. 80. The composition according to item 77 or 78, for use in treating a medical condition associated with the GDF15-GFRAL pathway in a patient. 81. Use of the composition according to item 77 or item 78 for the manufacture of a medicament for treating a medical condition associated with the GDF15-GFRAL pathway in a patient. 82. The method according to item 79, the composition according to item 80, or the use of the composition according to item 81, wherein the medical condition is hyperemesis gravidarum, anorexia, cachexia, conditioned taste aversion, and / or a side effect of chemotherapy treatment for cancer. 83. The method, composition or use according to item 82, wherein the patient is also undergoing or has undergone treatment with an anti-cancer chemotherapeutic agent. 84. The method, composition or use according to item 82 or item 83, wherein the medical condition is anorexia in cancer patients. 85. The method, composition or use according to item 82 or item 83, wherein the medical condition is cachexia in cancer patients. 86. The method, composition or use according to any of items 82 to 85, wherein the medical condition is a side effect of treatment with an anti-cancer chemotherapeutic agent. 87. The method, composition or use according to item 86, wherein the treatment comprises preventing conditioned taste aversion caused by treatment with an anti-cancer chemotherapeutic agent. 88. The method, composition or use according to any of items 82 to 87, wherein the treatment comprises reducing weight loss and extending survival of a cancer patient. 89. A method for reducing glucocorticoid (e.g., cortisol) levels in a patient, comprising administering to the patient an inhibitor of GDF15 signaling. 90. An inhibitor of GDF15 signaling for use in reducing glucocorticoid (e.g., cortisol) levels in a patient. 91. Use of a GDF15 signaling inhibitor for the manufacture of a medicament for reducing glucocorticoid (e.g., cortisol) levels in a patient. 92. The method according to item 89, the inhibitor for use according to item 90, or the use of the inhibitor according to item 91, for normalizing blood levels of glucocorticoids (e.g., cortisol) in a patient determined to have elevated blood glucocorticoid levels. 93. The method according to item 89, the inhibitor for use according to item 90, or the use of the inhibitor according to item 91, wherein the patient is a cancer patient. 94. The method, the inhibitor for use, or the use of the inhibitor according to any of items 89 to 93, wherein the patient is also receiving or has received treatment with a cytotoxic and / or antineoplastic agent. 95. The method, the inhibitor for use, or the use of the inhibitor according to any of items 89 to 94, wherein the inhibitor inhibits the GDF15-GFRAL-RET signaling complex. 96. The method, the inhibitor for use, or the use of the inhibitor according to item 95, wherein the inhibitor is an anti-GFRAL antibody. 97. The method, inhibitor for use, or use of the inhibitor according to item 96, wherein the anti-GFRAL antibody is an antibody according to any one of items 1 to 74. EXAMPLES

[0188] In these examples, we present the characterization of anti-GFRAL antagonist antibodies QUEL-0101 (and its variants QUEL-0102, QUEL-0103, QUEL-0104 and QUEL-0105), QUEL-0201, and QUEL-0301 (and its variants QUEL-0302, QUEL-0303 and QUEL-0304). We demonstrate the ability of anti-GFRAL antagonist antibodies to bind and potently inhibit GFRAL. We show that such blockade of GFRAL inhibits the effects of GDF15 on markers of food intake, body weight and muscle atrophy in vivo in mice. Furthermore, we show that at the same dose, anti-GFRAL antibodies also inhibit the effect of GDF15 acting through GFRAL to increase circulating glucocorticoid levels. This study shows that inhibition of GDF15:GFRAL interaction inhibits the neuroendocrine response to GDF15, including elevated glucocorticoids, demonstrating the potential for therapeutic use of anti-GFRAL antagonist antibodies to lower glucocorticoid levels in patients, e.g., to lower elevated blood levels of glucocorticoids (e.g., cortisol) to approach normal physiological levels in patients. EXAMPLES

[0189] In vitro ERK phosphorylation assay for inhibition of GFRAL signaling HEK293 cells were engineered to express DNA encoding human GFRAL SEQ ID NO:31 and / or human RET on the cell surface. Human GDF15 (recombinant human GDF15 protein with a 6-His tag (R&D Systems, 957-GD)) was added to 96-well cell cultures containing the following HEK293 cell lines: HEK293 cells expressing only human GFRAL HEK293 cells expressing only human RET HEK293 cells co-expressing human GFRAL and RET.

[0190] After incubating cells with 4 nM GDF15, the medium was removed and the cell samples were lysed. The cell lysates were transferred to 384-well plates and HTRF assay was performed to measure the phosphorylation of ERK gene product, which represents the downstream signal of GDF15-GFRAL-RET pathway. When RET is activated by GDF15-GFRAL tetramer, it causes phosphorylation of ERK, which can be detected by HTRF. We used Cisbio Advanced Phospho-ERK assay kit to detect phosphorylated ERK in a sandwich assay format using two different antibodies specific for phosphorylated ERK, one labeled with Eu3+-cryptate (donor) and the other labeled with d2 (acceptor). When the dyes are in close proximity, excitation of the donor by a light source causes fluorescence resonance energy transfer (FRET) to the acceptor, which then fluoresces at a specific wavelength (665 nm) when the two antibodies bind to phosphorylated ERK. The change in fluorescence (ΔF) represents the effect of downstream signaling of GDF15. A plot of ΔF against the logarithm of the concentration of GDF15 produces a standard sigmoidal curve in cells coexpressing GFRAL and RET but shows no signal in cells expressing GFRAL or RET alone (Figure 3).

[0191] As a positive control, the anti-GFRAL antagonist antibody mAb Q was included for comparison.

[0192] Results for selected QUEL-0101, QUEL-0202, and QUEL-0301 antibodies and mAb Q are shown in the table below and in FIG.

[0193] [Table 1] EXAMPLES

[0194] Affinity for human GFRAL determined by SPR QUEL-0101, QUEL-0201 and QUEL-0301 showed a good range of affinities for human GFRAL as determined by SPR.

[0195] Binding of antibodies to human GFRAL was measured by SPR using His-tagged human GFRAL as the analyte, with test antibodies bound to the CM4 biosensor chip surface via immobilized anti-human Fc antibody. The running buffer was 1 mM CaCl 2 The buffer was HBS-P+ buffer pH 7.4 (GE BR100671) containing 100 mM Glycine. Antibodies were captured at 1 μg / ml for 60 seconds (approximately 80-140 RU captured, depending on the binder). Analyte (recombinant soluble GFRAL protein, R&D catalogue no. 9647-GR-050) was injected at 100, 25, 6.25, 1.56, 0.39, 0.098, 0.024, 0 nM at 30 μg / min for an association time of 120 seconds and dissociation was monitored for 1200 seconds. The chip surface was regenerated with 10 mM glycine pH 1.5. All experiments were performed at 25 °C. Sensorgrams for each antibody were double subtracted (reference cell and concentration 0). Data were fitted to a 1:1 binding interaction model. Sensorgrams were fitted using the multi-cycle kinetic mode. QUEL-0101, QUEL-0201, and QUEL-0301 each exhibited fast on-rates and slow dissociation.

[0196] The KD and other kinetic constants for binding to human GFRAL were calculated and the results are shown in the table below.

[0197] [Table 2]

[0198] Of these antibodies, QUEL-0301 showed the highest affinity for human GFRAL, approximately 1 picomolar (pM), stronger than the reference antibody mAb Q, which measured an affinity of 4.3 pM. The approximately four-fold difference between these antibodies indicates comparable affinities (a two-fold difference is within the experimental error of the Biacore instrument). The KD values ​​shown are only estimates, as antibody QUEL-0301 dissociates very slowly, with a kd approaching the measurement limit of the instrument. QUEL-0201 bound human GFRAL with an affinity of approximately 84 pM. QUEL-0101 bound human GFRAL with an affinity of 140 pM.

[0199] The QUEL antibody has very good kinetic properties. The kinetic constants are in the range expected for a potent therapeutic antibody against the receptor. The Koff and Kon values ​​are very informative, showing that the antibody binds to GFRAL very quickly and remains bound for a long time. EXAMPLES

[0200] Affinity for mouse GFRAL determined by SPR Affinity to mouse GFRAL was measured by SPR in a manner similar to Example 2, using mouse GFRAL-Fc fusion (R&D catalog number 9844-GR-050050) as the analyte.

[0201] The KD and other kinetic constants for binding to mouse GFRAL were calculated and the results are shown in the table below.

[0202] [Table 3]

[0203] Of these antibodies, QUEL-0201 had the highest affinity for mouse GFRAL at approximately 64.8 pM, QUEL-0101 had an affinity of approximately 1.2 nanomolar (nM), and QUEL-0301 had a relatively low affinity of less than 5 nM.

[0204] Comparing the measured affinities of these antibodies to human and mouse GFRAL, QUEL-0201 was the most cross-reactive, with affinities for human and mouse GFRAL within 1.5-fold (human: 84 pM; mouse: 65 pM). QUEL-0101 also showed good cross-reactivity within 10-fold, but with lower absolute affinity (human: 0.14 nM; mouse: 1.2 nM). QUEL-0301 also showed cross-reactivity, but in contrast to its high affinity for human GFRAL, its binding to mouse GFRAL was relatively weak (human: 1 pM; mouse: 5 nM). EXAMPLES

[0205] Analysis of antibody inhibition of GDF15-GFRAL-RET complex formation Epitope mapping assays were performed on selected antagonist mAbs to explore the possible mechanisms of inhibiting GDF15-GFRAL-RET signaling.

[0206] (a) Sandwich assay In this assay, pairs of antibodies are tested for their ability to simultaneously bind antigen. A first antibody is bound to a solid support via its Fc region, and GFRAL antigen is added in solution, allowing the formation of an antibody-antigen complex. A second antibody is then added in solution. Detected binding of the second antibody indicates that the first and second antibodies bind to different epitopes. No detection of second antibody binding indicates that the first and second antibodies compete for binding to the same epitope.

[0207] The same principle can also be used to evaluate whether an antibody and a native ligand compete for binding to an antigen. Thus, a test antibody is bound to a solid support, and a GFRAL antigen is added in solution, allowing the formation of an antibody-GFRAL complex. GDF15 is then added in solution. If binding of GDF15 is detected, this indicates that the antibody does not inhibit the formation of the GDF15-GFRAL complex.

[0208] Here, we used surface plasmon resonance to detect binding to antibodies bound to a biosensor chip surface via a pre-immobilized anti-Fc capture antibody. The following results were obtained in a sandwich assay using test antibodies, GFRAL antigen, and GDF15 or mAbQ as analytes:

[0209] [Table 4]

[0210] The results in the table are presented in resonance units after injection of GDF15 or mAbQ.

[0211] Since a binding signal was observed after the addition of GDF15 to the antibody-GFRAL complex, none of QUEL-0101, QUEL-0201, QUEL-0301, and the reference antibody mAb Q appeared to inhibit the binding of GDF15 to GFRAL. Since none of QUEL-0101, QUEL-0201, and QUEL-0301 were observed to compete with GDF15 for binding to GFRAL, these antibodies suggest that they inhibit GFRAL signaling through different mechanisms, such as by inhibiting the association of RET with GDF15-GFRAL, thus inhibiting the formation of the GDF15-GFRAL-RET active signaling complex.

[0212] Each of QUEL-0101, QUEL-0201, and QUEL-0301 appears to bind to a different epitope on GFRAL compared to mAb Q, since a binding signal was observed after mAb Q was added to the antibody-GFRAL complex.

[0213] (b) Tandem binding assay The tandem assay is an alternative to the sandwich assay described in (a) above. In the tandem assay, it is the antigen that binds to the surface, rather than the antibody. Human GFRAL with a 6His tag was captured on an anti-His antibody immobilized on the biosensor chip surface, and the first antibody was injected onto the antigen, followed by the second antibody. Using the tandem method, the QUEL antibodies were separated into two epitope bins. QUEL-0201 recognizes a different epitope compared to QUEL-0101 and QUEL-0301, which share one epitope bin. None of the QUEL antibodies competed with mAb Q in this assay (confirming the sandwich assay results), placing mAb Q in the third epitope bin. EXAMPLES

[0214] In vivo activity of QUEL-0201 and QUEL-0301 For use in mouse studies, QUEL-0201 and QUEL-0301 were generated as mouse IgG1 isotype, i.e., having a mouse IgG1 heavy chain constant region, while retaining human variable domains with sequences defined herein.

[0215] C57BL / 6 mice housed at 22° C. were injected with QUEL-0201 or QUEL-0301, followed by GDF15, and their food intake and body weight were monitored. Treatment groups were as follows: 1. Two injections of 10 mg / kg QUEL-0301 on Thursday and Saturday 2. Two injections of 10 mg / kg QUEL-0201 on Thursday and Saturday 3. One injection of 20 mg / kg QUEL-0301 on Saturday 4. One injection of 20 mg / kg QUEL-0201 on Saturday 5. Two injections of isotype control antibody on Thursday and Saturday.

[0216] All groups were injected with recombinant human GDF15 on Sunday.

[0217] Mice treated with the isotype control antibody had reduced food intake over the study period. Mice treated with the anti-GFRAL antibody QUEL-0201 also had reduced food intake, but to a lesser extent than the isotype control group. Mice treated with the anti-GFRAL antibody QUEL-0301 maintained food intake. FIG. 7A.

[0218] Consistent with the effect on food intake, control mice exhibited a loss in body weight, whereas mice treated with QUEL-0301 lost less body weight and mice treated with QUEL-0201 showed no loss in body weight.

[0219] The increased efficacy of QUEL-0201 in this study is due to its higher affinity for mouse GFRAL compared to QUEL-0301 (Example 3).

[0220] In a further mouse study (MS15), QUEL-0201 was confirmed to block GDF15-induced decreased food intake and weight loss when administered in two injections at a dose of 20 mg / kg each (Figures 8A and 8B). EXAMPLES

[0221] Exogenous GDF15 activates the HPA axis in a GFRAL-dependent manner To determine whether GDF15 acting through hindbrain receptors increases corticosterone levels in mice, we carried out an experiment using anti-GFRAL antibody QUEL-0201, which validated its efficacy against classical GDF15 response in mice (MS15) (Example 5).QUEL-0201 completely prevented GDF15-induced corticosterone levels, while control isotype antibody showed no effect (Figure 9).The concentration of human circulating GDF15 achieved in the study was not different between anti-GFRAL group and control isotype (Figure 10).

[0222] Thus, at the same doses that were effective in completely blocking the effects of GDF15 on food intake and body weight, anti-GFRAL antibodies also completely blocked the corticosterone response to GDF15. EXAMPLES

[0223] Sequence variants of QUEL-0101 Based on the high sequence homology with the VH and VL domains of QUEL-0101, the present inventors isolated antibodies from immunized mice with sequences that are believed to share the same or parallel evolutionary lineage as QUEL-0101 from the germline. These were named QUEL-0102, QUEL-0103, QUEL-0104 and QUEL-0105. Each of these clones demonstrated binding to human GFRAL. EXAMPLES

[0224] Sequence variants of QUEL-0301 Based on the high sequence homology with the VH and VL domains of QUEL-0301, the present inventors isolated antibodies from immunized mice with sequences that are believed to share the same or parallel evolutionary lineage as QUEL-0301 from the germline. These were named QUEL-0302, QUEL-0303, and QUEL-0304, respectively. Each of these clones showed binding to human GFRAL. QUEL-0304 was further confirmed to recognize human GFRAL in an in vitro primary screening assay. EXAMPLES

[0225] Inhibition of GFRAL reduces biomarkers of muscle atrophy We analyzed the effects of GDF15 and anti-GFRAL antibodies on the expression of Mafbx, Murf1, and Foxo1. These genes are transcriptionally increased in skeletal muscle under atrophy-inducing conditions and are excellent markers of muscle atrophy [52, 53]. We show here that treatment with the anti-GFRAL antibody QUEL-0201 inhibited the GDF15-induced increase in each of these markers in vivo in mouse skeletal muscle, thus providing further support for the use of GDF15 inhibitors in the treatment of conditions involving loss of skeletal muscle mass, such as cachexia.

[0226] Materials and Methods BL6 / C57 mice housed at 22 °C were intraperitoneally injected with either the anti-GFRAL antibody QUEL-0201 (at a dose of 20 mg / kg) or an isotype control once daily for 4 days. The day after the last injection, mice were subcutaneously injected with either human recombinant GDF15 (0.1 mg / kg, Qkine) or vehicle control. All groups (n = 4–5 mice) were sacrificed 6 h later and four muscles were harvested: tibialis anterior, extensor digitorum longus, gastrocnemius, and soleus. RNA was extracted, purified, and analyzed as previously described

[49] . Three well-recognized markers of skeletal muscle atrophy, Mafbx, Murf1, and Foxo1, were analyzed.

[0227] result At the time points analyzed, tissues from mice treated with human recombinant GDF15 and isotype control antibody showed a significant increase in the expression of markers of muscle atrophy. No such effect was seen in any of the muscle tissues analyzed when GDF15 was administered after pretreatment with anti-GFRAL antibody (Figure 11).

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[0229] Sequence information Table A Table A below shows the variable domain sequences of the antibodies described herein. All QUEL VH domains, QUEL VL domains, QUEL CDRs, QUEL heavy chains and QUEL light chains, antibodies comprising them, and nucleic acids encoding them, represent embodiments of the present invention.

[0230] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] [Table 5-8] [Table 5-9] [Table 5-10]

[0231] Table C Table C below shows the sequences of antibody heavy and light chain constant regions and the nucleic acids encoding same. These are examples of constant regions that may be used in the antibodies of the invention.

[0232] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] [Table 6-8] [Table 6-9] [Table 6-10] [Table 6-11] [Table 6-12] [Table 6-13] [Table 6-14]

[0233] Table G Human germline V, D and J gene segments recombine to produce the VH domain of an antibody, and human germline V and J gene segments recombine to produce the VL domain of an antibody. The nucleotide and / or amino acid sequences of the antibody variable domains can be compared to the human germline gene segments to identify the most closely matching gene segments.

[0234] Table G1 below shows the germline gene segments identified for the QUEL antibody.

[0235] [Table 7]

[0236] Table G2 below shows the variable domains with germline v and j framework regions corresponding to QUEL-0101, QUEL-0201 and QUEL-0301.

[0237] The antibodies of the invention optionally comprise framework regions having germline residues as shown herein.

[0238] [Table 8]

Claims

1. An antibody that binds to human GFRAL, comprising an antibody heavy chain variable (VH) domain and an antibody light chain variable (VL) domain, (i) the VH domain comprises the QUEL-0201 set of heavy chain complementarity determining regions (HCDRs) HCDR1 SEQ ID NO: 13, HCDR2 SEQ ID NO: 14 and HCDR3 SEQ ID NO: 15; and The VL domain comprises the QUEL-0201 set of light chain complementarity determining regions (LCDRs) LCDR1 SEQ ID NO: 18, LCDR2 SEQ ID NO: 19 and LCDR3 SEQ ID NO: 20; (ii) the VH domain comprises the QUEL-0301 set of heavy chain complementarity determining regions (HCDRs) HCDR1 SEQ ID NO:23, HCDR2 SEQ ID NO:24 and HCDR3 SEQ ID NO:25; and the VL domain comprises the QUEL-0301 set of light chain complementarity determining regions (LCDRs) LCDR1 SEQ ID NO:28, LCDR2 SEQ ID NO:29 and LCDR3 SEQ ID NO:30; or (iii) the VH domain comprises the QUEL-0101 set of heavy chain complementarity determining regions (HCDRs) HCDR1 SEQ ID NO:3, HCDR2 SEQ ID NO:4 and HCDR3 SEQ ID NO:5; and The VL domain contains the light chain complementarity determining region (LCDR) LCDR1 SEQ ID NO: 8, LCDR2 SEQ ID NO: 9 and LCDR3 SEQ ID NO: 10, including the QUEL-0101 set; antibody.

2. An antibody that binds to human GFRAL, (i) a VH domain having at least 90% amino acid sequence identity to the QUEL-0201 VH domain SEQ ID NO: 12; and QUEL-0201 VL domain A VL domain having at least 90% amino acid sequence identity to SEQ ID NO: 17; (ii) QUEL-0301 VH domain with at least 90% a VH domain having amino acid sequence identity; and QUEL-0301 VL domain A VL domain having at least 90% amino acid sequence identity to SEQ ID NO:27, or (iii) a VH domain having at least 90% amino acid sequence identity to the QUEL-0101 VH domain SEQ ID NO:2; and QUEL-0101 VL domain A VL domain having at least 90% amino acid sequence identity to SEQ ID NO:7 An antibody comprising:

3. (i) binds to human GFRAL with an affinity of 1 nM or greater as determined by surface plasmon resonance; (ii) cross-reacts with mouse GFRAL and has an affinity for mouse GFRAL within 10-fold of its affinity for human GFRAL; and / or (iii) inhibits GFRAL with a potency of 15 nM or greater, the potency being determined as an IC50 in an in vitro assay of ERK phosphorylation in response to GDF15; The antibody according to claim 1 or 2.

4. The VH domain comprises the QUEL-0201 set of heavy chain complementarity determining regions (HCDRs) HCDR1 SEQ ID NO: 13, HCDR2 SEQ ID NO: 14, and HCDR3 SEQ ID NO: 15, and the VL domain comprises the QUEL-0201 set of light chain complementarity determining regions (LCDRs) LCDR1 SEQ ID NO: 18, LCDR2 SEQ ID NO: 19, and LCDR3 SEQ ID NO: 20; 4. The antibody of claim 3, wherein the antibody binds to human GFRAL with an affinity of 100 pM or greater, or with an affinity in the range of 50 pM to 200 pM, as determined by surface plasmon resonance.

5. The VH domain comprises the QUEL-0301 set of heavy chain complementarity determining regions (HCDRs) HCDR1 SEQ ID NO:23, HCDR2 SEQ ID NO:24, and HCDR3 SEQ ID NO:25, and the VL domain comprises the QUEL-0301 set of light chain complementarity determining regions (LCDRs) LCDR1 SEQ ID NO:28, LCDR2 SEQ ID NO:29, and LCDR3 SEQ ID NO:30; the antibody binds to human GFRAL with an affinity of 1 pM or greater, or with an affinity in the range of 0.5 pM to 2 pM, as determined by surface plasmon resonance, and / or cross-reacts with mouse GFRAL and has an affinity for mouse GFRAL of 10 nM or greater, as determined by surface plasmon resonance; The antibody described in claim 3.

6. The VH domain contains the heavy chain complementarity determining region (HCDR) HCDR1 SEQ ID NO: 3, HCDR2 The QUEL-0101 set includes a light chain complementarity determining region (LCDR) LCDR1 SEQ ID NO: 8, LCDR2 SEQ ID NO: 5, and a VL domain includes a light chain complementarity determining region (LCDR) LCDR1 SEQ ID NO: 8, LCDR3 SEQ ID NO: 5, and a VL domain includes a light chain complementarity determining region (LCDR) LCDR1 SEQ ID NO: 8, LCDR2 SEQ ID NO: 5, and a VL domain comprising the QUEL-0101 set of SEQ ID NO: 9 and LCDR3 SEQ ID NO: 10; the antibody binds to human GFRAL with an affinity of 200 pM or greater, or in the range of 100 pM to 400 pM, as determined by surface plasmon resonance, and / or inhibits GFRAL with a potency of 5 nM or greater; The antibody described in claim 3.

7. The antibody of claim 3, which inhibits GFRAL with a potency of 10 nM or greater.

8. (i) a VH domain having at least 95% amino acid sequence identity to the QUEL-0201 VH domain SEQ ID NO: 12; and QUEL-0201 VL domain at least 95% amino acid identity to SEQ ID NO:17 VL domains with sequence identity (ii) a VH domain having at least 95% amino acid sequence identity to the QUEL-0301 VH domain SEQ ID NO:22; and QUEL-0301 VL domain A VL domain having at least 95% amino acid sequence identity to SEQ ID NO:27, or (iii) a VH domain having at least 95% amino acid sequence identity to the QUEL-0101 VH domain SEQ ID NO:2; and QUEL-0101 VL domain A VL domain having at least 95% amino acid sequence identity to SEQ ID NO:7 The antibody according to any one of claims 1 to 7, comprising:

9. (i) a VH domain having at least 98% amino acid sequence identity to the QUEL-0201 VH domain SEQ ID NO: 12; and QUEL-0201 VL domain A VL domain having at least 98% amino acid sequence identity to SEQ ID NO: 17; (ii) a VH domain having at least 98% amino acid sequence identity to the QUEL-0301 VH domain SEQ ID NO:22; and QUEL-0301 VL domain A VL domain having at least 98% amino acid sequence identity to SEQ ID NO:27, or (iii) a VH domain having at least 98% amino acid sequence identity to the QUEL-0101 VH domain SEQ ID NO:2; and QUEL-0101 VL domain A VL domain having at least 98% amino acid sequence identity to SEQ ID NO:7 The antibody according to any one of claims 1 to 8, comprising:

10. (i) the QUEL-0201 VH domain SEQ ID NO: 12, optionally comprising one or two amino acid modifications; and QUEL-0201 VL domain SEQ ID NO: 17, optionally with one or two amino acid modifications; (ii) the QUEL-0301 VH domain SEQ ID NO:22, optionally comprising one or two amino acid modifications; and QUEL-0301 VL domain SEQ ID NO:27, optionally containing one or two amino acid modifications; or (iii) the QUEL-0101 VH domain SEQ ID NO:2, optionally comprising one or two amino acid modifications; and QUEL-0101 VL domain SEQ ID NO:7, optionally with one or two amino acid modifications. The antibody of claim 9 .

11. (i) the QUEL-0201 VH domain SEQ ID NO: 12, comprising the QUEL-0201 set of HCDRs: HCDR1 SEQ ID NO: 13, HCDR2 SEQ ID NO: 14, and HCDR3 SEQ ID NO: 15, and optionally comprising one or two amino acid modifications in the VH domain framework; and QUEL-0201 VL domain SEQ ID NO: 17, comprising the QUEL-0201 set of LCDRs: LCDR1 SEQ ID NO: 18, LCDR2 SEQ ID NO: 19 and LCDR3 SEQ ID NO: 20, optionally with one or two amino acid modifications in the VL domain framework. (ii) the QUEL-0301 VH domain comprising the QUEL-0301 set of HCDRs: HCDR1 SEQ ID NO: 23, HCDR2 SEQ ID NO: 24, and HCDR3 SEQ ID NO: 25, optionally with one or two amino acid modifications in the VH domain framework; SEQ ID NO: 22, and QUEL-0301 VL domain SEQ ID NO:27, comprising the QUEL-0301 set of LCDRs: LCDR1 SEQ ID NO:28, LCDR2 SEQ ID NO:29 and LCDR3 SEQ ID NO:30, optionally with one or two amino acid modifications in the VL domain framework; or (iii) the QUEL-0101 VH domain SEQ ID NO:2, comprising the QUEL-0101 set of HCDRs: HCDR1 SEQ ID NO:3, HCDR2 SEQ ID NO:4, and HCDR3 SEQ ID NO:5, and optionally comprising one or two amino acid modifications in the VH domain framework; and 11. The antibody of claim 10, comprising the QUEL-0101 set of LCDRs: LCDR1 SEQ ID NO:8, LCDR2 SEQ ID NO:9, and LCDR3 SEQ ID NO:10, and optionally comprising the QUEL-0101 VL domain SEQ ID NO:7, which optionally contains one or two amino acid modifications in the VL domain framework.

12. The antibody of claim 10 or claim 11, wherein one or two amino acid changes are conservative substitutions.

13. (i) QUEL-0201 VH domain SEQ ID NO: 12 and QUEL-0201 VL domain SEQ ID NO: 17; (ii) the QUEL-0301 VH domain SEQ ID NO:22 and the QUEL-0301 VL domain SEQ ID NO:27; or (iii) QUEL-0101 VH domain SEQ ID NO: 2 and QUEL-0301 VL domain SEQ ID NO: 7 The antibody according to any one of claims 1 to 12, comprising:

14. The antibody of any one of claims 1 to 13, comprising a heavy chain Fc region.

15. The antibody of claim 14 which is a human IgG.

16. The antibody of claim 15 which is human IgG4.

17. The antibody of claim 16, comprising a heavy chain constant region sequence number 60.

18. A nucleic acid encoding the antibody according to any one of claims 1 to 17.

19. 20. An in vitro host cell comprising the nucleic acid of claim 18.

20. A composition comprising the antibody of any one of claims 1 to 17 or the nucleic acid of claim 18, formulated together with a pharma- ceutically acceptable excipient.

21. 21. The composition of claim 20 formulated for intravenous or subcutaneous injection.

22. 22. The composition of claim 20 or 21 for use in treating a medical condition associated with the GDF15-GFRAL pathway in a patient.

23. 22. Use of the composition of claim 20 or 21 for the manufacture of a medicament for treating a medical condition associated with the GDF15-GFRAL pathway in a patient.

24. 23. The composition for use according to claim 22, wherein the medical condition is hyperemesis gravidarum, anorexia, cachexia, conditioned taste aversion, and / or a side effect of chemotherapy treatment for cancer; or Use of the composition according to claim 23.

25. 25. The composition or use of claim 24, wherein the patient is also undergoing or has undergone treatment with an anti-cancer chemotherapeutic agent.

26. 26. The composition or use of claim 24 or claim 25, wherein the medical condition is anorexia or cachexia in cancer patients.

27. 27. The composition or use of any one of claims 24 to 26, wherein the medical condition is a side effect of treatment with an anti-cancer chemotherapeutic agent, and optionally the treatment comprises preventing conditioned taste aversion caused by treatment with an anti-cancer chemotherapeutic agent.

28. The composition or use according to any one of claims 24 to 27, wherein the treatment comprises reducing weight loss and increasing survival time in cancer patients.

29. 20. An inhibitor of GDF15 signaling for use in reducing glucocorticoid (eg cortisol) levels in a patient, the inhibitor being an anti-GFRAL antibody according to any one of claims 1 to 17.

30. 20. Use of a GDF15 signaling inhibitor for the manufacture of a medicament for reducing glucocorticoid (e.g. cortisol) levels in a patient, wherein the inhibitor is an anti-GFRAL antibody as defined in any one of claims 1 to 17.

31. An inhibitor for use as described in claim 29, or use of an inhibitor as described in claim 30, for normalizing blood levels of glucocorticoids (e.g., cortisol) in a patient determined to have elevated blood glucocorticoid levels.

32. 31. The inhibitor for use according to claim 29 or the use of the inhibitor according to claim 30, wherein the patient is a cancer patient.

33. The inhibitor for use or the use of an inhibitor according to any one of claims 29 to 32, wherein the patient is also receiving or has been receiving treatment with a cytotoxic and / or antineoplastic agent.

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