Anti-GDF15 antibody, and method and use therefor

Abstract

A GDF15 binding molecule, in particular an antibody specifically recognizing GDF15 and a fragment thereof. A nucleic acid or host cell containing the antibody or fragment thereof, a drug containing the antibody or fragment thereof, and a therapeutic and diagnostic method or use applying the antibody and fragment.

Description

Antibodies against GDF15 and methods and uses thereof Technical Field

[0001] The present invention relates to the field of antibodies, and specifically to GDF15-binding molecules, particularly antibodies and fragments thereof that specifically recognize GDF15. Furthermore, the present invention relates to nucleic acids or host cells containing such antibodies or fragments thereof, medicaments containing such antibodies or fragments thereof, and therapeutic and diagnostic methods or uses utilizing such antibodies and fragments. Background Art

[0002] Growth differentiation factor 15 (GDF15) is a member of the transforming growth factor β (TGFβ) superfamily. GDF15 is secreted as a 40kDa propeptide and released as a 25kDa dimeric protein active substance after cleavage. GDF15 is a stress response cytokine whose expression can be significantly increased under various stress conditions, such as inflammation, hypoxia, tissue damage, myocardial ischemia and various malignant tumors. It has been reported that circulating GDF15 levels are elevated in patients with various cancers, including prostate cancer, pancreatic cancer, endometrial cancer and colorectal cancer. In the tumor microenvironment, GDF15 can be expressed by tumor cells, cancer-associated fibroblasts and tumor-associated macrophages. In addition, chemotherapy can also induce a significant increase in GDF15 levels in patients (Suriben R et al., Nature medicine, 2020, 26(8):1264-1270.).

[0003] Cachexia is a condition that causes loss of lean body mass and occurs during the progression of many diseases. It is estimated that cachexia causes 2 million deaths per year worldwide and is one of the leading causes of death in humans (Ferrer M et al., Cell, 2023, 186(9):1824-1845.). Several recent studies (Mullican SE et al., Nature medicine, 2017, 23(10):1150-1157; Yang L et al., Nature medicine, 2017, 23(10):1158-1166; Emmerson PJ et al., Nature medicine, 2017, 23(10):1215-1219.) have found that GDF15 can bind to the glial cell line derived neurotrophic factor (GDNF) family receptor α-like (GFRAL) protein, a receptor mainly expressed in the hindbrain, and then form a heterohexamer with the rearranged during transfection (RET) molecule to activate the GFRAL-RET signaling pathway, transmit anorexia neural signals, reduce body weight, affect fat and muscle degradation, and induce cachexia. GDF15 / GFRAL antibodies can reverse this reaction and are a potential target for the treatment of cachexia.

[0004] Ponsegromab (CN112912395A), a GDF15 antibody developed by Pfizer, and NGM-120 (WO2023 / 039359Al), a GFRAL antibody developed by NGM, have entered Phase 2 clinical trials for improving tumor cachexia and have shown the potential to increase patient weight.

[0005] However, the specificity, efficacy, and drugability of current GDF15 antibodies need to be improved. Summary of the Invention

[0006] In one aspect, the present invention provides a GDF15 antibody having high binding affinity to GDF15.

[0007] In some embodiments, the anti-GDF15 antibodies or antigen-binding fragments thereof of the present invention bind to GDF15 (e.g., human GDF15 or monkey GDF15, such as cynomolgus monkey GDF15, or murine GDF15, such as mouse GDF15) with higher affinity. In some embodiments, the anti-GDF15 antibodies or antigen-binding fragments thereof of the present invention are capable of binding to both human GDF15 and monkey GDF15, such as cynomolgus monkey GDF15, and optionally also to murine GDF15, such as mouse GDF15. In some embodiments, the affinity of the antibody is determined by thin-layer interferometry or surface plasmon resonance. In some embodiments, the anti-GDF15 antibodies of the invention bind to human GDF15 or monkey GDF15, e.g., cynomolgus monkey GDF15, with an equilibrium dissociation constant (KD) of less than about 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, 0.01 nM, or 1 pM, or between any of the foregoing values. In some embodiments, an anti-GDF15 antibody of the invention binds to murine GDF15, e.g., mouse GDF15, with an equilibrium dissociation constant (KD) of less than about 20 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, or 0.5 nM, or between any of the foregoing values, e.g., between 0.5 nM and 15 nM, e.g., between 1 nM and 15 nM.

[0008] In some embodiments, the anti-GDF15 antibodies or antigen-binding fragments thereof of the present invention bind to human, cynomolgus monkey, or mouse GDF15 expressed in cells. In some embodiments, the affinity of the anti-GDF15 antibodies to human, cynomolgus monkey, or mouse GDF15 expressed in cells is determined by flow cytometry.

[0009] In some embodiments, the anti-GDF15 antibodies or antigen-binding fragments thereof of the present invention do not cross-react with human TGFβ family proteins, for example, they do not bind to human GDF1, GDF2, GDF3, AMH, BMP4, GDF11, GDF8, GDNF, TGFβ1, GFRα1, ActivinA, ActivinB, and / or BMP2 proteins.

[0010] In some embodiments, the anti-GDF15 antibodies or antigen-binding fragments thereof of the invention are known to have free GDF15 in blood, such as serum, for example, better than a known control, such as Ponsegromab.

[0011] In some embodiments, the anti-GDF15 antibodies or antigen-binding fragments thereof of the present invention block GDF15 activation of stress signaling pathways, such as activation of serum response elements (SREs), for example, in a dose-dependent manner, for example, with greater efficacy than a control such as Ponsegromab.

[0012] In some embodiments, the anti-GDF15 antibodies or antigen-binding fragments thereof of the present invention inhibit the level of ERK phosphorylation downstream of GDF15 / GFRAL / RET, for example, better than a known control, such as Ponsegromab.

[0013] In some embodiments, the anti-GDF15 antibodies or antigen-binding fragments thereof of the invention reverse cachexia induced by a tumor (eg, tumor cachexia), such as a solid tumor, such as a fibrosarcoma, for example, better than a known control, such as Ponsegromab.

[0014] In some embodiments, the anti-GDF15 antibodies or antigen-binding fragments thereof of the invention reverse cachexia induced by a chemotherapeutic agent, such as cisplatin, eg, better than a known control, such as Ponsegromab.

[0015] In some embodiments, the anti-GDF15 antibodies or antigen-binding fragments thereof of the present invention have good drug exposure levels, for example, better than known controls, such as Ponsegromab.

[0016] In some embodiments, the anti-GDF15 antibodies or antigen-binding fragments thereof of the present invention have pharmacokinetic characteristics suitable for drug development.

[0017] In some embodiments, an anti-GDF15 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region.

[0018] In some embodiments, the anti-GDF15 antibodies of the invention are full-length antibodies or antigen-binding fragments thereof.

[0019] In some embodiments, the anti-GDF15 antibody of the present invention is a VHH antibody or a heavy chain antibody comprising the same.

[0020] The present invention also relates to nucleic acid molecules encoding the antibodies, as well as therapeutic methods and uses using the antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1A and Figure 1B show the reporter gene activity of humanized anti-GDF15 antibodies.

[0022] FIG1C shows the reporter gene activity of a humanized anti-GDF15 antibody having a YTE mutation in the Fc region.

[0023] FIG2A shows a PAGE image showing the inhibition of ERK1 / 2 phosphorylation by humanized anti-GDF15 antibody as determined by Western blot.

[0024] Figure 2B shows the grayscale value analysis results of Western blot analysis of the inhibition of ERK1 / 2 phosphorylation by humanized anti-GDF15 antibody.

[0025] FIG3A shows a PAGE image showing the inhibition of ERK1 / 2 phosphorylation by VHH humanized anti-GDF15 antibody as determined by Western blot.

[0026] Figure 3B shows the grayscale value analysis results of Western blot analysis of the inhibition of ERK1 / 2 phosphorylation by VHH humanized anti-GDF15 antibody.

[0027] FIG4 shows the binding activities of a control antibody, a humanized anti-GDF15 antibody, and an IgG Control to GDF2.

[0028] FIG5 shows the binding activities of a control antibody, a humanized anti-GDF15 antibody, and an IgG Control to GDNF.

[0029] Figures 6A and 6B show the changes in the net body weight of mice on day 6 (10 mg / kg) and day 7 (3 mg / kg, 1 mg / kg) after administration.

[0030] Figure 6C shows the average daily food intake after drug administration.

[0031] FIG6D shows the ovarian fat weight of mice on day 7 after administration.

[0032] Figure 7 shows the free drug levels in mice after a single dose for 72 hours.

[0033] FIG8 shows the changes in the net body weight of mice after administration in the cisplatin model.

[0034] FIG9 shows the blood drug concentration levels at the endpoint in the cisplatin efficacy model.

[0035] FIG10 shows the blood concentration-time curve of anti-GDF15 antibodies in the serum of humanized FcRn mice. DETAILED DESCRIPTION

[0036] Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific methods and experimental conditions in this specification, because the methods and conditions can be varied. In addition, the terminology used herein is only for describing specific embodiments and is not intended to be limiting.

[0037] Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific methods and experimental conditions in this specification, because the methods and conditions can be varied. In addition, the terminology used herein is only for describing specific embodiments and is not intended to be limiting.

[0038] I. Definition

[0039] For the purpose of interpreting this specification, the following definitions will be used, and whenever appropriate, terms used in the singular may also include the plural, and vice versa. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0040] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0041] As used herein, the term "and / or" means any one of the alternatives or two or more or all of the alternatives.

[0042] As used herein, the term "comprising" means including the recited elements, integers, or steps, but does not exclude any other elements, integers, or steps. In this article, when the term "comprising" is used, unless otherwise indicated, it also encompasses the situation of consisting of the recited elements, integers, or steps. For example, when it is mentioned that an antibody variable region "comprising" a specific sequence, it is also intended to encompass an antibody variable region consisting of the specific sequence.

[0043] Growth differentiation factor 15 (GDF15) is a diverse member of the TGFβ superfamily and is also known as macrophage inhibitory cytokine 1 (M1C1) (Bootcov MR, 1997, Proc Natl Acad Sci 94: 11514-9), placental bone morphogenetic factor (PLAB) (Hromas R 1997, Biochim Biophys Acta. 1354: 40-4), placental transforming growth factor beta (PTGFB) (Lawton LN 1997, Gene. 203: 17-26), prostate-derived factor (PDF) (Paralkar VM 1998, J Biol Chem. 273: 13760-7) and nonsteroidal anti-inflammatory drug-activated gene (NAG-1) (Baek SJ 2001, J Biol Chem. 276: 33384-92). The mature GDF15 peptide has low homology with other family members (Katoh M 2006, Int J Mol Med. 17: 951-5). GDF15 is synthesized as a large precursor protein that is cleaved at a dibasic cleavage site to release the carboxyl-terminal mature peptide. The human full-length precursor contains 308 amino acids and is cleaved at the RGRRRAR (SEQ ID NO: 185) cleavage site to produce the mature GDF peptide. Naturally occurring GDF15 is a 25KD homodimer of mature peptides covalently linked by an interchain disulfide bond. In some embodiments, GDF15, for example, human GDF15 or cynomolgus monkey GDF15 or mouse GDF15 protein, such as the human GDF15 protein under accession number Q99988-1 or the cynomolgus monkey GDF15 protein under G7PWZ3 or the mouse GDF15 protein under Q9Z0J7. In one embodiment, the human GDF15 protein of the present invention comprises the amino acid sequence of SEQ ID NO: 40 (or its amino acid sequence without the His tag), or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consists of said sequence. In one embodiment, the cynomolgus monkey GDF15 protein of the present invention comprises the amino acid sequence of SEQ ID NO: 41 (or its amino acid sequence without the His tag), or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consists of said sequence.In one embodiment, the mouse GDF15 protein of the present invention comprises the amino acid sequence shown in SEQ ID NO:42 (or its amino acid sequence without the His tag), or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of the said sequence.

[0044] As used herein, the terms "anti-GDF15 antibody," "anti-GDF15," "GDF15 antibody," or "antibody that binds to GDF15" refer to an antibody that is capable of binding to a GDF15 protein or a fragment thereof with sufficient affinity. In one embodiment, the extent of binding of an anti-GDF15 antibody to a non-GDF15 protein is less than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% or more of the binding of the antibody to GDF15. In some embodiments, the binding is measured, for example, by radioimmunoassay (RIA), thin-layer interferometry (BLI), biointerferometry, MSD assay, surface plasmon resonance (SPR), or flow cytometry.

[0045] In one embodiment, the binding molecules of the present invention that specifically bind to GDF15, such as anti-GDF15 antibodies or antigen-binding fragments thereof, have high affinity binding activity to cells expressing human GDF15 or cynomolgus monkey GDF15. In one embodiment, the binding molecules of the present invention that specifically bind to GDF15, such as anti-GDF15 antibodies or antigen-binding fragments thereof, have cross-reactivity to human and monkey (e.g., cynomolgus monkey) GDF15 or their extracellular domains, i.e., they can bind to human and monkey (e.g., cynomolgus monkey) GDF15. In one embodiment, the binding molecules of the present invention that specifically bind to GDF15, such as anti-GDF15 antibodies or antigen-binding fragments thereof, can also bind to mouse GDF15.

[0046] The terms "whole antibody," "full-length antibody," "complete antibody," and "intact antibody" are used interchangeably herein to refer to a naturally occurring glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, known as complementarity determining regions (CDRs), interspersed with more conserved regions, known as framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region is not directly involved in binding the antibody to the antigen, but exhibits various effector functions.

[0047] In some embodiments, the heavy chain constant region HC of the antibody of the present invention is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of IgG1.

[0048] In some embodiments, the light chain constant region LC of an antibody of the present invention is a Lambda or Kappa light chain constant region.

[0049] The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0050] The term "antigen-binding fragment" refers to a portion or segment of an intact or complete antibody that has fewer amino acid residues than the intact or complete antibody and that is capable of binding to an antigen or competing for antigen binding with the intact antibody (i.e., the intact antibody from which the antigen-binding fragment is derived). Antigen-binding fragments can be prepared by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabodies, and single-domain antibodies (sdAbs). The Fab fragment is a monovalent fragment composed of the VL, VH, CL, and CH1 domains. For example, Fab fragments can be obtained by papain digestion of an intact antibody. In addition, pepsin digestion of an intact antibody below the disulfide bonds in the hinge region produces F(ab')2, a divalent antibody fragment that is a dimer of Fab'. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into a Fab' monomer. A Fab' monomer is essentially a Fab fragment with a hinge region. The Fv fragment is composed of the VL and VH domains of a single antibody arm. In addition, although the two domains VL and VH of the Fv fragment are encoded by independent genes, using recombinant methods, they can be connected by a synthetic connecting peptide that enables the two domains to be produced as a single protein chain, in which the VL region and the VH region are paired to form a single-chain Fv (scFv). The antibody fragment can be obtained by chemical methods, recombinant DNA methods or protease digestion methods.

[0051] "Fab fragment" or "Fab" is used interchangeably herein to refer to an immunoglobulin fragment composed of two polypeptide chains comprising an immunoglobulin heavy chain variable domain VH, a heavy chain constant domain CH1, a light chain variable domain VL, and a light chain constant domain CL, wherein one polypeptide chain comprises, from N-terminus to C-terminus, VH and a constant region selected from CH1 and CL, and the other polypeptide chain comprises, from N-terminus to C-terminus, VL and another constant region selected from CL and CH1, wherein the VH domain and the VL domain pair to form an antigen binding site. The Fab chain comprising the heavy chain constant region CH1 is also referred to as a "Fab heavy chain"; accordingly, the Fab chain comprising the light chain constant region CL is also referred to as a "Fab light chain."

[0052] The term "antigen" refers to a molecule that elicits an immune response. This immune response may involve the production of antibodies or the activation of specific immune cells, or both. The skilled artisan will appreciate that any macromolecule, including essentially all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA.

[0053] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable regions of the heavy and light chains of natural antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions.

[0054] "Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment schemes, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImmunoGene Tics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and the definition of NorthCDRs based on affinity propagation clustering using a large number of crystal structures.

[0055] The following are the regional ranges of CDRs defined using the Kabat, AbM, Chothia, Contact, and IMGT schemes.

[0056] Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways. CDRs can also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the exemplary CDRs of the present invention). Unless otherwise indicated, in the present invention, when referring to residue positions in an antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the numbering position of the Kabat numbering system according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0057] In one embodiment, the HCDRs and LCDRs in an antibody of the invention are determined according to the Kabat scheme.

[0058] In some embodiments, the HCDRs in the VHH antibodies of the invention or heavy chain antibodies comprising the same are determined according to IMGT.

[0059] The term "Fc domain" or "Fc region" or "Fc fragment" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" comprises two or three constant domains, namely a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in a native antibody, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) of two heavy chains derived from IgG, IgA, and IgD classes of antibodies; or the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of two heavy chains derived from IgM and IgE classes of antibodies. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or heavy chain constant region is according to the EU numbering system (also known as the EU index) as described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) (https: / / pubmed.ncbi.nlm.nih.gov / 5257969 / ), see also http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html. As used herein, the term "Fc domain" or "Fc region" or "Fc fragment" excludes the heavy chain variable region VH and light chain variable region VL of an immunoglobulin, as well as the heavy chain constant region CH1 (without the hinge region) and the light chain constant region CL, but may include the hinge region at the N-terminus of the heavy chain constant region, such as EPKSS or EPKSC. In some embodiments, the heavy chain constant region Fc suitable for use in the present invention is derived from an antibody heavy chain constant region, such as the constant region of human IgG1, IgG2, IgG3, or IgG4, preferably the constant region of IgG1. In some embodiments, the Fc region comprises the amino acid sequence shown in SEQ ID NO: 46, or comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consists of said amino acid sequence.

[0060] Examples of "effector functions" of immunoglobulins include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B-cell receptor), and B-cell activation.

[0061] The term "chimeric antibody" is an antibody molecule in which (a) the constant region or a portion thereof is changed, replaced or exchanged so that the antigen binding site is connected to a constant region of a different or altered class, effector function and / or species or a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug) that imparts new properties to the chimeric antibody; or (b) the variable region or a portion thereof is changed, replaced or exchanged with a variable region having a different or altered antigenic specificity. For example, a mouse antibody can be modified by replacing its constant region with a constant region from a human immunoglobulin. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity in recognizing the antigen while having reduced immunogenicity in humans as compared to the original mouse antibody.

[0062] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody (e.g., a murine antibody) while being less immunogenic when administered to humans as a therapeutic agent. This can be achieved, for example, by retaining the non-human antigen-binding site and replacing the remaining portions of the antibody with their human counterparts (i.e., replacing portions of the variable region that are not involved in binding with the corresponding portions of a human antibody).

[0063] As used herein, the terms "anti," "binding," or "specific binding" mean that the binding is selective for the target or antigen and can be distinguished from unwanted or non-specific interactions. The ability of a binding site to bind to a specific target or antigen can be determined by flow cytometry or enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as by radioimmunoassay (RIA) or thin-layer interferometry or MSD assays or surface plasmon resonance (SPR).

[0064] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K D ) indicates that the dissociation constant is the dissociation rate constant and the association rate constant (K dis and K on Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay.

[0065] "Percent identity (%)" of an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the specific amino acid sequence set forth in this specification, after aligning the candidate sequence with the specific amino acid sequence set forth in this specification and introducing gaps, if necessary, to achieve the maximum percentage identity, and not considering any conservative substitutions as part of the sequence identity. In some embodiments, the present invention contemplates variants of the antibody molecules of the present invention that have a substantial degree of identity, e.g., at least 80%, 85%, 90%, 95%, 97%, 98% or 99% or more, relative to the antibody molecules and sequences thereof specifically disclosed herein. The variants may comprise conservative changes or be conservatively modified variants.

[0066] For polypeptide sequences, "conservative changes" include replacements, deletions or additions to the polypeptide sequence, but do not substantially change the desired functional activity of the polypeptide sequence. For example, conservative substitutions often result in a certain amino acid being replaced with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following lists 8 groups of amino acids containing mutually conservative replacements: 1) alanine (A), glycine (G); 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); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M). In some embodiments, the term "conservative sequence change" is used to refer to amino acid modifications that do not significantly affect or change the target antigen binding characteristics of the antibody molecule or binding protein molecule of the present invention containing the amino acid sequence. For example, a conservatively modified variant retains at least 80%, 85%, 90%, 95%, 98%, 99% or more, such as 100-110% or more, binding affinity for the antigen of interest relative to the parent antibody or binding protein.

[0067] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom. Host cells are any type of cell system that can be used to produce the antibody molecules of the present invention, including eukaryotic cells, e.g., mammalian cells, insect cells, yeast cells; and prokaryotic cells, e.g., E. coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.

[0068] The term "vector" when used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is attached. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which it has been introduced. The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences that are operatively linked to the nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) into which recombinant polynucleotides are incorporated.

[0069] The terms "individual" or "subject" are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual is a human.

[0070] The term "treat," ..."

[0071] The term "prevent" includes the inhibition of the development or progression of a disease or condition or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a preventative regimen. Generally, in the context of cancer, the term "prevent" refers to the administration of a drug before the development of signs or symptoms of cancer, particularly in a subject at risk for cancer.

[0072] The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies (such as immune checkpoint molecule antibodies), small molecule drugs, or immunomodulators (such as immunosuppressants or agonists).

[0073] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0074] "Chemotherapeutic agents" include chemical compounds useful in treating cancer or immune system disorders.

[0075] The term "small molecule drug" refers to low molecular weight organic compounds that are capable of regulating biological processes. "Small molecules" are defined as molecules with a molecular weight of less than 10 kD, typically less than 2 kD, and preferably less than 10 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimics. As therapeutic agents, small molecules can be more cell-permeable, less susceptible to degradation, and less prone to eliciting an immune response than macromolecules.

[0076] As used herein, the term "immunomodulator" refers to a natural or synthetic agent or drug that inhibits or modulates an immune response. The immune response can be a humoral response or a cellular response. Immunomodulators include immunosuppressants or agonists. In some embodiments, the immunomodulators of the present invention include immune checkpoint inhibitors or immune checkpoint agonists.

[0077] The term "effective amount" refers to that amount or dosage of an antibody or fragment or composition or combination of the present invention which, after single or multiple doses, produces the desired effect in a patient in need of treatment or prevention.

[0078] A "therapeutically effective amount" is an amount effective to achieve the desired therapeutic outcome at the required dosage and for the required period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or composition or combination are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter or improves a measurable parameter by at least about 40%, even more preferably by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100%, relative to an untreated subject.

[0079] A "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result, at the required dosage and for the required period of time. Typically, a prophylactic amount will be less than a therapeutically effective amount because a prophylactic dose is used in a subject prior to or at an earlier stage of disease.

[0080] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when referred to herein. The term "tumor" encompasses solid tumors and hematologic tumors. The term "anti-tumor effect" or "tumor inhibitory effect" or "tumor suppression effect" refers to a biological effect that can be demonstrated by a variety of means, including but not limited to, for example, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0081] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.

[0082] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0083] The term "drug combination or combination product" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit. The term "non-fixed combination" means that the active ingredients (e.g., (i) the binding molecules of the present invention and (ii) other therapeutic agents) are administered to a patient simultaneously, without specific time restrictions, or at the same or different time intervals, in a separate entity, wherein such administration provides two or more active agents with effective levels of prevention or treatment in the patient's body. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. The dosage and / or time interval of the two or more active agents are preferably selected so that the combined use of the parts can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in the form of a separate formulation, which can be the same or different.

[0084] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiotherapy or surgery) to treat diseases described herein. This administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of active ingredients. Alternatively, this administration includes co-administration of each active ingredient in a variety of or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids can be reconstituted or diluted to the desired dose before administration. In addition, this administration also includes using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the therapeutic regimen will provide the beneficial effects of the drug combination in treating disorders or conditions described herein.

[0085] "Subject / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample can be solid tissue, such as from fresh, frozen and / or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood component; body fluids, such as tears, vitreous humor, cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time during the subject's pregnancy or development. In some embodiments, the tissue sample is tumor tissue. The tissue sample may contain compounds that are not naturally mixed with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0086] II. Antibodies of the Invention

[0087] A specific aspect of the present invention relates to an anti-GDF15 antibody or an antigen-binding fragment thereof.

[0088] In some embodiments, the anti-GDF15 antibodies of the invention are full-length antibodies or antigen-binding fragments thereof.

[0089] In some embodiments, the anti-GDF15 antibodies or antigen-binding fragments thereof of the present invention comprise three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3.

[0090] In some embodiments, the anti-GDF15 antibodies or antigen-binding fragments thereof of the present invention comprise three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0091] In some embodiments, an anti-GDF15 antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (HCDRs) from a heavy chain variable region and three complementarity determining regions (LCDRs) from a light chain variable region.

[0092] In some aspects, the anti-GDF15 antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH). In some aspects, the anti-GDF15 antibodies or antigen-binding fragments thereof of the present invention comprise a light chain variable region (VL). In some aspects, the anti-GDF15 antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementary determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementary determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0093] In some embodiments, the anti-GDF15 antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody heavy chain constant region HC. In some embodiments, the anti-GDF15 antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody light chain constant region LC. In some embodiments, the anti-GDF15 antibodies or antigen-binding fragments thereof of the present invention further comprise a heavy chain constant region HC and a light chain constant region LC.

[0094] In some embodiments, the heavy chain variable region VH of the anti-GDF15 antibody of the present invention is

[0095] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 7, 14, 23 or 25; or

[0096] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 7, 14, 23 or 25; or

[0097] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 7, 14, 23 or 25, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0098] In some embodiments, the light chain variable region VL of the anti-GDF15 antibody of the present invention is

[0099] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 8, 15, 24 or 26; or

[0100] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 8, 15, 24 or 26; or

[0101] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 8, 15, 24 or 26, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0102] In some embodiments, the three complementarity determining regions (HCDRs) from the heavy chain variable region of the anti-GDF15 antibody of the present invention, HCDR1, HCDR2, and HCDR3 are

[0103] (i) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 7, 14, 23 or 25, or

[0104] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in the three HCDR regions relative to the sequence of (i),

[0105] Preferably, the HCDRs are defined according to Kabat.

[0106] In some embodiments, the three complementarity determining regions (LCDRs), LCDR1, LCDR2, and LCDR3, of the anti-GDF15 antibodies of the present invention are from the light chain variable region.

[0107] (i) three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 8, 15, 24 or 26, or

[0108] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three LCDR regions relative to the sequence of (i);

[0109] Preferably, the LCDR is determined according to Kabat.

[0110] In some embodiments, HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having 1, 2 or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therein.

[0111] In some embodiments, HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 2, 9 or 20, or an amino acid sequence having 1, 2 or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therein.

[0112] In some embodiments, HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 3 or 10, or an amino acid sequence having 1, 2 or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therein.

[0113] In some embodiments, LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4, 11 or 21, or an amino acid sequence having 1, 2 or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therein.

[0114] In some embodiments, LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 5, 12 or 22, or an amino acid sequence having 1, 2 or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therein.

[0115] In some embodiments, LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6 or 13, or an amino acid sequence having 1, 2 or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) therein.

[0116] In some embodiments, in the anti-GDF15 antibodies of the present invention,

[0117] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 4; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 5; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 6;

[0118] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 9; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 10; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 11; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 12; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 13; or

[0119] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 20; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 21; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 22; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 6.

[0120] In some specific embodiments of the present invention, the anti-GDF15 antibody or antigen-binding fragment thereof comprises VH and VL, wherein

[0121] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0122] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0123] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0124] The VH comprises the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of said amino acid sequence, and the VL comprises the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of said amino acid sequence.

[0125] In some specific embodiments of the present invention, the anti-GDF15 antibody or antigen-binding fragment thereof comprises

[0126] (i) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 7, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 8;

[0127] (ii) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 14, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 15;

[0128] (iii) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 23, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 24; or

[0129] (iv) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 25, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 26.

[0130] In some embodiments of the present invention, the anti-GDF15 antibody or antigen-binding fragment thereof comprises:

[0131] HCDR1 as set forth in SEQ ID NO: 1, HCDR2 as set forth in SEQ ID NO: 2, HCDR3 as set forth in SEQ ID NO: 3; LCDR1 as set forth in SEQ ID NO: 4, LCDR2 as set forth in SEQ ID NO: 5, and LCDR3 as set forth in SEQ ID NO: 6;

[0132] HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 9, HCDR3 as shown in SEQ ID NO: 10; LCDR1 as shown in SEQ ID NO: 11, LCDR2 as shown in SEQ ID NO: 12, and LCDR3 as shown in SEQ ID NO: 13; or

[0133] HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 20, HCDR3 as shown in SEQ ID NO: 3; LCDR1 as shown in SEQ ID NO: 21, LCDR2 as shown in SEQ ID NO: 22 and LCDR3 as shown in SEQ ID NO: 6.

[0134] In some specific embodiments of the present invention, the anti-GDF15 antibody or antigen-binding fragment thereof comprises VH and VL, wherein

[0135] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 7, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 8;

[0136] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 14, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 15;

[0137] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 23, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 24; or

[0138] The VH contains or consists of the amino acid sequence shown in SEQ ID NO: 25, and the VL contains or consists of the amino acid sequence shown in SEQ ID NO: 26.

[0139] In one embodiment of the present invention, the amino acid changes described herein include amino acid substitutions, insertions, or deletions. In preferred embodiments, the amino acid changes described herein occur in regions outside the CDRs (e.g., in the FRs). More preferably, the amino acid changes described herein occur in regions outside the heavy chain variable region and / or outside the light chain variable region. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

[0140] In some embodiments, the anti-GDF15 antibodies or antigen-binding fragments thereof of the present invention have one or more of the following properties:

[0141] (i) exhibiting the same or similar binding affinity and / or specificity for GDF15 as the antibodies of the present invention;

[0142] (ii) inhibiting (e.g., competitively inhibiting) the binding of an antibody of the present invention to GDF15;

[0143] (iii) binds to the same or overlapping epitope as an antibody of the invention;

[0144] (iv) competing with the antibodies of the present invention for binding to GDF15;

[0145] (v) possess one or more biological properties of an antibody of the invention.

[0146] In some embodiments, the anti-GDF15 antibody or antigen-binding fragment thereof of the present invention is an antibody in the form of IgG1, IgG2, IgG3, or IgG4, or an antigen-binding fragment thereof, for example, an antibody in the form of IgG1. In some embodiments, the heavy chain constant region of the anti-GDF15 antibody of the present invention is a constant region of IgG1, IgG2, IgG3, or IgG4, for example, a constant region of IgG1. "Constant region" includes native constant regions, allotypes, or naturally occurring variants, such as D356E and L358M.

[0147] In some embodiments, the IgG1 constant region of the present invention comprises the Fc district. As will be appreciated by those skilled in the art, according to the intended use of antibody molecule of the present invention, antibody molecule of the present invention may also comprise one or more modifications in the Fc district. In one embodiment, the Fc district is modified on the characteristic of the effector function (for example, complement activation function in the Fc district) in the Fc district. In one embodiment, the effector function has been reduced or eliminated relative to the wild isotype Fc district. In one embodiment, the effector function is reduced or eliminated by being selected from the following method: use natural Fc isotype and Fc district modifications with the effector function reduced or eliminated. In a preferred embodiment, the Fc district has the effector function mediated by the Fc district that reduces, for example, ADCC or ADCP or CDC effector function reduced or eliminated, for example, comprising the sudden change realizing above-mentioned function.

[0148] In some embodiments, the antibody molecules of the present invention may also include modifications in the Fc region that alter binding affinity to one or more Fc receptors. In one embodiment, the Fc receptor is an Fcγ receptor, particularly a human Fcγ receptor. In some embodiments, the Fc region includes mutations that reduce binding to Fcγ receptors. For example, in some embodiments, the Fc region used in the present invention has one or more of the L234A / L235A mutation or the G237A mutation that reduces binding to Fcγ receptors. In some embodiments, the Fc region used in the present invention has both the L234A / L235A mutation and the G237A mutation that reduce binding to Fcγ receptors.

[0149] In another preferred embodiment, the Fc region may have a mutation that results in increased serum half-life, such as a mutation that improves binding of the Fc fragment to FcRn. In some embodiments, the Fc region of the present invention has a mutation that extends half-life, such as an M252Y / S254T / T256E mutation (YTE mutation). In some embodiments, the Fc region of the present invention comprises an M252Y / S254T / T256E mutation.

[0150] In some embodiments, the antibody molecules of the present invention have mutations in the Fc region that can reduce binding to Fcγ receptors and / or extend half-life and / or increase affinity for FcRn, such as L234A / L235A mutations and / or G237A mutations and / or M252Y / S254T / T256E mutations. In some embodiments, the Fc region of the present invention comprises L234A / L235A mutations, G237A mutations and M252Y / S254T / T256E mutations.

[0151] In some embodiments, the Fc region amino acid sequence suitable for use in an antibody molecule of the invention comprises or consists of SEQ ID NO: 43 or an amino acid sequence having at least 90% identity thereto, such as 95%, 96%, 97%, 99% or more identity. In some embodiments, the Fc region comprises an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or more identity to SEQ ID NO: 43 and comprises L234A / L235A mutations and G237A mutations.

[0152] In some embodiments, the Fc region amino acid sequence suitable for use in an antibody molecule of the invention comprises or consists of SEQ ID NO: 47 or an amino acid sequence having at least 90% identity thereto, such as 95%, 96%, 97%, 99% or more identity. In some embodiments, the Fc region comprises an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or more identity to SEQ ID NO: 47 and comprises L234A / L235A mutations, G237A mutations, and M252Y / S254T / T256E mutations.

[0153] In some embodiments, the IgG1 heavy chain constant region suitable for use in the antibody molecules of the invention comprises or consists of the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence having at least 90% identity thereto, such as 95%, 96%, 97%, 99% or more identity thereto. In some embodiments, the heavy chain constant region comprises an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or more identity thereto, and comprises L234A / L235A mutations and G237A mutations.

[0154] In some embodiments, the IgG1 heavy chain constant region suitable for use in the antibody molecules of the invention comprises or consists of the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence having at least 90% identity thereto, such as 95%, 96%, 97%, 99% or more identity thereto. In some embodiments, the heavy chain constant region comprises an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or more identity thereto, and comprises L234A / L235A mutations, G237A mutations, and M252Y / S254T / T256E mutations.

[0155] In some embodiments, the light chain constant region of an anti-GDF15 antibody of the invention is a kappa or lambda light chain constant region, such as a kappa light chain constant region.

[0156] In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 45, or an amino acid sequence that is at least 90% identical thereto, such as 95%, 96%, 97%, 99% or more identical thereto.

[0157] In some embodiments, the anti-GDF15 antibodies or antigen-binding fragments thereof of the present invention comprise an antibody heavy chain. In some embodiments, the anti-GDF15 antibodies or antigen-binding fragments thereof of the present invention comprise an antibody light chain. In some embodiments, the anti-GDF15 antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain and a light chain. In some embodiments, the anti-GDF15 antibodies or antigen-binding fragments thereof of the present invention comprise two heavy chains and two light chains.

[0158] In one embodiment, the heavy chain of the anti-GDF15 antibody of the present invention

[0159] (i) comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28, 30, 33, 35 or 49; or

[0160] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 28, 30, 33, 35 or 49; or

[0161] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 28, 30, 33, 35 or 49, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions, more preferably said amino acid changes do not occur in the variable regions.

[0162] In some embodiments, the light chain of the anti-GDF15 antibody of the invention

[0163] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 29, 31, 34 or 36; or

[0164] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 29, 31, 34 or 36; or

[0165] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 29, 31, 34 or 36, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions, more preferably said amino acid changes do not occur in the variable regions.

[0166] In some specific embodiments of the present invention, the anti-GDF15 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain, wherein

[0167] (i) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0168] (ii) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0169] (iii) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 34, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0170] (iv) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 35, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 36, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0171] (v) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 34, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0172] In some specific embodiments of the present invention, the anti-GDF15 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain, wherein

[0173] (i) The heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 28, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 29;

[0174] (ii) the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 30, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 31;

[0175] (iii) the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 33, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 34;

[0176] (iv) the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 35, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 36; or

[0177] (v) The heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 49, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 34.

[0178] In some embodiments, the anti-GDF15 antibody is a monoclonal antibody.

[0179] In some embodiments, the anti-GDF15 antibody is humanized. Humanization can be achieved by replacing one or more amino acid residues, particularly framework sequences, in the heavy and light chain variable regions of a non-human native antibody with residues from the corresponding positions in the variable region of a conventional human antibody. Methods for humanizing antibodies are well known in the art. Typically, humanizing substitutions are performed in a manner that maintains the favorable binding properties of the antibody. Assays for determining the biological properties of humanized antibodies, such as binding affinity, are well known in the art to identify and select appropriate humanizing residue mutations or combinations of mutations.

[0180] In some embodiments, the humanized antibodies of the present invention can be obtained by a method comprising the following steps:

[0181] 1) determining the CDR loop structure of a parent antibody (e.g., an antibody produced from a hybridoma);

[0182] 2) Find the closest homologous sequence for each V / J region in a human germline sequence database (e.g., IMGT database);

[0183] 3) Screening for the human germline framework that best matches the heavy chain and the lowest amount of back mutations. In one embodiment of the present invention, the human germline gene sequence IGHV1-3*01 is used as the humanized framework for the heavy chain variable region; the CDR regions of the chimeric antibody are constructed onto the human framework regions;

[0184] 4) Screening for the human germline framework that best matches the light chain and the lowest amount of back mutations. In one embodiment of the present invention, the human germline gene is IGKV6-21*01; constructing the CDR regions of the chimeric antibody onto the human framework regions;

[0185] 5) Using sequence and structural features, determine the amino acid positions in the framework regions that maintain CDR function;

[0186] 6) Perform back mutations (return to the input amino acid type) at sequence positions identified as important;

[0187] 7) optionally, optimizing amino acids at risk sites; and

[0188] 8) Obtaining humanized antibodies and optionally sequencing the antibody sequence.

[0189] In one embodiment, the anti-GDF15 antibodies of the present invention also encompass antibody fragments thereof (e.g., antigen-binding fragments), preferably selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies such as VHH, dAb (domain antibody), heavy chain antibodies, or linear antibodies. In one embodiment, the anti-GDF15 antibody fragment of the present invention is Fab, comprising VH and VL as described herein, as well as CH1 and CL.

[0190] In one embodiment, the anti-GDF15 antibody of the present invention may also be a bispecific antibody or a multispecific antibody that specifically binds to GDF15 and one or more other antigens.

[0191] Another specific aspect of the present invention relates to an anti-GDF15 antibody, which is an anti-GDF15 VHH antibody or a heavy chain antibody comprising the same.

[0192] Single domain antibodies

[0193] In some embodiments, the anti-GDF15 antibodies of the invention are single domain antibodies, particularly VHH antibodies.

[0194] Single-domain antibodies, or VHH antibodies, have a molecular weight approximately one-tenth that of a human IgG molecule and a physical diameter of only a few nanometers. Due to their small molecular size, single-domain monoclonal antibodies offer advantages over conventional four-chain antibodies: high stability and solubility, as well as the ability to recognize hidden antigenic sites. Furthermore, single-domain antibodies are cheaper to prepare than conventional four-chain antibodies. In addition to their use as standalone molecules, single-domain antibodies are also suitable components for constructing multispecific molecules.

[0195] In some embodiments, the anti-GDF15 single-domain antibody of the present invention is a VHH antibody comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region generally has the following structure: FR1-VHH CDR1-FR2-VHH CDR2-FR3-VHH CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4; VHH CDR1 to VHH CDR3 refer to complementarity determining regions 1 to 3. The CDR sequences in the VHH variable region can be determined according to any of the CDR definition schemes described in the "Definitions" section, and preferably, the boundaries of the three CDRs in the VHH sequence can be defined using IMGT.

[0196] In some embodiments, the anti-GDF15 VHH antibody of the present invention comprises

[0197] (i) three complementarity determining regions (CDRs) contained in the VH represented by any one of SEQ ID NO: 19 or 27, or

[0198] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three CDR regions relative to the sequence of (i);

[0199] Preferably, the CDR sequences are defined according to IMGT.

[0200] In some embodiments, the anti-GDF15 VHH antibody of the present invention comprises or consists of a heavy chain variable region comprising

[0201] (i) three complementarity determining regions (CDRs) contained in the VH represented by any one of SEQ ID NO: 19 or 27, or

[0202] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three CDR regions relative to the sequence of (i);

[0203] Preferably, the CDR sequences are defined according to IMGT.

[0204] In some embodiments, the anti-GDF15 VHH antibodies of the present invention comprise complementarity determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3. In some embodiments, the anti-GDF15 VHH antibodies of the present invention comprise or consist of a heavy chain variable region comprising complementarity determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3.

[0205] In some embodiments, the VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 16, or the VHH CDR1 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 16.

[0206] In some embodiments, the VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 17, or the VHH CDR2 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 17.

[0207] In some embodiments, the VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 18, or the VHH CDR3 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 18.

[0208] In one embodiment, the anti-GDF15 VHH antibody of the present invention comprises complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein

[0209] VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 16, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 17, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 18.

[0210] In one embodiment, an anti-GDF15 VHH antibody of the invention comprises or consists of a heavy chain variable region, wherein said heavy chain variable region comprises complementarity determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, wherein

[0211] VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 16, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 17, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 18.

[0212] In some embodiments, the anti-GDF15 VHH antibodies of the present invention comprise or consist of a heavy chain variable region, wherein the heavy chain variable region

[0213] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in any one of SEQ ID NO: 19 or 27; or

[0214] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NO: 19 or 27; or

[0215] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in any one of SEQ ID NOs: 19 or 27, preferably, the amino acid changes do not occur in the CDR regions.

[0216] In some embodiments, the anti-GDF15 VHH antibody of the present invention comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 19 or 27.

[0217] In some embodiments, the VHH antibodies of the present invention comprise CDR amino acid sequences and / or framework (FR) amino acid sequences derived from camelid heavy chain antibodies produced by immunizing camelids (e.g., alpacas). In some embodiments, the VHH monoclonal antibodies of the present invention derived from camelid heavy chain antibodies can be engineered, for example, to comprise framework region sequences derived from human amino acid sequences (i.e., human antibodies) or other non-camelid mammalian species. In one embodiment, to further improve the properties (e.g., affinity) of the engineered antibodies, camelid amino acid residues located at corresponding positions in the parent camelid antibody can be introduced into the engineered antibodies at one or more positions (e.g., framework regions) by back mutation.

[0218] In one embodiment, the VHH antibody of the present invention is a humanized antibody. Humanization can be achieved by replacing one or more amino acid residues, especially framework region sequences, in a non-human natural VHH sequence (e.g., a VHH sequence from camelids or alpacas immunization) with residues at corresponding positions in the heavy chain VH of a conventional human antibody. Methods for humanizing VHHs are well known in the art, such as the method described in Example 3. Typically, humanizing substitutions are performed in a manner that maintains the favorable binding properties of single-domain antibodies. Tests for determining the biological properties of humanized single-domain antibodies, such as binding affinity, are well known in the art to determine and select suitable humanized residue mutations or combinations of mutations.

[0219] In some embodiments, the humanized single domain antibody of the present invention can be obtained by a method comprising the following steps:

[0220] Determine the CDR loop structure of a parent single domain antibody (e.g., a camelid VHH antibody screened from a phage display library);

[0221] Find the closest homologous sequence for each V / J region in the human germline sequence database as a template, for example, by aligning with the IMGT human antibody heavy chain variable region germline gene database (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi), and select the heavy chain variable region germline gene with high homology to the VHH antibody as the template;

[0222] The CDRs of the VHH antibody are respectively transplanted into the corresponding selected human templates to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CR3-FR4. Preferably, the framework sequence used for replacement has structural similarity with the framework sequence of the antibody to be humanized, for example, having a sequence identity of at least 80%, 85%, 90%, or 95%, 96%, 97%, 98%, or 99% or more;

[0223] If necessary, key amino acids in the FR region are back-mutated to amino acids corresponding to the VHH antibody to maintain the original affinity, thereby obtaining a humanized anti-GDF15 VHH antibody. The VHH antibody is optionally sequenced.

[0224] In some embodiments, the back mutation site is selected from one or more of the framework (FR).

[0225] In some embodiments, the heavy chain variable region germline gene suitable for humanization of the VHH antibodies of the present invention is selected from IGHV3-23*04.

[0226] In some embodiments, the present invention also provides functional variants of single domain antibodies of the present invention (particularly VHH antibodies). The functional variants can be introduced into the encoding nucleic acid sequence of the exemplary single domain antibody of the present invention by methods well known in the present invention, such as by random or site-directed mutagenesis, for example, by introducing CDR sequences and / or FR sequences, and then screening (for example, by phage display library screening) variants that maintain the desired properties to obtain functional variants. Typically, functional variants maintain significant sequence identity with the parent single domain antibody (or VHH). Preferably, the functional variant maintains the desired biological properties of the parent single domain antibody (or VHH), for example, relative to the biological activity of the parent, the variant has a comparable (for example, at least 50%, 60%, 70%, 80%, preferably more than 90%) biological activity, or improved biological activity (for example, 110-150% or higher). The desired biological properties include, for example, but are not limited to, binding affinity to the target antigen (e.g., GDF15) (as measured by a KD value), activity in blocking binding of the target antigen to a receptor (e.g., as measured by an IC50 value), activity in activating T cells in in vitro or in vivo experiments (e.g., as measured by released cytokines), and inhibition of tumor growth / survival in in vitro or in vivo experiments.

[0227] In some embodiments, the present invention provides affinity variants of the VHH polypeptides of the present invention. Preferably, the affinity variants exhibit one or more amino acid changes in the amino acid sequence relative to the parent single domain antibody from which they are derived, wherein the affinity variants have altered binding affinity for the target antigen compared to the parent antibody.

[0228] Heavy chain antibodies

[0229] In another aspect of the present invention, the present invention also provides a heavy chain antibody comprising the heavy chain variable region of the VHH antibody of the present invention.

[0230] In some embodiments, a single domain antibody or VHH of the present invention (e.g., a camel-derived VHH or a humanized form thereof) can be linked to a constant region of a human antibody or a portion thereof, such as an Fc region, to produce a heavy chain antibody comprising a VHH-constant region or VHH-CH1-Fc or VHH-Fc. In one embodiment, the heavy chain antibody comprises a VHH antibody of the present invention and an Fc region at its C-terminus. In some embodiments, VHH and Fc are linked by a hinge region or a portion thereof, such as a hinge region from IgG (e.g., a hinge region of IgG1, 2, 3, or 4) or a portion thereof.

[0231] In some embodiments, the anti-GDF15 heavy chain antibodies of the present invention comprise a VHH as defined herein, and a heavy chain constant region or an Fc region of a heavy chain constant region as defined herein. In some embodiments, the Fc region comprises an antibody hinge region or a portion thereof, such as a hinge region or a portion thereof from an IgG. The hinge region may contain mutations. In some embodiments, the hinge region may be EPKSS or EPKSC.

[0232] In one embodiment, the heavy chain antibody comprises an Fc region or portion thereof from a camelid (e.g., an alpaca). In one embodiment, the heavy chain antibody is produced and isolated by immunizing the camelid, e.g., an alpaca. Various methods are known in the art for immunizing camelids and isolating the resulting VHH antibodies or heavy chain antibodies against an antigen of interest.

[0233] In some embodiments, the heavy chain antibody comprises a constant region from a human or non-human primate (eg, cynomolgus monkey) antibody, eg, a constant region from human IgG1, human IgG2, human IgG3, or human IgG4, eg, as defined herein.

[0234] In some embodiments, the heavy chain antibody comprises an Fc portion from a human or non-human primate (e.g., cynomolgus monkey). In yet another embodiment, the heavy chain antibody comprises a human IgG Fc region, e.g., a human IgG1, human IgG2, human IgG3, or human IgG4 Fc region, preferably a human IgG1 or human IgG4 Fc region, such as a human IgG1 Fc region, e.g., as defined herein.

[0235] In one embodiment, the heavy chain antibody according to the present invention can dimerize with another polypeptide chain (e.g., the same or different heavy chain antibody) comprising an Fc region through the Fc region. Therefore, in one embodiment, the present invention also provides a homologous or heterologous multimeric protein comprising a heavy chain antibody of the present invention. In a preferred embodiment, the protein preferably comprises a heavy chain antibody formed by pairing two identical heavy chain antibody chains.

[0236] The Fc region of the present invention may be mutated to obtain desired properties. Mutations to the Fc region are known in the art and are as defined herein.

[0237] In some embodiments, an anti-GDF15 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain comprising or consisting of a heavy chain variable region and an Fc region.

[0238] In some embodiments, the anti-GDF15 antibodies or antigen-binding fragments thereof of the present invention comprise or consist of a heavy chain comprising a heavy chain variable region of a VHH of the present invention and an Fc region (e.g., an Fc region comprising part of a hinge region or a hinge region), wherein the heavy chain

[0239] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 32 or 37; or

[0240] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 32 or 37; or

[0241] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 32 or 37, preferably, the amino acid changes do not occur in the CDR regions.

[0242] III. Encoding nucleic acids and host cells containing the same

[0243] In one aspect, the present invention provides nucleic acids encoding any of the above anti-GDF15 antibodies or antigen-binding fragments thereof, or either chain thereof.

[0244] For example, the nucleic acids of the present invention include nucleic acids encoding an amino acid sequence selected from any one of SEQ ID NOs: 7, 8, 14, 15, 19, 23-37, and 49, or nucleic acids encoding an amino acid sequence selected from any one of SEQ ID NOs: 7, 8, 14, 15, 19, 23-37, and 49 that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 7, 8, 14, 15, 19, 23-37, and 49.

[0245] As will be appreciated by those skilled in the art, because of codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by a variety of nucleic acid sequences. Nucleic acid sequences encoding molecules of the present invention can be produced by methods well known in the art, for example, by de novo solid phase DNA synthesis, or by PCR amplification.

[0246] In one aspect, the present invention provides nucleic acids encoding any of the above antibodies or antibody chains. When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid can exhibit human and / or monkey (eg, cynomolgus monkey) GDF15 antigen binding ability.

[0247] In one embodiment, the nucleic acids encoding each chain of an antibody or antigen-binding fragment thereof of the present invention can be in the same vector or in different vectors. In another embodiment, the nucleic acids encoding each chain of an antibody or antigen-binding fragment thereof of the present invention can be introduced into the same or different host cells for expression. Therefore, in some embodiments, the method for producing an antibody or antigen-binding fragment thereof of the present invention comprises the steps of culturing a host cell containing nucleic acids encoding each chain under conditions suitable for expressing each chain of the molecule to produce the antibody or antigen-binding fragment thereof of the present invention.

[0248] In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). In one embodiment, the expression vector is, for example, a pcDNA vector, such as pcDNA3.1.

[0249] In one embodiment, a host cell comprising the nucleic acid or the vector is provided, for example, a vector for cloning or expressing an anti-GDF15 antibody or antigen-binding fragment thereof. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells such as CHO cells (e.g., CHO-S, such as ExpiCHO-S) or 293 cells (e.g., 293F or HEK293 cells) or other cells suitable for preparing antibodies or fragments thereof. In one embodiment, the host cell is prokaryotic, for example, a bacterium, such as Escherichia coli.

[0250] In some embodiments, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. For example, fungal and yeast strains whose glycosylation pathways have been "humanized" result in the production of antibodies with partially or fully human glycosylation patterns. Host cells suitable for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines modified to be suitable for suspension growth can be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 lines (COS-7) transformed with SV40; human embryonic kidney lines (HEK293, 293F or 293T cells), etc. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells, CHO-S cells, ExpiCHO, etc.; and myeloma cell lines such as Y0, NS0 and Sp2 / 0. Mammalian host cell lines suitable for producing antibodies are known in the art.

[0251] IV. Production and Purification of Anti-GDF15 Antibodies or Antigen-Binding Fragments thereof of the Invention

[0252] In one embodiment, a method for preparing an anti-GDF15 antibody or antigen-binding fragment thereof of the present invention is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the anti-GDF15 antibody or antigen-binding fragment thereof (e.g., any one polypeptide chain and / or multiple polypeptide chains) or an expression vector comprising the nucleic acid under conditions suitable for the expression of the anti-GDF15 antibody or antigen-binding fragment thereof or its chains, as provided above, and optionally recovering the anti-GDF15 antibody or antigen-binding fragment thereof from the host cell (or host cell culture medium).

[0253] The polynucleotide encoding the polypeptide chains of the anti-GDF15 antibodies or antigen-binding fragments thereof of the present invention can be inserted into one or more vectors for further cloning and / or expression in host cells. Methods well known to those skilled in the art can be used to construct expression vectors. Once an expression vector comprising one or more nucleic acid molecules of the present invention has been prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this goal, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection, or other conventional techniques.

[0254] Anti-GDF15 antibodies or antigen-binding fragments thereof prepared as described herein can be purified by known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these will be apparent to those skilled in the art.

[0255] The purity of the anti-GDF15 antibodies or antigen-binding fragments thereof of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

[0256] V. Assays for Anti-GDF15 Antibodies or Antigen-Binding Fragments thereof

[0257] The anti-GDF15 antibodies or antigen-binding fragments thereof provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by various assays known in the art.

[0258] In one aspect, the anti-GDF15 antibodies or antigen-binding fragments thereof of the present invention are tested for their target (e.g., antigen, such as free antigen or antigen expressed on cells) binding activity, for example, by known methods such as thin-layer interferometry, ELISA, flow cytometry, etc. Binding to VEGF and / or GDF15 (or VEGF and / or GDF15 expressed on cells) can be determined using methods known in the art, exemplary methods are disclosed herein, such as those described in Example 7. In some embodiments, radioimmunoassay (RIA), thin-layer interferometry (BLI), electrochemiluminescence (ECL), surface plasmon resonance (SPR), or flow cytometry (FACS) is used for measurement.

[0259] The present invention also provides assays for identifying the biological activity of an anti-GDF15 antibody or antigen-binding fragment thereof. The biological activity is selected from the properties of the anti-GDF15 antibody or antigen-binding fragment thereof of the present invention.

[0260] For example, cross-reactivity of the molecules of the present invention with human TGFβ family proteins can be determined by methods known in the art, such as ELISA, or by exemplary methods disclosed in the Examples herein, such as the method shown in Example 8.

[0261] For example, the activity of the molecules of the present invention in blocking GDF15 activation can be determined by methods known in the art, such as the method described in Example 9.

[0262] For example, the activity of the molecules of the present invention in inhibiting the phosphorylation level of ERK can be determined by methods known in the art, such as the method shown in Example 10.

[0263] For example, the reversal or treatment of cachexia, such as tumor-induced cachexia or cachexia induced by chemotherapeutic agents such as cisplatin, by the molecules of the present invention can be determined by methods known in the art, such as in vivo efficacy experiments conducted on tumor models, such as the methods described in Example 11 or 12.

[0264] For example, the pharmacokinetic analysis of the molecules of the present invention can be performed by methods known in the art, such as pharmacokinetic experiments, such as the method shown in Example 13.

[0265] Cells used in any of the above in vitro assays are primary cells or cell lines, including cells that naturally express or overexpress VEGF (e.g., human or monkey (e.g., cynomolgus monkey)) or GDF15 (e.g., human or monkey (e.g., cynomolgus monkey) GDF15), such as cells that overexpress GDF15, such as 293 cells or CHO cells or HT-1080 cells.

[0266] It will be appreciated that any of the above assays can be performed using a combination of the antibodies of the invention and additional active agents.

[0267] VI. Immunoconjugates, Pharmaceutical Compositions, Pharmaceutical Combination Products, and Kits of Anti-GDF15 Antibodies or Antigen-Binding Fragments thereof

[0268] In some embodiments, the present invention provides an immunoconjugate comprising any of the anti-GDF15 antibodies or antigen-binding fragments thereof described herein. Preferably, the immunoconjugate comprises one or more other therapeutic agents (e.g., cytotoxins or small molecule compounds) or markers. In some embodiments, the immunoconjugate is an antibody drug conjugate (ADC).

[0269] In some embodiments, the present invention provides a composition, medicament, or formulation comprising any of the anti-GDF15 antibodies, or antigen-binding fragments thereof, or immunoconjugates described herein. Preferably, the composition is a pharmaceutical composition.

[0270] In one embodiment, a composition, eg, a pharmaceutical composition, comprises an anti-GDF15 antibody or antigen-binding fragment or immunoconjugate of the invention in combination with one or more additional therapeutic agents.

[0271] The composition, medicament or preparation of the present invention may further comprise suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers. As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents and absorption delaying agents, etc.

[0272] For the use of pharmaceutical excipients and their applications, see also "Hand book of Pharmaceutical Excipients", 8th edition, RC Rowe, PJ Eskey and S C Owen, Pharmaceutical Press, London, Chicago.

[0273] The compositions, medicaments or preparations of the present invention can be in various forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injections or eye drops), powders or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

[0274] Medicaments or formulations comprising the anti-GDF15 antibodies or antigen-binding fragments thereof described herein can be prepared by mixing the anti-GDF15 antibodies or antigen-binding fragments thereof or immunoconjugates of the present invention having the desired purity with one or more optional pharmaceutical excipients, for example, in the form of lyophilized formulations or aqueous solutions.

[0275] The compositions or medicaments or formulations of the present invention may also contain more than one active ingredient as required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to also provide other therapeutic agents.

[0276] The present invention also provides a pharmaceutical combination or pharmaceutical combination product comprising an anti-GDF15 antibody or antigen-binding fragment or immunoconjugate thereof of the present invention and one or more other therapeutic agents.

[0277] The present invention also provides a complete kit comprising the drug combination, for example, the complete kit comprises in the same package:

[0278] - a first container containing a pharmaceutical composition comprising an anti-GDF15 antibody, or an antigen-binding fragment thereof, or an immunoconjugate of the present invention;

[0279] - A second container containing a pharmaceutical composition comprising an additional therapeutic agent.

[0280] In some embodiments, the additional therapeutic agent is, for example, a chemotherapeutic agent, a cytokine, a cytotoxic agent, another antibody, a small molecule drug, or an immunomodulatory agent (eg, an immunosuppressant).

[0281] VII. Uses of anti-GDF15 antibodies or antigen-binding fragments thereof and methods of using the same

[0282] In one aspect, the present invention relates to an anti-GDF15 antibody or antigen-binding fragment thereof of the present invention (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, combination products, etc. comprising the same) for use in therapy.

[0283] In some embodiments, the present invention relates to an anti-GDF15 antibody or antigen-binding fragment thereof of the present invention (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, combination products, etc. comprising the same) for use as a medicament.

[0284] In another aspect, the present invention provides a method for preventing or treating a disease or condition in an individual, comprising administering to the individual an anti-GDF15 antibody or antigen-binding fragment thereof of the present invention, or an immunoconjugate, composition, medicament, or formulation comprising the same.

[0285] In some embodiments, the condition is cachexia.

[0286] The term "cachexia" as used herein includes metabolic diseases and complications that occur with (or are induced by) various diseases (e.g., chronic diseases), or metabolic diseases or complications induced by certain therapies. In some embodiments, the disease is selected from tumors, chronic heart failure, congestive heart failure, sarcopenia, chronic obstructive pulmonary disease (COPD), sarcopenia, or chronic kidney disease (CKD). In some embodiments, the tumor is cancer. In some embodiments, the therapy is, for example, chemotherapy or chemotherapy combined with immunotherapy for tumors. In some embodiments, chemotherapy includes administering a chemotherapeutic agent, such as cisplatin, to the patient.

[0287] In some embodiments, the disease is a tumor. In some embodiments, the disease is cancer. In some embodiments, the cancer can be in the early, middle, or late stages or be metastatic. In some embodiments, the tumor can be a solid tumor or a blood tumor, such as a malignant solid tumor or a blood tumor. In some embodiments, the cancer is fibrosarcoma, prostate cancer, pancreatic cancer, endometrial cancer, or colorectal cancer.

[0288] In some embodiments, the sample from the individual with cachexia has GDF15, e.g., has or has elevated GDF15 protein and / or nucleic acid levels, e.g., compared to GDF15 protein and / or nucleic acid levels in the same tissue from a healthy individual. In some embodiments, the sample is a body fluid, e.g., blood, serum, or plasma.

[0289] In some embodiments, the cancer is a GDF15-positive cancer. In some embodiments, the cancer is characterized by having or having elevated protein and / or nucleic acid levels of GDF15 in the patient (e.g., in the patient's cancer tissue or cells) (e.g., compared to the protein and / or nucleic acid levels of GDF15 in the same tissue of a healthy individual, or compared to the protein and / or nucleic acid levels of GDF15 in adjacent healthy tissue of the patient), for example, having or having elevated protein and / or nucleic acid levels of GDF15 in tumor cells of the cancer (e.g., compared to the protein and / or nucleic acid levels of GDF15 in cells of the same tissue of a healthy individual, or compared to the protein and / or nucleic acid levels of GDF15 in healthy cells of the same tissue of the patient or in cells of adjacent healthy tissue).

[0290] The anti-GDF15 antibodies or antigen-binding fragments thereof of the present invention (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, combination products, etc. comprising the same) can be administered by any suitable method, including parenteral administration, intrapulmonary administration, and intranasal administration, and, if desired for local treatment, intralesional administration. Parenteral injection or infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or infusion. Depending to some extent on whether the administration is short-term or long-term, the administration can be by any suitable route, for example, by injection, such as intravenous injection. Various administration schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

[0291] For the prevention or treatment of a disease or condition, the appropriate dosage of the anti-GDF15 antibodies or antigen-binding fragments thereof (and immunoconjugates, compositions, pharmaceutical compositions, formulations, combination products, etc. comprising the same) of the present invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the administration is for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient as a single treatment or over a series of treatments.

[0292] In other aspects, the present invention provides the anti-GDF15 antibodies or antigen-binding fragments thereof, or immunoconjugates or compositions or combination products comprising the same, for use as described herein, such as for preventing or treating the relevant diseases or conditions mentioned herein.

[0293] In other aspects, the present invention provides use of the anti-GDF15 antibodies or antigen-binding fragments thereof, or immunoconjugates or compositions or combination products comprising the same, in the production or preparation of medicaments for the uses described herein, such as for preventing or treating the relevant diseases or conditions mentioned herein.

[0294] In some embodiments, anti-GDF15 antibodies or antigen-binding fragments thereof (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, etc. comprising the same) can also be administered in combination with one or more other therapies, such as treatment modalities and / or other therapeutic agents, for the uses described herein, e.g., for preventing and / or treating the relevant diseases or conditions mentioned herein.

[0295] In some embodiments, the treatment modality is, for example, surgery or radiation therapy.

[0296] In some embodiments, the other therapeutic agent is, for example, a drug effective in cancer, such as a chemotherapeutic agent, a cytokine, a cytotoxic agent, other antibodies, a small molecule drug, or an immunomodulatory agent (eg, an immunosuppressant).

[0297] VIII. Diagnosis and Testing

[0298] In one aspect, the present invention also relates to methods for diagnosis and detection (eg, for diagnostic or non-diagnostic purposes) of anti-GDF15 antibodies or antigen-binding fragments thereof and compositions for diagnosis and detection comprising the same.

[0299] In certain embodiments, the anti-GDF15 antibodies provided herein can be used to detect the presence of GDF15 in a biological sample.

[0300] The term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods can involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays, PCR-techniques (e.g., RT-PCR). In certain embodiments, the biological sample is a body fluid, such as blood, serum, or plasma.

[0301] In certain embodiments, the method comprises contacting a biological sample with an anti-GDF15 antibody or antigen-binding fragment thereof as described herein under conditions that allow it to bind to GDF15, and detecting whether a complex is formed between the anti-GDF15 antibody or antigen-binding fragment thereof and GDF15. The formation of a complex indicates the presence of GDF15. This method can be an in vitro or in vivo method.

[0302] In certain embodiments, a labeled anti-GDF15 antibody or antigen-binding fragment thereof is provided. Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as indirectly detectable moieties such as enzymes or ligands, e.g., via an enzymatic reaction or molecular interaction. In some embodiments, the label is, for example, a marker such as biotin or hFc.

[0303] In some embodiments provided herein, the sample is obtained prior to treatment with an anti-GDF15 antibody or antigen-binding fragment thereof of the present invention. In some embodiments, the sample is obtained prior to treatment with another therapy. In some embodiments, the sample is obtained during or after treatment with another therapy.

[0304] In some embodiments, GDF15 is detected prior to treatment, eg, prior to initiation of treatment or prior to a treatment following a treatment interval.

[0305] In some specific embodiments, the present invention relates to:

[0306] 1. An anti-GDF15 antibody or an antigen-binding fragment thereof, comprising

[0307] (i) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 7, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 8;

[0308] (ii) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 14, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 15;

[0309] (iii) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 23, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 24; or

[0310] (iv) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 25, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 26.

[0311] 2. An anti-GDF15 antibody or an antigen-binding fragment thereof comprising HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3, wherein

[0312] (i) HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 4; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 5; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 6;

[0313] (ii) HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 9; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 10; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 11; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 12; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 13; or

[0314] (iii). HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 20; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 21; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 22; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 6.

[0315] 3. The antibody or antigen-binding fragment thereof according to embodiment 1 or 2, comprising a heavy chain variable region VH, wherein the heavy chain variable region

[0316] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 7, 14, 23 or 25; or

[0317] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 7, 14, 23 or 25.

[0318] 4. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 3, comprising a light chain variable region VL, wherein the light chain variable region

[0319] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 8, 15, 24 or 26; or

[0320] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 8, 15, 24 or 26.

[0321] 5. The antibody or antigen-binding fragment thereof according to embodiment 1 or 2, comprising a heavy chain variable region VH and a light chain variable region VL, wherein

[0322] (i) the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0323] (ii) the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0324] (iii) the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0325] (iv) the VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0326] 6. The antibody or antigen-binding fragment thereof according to embodiment 1 or 2, comprising a heavy chain variable region VH and a light chain variable region VL, wherein

[0327] (i) The VH comprises the amino acid sequence shown in SEQ ID NO: 7 or consists of the amino acid sequence, and the VL comprises the amino acid sequence shown in SEQ ID NO: 8 or consists of the amino acid sequence;

[0328] (ii) the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 14, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 15;

[0329] (iii) the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 23, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 24; or

[0330] (iv) the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 25, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 26.

[0331] 7. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 6, further comprising a heavy chain constant region HC, for example, the antibody heavy chain constant region HC is a heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably a heavy chain constant region of IgG1, optionally, the heavy chain constant region has an Fc region amino acid modification, such as a modification that reduces or eliminates effector function, a modification that increases affinity for FcRn, a modification that extends half-life, etc.; preferably, the heavy chain constant region has one or more of L234A / L235A mutation, G237A mutation and M252Y / S254T / T256E mutation; more preferably, the heavy chain constant region has L234A / L235A mutation, G237A mutation and M252Y / S254T / T256E mutation.

[0332] 8. The antibody or antigen-binding fragment thereof of embodiment 7, wherein the heavy chain constant region

[0333] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 44 or 48; or

[0334] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 44 or 48.

[0335] 9. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-8, which comprises a light chain constant region, for example, the light chain constant region is a lambda or kappa light chain constant region.

[0336] 10. The antibody or antigen-binding fragment thereof according to embodiment 9, wherein the light chain constant region

[0337] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 45; or

[0338] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 45.

[0339] 11. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 10, wherein the antibody comprises a heavy chain, wherein the heavy chain

[0340] (i) comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28, 30, 33, 35 or 49; or

[0341] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 28, 30, 33, 35 or 49.

[0342] 12. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 11, wherein the antibody comprises a light chain, wherein the light chain

[0343] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 29, 31, 34 or 36; or

[0344] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 29, 31, 34 or 36.

[0345] 13. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-12, comprising a heavy chain and a light chain, wherein

[0346] (i) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0347] (ii) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0348] (iii) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 34, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0349] (iv) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 35, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 36, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0350] (v) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 34, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0351] 14. The antibody or antigen-binding fragment thereof of any one of embodiments 1-13, comprising a heavy chain and a light chain, wherein

[0352] (i) The heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 28, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 29;

[0353] (ii) the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 30, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 31;

[0354] (iii) the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 33, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 34;

[0355] (iv) the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 35, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 36; or

[0356] (v) The heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 49, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 34.

[0357] 15. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-14, wherein the antibody is a chimeric antibody or a humanized antibody.

[0358] 16. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-15, wherein the antibody is a monoclonal antibody.

[0359] 17. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-16, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single-chain antibody, (Fab')2, single-domain antibody such as heavy chain variable region VHH, dAb (domain antibody), heavy chain antibody or linear antibody.

[0360] 18. The antibody or antigen-binding fragment thereof of embodiment 17, wherein the single-chain antibody is scFv.

[0361] 19. A VHH antibody that specifically binds to GDF15, comprising the three complementarity determining regions (CDRs) contained in the VHH shown in any one of SEQ ID NO: 19 or 27,

[0362] Preferably, the CDR sequences are defined according to IMGT.

[0363] 20. The VHH antibody of embodiment 19, comprising complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein

[0364] VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 16, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 17, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 18.

[0365] 21. The VHH antibody of embodiment 19, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region

[0366] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in any one of SEQ ID NO: 19 or 27; or

[0367] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NO: 19 or 27.

[0368] 22. A heavy chain antibody that specifically binds to GDF15, comprising the VHH antibody of any one of embodiments 19-21.

[0369] 23. The heavy chain antibody of embodiment 22, comprising the VHH antibody of any one of embodiments 19-21 linked to an antibody constant region or Fc region, preferably, the antibody constant region or Fc region is from human IgG1, human IgG2, human IgG3 or human IgG4, preferably, the Fc region

[0370] (i) comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 46; or

[0371] (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 46.

[0372] 24. The heavy chain antibody of embodiment 22, comprising the VHH antibody of any one of embodiments 1-3 linked to an antibody Fc region, wherein the Fc region is an Fc region from human IgG1, IgG2, IgG3 or IgG4, optionally comprising L234A / L235A mutations and G237A mutations, preferably, the Fc region

[0373] (i) comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:43; or

[0374] (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 43.

[0375] 25. The heavy chain antibody according to embodiment 22, wherein

[0376] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 32 or 37; or

[0377] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 32 or 37; or

[0378] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 32 or 37, preferably, the amino acid changes do not occur in the CDR regions.

[0379] 26. The VHH antibody of any one of embodiments 19-21, or the heavy chain antibody of any one of embodiments 22-25, wherein the antibody is a chimeric antibody or a humanized antibody.

[0380] 27. A multispecific antibody, such as a bispecific antibody, comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-18, or the VHH antibody of any one of embodiments 19-21, or the heavy chain antibody of any one of embodiments 22-26.

[0381] 28. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of embodiments 1-18, or the VHH antibody of any one of embodiments 19-21, or the heavy chain antibody of any one of embodiments 22-26, or any chain of the multispecific antibody of embodiment 27, or consisting of said nucleic acid sequence.

[0382] 29. An expression vector comprising the nucleic acid molecule of embodiment 28, preferably, the expression vector is pCDNA, such as pCDNA3.1.

[0383] 30. A host cell comprising the nucleic acid molecule of embodiment 28 or the expression vector of embodiment 29. Preferably, the host cell is prokaryotic or eukaryotic, such as 293 cells or CHO cells.

[0384] 31. A method for preparing the antibody or antigen-binding fragment thereof of any one of embodiments 1-18, or the VHH antibody of any one of embodiments 19-21, or the heavy chain antibody of any one of embodiments 22-26, or the multispecific antibody of embodiment 27, the method comprising culturing a host cell comprising the nucleic acid molecule of embodiment 28 or the expression vector of embodiment 29 or the host cell of embodiment 30 under conditions suitable for expression of the chains of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0385] 32. The antibody or antigen-binding fragment thereof or multispecific binding molecule prepared by the method described in embodiment 31.

[0386] 33. An immunoconjugate comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-18, or the VHH antibody of any one of embodiments 19-21, or the heavy chain antibody of any one of embodiments 22-26, or the multispecific antibody of embodiment 27, e.g., the immunoconjugate is an ADC.

[0387] 34. A pharmaceutical composition, medicament or formulation comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-18, or the VHH antibody of any one of embodiments 19-21, or the heavy chain antibody of any one of embodiments 22-26, or the multispecific antibody of embodiment 27, or the immunoconjugate of embodiment 33, and optionally a pharmaceutically acceptable excipient.

[0388] 35. A pharmaceutical combination product comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-18, or the VHH antibody of any one of embodiments 19-21, or the heavy chain antibody of any one of embodiments 22-26, or the multispecific antibody of embodiment 27, or the immunoconjugate of embodiment 33, and one or more other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulatory agents).

[0389] 36. A method for preventing or treating cachexia in a subject, comprising administering to the individual an effective amount of the antibody or antigen-binding fragment thereof of any one of embodiments 1-18, or the VHH antibody of any one of embodiments 19-21, or the heavy chain antibody of any one of embodiments 22-26, or the multispecific antibody of embodiment 27, or the immunoconjugate of embodiment 33, or the pharmaceutical composition or formulation of embodiment 34; or the pharmaceutical combination product of embodiment 35.

[0390] 37. The method of embodiment 36, wherein the cachexia occurs with a disease selected from a tumor such as cancer, chronic heart failure, congestive heart failure, sarcopenia, chronic obstructive pulmonary disease (COPD), sarcopenia or chronic kidney disease (CKD), for example, the tumor is a solid tumor or a blood tumor, such as a malignant solid tumor or a blood tumor, such as fibrosarcoma, prostate cancer, pancreatic cancer, endometrial cancer or colorectal cancer.

[0391] 38. The method of embodiment 36, wherein the cachexia is induced by chemotherapy or chemotherapy combined with immuno-oncology therapy, for example, the chemotherapy comprises administration of a chemotherapeutic agent such as cisplatin.

[0392] 39. The method of any one of embodiments 36-38, wherein the individual has GDF15 in a sample, e.g., has or has elevated protein levels and / or nucleic acid levels of GDF15, e.g., compared to protein levels and / or nucleic acid levels of GDF15 in the same tissue of a healthy individual, e.g., the sample is a body fluid, e.g., blood, serum, or plasma.

[0393] 40. The method of any one of embodiments 36-39, wherein the method further comprises administering the drug in combination with other therapies, such as treatment modalities (e.g., surgery or radiotherapy) and / or other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint molecule inhibitors or agonists).

[0394] 41. A method for detecting the presence of GDF15 in a biological sample, comprising:

[0395] (i) contacting a biological sample with the antibody or antigen-binding fragment thereof of any one of embodiments 1-18, or the VHH antibody of any one of embodiments 19-21, or the heavy chain antibody of any one of embodiments 22-26, or the multispecific antibody of embodiment 27, or the immunoconjugate of embodiment 33 under conditions that allow binding to GDF15,

[0396] (ii) detecting whether a complex is formed between the antibody or multispecific binding molecule and GDF15,

[0397] The formation of the complex indicates the presence of GDF15.

[0398] Example

[0399] Example 1: Obtaining murine antibodies by immunizing mouse hybridomas

[0400] 1.1 Animal immunization

[0401] Five BALB / c mice (Nanjing Pengbo Biotechnology) were immunized with human GDF15 protein (Kaixia Biotechnology, Cat. No. GDF-HE115) every two weeks. Starting after the second immunization, venous blood was collected from the mice seven days after each immunization. The immune sera were assayed by enzyme-linked immunosorbent assay (ELISA) using human GDF15 protein (Kaixia Biotechnology, Cat. No. GDF-HE115), Cynomolgus GDF15 protein (Kaixia Biotechnology, Cat. No. GDF-CE115), and mouse GDF15 protein (Kaixia Biotechnology, Cat. No. GDF-ME115). Two mice were selected for animal fusion. Four days after the final booster immunization, spleens were harvested and lymphocytes isolated from the spleens were used for fusion to generate hybridomas.

[0402] 1.2 Hybridoma Screening

[0403] The culture supernatants of the hybridomas were subjected to primary screening tests by ELISA. In this assay, 30 clones that bound to Human GDF15 protein, Cynomolgus GDF15 protein, and Mouse GDF15 protein were selected for subcloning.

[0404] 1.3 Subcloning

[0405] The selected hybridoma cell line (parent clone) was subcloned to ensure monoclonality. Subcloning was performed by re-plating the parent clone using a single-step cloning system. 30 subclones were transferred to 96-well culture plates. The subclones were screened by ELISA for antibodies that simultaneously bound to human GDF15, cynomolgus GDF15 protein, and mouse GDF15 protein, as well as the binding antibodies.

[0406] 1.4 Hybridoma Sequencing

[0407] The hybridomas screened in the previous step were expanded and some cells were frozen for sequencing. The obtained antibody variable region amino acid sequences are shown in Table 1 below, where the CDRs were determined using Kabat.

[0408] Example 2: Production of anti-GDF15 antibodies by alpaca immunization

[0409] 2.1 Animal immunization

[0410] Two alpacas (Chengdu Apak Biotechnology Co., Ltd.) were immunized with human GDF15 protein (Kaixia Bio, Cat. No. GDF-HE115). Four 21-day immunizations were administered to the alpacas (Table 1). Venous blood was collected from the alpacas 10 days after the second and third booster immunizations. The immune serum was assayed by ELISA using recombinant human GDF15 protein. One alpaca was selected for blood collection and library establishment.

[0411] Table 2. Alpaca immunization schedule

[0412] 2.2 Construction and screening of alpaca immune library

[0413] Total RNA was extracted from alpaca peripheral blood using the Trizol method and reverse transcribed using the PrimeScript II 1st Strand cDNA Synthesis Kit (Takara, Cat. No. 6210A) to obtain cDNA. Nested PCR amplification of the cDNA was performed to recover a 750 bp product containing the nanobody, which was then purified by a second round of PCR amplification. The VHHs were then ligated into phage vectors using enzyme digestion and ligation, followed by electroporation, transformation, and plate plating. The next day, the library capacity was determined to be 2 × 10e8 CFU, with an insertion rate of 100%. After two rounds of solid-phase screening with human GDF15 recombinant protein, positive clones were identified by sequencing. The partial amino acid sequences obtained are shown in Table 3, with the CDRs determined using IMGT.

[0414] Table 3. Amino acid sequences of camel-derived VHHs of anti-GDF15 antibodies

[0415] Example 3: Synthesis and expression of anti-GDF15 chimeric antibodies

[0416] 3.1 Synthesis of anti-GDF15 chimeric antibodies

[0417] The variable region amino acid sequences of the two murine antibodies in Table 1 and the one camelid antibody in Table 3 were sent to Tongyong Biotech Co., Ltd. for codon optimization and gene synthesis. The genes encoding the VH and VL regions of the antibodies were sequentially inserted into expression vectors containing the human IgG1 heavy chain constant region (SEQ ID NO: 44, with L234A, L235A, G237A) and the kappa light chain constant region (SEQ ID NO: 45) to obtain murine chimeric antibodies.

[0418] The sequence of the control antibody Ponsegromab is from patent CN 112912395 A.

[0419] The amino acid sequence of the heavy chain of the control antibody Ponsegromab is as follows: (SEQ ID NO: 38)

[0420] The amino acid sequence of the light chain of the control antibody Ponsegromab is as follows: (SEQ ID NO: 39)

[0421] 3.2 Expression and purification of anti-GDF15 chimeric antibodies

[0422] The plasmids encoding the heavy and light chains of the anti-GDF15 antibody were co-transfected into ExpiCHO-S cells to express the murine chimeric antibodies and the control antibody Prosegromab as described in Table 4, and the corresponding purification was performed. TM ExpiCHO-S cells were co-transfected with the plasmids encoding the heavy and light chains of the anti-GDF15 antibodies described in Table 4 using an expression system (ThermoFisher, Cat# A29133) to express the anti-GDF15 chimeric antibodies, as well as a similarly expressed control antibody, Prosegromab. Cells were cultured for 10-12 days after transfection. When cell viability dropped to 60% to 70%, the supernatant was collected and the antibody expressed in the supernatant was purified using the MabSelect Sure Protein A affinity chromatography system (GE healthcare). The purified antibody was concentrated and sterile filtered. The antibody protein purity was determined by SDS-PAGE and size exclusion. The results showed that the antibody purity was >90% and was suitable for use in the next step.

[0423] Table 4: Names of murine chimeric antibodies and corresponding heavy and light chain combinations

[0424] Table 5: Camel-derived chimeric antibody names and corresponding heavy chains

[0425] Example 4: Humanization of murine anti-GDF15 antibody and construction of humanized anti-GDF15 antibody with YTE mutation in the Fc region

[0426] 4.1 CDR grafting technology was used to humanize the mouse antibodies 19C4C7 and 21H6D2. The heavy and light chain variable region sequences of 19C4C7 were searched and aligned in the IMGT database. The human germline gene sequence IGHV1-69*02, which showed high homology to the 19C4C7 heavy chain variable region, was obtained as the humanized framework for the heavy chain variable region. The human germline gene sequence IGKV3-11*01, which showed high homology to the light chain variable region, was selected as the humanized framework for the light chain variable region. The CDRs of the 19C4C7 heavy and light chain variable regions were transplanted into the corresponding humanized frameworks to form the humanized 19C4C7 antibody variable regions. To maintain the affinity of antibody 19C4C7, the variable regions of the obtained humanized antibody required back mutations. The heavy and light chain variable region sequences of 21H6D2 were searched and aligned in the IMGT database. The human germline gene sequence IGHV1-3*01, which showed high homology to the 21H6D2 heavy chain variable region, was obtained as the humanized framework for the heavy chain variable region. The human germline gene sequence IGKV6-21*01, which showed high homology to the light chain variable region, was selected as the humanized framework for the light chain variable region. The CDRs of the 21H6D2 heavy and light chain variable regions were transplanted into the corresponding humanized frameworks to form the humanized variable regions of the 21H6D2 antibody. To maintain the affinity of the antibody 21H6D2, the variable regions of the obtained humanized antibody required back mutations.

[0427] The humanized heavy chain variable region and light chain variable region sequences of 19C4C7 and 21H6D2 were obtained, respectively. Table 6 lists the amino acid sequences of the humanized variable regions of the murine antibodies 19C4C7 and 21H6D2. The variable region amino acid sequences were sent to Tongyong Biotech Co., Ltd. (Anhui) for codon optimization and gene synthesis. The genes encoding the VH and VL regions of the antibodies were sequentially inserted into expression vectors containing the genes encoding the human IgG1 heavy chain constant region (SEQ ID NO: 44, with L234A, L235A, G237A) and the kappa light chain constant region (SEQ ID NO: 45) to obtain plasmids expressing the full-length heavy chain and full-length light chain of the anti-GDF15 humanized antibody. Table 6 lists the names, variable region sequences, and heavy and light chain sequences of each antibody.

[0428] 4.2 Construction of a humanized anti-GDF15 antibody with a YTE mutation in the Fc region

[0429] Using the constructed vector containing the human IgG1 heavy chain constant region (SEQ ID NO: 44, with L234A, L235A, and G237A) as a template, point mutagenesis was performed. Mutations were made at M252Y, S254T, and T256E (according to EU numbering) using the PCR overlap technique. The genes encoding the VH and VL regions of the antibody Hu19C4C7-YTE were then sequentially inserted into expression vectors containing the gene encoding the human IgG1 heavy chain constant region (SEQ ID NO: 48, with L234A, L235A, G237A, M252Y, S254T, and T256E) and the gene encoding the kappa light chain constant region (SEQ ID NO: 45) to obtain plasmids expressing the full-length heavy chain and plasmids encoding the full-length light chain of the anti-GDF15 humanized antibody.

[0430] Table 6: Humanized antibody names and corresponding SEQ ID NOs.

[0431] Example 5: Humanization of camel-derived anti-GDF15 antibody

[0432] The camel-derived antibody VHH43 was humanized using CDR grafting technology. The heavy and light chain variable region sequences of VHH43 were searched and compared in the IMGT database, and the human germline gene sequence IGHV3-23*04 with high homology to the VHH43 heavy chain variable region was obtained as the humanized framework for the heavy chain variable region. The CDRs of the VHH43 heavy chain variable region were transplanted into the corresponding humanized framework to form the humanized VHH43 antibody variable region. In order to maintain the affinity of the antibody VHH43-3, the obtained humanized antibody variable region needs to be reverse mutated.

[0433] The VHH43 humanized heavy chain variable region sequence was thus obtained. Table 7 lists the amino acid sequence of the humanized variable region of the camelid antibody VHH43. The variable region amino acid sequence was sent to Tongyong Biotech Co., Ltd. (Anhui) for codon optimization and gene synthesis. The gene encoding the VHH region of the antibody was inserted into an expression vector containing the gene encoding human IgG1-Fc (with L234A, L235A, G237A) (SEQ ID NO: 52) to obtain a plasmid expressing the full-length heavy chain of the anti-GDF15 humanized antibody. Table 7 lists the name, variable region sequence, and heavy and light chain sequences of each antibody, where the HCDRs were determined using IMGT.

[0434] Table 7: Humanized antibody names and corresponding SEQ ID NOs.

[0435] Example 6: Expression and purification of humanized anti-GDF15 antibodies

[0436] Plasmids encoding the heavy and light chains of the humanized antibodies shown in Table 6 and the heavy chains of the humanized antibodies shown in Table 7 were transfected into ExpiCHO-S cells using the method of Example 3.2 to express the anti-GDF15 humanized antibodies, and purified accordingly as described in Example 3.2. The purified antibodies were concentrated, sterile filtered, and assayed for purity by SDS-PAGE and size exclusion chromatography (SEC).

[0437] Example 7: Binding affinity of humanized anti-GDF15 antibodies to human, cynomolgus monkey and mouse GDF15 proteins

[0438] To evaluate the binding affinity of humanized anti-GDF15 antibodies to human, cynomolgus monkey, and mouse GDF15, Fortebio was used for the assay.

[0439] Pre-equilibrate an AHC sensor (Fortebio, Cat. No. 18-5060) by adding Running Buffer (PBS, 0.1% BSA, 0.02% Tween 20, pH 7.0) to a pre-wetted plate. Equilibrate the baseline with Running Buffer for 300 seconds before loading. For anti-GDF15 antibody-coupled AHC sensor: Dilute humanized anti-GDF15 antibody to 5 μg / mL in Running Buffer and allow to solidify for 200 seconds. Equilibrate the baseline with Running Buffer for 200 seconds. Human GDF15 protein (Kaixia Biotechnology, Catalog No. GDF-HE115), cynomolgus macaque GDF15 protein (Kaixia Biotechnology, Catalog No. GDF-CE115), and mouse GDF15 protein (Beijing Biosciences, Catalog No. GD5-M5149) were diluted in Running Buffer to 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.13 nM, respectively. Binding was performed for 200 s and dissociation for 600 s. Regeneration was performed in regeneration buffer for 30 s. Data were fitted to a 1:1 binding model using Octet software and calculated using local fitting.

[0440] Table 10 shows the KD values ​​of humanized anti-GDF15 antibodies binding to human, cynomolgus monkey, and mouse GDF15. Humanized anti-GDF15 antibodies bind strongly to human and cynomolgus monkey GDF15, and also bind to mouse GDF15.

[0441] Table 10: Binding affinity of humanized anti-GDF15 antibodies to human, cynomolgus monkey and mouse GDF15

[0442] Example 8: Humanized anti-GDF15 antibodies and humanized anti-GDF15 antibodies with a YTE mutation in the Fc region do not cross-react with human TGFβ family proteins

[0443] 8.1 To evaluate the binding specificity of humanized anti-GDF15 antibodies to GDF15, the binding activity of humanized anti-GDF15 antibodies to human GDF1, GDF2, GDF3, AMH, BMP4, GDF11, GDF8, GDNF, TGFβ1, GFRα1, ActivinA, ActivinB, and BMP2 was determined by ELISA.

[0444] Human GDF1 protein (Wuhan Feien Biotechnology, catalog number: P0298), human GDF2 protein (Kaixia Biotechnology, catalog number: BMP-HM209), human GDF3 protein (Wuhan Feien Biotechnology, catalog number: P0300), human AMH protein (Jinan Biotechnology, catalog number: DRA15), human BMP4 protein (Kaixia Biotechnology, catalog number: BMP-HE004), human GDF11 protein (Wuhan Aibote, catalog number: RP00378), human GDF8 protein (Beijing Baipusaisi, catalog number: GD8-H5243), human GDNF protein (Kaixia Biotechnology, catalog number: Human TGFβ1 protein (Beijing Biopsies, Catalog No. TG1-H4212), human GFRα1 protein (Kaixia Biotechnology, Catalog No. GFR-HM11A), human ActivinA protein (Kaixia Biotechnology, Catalog No. ACV-HM001), human ActivinB protein (Jinan Biotechnology, Catalog No. C905), human BMP2 protein (Kaixia Biotechnology, Catalog No. BMP-HE002), and human GDF15 protein (Kaixia Biotechnology, Catalog No. GDF-HE115) were coated onto 96-well plates and incubated overnight at 4°C. Nonspecific binding sites were blocked by incubation at 37°C with PBS containing 1% BSA for 1 hour. After blocking, the plates were washed three times with PBST (PBS containing 0.05% Tween 20). Humanized anti-GDF15 antibody and IgG control (Beijing Sino Biological, Cat. No. HG1K) were diluted to 4 μg / mL in binding buffer (PBS containing 0.05% Tween 20 and 0.5% BSA) and incubated with the coated protein at 37°C for 1 hour. After incubation, the plate was washed three times with PBST and a peroxidase-conjugated goat anti-human Fc secondary antibody (Jackson Immuno Research, Cat. No.

[0445] 109-035-098) was diluted to 1 / 20,000 with binding buffer, incubated at 37°C for 1 hour, washed again, developed with TMB, and terminated with 1 M H2SO4. The absorbance (OD value) at 450 nm to 620 nm was measured using a microplate reader (MD, SpectraMax_i3x).

[0446] Table 11-1 shows the OD values ​​of humanized anti-GDF15 antibody binding to human TGFβ family proteins. Humanized anti-GDF15 antibody exhibits excellent specificity by not binding to human GDF1, GDF2, GDF3, AMH, BMP4, GDF11, GDF8, GDNF, TGFβ1, GFRα1, ActivinA, ActivinB, or BMP2. Figures 4 and 5 show that the control antibody, Ponsegromab, nonspecifically binds to GDNF and GDF-2. Considering that GDF-2 is involved in cartilage and skeletal development and that GDNF promotes neuronal survival, the binding of Ponsegromab to GDNF and GDF-2 may raise safety concerns.

[0447] Table 11-1: OD values ​​of humanized anti-GDF15 antibodies binding to human TGFβ family proteins

[0448] 8.2 To evaluate the binding specificity of anti-GDF15 antibodies with a YTE mutation in the Fc region to GDF15, the binding activity of anti-GDF15 antibodies with a YTE mutation in the Fc region to human GDF1, GDF2, GDF3, AMH, BMP4, GDF11, GDF8, GDNF, TGFβ1, GFRα1, ActivinA, ActivinB, and BMP2 was determined by ELISA.

[0449] The experimental method is the same as Example 8.1, and the results are shown in Table 11-2.

[0450] The results showed that the anti-GDF15 antibodies hu19C4C7 and hu19C4C7-YTE did not bind to human GDF1, GDF2, GDF3, AMH, BMP4, GDF11, GDF8, GDNF, TGFβ1, GFRα1, ActivinA, ActivinB, and BMP2 proteins, while the control antibody strongly bound to GDNF and GDF2. This indicates that the specificity of hu19C4C7 and hu19C4C7-YTE is superior to that of the control antibody.

[0451] Table 11-2: OD values ​​of binding of anti-GDF15 antibodies to human TGFβ family proteins

[0452] Example 9: Reporter gene activity of humanized anti-GDF15 antibodies and humanized anti-GDF15 antibodies with a YTE mutation in the Fc region

[0453] 9.1 HEK293 cells (ATCC, catalog number: CRL-1573) were maintained in DMEM medium supplemented with 10% FBS in a humidified incubator at 37°C with 5% CO2. The plasmids GFRAL (Gene ID: NM_207410.2), RET9 (Gene ID: NM_020975.6), and SRE-Luc2P (Sequence ID: AAV52871.1) were co-transfected into HEK293 cells via lentivirus and selected with puromycin (Gibco, catalog number: A1113802), hygromycin (ThermoFisher, catalog number: 10687010), and bleomycin (Beyotime, catalog number ST1450-1ml) to obtain polyclonal cells expressing human GFRAL, RET9, and SRE-Luc2P (HEK293 / GFRAL / RET9 / SRE-Luc2P cells). HEK293 / GFRAL / RET9 / SRE-Luc2P cells were resuspended in culture medium (99.375% DMEM + 0.625% FBS) containing 12.5 nM GDF15 (ACRO, catalog number GD5-H5149) and seeded into 384-well plates (20,000 cells / well, 40 μL). A 5X serial dilution of the antibody (final concentration starting at 30 nM, 2-fold dilution, 8 points) was prepared in empty DMEM medium, and 10 μL of the antibody was added to each well. The culture plate was incubated in a 37°C, 5% CO2 incubator for five hours. After incubation, 25 μL of One-Glo™ reagent (Promega, catalog number: E6130) was added to the wells of the assay plate, and luminescence was measured using a luminometer (Tecan F200 Pro).

[0454] The results can be seen in Figures 1A and 1B. The results showed that GDF15 protein can activate the SRE-Luc2P reporter gene activity, and Hu21H6D2, Hu19C4C7, and HuVHH43-3 can block this process and inhibit the reporter gene activity in a dose-dependent manner. Furthermore, the EC50 values ​​of Hu21H6D2, Hu19C4C7, and HuVHH43-3 were superior to those of Ponsegromab, demonstrating stronger in vitro efficacy.

[0455] 9.2 Detection of reporter gene activity of humanized anti-GDF15 antibodies with a YTE mutation in the Fc region

[0456] HEK293 cells (ATCC, catalog number: CRL-1573) were maintained in DMEM medium supplemented with 10% FBS and placed in a humidified incubator at 37°C with 5% CO2. The plasmids GFRAL (Gene ID: NM_207410.2), RET9 (Gene ID: NM_020975.6), and SRE-Luc2P (Sequence ID: AAV52871.1) were co-transfected into HEK293 cells using lentiviral vectors. Polyclonal cells expressing human GFRAL, RET9, and SRE-Luc2P were selected using puromycin (Gibco, catalog number: A1113802), hygromycin (ThermoFisher, catalog number: 10687010), and bleomycin (Beyotime, catalog number ST1450-1ml). Finally, monoclonal cell lines expressing human GFRAL, RET9, and SRE-Luc2P were obtained by limiting dilution. HEK293 / GFRAL / RET9 / SRE-Luc2P cells were resuspended in culture medium (99.375% DMEM + 0.625% FBS) containing 10 nM GDF-15 (ACRO, Catalog No. GD5-H5149) and seeded into 384-well plates (20,000 cells / well, 40 μL). A 5X serial dilution of the antibody (starting at a final concentration of 100 nM, 2-fold dilution, 12 points) was prepared in empty DMEM medium, and 10 μL of the antibody was added to each well. The culture plate was incubated in a 37°C, 5% CO2 incubator for five hours. After incubation, 25 μL of Bio-Lite Luciferase Assay System reagent (Vazyme, Catalog No. DD1201) was added to the wells of the assay plate, and luminescence was measured using a luminometer (Tecan F200 Pro).

[0457] The results can be seen in Figure 1C. The results showed that GDF-15 protein can activate the activity of the SRE-Luc2P reporter gene, and hu19C4C7 and 19C4C7-YTE can block this process, inhibiting the reporter gene activity in a dose-dependent manner, and are superior to the control antibody Ponsegromab.

[0458] Example 10: Humanized anti-GDF15 antibody inhibits ERK phosphorylation levels

[0459] To evaluate the inhibitory effect of anti-GDF15 antibody on ERK phosphorylation downstream of GDF15 / GFRAL / RET, 12.5 nM GDF15 and 10 μl anti-human GDF15 antibody were co-incubated at 37°C for 30 minutes and then added to HEK293 / GFRAL / RET9 / SRE-Luc2P cells (2 × 10 6The cells were incubated at 37°C for 10 minutes, the supernatant was removed, 200 μL of lysis buffer (Solarbio, catalog number: R0010) was added to each well, and the cells were lysed at 4°C for 10 minutes. The levels of ERK and p-ERK were detected by Western blot.

[0460] After cell lysis, total protein concentration was determined using a BCA protein assay kit (Biyuntian, Catalog No. P0010S). 80 μl of cell lysate was mixed with 20 μl of 5X Loading Buffer, incubated in a metal bath at 100°C for 5 minutes, mixed, and centrifuged. Samples were loaded at 20 μg / well and electrophoresed with a marker (Thermo Scientific, Catalog No. 26619). After electrophoresis, PVDF membranes (Merck, Catalog No. ISEQ00010) were cut according to gel size and activated by soaking in methanol for 30 seconds. The membranes were then transferred to equilibration buffer (Nanjing GenScript, Catalog No. L00734C) for transfer. After transfer, the membranes were washed with PBST and then placed in blocking buffer (5% BSA, 0.05% Tween 20 in PBS) for 1 hour at room temperature on a shaker. After blocking, ERK1 / 2 antibody (Aibotek, Catalog No. A4782) and p-ERK1 / 2 antibody (Aibotek, Catalog No. AP0974) were diluted 1:1000 and the blocked membrane was placed in the prepared solution and incubated overnight at 4°C. The incubated membrane was washed three times with PBST for 5 minutes each. Secondary antibody (Cell Signaling, Catalog No. 7074S) was diluted 1:2000 and placed in the prepared solution. The washed membrane was incubated on a shaker at room temperature for 1 hour and then washed three times with PBST for 5 minutes each. Luminescent solution A and B were diluted and mixed in a 1:1 ratio. The membrane was lifted from one corner with tweezers and the excess liquid was absorbed with filter paper. The developer solution was then dripped onto the membrane and photographed using a chemiluminescence imaging system for grayscale analysis.

[0461] Figures 2A, B and 3A, B show Western blot analysis of p-ERK / ERK grayscale values ​​of humanized anti-GDF15 antibodies. The results showed that all humanized anti-GDF15 antibodies inhibited ERK phosphorylation. Hu21H6D2 and Hu19C4C7 inhibited ERK1 / 2 phosphorylation at levels comparable to ponegromab, while HuVHH43-3 was superior to ponegromab.

[0462] Example 11: Humanized anti-GDF15 antibody reverses HT-1080-induced tumor cachexia

[0463] The ability of humanized anti-GDF15 antibodies to reverse tumor cachexia was investigated using the HT-1080 tumor cachexia model in CB17-SCID mice.

[0464] 80 CB17-SCID mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0465] Human fibrosarcoma HT-1080 cells were purchased from the Cell Bank of the Chinese Academy of Sciences. The cells were cultured in a 37°C, 5% CO2 incubator in DMEM containing 10% inactivated fetal bovine serum.

[0466] Eight mice were randomly selected to be not inoculated with tumor cells (NTB), and the remaining 72 CB17-SCID mice were subcutaneously implanted with 2×10 6 HT-1080 cells / 0.1 mL serum-free DMEM / 0.1 mL Matrigel matrix / mouse.

[0467] Experiment 1: Use a vernier caliper to measure the tumor volume of tumor-bearing mice. 3 The net body weight (actual body weight - tumor weight) is obtained by deducting the weight of the tumor from the weight of about 1g. When the net body weight decreases by about 8% compared with the 0th day of inoculation, suitable mice are selected and randomly assigned to 5 experimental groups, with 6 mice in each group. On the 0th and 3rd days after grouping, blank control (PBS), Ponsegromab (10 mg / kg), Hu19C4C7 (10 mg / kg), Hu21H6D2 (10 mg / kg), and HuVHH43-3 (5.3 mg / kg) are intraperitoneally administered to the mice, respectively. All mice are weighed every other day after grouping, and the maximum long axis (L) and maximum wide axis (W) of the tumor in the tumor-bearing mice are measured. The tumor volume is calculated according to the following formula: V = L × W 2 / 2.

[0468] 72 hours after the first administration (before the second administration) and at the end of the experiment, the serum of mice was collected to detect the free GDF15 level and the free drug level.

[0469] Experiment 2: Similar to the above experiment, 8 mice were randomly selected without tumor cell inoculation (G1 NTB), and the remaining 53 CB17-SCID mice were subcutaneously implanted with 2×10 6 HT-1080 cells / 0.1 mL serum-free DMEM / 0.1 mL Matrigel matrix / mouse.

[0470] The tumor volume of tumor-bearing mice was measured using a vernier caliper. 3The net body weight (actual body weight - tumor weight) was calculated by deducting the weight of the tumor from the weight of about 1 g. When the net body weight decreased by about 3% compared with the 0th day of inoculation, appropriate mice were selected and randomly assigned to 7 experimental groups, with 5 mice in each group. On the 0th and 3rd days after grouping, blank control (PBS) (G2), Ponsegromab (1 mg / kg) (G3), Ponsegromab (3 mg / kg) (G4), Hu19C4C7 (1 mg / kg) (G5), Hu19C4C7 (3 mg / kg) (G6), Hu21H6D2 (1 mg / kg) (G7) Hu21H6D2 (3 mg / kg) (G8) were intraperitoneally administered to the mice, respectively. On the 0th, 2nd, 5th and 7th days after grouping, all mice were weighed and the maximum long axis (L) and maximum wide axis (W) of the tumor in the tumor-bearing mice were measured. The tumor volume was calculated according to the following formula: V = L × W 2 After grouping, the total food intake of mice in each group was monitored every 2-3 days to calculate the daily food intake of each mouse. This was monitored three times during the experimental period. At the end of the experiment (7 days after grouping), all mice were euthanized, and bilateral ovarian fat was collected and weighed.

[0471] Free Drug Assay: Human GDF15 protein (Kaixia Biotechnology, Cat. No. GDF-HE115) was diluted to 0.5 μg / mL in PBS, coated at 50 μl / well in a 96-well plate, and incubated overnight at 4°C. Nonspecific binding sites were blocked by incubation with PBS containing 1% BSA at 37°C for 1 hour. After blocking, the plate was washed three times with PBST (PBS containing 0.05% Tween 20). Ponsegromab, Hu19C4C7, Hu21H6D2, and HuVHH43-3 were diluted to 20 ng / mL in 1% blank mouse serum. The plates were then serially diluted two-fold to final concentrations of 20 ng / mL, 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.63 ng / mL, and 0.31 ng / mL, respectively. The diluted mouse serum samples were then added to the plate and incubated at 37°C for 1 hour. After incubation, the plate was washed three times with PBST and diluted to 1 / 20,000 with peroxidase-conjugated goat anti-human Fc secondary antibody (Jackson Immuno Research, Cat# 109-035-098) in binding buffer. The plate was incubated at 37°C for 1 hour, followed by further washing. Color was developed with TMB and terminated with 1 M H₂SO₄. The absorbance (OD) at 450–620 nm was measured using a microplate reader.

[0472] Figure 6A shows the net body weight changes of mice on day 6 after dosing in Experiment 1. Humanized GDF15 antibody treatment significantly improved cachexia-induced weight loss in mice compared to PBS. The body weights of mice in the Hu19C4C7, Hu21H6D2, HuVHH43-3, and Ponsegromab treatment groups recovered from -19.6% of the PBS group to -1.5%, +2.0%, -1.0%, and -0.1%, respectively. Hu21H6D2 and HuVHH43-3 outperformed Ponsegromab, while Hu19C4C7 was comparable to Ponsegromab.

[0473] As shown in Figure 6B, on day 7 after grouping, the body weight of the PBS group decreased to -13.0%. Each treatment group showed dose-dependent inhibition of body weight loss. At 1 mg / kg, body weights of mice in the ponegromab, Hu19C4C7, and Hu21H6D2 groups recovered to -11.0%, -2.6%, and -10.2%, respectively. At 3 mg / kg, body weights of mice in the ponegromab, Hu19C4C7, and Hu21H6D2 groups recovered to -1.8%, 0.4%, and 3.6%, respectively. Compared to the control antibody ponegromab, Hu19C4C7 and Hu21H6D2 were more potent in reversing tumor-induced weight loss.

[0474] As shown in FIG6C , each administration group could increase the food intake of mice in a dose-dependent manner.

[0475] As shown in Figure 6D, on day 7 after grouping, ovarian fat weight was significantly reduced in the PBS group. All drug administrations increased ovarian fat weight at different doses. Compared to the control antibody Ponsegromab, Hu19C4C7 at a dose of 1 mg / kg and Hu21H6D2 at a dose of 3 mg / kg significantly increased ovarian fat weight.

[0476] Figure 7 shows the free drug concentration 72 hours after a single dose, with Hu19C4C7, Hu21H6D2, and HuVHH43-3 demonstrating good drug exposure levels.

[0477] Example 12: Humanized anti-GDF15 antibody reverses cisplatin-induced cachexia

[0478] Eighteen B-hGDF15 mice were purchased from Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd.

[0479] Three mice were randomly selected and left untreated (G1 NT). The remaining 15 mice were randomly divided into three groups of five mice each. 5 mg / kg cisplatin was administered intraperitoneally to the mice on days 0, 4, 8, 12, and 15, respectively. A blank control (PBS) (G2), Ponsegromab (10 mg / kg) (G3), or Hu19C4C7 (10 mg / kg) (G4) were administered intraperitoneally on days 0, 4, 7, 11, and 14 after grouping. The body weights of the mice were monitored on days 0, 1, 4, 6, 8, 11, 13, 15, and 18, and blood was collected at the endpoint to measure free drug levels (as described in Example 11).

[0480] Figure 8 shows changes in mouse body weight. On day 18 after dosing, all treatment groups significantly improved cachexia endpoints compared to PBS. The Hu19C4C7 and Ponsegromab groups recovered their body weight from -14.2% of the PBS group to +1.7% and -0.1%, respectively. Hu19C4C7 was superior to Ponsegromab. Figure 9 shows free drug levels at endpoint, with Hu19C4C7 showing a higher level than Ponsegromab.

[0481] Example 13: Pharmacokinetic Study of Humanized Anti-GDF15 Antibody in Rats

[0482] The pharmacokinetic characteristics of the humanized anti-GDF15 antibody Hu19C4C7 in rats (Pengli Bio) were evaluated. The procedures involving the care and use of animals in this study were reviewed and approved. Three naive rats were used for each antibody. In this study, the animals were injected intravenously with Hu19C4C7 antibody at a dose of 10 mg / kg, and blood samples were obtained at various time points between 0 hours and 336 hours (0 days-14 days). All samples were processed into serum and stored frozen at -70°C to -86°C until analysis. The concentration of Hu19C4C7 antibody in the serum was determined.

[0483] The pharmacokinetic results of the Hu19C4C7 antibody were analyzed using human GDF15 protein for capture and peroxidase-labeled goat anti-human Fc secondary antibody for detection (as shown in Example 11). Pharmacokinetic parameters were analyzed using WinNonlin 8.3 and are shown in Table 12. The humanized anti-GDF15 antibody Hu19C4C7 exhibited excellent pharmacokinetic properties, with a half-life of 12.6 days in rats.

[0484] Table 12: Pharmacokinetic parameters of Hu19C4C7 antibody in rats

[0485] Example 14: Affinity Detection of Anti-GDF15 Antibodies with YTE Mutation in the Fc Region to Human FcRn

[0486] The binding affinity of hu19C4C7 and hu19C4C7-YTE antibodies to human FcRn protein (ACRO Biosystems, Catalog No.: FCM-H82W4) was detected using ForteBio Octet RED96e.

[0487] Briefly, a SA sensor (ForteBio, Catalog No. 18-5019) was placed in loading buffer (1X PBS, Cytiva, Catalog No. SH30256.01, containing 0.05% Tween 20, pH 7.0) and pre-equilibrated at room temperature for 10 minutes. FcRn kinetics assays were performed in a 96-well plate according to the following steps:

[0488] a) Equilibrate the baseline with loading buffer for 180 s;

[0489] b) Add human FcRn protein diluted in loading buffer to a final concentration of 1 μg / mL, and stop when the protein solidifies to 0.3 nm;

[0490] c) The baseline was equilibrated with blocking solution (1X PBS, Cytiva, Catalog No.: SH30256.01, containing 0.05% Tween 20, 0.1% BSA, pH 7.0) for 180 seconds.

[0491] d) Add hu19C4C7 or hu19C4C7-YTE antibodies diluted in running buffer (1X PBS, Cytiva, Cat. No. SH30256.01, containing 0.05% Tween 20, 0.1% BSA, pH 6.0 / 7.4) to each well at the following concentrations: 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.13 nM. Bind for 60 s (pH 6.0) and dissociate for 60 s (pH 6.0 / 7.4).

[0492] e) Regeneration buffer (1X PBS, Cytiva, Catalog No. SH30256.01, pH 7.7) was used for 30 seconds. Baseline equilibration with blocking buffer was performed for 60 seconds. Experimental data were fitted and calculated using a 1:1 binding model using Fortebio Data Analysis software.

[0493] Table 13 summarizes the binding affinity of hu19C4C7 and hu19C4C7-YTE antibodies to human FcRn protein.

[0494] Table 13 Binding affinity of hu19C4C7 and hu19C4C7-YTE antibodies to human FcRn protein

[0495] Table 13 shows that the antibodies of the present invention have an increased affinity for human FcRn compared to hu19C4C7 under the conditions of association for 60 s (pH 6.0) and dissociation for 60 s (pH 6.0) in the running buffer.

[0496] Example 15: Pharmacokinetic Study of Anti-GDF15 Antibodies in Humanized FcRn Mice

[0497] Humanized FcRn mice (Biocytogen) were injected intravenously with anti-GDF15 antibodies at a dose of 10 mg / kg. Blood samples were obtained at various time points between 0 hours and 336 hours. All samples were centrifuged into serum and stored frozen at -80°C until analysis. The concentration of anti-GDF15 antibodies present in the serum was determined by ELISA. The ELISA detection method is as follows: 0.5 μg / ml human GDF15 protein (KACTUS, Cat#GDF-HE115) was coated on a 96-well plate at 50 μL / well and incubated overnight at 4°C. The plate was incubated at 37°C with blocking buffer (PBS solution containing 1% BSA) and blocked for 1 hour. After blocking, the plate was washed once with PBST solution (PBS solution containing 0.05% Tween 20). Anti-GDF15 antibodies (hu19C4C7, hu19C4C7-YTE) were serially diluted with 2% mouse blank serum diluent (PBS solution containing 0.05% Tween 20 and 0.5% BSA) as standards. Serum samples were diluted with mouse blank serum and diluent at various dilution ratios, added to the plate, and incubated at 37°C for 1 hour. After incubation, the plate was washed twice with PBST solution. Secondary antibody (horseradish peroxidase HRP-conjugated goat anti-human Fc secondary antibody, Jackson Immuno Research, Cat#109-035-098) was diluted in diluent and added to the plate for incubation at 37°C for 1 h. After incubation, the plate was washed again and developed with TMB (Thermo Scientific, Cat#34029). The color was then terminated with 1 M H2SO4, and the absorbance values ​​at OD450nm-OD620nm were read in a microplate reader. Pharmacokinetic parameters were calculated using WinNonlin 8.3 software. The analysis results are shown in Table 14 and Figure 10.

[0498] Table 14 Pharmacokinetic parameters of anti-GDF15 antibodies in humanized FcRn mice Cmax: Maximum observed concentration Cl: Clearance T1 / 2: Terminal elimination half-life Vz: Apparent volume of distribution AUClast: Area under the plasma concentration-time curve from t=0 to time t

[0499] Pharmacokinetic studies of anti-GDF15 antibodies in humanized FcRn mice showed that hu19C4C7-YTE had a superior half-life to hu19C4C7.

[0500] Sequence Listing:

Claims

1. An anti-GDF15 antibody or an antigen-binding fragment thereof, comprising (i) three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 7, and three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 8; (ii) three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 14, and three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 15; (iii) three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 23, and three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 24; or (iv). The three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 25, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:

26.

2. An anti-GDF15 antibody or an antigen-binding fragment thereof, comprising HCDR1, HCDR2 and HCDR3, and LCDR1, LCDR2 and LCDR3, wherein (i) HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 4; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 5; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 6; (ii). HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 9; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 10; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 11; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 12; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 13; or (iii). HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 20; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 21; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 22; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:

6.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, comprising a heavy chain variable region VH, wherein the heavy chain variable region (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 7, 14, 23 or 25; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 7, 14, 23 or 25.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising a light chain variable region VL, wherein the light chain variable region (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 8, 15, 24 or 26; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 8, 15, 24 or 26.

5. The antibody or antigen-binding fragment thereof of claim 1 or 2, comprising a heavy chain variable region VH and a light chain variable region VL, wherein (i) the VH comprises or consists of the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the VL comprises or consists of the amino acid sequence of SEQ ID NO:8, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; (ii) the VH comprises or consists of the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; (iii). the VH comprises or consists of the amino acid sequence of SEQ ID NO:23, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises or consists of the amino acid sequence of SEQ ID NO:24, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or (iv). the VH comprises the amino acid sequence shown in SEQ ID NO:25, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of the amino acid sequence, and the VL comprises the amino acid sequence shown in SEQ ID NO:26, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of the amino acid sequence.

6. The antibody or antigen-binding fragment thereof of claim 1 or 2, comprising a heavy chain variable region VH and a light chain variable region VL, wherein (i) the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 7, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 8; (ii) the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 14, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 15; (iii) the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 23, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 24; or (iv). The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 25, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO:

26.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, further comprising a heavy chain constant region HC, for example, the antibody heavy chain constant region HC is a heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably a heavy chain constant region of IgG1, optionally, the heavy chain constant region has an Fc region amino acid modification, such as a modification that reduces or eliminates ADCC effector function, a modification that extends the half-life, a modification that increases affinity for FcRn, etc.; preferably, the heavy chain constant region has one or more of L234A / L235A mutation, G237A mutation and M252Y / S254T / T256E mutation; more preferably, the heavy chain constant region has L234A / L235A mutation, G237A mutation and M252Y / S254T / T256E mutation.

8. The antibody or antigen-binding fragment thereof of claim 7, wherein the heavy chain constant region (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 44 or 48; or (ii) comprises the amino acid sequence of SEQ ID NO: 44 or 48 or consists of said amino acid sequence.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, comprising a light chain constant region, for example, the light chain constant region is a lambda or kappa light chain constant region.

10. The antibody or antigen-binding fragment thereof of claim 9, wherein the light chain constant region (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:45; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO:

45.

11. The antibody or antigen-binding fragment thereof of any one of claims 1 to 10, wherein the antibody comprises a heavy chain, the heavy chain (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28, 30, 33, 35 or 49; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 28, 30, 33, 35 or 49.

12. The antibody or antigen-binding fragment thereof of any one of claims 1 to 11, wherein the antibody comprises a light chain (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 29, 31, 34 or 36; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 29, 31, 34 or 36.

13. The antibody or antigen-binding fragment thereof of any one of claims 1 to 12, comprising a heavy chain and a light chain, wherein (i) the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO:28, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO:29, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; (ii). the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO:30, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO:31, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; (iii). the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO:33, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO:34, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; (iv). the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO:35, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO:36, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or (v). the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:49, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:34, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

14. The antibody or antigen-binding fragment thereof of any one of claims 1 to 13, comprising a heavy chain and a light chain, wherein (i) The heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 28, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 29; (ii) the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 30, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 31; (iii) the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 33, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 34; (iv) the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 35, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 36; or (v). The heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO:49, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:

34.

15. The antibody or antigen-binding fragment thereof of any one of claims 1 to 14, wherein the antibody is a chimeric antibody or a humanized antibody.

16. The antibody or antigen-binding fragment thereof of any one of claims 1-15, wherein the antibody is a monoclonal antibody.

17. The antibody or antigen-binding fragment thereof of any one of claims 1 to 16, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single-chain antibody, (Fab')2, single-domain antibody such as heavy chain variable region VHH, dAb (domain antibody), heavy chain antibody or linear antibody.

18. The antibody or antigen-binding fragment thereof of claim 17, wherein the single-chain antibody is a scFv.

19. A VHH antibody that specifically binds to GDF15, comprising three complementarity determining regions (CDRs) contained in the VHH shown in any one of SEQ ID NO: 19 or 27, Preferably, the CDR sequences are according to the IMGT definition.

20. The VHH antibody of claim 19, comprising complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 16, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 17, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:

18.

21. The VHH antibody of claim 19, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 19 or 27; or (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NO: 19 or 27.

22. A heavy chain antibody that specifically binds to GDF15, comprising the VHH antibody of any one of claims 19 to 21.

23. The heavy chain antibody of claim 22, comprising the VHH antibody of any one of claims 19 to 21 linked to an antibody constant region or Fc region, preferably, the antibody constant region or Fc region is from human IgG1, human IgG2, human IgG3 or human IgG4, preferably, the Fc region (i) comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:46; or (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO:

46.

24. The heavy chain antibody of claim 22, comprising the VHH antibody of any one of claims 19 to 21 linked to an antibody Fc region, wherein the Fc region is an Fc region from human IgG1, IgG2, IgG3 or IgG4, optionally comprising L234A / L235A mutations and G237A mutations, preferably, the Fc region (i) comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:43; or (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO:

43.

25. The heavy chain antibody of claim 22, (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 32 or 37; or (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 32 or 37; or (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 32 or 37, preferably, the amino acid changes do not occur in the CDR region.

26. The VHH antibody of any one of claims 19-21, or the heavy chain antibody of any one of claims 22-25, wherein the antibody is a chimeric antibody or a humanized antibody.

27. A multispecific antibody, such as a bispecific antibody, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, or the VHH antibody according to any one of claims 19 to 21, or the heavy chain antibody according to any one of claims 22 to 26.

28. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, or the VHH antibody according to any one of claims 19 to 21, or the heavy chain antibody according to any one of claims 22 to 26, or any chain of the multispecific antibody according to claim 27, or consisting of said nucleic acid sequence.

29. An expression vector comprising the nucleic acid molecule according to claim 28, preferably, the expression vector is pCDNA, such as pCDNA3.

1.

30. A host cell comprising the nucleic acid molecule of claim 28 or the expression vector of claim 29, preferably, the host cell is prokaryotic or eukaryotic, such as 293 cells or CHO cells.

31. A method for preparing the antibody or antigen-binding fragment thereof of any one of claims 1-18, or the VHH antibody of any one of claims 19-21, or the heavy chain antibody of any one of claims 22-26, or the multispecific antibody of claim 27, the method comprising culturing a host cell comprising the nucleic acid molecule of claim 28 or the expression vector of claim 29 or the host cell of claim 30 under conditions suitable for chain expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

32. The antibody or antigen-binding fragment thereof or multispecific binding molecule prepared by the method of claim 31.

33. An immunoconjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-18, or the VHH antibody of any one of claims 19-21, or the heavy chain antibody of any one of claims 22-26, or the multispecific antibody of claim 27, e.g., the immunoconjugate is an ADC.

34. A pharmaceutical composition or medicament or formulation comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, or the VHH antibody according to any one of claims 19 to 21, or the heavy chain antibody according to any one of claims 22 to 26, or the multispecific antibody according to claim 27, or the immunoconjugate according to claim 33, and optionally a pharmaceutically acceptable excipient.

35. A pharmaceutical combination product comprising the antibody or antigen-binding fragment thereof of any one of claims 1-18, or the VHH antibody of any one of claims 19-21, or the heavy chain antibody of any one of claims 22-26, or the multispecific antibody of claim 27, or the immunoconjugate of claim 33, and one or more other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators).

36. A method for preventing or treating cachexia in a subject, comprising administering to the individual an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-18, or the VHH antibody of any one of claims 19-21, or the heavy chain antibody of any one of claims 22-26, or the multispecific antibody of claim 27, or the immunoconjugate of claim 33, or the pharmaceutical composition or formulation of claim 34; or the pharmaceutical combination product of claim 35.

37. The method of claim 36, wherein the cachexia occurs with a disease selected from a tumor such as cancer, chronic heart failure, congestive heart failure, sarcopenia, chronic obstructive pulmonary disease (COPD), sarcopenia or chronic kidney disease (CKD), for example the tumor is a solid tumor or a blood tumor, such as a malignant solid tumor or a blood tumor, such as fibrosarcoma, prostate cancer, pancreatic cancer, endometrial cancer or colorectal cancer.

38. The method of claim 36, wherein the cachexia is induced by chemotherapy or chemotherapy combined with immuno-oncology therapy, such as chemotherapy comprising administration of a chemotherapeutic agent such as cisplatin.

39. The method of any one of claims 36-38, wherein the individual has GDF15 in a sample, e.g., has or has elevated protein levels and / or nucleic acid levels of GDF15, e.g., compared to protein levels and / or nucleic acid levels of GDF15 in the same tissue of a healthy individual, e.g., the sample is a body fluid, e.g., blood, serum, or plasma.

40. The method of any one of claims 36-39, wherein the method further comprises administering in combination with other therapies such as treatment modalities (e.g., surgery or radiotherapy) and / or other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint molecule inhibitors or agonists).

41. A method for detecting the presence of GDF15 in a biological sample, comprising (i) contacting a biological sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, or the VHH antibody according to any one of claims 19 to 21, or the heavy chain antibody according to any one of claims 22 to 26, or the multispecific antibody according to claim 27, or the immunoconjugate according to claim 33 under conditions that allow binding thereof to GDF15, (ii) detecting whether a complex is formed between the antibody or multispecific binding molecule and GDF15, The formation of the complex indicates the presence of GDF15.

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