ZIP12 antibody

Abstract

To provide an improved means of treating pulmonary hypertension. [Solution] The present invention relates to the ZIP12 antibody. The present invention extends to compositions comprising the antibody, including pharmaceutical compositions and kits. The present invention also extends to methods for producing the antibody, and methods for using the antibody in the treatment and diagnosis of hypoxia-related diseases, such as pulmonary hypertension and cancer.

Description

[Technical Field]

[0001] The present invention relates to ZIP12 antibodies. The present invention extends to compositions comprising said antibodies, including pharmaceutical compositions and kits. The present invention also extends to methods of producing said antibodies, and methods of using said antibodies in the treatment and diagnosis of hypoxia-related diseases such as pulmonary hypertension and lung cancer, for example. [Background Art]

[0002] Pulmonary hypertension (PH) is a pathological condition characterized by elevated pulmonary artery pressure and structurally remodeled pulmonary blood vessels. Current treatments for pulmonary hypertension center on pharmacological manipulation of signaling mechanisms utilized by vasoactive factors, and have limited therapeutic effects. There is a significant unmet medical need for new therapeutics and diagnostics for this potentially lethal condition.

[0003] The typical response of the pulmonary circulation of adult mammals to a hypoxic environment is vasoconstriction and structural remodeling of pulmonary arterioles, resulting in a chronic increase in pulmonary artery pressure (pulmonary hypertension) and right ventricular hypertrophy. However, some mammals exhibit genetic resistance to hypoxia-induced pulmonary hypertension (WILKINS, M.R. et al., Pathophysiology and treatment of high-altitude pulmonary vascular disease.Circulation.2015, 131, 582~590; ZHAO, L. et al., Right ventricular hypertrophy secondary to pulmonary hypertension is linked to rat chromosome 17: evaluation of cardiac ryanodine Ryr2 receptor as a candidate.Circulation.2001, 103, 442~447; RHODES, J., Comparative physiology of hypoxic pulmonary hypertension: historical clues from brisket disease.Journal of applied physiology.2005, 98, 1092~1100). The inventors previously reported that the Fisher 344 (F344) rat strain is more resistant to hypoxia-induced pulmonary hypertension compared to the Wistar Kyoto (WKY) strain (ZHAO, L. et al., Right ventricular hypertrophy secondary to pulmonary hypertension is linked to rat chromosome 17: evaluation of cardiac ryanodine Ryr2 receptor as a candidate. Circulation. 2001, 103, 442-447). However, the cause of this resistance has not been identified.

[0004] The inventors previously utilized genetic breeding programs and comparative genomic analysis to leverage this variation in rats and identified the gene Slc39a12 as a major regulator of hypoxia-induced pulmonary vascular remodeling. Slc39a12 encodes the zinc transporter, ZIP12. The inventors found that ZIP12 expression was increased in many cell types, including endothelial cells, smooth muscle cells, and stromal cells, in remodeled pulmonary arterioles of rats, cattle, and humans susceptible to hypoxia-induced pulmonary hypertension. The inventors showed that ZIP12 expression in pulmonary vascular smooth muscle cells is hypoxia-dependent, and that targeted inhibition of ZIP12 inhibits the increase in intracellular unstable zinc and their proliferation in culture in hypoxia-exposed pulmonary vascular smooth muscle cells. The inventors also previously demonstrated that gene disruption of ZIP12 expression reduces the development of pulmonary hypertension in rats housed in a hypoxic atmosphere.

[0005] However, specific treatments targeting the ZIP12 protein, which the inventors believe could provide an improved means of treating pulmonary hypertension, have not been available to date. Furthermore, the diagnosis of pulmonary hypertension often requires invasive procedures such as right heart catheterization and echocardiography. Therefore, the identification of novel markers of pulmonary hypertension that can be measured through simpler and less invasive procedures is also desirable. [Overview of the project]

[0006] The inventors hypothesized that ZIP12 inhibition could prevent hypoxia-induced pulmonary vascular smooth muscle cell (PVSMC) proliferation in vitro and pulmonary angiogenesis ex vivo. This identified ZIP12 as a promising novel therapeutic target for treating the underlying disease mechanism of pulmonary hypertension, which led to further research by the inventors in developing antibodies capable of targeting the extracellular domain of ZIP12 and inhibiting its function. The anti-ZIP12 activity of these antibodies means that they are themselves useful as therapeutic agents and can be used to treat, improve, or prevent any hypoxia-induced or hypoxia-related conditions, and, in particular, pulmonary hypertension, though not exclusively.

[0007] Therefore, in a first aspect of the present invention, an antibody or an antigen-binding fragment thereof that specifically binds to the extracellular region of ZIP12 is provided. As shown in the examples, the inventors identified an extracellular region or domain of the ZIP12 protein as key to its function and accordingly developed antibodies capable of binding to ZIP12 and inhibiting its function. For example, as shown in Figures 2 and 3, the inventors developed a number of antibodies and demonstrated that they specifically target the extracellular domain and inhibit ZIP12 function. Furthermore, as shown in Figures 6, 7, and 9, the inventors demonstrated that these antibodies not only inhibit ZIP12 function but also do not target the ZIP4 protein.

[0008] Preferably, the antibody or antigen-binding fragment of the present invention has the ability to inhibit ZIP12 function. Preferably, the antibody or antigen-binding fragment of the present invention has the ability to inhibit ZIP12 function, thereby inhibiting the increase of intracellular unstable zinc in pulmonary vascular smooth muscle cells exposed to hypoxia and their proliferation in culture.

[0009] ZIP12, ZIP13, and ZIP4 are all zinc transporters. However, ZIP4 is not involved in the pathogenesis of pulmonary hypertension and plays an important role in tissue homeostasis, metabolism, development, and immunity. Furthermore, ZIP13 has been shown to regulate intracellular zinc and smad signaling, which is important for BMP / TGF-beta signaling (Fakuda et al., J Biol Inorg Chem, 2011). Impairment of BMP signaling is known to be associated with pulmonary hypertension, as shown by Morrell et al., Nature Reviews Cardiology, 2015. Therefore, inhibition of ZIP13 leads to lower zinc concentrations, impairing BMP signaling and causing, rather than curing, pulmonary hypertension.

[0010] Therefore, it is important that the antibody of the present invention, which targets ZIP12, acts specifically in this manner and has little to no cross-reactivity with ZIP13 and / or ZIP4, because this cross-reactivity can result in significant and undesirable off-target effects.

[0011] Therefore, preferably, the antibody of the present invention or its antigen-binding fragment does not substantially bind to human ZIP13. Preferably, the antibody of the present invention or its antigen-binding fragment does not exhibit cross-reactivity with human ZIP13.

[0012] In addition, preferably, the antibody of the present invention or its antigen-binding fragment does not substantially bind to human ZIP4. Preferably, the antibody of the present invention or its antigen-binding fragment does not exhibit cross-reactivity with human ZIP4.

[0013] In one embodiment, ZIP12 may be represented by Genbank ID No: NP-001138667, which is provided herein as Sequence ID 1: [Sequence ID 1] The antibody or its antigen-binding fragment can bind to the region between amino acid positions 1 to 202 of Sequence ID No. 1, which corresponds to the extracellular domain of ZIP12.

[0014] Therefore, preferably, the antibody or its antigen-binding fragment may bind to one or more amino acids between amino acid positions 1 to 202 of ZIP12, which is provided herein as Sequence ID No. 2: MCFRTKLSVSWVPLFLLLSRVFSTETDKPSAQDSRSRGSSGQPADLLQVLSAGDHPPHNHSRSLIKTLLEKTGCPRRRNGMQGDCNLCFEPDALLLIAGGNFEDQLREEVVQRVSLLLLYYIIHQEEICSSKLNMSNKEYKFYLHSLLSLRQDEDSSFLSQNETEDILAFTRQYFDTSQSQCMETKTLQKKSGIVSSEGANE[Sequence ID 2] Therefore, preferably, the antibody or its antigen-binding fragment binds to an epitope in a sequence that includes or consists of a sequence substantially like that shown in SEQ ID NO: 2 or its variant or fragment.

[0015] Preferably, the antibody or its antigen-binding fragment binds to one or more amino acids in SEQ ID NO: 2 or its fragment or variant. Preferably, the antibody or its antigen-binding fragment binds to any sequence of 5, 10, 15, 20, 25, 30, 35, 40, or 45 amino acids present in SEQ ID NO: 2 or its variant or fragment.

[0016] In one embodiment, the antibody or its antigen-binding fragment may bind to one or more amino acids between amino acid positions 20 to 202 of SEQ ID NO: 2. The antibody or its antigen-binding fragment may bind to one or more amino acids between amino acid positions 20 to 180, or between amino acid positions 20 to 160, or between amino acid positions 20 to 140, or between amino acid positions 20 to 120, or between amino acid positions 20 to 110, or between amino acid positions 20 to 108 of SEQ ID NO: 2. The antibody or its antigen-binding fragment may bind to one or more amino acids between amino acid positions 20 to 104 of SEQ ID NO: 2.

[0017] The antibody or its antigen-binding fragment may bind to one or more amino acids between amino acid positions 40 to 202, or between amino acid positions 60 to 202, or between amino acid positions 80 to 202, or between amino acid positions 100 to 202, or between amino acid positions 120 to 202, or between amino acid positions 140 to 202, or between amino acid positions 150 to 202 of SEQ ID NO: 2. The antibody or its antigen-binding fragment may bind to one or more amino acids between amino acid positions 156 to 202 of SEQ ID NO: 2.

[0018] Epitopes can be linear or three-dimensional. The term "linear epitope" may refer to an epitope consisting of amino acid residues that form a sequence together in the primary sequence of the protein antigen, i.e., a sequential or continuous epitope. The term "three-dimensional epitope" may refer to an epitope consisting of amino acid residues in the primary sequence of the protein antigen in which at least some of the amino acid residues are separated from other amino acid residues, but together they form a 3D structure that is recognized by the antibody, i.e., a discontinuous epitope.

[0019] In one embodiment, ZIP4 may be represented by Genbank ID No: NP-570901, which is provided herein as Sequence ID No. 19: [Sequence ID 19] Therefore, preferably, the antibody or its antigen-binding fragment does not bind to sequences substantially represented in SEQ ID NO: 19 or its variants or fragments.

[0020] In one embodiment, ZIP13 may be represented by Gene ID No: 91252, which is provided herein as Sequence ID No. 75: MPGCPCPGCGMAGPRLLFLTALALELLERAGGSQPALRSRGTATACRLDNKESESWGALLSGERLDTWICSLLGSLMVGLSGVFPLLVIPLEMGTMLRSEAGAWRLKQLLSFALGGLLGNVFLHLLPEAWAYTCSASPGGEGQSLQQQQQLGLWVIAGILTFLALEKMFLDSKEEGTSQAPNKDPTAAAAALNGGHCLAQPAAEPGLGAVVRSIKVSGYLNLLANTIDNFTHGLAVAASFLVSKKIGLLTTMAILLHEIPHEVGDFAILLRAGFDRWSAAKLQLSTALGGLLGAGFAICTQSPKGVVGCSPAAEETAAWVLPFTSGGFLYIALVNVLPDLLEEEDPWRSLQQLLLLCAGIVVMVLFSLFVD[Sequence ID 75] Therefore, preferably, the antibody or its antigen-binding fragment does not bind to a sequence substantially like that shown in SEQ ID NO: 75 or its variant or fragment.

[0021] The present invention extends to both the entire antibody (i.e., immunoglobulin) having immunospecificity against the extracellular portion of ZIP12, and, in addition, to the antigen-binding fragment or region of the corresponding full-length antibody.

[0022] Antibodies or their antigen-binding fragments can be monovalent, divalent, or polyvalent. A monovalent antibody is a dimer (HL) containing a light chain (L) and a heavy chain (H) associated by a disulfide crosslink. A divalent antibody is a tetramer (H2L2) containing two dimers associated by at least one disulfide crosslink. Polyvalent antibodies can also be produced, for example, by linking multiple dimers. The basic structure of an antibody molecule consists of two identical light chains and two identical heavy chains that are non-covalently associated and can be linked by disulfide bonds. Each heavy and light chain contains an amino-terminal variable region of about 110 amino acids and a constant sequence in the rest of the chain. The variable region includes several hypervariable regions, or complementarity-determining regions (CDRs), that form the antigen-binding site of the antibody molecule and determine its specificity to the antigen, i.e., the extracellular portion of ZIP12 or its variants or fragments (e.g., epitopes). On both the heavy and light chains of the CDR, there is a framework region, which is a relatively conserved sequence of amino acids that anchors and directs the CDR. Antibody fragments may include bispecific antibodies (BsAbs) or chimeric antigen receptors (CDRs).

[0023] The heavy chain constant region typically consists of three domains, C H1 , C H2 , and C H3 Each light chain typically has a light chain variable region (V L ) and the light chain steady region. The light chain steady region is typically C L It includes one domain that is abbreviated as [domain name].

[0024] Each heavy and light chain typically contains three CDRs and four FRs arranged in the following order (N-terminus to C-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs are involved in antigen binding, conferring antigen specificity and binding affinity to the antibody. The entire sequence was incorporated by reference, as described by Kabat et al., Sequences. See *Proteins of Immunological Interest*, 5th edition (1991), Public Health Service, National Institutes of Health, Bethesda, MD.

[0025] Heavy chains from any vertebrate species can be assigned to one of five distinct classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also named α, δ, ε, γ, and μ, respectively. The IgG and IgA classes are further divided into subclasses based on differences in sequence and function. Humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0026] Light chains derived from any vertebrate species can be assigned to one of two types, called kappa and lambda, based on the sequence of their constant domains. The constant region consists of one of five heavy chain sequences (μ, γ, ζ, α, or ε) and one of two light chain sequences (κ or λ). The heavy chain constant region sequence determines the antibody isotype and the effector function of that molecule.

[0027] Preferably, the antibody or its antigen-binding properties are isolated or purified. In one preferred embodiment, the antibody or its antigen-binding fragment comprises a polyclonal antibody or its antigen-binding fragment. The antibody or its antigen-binding fragment may be produced in rabbits, mice, or rats.

[0028] Preferably, the antibody or its antigen-binding fragment is obtained by immunizing a host animal with the extracellular component of ZIP12 or a variant or fragment thereof, and then collecting the antibody or its antigen-binding fragment. The host animal may be a rabbit.

[0029] In another preferred embodiment, the antibody or its antigen-binding fragment comprises a monoclonal antibody or its antigen-binding fragment. Preferably, the antibody of the present invention is a human antibody. As used herein, the term “human antibody” may mean an antibody, such as a monoclonal antibody, that contains substantially the same heavy-chain CDR amino acid sequence and light-chain CDR amino acid sequence as found in a particular human antibody exhibiting immunospecificity to the extracellular portion of ZIP12 or its variants or fragments. Amino acid sequences that are substantially the same as the heavy-chain CDR or light-chain CDR exhibit a considerable degree of sequence identity when compared to a reference sequence. Such identity can be definitively understood or recognized as representing the amino acid sequence of a particular human antibody. Substantially identical heavy-chain CDR amino acid sequences and light-chain CDR amino acid sequences may, for example, have a small number of amino acid modifications or conserved substitutions. Such a human antibody maintains its function of selectively binding to the extracellular portion of ZIP12 or its variants or fragments.

[0030] The term "human monoclonal antibody" may include monoclonal antibodies having substantially or completely human CDR amino acid sequences, produced by lymphocytes or hybridoma cells, for example, by recombinant methods such as production using a phage library.

[0031] The term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies. This substantially homogeneous population contains antibodies that are substantially similar and bind to the same epitopes, with the exception of variants that may typically arise during the production of monoclonal antibodies. Such variants are generally present only in trace amounts. Monoclonal antibodies are typically obtained by a process involving the selection of a single antibody from multiple antibodies. For example, the selection process could be the selection of a single clone from multiple clones, such as a hybridoma clone, phage clone, yeast clone, bacterial clone, or a pool of other recombinant DNA clones. The selected antibody can be further modified, for example, to improve its affinity for a target (through so-called "affinity maturation"), to humanize the antibody, to improve its production in cell culture, and / or to reduce its immunogenicity in a target.

[0032] The term "humanized antibody" can refer to an antibody derived from a non-human species (e.g., mouse or rabbit) whose protein sequence has been modified to increase its similarity to antibodies naturally produced in humans.

[0033] Antibodies may be recombinant antibodies. The term "recombinant human antibody" may include human antibodies produced using recombinant DNA technology. The term “antigen-binding region” may refer to a region of an antibody having a specific binding affinity to a target antigen, such as the extracellular portion of ZIP12 or its variants or fragments. Preferably, the fragment is an epitope. The antigen-binding region may be a hypervariable CDR or its functional portion. The term “functional portion” of a CDR may refer to a sequence within the CDR that exhibits a specific affinity for the target antigen. The functional portion of a CDR may include a ligand that specifically binds to the extracellular portion of ZIP12 or its fragments.

[0034] The term "CDR" can refer to hypervariable regions in the heavy chain variable region and the light chain variable region. Each of the heavy and light chains of an antibody can have one, two, three, or more CDRs. Typically, each chain has at least three CDRs, which, when combined, form antigen-binding sites, i.e., three-dimensional binding sites to which antigens bind or specifically react. However, it is hypothesized that some antibodies may have four CDRs in the heavy chain.

[0035] The definition of a CDR also includes duplicates or subsets of amino acid residues when compared to one another. The exact number of residues that make up a particular CDR or its functional portion varies depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues constitute a particular CDR, assuming the variable region amino acid sequence of an antibody.

[0036] The amino acid sequence boundaries of CDRs can be determined by using one of several known numbering schemes, including Kabat et al., the above ("Kabat" numbering scheme); Al-Lazikani et al., 1997, J.Mol.Biol., 273:927~948 ("Chothia" numbering scheme); MacCallum et al., 1996, J.Mol.Biol. 262:732~745 ("Contact" numbering scheme); Lefranc et al., Dev.Comp.Immunol., 2003, 27:55~77 ("IMGT" numbering scheme); and Honegge and Plueckthun, J.Mol.Biol., 2001, 309:657~70 ("AHo" numbering scheme).

[0037] The term “functional fragment” of an antibody may mean a portion of the antibody that retains functional activity. Functional activity may be, for example, antigen-binding activity or specificity. Functional activity may also be, for example, effector function provided by the constant region of the antibody. The term “functional fragment” is also intended to include fragments produced, for example, by protease digestion or reduction of human monoclonal antibodies and by recombinant DNA methods known to those skilled in the art. Examples of functional fragments of human monoclonal antibodies include individual heavy or light chains and their fragments, such as VL, VH, and Fd; monovalent fragments, such as Fv, Fab, and Fab'; bivalent fragments, such as F(ab')2; single-stranded Fv(scFv); and Fc fragments.

[0038] The term "VL fragment" may refer to a fragment of the light chain of a human monoclonal antibody that contains all or part of the light chain variable region, including the CDR. The VL fragment may further contain the light chain constant region sequence.

[0039] The term "VH fragment" can refer to a fragment of the heavy chain of a human monoclonal antibody that contains all or part of the heavy chain variable region, including the CDR. The term "Fd fragment" can refer to the heavy chain variable region linked to the first heavy chain steady region, namely VH and CH-1. The "Fd fragment" does not include the light chain or the second and third steady regions of the heavy chain.

[0040] The term "Fv fragment" may refer to a monovalent antigen-binding fragment of a human monoclonal antibody, which includes all or part of the variable regions of the heavy and light chains and lacks the constant regions of the heavy and light chains. The variable regions of the heavy and light chains include, for example, the CDR. For example, an Fv fragment includes all or part of the amino-terminal variable regions of both the heavy and light chains, which consist of approximately 110 amino acids.

[0041] The term "Fab fragment" can refer to a monovalent antigen-binding fragment of a human monoclonal antibody that is larger than the Fv fragment. For example, a Fab fragment includes all or part of the variable regions of the heavy and light chains, and the first constant domain. Therefore, a Fab fragment may also include, for example, approximately 110 to 220 amino acid residues from the heavy and light chains.

[0042] The term "Fab' fragment" can refer to a monovalent antigen-binding fragment of a human monoclonal antibody that is larger than a Fab fragment. For example, a Fab' fragment may include the entire light chain, the entire variable region of the heavy chain, and all or part of the first and second constant domains of the heavy chain. For example, a Fab' fragment may also include some or all of 220 to 330 amino acid residues of the heavy chain.

[0043] The term "F(ab')2 fragment" may refer to a bivalent antigen-binding fragment of a human monoclonal antibody. The F(ab')2 fragment may include, for example, all or part of the variable regions of two heavy chains and two light chains, and may further include all or part of the first constant domains of two heavy chains and two light chains.

[0044] The term "single-chain Fv (scFv)" can refer to a fusion of the variable region (VH) of a heavy chain and the variable region (VL) of a light chain, linked by a short linker peptide. The term "bispecific antibody (BsAb)" can refer to a bispecific antibody containing two scFvs linked to each other by a short linker peptide.

[0045] Those skilled in the art know that the precise boundaries of antibody fragments are not important as long as the fragments maintain their functional activity. Using well-known recombination methods, those skilled in the art can manipulate polynucleotide sequences to express functional fragments having any endpoints desired for a particular application. Functional antibody fragments may contain or consist of fragments having substantially the same heavy-chain and light-chain variable regions as those of human antibodies.

[0046] Preferably, with respect to the first aspect of the present invention, the antigen-binding fragment is immunospecific to an epitope in the extracellular portion of ZIP12. The antigen-binding fragment may include or consist of any fragment selected from the group consisting of VH, VL, Fd, Fv, Fab, Fab', scFv, F(ab')2, and Fc fragments.

[0047] The antigen-binding fragment may be a single-domain antibody (sdAb) (also known as a nanobody), which a person skilled in the art will understand to be an antibody fragment consisting of a single monomeric variable antibody domain.

[0048] The antigen-binding fragment may include or consist of one of the VL antigen-binding region sequences of a human antibody, one of the VH antigen-binding region sequences, or a combination of the VL and VH antigen-binding regions. The appropriate number and combination of VH and VL antigen-binding region sequences can be determined by those skilled in the art, depending on the desired affinity and specificity of the antigen-binding fragment and its intended use. Functional or antigen-binding fragments of antibodies can be readily prepared and isolated using methods well known to those skilled in the art. Such methods include, for example, proteolytic methods, recombinant methods, and chemical synthesis. Proteolytic methods for the isolation of functional fragments involve using human antibodies as starting materials. Suitable enzymes for proteolytic degradation of human immunoglobulins include, for example, papain and pepsin. A suitable enzyme can be readily selected by those skilled in the art, depending, for example, whether a monovalent or bivalent fragment is required. For example, papain cleavage yields two monovalent Fab' and Fc fragments that bind the antigen. For example, pepsin cleavage yields a divalent F(ab') fragment. The F(ab')2 fragment of the present invention can be further reduced, for example, with DTT or 2-mercaptoethanol, to yield two monovalent Fab' fragments.

[0049] A functional or antigen-binding fragment of an antibody produced by proteolysis can be purified by affinity and column chromatography procedures. For example, undigested antibodies and Fc fragments can be removed by binding to protein A. Additionally, the functional fragments can be purified according to their charge and size, for example, using ion exchange chromatography and gel filtration chromatography. Such methods are well known to those skilled in the art.

[0050] An antibody or an antigen-binding fragment thereof can be produced by recombinant technology. Preferably, first, a polynucleotide encoding the desired regions of antibody heavy chain and light chain is isolated. Such regions may include, for example, all or part of the variable regions of heavy and light chains. Preferably, such regions may particularly include the antigen-binding regions of heavy chains and light chains, preferably antigen-binding sites, and most preferably CDRs.

[0051] The polynucleotide encoding an antibody or an antigen-binding fragment thereof according to the present invention can be prepared using methods known to those skilled in the art. The polynucleotide encoding an antibody or an antigen-binding fragment thereof can be directly synthesized by oligonucleotide synthesis methods known in the art. Alternatively, smaller fragments can be synthesized and ligated using recombinant methods known in the art to form larger functional fragments.

[0052] As used herein, the term "immunospecificity" may mean that the binding region of an antibody or an antigen-binding fragment thereof is capable of specifically binding to the extracellular portion of ZIP12 or a variant or fragment thereof, thereby being capable of immunoreacting with the same. The antibody or antigen-binding fragment thereof preferably has an affinity of approximately 10 -5 ~10 -13 M -1 , preferably 10 -6 ~10 -9 M -1 , even more preferably 10 -10 ~10 -12 M-1 affinity constant Therefore, it can selectively interact with the antigen (the extracellular component of ZIP12). The antibody or its antigen-binding fragment preferably has an affinity constant of approximately 10 -10 M -1 Higher, 10 -9 M -1 Higher, 10 -8 M -1 Higher, 10 -7 M -1 Higher, or 10 -6 M -1 Preferably 10 -5 M -1 Higher, 10 -4 M -1 , or 10 -3 M - 1 Higher, and even more preferably 10 -2 M -1 , 10 -1 M -1 , or 10 -2 M -1 , most preferably 10 +1 M -1 , 10 +2 M -1 , or 10 +3 M -1 That's right. Therefore, it is virtually incompatible with ZIP4 and / or ZIP13.

[0053] The term "immunely responsive" may mean that the binding domain has the ability to induce an immune response by binding to the extracellular domain or epitope of ZIP12. The term "epitope" can refer to any region of an antigen that has the ability to induce and bind to the binding domain of an antibody or its antigen-binding fragment.

[0054] Therefore, the antibody or its antigen-binding fragment may contain a heavy chain. The heavy chain may be selected from the group consisting of IgA; IgD; IgE; IgG, and IgM. Preferably, the heavy chain is IgG. Preferably, the heavy chain is IgA.

[0055] The heavy chain can be IgG1. The heavy chain can be IgG2. The heavy chain can be IgG3. The heavy chain can be IgG4. The heavy chain can be IgA1. The heavy chain can be IgA2. As described in the examples and shown in Figure 2, the inventors have surprisingly demonstrated that all antibodies referred to herein as 19212 ("38F02"), 19213 ("51B12"), 19214 ("63A11"), and 19218 ("89G11") can significantly target and inhibit the function of ZIP12. The CDR / VH / VL, HC, and LC sequences of these four antibodies are conveniently summarized in the table shown in Figure 13. Advantageously, and preferably, the inventors have also demonstrated that both the parental and humanized antibodies of the 63A11 and 51B12 series retain activity and do not exhibit cross-reactivity with ZIP4.

[0056] 19214(「63A11」) Therefore, in one embodiment, the antibody or its antigen-binding fragment is referred to herein as 63A11. The antibody or its antigen-binding fragment may contain the CDR-H1 domain of SEQ ID NO: 3, which is provided herein as follows: DHGMH [Sequence ID 3] Preferably, the antibody or its antigen-binding fragment comprises a CDR-H1 domain consisting of a sequence substantially represented in SEQ ID NO: 3 or its variant or fragment.

[0057] The antibody or its antigen-binding fragment may contain the CDR-H2 domain of SEQ ID NO: 4, which is provided herein as follows: YISSGSSAIFYADTVKG[Sequence ID 4] Preferably, the antibody or its antigen-binding fragment comprises a CDR-H2 domain comprising a sequence substantially as shown in SEQ ID NO: 4 or its variant or fragment.

[0058] The antibody or its antigen-binding fragment may contain the CDR-H3 domain of SEQ ID NO: 5, which is provided herein as follows: WTNLYAMDY[SEQ ID NO: 5] Preferably, the antibody or its antigen-binding fragment comprises a CDR-H3 domain consisting of a sequence substantially as shown in SEQ ID NO: 5 or its variant or fragment.

[0059] Preferably, the antibody or its antigen-binding fragment comprises a CDR-H1 domain containing or consisting of SEQ ID NO: 3, a CDR-H2 domain containing or consisting of SEQ ID NO: 4, and / or a CDR-H3 domain containing or consisting of SEQ ID NO: 5. However, preferably, the antibody or its antigen-binding fragment comprises a CDR-H1 domain containing or consisting of SEQ ID NO: 3, a CDR-H2 domain containing or consisting of SEQ ID NO: 4, and a CDR-H3 domain containing or consisting of SEQ ID NO: 5.

[0060] The antibody or its antigen-binding fragment may contain a heavy chain variable (VH) sequence, such as the sequence number 41, which is provided herein as follows: EVQLVESGGGLVKPGGSRKLSCAASGFTFSDHGMHWVRQAPEKGLEWVAYISSGSSAIFYADTVKGRFTMSRDNAKNTLFLQMTSLRSEDTAMYFCARWTNLYAMDYWGQGTSVTVSS [Sequence ID 41] Preferably, the antibody or its antigen-binding fragment includes a heavy chain variable (VH) region comprising a sequence substantially as shown in SEQ ID NO: 41 or its variant or fragment.

[0061] The antibody or its antigen-binding fragment may contain a heavy chain sequence such as that shown in SEQ ID NO: 6, which is provided herein as follows: EVQLVESGGGLVKPGGSRKLSCAASGFTFSDHGMHWVRQAPEKGLEWVAYISSGSSAIFYADTVKGRFTMSRDNAKNTLFLQMTSLRSEDTAMYFCARWTNLYAMDYWGQGTS VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK[Sequence ID 6] Preferably, the antibody or its antigen-binding fragment includes a heavy chain region comprising a sequence substantially as shown in SEQ ID NO: 6 or its variant or fragment.

[0062] The antibody or its antigen-binding fragment may contain a heavy chain sequence such as that shown in SEQ ID NO: 49, which is provided herein as follows: QVQLVESGGGVVQPGRSLRLSCAASGFTFSDHGMHWVRQAPGKGLEWVAYISSGSSAIFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWTNLYAMDYWGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK[Sequence No. 49] Preferably, the antibody or its antigen-binding fragment includes a heavy chain region comprising a sequence substantially as shown in SEQ ID NO: 49 or its variant or fragment.

[0063] The antibody or its antigen-binding fragment may contain a heavy chain sequence such as that shown in SEQ ID NO: 50, which is provided herein as follows: QVQLVESGGGVVQPGRSLRLSCAASGFTFSDHGMHWVRQAPGKGLEWVAYISSGSSAIFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCARWTNLYAMDYWGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK[Sequence No. 50] Preferably, the antibody or its antigen-binding fragment includes a heavy chain region comprising a sequence substantially as shown in SEQ ID NO: 50 or its variant or fragment.

[0064] The antibody or its antigen-binding fragment may contain a heavy chain sequence such as that shown in SEQ ID NO: 51, which is provided herein as follows: QVQLVESGGGVVQPGRSLRLSCAASGFTFSDHGMHWVRQAPGKGLEWVAYISSGSSAIFYADTVKGRFTMSRDNSKNTLYLQMNSLRAEDTAVYYCARWTNLYAMDYWGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK[Sequence No. 51] Preferably, the antibody or its antigen-binding fragment includes a heavy chain region comprising a sequence substantially as shown in SEQ ID NO: 51 or its variant or fragment.

[0065] The antibody or its antigen-binding fragment may contain a heavy chain sequence such as that shown in SEQ ID NO: 52, which is provided herein as follows: QVQLVESGGGVVQPGRSLRLSCAASGFTFSDHGMHWVRQAPGKGLEWVAYISSGSSAIFYADTVKGRFTMSRDNSKNTLYLQMNSLRAEDTAVYFCARWTNLYAMDYWGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK[Sequence ID 52] Preferably, the antibody or its antigen-binding fragment includes a heavy chain region comprising a sequence substantially as shown in SEQ ID NO: 52 or its variant or fragment.

[0066] The antibody or its antigen-binding fragment may contain the light chain CDR-L1 domain of SEQ ID NO: 7, which is provided herein as follows: RASKSVSTSGYSYMH[Sequence ID 7] Preferably, the antibody or its antigen-binding fragment contains a light chain CDR-L1 domain comprising a sequence substantially as shown in SEQ ID NO: 7 or its variant or fragment.

[0067] The antibody or its antigen-binding fragment may contain the light chain CDR-L2 domain of SEQ ID NO: 8, which is provided herein as follows: LASNLES[SEQ ID NO: 8] Preferably, the antibody or its antigen-binding fragment contains a light chain CDR-L2 domain comprising a sequence substantially as shown in SEQ ID NO: 8 or its variant or fragment.

[0068] The antibody or its antigen-binding fragment may contain the light chain CDR-L3 domain of SEQ ID NO: 9, which is provided herein as follows: QHSRELPLT[Sequence ID 9] Preferably, the antibody or its antigen-binding fragment comprises a light chain CDR-L3 domain consisting of a sequence substantially represented by SEQ ID NO: 9 or its variant or fragment.

[0069] Preferably, the antibody or its antigen-binding fragment comprises a CDR-L1 domain containing or consisting of SEQ ID NO: 7, a CDR-L2 domain containing or consisting of SEQ ID NO: 8, and / or a CDR-L3 domain containing or consisting of SEQ ID NO: 9. However, preferably, the antibody or its antigen-binding fragment comprises a CDR-L1 domain containing or consisting of SEQ ID NO: 7, a CDR-L2 domain containing or consisting of SEQ ID NO: 8, and a CDR-L3 domain containing or consisting of SEQ ID NO: 9.

[0070] The antibody or its antigen-binding fragment may contain a light chain variable (VL) sequence, such as that shown in SEQ ID NO: 42, which is provided herein as follows: DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTKLELK [Sequence ID 42] Preferably, the antibody or its antigen-binding fragment includes a light chain variable region comprising a sequence substantially as shown in SEQ ID NO: 42 or its variant or fragment.

[0071] The antibody or its antigen-binding fragment may contain a light chain sequence such as that shown in SEQ ID NO: 10, which is provided herein as follows: DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[Sequence ID 10] Preferably, the antibody or its antigen-binding fragment includes a light chain region comprising a sequence substantially as shown in SEQ ID NO: 10 or its variant or fragment.

[0072] The antibody or its antigen-binding fragment may contain a light chain sequence such as that shown in SEQ ID NO: 53, which is provided herein as follows: DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSRELPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[Sequence ID 53] Preferably, the antibody or its antigen-binding fragment includes a light chain region comprising a sequence substantially as shown in SEQ ID NO: 53 or its variant or fragment.

[0073] The antibody or its antigen-binding fragment may contain a light chain sequence such as that shown in SEQ ID NO: 54, which is provided herein as follows: DIVLTQSPDSLAVSLGERATINCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSRELPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[Sequence ID 54] Preferably, the antibody or its antigen-binding fragment includes a light chain region comprising a sequence substantially as shown in SEQ ID NO: 54 or its variant or fragment.

[0074] Preferably, the antibody or its antigen-binding fragment contains at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or its antigen-binding fragment contains at least CDR-H3.

[0075] Preferably, the antibody or its antigen-binding fragment comprises a CDR-H1 domain containing or consisting of SEQ ID NO: 3, a CDR-H2 domain containing or consisting of SEQ ID NO: 4, a CDR-H3 domain containing or consisting of SEQ ID NO: 5, a CDR-L1 domain containing or consisting of SEQ ID NO: 7, a CDR-L2 domain containing or consisting of SEQ ID NO: 8, and a CDR-L3 domain containing or consisting of SEQ ID NO: 9.

[0076] Preferably, the antibody or its antigen-binding fragment includes a heavy chain variable region containing or consisting of SEQ ID NO: 41, and a light chain variable region containing or consisting of SEQ ID NO: 42.

[0077] Preferably, the antibody or its antigen-binding fragment includes a heavy chain region containing or consisting of SEQ ID NO: 6, and a light chain region containing or consisting of SEQ ID NO: 10. Preferably, the antibody or its antigen-binding fragment includes a heavy chain region selected from the group consisting of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, and a light chain region consisting of SEQ ID NO: 53 or SEQ ID NO: 54.

[0078] Preferably, the antibody or its antigen-binding fragment includes a heavy chain region containing or consisting of SEQ ID NO: 49, and a light chain region containing or consisting of SEQ ID NO: 53 or SEQ ID NO: 54. Preferably, the antibody or its antigen-binding fragment includes a heavy chain region containing or consisting of SEQ ID NO: 50, and a light chain region containing or consisting of SEQ ID NO: 53 or SEQ ID NO: 54. Preferably, the antibody or its antigen-binding fragment includes a heavy chain containing or consisting of SEQ ID NO: 51, and a light chain region containing or consisting of SEQ ID NO: 53 or SEQ ID NO: 54. Preferably, the antibody or its antigen-binding fragment includes a heavy chain region containing or consisting of SEQ ID NO: 52, and a light chain region containing or consisting of SEQ ID NO: 53 or SEQ ID NO: 54.

[0079] 19213(「51B12」) In one embodiment, the antibody or its antigen-binding fragment is referred to herein as 51B12. The antibody or its antigen-binding fragment may contain the CDR-H1 domain of SEQ ID NO: 11, which is provided herein as follows: DYGMH[SEQ ID NO: 11] Preferably, the antibody or its antigen-binding fragment comprises a CDR-H1 domain consisting of a sequence substantially represented in SEQ ID NO: 11 or its variant or fragment.

[0080] The antibody or its antigen-binding fragment may contain the CDR-H2 domain of SEQ ID NO: 12, which is provided herein as follows: YISSGGTTIYYADTVKG[Sequence ID 12] Preferably, the antibody or its antigen-binding fragment contains a CDR-H2 domain comprising a sequence substantially as shown in SEQ ID NO: 12 or its variant or fragment.

[0081] The antibody or its antigen-binding fragment may contain the CDR-H3 domain of SEQ ID NO: 13, which is provided herein as follows: WTNLYAMDY[Sequence ID 13] Preferably, the antibody or its antigen-binding fragment contains a CDR-H3 domain comprising a sequence substantially as shown in SEQ ID NO: 13 or its variant or fragment.

[0082] Preferably, the antibody or its antigen-binding fragment comprises a CDR-H1 domain containing or consisting of SEQ ID NO: 11, a CDR-H2 domain containing or consisting of SEQ ID NO: 12, and / or a CDR-H3 domain containing or consisting of SEQ ID NO: 13. However, preferably, the antibody or its antigen-binding fragment comprises a CDR-H1 domain containing or consisting of SEQ ID NO: 11, a CDR-H2 domain containing or consisting of SEQ ID NO: 12, and a CDR-H3 domain containing or consisting of SEQ ID NO: 13.

[0083] The antibody or its antigen-binding fragment may contain a heavy chain variable (VH) sequence, such as the sequence number 43, which is provided herein as follows: EVQLVESGGGLVKPGGSRKLSCAASGFTFSDYGMHWVRQAPEKGLEWVAYISSGGTTIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARWTNLYAMDYWGQGTSVTVSS [Sequence ID 43] Preferably, the antibody or its antigen-binding fragment includes a heavy chain variable (VH) region comprising a sequence substantially as shown in SEQ ID NO: 43 or its variant or fragment.

[0084] The antibody or its antigen-binding fragment may contain a heavy chain sequence such as that shown in SEQ ID NO: 14, which is provided herein as follows: EVQLVESGGGLVKPGGSRKLSCAASGFTFSDYGMHWVRQAPEKGLEWVAYISSGGTTIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARWTNLYAMDYWGQGTSV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK[Sequence ID 14] Preferably, the antibody or its antigen-binding fragment includes a heavy chain region comprising a sequence substantially as shown in SEQ ID NO: 14 or its variant or fragment.

[0085] The antibody or its antigen-binding fragment may contain a heavy chain sequence such as that shown in SEQ ID NO: 55, which is provided herein as follows: QVQLVESGGGVVQPGRSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAYISSGGTTIYYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWTNLYAMDYWGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK[Sequence ID 55] Preferably, the antibody or its antigen-binding fragment includes a heavy chain region comprising a sequence substantially as shown in SEQ ID NO: 55 or its variant or fragment.

[0086] The antibody or its antigen-binding fragment may contain the light chain CDR-L1 domain of SEQ ID NO: 15, which is provided herein as follows: RASKSVSTSGYSYMH[Sequence ID 15] Preferably, the antibody or its antigen-binding fragment contains a light chain CDR-L1 domain comprising a sequence substantially as shown in SEQ ID NO: 15 or its variant or fragment.

[0087] The antibody or its antigen-binding fragment may contain the light chain CDR-L2 domain of SEQ ID NO: 16, which is provided herein as follows: LASNLES[SEQ ID NO: 16] Preferably, the antibody or its antigen-binding fragment contains a light chain CDR-L2 domain comprising a sequence substantially as shown in SEQ ID NO: 16 or its variant or fragment.

[0088] The antibody or its antigen-binding fragment may contain the light chain CDR-L3 domain of SEQ ID NO: 17, which is provided herein as follows: QHSREVPYT[Sequence ID 17] Preferably, the antibody or its antigen-binding fragment contains a light chain CDR-L3 domain comprising a sequence substantially as shown in SEQ ID NO: 17 or its variant or fragment.

[0089] Preferably, the antibody or its antigen-binding fragment comprises a CDR-L1 domain containing or consisting of SEQ ID NO: 15, a CDR-L2 domain containing or consisting of SEQ ID NO: 16, and / or a CDR-L3 domain containing or consisting of SEQ ID NO: 17. However, preferably, the antibody or its antigen-binding fragment comprises a CDR-L1 domain containing or consisting of SEQ ID NO: 15, a CDR-L2 domain containing or consisting of SEQ ID NO: 16, and a CDR-L3 domain containing or consisting of SEQ ID NO: 17.

[0090] The antibody or its antigen-binding fragment may contain a light chain variable (VL) sequence, such as that shown in SEQ ID NO: 44, which is provided herein as follows: DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKFLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSREVPYTFGGGTKLEIK [Sequence ID 44] Preferably, the antibody or its antigen-binding fragment includes a light chain variable (VL) region comprising a sequence substantially as shown in SEQ ID NO: 44 or its variant or fragment.

[0091] The antibody or its antigen-binding fragment may contain a light chain sequence such as that shown in SEQ ID NO: 18, which is provided herein as follows: DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKFLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSREVPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[Sequence ID 18] Preferably, the antibody or its antigen-binding fragment includes a light chain region comprising a sequence substantially as shown in SEQ ID NO: 18 or its variant or fragment.

[0092] The antibody or its antigen-binding fragment may contain a light chain sequence such as that shown in SEQ ID NO: 56, which is provided herein as follows: DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSREVPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[Sequence ID 56] Preferably, the antibody or its antigen-binding fragment includes a light chain region comprising a sequence substantially as shown in SEQ ID NO: 56 or its variant or fragment.

[0093] The antibody or its antigen-binding fragment may contain a light chain sequence such as that shown in SEQ ID NO: 57, which is provided herein as follows: DIVLTQSPDSLAVSLGERATINCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSREVPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[Sequence ID 57] Preferably, the antibody or its antigen-binding fragment includes a light chain region comprising a sequence substantially as shown in SEQ ID NO: 57 or its variant or fragment.

[0094] The antibody or its antigen-binding fragment may contain a light chain sequence such as that shown in SEQ ID NO: 58, which is provided herein as follows: DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSYMHWYQQKPGQPPKFLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSREVPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[Sequence ID 58] Preferably, the antibody or its antigen-binding fragment includes a light chain region comprising a sequence substantially as shown in SEQ ID NO: 58 or its variant or fragment.

[0095] The antibody or its antigen-binding fragment may contain a light chain sequence such as that shown in SEQ ID NO: 59, which is provided herein as follows: DIVLTQSPDSLAVSLGERATINCRASKSVSTSGYSYMHWYQQKPGQPPKFLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSREVPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[Sequence ID 59] Preferably, the antibody or its antigen-binding fragment includes a light chain region comprising a sequence substantially as shown in SEQ ID NO: 59 or its variant or fragment.

[0096] Preferably, the antibody or its antigen-binding fragment contains at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or its antigen-binding fragment contains at least CDR-H3.

[0097] Preferably, the antibody or its antigen-binding fragment comprises a CDR-H1 domain containing or consisting of SEQ ID NO: 11, a CDR-H2 domain containing or consisting of SEQ ID NO: 12, a CDR-H3 domain containing or consisting of SEQ ID NO: 13, a CDR-L1 domain containing or consisting of SEQ ID NO: 15, a CDR-L2 domain containing or consisting of SEQ ID NO: 16, and a CDR-L3 domain containing or consisting of SEQ ID NO: 17.

[0098] Preferably, the antibody or its antigen-binding fragment includes a heavy chain variable region containing or consisting of SEQ ID NO: 43, and a light chain variable region containing or consisting of SEQ ID NO: 44.

[0099] Preferably, the antibody or its antigen-binding fragment includes a heavy chain region containing or consisting of SEQ ID NO: 14, and a light chain region containing or consisting of SEQ ID NO: 18. Preferably, the antibody or its antigen-binding fragment includes a heavy chain region comprising or consisting of SEQ ID NO: 55, and a light chain region selected from the group comprising or consisting of SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, and SEQ ID NO: 59.

[0100] Preferably, the antibody or its antigen-binding fragment includes a heavy chain region containing or consisting of SEQ ID NO: 55, and a light chain region containing or consisting of SEQ ID NO: 56. Preferably, the antibody or its antigen-binding fragment includes a heavy chain region containing or consisting of SEQ ID NO: 55, and a light chain region containing or consisting of SEQ ID NO: 57. Preferably, the antibody or its antigen-binding fragment includes a heavy chain region containing or consisting of SEQ ID NO: 55, and a light chain region containing or consisting of SEQ ID NO: 58. Preferably, the antibody or its antigen-binding fragment includes a heavy chain region containing or consisting of SEQ ID NO: 55, and a light chain region containing or consisting of SEQ ID NO: 59.

[0101] 38F02(19212) In one embodiment, the antibody or its antigen-binding fragment is referred to herein as 19212. The antibody or its antigen-binding fragment may contain the CDR-H1 domain of SEQ ID NO: 20, which is provided herein as follows: DYAMH[Sequence ID 20] Preferably, the antibody or its antigen-binding fragment comprises a CDR-H1 domain comprising a sequence substantially as shown in SEQ ID NO: 20 or its variant or fragment.

[0102] The antibody or its antigen-binding fragment may contain the CDR-H2 domain of SEQ ID NO: 21, which is provided herein as follows: VIRTNYGGASYNQKFKG[Sequence ID 21] Preferably, the antibody or its antigen-binding fragment contains a CDR-H2 domain comprising a sequence substantially as shown in SEQ ID NO: 21 or its variant or fragment.

[0103] The antibody or its antigen-binding fragment may contain the CDR-H3 domain of SEQ ID NO: 22, which is provided herein as follows: GVGRAWFAY[SEQ ID NO: 22] Preferably, the antibody or its antigen-binding fragment contains a CDR-H3 domain comprising a sequence substantially as shown in SEQ ID NO: 22 or its variant or fragment.

[0104] Preferably, the antibody or its antigen-binding fragment comprises a CDR-H1 domain containing or consisting of SEQ ID NO: 20, a CDR-H2 domain containing or consisting of SEQ ID NO: 21, and / or a CDR-H3 domain containing or consisting of SEQ ID NO: 22. However, preferably, the antibody or its antigen-binding fragment comprises a CDR-H1 domain containing or consisting of SEQ ID NO: 20, a CDR-H2 domain containing or consisting of SEQ ID NO: 21, and a CDR-H3 domain containing or consisting of SEQ ID NO: 22.

[0105] The antibody or its antigen-binding fragment may contain a heavy chain variable (VH) sequence, such as that shown in SEQ ID NO: 45, which is provided herein as follows: QVQLQQSGAELVRPGVSVKISCKGSGYTFTDYAMHWVKQSHAKSLEWIGVIRTNYGGASYNQKFKGKATMTVDKSSSTAYMELARLTSEDSAIYYCARGVGRAWFAYWGQGTLVTVSA [Sequence ID 45] Preferably, the antibody or its antigen-binding fragment includes a heavy chain variable (VH) region comprising a sequence substantially as shown in SEQ ID NO: 45 or its variant or fragment.

[0106] The antibody or its antigen-binding fragment may contain a heavy chain sequence such as that shown in SEQ ID NO: 23, which is provided herein as follows: QVQLQQSGAELVRPGVSVKISCKGSGYTFTDYAMHWVKQSHAKSLEWIGVIRTNYGGASYNQKFKGKATMTVDKSSSTAYMELARLTSEDSAIYYCARGVGRAWFAYWGQGTLV TVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK[Sequence ID 23] Preferably, the antibody or its antigen-binding fragment includes a heavy chain region comprising a sequence substantially as shown in SEQ ID NO: 23 or its variant or fragment.

[0107] The antibody or its antigen-binding fragment may contain the light chain CDR-L1 domain of SEQ ID NO: 24, which is provided herein as follows: KASQSVDYDGDSYMN[Sequence ID 24] Preferably, the antibody or its antigen-binding fragment contains a light chain CDR-L1 domain comprising a sequence substantially as shown in SEQ ID NO: 24 or its variant or fragment.

[0108] The antibody or its antigen-binding fragment may contain the CDR-L2 domain of SEQ ID NO: 25, which is provided herein as follows: AASNLES[SEQ ID NO: 25] Preferably, the antibody or its antigen-binding fragment contains a CDR-L2 domain comprising a sequence substantially as shown in SEQ ID NO: 25 or its variant or fragment.

[0109] The antibody or its antigen-binding fragment may contain the CDR-L3 domain of SEQ ID NO: 26, which is provided herein as follows: QQSYDDPYT[Sequence ID 26] Preferably, the antibody or its antigen-binding fragment contains a CDR-L3 domain comprising a sequence substantially as shown in SEQ ID NO: 26 or its variant or fragment.

[0110] Preferably, the antibody or its antigen-binding fragment comprises a CDR-L1 domain containing or consisting of SEQ ID NO: 24, a CDR-L2 domain containing or consisting of SEQ ID NO: 25, and / or a CDR-L3 domain containing or consisting of SEQ ID NO: 26. However, preferably, the antibody or its antigen-binding fragment comprises a CDR-L1 domain containing or consisting of SEQ ID NO: 24, a CDR-L2 domain containing or consisting of SEQ ID NO: 25, and a CDR-L3 domain containing or consisting of SEQ ID NO: 26.

[0111] The antibody or its antigen-binding fragment may contain a light chain variable (VL) sequence, such as that shown in SEQ ID NO: 46, which is provided herein as follows: DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSYDDPYTFGGGTKLEIK [Sequence ID 46] Preferably, the antibody or its antigen-binding fragment includes a light chain variable (VL) region comprising a sequence substantially as shown in SEQ ID NO: 46 or its variant or fragment.

[0112] The antibody or its antigen-binding fragment may contain a light chain sequence such as that shown in SEQ ID NO: 27, which is provided herein as follows: DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSYDDPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[Sequence ID 27] Preferably, the antibody or its antigen-binding fragment includes a light chain region comprising a sequence substantially as shown in SEQ ID NO: 27 or its variant or fragment.

[0113] Preferably, the antibody or its antigen-binding fragment contains at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or its antigen-binding fragment contains at least CDR-H3.

[0114] Preferably, the antibody or its antigen-binding fragment comprises a CDR-H1 domain containing or consisting of SEQ ID NO: 20, a CDR-H2 domain containing or consisting of SEQ ID NO: 21, a CDR-H3 domain containing or consisting of SEQ ID NO: 22, a CDR-L1 domain containing or consisting of SEQ ID NO: 24, a CDR-L2 domain containing or consisting of SEQ ID NO: 25, and a CDR-L3 domain containing or consisting of SEQ ID NO: 26.

[0115] Preferably, the antibody or its antigen-binding fragment includes a heavy chain variable region containing or comprising SEQ ID NO: 45, and a light chain variable region containing or comprising SEQ ID NO: 46.

[0116] Preferably, the antibody or its antigen-binding fragment includes a heavy chain region containing or comprising SEQ ID NO: 23, and a light chain region containing or comprising SEQ ID NO: 27. 89G11(19218) In one embodiment, the antibody or its antigen-binding fragment is referred to herein as 19218. The antibody or its antigen-binding fragment may contain the CDR-H1 domain of SEQ ID NO: 28, which is provided herein as follows: DYEMF[SEQ ID NO: 28] Preferably, the antibody or its antigen-binding fragment comprises a CDR-H1 domain consisting of a sequence substantially represented by SEQ ID NO: 28 or its variant or fragment.

[0117] The antibody or its antigen-binding fragment may contain the CDR-H2 domain of SEQ ID NO: 29, which is provided herein as follows: VIDPETGGTAYNQKFKG[Sequence ID 29] Preferably, the antibody or its antigen-binding fragment contains a CDR-H2 domain comprising a sequence substantially as shown in SEQ ID NO: 29 or its variant or fragment.

[0118] The antibody or its antigen-binding fragment may contain the CDR-H3 domain of SEQ ID NO: 30, which is provided herein as follows: GYGDYPFAY [SEQ ID NO: 30] Preferably, the antibody or its antigen-binding fragment contains a CDR-H3 domain comprising a sequence substantially as shown in SEQ ID NO: 30 or its variant or fragment.

[0119] Preferably, the antibody or its antigen-binding fragment comprises a CDR-H1 domain containing or consisting of SEQ ID NO: 28, a CDR-H2 domain containing or consisting of SEQ ID NO: 29, and / or a CDR-H3 domain containing or consisting of SEQ ID NO: 30. However, preferably, the antibody or its antigen-binding fragment comprises a CDR-H1 domain containing or consisting of SEQ ID NO: 28, a CDR-H2 domain containing or consisting of SEQ ID NO: 29, and a CDR-H3 domain containing or consisting of SEQ ID NO: 30.

[0120] The antibody or its antigen-binding fragment may contain a heavy chain variable (VH) sequence, such as the sequence number 47, which is provided herein as follows: QVQLQQSGAELVRPGASVKLSCKASGYTFTDYEMFWVKQTPVYGLEWIGVIDPETGGTAYNQKFKGKATLTADKSSSTAYMELRSLTSEDSAVYYCTRGYDGYPFAYWGQGTLVTVSA [Sequence ID 47] Preferably, the antibody or its antigen-binding fragment includes a heavy chain variable (VH) region comprising a sequence substantially as shown in SEQ ID NO: 47 or its variant or fragment.

[0121] The antibody or its antigen-binding fragment may contain a heavy chain sequence such as that shown in SEQ ID NO: 31, which is provided herein as follows: QVQLQQSGAELVRPGASVKLSCKASGYTFTDYEMFWVKQTPVYGLEWIGVIDPETGGTAYNQKFKGKATLTADKSSSTAYMELRSLTSEDSAVYYCTRGYDGYPFAYWGQGTLV TVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK[Sequence ID 31] Preferably, the antibody or its antigen-binding fragment includes a heavy chain region comprising a sequence substantially as shown in SEQ ID NO: 31 or its variant or fragment.

[0122] The antibody or its antigen-binding fragment may contain the light chain CDR-L1 domain of SEQ ID NO: 32, which is provided herein as follows: KSSQLYSSNQKNFLA[Sequence ID 32] Preferably, the antibody or its antigen-binding fragment comprises a CDR-L1 domain consisting of a sequence substantially represented by SEQ ID NO: 32 or its variant or fragment.

[0123] The antibody or its antigen-binding fragment may contain the CDR-L2 domain of SEQ ID NO: 33, which is provided herein as follows: WASTRES [Sequence ID 33] Preferably, the antibody or its antigen-binding fragment contains a CDR-L2 domain comprising a sequence substantially as shown in SEQ ID NO: 33 or its variant or fragment.

[0124] The antibody or its antigen-binding fragment may contain the CDR-L3 domain of SEQ ID NO: 34, which is provided herein as follows: LQYLSSYT[Sequence ID 34] Preferably, the antibody or its antigen-binding fragment comprises a CDR-L3 domain consisting of a sequence substantially represented by SEQ ID NO: 34 or its variant or fragment.

[0125] Preferably, the antibody or its antigen-binding fragment comprises a CDR-L1 domain containing or consisting of SEQ ID NO: 32, a CDR-L2 domain containing or consisting of SEQ ID NO: 33, and / or a CDR-L3 domain containing or consisting of SEQ ID NO: 34. However, preferably, the antibody or its antigen-binding fragment comprises a CDR-L1 domain containing or consisting of SEQ ID NO: 32, a CDR-L2 domain containing or consisting of SEQ ID NO: 33, and a CDR-L3 domain containing or consisting of SEQ ID NO: 34.

[0126] The antibody or its antigen-binding fragment may contain a light chain variable (VL) sequence, such as that shown in SEQ ID NO: 48, which is provided herein as follows: NIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNFLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVRAEDLAVYYCLQYLSSYTFGGGTRLDIK [Sequence ID 48] Preferably, the antibody or its antigen-binding fragment includes a light chain variable (VL) region comprising a sequence substantially as shown in SEQ ID NO: 48 or its variant or fragment.

[0127] The antibody or its antigen-binding fragment may contain a light chain sequence such as that shown in SEQ ID NO: 35, which is provided herein as follows: NIMMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNFLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVRAEDLAVYYCLQYLSSYTFGGGTRLDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[Sequence ID 35] Preferably, the antibody or its antigen-binding fragment includes a light chain region comprising a sequence substantially as shown in SEQ ID NO: 35 or its variant or fragment.

[0128] Preferably, the antibody or its antigen-binding fragment contains at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or its antigen-binding fragment contains at least CDR-H3.

[0129] Preferably, the antibody or its antigen-binding fragment comprises a CDR-H1 domain containing or consisting of SEQ ID NO: 28, a CDR-H2 domain containing or consisting of SEQ ID NO: 29, a CDR-H3 domain containing or consisting of SEQ ID NO: 30, a CDR-L1 domain containing or consisting of SEQ ID NO: 32, a CDR-L2 domain containing or consisting of SEQ ID NO: 33, and a CDR-L3 domain containing or consisting of SEQ ID NO: 34.

[0130] Preferably, the antibody or its antigen-binding fragment includes a heavy chain variable region containing or consisting of SEQ ID NO: 47, and a light chain variable region containing or consisting of SEQ ID NO: 48.

[0131] Preferably, the antibody or its antigen-binding fragment includes a heavy chain region containing or comprising SEQ ID NO: 31, and a light chain region containing or comprising SEQ ID NO: 35. As shown in Figure 5, the antibodies 51B12 and 63A11 share a high degree of sequence identity, which allowed us to identify amino acid residues that we believe are important for maintaining anti-ZIP12 activity and to generate a consensus sequence.

[0132] Therefore, preferably, the antibody or its antigen-binding fragment may contain the CDR-H1 domain of SEQ ID NO: 36, which is provided herein as follows: DXGMH[SEQ ID NO: 36] Preferably, the antibody or its antigen-binding fragment comprises a CDR-H1 domain consisting of a sequence substantially as shown in SEQ ID NO: 36 or its variant or fragment, where X can be any amino acid in the sequence of SEQ ID NO: 36. Preferably, X is H or Y.

[0133] The antibody or its antigen-binding fragment may contain the CDR-H2 domain of SEQ ID NO: 37, which is provided herein as follows: YISSGX 1 X 2 X 3 IX 4 YADTVKG[Sequence ID 37] Preferably, the antibody or its antigen-binding fragment comprises a CDR-H2 domain containing or consisting of a sequence substantially as shown in SEQ ID NO: 37 or its variant or fragment, and in the sequence of SEQ ID NO: 37, X 1 , X 2 , X 3 , and X 4 X can be any amino acid. Preferably, X 1 It can be S or G. Preferably, X 2 It can be S or T. Preferably, X 3 can be A or T, and X 4It could be F or Y.

[0134] The antibody or its antigen-binding fragment may contain the CDR-H3 domain of SEQ ID NO: 5, which is provided herein as follows: WTNLYAMDY[SEQ ID NO: 5] Preferably, the antibody or its antigen-binding fragment comprises a CDR-H3 domain consisting of a sequence substantially as shown in SEQ ID NO: 5 or its variant or fragment.

[0135] The antibody or its antigen-binding fragment may contain a heavy chain variable domain as shown in SEQ ID NO: 38, which is provided herein as follows: EVQLVESGGGLVKPGGSRKLSCAASGFTFSDX 1 GMHWVRQAPEKGLEWVAYISSGX 2 X 3 X 4 IX 5 YADTVKGRFTX 6 SRDNAKNTLFLQMTSLRSEDTAMYX 7 CARWTNLYAMDYWGQGTSVTVSS [Sequence ID 38] Therefore, preferably, the antibody or its antigen-binding fragment, or the fragment or variant thereof, comprises or consists of SEQ ID NO: 38, and in the sequence of SEQ ID NO: 38, X 1 ~X 7 X can be any amino acid. Preferably, X 1 This can be Y or H. Preferably, X 2 It can be G or S. Preferably, X 3 can be T or S, X 4 can be T or A, X 5 This can be Y or F. Preferably, X 6 It can be I or M. Preferably, X 7 It can be Y or F.

[0136] The antibody or its antigen-binding fragment may contain the light chain CDR-L1 domain of SEQ ID NO: 7, which is provided herein as follows: RASKSVSTSGYSYMH[Sequence ID 7] Preferably, the antibody or its antigen-binding fragment comprises a CDR-L1 domain consisting of a sequence substantially represented by SEQ ID NO: 7 or its variant or fragment.

[0137] The antibody or its antigen-binding fragment may contain the CDR-L2 domain of SEQ ID NO: 8, which is provided herein as follows: LASNLES[SEQ ID NO: 8] Preferably, the antibody or its antigen-binding fragment comprises a CDR-L2 domain consisting of a sequence substantially represented by SEQ ID NO: 8 or its variant or fragment.

[0138] The antibody or its antigen-binding fragment may contain the CDR-L3 domain of SEQ ID NO: 39, which is provided herein as follows: QHSREX 1 PX 2 T [Sequence ID 39] Thus, preferably, the antibody or its antigen-binding fragment comprises a CDR-L3 domain consisting of a sequence substantially represented in SEQ ID NO: 39 or its variant or fragment, and in the sequence of SEQ ID NO: 39, X 1 and X 2 X can be any amino acid. Preferably, X 1 It can be L or V. Preferably, X 2 It can be L or Y.

[0139] The antibody or its antigen-binding fragment may contain a light chain variable sequence such as that shown in SEQ ID NO: 40, which is provided herein as follows: DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSREX 1 PX 2 TFGX 3 GTKLELK[SEQ ID NO: 40] Preferably, the antibody or its antigen-binding fragment includes a light chain variable region comprising a sequence substantially shown in SEQ ID NO: 40 or its variant or fragment, and in the sequence of SEQ ID NO: 40, X 1 and X 2 X can be any amino acid. Preferably, X 1 It can be L or V. Preferably, X 2 It can be L or Y, and X 3 It could be G or A.

[0140] The antibody of the present invention may contain or consist of any sequence as defined in any one of Figures 14-19. For example, an antibody or its antigen-binding fragment may include a heavy chain variable sequence containing or consisting of a sequence substantially shown in SEQ ID NO: 69, 71, or 72, or its variants or fragments. Thus, preferably, an antibody or its antigen-binding fragment may include a heavy chain variable sequence containing or consisting of a sequence substantially shown in SEQ ID NO: 69, or its variants or fragments. Preferably, an antibody or its antigen-binding fragment may include a heavy chain variable sequence containing or consisting of a sequence substantially shown in SEQ ID NO: 71, or its variants or fragments. Preferably, an antibody or its antigen-binding fragment may include a heavy chain variable sequence containing or consisting of a sequence substantially shown in SEQ ID NO: 72, or its variants or fragments.

[0141] For example, an antibody or its antigen-binding fragment may include a light chain variable sequence containing a sequence substantially shown in SEQ ID NO: 70, 73, or 74, or a variant or fragment thereof. Thus, preferably, an antibody or its antigen-binding fragment may include a light chain variable sequence containing a sequence substantially shown in SEQ ID NO: 70, or a variant or fragment thereof. Preferably, an antibody or its antigen-binding fragment may include a light chain variable sequence containing a sequence substantially shown in SEQ ID NO: 73, or a variant or fragment thereof. Preferably, an antibody or its antigen-binding fragment may include a light chain variable sequence containing a sequence substantially shown in SEQ ID NO: 74, or a variant or fragment thereof.

[0142] In one embodiment, the antibody or its antigen-binding fragment may include a heavy chain variable sequence containing or comprising a sequence substantially as shown in SEQ ID NO: 69, 71, or 72, and a light chain variable sequence containing or comprising a sequence substantially as shown in SEQ ID NO: 70, 73, or 74.

[0143] Thus, advantageously, the anti-ZIP12 activity of the antibody or its antigen-binding fragment according to the first aspect of the present invention means that it has therapeutic utility on its own and can be used in the treatment, improvement, or prevention of hypoxia-induced or hypoxia-related conditions, and in particular pulmonary hypertension.

[0144] Accordingly, in a second aspect of the present invention, an antibody or an antigen-binding fragment thereof according to the first aspect is provided for therapeutic use. In a third aspect of the present invention, an antibody or an antigen-binding fragment thereof according to the first aspect is provided for use in treating, preventing, or improving hypoxia-related conditions.

[0145] A fourth aspect of the present invention provides a method for treating, preventing or improving a hypoxia-related condition in a subject, comprising the step of administering, or being administered to, a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to the first aspect to a patient in need of such treatment.

[0146] Hypoxia-related conditions may be selected from the following group: ischemic reperfusion injury (IRI), cardiovascular disease, ischemic heart disease, ischemic cerebral state, macular degeneration, ocular ischemic syndrome, ischemic optic neuropathy (ION), diabetic retinopathy, arthritis, inflammation, sepsis, septic-induced shock, renal disease, histofibrosis, gastrointestinal disease, neurodegenerative disease, respiratory distress syndrome, bronchopulmonary dysplasia, pulmonary hypertension, hypoxic pulmonary hypertension, severe pulmonary hypertension, COPD, idiopathic pulmonary fibrosis (IPF), diabetic retinopathy, diabetes mellitus, corneal neovascularization, pathogenic blood vessel growth, cancer, and musculoskeletal disorders.

[0147] The inventors have demonstrated that the hypoxic core of a tumor is the site of hypoxic-induced angiogenesis (Zhao et al., 2015, The zinc transporter, ZIP12, regulates the pulmonary vascular response to chronic hypoxia, Nature, vol:524, ISSN:0028-0836, pp:356-360), and hypoxia is known to be a universal feature of tumors and contributes to resistance to radiation and chemotherapy. Therefore, ZIP12, which the inventors have demonstrated is upregulated in hypoxic conditions and is a key factor in disease progression, has been identified by the inventors as an ideal therapeutic target for cancer. Inhibition of ZIP12 in the tumor microenvironment can restrict blood supply to the tumor, thereby limiting tumor growth and / or inducing hypoxic tumor cell death.

[0148] Thus, in one embodiment, the hypoxia-related state is cancer. Preferably, the use or method in treating, preventing, or improving cancer includes inhibiting angiogenesis. Preferably, the use or method includes restricting the blood supply to the tumor.

[0149] However, preferably, the hypoxia-related state is pulmonary hypertension. Most preferably, the oxygen-related state is hypoxic pulmonary hypertension. It will be understood that the antibodies and fragments thereof according to the present invention (collectively referred to herein as “active agents”) may be used in monotherapy (e.g., the use of the antibody or its antigen-binding fragment alone) to treat, improve, or prevent hypoxia-associated conditions, preferably pulmonary hypertension. Alternatively, the active agents according to the present invention may be used as adjuncts to, or in combination with, known treatments for treating, improving, or preventing hypoxia-associated conditions, preferably pulmonary hypertension, such as anticoagulants such as warfarin, diuretics, endothelin receptor antagonists such as digoxin, bosentan, ambrisentan, and macitentan, phosphodiesterase 5 inhibitors such as sildenafil and tadalafil, prostaglandins such as epoprostenol, iloprost, and treprostinil, soluble guanylate cyclase stimulants such as riociguat, and calcium channel blockers such as nifedipine, diltiazem, nicardipine, and amlodipine.

[0150] The active substances according to the present invention can be combined in several different forms of compositions, particularly depending on the mode in which the composition is to be used. Thus, for example, the composition may take the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micelle solution, transdermal patch, liposome suspension, or any other suitable form, which can be administered to a person or animal in need of treatment. It will be recognized that the medium of the drug according to the present invention should be well acceptable to the subject to which it is given.

[0151] The drug containing the active substance of the present invention can be used in several forms. For example, oral administration may be required, in which case the active substance may be contained in a composition that can be taken orally, for example, in the form of a tablet, capsule, or liquid. Compositions containing the active substance and drug of the present invention can be administered by inhalation (for example, intranasally). Compositions can also be formulated for topical use. For example, a cream or ointment may be applied to the skin.

[0152] The active substances and drugs according to the present invention may also be incorporated into sustained-release or delayed-release devices. Such devices may, for example, be inserted above or below the skin, and the drug may be released over several weeks or even months. The device may be located at least adjacent to the treatment site. Such devices may be particularly advantageous when long-term treatment with the active substance used according to the present invention is required and which typically requires frequent administration (e.g., at least daily injections).

[0153] In a preferred embodiment, the active substances and drugs according to the present invention may be administered to a subject by injection into the bloodstream or directly to a site requiring treatment. The injection may be intravenous (bolus or infusion), subcutaneous (bolus or infusion), or intradermal (bolus or infusion).

[0154] The required amount of antibodies and fragments (i.e., active agents) is determined by their bioactivity and bioavailability, and it will be recognized that they, in turn, depend on the mode of administration, the physiochemical properties of the active agent, and whether it is to be used as monotherapy or in combination therapy. The frequency of administration is also influenced by the half-life of the active agent in the subject to be treated. The optimal dosage to be administered can be determined by those skilled in the art and will vary depending on the specific active agent used, the strength of the pharmaceutical composition, the mode of administration, and the progression of pulmonary hypertension. Additional factors specific to the subject to be treated, including the subject's age, weight, sex, diet, and timing of administration, may necessitate adjustments to the dosage.

[0155] Generally, a daily dose of the active substance according to the present invention, ranging from 0.01 μg / kg body weight to 100 mg / kg body weight, can be used to treat, improve, or prevent coronavirus infection, depending on the active substance. More preferably, the daily dose of the active substance is between 1 μg / kg body weight and 100 mg / kg body weight, more preferably between 10 μg / kg and 10 mg / kg body weight, and most preferably between approximately 100 μg / kg and 10 mg / kg body weight.

[0156] The active agent may be administered before, during, or after the onset of pulmonary hypertension. The daily dose may be given as a single dose (e.g., once daily injection). Alternatively, the active agent may require administration two or more times a day. For example, the active agent may be administered in doses between 0.07 μg and 700 mg (i.e., assuming a body weight of 70 kg) twice daily (or more, depending on the severity of the pulmonary hypertension to be treated). The patient receiving treatment may take the first dose upon waking, and then the second dose (in the case of a two-dose regimen) at night, or after an interval of three or four hours thereafter. Alternatively, a sustained-release device may be used to deliver the optimal dose of the active agent according to the present invention to the patient without the need for repeated administration. Known procedures, such as those commonly used by the pharmaceutical industry (e.g., in vivo experiments, clinical trials, etc.), may be used to form specific formulations of the active agent according to the present invention and precise treatment regimens (such as the daily dose and frequency of administration of the active agent).

[0157] In a fifth embodiment of the present invention, a pharmaceutical composition is provided comprising an antibody or an antigen-binding fragment thereof according to the first embodiment, and optionally, a pharmaceutically acceptable medium. The pharmaceutical composition is preferably an antihypertensive agent, i.e., a pharmaceutical preparation used for the therapeutic improvement, prevention, or treatment of pulmonary hypertension.

[0158] The present invention also provides, in a sixth embodiment, a method for preparing a pharmaceutical composition according to the fifth embodiment, comprising the step of combining a therapeutically effective amount of an antibody or an antigen-binding fragment thereof, as defined in the first embodiment, with a pharmaceutically acceptable medium.

[0159] The antibody or its antigen-binding fragment may be as defined in relation to the first embodiment. The "subject" may be a vertebrate, mammal, or domesticated animal. Therefore, the drug according to the present invention may be used to treat any mammal, e.g., livestock (e.g., horses), pets, or for other veterinary applications. Most preferably, the subject is a human.

[0160] The "therapeutic effective dose" of an antibody or its antigen-binding fragment is any amount that, when administered to a subject, is required to treat pulmonary hypertension or produce the desired effect.

[0161] For example, the therapeutically effective amount of the antibody or antigen-binding fragment used may be from about 0.1 ng / kg to about 100 mg / kg, preferably from about 1 ng / kg to about 10 mg / kg. It is preferable that the amount of antibody or fragment be from about 10 ng / kg to about 10 mg / kg, most preferably from about 50 ng / kg to about 5 mg / kg.

[0162] Where used herein, “pharmaceutically acceptable medium” is any known compound or combination of known compounds that is useful to those skilled in the art in formulating a pharmaceutical composition.

[0163] In one embodiment, the pharmaceutically acceptable medium may be a solid, and the composition may take the form of a powder or a tablet. A solid pharmaceutically acceptable medium may contain one or more substances that can act as a flavoring agent, lubricant, solubilizer, suspending agent, colorant, bulking agent, flow enhancer, compression aid, inert binder, sweetener, preservative, colorant, coating agent, or tablet disintegrant. The medium may also be an encapsulating material. In the case of a powder, the medium is a micronized solid in a mixed state with the micronized active substance according to the present invention. In the case of a tablet, the active substance can be mixed in an appropriate proportion with a medium having the required compressibility and compressed into the desired shape and size. The powder and tablet preferably contain up to 99% of the active substance. Suitable solid mediums include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low-melting-point waxes, and ion-exchange resins. In another embodiment, the pharmaceutical medium may be a gel, and the composition may take the form of a cream or other similar substance.

[0164] However, the pharmaceutical medium may be a liquid, and the pharmaceutical composition may take the form of a solution. Liquid mediums are used in preparing solutions, suspensions, emulsions, syrups, elixirs, and pressurized compositions. The active ingredients according to the present invention may be dissolved or suspended in water, organic solvents, mixtures of both, or pharmaceutically acceptable liquid mediums such as pharmaceutically acceptable oils or fats. Liquid mediums may contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavorings, suspending agents, thickeners, colorants, viscosity modifiers, stabilizers, or osmotic pressure modifiers. Suitable examples of liquid mediums for oral and parenteral administration include water (partially containing the above-mentioned additives, e.g., cellulose derivatives, preferably carboxymethylcellulose solutions), alcohols (monohydric and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and peanut oil). For parenteral administration, the medium may also be oily esters such as ethyl oleate and isopropyl myristate. Sterile liquid media are useful in sterile liquid compositions for parenteral administration. For pressurized compositions, the liquid media may be halogenated hydrocarbons or other pharmaceutically acceptable sprays.

[0165] Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be administered, for example, by intramuscular, intrathecal, epidural, intraperitoneal, intravenous, and especially subcutaneous injection. The active ingredient may be prepared as a sterile solid composition, which can be dissolved or suspended at the time of administration using sterile water, saline, or other suitable sterile injection medium.

[0166] The active substances and compositions of the present invention may be administered orally in the form of sterile solutions or suspensions containing other solutes or suspending agents (e.g., saline or glucose sufficient to make the solution isotonic), bile salts, acacia gum, gelatin, sorbitan monooleate, polysorbate 80 (oleic acid esters of sorbitol and its anhydride copolymerized with ethylene oxide) and other similar substances. The active substances used in the present invention may also be administered orally in the form of either liquid or solid compositions. Compositions suitable for oral administration include solid forms such as pills, capsules, granules, tablets, and powders, as well as liquid forms such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

[0167] The present invention also extends to a method for producing an antibody according to a first embodiment and to an antibody produced in this manner. In a seventh embodiment, an antibody or its antigen-binding fragment obtained by a method comprising the following steps is provided: (i) a step of immunizing a host organism with the extracellular domain of ZIP12 or a region of the ZIP12 protein, as defined in the first embodiment; and (ii) A step of collecting an antibody or an antigen-binding fragment thereof from the host.

[0168] The host can be a mammal, and could be a human, rabbit, or mouse. Preferably, the method includes the steps of collecting blood from a host animal and then collecting antibodies or antigen-binding fragments from the blood, most preferably from serum. Preferably, the serum is passed through a gravity column having a covalently bound peptide support. After washing, the antibodies or antigen-binding fragments are preferably eluted in a buffer (preferably an acidic buffer), and the solution can then be neutralized. The method may further include dialysis in a suitable buffer (e.g., PBS) and, optionally, lyophilization.

[0169] In an eighth aspect of the present invention, a polynucleotide encoding an antibody or an antigen-binding fragment thereof, as defined in the first aspect, is provided. In a ninth embodiment of the present invention, an expression cassette comprising a polynucleotide sequence according to the eighth embodiment is provided.

[0170] The polynucleotide sequence encoding the antibody or its antigen-binding fragment of the present invention is preferably contained within a recombinant vector, for example, a recombinant vector for delivery to a host cell of interest, in order to enable the production of the antibody or its antigen-binding fragment.

[0171] Accordingly, in a tenth embodiment of the present invention, a recombinant vector comprising an expression cassette according to the ninth embodiment is provided. The vector encoding the antibody or its antigen-binding fragment according to the first embodiment may be, for example, a plasmid, cosmid, or phage, and / or a viral vector. Such recombinant vectors are very useful in the delivery system of the present invention for transforming cells with a nucleotide sequence. The nucleotide sequence may preferably be a DNA sequence, which is the DNA sequence encoding the antibody or its antigen-binding fragment that forms the antibody or its antigen-binding fragment according to the first embodiment.

[0172] Recombinant vectors encoding an antibody or its antigen-binding fragment according to the first embodiment may also include other functional elements. For example, they may further include a variety of other functional elements, including a suitable promoter for initiating transgene expression upon introduction of the vector into a host cell. For example, the vector may preferably have the ability to autonomously replicate in the nucleus of a host cell. In this case, elements that induce or control DNA replication may be required in the recombinant vector. Alternatively, the recombinant vector may be designed to integrate into the genome of a host cell. In this case, a DNA sequence that works favorably for the targeted integration (e.g., by homologous recombination) is conceived. Suitable promoters may include the SV40 promoter, CMV, EF1a, PGK, viral terminal repeat sequences, and inductive promoters such as the tetracycline induction system, for example. The cassette or vector may also include a terminator such as betaglobin, an SV40 polyadenylated sequence, or a synthetic polyadenylated sequence. The recombinant vector may also include promoters or regulators or enhancers to modulate nucleic acid expression, as needed.

[0173] The vector may also contain DNA encoding a gene that can be used as a selection marker in the cloning process, i.e., to enable the selection of cells being transfected or transformed, and to enable the selection of cells having a vector incorporating heterologous DNA. For example, ampicillin, neomycin, puromycin, or chloramphenicol resistance are conceivable. Alternatively, the selection marker gene may be in a different vector used simultaneously with the vector containing the transgene. The cassette or vector may also contain DNA involved in controlling the expression of nucleotide sequences, or DNA for targeting expressed polypeptides to a specific region of the host cell.

[0174] The purified vector can be directly inserted into host cells by appropriate means, such as direct endocytosis. The vector can be directly introduced into host cells (e.g., eukaryotic or prokaryotic cells) by transfection, infection, electroporation, microinjection, cell fusion, protoplast fusion, calcium phosphate method, cationic lipid-based lipofection, polymer or dendrimer-based methods, or ballistic bombardment. Alternatively, the vector of the present invention can be directly introduced into host cells using a particle gun.

[0175] Alternatively, the delivery system may deliver polynucleotides to host cells without them being incorporated into a vector. For example, nucleic acid molecules may be incorporated into liposomes or viral particles. Or, "naked" polynucleotides may be inserted into host cells by appropriate means, such as direct endocytosis.

[0176] In an eleventh embodiment of the present invention, a host cell is provided comprising a polynucleotide sequence according to the eighth embodiment, an expression cassette according to the ninth embodiment, or a vector according to the tenth embodiment. The host cell may be a eukaryotic or prokaryotic host cell. Preferably, the host cell is a eukaryotic host cell. More preferably, the host cell is a mammalian host cell, such as NS0 mouse myeloma cells, PER.C6® human cells, human fetal kidney 293 cells, or Chinese hamster ovary (CHO) cells. Most preferably, the host cell is a CHO cell.

[0177] In a twelfth embodiment, a method for preparing an antibody or an antigen-binding fragment thereof according to a first embodiment is provided, comprising the following steps: a) the step of introducing the vector of the tenth embodiment into a host cell; and b) A step of culturing the host cells under conditions that can produce an antibody or an antigen-binding fragment thereof according to the first embodiment.

[0178] The host cell in step a) may be a eukaryotic or prokaryotic host cell. Preferably, the host cell is a eukaryotic host cell. More preferably, the host cell is a mammalian host cell, such as NS0 mouse myeloma cells, PER.C6® human cells, human fetal kidney 293 cells, or Chinese hamster ovary (CHO) cells. Most preferably, the host cell is a CHO cell.

[0179] The method may further include the step of (c) collecting the cell culture medium, centrifuging it, and / or filtering it to obtain a cell culture supernatant containing the antibody or its antigen-binding fragment. The method may further include (d) separating and purifying the antibody or its antigen-binding fragment from the cell culture supernatant. Preferably, the purification is carried out by at least one chromatographic step.

[0180] Appropriate chromatographic steps include affinity chromatography and / or ion exchange chromatography. Preferably, the affinity chromatography is protein A chromatography. The ion exchange chromatography may be anion exchange chromatography and / or cation exchange chromatography.

[0181] Preferably, step (d) includes separating and purifying the antibody or its antigen-binding fragment from the cell culture supernatant as follows: i) Protein A chromatography; ii) Anion exchange chromatography; and / or iii) Cation exchange chromatography.

[0182] The method may further include a step of filtering the purified antibody or antigen-binding fragment obtained from step (d). Preferably, step (e) includes viral filtration. Therefore, preferably, the purified antibody or antigen-binding fragment obtained from step (d) is filtered using a viral filtration membrane. Suitable membranes are known to those skilled in the art.

[0183] As discussed herein, ZIP12 expression is increased in many cell types, including endothelial cells, smooth muscle cells, and stromal cells, in reconstructed pulmonary arterioles of rats, cattle, and humans susceptible to hypoxia-induced pulmonary hypertension. Therefore, assuming that the antibody of the present invention can bind to the extracellular domain of ZIP12, the antibody or its antigen-binding fragment can be used as a robust diagnostic tool by detecting the presence of ZIP12 and determining its concentration.

[0184] Accordingly, in a thirteenth embodiment, an antibody or its antigen-binding fragment according to the first embodiment is provided for use in diagnostic or prognostic diagnosis. According to a fourteenth aspect of the present invention, an antibody or an antigen-binding fragment thereof according to a first aspect is provided for use in diagnosing or prognosticating hypoxia-related conditions.

[0185] According to the 15th aspect, a method is provided for diagnosing or prognosing a hypoxia-related condition in a subject, comprising the step of detecting ZIP12 in a biological sample obtained from the subject using an antibody or its antigen-binding fragment according to the first aspect.

[0186] Preferably, hypoxia-related conditions are selected from the group consisting of: ischemic reperfusion injury (IRI), cardiovascular disease, ischemic heart disease, ischemic cerebral state, macular degeneration, ocular ischemic syndrome, ischemic optic neuropathy (ION), diabetic retinopathy, arthritis, inflammation, sepsis, septic-induced shock, renal disease, histofibrosis, gastrointestinal disease, neurodegenerative disease, respiratory distress syndrome, bronchopulmonary dysplasia, pulmonary hypertension, hypoxic pulmonary hypertension, severe pulmonary hypertension, COPD, idiopathic pulmonary fibrosis (IPF), diabetic retinopathy, diabetes, corneal neovascularization, pathological vascular proliferation, cancer, and musculoskeletal disorders.

[0187] Preferably, the hypoxia-related condition is cancer or pulmonary hypertension. Most preferably, the hypoxia-related condition is pulmonary hypertension. The method may be in vitro or ex vivo. Preferably, the method is in vitro.

[0188] The use or method may include a step of determining the level of ZIP12 expression in the subject, preferably an increase in the concentration of ZIP12 in the biological sample compared to a reference concentration from a healthy control population, indicating pulmonary hypertension or poor prognosis.

[0189] In one embodiment, the presence of ZIP12 in the lungs may indicate pulmonary hypertension or a poor prognosis. The presence of ZIP12 can be detected by immunocytochemistry. In one embodiment, a 1x increase in ZIP12 compared to a reference from a healthy control population indicates pulmonary hypertension or poor prognosis. In one embodiment, a 2x, 3x, 4x, or 5x increase in ZIP12 compared to a reference from a healthy control population indicates pulmonary hypertension or poor prognosis. In one embodiment, a 10x, 50x, or 100x increase in ZIP12 compared to a reference from a healthy control population indicates pulmonary hypertension or poor prognosis.

[0190] According to a sixteenth aspect of the present invention, a kit is provided for diagnosing a subject suffering from a hypoxia-related condition or for providing a prognostic diagnosis of the subject's condition, comprising an antibody or antigen-binding fragment thereof according to a first aspect for detecting ZIP12 in a sample derived from a subject to be tested.

[0191] The kit may further include instructions for use and / or a container for collecting biological samples from the subject. Preferably, the hypoxia-related condition is as defined in the 14th embodiment, and preferably pulmonary hypertension.

[0192] Prognostic diagnosis may be relevant to determining the effectiveness of treatment in patients diagnosed with pulmonary hypertension. Prognostic diagnosis may be relevant to predicting the rate and / or duration of progression or improvement of pulmonary hypertension, survival rate, and / or the efficacy of various treatment regimens in patients. Therefore, a poor prognosis may indicate progression of pulmonary hypertension, low survival rate, and reduced efficacy of treatment regimens. A good prognosis may indicate resolution of pulmonary hypertension, high survival rate, and increased efficacy of treatment regimens.

[0193] Preferably, the sample includes a biological sample. The sample may be any material obtainable from the subject from which proteins can be obtained. A biological sample may be tissue or biological fluid. A biological sample may be any material obtainable from the subject from which endothelial cells, smooth muscle cells, and / or stromal cells can be obtained. Furthermore, a sample may be blood, plasma, serum, cerebrospinal fluid, urine, sweat, saliva, tears, mammary gland fluid, breast milk, prostatic fluid, semen, vaginal fluid, feces, cervical swabs, cells, amniotic fluid, intraocular fluid, mucus, respiratory fluid, animal tissue, cell solubilization, tumor tissue, hair, skin, buccal swabs, lymph, interstitial fluid, nails, bone marrow, cartilage, prions, bone meal, earwax, lymph, granuloma, cancer biopsy, or a combination thereof.

[0194] The sample may be a liquid aspirate. For example, the sample may be bronchoalveolar lavage fluid (BAL), ascites, pleural lavage fluid, or pericardial lavage fluid. The sample may include blood, urine, tissue, etc. In one preferred embodiment, the biological sample includes a blood sample. The blood may be venous or arterial blood. The blood sample may be assayed immediately. Alternatively, the blood sample may be stored at a low temperature, for example in a refrigerator, or even frozen, before the method is performed. Alternatively, the blood sample may be stored at room temperature, for example between 18°C ​​and 22°C, before the method is performed. The blood sample may include serum. The blood sample may include plasma. However, preferably, the detection is performed on whole blood, and most preferably, the blood sample is peripheral blood.

[0195] The blood may be further treated before use according to the first embodiment. For example, an anticoagulant such as citrate (e.g., sodium citrate), hirudin, heparin, PPACK, or sodium fluoride may be added. Thus, the sample collection container may contain an anticoagulant to prevent the blood sample from clotting.

[0196] Preferably, the sample may include endothelial cells, smooth muscle cells, and / or interstitial cells, preferably lung endothelial cells, smooth muscle cells, and / or interstitial cells. It will be recognized that the present invention extends to any nucleic acid or peptide or its variants, derivatives, or analogues substantially comprising any amino acid sequence or nucleic acid sequence of any of the sequences (including variants or fragments thereof) referred to herein. The terms “substantially amino acid / nucleotide / peptide sequence,” “variant,” and “fragment” may be sequences having at least 40% sequence identity with any one of the amino acid / nucleotide / peptide sequences referred to herein, such as 40% identity with the sequences identified as SEQ ID NOs. 1 to 75.

[0197] Amino acid / polynucleotide / polypeptide sequences having sequence identity of higher than 65%, more preferably higher than 70%, even more preferably higher than 75%, and still more preferably higher than 80% with any of the sequences mentioned herein are also conceivable. Preferably, the amino acid / polynucleotide / polypeptide sequences have at least 85% identity with any of the sequences mentioned herein, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, and most preferably at least 99% identity.

[0198] A skilled technician would know how to calculate the percentage identity between two amino acid / polynucleotide / polypeptide sequences. To calculate the percentage identity between two amino acid / polynucleotide / polypeptide sequences, first, an alignment of the two sequences must be prepared, and then the sequence identity value is calculated. The percentage identity between two sequences can take different values ​​depending on: (i) the method used to align the sequences, e.g., structural alignment from ClustalW, BLAST, FASTA, Smith-Waterman, or 3D comparison (implemented in different programs); and (ii) the parameters used by the alignment method, e.g., local vs. global alignment, the pair score matrix used (e.g., BLOSUM62, PAM250, Gonnet, etc.), and the gap penalty, e.g., functional form and constant.

[0199] If alignment is performed, there are many different ways to calculate the percentage identity between two sequences. For example, the number of identities can be divided by: (i) the length of the shortest sequence; (ii) the length of the alignment; (iii) the average length of the sequences; (iv) the number of non-gap positions; or (v) the number of equivalent positions excluding overhangs. Furthermore, it will be recognized that percentage identity also strongly depends on length. Therefore, the shorter the pair of sequences, the higher the sequence identity will be, which may be expected to occur by chance.

[0200] Therefore, it will be recognized that the precise alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred method for producing multiple alignments of protein or DNA according to the present invention. Appropriate parameters for ClustalW may be as follows: For DNA alignment: Gap start penalty = 15.0, gap extension penalty = 6.66, and matrix = identity. For protein alignment: Gap start penalty = 10.0, gap extension penalty = 0.2, and matrix = Gonnet. For DNA and protein alignment: ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will know that it may be necessary to change these and other parameters for optimal sequence alignment.

[0201] Preferably, the percentage identity between two amino acid / polynucleotide / polypeptide sequences can then be calculated from such alignment as (N / T)*100 (wherein N is the number of positions in which the sequences share identical residues, and T is the total number of positions compared, either including gaps and including or excluding overhangs). Preferably, overhangs are included in the calculation. Thus, the most preferred method for calculating percentage identity between two sequences includes the steps of (i) preparing a sequence alignment using a ClustalW program with an appropriate set of parameters, such as set above, and (ii) inserting the values ​​of N and T into the following formula: Sequence Identity = (N / T)*100.

[0202] Alternative methods for identifying similar sequences are known to those skilled in the art. For example, substantially similar nucleotide sequences are encoded by sequences that hybridize with DNA sequences or their complements under stringent conditions. Stringent conditions, as the inventors define them, mean that the nucleotide hybridizes with filter-bound DNA or RNA in 3× sodium chloride / sodium citrate (SSC) at approximately 45°C, followed by at least one wash in 0.2× SSC / 0.1% SDS at approximately 20–65°C. Alternatively, substantially similar polypeptides may differ from sequences shown in sequence numbers 1–75, which are amino acid sequences, by at least one amino acid, but by fewer than five, ten, twenty, fifty, or 100 amino acids.

[0203] Due to the degeneracy of the genetic code, it is evident that any nucleic acid sequences described herein may differ or be altered to provide functional variants of the protein they encode without substantially affecting the sequence of the protein they encode. Suitable nucleotide variants have a sequence altered by the substitution of a different codon encoding the same amino acid, thus resulting in a silent (synonymous) change. Other suitable variants include all or part of a sequence that has a homologous nucleotide sequence but is altered by the substitution of a different codon encoding an amino acid having a side chain with biophysical properties similar to the substituted amino acid, resulting in a conserved change. Examples of small, nonpolar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Examples of large, nonpolar, hydrophobic amino acids include phenylalanine, tryptophan, and tyrosine. Examples of polar, neutral amino acids include serine, threonine, cysteine, asparagine, and glutamine. Positively charged (basic) amino acids include lysine, arginine, and histidine. Positively charged (acidic) amino acids include aspartic acid and glutamic acid. Therefore, it is understood which amino acids can be substituted with those having similar biophysical properties, and skilled technicians know the nucleotide sequences encoding these amino acids.

[0204] All of the features described herein (including any appended claims, abstracts, and drawings) and / or all of the steps of any method or process disclosed herein may be combined with any of the above embodiments in any combination, except for any combination in which at least part of such features and / or steps are mutually exclusive.

[0205] For a better understanding of the present invention and to illustrate how embodiments of the present invention can be put into practice, the attached figures are referenced here as an example, in which the following: [Brief explanation of the drawing]

[0206] [Figure 1A] Figure 1A provides a summary of various ZIP12 immunogens used to stimulate immune responses in several mouse strains, and the responses of these mice to said immunogens, measured by ELISA and flow cytometry and graphed in Figure 1B. [Figure 1B] Same as described for Figure 1A. [Figure 2] This figure shows the initial FLIPR screening of antibodies developed by the present inventors, and indicates that some of the antibodies exhibit ZIP12 inhibitory activity. Data were calculated as maximum fluorescence signal minus minimum fluorescence signal, obtained 30 seconds before Zn addition (minimum signal) and 5 minutes after addition of Zn+ test agent (maximum signal). [Figure 3] This figure shows that the four most promising ZIP12 antibodies identified in the initial screening exhibit dose-dependent inhibition of zinc uptake in a FLIPR assay. Antibody 19220 was used as a negative control. Antibody lots 19213 and 19214 correspond to lead antibody hybridomas 63A11 (19214) and 51B12 (19213). [Figure 4] This is an expanded dose-response graph for 63A11 corresponding to Figure 3. [Figure 5] This figure shows an alignment between the VH sequence of 51B12 antibody and the VH sequence of 63A11 antibody, and demonstrates a high degree of sequence homology, including 100% sequence identity for the CDRH3 region. The consensus sequence resulting from the alignment is also shown. [Figure 6] 63A11 series: This figure shows ELISA-based reactivity screening, and indicates that the parent antibody and also the humanized heavy and light chains exhibit anti-ZIP12 activity and are not reactive with ZIP4. [Figure 7]63A11 series: This figure shows a reactivity screening based on flow cytometry, indicating that the parent antibody, as well as the humanized heavy and light chains, exhibit anti-ZIP12 activity and are not reactive with ZIP4. [Figure 8] This figure summarizes the properties of the 63A11 series antibodies. [Figure 9] 51B12 series: This figure shows a reactivity screening based on ELISA, indicating that the parent antibody, as well as the humanized heavy and light chains, exhibit anti-ZIP12 activity and are not reactive with ZIP4. [Figure 10] This figure summarizes the properties of the 51B12 series antibodies. [Figure 11] This figure shows the BIAcore data for 63A11. [Figure 12] This figure shows the BIAcore data for 51B12. [Figure 13] This table shows the sequences of four embodiments of the anti-ZIP12 antibody of the present invention. [Figure 14A] This figure shows the alignment and consensus sequences between heavy chain sequences (A) and light chain sequences (B) of 89G11, 38F02, 51B12, and 63A11 (all embodiments of the present invention). The amino acid sequence indicated by X in the majority sequence (SEQ ID NO: 69) may include any amino acid sequence or one of the amino acid sequences indicated at the same position in the sequences of 89G11, 38F02, 51B12, and 63A11. [Figure 14B] The explanation is the same as in Figure 14A. [Figure 15] This figure shows the alignment and consensus sequences between the heavy chain variable sequences 89G11, 38F02, 51B12, and 63A11 (all forming part of the present invention). The amino acid sequence indicated by X in the majority sequence (SEQ ID NO: 70) may include any amino acid sequence or the amino acids indicated at the same position in the sequences 89G11, 38F02, 51B12, and 63A11. [Figure 16]This figure shows the alignment and consensus sequences between the heavy chain variable sequences 51B12 and 63A11 (both forming part of the present invention). The amino acid sequence indicated by X in the majority sequence (SEQ ID NO: 71) may include any amino acid sequence or the amino acids shown at the same positions in the sequences 51B12 and 63A11. [Figure 17] This figure shows the alignment and consensus sequences between the heavy chain variable sequences of 89G11 and 38F02 (both forming part of the present invention). The amino acid sequence indicated by X in the majority sequence (SEQ ID NO: 72) may include any amino acid sequence or the amino acids shown at the same positions in the sequences of 89G11 and 38F02. [Figure 18] This figure shows the alignment and consensus sequences between the light chain variable sequences 89G11, 38F02, 51B12, and 63A11 (all forming part of the present invention). The amino acid sequence indicated by X in the majority sequence (SEQ ID NO: 73) may include any amino acid sequence or the amino acid indicated at the same position in the sequences 89G11, 38F02, 51B12, and 63A11. [Figure 19] This figure shows the alignment and consensus sequences between the light chain variable sequences 38F02, 51B12, and 63A11 (all forming part of the present invention). The amino acid sequence indicated by X in the majority sequence (SEQ ID NO: 74) may include any amino acid sequence or the amino acids shown at the same positions in the sequences 38F02, 51B12, and 63A11. [Figure 20] This figure shows that the parent antibody exhibits dose-dependent inhibition of zinc uptake at 1.25 μM and 500 μM zinc ions in the FLIPR assay. [Figure 21] Figures 21A and 21B show that humanized antibodies (63A11 and 51B12) exhibit dose-dependent inhibition of zinc uptake in the FLIPR assay. [Modes for carrying out the invention]

[0207] ExamplesZIP12 has been linked to the development of pulmonary hypertension, and increased expression of ZIP12 is associated with this condition. Therefore, the inventors have attempted to develop an antibody that specifically binds to ZIP12 and has the ability to inhibit its function in order to provide or improve the treatment and diagnosis of pulmonary hypertension. [Examples]

[0208] Example 1 - Preparation of ZIP12 antibody Anti-human ZIP12 mAbs were prepared from mice (BALB / c, C3H, AIC, or C57BL / 6) immunized with human ZIP12-ECD-Fc, human ZIP12-DNA, human ZIP12-VLP, or a stable expression strain of human ZIP12-ModiVacc(MV) (Figure 1A). Mice were immunized with the antigen and boosted 1 to 3 times at approximately 1-month intervals.

[0209] Serum reactivity screening based on ELISA against hZIP12-ECD-Fc and hZIP12-ECD-GST (coated at 5 μg / ml) and corresponding unrelated control proteins, human IgG and hZIP4-ECD-GST (coated at 1 μg / ml and 5 μg / ml, respectively). Serum from immunocollected blood at day 37 of VLP-immunized mice was also screened using ELISA against hZIP12-VLP and null particles (coated at 2 U / well). Serum samples were serially diluted (starting at 1:100 or 1:1,000 dilutions, then 3-fold and 7-fold dilutions, respectively) and detected with anti-mouse IgG-HRP secondary antibody (1:5,000). Correct coating was confirmed using anti-ZIP12 (1:5,000) and anti-ZIP4 (1:2,500), and detected with anti-rabbit IgG-HRP (1:5,000) or anti-human IgG-HRP (1:5,000). Serum reactivity screening based on flow cytometry for MV(-)-hZIP12-3A11, MV(-) parent, MV(+) parent, and CHO-k1-huZIP4-HA-3C4. Serum from mice immunized with MV(+)-hZIP12 stable expression strains was also screened by flow cytometry for MV(+)-hZIP12-1E8, MV(+)-hZIP12-2G3, and MV(+)-hZIP12-4D6 stable expression strains. Serum samples were serially diluted (starting at 1:100 dilution, then 3-fold dilution, and 7 dilutions, respectively) and detected with anti-mouse IgG-PE secondary antibody (1:300).

[0210] A summary of mouse strains and numbers, immunogens, and serum titers, expressed as EC50 values ​​(referring to the serum dilution ratio that produces the maximum half-volume signal), is shown in Figure 1A. NA indicates not applicable; EC50 values ​​could not be calculated due to too low a response. Four mice were selected for splenocyte and bone marrow collection and preservation.

[0211] Hybridomas were produced and cloned either using the ClonaCell-HY Hybridoma Cloning Kit (StemCell Technologies, Vancouver, BC) or conventional methods. In the conventional method, B cells derived from the spleen of an immunized animal were fused with Sp2 / 0 myeloma cells by electrofusion using Modifuse technology in the presence of PEG (Sigma-Aldrich, St. Louis, MO). After overnight recovery, the fused cells were plated by limiting dilution in 96-well plates and subjected to hypoxanthine-aminopterin-thymidine selection. Hybridoma culture supernatants were examined for the presence of anti-ZIP12 antibodies by ELISA and flow cytometry (Figure 1B). Immune serum activity was observed in hybridomas derived from BALB / c and C3H mice immunized with human ZIP12 virus-like particles, as well as hybridomas derived from BALB / c, AIP and C3H mice immunized with the human ZIP12 extracellular domain fused to human Fc. [Examples]

[0212] Example 2 - Hybridoma Variable Gene Sequencing RNA was isolated from TRIzol samples. cDNA was generated, and V-region amplification was completed according to standard procedures. PCR amplification of VH and VL gene fragments was performed using MQR standard procedures or by using degenerate forward primers. The amplified V-genes were gel-purified and cloned into a human IgG1 / IgK vector using T4 ligase for DNA sequencing. The ligation mixture (approximately 25 ng of vector) was transformed into chemocompetent E. coli XL1-Blue cells. Miniprep DNA was isolated from clones containing full-length inserts. The isolated DNA (up to 10 VH and VL fragments per hybridoma) was sequenced and analyzed using standard methods. Plasmids containing the correct gene were stored as glycerol stocks. [Examples]

[0213] Example 3 - Preparation of Chimeric Antibodies DNA expression constructs encoding chimeric antibodies were prepared using restriction sites for cloning into mammalian expression vectors, in addition to human signal sequences. BsiWI and BsmI restriction sites were introduced to incorporate variable domains containing signal sequences for cloning into mammalian expression vectors containing human γ1 or human kappa constant region.

[0214] The correct clones were confirmed using DNA sequencing. Plasmid DNA was transfected into HEK293 cells using FectoPro. After approximately 5 days, the supernatant was collected. Antibody concentration was measured in the culture supernatant, and the yield was calculated using Octet or ELISA. The antibodies were purified using Protein A (Mab Select SuRe) resin, and the antibody concentration was measured using Nanodrop. Antibody integrity and purity were confirmed using reduced SDS-PAGE. Antibody target reactivity was determined using ELISA and FACS. [Examples]

[0215] Example 4-Zn 2+ Antibody activity in 2+ FLIPR assay Production of Inducible ZIP12 Cell Lines: Inducible ZIP12 cell lines were produced by transfection of HEK293 or CHO cells with ZIP12 fused to a FLAG tag. ZIP12 expression was confirmed by PCR and Western blotting. In subsequent assays, cells were induced with tetracycline before screening.

[0216] Dye loading procedure: After cell induction and seeding, antibodies were prepared for pre-incubation. BSA was added to the growth medium at a final concentration of 0.1%, and tetracycline at a concentration of 1 μg / ml. Antibodies were added at the desired concentration and incubated at 37°C for either 4 hours or 24 hours. After approximately 24 hours, the medium was removed. EBSS, probenecid, and Flozin-3 were added to the reaction wells and incubated in the dark for 1 hour. The dye solution was removed, and Ca 2+ EBSS without [unclear] + 2.5 mM probenecid was added and transferred to FLIPR.

[0217] FLIPR assay procedure: 10 μL of assay buffer (Ca) per well 2+ EBSS (without 2.5 mM probenecid) was added. 20 μL of assay buffer per well was added along with ZnSO4 at 2 × the final desired concentration. Fluorescence was monitored for 10 minutes. % inhibition was calculated as the maximum fluorescence signal minus the minimum fluorescence signal, obtained 30 seconds before Zn addition (minimum signal) and 5 minutes after Zn+ test agent addition (maximum signal). [Examples]

[0218] Example 5 - Humanization of ZIP12 antibody (i) Cloning of humanized 51B12 and 63A11 variants: DNA expression constructs encoding humanized antibody variants were newly prepared by building up duplicate oligonucleotides containing restriction sites for cloning into mammalian expression vectors, as well as human signal sequences. HindIII (Hindlll) and SpeI (Spel) restriction sites were introduced to incorporate a VH domain containing a signal sequence for cloning into a mammalian expression vector containing the human γ1 constant region. HindIII and BsiWI restriction sites were introduced to incorporate a VL domain containing a signal sequence for cloning into a mammalian expression vector containing the human kappa constant region.

[0219] (ii) Expression of recombinant 51B12 and 63A11 antibodies (including antibody quantification) Expression plasmids encoding the heavy and light chains, respectively, were transiently and simultaneously transfected into HEK293 6E cells and expressed on a small scale to produce antibodies. The antibodies were quantified by ELISA. ELISA plates were coated with 1 mg / ml anti-human IgG (Sigma I3382) and blocked with blocking solution (4% BSA in Tris buffered saline). Tissue culture supernatants at various dilutions were added, and the plates were incubated at room temperature for 1 hour. Dilutions of known standard antibodies were also added to the plates. The plates were washed in TBST, and binding was detected by adding peroxidase-labeled anti-human kappa light chain antibody (Sigma A7164) at a dilution of 1 / 1000 in the blocking solution. The plates were incubated at room temperature for 1 hour before washing in TBST. The plates were colored by adding OPD substrate (Sigma P9187), and the color development was stopped by adding 2M H2S04. Absorbance was measured at 490 nm, and a standard curve was plotted using data for known standard dilutions. The antibody concentration in the tissue culture supernatant was estimated using the standard curve. For larger-scale antibody preparation, the antibodies were purified using Protein A, and the concentration was measured using Nanodrop (Thermo Scientific). [Examples]

[0220] Example 6 - ELI showing binding of humanized 63A11 to recombinant human ZIP12 protein SA experiment The binding of humanized 63A11 antibody to the extracellular domain of human ZIP12 (expressed as a GST fusion protein) was tested. Human ZIP12 extracellular domains were coated onto ELISA plates, and the plates were blocked with BSA to reduce nonspecific binding. Humanized antibodies were added to the human ZIP12-coated ELISA plates at concentrations ranging from 10 ug / ml to 0.1 ug / ml. Any bound humanized antibodies were detected using an anti-human IgG HRP conjugated secondary antibody as needed. HRP substrate (TMB) was added to develop the ELISA. This demonstrated that the 63A11 humanized antibody bound to recombinant human ZIP12 in the ELISA assay. The results are shown in Figures 6 and 8. [Examples]

[0221] Example 7 - Humanized antibody binds to ZIP12-transfected cells. Binding of humanized 63A11 to ZIP12-transfected cells as determined by FACS.

[0222] Human ZIP12-expressing transfectant cells were stained with humanized variants of 63A11, named H0L0, H0L1, H1L0, H1L1, H2L0, H2L1, H3L0, and H3L1, at various concentrations for 20 minutes at room temperature. The cells were then washed with FACS buffer (PBS + 0.5% BSA + 0.1% sodium azide) to remove unbound antibodies. The cells were incubated with secondary PE-labeled anti-human IgG antibody at 4°C in the dark for 1 hour, and then washed with FACS buffer to remove unbound antibodies. Cells were analyzed by FACS, and binding was determined by measuring the mean fluorescence intensity (MFI) value. The results showed that all tested antibodies bound to human ZIP12-expressing transfectant cells in a dose-dependent manner. The results are shown in Figures 7 and 8. [Examples]

[0223] Example 8 - E showing the binding of humanized 51B12 antibody to recombinant human ZIP12 protein. LISA experiment The binding of humanized 51B12 antibody to the extracellular domain of human ZIP12 (expressed as a GST fusion) was tested. The extracellular domain of human ZIP12 was coated onto ELISA plates, and the plates were blocked with BSA to reduce nonspecific binding. Humanized antibody was added to the human ZIP12-coated ELISA plates at concentrations ranging from 10 ug / ml to 0.1 ug / ml. Anti-human IgG was added as needed. Any conjugated humanized antibody was detected using an HRP-conjugated secondary antibody. The ELISA was color-developed by adding an HRP substrate (TMB). This demonstrated that the 51B12 humanized antibody bound to recombinant human ZIP12 in the ELISA assay. The results are shown in Figure 9. [Examples]

[0224] Example 9 - ZIP12 transformer of humanized 51B12 as determined by FACS Binding with fected cells . Human ZIP12-expressing transfectant cells were stained with humanized variants of 51B12, named H0L0, H0L1, H0L2, and H0L3, at various concentrations for 20 minutes at room temperature. The cells were then washed with FACS buffer (PBS + 0.5% BSA + 0.1% sodium azide) to remove unbound antibodies. The cells were incubated with secondary PE-labeled anti-human IgG antibody at room temperature for 15 minutes, and then washed with FACS buffer to remove unbound antibodies. Cells were analyzed by FACS, and binding was determined by measuring the mean fluorescence intensity (MFI) value. The results showed that all tested antibodies bound to human ZIP12-expressing transfectant cells in a dose-dependent manner. The results are shown in Figure 10. [Examples]

[0225] Example 10 - BIAcore analysis of humanized constructs of anti-ZIP12 63A11 A single concentration of hZIP12-ECD-GST (100 nM) was tested against the 63A11 clone. This step confirmed the validity of the antibody's capture level and allowed observation of the binding of the hZIP12-ECD-GST fusion protein before proceeding with the assay coloration. The results also confirmed that the regeneration conditions (i.e., complete removal of hZIP12-ECD-GST from the surface by injection of a solution of 10 mM glycine pH 1.5 and 10 mM NaOH) were appropriate, with a complete return to baseline.

[0226] Protein A was immobilized onto a Series S Protein A sensor tip (Cyvita) by primary amine coupling, and this surface was then used to capture antibody molecules. All measurements were performed at 25°C, and the compartment temperature was maintained at 10°C to help maintain reagent stability. Data were collected at 10 Hz. Single-cycle experiments were performed in double-cycle at 25°C. A 2-fold dilution series of huZIP12-ECD-GST and huZIP4-ECD-GST, ranging from 6.25 nM to 400 nM for the 63A11 clone, was injected onto the sensor surface at 30 μl / min in running buffer (1 × HBS-EP+ pH 7.4) with a contact time of 120 seconds and a dissociation time of 2100 seconds for the 63A11 clone. The sensor surface was regenerated between cycles with 10 mM glycine pH 1.5 at 50 μl / min for 60 seconds, and with 10 mM NaOH at 50 μl / min for 60 seconds. The association rate constant (ka) and dissociation rate constant (kd) were calculated using Biacore Insight Evaluation software (version 3.0.11.15423 Cytiva) by fitting a 1:1 binding model to sensorgrams subtracting reference and blank values. Both runs identified 63A11 H0L1 as the best antibody in terms of overall affinity to human ZIP12. The data obtained from this experiment are shown in Figure 11. [Examples]

[0227] Example 11 - BIAcore analysis of humanized constructs of anti-ZIP12 51B12 Protein A was immobilized onto a Series S Protein A sensor tip (Cyvita) by primary amine coupling, and this surface was then used to capture antibody molecules. All measurements were performed at 25°C, and the compartment temperature was maintained at 10°C to help maintain reagent stability. Data were collected at 10 Hz. Single-cycle experiments were performed in double-cycle at 25°C. A 2x dilution series of huZIP12-ECD-GST and huZIP4-ECD-GST, ranging from 12.5 nM to 800 nM for the 51B12 clone, was injected onto the sensor surface at 30 μl / min in running buffer (1 × HBS-EP+ pH 7.4) with a contact time of 120 seconds and a dissociation time of 2520 seconds for the 51B12 clone. The sensor surface was regenerated between cycles with 10 mM glycine pH 1.5 at 50 μl / min for 60 seconds, and with 10 mM NaOH at 50 μl / min for 60 seconds. The association rate constant (ka) and dissociation rate constant (kd) were calculated using Biacore Insight Evaluation software (version 3.0.11.15423 Cytiva) by fitting a 1:1 binding model to sensorgrams subtracting reference and blank values. Both runs identified 51B12 H0L3 as the best antibody in terms of overall affinity to human ZIP12. The data obtained from this experiment are shown in Figure 13.

[0228] Discussion and Conclusion The inventors identified the extracellular region or domain of the ZIP12 protein as key to its function and accordingly developed antibodies capable of binding to and inhibiting ZIP12 function. For example, as shown in Figures 2 and 3, the inventors developed numerous antibodies and demonstrated that they specifically target its extracellular domain and inhibit ZIP12 function. Furthermore, as shown in Figures 6, 7, and 9, the inventors demonstrated that these antibodies not only inhibit ZIP12 function but also do not target the ZIP4 protein.

[0229] The present inventors have previously demonstrated that ZIP12 is a novel therapeutic target for the treatment of disease mechanisms underlying hypoxia-associated conditions, particularly pulmonary hypertension. Accordingly, the antibodies developed by the present invention are useful as therapeutic agents in their own right and can be used in the treatment, amelioration or prevention of hypoxia-induced or hypoxia-associated conditions, in particular pulmonary hypertension.

Examples

[0230] Example 12-Zn 2+ Antibody activity in 2+ FLIPR assay (2) method Cells were seeded and ZIP12 expression was induced with 1 μg / ml tetracycline. 4 to 6 hours after cell induction and seeding, cells were prepared for pre-incubation with antibodies. No tetracycline was added to inducible controls. 25 μl / well of antibody was added to a cell proliferation plate already containing 25 μl / well of medium. The final BSA concentration is 0.1%, the final tetracycline concentration is 1 μg / ml, and the final antibody concentration is a 1:10 dilution of the stock. Serial dilutions of antibody were prepared in medium in a separate plate prior to addition to the cell plate. Antibody and cells were incubated together at 37°C for 1 to 72 hours, after which the cell culture medium was removed following antibody pre-incubation. For 384-well plates, 10 μL / well of EBSS + 2.5 mM probenecid + 5 μM FluoZin-3 was added according to the manufacturer's instructions. After 1 hour of incubation in the dark, the dye solution was removed, and 10 μL / well of Ca 2+ -free BSS supplemented with 2.5 mM probenecid was added, and the plate was analyzed.

[0231] At time 0, 10 μL of assay buffer was added to each well. After 1 minute, 20 μL of assay buffer containing ZnSO4 at twice the final desired concentration was added to each well. Fluorescence was monitored for 10 minutes. Data were calculated as maximum fluorescence signal - minimum fluorescence signal, obtained 30 seconds before Zn addition (minimum signal) and 5 minutes after Zn+ test agent addition (maximum signal). % inhibition was calculated as (1 - (test well - plate low control) / (plate high control - plate low control)) * 100. Inhibition relative to maximum inhibition was also calculated.

[0232] result Figure 20 shows that two promising ZIP12 antibodies identified in the initial screening exhibit dose-dependent inhibition of zinc uptake in the FLIPR assay. An unrelated human antibody with the same Fc region was used as a negative control.

[0233] Figures 21A and 21B show humanized antibodies (i.e., 63A11 and 51B12) derived from two promising ZIP12 antibodies identified in the initial screening. These exhibit dose-dependent inhibition of zinc uptake in the FLIPR assay.

Claims

1. An antibody or its antigen-binding fragment that specifically binds to the extracellular region of ZIP12.

2. The antibody or its antigen-binding fragment according to claim 1, which does not substantially bind to human ZIP4 and / or ZIP13.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, which binds to a region between amino acid positions 1 to 202 of ZIP 12, substantially as shown in Sequence ID No.

1.

4. An antibody or antigen-binding fragment according to any one of claims 1 to 3, comprising a sequence substantially shown in SEQ ID NO: 2 or a variant or fragment thereof, or binding to an epitope in a sequence consisting of such a sequence.

5. An antibody or antigen-binding fragment according to any one of claims 1 to 4, which binds to one or more amino acids in SEQ ID NO: 2 or a fragment or variant thereof, preferably one or more amino acids between amino acid positions 20 to 202 of SEQ ID NO: 2, or (i) between amino acid positions 20 to 104 of SEQ ID NO: 2, or (ii) between amino acid positions 156 to 202 of SEQ ID NO: 2, and more preferably binds to any 5, 10, 15, 20, 25, 30, 35, 40, or 45 amino acid sequences present in SEQ ID NO: 2 or a variant or fragment thereof.

6. The antibody or antigen-binding fragment according to any one of claims 1 to 5, which is a monoclonal antibody or an antigen-binding fragment thereof.

7. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, comprising: (i) a CDR-H1 domain comprising or consisting of a sequence substantially shown in SEQ ID NO: 36 or a variant or fragment thereof, wherein in the sequence of SEQ ID NO: 36, X is any amino acid, and optionally X is H or Y; (ii) A CDR-H2 domain comprising or consisting of the sequence substantially shown in Sequence ID No. 37 or a variant or fragment thereof, wherein in the sequence of Sequence ID No. 37, X １ , X ２ , X ３ , and X ４ is any amino acid, and is optional, X １ is S or G; X ２ is S or T; X ３ is A or T; and / or X ４ CDR-H2 domain where is F or Y; (iii) A CDR-H3 domain comprising or consisting of the sequence substantially shown in Sequence ID No. 5 or its variant or fragment; (iv) A CDR-L1 domain comprising or consisting of the sequence substantially shown in Sequence ID No. 7 or its variant or fragment; (v) A CDR-L2 domain comprising or consisting of the sequence substantially shown in Sequence ID No. 8 or its variant or fragment, and / or (v) a CDR-L3 domain comprising or consisting of a sequence substantially set forth in SEQ ID NO: 39 or a variant or fragment thereof, wherein in the sequence of SEQ ID NO: 39, X １ and X ２ is any amino acid, optionally, X １ is L or V; and / or X ２ is L or Y, the CDR-L3 domain.

8. An antibody or antigen-binding fragment according to any one of claims 1 to 7, comprising a CDR-H1 domain containing SEQ ID NO: 3, a CDR-H2 domain containing SEQ ID NO: 4, a CDR-H3 domain containing SEQ ID NO: 5, a CDR-L1 domain containing SEQ ID NO: 7, a CDR-L2 domain containing SEQ ID NO: 8, and / or a CDR-L3 domain containing SEQ ID NO: 9, and optionally comprising at least one, at least two, at least three, at least four, at least five, or at least six of the CDRs.

9. An antibody or antigen-binding fragment according to any one of claims 1 to 8, comprising a heavy chain variable region comprising or consisting of SEQ ID NO: 41, and a light chain variable region comprising or consisting of SEQ ID NO: 42, and optionally comprising a heavy chain region comprising or consisting of SEQ ID NO: 6, and a light chain region comprising or consisting of SEQ ID NO:

10.

10. An antibody or antigen-binding fragment according to any one of claims 1 to 9, comprising a heavy chain region comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 49, 50, 51, and 52, and a light chain region comprising or consisting of SEQ ID NOs: 53 or 54.

11. An antibody or antigen-binding fragment according to any one of claims 1 to 7, comprising a CDR-H1 domain containing SEQ ID NO: 11, a CDR-H2 domain containing SEQ ID NO: 12, a CDR-H3 domain containing SEQ ID NO: 13, a CDR-L1 domain containing SEQ ID NO: 15, a CDR-L2 domain containing SEQ ID NO: 16, and / or a CDR-L3 domain containing SEQ ID NO: 17, and optionally comprising at least one, at least two, at least three, at least four, at least five, or at least six of the CDRs.

12. An antibody or antigen-binding fragment according to any one of claims 1 to 7, comprising a heavy chain variable region comprising or consisting of SEQ ID NO: 43, and a light chain variable region comprising or consisting of SEQ ID NO: 44, and optionally comprising a heavy chain region comprising or consisting of SEQ ID NO: 14, and a light chain region comprising or consisting of SEQ ID NO:

18.

13. An antibody or antigen-binding fragment according to any one of claims 1 to 7, comprising a heavy chain region comprising or consisting of SEQ ID NO: 55, and a light chain region comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 56, 57, 58, and 59.

14. An antibody or antigen-binding fragment according to any one of claims 1 to 7, comprising a CDR-H1 domain containing SEQ ID NO: 20, a CDR-H2 domain containing SEQ ID NO: 21, a CDR-H3 domain containing SEQ ID NO: 22, a CDR-L1 domain containing SEQ ID NO: 24, a CDR-L2 domain containing SEQ ID NO: 25, and / or a CDR-L3 domain containing SEQ ID NO: 26, and optionally comprising at least one, at least two, at least three, at least four, at least five, or at least six of the CDRs.

15. An antibody or antigen-binding fragment according to any one of claims 1 to 7, comprising a heavy chain variable region comprising or consisting of SEQ ID NO: 45, and a light chain variable region comprising or consisting of SEQ ID NO: 46, and optionally comprising a heavy chain region comprising or consisting of SEQ ID NO: 23, and a light chain region comprising or consisting of SEQ ID NO:

27.

16. An antibody or antigen-binding fragment according to any one of claims 1 to 7, comprising a CDR-H1 domain containing SEQ ID NO: 28, a CDR-H2 domain containing SEQ ID NO: 29, a CDR-H3 domain containing SEQ ID NO: 30, a CDR-L1 domain containing SEQ ID NO: 32, a CDR-L2 domain containing SEQ ID NO: 33, and / or a CDR-L3 domain containing SEQ ID NO: 34, and optionally comprising at least one, at least two, at least three, at least four, at least five, or at least six of the CDRs.

17. An antibody or antigen-binding fragment according to any one of claims 1 to 7, comprising a heavy chain variable region comprising or consisting of SEQ ID NO: 47, and a light chain variable region comprising or consisting of SEQ ID NO: 48, and optionally comprising a heavy chain region comprising or consisting of SEQ ID NO: 31, and a light chain region comprising or consisting of SEQ ID NO:

35.

18. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, for use in therapeutic purposes.

19. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, for use in treating, preventing, or improving hypoxia-related conditions.

20. An antibody or its antigen-binding fragment for use according to claim 19, wherein the hypoxia-related condition is pulmonary hypertension or cancer.

21. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to claims 1 to 17, and optionally a pharmaceutically acceptable medium.

22. An antibody or antigen-binding fragment obtained by a method comprising the following steps: (i) immunizing a host organism with the extracellular domain of ZIP12 or a region of the ZIP12 protein as defined in any one of claims 1 to 17; and (ii) A step of collecting an antibody or an antigen-binding fragment thereof from the host.

23. A polynucleotide sequence encoding an antibody or an antigen-binding fragment thereof, as defined in any one of claims 1 to 17.

24. An expression cassette comprising the polynucleotide sequence described in claim 23.

25. A recombinant vector comprising the expression cassette described in claim 24.

26. A host cell comprising the polynucleotide sequence described in claim 23, the expression cassette described in claim 24, or the vector described in claim 25.

27. A method for preparing an antibody or antigen-binding fragment according to any one of claims 1 to 17, comprising the following steps: a) the step of introducing the vector according to claim 25 into a host cell; and b) A step of culturing the host cells under conditions that can produce the antibody or antigen-binding fragment described in any one of claims 1 to 17.

28. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, for use in diagnosis or prognosis.

29. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, for use in diagnosing or prognosing hypoxia-related conditions.

30. A method for diagnosing or prognosing a hypoxia-related condition in a subject, comprising the step of detecting ZIP 12 in a biological sample obtained from the subject using an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 17.

31. A kit for diagnosing a subject suffering from a hypoxia-related condition, or for providing a prognosis for the condition of the subject, comprising an antibody or antigen-binding fragment according to any one of claims 1 to 17 for detecting ZIP 12 in a sample derived from a test subject.

32. The use according to claim 29, the method according to claim 30, or the kit according to claim 31, wherein the hypoxia-related condition is pulmonary hypertension or cancer.

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