ZIP12 Antibody

Abstract

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 making said antibodies, and to methods of using said antibodies 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 making said antibodies, and to methods of using said antibodies in the treatment and diagnosis of hypoxia-related diseases, such as pulmonary hypertension and lung cancer. [Background technology]

[0002] Pulmonary hypertension (PH) is a pathological condition characterized by elevated pulmonary arterial pressure and structurally remodeled pulmonary vasculature. Current treatments for pulmonary hypertension focus on pharmacological manipulation of signaling mechanisms used by vasoactive factors and have limited therapeutic efficacy. There is a significant unmet medical need for new therapeutics and diagnostics for this potentially fatal condition.

[0003] The typical response of adult mammalian pulmonary circulation to a hypoxic environment is vasoconstriction and structural remodeling of pulmonary arterioles, resulting in chronic elevation of pulmonary arterial pressure (pulmonary hypertension) and right ventricular hypertrophy. However, some mammals show genetic resistance to hypoxia-induced pulmonary hypertension (WILKINS, MR 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 present inventors previously reported that the Fisher 344 (F344) rat strain is resistant to hypoxia-induced pulmonary hypertension compared with 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] We also previously used a genetic breeding program and comparative genomics to exploit this variation in rats and identified the gene Slc39a12 as a key regulator of hypoxia-induced pulmonary vascular remodeling. Slc39a12 encodes the zinc transporter, ZIP12. We found that ZIP12 expression was increased in many cell types, including endothelial, smooth muscle, and interstitial cells, in remodeled pulmonary arterioles from rats, cows, and humans that are highly susceptible to hypoxia-induced pulmonary hypertension. We showed that ZIP12 expression in pulmonary vascular smooth muscle cells is hypoxia-dependent, and that targeted inhibition of ZIP12 inhibits the elevation of intracellular labile zinc in pulmonary vascular smooth muscle cells exposed to hypoxia and their proliferation in culture. We also previously demonstrated that genetic disruption of ZIP12 expression reduces the development of pulmonary hypertension in rats housed in a hypoxic atmosphere.

[0005] However, the specific treatment for targeting ZIP12 protein, which the present inventors believe may provide an improved means of treating pulmonary hypertension, has not been available until now.In addition, the diagnosis of pulmonary hypertension often requires invasive procedures such as right heart catheterization and echocardiogram.Therefore, it is also desirable to identify new markers of pulmonary hypertension that can be measured through simple and less invasive procedures. Summary of the Invention [Means for solving the problem]

[0006] We 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 new therapeutic target for the treatment of the disease mechanism underlying pulmonary hypertension, which led us to further research 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 useful as therapeutic agents themselves, and can be used to treat, ameliorate, or prevent any hypoxia-induced or hypoxia-related condition, and in particular, but not exclusively, pulmonary hypertension.

[0007] Thus, in a first aspect of the present invention, there is provided an antibody or an antigen-binding fragment thereof which specifically binds to the extracellular domain of ZIP12 and recognises an epitope comprising or consisting of ADLLQVLSAGDHPPHNHSRS (SEQ ID NO: 252) or a variant or fragment thereof, or preferably an epitope comprising or consisting of LLQVLSAGDHPPHNHSRS (SEQ ID NO: 1) or a variant or fragment thereof.

[0008] In another aspect, an antibody or antigen-binding fragment thereof is provided that specifically binds to the extracellular domain of ZIP12 and recognizes an epitope comprising or consisting of LLQVLSAGDHPPHNHSRS (SEQ ID NO: 1) or a variant or fragment thereof.

[0009] As shown in the Examples (and in Figure 8), the present inventors have identified a novel epitope on the extracellular region or domain of ZIP12 protein as key to the function of ZIP12 protein, and have therefore developed antibodies capable of binding to ZIP12 and inhibiting ZIP12 function. For example, as shown in Figure 9b, the present inventors have developed a number of antibodies that specifically target this extracellular epitope and inhibit ZIP12 function, as shown.

[0010] Preferably, the antibody or antigen-binding fragment of the present invention is capable of inhibiting ZIP12 function. Preferably, the antibody or antigen-binding fragment of the present invention is capable of inhibiting ZIP12 function, thereby inhibiting the increase of intracellular labile zinc in pulmonary vascular smooth muscle cells exposed to hypoxia and their proliferation in culture.

[0011] The epitope is SEQ ID NO:252: ADLLQVLSAGDHPPHNHSRS [SEQ ID NO: 252] may be provided herein as

[0012] Preferably, the epitope can be provided herein as SEQ ID NO:1, as follows: LLQVLSAGDHPPHNHSRS [SEQ ID NO: 1] As shown in FIG. 10C, the inventors identified the sequence ADLLQ (SEQ ID NO:253) as a common motif found in several of the candidate epitopes.

[0013] Thus, preferably, the epitope may be provided herein as SEQ ID NO: 253, as follows: ADLLQ [SEQ ID NO: 253] Thus, in one embodiment, the antibody or antigen-binding fragment thereof recognizes an epitope comprising or consisting of ADLLQ (SEQ ID NO: 253) or a variant or fragment thereof.

[0014] In one embodiment, ZIP12 can be represented by Genbank ID No: NP-001138667, which is provided herein as SEQ ID NO: 2, as follows: [SEQ ID NO:2] The antibody or antigen-binding fragment thereof may bind to an epitope that is part of SEQ ID NO:2 or a fragment or variant thereof.

[0015] Preferably, the antibody or antigen-binding fragment thereof binds to an epitope contained in the amino acid sequence of SEQ ID NO:252 and / or SEQ ID NO:1. Preferably, the antibody or antigen-binding fragment thereof binds to one or more amino acids in SEQ ID NO:252 and / or SEQ ID NO:1, or a fragment or variant thereof. Preferably, the antibody or antigen-binding fragment thereof binds to any 5, 6, or 7 amino acids present in SEQ ID NO:252 and / or SEQ ID NO:1, or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof binds to any 8, 9, or 10 amino acids present in SEQ ID NO:252 and / or SEQ ID NO:1, or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof binds to any 11, 12, or 13 amino acids present in SEQ ID NO:252 and / or SEQ ID NO:1, or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof binds to any 14, 15, or 16 amino acids present in SEQ ID NO:252 and / or SEQ ID NO:1, or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof binds to any of 17 or 18 amino acids present in SEQ ID NO: 252 and / or SEQ ID NO: 1, or a variant or fragment thereof.

[0016] Epitopes can be linear or conformational. The term "linear epitope" can refer to epitopes that include or consist of amino acid residues that form a sequence together in the primary sequence of the protein antigen, i.e., sequential or continuous epitopes. Linear epitopes can be present on folded ZIP12.

[0017] The term "conformational epitope" may refer to an epitope consisting of amino acid residues, at least some of which are separated from other amino acid residues in the primary sequence of the protein antigen, but which together form a 3D structure and are recognized by an antibody, i.e., a discontinuous or non-sequential epitope.

[0018] From the data obtained in Figure 8, the inventors believe that the epitope of the antibody or antigen-binding fragment thereof is at least partially linear. In fact, the antibody is likely to bind to a predominantly linear epitope. However, the antibody or antigen-binding fragment thereof may not bind to every single amino acid in the amino acid sequence of SEQ ID NO:252 and / or SEQ ID NO:1.

[0019] ZIP12, ZIP13, and ZIP4 are all zinc transporters. However, ZIP4 is not involved in the pathogenesis of pulmonary hypertension and plays important roles in tissue homeostasis, metabolism, development, and immunity. In addition, ZIP13 has been shown to control intracellular zinc and smad signaling, which is important for BMP / TGF-beta signaling (Fakuda et al., J Biol Inorg Chem, 2011). Impaired BMP signaling is known to be associated with pulmonary hypertension, as shown in Morrell et al., Nature Reviews Cardiology, 2015. Thus, inhibition of ZIP13 leads to lower zinc concentrations, impairing BMP signaling and causing, rather than curing, pulmonary hypertension.

[0020] Therefore, it is important that the antibodies of the present invention targeting ZIP12 do so specifically and have little or no cross-reactivity with ZIP13 and / or ZIP4, since this cross-reactivity could result in significant undesirable off-target effects.

[0021] Therefore, preferably, the antibody or antigen-binding fragment thereof of the present invention does not substantially bind to human ZIP13. Preferably, the antibody or antigen-binding fragment thereof of the present invention has no cross-reactivity with human ZIP13.

[0022] In one embodiment, ZIP13 can be represented by Gene ID No:91252, which is provided herein as SEQ ID NO:3, as follows: MPGCPCPGCGMAGPRLFLTALALELLERAGGSQPALRSRGTATACRLDNKESESWGALLSGERLDTWICSLLGSLMVGLSGVFPLLVIPLEMGTMLRSEAGAWRLKQLLSFALGGLLGNVFLHLLPEAWAYTCSASPGGEGQSLQQQQLGLWVIAGILTFLALEKMFLDSKEEGTSQAPNKDPTAAAAALNGGHCLAQPAAEPGLGAVVRSIKVSGYLNLLANTIDNFTHGLAVAASFLVSKKIGLLTTMAILLHEIPHEVGDFAILLRAGFDRWSAAKLQLSTALGGLLGAGFAICTQSPKGVVGCSPAAEETAAWVLPFTSGGFLYIALVNVLPDLLEEEDPWRSLQQLLLLCAGIVVMVLFSLFVD [SEQ ID NO: 3] Thus, preferably the antibody or antigen-binding fragment thereof does not bind to a sequence substantially as shown in SEQ ID NO: 3 or a variant or fragment thereof.

[0023] In addition, the antibody or antigen-binding fragment of the present invention preferably does not substantially bind to human ZIP4. Preferably, the antibody or antigen-binding fragment of the present invention has no cross-reactivity with human ZIP4.

[0024] In one embodiment, ZIP4 can be represented by Genbank ID No: NP-570901, and is provided herein as SEQ ID NO: 4, as follows: [SEQ ID NO:4] Thus, preferably, the antibody or antigen-binding fragment thereof does not bind to a sequence substantially as shown in SEQ ID NO: 4 or a variant or fragment thereof.

[0025] Preferably, the antibody or antigen-binding fragment thereof binds to human and / or cynomolgus monkey ZIP12. Preferably, the antibody or antigen-binding fragment thereof does not bind to dog and / or pig ZIP12.

[0026] The inventors cover both whole antibodies (i.e., immunoglobulins) having immunospecificity for a ZIP12 extracellular epitope comprising or consisting of SEQ ID NO:252 and / or SEQ ID NO:1, or a variant or fragment thereof, as well as antigen-binding fragments or regions of the corresponding full-length antibodies.

[0027] An antibody or antigen-binding fragment thereof can be monovalent, bivalent, or multivalent. A monovalent antibody is a dimer (HL) that includes a light chain (L) and a heavy chain (H) associated by a disulfide bridge. A bivalent antibody is a tetramer (H2L2) that includes two dimers associated by at least one disulfide bridge. Multivalent antibodies can also be made, 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 remainder 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 for the antigen, i.e., the extracellular portion of ZIP12 or its variants or fragments (e.g., epitopes). On either side of the heavy and light chain CDRs are framework regions, which are relatively conserved sequences of amino acids that anchor and orient the CDRs. Antibody fragments can include bispecific antibodies (BsAbs) or chimeric antigen receptors (CDRs).

[0028] The heavy chain constant region typically comprises three domains, C H1 , C H2 , and C H3 Each light chain typically comprises a light chain variable region (V L ) and a light chain constant region. The light chain constant region typically comprises L It contains one domain, abbreviated as .

[0029] Each heavy and light chain generally 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 and confer antigen specificity and binding affinity to the antibody. See Kabat et al., Sequences of Proteins of Immunological Interest 5th ed. (1991) Public Health Service, National Institutes of Health, Bethesda, MD, incorporated by reference in its entirety.

[0030] Heavy chains from any vertebrate species can be assigned to one of five different 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 sequence and functional differences. Humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0031] Light chains from any vertebrate species can be assigned to one of two types, called kappa and lambda, based on the sequence of the constant domain. 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 functions of the molecule.

[0032] Preferably, the antibody or its antigen-binding portion is isolated or purified. In one preferred embodiment, the antibody or antigen-binding fragment thereof comprises a polyclonal antibody or antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof may be produced in rabbits, mice, or rats.

[0033] Preferably, the antibody or antigen-binding fragment thereof is obtained by immunizing a host animal with a ZIP12 extracellular epitope comprising or consisting of SEQ ID NO:252 and / or SEQ ID NO:1, or a variant or fragment thereof, and then collecting the antibody or antigen-binding fragment thereof. The host animal may be a rabbit.

[0034] 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 refer to an antibody, such as a monoclonal antibody, that comprises substantially the same heavy chain CDR amino acid sequence and light chain CDR amino acid sequence as found in a particular human antibody that exhibits immunospecificity for the extracellular portion of ZIP12 or a variant or fragment thereof. The amino acid sequence that is substantially the same as the heavy chain CDR or light chain CDR shows a significant amount of sequence identity when compared to a reference sequence. Such identity can be categorically understood or recognized as representing the amino acid sequence of a particular human antibody. The substantially same heavy chain CDR amino acid sequence and light chain CDR amino acid sequence may have, for example, a small number of modifications or conservative substitutions of amino acids. Such a human antibody maintains its function of selectively binding to a ZIP12 extracellular epitope that comprises or consists of SEQ ID NO:252 and / or SEQ ID NO:1, or a variant or fragment thereof.

[0035] The term "human monoclonal antibody" can include monoclonal antibodies having substantially or completely human CDR amino acid sequences produced, for example, by recombinant methods, such as production by a phage library, by lymphocytes, or by hybridoma cells.

[0036] The term "monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies includes antibodies that are substantially similar and bind the same epitope, except for variants that may normally arise during the production of monoclonal antibodies. Such variants are generally present in only trace amounts. Monoclonal antibodies are typically obtained by a process that includes the selection of a single antibody from a plurality of antibodies. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones. The selected antibody can be further altered, for example, to improve its affinity for the target (by so-called "affinity maturation"), to humanize the antibody, to improve its production in cell culture, and / or to reduce its immunogenicity in a subject.

[0037] The term "humanized antibody" may refer to antibodies from non-human species (e.g., mouse or rabbit) whose protein sequences have been modified to increase their similarity to antibodies naturally produced in humans.

[0038] The antibody may be a recombinant antibody. 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 that has specific binding affinity to the target antigen of the antibody, for example, a ZIP12 extracellular epitope that comprises or consists of SEQ ID NO:252 and / or SEQ ID NO:1, or a variant or fragment thereof. Preferably, the fragment is an epitope. The antigen-binding region may be a hypervariable CDR or a functional part thereof. The term "functional part" of a CDR may refer to a sequence within the CDR that exhibits specific affinity to the target antigen. The functional part of a CDR may include a ligand that specifically binds to a ZIP12 extracellular epitope that comprises or consists of SEQ ID NO:252 and / or SEQ ID NO:1, or a variant or fragment thereof.

[0039] The term "CDR" can refer to the hypervariable region in the heavy and light chain variable regions. There can be one, two, three or more CDRs in each of the heavy and light chains of an antibody. Usually, there are at least three CDRs in each chain, which, when assembled together, form an antigen-binding site, i.e., a three-dimensional binding site to which an antigen binds or specifically reacts. However, it is hypothesized that there can be four CDRs in the heavy chain of some antibodies.

[0040] The definition of CDR also includes overlapping or subsets of amino acid residues when compared against each other. The exact number of residues that encompass 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, given the variable region amino acid sequence of an antibody.

[0041] The amino acid sequence boundaries of the CDRs can be determined using any of a numbering scheme known in the art, including Kabat et al., supra (the "Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (the "Chothia" numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (the "Contact" numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (the "IMGT" numbering scheme); and Honegge and Plueckthun, J. Mol. Biol., 2001, 309:657-70 (the "AHo" numbering scheme).

[0042] The term "functional fragment" of an antibody may refer to a portion of an antibody that retains a functional activity. A functional activity may be, for example, antigen binding activity or specificity. A functional activity may also be, for example, an effector function provided by an antibody constant region. The term "functional fragment" is also intended to include fragments generated, for example, by protease digestion or reduction of human monoclonal antibodies and by recombinant DNA methods known to those skilled in the art. Human monoclonal antibody functional fragments include, for example, individual heavy or light chains and fragments thereof, such as VL, VH, and Fd; monovalent fragments, such as Fv, Fab, and Fab'; F(ab') 2 such as Fv; single chain Fv (scFv); and Fc fragments.

[0043] The term "VL fragment" may refer to a fragment of the light chain of a human monoclonal antibody that contains all or a portion of the light chain variable region, including the CDRs. The VL fragment may further contain the light chain constant region sequence.

[0044] The term "VH fragment" may refer to a fragment of the heavy chain of a human monoclonal antibody that contains all or a portion of the heavy chain variable region, including the CDRs. The term "Fd fragment" may refer to the heavy chain variable region, i.e., VH and CH-1, linked to the first heavy chain constant region. The "Fd fragment" does not include the light chain or the second and third constant regions of the heavy chain.

[0045] The term "Fv fragment" may refer to a monovalent antigen-binding fragment of a human monoclonal antibody, which contains all or a portion of the heavy and light chain variable regions and lacks the heavy and light chain constant regions. The heavy and light chain variable regions include, for example, the CDRs. For example, the Fv fragment contains all or a portion of the amino-terminal variable region of about 110 amino acids of both the heavy and light chains.

[0046] The term "Fab fragment" may refer to a monovalent antigen-binding fragment of a human monoclonal antibody that is larger than an Fv fragment. For example, a Fab fragment contains the variable regions of the heavy and light chains and all or part of the first constant domain. Thus, a Fab fragment additionally contains, for example, about 110 to about 220 amino acid residues of the heavy and light chains.

[0047] The term "Fab' fragment" may refer to a monovalent antigen-binding fragment of a human monoclonal antibody that is larger than the Fab fragment. For example, a Fab' fragment may contain all of the light chain, all of the variable region of the heavy chain, and all or a portion of the first and second constant domains of the heavy chain. For example, a Fab' fragment may additionally contain some or all of the 220-330 amino acid residues of the heavy chain.

[0048] The term "F(ab') 2 "Fragment" may refer to a bivalent antigen-binding fragment of a human monoclonal antibody. F(ab') 2 The fragment may, for example, contain all or part of the variable regions of two heavy chains and two light chains, and may further contain all or part of the first constant domain of two heavy chains and the first constant domain of two light chains.

[0049] The term "single-chain Fv (scFv)" may refer to a fusion of the variable domain of the heavy chain (VH) and the variable domain of the light chain (VL) connected by a short linker peptide. The term "bispecific antibody (BsAb)" may refer to a bispecific antibody comprising two scFvs linked to each other by a short linker peptide.

[0050] Those skilled in the art know that the exact boundaries of an antibody fragment are not important, so long as the fragment maintains its functional activity. Using well-known recombinant methods, those skilled in the art can manipulate polynucleotide sequences to express functional fragments with any endpoints desired for a particular application. A functional antibody fragment can include or consist of a fragment having substantially the same heavy and light chain variable regions as a human antibody.

[0051] Preferably, the antigen-binding fragment of the first aspect of the invention is immunospecific for a ZIP12 extracellular epitope comprising or consisting of SEQ ID NO:252 and / or SEQ ID NO:1, or a variant or fragment thereof. The antigen-binding fragment may be any of the following: VH, VL, Fd, Fv, Fab, Fab', scFv, F(ab') 2 and an Fc fragment.

[0052] The antigen-binding fragment may be a single domain antibody (sdAb), otherwise known as a nanobody, which those of skill in the art understand to be an antibody fragment consisting of a single monomeric variable antibody domain.

[0053] The antigen-binding fragment may comprise or consist of any one of the VL antigen-binding region sequences, any one of the VH antigen-binding region sequences, or a combination of the VL and VH antigen-binding regions of a human antibody. The appropriate number and combination of VH and VL antigen-binding region sequences can be determined by one skilled in the art depending on the desired affinity and specificity of the antigen-binding fragment and the intended use. Functional or antigen-binding fragments of antibodies can be easily produced 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 include using human antibodies as starting materials. Enzymes suitable for proteolysis of human immunoglobulins can include, for example, papain and pepsin. Appropriate enzymes can be easily selected by one skilled in the art depending, for example, on whether a monovalent or bivalent fragment is required. For example, papain cleavage produces two monovalent Fab' fragments and an Fc fragment that bind the antigen. For example, pepsin cleavage produces a bivalent F(ab') fragment. 2 The fragment can be further reduced, for example with DTT or 2-mercaptoethanol, to yield two monovalent Fab' fragments.

[0054] Functional or antigen-binding fragments of antibodies generated by proteolysis can be purified by affinity and column chromatography procedures. For example, undigested antibodies and Fc fragments can be removed by binding with Protein A. Additionally, functional fragments can be purified according to their charge and size, for example, using ion exchange and gel filtration chromatography. Such methods are well known to those skilled in the art.

[0055] The antibody or antigen-binding fragment thereof can be produced by recombinant technology. Preferably, first, polynucleotides encoding the desired regions of the antibody heavy and light chains are isolated. Such regions can include, for example, all or part of the variable regions of the heavy and light chains. Preferably, such regions can include, in particular, the antigen-binding regions of the heavy and light chains, preferably the antigen-binding sites, most preferably the CDRs.

[0056] Polynucleotides encoding the antibodies or antigen-binding fragments thereof according to the present invention can be produced using methods known to those skilled in the art. Polynucleotides encoding the antibodies or antigen-binding fragments thereof can be directly synthesized by methods of oligonucleotide synthesis known in the art. Alternatively, smaller fragments can be synthesized and linked to form larger functional fragments using recombinant methods known in the art.

[0057] As used herein, the term "immunospecific" may mean capable of immunoreacting with a ZIP12 extracellular epitope comprising or consisting of SEQ ID NO:252 and / or SEQ ID NO:1, or a variant or fragment thereof, by specifically binding thereto. The antibody or antigen-binding fragment thereof preferably has a specific binding activity of approximately 10 -5 ~10 -13 M -1 , preferably 10 -6 ~10 -9 M -1 , and even more preferably 10 -10 ~10 -12 M-1 The antibody or antigen-binding fragment thereof is preferably capable of selectively interacting with an antigen (i.e., a ZIP12 extracellular epitope comprising or consisting of SEQ ID NO:252 and / or SEQ ID NO:1, or a variant or fragment thereof) with 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, 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 As such, there is substantially no binding to ZIP4 and / or ZIP13.

[0058] The term "immunoreact" may mean capable of eliciting an immune response by binding to a ZIP12 extracellular epitope comprising or consisting of SEQ ID NO:252 and / or SEQ ID NO:1, or a variant or fragment thereof.

[0059] The term "epitope" may refer to any region of an antigen that has the ability to elicit and bind to the binding domain of an antibody or antigen-binding fragment thereof. Thus, the antibody or antigen-binding fragment thereof may comprise 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.

[0060] 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 1, the present inventors have developed an antibody candidate that binds to the extracellular domain of ZIP12, referred to herein as 51B12, which can target ZIP12 and inhibit its function. The six CDR, VH, and VL sequences of this so-called "51B12 parent" antibody are summarized in the table shown in Figure 9a. The present inventors then subjected the "51B12 parent antibody" to affinity maturation to generate novel variants or mutants.

[0061] Eight or nine variants per CDR residue in the three heavy and three light chain CDRs were designed as described in Example 1. For the heavy chain variable region (VH) CDR variants, the variable regions were synthesized and cloned into an expression vector containing an Fc null human IgG1. For the light chain variable region (VL) CDR variants, the variable regions were synthesized and cloned into an expression vector containing a human kappa light chain constant region.

[0062] Figure 9b shows a table summarizing the sequences of 26 different embodiments of affinity matured antibodies derived from the "51B12 parent". The first row in the table displays the corresponding sequence (6 CDRs, VH, and VL) for the affinity matured variant of the 51B12 parent, designated "H3_W99H_L1", in which the CDR-H3 sequence is the same as that of the parent except for the W99H amino acid replacement, and the CDR-L1 CDR is as indicated. The second row in the table displays the corresponding sequence for the affinity matured variant of the 51B12 parent, designated "H3_W99H_L2", in which the CDR-H3 sequence is the same as that of the parent except for the W99H amino acid replacement, and the CDR-L2 CDR is as indicated, and so on.

[0063] Each of these affinity matured variants is now described with respect to their sequences. H3_W99H_L1 Thus, in one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as H3_W99H_L1.

[0064] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 13 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 14 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 15 or a variant or fragment thereof.

[0065] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 13, a CDR-H2 domain comprising or consisting of SEQ ID NO: 14, and / or a CDR-H3 domain comprising or consisting of SEQ ID NO: 15. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 13, a CDR-H2 domain comprising or consisting of SEQ ID NO: 14, and a CDR-H3 domain comprising or consisting of SEQ ID NO: 15.

[0066] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region comprising or consisting of a sequence substantially as set out in SEQ ID NO: 19, or a variant or fragment thereof.

[0067] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 16 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 17 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 18 or a variant or fragment thereof.

[0068] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 16, a CDR-L2 domain comprising or consisting of SEQ ID NO: 17, and / or a CDR-L3 domain comprising or consisting of SEQ ID NO: 18. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 16, a CDR-L2 domain comprising or consisting of SEQ ID NO: 17, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 18.

[0069] Preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of a sequence substantially as set out in SEQ ID NO: 20, or a variant or fragment thereof.

[0070] Preferably, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0071] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 13, a CDR-H2 domain comprising or consisting of SEQ ID NO: 14, a CDR-H3 domain comprising or consisting of SEQ ID NO: 15, a CDR-L1 domain comprising or consisting of SEQ ID NO: 16, a CDR-L2 domain comprising or consisting of SEQ ID NO: 17, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 18.

[0072] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID NO:19, and a light chain variable region comprising or consisting of SEQ ID NO:20.

[0073] H3_W99H_L2 In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as H3_W99H_L2.

[0074] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 21 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 22 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 23 or a variant or fragment thereof.

[0075] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 21, a CDR-H2 domain comprising or consisting of SEQ ID NO: 22, and / or a CDR-H3 domain comprising or consisting of SEQ ID NO: 23. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 21, a CDR-H2 domain comprising or consisting of SEQ ID NO: 22, and a CDR-H3 domain comprising or consisting of SEQ ID NO: 23.

[0076] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region comprising or consisting of a sequence substantially as set out in SEQ ID NO: 27, or a variant or fragment thereof.

[0077] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 24 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 25 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 26 or a variant or fragment thereof.

[0078] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 24, a CDR-L2 domain comprising or consisting of SEQ ID NO: 25, and / or a CDR-L3 domain comprising or consisting of SEQ ID NO: 26. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 24, a CDR-L2 domain comprising or consisting of SEQ ID NO: 25, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 26.

[0079] Preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence substantially as set forth in SEQ ID NO:28, or a variant or fragment thereof.

[0080] Preferably, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0081] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO:21, a CDR-H2 domain comprising or consisting of SEQ ID NO:22, a CDR-H3 domain comprising or consisting of SEQ ID NO:23, a CDR-L1 domain comprising or consisting of SEQ ID NO:24, a CDR-L2 domain comprising or consisting of SEQ ID NO:25, and a CDR-L3 domain comprising or consisting of SEQ ID NO:26.

[0082] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID NO:27, and a light chain variable region comprising or consisting of SEQ ID NO:28.

[0083] H3_W99H_VL-par In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as H3_W99H_VL-par.

[0084] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 29 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 30 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 31 or a variant or fragment thereof.

[0085] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 29, a CDR-H2 domain comprising or consisting of SEQ ID NO: 30, and / or a CDR-H3 domain comprising or consisting of SEQ ID NO: 31. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 29, a CDR-H2 domain comprising or consisting of SEQ ID NO: 30, and a CDR-H3 domain comprising or consisting of SEQ ID NO: 31.

[0086] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of a sequence substantially as set out in SEQ ID NO: 35, or a variant or fragment thereof.

[0087] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 32 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 33 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 34 or a variant or fragment thereof.

[0088] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 32, a CDR-L2 domain comprising or consisting of SEQ ID NO: 33, and / or a CDR-L3 domain comprising or consisting of SEQ ID NO: 34. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 32, a CDR-L2 domain comprising or consisting of SEQ ID NO: 33, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 34.

[0089] Preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 36, or a variant or fragment thereof.

[0090] Preferably, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0091] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO:29, a CDR-H2 domain comprising or consisting of SEQ ID NO:30, a CDR-H3 domain comprising or consisting of SEQ ID NO:31, a CDR-L1 domain comprising or consisting of SEQ ID NO:32, a CDR-L2 domain comprising or consisting of SEQ ID NO:33, and a CDR-L3 domain comprising or consisting of SEQ ID NO:34.

[0092] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID NO:35, and a light chain variable region comprising or consisting of SEQ ID NO:36.

[0093] H2_T56H_L1 In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as H2_T56H_L1.

[0094] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 37 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 38 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 39 or a variant or fragment thereof.

[0095] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 37, a CDR-H2 domain comprising or consisting of SEQ ID NO: 38, and / or a CDR-H3 domain comprising or consisting of SEQ ID NO: 39. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 37, a CDR-H2 domain comprising or consisting of SEQ ID NO: 38, and a CDR-H3 domain comprising or consisting of SEQ ID NO: 39.

[0096] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of a sequence substantially as set out in SEQ ID NO: 43, or a variant or fragment thereof.

[0097] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 40 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 41 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 42 or a variant or fragment thereof.

[0098] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 40, a CDR-L2 domain comprising or consisting of SEQ ID NO: 41, and / or a CDR-L3 domain comprising or consisting of SEQ ID NO: 42. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 40, a CDR-L2 domain comprising or consisting of SEQ ID NO: 41, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 42.

[0099] Preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 44, or a variant or fragment thereof.

[0100] Preferably, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0101] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 37, a CDR-H2 domain comprising or consisting of SEQ ID NO: 38, a CDR-H3 domain comprising or consisting of SEQ ID NO: 39, a CDR-L1 domain comprising or consisting of SEQ ID NO: 40, a CDR-L2 domain comprising or consisting of SEQ ID NO: 41, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 42.

[0102] Preferably, the antibody or antigen-binding fragment thereof comprises 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.

[0103] H2_T57H_L1 In one embodiment, the antibody or antigen-binding fragment thereof is designated herein as H2_T57H_L1.

[0104] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 45 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 46 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 47 or a variant or fragment thereof.

[0105] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 45, a CDR-H2 domain comprising or consisting of SEQ ID NO: 46, and / or a CDR-H3 domain comprising or consisting of SEQ ID NO: 47. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 45, a CDR-H2 domain comprising or consisting of SEQ ID NO: 46, and a CDR-H3 domain comprising or consisting of SEQ ID NO: 47.

[0106] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of a sequence substantially as set out in SEQ ID NO:51, or a variant or fragment thereof.

[0107] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 48 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 49 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 50 or a variant or fragment thereof.

[0108] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 48, a CDR-L2 domain comprising or consisting of SEQ ID NO: 49, and / or a CDR-L3 domain comprising or consisting of SEQ ID NO: 50. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 48, a CDR-L2 domain comprising or consisting of SEQ ID NO: 49, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 50.

[0109] Preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of a sequence substantially as set forth in SEQ ID NO:52, or a variant or fragment thereof.

[0110] Preferably, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0111] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 45, a CDR-H2 domain comprising or consisting of SEQ ID NO: 46, a CDR-H3 domain comprising or consisting of SEQ ID NO: 47, a CDR-L1 domain comprising or consisting of SEQ ID NO: 48, a CDR-L2 domain comprising or consisting of SEQ ID NO: 49, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 50.

[0112] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID NO:51, and a light chain variable region comprising or consisting of SEQ ID NO:52.

[0113] H2_Y60K_L1 In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as H2_Y60K_L1.

[0114] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 53 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 54 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 55 or a variant or fragment thereof.

[0115] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 53, a CDR-H2 domain comprising or consisting of SEQ ID NO: 54, and / or a CDR-H3 domain comprising or consisting of SEQ ID NO: 55. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 53, a CDR-H2 domain comprising or consisting of SEQ ID NO: 54, and a CDR-H3 domain comprising or consisting of SEQ ID NO: 55.

[0116] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of a sequence substantially as set forth in SEQ ID NO:59, or a variant or fragment thereof.

[0117] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 56 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 57 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 58 or a variant or fragment thereof.

[0118] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 56, a CDR-L2 domain comprising or consisting of SEQ ID NO: 57, and / or a CDR-L3 domain comprising or consisting of SEQ ID NO: 58. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 56, a CDR-L2 domain comprising or consisting of SEQ ID NO: 57, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 58.

[0119] Preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 60, or a variant or fragment thereof.

[0120] Preferably, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0121] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO:53, a CDR-H2 domain comprising or consisting of SEQ ID NO:54, a CDR-H3 domain comprising or consisting of SEQ ID NO:55, a CDR-L1 domain comprising or consisting of SEQ ID NO:56, a CDR-L2 domain comprising or consisting of SEQ ID NO:57, and a CDR-L3 domain comprising or consisting of SEQ ID NO:58.

[0122] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID NO:59, and a light chain variable region comprising or consisting of SEQ ID NO:60.

[0123] H2_T63Y_L1 In one embodiment, the antibody or antigen-binding fragment thereof is designated herein as H2_T63Y_L1.

[0124] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 61 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 62 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 63 or a variant or fragment thereof.

[0125] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 61, a CDR-H2 domain comprising or consisting of SEQ ID NO: 62, and / or a CDR-H3 domain comprising or consisting of SEQ ID NO: 63. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 61, a CDR-H2 domain comprising or consisting of SEQ ID NO: 62, and a CDR-H3 domain comprising or consisting of SEQ ID NO: 63.

[0126] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of a sequence substantially as set forth in SEQ ID NO:67, or a variant or fragment thereof.

[0127] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 64 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 65 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 66 or a variant or fragment thereof.

[0128] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 64, a CDR-L2 domain comprising or consisting of SEQ ID NO: 65, and / or a CDR-L3 domain comprising or consisting of SEQ ID NO: 66. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 64, a CDR-L2 domain comprising or consisting of SEQ ID NO: 65, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 66.

[0129] Preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of a sequence substantially as set forth in SEQ ID NO:68, or a variant or fragment thereof.

[0130] Preferably, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0131] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO:61, a CDR-H2 domain comprising or consisting of SEQ ID NO:62, a CDR-H3 domain comprising or consisting of SEQ ID NO:63, a CDR-L1 domain comprising or consisting of SEQ ID NO:64, a CDR-L2 domain comprising or consisting of SEQ ID NO:65, and a CDR-L3 domain comprising or consisting of SEQ ID NO:66.

[0132] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID NO:67, and a light chain variable region comprising or consisting of SEQ ID NO:68.

[0133] H2_K65P_L1 In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as H2_K65P_L1.

[0134] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 69 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 70 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 71 or a variant or fragment thereof.

[0135] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 69, a CDR-H2 domain comprising or consisting of SEQ ID NO: 70, and / or a CDR-H3 domain comprising or consisting of SEQ ID NO: 71. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 69, a CDR-H2 domain comprising or consisting of SEQ ID NO: 70, and a CDR-H3 domain comprising or consisting of SEQ ID NO: 71.

[0136] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 75, or a variant or fragment thereof.

[0137] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 72 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 73 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 74 or a variant or fragment thereof.

[0138] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 72, a CDR-L2 domain comprising or consisting of SEQ ID NO: 73, and / or a CDR-L3 domain comprising or consisting of SEQ ID NO: 74. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 72, a CDR-L2 domain comprising or consisting of SEQ ID NO: 73, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 74.

[0139] Preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 76, or a variant or fragment thereof.

[0140] Preferably, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0141] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO:69, a CDR-H2 domain comprising or consisting of SEQ ID NO:70, a CDR-H3 domain comprising or consisting of SEQ ID NO:71, a CDR-L1 domain comprising or consisting of SEQ ID NO:72, a CDR-L2 domain comprising or consisting of SEQ ID NO:73, and a CDR-L3 domain comprising or consisting of SEQ ID NO:74.

[0142] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID NO:75, and a light chain variable region comprising or consisting of SEQ ID NO:76.

[0143] H2_G66Q_L1 In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as H2_G66Q_L1.

[0144] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 77 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 78 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 79 or a variant or fragment thereof.

[0145] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 77, a CDR-H2 domain comprising or consisting of SEQ ID NO: 78, and / or a CDR-H3 domain comprising or consisting of SEQ ID NO: 79. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 77, a CDR-H2 domain comprising or consisting of SEQ ID NO: 78, and a CDR-H3 domain comprising or consisting of SEQ ID NO: 79.

[0146] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of a sequence substantially as set forth in SEQ ID NO:83, or a variant or fragment thereof.

[0147] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 80 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 81 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 82 or a variant or fragment thereof.

[0148] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 80, a CDR-L2 domain comprising or consisting of SEQ ID NO: 81, and / or a CDR-L3 domain comprising or consisting of SEQ ID NO: 82. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 80, a CDR-L2 domain comprising or consisting of SEQ ID NO: 81, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 82.

[0149] Preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 84, or a variant or fragment thereof.

[0150] Preferably, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0151] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 77, a CDR-H2 domain comprising or consisting of SEQ ID NO: 78, a CDR-H3 domain comprising or consisting of SEQ ID NO: 79, a CDR-L1 domain comprising or consisting of SEQ ID NO: 80, a CDR-L2 domain comprising or consisting of SEQ ID NO: 81, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 82.

[0152] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID NO:83, and a light chain variable region comprising or consisting of SEQ ID NO:84.

[0153] H3_M105P_L1 In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as H3_M105P_L1.

[0154] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 85 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 86 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 87 or a variant or fragment thereof.

[0155] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 85, a CDR-H2 domain comprising or consisting of SEQ ID NO: 86, and / or a CDR-H3 domain comprising or consisting of SEQ ID NO: 87. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 85, a CDR-H2 domain comprising or consisting of SEQ ID NO: 86, and a CDR-H3 domain comprising or consisting of SEQ ID NO: 87.

[0156] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 91, or a variant or fragment thereof.

[0157] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 88 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 89 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 90 or a variant or fragment thereof.

[0158] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 88, a CDR-L2 domain comprising or consisting of SEQ ID NO: 89, and / or a CDR-L3 domain comprising or consisting of SEQ ID NO: 90. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 88, a CDR-L2 domain comprising or consisting of SEQ ID NO: 89, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 90.

[0159] Preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 92, or a variant or fragment thereof.

[0160] Preferably, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0161] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO:85, a CDR-H2 domain comprising or consisting of SEQ ID NO:86, a CDR-H3 domain comprising or consisting of SEQ ID NO:87, a CDR-L1 domain comprising or consisting of SEQ ID NO:88, a CDR-L2 domain comprising or consisting of SEQ ID NO:89, and a CDR-L3 domain comprising or consisting of SEQ ID NO:90.

[0162] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID NO:91, and a light chain variable region comprising or consisting of SEQ ID NO:92.

[0163] 51B12_VH_L1 In one embodiment, the antibody or antigen-binding fragment thereof is designated herein as 51B12_VH_L1.

[0164] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 93 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 94 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 95 or a variant or fragment thereof.

[0165] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 93, a CDR-H2 domain comprising or consisting of SEQ ID NO: 94, and / or a CDR-H3 domain comprising or consisting of SEQ ID NO: 95. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 93, a CDR-H2 domain comprising or consisting of SEQ ID NO: 94, and a CDR-H3 domain comprising or consisting of SEQ ID NO: 95.

[0166] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 99, or a variant or fragment thereof.

[0167] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 96 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 97 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 98 or a variant or fragment thereof.

[0168] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 96, a CDR-L2 domain comprising or consisting of SEQ ID NO: 97, and / or a CDR-L3 domain comprising or consisting of SEQ ID NO: 98. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 96, a CDR-L2 domain comprising or consisting of SEQ ID NO: 97, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 98.

[0169] Preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of a sequence substantially as set out in SEQ ID NO: 100, or a variant or fragment thereof.

[0170] Preferably, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0171] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO:93, a CDR-H2 domain comprising or consisting of SEQ ID NO:94, a CDR-H3 domain comprising or consisting of SEQ ID NO:95, a CDR-L1 domain comprising or consisting of SEQ ID NO:96, a CDR-L2 domain comprising or consisting of SEQ ID NO:97, and a CDR-L3 domain comprising or consisting of SEQ ID NO:98.

[0172] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID NO:99, and a light chain variable region comprising or consisting of SEQ ID NO:100.

[0173] H2_T56H_L2 In one embodiment, the antibody or antigen-binding fragment thereof is designated herein as H2_T56H_L2.

[0174] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 101 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 102 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 103 or a variant or fragment thereof.

[0175] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 101, a CDR-H2 domain comprising or consisting of SEQ ID NO: 102, and / or a CDR-H3 domain comprising or consisting of SEQ ID NO: 103. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 101, a CDR-H2 domain comprising or consisting of SEQ ID NO: 102, and a CDR-H3 domain comprising or consisting of SEQ ID NO: 103.

[0176] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 107, or a variant or fragment thereof.

[0177] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 104 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 105 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 106 or a variant or fragment thereof.

[0178] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 104, a CDR-L2 domain comprising or consisting of SEQ ID NO: 105, and / or a CDR-L3 domain comprising or consisting of SEQ ID NO: 106. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 104, a CDR-L2 domain comprising or consisting of SEQ ID NO: 105, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 106.

[0179] Preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 108, or a variant or fragment thereof.

[0180] Preferably, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0181] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO:101, a CDR-H2 domain comprising or consisting of SEQ ID NO:102, a CDR-H3 domain comprising or consisting of SEQ ID NO:103, a CDR-L1 domain comprising or consisting of SEQ ID NO:104, a CDR-L2 domain comprising or consisting of SEQ ID NO:105, and a CDR-L3 domain comprising or consisting of SEQ ID NO:106.

[0182] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID NO:107, and a light chain variable region comprising or consisting of SEQ ID NO:108.

[0183] H2_T57H_L2 In one embodiment, the antibody or antigen-binding fragment thereof is designated herein as H2_T57H_L2.

[0184] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 109 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 110 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 111 or a variant or fragment thereof.

[0185] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 109, a CDR-H2 domain comprising or consisting of SEQ ID NO: 110, and / or a CDR-H3 domain comprising or consisting of SEQ ID NO: 111. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 109, a CDR-H2 domain comprising or consisting of SEQ ID NO: 110, and a CDR-H3 domain comprising or consisting of SEQ ID NO: 111.

[0186] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of a sequence substantially as set out in SEQ ID NO: 115, or a variant or fragment thereof.

[0187] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 112 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 113 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 114 or a variant or fragment thereof.

[0188] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 112, a CDR-L2 domain comprising or consisting of SEQ ID NO: 113, and / or a CDR-L3 domain comprising or consisting of SEQ ID NO: 114. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 112, a CDR-L2 domain comprising or consisting of SEQ ID NO: 113, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 114.

[0189] Preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 116, or a variant or fragment thereof.

[0190] Preferably, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0191] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 109, a CDR-H2 domain comprising or consisting of SEQ ID NO: 110, a CDR-H3 domain comprising or consisting of SEQ ID NO: 111, a CDR-L1 domain comprising or consisting of SEQ ID NO: 112, a CDR-L2 domain comprising or consisting of SEQ ID NO: 113, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 114.

[0192] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID NO:115, and a light chain variable region comprising or consisting of SEQ ID NO:116.

[0193] H2_Y60K_L2 In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as H2_Y60K_L2.

[0194] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 117 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 118 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 119 or a variant or fragment thereof.

[0195] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 117, a CDR-H2 domain comprising or consisting of SEQ ID NO: 118, and / or a CDR-H3 domain comprising or consisting of SEQ ID NO: 119. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 117, a CDR-H2 domain comprising or consisting of SEQ ID NO: 118, and a CDR-H3 domain comprising or consisting of SEQ ID NO: 119.

[0196] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of a sequence substantially as set out in SEQ ID NO: 123, or a variant or fragment thereof.

[0197] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 120 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 121 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 122 or a variant or fragment thereof.

[0198] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 120, a CDR-L2 domain comprising or consisting of SEQ ID NO: 121, and / or a CDR-L3 domain comprising or consisting of SEQ ID NO: 122. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 120, a CDR-L2 domain comprising or consisting of SEQ ID NO: 121, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 122.

[0199] Preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 124, or a variant or fragment thereof.

[0200] Preferably, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0201] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO:117, a CDR-H2 domain comprising or consisting of SEQ ID NO:118, a CDR-H3 domain comprising or consisting of SEQ ID NO:119, a CDR-L1 domain comprising or consisting of SEQ ID NO:120, a CDR-L2 domain comprising or consisting of SEQ ID NO:121, and a CDR-L3 domain comprising or consisting of SEQ ID NO:122.

[0202] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID NO:123, and a light chain variable region comprising or consisting of SEQ ID NO:124.

[0203] H2_T63Y_L2 In one embodiment, the antibody or antigen-binding fragment thereof is designated herein as H2_T63Y_L2.

[0204] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 125 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 126 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 127 or a variant or fragment thereof.

[0205] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 125, a CDR-H2 domain comprising or consisting of SEQ ID NO: 126, and / or a CDR-H3 domain comprising or consisting of SEQ ID NO: 127. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 125, a CDR-H2 domain comprising or consisting of SEQ ID NO: 126, and a CDR-H3 domain comprising or consisting of SEQ ID NO: 127.

[0206] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of a sequence substantially as set out in SEQ ID NO: 131, or a variant or fragment thereof.

[0207] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 128 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 129 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 130 or a variant or fragment thereof.

[0208] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 128, a CDR-L2 domain comprising or consisting of SEQ ID NO: 129, and / or a CDR-L3 domain comprising or consisting of SEQ ID NO: 130. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 128, a CDR-L2 domain comprising or consisting of SEQ ID NO: 129, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 130.

[0209] Preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 132, or a variant or fragment thereof.

[0210] Preferably, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0211] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 125, a CDR-H2 domain comprising or consisting of SEQ ID NO: 126, a CDR-H3 domain comprising or consisting of SEQ ID NO: 127, a CDR-L1 domain comprising or consisting of SEQ ID NO: 128, a CDR-L2 domain comprising or consisting of SEQ ID NO: 129, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 130.

[0212] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID NO:131, and a light chain variable region comprising or consisting of SEQ ID NO:132.

[0213] H3_M105P_VL-par In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as H3_M105P_VL-par.

[0214] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 133 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 134 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 135 or a variant or fragment thereof.

[0215] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 133, a CDR-H2 domain comprising or consisting of SEQ ID NO: 134, and / or a CDR-H3 domain comprising or consisting of SEQ ID NO: 135. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 133, a CDR-H2 domain comprising or consisting of SEQ ID NO: 134, and a CDR-H3 domain comprising or consisting of SEQ ID NO: 135.

[0216] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 139, or a variant or fragment thereof.

[0217] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 136 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 137 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 138 or a variant or fragment thereof.

[0218] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 136, a CDR-L2 domain comprising or consisting of SEQ ID NO: 137, and / or a CDR-L3 domain comprising or consisting of SEQ ID NO: 138. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 136, a CDR-L2 domain comprising or consisting of SEQ ID NO: 137, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 138.

[0219] Preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 140, or a variant or fragment thereof.

[0220] Preferably, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0221] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 133, a CDR-H2 domain comprising or consisting of SEQ ID NO: 134, a CDR-H3 domain comprising or consisting of SEQ ID NO: 135, a CDR-L1 domain comprising or consisting of SEQ ID NO: 136, a CDR-L2 domain comprising or consisting of SEQ ID NO: 137, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 138.

[0222] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID NO:139, and a light chain variable region comprising or consisting of SEQ ID NO:140.

[0223] H2_K65P_L2 In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as H2_K65P_L2.

[0224] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 141 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 142 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 143 or a variant or fragment thereof.

[0225] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 141, a CDR-H2 domain comprising or consisting of SEQ ID NO: 142, and / or a CDR-H3 domain comprising or consisting of SEQ ID NO: 143. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 141, a CDR-H2 domain comprising or consisting of SEQ ID NO: 142, and a CDR-H3 domain comprising or consisting of SEQ ID NO: 143.

[0226] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 147, or a variant or fragment thereof.

[0227] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 144 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 145 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 146 or a variant or fragment thereof.

[0228] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 144, a CDR-L2 domain comprising or consisting of SEQ ID NO: 145, and / or a CDR-L3 domain comprising or consisting of SEQ ID NO: 146. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 144, a CDR-L2 domain comprising or consisting of SEQ ID NO: 145, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 146.

[0229] Preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 148, or a variant or fragment thereof.

[0230] Preferably, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0231] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 141, a CDR-H2 domain comprising or consisting of SEQ ID NO: 142, a CDR-H3 domain comprising or consisting of SEQ ID NO: 143, a CDR-L1 domain comprising or consisting of SEQ ID NO: 144, a CDR-L2 domain comprising or consisting of SEQ ID NO: 145, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 146.

[0232] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID NO:147, and a light chain variable region comprising or consisting of SEQ ID NO:148.

[0233] H2_G66Q_L2 In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as H2_G66Q_L2.

[0234] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 149 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 150 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 151 or a variant or fragment thereof.

[0235] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 149, a CDR-H2 domain comprising or consisting of SEQ ID NO: 150, and / or a CDR-H3 domain comprising or consisting of SEQ ID NO: 151. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 149, a CDR-H2 domain comprising or consisting of SEQ ID NO: 150, and a CDR-H3 domain comprising or consisting of SEQ ID NO: 151.

[0236] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 155, or a variant or fragment thereof.

[0237] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 152 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 153 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 154 or a variant or fragment thereof.

[0238] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 152, a CDR-L2 domain comprising or consisting of SEQ ID NO: 153, and / or a CDR-L3 domain comprising or consisting of SEQ ID NO: 154. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 152, a CDR-L2 domain comprising or consisting of SEQ ID NO: 153, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 154.

[0239] Preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 156, or a variant or fragment thereof.

[0240] Preferably, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0241] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 149, a CDR-H2 domain comprising or consisting of SEQ ID NO: 150, a CDR-H3 domain comprising or consisting of SEQ ID NO: 151, a CDR-L1 domain comprising or consisting of SEQ ID NO: 152, a CDR-L2 domain comprising or consisting of SEQ ID NO: 153, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 154.

[0242] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID NO:155, and a light chain variable region comprising or consisting of SEQ ID NO:156.

[0243] H3_M105P_L2 In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as H3_M105P_L2.

[0244] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 157 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 158 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 159 or a variant or fragment thereof.

[0245] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 157, a CDR-H2 domain comprising or consisting of SEQ ID NO: 158, and / or a CDR-H3 domain comprising or consisting of SEQ ID NO: 159. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 157, a CDR-H2 domain comprising or consisting of SEQ ID NO: 158, and a CDR-H3 domain comprising or consisting of SEQ ID NO: 159.

[0246] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 163, or a variant or fragment thereof.

[0247] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 160 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 161 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 162 or a variant or fragment thereof.

[0248] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 160, a CDR-L2 domain comprising or consisting of SEQ ID NO: 161, and / or a CDR-L3 domain comprising or consisting of SEQ ID NO: 162. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 160, a CDR-L2 domain comprising or consisting of SEQ ID NO: 161, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 162.

[0249] Preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 164, or a variant or fragment thereof.

[0250] Preferably, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0251] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 157, a CDR-H2 domain comprising or consisting of SEQ ID NO: 158, a CDR-H3 domain comprising or consisting of SEQ ID NO: 159, a CDR-L1 domain comprising or consisting of SEQ ID NO: 160, a CDR-L2 domain comprising or consisting of SEQ ID NO: 161, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 162.

[0252] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID NO:163, and a light chain variable region comprising or consisting of SEQ ID NO:164.

[0253] 51B12_VH_L2 In one embodiment, the antibody or antigen-binding fragment thereof is designated herein as 51B12_VH_L2.

[0254] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 165 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 166 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 167 or a variant or fragment thereof.

[0255] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 165, a CDR-H2 domain comprising or consisting of SEQ ID NO: 166, and / or a CDR-H3 domain comprising or consisting of SEQ ID NO: 167. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 165, a CDR-H2 domain comprising or consisting of SEQ ID NO: 166, and a CDR-H3 domain comprising or consisting of SEQ ID NO: 167.

[0256] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 171, or a variant or fragment thereof.

[0257] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 168 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 169 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 170 or a variant or fragment thereof.

[0258] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 168, a CDR-L2 domain comprising or consisting of SEQ ID NO: 169, and / or a CDR-L3 domain comprising or consisting of SEQ ID NO: 170. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 168, a CDR-L2 domain comprising or consisting of SEQ ID NO: 169, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 170.

[0259] Preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 172, or a variant or fragment thereof.

[0260] Preferably, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0261] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 165, a CDR-H2 domain comprising or consisting of SEQ ID NO: 166, a CDR-H3 domain comprising or consisting of SEQ ID NO: 167, a CDR-L1 domain comprising or consisting of SEQ ID NO: 168, a CDR-L2 domain comprising or consisting of SEQ ID NO: 169, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 170.

[0262] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID NO:171, and a light chain variable region comprising or consisting of SEQ ID NO:172.

[0263] H2_T56H_VL-par In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as H2_T56H_VL-par.

[0264] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 173 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 174 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 175 or a variant or fragment thereof.

[0265] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 173, a CDR-H2 domain comprising or consisting of SEQ ID NO: 174, and / or a CDR-H3 domain comprising or consisting of SEQ ID NO: 175. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 173, a CDR-H2 domain comprising or consisting of SEQ ID NO: 174, and a CDR-H3 domain comprising or consisting of SEQ ID NO: 175.

[0266] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 179, or a variant or fragment thereof.

[0267] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 176 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 177 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 178 or a variant or fragment thereof.

[0268] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 176, a CDR-L2 domain comprising or consisting of SEQ ID NO: 177, and / or a CDR-L3 domain comprising or consisting of SEQ ID NO: 178. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 176, a CDR-L2 domain comprising or consisting of SEQ ID NO: 177, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 178.

[0269] Preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 180, or a variant or fragment thereof.

[0270] Preferably, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0271] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 173, a CDR-H2 domain comprising or consisting of SEQ ID NO: 174, a CDR-H3 domain comprising or consisting of SEQ ID NO: 175, a CDR-L1 domain comprising or consisting of SEQ ID NO: 176, a CDR-L2 domain comprising or consisting of SEQ ID NO: 177, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 178.

[0272] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID NO:179, and a light chain variable region comprising or consisting of SEQ ID NO:180.

[0273] H2_T57H_VL-par In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as H2_T57H_VL-par.

[0274] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 181 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 182 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 183 or a variant or fragment thereof.

[0275] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 181, a CDR-H2 domain comprising or consisting of SEQ ID NO: 182, and / or a CDR-H3 domain comprising or consisting of SEQ ID NO: 183. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 181, a CDR-H2 domain comprising or consisting of SEQ ID NO: 182, and a CDR-H3 domain comprising or consisting of SEQ ID NO: 183.

[0276] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 187, or a variant or fragment thereof.

[0277] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 184 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 185 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 186 or a variant or fragment thereof.

[0278] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 184, a CDR-L2 domain comprising or consisting of SEQ ID NO: 185, and / or a CDR-L3 domain comprising or consisting of SEQ ID NO: 186. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 184, a CDR-L2 domain comprising or consisting of SEQ ID NO: 185, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 186.

[0279] Preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 188, or a variant or fragment thereof.

[0280] Preferably, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0281] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 181, a CDR-H2 domain comprising or consisting of SEQ ID NO: 182, a CDR-H3 domain comprising or consisting of SEQ ID NO: 183, a CDR-L1 domain comprising or consisting of SEQ ID NO: 184, a CDR-L2 domain comprising or consisting of SEQ ID NO: 185, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 186.

[0282] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID NO:187, and a light chain variable region comprising or consisting of SEQ ID NO:188.

[0283] H2_Y60K_VL-par In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as H2_Y60K_VL-par.

[0284] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 189 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 190 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 191 or a variant or fragment thereof.

[0285] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 189, a CDR-H2 domain comprising or consisting of SEQ ID NO: 190, and / or a CDR-H3 domain comprising or consisting of SEQ ID NO: 191. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 189, a CDR-H2 domain comprising or consisting of SEQ ID NO: 190, and a CDR-H3 domain comprising or consisting of SEQ ID NO: 191.

[0286] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 195, or a variant or fragment thereof.

[0287] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 192 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 193 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 194 or a variant or fragment thereof.

[0288] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 192, a CDR-L2 domain comprising or consisting of SEQ ID NO: 193, and / or a CDR-L3 domain comprising or consisting of SEQ ID NO: 194. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 192, a CDR-L2 domain comprising or consisting of SEQ ID NO: 193, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 194.

[0289] Preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 196, or a variant or fragment thereof.

[0290] Preferably, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0291] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 189, a CDR-H2 domain comprising or consisting of SEQ ID NO: 190, a CDR-H3 domain comprising or consisting of SEQ ID NO: 191, a CDR-L1 domain comprising or consisting of SEQ ID NO: 192, a CDR-L2 domain comprising or consisting of SEQ ID NO: 193, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 194.

[0292] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID NO:195, and a light chain variable region comprising or consisting of SEQ ID NO:196.

[0293] H2_T63Y_VL-par In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as H2_T63Y_VL-par.

[0294] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 197 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 198 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 199 or a variant or fragment thereof.

[0295] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 197, a CDR-H2 domain comprising or consisting of SEQ ID NO: 198, and / or a CDR-H3 domain comprising or consisting of SEQ ID NO: 199. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 197, a CDR-H2 domain comprising or consisting of SEQ ID NO: 198, and a CDR-H3 domain comprising or consisting of SEQ ID NO: 199.

[0296] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 203, or a variant or fragment thereof.

[0297] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 200 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 201 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 202 or a variant or fragment thereof.

[0298] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 200, a CDR-L2 domain comprising or consisting of SEQ ID NO: 201, and / or a CDR-L3 domain comprising or consisting of SEQ ID NO: 202. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 200, a CDR-L2 domain comprising or consisting of SEQ ID NO: 201, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 202.

[0299] Preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 204, or a variant or fragment thereof.

[0300] Preferably, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0301] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 197, a CDR-H2 domain comprising or consisting of SEQ ID NO: 198, a CDR-H3 domain comprising or consisting of SEQ ID NO: 199, a CDR-L1 domain comprising or consisting of SEQ ID NO: 200, a CDR-L2 domain comprising or consisting of SEQ ID NO: 201, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 202.

[0302] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID NO:203, and a light chain variable region comprising or consisting of SEQ ID NO:204.

[0303] H2_K65P_VL-par In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as H2_K65P_VL-par.

[0304] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 205 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 206 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 207 or a variant or fragment thereof.

[0305] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 205, a CDR-H2 domain comprising or consisting of SEQ ID NO: 206, and / or a CDR-H3 domain comprising or consisting of SEQ ID NO: 207. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 205, a CDR-H2 domain comprising or consisting of SEQ ID NO: 206, and a CDR-H3 domain comprising or consisting of SEQ ID NO: 207.

[0306] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of a sequence substantially as set out in SEQ ID NO: 211, or a variant or fragment thereof.

[0307] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 208 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 209 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 210 or a variant or fragment thereof.

[0308] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 208, a CDR-L2 domain comprising or consisting of SEQ ID NO: 209, and / or a CDR-L3 domain comprising or consisting of SEQ ID NO: 210. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 208, a CDR-L2 domain comprising or consisting of SEQ ID NO: 209, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 210.

[0309] Preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 212, or a variant or fragment thereof.

[0310] Preferably, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0311] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO:205, a CDR-H2 domain comprising or consisting of SEQ ID NO:206, a CDR-H3 domain comprising or consisting of SEQ ID NO:207, a CDR-L1 domain comprising or consisting of SEQ ID NO:208, a CDR-L2 domain comprising or consisting of SEQ ID NO:209, and a CDR-L3 domain comprising or consisting of SEQ ID NO:210.

[0312] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID NO:211, and a light chain variable region comprising or consisting of SEQ ID NO:212.

[0313] H2_G66Q_VL-par In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as H2_G66Q_VL-par.

[0314] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 213 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 214 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 215 or a variant or fragment thereof.

[0315] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 213, a CDR-H2 domain comprising or consisting of SEQ ID NO: 214, and / or a CDR-H3 domain comprising or consisting of SEQ ID NO: 215. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO: 213, a CDR-H2 domain comprising or consisting of SEQ ID NO: 214, and a CDR-H3 domain comprising or consisting of SEQ ID NO: 215.

[0316] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 219, or a variant or fragment thereof.

[0317] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 216 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 217 or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence substantially as set out in SEQ ID NO: 218 or a variant or fragment thereof.

[0318] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 216, a CDR-L2 domain comprising or consisting of SEQ ID NO: 217, and / or a CDR-L3 domain comprising or consisting of SEQ ID NO: 218. However, preferably, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID NO: 216, a CDR-L2 domain comprising or consisting of SEQ ID NO: 217, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 218.

[0319] Preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising or consisting of a sequence substantially as set forth in SEQ ID NO: 220, or a variant or fragment thereof.

[0320] Preferably, the antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs. Preferably, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0321] Preferably, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID NO:213, a CDR-H2 domain comprising or consisting of SEQ ID NO:214, a CDR-H3 domain comprising or consisting of SEQ ID NO:215, a CDR-L1 domain comprising or consisting of SEQ ID NO:216, a CDR-L2 domain comprising or consisting of SEQ ID NO:217, and a CDR-L3 domain comprising or consisting of SEQ ID NO:218.

[0322] Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID NO:219, and a light chain variable region comprising or consisting of SEQ ID NO:220.

[0323] In a second aspect of the invention, there is provided an antibody or antigen-binding fragment thereof which specifically binds ZIP12 and comprises the six CDR sequences as shown for each antibody in Figure 9b.

[0324] For example, in an embodiment, the antibody or antigen-binding fragment thereof preferably comprises six CDR sequences as shown for the antibody designated "H3_W99H_L1" as shown in Figure 9b, i.e., CDR-H1 = SEQ ID NO: 13; CDR-H2 = SEQ ID NO: 14; CDR-H3 = SEQ ID NO: 15; CDR-L1 = SEQ ID NO: 16; CDR-L2 = SEQ ID NO: 17; and CDR-L3 = SEQ ID NO: 18. The same applies to the other CDR sequences for each of the other antibodies shown in Figure 9b.

[0325] Further, the antibody or antigen-binding fragment thereof preferably comprises a VH / VL sequence pair as shown for each antibody shown in Figure 9b. For example, in an embodiment, the antibody or antigen-binding fragment thereof preferably comprises VH / VL sequences as shown for the antibody clone designated "H3_W99H_L1" as shown in Figure 9b, i.e., VH=SEQ ID NO: 19; and VL=SEQ ID NO: 20. The same applies to the other VH / VL sequences for each of the other antibodies shown in Figure 9b.

[0326] An antibody or antigen-binding fragment thereof of the present invention may not comprise the six CDR sequences (or VH / VL sequences) as shown in Figure 9a for the antibody clone referred to as "51B12 parent".

[0327] The antibody or antigen-binding fragment thereof of the second aspect specifically binds to the extracellular domain of ZIP12 and recognizes an epitope comprising or consisting of ADLLQVLSAGDHPPHNHSRS (SEQ ID NO: 252) or a variant or fragment thereof. Preferably, the antibody or antigen-binding fragment thereof of the second aspect specifically binds to the extracellular domain of ZIP12 and recognizes an epitope comprising or consisting of LLQVLSAGDHPPHNHSRS (SEQ ID NO: 1) or a variant or fragment thereof. The epitope of the antibody or antigen-binding fragment thereof may be as defined for the first aspect.

[0328] Advantageously, the anti-ZIP12 activity of the antibody or antigen-binding fragment thereof according to the first and second aspects of the invention means that it has utility as a therapeutic agent in its own right and may be used in the treatment, amelioration or prevention of hypoxia-induced or hypoxia-related conditions, and in particular pulmonary hypertension.

[0329] Thus, in a third aspect of the invention there is provided an antibody or antigen-binding fragment thereof according to the first or second aspect for use in therapy. In a fourth aspect of the invention there is provided an antibody or antigen-binding fragment thereof according to the first or second aspect for use in treating, preventing or ameliorating a hypoxia-associated condition.

[0330] According to a fifth aspect of the invention there is provided a method of treating, preventing or ameliorating a hypoxia-related condition in a subject comprising administering, or having administered to a patient in need of such treatment, a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to the first or second aspect.

[0331] The hypoxia-associated condition may be selected from the group consisting of ischemia-reperfusion injury (IRI), cardiovascular disease, ischemic heart disease, ischemic brain conditions, macular degeneration, ocular ischemic syndrome, ischemic optic neuropathy (ION), diabetic retinopathy, arthritis, inflammation, sepsis, sepsis-induced shock, renal disease, tissue fibrosis, 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, pathogenic blood vessel growth, cancer, and musculoskeletal disorders.

[0332] The inventors have demonstrated that the hypoxic core of tumors is the site of hypoxia-stimulated 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 as a universal feature of tumors, contributing to resistance to radiation and chemotherapy. Thus, ZIP12, which the inventors have demonstrated to be upregulated in hypoxic conditions and 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 may limit blood supply to tumors and thus limit tumor growth and / or induce hypoxic tumor cell death.

[0333] Thus, in one embodiment, the hypoxia-associated condition is cancer. Preferably, the use or method in treating, preventing or ameliorating cancer comprises inhibiting angiogenesis. Preferably, said use or method comprises restricting the blood supply to a tumor.

[0334] However, preferably, the hypoxia-related condition is pulmonary hypertension. Most preferably, the oxygen-related condition is hypoxic pulmonary hypertension. It will be understood that the antibodies, fragments thereof (collectively referred to herein as "agents") according to the present invention may be used in monotherapy (e.g., the use of the antibody or antigen-binding fragment thereof alone) to treat, ameliorate, or prevent hypoxia-related conditions, preferably pulmonary hypertension. Alternatively, the agents according to the present invention may be used as an adjunct to or in combination with known therapies for treating, ameliorating, or preventing hypoxia-related 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 stimulators such as riociguat, and calcium channel blockers nifedipine, diltiazem, nicardipine, and amlodipine.

[0335] The agents according to the invention can be combined in a composition in several different forms, depending in particular on the manner in which the composition is to be used. Thus, for example, the composition can be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposomal suspension, or any other suitable form that can be administered to a person or animal in need of treatment. It will be recognized that the vehicle of the drug according to the invention should be well tolerated by the subject to which it is given.

[0336] The drug containing the agent of the present invention can be used in several ways.For example, oral administration may be required, in which case the agent can be contained in a composition that can be taken orally, for example, in the form of a tablet, capsule, or liquid.The composition containing the agent and drug of the present invention can be administered by inhalation (for example, intranasally).The composition can also be formulated for topical use.For example, cream or ointment can be applied to the skin.

[0337] The agent and drug according to the present invention can also be incorporated into a sustained or delayed release device. Such a device can be inserted, for example, above or below the skin, and the drug can be released over a period of weeks or even months. The device can be located at least adjacent to the treatment site. Such a device can be particularly advantageous when long-term treatment with the agent used according to the present invention is required and it usually requires frequent administration (e.g., at least daily injections).

[0338] In a preferred embodiment, the agents and drugs according to the invention may be administered to a subject by injection into the bloodstream or directly to the site requiring treatment. Injection may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion) or intradermal (bolus or infusion).

[0339] It will be appreciated that the amount of antibody and fragment (i.e., agent) required will be determined by its biological activity and bioavailability, which in turn will depend on the mode of administration, the physiochemical properties of the agent, and whether it is to be used as a monotherapy or in a combination therapy. The frequency of administration will also be affected by the half-life of the 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 agent used, the strength of the pharmaceutical composition, the mode of administration, and the progression of pulmonary hypertension. Additional factors that depend on the specific subject to be treated, including the subject's age, weight, sex, diet, and time of administration, will result in the need to adjust the dosage.

[0340] Generally, a daily dose of between 0.01 μg / kg body weight and 100 mg / kg body weight of an agent according to the invention can be used to treat, ameliorate, or prevent conditions such as pulmonary hypertension, depending on which agent. More preferably, the daily dose of the agent 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 about 100 μg / kg and 10 mg / kg body weight.

[0341] The 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., a single injection per day). Alternatively, the agent may require administration more than once per day. By way of example, the agent may be administered as a dose between 0.07 μg and 700 mg (i.e., assuming a body weight of 70 kg) twice a day (or more, depending on the severity of the pulmonary hypertension to be treated). The patient to be treated may take a first dose upon waking and then a second dose (in the case of a two-dose regimen) at night or at an interval of 3 or 4 hours thereafter. Alternatively, a sustained release device may be used to provide the patient with an optimal dose of the agent according to the invention without the need for repeated administration. Known procedures, such as those normally used by the pharmaceutical industry (e.g., in vivo experiments, clinical trials, etc.), may be used to formulate a particular formulation of the agent according to the invention and a precise treatment regimen (such as the daily amount and frequency of administration of the agent).

[0342] In a sixth aspect of the invention there is provided a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to the first or second aspect, and optionally a pharma- ceutically acceptable vehicle. The pharmaceutical composition is preferably an antihypertensive, ie, a pharmaceutical preparation used in the therapeutic amelioration, prevention, or treatment of pulmonary hypertension.

[0343] The present invention also provides in a seventh aspect a method for making a pharmaceutical composition according to the sixth aspect, comprising combining a therapeutically effective amount of an antibody or antigen-binding fragment thereof as defined in the first or second aspect with a pharma- ceutically acceptable vehicle.

[0344] The antibody or antigen-binding fragment thereof may be as defined in relation to the first or second aspect. A "subject" may be a vertebrate, a mammal, or a domesticated animal. Thus, the medicament according to the present invention may be used to treat any mammal, for example, livestock (e.g., horses), pets, or other veterinary applications. Most preferably, the subject is a human being.

[0345] A "therapeutically effective amount" of an antibody or antigen-binding fragment thereof is any amount that, when administered to a subject, is the amount of agent needed to treat pulmonary hypertension or produce a desired effect.

[0346] For example, a therapeutically effective amount of an antibody or antigen-binding fragment thereof 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 preferred that the amount of antibody or fragment is from about 10 ng / kg to about 10 mg / kg, most preferably from about 50 ng / kg to about 5 mg / kg.

[0347] As referred to herein, a "pharmaceutically acceptable vehicle" is any known compound or combination of known compounds known to those of skill in the art to be useful in formulating a pharmaceutical composition.

[0348] In one embodiment, the pharma- ceutically acceptable medium may be solid and the composition may take the form of a powder or tablet. The solid pharma- ceutically acceptable medium may contain one or more substances that may act as flavoring agents, lubricants, solubilizers, suspending agents, dyes, bulking agents, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coating agents, or tablet disintegrating agents. The medium may also be an encapsulating material. In powders, the medium is a finely divided solid in admixture with the finely divided active agent according to the present invention. In tablets, the active agent may be mixed with the medium having the necessary compression properties in appropriate proportions and compressed into the desired shape and size. Powders and tablets preferably contain up to 99% of the active agent. Suitable solid mediums include, for example, calcium phosphate, magnesium stearate, talc, sugar, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes, and ion exchange resins. In another embodiment, the pharmaceutical vehicle may be a gel and the composition may take the form of a cream or the like.

[0349] However, the pharmaceutical medium may be liquid and the pharmaceutical composition is in the form of a solution. Liquid media are used in preparing solutions, suspensions, emulsions, syrups, elixirs, and pressurized compositions. The active agent according to the invention may be dissolved or suspended in a pharma- ceutically acceptable liquid medium, such as water, an organic solvent, a mixture of both, or a pharma- ceutically acceptable oil or fat. The liquid medium may contain other suitable pharmaceutical additives, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavorings, suspending agents, thickeners, colorants, viscosity regulators, stabilizers, or osmolality regulators. Suitable examples of liquid media for oral and parenteral administration include water (some of which contain additives as described above, such as cellulose derivatives, preferably carboxymethylcellulose solutions), alcohols (including monohydric and polyhydric alcohols, such as glycols) and their derivatives, and oils (e.g., fractionated coconut oil and peanut oil). For parenteral administration, the medium may also be an oily ester, such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid vehicle for pressurized compositions can be halogenated hydrocarbon or other pharma- ceutically acceptable propellant.

[0350] Liquid pharmaceutical compositions that are sterile solutions or suspensions can be utilized, for example, by intramuscular, intrathecal, epidural, intraperitoneal, intravenous, and especially subcutaneous injection. The agent can be prepared as a sterile solid composition that can be dissolved or suspended at the time of administration using sterile water, saline, or other suitable sterile injectable medium.

[0351] The agents and compositions of the present invention may be orally administered in the form of a sterile solution or suspension containing other solutes or suspending agents (e.g., sufficient saline or glucose to make the solution isotonic), bile salts, gum acacia, gelatin, sorbitan monooleate, polysorbate 80 (oleic acid ester of sorbitol and its anhydrides, copolymerized with ethylene oxide), and the like. The agents used according to the present invention may also be orally administered 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, and liquid forms such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

[0352] The present invention also extends to methods for producing the antibodies of the first or second aspects and to the antibodies so produced. In an eighth aspect, there is provided an antibody or antigen-binding fragment thereof obtained by a method comprising the steps of: (i) immunizing a host organism with a ZIP12 extracellular epitope comprising or consisting of SEQ ID NO: 252 or SEQ ID NO: 1, or a variant or fragment thereof; and (ii) harvesting the antibody or antigen-binding fragment thereof from the host.

[0353] The host may be a mammal, and may be a human, rabbit, or mouse. Epitopes may be linear or conformational. Epitopes may be denatured or folded. However, preferably, folded epitopes are used to immunize the host.

[0354] Preferably, the method includes a step of bleeding the host animal, and then preferably collecting the antibody or antigen-binding fragment thereof from the blood, most preferably the serum. Preferably, the serum is passed through a gravity column with a covalently bound peptide-support. After washing, the antibody or antigen-binding fragment thereof is preferably eluted in a buffer, preferably an acidic buffer, and the solution may then be neutralized. The method may further include dialysis against a suitable buffer (e.g., PBS), and optionally, lyophilization.

[0355] In a ninth aspect of the invention there is provided a polynucleotide encoding an antibody or antigen-binding fragment thereof as defined in the first or second aspect. In a tenth aspect of the present invention there is provided an expression cassette comprising a polynucleotide sequence according to the ninth aspect.

[0356] The polynucleotide sequence encoding the antibody or antigen-binding fragment of the present invention is preferably included within a recombinant vector, e.g., for delivery to a host cell of interest, to enable production of the antibody or antigen-binding fragment thereof.

[0357] Thus, in an eleventh aspect of the present invention there is provided a recombinant vector comprising an expression cassette according to the tenth aspect. The vector encoding the antibody or antigen-binding fragment thereof of the first or second aspect can 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 can preferably be a DNA sequence, and it is the DNA sequence that encodes the antibody or antigen-binding fragment thereof that forms the antibody or antigen-binding fragment thereof of the first or second aspect.

[0358] Recombinant vectors encoding the antibody or antigen-binding fragment thereof of the first or second embodiment may also include other functional elements. For example, they may further include various other functional elements, including a suitable promoter for initiating transgene expression upon introduction of the vector in a host cell. By way of example, the vector is preferably capable of autonomously replicating in the nucleus of the 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 such that it integrates into the genome of the host cell. In this case, DNA sequences that favor targeted integration (e.g., by homologous recombination) are envisioned. Suitable promoters may include SV40 promoter, CMV, EF1a, PGK, viral terminal repeats, as well as inducible promoters such as, for example, the tetracycline inducible system. The cassette or vector may also include a terminator, such as beta globin, SV40 polyadenylation sequence, or a synthetic polyadenylation sequence. The recombinant vector may also include a promoter or regulator or enhancer for regulating expression of the nucleic acid, as required.

[0359] Vectors may also contain DNA that codes for genes that can be used as selection markers during cloning processes, i.e., to allow the selection of cells that have been transfected or transformed, and to allow the selection of cells that have vectors that incorporate heterologous DNA. For example, ampicillin, neomycin, puromycin, or chloramphenicol resistance are envisioned. Alternatively, the selection marker gene may be in a different vector that is used simultaneously with the vector that contains the transgene. Cassettes or vectors may also contain DNA that is involved in controlling the expression of nucleotide sequences, or DNA that targets the expressed polypeptide to a certain part of the host cell.

[0360] The purified vector can be directly inserted into the host cell by suitable means, for example, direct endocytotic uptake. The vector can be directly introduced into the host cell (e.g., eukaryotic or prokaryotic cell) by transfection, infection, electroporation, microinjection, cell fusion, protoplast fusion, calcium phosphate method, cationic lipid-based lipofection, polymer or dendrimer-based method, or ballistic bombardment. Alternatively, the vector of the present invention can be directly introduced into the host cell using a particle gun.

[0361] Alternatively, the delivery system can provide the polynucleotide to a host cell without it being incorporated into a vector. By way of example, the nucleic acid molecule can be incorporated into a liposome or viral particle. Alternatively, a "naked" polynucleotide can be inserted into the host cell by suitable means, such as direct endocytic uptake.

[0362] In a twelfth aspect of the invention there is provided a host cell comprising a polynucleotide sequence according to the ninth aspect, an expression cassette according to the tenth aspect, or a vector according to the eleventh aspect.

[0363] The host cell can 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 an NS0 mouse myeloma cell, a PER.C6® human cell, a human embryonic kidney 293 cell, or a Chinese hamster ovary (CHO) cell. Most preferably, the host cell is a CHO cell.

[0364] In a thirteenth aspect, there is provided a method of preparing an antibody or antigen-binding fragment thereof according to the first or second aspect, comprising the steps of: a) introducing the vector of the eleventh aspect into a host cell; and b) culturing said host cell under conditions that may result in the production of an antibody or antigen-binding fragment thereof according to the first or second aspect.

[0365] The host cell in step a) can 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 an NS0 mouse myeloma cell, a PER.C6® human cell, a human embryonic kidney 293 cell, or a Chinese hamster ovary (CHO) cell. Most preferably, the host cell is a CHO cell.

[0366] The method may further comprise the step of (c) harvesting, centrifuging, and / or filtering the cell culture medium to obtain a cell culture supernatant comprising said antibody or antigen-binding fragment thereof. The method may further comprise the step of (d) separating and purifying said antibody or antigen-binding fragment thereof from said cell culture supernatant. Preferably, purification is carried out by at least one chromatographic step.

[0367] Suitable chromatography steps include affinity chromatography and / or ion exchange chromatography. Preferably, the affinity chromatography is Protein A chromatography. The ion exchange chromatography can be anion exchange chromatography and / or cation exchange chromatography.

[0368] Preferably, step (d) comprises isolating and purifying said antibody or antigen-binding fragment thereof from said cell culture supernatant by: i) Protein A chromatography; ii) anion exchange chromatography; and / or iii) Cation exchange chromatography.

[0369] The method may further comprise the step of (e) filtering the purified antibody or antigen-binding fragment thereof resulting from step (d). Preferably, step (e) comprises virus filtration. Thus, preferably, the purified antibody or antigen-binding fragment thereof resulting from step (d) is filtered using a virus filtration membrane. Suitable membranes are known to those skilled in the art.

[0370] As discussed herein, ZIP12 expression is increased in many cell types, including endothelial cells, smooth muscle cells, and interstitial cells, in reconstituted pulmonary arterioles of rats, cows, and humans, which are susceptible to hypoxia-induced pulmonary hypertension. Therefore, assuming that the antibody of the present invention can bind to the ZIP12 extracellular epitope comprising or consisting of SEQ ID NO:252 and / or SEQ ID NO:1, or a variant or fragment thereof, the antibody or its antigen-binding fragment can be used as a robust diagnostic tool by detecting the presence of ZIP12 and determining the concentration of ZIP12.

[0371] Thus, in a fourteenth aspect there is provided an antibody or antigen-binding fragment thereof according to the first or second aspect for use in diagnosis or prognosis. According to a fifteenth aspect of the invention there is provided an antibody or antigen-binding fragment thereof according to the first or second aspect for use in diagnosing or prognosing a hypoxia-related condition.

[0372] According to a sixteenth aspect, there is provided a method for diagnosing or prognosing a hypoxia-related condition in a subject, the method comprising detecting ZIP12 in a biological sample obtained from the subject using an antibody or antigen-binding fragment thereof according to the first or second aspect.

[0373] Preferably, the hypoxia-associated condition is selected from the group consisting of ischemia-reperfusion injury (IRI), cardiovascular disease, ischemic heart disease, ischemic brain conditions, macular degeneration, ocular ischemic syndrome, ischemic optic neuropathy (ION), diabetic retinopathy, arthritis, inflammation, sepsis, sepsis-induced shock, renal disease, tissue fibrosis, 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.

[0374] Preferably, the hypoxia-associated condition is cancer or pulmonary hypertension. Most preferably, the hypoxia-associated condition is pulmonary hypertension. The method can be an in vitro or ex vivo method. Preferably, the method is an in vitro method.

[0375] The use or method may include determining the level of expression of ZIP12 in a subject, preferably where an increased concentration of ZIP12 in a biological sample compared to a reference concentration from a healthy control population is indicative of pulmonary hypertension or poor prognosis.

[0376] In one embodiment, the presence of ZIP12 in the lung may be considered to indicate pulmonary hypertension or poor prognosis. The presence of ZIP12 may be detected by immunocytochemistry. In one embodiment, a 1-fold increase in ZIP12 compared to a reference from a healthy control population indicates pulmonary hypertension or poor prognosis. In one embodiment, a 2-fold, 3-fold, 4-fold, or 5-fold increase in ZIP12 compared to a reference from a healthy control population indicates pulmonary hypertension or poor prognosis. In one embodiment, a 10-fold, 50-fold, or 100-fold increase in ZIP12 compared to a reference from a healthy control population indicates pulmonary hypertension or poor prognosis.

[0377] According to a seventeenth aspect of the present invention there is provided a kit for diagnosing a subject suffering from a hypoxia-associated condition or for providing a prognosis of the condition in a subject comprising an antibody or antigen-binding fragment thereof according to the first or second aspect for detecting ZIP12 in a sample from a test subject.

[0378] The kit may further include instructions for use and / or containers for collecting a biological sample from a subject. Preferably, the hypoxia-related condition is as defined herein, preferably pulmonary hypertension.

[0379] Prognosis can be related to determining the therapeutic effect in subjects diagnosed with pulmonary hypertension.Prognosis can be related to predicting the speed and / or duration of progression or reversal of pulmonary hypertension in subjects, survival rate, and / or efficacy of various treatment regimens.Thus, poor prognosis can indicate pulmonary hypertension progression, low survival rate, and reduced efficacy of treatment regimens.Good prognosis can indicate pulmonary hypertension resolution, high survival rate, and increased efficacy of treatment regimens.

[0380] Preferably, the sample comprises a biological sample. The sample may be any material obtainable from a subject from which proteins can be obtained. The biological sample may be a tissue or biological fluid. The biological sample may be any material available from a subject from which endothelial cells, smooth muscle cells, and / or stromal cells can be obtained. Furthermore, the sample may be blood, plasma, serum, cerebrospinal fluid, urine, sweat, saliva, tears, breast aspirate, breast milk, prostatic fluid, semen, vaginal fluid, feces, cervical scraping, cells, amniotic fluid, ocular fluid, mucus, respiratory moisture, animal tissue, cell lysate, tumor tissue, hair, skin, buccal scraping, lymph, interstitial fluid, nail, bone marrow, cartilage, prion, bone powder, ear wax, lymph, granuloma, cancer biopsy, or a combination thereof.

[0381] The sample can be a fluid aspirate, for example, the sample can be bronchoalveolar lavage (BAL), peritoneal fluid, pleural lavage, or pericardial lavage. 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 low temperature, e.g., in a refrigerator, or even frozen, before the method is performed. Alternatively, the blood sample may be stored at room temperature, e.g., 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.

[0382] Blood may be further treated before it is contacted with the antibody of the present invention. 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.

[0383] Preferably, the sample may comprise endothelial cells, smooth muscle cells and / or interstitial cells, preferably pulmonary endothelial cells, smooth muscle cells and / or interstitial cells. It will be appreciated that the present invention extends to any nucleic acid or peptide comprising substantially the amino acid or nucleic acid sequence of any of the sequences mentioned herein (including variants or fragments thereof), or variants, derivatives or analogues thereof. The terms "substantially an amino acid / nucleotide / peptide sequence", "variant" and "fragment" may refer to a sequence having at least 40% sequence identity with the amino acid / nucleotide / peptide sequence of any one of the sequences mentioned herein, for example 40% identity with any of the sequences identified herein.

[0384] Also envisaged are amino acid / polynucleotide / polypeptide sequences having sequence identity of greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and even more preferably greater than 80% sequence identity with any of the sequences mentioned herein. Preferably, the amino acid / polynucleotide / polypeptide sequence has 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 with any of the sequences mentioned herein.

[0385] A skilled artisan will 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, the alignment of the two sequences must be prepared first, and then the sequence identity value is calculated. The percentage identity for two sequences can take different values ​​depending on: (i) the method used to align sequences, such as ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, such as local vs. global alignment, the pair score matrix used (e.g., BLOSUM62, PAM250, Gonnet, etc.), and gap penalties, such as function form and constants.

[0386] Once aligned, 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-gapped positions; or (v) the number of equivalent positions excluding overhangs. Furthermore, it will be recognized that the percentage identity is also strongly length-dependent. Thus, the shorter the sequence pair, the higher the sequence identity, which may be expected to have arisen by chance.

[0387] It will therefore be appreciated that accurate 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 the preferred method for generating protein or DNA multiple alignments according to the present invention. Suitable parameters for ClustalW may be as follows: for DNA alignment: gap opening penalty = 15.0, gap extension penalty = 6.66, and matrix = identity. For protein alignment: gap opening 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 vary these and other parameters for optimal sequence alignment.

[0388] Preferably, the percentage identity calculation between two amino acid / polynucleotide / polypeptide sequences can then be calculated from such alignment as (N / T)*100, where N is the number of positions where the sequences share identical residues, and T is the total number of positions compared, including gaps, and either including or excluding overhangs. Preferably, overhangs are included in the calculation. Thus, the most preferred method for calculating the percentage identity between two sequences includes (i) preparing a sequence alignment using the ClustalW program with an appropriate set of parameters, for example as set out above, and (ii) inserting the values ​​of N and T into the following formula: sequence identity=(N / T)*100.

[0389] Alternative methods for identifying similar sequences are known to those of skill in the art. For example, a substantially similar nucleotide sequence is encoded by a sequence that hybridizes to a DNA sequence or its complement under stringent conditions. By stringent conditions, we mean that the nucleotide hybridizes to filter-bound DNA or RNA in 3x sodium chloride / sodium citrate (SSC) at approximately 45°C, followed by at least one wash in 0.2x SSC / 0.1% SDS at approximately 20-65°C. Alternatively, a substantially similar polypeptide may differ from any of the sequences described herein by at least one, but fewer than 5, fewer than 10, fewer than 20, fewer than 50, or fewer than 100 amino acids.

[0390] Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein may be different or altered to provide a functional variant thereof without substantially affecting the sequence of the protein encoded by them. Suitable nucleotide variants are those that have sequences that are altered by the substitution of different codons that code for the same amino acid within the sequence, thus resulting in silent (synonymous) changes. Other suitable variants include all or a portion of a sequence that has a homologous nucleotide sequence, but is altered by the substitution of different codons that code for amino acids with side chains of similar biophysical properties to the amino acid that is substituted, resulting in conservative changes. For example, small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include serine, threonine, cysteine, asparagine, and glutamine. Positively charged (basic) amino acids include lysine, arginine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Thus, it will be understood, and the skilled artisan will know the nucleotide sequences encoding, which amino acids may be replaced with amino acids having similar biophysical properties.

[0391] All of the features described in this specification (including any accompanying claims, abstract, and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive.

[0392] For a better understanding of the present invention, and to show how embodiments thereof may be carried into effect, reference will now be made, by way of example, to the accompanying drawings, in which: [Brief description of the drawings]

[0393] [Figure 1] FIG. 1 shows the affinity maturation strategy used in the development of the antibody embodiments described herein (Rajpal et al., 2005, PNAS 102:24 8466-8471). Nine amino acids were selected to be substituted at each CDR residue: alanine, serine, histidine, leucine, proline, tyrosine, aspartic acid, glutamine, and lysine. The heavy chain variable region (VH) and light chain variable region (VL) are also shown, with their respective CDRs highlighted by boxes: CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. [Diagram 2] Figure 1 shows the results of screening 51B12 parent VH-CDR variant production supernatants in HTRF and ELISA assays. Diluted production supernatants were tested. For ELISA, antibody binding was detected with anti-human IgG-HRP and visualized with TMB. Reactivity data was plotted on the x-axis from left to right, sorted based on position in the CDR and alphabetically within the CDR residue position (n=8 or 9 variants per CDR residue). The dotted line indicates the average HTRF signal of the 51B12 parent antibody. [Diagram 3] Figure 1 shows the results of screening 51B12 parent VL-CDR mutant production supernatants in HTRF and ELISA assays. Diluted production supernatants were tested. For ELISA, antibody binding was detected with anti-human IgG-HRP and visualized with TMB. Reactivity data was plotted on the x-axis from left to right, sorted based on position in the CDR and alphabetically within the CDR residue position (n=8 or 9 mutants per CDR residue). The dotted line indicates the average HTRF signal of the 51B12 parent antibody. [Figure 4] FIG. 1 shows an example of an overlay of selected antibody clones showing their reactivity profiles in an HTRF inhibition assay. [Diagram 5] FIG. 1 shows the mean data for control parent antibodies. [Figure 6]FIG. 1 shows data for a single cycle kinetic screen using 3.12 nM to 800 nM huZIP12-ECD-GST. [Figure 7] FIG. 1 shows data for a single cycle kinetic screen using 2.34 nM to 600 nM huZIP12-ECD-GST. [Figure 8] FIG. 1 shows epitope mapping of the 51B12 antibody. [Figure 9a] 1 is a table summarizing the six CDR, VH, and VL sequences of the "51B12 parent" antibody. [Figure 9b-1] 1 is a table summarizing six CDR, VH, and VL sequences of 26 different embodiments of affinity matured antibodies described herein. [Figure 9b-2] 1 is a table summarizing six CDR, VH, and VL sequences of 26 different embodiments of affinity matured antibodies described herein. [Figure 9b-3] 1 is a table summarizing six CDR, VH, and VL sequences of 26 different embodiments of affinity matured antibodies described herein. [Figure 9b-4] 1 is a table summarizing six CDR, VH, and VL sequences of 26 different embodiments of affinity matured antibodies described herein. [Figure 9b-5] 1 is a table summarizing six CDR, VH, and VL sequences of 26 different embodiments of affinity matured antibodies described herein. [Figure 9b-6] 1 is a table summarizing six CDR, VH, and VL sequences of 26 different embodiments of affinity matured antibodies described herein. [Figure 9b-7] 1 is a table summarizing six CDR, VH, and VL sequences of 26 different embodiments of affinity matured antibodies described herein. [Figure 10-1] Figure 10 shows epitope mapping of affinity matured 51B12 W99H_L1 (also called H3_W99H_L1), Figure 10A shows binding at low concentration (5ng / ml) and Figure 10B shows binding at higher concentration (1ug / ml). [Figure 10-2] FIG. 10C shows a list of epitope candidates for 51B12_W99H_L1. [Figure 11] FIG. 11A shows binding of antibodies 51B12, 63A11, 6B11, 38F02, 75E05, and 68G01 to CHO-K1 cells overexpressing ZIP12 (FIG. 11A), and internalization of antibodies 51B12 and 63A11 as measured by using direct conjugation (FIG. 11B). [Figure 12] FIG. 1 shows average data for binding of 51B12_W99H_L1 to human, cynomolgus monkey, dog, and pig ZIP12-ECD-GST. [Figure 13] FIG. 1 shows a set of histograms showing results from the Sugen model used to analyze the effect of dosing with 51B12_W99H_L1 on reversal of pulmonary arterial hypertension pathology. Bars represent the average activity from 6 animals. [Figure 14] FIG. 14A shows the number of remodeled and occluded vessels by treatment with 51B12_W99H_L1 (FIG. 14A), and representative images of remodeled and occluded vessels and their recovery in isotype control and 51B12_W99H_L1 treated groups (FIG. 14B). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0394] Working Example ZIP12 has been implicated in the pathogenesis of pulmonary hypertension, and increased expression of ZIP12 is associated with the condition. Therefore, the present inventors have attempted to develop an antibody capable of specifically binding to ZIP12 and inhibiting its function, in order to provide or improve the treatment and diagnosis of pulmonary hypertension. The present inventors have taken a ZIP12-binding antibody, called 51B12 parent, and subjected it to affinity maturation to produce a novel variant that exhibits improved properties compared to the parent.

[0395] material and method Cloning and expression Cloning of 51B12 affinity maturation variants The DNA expression construct encoding the antibody was newly prepared by building up overlapping 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 the VH domain containing the signal sequence for cloning into mammalian expression vectors containing modified Fc null human gamma 1 constant regions. HindIII and BsiWI restriction sites were introduced to incorporate the VL domain containing the signal sequence for cloning into mammalian expression vectors containing human kappa constant regions.

[0396] Expression of recombinant antibodies (including antibody quantification) Expression plasmids encoding the heavy and light chains, respectively, were transiently co-transfected into HEK293 6E cells and expressed on a small scale to produce antibodies. Antibodies were quantified by ELISA. ELISA plates were coated with anti-human IgG (Sigma I3382) at 1 mg / ml and blocked with blocking solution (4% BSA in Tris-buffered saline). Various dilutions of tissue culture supernatants were added and the plates were incubated for 1 hour at room temperature. Dilutions of known standard antibodies were also added to the plates. Plates were washed in TBST and binding was detected by the addition of peroxidise-labeled anti-human kappa light chain antibody (Sigma A7164) at a dilution of 1 / 1000 in blocking solution. Plates were incubated for 1 hour at room temperature before washing in TBST. Plates were developed by the addition of OPD substrate (Sigma P9187) and color development was stopped by the addition of 2M H2S04. Absorbance was measured at 490 nm and a standard curve was plotted using data for known standard dilutions. The standard curve was used to estimate the concentration of antibody in tissue culture supernatants. Larger scale antibody preparations were purified using Protein A and concentrations were measured using a Nanodrop (Thermo Scientific).

[0397] Assay ELISA experiments showing binding of ZIP12 antibodies to recombinant human ZIP12 protein hZIP12-ECD-GST (5 μg / ml) was coated onto ELISA plates, blocked, and incubated with a semi-log dilution series of antibodies. Antibody binding was detected with anti-human IgG-HRP and TMB. Protein coating was confirmed using incubation with anti-ZIP12 and anti-rabbit IgG-HRP and TMB staining.

[0398] HTRF experiments showing competition between parental and affinity matured antibodies in recombinant human ZIP12 protein The antibodies were analyzed using a homogeneous time resolved fluorescence (HTRF) assay. A checkerboard assay was performed to confirm the binding of terbium-labeled antibodies to hZIP12-ECD-GST. Serial dilutions of terbium-labeled 51B12 parent (starting from 1000 ng / ml, semi-log dilutions) were incubated with serial dilutions of hZIP12-ECD-GST (starting from 5000 ng / ml, semi-log dilutions) followed by incubation with anti-GST-XL665. After excitation (320 nm), emission signals were obtained and HTRF ratios were calculated (665 nm / 615 nm, panel A). Signal / background ratios (S / B) were calculated from the HTRF ratios (0 ng / ml hZIP12-ECD-GST as background signal) and plotted against the Tb-51B12 parent concentration. The combinations 500ng / ml hZIP12-ECD-GST+50ng Tb-Ab, 150ng / ml hZIP12-ECD-GST+50ng Tb-Ab, and 50ng / ml hZIP12-ECD-GST+50ng Tb-Ab were selected as ranges for the competitive assay. For the HTRF assay, 150ng / ml hZIP12-ECD-GST+30ng / ml Tb-51B12 was selected.

[0399] SPR Experiments – Single Cycle Kinetics Single cycle kinetics (SCK) involves first capturing mAb onto a Protein A sensor surface, then sequentially injecting a series of concentrations of huZIP12-ECD-GST over the surface, starting with the lowest concentration and ending with the highest. The final concentration is followed by a dissociation phase, in which only buffer is passed over the surface, followed by regeneration, in which remaining bound huZIP12-ECD-GST is removed from the surface by injection of a solution of 10 mM glycine pH 1.5 and 50 mM NaOH. SCK is repeated for each monoclonal antibody to provide duplicate data, and the kinetics of each interaction is evaluated by the Biacore software.

[0400] The range of concentrations of huZIP12-ECD-GST injected was selected based on the affinity of a control mAb established during previous studies. The data for each mAb was examined. Ideally, the concentration series of huZIP12-ECD-GST should reach approximately 80% saturation. Saturation was observed for some of the mAb samples at the highest concentration of huZIP12-ECD-GST tested. In those cases, the concentration series of huZIP12-ECD-GST was lowered to obtain optimal binding kinetics data.

[0401] The parental clone 51B12_VH_VL-par was screened as a positive control at the start of each cycle of the SCK series and was included twice in duplicate throughout the 17 hour run to provide a measure of sample stability during the 17 hour run.

[0402] Epitope mapping Peptide sequences corresponding to the sequence of the ZIP12 extracellular domain were directly synthesized with support in a hydrogel matrix. This results in highly sensitive arrays, allowing reliable detection of even the weakest binding signals. These immobilized peptide arrays are reusable and can be tested multiple times. This therefore allows the screening of a series of antibodies or sera on a single array, making linear epitope mapping a rapid and cost-effective option for best candidate selection, antibody characterization, antibody profiling, and further development. EXAMPLES

[0403] Example 1: Generation of affinity matured variants The inventors took a candidate parent antibody, called 51B12 parent (or 51B12 par), and subjected it to affinity maturation to produce novel variants or mutants that are expected to exhibit improved properties compared to the parent.

[0404] Eight or nine mutations per CDR residue in the three heavy chain and three light chain CDRs were designed as shown in FIG. 1 using the methodology described in Rajpal et al., 2005.

[0405] For the heavy chain variable region (VH) CDR variants, the variable regions were synthesized and cloned into an expression vector containing an Fc null human IgG1. For the light chain variable region (VL) CDR variants, the variable regions were synthesized and cloned into an expression vector containing the human kappa light chain constant region.

[0406] Figure 9a shows the respective amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3, VH, and VL for the 51B12 parent. Figure 9b shows a table summarizing the sequences of 26 different embodiments of affinity matured antibodies derived from the 51B12 parent. The first row in the table displays the corresponding sequence for the affinity matured variant of the 51B12 parent, H3_W99H_L1, in which the CDR-H3 sequence is the same as that of the parent except for the W99H amino acid replacement, and the CDR-L1 CDR is as shown. The second row in the table displays the corresponding sequence for the affinity matured variant of the 51B12 parent, H3_W99H_L2, in which the CDR-H3 sequence is the same as that of the parent except for the W99H amino acid replacement, and the CDR-L2 CDR is as shown, etc. EXAMPLES

[0407] Example 2: Screening for 51B12 variants / mutants Referring to Figures 2 and 3, the results of screening 51B12 parent VH-CDR (Figure 2) and VL-CDR (Figure 3) mutant production supernatants in HTRF and ELISA assays are shown. Diluted production supernatants were tested. For ELISA, antibody binding was detected with anti-human IgG-HRP and visualized with TMB. Reactivity data was plotted on the x-axis from left to right, sorted based on position in the CDR and alphabetically within the CDR residue position (n=8 or 9 mutants per CDR residue). The dotted line indicates the average HTRF signal of the 51B12 parent antibody.

[0408] Screening demonstrated that amino acids G33, Y50, S53, W99, T100, Y103, and D106 in the heavy chain CDRs appear to be particularly important for target epitope recognition, because several clones with substitutions of these amino acids in ELISA and HTRF assay formats resulted in loss of target recognition. Amino acids A24, V29, Y36, H94, S95, and P99 in the light chain CDRs also appear to be particularly important for target recognition, because several clones with substitutions of these amino acids in ELISA and HTRF assay formats also resulted in loss of target recognition.

[0409] As a result of extensive screening, 78 mutants were selected for dose-response screening, a decision based on experimental data from HTRF-based antibody concentration analysis and HTRF competition assays. EXAMPLES

[0410] Example 3: Screening of 51B12 variants / mutants in a dose-response HTRF assay FIG. 4 shows an example of an overlay of selected antibody clones showing their reactivity profiles in the HTRF inhibition assay.

[0411] Clones H3_W99H_VL-par and H2_Y60K_L2 showed stronger inhibition in HTRF compared to 51B12_VH_VL-par. Clones H3_M105P_L2 and H2_T63Y_VL-par showed slightly lower inhibition in HTRF compared to 51B12_VH_VL-par. Clone H3_W99H_VL-par showed improved reactivity profile in all three assays. Heavy and light chain sequences were then selected and combined to generate 27 affinity matured antibodies, which were then tested in SPR assays. EXAMPLES

[0412] Example 4: Screening of purified affinity matured antibodies in an SPR assay Referring to FIG. 5, the mean data for the control 51B12_VH_VL parent antibody is shown.

[0413] Referring to Figure 6, the kinetic parameters obtained for all antibody variants when screened with 3.12 nM - 800 nM huZIP12-ECD-GST are shown. The data shows that the on-rates (ka) of variants H3_W99H_L1, H3_W99H_L2 and H3_W99H_par are superior to the parent antibody, 51B12_par (shown in Figure 5; 2.75e+04). The on-rates of antibodies H2_T57H_L1, H2_Y60K_L1, H2_T63Y_L1, H2_K65P_L1, H2_G66Q_L1, H3_M105P_L1, 51B12_VH_L1, H2_T57H_L2, H2_Y60K_L2, H2_G66Q_L2, H2_T57H_VL_par, H2_Y60K_VL_par, H2, T63Y_VL_par, and H2_G66Q_VL_par were also superior to the parental antibodies. Affinity. All antibodies listed in Figure 6 showed excellent off-rate kinetics (kd). In particular, antibodies H3_W99H_L1, H3_W99H_L2, and H3_W99H_par showed excellent binding affinity, defined as KD(M).

[0414] As can be seen, all three antibody variants, H3_W99H_L1, H3_W99HL2, and H3_W99H_VL-par, appear to have a higher affinity (KD) of approximately 500 pM.

[0415] A selection of higher affinity antibody variants were retested at lower concentration series with extended dissociation times to obtain a better assessment of binding kinetics. Referring now to FIG. 7, data is shown for the monoclonal antibodies retested to optimize binding kinetics data. Again, all three antibody variants, H3_W99H_L1, H3_W99HL2, and H3_W99H_VL-par, appear to have higher affinity than the parent antibody. On-rates (ka), off-rates (kd), and overall affinity measurements (KD) are all improved compared to the 51B12 VH_VL_par parent antibody. Affinities of about 300 pM were obtained for the affinity matured variants, compared to 1.1 nM for the parent antibody (shown in the KD column in FIG. 7). EXAMPLES

[0416] Example 5: Epitope Mapping The inventors then set out to determine the exact epitope on the extracellular sequence of the ZIP12 protein to which the antibody variants bind.

[0417] The plot shown in Figure 8 compares the binding profile of the sample (51B12 variant antibody, the intensity of the interaction is shown by the blue bar) with the isotype control sample (human IgG1 Fc null, the intensity of the interaction is shown by the red bar) under the same conditions (in PBST buffer). Linear (LIN10 and LIN15) and looped (LOOP10, LOOP15) overlapping peptides (as well as HELix 19 and BETa sheet 22) were generated based on the human ZIP12 extracellular domain sequence and immobilized on a hydrogel matrix. The peptides are defined as follows: LIN10 = a 10-mer linear peptide starting at position 1 of the ZIP12 extracellular domain. The position on the y-axis in Figure 8 is defined as the last residue number of that peptide. For example, peptide 1 covers amino acids 1-10, therefore the last residue number is 10, etc.

[0418] LIN15 = a 15-mer linear peptide starting at position 1 of the ZIP12 extracellular domain. The position on the y-axis in Figure 8 is defined as the last residue number of that peptide. For example, peptide 1 covers amino acids 1-15, therefore the last residue number is 15, etc.

[0419] LOOP10 = a 10-mer peptide starting at position 1 of the ZIP12 extracellular domain. The position on the y-axis in Figure 8 is defined as the last residue number of the peptide. For example, peptide 1 covers amino acids 1-10, and therefore the last residue number is 10, etc. The LOOP peptide differs from the LIN peptide because it is structurally locked into a defined single loop structure using CLIPS scaffold technology (Pepscan).

[0420] LOOP15 = a 15-mer peptide starting at position 1 of the ZIP12 extracellular domain. The position on the y-axis in Figure 8 is defined as the last residue number of the peptide. For example, peptide 1 covers amino acids 1-15, therefore the last residue number is 15, etc. The LOOP peptide differs from the LIN peptide because it is structurally locked into a defined single loop structure using CLIPS scaffold technology (Pepscan).

[0421] HEL19 = a 19-mer peptide starting at position 1 of the ZIP12 extracellular domain. The position on the y-axis in Figure 8 is defined as the last residue number of the peptide. For example, peptide 1 covers amino acids 1-19, therefore the last residue number is 19, etc. The HEL peptide differs from the LIN peptide because it is structurally locked into a defined alpha-helical structure using CLIPS scaffold technology (Pepscan).

[0422] BET22 = a 19-mer peptide starting at position 1 of the ZIP12 extracellular domain. The position on the y-axis in Figure 8 is defined as the last residue number of that peptide. For example, peptide 1 covers amino acids 1-22, and therefore the last residue number is 22, etc. The HEL peptide differs from the LIN peptide because it is structurally locked into a defined beta-sheet structure using CLIPS scaffold technology (Pepscan).

[0423] By using both linear and structurally defined peptides, both linear and conformational epitopes were explored. Antibody binding to each peptide was measured and plotted as in Figure 8. For sample 51B12 antibody, strong binding peaks were observed between amino acids 46-63 in linear peptides (LIN10 and LIN15) as well as in conformational peptide (LOOP10, LOOP15, HEL19, BET22) mimics and were absent in isotypes. From the data obtained in Figure 8, it can be determined that the epitopes of the 51B12 antibody variants are at least partially linear. The peptides showing intensity above that of the isotype control in Figure 8 are candidate epitopes for the 51B12 antibody. This antibody is functionally active in the Zn FLIPR assay, which indicates that it binds Zn 2+ It has been demonstrated that it inhibits transport.

[0424] The sequence of the epitope is: ADLLQVLSAGDHPPHNHSRS [SEQ ID NO: 252] More preferably, the sequence of the epitope is: LLQVLSAGDHPPHNHSRS [SEQ ID NO: 1] EXAMPLES

[0425] Example 6: Epitope mapping of affinity matured antibodies To reconstruct the epitopes of the target molecules, a library of peptide-based mimetics was synthesized using Fmoc-based solid-phase peptide synthesis. An amino-functionalized polypropylene support was grafted with a proprietary hydrophilic polymer formulation, followed by reaction with t-butyloxycarbonyl-hexamethylenediamine (BocHMDA) using dicyclohexylcarbodiimide (DCC) together with N-hydroxybenzotriazole (HOBt), followed by cleavage of the Boc group using trifluoroacetic acid (TFA). Peptides were synthesized on the amino-functionalized solid phase using standard Fmoc-peptide synthesis on a custom-modified JANUS liquid handling station (Perkin Elmer).

[0426] A different set of peptides was designed as follows:

[0427] [Table 1]

[0428] The binding of the antibodies to each of the synthetic peptides was tested in a peptide-based ELISA. The peptide array was incubated with the primary antibody solution (overnight at 4°C). After washing, the peptide array was incubated with a 1 / 1000 dilution of the peroxidase conjugate of the appropriate antibody at 25°C for 1 hour. After washing, the peroxidase substrate 2,2'-azino-di-3-ethylbenzthiazoline sulfonic acid (ABTS) and 20 μl / ml of 3 percent H2O2 were added. After 1 hour, color development was measured. Color development was quantified with a charge-coupled device camera and image processing system.

[0429] Sample 907672.1 (51B12 W99H_L1, also called H3_W99H_L1) was screened under various conditions. Consistently specific binders were observed in the various screens in the amino acid region 35-48. The samples showed strong binding with this particular region of the peptide even at very low concentrations such as 5 ng / ml (Figure 10A). As shown in Figure 10A, the high-strength binders identified in the region 35-48 did not show clear peaks in the LIN10 and LOOP10 peptidomimetics, each of which contains a peptide consisting of 10 residues. By linear and conformational mapping, several additional binding peaks were observed at higher concentrations that were distinct from the isotype control (Figure 10B). These peaks are determined to be minor epitope candidates since they only appeared at very higher concentrations. These binders could be non-specific.

[0430] To identify sample-specific binders, isotype controls were screened under similar conditions. Binding peaks distinct from the isotype controls were selected based on the criterion that the sample peptide intensity was at least four times that of the control peptide intensity. As shown in Figure 10A, the high-intensity binders identified in region 35-48 did not show distinct peaks in the LIN10 and LOOP10 peptidomimetics, each of which contains a 10-residue peptide.

[0431] FIG. 10C shows a list of epitope candidates for 51B12_W99H_L1. EXAMPLES

[0432] Example 7: 51B12_W99H_L1 is internalized in cells overexpressing ZIP12 full-length protein Antibody internalization was measured using direct conjugation flow cytometry over a time course. The following antibodies were selected to test for internalization: 51B12, 63A11, 6B11, 38F02, 75E05, and 68G01.

[0433] Figure 11A shows the binding of antibodies 51B12, 63A11, 6B11, 38F02, 75E05, and 68G01 to CHO-K1 cells overexpressing ZIP12. Non-inhibitors 75E05 and 68G01 did not bind to CHO-K1 cells as expected. These antibodies were not tested further. Figure 11B shows the internalization of antibodies 51B12 and 63A11 as measured by using direct conjugation. Only a slight difference in the internalization rate could be observed between 51B12 and 63A11. No internalization could be observed for 6B11 or 38F02 using direct conjugation. EXAMPLES

[0434] Example 8: 51B12_W99H_L1 binds to human and cynomolgus ZIP12-GST-ECD but does not cross-react with dog or pig proteins Surface plasmon resonance (SPR) is a label-free biophysical technique that allows for the direct measurement of biomolecular interactions. A single cycle kinetics (SCK) SPR direct binding assay was developed to characterize antibody binding to human, cynomolgus monkey, porcine, and canine ZIP12 fusion proteins. This assay was used to characterize the binding kinetics (defined by kon, koff, KD) of monoclonal antibody 51B12_W99H_L1 to each of the four proteins. Single cycle kinetics (SCK) involves first capturing mAb onto a Protein A sensor surface, and then sequentially injecting a series of concentrations of ZIP12-ECD-GST over the surface, starting with the lowest concentration and ending with the highest concentration. The final concentration is followed by a dissociation phase in which only buffer is passed over the surface, followed by regeneration in which remaining bound huZIP12-ECD-GST is removed from the surface by injection of a solution of 10 mM glycine pH 1.5 and 50 mM NaOH. SCK is repeated for each monoclonal antibody to provide duplicate data and the kinetics of each interaction are evaluated by the Biacore software.

[0435] Figure 12 shows the average data for binding of 51B12_W99H_L1 to human, cynomolgus monkey, dog, and pig ZIP12-ECD-GST. 51B12_W99H_L1 bound to the human and cynomolgus monkey proteins with similar ka, kd, and KD (4.02 x 10, respectively). -9 and 6.31×10 -9 ). EXAMPLES

[0436] Example 9: 51B12_W99H_L1 ameliorates pulmonary arterial hypertension (PAH) in the Sugen model of pulmonary remodeling FIG. 13 shows a set of histograms showing results from the Sugen model used to analyze the effect of dosing with 51B12_W99H_L1 on reversal of pulmonary arterial hypertension pathology. Humanized ZIP12 Sprague-Dawley rats were treated with 20 mg / kg Sugen5416 and placed in a hypoxic chamber with an oxygen level of 10%. The rats were left in the chamber for 24 days and then returned to room air for an additional 8 days. After this time point, PAH pathology is expected to have developed. The rats were then randomized into two groups and treated with either 10 mg / kg human IgG1 isotype control antibody (control) or 10 mg / kg 51B12_W99H_L1. To quantify the reversal of PAH pathology, multiple hemodynamic measurements were taken. Bars represent the average activity from 6 animals.

[0437] Figure 13 shows that humanized rats treated with 51B12_W99H_L1 have reduced mean pulmonary artery pressure (mPAP) compared to isotype control.Right ventricular hypertrophy was also reduced in rats treated with 51B12_W99H_L1 compared to isotype control.

[0438] Figure 14A shows that the number of remodeled and obstructed vessels is reduced with treatment with 51B12_W99H_L1. Remodeled vessels are defined as pulmonary vasculature vessels that exhibit thickening of the vessel wall, and obstructed vessels are defined as vessels that are closed.

[0439] Figure 14B shows representative examples of remodeled and occluded vessels and their recovery in the isotype control and 51B12_W99H_L1 treated groups. Remodeled vessels are indicated by black arrows, and occluded vessels are indicated by white arrows.

[0440] conclusion In summary, we took a ZIP12-binding antibody, called 51B12 parent, and subjected it to affinity maturation to produce a series of 26 novel variants. Using epitope mapping, as shown in Figure 8, we have precisely determined the epitope on the extracellular domain of ZIP12 to which each of these antibodies bind. Furthermore, the variant antibodies have surprisingly high binding affinity to this epitope, with improved on-rates (ka), off-rates (kd), and overall affinity (KD). Furthermore, the variants are functionally active in the Zn FLIPR assay, indicating that they inhibit zinc ion transport. As a result, these antibodies would be strong therapeutic candidates for use in treating, preventing, or ameliorating hypoxia-related conditions, such as pulmonary hypertension or cancer.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to the extracellular domain of ZIP12 and recognizes an epitope comprising or consisting of ADLLQVLSAGDHPPHNHSRS (SEQ ID NO: 252) or a variant or fragment thereof.

2. The antibody or antigen-binding fragment thereof of claim 1, which recognizes (i) an epitope comprising or consisting of LLQVLSAGDHPPHNHSRS (SEQ ID NO: 1) or a variant or fragment thereof; or (ii) an epitope comprising or consisting of ADLLQ (SEQ ID NO: 253) or a variant or fragment thereof.

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

4. The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, comprising: (i) a CDR-H1 domain comprising or consisting of SEQ ID NO: 13, a CDR-H2 domain comprising or consisting of SEQ ID NO: 14, a CDR-H3 domain comprising or consisting of SEQ ID NO: 15, a CDR-L1 domain comprising or consisting of SEQ ID NO: 16, a CDR-L2 domain comprising or consisting of SEQ ID NO: 17, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 18; (ii) a CDR-H1 domain comprising or consisting of SEQ ID NO:21, a CDR-H2 domain comprising or consisting of SEQ ID NO:22, a CDR-H3 domain comprising or consisting of SEQ ID NO:23, a CDR-L1 domain comprising or consisting of SEQ ID NO:24, a CDR-L2 domain comprising or consisting of SEQ ID NO:25, and a CDR-L3 domain comprising or consisting of SEQ ID NO:26; (iii) a CDR-H1 domain comprising or consisting of SEQ ID NO:29, a CDR-H2 domain comprising or consisting of SEQ ID NO:30, a CDR-H3 domain comprising or consisting of SEQ ID NO:31, a CDR-L1 domain comprising or consisting of SEQ ID NO:32, a CDR-L2 domain comprising or consisting of SEQ ID NO:33, and a CDR-L3 domain comprising or consisting of SEQ ID NO:34; (iv) a CDR-H1 domain comprising or consisting of SEQ ID NO:37, a CDR-H2 domain comprising or consisting of SEQ ID NO:38, a CDR-H3 domain comprising or consisting of SEQ ID NO:39, a CDR-L1 domain comprising or consisting of SEQ ID NO:40, a CDR-L2 domain comprising or consisting of SEQ ID NO:41, and a CDR-L3 domain comprising or consisting of SEQ ID NO:42; (v) a CDR-H1 domain comprising or consisting of SEQ ID NO:45, a CDR-H2 domain comprising or consisting of SEQ ID NO:46, a CDR-H3 domain comprising or consisting of SEQ ID NO:47, a CDR-L1 domain comprising or consisting of SEQ ID NO:48, a CDR-L2 domain comprising or consisting of SEQ ID NO:49, and a CDR-L3 domain comprising or consisting of SEQ ID NO:50; (vi) a CDR-H1 domain comprising or consisting of SEQ ID NO:53, a CDR-H2 domain comprising or consisting of SEQ ID NO:54, a CDR-H3 domain comprising or consisting of SEQ ID NO:55, a CDR-L1 domain comprising or consisting of SEQ ID NO:56, a CDR-L2 domain comprising or consisting of SEQ ID NO:57, and a CDR-L3 domain comprising or consisting of SEQ ID NO:58; (vii) a CDR-H1 domain comprising or consisting of SEQ ID NO:61, a CDR-H2 domain comprising or consisting of SEQ ID NO:62, a CDR-H3 domain comprising or consisting of SEQ ID NO:63, a CDR-L1 domain comprising or consisting of SEQ ID NO:64, a CDR-L2 domain comprising or consisting of SEQ ID NO:65, and a CDR-L3 domain comprising or consisting of SEQ ID NO:66; (viii) a CDR-H1 domain comprising or consisting of SEQ ID NO:69, a CDR-H2 domain comprising or consisting of SEQ ID NO:70, a CDR-H3 domain comprising or consisting of SEQ ID NO:71, a CDR-L1 domain comprising or consisting of SEQ ID NO:72, a CDR-L2 domain comprising or consisting of SEQ ID NO:73, and a CDR-L3 domain comprising or consisting of SEQ ID NO:74; (ix) a CDR-H1 domain comprising or consisting of SEQ ID NO:77, a CDR-H2 domain comprising or consisting of SEQ ID NO:78, a CDR-H3 domain comprising or consisting of SEQ ID NO:79, a CDR-L1 domain comprising or consisting of SEQ ID NO:80, a CDR-L2 domain comprising or consisting of SEQ ID NO:81, and a CDR-L3 domain comprising or consisting of SEQ ID NO:82; (x) a CDR-H1 domain comprising or consisting of SEQ ID NO:85, a CDR-H2 domain comprising or consisting of SEQ ID NO:86, a CDR-H3 domain comprising or consisting of SEQ ID NO:87, a CDR-L1 domain comprising or consisting of SEQ ID NO:88, a CDR-L2 domain comprising or consisting of SEQ ID NO:89, and a CDR-L3 domain comprising or consisting of SEQ ID NO:90; (xi) a CDR-H1 domain comprising or consisting of SEQ ID NO:93, a CDR-H2 domain comprising or consisting of SEQ ID NO:94, a CDR-H3 domain comprising or consisting of SEQ ID NO:95, a CDR-L1 domain comprising or consisting of SEQ ID NO:96, a CDR-L2 domain comprising or consisting of SEQ ID NO:97, and a CDR-L3 domain comprising or consisting of SEQ ID NO:98; (xii) a CDR-H1 domain comprising or consisting of SEQ ID NO:101, a CDR-H2 domain comprising or consisting of SEQ ID NO:102, a CDR-H3 domain comprising or consisting of SEQ ID NO:103, a CDR-L1 domain comprising or consisting of SEQ ID NO:104, a CDR-L2 domain comprising or consisting of SEQ ID NO:105, and a CDR-L3 domain comprising or consisting of SEQ ID NO:106; (xiii) a CDR-H1 domain comprising or consisting of SEQ ID NO: 109, a CDR-H2 domain comprising or consisting of SEQ ID NO: 110, a CDR-H3 domain comprising or consisting of SEQ ID NO: 111, a CDR-L1 domain comprising or consisting of SEQ ID NO: 112, a CDR-L2 domain comprising or consisting of SEQ ID NO: 113, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 114; (xiv) a CDR-H1 domain comprising or consisting of SEQ ID NO: 117, a CDR-H2 domain comprising or consisting of SEQ ID NO: 118, a CDR-H3 domain comprising or consisting of SEQ ID NO: 119, a CDR-L1 domain comprising or consisting of SEQ ID NO: 120, a CDR-L2 domain comprising or consisting of SEQ ID NO: 121, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 122; (xv) a CDR-H1 domain comprising or consisting of SEQ ID NO: 125, a CDR-H2 domain comprising or consisting of SEQ ID NO: 126, a CDR-H3 domain comprising or consisting of SEQ ID NO: 127, a CDR-L1 domain comprising or consisting of SEQ ID NO: 128, a CDR-L2 domain comprising or consisting of SEQ ID NO: 129, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 130; (xvi) a CDR-H1 domain comprising or consisting of SEQ ID NO: 133, a CDR-H2 domain comprising or consisting of SEQ ID NO: 134, a CDR-H3 domain comprising or consisting of SEQ ID NO: 135, a CDR-L1 domain comprising or consisting of SEQ ID NO: 136, a CDR-L2 domain comprising or consisting of SEQ ID NO: 137, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 138; (xvii) a CDR-H1 domain comprising or consisting of SEQ ID NO: 141, a CDR-H2 domain comprising or consisting of SEQ ID NO: 142, a CDR-H3 domain comprising or consisting of SEQ ID NO: 143, a CDR-L1 domain comprising or consisting of SEQ ID NO: 144, a CDR-L2 domain comprising or consisting of SEQ ID NO: 145, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 146; (xviii) a CDR-H1 domain comprising or consisting of SEQ ID NO: 149, a CDR-H2 domain comprising or consisting of SEQ ID NO: 150, a CDR-H3 domain comprising or consisting of SEQ ID NO: 151, a CDR-L1 domain comprising or consisting of SEQ ID NO: 152, a CDR-L2 domain comprising or consisting of SEQ ID NO: 153, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 154; (xix) a CDR-H1 domain comprising or consisting of SEQ ID NO: 157, a CDR-H2 domain comprising or consisting of SEQ ID NO: 158, a CDR-H3 domain comprising or consisting of SEQ ID NO: 159, a CDR-L1 domain comprising or consisting of SEQ ID NO: 160, a CDR-L2 domain comprising or consisting of SEQ ID NO: 161, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 162; (xx) a CDR-H1 domain comprising or consisting of SEQ ID NO: 165, a CDR-H2 domain comprising or consisting of SEQ ID NO: 166, a CDR-H3 domain comprising or consisting of SEQ ID NO: 167, a CDR-L1 domain comprising or consisting of SEQ ID NO: 168, a CDR-L2 domain comprising or consisting of SEQ ID NO: 169, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 170; (xxi) a CDR-H1 domain comprising or consisting of SEQ ID NO: 173, a CDR-H2 domain comprising or consisting of SEQ ID NO: 174, a CDR-H3 domain comprising or consisting of SEQ ID NO: 175, a CDR-L1 domain comprising or consisting of SEQ ID NO: 176, a CDR-L2 domain comprising or consisting of SEQ ID NO: 177, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 178; (xxii) a CDR-H1 domain comprising or consisting of SEQ ID NO: 181, a CDR-H2 domain comprising or consisting of SEQ ID NO: 182, a CDR-H3 domain comprising or consisting of SEQ ID NO: 183, a CDR-L1 domain comprising or consisting of SEQ ID NO: 184, a CDR-L2 domain comprising or consisting of SEQ ID NO: 185, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 186; (xxiii) a CDR-H1 domain comprising or consisting of SEQ ID NO: 189, a CDR-H2 domain comprising or consisting of SEQ ID NO: 190, a CDR-H3 domain comprising or consisting of SEQ ID NO: 191, a CDR-L1 domain comprising or consisting of SEQ ID NO: 192, a CDR-L2 domain comprising or consisting of SEQ ID NO: 193, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 194; (xxiv) a CDR-H1 domain comprising or consisting of SEQ ID NO: 197, a CDR-H2 domain comprising or consisting of SEQ ID NO: 198, a CDR-H3 domain comprising or consisting of SEQ ID NO: 199, a CDR-L1 domain comprising or consisting of SEQ ID NO: 200, a CDR-L2 domain comprising or consisting of SEQ ID NO: 201, and a CDR-L3 domain comprising or consisting of SEQ ID NO: 202; (xxv) a CDR-H1 domain comprising or consisting of SEQ ID NO:205, a CDR-H2 domain comprising or consisting of SEQ ID NO:206, a CDR-H3 domain comprising or consisting of SEQ ID NO:207, a CDR-L1 domain comprising or consisting of SEQ ID NO:208, a CDR-L2 domain comprising or consisting of SEQ ID NO:209, and a CDR-L3 domain comprising or consisting of SEQ ID NO:210; or (xxvi) a CDR-H1 domain comprising or consisting of SEQ ID NO:213, a CDR-H2 domain comprising or consisting of SEQ ID NO:214, a CDR-H3 domain comprising or consisting of SEQ ID NO:215, a CDR-L1 domain comprising or consisting of SEQ ID NO:216, a CDR-L2 domain comprising or consisting of SEQ ID NO:217, and a CDR-L3 domain comprising or consisting of SEQ ID NO:

218.

5. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, comprising: (i) a heavy chain variable region comprising, or consisting of, SEQ ID NO: 19, and a light chain variable region comprising, or consisting of, SEQ ID NO: 20; (ii) a heavy chain variable region comprising, or consisting of, SEQ ID NO:27, and a light chain variable region comprising, or consisting of, SEQ ID NO:28; (iii) a heavy chain variable region comprising, or consisting of, SEQ ID NO: 35, and a light chain variable region comprising, or consisting of, SEQ ID NO: 36; (iv) 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; (v) a heavy chain variable region comprising, or consisting of, SEQ ID NO:51, and a light chain variable region comprising, or consisting of, SEQ ID NO:52; (vi) a heavy chain variable region comprising, or consisting of, SEQ ID NO:59, and a light chain variable region comprising, or consisting of, SEQ ID NO:60; (vii) a heavy chain variable region comprising, or consisting of, SEQ ID NO:67, and a light chain variable region comprising, or consisting of SEQ ID NO:68; (viii) a heavy chain variable region comprising, or consisting of, SEQ ID NO: 75, and a light chain variable region comprising, or consisting of, SEQ ID NO: 76; (ix) a heavy chain variable region comprising, or consisting of, SEQ ID NO: 83, and a light chain variable region comprising, or consisting of, SEQ ID NO: 84; (x) a heavy chain variable region comprising, or consisting of, SEQ ID NO: 91, and a light chain variable region comprising, or consisting of, SEQ ID NO: 92; (xi) a heavy chain variable region comprising, or consisting of, SEQ ID NO: 99, and a light chain variable region comprising, or consisting of, SEQ ID NO: 100; (xii) a heavy chain variable region comprising, or consisting of, SEQ ID NO: 107, and a light chain variable region comprising, or consisting of, SEQ ID NO: 108; (xiii) a heavy chain variable region comprising, or consisting of, SEQ ID NO: 115, and a light chain variable region comprising, or consisting of, SEQ ID NO: 116; (xiv) a heavy chain variable region comprising, or consisting of, SEQ ID NO: 123, and a light chain variable region comprising, or consisting of, SEQ ID NO: 124; (xv) a heavy chain variable region comprising, or consisting of, SEQ ID NO: 131, and a light chain variable region comprising, or consisting of, SEQ ID NO: 132; (xvi) a heavy chain variable region comprising, or consisting of, SEQ ID NO: 139, and a light chain variable region comprising, or consisting of, SEQ ID NO: 140; (xvii) a heavy chain variable region comprising, or consisting of, SEQ ID NO: 147, and a light chain variable region comprising, or consisting of, SEQ ID NO: 148; (xviii) a heavy chain variable region comprising, or consisting of, SEQ ID NO: 155, and a light chain variable region comprising, or consisting of, SEQ ID NO: 156; (xix) a heavy chain variable region comprising, or consisting of, SEQ ID NO: 163, and a light chain variable region comprising, or consisting of, SEQ ID NO: 164; (xx) a heavy chain variable region comprising, or consisting of, SEQ ID NO: 171, and a light chain variable region comprising, or consisting of, SEQ ID NO: 172; (xxi) a heavy chain variable region comprising, or consisting of, SEQ ID NO: 179, and a light chain variable region comprising, or consisting of, SEQ ID NO: 180; (xxii) a heavy chain variable region comprising, or consisting of, SEQ ID NO: 187, and a light chain variable region comprising, or consisting of, SEQ ID NO: 188; (xxiii) a heavy chain variable region comprising, or consisting of, SEQ ID NO: 195, and a light chain variable region comprising, or consisting of, SEQ ID NO: 196; (xxiv) a heavy chain variable region comprising, or consisting of, SEQ ID NO: 203, and a light chain variable region comprising, or consisting of, SEQ ID NO: 204; (xxv) a heavy chain variable region comprising, or consisting of, SEQ ID NO:211, and a light chain variable region comprising, or consisting of, SEQ ID NO:212; or (xxvi) a heavy chain variable region comprising, or consisting of, SEQ ID NO:219, and a light chain variable region comprising, or consisting of, SEQ ID NO:

220.

6. An antibody or antigen-binding fragment thereof that specifically binds ZIP12 and comprises the six CDR sequences shown for each antibody in Figure 9b.

7. 7. An antibody or antigen-binding fragment thereof according to claim 6, comprising the VH / VL sequence pair shown for each antibody shown in Figure 9b.

8. The antibody or antigen-binding fragment thereof according to claim 6 or 7, which specifically binds to the extracellular domain of ZIP12 and recognizes an epitope comprising or consisting of ADLLQVLSAGDHPPHNHSRS (sequence number 252).

9. The antibody or antigen-binding fragment thereof according to claim 8, which specifically binds to the extracellular domain of ZIP12 and recognizes an epitope comprising or consisting of LLQVLSAGDHPPHNHSRS (SEQ ID NO: 1) or a variant or fragment thereof.

10. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 for use in therapy.

11. The antibody or antigen-binding fragment thereof of any one of claims 1 to 9 for use in treating, preventing or ameliorating a hypoxia-related condition.

12. The antibody or antigen-binding fragment thereof for use according to claim 11, wherein the hypoxia-associated condition is pulmonary hypertension or cancer.

13. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, and optionally a pharma- ceutically acceptable vehicle.

14. A method for making the pharmaceutical composition of claim 13, comprising combining a therapeutically effective amount of an antibody or antigen-binding fragment thereof as defined in any one of claims 1 to 9 with a pharma- ceutically acceptable vehicle.

15. An antibody or antigen-binding fragment thereof obtained by a method comprising the steps of: (i) immunizing a host organism with a ZIP12 extracellular epitope comprising or consisting of SEQ ID NO:252 or SEQ ID NO:1, or a variant or fragment thereof; and (ii) harvesting the antibody or antigen-binding fragment thereof from the host.

16. A polynucleotide sequence encoding an antibody or an antigen-binding fragment thereof as defined in any one of claims 1 to 9.

17. 17. An expression cassette comprising the polynucleotide sequence of claim 16.

18. A recombinant vector comprising the expression cassette of claim 17.

19. 19. A host cell comprising the polynucleotide sequence of claim 16, the expression cassette of claim 17, or the vector of claim 18.

20. A method for preparing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, comprising the steps of: a) introducing the vector of claim 18 into a host cell; and b) culturing said host cell under conditions that may result in the production of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

21. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 for use in diagnosis or prognosis.

22. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 for use in diagnosing or prognosing a hypoxia-related condition.

23. A method for diagnosing or prognosing a hypoxia-related condition in a subject, comprising detecting ZIP12 in a biological sample obtained from the subject using an antibody or antigen-binding fragment thereof described in any one of claims 1 to 9.

24. A kit for diagnosing a subject suffering from a hypoxia-related condition or for providing a prognosis of the condition in said subject, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 for detecting ZIP12 in a sample from a test subject.

25. 25. The use according to claim 21, the method according to claim 23 or the kit according to claim 24, wherein the hypoxia-related condition is pulmonary hypertension or cancer.

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