Zip12 antibody

Abstract

The invention relates to ZIP12 antibodies, and their use in the treatment of hypoxia- related diseases in humans, in particular, pulmonary hypertension.

Description

[0001] ZIP12 Antibody

[0002] The invention relates to ZIP12 antibodies, and their use in the treatment of hypoxia- related diseases in humans, in particular, pulmonary hypertension.

[0003] Pulmonary hypertension (PH) is a rare, progressive disease of the pulmonary vasculature, characterised by increased pulmonary vascular resistance that can ultimately lead to right heart failure and death. Patients typically present with nonspecific symptoms such as shortness of breath, fatigue, angina, and syncope. Newly diagnosed cases of pulmonary hypertension are estimated to occur in 2.4 to 6 individuals per million each year, and the current prevalence in adults is 15 to 55 per million.

[0004] The remodelling of the pulmonary vascular that occurs in pulmonary hypertension is the pathogenic driver of disease, resulting in compromised vascular compliance leading to increased pulmonary vascular resistance and subsequent right ventricular hypertrophy. ZIP12 (SLC39A12) is a non-voltage gated zinc ion (Zn2+) transporter belonging to the solute carrier gene family. In response to hypoxia, ZIP12 is overexpressed in pulmonary artery smooth muscle cells in the remodelled arterioles of rats, cows, and humans.

[0005] Silencing of the SLC39A12 gene or inhibition of protein production as well as pharmacological inhibition of ZIP12 protein function have all demonstrated a therapeutic potential using in vivo models of pulmonary hypertension. Based on this evidence, the inventors developed a function-blocking monoclonal antibody (mAb) that inhibits the intracellular influx of Zn2+ through binding to the extracellular domain of the ZIP12 protein (WO 2022 / 064216 Al). The inventors have demonstrated that inhibition of ZIP12 function by the anti-ZIP2 antibody results in therapeutic effects on hemodynamic and histological endpoints in both hypoxia-driven and Sugen / hypoxia- driven in vivo models. The inventors then subjected the anti-ZIP12 antibody to affinity maturation, producing novel variants that have surprisingly high binding affinities to the ZIP12 epitope (WO 2023 / 180743 Al).

[0006] Based on these data, the inventors now propose inhibition of ZIP12 by the ZIP12 monoclonal antibody as a novel clinical therapeutic strategy to treat pulmonary hypertension in humans. In particular, the inventors are proposing treatment of pulmonary hypertension with a subcutaneous formulation of the ZIP12 antibody, that has a surprisingly long half-life. Accordingly, in a first aspect of the invention, there is provided an antibody or antigen-binding fragment thereof, comprising:

[0007] (i) a CDR-H1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 1, or a variant or fragment thereof;

[0008] (ii) a CDR-H2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 2, or a variant or fragment thereof;

[0009] (iii) a CDR-H3 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 3, or a variant or fragment thereof;

[0010] (iv) a CDR-L1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 4, or a variant or fragment thereof;

[0011] (v) a CDR-L2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 5, or a variant or fragment thereof; and

[0012] (vi) a CDR-L3 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 6, or a variant or fragment thereof, for use in treating, preventing or ameliorating pulmonary hypertension in a human.

[0013] In a second aspect of the invention, there is provided a method of treating, preventing or ameliorating pulmonary hypertension in a human, the method comprising administering, or having administered, to a human in need of such treatment, a therapeutically effective amount of an antibody or antigen-binding fragment thereof, comprising:

[0014] (i) a CDR-H1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 1, or a variant or fragment thereof;

[0015] (ii) a CDR-H2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 2, or a variant or fragment thereof;

[0016] (iii) a CDR-H3 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 3, or a variant or fragment thereof;

[0017] (iv) a CDR-L1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 4, or a variant or fragment thereof;

[0018] (v) a CDR-L2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 5, or a variant or fragment thereof; and

[0019] (vi) a CDR-L3 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 6, or a variant or fragment thereof.

[0020] As shown in the examples, the inventors have investigated the pharmacokinetics of different dosages of the anti-ZIP12 antibody (0.3 mg / kg, 1 mg / kg, 3 mg / kg, 6 mg / kg and 12 mg / kg), in adult humans, and have surprisingly discovered that these doses of the anti-ZIP12 antibody can achieve a long half-life when administered subcutaneously.

[0021] In one embodiment, ZIP12 may be represented by Genbank ID No: NP-001138667, which is provided herein as SEQ ID No: 11, as follows:

[0022] MCFRTKLSVSWVPLFLLLSRVFSTETDKPSAQDSRSRGSSGQPADLLQVLSAGDHPPHNHSRSLI KTLLEKTGCPRRRNGM QGDCNLCFEPDALLLIAGGNFEDQLREEWQRVSLLLLYYI IHQEEICSSKLNMSNKEYKFYLHSLLSLRQDEDS SFLSQN ETEDILAFTRQYFDTSQSQCMETKTLQKKSGIVS SEGANESTLPQLAAMI ITLSLQGVCLGQGNLPS PDYFTEYI FS SLNR TNTLRLSELDQLLNTLWTRSTCIKNEKIHQFQRKQNNI ITHDQDYSNFSS SMEKESEDGPVSWDQTCFSARQLVEI FLQKG LSLI SKEDFKQMSPGI IQQLLSCSCHLPKDQQAKLPPTTLEKYGYSTVAVTLLTLGSMLGTALVLFHSCEENYRLILQLFV GLAVGTLSGDALLHLI PQVLGLHKQEAPEFGHFHESKGHIWKLMGLIGGIHGFFLIEKCFI LLVS PNDKQGLSLVNGHVGH SHHLALNSELSDQAGRGKSASTIQLKSPEDSQAAEMPI GSMTASNRKCKAI SLLAIMILVGDSLHNFADGLAI GAAFSS SS ESGVTTTIAI LCHEI PHEMGDFAVLLSSGLSMKTAI LMNFI S SLTAFMGLYIGLSVSADPCVQDWI FTVTAGMFLYLSLVE MLPEMTHVQTQRPWMMFLLQNFGLILGWLSLLLLAI YEQNIKI

[0023] [SEQ ID No: 11]

[0024] The antibody or antigen-binding fragment thereof may bind to a region between amino acid positions 1 and 202 of SEQ ID No: 11, which corresponds to the extracellular domain of ZIP12.

[0025] Thus, in one embodiment the antibody or antigen-binding fragment thereof may bind to one or more amino acids between amino acid positions 1 and 202 of ZIP12, which is provided herein as SEQ ID No: 12, as follows:

[0026] MCFRTKLSVSWVPLFLLLSRVFSTETDKPSAQDSRSRGSSGQPADLLQVLSAGDHPPHNHSRSLI KTLLEKTGCPRRRNGM QGDCNLCFEPDALLLIAGGNFEDQLREEWQRVSLLLLYYI IHQEEICSSKLNMSNKEYKFYLHSLLSLRQDEDS SFLSQN ETEDILAFTRQYFDTSQSQCMETKTLQKKSGIVS SEGANE

[0027] [SEQ ID No: 12]

[0028] Thus, in one embodiment, the antibody or antigen-binding fragment thereof binds to an epitope within a sequence comprising or consisting of a sequence as substantially set out in SEQ ID No: 12, or a variant or fragment thereof.

[0029] In one embodiment the antibody or antigen-binding fragment thereof recognises an epitope comprising or consisting of ADLLQVLSAGDHPPHNHSRS (SEQ ID No: 13), or a variant or fragment thereof, or an epitope comprising or consisting of

[0030] LLQVLSAGDHPPHNHSRS (SEQ ID No: 14), or a variant or fragment thereof.

[0031] The epitope may be linear or conformational. The term "linear epitope" can mean an epitope comprising or consisting of amino acid residues that form a sequence together in the primary sequence of the protein antigen, i.e. sequential or continuous epitope.

[0032] The linear epitope may be present on folded ZIP12.

[0033] The term "conformational epitope" can mean an epitope consisting of amino acid residues, at least some of which are separated from others in the primary sequence of the protein antigen, but which together assemble in the 3D structure and are recognised by an antibody, i.e. discontinuous or non-sequential epitope.

[0034] It is important that the antibody or antigen-binding fragment thereof of the invention, which targets ZIP12, does so specifically, and has no or little cross- reactivity with ZIP13 and / or ZIP4, because this could result in significant unwanted off-target effects.

[0035] Accordingly, in one embodiment the antibody or antigen binding fragment thereof of the invention does not substantially bind to human ZIP13. In one embodiment, the antibody or antigen binding fragment thereof of the invention has no cross- reactivity with human ZIP13.

[0036] In addition, in one embodiment the antibody or antigen binding fragment thereof of the invention does not substantially bind to human ZIP4. In one embodiment, the antibody or antigen binding fragment thereof of the invention has no cross- reactivity with human ZIP4.

[0037] In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as H3_W99H_L1.

[0038] The antibody or antigen-binding fragment comprises a CDR-H1 domain of SEQ ID No: 1, which is provided herein, as follows:

[0039] DYGMH

[0040] [SEQ ID No: 1]

[0041] The antibody or antigen-binding fragment thereof comprises a CDR-H2 domain of SEQ ID No: 2, which is provided herein, as follows:

[0042] YI S S GGTT I YYADTVKG

[0043] [SEQ ID No: 2]

[0044] The antibody or antigen-binding fragment thereof comprises a CDR-H3 domain of SEQ ID No: 3, which is provided herein, as follows: HTNLYAMDY

[0045] [SEQ ID No: 3]

[0046] The antibody or antigen-binding fragment thereof may comprise a heavy chain variable (VH) sequence as set out in SEQ ID No: 7, which is provided herein, as follows:

[0047] QVQLVESGGGWQPGRSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAYISSGGTTIYYADTVKGRFTI SRD

[0048] NSKNTLYLQMNSLRAEDTAVYYCARHTNLYAMDYWGQGTTVTVSS

[0049] [SEQ ID No: 7]

[0050] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 7, or a variant or fragment thereof.

[0051] The antibody or antigen-binding fragment thereof may comprise a heavy chain sequence as set out in SEQ ID No: 9, which is provided herein, as follows:

[0052] QVQLVESGGGWQPGRSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAYISSGGTTIYYADTVKGRFTI SRD NSKNTLYLQMNSLRAEDTAVYYCARHTNLYAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSS SLGTQTYI CNVNHKPSNTKVDKKVEPK

[0053] SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTI SKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLS PGK

[0054] [SEQ ID No: 9]

[0055] Accordingly, in one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain region comprising or consisting of a sequence as substantially set out in SEQ ID No: 9, or a variant or fragment thereof.

[0056] The antibody or antigen-binding fragment thereof comprises a CDR-L1 domain of SEQ ID No: 4, which is provided herein, as follows:

[0057] RASKSVSTQGYSYMH

[0058] [SEQ ID No: 4]

[0059] The antibody or antigen-binding fragment thereof comprises a CDR-L2 domain of SEQ ID No: 5, which is provided herein, as follows:

[0060] LASNLES

[0061] [SEQ ID No: 5] The antibody or antigen-binding fragment thereof comprises a CDR-L3 domain of SEQ ID No: 6, which is provided herein, as follows:

[0062] QHSREVPYT

[0063] [SEQ ID No: 6]

[0064] The antibody or antigen-binding fragment thereof may comprise a light chain variable (VL) sequence as set out in SEQ ID No: 8, which is provided herein, as follows:

[0065] DIVLTQS PDSLAVSLGERATINCRASKSVSTQGYSYMHWYQQKPGQPPKFLIYLASNLESGVPDRFSGSGSGT DFTLTI S SLQAEDVAVYYCQHSREVPYTFGGGTKVEI KR

[0066] [SEQ ID No: 8]

[0067] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising or consisting of a sequence as substantially set out in SEQ ID No: 8, or a variant or fragment thereof.

[0068] The antibody or antigen-binding fragment thereof may comprise a light chain sequence as set out in SEQ ID No: 10, which is provided herein, as follows:

[0069] DIVLTQS PDSLAVSLGERATINCRASKSVSTQGYSYMHWYQQKPGQPPKFLIYLASNLESGVPDRFSGSGSGT DFTLTI S SLQAEDVAVYYCQHSREVPYTFGGGTKVEI KRTVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0070] [SEQ ID No: 10]

[0071] Accordingly, in one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain region comprising or consisting of a sequence as substantially set out in SEQ ID No: 10, or a variant or fragment thereof.

[0072] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 7 and a light chain variable region comprising or consisting of SEQ ID No: 8.

[0073] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain region comprising or consisting of a sequence as substantially set out in SEQ ID No: 9, or a variant or fragment thereof, and a light chain region comprising or consisting of a sequence as substantially set out in SEQ ID No: 10, or a variant or fragment thereof. In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof, consisting of a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein: the heavy chain variable region CDR1 amino acid sequence is SEQ ID NO: 1, or variant sequences thereof comprising 1, 2, 3, 4 or 5 amino acid changes, and conservative modifications thereof; the heavy chain variable region CDR2 amino acid sequence is SEQ ID NO: 2, or variant sequences thereof comprising 1, 2, 3, 4 or 5 amino acid changes, and conservative modifications thereof; the heavy chain variable region CDR3 amino acid sequence is SEQ ID NO: 3, or variant sequences thereof comprising 1, 2, 3, 4 or 5 amino acid changes, and conservative modifications thereof; the light chain variable regions CDR1 amino acid sequence is SEQ ID NO: 4, or variant sequences thereof comprising 1, 2, 3, 4 or 5 amino acid changes, and conservative modifications thereof; the light chain variable regions CDR2 amino acid sequence is SEQ ID NO: 5, or variant sequences thereof comprising 1 , 2, 3, 4 or 5 amino acid changes, and conservative modifications thereof; the light chain variable regions of CDR3 amino acid sequence is SEQ ID NO: 6, or variant sequences thereof comprising 1, 2, 3, 4 or 5 amino acid changes, and conservative modifications thereof.

[0074] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises an amino acid sequence that is at least 80%, or at least 90% (preferably at least 95, 97 or 99%) identical to SEQ ID NO: 7; the light chain variable region comprises an amino acid sequence that is at least 80%, or at least 90% (preferably at least 95, 97 or 99%) identical to SEQ ID NO: 8. Alternatively the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises no more than 5 amino acid, or no more than 4 amino acid, or no more than 3 amino acid, or no more than 2 or no more than 1 amino acid change compared to the amino acid sequence of SEQ ID NO: 7; the light chain variable region comprises no more than 5 amino acid, or no more than 4 amino acid, or no more than 3 amino acid, or no more than 2 or no more than 1 amino acid change compared to the amino acid sequence of SEQ ID NO: 8. In this context, the term "change" refers to insertions, deletions and / or substitutions.

[0075] The invention extends to both whole antibodies (i.e. immunoglobulins) with immunospecificity for ZIP12, as well as to antigen-binding fragments or regions of the corresponding full-length antibody. The antibody or antigen-binding fragment thereof may be monovalent, divalent or polyvalent. Monovalent antibodies are dimers (HL) comprising a heavy (H) chain associated by a disulphide bridge with a light chain (L). Divalent antibodies are tetramer (H2L2) comprising two dimers associated by at least one disulphide bridge. Polyvalent antibodies may also be produced, for example by linking multiple dimers. The basic structure of an antibody molecule consists of two identical light chains and two identical heavy chains which associate non-covalently and can be linked by disulphide bonds. Each heavy and light chain contains an amino-terminal variable region of about 110 amino acids, and constant sequences 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 variant or fragment thereof (e.g. an epitope). On either side of the CDRs of the heavy and light chains is a framework region, a relatively conserved sequence of amino acids that anchors and orients the CDRs. Antibody fragments may include a bispecific antibody (BsAb) or a chimeric antigen receptor (CAR).

[0076] The heavy chain constant region typically comprises three domains, CHI, CH2, and CHS. Each light chain typically comprises a light chain variable region (VL) and a light chain constant region. The light chain constant region typically comprises one domain, abbreviated CL.

[0077] Each heavy chain and light chain generally comprise three CDRs and four FRs, arranged in the following order (from 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.

[0078] The heavy chain 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 designated a, 6, e, y, and p, respectively. The IgG and IgA classes are further divided into subclasses on the basis of differences in sequence and function. Humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.

[0079] The light chain 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 (p, y, a, or e) and one of two light chain sequences (K or A). The heavy chain constant region sequences determine the isotype of the antibody and the effector functions of the molecule.

[0080] The antibody or antigen-binding fragment thereof may be isolated or purified.

[0081] In one embodiment, the antibody or antigen-binding fragment thereof comprises a polyclonal antibody, or an antigen-binding fragment thereof. The antibody or antigenbinding fragment thereof may be generated in a rabbit, mouse or rat.

[0082] In one embodiment, the antibody or antigen-binding fragment thereof is obtained by immunising a host animal with ZIP12, and then collecting the antibody or antigenbinding fragment thereof. The host animal may be a rabbit.

[0083] In another embodiment, the antibody or antigen-binding fragment thereof comprises a monoclonal antibody or an antigen-binding fragment thereof. In one embodiment, the antibody of the invention is a human antibody. As used herein, the term "human antibody" can mean an antibody, such as a monoclonal antibody, which comprises substantially the same heavy and light chain CDR amino acid sequences as found in a particular human antibody exhibiting immunospecificity for the extracellular portion of ZIP12, or a variant or fragment thereof. An amino acid sequence, which is substantially the same as a heavy or light chain CDR, exhibits a considerable amount of sequence identity when compared to a reference sequence. Such identity is definitively known or recognizable as representing the amino acid sequence of the particular human antibody. Substantially the same heavy and light chain CDR amino acid sequence can have, for example, minor modifications or conservative substitutions of amino acids. Such a human antibody maintains its function of selectively binding to ZIP12.

[0084] The term "human monoclonal antibody" can include a monoclonal antibody with substantially or entirely human CDR amino acid sequences produced, for example by recombinant methods such as production by a phage library, by lymphocytes or by hybridoma cells.

[0085] The term "monoclonal antibody" refers to an antibody from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies comprises antibodies that are substantially similar and that bind the same epitope(s), except for variants that may normally arise during production of the monoclonal antibody. Such variants are generally present in only minor amounts. A monoclonal antibody is 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 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.

[0086] The term "humanised antibody" can mean an antibody from a non-human species (e.g. mouse or rabbit) whose protein sequences have been modified to increase their similarity to antibodies produced naturally in humans.

[0087] The antibody may be a recombinant antibody. The term "recombinant human antibody" can include a human antibody produced using recombinant DNA technology.

[0088] The term "antigen-binding region" can mean a region of the antibody having specific binding affinity for its target antigen. The antigen-binding region may be a hypervariable CDR or a functional portion thereof. The term "functional portion" of a CDR can mean a sequence within the CDR which shows specific affinity for the target antigen. The functional portion of a CDR may comprise a ligand which specifically binds to ZIP12.

[0089] The term "CDR" can mean a hypervariable region in the heavy and light variable chains. There may be one, two, three or more CDRs in each of the heavy and light chains of the antibody. Normally, there are at least three CDRs on each chain which, when configured together, form the antigen-binding site, i.e. the three-dimensional combining site with which the antigen binds or specifically reacts. It has however been postulated that there may be four CDRs in the heavy chains of some antibodies.

[0090] The definition of CDR also includes overlapping or subsets of amino acid residues when compared against each other. The exact residue numbers which encompass a particular CDR or a functional portion thereof will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody. The amino acid sequence boundaries of a CDR can be determined by using any of a number of known numbering schemes, including those described by Kabat et al., supra ("Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927- 948 ("Chothia" numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 ("Contact" numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27: 55-77 ("IMGT" numbering scheme); and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 ("AHo" numbering scheme).

[0091] The term "functional fragment" of an antibody can mean a portion of the antibody which retains a functional activity. A functional activity can be, for example antigen binding activity or specificity. A functional activity can also be, for example, an effector function provided by an antibody constant region. The term "functional fragment" is also intended to include, for example, fragments produced by protease digestion or reduction of a human monoclonal antibody 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'; bivalent fragments such as F(ab')2; single chain Fv (scFv); and Fc fragments.

[0092] The term "VL fragment" can mean a fragment of the light chain of a human monoclonal antibody which includes all or part of the light chain variable region, including the CDRs. A VL fragment can further include light chain constant region sequences.

[0093] The term "VH fragment" can mean a fragment of the heavy chain of a human monoclonal antibody which includes all or part of the heavy chain variable region, including the CDRs.

[0094] The term "Fd fragment" can mean the heavy chain variable region coupled to the first heavy chain constant region, i.e. VH and CH-1. The "Fd fragment" does not include the light chain, or the second and third constant regions of the heavy chain.

[0095] The term "Fv fragment" can mean a monovalent antigen-binding fragment of a human monoclonal antibody, including all or part of the variable regions of the heavy and light chains, and absent of the constant regions of the heavy and light chains. The variable regions of the heavy and light chains include, for example, the CDRs. For example, an Fv fragment includes all or part of the amino terminal variable region of about 110 amino acids of both the heavy and light chains.

[0096] The term "Fab fragment" can mean a monovalent antigen-binding fragment of a human monoclonal antibody that is larger than an Fv fragment. For example, a Fab fragment includes the variable regions, and all or part of the first constant domain of the heavy and light chains. Thus, a Fab fragment additionally includes, for example, amino acid residues from about 110 to about 220 of the heavy and light chains.

[0097] The term "Fab' fragment" can mean a monovalent antigen-binding fragment of a human monoclonal antibody that is larger than a Fab fragment. For example, a Fab' fragment includes all of the light chain, all of the variable region of the heavy chain, and all or part of the first and second constant domains of the heavy chain. For example, a Fab' fragment can additionally include some or all of amino acid residues 220 to 330 of the heavy chain.

[0098] The term "F(ab')2 fragment" can mean a bivalent antigen-binding fragment of a human monoclonal antibody. An F(ab')2 fragment includes, for example, all or part of the variable regions of two heavy chains-and two light chains, and can further include all or part of the first constant domains of two heavy chains and two light chains.

[0099] The term "single chain Fv (scFv)" can mean a fusion of the variable regions of the heavy (VH) and light chains (VL) connected with a short linker peptide.

[0100] The term "bispecific antibody (BsAb)" can mean a bispecific antibody comprising two scFv linked to each other by a shorter linked peptide.

[0101] One skilled in the art knows that the exact boundaries of a fragment of an antibody are not important, so long as the fragment maintains a functional activity. Using well- known recombinant methods, one skilled in the art can engineer a polynucleotide sequence to express a functional fragment with any endpoints desired for a particular application. A functional fragment of the antibody may comprise or consist of a fragment with substantially the same heavy and light chain variable regions as the human antibody.

[0102] Typically, the antigen-binding fragment thereof, with respect to the first aspect of the invention, is immunospecific for ZIP12. The antigen-binding fragment thereof may comprise or consist of any of the fragments selected from a group consisting of VH, VL, Fd, Fv, Fab, Fab', scFv, F (ab')2 and Fc fragment. The antigen-binding fragment thereof may be a single domain antibody (sdAb), otherwise referred to as a nanobody, which the skilled person would understand is an antibody fragment consisting of a single monomeric variable antibody domain.

[0103] The antigen-binding fragment thereof may comprise or consist of any one of the antigen binding region sequences of the VL, any one of the antigen binding region sequences of the VH, or a combination of VL and VH antigen binding regions of a human antibody. The appropriate number and combination of VH and VL antigen binding region sequences may be determined by those skilled in the art depending on the desired affinity and specificity and the intended use of the antigen-binding fragment. Functional fragments or antigen-binding fragments of antibodies may be readily 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 comprise using human antibodies as a starting material. Enzymes suitable for proteolysis of human immunoglobulins may include, for example, papain, and pepsin. The appropriate enzyme may be readily chosen by one skilled in the art, depending on, for example, whether monovalent or bivalent fragments are required. For example, papain cleavage results in two monovalent Fab' fragments that bind antigen and an Fc fragment. Pepsin cleavage, for example, results in a bivalent F (ab') fragment. An F (ab')2 fragment of the invention may be further reduced using, for example, DTT or 2-mercaptoethanol to produce two monovalent Fab' fragments.

[0104] Functional or antigen-binding fragments of antibodies produced by proteolysis may be purified by affinity and column chromatographic procedures. For example, undigested antibodies and Fc fragments may be removed by binding to protein A. Additionally, functional fragments may be purified by virtue of their charge and size, using, for example, ion exchange and gel filtration chromatography. Such methods are well known to those skilled in the art.

[0105] The antibody or antigen-binding fragment thereof may be produced by recombinant methodology. In one embodiment, one initially isolates a polynucleotide encoding desired regions of the antibody heavy and light chains. Such regions may include, for example, all or part of the variable region of the heavy and light chains. In one embodiment, such regions can particularly include the antigen binding regions of the heavy and light chains, typically the antigen binding sites, such as the CDRs. The polynucleotide encoding the antibody or antigen-binding fragment thereof according to the invention may be produced using methods known to those skilled in the art. The polynucleotide encoding the antibody or antigen-binding fragment thereof may be directly synthesized by methods of oligonucleotide synthesis known in the art. Alternatively, smaller fragments may be synthesized and joined to form a larger functional fragment using recombinant methods known in the art.

[0106] As used herein, the term "immunospecificity" can mean the binding region of the antibody or antigen-binding fragment thereof is capable of immunoreacting with the ZIP12 extracellular epitope, by specifically binding therewith. The antibody or antigenbinding fragment thereof can typically selectively interact with an antigen (i.e. the ZIP12 extracellular epitope) with an affinity constant of approximately 10'5to 10'13M’x, typically 10'6to 10'9M’1, even more typically, 10'10to 10'12M’1. The antibody or antigen-binding fragment thereof typically does not substantially bind to ZIP4 and / or ZIP13, such that the affinity constant is approximately more than 10'10M’1, 10'9M-1, 10-8M-i;io-7M-1, or 10'6M’1' typically more than IO-5M’1, 10'4M-1or 10'3M-1and even more typically 10'2M-110-1M-1or 10'2M-1and most typically 10+1M-1, 10+2M-1 or10+3M-1.

[0107] The term "immunoreact" can mean the binding region is capable of eliciting an immune response upon binding with the ZIP12 extracellular epitope.

[0108] The term "epitope" can mean any region of an antigen with the ability to elicit, and combine with, a binding region of the antibody or antigen-binding fragment thereof.

[0109] Thus, the antibody or 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. In one embodiment, the heavy chain is an IgG. In one embodiment, the heavy chain is an IgA.

[0110] The heavy chain may be an IgGl. The heavy chain may be an IgG2. The heavy chain may be an IgG3. The heavy chain may be an IgG4. The heavy chain may be an IgAl. The heavy chain may be an IgA2. Typically, the heavy chain is an IgGl.

[0111] In one embodiment, the Fc fragment comprises one or more amino acid substitution selected from the group consisting of: L234A and L235A, according to EU numbering. In one embodiment, the Fc fragment comprises the amino acid substitutions L234A and L235A. In one embodiment, the antibody or antigen-binding fragment thereof is administered at a dose of between > 0.3 mg / kg and 13 mg / kg, between 0.4 mg / kg and 12 mg / kg, between 0.5 mg / kg and 11 mg / kg, between 0.6 mg / kg and 10 mg / kg, between 0.7 mg / kg and 9 mg / kg, between 0.8 mg / kg and 8 mg / kg, between 0.9 mg / kg and 7 mg / kg, or between > 1 mg / kg and < 6 mg / kg.

[0112] In one embodiment, the antibody or antigen-binding fragment thereof is administered at a dose of between > 0.3 mg / kg and 12 mg / kg, between > 0.3 mg / kg and 11 mg / kg, between > 0.3 mg / kg and 10 mg / kg, between > 0.3 mg / kg and 9 mg / kg, between > 0.3 mg / kg and 8 mg / kg, between > 0.3 mg / kg and 7 mg / kg, or between > 0.3 mg / kg and < 6 mg / kg.

[0113] In one embodiment, the antibody or antigen-binding fragment thereof is administered at a dose of between > 0.3 mg / kg and 12 mg / kg, between 0.4 mg / kg and 12 mg / kg, between 0.5 mg / kg and 12 mg / kg, between 0.6 mg / kg and 12 mg / kg, between 0.7 mg / kg and 12 mg / kg, between 0.8 mg / kg and 12 mg / kg, between 0.9 mg / kg and 12 mg / kg, or between > 1 mg / kg and < 12 mg / kg.

[0114] In one embodiment, the antibody or antigen-binding fragment thereof is administered at a dose of between > 0.3 mg / kg and < 6 mg / kg, between 0.4 mg / kg and < 6 mg / kg, between 0.5 mg / kg and < 6 mg / kg, between 0.6 mg / kg and < 6 mg / kg, between 0.7 mg / kg and < 6 mg / kg, between 0.8 mg / kg and < 6 mg / kg, between 0.9 mg / kg and < 6 mg / kg, or between > 1 mg / kg and < 6 mg / kg.

[0115] In one embodiment, the antibody or antigen-binding fragment thereof is administered at a dose of between 1.5 mg / kg and < 6 mg / kg, between 2 mg / kg and < 6 mg / kg, between 2.5 mg / kg and < 6 mg / kg, between 3 mg / kg and < 6 mg / kg, between 3.5 mg / kg and < 6 mg / kg, between 4 mg / kg and < 6 mg / kg, between 4.5 mg / kg and < 6 mg / kg, or between 5 mg / kg and < 6 mg / kg.

[0116] In one embodiment, the antibody or antigen-binding fragment thereof is administered at a dose of between > 0.3 mg / kg and < 6 mg / kg, between > 0.3 mg / kg and 5.5 mg / kg, between > 0.3 mg / kg and 5 mg / kg, between > 0.3 mg / kg and 4.5 mg / kg, between > 0.3 mg / kg and 4 mg / kg, between > 0.3 mg / kg and 3.5 mg / kg, or between > 0.3 mg / kg and 3 mg / kg. Typically, the antibody or antigen-binding fragment thereof is administered at a dose of between > 1 mg / kg and < 6 mg / kg.

[0117] In one embodiment, the antibody or antigen-binding fragment thereof is administered at a dose of at least 0.3 mg / kg, at least 0.4 mg / kg, at least 0.5 mg / kg, at least 0.6 mg / kg, at least 0.7 mg / kg, at least 0.8 mg / kg, at least 0.9 mg / kg, or at least 1 mg / kg.

[0118] In one embodiment, the antibody or antigen-binding fragment thereof is administered at a dose of at least 1.5 mg / kg, at least 2 mg / kg, at least 2.5 mg / kg, at least 3 mg / kg, at least 3.5 mg / kg, at least 4 mg / kg, at least 4.5 mg / kg, at least 5 mg / kg, at least 5.5 mg / kg, or at least 6 mg / kg.

[0119] In one embodiment, the antibody or antigen-binding fragment thereof is administered at a dose of less than 12.5 mg / kg, less than 12 mg / kg, less than 11.5 mg / kg, less than 11 mg / kg, less than 10.5 mg / kg, less than 10 mg / kg, less than 9.5 mg / kg, less than 9 mg / kg, less than 8.5 mg / kg, less than 8 mg / kg, less than 7.5 mg / kg, less than 7 mg / kg, or less than 6.5 mg / kg.

[0120] In one embodiment, the antibody or antigen-binding fragment thereof is administered at a dose of 1 mg / kg, 3 mg / kg, or 6 mg / kg.

[0121] In one embodiment, the antibody or antigen-binding fragment thereof is administered at a dose of Img / kg.

[0122] In one embodiment, the antibody or antigen-binding fragment thereof is administered at a dose of 3mg / kg.

[0123] In one embodiment, the antibody or antigen-binding fragment thereof is administered at a dose of 6mg / kg.

[0124] In some embodiments, the antibody or antigen-binding fragment thereof is administered once every four weeks, once every five weeks, or once every six weeks. In some embodiments, the antibody or antigen-binding fragment thereof is administered once every seven weeks, once every eight weeks, or once every nine weeks. In some embodiments, the antibody or antigen-binding fragment thereof is administered once every ten weeks, once every eleven weeks, or once every twelve weeks. Typically, the antibody or antigen-binding fragment thereof is administered once every four weeks.

[0125] In some embodiments, the anti-ZIP12 antibody or antigen-binding fragment thereof is administered in the form of a pharmaceutically acceptable composition.

[0126] In one embodiment, the antibody or antigen-binding fragment thereof is administered to a subject by injection into the blood stream or directly into a site requiring treatment. Injections may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion), or intradermal (bolus or infusion).

[0127] In one embodiment, the antibody or antigen-binding fragment thereof is administered subcutaneously.

[0128] As shown in the examples, the inventors have investigated the pharmacokinetics of different subcutaneous dosages of the anti-ZIP12 antibody (0.3 mg / kg, 1 mg / kg, 3 mg / kg, 6 mg / kg and 12 mg / kg), in adult humans, and have surprisingly discovered that these doses of the anti-ZIP12 antibody can achieve a surprisingly long half-life.

[0129] Accordingly, in some embodiments, the anti-ZIP12 antibody or antigen-binding fragment thereof comprises a half-life of at least 30 days, at least 35 days, at least 40 days, at least 45 days, or at least 50 days.

[0130] Anti-drug antibodies (ADAs) are antibodies produced by the immune system in response to a therapeutic drug (e.g., an antibody), which can interfere with the drug's effectiveness or cause adverse reactions. Advantageously, the inventors discovered that subjects administered with the anti-ZIP12 antibody according to the invention were anti-drug antibody (ADA) negative.

[0131] Accordingly, in some embodiments, the anti-ZIP12 antibody or antigen-binding fragment thereof does not produce an anti-drug antibody response in a subject.

[0132] Pulmonary hypertension may be as defined by the World Health Organisation (WHO) Groups 1-5.

[0133] Accordingly, pulmonary hypertension may be pulmonary arterial hypertension (PAH).

[0134] Pulmonary hypertension may be pulmonary hypertension due to left heart disease. Pulmonary hypertension may be pulmonary hypertension due to lung disease. Pulmonary hypertension may be pulmonary hypertension due to chronic blood clots in the lungs. Pulmonary hypertension may be pulmonary hypertension due to unknown causes. Pulmonary hypertension may be hypoxic pulmonary hypertension.

[0135] As discussed in the Examples, following administration of the anti-ZIP12 antibody, the inventors are measuring a number of clinical endpoints. For example, the effects of the anti-ZIP12 antibody on mean pulmonary artery pressure (mPAP), total pulmonary resistance, cardiac output and stroke volume will be monitored via the CardioMEMS™ telemeter. Additional clinical endpoints include 6MWD, NT-proB-type natriuretic peptide (NT-proBNP), QoL measures, WHO functional class, European Respiratory Society (ERS) risk scores and cardiac magnetic resonance imaging (MRI) parameters.

[0136] Accordingly, in a third aspect of the invention, there is provided a method of evaluating the efficacy of an antibody or antigen-binding fragment thereof in treating pulmonary hypertension, comprising administering the antibody or antigen-binding fragment to a patient with pulmonary hypertension and measuring a primary clinical endpoint, wherein the clinical endpoint is selected from the group consisting of: a reduction in mean pulmonary artery pressure (mPAP); a reduction in total pulmonary resistance (TPR); an increase in cardiac output; an increase in stroke volume; an increase in six-minute walking distance (6MWD); and a decrease in NT-proB-type natriuretic peptide, and wherein the antibody or antigen-binding fragment thereof, comprises:

[0137] (vii) a CDR-H1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 1, or a variant or fragment thereof;

[0138] (viii) a CDR-H2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 2, or a variant or fragment thereof;

[0139] (ix) a CDR-H3 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 3, or a variant or fragment thereof;

[0140] (x) a CDR-L1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 4, or a variant or fragment thereof;

[0141] (xi) a CDR-L2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 5, or a variant or fragment thereof; and

[0142] (xii) a CDR-L3 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 6, or a variant or fragment thereof.

[0143] It will be appreciated that antibodies and fragments thereof according to the invention (collectively referred to herein as "agents") may be used in a monotherapy (e.g. the use of an antibody or antigen binding fragment thereof alone), for treating, ameliorating or preventing pulmonary hypertension. Alternatively, agents according to the invention may be used as an adjunct to, or in combination with, known therapies for treating, ameliorating, or preventing pulmonary hypertension, such as anticoagulants such as warfarin, diuretics, digoxin, endothelin receptor antagonists such as 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, sotatercept, and amlodipine.

[0144] The agents according to the invention may be combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used. Thus, for example, the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension or any other suitable form that may be administered to a person or animal in need of treatment. It will be appreciated that the vehicle of medicaments according to the invention should be one which is well-tolerated by the subject to whom it is given.

[0145] Medicaments comprising agents of the invention may be used in a number of ways. For instance, oral administration may be required, in which case the agents may be contained within a composition that may, for example, be ingested orally in the form of a tablet, capsule or liquid. Compositions comprising agents and medicaments of the invention may be administered by inhalation (e.g. intranasally). Compositions may also be formulated for topical use. For instance, creams or ointments may be applied to the skin.

[0146] Agents and medicaments according to the invention may also be incorporated within a slow- or delayed-release device. Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months. The device may be located at least adjacent the treatment site. Such devices may be particularly advantageous when long-term treatment with agents used according to the invention is required and which would normally require frequent administration (e.g. at least daily injection).

[0147] It will be appreciated that the amount of the antibodies and fragments (i.e. agent) that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of the agent, and whether it is being used as a monotherapy or in a combined therapy. The frequency of administration will also be influenced by the half-life of the agent within the subject being treated. Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular agent in use, the strength of the pharmaceutical composition, the mode of administration, and the advancement of pulmonary hypertension. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.

[0148] The agents may be administered before, during or after onset of pulmonary hypertension. Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials, etc.), may be used to form specific formulations of the agents according to the invention and precise therapeutic regimes (such as daily doses of the agents and the frequency of administration).

[0149] It will be appreciated that the invention extends to any nucleic acid or peptide or variant, derivative or analogue thereof, which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including variants or fragments thereof. The terms "substantially the amino acid / nucleotide / peptide sequence", "variant" and "fragment", can be a sequence that has at least 40% sequence identity with the amino acid / nucleotide / peptide sequences of any one of the sequences referred to herein, for example 40% identity with any of the sequence identified herein.

[0150] Amino acid / polynucleotide / polypeptide sequences with a sequence identity which is greater than 65%, or greater than 70%, or greater than 75%, or greater than 80% sequence identity to any of the sequences referred to are also envisaged. In one embodiment, the amino acid / polynucleotide / polypeptide sequence has at least 85% identity with any of the sequences referred to, or at least 90% identity, or at least 92% identity, or at least 95% identity, or at least 97% identity, or at least 98% identity, or at least 99% identity with any of the sequences referred to herein.

[0151] The skilled technician will appreciate how to calculate the percentage identity between two amino acid / polynucleotide / polypeptide sequences. In order to calculate the percentage identity between two amino acid / polynucleotide / polypeptide sequences, an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value. The percentage identity for two sequences may take different values depending on:- (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional form and constants.

[0152] Having made the alignment, there are many different ways of calculating percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (v) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.

[0153] Hence, it will be appreciated that the 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 one way for generating multiple alignments of proteins or DNA in accordance with the invention. Suitable parameters for ClustalW may be as follows: For DNA alignments: Gap Open Penalty = 15.0, Gap Extension Penalty = 6.66, and Matrix = Identity. For protein alignments: Gap Open Penalty = 10.0, Gap Extension Penalty = 0.2, and Matrix = Gonnet. For DNA and Protein alignments: ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will be aware that it may be necessary to vary these and other parameters for optimal sequence alignment.

[0154] In one embodiment, calculation of percentage identities between two amino acid / polynucleotide / polypeptide sequences may then be calculated from such an alignment as (N / T)*100, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps and either including or excluding overhangs. In one embodiment, overhangs are included in the calculation. Hence, one method for calculating percentage identity between two sequences comprises (i) preparing a sequence alignment using the ClustalW program using a suitable 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. Alternative methods for identifying similar sequences will be known to those skilled in the art. For example, a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to DNA sequences or their complements under stringent conditions. By stringent conditions, the inventors mean the nucleotide hybridises 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 by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from any of the sequences described herein.

[0155] Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof. Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent (synonymous) change. Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change. 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. The polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. The positively charged (basic) amino acids include lysine, arginine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids.

[0156] All of the features described herein (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 where at least some of such features and / or steps are mutually exclusive.

[0157] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:- Figure 1 shows an overview of a Phase 2 clinical trial investigating the anti-ZIP12 monoclonal antibody according to the invention, in adults with pulmonary hypertension (PH), implanted with CardioMEMS™ sensors. The trial is evaluating the anti-ZIP12 antibody in a Phase 2, open-label, multicentre, multiple ascending dose (subcutaneous monthly dosing for 24 weeks). The trial is being conducted in the UK in World Health Organisation (WHO) Group 1 / 3 patients previously implanted with the CardioMEMS™ sensor. The trial is employing a Bayesian Optimal Interval design to determine the safety of up to three dose levels of the anti-ZIP12 antibody (1 mg / kg, 3 mg / kg and 6 mg / kg), with up to N = 12 participants treated at each dose level.

[0158] Following the completion of treatment, participants are being monitored through an up to 12-month washout period to assess long-term pharmacodynamics. The primary endpoints of the trial are safety related; the secondary endpoints focus on efficacy, PK and PD. The effects of the anti-ZIP12 antibody on mean PAP, total pulmonary resistance, cardiac output and stroke volume are being monitored via the CardioMEMS™ telemeter. Additional clinical endpoints include 6MWD, NT-proB-type natriuretic peptide (NT-proBNP), QoL measures, WHO functional class, European Respiratory Society (ERS) risk scores and cardiac magnetic resonance imaging (MRI) parameters.

[0159] Figure 2 shows single dose anti-ZIP12 antibody plasma concentration-time profiles following subcutaneous administration in humans in the clinical study (AP13CP01). Abbreviations: EC50=half maximal effective concentration; SAD=single ascending dose. SADl=0.3 mg / kg; SAD2=1 mg / kg; SAD3=3 mg / kg dose cohorts. Available interim data from SAD Cohorts 1 to 3 are presented. Data are shown for Cohort 1 (0.3 mg / kg) up to Day 96, Cohort 2 (1 mg / kg) up to Day 72 and Cohort 3 (3 mg / kg) up to 504 hours.

[0160] ZIP12 has been implicated in the pathogenesis of pulmonary hypertension, with an increase in expression of ZIP12 associated with the condition. Therefore, the inventors set out to test a monoclonal humanized immunoglobulin G1 (IgGl), fragment crystallizable (Fc) null (IgGl, L234A, L235A) antibody directed against human ZIP12, a protein that is encoded by the solute carrier 39 member 12 (SLC39A12) gene. In particular, the inventors set out to investigate the long-term pharmacokinetics of different dosages of the anti-ZIP12 antibody (1 mg / kg, 3 mg / kg and 6 mg / kg), in adults with pulmonary hypertension (PH), implanted with CardioMEMS™ sensors. Materials and Methods

[0161] Formulation

[0162] The anti-ZIP12 antibody according to the invention is presented as a sterile, preservative-free solution, formulated to a concentration of 100 mg / mL in 20 mM histidine buffer, 6.5% (weight per volume [w / v]) sucrose, 0.04% polysorbate 80 (PS80) (w / v), pH 5.5.

[0163] Pharmacokinetics and Product Metabolism in Animals

[0164] The single dose pharmacokinetics of the anti-ZIP12 antibody have been investigated in vivo following intravenous administration in rats and both intravenous and subcutaneous administration in non-human primates. Repeat dose pharmacokinetics via both routes have further been investigated as part of the 4-week dose range finding and 13-week GLP toxicology study in non-human primates.

[0165] Methods of Analysis

[0166] A qualified enzyme-linked immunosorbent assay (ELISA) was used to determine the anti-ZIP12 antibody concentrations in serum samples from single dose studies performed in rats and monkeys and repeat dose studies performed in monkeys. All assay runs met the acceptance criteria. The assay ranges were 50.0 ng / mL to 1200 ng / mL in rats and 25.0 ng / mL to 1000 ng / mL in monkeys in the single dose studies and 0.1 pg / mL to 15 pg / mL in monkeys in the repeat dose studies. The repeat dose toxicokinetic analyses were conducted using GLP validated methods. Standard non-compartmental analysis was performed using Phoenix WinNonlin (Pharsight) pharmacokinetic software to calculate pharmacokinetic parameters.

[0167] Single and Repeat Dose Pharmacokinetics

[0168] Single Dose Pharmacokinetics

[0169] Non-GLP studies were performed to investigate the single dose pharmacokinetics of 10 mg / kg of the anti-ZIP12 antibody following intravenous bolus injection to three male 7 to 10-week old Sprague-Dawley rats and three female cynomolgus monkeys and following subcutaneous injection to three further female monkeys. All animals were non-fasted. Sampling for concentration of the anti-ZIP12 antibody was performed at the following timepoints:

[0170] Rat intravenous administration: pre-dose and 0.5, 2, 12, 24, 48, 72, 96, 120, 144, 168, 240, 336 and 504 hours post-dose;

[0171] Monkey intravenous administration: pre-dose and 0.083, 0.25 0.5, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, 240, 336 and 504 hours post-dose; Monkey subcutaneous administration: pre-dose and 0.25 0.5, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, 240, 336 and 504 hours post-dose.

[0172] The anti-ZIP12 antibody was dosed as supplied in either a 2 mg / mL (rats) or 10 mg / mL (monkey) formulation. The concentration of the anti-ZIP12 antibody was below the lower limit of quantification pre-dose and the anti-ZIP12 antibody was detected in all samples taken post-dose. The calculated pharmacokinetic parameter results for intravenous and subcutaneous administration are summarized in Table 1.

[0173] Table 1. Mean Single Dose Pharmacokinetic Parameters of the anti -ZIP 12 antibody in

[0174] Sprague-Dawley Rats and Cynomolgus Monkeys

[0175] Abbreviations: BQL=below the lower limit of quantification; NA=not applicable BQL was excluded in the pharmacokinetic parameter calculations.

[0176] [a] Calculated using AUCO-last with nominal dose.

[0177] Results from all animals following single intravenous administration of the anti-ZIP12 antibody to non-fasted rats and monkeys were similar and consistent with the standard two-phase pharmacokinetic profile of an antibody in the respective model. The initial concentration was similar in both species. A single dose of 10 mg / kg resulted in a low volume of distribution (0.101 and 0.0804 L / kg) and low clearance 0.00359 and 0.00262 mL / min / kg and corresponding long half-life (334 and 364 hours) in rats and monkeys, respectively.

[0178] Following single subcutaneous administration of the anti-ZIP12 antibody to non-fasted monkeys, the maximum concentration of 99,767 ng / mL was achieved at 112 hours.

[0179] Surprisingly, however, the half-life was comparable to intravenous administration (341 hours). The mean bioavailability following extravascular administration was 83.1%. Repeat Dose Toxicokinetics

[0180] In the non-GLP 4-week dose range finding study the toxicokinetics of the anti-ZIP12 antibody was evaluated in male and female non-fasted cynomolgus monkeys (two / sex / group) following once weekly administration at 10 or 30 mg / kg / week by subcutaneous injection or 100 mg / kg / week by intravenous infusion. Sampling for concentration of the anti-ZIP12 antibody was performed pre-dose and 0.5, 4, 24, 72 and 144 hours post-dose on Days 1 and 22, and pre-dose only on Days 8, 15 and 29.

[0181] The calculated pharmacokinetic parameter results for intravenous and subcutaneous administration are summarised in Table 2. Data from male and female animals were combined as there were no apparent gender differences (gender ratios were less than two-fold) following both intravenous and subcutaneous administration of the anti- ZIP12 antibody.

[0182] Exposure, as assessed by the anti-ZIP12 antibody mean maximum concentration (Cmax) and area under the concentration-time curve (AUC)o-i68, increased in a generally dose proportional manner with the increase in dose from 10 to 30 mg / kg following subcutaneous administration. Accumulation of the anti-ZIP12 antibody was observed after multiple subcutaneous and intravenous doses in monkeys, with accumulation slightly more notable after subcutaneous administration than intravenous administration, although some variation in concentration values due to inter-animal variation may explain possible differences in Cmax and AUCo-ies between Day 1 and 22.

[0183] Mean accumulation ratio values ranged from 2.60 to 2.69 for Cmax and from 2.65 to 2.81 for AUCo-ies, following subcutaneous administration and 1.62 for Cmax and 2.11 for AUCo -168, following intravenous administration.

[0184] The absolute bioavailability was 0.796 and 0.650 on Day 1 following subcutaneous administration of 10 and 30 mg / kg, respectively indicating moderate bioavailability of the anti-ZIP12 antibody following subcutaneous relative to intravenous administration. le 2. Repeat Dose Pharmacokinetic Parameters of the anti-ZIP12 antibody in Cynomolgus Monkeys in the 4-week Dose Range Finding dy reviations: BQL=below the lower limit of quantification; NC=not calculated (due to inability to appropriately define the elimination phase) was excluded in the pharmacokinetic parameter calculations. a from male and female animals were combined as there were no apparent gender differences following either intravenous or subcutaneous inistration of the anti-ZIP12 antibody. ata reported are mean values except for Tmax where the median is reported. Absolute bioavailability (Day 1 only), calculated as AUCO-last (subcutaneous) / AUCO-last (intravenous) x Dose (intravenous) / Dose (subcutaneous).

[0185] In the GLP 13-week repeat dose toxicity study, the toxicokinetics of the anti-ZIP12 antibody was evaluated in male and female non-fasted cynomolgus monkeys following once weekly administration at 0, 10 or 30 mg / kg / week by subcutaneous injection or 100 mg / kg / week by intravenous infusion (three / sex / group plus two / sex in the control and high dose groups for assessment of recovery). Sampling for concentration of the anti-ZIP12 antibody was performed pre-dose and 0.5, 4, 24, 72 and 168 hours postdose on Days 1 and 85 following subcutaneous administration and pre-dose and 1, 4.5, 24.5, 72.5 and 168.5 hours post-dose on Days 1 and 85 following intravenous administration.

[0186] The calculated pharmacokinetic parameter results for intravenous and subcutaneous administration are summarized in Table 3. Data from male and female animals were combined as there were no apparent gender differences (gender ratios were less than two-fold) following both intravenous and subcutaneous administration of the anti- ZIP12 antibody.

[0187] There were no quantifiable concentrations of the anti-ZIP12 antibody in the control group. Following the first and subsequent weekly subcutaneous administrations of the anti-ZIP12 antibody, serum antibody concentrations increased up to 72 hours and were quantifiable up to the last sampling timepoint. Following the first and subsequent weekly intravenous administrations of the anti-ZIP12 antibody, serum antibody concentrations slowly decreased in a bi-exponential manner and were quantifiable up to the last sampling timepoint. Steady-state was reached by approximately Day 57 following weekly subcutaneous administration, whereas serum antibody concentrations were still increasing up to Day 85 following weekly intravenous administration.

[0188] Drug exposures achieved in the 13-week repeat dose toxicity study were as anticipated and aligned with those observed in the 4-week dose range finding study. The anti-ZIP12 antibody Cmax and AUCo-ies or AUCo-ies.s values increased with dose. The increases in Cmax and AUCo-ies values were generally dose proportional following the increase in dose from 10 to 30 mg / kg / week by subcutaneous administration.

[0189] The relative bioavailability was 0.675 and 0.747 on Day 1 and 0.970 and 0.907 on Day 85 following subcutaneous administration of 10 and 30 mg / kg, respectively. le 3. Mean Repeat Dose Pharmacokinetic Parameters of the anti-ZIP 12 antibody in Cynomolgus Monkeys in the 13- week Repeat Dose icity Study reviations: BQL=below the lower limit of quantification; NA=not applicable; NC=not calculated was excluded in the pharmacokinetic parameter calculations. a from male and female animals were combined as there were no apparent gender differences following either intravenous or subcutaneous inistration of the anti-ZIP12 antibody. ata reported are mean values except for Tmax where the median is reported. Calculated as Mean AUCo-ies (subcutaneous) / Mean AUCo-ies (intravenous) x Dose (intravenous) / Dose (subcutaneous)

[0190] The nonclinical toxicology programme for the anti-ZIP12 antibody has included repeat dose toxicity studies in cynomolgus monkeys. An overview of the toxicity studies is shown in Table 4. Based on the results of this study, the no observed adverse effect level (NOAEL) was 100 mg / kg / week by the intravenous route of administration.

[0191] Table 4. Summary of Toxicity Studies with the anti-ZIP 12 antibody

[0192] Effects in humans

[0193] Clinical Plan

[0194] A first-in-human clinical study (AP13CP01) to assess the pharmacokinetics, safety and tolerability of ascending doses of the anti-ZIP12 antibody in healthy adult participants is ongoing and dosing is complete. The antibody has been administered to a total of 39 participants (six participants each at 0.3, 1, 6, and 12 mg / kg, five participants at 3 mg / kg); eight have received matched placebo.

[0195] Overview of Clinical Program

[0196] The clinical program was initiated with a Phase 1 study with a single ascending dose (SAD) in healthy adult participants (Study AP13CP01). Up to five cohorts of eight participants (randomised to receive the anti-ZIP12 antibody or placebo in a 3: 1 ratio) at escalating dose levels of 0.3, 1, 3, 6, and 12 mg / kg were evaluated in the SAD, with dosing on Day 1 and follow-up at least until Day 96. Escalating multiple doses of the anti-ZIP12 antibody (estimated between 1 and 6 mg / kg) administered as six subcutaneous administrations doses every 4 weeks (Q4W) are being evaluated in telemetered adults with WHO Group 1 pulmonary arterial hypertension receiving stable therapy as part of AP13CP02. An optional second part of the study is further evaluating one or more dose levels of the anti-ZIP12 antibody in participants with WHO Group 3 pulmonary hypertension associated with interstitial lung disease.

[0197] Prediction of Human Pharmacokinetics

[0198] The pharmacokinetic data from cynomolgus monkeys were characterized using a two- compartmental model to describe the observed linear pharmacokinetics.

[0199] Subsequently, a human pharmacokinetic model was established by allometrically scaling the pharmacokinetic parameters from monkey to human. The scaling was performed using an exponent of 0.85 for clearance and blood flow and an exponent of 1.0 for volume (Deng, 2011). In humans the projected serum clearance is 7.42 mL / hour and the terminal half-life is estimated to be approximately 24 days based on nonclinical data.

[0200] Prediction of Human Efficacious Exposure

[0201] The anti-ZIP12 antibody has been characterized in the gold standard models of pulmonary hypertension. The models used were the chronic hypoxia-induced pulmonary hypertension model and the Sugen 5416 / hypoxia model. In both models, the effective dose range was 1 to 10 mg / kg by intraperitoneal administration, achieving exposures in the range of 6.59 pg / mL and 71.0 pg / mL, respectively.

[0202] Pharmacokinetics and Product Metabolism in Humans

[0203] Single dose pharmacokinetics of the antibody according to the invention were evaluated in the first clinical study in healthy adult participants (AP13CP01). A body weight-based dosing approach (mg / kg) was used in the AP13CP01 study with the goal to reduce inter-individual variations in pharmacokinetic exposure.

[0204] Serum concentrations of antibody were measured using a validated Meso Scale Discovery method and used for the calculation of pharmacokinetic parameters using noncompartmental methods. Following single subcutaneous dosing, serial blood sampling for concentration of serum antibody was performed pre-dose and at the following timepoints post-dose: 1, 2, 4, 8, 12, 24, 48, 72, 120, 168, 216, 336 and 504 hours, plus Days 43, 72 and 96. Serum concentrations of antibody at Day 29 (i.e. representing trough levels with a Q4W dosing schedule) are summarized for participants receiving active treatment in Table 5 and plasma concentration-time profiles are shown on a logarithmic scale in Figure 2. Based on data available to date, the Day 29 concentrations increased approximately dose proportionally across the 0.3 to 3 mg / kg dose range.

[0205] Table 5. The anti-ZIP12 antibody Plasma Concentrations on Day 29 Following Single

[0206] Subcutaneous Administration in AP13CP01

[0207] Abbreviations: Max=maximum; Min=minimum; SAD=single ascending dose; SD=standard deviation

[0208] Interim data from SAD Cohorts 1 to 3.

[0209] N is one less than anticipated in Cohorts 1 and 3 due to data only being available out to Day 15.

[0210] Pharmacokinetic parameters are summarised in Table 6. Based on data available to date, antibody exposure (Cmax and area under the concentration-time curve from time zero to Day 29 [AUC0-29]) increased approximately dose proportionally across the 0.3 to 3 mg / kg dose range. The mean time to maximum concentration (Tmax) was approximately 6 to 7.5 days (139 to 180 hours) and surprisingly, the mean half-life 50 to 67 days, which was better than the prediction that was based on nonclinical data.

[0211] Table 6. Summary of Pharmacokinetic Parameters (Mean ± Standard Deviation) in

[0212] AP13CP01

[0213] Abbreviations: NC=not calculable; SAD=single ascending dose

[0214] Available interim data from SAD Cohorts 1 to 3. Available data for Cohort 1 (0.3 mg / kg) up to Day 96, Cohort 2 (1 mg / kg) up to Day 72 and Cohort 3 (3 mg / kg) up to 504 hours are included.

[0215] [a] Values are based on different follow-up times for the three cohorts

[0216] Updated half-life for all dose groups following the end of the study are summarised in Table 7. Surprisingly, the mean half-life of the anti-ZIP12 antibody was 935 hours / 39 days (0.3 mg / kg), 821 hours / 34.2 days (1 mg / kg), 1280 hours / 53.3 days (3 mg / kg), 1000 hours / 41.7 days (6 mg / kg) and 990 hours / 41.3 days (12 mg / kg). Table 7. Summary of half-life by Dose group in AP13CP01

[0217] 5 Key: SD = Standard Deviation; CV = Coefficient of Variation; SAD = Single Ascending Dose.

[0218] Advantageously, at the end of the study, it was also shown that the majority of subjects were Anti-drug Antibody (ADA) negative, as shown in Table 8. For the 3 mg / kg dose, 100% of subjects were ADA negative.

[0219] 10

[0220] Table 8. Summary of Anti-drug Antibody - End of Study Key: ADA = Anti-drug Antibody; SAD = Single Ascending Dose.

[0221] ADA Categories Definition:

[0222] 1. ADA-Positive: A subject is considered ADA-positive in an assay designed to test for ADAs if they have at least one sample that tests positive for anti-drug antibodies during the study (usually postbaseline).

[0223] 2. Treatment-Emergent ADA (TE ADA): ADA responses that emerge after treatment initiation in subjects who were ADA-negative at baseline.

[0224] 3. Treatment boosted ADA positive: Participants with an ADA-positive predose sample and at least 1 ADA-positive post-treatment sample that is increased >=4x over the baseline titer

[0225] 4. Treatment induced ADA: Those who are negative at baseline and have at least 1 positive post dose ADA sample.

[0226] NOTE: ADA treatment emergent (TE) positive, is the sum of treatment induced ADA + treatment boosted ADA

[0227] 5. Transient ADA: Subjects who temporarily develop ADAs, but then return to ADA-negative status without sustained immune response.

[0228] 6. Persistent ADA: Subjects who are ADA positive at baseline and ADA positive at 2 or more postbaseline assessments

[0229] (with > 16 weeks between first and last positive) or ADA positive at the last post-baseline assessment.

[0230] 7. ADA treatment unaffected: Participants with an ADA-positive predose sample and ADA-positive post-treatment samples that are increased <4x over the baseline titer

[0231] 8. ADA negative: The sum of ADA never positive + treatment unaffected ADA.

[0232] The multiple dosing regimen for the Phase 2 study in participants with pulmonary hypertension (AP13CP02) is six subcutaneous doses Q4W.

[0233] The starting dose is 1 mg / kg Q4W, supported by the acceptable safety and tolerability of single subcutaneous doses up to 6.0 mg / kg observed in healthy participants in the AP13CP01 study. Based on the pharmacokinetic data from AP13CP01, a 1 mg / kg Q4W dosing regimen is expected to provide antibody serum concentrations that are maintained above the nonclinical EC50 of 6.69 pg / mL (change in mPAP from the induced-hypoxia rat model of pulmonary hypertension) throughout the dosing interval once steady state is achieved. Based on data available, the mean antibody serum concentrations observed at a nominal time of 28 days post-dose in the AP13CP01 study are 2.36, 8.41 and 34.5 pg / mL for the 0.3, 1 and 3 mg / kg dose levels, respectively. A starting dose of 1 mg / kg Q4W is therefore expected to provide mean steady state trough concentrations above the nonclinical EC50 based on a target trough concentration of 6.69 pg / mL. Differences in target binding between healthy participants and patients would not be expected to result in differences in systemic exposure.

[0234] AP13CP02 Clinical Trial Protocol

[0235] AP13CP02 is a Phase 2 trial designed to evaluate the safety and efficacy of multiple doses of the anti-ZIP12 antibody. AP13CP02 is also evaluating the impact of inhibition of ZIP12 on haemodynamic measures and cardiovascular functional capacity in adults with PH who are implanted with haemodynamic monitors (CardioMEMS™ HF System; Abbott, Sylmar, CA, USA). CardioMEMS™ is a wireless, CE-marked PAP monitor implanted into the distal left pulmonary artery at the time of right heart catheterisation (RHC). CardioMEMS™ provides non-invasive, real-time measurement of haemodynamic data which includes the PAP waveform, systolic / diastolic / mean PAP and heart rate, along with derived cardiac output and pulmonary resistance. Trials conducted on the use of CardioMEMS™ in PH demonstrate that this telemetry device can provide real-time data on treatment effects on haemodynamic parameters, with lower variability than RHC, and without the need for repeated RHC procedures to assess haemodynamics (Benza 2019; Middleton 2024). The recruitment of a telemetered PH patient population in this trial enables a more precise assessment of the therapeutic effect of the anti-ZIP12 antibody and permits the determination of safe and effective doses of the anti-ZIP12 antibody while limiting both the number of participants and the need for invasive RHC procedures.

[0236] AP13CP02 is employing a Bayesian Optimal Interval (BOIN) design to determine tolerability of the anti-ZIP12 antibody in participants with WHO Group 1 PH. Target nominal dose levels are 1 mg / kg Q4W, 3 mg / kg Q4W and 6 mg / kg Q4W. Dose limiting toxicities (DLTs) are being evaluated during the first 28 days of treatment. Participants are being treated through Day 169 to further evaluate safety and efficacy of the anti- ZIP12 antibody. Data from this participant group may be used to determine the dose level of the anti-ZIP12 antibody for an optional cohort of participants with WHO Group 3 PH-ILD.

[0237] Brief Summary

[0238] The purpose of this trial is to assess the safety, tolerability, efficacy and PK of the anti-ZIP12 antibody, at multiple dose levels, in participants with PH.

[0239] Trial details include:

[0240] - The trial duration is up to 508 days (approximately 18 months) for an individual participant.

[0241] - The treatment duration is up to Day 169.

[0242] - The visit frequency is approximately every 28 days during the treatment period, with 2 PK blood draw visits approximately 14 days after Day 1 (first dose) and Day 85 (fourth dose). There are 3 visits during the 339-day (approximately 12 months) washout period; the last of these 3 visits are the End of Trial (EoT) visit. TRIAL DESIGN

[0243] Overall Design

[0244] AP13CP02 is a Phase 2, open-label, dose escalation trial to determine the safety, tolerability, efficacy, and PK of multiple subcutaneous (SC) injections of the anti-ZIP12 antibody in telemetered adults with PH receiving stable therapy. Trial sites are enrolling participants with pre-implanted CardioMEMS™ devices.

[0245] The trial is being conducted in two parts:

[0246] Part A: BOIN design in adults with WHO Group 1 PAH.

[0247] Part B (optional): Sequential cohorts of N=4, up to a total of N = 12 adults with WHO Group 3 PH-ILD.

[0248] Justification for Dose

[0249] Dose Rationale and Dosina Schedule

[0250] All participants are being treated with the anti-ZIP12 antibody at the assigned dose level with subcutaneous (SC) administration Q4W (i.e., on Days 1, 29, 57, 85, 113 and 141).

[0251] The starting dose for Cohort 1 is 1 mg / kg Q4W, supported by the acceptable safety and tolerability of a single SC administration of the anti-ZIP12 antibody at dose levels up to 6.0 mg / kg observed in healthy participants from the AP13CP01 Phase 1 trial.

[0252] The highest dosing regimen expected to be studied is 6 mg / kg.

[0253] TRIAL POPULATION

[0254] Inclusion Criteria

[0255] Participants have been included in the trial only if all of the following criteria apply prior to dosing on Day 1 :

[0256] Age

[0257] 1. Participant must be 18 to 80 years of age inclusive, at the time of signing the informed consent.

[0258] Type of Participant and Disease Characteristics

[0259] 2. Participants who are diagnosed with pulmonary hypertension via right heart catheterisation (RHC), documented at any time prior to Screening: a) Part A: WHO Group 1 PAH which is idiopathic, heritable or associated with drugs, toxins or connective tissue disease b) Optional Part B: WHO Group 3 PH associated with ILD, as confirmed by historical diagnostic CT (or other imaging modality) 3. WHO Functional Class II or III

[0260] 4. Participant has the CardioMEMS PA Sensor implanted.

[0261] 5. PH therapy at stable dose levels for 30 days prior to Screening that remain stable to Day 1 (unless otherwise described below). Dose modification of diuretic treatment is permitted. Permitted therapies include: a) Endothelin-receptor antagonist (ERA) b) Phosphodiesterase 5 (PDE5) inhibitors c) Prostacyclin analogue or receptor agonist (SC / inhaled / PO). Prostacyclin dose may be adjusted but should be maintained within ± 10% of the dose recorded at screening. d) Soluble guanylate cyclase stimulator e) Sotatercept, at a stable dose for 180 days

[0262] 6. The following as measured by telemetry during Screening: a) Resting mPAP > 25 mmHg b) Total pulmonary resistance (TPR) > 6 Wood Units

[0263] 7. 6MWD > 100 metres during the Screening period

[0264] 8. Pulmonary function tests (PFTs) within 6 months prior to the Screening Visit as follows: a) Part A: Total lung capacity > 60% predicted b) Part B: A diffusing capacity of the lungs for carbon monoxide (DLCO) of < 60%

[0265] Trial Intervention(s) Administered

[0266] Table 9. Trial Intervention(s) Administered

[0267] Table 10. Tria! Arm(s)

[0268] Conclusions

[0269] The inventors have previously developed a function-blocking monoclonal antibody (mAb) that inhibits the intracellular influx of Zn2+ through binding to the extracellular domain of the ZIP12 protein (WO 2022 / 064216 Al). The inventors then subjected the anti-ZIP12 antibody to affinity maturation, producing novel variants that have surprisingly high binding affinities to the ZIP12 epitope (WO 2023 / 180743 Al). Based on these previous findings, the inventors now propose inhibition of ZIP12 by the ZIP12 monoclonal antibody according to the invention, as a novel clinical therapeutic strategy to treat pulmonary hypertension in humans. In particular, the inventors are now proposing treatment of pulmonary hypertension with a ZIP12 antibody, that has a surprisingly long half-life when administered subcutaneously.

Claims

Claims1. An antibody or antigen-binding fragment thereof, comprising:(i) a CDR-H1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 1, or a variant or fragment thereof;(ii) a CDR-H2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 2, or a variant or fragment thereof;(iii) a CDR-H3 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 3, or a variant or fragment thereof;(iv) a CDR-L1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 4, or a variant or fragment thereof;(v) a CDR-L2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 5, or a variant or fragment thereof; and(vi) a CDR-L3 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 6, or a variant or fragment thereof, for use in treating, preventing or ameliorating pulmonary hypertension in a human.

2. The antibody or antigen-binding fragment thereof, for use according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 7, or a variant or fragment thereof, and / or a light chain variable region comprising or consisting of a sequence as substantially set out in SEQ ID No: 8, or a variant or fragment thereof.

3. The antibody or antigen-binding fragment thereof, for use according to claim 1 or claim 2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain region comprising or consisting of a sequence as substantially set out in SEQ ID No: 9, or a variant or fragment thereof, and / or a light chain region comprising or consisting of a sequence as substantially set out in SEQ ID No: 10, or a variant or fragment thereof.

4. The antibody or antigen-binding fragment thereof, for use according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises an Fc fragment comprising one or more amino acid substitution selected from the group consisting of: L234A and L235A, according to EU numbering.

5. The antibody or antigen-binding fragment thereof, for use according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof isadministered at a dose of between > 0.3 mg / kg and 13 mg / kg, between 0.4 mg / kg and 12 mg / kg, between 0.5 mg / kg and 11 mg / kg, or between 0.6 mg / kg and 10 mg / kg.

6. The antibody or antigen-binding fragment thereof, for use according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is administered at a dose of between 0.7 mg / kg and 9 mg / kg, between 0.8 mg / kg and 8 mg / kg, between 0.9 mg / kg and 7 mg / kg, or between > 1 mg / kg and < 6 mg / kg.

7. The antibody or antigen-binding fragment thereof, for use according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is administered at a dose of between > 0.3 mg / kg and 12 mg / kg, between > 0.3 mg / kg and 11 mg / kg, between > 0.3 mg / kg and 10 mg / kg, or between > 0.3 mg / kg and 9 mg / kg.

8. The antibody or antigen-binding fragment thereof, for use according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is administered at a dose of between > 0.3 mg / kg and 8 mg / kg, between > 0.3 mg / kg and 7 mg / kg, or between > 0.3 mg / kg and < 6 mg / kg.

9. The antibody or antigen-binding fragment thereof, for use according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is administered at a dose of between > 0.3 mg / kg and < 6 mg / kg, between 0.4 mg / kg and < 6 mg / kg, between 0.5 mg / kg and < 6 mg / kg, or between 0.6 mg / kg and < 6 mg / kg.

10. The antibody or antigen-binding fragment thereof, for use according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is administered at a dose of between 0.7 mg / kg and < 6 mg / kg, between 0.8 mg / kg and < 6 mg / kg, between 0.9 mg / kg and < 6 mg / kg, or between > 1 mg / kg and < 6 mg / kg.

11. The antibody or antigen-binding fragment thereof, for use according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is administered at a dose of between > 1 mg / kg and < 6 mg / kg.

12. The antibody or antigen-binding fragment thereof, for use according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is administered at a dose of 1 mg / kg, 3 mg / kg, or 6 mg / kg.

13. The antibody or antigen-binding fragment thereof, for use according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is administered once every four weeks, once every five weeks, or once every six weeks.

14. The antibody or antigen-binding fragment thereof, for use according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is administered once every seven weeks, once every eight weeks, or once every nine weeks.

15. The antibody or antigen-binding fragment thereof, for use according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is administered once every four weeks.

16. The antibody or antigen-binding fragment thereof, for use according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is administered subcutaneously.

17. The antibody or antigen-binding fragment thereof, for use according to any one of the preceding claims, wherein pulmonary hypertension is pulmonary arterial hypertension (PAH), pulmonary hypertension due to left heart disease, pulmonary hypertension due to lung disease, pulmonary hypertension due to chronic blood clots in the lungs, pulmonary hypertension due to unknown causes, or hypoxic pulmonary hypertension.

18. A method of evaluating the efficacy of an antibody or antigen-binding fragment thereof in treating pulmonary hypertension, comprising administering the antibody or antigen-binding fragment to a patient with pulmonary hypertension and measuring a primary clinical endpoint, wherein the clinical endpoint is selected from the group consisting of: a reduction in mean pulmonary artery pressure (mPAP); a reduction in total pulmonary resistance (TPR); an increase in cardiac output; an increase in stroke volume; an increase in six-minute walking distance (6MWD); and a decrease in NT- proB-type natriuretic peptide, and wherein the antibody or antigen-binding fragment thereof, comprises:(i) a CDR-H1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 1, or a variant or fragment thereof;(ii) a CDR-H2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 2, or a variant or fragment thereof;(iii) a CDR-H3 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 3, or a variant or fragment thereof;(iv) a CDR-L1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 4, or a variant or fragment thereof;(v) a CDR-L2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 5, or a variant or fragment thereof; and(vi) a CDR-L3 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 6, or a variant or fragment thereof.

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