TLR4 antibody

Abstract

The present invention relates to TLR4 antibodies. The invention extends to compositions comprising the antibodies, including pharmaceutical compositions and kits. The invention also extends to methods of making and using the antibodies, for example in therapy and diagnosis of TLR4-mediated conditions, such as neuroinflammation and lung inflammation. The invention also extends to novel biomarkers that are associated with a TLR4-mediated condition, and methods and kits for diagnosing and prognosing a subject with a TLR4-mediated condition.

Description

[0001] TLR4 Antibody

[0002] The present invention relates to TLR4 antibodies. The invention extends to compositions comprising the antibodies, including pharmaceutical compositions and kits. The invention also extends to methods of making and using the antibodies, for example in therapy and diagnosis of TLR4-mediated conditions, such as neuroinflammation and lung inflammation. The invention also extends to novel biomarkers that are associated with a TLR4-mediated condition, and methods and kits for diagnosing and prognosing a subject with a TLR4-mediated condition. TLR4 (Toll-like receptor 4) is a member of a family of related TLR proteins that play pivotal roles in the innate immune response. TLRs are a central component of the innate immune response, forming an early line of defence against invading pathogens directly, whilst also acting to orchestrate the adaptive (B-cell and T-cell mediated) immune response.

[0003] TLR4 is a receptor for pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). In fact, TLR4 molecule has the broadest spectrum of endogenous ligands (DAMPs) of all the TLR family. Examples of PAMPs include LPS and other bacterial proteins, nucleic acids or lipid-containing complexes which exist in forms not observed in eukaryotic organisms. Examples of PAMPs also include viral products such as RSV F protein. TLRs and other pattern recognition receptors (PRRs) can also respond to the presence of components released from damaged host cells (damage-associated molecular pattern receptors; DAMPs). DAMPS include extracellular matrix molecules such as hyaluronan, biglycan, fibronectin, and heparan, and intracellular and secreted molecules such as HMGB1, HSP22, 60, 70, 72, calcineurin B, [3-defensin 2, S100 proteins, surfactant protein A, fibrinogen, and amyloid-3.

[0004] TLR4 is principally known as the receptor for bacterial lipopolysaccharide (LPS; endotoxin). LPS is known to be a potent stimulator of the immune response through its interaction with TLR4, although the activation of immune signalling by LPS via TLR4 requires the presence of a number of other molecules. LPS-binding protein (LBP) is present in serum and extracts LPS from the bacterial outer membrane and transfers it to the CD14 protein which is either found on the surface of monocytes and macrophages as a glycosylphosphatidylinositol (GPI)-linked receptor or free in the serum. CD14 in turn presents LPS monomers to a complex of TLR4 and the MD-2 (Ly96) protein on the cell surface which promotes multimerization and signalling, ultimately via NFkB & IRF3, resulting in the release of pro-inflammatory cytokines.

[0005] TLR4 has been implicated in the aetiology of many diseases and conditions involving both pathogenic infections and the perturbation of normal cellular homeostasis. However, the involvement of TLR4 pathways in several conditions, such as lung inflammation diseases such as asthma, acute kidney injury (AKI), decompensated liver diseases including, but not limited to, acute-on-chronic liver failure (ACLF), is poorly understood.

[0006] There is, therefore, the need for improved therapies that target TLR4, in order to provide treatments for several conditions that are mediated by TLR4.

[0007] Accordingly, in a first aspect of the invention, there is provided an antibody, or an antigen-binding fragment thereof that specifically binds to TLR4 or a TLR4 / MD2 complex.

[0008] As shown in the Examples, the inventors have developed antibodies that are capable of binding to, and inhibiting, TLR4 function. For example, as illustrated in Figures 1 to 3, the inventors have shown that the antibodies according to the invention surprisingly inhibit TNF-a release (a cytokine that would otherwise be released upon the activation of TLR4 by LPS). The inventors have also demonstrated that the anti-TLR4 antibodies according to the invention surprisingly inhibit dextran-permeability in human bronchial epithelial (16HBE) cells when treated with HDM, illustrating that the claimed antibodies will provide an effective treatment for lung inflammation by preserving disruption or loss of the epithelial cell barrier function and / or cell-to-cell adhesion. Blocking of different ligands such as LPS and HDM is indicative that antibodies have an agnostic ligand inhibitory function.

[0009] The TLR4 gene is located on human chromosome 9 and encodes a protein product of 839 amino acids with a calculated molecular mass of 95,680 Da.

[0010] In one embodiment, the amino acid sequence of TLR4 (Accession number: NP_612564) may be provided herein as SEQ ID No: 1, as follows:

[0011] MMSASRLAGT LI PAMAFLSC VRPESWEPCV EWPNITYQC MELNFYKI PD NLPFSTKNLD LS FNPLRHLG SYSFFS FPEL QVLDLSRCEI QTIEDGAYQS LSHLSTLI LT GNPIQSLALG AFSGLSSLQK LVAVETNLAS LENFPIGHLK TLKELNVAHN LIQS FKLPEY FSNLTNLEHL DLSSNKIQS I YCTDLRVLHQ MPLLNLSLDL SLNPMNFIQP GAFKEI RLHK LTLRNNFDSL NVMKTCIQGL AGLEVHRLVL GEFRNEGNLE KFDKSALEGL CNLTIEEFRL AYLDYYLDDI IDLFNCLTNV SS FSLVSVTI ERVKDFSYNF GWQHLELVNC KFGQFPTLKL KSLKRLTFTS NKGGNAFSEV DLPSLEFLDL SRNGLSFKGC CSQSDFGTTS LKYLDLSFNG VITMSSNFLG LEQLEHLDFQ HSNLKQMSEF SVFLSLRNLI YLDI SHTHTR VAFNGI FNGL S SLEVLKMAG NS FQENFLPD I FTELRNLTF LDLSQCQLEQ LS PTAFNSLS SLQVLNMSHN NFFSLDTFPY KCLNSLQVLD YSLNHIMTSK KQELQHFPS S LAFLNLTQND FACTCEHQSF LQWI KDQRQL LVEVERMECA TPSDKQGMPV LSLNITCQMN KTI I GVSVLS VLWSWAVL VYKFYFHLML LAGCI KYGRG ENIYDAFVIY SSQDEDWVRN ELVKNLEEGV PPFQLCLHYR DFI PGVAIAA NI IHEGFHKS RKVIVWSQH FIQSRWCI FE YEIAQTWQFL SSRAGI I FIV LQKVEKTLLR QQVELYRLLS RNTYLEWEDS VLGRHI FWRR LRKALLDGKS WNPEGTVGTG CNWQEATS I

[0012] [SEQ ID No: 1] Thus, in one embodiment, the antibody or antigen-binding fragment thereof binds to one or more amino acids within a sequence comprising or consisting of a sequence as substantially set out in SEQ ID No: 1, or a variant or fragment thereof.

[0013] In one embodiment, the amino acid sequence of MD2 (Accession number NP_056179) may be provided herein as SEQ ID No: 232, as follows:

[0014] MLPFLFFSTLFSS I FTEAQKQYWVCNSSDAS I SYTYCDKMQYPI SINVNPCI ELKRSKGL LHI FYI PRRDLKQLYFNLYITVNTMNLPKRKEVICRGSDDDYSFCRALKGETVNTTI SFS FKGIKFSKGKYKCWEAI SGSPEEMLFCLEFVILHQPNSN

[0015] [SEQ ID No: 232]

[0016] Thus, it will be appreciated that the TLR4 / MD2 complex comprises or consists of a sequence as substantially set out in SEQ ID No: 1 and 232, or a variant or fragment thereof. Thus, in one embodiment, the antibody or antigen-binding fragment thereof binds to one or more amino acids within a sequence comprising or consisting of a sequence as substantially set out in SEQ ID No: 1 and 232, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof specifically binds to TLR4 alone (i.e. TLR4 when not complexed with MD2).

[0017] In some embodiments, the antibody or antigen-binding fragment thereof does not bind to TLR2. In some embodiments, the antibody or antigen-binding fragment thereof does not bind to MD2 alone (i.e. MD2 when not complexed with TLR4). In one embodiment, the antibody or antigen-binding fragment thereof recognises an epitope comprising or consisting of any one of the sequences listed in Figure 7, or a variant or fragment thereof. In one embodiment, the antibody or antigen binding fragment thereof recognises an epitope comprising or consisting of any one of SEQ ID Nos: 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191,

[0018] 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,

[0019] 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 223, 224, 225, 226, 227, 228, 229, 230 or 231, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof binds to one or more amino acids within a sequence comprising or consisting of a sequence as substantially set out in any one of SEQ ID Nos: 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,

[0020] 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 223, 224, 225, 226, 227,

[0021] 228, 229, 230 or 231, or a variant or fragment thereof. In one embodiment, the epitope may be provided herein as SEQ ID No: 179:

[0022] FNPLRHLGSYSFF

[0023] [SEQ ID No: 179] In another embodiment, the epitope may be provided herein as SEQ ID No: 180:

[0024] EFRLAYLDY

[0025] [SEQ ID No: 180] In another embodiment, the epitope may be provided herein as SEQ ID No: 210:

[0026] IEEFRLAYLDYYLD

[0027] [SEQ ID No: 210] Accordingly, in one embodiment, the antibody or antigen binding fragment thereof recognises an epitope comprising or consisting of SEQ ID No: 179, 180 or 210, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof binds to one or more amino acids within a sequence comprising or consisting of a sequence as substantially set out in SEQ ID No: 179, 180 or 210, or a variant or fragment thereof. 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. The linear epitope may be present on folded TLR4 or a TLR4 / MD2 complex.

[0028] 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.

[0029] The invention extends to both whole antibodies (i.e. immunoglobulins) with immunospecificity for TLR4, 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. TLR4, 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 bi-specific antibody (BsAb) or a chimeric antigen receptor (CAR). The heavy chain constant region typically comprises three domains, CHI, CH2, and CH3. 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.

[0030] 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

[0031] Institutes of Health, Bethesda, MD, incorporated by reference in its entirety.

[0032] 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, £, y, and p, respectively. The IgG and IgA classes are further divided into subclasses on the basis of differences in sequence and function.

[0033] Humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. In one embodiment, the antibody is of the IgG antibody class. 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.

[0034] The constant region consists of one of five heavy chain sequences (p, y, , a, or s) 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.

[0035] In some embodiments, the antibody or antigen-binding fragment thereof is isolated or purified.

[0036] In one embodiment, the antibody or antigen-binding fragment thereof comprises a polyclonal antibody, or an antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof may be generated in a rabbit, mouse or rat. In some embodiments, the antibody or antigen-binding fragment thereof is obtained by immunising a host animal with a TLR4-Fc protein, or a variant or fragment thereof, and then collecting the antibody or antigen-binding fragment thereof. The host animal may be a rabbit.

[0037] In another embodiment, the antibody or antigen-binding fragment thereof comprises a monoclonal antibody or an antigen-binding fragment thereof. The antibody or fragment thereof of may be mammalian. 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 TLR4, 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 recognisable 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 or fragment thereof maintains its function of selectively binding to TLR4, or a variant or fragment thereof. 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. 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. 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.

[0038] The antibody may be a recombinant antibody. The term "recombinant human antibody" can include a human antibody produced using recombinant DNA technology. The term "antigen-binding fragment" can mean a region of the antibody having specific binding affinity for its target antigen, for example, TLR4 or a variant or fragment thereof. In one embodiment, the fragment is an epitope. The epitope may be linear or conformational. 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 TLR4, or a fragment thereof.

[0039] 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.

[0040] 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.

[0041] 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). 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. Alternatively, as discussed hereinafter, and as exemplified, the Fc fragment of the antibody may be disabled by introducing amino acid substitutions into the Fc region, which silence or reduce the effector function of the antibody.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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. 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.

[0046] The term "Fab1fragment" 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.

[0047] 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.

[0048] 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. The term "bispecific antibody (BsAb)" can mean a bispecific antibody comprising two scFv linked to each other by a shorter linked peptide.

[0049] 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.

[0050] In one embodiment, the antibody or antigen-binding fragment thereof, with respect to the first aspect of the invention, is immunospecific for an epitope within TLR4. 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.

[0051] 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 (ab1) 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.

[0052] 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.

[0053] 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, optionally the antigen binding sites, and optionally the CDRs.

[0054] 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.

[0055] As used herein, the term "immunospecificity" can mean the binding region of the antibody or antigen-binding fragment thereof is capable of immunoreacting with

[0056] TLR4, or a variant or fragment thereof, by specifically binding therewith. The antibody or antigen-binding fragment thereof can selectively interact with an antigen (TLR4) with an affinity constant of approximately IO-5to IO-13M-1, optionally IO-6to IO-9M-1, or optionally, IO-10to IO-12M-1.

[0057] The term "immunoreact" can mean the binding region is capable of eliciting an immune response upon binding with TLR4, or an epitope thereof.

[0058] 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.

[0059] The epitope may be linear. This can mean that the antibody interacts with a plurality of continuous amino acids of the antigen, and so the epitope can consist of these defined amino acids. Alternatively, the epitope may be conformational, i.e. non-linear or discontinuous. This can mean that the antibody interacts with multiple, distinct segments from the primary amino acid sequence of the antigen.

[0060] 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;

[0061] IgG and IgM. In one embodiment, the heavy chain is an IgG. In another embodiment, the heavy chain is an IgA. 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.

[0062] As described in the Examples, the inventors have surprisingly demonstrated that the antibodies and antigen-binding fragments referred to herein, are able to significantly target TLR4, and each of these antibodies are defined below in detail. The CDR / VH / VL, HC and LC sequences of these eight antibodies are conveniently summarised in the table shown in Figure 9.

[0063] H05-56

[0064] In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as H05-56.

[0065] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 50, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 51, or a variant or fragment thereof.

[0066] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence as substantially set out in

[0067] SEQ ID No: 52, or a variant or fragment thereof.

[0068] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 50, a CDR-H2 domain comprising or consisting of SEQ ID No: 51 and / or a CDR-H3 domain comprising or consisting of SEQ ID No: 52.

[0069] In one embodiment, 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: 56, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 53, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 54, or a variant or fragment thereof.

[0070] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence as substantially set out in

[0071] SEQ ID No: 55, or a variant or fragment thereof.

[0072] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID No: 53, a CDR-L2 domain comprising or consisting of SEQ ID No: 54, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 55.

[0073] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 57, or a variant or fragment thereof.

[0074] In one embodiment, 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. In one embodiment, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0075] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 50, a CDR-H2 domain comprising or consisting of SEQ ID No: 51, a CDR-H3 domain comprising or consisting of SEQ ID No: 52, a CDR-L1 domain comprising or consisting of SEQ ID No: 53, a CDR-L2 domain comprising or consisting of SEQ ID No: 54, and a CDR-L3 domain comprising or consisting of SEQ ID No: 55.

[0076] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 56, and a light chain variable region comprising or consisting of SEQ ID No: 57. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 233, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 234, or a variant or fragment thereof.

[0077] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 233 and a light chain constant region comprising or consisting of SEQ ID No: 234.

[0078] D04-23

[0079] In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as D04-23.

[0080] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 2, or a variant or fragment thereof.

[0081] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 3, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 4, or a variant or fragment thereof.

[0082] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 2, a CDR-H2 domain comprising or consisting of SEQ ID No: 3 and / or a CDR-H3 domain comprising or consisting of SEQ ID No: 4.

[0083] In one embodiment, 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: 8, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 5, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 6, or a variant or fragment thereof.

[0084] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence as substantially set out in

[0085] SEQ ID No: 7, or a variant or fragment thereof.

[0086] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID No: 5, a CDR-L2 domain comprising or consisting of SEQ ID No: 6, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 7.

[0087] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 9, or a variant or fragment thereof.

[0088] In one embodiment, 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. In one embodiment, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0089] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 2, a CDR-H2 domain comprising or consisting of SEQ ID No: 3, a CDR-H3 domain comprising or consisting of SEQ ID No: 4, a CDR-L1 domain comprising or consisting of SEQ ID No: 5, a CDR-L2 domain comprising or consisting of SEQ ID No: 6, and a CDR-L3 domain comprising or consisting of SEQ ID No: 7.

[0090] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 8, and a light chain variable region comprising or consisting of SEQ ID No: 9. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 233, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 234, or a variant or fragment thereof.

[0091] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 233 and a light chain constant region comprising or consisting of SEQ ID No: 234.

[0092] C07-39

[0093] In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as C07-39.

[0094] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 10, or a variant or fragment thereof.

[0095] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 11, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 12, or a variant or fragment thereof.

[0096] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 10, a CDR-H2 domain comprising or consisting of SEQ ID No: 11 and / or a CDR-H3 domain comprising or consisting of SEQ ID No: 12.

[0097] In one embodiment, 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: 16, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 13, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 14, or a variant or fragment thereof.

[0098] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence as substantially set out in

[0099] SEQ ID No: 15, or a variant or fragment thereof.

[0100] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID No: 13, a CDR-L2 domain comprising or consisting of SEQ ID No: 14, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 15.

[0101] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 17, or a variant or fragment thereof.

[0102] In one embodiment, 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. In one embodiment, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0103] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 10, a CDR-H2 domain comprising or consisting of SEQ ID No: 11, a CDR-H3 domain comprising or consisting of SEQ ID No: 12, a CDR-L1 domain comprising or consisting of SEQ ID No: 13, a CDR-L2 domain comprising or consisting of SEQ ID No: 14, and a CDR-L3 domain comprising or consisting of SEQ ID No: 15.

[0104] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 16, and a light chain variable region comprising or consisting of SEQ ID No: 17. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 233, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 234, or a variant or fragment thereof.

[0105] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 233 and a light chain constant region comprising or consisting of SEQ ID No: 234.

[0106] D03-45

[0107] In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as D03-45.

[0108] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 18, or a variant or fragment thereof.

[0109] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 19, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 20, or a variant or fragment thereof.

[0110] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 18, a CDR-H2 domain comprising or consisting of SEQ ID No: 19 and / or a CDR-H3 domain comprising or consisting of SEQ ID No: 20.

[0111] In one embodiment, 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: 24, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 21, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 22, or a variant or fragment thereof.

[0112] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence as substantially set out in

[0113] SEQ ID No: 23, or a variant or fragment thereof.

[0114] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID No: 21, a CDR-L2 domain comprising or consisting of SEQ ID No: 22, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 23.

[0115] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 25, or a variant or fragment thereof.

[0116] In one embodiment, 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. In one embodiment, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0117] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 18, a CDR-H2 domain comprising or consisting of SEQ ID No: 19, a CDR-H3 domain comprising or consisting of SEQ ID No: 20, a CDR-L1 domain comprising or consisting of SEQ ID No: 21, a CDR-L2 domain comprising or consisting of SEQ ID No: 22, and a CDR-L3 domain comprising or consisting of SEQ ID No: 23.

[0118] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 24, and a light chain variable region comprising or consisting of SEQ ID No: 25. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 233, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 234, or a variant or fragment thereof.

[0119] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 233 and a light chain constant region comprising or consisting of SEQ ID No: 234.

[0120] E05-49

[0121] In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as E05-49.

[0122] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 26, or a variant or fragment thereof.

[0123] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 27, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 28, or a variant or fragment thereof.

[0124] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 26, a CDR-H2 domain comprising or consisting of SEQ ID No: 27 and / or a CDR-H3 domain comprising or consisting of SEQ ID No: 28.

[0125] In one embodiment, 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: 32, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 29, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 30, or a variant or fragment thereof.

[0126] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence as substantially set out in

[0127] SEQ ID No: 31, or a variant or fragment thereof.

[0128] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID No: 29, a CDR-L2 domain comprising or consisting of SEQ ID No: 30, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 31.

[0129] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 33, or a variant or fragment thereof.

[0130] In one embodiment, 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. In one embodiment, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0131] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 26, a CDR-H2 domain comprising or consisting of SEQ ID No: 27, a CDR-H3 domain comprising or consisting of SEQ ID No: 28, a CDR-L1 domain comprising or consisting of SEQ ID No: 29, a CDR-L2 domain comprising or consisting of SEQ ID No: 30, and a CDR-L3 domain comprising or consisting of SEQ ID No: 31.

[0132] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 32, and a light chain variable region comprising or consisting of SEQ ID No: 33. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 233, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 234, or a variant or fragment thereof.

[0133] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 233 and a light chain constant region comprising or consisting of SEQ ID No: 234.

[0134] E04-50

[0135] In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as E04-50.

[0136] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 34, or a variant or fragment thereof.

[0137] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 35, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 36, or a variant or fragment thereof.

[0138] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 34, a CDR-H2 domain comprising or consisting of SEQ ID No: 35 and / or a CDR-H3 domain comprising or consisting of SEQ ID No: 36.

[0139] In one embodiment, 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: 40, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 37, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 38, or a variant or fragment thereof.

[0140] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence as substantially set out in

[0141] SEQ ID No: 39, or a variant or fragment thereof.

[0142] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID No: 37, a CDR-L2 domain comprising or consisting of SEQ ID No: 38, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 39.

[0143] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 41, or a variant or fragment thereof.

[0144] In one embodiment, 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. In one embodiment, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0145] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 34, a CDR-H2 domain comprising or consisting of SEQ ID No: 35, a CDR-H3 domain comprising or consisting of SEQ ID No: 36, a CDR-L1 domain comprising or consisting of SEQ ID No: 37, a CDR-L2 domain comprising or consisting of SEQ ID No: 38, and a CDR-L3 domain comprising or consisting of SEQ ID No: 39.

[0146] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 40, and a light chain variable region comprising or consisting of SEQ ID No: 41. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 233, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 234, or a variant or fragment thereof.

[0147] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 233 and a light chain constant region comprising or consisting of SEQ ID No: 234.

[0148] D07-56

[0149] In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as D07-56.

[0150] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 42, or a variant or fragment thereof.

[0151] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 43, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 44, or a variant or fragment thereof.

[0152] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 42, a CDR-H2 domain comprising or consisting of SEQ ID No: 43 and / or a CDR-H3 domain comprising or consisting of SEQ ID No: 44.

[0153] In one embodiment, 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: 48, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 45, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 46, or a variant or fragment thereof.

[0154] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence as substantially set out in

[0155] SEQ ID No: 47, or a variant or fragment thereof.

[0156] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID No: 45, a CDR-L2 domain comprising or consisting of SEQ ID No: 46, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 47.

[0157] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 49, or a variant or fragment thereof.

[0158] In one embodiment, 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. In one embodiment, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0159] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 42, a CDR-H2 domain comprising or consisting of SEQ ID No: 43, a CDR-H3 domain comprising or consisting of SEQ ID No: 44, a CDR-L1 domain comprising or consisting of SEQ ID No: 45, a CDR-L2 domain comprising or consisting of SEQ ID No: 46, and a CDR-L3 domain comprising or consisting of SEQ ID No: 47.

[0160] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 48, and a light chain variable region comprising or consisting of SEQ ID No: 49. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 233, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 234, or a variant or fragment thereof.

[0161] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 233 and a light chain constant region comprising or consisting of SEQ ID No: 234.

[0162] B02-62 VH

[0163] In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as B02-62 VH.

[0164] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 58, or a variant or fragment thereof.

[0165] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 59, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 60, or a variant or fragment thereof.

[0166] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 58, a CDR-H2 domain comprising or consisting of SEQ ID No: 59 and / or a CDR-H3 domain comprising or consisting of SEQ ID No: 60.

[0167] In one embodiment, 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: 64, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 61, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 62, or a variant or fragment thereof.

[0168] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence as substantially set out in

[0169] SEQ ID No: 63, or a variant or fragment thereof.

[0170] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID No: 61, a CDR-L2 domain comprising or consisting of SEQ ID No: 62, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 63.

[0171] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 65, or a variant or fragment thereof.

[0172] In one embodiment, 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. In one embodiment, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0173] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 58, a CDR-H2 domain comprising or consisting of SEQ ID No: 59, a CDR-H3 domain comprising or consisting of SEQ ID No: 60, a CDR-L1 domain comprising or consisting of SEQ ID No: 61, a CDR-L2 domain comprising or consisting of SEQ ID No: 62, and a CDR-L3 domain comprising or consisting of SEQ ID No: 63.

[0174] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 64, and a light chain variable region comprising or consisting of SEQ ID No: 65. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 233, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 234, or a variant or fragment thereof.

[0175] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 233 and a light chain constant region comprising or consisting of SEQ ID No: 234.

[0176] B02-62 VH (2 / 2)

[0177] In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as B02-62 VH (2 / 2).

[0178] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 66, or a variant or fragment thereof.

[0179] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 67, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 68, or a variant or fragment thereof.

[0180] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 66, a CDR-H2 domain comprising or consisting of SEQ ID No: 67 and / or a CDR-H3 domain comprising or consisting of SEQ ID No: 68.

[0181] In one embodiment, 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: 72, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 69, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 70, or a variant or fragment thereof.

[0182] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence as substantially set out in

[0183] SEQ ID No: 71, or a variant or fragment thereof.

[0184] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID No: 69, a CDR-L2 domain comprising or consisting of SEQ ID No: 70, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 71.

[0185] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 73, or a variant or fragment thereof.

[0186] In one embodiment, 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. In one embodiment, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0187] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 66, a CDR-H2 domain comprising or consisting of SEQ ID No: 67, a CDR-H3 domain comprising or consisting of SEQ ID No: 68, a CDR-L1 domain comprising or consisting of SEQ ID No: 69, a CDR-L2 domain comprising or consisting of SEQ ID No: 70, and a CDR-L3 domain comprising or consisting of SEQ ID No: 71.

[0188] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 72, and a light chain variable region comprising or consisting of SEQ ID No: 73. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 233, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 234, or a variant or fragment thereof.

[0189] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 233 and a light chain constant region comprising or consisting of SEQ ID No: 234.

[0190] C12-73

[0191] In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as C12-73.

[0192] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 74, or a variant or fragment thereof.

[0193] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 75, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 76, or a variant or fragment thereof.

[0194] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 74, a CDR-H2 domain comprising or consisting of SEQ ID No: 75 and / or a CDR-H3 domain comprising or consisting of SEQ ID No: 76.

[0195] In one embodiment, 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: 80, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 77, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 78, or a variant or fragment thereof.

[0196] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence as substantially set out in

[0197] SEQ ID No: 79, or a variant or fragment thereof.

[0198] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID No: 77, a CDR-L2 domain comprising or consisting of SEQ ID No: 78, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 79.

[0199] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 81, or a variant or fragment thereof.

[0200] In one embodiment, 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. In one embodiment, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0201] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 74, a CDR-H2 domain comprising or consisting of SEQ ID No: 75, a CDR-H3 domain comprising or consisting of SEQ ID No: 76, a CDR-L1 domain comprising or consisting of SEQ ID No: 77, a CDR-L2 domain comprising or consisting of SEQ ID No: 78, and a CDR-L3 domain comprising or consisting of SEQ ID No: 79.

[0202] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 80, and a light chain variable region comprising or consisting of SEQ ID No: 81. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 233, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 234, or a variant or fragment thereof.

[0203] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 233 and a light chain constant region comprising or consisting of SEQ ID No: 234.

[0204] H02-68

[0205] In one embodiment, the antibody or antigen-binding fragment thereof is referred to herein as H02-68.

[0206] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 82, or a variant or fragment thereof.

[0207] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 83, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 84, or a variant or fragment thereof.

[0208] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 82, a CDR-H2 domain comprising or consisting of SEQ ID No: 83 and / or a CDR-H3 domain comprising or consisting of SEQ ID No: 84.

[0209] In one embodiment, 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: 88, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 85, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 86, or a variant or fragment thereof.

[0210] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence as substantially set out in

[0211] SEQ ID No: 87, or a variant or fragment thereof.

[0212] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID No: 85, a CDR-L2 domain comprising or consisting of SEQ ID No: 86, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 87.

[0213] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 89, or a variant or fragment thereof.

[0214] In one embodiment, 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. In one embodiment, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0215] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 82, a CDR-H2 domain comprising or consisting of SEQ ID No: 83, a CDR-H3 domain comprising or consisting of SEQ ID No: 84, a CDR-L1 domain comprising or consisting of SEQ ID No: 85, a CDR-L2 domain comprising or consisting of SEQ ID No: 86, and a CDR-L3 domain comprising or consisting of SEQ ID No: 87.

[0216] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 88, and a light chain variable region comprising or consisting of SEQ ID No: 89. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 233, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 234, or a variant or fragment thereof.

[0217] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 233 and a light chain constant region comprising or consisting of SEQ ID No: 234.

[0218] Humanised antibody sequences

[0219] The inventors then set out to generate humanised antibodies of H05-56, and the sequences of the humanised antibodies are illustrated in Figure 10. Unless stated otherwise, the six CDR sequences of all of the humanised antibodies are identical to the six CDR sequences of the antibody W30047-cAbl (H05-56), i.e. SEQ ID Nos: 90-95. W30047-cAbl (H05-56)

[0220] In one embodiment, the humanised antibody or antigen-binding fragment thereof is referred to herein as W30047-cAbl (H05-56).

[0221] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 90, or a variant or fragment thereof.

[0222] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 91, or a variant or fragment thereof.

[0223] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H3 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 92, or a variant or fragment thereof.

[0224] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 90, a CDR-H2 domain comprising or consisting of SEQ ID No: 91 and / or a CDR-H3 domain comprising or consisting of SEQ ID No: 92.

[0225] In one embodiment, 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: 96, or a variant or fragment thereof.

[0226] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 93, or a variant or fragment thereof.

[0227] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L2 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 94, or a variant or fragment thereof.

[0228] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L3 domain comprising or consisting of a sequence as substantially set out in SEQ ID No: 95, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-L1 domain comprising or consisting of SEQ ID No: 93, a CDR-L2 domain comprising or consisting of SEQ ID No: 94, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 95. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 97, or a variant or fragment thereof.

[0229] In one embodiment, 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. In one embodiment, the antibody or antigen-binding fragment thereof comprises at least CDR-H3.

[0230] In one embodiment, the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 90, a CDR-H2 domain comprising or consisting of SEQ ID No: 91, a CDR-H3 domain comprising or consisting of SEQ ID No: 92, a CDR-L1 domain comprising or consisting of SEQ ID No: 93, a CDR-L2 domain comprising or consisting of SEQ ID No: 94, and a CDR-L3 domain comprising or consisting of SEQ ID No: 95.

[0231] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 96, and a light chain variable region comprising or consisting of SEQ ID No: 97.

[0232] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 98, or a variant or fragment thereof.

[0233] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 99, or a variant or fragment thereof.

[0234] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 98 and a light chain constant region comprising or consisting of SEQ ID No: 99. W30047-cAbl-z0

[0235] In one embodiment, the humanised antibody or antigen-binding fragment thereof is referred to herein as W30047-cAbl-z0.

[0236] In one embodiment, 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: 100, or a variant or fragment thereof.

[0237] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 101, or a variant or fragment thereof.

[0238] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 100, and a light chain variable region comprising or consisting of SEQ ID No: 101. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 102, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 103, or a variant or fragment thereof.

[0239] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 102 and a light chain constant region comprising or consisting of SEQ ID No: 103.

[0240] W30047-cAbl-zl

[0241] In one embodiment, the humanised antibody or antigen-binding fragment thereof is referred to herein as W30047-cAbl-zl.

[0242] In one embodiment, 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: 104, or a variant or fragment thereof.

[0243] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 105, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 104, and a light chain variable region comprising or consisting of SEQ ID No: 105.

[0244] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 106, or a variant or fragment thereof.

[0245] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 107, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 106 and a light chain constant region comprising or consisting of SEQ ID No: 107. W30047-cAbl-z2

[0246] In one embodiment, the humanised antibody or antigen-binding fragment thereof is referred to herein as W30047-cAbl-z2.

[0247] In one embodiment, 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: 108, or a variant or fragment thereof.

[0248] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 109, or a variant or fragment thereof.

[0249] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 108, and a light chain variable region comprising or consisting of SEQ ID No: 109.

[0250] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 110, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 111, or a variant or fragment thereof.

[0251] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 110 and a light chain constant region comprising or consisting of SEQ ID No: 111.

[0252] W30047-cAbl-z3

[0253] In one embodiment, the humanised antibody or antigen-binding fragment thereof is referred to herein as W30047-cAbl-z3. In one embodiment, 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: 112, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 113, or a variant or fragment thereof.

[0254] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 112, and a light chain variable region comprising or consisting of SEQ ID No: 113.

[0255] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 114, or a variant or fragment thereof.

[0256] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 115, or a variant or fragment thereof.

[0257] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 114 and a light chain constant region comprising or consisting of SEQ ID No: 115. W30047-cAbl-z4

[0258] In one embodiment, the humanised antibody or antigen-binding fragment thereof is referred to herein as W30047-cAbl-z4.

[0259] In one embodiment, 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: 116, or a variant or fragment thereof.

[0260] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 117, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 116, and a light chain variable region comprising or consisting of SEQ ID No: 117. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 118, or a variant or fragment thereof.

[0261] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 119, or a variant or fragment thereof.

[0262] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 118 and a light chain constant region comprising or consisting of SEQ ID No: 119.

[0263] W30047-cAbl-z5

[0264] In one embodiment, the humanised antibody or antigen-binding fragment thereof is referred to herein as W30047-cAbl-z5.

[0265] In one embodiment, 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: 120, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 121, or a variant or fragment thereof.

[0266] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 120, and a light chain variable region comprising or consisting of SEQ ID No: 121.

[0267] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 122, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 123, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 122 and a light chain constant region comprising or consisting of SEQ ID No: 123.

[0268] W30047-cAbl-z6 In one embodiment, the humanised antibody or antigen-binding fragment thereof is referred to herein as W30047-cAbl-z6.

[0269] In one embodiment, 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: 124, or a variant or fragment thereof.

[0270] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 125, or a variant or fragment thereof.

[0271] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 124, and a light chain variable region comprising or consisting of SEQ ID No: 125. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 126, or a variant or fragment thereof.

[0272] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 127, or a variant or fragment thereof.

[0273] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 126 and a light chain constant region comprising or consisting of SEQ ID No: 127.

[0274] W30047-cAbl-z7 In one embodiment, the humanised antibody or antigen-binding fragment thereof is referred to herein as W30047-cAbl-z7.

[0275] In one embodiment, 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: 128, or a variant or fragment thereof.

[0276] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 129, or a variant or fragment thereof.

[0277] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 128, and a light chain variable region comprising or consisting of SEQ ID No: 129.

[0278] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 130, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 131, or a variant or fragment thereof.

[0279] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 130 and a light chain constant region comprising or consisting of SEQ ID No: 131.

[0280] W30047-cAbl-z8

[0281] In one embodiment, the humanised antibody or antigen-binding fragment thereof is referred to herein as W30047-cAbl-z8.

[0282] In one embodiment, 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: 132, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 133, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 132, and a light chain variable region comprising or consisting of SEQ ID No: 133.

[0283] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 134, or a variant or fragment thereof.

[0284] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 135, or a variant or fragment thereof.

[0285] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 134 and a light chain constant region comprising or consisting of SEQ ID No: 135.

[0286] W30047-cAbl-z9

[0287] In one embodiment, the humanised antibody or antigen-binding fragment thereof is referred to herein as W30047-cAbl-z9. In one embodiment, 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: 136, or a variant or fragment thereof.

[0288] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 137, or a variant or fragment thereof.

[0289] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 136, and a light chain variable region comprising or consisting of SEQ ID No: 137. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 138, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 139, or a variant or fragment thereof.

[0290] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 138 and a light chain constant region comprising or consisting of SEQ ID No: 139.

[0291] W30047-cAbl-zl0

[0292] In one embodiment, the humanised antibody or antigen-binding fragment thereof is referred to herein as W30047-cAbl-zl0.

[0293] In one embodiment, 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: 140, or a variant or fragment thereof.

[0294] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 141, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 140, and a light chain variable region comprising or consisting of SEQ ID No: 141.

[0295] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 142, or a variant or fragment thereof.

[0296] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 143, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 142 and a light chain constant region comprising or consisting of SEQ ID No: 143. W30047-cAbl-zll

[0297] In one embodiment, the humanised antibody or antigen-binding fragment thereof is referred to herein as W30047-cAbl-zll.

[0298] In one embodiment, 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: 144, or a variant or fragment thereof.

[0299] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 145, or a variant or fragment thereof.

[0300] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 144, and a light chain variable region comprising or consisting of SEQ ID No: 145.

[0301] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 146, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 147, or a variant or fragment thereof.

[0302] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 146 and a light chain constant region comprising or consisting of SEQ ID No: 147.

[0303] W30047-cAbl-zl2

[0304] In one embodiment, the humanised antibody or antigen-binding fragment thereof is referred to herein as W30047-cAbl-zl2. In one embodiment, 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: 148, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 149, or a variant or fragment thereof.

[0305] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 148, and a light chain variable region comprising or consisting of SEQ ID No: 149.

[0306] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 150, or a variant or fragment thereof.

[0307] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 151, or a variant or fragment thereof.

[0308] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 150 and a light chain constant region comprising or consisting of SEQ ID No: 151. W30047-cAbl-zl3

[0309] In one embodiment, the humanised antibody or antigen-binding fragment thereof is referred to herein as W30047-cAbl-zl3.

[0310] In one embodiment, 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: 152, or a variant or fragment thereof.

[0311] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 153, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 152, and a light chain variable region comprising or consisting of SEQ ID No: 153. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 154, or a variant or fragment thereof.

[0312] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 155, or a variant or fragment thereof.

[0313] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 154 and a light chain constant region comprising or consisting of SEQ ID No: 155.

[0314] W30047-cAbl-zl4

[0315] In one embodiment, the humanised antibody or antigen-binding fragment thereof is referred to herein as W30047-cAbl-zl4.

[0316] In one embodiment, 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: 156, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 157, or a variant or fragment thereof.

[0317] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 156, and a light chain variable region comprising or consisting of SEQ ID No: 157.

[0318] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 158, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 159, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 158 and a light chain constant region comprising or consisting of SEQ ID No: 159.

[0319] W30047-cAbl-zl5 In one embodiment, the humanised antibody or antigen-binding fragment thereof is referred to herein as W30047-cAbl-zl5.

[0320] In one embodiment, 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: 160, or a variant or fragment thereof.

[0321] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 161, or a variant or fragment thereof.

[0322] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 160, and a light chain variable region comprising or consisting of SEQ ID No: 161. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 162, or a variant or fragment thereof.

[0323] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 163, or a variant or fragment thereof.

[0324] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 162 and a light chain constant region comprising or consisting of SEQ ID No: 163.

[0325] W30047-cAbl-zl6 In one embodiment, the humanised antibody or antigen-binding fragment thereof is referred to herein as W30047-cAbl-zl6.

[0326] In one embodiment, 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: 164, or a variant or fragment thereof.

[0327] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 165, or a variant or fragment thereof.

[0328] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 164, and a light chain variable region comprising or consisting of SEQ ID No: 165.

[0329] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 166, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 167, or a variant or fragment thereof.

[0330] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 166 and a light chain constant region comprising or consisting of SEQ ID No: 167.

[0331] W30047-cAbl-zl7

[0332] In one embodiment, the humanised antibody or antigen-binding fragment thereof is referred to herein as W30047-cAbl-zl7.

[0333] In one embodiment, 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: 168, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable (VL) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 169, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of SEQ ID No: 168, and a light chain variable region comprising or consisting of SEQ ID No: 169.

[0334] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant (HC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 170, or a variant or fragment thereof.

[0335] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant (LC) region comprising or consisting of a sequence as substantially set out in SEQ ID No: 171, or a variant or fragment thereof.

[0336] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising or consisting of SEQ ID No: 170 and a light chain constant region comprising or consisting of SEQ ID No: 171.

[0337] 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 sequences are selected from the group consisting of SEQ ID NOs: 2, 10, 18, 26, 34, 42, 50, 58, 66, 74, 82 and 90, 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 sequences are selected from the group consisting of SEQ ID NOs: 3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 83 and 91, 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 sequences are selected from the group consisting of SEQ ID NOs: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84 and 92, 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 sequences are selected from the group consisting of SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85 and 93, 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 sequences are selected from the group consisting of SEQ ID NOs: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 86 and 94, 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 sequences are selected from the group consisting of SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87 and 95, or variant sequences thereof comprising 1, 2, 3, 4 or 5 amino acid changes, and conservative modifications thereof. 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 an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164 and 168; 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 an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149,

[0338] 153, 157, 161, 165 and 169. 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 selected from the group consisting of SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164 and 168; 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 selected from the group consisting of SEQ ID NOs: 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165 and 169. In this context, the term "change" refers to insertions, deletions and / or substitutions.

[0339] The antibody of the invention may comprise or consist of any sequence as defined in any one of Figures 8 to 9. In one embodiment, the antibody or antigen-binding fragment according to the first aspect, may comprise a mutated Fc fragment. For example, the antibody or antigen-binding fragment may be mutated by introducing amino acid substitutions into the Fc region, which silence or reduce the effector function of the antibody, and / or enhance Fc function. Accordingly, in one embodiment, the Fc fragment comprises one or more amino acid substitution that silences or reduces the effector function of the antibody or antigen-binding fragment thereof. In one embodiment, the Fc fragment comprises one or more amino acid substitution selected from the group consisting of: L234A, L235A, and P329G, according to EU numbering. In one embodiment, the Fc fragment comprises the amino acid substitutions L234A, L235A, and P329G. In one embodiment, the Fc fragment comprises the amino acid substitutions L234A and L235A.

[0340] In another embodiment, the Fc fragment comprises one or more amino acid substitution selected from the group consisting of: N325S and L328F, according to EU numbering. In one embodiment, the Fc fragment comprises the amino acid substitutions N325S and L328F.

[0341] In another embodiment, the Fc fragment comprises one or more amino acid substitution selected from the group consisting of: M252Y, S254T, and T256E, according to EU numbering. In one embodiment, the Fc fragment comprises the amino acid substitutions M252Y, S254T, and T256E. Advantageously, these amino acid substitutions improve antibody affinity.

[0342] In one embodiment, the antibody or antigen binding fragment thereof of the invention is capable of inhibiting TLR4 function. In one embodiment, the antibody or antigen binding fragment thereof of the invention is capable of inhibiting the TLR4 / MD2 signalling cascade. In one embodiment, inhibition of the TLR4 / MD2 signalling cascade includes inhibition of MyD88-dependent and MyD88-independent TLR4 signalling pathways. These pathways mediate the activation of proinflammatory cytokines and Type I interferon genes following activation of several transcription factors (NF-kB, CREB, AP-1 and IRF). In another embodiment, inhibition of the TLR4 / MD2 signalling cascade can include inhibition of pro- inflammatory molecules release, inhibition of inflammasome activation, inhibition of immune cell infiltration to tissues, inhibition of immune-mediated tissue damage that can lead to organ failure, inhibition of immune cell proliferation, inhibition of immune cell differentiation, and / or inhibition of the generation of DAMPs. In one embodiment, the antibody or antigen binding fragment thereof of the invention is capable of inhibiting LPS, exogenous and endogenous DAMPs induced cytokine release.

[0343] LPS induces changes in electric field stimulation (EFS), which can be reversed by the antibody according to the invention. Accordingly, in one embodiment, the antibody or antigen binding fragment thereof of the invention is capable of reversing changes in EFS induced by LPS.

[0344] In one embodiment, the antibody or antigen binding fragment thereof of the invention is capable of inhibiting HDM. In one embodiment, the antibody or antigen-binding fragment thereof is ligandagnostic.

[0345] In one embodiment, the antibody or antigen binding fragment thereof specifically binds to cynomolgus TLR4 or a cynomolgus TLR4 / MD2 complex. In another embodiment, the antibody or antigen binding fragment thereof specifically binds to human TLR4 or a human TLR4 TLR4 / MD2 complex.

[0346] In some embodiments, the affinity of the antibody or antigen binding fragment thereof for TLR4 as indicated by KD, is less than about IO-5M, less than about IO-6M, less than about IO-7M, less than about IO-8M, less than about IO-9M, less than about IO-10M, less than about IO-11M, less than about IO-12M, or less than about 1013M.

[0347] In one embodiment, the affinity of the antibody or antigen binding fragment thereof for TLR4 is less than 6000 pM, less than 5000 pM, less than 4000 pM, or less than 3000 pM. In another embodiment, the affinity of the antibody or antigen binding fragment thereof for TLR4 is less than 2800 pM, less than 2600 pM, less than 2400 pM, less than 2200 pM, or less than 2000 pM. In another embodiment, the affinity of the antibody or antigen binding fragment thereof for TLR4 is less than 1800 pM, less than 1600 pM, less than 1400 pM, less than 1200 pM, or less than 1000 pM. In another embodiment, the affinity of the antibody or antigen binding fragment thereof for TLR4 is less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, less than 400 pM, or less than 300 pM.

[0348] In one embodiment, the affinity of the antibody or antigen binding fragment thereof for TLR4 is between 200 and 3500 pM, between 200 and 3000 pM, between 200 and 2500 pM, between 200 and 2000 pM, between 200 and 1500 pM, between 200 and 1000 pM, or between 200 and 500 pM.

[0349] In some embodiments, the affinity of the antibody or antigen binding fragment thereof for the TLR4 / MD2 complex as indicated by KD, is less than about IO-5M, less than about IO-6M, less than about 10-7M, less than about IO-8M, less than about 10-9M, less than about 10-10M, less than about 10-11M, less than about 10-12M, or less than about IO-13M. In one embodiment, the affinity of the antibody or antigen binding fragment thereof for the TLR4 / MD2 complex is less than 10-12M.

[0350] In one embodiment, the affinity of the antibody or antigen binding fragment thereof for the TLR4 / MD2 complex is less than 7000 pM, less than 6000 pM, less than 5000 pM, less than 4000 pM, or less than 3000 pM. In another embodiment, the affinity of the antibody or antigen binding fragment thereof for TLR4 is less than 2800 pM, less than 2600 pM, less than 2400 pM, less than 2200 pM, or less than 2000 pM. In another embodiment, the affinity of the antibody or antigen binding fragment thereof for TLR4 is less than 1800 pM, less than 1600 pM, less than 1400 pM, less than 1200 pM, or less than 1000 pM. In another embodiment, the affinity of the antibody or antigen binding fragment thereof for TLR4 is less than 800 pM, less than 700 pM, less than 600 pM, less than 500 pM, or less than 400 pM.

[0351] In one embodiment, the affinity of the antibody or antigen binding fragment thereof for the TLR4 / MD2 complex is between 300 and 4500 pM, between 300 and 4000 pM, between 300 and 3500 pM, between 300 and 3000 pM, between 300 and 2500 pM, between 300 and 2000 pM, between 300 and 1500 pM, between 300 and 1000 pM, or between 300 and 500 pM.

[0352] In one embodiment, the antibody or antigen binding fragment thereof is capable of inhibiting cytokine release, e.g. TNF-a, IL-6.

[0353] In one embodiment, the antibody or antigen binding fragment thereof is capable of inhibiting TNF-a release. In some embodiments, the antibody or antigen binding fragment thereof inhibits TNF-a release by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%. In one embodiment, the antibody or antigen binding fragment thereof inhibits TNF- a release, such that the concentration of TNF-a is less than 4000 pg / ml, less than 3500 pg / ml, less than 3000 pg / ml, less than 2500 pg / ml, less than 2000 pg / ml, less than 1500 pg / ml, or less than 1000 pg / ml. In one embodiment, the antibody or antigen binding fragment thereof inhibits TNF-a release in human PBMCs, such that the concentration of TNF-a is between 1000 and 3000 pg / ml. In one embodiment, the antibody or antigen binding fragment thereof inhibits TNF-a release in microglia, such that the concentration of TNF-a is between 2000 and 4000 pg / ml. The inventors have also demonstrated that the anti-TLR4 antibodies according to the invention surprisingly inhibit dextran-permeability in human bronchial epithelial (16HBE) cells when treated with HDM, illustrating that the claimed antibodies will provide an effective treatment for lung inflammation by preserving disruption or loss of the epithelial cell barrier function and / or cell-to-cell adhesion. Epithelial cells form "tight junctions" and control the access of molecules from the lumen. They also control the infiltration of immune cells, i.e. neutrophils. Barrier epithelial cells (ECs) represent the very first line of defence and express pattern recognition receptors, such as TLR4, to recognize type-2-cell-mediated immune insults like proteolytic allergens. The Toll-like receptor-4 (TLR4) recognizes the pathogen- and damage-associated molecular patterns released during lung injury and inflammation and coordinates cellular responses, including neutrophil and type-2- cell-mediated immunity, characterized by the presence of eosinophils, basophils, mast cells, CD4+ T helper 2 (Th2) cells, and type 2 innate lymphoid cells (ILC2s). Accordingly, in another embodiment, the antibody or antigen binding fragment thereof is capable of inhibiting dextran-permeability. In some embodiments, the antibody or antigen binding fragment thereof inhibits dextran-permeability by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%.

[0354] The inventors have also demonstrated that the anti-TLR4 antibodies according to the invention can be used for the treatment of several liver indications. In one embodiment, the antibody or antigen binding fragment thereof is capable of improving hepatocyte viability in the liver. In one embodiment, the antibody or antigen binding fragment thereof inhibits the release of serum alanine transaminase (ALT) and creatinine in the liver.

[0355] The inventors have also demonstrated that the anti-TLR4 antibodies according to the invention can be used for the treatment of respiratory diseases. In one embodiment, the antibody or antigen binding fragment thereof is capable of reducing lympho-plasmacytic and alveolar macrophage infiltration. In one embodiment, the antibody or antigen binding fragment thereof inhibits recruitment of immune cells including interstitial macrophages, neutrophils and eosinophils, and y6 T-cells, to the lungs. In one embodiment, the antibody or antigen binding fragment thereof inhibits recruitment of CD68+ CD206+ cells and CD20+ cells to the lungs. In one embodiment, the antibody or antigen binding fragment thereof inhibits recruitment of CD45+CD68+CD44+ cells, to the lungs. In one embodiment, the antibody or antigen binding fragment thereof inhibits perivascular recruitment of CD45+CD44+CD206+CD68+ cells, to the lungs. In one embodiment, the antibody or antigen binding fragment thereof inhibits perivascular recruitment of CD45+CD20+ cells, to the lungs. In one embodiment, the antibody or antigen binding fragment thereof inhibits bronchiolar hypertrophy / hyperplasia and smooth muscle cell hyperplasia / hypertrophy. In one embodiment, the antibody or antigen binding fragment thereof inhibits mucus production in the lungs.

[0356] In some embodiments, the antibody or antigen binding fragment thereof is thermally stable. In some embodiments, the antibody or antigen binding fragment thereof has a Tml of greater than 65°C, 66°C, 67°C, 68°C, or 69°C. In some embodiments, the antibody or antigen binding fragment thereof has a Tml of greater than 70°C, 71°C, 72°C, or 73°C. Advantageously, the anti-TLR4 activity of the antibody or antigen-binding fragment according to the first aspect of the invention means that it has significant utility as a therapeutic agent in its own right, and may be used in the treatment, amelioration or prevention of a condition involving TLR4 activity. Accordingly, in a second aspect of the invention, there is provided an antibody or antigen-binding fragment thereof according to the first aspect, for use in therapy. In a third aspect of the invention, there is provided an antibody or antigen-binding fragment thereof according to the first aspect, for use in treating, preventing or ameliorating a TLR4-mediated condition. According to a fourth aspect of the invention, there is provided a method of treating, preventing or ameliorating a TLR4-mediated condition in a subject, the method comprising administering, or having administered, to a patient in need of such treatment, a therapeutically effective amount of an antibody or antigenbinding fragment thereof according to the first aspect.

[0357] It will be appreciated by the skilled person that a "TLR4-mediated condition" may be a condition where TLR4 recognises the pathogen- and damage-associated molecular pattern like PAMPs and DAMPs in inflammation and coordinates cellular responses, including neutrophil and type-2-cell-mediated immunity, characterized by the presence of eosinophils, basophils, mast cells, CD4+ T helper 2 (Th2) cells, and type 2 innate lymphoid cells (ILC2s). Additionally, a "TLR4-mediated condition" can also be defined as a condition involving inflammation driving tissue damage, barrier damage, loss of cell-cell adhesion, loss of epithelial barrier function, immune cell infiltration (e.g. neutrophils), and / or where epithelial cell biology plays a key role.

[0358] A "TLR4 mediated condition" may be one where TLR4 expression is increased. This increased expression of TLR4 may lead to the disruption of tight junction proteins (e.g. coxsackievirus and adenovirus receptor), and / or compromised barrier function and epithelial function during lung or skin injury.

[0359] A "TLR4 mediated condition" may also be defined as a condition that is triggered by exogenous and endogenous ligands that activate and increase TLR4 expression, cause systemic inflammation, release of pro-inflammatory molecules, immune cell triggered tissue damage, and / or immune mediated tissue damage, including cell death or organ failure.

[0360] The TLR4-mediated condition may be selected from a group of conditions consisting of: neuroinflammation including infection (e.g. encephalitis or meningitis) and neurodegenerative disorders (e.g. Alzheimer's and Parkinson's disease); skin inflammation such as atopic dermatitis or psoriasis; an infectious disease (e.g. a bacterial or viral infection), such as sepsis; lung inflammation and allergic airway diseases such as asthma, COPD, idiopathic pulmonary fibrosis; acute lung injury; acute respiratory distress syndrome; long COVID; Pulmonary hypertension; other inflammatory diseases such as Crohn's / IBD, systemic sclerosis, and rheumatoid arthritis; acute pancreatitis; Hepatic fibrosis; Metabolic dysfunction-associated steatotic liver disease (MASLD); compensated liver diseases; decompensated liver diseases; liver failure; acute liver injury; liver cirrhosis, acute-on-chronic liver failure (ACLF); alcohol-associated liver disease (ALD); steatotic liver disease (SLD); metabolic dysfunction and alcohol-associated liver disease (MetALD); metabolic dysfunction-associated steatohepatitis (MASH); cryptogenic SLD; alcohol liver disease; hepatocellular carcinoma; renal fibrosis; renal failure; acute kidney injury; chronic kidney disease; ischaemia / reperfusion injury; end stage renal failure; and cardiac disease. The cardiac disease may be myocardial inflammation, including myocarditis, MI, I / R injury, heart failure, aortic valve diseases, atherosclerosis, viral myocarditis (VMC), and / or thrombosis.

[0361] It will be appreciated that antibodies or antigen-binding fragments thereof according to the invention (referred to herein as "agents") may be used in a monotherapy (e.g. the use of an antibody alone), for treating, ameliorating or preventing the TLR-mediated condition. Alternatively, agents according to the invention may be used as an adjunct to, or in combination with, known therapies for treating, ameliorating, or preventing a TLR-mediated condition, such as in combination with drugs that promote hepatocyte regeneration.

[0362] 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.

[0363] Medicaments comprising agents of the invention may be used in a number of ways.

[0364] 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.

[0365] 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).

[0366] In one embodiment, agents and medicaments according to the invention may be 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).

[0367] It will be appreciated that the amount of the antibody (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 the condition. 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.

[0368] Generally, a daily dose of between O.Olpg / kg of body weight and lOOmg / kg of body weight of agent according to the invention may be used for treating, ameliorating, or preventing the TLR4-mediated condition, depending upon which agent. In some embodiments, the daily dose of agent is between 1 p g / kg of body weight and lOOmg / kg of body weight, optionally between lOpg / kg and lOmg / kg body weight, and optionally between approximately 100 p g / kg and lOmg / kg body weight. The agent may be administered before, during or after onset of the TLR-mediated condition. Daily doses may be given as a single administration (e.g. a single daily injection). Alternatively, the agent may require administration twice or more times during a day. As an example, agents may be administered as two (or more depending upon the severity of the condition being treated) daily doses of between 0.07pg and 700 mg (i.e. assuming a body weight of 70 kg). A patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3- or 4-hourly intervals thereafter.

[0369] Alternatively, a slow release device may be used to provide optimal doses of agents according to the invention to a patient without the need to administer repeated doses. 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).

[0370] In a fifth aspect of the invention, there is provided a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to the first aspect, and optionally a pharmaceutically acceptable vehicle.

[0371] The pharmaceutical composition may be anti-TLR4, i.e. a pharmaceutical formulation used in the therapeutic amelioration, prevention or treatment of a TLR- mediated condition. The invention also provides in a sixth aspect, a process for making the pharmaceutical composition according to the fifth aspect, the process comprising combining a therapeutically effective amount of an antibody or antigen-binding fragment thereof as defined in the first aspect, with a pharmaceutically acceptable vehicle.

[0372] The antibody or antigen-binding fragment thereof may be as defined with respect to the first aspect.

[0373] A "subject" may be a vertebrate, mammal, or domestic animal. Hence, medicaments according to the invention may be used to treat any mammal, for example livestock (e.g. a horse), pets, or may be used in other veterinary applications. In some embodiments, the subject is a human being. A "therapeutically effective amount" of the antibody or antigen-binding fragment thereof is any amount which, when administered to a subject, is the amount of agent that is needed to treat the TLR-mediated condition, or produce the desired effect.

[0374] For example, the therapeutically effective amount of antibody or antigen-binding fragment thereof used may be from about 0.1 ng / kg to about 100 mg / kg, and optionally from about 1 ng / kg to about 10 mg / kg. In one embodiment, the amount of antibody or antigen-binding fragment thereof is an amount from about 10 ng / kg to about 10 mg / kg, and optionally from about 50 ng / kg to about 5 mg / kg.

[0375] A "pharmaceutically acceptable vehicle" as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.

[0376] In one embodiment, the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet. A solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet-disintegrating agents. The vehicle may also be an encapsulating material. In powders, the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention. In tablets, the active agent may be mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets may contain up to 99% of the active agents. Suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, 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 be in the form of a cream or the like.

[0377] However, the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution. Liquid vehicles 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 pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, such as sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). For parenteral administration, the vehicle can 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 a halogenated hydrocarbon or other pharmaceutically acceptable propellant. Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and particularly subcutaneous injection. The agent may be prepared as a sterile solid composition that may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.

[0378] The agents and compositions of the invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like. The agents used according to the invention can also be administered orally either in liquid or solid composition form. 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.

[0379] In a seventh aspect of the invention, there is provided a polynucleotide sequence encoding the antibody, or antigen-binding fragment thereof as defined in the first aspect. In an eighth aspect of the invention, there is provided an expression cassette comprising a polynucleotide sequence according to the seventh aspect.

[0380] The polynucleotide sequence encoding the antibody, or antigen-binding fragment of the invention may be harboured in a recombinant vector, for example a recombinant vector for delivery into a host cell of interest to enable production of the antibody, or antigen-binding fragment thereof.

[0381] Accordingly, in a ninth aspect of the invention, there is provided a recombinant vector comprising the expression cassette according to the eighth aspect.

[0382] The vector encoding the antibody, or antigen-binding fragment may for example be a plasmid, cosmid or phage and / or be a viral vector. Such recombinant vectors are highly useful in the delivery systems of the invention for transforming cells with the nucleotide sequences. The nucleotide sequences may be a DNA sequence, and it is this DNA sequence which encodes the antibody, or antigen-binding fragment.

[0383] Recombinant vectors encoding the antibody, or antigen-binding fragment may also include other functional elements. For example, they may further comprise a variety of other functional elements including a suitable promoter for initiating transgene expression upon introduction of the vector in a host cell. For instance, the vector may be capable of autonomously replicating in the nucleus of the host cell. In this case, elements which induce or regulate DNA replication may be required in the recombinant vector. Alternatively, the recombinant vector may be designed such that it integrates into the genome of a host cell. In this case, DNA sequences which favour targeted integration (e.g. by homologous recombination) are envisaged. Suitable promoters may include the SV40 promoter, CMV, EFla, PGK, viral long terminal repeats, as well as inducible promoters, such as the Tetracycline inducible system, as examples. The cassette or vector may also comprise a terminator, such as the Beta globin, SV40 polyadenylation sequences or synthetic polyadenylation sequences. The recombinant vector may also comprise a promoter or regulator or enhancer to control expression of the nucleic acid as required. The vector may also comprise DNA coding for a gene that may be used as a selectable marker in the cloning process, i.e. to enable selection of cells that have been transfected or transformed, and to enable the selection of cells harbouring vectors incorporating heterologous DNA. For example, ampicillin, neomycin, puromycin or chloramphenicol resistance is envisaged. Alternatively, the selectable marker gene may be in a different vector to be used simultaneously with the vector containing the transgene. The cassette or vector may also comprise DNA involved with regulating expression of the nucleotide sequence, or for targeting the expressed polypeptide to a certain part of the host cell.

[0384] Purified vector may be inserted directly into a host cell by suitable means, e.g. direct endocytotic uptake. The vector may be introduced directly into a host cell (e.g. a eukaryotic or prokaryotic cell) by transfection, infection, electroporation, microinjection, cell fusion, protoplast fusion, calcium phosphate, cationic lipid-based lipofection, polymer or dendrimer-based methods or ballistic bombardment.

[0385] Alternatively, vectors of the invention may be introduced directly into a host cell using a particle gun.

[0386] Alternatively, the delivery system may provide the polynucleotide to the host cell without it being incorporated in a vector. For instance, the nucleic acid molecule may be incorporated within a liposome or virus particle. Alternatively, a "naked" polynucleotide may be inserted into a host cell by a suitable means e.g. direct endocytotic uptake.

[0387] In a tenth aspect of the invention, there is provided a host cell comprising the polynucleotide sequence according to the seventh aspect, the expression cassette according to the eighth aspect, or the vector according to the ninth aspect.

[0388] The host cell may be a eukaryotic or prokaryotic host cell. In one embodiment, the host cell is a eukaryotic host cell. In one embodiment, the host cell is a mammalian host cell such as NSO murine myeloma cells, PER.C6® human cells, Human embryonic kidney 293 cells or Chinese hamster ovary (CHO) cells. In one embodiment, the host cell is a CHO cell.

[0389] In an eleventh aspect, there is provided a method of preparing the antibody, or antigen-binding fragment according to the first aspect, the method comprising: a) introducing, into a host cell, the vector of the ninth aspect; and b) culturing the host cell under conditions to result in the production of the antibody, or antigen-binding fragment according to the first aspect. The host cell of step a) may be a eukaryotic or prokaryotic host cell. In one embodiment, the host cell is a eukaryotic host cell. In one embodiment, the host cell is a mammalian host cell such as NSO murine myeloma cells, PER.C6® human cells, Human embryonic kidney 293 cells or Chinese hamster ovary (CHO) cells. In one embodiment, the host cell is a CHO cell.

[0390] The method may further comprise (c) harvesting, centrifuging and / or filtering the cell culture media to obtain a cell culture supernatant comprising the antibody or antigen binding fragment thereof.

[0391] The method may further comprise (d) separating and purifying the antibody or antigen binding fragment thereof from the cell culture supernatant. In one embodiment, purification is performed by at least one chromatographic step.

[0392] Suitable chromatographic steps include affinity chromatography and / or ion exchange chromatography. In some embodiments, affinity chromatography is protein A chromatography. Ion exchange chromatography may be anionic exchange chromatography and / or cationic exchange chromatography.

[0393] In some embodiments, step (d) comprises separating and purifying the antibody or antigen binding fragment thereof from the cell culture supernatant by: i) protein A chromatography; ii) anionic exchange chromatography; and / or iii) cationic exchange chromatography.

[0394] The method may further comprise (e) filtering the purified antibody or antigen binding fragment thereof resulting from step (d). In some embodiments, step (e) comprises virus filtration. Thus, in some embodiments the purified antibody or antigen binding fragment thereof resulting from step (d) is filtered using a virus filtration membrane. Suitable membranes would be known to those skilled in the art.

[0395] As discussed herein, TLR4 plays a role in many conditions. Thus, given that the antibodies of the invention are able to bind to TLR4, the antibodies or antigenbinding fragments thereof may be used as a robust diagnostic tool by detecting the presence, and determining the concentration of TLR4. Thus, in a twelfth aspect, there is provided the antibody or antibody binding fragment of according to the first aspect, for use in diagnosis or prognosis. According to a thirteenth aspect of the invention, there is provided the antibody or antibody binding fragment of according to the first aspect, for use in diagnosing or prognosing a TLR4-mediated condition.

[0396] According to the fourteenth aspect, there is provided a method of diagnosing or prognosing a TLR4-mediated condition in a subject, the method comprising detecting TLR4 in a biological sample obtained from the subject with the antibody or antibody binding fragment of according to the first aspect.

[0397] It will be appreciated by the skilled person that a "TLR4-mediated condition" may be a condition where TLR4 recognises the pathogen- and damage-associated molecular pattern like PAMPs and DAMPs in inflammation and coordinates cellular responses, including neutrophil and type-2-cell-mediated immunity, characterized by the presence of eosinophils, basophils, mast cells, CD4+ T helper 2 (Th2) cells, and type 2 innate lymphoid cells (ILC2s). Additionally, a "TLR4-mediated condition" can also be defined as a condition involving inflammation driving tissue damage, barrier damage, loss of cell-cell adhesion, loss of epithelial barrier function, immune cell infiltration (e.g. neutrophils), and / or where epithelial cell biology plays a key role. ! "TLR4 mediated condition" may be one where TLR4 expression is increased. This increased expression of TLR4 may lead to the disruption of tight junction proteins (e.g. coxsackievirus and adenovirus receptor), and / or compromised barrier function and epithelial function during lung or skin injury. "TLR4 mediated condition" may also be defined as a condition that is triggered by exogenous and endogenous ligands that activate and increase TLR4 expression, cause systemic inflammation, release of pro-inflammatory molecules, immune cell triggered tissue damage, and / or immune mediated tissue damage, including cell death or organ failure.

[0398] The TLR4-mediated condition may be selected from a group of conditions consisting of: neuroinflammation including infection (e.g. encephalitis or meningitis) and neurodegenerative disorders (e.g. Alzheimer's and Parkinson's disease); skin inflammation such as atopic dermatitis or psoriasis; an infectious disease (e.g. a bacterial or viral infection), such as sepsis; lung inflammation and allergic airway diseases such as asthma, COPD, idiopathic pulmonary fibrosis; acute lung injury; acute respiratory distress syndrome; long COVID; pulmonary hypertension; other inflammatory diseases such as Crohn's / IBD, systemic sclerosis, rheumatoid arthritis; acute pancreatitis; Hepatic fibrosis; Metabolic dysfunction-associated steatotic liver disease (MASLD); liver failure; compensated liver diseases; decompensated liver diseases; acute liver injury; liver cirrhosis, acute-on-chronic liver failure (ACLF); alcohol-associated liver disease (ALD); steatotic liver disease (SLD); metabolic dysfunction and alcohol-associated liver disease (MetALD); metabolic dysfunction-associated steatohepatitis (MASH); cryptogenic SLD; alcohol liver disease; hepatocellular carcinoma; renal fibrosis; renal failure, acute kidney injury; chronic kidney disease; ischaemia / reperfusion injury; end stage renal failure; and cardiac disease. The cardiac disease can be myocardial inflammation, including myocarditis, MI, I / R injury, heart failure, aortic valve diseases, atherosclerosis, viral myocarditis (VMC), and / or thrombosis.

[0399] The method may be an in vitro or ex vivo method. In some embodiments, the method is an in vitro method.

[0400] The use or method may comprise determining the level of expression of TLR4 in a subject, optionally wherein an increase in the concentration of TLR4 in the biological sample when compared to a reference concentration from a healthy control population is indicative of a TLR4-mediated condition or a poor prognosis.

[0401] According to the fifteenth aspect of the invention, there is provided a kit for diagnosing a subject suffering from a TLR4-mediated condition, or for providing a prognosis of the subject's condition, the kit comprising an antibody or antigen- binding fragment thereof according to the first aspect for detecting TLR4 in a sample from a test subject.

[0402] The kit may further comprise instructions for use and / or a receptacle for obtaining a biological sample from a subject.

[0403] It will be appreciated by the skilled person that a "TLR4-mediated condition" may be a condition where TLR4 recognises the pathogen- and damage-associated molecular pattern like PAMPs and DAMPs in inflammation and coordinates cellular responses, including neutrophil and type-2-cell-mediated immunity, characterized by the presence of eosinophils, basophils, mast cells, CD4+ T helper 2 (Th2) cells, and type 2 innate lymphoid cells (ILC2s). Additionally, a "TLR4-mediated condition" can also be defined as a condition involving inflammation driving tissue damage, barrier damage, loss of cell-cell adhesion, loss of epithelial barrier function, immune cell infiltration (e.g. neutrophils), and / or where epithelial cell biology plays a key role. A "TLR4 mediated condition" may be one where TLR4 expression is increased. This increased expression of TLR4 may lead to the disruption of tight junction proteins (e.g. coxsackievirus and adenovirus receptor), and / or compromised barrier function and epithelial function during lung or skin injury. "TLR4 mediated condition" may also be defined as a condition that is triggered by exogenous and endogenous ligands that activate and increase TLR4 expression, cause systemic inflammation, release of pro-inflammatory molecules, immune cell triggered tissue damage, and / or immune mediated tissue damage, including cell death or organ failure.

[0404] The TLR4-mediated condition may be selected from a group of conditions consisting of: neuroinflammation including infection (e.g. encephalitis or meningitis) and neurodegenerative disorders (e.g. Alzheimer's and Parkinson's disease); skin inflammation such as atopic dermatitis or psoriasis; an infectious disease (e.g. a bacterial or viral infection), such as sepsis; lung inflammation and allergic airway diseases such as asthma, COPD, idiopathic pulmonary fibrosis; acute lung injury; acute respiratory distress syndrome, long COVID; pulmonary hypertension; other inflammatory diseases such as Crohn's / IBD, systemic sclerosis, rheumatoid arthritis; acute pancreatitis; Hepatic fibrosis; Metabolic dysfunction-associated steatotic liver disease (MASLD); compensated liver diseases; decompensated liver diseases; liver failure, acute liver injury; liver cirrhosis, acute-on-chronic liver failure (ACLF); alcohol-associated liver disease (ALD); steatotic liver disease (SLD); metabolic dysfunction and alcohol-associated liver disease (MetALD); metabolic dysfunction-associated steatohepatitis (MASH); cryptogenic SLD; alcohol liver disease; hepatocellular carcinoma; renal fibrosis; renal failure, acute kidney injury; chronic kidney disease; ischaemia / reperfusion injury; end stage renal failure; and cardiac disease. The cardiac disease can be myocardial inflammation, including myocarditis, MI, I / R injury, heart failure, aortic valve diseases, atherosclerosis, viral myocarditis (VMC), and / or thrombosis.

[0405] Prognosis may relate to determining the therapeutic outcome in a subject that has been diagnosed with the TLR4-mediated condition. Prognosis may relate to predicting the rate of progression or improvement and / or the duration of the TLR4- mediated condition in a subject, the probability of survival, and / or the efficacy of various treatment regimes. Thus, a poor prognosis may be indicative of progression of the TLR4-mediated condition, low probability of survival and reduced efficacy of a treatment regime. A favourable prognosis may be indicative of resolution of the TLR4-mediated condition, high probability of survival and increased efficacy of a treatment regime.

[0406] In some embodiments, the sample comprises a biological sample. The sample may be any material that is obtainable from a subject from which protein is obtainable.

[0407] The biological sample may be tissue or a biological fluid. The biological sample may be any material that is obtainable from the subject from which endothelial, smooth muscle and / or interstitial cells are obtainable. Furthermore, the sample may be blood, plasma, serum, spinal fluid, urine, sweat, saliva, tears, breast aspirate, breast milk, prostate fluid, seminal fluid, vaginal fluid, stool, cervical scraping, cytes, amniotic fluid, intraocular fluid, mucous, moisture in breath, animal tissue, cell lysates, tumour tissue, hair, skin, buccal scrapings, lymph, interstitial fluid, nails, bone marrow, cartilage, prions, bone powder, ear wax, lymph, granuloma, cancer biopsy or combinations thereof.

[0408] The sample may comprise blood, urine, tissue etc. In one embodiment, the biological sample comprises a blood sample. The blood may be venous or arterial blood. Blood samples may be assayed immediately. Alternatively, the blood sample may be stored at low temperatures, for example in a fridge or even frozen before the method is conducted. Alternatively, the blood sample may be stored at room temperature, for example between 18 to 22 degrees Celsius, before the method is conducted. The blood sample may comprise comprises blood serum. The blood sample may comprise blood plasma. The detection may be carried out on whole blood or peripheral blood. The blood may be further processed before the use of the first aspect is performed. For instance, an anticoagulant, such as citrate (such as sodium citrate), hirudin, heparin, PPACK, or sodium fluoride may be added. Thus, the sample collection container may contain an anticoagulant in order to prevent the blood sample from clotting.

[0409] As discussed in the Examples, the inventors have identified that LPS + D-Gal or HDM challenge (stimulators of TLR4), results in the up-regulation of genes highlighted in Figures 17A and 30A. The inventors also identified that treatment with the anti-TLR4 antibody (or other TLR4 antagonists) results in the downregulation of genes highlighted in Figures 17B and 30B.

[0410] Accordingly, in another aspect of the invention, there is provided the use of at least one gene selected from the group consisting of: CCL7, IL1B, CCL5, CCL3, CCL19, CCL2, CCL4, ITGB2, CHI3L3, CXCL2, CXCL9, ILIA, FOS, CXCR2, MYC, PTGS1,

[0411] NLRP3, CXCL10, TNFAIP3, CXCL1, CXCL5, CFB, TYROBP, C3, CCL17, CCL8, CCL11, CCL20, OAS1A, H2-EB1, MRC1, TLR6, ARG1, CSF2, CXCL3, LIGP1, CCR2, CCR3, OAS2, RETNLA, CD86, IL17A, CCL24, IL1RN, IL-6 and TNF-a, or a variant or fragment thereof, or a polypeptide encoded by the at least one gene, or a variant or fragment thereof, as a biomarker for a TLR4-mediated condition.

[0412] The biomarkers described herein may be used to diagnose or prognose a subject having a TLR4-mediated condition using a sample obtained from the subject. Therefore, in another aspect, there is provided a method for diagnosing a subject suffering from a TLR4-mediated condition, or a pre-disposition thereto, or for providing a prognosis of the subject's condition, the method comprising detecting the expression level of at least one gene selected from the group consisting of: CCL7, IL1B, CCL5, CCL3, CCL19, CCL2, CCL4, ITGB2, CHI3L3, CXCL2, CXCL9, ILIA, FOS, CXCR2, MYC, PTGS1, NLRP3, CXCL10, TNFAIP3, CXCL1, CXCL5, CFB, TYROBP, C3, CCL17, CCL8, CCL11, CCL20, OAS1A, H2-EB1, MRC1, TLR6, ARG1, CSF2, CXCL3, LIGP1, CCR2, CCR3, OAS2, RETNLA, CD86, IL17A, CCL24, IL1RN, IL-6 and TNF-a, or a variant or fragment thereof, or the concentration of a polypeptide encoded by the at least one gene, or a variant or fragment thereof, in a sample obtained from a test subject, and comparing this expression level or concentration, with a reference for the expression level or concentration of the at least one gene or polypeptide, in an individual who does not suffer from a TLR4-mediated condition, wherein : i) an increase in the expression level or concentration of the at least one gene or polypeptide in the sample obtained from the test subject compared to the reference suggests that the subject is suffering from a TLR4- mediated condition, or has a pre-disposition thereto, or provides a negative prognosis of the subject's condition; and / or ii) a decrease in the expression level or concentration of the at least one gene or polypeptide in the sample obtained from the test subject compared to the reference suggests that the subject is not suffering from a TLR4- mediated condition, or does not have a pre-disposition thereto, or provides a positive prognosis of the subject's condition.

[0413] It will be appreciated that genomic DNA encoding each of the biomarkers, is expressed at increased levels in subjects suffering from a TLR4-mediated condition, or in those with a negative prognosis. Accordingly, it will also be appreciated that this genomic DNA is transcribed into RNA, and subsequently translated into a polypeptide or protein. Indeed, the RNA transcript and / or the translated polypeptide may be detectable in a sample obtained from a test subject suffering from a TLR4-mediated condition and may therefore serve as biomarkers. In one embodiment, therefore, the method may comprise detecting the expression level of DNA, or an expressed transcript (e.g., RNA or mRNA).

[0414] In one embodiment, the polypeptide may comprise the full-length protein encoded by the biomarker gene, or a biologically active fragment thereof.

[0415] In one embodiment, the polypeptide biomarker may comprise post-translational modifications. The post-translational modifications may be selected from a group of post-translational modifications consisting of: phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation, and proteolysis.

[0416] In one embodiment, the use or method may comprise at least two, three, four, five, six, seven, eight, nine, or ten genes selected from the group consisting of: CCL7, IL1B, CCL5, CCL3, CCL19, CCL2, CCL4, ITGB2, CHI3L3, CXCL2, CXCL9, ILIA, FOS, CXCR2, MYC, PTGS1, NLRP3, CXCL10, TNFAIP3, CXCL1, CXCL5, CFB, TYROBP, C3,

[0417] CCL17, CCL8, CCL11, CCL20, OAS1A, H2-EB1, MRC1, TLR6, ARG1, CSF2, CXCL3, LIGP1, CCR2, CCR3, OAS2, RETNLA, CD86, IL17A, CCL24, IL1RN, IL-6 and TNF-a, or a variant or fragment thereof, or a polypeptide encoded by the gene, or a variant or fragment thereof. In another embodiment, the use or method may comprise at least twelve, fourteen, sixteen, eighteen, twenty, twenty-five, thirty, thirty-five, or forty genes selected from the group consisting of: CCL7, IL1B, CCL5, CCL3, CCL19, CCL2, CCL4, ITGB2, CHI3L3, CXCL2, CXCL9, ILIA, FOS, CXCR2, MYC, PTGS1,

[0418] NLRP3, CXCL10, TNFAIP3, CXCL1, CXCL5, CFB, TYROBP, C3, CCL17, CCL8, CCL11, CCL20, OAS1A, H2-EB1, MRC1, TLR6, ARG1, CSF2, CXCL3, LIGP1, CCR2, CCR3, OAS2, RETNLA, CD86, IL17A, CCL24, IL1RN, IL-6 and TNF-a, or a variant or fragment thereof, or polypeptide encoded by the gene, or a variant or fragment thereof.

[0419] The term "detecting" can refer, but is not limited, to the use of any one of the following conventional assays for detecting the presence of one or more of the biomarkers, or variants or fragments thereof: flow cytometry; immunoassays, such as enzyme-linked immunosorbent assays (ELISAs), an enzyme immunoassay (EIAs), radioimmunoassay (RIAs), Western Blots, immuno-precipitation or immunohistochemistry; chromogenic (enzyme activity) assays; fluorometric imaging plate reader (FLIPR) assay; or high performance liquid chromatography (HPLC) tandem mass spectrometry (MS / MS).

[0420] Alternatively, the expression level or concentration of one or more of the biomarkers may be detected in the sample by measuring their functional activity, e.g., by enzyme assay. Alternatively, to detect the expression level, of the nucleic acid sequence encoding one of the biomarkers, primers may be designed to amplify test sequences using a quantitative form of Polymerase Chain Reaction.

[0421] The "sample" is typically a bodily sample taken from a test subject. Detection for the expression level or concentration of at least one biomarker in the sample, is therefore typically carried out in vitro. The sample may comprise blood, plasma, serum, spinal fluid, urine, sweat, saliva, tears, breast aspirate, prostate fluid, seminal fluid, vaginal fluid, stool, cervical scraping, cytes, amniotic fluid, intraocular fluid, mucous, moisture in breath, animal tissue, cell lysates, tumour tissue, hair, skin, buccal scrapings, nails, bone marrow, cartilage, prions, bone powder, ear wax, or combinations thereof. In another embodiment, the sample may be contained within the test subject, which may be an experimental animal (e.g., a mouse or rat) or a human, wherein the method is an in vivo based test. Alternatively, the sample may be an ex vivo sample or an in vitro sample. Therefore, for example, the sample being tested may be in a tissue sample (for ex vivo based tests) or grown in culture (an in vitro sample). Typically, the biological sample is an ex vivo sample.

[0422] The method may comprise detecting the expression level or concentration, in the sample, of the at least one gene or polypeptide, wherein an increased level of expression or concentration of the at least one gene or polypeptide, or a variant or fragment thereof, relative to the level of expression or concentration detected in the reference sample, is an indication that the subject is suffering from a TLR4- mediated condition, or has a pre-disposition thereto, or provides a negative prognosis of the subject's condition.

[0423] Typically, the reference sample (i.e., a control) is obtained, or obtainable, from an individual who does not suffer from a TLR4-mediated condition, and so substantially does not express any of the biomarkers, or only very low or undetectable expression levels thereof.

[0424] It will be appreciated that if the expression level or concentration of any one of the biomarkers is greater in the sample obtained from the test subject than the reference, then the test subject may be diagnosed with a TLR4-mediated condition, and / or the test subject may be given a bad prognosis for with a TLR4-mediated condition.

[0425] It will also be appreciated that if the expression level or concentration of any one of the biomarkers is lower in the sample obtained from the test subject than the reference, then the test subject may not be diagnosed with a TLR4-mediated condition, and / or the test subject may be given a good prognosis for a TLR4- mediated condition.

[0426] The inventors believe that the biomarkers described herein may be used in diagnostic or prognostic methods, which may be performed ex vivo, in vitro, or in vivo, i.e. performed directly on the human body (and not necessarily on a bodily sample obtained from the test subject). Thus, according to another aspect, there is provided a method for prognosing a subject suffering from a TLR4-mediated condition, the method comprising detecting, in a test subject, the expression level of at least one gene selected from the group consisting of: CCL7, IL1B, CCL5, CCL3, CCL19, CCL2, CCL4, ITGB2, CHI3L3, CXCL2, CXCL9, ILIA, FOS, CXCR2, MYC, PTGS1, NLRP3, CXCL10, TNFAIP3, CXCL1, CXCL5, CFB, TYROBP, C3, CCL17, CCL8, CCL11, CCL20, OAS1A, H2-EB1, MRC1, TLR6, ARG1, CSF2, CXCL3, LIGP1, CCR2, CCR3, OAS2, RETNLA, CD86, IL17A, CCL24, IL1RN, IL-6 and TNF-a, or a variant or fragment thereof, or the concentration of a polypeptide encoded by the at least one gene, or a variant or fragment thereof, and comparing this expression level or concentration with a reference for the expression level or concentration of the at least one gene or polypeptide in an individual who does not suffer from a TLR4-mediated condition, wherein :

[0427] (i) an increase in the expression level or concentration of the at least one gene or polypeptide in the test subject compared to the reference provides a negative prognosis of the subject's condition; and / or

[0428] (ii) a decrease in the expression level or concentration of the at least one gene or polypeptide in the test subject compared to the reference provides a positive prognosis of the subject's condition.

[0429] The inventors have developed a kit which is useful for diagnosing a subject suffering from a TLR4-mediated condition, or a pre-disposition thereto, or for providing a prognosis of the subject's condition. According to another aspect, therefore, there is provided a kit for diagnosing a subject suffering from a TLR4-mediated condition, or a pre-disposition thereto, or for providing a prognosis of the subject's condition, the kit comprising means for detecting the expression level, in a sample from a test subject, of at least one gene selected from the group consisting of: CCL7, IL1B, CCL5, CCL3, CCL19, CCL2, CCL4, ITGB2, CHI3L3, CXCL2, CXCL9, ILIA, FOS, CXCR2, MYC, PTGS1, NLRP3, CXCL10, TNFAIP3, CXCL1, CXCL5, CFB, TYROBP, C3, CCL17, CCL8, CCL11, CCL20, OAS1A, H2-EB1, MRC1, TLR6, ARG1, CSF2, CXCL3, LIGP1, CCR2, CCR3, OAS2, RETNLA, CD86, IL17A, CCL24, IL1RN, IL-6 and TNF-a, or a variant or fragment thereof, or the concentration of a polypeptide encoded by the at least one gene, or a variant or fragment thereof. Typically, the kit comprises at least one control or reference sample. The kit may comprise a negative control and / or a positive control. A negative control may comprise any sample that does not contain any of the biomarkers according to the invention, or only very low or undetectable expression levels thereof. A positive control may comprise any sample that does contain one or more of the biomarkers according to the invention.

[0430] The kit may comprise a means to compare the expression level or concentration of the biomarkers in the negative control sample to the expression level or concentration of the equivalent biomarkers in a biological sample from an unknown subject, wherein an increased expression level or concentration of one or more of the biomarkers relative to that detected in the negative control is an indication that the subject from which the sample is obtained suffers from a TLR4-mediated condition, or has a pre-disposition thereto. Hence, by way of example, the expression level or concentration of the biomarker, or a fragment or variant thereof, in a sample may be at least 1-, 2-, 5-, or 10- fold high than in the negative control, which would mean that the test subject suffers from a TLR4-mediated condition, or has a pre-disposition thereto, or has a negative prognosis.

[0431] Alternatively, the expression level or concentration of the biomarker, or a fragment or variant thereof, in a sample may be at least 1-, 2-, 5-, or 10- fold lower than in the negative control, which would mean that the test subject does not suffer from a TLR4-mediated condition, or does not have a pre-disposition thereto, or have a negative prognosis. Typically, the kit comprises instructions for use.

[0432] Biomarkers according to the invention may be detected using conventional techniques known in the art that are capable of detecting expression of a protein, such as flow cytometry; immunoassays, such as enzyme-linked immunosorbent assays (ELISAs), an enzyme immunoassay (EIAs), radioimmunoassay (RIAs), Western Blots, immuno-precipitation or immunohistochemistry; chromogenic (enzyme activity) assays; fluorometric imaging plate reader (FLIPR) assay; or high performance liquid chromatography (HPLC) tandem mass spectrometry (MS / MS). Methods of detecting gene expression level will be well-known to those in the art. For example, the detection of gene expression level may be measured using a microarray, DNA or RNA arrays, RNA-seq, or PCR, such as RT-PCR. In one embodiment, the PCR may be multiplex PCR. As such, the means for detecting gene expression level in the kit, may include oligonucleotides that specifically hybridise to RNA of the genes. Such oligonucleotides can be used as PCR primers in RT-PCR reactions, or hybridization probes. The oligonucleotides in the kit may be labelled with any suitable detection marker including but not limited to, radioactive isotopes, fluorophores, biotin, enzymes (e.g., alkaline phosphatase), enzyme substrates, ligands and antibodies.

[0433] Advantageously, the use, methods, and kit of the invention, as described herein, enable the diagnosis or prognosis of a subject who suffers from a TLR4-mediated condition. Thus, subjects diagnosed with a TLR4-mediated condition, or have a negative prognosis, according to the use, methods, and kit of the invention, can benefit from earlier therapeutic treatment. Therefore, methods of the invention may comprise administering or having administered, to the subject, a therapeutic agent or putting the subject on a specialised diet, wherein the therapeutic agent or the specialised diet prevents, treats, or ameliorates the TLR4-mediated condition. Thus, in another aspect, there is provided a method of treating a subject suffering from a TLR4-mediated condition, the method comprising: i) detecting, in a sample obtained from a test subject, the expression level of at least one gene selected from the group consisting of: CCL7, IL1B, CCL5, CCL3, CCL19, CCL2, CCL4, ITGB2, CHI3L3, CXCL2, CXCL9, ILIA, FOS, CXCR2, MYC, PTGS1, NLRP3, CXCL10, TNFAIP3, CXCL1, CXCL5, CFB,

[0434] TYROBP, C3, CCL17, CCL8, CCL11, CCL20, OAS1A, H2-EB1, MRC1, TLR6, ARG1, CSF2, CXCL3, LIGP1, CCR2, CCR3, OAS2, RETNLA, CD86, IL17A, CCL24, IL1RN, IL-6 and TNF-a, or a variant or fragment thereof, or the concentration of a polypeptide encoded by the at least one gene, or a variant or fragment thereof, and comparing this expression level or concentration with a reference for the expression level or concentration of the at least one gene or polypeptide in an individual who does not suffer from a TLR4-mediated condition, wherein an increase in the expression level or concentration of the at least one gene or polypeptide in the sample obtained from the test subject compared to the reference suggests that the subject is suffering from a TLR4-mediated condition, or has a pre-disposition thereto, or provides a negative prognosis of the subject's condition; and ii) administering or having administered, to the test subject, a therapeutic agent or putting the test subject on a specialised diet, wherein the therapeutic agent or the specialised diet prevents, treats, or ameliorates the TLR4-mediated condition.

[0435] Suitable therapeutic agents for treating the TLR4-mediated condition include the antibody or antigen-binding fragment thereof according to the first aspect.

[0436] It will be appreciated that the expression level or concentration of the at least one gene or polypeptide in patients suffering from a TLR4-mediated condition is highly dependent on a number of factors, for example how far the disease has progressed, and the age and gender of the subject. It will also be appreciated that the reference expression level or concentration of the at least one gene or polypeptide in subjects who do not suffer from a TLR4-mediated condition may fluctuate to some degree, but that on average, over a given period of time, the expression level tends to be substantially constant. In addition, it should be appreciated that the expression level or concentration of the at least one gene or polypeptide in one group of subjects who do not suffer from a TLR4-mediated condition may be different to the expression level of the at least one gene or polypeptide in another group of subjects who do not suffer from a TLR4-mediated condition. However, it is possible to determine the average expression level of the at least one gene or polypeptide in subjects who do not suffer from a TLR4-mediated condition, and this is referred to as the reference or 'normal' expression level or concentration of the at least one gene or polypeptide. The normal expression level or concentration corresponds to the reference values discussed above.

[0437] The inventors also discovered that treatment with the anti-TLR4 antibody (or other TLR4 antagonists) results in the down-regulation of genes highlighted in Figures 17B and 30B.

[0438] Therefore, in another aspect of the invention, there is provided a method for determining the efficacy of treating a subject suffering from a TLR4-mediated condition with a therapeutic agent, the method comprising analysing the expression level of at least one gene selected from the group consisting of: CCL7, IL1B, CCL5, CCL3, CCL19, CCL2, CCL4, ITGB2, CHI3L3, CXCL2, CXCL9, ILIA, FOS, CXCR2, MYC, PTGS1, NLRP3, CXCL10, TNFAIP3, CXCL1, CXCL5, CFB, TYROBP, C3, CCL17, CCL8, CCL11, CCL20, OAS1A, H2-EB1, MRC1, TLR6, ARG1, CSF2, CXCL3, LIGP1, CCR2, CCR3, 0AS2, RETNLA, CD86, IL17A, CCL24, IL1RN, IL-6 and TNF-a, or a variant or fragment thereof, or the concentration of a polypeptide encoded by the at least one gene, or a variant or fragment thereof, in a bodily sample from the subject and comparing the expression level or concentration with a reference for the expression level or concentration of the at least one gene or polypeptide in an individual who does suffer from a TLR4-mediated condition, wherein a decrease in the expression level or concentration of the at least one gene or polypeptide in the bodily sample from the subject, compared to the reference, suggests that the treatment with the therapeutic agent is effective, or wherein an increase in the expression level or concentration of the at least one gene or polypeptide in the bodily sample from the subject, compared to the reference, suggests that the treatment with the therapeutic agent is ineffective.

[0439] In one embodiment, the therapeutic agent is the antibody or antigen-binding fragment thereof according to the first aspect.

[0440] The TLR4-mediated condition may be as described throughout the specification.

[0441] In one embodiment, (i) the TLR4-mediated condition may be selected from a group of conditions consisting of: Metabolic dysfunction-associated steatotic liver disease (MASLD); compensated liver diseases; decompensated liver diseases; liver failure; acute liver injury; liver cirrhosis, acute-on-chronic liver failure (ACLF); alcohol- associated liver disease (ALD); steatotic liver disease (SLD); metabolic dysfunction and alcohol-associated liver disease (MetALD); metabolic dysfunction-associated steatohepatitis (MASH); cryptogenic SLD; alcohol liver disease; and hepatocellular carcinoma; and (ii) the at least one gene may be selected from the group consisting of: CCL7, IL1B, CCL5, CCL3, CCL19, CCL2, CCL4, ITGB2, CHI3L3, CXCL2, CXCL9, ILIA, FOS, CXCR2, MYC, PTGS1, NLRP3, CXCL10, TNFAIP3, CXCL1, IL-6 and TNF-a, or a variant or fragment thereof, or a polypeptide encoded by the at least one gene, or a variant or fragment thereof.

[0442] In another embodiment, (i) the TLR4-mediated condition may be selected from a group of conditions consisting of: lung inflammation and allergic airway diseases such as asthma, COPD, idiopathic pulmonary fibrosis; acute lung injury; acute respiratory distress syndrome, and long COVID; and (II) the at least one gene may be selected from the group consisting of: CXCL5, CFB, TYROBP, C3, CXCL10, CCL17, CCL8, CCL11, CHI3L3, CCL20, ITGB2, OAS1A, H2-EB1, MRC1, TLR6, CCL2, ARG1, CXCR2, CSF2, CXCL3, LIGP1, CCR2, CCR3, 0AS2, CXCL9, RETNLA, CD86, IL17A, CXCL1, CCL3, CCL24, CCL7, IL1RN, IL-6 and TNF-a, or a variant or fragment thereof, or a polypeptide encoded by the at least one gene, or a variant or fragment thereof.

[0443] In one embodiment, the decrease in the expression level or concentration of the at least one gene or polypeptide in the bodily sample from the subject, compared to the reference, may be a decrease of at least 1%, 5% 10%, 20%, 40%, 60%, 80% or 100%. In one embodiment, the increase in the expression level or concentration of the at least one gene or polypeptide in the bodily sample from the subject, compared to the reference, may be an increase of at least 1%, 5% 10%, 20%, 40%, 60%, 80% or 100%.

[0444] 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 the sequence identified as SEQ ID Nos: 1-234 and so on.

[0445] Amino acid / polynucleotide / polypeptide sequences with a sequence identity which is greater than 65%, optionally greater than 70%, optionally greater than 75%, and optionally greater than 80% sequence identity to any of the sequences referred to are also envisaged. In some embodiments, the amino acid / polynucleotide / polypeptide sequence has at least 85% identity with any of the sequences referred to, optionally at least 90% identity, optionally at least 92% identity, optionally at least 95% identity, optionally at least 97% identity, optionally at least 98% identity and, optionally at least 99% identity with any of the sequences referred to herein.

[0446] 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.

[0447] 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.

[0448] 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.

[0449] In some embodiments, 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 some embodiments, 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.

[0450] 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 the sequences shown in, for example, in those of SEQ ID Nos: 1 to 234 that are amino acid sequences. 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. 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.

[0451] 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 :-

[0452] Figure 1 shows the percentage of inhibition of TNF-a release by the anti-TLR4 antibodies according to the invention, i.e. 45-D03, 49-E05, 56-H05, 62-H02, and 68-H02. The graph shows example data of three independent experiments using distinct human PBMC donors treated with LPS (i.e. a stimulator of TLR4).

[0453] Figure 2 shows the percentage of TNF-a release by human IPSC-derived microglia with LPS, following treatment with the anti-TLR4 antibody 56-H05 and matching isotype control. The graph shows example data of two independent experiments.

[0454] Figure 3 shows the percentage of TNF-a release by a chimeric human iPSC- microglia and mouse cortex neuron and astrocyte neuroinflammatory model treated with LPS and the anti-TLR4 antibody 56-H05 and matching isotype control.

[0455] Figure 4 shows the changes in morphology and reversal of microglia stimulated with the anti-TLR4 antibody 56-H05 before addition of LPS. Scale bar in images = 20 pM Figure 5 shows the microglia cell ratio width to length following treatment with the anti-TLR4 antibody according to the invention, i.e. 56-H05 (top plot), or an isotype control antibody (bottom plot).

[0456] Figure 6 shows the effect of the anti-TLR4 antibody (56-H05) and matching isotype control on dextran-permeability in human bronchial epithelial (16HBE) cells when treated with HDM. 16HBE are cells used to model barrier function of the airway epithelium. The graph shows example data of three independent experiments. EDTA treated cells are used as positive control for loss of the barrier integrity.

[0457] Figure 7 is a table showing the main epitope candidates of the antibodies according to the invention. Figure 8 shows the results of SPR kinetics analysis to determine the binding affinity of the parental antibody and humanised leads towards A) W30047- hProl.His (i.e. TLR4), B) W30047-hPro2.His (i.e. recombinant human TLR4 / MD2 complex protein), and C) W30047-hPro3.His (i.e. TLR2 protein). The data was fitted by 1 : 1 binding model.

[0458] Figure 9 is a table showing the various sequences of eleven embodiments of the anti-TLR4 antibody of the invention. CDR = complementarity determining region; VH = variable heavy chain sequence; VL = variable light chain sequence; HC = constant heavy chain sequence; LC = constant light chain sequence.

[0459] Figure 10 is a table showing the humanised mAb sequences. CDR = complementarity determining region; VH = variable heavy chain sequence; VL = variable light chain sequence; HC = constant heavy chain sequence; LC = constant light chain sequence.

[0460] Figure 11 shows LPS stimulation in the chimeric human / rat cortex neuroinflammatory model Microglia add 7 DIV, compound and LPS (10 ng / ml) add 10 days in vitro (DIV), electric field stimulation (EFS) at 15 DIV. 56-H05 was tested at different concentrations. One way ANOVA with Dunnett's correction for multiple comparisons (comparing all groups with LPS). The highest concentration of therapeutic antibody tested (0.5 pg / ml) significantly decreased the response ratio compared to the LPS-treated group with microglia (MG) added.

[0461] Figure 12 shows LPS stimulation in the chimeric human / rat cortex neuroinflammatory model Microglia add 7 DIV, compound and LPS (10 ng / ml) add 10 DIV, EFS at 15 DIV. Isotype control was tested at different concentrations. One way ANOVA with Dunnett's correction for multiple comparisons (comparing all groups with LPS). No effects of the isotype control were seen in the same concentration range as targeted antibody.

[0462] Figure 13 shows example images from H&E liver sections. From left to right 1) PBS; 2) D-Gal + LPS+ isotype control and 3) D-Gal + LPS + anti-TLR4 antibody. Treatment of male C57BL / 6 mice with D-Gal and LPS induced acute, severe hepatocellular necrosis in the majority of animals treated (highlighted in the image by black arrows). Dosing with anti-TLR4 antibody showed microscopic evidence of a therapeutic response with anti-TLR4 antibody but no response with isotype control was observed.

[0463] Figure 14 shows the histopathological score of liver and kidney H&E images, where scoring is a composite of the following parameters: 1) liver: hyperaemia, apoptosis, inflammatory cell infiltration, hepatocyte degeneration and necrosis; 2) kidney: loss of brush border / degeneration, necrosis, hyperaemia / congestion, tubular hypertrophy, apoptosis, inflammatory cell infiltration. Severity score grading from none (0) to severe (4).

[0464] Figure 15 shows that an anti-TLR4 antibody inhibits the increase of serum alanine transaminase (ALT) and serum creatine levels following D-Gal + LPS challenge back to baseline levels. Figure 16 shows that an anti-TLR4 antibody completely inhibits TNF-a and IL-6 release following challenge with LPS + D-Gal.

[0465] Figure 17A shows the changes to expression of genes associated with response to inflammation following LPS + D-Gal administration. Up-regulated genes were identified by comparing the gene expression profile of the D-Gal + LPS + isotype control mice with the PBS treated group. Figure 17B shows genes that are downregulated by the anti-TLR4 antibody treatment identified by comparing gene expression profile of D-Gal + LPS + anti-TLR4 antibody with the D-Gal + LPS + isotype control group.

[0466] Figure 18A shows spatial antibody multiplexing imaging (Anoka Biosciences, Immuno-Oncology panel for mouse FFPE tissues). Marked haemorrhage (Terll9 staining, erythrocytes) observed in liver sections of mice treated with D-Gal + LPS + isotype control show a (left image), is inhibited by anti-TLR4 antibody treatment (right image). Figure 18B shows spatial antibody multiplexing imaging (Anoka

[0467] Biosciences, Immuno-Oncology panel for mouse FFPE tissues). Significant increase in S009A+ staining (myeloid differentiation marker) is observed in liver sections of mice treated with D-Gal + LPS + isotype control (left image) is inhibited by anti- TLR4 antibody treatment (right image). Figure 18C shows spatial antibody multiplexing imaging (Anoka Biosciences, Immuno-Oncology panel for mouse FFPE tissues). Significant increase of the recruitment and activation of immune cells (CD44+ staining) observed in liver sections of mice treated with D-Gal + LPS + isotype control (top image) is inhibited by anti-TLR4 antibody treatment (bottom image).

[0468] Figure 19 shows treatment with an anti-TLR4 antibody completely prevents mortality in a mouse model of acute liver failure (ALF). While the anti-TLR4 antibody-treated group (G5) exhibited 100% survival, both the D-Gal + LPS (G3) and the D-Gal + LPS + isotype control (G4) groups showed a 90% mortality rate by the experimental endpoint at 36 hours. Figure 20 shows an example H&E figure demonstrating that the anti-TLR4 antibody reduces lympho-plasmacytic and alveolar macrophage infiltration. From left to right: 1) isotype control + HDM; 2) anti-TLR4 antibody + HDM.

[0469] Figure 21 shows flow cytometry analysis of lung tissue samples shows that anti- TLR4 antibody inhibits recruitment immune cells, including 1) interstitial macrophages, 2) neutrophils and eosinophils, 3) y6 T-cells.

[0470] Figure 22 shows spatial antibody multiplexing imaging (Anoka Biosciences, Immuno-Oncology panel for mouse FFPE tissues). Anti-TLR4 antibody inhibits HDM- driven recruitment of CD68+ CD206+ cells (macrophages, top panel highlighted by arrows) and CD20+ cells (B-cells, bottom panel highlighted by arrows).

[0471] Figure 23 shows an example of quantification using spatial antibody multiplexing (Anoka Biosciences, Immuno-Oncology panel for mouse FFPE tissues) data. Anti- TLR4 antibody inhibits HDM-driven recruitment of CD45+CD68+CD44+ cells showing proximal localization to blood vessels ((activated macrophages, global view, top panel; co-localization with blood vessels, bottom panel).

[0472] Figure 24 shows an example of quantification using spatial antibody multiplexing (Anoka Biosciences, Immuno-Oncology panel for mouse FFPE tissues) data. Anti- TLR4 antibody inhibits perivascular HDM-driven recruitment of CD45+CD20+ cells (B-cells, global view, top panel; co-localization with blood vessels, bottom panel).

[0473] Figure 25A shows an example H8iE figure demonstrating that bronchiolar hypertrophy / hyperplasia and smooth muscle hypertrophy are reversed by an anti- TLR4 antibody. From left to right: 1) isotype control + PBS; 2) isotype control + HDM; 3) anti-TLR4 antibody + HDM (low magnification). Figure 25B shows an example H&E figure demonstrating that bronchiolar hypertrophy / hyperplasia and smooth muscle cell hypertrophy following HDM are reversed by an anti-TLR4 antibody. From left to right: 1) isotype control + HDM; 2) anti-TLR4 antibody + HDM (high magnification). Figure 25C shows spatial antibody multiplexing quantification (Anoka Biosciences, Immuno-Oncology panel for mouse FFPE tissues) of a-SMA positive cells demonstrating that anti-TLR4 antibody inhibits HDM-driven smooth muscle cell hyperplasia.

[0474] Figure 26A shows an example H&E stained with PAS for the detection of mucoproteins. From left to right: 1) isotype control + PBS; 2) isotype control +

[0475] HDM; 3) anti-TLR4 antibody + HDM. Figure 26B shows digital image analysis of H&E stained tissue, from left to right: 1) anti-TLR4 antibody reduces the area of mucus in bronchiole airways; 2) the luminal area of the bronchioles increases following anti-TLR4 antibody treatment; 3) the epithelium area of the bronchioles decreases following anti-TLR4 antibody treatment.

[0476] Figure 27 shows an example H&E figure demonstrating that anti-TLR4 antibody reduces HDM-induced remodelling and fibrosis that contributes to airway restriction, quantified by Masson's Trichrome staining. From left to right: 1) isotype control + PBS; 2) isotype control + HDM; 3) anti-TLR4 antibody + HDM.

[0477] Figure 28 shows qPCR analysis of mRNA extracted from lung samples demonstrating that treatment with an anti-TLR4 antibody suppresses the HDM- driven upregulation of IL-4 (top graft) and CCL20 (bottom graft) gene expression. Data is presented as 2-AACT Fold change normalized to 18Rpsp.

[0478] Figure 29A shows the changes to gene expression following HDM challenge. Up- regulated genes were identified by comparing the gene expression profile of the isotype control + HDM mice with the isotype control + PBS treated group. Figure 29B shows the gene expression profile of the anti-TLR4 antibody + HDM treated animals. Down-regulated genes by the anti-TLR4 antibody treatment are highlighted.

[0479] Examples TLR4 has been implicated in the pathogenesis of several conditions, such as neuroinflammation and lung inflammation. Therefore, the inventors set out to develop antibodies that are capable of specifically binding to, and inhibiting the function of, TLR4, to improve the treatment and diagnosis of these conditions.

[0480] Example 1 - Immunisation and hvbridoma screening

[0481] Introduction

[0482] Anti-TLR4 antibodies were generated following two immunisation strategies, with recombinant TLR4 / MD2 complex or HEK293T / TLR4-MD2-CD14 cells, followed with boosts with recombinant TLR4 / MD2. A sustained immune response towards plate- immobilised recombinant TLR4 / MD2 and cell-expressed TLR4-MD2-CD14 was observed. Before tissue harvest, the best responders received a final boost with recombinant TLR4 / MD2 complex. Isolated splenocytes from harvested spleens were electrofused using ModiFuse technology and cells plated to allow hybridoma growth. Hybridoma supernatants were screened by ELISA against TLR4 / MD2, and by flow cytometry using HEK293 / null, HEK293 / TLR4-MD2-CD14 and HEK293 / pIPA-hCD14 cells. The top 200 hybridomas in both assays were selected for functional screening using NfkB reporter cell line treated with LPS / TNFa, resulting in identification of 42 hybridoma showing 30% or more inhibition when treated with LPS and no inhibition of TNFa response. A total of 28 hybridomas were selected for variable gene sequencing, of which 10 VH / VL pairs were selected for cloning and expression. The sequences selected were diverse and included the best hybridomas from a functional perspective. Method for hybridoma screening (Reporter cell assays)

[0483] The reporter cell system used to screen hybridoma supernatants is based on the transient transfection of human embryonic kidney (HEK)-293 cell line with the set of 6 plasmids (plasmids encoding TLR4, myeloid differentiation factor 2 (MD-2), and cluster of differentiation (CD) 14 under the cytomegalovirus (CMV) promoter, as well as PNF-KB-LUC, phRG-TK and empty pcDNA3). The reporter system offers expression of two different luciferase enzymes - firefly and renilla luciferase. These enzymes differ in their substrate and cofactor requirements which enables quantification of transfection efficiency. The renilla luciferase expression and activation is under the control of a housekeeping gene promoter (irrespective of factors treatment) which allows to measure the cell viability and normalize data to account for variability in transfection efficiency. The protocol used is hereby described. HEK-293 cells were cultured as per manufacturer's recommendations and incubated at 37°C ± 2°C with 5% CO2 throughout all steps of the transfection protocol and after incubation with hybridoma supernatants. Briefly, cells were seeded at 25,000 cells per well and cultured for 24 hours prior transfection. The transfection mixture was prepared with pcDNA3: hTLR4, pEFIRES: hMD-2, pcDNA3: hCD14, pcDNA3, PN FKB-IUC and phRG-TK DNA and jetPEI for 30 minutes before adding to wells for 48 hours. Following this incubation step, wells were aspirated gently before adding hybridoma supernatants or antibody titrations in 1% FBS growth medium for 30 minutes. Cells were then treated with LPS or TNFa at lOng / ml final assay concentration and antibodies for 6 hours, washed and thoroughly lysed. Cell lysates were mixed with either Firefly (reporter gene) or Renilla substrate (control plasmid for transfection efficiency) for 10 minutes before reading plates in a luminometer. For each well, normalized Firefly luminescence / / ?en / 7 / a luminescence ratio was calculated. The percentage of inhibition of LPS stimulation was calculated using LPS- and 10 ng / mL LPS + 1 pM TAK-242- treated wells as controls for 0 and 100% inhibition, respectively. The percentage of inhibition of TNFa stimulation was calculated using TNFa and 10 ng / mL TNFa + 1 pM BI605906 treated wells as controls for 0 and 100% inhibition, respectively. Reporter cell assays

[0484] A panel of antibodies with a unique sequence were tested in the reporter cell assay, where cells were treated with LPS (TLR4 response) or TNFa (TNFaR response). Assays were run using the same protocol as described previously for screening of hybridoma supernatants. Table 1 below is the outcome of 3 independent experiments.

[0485] Table 1: Reporter cell assay - inhibition of LPS or TNFa THP1 assays

[0486] The panel of clones with confirmed functional activity were tested in THP1 cells treated with either LPS (TLR4 response) or PAM2CSK4 (TLR2 response).

[0487] Methods

[0488] The differentiation of THP-1 monocytes into macrophages is conducted by a phorbol 12-myristate 13-acetate (PMA). Following treatment with PMA, cells acquire macrophage-like phenotype, cells adherence and responsiveness to pro- inflammatory stimulus. Resultant THP-1 macrophage populations are characterized by high expression levels of TLR4 and TLR2 receptors, which mediate the innate immune response to bacterial lipopolysaccharides and lipopeptides, respectively. The TLR2 receptor recognizes its synthetic agonist: Pam2CSk4, whereas TLR4 recognizes LPS. Both receptors act via MYD88 and TRAF6, leading to NF-kappa-B activation and cytokine secretion. Secretion of TNFa into growth medium by differentiated THP-1 cells in response to stimulation with Pam2CSK4 or LPS is a measurable way to assess the level of pro-inflammatory response. This protocol was developed in order to evaluate the biological activity of compounds against TLR4. Their potency is expressed as IC50 value. The TLR4 downstream signalling is initiated with stimulating cells with LPS. The concurrent treatment with tested compounds can potentially inhibit the downstream signalling which affects the TNFa secretion level. The end-point measurement involves quantifying the concentration of secreted TNFa by ELISA assay. Data are normalized to positive and negative controls and calculated inhibition percentage is used to estimate IC50 value basing on four-parametric log(inhibitor) vs. response model. THP1 cells (ATCC, catalogue number TIB-202) were cultured as per manufacturer's recommendations and differentiated into adherent macrophages after treatment with PMA for 72 hours. Cell culture is incubated at 37°C ± 2°C with 5% CO2 throughout all steps hereby described for the assessment of the inhibition of LPS- and PAM2CSK4- driven response.

[0489] THP1 cells were then treated with antibody titrations for 30' before adding LPS or PAM2CSK4 at 5ng / ml final assay concentration and antibodies for 24 hours. Supernatants were collected and kept at -70oC until analysis. TNFa was quantified using the Human TNF-alpha DuoSet ELISA (R&D systems, DY210) as per manufacturer's recommendations. The percentage of inhibition of LPS stimulation was calculated using LPS- and 5 ng / mL LPS + 1 pM TAK-242- treated wells as controls for 0 and 100% inhibition, respectively. The percentage of inhibition of PAM2CSK4 stimulation was calculated using PAM2CSK4 and 10 ng / mL PAM2CSK4 + 1 pM BI605906 treated wells as controls for 0 and 100% inhibition, respectively.

[0490] Table 2: Inhibition of THP1 + LPS, or THP1 + PAM2CSK4

[0491] As illustrated above, all of the antibody clones that were tested inhibited TNFa- release. TNFa is normally released upon activation of TLR4 by LPS. Thus, the fact that TNFa-release was inhibited demonstrates that the claimed antibodies target and inhibit TLR4.

[0492] Example 2 - Epitope mapping

[0493] Methods - Introduction The epitopes recognised by the panel of anti-TLR4 antibodies on the human TLR4 were determined using linear and conformational epitope mapping. The experimental works starts by the synthesis of libraries of linear and conformational epitopes. To generate a library of linear peptides, the amino acid sequence of the target protein is split in overlapping fragments in silico, which are then synthesized on a solid support. The construction of a conformational peptide library is performed using the Biosynth's proprietary Chemically Linked Peptides on Scaffolds (CLIPS) technology, which allows to structure peptides into single loops, doubleloops, triple loops, sheet-like folds, helix-like folds, and combinations thereof. Binding of antibodies is then quantified using an automated ELISA-type read-out. A more detailed description of methods is below.

[0494] Synthesis of peptides To reconstruct epitopes of the target molecule a library of peptide-based mimics was synthesized using Fmoc-based solid-phase peptide synthesis. An amino functionalized polypropylene support was obtained by grafting with a proprietary hydrophilic polymer formulation, followed by reaction with tbutyloxycarbonyl- hexamethylenediamine (BocHMDA) using dicyclohexylcarbodiimide (DCC) with N- hydroxybenzotriazole (HOBt) and subsequent cleavage of the Boc-groups using trifluoroacetic acid (TFA). Standard Fmoc-peptide synthesis was used to synthesize peptides on the amino-functionalized solid support by custom modified JANUS liquid handling stations (Perkin Elmer). If constrained peptides were used in the study, synthesis of such structural mimics was done using Biosynth's proprietary Chemically Linked Peptides on Scaffolds (CLIPS) technology. CLIPS technology allows to structure peptides into single loops, double-loops, triple loops, sheet-like folds, helix-like folds, and combinations thereof. CLIPS scaffolds are coupled to cysteine residues. The side-chains of multiple cysteines in the peptides are coupled to a CLIPS scaffold with two or three reactive groups. For example, a 0.5 mM solution of the P2 CLIPS (2,6-bis(bromomethyl)pyridine) is dissolved in ammonium bicarbonate (20 mM, pH 7.8) / acetonitrile (l :3(v / v)). This solution is added onto the peptide arrays. The CLIPS template will bind to side-chains of two cysteines introduced in the solid-phase bound peptides of the peptide-arrays (455 wells plate with 3 pl wells). The peptide arrays are gently shaken in the solution for 30 to 60 minutes while completely covered in solution. Finally, the peptide arrays are washed extensively with excess of H2O and sonicated in disrupt-buffer containing 1 % SDS / 0.1 % 2,2'-(Ethylenedioxy)diethanethiol in PBS (pH 7.2) at 70°C for 30 minutes, followed by sonication in H2O for another 45 minutes. The T3 CLIPS carrying peptides were made in a similar way but now with three cysteines.

[0495] ELISA screening

[0496] The binding of antibody to each of the synthesized peptides was tested in a Biosynth-based ELISA. The peptide arrays were either incubated with primary antibody solution directly covering the mini-card overnight at 4°C (Normal Procedure), or in a larger container whilst agitating overnight at RT (BOX procedure). After washing, the peptide arrays were incubated with a 1 / 1000 dilution of an appropriate antibody peroxidase conjugate (goat anti-human HRP conjugate, Southern Biotech) for one hour at 25°C. After washing, the peroxidase substrate 2,2'-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 20 pl / ml of 3 percent H2O2 were added. After one hour, the colour development was measured. The colour development was quantified with a charge coupled device (CCD) - camera and an image processing system.

[0497] The values obtained from the CCD camera range from 0 to 3000 mAU, similar to a standard 96-well plate ELISA-reader. The results are quantified and stored into the Peplab database. Occasionally a well contains an air-bubble resulting in a false- positive value, the cards are manually inspected, and any values caused by an airbubble are scored as 0.

[0498] Data analysis

[0499] Peaks are defined as at least two times the background value (defined as the basis of the peaks for the specific peptide mimic). In addition, a binding event was only noted if multiple overlapping peaks were present within this binding region.

[0500] Putative core epitopes were identified from adjacent peptides with similar or maximally 30% lower intensity as the top peak within a binding region. In addition, if peaks were well defined containing multiple overlapping peptides, also lower intensity peaks may be indicated as well. For full evaluation of the putative epitope, information is taken from multiple mimic types. When no additional information can be extracted from these mimic types, these may not be listed in the epitope tables.

[0501] Results The list of main epitope candidates found are summarised in Figure 7. Different epitopes were detected by different samples in this study. The most frequently detected sequence 285 IEEFRLAYLDYYLD 298 (SEQ ID No: 210) could be a highly immunogenic region for the target. In many cases, several binding sequences were identified, which suggest that the epitopes may be discontinuous.

[0502] SPR

[0503] SPR experiments were carried out using a Biacore 8K (Cytiva) at 25°C with the sample compartment set to 10°C. Experiments were carried out using PBS-P+ (20mM phosphate buffer 2.7 mM KCI, 137 M NaCI, 0.05% Surfactant P20, pH 7.4) as running buffer. A Series S Protein A chip was docked and equilibrated with running buffer prior to 3 priming cycles of 60 second injections of Glycine pH1.5 and 50mM NaOH. Antibody NI-0101 was prepared from a stock of 18pM to 5nM in running buffer and captured by a 30s injection at a flow rate of lOpL / min resulting in a capture level of 65Ru.

[0504] A 6 point 1 in 2 dilution series from a top concentration of lOOnM of TLR4 and TLR4 / MD2 were prepared in a 96 well plate. A single cycle kinetic method consisting of 6 x 60 second injections and one dissociation step of 600s was carried out. All flow cells were regenerated with Glycine pH1.5 and 50mM NaOH for 60 seconds to regenerate the surface for other antibody testing. All data was analysed using the Biacore Insight Evaluation software.

[0505] Table 3: Assay conditions

[0506] Table 4: SPR Results

[0507] Antibodies were run at least in one independent experiment.

[0508] Example 3 - Human and cvno PBMC data

[0509] Methods Lead panel was tested in the cyno PBMC assays as described. PBMCs isolated from cynomolgus monkey were added at 2 million cells / well to a 96-well plate and treated with antibody titrations / isotype control for 30 minutes. After this preincubation step, LPS was added to wells at EC80 concentration for 24 hours. Cell supernatant was then collected for TNFa quantification, performed using the Human TNF-alpha ELISA kit, as per manufacturer's recommendations. The percentage of inhibition of TNF-alpha following LPS stimulation was calculated using LPS and untreated wells as controls for 0 and 100% inhibition, respectively. Table 5: Inhibition of TNFa release in cvno PBMC assay

[0510] As illustrated in Figure 1, the anti-TLR4 antibodies inhibit TNF-alpha release by human PBMC treated with LPS. In contrast, the control (human IgGl) does not prevent the release of TNF-alpha upon the activation of TLR4 by LPS.

[0511] Human and cyno cross- reactive clones were tested in the human PBMC assay.

[0512] PBMCs isolated from three donors were added at 2 million cells / well to a 96-well plate and treated with antibody titrations / isotype control for 30 minutes. After this pre-incubation step, LPS was added to wells at EC80 concentration for 24 hours.

[0513] Cell supernatant was then collected for TNFa quantification, performed using the Human TNF-alpha ELISA kit , as per manufacturer's recommendations. The percentage of inhibition of TNF-alpha following LPS stimulation was calculated using LPS and untreated wells as controls for 0 and 100% inhibition, respectively. IC50 data for 3 donors is available and summarized in Table 6.

[0514] Table 6: TNF-aloha inhibition following LPS stimulation in 3 human PBMC donors

[0515] As illustrated in Table 6, the antibody clones according to the invention inhibit TNF- alpha release with an IC50 of 2-7pM. In contrast, the IC50 of Nl-0101 was 6.23.

[0516] Example 4 - In vitro disease models - Neuroinflammation iMicroglia LPS assay

[0517] Human iPSC-derived microglia were cultured and matured as per manufacturer's recommendations. Microglia upon maturation are functionally active (cytokine profile upon stimulation and ability to phagocytise) and express TLR4, MD2 and key microglial cell surface markers, including TREM2. Cells are seeded at 5,00 cells per well in 50 pl culture media and treated with a serial dilution of test antibodies for one hours at 37°C, 5% CO2. After this pre-incubation step, LPS was added to wells at EC80 concentration for 24 hours. Cell supernatant was then collected for TNFa quantification, performed using the Human TNF-alpha HTRF kit, as per manufacturer's recommendations. The percentage of inhibition of TNF-alpha following LPS stimulation was calculated using LPS and untreated wells as controls for 0 and 100% inhibition, respectively.

[0518] As illustrated in Figure 2, the anti-TLR4 antibody 56-H05 inhibits TNF-alpha release by human PBMC treated with LPS.

[0519] Chimeric human iPSC-microglia and mouse cortex neuron and astrocyte neuroinfiammatory model

[0520] Mouse cortical cultures, which include cortex neurons and astrocytes, were seeded in 384-well plates. After seven days of culture, hiPSC-derived microglia were added at 2000 cells per well. At day 14 of culture, anti-TLR4 antibodies and isotype control dilution series were added to wells and incubated for 1 h prior to addition of LPS. LPS was added at 10 ng / ml to cells for 24 h. At day 15, conditioned medium was collected for analysis of released TNFa by ELISA.

[0521] After medium collection, cultures were fixated and processed for immunocytochemistry using antibodies binding to MAP2 (neuronal networks) GFAP (astrocytic networks) and Ibal (microglia). High content analysis (HCA) was analysed for quantitative measurements of neuronal-and astrocytic networks and microglia morphology, including measurement of alterations morphology due to microglial activation induced by LPS.

[0522] Conditioned medium from 6 wells were pooled and used for analysis of released TNFa by ELISA. Quantification was performed in technical duplicates (2 wells per sample) and in two biological replicates per condition (medium from six well each). Average ±SD from the biological duplicates were calculated and plotted as total TNFa (ng / ml). As illustrated in Figure 3, anti-TLR4 antibody 56-H05 inhibit TNF-a release in Chimeric human iPSC-microglia and mouse cortex neuron and astrocyte neuroinflammatory model treated with LPS.

[0523] Figure 4 illustrates the changes in morphology and reversal. In control cultures, with no activation, microglia appeared with morphology resembling unstimulated microglia, i.e. elongated cells with processes. However, cells with a rounded morphology could also be identified. In cultures stimulated with LPS for 24 h, the morphology was altered with the majority of microglia appearing rounder and could also be found in groups of cells. Incubation with the anti-TLR4 antibody H05-56 before addition of LPS appeared to prevent the LPS-induced change in morphology and cultures appeared to be similar to unstimulated cultures. In contrast, preincubation with an isotype control antibody did not prevent the LPS-induced altered morphology. In the HCA, an area covering 27% of the well area was analysed. Average ±SD from 2-6 wells per condition is presented, as well as representative images of untreated and treated wells. In the presence of LPS, an increase in TNFa release and in the HCA parameter, cell ratio width to length was detected in the cultures. Anti-TLR4 antibody efficiently inhibited the LPS-induced change in both TNFa release and in the ratio width to length, at all concentrations evaluated (Figure 5, top plot). In contrast, no effects on those parameters were detected in presence of the isotype control antibody (Figure 5, bottom plot). Functional assessment using optical electrophysiology

[0524] The electric field stimulation (EFS) experiments were performed on Cellectricon's Optical Electrophysiology Platform. The setup resembles an inverted microscope where the cell culture plate is imaged from below. The platform is equipped with an electrostimulation module that enters the plate from above. The temperature in the instrument was kept at approximately 32°C. The system was zoomed-in in order to image 48 wells in the first quadrant in the center of a 384-well plate. EFS experiments were performed 15 days after plating. On the day of the experiment, the calcium indicator Ca5 was dissolved in Neurobasal Plus. Cell cultures were stained with Ca5, and the cells were incubated at 37°C, 5% CO2. One hour after Ca5 addition, the cell plate was inserted in the Optical Electrophysiology Platform. After a short equilibrium time, the EFS electrode module was entered into the cell culture plate and an EFS protocol was applied delivering three pulse trains (Table 2). The neuronal response was simultaneously monitored. Images were captured at 20 Hz, binning 4x4.

[0525] During the recording of the EFS experiments, image stacks were generated. In defined ROIs, one ROI per cell culture well, the average fluorescence intensity per ROI and image frame was recorded. The EFS-evoked responses were analysed using KNIME, an integrated open-source platform for large scale multiparametric data. In KNIME, several wave form parameters were extracted (Figure 1). In a data exploration, it was concluded that the response ratio (the ratio between the maximum and minimum fluorescence intensity per EFS pulse) for the 20 V protocol provided the best assay window for the LPS-induced activation and the ratio was therefore used for analysis in the study.

[0526] For evaluation of the EFS response, the response ratio was calculated per peak and the median value of the peaks recorded was delivered for each well. The number of peaks in the recorded interval was also analysed and was used to determine whether the cultures responded to EFS or not. In wells where the number of peaks was lower than anticipated due to EFS, the result was omitted. On 10 DIV, 56-H05, isotype control, were added to the cultures followed by LPS stimulation.

[0527] Example 5 - In vitro disease models - Lung inflammation Permeability assay

[0528] 16HBE human bronchial epithelial (Sigma, catalogue number SCC150) cells were plated at 25,000 cells per well in 150 ml normal growth media in the upper chamber of 24-well 0.4 pm pore size Transwell pre-coated with collagen (1 : 500 for 1 hr at room temp), with 600 ml of growth media in the lower well. After 24h, cells are checked under the TC microscope to ensure a complete cell monolayer. Normal growth media is replaced by MEM without serum in apical and basal wells. To HDM wells, media was added with lOOul HDM (derpl / 2 enhanced, Citeq) diluted 1:40 from stock in media. Following the addition of 10 pl of 2kDa TRITC-dextran solution to the upper chamber of all wells, the plate is incubated overnight. For antibody treatments, lOmg / ml final concentration of control IgG or anti-TLR4 antibodies was added to relevant wells. The following day, in positive control wells, 5 mM EDTA was added for 30 mins at 37°C. 100 pl of media from the lower chamber was collected for analysis using a fluorescence plate reader (Aex 550 nm; Aem 577 nm). 3 x 100 pl samples were collected from each well for measurement and take the mean of this value to plot.

[0529] 16HBE are cells used to model barrier function of the airway epithelium. As illustrated in Figure 6, the anti-TLR4 antibody HO5-566, inhibits dextran- permeability in human bronchial epithelial (16HBE) cells when treated with HDM.

[0530] Example 6 - Antibody Humanisation

[0531] The mouse antibody, H05-56 (W30047-cAbl) targeting TLR4, underwent humanization The sequence of the mouse antibody (W30047-cAbl) was aligned to the human germline sequence repertoire of IMGT (Lefranc 2003). The human germline sequence with the least number of amino acid differences at counterpart positions in framework with respect to W30047-cAbl was selected as the humanization template. Mouse CDRs were in silico grafted into selected human framework template to create humanized version of W30047-cAbl-z0. Several human-to-mouse back mutation sites that could be important for maintaining CDR conformation were selected, and corresponding mutations were introduced and combined to create various humanized variants. Small scale transient expression was used to produce each variant, which was used to test in different assays to ascertain binding to target (eg SPR) and functional profile (PBMC assays).

[0532] SPR full kinetics analysis was performed to determine the binding affinity of the parental antibody and humanized constructs towards W30047-hProl.His (i.e. TLR4 protein), W30047-hPro2.His (i.e. recombinant human TLR4 / MD2 complex protein), and W30047-hPro3.His (i.e. TLR2 protein). The data was fitted by 1: 1 binding model. PBMCs isolated from two human and cyno donors were added at 2 million cells / well to a 96-well plate and treated with antibody titrations / isotype control for 30 minutes. After this pre-incubation step, LPS was added to wells at EC80 concentration for 24 hours. Cell supernatant was then collected for TNFa quantification, performed using the Human TNF-alpha ELISA kit as per manufacturer's recommendations. The percentage of inhibition of TNF-alpha following LPS stimulation was calculated using LPS and untreated wells as controls for 0 and 100% inhibition, respectively. Data for 2 donors is available and summarized in Table 7. Results

[0533] Binding to target - SPR

[0534] Table 7. SPR full kinetics result against W30047-hProl .His (1 : 1 binding model)

[0535] Table 8. SPR sensorgram of full curve of parental and humanized leads against W30047-hPro2.His

[0536] Table 9. SPR full kinetics result against W30047-hPro3.His

[0537] Humanized variants bind to target in a comparable manner to parental clone as measured by ELISA and SPR (see Figure 8B). The parental antibody and the humanized constructs showed no or weak binding to W30047-hPro3.His, i.e. TLR2 (Figure 8C). The results are in line with the expectation that after humanisation the variants still keep their specificity against TLR4, TLR4 / MD2 complex but not TLR2.

[0538] Functional profile

[0539] Table 10: TNF-aloha inhibition by W30047-cAbl-z2 following LPS stimulation in two human and cvno PBMC donors Example 7 - In vivo liver model

[0540] Experiment 1 run in an Acute Liver Failure mouse model (including histopathology, liver and kidney function and cytokine response as the experimental endpoints) Acute liver failure was induced via intraperitoneal co-administration of D- galactosamine (500 mg / kg) and lipopolysaccharide (LPS, 10 pg / kg) in C57BL / 6 mice, aged 8-9 weeks. The animals were assigned to five experimental groups: Gl: Vehicle control (PBS only) G2: D-galactosamine alone G3: D-galactosamine + LPS

[0541] G4: D-galactosamine + LPS + isotype control

[0542] G5: D-galactosamine + LPS + anti-TLR4 antibody Antibodies (20 mg / kg, intraperitoneally) were administered on days 7 and day 1 prior to D-GAL + LPS injection. Mice were euthanized 6 hours after induction of acute liver failure. Blood, liver, and kidney tissues were collected for subsequent analyses, including histopathology, measurement of cytokines, ALT and creatinine levels.

[0543] These data support the use of an anti-TLR4 antibody for the treatment of liver indications (for example, but not limited to Metabolic Dysfunction-Associated Steatohepatitis (MASH), Alcohol-Associated Liver Disease (ALD), Metabolic Dysfunction and Alcohol-Associated Liver Disease (MetALD), Cryptogenic SLD, compensated and decompensated liver disease, acute-on-chronic liver disease, Alcohol liver disease, hepatocellular carcinoma). These data utilise an anti-TLR4 surrogate antibody that has equivalent functionality to the claimed anti-TLR4 antibodies. As such, it will be expected that the anti-TLR4 antibodies according to the invention will demonstrate the same activity and will effectively treat liver indications.

[0544] The anti-TLR4 antibody shows in vivo therapeutic efficacy and improves histopathology including hepatocyte viability following LPS + D-galactosamine (D- Gal) challenge (Figure 13).

[0545] The histopathology examination was performed using Aperio ImageScope software (Leica Biosystems, Version 12.4.6). Key findings are summarised as follows:

[0546] PBS control group shows minimal inflammatory cell infiltration and variable amounts of hepatocyte cytoplasmic rarefaction (glycogen accumulation).

[0547] Treatment with D-Gal and LPS resulted in moderate to very marked necrosis in 4 / 5 animals, accompanied by marked congestion / hemorrhage, and absence of hepatocyte cytoplasmic rarefaction.

[0548] Necrosis was not accompanied by any inflammatory cell infiltration, likely due to the acute nature of this ALF model and early timepoint of the analysis

[0549] (6h).

[0550] Isotype control group showed no evidence of therapeutic response. The incidence and grade of necrosis and congestion / hemorrhage were similar to (if not higher than) LPS + D-Gal group. - Anti-TLR4 antibody showed therapeutic efficacy. Necrosis is reduced in 4 / 5 animals. Congestion / hemorrhage were absent, and the amount of hepatocyte cytoplasmic rarefaction returned to control levels. The anti-TLR4 antibody also improves the histopathology scoring of liver and kidney (Figure 14). Additionally, the anti-TLR4 antibody inhibits the increase of clinically relevant endpoints for liver and kidney function (serum ALT and creatinine) following LPS and D-Gal challenge to baseline levels (Figure 15). Further, the anti- TLR4 antibody inhibits pro-inflammatory cytokine release (TNF-a and IL-6), a hallmark inflammation that drives disease progression (Figure 16).

[0551] Treatment with the anti-TLR4 antibody also results in changes to gene expression associated with response to inflammation following LPS + D-Gal challenge. mRNA was extracted from liver sections using the Qiagen RNeasy FFPE Kit. Samples were run using the NanoString Mouse inflammation, for the multiplex gene expression analysis of more than 200 genes, covering a broad range of relevant pathways related to inflammation. LPS + D-Gal challenge results in the increase of genes highlighted in Figure 17A. Up-regulated genes were identified by comparing the gene expression profile of the D-Gal + LPS + isotype control mice with the PBS treated group.

[0552] The gene expression profile of the anti-TLR4 antibody + LPS + D-Gal animals was compared with the respective control group, composed of animals treated with D- Gal + LPS + isotype control. Anti-TLR4 antibody treatment results in the downregulation of genes highlighted in Figure 17B, which code for several chemokines with a crucial role in recruitment and migration of immune cells, including T cells, eosinophils, and basophils and neutrophils, to site of inflammation. This list of genes are potential biomarkers of a clinical response to anti-TLR4 antibody treatment or other TLR4 antagonists.

[0553] Spatial antibody multiplexing imaging data is supportive of histopathology analysis in that the role of the anti-TLR4 antibody in inhibiting LPS + D-gal driven damage to the liver is demonstrated by the absence of marked haemorrhage seen in the D- Gal + LPS + isotype control (Figure 18A). Furthermore, this analysis shows the role of the anti-TLR4 antibody in modulating myeloid differentiation and recruitment and activation of immune cells, as per S009A+ staining and CD44+ staining, respectively (Figure 18B and C, respectively).

[0554] Experiment 2 run in an Acute Liver Failure mouse model (mortality as the experimental endpoint) Acute liver failure was induced in mice via intraperitoneal co-administration of D-gal (500 mg / kg) and lipopolysaccharide (LPS, 5 pg / kg). C57BL / 6 mice, aged 8-9 weeks, were assigned to six experimental groups: Gl: Vehicle control (PBS only)

[0555] G2: D-galactosamine alone

[0556] G3: D-galactosamine + LPS

[0557] G4: D-galactosamine + LPS + isotype control antibody

[0558] G5: D-galactosamine + LPS + anti-TLR4 antibody

[0559] Antibodies (20 mg / kg, intraperitoneally) were administered on days 7 and day 1 prior to LPS injection. Mice were monitored for clinical signs for 36 hours and the time of mortality following LPS administration was recorded. The anti-TLR4 antibody prevents mortality that results from liver failure (Figure 19). While the anti-TLR4-treated group (G5) exhibited 100% survival, both the D- Gal + LPS (G3) and the D-Gal + LPS + isotype control (G4) control groups showed a 90% mortality rate by the experimental endpoint at 36 hours. Example 8 - In vivo lung model

[0560] Methods - HDM mouse model

[0561] Six-week-old C57BL / 6 mice were administered anti-TLR4 antibody or isotype control via intraperitoneal (IP) injection for one week, starting eight days prior to house dust mite (HDM) exposure (or PBS control). HDM challenge began on day 0. From day 3 onward, mice continued to receive anti-TLR4 antibody or isotype control twice weekly for five weeks.

[0562] Mice were challenged intranasally with HDM (a purified mixture of Dermatophagoides pteronyssinus 1 / 2 and Dermatophagoides farinae 1; Citeq Biologies) once daily for five consecutive days, followed by a two-day rest period. This cycle was repeated until the experimental endpoint at five weeks.

[0563] At termination, mice were euthanized via terminal anaesthesia using sodium pentobarbital administered IP, followed by exsanguination. Bronchoalveolar lavage fluid (BAL) and lung tissue were collected for flow cytometry and mRNA extraction. For histological analysis, lungs were harvested, fixed, and processed into FFPE sections. Tissue slides were stained with hematoxylin and eosin (H8iE), Periodic Acid-Schiff (PAS), and Masson's Trichrome. Whole-slide digital images were acquired using the Aperio ImageScope software (Leica Biosystems, Version 12.4.6) and evaluated by a certified pathologist. These data support of the use of an anti-TLR4 antibody for the treatment of respiratory indications (for example, but not limited to, asthma, COPD, ARDS, Idiopathic Pulmonary Fibrosis). These data utilise an anti-TLR4 surrogate antibody that has equivalent functionality to the claimed anti-TLR4 antibodies. As such, it will be expected that the anti-TLR4 antibodies according to the invention will demonstrate the same activity and will effectively treat respiratory indications.

[0564] The anti-TLR4 antibody inhibits key aspects of lung inflammation including significant immune cell infiltration following HDM challenge. The anti-TLR4 antibody reduces lympho-plasmacytic and alveolar macrophage infiltration (Figure 20). The anti-TLR4 antibody inhibits recruitment immune cells including interstitial macrophages, neutrophils and eosinophils, and y6 T-cells (Figure 21). The anti- TLR4 antibody inhibits HDM-driven recruitment of macrophages (CD68+ CD206+ cells) and B-cells (CD20+ cells) (Figure 22). The anti-TLR4 antibody inhibits HDM- driven recruitment of macrophages (CD45+CD68+CD44+ cells) (Figure 23) showing proximal localization to blood vessels. The anti-TLR4 antibody inhibits perivascular HDM-driven recruitment of B-cells (CD45+CD20+ cells) (Figure 24).

[0565] The anti-TLR4 antibody inhibits key aspects of lung inflammation including bronchiolar hypertrophy / hyperplasia and smooth muscle cell hyperplasia / hypertrophy following HDM challenge (Figure 25). The anti-TLR4 antibody inhibits key aspects of lung inflammation including mucus production, increases the luminal area and decreases the epithelial area per bronchiole airway following HDM challenge (Figure 26). The anti-TLR4 antibody inhibits HDM-induced tissue remodelling, including fibrosis, following HDM challenge (Figure 27). The anti-TLR4 antibody inhibits Th2 cytokine release and CCL20 upon HDM challenge (Figure 28).

[0566] Treatment with anti-TLR4 antibody results in changes to gene expression associated with response to HDM challenge. mRNA was extracted from lung sections using Qiagen RNeasy FFPE Kit. Samples were run using the NanoString

[0567] Mouse inflammation, which includes more than 200 genes focused on inflammation, covering a broad range of relevant pathways related to inflammation. HDM treatment results in the upregulation of genes highlighted in Figure 29A. Increase of gene expression was identified by comparing the gene expression profile of the isotype control + HDM mice with the isotype control + PBS treated group.

[0568] The gene expression profile of the anti-TLR4 antibody + HDM animals was compared with the respective control group, composed of animals treated with isotype control + HDM. Anti-TLR4 antibody treatment results in the downregulation of genes highlighted in Figure 29B that code for several chemokines and cytokines with a crucial role in recruitment and migration of immune cells, as well as driving an inflammatory response. This list of genes are potential biomarkers of a clinical response to anti-TLR4 antibody treatment or other TLR4 antagonists.

[0569] Conclusions In summary, the inventors have developed antibodies that are capable of binding to, and inhibiting, TLR4 function. For example, as shown in Figures 8 and 9, the inventors have developed a number of antibodies and antigen-binding fragments thereof that have demonstrated the ability to specifically target TLR4. Additionally, the inventors have generated humanised versions of the antibodies according to the invention, and have demonstrated that the humanised antibodies can bind to TLR4 with high specificity, high potency, and are functionally active in inhibiting TLR4 signalling. Furthermore, as illustrated in Figures 1 to 3, the inventors have shown that the antibodies according to the invention inhibit TNF-a release (a cytokine that would otherwise be released upon the activation of TLR4 by LPS). Additionally, the inventors have demonstrated that the anti-TLR4 antibody according to the invention can inhibit dextran-permeability in human bronchial epithelial (16HBE) cells when treated with HDM, illustrating that the claimed antibodies will provide an effective treatment for lung inflammation. Therefore, in summary, the inventors have identified improved antibody therapies for the treatment of TLR4- mediated diseases.

Claims

1. Claims1. An antibody, or an antigen-binding fragment thereof that specifically binds to TLR4 or a TLR4 / MD2 complex.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof specifically binds to TLR4 alone.

3. The antibody or antigen-binding fragment thereof according to either claim 1 or claim 2, wherein the antibody or antigen-binding fragment thereof does not bind to TLR2, and / or wherein the antibody or antigen-binding fragment thereof does not bind to MD2 alone.

4. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen binding fragment thereof recognises an epitope comprising or consisting of SEQ ID No: 179, 180 or 210, or a variant or fragment thereof.

5. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises:(i) a CDR-H1 domain comprising or consisting of SEQ ID No: 50, a CDR-H2 domain comprising or consisting of SEQ ID No: 51, a CDR-H3 domain comprising or consisting of SEQ ID No: 52, a CDR-L1 domain comprising or consisting of SEQ ID No: 53, a CDR-L2 domain comprising or consisting of SEQ ID No: 54, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 55;(ii) a CDR-H1 domain comprising or consisting of SEQ ID No: 2, a CDR-H2 domain comprising or consisting of SEQ ID No: 3, a CDR-H3 domain comprising or consisting of SEQ ID No: 4, a CDR-L1 domain comprising or consisting of SEQ ID No: 5, a CDR-L2 domain comprising or consisting of SEQ ID No: 6, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 7;(iii) a CDR-H1 domain comprising or consisting of SEQ ID No: 10, a CDR-H2 domain comprising or consisting of SEQ ID No: 11, a CDR-H3 domain comprising or consisting of SEQ ID No: 12, a CDR-L1 domain comprising or consisting of SEQ ID No: 13, a CDR-L2 domain comprising or consisting of SEQ ID No: 14, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 15;(iv) a CDR-H1 domain comprising or consisting of SEQ ID No: 18, a CDR-H2 domain comprising or consisting of SEQ ID No: 19, a CDR-H3 domain comprising or consisting of SEQ ID No: 20, a CDR-L1 domain comprising or consisting of SEQ ID No: 21, a CDR-L2 domain comprising or consisting of SEQ ID No: 22, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 23;(v) a CDR-H1 domain comprising or consisting of SEQ ID No: 26, a CDR-H2 domain comprising or consisting of SEQ ID No: 27, a CDR-H3 domain comprising or consisting of SEQ ID No: 28, a CDR-L1 domain comprising or consisting of SEQ ID No: 29, a CDR-L2 domain comprising or consisting of SEQ ID No: 30, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 31;(vi) a CDR-H1 domain comprising or consisting of SEQ ID No: 34, a CDR-H2 domain comprising or consisting of SEQ ID No: 35, a CDR-H3 domain comprising or consisting of SEQ ID No: 36, a CDR-L1 domain comprising or consisting of SEQ ID No: 37, a CDR-L2 domain comprising or consisting of SEQ ID No: 38, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 39;(vii) a CDR-H1 domain comprising or consisting of SEQ ID No: 42, a CDR-H2 domain comprising or consisting of SEQ ID No: 43, a CDR-H3 domain comprising or consisting of SEQ ID No: 44, a CDR-L1 domain comprising or consisting of SEQ ID No: 45, a CDR-L2 domain comprising or consisting of SEQ ID No: 46, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 47;(viii) a CDR-H1 domain comprising or consisting of SEQ ID No: 58, a CDR- H2 domain comprising or consisting of SEQ ID No: 59, a CDR-H3 domain comprising or consisting of SEQ ID No: 60, a CDR-L1 domain comprising or consisting of SEQ ID No: 61, a CDR-L2 domain comprising or consisting of SEQ ID No: 62, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 63;(ix) a CDR-H1 domain comprising or consisting of SEQ ID No: 66, a CDR-H2 domain comprising or consisting of SEQ ID No: 67, a CDR-H3 domain comprising or consisting of SEQ ID No: 68, a CDR-L1 domain comprising or consisting of SEQ ID No: 69, a CDR-L2 domain comprising or consisting of SEQ ID No: 70, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 71;(x) a CDR-H1 domain comprising or consisting of SEQ ID No: 74, a CDR-H2 domain comprising or consisting of SEQ ID No: 75, a CDR-H3 domain comprising or consisting of SEQ ID No: 76, a CDR-L1 domain comprising or consisting of SEQ ID No: 77, a CDR-L2 domain comprising or consisting of SEQ ID No: 78, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 79; or(xi) a CDR-H1 domain comprising or consisting of SEQ ID No: 82, a CDR-H2 domain comprising or consisting of SEQ ID No: 83, a CDR-H3 domain comprising orconsisting of SEQ ID No: 84, a CDR-L1 domain comprising or consisting of SEQ ID No: 85, a CDR-L2 domain comprising or consisting of SEQ ID No: 86, and / or a CDR-L3 domain comprising or consisting of SEQ ID No:

87.

6. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises:(i) a heavy chain variable region comprising or consisting of SEQ ID No: 56, and a light chain variable region comprising or consisting of SEQ ID No: 57; (ii) a heavy chain variable region comprising or consisting of SEQ ID No: 8, and a light chain variable region comprising or consisting of SEQ ID No: 9;(iii) a heavy chain variable region comprising or consisting of SEQ ID No: 16, and a light chain variable region comprising or consisting of SEQ ID No: 17;(iv) a heavy chain variable region comprising or consisting of SEQ ID No: 24, and a light chain variable region comprising or consisting of SEQ ID No: 25;(v) a heavy chain variable region comprising or consisting of SEQ ID No: 32, and a light chain variable region comprising or consisting of SEQ ID No: 33;(vi) a heavy chain variable region comprising or consisting of SEQ ID No:40, and a light chain variable region comprising or consisting of SEQ ID No: 41; (vii) a heavy chain variable region comprising or consisting of SEQ ID No:48, and a light chain variable region comprising or consisting of SEQ ID No: 49;(viii) a heavy chain variable region comprising or consisting of SEQ ID No:64, and a light chain variable region comprising or consisting of SEQ ID No: 65;(ix) a heavy chain variable region comprising or consisting of SEQ ID No: 72, and a light chain variable region comprising or consisting of SEQ ID No: 73;(x) a heavy chain variable region comprising or consisting of SEQ ID No: 80, and a light chain variable region comprising or consisting of SEQ ID No: 81; or(xi) a heavy chain variable region comprising or consisting of SEQ ID No:88, and a light chain variable region comprising or consisting of SEQ ID No: 89.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof comprises a CDR-H1 domain comprising or consisting of SEQ ID No: 90, a CDR-H2 domain comprising or consisting of SEQ ID No: 91, a CDR-H3 domain comprising or consisting of SEQ ID No: 92, a CDR-L1 domain comprising or consisting of SEQ ID No: 93, a CDR-L2 domain comprising or consisting of SEQ ID No: 94, and / or a CDR-L3 domain comprising or consisting of SEQ ID No: 95.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or 7, wherein the antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable region comprising or consisting of SEQ ID No: 96, and a light chain variable region comprising or consisting of SEQ ID No: 97;(ii) a heavy chain variable region comprising or consisting of SEQ ID No: 100, and a light chain variable region comprising or consisting of SEQ ID No: 101;(iii) a heavy chain variable region comprising or consisting of SEQ ID No: 104, and a light chain variable region comprising or consisting of SEQ ID No: 105;(iv) a heavy chain variable region comprising or consisting of SEQ ID No: 108, and a light chain variable region comprising or consisting of SEQ ID No: 109;(v) a heavy chain variable region comprising or consisting of SEQ ID No: 112, and a light chain variable region comprising or consisting of SEQ ID No: 113; (vi) a heavy chain variable region comprising or consisting of SEQ ID No:116, and a light chain variable region comprising or consisting of SEQ ID No: 117;(vii) a heavy chain variable region comprising or consisting of SEQ ID No: 120, and a light chain variable region comprising or consisting of SEQ ID No: 121;(viii) a heavy chain variable region comprising or consisting of SEQ ID No: 124, and a light chain variable region comprising or consisting of SEQ ID No: 125;(ix) a heavy chain variable region comprising or consisting of SEQ ID No: 128, and a light chain variable region comprising or consisting of SEQ ID No: 129;(x) a heavy chain variable region comprising or consisting of SEQ ID No: 132, and a light chain variable region comprising or consisting of SEQ ID No: 133; (xi) a heavy chain variable region comprising or consisting of SEQ ID No:136, and a light chain variable region comprising or consisting of SEQ ID No: 137;(xii) a heavy chain variable region comprising or consisting of SEQ ID No: 140, and a light chain variable region comprising or consisting of SEQ ID No: 141;(xiii) a heavy chain variable region comprising or consisting of SEQ ID No: 144, and a light chain variable region comprising or consisting of SEQ ID No: 145;(xiv) a heavy chain variable region comprising or consisting of SEQ ID No: 148, and a light chain variable region comprising or consisting of SEQ ID No: 149;(xv) a heavy chain variable region comprising or consisting of SEQ ID No: 152, and a light chain variable region comprising or consisting of SEQ ID No: 153; (xvi) a heavy chain variable region comprising or consisting of SEQ ID No:156, and a light chain variable region comprising or consisting of SEQ ID No: 157;- Il l -(xvii) a heavy chain variable region comprising or consisting of SEQ ID No:160, and a light chain variable region comprising or consisting of SEQ ID No: 161;(xviii) a heavy chain variable region comprising or consisting of SEQ ID No:164, and a light chain variable region comprising or consisting of SEQ ID No: 165; or(xix) a heavy chain variable region comprising or consisting of SEQ ID No: 168, and a light chain variable region comprising or consisting of SEQ ID No: 169.

9. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises a mutated Fc fragment, optionally wherein the Fc fragment comprises one or more amino acid substitution that silences or reduces the effector function of the antibody or antigen-binding fragment thereof.

10. The antibody or antigen-binding fragment thereof according to claim 9, wherein :(i) the Fc fragment comprises one or more amino acid substitution selected from the group consisting of: L234A, L235A, and P329G;(ii) the Fc fragment comprises one or more amino acid substitution selected from the group consisting of: N325S and L328F; and / or(iii) the Fc fragment comprises one or more amino acid substitution selected from the group consisting of: M252Y, S254T, and T256E.

11. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is capable of inhibiting TLR4 function, and / or wherein the antibody or antigen binding fragment thereof of is capable of inhibiting the TLR4 / MD2 signalling cascade.

12. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof specifically binds to cynomolgus TLR4 or a cynomolgus TLR4 / MD2 complex, and / or wherein the antibody or antigen binding fragment thereof specifically binds to human TLR4 or a human TLR4 TLR4 / MD2 complex.

13. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the affinity of the antibody or antigen-binding fragment thereof for TLR4 as indicated by KD, is less than about IO-5M, less thanabout IO-6M, less than about IO-7M, less than about IO-8M, less than about IO-9M, less than about IO-10M, less than about IO-11M, less than about IO-12M, or less than about IO-13M.

14. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen binding fragment thereof is capable of inhibiting TNF-a release, optionally wherein the antibody or antigen binding fragment thereof inhibits TNF-a release by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%.

15. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen binding fragment thereof is capable of inhibiting dextran-permeability, optionally wherein the antibody or antigen binding fragment thereof inhibits dextran-permeability by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%.

16. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, for use in therapy.

17. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, for use in treating, preventing or ameliorating a TLR4-mediated condition.

18. The antibody or antigen-binding fragment thereof for use according to claim 17, wherein the TLR4-mediated condition is a condition where TLR4 recognises the pathogen- and damage-associated molecular pattern like PAMPs and DAMPs in inflammation and coordinates cellular responses, and / or is a condition involving inflammation driving tissue damage, barrier damage, loss of cell-cell adhesion, loss of epithelial barrier function, immune cell infiltration, and / or where epithelial cell biology plays a key role.

19. The antibody or antigen-binding fragment thereof for use according to either claim 17 or claim 18, wherein the TLR4-mediated condition is one where TLR4 expression is increased, optionally wherein the increased expression of TLR4 leadsto the disruption of tight junction proteins, and / or compromised barrier function and epithelial function during lung or skin injury.

20. The antibody or antigen-binding fragment thereof for use according to any one of claims 17 to 19, wherein the TLR4-mediated condition is a condition that is triggered by exogenous and endogenous ligands that activate and increase TLR4 expression, and cause systemic inflammation, release of pro-inflammatory molecules, immune cell triggered tissue damage, immune mediated tissue damage, and / or cell death or organ failure.

21. The antibody or antigen-binding fragment thereof for use according to any one of claims 17 to 20, wherein the TLR4-mediated condition is selected from a group of conditions consisting of: neuroinflammation; encephalitis; meningitis; neurodegenerative disorders; Alzheimer's disease; Parkinson's disease; skin inflammation; atopic dermatitis; psoriasis; an infectious disease; sepsis; lung inflammation; allergic airway diseases; asthma; COPD; idiopathic pulmonary fibrosis; acute lung injury; acute respiratory distress syndrome; long COVID; inflammatory diseases; Crohn's / IBD; systemic sclerosis; rheumatoid arthritis; acute pancreatitis; Hepatic fibrosis; metabolic dysfunction-associated steatotic liver disease (MASLD); compensated liver disease; decompensated liver disease; liver failure; acute liver injury; liver cirrhosis; acute-on-chronic liver failure (ACLF); alcohol-associated liver disease (ALD); steatotic liver disease (SLD); metabolic dysfunction and alcohol-associated liver disease (MetALD); metabolic dysfunction- associated steatohepatitis (MASH); cryptogenic SLD; alcohol liver disease; hepatocellular carcinoma; renal fibrosis; renal failure; acute kidney injury; chronic kidney disease; ischaemia / reperfusion injury; end stage renal failure; and cardiac disease.

22. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, and optionally a pharmaceutically acceptable vehicle.

23. A process for making the pharmaceutical composition according to claim 22, the process comprising combining a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to15, with a pharmaceutically acceptable vehicle.

24. A polynucleotide sequence encoding the antibody, or antigen-binding fragment thereof according to any one of claims 1 to 15.

25. An expression cassette comprising the polynucleotide sequence according to claim 24.

26. A recombinant vector comprising the expression cassette according to claim 25.

27. A host cell comprising the polynucleotide sequence according to claim 24, the expression cassette according to claim 25, or the vector according to claim 26.

28. A method of preparing the antibody, or antigen-binding fragment according to any one of claims 1 to 15, the method comprising: a) introducing, into a host cell, the vector according to claim 26; and b) culturing the host cell under conditions to result in the production of the antibody, or antigen-binding fragment according to any one of claims 1 to 15.

29. The antibody or antibody binding fragment of according to any one of claims 1 to 15, for use in diagnosis or prognosis.

30. The antibody or antibody binding fragment of according to any one of claims 1 to 15, for use in diagnosing or prognosing a TLR4-mediated condition.

31. A method of diagnosing or prognosing a TLR4-mediated condition in a subject, the method comprising detecting TLR4 in a biological sample obtained from the subject with the antibody or antibody binding fragment of according to any one of claims 1 to 15.

32. A kit for diagnosing a subject suffering from a TLR4-mediated condition, or for providing a prognosis of the subject's condition, the kit comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 for detecting TLR4 in a sample from a test subject.

33. The antibody or antibody binding fragment for use according to claim 30, the method according to claim 31, or the kit according to claim 32, wherein the TLR4- mediated condition is a condition where TLR4 recognises the pathogen- anddamage-associated molecular pattern like PAMPs and DAMPs in inflammation and coordinates cellular responses, and / or is a condition involving inflammation driving tissue damage, barrier damage, loss of cell-cell adhesion, loss of epithelial barrier function, immune cell infiltration, and / or where epithelial cell biology plays a key role.

34. The antibody or antibody binding fragment for use according to claim 30, the method according to claim 31, or the kit according to claim 32, wherein the TLR4- mediated condition is one where TLR4 expression is increased, optionally wherein the increased expression of TLR4 leads to the disruption of tight junction proteins, and / or compromised barrier function and epithelial function during lung or skin injury.

35. The antibody or antibody binding fragment for use according to claim 30, the method according to claim 31, or the kit according to claim 32, wherein the TLR4- mediated condition is a condition that is triggered by exogenous and endogenous ligands that activate and increase TLR4 expression, and cause systemic inflammation, release of pro-inflammatory molecules, immune cell triggered tissue damage, immune mediated tissue damage, and / or cell death or organ failure.

36. The antibody or antibody binding fragment for use according to claim 30, the method according to claim 31, or the kit according to claim 32, wherein the TLR4- mediated condition is selected from a group of conditions consisting of: neuroinflammation; encephalitis; meningitis; neurodegenerative disorders; Alzheimer's disease; Parkinson's disease; skin inflammation; atopic dermatitis; psoriasis; an infectious disease; sepsis; lung inflammation; allergic airway diseases; asthma; COPD; idiopathic pulmonary fibrosis; acute lung injury; acute respiratory distress syndrome; long COVID; inflammatory diseases; Crohn's / IBD; systemic sclerosis; rheumatoid arthritis; acute pancreatitis; Hepatic fibrosis; metabolic dysfunction-associated steatotic liver disease (MASLD); compensated liver disease; decompensated liver disease; liver failure; acute liver injury; liver cirrhosis; acute- on-chronic liver failure (ACLF); alcohol-associated liver disease (ALD); steatotic liver disease (SLD); metabolic dysfunction and alcohol-associated liver disease (MetALD); metabolic dysfunction-associated steatohepatitis (MASH); cryptogenic SLD; alcohol liver disease; hepatocellular carcinoma; renal fibrosis; renal failure; acute kidney injury; chronic kidney disease; ischaemia / reperfusion injury; end stage renal failure; and cardiac disease.

37. Use of at least one gene selected from the group consisting of: CCL7, IL1B, CCL5, CCL3, CCL19, CCL2, CCL4, ITGB2, CHI3L3, CXCL2, CXCL9, ILIA, FOS, CXCR2, MYC, PTGS1, NLRP3, CXCL10, TNFAIP3, CXCL1, CXCL5, CFB, TYROBP, C3, CCL17, CCL8, CCL11, CCL20, OAS1A, H2-EB1, MRC1, TLR6, ARG1, CSF2, CXCL3,LIGP1, CCR2, CCR3, OAS2, RETNLA, CD86, IL17A, CCL24, IL1RN, IL-6 and TNF-a, or a variant or fragment thereof, or a polypeptide encoded by the at least one gene, or a variant or fragment thereof, as a biomarker for a TLR4-mediated condition.

38. A method for diagnosing a subject suffering from a TLR4-mediated condition, or a pre-disposition thereto, or for providing a prognosis of the subject's condition, the method comprising detecting the expression level of at least one gene selected from the group consisting of: CCL7, IL1B, CCL5, CCL3, CCL19, CCL2, CCL4, ITGB2, CHI3L3, CXCL2, CXCL9, ILIA, FOS, CXCR2, MYC, PTGS1, NLRP3,CXCL10, TNFAIP3, CXCL1, CXCL5, CFB, TYROBP, C3, CCL17, CCL8, CCL11, CCL20,OAS1A, H2-EB1, MRC1, TLR6, ARG1, CSF2, CXCL3, LIGP1, CCR2, CCR3, OAS2, RETNLA, CD86, IL17A, CCL24, IL1RN, IL-6 and TNF-a, or a variant or fragment thereof, or the concentration of a polypeptide encoded by the at least one gene, or a variant or fragment thereof, in a sample obtained from a test subject, and comparing this expression level or concentration, with a reference for the expression level or concentration of the at least one gene or polypeptide, in an individual who does not suffer from a TLR4-mediated condition, wherein : i) an increase in the expression level or concentration of the at least one gene or polypeptide in the sample obtained from the test subject compared to the reference suggests that the subject is suffering from a TLR4- mediated condition, or has a pre-disposition thereto, or provides a negative prognosis of the subject's condition; and / or ii) a decrease in the expression level or concentration of the at least one gene or polypeptide in the sample obtained from the test subject compared to the reference suggests that the subject is not suffering from a TLR4- mediated condition, or does not have a pre-disposition thereto, or provides a positive prognosis of the subject's condition.

39. A method for prognosing a subject suffering from a TLR4-mediated condition, the method comprising detecting, in a test subject, the expression level of at least one gene selected from the group consisting of: CCL7, IL1B, CCL5, CCL3,CCL19, CCL2, CCL4, ITGB2, CHI3L3, CXCL2, CXCL9, ILIA, FOS, CXCR2, MYC, PTGS1, NLRP3, CXCL10, TNFAIP3, CXCL1, CXCL5, CFB, TYROBP, C3, CCL17, CCL8, CCL11, CCL20, OAS1A, H2-EB1, MRC1, TLR6, ARG1, CSF2, CXCL3, LIGP1, CCR2, CCR3, OAS2, RETNLA, CD86, IL17A, CCL24, IL1RN, IL-6 and TNF-a, or a variant or fragment thereof, or the concentration of a polypeptide encoded by the at least one gene, or a variant or fragment thereof, and comparing this expression level or concentration with a reference for the expression level or concentration of the at least one gene or polypeptide in an individual who does not suffer from a TLR4- mediated condition, wherein : (i) an increase in the expression level or concentration of the at least one gene or polypeptide in the test subject compared to the reference provides a negative prognosis of the subject's condition; and / or(ii) a decrease in the expression level or concentration of the at least one gene or polypeptide in the test subject compared to the reference provides a positive prognosis of the subject's condition.

40. A kit for diagnosing a subject suffering from a TLR4-mediated condition, or a pre-disposition thereto, or for providing a prognosis of the subject's condition, the kit comprising means for detecting the expression level, in a sample from a test subject, of at least one gene selected from the group consisting of: CCL7, IL1B,CCL5, CCL3, CCL19, CCL2, CCL4, ITGB2, CHI3L3, CXCL2, CXCL9, ILIA, FOS, CXCR2, MYC, PTGS1, NLRP3, CXCL10, TNFAIP3, CXCL1, CXCL5, CFB, TYROBP, C3, CCL17, CCL8, CCL11, CCL20, OAS1A, H2-EB1, MRC1, TLR6, ARG1, CSF2, CXCL3, LIGP1, CCR2, CCR3, OAS2, RETNLA, CD86, IL17A, CCL24, IL1RN, IL-6 and TNF-a, or a variant or fragment thereof, or the concentration of a polypeptide encoded by the at least one gene, or a variant or fragment thereof.

41. A method for determining the efficacy of treating a subject suffering from a TLR4-mediated condition with a therapeutic agent, the method comprising analysing the expression level of at least one gene selected from the group consisting of: CCL7, IL1B, CCL5, CCL3, CCL19, CCL2, CCL4, ITGB2, CHI3L3, CXCL2, CXCL9, ILIA, FOS, CXCR2, MYC, PTGS1, NLRP3, CXCL10, TNFAIP3, CXCL1, CXCL5, CFB, TYROBP, C3, CCL17, CCL8, CCL11, CCL20, OAS1A, H2-EB1, MRC1, TLR6, ARG1, CSF2, CXCL3, LIGP1, CCR2, CCR3, OAS2, RETNLA, CD86, IL17A, CCL24, IL1RN, IL-6 and TNF-a, or a variant or fragment thereof, or the concentration of a polypeptide encoded by the at least one gene, or a variant or fragment thereof, in a bodily sample from the subject and comparing the expression level or concentrationwith a reference for the expression level or concentration of the at least one gene or polypeptide in an individual who does suffer from a TLR4-mediated condition, wherein a decrease in the expression level or concentration of the at least one gene or polypeptide in the bodily sample from the subject, compared to the reference, suggests that the treatment with the therapeutic agent is effective, or wherein an increase in the expression level or concentration of the at least one gene or polypeptide in the bodily sample from the subject, compared to the reference, suggests that the treatment with the therapeutic agent is ineffective.

42. The use according to claim 37, the method according to any one of claims38, 39 or 41, or the kit according to claim 40,(a) wherein (i) the TLR4-mediated condition is selected from the group of conditions consisting of: Metabolic dysfunction-associated steatotic liver disease (MASLD); compensated liver diseases; decompensated liver diseases; liver failure; acute liver injury; liver cirrhosis, acute-on-chronic liver failure (ACLF); alcohol- associated liver disease (ALD); steatotic liver disease (SLD); metabolic dysfunction and alcohol-associated liver disease (MetALD); metabolic dysfunction-associated steatohepatitis (MASH); cryptogenic SLD; alcohol liver disease; and hepatocellular carcinoma; and (ii) the at least one gene is selected from the group consisting of: CCL7, IL1B, CCL5, CCL3, CCL19, CCL2, CCL4, ITGB2, CHI3L3, CXCL2, CXCL9, ILIA, FOS, CXCR2, MYC, PTGS1, NLRP3, CXCL10, TNFAIP3, CXCL1, IL-6 and TNF-a, or a variant or fragment thereof, or a polypeptide encoded by the at least one gene, or a variant or fragment thereof; or(b) wherein (i) the TLR4-mediated condition is selected from the group of conditions consisting of: lung inflammation and allergic airway diseases such as asthma, COPD, idiopathic pulmonary fibrosis; acute lung injury; acute respiratory distress syndrome, and long COVID; and (II) the at least one gene is selected from the group consisting of: CXCL5, CFB, TYROBP, C3, CXCL10, CCL17, CCL8, CCL11, CHI3L3, CCL20, ITGB2, OAS1A, H2-EB1, MRC1, TLR6, CCL2, ARG1, CXCR2, CSF2, CXCL3, LIGP1, CCR2, CCR3, OAS2, CXCL9, RETNLA, CD86, IL17A, CXCL1, CCL3, CCL24, CCL7, IL1RN, IL-6 and TNF-a, or a variant or fragment thereof, or a polypeptide encoded by the at least one gene, or a variant or fragment thereof.

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