WO2025238135A2

WO2025238135A2 - Antibody with binding specificity for il-11

Info

Publication number: WO2025238135A2
Application number: PCT/EP2025/063349
Authority: WO
Inventors: Pallavi BHATTA; Leo Alexander BOWSHER; Hannah Elizabeth EDWARDS; Elisa MAGGIOLI; Zofia WHITTAKER
Original assignee: UCB Biopharma SRL
Current assignee: UCB Biopharma SRL
Priority date: 2024-05-17
Filing date: 2025-05-15
Publication date: 2025-11-20
Anticipated expiration: 2026-11-17

Abstract

The present technology relates to antibodies or antigen-binding fragments thereof that bind to IL-11 and their therapeutic use. Additionally, the present technology also relates to agents that inhibit IL-11 mediated signaling for the treatment of hidradenitis suppurativa.

Description

ANTIBODY WITH BINDING SPECIFICITY FOR IL-11

FIELD

The present technology relates to antibodies and antigen-binding fragments thereof that mediate interleukin-11 (IL-11) signaling. Such antibodies and pharmaceutical compositions as provided herein are useful in the therapeutic treatment of subjects suffering from a number of diseases, in particular, from hidradenitis suppurativa.

BACKGROUND

IL-11 is a 19 kDa member of the IL-6 cytokine family, which comprise oncostatin-M (OSM), IL- 6 itself, ciliary neurotrophic factor (CNTF), leukaemia inhibitory factor (LIF), cardiotrophin 1 (CT-1), IL-31 and IL-27. These cytokines mostly act by binding to varied, and sometimes shared, alpha receptors and then complex with the common gp130 coreceptor to initiate intracellular signaling.

To signal in cis IL-11 uses its cognate alpha receptor - IL-11 RA in humans, 1111 ra1 in mice - and the ubiquitously expressed gp130 (or IL6ST) co-receptor to activate downstream signaling pathways. The IL-11 : 1 L- 11 RA:gp130 complex needs to dimerise with another equivalent trimer to form a hexameric signaling complex. This initiates canonical gp130-mediated signaling via JAK/STAT, notably JAK2/STAT3, which is thought to be the primary IL-11 pathway.

A number of studies have reported IL-11 can also signal via the non-canonical MEK/ERK activation, which has been identified as particularly important for fibroblast mesenchymal transition (FMT) and also vascular smooth cell mesenchymal transition (VMT), a process also referred to as phenotypic switching. MEK/ERK is a recognised non-canonical signaling pathway downstream of gp130.

Trans-signaling is known to be part of the IL-6 family functional biology and proposes that IL- 6 family cytokines, when complexed with the soluble form of their cognate receptor can signal on most cells expressing the gp130 co-receptor. There have been conflicting reports on the biological relevance of such alternative signaling for IL-11 , with the available data in the literature supporting IL-11 cis-signaling as the dominant pathway driving IL-11 activity.

IL-11 receptor alpha (also referred to herein as “IL-11 RA”, “IL-11 Ra” or “IL-11 R”) is highly expressed on stromal cells, including fibroblasts, vascular smooth muscle cells (VSMCs), adipocytes, hepatic/pancreatic stellate cells or pericytes, epithelial and polarized cells. The same cells are also able to secrete IL-11 upon tissue injury, which then triggers both autocrine and paracrine signaling and drives the three pathologies common to all fibro-inflammatory diseases: myofibroblast activation, parenchymal cell dysfunction, and inflammation - while also inhibiting tissue regeneration.

IL-11 can also be secreted by multiple immune cell types, including CD8+ T cells, B-cells, natural killer (NK) cells, macrophages, y<5T cells, and eosinophils, and has been shown to have wide range of biological activities, including anti-tumour responses, and differentiation of B- cells and T-cells.

IL-11 has been reported to be upregulated in a wide variety of fibro-inflammatory diseases and solid malignancies. Elevated IL-11 expression is also associated with several non-malignant inflammatory diseases including Multiple sclerosis, Periodontitis, Asthma, Inflammatory Bowel Disease (IBD) and Arthritis, where its function remains less well-characterized.

Hidradenitis suppurativa (HS), also known as acne inversa, is a chronic, disabling and debilitating inflammatory skin disease, mainly characterized by the occurrence of painful nodules caused by occlusion and inflammation of hair follicles, which progress into abscesses, and chronically pus-draining fistulas in apocrine gland-bearing areas of the body, such as in the armpits and groin. Over time, the chronic, uncontrolled inflammation which characterizes HS results in irreversible tissue destruction and scarring. HS has profound, wide-ranging, negative consequences for patients, including chronic pain, mobility deficits, depression and anxiety, suicide and suicidality, stigmatization, impaired body image and sex life, unemployment and socio-economic consequences. Moreover, the high frequency of comorbidity and concomitant disease in HS, in particular metabolic syndrome, contributes to increased cardiovascular disease and morbidity and a reduction in life expectancy. The exact cause of HS is unknown, but it is a complex disease with contributory genetic and epigenetic changes, and hormonal, mechanical, microbial and lifestyle factors such as obesity and smoking (Krueger J et al, BJD, 2024¹).

The treatment landscape of HS is challenging due to wide clinical manifestations of the disease, which makes the disease diagnosis very challenging, and the complex, still poorly understood, pathogenesis. Current treatment options to reduce symptom burden include topical antibiotic treatments, local steroid injections, systemic therapies and repeated and/or rotational courses of systemic antibiotics, retinoids and hormonal therapies. Various physical, like light therapy, and surgical procedures exist, with guidelines generally recommending concomitant use of both surgical and medical treatments, particularly in advanced HS.

Multiple immunological pathways have been found to be associated with HS, however to date only the TNF-a and IL-17 pathways have been confirmed as pathological drivers of the disease. Elevated levels of TNF-a and IL-17 can be found in skin and/or serum of patients with HS and correlate with HS severity. The anti-TNFa antibody Humira (adalimumab), was for a long time the only approved biologic for treatment of moderate-to-severe HS patients. In the PIONEER phase III trials, about 50% of patients achieved HiSCR50 response (HiSCR: Hidradenitis Suppurative Clinical Response, HISCR50/90: patients with more than 50 or 90% reduction of in inflammatory nodules and abscess counts, with no increase in abscess or tunnel count), however a significant number of abscesses and draining fistulas remain after treatment. Only 30.5% of patients remain on Humira after 24 months, due to loss of efficacy (Prens M et al, Br J Dermatol. 2021²).

More recently, Secukinumab (an inhibitor of IL-17A) and Bimekizumab (an inhibitor of IL-17F in addition to IL-17A) received approval for HS treatment.

In two Phi II trials (BE HEARD I & II), Bimekizumab demonstrated significant clinical improvement in people affected by moderate to severe HS vs. placebo, with up to 55% of patients achieving HiSCR50 at Week 16 (mNRI). Response was maintained, or increased through to Wk48, particularly for more stringent outcomes such as HiSCR75, HiSCR90 and HiSCRIOO. Furthermore, Bimekizumab was shown to reduce the number of all types of inflammatory lesions, with ~ 50% reduction from baseline in draining tunnel count over 48 weeks. Although there are no head to head studies, in indirect comparisons to the IL-17A inhibitor Secukinumab, which is approved for moderate to severe HS in the US and EU, Bimekizumab demonstrated superior HiSCR50 responses at Week 16 and Week 48, providing evidence for both IL-17A and IL-17F playing an important role in disease pathogenesis. Furthermore, another therapeutic which inhibits IL-17A and IL-17F, the nanobody Sonelokimab, has recently completed a Phil trial in HS, meeting the primary endpoint of HiSCR75 and showing positive response across lesion types including draining tunnels through to Wk24. Similarly, IL17 blockade with the anti-IL17RA antibody Brodalumab has showed a positive clinical response resulted in decreased tunnel size and drainage in HS patients in a small study.

Although IL- 17 blockade has been shown to be an important advancement in the treatment of HS, many patients do not achieve complete disease control with current therapies. Given the disfiguring nature of the disease, there is an unmet need for new treatments to provide disease control and prevent disease progression and irreversible scaring.

¹ Krueger JG, Frew J, Jemec GBE, Kimball AB, Kirby B, Bechar F, Navrazhina K, Prens E, Reich K, Cullen E, Wolk K. Hidradenitis suppurativa: new insights into disease mechanisms and an evolving treatment landscape. Br J Dermatol. 2024; 190 (2): 149-162.

² Prens LM, Bouwman K, Aarts P, Arends S, van Straalen KR, Dudink K, Horvath B, Prens E P. Adalimumab and infliximab survival in patients with hidradenitis suppurativa: a daily practice cohort study. Br J Dermatol. 2021 ; 185 (1): 177-184. SUMMARY

The present technology addresses the need for new treatments of hidradenitis suppurativa by providing antibodies that are capable of inhibiting IL- 11 mediated signaling. The inventors have established for the first time that IL-11 plays a role in HS biology and that blockade of the IL- 11 signaling pathway thus has the potential to result in clinical responses.

The present technology further provides antibodies capable of binding to human IL-11.

In a first aspect, the present technology provides an antibody or an antigen-binding fragment thereof that specifically binds to IL-11, defined by a set of specific CDR sequences and/or variable region sequences.

In a second aspect, the present technology provides an agent capable of inhibiting IL-11 mediated signaling for use in the treatment of hidradenitis suppurativa.

BRIEF DESCRIPTION OF THE DRAWINGS

The present technology is described below by reference to the following drawings, in which:

Figure 1 Alignments of the rat antibody (donor) V-region sequences with the human germline (acceptor) V-region sequences for the light chain graft of 19439

Figure 2 Alignments of the rat antibody (donor) V-region sequences with the human germline (acceptor) V-region sequences for the heavy chain graft of 19439

Figure 3 Alignment of light chain 19439 variants

Figure 4 Alignment of heavy chain 19439 variants

Figure 5 Alignments of the rat antibody (donor) V-region sequences with the mouse germline (acceptor) V-region sequences for the light chain graft of 19439

Figure 6 Alignments of the rat antibody (donor) V-region sequences with the mouse germline (acceptor) V-region sequences for the light chain graft of 19439

Figure 719439gL1 gH1 Fab does not bind to IL-11 RA or IL-11 RA+ gp130 expressing cells in the presence of IL-11 indicating non internalization properties. VR19882gH1gL1 Fab (with non-blocking VR) does bind in the presence of IL-11.

Figure 8 VR 19439gL1gH1 Fab and reference mAb1 and derived Fab fragment in the human IL-11 induced cis-STAT3 signaling assay using a human HepG2 IL-11R/STAT3 reporter cell line. Representative 4PL-fitted inhibition curves are shown, the error bars represent the standard deviation of the data relative to the arithmetic mean. Figure 9 19439gl_1gH1 Fab, reference mAb1 and derived Fab fragment in the human IL- 11/IL-11RA mediated STAT3 trans-signaling assay in the ‘non-displacement’ format. Representative 4PL-fitted inhibition curves are shown, the error bars represent the standard deviation of the data relative to the arithmetic mean.

Figure 10 19439gl_1gH1 Fab and Fab derived from reference mAb1 in the IL-11 and IL- 17AA mediated CXCL1 release assay on primary human dermal fibroblasts. Representative 4PL-fitted inhibition curves are shown, the error bars represent the standard deviation of the data relative to the arithmetic mean.

Figure 11 Expression profile by qPCR of IL-11RA and gp130 (IL6ST) from human and cynomolgus dermal fibroblasts. A. Raw CT values B. Fold change expression level relative to human dermal fibroblast GAPDH.

Figure 12 Primary human dermal fibroblast stimulation with rhlL-11 at 10ng/ml (518pM) or rhlL-17AA at 10ng/ml (311pM) with IL-17FF at 100ng/ml (3.36nM) independently induced secretion of CCL2. Combined stimulation of dermal fibroblast with rhlL-11 and rhlL-17AA/FF resulted in synergistic secretion of CCL2. 19439gL1gH1 / null KiH hlgG1 LA LA (in vitro transient material) at 10pg/ml (66.7nM) functionally inhibited rhlL-11 and rhlL-11 + rhlL-17AA/FF mediated CCL2 secretion from primary human dermal fibroblasts. Geomean, n=3 donors/ 3 replicates per donor per condition.

Figure 13 Primary human dermal fibroblast stimulation with rhlL-11 at 10ng/ml (518pM) or rhlL-17AA at 10ng/ml (311pM) with IL-17FF at 100ng/ml (3.36nM) independently induced secretion of IL-6. Combined stimulation of dermal fibroblast with rhlL-11 and rhlL-17AA/FF resulted in synergistic secretion of IL-6. 19439gL1gH1 / null KiH hlgG1 LA LA (in vitro transient material) at 10pg/ml (66.7nM) functionally inhibited rhlL-11 and rhlL-11 + rhlL-17AA/FF mediated IL-6 secretion from primary human dermal fibroblasts. Geomean, n=3 donors/ 3 replicates per donor per condition.

Figure 14 Primary human dermal fibroblast stimulation with rhlL-11 at 10ng/ml (518pM) with rhlL-17AA at 10ng/ml (311pM) and IL-17FF at 100ng/ml (3.36nM) induced secretion of MMP2. 19439gL1gH1 / null KiH hlgG1 LALA (in vitro transient material) at 10pg/ml (66.7nM) functionally inhibited rhlL-11+ rhlL-17AA/FF mediated MMP2 secretion from primary human dermal fibroblasts. Geomean, n=3 donors/ 2-3 replicates per donor per condition.

Figure 15 Left: number of genes upregulated or downregulated (FDR < 0.05) upon IL-11 stimulation, as a histogram separated by cell type. Right: Gene sets enriched (Mitch Framework, FDR < 0.01) and mean effect sizes by biological theme. Figure 16 HS Lesional and Non-Lesional Fibroblast & Pericyte Single Cell RNAseq clusters on a Uniform Manifold Approximation and Projection (UMAP) embedding. Points are coloured by normalised expression values, and highlight an area where cells are lesion specific. Blue colour highlights IL-11RA expression.

Figure 17 RNAscope analysis shows differential expression of IL-11 in healthy vs HS lesional skin. IL-11 expression demonstrated low level in both follicular and interfollicular epidermis of normal skin (A). Increased level of IL-11 RNAscope signal was observed in a number of different cell types such as epidermal, dermal and immune cells near de-epithelialized HS lesion (B). Blood vessels are marked by asterisks. RNAscope signal is marked by arrowheads.

Figure 18 RNAscope analysis shows differential expression of IL-11R in healthy vs HS lesional skin. Representative examples of IL-11R RNAscope signal distribution in hair follicles and interfollicular epidermis of normal skin (A) and at de-epithelialized HS lesion (B). IL-11 R expression increased in infundibular epidermis and in immune infiltrates populating the de-epithelialized lesion. RNAscope signal is marked by arrowheads. Outer root sheet of hair follicle (ORS).

Figure 19 Normalised H-score for IL-11 and IL-11R RNAscope staining of HS lesions. Patient samples are classified as mild and moderate-severe. Symbols and patient ID numbers representing individual patients, 10479-10483 (mild), 10484-10493 (moderate- severe).

Figure 20 (A) Stimulation of HDF with rhlL-11 at 10ng/ml in combination with rhlL-17AA at 10ng/ml and IL-17FF at 100ng/ml, or TNFa at 1ng/ml, or IL-1 10pg/m induced synergistic secretion of CXCL-1 (indicated by arrows). Geomean, N=1/3 donors, 3 replicates/donor. (B) Stimulation of HDF with rhlL-11 at 10ng/ml in combination with TNFa at 1ng/ml, or IL-10 10pg/m induced synergistic secretion of IL-8 (indicated by arrows). Geomean, n=1 donor, 3 replicates/donor.

Figure 21 MMP-1, MMP-2, MMP-3, MMP-7, MMP-9 and MMP-10 levels in supernatants post stimulation with IL-17AA at 100ng/ml and IL-17FF at 1000ng/ml (referred to as IL- 17A/F) or rhlL-17AA at 100ng/ml and rhlL-17FF at 1000ng/ml together with 100ng/ml of rhlL-11 (referred to as IL-17A/F + IL-11) in the ex vivo hair follicle organ culture model. Data represented as Geomean, n=2 donors.

Figure 22 (A) Representative images of immunofluorescence IL-11Ra protein expression in the hair follicle and skin samples from healthy individuals. HF- hair follicle; Scale bars 100pm. (B) Percentage of Ki67 expressing cells in the distal outer root sheet in the ex vivo hair follicle organ culture model; n=1 donors, Mean ± SD, Statistical analysis: ANOVA test with Dunnett’s multiple comparison test , *-p<0.05.

Figure 23 Representative examples of SSc and normal skin samples stained for IL-11 by RNAscope. RNAscope labelling demonstrated increased number of IL-11 positive cells in the epidermis of the SSc skin, particularly in areas of profound hyperkeratosis while normal skin expressed low levels of IL-11 in interfollicular epidermal areas. RNAscope staining is marked by arrow heads, framed areas are enlarged in A-C.

Figure 24 Barplots showing total number of differentially expressed (DE) genes in HS skin biopsies (left) and stimulated skin model (right), and concordantly and discordantly dysregulated genes between HS skin biopsies and the HS model.

Figure 25 Concordant upregulation of hallmarks of inflammatory biology between the ex vivo disease model (Stimulated vs. Non-stimulated) and HS0001 skin biopsies (Lesional vs. Non-lesional).

Figure 26 Barplots showing Mean logFC change in key biological pathways after anti- IL-11 treatment of the skin model compared to the control non-treated stimulated skin.

DETAILED DESCRIPTION

Abbreviations

Definitions

The following terms are used throughout the specification.

The term "acceptor human framework" as used herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework derived from a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes.

The term “affinity” refers to the strength of all noncovalent interactions between an antibody and the target protein. Unless indicated otherwise, as used herein, the term "binding affinity" refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule for its binding partner can be generally represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Binding affinity may be measured by standard assays, for example surface plasmon resonance, such as BIAcore. The term "affinity matured" in the context of an antibody refers to an antibody with one or more alterations in the hypervariable regions, compared to a parent antibody which does not possess such alterations, where such alterations result in an improvement in the affinity of the antibody for antigen.

The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and multispecific antibodies as long as they exhibit the desired antigen-binding activity. The term antibody as used herein relates to whole (full-length) antibodies (i.e. comprising the elements of two heavy chains and two light chains) and functionally active fragments thereof (i.e., molecules that contain an antigen-binding domain that specifically binds to an antigen, also termed antibody fragments or antigen-binding fragments). Features described herein with respect to antibodies also apply to antibody fragments unless context dictates otherwise. An antibody may comprise a Fab linked to two scFvs or dsscFvs, each scFv or dsscFv binding the same or a different target (e.g., one scFv or dsscFv binding a therapeutic target and one scFv or dsscFv that increases half-life by binding, for instance, albumin). Such antibodies are described in WO2015/197772. The term "antibody" encompasses monovalent, i.e., antibodies comprising only one antigen-binding domain (e.g. one-armed antibodies comprising a full- length heavy chain and a full-length light chain interconnected, also termed “half-antibody”), and multivalent antibodies, i.e. antibodies comprising more than one antigen-binding domain, e.g bivalent.

The term “antibody-dependent cellular cytotoxicity” or “ADCC” is a mechanism for inducing cell death that depends upon the interaction of antibody-coated target cells with effector cells possessing lytic activity, such as natural killer cells, monocytes, macrophages and neutrophils via Fc gamma receptors (FcyR) expressed on effector cells.

The term “antigen-binding domain” or “binding domain” as employed herein refers to a portion of the antibody, which comprises a part or the whole of one or more variable domains, for example a part or the whole of a pair of variable domains VH and VL, that interact specifically with a target antigen. In the context of the present technology the term is used in relation to IL-11. This antigen-binding domains are also referred to as “IL-11 binding domain”. The IL-11 binding domain specifically binds to IL-11. The binding domain may comprise no more than one VH and one VL. The antigen-binding domain may comprise or consist of an antibody or antigen-binding fragment of an antibody. An example of an antigen-binding domain is a VH/VL unit comprised of a heavy chain variable domain (VH) and a light chain variable domain (VL).

The term "antigen-binding fragment" as employed herein refer to functionally active antibody binding fragments including but not limited to Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single domain antibodies, scFv, Fv, bi, tri or tetra-valent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies and epitope-binding fragments of any of the above (see for example Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews - Online 2(3), 209-217). A "binding fragment" as employed herein refers to a fragment capable of binding a target peptide or antigen with sufficient affinity to characterize the fragment as specific for the peptide or antigen.

The term “bispecific” or “bispecific antibody” as employed herein refers to an antibody with two antigen specificities.

The term "CDRs" refers to "complementarity determining regions". Generally, antibodies comprise six CDRs: three in the VH (CDR-H1 , CDR-H2, CDR-H3), and three in the VL (CDR- L1 , CDR-L2, CDR-L3). The CDRs of the heavy chain variable domain are located at residues 31-35 (CDR-H1), residues 50-65 (CDR-H2) and residues 95-102 (CDR-H3) according to the Kabat numbering system. However, according to Chothia (Chothia, C. and Lesk, A.M. J. Mol. Biol., 196, 901-917 (1987)), the loop equivalent to CDR-H1 extends from residue 26 to residue 32. Thus, unless indicated otherwise “CDR-H1” as employed herein is intended to refer to residues 26 to 35, as described by a combination of the Kabat numbering system and Chothia’s topological loop definition. The CDRs of the light chain variable domain are located at residues 24-34 (CDR-L1), residues 50-56 (CDR-L2) and residues 89-97 (CDR-L3) according to the Kabat numbering system. Unless indicated otherwise, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat.

The term "chimeric" antibody refers to an antibody in which the variable domain (or at least a portion thereof) of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain (i.e. the constant domains) is derived from a different source or species. (Morrison; PNAS 81 , 6851 (1984)). Chimeric antibodies can for instance comprise non-human variable domains and human constant domains. Chimeric antibodies are typically produced using recombinant DNA methods. A subcategory of “chimeric antibodies” is “humanized antibodies”.

The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 , and lgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, E, y, and p, respectively.

The term “complement-dependent cytotoxicity”, or “CDC” refers to a mechanism for inducing cell death in which an Fc effector domain of a target-bound antibody binds to and activates complement component C1 q which in turn activates the complement cascade leading to target cell death.

The terms “constant domain(s)” or “constant region”, as used herein are used interchangeably to refer to the domain(s) of an antibody which is outside the variable regions. The constant domains are identical in all antibodies of the same isotype but are different from one isotype to another. Typically, the constant region of a heavy chain is formed, from N to C terminal, by CH 1 -hinge -CH2-CH3-, optionally CH4, comprising three or four constant domains.

The term “competing antibody” or “cross-competing antibody” shall be interpreted as meaning that the claimed antibody binds to either (i) the same position on the antigen to which the reference antibody binds, or (ii) a position on the antigen where the antibody sterically hinders the binding of the reference antibody to the antigen.

The term “comprising” is to be interpreted as “including”. Aspects of the technology comprising certain elements are also intended to extend to alternative embodiments “consisting” or “consisting essentially” of the relevant elements.

The term “derivatives” as used herein is intended to include reactive derivatives, for example thiol-selective reactive groups such as maleimides and the like. The reactive group may be linked directly or through a linker segment to the polymer. It will be appreciated that the residue of such a group will in some instances form part of the product as the linking group between the antibody fragment and the polymer.

The term “derived from” in the context of generating variable sequences refers to the fact that the sequence employed or a sequence highly similar to the sequence employed was obtained from the original genetic material, such as the light or heavy chain of an antibody.

The term “diabody” as employed herein refers to two Fv pairs, a first VH/VL pair and a further VH/VL pair which have two inter-Fv linkers, such that the VH of a first Fv is linked to the VL of the second Fv and the VL of the first Fv is linked to the VH of the second Fv.

The term “DiFab” as employed herein refers to two Fab molecules linked via their C-terminus of the heavy chains.

The term “a diagnostic agent” with reference to an IL-11 binding antibody or binding fragment thereof refers to the use of an IL-11 binding antibody or binding fragment thereof in the diagnosis of a disease.

A "diagnostically effective amount" refers to the amount of the antibody or binding fragment thereof that, when used in a diagnostic test on a biological sample is sufficient to allow identification of a disease or of monitoring the amount of disease tissue as a means of monitoring the efficacy of therapeutic intervention. The term “dsscFv” or “disulfide-stabilised single chain variable fragment” as employed herein refer to a single chain variable fragment which is stabilised by a peptide linker between the VH and VL variable domain and also includes an inter-domain disulfide bond between VH and VL. (see for example, Weatherill et al., Protein Engineering, Design & Selection, 25 (321-329), 2012, W02007109254.

The term “DVD-lg” (also known as dual V domain IgG) refers to a full-length antibody with 4 additional variable domains, one on the N-terminus of each heavy and each light chain.

The “Ell index” or “Ell index as in Kabat” or “Ell numbering scheme” refers to the numbering of the Ell antibody (Edelman etal., 1969, Proc Natl Acad Sci USA 63:78-85). Such is generally used when referring to a residue in an antibody heavy chain constant region (e.g., as reported in Kabat et al.). Unless stated otherwise, the EU numbering scheme is used to refer to residues in antibody heavy chain constant regions described herein.

The term “Fab” as used herein refers to an antibody fragment comprising a light chain fragment comprising a VL (variable light) domain and a constant domain of a light chain (CL), and a heavy chain fragment comprising a VH (variable heavy) domain and a first constant domain (CH1) of a heavy chain.

The term “Fab’ fragment” as used herein refers to an antibody fragment comprising a heavy and a light chain pair in which the heavy chain comprises a variable region VH, a constant domain CH1 and a natural or modified hinge region and the light chain comprises a variable region VL and a constant domain CL. Dimers of a Fab’ according to the present disclosure create a F(ab’)2 where, for example, dimerisation may be through the hinge.

The term “Fab-dsFv” as employed herein refers to a FabFv wherein an intra-Fv disulfide bond stabilises the appended C-terminal variable regions. The format may be provided as a PEGylated version thereof.

The term “Fab-Fv” as employed herein refers to a Fab fragment with a variable region appended to the C-terminal of each of the following, the CH1 of the heavy chain and CL of the light chain. The format may be provided as a PEGylated version thereof.

The term “Fab-scFv” as employed herein is a Fab molecule with a scFv appended on the C- terminal of the light or heavy chain.

The term “Fc”, “Fc fragment”, and “Fc region” are used interchangeably to refer to the C- terminal region of an antibody comprising the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus, Fc refers to the last two constant domains, CH2 and CH3, of IgA, IgD, and IgG, or the last three constant domains of IgE and IgM, and the flexible hinge N-terminal to these domains. The human lgG1 heavy chain Fc region is defined herein to comprise residues C226 to its carboxyl-terminus, wherein the numbering is according to the Ell index. In the context of human lgG1 , the lower hinge refers to positions 226-230, the CH2 domain refers to positions 231-340 and the CH3 domain refers to positions 341-447 according to the Ell index. The corresponding Fc region of other immunoglobulins can be identified by sequence alignments.

The term "Framework" or "FR" refers to variable domain residues other than hypervariable region residues. The FR of a variable domain generally consists of four FR domains: FR1 , FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1 (L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The term “Fv” refers to two variable domains of full length antibodies, for example co-operative variable domains, such as a cognate pair or affinity matured variable domains, i.e. a VH and VL pair.

The term “highly similar” as employed in the context of amino-acid sequences is intended to refer to an amino acid sequence which over its full length is 95% similar or more, such as 96, 97, 98 or 99% similar.

The term "human antibody" refers to an antibody which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibodyencoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

The term "human consensus framework" refers to a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In some embodiments, for the VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In some embodiments, for the VH, the subgroup is subgroup I, III or IV as in Kabat et al.

The term “humanized” antibody refers to an antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. Typically the heavy and/or light chain contains one or more CDRs (including, if desired, one or more modified CDRs) from a donor antibody (e.g. a non-human antibody such as a murine or rabbit monoclonal antibody) and is grafted into a heavy and/or light chain variable region framework of an acceptor antibody (a human antibody)( see e.g. Vaughan et al, Nature Biotechnology, 16, 535-539, 1998). The advantage of such humanized antibodies is to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Rather than the entire CDR being transferred, only one or more of the specificity determining residues from any one of the CDRs described herein above can be transferred to the human antibody framework (see e.g., Kashmiri et al., 2005, Methods, 36, 25-34). A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

The term "hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence ("complementarity determining regions" or "CDRs") and/or form structurally defined loops ("hypervariable loops") and/or contain the antigen-contacting residues ("antigen contacts").

The term ”IC50” as used herein refers to the half maximal inhibitory concentration which is a measure of the effectiveness of a substance, such as an antibody, in inhibiting a specific biological or biochemical function. The IC50 is a quantitative measure which indicates how much of a particular substance is needed to inhibit a given biological process by 50%.

The term “inhibiting” (or “inhibit”) or “neutralizing” (or “neutralize”) in the context of antibodies and antigen-binding domains describes an antibody (or an antigen-binding domain) that is capable of inhibiting or attenuating the biological signaling activity of its target (target protein).

The term “isolated” means, throughout this specification, that the antibody, or polynucleotide, as the case may be, exists in a physical milieu distinct from that in which it may occur in nature. The term “isolated” nucleic acid refers to a nucleic acid molecule that has been isolated from its natural environment or that has been synthetically created. An isolated nucleic acid may comprise synthetic DNA, for instance produced by chemical processing, cDNA, genomic DNA or any combination thereof.

The term “Kabat residue designations” or “Kabat” refer to the residue numbering scheme commonly used for antibodies. Such do not always correspond directly with the linear numbering of the amino acid residues. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure. The correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a “standard” Kabat numbered sequence. For details see Kabat eta/., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Unless indicated otherwise, Kabat numbering is used throughout the specification. The term “KD” as used herein refers to the constant of dissociation which is obtained from the ratio of Kd to Ka (i.e. Kd/Ka) and is expressed as a molar concentration (M). Kd and Ka refers to the dissociation rate and association rate, respectively, of a particular antigen-antibody interaction. KD values for antibodies can be determined using methods well established in the art.

The term "monoclonal antibody" (or “mAb”) refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. each individual of a monoclonal antibody preparation is identical except for possible mutations (e.g., naturally occurring mutations), that may be present in minor amounts. Certain differences in the protein sequences linked to post- translational modifications (for example, cleavage of the heavy chain C-terminal lysine, deamidation of asparagine residues and/or isomerisation of aspartate residues) may nevertheless exist between the various different antibody molecules present in the composition. Contrary to polyclonal antibody preparations, each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.

The term “multispecific” or “multispecific antibody” as employed herein refers to an antibody as described herein which has at least two binding domains, i.e. two or more binding domains, for example two or three binding domains, wherein the at least two binding domains independently bind two different antigens or two different epitopes on the same antigen. Multispecific antibodies are generally monovalent for each specificity (antigen). Multispecific antibodies described herein encompass monovalent and multivalent, e.g. bivalent, trivalent, tetravalent multispecific antibodies.

The term “paratope” refers to a region of an antibody which recognizes and binds to an antigen.

The term “polyclonal antibody” refers to a mixture of different antibody molecules which bind to (or otherwise interact with) more than one epitope of an antigen.

The term “scDiabody” refers to a diabody comprising an intra-Fv linker, such that the molecule comprises three linkers and forms a normal scFv whose VH and VL terminals are each linked to one of the variable regions of a further Fv pair.

The term “Scdiabody-CH3” as employed herein refers to two scdiabody molecules each linked, for example via a hinge to a CH3 domain.

The term “ScDiabody-Fc” as employed herein is two scdiabodies, wherein each one is appended to the N-terminus of a CH2 domain, for example via a hinge, of constant region fragment -CH2CH3. The term “single chain variable fragment” or “scFv” as employed herein refers to a single chain variable fragment which is stabilised by a peptide linker between the VH and VL variable domains.

The term “scFv-IgG” as employed herein is a full-length antibody with a scFv on the N-terminus of each of the heavy chains or each of the light chains.

The term "single domain antibody" as used herein refers to an antibody fragment consisting of a single monomeric variable domain. Examples of single domain antibodies include VH or VL or VHH or V-NAR.

The terms "subject" or “individual” in the context of the treatments and diagnosis generally refer to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). More specifically, the individual or subject is a human.

The term “Tandem scFv” as employed herein refers to at least two scFvs linked via a single linker such that there is a single inter-Fv linker.

The term “Tandem scFv-Fc” as employed herein refers to at least two tandem scFvs, wherein each one is appended to the N-terminus of a CH2 domain, for example via a hinge, of constant region fragment -CH2CH3.

The term “target” or “antibody target” as used herein refers to target antigen to which the antibody binds .

The term “therapeutically effective amount” refers to the amount of an antibody thereof that, when administered to a subject for treating a disease, is sufficient to produce such treatment for the disease. The therapeutically effective amount will vary depending on the antibody, the disease and its severity and the age, weight, etc., of the subject to be treated.

The term “trispecific or trispecific antibody” as employed herein refers to an antibody with three antigen-binding specificities. For example, the antibody is an antibody with three antigenbinding domains (trivalent), which independently bind three different antigens or three different epitopes on the same antigen, i.e. each binding domain is monovalent for each antigen. One of the examples of a trispecific antibody format is TrYbe.

The terms "prevent", or "preventing" and the like, refer to obtaining a prophylactic effect in terms of completely or partially preventing a disease or symptom thereof. Preventing thus encompasses stopping the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having the disease. The terms “treatment”, “treating” and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. Treatment thus encompasses (a) inhibiting the disease, i.e. , arresting its development; and (b) relieving the disease, i.e. , causing regression of the disease.

The term “TrYbe” as employed herein refers to a Fab fragment with a first dsscFv appended to the C-terminal of the light chain and a second dsscFv appended to the C-terminal of the heavy chain.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain (VH) and light chain (VL) of a full length antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. The CDRs and the FR together form a variable region. By convention, the CDRs in the heavy chain variable region of an antibody are referred as CDR-H1 , CDR-H2 and CDR-H3 and in the light chain variable regions as CDR-L1 , CDR-L2 and CDR-L3. They are numbered sequentially in the direction from the N-terminus to the C-terminus of each chain. CDRs are conventionally numbered according to a system devised by Kabat.

The term “VH” refers to the variable domain (or the sequence) of the heavy chain.

The term “VL” refers to the variable domain (or the sequence) of the light chain.

Antibodies and antigen-binding domains that inhibit IL-11 mediated signaling

The present technology aims at providing a novel type of drug to treat hidradenitis suppurativa.

In the present disclosure, the inventors establish that IL-11 plays a role in HS (hidradenitis suppurativa) biology. IL-11 was found to be upregulated in HS lesions, to impact hair follicle biology, and to contribute to chronic inflammation, the three key pathological processes in HS. Blockade of the IL-11 pathway thus has the potential to result in clinical responses.

Subsequently, antibodies were developed that are capable of inhibiting IL-11 mediated signaling. Such antibodies can also be used for the treatment of alternative diseases.

In a first general aspect, the present technology therefore provides an agent for use in the treatment of HS. In a further particular embodiment the agent is an antibody capable of inhibiting IL-11 mediated signaling.

In a further particular embodiment the agent is an antibody that specifically binds to IL-11.

In a second general aspect, the present technology provides an antibody that specifically binds to IL-11 and has functional and structural properties as further described herein.

Interleukin 11 (IL-11)

The term “IL-11 signaling” or “IL-11 mediated signaling” refers to signaling mediated by binding of IL-11 to the IL-11 R. In one embodiment, the IL-11 R is provided in a soluble form. In another embodiment, the IL-11 R is membrane-bound.

In all aspects of the current technology, antibodies are being used that are capable of inhibiting IL-11 mediated signaling.

In one embodiment, inhibition of IL-11 mediated signaling can be obtained through binding of the antibody to IL-11 R.

Alternatively, inhibition of IL-11 mediated signaling is obtained through binding of the antibody to IL-11.

The antibody may be capable of binding to IL-11. In a preferred embodiment, the antibody comprises an antigen-binding domain that specifically binds to IL-11.

More specifically, such an antigen-binding domain specifically binds to human, cynomolgus and/or mouse IL-11.

In one embodiment, IL-11 is human IL-11. In one embodiment, IL-11 is cynomolgus IL-11. In one embodiment, IL-11 is mouse IL-11.

In one embodiment, human IL-11 has the amino acid sequence of SEQ ID NO: 100. In one embodiment, cynomolgus IL-11 has the amino acid sequence of SEQ ID NO: 101.

In one embodiment, the antigen-binding domain specifically binds to human IL-11 with a KD of less than 100, 50, or 20 pM. In one embodiment, the antigen-binding domain specifically binds to human IL-11 with a KD of <100pM. In one embodiment, the antigen-binding domain specifically binds to human IL-11 with a KD of <50pM. In one embodiment, the antigen-binding domain specifically binds to human IL-11 with a KD of <20pM.

In one embodiment, the antigen-binding domain specifically binds to cynomolgus IL-11 with a KD of less than 200, 100, or 40 pM. In one embodiment, the antigen-binding domain specifically binds to cynomolgus IL-11 with a KD of <200pM. In one embodiment, the antigen-binding domain specifically binds to cynomolgus IL-11 with a KD of <100pM. In one embodiment, the antigen-binding domain specifically binds to cynomolgus IL-11 with a KD of <40pM.

In one embodiment, the antigen-binding domain specifically binds to mouse IL-11 with a KD of less than 250, or 100 pM. In one embodiment, the antigen-binding domain specifically binds to mouse IL-11 with a KD of <250pM. In one embodiment, the antigen-binding domain specifically binds to mouse IL- 11 with a KD of <100pM.

The binding properties described here in relation to antigen-binding domains also apply to antibodies that contain those domains.

The present technology provides a novel family of binding proteins, CDR grafted antibodies, humanised antibodies and fragments thereof, capable of inhibiting IL-11 mediated signaling.

Hidradenitis suppurativa

HS is a dermal disease driven by three key pathological processes. Early HS is associated with follicular occlusion caused by hyperkeratinisation of the upper hair follicle. Formation of nodules and abscesses involves inflammation and swelling of the follicular units, a process that is driven by accumulation of keratin debris, bacterial dysbiosis and that leads to cyst formation. Rupture of the cyst drives tissue inflammation, immune cell recruitment and contributes to the formation of early dermal tunnels. Later-stage HS lesions are associated with progressive extension of dermal tunnels: epithelial-lined, duct-like structures that fuse with the skin surface to form ostia and discharge pus to the skin surface. Tunnel-associated chronic inflammation leads to significant epidermal and dermal remodeling, which includes extracellular matrix deposition. In ‘end-stage’ disease, considerable scarring may be present, as well as multiple interconnected tunnels, which restrict motility of affected skin regions.

The inventors set out to firstly perform comparative transcriptomic analysis of HS lesional skin samples against non-lesional skin of the same subject to better understand the biological pathways driving the lesional phenotype. During the analysis, groups (also referred to as modules) of lesion-specific genes were identified that participate in similar biological function and are dysregulated in HS lesional tissue. Out of these modules, three were found to be enriched for genes that are regulated by IL-11 pathway genes.

Further transcriptomic analysis of HS lesional skin samples taken before and after treatment with Bimekizumab (an inhibitor of both IL-17A and IL-17F) identified that these three IL-11- regulated gene modules were only partially normalized after Bimekizumab treatment. Collectively, these data suggest that IL-11 plays a role in HS pathobiology, and that blockade of the IL-11 signaling pathway thus has the potential to result in clinical responses.

Further experiments were performed to strengthen this hypothesis. First, the expression levels of IL-11 and IL-11 RA were examined in early HS lesional tissue of different disease stage using single cell transcriptomics, and compared to non-lesional skin taken in close proximity to the lesion.

Focused cell type analysis of the HS lesional tissue identified an expanded, lesion-specific fibroblast population displaying more than 3-fold increase in IL-11RA expression in comparison to non-lesion specific fibroblasts. In addition, a number of genes that are activated by IL-11 stimulation of fibroblasts in vitro were found to be upregulated in the lesion-specific fibroblasts, suggesting that these cells are responding to IL- 11 in vivo.

Increased IL-11 and IL-11 R expression in HS lesions compared to healthy skin (non-HS subjects) was confirmed by semi-quantitative imaging analysis. The results indicated that the IL-11 and IL-11 RA levels increase as the HS pathology progresses from mild to moderate-to- severe.

It was thus found that IL-11 and IL-11 R expression is significantly higher in HS than in healthy skin and that the level of expression correlates with disease severity.

Next, dermal fibroblasts and hair follicle dermal papilla cells, which express the highest level of IL-11 RA in skin, were stimulated with IL-11 and the functional gene expression response was analysed. Different types of genesets were found to be upregulated in these different cell types. While in dermal fibroblasts an enrichment of pro-inflammatory genesets could be observed, the enriched genesets in dermal papilla cells regulated cell cycle and proliferation.

IL-11 by driving the unique functional response in hair follicle cells could thus contribute to HS pathology by affecting normal hair follicle biology.

Subsequently, the inventors demonstrated that IL-11 alone was capable of stimulating dermal fibroblast to induce HS relevant inflammatory mediators, such as CXCL1 and IL-8. Moreover, it was shown that IL-11 could synergize with other HS relevant proinflammatory cytokines, such as IL-17A, IL-17F, TNF-a and I L-1 to amplify the proinflammatory immune response.

In an ex vivo human hair follicle organ culture model, only the combination addition of IL-11 on top of IL-17AA and IL-17FF stimulation resulted in an increased secretion of MMP-1 , MMP- 2, MMP-3, MMP-7, MMP-9 and MMP-10, whose increase was not significantly induced when treated with IL-17AA and IL-17FF alone.

In the same model, the combination of IL-11 and IL-17AA and IL-17FF stimulation resulted in decreased expression of genes associated with modulation of hair follicle cycling, which was not seen with IL-17AA and IL-17FF alone. A change in expression of these particular genes has been reported to lead to loss of cell polarity and reduced proliferation of affected cells. To study the consequence of IL-11 inhibition in HS-relevant preclinical models, the inventors explored the effect of an anti-IL-11 Fab in an ex vivo human full thickness skin explant model. Upon treatment of the HS-like skin with anti-IL-11 Fab, the transcriptomic changes observed highlighted a clear normalization of gene pathways linked to inflammation and epithelial mesenchymal transition (ETM).

Overall the obtained data demonstrate for the first time that IL-11 is upregulated in HS lesions, impacts hair follicle biology, contributes to chronic inflammation and has a role in driving the dermal and epidermal tissue remodeling which characterizes the more severe disease stages.

Antibodies

In all aspects of the present technology, the antibody might be a full-length antibody or a fragment of a full-length antibody.

The antibodies may be (or derived from) polyclonal, monoclonal, fully human, humanized or chimeric.

The antibodies described further serve as a reference and example only and do not limit the scope of the embodiments of the present technology.

An antibody used according to the present technology may be a chimeric antibody, a CDR- grafted antibody, a single domain antibody, a nanobody, a human or humanized antibody. For the production of both monoclonal and polyclonal antibodies, the animal used to raise such antibodies is typically a non-human mammal such as a goat, rabbit, rat or mouse but the antibody may also be raised in other species.

Polyclonal antibodies may be produced by routine methods such as immunization of a suitable animal with an antigen of interest. Blood may be subsequently removed from such animal and the produced antibodies purified.

Monoclonal antibodies may be made by a variety of techniques, including but not limited to, the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or a part of the human immunoglobulin loci. Some exemplary methods for making monoclonal antibodies are described herein.

For example, monoclonal antibodies may be prepared using the hybridoma technique (Kohler & Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today, 4:72) and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan R Liss, Inc., 1985).

Monoclonal antibodies may also be generated using single lymphocyte antibody methods by cloning and expressing immunoglobulin variable region cDNAs generated from single lymphocytes selected for the production of specific antibodies by for example the methods described in WO9202551 , W02004051268 and W02004106377.

Antibodies generated against the target polypeptide may be obtained, where immunization of an animal is necessary, by administering the polypeptide to an animal, preferably a non-human animal, using well-known and routine protocols, see for example Handbook of Experimental Immunology, D. M. Weir (ed.), Vol 4, Blackwell Scientific Publishers, Oxford, England, 1986). Many animals, such as rabbits, mice, rats, sheep, cows, camels or pigs may be immunized. However, mice, rabbits, pigs and rats are generally used.

Monoclonal antibodies can also be generated using various phage display methods known in the art and include those disclosed by Brinkman et al. (in J. Immunol. Methods, 1995, 182: 41- 50), Ames et al. (J. Immunol. Methods, 1995, 184:177-186), Kettleborough et al. (Eur. J. Immunol. 1994, 24:952-958), Persic et al. (Gene, 1997 187 9-18), Burton et al. (Advances in Immunology, 1994, 57:191-280). In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol, 12: 433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths etal., EMBO J 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol, 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example: US 5,750,373, and US 2005/0079574, US 2005/0119455, US 2005/0266000, US 2007/0117126, US 2007/0160598, US 2007/0237764, US 2007/0292936, and US 2009/0002360.

Screening for antibodies can be performed using assays to measure binding to the target polypeptide and/or assays to measure the ability of the antibody to block a particular interaction. An example of a binding assay is an ELISA, for example, using a fusion protein of the target polypeptide, which is immobilized on plates, and employing a conjugated secondary antibody to detect the antibody bound to the target. An example of a blocking assay is a flow cytometry based assay measuring the blocking of a ligand protein binding to the target polypeptide. A fluorescently labelled secondary antibody is used to detect the amount of such ligand protein binding to the target polypeptide. Antibodies may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics.

Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments.

The antibody may be a full length antibody. More particularly the antibody may be of the IgG isotype. More particularly the antibody may be an IgG 1 or lgG4.

The constant region domains of the antibody, if present, may be selected having regard to the proposed function of the antibody molecule, and in particular the effector functions which may be required. For example, the constant region domains may be human IgA, IgD, IgE, IgG or IgM domains. In particular, human IgG constant region domains may be used, especially of the lgG1 and lgG3 isotypes when the antibody molecule is intended for therapeutic uses and antibody effector functions are required. Alternatively, lgG2 and lgG4 isotypes may be used when the antibody molecule is intended for therapeutic purposes and antibody effector functions are not required. It will be appreciated that sequence variants of these constant region domains may also be used. It will also be known to the person skilled in the art that antibodies may undergo a variety of posttranslational modifications. The type and extent of these modifications often depends on the host cell line used to express the antibody as well as the cell culture conditions. Such modifications may include variations in glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization and asparagine deamidation. A frequent modification is the loss of a carboxy-terminal basic residue (such as lysine or arginine) due to the action of carboxypeptidases (as described in Harris, RJ. Journal of Chromatography 705:129-134, 1995). Accordingly, the C-terminal lysine of the antibody heavy chain may be absent.

Alternatively, the antibody is an antigen-binding fragment.

For a review of certain antigen-binding fragments, see Hudson et al. Nat. Med. 9: 129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer- Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and US 5,571 ,894 and US 5,587,458. Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life are disclosed in US 5,869,046.

Antigen-binding fragments and methods of producing them are well known in the art, see for example Verma et al., 1998, Journal of Immunological Methods, 216, 165-181 ; Adair and Lawson, 2005. Therapeutic antibodies. Drug Design Reviews — Online 2(3):209-217. The Fab- Fv format was first disclosed in W02009/040562 and the disulfide stabilized version thereof, the Fab-dsFv, was first disclosed in WO2010/035012, and TrYbe format is disclosed in WO2015/197772.

Various techniques have been developed for the production of antibody fragments. Such fragments might be derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) and Brennan et al, Science 229:81 (1985)). However, antibody fragments can also be produced directly by recombinant host cells. For example, antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio/Technology 10: 163-167 (1992)).

F(ab')2 fragments can be isolated directly from recombinant host cell culture. The antibody may be a single chain Fv fragment (scFv). Such are described in WO 93/16185; US 5,571 ,894; and US 5,587,458. The antibody fragment may also be a "linear antibody," e.g., as described in US 5,641 ,870. Such linear antibody fragments may be monospecific or bispecific.

The antibody may be a Fab, Fab’, F(ab’)₂, Fv, dsFv, scFv.or dsscFv. The antibody may be a single domain antibody or a nanobody, for example VH or VL or VHH or VNAR. The antibody may be Fab or Fab’ fragment described in WO2011/117648, W02005/003169, W02005/003170 and W02005/003171.

The antibody may be a disulfide - stabilized single chain variable fragment (dsscFv).

The disulfide bond between the variable domains VH and VL may be between two of the residues listed below:

• VH37 + VL95 see for example Protein Science 6, 781-788 Zhu et al (1997);

• VH44 + VL100 see for example Weatherill et al., Protein Engineering, Design & Selection, 25 (321-329), 2012;

• VH44 + VL105 see for example J Biochem. 118, 825-831 Luo et al (1995);

• V_H45 + VL87 see for example Protein Science 6, 781-788 Zhu et al (1997);

• VH55 + Vi_101 see for example FEBS Letters 377 135-139 Young et al (1995);

• VH100 + VL50 see for example Biochemistry 29 1362-1367 Glockshuber et al (1990);

• VnilOOb + VL49; see for example Biochemistry 29 1362-1367 Glockshuber et al (1990);

• VH98 + VL 46 see for example Protein Science 6, 781-788 Zhu et al (1997);

• VH101 + VL46; see for example Protein Science 6, 781-788 Zhu et al (1997);

• V_H105 + VL43 see for example; Proc. Natl. Acad. Sci. USA Vol. 90 pp.7538-7542 Brinkmann et al (1993); or Proteins 19, 35-47 Jung et al (1994), • V_H106 + VL57 see for example FEBS Letters 377 135-139 Young et al (1995) and a position or positions corresponding thereto in a variable region pair located in the molecule.

The disulfide bond may be formed between positions VH44 and VL100.

It will be appreciated by the skilled person that antigen-binding fragments described herein may also be characterized as monoclonal, chimeric, humanized, fully human, multispecific, bispecific etc., and that discussion of these terms also relate to such fragments.

Multispecific antibodies

The antibody of the present technology may be a multispecific antibody.

Examples of multispecific antibodies, which also are contemplated for use in the context of the disclosure, include bi, tri or tetra-valent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies, bibodies and tribodies (see for example Holliger and Hudson, 2005, Nature Biotech 23(9): 1126-1136; Schoonjans etal. 2001 , Biomolecular Engineering, 17(6), 193-202).

A variety of different multispecific antibody formats are known in the art. Different classifications have been proposed, but multispecific IgG antibody formats generally include bispecific IgG, appended IgG, multispecific (e.g. bispecific) antibody fragments, multispecific (e.g. bispecific) fusion proteins, and multispecific (e.g. bispecific) antibody conjugates, as described for example in Spiess et al., Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol Immunol. 67(2015):95-106.

In one embodiment the multispecific antibody is a bispecific antibody. In one embodiment, the antibody comprises two antigen-binding domains wherein one binding domain specifically binds to IL-11 and the other binding domain specifically to another antigen, i.e. each binding domain is monovalent for each antigen.

In one embodiment the multispecific antibody is a trispecific antibody. In one embodiment, the antibody comprises three antigen-binding domains wherein one antigen-binding domain specifically binds to IL-11 , one antigen-binding domain specifically binds to another antigen, and the other antigen-binding domain specifically binds to yet another antigen.

In one embodiment, each binding domain is monovalent. Preferably each binding domain comprises two antibody variable domains. More preferably each binding domain comprises no more than one VH and one VL.

More particularly the binding domain which specifically binds to IL-11 is selected from a Fab, scFv, Fv, dsFv and dsscFv. Appended IgG classically comprise full-length IgG engineered by appending additional antigen-binding fragment to the N- and/or C-terminus of the heavy and/or light chain of the IgG. Examples of such additional antigen-binding fragments include sdAb antibodies (e.g. VH or VL), Fv, scFv, dsscFv, Fab, scFab. Appended IgG antibody formats include in particular DVD-IgG, lgG(H)-scFv, scFv-(H)lgG, lgG(L)-scFv, scFv-(L)lgG, lgG(L,H)-Fv, lgG(H)-V, V(H)- IgG, lgC(L)-V, V(L)-lgG, KIH IgG-scFab, scFv-IgG, lgG-2scFv, scFv4-lg, Zybody and DVI-IgG (four-in-one), for example as described in Spiess et al., Mol Immunol. 67(2015):95-106.

Multispecific antibodies include single domain antibody, nanobody, nanobody-HSA, BiTEs, diabody, DART, TandAb, scDiabody, sc-Diabody-CH3, Diabody-CH3, Triple Body, Miniantibody; Minibody, Tri Bi minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, Tetravalent HCAb, scDiabody-Fc, Diabody-Fc, Tandem scFv-Fc; and intrabody, as described, for example, Spiess et al., Mol Immunol. 67(2015): 95- 106.

Multispecific fusion proteins include Dock and Lock, ImmTAC, HSAbody, scDiabody-HSA, and Tandem scFv-Toxin.

Multispecific antibody conjugates include IgG-IgG; Cov-X-Body; and scFvl -PEG-scFv₂.

Additional multispecific antibody formats have been described for example in Brinkmann and Kontermann, mAbs, 9:2, 182-212 (2017), for example tandem scFv, triplebody, Fab-VHH, taFv-Fc, scFv4-lg, scFv₂-Fcab, scFv4-lgG. Bibodies, tribodies and methods for producing the same are disclosed for example in WO99/37791.

Techniques for making bispecific antibodies include, but are not limited to, CrossMab technology (Klein et al. Engineering therapeutic bispecific antibodies using CrossMab technology, Methods 154 (2019) 21-31), Knobs-into-holes engineering (e.g. W01996027011 , WO1998050431), DuoBody technology (e.g. WO2011131746), Azymetric technology (e.g. WO20 12058768). Further technologies for making bispecific antibodies have been described for example in Godar et al., 2018, Therapeutic bispecific antibody formats: a patent applications review (1994-2017), Expert Opinion on Therapeutic Patents, 28:3, 251-276. Bispecific antibodies include in particular CrossMab antibodies, DAF (two-in-one), DAF (four- in-one), DutaMab, DT-IgG, Knobs-into-holes common LC, Knobs-into-holes assembly, Charge pair, Fab-arm exchange, SEEDbody, Triomab, LLIZ-Y, Fcab, K -body and orthogonal Fab.

A bispecific antibody for use in the present technology is a Knobs-into-holes antibody (“KiH”). Generally, such technology involves introducing a protuberance ("knob") at the interface of a first polypeptide (such as a first CH3 domain in a first antibody heavy chain) and a corresponding cavity ("hole") in the interface of a second polypeptide (such as a second CH3 domain in a second antibody heavy chain), such that the protuberance can be positioned in the cavity so as to assist the formation bispecific antibody. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide (such as a first CH3 domain in a first antibody heavy chain) with larger side chains (e.g. arginine, phenylalanine, tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide (such as a second CH3 domain in a second antibody heavy chain) by replacing large amino acid side chains with smaller ones (e.g. alanine, serine, valine, or threonine). The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis. Further details regarding "Knobs-into-holes" technology are described in, e.g., US5731168; US7695936; W02009/089004; US2009/0182127; Marvin md Z u, Acta Pharmacologica Sincia (2005) 26(6):649-658; Kontermann Acta Pharmacologica Sincia (2005) 26: 1-9; Ridgway et al, Prot Eng 9, 617-621 (1996);and Carter, J Immunol Meth 248, 7-15 (2001).

Another antibody for use in the present technology comprises a Fab linked to two scFvs or dsscFvs, each scFv or dsscFv binding a different target (e.g., one scFv or dsscFv binding a therapeutic target and one scFv or dsscFv that increases half-life by binding, for instance, albumin). Such multispecific antibodies are described in WO2015/197772. Another antibody for use in the present technology comprises a Fab linked to only one scFv or dsscFv, as described for example in WO2013/068571 , and Dave et al, Mabs, 8(7) 1319-1335 (2016).

Humanized, human, and chimeric antibodies and methods of producing such

In certain embodiments, an antibody provided herein is a chimeric antibody. Examples of chimeric antibodies are described, e.g., in US 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)). In one example, a chimeric antibody comprises a nonhuman variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a "class switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In one embodiment, the antibody is a humanized antibody.

Humanized antibodies may optionally further comprise one or more framework residues derived from the non-human species from which the CDRs were derived. It will be appreciated that it may only be necessary to transfer the specificity determining residues of the CDRs rather than the entire CDR (see for example, Kashmiri et al., 2005, Methods, 36, 25-34). Suitably, the humanized antibody according to the present technology has a variable domain comprising human acceptor framework regions as well as one or more of the CDRs and optionally further including one or more donor framework residues.

Thus, provided in one embodiment is a humanized antibody wherein the variable domain comprises human acceptor framework regions and non-human donor CDRs.

When the CDRs or specificity determining residues are grafted, any appropriate acceptor variable region framework sequence may be used having regard to the class/type of the donor antibody from which the CDRs are derived, including mouse, primate and human framework regions.

Examples of human frameworks which can be used in the present technology are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Kabat et al). For example, KOL and NEWM can be used for the heavy chain, REI can be used for the light chain and EU, LAY and POM can be used for both the heavy chain and the light chain. Alternatively, human germline sequences may be used; these are available at: www.imgt.org. In embodiments, the acceptor framework is IGHV3-07 human germline, and/or IGKV1-12 human germline. In embodiments, the human framework contains 1-5, 1-4, 1-3 or 1-2 donor antibody amino acid residues.

In a humanized antibody of the present technology, the acceptor heavy and light chains do not necessarily need to be derived from the same antibody and may, if desired, comprise composite chains having framework regions derived from different chains.

In certain embodiments, an antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art.

Human antibodies comprise heavy or light chain variable regions or full length heavy or light chains that are "the product of" or "derived from" a particular germline sequence if the variable regions or full-length chains of the antibody are obtained from a system that uses human germline immunoglobulin genes. Such systems include immunizing a transgenic mouse carrying human immunoglobulin genes with the antigen of interest or screening a human immunoglobulin gene library displayed on phage with the antigen of interest. A human antibody or fragment thereof that is "the product of' or "derived from" a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequences of human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is closest in sequence (i.e., greatest % identity) to the sequence of the human antibody. A human antibody that is "the product of" or "derived from" a particular human germline immunoglobulin sequence may contain amino acid differences as compared to the germline sequence, due to, for example, naturally occurring somatic mutations or intentional introduction of site-directed mutations. However, a selected

T1 human antibody typically is at least 90% identical in amino acid sequence to an amino acid sequence encoded by a human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as being human when compared to the germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences). In certain cases, a human antibody may be at least 60%, 70%, 80%, 90%, or at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a human antibody derived from a particular human germline sequence will display no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, the human antibody may display no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.

Structural features of the antibody

The antibodies of all aspects of the present technology may comprise an IL-11 binding domain.

An antigen-binding domain will generally comprise 6 CDRs, three from a heavy chain and three from a light chain. In one embodiment, the CDRs are in a framework and together form a variable region. Thus, in one embodiment, the binding domain specific for antigen comprises a light chain variable region and a heavy chain variable region.

The IL-11 binding domain may comprise a heavy chain variable region (VH) and light chain variable region (VL). VH and VL may form a VH/VL pair (VH/VL).

SEQ ID NO’s of sequences related to specific examples of antibody sequences that can be used in the antibodies of any aspect of the present technology are listed in Table 1 .

Table 1. Summary of sequences of IL-11 binding antibodies.

* i.e. with cysteines as to allow for disulfide bond formation formed between positions VH44 and VL100

In one embodiment, the antibody that specifically binds to IL-11 comprises a heavy chain variable region (VH) comprising: a CDR-H1 comprising the amino acid sequence of SEQ ID NO:1 , a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:3.

In one embodiment, the antibody that specifically binds to IL-11 comprises a light chain variable region (VL) comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:6.

In one embodiment, the antibody that specifically binds to IL-11 comprises a VH comprising: a CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:3; and VL comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:6.

In one embodiment, the antibody that specifically binds to IL-11 comprises a VH comprising the amino acid sequence of SEQ ID NO:7.

In one embodiment, the antibody that specifically binds to IL-11 comprises a VL comprising the amino acid sequence of SEQ ID NO:9. In one embodiment, the antibody that specifically binds to IL-11 comprises a VH comprising the amino acid sequence of SEQ ID NO:7, and a VL comprising the amino acid sequence of SEQ ID NO:9.

In one embodiment, the antibody that specifically binds to IL-11 is a Fab, Fv, scFv, or an IgG.

In one embodiment, the antibody is a Fab which comprises a light chain comprising the amino acid sequence of SEQ ID NO:11 , and a heavy chain comprising the amino acid sequence of SEQ ID NO:13.

In another embodiment, the antibody is a scFv which comprises the amino acid sequence of SEQ ID NO:17.

In another embodiment, the antibody is an IgG 1 which comprises a light chain comprising the amino acid sequence of SEQ ID NO:11 , and a heavy chain comprising the amino acid sequence of SEQ ID NO:98.

As further described herein, antibodies can comprise substitutions in the Fc region to reduce effector function. In one embodiment of the present technology, the antibody comprises the L234A and L235A substitutions, wherein the numbering is according to EU as in Kabat.

In one particular embodiment, the antibody is an IgG 1 comprising a light chain comprising the amino acid sequence of SEQ ID NO:11 , and a heavy chain comprising the amino acid sequence of SEQ ID NO:19.

In another embodiment, the antibody is an lgG4P which comprises a light chain comprising the amino acid sequence of SEQ ID NO:11 , and a heavy chain comprising the amino acid sequence of SEQ ID NO:99.

In an alternative embodiment, the antibody that specifically binds to IL-11 comprises a VH comprising the amino acid sequence of SEQ ID NO: 15.

In an alternative embodiment, the antibody that specifically binds to IL-11 comprises a VL comprising the amino acid sequence of SEQ ID NO: 16.

In an alternative embodiment, the antibody that specifically binds to IL-11 comprises a VH comprising the amino acid sequence of SEQ ID NO: 15, and a VL comprising the amino acid sequence of SEQ ID NO:16.

In an alternative embodiment, the antibody that specifically binds to IL-11 is a dsFv, or a dsscFv.

In an alternative embodiment, the antibody is a dsscFv which comprises the amino acid sequence of SEQ ID NO:18. Functional properties of the antibodies

The properties described here in relation to antigen-binding domains also apply to antibodies, that contain those domains.

The IL-11 binding domain is inhibiting one or more of IL-11 activities.

The IL-11 binding domain may: i. bind to IL-11 and prevent binding of IL-11 to IL-11 Ra and as a result also block subsequent interaction with gp130; or ii. bind to IL-11 in such a way that it allows IL- 11 binding to IL-11 Ra but prevents recruitment of gp130 into the complex.

In preferred embodiments, the IL-11 binding domain specifically binds to IL-11 and prevents binding of IL-11 to IL-11 Ra and as a result also blocks subsequent interaction with gp130. In such embodiments, the IL-11 binding domain inhibits IL-11 interaction with IL-11 Ra. Inhibition of IL-11 binding to IL-11 Ra therefore prevents the formation of the IL-11/IL-11 Ra/gp130 receptor complex.

In one embodiment, the IL-11 binding domain specifically binds to IL-11 and prevents binding of IL-11 to soluble IL-11 Ra. In such an embodiment, the IL-11 binding domain may inhibit trans-STAT3 signaling. This property can be measured in cells which express gp130 but lack expression of IL-11 Ra. A representative assay has been exemplified herein.

In one embodiment, the IL-11 binding domain specifically binds to IL-11 and prevents binding of IL-11 to membrane-bound IL-11 Ra. In such an embodiment, the IL-11 binding domain may inhibit cis-STAT3 signaling. This property can be measured in cells which express both gp130 and IL-11 Ra. A representative assay has been exemplified herein.

In one embodiment, the IL-11 binding domain may inhibit CCL-2 release in cells. This property can be measured in a human dermal fibroblast assay. A representative assay has been exemplified herein. The term “CCL-2” refers to C-C motif chemokine ligand 2.

In one embodiment, the IL-11 binding domain may inhibit IL-6 release in cells. This property can be measured in a human dermal fibroblast assay. A representative assay has been exemplified herein. The term “IL-6” refers to interleukin 6.

In one embodiment, the IL-11 binding domain may inhibit MMP2 release in cells. This property can be measured in a reporter cell line, as has been exemplified herein. The term “MM P-2” refers to matrix metalloproteinase-2.

In one embodiment, the IL-11 binding domain has a stronger binding affinity for IL-11 as compared to the affinity of the IL-11 R to gp130. This is characterized by a constant of dissociation (KD) for binding of the IL-11 binding domain to IL-11 which is at least 10-fold higher than for binding of the IL-11 Ra to gp130. Specifically such is measured using BIACore technique.

Antibodies binding to the same epitope

Antibodies may compete for binding to IL- 11 with, or bind to the same epitope as, an antibody defined above in terms of light chain, heavy chain, light chain variable region, heavy chain variable region or CDR sequences.

In particular, the present technology provides an isolated antibody that competes for binding to IL-11 with, or binds to the same epitope as, an antibody which comprises a CDR-H1/CDR- H2/CDR-H3/CDR-L1/CDR-L2/CDR-L3 sequence combination of SEQ ID N Os :172/3/4/5/6.

The isolated antibody may compete for binding to IL-11 with, or bind to the same epitope as, an antibody which comprises a VH/VL sequence combination of SEQ ID NOs:7/9.

The isolated antibody may compete for binding to IL-11 with, or bind to the same epitope as, a Fab which comprises a light chain/heavy chain sequence combination of SEQ ID NOs:11/13.

The term "epitope" or “binding site” in the context of antibodies refer to a site (or a part) on an antigen to which the paratope of an antibody binds or recognizes. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes can be formed both from contiguous amino acids (also often called “linear epitopes”) or noncontiguous amino acids formed by tertiary folding of a protein (often called “conformational epitopes”). Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5-10 amino acids in a unique spatial conformation. Epitopes usually consist of chemically active surface groups of molecules such as amino acids, sugar side chains and usually have specific 3D structural and charge characteristics. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics.

One can easily determine whether an antibody binds to the same epitope as, or competes for binding with, a reference antibody by using routine methods known in the art. For example, to determine if a test antibody binds to the same epitope as a reference antibody of the technology, the reference antibody is allowed to bind to a protein or peptide under saturating conditions. Next, the ability of a test antibody to bind to the protein or peptide is assessed. If the test antibody is able to bind to the protein or peptide following saturation binding with the reference antibody, it can be concluded that the test antibody binds to a different epitope than the reference antibody. On the other hand, if the test antibody is not able to bind to protein or peptide following saturation binding with the reference antibody, then the test antibody may bind to the same epitope as the epitope bound by the reference antibody of the technology.

The term "antibody binding to the same epitope as a reference antibody” refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more.

To determine if an antibody competes for binding with a reference antibody, the abovedescribed binding methodology is performed in two orientations. In a first orientation, the reference antibody is allowed to bind to a protein/peptide under saturating conditions followed by assessment of binding of the test antibody to the protein/peptide molecule. In a second orientation, the test antibody is allowed to bind to the protein/peptide under saturating conditions followed by assessment of binding of the reference antibody to the protein/peptide. If, in both orientations, only the first (saturating) antibody is capable of binding to the protein/peptide, then it is concluded that the test antibody and the reference antibody compete for binding to the protein/peptide. As will be appreciated by the skilled person, an antibody that competes for binding with a reference antibody may not necessarily bind to the identical epitope as the reference antibody, but may sterically block binding of the reference antibody by binding an overlapping or adjacent epitope.

Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a 1 -, 5-, 10-, 20- or 100-fold excess of one antibody inhibits binding of the other by at least 50%, 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res, 1990:50:1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

Additional routine experimentation (e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art.

Antibody variants

In one embodiment, rather than the specific sequence set out herein, an antibody or antibody binding domain provided by the present technology may have a specific level of sequence identity or number of amino acid sequence changes compared to that specific sequence, so long as the antibody or antibody binding domain is still able to specifically bind to IL-11. Such an antibody is referred to herein as an “antibody variant”. In another embodiment, a nucleic acid sequence may have a particular level of sequence identity compared to one of the specific sequences set out herein, provided that it still encodes an antibody or binding domain, or a constituent of those, which can still specifically bind to IL-11.

Degrees of identity and similarity can be readily calculated (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1 , Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987, Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 , the BLAST™ software available from NCBI (Altschul, S.F. et al., 1990, J. Mol. Biol. 215:403-410; Gish, W. & States, D.J. 1993, Nature Genet. 3:266-272. Madden, T.L. et al., 1996, Meth. Enzymol. 266:131-141 ; Altschul, S.F. et al., 1997, Nucleic Acids Res. 25:3389- 3402; Zhang, J. & Madden, T.L. 1997, Genome Res. 7:649-656,).

The term "percent (%) sequence identity (or similarity)" with respect to the polypeptide and antibody sequences is defined as the percentage of amino acid residues in a candidate sequence that are identical (or similar) to the amino acid residues in the polypeptide being compared, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity .

One or more amino acid substitutions, additions and/or deletions may be made to the CDRs of the IL-11 binding domain provided by the present technology without significantly altering the ability of the antibody to bind to IL- 11 and to inhibit its biological activity.

Consequently, in certain embodiments of the variant VH and VL sequences of the IL-11 binding domain, each CDR either contains no more than one, two or three amino acid substitutions, and wherein the IL-11 binding domain retains its binding properties to IL-11 and blocks IL-11 binding to IL-11 R. Accordingly, in one embodiment, the IL-11 antibody comprises CDRs as defined by the sequences given in SEQ ID NO:1 , 2, 3, 4, 5, and 6 in which one or more amino acids in one or more of the CDRs has been substituted with another amino acid, for example a similar amino acid as defined herein below.

In one embodiment, the CDRs of the IL-11 antibody comprise sequences which have at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequences given in SEQ ID NOs: 1 , 2, 3, 4, 5, and 6.

In embodiments, the IL-11 antibody comprises a CDR-H2 wherein 1 , or 2 amino acids in the CDR-H2 of SEQ ID NO:2 have been substituted with another amino acid, wherein at position 6 the G has changed into S, or A, at position 7 the S has changed into G, at position 9 the T has changed into S, and/or at position 17 the S has changed into R.

The term “position” with respect to a CDR sequence indicates which amino acid residue of the CDR is being substituted when starting from the left of the amino acid sequence of that respective CDR sequence. As an example, the CDR-H2 of SEQ ID NO:2 is TIVYDGSDTYYRDSVKS and has at position 1 the amino acid T, at position 2, the amino acid I, at position 3 the amino acid V, at position 4 the amino acid Y, at position 5 the amino acid D, at position 6 the amino acid G, and so forth. When the amino acid residue at the position 6 has changed into S or A, it means that G at position 6 will be substituted into S or A. Said CDR would then be one of the two following sequences: TIVYDSSDTYYRDSVKS (SEQ ID NO: 22) or TIVYDASDTYYRDSVKS (SEQ ID NO: 23).

In embodiments of the IL-11 antibody, one or more amino acid substitutions in one or more CDRs modifies a potential Aspartic acid isomerization site. In one such an embodiment, the IL-11 antibody comprises a CDR-H2 wherein 1 , or 2 amino acids in the CDR-H2 of SEQ ID NO:2 have been substituted with another amino acid, wherein the G at position 6 has changed to S, or A.

In one embodiment, the IL-11 antibody comprises a CDR-H2 chosen from the group consisting of SEQ ID NO:21 , 22, 23, 56 or 57.

In embodiments, the IL-11 antibody comprises a CDR-H3 wherein 1 amino acid in the CDR- H3 of SEQ ID NO:3 has been substituted with another amino acid, wherein at position 5 the T has changed into A.

In one embodiment, the IL-11 antibody comprises a CDR-H3 of SEQ ID NO:58. In embodiments, the IL-11 antibody comprises a CDR-L1 wherein 1 amino acid in the CDR- L1 of SEQ ID NO:4 has been substituted with another amino acid, wherein at position 1 the K has changed into R, and/or at position 9 the Y has changed into H.

In one embodiment, the IL-11 antibody comprises a CDR-L1 chosen from the group consisting of SEQ ID NO:42, or 43.

In embodiments, the IL-11 antibody comprises a CDR-L2 wherein 1 , or 2 amino acids in the CDR-L2 of SEQ ID NO:5 have been substituted with another amino acid, wherein at position 5 the L has changed into R, and/or at position 6 the Y has changed into N or D.

In one embodiment, the IL-11 antibody comprises a CDR-L2 chosen from the group consisting of SEQ ID NO:44, 45, or 46.

In one embodiment, the IL-11 antibody comprises a heavy chain variable region (VH) comprising: a CDR-H1 comprising the amino acid sequence of SEQ ID NO:1 , a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, 21 , 22, 23, 56 or 57, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:3, or 58; and a light chain variable region (VL) comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, 42, or 43, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, 44, 45, or 46, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:6.

In one embodiment, the IL-11 antibody comprises a heavy chain variable domain which comprises a sequence which has at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence given in SEQ ID NO:7 and a light chain variable domain which comprises a sequence which has at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequence given in SEQ ID NO:9.

In some embodiments, the IL-11 antibody is a Fab comprising a light chain comprising a sequence which has at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence given in SEQ ID NO: 11 and/or a heavy chain comprising a sequence which has at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence given in SEQ ID NO: 13. In some embodiments, the IL-11 antibody is a scFv comprising a sequence which has at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence given in SEQ ID NO: 17 or a dsscFv comprising a sequence which has at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence given in SEQ ID NO: 18.

In some embodiments, the IL-11 antibody is an lgG1 comprising a light chain comprising a sequence which has at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence given in SEQ ID NO:11 and/or a heavy chain comprising a sequence which has at least 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence given in SEQ ID NO:98.

In some embodiments, the IL-11 antibody is an lgG1 comprising a light chain comprising a sequence which has at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence given in SEQ ID NO:11 and/or a heavy chain comprising a sequence which has at least 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence given in SEQ ID NO: 19.

In some embodiments, the IL-11 antibody is an lgG4P comprising a light chain comprising a sequence which has at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence given in SEQ ID NO:11 and/or a heavy chain comprising a sequence which has at least 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence given in SEQ ID NO:99.

In some embodiments, the IL-11 antibody comprises CDR-H1/CDR-H2/CDR-H3/CDR- L1/CDR-L2/CDR-L3 sequences comprising SEQ ID NOs:1 , 2, 3, 4, 5, and 6 respectively, and the remainder of the heavy chain and light chain variable regions have at least 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to SEQ ID NO:7 and 9 respectively.

Constant region variants

In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human lgG1 , lgG2, lgG3 or lgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.

Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001). Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn) are described in US2005/0014934A1. Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn.

In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (Ell numbering of residues).

Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 234, 235, 237, 238, 265, 269, 270, 297, 327 and 329 (see, e.g., U.S. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327 wherein the amino acid residue is numbered according to the EU numbering system. In a preferred embodiment of the present technology, the antibody comprises the L234A and L235A substitutions.

In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in US5,500,362; US5,821 ,337. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat I Acad. Sci. USA 95:652-656 (1998). Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al, J. Immunol. Methods 202: 163 (1996); Cragg, M.S. et al, Blood 101 : 1045-1052 (2003); and Cragg, M.S. and M.l Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al, Int I. Immunol. 18(12): 1759- 1769 (2006)).

In one embodiment, the IL-11 antibody is an lgG1 comprising a heavy chain which comprises an amino acid sequence with a sequence identity of more than 90%, or more than 95%, more than 96%, more than 97%, more than 98% or more than 99% with SEQ ID NO:98. In one embodiment, the IL-11 antibody is an lgG1 comprising a heavy chain which comprises an amino acid sequence with a sequence identity of more than 90%, or more than 95%, more than 96%, more than 97%, more than 98% or more than 99% with SEQ ID NO: 19.

In one embodiment, the IL-11 antibody is an lgG4P comprising a heavy chain which comprises an amino acid sequence with a sequence identity of more than 90%, or more than 95%, more than 96%, more than 97%, more than 98% or more than 99% with SEQ ID NO:99.

Effector molecules

If desired an antibody may be conjugated to one or more effector molecule(s). In one embodiment the antibody is attached to an effector molecule.

It will be appreciated that the effector molecule may comprise a single effector molecule or two or more such molecules so linked as to form a single moiety that can be attached to the antibodies of the present technology. Where it is desired to obtain an antibody linked to an effector molecule, this may be prepared by standard chemical or recombinant DNA procedures in which the antibody fragment is linked either directly or via a coupling agent to the effector molecule. Techniques for conjugating such effector molecules to antibodies are well known in the art (see, Hellstrom et al., Controlled Drug Delivery, 2nd Ed., Robinson et al., eds., 1987, pp. 623-53; Thorpe et al., 1982, Immunol. Rev., 62:119-58 and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67-123). Particular chemical procedures include, for example, those described in WO 93/06231 , WO 92/22583, WO 89/00195, WO 89/01476 and WO 03/031581. Alternatively, where the effector molecule is a protein or polypeptide the linkage may be achieved using recombinant DNA procedures, for example as described in WO 86/01533 and EP0392745.

The term "effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down regulation of cell surface receptors (e.g. B cell receptor), and B cell activation.

The term “effector molecule” as used herein includes, for example, antineoplastic agents, drugs, toxins, biologically active proteins, for example enzymes, other antibody or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof e.g. DNA, RNA and fragments thereof, radionuclides, particularly radioiodide, radioisotopes, chelated metals, nanoparticles and reporter groups such as fluorescent compounds or compounds which may be detected by NMR or ESR spectroscopy. Examples of effector molecules may include cytotoxins or cytotoxic agents including any agent that is detrimental to (e.g. kills) cells. Examples include combrestatins, dolastatins, epothilones, staurosporin, maytansinoids, spongistatins, rhizoxin, halichondrins, roridins, hemiasterlins, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.

Effector molecules also include, but are not limited to, antimetabolites (e.g. methotrexate, 6- mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g. mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNll) and lomustine (CCNll), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g. daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g. dactinomycin (formerly actinomycin), bleomycin, mithramycin, anthramycin (AMC), calicheamicins or duocarmycins), and antimitotic agents (e.g. vincristine and vinblastine).

Other effector molecules may include chelated radionuclides such as 1111n and 90Y, Lu177, Bismuth213, Californium252, Iridium192 and Tungsten188/Rhenium188; or drugs such as but not limited to, alkylphosphocholines, topoisomerase I inhibitors, taxoids and suramin.

Other effector molecules include proteins, peptides and enzymes. Enzymes of interest include, but are not limited to, proteolytic enzymes, hydrolases, lyases, isomerases, transferases. Proteins, polypeptides and peptides of interest include, but are not limited to, immunoglobulins, toxins such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin, a protein such as insulin, tumour necrosis factor, a-interferon, p-interferon, nerve growth factor, platelet derived growth factor or tissue plasminogen activator, a thrombotic agent or an anti- angiogenic agent, e.g. angiostatin or endostatin, or, a biological response modifier such as a lymphokine, interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), nerve growth factor (NGF) or other growth factor and immunoglobulins.

Other effector molecules may include detectable substances useful for example in diagnosis. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive nuclides, positron emitting metals (for use in positron emission tomography), and nonradioactive paramagnetic metal ions. See generally US4,741 ,900 for metal ions which can be conjugated to antibodies for use as diagnostics. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta galactosidase, or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin and biotin; suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin, and aequorin; and suitable radioactive nuclides include 1251, 1311, 1111n and 99Tc.

In another example the effector molecule may increase the half-life of the antibody in vivo, and/or reduce immunogenicity of the antibody and/or enhance the delivery of an antibody across an epithelial barrier to the immune system. Examples of suitable effector molecules of this type include polymers, albumin, albumin binding proteins or albumin binding compounds such as those described in W02005/117984.

Where the effector molecule is a polymer it may, in general, be a synthetic or a naturally occurring polymer, for example an optionally substituted straight or branched chain polyalkylene, polyalkenylene or polyoxyalkylene polymer or a branched or unbranched polysaccharide, e.g. a homo- or hetero- polysaccharide.

Specific optional substituents which may be present on the above-mentioned synthetic polymers include one or more hydroxy, methyl or methoxy groups.

Specific examples of synthetic polymers include optionally substituted straight or branched chain poly(ethyleneglycol), poly(propyleneglycol) poly(vinylalcohol) or derivatives thereof, especially optionally substituted poly(ethyleneglycol) such as methoxypoly(ethyleneglycol) or derivatives thereof.

Specific naturally occurring polymers include lactose, amylose, dextran, glycogen or derivatives thereof.

In one embodiment, the polymer is albumin or a fragment thereof, such as human serum albumin or a fragment thereof.

The size of the polymer may be varied as desired, but will generally be in an average molecular weight range from 500Da to 50000Da, for example from 5000 to 40000Da such as from 20000 to 40000Da. The polymer size may in particular be selected on the basis of the intended use of the product for example ability to localize to certain tissues such as tumors or extend circulating half-life (for review see Chapman, 2002, Advanced Drug Delivery Reviews, 54, 531- 545). Thus, for example, where the product is intended to leave the circulation and penetrate tissue, for example for use in the treatment of a tumor, it may be advantageous to use a small molecular weight polymer, for example with a molecular weight of around 5000Da. For applications where the product remains in the circulation, it may be advantageous to use a higher molecular weight polymer, for example having a molecular weight in the range from 20000Da to 40000Da. Suitable polymers include a polyalkylene polymer, such as a poly(ethyleneglycol) or, especially, a methoxypoly(ethyleneglycol) or a derivative thereof, and especially with a molecular weight in the range from about 15000Da to about 40000Da.

In one example, the antibody are attached to poly(ethyleneglycol) (PEG) moieties. In one particular embodiment, the antigen-binding fragment according to the present technology and the PEG molecules may be attached through any available amino acid side-chain or terminal amino acid functional group located in the antibody fragment, for example any free amino, imino, thiol, hydroxyl or carboxyl group. Such amino acids may occur naturally in the antibody fragment or may be engineered into the fragment using recombinant DNA methods (see for example US 5,219,996; US 5,667,425; WO98/25971 , WG2008/038024). In one example the antibody molecule of the present technology is a modified Fab fragment wherein the modification is the addition to the C-terminal end of its heavy chain one or more amino acids to allow the attachment of an effector molecule. Suitably, the additional amino acids form a modified hinge region containing one or more cysteine residues to which the effector molecule may be attached. Multiple sites can be used to attach two or more PEG molecules.

Suitably PEG molecules are covalently linked through a thiol group of at least one cysteine residue located in the antibody fragment. Each polymer molecule attached to the modified antibody fragment may be covalently linked to the sulfur atom of a cysteine residue located in the fragment. The covalent linkage will generally be a disulfide bond or, in particular, a sulfurcarbon bond. Where a thiol group is used as the point of attachment appropriately activated effector molecules, for example thiol selective derivatives such as maleimides and cysteine derivatives may be used. An activated polymer may be used as the starting material in the preparation of polymer-modified antibody fragments as described above. The activated polymer may be any polymer containing a thiol reactive group such as an a-halocarboxylic acid or ester, e.g. iodoacetamide, an imide, e.g. maleimide, a vinyl sulfone or a disulfide. Such starting materials may be obtained commercially (for example from Nektar, formerly Shearwater Polymers Inc., Huntsville, AL, USA) or may be prepared from commercially available starting materials using conventional chemical procedures. Particular PEG molecules include 20K methoxy-PEG-amine (obtainable from Nektar, formerly Shearwater; Rapp Polymere; and SunBio) and M-PEG-SPA (obtainable from Nektar, formerly Shearwater).

In one embodiment, the antibody comprises a modified Fab fragment, Fab’ fragment or diFab which is PEGylated, i.e. has PEG (poly(ethyleneglycol)) covalently attached thereto, e.g. according to the method disclosed in EP 0948544 or EP1090037 [see also "Poly(ethyleneglycol) Chemistry, Biotechnical and Biomedical Applications", 1992, J. Milton Harris (ed), Plenum Press, New York, "Poly(ethyleneglycol) Chemistry and Biological Applications", 1997, J. Milton Harris and S. Zalipsky (eds), American Chemical Society, Washington DC and "Bioconjugation Protein Coupling Techniques for the Biomedical Sciences", 1998, M. Aslam and A. Dent, Grove Publishers, New York; Chapman, A. 2002, Advanced Drug Delivery Reviews 2002, 54:531-545], In one example PEG is attached to a cysteine in the hinge region. In one example, a PEG modified Fab fragment has a maleimide group covalently linked to a single thiol group in a modified hinge region. A lysine residue may be covalently linked to the maleimide group and to each of the amine groups on the lysine residue may be attached a methoxypoly(ethyleneglycol) polymer having a molecular weight of approximately 20,000Da. The total molecular weight of the PEG attached to the Fab fragment may therefore be approximately 40,000Da.

In one embodiment the antibody is not attached an effector molecule.

Polynucleotides and vectors

The present technology also provides an isolated polynucleotide encoding the antibody or part thereof according to the present technology. The isolated polynucleotide according to the present technology may comprise synthetic DNA, for instance produced by chemical processing, cDNA, genomic DNA or any combination thereof.

Examples of suitable sequences are provided herein in Tables A-1 and A-2.

Standard techniques of molecular biology may be used to prepare DNA sequences coding for the antibody or antigen-binding fragment thereof of the present technology. Desired DNA sequences may be synthesized completely or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used as appropriate.

Preferably, the encoding nucleic acid sequences are operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells. Expression of said polynucleotide comprises transcription of the polynucleotide into a translatable mRNA. Regulatory elements ensuring expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally associated or heterologous promoter regions.

In a further aspect, the present technology provides for a cloning or expression vector comprising one or more of the isolated polynucleotides encoding for the antibodies or a part thereof of the present technology.

A "vector" is any molecule or composition that has the ability to carry a nucleic acid sequence into a suitable host cell where e.g. synthesis of the encoded polypeptide can take place. Typically and preferably, a vector is a nucleic acid that has been engineered, using recombinant DNA techniques that are known in the art, to incorporate a desired nucleic acid sequence (e.g., a nucleic acid of the technology). Expression vectors typically contain one or more of the following components (if they are not already provided by the nucleic acid molecules): a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a leader sequence for secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.

Vectors are typically selected to be functional in the host cell in which the vector will be used (the vector is compatible with the host cell machinery such that amplification of the gene and/or expression of the gene can occur).

General methods by which the vectors may be constructed, transfection methods and culture methods are well known to those skilled in the art. In this respect, reference is made to “Current Protocols in Molecular Biology”, 1999, F. M. Ausubel (ed), Wiley Interscience, New York and the Maniatis Manual produced by Cold Spring Harbor Publishing.

Host cells for production of the antibodies

Also provided is a host cell comprising one or more isolated polynucleotide sequences according to the present technology encoding an antibody thereof of the present technology. Also provided is a host cell comprising one or more vectors according to the present technology encoding an antibody of the present technology. Any suitable host cell/vector system may be used for expression of the polynucleotide sequences encoding the antibody or antigen-binding fragment thereof of the present technology. Bacterial, for example E. coli, and other microbial systems may be used or eukaryotic, for example mammalian, host cell expression systems may also be used. Suitable mammalian host cells include CHO, myeloma or hybridoma cells.

In a further embodiment, a host cell comprising such polynucleotide(s) or vector(s) or a combination thereof according to present technology is provided. In one such embodiment, a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleotide that encodes an amino acid sequence comprising the VL of the anti-IL11 antibody and an amino acid sequence comprising the VH of the anti-l L11 antibody, or (2) a first vector comprising a polynucleotide that encodes an amino acid sequence comprising the VL of the anti-IL 11 antibody and a second vector comprising a polynucleotide that encodes an amino acid sequence comprising the VH of the anti-l L11 antibody. In one embodiment, the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., YO, NSO, Sp20 cell). In one embodiment, the host cell is prokaryotic, e.g. an E. coli cell. In one embodiment, a method of making an anti-IL-11 antibody is provided, wherein the method comprises culturing a host cell comprising a polynucleotide encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

Suitable host cells for cloning or expression of vectors encoding antibodies or components thereof include prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., US 5,648,237, 5,789,199, and 5,840,523. (See for example Charlton, Methods in Molecular Biology, Vol. 248, B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003, pp. 245-254),). After expression, the antibody may be isolated and can be further purified.

Eukaryotic microbes such as fungi or yeast are suitable cloning and/or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of an antibody with a partially or fully human glycosylation pattern. (Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006)).

Suitable types of Chinese Hamster Ovary (CHO cells) for use in the present technology may include CHO and CHO-K1 cells including dhfr- CHO cells, such as CHO-DG44 cells and CHO- DXB11 cells and which may be used with a DHFR selectable marker or CHOK1-SV cells which may be used with a glutamine synthetase selectable marker. Other cell types of use in expressing antibodies include lymphocytic cell lines, e.g., NSO myeloma cells and SP2 cells, COS cells. The host cell may be stably transformed or transfected with the isolated polynucleotide sequences or the expression vectors according to the present technology.

A method of producing the antibody of the present technology is also provided. In a further embodiment, a method of producing the antibody of the present technology is provided, comprising culturing the host cell of under conditions permitting production of the antibody, and recovering the antibody produced.

Purified antibodies

In one embodiment there is provided a purified antibody, for example a humanized antibody, in particular an antibody according to the present technology, in substantially purified from, in particular free or substantially free of endotoxin and/or host cell protein or DNA. Substantially free of endotoxin is generally intended to refer to an endotoxin content of 1 Ell per mg antibody product or less such as 0.5 or 0.1 Ell per mg product.

Substantially free of host cell protein or DNA is generally intended to refer to host cell protein and/or DNA content 400 pg per mg of antibody product or less such as 100 pg per mg or less, in particular 20 pg per mg, as appropriate.

Therapeutic uses

The present technology provides an agent capable of inhibiting IL-11 mediated signaling for use in the treatment of hidradenitis suppurativa. The method may comprise administering to a human subject in need thereof a therapeutically effective amount of an agent capable of inhibiting IL-11 mediated signaling.

In a particular embodiment the agent is an antibody capable of inhibiting IL-11 mediated signaling.

In a particular embodiment the agent is an antibody that specifically binds to IL-11.

In a particular embodiment the agent is an antibody that specifically binds to IL-11 and has the functional and/or structural properties as described herein.

Therapeutic use of the antibodies

The antibodies according to the present technology or pharmaceutical compositions thereof may be administered for prophylactic and/or therapeutic treatments.

The present technology provides an antibody according to the technology or pharmaceutical composition thereof for use as a medicament.

In prophylactic applications, the antibodies or pharmaceutical compositions thereof are administered to a subject at risk of a disorder or condition as described herein, in an amount sufficient to prevent or reduce the subsequent effects of the condition or one or more of its symptoms.

In therapeutic applications, the antibodies or pharmaceutical compositions thereof are administered to a subject already suffering from a disorder or condition as described herein, in an amount sufficient to cure, alleviate or partially arrest the condition or one or more of its symptoms. Such therapeutic treatment may result in a decrease in severity of disease symptoms, or an increase in frequency or duration of symptom-free periods.

The subjects to be treated can be animals. Preferably, the pharmaceutical compositions according to the present technology are adapted for administration to human subjects. The present technology provides a method of treating a disorder or condition as described herein in a subject in need thereof, the method comprising administering to the subject an antibody according to the present technology or a pharmaceutical composition thereof. The antibody is administered in a therapeutically effective amount.

The present technology also provides an antibody according to the present technology, or a pharmaceutical composition thereof for use in the treatment of a disorder or condition as described herein.

Therapeutic indications

The antibodies, and the pharmaceutical compositions of the antibodies of present technology may be used in treating, preventing or ameliorating conditions that are associated with IL-11, mediated signaling, for example any condition which results in whole or in part from signaling through the IL-11/IL-11 Ra/gp130 complex.

As described and exemplified herein, the inventors have established that IL-11 is involved in HS biology. More particularly, it has been demonstrated that IL-11 is upregulated in HS lesions, impacts hair follicle biology, contributes to chronic inflammation, and has a role in driving the dermal and epidermal tissue remodeling which characterizes the more severe disease stages. This suggests that blockade of the IL-11 signaling pathways could result in clinical responses.

In a preferred embodiment, the antibody, and the pharmaceutical compositions of the present technology are thus used to treat hidradenitis suppurativa.

Diseases or conditions such as cancer, fibrosis, autoimmune disease, an inflammatory disease, a metabolic disease, a wasting disease, a bone disease, or a disease in which smooth muscle cells (SMCs) are pathologically implicated may be associated with IL-11 mediated signaling.

In another embodiment, the antibody, and the pharmaceutical compositions of the present technology are thus used to treat cancer, fibrosis, an autoimmune disease, an inflammatory disease, a metabolic disease, a wasting disease, a bone disease, or a disease in which smooth muscle cells (SMCs) are pathologically implicated.

In one embodiment, the antibody, and the pharmaceutical compositions of the present technology are used for treatment of inflammatory skin conditions, systemic sclerosis, inflammatory fibrotic diseases of the lung (such as I PF, COPD, and asthma), inflammatory fibrotic diseases of the liver (such as MASLD, MAFLD, MAFL, and MASH), inflammatory fibrotic diseases of the heart (such as congestive heart failure, myocardial infarction, and ischemic heart disease), endometrial disease (such as endometriosis, and adenomyosis), or cancer (such as colon, liver, and lung cancer). In one embodiment, the antibody, and the pharmaceutical compositions of the present technology are used for treatment of systemic sclerosis, diabetes, hyperglycemia, sarcopenia, hyperlipidaemia, hypertriglyceridemia hypercholesterolemia, pancreatitis, steatosis nonalcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), cholestatic liver disease, wasting, cachexia, chemotherapy- associated weight loss, pancreatic insufficiency, pancreatitis, lipotoxicity, lipodystrophy, lipohypertrophy, lipoatrophy, insulin resistance, hyperglucagonemia, hypertension; abnormal uterine bleeding (AIIB), dysmenorrhea, leiomyoma, endometriosis; cancer (such as liver cancer, colon cancer, bone cancer, prostate cancer, melanoma (e.g., metastatic melanoma), pancreatic cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (e.g., lymphocytic leukemia, chronic myelogenous leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia, hairy cell leukemias, acute lymphoblastic leukemias), lymphoma (e.g., non-Hodgkin’s lymphomas, Hodgkin’s lymphoma), hepatoma (hepatocellular carcinoma), sarcoma, B-cell malignancy, breast cancer, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), kidney cancer (e.g., renal cell carcinoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancers, cervical cancer, testicular cancer, thyroid cancer, stomach cancer, metastatic cancer); fibrosis of the cardiovascular system (such as from hypertrophic cardiomyopathy, dilated cardiomyopathy (DCM), fibrosis of the atrium, atrial fibrillation, fibrosis of the ventricle, ventricular fibrillation, myocardial fibrosis, Brugada syndrome, myocarditis, endomyocardial fibrosis, myocardial infarction, fibrotic vascular disease, hypertensive heart disease, arrhythmogenic right ventricular cardiomyopathy (ARVC), tubulointerstitial and glomerular fibrosis, atherosclerosis, varicose veins or cerebral infarct), fibrosis of the liver (such as from chronic liver disease, non-alcoholic steatohepatitis (NASH), primary biliary cirrhosis (PBC), schistosomal liver disease and liver cirrhosis), fibrosis of the kidney (such as from nephritic syndrome, Alport's syndrome, HIV associated nephropathy, polycystic kidney disease, Fabry's disease, diabetic nephropathy, chronic or crescentic glomerulonephritis), fibrosis of the eye (such as from retinal fibrosis, epiretinal fibrosis, or subretinal fibrosis, Grave's opthalmopathy, subretinal fibrosis associated with macular degeneration (AMD), subretinal fibrosis associated with wet AMD, diabetic retinopathy, glaucoma, corneal fibrosis, post-surgical fibrosis, fibrosis of the posterior capsule following cataract surgery, fibrosis of the bleb following trabeculectomy for glaucoma, conjunctival fibrosis and subconjunctival fibrosis), fibrosis of the skin (such as from scleroderma, nephrogenic systemic fibrosis and cutis keloid), fibrosis of an organ of the gastrointestinal system (such as from Crohn's disease, microscopic colitis and primary sclerosing cholangitis (PSC)), or of the lung (such as from pulmonary fibrosis, idiopathic pulmonary fibrosis (I PF), cystic fibrosis, progressive massive fibrosis, scleroderma, obliterative bronchiolitis, Hermansky-Pudlak syndrome, asbestosis, silicosis, chronic pulmonary hypertension, AIDS associated pulmonary hypertension, sarcoidosis, tumor stroma in lung disease and asthma); fibrosis from neurological conditions (such as gliosis and Alzheimer's disease), muscular dystrophy (such as Duchenne muscular dystrophy (DMD) or Beckers muscular dystrophy (BMD)), arthrofibrosis, Dupuytren's contracture, mediastinal fibrosis; retroperitoneal fibrosis, myelofibrosis, Peyronie's disease, adhesive capsulitis, chronic graft versus host disease, arthriti, fibrotic pre-neoplastic and fibrotic neoplastic disease, and fibrosis induced by chemical or environmental insult (e.g., cancer chemotherapy, pesticides, radiation/cancer radiotherapy); disease in which smooth muscle cells (SMCs) are pathologically implicated (such as vascular aneurysm, Marian's syndrome, aortic aneurysm, Furlong's syndrome, Sphrintzen-Goldberg syndrome, Loeys-Dietz syndrome, familial thoracic aortic aneurysm syndrome, arterial tortuosity syndrome, cerebral aneurysm, vascular stenosis and restenosis, fibromuscular dysplasia (FMD), supravalvular stenosis, renal artery stenosis, pulmonary artery hypertension (PAH), plexiform lesions, telangiectasia, achalasia, dysphagia, diarrhoea, constipation, inflammatory bowel disease (IBD), bowel stricture, pyloric stenosis, coeliac disease, irritable bowel syndrome, diverticulitis, ulcerative colitis, renal disease, focal and segmental glomerulosclerosis (FSGS), IgA nephropathy, , lupus nephritis, bladder disease, lung disease, , acute respiratory distress syndrome (ARDS), systemic sclerosis, Hutchinson-Gilford Progeria Syndrome (HGPS), leiomyoma, leiomyosarcoma, and Hermansky-Pudlak Syndrome (HPS)); chronic obstructive pulmonary disease (COPD), rhinitis, allergies, atopic dermatitis.

NAFLD, NAFL and NASH are also known as, and referred to herein, as MASLD (metabolic dysfunction-associated steatotic liver disease) or MAFLD (metabolic dysfunction-associated fatty liver disease), MAFL (metabolic dysfunction-associated fatty liver) and MASH (metabolic dysfunction-associated steatohepatitis) respectively. Rinella ME et al A multisociety Delphi consensus statement on new fatty liver disease nomenclature. Hepatology 2023: 78(6): 1966- 1986.

Diagnostic use of the antibodies and antigen-binding fragments thereof

The present technology also provides the use of the antibodies of the present technology as diagnostically active agents or in diagnostic assays, for example, for diagnosing skin inflammatory diseases or their severity. In one embodiment, the present technology thus provides an antibody of the present technology for use as a diagnostic agent.

The antibodies may be used to diagnose a disorder or condition as described herein.

The diagnosis may preferably be performed on biological samples. A “biological sample” encompasses a variety of sample types obtained from an individual and can be used in a diagnostic or monitoring assay. The definition encompasses cerebrospinal fluid, blood such as plasma and serum, and other liquid samples of biological origin such as urine and saliva, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as polynucleotides.

Diagnostic testing may preferably be performed on biological samples which are not in contact with the human or animal body. Such diagnostic testing is also referred to as in vitro testing. In vitro diagnostic testing may rely on an in vitro method of detecting free IL-11 (e.g. not bound to IL- 11 R) in a biological sample, which has been obtained from a subject.

In vitro diagnostic testing may rely on an in vitro method of detecting IL-11 in a biological sample which has been obtained from an individual comprising the steps of i) contacting the biological sample with an IL-11 binding antibody or binding fragment thereof as described herein; and ii) detecting binding of the IL-11 binding antibody or binding fragment thereof as described herein to IL-11. By comparing the detected IL-11 level with a suitable control, one can then diagnose the presence or likely occurrence of a disease associated with IL-11 mediated signaling as described herein. Such a detection method can thus be used to determine whether a subject has, or is at risk of developing, a disease associated with IL-11 mediated signaling including determining the stage (severity) of said disease.

The present disclosure thus provides an in vitro method of diagnosing a disease associated with IL-11 mediated signaling in a subject comprising the steps of i) assessing the level or state of IL-11 in a biological sample obtained from the subject by using an IL-11 binding antibody or binding fragment thereof as described herein; and ii) comparing the level or state of IL-11 to a reference, a standard, or a normal control value that indicates the level or state of IL-11 in normal control subjects. A significant difference between the level and/or state of the IL-11 polypeptide in the biological sample and the normal control value indicates that the individual has a disease associated with IL-11 mediated signaling.

Pharmaceutical and diagnostic compositions

An antibody of the present technology may be formulated in a pharmaceutical or diagnostic composition. The pharmaceutical composition will normally be sterile and will typically include a pharmaceutically acceptable agent.

As the antibodies of the present technology are useful in the diagnosis of a disorder or condition as described herein, the present technology also provides for a diagnostic composition comprising an antibody or antigen-binding fragment thereof according to the present technology and a diagnostically acceptable agent. In one embodiment, the present technology thus provides a diagnostic composition comprising the antibody of the present technology and a diagnostically acceptable carrier. Diagnostic compositions comprise a diagnostically effective amount of the antibody of the present technology.

As the antibodies of the present technology are useful in the treatment and/or prophylaxis of a disorder or condition as described herein, the present technology also provides for a pharmaceutical comprising an antibody or antigen-binding fragment thereof according to the present technology and a pharmaceutically acceptable agent. In one embodiment, the present technology thus provides a pharmaceutical composition comprising the antibody of the present technology and a pharmaceutically acceptable carrier.

A pharmaceutically acceptable agent for use in the present pharmaceutical compositions include carriers, excipients, diluents, antioxidants, preservatives, coloring, flavoring and diluting agents, emulsifying agents, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, tonicity agents, cosolvents, wetting agents, complexing agents, buffering agents, antimicrobials, and surfactants.

The pharmaceutical composition can be in liquid form (see for example US 6,171 ,586 and W02006/044908) or in a lyophilized or freeze-dried form and may include one or more lyoprotectants, excipients, surfactants, high molecular weight structural additives and/or bulking agents (see for example US Patents 6,685,940, 6,566,329, and 6,372,716).

Compositions can be suitable for parenteral administration. Exemplary compositions are suitable for injection or infusion into an animal by any route available to the skilled worker, such as intraarticular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, or intralesional routes. A parenteral formulation typically will be a sterile, pyrogen-free, isotonic aqueous solution, optionally containing pharmaceutically acceptable preservatives.

Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringers' dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, anti-microbials, antioxidants, chelating agents, inert gases and the like. See generally, Remington's Pharmaceutical Science, 16th Ed., Mack Eds., 1980, which is incorporated herein by reference. Pharmaceutical compositions described herein can be formulated for controlled or sustained delivery in a manner that provides local concentration of the product (e.g., bolus, depot effect) and/or increased stability or half-life in a particular local environment. The pharmaceutical compositions can include the formulation of antibodies, antigen-binding fragments, nucleic acids, or vectors of the present technology with particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc., as well as agents such as a biodegradable matrix, injectable microspheres, microcapsular particles, microcapsules, bioerodible particle beads, liposomes, and implantable delivery devices that provide for the controlled or sustained release of the active agent which can then be delivered as a depot injection.

Alternatively or additionally, the pharmaceutical compositions can be administered locally via implantation into the affected area of a membrane, sponge, or other appropriate material on to which an antibody, binding fragment, nucleic acid, or vector of the present technology has been absorbed or encapsulated. Where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of an antibody, binding fragment, nucleic acid, or vector of the present technology can be directly through the device via bolus, or via continuous administration, or via catheter using continuous infusion. A pharmaceutical composition can be formulated for inhalation, such as for example, as a dry powder. Inhalation solutions also can be formulated in a liquefied propellant for aerosol delivery. In yet another formulation, solutions may be nebulized.

Therapeutically effective amount and dosage

The antibodies and pharmaceutical compositions of the present technology may be administered suitably to a patient to identify the therapeutically effective amount required. For any antibody, the therapeutically effective amount can be estimated initially either in cell culture assays or in animal models, usually in rodents, rabbits, dogs, pigs or primates. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

The precise therapeutically effective amount for a human subject will depend upon the severity of the disease state, the general health of the subject, the age, weight and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities and tolerance/response to therapy. Compositions may be conveniently presented in unit dose forms containing a predetermined amount of an active agent of the disclosure per dose. Dose ranges and regimens for any of the embodiments described herein include, but are not limited to, dosages ranging from 1 mg-1000 mg unit doses. A suitable dosage of an antibody/modulatory agent or pharmaceutical composition of the technology may be determined by a skilled medical practitioner. Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present technology may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present technology employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A suitable dose may be, for example, in the range of from about 0.01 pg/kg to about 10OOmg/kg body weight, typically from about 0.1 pg/kg to about 100mg/kg body weight, of the patient to be treated.

Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single dose may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical agent.

Administration of pharmaceutical compositions or formulations

Antibodies, or pharmaceutical compositions thereof may be administered for prophylactic and/or therapeutic treatments.

An antibody or pharmaceutical composition may be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Examples of routes of administration for compounds or pharmaceutical compositions of the technology include intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. Alternatively, antibody/modulatory agent or pharmaceutical composition of the technology can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration. The antibody/modulatory agent or pharmaceutical composition of the technology may be for oral administration. Suitable forms for administration include forms suitable for parenteral administration, e.g. by injection or infusion, for example by bolus injection or continuous infusion, in intravenous, inhalable or sub-cutaneous form. Where the product is for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain additional agents, such as suspending, preservative, stabilizing and/or dispersing agents. Alternatively, the antibody or antigen-binding fragment thereof according to the present technology may be in dry form, for reconstitution before use with an appropriate sterile liquid. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.

Once formulated, the compositions of the technology can be administered directly to the subject.

Articles of manufacture and kits

The present disclosure also provides kits comprising the antibodies of the present technology and instructions for use. The kit may further contain one or more additional reagents, such as an additional therapeutic or prophylactic agent as discussed above.

The present technology provides use of an antibody according to the present technology or a pharmaceutical composition thereof for the manufacture of a medicament.

The present technology also provides use of an antibody according to the present technology or a pharmaceutical composition thereof for the manufacture of a medicament for the treatment of a disorder or condition as described herein.

In certain embodiments, the article of manufacture or kit comprises a container containing one or more of the antibodies of the technology, or the compositions described herein.

In certain embodiments, the article of manufacture or kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treatment, prevention and/or diagnosis and may have a sterile access port. At least one agent in the composition is an antibody of the present technology. The label or package insert indicates that the composition is used for the treatment of an inflammatory skin condition, more specifically hidradenitis suppurativa.

With respect to these various aspects and embodiments which have been described herein, the present disclosure contemplates inter alia: 1. An antibody or an antigen-binding fragment thereof that specifically binds to IL-11 , which comprises a heavy chain variable region comprising: a CDR-H1 comprising the amino acid sequence of SEQ ID NO:1 , a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, or a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2 wherein 1 , or 2 amino acids have been substituted with another amino acid, wherein at position 6 the G has changed into S, or A, at position 7 the S has changed into G, at position 9 the T has changed into S, and/or at position 17 the S has changed into R, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:3, or a CDR-H3 comprising the amino acid sequence of SEQ ID NO:3 wherein 1 amino acid has been substituted with another amino acid, wherein at position 5 the T has changed into A; and a light chain variable region comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, or a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4 wherein 1 amino acid has been substituted with another amino acid, wherein at position 1 the K has changed into R, and/or at position 9 the Y has changed into H, and a CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, or a CDR-L2 comprising the amino acid sequence of SEQ ID NO:5 wherein 1 , or 2 amino acids have been substituted with another amino acid, wherein at position 5 the L has changed into R, and/or at position 6 the Y has changed into N or D, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:6.

2. The antibody or the antigen-binding fragment thereof according to embodiment 1 , which comprises a heavy chain variable region comprising: a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1 , a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, 21 , 22, 23, 56 or 57, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:3, or 58; and a light chain variable region comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, 42, or 43, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, 44, 45, or 46, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:6.

3. The antibody or the antigen-binding fragment thereof according to embodiment 1 , or 2, which comprises a heavy chain variable region comprising: a CDR-H1 comprising the amino acid sequence of SEQ ID NO:1 , a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:3; and a light chain variable region comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:6.

4. The antibody or the antigen-binding fragment thereof according to any one of embodiments

1 to 3, wherein the heavy chain variable region comprises an amino acid sequence with a sequence identity of more than 90% with SEQ ID NO:7.

5. The antibody or the antigen-binding fragment thereof according to any one of embodiments 1 to 4, wherein the light chain variable region comprises an amino acid sequence with a sequence identity of more than 90% with SEQ ID NO:9.

6. The antibody or the antigen-binding fragment thereof according to any one of embodiments 1 to 5, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:7

7. The antibody or the antigen-binding fragment thereof according to any one of embodiments 1 to 6, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:9.

8. The antibody according to any one of embodiments 1 to 7, wherein the antibody is an antibody fragment.

9. The antibody according to any one of embodiments 1 to 8, wherein the antibody is a Fab, Fab’, F(ab’)₂, Fv, or scFv. 10. The antibody according to any one of embodiments 1 to 9, wherein the antibody is a Fab comprising a light chain comprising an amino acid sequence which has at least 90% identity or similarity to the amino acid sequence of SEQ ID NO:11 , and a heavy chain comprising an amino acid sequence which has at least 90% identity or similarity to the amino acid sequence of SEQ ID NO:13.

11 . The antibody according to embodiment 10, wherein the antibody is a Fab which comprises a light chain comprising the amino acid sequence of SEQ ID NO:11 , and a heavy chain comprising the amino acid sequence of SEQ ID NO: 13.

12. The antibody according to any one of embodiments 1 to 7, wherein the antibody is a full length antibody.

13. The antibody according to embodiment 12, wherein the antibody is an lgG1 comprising a light chain comprising an amino acid sequence which has at least 90% identity or similarity to the amino acid sequence of SEQ ID NO:11 , and a heavy chain comprising an amino acid sequence which has at least 90% identity or similarity to the amino acid sequence of SEQ ID NO:98.

14. The antibody according to embodiment 13, wherein the antibody is an lgG1 which comprises a light chain comprising the amino acid sequence of SEQ ID NO:11 , and a heavy chain comprising the amino acid sequence of SEQ ID NO:98.

15. The antibody according to embodiment 12, wherein the antibody is an lgG1 comprising a light chain comprising an amino acid sequence which has at least 90% identity or similarity to the amino acid sequence of SEQ ID NO:11 , and a heavy chain comprising an amino acid sequence which has at least 90% identity or similarity to the amino acid sequence of SEQ ID NO:19.

16. The antibody according to embodiment 15, wherein the antibody is an lgG1 comprising a light chain comprising the amino acid sequence of SEQ ID NO:11 , and a heavy chain comprising the amino acid sequence of SEQ ID NO: 19.

17. The antibody according to embodiment 12, wherein the antibody is an lgG4P comprising a light chain comprising an amino acid sequence which has at least 90% identity or similarity to the amino acid sequence of SEQ ID NO:11 , and a heavy chain comprising an amino acid sequence which has at least 90% identity or similarity to the amino acid sequence of SEQ ID NO:99.

18. The antibody according to embodiment 17, wherein the antibody is an lgG4P which comprises a light chain comprising the amino acid sequence of SEQ ID NO:11 , and a heavy chain comprising the amino acid sequence of SEQ ID NO:99. 19. An isolated antibody that competes for binding to IL-11 with the antibody or the antigenbinding fragment thereof according to any one of embodiments 1 to 18.

20. An isolated antibody that binds to the same epitope of IL- 11 as the antibody or the antigenbinding fragment thereof according to any one of embodiments 1 to 19.

21. An isolated polynucleotide encoding the antibody or the antigen-binding fragment thereof according to any one of embodiments 1 to 20.

22. An expression vector carrying the polynucleotide according to embodiment 21.

23. A host cell comprising the vector according to embodiment 22.

24. A method of producing the antibody or the antigen-binding fragment thereof according to any one of embodiments 1 to 20, comprising culturing the host cell according to embodiment 23 under conditions permitting production of the antibody or the antigen-binding fragment thereof, and recovering the antibody or the antigen-binding fragment thereof produced.

25. A diagnostic composition comprising the antibody or the antigen-binding fragment thereof according to any one of embodiments 1 to 20 and a diagnostically acceptable agent.

26. The antibody according to any one of embodiments 1 to 20 for use as a diagnostic agent.

27. A pharmaceutical composition comprising the antibody or the antigen-binding fragment thereof according to any one of embodiments 1 to 20 and a pharmaceutically acceptable agent.

28. The antibody or the antigen-binding fragment thereof according to any one of embodiments 1 to 20, or the pharmaceutical composition according to embodiment 27, for use as a medicament.

29. The antibody or the antigen-binding fragment thereof according to any one of embodiments 1 to 20, or the pharmaceutical composition according to embodiment 27, for use in the treatment or prevention of cancer, fibrosis, an autoimmune disease, an inflammatory disease, a metabolic disease, a wasting disease, a bone disease, or a disease in which smooth muscle cells (SMCs) are pathologically implicated.

30. The antibody or the antigen-binding fragment thereof according to any one of embodiments 1 to 20, or the pharmaceutical composition according to embodiment 27, for use in the treatment or prevention of hidradenitis suppurativa.

31. Use of the antibody or the antigen-binding fragment thereof according to any one of embodiments 1 to 20, or the pharmaceutical composition according to embodiment 27, for the manufacture of a medicament. 32. Use of the antibody or the antigen-binding fragment thereof according to any one of embodiments 1 to 20, or the pharmaceutical composition according to embodiment 27, for the manufacture of a medicament for the treatment of cancer, fibrosis, an autoimmune disease, an inflammatory disease, a metabolic disease, a wasting disease, a bone disease, or a disease in which smooth muscle cells (SMCs) are pathologically implicated.

33. Use of the antibody or the antigen-binding fragment thereof according to any one of embodiments 1 to 20, or the pharmaceutical composition according to embodiment 27, for the manufacture of a medicament for the treatment hidradenitis suppurativa.

34. A method of treating or preventing cancer, fibrosis, an autoimmune disease, an inflammatory disease, a metabolic disease, a wasting disease, a bone disease, or a disease in which smooth muscle cells (SMCs) are pathologically implicated, comprising administering a therapeutically effective amount of the antibody or the antigen-binding fragment thereof according to any one of embodiments 1 to 20, or the pharmaceutical composition according to embodiment 27.

35. A method of treating or preventing hidradenitis suppurativa, comprising administering a therapeutically effective amount of the antibody or the antigen-binding fragment thereof according to any one of embodiments 1 to 20, or the pharmaceutical composition according to embodiment 27.

36. An agent capable of inhibiting IL-11 mediated signaling for use in the treatment of hidradenitis suppurativa.

37. Use of an agent capable of inhibiting IL-11 mediated signaling for the manufacture of a medicament for the treatment of hidradenitis suppurativa.

38. A method of treating or preventing hidradenitis suppurativa, comprising administering a therapeutically effective amount of an agent capable of inhibiting IL-11 mediated signaling.

39. The agent for use according to embodiment 36, use of the agent according to embodiment 37, or the method according to embodiment 38, wherein the agent is an antibody or an antigenbinding fragment thereof capable of inhibiting IL-11 mediated signaling.

40. The agent for use according to embodiment 39, use of the agent according to embodiment 39, or the method according to embodiment 39, wherein the antibody or the antigen-binding fragment thereof specifically binds to IL-11.

41. The agent for use according to embodiment 39 or 40, use of the agent according to embodiment 39 or 40, or the method according to embodiment 39 or 40, wherein the antibody is a Fab, scFv, Fv, dsFv, dsscFv, or an IgG. 42. The agent for use according to any one of embodiments 39 to 41, use of the agent according to any one of embodiments 39 to 41, or the method according to any one of embodiments 39 to 41 , wherein the antibody is an I gG 1 , or an lgG4P. It should be noted that the above-mentioned embodiments illustrate rather than limit the technology, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the claims.

The sequences included in the present technology are shown in Tables A-1 to A-3. Table A-1. Sequences related to IL-11 binding domain 19439gL1gH1

* Mutated with cysteines engineered for a disulfide bond

Table A-2. Sequences related to variants of the IL-11 binding domain 19439

Table A-3. Other sequences

Table A-4. Sequences of IL-11 proteins

EXAMPLES

Example 1. Generation of IL-11 tool proteins IL-11 tool proteins were generated according to one of the methods below. Description of the generated proteins, their sequence and used production method are listed in Table X-1.

Method 1 :

DNA was optimised for expression in E. coli and cloned into a modified pET28b vector (ATLIM) using BamHI/Xhol, generating a vector encoding the desired protein sequence with N-terminal Thioredoxin, a His tag and a TEV cleavage site. Plasmid DNA was used to transform BL21(DE3) cells (NEB), briefly, 200 - 500 ng of DNA was added to 100uL of BL21 (DE3) competent cells and incubated on ice for 20 minutes before heat shocking for 20 secs at 42°C. The cells were then incubated on ice for 5 minutes before adding 200 uL of S.O.C media (Invitrogen) and incubating shaking at 37°C for 30-60 mins. 50 uL of cell suspension was added to an LB agar plate made with carbenicillin antibiotic. The plate was incubated at 37°C overnight. A single ampicillin resistant colony was picked from the plate and used to inoculate a 100ml starter culture of LB with carbenicillin antibiotic. The starter culture was used to inoculate LB/Carb media (starting OD600 of 0.01) and the culture was shaken (250rpm) at 37 °C until an OD600 of 3.0 was achieved. Protein expression was induced with 100pM IPTG and feed (50 mM MOPS, 1 mM Mg salts, 2% glycerol) added to support cell growth. Cells were further incubated at 18°C for 25 hours before harvesting via centrifugation at 4000 rpm for 30 mins (4°C). Cell pellet was frozen at -80 until needed.

Pellet was defrosted in water and diluted in lysis buffer (PBS pH 7.4, 500 mM NaCI, 20 mM Imidazole, 1x per 50 mL protease inhibitor cocktail pills, 15 units/mL benzonase, 2 mM MgCI2). Cells were then lysed using a cell disrupter at 40 kpsi. Cell lysate was centrifuged at 18 krpm for 30 minutes and supernatant filtered at 0.2 pm. Filtered supernatant was loaded onto a washed 5mL Histrap HP (Cytiva) (PBS pH 7.4, 500 mM NaCI, 20 mM Imidazole) using an AKTA Pure system. The protein was eluted from the Histrap using a high imidazole buffer (PBS pH 7.4, 500 mM NaCI, 0.5 M Imidazole) in a gradient elution over 5 column volumes. Protein containing fractions were pooled and the His tag cleaved using TEV protease in a 1 :50 w/w ratio. Tag and protease were removed using a Histrap. The subsequent protein product was concentrated and loaded onto a Superdex 75 16/600 column for size exclusion chromatography. Protein containing fractions were pooled and tested for purity using mass spectrometry and SDS-PAGE. The protein was frozen at -80 until required.

Method 2:

DNA was optimised for expression in mammlian cells and cloned into a modified pMH vector (ATUM) using BamHI/EcoRI, generating a vector encoding the desired protein sequence with N-terminal His tag and a TEV cleavage site. Plasmid DNA was used to transfect Expi293 HEK cells, briefly, 0.5 mg of DNA per 1 L of 2.5 X 106 cells was diluted in 1 mL OptiMEM and incubated with Expifectamine 293 (ThermoFisher) for 20 mins at room temp. DNA and Expifectamine mix was added to Expi293 HEK cells and incubated shaking at 37°C for 4 days. Enhancers (ThermoFisher) were added after 1 day. Cell supernatant was harvested via centrifugation at 6000 rpm for 3 hrs. Supernatant was filtered at 0.2um and loaded onto 2x 5mL Histrap Excel (Cytiva) columns using an AKTA Pure system. Columns were washed with PBS pH 7.4, 0.5 M NaCI, 20 mM Imidazole. Protein was eluted using PBS pH 7.4, 0.5 M NaCI, 0.5 M Imidazole over 5 column volumes using a gradient. Protein containing fractions were pooled, concentrated and loaded onto a Superdex 200 26/600 column washed in PBS pH 7.4 for size exclusion chromatography. Protein containing fractions were pooled and tested for purity using mass spectrometry and SDS-PAGE. The protein was frozen at -80 until required.

Tag removal was carried out on a portion of the protein. Briefly, Tev protease was added to purified protein in a 1 :50 w/w ratio. Tag and protease were removed using a Histrap, and the protein buffer exchanged into PBS pH 7.4 using PD10 desalting columns (Cytiva). Protein purity was verified using mass spectrometry and SDS-PAGE.

Method 3:

DNA was optimised for expression in mammalian cells and cloned into a modified pMH vector (ATLIM) using Hindll l/Xhol , generating a vector encoding the desired protein sequence with C-terminal human FC tag and TEV cleavage site. Plasmid DNA was used to transfect Expi293 HEK cells, briefly, 0.5 mg of DNA per 1 L of 2.5 X 106 cells was diluted in 1 mL OptiMEM and incubated with Expifectamine 293 (ThermoFisher) for 20 mins at room temp. DNA and Expifectamine mix was added to Expi293 HEK cells and incubated shaking at 37°C for 4 days. Enhancers (ThermoFisher) were added after 1 day. Cell supernatant was harvested via centrifugation at 6000 rpm for 3 hrs. Supernatant was filtered at 0.2um and loaded onto 5mL HiTrap Protein A (Cytiva) column using an AKTA Pure system. The column was washed with PBS pH 7.4. Protein was eluted using 0.1M citric acid pH2 in 1.5 mL fractions, 0.4 mL 2M Tris pH 8 was then added to each protein containing fraction to neutralise. Protein containing fractions were pooled, concentrated and loaded onto a Superdex 200 26/600 column washed in PBS pH 7.4 for size exclusion chromatography. Protein containing fractions were pooled and tested for purity using mass spectrometry and SDS-PAGE. The protein was frozen at -80 until required.

Method 4:

DNA was optimised for expression in mammalian cells and cloned into a modified pMH vector (ATLIM) using BamHI/EcoRI, generating a vector encoding the desired protein sequence with an N-terminal His tag and a TEV cleavage site. Plasmid DNA was used to transfect Expi293 HEK cells, briefly, 0.5 mg of DNA per 1 L of 2.5 X 106 cells was diluted in 1 mL OptiMEM and incubated with Expifectamine 293 (ThermoFisher) for 20 mins at room temp. DNA and Expifectamine mix was added to Expi293 HEK cells and incubated shaking at 37°C for 4 days. Enhancers (ThermoFisher) were added after 1 day. Cell supernatant was harvested via centrifugation at 6000 rpm for 3 hrs. Supernatant was filtered at 0.2um and loaded onto 2x 5mL Histrap Excel (Cytiva) columns using an AKTA Pure system. Columns were washed with PBS pH 7.4, 0.5 M NaCI, 20 mM Imidazole, 5% Glycerol. Protein was eluted using PBS pH 7.4, 0.5 M NaCI, 0.5 M Imidazole, 5% Glycerol over 5 column volumes using a gradient. Protein containing fractions were pooled and the His tag cleaved using TEV protease in a 1 :50 w/w ratio, incubated overnight at 4°C rolling. The cleaved protein was concentrated and loaded onto a Superdex 200 26/600 column washed in PBS pH 7.4, 300 mM NaCI, 5% glycerol for size exclusion chromatography. Protein containing fractions were pooled and tested for purity using mass spectrometry and SDS-PAGE. The protein was frozen at -80 until required.

Table X-1. Sequences of IL-11 related proteins TEV protease cleaved tag, which is not part of final protein product, is indicated in bold italic.

Example 2. Discovery and selection of therapeutic anti-IL-11 antibody VR19439

Immunisations

Five female Sprague Dawley rats were immunised sub-cutaneously with 50pg/rat of recombinant Human IL-11 in a 1 :1 emulsion with complete Freunds adjuvant. Rats were given 4 booster injections at 14 day intervals with recombinant Human IL-11 (SEQ ID NO:86) and recombinant Cyno IL-11 protein (SEQ ID NO:90) in a 1 :1 emulsion with incomplete Freunds adjuvant. Non-heparinised bleeds (50ul) were taken from the tail vein prior to each immunisation. Sera was collected from the bleeds and monitored for binding to the immunogens by ELISA throughout the duration of the immunisation campaign. Termination occurred 14 days after the final boost and single cell suspensions of spleen, lymph node, bone marrow and peripheral blood mononuclear cells were prepared in addition to a terminal sera bleed.

Discovery to TAP Stage

B-cell cultures were set up using a similar method as described by Tickle et al, 2015. Culture supernatants were screened for the presence of antibodies which bound to human and cynomolgus IL-11 using a high-throughput flow cytometry assay. Streptavidin-coupled fluorescent beads were coated with biotinylated human IL-11 , cynomolgus IL- 11 or an irrelevant protein control and binding was detected using an anti-species Fc antibody conjugated to DyLight 405 (Jackson). Approximately 40 human and cynomolgus IL-11 cross- reactive hits were identified through B-cell cultures. Single antigen-specific B-cells were isolated from hit culture wells using a similar method as described by Clargo et al. (2014), the fluorescent foci method. B-cells from hit wells were picked into PCR plates for reverse transcription (RT) reactions, followed by V-region-specific PCRs to generate approximately 100 transcriptionally active PCR (TAP) products (Clargo et al, 2014). In addition to B-cell cultures, single antigen-specific B-cells were isolated directly from unstimulated cells using a variation of the fluorescent foci method. Briefly, cryopreserved immune cells from lymph node or bone marrow samples were incubated with streptavidin beads coated with biotinylated human or cynomolgus IL-11 protein and a secondary AF647-conjugated anti-species Fc antibody. Individual cells were then picked as above directly into RT mix and PCR reactions carried out to generate TAP products. Around 500 TAP products were generated using this method. TAP products from B-cell culture and direct foci experiments were transiently transfected into Expi293F cells (Thermo Fisher) at a 1ml scale. Resultant supernatants containing recombinant antibodies were screened for their ability to bind human and cynomolgus IL-11 protein using the same high-throughput flow cytometry assay as described above. Supernatants containing antibodies which showed cross- reactive binding to human and cynomolgus IL-11 underwent binding kinetics assessment by Biacore. For progression at this stage, antibodies had to achieve an affinity of less than 100pM for human IL-11 with a cynomolgus IL- 11 affinity within 10-fold of the human affinity.

Progression to Cloned Antibodies

Heavy and light chain variable region (VR) pairs from TAP products that met the binding and affinity criteria were cloned into vectors for expression as chimeric human Fab fragments. These were transiently transfected into Expi293F cells and tested for binding to human and cynomolgus IL-11 using flow cytometry. Binding kinetics of cloned transient supernatants were confirmed by Biacore. Supernatants were also tested in a STAT3 inhibition assay to study function. Briefly, supernatants were incubated with IL-11 protein and a STAT3 reporter cell line expressing IL-11 R (Eurofins DiscoverX). Functionally active antibodies showing inhibition of IL-11-induced STAT3 activation which had an affinity for human IL-11 of ~10pM or lower, a cynomolgus affinity of ~100pM or lower, and showed blocking of IL-11 to IL-11 R were considered for selection. A total of 51 cloned antibodies were tested for function, and only three antibodies comprising unique variable regions were identified which met the required criteria. VR19439 was selected as the lead molecule.

Example 3. Humanization of VR19439

Antibody 19439 was humanized by grafting CDRs from the rat V-region onto human germline antibody V-region frameworks. To attempt to recover the activity of the antibody, a number of framework residues from the rat V-region were also retained in the humanized sequence. These residues were selected using the protocol outlined by Adair et al. (1991) (WO91/09967). Alignments of the rat antibody (donor) V-region sequences with the human germline (acceptor) V-region sequences are shown in Figures 1 and 2 for the light chain and the heavy chain graft respectively, together with the designed humanized sequences. The CDRs grafted from the donor to the acceptor sequence are as defined by Kabat (Kabat et al., 1987), with the exception of CDRH1 where the combined Chothia/Kabat definition is used (see Adair et al., WO91/09967). A number of related variant V-regions were discovered alongside antibody 19439; alignments of these rat V-region sequences are shown in Figures 3 and 4 for the light chain and the heavy chain variable sequences respectively.

Human V-region IGKV1-12 plus IGKJ2 J-region (IMGT, http://www.imgt.org/) was chosen as the acceptor for antibody 19439 light chain CDRs. The light chain framework residues in the humanized graft variants are all from the human germline gene, with the exception of zero, one or more residues from the group comprising residues 60 and 63 (with reference to SEQ ID NO:9), where the donor residues Aspartic Acid 60 (D60) and Threonine 63 (T63) were retained, respectively. The different mutations are depicted in Figure 1.

Human V-region IGHV3-07 plus IGHJ4 J-region (IMGT, http://www.imgt.org/) was chosen as the acceptor for the heavy chain CDRs of antibody 19439. The heavy chain framework residues in the humanized graft variants are all from the human germline gene, with the exception of zero, one or more residues from the group comprising residues 77 and 98 (with reference to SEQ ID NO:7), where the donor residues Serine 77 (S77) and Threonine 98 (T98) were retained, respectively. In some humanized graft variants, a potential Aspartic Acid isomerisation site in CDRH2 was modified by replacing the Glycine residue at position 55 with either a Serine (G55S), or Alanine (G55A). The different mutations are depicted in Figure 2.

Genes encoding variant heavy and light chain V-region sequences were designed and constructed. For transient expression in mammalian cells, the humanized light chain V-region genes were cloned into a light chain expression vector pMhCK, which contains DNA encoding the human Kappa chain constant region (Km3 allotype). The humanized heavy chain V-region genes were cloned into either a human Fab 10His heavy chain expression vector pMhFablOHis, which contains DNA encoding the human CH1 heavy chain constant region with a C-terminal 10x histidine tag for purification; a human gamma-1 heavy chain expression vector pMhyl LALA, which contains DNA encoding the human gamma-1 heavy chain constant region (G1m17, 1 allotype) with additional Fey receptor binding inactivating mutations L234A and L235A (Tamm A & Schmidt RE (1997) IgG Binding Sites on Human Fey Receptors, International Reviews of Immunology, 16:1-2, 57-85); a human gamma-1 heavy chain expression vector pMhyl LALA K which contains an additional ‘knob’ mutation (T366W) to promote knob into hole heavy chain heterodimerization; or a human gamma- 1 heavy chain expression vector pMhyl LALA H which contains the counterpart additional ‘hole’ mutations (T366S, L368A, and Y407V) to promote knob into hole heavy chain heterodimerization (Ridgway JB, Presta LG, Carter P. 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng. 1996 Jul;9(7):617-21).

Co-transfection of the resulting heavy and light chain vectors into CHO-SXE suspension cells was achieved using ExpiFectamine TM CHO transfection reagent (A29130, ThermoFisher Scientific), and gave expression of the humanized, recombinant Fab or lgG1 antibodies. The variant humanized antibody chains, and combinations thereof, were expressed and assessed for their binding affinity for human IL-11 relative to the parent antibody by surface plasmon resonance. The humanised antibody containing all donor residues (gL1gH1) showed a comparable binding affinity compared to the parental antibody (Table X-2, gL1gH1 (14.8 pM) compared to 19439 (4.8 pM)). The donor residues were removed individually and in combinations from the light (gL2, gL3, and gL4) and heavy chains (gH9, gH10, and gH11). All but two of these humanised graft combinations tested showed comparable binding affinity compared to the parent antibody 19439 and the humanised graft with all donor residues (gL1gH1). The two humanised graft combinations (gL1gH10 and gL1gH11) in which T98 had been removed resulted in significant loss of binding affinity (Table X-2, gH10: 7870.0 pM, gH11 : 1740.0 pM) indicating this residue is essential for high affinity binding of this V-region to IL-11.

The G55S and G55A modifications made in CDRH2 to modify a potential Aspartic acid isomerisation site did not result in a significant change in the measured affinity (Table X-2, gL3gH1 (16.7 pM) and gL4gH1 (15.7 pM) compared to gL1gH1 (14.8 pM).

Table X-2. Binding affinity of different generated variants in comparison to VR19439 as measured by SPR

Run in a different SPR assay

The CDR variant sequences shown in Figures 3 and 4 were also expressed and assessed for binding affinity. These variants all showed comparable binding affinity to 19439 (CDR variants (6.7 - 16.7 pM) compared to 19439 (9.3 pM); the SPR results are shown in Table X-3.

Table X-3. Binding affinity of CDR variants in comparison to VR19439 as measured by SPR

A disulfide bond stabilised single chain Fv format of the humanised V-region containing all donor residues (gL1gH1) was also expressed and assessed for binding affinity for human IL- 11 relative to the parent antibody by surface plasmon resonance. The mutations to introduce the stabilising disulfide bond (LC: Q100C, HC: G44C) are shown in Figures 1 and 2 as an alignment with the parental V-region sequence. A gene encoding the disulfide stabilised single chain Fv sequence as part of a Fab-dsscFv BYbe construct was designed and constructed. The disulfide stabilized single chain Fv antibody containing all donor residues showed a comparable binding affinity compared to the Fab antibody containing all donor residues (Table X-4, 19439gL1gH1 dsscFv (15.9 pM) compared to 19439gL1gH1 Fab (16.6 pM)) as well as the parental antibody (Table X-4, 19439 (9.3 pM)).

Table X-4. Binding affinity of Fab and dsscFv in comparison to VR19439 as measured by SPR

The final selected variable graft sequences gL1 and gH1 are shown in Figures 1 and 2 respectively (19439gL1gH1) and the corresponding sequences are listed in Table A-1.

Example 4: Generation of anti-IL-11 reference mAb1 and the anti-IL-11 Fabs derived from 19439gL1gH1 and reference mAb1

Generation of Fab derived from anti-IL-11 reference mAb1

The variable region (V-region) sequences of Enx203 were taken from patent application US 2020/0270340 A1 for the generation of anti-human-IL-11 reference mAb1. Genes encoding the heavy and light chain V-region sequences were designed and constructed. The synthetic VL-region gene was supplied cloned into a mouse light chain expression vector pMmCK, which contains DNA encoding the mouse Kappa chain constant region, to create pMmCK (reference mAb1). The synthetic VH-region gene was sub-cloned into a mouse gamma-1 Fab expression vector pM mg I Fabnh, which contains DNA encoding the mouse gamma-1 CH1 constant region with a truncated hinge, to create pMmgl Fabnh (reference mAb1). Heavy and light chain vectors encoding the anti-human-IL-11 reference mAb1 Fab fragment were transfected into CHO-SXE suspension cells by electroporation. Following electroporation, transfected cells were cultured in enriched PROCHO™ 5 medium (Lonza) at 32°C for 14 days in shaken culture. Cultures were harvested and clarified by centrifugation followed by 0.22pm filtration to recover the cell culture supernatant containing expressed Fab. Expression titre was determined by HiTrap™ Protein G HP (Cytiva) quantification HPLC (Agilent) assay prior to standard affinity capture chromatography using a GammaBind Plus Sepharose™ column (Cytiva) and AKTA Pure™ 25L chromatography system (Cytiva). The anti-human-IL-11 reference mAb1 Fab fragment was captured onto the column under neutral conditions and eluted in 0.1 M Glycine-HCI, pH2.7 followed by direct neutralisation to pH7.0- 7.5 with Tris-HCI solution. Peak fractions were pooled, sterile filtered and concentration was determined by A280 measurement (Nanodrop™ 2000). The purified Fab was concentrated prior to loading onto a HiLoad® Superdex® 200 prep, grade column (Cytiva) to remove residual high and low molecular weight product related impurities and to buffer exchange the product into Phosphate Buffered Saline, pH7.4 formulation buffer. The final product was concentrated using a 10KDa MWCO membrane, final concentration was determined by A280 (Nanodrop™ 2000), monomer content was determined by analytical size exclusion HPLC, correct banding pattern was determined by SDS-PAGE, endotoxin level was determined using the Charles River Endosafe® LAL Reagent Cartridge technology, and intact mass and expected post translational modifications were confirmed by mass spectrometry.

Generation of Fab derived from 19439gL1gH1

Fab 19439gL1gH1 was generated as described in Example 3. Heavy and light chain vectors encoding the anti-human-IL-11 19439 humanized Fab were transfected into CHO-SXE suspension cells using ExpiFectamine™ CHO transfection reagent (ThermoFisher Scientific). Following transfection, cells were cultured in ExpiCHO™ expression medium at 32°C for 7 days in shaken culture. Cultures were harvested and clarified, and Fab was purified and analysed as detailed above.

Example 5: Murinization of antibody 19439

Antibody 19439 was murinized by grafting CDRs from the rat V-region onto mouse germline antibody V-region frameworks. In order to recover the activity of the antibody, a number of framework residues from the rat V-region were also retained in the murinized sequences. These residues were selected using the protocol outlined by Adair et al. (1991) (Humanised antibodies. WO91/09967). Alignments of the rat antibody (donor) V-region sequences with the mouse germline (acceptor) V-region sequences are shown in Figures 5 and 6, together with the designed murinized sequences. The CDRs grafted from the donor to the acceptor sequences are as defined by Kabat (Kabat et al., 1987), with the exception of CDR-H1 where the combined Chothia/Kabat definition is used (see Adair et al., 1991 Humanised antibodies. WO91/09967).

Two alternative mouse kappa chain frameworks, IGKV6-25 and IGKV8-30 were selected as the acceptor for antibody 19439 CDRs, mouse IGKJ1 was used as the J-region. For the IGKV6-25 light chain graft (19439mL1), a single donor residue at position71 (Phenylalanine, F71) was retained, whilst for the IGKV8-30 light chain graft (19439mL1.1), a single donor residue at position 85 (Leucine, L85) was retained.

The heavy chain CDRs from antibody 19439 were grafted onto two alternative mouse heavy chain frameworks, IGHV5S3 and IGHV6S1 , mouse IGHJ3 was used as the J-region. For the IGHV5S3 heavy chain graft (19439mH1), donor residues were retained at positions 2 (methionine, M2), 3 (Glutamine, Q3), 44 (Glycine, G44), 77 (Serine, S77), 93 (Threonine, T93), 98 (Threonine, T98) and 119 (Serine, S119), whilst for the IGHV6S1 heavy chain graft (19439mH1.1), donor residues were retained at positions 2 (methionine, M2), 3 (Glutamine, Q3), 23 (Alanine, A23), 74 (Asparagine, N74), 79 (Leucine 79), 93 (Threonine, T93), 97 (Alanine, A97), 98 (Threonine, T98) and 119 (Serine, S119).

Genes encoding the heavy and light chain murinized V-region sequences were designed and constructed. For transient expression in mammalian cells, the murinized light chain V-region genes were cloned into a light chain expression vector pMmCK, which contains DNA encoding the mouse Kappa chain constant region. The murinized heavy chain V-region genes were cloned into a mouse gamma-1 heavy chain expression vector pMmgl FL, which contains DNA encoding the mouse gamma-1 heavy chain constant region. The rat V-region genes of antibody 19439 were also cloned into mouse antibody expression vectors. Co-transfection of the resulting heavy and light chain vectors into CHOS-XE suspension cells gave expression of the murinized and chimeric recombinant antibodies in the mouse lgG1 format. The recombinant Abs were assessed for their binding affinity for mouse IL-11 relative to the parent antibody by surface plasmon resonance: all four combinations of murinized light and heavy chain grafts retained affinity (Table X-5).

Table X-5. Binding affinity of murinized antibody 19439 in comparison to VR19439 as measured by SPR | mL1.1 | mH1.1 | 2.64E+06 | 4.05E-05 | 15.3 |

Example 6: Generation of the IL-11 mouse lgG1 Ab

Two versions of the anti-IL-11 mouse lgG1 antibody were generated. The first being a chimeric mouse lgG1 antibody containing the originally discovered 19439 rat variable region and the latter being a murinized 19439 mlgG1 antibody.

Heavy and light chain vectors encoding the chimeric mouse IgG 1 were transfected into CHO- SXE suspension cells using ExpiFectamine™ CHO transfection reagent (ThermoFisher Scientific). Following transfection, cells were cultured in ExpiCHO™ expression medium at 32°C for 7 days in shaken culture. Cultures were harvested and clarified by centrifugation followed by 0.22|jm filtration to recover the cell culture supernatant containing expressed antibody. Expression titre was determined by HiTrap™ Protein G HP (Cytiva) quantification HPLC (Agilent) assay prior to affinity capture chromatography using a MabSelect™ SuRe™ column (Cytiva) and AKTA Pure™ 25L chromatography system (Cytiva). The chimeric antibody was captured onto the column under mildly basic conditions (pH8.6) and strong Sodium Chloride concentration (4M). Elution was achieved using a 0.1M Sodium Acetate buffer, pH4.1 followed by direct neutralisation to pH5.0-6.0 with Tris-HCI solution. Peak fractions were pooled, sterile filtered and concentration was determined by A280 measurement (Nanodrop™ 2000). The purified chimeric antibody was concentrated prior to loading onto a HiLoad® Superdex® 200 prep, grade column (Cytiva) to remove residual high and low molecular weight product related impurities and to buffer exchange the product into Phosphate Buffered Saline, pH7.4 formulation buffer. The final product was concentrated using a 30KDa MWCO membrane, final concentration was determined by A280 (Nanodrop™ 2000), monomer content was determined by analytical size exclusion HPLC, correct banding pattern was determined by SDS-PAGE, endotoxin level was determined using the Charles River Endosafe® LAL Reagent Cartridge technology, and intact mass and expected post translational modifications were confirmed by mass spectrometry.

Heavy and light chain vectors encoding the anti-IL-11 murinized 19439 mlgG1 antibody were transfected into CHO-SXE suspension cells by electroporation. Following electroporation, transfected cells were cultured in enriched PROCHO™ 5 medium (Lonza) at 32°C for 14 days in shaken culture. Cultures were harvested and clarified by centrifugation followed by 0.22|jm filtration to recover the cell culture supernatant containing murinized antibody. Expression titre was determined by HiTrap™ Protein G HP (Cytiva) quantification HPLC (Agilent) assay prior to affinity capture chromatography using a PROchievA™ column (VWR) and AKTA Pure™ 25L chromatography system (Cytiva). The murinized antibody was captured onto the column under manufacturer recommended conditions and was eluted using a 0.1M Sodium Acetate buffer, pH3.8 followed by direct neutralisation to pH7.0-7.5 with Tris-HCI solution. Peak fractions were pooled, sterile filtered and concentration was determined by A280 measurement (Nanodrop™ 2000). The purified murinized antibody was concentrated prior to loading onto a HiLoad® Superdex® 200 prep, grade column (Cytiva) to remove residual high and low molecular weight product related impurities and to buffer exchange the product into Phosphate Buffered Saline, pH7.4 formulation buffer. The final product was concentrated using a 30KDa MWCO membrane, final concentration was determined by A280 (Nanodrop™ 2000), monomer content was determined by analytical size exclusion HPLC, correct banding pattern was determined by SDS-PAGE, endotoxin level was determined using the Charles River Endosafe® LAL Reagent Cartridge technology, and intact mass and expected post translational modifications were confirmed by mass spectrometry.

Example 7: Generation of anti-IL-11 mAb 19439gL1gH1 lgG1 LALA

The heavy chain variable region gene for humanized 19439gH1 was cloned into a gamma-1 heavy chain expression vector pMhyl LALA, which contains DNA encoding the human gamma-1 heavy chain constant region (G1m17, 1 allotype) with additional Fey receptor binding inactivating mutations L234A and L235A (Tamm A & Schmidt RE (1997) IgG Binding Sites on Human Fey Receptors, International Reviews of Immunology, 16:1-2, 57-85). The light chain variable region gene for humanized 19439gL1 was cloned into a kappa light chain expression vector pMhCK, which contains DNA encoding the human Kappa chain constant region (Km3 allotype). The resulting heavy and light chain vectors were co-transfected into CHO-SXE suspension cells using ExpiFectamine TM CHO transfection reagent (A29130, ThermoFisher Scientific), to achieve expression of the humanized, recombinant lgG1 LALA antibody. Purification of the antibody was by Protein A affinity chromatography (MabSelect™ Sure™), as described for the murinized antibody in Example 6, except that binding buffer was PBS, pH7.4 and elution buffer was 0.1M Sodium Citrate, pH3.4. Prior to Size Exclusion Chromatography the affinity capture pool was neutralised with Tris-HCI solution to pH7.0-7.5.

Example 8: Binding kinetics of 19439gL1gH1 to human and cynomolgus monkey IL-11

The kinetics of 19439gL1gH1 Fab or 19439gL1gH1 IgG LALA binding to human and cynomolgus monkey IL-11 were measured at 25°C by surface plasmon resonance on a Biacore T200 or Biacore 8K+ instrument (Cytiva). A goat anti-human F(ab’)2 specific F(ab’)2 fragment (Jackson ImmunoResearch) was immobilised on a CM5 sensorchip to a level of approximately 5000 RU. Each analysis cycle consisted of capture of approximately 250RU of 19439 gL1gH1 Fab or 19439 gL1gH1 IgG LALA to the anti F(ab’)2 surface, injection of analyte for 180 or 200s (at 25°C at a flow rate of 30pl/min or 50pl/min), dissociation of the analyte for 1800s and finally surface regeneration (with a 60 s injection of 50 mM HCI, a 30 s injection of 5 mM NaOH, and a further 60 s injection of 50 mM HCI). HBS-EP+ was used as running buffer and analyte diluent. Analyte concentrations varied between experiments as outlined below. The binding response of the reference flow cell was subtracted from that of the active flow cell and buffer blank injections were included to subtract instrument noise and drift.

Kinetic parameters were determined using a 1 :1 binding model using Biacore Insight Evaluation software (versions 4.0 and 5.0) and Biacore T200 Evaluation software (version 3.0) as appropriate. Data were analysed from an analyte concentration range of 50 to 0.4nM (5- fold dilutions), 50 to 0.2nM (4-fold dilutions) or 20 to 0.08nM (3-fold dilutions).

As summarised in Table X-6, 19439gL1gH1 Fab and 19439gL1gH1 IgG LALA were shown to bind with high affinity to human IL-11 (KD of 14.5pM, and 12.6pM respectively), and to cross react with cynomolgus monkey IL-11 (KD of 31.8pM, and 23.4pM respectively).

Table X-6. Binding affinity of 19439gL1gH1 Fab and 19439gL1gH1 IgG LALA

The kinetics of anti-IL-11 reference mAb1 binding to human, and cynomolgus monkey IL-11 were measured at 25°C by surface plasmon resonance on a Biacore T200 instrument (Cytiva). A goat anti-mouse Fc specific F(ab’)2 fragment (Jackson ImmunoResearch) was immobilised on a CM5 sensorchip to a level of approximately 7000 RU. Each analysis cycle consisted of capture of approximately 150RU of sample to the anti-Fc surface, injection of analyte for 180s (at 25°C at a flow rate of 30p/min), dissociation of the analyte for 300s and finally surface regeneration (with a 60 s injection of 50 mM HCI, a 30 s injection of 5 mM NaOH, and a further 60 s injection of 50 mM HCI). HBS-EP+ was used as running buffer and analyte diluent. Human IL-11 was titrated from 66.6 to 0.8nM and cynomolgus monkey IL-11 was titrated from 200 to 0.8nM (3-fold dilutions). The binding response of the reference flow cell was subtracted from that of the active flow cell and buffer blank injections were included to subtract instrument noise and drift.

Kinetic parameters were determined using a 1 :1 binding model using Biacore T200 Evaluation software (version 3.0).

As summarised in Table X-7, anti-IL-11 reference mAb1 showed a substantial drop in affinity for cynomolgus monkey IL-11 (263.5nM) compared to human IL-11 (3.7nM). Table X-7. Binding affinity of anti-IL-11 reference mAb1

Example 9: Blocking of the IL-11 :slL-11 R interaction by VR19439

SPR was used to demonstrate that VR19439, when bound to IL-11 blocks its interaction with IL-11 Ra. A chimeric Fab which comprises the parental rat VR19439 with mouse constant regions was used during the experiment. Experiments were conducted on a Biacore T200 (Cytiva) at 25°C, by immobilising a goat anti-mouse F(ab’)2 specific F(ab’)2 fragment (Jackson ImmunoResearch) on flow cell 2 and flow cell 4 of a CM5 sensorchip to a level of approximately 6000 RU. Flow cells 1 and 3 remained blank and were used for reference subtraction for flow cells 2 and 4 respectively. Approximately 150 to 200RU of Fabs were then captured to the anti-mouse surface on either flow cell 2 or flow cell 4 by injecting each sample over the relevant flow cell for 1 min at lO l/min. Analysis consisted of injection of 5nM IL-11 or buffer blank for 180s, followed by 50nM IL-11 Ra (R&D Systems) or buffer blank for 180s at 30 pl/min using the dual injection function over all four flow cells. Using BiaEvaluation Software 3.0, the binding response of each sample was determined after subtraction of the relevant reference surface response and the buffer blanks. As summarised in Table X-8, IL-11 Ra was unable to bind IL-11 in the presence of VR19439 Fab but bound (with responses ranging from 50.9 to 64.9RU) in the presence of non-blocking control Fabs VR19782, VR19783 and VR19175.

Table X-8. Binding of IL-11 Ra to IL-11 in the presence of anti-IL-11 Fabs

Example 10: Confirmation of 19439gL1gH1 Fab non-binding to cell surface, thus indicating non-internalisation properties

ExpiHek cells were transiently transfected with IL-11 R, IL-11 R + gp130 or mock (PBS) at 1 g each using ExpiFectamine™ protocol (Thermo). Cells expressing IL-11 R and gp130 were harvested after 24hr incubation, and IL-11 APC binding was confirmed by flow cytometry on BD FACS Canto. Binding assay was performed with 1 :1 molar ratio of Antibody to IL-11 (IL-11 @ 100ng/ml -5.18 nM, Fab fragments 5.18 nM = 259 ng/ml final concentrations). Transfected cells were resuspended in cell staining buffer containing BSA and NaN₃ (Biolegend®) and chilled on ice before use. 19439gL1gH1 Fab, 19882gL1gH1 Fab and human F(ab)2 control were prebound with unlabelled IL-11 (or buffer alone) at 1 :1 Molar ratio in cell staining buffer for 1 hr on ice. Cells and antibody : IL-11 mixes were then combined and incubated on ice for 30mins. Cells were washed with PBS and stained with Gt anti Human F(ab)2 fragment AF647 (Jackson®) diluted in cell staining buffer for 30mins. Cells were washed for a final time in PBS and resuspended with PBS + DAPI and read using the BD FACS Canto. Anti IL-11 RA (MAB1977) + Gt anti Mouse IgG F(ab)2 AF647 (Jackson®), anti gp130 APC were included in the experiment to confirm receptor expression in the assay. IL-11 labelled with AF647 was used to confirm cytokine binding to the receptor. Analysis was performed using FlowJo™10 with Geometric Mean values plotted using GraphPad Prism 9.2.0.

The results are depicted in Figure 7.

19439gL1gH1 Fab does not bind to IL-11 R or IL-11 RA+ gp130 expressing cells in the presence of IL- 11 indicating non internalization properties. 19882gL1gH1 Fab (with nonblocking VR) does bind in the presence of IL-11.

Example 11 : Inhibition of human IL-11 induced cis-STAT3 signaling by 19439gL1gH1 Fab, reference mAb1 and derived Fab fragment using a human HepG2 IL-11 R/STAT3 reporter cell line

Antibody functional activity was assessed by the ability of antibodies to inhibit IL-11 induced cis-signaling in a PathHunter® Hepg2 IL-11 R STAT3 signaling pathway reporter cell line. These cells stably express human IL-11 RA as well as a synthetic DNA reporter construct, comprised of a STAT3 transcription factor response element that drives expression of ePL- tagged reporter protein. The addition of IL-11 activated the signaling pathway, and this activated signaling pathway induced expression of the ePL-tagged reporter protein, which was measured by addition of the detection reagent containing EA, resulting in complementation of the two enzyme fragments, and production of an active enzyme that hydrolysed the substrate and generated a chemiluminescent signal. A reduction in the luminescent signal demonstrates the functional activity of the tested antibodies in this assay. The assay is described in more detail below.

PathHunter® Hepg2 IL-11 R STAT3 signaling pathway reporter cells (Eurofins DiscoverX #93- 11680044) were cultured in AssayComplete™ thawing reagent (Eurofins DiscoverX #92- 4103TR) using standard tissue culture techniques. Three days before assay set up, 2 x 10⁶ cells were seeded into 30ml of AssayComplete™ thawing reagent in a T175 tissue-culture treated flask, placed flat in the incubator. On the day of the assay, the AssayComplete™ thawing reagent was removed from the flask and the cells were washed with Dulbecco’s phosphate buffered saline (DPBS). The DPBS was removed and 5ml of AssayComplete™ cell detachment reagent (Eurofins DiscoverX #92-0009) was added to the cells. The cells were transferred to a 37°C I 5% CO2 incubator for 10 minutes to allow for the cells to detach from the flask. Subsequently, 10ml of AssayComplete™ Cell Plating 5 (CP5) reagent (Eurofins DiscoverX #93-0563R5A) was added to the cells, and the contents of the flask were transferred to a 50ml falcon tube. The falcon tube was then centrifuged at 150 x g for 5 minutes, the supernatant was discarded, and the cell pellet was resuspended in 10ml of fresh CP5 and counted. Cells were resuspended at 0.625x10⁵ cells/ml by adding cell suspension to CP5, and 80pl/well was added to the assay plates (Corning #3917). Antibodies were serially diluted in CP5 in a 96-well dilution plate (Thermo Scientific, Nunc #249946). The serial dilution of antibodies was then transferred to another 96-well dilution plate (Thermo Scientific, Nunc #249946) containing recombinant human IL-11 (in-house material). The antibody titration/IL- 11 mixture-containing dilution plate was incubated in a 37°C 15% CO2 incubator for 30 minutes. After the incubation, the antibody titration/l L- 11 mixture was transferred from the dilution plate to the assay plates containing cells, to an assay final concentration of 10ng/ml (520pM) IL-11. The plate controls (no antibody added) included IL-11 alone and CP5 alone, as assay maximum and minimum values, respectively. The assay plates were incubated in a 37°C 15% CO2 incubator for 24 hours ± 2 hours. Following this incubation, the level of STAT3 activation was assessed using the PathHunter® ProLabel®/ProLink® Detection kit (Eurofins DiscoverX #93-0812) according to the manufacturer's instructions. Luminescence was then measured using the PHERAstar FSX plate reader and the raw luminescence values were used to determine the relative percentage inhibition as compared to the control wells. 4PL curve fitting and the calculation of IC50 values was performed using Activity Base 9.4 or GraphPad Prism 7.0.

The results are summarised in Table X-9 and Figure 8.

19439gL1gH1 Fab was found to be a potent and efficacious inhibitor of human IL-11 cis signaling in this assay. Reference mAb1 and derived Fab fragment had only weak activity in the assay and in the concentration range it was assessed at and neither an accurate IC50 or Emax could be reported. Table X-9 Summary of potency and efficacy values for VR 19439gl_1gH1 Fab, reference mAb1 and derived Fab fragment against the human IL-11 induced cis-signaling in the Hepg2 IL-11 R reporter cell assay. ND = Not determined.

Example 12: Inhibition of IL-11 mediated trans-STAT3 signaling by 19439gL1gH1 Fab, reference mAb1 and derived Fab fragment.

For the evaluation of IL-11 trans-signaling inhibition by antibodies, primary human dermal fibroblasts (HDF) with IL-11 RA stably knocked out using CRISPR/Cas9 were used. Successful knockout of IL-11 RA was confirmed on the sequence level as well as on the functional level. Knockout of IL-11 RA on the functional level was confirmed by showing that IL-11 alone did not result in an increase in phospho-STAT3 compared to cells treated with media alone, whereas the complex of IL-11 with soluble IL-11 RA was able to increase phospho-STAT3 levels relative to media alone. In the same HDF without IL-11 RA knocked out, IL-11 alone was able to increase the level of phospho-STAT3 independently of soluble IL-11 RA. To activate STAT3 trans-signaling, soluble IL-11 RA complexed with IL-11 was added to the IL-11 RA knockout cells. The level of phospho-STAT3 was assessed using the Phospho-STAT3 (Tyr705) cellular kit (Perkin Elmer Cisbio #62AT3PEH). After lysis of the cell membranes, phospho-STAT3 (Tyr705) levels were measured (proportional to the Fluorescence Resonance Energy Transfer (FRET) fluorescent signal obtained). A reduction in the FRET signal demonstrates the functional activity of the tested antibodies. The assay is described in more detail below.

Adult human dermal fibroblasts (HDF) with IL-11 RA knocked out (KO) using CRISPR/Cas9 were used for these experiments, knock out cells were made using standard methods (genetic modification was performed in-house, unmodified primary cells sourced from Promocell #012302, lot #472Z001.3). These IL-11 RA KO HDF were cultured in growth media, consisting of Fibroblast growth medium 2 supplemented with the contents of the Growth medium 2 kit (Promocell #023120), using standard tissue culture techniques. Six days before assay set up, 0.5 x 10⁶ cells were seeded into 25ml of growth media and transferred to a T175 flask, placed flat in the incubator. On the day of the assay, the growth media was removed from the flask and the cells were washed with DPBS. The DPBS was removed and 5ml of TrypLE Express enzyme (ThermoFisher Scientific #12604021) was added to the cells. The cells were transferred to a 37°C 15% CO2 incubator for 5 minutes to allow for the cells to detach from the flask. Subsequently, approximately 10ml of growth media was added to the cells, and the contents of the flask were transferred to a 50ml falcon tube. The falcon tube was then centrifuged at 300 x g for 5 minutes, the supernatant was discarded, and the cell pellet was resuspended in ~5ml of fresh growth medium and counted. Cells were then resuspended at 0.6x10⁶ cells/ml by adding cell suspension to growth media, and 100pl/well was added to the assay plates (Corning #353072). The assay plates were then incubated for 16 hours ± 2 hours in a 37°C I 5% CO2 incubator. After this incubation, the growth media was carefully removed from the assay plate and 80pl/well of serum free media media (Fibroblast growth medium 2 without the contents of the Growth medium 2 kit added, Promocell #C23120) was added to the assay plates. The assay plates were then returned to a 37°C I 5% CO2 incubator for 6-8 hours. Antibodies were serially diluted in serum free media in a 96-well dilution plate (Thermo Scientific, Nunc #249946).

Next, the antibodies were assessed for their ability to block trans-signaling when first preincubated with IL-11 before the addition of soluble IL-11 RA. The serial dilution of antibodies was transferred to 96-well dilution plates containing recombinant human IL-11 (in-house material) and incubated at 37°C 15% CO2for 30 minutes. Next, the antibody/IL-11 mixture was transferred to another dilution plate containing soluble IL-11 RA and incubated for 60 minutes at 37°C 1 5% CO2. Next, the antibody titration/l L-11/IL-11 RA mixture was transferred from the dilution plate to the assay plates containing cells, resulting in an assay final concentration of 10ng/ml (520pM) IL-11 and 60ng/ml (1560pM) IL-11 RA. The plate controls (no antibody added) included I L-11/1 L-11 RA complex and serum free media alone, as assay maximum and minimum values, respectively. The assay plates were incubated in a 37°C 15% CO2 incubator for 30 minutes ± 5 minutes. Following this incubation, the assay plates were quickly inverted to remove the liquid contents of the wells, and 50pl/well lysis buffer (Perkin Elmer Cisbio, Phospho-STAT3 (Tyr705) cellular kit #62AT3PEH) was immediately added. The assay plates were incubated for 30 minutes at RT, then 16pl/well of cell lysate was transferred from the assay plates to a 384-well HTRF plate (Corning #784075). Next, the antibodies from the Phospho-STAT3 (Tyr705) cellular kit were diluted in detection buffer. For one 384-well HTRF plate, 50 l of the Eu-cryptate antibody was mixed with 50 pl of the d2 antibody and diluted with 1900pl of detection buffer. 4pl/well of this antibody mixture was added to the 384-well HTRF plate. The 384-well HTRF plate was sealed with a foil plate seal and incubated overnight at RT. The following day, the plate was read on a Synergy Neo 2 as per the manufacturer’s instructions measuring the fluorescence at reads of 330/620nm and 330/665nm. The ratio values were then calculated using the following equation: (330/665nm divided by 330/620nm) X 10,000 and used to determine the relative percentage inhibition as compared to the control wells, using Microsoft Excel. 4PL curve fitting and calculation of IC50 values was performed using Graphpad Prism® 7.0.

The results of these experiments are summarised in Table X-10 and Figure 9.

19439gL1gH1 Fab was found to be a potent and efficacious inhibitor of IL-11 trans-signaling. Reference mAb1 and derived Fab fragment were also found to inhibit in this assay but they were not as potent as 19439gL1gH1 Fab and an accurate IC50 and Emax could not be determined in the concentration range they were assessed at.

Table X-10 Summary of potency and efficacy values for 19439gl_1gH1 Fab, reference mAb1 and derived Fab fragment in the STAT3 trans-signaling assay. ND = Not determined.

Example 13: Inhibition of IL-11 and IL-17AA mediated CXCL1 release by 19439gL1gH1 Fab, and Fab derived from reference mAb1 using primary human dermal fibroblasts

Another primary HDF assay looked at the functional activity of antibodies to inhibit CXCL1 release in response to IL- 11 and IL-17A stimulation. CXCL1 release in response to IL- 11 or IL-17A alone was low, however, together a synergistic effect was observed. HDFs were stimulated with IL-11 in combination with IL-17A. The resultant CXCL1 response was then measured using a CXCLI/GRO-a kit. A reduction in CXCL1 levels (as determined by ECL signal) demonstrates the functional activity of the tested antibodies. The assay is described in more detail below.

Primary human dermal fibroblasts (HDF, Promocell #C-12302, lot#469Z026.2) were defrosted from cryovials using standard techniques, resuspended in growth media (basal media with the contents of the growth medium 2 kit added - Promocell #C-23120), centrifuged for 5 minutes at 400 x g and resuspended at approximately 0.015 x 10⁶ in growth media. They were then seeded at approximately 3x10³ cells/well by adding 200pl cells/well into 96-well tissue culture plates. The plates were incubated at 37°C I 5% CO₂ for 96 hours. After the incubation, the growth media was aspirated, and the media was changed to basal media in an 80pl/well volume. Antibodies were diluted with basal media to 10X assay final concentration and added in a 90pl/well volume to column 3 of a 96-well plate. Basal media alone was also added to wells A3 and H3 in a 90pl/well volume. Next, 60pl/well of basal media was added to all other columns of the 96-well plate (excluding column 3) and an 8-point titration with 1 in 3 dilutions was run, transferring 30pl between columns.

Another 96-well plate was then prepared containing human IL-11 and human IL-17AA diluted to 10X assay final concentration (160pM IL-11 and 2780pM IL-17AA - for an assay final concentration of 16pM IL-11 and 278pM IL-17AA). The solution was prepared by diluting the stocks of human IL-11 and human IL-17AA in basal media, separately, and then combining them in equal volumes. The IL-17AA solution was also diluted in an equal volume of media for the ‘IL-17AA’ alone condition. Then, 40pl/well of these solutions were added to a 96-well plate. 40pl/well was then transferred from the antibody titration plate into the plate containing IL- 11/IL-17AA and this plate containing IL-11/IL-17AA/antibody titration was incubated at 37°C I 5% CO2 for 30 minutes. After the incubation, 20pl was transferred from the IL-11/IL- 17AA/antibody titration containing plate into the 96-well plate containing the cells. The cell plates were then further incubated at 37°C 15% CO2 for 48 hours. After this incubation, CXCL1 levels were measured in the HDF supernatants using the ll-PLEX Human Gro-a assay kit from MSD (MSD #K151 UXK-2). The MSD plates were read and a reduction in CXCL1 levels (as determined by ECL signal) demonstrated the functional activity of the tested antibodies. Percentage inhibition values were calculated using Microsoft Excel and 4PL curve fitting was performed using GraphPad Prism.

The activity of 19439gL1gH1 Fab and a Fab derived from reference mAb1 were measured in this assay.

19439gL1gH1 Fab was a potent and efficacious inhibitor of IL-11/IL-17 mediated CXCL1 release on primary HDF. The Fab fragment derived from reference mAb1 was also assessed in this assay and was found to inhibit, however, it was less potent and efficacious when compared to 19439gL1gH1 Fab (Table X-11 , Figure 10).

Table X-11 Summary of potency and efficacy values for 19439gL1gH1 Fab and Fab derived from reference mAb1 in the cis human IL-11 and IL-17AA CXCL1 release assay.

Example 14: Assessment of IL-11 R and gp130 expression in human and cynomolgus primary fibroblasts

Primary Normal Human Dermal Fibroblasts (PromoCell) and Primary Cynomolgus Dermal Fibroblasts (Primacyt CF063-B- 190805) were used to assess expression of IL-11 R and gp130 using qPCR. Stock vials were thawed at 37°C and 200K cells were pelleted in an Eppendorf and 600ul RLT buffer was added to lyse cells. RNA was extracted using RNeasy mini prep plus (Qiagen) and eluted in 40ul RNase free water. RNA was quantified using the Nanodrop. A standardised ng of RNA was used to generate cDNA with SuperScript™ IV VI LO™ Master Mix (Thermo). qPCR was performed using Quant Studio (Thermo) with TaqMan™ Fast Advanced Master Mix (Thermo) and the following species specific Taqman primers : HUMAN Hs00234415_m1 IL-11RA, Hs00174360_m1 IL6ST, Hs02786624_g1GAPDH, CYNO Mf02854633_g1 IL-11 RA, Mf02787830_m1 IL6ST, and Mf04392546_g1 GAPDH.

AACT method was used to calculate fold change relative to GAPDH.

The expression profiles are depicted in Figure 11A and Figure 11 B.

Example 15: CCL2, IL-6 and MMP2 inhibition by 19439gL1gH1 / null KiH hlgG1 LALA in cis using primary human dermal fibroblasts following rhlL-11, rhlL-17AA and rhlL-17FF stimulations

19439 gL1gH1 was produced as a KiH antibody, i.e. 19439gL1gH1 / 18136 (null) KiH hlgG1 LALA, with the 19439 gL1gH1 Fab on the Knob chain and a VH/VL pair which binds an irrelevant antigen (i.e. 19136 (null)) on the Hole chain. Antibody 19439gL1gH1 1 18136 (null) KiH hlgG1 LALA alongside an isotype control lgG1 LALA (null/null 18136 KiH lgG1 LALA) were tested in an in vitro cell assay against activity of human recombinant IL-11 , IL-17AA and IL-17FF (in house proteins, IL-11 , IL-17AA and IL-17FF). The primary human dermal fibroblasts were ethically sourced from different donors (HDF, Promocell #0-12302, lot #472Z001.3, 469Z015, 469Z026.2) and cells were expanded in culture for the assay. HDF cells respond to IL-11 stimulation and IL-17AA/FF stimulation by secretion of proinflammatory soluble molecules such as CCL-2 or IL-6 and upregulation of matrix metalloproteinases such as MMP2 that can play role in ECM degradation and remodeling. CCL-2, IL-6 and MMP2 levels in cell supernatants have been used in the assay to assess the activity of 19439gL1gH1 I 18136 (null) KiH hlgG1 LALA.

HDF cells were quickly defrosted in a water bath until a small pellet remained and added into a 15 ml Falcon containing 10 ml of pre-warmed growth media (Promocell, #C-23120). Cells were centrifuged for 5 min at 400 x g, supernatant was removed, and cells were resuspended in 1 ml growth media. Cells were counted, transferred to 50ml Falcon tube, and resuspended at approximately 0.025 x 10⁶ cells/ml. Cells were seeded at approximately 5x10³ cells per well by adding 200pl cells in growth media per well into 96-well culture plates (Corning, #353072). Seeding of cells resulted in passage 4, 5th culture. Cells were incubated at standard conditions (37°C, 5% CO2, 100% humidity) until they reached confluence on day 4, then media was aspirated, and plates were washed with 200pl pre-warmed (37°C) fibroblast cell basal media (Fibroblast growth medium 2 without growth kit, # C-23120). IL-11 and IL-17AA cytokines for stimulation were prepared at 40ng/ml and IL-17FF at 400ng/ml, and antibodies were prepared at 40|jg/ml in basal media. Antibodies, cytokines, and basal media were combined in a plate for the final 1 :4 dilution and incubated at 37°C for 30 minutes, then 200pl of stimulation was added per well to confluent cells. After 2 days (48hrs) supernatants were collected and stored at -20°C for further analysis. CCL-2 and IL-6 levels were measured in the HDF supernatants using the ll-PLEX Custom Biomarker (NHP) assay (MSD, #K15068M-2). The MSD plates were read and analysed on an MSD instrument. MMP2 levels were measured using human MMP2 kit (Cisbio, cat #62MMP2PEG) and read using a HTRF compatible plate reader. CCL- 2, IL-6 and MMP2 levels were plotted using GraphPad Prism and percentage inhibition of CCL- 2, IL-6 and MMP2 levels compare to the relevant isotype control were calculated using Microsoft Excel.

The results are summarised in Figure 12 (CCL2), Figure 13 (IL-6) and Figure 14 (MMP2) and Table X-12.

19439gL1gH1 I null KiH hlgG1 LALA showed efficacious inhibition of IL-11 mediated CCL-2 and IL-6 release and IL-11 + IL-17AA/FF mediated CCL-2, IL-6 and MMP2 release in the in vitro primary HDF assay.

Table X-12 Mean percentage inhibition of CCL2, IL-6 and MMP2 secretion from dermal fibroblasts compared to the relevant Isotype control group.

Example 16: IL-11 drives a distinct functional signature in different types of dermal cells

The primary adult human dermal fibroblasts (HDF, Promocell #C-12302) and adult normal human follicle dermal papilla cells (HFDPC, Promocell #C-12302) were ethically sourced from different donors (n=3) and cells were expanded in culture for the assay. HDF and HFDPC cells were quickly defrosted in a water bath until a small pellet remained and added into a 15 ml Falcon containing 10 ml of pre-warmed growth media (Promocell, #C-23120 and C-26500 respectively). Cells were centrifuged for 5min at 400 x g, supernatant was removed, and cells were resuspended in 1ml growth media. Cells were counted, transferred to 50ml Falcon tube, and resuspended at approximately 0.05 x 10⁶ cells/ml. Cells were seeded at approximately 10⁵ cells per well by adding 2ml cells in growth media per well into 6-well culture plates (Corning,# 3516). Seeding of cells resulted in passage 5, 6th culture. Cells were incubated at standard conditions (37°C, 5% CO2, 100% humidity) until they reached confluence on day 4, then media was aspirated, and plates were washed with 1ml pre-warmed (37°C) cell basal media without growth kits. HDF were kept for 3hrs in basal media before stimulation. Cells were stimulated with 100ng/ml of rhlL-11 in basal media for 24hrs. Media was aspirated and 600pL of RLT buffer (Qiagen, cat#79216) was added to the wells containing cells and the plates were frozen and stored at -80°C until RNA was extracted. Total RNA was extracted from the buffer RLT treated cells using RNeasy Mini Kit (Qiagen, cat#74136) according to manufacturer instructions. The transcriptome was sequence in samples using the NovaSeq6000_150 at the Oxford Genomics Centre. Briefly the total RNA was converted to cDNA, the cDNA was end-repaired, A-tailed and adapter-ligated. Samples were uridine digested prior to amplification. The prepared libraries were size selected, multiplexed and QC’ed before paired end sequencing over four units of a flow cell. Data was aligned to the reference and quality checked. Obtained raw data was of high quality with over 90% alignment rates and homogeneous libraries on key metric (e.g fragment lengths). Fastq files obtained from the experiment were quantified using the pseudo-aligner Salmon (v1.8) against GENCODE (v38). Quantifications were quality assessed before being read into R (v4.1) using the tximport package (v1.28.0) to the logCPM metric as recommended by the authors. Imported quantifications were input into the Limma framework for linear modelling (v3.56.2) where design matrix I contrast matrix were constructed prior to model fit with empirical Bayes moderation.

IL-11 stimulations were tested within each cell type relative to respective controls, showing that responses were only measurable in dermal papilla and fibroblasts. All differential expression results were tested against functional genesets (MSigDB) using the mitch framework (v1.14), with a geneset wise, and sample wise FDR threhold of 0.01. Significant genesets were summarised based on a tree cutoff (k=6) into common biological themes, and effect sizes averaged (mean). This analysis suggested pro-inflammatory genesets were enriched upon IL-11 stimulation in fibroblasts, whilst genesets regulating cell cycle and proliferation were found enriched in dermal papilla cells. The results are summarised in Figure 15.

Example 17: Transcriptomic analysis of HS skin samples at baseline and after treatment with an antibody that inhibits IL-17A and IL-17F demonstrates an overlap between IL-11 driven biology in dermal cells and lesion-specific pathobiology

Skin biopsy collection, sequencing, and processing RNA sequencing was conducted on 189 skin biopsies taken at baseline and 12 weeks after treatment with Bimekizumab in the phase 2 proof of concept study of Bimekizumab in patients with moderate to severe HS (NCT03248531). For full details on the phase 2, double-blind, placebo-controlled randomized clinical trial, see Glatt et al 2021. At baseline, 6 mm punch skin biopsies were taken from HS lesions and paired with non-lesional skin samples. Additional lesional skin biopsies were taken after 12 weeks of Bimekizumab treatment (treatment group: 640 mg at Week 0, then 320 mg every 2 weeks).

Biopsies were immersed in RNALater solution and frozen in preparation for gene expression profiling. The skin biopsies were disrupted with TissueLyser and QIAzol (Qiagen), and total RNA was extracted with the RNeasy Micro Kit (Qiagen), using reagents from the same manufacturing batch. The RNA samples were checked for purity (260/280 nm ratio, DropSense96, Trinean), quantified and checked for integrity using the Fragment Analyzer (Advanced Analytical). cDNA libraries were prepared with SENSE mRNA Seq (Lexogen) for the Illumina platform, with a spike-in control. The libraries were quality-checked with capillary electrophoresis (Fragment Analyzer, Advanced Analytical) and quantified with Picogreen (ThermoFisher Scientific). Next, the libraries were normalised and pooled before NextSeq500 sequencing (10-12 samples/run), which was performed in 20 runs using 2 x 75 base pairs (bp) high output.

Each pair of FASTQ files was checked to ensure orphaned reads were removed and that partnered reads were correctly ordered. Prior to quantification, reads were trimmed and filtered. Reads were filtered first using an entropy filter, and then trimmed at both ends using a quality threshold of 20 bases. In addition, k-mer filtering of reads was performed using k- mers of 20 bases from well-known contaminants. Finally, any read with <31 bp remaining was filtered from the dataset. Sample quality was assessed using FastQC.

Reads were quantified using Salmon vO.11.3 using a guanine-cytosine (GC) content bias correction and library autodetection. The human reference was taken from GENCODE v29.0.

Transcript abundances from quantification were imported using the tximport package in R v4.0.2 and normalised by library size to generate a counts per million (CPM) matrix at the gene level. Genes with <10 counts in each sample were removed. Data were transformed using their mean variance trend using linear models for microarray data (limma) voom.

Weighted co-expression network analysis to identify disease specific transcriptionally coregulated genes modules.

Weighted gene co-expression network analysis (WGCNA) was applied to the 64 baseline lesional samples using the WGCNA package in R. Mean absolute deviation (MAD) of gene expression values was used to filter out the 25% least variable genes. Ward’s linkage was used as the agglomeration method within a hierarchical clustering approach to module identification, and a soft power of 6 was used. Genes placed into the module with the lowest average correlation were further eliminated from downstream analysis. Each module was further split into a maximum of two modules using the direction of pairwise correlations between member genes. Overall, 9798 genes were assigned to 47 modules.

Modules were characterized by cell type and function using functional enrichment tests. Hypergeometric tests were used to test for significant overlaps between module genes and gene sets belonging to pathway, functional and cell type ontologies.

Differential expression analysis was performed using limma. A linear model was specified with treatment arm, timepoint, response, and sample type as model terms and with patient as a blocking factor to compute moderated paired t-tests for within-patient comparisons. Differentially expressed genes between lesional and nonlesional skin were determined using a false discovery rate (FDR) adjusted p<0.05 and an absolute fold change cut-off of >2. The effect of Bimekizumab treatment on each module was calculated using the percentage improvement of genes differentially expressed at baseline. Percentage improvement (PI) was defined as:

Examining the overlap between disease specific modules and IL-11 dermal biology

Coregulated modules associated with dermal biology were identified as those enriched with fibroblast specific signatures. Signatures were taken from IL-11 stimulated fibroblasts (see Example 16), and Gene Set Enrichment Analysis (GSEA) was used to assess the enrichment of these signatures in each dermal module using the fgsea package in R. Three out of five dermal modules were found to be significantly enriched for genes upregulated in IL-11 stimulated fibroblasts. These modules were only partially normalized under Bimekizumab treatment with percentage improvements no greater than 21 %. In particular, one of these (LS.20.N) was the module with the lowest percentage improvement of -8.2% (see Table X- 13).

Table X-13 - Summary statistics for coregulated modules enriched for genes upregulated in IL-11 stimulated fibroblasts. Correlation refers to the average pairwise pearson correlation of genes within that module.

Example 18: Single-cell sequencing of early lesion HS biopsies shows a prominent, aberrant cell population in HS lesions which is IL-11RA positive

Punch biopsies were taken from 6 HS patients with systemic disease scores of Hurley Stage l/ll, ensuring that patients had established disease. Biopsies were specifically taken from lesions which had formed in <1 week (self-reported by the patient) in order to understand early lesion pathogenesis. An additional biopsy was taken from a non-lesional area in close proximity to the lesion and defined as unaffected skin.

Punch biopsies were processed as follows: each skin biopsy sample was separated into epidermis, upper dermis and lower dermis with subcutaneous tissue, which were then digested separately and FACS sorted (live, CD45+ve and CD45-ve). Sorted cells were processed into single cell cDNA libraries using the 10X Genomics 5’ RNA kit and sequenced on an Illumina NovaSeq 6000.

Raw sequencing data was converted to fastq format, and subsequently quantified using 10X Genomics CellRanger Count tool (v7.1.0). Raw counts in the h5 format were read into R (v4.1) and classed into filled or empty droplets (emptydrops v1.2), where UMI barcodes classed as empty were discarded. Files were merged into a single UMI by gene sparse count matrix and a Seurat object created (v4.4.0). Doublet I Multiplets were identified using the scDblFinder framework (v1.14), and only confident singlets retained. Standard pre-processing steps were followed akin to Seurat vignettes, which briefly includes: log normalisation, highly variable feature identification, scaling, PCA, knn/snn creation, clustering (Louvain), and UMAP.

Dimensionality reductions (PCA/ UMAP) were assessed for variance associated with known variables to determine if corrections of lower dimensional space were necessary.

Cell type I Cell state inference was performed at a cluster level (resolution 1 ,2) and was a multi-step procedure. Firstly, an initial scaffold was formed using the SingleR framework with a custom panel derived from Reynolds et al 2021 , which helped to identify previously characterised cell population. Secondly, marker genes were calculated using the Presto (v1.0) Wilcoxon test framework, and manually curated against unknown clusters. A final consensus set of cluster identities was set through a manual process.

In order to identify global cell states which are specific to HS lesions, the MiloR (v1.99.12) framework was used with default parameters, constructing a model of Lesional vs Non- Lesional cells. Several communities of cells passed a spatial FDR < 0.05, including a set of fibroblasts with some similarity to vascular mural smooth muscle cells, these were a specific cell state to HS lesions, and are hypothesised to be part of the epithelial tunnels, a hallmark of the disease.

In order to identify markers of these lesion-specific fibroblasts, the Presto Wilcoxon test was used to test expanded fibroblasts relative to unchanging fibroblasts, i.e. fibroblasts which appear to have the same cellular phenotype in both lesional and non-lesional tissue as a background. These cells exhibited increased expression of tissue remodeling genes such as collagens, MMPs and fibronectin. Additionally, these cells had a 3.27 fold increase in the proportion of cells expressing IL-11 RA relative to unchanging fibroblasts. Results are summarized in Figure 16.

In addition, a number of genes activated upon IL-11 stimulation of fibroblasts in vitro (see Example 16) and which include SLILF1 , NPC1 , SLCA3, DLISP1 , ICAM1 and HIF1A, were also found to be upregulated in the HS lesional expanded fibroblasts, suggesting these cells are responding to IL-11 in vivo.

Example 19: RNAscope imaging of healthy and HS skin samples show increased IL-11 and IL-11 R expression in HS lesional skin

IL-11 and IL-11 receptor (IL-11 R) distributions in skin tissue were determined by chromogenic In Situ Hybridization (ISH)-based RNAscope assay. RNAscope staining was conducted on Healthy Volunteer or Hidradenitis Suppurativa (HS) patient lesional skin samples. HS lesional samples were further classified as mild, or moderate-severe. The samples derived from surgical excisions, with consent and ethical approval from commercial biobanks (National BioService and Precision for Medicine).

For the Singleplex RNAscope assay, tissue samples were labelled using Leica Bond RX processor, Advanced Cell Diagnostics (ACD) RNAscope® 2.5 LS Reagent Kit-RED (Cat No. 322150), along with Leica Bond Polymer Refine and Refine Red Detection Kits (Cat No. DS9800, DS9390) according to the manufacturer’s instructions. Tissue RNA quality was first assessed by performing RNAscope analysis for the housekeeping gene Homo sapiens ubiquitin C mRNA and background was confirmed by DapB negative probes (Table X-14). To detect single mRNA molecules, formalin-fixed, paraffin-embedded (FFPE) tissue sections (5pm) were cut and mounted on Superfrost Plus Gold slides and allowed to dry overnight at 37°C followed by Leica Bond RX routine factory based “Bake and Dewax” protocol. The slides were placed on the staining rack of the Leica BOND RX instrument without any pre-treatment and baked in position at 60°C and then dewaxed before being rehydrated on board using ethanol before pre-treatments. Heat-induced RNA retrieval was conducted by incubation in Epitope Retrieval Solution 2 (pH9, AR9640 Leica) for 15 min at 95°C, followed by protease treatment (ACD) from the LS Reagent kit for 15 min and peroxidase blocking with two rinses in distilled water between pre-treatments. Probe hybridization and signal amplification was performed according to manufacturer’s instructions. Briefly, 20 ZZ probe pairs targeting the relevant genomic nucleoprotein genes were designed and synthesized by ACD BioTechne. Sections were exposed to ISH target probes (Table X-14) and incubated at 42 °C for 2 hr. After rinsing, the ISH signal was amplified using company-provided Pre-amplifier and Amplifier conjugated to alkaline phosphatase (AP) and incubated with a red substrate-chromogen solution using the Bond Polymer Refine Red Detection Kit (Leica Biosystems, Cat No. DS9390) according to ACD protocol for 10 min at room temperature. Sections were then counterstained with hematoxylin then removed from the Bond RX and were heated at 60 °C for 1 h, washed in xylene and mounted using EcoMount Permanent Mounting Medium (Biocare Medical). The stained slides were imaged with the Olympus VS120 slide scanner using 40X super apochromat objective to create whole slide images for qualitative and quantitative analyses.

Table X-14 List of control and target RNAscope probes (ACD BioTechne)

Images show that both IL-11 and IL-11 R expression were increased in HS lesional skin compared to healthy volunteer skin (Figure 17 and 18).

Example 20: Semiquantitative analysis of HS skin lesional samples show IL-11 and IL- 11R expression is correlated with lesion severity

Analysis of RNAscope signal was performed with QuPath and Python software packages.

Cells were segmented from scanned whole tissue images utilizing haematoxylin counter stain. Signal segmentation was conducted by trained signal classifier to recognize RNAscope signal and annotated to enable heatmap generation for signal density assessment. Cells were then binned into different cell classes according to the area mm² of RNAscope signal they contained per cell. Cells were classed as negative if <0.3 mm², 1+ if >=0.3 mm², 2+ if >=1.2 mm2 and 3+ if >= 3.0 mm2 of RNAscope signal was present within the cell. These thresholds were based on the positive control stain (UBC) RNAscope signal, to distribute cell populations evenly between the three bins.

A summary H-score was generated per image to enable IL-11 and IL-11 RA signal comparison between patients. H-scores were generated with the following equation;

H_score = (percentage ¹⁺ * 1) + (percentage²* * 2) + (percentage³* * 3)

This approach was based on RNAscope quantification guidance issued by ACD BioTechne. Each whole slide image was scored 0-300 based on the percentage presence of each positive cell class as per Equation 1. To correct the tissue quality differences H-scores for IL-11 and IL-11 RA were normalised by subtracting a matched sections negative control H-score (which represents background staining) and then scoring relative to a matched section positive control UBC H-score:

Normalised H-score Statistical comparison was conducted using EasyBayes. For the analysis the moderate and severe cohort groups were pooled and compared to tissue samples with mild grading. We found that the IL-11 and IL-11 RA RNA transcript levels increase as HS pathology progresses from mild to moderate-severe grading normalised to UBC (positive control) and background was subtracted (BapB- negative control).

Results are summarized in Figure 19.

Example 21 : The role of IL-11, both alone and in combination with other cytokines, in promoting the inflammatory cascade in primary dermal fibroblasts

The primary human dermal fibroblasts were ethically sourced from different donors (HDF, Promocell #C-12302) and cells were expanded in culture for the assay. HDF cells were quickly defrosted in a water bath until a small pellet remained and added into a 15 ml Falcon containing 10 ml of pre-warmed growth media (Promocell, #C-23120). Cells were centrifuged for 5 min at 400 x g, supernatant was removed, and cells were resuspended in 1 ml growth media. Cells were counted, transferred to 50ml Falcon tube, and resuspended at approximately 0.025 x 10⁶ cells/ml. Cells were seeded at approximately 5x10³ cells per well by adding 200pl cells in growth media per well into 96-well culture plates (Corning, #353072). Seeding of cells resulted in passage 4, 5th culture. Cells were incubated at standard conditions (37°C, 5% CO2, 100% humidity) until they reached confluence on day 4, then media was aspirated, and plates were washed with 200pl pre-warmed (37°C) fibroblast cell basal media (Fibroblast growth medium 2 without growth kit, # C-23120). The confluent cells were stimulated with single cytokine or combination of cytokines. RhlL-11 and rhlL-17AA were used at 10ng/ml, rhlL-17FF at 100ng/ml, rhTNFa at 1ng/ml and rhl L-1 p at 10pg/ml. After 2 days (48hrs) supernatants were collected and stored at -20°C for further analysis. CXCL-1 levels were measured in the HDF supernatants using the ll-PLEX Custom Biomarker (NHP) assay (MSD, #K15067L-2), plates were read and analysed on an MSD instrument. IL-8 levels were measured using human IL-8 HTRF kit (Cisbio, #62HIL08PET) and read using a HTRF compatible plate reader. CXCL-1 and IL-8 levels were plotted using GraphPad Prism.

The results are summarised in Figure 20. IL-11 synergises with HS-relevant pro-inflammatory cytokines such as IL-17, TNF-a and IL-1 to amplify inflammatory responses by synergistic induction of secretion of a range of chemokines including CXCL-1 (Figure 20A) and IL-8 (Figure 20B). The augmented secretion of proinflammatory cytokines and chemokines from dermal fibroblasts could increase infiltration of immune cells, especially neutrophils, and exacerbate the severe inflammatory process associated with HS lesions.

Example 22: IL-11 synergises with IL-17AA/FF to induce secretion of M MPs in an ex vivo hair follicle organ culture model.

Hair follicle samples from elective surgeries were obtained after informed, written patient consent according to ethics committee approval (University of Muenster and ML Biobank). Experiments and data analysis were carried out at the Monasterium Laboratory, Skin and Hair research Solutions GmbH, Munster, Germany. Microdissected human anagen scalp hair follicles were cultured for 24 hrs at 37°C with 5% CO₂ in a hair follicle optimised media. Media was aspirated and hair follicles were stimulated with rhlL-17AA at 100ng/ml and rhl L- 17FF at 1000ng/ml (referred to as IL-17A/F) or rhlL-17AA at 100ng/ml and rhlL-17FF at 1000ng/ml together with 100ng/ml of rhlL-11 (referred to as IL-17A/F + IL-11) in the hair follicle media. Culture media was collected on day 1 and stored at -80°C until further processing. Samples were centrifuged for 5 min at 13000 rpm using a tabletop centrifuge to get rid of the debris. Samples were analysed using the human MMP and TIMP Discovery Array® for cell culture (Eve Technologies). Measurements were carried out in duplicates and average values were used to plot corresponding graphs using GraphPad 9.0.

The results from two hair follicle donors are summarised in Figure 21. The 24h hair follicles stimulation with IL-17A/F didn’t induce significant secretion of MMPs from the hair follicle cultures. Combining IL-11 and IL-17A/F stimulation increased MMP-1 , MMP-2, MMP-3, MMP- 7, MMP-9 and MMP-10 secretion from the hair follicles from both donors used in the assay. Example 23: IL-11 Ra is expressed by human healthy hair follicle cells and IL-11 can impact hair follicle biology by decreasing follicular keratinocyte proliferation as shown in the ex vivo hair follicle organ culture model.

Hair follicle and skin samples from elective surgeries have been obtained after informed, written patient consent according to ethics committee approval (University of Muenster and ML Biobank). Experiments and data analysis were carried out at the Monasterium Laboratory, Skin and Hair research Solutions GmbH, Munster, Germany.

The IL-11 Ra expression in the dermal and epidermal cells in the scalp skin and the hair follicles was tested with the monoclonal anti-IL-11 Ra antibody (Abeam, #Ab125015). The tissue was cryosectioned and processed for immunofluorescence visualisation of the IL-11 Ra protein on the sections using a HRP tagged secondary antibody and a TSA substrate. For the negative control, the secondary antibody for rabbit IgG was applied without the pre-incubation with the primary antibody. DAPI counterstain was used to visualize the nuclei.

Microdissected human anagen scalp hair follicles were cultured for 24 hrs at 37°C with 5% CO2 in a hair follicle optimised media. Media was aspirated and hair follicles were stimulated with rhlL-17AA at 100ng/ml and rhlL-17FF at 1000ng/ml (referred to as IL-17A/F) or rhlL-17AA at 100ng/ml and rhlL-17FF at 1000ng/ml together with 100ng/ml of rhlL-11 (referred to as IL- 17A/F+IL-11) in the hair follicle media. After 24 hrs hair follicles were collected for the RNAseq analysis. For the immunofluorescent imaging analysis hair follicle cultures were first stimulated with cytokines for 48 hrs, at which point the media was renewed with the same reagents for another 24 hours before the hair follicles were frozen in a cryomatrix for cryosectioning.

RNA was extracted using the Arcturus PicoPure RNA isolation kit (Thermofisher) according to manufacturer’s instructions. Sequencing was performed on a NovaSeq 6000 using 2x100 bp read length with an output of appr. 30 M clusters per sample. Demultiplexing of the sequence reads was performed with Illumina bcl2fastq (2.20). Adapters and Pico v2 SMART adapters were trimmed with Skewer and trimmed raw reads were aligned to a GRCh38.105 using STAR. The quality of the FASTQ files was analysed with FastQC. Normalised counts were calculated with DESeq2 and p values and p adjusted values were generated.

To determine the expression of the Ki-67, Caspase-3 and KRT1 proteins in the hair follicles, 7 pm sections of cryopreserved hair follicles were blocked with 10% goat serum and incubated overnight with the primary antibodies against Ki67 (Cell Signaling), Caspase-3 (cell Signalling) and KRT1 (Progen), followed by appropriate goat-raised fluorescent secondary antibodies. All immunostained sections were imaged with Keyence BZX microscope. Quantification was performed using Imaged software. Percentage of Ki67 expressing cells was calculated based on the total number of DAPI positive cells. The data was plotted in GraphPad 9.0. Data from the experiments is summarised in Figure 22. IL-11 Ra was expressed by epidermis (notably basal layer of epidermis), endothelial cells, some dermal cells and in the hair follicle epithelium and mesenchymal cell in the connective tissue sheath (Figure 22 A).

At the transcriptomic level, treatment of ex vivo hair follicles with IL-11 in combination with IL- 17A/F uniquely decreases WNT3 expression (regulator of Wnt/b-catenin pathway, adjusted p value p = 0.012) and KRT19 expression (regulator of Notch, adjusted p value p=0.003) that couldn’t not be seen with IL-17A/F stimulation alone. Those genes are associated with modulation of hair follicle cycling and a change in their expression could lead to loss of cell polarity and reduced proliferation of affected cells.

In combination with IL-11 , IL-17A/F significantly decrease Ki-67 expressing cells in the distal hair follicle outer root sheet (ORS) (Figure 22 B). Ki-67 downregulation is linked to reduced cellular proliferation, which means the combination reduced the percentage of proliferating cells in the epithelial ORS region of the hair follicle near the epidermis (site of hyperkeratinisation and follicular occlusion). There weren’t many caspase-3 positive cells detected in the hair follicles culture, suggesting that cytokines stimulations didn’t induce cell apoptosis.

Example 24: RNAscope analysis of IL-11 expression in Systemic sclerosis samples vs healthy shows an upregulation of IL-11 in disease

IL-11 RNA distributions were visualized in Systemic sclerosis (SSc) patients’ skin biopsies and normal tissue samples by RNAscope-based ISH. The identical chromogenic RNAscope protocol and ancillary reagents were used as described in Example 19. The tissue quality was first assessed by performing RNAscope analysis for mRNA of the housekeeping genes (Example 19, Table X-14). Tissue sections were processed on Leica® Bond™ RX and treated with routine, factory-based Bake and Dewax protocol before being rehydrated. The RNA retrieval (heat-induced) was conducted by incubation in retrieval buffer ER2 (pH 9) followed by protease treatment and peroxidase blocking. On-target RNA hybridization was carried out using highly selective, complementary RNA probe pairs (Table X-14) targeting the relevant genomic nucleoprotein genes. Multi-step signal amplification and background suppression were achieved by incubation with a fast-red substrate-chromogen solution. The sections were counterstained with hematoxylin, air-dried, xylene washed, and cover slipped with EcoMount (Biocare Medical). Microscopic images were captured by Olympus VS120-L100-W-12 slide scanner using 40X objective. Diseased and control tissue samples were acquired from skin biopsies with consent and ethical approval from commercial tissue depository (National BioService). RNAscope labelling demonstrated increased number of positive cells in the epidermis of the SSc skin, and particularly in areas of profound hyperkeratosis while normal skin expressed low levels of IL-11 in interfollicular epidermal areas. The results are shown in Figure 23.

Example 25: Inhibition of IL-11 with Fab antibodies downregulates HS-like activation signature in the ex vivo human full thickness skin explant model

A cytokine cocktail derived from human PBMCs activated with HS relevant stimuli (anti- CD3/CD28 with PGN-SA) pulled from 3 PBMC donors was used to stimulate healthy human skin from 3 further donors. Full thickness 11mm skin biopsies were acquired from Genoskin (NativeSkin access, NSA11). Skin was donated post abdominoplasty with informed ethical consent. 1 mL of provided skin media (Genoskin, NSMED2) was placed in the bottom of the transwell per biopsy and the skin was transferred into the incubator (37°C, 5% CO2, 100% humidity) and rested for >3 hours to allow acclimatisation. Stimulation with and without the inhibitory anti-IL-11 antibody 19439gL1gH1 Fab, 10pg/mL was performed. Solutions were prepared in advance. Briefly, PBMC supernatant was combined from 3 donors in a 1 :1 :1 ratio. Stimulation mix containing the antibody was prepared >30 minutes in advance of addition to culture and brought to approximately 37°C. On Day 0 of the study, the culture medium was removed from acclimatized skin samples and replaced with 1 mL of medium, with 10% PBMC cytokine stimulation ± inhibitory antibodies, or Control media (10% Complete RPMI medium (RPMI 1640 Medium, (Life technologies, 11875093) containing 10% FBS (Life Technologies, 16140-071), 50 U/mL Penicillin-Streptomycin (Life Technologies, 15070063) and 2mM L- Glutamine (life Technologies, A2916801)) + 90% skin media).

On day 6 the skin explants were dissected, one quarter of the biopsy was transferred into RNA later and stored in a -80°C freezer. RNA was extracted from the skin samples. Briefly, skin samples were homogenised in RLT plus buffer (Qiagen, 74136) containing BME. Proteinase K was added for proteins digestion and samples were run via QIAshredder columns, then RNA extracted following manufactures protocol (fibrous kit, Qiagen, 74704). RNA quantity (ng/pL) and 260/280, 260/230 values were determined using a nanodrop 2000 spectrophotometer following manufacturers guidelines. RNA underwent further quality control (QC) analysis and RNA sequencing.

Fastq files were generated using bcl2fastq and quality assessed prior to quantification using FastQC & MultiQC tools. Adapter sequences and polyG sequence tails (empty signal in two colour chemistry) were trimmed using bbduk (v38.86). Quantifications were performed using Salmon (version 1.9) against a GENCODE 38 reference. Counts were imported to R (v4.3.3) using the tximport package (v1.28) and summarised to gene level at the unit LengthScaledTPM. The raw count matrix was transformed under the Limma (v3.56.2) voom procedure, with lowly expressed genes removed using the filterByExpr function from edgeR (v3.42.4) using default parameters in a design aware mode. Linear models were fit using the limma framework using a non-intercept model and defined contrast matrix for pairwise comparisons, leveraging empirical bayes moderation. Differential expression was calculated for each contrast of interest with false discovery correction (FDR I Benjamini Hochberg), and a statistical significance threshold of FDR<0.05.

To confirm that the model captured inflammatory phenotype of HS skin, gene profile from the control samples was compared to that from samples of the HS0001 study. HS0001 is a bulk RNAseq study of matched lesional and non-lesional biopsies at baseline from a cohort of moderate to severe HS patients (NCT03248531). The data was processed in an identical manner as described above and as in example 17. Concordant dysregulation was observed between the ex vivo disease model (Stimulated vs. Non-stimulated) and HS0001 skin biopsies (Lesional vs. Non-lesional) with -50% concordant differential expression for the skin model (Figure 24) which broadly captures inflammatory biology similar to that observed in HS biopsies (Figure 25).

Treatment with anti-IL-11 Fabs in the skin model showed inhibition of gene pathways linked to inflammation and epithelial mesenchymal transition (ETM) (Figure 26).

Claims

WHAT IS CLAIMED IS:

1. An antibody or an antigen-binding fragment thereof that specifically binds to IL-11 , which comprises a heavy chain variable region comprising: a CDR-H1 comprising the amino acid sequence of SEQ ID NO:1 , a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:3; and a light chain variable region comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:6.

2. The antibody or an antigen-binding fragment thereof according to claim 1 , wherein the CDR-H2 comprises the amino acid sequence of SEQ ID NO:2 wherein 1 , or 2 amino acids have been substituted with another amino acid, wherein at position 6 the G has changed into S, or A, at position 7 the S has changed into G, at position 9 the T has changed into S, and/or at position 17 the S has changed into R, and/or the CDR-H3 comprises the amino acid sequence of SEQ ID NO:3 wherein 1 amino acid has been substituted with another amino acid, wherein at position 5 the T has changed into A; and/or the CDR-L1 comprises the amino acid sequence of SEQ ID NO:4 wherein 1 amino acid has been substituted with another amino acid, wherein at position 1 the K has changed into R, and/or at position 9 the Y has changed into H, and/or the CDR-L2 comprises the amino acid sequence of SEQ ID NO:5 wherein 1 , or 2 amino acids have been substituted with another amino acid, wherein at position 5 the L has changed into R, and/or at position 6 the Y has changed into N or D.

3. The antibody or the antigen-binding fragment thereof according to claim 1 or 2, wherein the heavy chain variable region comprises an amino acid sequence of SEQ ID NO:7 or with a sequence identity of more than 90% with SEQ ID NO:7, and wherein the light chain variable region comprises an amino acid sequence of SEQ ID NO:9 or with a sequence identity of more than 90% with SEQ ID NO:9.

4. The antibody according to any one of claims 1 to 3, wherein the antibody is a Fab, Fab’, F(ab’)2, Fv, a scFv or a full length antibody.

5. The antibody according to any one of claims 1 to 4, wherein the antibody is an IgG.

6. The antibody according to claim 5, wherein the antibody is an IgG 1 comprising a light chain comprising an amino acid sequence of SEQ ID NO:11 or which has at least 90% identity or similarity to the amino acid sequence of SEQ ID NO:11 , and a heavy chain comprising an amino acid sequence of SEQ ID NO: 19 or which has at least 90% identity or similarity to the amino acid sequence of SEQ I D NO: 19.

7. An isolated antibody or an antigen-binding fragment thereof that competes for binding to I L- 11 with an antibody or the antigen-binding fragment thereof according to any one of claims 1 to 6.

8. An isolated polynucleotide encoding the antibody or the antigen-binding fragment thereof according to any one of claims 1 to 7.

9. An expression vector carrying the polynucleotide according to claim 8.

10. A host cell comprising the vector according to claim 9.

11. A pharmaceutical composition comprising the antibody or the antigen-binding fragment thereof according to any one of claims 1 to 7 and a pharmaceutically acceptable agent.

12. The antibody or the antigen-binding fragment thereof according to any one of claims 1 to 7, or the pharmaceutical composition according to claim 11 , for use as a medicament.

13. The antibody or the antigen-binding fragment thereof according to any one of claims 1 to 7, or the pharmaceutical composition according to claim 11 , for use in the treatment or prevention of cancer, fibrosis, an autoimmune disease, an inflammatory disease, a metabolic disease, a wasting disease, a bone disease, or a disease in which smooth muscle cells (SMCs) are pathologically implicated.

14. The antibody or the antigen-binding fragment thereof according to any one of claims 1 to 7, or the pharmaceutical composition according to claim 11 , for use in the treatment or prevention of hidradenitis suppurativa.

15. An agent capable of inhibiting IL-11 mediated signaling for use in the treatment of hidradenitis suppurativa.

16. The antibody or the antigen-binding fragment thereof according to any one of claims 1 to 7 for use as a diagnostic agent.