WO2025238133A1 - Multispecific antibody with binding specificity for il-11 and il-17 - Google Patents

Abstract

The present technology relates to multispecific antibodies that inhibit both IL-11 mediated signaling and IL-17A and/or IL-17F mediated signaling. The technology provides pharmaceutical compositions comprising an antibody that inhibits IL-11 mediated signaling and an antibody that inhibits IL-17A and/or IL-17F mediated signaling. The technology further relates to therapeutic uses of a combination of IL-11 and IL-17A and/or IL-17F inhibiting antibodies or multispecific antibodies or agents that inhibit both IL-11 mediated signaling and IL-17A and/or IL-17F mediated signaling.

Description

MULTISPECIFIC ANTIBODY WITH BINDING SPECIFICITY FOR IL-11 AND IL-17

FIELD

The present technology relates to multispecific antibodies that inhibit both IL-11 mediated signaling and IL-17A and/or IL-17F mediated signaling. The technology provides pharmaceutical compositions comprising an antibody that inhibits IL-11 mediated signaling and an antibody that inhibits IL-17A and/or IL-17F mediated signaling. The technology further relates to therapeutic uses of a combination of IL-11 and IL-17A and/or IL-17F inhibiting antibodies or multispecific antibodies or agents that inhibit both IL-11 mediated signaling and IL-17A and/or IL-17F mediated 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 (HS).

BACKGROUND

The IL- 17 family of cytokines consists of 6 members based on structural similarities, with a molecular mass of 23-36 kDa and a dimer structure. The founding member IL- 17A shares 16% - 50% amino acid sequence identity with other members: IL-17B, IL-17C, IL-17D, IL-17E (also known as IL-25) and IL-17F. IL-17A and IL-17F share the greatest homology (50%) and bind to the same receptor complex, thus shared biological activities have been noted between these two cytokines. In addition, IL-17A and IL-17F exist not only as homodimers (also referred to herein as “IL-17AA” and “IL-17FF” respectively), but also as an IL-17AF heterodimer (also referred to herein as “IL-17AF”). IL-17E (IL-25) has the least similarity with IL-17A. Of significance and relevance to the biological activity of IL-17A and IL-17F is the finding that they share the same IL-17RA/IL-17RC receptor complex, with IL-17A having greatest affinity for IL- 17RA, whereas IL-17F binds more strongly to IL-17RC. The other family member to utilise IL- 17RA is IL-17E, which signals via the IL-17RA/IL-17RB receptor complex.

I L-17A and IL-17F are produced by the Th17 subset of CD4+ T cells. In addition, other T cell subsets produce IL-17A and IL-17F including cytotoxic CD8+ T cells (Tc17), gdT cells and NK T cells. Other cell populations reported to secrete IL-17A include neutrophils, monocytes, NK cells, lymphoid tissue inducer-like (LTi-like) cells, intestinal paneth cells and even B cells and mast cells. In addition, epithelial cells have been reported to secrete IL-17F.

The cell types which respond to IL-17 cytokines are reflected by the expression of the different receptors. IL-17RA is ubiquitously expressed, with particularly high levels in haematopoietic tissues whereas IL-17RC is more highly expressed in non-immune cells of joints, liver, kidney, thyroid and prostate. This differential expression could explain differences in IL-17A and IL- 17F biological activity as cells expressing high levels of IL-17RC could be more responsive to IL-17F whereas cells with higher expression of IL-17RA than IL-17RC may respond more readily to IL-17A. Specific cell types that are responsive to IL-17A and F include fibroblasts, epithelial cells, keratinocytes, synoviocytes and endothelial cells with IL-17A also reported to act on T and B cells and macrophages.

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 IL-6ST) co-receptor to activate downstream signaling pathways. The IL-11 :IL-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 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, 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 multifactorial 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 diagnosis very difficult, 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 Phil I 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 with the potential to provide higher levels of disease control for more patients 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 inflammatory conditions, such as HS, by providing antibodies that are capable of inhibiting IL-11 and IL-17A and/or IL- 17F mediated signaling. The inventors have established for the first time that IL- 11 plays a role in HS biology and that some of that biology remains unaddressed upon treatment of patients with antibodies that inhibit IL-17A and IL-17F mediated signaling and that blockade of the IL- 11 signaling pathway in combination with blockade of the IL-17A and/or IL-17F signaling pathway(s) thus has the potential to result in deeper, longer lasting clinical responses.

In a first aspect, the present technology provides a multispecific antibody comprising at least two antigen-binding domains, wherein the first antigen-binding domain inhibits IL-11 mediated signaling and the second antigen-binding domain inhibits IL-17A and/or IL-17F mediated signaling.

In a second aspect, the present technology provides a pharmaceutical composition that comprises a first antibody that inhibits IL-11 mediate signaling and a second antibody that inhibits IL-17A and/or IL-17F mediated signaling and a pharmaceutically acceptable agent.

In a third aspect, the present technology provides a combination of a first antibody that inhibits IL-11 mediated signaling and a second antibody that inhibits IL-17A and/or IL-17F mediated signaling for use as a medicament.

In a fourth aspect, the present technology provides an agent capable of inhibiting IL-11 and IL-17A and/or IL-17F mediated signaling for use in the treatment of an inflammatory disease.

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 7 19439gL1gH 1/4211 KiH hlgG1 LA LA was analysed by analytical size exclusion chromatography after preparative S200 chromatography (A, 99% monomeric) of the KiH parental mix after mild reduction. The S200 purified product was analysed on HIC with a single peak eluting at 24 minutes (B) and analysed by SDS-Page (C, gel image) as reduced sample (Lane 1) and non-reduced sample (Lane 2) using a SeeBluePlus2 pre- stained protein standard (Lane M, Thermo Fisher Scientific). (D) Intact mass analysis of a deglycosylated sample.

Figure 8 19439gL1gH1 / 4211 KiH hlgG1 LALA does not bind to IL-11R or IL-11RA+ gp130 expressing cells in the presence of IL-11 indicating non internalization properties. VR24979 and VR20008 (non-blocking VR) do bind in the presence of IL-11.

Figure 919439gL1 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 10 VR 19439gL1gH1 in different formats (Fab and lgG1 LALA) in the human IL- 11 induced cis-STAT3 signaling assay using a human HepG2 IL-11 R/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 11 19439gL1gH1/4211 KiH lgG1 LALA produced by different methods {in vitro stable and in vitro transient) 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 12 19439gL1gH 1/4211 KiH lgG1 LALA alongside several relevant control molecules (18136 (Null)/4211 lgG1 KiH LALA, 19439gL1gH1/18136 (Null) KiH lgG1 LALA, and an lgG1 LALA isotype control) 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 13 19439gL1gH1/4211 KiH lgG1 LALA alongside the isotype control lgG1 LALA in the cynomolgus IL-11 induced cis-STAT3 signaling assay using a human HepG2 IL- 11 R/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 14 19439gL1gH1/4211 KiH lgG1 LALA and the isotype control lgG1 LALA in the murine 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 15 19439gL1gH1/4211 KiH lgG1 LALA, 19439gL1gH1 Fab and the isotype control lgG1 LALA in the human IL-11/IL-11 RA 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 16 19439gl_1gH 1/4211 KiH lgG1 LALA and isotype control lgG1 LALA in the human IL-11/IL-11 RA mediated STAT3 trans-signaling assay in the ‘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 17 19439gl_1gH1/4211 KiH lgG1 LALA (in vitro transient material) and isotype control lgG1 LALA in the human IL-17AA mediated IL-6 release assay with primary human dermal fibroblasts.

Figure 18 19439gL1gH1/4211 KiH lgG1 LALA produced by different methods {in vitro stable and in vitro transient) and isotype control lgG1 LALA in the cis IL-11 induced CCL2 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 19 19439gL1gH1 Fab 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 20 19439gL1gH1/4211 KiH lgG1 LALA produced by different methods {in vitro stable and in vitro transient methods) and control molecules in the human IL-17AA induced NF-KB activation assay using a HEK Blue IL-17R 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 21 19439gL1gH1/4211 KiH lgG1 LALA produced by different methods {in vitro stable and in vitro transient methods) and control molecules in the cynomolgus monkey IL-17AA induced NF-KB activation assay using a HEK Blue IL-17R 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 22 19439gL1gH1/4211 KiH lgG1 LALA produced by different methods {in vitro stable and in vitro transient methods) and control molecules in the human IL-17FF induced NF-KB activation assay using a HEK Blue IL-17R 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 23 19439gl_1gH1/4211 KiH lgG1 LALA produced by different methods (in vitro stable and in vitro transient methods) and control molecules in the cynomolgus monkey IL-17FF induced NF-KB activation assay using a HEK Blue IL-17R 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 24 Expression profile by qPCR of IL-11RA, gp130 (IL-6ST), IL-17RA and IL-17RC from human and cynomolgus dermal fibroblasts. A. Raw CT values B. Fold change expression level relative to human dermal fibroblast GAPDH.

Figure 25 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. 19439gl_1gH1 / 4211 KiH hlgG1 LALA (in vitro transient material) at 10pg/ml (66.7nM) functionally inhibited rhlL-11 and rhlL-17AA/FF mediated CCL2 secretion from primary human dermal fibroblasts. Dual blockade of rhlL-11 and rhlL-17AA/FF with 19439gL1gH1 Z4211 KiH hlgG1 LALA resulted in greater inhibition of CCL2 secretion than inhibition of individual cytokines. Geomean, n=3 donors/ 3 replicates per donor per condition.

Figure 26 Primary human dermal fibroblast stimulation with rhlL-11 at 10ng/ml (518pM) or rhlL-17AA at 10ng/ml (311pM) with IL-17FF at Ong/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 / 4211 KiH hlgG1 LALA (in vitro transient material) at lOpg/ml (66.7nM) functionally inhibited rhlL-11 and rhlL-17AA/FF mediated IL-6 secretion from primary human dermal fibroblasts. Dual blockade of rhlL-11 and rhlL-17AA/FF with 19439gL1gH1 Z4211 KiH hlgG1 LALA resulted in greater inhibition of IL-6 secretion than inhibition of individual cytokines. Geomean, n=3 donors/ 3 replicates per donor per condition.

Figure 27 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 / 4211 KiH hlgG1 LALA (in vitro transient material) at 10pg/ml (66.7nM) functionally inhibited rhlL-11 and rhlL-17AA/FF mediated MMP2 secretion from primary human dermal fibroblasts. Dual blockade of rhlL-11 and rhlL-17AA/FF with 19439gL1gH1 / 4211 KiH hlgG1 LALA resulted in greater inhibition of MMP2 secretion than inhibition of individual cytokines. Geomean, n=3 donors/ 2-3 replicates per donor per condition. Figure 28 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 29 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 30 RNAscope analysis shows differential expression of IL-11 in healthy vs HS lesional skin. IL-11 expression demonstrated low level in both follicular and interfoil icular 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 31 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-11R 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 32 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 33 Representative examples of IL-17A positive cells (arrowhead) distributed in proximity to IL-11 expressing cells (arrow) in de-epithelialized, active lesion. IL-17A positive cells are prevalent in the lesion at the site of dense immune infiltration. Lesion lumen is marked by an open arrow, framed areas are enlarged in bottom panels and in A-C, blood vessels are labeled by an asterisk.

Figure 34 A significant number of cells expressing IL-17F (arrowhead) was evident in lesional HS tissue among dense immune cell infiltrates, confirmed by RNAscope imaging. Lesion is marked by an open arrow, framed areas are enlarged in A-C, and blood vessels are labeled by asterisks.

Figure 35 (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-1 10pg/m induced synergistic secretion of IL-8 (indicated by arrows). Geomean, n=1 donor, 3 replicates/donor.

Figure 36 (A) H&E images from the 3D RHE model illustrating morphological changes including epidermal atrophy, aberrant keratinocyte differentiation and parakeratosis (indicated by arrows) post rhlL-11 at 300ng/ml, rhlL-17AA at 100ng/ml and rhlL-17FF at 1pg/ml stimulations. (B) Epidermal thickness measurements post stimulation with IL- 17AA at 100ng/ml and IL-17FF at 1pg/ml (referred to as IL-17A/F) or rhlL-17AA at 100ng/ml and rhlL-17FF at 1pg/ml together with 300ng/ml of rhlL-11 (referred to as IL- 17A/F + IL-11) in in the RHE model, data represented as mean, n=5/6 measurements per section.

Figure 37 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 38 (A) Representative images of immunofluorescence IL-11Ra protein expression in the epidermis, dermis and hair follicle structures of 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 39 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 40 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 41 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 42 Barplots showing Mean logFC change in key biological pathways after anti- IL-11, anti-IL-17A/F or combined anti-IL-11 and anti-IL-17A/F 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 different antigens: IL-11 , IL-17A, IL-17F, and albumin. These antigen-binding domains are also referred to as “IL-11 binding domain”, “IL-17 binding domain”, and “albumin binding domain”. The IL-17 binding domain specifically binds to IL-17A and/or IL-17F. The IL-11 binding domain specifically binds to IL-11. The albumin binding domain specifically binds to albumin. The binding domain may comprise a single domain antibody. Each binding domain may be monovalent. Each 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 “DiFab”’ as employed herein refers to two Fab’ molecules linked via one or more disulfide bonds in the hinge region thereof.

The term “a diagnostic agent” with reference to an antibody (or binding fragment thereof) or a combination of antibodies refers to the use of such an antibody (or binding fragment thereof) or such a combination of antibodies 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 “dsFab” as employed herein refers to a Fab with an intra-variable region disulfide bond.

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 EU 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 EU 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 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 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-Fc-scFv” as employed herein refers to four scFvs, wherein one of each is appended to the N-terminus and the C-terminus of both the heavy chains of a -CH2CH3 fragment.

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 term “tribody” (also referred to a Fab(scFv)₂) as employed herein refers to a Fab fragment with a first scFv appended to the C-terminal of the light chain and a second scFv appended to the C-terminal of the heavy chain.

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 “IL-11/IL-17 TrYbe” as employed herein refers to a TrYbe that comprises an IL-11 binding domain, an IL-17 binding domain and an albumin binding domain.

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 and IL-17A and/or IL-17F mediated signaling

The present technology aims at providing a novel type of drug to treat particular inflammatory diseases, such as hidradenitis suppurativa.

In the present disclosure, the inventors establish that IL-11 plays a role in HS (hidradenitis suppurativa) biology and that some of that biology remains unaddressed upon treatment of patients with antibodies that inhibit IL-17A and IL-17F mediated signaling. 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. Dual blockade of the IL-11 and the IL-17A and/or IL-17F signaling pathways thus has the potential to result in deeper, longer lasting clinical responses.

Subsequently, antibodies were developed that are capable of inhibiting IL-11 and IL-17A and/or IL-17F mediated signaling. Such antibodies can also be used for the treatment of alternative inflammatory diseases, such as systemic sclerosis, idiopathic pulmonary fibrosis (IFF), non-alcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL), or nonalcoholic steatohepatitis (NASH).

The antibodies can be used for the treatment of inflammatory skin conditions, systemic sclerosis, inflammatory fibrotic diseases of the lung (such as I PF, chronic obstructive pulmonary disease (COPD), and asthma), inflammatory fibrotic diseases of the liver (such as metabolic dysfunction-associated steatotic liver disease (MASLD), metabolic dysfunction- associated fatty liver disease (MAFLD), metabolic dysfunction-associated fatty liver (MAFL), and metabolic dysfunction-associated steatohepatitis (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 a first general aspect, the present technology therefore provides an agent for use in in the treatment of an inflammatory disease, such as HS.

In a further particular embodiment the agent is an antibody or combination of antibodies capable of inhibiting IL-11 and IL-17A and/or IL-17F mediated signaling.

The treatment of such inflammatory diseases may be achieved by inhibiting IL-11 and IL-17A and/or IL-17F mediated signaling via a single antibody. In such case antigen-binding domains that inhibit IL-11 and IL-17A and/or IL-17F mediated signaling, may each be present on a single antibody.

In a further particular embodiment the agent is a multispecific antibody that comprises a first antigen-binding domain that inhibits IL-11 mediated signaling and a second antigen-binding domain that inhibits IL-17A and/or IL-17F mediated signaling.

In an alternative embodiment the agent comprises an antibody that inhibits IL-11 mediated signaling in combination with another antibody that inhibits IL-17A and/or IL-17F mediated signaling. In one particular embodiment both antibodies are present in the same pharmaceutical composition. In another particular embodiment each antibody is present in a separate pharmaceutical composition.

In a second general aspect, the present technology thus provides a multispecific antibody that comprises a first antigen-binding domain that inhibits IL-11 mediated signaling and a second antigen-binding domain that inhibits IL-17A and/or IL-17F mediated signaling.

In a third general aspect, the technology thus provides a pharmaceutical composition comprising a multispecific antibody or a combination of antibodies that inhibit IL-11 and IL-17A and/or IL-17F mediated signaling.

Interleukin 17 (IL-17) The term “IL-17A signaling” or “IL-17A mediated signaling” refers to signaling mediated by binding of IL-17A to the IL-17RA/IL-17RC receptor complex. The term “IL-17F signaling” or “IL-17F mediated signaling” refers to signaling mediated by binding of IL-17F to the IL-17RA/IL- 17RC receptor complex. “Signaling” as used herein refers to signal transduction and other cellular processes governing cellular activity.

In all aspects of the current technology, antibodies are being used that are capable of inhibiting IL-17A and/or IL-17F mediated signaling.

In one embodiment, inhibition of IL-17A and/or IL-17F mediated signaling can be obtained through binding of the antibody to the IL-17RA/IL-17RC receptor complex.

Alternatively, inhibition of IL-17A and/or IL-17F mediated signaling is obtained through binding of the antibody to IL-17A and/or IL-17F.

In a further alternative embodiment, the antibody comprises an antigen-binding domain that specifically binds to IL-17A. In one embodiment, the antigen-binding domain specifically binds to IL-17F. In one embodiment, the antigen-binding domain specifically binds to IL-17A and IL- 17F.

Such an antibody comprises an antigen-binding domain that specifically binds to IL-17A homodimer, IL-17F homodimer and/or IL-17AF heterodimer. More specifically, such an antigen-binding domain can be capable of binding to human and/or cynomolgus IL-17A and/or IL-17F.

In one embodiment, human IL-17A has the amino acid sequence of SEQ ID NO: 147. In one embodiment, cynomolgus IL-17A has the amino acid sequence of SEQ ID NO: 149. In one embodiment, human IL-17F has the amino acid sequence of SEQ ID NO: 148. In one embodiment, cynomolgus IL-17F has the amino acid sequence of SEQ ID NO: 150.

In one embodiment, the antigen-binding domain specifically binds to human IL-17A with a KD of less than 50, 25, or 10pM. In one embodiment, the antigen-binding domain specifically binds to human IL-17A with a KD of <50pM. In one embodiment, the antigen-binding domain specifically binds to human IL-17A with a KD of <25pM. In one embodiment, the antigenbinding domain specifically binds to human IL-17A with a KD of <10pM.

In one embodiment, the antigen-binding domain specifically binds to human IL-17F with a KD of less than 200, or 100pM. In one embodiment, the antigen-binding domain specifically binds to human IL-17F with a K_D of <200pM. In one embodiment, the antigen-binding domain specifically binds to human IL-17F with a KD of <100pM.

In one embodiment, the antigen-binding domain specifically binds to the human IL-17AF heterodimer with a KD of less than 100, 50 or 20pM. In one embodiment, the antigen-binding domain specifically binds to the human IL-17AF heterodimer with a KD of <100pM. In one embodiment, the antigen-binding domain specifically binds to the human IL-17AF heterodimer with a KD of <50pM. In one embodiment, the antigen-binding domain specifically binds to the human IL-17AF heterodimer with a KD of <20pM.

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

The affinity of an antibody, as well as the extent to which an antibody inhibits binding, can be determined by the skilled person using conventional techniques, for example those described by Scatchard et al. (Ann. KY. Acad. Sci. 51 :660-672 (1949)) or by surface plasmon resonance (SPR) using systems such as BIAcore. For surface plasmon resonance, target molecules are immobilized on a solid phase and exposed to ligands in a mobile phase running along a flow cell. If ligand binding to the immobilized target occurs, the local refractive index changes, leading to a change in SPR angle, which can be monitored in real time by detecting changes in the intensity of the reflected light. The rates of change of the SPR signal can be analyzed to yield apparent rate constants for the association and dissociation phases of the binding reaction. The ratio of these values gives the apparent equilibrium constant (affinity) (see, e.g., Wolff et al, Cancer Res. 53:2560-65 (1993)).

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: 145. In one embodiment, cynomolgus IL-11 has the amino acid sequence of SEQ ID NO: 146.

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, including multispecific 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 IL17A 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 given the fact that significant IL-11 biology remains unaddressed upon treatment with Bimekizumab also led to the hypothesis that dual blockade of the IL-11 and the IL-17A and/or IL-17F signaling pathways could result in deeper, longer lasting 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-11 RA 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. Additionally, high expression of IL-17A and IL-17F was found to colocalize with IL-11 expression in moderate to severe lesional skin.

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.

Dermal papilla cells were thus shown to have a unique functional response to IL-11 , which is different to the functional response triggered by IL-17 on the same cells. These data represent a critical example of why IL-11 driven HS pathobiology is not addressed by anti-IL-17A and/or IL17-F treatment.

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 p to amplify the proinflammatory immune response.

In an in vitro 3D human skin model, exacerbated epidermal atrophy was observed upon stimulation with IL-11 in combination with IL-17AA and IL-17FF in comparison with the combination of IL-17AA and IL-17FF alone. Also, parakeratosis was promoted, which indicates the potential contribution of IL-11 to keratinocyte proliferation and dedifferentiation, a pathological phenomenon observed during tunnel formation in HS.

In an ex vivo human hair follicle organ culture model, the stimulation with IL-11 in combination with IL-17AA and IL-17FF 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. 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 and IL-17A/F inhibition in HS-relevant preclinical models, the inventors firstly explored the effect of anti-IL-11 and/or anti-IL-17A/F Fabs in an ex vivo human full thickness skin explant model. Treatment with the different Fabs showed inhibition of gene pathways linked to inflammation and epithelial to mesenchymal transition (ETM), which are reported to be upregulated in HS patients. In particular, combined blockade of IL-11 and IL-17A/F resulted in a greater normalisation of the HS-relevant gene signature compared to inhibition with either IL-11 or IL-17A/F inhibition alone, suggesting that combined inhibition has potential to have greater impact than individual pathway blockade.

To confirm this data, the effect of anti-IL-11 and/or anti-IL-17A/F Fabs was explored in the HS ex vivo biopsy explant model. Consistently with the previous experimental system, treatment of diseased skin with anti-IL-11 + anti-IL17A/F antibodies showed greater number of significantly modulated genes compared to treatment with anti-IL17A/F alone. The inventors were able to identify the anti-IL-11 specific genes inhibited in the HS biopsies, which included genes linked to immune response (SERPINB4, IDO1), barrier function (CLDN17, SLC9A1), tissue remodelling (PRSS22), stress and apoptosis (PML), cellular signal transduction (TMEM171) and metabolic pathways (ALAS1 , SEL1 L3). 1

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. Moreover, pathway blockade exeriments have shown that combined blockade of the IL-11 and IL- 17 pathway exert an additive and/or synergistic effect in normalizing multiple disease drivers, thus offering the opportunity for a differentiated mechanism of action which could benefit HS patients in the future.

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;

• V_H44 + VL105 see for example J Biochem. 118, 825-831 Luo et al (1995);

• VH45 + 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),

• VH106 + 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.

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 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.

IL-11 and IL-17 antigen-binding domains and their sequences

In the context of the binding properties of the antibodies of all aspects of the present technology, different types of antigen-binding domains are being referred to: an IL-11 binding domain, and an IL-17 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 the antigen comprises a light chain variable region and a heavy chain variable region. The IL-11 binding domain is also referred to herein as the first antigen-binding domain and may comprise a heavy chain variable region (VH1) and light chain variable region (VL1). VH1 and VL1 may form a VH/VL pair (VH1/VL1).

The IL-17 binding domain is also referred to herein as the second antigen-binding domain and may comprise a heavy chain variable region (VH2) and light chain variable region (VL2). VH2 and VL2 may form a VH/VL pair (VH2/VL2).

SEQ ID NO’s of sequences related to specific examples of antigen-binding domains 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 , and IL-17 binding domains. * i.e. with cysteines as to allow for disulfide bond formation formed between positions VH44 and VL100

In one embodiment, the antibody comprises an antigen-binding domain that specifically binds to IL- 11 comprising a heavy chain variable region (VH1) comprising: a CDR-H1 comprising the amino acid sequence of SEQ ID NO:18, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:19, and a CDR-H3 comprising the amino acid sequence of SEQ ID NQ:20; and a light chain variable region (VL1) comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO:21 , a CDR-L2 comprising the amino acid sequence of SEQ ID NO:22, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:23.

In one embodiment, the antigen-binding domain that specifically binds to IL-11 comprises a

VH1 comprising the amino acid sequence of SEQ ID NO:24, and a VL1 comprising the amino acid sequence of SEQ ID NO:26.

In one embodiment, the antigen-binding domain that specifically binds to IL-11 is a Fab, scFv, or Fv comprising a VH1 comprising the amino acid sequence of SEQ ID NO: 24, and a VL1 comprising the amino acid sequence of SEQ ID NO:26.

In one embodiment, the Fab comprises a light chain comprising the amino acid sequence of SEQ ID NO:28, and a heavy chain comprising the amino acid sequence of SEQ ID NQ:30.

In one embodiment, the scFv comprises the amino acid sequence of SEQ ID NO:34.

In one embodiment, the antigen-binding domain that specifically binds to IL-11 is a dsscFv, or dsFv comprising a VH1 comprising the amino acid sequence of SEQ ID NO:32, and a VL1 comprising the amino acid sequence of SEQ ID NO:33.

In one embodiment, the dsscFv comprises the amino acid sequence of SEQ ID NO: 35.

In one embodiment, the antibody comprises an antigen-binding domain that specifically binds to IL-17A and/or IL17F comprising a heavy chain variable region (VH2) 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 (VL2) 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 antigen-binding domain that specifically binds to IL-17A and/or IL17F comprises a VH2 comprising the amino acid sequence of SEQ ID NO:7, and a VL2 comprising the amino acid sequence of SEQ ID NO:9. In one embodiment, the antigen-binding domain that specifically binds to IL-17A and/or IL-17F is a Fab comprising a VH2 comprising the amino acid sequence of SEQ ID NO:7, and a VL2 comprising the amino acid sequence of SEQ ID NO:9.

In one embodiment, the Fab 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.

Albumin binding domains and their sequences

In some embodiments, the multispecific antibody of the present technology lacks an Fc- domain and half-life is provided by an antigen-binding domain that binds to serum albumin. Such an antigen-binding domain is also referred to herein as “albumin binding domain”.

The albumin 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 albumin binding domain comprises a light chain variable region and a heavy chain variable region.

The albumin binding domain is also referred to herein as the third antigen-binding domain and may comprise a heavy chain variable region (VH3) and light chain variable region (VL3). VH3 and VL3 may form a VH/VL pair (VH3/VL3).

SEQ ID NOs of sequences related to specific examples of albumin binding domains that can be used in the antibodies of any aspect of the present technology are listed in Table 2.

Table 2. Summary of sequences of albumin binding domains. * i.e. with cysteines as to allow for disulfide bond formation formed between positions VH44 and VL100

In one embodiment, the multispecific antibody of the present technology comprises an albumin binding domain comprising a heavy chain variable region (VH3) comprising: a CDR-H1 comprising the amino acid sequence of SEQ ID NO:110, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:111 , and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:112; and a light chain variable region (VL3) comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO:113, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 114, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 115.

In one embodiment, the albumin binding domain comprises a VH3 comprising the amino acid sequence of SEQ ID NO: 116, and a VL3 comprising the amino acid sequence of SEQ ID NO:118.

In one embodiment, the albumin binding domain is a scFv comprising the amino acid sequence of SEQ ID NO:124.

In one embodiment, the albumin binding domain comprises a VH3 comprising the amino acid sequence of SEQ ID NQ:120, and a VL3 comprising the amino acid sequence of SEQ ID NO:122.

In one embodiment, the albumin binding domain is a dsscFv comprising the amino acid sequence of SEQ ID NO: 126.

Multispecific antibodies that bind to IL-11 and IL-17A and/or IL-17F

In some embodiments of the present technology, a multispecific antibody is provided that comprises at least two antigen-binding domains, wherein at least one antigen-binding domain specifically binds to IL-11 (“IL-11 binding domain”) and at least one antigen-binding domain specifically binds to IL-17A and/or IL-17F (“IL-17 binding domain”).

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 eta/. 2001 , Biomolecular Engineering, 17(6), 193-202).

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 binds to IL17A and/or IL17F, i.e. each binding domain is monovalent for each antigen. In one embodiment, the antibody is a trivalent bispecific antibody.

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 IL-17A, and the other antigen-binding domain specifically binds to albumin. 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 IL-17A, and the other antigenbinding domain specifically binds to IL-17F.

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 and the binding domain which specifically binds to IL-17A and/or IL-17F are independently selected from a Fab, scFv, Fv, dsFv and dsscFv.

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.

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 , W0 1998050431), DuoBody technology (e.g. WO2011131746), Azymetric technology (e.g. WO2012058768). 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.

In one embodiment, the present technology provides a multispecific antibody comprising a first antigen-binding domain that specifically binds to IL-11 comprising a heavy chain variable region (VH1) comprising: a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:19, and a CDR-H3 comprising the amino acid sequence of SEQ ID NQ:20; and a light chain variable region (VL1) comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO:21 , a CDR-L2 comprising the amino acid sequence of SEQ ID NO:22, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:23; and a second antigen-binding domain that specifically binds to IL-17A and/or IL-17F comprising a heavy chain variable region (VH2) 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 (VL2) 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 present technology provides a multispecific antibody comprising:

(i) a first antigen-binding domain that specifically binds to IL-11 comprising a VH1 comprising the amino acid sequence of SEQ ID NO:24, and a VL1 comprising the amino acid sequence of SEQ ID NO:26; and

(ii) a second antigen-binding domain that specifically binds to IL-17A and/or IL17F comprising a VH2 comprising the amino acid sequence of SEQ ID NO:7, and a VL2 comprising the amino acid sequence of SEQ ID NO:9.

A preferred 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; WQ2009/089004; 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). Where a suitably positioned and dimensioned protuberance or cavity exists at the interface of either the first or second CH3 polypeptide, it is only necessary to engineer a corresponding cavity or protuberance, respectively, at the adjacent interface.

In a further embodiment, the technology provides a bispecific antibody with a first antigenbinding domain that specifically binds to IL-11 and a second antigen-binding domain that specifically binds to IL-17A and/or IL-17F which comprises at least two polypeptides, each polypeptide comprising a CH3 domain (“CH3 polypeptide”), engineered with the Knobs-into- holes technology.

The resulting heterodimeric Fc-region can be further stabilized by the introduction/formation of artificial disulfide bridges. Non-naturally occurring disulfide bonds are constructed by replacing on the first CH3 polypeptide a naturally occurring amino acid with a free thiol-containing residue, such as cysteine, such that the free thiol interacts with another free thiol-containing residue on the second CH3 polypeptide such that a disulfide bond is formed between the first and second CH3 polypeptides.

The following substitutions resulting in appropriately spaced apart cysteine residues for the formation of new intra-chain disulfide bonds in the individual heavy chains of an Fc-region of an IgG antibody of subclass I gG 1 have been found to increase heterodimer formation: Y349C in one chain and S354C in the other; Y349C in one chain and E356C in the other; Y349C in one chain and E357C in the other; L351C in one chain and S354C in the other; T394C in one chain and E397C in the other; or D399C in one chain and K392C in the other (numbering of the residues according to the Kabat Ell index numbering system).

In one embodiment, the CH3 polypeptide is a heavy chain of an antibody. In one embodiment, the multispecific antibody is a bispecific full-length immunoglobulin (Ig), e.g. an IgG, comprising two heavy chains, wherein the CH3 domain of at least one of the two heavy chains is engineered with the Knobs-into-holes technology, and wherein each heavy chain is paired with a light chain to form an antigen-binding domain. In such embodiment, each antigen-binding domain formed by a pair of heavy and light chain binds to a separate epitope on the same or different antigen. The heavy chain engineered to introduce a knob may be termed the “knob chain”. The heavy chain engineered to introduce the complementary hole may be termed the “hole chain”.

In embodiments, the multispecific antibody of the present technology comprises one of the combinations of knobs and holes mutations (substitutions) as described in Table 3 (numbering of the residues according to the Ell index numbering system). Alternatively, a knob and a hole mutation can be introduced to one heavy chain and a complementary knob and hole mutation can be introduced in the second heavy chain.

Table 3. Exemplary knobs and holes mutations (substitutions). The numbering is according to Ell as in Kabat.

Mutations may be introduced into the constant domain of a heavy chain or light chain by methods well known in the art, for example by site-directed mutagenesis.

In one embodiment, the light chains of the multispecific antibody are identical to each other, and a first heavy chain and a first light chain form a binding domain that binds to a first antigen, and the second heavy chain and the second light chain that is identical to the first light chain form a binding domain that binds to a different antigen. In such embodiment, a host cell may be co-transfected with one or more vectors comprising the nucleic acids coding for the hole heavy chain, the knob heavy chain, and the common light chain. Methods of preparing a bispecific antibody comprising two common light chains have been described for example in US9409989.

In another embodiment, the multispecific antibody engineered with the Knobs-into-holes technology comprises two light chains that are different.

Methods of preparing a bispecific antibody engineered with the Knobs-into-holes technology comprising two antibody heavy chains and two different light chains, each heavy chain pairing with a light chain to form a distinct antigen-binding domain, have been described for example in WO2011/133886, WO2013/055958 and WO2015/171822.

More specifically, the present technology provides a multispecific antibody which comprises a first antigen-binding domain that specifically binds to IL-11 and a second antigen-binding domain that specifically binds to IL-17A and/or IL17-F comprising a) a polypeptide chain of formula (I):

VH-I-CH-I- CH₂ -CH_3; a) a polypeptide chain of formula (II):

VL-|-C_L; b) a polypeptide chain of formula (III):

VH₂-CH-|- CH₂ -CH_3; and c) a polypeptide chain of formula (IV):

VL₂-C_L; wherein: VHi and VH₂ represent a heavy chain variable domain; represents domain 1 of a heavy chain constant region; represents domain 2 of a heavy chain constant region; represents domain 3 of a heavy chain constant region;

VL-| and VL₂ represent a light chain variable domain; represents a domain from a light chain constant region, such as Ckappa; and wherein the VH1 and VL1 form a VH/VL pair that specifically binds to IL-11 , and wherein the VH2 and VL2 form a VH/VL pair that specifically binds to IL-17A and/or IL-17F, and wherein CH3 domains of the polypeptides of Formula I and III comprise one or more substitutions listed in Table 3.

In one embodiment, the antibody of the present technology comprises the knob substitution T366W in the heavy chain (i.e. polypeptide of Formula I or III) and the hole substitutions T366S, L368A, Y407V in the second heavy chain (i.e. polypeptide of Formula III or I respectively).

In a preferred embodiment, the antibody of the present technology comprises the knob substitution T366W in the polypeptide chain of Formula I and the hole substitutions T366S, L368A, Y407V in the polypeptide chain of Formula III.

In one embodiment, VH-j comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, and a CDR-H3 comprising the amino acid sequence of SEQ ID NQ:20; and VL-] comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:21, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:22, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:23.

In one embodiment, VH2 comprises 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 VL2 comprises 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, VH1 comprises the amino acid sequence of SEQ ID NO: 24, VL1 comprises the amino acid sequence of SEQ ID NO:26; VH2 comprises the amino acid sequence of SEQ ID NO:7, and VL2 comprises the amino acid sequence of SEQ ID NO:9.

The Fc region may comprise a human I gG 1 , 1 gG2 , lgG3 or lgG4 Fc region. In one embodiment of the present technology, the antibody is an lgG1 , lgG2, lgG3 or lgG4. In a preferred embodiment, the antibody is an lgG1.

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

In one embodiment, the polypeptide chain of formula (I) comprises the amino acid sequence of SEQ ID NQ:40, the polypeptide chain of formula (II) comprises the amino acid sequence of SEQ ID NO:28, the polypeptide chain of formula (III) comprises the amino acid sequence of SEQ ID NO: 17, and the polypeptide chain of formula (IV) comprises the amino acid sequence of SEQ ID NO:11.

In a preferred embodiment, the polypeptide chain of formula (I) comprises the amino acid sequence of SEQ ID NO:38, the polypeptide chain of formula (II) comprises the amino acid sequence of SEQ ID NO:28, the polypeptide chain of formula (III) comprises the amino acid sequence of SEQ ID NO: 15, and the polypeptide chain of formula (IV) comprises the amino acid sequence of SEQ I D NO: 11.

Tribody or TrYbe

Other preferred multispecific antibodies for use in the present technology comprise 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). Examples of such multispecific antibodies are described in WQ2015/197772. In one embodiment of the present technology, the multispecific antibody comprises a third antigen-binding domain that specifically binds to albumin.

In one embodiment, the technology thus provides a trispecific antibody with a first antigenbinding domain that specifically binds to IL-11 , a second antigen-binding domain that specifically binds to IL-17A and/or IL-17F, and a third antigen-binding domain that specifically binds albumin. More specifically, the present technology provides a multispecific antibody which comprises a first antigen-binding domain that specifically binds to IL-11 , a second antigen-binding domain that specifically binds to IL-17A and/or IL17-F, and a third antigen-binding domain comprising or consisting of: a) a polypeptide chain of formula (V):

V_H-CH-|-X-V_{1 ;} and b) a polypeptide chain of formula (VI):

V_L-C|_-Y-V₂; wherein:

V|_| represents a heavy chain variable domain;

CH-] represents domain 1 of a heavy chain constant region;

X represents a bond or linker;

Y represents a bond or linker;

V-j represents a scFv, or a dsscFv;

V|_ represents a light chain variable domain;

C|_ represents a domain from a light chain constant region, such as Ckappa; and

V₂ represents a scFv, or a dsscFv.

In one embodiment, the polypeptide chain of formula (V) comprises a protein A binding domain, and the polypeptide chain of formula (VI) does not bind protein A.

Methods of preparing a TrYbe comprising a polypeptide chain of formula (V) and (VI) have been described for example in WO2015/197772, WO2021/123186 and WO2022/122654.

Generally, the multispecific antibody is provided as a dimer of the polypeptides of Formula (V) and (VI) respectively, wherein the V|_|-CH-| portion together with the V|_-C|_ portion form a functional Fab or Fab’ fragment.

In one embodiment, the CH1 domain is a naturally occurring domain 1 from an antibody heavy chain or a derivative thereof.

In one embodiment, the CL domain is a constant kappa sequence or a derivative thereof. In one embodiment, the CL domain is a constant lambda sequence or a derivative thereof.

A derivative of a naturally occurring domain as employed herein is intended to refer to where at least one amino acid in a naturally occurring sequence have been replaced or deleted, for example to optimize the properties of the domain such as by eliminating undesirable properties but wherein the characterizing feature(s) of the domain is/are retained.

In one embodiment, one or more natural or engineered inter chain (i.e. inter light and heavy chain) disulfide bonds are present in the functional Fab or Fab’ fragment.

In one embodiment, a “natural” disulfide bond is present between a CH1 and CL in the polypeptide chains of Formula (V) and (VI).

When the CL domain is derived from either Kappa or Lambda, the natural position for a bond forming cysteine is 214 in human cKappa and cLambda (Kabat numbering 4th edition 1987).

The exact location of the disulfide bond forming cysteine in CH1 depends on the particular domain actually employed. Thus, for example in human gamma-1 the natural position of the disulfide bond is located at position 220 (EU numbering). The position of the bond forming cysteine for other human isotypes such as gamma 2, 3, 4, IgM and I g D are known, for example position 127 for human IgM, IgE, lgG2, lgG3, lgG4 and 131 of the heavy chain of human IgD and lgA2B (EU numbering).

In one embodiment, the multispecific antibody according to the disclosure has a disulfide bond in a position equivalent or corresponding to that naturally occurring between CH-] and C|_.

In one embodiment, a constant region comprising CH-] and a constant region such as C|_ has a disulfide bond which is in a non-naturally occurring position. This may be engineered into the molecule by introducing cysteine(s) into the amino acid chain at the position or positions required. This non-natural disulfide bond is in addition to or as an alternative to the natural disulfide bond present between CH-j and C|_. The cysteine(s) in natural positions can be replaced by an amino acid such as serine which is incapable of forming a disulfide bridge.

In one embodiment, a disulfide bond between CH-] and C|_ is completely absent, for example the interchain cysteines may be replaced by another amino acid, such as serine. Thus, in one embodiment there are no interchain disulfide bonds in the functional Fab fragment of the molecule. Disclosures such as W02005/003170, incorporated herein by reference, describe how to provide Fab fragments without an inter chain disulfide bond.

In one embodiment when V1 and/or V2 are a dsscFv, the variable domains VH and VL of V1 and/or the variable domains VH and VL of V2 , may be linked by a disulfide bond between two cysteine residues, one in VH and one in VL, which are outside of the CDRs wherein the position of the pair of cysteine residues is selected from the group consisting of VH37 and VL95, VH44 and VL100, VH44 and VL105, VH45 and VL87, VH100 and VL50, VH98 and VL46, VH105 and VL43 and VH106 and VL57. In one embodiment when V1 is a dsscFv, the variable domains VH and VL of V1 are linked by a disulfide bond between two engineered cysteine residues, one at position VH44 and the other at VL100. In one embodiment when V2 is a dsscFv, the variable domains VH and VL of V2 are linked by a disulfide bond between two engineered cysteine residues, one at position VH44 and the other at VL100.

In one embodiment, the VH domain of V1 is attached to X.

In one embodiment, the VL domain of V1 is attached to X.

In one embodiment, the VH domain of V2 is attached to Y.

In one embodiment, the VL domain of V2 is attached to Y.

In one embodiment, the antigen-binding domain formed by VH and VL is specific to a first antigen, the binding domain of V1 is specific to a second antigen, and the binding domain of V2 is specific to a third antigen.

In a preferred embodiment the multispecific antibody comprises a Fab binding to human IL- 17A and/or IL-17F linked to two scFv or dsscFv, where one scFv or dsscFv specifically binds to IL-11 and one scFv or dsscFv specifically binds to albumin.

In a preferred embodiment, the VH and VL form the VH/VL pair of the second antigen-binding domain that specifically binds to IL-17A and/or IL-17F, V1 comprises the third antigen-binding domain that specifically binds to albumin, and V2 comprises the first antigen-binding domain that specifically binds to IL-11.

In one embodiment, VH comprises 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 V|_ comprises 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, VH comprises the amino acid sequence of SEQ ID NO:7, and VL comprises the amino acid sequence of SEQ ID NO:9.

In one embodiment, the antigen-binding domain that specifically binds to IL-17A and/or IL17F is a Fab comprising a VH1 comprising the amino acid sequence of SEQ ID NO:7, and a VL1 comprising the amino acid sequence of SEQ ID NO:9. In one embodiment, V-] comprises a heavy chain variable region comprising: a CDR-H1 comprising the amino acid sequence of SEQ ID NO:110, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:111 , and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:112; and a light chain variable region comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 113, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 114, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 115.

In one embodiment, V1 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 116, and a light chain variable region comprising the amino acid sequence of SEQ I D NO: 118.

In a preferred embodiment, V1 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NQ:120 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 122.

In one embodiment, V2 comprises a heavy chain variable region comprising: a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, and a CDR-H3 comprising the amino acid sequence of SEQ ID NQ:20; and a light chain variable region comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO:21 , a CDR-L2 comprising the amino acid sequence of SEQ ID NO:22, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:23.

In one embodiment, V2 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:24 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:26.

In a preferred embodiment, V2 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:32 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:33.

In a particular embodiment, the present technology provides a multispecific antibody comprising: (i) a first antigen-binding domain V2 that specifically binds to IL-11 , wherein said antigen-binding domain is a scFv comprising a VH1 comprising the amino acid sequence of SEQ ID NO: 24, and a VL1 comprising the amino acid sequence of SEQ ID NO:26, or a dsscFv comprising a VH1 comprising the amino acid sequence of SEQ ID NO:32, and a VL1 comprising the amino acid sequence of SEQ ID NO:33; and

(ii) a second antigen-binding domain that specifically binds to IL-17A and/or IL-17F, wherein said antigen-binding domain is a Fab comprising a VH2 comprising the amino acid sequence of SEQ ID NO:7, and a VL2 comprising the amino acid sequence of SEQ ID NO:9, and

(iii) a third antigen-binding domain V1 that specifically binds to albumin, wherein said antigen-binding domain is a scFv comprising a VH3 comprising the amino acid sequence of SEQ ID NO: 116, and a VL3 comprising the amino acid sequence of SEQ ID NO: 118, or a dsscFv comprising a VH3 comprising the amino acid sequence of SEQ ID NQ:120, and a VL3 comprising the amino acid sequence of SEQ ID NO: 122.

In a preferred embodiment, V1 and V2 are a dsscFv.

In one embodiment, the Fab 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 one embodiment, the light chain variable region and heavy chain variable region of V1 are connected by a linker, said linker comprising the sequence given in SEQ ID NO:42.

In one embodiment, V1 is a scFv comprising the amino acid sequence of SEQ ID NO: 124 or a dsscFv comprising the sequence given in SEQ ID NO:126.

In a preferred embodiment, V1 is a dsscFv.

In one embodiment, the light chain variable region and heavy chain variable region of V2 are connected by a linker, said linker comprising the sequence given in SEQ ID NO:42.

In one embodiment, V2 is a scFv comprising the amino acid sequence of SEQ ID NO:34 or a dsscFv comprising the amino acid sequence of SEQ ID NO:35.

In a preferred embodiment, V2 is a dsscFv.

In one embodiment, X is a linker comprising the amino acid sequence of SEQ ID NO:43.

In one embodiment, Y is a linker comprising the amino acid sequence of SEQ ID NO:43. In one embodiment, the polypeptide chain of formula (V) comprises the sequence given in SEQ ID NO:129 or SEQ ID NO:130.

In one embodiment, the polypeptide chain of formula (VI) comprises the sequence given in SEQ ID NO:131 or SEQ ID NO:132.

In preferred embodiment, the polypeptide chain of formula (V) comprises the sequence given in SEQ ID NO: 130 and the polypeptide chain of formula (VI) comprises the sequence given in SEQ ID NO:132.

Functional properties of the antibodies

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

The IL-17 binding domain is inhibiting one or more of IL-17 activities.

The IL-17 binding domain may bind to IL-17A and/or IL-17F and prevent binding of IL-17A and/or IL-17F to IL-17RA/IL-17RC. In particular, the antigen-binding domain that specifically binds to IL-17A is capable of inhibiting IL-17AA and IL-17AF binding to IL-17RA/IL-17RC. The antigen-binding domain that specifically binds to IL-17F is capable of inhibiting IL-17AF and IL-17FF binding to IL-17RA/IL-17RC. The antigen-binding domain that specifically binds to IL- 17A and IL-17F is capable of inhibiting IL-17AA, IL-17AF and IL-17FF binding to IL-17RA/IL- 17RC.

In one embodiment, the IL-17 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-17 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-17 binding domain may inhibit MMP-2 release in cells. This property can be measured in a human dermal fibroblast assay. A representative assay has been exemplified herein. The term “MMP-2” refers to matrix metalloproteinase-2.

In one embodiment, the IL-17 binding domain may inhibit NF-KB activation in cells. This property can be measured in a reporter cell line, as has been exemplified herein. The term “NF-KB” refers to Nuclear Factor Kappa B.

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.

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.

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.

In one embodiment, 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.

The multispecific antibody, the combination of antibodies and the pharmaceutical compositions of the present technology are also capable of inhibiting synergistic IL-11 and IL- 17 signaling in cells.

As described in the examples, IL-11 and IL-17AA mediated signaling resulted in CXCL1 release in human dermal fibroblasts. The multispecific antibody of the present technology is capable of inhibiting CXCL1 release in such cells. The term “CXCL1” refers to C-X-C motif chemokine ligand 1.

As described in the examples, IL-11 , IL-17AA and IL-17FF mediated signaling resulted in increased release of CCL-2, IL-6 and MMP2, in human dermal fibroblasts, as compared to IL- 11 signaling or signaling with both IL-17AA and IL-17FF. The multispecific antibody of the present technology is capable of inhibiting CCL-2, IL-6 and MMP2 release in such cells.

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 whichever of IL-11 or IL-17A and/or IL-17F it is intended to be specific for. 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 whichever IL-11 or IL-17A and/or IL-17F it is intended to be specific for.

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 .

IL- 11 binding domain variants 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 binding domain comprises CDRs as defined by the sequences given in SEQ ID NO:18, 19, 20, 21 , 22, and 23 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 binding domain comprise sequences which have at least 70%, 80%, 90%, 95% or 98% identity or similarity to the sequences given in SEQ ID NOs:18, 19, 20, 21 , 22, and 23.

In embodiments, the IL-11 binding domain comprises a CDR-H2 wherein 1 , or 2 amino acids in the CDR-H2 of SEQ ID NO: 19 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: 19 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: 46) or TIVYDASDTYYRDSVKS (SEQ ID NO: 47).

In embodiments of the IL-11 binding domain, 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 binding domain comprises a CDR-H2 wherein 1 , or 2 amino acids in the CDR-H2 of SEQ ID NO: 19 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 binding domain comprises a CDR-H2 chosen from the group consisting of SEQ ID NO:45, 46, 47, 80 or 81.

In embodiments, the IL-11 binding domain comprises a CDR-H3 wherein 1 amino acid in the CDR-H3 of SEQ ID NO: 20 has been substituted with another amino acid, wherein at position 5 the T has changed into A.

In one embodiment, the IL-11 binding domain comprises a CDR-H3 of SEQ ID NO:82.

In embodiments, the IL-11 binding domain comprises a CDR-L1 wherein 1 amino acid in the CDR-L1 of SEQ ID NO:21 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 binding domain comprises a CDR-L1 chosen from the group consisting of SEQ ID NO:66, or 67.

In embodiments, the IL-11 binding domain comprises a CDR-L2 wherein 1 , or 2 amino acids in the CDR-L2 of SEQ ID NO: 22 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 binding domain comprises a CDR-L2 chosen from the group consisting of SEQ ID NO:68, 69, or 70.

In one embodiment, the IL-11 binding domain comprises a heavy chain variable region (VH1) comprising: a CDR-H1 comprising the amino acid sequence of SEQ ID NO:18, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, 45, 46, 47, 80 or 81 , and a CDR-H3 comprising the amino acid sequence of SEQ ID NQ:20, or 82; and a light chain variable region (VL1) comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO:21 , 66, or 67, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:22, 68, 69, or 70, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:23.

In one embodiment, the IL-11 binding domain 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:24 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:26.

In some embodiments, the IL-11 binding domain 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:28 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 NQ:30.

In some embodiments, the IL-11 binding domain 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:34 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:35.

In some embodiments, the IL-11 binding domain comprises CDR-H1/CDR-H2/CDR-H3/CDR- L1/CDR-L2/CDR-L3 sequences comprising SEQ ID NOs: 18, 19, 20, 21 , 22, and 23 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: 24 and 26 respectively.

The variants described here in relation to the IL-11 binding domains can be comprised in the antibodies, including multispecific antibodies, that contain an IL-11 binding domain.

IL- 17 antigen-binding domain variants

In some embodiments, the IL-17 binding domain comprises a heavy chain variable region 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:7 and/or a light chain variable region 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:9.

In some embodiments, the IL-17 binding domain 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-17 binding domain 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.

The variants described here in relation to the IL-17 binding domains can be comprised in the antibodies, including multispecific antibodies, that contain an IL-17 binding domain.

Albumin-binding domain variants

In some embodiments, the albumin binding domain comprises a heavy chain variable region 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:116 and/or a light chain variable region 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:118.

In some embodiments, the albumin binding domain 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: 124 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: 126.

In some embodiments, the albumin binding domain comprises CDR-H1/CDR-H2/CDR- H3/CDR-L1/CDR-L2/CDR-L3 sequences comprising SEQ ID NQs:110, 111 , 112, 113, 114, and 115 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: 116 and 118 respectively.

The variants described here in relation to the albumin binding domains can be comprised in the antibodies, including multispecific antibodies, that contain an albumin binding domain.

Multispecific antibody 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 IgGI, 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 polypeptide chain of formula (I) and formula (III) which comprise 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 polypeptide chain of formula (I) 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:38, the polypeptide chain of formula (II) comprises an amino acid sequence with a sequence identity of more than 90% with SEQ ID NO:28, the polypeptide chain of formula (III) 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: 15, and the polypeptide chain of formula (IV) comprises an amino acid sequence with a sequence identity of more than 90% with SEQ ID NO:11.

In one embodiment, the polypeptide of formula (V) 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: 129 or 130.

In one embodiment, the polypeptide of formula (VI) 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: 131 or 132.

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 multispecific 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 is 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 sidechain 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 or a combination of isolated polynucleotides encoding the antibodies 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 to A-7.

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 cloning or expression vectors or a combination of cloning or expression vectors comprising the nucleic acid sequences encoding for the antibodies or a component 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 multispecific antibodies

Also provided is a host cell comprising an isolated polynucleotide sequence or a combination of isolated polynucleotide sequences according to the present technology encoding an antibody thereof of the present technology. Also provided is a host cell comprising a vector or a combination of 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 polynucleotides or vector(s) or a combination thereof according to present technology is provided.

When the multispecific antibody is a knobs-into-holes antibody, 2 independent host cells may be used wherein one host cell comprises a polynucleotide that encodes the hole heavy chain and its corresponding light chain and the other host cell comprises a polynucleotide that encodes the knob heavy chain and its corresponding light chain. In one such embodiment, a combination of host cells is provided wherein (1) the first host cell comprises (e.g., has been transformed with) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the anti-IL-11 antibody and an amino acid sequence comprising the VH of the anti-IL-11 antibody, and (2) the second host cell comprises (e.g., has been transformed with) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the anti-IL-17 antibody and an amino acid sequence comprising the VH of the anti-IL-17 antibody.

When the multispecific antibody is a TrYbe, one host cell may be used which comprises a polynucleotide or a combination of polynucleotides that encodes for the TrYbe. In one particular embodiment, the host cell comprises (e.g., has been transformed with): (1) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the anti-l L17 antibody, an amino acid sequence comprising the CH1 , an amino acid sequence comprising the VH of the anti-albumin antibody, and an amino acid sequence comprising the VL of the anti-albumin antibody, and (2) a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the anti-IL17 antibody, an amino acid sequence comprising the CL, an amino acid sequence comprising the VH of the anti-IL11 antibody, and an amino acid sequence comprising the VL 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., Y0, 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 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid 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 400pg per mg of antibody product or less such as 100 pg per mg or less, in particular 20pg per mg, as appropriate.

Therapeutic uses

The present technology provides an agent capable of inhibiting IL-11 and IL-17A and/or IL- 17F mediated signaling for use in the treatment of an inflammatory disease. The method may comprise administering to a human subject in need thereof a therapeutically effective amount of an agent capable of inhibiting IL-11 and IL-17A and/or IL-17F mediated signaling.

In a particular embodiment the agent according to the present technology is used in the treatment of inflammatory skin conditions, systemic sclerosis, idiopathic pulmonary fibrosis (IPF), non-alcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL), or nonalcoholic steatohepatitis (NASH).

In a particular embodiment, the agent according to the present technology is used in the treatment of inflammatory skin conditions, systemic sclerosis, inflammatory fibrotic diseases of the lung (such as IPF, 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 a further particular embodiment the agent is used in the treatment of hidradenitis suppurativa.

In a particular embodiment the agent is an antibody or combination of antibodies capable of inhibiting IL-11 and IL-17A and/or IL-17F mediated signaling.

In a particular embodiment the agent is an antibody or combination of antibodies that specifically bind to IL-11 and IL-17A and/or IL-17F, according to the present technology.

In a particular embodiment the agent is a multispecific antibody that comprises a first antigenbinding domain that inhibits IL-11 mediated signaling and a second antigen-binding domain that inhibits IL-17A and/or IL-17F mediated signaling.

In a further particular embodiment the agent is a multispecific antibody that comprises a first antigen-binding domain that specifically binds to IL-11 and a second antigen-binding domain that specifically binds to IL-17A and/or IL-17F.

In an alternative embodiment, the agent comprises an antibody that inhibits IL-11 mediated signaling in combination with another antibody that inhibits IL-17A and/or IL-17F mediated signaling. In one particular embodiment both antibodies are present in the same pharmaceutical composition. In another particular embodiment each antibody is present in a separate pharmaceutical composition.

When each antibody is present in a separate pharmaceutical composition, the first and the second antibody might be administered either simultaneously or subsequently.

Therapeutic use of the multispecific antibodies

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

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

In prophylactic applications, the multispecific 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 multispecific 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 a multispecific antibody according to the present technology or a pharmaceutical composition thereof. The multispecific antibody is administered in a therapeutically effective amount.

The present technology also provides a multispecific 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 use of a combination of antibodies

The present technology also provides a therapeutic use of an antibody that inhibits IL-11 mediated signaling in combination with an antibody that inhibits IL-17A and/or IL-17F mediated signaling. In such combination the antibodies may be present in the same pharmaceutical composition or alternatively each antibody may be present in a separate pharmaceutical composition.

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

The present technology provides a combination of a first antibody that inhibits IL-11 mediated signaling and a second antibody that inhibits IL-17A and/or IL-17F mediated signaling or pharmaceutical compositions thereof for use as a medicament.

In prophylactic applications, the combination of 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 combination of 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 a combination of a first antibody that inhibits IL-11 mediated signaling and a second antibody that inhibits IL-17A and/or IL-17F mediated signaling or a pharmaceutical composition thereof. The combination of antibodies is administered in a therapeutically effective amount.

The present technology also provides a combination of a first antibody that inhibits IL-11 mediated signaling and a second antibody that inhibits IL-17A and/or IL-17F mediated signaling according to the present technology, or a pharmaceutical composition thereof for use in the treatment of a disorder or condition as described herein.

When each antibody is present in a separate pharmaceutical composition, the first and the second antibody might be administered either simultaneously or subsequently.

Diagnostic use of a combination of antibodies

The present technology also provides a diagnostic use of an antibody that inhibits IL-11 mediated signaling in combination with an antibody that inhibits IL-17A and/or IL-17F mediated signaling, for example, for diagnosing inflammatory diseases or their severity. In one embodiment, the present technology thus provides a combination of an antibody that inhibits IL-11 mediated signaling or an antigen-binding fragment thereof and an antibody that inhibits IL-17A and/or IL-17F mediated signaling or an antigen-binding fragment thereof for use as a diagnostic agent. In one specific embodiment, the present technology provides a combination of an antibody that specifically binds to IL-11 or an antigen-binding fragment thereof and an antibody that specifically binds to IL-17A and/or IL-17F or an antigen-binding fragment thereof for use as a diagnostic agent.

In such combination the antibodies may be present in the same diagnostic composition or alternatively each antibody may be present in a separate diagnostic composition.

The combination of 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) and IL-17A and/or IL-17F 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 and IL-17A and/or IL-17F 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 and an IL-17 binding antibody or binding fragment thereof as described herein; and ii) detecting binding of the IL-11 binding antibody or binding fragment thereof to IL-11 and of the IL-17 binding antibody or binding fragment thereof as described herein to IL-17A and/or IL-17F. By comparing the detected IL-11 and IL-17A and/or IL-17F level with a suitable control, one can then diagnose the presence or likely occurrence of an inflammatory disease associated with IL-11 and IL-17A and/or IL-17F 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, an inflammatory disease associated with IL-11 and IL-17A and/or IL-17F mediated signaling including determining the stage (severity) of said inflammatory disease.

The present disclosure thus provides an in vitro method of diagnosing an inflammatory disease associated with IL- 11 and IL-17A and/or IL-17F mediated signaling in a subject comprising the steps of i) assessing the level or state of IL- 11 and IL-17A and/or IL-17F in a biological sample obtained from the subject by using a combination of an antibody that specifically binds to IL- 11 or a binding fragment thereof and an antibody that binds to IL-17A and/or IL-17F or a binding fragment thereof as described herein; and ii) comparing the level or state of IL-11 and IL-17A and/or IL-17F to a reference, a standard, or a normal control value that indicates the level or state of IL-11 and IL-17A and/or IL-17F in normal control subjects. A significant difference between the level and/or state of the IL-11 and IL-17A and/or IL-17F polypeptide in the biological sample and the normal control value indicates that the individual has an inflammatory disease associated with IL-11 and IL-17A and/or IL-17F mediated signaling.

In one embodiment, the level or state of IL-11 and IL-17A and/or IL-17F in the biological sample is assessed separately. In another embodiment, the level or state of IL- 11 and IL-17A and/or IL-17F in the biological sample is assessed simultaneously.

Therapeutic indications

The multispecific antibodies, the combinations of 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 , IL-17A or IL-17F mediated signaling, for example any condition which results in whole or in part from signaling through the IL-11/1 L- 11 Ra/gp130 complex or the IL-17RA/IL-17RC complex. The antibodies and compositions of the present technology can be used to treat inflammatory skin conditions, systemic sclerosis, idiopathic pulmonary fibrosis (IPF), non-alcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL), or non-alcoholic steatohepatitis (NASH).

NAFLD, NAFL and NASH are also known, 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.

The antibodies and compositions of the present technology can be used to treat inflammatory skin conditions, systemic sclerosis, inflammatory fibrotic diseases of the lung (such as IPF, chronic obstructive pulmonary disease (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).

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. Additionally, it has been found that some IL-11 biology remains unaddressed upon treatment with an antibody that inhibits IL-17A and IL-17F mediated signaling, which suggests that dual blockade of the IL- 11 and IL-17A and/or IL-17F signaling pathways could result in deeper, longer lasting clinical responses.

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

Pharmaceutical and diagnostic compositions

Antibodies 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 multispecific antibodies and the combinations of 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 a pharmaceutical composition comprising a multispecific antibody or a combination of antibodies according to the present technology and a pharmaceutically acceptable agent.

As the combinations of 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 a combination of antibodies 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 combination of antibodies of the present technology or antigen-binding fragments thereof and a diagnostically acceptable carrier. Diagnostic compositions comprise a diagnostically effective amount of the antibody of the present technology.

In one embodiment, the combination of antibodies comprises an antibody that inhibits IL-11 mediated signaling or an antigen-binding fragment thereof and an antibody that inhibits IL-17A and/or IL-17F mediated signaling or an antigen-binding fragment thereof. In one specific embodiment, the combination of antibodies comprises an antibody that specifically binds to IL- 11 or an antigen-binding fragment thereof and an antibody that specifically binds to IL-17A and/or IL-17F or an antigen-binding fragment thereof.

In one embodiment, the present technology thus provides a pharmaceutical composition comprising the multispecific antibody comprising at least two antigen-binding domains, wherein the first antigen-binding domain inhibits IL-11 mediated signaling and the second antigen-binding domain inhibits IL-17A and/or IL-17F mediated signaling and a pharmaceutically acceptable carrier.

In another embodiment, the present technology thus provides a pharmaceutical composition comprising a first antibody that inhibits IL-11 mediated signaling and a second antibody inhibits IL-17A and/or IL-17F mediated signaling.

When a combination of antibodies is used for the treatment and/or prophylaxis of a disorder or condition as described herein, each antibody may also be present in a separate pharmaceutical composition. Such a pharmaceutical composition comprises the individual antibody and a pharmaceutically active 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 multispecific antibodies, combination of 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

Multispecific antibodies, combinations of 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 a multispecific antibody according to the present technology or a pharmaceutical composition thereof for the manufacture of a medicament.

The present technology also provides use of a multispecific 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.

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

The present technology provides use of a combination of antibodies according to the present technology for the manufacture of a medicament wherein each of the antibodies of the combination is separately provided as a pharmaceutical composition comprising a pharmaceutically acceptable agent.

The present technology provides use of a combination of antibodies 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.

The present technology provides use of a combination of antibodies according to the present technology for the manufacture of a medicament for the treatment of a disorder or condition as described herein wherein each of the antibodies of the combination is separately provided as a pharmaceutical composition comprising a pharmaceutically acceptable agent.

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. A multispecific antibody comprising at least two antigen-binding domains, wherein the first antigen-binding domain inhibits IL-11 mediated signaling and the second antigen-binding domain inhibits IL-17A and/or IL-17F mediated signaling. 2. A multispecific antibody according to embodiment 1 , wherein the first antigen-binding domain specifically binds to IL-11 and the second antigen-binding domain specifically binds to IL-17A and/or IL17-F.

3. The multispecific antibody according to embodiment 1 , or 2, wherein the second antigenbinding domain specifically binds to IL-17A.

4. The multispecific antibody according to embodiment 1 , or 2, wherein the second antigenbinding domain specifically binds to IL-17F.

5. The multispecific antibody according to any one of embodiments 1 to 4, wherein the second antigen-binding domain specifically binds to IL-17A and IL-17F.

6. The multispecific antibody according to any one of embodiments 1 to 5, wherein the second antigen-binding domain specifically binds to the IL-17AF heterodimer.

7. The multispecific antibody according to any one of embodiments 1 to 6, wherein IL-11 is human and/or cynomolgus IL-11 ; IL-17A is human and/or cynomolgus IL-17A; and IL-17F is human and/or cynomolgus IL-17F.

8. The multispecific antibody according to any one of embodiments 1 to 7, wherein said first antigen-binding domain specifically binds to human IL-11 and/or the human IL-17AF heterodimer with a KD of less than 100, 50, or 20pM.

9. The multispecific antibody according to any one of embodiments 1 to 8, wherein the first and the second antigen-binding domain each comprise two antibody variable domains.

10. The multispecific antibody according to any one of embodiments 1 to 9, wherein the first antigen-binding domain comprises a VH/VL pair (VH1/VL1) and the second antigen-binding domain comprises a VH/VL pair (VH2/VL2).

11. The multispecific antibody according to any one of embodiments 1 to 10, wherein the first antigen-binding domain comprises a heavy chain variable region (VH1) comprising: a CDR-H1 comprising the amino acid sequence of SEQ ID NO:18, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:19, or a CDR- H2 comprising the amino acid sequence of SEQ ID NO: 19 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:20, or a CDR- H3 comprising the amino acid sequence of SEQ ID NO:20 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 (VL1) comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO:21 , or a CDR- L1 comprising the amino acid sequence of SEQ ID NO:21 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:22, or a CDR- L2 comprising the amino acid sequence of SEQ ID NO:22 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:23.

12. The multispecific antibody according to any one of embodiments 1 to 11 , wherein the first antigen-binding domain comprises a heavy chain variable region (VH1) comprising: a CDR-H1 comprising the amino acid sequence of SEQ ID NO:18, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, 45, 46, 47, 80 or 81 , and a CDR-H3 comprising the amino acid sequence of SEQ ID NQ:20, or 82; and a light chain variable region (VL1) comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO:21 , 66, or 67, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:22, 68, 69, or 70, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:23.

13. The multispecific antibody according to any one of embodiments 1 to 12, wherein the first antigen-binding domain comprises a heavy chain variable region (VH1) comprising: a CDR-H1 comprising the amino acid sequence of SEQ ID NO:18, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:19, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:20; and a light chain variable region (VL1) comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO:21 , a CDR-L2 comprising the amino acid sequence of SEQ ID NO:22, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:23.

14. The multispecific antibody according to any one of embodiments 10 to 13, wherein the

VH1 comprises an amino acid sequence with a sequence identity of more than 90% with SEQ ID NO:24, and the VL1 comprises an amino acid sequence with a sequence identity of more than 90% with SEQ ID NO:26.

15. The multispecific antibody according to any one of embodiments 10 to 14, wherein the VH1 comprises the amino acid sequence of SEQ ID NO:24, and the VL1 comprises the amino acid sequence of SEQ ID NO:26.

16. The multispecific antibody according to any one of embodiments 1 to 15, wherein the second antigen-binding domain comprises a heavy chain variable region (VH2) 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 (VL2) 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.

17. The multispecific antibody according to any one of embodiments 10 to 16, wherein the VH2 comprises an amino acid sequence with a sequence identity of more than 90% with SEQ ID NO:7, and the VL2 comprises an amino acid sequence with a sequence identity of more than 90% with SEQ ID NO:9.

18. The multispecific antibody according to any one of embodiments 10 to 17, wherein the VH2 comprises the amino acid sequence of SEQ ID NO:7, and the VL2 comprises the amino acid sequence of SEQ ID NO:9.

19. The multispecific antibody according to any one of embodiments 1 to 18, wherein the at least two antigen-binding domains are independently selected from a Fab, scFv, Fv, dsFv and dsscFv.

20. The multispecific antibody according to any one of embodiments 1 to 19, comprising: a) a polypeptide chain of formula (I):

VH-i-CH-i- CH₂ -CH_3; b) a polypeptide chain of formula (II):

VL-|-C_L; c) a polypeptide chain of formula (III):

VH₂-CH-|- CH₂ -CH_3; and d) a polypeptide chain of formula (IV):

VL₂-C_L; wherein:

VH-] _anc| VH₂ represent a heavy chain variable domain;

CH-j represents domain 1 of a heavy chain constant region;

CH₂ represents domain 2 of a heavy chain constant region;

CH₃ represents domain 3 of a heavy chain constant region;

VL-] and VL₂ represent a light chain variable domain;

C|_ represents a domain from a light chain constant region, such as

Ckappa; wherein the VH1 and VL1 form a VH/VL pair that specifically binds to IL-11 , wherein the VH₂ and VL₂ form a VH/VL pair that specifically binds to IL-17A and/or IL- 17F, and wherein the polypeptides of Formula I and III are a pair of heavy chain polypeptides in which one polypeptide comprises the knob substitution T366W in the CH₃ domain and the other polypeptide comprises the hole substitutions T366S, L368A and Y407V in the CH₃ domain, wherein the numbering is according to EU as in Kabat.

21. The multispecific antibody according to embodiment 20, wherein the polypeptides of Formula I and III comprise the L234A and L235A substitutions, wherein the numbering is according to EU as in Kabat.

22. The multispecific antibody according to embodiment 20, or 21 , wherein the antibody is an lgG1 isotype.

23. The multispecific antibody according to any one of embodiments 20 to 22, wherein the polypeptide of Formula I comprises the knob substitution T366W in the CH3 domain, and the polypeptide of Formula III comprises the hole substitutions T366S, L368A, Y407V in the CH3 domain.

24. The multispecific antibody according to any one of embodiments 20 to 23, wherein the polypeptide chain of formula (I) comprises an amino acid sequence with a sequence identity of more than 90% with SEQ ID NO:40, the polypeptide chain of formula (II) 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:28, the polypeptide chain of formula (III) comprises an amino acid sequence with a sequence identity of more than 90% with SEQ ID NO: 17, and the polypeptide chain of formula (IV) 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:11.

25. The multispecific antibody according embodiment 24, wherein the polypeptide chain of formula (I) comprises an amino acid sequence of SEQ ID NQ:40, the polypeptide chain of formula (II) comprises an amino acid sequence of SEQ ID NO:28, the polypeptide chain of formula (III) comprises an amino acid sequence of SEQ ID NO:17, and the polypeptide chain of formula (IV) comprises an amino acid sequence of SEQ ID NO:11.

26. The multispecific antibody according to any one of embodiments 20 to 23, wherein the polypeptide chain of formula (I) 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:38, the polypeptide chain of formula (II) 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:28, the polypeptide chain of formula (III) comprises an amino acid sequence with a sequence identity of more than 90% with SEQ ID NO: 15, and the polypeptide chain of formula (IV) comprises an amino acid sequence with a sequence identity of more than 90% with SEQ ID NO:11.

27. The multispecific antibody according to embodiment 26, wherein the polypeptide chain of formula (I) comprises an amino acid sequence of SEQ ID NO:38, the polypeptide chain of formula (II) comprises an amino acid sequence of SEQ ID NO:28, the polypeptide chain of formula (III) comprises an amino acid sequence of SEQ ID NO:15, and the polypeptide chain of formula (IV) comprises an amino acid sequence of SEQ ID NO:11.

28. The multispecific antibody according to any one of embodiments 1 to 19, comprising a) a polypeptide chain of formula (V):

VH-CH-I -X-V-I ; and b) a polypeptide chain of formula (VI):

V|_-C_L-Y-V₂; wherein:

V|_| represents a heavy chain variable domain;

CH-j represents domain 1 of a heavy chain constant region;

X represents a bond or linker; Y represents a bond or linker;

V-j represents a scFv, or a dsscFv;

V|_ represents a light chain variable domain;

C|_ represents a domain from a light chain constant region, such as Ckappa;

V2 represents a scFv, or a dsscFv.

29. The multispecific antibody according to embodiment 28, wherein:

VH and VL form a VH/VL pair of the second antigen-binding domain that specifically binds to IL-17A and/or IL-17F,

V1 comprises a third antigen-binding domain that binds to serum albumin, and V2 comprises the first antigen-binding domain that binds to IL-11.

30. The multispecific antibody according to embodiment 28, or 29, wherein the third antigenbinding domain comprises a heavy chain variable region (VH3) comprising: a CDR-H 1 comprising the amino acid sequence of SEQ I D NO: 110, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:111 , and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:112; and a light chain variable region (VL3) comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO:113, a CDR-L2 comprising the amino acid sequence of SEQ ID NO/114, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 115.

31. The multispecific antibody according to embodiment 30, wherein the VH3 comprises the amino acid sequence of SEQ ID NO:116 or 120, and the VL3 comprises the amino acid sequence of SEQ I D NO: 118 or 122.

32. The multispecific antibody according to embodiment 30, or 31 , wherein the VH3 and VL3 of the third antigen-binding domain are connected by a linker, said linker comprising the sequence given in SEQ ID NO:42.

33. The multispecific antibody according to any one of embodiments 29 to 32, wherein the third antigen-binding domain is a scFv comprising the sequence given in SEQ ID NO:124 or a dsscFv comprising the sequence given in SEQ ID NO: 126.

34. The multispecific antibody according to any one of embodiments 28 to 33, wherein the second antigen-binding domain is a Fab comprising a light chain comprising the sequence given in SEQ I D NO: 11 and a heavy chain comprising the sequence given in SEQ I D NO: 13. 35. The multispecific antibody according to any one of embodiments 28 to 34, wherein the VH1 and VL1 of the first antigen-binding domain are connected by a linker, said linker comprising the sequence given in SEQ ID NO:42.

36. The multispecific antibody according to any one of embodiments 28 to 35, wherein the first antigen-binding domain is a scFv comprising the sequence given in SEQ ID NO:34 or a dsscFv comprising the sequence given in SEQ ID NO:35.

37. The multispecific antibody according to any one of embodiments 28 to 36, wherein Y and/or X is a linker comprising the sequence given in SEQ ID NO:43.

38. The multispecific antibody according to any one of embodiments 28 to 37, wherein the polypeptide of formula (V) comprises an amino acid sequence with a sequence identity of more than 90% with SEQ ID NO:129, or 130, and the polypeptide chain of formula (VI) comprises an amino acid sequence with a sequence identity of more than 90% with SEQ ID NO: 131 , or 132.

39. The multispecific antibody according to any one of embodiments 28 to 39, wherein the polypeptide of formula (V) comprises an amino acid sequence of SEQ ID NO: 130, and the polypeptide chain of formula (VI) comprises an amino acid sequence of SEQ ID NO: 132.

40. An isolated polynucleotide or a combination of isolated polynucleotides encoding the multispecific antibody according to any one of embodiments 1 to 39.

41. An expression vector carrying the polynucleotide according to embodiment 40 or a combination of expression vectors carrying the combination of polynucleotides according to embodiment 40.

42. A host cell or a combination of host cells comprising the vector or the combination of vectors according to embodiment 41.

43. A method of producing the multispecific antibody according to any one of embodiments 1 to 39, comprising culturing the host cell or combination of host cells according to embodiment 42 under conditions permitting production of the antibody, and recovering the antibody produced.

44. A pharmaceutical composition comprising the multispecific antibody according to any one of embodiments 1 to 39 and a pharmaceutically acceptable agent.

45. A pharmaceutical composition comprising a first antibody that inhibits IL-11 mediated signaling and a second antibody that inhibits IL-17A and/or IL-17F mediated signaling and a pharmaceutically acceptable agent, or a diagnostic composition comprising a first antibody that inhibits IL-11 mediated signaling and a second antibody that inhibits IL-17A and/or IL-17F mediated signaling and a diagnostically acceptable agent. 46. The pharmaceutical or diagnostic composition according to embodiment 45, wherein the first antibody specifically binds to IL-11 and the second antibody specifically binds to IL-17A and/or IL-17F, or a diagnostic composition comprising a first antibody that specifically binds to IL- 11 and a second antibody that specifically binds to IL-17A and/or IL- 17F and a diagnostically acceptable agent.

47. The pharmaceutical or diagnostic composition according to embodiment 45, or 46, wherein the second antibody specifically binds to IL-17A.

48. The pharmaceutical or diagnostic composition according to embodiment 45, or 46, wherein the second antibody specifically binds to IL-17F.

49. The pharmaceutical or diagnostic composition according to any one of embodiments 45 to

48, wherein the second antibody specifically binds to IL-17A and IL-17F.

50. The pharmaceutical or diagnostic composition according to any one of embodiments 45 to

49, wherein the second antibody specifically binds to the IL-17A/IL-17F heterodimer.

51. The pharmaceutical or diagnostic composition according to any one of embodiments 45 to

50, wherein the first antibody specifically binds to human IL-11 and/or the human IL-17AF heterodimer with a KD of less than 100, 50, or 20pM.

52. The pharmaceutical or diagnostic composition according to any one of embodiments 45 to

51 , wherein the first and the second antibody each comprise two antibody variable domains.

53. The pharmaceutical or diagnostic composition according to any one of embodiments 45 to

52, wherein the first antibody comprises a VH/VL pair (VH1/VL1) and the second antibody comprises a VH/VL pair (VH2/VL2).

54. The pharmaceutical or diagnostic composition according to any one of embodiments 45 to

53, wherein the first antibody comprises a heavy chain variable region (VH1) comprising: a CDR-H1 comprising the amino acid sequence of SEQ ID NO:18, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:19, and a CDR-H3 comprising the amino acid sequence of SEQ ID NQ:20; and a light chain variable region (VL1) comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO:21 , a CDR-L2 comprising the amino acid sequence of SEQ ID NO:22, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:23.

55. The pharmaceutical or diagnostic composition according to embodiment 53, or 54, wherein the VH1 comprises an amino acid sequence with a sequence identity of more than 90% with SEQ ID NO:24, and the VL1 comprises an amino acid sequence with a sequence identity of more than 90% with SEQ ID NO:26. 56. The pharmaceutical or diagnostic composition according to any one of embodiments 53 to 55, wherein the VH1 comprises the amino acid sequence of SEQ ID NO:24, and the VL1 comprises the amino acid sequence of SEQ ID NO:26.

57. The pharmaceutical or diagnostic composition according to any one of embodiments 45 to

56, wherein the second antibody comprises a heavy chain variable region (VH2) 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 (VL2) 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.

58. The pharmaceutical or diagnostic composition according to any one of embodiments 53 to

57, wherein the VH2 comprises an amino acid sequence with a sequence identity of more than 90% with SEQ ID NO:7, and the VL2 comprises an amino acid sequence with a sequence identity of more than 90% with SEQ ID NO:9.

59. The pharmaceutical or diagnostic composition according to any one of embodiments 53 to

58, wherein the VH2 comprises the amino acid sequence of SEQ ID NO:7, and the VL2 comprises the amino acid sequence of SEQ ID NO:9.

60. The pharmaceutical or diagnostic composition according to any one of embodiments 53 to

59, wherein the first antibody comprises a VH1 comprising the amino acid sequence of SEQ ID NO:24, and a VL1 comprising the amino acid sequence of SEQ ID NO:26; and the second antibody comprises a VH2 comprising the amino acid sequence of SEQ ID NO:7, and a VL2 comprising the amino acid sequence of SEQ ID NO:9.

61. The multispecific antibody according to any one of embodiments 1 to 39, or the pharmaceutical composition according to any one of embodiments 44 to 60, for use as a medicament.

62. The multispecific antibody according to any one of embodiments 1 to 39, or the pharmaceutical composition according to any one of embodiments 44 to 60, for use in the treatment or prevention of an inflammatory skin condition, systemic sclerosis, idiopathic pulmonary fibrosis (I PF), non-alcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL), or non-alcoholic steatohepatitis (NASH).

63. The multispecific antibody according to any one of embodiments 1 to 39, or the pharmaceutical composition according to any one of embodiments 44 to 60, for use in the treatment or prevention of an inflammatory skin condition, systemic sclerosis, an inflammatory fibrotic disease of the lung (such as I PF, COPD, and asthma), an inflammatory fibrotic disease of the liver (such as MASLD, MAFLD, MAFL, and MASH), an inflammatory fibrotic disease 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).

64. The multispecific antibody according to any one of embodiments 1 to 39, or the pharmaceutical composition according to any one of embodiments 44 to 60, for use in the treatment or prevention of hidradenitis suppurativa.

65. Use of the multispecific antibody according to any one of embodiments 1 to 39, or the pharmaceutical composition according to any one of embodiments 44 to 60, for the manufacture of a medicament.

66. Use of the multispecific antibody according to any one of embodiments 1 to 39, or the pharmaceutical composition according to any one of embodiments 44 to 60, for the manufacture of a medicament for the treatment of an inflammatory skin condition, systemic sclerosis, idiopathic pulmonary fibrosis (I PF), non-alcoholic fatty liver disease (NAFLD), nonalcoholic fatty liver (NAFL), or non-alcoholic steatohepatitis (NASH).

67. Use of the multispecific antibody according to any one of embodiments 1 to 39, or the pharmaceutical composition according to any one of embodiments 44 to 60, for the manufacture of a medicament for the treatment of an inflammatory skin condition, systemic sclerosis, an inflammatory fibrotic disease of the lung (such as IPF, COPD, and asthma), an inflammatory fibrotic disease of the liver (such as MASLD, MAFLD, MAFL, and MASH), an inflammatory fibrotic disease 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).

68. Use of the multispecific antibody according to any one of embodiments 1 to 39, or the pharmaceutical composition according to any one of embodiments 44 to 60, for the manufacture of a medicament for the treatment hidradenitis suppurativa.

69. A method of treating or preventing an inflammatory skin condition, systemic sclerosis, idiopathic pulmonary fibrosis (IPF), non-alcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL), or non-alcoholic steatohepatitis (NASH), comprising administering a therapeutically effective amount of the multispecific antibody according to any one of embodiments 1 to 39, or the pharmaceutical composition according to any one of embodiments 44 to 60.

70. A method of treating or preventing an inflammatory skin condition, systemic sclerosis, an inflammatory fibrotic disease of the lung (such as IPF, COPD, and asthma), an inflammatory fibrotic disease of the liver (such as MASLD, MAFLD, MAFL, and MASH), an inflammatory fibrotic disease 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).

71. A method of treating or preventing hidradenitis suppurativa, comprising administering a therapeutically effective amount of the multispecific antibody according to any one of embodiments 1 to 39, or the pharmaceutical composition according to any one of embodiments 44 to 60.

72. A combination of a first antibody that inhibits IL-11 mediated signaling and a second antibody that inhibits IL-17A and/or IL-17F mediated signaling for use as a medicament.

73. A combination of a first antibody that inhibits IL-11 mediated signaling and a second antibody that inhibits IL-17A and/or IL-17F mediated signaling for use in the treatment or prevention of an inflammatory skin condition, systemic sclerosis, idiopathic pulmonary fibrosis (IPF), non-alcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL), or nonalcoholic steatohepatitis (NASH).

74. A combination of a first antibody that inhibits IL-11 mediated signaling and a second antibody that inhibits IL-17A and/or IL-17F mediated signaling for use in the treatment or prevention of an inflammatory skin condition, systemic sclerosis, an inflammatory fibrotic disease of the lung (such as IPF, COPD, and asthma), an inflammatory fibrotic disease of the liver (such as MASLD, MAFLD, MAFL, and MASH), an inflammatory fibrotic disease 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).

75. A combination of a first antibody that inhibits IL-11 mediated signaling and a second antibody that inhibits IL-17A and/or IL-17F mediated signaling for use in the treatment or prevention of hidradenitis suppurativa.

76. Use of a combination of a first antibody that inhibits IL-11 mediated signaling and a second antibody that inhibits IL-17A and/or IL-17F mediated signaling for the manufacture of a medicament. 77. Use of a combination of a first antibody that inhibits IL-11 mediated signaling and a second antibody that inhibits IL-17A and/or IL-17F mediated signaling for the manufacture of a medicament for the treatment of an inflammatory skin condition, systemic sclerosis, idiopathic pulmonary fibrosis (I PF), non-alcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL), or non-alcoholic steatohepatitis (NASH).

78. Use of a combination of a first antibody that inhibits IL-11 mediated signaling and a second antibody that inhibits IL-17A and/or IL-17F mediated signaling for the manufacture of a medicament for the treatment of an inflammatory skin condition, systemic sclerosis, an inflammatory fibrotic disease of the lung (such as IPF, COPD, and asthma), an inflammatory fibrotic disease of the liver (such as MASLD, MAFLD, MAFL, and MASH), an inflammatory fibrotic disease 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).

79. Use of a combination of a first antibody that inhibits IL-11 mediated signaling and a second antibody that inhibits IL-17A and/or IL-17F mediated signaling for the manufacture of a medicament for the treatment hidradenitis suppurativa.

80. A method of treating or preventing an inflammatory skin condition, systemic sclerosis, idiopathic pulmonary fibrosis (IPF), non-alcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL), or non-alcoholic steatohepatitis (NASH), comprising administering a therapeutically effective amount of a combination of a first antibody that inhibits IL-11 mediated signaling and a second antibody that inhibits IL-17A and/or IL-17F mediated signaling.

81. A method of treating or preventing an inflammatory skin condition, systemic sclerosis, an inflammatory fibrotic disease of the lung (such as IPF, COPD, and asthma), an inflammatory fibrotic disease of the liver (such as MASLD, MAFLD, MAFL, and MASH), an inflammatory fibrotic disease 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).

82. A method of treating or preventing hidradenitis suppurativa, comprising administering a therapeutically effective amount of a combination of a first antibody that inhibits IL-11 mediated signaling and a second antibody that inhibits IL-17A and/or IL-17F mediated signaling.

83. A combination of a first antibody that inhibits IL-11 mediated signaling and a second antibody that inhibits IL-17A and/or IL-17F mediated signaling for use as a diagnostic agent.

84. A combination of a first antibody that specifically binds to IL-11 and a second antibody that specifically binds to IL-17A and/or IL-17F for use as a diagnostic agent. 85. The combination for use according to any one of embodiments 72 to 75, 83, or 84, the use according to any one of embodiments 76 to 79, or the method according to any one of embodiments 80 to 82, wherein the first antibody specifically binds to IL-11 and the second antibody specifically binds to IL-17A and/or IL-17F.

86. The combination for use according to any one of embodiments 72 to 75, 83, or 84, the use according to any one of embodiments 76 to 79, or the method according to any one of embodiments 80 to 82, wherein the second antibody specifically binds to IL-17A.

87. The combination for use according to any one of embodiments 72 to 75, 83, or 84, the use according to any one of embodiments 76 to 79, or the method according to any one of embodiments 80 to 82, wherein the second antibody specifically binds to IL-17F.

88. The combination for use according to any one of embodiments 72 to 75, 83, or 84, the use according to any one of embodiments 76 to 79, or the method according to any one of embodiments 80 to 82, wherein the second antibody specifically binds to IL-17A and IL-17F.

89. The combination for use according to any one of embodiments 72 to 75, 83, or 84, the use according to any one of embodiments 76 to 79, or the method according to any one of embodiments 80 to 82, wherein the second antibody specifically binds to the IL-17AF heterodimer.

90. The combination for use according to any one of embodiments 72 to 75, 83, or 84, the use according to any one of embodiments 76 to 79, or the method according to any one of embodiments80 to 82, wherein each antibody of the combination is independently selected from a full length antibody, Fab, scFv, Fv, dsFv and dsscFv.

91 . The combination for use according to any one of embodiments 72 to 75, the use according to any one of embodiments 76 to 79, or the method according to any one of embodiments80 to 82, wherein each of the antibodies of the combination is separately provided as a pharmaceutical composition comprising a pharmaceutically acceptable agent.

92. The combination for use according to embodiment 83, or 84, wherein each of the antibodies of the combination is separately provided as a diagnostic composition comprising a diagnostically acceptable agent.

93. An agent capable of inhibiting IL-11 and IL-17A and/or IL-17F mediated signaling for use in the treatment of an inflammatory disease.

94. Use of an agent capable of inhibiting IL-11 and IL-17A and/or IL-17F mediated signaling for the manufacture of a medicament.

95. Use of an agent capable of inhibiting IL-11 and IL-17A and/or IL-17F mediated signaling for the manufacture of a medicament for the treatment of an inflammatory disease. 96. A method of treating an inflammatory disease, comprising administering a therapeutically effective amount of an agent capable of inhibiting IL-11 and IL-17A and/or IL-17F mediated signaling.

97. An agent for use according to embodiment 93, use of an agent according to embodiment 95, or the method according to embodiment 96, wherein the inflammatory disease is an inflammatory skin condition, systemic sclerosis, idiopathic pulmonary fibrosis (IPF), nonalcoholic fatty liver disease (NAFLD), non-alcoholic fatty liver (NAFL), or non-alcoholic steatohepatitis (NASH).

98. An agent for use according to embodiment 93, use of an agent according to embodiment 95, or the method according to embodiment 96, wherein the inflammatory disease is an inflammatory skin condition, systemic sclerosis, an inflammatory fibrotic disease of the lung (such as IPF, COPD, and asthma), an inflammatory fibrotic disease of the liver (such as MASLD, MAFLD, MAFL, and MASH), an inflammatory fibrotic disease of the heart (such as congestive heart failure, myocardial infarction, and ischemic heart disease), or an endometrial disease (such as endometriosis, and adenomyosis).

99. An agent for use according to any one of embodiments 93, 97 or 98, use of an agent according to any one of embodiments 95, 97 or 98, or the method according to any one of embodiments 96, 97 or 98, wherein the inflammatory disease is hidradenitis suppurativa.

100. An agent for use according to any one of embodiments 93 or 97 to 99, use of an agent according to any one of embodiments 94, 95, or97 to 99, or the method according to any one of embodiments 96 to 99, wherein the agent is an antibody or a combination of antibodies capable of inhibiting IL-11 and IL-17A and/or IL-17F mediated signaling.

101 . An agent for use according to embodiment 100, use of an agent according to embodiment 100, or the method according to embodiment 100, wherein the agent is an antibody that specifically binds to IL-11 and IL-17A and/or IL-17F.

102. An agent for use according to embodiment 100 or 101 , use of an agent according to embodiment 100 or 101 , or the method according to embodiment 100 or 101 , wherein the agent is a multispecific antibody according to any one of embodiments 1 to 39.

103. An agent for use according to embodiment 100, use of an agent according to embodiment 100, or the method according to embodiment 100, wherein the combination of antibodies comprises a first antibody that inhibits IL-11 mediated signaling and a second antibody that inhibits IL17-A and/or IL-17F mediated signaling.

104. An agent for use according to embodiment 103, use of an agent according to embodiment 103, or the method according to embodiment 103, wherein the first antibody specifically binds to IL-11 and the second antibody specifically binds to IL-17A and/or IL-17F. 105. An agent for use according to embodiment 103 or 104, use of an agent according to embodiment 103 or 104, or the method according to embodiment 103 or 104, wherein the first and the second antibody are present in a separate pharmaceutical composition.

106. An agent for use according to embodiment 103 or 104, use of an agent according to embodiment 103 or 104, or the method according to embodiment 103 or 104, wherein the first and the second antibody are present in the same pharmaceutical composition.

107. An agent for use according to embodiment 106, use of an agent according to embodiment 106, or the method according to embodiment 106, wherein the pharmaceutical composition is a composition according to any one of embodiments 45 to 60.

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-7.

Table A-1. Sequences related to IL-17 binding domain 496. g3 (also referred to herein as 4211)

Table A-2. Sequences related to IL-11 binding domain 19439gL1gH1

* Mutated with cysteines engineered for a disulfide bond

Table A-3. Sequences related to albumin binding domain 645 * Mutated with cysteines engineered for a disulfide bond

Table A-4. Sequences related to linkers

Table A-5. Sequences related to Tribody and TrYbe molecules

Table A-6. Sequences related to variants of the IL-11 binding domain 19439

Table A-7. Other sequences

Table A-8. Sequences of IL-11 and IL-17 proteins

EXAMPLES

It should be noted that in the examples below when IL-17A or IL-17F is being used as a tool protein, IL-17A refers to the IL-17AA homodimer and IL-17F refers to the IL-17FF homodimer.

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 (ATUM) 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 (ATLIM) 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 Ill

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:133) and recombinant Cyno IL-11 protein (SEQ ID NO:137) 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. These three antibodies were tested in a Hydrophobic Interaction Chromatography (HIC) assay to determine the suitability of the VRs for the bispecific format. This aided in the selection of one antibody, referred to as VR19439 as the lead molecule, as in contrast to the two other antibodies, it showed a favourable separation profile from the anti-IL-17 arm to be used in the bispecific as well as being a high affinity, potent inhibitor of IL-11 induced STAT3 activation.

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:26), 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:24), 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-2.

Example 4. Generation of anti-IL17 antibody The production of the antibody CA028_00496.g3 (also referred to herein as antibody 496. g3 or 4211) against human IL-17A and human IL-17 has been previously described in WO20 12/095662. The antibody binds human IL-17A, IL-17F and IL-17AF heterodimer with pM affinity. The amino acid and DNA sequences encoding the CDRs, heavy and light variable regions and light chain and heavy chain of the Fab format of antibody 496. g3 are shown in Table A-1. The 496. g3 Fab constant regions comprised the human C-kappa constant region (K1 m3 allotype) and the human gamma-1 CH1 constant region and hinge (G1m17 allotype).

Example 5. Generation of anti-human albumin antibody 645

The production of the anti-human albumin antibody 645 has been previously described in WO2013/068571. The amino acid and DNA sequences encoding the CDRs, heavy and light variable regions, scFv and dsscFV formats of antibody 645 are listed in Table A-3.

Example 6: Generation of the 19439gL1gH1 / 4211 KiH hlgG1 LALA bispecific (transient expression)

Parental monoclonal antibodies (mAbs) human lgG1 with heavy chain mutations L234A L235A and either T366W (knob) or L366S L368A, and Y407V (hole) were expressed from CHO-SXE cells and purified by standard Protein A affinity chromatography. The bispecific, parental mAbs were mixed at a 1 :1 ratio in the presence of 5 mM beta-mercaptoethylamine and incubated for 16-18 h at room temperature. Subsequently, high molecular weight species and I or parental mAbs were removed by preparative size exclusion chromatography (SEC) using a HiLoad Superdex 200, 16/600 column (Cytiva) equilibrated in PBS pH 7.4. Percentage bispecific was determined by analytical HIC chromatography and percentage monomeric purity was determined by analytical SEC (BEH200) and SDS-PAGE. Endotoxin levels were determined for all material and if necessary reduced using High-Capacity Endotoxin Removal Spin Columns (Generon) to a final level of less than 1 Ell/rng (LAL assay).

Mass spectra were obtained on a reduced deglycosylated sample and the LC masses 23409 Da (expected 23414 Da) and 23611 Da (expected 23611 Da) were observed as well as the major lysine clipped HC masses 49699 Da (expected 49698 Da) and 48969 Da (expected 48971 Da) and minor mass of 49097 Da (expected 49099 Da) representing approximately 10% full length HC. Intact mass was determined after treating the sample with PNGase F under non-reducing conditions. SEC chromatography showing a crude mixture prior to preparative gel filtration had a purity around 100% of the desired bispecific observed molecular weight 145661 Da (145663 Da calculated). Figure 7 depicts the results obtained in the various assays used for analysis.

Analytical hydrophobic interaction Chromatography (HIC) assay The following HIC method used a Dionex ProPac HIC-10 column (100mmx4.6 mm). The column stationary phase consisted of a mixed population of ethyl and amide functional groups bonded to silica. All separations were carried out on an Agilent 1260 HPLC (Agilent, Santa Clara, CA, USA) equipped with a fluorescence detector. The column temperature was maintained at 20°C throughout the run and the flow rate was 0.8 mL/min. The mobile phase for the HIC method consists of 0,8M ammonium sulfate, 50 mM phosphate pH 7,4 (Buffer A) and 50 mM phosphate pH 7,4 (Buffer B). Following a 2 min hold at 0% B, bound protein is eluted using a linear gradient from 0 to 100% B in 45 min and the column is washed with 100% B for 2 min and re-equilibrated in 0% B for 10 min prior to the next sample. The separation is monitored by intrinsic fluorescence with excitation occurring at 280 nm and emission at 340 nm.

Analytical SEC

An Acquity UPLC Protein BEH SEC 200A, 1.7um, 4.6 X 150 mm column (Waters) was attached to an Acquity UPLC system equipped with TUV detector (Waters) and column temperature was maintained at 20°C. The mobile phase (200 mM Sodium Phosphate pH7,3 buffer) was passed over the column at a constant flow rate of 0,35 ml/min.

Mass spectrometry

Protein in PBS was treated with PNGase F under reducing conditions (NEB, P0710S) according to manufacturer’s instructions. Mass spectra were acquired on a LC/MS system comprised of a Waters Acquity UPLC and Waters Xevo G2 QTof MS. Approximately 2mg protein was loaded onto a reverse phase chromatography column (BioResolve RP mAB Polyphenyl 450 A, 2.7 pm, 2.1 x 150 mm) at 80°C, solvent flow at 0.6 mL I min and a gradient from 5% to 50% acetonitrile with 0.02 % TFA modifier was applied. MS data were recorded in positive ESI mode over a mass range from 400 to 5000 m/z, and data were deconvoluted using MassLynx and MaxEnt software.

Example 7: Generation of the anti-IL-11 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 8: 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

Example 9: 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 10: 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, as described for the murinized antibody in Example 9, 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 11 : Binding kinetics of 19439gL1gH1 / 4211 KiH hlgG1 LALA to human and cynomolgus monkey IL-11, IL-17A, IL-17F, and mouse IL-11 The kinetics of 19439gL1gH1 I 4211 KiH hlgG1 LALA binding to human and cynomolgus monkey IL-11 , IL-17A, IL-17F and IL-17AF, and murine IL-11 were measured at 25°C by surface plasmon resonance on a Biacore T200 or Biacore 8K+ instrument (Cytiva). The kinetics of both 19439gL1gH1 Fab or 19439gL1gH1 IgG LALA to human and cynomolgus monkey IL-11 were also assessed. 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 19439gL1gH1

I 4211 KiH hlgG1 LALA, 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) for IL-11 , and a concentration range of 5 to 0.16nM (3-fold dilutions) for IL-17A, IL-17F and IL-17AF. In individual measurements where the reported dissociation rate was below 1 E-5 (the limit of sensitivity of the Biacore T200), the dissociation rate was fixed at 1 E-5 and used to estimate the KD.

As summarised in Table X-6, 19439gL1gH1 I 4211 KiH hlgG1 LALA was shown to bind with high affinity to human IL-11 , IL-17A, IL-17AF and IL-17F (KD of 13.8pM, 5.9pM, 13.2pM and 83.5pM respectively), to cross react with cynomolgus monkey IL-11 , IL-17A, IL-17AF, IL-17F (KD of 26.6pM, 5.6pM, 13.8pM and 212. OpM respectively) and to retain binding to mouse IL-

I I with an approximate 6-fold decrease in affinity (77.1 pM, n=1) compared to human. The affinities of the 19439gL1gH1 Fab and 19439gL1gH1 IgG LALA to human and cynomolgus monkey IL-11 were in line with those of the bispecific 19439gL1gH1 14211 KiH hlgG1 LALA.

Table X-6. Binding affinity of 19439gL1gH1 I 4211 KiH hlgG1 LALA, 19439gL1gH1 Fab and 19439gL1gH1 IgG LALA

Example 12: Blocking of the IL-11 :slL-11 R interaction by VR19439 and 19439gl_1gH1 / 4211 KiH hlgG1 LALA

IL-11 Ra Receptor Blocking by Parental 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 on a mouse backbone 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 Rll. Flow cells 1 and 3 remained blank and were used for reference subtraction for flow cells 2 and 4 respectively. Approximately 150 to 200RLI 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-7, 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-7. Binding of IL-11 Ra to IL-11 in the presence of anti-IL-11 Fabs

IL-11 Ra Receptor Blocking by Bispecific 19439gL1gH1 14211 KiH hlgG1 LALA Experiments were conducted on a Biacore T200 (Cytiva) at 25°C, by immobilsing a goat antihuman F(ab’)2 specific F(ab’)2 fragment (Jackson ImmunoResearch) on flow cell 2 of a CM5 sensorchip to a level of approximately 6000 Rll. A control surface was prepared on the same chip by immobilising a goat anti-mouse F(ab’)2 specific F(ab’)2 (Jackson ImmunoResearch) to flow cell 4 to approximately 3500RLI. Flow cells 1 and 3 remained blank and were used for reference subtraction for flow cells 2 and 4 respectively. Approximately 300RLI of 19439gL1gH1 I 4211 KiH hlgG1 LALA was then captured to the anti-human surface and approximately 150RLI of a non-blocking anti-IL-11 mouse Fab control (VR20008 generated inhouse) was captured to the anti-mouse surface by injecting each sample over the relevant flow cell for 1 min at 10pl/min. Analysis consisted of injection of 50nM IL-11 or buffer blank for 180s, followed by 100nM 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 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 19439gL1gH1 I 4211 KiH hlgG1 LALA but bound (with a response of 54.8RU) in the presence of the VR20008 non-blocking control Fab.

Table X-8. Binding of IL-11 Ra to IL-11 in the presence of 19439gL1gH1 / 4211 KiH hlgG1 LALA

Example 13: Confirmation of 19439gL1gH1 1 4211 KiH hlgG1 LALA 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 , and 10:1 molar ratio of Antibody to IL-11(IL-11 @ 100ng/ml ~5.18nM, KiH molecules 5.18nM = 777ng/ml or 51.8nM = 7.77pg/ml). Transfected cells were resuspended in cell staining buffer containing BSA and NaN₃ (Biolegend®) and chilled on ice before use. Isotype control hlgG1 LALA, 19439gL1gH1 / 4211 KiH hlgG1 LALA, VR20008 IgG and VR24979 IgG molecules were prebound with unlabelled IL-11 (or buffer alone) at 1 :1 or 10:1 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 8.

19439gL1gH1 / 4211 KiH hlgG1 LALA does not bind to IL-11 R or IL-11 RA+ gp130 expressing cells in the presence of IL- 11 indicating non internalization properties. VR24979 and VR20008 (non-blocking VR) do bind in the presence of IL-11.

Example 14: 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 ~5.18nM, Fab fragments 5.18nM = 259ng/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 9.

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 15: 19439gl_1gH1 / 4211 KiH hlgG1 LA LA mammalian cell line development

To demonstrate the stable expression of 19439gL1gH1 I 4211 KiH hlgG1 LALA, two stably expressing mammalian cell lines were created: one expressing the anti-IL-11 knob-knob homodimer antibody and one expressing the anti-IL-17A and F hole-hole homodimer antibody. The antibodies harvested from the cell lines subsequently underwent an in vitro exchange step, as described in Example 6 above, to form the correct heterodimer knob-hole antibody product.

Separate CHO host cell lines were transfected with the plasmids 19439gL1gH1 hg1 LALA Hole IgG and VR4211 Kappa LALA Knob IgG. Polyclonal pool cell lines were generated, they were then cloned and evaluated for fit to a suitable manufacturing process. To assess the quality and quantity of the protein of interest and to ensure the optimal cell line was selected, the cell line was evaluated in a micro-scale model of a manufacturing fed-batch bioreactor. Lead clonal CHO cell lines were selected that express the antibodies at acceptable levels.

Example 16: Inhibition of human, cynomolgus monkey, and murine IL-11 induced cis- STAT3 signaling by 19439gL1gH1 14211 KiH hlgG1 LALA, and control molecules using a human HepG2 IL-11R/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 gfor 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), recombinant cynomolgus monkey IL-11 (in-house material) or recombinant murine IL-11 (in-house material). The antibody titration/l L-11 mixture-containing dilution plate was incubated in a 37°C I 5% 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 I 5% 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 of these experiments are summarised in Table X-9 (human IL-11), Table X-10 (cyno IL-11) and Table X-11 (murine IL-11) and Figures 10 to 12 (human IL-11), Figure 13 (cyno IL-11) and Figure 14 (murine IL-11).

Different antibody formats which comprised the VR 19439 gL1/gH1 (Fab and lgG1 LALA) were found to be potent and efficacious inhibitors of human IL-11 cis signaling in this assay, with comparable potency and efficacy between the different formats (Figure 10, Table X-9). There was also comparable potency and efficacy between 19439gL1gH1 /4211 KiH lgG1 LALA produced by different bispecific production methods (in vitro transient and in vitro stable; Figure 11 , Table X-9). The various control molecules behaved as anticipated, with the lgG1 LALA isotype control and the 18136 (Null)/4211 KiH lgG1 LALA antibody having no activity. The 19439gL1gH1 /18136 (Null) KiH lgG1 LALA antibody had similar activity to 19439gL1gH1 /4211 KiH lgG1 LALA. (Figure 12, Table X-9) VR18136 (Null) is a VR region which binds to an irrelevant protein.

Table X-9 Summary of potency and efficacy values for different antibody formats with VR 19439gl_1gH1 (various formats) and control molecules against the human IL-11 induced cis-signaling in the Hepg2 IL-11 R reporter cell assay. ND = Not determined.

Against cynomolgus monkey IL-11 induced cis-signaling, 19439gL1gH1 /4211 KiH lgG1 LALA was a potent and efficacious inhibitor. Again, the isotype control lgG1 LALA had no activity (Figure 13, Table X-10).

Table X-10 Summary of potency and efficacy values for 19439gL1gH 1/4211 KiH lgG1 LALA and the isotype control lgG1 LALA against cynomolgus monkey IL-11 induced cis-signaling in the Hepg2 IL-11 R reporter cell assay. Lastly, 19439gL1gH1 /4211 KiH lgG1 LALA was a potent and efficacious inhibitor of murine IL-11 induced cis-signaling while the isotype control had no activity. (Figure 14, Table X-11).

Table X-11 Summary of potency and efficacy values for 19439gl_1gH1 74211 KiH lgG1 LALA and the isotype control lgG1 LALA against murine IL-11 induced cis-signaling in the Hepg2 IL-11R reporter cell assay. ND = Not determined.

Example 17: Inhibition of IL-11 mediated trans-STAT3 signaling by 19439gL1gH1 / 4211 KiH hlgG1 LALA and 19439gL1gH1 Fab.

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 ‘non-displacement’ format) or assessed for their ability to block trans-signaling in the ‘displacement’ format, where IL-11 was first incubated with soluble IL-11 RA to enable IL-11/IL-11 RA complex formation, with subsequent addition of antibody to the complex. In the ‘non-displacement’ format assay, 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 I 5% CCh for 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 15% CO2. In the ‘displacement’ format, human IL-11 (inhouse material) was first incubated at 37°C 15% CO2with recombinant soluble IL-11 RA (R&D #8895-MR-050) for 60 minutes at 37°C I 5% CO2, then, the serial dilution of antibodies was added to the solution containing the soluble IL-11 RA/IL-11 complex and incubated for 30 minutes at 37°C 1 5% CO2. Next, in both formats, 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/IL-11 RA complex and serum free media alone, as assay maximum and minimum values, respectively. The assay plates were incubated in a 37°C I 5% 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, 50pl 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-12 (non-displacement format) and Table X-13 (displacement format) and Figure 15 (non-displacement format) and Figure 16 (displacement format).

19439gL1gH1 I 4211 KiH hlgG1 LALA and 19439gL1gH1 Fab were found to be potent and efficacious inhibitors of IL-11 trans-signaling in the non-displacement format. (Figure 15, Table X-12). In the displacement format, 19439gL1gH1 / 4211 KiH hlgG1 LALA was still found to be a potent and efficacious inhibitor of IL-11 trans-signaling, with comparable potency and efficacy to the results in the ‘non-displacement’ format. (Figure 16, Table X-13).

Table X-12 Summary of potency and efficacy values for 19439gL1gH 1/4211 KiH lgG1 LALA, 19439gL1gH1 Fab, and isotype control lgG1 LALA in the ‘non-displacement’ STAT3 trans-signaling assay. ND = Not determined.

Table X-13 Summary of potency and efficacy values for 19439gL1gH 1/4211 KiH lgG1 LALA and isotype control lgG1 LALA in the ‘displacement’ STAT3 trans-signaling assay. ND = Not determined.

Example 18: Inhibition of IL-17 mediated IL-6 release by 19439gL1gH1 14211 KiH hlgG1 LALA using primary human dermal fibroblasts

For the evaluation of IL-17 signaling inhibition by antibodies, a primary HDF assay was run in which the cells were stimulated with both IL-17AA and TNF-a. Stimulation of normal HDF with IL-17 alone resulted in only a small amount of IL-6 release, but in combination with TNF-a, a synergistic response occurred resulting in sufficient IL-6 release for a reliable and robust screening assay readout. This resultant IL-6 response was measured using a homogenous time-resolved FRET kit. The kit utilised two monoclonal antibodies, one labelled with Eu- Cryptate (Donor) and the second with d2 or XL665 (Acceptor). The intensity of the signal is proportional to the concentration of IL-6 present in the sample (ratio is calculated by 665/620 x 10⁴). A reduction in the FRET signal demonstrates the functional IL-17 neutralising activity of the tested antibodies. The assay is described in more detail below.

Primary normal neonate human dermal fibroblasts (Sigma/Public Health England #106-05n) were used and cultured using standard sterile cell culture techniques. On the day of the assay they were detached from the flasks by first removing the media, washing the cells in DPBS, removing the DPBS, and adding 3ml of TrypLE detachment reagent. The cells were then incubated at 37°C I 5% CO2 for 2-3 minutes, until the cells had detached from the plastic. Assay media was added, and the cells were centrifuged at 300 x g for 3 minutes at room temperature. The media was then removed from the cell pellet and the cells were resuspended in 5ml of fresh assay media and counted. The cells were resuspended to a density of 3.125x10⁴ cells/ml and seeded into tissue-culture treated 384-well plates (Corning #3701) in a 40pl/well volume. The plates were then transferred to a 37°C 15% CO2 incubator and placed on a heat block for 3-4 hours. The antibodies were diluted in assay media and titrated in low- binding 384-well plates (Greiner #781280) for a total of a 20-point serial dilution. The antibodies were diluted to 30X desired assay final concentration and added in a 30pl/well volume to column three of the low-binding 384-well plates and 18pl of DPBS was added to all other wells. 12pl was transferred between columns with a 15pl/well mix volume using a liquid handling robot. The cytokines IL-17AA and TNF-a were diluted in assay media and added to low-binding 384-well plates (Greiner #781280) in a 30pl/well volume. They were made up at a concentration so that upon dilution in the assay the final assay concentration of IL-17AA was 50pM and the finally assay concentration of TNF-a was 25pM. Manually, 10pl/well was transferred from the antibody titration plates to the ligand plates and antibody/ligand plates were transferred to a 37°C 15% CO2 incubator for 5 hours. After this incubation, 10pl/well was transferred from the antibody/ligand plate to the cell plates. The cell plates were then incubated on a heat block at 37°C I 5% CO2 for 18 ± 2 hours. After this incubation, antibodies from the Human IL-6 HTRF detection kit (Cisbio #62HIL06PEH) were combined in dilution buffer as described in the manufacturer’s instructions and added to a 384-well white HTRF plate (Greiner #784075). The kit utilises two monoclonal antibodies, one labelled with Eu-Cryptate (Donor) and the second with d2 or XL665 (Acceptor). Media from the cell plate was then diluted 1 in 3 with assay media and then 10pl/well was transferred to the HTRF plate containing the diluted antibodies. The HTRF plate was incubated for 2 hours at RT, sealed with a foil plate seal, on a 300RPM plate shaker. After the incubation, the plate was read on the Biotek Synergy Neo 2 using the Gen 5 software using the protocol: IL-17_HTRF_Ex330_Em620_665. The intensity of the signal is proportional to the concentration of IL-6 present in the sample (ratio is calculated by 665/620 x 10⁴). A reduction in the FRET signal demonstrated the functional activity of the tested antibodies. The ratio values were used to calculate the percentage inhibition values using Microsoft Excel, with the maximum values calculated from the average ratio value for the ‘IL-17A + TNF-a’ condition and the minimum values calculated from the average ratio value for the ‘TNF-a’ alone condition. 4PL-curve fitting was performed using GraphPad Prism.

The results are summarised in Table X-14 and Figure 17.

19439gL1gH1/4211 KiH lgG1 LALA (in vitro transient material) completely inhibited IL-17 mediated IL-6 release with an average Emax of 103% (N=2). An accurate IC50 could not be determined in the two experiments that were run, however, as the hill slopes were too steep for accurate determination of potency. It is likely that 19439gL1gH 1/4211 KiH IgG 1 LALA is at the biochemical limit of this assay, and for accurate IC50 determination a more sensitive assay with a lower IL-17 concentration should be used. The isotype control lgG1 LALA had no functional activity in this assay.

Table X-14 Summary of potency and efficacy values for 19439gL1gH 1/4211 KiH lgG1 LALA and the isotype control lgG1 LALA in the IL-17 mediated IL-6 release assay on primary human dermal fibroblasts. ND = not determined.

Example 19: Inhibition of cis IL-11 induced CCL2 release by 19439gL1gH1 / 4211 KiH hlgG1 LALA on primary human dermal fibroblasts

To characterise the ability of the antibodies to functionally inhibit cis-IL-11 mediated CCL2 release in a primary cell system, primary HDF were used. The addition of IL-11 to these cells resulted in the release of CCL2 which was subsequently measured in the HDF supernatants using a detection kit. These kits use sandwich ELISA methods coupled with electro-chemiluminescence (ECL) detection and plate array technology to provide highly sensitive and multiplexed detection of analytes. A reduction in the CCL2 levels (as determined by ECL signal) demonstrates the functional activity of the tested antibodies. The assay is described in more detail below.

Primary HDF from three separate donors were used (Promocell #C12302, lot #472Z001.3, #469Z015 and #469Z026.2). The cells were thawed from a frozen vial using standard techniques and seeded at 5,000 cells/well in flat-bottomed clear 96-well plates (Corning #353072) in growth media containing serum (Fibroblast growth medium 2 containing the contents of the Growth Medium 2 kit, Promocell #C-23120) and then incubated for 72 hours at 37°C, 5% CO2. Recombinant IL-11 (in-house material) was then diluted in basal media, ready to be added to the assay plates. Antibodies were serially diluted in the IL-11 containing basal media for a total of a 9-point 1 :3.5-fold dilution series using 96-well deep well plates (VWR #MATR4222). IL-11 alone and basal media alone were also added to the plates as the assay maximum and minimum conditions, respectively. The deep well plates containing the serial dilution of antibody and IL-11 were incubated for 30 minutes at 37°C, 5% CO2. The growth media was removed from assay plates, the plates were washed once with basal media (Fibroblast growth medium 2 without the contents of the Growth Medium 2 kit added), and then 100 pl of IL-11/antibody sample was transferred to each well from the deep well plates by hand. The assay plates were then returned to the 37°C I 5% CO2 incubator for 48 hours. The final assay concentration of IL-11 was 5ng/ml (259pM). After the 48-hour incubation, the cell supernatants were measured for CCL2 (also known as MCP-1) using a U-PLEX Human MCP- 1 Assay kit from MSD (MSD #K151 UGK-4) according to the manufacturer’s instructions. Percentage inhibition was calculated with Microsoft Excel using IL-11 stimulated and basal media alone stimulated cells as the maximum and minimum values, respectively. 4PL curve fitting and the calculation of IC50 and efficacy (Emax) values was performed using GraphPad Prism.

The activity of 19439g L1gH 1/4211 KiH I gG 1 LALA in vitro transient material, and in vitro stable material) and an lgG1 LALA isotype control were measured in this cis-IL-11 mediated CCL2 release assay. The results are summarized in Table X-15 and Figure 18.

19439gL1gH1/4211 KiH lgG1 LALA is a potent and efficacious inhibitor of IL-11 induced cissignaling in a primary cell system with equivalent functional activity between the different production methods. The lgG1 LALA isotype control did not functionally inhibit in this assay. Figure 18 shows a representative example from one experiment in one donor (#472Z001.3) where 19439gL1gH 1/4211 KiH lgG1 LALA produced by the two different methods as well as the lgG1 LALA isotype control were tested head-to-head. For each experiment, the three individual IC50 values from each donor were used to calculate an experimental geomean IC50. Additionally, the three individual Emax values were used to calculate an average experimental Emax. Then, from the experiments, the overall geomean IC50 and average Emax was calculated. These values are summarised in Table X-15.

Table X-15 Summary of potency and efficacy values for 19439gl_1gH1/4211 KiH lgG1 LALA {in vitro transient material, and in vitro stable material) and the isotype control lgG1 LALA in the cis IL-11 CCL2 release assay.

Example 20: Inhibition of IL-11 and IL-17AA mediated CXCL1 release by 19439gL1gH1 Fab 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 U-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 was measured in this assay.

19439gL1gH1 Fab was a potent and efficacious inhibitor of IL-11/IL-17 mediated CXCL1 release on primary HDF. (Table X-16, Figure 19).

Table X-16 Summary of potency and efficacy values for 19439gL1gH1 Fab in the cis human IL-11 and IL17AA CXCL1 release assay.

Example 21 : Inhibition of human and cynomolgus monkey IL-17 induced NF-kB activation by 19439gL1gH1 / 4211 KiH hlgG1 LA LA and relevant control antibodies using a HEK Blue IL-17 reporter cell line

The HEK Blue IL- 17 reporter cells were generated by stable transfection of the human genes encoding the IL-17RA/IL-17RC heterodimeric receptor. HEK-Blue IL-17 cells also express an NF-KB- and AP-1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. When IL-17AA or IL-17FF were added to the cells, a downstream signaling pathway was activated which resulted in the production of SEAP. The level of SEAP was therefore used to determine the level of IL-17 signaling pathway activation. SEAP can be detected using the QUANTI-Blue system by measuring absorbance on a plate reader, with a reduction in absorbance demonstrating the functional activity of the tested antibodies.

The HEK Blue IL-17 reporter cells (Invivogen #hkb-il17) were generated by stable transfection of the human genes encoding the IL-17RA/IL-17RC heterodimeric receptor. HEK-Blue IL-17 cells also express an NF-KB- and AP-1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. In the assay, the HEK Blue IL-17 cells were cultured using standard techniques until approximately 70-80% confluent. They were then detached from the cell culture flasks using TrypLE Express enzyme (ThermoFisher Scientific #12604021) and counted and resuspended in HEK Blue Assay medium (DMEM, 4.5 g/l glucose, 2mM L- Glutamine, 10% (v/v) heat-inactivated FBS, 100U/ml penicillin, 100pg/ml streptomycin, 100pg/ml Normocin, with HEK-Blue™ Selection (Invivogen, hb-sel) using standard techniques to a final cell concentration of 2.5E5 cells/ml. The cells were then added in a 40pl/well volume to a 384-well clear tissue culture treated plate (Corning #3701). The assay plates were then incubated for 4 hours at 37°C I 5% CO2 before the addition of antibody/IL-17. During this incubation, human IL-17AA, cynomolgus monkey IL-17AA, human IL-17FF and cynomolgus monkey IL-17FF was diluted in HEK Blue assay medium so that their final assay concentration would be 30pM (IL-17AA) or 300pM (IL-17FF) and added to 384-well dilution plates (Greiner #781280). The antibodies were serially diluted in 384-well dilution plates in assay medium for a total of a 10-point dilution series (1 :3 dilutions). Antibodies were either diluted from 10nM top concentration or from 1 nM top concentration. Next, 5pl/well was transferred from the plates containing IL-17 cytokines to the plates containing the antibody dilution. The plate containing IL-17 and antibody was then incubated for 60 minutes at 37°C, 5% CO2. Next, 10pl was transferred from the ligand/antibody plate to each cell assay plate prepared earlier. These assay plates were then incubated for 17 hours +/- 2hrs (37°C, 5% CO2). After the incubation, QUANTI-Blue SEAP detection reagent (Invivogen #req-qbs) was prepared according to the manufacturer’s instructions and 40pl/well was added to a new 384-well plate (Corning #3701). 10pl/well of supernatant was then transferred from the assay plates to the QUANTI-Blue plates and these plates were incubated at 37°C 1 5% CO2 for 2 hours. At the end of this incubation, absorbance was measured on a plate reader at 620-655nm. Higher absorbance reads indicate a higher level of SEAP in the supernatant, and therefore higher levels of IL-17-mediated NF- KB signaling. A reduction in the SEAP levels therefore indicate functional inhibition of IL- 17 signaling. Percentage inhibition values were calculated using the raw absorbance values relative to assay minimum and maximum values, respectively, using Microsoft Excel. 4PL curve fitting and the determination of potency and efficacy values for each antibody was performed using GraphPad Prism. The results are summarised in Table X-17 (human IL-17AA), Table X-18 (cyno IL-17AA), Table X-19 (human IL-17FF) and Table X-20 (cyno IL-17FF) and Figure 20 (human IL-17AA), Figure 21 (cyno IL-17AA), Figure 22 (human IL-17FF) and Figure 23 (cyno IL-17FF).

Equivalent activity was observed for 19439gL1gH1/4211 KiH lgG1 LALA produced by the two different production methods (in vitro transient or in vitro stable) against the different cytokines tested. 18136 (Null)/4211 lgG1 LALA had equivalent activity to 19439gL1gH1/4211 KiH lgG1 LALA against all cytokines tested, and both the 19439gL1gH1/18136 (Null) KiH lgG1 LALA and the isotype control lgG1 LALA had no functional activity against any cytokine tested, as expected. 19439gL1gH1/4211 KiH lgG1 LALA had similar activity against human and cyno IL- 17AA in terms of potency and efficacy, but there was a slight drop off in potency against cyno IL-17FF as compared to human IL-17FF, which could be explained by the fact that the affinity against cyno IL-17FF is lower than against human IL-17FF as determined by SPR (KD of 83.5pM for human IL-17FF and 212. OpM for cyno IL-17FF). Additionally, there was reduced potency against IL-17FF as compared to IL-17AA for both the human and cynomolgus cytokines. This could be explained by the fact that 19439gL1gH1/4211 KiH lgG1 LALA has higher affinity for IL-17AA as compared to IL-17FF (as determined by SPR). The KD for IL- 17AA was 5.9pM for human and 5.6pM for cynomolgus, for IL-17FF the KD was 83.5pM for human and 212. OpM for cynomolgus. Additionally, the IL-17FF ligand concentration used was 10-fold higher than the IL-17FF concentration in these reporter cell experiments due to reduced signalling activity, and this could underestimate the potency of19439gL1gH1/4211 KiH lgG1 LALA against IL-17FF.

Table X-17 Summary of potency and efficacy values for 19439gL1gH 1/4211 KiH lgG1 LALA (various bi-specific production methods) and control molecules against human IL-17AA induced NF-KB activation in a HEK Blue IL-17R reporter cell line.

Table X-18 Summary of potency and efficacy values for 19439gl_1gH 1/4211 KiH lgG1 LALA (various bi-specific production methods) and control molecules against cynomolgus monkey IL-17AA induced NF-KB activation in a HEK Blue IL-17R reporter cell line.

Table X-19 Summary of potency and efficacy values for 19439gl_1gH 1/4211 KiH lgG1 LALA (various bi-specific production methods) and control molecules against human IL-17FF induced NF-KB activation in a HEK Blue IL-17R reporter cell line.

Table X-20 Summary of potency and efficacy values for 19439gl_1gH 1/4211 KiH lgG1 LALA (various bi-specific production methods) and control molecules against cynomolgus monkey IL-17FF induced NF-KB activation in a HEK Blue IL-17R reporter cell line.

Example 22: Assessment of IL-11 R, gp130, IL-17RA and IL-17RC 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, gp130, IL- 17RA and IL-17RC 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 VILO™ 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-11 RA, Hs00174360_m1 IL6ST, Hs01056316_m1 IL17RA, Hs00994305_m1 IL17RC, Hs02786624_g1GAPDH,; CYNO Mf02854633_g1 IL-11 RA, Mf02787830_m1 IL6ST, Mf01064648_m1 IL17RA, Mf02793477_m1 IL17RC, Mf04392546_g1 GAPDH.

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

The expression profiles are depicted in Figure 24A and Figure 24B.

Example 23: CCL2, IL-6 and MMP2 inhibition by 19439gL1gH1 / 4211 KiH hlgG1 LALA in cis using primary human dermal fibroblasts following rhlL-11, rhlL-17AA and rhlL-17FF stimulations Antibody 19439gL1gH1 I 4211 KiH hlgG1 LALA alongside relevant assay controls (Isotype control lgG1 LALA: null/null VR18136 KiH lgG1 LALA, Anti-IL-11/null control: 19439gL1gH1/18136 KiH hlgGI LALA, Anti-IL-17/null control: 18136/4211 KiH hlgG1 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 / 4211 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 40pg/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 U-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 25 (CCL2), Figure 26 (IL-6) and Figure 27 (MMP2) and Table X-21. 19439gL1gH1 14211 KiH hlgG1 LALA showed efficacious inhibition of IL-11 mediated CCL-2 and IL-6 release and IL-17AA/FF mediated CCL-2 and IL-6 release in the in vitro primary HDF assay. 19439gL1gH1 / 4211 KiH hlgG1 LALA was able to simultaneously block both IL- 11 and IL-17AA/FF induced CCL-2 and IL-6 release and dual blockade of IL- 11 and IL-17AA/FF resulted in greater inhibition of CCL-2 and IL-6 secretion than inhibition of individual cytokines alone. 19439gL1gH1 / 4211 KiH hlgG1 LALA was also able to simultaneously block IL-11 and IL-17AA/FF induced MMP2 release and dual blockade of IL-11 and IL-17AA/FF resulted in greater inhibition of MMP2 secretion than inhibition of individual cytokines alone. These data indicate that in vitro 19439gL1gH1 / 4211 KiH hlgG1 LALA can simultaneously neutralize the biological function of both IL-11 and IL-17AA/FF.

Table X-21 Mean percentage inhibition of CCL2, IL-6 and MMP2 secretion from dermal fibroblasts compared to the relevant Isotype control group. Example 24: IL-11 drives a distinct functional signature in different types of dermal cells The primary adult human dermal fibroblasts (HDF, Promocell #012302) and adult normal human follicle dermal papilla cells (HFDPC, Promocell #012302) 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, #023120 and 026500 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 28.

Example 25: 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

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 co¬

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:

PI 100

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 24), 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- 22).

Table X-22 - 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 26: 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 29.

In addition, a number of genes activated upon IL-11 stimulation of fibroblasts in vitro (see Example 24) and which include SULF1 , NPC1. SLCA3, DUSP1 , 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 27: RNAScope imaging of healthy and HS skin samples show increased IL-11 and IL-11R 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 also 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 both Singleplex and Duplex RNAscope assays, tissue samples were labelled using Leica Bond RX processor, Advanced Cell Diagnostics (ACD) RNAscope® 2.5 LS Reagent Kit-RED (Cat No. 322150), and RNAscope® 2.5 LS Reagent Kit-RED/BROWN (Cat No. 322440) 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 or PPIB/PolR2a and background was confirmed by DapB or DupDabB negative probes (Table X-23). 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-23) 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 substratechromogen solution using the Bond Polymer Refine Red Detection Kit (Leica Biosystems, Cat No. DS9390) and/or Refine Detection kit (DAB) Brown (Cat No. DS9800) 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-23 List of control and target RNAscope probes (ACD BioTechne)

Images showed that both IL-11 and IL-11 R expression are increased in HS lesional skin compared to healthy volunteer skin (Figure 30 and 31).

Example 28: Semiquantitative analysis of HS skin lesional samples show IL-11 and IL- U R 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;

Hscore = (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 32.

Example 29: RNAScope imaging confirms high expression of IL-17A and IL-17F in HS moderate to severe lesional skin, and often co-localises with IL-11 expression

IL-17A and F expression was visualized in moderate-severe HS lesional tissue samples and related to IL-11 distribution using duplex RNAscope assay. A significant number of IL-17A expressing cells were populating the de-epithelialized, active HS lesion. The highly IL-11 positive cells were distributed around blood vessels, with IL-17A positive cells in their close proximity (Figure 33). Similarly, a significant number of IL-17F expressing cells were distributed in the de-epithelialized HS lesion at the site of dense immune infiltration (Figure 34). Both IL-17A and IL-17F signal was profound in the lesion and no IL-17 signature was present in HS peri-lesional skin or in normal control skin (not shown).

Example 30: 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 #012302) 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, #023120). 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 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 U-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 35. IL-11 synergises with HS-relevant pro-inflammatory cytokines such as IL-17, TNF-a and IL-1 p to amplify inflammatory responses by synergistic induction of secretion of a range of chemokines including CXCL-1 (Figure 35A) and IL-8 (Figure 35B). 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 31 : IL-11 synergises with IL-17AA/FF to influence epidermal tissue remodeling in complex, mechanistic functional assays.

3D epidermis full thickness in vitro skin model of reconstituted human epidermis (RHE) was acquired from Mattek (#EFT-400). RHE skin model consists of both a dermal layer of collagen matrix containing human fibroblasts and an epidermal layer consisting of differentiated keratinocytes, grown at an air liquid interface. Transwells containing tissue were transferred to a fresh 6-well plate and 2.5 mis media per well was added to the basal compartment (Mattek, #EFT400). Tissue was kept in the air liquid interface and plates were kept in an incubator at 37°C, 5% C02 overnight. Following acclimatisation wells were stimulated for 6 days with rhlL- 11 at 300ng/ml, rhlL-17AA at 100ng/ml and rhlL-17FF at 1000ng/ml in standard culture conditions (37°C, 5% CO2, 100% humidity). All cytokines were added to the basal compartment of the plate in 2.5 mis of media and stimulation was refreshed every other day. On day 6 the tissue was retrieved from the tissue culture insert/microporous membrane and placed in 10% formalin solution until ready for further histology processing. Hematoxylin & Eosin (H&E) was performed using standard methods, images were obtained using a Zeiss Axioscan z.1 microscope and analysed using Zen 2.6 software.

The results are summarised in Figure 36. IL-11 stimulation alone didn’t lead to significant morphological changes that could be seen by H&E (data not shown), whilst IL-17AA together with IL-17FF caused epidermal atrophy (thinning of epidermis) in the model. IL-11 in combination with IL-17AA and IL-17FF exacerbated epidermal atrophy and promoted parakeratosis indicating the potential contribution of IL-11 to keratinocyte proliferation and dedifferentiation, a pathological phenomenon often observed during tunnel formation in HS.

Example 32: 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 37. 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 33: IL-11Ra 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 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. 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 38. 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 38 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 38 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 34: 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 27. The tissue quality was first assessed by performing RNAscope analysis for mRNA of the housekeeping genes (Example 27, Table X-23). 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-23) 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 39.

Example 35: Inhibition of IL-11 and IL-17A/F 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 inhibitory antibodies Anti-IL-11 (19439gL1gH1 Fab, 10pg/mL), Anti-IL-17A (CDP435, WG2006/054059, hFab, 10pg/mL) and Anti- IL-17F (rbFab.10 HIS, 10pg/mL) alone and in combination was performed. Solutions were prepared in advance. Briefly, PBMC supernatant was combined from 3 donors in a 1 :1 :1 ratio. Stimulation mix containing antibodies were 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 25. 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 40) which broadly captures inflammatory biology similar to that observed in HS biopsies (Figure 41).

Treatment with anti-IL-11 and/or anti-IL-17A/F Fabs in the skin model showed inhibition of gene pathways linked to inflammation and epithelial mesenchymal transition (ETM). Combined blockade of IL-11 and IL-17A/F resulted in a greater normalisation of the gene signature compared to inhibition with either IL-11 or IL-17A/F inhibition alone (Figure 42) suggesting that combined inhibition has potential to have greater impact than individual pathway blockade.

Example 36: Anti-IL-11 and anti-IL-17A/F Fab antibodies show modulation of HS signature in the HS ex vivo biopsy explant model Lesional skin biopsies were collected from patients with a mild-to-moderate or moderate-to- severe HS (3 biopsies per donor, n=10 donors). Informed consent was obtained from all participants and all samples were collected, stored, and used in agreement with the ethical and research guidelines set by the clinical site, Reprocell and UCB. Briefly, 30 isolated skin biopsies (~3 mm full thickness skin) were cultured in air-liquid interface for 48 hours in the presence or absence of inhibiting antibodies against IL-11 (19439gL1gH1 Fab, , 10pg/mL), IL- 17A (CDP435 hFab, W02006/054059, 10pg/mL), and IL-17F (rabbit Fab.10 HIS, 10pg/mL) at standard conditions (37°C, 5% CO2). After 48 hours of culture biopsies were transferred into 1mL of RNAIater (Sigma, Cat. No. R0901-500ML) overnight, or up to 72 hrs stored at 2-8 °C. After removal from RNAIater the biopsies were snap frozen. RNA was extracted as previously described (example 35). Further RNA QC and RNA-sequencing was performed. Analysis of samples was carried out as previously described (example 35). The obtained data passed QC check with approximately 85% alignment rates with normal GC distributions (the ratio of guanine (G) and cytosine (C) bases). Treatment with anti-IL17A/F showed clear inhibitory effect in the HS skin, downregulating genes linked to inflammations that have been shown to be upregulated in HS lesional skin, such as IL36A/G, S100A12 and DEFB4A. Treatment with anti-IL-11 + anti-IL17A/F showed greater number of significantly modulated genes than treatment with anti-IL17A/F. The IL- 11 specific genes inhibited in the HS biopsies, that have also been shown to be upregulated in HS lesions, included genes linked to immune response (SERPINB4, IDO1), barrier function (CLDN17, SLC9A1), tissue remodelling (PRSS22), stress and apoptosis (PML), cellular signal transduction (TMEM171) and metabolic pathways (ALAS1 , SEL1 L3). This data suggests that combined inhibition has potential to have greater impact than individual pathway blockade.

Claims

WHAT IS CLAIMED IS:

1. A multispecific antibody comprising at least two antigen-binding domains, wherein the first antigen-binding domain inhibits IL-11 mediated signaling and the second antigen-binding domain inhibits IL-17A and/or IL-17F mediated signaling.

2. A multispecific antibody according to claim 1 , wherein the first antigen-binding domain specifically binds to IL-11 and the second antigen-binding domain specifically binds to IL-17A and/or I L17-F.

3. The multispecific antibody according to any one of claims 1 to 2, wherein the first antigenbinding domain comprises a heavy chain variable region (VH1) comprising: a CDR-H1 comprising the amino acid sequence of SEQ ID NO:18, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:19, or a CDR- H2 comprising the amino acid sequence of SEQ ID NO: 19 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 NQ:20, or a CDR- H3 comprising the amino acid sequence of SEQ ID NQ:20 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 (VL1) comprising: a CDR-L1 comprising the amino acid sequence of SEQ ID NO:21 , or a CDR- L1 comprising the amino acid sequence of SEQ ID NO:21 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:22, or a CDR- L2 comprising the amino acid sequence of SEQ I D NO:22 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:23.

4. The multispecific antibody according to claim 3, wherein the VH1 comprises an amino acid sequence with a sequence identity of more than 90% with SEQ ID NO:24, and the VL1 comprises an amino acid sequence with a sequence identity of more than 90% with SEQ ID NO:26.

5. The multispecific antibody according to any one of claims 1 to 4, wherein the second antigen-binding domain comprises a heavy chain variable region (VH2) 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 (VL2) 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.

6. The multispecific antibody according to claim 5, wherein the VH2 comprises an amino acid sequence with a sequence identity of more than 90% with SEQ ID NO:7, and the VL2 comprises an amino acid sequence with a sequence identity of more than 90% with SEQ ID NO:9.

7. The multispecific antibody according to any one of claims 1 to 6 comprising: a) a polypeptide chain of formula (I):

VH-I-CH-I- CH₂ -CH_3; b) a polypeptide chain of formula (II):

VL-I-CL; c) a polypeptide chain of formula (III):

VH₂-CH-|- CH₂ -CH_3; and d) a polypeptide chain of formula (IV):

VL₂-C_L; wherein:

VH-j _anc| VH₂ represent a heavy chain variable domain;

CH-] represents domain 1 of a heavy chain constant region;

CH₂ represents domain 2 of a heavy chain constant region;

CH₃ represents domain 3 of a heavy chain constant region; VL-] and VL2 represent a light chain variable domain;

C|_ represents a domain from a light chain constant region, such as

Ckappa; wherein the VH1 and VL1 form a VH/VL pair that specifically binds to IL-11 , wherein the VH2 and VL2 form a VH/VL pair that specifically binds to IL-17A and/or IL- 17F, and wherein the polypeptides of Formula I and III are a pair of heavy chain polypeptides in which one polypeptide comprises the knob substitution T366W in the CH3 domain and the other polypeptide comprises the hole substitutions T366S, L368A and Y407V in the CH3 domain, wherein the numbering is according to EU as in Kabat.

8. An isolated polynucleotide or a combination of isolated polynucleotides encoding the multispecific antibody according to any one of claims 1 to 7.

9. An expression vector carrying the polynucleotide according to claim 8 or a combination of expression vectors carrying the combination of polynucleotides according to claim 8.

10. A host cell or a combination of host cells comprising the vector or the combination of vectors according to claim 9.

11. A pharmaceutical composition comprising the multispecific antibody according to any one of claims 1 to 7 and a pharmaceutically acceptable agent.

12. A pharmaceutical composition comprising a first antibody that inhibits IL-11 mediated signaling and a second antibody that inhibits IL-17A and/or IL-17F mediated signaling and a pharmaceutically acceptable agent.

13. The multispecific antibody according to any one of claims 1 to 7, or the pharmaceutical composition according to claim 11 , or 12, for use as a medicament.

14. The multispecific antibody according to any one of claims 1 to 7, or the pharmaceutical composition according to claim 11 , or 12, for use in the treatment or prevention of an inflammatory skin condition, systemic sclerosis, an inflammatory fibrotic disease of the long (such as I PF, COPD, and asthma), an inflammatory fibrotic disease of the liver (such as MASLD, MAFLD, MAFL, and MASH), an inflammatory fibrotic disease 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).

15. The multispecific antibody according to any one of claims 1 to 7, or the pharmaceutical composition according to claim 11 , or 12, for use in the treatment or prevention of hidradenitis suppurativa.

16. A combination of a first antibody that inhibits IL-11 mediated signaling and a second antibody that inhibits IL-17A and/or IL-17F mediated signaling for use as a medicament.

17. An agent capable of inhibiting IL-11 and IL-17A and/or IL-17F mediated signaling for use in the treatment of an inflammatory disease.

18. An agent for use according to claim 17, wherein the inflammatory disease is hidradenitis suppurativa.