Therapeutic variable domains of heavy chain (VHH) antibodies that cross-neutralize interleukin-17 (IL-17) polypeptides

Abstract

The present invention provides VHH antibodies that neutralize the pro-inflammatory interleukin IL-17 in the form of IL-17A and IL-17F homodimers and IL-17AF heterodimers. Pharmaceutical compositions comprising the VHH antibodies and uses for the treatment of inflammatory and immune-related diseases and disorders are also provided.

Description

[Technical Field]

[0001] The present invention relates to the fields of antibody technology, medicine, pharmacology, infection biology, and medical diagnostics. More specifically, the present invention provides VHH antibodies that neutralize the pro-inflammatory interleukin 17 (IL-17) in the form of IL-17A and IL-17F homodimers and IL-17AF heterodimers. [Background technology]

[0002] Interleukin-17 (IL-17) is a multifunctional cytokine. It is produced by a specific subclass of CD4+ T helper cells, i.e., Th17 cells, but also by other CD4+ or CD8+ T cells and gamma / delta T cells. The primary effects of IL-17 consist of attracting neutrophils and monocytes and inducing the production of additional cytokines and chemokines by its target cells. Collectively, IL-17 is a key mediator of inflammatory responses. These responses can be of great benefit to patients, for example, in building defenses against various types of infection (Mills, 2022). However, excessive or unregulated production of IL-17 can also induce or exacerbate autoimmune diseases such as psoriasis, rheumatoid arthritis, asthma, or inflammatory bowel disease.

[0003] Upstream signaling pathways tightly regulate the synthesis and secretion of IL-17 during mucosal infection, most notably interleukin 23 (IL-23) and IL-1beta released from activated dendritic cells or macrophages, which stimulate Th17 cells to release IL-17.

[0004] The target cells of IL-17 can be varied, as receptors are found on a wide array of human cell types. Therefore, responses can be heterogeneous. However, most importantly, epithelial cells exposed to IL-17 release chemokines such as CXCL1 and CXCL8, thereby attracting neutrophilic granulocytes and macrophages to maintain epithelial barrier homeostasis.

[0005] In autoimmune diseases, elevated levels of IL-17 at sites of inflammation activate the release of interleukin-6, tumor necrosis factor alpha (TNF-alpha), and matrix metalloproteinases (MMPs), which result in tissue damage and amplify the inflammatory response (Mills, 2022).

[0006] IL-17 proteins form dimers. They currently comprise a family of six members, IL-17A through IL-17F. Of these, IL-17A and IL-17F are considered the primary and most related and are also the most well-studied. They bind to five receptors, IL-17RA through IL-17E, forming heterodimeric receptor complexes. IL-17A and IL-17F bind to and activate the IL-17RA and IL-17RC receptors as homodimers or heterodimers (IL-17A / F) (Nies and Panzer, 2020). IL-17 binding requires the adaptor protein TRAF3IP2 and can lead to lateral receptor assembly, which at least partially accounts for the initiation of intracellular signaling, such as activation of NF-kappa-B and mitogen-activated protein (MAP) kinases (Wilson et al., 2022).

[0007] Several approaches have been developed to disrupt the production and / or activity of IL-17. Specifically, monoclonal antibodies can target upstream IL-23, but most importantly, IL-17 itself or its receptor. Neutralization of IL-17 then attenuates the inflammatory response, which is beneficial when treating autoimmune diseases.

[0008] Antagonists, particularly antibodies, against IL-17 have been successfully applied to treat many diseases and have led to FDA-approved drugs. Most notably, inflammatory skin diseases, namely psoriasis and psoriatic arthritis, and hidradenitis suppurativa ("acne suppurativa"), have been successfully treated with IL-17-neutralizing antibodies (Skroza et al., 2017; summarized in Mills, 2022). These include ixekizumab and secukinumab, which target IL-17A alone in plaque psoriasis (Langley et al., 2014) and psoriatic arthritis (Mease et al., 2015), and bimekizumab, which targets both IL-17A and IL-17F in plaque psoriasis (Reich et al., 2021; Warren et al., 2021) and psoriatic arthritis (Glatt et al., 2018).

[0009] However, such antibodies are typically applied systemically with little or no spatial restriction to the actual site of inflammation. In this context, it should be noted that such indiscriminate blockade of IL-17 may also exacerbate bacterial or fungal infections, as reported for bimekizumab, for example (Reich et al., 2021; Warren et al., 2021). Ideally, when treating autoimmune diseases, IL-17 neutralization should be localized and limited to the site of disease rather than systemically applied. Using more compact, thermostable, and versatile antibody versions, topical application may become feasible.

[0010] Because IL-17 plays a major role in the development of psoriasis vulgaris, it is the molecular target of several recently launched biologic therapies, primarily monoclonal antibodies (mAbs), such as Cosentyx (Novartis) and Taltz (Lilly), both of which target IL-17A. Research has shown that targeting the IL-17F isoform in addition to IL-17A provides greater efficacy in treating psoriasis vulgaris. The recently launched Bimzelx (UCB) was the first mAb targeting both IL-17A and IL-17F to be approved by the European Medicines Agency (EMA). MoonLake Immunotherapeutics, which is developing a nanobody targeting both IL-17A and IL-17F, reported excellent results for Cosentyx in a phase 2 clinical trial for psoriasis vulgaris. All of the above antibodies, which are only suitable for patients with moderate to severe psoriasis, are administered by subcutaneous injection for systemic drug distribution, and are associated with significant risk of side effects.These drugs are also expensive, costing several thousand dollars each, because they require chronic, lifelong, biweekly injections.Unfortunately, there are no safe and affordable biological drugs available for mild psoriasis, which accounts for 50% of psoriasis vulgaris patients.Experience has shown that even 28% of patients with moderate psoriasis vulgaris tend to avoid or delay the initiation of these biological treatments due to the associated risks.

[0011] Nanobodies (VHH antibodies) comprise a novel class of therapeutic proteins based on monovalent camelid-derived heavy chain-only antibodies. In contrast to monoclonal IgG, which are produced in mammalian cells, nanobodies can be produced in bacteria or yeast. In addition to their low molecular weight, certain nanobodies have proven to be ultrathermostable and exhibit particularly high affinity (Guttler et al., 2021).

[0012] One IL-17 antagonist, soneloximab, was engineered based on a single-chain antibody / nanobody. It is a trimer of nanobodies targeting IL-17A, IL-17F, and albumin. The idea is to absorb both versions of IL-17 and increase the half-life of the antagonist by binding to albumin. However, due to its size and albumin-binding entity, this compound is still suitable for systemic rather than topical application. Similar to classic antibody treatments, the incidence of fungal Candida infections increased in study participants (Papp et al., 2021).

[0013] WO 2012 / 156219 discloses single domain antibodies that specifically bind to human IL-17A, human IL-17F, and / or human IL-17A / F, as well as tandem fusions thereof, although the affinity of individual VHHs for IL-17F isoforms was rather weak.

[0014] In addition to skin diseases, other inflammatory disorders may be treatable with IL-17 antagonists. These include, but are not limited to, ankylosing spondylitis (depending on the approved application), multiple sclerosis, rheumatoid arthritis, asthma, graft-versus-host disease, and even diseases such as Alzheimer's disease, fatty liver disease, and COVID-19 (all clinical studies are ongoing, as outlined in Mills, 2022). Future applications may include the treatment of autism, Parkinson's disease, atherosclerosis, stroke, and sepsis. Most of these applications would benefit from more versatile antibody types with high affinity, stability, and versatility in terms of application route and fusion to additional / stabilizing entities. This demonstrates the enormous range of benefits expected from VHH antibodies targeting IL-17A / F.

[0015] There is a clear unmet need for novel immunotherapeutic agents that target both IL-17A and IL-17F and that can be safely used to treat inflammatory, immune, and autoimmune disorders and diseases. Such agents could be advantageously used to treat mild and moderate plaque psoriasis by topical administration. Summary of the Invention

[0016] The present invention provides compositions and methods for the amelioration of symptoms associated with overactivity of the cytokine IL-17, and provides a novel class of human cytokine-neutralizing single domain VHH antibodies for treating patients with inflammatory and / or immune-related disorders caused by and / or associated with overactivity of this cytokine. The VHH antibodies of the present invention are provided for the treatment of immune, autoimmune, and inflammatory diseases and disorders, including skin diseases and disorders.

[0017] The novel VHH antibodies of the present invention are divided into four classes based not only on their sequences but also on data obtained from crystal and modeled structures of complexes of novel VHH antibodies with human IL-17 isoforms, which have been used to define the amino acid residues in the VHH sequences that interact with IL-17 isoforms and are primarily responsible for binding to and blocking of binding to the human IL-17 receptor, thus preventing or inhibiting receptor activation.

[0018] In certain embodiments, these VHH antibodies are in monovalent form, e.g., as single VHH domains or as fusions with heterologous proteins such as serum albumin, which can be used in a wide variety of formats. In certain embodiments, VHH antibodies are in multivalent form, e.g., as bivalent Fc fusions. Use in monovalent formats is possible due to their very high affinity. In certain embodiments, VHH antibodies have picomolar or even low picomolar target affinities in monovalent formats. In certain embodiments, VHH antibodies neutralize all major IL-17 isoforms, particularly human IL-17A homodimers, human IL-17F homodimers, and human IL-17A / F heterodimers, with similar high potency.

[0019] The present invention is based in part on the discovery that representative VHH antibodies unexpectedly cross-react with sub-nanomolar affinities between the IL-17A and IL-17F isoforms. The present invention is further based on the results of several preclinical studies of anti-IL-17 VHH antibodies for the treatment of psoriasis vulgaris, demonstrating their therapeutic potential to alleviate the symptoms of this disease.

[0020] The VHH antibodies of the invention, according to some embodiments, are designed to be administered locally to the dermis and eliminated in a manner that should prevent systemic side effects. Results of ex vivo studies using the VHH antibodies of the invention demonstrate the potential for a highly effective and specific, yet safer and more convenient, treatment for a large and underserved population of patients with mild to moderate plaque psoriasis.

[0021] The present invention provides VHH antibodies that recognize human IL-17 polypeptides, cross-react with multiple different human IL-17 polypeptides, including (i) human IL-17A homodimer, (ii) human IL-17F homodimer, and (iii) human IL-17A / F heterodimer, and prevent or neutralize activation of the human IL-17 receptor. The VHH antibodies of the present invention bind with extremely high affinity to different IL-17 isoforms that vary in epitope.

[0022] According to some embodiments, the binding affinity of the monomeric VHH, expressed as the dissociation constant KD, to immobilized human IL-17A or IL-17F homodimer is 5 nM, 1 nM, 500 pM, 300 pM, 100 pM, 50 pM or less.

[0023] According to one aspect, the present invention provides a VHH antibody that binds to IL-17A, IL-17F, and IL17AF and neutralizes their binding to and activation of the human IL-17 receptor, wherein the VHH antibody comprises an amino acid sequence selected from SEQ ID NOs: 19, 15, 5, 23, 12, 13, 20, 27, 28, 32, 35, 39, 40, 44, 45, 49, 53, and 54, or a variant thereof having at least 80% identity to any of these sequences.

[0024] According to some embodiments, the present invention provides a VHH antibody that binds to and neutralizes IL-17A, IL-17F, and IL17AF, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 15, 39, 35, 5, 23, 12, 13, 20, 27, 28, 32, 40, 44, 45, 49, 53, and 54, or a variant thereof having at least 85% identity to any of these sequences.

[0025] According to some embodiments, the present invention provides a VHH antibody that binds to and neutralizes IL-17A, IL-17F, and IL17AF, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 15, 39, 35, 5, 23, 12, 13, 20, 27, 28, 32, 40, 44, 45, 49, 53, and 54, or a variant thereof having at least 90% identity to any of these sequences.

[0026] According to some embodiments, the VHH antibody that binds to and neutralizes the activity of IL-17A, IL-17F, and IL17AF comprises an amino acid sequence selected from SEQ ID NOs: 19, 15, 5, 23, 12, 13, 20, 27, 28, and 32 (Class A), SEQ ID NOs: 39, 35, 40, 44, and 45 (Class B), SEQ ID NOs: 49 and 53 (Class C), and SEQ ID NO: 54 (Class D), or a variant thereof having at least 80% identity to any of these sequences.

[0027] According to some embodiments, the variants have at least 91%, 92%, 93%, 94%, or 95% sequence identity to the VHH antibody. According to more specific embodiments, the variants have at least 95%, 96%, 97%, 98%, or 99% sequence identity to the VHH antibody.

[0028] Variants containing substitutions of 1 to 10 amino acid residues are also included within the scope of the present invention. The substitutions may be selected from conservative substitutions, non-conservative substitutions, and combinations thereof.

[0029] According to some embodiments, variants of the above-described VHH antibodies are provided in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids have been substituted, deleted, or added. According to specific embodiments, 1 to 5 amino acids in the VHH antibody are substituted, deleted, or added. According to some specific embodiments, the substitution, deletion, or addition retains or improves at least one physical property of the VHH antibody. According to some embodiments, the substitution or combination of 2 to 5 substitutions, addition, or deletion improves the stability and / or productivity of certain VHH antibodies according to the present invention.

[0030] According to some embodiments, substitution of Leu (Leu) to Gln (Q) at non-CDR residue 115 of VHH antibody Re42B04 improves the physical properties of the resulting VHH antibody Re42B04a.

[0031] In a specific embodiment, at least one residue in an N-glycosylation site is substituted or deleted to prevent possible glycosylation of the VHH antibody. According to a specific embodiment, a residue selected from asparagine (Asn, N), threonine (Thr, T), and serine (Ser, S) is substituted or deleted.

[0032] Variants containing one to three amino acid substitutions in one, two, or three CDR sequences are also within the scope of the present invention. According to some embodiments, the substitutions are conservative. According to other embodiments, the substitutions are non-conservative.

[0033] The present invention provides VHH antibodies that bind to human IL-17A, IL-17F, and IL17AF dimers and neutralize their binding to the human IL-17 receptor, and the VHH antibodies belong to a class of structurally related antibodies, the class comprising: i. Class A comprising a CDR3 sequence of the formula NDMPYGX1X2TX3MDX4YX5X6W, wherein X1 is selected from L and M, X2 is selected from D and E, X3 is selected from R and T, X4 is selected from E and D, X5 is selected from A, V, E, D, and K, and X6 is selected from Y and S; ii. Class B comprising a CDR3 sequence of the formula X1HNEPGX2LYM, wherein X1 is selected from V and T, and X2 is selected from H and D; iii. Class C comprising the CDR3 sequence MAVRGLYGSNWYDYPFELW (SEQ ID NO: 52), and iv. selected from class D comprising the CDR3 sequence YIDSGSDRYY (SEQ ID NO: 57); The sequence identity between different VHH antibodies within a particular class is 80% or more.

[0034] According to some embodiments, the class A VHH comprises a CDR3 having a sequence selected from SEQ ID NOs: 8, 11, 18, 26, 31, and 34, a CDR2 having a sequence selected from SEQ ID NOs: 7, 14, 17, 22, 25, and 30, and a CDR1 having a sequence selected from SEQ ID NOs: 6, 10, 16, 21, 24, 29, and 33.

[0035] According to some embodiments, the VHH antibody or variant thereof comprises a CDR3 sequence set forth in any one of SEQ ID NOs: 8, 11, 18, 26, 31, 31, 38, 43, 52, and 57, or a sequence having at least 80%, at least 90%, or at least 95% identity to the CDR3 sequence.

[0036] According to certain embodiments, there is provided a class A VHH antibody that recognizes and neutralizes IL-17, wherein the VHH antibody comprises a set of three CDR sequences selected from SEQ ID NOs: 16, 17, and 18, SEQ ID NOs: 6, 7, and 8, SEQ ID NOs: 10, 7, and 11, SEQ ID NOs: 10, 14, and 11, SEQ ID NOs: 21, 22, and 18, SEQ ID NOs: 24, 25, and 26, SEQ ID NOs: 29, 30, and 31, and SEQ ID NOs: 33, 7, and 34. According to certain embodiments, there is provided a class B VHH antibody that recognizes and neutralizes IL-17, wherein the VHH antibody comprises a set of three CDR sequences selected from SEQ ID NOs: 36, 37, and 38, SEQ ID NOs: 41, 42, and 43, and SEQ ID NOs: 46, 42, and 43.

[0037] According to some specific embodiments, there is provided a class C VHH antibody that recognizes and neutralizes IL-17, wherein the VHH antibody comprises a set of three CDR sequences, the set comprising SEQ ID NOs: 50, 51, and 52.

[0038] According to some specific embodiments, a class D VHH antibody that recognizes and neutralizes IL-17 is provided, wherein the VHH antibody comprises a set of three CDR sequences, the set comprising SEQ ID NOs: 56, 57, and 58.

[0039] According to some specific embodiments, a class A or class B VHH antibody is provided that comprises a set of three CDR sequences, the set being selected from SEQ ID NOs: 16, 17, and 18, and SEQ ID NOs: 36, 37, and 38.

[0040] According to some embodiments, the VHH antibody is selected from RE42B04a (SEQ ID NO: 19), Re42B04 (SEQ ID NO: 15), Re42F08 (SEQ ID NO: 39), and Bm17B02 (SEQ ID NO: 35).

[0041] According to a specific embodiment, the present invention provides a VHH antibody Re42B04a comprising the VHH sequence shown in SEQ ID NO: 19, or a variant thereof having at least 90% identity.

[0042] According to a specific embodiment, the present invention provides a VHH antibody Re42B04 comprising the VHH sequence shown in SEQ ID NO: 15, or a variant thereof having at least 90% identity.

[0043] According to a specific embodiment, the present invention provides a VHH antibody Bm17B02 comprising the VHH sequence shown in SEQ ID NO: 35, or a variant thereof having at least 90% identity.

[0044] According to some embodiments, the Class A VHH antibody comprises the following positions that interact with IL-17A: position 1 is Q, position 29 is A, G, V, F or P, position 30 is S, position 31 is S or G, position 32 is Y, position 33 is A, position 50 is A, position 51 is I, position 52 is S, position 54 is I, S or V, position 55 is S or G, position 57 is G, S or D, position 58 is T, S or A, position 59 is K, R or V, position 100 is P, position 101 is Y, position 103 is L or M, position 104 is D or E, position 106 is R, and position 109 is E or D.

[0045] According to some embodiments, the class A VHH antibody comprises the following positions that interact with IL-17F: position 29 is A, GV, F or P, position 30 is S, position 31 is S or G, position 32 is Y, position 33 is A, position 50 is A, position 51 is I, position 52 is S, position 54 is S, I or V, position 55 is S or G, position 57 is G, S or D, position 58 is T, S or A, position 59 is K, R or V, position 100 is P, position 101 is Y, position 103 is L or M, position 104 is D or E, position 106 is R, and position 109 is E or D.

[0046] According to some embodiments, the Class B VHH antibody comprises the following positions that interact with IL-17A: position 3 is Q, position 31 is I or Q, position 32 is S, position 33 is A, position 37 is Y, position 45 is R, position 52 is H, position 59 is H or Y, position 99 is N, position 100 is E, position 101 is P, position 102 is G, position 103 is H or D, position 104 is L, position 105 is Y, and position 106 is M.

[0047] According to some embodiments, the Class B VHH antibody comprises the following positions that interact with IL-17F: position 3 is Q, position 31 is I or Q, position 32 is S, position 33 is A, position 37 is Y, position 45 is R, position 47 is L, position 50 is L or M, position 52 is T or H, position 59 is H or Y, position 99 is N, position 100 is E, position 101 is P, position 102 is G, position 103 is H or D, position 104 is L, position 105 is Y, and position 106 is M.

[0048] A further aspect of the invention relates to a set of two or more different VHH antibodies, at least one VHH antibody being as described above.

[0049] In certain embodiments, the above-described VHH antibodies are covalently or non-covalently conjugated to a heterologous moiety, which may be selected from a label group, a capture group, or an effector group.

[0050] In certain embodiments, the VHH antibodies described above are fused to a heterologous polypeptide moiety, such as an IgG Fc fragment, serum albumin, or an albumin-binding moiety. In certain embodiments, the VHH antibodies are conjugated to one or several polymer moieties, particularly hydrophilic polymer moieties such as polyethylene glycol (PEG), to increase the molecular weight of the antibody conjugate and thereby delay renal clearance. The molecular weight of the polymer moiety can vary over a wide range, for example, from about 5 kDa to about 80 kDa. Such coupling can be carried out, for example, through amino or carboxyl groups already present in the VHH (e.g., the amino and carboxy termini) and / or through the side chains of lysine, aspartic acid, glutamic acid, or cysteine ​​residues, or through engineered backbones or side chains of other amino acids, and can involve known chemistries for forming amide, secondary amine, urea, or thioether bonds.

[0051] According to some embodiments, the VHH antibody neutralizes the binding of human IL-17 dimers to the human IL-17 receptor. According to particular embodiments, the VHH antibody neutralizes the binding of (i) human IL-17A homodimer, (ii) human IL-17F homodimer, and (iii) human IL17A / F heterodimer to the human IL-17 receptor. According to more particular embodiments, the VHH antibody neutralizes the binding of at least one of (i) human IL-17A homodimer, (ii) human IL-17F homodimer, and (iii) human IL17A / F heterodimer to the human IL-17 receptor at a concentration of about 10 nM or less, about 3 nM or less, about 1 nM or less, or about 0.3 nM or less when tested in a cell-based assay under affinity-limited test conditions.

[0052] Also within the scope of the present invention are VHH antibodies that are variants of the above disclosed VHH antibodies and that compete with any of the above disclosed VHH antibodies for IL-17 binding.

[0053] According to some embodiments, the VHH antibodies are stable, in particular thermostable or hyperthermostable, and according to some particular embodiments, the VHH antibodies have a melting temperature of at least about 65°C, at least about 80°C, at least 90°C, or at least about 95°C when measured under non-reducing conditions.

[0054] According to some embodiments, the VHH antibody has an aggregation temperature of at least about 60°C, at least 70°C, at least about 80°C, at least about 90°C, or at least about 95°C when measured under non-reducing conditions.

[0055] The VHH antibody of any one of the previous embodiments, which is aglycosylated or glycosylated.

[0056] According to some embodiments, the VHH antibody or variant thereof is in a monovalent format.

[0057] According to some embodiments, the VHH antibody or variant thereof is in a dimeric or multimeric format.

[0058] The present invention also provides a set of two or more different VHH antibodies that recognize human IL-17 polypeptides, particularly IL-17A homodimer, IL-17F homodimer, and IL17A / F heterodimer, comprising at least one VHH antibody or variant thereof disclosed above, particularly a VHH antibody in a monovalent format.

[0059] The above VHH antibodies or sets of VHH antibodies are suitable for use in medicine, e.g., human medicine, in particular therapy, e.g., in the prevention or treatment of disorders caused by and / or associated with IL-17 overactivity, in particular IL-17A and / or IL-17F overactivity, or in diagnostics, e.g., for detecting IL-17 in patient samples, e.g., body fluid or tissue samples, or in research.

[0060] According to another aspect, the present invention also provides a nucleic acid molecule encoding the above-mentioned VHH antibody or a subunit of a VHH antibody, preferably operably linked to a heterologous expression control sequence or contained in a vector.

[0061] The present invention also provides recombinant cells or non-human organisms transformed or transfected with the nucleic acid molecules or vectors.

[0062] According to some embodiments, the cell or organism is selected from a bacterium, e.g., E. coli Bacillus sp., a unicellular eukaryote, e.g., a yeast, e.g., Pichia pastoris or Leishmania, an insect cell, a mammalian cell, and a plant cell.

[0063] According to some embodiments, the VHH antibody or variant thereof is produced in bacteria, such as E. coli, or yeast, such as Pichia pastoris.

[0064] The present invention also provides a method for the recombinant production of the above-mentioned VHH antibodies, which comprises culturing a cell or organism in a suitable medium and obtaining the VHH antibody from the cell or organism or from the medium.

[0065] In some embodiments, the method includes culturing cells of bacterial, yeast, insect, mammalian, and plant origin. In some embodiments, the cells are mammalian cells. In some embodiments, the mammalian cells are Chinese Hamster Ovary (CHO) cells.

[0066] According to some embodiments, the method comprises culturing yeast, for example Pichia pastoris, and obtaining a VHH antibody from the culture medium.

[0067] According to another aspect, the present invention further provides a pharmaceutical composition comprising at least one VHH antibody as defined above and a pharmaceutically acceptable carrier, excipient or diluent.

[0068] According to some embodiments, the pharmaceutical composition comprises a plurality of VHH antibodies as described above, eg a set of specific VHH antibodies.

[0069] According to some embodiments, the formulation is for topical administration. According to some embodiments, the formulation is for topical administration. The pharmaceutical compositions provided in accordance with the present invention may be formulated as a liquid, solid, or semi-solid state. According to some embodiments, the pharmaceutical composition is formulated as a cream, paste, gel, hydrogel, ointment, lotion, and emulsion. According to other embodiments, the pharmaceutical composition is a liquid formulation. According to some embodiments, the pharmaceutical composition is formulated for parenteral administration, for example, by injection or infusion. According to some embodiments, the pharmaceutical composition is formulated for intradermal injection.

[0070] According to some embodiments, the pharmaceutical composition is formulated as sustained, slow, or delayed release.

[0071] According to another aspect, the present invention further provides a diagnostic composition comprising at least one VHH antibody as defined above and an acceptable carrier, excipient or diluent.

[0072] Pharmaceutical and diagnostic kits comprising at least one VHH antibody and instructions for use are also provided.

[0073] The present invention also provides a pharmaceutical composition comprising at least one VHH antibody as defined above for use in medicine, in particular for use in therapy or diagnosis.

[0074] According to some embodiments, the pharmaceutical composition is for use in the prevention or treatment of disorders caused by and / or associated with IL-17 overactivity, in particular IL-17A and / or IL-17F overactivity.

[0075] According to some embodiments, the pharmaceutical composition is for use in the prevention or treatment of an inflammatory and / or immune-related disorder.

[0076] According to some embodiments, the pharmaceutical composition is for use in the prevention or treatment of inflammatory and / or immune-related skin disorders.

[0077] According to some embodiments, the pharmaceutical composition is for use in treating asthma, psoriasis, arthritis, hidradenitis suppurativa, inflammatory bowel disease (Crohn's disease, ulcerative colitis), multiple sclerosis, skin cancer, ankylosing spondylitis, uveitis, atopic dermatitis, graft-versus-host disease, Alzheimer's disease, fatty liver disease, sepsis, ischemic stroke, Parkinson's disease, axial spondyloarthritis not meeting radiographic criteria for activity with objective clinical signs of inflammation, systemic lupus erythematosus (SLE), familial mediterranean fever (FMF), tumor necrosis factor receptor-associated periodic syndrome (TRAPS), hidradenitis suppurativa (HS), pemphigus vulgaris (PV), pityriasis rubra pilaris (Pityriasis pilaris), and / or other conditions. rubra pilaris (PRP), alopecia areata, systemic sclerosis, and the infectious diseases lichen planus and impetigo herpetiformis.

[0078] According to some embodiments, the psoriasis is selected from plaque psoriasis, moderate to severe psoriasis, palmoplantar psoriasis, pustular psoriasis, and pustular psoriasis.

[0079] According to some embodiments, the arthritis is selected from rheumatoid arthritis, psoriatic arthritis, and enthesitis-associated arthritis.

[0080] According to some embodiments, the infectious disease is a viral, bacterial, or fungal disease.

[0081] According to some embodiments, the viral disease is caused by influenza virus infection or SARS-CoV-2 infection (COVID-19).

[0082] According to some embodiments, the disease or disorder is psoriasis or arthritis.

[0083] According to some particular embodiments, the disease or disorder is selected from moderate to severe psoriasis, hypertrophic palmoplantar psoriasis, generalized pustular psoriasis, psoriatic arthritis, enthesitis-associated arthritis, axial spondylitis not meeting radiographic criteria for activity with objective clinical signs of inflammation, ankylosing spondylitis, rheumatoid arthritis, systemic lupus erythematosus (SLE), familial Mediterranean fever, and tumor necrosis factor receptor-associated periodic syndrome (TRAPS).

[0084] According to some embodiments, the psoriasis is psoriasis vulgaris. According to more particular embodiments, the pharmaceutical composition is for use in the prevention or treatment of mild to moderate psoriasis vulgaris.

[0085] A still further aspect of the present invention relates to a method for the prevention or treatment of disorders caused by and / or associated with IL-17 overactivity, in particular IL-17A and / or IL-17F overactivity, comprising administering an effective dose of the above-mentioned VHH antibody or the above-mentioned set of at least two different VHH antibodies, or the above-mentioned pharmaceutical composition to a subject in need thereof.

[0086] According to some embodiments, the subject is a human subject suffering from a disorder caused by and / or associated with IL-17.

[0087] According to some embodiments, the disorder is an inflammatory or immune disorder.

[0088] According to some embodiments, the pharmaceutical composition is for use in the prevention or treatment of asthma, psoriasis, arthritis, hidradenitis suppurativa, inflammatory bowel disease (Crohn's disease, ulcerative colitis), multiple sclerosis, skin cancer, ankylosing spondylitis, uveitis, atopic dermatitis, graft-versus-host disease, Alzheimer's disease, fatty liver disease, sepsis, ischemic stroke, Parkinson's disease, axial spondyloarthritis not meeting radiographic criteria for activity with objective clinical signs of inflammation, systemic lupus erythematosus (SLE), familial Mediterranean fever (FMF), tumor necrosis factor receptor-associated periodic syndromes (TRAPS), hidradenitis suppurativa (HS), pemphigus vulgaris (PV), pityriasis rubra pilaris (PRP), alopecia areata, systemic sclerosis, and infectious diseases, lichen planus, and impetigo herpetiformis.

[0089] According to some embodiments, the psoriasis is selected from plaque psoriasis, moderate to severe psoriasis, palmoplantar psoriasis, and pustular psoriasis.

[0090] According to some embodiments, the arthritis is selected from rheumatoid arthritis, psoriatic arthritis, and enthesitis-associated arthritis.

[0091] According to some embodiments, the infectious disease is a viral, bacterial, or fungal disease.

[0092] According to some embodiments, the viral disease is caused by influenza virus infection or SARS-CoV-2 infection (COVID-19).

[0093] According to some particular embodiments, the disease or disorder is selected from moderate to severe plaque psoriasis, hypertrophic palmoplantar psoriasis, generalized pustular psoriasis, psoriatic arthritis, enthesitis-associated arthritis, axial spondylitis not meeting radiographic criteria for activity with objective clinical signs of inflammation, ankylosing spondylitis, rheumatoid arthritis, systemic lupus erythematosus (SLE), familial Mediterranean fever, and tumor necrosis factor receptor-associated periodic syndrome (TRAPS).

[0094] According to some embodiments, the disease or disorder is psoriasis or arthritis.

[0095] According to some embodiments, the disorder is psoriasis. According to more specific embodiments, the psoriasis is plaque psoriasis. According to even more specific embodiments, the disorder is mild to moderate plaque psoriasis.

[0096] According to some embodiments, the composition is administered topically.

[0097] According to some embodiments, the composition is administered topically.

[0098] According to some embodiments, administration is by injection. According to particular embodiments, administration is by intradermal injection.

[0099] According to another aspect of the present invention, there is also provided a method for delivering a VHH antibody to a cell, comprising contacting the cell with at least one VHH antibody or set of VHH antibodies as described above.

[0100] According to some embodiments, the cells are from a human subject suffering from a disorder caused by and / or associated with overactivity of IL-17, particularly associated with overactivity of IL-17A and / or IL-17F.

[0101] The invention is explained in more detail by the following figures and examples. [Brief explanation of the drawings]

[0102] Some embodiments of the present invention are herein described, by way of example only, with reference to the accompanying drawings. With particular reference now to the drawings in detail, it is emphasized that the details shown are by way of example and are for the purpose of explaining the embodiments of the invention. In this regard, the description taken in conjunction with the drawings will make apparent to those skilled in the art how embodiments of the invention may be practiced. [Figure 1-1] Sequences of selected anti-IL-17 VHH antibodies. The figure shows the alignment of sequences from selected VHH antibodies. Residues that deviate from the consensus are highlighted by a grey background. The three CDR regions are indicated. [Figure 1-2] Sequences of selected anti-IL-17 VHH antibodies. The figure shows the alignment of sequences from selected VHH antibodies. Residues that deviate from the consensus are highlighted by a grey background. The three CDR regions are indicated. [Figure 2] Affinity of Re42H11 class VHHs for IL-17 isoforms measured by biolayer interferometry (BLI). The graph shows the binding of the indicated IL-17 species to biotinylated VHH antibodies of the Re42H11 class (Class A) immobilized on a high-precision streptavidin biosensor. Association and dissociation were recorded as wavelength shifts (nm) on an Octet RED96e instrument (ForteBio / Sartorius). Baselines were recorded by measuring a "minus VHH control" in parallel. On-rates, off-rates, and apparent dissociation constants (KD) were calculated using Octet Data Analysis HT 12.0 software, using a mass transport model to fit the data. Note that immobilization of VHHs to the sensor chip allows for avidity effects, i.e., adjacent VHH molecules may simultaneously bind to the same IL-17 dimer, resulting in off-rates that are lower than the actual off-rate. This simulates the binding of bivalent IgG to IL-17 dimers. Note that Figures 4-8 and 11 measure monovalent affinity by using immobilized IL-17 and monovalent VHH antibodies as analytes. All VHH antibodies characterized in BLI experiments were produced by periplasmic expression in E. coli. [Figure 3] Affinity of Bm17B02 class VHH antibodies for IL-17 isoforms. BLI measurements as in Figure 2, except for Bm17B02 and Re42F08 VHH antibodies in class B. [Figure 4] Affinity of control VHHs, VHH662 and VHH664, for IL-17 isoforms. Human IL-17A and IL-17F were produced with a C-terminal Avi tag and enzymatically biotinylated with recombinant BirA (Beckett et al., 1999). Immobilized at 100 nM on a High Precision Streptavidin biosensor on an Octet RED96e instrument (Sartorius) for 200 seconds using phosphate-buffered saline (PBS) pH 7.4, 0.02% (w / v) Tween 20, and 0.1% (w / v) bovine serum albumin (BSA) as the assay buffer. Then, 20 and 100 nM VHH662 or VHH664 antibodies were allowed to bind for 450 seconds and then dissociated for 900 seconds. Binding and dissociation were recorded as wavelength shifts (nm). A baseline was recorded by measuring a "minus VHH control" in parallel. Curves (gray) were fitted with a mass transport model (for Re42H11 class VHH, class A) and a 2:1 heterogeneous model (for Bm17B02 class VHH, class B). The fitted curves and calculated dissociation constants (KD) are shown in black. Note that this setup measures affinity independently of avidity effects, since the VHH antibody was provided as the analyte and thus was monomeric prior to target binding. [Figure 5] Affinity of first-generation Re42H11 and Bm17B02 class VHHs for IL-17A. BLI experiments were performed with first-generation VHH antibodies (using 25, 50, and 100 nM VHHs) as analytes and biotinylated IL-17A immobilized on a sensor chip, as described in Figure 4. For dissociation constant (KD) calculations, curves (gray) were fitted with a mass transport model (for Re42H11 class VHHs, class A) or a 2:1 heterogeneous model (for Bm17B02 class VHHs, class B). The lower affinity component for class B VHHs is likely due to excess VHH molecules binding to secondary sites on IL-17. [Figure 6]Affinity of second generation Re42H11 class VHHs for IL-17 isoforms. BLI experiments were performed as described in Figure 4 to analyze the binding of Re42H11 class VHHs, indicated as analytes, to biotinylated IL-17A or IL-17F. [Figure 7] Affinity of second generation Bm17B02 class VHHs for IL-17 isoforms. The indicated VHHs (class B) were analyzed by BLI as described in Figure 4. [Figure 8] Affinity of other VHHs (classes C and D) for IL-17 isoforms. VHHs Bm42A03, Bm44G10 (class C), and Bm45G07 (class D) were analyzed by BLI as described in FIG. [Figure 9] Thermal stability of the disclosed VHH antibodies. The VHH antibodies were subjected to differential scanning fluorimetry (DSF) as detailed in Example 4. Thermal unfolding is measured here as the enhanced fluorescence of added SYBR Orange after a stepwise increase in temperature using 532 nm excitation and a 555 nm long-pass filter. The melting temperature is defined as the inflection point of the first melting peak. Re42B04 is the only VHH in this series that shows a melting peak; however, this peak is rather low, indicating only localized melting rather than global melting of the VHH. [Figure 10] Thermostability under non-reducing and disulfide bond reducing conditions. Thermostability assays were performed as in Figure 9, except here a control is shown in the presence of 10 mM dithiothreitol (DTT). DTT reduces structurally stabilizing disulfide bonds. [Figure 11A] Thermostability of VHHs determined by BLI. The indicated VHH antibodies (1 μM) of classes A (FIG. 11A) and C (FIG. 11B) were incubated at room temperature or 95°C for 10 min and centrifuged at 20,000 g for 20 min. Supernatants were diluted 20-fold (to 50 nM) and analyzed for IL-17A binding by BLI. [Figure 11B] Thermostability of VHHs determined by BLI. The indicated VHH antibodies (1 μM) of classes A (FIG. 11A) and C (FIG. 11B) were incubated at room temperature or 95°C for 10 min and centrifuged at 20,000 g for 20 min. Supernatants were diluted 20-fold (to 50 nM) and analyzed for IL-17A binding by BLI. [Figure 12] Dose response of HEK-Blue™ IL-17 reporter cells to recombinant IL-17 cytokine. Cells of the HEK-Blue™ IL-17 reporter line were stimulated with increasing concentrations of human IL-17A, IL-17F, and IL-17AF. Stimulation resulted in the secretion of an embryonic alkaline phosphatase (SEAP) reporter, whose activity was determined colorimetrically by measuring the absorbance at 620 nm using QUANTI-Blue™ as a substrate. OD620 values ​​are plotted in (A). The plot in (B) shows the range of induction, calculated by dividing the OD620 reading at each IL-17 dilution by the OD620 value of the IL-17 control minus the OD620 value. Results are the mean ± SD of one experiment measured in triplicate. The vertical dashed lines indicate the IL-17 concentrations used in subsequent VHH neutralization experiments (Figures 13-14). [Figure 13] Neutralization of IL-17A and IL-17F by first-generation VHH antibodies. The plot shows the expression of a phosphatase reporter by HEK-Blue™ IL-17 cells after induction with 0.17 nM IL-17A homodimer, 0.85 nM IL-17F homodimer, or 0.85 nM IL-17AF heterodimer preincubated with the indicated concentrations of anti-IL-17 VHH antibodies. A number of 1.0 indicates (full) induction in the absence of VHH, and a number of 0 indicates no induction above background of the minus VHH control. The graph shows neutralization by Re42H11 and Bm17B02 class members (classes A and B, respectively), each measured in triplicate. [Figure 14A]Neutralization of IL-17A, F, and AF by VHH antibodies of classes A–D. Plots show normalized expression of a phosphatase reporter induced by 0.17 nM IL-17A homodimer, 0.85 nM IL-17F homodimer, or 0.85 nM IL-17AF heterodimer preincubated with the indicated concentrations of anti-IL-17 VHH antibodies. A value of 1.0 indicates (complete) induction in the absence of VHH, and a value of 0 indicates no induction above background in untreated cells. Graphs show neutralization by (A) class A (Re42B04a), (B) class B (Re42F08), (C) class C (Bm42A03), and (D) class D (Bm45G07). Bm18F11 is included for reference in all graphs. Results are the mean ± SD of three independent experiments, each measured in triplicate. [Figure 14B] Neutralization of IL-17A, F, and AF by VHH antibodies of classes A–D. Plots show normalized expression of a phosphatase reporter induced by 0.17 nM IL-17A homodimer, 0.85 nM IL-17F homodimer, or 0.85 nM IL-17AF heterodimer preincubated with the indicated concentrations of anti-IL-17 VHH antibodies. A value of 1.0 indicates (complete) induction in the absence of VHH, and a value of 0 indicates no induction above background in untreated cells. Graphs show neutralization by (A) class A (Re42B04a), (B) class B (Re42F08), (C) class C (Bm42A03), and (D) class D (Bm45G07). Bm18F11 is included for reference in all graphs. Results are the mean ± SD of three independent experiments, each measured in triplicate. [Figure 14C]Neutralization of IL-17A, F, and AF by VHH antibodies of classes A–D. Plots show normalized expression of a phosphatase reporter induced by 0.17 nM IL-17A homodimer, 0.85 nM IL-17F homodimer, or 0.85 nM IL-17AF heterodimer preincubated with the indicated concentrations of anti-IL-17 VHH antibodies. A value of 1.0 indicates (complete) induction in the absence of VHH, and a value of 0 indicates no induction above background in untreated cells. Graphs show neutralization by (A) class A (Re42B04a), (B) class B (Re42F08), (C) class C (Bm42A03), and (D) class D (Bm45G07). Bm18F11 is included for reference in all graphs. Results are the mean ± SD of three independent experiments, each measured in triplicate. [Figure 14D] Neutralization of IL-17A, F, and AF by VHH antibodies of classes A–D. Plots show normalized expression of a phosphatase reporter induced by 0.17 nM IL-17A homodimer, 0.85 nM IL-17F homodimer, or 0.85 nM IL-17AF heterodimer preincubated with the indicated concentrations of anti-IL-17 VHH antibodies. A value of 1.0 indicates (complete) induction in the absence of VHH, and a value of 0 indicates no induction above background in untreated cells. Graphs show neutralization by (A) class A (Re42B04a), (B) class B (Re42F08), (C) class C (Bm42A03), and (D) class D (Bm45G07). Bm18F11 is included for reference in all graphs. Results are the mean ± SD of three independent experiments, each measured in triplicate. [Figure 15]Neutralization of IL-17A, IL-17F, and IL-17AF by control VHH antibodies. Plots show normalized expression of phosphatase reporters induced by 0.17 nM IL-17A homodimer, 0.85 nM IL-17F homodimer, or 0.85 nM IL-17AF heterodimer, each preincubated with the indicated concentrations of anti-IL-17 VHH antibodies. A value of 1.0 indicates (complete) induction in the absence of VHH, and a value of 0 indicates no induction above background in untreated cells. (A) Graphs show neutralization of representative members of class A (Re42B04a), class C (Bm42A03), and class D (Bm45G07) compared with control VHH antibodies VHH662, VHH664, and Sonelokimab Biosimilar (Proteogenix PX-TA1606). Soneloximab is a three-VHH tandem fusion to IL-17A, IL-17F, and albumin. Due to its higher molecular weight and the presence of two IL-17-binding sites in soneloximab, identical mass concentrations (ng / mL) were compared. Results are the mean ± SD of three independent experiments, each measured in triplicate. (B) Neutralization of IL-17A by secukinumab / Cosentyx® Biosimilar (Proteogenix PX-TA1234) and a research-grade anti-IL-17A antibody (R&D Systems AF-317-NA) as a control. Results are the mean ± SD of two independent experiments, each measured in triplicate. [Figure 16]Crystal structure of the VHH class A member Re42H11 bound to an IL-17F dimer. His14-ScSUMO-tagged Re42H11 was coexpressed with His14-BdNEDD8-tagged human IL-17F (residues 39–163) in E. coli SHuffle Express (New England Biolabs). The complex was purified by Ni-chelate chromatography using immobilization of the complex components via the His14 tag followed by two sequential tag cleavage elutions using bdNEDP1 protease (cleaving NEDD8) and ScUlp1 protease (cleaving SUMO) (Frey and Gorlich, 2014). The complex Re42H11·IL-17F was recovered in the second elution step and further purified by size-exclusion chromatography. The complex was crystallized, and an X-ray diffraction dataset was recorded at the Swiss Light Source synchrotron. The structure was solved by molecular replacement to a resolution of 2.8 Å and an R of 0.28. (Figure 16A) Crystal structure of the tetrameric Re42H11·IL-17F complex in ribbon representation. CDR regions (defined in Figure 1) are highlighted in yellow and indicated accordingly. (Figure 16B) The IL-17 receptor IL17-RC (translucent gray surface) was docked onto the IL-17F dimer based on its alignment with the IL-17RC ECD·IL-17F complex (PDB ID 6HG4, Goepfert et al., 2020). [Figure 17] Sequence alignment of class A anti-IL-17 VHHs with the IL-17 interacting residues indicated. The IL-17 interacting residues of class A VHH antibodies were identified from the crystal structure (Figure 16) or by homology modeling based on this structure. IL-17A interacting residues are underlined, and IL-17F interacting residues are printed in bold. [Figure 18]Crystal structure of class B VHH Bm17B02 bound to an IL-17F dimer. Complexation and structure determination were as described in Figure 16, with the only difference being the His14-ScSUMO-tagged Bm17B02 used for co-expression and complexation. Figure 18A. Crystal structure of the tetrameric Bm17B02·IL-17F complex in ribbon representation. CDR regions (shown in Figure 1) are highlighted in yellow and indicated accordingly. Figure 18B. IL17-RC (translucent grey surface) was docked onto the IL-17F dimer as described in Figure 16. [Figure 19] Sequence alignment of class B VHHs highlighting IL-17 interacting residues. IL-17 interacting residues of class B VHH antibodies were identified from the crystal structure (Figure 18) or by homology modeling based on this structure. IL-17A interacting residues are underlined and IL-17F interacting residues are printed in bold. [Figure 20A] Production of VHH antibodies in Pichia pastoris. [Figure 20B] Production of VHH antibodies in Pichia pastoris. The scheme describes the production of VHH antibodies Re42B04a and Re42F08 in Pichia pastoris. Figure 20A, upstream process. Figure 20B, downstream process. Abbreviations used: MeOH - methanol, t - time, RCF - relative centrifugal force, HF - hollow fiber, TFF - tangential flow filtration, HIC - hydrophobic interaction chromatography, AIEX - anion exchange, CIEX - cation exchange, UF / DF - ultrafiltration / diafiltration. [Figure 21] Full-thickness human ex vivo skin model. Schematic diagram of the "Inflammaskin" human ex vivo skin model, which reproduces key features and inflammatory responses observed in psoriatic lesions. [Figure 22]Evaluation of IL-17A release in an ex vivo psoriasis model. This model consists of healthy human skin samples treated with a Th-polarizing cocktail to induce a psoriasis phenotype in Hypo InflammaSkin samples. Details of the study design are shown in Table 4. VHH antibodies Re42B04a and Re42F08 were injected intradermally into the skin samples once or three times. Positive control samples included betamethasone and secukinumab, both used in the treatment of psoriasis. Betamethasone is an anti-inflammatory steroid, and secukinumab is a monoclonal antibody targeting IL-17A. An unrelated VHH antibody, Re32D03 (SEQ ID NO: 64, directed against the spike protein of SARS-CoV2), was used as a negative control. HypoSkin and HypoInflammaSkin samples were cultured for 7 days. On day 7 of culture, supernatants were sampled, and IL-17A concentrations were measured using an MSD kit (K15076K, MesoScale Discovery). Concentrations are expressed in pg / mL, and values ​​(dots) for each replicate are plotted, with the mean and standard error of the mean (SEM) shown for each condition. Mean values ​​were calculated from detectable / QC control values. Graphs show all replicates for each condition. Statistical analysis (by one-way ANOVA) was performed between each condition and the untreated HypoInflammaSkin control. Additionally, one-way ANOVA tests were performed to assess differences between each condition and the unrelated VHH Re32D03. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 23]Evaluation of human ex vivo psoriatic skin samples after treatment with anti-IL17 VHH. Skin samples from the study described in Table 4 and analyzed in Figure 22 were collected on day 7, fixed, stained with H&E, and analyzed by microscopy. (Figure 23A) HypoSkin sample (not treated with pro-inflammatory cytokines). Images B-H show InflammoSkin samples treated with a Th-polarizing cocktail. (Figure 23B) Untreated psoriasis-induced tissue (negative control), (Figure 23C) treated with betamethasone (positive control), (Figure 23D) treated with secukinumab (positive control), (Figure 23E) one injection of VHH antibody Re42B04a, (Figure 23F) three injections of VHH antibody Re42B04a, (Figure 23G) one injection of VHH antibody Re42F08, and (Figure 23H) three injections of VHH antibody Re42F08. DETAILED DESCRIPTION OF THE INVENTION

[0103] The present invention relates to VHH antibodies that recognize human IL-17 polypeptides, including IL-17A, IL-17F, and IL-17AF.

[0104] Because the neutralizing epitope has a small number of amino acid exchanges between 17A and 17F, obtaining a VHH antibody that blocks IL-17 and tightly binds to both IL17A and IL17F is challenging and non-trivial. The present invention provides VHH antibodies (e.g., Bm43B02) with picomolar affinity (measured as the binding affinity of a monomeric VHH to immobilized human IL-17A or IL-17F homodimers) for both 17A and 17F. It is noteworthy to note that the VHH antibodies of the present invention have higher affinity for IL-17 isoforms, particularly IL-17F, when compared to previously published VHH antibodies targeting IL-17 (e.g., those disclosed in WO2012156219).

[0105] The binding data for the novel VHH antibodies of the present invention have been confirmed in cell-based assays for blocking IL-17 receptor activation and in an ex vivo human skin model.

[0106] VHH antibody The sequences of the CDRs of the VHH antibodies of the invention and control antibodies are listed in Table 1 below, while Table 2 lists the sequences of the full-length VHH antibodies.

[0107] [Table 1] * These VHH antibodies of Ablynx are disclosed in WO2012156219 and are provided herein for comparison purposes only, as they were compared in some of the experiments with the VHH antibodies of the present invention.

[0108] [Table 2-1]

[0109] [Table 2-2]

[0110] The present invention relates to VHH antibodies, which are monovalent heavy chain-only antibodies comprising CDR1, CDR2 and CDR3 domains connected by framework regions, including, but not limited to, whole VHH antibodies, e.g., natural VHH antibodies comprising framework regions of camelid origin, and modified VHH antibodies comprising modified framework regions, VHH antibody fragments and VHH antibody fusion proteins, e.g., fusion proteins with immunoglobulin or non-immunoglobulin peptides or polypeptides, so long as they exhibit the characteristics according to the present invention.

[0111] Although several methods are known in the art for determining the CDR sequences of a given antibody molecule, there is no standard, definitive method. Determining CDR sequences from antibody heavy chain variable regions can be performed according to any method known in the art, including, but not limited to, the KABAT, Chothia, and IMGT methods. Because VHH antibody binding sites (paratopes) often include scaffold residues and residues outside the narrowly defined CDR regions, common determination methods such as KABAT will exclude essential portions of the variable regions. This also distinguishes nanobodies from conventional antibodies. Other methods for identifying VHH antibody binding site and CDR sequences include the use of custom reference databases that utilize large collections of VHH antibody sequencing data. The selected set of CDRs can include sequences identified by more than one method. CDRs can also be defined through multiple alignments (with many other VHH antibodies) to identify hotspots of variability and relate them to standard VHH antibody structures. It is also possible to define CDRs by analyzing the structure of VHH antibodies and determining what the loops and antibody scaffolds are. In some cases, CDR-flanking residues are also variable and therefore included in the CDR definition. According to some embodiments of the present invention, the CDR sequences of VHH antibody variable regions are determined using a custom reference database containing sequencing data for more than 10,000 VHH antibodies.

[0112] The present invention also relates to covalent or non-covalent conjugates of VHH antibody molecules to non-proteinaceous structures, e.g., label groups, capture groups such as solid phase binding groups, or effector groups such as toxins. For example, the heterologous moiety can be derived from a fluorescent group, an enzyme such as biotin, peroxidase, phosphatase, or luciferase, a hapten, an affinity tag, or a nucleic acid such as an oligonucleotide.

[0113] The VHH antibodies of the present invention are particularly monoclonal VHH antibodies characterized by a specific amino acid sequence. VHH antibodies can be produced in prokaryotic host cells, yeast cells, or mammalian cells. In certain embodiments, the VHH antibodies are non-glycosylated. In some embodiments, glycosylation sites in the parent VHH antibody sequence are mutated to eliminate predicted glycosylation. In a specific embodiment, the mutation comprises the substitution of an asparagine (Asn, N) residue at the N-glycosylation site to prevent potential glycosylation of the VHH antibody. In more specific embodiments, Asn residues are substituted to prevent or eliminate glycosylation in a VHH antibody selected from Bm43B02 (position 19 of SEQ ID NO: 23), Bm44B11 (position 19 of SEQ ID NO: 27), Bm44B04 (position 76 of SEQ ID NO: 40), Bm44G07 (position 76 of SEQ ID NO: 44), and Bm42A09 (position 76 of SEQ ID NO: 45).

[0114] In certain embodiments, the VHH antibody is glycosylated, and the carbohydrate structure may derive from a glycosylation site introduced into the VHH sequence and / or from a fusion partner.

[0115] VHH antibodies according to the invention are characterized by (i) a CDR3 sequence, (ii) a combination of CDR1, CDR2 and CDR3 sequences, (iii) a complete VHH sequence, or (iv) competition with a specific reference antibody. Specific CDR and VHH sequences are provided in the tables, figures and sequence listing.

[0116] The present invention encompasses sequences related to the above sequences. These related sequences are defined by having a minimum identity to a particular amino acid sequence, e.g., a CDR or VHH sequence. This identity is shown over the entire length of the respective reference sequence and can be determined using well-known algorithms such as BLAST.

[0117] In specific embodiments, the related CDR3 sequence has at least 80%, or at least 90%, or at least 95% identity to the specifically indicated CDR3 sequence, eg, 1, 2, or 3 amino acid substitutions.

[0118] In specific embodiments, the relevant combination of CDR1, CDR2, and CDR3 sequences has at least 80%, or at least 90%, or at least 95% identity to the specifically indicated combination of CDR1, CDR2, and CDR3 sequences, e.g., substitution of 1, 2, 3, 4, 5, or 6 amino acids with different amino acids.

[0119] In specific embodiments, the related VHH sequence has at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity to the VHH sequence, e.g., 1, 2, 3, 4, 5, or up to 20 amino acid substitutions.

[0120] Furthermore, the present invention relates to VHH antibodies that compete with specific VHH antibodies disclosed herein for binding to human IL-17 polypeptide. In certain embodiments, competing VHH antibodies bind to the same or overlapping epitope on human IL-17 polypeptide. For example, the present invention refers to VHH antibodies that compete with a reference antibody, e.g., VHH antibody Re42H11 or VHH antibody Bm17B02. Competition can be determined by label-free biolayer interferometry performed as a cross-competition or epitope binning assay using a label-free detection system, e.g., the Octet® system from Sartorius, according to the manufacturer's instructions.

[0121] In specific embodiments, at least one amino acid of a reference sequence, including an amino acid in the CDR1, CDR2, or CDR3 sequence and / or an amino acid in a framework region, is replaced with another amino acid while preserving the structural integrity and epitope binding of the VHH antibody. These replacements can be conservative (i.e., with a similar amino acid) or non-conservative.

[0122] In further specific embodiments, at least one amino acid of the reference sequence, including an amino acid in the CDR1, CDR2, or CDR3 sequence and / or an amino acid in the framework region, is replaced by a conservative amino acid substitution, i.e., substitution of an amino acid with another amino acid having similar biochemical properties, such as substitution of an aliphatic amino acid, e.g., Gly, Ala, Val, Leu, or Ile, for another aliphatic amino acid; substitution of a basic amino acid, e.g., His, Lys, or Arg, for another basic amino acid or Met; substitution of an acidic amino acid or its amide, e.g., Asp, Glu, Asn, or Gln, for another acidic amino acid or its amide; substitution of an aromatic amino acid, e.g., Phe, Tyr, or Trp, for another aromatic amino acid.

[0123] In a further specific embodiment, the VHH antibody is selected from the VHH antibody Re42B04a comprising the VHH sequence set forth in SEQ ID NO: 19 or a variant thereof. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 19 are replaced by another amino acid.

[0124] In a further specific embodiment, the VHH antibody is selected from antibody R242B04 comprising the VHH sequence set forth in SEQ ID NO: 15 or a VHH antibody which is a variant thereof. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 15 are replaced by another amino acid.

[0125] In a further specific embodiment, the VHH antibody is selected from the VHH antibody Re42F08 comprising the VHH sequence set forth in SEQ ID NO: 39 or a variant thereof. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 39 are replaced by another amino acid.

[0126] In a further specific embodiment, the VHH antibody is selected from antibody Bm17B02 comprising the VHH sequence set forth in SEQ ID NO: 35 or a VHH antibody which is a variant thereof. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of SEQ ID NO: 35 are replaced by another amino acid.

[0127] The present invention provides four classes of VHH antibodies based on sequence similarity (80% or greater sequence identity for VHHH antibodies of the same class) and data obtained from crystal structures of VHH antibodies complexed with human IL-17. These crystal structures have been used to define the amino acid residues in the VHH sequence that primarily contribute to binding to IL-17 and neutralizing its binding to the receptor. Each VHH antibody of a particular class has at least 80% sequence identity to other members of the same class.

[0128] The present invention further relates to a nucleic acid molecule, e.g., a DNA molecule, encoding the above-described VHH, or a vector comprising such a nucleic acid molecule operably linked to an expression control sequence, particularly a heterologous expression control sequence. Furthermore, the present invention also relates to a cell comprising the above-described nucleic acid molecule or vector. Vectors for the recombinant production of VHH antibodies are well known in the art. In certain embodiments, the vector is an extrachromosomal vector. In other embodiments, the vector is a vector for genomic integration. The cell can be any known host cell for producing antibodies or antibody fragments, for example, a prokaryotic cell such as an E. coli or Bacillus sp. cell, a yeast cell, particularly a Pichia yeast cell, an insect cell, or a mammalian cell, e.g., a CHO cell, or a plant cell. In certain embodiments, the cell comprises the nucleic acid or vector extrachromosomally. In other embodiments, the cell comprises the nucleic acid or vector integrated into the genome, for example, as an expression cassette integrated into the genome.

[0129] A further aspect of the present invention is a method for recombinantly producing a VHH antibody by growing the above-described cells in a culture medium and obtaining the VHH antibody from the cells or the culture medium. Suitable culture media and conditions are well known in the art.

[0130] Binding to human IL-17 The VHH antibodies of the present invention bind to human IL-17 polypeptide. As shown in Table 3, the present inventors have identified VHH antibodies that bind to human IL-17 polypeptide with high affinity and cross-react with (i) human IL-17A homodimer and (ii) human IL-17F homodimer.

[0131] In the context of the present disclosure, the term "human IL-17" encompasses multiple different human IL-17 family members, or isoforms, including, but not limited to: -IL-17A (UniProt accession number Q16552), -IL-17B (UniProt accession number Q9UHF), -IL-17C (UniProt accession number Q9P0M4), -IL-17D (UniProt accession number Q8TAD2), -IL-17E (UniProt accession number Q9H293), - Contains IL-17F (UniProt accession number Q96PD4).

[0132] The term "human IL-17" encompasses human IL-17A (UniProt accession number Q16552), in particular in the form of a homodimer comprising two IL-17A units, IL-17F (UniProt accession number Q96PD4), in particular in the form of a homodimer comprising two IL-17F units, and IL-17AF, in the form of a heterodimer comprising one IL-17A unit and one IL-17F unit.

[0133] However, it should be noted that the term "human IL-17" also encompasses naturally occurring variants of the human IL-17 polypeptide and genetically modified constructs as described herein.

[0134] We performed selection and binding experiments using genetically modified IL-17A, IL-17F, and IL-17A / F constructs as follows: - IL-17A homodimer containing amino acid residues 40-155 of human IL-17A (UniProt Q16552), which corresponds to the ordered region in the crystal structure. The first five amino acids (GSEDS) are spacer residues. Furthermore, it contains two point mutations: N68D (eliminating an N-glycosylation site) and C192S (eliminating an unpaired cysteine ​​that would otherwise cause problems in recombinant expression). The amino acid sequence of this polypeptide is shown in SEQ ID NO:1. - IL-17F homodimer containing amino acid residues 39 to 163 of human IL-17F (UniProt Q96PD4). N83D mutation eliminates the N-glycosylation site. The first five residues (GSEGE) are a linker. The amino acid sequence of this polypeptide is shown in SEQ ID NO:2. - an IL17A / F heterodimer consisting of two polypeptides. The IL-17A unit contains amino acid residues 40-155 of human IL-17A (UniProt Q16552) preceded by five spacer residues (GSEDS) and the N68D mutation. The amino acid sequence of this polypeptide is shown in SEQ ID NO:3. The IL-17F unit contains amino acid residues 39-163 of human IL-17F (UniProt Q96PD4) preceded by five spacer residues (GSEDS) and the N83D and C137S mutations. The amino acid sequence of this polypeptide is shown in SEQ ID NO:4.

[0135] The bacterially expressed IL-17A, IL-17F, and IL-17AF proteins described above were used during initial immunization, selection, characterization, and crystallization.

[0136] For the immunization and selection of second generation VHH antibodies and for their characterization, mammalian expressed versions of IL-17A and IL-17F lacking the indicated modifications were used as follows: - Mature human IL-17A in mammalian expression (UniProt Q16552). The amino acid sequence of this polypeptide is shown in SEQ ID NO: 77. - Mature human IL-17F (UniProt Q96PD4) in mammalian expression. The amino acid sequence of this polypeptide is shown in SEQ ID NO: 78.

[0137] In certain embodiments, a VHH antibody of the invention binds to (i) human IL-17A homodimer, (ii) human IL-17F homodimer, and (iii) human IL17A / F heterodimer, and has a binding affinity, expressed as a dissociation constant KD, for each of (i), (ii), and (iii) of about 1 nM or less, about 100 pM or less, about 50 pM or less, about 20 pM or less, or about 10 pM or less. Binding affinities can be determined as described in detail herein in the Examples and Figures, for example, using the polypeptides of SEQ ID NOs: 1-4 and 77-78 as described above.

[0138] IL-17 neutralization The VHH antibodies of the present invention can neutralize the binding of human IL-17 dimers to the human IL-17 receptor and prevent or inhibit receptor activation. As shown in Table 3, the present inventors have identified VHH antibodies that cross-neutralize (i) human IL-17A homodimer, (ii) human IL-17F homodimer, and (iii) human IL17A / F heterodimer.

[0139] In certain embodiments, the VHH antibodies of the invention cross-neutralize the binding of human IL-17 polypeptides, particularly (i) human IL-17A homodimer, (ii) human IL-17F homodimer, and (iii) human IL17A / F heterodimer, to the human IL-17 receptor. Neutralizing potency can be determined as described in detail herein in the Examples.

[0140] stability For their intended therapeutic use, anti-IL-17 VHH antibodies should not only be highly potent in interleukin neutralization, but also be developable as biological drugs, which includes being sufficiently stable to withstand lengthy large-scale production processes as well as transportation and storage (ideally for several years in liquid, semi-liquid, or solid formulations) without aggregation or loss of activity.

[0141] A good predictor of stability is thermostability, which can be measured, for example, by thermal shift assays or, in particular, by differential scanning fluorimetry. The inventors have identified several thermostable or ultrathermostable VHH antibodies, as shown in Table 3.

[0142] In a particular embodiment, the present invention relates to stable, in particular thermostable or ultrathermostable, VHH antibodies. Preferably, the VHH antibodies have a melting point (melting temperature) of at least about 65°C, at least about 80°C, at least 90°C, or at least about 95°C, when measured under non-reducing conditions, and / or an aggregation temperature of at least about 50°C, at least about 60°C, at least 70°C, or at least about 80°C. Melting and aggregation temperatures are determined as described herein.

[0143] In certain embodiments, the present invention relates to stable, particularly thermostable or superthermostable, VHH antibodies. Specifically, the VHH antibodies have a melting point (melting temperature) of at least about 65°C, at least about 80°C, at least 90°C, or at least about 95°C, and / or an aggregation temperature of at least about 50°C, at least about 60°C, at least 70°C, or at least about 80°C, when measured under non-reducing conditions. Melting and aggregation temperatures are determined as described herein. According to some embodiments, stable VHH antibodies have a melting point (melting temperature) of at least about 65°C and / or an aggregation temperature of at least 50°C, and superthermostable VHH antibodies have a melting point (melting temperature) of at least 95°C and / or an aggregation temperature of at least about 80°C, when measured under non-reducing conditions. However, it should be noted that the results of stability measurements are condition-dependent. Aggregation is promoted, for example, by high protein concentration and by a pH close to the isoelectric point of the protein. Stability is also affected by the buffer composition and additives used with the component being tested.

[0144] VHH antibody set In a further aspect, the present invention relates to a set comprising at least two, three, four, or more of the above-described VHH antibodies. In such a set, the individual VHH antibodies are present at a suitable molar ratio. Typically, the molar ratio is in the range of about 2:1 to about 1:2, particularly about 1.5:1 to about 1:1.5, and even more particularly about 1:1. In certain embodiments, the VHH antibody set may comprise a single composition, and the VHH antibodies in the set consist of a predetermined number of VHH antibodies of different species as described above. The VHH antibody set may also comprise multiple compositions, each comprising a VHH antibody of a different species as described above. The set of the present invention may not comprise other VHH antibodies.

[0145] Monovalent and polyvalent VHH antibodies In certain embodiments, the VHH antibodies of the invention are in a monovalent format, i.e., have a single binding site for a human IL-17 polypeptide. In these embodiments, the VHH antibodies may be present as such or may be covalently or non-covalently linked to a heterologous moiety, such as a peptide or non-peptide moiety.

[0146] In further embodiments, the VHH antibodies of the invention are in a multimeric, e.g., dimeric, or trimeric format. In these embodiments, several VHH antibody units may be covalently or non-covalently linked together via linkers and / or multimerizing, e.g., dimerizing or trimerizing, moieties.

[0147] In certain embodiments, the VHH antibody is a homodimeric VHH antibody in which the VHH antibody unit is covalently linked to a dimerization moiety, such as an immunoglobulin Fc fragment.

[0148] In certain embodiments, the VHH antibody is a heterodimeric VHH antibody, in particular a covalent VHH heterodimer comprising a first VHH antibody and a second VHH antibody, wherein the first VHH antibody and the second VHH antibody bind to different epitopes on IL-17, or the first VHH antibody binds to IL-17 and the second VHH antibody binds to a different target.

[0149] VHH antibody production VHH antibodies, including monomeric and multimeric VHH antibodies, may be produced as described in WO 2022 / 023483 and WO 2022 / 023484, the contents of which are incorporated herein by reference, or by other methods known in the art.

[0150] VHH antibodies can be recombinantly produced in suitable host cells, such as prokaryotic or eukaryotic host cells or host organisms. To this end, a nucleic acid molecule encoding the VHH antibody is introduced into the host cell or host organism and expressed in the host cell or host organism. The nucleic acid molecule can encode a monomeric VHH antibody or a subunit of a multimeric VHH antibody.

[0151] In certain embodiments, VHH antibodies are recombinantly produced in bacteria, such as E. coli or Bacillus. For example, expression in bacteria can involve cytoplasmic and / or periplasmic expression of the VHH antibody and purification from the host cell, or secretory expression and purification of the VHH antibody from the culture medium. In certain embodiments, the nucleic acid sequence encoding the VHH antibody is fused to at least one sequence that directs expression into the periplasm and / or culture medium.

[0152] In further particular embodiments, the VHH antibody is recombinantly produced in a eukaryotic host cell or host organism, preferably in yeast, such as Pichia pastoris, Saccharomyces cerevisiae, or Hansenula polymorpha, or in animal cells, particularly mammalian, such as human or hamster cells. For example, expression in a eukaryotic host cell or host organism, such as yeast, may involve cytoplasmic and / or periplasmic expression and purification of the VHH antibody from the host cell, or preferably secretion from the host cell and purification of the VHH antibody from the culture medium. In certain embodiments, the nucleic acid sequence encoding the VHH antibody is fused to at least one sequence that directs expression into the culture medium.

[0153] VHH antibodies can be produced using any method known in the art for producing proteins, antibodies, and nanobodies. Typically, VHH antibodies are recombinantly produced in prokaryotic or eukaryotic host cell systems or host organisms, such as bacteria, yeast, plant cells, or in mammalian cells. According to some embodiments, VHH antibodies are recombinantly produced in Pichia pastoris.

[0154] In an even more specific embodiment, the VHH antibodies are produced using the methods shown in Figures 20A and 20B.

[0155] Therapeutic Uses and Methods of Use A still further aspect of the present invention is the use of the above VHH antibodies in medicine, in particular for therapeutic and / or in vitro or in vivo diagnostic uses. In certain particular embodiments, the VHH antibodies are used in human medicine.

[0156] The VHH antibodies of the present invention are useful in the prevention or treatment of disorders caused by and / or associated with overactivity of IL-17, particularly overactivity of IL-17A and / or IL-17F.

[0157] In certain embodiments, the VHH antibodies are useful in the prevention or treatment of inflammatory and / or immune-related disorders, such as inflammatory and / or immune-related skin disorders.

[0158] Exemplary disorders are asthma, psoriasis, e.g., plaque psoriasis, arthritis, e.g., rheumatoid arthritis or psoriatic arthritis, hidradenitis suppurativa, inflammatory bowel disease (Crohn's disease, ulcerative colitis), multiple sclerosis, skin cancer, ankylosing spondylitis, uveitis, atopic dermatitis, graft versus host disease, Alzheimer's disease, fatty liver disease, COVID-19, sepsis, ischemic stroke, Parkinson's disease, influenza virus infection.

[0159] In some embodiments, the disorder is psoriasis vulgaris. In particular embodiments, the disorder is mild to moderate psoriasis vulgaris.

[0160] In therapeutic applications, VHH antibodies are administered to a subject in need thereof, particularly a human subject, in an effective amount. The dosage will depend on the specific type of agent, e.g., monovalent or multimeric VHH antibody, the type of disease, and the route of administration.

[0161] Typically, VHH antibodies are administered as a pharmaceutical composition comprising an active agent and a pharmaceutically acceptable carrier or excipient. Examples of suitable carriers and excipients for formulating antibodies or antibody fragments are well known in the art.

[0162] The present invention further provides a method for treating diseases and disorders associated with overexpression or hyperactivity of human IL-17, comprising administering to a subject in need of such treatment a pharmaceutical composition comprising an effective amount of a VHH antibody that neutralizes IL-17 receptor activation and a pharmaceutically acceptable carrier, thereby treating the disease or disorder.

[0163] In certain embodiments, the disease or disorder is an inflammatory and / or immune-related disorder. According to more particular embodiments, the disease or disorder is an inflammatory and / or immune-related skin disorder.

[0164] Exemplary disorders that can be treated with the VHH antibodies of the invention are psoriasis, e.g., plaque psoriasis, asthma, arthritis, e.g., rheumatoid arthritis or psoriatic arthritis, hidradenitis suppurativa, inflammatory bowel disease (Crohn's disease, ulcerative colitis), multiple sclerosis, skin cancer, ankylosing spondylitis, uveitis, atopic dermatitis, graft versus host disease, Alzheimer's disease, fatty liver disease, COVID-19, sepsis, ischemic stroke, Parkinson's disease, influenza virus infection.

[0165] In some embodiments, the disorder is psoriasis vulgaris. In particular embodiments, the disorder is mild to moderate psoriasis vulgaris.

[0166] According to some embodiments, a pharmaceutical composition comprising an effective amount of an IL-17-neutralizing VHH antibody and a pharmaceutically acceptable carrier is administered locally to the site of a subject in need of such treatment, thereby treating the disease or disorder.

[0167] The terms "administering locally", "topical administration" or "transdermal administration" mean that the VHH antibody is not administered systemically. The term includes topical application, in particular onto the surface of the skin. Topical administration also encompasses administration of the composition by injection, in particular intraepidermal and / or intradermal and / or subcutaneous and / or subcutaneous injection and / or microinjection.

[0168] The compositions of the present invention are, according to some embodiments, suitable for administration by topical or injectable routes of administration (particularly intraepidermal and / or intradermal and / or microinjection) and / or by systemic injection, e.g., subcutaneous injection.

[0169] The method of the present invention involves topically administering a pharmaceutical composition to a defined area of ​​the skin.

[0170] The treatment period is ideally long enough to provide improvement of the disease or disorder being treated. The treatment period can be at least one week, and in some embodiments, the treatment period can last about four, eight, or twelve weeks. In certain embodiments, the treatment period spans multiple months (i.e., 3 to 12 months) or years. In one embodiment, the pharmaceutical composition is applied at least once daily for a treatment period of at least four, eight, or twelve weeks. In one embodiment, the pharmaceutical composition is applied twice daily for a treatment period of at least four, eight, or twelve weeks. Alternatively, the pharmaceutical composition is administered once every other day, once every three days, or once weekly for one week, one month, or as long as improvement is achieved.

[0171] Although subjects for the pharmaceutical methods may be of any suitable age, the subject in some embodiments is a pediatric, adult, or geriatric subject.

[0172] Pharmaceutical compositions suitable for dermal administration can be used in combination with mechanical devices, such as massage roller devices with mechanical and frictional action to promote the penetration of the active agent, or wave-emitting systems (light, low frequency, infrared frequency, etc.) that activate a skin response. Additional means that can be used or combined with other administration routes are patches and microneedles.

[0173] The pharmaceutical composition of the present invention suitable for injection, particularly for intraepidermal and / or intradermal and / or subcutaneous injection and / or microinjection, can be injected by a device containing a needle or microneedle, or by a needleless injection device. Such devices are well known in mesotherapy. Alternatively, the pharmaceutical composition can be directly administered by iontophoresis to achieve greater penetration of the active agent.

[0174] The terms "treat," "treating," and "treatment" are all intended to refer to the improvement or reversal of at least one measurable physical parameter associated with an amendable disease or disorder.

[0175] The terms "treatment" or "treating" may be used interchangeably herein and refer to inhibiting, preventing or arresting the onset of a disease or disorder and / or causing the reduction, remission or regression of a disease or disorder.

[0176] As used herein, the phrase "inflammatory disease or disorder" refers to a disease, condition, or disorder associated with inflammation. As used herein, the term "inflammation" refers to the process by which a subject's immune system coordinates a response to tissue injury, infection, antigenic challenge, etc. Inflammation can be associated with an increased blood supply to the tissue, increased capillary permeability of the tissue, and / or increased migration of leukocytes into the tissue.

[0177] According to some embodiments of the present invention, the inflammatory disease or disorder is an autoimmune disease or disorder, which may be, but is not limited to, psoriasis, arthritis, systemic lupus erythematosus (lupus, SLE), multiple sclerosis, and inflammatory bowel disease.

[0178] Pharmaceutical Composition The VHH antibodies of the present invention can be formulated into any form of pharmaceutical composition according to the intended use. Any form of pharmaceutical composition according to the present invention includes liquid, semi-liquid, solid, and semi-solid formulations.

[0179] According to some embodiments, the VHH antibodies are formulated for topical administration, for example as creams, pastes, gels, hydrogels, ointments, lotions, and emulsions.

[0180] According to some embodiments, the pharmaceutical composition comprises one or more excipients, carriers, or buffers. According to some embodiments, the pharmaceutical composition comprises an excipient selected from the group consisting of an emulsifier, a pH buffering agent, a preservative, a chelating agent, a tonicity agent, a humectant, an antioxidant, and a gelling agent.

[0181] According to additional embodiments, the pharmaceutical composition is in a form selected from the group consisting of a solution, emulsion, nanoemulsion, suspension, lipid nanoparticles (e.g., liposomes), microparticles, ointment, cream, lotion, paste, gel, hydrogel, spray, powder, stick, and patch, with each possibility representing a separate embodiment of the present invention.

[0182] The present invention provides pharmaceutical compositions comprising at least one VHH antibody that neutralizes IL-17 as an active agent and a pharmaceutically acceptable carrier, diluent, or excipient.

[0183] The term "pharmaceutical composition" as used herein refers to a composition suitable for treating a disease or disorder caused by and / or associated with overactivity of IL-17, particularly associated with overactivity of IL-17A and / or IL-17F. In particular, the pharmaceutical composition is useful for the prevention or treatment of inflammatory and / or immune-related disorders, e.g., inflammatory and / or immune-related skin disorders. Exemplary disorders include asthma, psoriasis, e.g., plaque psoriasis, arthritis, e.g., rheumatoid arthritis or psoriatic arthritis, hidradenitis suppurativa, inflammatory bowel disease (Crohn's disease, ulcerative colitis), multiple sclerosis, skin cancer, ankylosing spondylitis, uveitis, atopic dermatitis, graft-versus-host disease, Alzheimer's disease, fatty liver disease, COVID-19, sepsis, ischemic stroke, Parkinson's disease, and influenza virus infection.

[0184] The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0185] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in humans. The components of the pharmaceutical compositions of the present invention are all pharmaceutically acceptable agents.

[0186] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. A carrier can be a liquid, preferably a sterile liquid, such as water, oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.

[0187] The compositions can also contain, if desired, minor amounts of emulsifiers, anionic emulsifiers, synthetic polymers, lipid-based excipients, lecithin, or pH buffers, such as acetates, citrates, or phosphates. Preservatives such as benzyl alcohol or methylparabens, chelating agents such as ethylenediaminetetraacetic acid, and agents for adjusting tonicity, such as sodium chloride or dextrose, are also contemplated.

[0188] Other optional excipients include, but are not limited to, humectants such as water-soluble liquid polyols, e.g., glycerin, propylene glycol, hexylene glycol, butylene glycol, pentylene glycol, dipropylene glycol, and mixtures thereof, antioxidants such as glycolic acid, citric acid, lactic acid, malic acid, mandelic acid, ascorbic acid, sodium bisulfite, vitamin E, and derivatives thereof, humectants, suspending agents, gelling agents, emollients and skin moisturizers, antimicrobial (e.g., antibacterial) agents, antifungal agents, analgesics, UV absorbers, wound healing promoters, growth factors, reactive oxygen species, anti-inflammatory agents, vitamins such as vitamin C, vitamin B, and derivatives thereof, thiamine, riboflavin, niacin, pantothenate, pyridoxine, folic acid, cobalamin, biotin, choline, inositol, niacin ... amino acids and their derivatives such as alanine, arginine, asparagine, aspartic acid, carnitine, citrulline, cysteine, dimethylglycine, gamma-aminobutyric acid, glutamic acid, glutamine, glutathione, glycine, histidine, isoleucine, leucine, lysine, methionine, ornithine, phenylalanine, praline, serine, taurine, threonine, tryptophan, tyrosine, valine; minerals such as boron, calcium, chromium, cobalt, copper, fluoride, germanium, iodine, iron, lithium, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, silicon, sodium, sulfur, vanadium, zinc; herbal extracts such as green tea, algae, aloe, etc.; retinoids, flavonoids, and mixtures thereof.

[0189] The composition may be in the form of a solution, emulsion (e.g., oil-in-water, water-in-oil-in-water, water-in-oil, or oil-in-water-in-oil), nanoemulsion, suspension, microparticles, oil, ointment, cream, lotion, paste, gel, hydrogel, spray, powder, stick, or combinations thereof.

[0190] The pharmaceutical compositions of the present invention may be formulated for sustained, slow, or delayed release using methods and ingredients well known in the art. Thus, depot formulations of the VHH antibodies of the present invention are included within their scope.

[0191] In some embodiments, a tape or other support structure can be applied to the skin. In some embodiments, the composition is applied to the skin before the tape is placed. In that situation, the tape may or may not be porous.

[0192] In some embodiments, the tape is a polymer matrix or gel that allows the composition to contact the skin when applied onto the tape. Such tapes, also called patches or transdermal patches, have the additional advantage of providing controlled delivery of compounds to the body. Transdermal patches can be made by dissolving or dispersing the compound in a suitable medium and then applying it to the tape. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by the polymer matrix or gel, and optionally by a rate-controlling membrane. In one embodiment, the polymer matrix can be hyaluronic acid, which has the property of trapping water to form a gel.

[0193] For topical application, the composition can be formulated in the form of an ointment containing the VHH antibody dissolved or suspended in a suitable carrier, including, but not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene, emulsifier wax, and water. Alternatively, the composition can be formulated as a cream or lotion containing the VHH antibody dissolved or suspended in a suitable carrier, including, but not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water.

[0194] For injection, pharmaceutical compositions can be formulated as liquids such as solutions, emulsions, suspensions, or gels. According to a preferred embodiment, the pharmaceutical composition is a sterile solution or suspension. Acceptable solvents and carriers include, but are not limited to, water, Ringer's solution, and isotonic sodium chloride. In addition, sterile oils are often used as solvents. For this purpose, any oil, such as mono- and diglycerides, can be used. Fatty acids such as oleic acid and its glyceride derivatives can also be used to prepare injectable compositions, as can pharmaceutically acceptable natural oils such as olive oil and castor oil, especially their polyoxyethylated forms. These oily solutions can contain suspending agents or diluents, such as carboxymethylcellulose, for the formulation of emulsions and suspensions. Surfactants and emulsifiers, such as Tween, can also be included. Aqueous or oily suspensions can be formulated using wetting agents, dispersing agents, and suspending agents, according to methods well known to those skilled in the art.

[0195] As used herein, "microparticles" refers to polymers or combinations of polymers fabricated into objects of various sizes. Microparticles can be of any shape, but they are often substantially spherical in shape, in which case they are referred to as "microspheres" or "microbeads." Incorporation of an active agent into the microparticles can be achieved by mixing the dry microparticles with a solution of the active agent in an aqueous or aqueous-organic solution. Microspheres are sterilized before injection or before being constituted into an injectable composition.

[0196] Various techniques can be used to prepare the pharmaceutical composition of the present invention.For example, the active agent can generally be incorporated into an acceptable carrier in the usual manner for preparing pharmaceutical products.Therefore, the active agent can first be dissolved or dispersed in water or another solvent or liquid that is incorporated into the acceptable carrier.Preferred compositions for use in this preparation approach are oil-in-water, water-in-oil, or water-in-oil-in-water emulsion.

[0197] In some embodiments, the active agent is maintained separately from the carrier, for example, as a dry powder, with or without excipients.The subject mixes the desired amount of active agent and the desired amount of carrier just before applying the pharmaceutical composition, ensuring that the active agent maintains its maximum effectiveness and allowing the efficacy of the composition to be adjusted to the subject's individual needs.The pharmaceutical composition obtained can then be applied to the skin.

[0198] According to further embodiments, the pharmaceutical composition is administered topically. According to certain embodiments, the pharmaceutical composition is administered topically, and the composition is in the form of an ointment, cream, lotion, paste, gel, hydrogel, spray, powder, stick, or patch.

[0199] Depending on the stage and severity of the disorder, the pharmaceutical composition may be administered once or several times during the course of the disorder, for example, once or several times daily, every other day, twice weekly, once weekly, once monthly, or every few months for a suitable period of time.

[0200] In certain embodiments, the pharmaceutical composition is administered parenterally, for example, by subcutaneous, intramuscular, or intravenous injection, or by infusion. In certain embodiments, the pharmaceutical composition is administered by injection. According to some embodiments, the injection is intraepidermal, intradermal, subcutaneous, microinjection, or any combination thereof. According to certain embodiments, the pharmaceutical composition administered by injection is in the form of a solution, emulsion suspension, or microparticles.

[0201] In further embodiments, the pharmaceutical composition is administered locally, eg, topically, orally, nasally, or pulmonary, eg, by inhalation as an aerosol.

[0202] The VHH antibodies may be administered alone or together with further active agents or treatments, in particular further agents useful for the prevention and / or treatment of disorders caused by and / or associated with IL-17 overactivity, in particular associated with overactivity of IL-17A and / or IL-17F.

[0203] Pharmaceutical compositions suitable for use in connection with the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredient effective to prevent, alleviate, or ameliorate symptoms of the disease or disorder being treated in the subject.

[0204] The term "effective amount" is the amount of active agent sufficient to provide a beneficial effect to the subject to which the composition is administered.

[0205] Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0206] For any preparation used in the methods of the present invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0207] The toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro in cell cultures or experimental animals. Data obtained from these in vitro and cell culture assays and animal tests can be used in formulating various dosages for use in humans. Dosages can vary depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration, and dosage can be chosen by the individual physician in consideration of the patient's condition. (See, for example, Fingl, et al. (1975), "The Pharmacological Basis of Therapeutics", Ch. 1 p. 1).

[0208] Dosage amount and interval can be individually adjusted to a level of active ingredient sufficient to achieve the minimum effective concentration (MEC). The MEC varies from preparation to preparation but can be estimated from in vitro data. The dosage required to achieve the MEC depends on individual characteristics and the route of administration. Detection assays can be used to determine plasma or tissue concentrations.

[0209] Depending on the severity and responsiveness of the condition to be treated, dosage may be single or multiple administrations, and the course of treatment may last from several days to several weeks, or a diminution of the disease state may be achieved.

[0210] The amount of a composition administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0211] Thus, compositions and / or articles of some embodiments of the present invention may be presented in a pack or dispenser device, such as an FDA-approved kit, which may contain one or more unit dosage forms containing the active ingredient, if desired. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also correspond to a notice associated with the container in a format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, reflecting approval by the agency of the composition or form of administration in human or veterinary medicine. Such notice may, for example, be a label approved by the U.S. Food and Drug Administration for prescription drugs or an approved product insert. Compositions comprising the preparations of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container (e.g., a lyophilized vial), and labeled for treatment of an indicated condition, as further detailed above.

[0212] In some embodiments, VHH antibodies and pharmaceutical compositions comprising them are administered to subjects suffering from psoriasis, particularly plaque psoriasis, more particularly mild to moderate plaque psoriasis, as a sole treatment or as part of a treatment regimen that includes at least one additional agent or treatment for psoriasis or at least one symptom associated with the disease. According to certain embodiments, the additional treatment includes a steroid.

[0213] In certain embodiments, the additional active agent is an active agent useful in the prevention and / or treatment of the exemplary disorders listed above.

[0214] The invention provides a kit comprising a composition of the invention and a syringe or injection device suitable for injection.

[0215] Diagnostic Use Diagnostic applications include in vitro methods in which VHH antibodies are used to detect IL-17 in a sample, for example, in a body fluid such as saliva, blood, serum, or plasma, in a fecal sample, or in a tissue or biopsy sample. Diagnostic applications further include in vivo methods in which VHH antibodies are used to detect IL-17 in a subject, particularly a human patient. For diagnostic applications, VHH antibodies may carry a label for direct detection or may be used in combination with a secondary detection reagent, for example, an antibody, including a conventional antibody or a VHH antibody, for indirect detection according to techniques established in the art.

[0216] Evaluation of the activity and safety of VHH antibodies for use in therapy The VHH antibodies and sets of VHH antibodies of the present invention can be tested for their activity in vitro, in vivo, and ex vivo in IL-17-related disease models.

[0217] In vitro disease models for psoriasis and atopic dermatitis were reviewed in Sarama et al., 2022.

[0218] Some animal models utilize animal or human skin. Because the amino acid sequence homology between human and porcine IL-17 is relatively high (73%), it is possible to evaluate VHH antibodies in a pig skin model. Human skin models, such as those for psoriasis, that utilize cultures of primary human epidermal keratinocytes isolated from neonatal foreskins can also be used to evaluate the potential therapeutic effects of VHH antibodies. The effects of VHH antibodies of the present invention on factors regulating psoriatic keratinocytes (Zhou, X. et al. 2022) can also be tested.

[0219] As a non-limiting example, in vivo studies in SCID / Beige mice implanted with human xenografts (Keren et al., 2018) can be used to test several toxicological parameters. Transplanted mice are considered a suitable in vivo model because normal human skin is transplanted onto SCID mice and disease is induced by injection of IL-2-activated PBMCs from human psoriasis patients. Blockade of the IL-17 downstream cascade by the VHH antibodies of the present invention is determined by improved levels of markers (S100A7, Ki-67, beta-defensin-2, etc.) and skin appearance as determined by H&E staining or qRT-PCR.

[0220] In this model, healthy human skin fragments are transplanted into SCID mice on day 0. Peripheral blood mononuclear cells (PBMCs) from the blood of psoriasis patients are isolated and cultured for 14 days in the presence of high doses of IL-2 to activate allogeneic T cells expressing high levels of NK cell receptors. These PBMCs are then injected into mice on day 28 post-transplant to generate psoriasis-like lesions that develop within the grafts and exhibit almost all of the key features of human psoriasis. These features include epidermal thickening, the presence of immune cells, edema, dilated blood vessels, and increased keratinocyte proliferation. Importantly, these psoriasis-like lesions respond to commonly used antipsoriatic treatments, highlighting the remarkable relevance of this preclinical model for psoriasis research (Keren et al., 2018). Treatment with VHH antibodies or controls is performed on day 42, and skin sampling and analysis are performed on day 56.

[0221] Preclinical safety studies include, for example, (i) human tissue cross reactivity (TCR) studies, (ii) pharmacokinetic / biodistribution (PK / BD) studies in rats, and (ii) GLP toxicology studies in minipigs.

[0222] GLP toxicology testing can be used to confirm an acceptable safety profile of the tested VHH antibody. Currently accepted toxicology animal models for mAbs against psoriasis are performed in non-human primates (NHPs), as described, for example, in Kolbinger et al., 2022. Furthermore, the minipig model used for the VHH antibodies of the present invention is more suitable for the following reasons: 1. VHH antibodies are administered topically or ID, not systemically like mAbs; therefore, the similar skin vascularization, structure, and neurological structure in minipigs and humans are more relevant. 2. VHH antibodies are cleared from the blood via the kidneys and liver within 1 hour and are not expected to significantly diffuse throughout the body for extended periods of time (Esparza et al., 2021). Upon ID administration of biotinylated VHH antibodies to mice and histopathological sampling, it was possible to detect them in the epidermis and dermis as well as in the blood immediately after administration. 3. VHH antibodies lack Fc receptors and are therefore not immunogenic. 4. Regulatory efforts to reduce the use of NHPs in preclinical research (Prior et al., 2017).

[0223] In vitro tissue cross-reactivity (TCR) studies can be used to identify non-specific and specific binding of VHH antibodies in different types of human tissues. This information is important in ensuring that experimental antibodies do not bind to epitopes other than the targeted site, thus minimizing the risk of treatment-related toxicity.

[0224] An immunohistochemistry-based tissue cross-reactivity (TCR) screening assay was developed to identify non-specific and specific binding of the VHH antibodies of the present invention. This test is considered important to demonstrate that the tested moiety does not bind to epitopes other than the targeted site. The assay was established using human tissues from three different donors.

[0225] Since the primary objective is safety assessment, the intensity of immunohistochemistry (IHC) staining can provide valuable insight into potential toxicity. IHC staining is investigated using biotinylated (and / or FITC) conjugated forms of VHH antibodies on positive and negative control systems (slides spotted with target protein or BSA). VHH antibodies are tested on human tissues (three donors) at two concentrations according to the EMA / FDA tissue list, and isotype controls are tested at one concentration. Following the experimental phase, pathology evaluation is performed. Tissue integrity is assessed using von Willebrand factor and by two independent histopathologists.

[0226] To examine histopathology (in standard organs), subchronic administration to minipigs can be performed for 28 days at three increasing doses.

[0227] A Phase I / IIa randomized, double-blind, placebo-controlled study can be conducted to assess safety in humans. The tolerability, immunogenicity, pharmacokinetics, pharmacodynamics, and efficacy of multiple ascending doses of the anti-IL-17A / F VHH antibodies of the present invention will be evaluated. The study can be conducted, for example, in male and female subjects with mild to moderate psoriasis using intradermal and / or topical administration modes.

[0228] As used herein, the phrase "subject in need thereof" refers to a mammalian male or female subject (e.g., a human) diagnosed with an inflammatory disease or disorder. In certain embodiments, the term encompasses individuals at risk of developing an inflammatory disease or disorder. Subjects may be of any gender or age, including newborns, infants, juveniles, adolescents, adults, and the elderly.

[0229] As used herein, the term "about" refers to about 10%.

[0230] The terms "comprises," "comprising," "includes," "including," "having," and variations thereof mean "including but not limited to."

[0231] The term "consisting of" means "including and limited to."

[0232] The term "consisting essentially of" means that a composition, method, or structure may include additional components, steps, and / or moieties, provided that the additional components, steps, and / or moieties do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0233] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" can include a plurality of compounds, including mixtures thereof.

[0234] Throughout this application, various embodiments of the present invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0235] Whenever a numerical range is given herein, it is intended to include any recited number (fractional or integer) within the range given. The phrases "ranging / ranges" from a first denoted number to a second denoted number and "ranging / ranges" from a first denoted number to a second denoted number are used interchangeably herein and are intended to include the first and second denoted numbers and all fractional and integer numbers therebetween.

[0236] As used herein, the term "method" refers to ways, means, techniques, and procedures for accomplishing a given task, and includes, but is not limited to, ways, means, techniques, and procedures known to or readily developed from known ways, means, techniques, and procedures by practitioners in the fields of chemistry, pharmacy, biology, biochemistry, and medicine.

[0237] It will be understood that certain features of the invention that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment may also be provided separately or in any suitable subcombination, or in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments, unless the embodiment cannot function without those elements.

[0238] The term "about" refers to a value 10% above or below the stated value.

[0239] It is noted that each possibility disclosed throughout this specification represents a separate embodiment of the present invention.

[0240] A non-limiting list of VHH antibodies of the present invention, as well as their affinities and utility for IL-17 neutralization, is provided in Table 3 below.

[0241] [Table 3] * Monovalent affinity as described in Example 3 and shown in Figures 4-8. ** These VHH antibodies from Ablynx were disclosed in WO2012156219. *** Two dissociation constants were calculated for the VHHs and their BLI curves were fitted with a "2:1 heterologous model". Only the high affinity components are reported in the table. #Neutralization efficacy was expressed as a ranking from 1=best to 4=good. None of the VHH antibodies showed aggregation at 95°C, demonstrating their stability.

[0242] Comparison of the VHH antibodies of the present invention with known VHHs (e.g., those disclosed in WO2012156219) using BLI revealed that the VHH antibodies of the present invention bind with significantly higher affinity to IL-17F.

[0243] The challenge in this project was to obtain VHH antibodies that block IL-17 and bind tightly to both IL-17A and IL-17F. This is no trivial task, as the neutralizing epitopes have a significant number of amino acid exchanges between IL-17A and IL-17F. Remarkably, binders with picomolar affinity for both IL-17A and IL-17F were identified, such as the VHH antibody designated Bm43B02 and its class A members.

[0244] The binding of some of the VHH antibodies of the present invention is listed in Table 3 in comparison with known VHH antibodies.

[0245] The affinity of VHH antibodies for IL-17 isoforms can be measured using methods known in the art. A specific measurement method uses a biolayer interferometry (BLI) instrument. According to some embodiments, biotinylated VHH antibodies are immobilized on a sensor chip, and their binding to homodimeric or heterodimeric IL17 species is measured. Because the VHH antibodies are immobilized at a high density, two adjacent VHH molecules can simultaneously bind to one IL-17 dimer. This results in an avidity effect similar to that of bivalent IgG binding to IL-17 dimers. In another setting, IL-17 isoforms are immobilized on a chip, and the binding of monomeric VHH antibodies to them is measured, which provides a more accurate KD because the avidity effect due to the dimeric nature of IL-17 is avoided. According to certain embodiments, the binding affinity of monomeric VHHs, expressed as the dissociation constant KD, to immobilized human IL-17A or IL-17F homodimers is determined.

[0246] The crystal structures of VHH antibody-IL17 complexes (Figures 16 and 18 for class A and class B VHH antibodies, respectively) demonstrate a deep understanding of the mode of action of VHH antibodies. Using structures and structural predictions, we defined the actual paratopes (at near-atomic resolution) and classified VHH antibodies into four classes.

[0247] Before describing at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or illustrated by way of example. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology or terminology used herein is for the purpose of description and should not be regarded as limiting.

[0248] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. [Example]

[0249] Example 1. Generation of VHH antibodies The generation of anti-IL-17 VHH antibodies that exhibit high-affinity cross-reactivity between several different IL-17 isoforms has been a formidable endeavor, given that sequence identity between IL-17 paralogs is limited to only about 50% identity between human IL-17A and IL-17F, and that most conserved residues are actually buried in the hydrophobic core of the disulfide-bridged IL-17 dimer. Thus, a desired VHH antibody will likely have to tolerate sequence exchanges in its epitope without loss of binding strength. The IL-17 receptor has a much larger interaction interface than the epitope of a VHH antibody might be able to, and therefore can more easily tolerate such sequence variability.

[0250] Therefore, the present inventors took these limitations into consideration in their VHH generation strategy. They recombinantly produced human IL-17A homodimers, IL-17F homodimers, and IL-17A / F heterodimers and injected the three into alpacas multiple times as immunogens. This strategy was chosen to promote an immune response that also included antibodies cross-reactive between IL-17 paralogs.

[0251] After the final immunization, blood samples were collected, lymphocytes were isolated, and mRNA was purified and reverse transcribed. VHH coding regions were amplified by nested PCR, reverse transcribed, and cloned as cDNA into M13 phagemids, resulting in a library of over 100 million independent clones.

[0252] In the next step, several rounds of phage display were performed, and the selected VHH antibodies were sorted according to sequence similarity, cloned into E. coli expression vectors, produced by periplasmic expression, purified, and finally characterized for IL-17 binding using biolayer interferometry (BLI; Abdiche et al., 2008).

[0253] In one arm of the selection approach, IL-17A homodimers and IL-17A / F heterodimers were used sequentially as bait. This resulted in considerable sequence diversity among the selected VHH antibodies—approximately eight major classes and numerous minor classes, as judged by sequence similarity. Most tested bound IL-17A with high affinity. "Binning" identified three complementary epitopes (epitopes 1 to 3); that is, epitope 1-binding VHH antibodies could simultaneously bind either epitope 2 or epitope 3-binding antibodies. Similarly, epitope 2 and 3-binding antibodies could simultaneously bind. However, epitope 2 and 3-binding antibodies were later found not to block IL-17 function.

[0254] In the second selection arm, IL-17A homodimers, IL-17F homodimers, and IL-17A / F heterodimers were used sequentially as baits. This direct selection for cross-reactivity significantly reduced sequence complexity. All epitope 2 and epitope 3 binders were lost. Two VHH classes became dominant, now accounting for 98% of all sequences. These two classes are disclosed below and are referred to as the Re42H11 and Bm17B02 classes (classes A and B, as defined in Tables 1 and 2), and include Re42H11, Re42B03, Re42B04, Bm18B05, Bm18F11 (class A), and Bm17B02 and Re42F08 (class B).

[0255] To generate "second-generation" VHH antibodies, which also allow for further affinity maturation of the antibodies, alpacas were re-immunized with IL-17A and IL-17F proteins 7 months after the last immunization. An immune library was prepared, and phage display was performed. Cross-selection was performed using alternating IL-17 versions, i.e., IL-17A and IL-17F, as bait at a low bait concentration of 100 pM, including an off-rate selection step. This strategy yielded additional VHH antibodies (Tables 1, 2, and 3), including new VHH classes (e.g., Bm42A03 in class C and Bm45G07 in class D). However, most of the newly discovered VHH antibodies were variants of previously isolated Re42H11 class (class A) and Bm17B02 class (class B) VHH antibodies.

[0256] Example 2. Affinity measurement via VHH immobilization First, five members of the Re42H11 class (Re42H11 itself, Re42B03, Re42B04, Bm18B05, and Bm18F11) and two members of the Bm17B02 class (Bm17B02 and Re42F08) were characterized in detail. For affinity measurements, they were produced with a C-terminal Avi-biotin tag and enzymatically biotinylated with recombinant BirA (Beckett et al., 1999). They were then immobilized at a concentration of 0.7 μg / ml on a High Precision Streptavidin biosensor on an Octet RED96e instrument (ForteBio / Sartorius) for 110 s using phosphate-buffered saline (PBS) pH 7.4, 0.02% (w / v) Tween 20, and 0.1% (w / v) bovine serum albumin (BSA) as the assay buffer. Then, 10, 20, and 40 nM of the indicated IL-17 species were allowed to bind for 800 seconds and then dissociate for another 800 seconds. Association and dissociation were recorded as wavelength shifts (nm). A baseline was recorded by measuring a "minus VHH control" in parallel. On-rates, off-rates, and dissociation constants (K D) was calculated by Octet Data Analysis HT 12.0 software using a mass transport model to fit the data.

[0257] These assays demonstrated complete cross-reactivity with IL-17A, IL-17F, and the IL17-A / F heterodimer (Figures 2 and 3), high on-rates (mostly around 10 5 ~10 6 M -1 ·s -1 ), an undetectable or nearly undetectable dissociation rate, and an apparent K of 20 pM or better D We demonstrated that the BLI setup does not allow for the discrimination of affinities below 10 pM. Also, note that this setup with immobilized VHHs involves avidity effects, whereby adjacent VHH molecules on the sensor chip can bind to the same IL-17 dimer, contributing to the very low off-rates observed. This simulates the situation of dimeric IgG binding to the IL-17 dimer.

[0258] Example 3. Affinity measurements via IL-17 immobilization To measure true monovalent affinity, we also performed BLI via immobilizing IL-17 species and used monovalent VHHs as analytes. The results are shown in Figures 4-8 and 11 and in Table 3. In the first set of experiments, VHH662 and VHH664 from Ablynx / Sanofi were tested as control antibodies. They had K values ​​of 115 pM and 150 pM (for VHH662 and VHH664, respectively). D showed high affinity for IL-17A at 1000 kJ / s, but a K of approximately 15 nM. D It bound poorly to IL-17F (Fig. 4).

[0259] When representatives of class A and class B VHH antibodies, namely Re42B04a and Re42F08, were probed for IL-17 binding under the same conditions, they showed better cross-reactivity, with examples showing K values ​​of 50 pM and 500 pM for IL-17A and IL-17F, respectively.D The most notable VHH was Re42B04, which has a nucleotide sequence similar to that of the IL-17A VHH (Figure 6). Some of the second-generation anti-IL-17 VHHs showed significant cross-reactivity between IL-17A and IL-17F, with affinities as high as 100 pM for both species (Figures 6, 7, and 8, Table 3).

[0260] Example 4. Testing the stability of VHH antibodies For intended therapeutic use, IL-17 VHH antibodies should not only bind to their target with high affinity, but also be developable as biologic drugs, which includes being sufficiently stable to withstand lengthy large-scale production processes as well as transportation and storage (ideally for several years in formulation) without aggregation or loss of activity.

[0261] A good predictor of stability is thermal stability, which can be measured, for example, by thermal shift assays or, in particular, by differential scanning fluorimetry (Goldberg et al., 2011). The method exploits the fact that protein melting (thermal unfolding) exposes aromatic / hydrophobic residues (from its hydrophobic core), which then bind and enhance the fluorescence of added SYPRO Orange dye.

[0262] VHH antibodies were subjected to differential scanning fluorimetry (DSF). Assays were performed in a volume of 20 μl at a VHH concentration of 1 mg / ml in 50 mM Tris / HCl, 150 mM NaCl (pH 8.0 at 20°C), and 1× SYBR Organge dye (5000× stock, Life Technologies). The plate was sealed with a clear MicroSeal® "B" Seal (Bio-Rad), briefly centrifuged to remove any air bubbles, and placed on a CFX96 Real-Time System (C1000 Thermal Cycler, BioRad). Samples were incubated at 20°C for 5 minutes. The temperature was then increased by 1°C every 45 seconds until reaching 95°C. At the end of each step, fluorescence was measured with 532 nm excitation and a 555 nm long-pass filter. The melting temperature was defined as the inflection point of the first melting peak. If the melting peak remained lower than the initial fluorescence at 20°C, this was interpreted as if no melting had occurred.

[0263] Figure 9 shows such an analysis for the disclosed VHH antibodies while slowly heating from 20°C to 95°C. The majority of the VHHs (Re42H11 itself, Re42B03, Re42B04, Re42B05, Bm18F11, and Re42F08) showed negligible unfolding signals and thus no melting (small fluorescence peaks that did not or barely exceeded the fluorescence measured at 20°C). The very low melting amplitudes of Re42H11, Re42B03, Bm18F11, Bm18B05, and Re42F08 indicate resistance to melting and thus complete thermostability. As a control, we repeated the experiment for some candidates in the presence of DTT (dithiothreitol) to reduce the structural disulfide bonds, where we observed a large unfolding peak indicating global melting of the respective VHH antibodies (Figure 10). Note the much higher magnitude of unfolding in the presence of DTT. These controls show what fluorescent signal would be expected if the VHH antibody were completely melted.

[0264] One Re42H11 class member (Re42B04) showed a small melting peak above the 20°C signal with an inflection point at 57°C (Figure 9), but even this VHH did not aggregate when heated to 95°C. Thus, the anti-IL-17 VHH antibodies disclosed herein fulfill three important criteria for their intended uses: extremely high affinity for their targets, complete cross-reactivity between IL17A, IL17A / F, and IL17F, and unusually high thermostability.

[0265] The second-generation VHH antibodies were also subjected to DSF as described in Example 2 and Figure 4. All Bm17B02 (class B) members (Bm44B04, Bm44G07, and Bm42A09), as well as Bm45G07 and Bm42B11, resisted melting even at 95°C, thus demonstrating their complete thermostability (Table 3). Other VHH antibodies (Bm43B02, Bm44B11, Bm44C09, Bm42A03, and Bm44G10), on the other hand, showed melting at 50-60°C. These VHHs also did not aggregate when heated to 95°C.

[0266] Example 5. Testing VHH stability by BLI BLI is highly sensitive to sample concentration and is therefore suitable for detecting any loss of active substance in solution, including loss of active nanobodies due to instability. To determine whether VHHs that already melt at 60 °C in DSF experiments retain their binding activity upon heat treatment, the antibodies were incubated at 95 °C for 10 min (at 1 μM concentration), cooled, and then centrifuged to remove any aggregates that may have formed. When tested for IL-17A binding by BLI, heated and untreated samples of Re42H11 and Bm42A03 class members (classes A and C) behaved similarly, showing no differences from the thermostable VHHs of class A, i.e., Re42B04a and Bm42B11 (Figure 11). Thus, these nanobodies either only partially melt or robustly refold to their native state after heat treatment.

[0267] Note that both the hyperthermostable VHHs (Re42B04a and Bm42B11) and VHHs with melting temperatures between 50 and 60 °C (Bm43B02, Bm44B11, Bm44C09, Bm42A03, and Bm44G10) showed no difference upon heat treatment, indicating that any melting (unfolding) was reversible.

[0268] Example 6. Cell-based IL17 stimulation assay and reporter activity measurement To monitor the biological activity of IL17 variants in a cell-based system, we used the HEK-Blue™ IL-17 reporter cell line (Invivogen hkb-il17). These cells are HEK293 cells stably expressing the human IL-17RA and IL-17RC receptors and the adaptor Act1 / TRAF3IP2. HEK-Blue™ IL-17 cells further encode a secreted embryonic alkaline phosphatase (SEAP) reporter driven by a promoter with NF-kB and AP-1 binding sites. Upon binding of the IL-17 ligand to its receptor on the cell surface, NF-kB and AP-1 are activated, producing SEAP, which is secreted into the cell supernatant. The amount of SEAP can be measured colorimetrically using QUANTI-Blue™ as a substrate.

[0269] Cells were cultured in DMEM medium supplemented with 10% (v / v) fetal bovine serum, 4.5 g / L glucose, 2 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, and 100 μg / mL Normocin™. Selection antibiotics (HEK-Blue™ Selection) were introduced after the second passage, and growth medium was refreshed at least twice per week. Cells were passaged at 70-80% confluency by scraping in PBS and were not cultured for longer than 20 passages.

[0270] To assess cellular responses, titration curves were performed with different IL-17 variants. To this end, HEK-Blue™ IL-17 cells were seeded in 96-well plates in selective antibiotic-free medium and stimulated with serial 3-fold dilutions of IL-17A (produced in HEK cells, Proteintech HZ-1113), IL-17F (produced in HEK cells, Proteintech HZ-1116), or IL-17A / F (produced in CHO cells, R&D Systems 5837-IL). Cell-free medium and untreated cells served as negative controls. After overnight incubation, 20 μl of cell supernatant from each well was mixed with 180 μl of QUANTI-Blue™ solution in a separate 96-well plate and incubated at 37°C for 30 minutes. SEAP levels in the supernatants were determined by measuring the absorbance at 620 nm (OD620). The OD620 of the medium-only control served as background and was subtracted from all sample wells. Each condition was assayed in triplicate. Induction was calculated from the ratio of OD620(sample) / OD620(untreated). Based on the titration curve, cells are more responsive to IL-17A (Figure 12). Therefore, higher concentrations of IL-17F and IL-17A / F were used to achieve a comparable range of induction in the neutralization assay described in Example 7.

[0271] Example 7. Cell-based IL17 neutralization assay To assess whether the disclosed anti-IL-17 VHH antibodies block the binding of IL-17 variants to their receptors, we performed a cell-based neutralization assay using HEK-Blue™ IL-17-expressing cells. These cells express the human IL-17RA and IL-17RC receptors and the adaptor Act1. They secrete a phosphatase reporter in response to receptor binding of IL-17 variants. IL17 receptor activation can be measured by quantifying the amount of secreted phosphatase, as described in Example 6 ( FIG. 12 ).

[0272] To test for neutralization, recombinant human IL-17A (5 ng / mL, corresponding to 0.17 nM, Proteintech HZ-1113), IL-17F (25 ng / mL, corresponding to 0.85 nM, Proteintech HZ-1116), or IL-17A / F (25 ng / mL, corresponding to 0.85 nM, R&D Systems 5837-IL) were preincubated with serial 3-fold dilutions of VHH antibodies for 2 hours at 37°C under constant shaking before being added to reporter cells. Cell-free medium, untreated cells, and cells treated with IL-17 variants in the absence of any VHH served as controls. Stimulated cells were incubated overnight at 37°C and 5% CO, and reporter activity was then measured as described in Example 6. The OD620 of medium alone served as background and was subtracted from all sample wells. Each condition was assayed in triplicate. Normalized induction was calculated from the ratio OD620(sample) / OD620(IL17 only).

[0273] From the first generation, the following VHH antibodies were tested: class A members Bm18B05, Bm18F11, Re42B03, Re42B04, and Re42H11 (IL-17A / IL-17F cross-reactive), class B (Bm17B02) members Bm17B02 and Re42F08 (IL-17A / IL-17F cross-reactive), Bm17D12 (IL-17A-preferring), and Bm17B11 class members Bm17B11 and Bm18F01 (targeting non-neutralizing epitope 3). The data show that pre-incubation with either class A or B members of IL-17A or IL-17F prevented receptor activation. Notably, subnanomolar IL-17A neutralization was observed for, for example, Re42B04, Re42B03, Bm17D12, and Re42F08. Similarly, IL-17A / F heterodimers were potently neutralized whenever VHH Bm18F11, Re42B03, Re42B04, Re42H11, Bm17B02, Re42F08, or Bm17D12 exceeded interleukin concentrations. Re42H11, Re42B03, and Re42B04 were particularly efficient at blocking IL-17F homodimers, with Re42H11 and Re42B03 showing essentially complete stoichiometric neutralization. Bm17D12 is a control VHH that binds tightly to IL-17A but only weakly to IL-17F. Bm17B11 and Bm18F01 bind only to IL-17A, which is the non-neutralizing epitope 3 (FIG. 13).

[0274] Second-generation VHHs also blocked receptor activation when preincubated with IL-17A, IL-17F, or IL-17A / F (Figure 14). The newly discovered VHH classes (Bm42A03 and Bm45G07) in particular demonstrated excellent cross-neutralization, even at picomolar antibody concentrations (Figure 14). In these assays, Re42B04a, Bm42A09, Bm42A03, and Bm45G07, representative members of VHH classes A-D, outperformed VHH662 and VHH664, particularly with regard to IL-17F neutralization (Figure 15). Note the excellent neutralization at low nanomolar or subnanomolar concentrations by members of all four VHH classes. For clarity, each graph lists VHHs in order of increasing neutralization potency.

[0275] The results shown in Figures 13 to 15 demonstrate that the VHH antibodies completely neutralize IL-17A, IL-17F, and IL-17AF at concentrations of <1 nM, <10 nM, and <1 nM, respectively.

[0276] Example 8 Structural characterization of IL-17·VHH complexes To understand the structural basis of IL-17 inhibition, we crystallized IL-17·VHH complexes. We solved X-ray crystal structures of tetrameric IL-17F complexes of VHH antibodies representing two major VHH classes: Re42H11 and Bm17B02 (Figures 16 and 18). Note that the IL-17RC receptor clashes with the VHH, explaining why the class A anti-IL17 VHH antibodies disclosed here block interleukin binding to the receptor. Comparison of these structures with the receptor-bound IL-17F structure (IL1-17RC·IL-17F, Goepfert et al., 2020) revealed that Re42H11 and Bm17B02 block receptor binding by directly competing for the IL-17 binding site.

[0277] To clarify the molecular details of VHH-IL-17 interactions, homology models were generated for all Re42H11 (class A) and Bm17B02 (class B) class members complexed with IL-17A and IL-17F. Analysis of these models allowed us to decipher the contribution of individual amino acids to interleukin binding and their influence on IL-17A / IL-17F cross-reactivity. All VHH residues identified in crystal or modeled structures that interact with IL-17 species, IL17A and IL-17F, are listed in Figures 17 and 19. Figure 17 shows the positions in class A VHH antibodies that interact with IL-17A or IL-17F. Figure 19 shows the positions in class B VHH antibodies that interact with IL-17A or IL-17F.

[0278] The following positions in the sequence of class A VHH antibodies were found to interact with IL-17A: position 1 is Q, position 29 is A, G, V, F or P, position 30 is S, position 31 is S or G, position 32 is Y, position 33 is A, position 50 is A, position 51 is I, position 52 is S, position 54 is I, S or V, position 55 is S or G, position 57 is G, S or D, position 58 is T, S or A, position 59 is K, R or V, position 100 is P, position 101 is Y, position 103 is L or M, position 104 is D or E, position 106 is R, and position 109 is E or D.

[0279] The following positions in the sequence of class A VHH antibodies were found to interact with IL-17F: position 29 is A, G, V, F or P, position 30 is S, position 31 is S or G, position 32 is Y, position 33 is A, position 50 is A, position 51 is I, position 52 is S, position 54 is S, I or V, position 55 is S or G, position 57 is G, S or D, position 58 is T, S or A, position 59 is K, R or V, position 100 is P, position 101 is Y, position 103 is L or M, position 104 is D or E, position 106 is R, and position 109 is E or D.

[0280] The following positions in the sequence of class B VHH antibodies were found to interact with IL-17A: position 3 is Q, position 31 is I or Q, position 32 is S, position 33 is A, position 37 is Y, position 45 is R, position 52 is H, position 59 is H or Y, position 99 is N, position 100 is E, position 101 is P, position 102 is G, position 103 is H or D, position 104 is L, position 105 is Y, and position 106 is M.

[0281] The following positions in the sequence of class B VHH antibodies were found to interact with IL-17F: position 3 is Q, position 31 is I or Q, position 32 is S, position 33 is A, position 37 is Y, position 45 is R, position 47 is L, position 50 is L or M, position 52 is T or H, position 59 is H or Y, position 99 is N, position 100 is E, position 101 is P, position 102 is G, position 103 is H or D, position 104 is L, position 105 is Y, and position 106 is M.

[0282] The differences in interaction are due to different residues in IL-17A and IL-17F. Using this information, we identified key residues in the VHHs. When classifying nanobodies, we can focus on the conservation of interacting residues rather than the complete sequence. For example, for the Bm17B02 class, new nanobodies have different CDR1 and CDR2 sequences, and therefore, it could be argued that they are of a different class, but by focusing on the interacting residues, we can see that they are nearly identical.

[0283] Example 9. Production of VHH antibodies in Pichia pastoris VHH antibodies Re42B04a and Re42F08 were recombinantly produced in Pichia pastoris. Figures 20A and 20B illustrate the upstream and downstream processes, respectively.

[0284] As depicted in the scheme in Figure 20A, the upstream steps were initiated by culturing an inoculum from a cell bank, followed by fermentation, induction of expression by adding methanol, and harvest. The downstream steps, shown in Figure 20B, included adjustment of pH, when necessary, and purification of the filtered harvest by passing it through several columns followed by a 20 mM phosphate buffer formulation.

[0285] Example 10. Ex vivo human skin model InflammaSkin® is a psoriasis-like model developed by Genoskin to recapitulate key features of the pro-Th17 / Th1 inflammation associated with psoriasis. The model, shown schematically in Figure 21, relies on the in situ activation of resident T cells in normal skin biopsies and their further polarization toward a Th17 / Th1 phenotype by supplementing the culture medium with a cocktail of pro-inflammatory cytokines. This model has been successfully validated with topically applied compounds, including steroids, delivered in either a prophylactic or therapeutic manner.

[0286] The model was used to evaluate effective doses and dosing regimens before conducting in vivo studies in mice implanted with psoriatic human skin. VHH antibodies Re42B04a and Re42F08 were compared with a monoclonal antibody targeting IL-17A (i.e., secukinumab) used clinically as a subcutaneous injection for the treatment of psoriasis, and steroid treatment with betamethasone.

[0287] Skin samples from female donors with no current inflammatory skin disease or history of treatment were cultured under standard cell culture conditions (37°C CO2 incubator, water saturation, daily medium changes). A psoriasis phenotype was then induced by in situ activation of skin-resident T cells with a pro-inflammatory cytokine cocktail and Th17 / Th1 polarization, followed by intravenous treatment with VHH antibodies once on day 3 or three times on days 3–5. Positive control groups included standard of care topical betamethasone or daily prophylactic treatment or a single SC administration of secukinumab on day 3, while negative control treatment included the unrelated VHH antibody Re32D03, which does not bind IL-17.

[0288] Anti-inflammatory effects were assessed by measuring cytokine secretion, including IL-17 family cytokines, interferon 1β (IFN-1β), and interferon γ (IFN-γ). Additionally, skin structure, integrity, and viability were assessed by histological analysis (e.g., H&E staining). The study design is shown in Table 4.

[0289] [Table 4]

[0290] The culture volume was 2 mL.

[0291] IL-17A emerged as the cytokine most significantly affected by the applied treatments (Figure 22). As expected, low levels of IL-17A were detected in the culture medium of untreated HypoSkin models (approximately 7 pg / ml was measured), whereas highly significant secretion was detected in untreated HypoInflammaSkin models, reaching an average of 130 pg / ml, confirming an efficient Th17-type response. Importantly, both positive control treatments, betamethasone and secukinumab, efficiently and significantly inhibited IL-17A secretion. The control, unrelated VHH Re32D03 (anti-SARS-CoV-2), did not significantly affect IL-17A secretion, although greater variability was observed between replicates (ranging from 39 to 135 pg / ml) when compared to other conditions. VHH Re42B04a, injected once and three times, almost completely abrogated IL-17A secretion to 6.8 and 5.7 pg / ml, respectively (one replicate for each condition was below the limit of detection). This was a 20-fold reduction compared to the HypoInflammaSkin condition and a 14-fold reduction compared to an irrelevant VHH. VHH Re42F08, injected once and three times, also significantly reduced IL-17A expression, but to a lesser extent than Re42B04a, resulting in average concentrations of 40 and 48 pg / ml, respectively.

[0292] Hematoxylin and eosin (H&E) staining was performed on day 7 to assess the structural integrity and cell viability of the skin samples at this time point. The following areas were assessed for basic features: epidermal cell apoptosis and loss of cell viability: This is typically indicated by pyknotic nuclei (dark, condensed) and / or eosinophilic cytoplasm (stained "pink" instead of purple) in the epidermis. Disrupted adipocytes in the subcutaneous layer may indicate loss of cell viability. Spongiform changes: Fluid infiltration into the epidermis. This typically appears as "white" spaces between keratinocytes, with interconnecting filaments still visible. Smaller white pockets / spaces may also be present, particularly near the epidermal / dermal junction. Loss of structural integrity: Separation of the epidermis and dermis, often associated with large areas of pyknotic nuclei in the basal epidermal layer.

[0293] As shown in Figures 23A-H, untreated normal tissue controls (Figure 23A) versus untreated psoriasis-induced tissue (Figure 23B) exhibited loss of cell viability, large areas of pyknotic and eosinophilic cells, and increased epidermal thickness. Treatment with betamethasone (Figure 23C) and secukinumab (Figure 23D) improved viability, but some signs of inflammation remained evident. When treated with VHH antibodies and injected once (Figure 23E) or three times (Figure 23F) with Re42b04a, or once (Figure 23G) or three times (Figure 23H) with Re42F08, pathological changes were significantly prevented, and skin histology improved compared to untreated psoriasis-induced tissue (Figure 23B). Compared to untreated controls, multiple cells exhibited pyknotic nuclei and loss of structural integrity, these phenomena were limited to samples treated with secukinumab and two VHHs. In Figures 24E and 24G, after a single treatment with VHH, the tissue appears intact with no psoriatic lesions occurring.

[0294] conclusion The purpose of this study was to evaluate the anti-inflammatory effects of VHH antibodies of the present invention using the InflammaSkin platform, in which T cell activation and stimulation leads to Th1 / Th17-mediated induction of psoriasis in the skin. The VHH antibodies Re42B04a and Re42F08 were injected intradermally into the HypoInflammaSkin model once or three times after the onset of inflammation. An unrelated VHH against the SARS-CoV-2 spike protein was used as a control for this experimental group and injected once. The topical steroid betamethasone was used as a positive control treatment. Additionally, secukinumab, a commercially available anti-IL-17A antibody used clinically to treat psoriasis, was injected intradermally to compare its anti-inflammatory potential with the tested VHH antibodies. All models were cultured for 7 days, after which the effects of the applied treatments were evaluated.

[0295] To assess the effect of a positive control, betamethasone treatment, on donor responsiveness to T cell activation, release of the cytokine IL-22 into the culture medium was assessed after 7 days. Additionally, H&E staining of HypoSkin and HypoInflammaSkin section samples was used to analyze skin structural integrity and cell viability. Finally, cytokine release using the MSD TH17 Combo 2 assay (K15076K, MesoScale Discovery) was assessed in all conditions on day 7.

[0296] Overall, no significant changes in skin characteristics were observed over the course of culture, and H&E staining indicated healthy, viable HypoInflammaSkin untreated models. Upon inflammation induction, the HypoInflammaSkin model showed signs of severe loss of cell viability, hyperkeratosis, the presence of spongiotic changes indicative of ongoing inflammation, and abnormalities in structural integrity. The positive control treatment, betamethasone, appeared to improve skin histology, particularly when assessing the degree of nuclear pyknosis and stratum corneum thickening. Secukinumab treatment appeared to improve skin characteristics even further, with less pronounced evidence of cell death and hyperkeratosis. An unrelated VHH did not affect the specific features of the HypoInflammaSkin model, and the degree of microscopic changes was comparable to that of the untreated HypoInflammaSkin group. The VHH antibodies tested, Re42B04a and Re42F08, significantly improved skin histology. In contrast to control conditions, the HypoInflammaSkin model, which received a single injection of the VHH antibodies Re42b04a and Re42f08, showed significantly fewer areas of pyknotic, eosinophilic keratinocytes, and no significant hyperkeratinization was observed. In these experimental conditions, spongiotic changes persisted, and the basal epidermis still showed signs of reduced cell viability. Collectively, the H&E results demonstrated an improvement in skin histology with the tested VHH antibodies Re42B04a and Re42F08, which appeared more efficient than betamethasone and at least as efficient as secukinumab in reducing inflammation-induced skin changes in this donor.

[0297] The most striking results were obtained for IL-17A secretion analysis. Its expression was significantly upregulated in response to T cell activation and stimulation. Both topical treatment with betamethasone and injection of secukinumab significantly reduced its levels. VHH antibody Re42B04a (injected once and three times) reduced IL-17A secretion to the basal level detected in untreated HypoSkin controls, producing a stronger effect when compared with betamethasone or secukinumab. VHH antibody Re42F08 (injected once and three times) also significantly reduced IL-17A release, albeit to a lesser extent than Re42B04a, betamethasone, and secukinumab. Importantly, the reduction in IL-17A secretion was significant for VHH Re42F08 (both treatment schemes) when compared with unrelated VHH-treated groups.

[0298] Overall, the results obtained demonstrated the strong potential of the VHH antibodies of the invention to modulate inflammation in the InflammaSkin model of psoriasis, as shown by improving skin viability and structural integrity, which was supported by cytokine release analysis, particularly IL-17A expression.

[0299] References Abdiche Y,Malashock D,Pinkerton A,Pons J(2008)Determining kinetics and affinities of protein interactions using a parallel real-time label-free biosensor,the Octet.Anal Biochem,377:209-217 Beckett D, Kovaleva E, Schatz PJ (1999) A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation. Protein Sci, 8:921-929 Su,S.;Esparza,T.J.;Nguyen,D.;Mastrogiacomo,S.;Kim,J.H.;Brody,D.L.Pharmacokinetics of Single Domain Antibodies and Conjugated Nanoparticles Using a Hybrid near Infrared Method.Int.J.Mol.Sci.2021,22,8695 Glatt S,Baeten D,Baker T,Griffiths M,Ionescu L,Lawson ADG,Maroof A,Oliver R,Popa S,Strimenopoulou F,Vajjah P,Watling MIL,Yeremenko N,Miossec P,Shaw S(2018)Dual IL-17A and IL-17F neutralisation by bimekizumab in psoriatic arthritis:evidence from preclinical experiments and a randomised placebo-controlled clinical trial that IL-17F contributes to human chronic tissue inflammation.Ann Rheum Dis,77:523-532 Goepfert,A.,Lehmann,S.,Blank,J.,Kolbinger,F.,& Rondeau,J.M.(2020).Structural Analysis Reveals that the Cytokine IL-17F Forms a Homodimeric Complex with Receptor IL-17RC to Drive IL-17RA-Independent Signaling.Immunity,52(3),499-512.e5 Goldberg DS,Bishop SM,Shah AU,Sathish HA(2011)Formulation development of therapeutic monoclonal antibodies using high-throughput fluorescence and static light scattering techniques:role of conformational and colloidal stability.J Pharm Sci,100:1306-1315 Guttler T,Aksu M,Dickmanns A,Stegmann KM,Gregor K,Rees R,Taxer W,Rymarenko O,Schunemann J,Dienemann C,Gunkel P,Mussil B,Krull J,Teichmann U,Groβ U,Cordes VC,Dobbelstein M,Gorlich D(2021)Neutralization of SARS-CoV-2 by highly potent,hyperthermostable,and mutation-tolerant nanobodies.EMBO J,e107985 Keren A,Shemer A,Ginzburg A,Ullmann Y,Schrum AG,Paus R,Gilhar A.Innate lymphoid cells 3 induce psoriasis in xenotransplanted healthy human skin.J Allergy Clin Immunol.2018 Jul;142(1):305-308 Frank Kolbinger,Franco Di Padova,Atul Deodhar,Jason E.Hawkes,Christine Huppertz,Torsten Kuiper,Iain B.McInnes,Christopher T.Ritchlin,David Rosmarin,Georg Schett,Jose M.Carballido,Peter Hausermann,Claudio Calonder,Beate Vogel,Jean-Michel Rondeau,Gerard Bruin,Secukinumab for the treatment of psoriasis,psoriatic arthritis,and axial spondyloarthritis:Physical and pharmacological properties underlie the observed clinical efficacy and safety,Pharmacology & Therapeutics,Volume 229,2022,107925, Langley RG,Elewski BE,Lebwohl M,Reich K,Griffiths CE,Papp K,Puig L,Nakagawa H,Spelman L,Sigurgeirsson B,Rivas E,Tsai TF,Wasel N,Tyring S,Salko T,Hampele I,Notter M,Karpov A,Helou S,Papavassilis C,ERASURE SG,FIXTURE SG(2014)Secukinumab in plaque psoriasis--results of two phase 3 trials.N Engl J Med,371:326-338 Mease PJ,McInnes IB,Kirkham B,Kavanaugh A,Rahman P,van der Heijde D,Landewe R,Nash P,Pricop L,Yuan J,Richards HB,Mpofu S,FUTURE SG(2015)Secukinumab Inhibition of Interleukin-17A in Patients with Psoriatic Arthritis.N Engl J Med,373:1329-1339 Mills KHG(2022)IL-17 and IL-17-producing cells in protection versus pathology.Nat Rev Immunol,1-17 Nies JF,Panzer U(2020)IL-17C / IL-17RE:Emergence of a Unique Axis in T H 17 Biology.Front Immunol,11:341 Papp KA,Weinberg MA,Morris A,Reich K(2021)IL17A / F nanobody sonelokimab in patients with plaque psoriasis:a multicentre,randomised,placebo-controlled,phase 2b study.Lancet,397:1564-1575 Helen Prior,Fiona Sewell,Jane Stewart, Overview of 3Rs opportunities in drug discovery and development using non-human primates,Drug Discovery Today:Disease Models,Volume 23,2017,Pages 11-16, Reich K,Warren RB,Lebwohl M,Gooderham M,Strober B,Langley RG,Paul C,De Cuyper D,Vanvoorden V,Madden C,Cioffi C,Peterson L,Blauvelt A(2021)Bimekizumab versus Secukinumab in Plaque Psoriasis, Engl J Med 2014:2014. Sarama et al.,Frontiers in Bioengineering and Biotechnology February 2022 | Volume 10|Article 803218. Skroza N,Proietti I,Bernardini N,Aquila E,Balduzzi V,La Viola G,Mambrin A,Muscianese M,Tolino E,Zuber S,Potenza C,Ferrazza P(2017)Il-17 and Its Role in Psoriasis,Hidradenitis Suppurativa And Ac Internal Medicine,16. Su,S.;Esparza,TJ;Nguyen,D.;Mastrogiacomo,S.;Kim,JH;Brody,DLPharmacokinetics of Single Domain Antibodies and Conjugated Nanoparticles Using a Hybrid near Infrared Method.Int.J.Mol.Sci.2021,269,895. Warren RB,Blauvelt A,Bagel J,Papp KA,Yamauchi P,Armstrong A,Langley RG,Vanvoorden V,De Cuyper D,Cioffi C,Peterson L,Cross N,Reich K(2021)Bimekizumab versus Adalimumab in Plaque Psoriasis.N Engl J Med,385:130-141 Wilson SC,Caveney NA,Yen M,Pollmann C,Xiang X,Jude KM,Hafer M,Tsutsumi N,Piehler J,Garcia KC(2022)Organizing structural principles of the IL-17 ligand-receptor axis.Nature,609:622-629 Zhang Y.Huo M.Zhou J.Xie S.PKSolver:an add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel.Comput Methods Programs Biomed.2010;99:306-314 Zhou,X.et al.Advances in the pathogenesis of psoriasis:from keratinocyte perspective.2022;Cell Death Dis 13,81

Claims

1. A VHH antibody recognizing a human IL-17 polypeptide, which cross-reacts with a plurality of different human IL-17 polypeptides, including (i) human IL-17A homodimer, (ii) human IL-17F homodimer, and (iii) human IL-17A / F heterodimer, and neutralizes IL-17 receptor activation, the VHH antibody comprising: i. A class A VHH antibody comprising a sequence selected from SEQ ID NOs: 19, 15, 5, 23, 12, 13, 20, 27, 28, and 32, or a variant thereof having at least 80% identity; ii. A class B VHH antibody comprising a sequence selected from SEQ ID NOs: 39, 35, 40, 44, and 45, or a variant thereof having at least 80% identity; iii. A class C VHH antibody comprising a sequence selected from SEQ ID NOs: 49 and 53, or a variant thereof having at least 80% identity; and iv. A VHH antibody selected from a class D VHH antibody comprising a sequence selected from SEQ ID NO: 54, or a variant thereof having at least 80% identity.

2. A VHH antibody as described in claim 1, having at least 90% identity to a sequence selected from SEQ ID NOs: 19, 15, 5, 23, 12, 13, 20, 27, 28, and 32 (Class A), SEQ ID NOs: 39, 35, 40, 44, and 45 (Class B), SEQ ID NOs: 49 and 53 (Class C), and SEQ ID NO: 54 (Class D).

3. A VHH antibody according to claim 1 or 2, comprising an amino acid sequence selected from SEQ ID NOs: 19, 15, 5, 39, 23, 12, 13, 20, 27, 28, 32, 35, 40, 44, 45, 49, 53, and 54.

4. i. Formula NDMPYGX １ X ２ TX ３ MDX ４ YX ５ X ６ W (in the formula, X １ is selected from L and M; X ２ is selected from D and E, and X ３ is selected from R and T; X ４ is selected from E and D, and X ５ is selected from A, V, E, D, and K; X ６ is selected from Y and S) (Class A); ii. Formula X １ HNEPGX ２ LYM sequence (wherein X １ is selected from V and T; X ２ is selected from H and D) (Class B); iii. The sequence MAVRGLYGSNWYDYPFELW (SEQ ID NO: 52), (Class C), and iv. A VHH antibody according to any one of claims 1 to 3, comprising a CDR3 sequence selected from the sequence YIDSGSDRYY (SEQ ID NO: 57), (Class D).

5. 5. A class A VHH antibody according to any one of claims 1 to 4, comprising a CDR3 having a sequence selected from SEQ ID NOs: 8, 11, 18, 26, 31, and 34, a CDR2 having a sequence selected from SEQ ID NOs: 7, 14, 17, 22, 25, and 30, and a CDR1 having a sequence selected from SEQ ID NOs: 6, 10, 16, 21, 24, 29, and 33.

6. A class A VHH antibody according to any one of claims 1 to 5, comprising a CDR3 sequence set forth in any one of SEQ ID NOs: 8, 11, 18, 26, 31, 31, 38, 43, 52, and 57, or a CDR3 sequence having at least 80% identity to any of said CDR3 sequences.

7. 7. A class A VHH antibody according to any one of claims 1 to 6, comprising a set of three CDR sequences selected from SEQ ID NOs: 16, 17, and 18; SEQ ID NOs: 6, 7, and 8; SEQ ID NOs: 10, 7, and 11; SEQ ID NOs: 10, 14, and 11; SEQ ID NOs: 21, 22, and 18; SEQ ID NOs: 24, 25, and 26; SEQ ID NOs: 29, 30, and 31; and SEQ ID NOs: 33, 7, and 34.

8. 8. The class A VHH antibody of claim 1, comprising the following sequence positions that interact with IL-17A: position 1 is Q, position 29 is A, G, V, F, or P, position 30 is S, position 31 is S or G, position 32 is Y, position 33 is A, position 50 is A, position 51 is I, position 52 is S, position 54 is I, S, or V, position 55 is S or G, position 57 is G, S, or D, position 58 is T, S, or A, position 59 is K, R, or V, position 100 is P, position 101 is Y, position 103 is L or M, position 104 is D or E, position 106 is R, and position 109 is E or D.

9. 8. The class A VHH antibody of claim 1, comprising the following sequence positions that interact with IL-17F: position 1 is Q, position 29 is A, G, V, F, or P, position 30 is S, position 31 is S or G, position 32 is Y, position 33 is A, position 50 is A, position 51 is I, position 52 is S, position 54 is I, S, or V, position 55 is S or G, position 57 is G, S, or D, position 58 is T, S, or A, position 59 is K, R, or V, position 100 is P, position 101 is Y, position 103 is L or M, position 104 is D or E, position 106 is R, and position 109 is E or D.

10. 5. A class B VHH antibody according to any one of claims 1 to 4, comprising a set of three CDR sequences, said set being selected from SEQ ID NOs: 36, 37, and 38; SEQ ID NOs: 41, 42, and 43; and SEQ ID NOs: 46, 42, and 43.

11. 11. A class B VHH antibody according to any one of claims 1 to 4 and 10, comprising a CDR3 having a sequence selected from SEQ ID NOs: 38 and 43, a CDR2 having a sequence selected from SEQ ID NOs: 37 and 42, and a CDR1 having a sequence selected from SEQ ID NOs: 36 and 41.

12. A class B VHH antibody according to any one of claims 1 to 4 and 10 to 11, comprising a CDR3 sequence set forth in any one of SEQ ID NOs: 38 and 43, or a CDR3 sequence having at least 80% identity to any of said CDR3 sequences.

13. 13. The Class B VHH antibody of any one of claims 1 to 4 and 10 to 12, comprising the following sequence positions that interact with IL-17A: position 3 which is Q, position 31 which is I or Q, position 32 which is S, position 33 which is A, position 37 which is Y, position 45 which is R, position 52 which is H, position 59 which is H or Y, position 99 which is N, position 100 which is E, position 101 which is P, position 102 which is G, position 103 which is H or D, position 104 which is L, position 105 which is Y, and position 106 which is M.

14. 12. The class B VHH antibody according to any one of claims 1 to 4 and 10 to 11, comprising the following sequence positions which interact with IL-17F: position 3 which is Q, position 31 which is I or Q, position 32 which is S, position 33 which is A, position 37 which is Y, position 45 which is R, position 47 which is L, position 50 which is L or M, position 52 which is T or H, position 59 which is H or Y, position 99 which is N, position 100 which is E, position 101 which is P, position 102 which is G, position 103 which is H or D, position 104 which is L, position 105 which is Y, and position 106 which is M.

15. A VHH antibody according to any one of claims 1 to 14, comprising a set of three CDR sequences, said set being selected from SEQ ID NOs: 16, 17 and 18, and SEQ ID NOs: 36, 37 and 38.

16. A VHH antibody according to any one of claims 1 to 15, selected from RE42B04a (SEQ ID NO: 19), Re42B04 (SEQ ID NO: 15), Re42F08 (SEQ ID NO: 39), Bm43B02 (SEQ ID NO: 23), and Bm17B02 (SEQ ID NO: 35), or a variant thereof having at least 90% identity to any of these sequences.

17. A VHH antibody according to any one of claims 1 to 4, comprising a set of three CDR sequences, said set comprising SEQ ID NOs: 50, 51, and 52 (class C) or SEQ ID NOs: 56, 57, and 58 (class D).

18. 18. The VHH antibody of any one of claims 1 to 17, which in monomeric form has a binding affinity, expressed as a dissociation constant KD, to immobilized human IL-17A or IL-17F homodimer of 5 nM, 1 nM, 500 pM, 300 pM, 100 pM, 50 pM or less.

19. 19. The VHH antibody of any one of claims 1 to 18, which neutralizes binding of at least one of (i) human IL-17A homodimer, (ii) human IL-17F homodimer, and (iii) human IL17A / F heterodimer to a human IL-17 receptor at a concentration of about 10 nM or less, about 3 nM or less, about 1 nM or less, or about 0.3 nM or less, when tested in a cell-based assay under affinity-limited test conditions.

20. 20. The VHH antibody of any one of claims 1 to 19, which is thermostable or ultrathermostable, having a melting temperature of at least about 65°C, at least about 80°C, at least 90°C, or at least about 95°C when measured under non-reducing conditions, and / or an aggregation temperature of at least about 60°C, at least 70°C, at least about 80°C, at least about 90°C, or at least about 95°C when measured under non-reducing conditions.

21. A VHH antibody according to any one of claims 1 to 20, which is conjugated or fused to a heterologous moiety.

22. A set of two or more different VHH antibodies that recognize human IL-17 polypeptides, particularly IL-17A homodimer, IL-17F homodimer, and IL17A / F heterodimer, comprising at least one VHH antibody according to any one of claims 1 to 21.

23. A nucleic acid molecule encoding a VHH antibody according to any one of claims 1 to 22.

24. 24. A recombinant cell or non-human organism transformed or transfected with the nucleic acid molecule of claim 23 or a vector comprising said nucleic acid molecule.

25. 25. The recombinant cell of claim 24, selected from a bacterial cell, a yeast cell, an insect cell, a mammalian cell, and a plant cell.

26. A method for the recombinant production of a VHH antibody according to any one of claims 1 to 21, comprising culturing a cell or organism in a suitable medium and obtaining the VHH antibody from said cell or organism or from said medium.

27. 27. The method of claim 26, wherein the cell is a Pichia pastoris cell.

28. Pharmaceutical composition: A VHH antibody or set of VHH antibodies according to any one of claims 1 to 22, and a pharmaceutically acceptable carrier, excipient or diluent.

29. 10. The pharmaceutical composition according to claim 9, which is formulated for local administration, in particular for topical administration or intradermal injection.

30. 30. The pharmaceutical composition of claim 28 or 29, formulated as a cream, paste, gel, hydrogel, ointment, lotion, or emulsion.

31. 29. The pharmaceutical composition of claim 28, formulated for parenteral administration.

32. 32. The pharmaceutical composition of any one of claims 28 to 31, formulated for sustained, slow, or delayed release.

33. 33. The pharmaceutical composition according to any one of claims 28 to 32 for use in the prevention or treatment of disorders caused by or associated with IL-17 overactivity, in particular IL-17A and / or IL-17F overactivity.

34. 34. The pharmaceutical composition for use according to claim 33, wherein the disorder is an inflammatory and / or immune-related disorder.

35. 35. The pharmaceutical composition for use according to claim 34, wherein the inflammatory and / or immune-related disorder is selected from psoriasis, arthritis, asthma, hidradenitis suppurativa, inflammatory bowel disease (Crohn's disease, ulcerative colitis), multiple sclerosis, skin cancer, ankylosing spondylitis, uveitis, atopic dermatitis, graft-versus-host disease, Alzheimer's disease, fatty liver disease, sepsis, ischemic stroke, Parkinson's disease, axial spondyloarthritis not meeting radiographic criteria of activity with objective clinical signs of inflammation, systemic lupus erythematosus (SLE), familial Mediterranean fever (FMF), tumor necrosis factor receptor-associated periodic syndromes (TRAPS), hidradenitis suppurativa (HS), pemphigus vulgaris (PV), pityriasis rubra pilaris (PRP), alopecia areata, systemic sclerosis, and infectious diseases, lichen planus, and impetigo herpetiformis.

36. 36. The method of claim 35, wherein the inflammatory and / or immune-related disorder is an autoimmune disease selected from psoriasis, arthritis, systemic lupus erythematosus (SLE), familial Mediterranean fever, and inflammatory bowel disease (IBD).

37. 35. The pharmaceutical composition for use according to claim 34, wherein the inflammatory and / or immune-related disorder is a skin disorder.

38. 38. The pharmaceutical composition for use according to any one of claims 33 to 37, wherein the disorder is psoriasis, in particular plaque psoriasis, moderate to severe psoriasis, hypertrophic palmoplantar psoriasis, pustular psoriasis, or pustular psoriasis.

39. 39. The pharmaceutical composition for use according to claim 38, wherein the disorder is mild to moderate plaque psoriasis.

40. 37. The pharmaceutical composition for use according to any one of claims 33 to 36, wherein the disorder is arthritis, in particular rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, or enthesitis-related arthritis.

41. A diagnostic composition comprising a VHH antibody according to any one of claims 1 to 21 and an acceptable carrier, excipient or diluent.

42. A method for the prevention or treatment of a disorder caused by and / or associated with IL-17 overactivity, particularly IL-17A and / or IL-17F overactivity, comprising administering an effective dose of a VHH antibody or set of VHH antibodies described in any one of claims 1 to 22, or a pharmaceutical composition described in any one of claims 28 to 32, to a subject in need thereof.

43. 43. The method of claim 42, wherein the disorder is an inflammatory and / or immune-related disorder.

44. 44. The method of claim 43, wherein the inflammatory and / or immune-related disorder is an autoimmune disease selected from psoriasis, arthritis, systemic lupus erythematosus (SLE), familial Mediterranean fever, and inflammatory bowel disease (IBD).

45. 45. The method of claim 44, wherein the inflammatory and / or immune-related disorder is a skin disorder.

46. 46. ​​The method of any one of claims 42 to 45, wherein the disorder is psoriasis, in particular plaque psoriasis, moderate to severe psoriasis, hypertrophic palmoplantar psoriasis, pustular psoriasis, or pustular psoriasis.

47. 47. The method of claim 46, wherein the psoriasis is mild to moderate plaque psoriasis.

48. 45. The method of any one of claims 42 to 44, wherein the disorder is arthritis, in particular rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, or enthesitis-related arthritis.

49. The method according to any one of claims 42 to 48, wherein the composition is administered topically, in particular topically.

50. The method according to any one of claims 42 to 49, wherein the composition is administered by injection, in particular by intradermal injection.

Images