Therapeutic heavy chain variable domain (VHH) antibodies that cross-neutralize interleukin 17 (IL-17) subtypes

Abstract

The present invention provides VHH antibodies in which the pro-inflammatory interleukin, IL-17, is neutralized in the form of a homodimer of IL-17A and IL-17F, and a heterodimer of IL-17AF. Pharmaceutical compositions comprising the VHH antibodies and uses for the treatment of inflammatory and immune related diseases and disorders are also provided.

Description

Field of the Invention

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

[0002] Interleukin 17 (IL-17) is a multifunctional cytokine. It is produced by a specific subset of CD4-positive helper T cells, namely Th17 cells, but also by other CD4 or CD8-positive T cells and γ / δ T cells. The main actions of IL-17 include attracting neutrophils and monocytes, as well as generating additional cytokines and chemokines by its target cells. In summary, IL-17 is a key mediator of the inflammatory response. These responses can be highly beneficial to the patient, for example, when establishing defenses against various types of infections (Mills, 2022). However, the excessive or uncontrolled production of IL-17 can also initiate or exacerbate autoimmune diseases, such as psoriasis, rheumatoid arthritis, asthma, or inflammatory bowel disease.

[0003] The synthesis and secretion of IL-17 during mucosal infection are strictly controlled by upstream signaling pathways. Most notably, interleukin 23 (IL-23) and IL-1β released by activated dendritic cells or macrophages stimulate Th17 cells to release IL-17.

[0004] Since the receptor is found in a variety of human cell types, the target cells of IL-17 can be diverse. Therefore, the responses can also be heterogeneous. However, most importantly, epithelial cells exposed to IL-17 release chemokines, such as CXCL1 and CXCL8, thereby attracting neutrophils and macrophages to maintain the homeostasis of the epithelial barrier.

[0005] In autoimmune diseases, elevated levels of IL-17 at the site of inflammation activate the release of interleukin-6, tumor necrosis factor α (TNF-α), and matrix metalloproteinases (MMPs). This leads to tissue damage and exacerbates the inflammatory response (Mills, 2022).

[0006] IL-17 proteins form dimers. Currently, they include a family of 6 members (IL-17A to IL-17F). Among them, IL-17A and IL-17F are considered the main and most relevant members; they are also the most intensively studied members. They bind to five receptors, IL-17RA-E, to form a heterodimeric receptor complex. IL-17A and IL-17F bind and activate the IL-17RA and IL-17RC receptors in the form of homodimers or as the heterodimer IL-17A / F (Nies and Panzer, 2020). IL-17 binding requires the adaptor protein TRAF3IP2 and can lead to the lateral assembly of the receptors, which at least partially explains the initiation of intracellular signaling, such as the activation of NF-κ-B and mitogen-activated protein (MAP) kinases (Wilson et al., 2022).

[0007] Numerous methods have been developed to interfere with the production and / or activity of IL-17. In particular, monoclonal antibodies can target upstream IL-23, but most importantly, they can target IL-17 itself or its receptor. Then, the neutralization of IL-17 inhibits the inflammatory response, which is beneficial in the treatment of autoimmune diseases.

[0008] Antagonists of IL-17, especially antibodies, have been successfully used to treat a variety of diseases, resulting in FDA-approved drugs. Most notably, antibodies that neutralize IL-17 have successfully treated inflammatory skin diseases, namely psoriasis and psoriatic arthritis, as well as hidradenitis suppurativa ("acne inversa") (reviewed in Skroza et al., 2017; Mills, 2022). Therapeutic agents include ixekizumab and secukinumab, which target IL-17A alone and are used in plaque psoriasis (Langley et al., 2014) and psoriatic arthritis (Mease et al., 2015), and bimekizumab, which targets both IL-17A and IL-17F and is used in plaque psoriasis (Reich et al., 2021; Warren et al., 2021) and psoriatic arthritis (Glatt et al., 2018).

[0009] However, such antibodies are typically administered systemically, with little or no spatial confinement to the actual site of inflammation. It should be noted that in such cases, this indiscriminate IL-17 blockade also exacerbates bacterial or fungal infections, as reported for example for secukinumab (Reich et al., 2021; Warren et al., 2021). Ideally, when treating autoimmune diseases, the neutralization of IL-17 should be locally confined to the disease site rather than applied systemically. Using smaller, thermostable, and versatile antibody versions, topical application may become possible.

[0010] Since IL-17 plays a major role in the development of plaque psoriasis, it is the molecular target of several recently launched biotherapeutics, which are mainly monoclonal antibodies (mAbs), such as Cosentyx (Novartis) and Taltz (Lilly), both of which target IL-17A. Studies have shown that targeting the IL-17F subtype in addition to IL-17A provides higher efficacy in treating plaque psoriasis. The recently launched Bimzelx (UCB) is the first mAb targeting both IL-17A and IL-17F to be approved by the European Medicines Agency (EMA). MoonLake Immunotherapeutics has developed a nanobody targeting both IL-17A and IL-17F, which reported superior results to Cosentyx in a phase 2 clinical trial of plaque psoriasis. All of the above antibodies are indicated only for patients with moderate to severe psoriasis, are administered subcutaneously for systemic drug distribution, and carry a significant risk of side effects. These drugs are also very expensive as they require long-term, lifelong, bi-weekly injections at a cost of thousands of dollars per injection. Unfortunately, there are no safe and affordable biologic drugs available for mild psoriasis (which accounts for 50% of plaque psoriasis patients). Experience has shown that even 28% of patients with moderate plaque psoriasis tend to avoid or delay starting these biotherapies due to the associated risks.

[0011] Nanobodies (VHH antibodies) comprise a class of novel therapeutic proteins based on monomeric, heavy-chain-only antibodies derived from camelids. Compared to monoclonal IgG produced in mammalian cells, nanobodies can be produced in bacteria or yeast. In addition to their low molecular weight, certain nanobodies have also proven to be super thermostable and show particularly high affinity (Güttler et al., 2021).

[0012] An IL-17 antagonist, sonelokimab, is based on single-chain antibodies / nanobodies. 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 application rather than topical application. Similar to classical antibody therapeutics, the incidence of fungal Candida infections increased among 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 their tandem fusions, but the affinity of a single VHH for the IL-17F subtype is rather weak.

[0014] In addition to skin diseases, other inflammatory disorders may be suitable for treatment with IL-17 antagonists. These disorders include, but are not limited to, ankylosing spondylitis (for which approval has been obtained), and also diseases such as multiple sclerosis, rheumatoid arthritis, asthma, graft-versus-host disease, and even Alzheimer's disease, fatty liver disease, and COVID-19 (as reviewed by Mills, 2022, and all clinical studies are ongoing). Future applications may include the treatment of autism, Parkinson's disease, atherosclerosis, stroke, and sepsis. Most of these applications would benefit from an antibody with a broader range of uses, which has high affinity, stability, and versatility in terms of the route of application and fusion with other / stable entities. This indicates a large benefit spectrum for VHH antibodies targeting IL-17A / F.

[0015] There is a significant unmet need for novel immunotherapeutic agents that target both IL-17A and IL-17F and are safe for the treatment of inflammatory, immune, and autoimmune disorders. Such agents can 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 ameliorating symptoms associated with the overactivity of the cytokine IL-17, and provides a new class of single-domain VHH antibodies that neutralize human cytokines to treat patients suffering from inflammatory and / or immune-related disorders caused by and / or associated with the overactivity of the 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] Based not only on sequences but also on data obtained from the crystal and modeled structures of complexes of novel VHH antibodies and human IL-17 subtypes, the novel VHH antibodies of the present invention are divided into four classes. These crystal and modeled structures have been used to define amino acid residues in the VHH sequences that interact with the IL-17 subtypes and that contribute predominantly to binding to the human IL-17 receptor and blocking the binding of the IL-17 subtypes to the human IL-17 receptor, thereby preventing or inhibiting receptor activation.

[0018] In certain embodiments, these VHH antibodies are in monovalent form, e.g., as a single VHH domain or as a fusion with a heterologous protein such as serum albumin, which can be used in multiple forms. In certain embodiments, the VHH antibodies are in multivalent form, e.g., as a bivalent Fc fusion. Due to their very high affinity, they can be used in monovalent form. In certain embodiments, the VHH antibodies in monovalent form have picomolar or even sub-picomolar target affinities. In certain embodiments, the VHH antibodies neutralize all major IL-17 subtypes, particularly human IL-17A homodimer, human IL-17F homodimer, and human IL-17A / F heterodimer, with similar high potency.

[0019] The present invention is in part based on the discovery that representative VHH antibodies unexpectedly show cross-reactivity between the IL-17A and IL-17F subtypes, with affinities in the sub-nanomolar range. The present invention is further based on the preclinical study results of some anti-IL-17 VHH antibodies for the treatment of plaque psoriasis, which indicate a therapeutic potential for alleviating the symptoms of the disease.

[0020] According to some embodiments, the VHH antibodies of the present invention are designed for topical administration to the dermis and are excreted in a manner that should prevent systemic side effects. The in vitro study results of the VHH antibodies of the present invention indicate the possibility of highly effective, specific, but safer and more convenient treatment for a large number and underserved population of mild to moderate plaque psoriasis patients.

[0021] The present invention provides a VHH antibody that recognizes human IL-17 polypeptides, cross-reacts with a variety of different human IL-17 polypeptides, and prevents or inhibits the activation of the human IL-17 receptor, wherein the human IL-17 polypeptides include: (i) human IL-17A homodimer, (ii) human IL-17F homodimer, and (iii) human IL-17A / F heterodimer. The VHH antibodies of the present invention bind to different IL-17 subtypes with extremely high affinity, and these IL-17 subtypes have different epitopes.

[0022] According to some embodiments, the binding affinity of the monomeric VHH for immobilized human IL-17A or IL-17F homodimers, expressed as the dissociation constant KD, 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 the binding of IL-17A, IL-17F, and IL17AF to the human IL-17 receptor and the activation of the human IL-17 receptor, the VHH antibody comprising 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 IL-17A, IL-17F, and IL17AF and neutralizes IL-17A, IL-17F, and IL17AF, the VHH antibody comprising an amino acid sequence selected from 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 IL-17A, IL-17F, and IL17AF and neutralizes IL-17A, IL-17F, and IL17AF, the VHH antibody comprising an amino acid sequence selected from 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 IL-17A, IL-17F, and IL17AF and neutralizes their activities comprises an amino acid sequence selected from SEQ ID NOs. 19, 15, 5, 23, 12, 13, 20, 27, 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 variant has at least 91%, 92%, 93%, 94% or 95% sequence identity with the VHH antibody. According to more specific embodiments, the variant has at least 95%, 96%, 97%, 98% or 99% sequence identity with the VHH antibody.

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

[0029] According to some embodiments, there are provided variants of the VHH antibody described above, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acids are substituted, deleted or added. According to specific embodiments, 1 - 5 amino acids in the VHH antibody are substituted, deleted or added. According to some specific embodiments, the substitution, deletion or addition preserves or improves at least one physical property of the VHH antibody. According to some embodiments, one substitution or a combination of 2 - 5 substitutions, additions or deletions improves the stability and / or producibility of the specific VHH antibody according to the present invention.

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

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

[0032] Variants comprising 1 - 3 amino acid substitutions in one, two or even three CDR sequences are also included within the scope of the present invention. According to some embodiments, the substitutions are conservative substitutions. According to other embodiments, the substitutions are non-conservative substitutions.

[0033] The present invention provides a VHH antibody that binds to human IL-17A, IL-17F and IL17AF dimer and neutralizes the binding of human IL-17A, IL-17F and IL17AF dimer to the human IL-17 receptor, and the VHH antibody belongs to a class of structurally related antibodies, wherein the class is selected from:

[0034] 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;

[0035] ii. Class B with the CDR3 sequence of inclusive X1HNEPGX2LYM, where X1 is selected from

[0036] V and T, and X2 is selected from H and D;

[0037] iii. Class C containing the CDR3 sequence MAVRGLYGSNWYDYPFELW (SEQ ID NO.

[0038] 52); and

[0039] iv. Class D containing the CDR3 sequence YIDSGSDRYY (SEQ ID NO. 57),

[0040] where the sequence identity between different VHH antibodies in a specific class is 80% or higher.

[0041] 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.

[0042] According to some embodiments, the VHH antibody or its variant comprises a CDR3 sequence as shown 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 with the CDR3 sequence.

[0043] According to some specific embodiments, a Class A VHH antibody that recognizes and neutralizes IL-17 is provided, where the VHH antibody comprises a set of 3 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; SEQ ID NOs. 33, 7, and 34. According to some specific embodiments, a Class B VHH antibody that recognizes and neutralizes IL-17 is provided, where the VHH antibody comprises a set of 3 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.

[0044] According to some specific embodiments, provided are VHH antibodies of class C that recognize and neutralize IL-17, wherein the VHH antibody comprises a set of 3 CDR sequences, the set comprising SEQ ID NOs. 50, 51, and 52.

[0045] According to some specific embodiments, provided are VHH antibodies of class D that recognize and neutralize IL-17, wherein the VHH antibody comprises a set of 3 CDR sequences, the set comprising SEQ ID NOs. 56, 57, and 58.

[0046] According to some specific embodiments, provided are VHH antibodies of class A or B that comprise a set of 3 CDR sequences, the set selected from: SEQ ID NOs. 16, 17, and 18, and SEQ ID NOs. 36, 37, and 38.

[0047] 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).

[0048] According to a specific embodiment, the present invention provides the VHH antibody Re42B04a, which comprises the VHH sequence as shown in SEQ ID NO. 19, or a VHH antibody that is a variant thereof having at least 90% identity.

[0049] According to a specific embodiment, the present invention provides the VHH antibody Re42B04, which comprises the VHH sequence as shown in SEQ ID NO. 15, or a VHH antibody that is a variant thereof having at least 90% identity.

[0050] According to a specific embodiment, the present invention provides the VHH antibody Bm17B02, which comprises the VHH sequence as shown in SEQ ID NO. 35, or a VHH antibody that is a variant thereof having at least 90% identity.

[0051] According to some embodiments, Class A VHH antibodies comprise the following positions that interact with IL-17A: position 1, which is Q; position 29, which is A, G, V, F or P; position 30, which is S; position 31, which is S or G; position 32, which is Y; position 33, which is A; position 50, which is A; position 51, which is I; position 52, which is S; position 54, which is I, S or V; position 55, which is S or G; position 57, which is G, S or D; position 58, which is T, S or A; position 59, which is K, R or V; position 100, which is P; position 101, which is Y; position 103, which is L or M; position 104, which is D or E; position 106, which is R; and position 109, which is E or D.

[0052] According to some embodiments, Class A VHH antibodies comprise the following positions that interact with IL-17F: position 29, which is A, G, V, F or P; position 30, which is S; position 31, which is S or G; position 32, which is Y; position 33, which is A; position 50, which is A; position 51, which is I; position 52, which is S; position 54, which is S, I or V; position 55, which is S or G; position 57, which is G, S or D; position 58, which is T, S or A; position 59, which is K, R or V; position 100, which is P; position 101, which is Y; position 103, which is L or M; position 104, which is D or E; position 106, which is R; and position 109, which is E or D.

[0053] According to some embodiments, Class B VHH antibodies comprise the following 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.

[0054] According to some embodiments, Class B VHH antibodies comprise the following positions that 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.

[0055] Another aspect of the invention relates to a set of two or more different VHH antibodies, wherein at least one VHH antibody is as described above.

[0056] In certain embodiments, the VHH antibody as described above is covalently or non-covalently conjugated to a heterologous moiety, which may be selected from a label group, a capture group or an effector group.

[0057] In certain embodiments, the VHH antibody as described above is fused to a heterologous polypeptide moiety, for example, fused to an IgG Fc fragment, to serum albumin or to an albumin-binding moiety. In certain embodiments, the VHH antibody is conjugated to one or several polymer moieties (especially hydrophilic polymer moieties, such as polyethylene glycol (PEG)) to increase the molecular weight of the antibody conjugate and thus delay renal clearance. The molecular weight of the polymer moiety may vary within a wide range, for example, within the range of about 5 kDa to about 80 kDa. Such conjugation can be carried out, for example, through amino or carboxyl groups already present in the VHH (e.g., amino and carboxyl termini) and / or through the side chains of lysine, aspartic acid, glutamic acid or cysteine residues, or through the engineered backbone or side chains of other amino acids, and involves known chemistry for forming amide bonds, secondary amine bonds, urea bonds or thioether bonds.

[0058] According to some embodiments, the VHH antibody neutralizes the binding of human IL-17 dimer to the human IL-17 receptor. According to specific 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 specific embodiments, when tested in a cell-based assay under affinity-limiting test conditions, the VHH antibody neutralizes the binding of at least one of the following to the human IL-17 receptor at a concentration of about 10 nM or lower, about 3 nM or lower, about 1 nM or lower, about 0.3 nM or lower: (i) human IL-17A homodimer, (ii) human IL-17F homodimer and (iii) human IL17A / F heterodimer.

[0059] VHH antibodies that are variants of the VHH antibodies disclosed above and compete with any of the VHH antibodies disclosed above for binding to IL-17 are also within the scope of the invention.

[0060] According to some embodiments, the VHH antibody is stable, especially thermally stable or super-thermally stable. According to some specific embodiments, when measured under non-reducing conditions, the melting temperature of the VHH antibody is at least about 65 °C, at least about 80 °C, at least 90 °C or at least about 95 °C.

[0061] According to some embodiments, when measured under non-reducing conditions, the aggregation temperature of the VHH antibody is 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.

[0062] The VHH antibody of any one of the previous embodiments, which is non-glycosylated or glycosylated.

[0063] According to some embodiments, the VHH antibody or a variant thereof is in a monovalent form.

[0064] According to some embodiments, the VHH antibody or a variant thereof is in a dimer or multimer form.

[0065] The present invention also provides a set of two or more different VHH antibodies that recognize human IL-17 polypeptides, particularly IL-17A homodimers, IL-17F homodimers, and IL17A / F heterodimers, the set comprising at least one of the VHH antibodies or variants thereof described above, particularly a VHH antibody in monovalent form.

[0066] The VHH antibody or a set of VHH antibodies described above is applicable in medicine, such as human medicine, particularly for treatment, such as for preventing or treating disorders caused by and / or associated with the overactivity of IL-17, particularly the overactivity of IL-17A and / or IL-17F, or for diagnosis, such as for detecting IL-17 in a patient sample (such as a body fluid or tissue sample), or for research.

[0067] According to another aspect, the present invention also provides a nucleic acid molecule that encodes the VHH antibody or a subunit of the VHH antibody described above, preferably operably linked to a heterologous expression control sequence or contained in a vector.

[0068] The present invention also provides a recombinant cell or non-human organism that is transformed or transfected with the nucleic acid molecule or vector.

[0069] According to some embodiments, the cell or organism is selected from bacteria, such as Escherichia coli (E. coli), Bacillus sp., single-celled eukaryotic organisms, such as yeast (such as Pichia pastoris) or Leishmania, insect cells, mammalian cells, and plant cells.

[0070] According to some embodiments, the VHH antibody or a variant thereof is produced in bacteria (such as Escherichia coli) or in yeast (such as Pichia pastoris).

[0071] The present invention also provides a method for recombinantly producing the VHH antibodies described above, which comprises culturing cells or organisms in a suitable medium and obtaining the VHH antibodies from the cells or organisms or from the medium.

[0072] According to some embodiments, the method comprises culturing cells from bacteria, yeast, insects, mammals, and plants. According to some embodiments, the cells are mammalian cells. According to some embodiments, the mammalian cells are Chinese Hamster Ovary (CHO) cells.

[0073] According to some embodiments, the method comprises culturing yeast, such as Pichia pastoris, and obtaining the VHH antibodies from the medium.

[0074] According to another aspect, the present invention also provides a pharmaceutical composition comprising at least one VHH antibody as defined above, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0075] According to some embodiments, the pharmaceutical composition comprises a plurality of the VHH antibodies described above, such as a set of specific VHH antibodies.

[0076] According to some embodiments, the formulation is for topical administration. According to some embodiments, the formulation is for surface administration. The pharmaceutical composition provided by the present invention can be formulated as a liquid, solid, or semi-solid. 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.

[0077] According to some embodiments, the pharmaceutical composition is formulated for sustained release, slow release, or delayed release.

[0078] According to another aspect, the present invention also provides a diagnostic composition comprising at least one VHH antibody as defined above, and an acceptable carrier, excipient, or diluent.

[0079] Also provided are pharmaceutical and diagnostic kits comprising at least one VHH antibody and instructions for use.

[0080] The present invention also provides a pharmaceutical composition comprising at least one of the VHH antibodies described above, which is for medical use, particularly for treatment or diagnosis.

[0081] According to some embodiments, the pharmaceutical composition is for preventing or treating a disorder caused by or associated with the overactivity of IL-17, particularly the overactivity of IL-17A and / or IL-17F.

[0082] According to some embodiments, the pharmaceutical composition is for preventing or treating an inflammatory disorder and / or an immune-related disorder.

[0083] According to some embodiments, the pharmaceutical composition is for preventing or treating an inflammatory skin disorder and / or an immune-related skin disorder.

[0084] According to some embodiments, the pharmaceutical composition is for preventing or 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, active non-radiographic axial spondyloarthritis 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 (PRP), alopecia areata, systemic sclerosis, as well as infectious diseases, lichen planus, and pustular psoriasis herpetiformis.

[0085] According to some embodiments, the psoriasis is selected from plaque psoriasis, moderate to severe psoriasis, palmoplantar psoriasis, pustular psoriasis, and pustular psoriasis.

[0086] According to some embodiments, the arthritis is selected from rheumatoid arthritis, psoriatic arthritis, and enthesitis-related arthritis.

[0087] According to some embodiments, the infectious disease is a viral, bacterial, or fungal disease.

[0088] According to some embodiments, the viral disease is caused by influenza virus infection or SARS-CoV-2 infection (COVID-19).

[0089] According to some embodiments, the disease or disorder is psoriasis or arthritis.

[0090] According to some specific embodiments, the disease or disorder is selected from moderate to severe psoriasis, palmoplantar psoriasis, generalized pustular psoriasis, psoriatic arthritis, enthesitis-related arthritis, active non-radiographic axial spondyloarthritis 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).

[0091] According to some embodiments, the psoriasis is plaque psoriasis. According to more specific embodiments, the pharmaceutical composition is for preventing or treating mild to moderate plaque psoriasis.

[0092] Another aspect of the invention relates to a method for preventing or treating a disorder caused by and / or associated with the overactivity of IL-17, in particular the overactivity of IL-17A and / or IL-17F, which comprises administering to a subject in need thereof an effective amount of a VHH antibody as described above or a group of at least two different VHH antibodies as described above or the pharmaceutical composition as described above.

[0093] According to some embodiments, the subject is a human subject suffering from a disorder caused by and / or associated with IL-17.

[0094] According to some embodiments, the disorder is an inflammatory or immune disorder.

[0095] According to some embodiments, the pharmaceutical composition is for preventing or 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, active non-radiographic axial spondyloarthritis 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 (PRP), alopecia areata, systemic sclerosis, and infectious diseases, lichen planus and herpes gestationis.

[0096] According to some embodiments, the psoriasis is selected from plaque psoriasis, moderate to severe psoriasis, hypertrophic palmoplantar psoriasis and pustular psoriasis.

[0097] According to some embodiments, the arthritis is selected from rheumatoid arthritis, psoriatic arthritis and enthesitis-related arthritis.

[0098] According to some embodiments, the infectious disease is a viral, bacterial or fungal disease.

[0099] According to some embodiments, the viral disease is caused by influenza virus infection or SARS-CoV-2 infection (COVID-19).

[0100] According to some specific embodiments, the disease or disorder is selected from moderate to severe psoriasis, palmoplantar pustular psoriasis, generalized pustular psoriasis, psoriatic arthritis, enthesitis-related arthritis, active non-radiographic axial spondyloarthritis 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).

[0101] According to some embodiments, the disease or disorder is psoriasis or arthritis.

[0102] According to some embodiments, the disorder is psoriasis. According to more specific embodiments, the psoriasis is plaque psoriasis. According to yet more specific embodiments, the disorder is mild to moderate plaque psoriasis.

[0103] According to some embodiments, the composition is administered topically.

[0104] According to some embodiments, the composition is administered superficially.

[0105] According to some embodiments, the administration is by injection. According to a specific embodiment, the administration is by intradermal injection.

[0106] According to another aspect of the invention, there is also provided a method of delivering a VHH antibody to a cell, which comprises contacting the cell with at least one VHH antibody or a group of VHH antibodies as described above.

[0107] According to some embodiments, the cell is a cell of a human subject suffering from a disorder caused by or associated with the overactivity of IL-17, particularly a disorder associated with the overactivity of IL-17A and / or IL-17F.

[0108] The present invention is explained in more detail by the following figures and examples. Description of the Drawings

[0109] Reference is now made to the drawings, which are by way of example only, to describe some embodiments of the present invention. In particular, with reference to the details of the drawings, it should be emphasized that the details shown are exemplary and are for the purpose of exemplarily illustrating the embodiments of the present invention. In this regard, the description in conjunction with the drawings makes it apparent to those skilled in the art how to implement the embodiments of the present invention.

[0110] Figure 1 : Sequences of selected anti-IL-17 VHH antibodies.

[0111] The figure shows a sequence alignment from selected VHH antibodies. Residues deviating from the consensus are highlighted with a grey background. The three CDR regions are indicated.

[0112] Figure 2 : Affinity of the VHH-like Re42H11 for IL-17 subtypes measured by Biolayer Interferometry (BLI).

[0113] The figure shows the binding of the indicated IL-17 species to biotinylated VHH antibody of the Re42H11 class (class A) immobilized on a high-precision streptavidin biosensor. Binding and dissociation were recorded as wavelength shift (in nm) on an Octet RED96e instrument (ForteBio / Sartorius). The baseline was recorded by parallel measurement of a “VHH-free control”. Association rate, dissociation rate and apparent dissociation constant (K D ) were calculated by fitting the data using a mass transport model by Octet DataAnalysis HT 12.0 software. It should be noted that immobilizing the VHH to the sensor chip creates an avidity effect, i.e. adjacent VHH molecules may simultaneously bind the same IL-17 dimer, resulting in a dissociation rate lower than the actual value. This mimics the binding of bivalent IgG to the IL-17 dimer. It should also be noted that Figures 4 - 8 Figure 11 measures the monovalent affinity by using immobilized IL-17 and monovalent VHH antibody as analytes. All VHH antibodies characterized in the BLI experiments were produced by periplasmic expression in E. coli.

[0114] Figure 3 : Affinity of the VHH antibody of the Bm17B02 class for IL-17 subtypes.

[0115] BLI measurements were as Figure 2 shown, but were used for class B Bm17B02 and Re42F08 VHH antibodies.

[0116] Figure 4 : Affinity of the control VHHs VHH662 and VHH664 for IL-17 subtypes.

[0117] Human IL-17A and IL-17F were produced with C-terminal Avi tags and enzymatically biotinylated by recombinant BirA (Beckett et al., 1999), and immobilized at a concentration of 100 nM on a high-precision streptavidin biosensor on an Octet RED96e instrument (Sartorius) for 200 s, where phosphate-buffered saline (PBS) pH 7.4, 0.02% (w / v) Tween 20, and 0.1% (w / v) bovine serum albumin (BSA) were used as assay buffer. Then 20 nM and 100 nM of VHH662 or VHH664 antibodies were allowed to bind for 450 s, followed by dissociation for 900 s. Binding and dissociation were recorded as wavelength shifts (in nm). Baselines were recorded by parallel measurement of a "VHH-free control". Curves (grey) were fitted using a mass transport model (class A for Re42H11-class VHHs) and a 2:1 heterogeneous model (class B for Bm17B02-class VHHs). The fitted curves and calculated dissociation constants (K D ) are shown in black. It should be noted that the affinity measured by this device is independent of the avidity effect, as the VHH antibodies are provided as analytes and are thus monomers prior to target binding.

[0118] Figure 5 : Affinity of first-generation Re42H11 and Bm17B02-class VHHs for IL-17A.

[0119] As Figure 4 described, BLI experiments were performed using first-generation VHH antibodies as analytes (using 25 nM, 50 nM, and 100 nM VHH) and biotinylated IL-17A immobilized on the sensor chip. For the calculation of the dissociation constant (K D ), curves (grey) were fitted using a mass transport model (class A for Re42H11-class VHHs) or a 2:1 heterogeneous model (class B for Bm17B02-class VHHs). The lower-affinity component in class B VHHs may be due to the binding of excess VHH molecules to secondary sites on IL-17.

[0120] Figure 6 : Affinity of second-generation Re42H11-class VHHs for IL-17 subtypes.

[0121] As Figure 4 described, BLI experiments were performed to analyze the binding of the designated Re42H11-class VHHs as analytes to biotinylated IL-17A or IL-17F.

[0122] Figure 7 : Affinity of second-generation Bm17B02-class VHHs for IL-17 subtypes.

[0123] The specified VHH (class B) was analyzed by BLI as Figure 4 described.

[0124] Figure 8 : Affinity of other VHHs (classes C and D) for IL-17 subtypes.

[0125] VHH Bm42A03, Bm44G10 (class C) and Bm45G07 (class D) were analyzed by BLI as Figure 4 described.

[0126] Figure 9 : Thermal stability of the disclosed VHH antibodies.

[0127] Differential scanning fluorimetry (DSF) was performed on the VHH antibodies as described in Example 4. Here, thermal unfolding was measured by the enhanced fluorescence of SYBR Orange added at 532 nm excitation and 555 nm long-pass filter after stepwise increasing the temperature. The melting temperature was defined as the inflection point of the first melting peak. Re42B04 was the only VHH in the series that showed a melting peak; however, this peak was rather low, indicating that the VHH was only locally unfolded, rather than globally unfolded.

[0128] Figure 10 : Thermal stability under non-reducing conditions and dithiol reduction conditions.

[0129] As Figure 9 shown, thermal stability was determined. However, a control in the presence of 10 mM dithiothreitol (DTT) is shown here. DTT reduces the structural, stabilizing disulfide bonds.

[0130] Figure 11: Thermal stability of VHHs determined by BLI.

[0131] The specified class A ( Figure 11A ) and class C ( Figure 11B ) VHH antibodies (1 μM) were incubated at room temperature or 95 °C for 10 minutes and centrifuged at 20,000 g for 20 minutes. The supernatant was diluted 20-fold (to 50 nM) and analyzed for IL-17A binding by BLI.

[0132] Figure 12 : Dose response of HEK-Blue TM IL-17 reporter cells to recombinant IL-17 cytokines.

[0133] HEK-Blue TMCells of the IL-17 reporter gene cell line were stimulated with increasing concentrations of human IL-17A, IL-17F, and IL-17AF. The stimulation led to the secretion of the embryonic alkaline phosphatase (SEAP) reporter gene, the activity of which was determined colorimetrically using QUANTI-BlueTM as a substrate and measuring the resulting absorbance at 620 nm. The OD620 values were plotted in (A). The graph in (B) depicts the induction range calculated by dividing the OD620 readings at each IL-17 dilution by the OD620 value of the IL-17-free control. The results are the mean ± SD of one experiment measured in triplicate. The vertical dashed line indicates the IL-17 concentration used in subsequent VHH neutralization experiments ( Figure 13 - 1 4).

[0134] Figure 13 : Neutralization of IL-17A and IL-17F by first-generation VHH antibodies.

[0135] The figure shows the expression of the phosphatase reporter gene in HEK-Blue TM 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 pre-incubated with the indicated concentrations of anti-IL-17 VHH antibodies. The number 1.0 represents induction in the absence of VHH (complete), and the number 0 represents no induction after subtracting the background of the VHH-free control. The figure shows the neutralization of members of the Re42H11 and Bm17B02 classes (class A and class B, respectively), each measured in triplicate.

[0136] Figure 14: Neutralization of IL-17A, IL-17F, and IL-17AF by class A-D VHH antibodies.

[0137] The figure shows the normalized expression of the phosphatase reporter gene induced by 0.17 nM IL-17A homodimer, 0.85 nM IL-17F homodimer, or 0.85 nM IL-17AF heterodimer pre-incubated with the indicated concentrations of anti-IL-17 VHH antibodies. The number 1.0 represents induction in the absence of VHH (complete), and the number 0 represents no induction after subtracting the background of untreated cells. The figure shows the neutralization of (A) class A (Re42B04a), (B) class B (Re42F08), (C) class C (Bm42A03), and (D) class D (Bm45G07). Bm18F11 is included in all figures for reference. The results are the mean ± SD of three independent experiments, each measured in triplicate.

[0138] Figure 15: Neutralizing effects of control VHH antibodies on IL-17A, IL-17F, and IL-17AF.

[0139] The figure shows the normalized expression of a phosphatase reporter gene induced by 0.17 nM IL-17A homodimer, 0.85 nM IL-17F homodimer, or 0.85 nM IL-17AF heterodimer pre-incubated with the indicated concentrations of anti-IL-17 VHH antibodies, respectively. The number 1.0 represents (complete) induction in the absence of VHH, and the number 0 represents no induction after subtracting the background of untreated cells. (A) This figure shows the neutralizing effects of representative members of class A (Re42B04a), class C (Bm42A03), and class D (Bm45G07) compared to control VHH antibodies VHH662, VHH664, and the secukinumab biosimilar (Proteogenix PX-TA1606). Secukinumab is a tandem fusion of three VHHs targeting IL-17A, IL-17F, and albumin. Due to its higher molecular weight and the presence of two IL-17 binding sites in secukinumab, the same mass concentration (ng / mL) was compared. The results are the mean ± SD of three independent experiments, each measured in triplicate. (B) Cosentyx / The neutralizing effect of the biosimilar (Proteogenix PX-TA1234) and the research-grade anti-IL-17A antibody (R&D Systems AF-317-NA) as controls on IL-17A. The results are the mean ± SD of two independent experiments, each measured in triplicate.

[0140] Figure 16 A - B: Crystal structure of class A VHH member Re42H11 bound to the IL-17F dimer.

[0141] His14-ScSUMO-tagged Re42H11 was co-expressed with His14-BdNEDD8-tagged human IL-17F (residues 39 - 163) in Escherichia coli SHuffle Express (New England Biolabs). The complex was purified as follows: Ni-chelation chromatography was performed to immobilize the complex components via the His14 tag, followed by two consecutive tag cleavage elutions with bdNEDP1 protease (cleaving NEDD8) and ScUlp1 protease (cleaving SUMO) (Frey and (in 2014). The complex Re42H11·IL-17F was recovered in the second elution step and further purified by size-exclusion chromatography. The complex was crystallized, an X-ray diffraction data set was recorded at the Swiss Light Source synchrotron, and the structure was solved by molecular replacement to a resolution of Rfree was 0.28.

[0142] Figure 16 A. Crystal structure of the tetrameric Re42H11·IL-17F complex represented as a ribbon diagram. The CDR regions (defined in Figure 1 ) are highlighted in yellow and represented accordingly.

[0143] Figure 16 B. Based on the alignment with the IL-17F complex of IL-17RC ECD·IL-17F (PDBID 6HG4, Goepfert et al., 2020), the IL-17 receptor IL17-RC (semi-transparent grey surface) is docked onto the IL-17F dimer.

[0144] Figure 17 : Sequence alignment of class A anti-IL-17 VHHs with the indicated IL-17 interacting residues.

[0145] 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. The IL-17A interacting residues are underlined and the IL-17F interacting residues are shown in bold.

[0146] Figure 18 : Crystal structure of the class B VHH Bm17B02 bound to the IL-17F dimer.

[0147] Complex formation and structure determination were as Figure 16 described, with the only difference being that His14-ScSUMO-tagged Bm17B02 was used for co-expression and complex formation.

[0148] Figure 18 A. Crystal structure of the tetrameric Bm17B02·IL-17F complex represented as a ribbon diagram. The CDR regions (as Figure 1 shown) are highlighted in yellow and represented accordingly.

[0149] Figure 18 B. As Figure 16 described, IL17-RC (semi-transparent grey surface) is docked onto the IL-17F dimer.

[0150] Figure 19: Sequence alignment of class B VHHs highlighting IL-17 interacting residues.

[0151] Identification of IL-17 interacting residues of class B VHH antibodies by crystal structure ( Figure 18 ) or by homology modeling based on this structure. IL-17A interacting residues are underlined and IL-17F interacting residues are in bold.

[0152] Figure 20A and 20B : Production of VHH antibodies in Pichia pastoris.

[0153] These protocols describe the production of VHH antibodies Re42B04a and Re42F08 in Pichia pastoris. Figure 20A For the upstream process. Figure 20B For the 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.

[0154] Figure 21 : Full - thickness ex vivo human skin model.

[0155] Schematic of the ex vivo human skin model "Inflammaskin", reproducing key features and inflammatory responses observed in psoriatic lesions.

[0156] Figure 22 : Evaluation of IL-17A release in the ex vivo psoriasis model.

[0157] The model consists of healthy human skin samples that are treated with a Th polarization mixture to induce the psoriatic phenotype of Hypo InfammaSkin samples. Details of the study design are shown in Table 4. The VHH antibodies Re42B04a and Re42F08 are injected intradermally one or three times into the skin samples. Positive control samples include betamethasone and secukinumab for the treatment of psoriasis. Betamethasone is an anti-inflammatory steroid, while secukinumab is a monoclonal antibody targeting IL-17A. The irrelevant VHH antibody Re32D03 (SEQ ID NO.64, against the spike protein of SARS-CoV2) is used as a negative control. HypoSkin and HypoInflammaSkin samples are cultured for 7 days. On day 7 of the culture, the supernatant is sampled and the concentration of IL-17A is measured using an MSD kit (K15076K, MesoScale Discovery). The concentration is expressed in pg / mL, the values of each parallel determination (points) are plotted, and the mean and standard error of the mean (SEM) for each case are shown. The mean is calculated from the detectable / QC control values. The figure shows all parallel determinations for each case. Statistical analysis (by one-way ANOVA) is performed between each case and the untreated HypoInflammaSkin control. In addition, a one-way ANOVA test is also performed to evaluate the differences between each case and the irrelevant VHH Re32D03; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0158] Figure 23 A-H: Ex vivo human psoriatic skin samples after treatment with anti-IL17 VHH

[0159] Skin samples from the study described in Table 4 and analyzed in Figure 22 are collected on day 7, fixed, stained with H&E and analyzed by microscopy. Figure 23 A: HypoSkin sample (not treated with pro-inflammatory cytokines). Panels B-H show InflammoSkin samples treated with a Th polarization mixture. Figure 23 B: Untreated psoriatic tissue (negative control), Figure 23 C: Treated with betamethasone (positive control), Figure 23 D: Treated with secukinumab (positive control), Figure 23 E: Injected with VHH antibody Re42B04a once, Figure 23 F: Injected with VHH antibody Re42B04a three times, Figure 23 G: Injected with VHH antibody Re42F08 once, Figure 23H: Inject VHH antibody Re42F08 three times. Detailed implementation mode

[0160] The present invention relates to VHH antibodies that recognize human IL-17 polypeptides, including IL-17A, IL-17F, and IL-17AF.

[0161] Obtaining VHH antibodies that block IL-17 and bind tightly to both IL17A and IL17F is challenging and not obvious because the neutralizing epitopes have some amino acid exchanges between 17A and 17F. The VHH antibodies provided by the present invention (e.g., Bm43B02) have picomolar affinities for both 17A and 17F (measured by the binding affinity of monomeric VHH to immobilized human IL-17A or IL-17F homodimers). Notably, compared to previously published VHH antibodies targeting IL-17 (such as those disclosed in WO2012156219), the VHH antibodies of the present invention have higher affinities for IL-17 subtypes, especially for IL-17F.

[0162] The binding data of the novel VHH antibodies of the present invention have been confirmed in cell-based assays that block IL-17 receptor activation and in ex vivo human skin models.

[0163] VHH antibody

[0164] The sequences of the CDRs of the VHH antibodies and control antibodies of the present invention are listed in Table 1 below, while Table 2 lists the sequences of the full-length VHH antibodies.

[0165] Table 1. Sequences of anti-IL-17 VHH antibodies.

[0166]

[0167]

[0168] * These VHH antibodies of Ablynx are disclosed in WO2012156219 and are provided herein only for comparison because they were compared with the VHH antibodies of the present invention in some experiments.

[0169] Table 2. Sequences of VHH antibodies

[0170]

[0171]

[0172] The present invention relates to a VHH antibody, which is a monovalent single-chain antibody, comprising a CDR1 domain, a CDR2 domain and a CDR3 domain connected by framework regions. The VHH antibodies include, but are not limited to, intact VHH antibodies, such as natural VHH antibodies comprising framework regions derived from camelids, and modified VHH antibodies comprising modified framework regions, VHH antibody fragments and VHH antibody fusion proteins, such as fusion proteins with immunoglobulins or non-immunoglobulin peptides or polypeptides, as long as they exhibit the characteristics described according to the present invention.

[0173] There are several known methods in the art for determining the CDR sequences of a given antibody molecule, but there is no clear standard method. The determination of the CDR sequences from the variable region of the antibody heavy chain can be carried out according to any method known in the art, including, but not limited to, methods known as KABAT, Chothia and IMGT. Common determination methods (such as KABAT) exclude key parts of the variable region because the VHH antibody binding site (paratope) usually includes residues of the framework and outside the narrowly defined CDR regions. This is also the difference between nanobodies and conventional antibodies. Other methods for identifying the binding site and CDR sequences of VHH antibodies include using a custom reference database utilizing a large amount of VHH antibody sequencing data. A selected set of CDRs may include sequences identified by more than one method. CDRs can also be defined by multiple alignment (with many other VHH antibodies) to identify variability hotspots and correlate them with the standard VHH antibody structure. CDRs can also be defined by analyzing the structure of the VHH antibody and determining what are the loops and what is the antibody framework. In some cases, residues adjacent to the CDRs are also variable and are thus included in the CDR definition. According to some embodiments of the present invention, the CDR sequences of the VHH antibody variable region are determined using a custom reference database containing sequencing data of ≥10,000 VHH antibodies.

[0174] The present invention also relates to covalent or non-covalent conjugates of VHH antibody molecules with non-protein structures, which are, for example, labeling groups, capture groups such as solid-phase binding groups, or effector groups such as toxins. For example, the heterologous moiety may be from a fluorophore, biotin, an enzyme (such as peroxidase, phosphatase or luciferase), a hapten, an affinity tag or a nucleic acid (such as an oligonucleotide).

[0175] The VHH antibodies of the present invention are in particular monoclonal VHH antibodies characterized by a specific amino acid sequence. The 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, the glycosylation sites in the parental VHH antibody sequence are mutated to eliminate predicted glycosylation. In a specific embodiment, the mutation includes replacement of the asparagine (Asn, N) residue of the N-glycosylation site to prevent potential glycosylation of the VHH antibody. In a more specific embodiment, the Asn residue is replaced to prevent or eliminate glycosylation in the 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).

[0176] In certain embodiments, the VHH antibodies are glycosylated, wherein the carbohydrate structure can be from the glycosylation sites introduced into the VHH sequence and / or from the fusion partner.

[0177] The VHH antibodies according to the present invention are characterized by: (i) the CDR3 sequence, (ii) the combination of the CDR1 sequence, the CDR2 sequence and the CDR3 sequence, (iii) the complete VHH sequence, or (iv) competing with a specific reference antibody. Specific CDR and VHH sequences are provided in the tables, figures and sequence listings.

[0178] According to the present invention, sequences related to the above sequences are covered. These related sequences are defined by having a minimum identity with a specifically designated amino acid sequence (such as a CDR or VHH sequence). This identity is indicated over the entire length of the corresponding reference sequence and can be determined by using well-known algorithms such as BLAST.

[0179] In a specific embodiment, the related CDR3 sequence has at least 80% or at least 90% or at least 95% identity with the specifically designated CDR3 sequence, for example, 1, 2 or 3 amino acids are replaced.

[0180] In a specific embodiment, the related combination of the CDR1 sequence, the CDR2 sequence and the CDR3 sequence has at least 80% or at least 90% or at least 95% identity with the combination of the specifically designated CDR1 sequence, the CDR2 sequence and the CDR3 sequence, for example, 1, 2, 3, 4, 5 or 6 amino acids are replaced by different amino acids.

[0181] In a specific embodiment, the relevant VHH sequence has at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity with the VHH sequence. For example, 1, 2, 3, 4, 5 or up to 20 amino acids are replaced.

[0182] Furthermore, the present invention refers to a VHH antibody that competes with the specific VHH antibodies disclosed herein for binding to the human IL-17 polypeptide. In certain embodiments, the competing VHH antibody binds to the same or overlapping epitopes on the human IL-17 polypeptide. For example, the present invention refers to a VHH antibody that competes with a reference antibody (such as the VHH antibody Re42H11) or with the VHH antibody Bm17B02. The competition can be determined by label-free biolayer interferometry performed in a cross-competition or epitope binning assay using a label-free detection system (such as the system) according to the manufacturer's instructions.

[0183] In a specific embodiment, at least one amino acid of the reference sequence, including the amino acids in the CDR1, CDR2 or CDR3 sequences and / or the amino acids in the framework region, is replaced by another amino acid while maintaining the structural integrity and epitope binding of the VHH antibody. These exchanges can be conservative (i.e., replaced by similar amino acids) or non-conservative.

[0184] In a further specific embodiment, at least one amino acid of the reference sequence, including the amino acids in the CDR1, CDR2 or CDR3 sequences and / or the amino acids in the framework region, is replaced by a conservative amino acid, i.e., an amino acid is replaced by another amino acid having similar biochemical properties. For example, one aliphatic amino acid (such as Gly, Ala, Val, Leu or Ile) is replaced by another aliphatic amino acid; one basic amino acid (such as His, Lys or Arg) is replaced by another basic amino acid or Met; one acidic amino acid or its amide (such as Asp, Glu, Asn or Gln) is replaced by another acidic amino acid or its amide; one aromatic amino acid (such as Phe, Tyr or Trp) is replaced by another aromatic amino acid.

[0185] In a further specific embodiment, the VHH antibody is selected from the antibody Re42B04a comprising the VHH sequence shown in SEQ ID NO.19 or a VHH antibody that is 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.

[0186] In a further specific embodiment, the VHH antibody is selected from antibody R242B04 comprising a VHH sequence as shown 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.

[0187] In a further specific embodiment, the VHH antibody is selected from antibody Re42F08 comprising a VHH sequence as shown in SEQ ID NO.39 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.39 are replaced by another amino acid.

[0188] In a further specific embodiment, the VHH antibody is selected from antibody Bm17B02 comprising a VHH sequence as shown 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.

[0189] Based on sequence identity (80% or higher sequence identity for the same class of VHH antibodies) and data obtained from the crystal structures of complexes of VHH antibodies and human IL-17, the present invention provides four classes of VHH antibodies. These crystal structures have been used to define amino acid residues in the VHH sequences that mostly contribute to binding to IL-17 and neutralizing its binding to the receptor. Each VHH antibody in a specific class has at least 80% sequence identity with other members of the same class.

[0190] In addition, the present invention relates to a nucleic acid molecule (e.g., a DNA molecule) encoding a VHH as described above, or a vector comprising the nucleic acid molecule as described above, wherein the nucleic acid molecule is operably linked to an expression control sequence, particularly a heterologous expression control sequence. In addition, the present invention relates to a cell comprising the nucleic acid molecule or vector as described above. 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 a known host cell for producing antibodies or antibody fragments, e.g., a prokaryotic cell (such as an Escherichia coli or Bacillus species 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 an extrachromosomal nucleic acid or vector. In other embodiments, the cell comprises a nucleic acid or vector integrated into the genome, e.g., as an expression cassette for genomic integration.

[0191] Another aspect of the present invention is a method for recombinantly producing a VHH antibody by growing a cell as described above in a culture medium and obtaining the VHH antibody from the cell or the culture medium. Suitable culture media and culture conditions are well known in the art.

[0192] Binds to human IL17

[0193] The VHH antibodies of the present invention bind to human IL-17 polypeptides. The inventors have identified VHH antibodies that bind to human IL-17 polypeptides with high affinity and cross-react with (i) human IL-17A homodimers and (ii) human IL-17F homodimers, as shown in Table 3.

[0194] In the context of the present disclosure, there is the term "human IL-17". This term encompasses a variety of different human IL-17 family members or subtypes, including but not limited to:

[0195] - IL-17A (UniProt accession number Q16552),

[0196] - IL-17B (UniProt accession number Q9UHF),

[0197] - IL-17C (UniProt accession number Q9P0M4),

[0198] - IL-17D (UniProt accession number Q8TAD2),

[0199] - IL-17E (UniProt accession number Q9H293),

[0200] -IL-17F (UniProt accession number Q96PD4).

[0201] The term "human IL-17" encompasses in particular human IL-17A (UniProt accession number Q16552) in the form of a homodimer comprising two IL-17A units, in particular IL-17F (UniProt accession number Q96PD4) in the form of a homodimer comprising 2 IL-17F units, and IL-17AF in the form of a heterodimer comprising one IL-17A unit and one IL-17F unit.

[0202] However, it should be noted that the term "human IL-17" also encompasses naturally occurring variants of human IL-17 polypeptides and genetically modified constructs as described herein.

[0203] The inventors have carried out selection and binding experiments using the following genetically modified IL-17A, IL-17F and IL-17A / F constructs:

[0204] - An IL-17A homodimer comprising amino acid residues 40-155 of human IL-17A (UniProt Q16552). This corresponds to the highly ordered part in the crystal structure. The first five amino acids (GSEDS) are spacer residues. In addition, it also contains two point mutations: N68D (eliminating the N-glycosylation site) and C192S (eliminating the unpaired cysteine, which would otherwise cause problems in recombinant expression). The amino acid sequence of this polypeptide is shown in SEQ ID NO.1.

[0205] - An IL-17F homodimer comprising amino acid residues 39-163 of human IL-17F (UniProt Q96PD4). The N83D mutation is used to eliminate the N-glycosylation site. The first five residues (GSEGE) are linkers. The amino acid sequence of this polypeptide is shown in SEQ ID NO.2.

[0206] - An IL17A / F heterodimer consisting of two polypeptides. The IL-17A unit comprises amino acid residues 40-155 of human IL-17A (UniProtQ16552), which has 5 spacer residues at the front (GSEDS) and has an N68D mutation. The amino acid sequence of this polypeptide is shown in SEQ ID NO.3. The IL-17F unit comprises amino acid residues 39-163 of human IL-17F (UniProt Q96PD4), which has 5 spacer residues at the front (GSEDS) and has N83D and C137S mutations. The amino acid sequence of this polypeptide is shown in SEQ ID NO.4.

[0207] The IL-17A, IL-17F, and IL-17AF proteins expressed by the above bacteria are used during initial immunization, selection, characterization, and crystallization.

[0208] For the immunization and selection of the second-generation VHH antibodies and for their characterization, mammalian-expressed versions of IL-17A and IL-17F lacking the specified modifications are used, as follows:

[0209] - Mature human IL-17A expressed by mammals (UniProt Q16552). The amino acid sequence of this polypeptide is shown in SEQ ID NO. 77.

[0210] - Mature human IL-17F expressed by mammals (UniProt Q96PD4). The amino acid sequence of this polypeptide is shown in SEQ ID NO. 78.

[0211] In certain embodiments, the VHH antibodies of the present invention bind to (i) human IL-17A homodimers, (ii) human IL-17F homodimers, and (iii) human IL17A / F heterodimers, wherein the binding affinity for each of (i), (ii), and (iii) is expressed as a dissociation constant KD of about 1 nM or lower, about 100 pM or lower, about 50 pM or lower, about 20 pM or lower, or about 10 pM or lower. The binding affinity can be determined using, for example, the polypeptides of SEQ:ID NO: 1-4 and 77-78 as described above, as detailed in the examples and figures herein.

[0212] IL-17 neutralization

[0213] The VHH antibodies of the present invention are capable of neutralizing the binding of human IL-17 dimers to the human IL-17 receptor and are capable of preventing or inhibiting receptor activation. The inventors have identified VHH antibodies that cross-neutralize (i) human IL-17A homodimers, (ii) human IL-17F homodimers, and (iii) human IL17A / F heterodimers, as shown in Table 3.

[0214] In certain embodiments, the VHH antibodies of the present invention cross-neutralize the binding of human IL-17 polypeptides to the human IL-17 receptor, wherein the human IL-17 polypeptides are particularly (i) human IL-17A homodimers, (ii) human IL-17F homodimers, and (iii) human IL17A / F heterodimers. The neutralization potency can be determined as detailed in the examples herein.

[0215] Stability

[0216] For the intended therapeutic applications, anti-IL-17 VHH antibodies should not only be highly potent in interleukin neutralization, but they should also be developable as biopharmaceuticals. This includes that they are stable enough to survive the long large-scale production process as well as shipping and storage (ideally for several years in liquid, semi-liquid or solid formulations) without aggregating or losing activity.

[0217] A good predictor of stability is thermal stability, which can be measured by thermal shift assays or especially by differential scanning fluorimetry. The inventors have identified several thermally stable or super-thermally stable VHH antibodies, as shown in Table 3.

[0218] In a specific embodiment, the invention relates to a VHH antibody that is stable, particularly thermally stable or super-thermally stable. Preferably, when measured under non-reducing conditions, the melting point (melting temperature) of the VHH antibody is at least about 65°C, at least about 80°C, at least 90°C or at least about 95°C, and / or the aggregation temperature is at least about 50°C, at least about 60°C, at least 70°C or at least about 80°C. The melting and aggregation temperatures are determined as described herein.

[0219] In a specific embodiment, the invention relates to a VHH antibody that is stable, particularly thermally stable or super-thermally stable. Specifically, when measured under non-reducing conditions, the melting point (melting temperature) of the VHH antibody is at least about 65°C, at least about 80°C, at least 90°C or at least about 95°C, and / or the aggregation temperature is at least about 50°C, at least about 60°C, at least 70°C or at least about 80°C. The melting and aggregation temperatures are determined as described herein. According to some embodiments, when measured under non-reducing conditions, the melting point (melting temperature) of a stable VHH antibody is at least about 65°C and / or the aggregation temperature is at least 50°C, while the melting point (melting temperature) of a super-thermally stable VHH antibody is at least 95°C, and / or the aggregation temperature is at least about 80°C. It should be noted, however, that the results of stability measurements depend on conditions. For example, high protein concentration, pH close to the isoelectric point of the protein favors aggregation. Stability is also affected by buffer composition and additives used with the test components.

[0220] VHH antibody group

[0221] In a further aspect, the invention relates to a group comprising at least 2, 3, 4 or more of the above-mentioned VHH antibodies. In such a group, the individual VHH antibodies are present in a suitable molar ratio. Generally, 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 specifically about 1:1. In certain embodiments, the group of VHH antibodies may comprise a single composition, wherein the VHH antibodies in the group consist of a predetermined number of different types of VHH antibodies as described above. The group of VHH antibodies may comprise multiple compositions, each composition comprising different types of VHH antibodies as described above. The groups of the invention may be free of other VHH antibodies.

[0222] Monovalent and multivalent VHH antibodies

[0223] In certain embodiments, the VHH antibodies of the invention are in monovalent form, i.e., it has a single human IL-17 polypeptide binding site. In these embodiments, the VHH antibody may be present per se or covalently or non-covalently linked to a heterologous moiety (e.g., a peptide or non-peptide moiety).

[0224] In other embodiments, the VHH antibodies of the invention are present in the form of a multimer (e.g., a dimer or trimer). In these embodiments, several VHH antibody units may be covalently or non-covalently linked to each other via a linker and / or a multimerization (e.g., dimerization or trimerization) moiety.

[0225] In certain embodiments, the VHH antibody is a homodimeric VHH antibody, wherein the VHH antibody unit is covalently linked to a dimerization moiety (e.g., an immunoglobulin Fc fragment).

[0226] In certain embodiments, the VHH antibody is a heterodimeric VHH antibody, particularly a covalently linked 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 wherein the first VHH antibody binds to IL-17 and the second VHH antibody binds to a different target.

[0227] Production of VHH antibodies

[0228] VHH antibodies, including monomeric and multimeric VHH antibodies, can 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.

[0229] VHH antibodies can be recombinantly produced in suitable host cells, e.g., in prokaryotic or eukaryotic host cells or host organisms. For this purpose, a nucleic acid molecule encoding a VHH antibody is introduced into the host cell or host organism and expressed therein. The nucleic acid molecule can encode a monomeric VHH antibody or a subunit of a multimeric VHH antibody.

[0230] In one specific embodiment, the VHH antibody is recombinantly produced in bacteria (e.g., Escherichia coli or Bacillus spp.). For example, expression in bacteria may involve cytoplasmic and / or periplasmic expression and purification of the VHH antibody 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 to the periplasm and / or into the culture medium.

[0231] In a further specific embodiment, the VHH antibody is recombinantly produced in a eukaryotic host cell or host organism or in an animal cell, preferably the eukaryotic host cell or host organism is yeast, such as Pichia pastoris, Saccharomyces cerevisiae or Hansenula polymorpha, and the animal cell is particularly a mammalian cell, such as a human or hamster cell. For example, expression in a eukaryotic host cell or host organism (e.g., yeast) may involve cytoplasmic and / or periplasmic expression and purification of the VHH antibody from the host cell, or preferably secretion by 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.

[0232] VHH antibodies can be produced using any method known in the art for producing proteins, antibodies and nanobodies. Generally, the VHH antibodies are recombinantly produced in prokaryotic or eukaryotic host cell systems or host organisms (such as bacteria, yeast, plant cells or mammalian cells). According to some embodiments, the VHH antibodies are recombinantly produced in Pichia pastoris.

[0233] In yet a more specific embodiment, the VHH antibody is produced using Figure 20A and 20B the methods shown in.

[0234] Therapeutic applications and methods of use

[0235] Another aspect of the invention is the use of the VHH antibodies as described above in medicine, particularly for therapeutic and / or in vitro or in vivo diagnostic applications. In certain embodiments, the VHH antibodies are used in human medicine.

[0236] The VHH antibodies of the present invention can be used for preventing or treating diseases caused by the overactivity of IL-17 or diseases associated therewith, particularly diseases associated with the overactivity of IL-17A and / or IL-17F.

[0237] In certain embodiments, the VHH antibodies can be used for preventing or treating inflammatory and / or immune-related diseases, such as inflammatory and / or immune-related skin diseases.

[0238] Typical diseases are asthma, psoriasis (such as plaque psoriasis), arthritis (such as 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.

[0239] In some embodiments, the disease is plaque psoriasis. In a specific embodiment, the disease is mild or moderate plaque psoriasis.

[0240] In a therapeutic application, the VHH antibody is administered to a subject in need thereof, particularly a human subject, in an effective amount. The dosage will depend on the specific type of the agent (such as a monomeric VHH antibody or a polymeric VHH antibody), the type of the disease, and the route of administration.

[0241] Generally, the VHH antibody is administered as a pharmaceutical composition, which comprises an active agent and a pharmaceutically acceptable carrier or excipient. Examples of carriers and excipients suitable for formulating antibodies or antibody fragments are well known in the art.

[0242] The present invention also provides a method for treating diseases and disorders associated with the overexpression or overactivity of human IL-17, which comprises administering to a subject in need of such treatment a pharmaceutical composition for treating the disease or disorder, the pharmaceutical composition comprising an effective amount of a VHH antibody that neutralizes IL-17 receptor activation and a pharmaceutically acceptable carrier.

[0243] In certain embodiments, the disease or disorder is an inflammatory and / or immune-related disease. According to a more specific embodiment, the disease or disorder is an inflammatory and / or immune-related skin disease.

[0244] Typical diseases that can be treated with the VHH antibodies of the present 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.

[0245] In some embodiments, the disease is plaque psoriasis. In a specific embodiment, the disease is mild or moderate plaque psoriasis.

[0246] According to some embodiments, the pharmaceutical composition is topically administered to a site of a subject in need of such treatment to treat the disease or disorder, and the pharmaceutical composition comprises an effective amount of a VHH antibody that neutralizes IL-17 and a pharmaceutically acceptable carrier.

[0247] The terms "administer topically", "topical administration" or "dermal administration" mean that the VHH antibody is not administered systemically. These terms specifically include surface application on the skin surface. Topical administration also encompasses administering the composition by injection, particularly by intradermal injection and / or intradermal injection and / or subdermal injection and / or subcutaneous injection and / or microinjection.

[0248] According to some embodiments, the compositions of the present invention are suitable for administration by surface or injection routes (particularly intradermal and / or intradermal and / or microinjection, and / or by systemic injection, such as subcutaneous injection).

[0249] The method of the present invention includes topically administering a pharmaceutical composition to a defined area of the skin.

[0250] Ideally, the treatment period lasts for a sufficient time to improve the disease or disorder being treated. The treatment period can be at least 1 week, and in some embodiments, the treatment period can last about 4 weeks, 8 weeks or 12 weeks. In certain embodiments, the treatment period will be extended for several months (i.e., 3 - 12 months) or years. In one embodiment, the pharmaceutical composition is administered at least once a day during a treatment period of at least 4 weeks, 8 weeks or 12 weeks. In one embodiment, the pharmaceutical composition is administered twice a day during a treatment period of at least 4 weeks, 8 weeks or 12 weeks. Alternatively, the pharmaceutical composition is administered every other day, once every three days, once a week, for one week, one month, or until improvement is achieved.

[0251] Although the subject undergoing the pharmaceutical method can be of any suitable age, in some embodiments, the subject is a child, an adult or an elderly subject.

[0252] A pharmaceutical composition suitable for dermal administration can be used in combination with a mechanical device or a wave emission system (light, low frequency, infrared frequency, etc.) that activates a skin response. The mechanical device is, for example, a massage roller device having a mechanical and frictional action to promote the penetration of the active agent. Other modalities that can be used or combined with other administration routes include patches and microneedles.

[0253] The pharmaceutical composition of the present invention suitable for injection, particularly for intradermal and / or intradermal and / or subcutaneous injection and / or microinjection, can be injected through a device comprising a needle or microneedle or through a needleless injection device. Such devices are well known in mesotherapy. Alternatively, the pharmaceutical composition can be administered directly by iontophoresis to achieve greater penetration of the active agent.

[0254] The terms "treat / treating / treatment" are all intended to refer to an improvement or reversal of at least one measurable physical parameter associated with a treatable disease or disorder.

[0255] The term "treatment" or "treating" can be used interchangeably herein and refers to inhibiting, preventing or arresting the development of a disease or disorder and / or reducing, alleviating or eliminating the disease or disorder.

[0256] The phrase "inflammatory disease or disorder" as used herein refers to a disease, condition or disorder associated with inflammation. The term "inflammation" as used herein refers to the process by which a subject's immune system coordinates a response to tissue damage, infection, antigen attack, etc. Inflammation may be associated with an increase in tissue blood supply, an increase in capillary permeability in the tissue and / or an increase in the migration of white blood cells into the tissue.

[0257] According to some embodiments of the present invention, the inflammatory disease or disorder is an autoimmune disease or disorder. Autoimmune diseases or disorders can be, but are not limited to, psoriasis, arthritis, systemic lupus erythematosus (lupus, SLE), multiple sclerosis and inflammatory bowel disease.

[0258] Pharmaceutical composition

[0259] The VHH antibodies of the present invention can be formulated into any form of pharmaceutical composition according to the intended use. Optional forms of the pharmaceutical composition according to the present invention include liquid, semi-liquid, solid and semi-solid preparations.

[0260] According to some embodiments, the VHH antibody is formulated for topical administration, for example as a cream, paste, gel, hydrogel, ointment, lotion and emulsion.

[0261] According to some embodiments, the pharmaceutical composition further comprises one or more excipients, carriers or buffers. According to some embodiments, the pharmaceutical composition comprises excipients selected from the group consisting of emulsifying agents, pH buffers, preservatives, chelating agents, tonicity agents, antioxidants and gelling agents.

[0262] According to other embodiments, the pharmaceutical composition is in a form selected from the group consisting of solutions, emulsions, nanoemulsions, suspensions, lipid nanoparticles (such as liposomes), microparticles, ointments, creams, lotions, pastes, gels, hydrogels, sprays, powders, sticks and patches. Each possibility represents a separate embodiment of the invention.

[0263] The pharmaceutical composition provided by the present invention comprises at least one VHH antibody that neutralizes IL-17 as an active agent and a pharmaceutically acceptable carrier, diluent or excipient.

[0264] As used herein, the term "pharmaceutical composition" refers to a composition suitable for treating a disease or disorder caused by and / or associated with the overactivity of IL-17, particularly associated with the overactivity of IL-17A and / or IL-17F. In particular, the pharmaceutical composition can be used for preventing or treating inflammatory and / or immune-related disorders, such as inflammatory and / or immune-related skin disorders. Exemplary disorders are asthma, psoriasis (such as plaque psoriasis), arthritis (such as 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.

[0265] The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0266] As used herein, the term "pharmaceutically acceptable" means approved by a federal or state government regulatory agency for use in humans, or listed in the United States Pharmacopeia or other recognized pharmacopeias for use in humans. The components of the pharmaceutical composition of the present invention are all pharmaceutically acceptable agents.

[0267] The term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which a compound is administered. The carrier can be a liquid, preferably a sterile liquid, such as water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc., polyethylene glycol, glycerol, propylene glycol or other synthetic solvents.

[0268] If desired, the composition may also contain small amounts of an emulsifying agent, an anionic emulsifier, a synthetic polymer, a lipid matrix excipient, lecithin, or a pH buffer (such as acetate, citrate, or phosphate). Preservatives such as benzyl alcohol or methylparaben are also contemplated; chelating agents such as ethylenediaminetetraacetic acid; and agents for adjusting osmotic pressure such as sodium chloride or glucose.

[0269] Other optional excipients include, but are not limited to, humectants such as water-soluble liquid polyols such as glycerol, 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 its derivatives; wetting agents; suspending agents; gelling agents; skin emollients and skin moisturizers; antibacterial agents (e.g., antibacterials); antifungal agents; analgesics; ultraviolet absorbers; wound healing promoters; growth factors; reactive oxygen species; anti-inflammatory agents; vitamins such as vitamin C, vitamin B and its derivatives; nutrients such as thiamine, riboflavin, niacin, pantothenate, pyridoxine, folic acid, cobalamin, biotin, choline, inositol, carnitine, etc.; 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, fructose, 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 vera, etc.; retinoids; flavonoids; and mixtures thereof.

[0270] The composition may be in the form of a solution, an emulsion (e.g., oil-in-water, water-in-oil-in-water, water-in-oil, or oil-in-water-in-oil), a nanoemulsion, a suspension, microparticles, an oil, an ointment, a cream, a lotion, a paste, a gel, a hydrogel, a spray, a powder, a stick, or a combination thereof.

[0271] The pharmaceutical composition of the present invention can be formulated for sustained release, slow release, or delayed release using methods and ingredients known in the art. Thus, depot formulations of the VHH antibodies of the present invention are included within its scope.

[0272] In some embodiments, a tape or other support structure may be applied to the skin. In some embodiments, the composition is applied to the skin before placing the tape. In this case, the tape may be porous or non-porous.

[0273] In some embodiments, the tape is a polymeric matrix or gel that allows the composition to contact the skin when the composition is applied to the tape. Such tapes, also known as patches or transdermal patches, have the additional advantage of delivering compounds to the body in a controlled manner. Transdermal patches can be made by dissolving or dispersing the compound in a suitable medium and then applying it to the tape. Absorption promoters can also be used to increase the flux of the compound through the skin. The rate of such flux can be controlled by the polymeric matrix or gel and optionally by a rate-controlling membrane. In one embodiment, the polymeric matrix can be hyaluronic acid, which has the property of trapping water and forming a gel.

[0274] For topical applications, the composition can be formulated in the form of an ointment containing the VHH antibody dissolved or suspended in a suitable carrier. Such carriers include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyethylene oxide, polypropylene oxide, emulsifying wax, and water. Alternatively, it can be formulated as a cream or lotion containing the VHH antibody dissolved or suspended in a suitable carrier. Such carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water.

[0275] For injection, the pharmaceutical composition can be formulated as a liquid, such as a solution, emulsion, suspension; or a gel. 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 commonly used as solvents. For this purpose, any oil can be used, such as glycerol monoesters and glycerol diesters. Fatty acids such as oleic acid and their glyceride derivatives are also used to prepare injectable compositions, and pharmaceutically acceptable natural oils such as olive oil, castor oil, especially their polyoxyethylated forms are also used to prepare injectable compositions. These oily solutions may contain suspending agents or diluents (such as carboxymethyl cellulose) to formulate emulsions and suspensions. Surfactants such as Tweens and emulsifiers can also be included. Aqueous or oily suspensions can be formulated by using wetting agents or dispersing agents and suspending agents by methods well known to those skilled in the art.

[0276] As used herein, "microparticles" means entities made of polymers or combinations of polymers in various sizes. Microparticles can be of any shape, although they are generally substantially spherical, in which case the microparticles are called "microspheres" or "microbeads". An active agent can be incorporated into the microparticles by mixing the dried microparticles with a solution of the active agent in an aqueous or water-organic solution. The microspheres are sterilized before injection or incorporation into an injectable composition.

[0277] To prepare the pharmaceutical composition of the present invention, a variety of techniques can be employed. For example, the active agent can generally be incorporated into an acceptable carrier in a manner commonly used for preparing pharmaceutical products. Thus, the active agent can first be dissolved or dispersed in a portion of water or other solvent or liquid for incorporation into the acceptable carrier. Preferred compositions for such production methods are oil-in-water, water-in-oil, or water-in-oil-in-water emulsions.

[0278] In some embodiments, the active agent, with or without excipients, remains separate from the carrier, for example, in the form of a dry powder. The subject mixes the required amount of the active agent with the required amount of the carrier immediately before applying the pharmaceutical composition, ensuring that the active agent retains its maximum efficacy and also allowing the potency of the composition to be adjusted according to the individual needs of the subject. The resulting pharmaceutical composition is then applied to the skin.

[0279] According to a further embodiment, 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.

[0280] Depending on the stage and severity of the condition, the pharmaceutical composition can be administered one or several times during the course of the condition. For example, it can be administered once or several times a day, once every two days, twice a week, once a week, once a month or several months, for an appropriate period of time.

[0281] 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 intradermal injection, intradermal injection, subcutaneous injection, microinjection, or any combination thereof. According to certain embodiments, the pharmaceutical composition in the form of a solution, emulsion, suspension, or microparticles is administered by injection.

[0282] In a further embodiment, the pharmaceutical composition is administered locally, for example, topically, orally, nasally, or intralungally, for example, by inhalation as an aerosol.

[0283] The VHH antibody can be administered alone, or in combination with another active pharmaceutical or therapeutic agent, particularly with another agent useful for preventing and / or treating a condition caused by and / or associated with the overactivity of IL-17, particularly associated with the overactivity of IL-17A and / or IL-17F.

[0284] Pharmaceutical compositions useful in the context of the present invention include compositions comprising an active ingredient in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of the active ingredient effective to prevent, alleviate or ameliorate the symptoms of a disease or disorder in a subject being treated.

[0285] The term "effective amount" refers to an amount of an active agent sufficient to provide a beneficial effect to a subject to which the composition is administered.

[0286] Determination of a therapeutically effective amount is entirely within the capabilities of those skilled in the art, particularly in view of the detailed disclosure provided herein.

[0287] For any preparation used in the methods of the present invention, a therapeutically effective amount or dose can initially be estimated from in vitro and cell culture assays. For example, a dose can be formulated in an animal model to achieve the desired concentration or titer. This information can be used to more accurately determine useful doses in humans.

[0288] The toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or in experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used to formulate a range of doses for use in humans. The dose may vary according to the dosage form employed and the route of administration used. The exact formulation, route of administration and dose can be chosen by each physician in view of the patient's condition. (See, for example, Fingl et al., 1975, "The Pharmacological Basis of Therapeutics", Chapter 1, p. 1).

[0289] The dose and interval can be adjusted individually to a level of the active ingredient sufficient to achieve the minimum effective concentration (MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. The dose required to reach the MEC will depend on the individual characteristics and the route of administration. Assay determinations can be used to measure plasma or tissue concentrations.

[0290] Depending on the severity and responsiveness of the condition to be treated, administration can be by single or multiple doses, and the treatment course can extend from several days to several weeks or until alleviation of the disease state is achieved.

[0291] Of course, the amount of the composition to be administered will depend on the subject to be treated, the severity of the affliction, the mode of administration, the judgment of the prescribing physician, etc.

[0292] Thus, if desired, the compositions and / or articles of some embodiments of the invention can be presented in a packaging or dispenser device, such as an FDA-approved kit, which can contain one or more unit dosage forms containing the active ingredient. For example, the packaging can include a metal or plastic foil, such as a blister pack. The packaging or dispenser can be accompanied by instructions for administration. The packaging or dispenser device can also be accompanied by a notice associated with the container, in a form prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceuticals, which notice reflects approval by the agency of the form of the composition or for human or veterinary administration. For example, such notice can be a prescription drug label approved by the U.S. Food and Drug Administration or an approved product insert. Compositions containing the preparations of the invention formulated in a compatible pharmaceutical carrier can also be prepared, placed in an appropriate container (e.g., a lyophilized vial), and labeled for treatment of the designated condition, as further detailed above.

[0293] In some embodiments, the VHH antibodies and pharmaceutical compositions containing them are administered to a subject, which subject has psoriasis, particularly plaque psoriasis, more particularly mild to moderate plaque psoriasis, as a single therapeutic agent or treatment regimen, which treatment regimen includes at least one additional agent or therapeutic agent for psoriasis or at least one symptom associated with the disease. According to a particular embodiment, the additional therapeutic agent includes a steroid.

[0294] In certain embodiments, the other active agent is an active agent useful for the prevention and / or treatment of the exemplary conditions listed above.

[0295] The kit provided by the invention contains the composition of the invention suitable for injection and a syringe or injection device.

[0296] Diagnostic applications

[0297] Diagnostic applications include in vitro methods in which the VHH antibodies are used to detect IL-17 in a sample, such as a body fluid such as saliva, blood, serum, or plasma, a fecal sample, or a tissue or biopsy sample. Diagnostic applications also include in vivo methods in which the VHH antibodies are used to detect IL-17 in a subject, particularly IL-17 in a human patient. For diagnostic applications, the VHH antibodies can be labeled for direct detection or used in conjunction with a secondary detection reagent (e.g., an antibody, including a conventional antibody or a VHH antibody) for indirect detection according to techniques established in the art.

[0298] Evaluating the activity and safety of VHH antibodies for treatment

[0299] The VHH antibodies and VHH antibody sets of the invention can be tested for their activity in IL-17-related disease models in vitro, in vivo, and ex vivo.

[0300] Sarama et al. reviewed in vitro disease models of psoriasis and atopic dermatitis in 2022.

[0301] Some animal models use animal or human skin. Since the homology between the amino acid sequences of human and porcine IL-17 is relatively high (73%), VHH antibodies can be evaluated in porcine skin models. Human skin models using cultures of primary human epidermal keratinocytes isolated from neonatal foreskins, such as psoriasis human skin models, are also used to evaluate the potential therapeutic effects of VHH antibodies. The effects of the VHH antibodies of the present invention on factors regulating psoriasis keratinocytes can also be tested (Zhou, X. et al., 2022).

[0302] As a non-limiting example, in vivo studies conducted in SCID / Beige mice transplanted with human xenografts (Keren et al., 2018) can be used to test several toxicological parameters. The transplanted mice are considered suitable in vivo models because normal human skin is implanted into SCID mice and the disease is induced by injecting PBMCs activated with IL-2 from human psoriasis patients. The VHH antibodies of the present invention block the cascade downstream of IL-17, as determined by improving marker levels (such as S100A7, Ki-67, β-defensin-2) and skin appearance, which is judged by H&E staining or qRT-PCR.

[0303] In this model, healthy human skin pieces are transplanted into SCID mice on day 0. Peripheral blood mononuclear cells (PBMCs) are isolated from the blood of psoriasis patients and cultured for 14 days in the presence of high-dose IL-2 to activate allogeneic T cells expressing high levels of NK cell receptors. On day 28 after transplantation, these PBMCs are injected into the mice, generating psoriasis-like lesions that develop within the grafts and almost display all the key features of human psoriasis. These features include epidermal thickening, appearance of immune cells, edema, vasodilation, and increased keratinocyte proliferation. Importantly, these psoriasis-like lesions respond to commonly used anti-psoriasis treatments. This highlights the important relevance of this preclinical model for psoriasis research (Keren et al., 2018). Treatment with VHH antibodies or a control is performed on day 42, while skin collection and analysis are performed on day 56.

[0304] Preclinical safety studies include, for example, (i) human tissue cross-reactivity (TCR) studies, (ii) rat pharmacokinetics / biodistribution (PK / BD) studies, and (iii) GLP toxicology studies in minipigs.

[0305] GLP toxicology studies can be used to confirm the acceptable safety profile of the VHH antibodies for the confirmatory testing. The currently accepted toxicology animal model for anti - psoriasis mAbs is conducted in non - human primates (NHPs) as described, for example, by Kolbinger et al., 2022. However, the minipig model for the VHH antibodies of the present invention is more suitable for the following reasons: 1. The VHH antibodies are administered superficially or intradermally (ID), rather than systemically like mAbs. Therefore, the skin vasculature formation, structure, and neurological structure of minipigs, which are similar to those of humans, are more relevant. 2. The VHH antibodies are cleared from the blood via the kidneys and liver within one hour, and it is expected that they will not significantly spread throughout the body for an extended period (Esparza et al., 2021). After intradermal (ID) administration of biotinylated VHH antibodies to mice and histopathological sampling, it can be detected immediately in the epidermis, dermis, and blood after administration. 3. The VHH antibodies lack Fc receptors and thus are not immunogenic. 4. Regulatory requirements to reduce the use of NHPs in pre - clinical studies (Prior et al., 2017).

[0306] In vitro tissue cross - reactivity (TCR) studies can be used to differentiate non - specific and specific binding of VHH antibodies in different types of human tissues. This information is crucial for ensuring that the experimental antibody does not bind to epitopes other than the target site, thereby minimizing the risk of treatment - related toxicity.

[0307] An immunohistochemistry - based tissue cross - reactivity (TCR) screening assay was developed to differentiate non - specific and specific binding of the VHH antibodies of the present invention. This study was considered important for demonstrating that the test portion does not bind to epitopes other than the target site. The establishment of the assay included human tissues from 3 different donors.

[0308] Since the primary objective is safety assessment, the intensity of immunohistochemistry (IHC) staining can provide valuable insights into potential toxicity. The IHC staining was studied using the biotinylated (and / or FITC) conjugated form of the VHH antibody on positive and negative control systems (target protein or BSA spotted slides). The VHH antibody was tested on human tissues (3 donors) at 2 concentrations and the isotype control was tested at 1 concentration according to the EMA / FDA tissue list. After the experimental phase, pathological evaluation was performed. Von Willebrand factor was used and the tissue integrity was evaluated by two independent histopathologists.

[0309] To test the histopathology (in standard organs), minipigs can be sub - chronically administered for 28 days at 3 escalating doses.

[0310] To evaluate safety in humans, it is possible to conduct Phase I / IIa, randomized, double-blind, placebo-controlled trials. The tolerability, immunogenicity, pharmacokinetics, pharmacodynamics, and efficacy of multiple escalating doses of the anti-IL-17A / F VHH antibody of the present invention were evaluated. For example, the trials can be conducted in male and female subjects with mild to moderate psoriasis using intradermal and / or topical administration modalities.

[0311] As used herein, the phrase "subject in need" refers to a mammalian male or female subject (e.g., a male or female subject of the human species) diagnosed with an inflammatory disease or disorder. In a specific embodiment, the term encompasses individuals at risk of developing an inflammatory disease or disorder. The subject can be of any gender or any age, including neonates, infants, children, adolescents, adults, and the elderly.

[0312] As used herein, the term "about" means ~10%.

[0313] The terms "comprising," "containing," "including," "including having," "having," and variations thereof mean "including, but not limited to."

[0314] The term "consisting of" means "including and limited to."

[0315] The term "consisting essentially of" means that a composition, method, or structure can include additional ingredients, steps, and / or parts, provided that the additional ingredients, steps, and / or parts do not materially alter the basic and novel features of the claimed composition, method, or structure.

[0316] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" can include multiple compounds, including mixtures thereof.

[0317] Throughout the application, various embodiments of the present invention may be presented in a range format. It should be understood that the description of the range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention. Thus, the description of a range should be considered to have expressly disclosed all possible sub-ranges as well as the individual numerical values within that range. For example, a description of a range such as 1 to 6 should be considered to have expressly disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as the individual numbers within that range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0318] Whenever a numerical range is specified herein, it is meant to include any recited numerical value (fractional or integral) within the specified range. The phrases “range between” the first specified number and the second specified number and “range from” the first specified number “to” the second specified number are used interchangeably herein and are meant to include the first and second specified numbers and all the fractional and integral values therebetween.

[0319] As used herein, the term “method” refers to a way, means, technique and procedure for accomplishing a given task, including but not limited to those ways, means, techniques and procedures known to or developed from known ways, means, techniques and procedures by practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine.

[0320] It is understood that certain features of the invention, which 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 described in the context of a single embodiment for brevity may also be provided separately, or in any suitable sub-combination, or in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments unless the embodiment cannot be practiced without those elements.

[0321] The term “about” means a value that is 10% higher or lower than the specified value.

[0322] It should be noted that each possibility disclosed throughout the specification represents a separate embodiment of the invention.

[0323] A non-limiting list of the VHH antibodies of the invention and their affinity and utility for IL-17 neutralization are shown in Table 3 below.

[0324] Table 3. Anti-IL-17A / F VHHs, their affinity (K D values expressed in pM), neutralizing potency and thermal stability.

[0325]

[0326] * Monovalent affinity as described in Example 3 and shown in Figures 4 - 8 .

[0327] ** These VHH antibodies of Ablynx are disclosed in WO2012156219.

[0328] *** Two dissociation constants were calculated for the VHHs that fit the BLI curves using the “2:1 heterogenous model”. Only the high affinity component is reported in the table.

[0329] # Neutralizing potency is expressed as a rank order, from 1 = best to 4.

[0330] At 95 °C, none of the VHH antibodies showed aggregation, indicating their stability.

[0331] Comparison of the VHH antibodies of the present invention with known VHHs (such as those disclosed in WO2012156219) using BLI indicated that the VHH antibodies of the present invention have significantly higher affinity for binding to IL-17F.

[0332] The challenge of this project was to obtain VHH antibodies that block IL-17 and bind tightly to both IL-17A and IL-17F. This was by no means an easy task because there are quite a number of amino acid exchanges in the neutralizing epitopes between IL-17A and IL-17F. Notably, binders with picomolar affinity for both IL-17A and IL-17F were identified, for example, the VHH antibody named Bm43B02 and its class A members.

[0333] The binding of some VHH antibodies of the present invention compared to known VHH antibodies is described in Table 3.

[0334] The affinity of the VHH antibodies for IL-17 subtypes 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 the binding of homologous or heterologous dimeric IL17 species to them is measured. Since the VHH antibodies are immobilized at a non-low density, two adjacent VHH molecules can simultaneously bind to an IL-17 dimer. This results in an avidity effect, similar to the binding of bivalent IgG to an IL-17 dimer. In another setup, the IL-17 subtypes are immobilized on the chip, and the binding of monomeric VHH antibodies to them is measured, showing a more accurate KD because the avidity effect due to the dimeric nature of IL-17 is avoided. According to specific embodiments, the binding affinity of monomeric VHH to immobilized human IL-17A or IL-17F homologous dimers is determined and expressed as the dissociation constant KD.

[0335] Crystal structures of the VHH antibody-IL17 complexes (class A and class B VHH antibodies are Figure 16 and Figure 18 respectively) provide a profound understanding of the mode of action of the VHH antibodies. Structures and structure predictions were used to define the actual complementarity determining regions (CDRs) (at near-atomic resolution) and to classify the VHH antibodies into four classes.

[0336] Before explaining in detail at least one embodiment of the present invention, it is to be understood that the applications of the present invention are not necessarily limited to the details set forth in the following description or illustrated by the examples. The present invention is capable of other embodiments or of being practiced or carried out in various ways. Further, it is to be understood that the phrases and terms used herein are for the purpose of description and should not be regarded as limiting.

[0337] As described above and as claimed in the following claims, various embodiments and aspects of the present invention find experimental support in the following examples.

[0338] Examples

[0339] Example 1. Generation of VHH antibodies

[0340] Generating anti-IL-17 VHH antibodies that display high-affinity cross-reactivity between several different IL-17 subtypes is a highly challenging task - given that the sequence identity between IL-17 paralogs is limited to ~50% identity between human IL-17A and IL-17F, and most of the conserved residues are actually buried deep within the hydrophobic core of the disulfide-bridged IL-17 dimer. Thus, the required VHH antibodies may have to tolerate sequence exchanges in their epitopes without loss of binding strength. IL-17 receptors can more readily tolerate this sequence variability because they have an interaction interface that is much larger than the possible epitopes of VHH antibodies.

[0341] Therefore, the inventors considered these limitations in their VHH generation strategy. They recombinantly produced human IL-17A homodimers, IL-17F homodimers, and IL-17A / F heterodimers and injected all three into alpacas multiple times as immunogens. This strategy was chosen to promote an immune response that also includes antibodies that cross-react between IL-17 paralogs.

[0342] After the final immunization, blood samples were collected, lymphocytes were isolated, mRNA was purified and reverse transcribed. The VHH coding regions were amplified by nested PCR, reverse transcribed, and cloned into an M13 phagemid in cDNA form, generating a library with >100 million independent clones.

[0343] In the next steps, several rounds of phage display were performed. The selected VHH antibodies were classified based on sequence similarity, cloned into an Escherichia coli expression vector, produced by periplasmic expression, purified, and finally characterized for IL-7 binding using biolayer interferometry (BLI; Abdiche et al., 2008).

[0344] In one set of selection methods, IL-17A homodimers and IL-17A / F heterodimers were successively used as baits. This generated substantial sequence diversity among the selected VHH antibodies - approximately 8 major classes and many minor classes, as judged by sequence similarity. Most of the antibodies tested bound IL-17A with high affinity. "Clustering" identified three complementary epitopes (epitopes 1 - 3), i.e., VHH antibodies that bind epitope 1 can bind to epitope 2 conjugates or epitope 3 conjugates simultaneously. Similarly, epitope 2 and 3 conjugates can also bind simultaneously. However, it was subsequently found that epitope 2 and 3 conjugates do not block IL-17 function.

[0345] In a second set of selections, IL-17A homodimers, IL-17F homodimers, and IL-17A / F heterodimers were successively used as baits. This direct selection for cross-reactivity substantially reduced sequence complexity. All epitope 2 and epitope 3 conjugates were eliminated. Two VHH classes became dominant and now account for 98% of all sequences. These two classes, designated as the Re42H11 and Bm17B02 classes (classes A and B as defined in Tables 1 and 2), are disclosed below and include Re42H11, Re42B03, Re42B04, Bm18B05, Bm18F11 (class A) and Bm17B02 and Re42F08 (class B).

[0346] To generate "second-generation" VHH antibodies, the antibodies were also further affinity matured. Seven months after the last immunization, alpacas were re-immunized with IL-17A and IL-17F proteins. Immunization libraries were prepared and phage display was performed, with alternating IL-17 versions as baits, i.e., cross-selection with IL-17A and IL-17F, at bait concentrations as low as 100 pM and including a dissociation rate selection step. This strategy generated additional VHH antibodies (Tables 1, 2, and 3), including new VHH classes (e.g., Bm42A03 of class C and Bm45G07 of class D). However, most of the newly discovered VHH antibodies are variants of the previously isolated Re42H11 class (class A) and Bm17B02 class (class B) VHH antibodies.

[0347] Example 2. Affinity measurements by VHH immobilization

[0348] Initially, five members of the Re42H11 class (Re42H11 itself, Re42B03, Re42B04, Bm18B05, and Bm18F11) and two members of the Bm17B02 class (Bm17B02 and Re42F08) were deeply characterized. For affinity measurements, antibodies were produced in the form of C-terminal Avi-biotin tags and enzymatically biotinylated by recombinant BirA (Beckett et al., 1999). Subsequently, 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, they were immobilized at a concentration of 0.7 μg / ml on a high-precision streptavidin biosensor on an Octet RED96e instrument (ForteBio / Sartorius) for 110 seconds. Then, 10, 20, and 40 nM of the designated IL-17 species were allowed to bind for 800 seconds, followed by dissociation for 800 seconds. The binding and dissociation were recorded as wavelength shifts (in nm). The baseline was recorded by parallel measurement of the "no VHH control". The binding rate, dissociation rate, and dissociation constant (K D ) were calculated by fitting the data using the mass transport model with Octet Data Analysis HT 12.0 software.

[0349] These measurements demonstrated perfect cross-reactivity with IL-17A, IL-17F, and IL17-A / F heterodimers ( Figure 2 and Figure 3 ), high binding rates (mainly around 10 5 -10 6 M -1 ·s -1 ), undetectable or almost undetectable dissociation rates, and an apparent K D of 20 pM or better. It should be noted that the BLI setup cannot distinguish affinities below 10 pM. It should also be noted that this setup using immobilized VHH includes avidity effects, so adjacent VHH molecules on the sensor chip can bind to the same IL-17 dimer, which contributes to the observed very low dissociation rates. This mimics the situation of dimer IgG binding to the IL-17 dimer.

[0350] Example 3. Affinity measurement by IL-17 immobilization

[0351] To measure the true monovalent affinity, the present inventors also performed BLI by immobilizing the IL-17 species and using monovalent VHH as the analyte. The results are shown in Figures 4 - 8as shown in FIGS. 11 and Table 3. In the first set of experiments, VHH662 and VHH664 from Ablynx / Sanofi were tested as control antibodies. They showed high affinity for IL-17A, with K D being 115 pM and 150 pM (for VHH662 and VHH664, respectively), but poor binding to IL-17F, with K D being approximately 15 nM ( Figure 4 ).

[0352] When probing the IL-17 binding of representatives of class A and class B VHH antibodies (i.e., Re42B04a and Re42F08) under the same conditions, they showed better cross-reactivity. One example, Re42B04, had K D values of 50 pM and 500 pM for IL-17A and IL-17F, respectively ( Figure 6 ). Several second-generation anti-IL-17 VHHs showed significant cross-reactivity between IL-17A and IL-17F, with affinities for both species as high as 100 pM ( Figure 6 , Figure 7 and Figure 8 ; Table 3).

[0353] Example 4. Testing the stability of VHH antibodies

[0354] For the intended therapeutic applications, IL-17 VHH antibodies should not only bind their targets with high affinity, but they should also be developable as biopharmaceuticals. This includes that they are stable enough to survive the long large-scale production process as well as transportation and storage (ideally for several years in formulation) without aggregating or losing activity.

[0355] A good predictor of stability is thermal stability, which can be measured, for example, by thermal shift assays or particularly by differential scanning fluorimetry (Goldberg et al., 2011). This method exploits the unfolding (thermal denaturation) of the protein to expose aromatic / hydrophobic residues (from its hydrophobic core), which then bind and enhance the fluorescence of the added SYPRO Orange dye.

[0356] Differential scanning fluorimetry (DSF) was performed on the VHH antibodies. The assay was carried out in a volume of 20 μl, with a VHH concentration of 1 mg / ml, in 50 mM Tris / HCl, 150 mM NaCl (pH 8.0 at 20 °C) and 1× SYBR Orange dye (diluted from a 5000× stock solution; Life Technologies). Using a transparent The 'B' seal (Bio-Rad) seals the plate, briefly centrifuged to remove any air bubbles, and then placed on a CFX96 Real-Time System (C1000 Thermal Cycler, BioRad). The samples were incubated at 20 °C for 5 minutes. Then the temperature was increased by 1 °C every 45 seconds until 95 °C was reached. Fluorescence was measured at the end of each step using 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 was still below the initial fluorescence at 20 °C, it was interpreted as no melting occurred.

[0357] Figure 9 Such an analysis of the disclosed VHH antibodies is shown, where heating was slowly increased from 20 °C to 95 °C. Most VHHs (Re42H11 itself, Re42B03, Re42B04, Re42B05, Bm18F11, and Re42F08) showed only negligible unfolding signals and thus no melting (small fluorescence peaks did not or hardly 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 thermal stability. As a control, the inventors repeated the experiment on some candidate drugs in the presence of DTT (dithiothreitol) that reduces structural disulfide bonds, and at this time, they observed large unfolding peaks, indicating overall melting of the corresponding VHH antibody ( Figure 10 ). It should be noted that in the presence of DTT, the unfolding amplitude is much higher. These controls show what kind of fluorescence signal should be expected if the VHH antibody completely unfolds.

[0358] One member of the Re42H11 class (Re42B04) showed a small melting peak above the 20 °C signal and had an inflection point at 57 °C ( Figure 9 ); however, even this VHH did not aggregate when heated to 95 °C. Thus, the anti-IL-17 VHH antibodies disclosed herein meet three key criteria for the intended application: extremely high affinity for its target, complete cross-reactivity between IL17A, IL17A / F, and IL17F, and extremely high thermal stability.

[0359] DSF was also performed on the second-generation VHH antibodies, as in Example 2 and Figure 4All members of Bm17B02 (class B) (Bm44B04, Bm44G07, and Bm42A09), as well as Bm45G07 and Bm42B11, are resistant to denaturation even at 95 °C and thus they have complete thermal stability (Table 3). On the other hand, the denaturation of other VHH antibodies (Bm43B02, Bm44B11, Bm44C09, Bm42A03, and Bm44G10) occurs between 50 - 60 °C. These VHHs also do not aggregate when heated to 95 °C.

[0360] Example 5. Testing VHH Stability by BLI

[0361] BLI is very sensitive to sample concentration and is thus suitable for detecting any loss of active substance in solution, including the loss of active nanobodies due to instability. To determine whether the VHHs that had denatured at 60 °C in the DSF experiment retained their binding activity after heat treatment, the antibodies were incubated at 95 °C for 10 minutes (at a concentration of 1 μM), cooled, and then centrifuged to remove any possible aggregates formed. When testing IL-17A binding by BLI, there was no difference between the heated and untreated samples of the Re42H11 and Bm42A03 class members (class A and C), and they behaved similarly to the thermostable class A VHHs, namely Re42B04a and Bm42B11 (Figure 11). Thus, these nanobodies either only partially denatured or robustly refolded into their native state after heat treatment.

[0362] It should be noted that there was no difference between the super-thermostable VHHs (Re42B04a and Bm42B11) and the VHHs with a denaturation temperature of 50 - 60 °C (Bm43B02, Bm44B11, Bm44C09, Bm42A03, and Bm44G10) after heat treatment, indicating that any denaturation (unfolding) is reversible.

[0363] Example 6. Cell-Based IL17 Stimulation Assay and Measurement of Reporter Gene Activity

[0364] To monitor the biological activity of IL17 variants in a cell-based system, HEK-Blue TM IL-17 reporter gene cell line (Invivogen hkb-il17) was used. These cells are HEK293 cells stably expressing the human IL-17RA and IL-17RC receptors as well as the adaptor Act1 / TRAF3IP2. HEK-Blue TMIL-17 cells also encode a secreted embryonic alkaline phosphatase (SEAP) reporter gene, which is controlled by a promoter with NF-kB and AP-1 binding sites. When the IL-17 ligand binds to its receptor on the cell surface, NF-kB and AP-1 are activated, producing SEAP and secreting it into the cell supernatant. The amount of SEAP can be measured colorimetrically using QUANTI-Blue TM as a substrate.

[0365] 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 TM . Selection antibiotics (HEK-Blue TM Selection) were introduced after the second passage, and the growth medium was updated at least twice a week. Cells were passaged at 70 - 80% confluence by scraping in PBS and cultured for no more than 20 passages.

[0366] To evaluate the cellular response, titration curves were performed with different IL-17 variants. For this, HEK-Blue TM IL-17 cells were seeded in medium without selection antibiotics in 96-well plates and stimulated with serial three-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 were used as negative controls. After overnight incubation, 20 μl of cell supernatant from each well was mixed with 180 μl of QUANTI-Blue TM solution in a separate 96-well plate and incubated at 37 °C for 30 minutes. The SEAP level in the supernatant was determined by measuring the absorbance at 620 nm (OD620). The OD 620 of the control with only medium was used as the background and subtracted from all sample wells. Each case was assayed in triplicate. Induction was calculated from the ratio OD 620 (sample) / OD 620 (untreated). Based on the titration curves, the cells responded more strongly to IL-17A( Figure 12 ). Therefore, a comparable induction range was achieved using higher concentrations of IL-17F and IL-17A / F in the neutralization assay described in Example 7.

[0367] Example 7. Cell-based IL17 Neutralization Assay

[0368] To evaluate whether the disclosed anti-IL-17 VHH antibodies block the binding of IL-17 variants to their receptors, the inventors performed a cell-based neutralization assay using HEK-Blue TM IL-17 expressing cells. These cells express the human IL-17RA and IL-17RC receptors as well as the adaptor Act1. They secrete a phosphatase reporter gene in response to receptor binding of IL-17 variants. By quantifying the amount of secreted phosphatase, activation of the IL17 receptor can be measured as described in Example 6( Figure 12 ).

[0369] To test for neutralization, recombinant human IL-17A (5 ng / mL, equivalent to 0.17 nM, Proteintech HZ-1113), IL-17F (25 ng / mL, equivalent to 0.85 nM, Proteintech HZ-1116) or IL-17A / F (25 ng / mL, equivalent to 0.85 nM, R&D Systems 5837-IL) were pre-incubated with serial three-fold dilutions of the VHH antibody for 2 hours at 37 °C with continuous shaking and then added to the reporter cells. Cell-free medium, untreated cells and cells treated with the IL-17 variant in the absence of any VHH were used as controls. The stimulated cells were incubated overnight at 37 °C, 5% CO2 and then the reporter gene activity was measured as described in Example 6. The OD620 of the medium was used only as background and was subtracted from all sample wells. Each case was assayed in triplicate. The normalized induction was calculated from the ratio OD620 (sample) / OD620 (IL17 only).

[0370] Since the first generation, the following VHH antibodies have been tested: members of class A, Bm18B05, Bm18F11, Re42B03, Re42B04, and Re42H11 (IL-17A / IL-17F cross-reactive); members of class B (Bm17B02), Bm17B02 and Re42F08 (IL-17A / IL-17F cross-reactive); Bm17D12 (preferably IL-17A); and members of class Bm17B11, Bm17B11 and Bm18F01 (targeting non-neutralizing epitope 3). The data show that pre-incubation of IL-17A or IL-17F with any class A or B member blocks receptor activation. Notably, IL-17A neutralization was observed at sub-nanomolar concentrations, e.g., Re42B04, Re42B03, Bm17D12, and Re42F08. Similarly, when the VHH Bm18F11, Re42B03, Re42B04, Re42H11, Bm17B02, Re42F08, or Bm17D12 exceeds the interleukin concentration, the IL-17A / F heterodimer is effectively neutralized. Re42H11, Re42B03, and Re42B04 are particularly efficient at blocking the IL-17F homodimer, with Re42H11 and Re42B03 showing substantially 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 and bind at non-neutralizing epitope 3 ( Figure 13 ).

[0371] The second-generation VHHs also blocked receptor activation when pre-incubated with IL-17A, IL-17F, or IL-17A / F (Figure 14). The newly discovered VHH classes (Bm42A03 and Bm45G07) particularly showed excellent cross-neutralization, already reaching picomolar antibody concentrations (Figure 14). In these assays, Re42B04a, Bm42A09, Bm42A03, and Bm45G07, as representative members of classes A-D VHHs, were superior to VHH662 and VHH664, especially for the neutralization of IL-17F ( Figure 15 ). It should be noted that members of all four VHH classes have excellent neutralization at low nanomolar or even sub-nanomolar concentrations. For clarity, each figure lists the VHHs in order of increasing neutralization potency.

[0372] Figures 13 - 15 The results shown indicate that the VHH antibodies completely neutralize IL-17A, IL-17F, and IL-17AF at concentrations of <1 nM, <10 nM, and <1 nM, respectively.

[0373] Example 8. Structural Characterization of the IL-17·VHH Complex

[0374] To understand the structural basis of IL-17 inhibition, the IL-17·VHH complex was crystallized. The X-ray crystal structures of the tetrameric IL-17F complexes of VHH antibodies representing two major VHH classes (Re42H11 and Bm17B02) were solved ( Figure 16 and 18 ). It should be noted that the IL-17RC receptor competes with VHH, which explains why the class A anti-IL17 VHH antibodies disclosed herein 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) shows that Re42H11 and Bm17B02 block receptor binding by directly competing for the IL-17 binding site.

[0375] To reveal the molecular details of the VHH-IL-17 interaction, homology models of complexes of all members of the Re42H11 class (class A) and Bm17B02 class (class B) with IL-17A and IL-17F were created. Analysis of these models enabled us to decipher the contribution of individual amino acids to interleukin binding and their impact on IL-17A / IL-17F cross-reactivity. Figure 17 and 19 Lists all VHH residues identified in the crystal or modeled structures that interact with the IL-17 species IL17A and IL-17F. 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.

[0376] The following positions in the sequence of class A VHH antibodies were found to interact with IL-17A: position 1, which is Q; position 29, which is A, G, V, F or P; position 30, which is S; position 31, which is S or G; position 32, which is Y; position 33, which is A; position 50, which is A; position 51, which is I; position 52, which is S; position 54, which is I, S or V; position 55, which is S or G; position 57, which is G, S or D; position 58, which is T, S or A; position 59, which is K, R or V; position 100, which is P; position 101, which is Y; position 103, which is L or M; position 104, which is D or E; position 106, which is R; position 109, which is E or D.

[0377] The following positions in the sequence of the Class A VHH antibody interact with IL-17F: position 29, which is A, G, V, F or P; position 30, which is S; position 31, which is S or G; position 32, which is Y; position 33, which is A; position 50, which is A; position 51, which is I; position 52, which is S; position 54, which is S, I or V; position 55, which is S or G; position 57, which is G, S or D; position 58, which is T, S or A; position 59, which is K, R or V; position 100, which is P; position 101, which is Y; position 103, which is L or M; position 104, which is D or E; position 106, which is R; position 109, which is E or D.

[0378] The following positions in the sequence of the Class B VHH antibody 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; position 106, which is M.

[0379] The following positions in the sequence of the Class B VHH antibody 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; position 106, which is M.

[0380] The differences in interactions are due to the different residues in IL-17A and IL-17F. This information is used to identify the key residues of the VHH. When classifying nanobodies, we can examine the conservation of the interacting residues rather than their complete sequences. For example, for the Bm17B02 class, although the new nanobodies have different CDR1 and CDR2 sequences, so one might argue that they are different classes, by examining the interacting residues, it can be seen that they are almost the same.

[0381] Example 9. Production of VHH Antibodies in Pichia pastoris

[0382] The VHH antibodies Re42B04a and Re42F08 were recombinantly produced in Pichia pastoris. Figure 20A and20B The upstream and downstream processes are described separately.

[0383] As Figure 20A described in the protocol of , the upstream stage is initiated by culturing an inoculum from a cell bank, followed by fermentation, induction of expression by addition of methanol, and collection. Figure 20B The downstream stage shown in includes adjusting the pH when necessary, purifying the filtered collection by passing it through several columns, and then formulating it with 20 mM phosphate buffer.

[0384] Example 10. Ex Vivo Human Skin Model

[0385] is a psoriasiform model developed by Genoskin to reproduce the key features of pro-Th17 / Th1 inflammation associated with psoriasis. This model, as Figure 21 shown in the schematic diagram of , relies on the in situ activation of resident T-cells in normal skin biopsies and their further polarization into the Th17 / Th1 phenotype in the presence of a medium supplemented with a mixture of pro-inflammatory cytokines. This model has been successfully validated with topically applied compounds (including steroids) delivered in a prophylactic or therapeutic manner.

[0386] This model was used to evaluate the effective dose and dosing regimen, and then in vivo studies were conducted in mice transplanted with psoriatic human skin. The VHH antibodies Re42B04a and Re42F08 were compared with the monoclonal antibody targeting IL-17A (i.e., secukinumab) clinically used for subcutaneous injection in the treatment of psoriasis, and with steroid treatment using betamethasone.

[0387] Skin samples from female donors - currently without a record of inflammatory skin diseases or treatment - were cultured under standard cell culture conditions (CO2 incubator, 37 °C, water saturation, daily medium renewal). Next, in situ activation of skin resident T-cells and Th17 / Th1 polarization with a mixture of pro-inflammatory cytokines were performed to induce the psoriatic phenotype, and then the VHH antibody was administered intradermally (ID) once on day 3 or three times on days 3 - 5. The positive control group included standard care with topical betamethasone, or daily prophylactic treatment or a single subcutaneous (SC) administration of secukinumab on day 3, while the negative control treatment included the irrelevant VHH antibody Re32D03 that does not bind to IL-17.

[0388] The anti-inflammatory effect was evaluated by measuring cytokine secretion, including cytokines of the IL-17 family, interferon 1β (IFN-1β), and interferon γ (IFN-γ). In addition, the structure, integrity, and viability of the skin were evaluated by histological analysis (e.g., H&E staining). The study design is shown in Table 4.

[0389] Table 4: In vitro study design

[0390]

[0391]

[0392] The culture volume was 2 mL.

[0393] IL-17A appears to be the cytokine most significantly affected by the therapeutic agents applied ( Figure 22 ). As expected, low levels of IL-17A (measured at approximately 7 pg / ml) were detected in the culture medium of the untreated HypoSkin model, while very pronounced secretions were detected in the untreated HypoInflammaSkin model, reaching an average of 130 pg / ml and confirming an effective Th17-type response. Importantly, the positive control therapeutic agents betamethasone and secukinumab were both effective and significantly inhibited the secretion of IL-17A. The control irrelevant VHH Re32D03 (anti-SARS-CoV-2) did not significantly affect the secretion of IL-17A, but more variability between parallel assays was observed compared to other cases (varying from 39 to 135 pg / ml). Injection of VHH Re42B04a once and three times almost completely eliminated IL-17A secretion, reaching 6.8 and 5.7 pg / ml respectively, with one parallel assay below the detection limit in each case. This represents a reduction of approximately 20-fold compared to the HypoInflammaSkin case and approximately 14-fold compared to the irrelevant VHH. Injection of VHH Re42F08 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.

[0394] Hematoxylin and eosin (H&E) staining was performed on day 7 to evaluate the structural integrity and cell viability of the skin samples at this time point. The following areas were evaluated for basic comments: Apoptosis and loss of epidermal cell viability: This is typically indicated by pycnotic nuclei (dark, condensed) in the epidermis and / or highly eosinophilic cytoplasm (stained "pink" instead of purple). Rupture of adipocytes in the subcutaneous layer may indicate loss of cell viability. Spongiotic edema: Infiltration of fluid into the epidermis. This typically appears as "white" spaces between keratinocytes, where the interconnected filaments are still visible. There may also be smaller white pockets / spaces, especially near the epidermal / dermal junction. Loss of structural integrity: Separation of the epidermis and dermis, usually associated with extensive pyknosis of the basal epidermal layer.

[0395] As Figure 23 shown in A-23H: Untreated normal tissue control ( Figure 23A) With untreated tissue that induced psoriasis ( Figure 23 B) showing loss of cell viability, extensive karyopyknosis and high eosinophilia, and increased epidermal thickness. With betamethasone ( Figure 23 C) and secukinumab ( Figure 23 D) treatment agents, cell viability was improved, but some signs of inflammation were still evident. When treated with the VHH antibody, after one injection ( Figure 23 E) or three injections ( Figure 23 F) Re42b04a or after one injection ( Figure 23 G) or three injections ( Figure 23 H) Re42F08, compared with the untreated tissue that induced psoriasis ( Figure 23 B), significantly prevented pathological changes and improved skin histology. Compared with multiple cells with karyopyknosis and loss of structural integrity in the untreated control, these phenomena were reduced in the samples treated with secukinumab and the two VHHs. In Figures 24E and 24G treated with the VHH once, the tissue looked intact and no psoriasis lesions occurred.

[0396] Conclusion

[0397] The aim of this study was to evaluate the anti-inflammatory effects of the VHH antibodies of the present invention using the InflammaSkin platform (where T cell activation and stimulation lead to Th1 / Th17-mediated induction of skin psoriasis). The VHH antibodies Re42B04a and Re42F08 were injected intradermally into the HypoInflammaSkin model 1 or 3 times after the onset of inflammation. An irrelevant VHH against the SARS-CoV-2 spike protein was used as a control for this experimental group and was injected once. Betamethasone (a topical steroid) was used as a positive control treatment agent. In addition, 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, and then the effects of the applied treatment agents were evaluated.

[0398] To evaluate the effect of the positive control betamethasone treatment agent on the donor's reactivity to T cell activation, the release of the cytokine IL-22 in the culture medium was evaluated after 7 days. In addition, H&E staining of HypoSkin and HypoInflammaSkin section samples was used to analyze skin structural integrity and cell viability. Finally, the cytokine release in all cases was evaluated using the MSD TH17 Combo 2 assay (K15076K, MesoScale Discovery) on day 7.

[0399] Overall, no significant changes in skin features were observed during the culture process, and H&E staining indicated that the untreated HypoSkin model was healthy and viable. After inducing inflammation, the HypoInflammaSkin model showed a severe loss of cell viability, hyperkeratosis, signs of spongiotic edema indicating inflammation, and abnormal structural integrity. Betamethasone (the positive control therapeutic agent) seemed to improve skin histology, especially when assessing the degree of pyknosis and thickening of the stratum corneum. The secukinumab therapeutic agent seemed to further improve skin features, with less obvious signs of cell death and hyperkeratosis. The irrelevant 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 tested VHH antibodies Re42B04a and Re42F08 significantly improved skin histology. Compared with the control situation, the HypoInflammaSkin models injected with the VHH antibodies Re42b04a and Re42f08 once showed a significantly smaller area of pyknosis, eosinophilic keratinocytes, and no obvious hyperkeratosis was observed. Spongiotic edema persisted in these experimental situations, and the basal epidermis still showed signs of reduced cell viability. In summary, the H&E results showed that the tested VHH antibodies Re42B04a and Re42F08 improved skin histology, and these antibodies seemed to be more effective than betamethasone in reducing skin changes caused by inflammation in this donor, and at least as effective as secukinumab.

[0400] The most significant and prominent results were obtained from the 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 level. The VHH antibody Re42B04a (injected once and three times) reduced IL-17A secretion to the basal level detected in the untreated HypoSkin control, with a stronger effect compared to betamethasone or secukinumab. The VHH antibody Re42F08 (injected once and three times) also significantly reduced the release of IL-17A, but to a lesser extent than Re42B04a, betamethasone, and secukinumab. Importantly, the reduction in IL-17A secretion by VHH Re42F08 (both treatment regimens) was significant compared to the irrelevant VHH treatment group.

[0401] Overall, the results obtained indicate that the VHH antibodies of the present invention have great potential to regulate inflammation in the psoriatic InflammaSkin model, as shown by the improvement in skin viability and structural integrity. The cytokine release analysis, especially the IL-17A expression, confirmed this.

[0402] References

[0403] 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

[0404] Beckett D, Kovaleva E, Schatz PJ (1999) A minimal peptide substrate in biotinholoenzyme synthetase-catalyzed biotinylation. Protein Sci, 8:921–929

[0405] 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

[0406] 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

[0407] 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

[0408] 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

[0409] Güttler T, Aksu M, Dickmanns A, Stegmann KM, Gregor K, Rees R, Taxer W, Rymarenko O, Schünemann J, Dienemann C, Gunkel P, Mussil B, Krull J, Teichmann U, Groß U, Cordes VC, Dobbelstein M D(2021)Neutralization of SARS-CoV-2 by highly potent, hyperthermostable, and mutation-tolerant nanobodies. EMBO J, e107985

[0410] 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

[0411] Frank Kolbinger, Franco Di Padova, Atul Deodhar, Jason E. Hawkes, Christine Huppertz, Torsten Kuiper, Iain B. McInnes, Christopher T. Ritchlin, David Rosmarin, Georg Schett, José M. Carballido, Peter 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,

[0412] 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

[0413] Mease PJ, McInnes IB, Kirkham B, Kavanaugh A, Rahman P, van der Heijde D, Landewé 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

[0414] Mills KHG (2022) IL-17 and IL-17-producing cells in protection versus pathology. Nat Rev Immunol, 1–17

[0415] Nies JF, Panzer U (2020) IL-17C / IL-17RE: Emergence of a Unique Axis in T H 17 Biology. Front Immunol, 11:341

[0416] 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

[0417] 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,

[0418] Reich K, Warren RB, Lebwohl M, Gooderham M, Strober B, Langley RG, Paul C, DeCuyper D, Vanvoorden V, Madden C, Cioffi C, Peterson L, Blauvelt A(2021) Bimekizumab versus Secukinumab in Plaque Psoriasis. N Engl J Med, 385:142–152

[0419] Sarama et al., Frontiers in Bioengineering and Biotechnology February 2022|Volume 10|Article 803218.

[0420] 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 Acne. Internal Medicine and Care, 1(2): 1 - 6

[0421] 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.

[0422] 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

[0423] 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

[0424] 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

[0425] Zhou, X. et al. Advances in the pathogenesis of psoriasis: from keratinocyte perspective. 2022; Cell Death Dis 13, 81.

Claims

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

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

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

4. The VHH antibody according to any one of claims 1-3, which comprises a CDR3 sequence selected from: i. A sequence of the formula NDMPYGX1X2TX3MDX4YX5X6W (Class A), 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. A sequence of the formula X1HNEPGX2LYM (Class B), wherein X1 is selected from V and T, X2 is selected from H and D; iii. The sequence MAVRGLYGSNWYDYPFELW (SEQ ID NO.52) (Class C); and iv. The sequence YIDSGSDRYY (SEQ ID NO.57) (Class D).

5. The Class A VHH antibody according to any one of claims 1-4, which comprises: a CDR3 having a sequence selected from SEQ ID NO.8, 11, 18, 26, 31 and 34, a CDR2 having a sequence selected from SEQ ID NO.7, 14, 17, 22, 25 and 30, and a CDR1 having a sequence selected from SEQ ID NO.6, 10, 16, 21, 24, 29 and 33.

6. The class A VHH antibody according to any one of claims 1-5, which comprises a CDR3 sequence as shown in any one of SEQ ID NO: 8, 11, 18, 26, 31, 31, 38, 43, 52 and 57, or a CDR3 sequence having at least 80% identity with any one of the CDR3 sequences.

7. The class A VHH antibody according to any one of claims 1-6, which comprises a set of 3 CDR sequences selected from the group consisting of: SEQ ID NO.16, 17 and 18; SEQ ID NO.6, 7 and 8; SEQ ID NO.10, 7 and 11; SEQ ID NO.10, 14 and 11; SEQ ID NO.21, 22 and 18; SEQ ID NO.24, 25 and 26; SEQ ID NO.29, 30 and 31; SEQ ID NO.33, 7 and 34.

8. An anti-IL-17A VHH antibody of class A according to any one of claims 1-7, comprising the following sequence positions that interact with IL-17A: position 1, which is Q; position 29, which is A, G, V, F or P; position 30, which is S; position 31, which is S or G; position 32, which is Y; position 33, which is A; position 50, which is A; position 51, which is I; position 52, which is S; position 54, which is I, S or V; position 55, which is S or G; position 57, which is G, S or D; position 58, which is T, S or A; position 59, which is K, R or V; position 100, which is P; position 101, which is Y; Position 103, which is L or M; Position 104, which is D or E; position 106, which is R; and position 109, which is E or D.

9. An anti-IL-17F VHH antibody of class A according to any one of claims 1-7, which comprises the following sequence positions that interact with IL-17F: position 1, which is Q; position 29, which is A, G, V, F or P; position 30, which is S; position 31, which is S or G; position 32, which is Y; position 33, which is A; position 50, which is A; position 51, which is I; position 52, which is S; position 54, which is I, S or V; position 55, which is S or G; position 57, which is G, S or D; position 58, which is T, S or A; position 59, which is K, R or V; position 100, which is P; position 101, which is Y; Position 103, which is L or M; Position 104, which is D or E; position 106, which is R; and position 109, which is E or D.

10. The class B VHH antibody according to any one of claims 1-4, which comprises a set of 3 CDR sequences selected from the group consisting of: SEQ ID NO.36, 37 and 38; SEQ ID NO.41, 42 and 43; and SEQ ID NO.46, 42 and 43.

11. The class B VHH antibody according to any one of claims 1-4 and 10, which comprises: a CDR3 having a sequence selected from SEQ ID NO.38 and 43, a CDR2 having a sequence selected from SEQ ID NO.37 and 42, and a CDR1 having a sequence selected from SEQ ID NO.36 and 41.

12. The class B VHH antibody according to any one of claims 1-4 and 10-11, which comprises a CDR3 sequence as shown in any one of SEQ ID NO.38 and 43, or a CDR3 sequence having at least 80% identity with any one of the CDR3 sequences.

13. The class B VHH antibody according to any one of claims 1-4 and 10-12, which comprises 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. The class B VHH antibody according to any one of claims 1-4 and 10-11, which comprises the following sequence positions that 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. The VHH antibody according to any one of claims 1-14, which comprises a set of 3 CDR sequences, wherein the set is selected from: SEQ ID NO.16, 17 and 18, and SEQ ID NO.36, 37 and 38.

16. The VHH antibody according to any one of claims 1-15, which is 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 is a VHH antibody that is a variant having at least 90% identity with any one of these sequences.

17. The VHH antibody according to any one of claims 1-4, which comprises a set of 3 CDR sequences, the set comprising SEQ ID NO.50, 51 and 52 (class C), or SEQ ID NO.56, 57 and 58 (class D).

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

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

20. The VHH antibody according to any one of the foregoing claims, which is thermostable or super-thermostable, 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 having 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 the preceding claims, 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, said human IL-17 polypeptides being in particular IL-17A homodimers, IL-17F homodimers and IL17A / F heterodimers, said set comprising at least one VHH antibody according to any one of the preceding claims.

23. A nucleic acid molecule that encodes a VHH antibody according to any one of the preceding claims.

24. A recombinant cell or non-human organism that is transformed or transfected with the nucleic acid molecule according to claim 23 or a vector containing said nucleic acid molecule.

25. The recombinant cell according to claim 24, which is selected from bacterial cells, yeast cells, insect cells, mammalian cells and plant cells.

26. A method for recombinantly producing a VHH antibody according to any one of claims 1-21, which comprises culturing a cell or organism in a suitable medium and obtaining the VHH antibody from said cell or organism or from said medium.

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

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

29. The pharmaceutical composition according to claim 28, which is formulated for topical administration, in particular for surface application or intradermal injection.

30. The pharmaceutical composition according to any one of claims 28-29, which is formulated as a cream, paste, gel, hydrogel, ointment, lotion or emulsion.

31. The pharmaceutical composition according to claim 28, which is formulated for parenteral administration.

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

33. The pharmaceutical composition according to any one of claims 28-32, which is used for preventing or treating a disorder caused by or associated with the overactivity of IL-17, in particular the overactivity of IL-17A and / or IL-17F.

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

35. The pharmaceutical composition for use according to claim 34, wherein the inflammatory disorder 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, active non-radiographic axial spondyloarthritis with objective clinical signs of inflammation, systemic lupus erythematosus (SLE), familial Mediterranean fever (FMF), TNF receptor-associated periodic syndrome (TRAPS), hidradenitis suppurativa (HS), pemphigus vulgaris (PV), pityriasis rubra pilaris (PRP), alopecia areata, systemic sclerosis, and infectious diseases, lichen planus, and herpes gestationis.

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

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

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

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

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

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

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

44. The method according to claim 43, wherein the inflammatory disorder 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. The method according to claim 44, wherein the inflammatory disorder and / or immune-related disorder is a skin disorder.

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

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

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

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

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

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