Polypeptides containing immunoglobulin single variable domains that target IL-13 and TSLP

Abstract

The present technology aims to provide a new type of drug for treating subjects suffering from inflammatory diseases. Specifically, the present technology provides a polypeptide comprising at least four immunoglobulin single variable domains (ISVDs), characterized in that at least two ISVDs bind to IL-13 and at least two ISVDs bind to TSLP. The present technology also provides nucleic acids, vectors, and compositions.

Description

[Technical field]

[0001] explanation 1 Field of this technology The present technology relates to a polypeptide that targets IL-13 and TSLP.The present technology also relates to a nucleic acid molecule that encodes the polypeptide and a vector that contains the nucleic acid, and a composition that contains the polypeptide, the nucleic acid, or the vector.The present technology further relates to these products for use in a method for treating a subject suffering from an inflammatory disease.The present technology also relates to a method for producing these products. [Background technology]

[0002] 2 Technical background Unchecked immune responses, while necessary for host defense, can lead to various inflammatory diseases such as asthma, atopic dermatitis and rheumatoid arthritis. The cascade of immune responses mediated by the innate and adaptive arms of the immune system (e.g., antigen recognition, antigen processing, antigen presentation, cytokine production, antibody production, target cell killing) promotes the initiation and spread of various immune diseases. Inflammatory diseases are often chronic and can even be life-threatening. Allergic and atopic diseases, e.g., asthma and atopic dermatitis, are predominantly driven by type 2 immune responses and are characterized by hallmarks of type 2 immunity such as high IgE production and eosinophilia.

[0003] Thymic stromal lymphopoietin (TSLP) and interleukin-13 (IL-13) are soluble cytokine targets produced by stromal and / or immune cells (Non-Patent Document 1, Non-Patent Document 2). Human TSLP and IL-13 promote distinct, overlapping, and synergistic aspects of type 2 immune, type 2 inflammatory diseases, such as asthma and atopic dermatitis, as well as various immune diseases.

[0004] TSLP signaling is initiated through a heterodimeric receptor complex composed of the thymic stromal lymphopoietin receptor (TSLPR) and the IL-7R alpha chain (IL-7Rα). Similarly, IL-13 signaling is initiated by binding to a heterodimeric receptor complex composed of the alpha IL-4 receptor (IL-4Rα) and the alpha interleukin-13 receptor (IL-13R1α). The high affinity of IL-13 for IL-13R1 leads to their complex formation, which further increases the likelihood of heterodimerization to IL-4Rα.

[0005] TSLP promotes dendritic cell maturation, mast cell development and proliferation, as well as activating other immune cells such as basophils and innate lymphoid cells (ILC2s). Similarly, IL-13 induces a variety of immunopathologies, including epithelial barrier breakdown, mucus production by mucosal to epithelial surfaces, and airway remodeling, as well as induction of eosinophils that recruit chemokines such as eotaxin. These mechanisms are central to the initiation and spread of type 2 inflammatory responses and the development of a variety of immunopathologies in diseases such as atopic dermatitis and asthma.

[0006] Currently, patients with mild / severe asthma respond inadequately to currently available standard of care treatments such as anti-IL4Rα monoclonal antibody Dupixent (dupilumab; commercially available), anti-IL5 (commercially available), and biologics such as anti-IgE monoclonal antibody Xolair (omalizumab; commercially available), especially in asthma patients with a low eosinophilic phenotype. Antagonist monoclonal antibodies against TSLP (tezepelumab) and IL-13 (lebrikizumab) are currently in clinical trials, but there are no active clinical development programs targeting both TSLP and IL-13. Dual targeting of TSLP and IL-13 with a single agent has the potential to confer efficacy in both low and high type 2 asthma as well as atopic dermatitis, in subpopulations within these indications where single monospecific agent therapy is less effective. Thus, there remains a continuing unmet medical need for treatments of type 2 inflammatory diseases such as asthma and atopic dermatitis that are not only more effective but also better adapted to patients.

[0007] Such therapy may involve targeting multiple disease factors, for example, IL-13 and TSLP.

[0008] Targeting multiple disease factors can be achieved, for example, by co-administration or combinatorial use of two separate biological agents, such as antibodies, that bind to different therapeutic targets. However, co-administration or combinatorial use of separate biological agents can be problematic from both practical and commercial perspectives. For example, two injections of separate products result in a more inconvenient and more painful treatment regimen for patients, which can negatively impact compliance. For a single injection of two separate products, it can be difficult or impossible to provide a formulation that allows acceptable viscosity at the required concentration and suitable stability of both products. In addition, co-administration and co-formulation can increase overall costs, since it requires the production of two separate drugs.

[0009] Bispecific antibodies, capable of binding to two different antigens, have been suggested as one strategy to address such limitations associated with the co-administration or combinatorial use of separate biologics, such as antibodies.

[0010] Bispecific antibody constructs have been proposed in several formats. For example, bispecific antibody formats may involve chemical conjugation of two antibodies or fragments thereof (Non-Patent Document 3; Non-Patent Document 4). [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Ziegler and Artis, Nat Rev Immunol (2010) 11:289 [Non-Patent Document 2] Gieseck III et al., Nat Rev Immunol (2018) 18:62 [Non-Patent Document 3] Brennan, M. et al., Science, 1985. 229(4708):81-83 [Non-Patent Document 4] Glennie, MJ et al., J Immunol, 1987.139(7):2367-2375 Summary of the Invention [Problem to be solved by the invention]

[0012] However, disadvantages of such bispecific antibody formats include high viscosity at high concentrations, which makes e.g. subcutaneous administration difficult, and impacts on polypeptide stability and production efficiency, in that each binding unit requires the interaction of two variable domains for specific and high affinity binding. Such bispecific antibody formats can also potentially cause chemistry, manufacturing and quality control (CMC) problems related to light chain mispairing or heavy chain mispairing. [Means for solving the problem]

[0013] 3 Summary of this technology In some embodiments, the technology relates to polypeptides that specifically target IL-13 and TSLP simultaneously, resulting in increased efficiency in modulating type 2 inflammatory responses in vitro compared to monospecific anti-IL-13 or anti-TSLP polypeptides. In some embodiments, the polypeptides are efficiently produced (e.g., in microbial hosts). Furthermore, in some embodiments, such polypeptides have limited reactivity with pre-existing antibodies in the subject to be treated (i.e., antibodies present in the subject before the first treatment with the antibody construct). In other embodiments, such polypeptides exhibit a sufficiently long half-life in the subject to be treated to allow for convenient spacing between successive treatments.

[0014] In one embodiment, the technology provides a polypeptide comprising or consisting of at least one immunoglobulin single variable domain (ISVD) that specifically binds to IL-13. In a further embodiment, the polypeptide of the technology comprises or consists of at least two ISVDs that specifically bind to IL-13, and the two ISVDs are optionally linked via a peptidic linker. In one embodiment, the two ISVDs that specifically bind to IL-13 are separate ISVDs. Furthermore, in one embodiment, the polypeptide further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers, which provide a polypeptide with an increased half-life compared to a corresponding polypeptide without the one or more other groups, residues, moieties or binding units. For example, the binding unit can be an ISVD that binds to a (human) serum protein, such as human serum albumin.

[0015] In another aspect, the polypeptide of the present technology comprises or consists of at least one ISVD that specifically binds to TSLP. In a further embodiment, the polypeptide of the present technology comprises or consists of at least two ISVDs that specifically bind to TSLP, and the two ISVDs are optionally linked via a peptidic linker. In one embodiment, the two ISVDs that specifically bind to TSLP are separate ISVDs. Furthermore, in one embodiment, the polypeptide further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers, which provide the polypeptide with an increased half-life compared to the corresponding polypeptide without the one or more other groups, residues, moieties or binding units. For example, the binding unit can be an ISVD that binds to (human) serum proteins, such as human serum albumin.

[0016] In another embodiment, the polypeptide of the present technology comprises or consists of at least four ISVDs, at least two ISVDs specifically bind to IL-13 and at least two ISVDs specifically bind to TSLP. In one embodiment, the at least two ISVDs specifically bind to IL-13 specifically bind to human IL-13 and the at least two ISVDs specifically bind to TSLP specifically bind to human TSLP. In one embodiment, the at least two ISVDs specifically bind to IL-13 are separate ISVDs and the at least two ISVDs that bind to TSLP are separate ISVDs. In another embodiment, the polypeptide comprising or consisting of at least four ISVDs further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers, which provide the polypeptide with an increased half-life compared to the corresponding polypeptide without the one or more other groups, residues, moieties or binding units. For example, the binding unit may be an ISVD that binds to a (human) serum protein, such as human serum albumin.

[0017] Also provided are nucleic acid molecules capable of expressing the polypeptides of the present technology, vectors comprising the nucleic acids or nucleic acids, and compositions comprising the polypeptides, nucleic acids or vectors. In one embodiment, the composition is a pharmaceutical composition.

[0018] Also provided are hosts or host cells comprising a nucleic acid or vector encoding a polypeptide according to the technology.

[0019] Further provided herein is a method for producing a polypeptide according to the present technology, comprising at least: a. expressing in a suitable host cell or host organism, or in another suitable expression system, a nucleic acid comprising a nucleotide sequence encoding a polypeptide of the present technology; optionally followed by: b. Isolating and / or purifying the polypeptide according to the present technology The method includes:

[0020] Furthermore, the present technology provides a composition comprising a polypeptide, a composition comprising a polypeptide, or a composition comprising a nucleic acid or vector comprising a nucleotide sequence encoding the polypeptide, for use as a medicine.In one embodiment, the polypeptide or composition is for use in treating inflammatory disease, such as type 2 inflammatory disease.In one embodiment, the type 2 inflammatory disease is selected from atopic dermatitis and asthma.

[0021] In addition, a method for treating inflammatory disease, such as type 2 inflammatory disease, is provided, comprising administering a pharmacologic active amount of the polypeptide or composition according to the present technology to a subject in need thereof.In one embodiment, the type 2 inflammatory disease is selected from atopic dermatitis and asthma.In one embodiment, the method further comprises administering one or more additional therapeutic agents.

[0022] Further provided is the use of the polypeptide or composition of the present technology in the preparation of a pharmaceutical composition for treating an inflammatory disease, such as type 2 inflammatory disease. In one embodiment, the type 2 inflammatory disease is selected from atopic dermatitis and asthma.

[0023] In particular, the present technology provides the following embodiments:

[0024] Embodiment 1. A polypeptide, a composition comprising said polypeptide, or a composition comprising a nucleic acid comprising a nucleotide sequence encoding said polypeptide, for use as a medicament, said polypeptide comprising or consisting of at least one immunoglobulin single variable domain (ISVD), said ISVD comprising three complementarity determining regions (CDR1 to CDR3 respectively), said at least one ISVD comprising: a) CDR1 which is the amino acid sequence of SEQ ID NO: 7 or has two or one amino acid difference from SEQ ID NO: 7; CDR2 is the amino acid sequence of SEQ ID NO: 12 or has 2 or 1 amino acid difference from SEQ ID NO: 12; and CDR3 which is the amino acid sequence of SEQ ID NO: 17 or has 2 or 1 amino acid difference from SEQ ID NO: 17; b) CDR1 which is the amino acid sequence of SEQ ID NO:8 or has 2 or 1 amino acid difference from SEQ ID NO:8; CDR2 is the amino acid sequence of SEQ ID NO: 13 or has two or one amino acid difference from SEQ ID NO: 13; and CDR3 which is the amino acid sequence of SEQ ID NO: 18 or has 2 or 1 amino acid difference from SEQ ID NO: 18; c) CDR1 which is the amino acid sequence of SEQ ID NO: 9 or has 2 or 1 amino acid difference from SEQ ID NO: 9; CDR2 is the amino acid sequence of SEQ ID NO: 14 or has 2 or 1 amino acid difference from SEQ ID NO: 14; and a CDR3 which is the amino acid sequence of SEQ ID NO: 19 or which has 2 or 1 amino acid difference from SEQ ID NO: 19; d) CDR1 which is the amino acid sequence of SEQ ID NO: 11 or has 2 or 1 amino acid difference from SEQ ID NO: 11; CDR2 is the amino acid sequence of SEQ ID NO: 16 or has 2 or 1 amino acid difference from SEQ ID NO: 16; and CDR3 having the amino acid sequence of SEQ ID NO:21 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO:21 The polypeptide or composition comprising:

[0025] Embodiment 2. At least one ISVD comprises: a) CDR1 having the amino acid sequence of SEQ ID NO: 7, CDR2 having the amino acid sequence of SEQ ID NO: 12, and CDR3 having the amino acid sequence of SEQ ID NO: 17; b) CDR1 having the amino acid sequence of SEQ ID NO: 8, CDR2 having the amino acid sequence of SEQ ID NO: 13, and CDR3 having the amino acid sequence of SEQ ID NO: 18; c) CDR1 having the amino acid sequence of SEQ ID NO: 9, CDR2 having the amino acid sequence of SEQ ID NO: 14 and CDR3 having the amino acid sequence of SEQ ID NO: 19, or d) CDR1 having the amino acid sequence of SEQ ID NO: 11, CDR2 having the amino acid sequence of SEQ ID NO: 16, and CDR3 having the amino acid sequence of SEQ ID NO: 21 2. A polypeptide or composition for use according to embodiment 1, comprising:

[0026] Embodiment 3. The amino acid sequence of at least one ISVD is: a) greater than 90% sequence identity with SEQ ID NO:2; b) greater than 90% sequence identity with SEQ ID NO:3; c) greater than 90% sequence identity to SEQ ID NO:4; or d) greater than 90% sequence identity with SEQ ID NO:6 3. A polypeptide or composition for use according to any of embodiments 1 or 2, comprising:

[0027]

[0023] Embodiment 4. The at least one ISVD comprises: a) the amino acid sequence of SEQ ID NO:2; b) the amino acid sequence of SEQ ID NO:3; c) the amino acid sequence of SEQ ID NO: 4, or d) the amino acid sequence of SEQ ID NO:6 A polypeptide or composition for use according to any one of embodiments 1 to 3, comprising:

[0028] Embodiment 5. The polypeptide comprises or consists of at least two ISVDs, each of which comprises three complementarity determining regions (CDR1 to CDR3, respectively), and the at least two ISVDs are optionally linked via one or more peptidic linkers: a) The first and second ISVDs are: i. CDR1 which is the amino acid sequence of SEQ ID NO:7 or has 2 or 1 amino acid difference from SEQ ID NO:7; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 12 or has two or one amino acid difference from SEQ ID NO: 12; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 17. Including, b) The first and second ISVDs are: i. CDR1 which is the amino acid sequence of SEQ ID NO:8 or has 2 or 1 amino acid difference from SEQ ID NO:8; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 13 or has two or one amino acid difference from SEQ ID NO: 13; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 18. Including, c) The first ISVD is: i. CDR1 which is the amino acid sequence of SEQ ID NO:7 or has 2 or 1 amino acid difference from SEQ ID NO:7; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 12 or has two or one amino acid difference from SEQ ID NO: 12; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 17. Including, The second ISVD is iv. CDR1 which is the amino acid sequence of SEQ ID NO:8 or has 2 or 1 amino acid difference from SEQ ID NO:8; v. CDR2 which is the amino acid sequence of SEQ ID NO:13 or has two or one amino acid difference from SEQ ID NO:13; and vi. CDR3 having the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 18 Including, d) The first ISVD is: i. CDR1 which is the amino acid sequence of SEQ ID NO:8 or has 2 or 1 amino acid difference from SEQ ID NO:8; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 13 or has two or one amino acid difference from SEQ ID NO: 13; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 18. Including, The second ISVD is iv. CDR1 which is the amino acid sequence of SEQ ID NO:7 or has 2 or 1 amino acid difference from SEQ ID NO:7; v. CDR2 which is the amino acid sequence of SEQ ID NO:12 or has two or one amino acid difference from SEQ ID NO:12; and vi. CDR3 having the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 17 Including, e) The first ISVD is: i. CDR1 which is the amino acid sequence of SEQ ID NO:11 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO:11; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 16 or has two or one amino acid difference from SEQ ID NO: 16; and iii. A CDR3 having the amino acid sequence of SEQ ID NO:21 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO:21. Including, The second ISVD is iv. CDR1 which is the amino acid sequence of SEQ ID NO:9 or has 2 or 1 amino acid difference from SEQ ID NO:9; v. CDR2 which is the amino acid sequence of SEQ ID NO:14 or has two or one amino acid difference from SEQ ID NO:14; and vi. CDR3 having the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 19 Contains, or f) The first ISVD is: i. CDR1 which is the amino acid sequence of SEQ ID NO:9 or has 2 or 1 amino acid difference from SEQ ID NO:9; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 14 or has two or one amino acid difference from SEQ ID NO: 14; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 19. Including, The second ISVD is iv. CDR1 which is the amino acid sequence of SEQ ID NO:11 or has 2 or 1 amino acid difference from SEQ ID NO:11; v. CDR2 which is the amino acid sequence of SEQ ID NO:16 or has two or one amino acid difference from SEQ ID NO:16; and vi. CDR3 having the amino acid sequence of SEQ ID NO:21 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO:21 Including, 2. A polypeptide or composition for use according to embodiment 1, wherein the order of the ISVDs indicates their relative position with respect to each other considered in the direction from the N-terminus to the C-terminus of the polypeptide.

[0029] Embodiment 6.a) the first and second ISVDs comprise a CDR1 having the amino acid sequence of SEQ ID NO:7, a CDR2 having the amino acid sequence of SEQ ID NO:12, and a CDR3 having the amino acid sequence of SEQ ID NO:17; b) the first and second ISVDs comprise a CDR1 having the amino acid sequence of SEQ ID NO:8, a CDR2 having the amino acid sequence of SEQ ID NO:13, and a CDR3 having the amino acid sequence of SEQ ID NO:18; c) the first ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO:7, a CDR2 having the amino acid sequence of SEQ ID NO:12, and a CDR3 having the amino acid sequence of SEQ ID NO:17, and the second ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO:8, a CDR2 having the amino acid sequence of SEQ ID NO:13, and a CDR3 having the amino acid sequence of SEQ ID NO:18; d) the first ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO:8, a CDR2 having the amino acid sequence of SEQ ID NO:13, and a CDR3 having the amino acid sequence of SEQ ID NO:18, and the second ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO:7, a CDR2 having the amino acid sequence of SEQ ID NO:12, and a CDR3 having the amino acid sequence of SEQ ID NO:17; e) the first ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO: 11, a CDR2 having the amino acid sequence of SEQ ID NO: 16, and a CDR3 having the amino acid sequence of SEQ ID NO: 21, and the second ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO: 9, a CDR2 having the amino acid sequence of SEQ ID NO: 14, and a CDR3 having the amino acid sequence of SEQ ID NO: 19, or f) A polypeptide or composition for use according to embodiment 5, wherein the first ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO: 9, a CDR2 having the amino acid sequence of SEQ ID NO: 14 and a CDR3 having the amino acid sequence of SEQ ID NO: 19, and the second ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO: 11, a CDR2 having the amino acid sequence of SEQ ID NO: 16 and a CDR3 having the amino acid sequence of SEQ ID NO: 21.

[0030] In embodiment 7.a) the amino acid sequence of the first and second ISVD comprises greater than 90% sequence identity with SEQ ID NO:2; b) the amino acid sequences of the first and second ISVDs comprise greater than 90% sequence identity with SEQ ID NO:3; c) the amino acid sequence of the first ISVD comprises greater than 90% sequence identity to SEQ ID NO:2 and the second ISVD comprises greater than 90% sequence identity to SEQ ID NO:3; d) the amino acid sequence of the first ISVD comprises greater than 90% sequence identity to SEQ ID NO:3 and the second ISVD comprises greater than 90% sequence identity to SEQ ID NO:2; e) the amino acid sequence of the first ISVD comprises greater than 90% sequence identity to SEQ ID NO:6 and the second ISVD comprises greater than 90% sequence identity to SEQ ID NO:4; f) A polypeptide or composition for use according to any of embodiments 5 or 6, wherein the amino acid sequence of the first ISVD comprises greater than 90% sequence identity with SEQ ID NO:4 and the second ISVD comprises greater than 90% sequence identity with SEQ ID NO:6.

[0031] In embodiment 8.a) the first and second ISVDs comprise the amino acid sequence of SEQ ID NO:2; b) the first and second ISVDs comprise the amino acid sequence of SEQ ID NO:3; c) the first ISVD comprises the amino acid sequence of SEQ ID NO:2 and the second ISVD comprises the amino acid sequence of SEQ ID NO:3; d) the first ISVD comprises the amino acid sequence of SEQ ID NO:3 and the second ISVD comprises the amino acid sequence of SEQ ID NO:2; e) the first ISVD comprises the amino acid sequence of SEQ ID NO:6 and the second ISVD comprises the amino acid sequence of SEQ ID NO:4; or f) A polypeptide or composition for use according to any of embodiments 5 to 7, wherein the first ISVD comprises the amino acid sequence of SEQ ID NO: 4 and the second ISVD comprises the amino acid sequence of SEQ ID NO: 6.

[0032] Embodiment 9. The polypeptide comprises: a) the amino acid sequence of SEQ ID NO: 148; b) the amino acid sequence of SEQ ID NO: 149; c) the amino acid sequence of SEQ ID NO: 150; d) the amino acid sequence of SEQ ID NO: 151; e) the amino acid sequence of SEQ ID NO: 152; f) the amino acid sequence of SEQ ID NO: 153; g) the amino acid sequence of SEQ ID NO: 154; h) the amino acid sequence of SEQ ID NO: 155; i) the amino acid sequence of SEQ ID NO: 156; j) the amino acid sequence of SEQ ID NO: 157; k) the amino acid sequence of SEQ ID NO: 158, or l) Amino acid sequence of SEQ ID NO: 159 9. The polypeptide according to any one of embodiments 5 to 8, comprising or consisting of:

[0033] Embodiment 10. The polypeptide comprises or consists of at least four ISVDs, each of which comprises three complementarity determining regions (CDR1 to CDR3, respectively), and the at least four ISVDs are optionally linked via one or more peptidic linkers: a) The first ISVD is: i. CDR1 which is the amino acid sequence of SEQ ID NO:7 or has 2 or 1 amino acid difference from SEQ ID NO:7; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 12 or has two or one amino acid difference from SEQ ID NO: 12; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 17. Includes; b) The second ISVD is: iv. CDR1 which is the amino acid sequence of SEQ ID NO:8 or has 2 or 1 amino acid difference from SEQ ID NO:8; v. CDR2 which is the amino acid sequence of SEQ ID NO:13 or has two or one amino acid difference from SEQ ID NO:13; and vi. CDR3 having the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 18 Includes; c) The third ISVD is: vii. CDR1 which is an amino acid sequence of SEQ ID NO:9 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO:9; viii. CDR2 is an amino acid sequence of SEQ ID NO: 14 or has two or one amino acid difference from SEQ ID NO: 14; and ix. CDR3 having the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 19 Includes; d) The fourth ISVD is: x. CDR1 which is the amino acid sequence of SEQ ID NO:11 or has 2 or 1 amino acid difference from SEQ ID NO:11; xi. CDR2 is an amino acid sequence of SEQ ID NO: 16 or has 2 or 1 amino acid difference from SEQ ID NO: 16; and xii. CDR3 having the amino acid sequence of SEQ ID NO:21 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO:21 Including, 6. The polypeptide or composition for use according to any of embodiments 1 or 5, wherein the order of the ISVDs indicates their relative position with respect to each other considered in the direction from the N-terminus to the C-terminus of the polypeptide.

[0034] Embodiment 11. A composition for use according to any one of embodiments 1 to 10, which is a pharmaceutical composition further comprising at least one pharma- ceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally one or more additional pharma- ceutically active polypeptides and / or compounds.

[0035] Embodiment 12.a) the first ISVD comprises a CDR1 which is the amino acid sequence of SEQ ID NO:7, a CDR2 which is the amino acid sequence of SEQ ID NO:12, and a CDR3 which is the amino acid sequence of SEQ ID NO:17; b) the second ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO:8, a CDR2 having the amino acid sequence of SEQ ID NO:13, and a CDR3 having the amino acid sequence of SEQ ID NO:18; c) the third ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO: 9, a CDR2 having the amino acid sequence of SEQ ID NO: 14, and a CDR3 having the amino acid sequence of SEQ ID NO: 19; d) A polypeptide or composition for use according to embodiment 10 or 11, wherein the fourth ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO: 11, a CDR2 having the amino acid sequence of SEQ ID NO: 16 and a CDR3 having the amino acid sequence of SEQ ID NO: 21.

[0036] Embodiment 13.a) the amino acid sequence of the first ISVD comprises greater than 90% sequence identity to SEQ ID NO:2; b) the amino acid sequence of the second ISVD comprises greater than 90% sequence identity to SEQ ID NO:3; c) the amino acid sequence of the third ISVD comprises greater than 90% sequence identity to SEQ ID NO:4; d) A polypeptide or composition for use according to any of embodiments 10 to 12, wherein the amino acid sequence of the fourth ISVD comprises more than 90% sequence identity with SEQ ID NO:6.

[0037] Embodiment 14.a) the first ISVD comprises the amino acid sequence of SEQ ID NO:2; b) the second ISVD comprises the amino acid sequence of SEQ ID NO:3; c) the third ISVD comprises the amino acid sequence of SEQ ID NO:4; d) A polypeptide or composition for use according to any one of embodiments 10 to 13, wherein the fourth ISVD comprises the amino acid sequence of SEQ ID NO: 6.

[0038] Embodiment 15. A polypeptide or composition for use according to any of embodiments 1 to 14, wherein the polypeptide further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers, which one or more other groups, residues, moieties or binding units provide the polypeptide with an increased half-life compared to a corresponding polypeptide without said one or more other groups, residues, moieties or binding units.

[0039] Embodiment 16. A polypeptide or composition for use according to embodiment 15, wherein said one or more other groups, residues, moieties or binding units providing a polypeptide with increased half-life are selected from the group consisting of polyethylene glycol molecules, serum proteins or fragments thereof, binding units capable of binding to serum proteins, Fc moieties, and small proteins or peptides capable of binding to serum proteins.

[0040] Embodiment 17. Polypeptide or composition for use according to any one of embodiments 15 to 16, wherein the one or more other binding units providing the polypeptide with an increased half-life are selected from the group consisting of binding units capable of binding to serum albumin (such as human serum albumin) or serum immunoglobulin (such as IgG).

[0041] Embodiment 18. A polypeptide or composition for use according to embodiment 17, wherein said binding unit providing a polypeptide with increased half-life is an ISVD capable of binding to human serum albumin.

[0042] Embodiment 19. The ISVD that binds to human serum albumin is i. CDR1 which is the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 10; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 15 or has two or one amino acid difference from SEQ ID NO: 15; and iii. A CDR3 having the amino acid sequence of SEQ ID NO:20 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO:20. 19. The polypeptide or composition for use according to embodiment 18, comprising:

[0043] Embodiment 20. A polypeptide or composition for use according to any of embodiments 18 to 19, wherein the ISVD that binds to human serum albumin comprises a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 15, and a CDR3 having the amino acid sequence of SEQ ID NO: 20.

[0044] Embodiment 21. A polypeptide or composition for use according to any of embodiments 18 to 20, wherein the amino acid sequence of the ISVD that binds to human serum albumin comprises more than 90% sequence identity with SEQ ID NO:5.

[0045] Embodiment 22. A polypeptide or composition for use according to any of embodiments 18 to 21, wherein the ISVD that binds to human serum albumin comprises the amino acid sequence of SEQ ID NO:5.

[0046] Embodiment 23. A polypeptide or composition for use according to any of embodiments 10 to 22, wherein the amino acid sequence of the polypeptide comprises more than 90% sequence identity with SEQ ID NO:1.

[0047] Embodiment 24. A polypeptide or composition for use according to any of embodiments 10 to 23, wherein the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:1.

[0048] Embodiment 25. A polypeptide or composition for use according to any of embodiments 1 to 24 for use in the treatment of an inflammatory disease, such as a type 2 inflammatory disease.

[0049] Embodiment 26. The polypeptide or composition for use according to embodiment 25, wherein the type 2 inflammatory disease is selected from asthma and atopic dermatitis.

[0050] Embodiment 27. A polypeptide comprising or consisting of at least one immunoglobulin single variable domain (ISVD), said ISVD comprising three complementarity determining regions (CDR1 to CDR3 respectively); at least one ISVD comprises: a) CDR1 which is the amino acid sequence of SEQ ID NO: 7 or has two or one amino acid difference from SEQ ID NO: 7; CDR2 is the amino acid sequence of SEQ ID NO: 12 or has 2 or 1 amino acid difference from SEQ ID NO: 12; and CDR3 which is the amino acid sequence of SEQ ID NO: 17 or has 2 or 1 amino acid difference from SEQ ID NO: 17; b) CDR1 which is the amino acid sequence of SEQ ID NO:8 or has 2 or 1 amino acid difference from SEQ ID NO:8; CDR2 is the amino acid sequence of SEQ ID NO: 13 or has two or one amino acid difference from SEQ ID NO: 13; and CDR3 which is the amino acid sequence of SEQ ID NO: 18 or has 2 or 1 amino acid difference from SEQ ID NO: 18; c) CDR1 which is the amino acid sequence of SEQ ID NO: 9 or has 2 or 1 amino acid difference from SEQ ID NO: 9; CDR2 is the amino acid sequence of SEQ ID NO: 14 or has 2 or 1 amino acid difference from SEQ ID NO: 14; and a CDR3 which is the amino acid sequence of SEQ ID NO: 19 or which has 2 or 1 amino acid difference from SEQ ID NO: 19; d) CDR1 which is the amino acid sequence of SEQ ID NO: 11 or has 2 or 1 amino acid difference from SEQ ID NO: 11; CDR2 is the amino acid sequence of SEQ ID NO: 16 or has 2 or 1 amino acid difference from SEQ ID NO: 16; and CDR3 having the amino acid sequence of SEQ ID NO:21 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO:21 The polypeptide comprising:

[0051] Embodiment 28. At least one ISVD comprises: a) CDR1 having the amino acid sequence of SEQ ID NO: 7, CDR2 having the amino acid sequence of SEQ ID NO: 12, and CDR3 having the amino acid sequence of SEQ ID NO: 17; b) CDR1 having the amino acid sequence of SEQ ID NO: 8, CDR2 having the amino acid sequence of SEQ ID NO: 13, and CDR3 having the amino acid sequence of SEQ ID NO: 18; c) CDR1 having the amino acid sequence of SEQ ID NO: 9, CDR2 having the amino acid sequence of SEQ ID NO: 14 and CDR3 having the amino acid sequence of SEQ ID NO: 19, or d) CDR1 having the amino acid sequence of SEQ ID NO: 11, CDR2 having the amino acid sequence of SEQ ID NO: 16, and CDR3 having the amino acid sequence of SEQ ID NO: 21 28. The polypeptide of embodiment 27, comprising:

[0052] Embodiment 29. The amino acid sequence of at least one ISVD is: a) greater than 90% sequence identity with SEQ ID NO:2; b) greater than 90% sequence identity with SEQ ID NO:3; c) greater than 90% sequence identity to SEQ ID NO:4; or d) greater than 90% sequence identity with SEQ ID NO:6 29. The polypeptide of any of embodiments 27 or 28, comprising:

[0053]

[0026] Embodiment 30. The at least one ISVD comprises: a) the amino acid sequence of SEQ ID NO:2; b) the amino acid sequence of SEQ ID NO:3; c) the amino acid sequence of SEQ ID NO: 4, or d) the amino acid sequence of SEQ ID NO:6 30. The polypeptide according to any one of embodiments 27 to 29, comprising:

[0054] Embodiment 31. The polypeptide comprises or consists of at least two ISVDs, each of which comprises three complementarity determining regions (CDR1 to CDR3, respectively), and the at least two ISVDs are optionally linked via one or more peptidic linkers: a) The first and second ISVDs are: i. CDR1 which is the amino acid sequence of SEQ ID NO:7 or has 2 or 1 amino acid difference from SEQ ID NO:7; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 12 or has two or one amino acid difference from SEQ ID NO: 12; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 17. Including, b) The first and second ISVDs are: i. CDR1 which is the amino acid sequence of SEQ ID NO:8 or has 2 or 1 amino acid difference from SEQ ID NO:8; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 13 or has two or one amino acid difference from SEQ ID NO: 13; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 18. Including, c) The first ISVD is: i. CDR1 which is the amino acid sequence of SEQ ID NO:7 or has 2 or 1 amino acid difference from SEQ ID NO:7; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 12 or has two or one amino acid difference from SEQ ID NO: 12; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 17. Including, The second ISVD is iv. CDR1 which is the amino acid sequence of SEQ ID NO:8 or has 2 or 1 amino acid difference from SEQ ID NO:8; v. CDR2 which is the amino acid sequence of SEQ ID NO:13 or has two or one amino acid difference from SEQ ID NO:13; and vi. CDR3 having the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 18 Including, d) The first ISVD is: i. CDR1 which is the amino acid sequence of SEQ ID NO:8 or has 2 or 1 amino acid difference from SEQ ID NO:8; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 13 or has two or one amino acid difference from SEQ ID NO: 13; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 18. Including, The second ISVD is iv. CDR1 which is the amino acid sequence of SEQ ID NO:7 or has 2 or 1 amino acid difference from SEQ ID NO:7; v. CDR2 which is the amino acid sequence of SEQ ID NO:12 or has two or one amino acid difference from SEQ ID NO:12; and vi. CDR3 having the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 17 Including, e) The first ISVD is: i. CDR1 which is the amino acid sequence of SEQ ID NO:11 or has 2 or 1 amino acid difference from SEQ ID NO:11; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 16 or has two or one amino acid difference from SEQ ID NO: 16; and iii. A CDR3 having the amino acid sequence of SEQ ID NO:21 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO:21. Including, The second ISVD is iv. CDR1 which is the amino acid sequence of SEQ ID NO:9 or has 2 or 1 amino acid difference from SEQ ID NO:9; v. CDR2 which is the amino acid sequence of SEQ ID NO:14 or has two or one amino acid difference from SEQ ID NO:14; and vi. CDR3 having the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 19 Contains, or f) The first ISVD is: i. CDR1 which is the amino acid sequence of SEQ ID NO:9 or has 2 or 1 amino acid difference from SEQ ID NO:9; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 14 or has two or one amino acid difference from SEQ ID NO: 14; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 19. Including, The second ISVD is iv. CDR1 which is the amino acid sequence of SEQ ID NO:11 or has 2 or 1 amino acid difference from SEQ ID NO:11; v. CDR2 which is the amino acid sequence of SEQ ID NO:16 or has two or one amino acid difference from SEQ ID NO:16; and vi. CDR3 having the amino acid sequence of SEQ ID NO:21 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO:21 Including, 30. The polypeptide of embodiment 1 or 27, wherein the order of the ISVDs indicates their relative position with respect to each other considering the N-terminus to C-terminus of the polypeptide.

[0055] Embodiment 32.a) the first and second ISVDs comprise a CDR1 having the amino acid sequence of SEQ ID NO:7, a CDR2 having the amino acid sequence of SEQ ID NO:12, and a CDR3 having the amino acid sequence of SEQ ID NO:17; b) the first and second ISVDs comprise a CDR1 having the amino acid sequence of SEQ ID NO:8, a CDR2 having the amino acid sequence of SEQ ID NO:13, and a CDR3 having the amino acid sequence of SEQ ID NO:18; c) the first ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO:7, a CDR2 having the amino acid sequence of SEQ ID NO:12, and a CDR3 having the amino acid sequence of SEQ ID NO:17, and the second ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO:8, a CDR2 having the amino acid sequence of SEQ ID NO:13, and a CDR3 having the amino acid sequence of SEQ ID NO:18; d) the first ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO:8, a CDR2 having the amino acid sequence of SEQ ID NO:13, and a CDR3 having the amino acid sequence of SEQ ID NO:18, and the second ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO:7, a CDR2 having the amino acid sequence of SEQ ID NO:12, and a CDR3 having the amino acid sequence of SEQ ID NO:17; e) the first ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO: 11, a CDR2 having the amino acid sequence of SEQ ID NO: 16, and a CDR3 having the amino acid sequence of SEQ ID NO: 21, and the second ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO: 9, a CDR2 having the amino acid sequence of SEQ ID NO: 14, and a CDR3 having the amino acid sequence of SEQ ID NO: 19, or f) A polypeptide as described in embodiment 31, wherein the first ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO: 9, a CDR2 having the amino acid sequence of SEQ ID NO: 14, and a CDR3 having the amino acid sequence of SEQ ID NO: 19, and the second ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO: 11, a CDR2 having the amino acid sequence of SEQ ID NO: 16, and a CDR3 having the amino acid sequence of SEQ ID NO: 21.

[0056] Embodiment 33.a) the amino acid sequence of the first and second ISVD comprises greater than 90% sequence identity with SEQ ID NO:2; b) the amino acid sequences of the first and second ISVDs comprise greater than 90% sequence identity with SEQ ID NO:3; c) the amino acid sequence of the first ISVD comprises greater than 90% sequence identity to SEQ ID NO:2 and the second ISVD comprises greater than 90% sequence identity to SEQ ID NO:3; d) the amino acid sequence of the first ISVD comprises greater than 90% sequence identity to SEQ ID NO:3 and the second ISVD comprises greater than 90% sequence identity to SEQ ID NO:2; e) the amino acid sequence of the first ISVD comprises greater than 90% sequence identity to SEQ ID NO:6 and the second ISVD comprises greater than 90% sequence identity to SEQ ID NO:4; f) A polypeptide according to any of embodiments 31 or 32, wherein the amino acid sequence of the first ISVD comprises greater than 90% sequence identity with SEQ ID NO:4 and the second ISVD comprises greater than 90% sequence identity with SEQ ID NO:6.

[0057] In embodiment 34.a) the first and second ISVDs comprise the amino acid sequence of SEQ ID NO:2; b) the first and second ISVDs comprise the amino acid sequence of SEQ ID NO:3; c) the first ISVD comprises the amino acid sequence of SEQ ID NO:2 and the second ISVD comprises the amino acid sequence of SEQ ID NO:3; d) the first ISVD comprises the amino acid sequence of SEQ ID NO:3 and the second ISVD comprises the amino acid sequence of SEQ ID NO:2; e) the first ISVD comprises the amino acid sequence of SEQ ID NO:6 and the second ISVD comprises the amino acid sequence of SEQ ID NO:4; or f) A polypeptide described in any one of embodiments 31 to 33, wherein the first ISVD comprises the amino acid sequence of SEQ ID NO: 4 and the second ISVD comprises the amino acid sequence of SEQ ID NO: 6.

[0058] Embodiment 35.a) The amino acid sequence of SEQ ID NO: 148, b) the amino acid sequence of SEQ ID NO: 149; c) the amino acid sequence of SEQ ID NO: 150; d) the amino acid sequence of SEQ ID NO: 151; e) the amino acid sequence of SEQ ID NO: 152; f) the amino acid sequence of SEQ ID NO: 153; g) the amino acid sequence of SEQ ID NO: 154; h) the amino acid sequence of SEQ ID NO: 155; i) the amino acid sequence of SEQ ID NO: 156; j) the amino acid sequence of SEQ ID NO: 157; k) the amino acid sequence of SEQ ID NO: 158, or l) Amino acid sequence of SEQ ID NO: 159 35. The polypeptide according to any one of embodiments 31 to 34, comprising or consisting of:

[0059] Embodiment 36. The polypeptide comprises or consists of at least four ISVDs, each of which comprises three complementarity determining regions (CDR1 to CDR3, respectively), and the at least four ISVDs are optionally linked via one or more peptidic linkers: a) The first ISVD is: i. CDR1 which is the amino acid sequence of SEQ ID NO:7 or has 2 or 1 amino acid difference from SEQ ID NO:7; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 12 or has two or one amino acid difference from SEQ ID NO: 12; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 17. Includes; b) The second ISVD is: iv. CDR1 which is the amino acid sequence of SEQ ID NO:8 or has 2 or 1 amino acid difference from SEQ ID NO:8; v. CDR2 which is the amino acid sequence of SEQ ID NO:13 or has two or one amino acid difference from SEQ ID NO:13; and vi. CDR3 having the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 18 Includes; c) The third ISVD is: vii. CDR1 which is an amino acid sequence of SEQ ID NO:9 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO:9; viii. CDR2 is an amino acid sequence of SEQ ID NO: 14 or has two or one amino acid difference from SEQ ID NO: 14; and ix. CDR3 having the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 19 Includes; d) The fourth ISVD is: x. CDR1 which is the amino acid sequence of SEQ ID NO:11 or has 2 or 1 amino acid difference from SEQ ID NO:11; xi. CDR2 is an amino acid sequence of SEQ ID NO: 16 or has 2 or 1 amino acid difference from SEQ ID NO: 16; and xii. CDR3 having the amino acid sequence of SEQ ID NO:21 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO:21 Including, 32. The polypeptide of any of embodiments 27 or 31, wherein the order of the ISVDs indicates their relative position with respect to each other considering the N-terminus to C-terminus direction of the polypeptide.

[0060] Embodiment 37.a) the first ISVD comprises a CDR1 which is the amino acid sequence of SEQ ID NO:7, a CDR2 which is the amino acid sequence of SEQ ID NO:12, and a CDR3 which is the amino acid sequence of SEQ ID NO:17; b) the second ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO:8, a CDR2 having the amino acid sequence of SEQ ID NO:13, and a CDR3 having the amino acid sequence of SEQ ID NO:18; c) the third ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO: 9, a CDR2 having the amino acid sequence of SEQ ID NO: 14, and a CDR3 having the amino acid sequence of SEQ ID NO: 19; d) The polypeptide of embodiment 36, wherein the fourth ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO: 11, a CDR2 having the amino acid sequence of SEQ ID NO: 16, and a CDR3 having the amino acid sequence of SEQ ID NO: 21.

[0061] Embodiment 38.a) the amino acid sequence of the first ISVD comprises greater than 90% sequence identity with SEQ ID NO:2; b) the amino acid sequence of the second ISVD comprises greater than 90% sequence identity to SEQ ID NO:3; c) the amino acid sequence of the third ISVD comprises greater than 90% sequence identity to SEQ ID NO:4; d) The polypeptide of any of embodiments 36 or 37, wherein the amino acid sequence of the fourth ISVD comprises more than 90% sequence identity with SEQ ID NO:6.

[0062] Embodiment 39.a) the first ISVD comprises the amino acid sequence of SEQ ID NO:2; b) the second ISVD comprises the amino acid sequence of SEQ ID NO:3; c) the third ISVD comprises the amino acid sequence of SEQ ID NO:4; d) The polypeptide according to any one of embodiments 36 to 38, wherein the fourth ISVD comprises the amino acid sequence of SEQ ID NO:6.

[0063] Embodiment 40. A polypeptide according to any of embodiments 27 to 39, wherein the polypeptide further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers, which one or more other groups, residues, moieties or binding units provide the polypeptide with an increased half-life compared to a corresponding polypeptide without said one or more other groups, residues, moieties or binding units.

[0064] Embodiment 41. The polypeptide of embodiment 40, wherein the one or more other groups, residues, moieties or binding units providing the polypeptide with increased half-life are selected from the group consisting of polyethylene glycol molecules, serum proteins or fragments thereof, binding units capable of binding to serum proteins, Fc moieties, and small proteins or peptides capable of binding to serum proteins.

[0065] Embodiment 42. A polypeptide according to any one of embodiments 40 to 41, wherein the one or more other binding units providing the polypeptide with an increased half-life are selected from the group consisting of binding units capable of binding to serum albumin (such as human serum albumin) or serum immunoglobulin (such as IgG).

[0066] Embodiment 43. The polypeptide of embodiment 42, wherein the binding unit that provides the polypeptide with increased half-life is an ISVD capable of binding to human serum albumin.

[0067] Embodiment 44. The ISVD that binds to human serum albumin is i. CDR1 which is the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 10; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 15 or has two or one amino acid difference from SEQ ID NO: 15; and iii. A CDR3 having the amino acid sequence of SEQ ID NO:20 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO:20. 44. The polypeptide of embodiment 43, comprising:

[0068] Embodiment 45. A polypeptide described in any of embodiments 43 to 44, wherein the ISVD that binds to human serum albumin comprises CDR1 having the amino acid sequence of SEQ ID NO: 10, CDR2 having the amino acid sequence of SEQ ID NO: 15, and CDR3 having the amino acid sequence of SEQ ID NO: 20.

[0069] Embodiment 46. A polypeptide described in any one of embodiments 43 to 45, wherein the amino acid sequence of the ISVD that binds to human serum albumin comprises more than 90% sequence identity with SEQ ID NO:5.

[0070] Embodiment 47. A polypeptide described in any one of embodiments 43 to 46, wherein the ISVD that binds to human serum albumin comprises the amino acid sequence of SEQ ID NO:5.

[0071] Embodiment 48. A polypeptide according to any one of embodiments 36 to 47, wherein the amino acid sequence of the polypeptide comprises more than 90% sequence identity with SEQ ID NO:1.

[0072] Embodiment 49. A polypeptide according to any one of embodiments 36 to 48, comprising or consisting of the amino acid sequence of SEQ ID NO:1.

[0073] Embodiment 50. A nucleic acid comprising a nucleotide sequence encoding a polypeptide according to any of embodiments 27 to 49, or a polypeptide according to embodiments 36 to 49.

[0074] Embodiment 51. A host or host cell comprising a nucleic acid according to embodiment 50.

[0075] Embodiment 52. A method for producing a polypeptide according to any of embodiments 27 to 49 or a polypeptide according to embodiments 36 to 49, comprising at least: a) expressing the nucleic acid according to embodiment 50 in a suitable host cell or host organism or in another suitable expression system; optionally followed by: b) isolating and / or purifying a polypeptide according to any of embodiments 27 to 49 or a polypeptide according to embodiments 36 to 49 The method comprising:

[0076] Embodiment 53. A composition comprising at least one polypeptide according to any of embodiments 27 to 49, or at least one polypeptide according to embodiments 36 to 49, or a nucleic acid according to embodiment 50.

[0077] Embodiment 54. The composition according to embodiment 53, which is a pharmaceutical composition further comprising at least one pharma- ceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally one or more additional pharma- ceutically active polypeptides and / or compounds.

[0078] Embodiment 55. A method for treating an inflammatory disease, such as a type 2 inflammatory disease, comprising administering to a subject in need thereof a pharma- ceutical active amount of a polypeptide according to any of embodiments 27-49, or a polypeptide according to embodiments 36-49, or a composition according to any of embodiments 53-54.

[0079] The method of embodiment 55, wherein the type 2 inflammatory disease is selected from asthma and atopic dermatitis.

[0080] Embodiment 57. Use of a polypeptide according to any of embodiments 27 to 49, or a polypeptide according to embodiments 36 to 49, or a composition according to any of embodiments 53 to 54 in the preparation of a pharmaceutical composition for treating an inflammatory disease, such as a type 2 inflammatory disease.

[0081] Embodiment 58. The use of a polypeptide or composition according to embodiment 57, wherein the type 2 inflammatory disease is selected from asthma and atopic dermatitis. [Brief description of the drawings]

[0082] [Figure 1] FIG. 13 is a sensorgram showing simultaneous binding of IL13 and HSA to TSLP-captured F027400161. [Diagram 2]FIG. 1 shows inhibition of human, cynomolgus and rhesus IL13 in an eotaxin release assay by VHH F027400161 and reference compounds anti-hIL-13mAb1 and anti-hIL-13mAb2. [Diagram 3] FIG. 1 shows inhibition of human and cynomolgus IL13 in a SEAP reporter assay by F027400161 and reference compounds anti-hIL-13mAb1 and anti-hIL-13mAb2. [Figure 4] FIG. 1 shows inhibition of human and cynomolgus TSLP induced BaF3 proliferation by F027400161 and the reference compound anti-hTSLP mAb 1. IRR00096 is a negative control Nb. [Diagram 5] Box plot showing binding of pre-existing antibodies present in 96 human serum samples to F027400161, 163 and 164 compared to the control F027301186. [Figure 6] Dose-response inhibition profile of F-27400161 (also called F027400161) and comparator antibody anti-hTSLP reference mAb1 against TSLP-induced CCL17 responses in human DCs. Enriched human DCs were treated with 4 ng / mL TSLP and incubated with eight concentrations of ISVD constructs and anti-hTSLP reference mAb1 for 36 hours. CCL17 concentrations in freshly collected supernatants were measured by ELISA. IC50 values ​​were calculated in Graphpad Prism 8.0 by nonlinear regression (four-parameter least-squares fitting of log inhibitor vs. response-variable slope) without constraints. Data are presented as mean ± standard error (SEM) of all eight donors combined from eight individual experiments. [Figure 7]Figure 1 shows the dose inhibitory response of F-27400161 (also referred to as F027400161) and comparators anti-hIL-13 reference mAb1 and anti-hTSLP reference mAb1 against 0.5ng / mL IL-13 and TSLP-induced synergistic production of CCL17 in human PBMCs. Healthy donor PBMCs were stimulated with 0.5ng / mL recombinant IL-13+TSLP and incubated with 10 doses of ISVD constructs (Nb) and comparators for 20 hours. CCL17 concentrations in cell culture supernatants were measured by MSD V-Plex kit. IC50 values ​​were calculated in Graphpad Prism 8.0 by nonlinear regression (four-parameter least-squares fitting of log inhibitor vs. response-variable slope) without constraints. Data are presented as mean ± standard error (SEM) of all donors combined from eight individual experiments. [Figure 8] Figure 1 shows the dose inhibitory response of F-27400161 (also referred to as F027400161) and comparator antibodies anti-hIL-13 ref mAb1 and anti-hTSLP ref mAb1 on the synergistic production of CCL17 induced by 5ng / mL IL-13 and TSLP in human PBMC. Healthy donor PBMC were stimulated with 5ng / mL recombinant IL-13+TSLP and incubated with 10 doses of ISVD constructs (Nb) and comparator antibodies for 20 hours. CCL17 concentrations in freshly collected supernatants were measured by MSD V-Plex kit. IC50 values ​​were calculated in Graphpad Prism 8.0 by nonlinear regression (four-parameter least-squares fitting of log inhibitor vs. response-variable slope) without constraints. Data are presented as mean ± standard error (SEM) of all donors combined from eight individual experiments. [Figure 9]Figure 1 shows the inhibition profile of F-27400161 (also called F027400161) and comparator antibodies anti-hIL-13 Reference mAb1 and anti-hTSLP Reference mAb1 against allergen Der P-induced IL-5, CCL17, and CCL26 production by human PBMC in an assay of three cultures. Normal donor PBMCs cultured with MRC5 fibroblasts and A549 epithelial cells were stimulated with 3 mg / mL Der P and incubated with 11.1 nM ISVD, anti-hIL-13 Reference mAb1, or anti-hTSLP Reference mAb1 in a 24-well plate for 6 days in a cell culture incubator at 37°C. IL-5, CCL17, and CCL26 concentrations in freshly collected supernatants were measured by Human Magnetic Luminex Assay. The percentage of inhibition was calculated relative to unstimulated (min) and stimulated (max) control samples that received neither ISVD polypeptide nor antibody. All calculations were performed using GraphPad Prism 8.0. Data are presented as the mean ± standard error (SEM) of all donors combined from three independent experiments. [Figure 10] F027400161 significantly reduced detectable levels of human TSLP in the plasma of NSG-SGM3 mice. Human plasma TSLP levels were reduced at 0.1 mg / kg F027400161 dose (45.772 pg / ml) and 10 mg / kg F027400161 dose (0.072 pg / ml) compared to vehicle-treated mice (376.166 pg / ml), demonstrating that the TSLP arm of F027400161 can bind human TSLP in the plasma of humanized NSG-SGM3 mice. [Figure 11]F027400161 significantly reduced detectable levels of human IL-13 in the plasma of NSG-SGM3 mice. Compared to vehicle-treated mice (274.052pg / ml), human IL-13 levels were reduced at 0.01mg / kg F027400161 dose (201.286pg / ml), 0.05mg / kg F027400161 dose (22.028pg / ml), 0.1mg / kg F027400161 dose (40.740pg / ml), and 10mg / kg F027400161 dose (1.777pg / ml), demonstrating that the IL-13 arm of F27400161 can bind human IL-13 in the plasma of humanized NSG-SGM3 mice. [Figure 12] F027400161 significantly reduced mouse Retnla and Clca1 transcriptional expression in the lungs of NSG-SGM3 mice receiving hydrodynamic delivery of hTSLP and hIL-4. Overexpression of human TSLP and IL-4 in NSG-SGM3 mice induces the production of hIL-13 from precursor CD34+ derived human immune cells. Neutralization of human IL-13 by F027400161 resulted in inhibition of mouse Retnla and Clca1 marker genes at a dose of 10mg / Kg. [Figure 13] F027400161 significantly reduced mouse Retnla and Clca1 transcriptional expression in the lungs of NSG-SGM3 mice receiving hydrodynamic delivery of hTSLP and hIL-4. Overexpression of human TSLP and IL-4 in NSG-SGM3 mice induces the production of hIL-13 from precursor CD34+ derived human immune cells. Neutralization of human IL-13 by F027400161 resulted in inhibition of mouse Retnla and Clca1 marker genes at a dose of 10mg / Kg. [Figure 14]Schematic representation of ISVD construct F027400161 showing from N-terminus to C-terminus monovalent building blocks / ISVDs 4B02, 4B06, 501A02, 529F10 plus albumin binder ALB23002, whereby 4B02 and 4B06 are connected via a 35GS linker and the remaining building blocks are linked via a 9GS linker. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0083] 5 Detailed description of this technology The present technology aims to provide a new type of drug for treating inflammatory diseases such as atopic dermatitis and asthma.

[0084] In some embodiments, the technology relates to polypeptides that simultaneously target IL-13 and TSLP in vitro and / or in vivo, resulting in increased efficiency of modulating type 2 inflammatory responses compared to monospecific anti-IL-13 or anti-TSLP polypeptides. In some embodiments, the polypeptides are efficiently produced (e.g., in microbial hosts). Furthermore, in some embodiments, such polypeptides can show only limited reactivity to pre-existing antibodies in the subject to be treated (i.e., antibodies present in the subject before the first treatment with the antibody construct). In other embodiments, such polypeptides exhibit a sufficiently long half-life in the subject to be treated to allow for convenient spacing between successive treatments.

[0085] 5.1 Polypeptide of the Present Technology Monospecific Monovalent Polypeptides In one embodiment, the polypeptides of the present technology are monospecific and monovalent.

[0086] The term "monospecific" refers to binding to one (specific) type of target molecule. Thus, the monospecific polypeptide of the present technology specifically binds to IL-13. The other monospecific polypeptide of the present technology specifically binds to TSLP.

[0087] The term "monovalent" indicates that there is only one binding unit / building block that (specifically) targets a molecule, e.g., an ISVD.

[0088] Thus, in one embodiment, the present technology provides a monospecific monovalent polypeptide comprising or consisting of one ISVD that specifically binds to IL-13, comprising three complementarity determining regions (CDR1-CDR3, respectively). a) a CDR1 which is the amino acid sequence of SEQ ID NO:7 or which has two or one amino acid difference from SEQ ID NO:7, a CDR2 which is the amino acid sequence of SEQ ID NO:12 or which has two or one amino acid difference from SEQ ID NO:12, and a CDR3 which is the amino acid sequence of SEQ ID NO:17 or which has two or one amino acid difference from SEQ ID NO:17; or b) CDR1 which is the amino acid sequence of SEQ ID NO:8 or has 2 or 1 amino acid difference from SEQ ID NO:8, CDR2 which is the amino acid sequence of SEQ ID NO:13 or has 2 or 1 amino acid difference from SEQ ID NO:13, and CDR3 which is the amino acid sequence of SEQ ID NO:18 or has 2 or 1 amino acid difference from SEQ ID NO:18. You can choose from ISVDs including:

[0089] In one embodiment of this aspect of the technology, the ISVD specifically binds human IL-13.

[0090] In a further embodiment, the ISVD that specifically binds IL-13 is: a) a CDR1 having the amino acid sequence of SEQ ID NO: 7, a CDR2 having the amino acid sequence of SEQ ID NO: 12, and a CDR3 having the amino acid sequence of SEQ ID NO: 17; or b) CDR1 having the amino acid sequence of SEQ ID NO:8, CDR2 having the amino acid sequence of SEQ ID NO:13, and CDR3 having the amino acid sequence of SEQ ID NO:18 The ISVDs are selected from those containing

[0091] In a further embodiment of this aspect of the technology, the ISVD that specifically binds IL-13 is: a) greater than 90% sequence identity to SEQ ID NO:2; or b) greater than 90% sequence identity with SEQ ID NO:3 The ISVDs are selected from those containing

[0092] In one embodiment, the ISVD that specifically binds to IL-13 is selected from an ISVD comprising the amino acid sequence of SEQ ID NO:2; or the amino acid sequence of SEQ ID NO:3.

[0093] In another embodiment, the present technology provides a monospecific monovalent polypeptide comprising or consisting of one ISVD that specifically binds to TSLP, the ISVD comprising three complementarity determining regions (CDR1-CDR3, respectively). a) a CDR1 which is the amino acid sequence of SEQ ID NO:9 or has 2 or 1 amino acid difference from SEQ ID NO:9, a CDR2 which is the amino acid sequence of SEQ ID NO:14 or has 2 or 1 amino acid difference from SEQ ID NO:14, and a CDR3 which is the amino acid sequence of SEQ ID NO:19 or has 2 or 1 amino acid difference from SEQ ID NO:19; or b) CDR1 which is the amino acid sequence of SEQ ID NO: 11 or has 2 or 1 amino acid difference from SEQ ID NO: 11, CDR2 which is the amino acid sequence of SEQ ID NO: 16 or has 2 or 1 amino acid difference from SEQ ID NO: 16, and CDR3 which is the amino acid sequence of SEQ ID NO: 21 or has 2 or 1 amino acid difference from SEQ ID NO: 21. You can choose from ISVDs including:

[0094] In one embodiment of this aspect of the technology, the ISVD specifically binds human TSLP.

[0095] In a further embodiment, the ISVD that specifically binds TSLP is: a) a CDR1 having the amino acid sequence of SEQ ID NO: 9, a CDR2 having the amino acid sequence of SEQ ID NO: 14, and a CDR3 having the amino acid sequence of SEQ ID NO: 19; or b) CDR1 having the amino acid sequence of SEQ ID NO: 11, CDR2 having the amino acid sequence of SEQ ID NO: 16, and CDR3 having the amino acid sequence of SEQ ID NO: 21 The ISVDs are selected from those containing

[0096] In a further embodiment of this aspect of the technology, the ISVD that specifically binds TSLP is: a) greater than 90% sequence identity to SEQ ID NO:4; or b) greater than 90% sequence identity with SEQ ID NO:6 The ISVDs are selected from those containing

[0097] In one embodiment, the ISVD that specifically binds to TSLP is selected from an ISVD comprising the amino acid sequence of SEQ ID NO:4; or the amino acid sequence of SEQ ID NO:6.

[0098] Monospecific Multivalent Polypeptides In another embodiment, the polypeptides of the present technology are monospecific and at least bivalent, but may be, for example, trivalent, tetravalent, pentavalent, hexavalent, etc.

[0099] The terms "bivalent", "trivalent", "tetravalent", "pentavalent", or "hexavalent" are all subclasses of the term "multivalent" and refer to the presence of two, three, four, five or six linkage units / building blocks, e.g., ISVDs, respectively.

[0100] Thus, in one aspect, the present technology provides a monospecific bivalent polypeptide comprising or consisting of two ISVDs that specifically bind to IL-13, each of the two ISVDs comprising three complementarity determining regions (CDR1-CDR3, respectively), the two ISVDs being optionally linked via one or more peptidic linkers: a) the first and second ISVDs comprise a CDR1 which is an amino acid sequence of SEQ ID NO:7 or has two or one amino acid difference from SEQ ID NO:7, a CDR2 which is an amino acid sequence of SEQ ID NO:12 or has two or one amino acid difference from SEQ ID NO:12, and a CDR3 which is an amino acid sequence of SEQ ID NO:17 or has two or one amino acid difference from SEQ ID NO:17; b) the first and second ISVDs comprise a CDR1 which is an amino acid sequence of SEQ ID NO:8 or has two or one amino acid difference from SEQ ID NO:8, a CDR2 which is an amino acid sequence of SEQ ID NO:13 or has two or one amino acid difference from SEQ ID NO:13, and a CDR3 which is an amino acid sequence of SEQ ID NO:18 or has two or one amino acid difference from SEQ ID NO:18; c) the first ISVD comprises a CDR1 which is an amino acid sequence of SEQ ID NO:7 or has two or one amino acid difference from SEQ ID NO:7, a CDR2 which is an amino acid sequence of SEQ ID NO:12 or has two or one amino acid difference from SEQ ID NO:12, and a CDR3 which is an amino acid sequence of SEQ ID NO:17 or has two or one amino acid difference from SEQ ID NO:17; the second ISVD comprises a CDR1 which is an amino acid sequence of SEQ ID NO:8 or has two or one amino acid difference from SEQ ID NO:8, a CDR2 which is an amino acid sequence of SEQ ID NO:13 or has two or one amino acid difference from SEQ ID NO:13, and a CDR3 which is an amino acid sequence of SEQ ID NO:18 or has two or one amino acid difference from SEQ ID NO:18; or d) the first ISVD comprises a CDR1 which is an amino acid sequence of SEQ ID NO:8 or has two or one amino acid difference from SEQ ID NO:8, a CDR2 which is an amino acid sequence of SEQ ID NO:13 or has two or one amino acid difference from SEQ ID NO:13, and a CDR3 which is an amino acid sequence of SEQ ID NO:18 or has two or one amino acid difference from SEQ ID NO:18; The second ISVD comprises a CDR1 which is an amino acid sequence of SEQ ID NO:7 or has two or one amino acid difference from SEQ ID NO:7, a CDR2 which is an amino acid sequence of SEQ ID NO:12 or has two or one amino acid difference from SEQ ID NO:12, and a CDR3 which is an amino acid sequence of SEQ ID NO:17 or has two or one amino acid difference from SEQ ID NO:17. Monospecific bivalent polypeptides are provided.

[0101] In one embodiment of this aspect of the technology, the ISVDs are linked via one or more peptidic linkers. In one embodiment, the two ISVDs specifically bind human IL13.

[0102] In a further embodiment, the monospecific bivalent polypeptide comprises or consists of two ISVDs that specifically bind to IL-13: a) the first and second ISVDs comprise a CDR1 that is the amino acid sequence of SEQ ID NO:7, a CDR2 that is the amino acid sequence of SEQ ID NO:12, and a CDR3 that is the amino acid sequence of SEQ ID NO:17; b) the first and second ISVDs comprise a CDR1 which is the amino acid sequence of SEQ ID NO:8, a CDR2 which is the amino acid sequence of SEQ ID NO:13, and a CDR3 which is the amino acid sequence of SEQ ID NO:18; c) the first ISVD comprises a CDR1 which is the amino acid sequence of SEQ ID NO:7, a CDR2 which is the amino acid sequence of SEQ ID NO:12, and a CDR3 which is the amino acid sequence of SEQ ID NO:17, and the second ISVD comprises a CDR1 which is the amino acid sequence of SEQ ID NO:8, a CDR2 which is the amino acid sequence of SEQ ID NO:13, and a CDR3 which is the amino acid sequence of SEQ ID NO:18; or d) the first ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO: 8, a CDR2 having the amino acid sequence of SEQ ID NO: 13, and a CDR3 having the amino acid sequence of SEQ ID NO: 18, and the second ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO: 7, a CDR2 having the amino acid sequence of SEQ ID NO: 12, and a CDR3 having the amino acid sequence of SEQ ID NO: 17.

[0103] In a further embodiment of this aspect of the technology, the monospecific bivalent polypeptide comprises or consists of two ISVDs that specifically bind to IL-13: a) the amino acid sequence of the first and second ISVDs comprises greater than 90% sequence identity with SEQ ID NO:2; b) the amino acid sequence of the first and second ISVDs comprises greater than 90% sequence identity with SEQ ID NO:3; c) the amino acid sequence of the first ISVD comprises greater than 90% sequence identity to SEQ ID NO:2 and the second ISVD comprises greater than 90% sequence identity to SEQ ID NO:3; or d) the amino acid sequence of the first ISVD comprises greater than 90% sequence identity with SEQ ID NO:3 and the second ISVD comprises greater than 90% sequence identity with SEQ ID NO:2.

[0104] In one embodiment, the monospecific bivalent polypeptide comprises or consists of two ISVDs that specifically bind to IL-13: a) the first and second ISVDs comprise the amino acid sequence of SEQ ID NO:2; b) the first and second ISVDs comprise the amino acid sequence of SEQ ID NO:3: c) the first ISVD comprises the amino acid sequence of SEQ ID NO:2 and the second ISVD comprises the amino acid sequence of SEQ ID NO:3; or d) the first ISVD comprises the amino acid sequence of SEQ ID NO:3 and the second ISVD comprises the amino acid sequence of SEQ ID NO:2.

[0105] In another aspect, the present technology provides a monospecific bivalent polypeptide comprising or consisting of two ISVDs that specifically bind to TSLP, each of the two ISVDs comprising three complementarity determining regions (CDR1-CDR3, respectively), the two ISVDs optionally being linked via one or more peptidic linkers: a) a first ISVD comprises a CDR1 which is an amino acid sequence of SEQ ID NO: 11 or has two or one amino acid difference from SEQ ID NO: 11, a CDR2 which is an amino acid sequence of SEQ ID NO: 16 or has two or one amino acid difference from SEQ ID NO: 16, and a CDR3 which is an amino acid sequence of SEQ ID NO: 21 or has two or one amino acid difference from SEQ ID NO: 21; The second ISVD comprises a CDR1 which is an amino acid sequence of SEQ ID NO:9 or has two or one amino acid difference from SEQ ID NO:9, a CDR2 which is an amino acid sequence of SEQ ID NO:14 or has two or one amino acid difference from SEQ ID NO:14, and a CDR3 which is an amino acid sequence of SEQ ID NO:19 or has two or one amino acid difference from SEQ ID NO:19; or b) the first ISVD comprises a CDR1 which is an amino acid sequence of SEQ ID NO:9 or has two or one amino acid difference from SEQ ID NO:9, a CDR2 which is an amino acid sequence of SEQ ID NO:14 or has two or one amino acid difference from SEQ ID NO:14, and a CDR3 which is an amino acid sequence of SEQ ID NO:19 or has two or one amino acid difference from SEQ ID NO:19; The second ISVD provides a monospecific bivalent polypeptide comprising a CDR1 which is the amino acid sequence of SEQ ID NO:11 or an amino acid sequence which has two or one amino acid difference from SEQ ID NO:11, a CDR2 which is the amino acid sequence of SEQ ID NO:16 or an amino acid sequence which has two or one amino acid difference from SEQ ID NO:16, and a CDR3 which is the amino acid sequence of SEQ ID NO:21 or an amino acid sequence which has two or one amino acid difference from SEQ ID NO:21.

[0106] In one embodiment of this aspect of the technology, the ISVDs are linked via one or more peptidic linkers. In one embodiment, the two ISVDs specifically bind human TSLP.

[0107] In a further embodiment, the monospecific bivalent polypeptide comprises or consists of two ISVDs that specifically bind TSLP: a) the first ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO: 11, a CDR2 having the amino acid sequence of SEQ ID NO: 16, and a CDR3 having the amino acid sequence of SEQ ID NO: 21, and the second ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO: 9, a CDR2 having the amino acid sequence of SEQ ID NO: 14, and a CDR3 having the amino acid sequence of SEQ ID NO: 19, or b) the first ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO: 9, a CDR2 having the amino acid sequence of SEQ ID NO: 14, and a CDR3 having the amino acid sequence of SEQ ID NO: 19, and the second ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO: 11, a CDR2 having the amino acid sequence of SEQ ID NO: 16, and a CDR3 having the amino acid sequence of SEQ ID NO: 21.

[0108] In a further embodiment of this aspect of the technology, the monospecific bivalent polypeptide comprises or consists of two ISVDs that specifically bind to TSLP: a) the amino acid sequence of the first ISVD comprises greater than 90% sequence identity to SEQ ID NO:6 and the second ISVD comprises greater than 90% sequence identity to SEQ ID NO:4; or b) the amino acid sequence of the first ISVD comprises greater than 90% sequence identity with SEQ ID NO:4 and the second ISVD comprises greater than 90% sequence identity with SEQ ID NO:6.

[0109] In one embodiment, the monospecific bivalent polypeptide comprises or consists of two ISVDs that specifically bind TSLP: a) the first ISVD comprises the amino acid sequence of SEQ ID NO:6 and the second ISVD comprises the amino acid sequence of SEQ ID NO:4; or b) the first ISVD comprises the amino acid sequence of SEQ ID NO:4 and the second ISVD comprises the amino acid sequence of SEQ ID NO:6.

[0110] In this regard, the terms "first ISVD" and "second ISVD" only indicate the relative positions of the specifically listed ISVDs that bind IL-13 / TSLP relative to each other, and the numbering starts from the N-terminus of the polypeptide of the present technology. Thus, the "first ISVD" is closer to the N-terminus than the "second ISVD". Correspondingly, the "second ISVD" is therefore closer to the C-terminus than the "first ISVD". Thus, the numbering is not absolute, but only indicates the relative positions of the two ISVDs, and does not exclude the possibility that additional binding units / building blocks, such as ISVDs, that bind IL-13 and TSLP, respectively, may be present in the polypeptide. Furthermore, it does not exclude the possibility that other binding units / building blocks, such as ISVDs, may be located therebetween. For example, as described further below (see in particular sections "Multispecific, multivalent polypeptides" and 5.4 "Extended half-life (in vivo)"), the polypeptide may further comprise another ISVD that binds to human serum albumin, which may also be located between the "first ISVD" and the "second ISVD" (such constructs are therefore referred to as multispecific, as described in the subsequent sections).

[0111] In one embodiment, the (at least two) ISVDs of a monospecific multivalent polypeptide, in particular of the monospecific bivalent polypeptides described above, are linked via a peptidic linker. The use of peptidic linkers to connect two or more (poly)peptides is well known in the art. Exemplary peptidic linkers that can be used with monospecific multivalent polypeptides, in particular with the monospecific bivalent polypeptides described above, are shown in Table A-5. One class of peptidic linkers that are often used are known as "Gly-Ser" or "GS" linkers. These are linkers that consist essentially of glycine (G) and serine (S) residues, and usually have a GGGGS (SEQ ID NO: 73) motif (e.g., the formula (Gly-Gly-Gly-Gly-Ser) n (wherein n can be 1, 2, 3, 4, 5, 6, 7 or more). Some frequently used examples of such GS linkers are the 9GS linker (GGGGSGGGS, SEQ ID NO: 76), the 15GS linker (n=3) and the 35GS linker (n=7). See Chen et al., Adv. Drug Deliv. Rev. 2013 Oct. 15; 65(10): 1357-1369; and Klein et al., Protein Eng. Des. Sel. (2014) 27(10): 325-330. In one embodiment of the present technology, the ISVDs of the monospecific multivalent polypeptide, in particular the monospecific bivalent polypeptide of the present technology, are linked via a linker as set forth in Table A-5. In one embodiment, (at least) two ISVDs are linked via a 35GS linker.

[0112] Thus, in one embodiment the monospecific bivalent polypeptide is: a) the amino acid sequence of SEQ ID NO: 148; b) the amino acid sequence of SEQ ID NO: 149; c) the amino acid sequence of SEQ ID NO: 150; d) the amino acid sequence of SEQ ID NO: 151; e) the amino acid sequence of SEQ ID NO: 152; f) the amino acid sequence of SEQ ID NO: 153; g) the amino acid sequence of SEQ ID NO: 154; h) the amino acid sequence of SEQ ID NO: 155; i) the amino acid sequence of SEQ ID NO: 156; j) the amino acid sequence of SEQ ID NO: 157; k) the amino acid sequence of SEQ ID NO: 158, or l) Amino acid sequence of SEQ ID NO: 159 It comprises or consists of:

[0113] Multispecific, multivalent polypeptides In further embodiments, the polypeptides of the present technology are at least bispecific, but may be, for example, trispecific, tetraspecific, pentaspecific, etc. Additionally, the polypeptides are at least bivalent, but may be, for example, trivalent, tetravalent, pentavalent, hexavalent, etc.

[0114] The terms "bispecific," "trispecific," "tetraspecific," "pentaspecific," etc., all fall under the term "having multiple specificities" and refer to binding to two, three, four, five, etc. different target molecules, respectively.

[0115] The terms "bivalent," "trivalent," "tetravalent," "pentavalent," "hexavalent," etc., are all referred to under the term "multivalent" and refer to the presence of two, three, four, five, six, etc. linking units / building blocks, e.g., ISVDs.

[0116] For example, the polypeptide may be bispecific tetravalent, e.g., a polypeptide comprising or consisting of at least four ISVDs, where at least two ISVDs specifically bind IL-13 and at least two ISVDs specifically bind TSLP. In one embodiment, the IL-13 and TSLP are human IL-13 and human TSLP. In another example, the polypeptide may be trispecific pentavalent, e.g., a polypeptide comprising or consisting of five ISVDs, where two ISVDs specifically bind human IL-13, two ISVDs specifically bind human TSLP, and one ISVD binds human serum albumin. Such a polypeptide may be simultaneously biparatopic, e.g., when two ISVDs bind two different epitopes on human IL-13 or human TSLP. The term "biparatopic" refers to binding to two different portions (e.g., epitopes) of the same target molecule. In one embodiment, the trispecific pentavalent polypeptide of the present technology is, for example, ISVD construct F027400161, which contains two ISVDs that specifically bind human IL-13, two ISVDs that specifically bind human TSLP, and one ISVD that binds human serum albumin, and is biparatopic with respect to binding to both IL-13 and TSLP.

[0117] In one embodiment, the multispecific multivalent polypeptide comprises or consists of at least four ISVDs, each of said ISVDs comprising three complementarity determining regions (CDR1 to CDR3, respectively), and the at least four ISVDs are optionally linked via one or more peptidic linkers: a) a first ISVD specifically binds to IL-13 and comprises a CDR1 which is an amino acid sequence of SEQ ID NO:7 or has two or one amino acid difference from SEQ ID NO:7, a CDR2 which is an amino acid sequence of SEQ ID NO:12 or has two or one amino acid difference from SEQ ID NO:12, and a CDR3 which is an amino acid sequence of SEQ ID NO:17 or has two or one amino acid difference from SEQ ID NO:17; b) a second ISVD specifically binds to IL-13 and comprises a CDR1 which is an amino acid sequence of SEQ ID NO:8 or has two or one amino acid difference from SEQ ID NO:8, a CDR2 which is an amino acid sequence of SEQ ID NO:13 or has two or one amino acid difference from SEQ ID NO:13, and a CDR3 which is an amino acid sequence of SEQ ID NO:18 or has two or one amino acid difference from SEQ ID NO:18; c) a third ISVD specifically binds to TSLP and comprises a CDR1 having an amino acid sequence of SEQ ID NO:9 or having two or one amino acid difference from SEQ ID NO:9, a CDR2 having an amino acid sequence of SEQ ID NO:14 or having two or one amino acid difference from SEQ ID NO:14, and a CDR3 having an amino acid sequence of SEQ ID NO:19 or having two or one amino acid difference from SEQ ID NO:19; d) The fourth ISVD specifically binds to TSLP and comprises a CDR1 that is an amino acid sequence of SEQ ID NO:11 or an amino acid sequence having two or one amino acid difference from SEQ ID NO:11, a CDR2 that is an amino acid sequence of SEQ ID NO:16 or an amino acid sequence having two or one amino acid difference from SEQ ID NO:16, and a CDR3 that is an amino acid sequence of SEQ ID NO:21 or an amino acid sequence having two or one amino acid difference from SEQ ID NO:21.

[0118] In one embodiment, the IL-13 and TSLP bound by the polypeptide are human IL-13 and human TSLP, respectively.

[0119] In a further embodiment of the multispecific multivalent polypeptide: a) the first ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO:7, a CDR2 having the amino acid sequence of SEQ ID NO:12, and a CDR3 having the amino acid sequence of SEQ ID NO:17; b) the second ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO:8, a CDR2 having the amino acid sequence of SEQ ID NO:13, and a CDR3 having the amino acid sequence of SEQ ID NO:18; c) the third ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO: 9, a CDR2 having the amino acid sequence of SEQ ID NO: 14, and a CDR3 having the amino acid sequence of SEQ ID NO: 19; d) the fourth ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO:11, a CDR2 having the amino acid sequence of SEQ ID NO:16, and a CDR3 having the amino acid sequence of SEQ ID NO:21.

[0120] In a further embodiment of the multispecific multivalent polypeptide: a) the amino acid sequence of the first ISVD comprises greater than 90% sequence identity to SEQ ID NO:2; b) the amino acid sequence of the second ISVD comprises greater than 90% sequence identity to SEQ ID NO:3; c) the amino acid sequence of the third ISVD comprises greater than 90% sequence identity to SEQ ID NO:4; d) the amino acid sequence of the fourth ISVD comprises greater than 90% sequence identity with SEQ ID NO:6.

[0121] In one embodiment of a multispecific, multivalent polypeptide: a) the first ISVD comprises the amino acid sequence of SEQ ID NO:2; b) the second ISVD comprises the amino acid sequence of SEQ ID NO:3; c) the third ISVD comprises the amino acid sequence of SEQ ID NO:4; d) The fourth ISVD comprises the amino acid sequence of SEQ ID NO:6.

[0122] In this regard, the terms "first ISVD", "second ISVD", "third ISVD", etc., only indicate the relative positions of the ISVDs to each other, and the numbering starts from the N-terminus of the polypeptide of the present technology. Thus, the "first ISVD" is closer to the N-terminus than the "second ISVD", whereas the "second ISVD" is closer to the N-terminus than the "third ISVD". Thus, when considered from the C-terminus, the arrangement of the ISVDs is reversed. Since the numbering is not absolute and only indicates the relative positions of at least four ISVDs, it does not exclude that other binding units / building blocks that bind to IL-13 or TSLP, such as additional ISVDs, or ISVDs that bind to another target, may be present in the polypeptide. Moreover, the numbering does not exclude the possibility that other binding units / building blocks, such as ISVDs, may be placed in between. For example, as described further below (see in particular Section 5.4 "Extended (in vivo) half-life" below), the polypeptide may further comprise another ISVD that binds to human serum albumin, which may also be located, for example, between the "third ISVD" and the "fourth ISVD".

[0123] In another aspect, the technology provides bispecific bivalent polypeptides comprising an ISVD that specifically binds IL-13 or TSLP, as described in detail for monospecific monovalent polypeptides above (Section 5.1; "Monospecific Monovalent Polypeptides"), and an ISVD that binds human serum albumin, as described in detail below (Section 5.4; "Extended (In Vivo) Half-Life").

[0124] In another embodiment, the technology provides bispecific trivalent polypeptides comprising an ISVD that binds human serum albumin as described above (Section 5.1; "Monospecific Bivalent Polypeptides") and below (Section 5.4; "Extended (In Vivo) Half-Life").

[0125] The components of said multispecific multivalent polypeptide, eg ISVDs, may be linked to each other by one or more suitable linkers, eg peptidic linkers.

[0126] The use of linkers to connect two or more (poly)peptides is well known in the art. Exemplary peptidic linkers are shown in Table A-5. One class of peptidic linkers that is frequently used is known as the "Gly-Ser" or "GS" linker. These are linkers that consist essentially of glycine (G) and serine (S) residues, and usually have a GGGGS (SEQ ID NO: 73) motif (e.g., the formula (Gly-Gly-Gly-Gly-Ser) n (wherein n can be 1, 2, 3, 4, 5, 6, 7 or more). Some frequently used examples of such GS linkers are the 9GS linker (GGGGSGGGS, SEQ ID NO: 76), the 15GS linker (n=3) (SEQ ID NO: 78) and the 35GS linker (n=7) (SEQ ID NO: 83). See Chen et al., Adv. Drug Deliv. Rev. 2013 Oct. 15; 65(10): 1357-1369; and Klein et al., Protein Eng. Des. Sel. (2014) 27(10): 325-330. In the polypeptide of the present technology, the 9GS (SEQ ID NO: 76) and 35GS (SEQ ID NO: 83) linkers are used to link the components of the polypeptide together.

[0127] In one embodiment of the multispecific multivalent polypeptide of the present technology, the polypeptide comprising or consisting of at least four ISVDs comprises at least two ISVDs that specifically bind to IL-13 and at least two ISVDs that specifically bind to TSLP. In this embodiment of the present technology, the at least two ISVDs that specifically bind to IL-13 are linked via a 35GS linker, whereas the at least two ISVDs that specifically bind to TSLP are linked via a 9GS linker. In one embodiment, the at least two ISVDs that specifically bind to TSLP are separated by an ISVD that binds to albumin (9GS-Alb-9GS) (as described in Section 5.4 "Extended half-life (in vivo)" below). The inventors have surprisingly found that such a configuration can increase the production yield of the polypeptide.

[0128] Thus, in one embodiment, the polypeptide comprises or consists of, in order starting from the N-terminus of the polypeptide: a first ISVD that specifically binds IL-13, a second ISVD that specifically binds IL-13, a first ISVD that specifically binds TSLP, an optional binding unit that provides a polypeptide with increased half-life as defined herein, and a second ISVD that specifically binds TSLP. In one embodiment, the binding unit that provides a polypeptide with increased half-life is an ISVD.

[0129] In a further embodiment, the polypeptide comprises or consists of, in order starting from the N-terminus of the polypeptide: an ISVD that specifically binds IL-13, a linker, a second ISVD that specifically binds IL-13, a linker, a first ISVD that specifically binds TSLP, a linker, an ISVD that binds human serum albumin, a linker, and a second ISVD that specifically binds TSLP. In a specific embodiment, the linker between the two ISVDs that bind IL-13 is a 35GS linker, whereas the other linker is a 9GS linker.

[0130] Such a configuration of the polypeptide can result in increased production yields, superior CMC characteristics, such as sufficient solubility and biophysical stability, strong potency for modulating type 2 immune responses, as well as reduced binding to pre-existing antibodies.

[0131] In one embodiment, the multispecific multivalent polypeptides of the present technology exhibit reduced binding by pre-existing antibodies in human serum. To this end, in one embodiment, the polypeptide comprises a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering) in at least one ISVD. In one embodiment, the polypeptide comprises a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering) in each ISVD. In another embodiment, the polypeptide comprises a stretch of 1-5 (naturally occurring) amino acids, such as a stretch of a single alanine (A) at the C-terminus of the ISVD. The C-terminus of the ISVD is typically VTVSS (SEQ ID NO: 138). In another embodiment, the polypeptide comprises a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) in at least one ISVD. In another embodiment, the ISVDs include a lysine (K) or glutamine (Q) at position 112 (according to Kabat numbering) in at least one ISVD. In these embodiments, the C-terminus of the ISVD is VKVSS (SEQ ID NO:139), VQVSS (SEQ ID NO:140), VTVKS (SEQ ID NO:166), VTVQS (SEQ ID NO:167), VKVKS (SEQ ID NO:168), VKVQS (SEQ ID NO:169), VQVKS (SEQ ID NO:170), or VQVQS (SEQ ID NO:171), such that after the addition of a single alanine, the C-terminus of the polypeptide comprises, for example, the sequence VTVSSA (SEQ ID NO:141), VKVSSA (SEQ ID NO:142), VQVSSA (SEQ ID NO:143), VTVKSA (SEQ ID NO:172), VTVQSA (SEQ ID NO:173), VKVKSA (SEQ ID NO:174), VKVQSA (SEQ ID NO:175), VQVKSA (SEQ ID NO:176), or VQVQSA (SEQ ID NO:177). In one embodiment, the C-terminus comprises VTVSSA (SEQ ID NO:141).In another embodiment, the polypeptide comprises in each ISVD a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering), and optionally in at least one ISVD a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering), and comprises a stretch of 1 to 5 (naturally occurring) amino acids, such as a single alanine (A) stretch at the C-terminus of the ISVD (such that the C-terminus of the polypeptide has, for example, the sequence VTVSSA (SEQ ID NO: 141), VKVSSA (SEQ ID NO: 142) or VQVSSA (SEQ ID NO: 143), e.g. VTVSSA (SEQ ID NO: 141). For further information on this, see, for example, WO2012 / 175741 and WO2015 / 173325.

[0132] In one embodiment, the multispecific, multivalent polypeptide of the present technology comprises or consists of an amino acid sequence that comprises more than 90% sequence identity, such as more than 95%, or more than 99% sequence identity, to SEQ ID NO:1, and the CDRs of the five ISVDs are as defined in items A to E (or A' to E', if using Kabat's definition) in sections "5.2 Immunoglobulin Single Variable Domains" and "5.4 Extension of (In Vivo) Half-Life", respectively, below, in particular: a first ISVD that specifically binds to IL-13 has a CDR1 that is the amino acid sequence of SEQ ID NO:7, a CDR2 that is the amino acid sequence of SEQ ID NO:12, and a CDR3 that is the amino acid sequence of SEQ ID NO:17; a second ISVD that specifically binds to IL-13 has a CDR1 that is the amino acid sequence of SEQ ID NO:8, a CDR2 that is the amino acid sequence of SEQ ID NO:13, and a CDR3 that is the amino acid sequence of SEQ ID NO:18; a third ISVD that specifically binds to TSLP has a CDR1 that is the amino acid sequence of SEQ ID NO:9, a CDR2 that is the amino acid sequence of SEQ ID NO:14, and a CDR3 that is the amino acid sequence of SEQ ID NO:19; a fourth ISVD that specifically binds to TSLP has a CDR1 that is the amino acid sequence of SEQ ID NO:11, a CDR2 that is the amino acid sequence of SEQ ID NO:16, and a CDR3 that is the amino acid sequence of SEQ ID NO:21; the ISVD that binds to human serum albumin has a CDR1 having the amino acid sequence of SEQ ID NO: 10, a CDR2 having the amino acid sequence of SEQ ID NO: 15, and a CDR3 having the amino acid sequence of SEQ ID NO: 20; Or alternatively, using Kabat's definition: a first ISVD that specifically binds to IL-13 has a CDR1 that is the amino acid sequence of SEQ ID NO: 37, a CDR2 that is the amino acid sequence of SEQ ID NO: 42, and a CDR3 that is the amino acid sequence of SEQ ID NO: 17; a second ISVD that specifically binds to IL-13 has a CDR1 that is the amino acid sequence of SEQ ID NO: 38, a CDR2 that is the amino acid sequence of SEQ ID NO: 43, and a CDR3 that is the amino acid sequence of SEQ ID NO: 18; a third ISVD that specifically binds to TSLP has a CDR1 that is the amino acid sequence of SEQ ID NO: 39, a CDR2 that is the amino acid sequence of SEQ ID NO: 44, and a CDR3 that is the amino acid sequence of SEQ ID NO: 19; a fourth ISVD that specifically binds to TSLP has a CDR1 that is the amino acid sequence of SEQ ID NO: 41, a CDR2 that is the amino acid sequence of SEQ ID NO: 46, and a CDR3 that is the amino acid sequence of SEQ ID NO: 21; The ISVD that binds to human serum albumin has a CDR1 that is the amino acid sequence of SEQ ID NO: 40, a CDR2 that is the amino acid sequence of SEQ ID NO: 45, and a CDR3 that is the amino acid sequence of SEQ ID NO: 20.

[0133] In another embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 1. In another embodiment, the polypeptide consists of the amino acid sequence of SEQ ID NO:1.

[0134] In one embodiment, the polypeptide of the present technology has at least half the binding affinity, or at least the same binding affinity, of human IL-13 and human TSLP when compared to a polypeptide consisting of the amino acids of SEQ ID NO:1, where the binding affinity is measured using the same method, e.g., surface plasmon resonance (SPR).

[0135] 5.2 Immunoglobulin Single Variable Domains The term "immunoglobulin single variable domain" (ISVD) is used synonymously with "single variable domain" and defines an immunoglobulin molecule in which an antigen binding site is present on and formed by a single immunoglobulin domain. This sets ISVDs apart from "conventional" immunoglobulins (e.g., monoclonal antibodies) or fragments thereof (e.g., Fab, Fab', F(ab')2, scFv, di-scFv), in which two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (V H ) and the light chain variable domain (V L ) interact with each other to form the antigen-binding site. In this case, V H and V L Both complementarity determining regions (CDRs) of the nucleotide sequence contribute to the antigen-binding site, i.e., a total of six CDRs are involved in forming the antigen-binding site.

[0136] In view of the above definition, the antigen-binding domains of traditional four-chain antibodies (e.g., IgG, IgM, IgA, IgD or IgE molecules; known in the art), or of Fab fragments, F(ab')2 fragments, Fv fragments, such as disulfide-linked Fv or scFv fragments, or diabodies derived from such traditional four-chain antibodies (all known in the art), are not typically considered ISVDs, because in these cases, binding to each epitope of an antigen typically does not occur by one (single) immunoglobulin domain, but by a pair (associated) immunoglobulin domains, e.g., a light chain and a heavy chain variable domain, i.e., the V domains of immunoglobulin domains that bind in conjunction to each epitope of an antigen. H -V L This is because it occurs in pairs.

[0137] In contrast, ISVDs are capable of specifically binding to an epitope of an antigen without pairing with an additional immunoglobulin variable domain. The binding site of an ISVD consists of a single V H , a single V HH or a single V L Formed by domains.

[0138] Thus, a single variable domain may be selected from the group consisting of a light chain variable domain sequence (e.g., a VV ... L -sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., V H -Array or V HH sequence) or a suitable fragment thereof.

[0139] The ISVD can be, for example, a heavy chain ISVD, for example a V H , V HH , e.g. camelized V H Or a humanized V HH In one embodiment, the ISVD may beHH For example, camelized V H or humanized V HH etc. The heavy chain ISVD may be derived from a traditional four chain antibody or a heavy chain antibody.

[0140] For example, an ISVD may be a single domain antibody (or a suitable amino acid sequence for use as a single domain antibody), a "dAb" or dAb (or a suitable amino acid sequence for use as a dAb), or a Nanobody® (as defined herein, examples of which include, but are not limited to, V HH ); other single variable domains, or any suitable fragment of any one of them.

[0141] In particular, ISVD is a Nanobody® (e.g., HH , e.g. humanized V HH or Camelization V H etc.) or a suitable fragment thereof. Nanobody®, Nanobodies® and Nanoclone® are registered trademarks of Ablynx NV.

[0142] "V HH Domain" is V HH , V HH Antibody fragments, and V HH Also known as antibodies, they were originally described as antigen binding variable domains of immunoglobulins that are "heavy chain antibodies" (i.e., "light chain deleted antibodies"; Hamers-Casterman et al., Nature 363:446-448, 1993). HH The "V domains" refer to these variable domains as those in a conventional four-chain antibody (referred to herein as "V H The heavy chain variable domains present in conventional four-chain antibodies (referred to herein as "V L The V domain was chosen to distinguish it from the light chain variable domains present in HHFor further discussion, reference is made to the review by Muyldermans (Reviews in Molecular Biotechnology 74:277-302, 2001).

[0143] Typically, production of immunoglobulins involves immunization of laboratory animals, fusing immunoglobulin-producing cells to generate hybridomas, and screening for the desired specificity. Alternatively, immunoglobulins may be produced by screening naive or synthetic libraries, for example by phage display.

[0144] The generation of immunoglobulin sequences, such as Nanobodies®, has been extensively described in various publications, among which WO94 / 04678, Hamers-Casterman et al., 1993 and Muyldermans et al., 2001 can be mentioned. In these methods, a camelid is immunized with a target antigen in order to induce an immune response against said target antigen. The repertoire of Nanobodies obtained from said immunization is further screened for Nanobodies that bind to the target antigen.

[0145] In these instances, the production of antibodies requires purified antigen for immunization and / or screening. The antigen may be purified from a natural source or may be purified during recombinant production.

[0146] Immunization and / or screening for immunoglobulin sequences can be carried out using peptide fragments of such antigens.

[0147] The technology can use immunoglobulin sequences of different origins, including mouse, rat, rabbit, donkey, human and camel immunoglobulin sequences. The technology also includes fully human, humanized or chimeric sequences. For example, the technology includes camel immunoglobulin sequences and humanized camel immunoglobulin sequences, or camelized domain antibodies, such as camelized dAbs, as described by Ward et al. (see, for example, WO 94 / 04678 and Davies and Riechmann (1994 and 1996)). In addition, the technology also includes fused immunoglobulin sequences, such as multivalent and / or multispecific constructs (one or more V HH Multivalent and multispecific polypeptides containing domains and their preparation (see Conrath et al., J. Biol. Chem., vol. 276, pp. 10.7346-7350, 2001; see also, e.g., WO 96 / 34103 and WO 99 / 23221), as well as fused immunoglobulin sequences forming immunoglobulin sequences containing tags or other functional moieties, e.g., toxins, labels, radiochemicals, etc., which are derivable from the immunoglobulin sequences of the present technology are also used.

[0148] "Humanized V HH " is a naturally occurring V HH domain, but is "humanized", i.e. HH One or more amino acid residues in the amino acid sequence of the sequence (particularly in the framework sequence) can be replaced with a V H It should be noted here that such humanized V domains include amino acid sequences that have been humanized by replacing them with one or more of the amino acid residues present at the corresponding positions in the V domain. This can be carried out in a manner known per se that will be clear to the skilled artisan, for example on the basis of the further explanations provided herein and in the prior art (e.g. WO2008 / 020079). HHcan be obtained in any suitable manner known per se and is therefore not strictly limited to polypeptides obtained using naturally occurring VHH domain-containing polypeptides as starting material.

[0149] "Camelization V H " is a naturally occurring V H The amino acid sequence of the V domain corresponds to that of the naturally occurring V from a conventional four-chain antibody, but has been "camelized", i.e. H One or more amino acid residues in the amino acid sequence of the domain are HH It includes amino acid sequences which have been camelized by replacing them with one or more of the amino acid residues present at the corresponding positions in the V domain. This can be carried out in a manner known per se that will be clear to the skilled person, for example on the basis of the further explanations given herein and in the prior art (e.g. WO2008 / 020079). Such "camelization" substitutions are usually H -V L and / or at amino acid positions forming and / or present at the boundaries of the camel V sequence and / or at the so-called camel hallmark residues as defined herein (see, for example, WO 94 / 04678 and Davies and Riechmann (1994 and 1996), supra). H V, which is used as a starting material or starting point for generating or designing H The sequences are V from mammals H sequence, or human V H Array, e.g. V H However, it is noteworthy that such camelized V H can be obtained in any suitable manner known per se and therefore does not necessarily have to be derived from the naturally occurring V H The present invention is not limited to polypeptides obtained using a polypeptide containing the domain.

[0150] The structure of an ISVD sequence can be considered to be composed of four framework regions ("FR"), which are referred to in the art and herein as "framework region 1" ("FR1"); "framework region 2" ("FR2"); "framework region 3" ("FR3"); and "framework region 4" ("FR4"); which are interrupted by three complementarity determining regions ("CDRs"), which are referred to in the art and herein as "complementarity determining region 1" ("CDR1"); "complementarity determining region 2" ("CDR2"); and "complementarity determining region 3" ("CDR3");

[0151] As further described in WO08 / 020079, pages 58 and 59, paragraph q), the amino acid residues of the ISVD are identical to those of the VSVD from camelids described in the article by Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240(1-2):185-195; see e.g. FIG. 2 of this publication). HH As applied to the domain, the V given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91) H It can be numbered according to the common numbering of domains. H Domains and V HH As is well known in the art for domains, the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions according to the Kabat numbering may be unoccupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat numbering). This means that, in general, the Kabat numbering may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. HDomains and V HH The total number of amino acid residues in a domain will usually be in the range of 110 to 120, often 112 to 115. However, it should be noted that shorter and longer sequences may also be suitable for the purposes described herein.

[0152] In this application, unless otherwise specified, the CDR sequences were determined according to the AbM definition as described in Kontermann and Dubel (Antibody Engineering, Vol. 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51, eds. 2010). According to this method, FR1 comprises amino acid residues at positions 1-25, CDR1 comprises amino acid residues at positions 26-35, FR2 comprises amino acid residues at positions 36-49, CDR2 comprises amino acid residues at positions 50-58, FR3 comprises amino acid residues at positions 59-94, CDR3 comprises amino acid residues at positions 95-102, and FR4 comprises amino acid residues at positions 103-113.

[0153] The determination of the CDR regions may be performed according to different methods: in the CDR determination according to Kabat, FR1 of the ISVD comprises amino acid residues at positions 1 to 30, CDR1 of the ISVD comprises amino acid residues at positions 31 to 35, FR2 of the ISVD comprises amino acid residues at positions 36 to 49, CDR2 of the ISVD comprises amino acid residues at positions 50 to 65, FR3 of the ISVD comprises amino acid residues at positions 66 to 94, CDR3 of the ISVD comprises amino acid residues at positions 95 to 102 and FR4 of the ISVD comprises amino acid residues at positions 103 to 113.

[0154] In such immunoglobulin sequences, the framework sequences may be any suitable framework sequence, and examples of suitable framework sequences will be clear to the skilled person based on, e.g., standard textbooks and the further disclosure and prior art set forth herein.

[0155] The framework sequences are immunoglobulin framework sequences or suitable combinations of framework sequences derived from immunoglobulin framework sequences (e.g., by humanization or camelization). For example, the framework sequences may be those of the light chain variable domain (e.g., V L sequence) and / or a heavy chain variable domain (e.g., V H Array or V HH In one embodiment, the framework sequence may be a framework sequence obtained from the V HH The framework sequences may be derived from the camel V sequences (wherein said framework sequences may optionally be partially or completely humanized) or may be derived from conventional camel V sequences. H A sequence (as defined herein) is either

[0156] In particular, the framework sequences present in the ISVD sequences used in the present technology are those that are suitable for use in the synthesis of Nanobodies®, such as humanized VSVs. HH or Camelization V H Contains V HH Some non-limiting examples of such framework sequences (suitable combinations thereof) will become clear from the further disclosure set forth herein.

[0157] Again, as generally described herein with respect to immunoglobulin sequences, it is also contemplated to use any suitable fragment (or combination of fragments) of the foregoing, for example a fragment containing one or more CDR sequences, preferably flanked by and / or linked via one or more framework sequences (e.g., these CDR and framework sequences in the same order as they may occur in the full-sized immunoglobulin sequence from which the fragment is derived).

[0158] Of note, however, the present technology is not limited by the origin of the ISVD sequence (or the nucleotide sequence used to express it), nor by the manner in which the ISVD sequence or nucleotide sequence is generated (or generated) or obtained. Thus, the ISVD sequence may be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In specific, but non-limiting, embodiments, the ISVD sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, examples of which include, but are not limited to, a "humanized" (as defined herein) immunoglobulin sequence (e.g., a partially or fully humanized mouse or rabbit immunoglobulin sequence, particularly a partially or fully humanized VHV sequence). HH immunoglobulin sequences), "camelized" (as defined herein) immunoglobulin sequences, as well as immunoglobulin sequences obtained by techniques such as affinity maturation (e.g., starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments obtained from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to those of skill in the art; or any suitable combination of any of the foregoing.

[0159] Likewise, the nucleotide sequence may be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence, for example a sequence isolated by PCR from a suitable naturally occurring template (e.g. DNA or RNA isolated from a cell), a nucleotide sequence isolated from a library (in particular an expression library), a nucleotide sequence prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, for example mismatch PCR), a nucleotide sequence prepared by PCR using overlapping primers, or a nucleotide sequence prepared using techniques for DNA synthesis known per se.

[0160] As mentioned above, the ISVD may be a Nanobody® or a suitable fragment thereof. For a general description of Nanobodies, reference is made to the further description below as well as to the prior art cited herein. However, in this respect, the description and the prior art are mainly directed to the so-called "V H 3 Class Nanobodies (i.e. V such as DP-47, DP-51 or DP-29) H It should be noted that this application describes three classes of Nanobodies with a high degree of sequence homology to human germline sequences. However, in its broadest sense, the present technology can generally be used with any type of Nanobody, including so-called "V Nanobodies," as described, for example, in WO2007 / 118670. H Nanobodies belonging to the "V 4 class" (i.e., DP-78, etc.) H It should be noted that four classes of Nanobodies with a high degree of sequence homology to human germline sequences are also used.

[0161] In general, Nanobodies (especially V HH Sequences such as (partially) humanized V HH Sequence and camelization V H A Nanobody may be characterized by the presence of one or more "hallmark residues" (as described herein) in one or more of the framework sequences (again as further described herein). Thus, in general, Nanobodies may be characterized by the (general) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 wherein FR1-FR4 refer to Framework Regions 1-4, respectively, and CDR1-CDR3 refer to Complementarity Determining Regions 1-3, respectively, and one or more of the Hallmark Residues are as further defined herein.

[0162] In particular, Nanobodies have the (general) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 where FR1-FR4 refer to framework regions 1-4, respectively, and CDR1-CDR3 refer to complementarity determining regions 1-3, respectively, and the framework sequences are as further defined herein.

[0163] More specifically, Nanobodies have the (general) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively: One or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are selected from the Hallmark Residues set forth in Table A-0 below.

[0164] [Table 1-1] [Table 1-2]

[0165] The technology employs, inter alia, ISVDs capable of binding to IL-13 or TSLP. In the context of the technology, "binding to" a particular target molecule has its normal meaning in the art as understood in the context of antibodies and their respective antigens.

[0166] The multispecific, multivalent polypeptides of the present technology may contain two or more ISVDs that specifically bind to IL-13 and two or more ISVDs that specifically bind to TSLP. For example, the polypeptide may contain two ISVDs that specifically bind to IL-13 and two ISVDs that specifically bind to TSLP.

[0167] In some embodiments, at least one ISVD can functionally block its target molecule. For example, the targeting moiety can block the interaction of IL-13 with IL-13Rα1 (interleukin 13 receptor, alpha 1) and / or the interaction of the IL-13 / IL-13Rα1 complex with IL-4Rα (alpha interleukin-4 receptor), or can block the interaction of TSLP with TSLPR (TSLP receptor) and / or the interaction of the TSLP / TSLPR complex with IL-7Rα (interleukin-7 receptor subunit alpha). Thus, in one embodiment, the polypeptide of the present technology comprises at least two ISVDs that specifically bind IL-13 and functionally block its interaction with IL-13Rα1 and / or the interaction of the IL-13 / IL-13Rα1 complex with IL-4Rα, and two ISVDs that specifically bind TSLP and functionally block its interaction with TSLPR and / or the interaction of the TSLP / TSLPR complex with IL-7Rα.

[0168] The ISVDs used in the present technology form part of the polypeptides of the present technology that comprise or consist of at least four ISVDs such that the polypeptides are capable of specifically binding to IL-13 and TSLP.

[0169] Therefore, the target molecules of at least four ISVDs used in the polypeptide of the present technology are IL-13 and TSLP.Examples thereof are mammalian IL-13 and TSLP.In addition to human IL-13 (Uniprot Accession No. P35225) and human TSLP (Uniprot Accession No. Q969D9), versions from other species are also suitable for the present technology, including, for example, IL-13 and TSLP from mouse, rat, rabbit, cat, dog, goat, sheep, horse, pig, non-human primate, such as cynomolgus monkey (also referred to as "cyno" herein), or camelid, such as llama or alpaca.

[0170] Specific examples of ISVDs that specifically bind to IL-13 that can be used in the present technology are described in sections A and B below: A. Binds specifically to human IL-13; i. CDR1 which is the amino acid sequence of SEQ ID NO:7 or has 2 or 1 amino acid difference from SEQ ID NO:7; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 12 or has two or one amino acid difference from SEQ ID NO: 12; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 17. ISVDs including:

[0171] In one embodiment, the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO:7, a CDR2 that is the amino acid sequence of SEQ ID NO:12, and a CDR3 that is the amino acid sequence of SEQ ID NO:17.

[0172] B. Binds specifically to human IL-13; i. CDR1 which is the amino acid sequence of SEQ ID NO:8 or has 2 or 1 amino acid difference from SEQ ID NO:8; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 13 or has two or one amino acid difference from SEQ ID NO: 13; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 18. ISVDs including:

[0173] In one embodiment, the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO:8, a CDR2 that is the amino acid sequence of SEQ ID NO:13, and a CDR3 that is the amino acid sequence of SEQ ID NO:18.

[0174] An example of such an ISVD that specifically binds human IL-13 has one or more, or all, of the framework regions (in addition to the CDRs defined above in items A and B, respectively) as shown for constructs 4B02 or 4B06, respectively, in Table A-2. In one embodiment, this is an ISVD that comprises or consists of the full-length amino acid sequence of construct 4B02 or construct 4B06 (SEQ ID NOs: 2 and 3, respectively; see Tables A-1 and A-2).

[0175] In another embodiment, the amino acid sequence of an ISVD that specifically binds to human IL-13 may have greater than 90%, such as greater than 95%, or greater than 99% sequence identity with SEQ ID NO: 2 or 3, respectively, and the CDRs are as defined above in items A or B, respectively. In one embodiment, an ISVD that specifically binds to IL-13 comprises or consists of the amino acid sequence of SEQ ID NO: 2 or 3.

[0176] When such an ISVD that binds to IL-13 has two or one amino acid difference in at least one CDR compared to the corresponding reference CDR sequence (items A or B above), the ISVD has at least half the binding affinity of human IL-13, or at least the same binding affinity, such as constructs 4B02 or 4B06 set forth in SEQ ID NO: 2 or 3, respectively, where the binding affinity is measured using the same method, such as SPR.

[0177] Specific examples of ISVDs that specifically bind to TSLP that can be used in the present technology are described in sections C and D below: C. Specific binding to human TSLP; i. CDR1 which is the amino acid sequence of SEQ ID NO:9 or has 2 or 1 amino acid difference from SEQ ID NO:9; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 14 or has two or one amino acid difference from SEQ ID NO: 14; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 19. ISVDs including:

[0178] In one embodiment, the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO:9, a CDR2 that is the amino acid sequence of SEQ ID NO:14, and a CDR3 that is the amino acid sequence of SEQ ID NO:19.

[0179] D. specifically binds to human TSLP; i. CDR1 which is the amino acid sequence of SEQ ID NO:11 or has 2 or 1 amino acid difference from SEQ ID NO:11; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 16 or has two or one amino acid difference from SEQ ID NO: 16; and iii. A CDR3 having the amino acid sequence of SEQ ID NO:21 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO:21. ISVDs including:

[0180] In one embodiment, the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO:11, a CDR2 that is the amino acid sequence of SEQ ID NO:16, and a CDR3 that is the amino acid sequence of SEQ ID NO:21.

[0181] An example of such an ISVD that specifically binds human TSLP has one or more, or all, of the framework regions (in addition to the CDRs defined above in items C and D) as shown for constructs 501A02 and 529F10, respectively, in Table A-2. In one embodiment, this is an ISVD that includes or consists of the full-length amino acid sequence of construct 501A02 or 529F10 (SEQ ID NO: 4 or 6, see Tables A-1 and A-2).

[0182] In another embodiment, the amino acid sequence of an ISVD that specifically binds human TSLP may have greater than 90%, e.g., greater than 95%, or greater than 99% sequence identity to SEQ ID NO:4 or 6, respectively, and the CDRs are as defined above in items C or D. In one embodiment, an ISVD that binds human TSLP comprises or consists of the amino acid sequence of SEQ ID NO:4 or 6.

[0183] When such an ISVD that binds to human TSLP has two or one amino acid difference in at least one CDR compared to the corresponding reference CDR sequence (items C or D above), the ISVD has at least half the binding affinity of human TSLP, or at least the same binding affinity as that of human TSLP, such as constructs 501A02 or 529F10 set forth in SEQ ID NOs: 4 and 6, respectively, where the binding affinity is measured using the same method, such as SPR.

[0184] In one embodiment, each of the ISVDs defined in items A-D above is included in the polypeptide of the present technology.

[0185] The polypeptides of the present technology including each of the ISVDs defined in items A to D above have at least half the binding affinity of human IL-13 and human TSLP, or at least the same binding affinity as a polypeptide consisting of the amino acid sequence of SEQ ID NO:1, where the binding affinity is measured using the same method, such as SPR.

[0186] The SEQ ID NOs referred to in items A-D above and in item E below (see section 5.4 "Extended half-life (in vivo)") are based on the AbM definition of CDRs (see Table A-2). Note that SEQ ID NOs defining the same CDRs according to the Kabat definition (see Table A-2.1) can similarly be used in items A-D above and in item E below (see section 5.4 "Extended half-life (in vivo)").

[0187] Thus, specific examples of ISVDs that specifically bind IL-13 or TSLP that can be used in the present technology are as described above using the AbM definition, and can also be described using the Kabat definition as described in sections A'-D' below: A'. Binds specifically to human IL-13; i. CDR1 which is the amino acid sequence of SEQ ID NO: 37 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 37; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 42 or has two or one amino acid difference from SEQ ID NO: 42; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 17. ISVDs including:

[0188] In one embodiment, the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO:37, a CDR2 that is the amino acid sequence of SEQ ID NO:42, and a CDR3 that is the amino acid sequence of SEQ ID NO:17.

[0189] B'. Binds specifically to human IL-13; i. CDR1 which is the amino acid sequence of SEQ ID NO: 38 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 38; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 43 or has two or one amino acid difference from SEQ ID NO: 43; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 18. ISVDs including:

[0190] In one embodiment, the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO:38, a CDR2 that is the amino acid sequence of SEQ ID NO:43, and a CDR3 that is the amino acid sequence of SEQ ID NO:18.

[0191] An example of such an ISVD that specifically binds human IL-13 has one or more, or all, of the framework regions as shown for constructs 4B02 or 4B06, respectively, in Table A-2-1 (in addition to the CDRs defined above in items A' and B', respectively). In one embodiment, this is an ISVD that comprises or consists of the full-length amino acid sequence of construct 4B02 or construct 4B06 (SEQ ID NOs: 2 and 3, respectively; see Tables A-1 and A-2-1).

[0192] C'. Binds specifically to human TSLP i. CDR1 which is the amino acid sequence of SEQ ID NO: 39 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 39; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 44 or has two or one amino acid difference from SEQ ID NO: 44; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 19. ISVDs including:

[0193] In one embodiment, the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO:39, a CDR2 that is the amino acid sequence of SEQ ID NO:44, and a CDR3 that is the amino acid sequence of SEQ ID NO:19.

[0194] D'. Binds specifically to human TSLP i. CDR1 which is the amino acid sequence of SEQ ID NO: 41 or has 2 or 1 amino acid difference from SEQ ID NO: 41; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 46 or has two or one amino acid difference from SEQ ID NO: 46; and iii. A CDR3 having the amino acid sequence of SEQ ID NO:21 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO:21. ISVDs including:

[0195] In one embodiment, the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO:41, a CDR2 that is the amino acid sequence of SEQ ID NO:46, and a CDR3 that is the amino acid sequence of SEQ ID NO:21.

[0196] An example of such an ISVD that specifically binds human TSLP has one or more, or all, of the framework regions (in addition to the CDRs defined in sections C' and D' above) as shown for constructs 501A02 and 529F10, respectively, in Table A-2-1. In one embodiment, this is an ISVD that includes or consists of the full-length amino acid sequence of construct 501A02 or 529F10 (SEQ ID NO: 4 or 6, see Tables A-1 and A-2-1).

[0197] The percentage of "sequence identity" between a first amino acid sequence and a second amino acid sequence can be calculated by dividing the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at corresponding positions in the second amino acid sequence by the total number of amino acid residues in the first amino acid sequence and multiplying by 100%, where each deletion, insertion, substitution or addition of an amino acid residue in the second amino acid sequence compared to the first amino acid sequence is considered as a difference at a single amino acid residue (i.e., at a single position).

[0198] Typically, for purposes of determining the percentage of "sequence identity" between two amino acid sequences according to the calculation method outlined above, the amino acid sequence having the greatest number of amino acid residues will be treated as the "first" amino acid sequence, and the other amino acid sequence will be treated as the "second" amino acid sequence.

[0199] An "amino acid difference," as used herein, refers to a deletion, insertion, or substitution of a single amino acid residue relative to a reference sequence. In one embodiment, an "amino acid difference" is a substitution.

[0200] In one embodiment, the amino acid substitution is a conservative substitution. Such a conservative substitution is a substitution in which one amino acid residue in the following groups (a) to (e) is replaced with another amino acid residue in the same group: (a) small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar positively charged residues: His, Arg and Lys; (d) large aliphatic, non-polar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0201] In one embodiment, conservative substitutions are as follows: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln, or Glu; Met to Leu, Tyr, or Ile; Phe to Met, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile, or Leu.

[0202] 5.3 Specificity The terms "specificity", "specifically binds" or "specific binding" refer to the number of different target molecules, such as antigens from the same organism, that a particular binding unit, e.g., an ISVD, can bind with sufficiently high affinity (see below). "Specificity", "specifically binds" or "specific binding" are used herein synonymously with "selectivity", "selectively binds" or "selective binding". A binding unit, e.g., an ISVD, specifically binds to its designated target.

[0203] The specificity / selectivity of a binding unit can be determined based on affinity. Affinity represents the strength or stability of a molecular interaction. Affinity is generally expressed by KD, or dissociation constant, which has units of moles / liter (or M). Affinity is also equal to 1 / KD, (moles / liter) -1 (or M -1 The association constant, KA, can also be expressed as the association constant, which has units of .

[0204] Affinity is a measure of the binding strength between a moiety and a binding site on a target molecule: the lower the KD value, the stronger the binding strength between the target molecule and the targeting moiety.

[0205] Typically, the binding units (e.g., ISVDs) used in the present technology bind to their targets at least 10 -5 ~10 -12 moles / liter or less, or 10 -7 ~10 -12 moles / liter or less, or 10 -8 ~10 -12 Dissociation constant (KD) in moles / liter (i.e. 10 5 ~10 12 Liters / mole or more, or 10 7 ~10 12 Liters / mole or more, or 10 8 ~10 12 with an association constant (KA) in liters / mol.

[0206] Generally, 10 -4 Any KD value greater than moles / liter (or 10 4 Any K A value lower than 1.0 liter / mol) is considered to indicate nonspecific binding.

[0207] The KD of a biological interaction, for example the KD of binding of an immunoglobulin sequence to an antigen that is considered specific, is typically greater than 10 -5 moles / liter (10,000 nM or 10 μM) to 10 -12 in the range of moles per liter (0.001 nM or 1 pM) or less.

[0208] Therefore, specific / selective binding can be achieved using the same measurement method, e.g., SPR, where a binding unit (or a polypeptide containing it) is 10 -5 ~10 -12 Binds to IL13 and / or TSLP with KD values ​​of 10 moles / liter or less and binds related cytokines -4 It may mean binding with a KD value of more than moles per liter. An example of a target related to IL13 is human IL4. An example of a cytokine related to TSLP is human IL7. Thus, in one embodiment of the present technology, at least two ISVDs contained in the polypeptide bind to IL13 at 10 -5 ~10 -12 It binds to IL4 of the same species with a KD value of 10 moles / liter or less. -4 The polypeptide binds to TSLP with a K value of greater than 10 moles per liter, and at least two ISVDs are contained in the polypeptide. -5 ~10 -12 It binds to human IL7 from the same species with a KD value of 10 -4 Binds with a KD value greater than moles / liter.

[0209] Thus, the polypeptides of the present technology have at least half the binding affinity, or at least the same binding affinity, of human IL13 and human TSLP when compared to a polypeptide consisting of the amino acids of SEQ ID NO:1, where the binding affinity is measured using the same method, e.g., SPR.

[0210] Specific binding to a particular target from a particular species does not exclude that the binding unit may also specifically bind to a similar target from a different species. For example, specific binding to human IL13 does not exclude that the binding unit (or a polypeptide comprising it) may also specifically bind to IL13 from cynomolgus monkeys. Similarly, specific binding to, for example, human TSLP does not exclude that the binding unit (or a polypeptide comprising it) may also specifically bind to TSLP from cynomolgus monkeys ("cyno").

[0211] Specific binding of a binding unit to its designated target can be determined in any suitable manner known per se, such as Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, as well as their various modifications known per se in the art; in addition to other techniques described herein.

[0212] The dissociation constant may be an actual dissociation constant or an apparent dissociation constant, as will be apparent to one of skill in the art. Methods for determining dissociation constants will be apparent to one of skill in the art, and examples include the techniques described below. In this regard, -4 moles / liter or 10 -3 Larger than moles / liter (e.g. 10 -2 It will also be apparent that the dissociation constant (in moles / liter) may not be measurable. In some cases, as will be apparent to one skilled in the art, the dissociation constant (actual or apparent) can be calculated based on the association constant (KA) (actual or apparent) according to the relationship: [KD=1 / KA].

[0213] The affinity of a molecular interaction between two molecules can be measured via various techniques known per se, such as the well-known surface plasmon resonance (SPR) biosensor technology (see, for example, Ober et al., 2001, Intern. Immunology 13:1551-1559). The term "surface plasmon resonance" as used herein refers to an optical phenomenon that allows the analysis of real-time biospecific interactions by detection of changes in protein concentration in a biosensor matrix, where one molecule is immobilized on a biosensor chip and the other molecule passes over the immobilized molecule under flow conditions, resulting in a change in k on , k off measurements, and therefore K D (or K A ) values ​​are obtained. This can be done, for example, using the well-known BIAcore® system (BIAcore International AB, GE Healthcare, Uppsala, Sweden and Piscataway, NJ). For further description, see Jonsson et al. (1993, Ann. Biol. Clin. 51:19-26), Jonsson et al. (1991 Biotechniques 11:620-627), Johnsson et al. (1995, J. Mol. Recognit. 8:125-131), and Johnnson et al. (1991, Anal. Biochem. 198:268-277).

[0214] Another well-known biosensor technique for determining affinity of biomolecular interactions is Biolayer Interferometry (BLI) (see, for example, Abdiche et al., 2008, Anal. Biochem. 377:209-217). The term "Biolayer Interferometry" or "BLI" as used herein refers to a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and a layer of immobilized proteins on a biosensor chip (signal beam). Changes in the number of molecules bound to the biosensor chip cause a shift in the interference pattern, reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the biosensor chip surface. Interactions can be measured in real time, so that association and dissociation rates as well as affinities can be determined. BLI can be performed, for example, using the well-known Octet® system (ForteBio, a division of Pall Life Sciences, Menlo Park, USA).

[0215] Alternatively, affinity can be measured by Kinetic Exclusion Assay (KinExA) (see, e.g., Drake et al., 2004, Anal. Biochem., 328:35-43) using the KinExA® platform (Sapidyne Instruments Inc, Boise, USA). The term "KinExA" as used herein refers to a solution-based method for measuring true equilibrium binding affinity and kinetics of unmodified molecules. An equilibrated solution of antibody / antigen complexes is passed over a column with beads pre-coated with antigen (or antibody), allowing free antibody (or antigen) to bind to the coated molecule. Detection of the antibody (or antigen) captured in this way is achieved using a fluorescently labeled protein that binds to the antibody (or antigen).

[0216] The GYROLAB® Immunoassay System provides a platform for automated biological analysis and rapid sample turnover (Fraley et al., 2013, Bioanalysis 5:1765-74).

[0217] 5.4 Half-life extension (in vivo) The polypeptide may further comprise one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers, which provide the polypeptide with an increased half-life (in vivo) compared to the corresponding polypeptide without said one or more other groups, residues, moieties or binding units. The increased half-life in vivo means, for example, that the polypeptide has an increased half-life in a mammalian, for example, human subject after administration. The half-life can be expressed, for example, as t1 / 2 beta.

[0218] The type of group, residue, moiety or binding unit is generally not limited and can be selected, for example, from the group consisting of polyethylene glycol molecules, serum proteins or fragments thereof, binding units capable of binding to serum proteins, Fc portions, and small proteins or peptides capable of binding to serum proteins.

[0219] More specifically, the one or more other groups, residues, moieties or binding units that provide a polypeptide with increased half-life can be selected from the group consisting of binding units that can bind to serum albumin, such as human serum albumin, or serum immunoglobulin, such as IgG. In one embodiment, the one or more other binding units that provide a polypeptide with increased half-life are binding units that can bind to human serum albumin. In one embodiment, the binding unit is an ISVD.

[0220] For example, WO04 / 041865 describes Nanobodies® that bind to serum albumin (in particular to human serum albumin), which may be linked to other proteins (e.g. one or more other Nanobodies that bind to a desired target) to increase the half-life of the protein.

[0221] International application WO06 / 122787 describes a number of Nanobodies® against (human) serum albumin. These Nanobodies® include Alb-1 (SEQ ID NO: 52 in WO06 / 122787) and their humanized variants, such as the Nanobody® designated Alb-8 (SEQ ID NO: 62 in WO06 / 122787). These can also be used to extend the half-life of therapeutic proteins and polypeptides and other therapeutic entities or moieties.

[0222] Furthermore, WO2012 / 175400 describes a further improved version of Alb-1, called Alb-23.

[0223] In one embodiment, the polypeptide comprises a serum albumin binding moiety selected from Alb-1, Alb-3, Alb-4, Alb-5, Alb-6, Alb-7, Alb-8, Alb-9, Alb-10 and Alb-23. In one embodiment, the serum albumin binding moiety is Alb-8 or Alb-23 or variants thereof as set forth on pages 7-9 of WO2012 / 175400, and albumin binders described in WO2012 / 175741, WO2015 / 173325, WO2017 / 080850, WO2017 / 085172, WO2018 / 104444, WO2018 / 134235, WO2018 / 134234. Some serum albumin binders are also set forth in Table A-4. In one embodiment, the further components of the polypeptide of the present technology are as described in section E: E. binds to human serum albumin i. CDR1 which is the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 10; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 15 or has two or one amino acid difference from SEQ ID NO: 15; and iii. A CDR3 having the amino acid sequence of SEQ ID NO:20 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO:20. ISVDs including:

[0224] In one embodiment, the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO:10, a CDR2 that is the amino acid sequence of SEQ ID NO:15, and a CDR3 that is the amino acid sequence of SEQ ID NO:20.

[0225] An example of such a human serum albumin binding ISVD has one or more, or all, of the framework regions (in addition to the CDRs defined in item E above) as shown for construct ALB23002 in Table A-2. In one embodiment, this is an ISVD that comprises or consists of the full length amino acid sequence of construct ALB23002 (SEQ ID NO:5, see Tables A-1 and A-2).

[0226] Item E can also be written using the Kabat definition as follows: E'. Binds to human serum albumin i. CDR1 which is the amino acid sequence of SEQ ID NO: 40 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 40; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 45 or has two or one amino acid difference from SEQ ID NO: 45; and iii. A CDR3 having the amino acid sequence of SEQ ID NO:20 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO:20. ISVDs including:

[0227] In one embodiment, the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO:40, a CDR2 that is the amino acid sequence of SEQ ID NO:45, and a CDR3 that is the amino acid sequence of SEQ ID NO:20.

[0228] An example of such a human serum albumin binding ISVD has one or more or all of the framework regions (in addition to the CDRs defined in item E' above) as shown for construct ALB23002 in Table A-2-1. In one embodiment, this is an ISVD that comprises or consists of the full length amino acid sequence of construct ALB23002 (SEQ ID NO:5, see Tables A-1 and A-2-1).

[0229] In a further embodiment, the amino acid sequence of the ISVD that binds human serum albumin may have greater than 90%, such as greater than 95% or greater than 99% sequence identity with SEQ ID NO:5, and the CDRs are as defined in item E or E' above. In one embodiment, the ISVD that binds human serum albumin has the amino acid sequence of SEQ ID NO:5.

[0230] If such an ISVD that binds to human serum albumin has two or one amino acid difference in at least one CDR compared to the corresponding reference CDR sequence (item E above), the ISVD has at least half the binding affinity of human serum albumin, such as construct ALB23002 set forth in SEQ ID NO:5, or at least the same binding affinity, where the binding affinity is measured using the same method, such as SPR.

[0231] In one embodiment, such ISVD that binds to human serum albumin, when it has a C-terminal position, exhibits a C-terminal alanine (A) or glycine (G) extension. In one embodiment, such ISVD is selected from SEQ ID NO: 59, 60, 62, 64, 65, 66, 67, 68, 69 and 71 (see Table A-4 below). In one embodiment, the ISVD that binds to human serum albumin is at a position other than the C-terminal position (i.e., it is not an ISVD at the C-terminus of the polypeptide of the present technology). In one embodiment, such ISVD is selected from SEQ ID NO: 5, 57, 58, 61 and 63 (see Table A-4 below).

[0232] 5.5 Nucleic acid molecules Nucleic acid molecules encoding the polypeptides of the present technology are also provided.

[0233] A "nucleic acid molecule" (used interchangeably with "nucleic acid") is a chain of nucleotide monomers linked together via a phosphate backbone to form a nucleotide sequence. The nucleic acid can be used to transform / transfect a host cell or host organism, for example, for expression and / or production of a polypeptide. Suitable hosts or host cells for production purposes will be apparent to the skilled artisan and can be, for example, any suitable fungal, prokaryotic or eukaryotic cell or cell line, or any suitable fungal, prokaryotic or eukaryotic organism. Hosts or host cells comprising a nucleic acid encoding a polypeptide of the present technology are also encompassed by the present technology.

[0234] The nucleic acid may be, for example, DNA, RNA, or a hybrid thereof, or may contain nucleotides modified (e.g., chemically) such as PNA. The nucleic acid may be single-stranded or double-stranded. In one embodiment, the nucleic acid is in the form of double-stranded DNA. For example, the nucleotide sequence of the present technology may be genomic DNA, cDNA.

[0235] The nucleic acid of the present technology may be prepared or obtained in a manner known per se and / or may be isolated from a suitable natural source.Nucleotide sequences encoding naturally occurring (poly)peptides may, for example, be subjected to site-directed mutagenesis to provide nucleic acid molecules encoding polypeptides with sequence changes.It will also be clear to those skilled in the art that, to prepare nucleic acid, several nucleotide sequences, such as at least one nucleotide sequence encoding a targeting moiety, such as the nucleic acid encoding one or more linkers, may also be linked together in a suitable manner.

[0236] Techniques for generating nucleic acids will be apparent to those of skill in the art and may include, but are not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and / or synthetic sequences (or two or more portions thereof), introducing mutations that result in expression of a truncated expression product; introducing one or more restriction sites (e.g., to create cassettes and / or regions that can be easily digested and / or ligated using suitable restriction enzymes), and / or introducing mutations by PCR reactions using one or more "mismatched" primers.

[0237] 5.6 Vectors Also provided is a vector that comprises the nucleic acid molecule that codes for the polypeptide of the present technology.A vector as used herein is a suitable vehicle for carrying genetic material into cells.Vector includes naked nucleic acid, such as plasmid or mRNA, or nucleic acid embedded in larger structure, such as liposome or virus vector.

[0238] A vector generally comprises at least one nucleic acid, optionally linked to one or more regulatory elements, such as one or more suitable promoters, enhancers, terminators, etc. In one embodiment, the vector is an expression vector, i.e. a vector suitable for expressing an encoded polypeptide or construct under suitable conditions, e.g. when the vector is introduced into a (e.g. human) cell. In the case of a DNA-based vector, this usually includes the presence of elements for transcription (e.g. promoter and polyA signal) and the presence of elements for translation (e.g. Kozak sequence).

[0239] In one embodiment, in a vector, the at least one nucleic acid and the regulatory element are "operably linked" to each other, which generally means that they are in a functional relationship with each other. For example, a promoter is considered to be "operably linked" to a coding sequence (wherein the coding sequence should be understood to be "under the control" of the promoter) if the promoter is capable of initiating or otherwise controlling / regulating the transcription and / or expression of the coding sequence. Generally, when two nucleotide sequences are operably linked, they are in the same orientation and usually in the same reading frame. They are also usually essentially contiguous, although this may not be necessary.

[0240] In one embodiment, any regulatory elements of the vector are such that they are capable of providing their intended biological function in the intended host cell or host organism.

[0241] For example, a promoter, enhancer or terminator should be "operable" in the intended host cell or host organism, meaning, for example, that said promoter should be capable of initiating or otherwise controlling / regulating the transcription and / or expression of a nucleotide sequence, e.g., a coding sequence, operably linked to it.

[0242] 5.7 Composition The present technology also provides a composition comprising at least one polypeptide of the present technology, at least one nucleic acid molecule encoding the polypeptide of the present technology, or at least one vector comprising such nucleic acid molecule.The composition can be a pharmaceutical composition.The composition can further comprise at least one pharma- ceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally comprises one or more additional pharma- ceutically active polypeptides and / or compounds.

[0243] 5.8 Host organisms The present technology also relates to host cells or host organisms comprising a polypeptide of the present technology, a nucleic acid encoding a polypeptide of the present technology, and / or a vector comprising a nucleic acid molecule encoding a polypeptide of the present technology.

[0244] Suitable host cells or host organisms are clear to those skilled in the art, for example, any suitable fungal, prokaryotic or eukaryotic cell or cell line, or any suitable fungal, prokaryotic or eukaryotic organism.Specific examples include HEK293 cells, CHO cells, Escherichia coli or Pichia pastoris.In one embodiment, the host is Pichia pastoris.

[0245] 5.9 Polypeptide Methods and Uses The present technology also provides methods for producing the polypeptides of the present technology, which may include transforming / transfecting a host cell or host organism with a nucleic acid encoding the polypeptide, expressing the polypeptide in the host, optionally followed by one or more isolation and / or purification steps. Specifically, the method comprises: a) expressing a nucleic acid sequence encoding the polypeptide in a suitable host cell or host organism, or in another suitable expression system; optionally followed by: b) isolating and / or purifying the polypeptide may also include

[0246] Suitable host cells or host organisms for production purposes are clear to those skilled in the art and can be, for example, any suitable fungal, prokaryotic or eukaryotic cell or cell line, or any suitable fungal, prokaryotic or eukaryotic organism. Specific examples include HEK293 cells, CHO cells, E. coli or Pichia pastoris. In one embodiment, the host is Pichia pastoris.

[0247] The described polypeptides, nucleic acid molecules or vectors of the present technology, or compositions comprising the polypeptides, nucleic acid molecules or vectors of the present technology, are useful as pharmaceuticals.

[0248] Thus, the present technology provides a composition comprising the described polypeptide, nucleic acid molecule, or vector of the present technology, or the polypeptide, nucleic acid molecule, or vector of the present technology, for use as a medicament.

[0249] Also provided is a composition comprising the described polypeptides, nucleic acid molecules or vectors of the present technology, or the polypeptides, nucleic acid molecules or vectors of the present technology, for use in the treatment (prophylactic or therapeutic) of inflammatory diseases.

[0250] Further provided is a method of treating (prophylactic and / or therapeutic) an inflammatory disease, comprising administering to a subject in need thereof a pharma- tically active amount of the polypeptide, nucleic acid molecule or vector of the present technology described, or a composition comprising the polypeptide, nucleic acid molecule or vector of the present technology.

[0251] Further provided is the use of the polypeptide of the present technology, the described nucleic acid molecule or vector, or a composition comprising the polypeptide, nucleic acid molecule or vector of the present technology in the preparation of a pharmaceutical composition. In one embodiment, the prepared pharmaceutical composition is for treating an inflammatory disease.

[0252] The inflammatory disease is a type 2 inflammatory disease, such as atopic dermatitis and asthma.

[0253] "Subject" as referred to in the present technology may be any animal, more specifically a mammal. Among mammals, a distinction may be made between humans and non-human mammals. Non-human animals may be, for example, companion animals (e.g. dogs, cats), livestock animals (e.g. bovine, equine, ovine, caprine, or porcine animals), or animals commonly used for research purposes and / or antibody production (e.g. mice, rats, rabbits, cats, dogs, caprines, ovine, equine, porcine, non-human primates, such as cynomolgus monkeys, or camelids, such as llamas or alpacas).

[0254] For prophylactic and / or therapeutic purposes, the subject may be any animal, and more particularly, any mammal. In one embodiment, the subject is a human subject.

[0255] Substances (such as polypeptides, nucleic acid molecules, and vectors) or compositions can be administered to a subject by any suitable route of administration, such as enteral (e.g., oral or rectal) or parenteral (e.g., epicutaneous, sublingual, buccal, nasal, intraarticular, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, subdermal, or mucosal) administration. In one embodiment, the substance is administered parenterally, such as intramuscular, subcutaneous, or intradermal administration. In one embodiment, subcutaneous administration is used.

[0256] An effective amount of the described polypeptides, nucleic acid molecules or vectors, or a composition comprising the polypeptides, nucleic acid molecules or vectors, can be administered to a subject to provide the intended therapeutic result.

[0257] One or more doses may be administered. If more than one dose is administered, the doses may be administered at suitable intervals to maximize the effect of the polypeptide, composition, nucleic acid molecule or vector.

[0258] [Table 2]

[0259] [Table 3]

[0260] [Table 4]

[0261] [Table 5]

[0262] [Table 6-1] [Table 6-2]

[0263] [Table 7]

[0264] [Table 8-1] [Table 8-2] [Table 8-3] EXAMPLES

[0265] 6. Working Example 6.1 Generation of monovalent ISVDs that specifically bind IL-13 and TSLP, respectively 6.1.1 Example 1: Immunization To induce heavy chain antibody-dependent humoral immune responses, three llamas were immunized with recombinant human IL-13 (Peprotech, Cat. No. 200-13, derived from E. coli) according to standard protocols and were boosted with human / cynomolgus IL-13 from another source (Sino Biological, Cat. Nos. 10369-HNAC and 11057-CNAH, derived from mammalian cells).

[0266] Another three llamas were immunized with recombinant hTSLP-Fc, and these llamas were boosted with cynomolgus TSLP-Fc. Two additional llamas were immunized with hTSLP alternating with cynomolgus TSLP-Fc.

[0267] Immune blood (PBL) samples were taken periodically and total RNA was prepared from isolated B cells. During the immunization process, humoral immune responses were monitored by comparing antigen-specific serum titers between samples collected before the start of immunization and serum samples collected routinely after multiple antigen challenges. Briefly, 96-well Maxisorp plates were coated with human IL-13 (Sino Biological, Cat. No. 10369-HNAC) or human TSLP. Recombinant human TSLP is commercially available, for example, from R&D Systems (Cat. No. 1398-TS). After blocking and addition of diluted serum samples, the presence of anti-IL-13 and anti-TSLP ISVD was demonstrated by using HRP (horseradish peroxidase)-conjugated goat anti-llama immunoglobulin (Bethyl Laboratories Inc.) and subsequent enzymatic reaction in the presence of the substrate TMB (3,3',5,5'-tetramethylbenzidine).

[0268] 6.1.2 Example 2: Library construction and phage display selection Peripheral blood mononuclear cells were prepared from blood samples using Ficoll-Hypaque according to the manufacturer's instructions. Total RNA extracted from these cells and lymph nodes was used as starting material for RT-PCR to amplify gene fragments encoding ISVD. These fragments were cloned into the phagemid vector pAX212. Phages were prepared according to standard protocols (Antibody Phage Display: Methods and Protocols (1st ed., 2002, O'Brian and Aitken, eds., Humana Press, Totowa, NJ) and stored after filter sterilization at 4°C until further use. Five phage libraries for IL13 were constructed, with a library size of 6.1 × 10 8 ~1.3×10 9 The percentage of inserts ranged from 87 to 96%. Five phage libraries for TSLP were constructed, with a library size of 4.2 × 108 ~9.8×10 8 , with the percentage of inserts ranging from 91 to 100%.

[0269] To identify ISVDs that recognize human and cynomolgus IL-13, the phage library was incubated with 50 nM soluble biotinylated hIL13-Fc in the presence of IgG from human serum (Sigma, I4506). Complexes of hIL-13-Fc and phage were captured from solution onto streptavidin-coated magnetic beads. After extensive washing with PBS / 0.05% Tween 20, bound phages were eluted by the addition of trypsin (1 mg / ml). These round 1 selection outputs were incubated with 0.05, 0.5 or 5 nM soluble biotinylated hIL-13-Fc or cynomolgus IL-13-Fc. In round 3, the unenriched output from round 2 selection was further incubated with 0.005, 0.05, 0.5 or 5 nM soluble biotinylated human or cynomolgus IL-13-Fc. Enriched individual clones from round 2 and round 3 selections were picked.

[0270] To identify ISVDs that recognize human and cynomolgus TSLP, the phage library was incubated with 50 nM soluble biotinylated hTSLP-Fc, or with 500 nM biotinylated hTSLP, or with 500 nM biotinylated cynomolgus TSLP in the presence of IgG from human serum (Sigma, I4506). Human and cynomolgus TSLP sequences are known (Uniprot accession number Q969D9 and NCBI reference sequence XP_005557555.1, respectively). The assay was performed using recombinant proteins. TSLP and phage complexes were captured from solution onto streptavidin-coated magnetic beads. After extensive washing with PBS / 0.05% Tween 20, bound phages were eluted by the addition of trypsin (1 mg / ml). These round 1 selection outputs were incubated with 5 nM soluble biotinylated hILTSLP-Fc or cynomolgus TLSP-Fc or 0.5 nM biotinylated hTSLP. Output from round 2 selection was incubated with 0.05, 0.5 or 5 nM soluble biotinylated human TSLP, TSLP-Fc or cynomolgus TSLP-Fc. Individual clones from the enriched output for each round were picked.

[0271] All individual clones were grown in 96 deep-well plates (1 ml volume). ISVD expression was induced by adding IPTG to a final concentration of 1 mM. Periplasmic extracts were prepared by freezing the cell pellet and dissolving it in 100 μl of PBS. Dead cell debris was removed by centrifugation.

[0272] As a control, selected periplasmic extracts were screened for binding to human and cynomolgus IL13-Fc, TSLP-Fc, respectively, by ELISA. Evaluation was performed in a 25 μl format on a Spectraplate 384-HB (PerkinElmer). Antigen was coated overnight at 1 μg / ml in PBS at 4° C. The wells were blocked with casein solution (1%). After addition of 5-fold dilutions of periplasmic extracts, ISVD binding was detected using mouse anti-Flag-HRP (Sigma) and a subsequent enzymatic reaction in the presence of the substrate esTMB (3,3',5,5'-tetramethylbenzidine).

[0273] 6.1.3 Example 3: Screening for blocking ISVD in periplasmic extracts by AlphaScreen assay using human IL13 and human TSLP To determine the blocking capacity of ISVD, crude periplasmic extracts were screened in different protein-based competitive assays using AlphaScreen technology (PerkinElmer, Waltham, MA USA). Fluorescence was measured using an EnVision multilabel plate reader (PerkinElmer) using an excitation wavelength of 680 nm and an emission wavelength of 520 nm.

[0274] We investigated whether the ISVD could block the interaction of hIL-13 with the hIL-13Rα1 extracellular domain in the hIL-13:hIL-13Rα1 binary complex AlphaScreen. To this end, dilutions of periplasmic extracts were preincubated with biotinylated hIL-13 (Peprotech catalogue no. 200-13). To this mixture, hIL-13Rα1-hFc (R&D Systems; catalogue no. 146-IR) and anti-hFc-conjugated acceptor beads were added and further incubated for 1 h at room temperature, followed by the addition of streptavidin-conjugated donor beads and incubation for an additional hour. Once the binary complex was formed, the acceptor and donor beads were brought into close proximity and laser excitation produced a detectable signal. A decrease in the AlphaScreen signal indicates that the binding of biotinylated hIL-13 to hIL-13Rα1 was blocked by the ISVD in the periplasmic extract. In a similar setting, we investigated whether ISVD could block the interaction of hTSLP (eBioscience, catalog number 10-8499) with the hTSLPR extracellular domain (R&D Systems catalog number 981-TR).

[0275] We screened whether ISVDs could block the recruitment of hIL4Rα to the hIL13:hIL13Rα1 binary complex in the hIL-13:hIL-13Rα1:hIL4Rα ternary complex AlphaScreen. hIL-13 binds to hIL-13Rα1, and this binary complex recruits hIL4Rα, resulting in the formation of the ternary complex hIL-13:hIL-13Rα1:hIL4Rα. Dilutions of periplasmic extracts were preincubated with hIL-13 (Peprotech Cat. No. 200-13) and biotinylated huIL4Rα (R&D Systems; Cat. No. 230-4R / CF). To this mixture, hIL-13Rα1-hFc (R&D Systems; Cat. No. 146-IR) and anti-hFc-conjugated acceptor beads were added and further incubated at room temperature for 1 h, followed by the addition of streptavidin-conjugated donor beads and incubation for an additional hour. Once the ternary complex was formed, the acceptor and donor beads were brought into close proximity and laser excitation produced a detectable signal. A decrease in AlphaScreen signal indicates that the formation of the ternary complex is blocked by the ISVD in the periplasmic extract. Similarly, we investigated whether the ISVD could block the formation of the hTSLP:hTSLPR:hIL7Rα complex (hIL7Rα source=Sino Biological Cat. No. 1095-H08H).

[0276] Based on the AlphaScreen analysis (Tables 3 and 4), a number of blocking ISVDs were selected and sequenced (Tables 1 and 2).

[0277] [Table 9]

[0278] [Table 10]

[0279] 6.1.4 Example 4: Surface Plasmon Resonance Analysis of Periplasmic Extracts for IL13 and TSLP The off-rates of periplasmic extracts containing anti-IL13 or anti-TSLP ISVD were measured by surface plasmon resonance (SPR) using a Proteon XPR36 instrument (Bio-Rad Laboratories, Inc.) Phosphate buffered saline (PBS), pH 7.4, supplemented with 0.005% Tween 20, was used as the running buffer and experiments were performed at 25°C.

[0280] hIL13-Fc, cynomolgus IL13-Fc, hTSLP-Fc and cynomolgus TSLP-Fc were immobilized on a ProteOn GLC sensor chip by amine coupling using EDC and NHS at a flow rate of 30 μl / min for activation. IL13 protein was injected at 10 μg / ml in ProteOn acetate buffer at pH 5.0. TSLP protein was injected at 5 μg / mL in ProteOn acetate buffer at pH 5.5. After immobilization, the surface was inactivated with ethanolamine.

[0281] Periplasmic extracts of ISVD candidates were diluted 10-fold in PBS-Tween 20 (0.1%) and injected at 45 μl / min for 2 min and allowed to dissociate for 900 s. Between the different samples, the surface was regenerated by a 2 min injection of phosphoric acid (0.425%) at 45 μl / min for IL13 or by a 1 min injection of glycine pH 3.0 (5 mM) / SDS (0.25%) at 45 μl / min for TSLP. Off-rates were calculated from the sensorgrams obtained for the different periplasmic extracts.

[0282] Off-rate analysis for hIL-13-Fc and cynomolgus IL-13-Fc is shown in Table 3.

[0283] [Table 11] Off-rate analysis for hTSLP-Fc and cynomolgus TSLP-Fc is shown in Table 4.

[0284] [Table 12]

[0285] 6.1.5 Example 5: Expression and Purification of Anti-IL-13 and Anti-TSLP ISVDs Anti-IL-13 and anti-TSLP ISVDs were selected for expression and purification based on their blocking ability in the AlphaScreen assay and off-rate values. Sequences are shown in Tables 1 and 2.

[0286] ISVD was expressed as a c-myc, His6 tagged protein in E. coli TG1 cells. Expression was induced by the addition of 1 mM IPTG and allowed to continue for 3 hours at 37°C. After spinning the cell cultures, periplasmic extracts were prepared by freeze-thawing the pellet and resuspending it in dPBS. These extracts were used as starting material for immobilized metal affinity chromatography (IMAC) using a Nickel Sepharose TM6 FF column (Atoll). ISVD was eluted from the column with 250 mM imidazole and then desalted against dPBS. For the cell-based assays described below, endotoxin was removed by gel filtration in the presence of 50 mM octyl β-D-glucopyranoside (OGP, Sigma). Endotoxin levels were determined using a standard LAL-assay.

[0287] 6.1.6 Example 6: Blocking Ability of Purified Anti-IL13 and Anti-TSLP ISVD in the AlphaScreen Assay Binary and ternary complex AlphaScreen assays were used as described in Example 3 to determine IC50 values ​​for anti-IL-13 and anti-TSLP ISVDs, purified as described in Example 5. Instead of dilutions of periplasmic extracts, a dilution series starting from 500 nM down to 1.8 pM of each purified ISVD was pre-incubated with IL-13 or TSLP.

[0288] The anti-IL-13 ISVDs tested inhibited the formation of the ternary complex and partially blocked the binary complex with IC50 values ​​as shown in Table 5.

[0289] [Table 13]

[0290] The anti-TSLP ISVDs tested completely inhibited ternary complex formation and also inhibited binary complex formation with IC50 values ​​as shown in Table 6.

[0291] [Table 14]

[0292] 6.1.7 Example 7: Blocking Potential of Purified Anti-IL-13 and Anti-TSLP ISVDs in Cell-Based Assays The inhibitory potency of anti-IL-13 ISVDs was determined in a cell-based assay monitoring IL-13-mediated proliferation of TF-1 cells. To this end, TF-1 cells were cultured in RPMI1640 medium supplemented with 1 / 5 HEPES, 1 / 500 Na-pyruvate, 1 / 500 Glutamax and 2ng / mL recombinant human GM-CSF. TF-1 cells were seeded at 40,000 cells per well in growth medium without GM-CSF. A dilution series of purified anti-IL-13 ISVDs or reference compounds were added. After 15 minutes of incubation at 37°C, 200pM IL-13 (Peprotech Cat. No. 200-13) was added. After 96 hours, proliferation of TF-1 cells was determined with Cell Titer 96 Aqueous One Solution (Promega #G3580) on an EnVision multilabel reader (Perkin Elmer).

[0293] The ISVDs shown in Table 7 inhibit IL-13-induced TF1 proliferation.

[0294] [Table 15]

[0295] The blocking potency of anti-TSLP ISVD was determined in a cell-based assay that monitored TSLP-mediated proliferation of BaF3 cells transfected with plasmids encoding hTSLPR and hIL7Ra. Cells were seeded at a density of 20,000 cells / well in RPMI1640 growth medium in cell culture-treated white 96-well plates. A dilution series of anti-TSLP ISVD was added, followed by 50 pM hTLSP-Fc or 50 pM cynomolgus TSLP-Fc for stimulation of cells. Human and cynomolgus TSLP sequences are known (Uniprot accession number Q969D9 and NCBI reference sequence XP_005557555.1, respectively). The assay was performed using recombinant proteins.

[0296] After 72 hours of incubation, cell density and viability were monitored using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, G7571 / G7572 / G7573) and read on an EnVision multilabel reader (Perkin Elmer). The results are shown in Table 8.

[0297] [Table 16]

[0298] 6.1.8 Example 8: Binding Affinities of Purified Anti-IL-13 and Anti-TSLP ISVDs to Human and Cynomolgus Monkey IL-13 and TSLP Full binding kinetics studies by SPR were performed on a BIAcore T100 instrument (GE Healthcare).

[0299] For IL-13, approximately 2000RU of hIL13-Fc or 4000RU of cynomolgus IL13-Fc were directly immobilized on a CM5 sensor chip. ISVDs were then injected at different concentrations (3μM-12nM) over 120 seconds and allowed to dissociate for 900 seconds. Regeneration of the hIL-13-Fc and cynomolgus IL-13-Fc surfaces was performed using a 47 second injection of 0.85% H3PO4:MilliQ (1:1).

[0300] For TSLP, approximately 8000 RU of anti-huIgG antibody (GE Healthcare) was directly immobilized on a CM5 sensor chip. hTSLP-Fc (at 1 μg / mL) or cynomolgus TSLP-Fc (at 0.75 μg / mL) was suspended and captured on the chip for 120 s. ISVD was then injected at different concentrations (0.4 nM to 3000 nM) for 120 s and allowed to dissociate for 900 s.

[0301] Binding curve evaluation was performed using BIAcore T100 evaluation software V2.0.3. Kinetic analysis was performed by fitting a 1:1 interaction model (Langmuir binding) (Rmax=global; RI=constant=0, offset=0). Interactions that may not meet the acceptance criteria of the 1:1 interaction model were fitted using a heterogeneous ligand fitting model (RI=constant=0, offset=0).

[0302] Kinetic data are shown in Table 9 for the IL-13 ISVD and Table 10 for the TSLP ISVD.

[0303] [Table 17]

[0304] [Table 18]

[0305] 6.2 Generation of monospecific multivalent polypeptides that bind IL-13 and TSLP, respectively 6.2.1 Example 10: Generation and in vitro characterization of bivalent or biparatopic ISVD constructs of wild-type anti-IL-13 The selected anti-IL-13 ISVDs (F0107004B02 and F0107004B06) were formatted into biparatopic and bivalent ISVD constructs. The building blocks in the constructs are genetically linked by a flexible 35GS (GlySer) linker. The ISVDs were expressed in Pichia pastoris as FLAG3-HIS6 tagged proteins (amino acid sequences are shown in Table 14). Induction of ISVD construct expression occurred by stepwise addition of methanol. The clarified medium containing the secreted ISVD constructs was used as starting material for immobilized metal affinity chromatography (IMAC) followed by desalting, resulting in 90% purity as assessed by SDS-PAGE.

[0306] The biparatopic and bivalent IL-13 constructs were characterized in the AlphaScreen assay (as described in Example 6) for blocking binary and ternary complexes, as well as in the TF-1 proliferation assay (as described in Example 7). A summary of the resulting constructs and their blocking potency in the binary and ternary AlphaScreen assays and the TF-1 proliferation assay is shown in Table 11. The biparatopic constructs showed good potency against hIL-13 and cyIL-13, similar to that of the anti-hIL-13 reference mAb1. The bivalent constructs also improved potency, but not to the same extent as the biparatopic construct F010700029 in the TF1 proliferation assay. In addition, the bivalent constructs do not reach complete inhibition in the AlphaScreen of IL13-IL13Rα1.

[0307] [Table 19]

[0308] The most potent anti-IL-13 biparatopic ISVD constructs were tested in an IL-13-induced A549 eotaxin release assay. To this end, A549 suspension cells were cultured in Ham's F12K medium supplemented with 10% FCS. Cells were seeded in 96-well plates at 200,000 cells / well. The following day, 200 pM of hIL-13 (Sino Biological Catalog No. 10369-HNAC) or cynomolgus IL-13 (Sino Biological Catalog No. 11057-CNAH) was added, followed by a dilution series of ISVD constructs. After 24 hours, eotaxin-3 was determined in the supernatants using an MSD ELISA (Human Eotaxin-3 Tissue Culture Kit (Meso Scale, K151ABB-1)). The results are shown in Table 12.

[0309] [Table 20]

[0310] A competitive ELISA was used to test whether anti-IL-13 monovalent and biparatopic ISVD constructs inhibited the binding of hIL-13 to IL13Rα2. IL13Rα2 (SinoBiological Catalog No. 10350-H08H) was coated onto a 384-well Spectraplate (Perkin Elmer). hIL13-Fc (SinoBiological, Catalog No. 10369-H01H) was mixed with serial dilutions of the ISVD constructs or the positive control compound IL-13Rα2 and incubated for 1 hour. After washing and blocking, the coated receptors were incubated with the IL13-Fc ISVD / control mix and incubated for 1 hour. After washing, the presence of bound IL-13-Fc was detected using an anti-human IgG-peroxidase antibody, followed by an enzymatic reaction in the presence of the substrate esTMB. When ISVD inhibits IL-13-IL-13Rα2 interaction, detection of IL-13-Fc is abolished in a dose-dependent manner. The results are shown in Table 13.

[0311] [Table 21]

[0312] [Table 22]

[0313] 6.2.2 Example 11: Generation and characterization of wild-type biparatopic anti-TSLP ISVD constructs Selected TSLPR blockers (F0107501A02 and F0107529F10) were combined into a biparatopic ISVD construct. The construct was expressed in Pichia pastoris as a FLAG3-HIS6 tagged protein (amino acid sequence shown in Table 16). Induction of ISVD construct expression occurred by stepwise addition of methanol. Clarified medium containing the secreted ISVD construct was used as starting material for immobilized metal affinity chromatography (IMAC) followed by desalting, resulting in 90% purity as assessed by SDS-PAGE.

[0314] In BaF3 proliferation assay (as described in Example 7), the biparatopic constructs were titrated as purified proteins against 50 or 5 pM hTSLP and 50 or 5 pM cynomolgus TSLP and compared with different anti-TSLP reference compounds (anti-hTSLP reference mAb2 and anti-hTSLP reference mAb1). The data are summarized in Table 15 (hTSLP and cynomolgus TSLP). The biparatopic constructs clearly outperform the reference mAbs. The biparatopic constructs with F0107501A02 at the N-terminus show improved reactivity to cynomolgus TSLP compared to the constructs with F0107501A02 at the C-terminus.

[0315] [Table 23]

[0316] [Table 24]

[0317] 6.3 Sequence optimization of anti-IL-13 and anti-TSLP monovalent ISVDs 6.3.1 Example 12: Sequence Optimization of Anti-IL-13 and Anti-TSLP Monovalent ISVDs Anti-IL-13 ISVDs F0107004B02 and F0107004B06 and anti-TSLP ISVDs F0107501A02 and F0107529F10 were further sequence optimized.

[0318] Sequence optimization involves replacing one or more specific amino acid residues in a sequence to improve one or more (desired) properties of the ISVD.

[0319] Some examples of such sequence optimizations are described further herein, and include, but are not limited to, the following: 1) Substitution in the parent wild-type ISVD sequence to obtain an ISVD sequence more identical to the human VH3-JH germline consensus sequence, a process called humanization. To achieve this goal, specific amino acids in the FRs that differ between the ISVD and the human VH3-JH germline consensus, except for the so-called hallmark residues, are changed to their human counterparts in such a way that the protein structure, activity and stability remain intact.

[0320] 2) Substitutions into the llama germline to increase the stability of the ISVD, defined as camelization. To this end, the parent wild-type ISVD amino acid sequence is aligned against the llama IGHV germline amino acid sequence of the ISVD (identified as the top hit from the BlastP analysis of the ISVD against the llama IGHV germline).

[0321] 3) Again depending on the desired host cell or host organism, substitutions that improve long-term stability or properties under storage, substitutions that increase expression levels in the desired host cell or host organism, and / or substitutions that remove or reduce (undesirable) post-translational modifications (e.g. glycosylation or phosphorylation). To avoid N-terminal pyroglutamate formation, typically E1D mutations are introduced into the N-terminal building blocks of multivalent Nbs without affecting potency or stability. Thus, E1D mutations are not consistently introduced during sequence optimization of building blocks.

[0322] 4) The mutation at position 11 to Val and at position 89 to Leu minimizes binding of any naturally occurring pre-existing antibody activity.

[0323] F0107004B02 and F0107004B06 Sequence optimization of anti-IL-13 ISVD F0107004B02 resulted in the final sequence-optimized variant F027100019, which contains eight amino acid substitutions (i.e., E1D, L11V, A14P, N64K, S65G, A74S, K83R, I89L) compared to the parent ISVD F107004B02. Sequence optimization of anti-IL-13 ISVD F0107004B06 resulted in the final sequence-optimized variant F027100183, which contains four amino acid substitutions (i.e., L11V, R74S, K83R, V89L) compared to the parent ISVD F107004B06.

[0324] Sequence-optimized variants were assembled from oligonucleotides using a PCR overlap extension method. The variants were expressed in E. coli and purified by IMAC and desalting. F027100019 and F027100183 were evaluated for their hIL-13 binding capacity by surface plasmon resonance using hIL-13 (cat. no. 200-13) from Peprotech. In addition, F027100019 was tested for its neutralizing activity in an eotaxin release assay. The monomeric behavior of both variants was monitored by size-exclusion HPLC (SE-HPLC). The thermal stability of the variants was tested in a thermal shift assay (TSA) using a Lightcycler (Roche). In this assay, the parent ISVD and its variants are incubated in the presence of cypro orange at different pHs and a temperature gradient is applied. Once the ISVD begins to unfold, Sypro Orange binds and the measured fluorescence suddenly increases, allowing the melting temperature to be determined for a particular pH. The results are summarized in Tables 17 and 18.

[0325] [Table 25]

[0326] F027100019 exhibited good potency in eotaxin release assays and its affinity for hIL-13 was determined to be 34 nM in SPR. The Tm of F027100019 is 3° C. higher than that of the parent ISVD F0107004B2. The % framework identity in the framework regions of F027100019 is 85% based on the AbM definition (see Kontermann and Dubel (eds.), Antibody Engineering, Vol. 2, Springer Verlag Heidelberg Berlin, 2010) and 86% based on the Kabat definition.

[0327] [Table 26]

[0328] The affinity of F027100183 is similar compared to the WT sequence, with the variant having a Tm of 61° C. The variant elutes as a 100% monomer peak in SE-HPLC. The % framework identity in the framework regions of F027100183 is 94.4% based on the AbM definition and 93.1% based on the Kabat definition.

[0329] F0107529F10 Sequence optimization of anti-TSLP ISVD F0107529F10 resulted in the final sequence-optimized variant F027400021, which contains eight amino acid substitutions (i.e., L11V, A14P, T60A, S71R, A74S, S79Y, K83R, V89L) compared to the parent ISVD F0107529F10. Sequence optimization of anti-TSLP ISVD F0107501A02 resulted in the final sequence-optimized variant F027400160, which contains six amino acid substitutions (i.e., L11V, A14P, E16G, A74S, K83R, V89L) compared to the parent ISVD F0107501A02.

[0330] Sequence-optimized variants were assembled from oligonucleotides using the PCR overlap extension method. Constructs were expressed in E. coli and purified by IMAC and desalting. Variants were evaluated for their binding ability to human and cynomolgus TSLP by surface plasmon resonance. The monomer behavior of all variants was monitored by size-exclusion HPLC (SE-HPLC) and thermal stability was monitored by thermal shift assay (TSA). In addition, variants of F0107501A02 were tested for their blocking activity against hTSLP in the ternary complex Alphascreen. The results are summarized in Table 19 and Table 20.

[0331] [Table 27]

[0332] The intermediate variant F010704099 with mutations A14P, T60A, S71R, A74S, S79Y, K83R showed similar affinity to hTSLP and half-fold lower affinity to cynomolgus TSLP compared to the parent ISVD F0107529F10. The Tm showed an overall increase of 11.4°C compared to the parent ISVD. Two additional mutations, namely L11V and V89L, were introduced into variant F010704099 to minimize pre-existing antibody binding, resulting in the final variant F027400021. The % framework identity in the framework regions of F027400021 is 89.9% based on the AbM definition and 88.5% based on the Kabat definition.

[0333] [Table 28]

[0334] The intermediate variant F010704076, with mutations A14P, E16G, A74S, K83R, showed similar off-rates against hTSLP and cynomolgus TSLP and similar potency in the ternary complex Alphascreen compared to the parent ISVD F0107501A02. The Tm showed an overall increase of 12.3 °C compared to the parent ISVD. Two additional mutations, namely L11V and V89L, were introduced into variant F010704076 to minimize pre-existing antibody binding, resulting in the final variant F027400160.

[0335] The percent framework identity in the framework regions of F027400160 is 83.1% based on the AbM definition and 80.5% based on the Kabat definition.

[0336] [Table 29]

[0337] 6.4 Multispecific ISVD construct F027400161 The above identified optimized ISVDs F027100019 (optimized variant of F0107004B02), F027100183 (optimized variant of F0107004B06), F027400021 (optimized variant of F0107529F10), and F027400160 (optimized version of F0107501A02) were used for the generation of the multispecific ISVD construct F027400161. The optimized monovalent building blocks used in F027400161 are named below according to their ISVD origin in abbreviated form as 04B02, 04B06, 529F10, and 501A02, respectively.

[0338] 6.4.1 Example 15: Generation of multispecific ISVD constructs Identification of a polypeptide F027400161 (SEQ ID NO: 1) containing an ISVD that binds IL-13 and TSLP was achieved by a data-driven bispecific engineering and formatting strategy that included several anti-TLSP building blocks, several anti-IL13 building blocks and the anti-HSA building block ALB23002. Different positions / orientations of the building blocks and different linker lengths (9GS vs. 35GS) were applied and proved to be important for different parameters (potency, cross-reactivity, expression yield, etc.).

[0339] A large panel of different ISVD constructs was transformed into Pichia pastoris for small-scale production. Induction of ISVD expression occurred by stepwise addition of methanol. Clarified medium containing secreted ISVD was used as starting material for purification via protein A affinity chromatography and subsequent desalting. Purified samples were used for functional characterization and expression evaluation.

[0340] Some constructs showed compromised potency depending on the linker length and relative position of the ISVD building blocks. For example: the potency of the anti-TSLP biparatopic combination 501A02-529F10 against cynomolgus TSLP was strongly compromised when linked with a short 9GS linker. Some constructs showed low expression levels depending on the combination and order of the building blocks. For the bispecific containing 4B02-4B06, expression was best when combined with 501A02-529F10.

[0341] [Table 30] Table 23 illustrates that six constructs containing the same building blocks but arranged differently and connected with different linker lengths gave different yields ranging from low to high titers. The highest expression titers were obtained with constructs (F027400161 and F027400163) containing IL-13 ISVD 4B02 and 4B06 linked via a 35GS linker. In addition, the solubility of F027400161 and F027400163 was higher than their respective counterparts F027400296 and 298, in which the building blocks were linked with four 9GS linkers and the ALB was located at the C-terminus.

[0342] The large bispecific panel was then reduced to a panel of two bispecific constructs consisting of ISVD constructs F027400161 and F027400163, which proved potent against both targets (human and cynomolgus monkey) and had the potential for high expression levels based on preliminary yield estimates.

[0343] Larger scale 2L and 5L production in Pichia pastoris was performed for expression yield determination and evaluation of biophysical properties and pre-existing antibody reactivity.

[0344] Table 24 and Example 22 demonstrate that pre-existing antibody reactivity is enhanced by building block orientation and linker length.

[0345] [Table 31]

[0346] [Table 32]

[0347] Finally, ISVD construct F027400161 was selected based on potency, reduced binding to pre-existing antibodies, and superior expression levels and CMC characteristics.

[0348] 6.4.2 Example 16: Binding Affinities of Multispecific ISVD Constructs for TSLP, IL13, and Serum Albumin The equilibrium dissociation constants (K D Affinity expressed as affinity (Human and Cynomolgus TSLP sequences are known (Uniprot accession number Q969D9 and NCBI reference sequence XP_005557555.1, respectively). Assays were performed using recombinant proteins and human IL-13 (Sino Biological catalog number 10369-HNAC), Cynomolgus IL-13 (Sino Biological catalog number 11057-CNAH) and Rhesus IL-13 (R&D Systems catalog number 2674-RM), as well as human (Sigma Aldrich, catalog number A8763), Cynomolgus and mouse (Albumin Bioscience catalog number N1204H1CM) serum albumins were quantified by affinity measurements in solution on a Gyrolab xP workstation (Gyros).

[0349] K DIn measurements controlled by, serial dilutions of TSLP or IL-13 (ranging from 1 μM to 0.25 fM) or serum albumin (ranging from 100 μM to 320 pM) and a fixed amount of F027400161 (5 pM or 10 pM for TSLP and IL13, and 20 nM for serum albumin) were mixed and allowed to interact and incubated for either 48 or 72 h (for TSLP and IL13) or 2 h (for serum albumin) to reach equilibrium.

[0350] In receptor-controlled measurements, serial dilutions of TSLP or IL-13 (ranging from 1 μM to 0.25 fM) or serum albumin (ranging from 100 μM to 320 pM) and fixed amounts of F027400161 (250 pM for TSLP, 10 nM for IL-13, and 1 μM for serum albumin) were mixed and allowed to interact and incubated for either 48 or 72 h (for TSLP and IL-13) or 2 h (for serum albumin) to reach equilibrium.

[0351] Biotinylated human TSLP / IL-13 / serum albumin was captured on a Gyrolab Bioaffy 1000CD microstructure containing a column of beads and used as a molecular probe to capture free F027400161 from the equilibrated solution. A mixture of TLSP / IL-13 / serum albumin and F027400161 (containing free TLSP / IL-13 / serum albumin, free F027400161 and TLSP / IL-13 / serum albumin-F027400161 complex) was flowed through the beads, capturing a small percentage of free F027400161 proportional to the free ISVD concentration. Fluorescently labeled anti-V HH The antibody, ABH0086-Alexa647, was injected to label all captured F027400161 and the change in fluorescence was determined after rinsing off excess fluorescent probe. Fitting of the dilution series was performed using Gyrolab Analysis software to determine the K Dand receptor-regulated curves were analyzed to determine KD values.

[0352] The results (Table 25) demonstrate that the multispecific ISVD constructs bind human / cynomolgus TSLP and human / cynomolgus / rhesus IL13 with high affinity.

[0353] [Table 33]

[0354] 6.4.3 Example 17: Multispecific ISVD constructs selectively bind TSLP and IL13 The absence of F027400161 binding to IL-4 and IL-7 as IL13 and TSLP-related cytokines, respectively, was assessed by SPR (Proteon XPR36).

[0355] Cytokines were immobilized on the proteon GLC sensor chip at 25 μg / mL for 600 seconds using amine coupling (ProteOn amine coupling kit, catalog number 176-2410) with an 80 second injection of EDC / NHS for activation and a 150 second injection of 1M ethanolamine HCl for deactivation. The flow rate during activation and deactivation was set to 30 μl / min, and the flow rate during ligand injection was set to 25 μl / min. The pH of the 10 mM acetate immobilization buffer was 6.0 for IL13 and IL4 (Peprotech catalog number 200-07) and 5.5 for TSLP and IL7 (R&D Systems catalog number 204-IL / CF).

[0356] Next, 1 μM of F027400161 was injected over 2 min and dissociated for 600 s at a flow rate of 45 μL / min. PBS (pH 7.4) + 0.005% Tween 20 was used as the running buffer. As positive controls, 100 nM of α-hIL4 Ab and 100 nM of α-IL7 Ab were injected. The interaction of F027400161 and the positive controls with the immobilized target was measured by detecting an increase in the immune indicator that occurs as a result of a mass change on the chip upon binding.

[0357] All positive controls did not bind to their respective targets. No binding of F027400161 to human IL4 and IL7 was detected.

[0358] In addition, we investigated whether F027400161 could bind to the short form of TSLP. To this end, TSLP and the short form of TSLP (as described in Fornasa, 2015) were immobilized on a proteon GLC sensor chip at 10 μg / mL using amine coupling as described above for 150 seconds at 5 μg / mL, respectively. The pH of the 10 mM acetate immobilization buffer was 5.5 for TSLP and 4.0 for the short form of TSLP.

[0359] Next, 500 nM of F027400161 was injected over 2 min and allowed to dissociate for 600 s at a flow rate of 45 μL / min. PBS (pH 7.4) + 0.005% Tween 20 was used as the running buffer. As a reference compound, 500 nM of anti-hTSLP reference mAb1 was injected, and as a positive control, 500 nM of α-hTSLP pAb (Abcam ab47943) was injected.

[0360] In contrast, the positive control did not bind to either the long (normal) or short form of TSLP, and binding of F027400161 and anti-hTSLP reference mAb1 to the short form of TSLP was undetectable.

[0361] 6.4.4 Example 18: Simultaneous binding of multispecific ISVD constructs to IL13, TSLP and HSA Using a Biacore T200 instrument, we determined whether F027400161 could bind hTSLP and hIL13 simultaneously. To this end, hTSLP (recombinant human TSLP is commercially available, e.g., from R&D Systems (catalog no. 1398-TS) was immobilized onto a CM5 sensor chip via amine coupling. To capture the ISVD construct via the TSLP building block 501A02-529F10, 100 nM F027400161 was injected over the TSLP surface at 10 μΙ / min for 2 min. Then, 100 nM hIL13 (Peprotec) was injected over the TSLP surface at 10 μΙ / min for 2 min. h, Cat. No. 200-13), either HSA or hOX40L or 1000 nM HSA was injected, or a mixture of 100 nM IL13 + 100 nM HSA, 100 nM IL13 + 1000 nM HSA, 100 nM OX40L + 100 nM HSA, 100 nM OX40L + 1000 nM HSA or 100 nM IL13 + 100 nM OX40L at a flow rate of 10 μl / min. The TSLP surface was regenerated with a 1 min injection of 0.5% SDS + 10 mM glycine pH 3 at 45 μl / min. The sensorgrams (FIG. 1) demonstrate that F027400161 can bind human IL13, human TSLP and HSA simultaneously as shown by the increase in response units after capture with TSLP: approx. 130 RU increase with IL13 alone, approx. 60 RU increase with 100 nM HSA, and approx. 350 RU with 1000 nM HSA alone, approx. 180 RU increase with IL13 and 100 nM HSA mixture, and approx. 500 RU with IL13 and 1000 nM HSA mixture. In the case of HSA, a higher concentration of HSA was required to see some RU increase level due to the lower affinity of F027400161 (see Example 16).

[0362] Example 19: Multispecific ISVD construct inhibition of IL13-induced eotaxin release in vitro The functional activities of soluble IL13 from different species of interest (human, rhesus and cynomolgus monkeys) and their inhibition by F027400161 were studied using a cell-based assay investigating eotaxin release by A549 human lung carcinoma cells.

[0363] To this end, A549 suspension cells were cultured in Ham's F12K medium supplemented with 10% FCS and seeded at 400,000 cells / well in 96-well plates. After 24 hours of incubation, a dilution series of F027100161 or reference compounds (anti-hIL-13 reference mAb1 and anti-hIL-13 reference mAb2) were added. After 20 minutes of incubation, human IL13 (Sino Biological Cat. No. 10369-HNAC), cynomolgus IL13 (Sino Biological Cat. No. 11057-CNAH) or rhesus IL13 (R&D Systems, Cat. No. 2674-RM-025) were added to a final concentration of 160 pM. After a further 24 h incubation in the presence of 30 μM HSA, heparin was added to a final concentration of 50 μg / ml to enhance eotaxin expression. After an additional 4 h incubation, eotaxin-3 secreted into the cell supernatant was quantified by using the Human CCL26 / Eotaxin-3 DuoSet ELISA (R&D Systems, DY346).

[0364] F027400161 inhibited eotaxin-3 release induced by human, cynomolgus and rhesus IL13 in a concentration-dependent manner with IC50 of 194 pM (for human IL13), 1040 pM (for cynomolgus IL13) and 713 pM (for rhesus IL13), which was comparable to the reference compound anti-hIL-13 refmAb1 and superior to the reference compound anti-hIL-13 refmAb2 (Table 26, Figure 2).

[0365] [Table 34]

[0366] 6.4.5 Example 20: Multispecific ISVD construct inhibition of IL13-induced STAT-6 activation in HEK-Blue IL4 / IL13 cells HEK-Blue™ IL-4 / IL-13 cells were generated by stable transfection of HEK293 cells with the human STAT6 gene and a STAT6-inducible SEAP reporter gene. Upon stimulation with IL-4 and IL-13, the cells produced STAT6-induced SEAP, which was secreted into the supernatant and quantified by QUANTI-Blue™.

[0367] HEK-Blue™ cells were cultured in DMEM supplemented with 10% FBS and seeded at 50,000 cells / well in 96-well plates. Serial dilutions of F027100161 or reference compounds (anti-hIL-13 reference mAb1 and anti-hIL-13 reference mAb2) were pre-incubated with 10 pM hIL13 (Sino Biological Cat. No. 10369-HNAC) or cynomolgus IL-13 (Sino Biological Cat. No. 11057-CNAH) for 1 h at room temperature and added to the cells. After 22-24 h of incubation in the presence of 30 μM HSA, 40 μl of cell supernatant was mixed with 160 μl of QUANTI-Blue™. Secreted SEAP was quantified by measuring absorbance at 620 nm on a Clariostar instrument.

[0368] F027400161 inhibited SEAP secretion induced by human and cynomolgus IL-13 in a concentration-dependent manner with IC50 of 32.8 pM (for human IL-13) and 53.4 pM (for cynomolgus IL-13), superior to the reference compound anti-hIL-13 Reference mAb2 (Table 27, Figure 3).

[0369] [Table 35]

[0370] 6.4.6 Example 21: Multispecific ISVD Construct Inhibition of TSLP-Induced Ba / F3 Cell Proliferation In Vitro The functional activities of soluble TSLP from different species of interest (human, rhesus and cynomolgus monkeys) and their inhibition by F027400161 were studied using a cell-based assay investigating the proliferation of BaF3 cells transfected with plasmids encoding hTSLPR and hIL7Ra.

[0371] Cells were seeded in white cell culture treated 96 well plates at a density of 15000 cells / well in RPMI1640 growth medium. A dilution series of F027100161 or a reference compound (anti-hTSLP reference mAb1) was added, followed by 5 pM of human or cynomolgus TLSP for cell stimulation. Human and cynomolgus TSLP sequences are known (Uniprot accession number Q969D9 and NCBI reference sequence XP_005557555.1, respectively). Assays were performed using recombinant proteins. After 48 hours of incubation in the presence of 30 μM HSA, cell density and viability were monitored using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, G7571 / G7572 / G7573) and read out on an EnVision multilabel reader (Perkin Elmer).

[0372] F027400161 inhibited human and cynomolgus TSLP-dependent proliferation of Ba / F3 cells in a concentration-dependent manner with IC50 of 7.8 pM (for human TSP) and 24 pM (for cynomolgus TSLP), thus outperforming the reference compound anti-hTSLP reference mAb1 (Table 28, Figure 4).

[0373] [Table 36]

[0374] 6.4.7 Example 22: Multispecific ISVD Constructs that Bind to Existing Antibodies Pre-existing antibody reactivity of ISVD construct F027400161 was assessed in normal human serum (n=96) using ProteOn XPR36 (Bio-Rad Laboratories, Inc.). PBS / Tween (phosphate buffered saline, pH 7.4, 0.005% Tween 20) was used as the running buffer and experiments were performed at 25°C.

[0375] ISVD was captured on the chip via binding of the ALB23002 building block to HSA immobilized on the chip. To immobilize HSA, the ligand lane of the ProteOn GLC sensor chip was activated with EDC / NHS (flow rate 30 μΙ / min) and HSA was injected at 100 μl / ml in ProteOn acetate buffer, pH 4.5, to an immobilization level of approximately 2900 RU. After immobilization, the surface was inactivated with ethanolamine HCl (flow rate 30 μΙ / min).

[0376] The ISVD construct was then injected over the HSA surface at 45 μl / min for 2 min, resulting in an ISVD capture level of approximately 800 RU. Samples containing pre-existing antibodies were centrifuged at 14,000 rpm for 2 min, and the supernatant was diluted 1:10 in PBS-Tween20 (0.005%) and then injected for 2 min at 45 μl / min, followed by a subsequent 400 s dissociation step. After each cycle (i.e. before a new ISVD capture and blood sample injection step), the HSA surface was regenerated with a 2 min injection of 45 μl / min of HCl (100 mM). After 1) ISVD-HSA dissociation and 2) double referencing by subtracting non-specific binding to the reference ligand lane, a sensorgram showing pre-existing antibody binding was obtained. The binding level of pre-existing antibodies was determined by setting the reporting point at 125 s (5 s after the end of association). The percentage reduction in existing antibody binding relative to the binding level at 125 seconds of the reference ISVD was calculated.

[0377] The pentavalent ISVD construct F027400161 was optimized for reduction of pre-existing antibody binding by introduction of the mutations L11V and V89L in each building block and a C-terminal alanine, and shows substantially less binding to pre-existing antibodies compared to the control non-optimized pentavalent ISVD construct F027301186 (Table 24 and Figure 5).

[0378] Pre-existing antibody binding depends on the orientation of the building blocks and the linker length present in the multispecific construct. Table 24 and Figure 5 demonstrate that construct F027400161 exhibits lower pre-existing antibody reactivity than construct F027400163 due to its specific orientation, but F027400164 exhibits lower reactivity than F027400161 due to the overall shorter linker.

[0379] 6.4.8 Example 23: F027400161 Blocks TSLP-Induced CCL17 in Human Dendritic Cells In Vitro The type 2 inflammatory cascade is initiated and propagated by the coordinated action of epithelial cells, dendritic cells, type 2 helper T cells (Th2 cells), mast cells, and innate lymphoid cells in an environment-dependent manner. The cytokine thymic stromal lymphopoietin (TSLP) has been shown to be involved in the initiation and progression of allergic inflammation through its ability to activate dendritic cells (DCs). Upon activation by TSLP, human DCs produce CCL17, a Th2-associated chemokine, and upregulate naive CD4 + Promotes Th2 cell differentiation from T cells. F027400161 targets both TSLP and IL-13 and can block the interaction of TSLP with DCs, thereby reducing CCL-17 production and is expected to be effective in type 2 inflammatory diseases, etc.

[0380] The ability of F027400161 to inhibit TSLP-induced CCL17 production in human DCs isolated from eight individual healthy donors was evaluated in comparison to a reference monospecific antibody, anti-hTSLP reference mAb1. Human DCs (CD3- CD14 - CD11c + HLA-DR high ) were isolated and enriched from healthy human PBMCs in buffy coat (human packed white blood cell) samples. A total of 0.5–0.8 × 10 6 DCs / well were incubated with eight 3-fold serially diluted concentrations of F027400161 (400 ng / mL or 5.714 nM top concentration) or eight 4-fold serially diluted concentrations of anti-hTSLP reference mAb1 (4000 ng / mL or 27 nM top concentration) prior to stimulation with 4 ng / mL recombinant human TSLP for 36 h in a 37° C. cell culture incubator. Recombinant human TSLP is commercially available, e.g., from R&D Systems (catalog no. 1398-TS). CCL17 production in freshly harvested cell culture supernatants was measured by ELISA and the IC of ISVD constructs and reference antibodies were calculated. 50 Values ​​were calculated with Graphpad Prism.

[0381] The collective results of the dose inhibitory responses of F027400161 and anti-hTSLP Reference mAb1 in human DCs are shown in Figure 6. The reference monospecific antibody, anti-hTSLP Reference mAb1, inhibited TSLP-induced CCL17 production with a mean IC of 793.4 pM. 50 concentrations, whereas F027400161 inhibited TSLP-induced CCL17 production with an average IC of 53.26 pM. 50 was inhibited.

[0382] In conclusion, these results demonstrate that the ISVD construct F027400161 is more effective in inhibiting TSLP-induced CCL17 responses in human DCs compared to the anti-hTSLP reference mAb1.

[0383] 6.4.9 Example 24: In Vitro, F027400161 Blocks Synergistic CCL17 Induced by 0.5 ng / mL [IL-13+TSLP] in Human PBMCs Type 2 cytokines, such as thymic stromal lymphopoietin (TSLP) and interleukin-13 (IL-13), exert unique, additive and synergistic responses to drive the pathophysiology of asthma and atopic dermatitis (AD). The role of TSLP as an epithelial cell-derived initiator of the type 2 immune cascade and IL-13 as a downstream effector cytokine have been extensively examined. F027400161 targets both TSLP and IL-13, thereby expected to confer efficacy in type 2-mediated inflammatory diseases, etc.

[0384] The ability of F027400161 to inhibit 0.5ng / mL IL-13 and TSLP-induced synergistic production of CCL17 (TARC) was evaluated in human PBMCs from eight individual healthy donors. The study was designed to evaluate the non-inferiority of the ISVD construct to monospecific biologics, anti-hTSLP reference mAb1 and anti-hIL-13 reference mAb1. One million cells per well of human PBMCs were stimulated with 0.5 ng / mL recombinant human TSLP (recombinant human TSLP is commercially available, e.g., from R&D Systems (cat. no. 1398-TS) plus IL-13 (R&D Systems, cat. no. 213-ILB-005 / CF) and incubated in a 37° C. cell culture incubator in 96-well plates with 10-3-fold serially diluted concentrations of ISVD constructs (10 nM top concentration), anti-hIL-13 reference mAb1 (10 nM top concentration) and anti-hTSLP reference mAb1 (100 nM top concentration) for 20 h. Assays were performed in technical triplicates for each donor for F027400161. Concentrations of cytokines used were within 2-fold standard error of reported literature values ​​from serum, BAL fluid, sputum and skin of normal humans, asthmatics and atopic dermatitis patients (Berraies A et al., Immunol Letter 178:85-91, 2016; Bellini A et al., Mucosal Immunology 5(2):140-9, 2012; Davoodi P et al., Cytokine 60(2):431-7, 2012; Szegedi K et al., J Eur Acad Dermatol Venereol 29(11):2136-44, 2015). CCL17 production in freshly collected cell culture supernatants was measured by the Meso Scale Diagnostics (MSD) V-PLEX human TARC kit.

[0385] The collective results of the dose inhibitory response of F027400161 and the reference antibodies, anti-hIL-13 Reference mAb1 and anti-hTSLP Reference mAb1, are shown in Figure 7. F027400161 induced 100% inhibition of synergistic CCL17 production induced by 0.5 ng / mL IL-13 + TSLP with an average IC of 0.0061 nM. 50 The reference antibody, anti-hIL-13mAb1, had a lower mean IC of 0.0028 nM. 50 Despite having a mean IC50 of 2.932 nM, equimolar doses of F027400161 were unable to completely block the synergistic CCL17 response, plateauing at approximately 80% inhibition. On the other hand, the anti-hTSLP reference mAb1 had a mean IC50 of 2.932 nM. 50 was able to block approximately 50% of CCL17 production.

[0386] In conclusion, these results demonstrate that F027400161 is more effective than anti-hTSLP reference mAb1 and is superior to anti-hIL-13 reference mAb1 in blocking the synergistic responses induced by pathophysiologically relevant concentrations of IL-13 and TSLP in human PBMCs, highlighting its therapeutic potential for the treatment of type 2 inflammatory diseases such as asthma and atopic dermatitis.

[0387] 6.4.10 Example 25: In Vitro, F027400161 Blocks Synergistic CCL17 Induced by 5 ng / mL [IL-13+TSLP] in Human PBMCs The ability of F027400161 to inhibit CCL17 production induced by 5ng / mL IL-13+TSLP was evaluated in human PBMCs from eight healthy individual donors. The study was designed to evaluate the non-inferiority of the ISVD construct to monospecific biologics, anti-hTSLP reference mAb1 and anti-IL-13 reference mAb1. The cytokine concentrations used were approximately 10-fold above the upper limit of the pathophysiological range of TSLP and IL-13 reported in the literature in normal humans, asthmatics, and atopic dermatitis patients, as hypothesized concentrations during transient inflammatory states (Berraies A et al., Immunol Letter 178:85-91, 2016; Bellini A et al., Mucosal Immunology 5(2):140-9, 2012; Davoodi P et al., Cytokine 60(2):431-7, 2012; Szegedi K et al., J Eur Acad Dermatol Venereol 29(11):2136-44, 2015). Human PBMCs, 1 million cells per well, were stimulated with 5 ng / mL recombinant human TSLP plus IL-13 (R&D Systems, Cat. No. 213-ILB-005 / CF) and incubated in a 96-well plate with 10-fold serially diluted concentrations of F-27400161 (10 nM top concentration), anti-hIL-13 reference mAb1 (10 nM top concentration) and anti-hTSLP reference mAb1 (100 nM top dose) for 20 h in a 37° C. cell culture incubator. Assays were performed in technical triplicates for each donor for F027400161. CCL17 production in freshly collected cell culture supernatants was measured by Meso Scale Diagnostics (MSD) V-PLEX human TARC kit.

[0388] The collective results of the dose inhibitory response of F027400161 and the reference antibodies, anti-hIL-13 Reference mAb1 and anti-hTSLP Reference mAb1, are shown in Figure 8. F027400161 induced 100% inhibition of synergistic CCL17 production induced by 5 ng / mL IL-13 + TSLP with an average IC of 0.0387 nM. 50PBMCs treated with an equimolar dose of the comparator antibody, anti-hIL-13 reference mAb1, had a lower mean IC of 0.01339 nM. 50 However, the inhibitory response never reached 100% and plateaued at approximately 90% inhibition. In contrast, anti-hTSLP reference mAb1 inhibited only approximately 40% of CCL17 production with an average IC of 19 nM. 50 did not partially block it.

[0389] In conclusion, these results demonstrate that F027400161 is superior to both anti-hTSLP and anti-hIL-13 reference mAb1 in blocking the synergistic CCL17 response induced by IL-13 and TSLP in human PBMCs at [TSLP+IL-13] concentrations 10-fold above the pathophysiological range of the cytokines, highlighting its therapeutic potential for the treatment of asthma and atopic dermatitis during acute or inflammatory periods.

[0390] 6.4.11 Experiment 27: T027400161 blocks allergen Der P-induced production of IL-5, CCL17, and CCL26 in three culture assay systems TSLP promotes type 2 immune responses by inducing CCL17, IL-5, and IL-13 production, which then initiates CCL26 production by local epithelial cells, resulting in collateral problems such as inflammatory diseases mediated by type 2 immune responses.

[0391] The ability of F027400161 to inhibit TSLP-induced IL-5 and CCL17, and IL-13-induced CCL26 production was evaluated in a three-culture assay system using MRC5 fibroblasts and A549 epithelial cells cultured for six days with Der P-stimulated human PBMCs from six individual normal donors. The study was designed to evaluate the non-inferiority of ISVD to monospecific biologics, anti-hTSLP reference mAb1 and anti-hIL-13 reference mAb1. In response to IL-13 produced by PBMCs with Der P and endogenous TSLP stimulation, MRC5 fibroblasts constitutively produced approximately 100 pg / mL of endogenous TSLP, and A549 epithelial cells produced CCL26. 75,000 MRC5 fibroblasts and A549 epithelial cells were plated per well one day prior to co-culture with human PBMCs. Human PBMCs were added at 1 million cells per well to plated MRC5 fibroblasts and A549 epithelial cells, stimulated with 3 μg / mL Der P, and incubated in 24-well plates with 11.1 nM ISVD, anti-hIL-13 reference mAb1, or anti-hTSLP reference mAb1 for 6 days in a 37° C. cell culture incubator. Assays were performed in technical triplicates for each donor for F027400161. IL-5, CCL17, and CCL26 production in freshly collected cell culture supernatants was measured by human magnetic Luminex assays from RnD System.

[0392] The collective results of the inhibitory responses of F027400161 and the reference antibodies, anti-hIL-13 Reference mAb1 and anti-hTSLP Reference mAb1, are shown in Figure 9. F027400161 demonstrated 60% inhibition of CCL17 production, 50% inhibition of IL-5 production, and 95% inhibition of CCL26 production. The reference antibody, anti-hTSLP Reference mAb1, showed 50% inhibition of CCL17 production, 50% inhibition of IL-5 production, and approximately 55% inhibition of CCL26 production. While anti-hIL-13 Reference mAb1 demonstrated a comparable 95% inhibition of CCL26 production, this reference antibody was only able to block approximately 35% of CCL17 production and less than 10% of IL-5 production (Figure 9). The lack of complete IL-5 and CCL17 inhibition by these tested molecules may suggest that Der P stimulation initiates pathways other than TSLP and IL-13 to promote IL-5 and CCL17 production by PBMCs.

[0393] In conclusion, these results demonstrate that anti-TSLP / IL-13 ISVD F027400161 is superior to anti-hTSLP reference mAb1 and anti-hIL-13 reference mAb1 by its ability to block three cytokines and chemokines (CCL17, IL-5 and CCL26) in a complex assay system including human PBMCs cultured with tissue-specific cells, highlighting its therapeutic potential for the treatment of type 2 inflammatory diseases such as asthma and atopic dermatitis as well as a wide range of immune disease indications.

[0394] 6.4.12 Example 26: NSG-SGM3 Mouse Model to Assess Target Occupancy and Pharmacodynamics Mediated by F027400161 In Vivo F027400161 targets both human TSLP and IL-13 and does not react with the mouse orthologues. Therefore, a xenografted humanized model system was used to evaluate the biological activity of F027400161. Female NSG-SGM3 (NOD / SCID-IL2Rγ- / -, NOD.Cg-PrkdcscidIl2rγtm1Wjl / SzJ) were obtained from Jackson labs, Bar Harbor, ME, USA. These mice express human hematopoietic cytokines: stem cell factor (SCF), granulocyte / macrophage stimulating factor (GM-CSF), and interleukin-3 (IL-3), all driven by human cytomegalovirus promoter / enhancer sequences. The triple transgenic mice constitutively produce the above cytokines, provide cell proliferation and survival signals, and maintain stable engraftment of CD33+ myeloid lineage, and several types of lymphoid cells. Briefly, the protocol followed for engraftment is as follows: On day 0 of the study, mice were irradiated with 150 centigrays at a rate of 120 rad / min for 1 min and 15 s. Mice were irradiated with 1 × 10 5Cord blood CD34+ stem / progenitor cells were engrafted by intravenous (IV) route approximately 6 hours after engraftment. One group of mice was irradiated in the same manner but not engrafted. These mice were considered as irradiated naive mice. On day 88 after engraftment, mice received hydrodynamic (HDD) iv injection of either saline (engrafted control mice) or a combination of 50 μg IL-4 minicircle DNA and 50 μg TSLP minicircle DNA. On day 91, submandibular blood sampling was performed and 100–150 μl of blood was collected from each mouse and placed into lithium heparin tubes. Engraftment checks were performed by flow cytometry to assess plasma concentrations of IL-4 and TSLP. Information from the engraftment checks and / or cytokine determinations were used to select (include or exclude) mice from the study. Mice from the engrafted control group with less than 25% human CD45+ cells were excluded from the study. Similarly, implanted mice that received minicircle DNA and showed plasma TSLP levels one standard deviation below the mean were also excluded from the study. Mice that received minicircle DNA by HDDi.v. injection received either vehicle (20 mM phosphate, 125 mM L-arginine HCL, and 0.01% Tween 20, pH 7.0) or F027400161 (0.01, 0.05, 0.1, or 10 mg / kg) subcutaneously on days 95, 97, 99, and 101. On day 103, mice were anesthetized by isoflurane anesthesia. While under isoflurane anesthesia, blood was collected by retroorbital bleeding. After blood collection, while still under isoflurane anesthesia, mice were sacrificed by cervical dislocation. Portions of lungs were harvested and placed in RNA later for gene expression assessment. Plasma concentrations of human TSLP from plasma samples on day 103 were determined by MSD kit assay (catalog number K15067-L-2, Meso Scale Diagnostics, Rockville, MD, USA).Plasma concentrations of human IL-13 from plasma samples on day 103 were determined by ELISA (catalog number 88-7439--88Human IL-13 ELISA kit, Invitrogen / Thermo-Fischer, Waltham, MA, USA). Internal assay validation experiments demonstrated that both human TSLP and IL-13 detection kits were unable to detect F027400161 bound to hTSLP and hIL-13.

[0395] The collective results of these experiments, shown in Figures 10 and 11, demonstrated that F027400161 could significantly inhibit detectable levels of human TSLP and IL-13 in the plasma of humanized NSG-SGM3 mice, demonstrating target occupancy for both human TSLP and human IL-13.

[0396] In the NSG-SGM3 mouse model, hydrodynamic delivery of cDNA for TSLP and IL-4 drives expression of human IL-13 (Figure 11). By taking advantage of the ability of human IL-13 to signal through the mouse IL-13 receptor, samples obtained from the NSG-SGM3 study were used to study the pharmacodynamic effects of F027400161 (Hershey GK. 2003, J Allergy Clin Immunol.;111(4):677-90). In these studies, the effects of F027400161 treatment on mouse gene transcripts regulated by human IL-13 were studied. Mouse lung tissue from the above study was used for this analysis.

[0397] Lungs from treated and control NSG-SGM3 mice were harvested and processed as detailed in the accompanying protocol to generate RNA. RNA was processed for quantification by TaqMan and data was analyzed as described in the protocol. Briefly, lungs harvested from mice were stored in RNALater, processed and purified according to standard protocols to generate high quality RNA. Purified lung RNA was then reverse transcribed into cDNA using Quanta Q-Script 5X master mix according to the manufacturer's protocol. The resulting lung cDNA was used to quantify the transcript expression levels of human IL-13 responsive mouse target genes (mouse Retnla and mouse Clca1) and an endogenous control (Rpl37a) using TaqMan assays according to the manufacturer's protocol. Data analysis was performed with Quantstudio 6&7 flex software. For each probe, C T Value and Delta C T Values ​​(relative to Rpl37a) were exported to Excel and the relative expression value of each gene was calculated using the following formula: Normalized relative expression = (power(2,-(delta CT))) x 1000.

[0398] The two human IL-13 regulated mouse genes evaluated were Retnla (resistin-like alpha) and Clca1 (chloride channel accessory 1), which play a role in pulmonary vascular remodeling (Lewis CC, 2009, J Allergy Clin Immunol.;123(4):795-804).

[0399] The collective results of these experiments, as shown in Figures 12 and 13, demonstrated that F027400161 was able to significantly inhibit mouse Retnla and mouse Clca1 transcriptional expression, demonstrating a pharmacodynamic effect of F027400161 on human IL-13-driven mouse transcriptional responses in vivo.

[0400] method: Preparation of sample homogenates: Lungs were harvested from mice and stored in RNA later. The lobes were then dried and transferred to fastprep lysis matrix A tubes (for homogenizing the lungs) containing 1 mL of RLT+2-ME. Samples were homogenized in an MP-Bio homogenizer using program 1 (two 40 second cycles separated by 5 minutes to avoid heating the sample). Samples were then spun at 10,000g for 3 minutes. 350ul of lysate [in RLT+2ME (1% v / v)] was collected. Cells were lysed by pipetting multiple times (approximately 20 times) using a multichannel.

[0401] RNA preparation: For RNA purification, 350ul of homogenate was used. 1x volume (350ul) of 70% ethanol was added, the homogenate mixed thoroughly and transferred to a 96-well RNeasy spin plate placed in an elution plate, and RNA was prepared using the Qiagen RNA mini-tissue RNA extraction protocol with the following modifications: The 96-well plate was covered with sealable aluminum foil and centrifuged at 4000xg for 2 minutes. To wash the column, 400ul of Buffer RWT was added to the RNeasy spin column and the spin column plate was centrifuged at 4000xg for 2 minutes at room temperature. DNase I digestion was performed by adding 80ul of 1x DNase I mix and incubating at room temperature for 15 minutes. DNase I was washed off by adding 400ul of Buffer RWT and spinning the plate at 4000xg for 2 minutes. This was followed by washing the spin plate with 500ul of Buffer RPE and 80% ethanol, respectively. Following this, the column membrane was dried by spinning at 4000×g for 4 min. RNA was then eluted in 40 μl of Tris-HCl (10 mM; pH 8.0). RNA was quantified using Nanodrop and 500 ng of RNA was used for cDNA preparation.

[0402] First strand synthesis: cDNA was synthesized using Quanta Q-Script 5X master mix using the manufacturer's protocol. The final concentration of cDNA was 25ng / ul. cDNA was stored at -20°C until the TaqMan assay was performed.

[0403] TaqMan Assays: TaqMan multiplex master mixes were prepared by adding the components in the following order in a 1.5 ml microcentrifuge tube: Three separate master mixes were made for each probe set along with an internal Rpl37a control. Each multiplex qPCR reaction was performed in a 10 μl reaction volume.

[0404] [Table 37]

[0405] A total of 8.8ul of master mix for each sample was added to the appropriate wells of a 384-well optical plate. 30ng (1.2ul) of cDNA sample was added to each well. TaqMan assays were configured on a QuantStudio 7K. Conditions in the thermocycler were as follows: pre-denaturation at 95°C for 3 minutes, 40 cycles of denaturation at 95°C for 2 seconds, and annealing and extension at 60°C for 5 seconds. Fluorescence measurements were taken during the extension step.

[0406] Data Analysis: Data analysis was performed with Quantstudio 6&7 flex software.

[0407] For each probe, the CT value and delta CT value (relative to Rpl37a) were exported into Excel and the relative expression value of each gene was calculated using the following formula: Normalized relative expression = (power(2,-(delta CT))) x 1000

[0408] References: Liu, YJ 2006, J Exp Med.;203(2):269-73. Thymic stromal lymphopoietin: master switch for allergic inflammation. Hershey GK. 2003, J Allergy Clin Immunol.;111(4):677-90. IL-13 receptors and signaling pathways: an evolving web. Lewis CC, Aronow B, Hutton J, Santeliz J, Dienger K, Herman N, Finkelman FD, Wills-Karp M. 2009, J Allergy Clin Immunol.;123(4):795-804. Unique and overlapping gene expression patterns driven by IL-4 and IL-13 in the mouse lung. [Industrial Applicability]

[0409] 7 Industrial Applicability The polypeptides described herein, nucleic acid molecules encoding them, vectors containing the nucleic acids and compositions can be used, for example, in the treatment of subjects suffering from inflammatory diseases.

Claims

1. A polypeptide comprising or consisting of at least four ISVDs, each of said ISVDs comprising three complementarity determining regions (CDR1 to CDR3, respectively): a) the first ISVD specifically binds to IL-13; i. CDR1 which is the amino acid sequence of SEQ ID NO:7; ii. a CDR2 having the amino acid sequence of SEQ ID NO: 12; and iii. CDR3 having the amino acid sequence of SEQ ID NO: 17 Including; b) the second ISVD specifically binds IL-13; iv. CDR1 which is the amino acid sequence of SEQ ID NO:8; v. a CDR2 having the amino acid sequence of SEQ ID NO: 13; and vi. CDR3 having the amino acid sequence of SEQ ID NO: 18 Including; c) the third ISVD specifically binds TSLP; vii. CDR1 which is the amino acid sequence of SEQ ID NO:9; viii. a CDR2 having the amino acid sequence of SEQ ID NO: 14; and ix. CDR3 having the amino acid sequence of SEQ ID NO: 19 Including; d) the fourth ISVD specifically binds TSLP; x. CDR1 having the amino acid sequence of SEQ ID NO:11; xi. CDR2 having the amino acid sequence of SEQ ID NO: 16; and xii. CDR3 having the amino acid sequence of SEQ ID NO:21 Including, The order of the ISVDs indicates their relative positions with respect to each other considered in the N-terminal to C-terminal direction of the polypeptide.

2. A polypeptide as described in claim 1, wherein the at least four ISVDs are linked via one or more peptidic linkers.

3. a) the amino acid sequence of the first ISVD comprises greater than 90% sequence identity with SEQ ID NO:2; b) the amino acid sequence of the second ISVD comprises greater than 90% sequence identity with SEQ ID NO:3; c) the amino acid sequence of the third ISVD comprises greater than 90% sequence identity with SEQ ID NO:4; d) The polypeptide of claim 1 or 2, wherein the amino acid sequence of the fourth ISVD comprises greater than 90% sequence identity with SEQ ID NO:

6.

4. a) the first ISVD comprises the amino acid sequence of SEQ ID NO:2; b) the second ISVD comprises the amino acid sequence of SEQ ID NO:3; c) the third ISVD comprises the amino acid sequence of SEQ ID NO:4; d) The polypeptide according to any one of claims 1 to 3, wherein the fourth ISVD comprises the amino acid sequence of SEQ ID NO:

6.

5. 5. The polypeptide of any one of claims 1 to 4, wherein the polypeptide further comprises one or more other groups, residues, moieties or binding units, which provide the polypeptide with an increased half-life compared to a corresponding polypeptide without said one or more other groups, residues, moieties or binding units.

6. A polypeptide described in any one of claims 1 to 5, wherein the one or more other groups, residues, moieties or binding units are linked via one or more peptidic linkers.

7. 7. The polypeptide of claim 5 or 6, wherein the one or more other groups, residues, moieties or binding units that provide the polypeptide with an increased half-life are selected from the group consisting of polyethylene glycol molecules, serum proteins or fragments thereof, binding units capable of binding to serum proteins, Fc moieties, and low molecular weight proteins or peptides capable of binding to serum proteins.

8. 8. The polypeptide according to any one of claims 5 to 7, wherein the one or more other groups, residues, moieties or binding units providing the polypeptide with increased half-life are selected from the group consisting of binding units capable of binding to serum albumin (such as human serum albumin) or serum immunoglobulins (such as IgG).

9. 9. The polypeptide of claim 8, wherein the binding unit that provides the polypeptide with an increased half-life is an ISVD capable of binding to human serum albumin.

10. The ISVD that binds to human serum albumin is i. CDR1 which is the amino acid sequence of SEQ ID NO: 10; ii. a CDR2 having the amino acid sequence of SEQ ID NO: 15; and iii. CDR3 having the amino acid sequence of SEQ ID NO: 20 The polypeptide of claim 9 .

11. The polypeptide of claim 10, wherein the amino acid sequence of the ISVD that binds to human serum albumin comprises greater than 90% sequence identity with SEQ ID NO:

5.

12. The polypeptide described in claim 11, wherein the ISVD that binds to human serum albumin comprises the amino acid sequence of SEQ ID NO:

5.

13. The polypeptide according to any one of claims 10 to 12, wherein the amino acid sequence of the polypeptide comprises more than 90% sequence identity with SEQ ID NO:

1.

14. A polypeptide according to any one of claims 1 to 13, comprising or consisting of the amino acid sequence of SEQ ID NO:

1.

15. A nucleic acid comprising a nucleotide sequence encoding a polypeptide according to any one of claims 1 to 14.

16. A composition comprising at least one polypeptide according to any one of claims 1 to 14 or a nucleic acid according to claim 15, said composition being a pharmaceutical composition further comprising at least one pharma- ceutically acceptable carrier, diluent or excipient and / or adjuvant.

17. The composition described in claim 16, wherein the composition comprises one or more further pharma- ceutical active polypeptides and / or compounds.

18. A polypeptide according to any one of claims 1 to 14, or a composition according to claim 16 or 17, for use in the treatment of an inflammatory disease, such as a type 2 inflammatory disease.

19. The polypeptide or composition for use according to claim 18, wherein the type 2 inflammatory disease is selected from asthma and atopic dermatitis.

Images