Polypeptides comprising immunoglobulin single variable domains targeting il-13 and tslp

Abstract

To provide a novel type of drug for treating a subject suffering from an inflammatory disease.SOLUTION: Specifically, the present technique provides polypeptides 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 binds to TSLP. The present technique also provides nucleic acids, vectors and compositions.SELECTED DRAWING: None

Description

Technical Field

[0001] Description 1 Field of the Technology The present technology relates to polypeptides that target IL-13 and TSLP. The present technology also relates to nucleic acid molecules encoding the polypeptides and vectors containing the nucleic acids, as well as compositions containing the polypeptides, nucleic acids or vectors. The present technology further relates to these products for use in methods of treating subjects suffering from inflammatory diseases. Additionally, the present technology relates to methods of producing these products.

Background Art

[0002] 2 Technical Background An unchecked immune response is necessary for host defense while also potentially causing 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 onset and spread of various immune diseases. Inflammatory diseases are often chronic and can even be life-threatening. Allergic and atopic diseases, such as asthma and atopic dermatitis, are predominantly promoted by type 2 immune responses and are characterized by prominent features 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 synergistic aspects of type 2 immunity, type 2 inflammatory diseases, such as asthma and atopic dermatitis, as well as various immune diseases.

[0004] TSLP signaling begins through a receptor complex of a heterodimer composed of the thymic stromal lymphopoietin receptor (TSLPR) and the IL-7R alpha chain (IL-7Rα). Similarly, IL-13 signaling begins by binding to a receptor complex of a heterodimer consisting 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 results in their complex formation, which further increases the possibility of heterodimer formation with IL-4Rα.

[0005] TSLP promotes the maturation of dendritic cells, the development and proliferation of mast cells, and in addition, activates other immune cells such as basophils and innate lymphoid cells (ILC2). Similarly, IL-13 causes various immunopathologies such as the breakdown of the epithelial barrier, mucus production from the mucosa to the epithelial surface, airway remodeling, and in addition, induces eosinophils that replenish chemokines such as eotaxin. These mechanisms are at the core of the initiation and spread of type 2 inflammatory responses and are at the core of the development of various immunopathologies in diseases such as atopic dermatitis and asthma.

[0006] Currently, patients with mild / moderate asthma, particularly those with asthma having a particularly low eosinophilic phenotype, respond inappropriately to currently available standard treatment regimens such as biologics such as Dupixent (dupilumab; commercially available), an anti-IL4Rα monoclonal antibody, anti-IL5 (commercially available), and Xolair (omalizumab; commercially available), an anti-IgE monoclonal antibody. TSLP (tezepelumab) and IL-13 (lebri Antagonist monoclonal antibodies against Kizumab) are currently in clinical trials, but there is no active clinical development program 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, and this potential is to confer efficacy in subsets within the range of these symptoms where single-agent monotherapy is not very effective. Therefore, there is still an unmet medical need for the treatment of type 2 inflammatory diseases such as asthma and atopic dermatitis that can be more effective and also applied well to patients.

[0007] Such therapies may involve targeting multiple disease factors, such as IL-13 and TSLP.

[0008] Targeting multiple disease factors can be achieved, for example, by co-administering or using combinatorially 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 treatment regimen that is more inconvenient and painful for the patient, which can have a negative impact on compliance. For a single injection of two separate products, it can be difficult or impossible to provide a formulation that allows for acceptable viscosity at the required concentrations and suitable stability for both products. In addition, co-administration and co-formulations can increase the overall cost as they require the production of two separate drugs.

[0009] As one strategy to address such limitations associated with co-administration or combinatorial use of separate biological agents, such as antibodies, bispecific antibodies that can bind to two different antigens have been suggested.

[0010] Bispecific antibody constructs have been proposed in multiple formats. For example, bispecific antibody formats may include chemical conjugation of two antibodies or their fragments (Non-Patent Document 3; Non-Patent Document 4).

Prior Art Documents

Non-Patent Documents

[0011]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0012] However, the disadvantages of such bispecific antibody formats include high viscosity at high concentrations, which makes, for example, subcutaneous administration difficult, and in terms of specific and high - affinity binding, each binding unit requires the interaction of two variable domains, including the impact on polypeptide stability and production efficiency. Such bispecific antibody formats also potentially cause chemical, manufacturing, and quality control (CMC) problems related to mispairing of light chains or mispairing of heavy chains.

Means for Solving the Problems

[0013] Summary of the Technology In some embodiments, the present technology relates to a polypeptide that simultaneously and specifically targets IL-13 and TSLP and results in an increased efficiency of modulating type 2 inflammatory responses in vitro as compared to a monospecific anti-IL-13 or anti-TSLP polypeptide. In some embodiments, the polypeptide is efficiently produced (e.g., in a microbial host). Further, in some embodiments, such a polypeptide has limited reactivity with existing antibodies (i.e., antibodies present in the subject prior to the first treatment with the antibody construct) in the subject to be treated. In other embodiments, such a polypeptide exhibits a sufficiently long half-life such that it can be successfully spaced between successive treatments in the subject to be treated.

[0014] In one embodiment, the present 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 present 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. Further, 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, and the one or more other groups, residues, moieties or binding units provide a polypeptide having an increased half-life as compared to the corresponding polypeptide that does not have 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. Further, in one embodiment, the polypeptide further comprises one or more other groups, residues, moieties or linking units, optionally linked via one or more peptidic linkers, wherein the one or more other groups, residues, moieties or linking units provide a polypeptide having an increased half-life compared to the corresponding polypeptide that does not have the one or more other groups, residues, moieties or linking units. For example, the linking unit can be an ISVD that binds to a (human) serum protein, 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 of the ISVDs specifically bind to IL-13, and at least two of the ISVDs specifically bind to TSLP. In one embodiment, at least two of the ISVDs that specifically bind to IL-13 specifically bind to human IL-13, and at least two of the ISVDs that specifically bind to TSLP specifically bind to human TSLP. In one embodiment, at least two of the ISVDs that specifically bind to IL-13 are separate ISVDs, and at least two of the 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 linking units, optionally linked via one or more peptidic linkers, wherein the one or more other groups, residues, moieties or linking units provide an increased Provided is a polypeptide having a half-life. For example, the binding unit can be an ISVD that binds to a (human) serum protein, such as human serum albumin.

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

[0018] Also provided are hosts or host cells containing nucleic acids or vectors encoding the polypeptide according to the present technology.

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

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

[0021] In addition, provided is a method for treating an inflammatory disease such as a type 2 inflammatory disease, comprising administering a pharmaceutically 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] Furthermore, 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 a type 2 inflammatory disease is provided. 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 composition comprising a polypeptide, a composition comprising the polypeptide, or a nucleic acid comprising a nucleotide sequence encoding the polypeptide for use as a medicament, wherein the polypeptide comprises or consists of at least one immunoglobulin single variable domain (ISVD), said ISVD comprises three complementarity determining regions (CDR1 to CDR3 respectively), and said at least one ISVD is: a) CDR1 which is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 7; CDR2 which is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 12; and CDR3 which is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 17, b) CDR1 which is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 8; CDR2 which is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 13; and CDR3 which is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 18, c) CDR1 which is the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 9; A CDR2 that is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 14; and A CDR3 that is the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 19, or d) A CDR1 that is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 11; A CDR2 that is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 16; and A CDR3 that is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 21 The polypeptide or composition comprising the same.

[0025] Embodiment 2. At least one ISVD is: a) 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) 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, c) 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, or d) 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 polypeptide or composition for use according to Embodiment 1, comprising the same.

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

[0027] Embodiment 4. The at least one ISVD is: 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 of embodiments 1 to 3, comprising:

[0028] Embodiment 5. The polypeptide comprises or consists of at least two ISVDs, each of the ISVDs 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 an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 7; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 12; and iii. CDR3 which is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 17 comprising, b) The first and second ISVDs are: i. CDR1 which is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 8; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 13; and iii. CDR3 which is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 14 iii. A CDR3 that is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 18 and includes c) The first ISVD i. A CDR1 that is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 7; ii. A CDR2 that is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 12; and iii. A CDR3 that is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 17 and includes The second ISVD iv. A CDR1 that is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 8; v. A CDR2 that is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 13; and vi. A CDR3 that is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 18 and includes d) The first ISVD i. A CDR1 that is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 8; ii. A CDR2 that is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 13; and iii. A CDR3 that is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 18 and includes The second ISVD iv. A CDR1 that is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 7; v. a CDR2 that is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 12; and vi. a CDR3 that is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 17 comprising e) The first ISVD i. a CDR1 that is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 11; ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 16; and iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having a difference of 2 or 1 amino acids from SEQ ID NO: 21 comprising The second ISVD iv. a CDR1 that is the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 9; v. a CDR2 that is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 14; and vi. a CDR3 that is the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 19 comprising, or f) The first ISVD i. a CDR1 that is the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 9; ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 14; and iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 19 comprising The second ISVD iv. A CDR1 that is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 11; v. A CDR2 that is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 16; and vi. A CDR3 that is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 21 comprising, A polypeptide or composition for use according to Embodiment 1, wherein the order of the ISVDs indicates their relative positions to each other as 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 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 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, c) The first 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, and the second 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, d) The first 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, and the second 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, 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) 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, a polypeptide or composition for use according to embodiment 5.

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

[0031] 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) 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, A polypeptide or composition for use according to any of embodiments 5 to 7.

[0032] Embodiment 9. The 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) The amino acid sequence of SEQ ID NO: 159 A polypeptide according to any of embodiments 5 to 8, comprising or consisting of.

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

[0034] Embodiment 11. A pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, optionally comprising one or more further pharmaceutically active polypeptides and / or compounds, the composition for use according to any one of Embodiments 1 to 10.

[0035] Embodiment 12. a) The first 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; b) The second 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; c) The third 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; d) The fourth 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, a polypeptide or composition for use according to Embodiment 10 or 11.

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

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

[0038] Embodiment 15. The polypeptide further includes one or more other groups, residues, moieties or linking units optionally linked via one or more peptidic linkers, and the one or more other groups, residues, moieties or linking units have an increased half-life compared to the corresponding polypeptide without the one or more other groups, residues, moieties or linking units, a polypeptide or composition for use according to any one of embodiments 1 to 14.

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

[0040] For the use according to any one of Embodiments 15 - 16, the polypeptide or composition, wherein the one or more other linking units for providing a polypeptide having an increased half - life consist of a linking unit capable of binding to serum albumin (such as human serum albumin) or serum immunoglobulin (such as IgG).

[0041] For the use according to Embodiment 17, the polypeptide or composition, wherein the linking unit for providing a polypeptide having an increased half - life is an ISVD capable of binding to human serum albumin.

[0042] 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 a difference of 2 or 1 amino acid from SEQ ID NO: 10; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 15; and iii. CDR3 which is the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 20 For the use according to Embodiment 18, the polypeptide or composition comprising the above.

[0043] For the use according to any of Embodiments 18 - 19, the polypeptide or composition, wherein the ISVD that binds to human serum albumin comprises CDR1 which is the amino acid sequence of SEQ ID NO: 10, CDR2 which is the amino acid sequence of SEQ ID NO: 15, and CDR3 which is the amino acid sequence of SEQ ID NO: 20. For the use according to any of Embodiments 18 - 20, the polypeptide or composition, 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.

[0044]

[0045] ​Embodiment 22. The ISVD that binds to human serum albumin is a polypeptide or composition for use according to any one of Embodiments 18 to 21, comprising the amino acid sequence of SEQ ID NO: 5.

[0046] Embodiment 23. The amino acid sequence of the polypeptide is a polypeptide or composition for use according to any one of Embodiments 10 to 22, comprising more than 90% sequence identity with SEQ ID NO: 1.

[0047] Embodiment 24. The polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 1, and is a polypeptide or composition for use according to any one of Embodiments 10 to 23.

[0048] Embodiment 25. A polypeptide or composition for use according to any one of Embodiments 1 to 24, for use in the treatment of inflammatory diseases such as type 2 inflammatory diseases.

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

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

[0051] Embodiment 28. At least one ISVD is: a) 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) 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, c) 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, or d) 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 polypeptide according to embodiment 27, comprising

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

[0053] Embodiment 30. The at least one ISVD is: 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 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 the ISVDs 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 an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 7; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 12; and iii. CDR3 which is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 17 comprising b) The first and second ISVDs are i. CDR1 that is the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO:8; ii. CDR2 that is the amino acid sequence of SEQ ID NO:13 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO:13; and iii. CDR3 that is the amino acid sequence of SEQ ID NO:18 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO:18 comprising c) The first ISVD i. CDR1 that is the amino acid sequence of SEQ ID NO:7 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO:7; ii. CDR2 that is the amino acid sequence of SEQ ID NO:12 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO:12; and iii. CDR3 that is the amino acid sequence of SEQ ID NO:17 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO:17 comprising The second ISVD iv. CDR1 that is the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO:8; v. CDR2 that is the amino acid sequence of SEQ ID NO:13 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO:13; and vi. CDR3 that is the amino acid sequence of SEQ ID NO:18 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO:18 comprising d) The first ISVD i. CDR1 that is the amino acid sequence of SEQ ID NO:8 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO:8; ii. CDR2 that is the amino acid sequence of SEQ ID NO:13 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO:13; and iii. A CDR3 that is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 18 comprising The second ISVD is iv. A CDR1 that is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 7; v. A CDR2 that is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 12; and vi. A CDR3 that is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 17 comprising e) The first ISVD is i. A CDR1 that is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 11; ii. A CDR2 that is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 16; and iii. A CDR3 that is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 21 comprising The second ISVD is iv. A CDR1 that is the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 9; v. A CDR2 that is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 14; and vi. A CDR3 that is the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 19 comprising, or f) The first ISVD is i. A CDR1 that is the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 9; ii. A CDR2 that is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 14; and iii. A CDR3 that is the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 19 comprising The second ISVD is iv. A CDR1 that is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 11; v. A CDR2 that is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 16; and vi. A CDR3 that is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 21 comprising The order of the ISVDs indicates their relative positions to each other as considered in the direction from the N-terminus to the C-terminus of the polypeptide, the polypeptide according to Embodiment 1 or 27.

[0055] Embodiment 32. 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 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, c) The first 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, and the second 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, d) The first ISVD includes 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 includes 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 includes 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 includes 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) The polypeptide according to Embodiment 31, wherein the first ISVD includes 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 includes 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 sequences of the first and second ISVDs include more than 90% sequence identity with SEQ ID NO: 2. b) The amino acid sequences of the first and second ISVDs include more than 90% sequence identity with SEQ ID NO: 3. c) The amino acid sequence of the first ISVD includes more than 90% sequence identity with SEQ ID NO: 2, and the second ISVD includes more than 90% sequence identity with SEQ ID NO: 3. d) The amino acid sequence of the first ISVD includes more than 90% sequence identity with SEQ ID NO: 3, and the second ISVD includes more than 90% sequence identity with SEQ ID NO: 2. e) The amino acid sequence of the first ISVD includes more than 90% sequence identity with SEQ ID NO: 6, and the second ISVD includes more than 90% sequence identity with SEQ ID NO: 4. f) The amino acid sequence of the first ISVD contains more than 90% sequence identity with SEQ ID NO: 4, and the second ISVD contains more than 90% sequence identity with SEQ ID NO: 6, the polypeptide according to any one of embodiments 31 or 32.

[0057] Embodiment 34. a) The first and second ISVDs contain the amino acid sequence of SEQ ID NO: 2, b) The first and second ISVDs contain the amino acid sequence of SEQ ID NO: 3, c) The first ISVD contains the amino acid sequence of SEQ ID NO: 2, and the second ISVD contains the amino acid sequence of SEQ ID NO: 3, d) The first ISVD contains the amino acid sequence of SEQ ID NO: 3, and the second ISVD contains the amino acid sequence of SEQ ID NO: 2, e) The first ISVD contains the amino acid sequence of SEQ ID NO: 6, and the second ISVD contains the amino acid sequence of SEQ ID NO: 4, or f) The first ISVD contains the amino acid sequence of SEQ ID NO: 4, and the second ISVD contains the amino acid sequence of SEQ ID NO: 6, the polypeptide according to any one of embodiments 31 - 33.

[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) The amino acid sequence of SEQ ID NO: 159 The polypeptide according to any one of embodiments 31 - 34, comprising or consisting of.

[0059] Embodiment 36. The polypeptide comprises or consists of at least 4 ISVDs, each of said ISVDs comprising three complementarity determining regions (CDR1 to CDR3 respectively), said at least 4 ISVDs optionally being 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 an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 7; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 12; and iii. CDR3 which is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 17 and comprises; b) The second ISVD is iv. CDR1 which is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 8; v. CDR2 which is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 13; and vi. CDR3 which is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 18 and comprises; c) The third ISVD is vii. CDR1 which is the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 9; viii. CDR2 which is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 14; and ix. CDR3 which is the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 19 and comprises; d) The fourth ISVD is x. A CDR1 that is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 11; xi. A CDR2 that is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 16; and xii. A CDR3 that is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 21 comprising; The order of the ISVDs indicates their relative positions with respect to each other as considered in the direction from the N-terminus to the C-terminus of the polypeptide, the polypeptide according to any of embodiments 27 or 31.

[0060] Embodiment 37. a) The first 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; b) The second 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; c) The third 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; d) The fourth 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, the polypeptide according to embodiment 36.

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

[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 fourth ISVD comprises the amino acid sequence of SEQ ID NO: 6, and the polypeptide according to any one of Embodiments 36 to 38.

[0063] Embodiment 40. The polypeptide further comprises one or more other groups, residues, moieties or linking units, optionally linked via one or more peptidic linkers, wherein the one or more other groups, residues, moieties or linking units are compared to the corresponding polypeptide having no one or more other groups, residues, moieties or linking units, and provides a polypeptide having an increased half-life, and the polypeptide according to any one of Embodiments 27 to 39.

[0064] Embodiment 41. The one or more other groups, residues, moieties or linking units that provide a polypeptide having an increased half-life are selected from the group consisting of polyethylene glycol molecules, serum proteins or fragments thereof, linking units capable of binding to serum proteins, Fc moieties, and small proteins or peptides capable of binding to serum proteins, and the polypeptide according to Embodiment 40.

[0065] Embodiment 42. The one or more other linking units that provide a polypeptide having an increased half-life are selected from the group consisting of linking units capable of binding to serum albumin (such as human serum albumin) or serum immunoglobulin (such as IgG), and the polypeptide according to any one of Embodiments 40 to 41.

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

[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 a difference of 2 or 1 amino acid from SEQ ID NO: 10; ii. CDR2 which is the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 15; and iii. CDR3 which is the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 20 The polypeptide according to Embodiment 43, comprising

[0068] Embodiment 45. The ISVD that binds to human serum albumin is the polypeptide according to any one of Embodiments 43 to 44, comprising CDR1 which is the amino acid sequence of SEQ ID NO: 10, CDR2 which is the amino acid sequence of SEQ ID NO: 15, and CDR3 which is the amino acid sequence of SEQ ID NO: 20.

[0069] Embodiment 46. The amino acid sequence of the ISVD that binds to the human serum albumin contains more than 90% sequence identity with SEQ ID NO: 5, the polypeptide according to any one of Embodiments 43 to 45.

[0070] Embodiment 47. The ISVD that binds to the human serum albumin is the polypeptide according to any one of Embodiments 43 to 46, comprising the amino acid sequence of SEQ ID NO: 5.

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

[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 polypeptide according to any one of Embodiments 27 to 49, or a nucleotide sequence encoding a polypeptide according to any one of Embodiments 36 to 49.

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

[0075] Embodiment 52. A method for producing a polypeptide according to any one of Embodiments 27 to 49, or a polypeptide according to any one of 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 one of Embodiments 27 to 49, or a polypeptide according to any one of Embodiments 36 to 49 The method as described above.

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

[0077] Embodiment 54. The composition according to Embodiment 53, further comprising at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally comprising one or more further pharmaceutically active polypeptides and / or compounds.

[0078] A method of treating an inflammatory disease such as a type 2 inflammatory disease, comprising administering to a subject in need thereof a pharmaceutically 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, said method.

[0079] Embodiment 56. The method according to embodiment 55, wherein the type 2 inflammatory disease is selected from asthma and atopic dermatitis.

[0080] Use 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, in the preparation of a pharmaceutical composition for treating an inflammatory disease such as a type 2 inflammatory disease.

[0081] Embodiment 58. Use of the 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

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

[0083] 5 DETAILED DESCRIPTION OF THE TECHNOLOGY The present technology aims to provide a novel type of drug for treating inflammatory diseases such as atopic dermatitis and asthma.

[0084] In some embodiments, the present technology relates to a polypeptide that simultaneously targets IL-13 and TSLP in vitro and / or in vivo, resulting in an increased efficiency of modulating type 2 inflammatory responses compared to a monospecific anti-IL-13 or anti-TSLP polypeptide. In some embodiments, the polypeptide is efficiently produced (e.g., in a microbial host). Further, in some embodiments, such a polypeptide can be shown to have limited reactivity against existing antibodies in the subject to be treated (i.e., antibodies present in the subject prior to the first treatment with the antibody construct). In other embodiments, such a polypeptide exhibits a sufficiently long half-life such that it can be successfully spaced between consecutive treatments in the subject to be treated.

[0085] 5.1 Polypeptides of the present technology Monospecific monovalent polypeptide In one embodiment, the polypeptide of the present technology is 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. Another 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, such as ISVD. block present.

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

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

[0090] In a further embodiment, the ISVD that specifically binds to IL-13 is: 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; 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 selected from ISVDs comprising.

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

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

[0093] In another aspect, the technology provides a monospecific monovalent polypeptide comprising or consisting of one ISVD that specifically binds to TSLP and that comprises three complementarity determining regions (CDR1 to CDR3 respectively). The ISVD is: a) CDR1 having the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 9, CDR2 having the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 14, and CDR3 having the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 19; or b) CDR1 which is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 11, CDR2 which is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 16, and CDR3 which is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 21 can be selected from an ISVD comprising the same.

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

[0095] In a further embodiment, the ISVD that specifically binds to TSLP is: a) CDR1 which is the amino acid sequence of SEQ ID NO: 9, CDR2 which is the amino acid sequence of SEQ ID NO: 14, and CDR3 which is the amino acid sequence of SEQ ID NO: 19; or b) CDR1 which is the amino acid sequence of SEQ ID NO: 11, CDR2 which is the amino acid sequence of SEQ ID NO: 16, and CDR3 which is the amino acid sequence of SEQ ID NO: 21 is selected from an ISVD comprising the same.

[0096] In a further embodiment of this aspect of the present technology, the ISVD that specifically binds to TSLP is: a) having a sequence identity of more than 90% with SEQ ID NO: 4; or b) having a sequence identity of more than 90% with SEQ ID NO: 6 is selected from an ISVD comprising the same.

[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 polypeptide In another aspect, the polypeptide of the present technology is monospecific and at least bivalent, but may be, for example, trivalent, tetravalent, pentavalent, hexavalent, etc.

[0099] The terms "divalent", "trivalent", "tetravalent", "pentavalent", or "hexavalent" all fall under the term "multivalent" and indicate the presence of two, three, four, five, or six binding units / building blocks, e.g., ISVDs, respectively.

[0100] Thus, in one aspect, the technology is a monospecific divalent polypeptide comprising or consisting of two ISVDs that specifically bind to IL-13, wherein each of the two ISVDs comprises three complementarity-determining regions (CDR1 to CDR3 respectively), and the two ISVDs are optionally linked via one or more peptidic linkers: a) The first and second ISVDs comprise a CDR1 that is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 7, a CDR2 that is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 12, and a CDR3 that is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 17; b) The first and second ISVDs comprise a CDR1 that is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 8, a CDR2 that is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 13, and a CDR3 that is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 18; c) The first ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 7, a CDR2 that is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 12, and a CDR3 that is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 17, The second ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 8, a CDR2 that is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 13, and a CDR3 that is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 18; or d) The first ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 8, a CDR2 that is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 13, and a CDR3 that is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 18, The second ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 7, a CDR2 that is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 12, and a CDR3 that is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 17. Provided is a monospecific bivalent polypeptide.

[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 to 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 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; 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 sequences of the first and second ISVDs comprise more than 90% sequence identity with SEQ ID NO: 2; b) The amino acid sequences of the first and second ISVDs comprise more than 90% sequence identity with SEQ ID NO: 3; c) The amino acid sequence of the first ISVD comprises more than 90% sequence identity with SEQ ID NO: 2, and the second ISVD comprises more than 90% sequence identity with SEQ ID NO: 3; or d) The amino acid sequence of the first ISVD comprises more than 90% sequence identity with SEQ ID NO: 3, and the second ISVD comprises more 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 technology is a monospecific bivalent polypeptide comprising or consisting of two ISVDs that specifically bind to TSLP, wherein each of the two ISVDs comprises three complementarity determining regions (CDR1 to CDR3 respectively), and the two ISVDs are optionally linked via one or more peptidic linkers: a) The first ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having 2 or 1 amino acid differences from SEQ ID NO: 11, a CDR2 that is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having 2 or 1 amino acid differences from SEQ ID NO: 16, and a CDR3 that is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having 2 or 1 amino acid differences from SEQ ID NO: 21, The second ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having 2 or 1 amino acid differences from SEQ ID NO: 9, a CDR2 that is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having 2 or 1 amino acid differences from SEQ ID NO: 14, and a CDR3 that is the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having 2 or 1 amino acid differences from SEQ ID NO: 19, or b) The first ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having 2 or 1 amino acid differences from SEQ ID NO: 9, a CDR2 that is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having 2 or 1 amino acid differences from SEQ ID NO: 14, and a CDR3 that is the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having 2 or 1 amino acid differences from SEQ ID NO: 19, The second ISVD provides a monospecific bivalent polypeptide comprising a CDR1 that is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 11, a CDR2 that is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 16, and a CDR3 that is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having a difference of 2 or 1 amino acid 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 to human TSLP.

[0107] In a further embodiment, the monospecific bivalent polypeptide comprises or consists of two ISVDs that specifically bind to TSLP: a) The first 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, and the second 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, or b) The first 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, and the second 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.

[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 TS LP: a) The amino acid sequence of the first ISVD comprises more than 90% sequence identity with SEQ ID NO: 6, and the second ISVD comprises more than 90% sequence identity with SEQ ID NO: 4, or b) The amino acid sequence of the first ISVD contains more than 90% sequence identity with SEQ ID NO: 4, and the second ISVD contains more 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 to TSLP: a) The first ISVD contains the amino acid sequence of SEQ ID NO: 6, and the second ISVD contains the amino acid sequence of SEQ ID NO: 4; or b) The first ISVD contains the amino acid sequence of SEQ ID NO: 4, and the second ISVD contains the amino acid sequence of SEQ ID NO: 6.

[0110] In this regard, the terms "first ISVD" and "second ISVD" indicate only the relative positions of the specifically listed ISVDs that bind to IL-13 / TSLP, 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". Accordingly, the "second ISVD" is thus closer to the C-terminus than the "first ISVD". Thus, the numbering is not absolute and only indicates the relative positions of the two ISVDs, and therefore does not exclude the possibility that additional binding units / building blocks, such as ISVDs that bind to 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 arranged between them. For example, as further described below (see in particular the section "Multispecific multivalent polypeptides" and 5.4 "Prolongation of half-life (in vivo)"), the polypeptide may further comprise an ISVD that binds to another human serum albumin, which may also be arranged between the "first ISVD" and the "second ISVD" (and thus such constructs are referred to as multispecific as described in the subsequent section).

[0111] In one embodiment, the (at least two) ISVDs of a monospecific multivalent polypeptide, particularly of the monospecific bivalent polypeptide described above, are linked via a peptidic linker. The use of peptidic linkers for connecting two or more (poly)peptides is well known in the art. Exemplary peptidic linkers that can be used with a monospecific multivalent polypeptide, particularly with the monospecific bivalent polypeptide described above, are shown in Table A-5. One class of peptidic linkers that are often used is known as "Gly-Ser" or "GS" linkers. These are linkers consisting essentially of glycine (G) and serine (S) residues and usually contain one or more repeats of a peptide motif such as GGGGS (SEQ ID NO: 73) (e.g., the formula (Gly-Gly-Gly-Gly-Ser) n where n can be 1, 2, 3, 4, 5, 6, 7 or more). Some often 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. October 15, 2013; 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 a monospecific multivalent polypeptide, particularly of the monospecific bivalent polypeptide of the present technology, are linked via the linkers described in Table A-5. In one embodiment, the (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) the amino acid sequence of SEQ ID NO: 159 comprising or consisting of.

[0113] Multispecific multivalent polypeptide In a further aspect, the polypeptide of the present technology is at least bispecific, but may be, for example, trispecific, quadrispecific, pentaspecific, etc. Further, the polypeptide is at least bivalent, but may be, for example, trivalent, tetravalent, pentavalent, hexavalent, etc.

[0114] The terms "bispecific", "trispecific", "quadrispecific", "pentaspecific", etc. all fall under the term "having multispecificity" and refer to binding to 2, 3, 4, 5, etc. different target molecules, respectively.

[0115] The terms "bivalent", "trivalent", "tetravalent", "pentavalent", "hexavalent", etc. all fall under the term "multivalent" and indicate the presence of 2, 3, 4, 5, 6, etc. binding units / building blocks, for example, ISVD, respectively.

[0116] For example, the polypeptide may be a bispecific tetravalent polypeptide, for example, comprising or consisting of at least 4 ISVDs, in which case at least 2 ISVDs specifically bind to IL-13 and at least 2 ISVDs specifically bind to TSLP. In one embodiment, IL-13 and TSLP are human IL-13 and human TSLP. In another example, the polypeptide may be a trispecific pentavalent polypeptide, for example, comprising or consisting of 5 ISVDs, in which case 2 ISVDs specifically bind to human IL-13, 2 ISVDs specifically bind to human TSLP, and 1 ISVD binds to human serum albumin. Such a polypeptide can be biparatopic, for example, when 2 ISVDs bind to 2 different epitopes on human IL-13 or human TSLP. The term "biparatopic" refers to binding to 2 different parts (e.g., epitopes) of the same target molecule. In one embodiment, the trispecific pentavalent polypeptide of the present technology is, for example, the ISVD construct F027400161, which comprises 2 ISVDs that specifically bind to human IL-13, 2 ISVDs that specifically bind to human TSLP, and 1 ISVD that binds to 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 4 ISVDs, each of said ISVDs comprising 3 complementarity determining regions (CDR1 to CDR3 respectively), and the at least 4 ISVDs are optionally linked via one or more peptidic linkers: a) The first ISVD specifically binds to IL-13 and has a CDR1 that is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 7, a CDR2 that is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having a difference of 2 or 1 a CDR2, which is an amino acid sequence having amino acid differences, and CDR3, which is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having 2 or 1 amino acid differences from SEQ ID NO: 17; b) The second ISVD specifically binds to IL-13 and comprises CDR1, which is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having 2 or 1 amino acid differences from SEQ ID NO: 8, CDR2, which is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having 2 or 1 amino acid differences from SEQ ID NO: 13, and CDR3, which is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having 2 or 1 amino acid differences from SEQ ID NO: 18; c) The third ISVD specifically binds to TSLP and comprises CDR1, which is the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having 2 or 1 amino acid differences from SEQ ID NO: 9, CDR2, which is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having 2 or 1 amino acid differences from SEQ ID NO: 14, and CDR3, which is the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having 2 or 1 amino acid differences from SEQ ID NO: 19, d) The fourth ISVD specifically binds to TSLP and comprises CDR1, which is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having 2 or 1 amino acid differences from SEQ ID NO: 11, CDR2, which is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having 2 or 1 amino acid differences from SEQ ID NO: 16, and CDR3, which is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having 2 or 1 amino acid differences 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 aspect of the multispecific multivalent polypeptide: a) The amino acid sequence of the first ISVD comprises more than 90% sequence identity with SEQ ID NO: 2; b) The amino acid sequence of the second ISVD comprises more than 90% sequence identity with SEQ ID NO: 3; c) The amino acid sequence of the third ISVD comprises more than 90% sequence identity with SEQ ID NO: 4; d) The amino acid sequence of the fourth ISVD comprises more than 90% sequence identity with SEQ ID NO: 6.

[0121] In one embodiment of the 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 with respect 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", and the "second ISVD" is closer to the N-terminus than the "third ISVD". Thus, when considering from the C-terminus, the arrangement of the ISVDs is reversed. The numbering is not absolute and only indicates the relative positions of at least four ISVDs, so it is not excluded that other binding units / building blocks that bind to IL-13 or TSLP, such as additional ISVDs, or ISVDs that bind to other targets, may be present in the polypeptide. Furthermore, the numbering does not exclude the possibility that other binding units / building blocks, such as ISVDs, may be arranged between them. For example, as further described below (in particular, see Section 5.4 "Prolongation of Half-Life (in Vivo)"), the polypeptide may further contain an ISVD that binds to another human serum albumin, which may also be arranged, for example, between the "third ISVD" and the "fourth ISVD".

[0123] In another aspect, the present technology provides a bispecific bivalent polypeptide comprising an ISVD that specifically binds to IL-13 or TSLP, which is described in detail with respect to the monospecific monovalent polypeptide above (Section 5.1; "Monospecific Monovalent Polypeptide"), and an ISVD that binds to human serum albumin, which is described in detail below (Section 5.4; "Prolongation of Half-Life (in Vivo)").

[0124] In another aspect, the present technology provides a bispecific trivalent polypeptide comprising the above monospecific bivalent polypeptide (Section 5.1; "Monospecific Bivalent Polypeptide") and an ISVD that binds to human serum albumin, which is described in detail below (Section 5.4; "Prolongation of Half-Life (in Vivo)").

[0125] The components of the multispecific multivalent polypeptide, such as ISVD, may be linked to each other by one or more suitable linkers, such as peptidic linkers.

[0126] The use of linkers for connecting two or more (poly)peptides is well known in the art. Exemplary peptidic linkers are shown in Table A-5. One of the most commonly used classes of peptidic linkers is known as the "Gly-Ser" or "GS" linker. These are linkers consisting essentially of glycine (G) and serine (S) residues and typically contain one or more repeats of a peptide motif such as GGGGS (SEQ ID NO: 73) (e.g., the formula (Gly-Gly-Gly-Gly-Ser) n and n can be 1, 2, 3, 4, 5, 6, 7 or more). Some of the most commonly 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. October 15, 2013; 65(10):1357-1369; and Klein et al., Protein Eng. Des. Sel. (2014) 27(10):325-330. In the polypeptides 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 to each other.

[0127] In one aspect of the multispecific multivalent polypeptide of the present technology, at least four ISVDs are The polypeptide comprising or consisting of comprises at least two ISVDs that specifically bind to IL-13 and at least two ISVDs that specifically bind to TSLP. In this aspect of the technology, the at least two ISVDs that 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 “Prolongation of half-life (in vivo)” below). The inventors have surprisingly found that such a conformation can increase the production yield of the polypeptide.

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

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

[0130] Such a conformation of the polypeptide can result in increased production yield, excellent CMC characteristics, such as sufficient solubility and biophysical stability, strong potency regarding modulation of the type 2 immune response, and in addition, low binding to existing antibodies.

[0131] In one embodiment, the multispecific multivalent polypeptide of the present technology exhibits reduced binding by existing antibodies in human serum. To achieve this purpose, in one embodiment, the polypeptide comprises valine (V) at amino acid position 11 and leucine (L) at amino acid position 89 (according to Kabat numbering) in at least one ISVD. In one embodiment, the polypeptide comprises valine (V) at amino acid position 11 and leucine (L) at amino acid position 89 (according to Kabat numbering) in each ISVD. In another embodiment, the polypeptide comprises an extension of 1 to 5 (naturally occurring) amino acids, for example, an extension of a single alanine (A) at the C-terminus of the ISVD. The C-terminus of the ISVD is usually VTVSS (SEQ ID NO: 138). In another embodiment, the polypeptide comprises lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) in at least one ISVD. In another embodiment, the ISVD comprises lysine (K) or glutamine (Q) at position 112 (according to Kabat numbering) in at least one ISVD. In these embodiments, after adding a single alanine, the C-terminus of the polypeptide is, for example, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, or SEQ ID NO: 177, and the C-terminus of the ISVD is SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, or SEQ ID NO: 171. In one embodiment In one state, the C-terminus contains VTVSSA (SEQ ID NO: 141). In another embodiment, the polypeptide contains valine (V) at position 11 and leucine (L) at position 89 (according to Kabat numbering) in each ISVD, and optionally contains lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) in at least one ISVD, and contains an extension of 1 to 5 (naturally occurring) amino acids, for example, an extension of a single alanine (A) 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), for example, 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 having more than 90% sequence identity, such as more than 95% or more than 99% sequence identity with SEQ ID NO: 1, and the CDRs of the five ISVDs are each as defined in items A to E (or A' to E' when using Kabat's definition) described in the following sections "5.2 Immunoglobulin single variable domain" and "5.4 Extension of half-life (in vivo)", in particular: · The first ISVD that specifically binds to IL-13 has CDR1 which is the amino acid sequence of SEQ ID NO: 7, CDR2 which is the amino acid sequence of SEQ ID NO: 12, and CDR3 which is the amino acid sequence of SEQ ID NO: 17; · The second ISVD that specifically binds to IL-13 has CDR1 which is the amino acid sequence of SEQ ID NO: 8, CDR2 which is the amino acid sequence of SEQ ID NO: 13, and CDR3 which is the amino acid sequence of SEQ ID NO: 18; · The third ISVD that specifically binds to TSLP has CDR1 which is the amino acid sequence of SEQ ID NO: 9, CDR2 which is the amino acid sequence of SEQ ID NO: 14, and CDR3 which is the amino acid sequence of SEQ ID NO: 19; ·The fourth ISVD that specifically binds to TSLP has CDR1 which is the amino acid sequence of SEQ ID NO: 11, CDR2 which is the amino acid sequence of SEQ ID NO: 16, and CDR3 which is the amino acid sequence of SEQ ID NO: 21; ·The ISVD that binds to human serum albumin has CDR1 which is the amino acid sequence of SEQ ID NO: 10, CDR2 which is the amino acid sequence of SEQ ID NO: 15, and CDR3 which is the amino acid sequence of SEQ ID NO: 20, or alternatively, when using Kabat's definition: ·The first ISVD that specifically binds to IL-13 has CDR1 which is the amino acid sequence of SEQ ID NO: 37, CDR2 which is the amino acid sequence of SEQ ID NO: 42, and CDR3 which is the amino acid sequence of SEQ ID NO: 17; ·The second ISVD that specifically binds to IL-13 has CDR1 which is the amino acid sequence of SEQ ID NO: 38, CDR2 which is the amino acid sequence of SEQ ID NO: 43, and CDR3 which is the amino acid sequence of SEQ ID NO: 18; ·The third ISVD that specifically binds to TSLP has CDR1 which is the amino acid sequence of SEQ ID NO: 39, CDR2 which is the amino acid sequence of SEQ ID NO: 44, and CDR3 which is the amino acid sequence of SEQ ID NO: 19; ·The fourth ISVD that specifically binds to TSLP has CDR1 which is the amino acid sequence of SEQ ID NO: 41, CDR2 which is the amino acid sequence of SEQ ID NO: 46, and CDR3 which is the amino acid sequence of SEQ ID NO: 21; ·The ISVD that binds to human serum albumin has CDR1 which is the amino acid sequence of SEQ ID NO: 40, CDR2 which is the amino acid sequence of SEQ ID NO: 45, and CDR3 which 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, or at least the same binding affinity as, the binding affinity of the polypeptide consisting of the amino acids of SEQ ID NO: 1 for human IL-13 and human TSLP, where the binding affinity is measured using the same method, such as surface plasmon resonance (SPR).

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

[0136] In view of the above definition, of a conventional four-chain antibody (e.g., IgG, IgM, IgA, IgD or IgE molecule; known in the art), or of a Fab fragment, F(ab’) 2Fragments, Fv fragments, e.g., Fv fragments linked by disulfide or scFv fragments, or antigen-binding domains of such conventional four-chain antibodies (all known in the art), are not normally considered ISVDs, as in these cases, binding to each epitope of the antigen usually occurs not by a single (monovalent) immunoglobulin domain, but by a pair of (associating) immunoglobulin domains, e.g., light and heavy chain variable domains, i.e., immunoglobulin domains that jointly bind to the epitope of each antigen, the V H -V L pairs.

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

[0138] Thus, as long as it is possible to form a single antigen-binding unit (i.e., a functional antigen-binding unit that consists essentially of a single variable domain and does not require interaction with another variable domain to form a functional antigen-binding unit), the single variable domain can be a light chain variable domain sequence (e.g., V L -sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., V H -sequence or V HH sequence) or a suitable fragment thereof.

[0139] The ISVD can be, for example, a heavy chain ISVD, e.g., V H , V HH , e.g., camelized V H or humanized V HH etc. In one embodiment, the ISVD is V HH , e.g., camelized V H or humanized V HHand the like. The heavy-chain ISVD may be derived from a conventional four-chain antibody or a heavy-chain antibody.

[0140] For example, the ISVD may be a single-domain antibody (or an amino acid sequence suitable for use as a single-domain antibody), a "dAb" or dAb (or an amino acid sequence suitable 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 of them.

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

[0142] "V HH domain" is also known as V HH V HH antibody fragments, and V HH antibodies, which were originally described as antigens that bind to the variable domain of an immunoglobulin of a "heavy-chain antibody" (i.e., an antibody lacking a light chain; Hamers-Casterman et al., Nature 363:446-448, 1993). The term "V HH domain" was chosen to distinguish these variable domains from the heavy-chain variable domains present in conventional four-chain antibodies (referred to herein as "V H domains") and the light-chain variable domains present in conventional four-chain antibodies (referred to herein as "V L domains"). For further explanation of V HH , see the review by Muyldermans (Reviews in Molecular Biotechnology 74:277-302, 2001).

[0143] Typically, the production of immunoglobulins involves immunizing experimental animals, fusing cells that produce immunoglobulins to create hybridomas, and screening for the desired specificity. Alternatively, immunoglobulins may be generated 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, including, for example, WO94 / 04678, Hamers-Casterman et al., 1993 and Muyldermans et al., 2001. In these methods, camelids are immunized with the 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 examples, the production of antibodies requires an antigen purified for immunization and / or screening. The antigen may be purified from a natural source or may be purified during the process of recombinant production.

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

[0147] This technology can use immunoglobulin sequences of different origins, including mouse, rat, rabbit, camel, human and llama immunoglobulin sequences. This technology also includes fully human, humanized or chimeric sequences. For example, this technology includes llama immunoglobulin sequences and humanized llama immunoglobulin sequences, or camelized domain antibodies, such as camelized dAbs as described by Ward et al. (see, for example, WO94 / 04678 and Davies and Riechmann (1994 and 1996)). Furthermore, this technology also uses fused immunoglobulin sequences, such as multivalent and / or multispecific constructs (multivalent and multispecific polypeptides containing one or more V HH domains and their preparation, see Conrath et al., J. Biol. Chem., Vol. 276, 10.7346 - 7350, 2001, in addition to, for example, WO96 / 34103 and WO99 / 23221), as well as fused immunoglobulin sequences that form immunoglobulin sequences containing tags or other functional parts, such as toxins, labels , radiochemicals, etc., which are derivable from the immunoglobulin sequences of this technology.

[0148] "Humanized V HH " corresponds to the amino acid sequence of a naturally occurring V HH domain, but is "humanized", i.e., one or more amino acid residues in the amino acid sequence of said naturally occurring V HH sequence (especially in the framework sequence) are replaced by one or more of the amino acid residues present at the corresponding positions in the V H domain from a conventional 4-chain antibody from humans (such as those shown above), and includes an amino acid sequence that has been humanized. This can be carried out in a manner known per se to those skilled in the art based on, for example, further explanations described herein and in the prior art (such as WO2008 / 020079). Also note here that such humanized V HHIt can be obtained by any suitable method known per se and thus is not strictly limited to polypeptides obtained using a polypeptide containing a naturally occurring VHH domain as starting material.

[0149] "Camelized V H " corresponds to the amino acid sequence of a naturally occurring V H domain but is "camelized", i.e., one or more amino acid residues in the amino acid sequence of the naturally occurring V H domain from a conventional four-chain antibody have been replaced by one or more of the amino acid residues present at the corresponding positions in the V HH domain of a heavy-chain antibody, and includes an amino acid sequence camelized in this way. This can be carried out in a manner known per se that is obvious to the person skilled in the art based on further explanations, for example, as described in the present specification and the prior art (e.g., WO2008 / 020079). Such "camelizing" substitutions are usually inserted at the boundary forming and / or present at the amino acid positions of V H -V L and / or at the characteristic residues of so-called camel as defined herein (see, for example, WO94 / 04678 and Davies and Riechmann (1994 and 1996), supra). In one embodiment, the V H sequence used as starting material or starting point for generating or designing camelized V H is a V H sequence from a mammal or a human V H sequence, such as a V H 3 sequence. However, it should be noted that such camelized V H can be obtained by any suitable method known per se and thus is not strictly limited to polypeptides obtained using a polypeptide containing a naturally occurring V H domain as starting material.

[0150] The structure of the ISVD array 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"), respectively. These framework regions have three complementarity-determining regions ("CDR") in between, 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"), respectively.

[0151] As further described on pages 58 and 59, paragraph q) of WO08 / 020079, the amino acid residues of ISVD are from the camelid V domains described in the paper by Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240(1-2):185-195; see, for example, Figure 2 of this publication) HH as applied to the Kabat et al. ( "Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91) H assigned V domain can be numbered according to the general numbering of the V domain. It should be noted that the V H domain and the V HH domain are well known in the art Furthermore, 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 Kabat numbering (i.e., one or more positions according to Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by Kabat numbering). This generally means that the numbering by Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. V H domain and V HH The total number of amino acid residues in the domain and the V domain is usually in the range of 110 - 120, and often 112 - 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 (2010 edition, Antibody Engineering, Volume 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pages 33 - 51). According to this method, FR1 contains amino acid residues at positions 1 - 25, CDR1 contains amino acid residues at positions 26 - 35, FR2 contains amino acid residues at positions 36 - 49, CDR2 contains amino acid residues at positions 50 - 58, FR3 contains amino acid residues at positions 59 - 94, CDR3 contains amino acid residues at positions 95 - 102, and FR4 contains 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 by Kabat, FR1 of the ISVD contains amino acid residues at positions 1 to 30, CDR1 of the ISVD contains amino acid residues at positions 31 to 35, FR2 of the ISVD contains amino acid residues at positions 36 to 49, CDR2 of the ISVD contains amino acid residues at positions 50 to 65, FR3 of the ISVD contains amino acid residues at positions 66 to 94, CDR3 of the ISVD contains amino acid residues at positions 95 to 102, and FR4 of the ISVD contains amino acid residues at positions 103 to 113.

[0154] In such immunoglobulin sequences, the framework sequence may be any suitable framework sequence, and examples of suitable framework sequences will be apparent to those skilled in the art based on, for example, standard textbooks and further disclosure and prior art described herein.

[0155] The framework sequence is a suitable combination of an immunoglobulin framework sequence or a framework sequence obtained from an immunoglobulin framework sequence (e.g., by humanization or camelization). For example, the framework sequence can be a framework sequence obtained from a light chain variable domain (e.g., V L sequence) and / or a heavy chain variable domain (e.g., V H sequence or V HH sequence). In one embodiment, the framework sequence is a framework sequence obtained from a V HH sequence (wherein the framework sequence may optionally be partially or fully humanized), or a conventional camelized V H sequence (as defined herein).

[0156] In particular, the framework sequence present in the ISVD sequence used in the present technology is such that the ISVD sequence contains a Nanobody®, e.g., a humanized V HH or a camelized V H in the V HHIt may contain one or more characteristic residues (as defined herein). Non-limiting examples (and suitable combinations thereof) of such framework sequences will become apparent from the further disclosure described herein.

[0157] Here too, as generally described herein with respect to immunoglobulin sequences, and any suitable fragment (or combination of fragments) of the foregoing, for example, preferably one or more framework sequences adjacent to and / or linked through one or more CDR sequences contained in a fragment (e.g., these CDRs and framework sequences in the same order as they may occur in the full-size immunoglobulin sequence from which the fragment is derived) may also be considered.

[0158] However, it should be noted that the present technology is not limited to the origin of the ISVD sequence (or the nucleotide sequence used to express it), nor to the method by which the ISVD sequence or nucleotide sequence is generated (or has been 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 a specific but non-limiting embodiment, 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, "humanized" (as defined herein) immunoglobulin sequences (e.g., partially or fully humanized mouse or rabbit immunoglobulin sequences, particularly, partially or fully humanized V HH sequences), "camelized" (as defined herein) immunoglobulin sequences, in addition to affinity maturation (e.g., starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, obtained from different immunoglobulin sequences combining the resulting fragments, PCR assembly using overlapping primers, and similar techniques for manipulating immunoglobulin sequences well known to those skilled in the art; or immunoglobulin sequences obtained by techniques such as any suitable combination of the foregoing.

[0159] Similarly, 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 cells), a nucleotide sequence isolated from a library (especially, an expression library), a nucleotide sequence prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as 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 described above, the ISVD can be a Nanobody® or a suitable fragment thereof. For a general description of Nanobodies, reference is made to the following further description, in addition to the prior art cited herein. However, in this regard, this description and the prior art mainly describe so-called "V H 3 class" Nanobodies (i.e., Nanobodies having a high degree of sequence homology to V H 3 class human germline sequences such as DP-47, DP-51 or DP-29). However, it should be noted that the technology can generally use any type of Nanobody in its broadest sense, for example, so-called "V H 4 class" Nanobodies (i.e., Nanobodies having a high degree of sequence homology to V H 4 class human germline sequences such as DP-78), as described, for example, in WO2007 / 118670.

[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 have the (general) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 In the formula, FR1 to FR4 are each Framework Regions 1 to 4 refer to CDR1 to CDR3 refer to Complementarity Determining Regions 1 to 3, respectively, in which 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] This technology uses, inter alia, an ISVD that can bind to IL-13 or TSLP. For this technology, "binding to" a particular target molecule has the ordinary meaning in the art as understood for an antibody and its respective antigen.

[0166] The multispecific multivalent polypeptide of this technology may include 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 include 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 between IL-13 and IL-13Rα1 (Interleukin-13 Receptor, Alpha 1), and / or the interaction between the IL-13 / IL-13Rα1 complex and IL-4Rα (Alpha Interleukin-4 Receptor), or the interaction between TSLP and TSLPR (TSLP Receptor), and / or the interaction between the TSLP / TSLPR complex and IL-7Rα (Interleukin-7 Receptor Subunit Alpha). Thus, in one embodiment, the polypeptide of the present technology specifically binds to IL-13 and functionally blocks its interaction with IL-13Rα1 and / or the interaction between the IL-13 / IL-13Rα1 complex and IL-4Rα, and at least two ISVDs that specifically bind to TSLP and functionally block its interaction with TSLPR and / or the interaction between the TSLP / TSLPR complex and IL-7Rα.

[0168] The ISVDs used in the present technology form part of the polypeptide of the present technology that comprises or consists of at least four ISVDs such that the polypeptide can specifically bind to IL-13 and TSLP.

[0169] Thus, 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 number P35225) and human TSLP (Uniprot accession number Q969D9), versions from other species are also suitable for the present technology, for example, IL-13 and TSLP from mouse, rat, rabbit, cat, dog, goat, sheep, horse, pig, non-human primates such as cynomolgus monkey (also referred to herein as "cyno"), or camelids such as llama or alpaca.

[0170] Specific examples of ISVDs that specifically bind to IL-13 and can be used in the present technology are as described in the following sections A and B: A. An ISVD that specifically binds to human IL-13 and i. CDR1 having the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 7; ii. CDR2 having the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 12; and iii. CDR3 having the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 17 An ISVD comprising the same.

[0171] In one embodiment, the ISVD comprises 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.

[0172] B. An ISVD that specifically binds to human IL-13 and i. CDR1 having the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 8; ii. CDR2 having the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 13; and iii. CDR3 having the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 18 An ISVD comprising the same.

[0173] In one embodiment, the ISVD comprises 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.

[0174] Examples of ISVDs that specifically bind to such human IL-13 are those having one or more, or all, of the framework regions (in addition to the CDRs defined in items A and B above, respectively), as shown for constructs 4B02 or 4B06 described in Table A-2. In one embodiment, this is an ISVD comprising or consisting 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 a sequence identity of more than 90%, such as more than 95%, or more than 99% with SEQ ID NO: 2 or 3, respectively, and the CDRs are as defined in item A or B above, respectively. In one embodiment, the ISVD that specifically binds to IL-13 comprises or consists of the amino acid sequence of SEQ ID NO: 2 or 3.

[0176] If such an ISVD that binds to IL-13 has a difference of 2 or 1 amino acid in at least one CDR compared to the corresponding reference CDR sequence (item A or B above), the ISVD has at least half, or at least the same, binding affinity for human IL-13 as constructs 4B02 or 4B06 described 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 and can be used in this technology are as described in items C and D below: C. Specifically binds to human TSLP and i. CDR1 that is the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 9; ii. CDR2 that is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having a difference of 2 or 1 amino acid 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 a difference of 2 or 1 amino acid from SEQ ID NO: 19 An ISVD comprising the same.

[0178] In one embodiment, the 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.

[0179] D. Specifically binds to human TSLP, i. A CDR1 having the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 11; ii. A CDR2 having the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having a difference of 2 or 1 amino acid 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 a difference of 2 or 1 amino acid from SEQ ID NO: 21 An ISVD comprising the same.

[0180] In one embodiment, the 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.

[0181] Examples of such ISVDs that specifically bind to human TSLP are shown for constructs 501A02 and 529F10 described in Table A-2, respectively, and have one or more, or all, of the framework regions (in addition to the CDRs defined in items C and D above). In one embodiment, this is an ISVD comprising or consisting of the full-length amino acid sequence of construct 501A02 or 529F10 (see SEQ ID NOs: 4 or 6, Tables A-1 and A-2).

[0182] In another embodiment, the amino acid sequences of the ISVDs that specifically bind to human TSLP may each have a sequence identity of more than 90%, such as more than 95%, or more than 99% with SEQ ID NO: 4 or 6, and the CDRs are as defined in item C or D above. In one embodiment, the ISVD that binds to human TSLP comprises or consists of the amino acid sequence of SEQ ID NO: 4 or 6.

[0183] If such an ISVD that binds to human TSLP has a difference of 2 or 1 amino acid in at least one CDR compared to the corresponding reference CDR sequence (item C or D above), the ISVD has at least half or at least the same binding affinity for human TSLP as constructs 501A02 or 529F10 described in SEQ ID NO: 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 polypeptide of the present technology comprising each of the ISVDs defined in items A - D above has at least half or at least the same binding affinity for human IL - 13 and human TSLP as a polypeptide consisting of the amino acids of SEQ ID NO: 1, where the binding affinity is measured using the same method such as SPR. The sequence numbers referred to in items A - D above and item E below (see Section 5.4 "Prolongation of half - life (in vivo)") are based on the definition of CDRs according to the definition of AbM (see Table A - 2). Note that the sequence numbers that define the same CDRs according to the Kabat definition (see Table A - 2.1) can similarly be used in items A - D above and item E below (see Section 5.4 "Prolongation of half - life (in vivo)").

[0186]

[0187] ​Therefore, specific examples of the ISVD that specifically binds to 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 the following items A' to D': A'. Specifically binds to human IL-13, i. CDR1 having the amino acid sequence of SEQ ID NO: 37 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 37; ii. CDR2 having the amino acid sequence of SEQ ID NO: 42 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 42; and iii. CDR3 having the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 17 An ISVD comprising the above.

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

[0189] B'. Specifically binds to human IL-13, i. CDR1 having the amino acid sequence of SEQ ID NO: 38 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 38; ii. CDR2 having the amino acid sequence of SEQ ID NO: 43 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 43; and iii. CDR3 having the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 18 An ISVD comprising the above.

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

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

[0192] C’. Specifically binds to human TSLP, i. CDR1 that is the amino acid sequence of SEQ ID NO: 39 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 39; ii. CDR2 that is the amino acid sequence of SEQ ID NO: 44 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 44; and iii. CDR3 that is the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 19 and an ISVD comprising the same.

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

[0194] D’. Specifically binds to human TSLP, i. CDR1 that is the amino acid sequence of SEQ ID NO: 41 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 41; ii. CDR2 that is the amino acid sequence of SEQ ID NO: 46 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 46; and iii. CDR3 that is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 21 and an ISVD comprising the same.

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

[0196] Examples of such ISVDs that specifically bind to human TSLP are shown, for constructs 501A02 and 529F10 described in Table A-2-1, respectively, as having one or more, or all, of the framework regions (in addition to the CDRs defined in items C' and D' above). In one embodiment, this is an ISVD that comprises or consists of the full-length amino acid sequence of construct 501A02 or 529F10 (see SEQ ID NO: 4 or 6, 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 the 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 as compared to the first amino acid sequence is considered as a difference at a single amino acid residue (i.e., a single position).

[0198] Typically, for the purpose 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 largest 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] "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, the "amino acid difference" is a substitution.

[0200] In one embodiment, the amino acid substitution is a conservative substitution. Such conservative substitutions are substitutions in which one amino acid within one of the following groups (a)-(e) is replaced with another amino acid residue within the same group: (a) small aliphatic, nonpolar, 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, nonpolar residues : Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

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

[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, to which a particular binding unit, e.g., ISVD, can bind with a 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., 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 indicated by KD, or dissociation constant, which has units of moles / liter (or M). Affinity is also equal to 1 / KD and can be expressed as the association constant, KA, which has units of (moles / liter) -1 (or M -1 ).

[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, a binding unit (e.g., ISVD) used in the present technology binds to its target with a dissociation constant (KD) of 10 -5 ~10 -12 moles / liter or less, or 10 -7 ~10 -12 moles / liter or less, or 10 -8 ~10 -12 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 liters / mole association constant (KA)).

[0206] Generally, any KD value greater than 10 -4 moles / liter (or any KA value lower than 10 4 liters / mol) is considered to exhibit non-specific binding.

[0207] The KD of a biological interaction, e.g., the KD of the binding of an immunoglobulin sequence considered specific to an antigen, is typically 10 -5 moles / liter (10000 nM or 10 μM) to 10 -12It is in the range of molar / liter (0.001 nM or 1 pM) or less.

[0208] Thus, for specific / selective binding, when using the same measurement method, e.g., SPR, the binding unit (or the polypeptide containing it) has a KD value of 10 -5 ~10 -12 molar / liter or less and binds to IL13 and / or TSLP, and may mean binding to related cytokines with a KD value greater than 10 -4 molar / liter. An example of a target related to IL13 is human IL4. Examples of cytokines related to TSLP are human IL7. Thus, in one embodiment of the present technology, at least two ISVDs contained in the polypeptide bind to IL13 with a KD value of 10 -5 ~10 -12 molar / liter or less and bind to the same species of IL4 with a KD value greater than 10 -4 molar / liter, and at least two ISVDs contained in the polypeptide bind to TSLP with a KD value of 10 -5 ~10 -12 molar / liter or less and bind to the same species of human IL7 with a KD value greater than 10 -4 molar / liter.

[0209] Thus, the polypeptide of the present technology has at least half the binding affinity of human IL13 and human TSLP, or at least the same binding affinity as the 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 preclude the binding unit from also specifically binding to a similar target from a different species. For example, specific binding to human IL13 does not preclude the binding unit (or polypeptide containing it) from also specifically binding to IL13 from cynomolgus monkeys. Similarly, for example, specific binding to human TSLP does not preclude the binding unit (or polypeptide containing it) from also specifically binding to TSLP from cynomolgus monkeys (``cyno'').

[0211] Specific binding of a binding unit to its designated target can be determined by any suitable method known per se, such as Scatchard analysis and / or competitive binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competitive assays, as well as various modified methods thereof known per se in the art; in addition by other techniques described herein.

[0212] The dissociation constant, although it will be apparent to those skilled in the art, may be the actual dissociation constant or the apparent dissociation constant. Methods for determining the dissociation constant will be apparent to those skilled in the art and examples thereof are described below. In this regard, it will also be apparent that dissociation constants greater than 10 -4 moles / liter or 10 -3 moles / liter (e.g., 10 -2 moles / liter) may not be measurable. Optionally, although it will be apparent to those skilled in the art, the (actual or apparent) dissociation constant can be calculated based on the (actual or apparent) association constant (KA) by the relationship [KD = 1 / KA].[[]END]]

[0213] The affinity of the intermolecular interaction between two molecules can be measured via various techniques known per se, for example, via 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 enables real-time analysis of a biospecific interaction by detecting a change in the protein concentration within a biosensor matrix, where one molecule is immobilized on a biosensor chip and the other molecule passes over the immobilized molecule under flowing conditions, whereby k on , k off measurement values, and thus K D (or K A ) values are obtained. This can be carried out, for example, using the well-known BIAcore® system (BIAcore International AB, GE Healthcare, Uppsala, Sweden and Piscataway, NJ). For further explanation, 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 technology for determining the affinity of biomolecular interactions is biolayer interferometry (BLI) (see, e.g., 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 interference fringes of light reflected from two surfaces: an internal reference layer (reference beam) and a layer of immobilized protein on the biosensor chip (signal beam). A change in the number of molecules bound to the biosensor chip causes a shift in the interference fringes, which is reported as a wavelength shift (nm), and the magnitude of which is a direct measure of the number of molecules bound to the biosensor chip surface. Since the interaction can be measured in real time, the association and dissociation rates as well as the affinity can be determined. BLI can be performed, for example, using the well-known Octet® system (a division of ForteBio, Pall Life Sciences, Menlo Park, USA).

[0215] Alternatively, affinity can be measured using a kinetic exclusion assay (KinExA) using the KinExA® platform (Sapidyne Instruments Inc, Boise, USA) (see, e.g., Drake et al., 2004, Anal. Biochem., 328:35-43). The term "KinExA", as used herein, refers to a solution-based method for measuring the true equilibrium binding affinity and kinetics of unmodified molecules. By passing an equilibrated solution of the antibody / antigen complex over a column having beads pre-coated with the antigen (or antibody), the free antibody (or antigen) can be bound to the coated molecules. Detection of the antibody (or antigen) thus captured is achieved using a fluorescently labeled protein that binds to the antibody (or antigen).

[0216] The GYROLAB (registered trademark) immunoassay system provides a platform for automated biological analysis and rapid sample rotation (Fraley et al., 2013, Bioanalysis 5:1765-74).

[0217] 5.4 Prolonged (in vivo) half-life The polypeptide may further comprise one or more other groups, residues, moieties or linking units, optionally linked via one or more peptidic linkers, said one or more other groups, residues, moieties or linking units providing a polypeptide having an increased (in vivo) half-life as compared to the corresponding polypeptide not having said one or more other groups, residues, moieties or linking units. Prolonged (in vivo) half-life means, for example, that the polypeptide has an increased half-life in a mammalian, e.g. human, subject after administration. The half-life can be expressed, for example, as t1 / 2 beta.

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

[0219] More specifically, said one or more other groups, residues, moieties or linking units providing a polypeptide having an increased half-life can be selected from the group consisting of linking units capable of binding to serum albumin, e.g. human serum albumin, or serum immunoglobulins, e.g. IgG. In one embodiment, said one or more other linking units providing a polypeptide having an increased half-life are linking units capable of binding to human serum albumin. In one embodiment, the linking unit is ISVD.

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

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

[0222] Furthermore, WO2012 / 175400 describes an improved version of Alb-1, designated 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 a variant thereof as shown 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 albumin binders are also shown in Table A-4. In one embodiment, further components of the polypeptides of the present technology are as described in item E: E. binds to human serum albumin, i. CDR1 that is the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 10; ii. CDR2 that is the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 15; and iii. CDR3 that is the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 20 An ISVD comprising the same.

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

[0225] Examples of such ISVDs that bind to human serum albumin are those having one or more, or all, of the framework regions (in addition to the CDRs defined in item E above), as shown for construct ALB23002 described 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 (see SEQ ID NO: 5, Tables A-1 and A-2).

[0226] Item E can also be described as follows using the Kabat definition: E’. Binds to human serum albumin, i. CDR1 that is the amino acid sequence of SEQ ID NO: 40 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 40; ii. CDR2 that is the amino acid sequence of SEQ ID NO: 45 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 45; and iii. CDR3 that is the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence having a difference of 2 or 1 amino acid from SEQ ID NO: 20 An ISVD comprising the same.

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

[0228] Examples of such ISVDs that bind to human serum albumin are shown with respect to construct ALB23002 described in Table A-2-1 and have one or more, or all, of the framework regions (in addition to the CDRs defined in item E' above). In one embodiment, this is an ISVD that comprises or consists of the full-length amino acid sequence of construct ALB23002 (see SEQ ID NO: 5, Tables A-1 and A-2-1).

[0229] In a further embodiment, the amino acid sequence of the ISVD that binds to human serum albumin may have a sequence identity of more than 90%, such as more than 95%, or more than 99% with SEQ ID NO: 5, and the CDRs are as defined in item E or E' above. In one embodiment, the ISVD that binds to 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 a difference of two or one amino acid 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, or at least the same binding affinity as that, like construct ALB23002 described in SEQ ID NO: 5, and in this case, the binding affinity is measured using the same method such as SPR.

[0231] In one embodiment, such an ISVD that binds to human serum albumin exhibits an extension of C-terminal alanine (A) or glycine (G) when it has a C-terminal position. In one embodiment, such an ISVD is selected from SEQ ID NOs: 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., not the C-terminal ISVD of the polypeptide of the present technology). In one embodiment, such an ISVD is selected from SEQ ID NOs: 5, 57, 58, 61, and 63 (see Table A-4 below).

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

[0233] A "nucleic acid molecule" (used interchangeably with "nucleic acid") is a chain of nucleotide monomers linked to each other via a phosphate backbone to form a nucleotide sequence. Nucleic acids can be used, for example, to transform / transfect a host cell or host organism for the expression and / or production of a polypeptide. Suitable hosts or host cells for production purposes will be apparent to those skilled in the art and can be, for example, any suitable fungus, prokaryotic or eukaryotic cell or cell line, or any suitable fungus, prokaryotic or eukaryotic organism. Also included in the present technology are hosts or host cells containing nucleic acids encoding the polypeptides of the present technology.

[0234] The nucleic acid may be, for example, DNA, RNA, or a hybrid thereof, or may contain modified nucleotides such as PNA (e.g., chemically). 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 acids of the present technology may be prepared or obtained in a manner known per se and / or isolated from suitable natural sources. Nucleotide sequences encoding naturally occurring (poly)peptides may be subjected to site-directed mutagenesis, for example, so as to provide nucleic acid molecules encoding polypeptides having sequence variations. Also, as will be apparent to those skilled in the art, for the preparation of nucleic acids, several nucleotide sequences, for example, at least one nucleotide sequence encoding a targeting moiety, for example, a nucleic acid encoding one or more linkers, may also be ligated together in a suitable manner.

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

[0237] 5.6 Vectors Vectors comprising nucleic acid molecules encoding the polypeptides of the present technology are also provided. As used herein, a vector is a suitable vehicle for carrying genetic material into cells. Examples of vectors include naked nucleic acids such as plasmids or mRNA, or nucleic acids embedded in larger structures such as liposomes or viral vectors.

[0238] Vectors generally contain, optionally, at least one nucleic acid 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 the encoded polypeptide or construct under suitable conditions, e.g., when the vector is introduced into a cell (e.g., a human cell). In the case of DNA-based vectors, this typically includes the presence of elements for transcription (e.g., a promoter and a polyA signal) and the presence of elements for translation (e.g., a Kozak sequence).

[0239] In one embodiment, in the 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 if the promoter can initiate or otherwise control / regulate the transcription and / or expression of the coding sequence (wherein the coding sequence is to be understood as being "under the control" of the promoter). Generally, when two nucleotide sequences are operably linked, they are in the same orientation and are usually within the same reading frame. They are also usually essentially continuous, although this may not necessarily be the case.

[0240] In one embodiment, any regulatory element of the vector is such that it can provide its 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, which means, for example, that the promoter should be capable of initiating the transcription and / or expression of a nucleotide sequence, such as a coding sequence operably linked thereto, or otherwise controlling / regulating them.

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

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

[0244] Suitable host cells or host organisms will be apparent to those skilled in the art and include, for example, any suitable fungus, prokaryotic or eukaryotic cell or cell line, or any suitable fungus, 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 Methods and Uses of Polypeptides The present technology also provides a method for producing the polypeptide of the present technology. The method comprises transforming / transfecting a host cell or host organism with a nucleic acid encoding the polypeptide, expressing the polypeptide in the host, and optionally subsequently including 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 subsequently: b) isolating and / or purifying the polypeptide which may be included.

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

[0247] The polypeptide, nucleic acid molecule or vector of the present technology as described, or a composition comprising the polypeptide, nucleic acid molecule or vector of the present technology is useful as a medicament.

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

[0249] Also provided is the polypeptide, nucleic acid molecule or vector of the present technology as described for use in (preventive or therapeutic) treatment of inflammatory diseases or a composition comprising the polypeptide, nucleic acid molecule or vector of the present technology.

[0250] Furthermore, provided is a method of treating (preventively and / or therapeutically) an inflammatory disease, the method comprising administering to a subject in need thereof a pharmaceutically active amount of a polypeptide, nucleic acid molecule or vector of the present technology as described, or a composition comprising a polypeptide, nucleic acid molecule or vector of the present technology.

[0251] Furthermore, provided is the use of a polypeptide of the present technology, a nucleic acid molecule or vector as described, or a composition comprising a 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] Inflammatory diseases are type 2 inflammatory diseases such as atopic dermatitis and asthma.

[0253] "Subject", when referred to in the context of 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 can be, for example, companion animals (e.g., dogs, cats), livestock (e.g., cows, horses, sheep, goats, or pigs), or animals generally used for research purposes and / or antibody production (e.g., mice, rats, rabbits, cats, dogs, goats, sheep, horses, pigs, non-human primates such as cynomolgus monkeys, or camelids such as llamas or alpacas).

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

[0255] A substance (such as a polypeptide, nucleic acid molecule, and vector, etc.) or composition can be administered to a subject by any suitable route of administration, such as enteral (e.g., oral or rectal) or parenteral (e.g., on the skin, sublingual, buccal, nasal, intra-articular, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, subdermal, or transmucosal) administration. In one embodiment, the substance is administered by parenteral administration, such as intramuscular, subcutaneous, or intradermal administration. In one embodiment, subcutaneous administration is used.

[0256] An effective amount of the described polypeptide, nucleic acid molecule, or vector, or a composition comprising a polypeptide, nucleic acid molecule, or vector can be administered to a subject to provide the intended treatment 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 action 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

Example

[0265] 6 Examples 6.1 Generation of monovalent ISVDs that specifically bind to IL-13 and TSLP respectively 6.1.1 Example 1: Immunization For the purpose of inducing a heavy-chain antibody-dependent humoral immune response, three llamas were immunized with recombinant human IL-13 (Peprotech, catalog number 200-13, derived from E. coli) according to a standard protocol. In addition, these llamas were boosted with human / cynomolgus IL-13 (Sino Biological, catalog numbers 10369-HNAC and 11057-CNAH, derived from mammalian cells) from another source.

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

[0267] Immune blood (PBL) samples were collected regularly and prepared from B cells isolated from total RNA. During the immunization process, the humoral immune response was monitored by comparing the antigen-specific serum titers between samples collected before the start of immunization and serum samples collected as usual after multiple antigen administrations. Briefly, 96-well Maxisorp plates were coated with human IL-13 (Sino Biological, catalog number 10369-HNAC) or human TSLP. Recombinant human TSLP is commercially available, for example, from R&D Systems (catalog number 1398-TS). After blocking and adding diluted serum samples, anti-IL-13 and anti-TSLP were detected by using an enzyme reaction in the presence of HRP (horseradish peroxidase)-conjugated goat anti-llama immunoglobulin (Bethyl Laboratories Inc.) and subsequent substrate TMB (3,3’,5,5’-tetramethylbenzidine). The presence of ISVD was demonstrated.

[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. These cells and total RNA extracted from lymph nodes were used as starting materials 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 edition, 2002, edited by O’Brian and Aitken, Humana Press, Totowa, NJ)), filtered and sterilized at 4°C, and stored until further use. Five phage libraries for IL13 were constructed, and the library sizes were 6.1×10 8 ~1.3×10 9and the percentage of inserts ranged from 87 to 96%. Five phage libraries for TSLP were constructed, and the library sizes were 4.2×10 8 ~9.8×10 8 and the percentage of inserts ranged from 91 to 100%.

[0269] To identify ISVDs that recognize human and cynomolgus monkey IL-13, phage libraries were incubated with 50 nM soluble biotinylated hIL13-Fc in the presence of IgG (Sigma, I4506) from human serum. The complex of hIL-13-Fc and phage was captured onto magnetic beads coated with streptavidin from the solution. After washing thoroughly with PBS / 0.05% Tween20, the bound phage was eluted by the addition of trypsin (1 mg / ml). The outputs of these round 1 selections were incubated with 0.05, 0.5 or 5 nM soluble biotinylated hIL-13-Fc or cynomolgus monkey IL-13-Fc. In round 3, the unamplified output from the round 2 selection was further incubated with 0.005, 0.05, 0.5 or 5 nM soluble biotinylated human or cynomolgus monkey IL-13-Fc. Individual clones from the amplified round 2 and round 3 selections were screened.

[0270] To identify ISVDs that recognize human and cynomolgus monkey TSLP, a phage library was incubated in the presence of IgG from human serum (Sigma, I4506), with 50 nM soluble biotinylated hTSLP-Fc, or with 500 nM biotinylated hTSLP, or with 500 nM biotinylated cynomolgus monkey TSLP. The human and cynomolgus monkey TSLP sequences are known (Uniprot accession numbers Q969D9 and NCBI reference sequence XP_005557555.1, respectively). The assay was performed using recombinant proteins. The TSLP and phage complex was captured onto magnetic beads coated with streptavidin from solution. After washing thoroughly with PBS / 0.05% Tween20, the bound phage was eluted by the addition of trypsin (1 mg / ml). The output from these round 1 selections was incubated with 5 nM soluble biotinylated hILTSLP-Fc or cynomolgus TLSP-Fc or 0.5 nM biotinylated hTSLP. The output from round 2 selections was incubated with 0.05, 0.5 or 5 nM soluble biotinylated human TSLP, TSLP-Fc or cynomolgus monkey TSLP-Fc. For each round, individual clones were picked from the enriched output.

[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. The cell pellet was frozen and the periplasmic extract was prepared by lysing it in 100 μl of PBS. Dead cell debris was removed by centrifugation.

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

[0273] 6.1.3 Example 3: Screening for blocking of ISVD in periplasmic extracts by AlphaScreen assay using human IL13 and human TSLP To determine the blocking ability 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] In the hIL-13:hIL-13Rα1 binary complex AlphaScreen, it was investigated whether ISVD could block the interaction between hIL-13 and the extracellular domain of hIL-13Rα1. To achieve this purpose, dilutions of the periplasmic extract were pre-incubated with biotinylated hIL-13 (Peprotech catalog number 200-13). hIL-13Rα1-hFc (R&D Systems; catalog number 146-IR) and anti-hFc binding acceptor beads were added to this mixture, and it was further incubated at room temperature for 1 hour. Subsequently, streptavidin-binding donor beads were added and incubated for an additional 1 hour. When the binary complex was formed, bringing the acceptor and donor beads into proximity and laser-exciting them generated a detectable signal. A decrease in the AlphaScreen signal indicates that the binding of biotinylated hIL-13 to hIL-13Rα1 was blocked by ISVD in the periplasmic extract. In a similar setting it was investigated whether ISVD could block the interaction between hTSLP (eBioscience, catalog number 10-8499) and the extracellular domain of hTSLPR (R&D Systems catalog number 981-TR).

[0275] In an AlphaScreen where the hIL-13:hIL-13Rα1:hIL4Rα ternary complex is present, it was screened whether ISVD could block the replenishment of hIL4Rα to the hIL13:hIL13Rα1 binary complex. hIL-13 binds to hIL-13Rα1, and this binary complex replenishes hIL4Rα, resulting in the formation of the ternary complex hIL-13:hIL-13Rα1:hIL4Rα. Dilutions of the periplasmic extract were pre-incubated with hIL-13 (Peprotech catalog number 200-13) and biotinylated huIL4Rα (R&D Systems; catalog number 230-4R / CF). To this mixture, hIL-13Rα1-hFc (R&D Systems; catalog number 146-IR) and anti-hFc binding acceptor beads were added, and it was further incubated at room temperature for 1 hour. Subsequently, streptavidin-binding donor beads were added and incubated for an additional 1 hour. When the ternary complex is formed, bringing the acceptor and donor beads into proximity and laser-exciting them generates a detectable signal. A decrease in the AlphaScreen signal indicates that the formation of the ternary complex is blocked by ISVD in the periplasmic extract. Similarly, it was investigated whether ISVD could block the formation of the hTSLP:hTSLPR:hIL7Rα complex (hIL7Rα source = Sino Biological catalog number 1095-H08H).

[0276] Based on 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 in 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% Tween20 was used as the running buffer and the experiment was 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. The IL13 protein was injected at pH 5.0 at 10 μg / ml in ProteOn acetate buffer. The TSLP protein was injected at pH 5.5 at 5 μg / mL in ProteOn acetate buffer. After immobilization, the surface was inactivated with ethanolamine.

[0281] The periplasmic extracts of the ISVD candidates were diluted 10-fold with PBS-Tween20 (0.1%) and injected at 45 μl / min for 2 minutes and dissociated for 900 seconds. Between different samples, the surface was regenerated by injection of phosphoric acid (0.425%) at 45 μl / min for 2 minutes in the case of IL13 or by injection of glycine pH 3.0 (5 mM) / SDS (0.25%) at 45 μL / min for 1 minute in the case of TSLP. The off-rates were calculated from the sensorgrams obtained for different periplasmic extracts.

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

[0283]

Table 11

[0284]

Table 12

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

[0286] The ISVDs were expressed in E. coli TG1 cells as c-myc, His6-tagged proteins. Expression was induced by the addition of 1 mM IPTG and continued for 3 hours at 37°C as such. After spinning the cell culture, the periplasmic extract was prepared by freeze-thawing the pellet and resuspending it in dPBS. These extracts were used as starting materials for immobilized metal affinity chromatography (IMAC) using a nickel sepharose TM6 FF column (Atoll). The ISVDs were 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 ISVDs in the AlphaScreen assay Dual and triple complex AlphaScreen assays were used as described in Example 3 to determine the IC50 values of anti-IL-13 and anti-TSLP ISVDs and purified as described in Example 5. Instead of dilutions of the periplasmic extract, a series of dilutions starting from 500 nM to 1.8 pM of each purified ISVD were pre-incubated with IL-13 or TSLP.

[0288] The tested anti-IL-13 ISVD 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 tested anti-TSLP ISVD completely inhibited the formation of the ternary complex and also inhibited the formation of the binary complex with IC50 values as shown in Table 6.

[0291]

Table 14

[0292] 6.1.7 Example 7: Blocking ability of purified anti-IL-13 and anti- TSLP ISVD in cell-based assays The inhibitory efficacy of anti-IL-13 ISVD was determined in a cell-based assay that monitored the IL-13-mediated proliferation of TF-1 cells. To achieve this, TF-1 cells were cultured in RPMI 1640 medium supplemented with 1 / 5 HEPES, 1 / 500 sodium pyruvate, 1 / 500 Glutamax, and 2 ng / mL recombinant human GM-CSF. TF-1 cells were seeded at 40,000 cells per well in GM-CSF-free growth medium. Serial dilutions of purified anti-IL-13 ISVD or a reference compound were added. After incubation at 37 °C for 15 minutes, 200 pM IL-13 (Peprotech catalog number 200-13) was added. After 96 hours, cell proliferation of TF-1 cells was determined using Cell Titer 96 Aqueous One Solution (Promega #G3580) on an EnVision multilabel reader (Perkin Elmer).

[0293] The ISVD shown in Table 7 inhibits TF1 proliferation induced by IL-13.

[0294]

Table 15

[0295] The blocking efficacy of anti-TSLP ISVD was determined in a cell-based assay that monitored the TSLP-mediated proliferation of BaF3 cells transfected with plasmids encoding hTSLPR and hIL7Ra. In a cell-cultured white 96-well plate, cells were seeded at a density of 20,000 cells / well in RPMI1640 growth medium. A series of dilutions of anti-TSLP ISVD were added, followed by the addition of 50 pM hTLSP-Fc or 50 pM cynomolgus TSLP-Fc for cell stimulation. The human and cynomolgus TSLP sequences are known (Uniprot accession numbers 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 out 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 IL-13 and TSLP A full binding kinetic study by SPR was performed on a BIAcore T100 instrument (GE Healthcare).

[0299] In the case of IL-13, approximately 2000 RU of hIL13-Fc or 4000 RU of cynomolgus monkey IL13-Fc was directly immobilized on the CM5 sensor chip. Then, ISVD was injected at different concentrations (3 μM to 12 nM) over 120 seconds and dissociated for 900 seconds. The regeneration of the hIL-13-Fc and cynomolgus monkey IL-13-Fc surfaces was carried out using a 47-second injection of 0.85% H 3 PO 4 : MilliQ (1:1) and performed using a 47-second injection.

[0300] In the case of TSLP, approximately 8000 RU of anti-huIgG antibody (GE Healthcare) was directly immobilized on the CM5 sensor chip. hTSLP-Fc (at 1 μg / mL) or cynomolgus monkey TSLP-Fc (at 0.75 μg / mL) was suspended and captured on the chip over 120 seconds. Then, ISVD was injected at different concentrations (0.4 nM to 3000 nM) over 120 seconds and dissociated for 900 seconds.

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

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

[0303]

Table 17

[0304]

Table 18

[0305] 6.2 Generation of monospecific multivalent polypeptides that bind to IL-13 and TSLP, respectively 6.2.1 Example 10: Generation and in vitro characterization of bivalent or bivalent paratopic ISVD constructs of wild-type anti-IL-13 Selected anti-IL-13 ISVDs (F0107004B02 and F0107004B06) were formatted into bivalent paratopic and bivalent ISVD constructs. The building blocks in the constructs were genetically linked by a flexible 35GS (GlySer) linker. The ISVDs were expressed as FLAG3-HIS6-tagged proteins in Pichia pastoris (the 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 construct was used as starting material for immobilized metal affinity chromatography (IMAC), followed by desalting, and as a result, a purity of 90% was obtained as evaluated by SDS-PAGE.

[0306] The bivalent paratopic and bivalent IL-13 constructs were characterized in an AlphaScreen assay that blocks binary and ternary complexes (as described in Example 6), in addition to a TF-1 proliferation assay (as described in Example 7). An overview of the generated constructs and their blocking potencies in the two-component and three-component AlphaScreen assays and the TF-1 proliferation assay is shown in Table 11. The bivalent paratopic construct showed excellent potency against hIL-13 and cyIL-13, similar to the potency of the anti-hIL-13 reference mAb1. The bivalent construct also improved potency, but not to the same extent as the bivalent paratopic construct F010700029 in the TF1 proliferation assay. In addition, the bivalent construct did not reach complete inhibition in the AlphaScreen of IL13-IL13Rα1.

[0307]

Table 19

[0308] The most potent anti-IL-13 bivalent ISVD constructs were tested in an IL-13-induced A549 eotaxin release assay. To achieve this, A549 suspension cells were cultured in Ham's F12K medium supplemented with 10% FCS. The cells were seeded into 96-well plates at 200,000 cells / well. The next day, 200 pM of hIL-13 (Sino Biological catalog number 10369-HNAC) or cynomolgus IL-13 (Sino Biological catalog number 11057-CNAH) was added, followed by a series of dilutions of the ISVD constructs. After 24 hours, eotaxin-3 was determined in the supernatant 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] Competitive ELISA was used to test whether the anti-IL-13 monovalent and bivalent ISVD constructs inhibit the binding of hIL-13 to IL13Rα2. IL13Rα2 (SinoBiological catalog number 10350-H08H) was coated on 384-well Spectraplates (Perkin Elmer). hIL13-Fc (SinoBiological, catalog number 10369-H01 H) 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 receptor was incubated with the IL13-Fc ISVD / control mix 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. If the ISVD inhibits the IL-13-IL-13Rα2 interaction, the detection of IL-13-Fc disappears 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 a Wild-Type Biparatopic Anti-TSLP ISVD Construct The selected TSLPR blockers (F0107501A02 and F0107529F10) were combined into a biparatopic ISVD construct. The construct was expressed as a FLAG3-HIS6-tagged protein in Pichia pastoris (amino acid sequences are shown in Table 16). Induction of ISVD construct expression occurred by the stepwise addition of methanol. The clarified medium containing the secreted ISVD construct was used as starting material for immobilized metal affinity chromatography (IMAC), followed by desalting and was evaluated by SDS-PAGE, resulting in 90% purity.

[0314] In a BaF3 proliferation assay (as described in Example 7), the biparatopic construct was titrated as purified protein against 50 or 5 pM of hTSLP and 50 or 5 pM of cynomolgus TSLP and compared to 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 construct clearly outperformed the reference mAbs. The biparatopic construct with F0107501A02 at the N-terminus showed improved reactivity against cynomolgus TSLP compared to the construct with F0107501A02 at the C-terminus.

[0315]

Table 23

[0316]

Table 24

[0317] 6.3 Array optimization of anti-IL-13 and anti-TSLP monovalent ISVDs 6.3.1 Example 12: Array optimization of anti-IL-13 and anti-TSLP monovalent ISVDs The anti-IL-13 ISVDs F0107004B02 and F0107004B06 and the anti-TSLP ISVDs F0107501A02 and F0107529F10 were further array optimized.

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

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

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

[0321] 3) Here too, depending on the desired host cell or host organism, substitutions that improve long-term stability or properties upon storage, substitutions that increase the expression level in the desired host cell or host organism, and / or substitutions that remove or reduce (undesirable) post-translational modifications (such as glycosylation or phosphorylation). To avoid N-terminal pyroglutamate formation, typically, the E1D mutation is introduced into the N-terminal building block of the multivalent Nb without affecting efficacy or stability. Thus, during the sequence optimization of the building block, the E1D mutation is not consistently introduced.

[0322] 4) Mutations to Val at position 11 and to Leu at position 89 minimize the binding of any existing naturally occurring antibody activity.

[0323] F0107004B02 and F0107004B06 The 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 parental ISVD F107004B02. The 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 parental ISVD F107004B06.

[0324] Array-optimized variants were assembled from oligonucleotides using the 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 ability by surface plasmon resonance using hIL-13 (Catalog No. 200-13) from Peprotech. In addition, F027100019 was tested for its neutralizing activity in an eosinophil release assay. The monomer behavior of both variants was monitored by size exclusion HPLC (SE-HPLC). The thermal stability of the variants was tested by a thermal shift assay (TSA) using a Lightcycler (Roche). In this assay, the parental ISVD and its variants are incubated at different pHs in the presence of SYPRO Orange and a temperature gradient is applied. When the ISVD starts to denature, SYPRO Orange binds and the measured fluorescence suddenly increases, allowing the melting temperature to be determined for a specific pH. The results are summarized in Tables 17 and 18.

[0325]

Table 25

[0326] F027100019 exhibited excellent potency in the eosinophil release assay, and its affinity for hIL-13 was determined to be 34 nM in SPR. The Tm of F027100019 was 3 °C higher than that of the parental ISVD F0107004B2. The % framework identity in the framework region of F027100019 was 85% based on the definition of AbM (see Antibody Engineering, Volume 2, Springer Verlag Heidelberg Berlin, 2010, edited by Kontermann and Dubel) and 86% based on the definition of Kabat.

[0327]

Table 26

[0328] The affinity of F027100183 is similar compared to the WT sequence, and the variant has a Tm of 61 °C. The variant elutes as a 100% monomer peak in SE-HPLC. The framework identity % in the framework region 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 containing eight amino acid substitutions (i.e., L11V, A14P, T60A, S71R, A74S, S79Y, K83R, V89L) compared to the parental ISVD F0107529F10. Sequence optimization of anti-TSLP ISVD F0107501A02 resulted in the final sequence-optimized variant F027400160 containing six amino acid substitutions (i.e., L11V, A14P, E16G, A74S, K83R, V89L) compared to the parental ISVD F0107501A02.

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

[0331]

Table 27

[0332] The intermediate variant F010704099 with mutations A14P, T60A, S71R, A74S, S79Y, K83R showed a similar affinity for hTSLP and a half affinity for cynomolgus TSLP compared to the parental ISVD F0107529F10. The Tm showed an overall increase of 11.4 °C compared to the parental ISVD. Two additional mutations, namely L11V and V89L, were introduced into variant F010704099 to minimize existing antibody binding, resulting in the final variant F027400021. The % framework identity in the framework region 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 for hTSLP and cynomolgus TSLP and similar potencies in the ternary complex Alphascreen compared to the parental ISVD F0107501A02. The Tm showed an overall increase of 12.3 °C compared to the parental ISVD. Two additional mutations, namely L11V and V89L, were introduced into variant F010704076 to minimize existing antibody binding, resulting in the final variant F027400160.

[0335] The % framework identity in the framework region 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 optimized ISVDs F027100019 (optimized variant of F0107004B02), F027100183 (optimized variant of F0107004B06), F027400021 (optimized variant of F0107529F10), and F027400160 (optimized version of F0107501A02) identified above were used for the generation of the multispecific ISVD construct F027400161. The optimized monovalent building blocks used in F027400161 are named below, in a form abbreviated as 04B02, 04B06, 529F10, and 501A02, respectively, according to their ISVD origin.

[0338] 6.4.1 Example 15: Generation of Multispecific ISVD Constructs The identification of the polypeptide F027400161 (SEQ ID NO: 1) containing an ISVD that binds to 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 / directions of the building blocks and different linker lengths (9GS vs. 35GS) were applied, demonstrating their importance for different parameters (potency, cross-reactivity, expression yield, etc.).

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

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

[0341]

Table 30

[0342] Subsequently, a large bispecific panel was narrowed down to a panel of two bispecific constructs consisting of ISVD constructs F027400161 and F027400163, which were proven to be potent against both targets (human and cynomolgus) and had the potential for high expression levels based on the prediction of preliminary yields.

[0343] For expression yield determination and evaluation of biophysical properties and existing antibody reactivity, larger - scale 2L and 5L production in Pichia pastoris was carried out.

[0344] Table 24 and Example 22 demonstrate that existing antibody reactivity is promoted by the orientation of the building blocks and the linker length.

[0345]

Table 31

[0346]

Table 32

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

[0348] 6.4.2 Example 16: Binding Affinity of Multispecific ISVD Constructs to TSLP, IL13, and Serum Albumin The affinity of F027400161 for human and cynomolgus TSLP, represented as the equilibrium dissociation constant (K D ), (the human and cynomolgus TSLP sequences are known (Uniprot accession numbers 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 albumin were quantified by affinity measurements in solution on a Gyrolab xP workstation (Gyros).

[0349] K DIn a measurement controlled by [relevant factor], serial dilutions of TSLP or IL-13 (in the range of 1 μM to 0.25 fM) or serum albumin (in the range of 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 hours or 72 hours (for TSLP and IL13) or 2 hours (for serum albumin) to reach equilibrium.

[0350] In a measurement controlled by the receptor, serial dilutions of TSLP or IL-13 (in the range of 1 μM to 0.25 fM) or serum albumin (in the range of 100 μM to 320 pM), and a fixed amount 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 hours (for TSLP and IL-13) or 2 hours (for serum albumin) to reach equilibrium.

[0351] Biotinylated human TSLP / IL-13 / serum albumin was captured in the microstructure of a Gyrolab Bioaffy 1000CD used as a molecular probe to capture free F027400161 from an equilibrated solution containing a bead column. 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. Then a fluorescently labeled anti-V HH antibody, ABH0086-Alexa647, was injected to label all captured F027400161, and after washing away the excess fluorescent probe, the change in fluorescence was determined. Fitting of a series of dilutions was performed using Gyrolab Analysis software, and K DThe KD value was determined by analyzing the curves controlled by the receptor.

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

[0353]

Table 33

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

[0355] The cytokines were immobilized at 25 μg / mL on the proteon GLC sensor chip for 600 seconds using amine coupling (ProteOn amine coupling kit. Catalog number 176-2410) with EDC / NHS injected for 80 seconds for activation and 1M ethanolamine HCl injected for 150 seconds for deactivation. The flow rate during activation and deactivation was set at 30 μl / min, and the flow rate during ligand injection was set at 25 μl / min. The pH of the 10 mM acetic acid 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 minutes and dissociated for 600 seconds 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 targets was measured by detecting the increase in the immunoassay signal resulting from the mass change on the chip upon binding.

[0357] None of the positive controls bound to their respective targets. Binding of F027400161 to human IL4 and IL7 was not detected.

[0358] In addition, it was investigated whether F027400161 could bind to the short form of TSLP. To achieve this, TSLP and the short form of TSLP (as described in Fornasà, 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. The pH of the 10 mM acetic acid 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 minutes and dissociated for 600 seconds 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) form or the short form of TSLP, and binding of F027400161 and anti-hTSLP reference mAb1 to the short form of TSLP was not detected.

[0361] 6.4.4 Example 18: Simultaneous Binding of a Multispecific ISVD Construct to IL13, TSLP, and HSA Using the Biacore T200 instrument, it was determined whether F027400161 could bind simultaneously to hTSLP and hIL13. To achieve this purpose, hTSLP (recombinant human TSLP is commercially available, for example, from R&D Systems (catalog number 1398-TS)) was immobilized on the CM5 sensor chip via amine coupling. To capture the ISVD construct via the TSLP building block 501A02-529F10, 100 nM of F027400161 was injected onto the TSLP surface at 10 μl / min for 2 minutes. Subsequently, either 100 nM of hIL13 (Peprotech, catalog number 200-13), HSA or hOX40L or 1000 nM of HSA was injected, or a mixture of 100 nM of IL13 + 100 nM of HSA, 100 nM of IL13 + 1000 nM of HSA, 100 nM of OX40L + 100 nM of HSA, 100 nM of OX40L + 1000 nM of HSA or 100 nM of IL13 + 100 nM of OX40L was injected at a flow rate of 10 μl / min for 2 minutes, followed by a subsequent dissociation step of 300 seconds. The TSLP surface was regenerated by injection of 0.5% SDS + 10 mM glycine pH 3 at 45 μl / min for 1 minute. The sensorgram (Figure 1) shows that F027400161 can bind simultaneously to human IL13, human TSLP and HSA as indicated by an increase in the response units after capture on TSLP: an increase of approximately 130 RU with IL13 alone, an increase of approximately 60 RU with 100 nM of HSA, and approximately 350 RU with 1000 nM of HSA alone, an increase of approximately 180 RU with the mixture of IL13 and 100 nM of HSA, and approximately 500 RU with the mixture of IL13 and 1000 nM of HSA. In the case of HSA, a higher concentration of HSA was required to see a certain level of RU increase because the affinity of F027400161 for HSA was lower (see Example 16).

[0362] Example 19: Inhibition of IL13-induced eosinophil chemotactic factor release by a multispecific ISVD construct in vitro The functional activities of soluble IL13 from species with different purposes (human, rhesus monkey, and cynomolgus monkey) and their inhibition by F027400161 were studied using a cell-based assay that investigated eosinophil chemotactic factor release by A549 human lung carcinoma cells.

[0363] To achieve this purpose, 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 series of dilutions 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 catalog number 10369-HNAC), cynomolgus monkey IL13 (Sino Biological catalog number 11057-CNAH), or rhesus monkey IL13 (R&D Systems, catalog number 2674-RM-025) was added to a final concentration of 160 pM. After an additional 24-hour incubation in the presence of 30 μM HSA, heparin was added at a final concentration of 50 μg / ml to enhance eosinophil chemotactic factor expression. After an additional 4-hour incubation, eosinophil chemotactic factor-3 secreted into the cell supernatant was quantified using the human CCL26 / eosinophil chemotactic factor-3 DuoSet ELISA (R&D Systems, DY346).

[0364] F027400161 inhibited eosinophil chemotactic factor-3 release induced by human, cynomolgus monkey, and rhesus monkey IL13 in a concentration-dependent manner, with IC50 values of 194 pM (for human IL13), 1040 pM (for cynomolgus monkey IL13), and 713 pM (for rhesus monkey IL13), which was comparable to the reference compound anti-hIL-13 reference mAb1 and superior to the reference compound anti-hIL-13 reference mAb2 (Table 26, Figure 2).

[0365]

Table 34

[0366] 6.4.5 Example 20: Inhibition of IL13-induced STAT-6 activation by a multispecific ISVD construct 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 the STAT6-inducible SEAP reporter gene. Stimulation with IL-4 and IL-13 caused the cells to produce SEAP induced by STAT6 secreted into the supernatant, which was 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. A series of dilutions of F027100161 or reference compounds (anti-hIL-13 reference mAb1 and anti-hIL-13 reference mAb2) were pre-incubated with 10 pM of hIL13 (Sino Biological catalog number 10369-HNAC) or cynomolgus IL-13 (Sino Biological catalog number 11057-CNAH) for 1 hour at room temperature and added to the cells. After incubation for 22 - 24 hours in the presence of 30 μM of HSA, 4 0 μl of cell supernatant was mixed with 160 μl of QUANTI-Blue™. The secreted SEAP was quantified by measuring the absorption 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 values of 32.8 pM (for human IL-13) and 53.4 pM (for cynomolgus IL-13), which was superior to the reference compound anti-hIL-13 reference mAb2 (Table 27, Figure 3).

[0369]

Table 35

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

[0371] Cells were seeded at a density of 15,000 cells / well in RPMI1640 growth medium in white 96-well plates that had been cell-cultured. A series of dilutions of F027100161 or a reference compound (anti-hTSLP reference mAb1) were added, followed by the addition of 5 pM of human or cynomolgus monkey TLSP for cell stimulation. The human and cynomolgus monkey TSLP sequences are known (Uniprot accession numbers Q969D9 and NCBI reference sequence XP_005557555.1, respectively). The assay was performed using recombinant proteins. After 48 hours of incubation in the presence of 30 μM of HSA, cell density and viability were monitored using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, G7571 / G7572 / G7573) and read out with an EnVision multilabel reader (Perkin Elmer).

[0372] F027400161 inhibited the human and cynomolgus monkey TSLP-dependent proliferation of Ba / F3 cells in a concentration-dependent manner, with IC50 values of 7.8 pM (for human TSP) and 24 pM (for cynomolgus monkey TSLP), and thus showed performance superior to that of 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 The existing antibody reactivity of the ISVD construct F027400161 was evaluated in normal human serum (n = 96) using a 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 the experiment was carried out at 25°C.

[0375] The ISVD was captured on the chip via the 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 μl / min), and HSA was injected at 100 μl / ml in ProteOn acetate buffer at pH 4.5 to a fixation level of approximately 2900 RU. After fixation, the surface was inactivated with ethanolamine HCl (flow rate 30 μl / min).

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

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

[0378] 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 shows lower existing antibody reactivity than construct F027400163 due to its specific orientation, however F027400164 shows lower reactivity than F027400161 due to its overall shorter linker.

[0379] 6.4.8 Example 23: In vitro, F027400161 blocks CCL17 induced by TSLP in human dendritic cells 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, depending on the environment. 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). When activated by TSLP, human DCs produce CCL17, a Th2-related chemokine, and promote Th2 cell differentiation from naive CD4 + T cells. F027400161 targets both TSLP and IL-13 and can block the interaction between TSLP and DCs, thereby reducing CCL-17 production and is expected to confer efficacy in type 2 inflammatory diseases and the like.

[0380] The ability of F027400161 to inhibit TSLP-induced CCL17 production in human DCs isolated from 8 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 the meninges (human leukocyte pack) samples. A total of 0.5 - 0.8×10 6 cells per well were incubated with eight 3-fold serially diluted concentrations of F027400161 (top concentration of 400 ng / mL or 5.714 nM) or eight 4-fold serially diluted concentrations of anti-hTSLP reference mAb1 (top concentration of 4000 ng / mL or 27 nM) prior to 36-hour stimulation with 4 ng / mL of recombinant human TSLP in a 37°C cell culture incubator. Recombinant human TSLP is commercially available, for example, from R&D Systems (catalog number 1398-TS). CCL17 production in freshly collected cell culture supernatants was measured by ELISA, and the IC 50 values of the ISVD construct and reference antibody were calculated with Graphpad Prism.

[0381] The collective results of the dose-inhibition responses of F027400161 and anti-hTSLP reference mAb1 in human DCs are shown in Figure 6. The anti-hTSLP reference mAb1, a reference monoclonal antibody, inhibited TSLP-induced CCL17 production at an average IC 50 concentration of 793.4 pM, while F027400161 inhibited TSLP-induced CCL17 production at an average IC 50 of 53.26 pM.

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

[0383] 6.4.9 Example 24: In vitro, F027400161 blocks the synergistic CCL17 induced by [IL-13 + TSLP] at 0.5 ng / mL in human PBMCs Type 2 cytokines, such as thymic stromal lymphopoietin (TSLP) and interleukin-13 (IL-13), exert unique, additive, and synergistic responses and promote the pathophysiology of asthma and atopic dermatitis (AD). The roles of TSLP as an epithelial cell-derived initiator and IL-13 as a downstream effector cytokine of the type 2 immune cascade have been extensively verified. F027400161 is expected to target both TSLP and IL-13, thereby conferring efficacy in type 2-mediated inflammatory diseases and the like.

[0384] The ability of F027400161 to inhibit the synergistic production of CCL17 (TARC) induced by 0.5 ng / mL of IL-13 and TSLP was evaluated in human PBMCs from 8 individual healthy donors. Single-specific biologics, anti-hTSLP reference mAb 1 and anti-hIL-13 reference mAb1 were designed to evaluate the non-inferiority of the ISVD construct. One million human PBMCs per well were stimulated with 0.5 ng / mL of recombinant human TSLP (recombinant human TSLP is commercially available, for example, from R&D Systems (catalog number 1398-TS), and in addition, IL-13 (R&D Systems, catalog number 213-ILB-005 / CF) and incubated in a 96-well plate with serially diluted concentrations of the ISVD construct (top concentration of 10 nM), anti-hIL-13 reference mAb1 (top concentration of 10 nM), and anti-hTSLP reference mAb1 (top concentration of 100 nM) in a cell culture incubator at 37°C for 20 hours. The assay was performed in technical triplicates for each donor for F027400161. The concentrations of cytokines used were within twice the standard error of the reported literature values from sera, BAL fluid, sputum, and skin of normal humans, asthmatic patients, 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 Meso Scale Diagnostics (MSD) V-PLEX human TARC kit.

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

[0386] In conclusion, these results demonstrate that F027400161 is more effective than anti-hTSLP reference mAb1 and is superior compared to anti-hIL-13 reference mAb1 with respect to blocking the synergistic response induced by pathophysiologically relevant concentrations of IL-13 and TSLP in human PBMC, 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 the synergistic CCL17 induced by 5 ng / mL of [IL-13 + TSLP] in human PBMC The ability of F027400161 to inhibit CCL17 production induced by 5 ng / mL of IL-13 + TSLP was evaluated in human PBMCs from 8 healthy individual donors. The study was designed to evaluate the non-inferiority of the ISVD construct to a monospecific biologic, anti-hTSLP reference mAb1 and anti-IL-13 reference mAb1. The concentrations of cytokines used were approximately 10-fold the upper limit of the pathophysiological ranges of TSLP and IL-13 reported in the literature in normal humans, asthmatic patients, and atopic dermatitis patients as the assumed 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). One million human PBMCs per well were stimulated with 5 ng / mL of recombinant human TSL P, plus IL-13 (R&D Systems, catalog number 213-ILB-005 / CF), and incubated for 20 hours in 96-well plates with serially diluted concentrations of F-27400161 (top concentration of 10 nM), anti-hIL-13 reference mAb1 (top concentration of 10 nM), and anti-hTSLP reference mAb1 (top dose of 100 nM) in a 3-fold dilution in a cell culture incubator at 37°C. The assay was performed in technical triplicates for each donor with respect to 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-inhibition responses of F027400161 and the reference antibodies, anti-hIL-13 reference mAb1 and anti-hTSLP reference mAb1, are shown in Figure 8. F027400161 inhibited 100% of the synergistic CCL17 production induced by 5 ng / mL of IL-13 + TSLP with a mean IC of 0.0387 nM 50was demonstrated. PBMCs treated with equimolar doses of the comparator antibody, anti-hIL-13 reference mAb1, showed a lower mean IC of 0.01339 nM 50 but the inhibitory response never reached 100% and plateaued at approximately 90% inhibition. On the other hand, anti-hTSLP reference mAb1 only partially blocked CCL17 production with a mean IC 50 of 19 nM at approximately 40%.

[0389] In conclusion, these results demonstrate that F027400161 is superior to both anti-hTSLP reference mAb1 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 within the pathophysiological range of the cytokines, highlighting its therapeutic potential for the treatment of asthma and atopic dermatitis during the acute or inflammatory phases.

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

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

[0392] The collective results of the inhibitory responses of F027400161 as well as 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. Anti-hIL-13 reference mAb1 demonstrated comparable 95% inhibition of CCL26 production, but 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 inhibition of IL-5 and CCL17 by these tested molecules may suggest that Der P stimulation initiates the induction of pathways other than TSLP and IL-13 to promote the production of IL-5 and CCL17 by PBMCs.

[0393] As a 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 containing human PBMCs cultured with stromal cells, and highlight its therapeutic potential for the treatment of type 2 inflammatory diseases such as asthma and atopic dermatitis, as well as signs of a broad range of immune diseases.

[0394] 6.4.12 Example 26: NSG-SGM3 mouse model to evaluate target occupancy and pharmacodynamics mediated by F027400161 in vivo F027400161 targets both human TSLP and IL-13 and does not react with the mouse ortholog. Therefore, to evaluate the biological activity of F027400161, a xenotransplanted humanized model system was used. 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 of which are driven by the human cytomegalovirus promoter / enhancer sequence. The triple transgenic mice constitutively produce the above cytokines, provide cell growth and survival signals, and maintain stable engraftment of the CD33+ myeloid lineage and several types of lymphoid cells. Briefly, the protocol for engraftment is as follows: On day 0 of the study, the mice were irradiated with radiation at 150 centigrays at a rate of 120 rad / min for 1 minute and 15 seconds. The mice were given 1 × 10 in 200 μl of Dulbecco's phosphate-buffered saline (DPBS) 5Individual cord blood CD34+ stem / progenitor cells were implanted approximately 6 hours post-implantation via the intravenous (IV) route. One group of mice was irradiated in the same manner, but implantation was not performed. These mice were considered irradiated naive mice. On day 88 post-engraftment, the mice received either a hydrodynamic (HDD) i.v. injection of saline (implanted control mice) or a combination of 50 μg of IL-4 minicircle DNA and 50 μg of TSLP minicircle DNA. On day 91, submandibular blood collection was performed, and 100 - 150 μl of blood was collected from each mouse and placed in lithium heparin tubes. Engraftment checks were performed by flow cytometry, and plasma concentrations of IL-4 and TSLP were evaluated. Mice were selected (included or excluded) from the study using information from engraftment checks and / or cytokine determinations. Mice from the implanted 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 less than 1 standard deviation of the mean were also excluded from the study. Mice that received minicircle DNA by HDD i.v. injection were given a vehicle (20 mM phosphate, 125 mM L-arginine HCL, and 0.01% Twe en20, pH 7.0) or F027400161 (0.01, 0.05, 0.1, or 10 mg / kg) by subcutaneous administration. On day 103, the mice were anesthetized with isoflurane. While under isoflurane anesthesia, blood was collected by retro-orbital bleeding. After blood collection, the mice were sacrificed by cervical dislocation while still under isoflurane anesthesia. A portion of the lung was harvested and placed in RNA later for gene expression evaluation. The plasma concentration of human TSLP from the plasma samples on day 103 was determined by MSD kit assay (Catalog number K15067-L-2, Meso Scale Diagnostics, Rockville, MD, USA). The plasma concentration of human IL-13 from the plasma samples on day 103 was determined by ELISA (Catalog number 88-7439--88 Human IL-13 ELISA kit, Invitrogen / Thermo-Fischer, Waltham, MA, USA). The internal assay validation experiment demonstrated that the detection kits for both human TSLP and IL-13 were unable to detect F027400161 bound to hTSLP and hIL-13.

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

[0396] In the NSG-SGM3 mouse model, hydrodynamic delivery of the cDNAs of TSLP and IL-4 activates the 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 NSG-SGM3 studies were used to study the pharmacodynamic effects of F027400161 (Hershey GK. 2003, J Allergy Clin Immunol.;111(4):677-90). In these studies, the effect of F027400161 treatment on mouse gene transcripts regulated by human IL-13 was investigated. Mouse lung tissue from the above studies was used for this analysis.

[0397] Lungs were harvested from treated and control NSG-SGM3 mice and processed to generate RNA as detailed in the attached protocol. The RNA was processed for quantification by TaqMan and the data were 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. The 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 transcriptional 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 using Quantstudio 6&7 Flex software. For each probe, the C T value and the delta C T value (relative to Rpl37a) were exported to Excel and the relative expression values for each gene were calculated using the following formula: Normalized relative expression = (power(2,-(delta CT))) × 1000.

[0398] The two human IL-13-regulated mouse genes evaluated were Retnla (resistin-like alpha) and Clca1 (chloride channel accessory 1), which play roles 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, which demonstrates the pharmacodynamic effect of F027400161 on the mouse transcriptional response driven by human IL-13 in vivo.

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

[0401] RNA preparation: For RNA purification, 350 ul of homogenate was used. 1× volume (350 ul) of 70% ethanol was added, the homogenate was thoroughly mixed, 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 4000×g for 2 minutes. To wash the column, 400 μl of buffer RWT was added to the RNeasy spin column, and the spin column plate was centrifuged at 4000×g for 2 minutes at room temperature. DNase I digestion was performed by adding 80 ul of 1× DNase I mix and incubating at room temperature for 15 minutes. DNase I was washed away by adding 400 ul of buffer RWT and spinning the plate at 4000×g for 2 minutes. Subsequently, the spin plate was washed with 500 ul each of buffer RPE and 80% ethanol. Subsequently, the column membrane was dried by spinning at 4000×g for 4 minutes. Then RNA was 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 25 ng / ul. cDNA was stored at -20 °C until TaqMan assay was performed.

[0403] TaqMan assay: TaqMan Multiplex Master Mix was 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 the internal Rpl37a control. Each multiplex qPCR reaction was performed in a 10 μl reaction volume.

[0404] [Table 37]

[0405] A total of 8.8 μl of master mix per sample was added to the appropriate wells of a 384-well optical plate. 30 ng (1.2 μl) of cDNA sample was added to each well. The TaqMan assay was configured on a QuantStudio 7K. The 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 using Quantstudio 6&7 Flex software.

[0407] For each probe, the CT value and the delta CT value (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))) × 1000

[0408] References: Liu, Y.J. 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, nucleic acid molecules encoding the same, vectors and compositions containing the nucleic acids described herein can be used, for example, in the treatment of subjects suffering from inflammatory diseases.

Claims

1. 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) that specifically binds to IL-13 or TSLP, said ISVD comprising three complementarity determining regions (CDR1 to CDR3, respectively), said at least one ISVD comprising: 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; 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 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) a CDR1 which is the amino acid sequence of SEQ ID NO:8 or which has two or one amino acid difference from SEQ ID NO:8; 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 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) a CDR1 which is an amino acid sequence of SEQ ID NO:9 or which has two or one amino acid difference from SEQ ID NO:9; 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 a CDR3 that is the amino acid sequence of SEQ ID NO: 19 or has two or one amino acid difference from SEQ ID NO: 19; or d) CDR1 which is the amino acid sequence of SEQ ID NO:11 or has two or one amino acid difference from SEQ ID NO:11; 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 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:

2. At least one ISVD or TSLP that specifically binds to IL-13: 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; b) 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) 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 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 claim 1 comprising:

3. The amino acid sequence of at least one ISVD that specifically binds to IL-13 or TSLP 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 with 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 claim 1 or 2, comprising:

4. The at least one ISVD that specifically binds to IL-13 or TSLP: 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 claims 1 to 3, comprising:

5. The polypeptide comprises or consists of at least two ISVDs, each of which comprises three complementarity determining regions (CDR1 to CDR3, respectively), said at least two ISVDs being optionally linked via one or more peptidic linkers: a) the first and second ISVDs specifically bind IL-13; i. 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; ii. CDR2 is an amino acid sequence of SEQ ID NO: 12 or has two or one amino acid difference from SEQ ID NO: 12; and iii. CDR3 having the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having two or one amino acid difference from SEQ ID NO:

17. Including, b) the first and second ISVDs specifically bind IL-13; i. CDR1 which is the amino acid sequence of SEQ ID NO:8 or has two or one amino acid difference from SEQ ID NO:8; ii. CDR2 is an amino acid sequence of SEQ ID NO: 13 or has two or one amino acid difference from SEQ ID NO: 13; and iii. 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 specifically binds to IL-13; i. 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; ii. CDR2 is an amino acid sequence of SEQ ID NO: 12 or has two or one amino acid difference from SEQ ID NO: 12; and iii. CDR3 having the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having two or one amino acid difference from SEQ ID NO:

17. Including, The second ISVD specifically binds IL-13; iv. CDR1 which is the amino acid sequence of SEQ ID NO:8 or has two or one amino acid difference from SEQ ID NO:8; v. CDR2 is an 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 specifically binds IL-13; i. CDR1 which is the amino acid sequence of SEQ ID NO:8 or has two or one amino acid difference from SEQ ID NO:8; ii. CDR2 is an amino acid sequence of SEQ ID NO: 13 or has two or one amino acid difference from SEQ ID NO: 13; and iii. 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 specifically binds IL-13; iv. 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; 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 specifically binds to TSLP; i. CDR1 which is the amino acid sequence of SEQ ID NO:11 or has two or one amino acid difference from SEQ ID NO:11; ii. CDR2 is an amino acid sequence of SEQ ID NO: 16 or has two or one amino acid difference from SEQ ID NO: 16; and iii. 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 specifically binds TSLP; iv. CDR1 which is the amino acid sequence of SEQ ID NO:9 or has two or one amino acid difference from SEQ ID NO:9; v. CDR2 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 specifically binds to TSLP; i. CDR1 which is the amino acid sequence of SEQ ID NO:9 or has two or one amino acid difference from SEQ ID NO:9; ii. 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 iii. 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 specifically binds TSLP; iv. CDR1 which is the amino acid sequence of SEQ ID NO:11 or has two or one amino acid difference from SEQ ID NO:11; v. CDR2 is an 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 claim 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.

6. a) the first and second ISVDs that specifically bind to IL-13 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 that specifically bind to IL-13 comprise 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; c) a first ISVD that specifically binds to IL-13 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, and a second ISVD that specifically binds to IL-13 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; d) a first ISVD that specifically binds to IL-13 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, and a second ISVD that specifically binds to IL-13 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; e) a first ISVD that specifically binds to TSLP 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, and a second ISVD that specifically binds to TSLP 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, or f) A polypeptide or composition for use according to claim 5, wherein a first ISVD that specifically binds to TSLP 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, and a second ISVD that specifically binds to TSLP 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.

7. a) the amino acid sequence of the first and second ISVDs that specifically bind IL-13 comprises greater than 90% sequence identity with SEQ ID NO:2; b) the amino acid sequence of the first and second ISVDs that specifically bind IL-13 comprises greater than 90% sequence identity with SEQ ID NO:3; c) the amino acid sequence of the first ISVD that specifically binds IL-13 comprises greater than 90% sequence identity with SEQ ID NO:2, and the second ISVD that specifically binds IL-13 comprises greater than 90% sequence identity with SEQ ID NO:3; d) the amino acid sequence of the first ISVD that specifically binds IL-13 comprises greater than 90% sequence identity with SEQ ID NO:3, and the second ISVD that specifically binds IL-13 comprises greater than 90% sequence identity with SEQ ID NO:2; e) the amino acid sequence of a first ISVD that specifically binds TSLP comprises greater than 90% sequence identity to SEQ ID NO:6, and a second ISVD that specifically binds TSLP comprises greater than 90% sequence identity to SEQ ID NO:4; f) A polypeptide or composition for use according to claim 5 or 6, wherein the amino acid sequence of a first ISVD that specifically binds to TSLP comprises greater than 90% sequence identity with SEQ ID NO:4 and a second ISVD that specifically binds to TSLP comprises greater than 90% sequence identity with SEQ ID NO:

6.

8. a) the first and second ISVDs that specifically bind IL-13 comprise the amino acid sequence of SEQ ID NO:2; b) the first and second ISVDs that specifically bind IL-13 comprise the amino acid sequence of SEQ ID NO:3; c) a first ISVD that specifically binds IL-13 comprises the amino acid sequence of SEQ ID NO:2, and a second ISVD that specifically binds IL-13 comprises the amino acid sequence of SEQ ID NO:3; d) a first ISVD that specifically binds IL-13 comprises the amino acid sequence of SEQ ID NO:3, and a second ISVD that specifically binds IL-13 comprises the amino acid sequence of SEQ ID NO:2; e) a first ISVD that specifically binds TSLP comprises the amino acid sequence of SEQ ID NO:6 and a second ISVD that specifically binds TSLP comprises the amino acid sequence of SEQ ID NO:4; or f) A polypeptide or composition for use according to any one of claims 5 to 7, wherein a first ISVD that specifically binds to TSLP comprises the amino acid sequence of SEQ ID NO: 4 and a second ISVD that specifically binds to TSLP comprises the amino acid sequence of SEQ ID NO:

6.

9. The 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) the amino acid sequence of SEQ ID NO: 159 A polypeptide according to any one of claims 5 to 8, comprising or consisting of:

10. The polypeptide comprises or consists of at least four ISVDs, each of which comprises three complementarity determining regions (CDR1 to CDR3, respectively), said at least four ISVDs being optionally linked via one or more peptidic linkers: a) the first ISVD specifically binds to IL-13; i. 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; ii. CDR2 is an amino acid sequence of SEQ ID NO: 12 or has two or one amino acid difference from SEQ ID NO: 12; and iii. 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 second ISVD specifically binds IL-13; 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; c) the third ISVD specifically binds TSLP; vii. CDR1 which is the amino acid sequence of SEQ ID NO:9 or has two or one 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 Including; d) the fourth ISVD specifically binds TSLP; 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. A polypeptide or composition for use according to claim 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.

11. The composition for use according to any one of claims 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 further pharma- ceutically active polypeptides and / or compounds.

12. a) said 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) said 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; c) said third ISVD comprises a CDR1 which is the amino acid sequence of SEQ ID NO:9, a CDR2 which is the amino acid sequence of SEQ ID NO:14, and a CDR3 which is 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.

13. 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 or composition for use according to any one of claims 10 to 12, wherein the amino acid sequence of the fourth ISVD comprises more than 90% sequence identity with SEQ ID NO:

6.

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) The polypeptide or composition for use according to any one of claims 10 to 13, wherein the fourth ISVD comprises the amino acid sequence of SEQ ID NO:

6.

15. The polypeptides are optionally linked via one or more peptidic linkers.

15. The polypeptide or composition for use according to any one of claims 1 to 14, further comprising one or more other groups, residues, moieties or binding units linked thereto, said one or more other groups, residues, moieties or binding units providing the polypeptide with an increased half-life compared to a corresponding polypeptide without said one or more other groups, residues, moieties or binding units.

16. 16. A polypeptide or composition for use according to claim 15, wherein the one or more other groups, residues, moieties or binding units providing 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 small proteins or peptides capable of binding to serum proteins.

17. 17. Polypeptide or composition for use according to claim 15 or 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).

18. 18. A polypeptide or composition for use according to claim 17, wherein the binding unit that provides a polypeptide with increased half-life is an ISVD capable of binding to human serum albumin.

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

20. A polypeptide or composition for use according to claim 18 or 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.

21. 21. The polypeptide or composition for use according to any one of claims 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.

22. The polypeptide or composition for use according to any one of claims 18 to 21, wherein the ISVD that binds to human serum albumin comprises the amino acid sequence of SEQ ID NO:

5.

23. The polypeptide or composition for use according to any one of claims 10 to 22, wherein the amino acid sequence of the polypeptide comprises more than 90% sequence identity with SEQ ID NO:

1.

24. A polypeptide or composition for use according to any one of claims 10 to 23, wherein the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:

1.

25. A polypeptide or composition for use according to any one of claims 1 to 24 for use in the treatment of an inflammatory disease, such as a type 2 inflammatory disease.

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

27. A polypeptide comprising or consisting of at least one immunoglobulin single variable domain (ISVD) that specifically binds to IL13 or TSLP, said ISVD comprising three complementarity determining regions (CDR1 to CDR3, respectively); said at least one ISVD comprising: 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; 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 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) a CDR1 which is the amino acid sequence of SEQ ID NO:8 or which has two or one amino acid difference from SEQ ID NO:8; 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 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) a CDR1 which is an amino acid sequence of SEQ ID NO:9 or which has two or one amino acid difference from SEQ ID NO:9; 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 a CDR3 that is the amino acid sequence of SEQ ID NO: 19 or has two or one amino acid difference from SEQ ID NO: 19; or d) CDR1 which is the amino acid sequence of SEQ ID NO:11 or has two or one amino acid difference from SEQ ID NO:11; 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 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:

28. At least one ISVD or TSLP that specifically binds to IL-13: 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; b) 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) 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 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 claim 27, comprising:

29. The amino acid sequence of at least one ISVD that specifically binds to IL-13 or TSLP 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 with SEQ ID NO:4; or d) greater than 90% sequence identity with SEQ ID NO:6 29. The polypeptide of claim 27 or 28, comprising:

30. The at least one ISVD that specifically binds to IL-13 or TSLP: 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 The polypeptide according to any one of claims 27 to 29, comprising:

31. The polypeptide comprises or consists of at least two ISVDs, each of which comprises three complementarity determining regions (CDR1 to CDR3, respectively), said at least two ISVDs being optionally linked via one or more peptidic linkers: a) the first and second ISVDs specifically bind IL-13; i. 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; ii. CDR2 is an amino acid sequence of SEQ ID NO: 12 or has two or one amino acid difference from SEQ ID NO: 12; and iii. CDR3 having the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having two or one amino acid difference from SEQ ID NO:

17. Including, b) the first and second ISVDs specifically bind IL-13; i. CDR1 which is the amino acid sequence of SEQ ID NO:8 or has two or one amino acid difference from SEQ ID NO:8; ii. CDR2 is an amino acid sequence of SEQ ID NO: 13 or has two or one amino acid difference from SEQ ID NO: 13; and iii. 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 specifically binds to IL-13; i. 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; ii. CDR2 is an amino acid sequence of SEQ ID NO: 12 or has two or one amino acid difference from SEQ ID NO: 12; and iii. CDR3 having the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having two or one amino acid difference from SEQ ID NO:

17. Including, The second ISVD specifically binds IL-13; iv. CDR1 which is the amino acid sequence of SEQ ID NO:8 or has two or one amino acid difference from SEQ ID NO:8; v. CDR2 is an 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 specifically binds IL-13; i. CDR1 which is the amino acid sequence of SEQ ID NO:8 or has two or one amino acid difference from SEQ ID NO:8; ii. the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 13 and two or one CDR2 having an amino acid sequence with an amino acid difference of iii. 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 specifically binds IL-13; iv. 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; 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 specifically binds to TSLP; i. CDR1 which is the amino acid sequence of SEQ ID NO:11 or has two or one amino acid difference from SEQ ID NO:11; ii. CDR2 is an amino acid sequence of SEQ ID NO: 16 or has two or one amino acid difference from SEQ ID NO: 16; and iii. 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 specifically binds TSLP; iv. CDR1 which is the amino acid sequence of SEQ ID NO:9 or has two or one amino acid difference from SEQ ID NO:9; v. CDR2 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 specifically binds to TSLP; i. CDR1 which is the amino acid sequence of SEQ ID NO:9 or has two or one amino acid difference from SEQ ID NO:9; ii. 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 iii. 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 specifically binds TSLP; iv. CDR1 which is the amino acid sequence of SEQ ID NO:11 or has two or one amino acid difference from SEQ ID NO:11; v. CDR2 is an 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, 28. The polypeptide of claim 27, wherein the order of the ISVDs indicates their relative positions with respect to each other considering the N-terminus to C-terminus of the polypeptide.

32. a) the first and second ISVDs that specifically bind to IL-13 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 that specifically bind to IL-13 comprise 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; c) a first ISVD that specifically binds to IL-13 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, and a second ISVD that specifically binds to IL-13 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; d) a first ISVD that specifically binds to IL-13 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, and a second ISVD that specifically binds to IL-13 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; e) a first ISVD that specifically binds to TSLP 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, and a second ISVD that specifically binds to TSLP 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, or f) A polypeptide according to claim 31, wherein a first ISVD that specifically binds to TSLP 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 a second ISVD that specifically binds to TSLP 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.

33. a) the amino acid sequence of the first and second ISVDs that specifically bind IL-13 comprises greater than 90% sequence identity with SEQ ID NO:2; b) the amino acid sequence of the first and second ISVDs that specifically bind IL-13 comprises greater than 90% sequence identity with SEQ ID NO:3; c) the amino acid sequence of the first ISVD that specifically binds IL-13 comprises greater than 90% sequence identity with SEQ ID NO:2, and the second ISVD that specifically binds IL-13 comprises greater than 90% sequence identity with SEQ ID NO:3; d) the amino acid sequence of the first ISVD that specifically binds IL-13 comprises greater than 90% sequence identity with SEQ ID NO:3, and the second ISVD that specifically binds IL-13 comprises greater than 90% sequence identity with SEQ ID NO:2; e) the amino acid sequence of a first ISVD that specifically binds TSLP comprises greater than 90% sequence identity to SEQ ID NO:6, and a second ISVD that specifically binds TSLP comprises greater than 90% sequence identity to SEQ ID NO:4; f) The polypeptide of claim 31 or 32, wherein the amino acid sequence of a first ISVD that specifically binds to TSLP comprises greater than 90% sequence identity to SEQ ID NO:4, and a second ISVD that specifically binds to TSLP comprises greater than 90% sequence identity to SEQ ID NO:

6.

34. a) the first and second ISVDs that specifically bind IL-13 comprise the amino acid sequence of SEQ ID NO:2; b) the first and second ISVDs that specifically bind IL-13 comprise the amino acid sequence of SEQ ID NO:3; c) a first ISVD that specifically binds IL-13 comprises the amino acid sequence of SEQ ID NO:2, and a second ISVD that specifically binds IL-13 comprises the amino acid sequence of SEQ ID NO:3; d) a first ISVD that specifically binds IL-13 comprises the amino acid sequence of SEQ ID NO:3, and a second ISVD that specifically binds IL-13 comprises the amino acid sequence of SEQ ID NO:

2. 、 e) a first ISVD that specifically binds TSLP comprises the amino acid sequence of SEQ ID NO:6 and a second ISVD that specifically binds TSLP comprises the amino acid sequence of SEQ ID NO:4; or f) A polypeptide according to any one of claims 31 to 33, wherein a first ISVD that specifically binds to TSLP comprises the amino acid sequence of SEQ ID NO: 4 and a second ISVD that specifically binds to TSLP comprises the amino acid sequence of SEQ ID NO:

6.

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) the amino acid sequence of SEQ ID NO: 159 A polypeptide according to any one of claims 31 to 34, comprising or consisting of:

36. The polypeptide comprises or consists of at least four ISVDs, each of which comprises three complementarity determining regions (CDR1 to CDR3, respectively), said at least four ISVDs being optionally linked via one or more peptidic linkers: a) the first ISVD specifically binds to IL-13; i. 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; ii. CDR2 is an amino acid sequence of SEQ ID NO: 12 or has two or one amino acid difference from SEQ ID NO: 12; and iii. 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 second ISVD specifically binds IL-13; 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; c) the third ISVD specifically binds TSLP; vii. CDR1 which is the amino acid sequence of SEQ ID NO:9 or has two or one 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 Including; d) the fourth ISVD specifically binds TSLP; 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 claim 27 or 31, wherein 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.

37. a) said 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) said 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; c) said third ISVD comprises a CDR1 which is the amino acid sequence of SEQ ID NO:9, a CDR2 which is the amino acid sequence of SEQ ID NO:14, and a CDR3 which is the amino acid sequence of SEQ ID NO:19; d) The polypeptide of claim 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.

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 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 36 or 37, wherein the amino acid sequence of the fourth ISVD comprises greater than 90% sequence identity with SEQ ID NO:

6.

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 claims 36 to 38, wherein the fourth ISVD comprises the amino acid sequence of SEQ ID NO:

6.

40. 40. The polypeptide of any one of claims 27 to 39, further comprising 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.

41. The one or more other groups, residues, moieties or binding units that provide a 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. The polypeptide of claim 40.

42. 42. The polypeptide of claim 40 or 41, wherein the one or more other groups, residues, moieties or binding units that provide 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 immunoglobulin (such as IgG).

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

44. The ISVD that binds to human serum albumin is i. CDR1 which is the amino acid sequence of SEQ ID NO: 10 or has two or one amino acid difference from SEQ ID NO: 10; ii. CDR2 is an amino acid sequence of SEQ ID NO: 15 or has two or one amino acid difference from SEQ ID NO: 15; and iii. 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 claim 43, comprising:

45. A polypeptide described in claim 43 or 44, 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.

46. The polypeptide of any one of claims 43 to 45, 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.

47. The polypeptide of any one of claims 43 to 46, wherein the ISVD that binds to human serum albumin comprises the amino acid sequence of SEQ ID NO:

5.

48. The polypeptide of any one of claims 36 to 47, wherein the amino acid sequence of the polypeptide comprises greater than 90% sequence identity with SEQ ID NO:

1.

49. A polypeptide according to any one of claims 36 to 48, comprising or consisting of the amino acid sequence of SEQ ID NO:

1.

50. A nucleic acid comprising a nucleotide sequence encoding a polypeptide according to any one of claims 27 to 49, or a polypeptide according to any one of claims 36 to 49.

51. 51. A host or host cell comprising the nucleic acid of claim 50.

52. A method for producing a polypeptide according to any one of claims 27 to 49 or a polypeptide according to any one of claims 36 to 49, comprising at least: a) expressing the nucleic acid according to claim 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 one of claims 27 to 49 or a polypeptide according to any one of claims 36 to 49 The method comprising:

53. At least one polypeptide according to any one of claims 27 to 49, or at least one polypeptide according to any one of claims 36 to 49, or claim 50 A composition comprising the nucleic acid described in.

54. 54. The composition of claim 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.

55. A method for treating an inflammatory disease, preferably a type 2 inflammatory disease, comprising administering to a subject in need thereof a pharma- ceutical active amount of a polypeptide according to any one of claims 27-49 or any one of claims 36-49, or a composition according to any one of claims 53-54.

56. 56. The method of claim 55, wherein the type 2 inflammatory disease is selected from asthma and atopic dermatitis.

57. Use of a polypeptide according to any one of claims 27 to 49, preferably a polypeptide according to any one of claims 36 to 49, or a composition according to any one of claims 53 to 54, in the preparation of a pharmaceutical composition for treating an inflammatory disease, such as a type 2 inflammatory disease.

58. 58. The use of a polypeptide or composition according to claim 57, wherein the type 2 inflammatory disease is selected from asthma and atopic dermatitis.

Images