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

Abstract

The present technology is directed to providing novel medicaments for treating a subject having an inflammatory disease. In particular, the present technology provides a polypeptide comprising at least four immunoglobulin single variable domains (ISVDs) characterized in that at least two ISVDs bind to IL-13 and at least two ISVDs bind to TSLP. The present technology also provides nucleic acids, vectors, and compositions.

Description

1. Technical Field

[0002] This invention relates to peptides targeting IL-13 and TSLP. It also relates to nucleic acid molecules encoding said peptides and carriers containing said nucleic acids, and to compositions containing said peptides, nucleic acids, or carriers. This invention further relates to these products in methods for treating subjects suffering from inflammatory diseases. Furthermore, this invention relates to methods for producing these products. 2 Background Technology

[0004] While essential for host defense, uncontrolled immune responses can lead to a range of inflammatory diseases, such as asthma, atopic dermatitis, and rheumatoid arthritis. A cascade of immune responses mediated by the innate and adaptive groups of the immune system (e.g., antigen recognition, antigen processing, antigen presentation, cytokine production, antibody production, target cell killing) drives the initiation and spread of a range of immune diseases. Inflammatory diseases are often chronic and can even be life-threatening. Allergic and atopic diseases (such as asthma and atopic dermatitis) are primarily driven by type 2 immune responses and are characterized by prominent features of type 2 immunity (such as high IgE production and eosinophilia).

[0005] Thymic stromal lymphopoietin (TSLP) and interleukin-13 (IL-13) are targets of soluble cytokines produced by stromal and / or immune cells (Ziegler and Artis, Nat Rev Immunol (2010) 11:289; Gieseck III et al., Nat Rev Immunol (2018) 18:62). Human TSLP and IL-13 drive unique, overlapping, and synergistic aspects of type 2 immunity, type 2 inflammatory diseases (such as asthma and atopic dermatitis), and a variety of immune diseases.

[0006] TSLP signaling is initiated via a heterodimeric receptor complex consisting of the thymic stromal lymphopoietin receptor (TSLPR) and the IL-7Rα chain (IL-7Rα). Similarly, IL-13 signaling is initiated via binding to a heterodimeric receptor complex consisting of the αIL-4 receptor (IL-4Rα) and the α-interleukin-13 receptor (IL-13R1α). The high affinity of IL-13 for IL-13R1 leads to the formation of its complex, which further increases the likelihood of heterodimer formation with IL-4Rα.

[0007] TSLP drives the maturation of dendritic cells, the development and proliferation of mast cells, and the initiation of other immune cells, such as basophils and innate lymphoid cells (ILC2). Similarly, IL-13 influences a range of immunopathologies, including epithelial barrier disruption, mucus production on muco-epithelial surfaces, airway remodeling, and the induction of eosinophil recruitment of chemokines, such as eosinophil chemokine. These mechanisms are important for the initiation and spread of type 2 inflammatory responses and for the development of a range of immunopathologies in diseases such as atopic dermatitis and asthma.

[0008] Currently, patients with moderate / severe asthma do not respond well to available standard of care treatments, including biologics such as the anti-IL4Rα monoclonal antibody Dupixent (dupixumab; marketed), anti-IL5 (marketed), and the anti-IgE monoclonal antibody Xolair (omalizumab; marketed), particularly in asthma patients with a low eosinophilic phenotype. Although antagonistic monoclonal antibodies against TSLP (terzolumab) and IL-13 (leregizumab) are currently undergoing clinical trials, there are no effective clinical development programs targeting both TSLP and IL-13. Dual targeting of TSLP and IL-13 with a single agent has the potential to confer efficacy in both low and high type 2 asthma, as well as atopic dermatitis, potentially in subgroups within these indications where monotherapy may not be fully effective. Therefore, there remains an unmet medical need for treatment of type 2 inflammatory diseases such as asthma and atopic dermatitis that is not only more effective but also convenient for patients.

[0009] This therapy may include targeting multiple disease factors, such as IL-13 and TSLP.

[0010] Targeting multiple disease factors can be achieved, for example, through the co-administration or combination of two separate biologics (e.g., antibodies binding to different therapeutic targets). However, from both a practical and commercial perspective, the co-administration or combination of separate biologics can be challenging. For instance, two injections of a single product present a more inconvenient and painful treatment regimen for patients, which can negatively impact adherence. Regarding a single injection of two separate products, providing a formulation that allows for acceptable viscosity and suitable stability of both products at the desired concentration may be difficult or impossible. Furthermore, co-administration and co-formulation require the production of two separate drugs, which can increase overall costs.

[0011] Bispecific antibodies capable of binding to two different antigens have been proposed as a strategy to address such limitations associated with the co-administration or combination of individual biologics (such as antibodies).

[0012] Various forms of bispecific antibody constructs have been proposed. For example, bispecific antibody forms may involve the chemical conjugation of two antibodies or fragments thereof (Brennan, M, et al., Science, 1985.229(4708): 81-83; Glennie, MJ, et al., J Immunol, 1987.139(7): 2367-2375).

[0013] However, drawbacks of this type of bispecific antibody include: high viscosity at high concentrations making subcutaneous administration challenging, for example; and the requirement for two variable domains to interact for specific, high-affinity binding per binding unit, which can impact peptide stability and production efficiency. This type of bispecific antibody may also potentially lead to chemistry, manufacturing, and control (CMC) problems related to light chain or heavy chain mismatches. 3. Summary of the Invention

[0015] In some embodiments, the present invention relates to peptides that simultaneously and specifically target IL-13 and TSLP, said peptides resulting in increased efficiency in modulating type 2 inflammatory responses in vitro compared to monospecific anti-IL-13 or anti-TSLP peptides. In some embodiments, said peptides are efficiently produced (e.g., in a microbial host). Furthermore, in some embodiments, such peptides exhibit limited reactivity to pre-existing antibodies in the subject to be treated (i.e., antibodies present in the subject prior to the first treatment with said antibody construct). In other embodiments, such peptides exhibit a sufficiently long half-life in the subject to be treated, allowing for convenient spacing of successive treatments.

[0016] In one embodiment, the present invention provides a polypeptide comprising or consisting of at least one immunoglobulin single variable domain (ISVD) that specifically binds to IL-13. In another embodiment, the present invention provides a polypeptide comprising or consisting of at least two ISVDs that specifically bind to IL-13, wherein the two ISVDs are optionally linked via peptide linkers. In one embodiment, the two ISVDs that specifically bind to IL-13 are different ISVDs. Furthermore, in one embodiment, the polypeptide also comprises one or more other groups, residues, portions, or binding units optionally linked via one or more peptide linkers, wherein the one or more other groups, residues, portions, or binding units provide an increased half-life to the polypeptide compared to a corresponding polypeptide without said one or more other groups, residues, portions, or binding units. For example, the binding unit may be an ISVD that binds to a (human) serum protein such as human serum albumin.

[0017] In another embodiment, the polypeptide of the present invention comprises or is composed of at least one ISVD that specifically binds to TSLP. In another embodiment, the polypeptide of the present invention comprises or is composed of at least two ISVDs that specifically bind to TSLP, wherein the two ISVDs are optionally linked via peptide linkers. In one embodiment, the two ISVDs that specifically bind to TSLP are different ISVDs. Furthermore, in one embodiment, the polypeptide also comprises one or more other groups, residues, portions, or binding units optionally linked via one or more peptide linkers, wherein the one or more other groups, residues, portions, or binding units provide an increased half-life to the polypeptide compared to a corresponding polypeptide without said one or more other groups, residues, portions, or binding units. For example, the binding unit may be an ISVD that binds to a (human) serum protein such as human serum albumin.

[0018] In another embodiment, the polypeptide of the present invention comprises or consists of at least four ISVDs, wherein at least two ISVDs specifically bind to IL-13 and at least two ISVDs specifically bind to TSLP. In one embodiment, the at least two ISVDs specifically binding to IL-13 specifically bind to human IL-13, and the at least two ISVDs specifically binding to TSLP specifically bind to human TSLP. In one embodiment, the at least two ISVDs specifically binding to IL-13 are different ISVDs, and the at least two ISVDs binding to TSLP are different ISVDs. In another embodiment, the polypeptide comprising or consists of at least four ISVDs further comprises one or more other groups, residues, portions, or binding units optionally linked via one or more peptide linkers, wherein the one or more other groups, residues, portions, or binding units provide an increased half-life to the polypeptide compared to a corresponding polypeptide without the one or more other groups, residues, portions, or binding units. For example, the binding unit may be an ISVD that binds to a (human) serum protein such as human serum albumin.

[0019] Also provided is a nucleic acid molecule capable of expressing the polypeptide of the present invention, a nucleic acid or carrier comprising the nucleic acid, and a composition comprising the polypeptide, the nucleic acid, or the carrier. In one embodiment, the composition is a pharmaceutical composition.

[0020] A host or host cell comprising a nucleic acid or vector encoding a polypeptide according to the present invention is also provided.

[0021] A method for generating a polypeptide according to the present invention is also provided, the method comprising at least the following steps:

[0022] a. Expressing a nucleic acid comprising a nucleotide sequence encoding a polypeptide of the present invention in a suitable host cell or host organism or in another suitable expression system; optionally followed by

[0023] b. Isolate and / or purify the polypeptide according to the technology of the present invention.

[0024] Furthermore, the present invention provides the polypeptide for use as a medicament, a composition comprising the polypeptide, or a composition comprising a nucleic acid or carrier containing a nucleotide sequence encoding the polypeptide. In one embodiment, the polypeptide or composition is used to treat inflammatory diseases, such as type 2 inflammatory diseases. In one embodiment, the type 2 inflammatory disease is selected from atopic dermatitis and asthma.

[0025] Additionally, a method for treating inflammatory diseases such as type 2 inflammatory disease is provided, wherein the method comprises administering a pharmaceutically active amount of a polypeptide or composition according to the technology of the invention to a subject in need. 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.

[0026] The invention also provides the use of the polypeptides or compositions of the present invention in the preparation of pharmaceutical compositions for treating inflammatory diseases such as type 2 inflammatory diseases. In one embodiment, the type 2 inflammatory disease is selected from atopic dermatitis and asthma.

[0027] Specifically, the present invention provides the following implementation schemes:

[0028] Implementation Scheme 1. A polypeptide for use as a drug, a composition comprising said polypeptide, or a composition comprising a nucleic acid containing a nucleotide sequence encoding said polypeptide, wherein said polypeptide comprises or is composed of at least one immunoglobulin single variable domain (ISVD), wherein said ISVD comprises three complementarity-determining regions (CDR1 to CDR3, respectively), and wherein said at least one ISVD comprises:

[0029] a) CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that differs from SEQ ID NO: 7 by 2 or 1 amino acids;

[0030] CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence differing from SEQ ID NO: 12 by 2 or 1 amino acids; and

[0031] CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence that differs from SEQ ID NO: 17 by 2 or 1 amino acids;

[0032] b) CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence that differs from SEQ ID NO: 8 by 2 or 1 amino acids;

[0033] CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence differing from SEQ ID NO: 13 by 2 or 1 amino acids; and

[0034] CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that differs from SEQ ID NO: 18 by 2 or 1 amino acids;

[0035] c) CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence that differs from SEQ ID NO: 9 by 2 or 1 amino acids;

[0036] CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence differing from SEQ ID NO: 14 by 2 or 1 amino acids; and

[0037] CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence differing from SEQ ID NO: 19 by 2 or 1 amino acids; or

[0038] d) CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence that differs from SEQ ID NO: 11 by 2 or 1 amino acids;

[0039] CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence differing from SEQ ID NO: 16 by 2 or 1 amino acids; and

[0040] CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence that differs from SEQ ID NO: 21 by 2 or 1 amino acids.

[0041] Implementation Scheme 2. The polypeptide or composition for the said use according to Implementation Scheme 1, wherein the at least one ISVD comprises:

[0042] a) CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7, CDR2 is the amino acid sequence of SEQ ID NO: 12 and CDR3 is the amino acid sequence of SEQ ID NO: 17;

[0043] b) CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8, CDR2 is the amino acid sequence of SEQ ID NO: 13 and CDR3 is the amino acid sequence of SEQ ID NO: 18;

[0044] c) CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 9, CDR2 is the amino acid sequence of SEQ ID NO: 14, and CDR3 is the amino acid sequence of SEQ ID NO: 19; or

[0045] d) CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 11, CDR2 is the amino acid sequence of SEQ ID NO: 16 and CDR3 is the amino acid sequence of SEQ ID NO: 21.

[0046] Implementation Scheme 3. A polypeptide or composition for the purpose according to any one of Implementation Schemes 1 or 2, wherein the amino acid sequence of the at least one ISVD comprises:

[0047] a) More than 90% sequence identity with SEQ ID NO: 2,

[0048] b) More than 90% sequence identity with SEQ ID NO: 3,

[0049] c) Sequence identity with SEQ ID NO: 4 exceeding 90% identity, or

[0050] d) Sequence identity with SEQ ID NO: 6, which is more than 90% identical.

[0051] Implementation Scheme 4. A polypeptide or composition for the purpose according to any one of Implementation Schemes 1 to 3, wherein the at least one ISVD comprises:

[0052] a) The amino acid sequence of SEQ ID NO: 2

[0053] b) The amino acid sequence of SEQ ID NO: 3

[0054] c) The amino acid sequence of SEQ ID NO: 4, or

[0055] d) The amino acid sequence of SEQ ID NO: 6.

[0056] Implementation Scheme 5. A polypeptide or composition for the purpose according to Implementation Scheme 1, wherein the polypeptide comprises or consists of at least two ISVDs, wherein each of the ISVDs comprises three complementarity-determining regions (CDR1 to CDR3, respectively), wherein the at least two ISVDs are optionally linked via one or more peptide linkers, and wherein:

[0057] a) The first ISVD and the second ISVD contain

[0058] i.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that differs from SEQ ID NO: 7 by 2 or 1 amino acids;

[0059] ii. CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence differing from SEQ ID NO: 12 by 2 or 1 amino acids; and

[0060] iii. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence that differs from SEQ ID NO: 17 by 2 or 1 amino acids;

[0061] b) The first ISVD and the second ISVD contain

[0062] i.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence that differs from SEQ ID NO: 8 by 2 or 1 amino acids;

[0063] ii. CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence differing from SEQ ID NO: 13 by 2 or 1 amino acids; and

[0064] iii. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that differs from SEQ ID NO: 18 by 2 or 1 amino acids;

[0065] c) The first ISVD contains

[0066] i.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that differs from SEQ ID NO: 7 by 2 or 1 amino acids;

[0067] ii. CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence differing from SEQ ID NO: 12 by 2 or 1 amino acids; and

[0068] iii. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence differing from SEQ ID NO: 17 by 2 or 1 amino acids; and

[0069] The second ISVD contains

[0070] iv.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence that differs from SEQ ID NO: 8 by 2 or 1 amino acids;

[0071] v.CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence differing from SEQ ID NO: 13 by 2 or 1 amino acids; and

[0072] vi. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that differs from SEQ ID NO: 18 by 2 or 1 amino acids;

[0073] d) The first ISVD contains

[0074] i.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence that differs from SEQ ID NO: 8 by 2 or 1 amino acids;

[0075] ii. CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence differing from SEQ ID NO: 13 by 2 or 1 amino acids; and

[0076] iii. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence differing from SEQ ID NO: 18 by 2 or 1 amino acids; and

[0077] The second ISVD contains

[0078] iv.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that differs from SEQ ID NO: 7 by 2 or 1 amino acids;

[0079] v.CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence differing from SEQ ID NO: 12 by 2 or 1 amino acids; and

[0080] vi. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence that differs from SEQ ID NO: 17 by 2 or 1 amino acids;

[0081] e) The first ISVD contains

[0082] i.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence that differs from SEQ ID NO: 11 by 2 or 1 amino acids;

[0083] ii. CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence differing from SEQ ID NO: 16 by 2 or 1 amino acids; and

[0084] iii. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence differing from SEQ ID NO: 21 by 2 or 1 amino acids; and

[0085] The second ISVD contains

[0086] iv.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence that differs from SEQ ID NO: 9 by 2 or 1 amino acids;

[0087] v.CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence differing from SEQ ID NO: 14 by 2 or 1 amino acids; and

[0088] vi. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence differing from SEQ ID NO: 19 by 2 or 1 amino acids; or

[0089] f) The first ISVD contains

[0090] i.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence that differs from SEQ ID NO: 9 by 2 or 1 amino acids;

[0091] ii. CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence differing from SEQ ID NO: 14 by 2 or 1 amino acids; and

[0092] iii. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence differing from SEQ ID NO: 19 by 2 or 1 amino acids; and / or

[0093] The second ISVD contains

[0094] iv.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence that differs from SEQ ID NO: 11 by 2 or 1 amino acids;

[0095] v.CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence differing from SEQ ID NO: 16 by 2 or 1 amino acids; and

[0096] vi. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence that differs from SEQ ID NO: 21 by 2 or 1 amino acids.

[0097] The order of the ISVDs indicates their perceived relative positions from the N-terminus to the C-terminus of the polypeptide.

[0098] Implementation Scheme 6. The polypeptide or composition for the said use according to Implementation Scheme 5, wherein:

[0099] a) The first ISVD and the second ISVD comprise CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7, CDR2 is the amino acid sequence of SEQ ID NO: 12 and CDR3 is the amino acid sequence of SEQ ID NO: 17;

[0100] b) The first ISVD and the second ISVD comprise CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8, CDR2 is the amino acid sequence of SEQ ID NO: 13 and CDR3 is the amino acid sequence of SEQ ID NO: 18;

[0101] c) The first ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7, CDR2 is the amino acid sequence of SEQ ID NO: 12, and CDR3 is the amino acid sequence of SEQ ID NO: 17; and the second ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8, CDR2 is the amino acid sequence of SEQ ID NO: 13, and CDR3 is the amino acid sequence of SEQ ID NO: 18;

[0102] d) The first ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8, CDR2 is the amino acid sequence of SEQ ID NO: 13, and CDR3 is the amino acid sequence of SEQ ID NO: 18; and the second ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7, CDR2 is the amino acid sequence of SEQ ID NO: 12, and CDR3 is the amino acid sequence of SEQ ID NO: 17;

[0103] e) The first ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 11, CDR2 is the amino acid sequence of SEQ ID NO: 16, and CDR3 is the amino acid sequence of SEQ ID NO: 21; and the second ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 9, CDR2 is the amino acid sequence of SEQ ID NO: 14, and CDR3 is the amino acid sequence of SEQ ID NO: 19; or

[0104] f) The first ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 9, CDR2 is the amino acid sequence of SEQ ID NO: 14, and CDR3 is the amino acid sequence of SEQ ID NO: 19; and the second ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 11, CDR2 is the amino acid sequence of SEQ ID NO: 16, and CDR3 is the amino acid sequence of SEQ ID NO: 21.

[0105] Implementation Scheme 7. A polypeptide or composition for the said use according to any one of Implementation Schemes 5 or 6, wherein:

[0106] a) The amino acid sequences of the first ISVD and the second ISVD contain more than 90% sequence identity with SEQ ID NO: 2.

[0107] b) The amino acid sequences of the first ISVD and the second ISVD contain more than 90% sequence identity with SEQ ID NO: 3.

[0108] c) The amino acid sequence of the first ISVD contains more than 90% sequence identity with SEQ ID NO: 2, and the second ISVD contains more than 90% sequence identity with SEQ ID NO: 3.

[0109] d) The amino acid sequence of the first ISVD contains more than 90% sequence identity with SEQ ID NO: 3, and the second ISVD contains more than 90% sequence identity with SEQ ID NO: 2.

[0110] e) The amino acid sequence of the first ISVD contains more than 90% sequence identity with SEQ ID NO: 6, and the second ISVD contains more than 90% sequence identity with SEQ ID NO: 4.

[0111] 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.

[0112] Implementation Scheme 8. A polypeptide or composition for the said use according to any one of Implementation Schemes 5 to 7, wherein:

[0113] a) The first ISVD and the second ISVD contain the amino acid sequence of SEQ ID NO: 2.

[0114] b) The first ISVD and the second ISVD contain the amino acid sequence of SEQ ID NO: 3.

[0115] 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.

[0116] 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.

[0117] 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

[0118] 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.

[0119] Implementation Scheme 9. The polypeptide according to any one of Implementation Schemes 5 to 8, wherein the polypeptide comprises or is composed of the following:

[0120] a) The amino acid sequence of SEQ ID NO: 148

[0121] b) The amino acid sequence of SEQ ID NO: 149

[0122] c) The amino acid sequence of SEQ ID NO: 150

[0123] d) The amino acid sequence of SEQ ID NO: 151

[0124] e) The amino acid sequence of SEQ ID NO: 152

[0125] f) The amino acid sequence of SEQ ID NO: 153

[0126] g) The amino acid sequence of SEQ ID NO: 154

[0127] h) The amino acid sequence of SEQ ID NO: 155

[0128] i) The amino acid sequence of SEQ ID NO: 156

[0129] j) The amino acid sequence of SEQ ID NO: 157

[0130] k) The amino acid sequence of SEQ ID NO: 158, or

[0131] l) The amino acid sequence of SEQ ID NO: 159.

[0132] Implementation Scheme 10. A polypeptide or composition for the purpose according to any one of Implementation Schemes 1 or 5, wherein the polypeptide comprises or consists of at least four ISVDs, wherein each of the ISVDs comprises three complementarity-determining regions (CDR1 to CDR3, respectively), wherein the at least four ISVDs are optionally linked via one or more peptide linkers, and wherein:

[0133] a) The first ISVD contains

[0134] i.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that differs from SEQ ID NO: 7 by 2 or 1 amino acids;

[0135] ii. CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence differing from SEQ ID NO: 12 by 2 or 1 amino acids; and

[0136] iii. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence that differs from SEQ ID NO: 17 by 2 or 1 amino acids;

[0137] b) The second ISVD contains

[0138] iv.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence that differs from SEQ ID NO: 8 by 2 or 1 amino acids;

[0139] v.CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence differing from SEQ ID NO: 13 by 2 or 1 amino acids; and

[0140] vi. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that differs from SEQ ID NO: 18 by 2 or 1 amino acids;

[0141] c) The third ISVD contains

[0142] vii.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence that differs from SEQ ID NO: 9 by 2 or 1 amino acids;

[0143] viii. CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence differing from SEQ ID NO: 14 by 2 or 1 amino acids; and

[0144] ix.CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence differing from SEQ ID NO: 19 by 2 or 1 amino acids; and

[0145] d) The fourth ISVD contains

[0146] x.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence that differs from SEQ ID NO: 11 by 2 or 1 amino acids;

[0147] xi.CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence differing from SEQ ID NO: 16 by 2 or 1 amino acids; and

[0148] xii.CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence that differs from SEQ ID NO: 21 by 2 or 1 amino acids.

[0149] The order of the ISVDs indicates their perceived relative positions from the N-terminus to the C-terminus of the polypeptide.

[0150] Implementation Scheme 11. A composition for the purpose according to any one of Implementation Schemes 1 to 10, wherein the composition is a pharmaceutical composition, the pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally comprising one or more additional pharmaceutically active peptides and / or compounds.

[0151] Implementation Scheme 12. A polypeptide or composition for the said use according to Implementation Scheme 10 or 11, wherein:

[0152] a) The first ISVD comprises CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7, CDR2 is the amino acid sequence of SEQ ID NO: 12 and CDR3 is the amino acid sequence of SEQ ID NO: 17;

[0153] b) The second ISVD comprises CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8, CDR2 is the amino acid sequence of SEQ ID NO: 13 and CDR3 is the amino acid sequence of SEQ ID NO: 18;

[0154] c) The third ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 9, CDR2 is the amino acid sequence of SEQ ID NO: 14, and CDR3 is the amino acid sequence of SEQ ID NO: 19; and

[0155] d) The fourth ISVD comprises CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 11, CDR2 is the amino acid sequence of SEQ ID NO: 16 and CDR3 is the amino acid sequence of SEQ ID NO: 21.

[0156] Implementation Scheme 13. A polypeptide or composition for the said use according to any one of Implementation Schemes 10 to 12, wherein:

[0157] a) The amino acid sequence of the first ISVD contains more than 90% sequence identity with SEQ ID NO: 2;

[0158] b) The amino acid sequence of the second ISVD contains more than 90% sequence identity with SEQ ID NO: 3;

[0159] c) The amino acid sequence of the third ISVD contains more than 90% sequence identity with SEQ ID NO: 4; and

[0160] d) The amino acid sequence of the fourth ISVD contains more than 90% sequence identity with SEQ ID NO: 6.

[0161] Implementation Scheme 14. A polypeptide or composition for the said use according to any one of Implementation Schemes 10 to 13, wherein:

[0162] a) The first ISVD contains the amino acid sequence of SEQ ID NO: 2;

[0163] b) The second ISVD contains the amino acid sequence of SEQ ID NO: 3;

[0164] c) The third ISVD contains the amino acid sequence of SEQ ID NO: 4; and

[0165] d) The fourth ISVD contains the amino acid sequence of SEQ ID NO: 6.

[0166] Implementation Scheme 15. A polypeptide or composition for the purpose according to any one of Implementation Schemes 1 to 14, wherein the polypeptide further comprises one or more other groups, residues, portions or binding units optionally linked via one or more peptide linkers, wherein the one or more other groups, residues, portions or binding units provide an increased half-life to the polypeptide compared to a corresponding polypeptide without the one or more other groups, residues, portions or binding units.

[0167] Implementation Scheme 16. A polypeptide or composition for the purpose according to Implementation Scheme 15, wherein the one or more other groups, residues, portions or binding units providing an increased half-life for the polypeptide are selected from polyethylene glycol molecules, serum proteins or fragments thereof, binding units that can bind to serum proteins, Fc portions and small proteins or peptides that can bind to serum proteins.

[0168] Implementation Scheme 17. A polypeptide or composition for the purpose according to any one of Implementation Schemes 15 to 16, wherein the one or more other binding units providing an increased half-life for the polypeptide are selected from binding units that can bind to serum albumin (such as human serum albumin) or serum immunoglobulin (such as IgG).

[0169] Implementation Scheme 18. A polypeptide or composition for the purpose according to Implementation Scheme 17, wherein the binding unit providing the increased half-life of the polypeptide is an ISVD that can bind to human serum albumin.

[0170] Implementation Scheme 19. A polypeptide or composition for the said use according to Implementation Scheme 18, wherein the ISVD bound to human serum albumin comprises

[0171] i.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that differs from SEQ ID NO: 10 by 2 or 1 amino acids;

[0172] ii. CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence differing from SEQ ID NO: 15 by 2 or 1 amino acids; and

[0173] iii. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence that differs from SEQ ID NO: 20 by 2 or 1 amino acids.

[0174] Implementation Scheme 20. A polypeptide or composition for the purpose according to any one of Implementation Schemes 18 to 19, wherein the ISVD that binds to human serum albumin comprises CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 10, CDR2 is the amino acid sequence of SEQ ID NO: 15 and CDR3 is the amino acid sequence of SEQ ID NO: 20.

[0175] Implementation Scheme 21. A polypeptide or composition for the purpose according to any one of Implementation Schemes 18 to 20, wherein the amino acid sequence of the ISVD that binds to human serum albumin contains more than 90% sequence identity with SEQ ID NO: 5.

[0176] Implementation Scheme 22. A polypeptide or composition for the purpose according to any one of Implementation Schemes 18 to 21, wherein the ISVD that binds to human serum albumin comprises the amino acid sequence of SEQ ID NO: 5.

[0177] Implementation Scheme 23. A polypeptide or composition for the purpose according to any one of Implementation Schemes 10 to 22, wherein the amino acid sequence of the polypeptide contains more than 90% sequence identity with SEQ ID NO: 1.

[0178] Implementation Scheme 24. A polypeptide or composition for the purpose according to any one of Implementation Schemes 10 to 23, wherein the polypeptide comprises or is composed of the amino acid sequence of SEQ ID NO: 1.

[0179] Implementation Scheme 25. A polypeptide or composition according to any one of Implementation Schemes 1 to 24 for the purpose of said use, which is used to treat inflammatory diseases, such as type 2 inflammatory diseases.

[0180] Implementation Scheme 26. The polypeptide or composition for the purpose according to Implementation Scheme 25, wherein the type 2 inflammatory disease is selected from asthma and atopic dermatitis.

[0181] Implementation Scheme 27. A polypeptide comprising at least one immunoglobulin single variable domain (ISVD) or thereof, wherein the ISVD comprises three complementarity-determining regions (CDR1 to CDR3, respectively); and wherein the at least one ISVD comprises:

[0182] a) CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that differs from SEQ ID NO: 7 by 2 or 1 amino acids;

[0183] CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence differing from SEQ ID NO: 12 by 2 or 1 amino acids; and

[0184] CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence that differs from SEQ ID NO: 17 by 2 or 1 amino acids;

[0185] b) CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence that differs from SEQ ID NO: 8 by 2 or 1 amino acids;

[0186] CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence differing from SEQ ID NO: 13 by 2 or 1 amino acids; and

[0187] CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that differs from SEQ ID NO: 18 by 2 or 1 amino acids;

[0188] c) CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence that differs from SEQ ID NO: 9 by 2 or 1 amino acids;

[0189] CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence differing from SEQ ID NO: 14 by 2 or 1 amino acids; and

[0190] CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence differing from SEQ ID NO: 19 by 2 or 1 amino acids; or

[0191] d) CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence that differs from SEQ ID NO: 11 by 2 or 1 amino acids;

[0192] CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence differing from SEQ ID NO: 16 by 2 or 1 amino acids; and

[0193] CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence that differs from SEQ ID NO: 21 by 2 or 1 amino acids.

[0194] Implementation Scheme 28. The polypeptide according to Implementation Scheme 27, wherein the at least one ISVD comprises:

[0195] a) CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7, CDR2 is the amino acid sequence of SEQ ID NO: 12 and CDR3 is the amino acid sequence of SEQ ID NO: 17;

[0196] b) CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8, CDR2 is the amino acid sequence of SEQ ID NO: 13 and CDR3 is the amino acid sequence of SEQ ID NO: 18;

[0197] c) CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 9, CDR2 is the amino acid sequence of SEQ ID NO: 14, and CDR3 is the amino acid sequence of SEQ ID NO: 19; or

[0198] d) CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 11, CDR2 is the amino acid sequence of SEQ ID NO: 16 and CDR3 is the amino acid sequence of SEQ ID NO: 21.

[0199] Implementation Scheme 29. The polypeptide according to any one of Implementation Schemes 27 or 28, wherein the amino acid sequence of the at least one ISVD comprises:

[0200] a) More than 90% sequence identity with SEQ ID NO: 2,

[0201] b) More than 90% sequence identity with SEQ ID NO: 3,

[0202] c) Sequence identity with SEQ ID NO: 4 exceeding 90% identity, or

[0203] d) Sequence identity with SEQ ID NO: 6, which is more than 90% identical.

[0204] Implementation Scheme 30. The polypeptide according to any one of Implementation Schemes 27 to 29, wherein the at least one ISVD comprises:

[0205] a) The amino acid sequence of SEQ ID NO: 2

[0206] b) The amino acid sequence of SEQ ID NO: 3

[0207] c) The amino acid sequence of SEQ ID NO: 4, or

[0208] d) The amino acid sequence of SEQ ID NO: 6.

[0209] Implementation Scheme 31. The polypeptide according to Implementation Scheme 1 or 27, wherein the polypeptide comprises or is composed of at least two ISVDs, wherein each of the ISVDs comprises three complementarity-determining regions (CDR1 to CDR3, respectively), wherein the at least two ISVDs are optionally linked via one or more peptide linkers, and wherein:

[0210] a) The first ISVD and the second ISVD contain

[0211] i.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that differs from SEQ ID NO: 7 by 2 or 1 amino acids;

[0212] ii. CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence differing from SEQ ID NO: 12 by 2 or 1 amino acids; and

[0213] iii. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence that differs from SEQ ID NO: 17 by 2 or 1 amino acids;

[0214] b) The first ISVD and the second ISVD contain

[0215] i.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence that differs from SEQ ID NO: 8 by 2 or 1 amino acids;

[0216] ii. CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence differing from SEQ ID NO: 13 by 2 or 1 amino acids; and

[0217] iii. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that differs from SEQ ID NO: 18 by 2 or 1 amino acids;

[0218] c) The first ISVD contains

[0219] i.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that differs from SEQ ID NO: 7 by 2 or 1 amino acids;

[0220] ii. CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence differing from SEQ ID NO: 12 by 2 or 1 amino acids; and

[0221] iii. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence differing from SEQ ID NO: 17 by 2 or 1 amino acids; and

[0222] The second ISVD contains

[0223] iv.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence that differs from SEQ ID NO: 8 by 2 or 1 amino acids;

[0224] v.CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence differing from SEQ ID NO: 13 by 2 or 1 amino acids; and

[0225] vi. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that differs from SEQ ID NO: 18 by 2 or 1 amino acids;

[0226] d) The first ISVD contains

[0227] i.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence that differs from SEQ ID NO: 8 by 2 or 1 amino acids;

[0228] ii. CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence differing from SEQ ID NO: 13 by 2 or 1 amino acids; and

[0229] iii. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence differing from SEQ ID NO: 18 by 2 or 1 amino acids; and

[0230] The second ISVD contains

[0231] iv.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that differs from SEQ ID NO: 7 by 2 or 1 amino acids;

[0232] v.CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence differing from SEQ ID NO: 12 by 2 or 1 amino acids; and

[0233] vi. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence that differs from SEQ ID NO: 17 by 2 or 1 amino acids;

[0234] e) The first ISVD contains

[0235] i.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence that differs from SEQ ID NO: 11 by 2 or 1 amino acids;

[0236] ii. CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence differing from SEQ ID NO: 16 by 2 or 1 amino acids; and

[0237] iii. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence differing from SEQ ID NO: 21 by 2 or 1 amino acids; and

[0238] The second ISVD contains

[0239] iv.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence that differs from SEQ ID NO: 9 by 2 or 1 amino acids;

[0240] v.CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence differing from SEQ ID NO: 14 by 2 or 1 amino acids; and

[0241] vi. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence differing from SEQ ID NO: 19 by 2 or 1 amino acids; or

[0242] f) The first ISVD contains

[0243] i.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence that differs from SEQ ID NO: 9 by 2 or 1 amino acids;

[0244] ii. CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence differing from SEQ ID NO: 14 by 2 or 1 amino acids; and

[0245] iii. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence differing from SEQ ID NO: 19 by 2 or 1 amino acids; and

[0246] The second ISVD contains

[0247] iv.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence that differs from SEQ ID NO: 11 by 2 or 1 amino acids;

[0248] v.CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence differing from SEQ ID NO: 16 by 2 or 1 amino acids; and

[0249] vi. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence that differs from SEQ ID NO: 21 by 2 or 1 amino acids.

[0250] The order of the ISVDs indicates their perceived relative positions from the N-terminus to the C-terminus of the polypeptide.

[0251] Implementation Scheme 32. The polypeptide according to Implementation Scheme 31, wherein:

[0252] a) The first ISVD and the second ISVD comprise CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7, CDR2 is the amino acid sequence of SEQ ID NO: 12 and CDR3 is the amino acid sequence of SEQ ID NO: 17;

[0253] b) The first ISVD and the second ISVD comprise CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8, CDR2 is the amino acid sequence of SEQ ID NO: 13 and CDR3 is the amino acid sequence of SEQ ID NO: 18;

[0254] c) The first ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7, CDR2 is the amino acid sequence of SEQ ID NO: 12, and CDR3 is the amino acid sequence of SEQ ID NO: 17; and the second ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8, CDR2 is the amino acid sequence of SEQ ID NO: 13, and CDR3 is the amino acid sequence of SEQ ID NO: 18;

[0255] d) The first ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8, CDR2 is the amino acid sequence of SEQ ID NO: 13, and CDR3 is the amino acid sequence of SEQ ID NO: 18; and the second ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7, CDR2 is the amino acid sequence of SEQ ID NO: 12, and CDR3 is the amino acid sequence of SEQ ID NO: 17;

[0256] e) The first ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 11, CDR2 is the amino acid sequence of SEQ ID NO: 16, and CDR3 is the amino acid sequence of SEQ ID NO: 21; and the second ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 9, CDR2 is the amino acid sequence of SEQ ID NO: 14, and CDR3 is the amino acid sequence of SEQ ID NO: 19; or

[0257] f) The first ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 9, CDR2 is the amino acid sequence of SEQ ID NO: 14, and CDR3 is the amino acid sequence of SEQ ID NO: 19; and the second ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 11, CDR2 is the amino acid sequence of SEQ ID NO: 16, and CDR3 is the amino acid sequence of SEQ ID NO: 21.

[0258] Implementation Scheme 33. The polypeptide according to any one of Implementation Schemes 31 or 32, wherein:

[0259] a) The amino acid sequences of the first ISVD and the second ISVD contain more than 90% sequence identity with SEQ ID NO: 2.

[0260] b) The amino acid sequences of the first ISVD and the second ISVD contain more than 90% sequence identity with SEQ ID NO: 3.

[0261] c) The amino acid sequence of the first ISVD contains more than 90% sequence identity with SEQ ID NO: 2, and the second ISVD contains more than 90% sequence identity with SEQ ID NO: 3.

[0262] d) The amino acid sequence of the first ISVD contains more than 90% sequence identity with SEQ ID NO: 3, and the second ISVD contains more than 90% sequence identity with SEQ ID NO: 2.

[0263] e) The amino acid sequence of the first ISVD contains more than 90% sequence identity with SEQ ID NO: 6, and the second ISVD contains more than 90% sequence identity with SEQ ID NO: 4.

[0264] 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.

[0265] Implementation Scheme 34. The polypeptide according to any one of Implementation Schemes 31 to 33, wherein:

[0266] a) The first ISVD and the second ISVD contain the amino acid sequence of SEQ ID NO: 2.

[0267] b) The first ISVD and the second ISVD contain the amino acid sequence of SEQ ID NO: 3.

[0268] 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.

[0269] 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.

[0270] 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

[0271] 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.

[0272] Implementation Scheme 35. The polypeptide according to any one of Implementation Schemes 31 to 34, wherein the polypeptide comprises or is composed of the following:

[0273] a) The amino acid sequence of SEQ ID NO: 148

[0274] b) The amino acid sequence of SEQ ID NO: 149

[0275] c) The amino acid sequence of SEQ ID NO: 150

[0276] d) The amino acid sequence of SEQ ID NO: 151

[0277] e) The amino acid sequence of SEQ ID NO: 152

[0278] f) The amino acid sequence of SEQ ID NO: 153

[0279] g) The amino acid sequence of SEQ ID NO: 154

[0280] h) The amino acid sequence of SEQ ID NO: 155

[0281] i) The amino acid sequence of SEQ ID NO: 156

[0282] j) The amino acid sequence of SEQ ID NO: 157

[0283] k) The amino acid sequence of SEQ ID NO: 158, or

[0284] l) The amino acid sequence of SEQ ID NO: 159.

[0285] Implementation Scheme 36. A polypeptide according to any one of Implementation Schemes 27 or 31, wherein the polypeptide comprises or is composed of at least four ISVDs, wherein each of the ISVDs comprises three complementarity-determining regions (CDR1 to CDR3, respectively), wherein the at least four ISVDs are optionally linked via one or more peptide linkers, and wherein:

[0286] a) The first ISVD contains

[0287] i.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that differs from SEQ ID NO: 7 by 2 or 1 amino acids;

[0288] ii. CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence differing from SEQ ID NO: 12 by 2 or 1 amino acids; and

[0289] iii. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence that differs from SEQ ID NO: 17 by 2 or 1 amino acids;

[0290] b) The second ISVD contains

[0291] iv.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence that differs from SEQ ID NO: 8 by 2 or 1 amino acids;

[0292] v.CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence differing from SEQ ID NO: 13 by 2 or 1 amino acids; and

[0293] vi. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that differs from SEQ ID NO: 18 by 2 or 1 amino acids;

[0294] c) The third ISVD contains

[0295] vii.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence that differs from SEQ ID NO: 9 by 2 or 1 amino acids;

[0296] viii. CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence differing from SEQ ID NO: 14 by 2 or 1 amino acids; and

[0297] ix.CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence differing from SEQ ID NO: 19 by 2 or 1 amino acids; and

[0298] d) The fourth ISVD contains

[0299] x.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence that differs from SEQ ID NO: 11 by 2 or 1 amino acids;

[0300] xi.CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence differing from SEQ ID NO: 16 by 2 or 1 amino acids; and

[0301] xii.CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence that differs from SEQ ID NO: 21 by 2 or 1 amino acids.

[0302] The order of the ISVDs indicates their perceived relative positions from the N-terminus to the C-terminus of the polypeptide.

[0303] Implementation Scheme 37. The polypeptide according to Implementation Scheme 36, wherein:

[0304] a) The first ISVD comprises CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7, CDR2 is the amino acid sequence of SEQ ID NO: 12 and CDR3 is the amino acid sequence of SEQ ID NO: 17;

[0305] b) The second ISVD comprises CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8, CDR2 is the amino acid sequence of SEQ ID NO: 13 and CDR3 is the amino acid sequence of SEQ ID NO: 18;

[0306] c) The third ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 9, CDR2 is the amino acid sequence of SEQ ID NO: 14, and CDR3 is the amino acid sequence of SEQ ID NO: 19; and

[0307] d) The fourth ISVD comprises CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 11, CDR2 is the amino acid sequence of SEQ ID NO: 16 and CDR3 is the amino acid sequence of SEQ ID NO: 21.

[0308] Implementation Scheme 38. The polypeptide according to any one of Implementation Schemes 36 or 37, wherein:

[0309] a) The amino acid sequence of the first ISVD contains more than 90% sequence identity with SEQ ID NO: 2;

[0310] b) The amino acid sequence of the second ISVD contains more than 90% sequence identity with SEQ ID NO: 3;

[0311] c) The amino acid sequence of the third ISVD contains more than 90% sequence identity with SEQ ID NO: 4; and

[0312] d) The amino acid sequence of the fourth ISVD contains more than 90% sequence identity with SEQ ID NO: 6.

[0313] Implementation Scheme 39. The polypeptide according to any one of Implementation Schemes 36 to 38, wherein:

[0314] a) The first ISVD contains the amino acid sequence of SEQ ID NO: 2;

[0315] b) The second -ISVD contains the amino acid sequence of SEQ ID NO: 3;

[0316] c) The third ISVD contains the amino acid sequence of SEQ ID NO: 4; and

[0317] d) The fourth ISVD contains the amino acid sequence of SEQ ID NO: 6.

[0318] Implementation Scheme 40. A polypeptide according to any one of Implementation Schemes 27 to 39, wherein the polypeptide further comprises one or more other groups, residues, portions or binding units optionally linked via one or more peptide linkers, wherein the one or more other groups, residues, portions or binding units provide an increased half-life to the polypeptide compared to a corresponding polypeptide without the one or more other groups, residues, portions or binding units.

[0319] Implementation Scheme 41. The polypeptide according to Implementation Scheme 40, wherein the one or more other groups, residues, portions or binding units providing the polypeptide with an increased half-life are selected from polyethylene glycol molecules, serum proteins or fragments thereof, binding units that can bind to serum proteins, Fc portions and small proteins or peptides that can bind to serum proteins.

[0320] Implementation Scheme 42. The polypeptide according to any one of Implementation Schemes 40 to 41, wherein the one or more other binding units providing the increased half-life of the polypeptide are selected from binding units that can bind to serum albumin (such as human serum albumin) or serum immunoglobulin (such as IgG).

[0321] Implementation Scheme 43. The polypeptide according to Implementation Scheme 42, wherein the binding unit providing the increased half-life of the polypeptide is an ISVD that can bind to human serum albumin.

[0322] Implementation Scheme 44. The polypeptide according to Implementation Scheme 43, wherein the ISVD bound to human serum albumin comprises

[0323] i.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that differs from SEQ ID NO: 10 by 2 or 1 amino acids;

[0324] ii. CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence differing from SEQ ID NO: 15 by 2 or 1 amino acids; and

[0325] iii. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence that differs from SEQ ID NO: 20 by 2 or 1 amino acids.

[0326] Implementation Scheme 45. The polypeptide according to any one of Implementation Schemes 43 to 44, wherein the ISVD bound to human serum albumin comprises CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 10, CDR2 is the amino acid sequence of SEQ ID NO: 15 and CDR3 is the amino acid sequence of SEQ ID NO: 20.

[0327] Implementation Scheme 46. The polypeptide according to any one of Implementation Schemes 43 to 45, wherein the amino acid sequence of the ISVD that binds to human serum albumin contains more than 90% sequence identity with SEQ ID NO: 5.

[0328] Implementation Scheme 47. The polypeptide according to any one of Implementation Schemes 43 to 46, wherein the ISVD that binds to human serum albumin comprises the amino acid sequence of SEQ ID NO: 5.

[0329] Implementation Scheme 48. The polypeptide according to any one of Implementation Schemes 36 to 47, wherein the amino acid sequence of the polypeptide contains more than 90% sequence identity with SEQ ID NO: 1.

[0330] Implementation Scheme 49. The polypeptide according to any one of Implementation Schemes 36 to 48, wherein the polypeptide comprises or is composed of the amino acid sequence of SEQ ID NO: 1.

[0331] Implementation Scheme 50. A nucleic acid comprising a nucleotide sequence encoding a polypeptide according to any one of Implementation Schemes 27 to 49 or a polypeptide according to Implementation Schemes 36 to 49.

[0332] Implementation Scheme 51. A host or host cell comprising the nucleic acid described in Implementation Scheme 50.

[0333] Implementation Scheme 52. A method for generating a polypeptide according to any one of Implementation Schemes 27 to 49 or a polypeptide according to Implementation Schemes 36 to 49, the method comprising at least the following steps:

[0334] a) Expressing the nucleic acid as described in embodiment 50 in a suitable host cell or host organism or in another suitable expression system; optionally followed by:

[0335] b) Isolate and / or purify the polypeptide according to any one of embodiments 27 to 49 or the polypeptide according to embodiments 36 to 49.

[0336] Implementation Scheme 53. A composition comprising at least one polypeptide according to any one of Implementation Schemes 27 to 49, or at least one polypeptide according to Implementation Schemes 36 to 49, or a nucleic acid according to Implementation Scheme 50.

[0337] Implementation Scheme 54. The composition according to Implementation Scheme 53, wherein the composition is a pharmaceutical composition, the pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally comprising one or more additional pharmaceutically active peptides and / or compounds.

[0338] Implementation Scheme 55. A method for treating an inflammatory disease such as type 2 inflammatory disease, wherein the method comprises administering to a subject in need an active amount of a polypeptide according to any one of Implementation Schemes 27 to 49 or according to Implementation Schemes 36 to 49 or a composition according to any one of Implementation Schemes 53 to 54.

[0339] Implementation Scheme 56. The method according to Implementation Scheme 55, wherein the type 2 inflammatory disease is selected from asthma and atopic dermatitis.

[0340] Implementation Scheme 57. Use of the polypeptide described in any one of Implementation Schemes 27 to 49 or 36 to 49, or the composition described in any one of Implementation Schemes 53 to 54, in the preparation of a pharmaceutical composition for treating inflammatory diseases such as type 2 inflammatory diseases.

[0341] Implementation Scheme 58. Use of the polypeptide or composition according to Implementation Scheme 57, wherein the type 2 inflammatory disease is selected from asthma and atopic dermatitis. 4. Attached Figure Descriptions

[0343] Figure 1 The image shows a sensory map combining IL13 and HSA with F027400161 captured via TSLP.

[0344] Figure 2 V HH F027400161 and reference compounds anti-hIL-13mAb1 and anti-hIL-13mAb2 inhibited IL13 in humans, cynomolgus monkeys and rhesus monkeys in an eosinophil chemokine release assay.

[0345] Figure 3 The inhibition of human and cynomolgus monkey IL13 by F027400161 and reference compounds anti-hIL-13mAb1 and anti-hIL-13mAb2 in SEAP reporter assay.

[0346] Figure 4 F027400161 and the reference compound antihTSLP mAb1 inhibited the proliferation of BaF3 cells induced by TSLP in humans and cynomolgus monkeys. IRR00096 was the negative control Nb.

[0347] Figure 5 The box plot shows the binding of pre-existing antibodies present in 96 human serum samples to F027400161, 163, and 164, compared to the control F027301186.

[0348] Figure 6 A profile of dose-response inhibition of TSLP-induced CCL17 responses in human DCs using F-27400161 (also known as F027400161) and the reference antibody anti-hTSLP reference mAb1. Enriched human DCs were treated with 4 ng / mL TSLP and incubated for 36 h with eight concentrations of ISVD constructs and anti-hTSLP reference mAb1. CCL17 concentrations in freshly collected supernatants were measured by ELISA. IC5050 The values ​​were calculated unconstrained in Graphpad Prism 8.0 via nonlinear regression (log inhibitors versus response-variable slope four-parameter least squares fit). Data are expressed as mean ± standard error (SEM) of the mean from all eight donors in eight individual experimental combinations.

[0349] Figure 7 The dose-inhibitory response of F-27400161 (also known as F027400161) and the reference anti-hIL-13 reference mAb1 and anti-hTSLP reference mAb1 to synergistic IL-13 and TSLP-induced CCL17 in human PBMCs was investigated. Healthy donor PBMCs were stimulated with 0.5 ng / mL recombinant IL-13 + TSLP and cultured for 20 h with 10 doses of ISVD construct (Nb) and the reference. CCL17 concentration in cell culture supernatant was measured using the MSD V-Plex kit. 50 The values ​​were calculated unconstrained in Graphpad Prism 8.0 via nonlinear regression (log inhibitors versus response-variable slope four-parameter least squares fit). Data are expressed as mean ± standard error (SEM) of all donors from 8 individual experimental combinations.

[0350] Figure 8 The dose-inhibitory response of F-27400161 (also known as F027400161) and reference antibodies anti-hIL-13 reference mAb1 and anti-hTSLP reference mAb1 to 5 ng / mL IL-13 and TSLP-induced CCL17 in human PBMCs was investigated. Healthy donor PBMCs were stimulated with 5 ng / mL recombinant IL-13 + TSLP and cultured for 20 h with 10 doses of ISVD construct (Nb) and reference antibody. The CCL17 concentration in freshly collected supernatant was measured using the MSD V-Plex kit. IC50 50 The values ​​were calculated unconstrained in Graphpad Prism 8.0 via nonlinear regression (log inhibitors versus response-variable slope four-parameter least squares fit). Data are expressed as mean ± standard error (SEM) of all donors from 8 individual experimental combinations.

[0351] Figure 9The inhibition profile of Der P-induced IL-5, CCL17, and CCL26 production from human PBMCs by allergen Der P in a ternary culture assay using F-27400161 (also known as F027400161) and reference antibodies anti-hIL-13 reference mAb1 and anti-hTSLP reference mAb1. Normal donor PBMCs co-cultured with MRC5 fibroblasts and A549 epithelial cells were stimulated with 3 mg / mL Der P and cultured for 6 days in 24-well plates at 37°C with 11.1 nM ISVD, anti-hIL-13 reference mAb1, or anti-hTSLP reference mAb1. IL-5, CCL17, and CCL26 concentrations in freshly collected supernatants were measured using a human Magnetic Luminex assay. Percentages of inhibition were calculated relative to unstimulated (min) and stimulated (max) control samples that did not receive ISVD peptides or antibodies. All calculations were performed using GraphPad Prism 8.0. Data are expressed as mean ± standard error (SEM) of all donors from three independent experimental combinations.

[0352] Figure 10 F027400161 significantly reduced detectable levels of human TSLP in the plasma of NSG-SGM3 mice. At doses of 0.1 mg / kg F027400161 (45.772 pg / ml) and 10 mg / kg F027400161 (0.072 pg / ml), human plasma TSLP levels were reduced compared to carboxytreated mice (376.166 pg / ml), demonstrating that the TSLP group from F027400161 can bind to human TSLP in the plasma of humanized NSG-SGM3 mice.

[0353] Figure 11 F027400161 significantly reduced detectable levels of human IL-13 in the plasma of NSG-SGM3 mice. Compared to carboxytreated mice (274.052 pg / ml), doses of 0.01 mg / kg F027400161 (201.286 pg / ml), 0.05 mg / kg F027400161 (22.028 pg / ml), 0.1 mg / kg F027400161 (40.740 pg / ml), and 10 mg / kg F027400161 (1.777 pg / ml) reduced human IL-13 levels, demonstrating that the IL-13 group of F027400161 can bind to human IL-13 in the plasma of humanized NSG-SGM3 mice.

[0354] Figure 12 and Figure 13F027400161 significantly reduced the expression of Retnla and Clca1 transcripts in the lungs of NSG-SGM3 mice that received hydrodynamic delivery of hTSLP and hIL-4. Overexpression of human TSLP and IL-4 in NSG-SGM3 mice induced the expression of CD34-derived precursors. + Human immune cells produce hIL-13. Neutralization of human IL-13 by F027400161 at a dose of 10 mg / kg resulted in inhibition of the Retnla and Clca1 marker genes in mice.

[0355] Figure 14 The diagram shows an ISVD construct F027400161 consisting of monovalent building blocks / ISVD 4B02, 4B06, 501A02, 529F10, and albumin binding agent ALB23002, from N-terminus to C-terminus. 4B02 and 4B06 are linked via 35GS connectors, while the remaining building blocks are linked via 9GS connectors. 5. Detailed Implementation

[0357] The present invention aims to provide novel drugs for the treatment of inflammatory diseases such as atopic dermatitis and asthma.

[0358] In some embodiments, the present invention relates to peptides that simultaneously target IL-13 and TSLP, said peptides causing increased regulation of type 2 inflammatory responses in vitro and / or in vivo compared to monospecific anti-IL-13 or anti-TSLP peptides. In some embodiments, said peptides are efficiently produced (e.g., in a microbial host). Furthermore, in some embodiments, such peptides may exhibit limited reactivity to antibodies pre-existing in the subject to be treated (i.e., antibodies present in the subject prior to the first treatment with said antibody construct). In other embodiments, such peptides exhibit a sufficiently long half-life in the subject to be treated, allowing for convenient spacing of successive treatments.

[0359] 5.1 The polypeptide of the present invention

[0360] Single-specific monovalent peptide

[0361] In one embodiment, the polypeptide of the present invention is single-specific and monovalent.

[0362] The term "single-specific" refers to binding to one or more target molecules of one (specific) type. Therefore, the single-specific peptide of this invention specifically binds to IL-13. Another single-specific peptide of this invention specifically binds to TSLP.

[0363] The term "monovalent" indicates that only one binding unit / building block of the target molecule (such as ISVD) is present (specifically)

[0364] Therefore, in one aspect, the present invention provides a monospecific monovalent polypeptide comprising an ISVD that specifically binds to IL-13, said ISVD comprising three complementarity-determining regions (CDR1 to CDR3, respectively). The ISVD may be selected from ISVDs comprising the following:

[0365] a) CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence differing from SEQ ID NO: 7 by 2 or 1 amino acids; CDR2 is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence differing from SEQ ID NO: 12 by 2 or 1 amino acids; and CDR3 is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence differing from SEQ ID NO: 17 by 2 or 1 amino acids; or

[0366] b) CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence that differs from SEQ ID NO: 8 by 2 or 1 amino acids, CDR2 is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence that differs from SEQ ID NO: 13 by 2 or 1 amino acids, and CDR3 is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that differs from SEQ ID NO: 18 by 2 or 1 amino acids.

[0367] In one embodiment of this aspect of the invention, the ISVD specifically binds to human IL-13.

[0368] In another embodiment, the ISVD that specifically binds to IL-13 is selected from ISVDs that include the following:

[0369] a) CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7, CDR2 is the amino acid sequence of SEQ ID NO: 12, and CDR3 is the amino acid sequence of SEQ ID NO: 17; or

[0370] b) CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8, CDR2 is the amino acid sequence of SEQ ID NO: 13 and CDR3 is the amino acid sequence of SEQ ID NO: 18.

[0371] In another embodiment of this aspect of the invention, the ISVD that specifically binds to IL-13 is selected from ISVDs comprising:

[0372] a) Sequence identity with more than 90% of SEQ ID NO: 2; or

[0373] b) More than 90% sequence identity with SEQ ID NO: 3.

[0374] In one embodiment, the ISVD that specifically binds to IL-13 is selected from the ISVD containing the amino acid sequence of SEQ ID NO: 2; or the amino acid sequence of SEQ ID NO: 3.

[0375] In another aspect of the invention, a monospecific monovalent polypeptide comprising an ISVD or thereof specifically binding to TSLP is provided, said ISVD comprising three complementarity-determining regions (CDR1 to CDR3, respectively). The ISVD may be selected from ISVDs comprising the following:

[0376] a) CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence differing from SEQ ID NO: 9 by 2 or 1 amino acids; CDR2 is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence differing from SEQ ID NO: 14 by 2 or 1 amino acids; and CDR3 is the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence differing from SEQ ID NO: 19 by 2 or 1 amino acids; or

[0377] b) CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence that differs from SEQ ID NO: 11 by 2 or 1 amino acids, CDR2 is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence that differs from SEQ ID NO: 16 by 2 or 1 amino acids, and CDR3 is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence that differs from SEQ ID NO: 21 by 2 or 1 amino acids.

[0378] In one embodiment of this aspect of the invention, the ISVD specifically binds to human TSLP.

[0379] In another implementation, the ISVD that specifically binds to the TSLP is selected from ISVDs that include the following:

[0380] a) CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 9, CDR2 is the amino acid sequence of SEQ ID NO: 14, and CDR3 is the amino acid sequence of SEQ ID NO: 19; or

[0381] b) CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 11, CDR2 is the amino acid sequence of SEQ ID NO: 16 and CDR3 is the amino acid sequence of SEQ ID NO: 21.

[0382] In another embodiment of this aspect of the invention, the ISVD specifically bound to TSLP is selected from ISVDs comprising:

[0383] a) More than 90% sequence identity with SEQ ID NO: 4; or

[0384] b) More than 90% sequence identity with SEQ ID NO: 6.

[0385] In one embodiment, the ISVD that specifically binds to TSLP is selected from the ISVD containing the amino acid sequence of SEQ ID NO: 4; or the amino acid sequence of SEQ ID NO: 6.

[0386] Single-specific multivalent peptide

[0387] On the other hand, the polypeptides of the present invention are single-specific and at least bivalent, but may also be, for example, trivalent, tetravalent, pentavalent, hexavalent, etc.

[0388] The terms “divalent,” “trivalent,” “quadrivalent,” “pentavalent,” or “hexavalent” all belong to the term “multivalent” and respectively indicate the presence of two, three, four, five, or six combining units / building blocks, such as ISVD.

[0389] Therefore, in one aspect, the present invention provides a single-specific divalent polypeptide comprising two ISVDs or composed thereof that specifically bind to IL-13, wherein each of the two ISVDs comprises three complementarity-determining regions (CDR1 to CDR3, respectively), wherein the two ISVDs are optionally linked via one or more peptide linkers, and wherein:

[0390] a) The first ISVD and the second ISVD comprise CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that differs from SEQ ID NO: 7 by 2 or 1 amino acids, CDR2 is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence that differs from SEQ ID NO: 12 by 2 or 1 amino acids, and CDR3 is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence that differs from SEQ ID NO: 17 by 2 or 1 amino acids;

[0391] b) The first ISVD and the second ISVD comprise CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence that differs from SEQ ID NO: 8 by 2 or 1 amino acids, CDR2 is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence that differs from SEQ ID NO: 13 by 2 or 1 amino acids, and CDR3 is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that differs from SEQ ID NO: 18 by 2 or 1 amino acids;

[0392] c) The first ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence differing from SEQ ID NO: 7 by 2 or 1 amino acids; CDR2 is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence differing from SEQ ID NO: 12 by 2 or 1 amino acids; and CDR3 is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence differing from SEQ ID NO: 17 by 2 or 1 amino acids; and

[0393] The second ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence differing from SEQ ID NO: 8 by 2 or 1 amino acids; CDR2 is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence differing from SEQ ID NO: 13 by 2 or 1 amino acids; and CDR3 is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence differing from SEQ ID NO: 18 by 2 or 1 amino acids; or

[0394] d) The first ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence differing from SEQ ID NO: 8 by 2 or 1 amino acids; CDR2 is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence differing from SEQ ID NO: 13 by 2 or 1 amino acids; and CDR3 is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence differing from SEQ ID NO: 18 by 2 or 1 amino acids; and

[0395] The second ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that differs from SEQ ID NO: 7 by 2 or 1 amino acids, CDR2 is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence that differs from SEQ ID NO: 12 by 2 or 1 amino acids, and CDR3 is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence that differs from SEQ ID NO: 17 by 2 or 1 amino acids.

[0396] In one embodiment of this aspect of the invention, the ISVDs are linked via one or more peptide linkers. In one embodiment, the two ISVDs specifically bind to human IL13.

[0397] In another embodiment, the single-specific divalent polypeptide comprises or consists of two ISVDs that specifically bind to IL-13, wherein:

[0398] a) The first ISVD and the second ISVD comprise CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7, CDR2 is the amino acid sequence of SEQ ID NO: 12 and CDR3 is the amino acid sequence of SEQ ID NO: 17;

[0399] b) The first ISVD and the second ISVD comprise CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8, CDR2 is the amino acid sequence of SEQ ID NO: 13 and CDR3 is the amino acid sequence of SEQ ID NO: 18;

[0400] c) The first ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7, CDR2 is the amino acid sequence of SEQ ID NO: 12, and CDR3 is the amino acid sequence of SEQ ID NO: 17; and the second ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8, CDR2 is the amino acid sequence of SEQ ID NO: 13, and CDR3 is the amino acid sequence of SEQ ID NO: 18; or

[0401] d) The first ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8, CDR2 is the amino acid sequence of SEQ ID NO: 13, and CDR3 is the amino acid sequence of SEQ ID NO: 18; and the second ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7, CDR2 is the amino acid sequence of SEQ ID NO: 12, and CDR3 is the amino acid sequence of SEQ ID NO: 17.

[0402] In another embodiment of this aspect of the invention, the single-specific divalent polypeptide comprises or consists of two ISVDs that specifically bind to IL-13, wherein:

[0403] a) The amino acid sequences of the first ISVD and the second ISVD contain more than 90% sequence identity with SEQ ID NO: 2;

[0404] b) The amino acid sequences of the first ISVD and the second ISVD contain more than 90% sequence identity with SEQ ID NO: 3;

[0405] c) The amino acid sequence of the first ISVD contains more than 90% sequence identity with SEQ ID NO: 2, and the second ISVD contains more than 90% sequence identity with SEQ ID NO: 3; or

[0406] d) The amino acid sequence of the first ISVD contains more than 90% sequence identity with SEQ ID NO: 3, and the second ISVD contains more than 90% sequence identity with SEQ ID NO: 2.

[0407] In one embodiment, the monospecific divalent polypeptide comprises or consists of two ISVDs that specifically bind to IL-13, wherein:

[0408] a) The first ISVD and the second ISVD contain the amino acid sequence of SEQ ID NO: 2;

[0409] b) The first ISVD and the second ISVD contain the amino acid sequence of SEQ ID NO: 3;

[0410] 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; or

[0411] 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.

[0412] On the other hand, the present invention provides a single-specific divalent polypeptide comprising two ISVDs or composed thereof that specifically bind to TSLP, wherein each of the two ISVDs comprises three complementarity-determining regions (CDR1 to CDR3, respectively), wherein the two ISVDs are optionally linked via one or more peptide linkers, and wherein:

[0413] a) The first ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence differing from SEQ ID NO: 11 by 2 or 1 amino acids; CDR2 is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence differing from SEQ ID NO: 16 by 2 or 1 amino acids; and CDR3 is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence differing from SEQ ID NO: 21 by 2 or 1 amino acids; and

[0414] The second ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence differing from SEQ ID NO: 9 by 2 or 1 amino acids; CDR2 is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence differing from SEQ ID NO: 14 by 2 or 1 amino acids; and CDR3 is the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence differing from SEQ ID NO: 19 by 2 or 1 amino acids; or

[0415] b) The first ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence differing from SEQ ID NO: 9 by 2 or 1 amino acids; CDR2 is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence differing from SEQ ID NO: 14 by 2 or 1 amino acids; and CDR3 is the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence differing from SEQ ID NO: 19 by 2 or 1 amino acids; and

[0416] The second ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence that differs from SEQ ID NO: 11 by 2 or 1 amino acids, CDR2 is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence that differs from SEQ ID NO: 16 by 2 or 1 amino acids, and CDR3 is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence that differs from SEQ ID NO: 21 by 2 or 1 amino acids.

[0417] In one embodiment of this aspect of the invention, the ISVDs are linked via one or more peptide linkers. In one embodiment, the two ISVDs specifically bind to human TSLP.

[0418] In another embodiment, the single-specific divalent polypeptide comprises or consists of two ISVDs that specifically bind to TSLP, wherein:

[0419] a) The first ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 11, CDR2 is the amino acid sequence of SEQ ID NO: 16, and CDR3 is the amino acid sequence of SEQ ID NO: 21; and the second ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 9, CDR2 is the amino acid sequence of SEQ ID NO: 14, and CDR3 is the amino acid sequence of SEQ ID NO: 19; or

[0420] b) The first ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 9, CDR2 is the amino acid sequence of SEQ ID NO: 14, and CDR3 is the amino acid sequence of SEQ ID NO: 19; and the second ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 11, CDR2 is the amino acid sequence of SEQ ID NO: 16, and CDR3 is the amino acid sequence of SEQ ID NO: 21.

[0421] In another embodiment of this aspect of the invention, the single-specific divalent polypeptide comprises or consists of two ISVDs that specifically bind to TSLP, wherein:

[0422] a) The amino acid sequence of the first ISVD contains more than 90% sequence identity with SEQ ID NO: 6, and the second ISVD contains more than 90% sequence identity with SEQ ID NO: 4; or

[0423] 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.

[0424] In one embodiment, the single-specific bivalent polypeptide comprises or consists of two ISVDs that specifically bind to TSLP, wherein:

[0425] 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

[0426] 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.

[0427] In this context, the terms "first ISVD" and "second ISVD" refer only to the relative positions of the ISVDs specifically described as binding to IL-13 / TSLP, wherein the numbering begins from the N-terminus of the polypeptide of the present invention. Thus, "first ISVD" is closer to the N-terminus than "second ISVD." Correspondingly, "second ISVD" is closer to the C-terminus than "first ISVD." Since the numbering is not absolute and only indicates the relative positions of the two ISVDs, the presence of additional binding units / building blocks, such as ISVDs, that bind to IL-13 and TSLP respectively within the polypeptide is not excluded. Furthermore, the possibility of placing other binding units / building blocks (such as ISVDs) in between is not excluded. For example, as further described below (see the “Multispecific Multivalent Peptides” section and 5.4 “(In vivo) Half-life Extension”), the peptide may also contain or even be located between the “first ISVD” and the “second ISVD”, another ISVD that binds to human serum albumin (such constructs will be referred to as multispecific in later sections).

[0428] In one embodiment, at least two ISVDs of the single-specific multivalent polypeptide, particularly the aforementioned single-specific divalent polypeptide, are linked via peptide linkers. The use of peptide linkers to link two or more (multi)peptides is well known in the art. Exemplary peptide linkers that can be used with the single-specific multivalent polypeptide, particularly the aforementioned single-specific divalent polypeptide, are shown in Tables A-5. A commonly used class of peptide linkers is called “Gly-Ser” or “GS” linkers. These are linkers consisting essentially of glycine (G) and serine (S) residues and typically contain one or more repetitions of a peptide motif such as the GGGGS (SEQ ID NO: 73) motif (e.g., containing the formula (Gly-Gly-Gly-Gly-Ser)n, where n can be 1, 2, 3, 4, 5, 6, 7, or greater). Some frequently used examples of such GS linkers are the 9GS linker (GGGGSGGGS, SEQ ID NO: 76), the 15GS linker (n=3), and the 35GS linker (n=7). See, for example, Chen et al., Adv. Drug Deliv. Rev. 15 Oct 2013; 65(10): 1357-1369; and Klein et al., Protein Eng. Des. Sel. (2014) 27(10): 325-330. In one embodiment of the present invention, the ISVDs of the monospecific multivalent polypeptides, particularly the ISVDs of the monospecific divalent polypeptides of the present invention, are linked via the linkers shown in Tables A-5. In one embodiment, the (at least) two ISVDs are linked via one or more 35GS linkers.

[0429] Therefore, in one embodiment, the single-specific divalent polypeptide comprises or consists of the following:

[0430] a) The amino acid sequence of SEQ ID NO: 148

[0431] b) The amino acid sequence of SEQ ID NO: 149

[0432] c) The amino acid sequence of SEQ ID NO: 150

[0433] d) The amino acid sequence of SEQ ID NO: 151

[0434] e) The amino acid sequence of SEQ ID NO: 152

[0435] f) The amino acid sequence of SEQ ID NO: 153

[0436] g) The amino acid sequence of SEQ ID NO: 154

[0437] h) The amino acid sequence of SEQ ID NO: 155

[0438] i) The amino acid sequence of SEQ ID NO: 156

[0439] j) The amino acid sequence of SEQ ID NO: 157

[0440] k) The amino acid sequence of SEQ ID NO: 158, or

[0441] l) The amino acid sequence of SEQ ID NO: 159.

[0442] Multispecific multivalent peptides

[0443] In another aspect, the polypeptide of the present invention is at least bispecific, but may also be, for example, trispecific, tetraspecific, pentaspecific, etc. Furthermore, the polypeptide is at least divalent, but may also be, for example, trivalent, tetravalent, pentavalent, hexavalent, etc.

[0444] The terms "bispecificity," "trispecificity," "quadrispecificity," and "pentaspecificity" all belong to the term "multispecificity," and refer to binding to two, three, four, or five different target molecules respectively.

[0445] The terms "divalent", "trivalent", "quadrivalent", "pentavalent", and "hexavalent" all belong to the term "multivalent" and respectively indicate the existence of two, three, four, five, and six combined units / building blocks, such as ISVD.

[0446] For example, the polypeptide may be bispecific tetravalent, such as a polypeptide comprising at least four ISVDs or composed thereof, wherein at least two ISVDs specifically bind to IL-13 and at least two 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 trispecific pentavalent, such as a polypeptide comprising five ISVDs or composed thereof, wherein two ISVDs specifically bind to human IL-13, two ISVDs specifically bind to human TSLP, and one ISVD binds to human serum albumin. For example, if two ISVDs bind to two different epitopes on human IL-13 or human TSLP, then such a polypeptide may be simultaneously bicomplementary. The term “bicomplementary” refers to binding to two different parts (e.g., epitopes) of the same target molecule. In one embodiment, the trispecific pentavalent polypeptide of the present invention is, for example, the ISVD construct F027400161, which comprises two ISVDs that specifically bind to human IL-13, two ISVDs that specifically bind to human TSLP, and one ISVD that binds to human serum albumin, and is doubly complementary for binding to both IL-13 and TSLP.

[0447] In one embodiment, the multispecific multivalent polypeptide comprises or consists of at least four ISVDs, wherein each of the ISVDs comprises three complementarity-determining regions (CDR1 to CDR3, respectively), wherein the at least four ISVDs are optionally linked via one or more peptide linkers, and wherein:

[0448] a) The first ISVD that specifically binds to IL-13 comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that differs from SEQ ID NO: 7 by 2 or 1 amino acids, CDR2 is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence that differs from SEQ ID NO: 12 by 2 or 1 amino acids, and CDR3 is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence that differs from SEQ ID NO: 17 by 2 or 1 amino acids;

[0449] b) The second ISVD that specifically binds to IL-13 comprises CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence that differs from SEQ ID NO: 8 by 2 or 1 amino acids, CDR2 is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence that differs from SEQ ID NO: 13 by 2 or 1 amino acids, and CDR3 is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that differs from SEQ ID NO: 18 by 2 or 1 amino acids;

[0450] c) The third ISVD that specifically binds to TSLP comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence differing from SEQ ID NO: 9 by 2 or 1 amino acids; CDR2 is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence differing from SEQ ID NO: 14 by 2 or 1 amino acids; and CDR3 is the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence differing from SEQ ID NO: 19 by 2 or 1 amino acids; and

[0451] d) The fourth ISVD that specifically binds to TSLP comprises CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence that differs from SEQ ID NO: 11 by 2 or 1 amino acids, CDR2 is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence that differs from SEQ ID NO: 16 by 2 or 1 amino acids, and CDR3 is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence that differs from SEQ ID NO: 21 by 2 or 1 amino acids.

[0452] In one embodiment, the IL-13 and TSLP bound by the polypeptide are human IL-13 and human TSLP, respectively.

[0453] In another embodiment of the multispecific multivalent peptide:

[0454] a) The first ISVD comprises CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7, CDR2 is the amino acid sequence of SEQ ID NO: 12 and CDR3 is the amino acid sequence of SEQ ID NO: 17;

[0455] b) The second ISVD comprises CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8, CDR2 is the amino acid sequence of SEQ ID NO: 13 and CDR3 is the amino acid sequence of SEQ ID NO: 18;

[0456] c) The third ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 9, CDR2 is the amino acid sequence of SEQ ID NO: 14, and CDR3 is the amino acid sequence of SEQ ID NO: 19; and

[0457] d) The fourth ISVD comprises CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 11, CDR2 is the amino acid sequence of SEQ ID NO: 16 and CDR3 is the amino acid sequence of SEQ ID NO: 21.

[0458] In another aspect of multispecific multivalent peptides:

[0459] a) The amino acid sequence of the first ISVD contains more than 90% sequence identity with SEQ ID NO: 2;

[0460] b) The amino acid sequence of the second ISVD contains more than 90% sequence identity with SEQ ID NO: 3;

[0461] c) The amino acid sequence of the third ISVD contains more than 90% sequence identity with SEQ ID NO: 4; and

[0462] d) The amino acid sequence of the fourth ISVD contains more than 90% sequence identity with SEQ ID NO: 6.

[0463] In one implementation of a multispecific multivalent polypeptide:

[0464] a) The first ISVD contains the amino acid sequence of SEQ ID NO: 2;

[0465] b) The second ISVD contains the amino acid sequence of SEQ ID NO: 3;

[0466] c) The third ISVD contains the amino acid sequence of SEQ ID NO: 4; and

[0467] d) The fourth ISVD contains the amino acid sequence of SEQ ID NO: 6.

[0468] In this context, the terms "first ISVD," "second ISVD," and "third ISVD," etc., indicate only the relative positions of the ISVDs, where the numbering begins from the N-terminus of the polypeptide of the present invention. Thus, "first ISVD" is closer to the N-terminus than "second ISVD," and "second ISVD" is closer to the N-terminus than "third ISVD." Therefore, when considered from the C-terminus, the ISVD arrangement is reversed. Since the numbering is not absolute and only indicates the relative positions of the at least four ISVDs, the presence of other binding units / building blocks, such as additional ISVDs binding to IL-13 or TSLP, or ISVDs binding to another target, is not excluded. Furthermore, the possibility of placing other binding units / building blocks (such as ISVDs) in between is not excluded. For example, as further described below (see Section 5.4, “(in vivo) half-life extension”), the polypeptide may also include or even be located between, for example, the “first ISVD” and the “fourth ISVD”, another ISVD that binds to human serum albumin.

[0469] On the other hand, the present invention provides a bispecific divalent polypeptide comprising an ISVD that specifically binds to IL-13 or TSLP, as described above in Section 5.1; “Monospecific monovalent polypeptide”, and an ISVD that binds to human serum albumin, as described below in Section 5.4; “(In vivo) half-life extension”.

[0470] On the other hand, the present invention provides a bispecific trivalent polypeptide comprising the monospecific bivalent polypeptide described above (Section 5.1; “monospecific bivalent polypeptide”) and the ISVD bound to human serum albumin as described in detail below (Section 5.4; “(in vivo) prolonged half-life”).

[0471] The components of the multispecific multivalent polypeptide (such as ISVD) can be linked together by one or more suitable linkers (such as peptide linkers).

[0472] The use of linkers to connect two or more (poly)peptides is well known in the art. Exemplary peptide linkers are shown in Table A-5. One commonly used class of peptide linkers is called “Gly-Ser” or “GS” linkers. These are linkers that consist essentially of glycine (G) and serine (S) residues and typically contain one or more repetitions of a peptide motif such as the GGGGS (SEQ ID NO: 73) motif (e.g., containing the formula (Gly-Gly-Gly-Gly-Ser)n, where n can be 1, 2, 3, 4, 5, 6, 7, or greater). Some frequently used examples of such GS linkers are the 9GS linker (GGGGSGGGS, SEQ ID NO: 76), the 15GS linker (n=3) (SEQ ID NO: 78), and the 35GS linker (n=7) (SEQ ID NO: 83). See, for example, Chen et al., Adv. Drug Deliv. Rev. 15 Oct 2013; 65(10): 1357-1369; and Klein et al., Protein Eng. Des. Sel. (2014) 27(10): 325-330. In one or more peptides of the present invention, the components of the peptide are linked together using 9GS (SEQ ID NO: 76) and 35GS (SEQ ID NO: 83) linkers.

[0473] In one aspect of the multispecific, multivalent polypeptide of the present invention, a polypeptide comprising or consisting of at least four ISVDs includes at least two ISVDs specifically binding to IL-13 and at least two ISVDs specifically binding to TSLP. In this aspect of the invention, the at least two ISVDs binding to IL-13 are linked via a 35GS linker, while the at least two ISVDs specifically binding to TSLP are linked via a 9GS linker. In one embodiment, the at least two ISVDs specifically binding to TSLP are separated by an albumin-bound ISVD (9GS-Alb-9GS) (as described in Section 5.4, “(In vivo) Half-life Extension” below). The inventors have surprisingly found that this configuration can increase the yield of the polypeptide.

[0474] Therefore, in one embodiment, the polypeptide comprises, or consists of, the following items in sequence starting from the N-terminus of the polypeptide: 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 an increased half-life to the polypeptide as defined herein, and a second ISVD that specifically binds to TSLP. In one embodiment, the binding unit that provides an increased half-life to the polypeptide is an ISVD.

[0475] In another embodiment, the polypeptide comprises, or consists of, the following items in sequence starting from the N-terminus of the polypeptide: 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 one particular embodiment, the linker between two ISVDs that bind to IL-13 is a 35GS linker, while the other linkers are 9GS linkers.

[0476] Such a configuration of the polypeptide can provide increased production yield, good CMC characteristics (such as sufficient solubility and biophysical stability), strong potency in modulating type 2 immune responses, and low binding to pre-existing antibodies.

[0477] In one embodiment, the multispecific, multivalent polypeptide of the present invention exhibits reduced binding to pre-existing antibodies in human serum. To this end, in one embodiment, the polypeptide contains valine (V) at amino acid position 11 and leucine (L) at amino acid position 89 in at least one ISVD (according to Kabat designation). In another embodiment, the polypeptide contains valine (V) at amino acid position 11 and leucine (L) at amino acid position 89 in each ISVD (according to Kabat designation). In another embodiment, the polypeptide contains an extension of 1 to 5 (naturally occurring) amino acids, such as a single alanine (A) extension, at the C-terminus of the C-terminus of the ISVD. The C-terminus of the ISVD is typically VTVSS (SEQ ID NO: 138). In another embodiment, the polypeptide contains lysine (K) or glutamine (Q) at position 110 in at least one ISVD (according to Kabat designation). In yet another embodiment, the ISVD contains lysine (K) or glutamine (Q) at position 112 in at least one ISVD (according to Kabat designation). In these embodiments, the C-terminus of the ISVD is VKVSS (SEQ ID NO: 139), VQVSS (SEQ ID NO: 140), VTVKS (SEQ ID NO: 166), VTVQS (SEQ ID NO: 167), VKVKS (SEQ ID NO: 168), VKVQS (SEQ ID NO: 169), VQVKS (SEQ ID NO: 170), or VQVQS (SEQ ID NO: 171), such that after the addition of a single alanine, the C-terminus of the polypeptide includes, for example, the sequences VTVSSA (SEQ ID NO: 141), VKVSSA (SEQ ID NO: 142), VQVSSA (SEQ ID NO: 143), VTVKSA (SEQ ID NO: 172), VTVQSA (SEQ ID NO: 173), VKVKSA (SEQ ID NO: 174), VKVQSA (SEQ ID NO: 175), or VQVKSA (SEQ ID NO: 175). NO: 176), or VQVQSA (SEQ ID NO: 177). In one embodiment, the C-terminus comprises VTVSSA (SEQ ID NO: 141).In another embodiment, the polypeptide comprises valine (V) at amino acid position 11 and leucine (L) at amino acid position 89 (according to Kabat designation) in each ISVD, optionally lysine (K) or glutamine (Q) at position 110 (according to Kabat designation) in at least one ISVD, and includes an extension of 1 to 5 (naturally occurring) amino acids, such as a single alanine (A) extension, at the C-terminus of the C-terminus of the polypeptide (such that the C-terminus of the polypeptide has the sequence VTVSSA (SEQ ID NO: 141), VKVSSA (SEQ ID NO: 142), or VQVSSA (SEQ ID NO: 143), such as VTVSSA (SEQ ID NO: 141)). See, for example, WO2012 / 175741 and WO2015 / 173325 for more information on this aspect.

[0478] In one embodiment, the multispecific multivalent polypeptide of the present invention comprises or is composed of an amino acid sequence having more than 90% (e.g., more than 95% or more than 99%) sequence identity with SEQ ID NO: 1, wherein the CDRs of the five ISVDs are as defined in items A to E (or A' to E', if Kabat's definition is used) as described in sections “5.2 Immunoglobulin Single Variable Domain” and “5.4 (In Vivo) Half-Life Extension” below, respectively, wherein in particular:

[0479] The first ISVD that specifically binds to IL-13 has CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7, CDR2 is the amino acid sequence of SEQ ID NO: 12 and CDR3 is the amino acid sequence of SEQ ID NO: 17;

[0480] The second ISVD that specifically binds to IL-13 has CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8, CDR2 is the amino acid sequence of SEQ ID NO: 13 and CDR3 is the amino acid sequence of SEQ ID NO: 18;

[0481] The third ISVD that specifically binds to TSLP has CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 9, CDR2 is the amino acid sequence of SEQ ID NO: 14 and CDR3 is the amino acid sequence of SEQ ID NO: 19;

[0482] The fourth ISVD that specifically binds to TSLP has CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 11, CDR2 is the amino acid sequence of SEQ ID NO: 16, and CDR3 is the amino acid sequence of SEQ ID NO: 21; and

[0483] The ISVD that binds to human serum albumin has CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 10, CDR2 is the amino acid sequence of SEQ ID NO: 15 and CDR3 is the amino acid sequence of SEQ ID NO: 20;

[0484] Alternatively, if Kabat is used:

[0485] The first ISVD that specifically binds to IL-13 has CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO:37, CDR2 is the amino acid sequence of SEQ ID NO:42 and CDR3 is the amino acid sequence of SEQ ID NO:17;

[0486] The second ISVD that specifically binds to IL-13 has CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 38, CDR2 is the amino acid sequence of SEQ ID NO: 43 and CDR3 is the amino acid sequence of SEQ ID NO: 18;

[0487] The third ISVD that specifically binds to TSLP has CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 39, CDR2 is the amino acid sequence of SEQ ID NO: 44 and CDR3 is the amino acid sequence of SEQ ID NO: 19;

[0488] The fourth ISVD that specifically binds to TSLP has CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 41, CDR2 is the amino acid sequence of SEQ ID NO: 46, and CDR3 is the amino acid sequence of SEQ ID NO: 21; and

[0489] The ISVD that binds to human serum albumin has CDR1, CDR2 and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 40, CDR2 is the amino acid sequence of SEQ ID NO: 45 and CDR3 is the amino acid sequence of SEQ ID NO: 20.

[0490] In another embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 1. In yet another embodiment, the polypeptide consists of the amino acid sequence of SEQ ID NO: 1.

[0491] In one embodiment, the polypeptide of the present invention has at least half or at least the same binding affinity for human IL-13 and human TSLP compared to the polypeptide composed of the amino acids of SEQ ID NO: 1, wherein said binding affinity is measured using the same method such as surface plasmon resonance (SPR).

[0492] 5.2 Immunoglobulin Single Variable Domain

[0493] The terms "immunoglobulin single variable domain" (ISVD) and "single variable domain" are used interchangeably, defining an immunoglobulin molecule in which the antigen-binding site is located on and formed by a single immunoglobulin domain. This distinguishes ISVD from "conventional" immunoglobulins (e.g., monoclonal antibodies) or fragments thereof (e.g., Fab, Fab', F(ab')2, scFv, di-scFv), where two immunoglobulin domains, particularly two variable domains, interact to form the antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (V... H ) and light chain variable structural domain (V L ) interact to form antigen-binding sites. In this case, V H and V L The complementary determinant regions (CDRs) of the two will facilitate the formation of antigen binding sites, meaning that a total of 6 CDRs will participate in the formation of antigen binding sites.

[0494] Given the above definitions, the antigen-binding domain of conventional four-chain antibodies (such as IgG, IgM, IgA, IgD, or IgE molecules; known in the art) or Fab fragments, F(ab′)2 fragments, Fv fragments (such as disulfide-linked Fv or scFv fragments) or biantibodies (all known in the art) derived from such conventional four-chain antibodies will generally not be considered ISVDs, because in these cases, it is not a (single) immunoglobulin domain that binds to the corresponding antigenic epitope, but rather a pair of (associated) immunoglobulin domains (such as light chain and heavy chain variable domains), i.e., the V of the immunoglobulin domain. H -V L The binding of the antigen to the corresponding epitope occurs, and they collectively bind to the corresponding epitope.

[0495] In contrast, ISVDs can specifically bind to antigenic epitopes without pairing with other immunoglobulin variable domains. The binding site of ISVDs consists of a single V... H A single V HH Or a single V L Domain formation.

[0496] Therefore, a single variable domain can be a sequence of light chain variable domains (e.g., V). L Sequences) or suitable fragments thereof; or heavy chain variable domain sequences (e.g., V H Sequence or V HH (sequence) or a suitable fragment thereof; as long as it can form a single antigen-binding unit (i.e., a functional antigen-binding unit consisting essentially of a single variable domain, such that a single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit).

[0497] ISVDs can be, for example, heavy chain ISVDs (such as V...). H V HH ), including camel-derived V H Or humanized V HH In one implementation, it is V HH Including camel-derived V H Or humanized V HH Heavy chain ISVDs can be derived from conventional four-chain antibodies or heavy chain antibodies.

[0498] For example, an ISVD can be a single-domain antibody (or an amino acid sequence suitable for use as a single-domain antibody), "dAb" or dAb (or an amino acid sequence suitable for use as a dAb) or (as defined herein, and including but not limited to V) HH ); any suitable fragment of other single variable structural domains or any one of them.

[0499] Specifically, ISVD can be (e.g. V) HH This includes humanized VHH or camel-derived VHH. H (or a suitable fragment thereof.) and It is a registered trademark of Ablynx NV.

[0500] “V HH "Structured domain" is also called V HH V HH Antibody fragments and V HHAntibodies, initially described as "heavy chain antibodies" (i.e., "antibodies without light chain"; Hamers-Casterman et al., Nature 363: 446-448, 1993), are antigen-binding immunoglobulin variable domains. The term "V" has been chosen. HH "V" refers to these variable domains in conjunction with the heavy chain variable domains present in conventional 4-chain antibodies (which are referred to in this paper as "V"). H The variable light chain domain (referred to in this paper as "V") and the light chain variable domain present in conventional 4-chain antibodies are also mentioned. L Distinguish between "structural domain" and "structural domain". Regarding V... HH For further description, please refer to Muyldermans' review article (Review, Molecular Biotechnology 74: 277-302, 2001).

[0501] Typically, immunoglobulin production involves immunizing laboratory animals, fusing immunoglobulin-producing cells to create hybridomas, and screening for the required specificity. Alternatively, immunoglobulins can be generated by screening natural or synthetic libraries (e.g., via phage display).

[0502] Immunoglobulin sequences (e.g.) The generation of nanobodies has been extensively described in various publications (with WO 94 / 04678, Hamers-Casterman et al. 1993, and Muyldermans et al. 2001 serving as examples). In these methods, camels are immunized with a target antigen to induce an immune response against the target antigen. The library of nanobodies obtained from the immunization is then further screened for nanobodies that bind to the target antigen.

[0503] In these cases, antibody generation requires purified antigens for immunization and / or screening. Antigens can be purified from natural sources or during recombinant production.

[0504] Immunization and / or screening of immunoglobulin sequences can be performed using peptide fragments of such antigens.

[0505] The present invention can utilize immunoglobulin sequences from various sources, including mouse, rat, rabbit, donkey, human, and camelid immunoglobulin sequences. The present invention also includes fully human, humanized, or chimeric sequences. For example, the present invention includes camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences or camel-derived domain antibodies, such as camel-derived dAbs described by Ward et al. (see, for example, WO 94 / 04678 and Davies and Riechmann (1994 and 1996)). Furthermore, the present invention also uses fused immunoglobulin sequences, thereby forming multivalent and / or multispecific constructs (for those containing one or more V...). HH For multivalent and multispecific polypeptides of the domains and their preparation, see also Conrath et al., J. Biol. Chem., Vol. 276, 10.7346-7350, 2001, and see, for example, WO 96 / 34103 and WO 99 / 23221; and immunoglobulin sequences containing markers or other functional parts (e.g., toxins, labels, radiochemical substances, etc.) that can be derived from the immunoglobulin sequences of the present invention.

[0506] "Humanization V" HH "Includes naturally occurring V" HH The amino acid sequence of the domain corresponds to, but has been "humanized," an amino acid sequence, i.e., by using the naturally occurring V... HH One or more amino acid residues in the amino acid sequence of the sequence (and especially the structural sequence) are used with V from a conventional 4-chain antibody from humans (e.g., shown above). H Humanization is achieved by substitution of one or more amino acid residues present at one or more corresponding positions within the domain. This can be done in ways known per se, as will be apparent to those skilled in the art, for example, based on further description herein and prior art (e.g., WO2008 / 020079). Furthermore, it should be noted that such humanized V can be obtained in any suitable manner known per se. HH Therefore, it is not strictly limited to peptides obtained using peptides that contain naturally occurring VHH domains as starting materials.

[0507] "Camel Source V" H "Includes naturally occurring V" H The amino acid sequence of the domain corresponds to, but has been "camelized," that is, by using the naturally occurring V from a conventional 4-chain antibody. H One or more amino acid residues in the amino acid sequence of the domain are used with the V of the heavy chain antibody. HHCamelization is achieved by substitution of one or more amino acid residues present at one or more corresponding positions within a domain. This can be done in ways known per se, as will be apparent to those skilled in the art, for example, based on further description herein and prior art (e.g., WO 2008 / 020079). As defined herein, such “camelization” substitution is typically performed in V H -V L Insertion at the amino acid position where the junction is formed and / or present, and / or insertion at the so-called cameloid marker residue, as defined herein (see, for example, WO 94 / 04678 and Davies and Riechmann (1994 and 1996), ibid.). In one embodiment, used as a camel-derived V H V of the starting material or starting point H The sequence is derived from V in mammals. H Sequence or human V H Sequences, such as V H 3. Sequence. However, it should be noted that such camel-derived V sequences can be obtained in any suitable manner known per se. H And therefore not strictly limited to those already using naturally occurring V H Peptides obtained from peptides whose structural domains are used as starting materials.

[0508] The structure of an ISVD sequence can be considered to consist of four architecture regions (“FR”), referred to in the art and herein as “architecture region 1” (“FR1”), “architecture region 2” (“FR2”), “architecture region 3” (“FR3”) and “architecture region 4” (“FR4”), respectively. The architecture regions are interrupted by three complementary decision regions (“CDR”), referred to in the art and herein as “complementary decision region 1” (“CDR1”), “complementary decision region 2” (“CDR2”) and “complementary decision region 3” (“CDR3”), respectively.

[0509] As further described in paragraphs q) on pages 58 and 59 of WO 08 / 020079, the amino acid residues of ISVD can be determined according to the V given by Kabat et al. H The domain's generic numbering is used for numbering ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, 91), as in Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240(1-2): 185-195; see, for example, that publication). Figure 2 The article applies to V from camelidae animals.HH Structural domain. It should be noted that, as in the art, V... H Domain and V HH As is well known in structural domains, the total number of amino acid residues in each CDR can vary and may not correspond to the total number of amino acid residues indicated by the Kabat number (that is, one or more positions according to the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat number). This means that, generally, the Kabat number may or may not correspond to the actual number of amino acid residues in the actual sequence. H Domain and V HH The total number of amino acid residues in the domain is typically between 110 and 120, often falling between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0510] In this application, unless otherwise stated, the CDR sequence is determined according to the AbM definition described in Kontermann and Dübel (edited 2010, Antibody Engineering, Vol. 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 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.

[0511] The determination of the CDR region can also be performed using different methods. In the CDR determination according to Kabat, FR1 of ISVD contains amino acid residues at positions 1-30, CDR1 of ISVD contains amino acid residues at positions 31-35, FR2 of ISVD contains amino acid residues at positions 36-49, CDR2 of ISVD contains amino acid residues at positions 50-65, FR3 of ISVD contains amino acid residues at positions 66-94, CDR3 of ISVD contains amino acid residues at positions 95-102, and FR4 of ISVD contains amino acid residues at positions 103-113.

[0512] In such immunoglobulin sequences, the architecture sequence can be any suitable architecture sequence, and examples of suitable architecture sequences will be clear to those skilled in the art, for example based on standard manuals and other disclosures and prior art mentioned herein.

[0513] The architecture sequence is an immunoglobulin architecture sequence or a suitable combination of architecture sequences already derived from immunoglobulin architecture sequences (e.g., through humanization or camelification). For example, the architecture sequence may be derived from a light chain variable domain (e.g., V). L Sequence) and / or heavy-chain variable structural domains (e.g., V) H Sequence or V HH The architecture sequence (sequence). In one aspect, the architecture sequence is derived from V. HH The sequence's architecture sequence (which may optionally be partially or fully humanized) or a camel-derived conventional V H Sequences (as defined in this article).

[0514] In particular, the architectural sequence present in the ISVD sequence used in this invention may contain one or more marker residues (as defined herein) such that the ISVD sequence is Such as V HH Including humanized V HH Or camel-derived V H Some non-limiting examples of such architectural sequences (appropriate combinations) will become clear from further disclosures in this paper.

[0515] In addition, as described herein in a general sense for immunoglobulin sequences, any suitable fragment (or combination of fragments) of any of the foregoing may be used, such as a fragment containing one or more architecture sequences appropriately side-joined and / or one or more CDR sequences linked via one or more architecture sequences (e.g., in the same order as these CDR and architecture sequences may appear in the full-size immunoglobulin sequence from which said fragment is derived).

[0516] However, it should be noted that the present invention is not limited in terms of the source of the ISVD sequence (or the nucleotide sequence used to express it), nor in the manner in which the ISVD sequence or nucleotide sequence is generated or obtained (or has been generated or obtained). Therefore, the ISVD sequence can be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In a specific but non-limiting aspect, the ISVD sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, including but not limited to "humanized" (as defined herein) immunoglobulin sequences (such as partially or fully humanized mouse or rabbit immunoglobulin sequences, and particularly partially or fully humanized V... HHThe sequence refers to, “camel-derived” (as defined herein) immunoglobulin sequences, and immunoglobulin sequences that have been obtained by techniques such as affinity maturation (e.g., starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR transplantation, mosaicking, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques known to those skilled in the art for engineered immunoglobulin sequences; or any suitable combination of the foregoing.

[0517] Similarly, the nucleotide sequence can be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence, and can be, for example, a sequence isolated from a suitable naturally occurring template (e.g., DNA or RNA isolated from a cell) by PCR, a nucleotide sequence that has been isolated from a library (and especially an expression library), a nucleotide sequence that has been prepared by introducing a mutation into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), a nucleotide sequence that has been prepared by PCR using overlapping primers, or a nucleotide sequence that has been prepared using DNA synthesis techniques known per se.

[0518] As mentioned above, ISVD can be Or a suitable fragment thereof. For a general description of nanobodies, see further description below and the prior art referenced herein. However, it should be noted in this regard that this specification and the prior art primarily describe the so-called "V" H "Class 3" nanobodies (i.e., those related to V) H Nanobodies with high sequence homology to human ancestral sequences of three types (such as DP-47, DP-51, or DP-29). However, it should be noted that the techniques of this invention can generally be used with any type of nanobody in its broadest sense, and, for example, also with those belonging to the so-called "V" class. H "Class 4" nanobodies (i.e., those with V H Nanobodies with high sequence homology to human germline sequences (such as DP-78), for example, as described in WO 2007 / 118670.

[0519] Typically, nanobodies (especially V) HH Sequence, including (partially) humanized V HH Sequence and camel-derived V H The characteristics of a nanobody can be the presence of one or more “marker residues” (as described herein) in one or more structural sequences (again, as further described herein). Therefore, nanobodies can generally be defined as immunoglobulin sequences having the following (general) structure:

[0520] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0521] FR1 to FR4 refer to architecture regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively, and one or more of the marker residues are as further defined herein.

[0522] In particular, nanobodies can be immunoglobulin sequences having the following (general) structure:

[0523] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0524] FR1 to FR4 refer to architecture regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determination regions 1 to 3, respectively, and the architecture sequence is as further defined herein.

[0525] More specifically, nanobodies can be immunoglobulin sequences having the following (general) structure:

[0526] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0527] FRI to FR4 refer to architecture regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determination regions 1 to 3, respectively.

[0528] According to the Kabat number, one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108 are selected from the marker residues mentioned in Table A-0 below.

[0529] Table A-0: Marker Residues in Nanobodies

[0530]

[0531]

[0532] The present invention particularly utilizes ISVDs that can bind to IL-13 or TSLP. In the context of the present invention, "binding" to a specific target molecule has the same meaning in the art as understood in the context of antibodies and their corresponding antigens.

[0533] The multispecific multivalent polypeptide of the present invention may contain two or more ISVDs that specifically bind to IL-13 and two or more ISVDs that specifically bind to TSLP. For example, the polypeptide may contain two ISVDs that specifically bind to IL-13 and two ISVDs that specifically bind to TSLP.

[0534] In some embodiments, the at least one ISVD can functionally block its target molecule. For example, the targeting portion can block the interaction between IL-13 and IL-13Rα1 (interleukin 13 receptor, α1) and / or the interaction between the IL-13 / IL-13Rα1 complex and IL-4Rα (α-interleukin 4 receptor), or can block the interaction between TSLP and TSLPR (TSLP receptor) and / or the TSLP / TSLPR complex and IL-7Rα (interleukin 7 receptor subunit α). Therefore, in one embodiment, the polypeptide of the present invention comprises at least two ISVDs that specifically bind to IL-13 and functionally block its interaction with IL-13Rα1 and / or the interaction between the IL-13 / IL-13Rα1 complex and IL-4Rα; and 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.

[0535] The ISVDs used in this invention form part of the polypeptide of this invention, the polypeptide containing at least four ISVDs or consisting of them, such that the polypeptide can specifically bind to IL-13 and TSLP.

[0536] Therefore, the target molecules of the at least four ISVDs used in the peptides of this invention are IL-13 and TSLP. Examples are mammalian IL-13 and TSLP. In addition to human IL-13 (Uniprot accession number P35225) and human TSLP (Uniprot accession number Q969D9), forms of IL-13 and TSLP from other species (e.g., from mice, rats, rabbits, cats, dogs, goats, sheep, horses, pigs, non-human primates (such as cynomolgus monkeys) (also referred to herein as "cyno") or camelids (such as llamas or alpacas) are also applicable to this invention.

[0537] Specific examples of ISVDs that specifically bind to IL-13 and can be used in the techniques of this invention are as described in items A and B below:

[0538] A. The following ISVD specifically binds to human IL-13 and contains

[0539] i.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that differs from SEQ ID NO: 7 by 2 or 1 amino acids;

[0540] ii. CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence differing from SEQ ID NO: 12 by 2 or 1 amino acids; and

[0541] iii. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence that differs from SEQ ID NO: 17 by 2 or 1 amino acids.

[0542] In one embodiment, the ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 7, CDR2 is the amino acid sequence of SEQ ID NO: 12, and CDR3 is the amino acid sequence of SEQ ID NO: 17.

[0543] B. The following ISVD specifically binds to human IL-13 and contains

[0544] i.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence that differs from SEQ ID NO: 8 by 2 or 1 amino acids;

[0545] ii. CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence differing from SEQ ID NO: 13 by 2 or 1 amino acids; and

[0546] iii. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that differs from SEQ ID NO: 18 by 2 or 1 amino acids.

[0547] In one embodiment, the ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 8, CDR2 is the amino acid sequence of SEQ ID NO: 13, and CDR3 is the amino acid sequence of SEQ ID NO: 18.

[0548] Examples of ISVDs that specifically bind to human IL-13 have one or more of the architectural regions shown in Table A-2 for constructs 4B02 or 4B06, respectively (and CDRs as defined in items A and B above). In one embodiment, it is an ISVD comprising or consisting of the complete amino acid sequence of construct 4B02 or construct 4B06 (SEQ ID NO: 2 and 3, respectively; see Tables A-1 and A-2).

[0549] In another embodiment, the amino acid sequence of the one or more ISVDs that specifically bind to human IL-13 may have more than 90% (e.g., more than 95% or more than 99%) sequence identity with SEQ ID NO: 2 or 3, wherein the CDR is as defined in item A or B above, respectively. In one embodiment, the ISVD that specifically binds to IL-13 comprises or is composed of the amino acid sequence of SEQ ID NO: 2 or 3.

[0550] When such an IL-13-binding ISVD has a difference of 2 or 1 amino acid relative to the corresponding reference CDR sequence in at least one CDR (item A or B above), the ISVD has at least half or at least the same binding affinity to human IL-13 compared to the constructs 4B02 or 4B06 shown in SEQ ID NO: 2 or 3, respectively, wherein the binding affinity is measured using the same method (e.g., SPR).

[0551] Specific examples of ISVDs that can be used in the technology of this invention and specifically combined with TSLP are as described in items C and D below:

[0552] C. The following ISVD, which specifically binds to human TSLP and contains

[0553] i.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence that differs from SEQ ID NO: 9 by 2 or 1 amino acids;

[0554] ii. CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence differing from SEQ ID NO: 14 by 2 or 1 amino acids; and

[0555] iii. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence that differs from SEQ ID NO: 19 by 2 or 1 amino acids.

[0556] In one embodiment, the ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 9, CDR2 is the amino acid sequence of SEQ ID NO: 14, and CDR3 is the amino acid sequence of SEQ ID NO: 19.

[0557] D. The following ISVD specifically binds to human TSLP and contains

[0558] i.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence that differs from SEQ ID NO: 11 by 2 or 1 amino acids;

[0559] ii. CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence differing from SEQ ID NO: 16 by 2 or 1 amino acids; and

[0560] iii. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence that differs from SEQ ID NO: 21 by 2 or 1 amino acids.

[0561] In one embodiment, the ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 11, CDR2 is the amino acid sequence of SEQ ID NO: 16, and CDR3 is the amino acid sequence of SEQ ID NO: 21.

[0562] Examples of ISVDs that specifically bind to human TSLP have one or more of the architectural regions (and CDRs as defined in items C and D above) shown in Table A-2 for constructs 501A02 and 529F10, respectively. In one embodiment, it is an ISVD comprising or consisting of the complete amino acid sequence of construct 501A02 or 529F10 (SEQ ID NO: 4 or 6; see Tables A-1 and A-2).

[0563] In another embodiment, the amino acid sequence of one or more ISVDs that specifically bind to human TSLP may have more than 90% (e.g., more than 95% or more than 99%) sequence identity with SEQ ID NO: 4 or 6, respectively, wherein the CDR is as defined in the preceding items C or D. In one embodiment, the ISVD that binds to human TSLP comprises or is composed of the amino acid sequence of SEQ ID NO: 4 or 6.

[0564] When such an ISVD that binds to human TSLP has a difference of 2 or 1 amino acid relative to the corresponding reference CDR sequence in at least one CDR (item C or D above), the ISVD has at least half or at least the same binding affinity to human TSLP compared to constructs 501A02 or 529F10 shown in SEQ ID NO: 4 and 6, respectively, wherein the binding affinity is measured using the same method (e.g., SPR).

[0565] In one implementation, each ISVD as defined by items A through D above is included in the polypeptide of the present invention.

[0566] Compared to the polypeptide composed of amino acids of SEQ ID NO: 1, such a polypeptide of the present invention comprising each ISVD defined under items A to D above has at least half or at least the same binding affinity for human IL-13 and human TSLP, wherein the binding affinity is measured using the same method (e.g., SPR).

[0567] The SEQ ID NO mentioned in items A through D above and item E below (see Section 5.4 “(In vivo) half-life extension”) is based on the CDR definition as defined by AbM (see Table A-2). It should be noted that the SEQ ID NO of the same CDR as defined by Kabat (see Table A-2.1) can also be used for items A through D above and item E below (see Section 5.4 “(In vivo) half-life extension”).

[0568] Therefore, the specific examples of ISVDs that specifically bind to IL-13 or TSLP and can be used in the present invention, as described above using the AbM definition, can also be described using the Kabat definition as shown in the following items A' to D':

[0569] A'. The following ISVD specifically binds to human IL-13 and contains

[0570] i.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 37 or an amino acid sequence that differs from SEQ ID NO: 37 by 2 or 1 amino acids;

[0571] ii. CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 42 or an amino acid sequence differing from SEQ ID NO: 42 by 2 or 1 amino acids; and

[0572] iii. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence that differs from SEQ ID NO: 17 by 2 or 1 amino acids.

[0573] In one embodiment, the ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 37, CDR2 is the amino acid sequence of SEQ ID NO: 42, and CDR3 is the amino acid sequence of SEQ ID NO: 17.

[0574] B'. The following ISVD specifically binds to human IL-13 and contains

[0575] i.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 38 or an amino acid sequence that differs from SEQ ID NO: 38 by 2 or 1 amino acids;

[0576] ii. CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 43 or an amino acid sequence differing from SEQ ID NO: 43 by 2 or 1 amino acids; and

[0577] iii. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that differs from SEQ ID NO: 18 by 2 or 1 amino acids.

[0578] In one embodiment, the ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 38, CDR2 is the amino acid sequence of SEQ ID NO: 43, and CDR3 is the amino acid sequence of SEQ ID NO: 18.

[0579] Examples of one or more ISVDs that specifically bind to human IL-13 have one or more of the architectural regions shown in Table A-2-1 for constructs 4B02 or 4B06, respectively (and CDRs as defined in the preceding items A' and B', respectively). In one embodiment, it is an ISVD comprising or consisting of the complete amino acid sequence of construct 4B02 or construct 4B06 (SEQ ID NO: 2 and 3, respectively; see Tables A-1 and A-2-1).

[0580] C'. The following ISVD specifically binds to human TSLP and contains

[0581] i.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 39 or an amino acid sequence that differs from SEQ ID NO: 39 by 2 or 1 amino acids;

[0582] ii. CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 44 or an amino acid sequence differing from SEQ ID NO: 44 by 2 or 1 amino acids; and

[0583] iii. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence that differs from SEQ ID NO: 19 by 2 or 1 amino acids.

[0584] In one embodiment, the ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 39, CDR2 is the amino acid sequence of SEQ ID NO: 44, and CDR3 is the amino acid sequence of SEQ ID NO: 19.

[0585] D'. The following ISVD specifically binds to human TSLP and contains

[0586] i.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 41 or an amino acid sequence that differs from SEQ ID NO: 41 by 2 or 1 amino acids;

[0587] ii. CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 46 or an amino acid sequence differing from SEQ ID NO: 46 by 2 or 1 amino acids; and

[0588] iii. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence that differs from SEQ ID NO: 21 by 2 or 1 amino acids.

[0589] In one embodiment, the ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 41, CDR2 is the amino acid sequence of SEQ ID NO: 46, and CDR3 is the amino acid sequence of SEQ ID NO: 21.

[0590] Examples of one or more ISVDs that specifically bind to human TSLP have one or more of the architectural regions (and CDRs as defined in items C' and D' above, respectively) shown in Table A-2-1 for constructs 501A02 and 529F10. In one embodiment, it is an ISVD comprising or consisting of the complete amino acid sequence of construct 501A02 or 529F10 (SEQ ID NO: 4 or 6; see Tables A-1 and A-2-1).

[0591] The percentage of “sequence identity” between the first amino acid sequence and the second amino acid sequence can be calculated as follows: ([the number of amino acid residues in the first amino acid sequence that are the same as the corresponding amino acid residues in the second amino acid sequence] / [the total number of amino acid residues in the first amino acid sequence]) × 100%, where each deletion, insertion, substitution or addition of amino acid residues in the second amino acid sequence compared to the first amino acid sequence is considered as a difference at a single amino acid residue (i.e., a single position).

[0592] Typically, in order to determine the percentage of “sequence identity” between two amino acid sequences according to the calculation method outlined above, the amino acid sequence with the largest number of amino acid residues is designated as the “first” amino acid sequence, and the other amino acid sequence is designated as the “second” amino acid sequence.

[0593] As used herein, "amino acid difference" refers to the deletion, insertion, or substitution of a single amino acid residue relative to a reference sequence. In one embodiment, "amino acid difference" is substitution.

[0594] In one implementation, the amino acid substitution is a conservative substitution. Such a conservative substitution refers to a substitution in which one amino acid in the following groups (a)-(e) is replaced by another amino acid residue in the same group: (a) small aliphatic, nonpolar or weakly 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.

[0595] In one implementation, the conservative substitutions are as follows: Ala is substituted with Gly or Ser; Arg is substituted with Lys; Asn is substituted with Gln or His; Asp is substituted with Glu; Cys is substituted with Ser; Gln is substituted with Asn; Glu is substituted with Asp; Gly is substituted with Ala or Pro; His is substituted with Asn or Gln; Ile is substituted with Leu or Val; Leu is substituted with Ile or Val; Lys is substituted with Arg, Gln, or Glu; Met is substituted with Leu, Tyr, or Ile; Phe is substituted with Met, Leu, or Tyr; Ser is substituted with Thr; Thr is substituted with Ser; Trp is substituted with Tyr; Tyr is substituted with Trp; and / or Phe is substituted with Val, Ile, or Leu.

[0596] 5.3 Specificity

[0597] The terms “specific,” “specifically binding,” or “specifically binding” refer to the number of different target molecules (such as antigens) from the same organism that a particular binding unit (such as an ISVD) can bind with a sufficiently high affinity (see below). “Specific,” “specifically binding,” or “specifically binding” are used interchangeably with “selective,” “selectively binding,” or “selectively binding” herein. A binding unit (such as an ISVD) binds specifically to its designated target.

[0598] The specificity / selectivity of binding units can be determined based on affinity. Affinity represents the strength or stability of molecular interactions. Affinity is usually given by KD or the dissociation constant, with units of mol / L (or M). Affinity can also be expressed as the association constant KA, which is equal to 1 / KD and has units of (mol / L). -1 (or M) -1 ).

[0599] Affinity is a measure of the strength of binding between a part of the target molecule and its binding site: the lower the KD value, the stronger the binding between the target molecule and the target part.

[0600] Typically, the binding unit (such as ISVD) used in this invention binds to its target with the following dissociation constant (KD): 10 -5 Up to 10 -12 mol / L or less, or 10 -7 Up to 10 -12 mol / L or less, or 10 -8 Up to 10 -12 mol / L (i.e., the association constant (KA) is 10) 5 Up to 10 12 L / mol or greater, or 10 7 Up to 10 12 L / mol or greater, or 10 8 Up to 10 12 L / mol).

[0601] It is generally considered that anything greater than 10 -4 KD value of mol / L (or any value less than 10 mol / L) 4 The KA value (L / mol) indicates nonspecific binding.

[0602] KD, which is considered to have specific biological interactions (such as the binding of immunoglobulin sequences to antigens), is typically in the range of 10. -5 mol / L (10000nM or 10μM) to 10 -12 Within the range of mol / L (0.001 nM or 1 pM) or smaller.

[0603] Therefore, specific / selective binding may mean that, using the same measurement method (e.g., SPR), the binding unit (or the polypeptide containing the binding unit) binds at 10... -5 Up to 10 -12 KD values ​​of mol / L or lower bind with IL13 and / or TSLP, and at a concentration greater than 10 mol / L. -4A KD value of mol / L binds to relevant cytokines. An example of an IL13-related target is human IL4. An example of a TSLP-related cytokine is human IL7. Therefore, in embodiments of the present invention, at least two ISVDs contained in the polypeptide bind at a concentration of 10 mol / L. -5 Up to 10 -12 KD values ​​of mol / L or lower bind to IL13 and at a concentration greater than 10 mol / L. -4 The KD value of mol / L binds to IL4 of the same species, and at least two ISVDs contained in the polypeptide are 10 -5 Up to 10 -12 KD values ​​of mol / L or less bind with TSLP and at a concentration greater than 10 -4 The KD value of mol / L is associated with the binding of human IL7 of the same species.

[0604] Therefore, compared with the polypeptide composed of the amino acids of SEQ ID NO: 1, the polypeptide of the present invention has at least half or at least the same binding affinity for human IL13 and human TSLP, wherein the binding affinity is measured using the same method (such as SPR).

[0605] Specific binding to a specific target from a particular species does not preclude the binding unit from specifically binding to similar targets from different species. For example, specific binding to human IL13 does not preclude the binding unit (or a polypeptide containing the binding unit) from specifically binding to IL13 from cynomolgus monkeys. Similarly, specific binding to human TSLP, for example, does not preclude the binding unit (or a polypeptide containing the binding unit) from specifically binding to TSLP from cynomolgus monkeys (“cyno”).

[0606] The binding unit to its designated target specificity can be determined by any suitable means known per se (including, for example, Scatchard analysis and / or competitive binding assays (such as radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competitive assays and their various variants known in the art); as well as other techniques mentioned herein.

[0607] The dissociation constant can be either actual or apparent, as will be apparent to those skilled in the art. Methods for determining the dissociation constant are clear to those skilled in the art and include, for example, the techniques mentioned below. In this regard, it will also be clear that it may be impossible to measure a value greater than 10. -4 mol / L or 10 -3 mol / L (e.g., 10 mol / L) -2The dissociation constant (mol / L). Optionally, as those skilled in the art will also know, the (actual or apparent) dissociation constant can be calculated based on the (actual or apparent) association constant (KA) through the relationship [KD = 1 / KA].

[0608] The affinity of molecular interactions between two molecules can be measured using various techniques known to them, such as the well-known surface plasmon resonance (SPR) biosensor technique (see, for example, Ober et al. 2001, Intern. Immunology 13: 1551-1559). As used herein, the term “surface plasmon resonance” refers to an optical phenomenon that allows for the analysis of real-time, biospecific interactions by detecting changes in protein concentration in a biosensor matrix, where one molecule is immobilized on a biosensor chip and another molecule flows through the immobilized molecule under flow conditions, thereby generating kon, k off Measured values, and thus K. D (or K) A ) value. For example, this can be done using well-known values. The system (BIAcore International AB, GE Healthcare, Uppsala, Sweden and Piscatave, New Jersey) was used. For further description, see Jonsson et al. (1993, Ann. Biol. Clin. 51: 19-26), Jonsson et al. (1991 Biotechniques 11: 620-627), Johnson et al. (1995, J. Mol. Recognit. 8: 125-131) and Johnson et al. (1991, Anal. Biochem. 198: 268-277).

[0609] Another well-known biosensor technique for determining the affinity of biomolecular interactions is biolayer interferometry (BLI) (see, for example, Abdiche et al. 2008, Anal. Biochem. 377: 209-217). As used herein, the term “biolayer interferometry” or “BLI” refers to a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and an immobilized protein layer on the biosensor tip (signal beam). Variations in the number of molecules bound to the biosensor tip result in a shift in the interference pattern, reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the surface of the biosensor tip. Because interactions can be measured in real time, association and dissociation rates, as well as affinity, can be determined. For example, BLI can use well-known... The system (ForteBio, a division of Pall Life Sciences, Menlo Park, USA) is used.

[0610] Alternatively, it can be used in kinetic repulsion determination (KinExA) (see, for example, Drake et al. 2004, Anal. Biochem., 328: 35-43). The platform (Sapidyne Instruments Inc., Boise, USA) measures affinity. As used herein, the term "KinExA" refers to a solution-based method for measuring the true equilibrium binding affinity and kinetics of unmodified molecules. An equilibrium solution of an antibody / antigen complex is passed through a column containing beads pre-coated with an antigen (or antibody) to allow the free antibody (or antigen) to bind to the coated molecule. Detection of the thus captured antibody (or antigen) is accomplished using a fluorescently labeled protein that binds to the antibody (or antigen).

[0611] Immunoassay systems provide a platform for automated bioanalysis and rapid sample turnaround (Fraley et al. 2013, Bioanalysis 5: 1765-74).

[0612] 5.4 (In vivo) prolonged half-life

[0613] The polypeptide may also include one or more other groups, residues, portions, or binding units optionally linked via one or more peptide linkers, wherein the one or more other groups, residues, portions, or binding units provide an increased (in vivo) half-life to the polypeptide compared to a corresponding polypeptide without said one or more other groups, residues, portions, or binding units. An extended in vivo half-life means, for example, that the polypeptide has an increased half-life in mammalian subjects such as humans after administration. Half-life may be expressed, for example, as t1 / 2β.

[0614] The types of groups, residues, parts or binding units are generally unrestricted and may be selected, for example, from polyethylene glycol molecules, serum proteins or fragments thereof, binding units that can bind to serum proteins, Fc parts and small proteins or peptides that can bind to serum proteins.

[0615] More specifically, the one or more other groups, residues, portions, or binding units that provide an increased half-life for the polypeptide may be selected from binding units that can bind to serum albumin (such as human serum albumin) or serum immunoglobulin (such as IgG). In one embodiment, the one or more other binding units that provide an increased half-life for the polypeptide are binding units that can bind to human serum albumin. In one embodiment, the binding unit is ISVD.

[0616] For example, WO 04 / 041865 describes binding to serum albumin (and specifically to human serum albumin). It can be linked to other proteins (such as one or more other nanobodies that bind to the desired target) to increase the half-life of the protein.

[0617] International application WO 06 / 122787 describes numerous studies targeting (human) serum albumin. These Including Alb-1 (SEQ ID NO: 52 in WO 06 / 122787) And its humanized variants, such as Alb-8 (SEQ ID NO: 62 in WO 06 / 122787). Furthermore, these can be used to extend the half-life of therapeutic proteins and peptides, as well as other therapeutic entities or portions.

[0618] In addition, WO 2012 / 175400 describes another improved form of Alb-1, called Alb-23.

[0619] 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 WO 2012 / 175400, and albumin binders are described in WO2012 / 175741, WO 2015 / 173325, WO 2017 / 080850, WO 2017 / 085172, WO2018 / 104444, WO 2018 / 134235, and WO 2018 / 134234. Some serum albumin binders are also shown in Table A-4. In one embodiment, an additional component of the polypeptide of the present invention is as described in item E:

[0620] E. The following ISVD, which binds to human serum albumin and contains

[0621] i.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that differs from SEQ ID NO: 10 by 2 or 1 amino acids;

[0622] ii. CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence differing from SEQ ID NO: 15 by 2 or 1 amino acids; and

[0623] iii. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence that differs from SEQ ID NO: 20 by 2 or 1 amino acids.

[0624] In one embodiment, the ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 10, CDR2 is the amino acid sequence of SEQ ID NO: 15, and CDR3 is the amino acid sequence of SEQ ID NO: 20.

[0625] Examples of such ISVDs that bind to human serum albumin have one or more or all of the architectural regions shown in Table A-2 for construct ALB23002 (as well as CDRs as defined in the preceding item E). In one embodiment, it is an ISVD comprising or consisting of the complete amino acid sequence of construct ALB23002 (SEQ ID NO: 5; see Tables A-1 and A-2).

[0626] Alternatively, the Kabat definition can be used to describe project E as follows:

[0627] E'. The following ISVD, which binds to human serum albumin and contains

[0628] i.CDR1, wherein CDR1 is the amino acid sequence of SEQ ID NO: 40 or an amino acid sequence that differs from SEQ ID NO: 40 by 2 or 1 amino acids;

[0629] ii. CDR2, wherein CDR2 is the amino acid sequence of SEQ ID NO: 45 or an amino acid sequence differing from SEQ ID NO: 45 by 2 or 1 amino acids; and

[0630] iii. CDR3, wherein CDR3 is the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence that differs from SEQ ID NO: 20 by 2 or 1 amino acids.

[0631] In one embodiment, the ISVD comprises CDR1, CDR2, and CDR3, wherein CDR1 is the amino acid sequence of SEQ ID NO: 40, CDR2 is the amino acid sequence of SEQ ID NO: 45, and CDR3 is the amino acid sequence of SEQ ID NO: 20.

[0632] Examples of ISVDs that bind to human serum albumin have one or more or all of the architectural regions (and CDRs as defined in the preceding item E') shown in Table A-2-1 for construct ALB23002. In one embodiment, it is an ISVD comprising or consisting of the complete amino acid sequence of construct ALB23002 (SEQ ID NO: 5; see Tables A-1 and A-2-1).

[0633] In another embodiment, the amino acid sequence of the ISVD that binds to human serum albumin may have more than 90% (e.g., more than 95% or more than 99%) sequence identity with SEQ ID NO: 5, wherein the CDR is as defined by 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.

[0634] When such an ISVD that binds to human serum albumin has a difference of 2 or 1 amino acid relative to the corresponding reference CDR sequence in at least one CDR (item E above), the ISVD has at least half or at least the same binding affinity to human serum albumin compared to the construct ALB23002 shown in SEQ ID NO: 5, wherein the binding affinity is measured using the same method (e.g., SPR).

[0635] In one embodiment, when the ISVD that binds to human serum albumin has a C-terminal position, it exhibits a C-terminal alanine (A) or glycine (G) extension. In one embodiment, the ISVD is selected from SEQ ID NO: 59, 60, 62, 64, 65, 66, 67, 68, 69, and 71 (see Table A-4 below). In one embodiment, the ISVD that binds to human serum albumin has a position other than the stated C-terminal position (i.e., a C-terminal ISVD that is not a peptide of the present invention). In one embodiment, the ISVD is selected from SEQ ID NO: 5, 57, 58, 61, and 63 (see Table A-4 below).

[0636] 5.5 Nucleic acid molecules

[0637] Nucleic acid molecules encoding the polypeptides of the present invention are also provided.

[0638] A “nucleic acid molecule” (used interchangeably with “nucleic acid”) is a chain of monomeric nucleotides linked together by a phosphate backbone to form a nucleotide sequence. Nucleic acids can be used to transform / transfect host cells or host organisms, for example, to express and / or produce polypeptides. Suitable hosts or host cells for the purpose of production will be clear 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. Hosts or host cells containing nucleic acids encoding polypeptides of the present invention are also covered by the present invention.

[0639] Nucleic acids can be, for example, DNA, RNA, or hybrids thereof, and may also contain (e.g., chemically modified) nucleotides, such as PNA. They can be single-stranded or double-stranded. In one embodiment, it is in the form of double-stranded DNA. For example, the nucleotide sequence of the present invention can be genomic DNA or cDNA.

[0640] The nucleic acids of the present invention can be prepared or obtained in a manner known per se, and / or isolated from suitable natural sources. Nucleotide sequences encoding naturally occurring (poly)peptides can, for example, be subjected to site-directed mutagenesis to provide nucleic acid molecules encoding polypeptides with sequence variations. Furthermore, it will be apparent to those skilled in the art that, in order to prepare nucleic acids, several nucleotide sequences (such as at least one nucleotide sequence encoding a target moiety) can be linked together in a suitable manner with nucleic acids, for example, encoding one or more adapters.

[0641] Techniques for generating nucleic acids will be clear to those skilled in the art and may include, for example, but not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and / or synthetic sequences (or two or more portions thereof) to introduce mutations that result in the expression of truncated expression products; introducing one or more restriction sites (e.g., to generate easily digestible and / or linked boxes and / or regions using suitable restriction enzymes); and / or introducing mutations via PCR reactions using one or more “mismatched” primers.

[0642] 5.6 Carrier

[0643] Vectors comprising nucleic acid molecules encoding polypeptides of the present invention are also provided. Vectors, as used herein, are suitable agents for carrying genetic material into cells. Vectors include naked nucleic acids (such as plasmids or mRNA) or nucleic acids embedded in larger structures (such as liposomes or viral vectors).

[0644] Vectors typically contain at least one nucleic acid optionally linked to one or more regulatory components, 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 a encoded polypeptide or construct under suitable conditions, such as when the vector is introduced into a cell (e.g., human). For DNA-based vectors, this typically includes the presence of components for transcription (e.g., promoters and poly-A signals) and translation (e.g., Kozak sequences).

[0645] In one embodiment, within the vector, the at least one nucleic acid and the regulatory component are "operably linked" to each other, which generally means that they are in a functional relationship. For example, a promoter is considered "operably linked" to a coding sequence (where the coding sequence should be understood as being "under the control" of the promoter) if it is capable of initiating or otherwise controlling / regulating transcription and / or expression of the coding sequence. Typically, when two nucleotide sequences are operably linked, they will be in the same orientation and usually in the same reading frame. They are usually also substantially sequential, although this may not be necessary.

[0646] In one embodiment, any regulatory components of the vector enable it to provide its intended biological function in the intended host cell or host organism.

[0647] For example, a promoter, enhancer, or terminator should be "operable" in the intended host cell or host organism, meaning, for example, that the promoter should be able to initiate or otherwise control / regulate the transcription and / or expression of a nucleotide sequence (e.g., a coding sequence) to which it is operably linked.

[0648] 5.7 Composition

[0649] The present invention also provides a composition comprising at least one polypeptide of the present invention, at least one nucleic acid molecule encoding the polypeptide of the present invention, or at least one carrier containing such nucleic acid molecule. The composition may be a pharmaceutical composition. The composition may further comprise at least one pharmaceutically acceptable carrier, diluent, or excipient and / or adjuvant, and optionally comprise one or more other pharmaceutically active polypeptides and / or compounds.

[0650] 5.8 Host organism

[0651] The present invention also relates to host cells or host organisms, wherein the host cells or host organisms contain polypeptides of the present invention, nucleic acids encoding polypeptides of the present invention, and / or carriers containing nucleic acid molecules encoding polypeptides of the present invention.

[0652] Suitable host cells or host organisms will be clear to those skilled in the art, and are, 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*.

[0653] 5.9 Methods and Applications of Peptides

[0654] The present invention also provides a method for generating the polypeptide of the present invention. The method may include transforming / transfecting a host cell or host organism with a nucleic acid encoding the polypeptide, expressing the polypeptide in the host, and optionally following one or more isolation and / or purification steps. Specifically, the method may include:

[0655] a) Expressing the nucleic acid sequence encoding the polypeptide in a suitable host cell or host organism or in another suitable expression system; optionally followed by:

[0656] b) Isolate and / or purify the polypeptide.

[0657] Suitable host cells or host organisms for the purpose of production will be clear 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.

[0658] The polypeptides, nucleic acid molecules or carriers described herein, or compositions comprising the polypeptides, nucleic acid molecules or carriers of the present invention can be used as pharmaceuticals.

[0659] Therefore, the present invention provides polypeptides of the present invention, nucleic acid molecules or carriers as described herein, or compositions comprising polypeptides, nucleic acid molecules or carriers of the present invention for use as pharmaceuticals.

[0660] Also provided are polypeptides of the present invention for (preventive or therapeutic) treatment of inflammatory diseases, nucleic acid molecules or carriers as described herein, or compositions comprising polypeptides, nucleic acid molecules or carriers of the present invention.

[0661] Methods for treating inflammatory diseases (preventive and / or therapeutic) are also provided, wherein the methods include administering to a subject in need a pharmaceutically active amount of a polypeptide of the present invention, a nucleic acid molecule or carrier as described herein, or a composition comprising a polypeptide, nucleic acid molecule or carrier of the present invention.

[0662] The invention also provides the use of polypeptides, nucleic acid molecules or carriers as described herein, or compositions comprising polypeptides, nucleic acid molecules or carriers of the invention in the preparation of pharmaceutical compositions. In one embodiment, the prepared pharmaceutical composition is used to treat inflammatory diseases.

[0663] The inflammatory diseases mentioned are type 2 inflammatory diseases, such as atopic dermatitis and asthma.

[0664] In the context of this invention, the term "subject" can refer to any animal, and more specifically, a mammal. Among mammals, a distinction can be made between human and non-human mammals. Non-human animals can be, for example, companion animals (e.g., dogs, cats), livestock (e.g., cattle, horses, sheep, goats, or pigs), or animals commonly used for research purposes and / or for antibody production (e.g., mice, rats, rabbits, cats, dogs, goats, sheep, horses, pigs, non-human primates (e.g., cynomolgus monkeys), or camelids (e.g., llamas or alpacas)).

[0665] In the context of preventative and / or therapeutic purposes, the subject can be any animal, and more specifically, any mammal. In one embodiment, the subject is a human subject.

[0666] Substances (including peptides, nucleic acid molecules, and carriers) or compositions may be administered to a subject via any suitable route of administration, such as enteric (e.g., oral or rectal) or parenteral (e.g., epidermal, sublingual, buccal, nasal, intra-articular, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, percutaneous, or transmucosal). In one embodiment, the substance is administered via parenteral administration (e.g., intramuscular, subcutaneous, or intradermal). In one embodiment, subcutaneous administration is used.

[0667] An effective amount of a polypeptide, a nucleic acid molecule or carrier as described herein, or a composition comprising said polypeptide, nucleic acid molecule or carrier may be administered to a subject to provide the desired therapeutic outcome.

[0668] One or more doses may be administered. If more than one dose is administered, the doses may be administered at appropriate intervals to maximize the effect of the polypeptide, composition, nucleic acid molecule, or carrier.

[0669] Table A-1: ​​Different monovalent Vs identified in pentavalent polypeptide F027400161 HHThe amino acid sequence of the building block (“ID” refers to SEQ ID NO as used herein)

[0670]

[0671]

[0672]

[0673]

[0674] Table A-4: ISVD sequences of serum albumin (“ID” refers to SEQ ID NO as used herein)

[0675]

[0676]

[0677] Table A-5: Connector Sequences (“ID” refers to SEQ ID NO as used herein)

[0678]

[0679]

[0680] Table A-6: Amino acid sequences of selected single-specific multivalent peptides (“ID” refers to the given SEQ ID NO)

[0681]

[0682]

[0683]

[0684]

[0685] 1 [] indicates the single-valent building blocks used for bivalent building blocks and their connections.

[0686] *() indicates the introduction of amino acid substitutions in the (parental) monovalent building block (e.g., 4B02(D1E) means that the monovalent building block 4B02(SEQ ID NO: 2) contains a D1E substitution).

[0687] 6 Examples

[0688] 6.1 Generation of monovalent ISVDs that specifically bind to IL-13 and TSLP, respectively

[0689] 6.1.1 Example 1: Immunization

[0690] Three llamas were immunized with recombinant human IL-13 (Peprotech, catalog number 200-13, derived from E. coli) according to standard protocol to induce a heavy chain antibody-dependent humoral immune response. These llamas were then boosted with human / cynomolgus monkey IL-13 (Sino Biological, catalog numbers 10369-HNAC and 11057-CNAH, derived from mammalian cells) from another source.

[0691] Three more llamas were immunized with recombinant hTSLP-Fc. These llamas were then boosted with cynomolgus monkey TSLP-Fc. Two more llamas were immunized alternately with hTSLP and cynomolgus monkey TSLP-Fc.

[0692] Immunoblood (PBL) samples were collected at regular intervals, and total RNA was prepared from isolated B cells. Humoral immune responses during immunization were monitored by comparing antigen-specific serum titers of samples collected before the start of immunization with those typically collected after multiple antigen administrations. Briefly, 96-well Maxisorp plates were coated with human IL-13 (SinoBiological, catalog 10369-HNAC) or human TSLP. Recombinant human TSLP is commercially available, such as from R&D Systems (catalog 1398-TS). The presence of anti-IL-13 and anti-TSLP ISVD was demonstrated by enzymatic reactions using HRP (horseradish peroxidase)-conjugated goat anti-lamb immunoglobulin (Bethyl Laboratories Inc.) and subsequently in the presence of the substrate TMB (3,3',5,5'-tetramethylbenzidine) after blocking and addition of diluted serum samples.

[0693] 6.1.2 Example 2: Library Construction and Phage Display Selection

[0694] Peripheral blood mononuclear cells were prepared from blood samples using Ficoll-Hypaque according to the manufacturer's instructions. Total RNA extracted from these cells and lymph nodes was used as starting material for RT-PCR to amplify gene fragments encoding ISVD. These fragments were cloned into the phage vector pAX212. Phages were prepared according to a standard protocol (Antibody Phage Display: Methods and Protocols (1st edition, 2002, edited by O'Brian and Aitken, Humana Press, Totoro, NJ)) and stored after filtration sterilization at 4°C until further use. Five phage libraries targeting IL13 were constructed, with a library size of 6.1 x 10⁻⁶. 8 With 1.3 x 10 9The percentage of inserted fragments ranged from 87% to 96%. Five phage libraries targeting TSLP were constructed, with library sizes ranging from 4.2 x 10⁻⁶. 8 With 9.8 x 10 8 The percentage of inserted segments is between 91% and 100%.

[0695] To identify the ISVD recognizing human and cynomolgus monkey IL-13, a phage library was cultured with 50 nM soluble biotinylated hIL13-Fc in the presence of IgG from human serum (Sigma, I4506). Complexes of hIL-13-Fc and phages were captured from the solution on streptavidin-coated magnetic beads. After extensive washing with PBS / 0.05% Tween 20, the bound phages were eluted by adding trypsin (1 mg / ml). These first-round selected outputs were cultured with 0.05 nM, 0.5 nM, or 5 nM soluble biotinylated hIL-13-Fc or cynomolgus monkey IL-13-Fc. In the third round, the unenriched outputs from the second-round selection were further cultured with 0.005 nM, 0.05 nM, 0.5 nM, or 5 nM soluble biotinylated human or cynomolgus monkey IL-13-Fc. Select individual clones from the enriched second and third rounds of selection.

[0696] To identify the ISVD recognizing human and cynomolgus monkey TSLPs, phage libraries were cultured with 50 nM soluble biotinylated hTSLP-Fc in the presence of human serum IgG (Sigma, I4506), or with 500 nM biotinylated hTSLP, or with 500 nM biotinylated cynomolgus monkey TSLP. Human and cynomolgus monkey TSLP sequences were known (Uniprot accession number Q969D9 and NCBI RefSeq XP_005557555.1, respectively). The assays were performed using recombinant proteins. TSLP-phage complexes were captured from solution on streptavidin-coated magnetic beads. After extensive washing with PBS / 0.05% Tween 20, bound phages were eluted by adding trypsin (1 mg / ml). These first-round selected outputs were cultured with 5 nM soluble biotinylated hTSLP-Fc, cynomolgus monkey TSLP-Fc, or 0.5 nM biotinylated hTSLP. The outputs from the second round of selection were cultured with 0.05 nM, 0.5 nM, or 5 nM soluble biotinylated human TSLP, TSLP-Fc, or cynomolgus monkey TSLP-Fc. From each round, individual clones were selected from the enriched outputs.

[0697] All individual clones were grown in 96-well plates (1 ml volume). ISVD expression was induced by adding IPTG to a final concentration of 1 mM. Periplasmic extract was prepared by freezing the cell pellet and dissolving it in 100 μl PBS. Cell debris was removed by centrifugation.

[0698] As a control, selected periplasmic extracts were screened in ELISA for binding to human and cynomolgus monkey IL13-Fc and TSLP-Fc, respectively. Evaluation was performed in 25 μl vials in Spectraplate 384-HB (PerkinElmer). The antigen was coated overnight at 4°C with 1 μg / ml in PBS. Wells were blocked with casein solution (1%). ISVD binding was detected using mouse anti-Flag-HRP (Sigma) followed by an enzymatic reaction in the presence of the substrate esTMB (3,3',5,5'-tetramethylbenzidine) after the addition of a 5-fold dilution of the periplasmic extract.

[0699] 6.1.3 Example 3: Screening for blocking ISVD in periplasmic extracts using human IL13 and human TSLP via AlphaScreen assay

[0700] To determine the blocking ability of ISVD, crude periplasmic extracts were screened in different protein-based competitive assays using AlphaScreen technology (PerkinElmer, Waltham, MA). Fluorescence was measured using an EnVision Multilabel reader (PerkinElmer) at an excitation wavelength of 680 nm and an emission wavelength of 520 nm.

[0701] In the hIL-13:hIL-13Rα1 binary complex AlphaScreen, we investigated whether ISVD could block the interaction between hIL-13 and the extracellular domain of hIL-13Rα1. For this purpose, a dilution of the periplasmic extract was pre-cultured with biotinylated hIL-13 (Peprotech catalog 200-13). To this mixture, hIL-13Rα1-hFc (R&Dsystems; catalog 146-IR) and anti-hFc-conjugated acceptor beads were added, and the mixture was further incubated at room temperature for 1 hour, followed by the addition of streptavidin-conjugated donor beads and incubation for another hour. When the binary complex formed, the acceptor and donor beads approached each other, and a detectable signal was generated upon laser excitation. The decrease in the AlphaScreen signal indicated that the binding of biotinylated hIL-13 to hIL-13Rα1 was blocked by ISVD present 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).

[0702] In the hIL-13:hIL-13Rα1:hIL4Rα ternary complex AlphaScreen, ISVD was screened to determine whether it could block the recruitment of hIL4Rα to the hIL13:hIL13Rα1 binary complex. hIL-13 binds to hIL-13Rα1, and this binary complex recruits hIL4Rα, resulting in the formation of the ternary complex hIL-13:hIL-13Rα1:hIL4Rα. A dilution of the periplasmic extract was pre-cultured with hIL-13 (Peprotech catalog 200-13) and biotinylated huIL4Rα (R&D Systems; catalog 230-4R / CF). This mixture was then added to hIL-13Rα1-hFc (R&D Systems; catalog 146-IR) and anti-hFc-conjugated acceptor beads, and further cultured at room temperature for 1 hour, followed by the addition of streptavidin-conjugated donor beads and an additional 1 hour of culture. When the ternary complex is formed, the acceptor and donor beads approach each other and generate a detectable signal upon laser excitation. A decrease in the AlphaScreen signal indicates that the formation of the ternary complex is blocked by ISVD present 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).

[0703] Based on Alphascreen analysis (Tables 3 and 4), many blocking ISVDs were selected and sequenced (Tables 1 and 2).

[0704] Table 1: Amino acid sequence of functional anti-IL-13ISVD.

[0705]

[0706] Table 2: Amino acid sequences of functional anti-TSLP ISVD.

[0707]

[0708] 6.1.4 Example 4: Surface plasmon resonance analysis of IL13 and TSLP periplasmic extracts

[0709] The dissociation rate of periplasmic extracts containing anti-IL13 or anti-TSLP ISVD was measured by surface plasmon resonance (SPR) using a Proteon XPR36 instrument (Bio-Rad Laboratories, Inc.). Phosphate-buffered saline (PBS) supplemented with 0.005% Tween 20 (pH 7.4) was used as the run buffer, and experiments were conducted at 25°C.

[0710] hIL13-Fc, cynomolgus monkey IL13-Fc, hTSLP-Fc, and cynomolgus monkey TSLP-Fc were immobilized on a ProteOn GLC sensor chip at a flow rate of 30 μl / min using EDC and NHS for activation. IL13 protein was injected at 10 μg / mL into ProteOn acetate buffer at pH 5.0. TSLP protein was injected at 5 μg / mL into ProteOn acetate buffer at pH 5.5. After immobilization, the surface was inactivated with ethanolamine.

[0711] The periplasmic extract of the ISVD candidate was diluted 10-fold in PBS-Tween 20 (0.1%) and injected at 45 μl / min for 2 min, allowing dissociation for 900 sec. Between different samples, surface regeneration was achieved by injecting phosphate (0.425%) at 45 μl / min for 2 min in the case of IL13, or by injecting glycine (5 mM) / SDS (0.25%) at pH 3.0 at 45 μl / min for 1 min in the case of TSLP. Dissociation rates were calculated from sensor maps obtained for different periplasmic extracts.

[0712] Table 3 shows the dissociation rate analysis of hIL-13-Fc and cynomolgus monkey IL-13-Fc.

[0713] Table 3: Summary of screening results for anti-IL-13ISVD F0107004B02 and F0107004B06.

[0714]

[0715] Table 4 shows the dissociation rate analysis of hTSLP-Fc and cynomolgus monkey TSLP-Fc.

[0716] Table 4: Summary of screening results for anti-TSLP ISVD F0107501A02 and F0107529F10.

[0717]

[0718] 6.1.5 Example 5: Expression and purification of anti-IL-13 and anti-TSLP ISVD

[0719] Anti-IL-13 ISVD and anti-TSLP ISVD were selected for expression and purification based on their blocking ability and dissociation rate values ​​in the AlphaScreen assay. The sequences are shown in Tables 1 and 2.

[0720] ISVD was expressed as a c-myc, His6-labeled protein in *E. coli* TG1 cells. Expression was induced by adding 1 mM IPTG and incubated at 37°C for 3 h. After rotating the cell culture, periplasmic extracts were prepared by freeze-thawing the precipitate and resuspending it in dPBS. These extracts were used as starting material for immobilized metal affinity chromatography (IMAC) using a Nickel Sepharose™ 6FF column (Atoll). ISVD was eluted from the column with 250 mM imidazole and subsequently desalted against dPBS. For the cell-based assays described below, endotoxins were 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.

[0721] 6.1.6 Example 6: Blocking ability of purified anti-IL13 and anti-TSLP ISVD in AlphaScreen assay

[0722] As described in Example 3, the IC50 values ​​of the purified anti-IL-13 and anti-TSLP ISVDs as described in Example 5 were determined using the binary and ternary complex AlphaScreen assay. Instead of diluents for the periplasmic extract, a series of dilutions of each purified ISVD (from 500 nM down to 1.8 pM) were pre-incubated with IL-13 or TSLP.

[0723] The tested anti-IL-13ISVD inhibitors inhibited the formation of the ternary complex and partially blocked the binary complex with IC50 values ​​as shown in Table 5.

[0724] Table 5: IC50 values ​​of blocking anti-IL13ISVD as determined in the AlphaScreen assay for binary and ternary complexes.

[0725]

[0726] The tested anti-TSLP ISVD completely inhibited the formation of ternary complexes and also inhibited the formation of binary complexes, with IC50 values ​​shown in Table 6.

[0727] Table 6: IC50 values ​​of blocking anti-TSLP ISVD determined in the AlphaScreen assay of binary and ternary complexes.

[0728]

[0729] 6.1.7 Example 7: Blocking ability of purified anti-IL-13 and anti-TSLP ISVD in cell-based assays

[0730] The inhibitory efficacy of anti-IL-13 ISVD was determined in a cell-based assay for monitoring IL-13-mediated TF-1 cell proliferation. For this purpose, 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 growth medium without GM-CSF. A serial dilution of purified anti-IL-13 ISVD or a reference compound was added. After culturing at 37°C for 15 min, 200 pM of IL-13 (Peprotech catalog number 200-13) was added. After 96 hours, the cells were analyzed using a Cell Titer 96 Aqueous One Solution assay on an EnVision Multilabel reader (Perkin Elmer).

[0731] (Promega#G3580) was used to measure the proliferation of TF-1 cells.

[0732] Table 7 shows that ISVD inhibits IL-13-induced TF1 proliferation.

[0733] Table 7: Overview of IC50 values ​​of monovalent ISVD against IL-13 in IL-13-induced TF-1 proliferation assays.

[0734] F0107004B02 2.7E-08 F0107004B06 1.0E-05 Anti-hIL-13 reference mAb2 4.1E-10 Anti-hIL-13 reference mAb3 9.2E-11 Anti-hIL-13 reference mAb1 2.2E-11

[0735] The blocking efficacy of anti-TSLP ISVD was determined in a cell-based assay monitoring TSLP-mediated proliferation of BaF3 cells transfected with plasmids encoding hTSLPR and hIL7Ra. Cells were seeded at 20,000 cells / well in RPMI 1640 growth medium in white 96-well plates treated with cell culture. A series of anti-TSLP ISVD dilutions were added, followed by stimulation with either 50 pM hTLSP-Fc or 50 pM cynomolgus TSLP-Fc. Human and cynomolgus TSLP sequences are known (Uniprot accession number Q969D9 and NCBI RefSeq XP-005557555.1, respectively). The assay was performed using recombinant proteins.

[0736] After 72 hours of incubation, use Cell density and viability were monitored using luminescent cell viability assays (Promega, G7571 / G7572 / G7573) and read out on an EnVision Multilabel reader (Perkin Elmer). The results are shown in Table 8.

[0737] Table 8: Potency and efficacy of anti-TSLP monovalent ISVD in TSLP-induced BaF3 cell proliferation assay. ND = Not measured

[0738]

[0739] 6.1.8 Example 8: Binding affinity of purified anti-IL-13 and anti-TSLP ISVD to human and cynomolgus monkey IL-13 and TSLP

[0740] The complete binding kinetics study via SPR was conducted on a BIAcore T100 instrument (GE Healthcare).

[0741] For IL-13, approximately 2000 RU of hIL13-Fc or 4000 RU of cynomolgus IL13-Fc was directly immobilized onto the CM5 sensor chip. ISVD was then injected at different concentrations (between 3 μM and 12 nM) for 120 s, followed by dissociation for 900 s. Regeneration of the hIL-13-Fc and cynomolgus IL-13-Fc surfaces was performed by injection of 0.85% H3PO4:MilliQ (1:1) for 47 s.

[0742] For TSLP, approximately 8000 RU of anti-huIgG antibody (GE Healthcare) was directly immobilized on the CM5 sensor chip. hTSLP-Fc (1 μg / mL) or cynomolgus monkey TSLP-Fc (0.75 μg / mL) was dispersed on the chip and captured for 120 s. ISVD was then injected at different concentrations (between 0.4 nM and 3000 nM) for 120 s, and dissociated for 900 s.

[0743] Binding curves were evaluated using BIAcore T100 evaluation software V2.0.3. Kinetic analysis was performed by fitting a 1:1 interaction model (Langmuir binding) (Rmax = global; RI = constant = 0, offset = 0). Interactions that did not meet the acceptance criteria of the 1:1 interaction model were fitted using a heteroligand fitting model (RI = constant = 0, offset = 0).

[0744] The kinetic data for IL-13 ISVD are shown in Table 9 and the kinetic data for TSLP ISVD are shown in Table 10.

[0745] Table 9: KD determination of monovalent anti-IL13 ISVD via SPR.

[0746]

[0747] Table 10: KD determination of monovalent anti-TSLP ISVD via SPR.

[0748]

[0749] 6.2 Generation of monospecific multivalent peptides that bind to IL-13 and TSLP, respectively

[0750] 6.2.1 Example 10: Generation and in vitro characterization of wild-type anti-IL-13 bivalent or dual complementary site ISVD constructs

[0751] The selected anti-IL-13 ISVDs (F0107004B02 and F0107004B06) were formatted as bicomplementary and divalent ISVD constructs. The construct blocks within these constructs were genetically linked via flexible 35GS (GlySer) linkers. The ISVDs were expressed in *Pichia pastoris* as a FLAG3-HIS6-tagged protein (amino acid sequences shown in Table 14). Induction of ISVD construct expression was achieved by stepwise addition of methanol. Clarified medium containing the secreted ISVD constructs was used as the starting material for immobilized metal affinity chromatography (IMAC), followed by desalting to produce a purity of 90%, as assessed by SDS-PAGE.

[0752] The bicomplementary and bivalent IL-13 constructs were characterized in binary and ternary complex blocking AlphaScreen assays (as described in Example 6) and in TF-1 proliferation assays (as described in Example 7). Table 11 shows an overview of the constructs generated and their blocking efficiencies in the binary and ternary AlphaScreen assays and in the TF-1 proliferation assay. The bicomplementary construct exhibited superior efficacy against both hIL-13 and cyIL-13, similar to the efficacy against hIL-13 reference mAb1. Furthermore, in the TF1 proliferation assay, the bivalent construct showed improved efficacy, but to a different extent than the bicomplementary construct F010700029. Additionally, the bivalent construct did not achieve complete inhibition in the IL13-IL13Rα1 Alphascreen.

[0753] Table 11: Overview of the potency of different anti-IL-13 constructs in the Alphascreen assay and an overview of their potency and efficacy in the TF-1 proliferation assay. ND = Not determined

[0754]

[0755] The most potent anti-IL-13 dual-complementary ISVD construct was tested in an IL-13-induced A549 eotaxin release assay. For this purpose, A549 suspension cells were cultured in Ham's F12K medium supplemented with 10% FCS. Cells were seeded at 200,000 cells / well in 96-well plates. On the second day, 200 pM hIL-13 (SinoBiological catalog 10369-HNAC) or cynomolgus monkey IL-13 (Sino Biological catalog 11057-CNAH) was added, followed by a serial dilution of the ISVD construct. After 24 h, eotaxin-3 was measured in the supernatant using an MSD ELISA (Human Eosinophil Chemokine-3 Tissue Culture Kit (Meso Scale, K151ABB-1)). The results are shown in Table 12.

[0756] Table 12: Potential of F010700029 in the IL-13-mediated Aa549 eosinophil chemokine release assay. Inhibition was 100%.

[0757]

[0758] A competitive ELISA was used to test whether monovalent and dual-complementary anti-IL-13 ISVD constructs inhibited the binding of hIL-13 to IL13Rα2. IL13Rα2 (SinoBiological catalog number 10350-H08H) was coated onto 384-well Spectraplate (PerkinElmer). hIL13-Fc (SinoBiological, catalog number 10369-H01H) was mixed with serial dilutions of the ISVD construct 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 mixture for 1 hour. After washing, the presence of bound IL-13-Fc was detected using an anti-human IgG-peroxidase antibody and subsequently an enzymatic reaction in the presence of the substrate esTMB. When ISVD inhibited the IL-13-IL-13Rα2 interaction, the detected IL-13-Fc disappeared in a dose-dependent manner. The results are shown in Table 13.

[0759] Table 13: Ability of anti-IL-13 monovalent and dual complementary ISVD constructs to inhibit the formation of the IL-13-IL-13Rα2 complex in competitive ELISA

[0760]

[0761] Table 14: Amino acid sequences of wild-type anti-IL13 double complementary sites and divalent ISVD constructs.

[0762]

[0763]

[0764] 6.2.2 Example 11: Generation and characterization of wild-type dual complementary site-specific anti-TSLPISVD construct

[0765] The selected TSLPR inhibitors (F0107501A02 and F0107529F10) were combined to form a dual-complementary ISVD construct. This construct was expressed in *Pichia pastoris* as a FLAG3-HIS6-tagged protein (amino acid sequences shown in Table 16). ISVD construct expression was induced by stepwise addition of methanol. Clarified medium containing the secreted ISVD construct was used as the starting material for immobilized metal affinity chromatography (IMAC), followed by desalting to produce a purity of 90%, as assessed by SDS-PAGE.

[0766] In BaF3 proliferation assays (as described in Example 7), the bicomplementary constructs were titrated against 50 pM or 5 pM hTSLP and 50 pM or 5 pM cynomolgus TSLP in purified protein form and compared with different anti-TSLP reference compounds (anti-hTSLP reference mAb2 and anti-hTSLP reference mAb1). The data are summarized in Table 15 (hTSLP and cynomolgus TSLP). The bicomplementary constructs were significantly superior to the reference mAbs. The bicomplementary construct with F0107501A02 at the N-terminus showed improved responsiveness to cynomolgus TSLP compared to the construct with F0107501A02 at the C-terminus.

[0767]

[0768] Table 16: Amino acid sequences of the double complementary anti-TSLP ISVD constructs F010703842 and F027400016.

[0769]

[0770] *() indicates the introduction of amino acid substitutions in the (parental) monovalent building block.

[0771] 6.3 Sequence Optimization for Anti-IL-13 and Anti-TSLP Monovalent ISVD

[0772] 6.3.1 Example 12: Sequence optimization for anti-IL-13 and anti-TSLP monovalent ISVD

[0773] The anti-IL-13ISVD F0107004B02 and F0107004B06 and the anti-TSLP ISVD F0107501A02 and F0107529F10 were further optimized.

[0774] Sequence optimization involves replacing one or more specific amino acid residues in the sequence to improve one or more (desired) properties of the ISVD.

[0775] Some examples of this sequence optimization are mentioned in further description in this paper, and are, for example, but not limited to:

[0776] 1) Substitution in the wild-type ISVD sequence of the parent produces an ISVD sequence that is more identical to the human VH3-JH germline common sequence; this process is called humanization. To achieve this, specific amino acids (excluding so-called marker residues) that differ between the ISVD and the human VH3-JH germline common sequence in the FR are changed to their human counterparts in a manner that preserves the protein's structure, activity, and stability.

[0777] 2) Replacement with llama lineage to increase the stability of ISVD, which is defined as camel-derived. To this end, the amino acid sequence of the parent wild-type ISVD was compared with the amino acid sequence of the llama IGHV lineage of ISVD (identified as the highest hit of BlastP analysis of ISVD for the llama IGHV lineage).

[0778] 3) Substitutions that improve long-term stability or properties during storage, increase expression levels in desired host cells or host organisms, and / or, depending on the desired host cell or host organism, eliminate or reduce one or more (unwanted) post-translational modifications (such as glycosylation or phosphorylation). To avoid N-terminal pyroglutamate formation, E1D mutations are typically introduced into the N-terminal building blocks of multivalent Nb without affecting potency or stability. Therefore, E1D mutations are not always introduced during the sequence optimization of the building blocks.

[0779] 4) The mutation at position 11 is changed to Val and the mutation at position 89 is changed to Leu, so as to minimize the activity of binding any naturally occurring pre-existing antibodies.

[0780] F0107004B02 and F0107004B06

[0781] Sequence optimization of anti-IL-13 ISVD F0107004B02 yielded a final sequence-optimized variant F027100019, which contains 8 amino acid substitutions (i.e., E1D, L11V, A14P, N64K, S65G, A74S, K83R, I89L) compared to the parental ISVD F107004B02. Sequence optimization of anti-IL-13 ISVD F0107004B06 yielded a final sequence-optimized variant F027100183, which contains 4 amino acid substitutions (i.e., L11V, R74S, K83R, V89L) compared to the parental ISVD F107004B06.

[0782] The variants were assembled from oligonucleotide sequences using a PCR overlap extension method. These variants were expressed in *E. coli* and purified by IMAC and desalting. The hIL-13 binding capacity of F027100019 and F027100183 was evaluated by surface plasmon resonance using hIL-13 (catalog number 200-13) from Peprotech. Additionally, the neutralizing activity of F027100019 was tested in an eosinophil chemokine release assay. The monomeric behavior of the two variants was monitored by size exclusion HPLC (SE-HPLC). The thermal stability of the variants was tested using a Lightcycler (Roche) thermal displacement assay (TSA). In this assay, the parental ISVD and its variants were cultured at different pH values ​​in the presence of sypro orange with a temperature gradient applied. Sypro orange bound when ISVD began to denature, and the measured fluorescence suddenly increased, thus allowing determination of the melting temperature at a specific pH. The results are summarized in Tables 17 and 18.

[0783] Table 17: Analysis results of sequence-optimized variant F027100019 of anti-IL-13 ISVD F0107004B02. nd = not determined, *measured with respect to hIL-13-Fc.

[0784]

[0785] F027100019 exhibited good potency in eosinophil chemokine release assays, 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 its parent, ISVD F0107004B2. Based on the AbM definition (see Kontermann and Dübel (ed.) Antibody Engineering, Vol. 2, Springer Verlag Heidelberg Berlin, 2010), the architectural identity % in the architectural region of F027100019 was 85%, and based on the Kabat definition, it was 86%.

[0786] Table 18: Analysis results of the sequence-optimized variant of anti-IL13 ISVD F0107004B06. Nd = Not determined

[0787]

[0788] The affinity of F027100183 is similar to that of the WT sequence, with a Tm of 61 °C for the variant. The variant eluted as a 100% monomer peak on SE-HPLC. The architectural identity % for the architectural region of F027100183 was 94.4% based on AbM definition and 93.1% based on Kabat definition.

[0789] F0107529F10

[0790] Sequence optimization of anti-TSLP ISVD F0107529F10 yielded a final sequence-optimized variant F027400021, which contains 8 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 yielded a final sequence-optimized variant F027400160, which contains 6 amino acid substitutions (i.e., L11V, A14P, E16G, A74S, K83R, V89L) compared to the parental ISVD F0107501A02.

[0791] Variants optimized from oligonucleotide assembly sequences were obtained using PCR overlap extension. These constructs were expressed in *E. coli* and purified by IMAC and desalting. The binding affinity of these variants to human and cynomolgus monkey TSLP was evaluated by surface plasmon resonance. The monomeric behavior of all variants was monitored by size exclusion HPLC (SE-HPLC), and thermal stability was monitored by thermal displacement assay (TSA). Additionally, the blocking activity of variant F0107501A02 against hTSLP was tested in the ternary complex Alphascreen. The results are summarized in Tables 19 and 20.

[0792] Table 19: Analysis results of sequence-optimized variants of anti-TSLP ISVD F0107529F10, nd = not determined

[0793]

[0794] Compared to the parental ISVD F0107529F10, the intermediate variant F010704099, with mutations A14P, T60A, S71R, A74S, S79Y, and K83R, exhibited similar affinity for hTSLP and half the affinity for cynomolgus monkey TSLP. Tm showed an overall increase of 11.4 °C compared to the parental ISVD. Two additional mutations, L11V and V89L, were introduced into variant F010704099 to minimize binding to pre-existing antibodies, resulting in the final variant F027400021. Based on the AbM definition, the architectural identity percentage in the architectural regions of F027400021 was 89.9%, and based on the Kabat definition, it was 88.5%.

[0795] Table 20: Analysis results of sequence optimization (SO) variants against TSLP ISVD F0107501A02.

[0796]

[0797] Compared to the parental ISVD F0107501A02, the intermediate variant F010704076, with mutations A14P, E16G, A74S, and K83R, exhibited similar dissociation rates against hTSLP and cynomolgus monkey TSLP, as well as similar potency in the ternary complex Alphascreen. Tm showed an overall increase of 12.3 °C compared to the parental ISVD. Introducing two additional mutations, L11V and V89L, into variant F010704076 to minimize pre-existing antibody binding yielded the final variant F027400160.

[0798] Based on the AbM definition, the architectural identity % in the architectural region of F027400160 is 83.1%, and based on the Kabat definition, it is 80.5%.

[0799] Table 21: Amino acid sequences in SO form of ISVD F0107004B02, F0107004B06, F0107501A02 and F107529F10.

[0800]

[0801] *() indicates the introduction of amino acid substitutions in the (parental) monovalent building block.

[0802] 6.4 Multispecific ISVD construct F027400161

[0803] The optimized ISVDs identified above—F027100019 (an optimized variant of F0107004B02), F027100183 (an optimized variant of F0107004B06), F027400021 (an optimized variant of F0107529F10), and F027400160 (an optimized form of F0107501A02)—were used to generate the multispecific ISVD construct F027400161. The optimized monovalent building blocks used in F027400161 are abbreviated below as 04B02, 04B06, 529F10, and 501A02, respectively, based on their ISVD origin.

[0804] 6.4.1 Example 15: Generation of Multispecific ISVD Constructs

[0805] The identification of the ISVD-containing peptide F027400161 (SEQ ID NO: 1) binding to IL-13 and TSLP was achieved through a data-driven bispecific engineering and formatting process involving several anti-TLSP building blocks, several anti-IL13 building blocks, and the anti-HSA building block ALB23002. Different positions / orientations of the building blocks and different linker lengths (9GS vs. 35GS) were applied, and these were demonstrated to be critical for various parameters (potency, cross-reactivity, expression yield, etc.).

[0806] A large group of different ISVD constructs were transformed in *Pichia pastoris* for small-scale production. ISVD expression was induced by stepwise addition of methanol. Clarified medium containing secreted ISVD was used as starting material for purification via protein A affinity chromatography, followed by desalting. The purified samples were used for characterization and expression evaluation.

[0807] Some constructs exhibited impaired potency depending on the connector length and the relative position of the ISVD building blocks. For example, the anti-TSLP dual complementary site combination 501A02-529F10 showed significantly impaired potency against cynomolgus monkey TSLP when conjugated with a short 9GS connector. Some constructs showed low expression levels depending on the combination and order of the building blocks. For the bispecific compound containing 4B02-4B06, optimal expression was observed when combined with 501A02-529F10.

[0808] Table 22: Selection of different multispecific ISVD formats for evaluation. BB = building block, ALB = AL, B23002

[0809]

[0810]

[0811] *() indicates the introduction of amino acid substitutions in the (parental) monovalent building block.

[0812] Table 23 shows that for six constructs containing the same building blocks but arranged in different ways and linked with different adapter lengths, different yields ranging from low to high titers were obtained. The highest expression titers (F027400161 and F027400163) were obtained for constructs containing IL-13ISVD 4B02 and 4B06 linked via 35GS adapters. Furthermore, the solubility of F027400161 and F027400163 was significantly higher than that of their respective counterparts F027400296 and 298, whose building blocks were linked with four 9GS adapters and had ALB at the C-terminus.

[0813] Subsequently, the large bispecific group was reduced to a set of two bispecific constructs consisting of the following: ISVD constructs F027400161 and F027400163, which were shown to be effective against both targets (human and cynomolgus monkey) and have the potential for high expression levels based on preliminary yield estimates.

[0814] Large-scale production in 2L and 5L of Pichia pastoris was carried out to determine expression yield and to assess biophysical properties and pre-existing antibody reactivity.

[0815] Table 24 and Example 22 demonstrate that the pre-existing antibody reactivity is driven by the orientation of the building block and the length of the linker.

[0816]

[0817] Finally, based on potency, reduced binding to pre-existing antibodies, and superior expression levels and CMC characteristics, the ISVD construct F027400161 was selected.

[0818] 6.4.2 Example 16: Binding affinity of the multispecific ISVD construct to TSLP, IL13 and serum albumin

[0819] The affinity of F027400161 for human and cynomolgus monkey TSLP (the human and cynomolgus monkey TSLP sequences are known (Uniprot accession number Q969D9 and NCBIRefSeqXP_005557555.1, respectively)) is expressed as the equilibrium dissociation constant (K). DThe assays were performed using recombinant proteins, and serum albumin in human IL-13 (Sino Biological catalog 10369-HNAC), cynomolgus monkey IL-13 (Sino Biological catalog 11057-CNAH), and rhesus monkey IL-13 (R&D systems catalog 2674-RM), as well as serum albumin in human (Sigma Aldrich, catalog A8763), cynomolgus monkey, and mouse (Albumin Bioscience catalog N1204H1CM) was quantified by in-solution affinity measurement on a Gyrolab xP Workstation (Gyros).

[0820] In K D Under controlled measurements, serial dilutions of TSLP or IL-13 (range 1 μM–0.25 fM) or serum albumin (range 100 μM–320 pM) were mixed with a fixed amount of F027400161 (5 pM or 10 pM for TSLP and IL-13, and 20 nM for serum albumin) to allow for interaction, and incubated for 48 or 72 hours (for TSLP and IL-13) or 2 hours (for serum albumin) to reach equilibrium.

[0821] Under receptor-controlled measurements, serial dilutions of TSLP or IL-13 (range 1 μM–0.25 fM) or serum albumin (range 100 μM–320 pM) were mixed with fixed amounts of F027400161 (250 pM for TSLP, 10 nM for IL-13, and 1 μM for serum albumin) to allow for interaction, and incubated for 48 or 72 hours (for TSLP and IL-13) or 2 hours (for serum albumin) to reach equilibrium.

[0822] Biotinylated human TSLP / IL-13 / serum albumin was captured in a microstructure of Gyrolab Bioaffy 1000 CD containing beaded columns and used as a molecular probe to capture free F027400161 from equilibrium solution. A mixture of TLSP / IL-13 / serum albumin and F027400161 (containing free TLSP / IL-13 / serum albumin, free F027400161, and the TLSP / IL-13 / serum albumin-F027400161 complex) was passed through the beads, capturing a small percentage of free F027400161 proportional to the concentration of free ISVD. Fluorescently labeled anti-V antibodies were then injected. HHAntibody ABH0086-Alexa647 was used to label any captured F027400161, and changes in fluorescence were measured after washing away excess fluorescent probe. Dilution series were fitted using Gyrolab analysis software, where K was analyzed. D The curves of receptor control were used to determine K. D value.

[0823] The results (Table 25) demonstrate that the multispecific ISVD construct binds human / cynomolgus monkey TSLP and human / cynomolgus monkey / rhesus monkey IL13 with high affinity.

[0824] Table 25: Binding affinity of F027400161 to human, rhesus monkey and cynomolgus monkey TSLP and IL13, as well as human, cynomolgus monkey and mouse serum albumin (the sequences of cynomolgus monkey TSLP and cynomolgus monkey albumin are identical).

[0825]

[0826] 6.4.3 Example 17: Multispecific ISVD construct selectively binds to TSLP and IL13

[0827] The presence or absence of binding between F027400161 and IL-4 and IL-7, which are IL-13 and TSLP-related cytokines, was assessed by SPR (Proteon XPR36).

[0828] Cytokines were immobilized on the Proteon GLC sensor chip at 25 μg / mL using amine conjugation for 600 s, including an 80-second injection of EDC / NHS for inactivation and a 150-second injection of 1 M ethanolamine hydrochloride for inactivation (ProteOn Amine Conjugation Kit, catalog number 176-2410). The flow rate was set to 30 μl / min during inactivation and 25 μl / min during ligand injection. For IL13 and IL4 (Peprotech catalog number 200-07), the pH of the 10 mM acetate immobilization buffer was 6.0, and for TSLP and IL7 (R&D systems catalog number 204-IL / CF) it was 5.5.

[0829] Next, 1 μM F027400161 was injected for 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 run buffer. 100 nM α-hIL4Ab and 100 nM α-IL7 Ab were injected as positive controls. The interaction between F027400161 and the positive controls with the immobilized target was measured by detecting an increase in refractive index, which was due to a change in mass on the chip after binding.

[0830] All positive controls did indeed bind to their respective targets. No binding of F027400161 to human IL4 and IL7 was detected.

[0831] Furthermore, the possibility of F027400161 binding to the short form of TSLP was investigated. To this end, TSLP and the short form of TSLP (as described in Fomasa, 2015) were immobilized on a Proteon GLC sensor chip at 10 μg / mL and 5 μg / mL, respectively, for 150 s using amine coupling, as described above. For TSLP, the pH of the 10 mM acetate immobilization buffer was 5.5, and for the short form of TSLP, it was 4.0.

[0832] Next, 500 nM F027400161 was injected for 2 minutes and dissociated at a flow rate of 45 μL / min for 600 s. PBS (pH 7.4) + 0.005% Tween 20 was used as the run buffer. 500 nM anti-hTSLP reference mAb1 was injected as the reference compound, and 500 nM α-hTSLPpAb (Abcamab47943) was injected as a positive control.

[0833] Although the positive control did bind to both the long (normal) and short forms of TSLP, no binding of F027400161 and anti-hTSLP reference mAb1 to the short form of TSLP was detected.

[0834] 6.4.4 Example 18: Simultaneous binding of multispecific ISVD construct with IL13, TSLP and HSA

[0835] The ability of F027400161 to bind simultaneously to hTSLP and hIL13 was determined using a Biacore T200 instrument. To this end, hTSLP (recombinant human TSLP is commercially available, such as from R&D Systems (catalog number 1398-TS)) was immobilized on a CM5 sensor chip via amine coupling. 100 nM of F027400161 was injected at 10 μL / min onto the TSLP surface for 2 min to capture the ISVD construct via TSLP building block 501A02-529F10. Subsequently, 100 nM hIL13 (PeproTech, catalog number 200-13), HSA, or hOX40L, or 1000 nM HSA, or 100 nM IL13 + 100 nM HSA, 100 nM IL13 + 1000 nM HSA, 100 nM OX40L + 100 nM HSA, 100 nM OX40L + 1000 nM HSA, or 100 nM IL13 + 100 nM OX40L for 2 min, followed by a 300-second dissociation step. The TSLP surface was regenerated by injecting 0.5% SDS + 10 mM glycine (pH 3) at 45 μl / min for 1 min. (Sensing diagram) Figure 1 The results show that F027400161 can simultaneously bind human IL13, human TSLP, and HSA, as indicated by the increase in response units after capture on TSLP: approximately 130 RU from IL13 alone, approximately 60 RU from 100 nM HSA alone, and approximately 350 RU from 1000 nM HSA alone, approximately 180 RU for a mixture of IL13 and 100 nM HSA, and approximately 500 RU for a mixture of IL13 and 1000 nM HSA. Because F027400161 has a low affinity for HSA, higher HSA concentrations are required to observe a significant increase in RU levels (see Example 16).

[0836] Example 19: Inhibition of IL13-induced eosinophil chemokine release in vitro by a multispecific ISVD construct.

[0837] The functional activity of soluble IL13 from different target species (human, rhesus monkey, and cynomolgus monkey) and its inhibition by F027400161 was investigated using a cell-based assay of eosinophil chemokine release from A549 human lung cancer cells.

[0838] For 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 culture, serial dilutions of F027100161 or reference compounds (anti-hIL-13 reference mAb1 and anti-hIL-13 reference mAb2) were added. After 20 minutes of culture, human IL13 (SinoBiological catalog 10369-HNAC), cynomolgus monkey IL13 (Sino Biological catalog 11057-CNAH), or rhesus monkey IL13 (R&D Systems, catalog 2674-RM-025) were added to a final concentration of 160 pM. After further 24 hours of culture in the presence of 30 μM HSA, heparin was added at a final concentration of 50 μg / ml to enhance the expression of eosinophil chemokines. After 4 hours of culture, the secretion of eosinophil chemokine-3 in the cell supernatant was quantified using the human CCL26 / eosinophil chemokine-3 DuoSet ELISA (R&D systems, DY346).

[0839] F027400161 inhibited IL13-induced eosinophil chemokine-3 release in a concentration-dependent manner in humans, cynomolgus monkeys, and rhesus monkeys, with IC50 values ​​of 194 pM (for human IL13), 1040 pM (for cynomolgus monkey IL13), and 713 pM (for rhesus monkey IL13). This was comparable to and superior to the reference compound anti-hIL-13 reference mAb1 (Table 26). Figure 2 ).

[0840] Table 26: IC50 values ​​of F027400161-mediated neutralization of IL13 in humans, cynomolgus monkeys, and rhesus monkeys in the eosinophil chemokine release assay, compared to reference compounds anti-hIL-13 reference mAb1 and anti-hIL-13 reference mAb2. nd = Not determined.

[0841]

[0842] 6.4.5 Example 20: Multispecific ISVD construct inhibits IL13-induced STAT-6 activation in HEK-Blue IL4 / IL13 cells

[0843] HEK-Blue TM IL-4 / IL-13 cells were generated by stably transfecting HEK293 cells with the human STAT6 gene and a STAT6-inducible SEAP reporter gene. Following stimulation with IL-4 and IL-13, cell production was enhanced using QUANTI-Blue. TMSTAT6-induced SEAP was quantitatively secreted into the supernatant.

[0844] HEK-Blue TM Cells were cultured in DMEM supplemented with 10% FBS and seeded at 50,000 cells / well in 96-well plates. Serial dilutions of F027100161 or reference compounds (anti-hIL-13 reference mAb1 and anti-hIL-13 reference mAb2) were pre-cultured with 10 pM hIL-13 (Sino Biological Catalogue No. 10369-HNAC) or cynomolgus monkey IL-13 (Sino Biological Catalogue No. 11057-CNAH) at room temperature for 1 hour and then added to the cells. After culturing for 22 to 24 hours in the presence of 30 μM HSA, 40 μl of cell supernatant was mixed with 160 μl of QUANTI-Blue. TM Mixed. Secreted SEAP was quantified by measuring the absorbance at 620 nm on a Clariostar instrument.

[0845] F027400161 inhibited IL-13-induced SEAP secretion in humans and cynomolgus monkeys in a concentration-dependent manner, with IC50 values ​​of 32.8 pM (for human IL-13) and 53.4 pM (for cynomolgus monkey IL-13), which was superior to the reference compound anti-hIL-13 reference mAb2 (Table 27). Figure 3 ).

[0846] Table 27: IC50 values ​​of neutralization of human and cynomolgus monkey IL-13 mediated by F027400161 in SEAP reporter assays compared with reference compounds anti-hIL-13 reference mAb1 and anti-hIL-13 reference mAb2.

[0847]

[0848] 6.4.6 Example 21: Multispecific ISVD construct inhibits TSLP-induced Ba / F3 cell proliferation in vitro

[0849] The functional activity of soluble TSLP from different target species (human, rhesus macaque, and cynomolgus monkey) and its inhibition by F027400161 was investigated using cell-based assays of the proliferation of BaF3 cells transfected with plasmids encoding hTSLPR and hIL7Ra.

[0850] Cells were seeded at 15,000 cells / well in RPMI 1640 growth medium in white 96-well plates treated with cell culture. A series of dilutions of F027100161 or a reference compound (anti-hTSLP reference mAb1) were added, followed by the addition of 5 pM human or cynomolgus monkey TSLP to stimulate the cells. Human and cynomolgus monkey TSLP sequences are known (Uniprot accession number Q969D9 and NCBI RefSeq XP_005557555.1, respectively). The assays were performed using recombinant protein. After culturing for 48 hours in the presence of 30 μM HSA, the assays were performed using… Cell density and viability were monitored using a luminescent cell viability assay (Promega, G7571 / G7572 / G7573) and read out on an EnVision Multilabel reader (PerkinElmer).

[0851] F027400161 inhibited TSLP-dependent proliferation of Ba / F3 cells in humans and cynomolgus monkeys in a concentration-dependent manner, with IC50 values ​​of 7.8 pM (for human TSP) and 24 pM (for cynomolgus monkey TSLP), thus the inhibition was significantly better than that of the reference compound against hTSLP reference mAb1 (Table 28). Figure 4 ).

[0852] Table 28: IC50 values ​​of F027400161-mediated neutralization of TSLP-induced BaF3 proliferation in humans and cynomolgus monkeys compared to the reference compound anti-hTSLP reference mAb1.

[0853]

[0854] 6.4.7 Example 22: Multispecific ISVD constructs that bind to pre-existing antibodies

[0855] The reactivity of the ISVD construct F027400161 to a pre-existing antibody was evaluated in normal human serum (n=96) using ProteOnXPR36 (Bio-Rad Laboratories, Inc.). PBS / Tween (phosphate-buffered saline (pH 7.4), 0.005% Tween 20) was used as the run buffer, and experiments were performed at 25°C.

[0856] ISVD was captured on the chip via binding to HSA immobilized on the chip using the ALB23002 building block. To immobilize the HSA, the ligand channels of the ProteOn GLC sensor chip were activated using an EDC / NHS (flow rate 30 μl / min), and the HSA was injected at 100 μl / ml into ProteOn acetate buffer at pH 4.5 to achieve an immobilization level of approximately 2900 RU. After immobilization, the surface was deactivated using ethanolamine hydrochloride (flow rate 30 μl / min).

[0857] Subsequently, the ISVD construct was injected onto the HSA surface at 45 μl / min for 2 min to achieve an ISVD capture level of approximately 800 RU. Samples containing the pre-existing antibody were centrifuged at 14,000 rpm for 2 min, and the supernatant was diluted 1:10 in PBS-Tween 20 (0.005%) and injected at 45 μl / min for 2 min, followed by a subsequent 400-second dissociation step. After each cycle (i.e., before a new ISVD capture and blood sample injection step), the HSA surface was regenerated by injecting HCl (100 mM) at 45 μl / min for 2 min. A sensor map showing the binding of the pre-existing antibody was obtained after subtracting two references: 1) ISVD-HSA dissociation and 2) non-specific binding with the reference ligand channel. The binding level of the pre-existing antibody was determined by setting the reporter point to 125 seconds (5 seconds after the end of association). The percentage reduction in pre-existing antibody binding was calculated relative to the binding level of the reference ISVD at 125 seconds.

[0858] The pentavalent ISVD construct F027400161, optimized for reducing binding to pre-existing antibodies by introducing mutants L11V and V89L and C-terminal alanine in each building block, showed significantly less binding to pre-existing antibodies compared to the unoptimized control pentavalent ISVD construct F027301186 (Table 24 and 2016). Figure 5 ).

[0859] Pre-existing antibody binding depends on the orientation of the building block and the linker length present in the multispecific construct. Table 24 and Figure 5 The results showed that construct F027400161 exhibited lower pre-existing antibody reactivity than construct F027400163 due to its specific orientation, but F027400164 exhibited lower reactivity than F027400161 due to the presence of short linkers throughout.

[0860] 6.4.8 Example 23: F027400161 blocks TSLP-induced CCL17 in human dendritic cells in vitro

[0861] The type 2 inflammatory cascade is initiated and propagated in an environment-dependent manner by the synergistic action of epithelial cells, dendritic cells, type 2 helper T cells (Th2 cells), mast cells, and innate lymphoid cells. The cytokine thymic stromal lymphopoietin (TSLP) has been involved in the initiation and development of allergic inflammation through its ability to activate dendritic cells (DCs). Following TSLP activation, human DCs produce CCL17 (a Th2-associated chemokine) and drive Th2 cells from immature CD4+ cells. + T cell differentiation. F027400161 targets both TSLP and IL-13, which can block the interaction between TSLP and DCs, thereby reducing CCL-17 production and potentially providing therapeutic effects in type 2 inflammatory diseases and other diseases.

[0862] The ability of F027400161 to inhibit TSLP-induced CCL17 production was evaluated in human DCs isolated from eight individual healthy donors, compared to a reference monospecific antibody anti-hTSLP reference mAb1. Human DCs (CD3+) were isolated and enriched from healthy human PBMCs in erythrocyte sedimentation rate (ESR) amber layer (human leukocyte package) samples. - CD14 - CD11c + HLA-DR 高 The total will be 0.5-0.8 x 10. 6 One DC / well was incubated with eight 3-fold serial dilutions of F027400161 (400 ng / mL or 5.714 nM maximum) or eight 4-fold serial dilutions of anti-hTSLP reference mAb1 (4000 ng / mL or 27 nM maximum) in a 37°C cell culture incubator for 36 hours, followed by stimulation with 4 ng / mL recombinant human TSLP. Recombinant human TSLP is commercially available, such as from R&D Systems (catalog number 1398-TS). CCL17 production in freshly collected cell culture supernatant was measured by ELISA, and the IC50 of the ISVD construct and the baseline antibody was calculated in GraphpadPrism. 50 value.

[0863] Figure 6 The combined results of dose-inhibitory response of F027400161 and anti-hTSLP reference mAb1 in human DCs are shown. The reference monospecific antibody anti-hTSLP reference mAb1 had a mean IC50 of 793.4 pM. 50 Concentration inhibited TSLP-induced CCL17 production, while F027400161 showed an average IC50 value of 53.26 pM. 50 Inhibit TSLP-induced CCL17 production.

[0864] In summary, these results demonstrate that the ISVD construct F027400161 is more effective than the anti-hTSLP reference mAb1 in inhibiting TSLP-induced CCL17 responses in human DCs.

[0865] 6.4.9 Example 24: F027400161 blocks synergistic CCL17 induced by 0.5 ng / mL [IL-13+TSLP] in human PBMCs in vitro.

[0866] Type II cytokines, such as thymic stromal lymphopoietin (TSLP) and interleukin-13 (IL-13), exert unique, additive, and synergistic effects to drive the pathophysiology of asthma and atopic dermatitis (AD). The roles of TSLP as an initiator of the epithelial cell-derived type II immune cascade and IL-13 as a downstream effector cytokine have been extensively validated. F027400161 targets both TSLP and IL-13, and therefore holds promise for efficacy in type II-mediated inflammatory diseases and other conditions.

[0867] The ability of F027400161 to inhibit the synergistic production of CCL17 (TARC) induced by 0.5 ng / mL IL-13 and TSLP was evaluated in human PBMCs from eight separate healthy donors. The study was designed to evaluate the non-inferiority of the ISVD construct compared to a single-specific biologic, an anti-hTSLP reference mAb1, and an anti-hIL-13 reference mAb1. Human PBMCs were stimulated with 0.5 ng / mL recombinant human TSLP (commercially available, such as from R&D Systems (catalog number 1398-TS)) and IL-13 (R&D Systems, catalog number 213-ILB-005 / CF) at 0.5 ng / mL per well and cultured in 96-well plates at 37°C for 20 h with ten 3-fold serial dilutions of ISVD constructs (maximum concentration 10 nM), anti-hIL-13 reference mAb1 (maximum concentration 10 nM), and anti-hTSLP reference mAb1 (maximum concentration 100 nM). The assays were performed in triplicate for each donor for F027400161. The cytokine concentrations used were reported literature values ​​from serum, BAL fluid, sputum, and skin samples from normal individuals, asthmatics, and patients with atopic dermatitis. The standard error was within 2 times that of the standard error of the study. CCL17 production in freshly collected cell culture supernatant was measured using the Meso Scale Diagnostics (MSD) V-PLEX Human TARC kit.

[0868] Figure 7 The combined results of dose-inhibitory responses of F027400161 and the baseline antibodies anti-hIL-13 reference mAb1 and anti-hTSLP reference mAb1 are shown. F027400161 showed 100% inhibition of 0.5 ng / mL IL-13+TSLP-induced co-CCL17 production, with an average IC50 of [missing data]. 50 The mean IC50 was 0.0061 nM. The reference antibody, anti-hIL-13 mAb1, although having a lower mean IC50 of 0.0028 nM... 50 However, at equimolar doses of F027400161, the synergistic CCL17 response could not be completely blocked, and the inhibition tended to stabilize at approximately 80%. On the other hand, anti-hTSLP reference mAb1 could only block approximately 50% of CCL17 production, with an average IC50 value of 1.5%. 50 It is 2.932 nM.

[0869] In summary, these results demonstrate that F027400161 is more effective than and superior to anti-hTSLP reference mAb1 in blocking the pathophysiologically relevant concentrations of IL-13 and TSLP-induced synergistic response in human PBMCs, highlighting its therapeutic potential for treating type 2 inflammatory diseases such as asthma and atopic dermatitis.

[0870] 6.4.10 Example 25: F027400161 blocks 5 ng / mL [IL-13+TSLP]-induced synergistic CCL17 in human PBMCs in vitro.

[0871] The ability of F027400161 to inhibit 5 ng / mL IL-13+TSLP-induced CCL17 production was evaluated in human PBMCs from eight separate healthy donors. The study was designed to evaluate the non-inferiority of the ISVD construct compared to monospecific biologics, anti-hTSLP reference mAb1, and anti-IL-13 reference mAb1. The cytokine concentrations used were approximately 10 times the upper limit of the hypothetical pathophysiological range of TSLP and IL-13 reported in the literature during transient inflammatory states in normal individuals, asthmatic patients, and patients with atopic dermatitis. et al., Immunol Letter 178: 85-91, 2016; Bellini A et al., Mucosal Immunology 5(2): 140-9, 2012; Davoodi P et al., Cytokine 60(2): 431-7, 2012; Szegedi K et al., J Eur Acad Dermatol Venereol 29(11): 2136-44, 2015). Human PBMCs were stimulated with 5 ng / mL recombinant human TSLP and IL-13 (R&D Systems, catalog number 213-ILB-005 / CF) at one million cells per well and cultured in 96-well plates at 37°C for 20 h with ten 3-fold serially diluted concentrations of F027400161 (maximum 10 nM), anti-hIL-13 reference mAb1 (maximum 10 nM), and anti-hTSLP reference mAb1 (maximum 100 nM). The assays for F027400161 were performed technically in triplicate for each donor. CCL17 production in freshly collected cell culture supernatant was measured using the Meso Scale Diagnostics (MSD) V-PLEX Human TARC kit.

[0872] Figure 8 The combined results of dose-inhibitory responses of F027400161 and the baseline antibodies anti-hIL-13 reference mAb1 and anti-hTSLP reference mAb1 are shown. F027400161 showed 100% inhibition of 5 ng / mL IL-13+TSLP-induced co-CCL17 production, with an average IC50 of [missing data]. 50 The mean IC50 was 0.0387 nM. Although PBMCs treated with an equimolar dose of the reference antibody anti-hIL-13 reference mAb1 showed a lower mean IC50 of 0.01339 nM. 50 However, the inhibitory response never reached 100% and tended to stabilize at approximately 90% inhibition. On the other hand, the anti-hTSLP reference mAb1 only partially blocked approximately 40% of CCL17 production, with an average IC50 value of 1.5%. 50It is 19 nM.

[0873] In summary, these results demonstrate that, at [TSLP+IL-13] concentrations 10 times the pathophysiological range of the cytokines, F027400161 is superior to 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, highlighting its therapeutic potential for treating asthma and atopic dermatitis in the acute or inflammatory phase.

[0874] 6.4.11 Experiment 27: T027400161 blocks the production of allergen DerP-induced IL-5, CCL17, and CCL26 in a ternary culture assay system.

[0875] TSLP drives a type 2 immune response by inducing the production of CCL17, IL-5, and IL-13. Subsequently, IL-13 triggers the production of CCL26 on local epithelial cells, leading to the spread of inflammatory diseases and other diseases mediated by a type 2 immune response.

[0876] In a ternary culture assay system, the ability of F027400161 to inhibit TSLP-induced IL-5 and CCL17 production and IL-13-induced CCL26 production was evaluated by co-culturing MRC5 fibroblasts and A549 epithelial cells with DerP-stimulated human PBMCs from six separate normal donors for 6 days. The study was designed to evaluate the non-inferiority of ISVD compared to single-specific biologics, anti-hTSLP reference mAb1, and anti-hIL-13 reference mAb1. MRC5 fibroblasts constitutively produced approximately 100 pg / mL of endogenous TSLP, and A549 epithelial cells responded to IL-13 produced by PBMCs via stimulation with DerP and endogenous TSLP to produce CCL26. One day prior to co-culturing with human PBMCs, 75,000 MRC5 fibroblasts and A549 epithelial cells were seeded per well. One million human PBMCs per well were added to plated MRC5 fibroblasts and A549 epithelial cells, stimulated with 3 μg / mL Der P, and cultured in 24-well plates at 37°C for 6 days with 11.1 nM ISVD, anti-hIL-13 reference mAb1, or anti-hTSLP reference mAb1. The assays were performed in triplicate for each donor for F027400161. The production of IL-5, CCL17, and CCL26 in freshly collected cell culture supernatant was measured using a Human Magnetic Luminex assay from RnD System.

[0877] Figure 9The combined results of the inhibitory responses of F027400161 and the reference antibodies anti-hIL-13 reference mAb1 and anti-hTSLP reference mAb1 are shown. F027400161 showed 60% inhibition of CCL17, 50% inhibition of IL-5, and 95% inhibition of CCL26 production. The reference antibody anti-hTSLP reference mAb1 showed 50% inhibition of CCL17, 50% inhibition of IL-5, and approximately 55% inhibition of CCL26 production. Although the anti-hIL-13 reference mAb1 showed a comparable 95% inhibition of CCL26 production, this reference antibody only blocked 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 indicate that Der P stimulation triggers PBMCs to induce pathways other than TSLP and IL-13 to drive the production of IL-5 and CCL17.

[0878] In summary, these results demonstrate that anti-TSLP / IL-13 ISVD F027400161 is superior to anti-hTSLP reference mAb1 and anti-hIL-13 reference mAb1 in blocking three cytokines and chemokines (CCL17, IL-5, and CCL26) in a complex assay system containing human PBMCs co-cultured with tissue-structuring cells, highlighting its therapeutic potential for treating type 2 inflammatory diseases such as asthma and atopic dermatitis, as well as a broad range of indications for immune diseases.

[0879] 6.4.12 Example 26: NSG-SGM3 mouse model used to evaluate F027400161-mediated target occupation and pharmacokinetics in vivo.

[0880] F027400161 targets human TSLP and IL-13 and does not cross-react with mouse orthologs. Therefore, to evaluate the bioactivity of F027400161, a xenograft humanized model system was used. Female NSG-SGM3 (NOD / SCID-IL2Rγ- / -, NOD.Cg-PrkdcscidIl2rγtm1Wjl / SzJ) were obtained from the Jackson Laboratory in Bar Harbor, Maine, USA. These mice expressed artificial hematopoietic cytokines: stem cell factor (SCF), granulocyte / macrophage stimulating factor (GM-CSF), and interleukin-3 (IL-3), all driven by human cytomegalovirus promoter / enhancer sequences. Triple transgenic mice constitutively produce the above cytokines, thereby providing cell proliferation and survival signals to support stable engraftment of CD33+ myeloid and several types of lymphoid cells. In short, the engraftment protocol is as follows:

[0881] On day 0 of the study, mice were irradiated with 150 centiGray at a rate of 120 rads / min for 1 minute and 15 seconds. Approximately 6 hours after implantation, 1 x 10⁻⁶ g of ... 5 Umbilical cord blood CD34+ stem / progenitor cells were implanted into mice. A group of mice were irradiated in the same manner but without implantation. These mice were considered irradiated but not implanted. Mice. On day 88 post-implantation, mice received either hydrodynamically controlled (HDD) intravenous injections of saline (for implanted control mice) or a combination of 50 μg IL-4 microcircular DNA and 50 μg TSLP microcircular DNA. On day 91, submandibular exsanguination was performed, and 100–150 μl of blood was collected from each mouse and placed in lithium heparin tubes. Implantation was examined by flow cytometry, and plasma levels of IL-4 and TSLP were evaluated. Information from the implantation examination and / or cytokine assays was used to select (assign or eliminate) mice from the study. Mice from the implanted control group with less than 25% human CD45+ cells were removed from the study. Similarly, implanted mice that received microcircular DNA and showed plasma TSLP levels below one standard deviation from the mean were also removed from the study. Mice receiving microcircular DNA via intravenous injection via HDD received subcutaneous doses of the carrier (20 mM phosphate, 125 mM L-arginine hydrochloride, and 0.01% Tween 20 (pH 7.0)) or F027400161 (0.01, 0.05, 0.1, or 10 mg / kg) on ​​days 95, 97, 99, and 101. On day 103, mice were anesthetized with isoflurane. Blood was collected via retroorbital pleophoresis while the mice were under isoflurane anesthesia. Mice were euthanized by cervical dislocation after blood collection and while still under isoflurane anesthesia. A portion of the lung was harvested and placed in an RNAlater for gene expression evaluation. Plasma levels of human TSLP were determined from plasma samples taken on day 103 using an MSD kit (catalog number K15067-L-2, Meso Scale Diagnostics, Rockville, MD, USA). Plasma levels of human IL-13 were measured from plasma samples taken on day 103 using an ELISA kit (catalog number 88-7439--88 Human IL-13 ELISA Kit, Invitrogen / Thermo-Fischer, Waltham, Massachusetts, USA). Internal assay validation experiments demonstrated that neither the human TSLP nor the IL-13 assay kit could detect hTSLP and hIL-13 bound to F027400161.

[0882] like Figure 10 and Figure 11The combined results of these experiments demonstrate that F027400161 can significantly inhibit the detectable levels of human TSLP and IL-13 in the plasma of humanized NSG-SGM3 mice, thus proving the target occupancy of both human TSLP and human IL-13.

[0883] In the NSG-SGM3 mouse model, the hydrodynamic delivery of TSLP and IL-4 cDNA initiated the expression of human IL-13. Figure 11 The pharmacokinetic effects of F027400161 were investigated using samples derived from the NSG-SGM3 study (Hershey GK. 2003, J Allergy Clin Immunol.; 111(4): 677-90), utilizing the ability of human IL-13 to signal via the mouse IL-13 receptor. In these studies, the effects of F027400161 treatment on human IL-13-regulated mouse gene transcripts were investigated. Mouse lung tissue from the aforementioned studies was used for this analysis.

[0884] Lungs were harvested from treated and control NSG-SGM3 mice and processed to prepare RNA according to the detailed instructions in the attached protocol. The processed RNA was quantified using TaqMan, and data were analyzed as described in the protocol. In short, lungs harvested from mice were stored in RNALater, processed and purified according to a standard protocol to produce high-quality RNA. The purified lung RNA was then reverse transcribed into cDNA using a Quanta Q-Script 5X master mix according to the manufacturer's protocol. The transcript expression levels of the obtained lung cDNA were determined using TaqMan to quantify the target genes of human IL-13-responsive mice (mouse Retnla and mouse Clcal) and the endogenous control (Rpl37a), according to the manufacturer's protocol. Data analysis was performed in Quantstudio 6 & 7flex software. For each probe, CT values ​​and ΔC were calculated. T Export the values ​​(for Rpl37a) to Excel and calculate the relative expression value for each gene using the following formula:

[0885] Normalized relative expression = (Power(2, -(ΔCT)))*1000.

[0886] The two mouse genes evaluated for IL-13 regulation are Retnla (resistin-like α) and Clca1 (chloride channel Annex 1), which play a role in pulmonary vascular remodeling (Lewis CC, 2009, J Allergy Clin Immunol.; 123(4): 795-804).

[0887] like Figure 12 and Figure 13 The combined results of these experiments demonstrate that F027400161 can significantly inhibit the expression of mouse Retnla and mouse Clca1 transcripts, thus demonstrating the in vivo pharmacokinetic effect of F027400161 on human IL-13-driven mouse transcript responses.

[0888] method:

[0889] Sample homogenate preparation:

[0890] Lungs were harvested from mice and stored in RNAlater. The lung lobes were then dried and transferred to FastPrep lysing Matrix A tubes containing 1 mL of RLT+2-ME (for lung homogenization). The samples were homogenized in an MP-Bio homogenizer using program 1 (two 40-second cycles, 5-minute intervals to avoid heating the samples). The samples were then rotated at 10,000 g for 3 minutes. 350 μL of lysate was collected [in RLT+2ME (1% v / v)]. Cells were lysed using multiple (approximately 20) aspirations via a multichannel system.

[0891] RNA preparation:

[0892] For RNA purification, use 350 μL homogenate. Add 1X volume (350 μL) of 70% ethanol, mix thoroughly, and transfer to a 96-well RNeasy spin plate placed in an elution plate. Prepare RNA using the Qiagen RNA Microtissue RNA Extraction Protocol with the following modifications. Cover the 96-well plate with sealable aluminum foil and centrifuge at 4000 x g for 2 min. To wash the column, add 400 μL of RWT buffer to the RNeasy spin column and centrifuge at 4000 x g for 2 min at room temperature. Perform DNase I digestion by adding 80 μL of 1X DNase I mixture and incubating at room temperature for 15 min. Wash away DNase I by adding 400 μL of RWT buffer and spinning the plate at 4000 x g for 2 min. Next, wash the spin plate with 500 μL each of RPE buffer and 80% ethanol. Then dry the column membrane by spinning at 4000 x g for 4 min. Then elute RNA 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.

[0893] First-chain synthesis:

[0894] cDNA was synthesized using Quanta Q-Script 5X master mix and the manufacturer's protocol. The final cDNA concentration was 25 ng / µL. The cDNA was stored at -20°C until TaqMan assay was performed.

[0895] TaqMan measurement:

[0896] TaqMan multiplex master mixtures were prepared in 1.5 ml microcentrifuge tubes by adding the components in the following order. Three separate master mixtures were prepared for each probe set and the internal Rp137a control. Each multiplex qPCR reaction was performed in a 10 μl reaction volume.

[0897]

[0898] For each sample, a total of 8.8 μL of the master mixture 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. TaqMan assays were set up on a QuantStudio 7K. Conditions in the thermal cycler were: pre-denaturation at 95 °C for 3 min, 40 cycles of denaturation at 95 °C for 2 s, followed by annealing and extension at 60 °C for 5 s. Fluorescence measurements were performed during the extension step.

[0899] Data Analysis:

[0900] Data analysis is performed using Quantstudio 6 & 7flex software.

[0901] For each probe, export the CT value and ΔCT value (for Rp137a) to Excel, and calculate the relative expression value of each gene using the following formula:

[0902] Normalized relative expression = (Power(2, -(ΔCT))) * 1000

[0903] References:

[0904] Liu, YJ2006, J Exp Med.; 203(2): 269-73. Thymic stromal lymphopoietin: master switch for allergic inflammation.

[0905] Hershey GK. 2003, JAllergy Clin Immuno1.;111(4):677-90. IL-13 receptors and signaling pathways: an evolving web.

[0906] 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-4and IL-13 in the mouselung.

[0907] 7. Industrial Applicability

[0908] The polypeptides, nucleic acid molecules encoding the polypeptides, carriers containing the nucleic acids, and compositions described herein can be used, for example, to treat subjects suffering from inflammatory diseases. sequence list <110> Alberlinx <110> Sanofi <120> Peptides containing immunoglobulin monovariable domains targeting IL-13 and TSLP <130> 228533 <150> US 62 / 945,391 <151> 2019-12-09 <150> EP 20 305 064.6 <151> 2020-01-27 <160> 177 <170> BiSSAP 1.3.6 <210> 1 <211> 671 <212> PRT <213> Artificial sequence <220> <223> F027400161 <400> 1 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30 Arg Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Leu Ser Gly Asp Gly Tyr Ser Thr Tyr Thr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Ala Lys Leu Gln Tyr Val Ser Gly Trp Ser Tyr Asp Tyr Pro Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val 145 150 155 160 Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu 165 170 175 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Tyr Ala Met 180 185 190 Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser 195 200 205 Ile Thr Thr Gly Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val Lys Gly 210 215 220 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 225 230 235 240 Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Asn 245 250 255 Val Pro Phe Gly Tyr Tyr Ser Glu His Phe Ser Gly Leu Ser Phe Asp 260 265 270 Tyr Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 275 280 285 Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val 290 295 300 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser 305 310 315 320 Gly Phe Gly Val Asn Ile Leu Tyr Trp Tyr Arg Gln Ala Ala Gly Ile 325 330 335 Glu Arg Glu Leu Ile Ala Ser Ile Thr Ser Gly Gly Ile Thr Asn Tyr 340 345 350 Val Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Glu 355 360 365 Asn Thr Met Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly 370 375 380 Leu Tyr Tyr Cys Ala Ser Arg Asn Ile Phe Asp Gly Thr Thr Glu Trp 385 390 395 400 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 405 410 415 Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 420 425 430 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 435 440 445 Arg Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro 450 455 460 Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala 465 470 475 480 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 485 490 495 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu 500 505 510 Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr 515 520 525 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu 530 535 540 Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser 545 550 555 560 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Asp Tyr Asp 565 570 575 Tyr Asp Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly 580 585 590 Val Ser Cys Ile Ser Asn Arg Asp Gly Ser Thr Tyr Tyr Ala Asp Ser 595 600 605 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val 610 615 620 Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr 625 630 635 640 Cys Ala Val Glu Ile His Cys Asp Asp Tyr Gly Val Glu Asn Phe Asp 645 650 655 Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 660 665 670 <210> 2 <211> 123 <212> PRT <213> artificial sequence <220> <223> 4B02 <400> 2 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30 Arg Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Leu Ser Gly Asp Gly Tyr Ser Thr Tyr Thr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Ala Lys Leu Gln Tyr Val Ser Gly Trp Ser Tyr Asp Tyr Pro Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 3 <211> 126 <212> PRT <213> Artificial sequence <220> <223> 4B06 <400> 3 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Tyr 20 25 30 Ala Met Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Thr Thr Gly Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Asn Val Pro Phe Gly Tyr Tyr Ser Glu His Phe Ser Gly Leu Ser 100 105 110 Phe Asp Tyr Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 4 <211> 117 <212> PRT <213> Artificial sequence <220> <223> 501A02 <400> 4 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Gly Phe Gly Val Asn 20 25 30 Ile Leu Tyr Trp Tyr Arg Gln Ala Ala Gly Ile Glu Arg Glu Leu Ile 35 40 45 Ala Ser Ile Thr Ser Gly Gly Ile Thr Asn Tyr Val Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Glu Asn Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Leu Tyr Tyr Cys Ala 85 90 95 Ser Arg Asn Ile Phe Asp Gly Thr Thr Glu Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 5 <211> 115 <212> PRT <213> Artificial sequence <220> <223> ALB23002 <400> 5 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 6 <211> 127 <212> PRT <213> artificial sequence <220> <223> 529F10 <400> 6 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Asp Tyr 20 25 30 Asp Tyr Asp Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Gly Val Ser Cys Ile Ser Asn Arg Asp Gly Ser Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr 85 90 95 Tyr Cys Ala Val Glu Ile His Cys Asp Asp Tyr Gly Val Glu Asn Phe 100 105 110 Asp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 7 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR1 <400> 7 Gly Arg Thr Phe Ser Ser Tyr Arg Met Gly 1 5 10 <210> 8 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR1 <400> 8 Gly Phe Thr Phe Asn Asn Tyr Ala Met Lys 1 5 10 <210> 9 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR1 <400> 9 Gly Ser Gly Phe Gly Val Asn Ile Leu Tyr 1 5 10 <210> 10 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR1 <400> 10 Gly Phe Thr Phe Arg Ser Phe Gly Met Ser 1 5 10 <210> 11 <211> 12 <212> PRT <213> Artificial sequence <220> <223> CDR1 <400> 11 Gly Phe Thr Phe Ala Asp Tyr Asp Tyr Asp Ile Gly 1 5 10 <210> 12 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR2 <400> 12 Ala Leu Ser Gly Asp Gly Tyr Ser Thr Tyr 1 5 10 <210> 13 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR2 <400> 13 Ser Ile Thr Thr Gly Gly Gly Ser Thr Asp 1 5 10 <210> 14 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CDR2 <400> 14 Ser Ile Thr Ser Gly Gly Ile Thr Asn 1 5 <210> 15 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR2 <400> 15 Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu 1 5 10 <210> 16 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR2 <400> 16 Cys Ile Ser Asn Arg Asp Gly Ser Thr Tyr 1 5 10 <210> 17 <211> 14 <212> PRT <213> Artificial sequence <220> <223> CDR3 <400> 17 Lys Leu Gln Tyr Val Ser Gly Trp Ser Tyr Asp Tyr Pro Tyr 1 5 10 <210> 18 <211> 17 <212> PRT <213> Artificial sequence <220> <223> CDR3 <400> 18 Val Pro Phe Gly Tyr Tyr Ser Glu His Phe Ser Gly Leu Ser Phe Asp 1 5 10 15 Tyr <210> 19 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CDR3 <400> 19 Arg Asn Ile Phe Asp Gly Thr Thr Glu 1 5 <210> 20 <211> 6 <212> PRT <213> Artificial sequence <220> <223> CDR3 <400> 20 Gly Gly Ser Leu Ser Arg 1 5 <210> twenty one <211> 16 <212> PRT <213> Artificial sequence <220> <223> CDR3 <400> twenty one Glu Ile His Cys Asp Asp Tyr Gly Val Glu Asn Phe Asp Phe Asp Pro 1 5 10 15 <210> twenty two <211> 25 <212> PRT <213> Artificial sequence <220> <223> FR1 <400> twenty two Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> twenty three <211> 25 <212> PRT <213> Artificial sequence <220> <223> FR1 <400> twenty three Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> twenty four <211> 14 <212> PRT <213> Artificial sequence <220> <223> FR2 <400> twenty four Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala 1 5 10 <210> 25 <211> 14 <212> PRT <213> Artificial sequence <220> <223> FR2 <400> 25 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 <210> 26 <211> 14 <212> PRT <213> Artificial sequence <220> <223> FR2 <400> 26 Trp Tyr Arg Gln Ala Ala Gly Ile Glu Arg Glu Leu Ile Ala 1 5 10 <210> 27 <211> 14 <212> PRT <213> Artificial sequence <220> <223> FR2 <400> 27 Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val Ser 1 5 10 <210> 28 <211> 14 <212> PRT <213> Artificial sequence <220> <223> FR2 <400> 28 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ser 1 5 10 <210> 29 <211> 39 <212> PRT <213> Artificial sequence <220> <223> FR3 <400> 29 Thr Ala Asn Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 1 5 10 15 Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr 20 25 30 Ala Leu Tyr Tyr Cys Ala Ala 35 <210> 30 <211> 39 <212> PRT <213> artificial sequence <220> <223> FR3 <400> 30 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 1 5 10 15 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr 20 25 30 Ala Leu Tyr Tyr Cys Ala Asn 35 <210> 31 <211> 39 <212> PRT <213> artificial sequence <220> <223> FR3 <400> 31 Tyr Val Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 1 5 10 15 Glu Asn Thr Met Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 20 25 30 Gly Leu Tyr Tyr Cys Ala Ser 35 <210> 32 <211> 39 <212> PRT <213> Artificial sequence <220> <223> FR3 <400> 32 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 1 5 10 15 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr 20 25 30 Ala Leu Tyr Tyr Cys Thr Ile 35 <210> 33 <211> 39 <212> PRT <213> Artificial sequence <220> <223> FR3 <400> 33 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 1 5 10 15 Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr 20 25 30 Ala Leu Tyr Tyr Cys Ala Val 35 <210> 34 <211> 11 <212> PRT <213> Artificial sequence <220> <223> FR4 <400> 34 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 35 <211> 11 <212> PRT <213> Artificial sequence <220> <223> FR4 <400> 35 Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 36 <211> 11 <212> PRT <213> Artificial sequence <220> <223> FR4 <400> 36 Ser Ser Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 37 <211> 5 <212> PRT <213> Artificial sequence <220> <223> CDR1 <400> 37 Ser Tyr Arg Met Gly 1 5 <210> 38 <211> 5 <212> PRT <213> Artificial sequence <220> <223> CDR1 <400> 38 Asn Tyr Ala Met Lys 1 5 <210> 39 <211> 5 <212> PRT <213> Artificial sequence <220> <223> CDR1 <400> 39 Val Asn Ile Leu Tyr 1 5 <210> 40 <211> 5 <212> PRT <213> Artificial sequence <220> <223> CDR1 <400> 40 Ser Phe Gly Met Ser 1 5 <210> 41 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CDR1 <400> 41 Asp Tyr Asp Tyr Asp Ile Gly 1 5 <210> 42 <211> 17 <212> PRT <213> Artificial sequence <220> <223> CDR2 <400> 42 Ala Leu Ser Gly Asp Gly Tyr Ser Thr Tyr Thr Ala Asn Ser Val Lys 1 5 10 15 Gly <210> 43 <211> 17 <212> PRT <213> Artificial sequence <220> <223> CDR2 <400> 43 Ser Ile Thr Thr Gly Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 44 <211> 16 <212> PRT <213> Artificial sequence <220> <223> CDR2 <400> 44 Ser Ile Thr Ser Gly Gly Ile Thr Asn Tyr Val Asp Ser Val Lys Gly 1 5 10 15 <210> 45 <211> 17 <212> PRT <213> Artificial sequence <220> <223> CDR2 <400> 45 Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 46 <211> 17 <212> PRT <213> Artificial sequence <220> <223> CDR2 <400> 46 Cys Ile Ser Asn Arg Asp Gly Ser Thr Tyr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 47 <211> 30 <212> PRT <213> Artificial sequence <220> <223> FR1 <400> 47 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser 20 25 30 <210> 48 <211> 30 <212> PRT <213> Artificial sequence <220> <223> FR1 <400> 48 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn 20 25 30 <210> 49 <211> 30 <212> PRT <213> Artificial sequence <220> <223> FR1 <400> 49 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Gly Phe Gly 20 25 30 <210> 50 <211> 30 <212> PRT <213> Artificial sequence <220> <223> FR1 <400> 50 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg 20 25 30 <210> 51 <211> 30 <212> PRT <213> artificial sequence <220> <223> FR1 <400> 51 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala 20 25 30 <210> 52 <211> 32 <212> PRT <213> artificial sequence <220> <223> FR3 <400> 52 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Ala 20 25 30 <210> 53 <211> 32 <212> PRT <213> artificial sequence <220> <223> FR3 <400> 53 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Asn 20 25 30 <210> 54 <211> 32 <212> PRT <213> Artificial sequence <220> <223> FR3 <400> 54 Arg Phe Thr Ile Ser Arg Asp Asn Ser Glu Asn Thr Met Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Leu Tyr Tyr Cys Ala Ser 20 25 30 <210> 55 <211> 32 <212> PRT <213> Artificial sequence <220> <223> FR3 <400> 55 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Thr Ile 20 25 30 <210> 56 <211> 32 <212> PRT <213> Artificial sequence <220> <223> FR3 <400> 56 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Val 20 25 30 <210> 57 <211> 115 <212> PRT <213> artificial sequence <220> <223> Alb8 <400> 57 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 58 <211> 115 <212> PRT <213> Artificial sequence <220> <223> Alb23 <400> 58 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 59 <211> 116 <212> PRT <213> artificial sequence <220> <223> Alb129 <400> 59 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala 115 <210> 60 <211> 116 <212> PRT <213> artificial sequence <220> <223> Alb132 <400> 60 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala 115 <210> 61 <211> 115 <212> PRT <213> Artificial sequence <220> <223> Alb11 <400> 61 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 62 <211> 116 <212> PRT <213> Artificial sequence <220> <223> Alb11 (S112K)-A <400> 62 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Lys 100 105 110 Val Ser Ser Ala 115 <210> 63 <211> 115 <212> PRT <213> artificial sequence <220> <223> Alb82 <400> 63 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 64 <211> 116 <212> PRT <213> Artificial sequence <220> <223> Alb82-A <400> 64 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala 115 <210> 65 <211> 117 <212> PRT <213> artificial sequence <220> <223> Alb82-AA <400> 65 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ala 115 <210> 66 <211> 118 <212> PRT <213> artificial sequence <220> <223> Alb82-AAA <400> 66 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ala Ala 115 <210> 67 <211> 116 <212> PRT <213> Artificial sequence <220> <223> Alb82-G <400> 67 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Gly 115 <210> 68 <211> 117 <212> PRT <213> Artificial sequence <220> <223> Alb82-GG <400> 68 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Gly Gly 115 <210> 69 <211> 118 <212> PRT <213> artificial sequence <220> <223> Alb82-GGG <400> 69 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Gly Gly Gly 115 <210> 70 <211> 115 <212> PRT <213> artificial sequence <220> <223> Alb23002 <400> 70 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 71 <211> 116 <212> PRT <213]] Artificial sequence <220> <223> Alb223 <400> 71 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala 115 <210> 72 <211> 3 <212> PRT <213> Artificial sequence <220> <223> 3A connector <400> 72 Ala Ala Ala 1 <210> 73 <211> 5 <212> PRT <213> Artificial sequence <220> <223> 5GS connector <400> 73 Gly Gly Gly Gly Ser 1 5 <210> 74 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 7GS connector <400> 74 Ser Gly Gly Ser Gly Gly Ser 1 5 <210> 75 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 8GS connector <400> 75 Gly Gly Gly Gly Ser Gly Gly Ser 1 5 <210> 76 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 9GS connector <400> 76 Gly Gly Gly Gly Ser Gly Gly Gly Ser 1 5 <210> 77 <211> 10 <212> PRT <213> Artificial sequence <220> <223> 10GS connector <400> 77 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 78 <211> 15 <212> PRT <213> Artificial sequence <220> <223> 15GS connector <400> 78 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 79 <211> 18 <212> PRT <213> Artificial sequence <220> <223> 18GS linker <400> 79 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Ser <210> 80 <211> 20 <212> PRT <213> Artificial sequence <220> <223> 20GS linker <400> 80 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 81 <211> 25 <212> PRT <213> Artificial sequence <220> <223> 25GS linker <400> 81 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 <210> 82 <211> 30 <212> PRT <213> Artificial sequence <220> <223> 30GS linker <400> 82 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 30 <210> 83 <211> 35 <212> PRT <213> Artificial sequence <220> <223> 35GS linker <400> 83 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser 35 <210> 84 <211> 40 <212> PRT <213> Artificial sequence <220> <223> 40GS linker <400> 84 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Ser 35 40 <210> 85 <211> 15 <212> PRT <213> Artificial sequence <220> <223> G1 hinge <400> 85 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 <210> 86 <211> twenty four <212> PRT <213> Artificial sequence <220> <223> 9GS-G1 hinge <400> 86 Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys 1 5 10 15 Thr His Thr Cys Pro Pro Cys Pro 20 <210> 87 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Upper hinge area of ​​the llama <400> 87 Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala 1 5 10 <210> 88 <211> 62 <212> PRT <213> Artificial sequence <220> <223> G3 hinge <400> 88 Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys 1 5 10 15 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 20 25 30 Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu 35 40 45 Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 50 55 60 <210> 89 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 89 Lys Glu Arg Glu 1 <210> 90 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 90 Lys Gln Arg Glu 1 <210> 91 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 91 Gly Leu Glu Trp 1 <210> 92 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 92 Lys Glu Arg Glu Leu 1 5 <210> 93 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 93 Lys Glu Arg Glu Phe 1 5 <210> 94 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 94 Lys Gln Arg Glu Leu 1 5 <210> 95 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 95 Lys Gln Arg Glu Phe 1 5 <210> 96 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 96 Lys Glu Arg Glu Gly 1 5 <210> 97 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 97 Lys Gln Arg Glu Trp 1 5 <210> 98 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 98 Lys Gln Arg Glu Gly 1 5 <210> 99 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 99 Thr Glu Arg Glu 1 <210> 100 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 100 Thr Glu Arg Glu Leu 1 5 <210> 101 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 101 Thr Gln Arg Glu 1 <210> 102 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 102 Thr Gln Arg Glu Leu 1 5 <210> 103 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 103 Lys Glu Cys Glu 1 <210> 104 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 104 Lys Glu Cys Glu Leu 1 5 <210> 105 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 105 Lys Glu Cys Glu Arg 1 5 <210> 106 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 106 Lys Gln Cys Glu 1 <210> 107 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 107 Lys Gln Cys Glu Leu 1 5 <210> 108 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 108 Arg Glu Arg Glu 1 <210> 109 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 109 Arg Glu Arg Glu Gly 1 5 <210> 110 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 110 Arg Gln Arg Glu 1 <210> 111 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 111 Arg Gln Arg Glu Leu 1 5 <210> 112 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 112 Arg Gln Arg Glu Phe 1 5 <210> 113 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 113 Arg Gln Arg Glu Trp 1 5 <210> 114 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 114 Gln Glu Arg Glu 1 <210> 115 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 115 Gln Glu Arg Glu Gly 1 5 <210> 116 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 116 Gln Gln Arg Glu 1 <210> 117 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 117 Gln Gln Arg Glu Trp 1 5 <210> 118 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 118 Gln Gln Arg Glu Leu 1 5 <210> 119 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 119 Gln Gln Arg Glu Phe 1 5 <210> 120 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 120 Lys Gly Arg Glu 1 <210> 121 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 121 Lys Gly Arg Glu Gly 1 5 <210> 122 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 122 Lys Asp Arg Glu 1 <210> 123 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 123 Lys Asp Arg Glu Val 1 5 <210> 124 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 124 Asp Glu Cys Lys Leu 1 5 <210> 125 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 125 Asn Val Cys Glu Leu 1 5 <210> 126 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 126 Gly Val Glu Trp 1 <210> 127 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 127 Glu Pro Glu Trp 1 <210> 128 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 128 Gly Leu Glu Arg 1 <210> 129 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 129 Asp Gln Glu Trp 1 <210> 130 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 130 Asp Leu Glu Trp 1 <210> 131 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 131 Gly Ile Glu Trp 1 <210> 132 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 132 Glu Leu Glu Trp 1 <210> 133 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 133 Gly Pro Glu Trp 1 <210> 134 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 134 Glu Trp Leu Pro 1 <210> 135 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 135 Gly Pro Glu Arg 1 <210> 136 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 136 Gly Leu Glu Arg 1 <210> 137 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 137 Glu Leu Glu Trp 1 <210> 138 <211> 5 <212> PRT <213> Artificial sequence <220> <223> C-terminus <400> 138 Val Thr Val Ser Ser 1 5 <210> 139 <211> 5 <212> PRT <213> Artificial sequence <220> <223> C-terminus <400> 139 Val Lys Val Ser Ser 1 5 <210> 140 <211> 5 <212> PRT <213> Artificial sequence <220> <223> C-terminus <400> 140 Val Gln Val Ser Ser 1 5 <210> 141 <211> 6 <212> PRT <213> Artificial sequence <220> <223> C-terminus <400> 141 Val Thr Val Ser Ser Ala 1 5 <210> 142 <211> 6 <212> PRT <213> Artificial sequence <220> <223> C-terminus <400> 142 Val Lys Val Ser Ser Ala 1 5 <210> 143 <211> 6 <212> PRT <213> artificial sequence <220> <223> C-terminus <400> 143 Val Gln Val Ser Ser Ala 1 5 <210> 144 <211> 123 <212> PRT <213> artificial sequence <220> <223> F0107004B02 <400> 144 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30 Arg Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Leu Ser Gly Asp Gly Tyr Ser Thr Tyr Thr Ala Asn Ser Val 50 55 60 Asn Ser Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Ala Lys Leu Gln Tyr Val Ser Gly Trp Ser Tyr Asp Tyr Pro Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 145 <211> 126 <212> PRT <213> Artificial sequence <220> <223> F0107004B06 <400> 145 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Tyr 20 25 30 Ala Met Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Thr Thr Gly Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Arg Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Val Pro Phe Gly Tyr Tyr Ser Glu His Phe Ser Gly Leu Ser 100 105 110 Phe Asp Tyr Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 146 <211> 117 <212> PRT <213> Artificial sequence <220> <223> F0107501A02 <400> 146 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Gly Phe Gly Val Asn 20 25 30 Ile Leu Tyr Trp Tyr Arg Gln Ala Ala Gly Ile Glu Arg Glu Leu Ile 35 40 45 Ala Ser Ile Thr Ser Gly Gly Ile Thr Asn Tyr Val Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Gly Val Tyr Tyr Cys Ala 85 90 95 Ser Arg Asn Ile Phe Asp Gly Thr Thr Glu Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 147 <211> 127 <212> PRT <213> Artificial sequence <220> <223> F0107529F10 <400> 147 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Asp Tyr 20 25 30 Asp Tyr Asp Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Gly Val Ser Cys Ile Ser Asn Arg Asp Gly Ser Thr Tyr Tyr Thr Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Ser Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Ser Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Val Glu Ile His Cys Asp Asp Tyr Gly Val Glu Asn Phe 100 105 110 Asp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 148 <211> 284 <212> PRT <213> Artificial sequence <220> <223> F010700003 <400> 148 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Tyr 20 25 30 Ala Met Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Thr Thr Gly Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Arg Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Val Pro Phe Gly Tyr Tyr Ser Glu His Phe Ser Gly Leu Ser 100 105 110 Phe Asp Tyr Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 145 150 155 160 Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly 165 170 175 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser 180 185 190 Tyr Arg Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe 195 200 205 Val Ala Ala Leu Ser Gly Asp Gly Tyr Ser Thr Tyr Thr Ala Asn Ser 210 215 220 Val Asn Ser Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val 225 230 235 240 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr 245 250 255 Cys Ala Ala Lys Leu Gln Tyr Val Ser Gly Trp Ser Tyr Asp Tyr Pro 260 265 270 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 275 280 <210> 149 <211> 281 <212> PRT <213> artificial sequence <220> <223> F010700014 <400> 149 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30 Arg Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Leu Ser Gly Asp Gly Tyr Ser Thr Tyr Thr Ala Asn Ser Val 50 55 60 Asn Ser Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Ala Lys Leu Gln Tyr Val Ser Gly Trp Ser Tyr Asp Tyr Pro Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val 145 150 155 160 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu 165 170 175 Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr Arg Met 180 185 190 Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala 195 200 205 Leu Ser Gly Asp Gly Tyr Ser Thr Tyr Thr Ala Asn Ser Val Asn Ser 210 215 220 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 225 230 235 240 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Ala 245 250 255 Lys Leu Gln Tyr Val Ser Gly Trp Ser Tyr Asp Tyr Pro Tyr Trp Gly 260 265 270 Gln Gly Thr Leu Val Thr Val Ser Ser 275 280 <210> 150 <211> 284 <212> PRT <213> artificial sequence <220> <223> F010700029 <400> 150 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30 Arg Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Leu Ser Gly Asp Gly Tyr Ser Thr Tyr Thr Ala Asn Ser Val 50 55 60 Asn Ser Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Ala Lys Leu Gln Tyr Val Ser Gly Trp Ser Tyr Asp Tyr Pro Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val 145 150 155 160 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 165 170 175 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Tyr Ala Met 180 185 190 Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser 195 200 205 Ile Thr Thr Gly Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val Lys Gly 210 215 220 Arg Phe Thr Ile Ser Arg Asp Asn Arg Lys Asn Thr Leu Tyr Leu Gln 225 230 235 240 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Asn 245 250 255 Val Pro Phe Gly Tyr Tyr Ser Glu His Phe Ser Gly Leu Ser Phe Asp 260 265 270 Tyr Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser 275 280 <210> 151 <211> 287 <212> PRT <213> Artificial sequence <220> <223> F010700031 <400> 151 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Tyr 20 25 30 Ala Met Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Thr Thr Gly Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Arg Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Val Pro Phe Gly Tyr Tyr Ser Glu His Phe Ser Gly Leu Ser 100 105 110 Phe Asp Tyr Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 145 150 155 160 Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 165 170 175 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn 180 185 190 Tyr Ala Met Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 195 200 205 Val Ser Ser Ile Thr Thr Gly Gly Gly Ser Thr Asp Tyr Ala Asp Ser 210 215 220 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Arg Lys Asn Thr Leu 225 230 235 240 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr 245 250 255 Cys Ala Asn Val Pro Phe Gly Tyr Tyr Ser Glu His Phe Ser Gly Leu 260 265 270 Ser Phe Asp Tyr Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser 275 280 285 <210> 152 <211> 279 <212> PRT <213> Artificial sequence <220> <223> F010703842 <400> 152 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Asp Tyr 20 25 30 Asp Tyr Asp Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Gly Val Ser Cys Ile Ser Asn Arg Asp Gly Ser Thr Tyr Tyr Thr Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Ser Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Ser Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Val Glu Ile His Cys Asp Asp Tyr Gly Val Glu Asn Phe 100 105 110 Asp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 145 150 155 160 Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala 165 170 175 Gly Glu Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Gly Phe Gly 180 185 190 Val Asn Ile Leu Tyr Trp Tyr Arg Gln Ala Ala Gly Ile Glu Arg Glu 195 200 205 Leu Ile Ala Ser Ile Thr Ser Gly Gly Ile Thr Asn Tyr Val Asp Ser 210 215 220 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Met 225 230 235 240 Tyr Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Gly Val Tyr Tyr 245 250 255 Cys Ala Ser Arg Asn Ile Phe Asp Gly Thr Thr Glu Trp Gly Gln Gly 260 265 270 Thr Leu Val Thr Val Ser Ser 275 <210> 153 <211> 279 <212> PRT <213> Artificial sequence <220> <223> F027400016 <400> 153 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Gly Phe Gly Val Asn 20 25 30 Ile Leu Tyr Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Ile 35 40 45 Ala Ser Ile Thr Ser Gly Gly Ile Thr Asn Tyr Val Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala 85 90 95 Ser Arg Asn Ile Phe Asp Gly Thr Thr Glu Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly 145 150 155 160 Gly Gly Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 165 170 175 Ser Gly Phe Thr Phe Ala Asp Tyr Asp Tyr Asp Ile Gly Trp Phe Arg 180 185 190 Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ser Cys Ile Ser Asn Arg 195 200 205 Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 210 215 220 Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu 225 230 235 240 Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Val Glu Ile His Cys 245 250 255 Asp Asp Tyr Gly Val Glu Asn Phe Asp Phe Asp Pro Trp Gly Gln Gly 260 265 270 Thr Leu Val Thr Val Ser Ser 275 <210> 154 <211> 284 <212> PRT <213> Artificial sequence <220> <223> F010700003-SO <400> 154 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Tyr 20 25 30 Ala Met Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Thr Thr Gly Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Asn Val Pro Phe Gly Tyr Tyr Ser Glu His Phe Ser Gly Leu Ser 100 105 110 Phe Asp Tyr Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 145 150 155 160 Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly 165 170 175 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser 180 185 190 Tyr Arg Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe 195 200 205 Val Ala Ala Leu Ser Gly Asp Gly Tyr Ser Thr Tyr Thr Ala Asn Ser 210 215 220 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val 225 230 235 240 Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr 245 250 255 Cys Ala Ala Lys Leu Gln Tyr Val Ser Gly Trp Ser Tyr Asp Tyr Pro 260 265 270 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 275 280 <210> 155 <211> 281 <212> PRT <213> artificial sequence <220> <223> F010700014-SO <400> 155 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30 Arg Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Leu Ser Gly Asp Gly Tyr Ser Thr Tyr Thr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Ala Lys Leu Gln Tyr Val Ser Gly Trp Ser Tyr Asp Tyr Pro Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val 145 150 155 160 Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu 165 170 175 Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr Arg Met 180 185 190 Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala 195 200 205 Leu Ser Gly Asp Gly Tyr Ser Thr Tyr Thr Ala Asn Ser Val Lys Gly 210 215 220 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln 225 230 235 240 Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Ala 245 250 255 Lys Leu Gln Tyr Val Ser Gly Trp Ser Tyr Asp Tyr Pro Tyr Trp Gly 260 265 270 Gln Gly Thr Leu Val Thr Val Ser Ser 275 280 <210> 156 <211> 284 <212> PRT <213> artificial sequence <220> <223> F010700029-SO <400> 156 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30 Arg Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Leu Ser Gly Asp Gly Tyr Ser Thr Tyr Thr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Ala Lys Leu Gln Tyr Val Ser Gly Trp Ser Tyr Asp Tyr Pro Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val 145 150 155 160 Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu 165 170 175 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Tyr Ala Met 180 185 190 Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser 195 200 205 Ile Thr Thr Gly Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val Lys Gly 210 215 220 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 225 230 235 240 Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Asn 245 250 255 Val Pro Phe Gly Tyr Tyr Ser Glu His Phe Ser Gly Leu Ser Phe Asp 260 265 270 Tyr Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser 275 280 <210> 157 <211> 287 <212> PRT <213> Artificial sequence <220> <223> F010700031-SO <400> 157 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Tyr 20 25 30 Ala Met Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Thr Thr Gly Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Asn Val Pro Phe Gly Tyr Tyr Ser Glu His Phe Ser Gly Leu Ser 100 105 110 Phe Asp Tyr Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 145 150 155 160 Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly 165 170 175 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn 180 185 190 Tyr Ala Met Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 195 200 205 Val Ser Ser Ile Thr Thr Gly Gly Gly Ser Thr Asp Tyr Ala Asp Ser 210 215 220 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 225 230 235 240 Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr 245 250 255 Cys Ala Asn Val Pro Phe Gly Tyr Tyr Ser Glu His Phe Ser Gly Leu 260 265 270 Ser Phe Asp Tyr Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser 275 280 285 <210> 158 <211> 279 <212> PRT <213> Artificial sequence <220> <223> F010703842-SO <400> 158 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ala Asp Tyr 20 25 30 Asp Tyr Asp Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Gly Val Ser Cys Ile Ser Asn Arg Asp Gly Ser Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr 85 90 95 Tyr Cys Ala Val Glu Ile His Cys Asp Asp Tyr Gly Val Glu Asn Phe 100 105 110 Asp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 145 150 155 160 Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro 165 170 175 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Gly Phe Gly 180 185 190 Val Asn Ile Leu Tyr Trp Tyr Arg Gln Ala Ala Gly Ile Glu Arg Glu 195 200 205 Leu Ile Ala Ser Ile Thr Ser Gly Gly Ile Thr Asn Tyr Val Asp Ser 210 215 220 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Glu Asn Thr Met 225 230 235 240 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Leu Tyr Tyr 245 250 255 Cys Ala Ser Arg Asn Ile Phe Asp Gly Thr Thr Glu Trp Gly Gln Gly 260 265 270 Thr Leu Val Thr Val Ser Ser 275 <210> 159 <211> 279 <212> PRT <213> Artificial sequence <220> <223> F027400016-SO <400> 159 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Gly Phe Gly Val Asn 20 25 30 Ile Leu Tyr Trp Tyr Arg Gln Ala Ala Gly Ile Glu Arg Glu Leu Ile 35 40 45 Ala Ser Ile Thr Ser Gly Gly Ile Thr Asn Tyr Val Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Glu Asn Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Leu Tyr Tyr Cys Ala 85 90 95 Ser Arg Asn Ile Phe Asp Gly Thr Thr Glu Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 ...

Claims

1. A polypeptide that specifically binds to IL-13 and TSLP, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

1.

2. A nucleic acid encoding the polypeptide according to claim 1.

3. A vector comprising nucleic acid, said nucleic acid comprising a nucleotide sequence encoding a polypeptide according to claim 1.

4. A host cell comprising the nucleic acid according to claim 2 or the vector according to claim 3.

5. A composition comprising the polypeptide of claim 1, the nucleic acid of claim 2, or the carrier of claim 3.

6. A pharmaceutical composition comprising a polypeptide according to claim 1, a nucleic acid according to claim 2, or a carrier according to claim 3; and the pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier or excipient and / or adjuvant, and optionally comprising one or more other pharmaceutically active polypeptides and / or compounds.

7. Use of the polypeptide of claim 1 or the composition of claim 6 in the preparation of a medicament for treating an inflammatory disease, wherein the inflammatory disease is asthma.

8. Use of the polypeptide of claim 1 or the composition of claim 6 in the preparation of a medicament for treating an inflammatory disease, wherein the inflammatory disease is atopic dermatitis.

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