CN116783214A

CN116783214A - Polypeptides comprising immunoglobulin single variable domains targeting IL-13 and OX40L

Info

Publication number: CN116783214A
Application number: CN202180065013.5A
Authority: CN
Inventors: H·隆美拉; A·博里塞; S·科内利斯; B·多姆布雷希特; K·厄伯; E·劳伦特; J·帕克; R·雷斯尼克; M·里格; B·韦格尔
Original assignee: Ablinks Co ltd; Sanofi Aventis France
Current assignee: Ablinks Co ltd; Sanofi Aventis France
Priority date: 2020-09-25
Filing date: 2021-09-24
Publication date: 2023-09-19

Abstract

The present disclosure provides a novel medicament for treating a subject suffering from an inflammatory disease. In particular, the present disclosure provides polypeptides comprising at least three Immunoglobulin Single Variable Domains (ISVD), characterized in that at least one ISV binds to OX40L and at least two ISVD binds to IL-13. The disclosure also provides nucleic acids, vectors, and compositions.

Description

Polypeptides comprising immunoglobulin single variable domains targeting IL-13 and OX40L

1 technical field

The present disclosure relates to polypeptides targeting interleukin-13 (IL-13) and OX 40L. The disclosure also relates to nucleic acid molecules encoding the polypeptides and vectors comprising the nucleic acids, as well as compositions comprising the polypeptides, nucleic acids, or vectors. The present disclosure also relates to these products in methods for treating subjects suffering from autoimmune and/or inflammatory diseases and/or fibrotic diseases. Furthermore, the present disclosure relates to a method of producing these products.

2 background art

Although essential for host defense, unrestricted immune responses may lead to a range of autoimmune and/or inflammatory diseases, such as asthma and atopic dermatitis, for example. A series of immune responses (e.g., antigen recognition, antigen processing, antigen presentation, cytokine production, antibody production, target cell killing) mediated by the innate and adaptive arms of the immune system drive the initiation and spread of a range of immune diseases. Autoimmune and inflammatory diseases are often chronic and may even be life threatening. Allergic and atopic diseases (such as asthma and atopic dermatitis) are driven mainly by type 2 immune responses and are characterized by significant type 2 immune characteristics such as hyperige production and eosinophilia.

Currently, patients with moderate to severe asthma do not respond adequately to the currently available standard of care.

In particular in asthmatic patients with low eosinophil phenotype, current standard therapies include treatment with biological agents such as the anti-IL 4 ra monoclonal antibody dipivumab (Dupilumab) (registered trade name of Sanofi Biotechnology)Sold), monoclonal anti-IL 5 antibody Mepolimab (registered trade name of GSK Group +.>Sold) or rayleigh bevacizumab (under the registered trade name Teva Pharmaceutical Industries Ltd ++>Sold), or an anti-IgE monoclonal antibody (registered trade name of Novartis AG ++>Sales).

While treatment of patients with conventional monoclonal antibodies as described above has been shown to be effective in blocking the type 2 pathway and significantly alleviating asthma symptoms and/or treating asthma, a portion of patients do not respond fully and optimally to these treatments.

For atopic dermatitis, many antagonistic antibodies show early clinical efficacy.

KY1005 (developed by Kymab) is a fully human monoclonal antibody that binds to OX40L and prevents it from activating OX40, thereby addressing potential immune system imbalance in patients with inflammatory and/or autoimmune diseases.

ISB 830 (previously GBR 830, developed by Glenmark Pharmaceuticals) is a humanized monoclonal antibody against OX 40. OX40 inhibition may have therapeutic effects in T cell mediated diseases, including atopic dermatitis.

KHK 4083 is an immunomodulatory anti-OX 40 monoclonal antibody (developed by Kyowa Kirin) for the treatment of atopic dermatitis and ulcerative colitis. Early clinical development of subcutaneous and intravenous formulations is underway in several countries.

Qu Luolu mab (Tralokinumab) is an IL-13 neutralizing human IgG4 monoclonal antibody developed by Leo Pharma for the treatment of Atopic Dermatitis (AD) and alopecia areata. Qu Luolu monoclonal antibodies bind to IL-13 helices A and D, thereby preventing IL-13 from interacting with IL-13Rα1 and IL-13Rα2. Qu Luolu mab is undergoing regulatory scrutiny in europe and the united states for the treatment of atopic dermatitis. Clinical development for atopic dermatitis is underway in a plurality of countries and clinical development for alopecia areata is underway in the united states.

Although some of the above-described antagonistic antibodies against OX40, OX40L or IL-13 show early clinical efficacy in atopic dermatitis, there is still an unmet medical need for improved agents for the treatment of such type 2 inflammatory diseases.

3 summary of the invention

The present inventors have developed new and improved agents for the treatment of autoimmune and/or inflammatory diseases such as, in particular, asthma and atopic dermatitis and/or fibrosis diseases. These agents target two or more disease factors, including IL-13 and OX40L, that mediate the biological mechanisms associated with autoimmune or inflammatory or fibrotic diseases.

Interleukin-13 (IL-13) is a cytokine secreted by type 2T helper (Th 2) cells, CD4 cells, natural killer T cells, mast cells, basophils, eosinophils, and nuocites. IL-13 is a central regulator of IgE synthesis, goblet cell proliferation, mucus hypersecretion, airway hyperresponsiveness and fibrosis. It is a key mediator of allergic inflammation and different diseases including asthma. IL-13 signaling is mediated through a multi-subunit receptor shared with IL-4. The receptor is a heterodimeric receptor complex consisting of IL-4 receptor alpha (IL-4 Ralpha) and IL-13 receptor alpha 1 (IL-13 Ralpha 1). The high affinity of IL-13 for IL-13Rα1 results in their formation of bonds, which further increases the likelihood of heterodimerization with IL-4Rα and the production of type 2 IL-4 receptors. Data from human and mouse studies show an important role for IL-13 in type 2 immune diseases including asthma and atopic dermatitis.

OX40L (also known as CD252 or TNFSF 4) is a member of the TNF superfamily and is an inducible co-stimulatory ligand for the OX40 receptor (also known as CD134 or TNFRSF 4). It is expressed predominantly on activated Antigen Presenting Cells (APCs) including dendritic cells, macrophages and B cells. OX40, on the other hand, is expressed primarily on activated T cells and natural killer T cells. OX40L is expressed primarily as a membrane-bound molecule, but can also be detected in a cleaved, soluble form. OX40L/OX40 has been considered an immune co-stimulatory modulator in many diseases, characterized by an activated T cell that plans an immune response. It triggers signaling through OX40, resulting in a range of events including the production and release of inflammatory cytokines, the expansion and accumulation of effector T cells (e.g., TH1, TH2, TH 17) and cytotoxic T cells. Data from human and mouse studies indicate that the OX40/OX40L axis has an important role in a variety of type 2 immune diseases including asthma and atopic dermatitis. It has been shown that OX40L or blocking of OX40 can alleviate disease in a mouse model of asthma, and that skin samples of patients with atopic dermatitis may be shown to contain elevated OX40 expressing T cells.

Without wishing to be bound by any particular theory, the above biological mechanisms play a central role in the initiation and transmission of type 2 inflammatory responses and are the basis of a range of immunopathological pathways that lead to diseases such as atopic dermatitis and asthma.

To date, there has been no active clinical development program for both IL-13 and OX 40L.

The inventors have now surprisingly found that dual targeting of OX40L and IL-13 with a single agent has the potential to confer adequate efficacy in low and high type 2 asthma and atopic dermatitis in a subpopulation in which single monospecific agent therapies for the same indication may not be sufficiently effective.

It is described that targeting multiple disease factors can be achieved, for example, by co-administration or combined use of two separate biological agents (e.g., antibodies that bind to different therapeutic targets). However, co-administration or combined use of separate biological agents can be challenging from both a practical and commercial perspective. For example, two injections of separate products makes the treatment regimen more inconvenient and painful for the patient, which can negatively impact compliance. For a single injection of two separate products, it may be difficult or impossible to provide a formulation that allows acceptable viscosity of the two products at the desired concentration, as well as proper stability and non-interference. In addition, co-administration and co-formulation require the production of two separate drugs, which can increase overall cost.

Thus, there is also a need for improved anti-autoimmune and/or anti-inflammatory and/or anti-fibrotic disease agents that can be conveniently administered to patients.

Bispecific antibodies capable of binding two different antigens have been proposed as a strategy to address the above limitations associated with co-administration or combined use of separate biological agents (e.g., antibodies).

Various forms of bispecific antibody constructs have been proposed. For example, bispecific antibody formats may involve chemical conjugation of two antibodies or fragments thereof (Brennan, M et al, science,1985.229 (4708): pages 81-83; glennie, M.J. et al, J Immunol,1987.139 (7): pages 2367-2375).

However, disadvantages of such bispecific antibody formats may include high viscosity at high concentrations, making for example subcutaneous administration challenging. Furthermore, each binding unit needs to interact with a different target with specificity and high affinity, which has implications for polypeptide stability and efficiency of production. For example, the generation of bispecific antibody forms can potentially lead to CMC (chemical manufacturing and control) problems associated with light chain mismatches or heavy chain mismatches.

Thus, there is a need for improved bispecific or multispecific antibody constructs that bind to both OX40L and IL-13 with sufficient affinity for two or more targets to modulate autoimmune and/or inflammatory responses. At the same time, it is desirable to be able to produce such constructs efficiently, e.g. in a microbial host, and to be able to conveniently administer such constructs to a patient. Ideally, such constructs should also have a sufficiently long half-life in the subject to be treated so that the number of consecutive treatments can be limited so as to be sufficiently spaced apart in time. Furthermore, it is desirable to limit the reactivity of such constructs to pre-existing antibodies in the subject to be treated (i.e., antibodies present in the subject prior to the first treatment with the antibody construct).

The inventors found that bispecific or multispecific polypeptides (also referred to as Immunoglobulin Single Variable Domain (ISVD) constructs in the context of the present disclosure) that specifically target OX40L and IL-13 simultaneously have increased efficiency in modulating a type 2 inflammatory response compared to monospecific anti-OX 40L and/or monospecific anti-IL-13 polypeptides. The polypeptide or ISVD construct can be produced efficiently (e.g., in a microbial host) and conveniently administered. Furthermore, such polypeptides or ISVD constructs can be demonstrated to have 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 the antibody construct). In some embodiments, such polypeptides or ISVD constructs exhibit a sufficiently long half-life in the subject to be treated such that the number of consecutive treatments can be limited and thus can be sufficiently spaced apart in time.

The polypeptides of the present disclosure (also referred to as Immunoglobulin Single Variable Domain (ISVD) constructs in the context of the present disclosure) comprise or consist of at least three Immunoglobulin Single Variable Domains (ISVD), wherein at least one ISVD specifically binds to OX40L and at least two ISVD specifically bind to IL-13. According to some embodiments, the at least one ISVD that binds to OX40L specifically binds to human OX40L and the at least two ISVD that binds to IL-13 specifically binds to human IL-13.

According to some embodiments, the polypeptide of the present disclosure further comprises one or more additional groups, residues, moieties or binding units optionally linked via one or more peptide linkers, wherein the one or more additional groups, residues, moieties or binding units provide the polypeptide with an increased half-life compared to a corresponding polypeptide without the one or more additional groups, residues, moieties or binding units. For example, the binding unit may be an ISVD that binds to a serum protein, e.g. to a human serum protein such as human serum albumin.

Also provided are nucleic acid molecules capable of expressing the polypeptides of the disclosure, nucleic acids or vectors comprising the nucleic acids, and compositions comprising the polypeptides, the nucleic acids or the vectors. In some embodiments, the composition is a pharmaceutical composition.

Also provided are hosts or host cells comprising nucleic acids or vectors encoding polypeptides according to the present disclosure.

Also provided is a method for producing a polypeptide according to the present disclosure, the method comprising at least the steps of:

a. expressing the nucleic acid sequence in a suitable host cell or host organism or another suitable expression system; optionally followed by:

b. Isolating and/or purifying a polypeptide according to the present disclosure.

Furthermore, the present disclosure provides the polypeptide, a composition comprising the polypeptide, or a composition comprising a nucleic acid or vector comprising a nucleotide sequence encoding the polypeptide for use as a medicament. In some embodiments, the polypeptide or composition is used to treat autoimmune and/or inflammatory diseases, such as type 2 inflammatory diseases. In some embodiments, the type 2 inflammatory disease is selected from atopic dermatitis and asthma. In some embodiments, the polypeptide or composition is used to treat a fibrotic disease.

In addition, a method of treating an autoimmune and/or inflammatory disease, such as inflammatory disease type 2, is provided, wherein the method comprises administering to a subject in need thereof a pharmaceutically active amount of a polypeptide or composition according to the present disclosure. In some embodiments, the type 2 inflammatory disease is atopic dermatitis and/or asthma. In addition, a method of treating a fibrotic disease is provided, wherein the method comprises administering to a subject in need thereof a pharmaceutically active amount of a polypeptide or composition according to the present disclosure. In some embodiments, the method further comprises administering one or more additional therapeutic agents.

Also provided is the use of a polypeptide or composition of the disclosure in the manufacture of a pharmaceutical composition for the treatment of autoimmune and/or inflammatory diseases, such as type 2 inflammatory diseases. In some embodiments, the type 2 inflammatory disease is atopic dermatitis and/or asthma. Also provided is the use of a polypeptide or composition of the disclosure in the manufacture of a pharmaceutical composition for the treatment of a fibrotic disease.

In particular, the present disclosure provides the following embodiments:

embodiment 1. A polypeptide, a composition comprising said polypeptide, or a composition comprising a nucleic acid comprising a nucleotide sequence encoding said polypeptide, wherein said polypeptide comprises or consists of at least three Immunoglobulin Single Variable Domains (ISVD), wherein each of said ISVD comprises three complementarity determining regions (CDR 1 to CDR3, respectively) optionally linked via one or more peptide linkers; and wherein:

a) The first ISVD includes

i. CDR1 having the amino acid sequence of SEQ ID NO. 6 or having a 2 or 1 amino acid difference from SEQ ID NO. 6;

CDR2 having the amino acid sequence of SEQ ID No. 10 or having 2 or 1 amino acid differences from SEQ ID No. 10; and

CDR3 having the amino acid sequence of SEQ ID No. 14 or having 2 or 1 amino acid differences from SEQ ID No. 14;

b) The second ISVD includes

CDR1 having the amino acid sequence of SEQ ID NO. 7 or having 2 or 1 amino acid differences from SEQ ID NO. 7;

v. CDR2 having the amino acid sequence of SEQ ID NO. 11 or having 2 or 1 amino acid differences from SEQ ID NO. 11; and

amino acid sequence with SEQ ID NO. 15 or CDR3 with 2 or 1 amino acid differences from SEQ ID NO. 15; and

c) The third ISVD includes

CDR1 having the amino acid sequence of SEQ ID NO 9 or having a 2 or 1 amino acid difference from SEQ ID NO 9;

CDR2 having the amino acid sequence of SEQ ID No. 13 or having 2 or 1 amino acid differences from SEQ ID No. 13; and

ix. a CDR3 having the amino acid sequence of SEQ ID NO:17 or having a 2 or 1 amino acid difference from SEQ ID NO:17,

wherein the sequence of the ISVD starts from the N-terminus.

Embodiment 2. The composition for the use according to embodiment 1, which is a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more other pharmaceutically active polypeptides and/or compounds.

Embodiment 3. The polypeptide or composition for use according to embodiment 1 or 2, wherein:

a) The first ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO. 6, a CDR2 having the amino acid sequence of SEQ ID NO. 10 and a CDR3 having the amino acid sequence of SEQ ID NO. 14;

b) The second ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO. 7, a CDR2 having the amino acid sequence of SEQ ID NO. 11 and a CDR3 having the amino acid sequence of SEQ ID NO. 15; and is also provided with

c) The third ISVD comprises CDR1 having the amino acid sequence of SEQ ID NO. 9, CDR2 having the amino acid sequence of SEQ ID NO. 13 and CDR3 having the amino acid sequence of SEQ ID NO. 17.

Embodiment 4. The polypeptide or composition for use according to any one of embodiments 1 to 3, wherein:

a) The amino acid sequence of the first ISVD has greater than 90% sequence identity to SEQ ID No. 2;

b) The amino acid sequence of the second ISVD has greater than 90% sequence identity to SEQ ID No. 3; and is also provided with

c) The amino acid sequence of the third ISVD has a sequence identity of greater than 90% identity to SEQ ID NO. 5.

Embodiment 5. The polypeptide or composition for use according to any one of embodiments 1 to 4, wherein:

a) The first ISVD has an amino acid sequence of SEQ ID NO. 2;

b) The second ISVD has the amino acid sequence of SEQ ID NO. 3; and is also provided with

c) The third ISVD has the amino acid sequence of SEQ ID NO. 5.

Embodiment 6. The polypeptide or composition for use according to any one of embodiments 1 to 5, wherein the polypeptide further comprises one or more additional groups, residues, moieties or binding units optionally linked via one or more peptide linkers, wherein the one or more additional groups, residues, moieties or binding units provide the polypeptide with an increased half-life compared to a corresponding polypeptide without the one or more additional groups, residues, moieties or binding units.

Embodiment 7. The polypeptide or composition for use according to embodiment 6, wherein the one or more other groups, residues, moieties or binding units providing the polypeptide with increased half-life are selected from the group consisting of polyethylene glycol molecules, serum proteins or fragments thereof, binding units that can bind to serum proteins, fc moieties and small proteins or peptides that can bind to serum proteins.

Embodiment 8. The polypeptide or composition for use according to any of embodiments 6 to 7, wherein the one or more other groups, residues, moieties or binding units providing the polypeptide with increased half-life are selected from binding units that can bind to serum albumin (such as human serum albumin) or serum immunoglobulin (such as IgG).

Embodiment 9. The polypeptide or composition for use according to embodiment 8, wherein the binding unit providing the polypeptide with increased half-life is ISVD which binds to human serum albumin.

Embodiment 10. The polypeptide or composition for use according to embodiment 9, wherein the ISVD that binds human serum albumin comprises

i. CDR1 having the amino acid sequence of SEQ ID NO. 8 or having a 2 or 1 amino acid difference from SEQ ID NO. 8;

CDR2 having the amino acid sequence of SEQ ID No. 12 or having 2 or 1 amino acid differences from SEQ ID No. 12; and

CDR3 having the amino acid sequence of SEQ ID No. 16 or having a 2 or 1 amino acid difference from SEQ ID No. 16.

Embodiment 11. The polypeptide or composition for use according to any of embodiments 9 to 10, wherein the ISVD which binds to human serum albumin comprises CDR1 with the amino acid sequence of SEQ ID No. 8, CDR2 with the amino acid sequence of SEQ ID No. 12 and CDR3 with the amino acid sequence of SEQ ID No. 16.

Embodiment 12. The polypeptide or composition for use according to any of embodiments 9 to 11, wherein the amino acid sequence of the ISVD which binds to human serum albumin has more than 90% sequence identity to SEQ ID No. 4.

Embodiment 13. The polypeptide or composition for use according to any of embodiments 9 to 12, wherein the ISVD which binds to human serum albumin has the amino acid sequence of SEQ ID No. 4.

Embodiment 14. The polypeptide or composition for use according to any of embodiments 1 to 13, wherein the amino acid sequence of the polypeptide has more than 90% sequence identity to SEQ ID No. 1.

Embodiment 15. The polypeptide or composition for use according to any one of embodiments 1 to 14, wherein the polypeptide comprises or consists of the amino acid sequence of SEQ ID No. 1.

Embodiment 16. The polypeptide or composition for use according to any one of embodiments 1 to 15 for use in the treatment of an inflammatory disease, such as type 2 inflammatory disease.

Embodiment 17. The polypeptide or composition for use according to embodiment 16, wherein the type 2 inflammatory disease is selected from asthma and atopic dermatitis.

Embodiment 18. A polypeptide comprising or consisting of at least three Immunoglobulin Single Variable Domains (ISVD), wherein each of said ISVD comprises three complementarity determining regions (CDR 1 to CDR3, respectively) optionally linked via one or more peptide linkers; and wherein:

a) The first ISVD binds to OX40L and comprises

b) The second ISVD binds IL-13 and comprises

c) Third ISVD binds IL-13 and comprises

wherein the sequence of the ISVD starts from the N-terminus.

Embodiment 19. The polypeptide of embodiment 18, wherein:

Embodiment 20. The polypeptide according to any of embodiments 18 or 19, wherein:

Embodiment 21 the polypeptide according to any one of embodiments 18 to 20, wherein:

a) The first ISVD has an amino acid sequence of SEQ ID NO. 2;

c) The third ISVD has the amino acid sequence of SEQ ID NO. 5.

Embodiment 22. The polypeptide according to any one of embodiments 18 to 21, wherein the polypeptide further comprises one or more additional groups, residues, moieties or binding units optionally linked via one or more peptide linkers, wherein the one or more additional groups, residues, moieties or binding units provide the polypeptide with an increased half-life compared to a corresponding polypeptide without the one or more additional groups, residues, moieties or binding units.

Embodiment 23. The polypeptide of embodiment 22, wherein the one or more additional groups, residues, moieties or binding units that provide the polypeptide with increased half-life are selected from the group consisting of polyethylene glycol molecules, serum proteins or fragments thereof, binding units that bind to serum proteins, fc portions, and small proteins or peptides that bind to serum proteins.

Embodiment 24. The polypeptide according to any one of embodiments 22 to 23, wherein the one or more other groups, residues, moieties or binding units providing the polypeptide with increased half-life are selected from binding units that can bind to serum albumin (such as human serum albumin) or serum immunoglobulin (such as IgG).

Embodiment 25. The polypeptide of embodiment 24, wherein the binding unit that provides the polypeptide with increased half-life is ISVD that binds to human serum albumin.

Embodiment 26. The polypeptide of embodiment 25, wherein the ISVD that binds human serum albumin comprises

Embodiment 27. The polypeptide of any one of embodiments 25 to 26, wherein the ISVD which binds to human serum albumin comprises CDR1 having the amino acid sequence of SEQ ID No. 8, CDR2 having the amino acid sequence of SEQ ID No. 12, and CDR3 having the amino acid sequence of SEQ ID No. 16.

Embodiment 28. The polypeptide of any one of embodiments 25 to 27, wherein the amino acid sequence of the ISVD that binds to human serum albumin has greater than 90% sequence identity to SEQ ID No. 4.

Embodiment 29. The polypeptide of any one of embodiments 25 to 28, wherein the ISVD that binds to human serum albumin has the amino acid sequence of SEQ ID No. 4.

Embodiment 30. The polypeptide according to any of embodiments 18 to 29, wherein the amino acid sequence of the polypeptide has more than 90% sequence identity to SEQ ID No. 1.

Embodiment 31 the polypeptide according to any one of embodiments 18 to 29, wherein said polypeptide comprises or consists of the amino acid sequence of SEQ ID No. 1.

Embodiment 32. A nucleic acid comprising a nucleotide sequence encoding the polypeptide according to any one of embodiments 18 to 31.

Embodiment 33. A host or host cell comprising a nucleic acid according to embodiment 32.

Embodiment 34. A method for producing a polypeptide according to any of embodiments 18 to 31, the method comprising at least the steps of:

a) Expressing the nucleic acid according to embodiment 32 in a suitable host cell or host organism or another suitable expression system; optionally followed by:

b) Isolating and/or purifying the polypeptide according to any one of embodiments 18 to 31.

Embodiment 35. A composition comprising at least one polypeptide according to any one of embodiments 18 to 31 or a nucleic acid according to embodiment 32.

Embodiment 36 the composition of embodiment 35, which is a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more other pharmaceutically active polypeptides and/or compounds.

Embodiment 37. A method of treating an inflammatory disease, such as inflammatory disease type 2, wherein the method comprises administering to a subject in need thereof a pharmaceutically active amount of the polypeptide according to any one of embodiments 18 to 31 or the composition according to any one of embodiments 35 to 36.

Embodiment 38. The method of embodiment 37, wherein the type 2 inflammatory disease is selected from asthma and atopic dermatitis.

Embodiment 39 the use of the polypeptide according to any one of embodiments 18 to 31 or the composition according to any one of embodiments 35 to 36 for the preparation of a pharmaceutical composition for the treatment of an inflammatory disease, such as an inflammatory disease of type 2.

Embodiment 40. The use of the polypeptide or composition of embodiment 39, wherein the type 2 inflammatory disease is selected from asthma and atopic dermatitis.

Embodiment 41. A polypeptide comprising or consisting of at least three Immunoglobulin Single Variable Domains (ISVD), wherein each of said ISVD comprises three complementarity determining regions (CDR 1 to CDR3, respectively) optionally linked via one or more peptide linkers; and wherein:

a) The first ISVD includes

b) The second ISVD includes

c) The third ISVD includes

wherein the sequence of the ISVD starts from the N-terminus.

Description of the drawings

Fig. 1: the simultaneous binding of soluble IL-13 and membrane-bound hOX40L to ISV construct F027100187 was shown by flow cytometry of CHO-Ki cells expressing human OX 40L. IRR00096 is negative control V _HH 。

Fig. 2: inhibition of human, cynomolgus and rhesus IL-13 in eosinophil chemokine release assays by ISVD F027100187 and reference anti-hIL-13 mAbs (designated as comparison 1 and comparison 2). Both comparative 1 and comparative 2 are standard conventional monoclonal antibodies directed against human IL-13. The data points are global averages (n=2), error bars represent +/-SD.

Fig. 3: inhibition of membrane-bound OX40L by ISVD construct F027100187 and reference compound anti-hOX 40L mAb (designated comparator 3) as determined in PBMC activity assay. Comparator 3 is a standard conventional monoclonal antibody directed against human OX 40L. The data points are global averages (n=2), error bars represent +/-SD.

Fig. 4: box plots (with median and quartile differences) showing the binding of pre-existing antibodies present in 96 human serum samples to ISVD construct F027100187 compared to control ISVD construct F027301186.

Fig. 5 and 6: inhibition profile of allergen house dust mite (Der P) induced IL-5 and CCL26 production by human PBMC by ISVD construct F027100187 as well as reference antibodies anti-hIL-13 mAb (designated as comparative 1) and anti-hOX 40L mAb (designated as comparative 3) in a triple culture assay. Normal donor PBMC co-cultured with MRC5 fibroblasts and A549 epithelial cells were stimulated with 3mg/mL house dust mite and incubated with 11.1nM of ISVD, anti-hIL-13 reference mAb (designated as comparator 1) or anti-hOX 40L reference mAb comparator 3 in a 24-well plate for 6 days at 37℃in a cell incubator. The IL-5 and CCL26 concentrations in freshly collected supernatants were measured by Human Magnetic Luminex Assay. Percent inhibition was calculated relative to unstimulated (min) and stimulated (max) control samples that did not receive ISVD polypeptides or antibodies. All calculations were performed using GraphPad Prism 8.0. Data are expressed as mean ± Standard Error of Mean (SEM) from all donors pooled from 3 independent experiments according to the above settings. Fig. 5: IL-5 inhibition in the triple culture assay was determined on day 7. Results from 4-position PBMC donor. Fig. 6: on day 7 CCL26 inhibition in triple culture assays. Results from 4-position PBMC donor.

Fig. 7: f27100187 significantly reduced human activation, effects and cellularity of central memory T cells in the lungs of NSG mice. A) F27100187 at any of the doses of 11.1mg/kg, 3.72mg/kg, 1.11mg/kg or 0.37mg/kg did not significantly decrease activated cells (CD4+CD45RA-cells) when compared to vehicle treated mice. B) When compared to vehicle treated mice (132,035 cells), the use of 11.1mg/kg F27100187 dose (16,609 cells), 3.72mg/kg F27100187 dose (17,779 cells), 1.11mg/kg F27100187 dose (14,808 cells), and 0.37mg/kg F27100187 dose (23,568 cells) reduced activated cells (cd4+hla-dr+cd38+ cells). C) The use of 11.1mg/kg F27100187 dose (151,974 cells), 3.72mg/kg F27100187 dose (164,639 cells), 1.11mg/kg F27100187 dose (156,677 cells) and 0.37mg/kg F27100187 dose (176,243 cells) reduced effector memory cells when compared to vehicle treated mice (685,726 cells). D) Central memory cells were reduced with the 11.1mg/kg F27100187 dose (106,497 cells), 3.72mg/kg F27100187 dose (97,465 cells), 1.11mg/kg F27100187 dose (95,135 cells) and 0.37mg/kg F27100187 dose (135,098 cells) when compared to vehicle treated mice (681,508 cells).

Fig. 8: f27100187 significantly reduced human activation (cd19+), memory B cells and plasmablasts in the lungs of NSG mice. A) The use of 11.1mg/kg F27100187 dose (11,581 cells), 3.72mg/kg F27100187 dose (8,236 cells), 1.11mg/kg F27100187 dose (9,948 cells) and 0.37mg/kg F27100187 dose (10,248 cells) reduced activated cells (cd19+), when compared to vehicle treated mice (60,393 cells). B) The use of the 11.1mg/kg F27100187 dose (1,636 cells), the 3.72mg/kg F27100187 dose (914 cells), the 1.11mg/kg F27100187 dose (1,243 cells) and the 0.37mg/kg F27100187 dose (11,268 3 cells) reduced memory B cells when compared to vehicle treated mice (6,467 cells). C) Plasmablasts were reduced with the 11.1mg/kg F27100187 dose (3,270 cells), 3.72mg/kg F27100187 dose (2,216 cells), 1.11mg/kg F27100187 dose (3,314 cells) and 0.37mg/kg F27100187 dose (2,559 cells) when compared to vehicle treated mice (26,148 cells).

Fig. 9: f27100187 significantly reduced detectable levels of human type 2 key cytokines IL-2, IL-4, IL-5 and IL-10 in plasma of NSG mice at all doses. The use of 11.1mg/kg F27100187 dose (0.7115 pg/ml), 3.72mg/kg F27100187 dose (0.689 pg/ml), 1.11mg/kg F27100187 dose (0.8593 pg/ml) and 0.37mg/kg F27100187 dose (1.659 pg/ml) reduced human IL-2 when compared to vehicle treated mice (2.203 pg/ml). The use of 11.1mg/kg F27100187 dose (1.074 pg/ml), 3.72mg/kg F27100187 dose (7.859 pg/ml), 1.11mg/kg F27100187 dose (3.920 pg/ml) and 0.37mg/kg F27100187 dose (7.415 pg/ml) reduced human IL-4 when compared to vehicle treated mice (44.42 pg/ml). The use of the 11.1mg/kg F27100187 dose (0 pg/ml), 3.72mg/kg F27100187 dose (1.388 pg/ml), 1.11mg/kg F27100187 dose (0.6192 pg/ml) and 0.37mg/kg F27100187 dose (0.6517 pg/ml) reduced human IL-5 when compared to vehicle treated mice (14.74 pg/ml). Human IL-10 was reduced using a dose of 11.1mg/kg F27100187 (9.324 pg/ml), 3.72mg/kg F27100187 (10.51 pg/ml), 1.11mg/kg F27100187 (12.94 pg/ml) and 0.37mg/kg F27100187 (13.47 pg/ml) when compared to vehicle treated mice (58.74 pg/ml).

Fig. 10: f27100187 significantly reduced detectable levels of human IgE in plasma of NSG mice at all doses. The use of the 11.1mg/kg F27100187 dose (0 pg/ml), 3.72mg/kg F27100187 dose (42.95 pg/ml), 1.11mg/kg F27100187 dose (11.55 pg/ml) and 0.37mg/kg F27100187 dose (11.91 pg/ml) reduced human IgE when compared to vehicle treated mice (383.2 pg/ml).

Fig. 11: f27100187 significantly reduced the detectable levels of human IL-13, IL-5, TARC and mouse eosinophil chemokines in plasma of NSG-SGM3 mice. The use of 3.72mg/kg F27100187 dose (28.94 pg/ml) and 1.11mg/kg F27100187 dose (25.89 pg/ml) reduced human IL-13 when compared to vehicle treated mice (981.7 pg/ml). The use of 3.72mg/kg F27100187 dose (1.158 pg/ml) and 1.11mg/kg F27100187 dose (1.079 pg/ml) reduced human IL-5 when compared to vehicle treated mice (1121 pg/ml). The use of a 3.72mg/kg F27100187 dose (259.7 pg/ml) and a 1.11mg/kg F27100187 dose (368.5 pg/ml) reduced human TARC when compared to vehicle treated mice (623.9 pg/ml). The use of 3.72mg/kg F27100187 dose (803.2 pg/ml) and 1.11mg/kg F27100187 dose (984.4 pg/ml) reduced mouse eosinophil chemokines when compared to vehicle treated mice (1107 pg/ml).

Fig. 12: schematic representation of ISVD construct F027100187 showing monovalent building blocks/ISVD 15B07AM, 4B02/1, ALB23002 and 4B06/1 linked via a 9GS linker from N-terminus to C-terminus.

FIG. 13.F27100187 significantly reduces the percentage of lung eosinophils, BAL IL-5, and eosinophils after HDM challenge. A) The use of 5.2mg/kg F27100187 dose (0.529 min) and 1.0mg/kg F27100187 dose (0.585 min) reduced lung eosinophils when compared to vehicle treated mice (2.37 min). B) BAL IL-5 was reduced with the 5.2mg/kg F27100187 dose (0.4178 pg/ml) and the 1.0mg/kg F27100187 dose (0.6825 pg/ml) when compared to vehicle treated mice (1.798 pg/ml). C) The use of 5.2mg/kg F27100187 dose (2.897%) and 1.0mg/kg F27100187 dose (2.836%) reduced the BAL eosinophil percentage when compared to vehicle treated mice (14.27%).

Fig. 14.F27100187 significantly reduces skin inflammation after HDM challenge. The use of 5.2mg/kg F27100187 dose (2.018 minutes) and 1.0mg/kg F27100187 dose (2.475 minutes) reduced skin inflammation when compared to vehicle treated mice (3.25 minutes).

Fig. 15.F27100187 significantly reduces IgE levels in serum. The use of 5.2mg/kg F27100187 dose (-1291 pg/ml) and 1.0mg/kg F27100187 dose (-180.2 pg/ml) reduced serum IgE levels when compared to vehicle treated mice (548.7 pg/ml).

5 detailed description of the preferred embodiments

The present disclosure provides a novel medicament for the treatment of autoimmune and/or inflammatory diseases (such as atopic dermatitis and asthma) and/or fibrotic diseases.

The inventors have found that polypeptides that target both OX40L and IL-13 result in increased efficiency in modulating a type 2 inflammatory response in vitro and/or in vivo as compared to a monospecific anti-OX 40L or anti-IL-13 polypeptide. The polypeptides may be produced efficiently (e.g., in a microbial host). Furthermore, such polypeptides may be demonstrated to have limited reactivity to pre-existing antibodies in the subject to be treated (i.e., antibodies present in the subject prior to first treatment with the antibody construct). In some embodiments, such polypeptides may be conveniently administered and exhibit a sufficiently long half-life in the subject to be treated such that a limited number of consecutive treatments are maintained and thus the treatments may be conveniently spaced apart in time.

The polypeptides are at least bispecific, but may also be, for example, trispecific, tetraspecific or penta-specific. Furthermore, the polypeptide is at least tetravalent, but may also be, for example, pentavalent or hexavalent, etc.

The terms "bispecific", "trispecific", "tetraspecific" or "penta-specific" are all within the scope of the term "multispecific" and refer to binding to two, three, four or five different target molecules, respectively. The terms "divalent", "trivalent", "tetravalent", "pentavalent" or "hexavalent" all fall within the scope of the term "multivalent" and mean the presence of two, three, four or five binding units (e.g., ISVD), respectively. For example, the polypeptide can be tri-specific tetravalent, such as a polypeptide comprising or consisting of four ISVD, wherein one ISVD binds to human OX40L, two ISVD binds to human IL-13 and one ISVD binds to human serum albumin (e.g., ISVD construct F027100187). Such polypeptides may be simultaneously biparatopic, for example, where two ISVD bind to two different epitopes on human OX40L or human IL-13. The term "biparatopic" refers to binding to two different portions (e.g., epitopes) of the same target molecule.

As used herein, the terms "first ISVD", "second ISVD", "third ISVD", etc., refer only to the relative positions of ISVD to each other, wherein numbering begins at the N-terminus of the polypeptides of the present disclosure. Thus, a "first ISVD" is closer to the N-terminus than a "second ISVD", a "second ISVD" is closer to the N-terminus than a "third ISVD", and so on. Thus, the ISVD arrangement is reversed when considered from the C-terminus. Since numbering is not absolute and only indicates the relative positions of the at least three ISVD, it is not excluded that other binding units/building blocks may be present in the polypeptide, such as additional ISVD that binds to OX40L or IL-13, or ISVD that binds to another target. Furthermore, the possibility that other binding units/building blocks (such as ISVD) may be placed in between is not excluded. For example, as described further below (see in particular section 5.3 "(in vivo) half-life extension"), the polypeptide may further comprise another ISVD that binds human serum albumin, which may even be located, for example, between "second ISVD" and "third ISVD".

In view of the foregoing, the present disclosure provides a polypeptide comprising or consisting of at least three ISVD, wherein at least one ISVD specifically binds to OX40L and at least two ISVD specifically binds to IL-13.

The components of the polypeptide, such as ISVD, can be linked to each other by one or more suitable linkers (e.g., peptide linkers).

The use of linkers to join two or more (poly) peptides is well known in the art. Exemplary peptide linkers are shown in Table A-5. One class of commonly used peptide linkers is known as "Gly-Ser" or "GS" linkers. These are linkers consisting essentially of glycine (G) and serine (S) residues, and typically comprise one or more repeats of a peptide motif, such as a GGGGS (SEQ ID NO: 65) motif (e.g., having the formula (Gly-Gly-Gly-Gly-Ser) n, where n may 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: 68), the 15GS linker (n=3) and the 35GS linker (n=7). Reference is made, for example, to Chen et al, adv. Drug deliv. Rev.2013oct 15;65 (10) 1357-1369; and Klein et al, protein Eng. Des. Sel. (2014) 27 (10): 325-330.

In the polypeptides of the present disclosure, in some embodiments, the components of the polypeptides may optionally be linked to each other using a 9GS linker.

In one embodiment, the ISVD that specifically binds to OX40L is located at the N-terminus of the polypeptide. The inventors have surprisingly found that this configuration can increase the product yield of the polypeptide.

Also in one embodiment, an ISVD that specifically binds IL-13 is located at the C-terminus of the polypeptide.

Thus, in some embodiments, the polypeptide comprises or consists of, in order from the N-terminus of the polypeptide: a first ISVD that specifically binds to OX40L, a first ISVD that specifically binds to IL-13, an optional binding unit that provides the polypeptide with increased half-life, and a second ISVD that specifically binds to IL-13. In one embodiment, the binding unit that provides the polypeptide with increased half-life is ISVD.

Provided that in some embodiments, the polypeptide comprises or consists of, in order from the N-terminus of the polypeptide: an ISVD that specifically binds to OX40L, a linker, a first ISVD that specifically binds to IL-13, a linker, an ISVD that binds to human serum albumin, a linker, and a second ISVD that specifically binds to IL-13. In some embodiments, the linker is a 9GS linker.

Such a configuration of the polypeptide may provide increased product yield, good CMC characteristics, and optimized functionality and greater efficacy with respect to immune response modulation.

In some embodiments, the polypeptides of the disclosure exhibit reduced binding to pre-existing antibodies in human serum. To this end, in one embodiment, the polypeptide has a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (numbering according to Kabat) in at least one or each ISVD. In another embodiment, the polypeptide has an extension of 1 to 5 amino acids (naturally occurring, non-naturally occurring, or mixtures thereof) at the C-terminus of the C-terminal ISVD, such as a single alanine (a) extension. The C-terminus of ISVD may be VTVSS (SEQ ID NO: 81). In another embodiment, the polypeptide has a lysine (K) or a glutamine (Q) at position 110 (numbered according to Kabat) in at least one or each ISVD. In another embodiment, the ISVD has a lysine (K) or a glutamine (Q) at position 112 (according to Kabat numbering) in at least one or each ISVD. In some embodiments, the ISVD is C-terminal to VKVSS (SEQ ID NO: 82), VQVSS (SEQ ID NO: 83), VTVKS (SEQ ID NO: 84), VTVQS (SEQ ID NO: 85), VKVKS (SEQ ID NO: 86), VKVQS (SEQ ID NO: 87), VQVKS (SEQ ID NO: 88) or VQVQS (SEQ ID NO: 89), such that after addition of a single alanine, the polypeptide has the sequence VTVSSA (SEQ ID NO: 90), VKVSSA (SEQ ID NO: 91), VQVSSA (SEQ ID NO: 92), VTVKSA (SEQ ID NO: 93), VTVQSA (SEQ ID NO: 94), VKVKSA (SEQ ID NO: 95), VKVKSA (SEQ ID NO: 96), VQVVKSA (SEQ ID NO: 97) or VQQQSA (SEQ ID NO: 98), for example. In one embodiment, the sequence is VKVSSA (SEQ ID NO: 91). In another embodiment, the polypeptide has valine (V) at amino acid position 11 and leucine (L) at amino acid position 89 (numbering according to Kabat) in each ISVD, optionally lysine (K) or glutamine (Q) at amino acid position 110 (numbering according to Kabat) in at least one ISVD, and has an extension of 1 to 5 amino acids (naturally occurring, non-naturally occurring or mixtures thereof) at the C-terminus of the C-terminal ISVD, such as a single alanine (a) extension (such that the C-terminus of the polypeptide has the sequence VTVSSA (SEQ ID NO: 90), VKVSSA (SEQ ID NO: 91) or VQVSSA (SEQ ID NO: 92), for example). For further information in this regard, see, for example, WO 2012/175741 and WO 2015/173325, which are incorporated herein by reference in their entirety.

In another embodiment, the polypeptide of the disclosure comprises or consists of an amino acid sequence having greater than 90%, such as greater than 95% or greater than 99%, sequence identity to SEQ ID No. 1, wherein the CDRs of the four ISVD are defined as items a to D (or a 'to D', if defined using Kabat) as set forth in the following sections "5.1 immunoglobulin single variable domain" and "5.3 (in vivo) half-life extension", respectively, wherein in particular:

ISVD specifically binding to OX40L has CDR1 comprising the amino acid sequence of SEQ ID NO. 6, CDR2 comprising the amino acid sequence of SEQ ID NO. 10 and CDR3 comprising the amino acid sequence of SEQ ID NO. 14;

the first ISVD specifically binding to IL-13 has a CDR1 comprising the amino acid sequence of SEQ ID NO. 7, a CDR2 comprising the amino acid sequence of SEQ ID NO. 11 and a CDR3 comprising the amino acid sequence of SEQ ID NO. 15;

a second ISVD that specifically binds IL-13 having a CDR1 comprising the amino acid sequence of SEQ ID NO:9, a CDR2 comprising the amino acid sequence of SEQ ID NO:13 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and is also provided with

ISVD binding to human serum albumin having a CDR1 comprising the amino acid sequence of SEQ ID NO. 8, a CDR2 comprising the amino acid sequence of SEQ ID NO. 12 and a CDR3 comprising the amino acid sequence of SEQ ID NO. 16,

Or alternatively if Kabat definition is used:

ISVD specifically binding to OX40L has CDR1 comprising the amino acid sequence of SEQ ID NO. 31, CDR2 comprising the amino acid sequence of SEQ ID NO. 35 and CDR3 comprising the amino acid sequence of SEQ ID NO. 14;

the first ISVD specifically binding to IL-13 has a CDR1 comprising the amino acid sequence of SEQ ID NO:32, a CDR2 comprising the amino acid sequence of SEQ ID NO:36 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 15;

a second ISVD that specifically binds IL-13 has a CDR1 comprising the amino acid sequence of SEQ ID NO:34, a CDR2 comprising the amino acid sequence of SEQ ID NO:38, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and is also provided with

ISVD binding to human serum albumin has a CDR1 comprising the amino acid sequence of SEQ ID NO. 33, a CDR2 comprising the amino acid sequence of SEQ ID NO. 37 and a CDR3 comprising the amino acid sequence of SEQ ID NO. 16.

In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO. 1. In one embodiment, the polypeptide consists of the amino acid sequence of SEQ ID NO. 1.

In some embodiments, the polypeptide of the present disclosure has at least half, at least the same, or even higher binding affinity for human O40L and human IL-13 as compared to a polypeptide consisting of the amino acids of SEQ ID No. 1, wherein the binding affinity is measured using the same method (e.g., SPR).

5.1 immunoglobulin Single variable Domains

The term "immunoglobulin single variable domain" (ISVD) is used interchangeably with "single variable domain" to define an immunoglobulin molecule in which an antigen binding site is present on and formed from a single immunoglobulin domain. This allows the immunoglobulin single variable domain to be compared to a "conventional" immunoglobulin (e.g., monoclonal antibody) or fragment thereof (e.g., fab ', F (ab') ₂ scFv, diascfv) wherein two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (V _H ) And a light chain variable domain (V _L ) The interactions form antigen binding sites. In this case V _H And V _L The Complementarity Determining Regions (CDRs) of both will contribute to the antigen binding site, i.e., a total of 6 CDRs will be involved in antigen binding site formation.

In view of the above definition, the antigen binding domain of a conventional 4-chain antibody (e.g., igG, igM, igA, igD or IgE molecule; known in the art) or a Fab fragment, F (ab') ₂ Antigen binding domains of fragments, fv fragments (such as disulfide-linked Fv or scFv fragments) or diabodies (all known in the art) are typically not considered immunoglobulin single variable domains, because in these cases, the binding of the corresponding epitope to the antigen typically occurs not through a single immunoglobulin domain, but through a pair of associated immunoglobulin domains, such as the light chain and heavy chain variable domains Domains, i.e. V through immunoglobulin domains that together bind to epitopes of the corresponding antigen _H -V _L The pairing occurs.

In contrast, an immunoglobulin single variable domain is capable of specifically binding to an epitope of an antigen without pairing with another immunoglobulin variable domain. The binding site of an immunoglobulin single variable domain consists of a single V _H Single V _HH Or a single V _L Domain formation.

Thus, the single variable domain can be a light chain variable domain sequence (e.g., V _L Sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., V _H Sequence or V _HH Sequence) or a suitable fragment thereof; so long as it is capable of forming 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 need not interact with another variable domain to form a functional antigen binding unit).

Immunoglobulin Single Variable Domains (ISVD) may be, for example, heavy chain ISVD, such as V _H 、V _HH Comprising camelized V _H Or humanized V _HH . According to some embodiments, the Immunoglobulin Single Variable Domain (ISVD) is V _HH Comprising camelized V _H Or humanized V _HH . The heavy chain ISVD may be derived from conventional four-chain antibodies or heavy chain antibodies.

For example, an immunoglobulin single variable domain 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 ) The method comprises the steps of carrying out a first treatment on the surface of the Other single variable domains, or any suitable fragment of any of these.

In particular, the immunoglobulin single variable domain may be(e.g. V _HH Including humanized V _HH Or camelized V _H ) Or a suitable fragment thereof. />And->Is a registered trademark of Ablynx n.v.

“V _HH Domain "(also known as V) _HH 、V _HH Antibody fragments and V _HH Antibodies) were initially described as "heavy chain antibodies" (i.e., "antibodies without light chains"; hamers-Casterman et al Nature363:446-448, 1993) antigen binding immunoglobulin variable domains. Select the term "V _HH Domains "in order to combine these variable domains with the heavy chain variable domains found in conventional 4-chain antibodies (referred to herein as" V _H Domain ") and a light chain variable domain (referred to herein as" V ") found in conventional 4-chain antibodies _L Domains ") are distinguished. For V _HH For further description, reference is made to the review article of Muyledermans (Reviews in Molecular Biotechnology 74:277-302,2001), and the following patent applications mentioned as general background: WO 94/04678, WO 95/04079 and WO 96/34103 of the university of Brussell freedom (Vrije Universiteit Brussel); WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Unilever; WO 97/49505, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of the Belgium Frand Biotechnology institute (Vlaams Instituut voor Biotechnologie, VIB); WO 03/050531 to Alganomics N.V. and Ablynx N.V.; WO 01/90190 of national institute of Canadian (National Research Council of Canada); WO 03/025020 (=ep 1433793) of the institute of antibodies (Institute of Antibodies); and Ablynx N.V. WO 04/041687, WO 04/041682, WO 04/041685, WO 04/041683, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO 06/122786, WO 06/122787 and WO 06/122825, each of which is incorporated by reference in its entirety The body is incorporated herein.

Typically, the production of immunoglobulins involves immunization of an experimental animal, fusion of immunoglobulin-producing cells to produce hybridomas, and screening for the desired specificity. Alternatively, immunoglobulins may be generated by screening a naive or synthetic library, for example by phage display.

Immunoglobulin sequences (e.g) The production of (C) has been widely described in various publications, of which WO 94/04678, hamers-Casterman et al 1993 and Muyldermans et al 2001 (Reviews in Molecular Biotechnology 74:277-302,2001), each of which is incorporated herein by reference in its entirety, may be taken as examples. In these methods, a camelid is immunized with a target antigen in order to induce an immune response against the target antigen. The pool of Nanobodies obtained from the immunization is further screened for Nanobodies that bind to the target antigen.

In these cases, the production of antibodies requires purified antigen for immunization and/or screening. The antigen may be purified from natural sources or during recombinant production.

Immunization and/or screening of immunoglobulin sequences may be performed using peptide fragments of such antigens.

Immunoglobulin sequences of different origins may be used, including mouse, rat, rabbit, donkey, human and camel immunoglobulin sequences. The disclosure also includes fully human, humanized or chimeric sequences. For example, the present disclosure includes camel immunoglobulin sequences and humanized camel immunoglobulin sequences or camelized domain antibodies, e.g., camelized dabs described by Ward et al (see, e.g., WO 94/04678 and Riechmann, febs lett.,339:285-290,1994 and prot.eng.,9:531-537,1996, each of which is incorporated herein in its entirety by reference). In addition, the present disclosure also uses fusion immunoglobulin sequences, e.g., to form multivalent and/or multispecific constructs (for polypeptides containing one or more V _HH Domain multivalent and multispecific polypeptides and their formulations, also referred to Conrath et al, J.biol.chem., volume 276, 10.7346-7350,2001 and, for example, WO 96/34103 and WO 99/23221, each of which is incorporated herein by reference in its entirety), as well as immunoglobulin sequences comprising tags or other functional moieties (e.g., toxins, labels, radiochemicals, etc.) that can be derived from the immunoglobulin sequences of the present disclosure.

"humanized V _HH "comprising a polypeptide corresponding to naturally occurring V _HH Amino acid sequence of a domain but which has been "humanized", i.e., by use in a V of a conventional 4-chain antibody from human (e.g., as indicated above) _H Substitution of one or more amino acid residues present at one or more corresponding positions of the domain for the naturally occurring V _HH One or more amino acid residues in the amino acid sequence of the sequence (and in particular in the framework sequence). This can be done in a manner known per se, for example based on the further description herein and as is clear to a person skilled in the art in the literature (e.g. WO 2008/020079). Also, note that such humanized V _HH Can be obtained in any suitable manner known per se and is therefore not strictly limited to the already use of a composition comprising naturally occurring V _HH A polypeptide obtained from a polypeptide having a domain as a starting material.

"camel derived V _H "comprising a polypeptide corresponding to naturally occurring V _H Amino acid sequence of the domain but which has been "camelized", i.e.by use in the V of heavy chain antibodies _HH Substitution of one or more amino acid residues present at one or more corresponding positions of the domain for naturally occurring V from conventional 4-chain antibodies _H One or more amino acid residues in the amino acid sequence of the domain. This can be done in a manner known per se, for example based on the further description herein and the literature (e.g. WO 2008/020079) as will be clear to a person skilled in the art. Such "camelized" substitutions, as defined herein, may be insertionally formed and/or present at V _H -V _L The amino acid position of the interface and/or at a so-called camelidae tag residue (see e.g. WO 94/04678 and Davies and Riechmann,1994 and 1996, supra). In some embodiments, as a production or design of camelized V _H V of the starting material or origin of (C) _H The sequence is V from a mammal _H V of a sequence or human _H Sequences, e.g. V _H 3 sequence. It should be noted, however, that such camelized V may be obtained in any suitable manner known per se _H And is thus not strictly limited to the use of a composition comprising naturally occurring V _H A polypeptide obtained from a polypeptide having a domain as a starting material.

It should be noted that one or more immunoglobulin sequences may be linked to each other and/or to other amino acid sequences (e.g., via disulfide bridges) to provide peptide constructs (e.g., fab 'fragments, F (ab') 2 fragments, scFv constructs, "diabodies" and other multispecific constructs) that may also be useful. For example, refer to Holliger and Hudson, nat biotechnol.2005, month 9; 23 A review of 1126-36. In general, when a polypeptide is intended for administration to a subject (e.g., for prophylactic, therapeutic, and/or diagnostic purposes), it can comprise an immunoglobulin sequence that does not occur naturally in the subject.

Non-limiting examples of structures of immunoglobulin single variable domain sequences may be considered to be made up of four framework regions ("FR"), which are referred to in the art and herein as "framework region 1" ("FR 1"), respectively; "frame region 2" ("FR 2"); "frame region 3" ("FR 3"); and "frame region 4" ("FR 4"); the framework region is interrupted by three complementarity determining regions ("CDRs") referred to in the art and herein, respectively, as "complementarity determining region 1" ("CDR 1"); "complementarity determining region 2" ("CDR 2"); and "complementarity determining region 3" ("CDR 3").

As further described in paragraph q) on pages 58 and 59 of WO 08/020079 (incorporated herein by reference), the amino acid residues of the immunoglobulin single variable domain may be as set forth in V by Kabat et al ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, publication No. 91) _H The general numbering of the domains is carried out, for example, in Riechmann and Muydermans, 2000 (J. Immunol. Methods 240 (1-2): 185-195; seeFor example figure 2) of the publication for application to V from camelids _HH A domain. It should be noted that, as is well known in the art, for V _H Domain and V _HH Domain-the total number of amino acid residues in each CDR may vary, and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e., one or more positions according to Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the Kabat numbering allows). This means that in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. V (V) _H Domain and V _HH The total number of amino acid residues in the domain will typically be in the range of 110 to 120, typically between 112 and 115. It should be noted, however, that smaller and longer sequences may also be suitable for the purposes described herein.

In the present application, unless otherwise indicated, the CDRs are determined according to AbM numbers as described in Kontermann and Dubel (edit 2010,Antibody Engineering, vol. 2, springer Verlag Heidelberg Berlin, martin, chapter 3, pages 33-51). According to this method, FR1 comprises the amino acid residues at positions 1-25, CDR1 comprises the amino acid residues at positions 26-35, FR2 comprises the amino acid residues at positions 36-49, CDR2 comprises the amino acid residues at positions 50-58, FR3 comprises the amino acid residues at positions 59-94, CDR3 comprises the amino acid residues at positions 95-102, and FR4 comprises the amino acid residues at positions 103-113.

Determination of CDR regions may also be performed according to different methods. In a CDR assay according to Kabat, FR1 of the immunoglobulin single variable domain comprises amino acid residues at positions 1-30, CDR1 of the immunoglobulin single variable domain comprises amino acid residues at positions 31-35, FR2 of the immunoglobulin single variable domain comprises amino acid residues at positions 36-49, CDR2 of the immunoglobulin single variable domain comprises amino acid residues at positions 50-65, FR3 of the immunoglobulin single variable domain comprises amino acid residues at positions 66-94, CDR3 of the immunoglobulin single variable domain comprises amino acid residues at positions 95-102, and FR4 of the immunoglobulin single variable domain comprises amino acid residues at positions 103-113.

In such immunoglobulin sequences, the framework sequences may be any suitable framework sequences, and examples of suitable framework sequences will be apparent to the skilled artisan, e.g., based on standard handbooks and further disclosures and the prior art mentioned herein.

The framework sequences may be immunoglobulin framework sequences or suitable combinations of framework sequences derived from immunoglobulin framework sequences (e.g., by humanization or camelization). For example, the framework sequence may be derived from a light chain variable domain (e.g., V _L Sequence) and/or heavy chain variable domains (e.g., V _H Sequence or V _HH Sequence) of the sequence. In one embodiment, the framework sequence is derived from V _HH A framework sequence of a sequence, wherein the framework sequence may optionally be partially or fully humanized; or conventional V which has been camelized _H Sequences (as defined herein).

In particular, the framework sequences present in an ISVD sequence as disclosed herein can contain one or more marker residues (as defined herein), such that the ISVD sequence is(e.g. V _HH Including humanized V _HH Or camelized V _H ). Some non-limiting examples of such framework sequences (suitable combinations) will become apparent from the further disclosure herein.

Also, as generally described herein for immunoglobulin sequences, any suitable fragment (or combination of fragments) of the foregoing may also be used, such as a fragment containing one or more CDR sequences, suitably flanked by and/or linked via one or more framework sequences (e.g., may be present in the full-size immunoglobulin sequence from which the fragment is derived in the same order as the CDRs and framework sequences).

It should be noted, however, that the present disclosure pertains to the origin of the ISVD sequence (or the nucleotide sequence used to express the ISVD sequence), and to the production or acquisition thereof The manner in which the ISVD sequence or nucleotide sequence is (or has been) generated or obtained is not limited. Thus, an ISVD sequence can be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In particular but non-limiting aspects, an ISVD sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semisynthetic 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 _HH Sequence), "camelized" (as defined herein) immunoglobulin sequences, and immunoglobulin sequences obtained by techniques such as: affinity maturation (e.g., starting from synthetic, random, or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences that are well known to the skilled artisan; or any suitable combination of any of the foregoing.

Similarly, the nucleotide sequence may be a naturally occurring nucleotide sequence or a synthetic or semisynthetic sequence, and may be a sequence isolated from a suitable naturally occurring template (e.g., DNA or RNA isolated from a cell), a nucleotide sequence that has been isolated from a library (and in particular, an expression library), a nucleotide sequence that has been prepared by introducing mutations 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.

As described above, the ISVD may beOr a suitable fragment thereof. For->(And->Is a general description of Ablynx n.v. (registered trademark of Sanofi Company), with reference to the following further description and the prior art cited herein. In this respect, however, it should be noted that this description and the prior art mainly describe so-called "V _H Class 3->(i.e. with V such as DP-47, DP-51 or DP-29 _H Human germline sequences of class 3 have a high degree of sequence homology +.>). It should be noted, however, that the present disclosure in its broadest sense may generally be used with any type +.>And also for example to use a component belonging to the so-called "V _H Class 4'(i.e. with V as DP-78) _H Human germline sequences of class 4 have a high degree of sequence homology) Examples are described in WO 2007/118670, incorporated herein by reference in its entirety.

In general, the number of the devices used in the system,(especially V _HH Sequences, including (partially) humanized V _HH Sequence and camelization V _H Sequences) may be characterized by the presence of one or more "tag residues" (as described herein) in one or more framework sequences (also as described further herein). Therefore, it is possible to always add +.>Defined as immunoglobulin sequences having a (general) structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

Wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and wherein one or more tag residues are as further defined herein.

In particular the number of the elements to be processed,may be an immunoglobulin sequence having a (general) structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

Wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and wherein the framework sequences are as further defined herein.

More particularly, it is possible to provide,may be an immunoglobulin sequence having a (general) structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

Wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and wherein:

one or more amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to Kabat numbering are selected from the marker residues mentioned in table a-0 below.

Table a-0:marker residues in (a)

In some embodiments, the tag residue at position 11 is L. In some embodiments, the tag residue at position 37 is F ⁽¹⁾ Or Y. In some embodiments, the tag residue at position 44 is G ⁽²⁾ Or Q ⁽³⁾ . In some embodiments, the tag residue at position 45 is L ⁽²⁾ Or R is ⁽³⁾ . In some embodiments, the tag residue at position 47 is F ⁽¹⁾ 、L ⁽¹⁾ Or W ⁽²⁾ . In some embodiments, the tag residue at position 83 is K. In some embodiments, the tag residue at position 84 is P. In some embodiments, the tag residue at position 103 is W. In some embodiments, the tag residue at position 104 is G. In some embodiments, the tag residue at position 108 is Q or L.

The present disclosure uses, among other things, ISVD that specifically binds to OX40L or IL-13. In the context of the present disclosure, "bind to a certain target molecule" has a common meaning in the art as understood in the context of antibodies and their corresponding antigens.

The polypeptides of the disclosure can comprise one or more ISVD that binds to OX40L and two or more ISVD that bind to IL-13. For example, the polypeptide can comprise one ISVD that binds to OX40L and two ISVD that binds to IL-13.

In some embodiments, at least one ISVD can functionally block its target molecule. For example, the targeting moiety may block the interaction between OX40L and OX40 (receptor), and in some embodiments may inhibit OX 40L-induced release of IL2 from T cells, or may block the interaction between IL-13 and IL-13Rα1 (Interleukin 13 receptor, α1) and/or the IL-13/IL-13Rα1 complex and IL-4Rα (αInterleukin-4 receptor). Thus, in one embodiment, the polypeptides of the present disclosure comprise at least one ISVD that specifically binds to OX40L and inhibits its interaction with OX40, and two ISVDs that specifically bind to IL-13 and functionally block its interaction with IL-13Rα1 and/or between the IL-13/IL-13Rα1 complex and IL-4Rα.

The ISVD used in the present disclosure forms part of the polypeptides of the present disclosure, which comprise or consist of at least three ISVD, such that the polypeptides can specifically bind to OX40L and IL-13.

Thus, the target molecules of the at least three ISVD as used in the polypeptides of the present disclosure are OX40L and IL-13. Examples are mammalian OX40L and IL-13. Although human OX40L (Uniprot accession No. P23510) and human IL-13 (Uniprot accession No. P35225) may be used, forms from other species are also suitable for use in the present disclosure, such as OX40L and IL-13 from mice, rats, rabbits, cats, dogs, goats, sheep, horses, pigs, non-human primates (e.g., cynomolgus monkey (also referred to herein as "cyno") or camelids (e.g., llama or alpaca).

Specific examples of ISVD that specifically bind to OX40L or IL-13 that can be used in the present disclosure are described in items a to C below:

A. ISVD that specifically binds to human OX40L and comprises

Amino acid sequence having SEQ ID NO. 14 or CDR3 having 2 or 1 amino acid differences from SEQ ID NO. 14,

in some embodiments, CDR1 has the amino acid sequence of SEQ ID NO. 6, CDR2 has the amino acid sequence of SEQ ID NO. 10, and CDR3 has the amino acid sequence of SEQ ID NO. 14.

Non-limiting examples of such ISVD that specifically binds to human OX40L have one or more or all of the framework regions (and CDRs as defined in item A above) as shown in Table A-2 for construct 15B07AM, such as ISVD having the complete amino acid sequence of construct 15B07AM (SEQ ID NO:2, see tables A-1 and A-2).

Furthermore, in one embodiment, the amino acid sequence of ISVD that specifically binds to human OX40L can have greater than 90%, such as greater than 95% or greater than 99% sequence identity to SEQ ID NO. 2, wherein the CDRs are optionally as defined in item A above. In some embodiments, the ISVD that specifically binds to OX40L has the amino acid sequence of SEQ ID NO. 2.

When such ISVD that specifically binds to OX40L has a 2 or 1 amino acid difference in at least one CDR relative to the corresponding reference CDR sequence (item a above), in some embodiments, the ISVD has at least half, at least the same, or even a higher binding affinity to human OX40L as compared to construct 15B07AM, wherein the binding affinity is measured using the same method (e.g., SPR).

B. ISVD that specifically binds to human IL-13 and comprises

i. CDR1 having the amino acid sequence of SEQ ID NO. 7 or having 2 or 1 amino acid differences from SEQ ID NO. 7;

CDR2 having the amino acid sequence of SEQ ID No. 11 or having 2 or 1 amino acid differences from SEQ ID No. 11; and

amino acid sequence having SEQ ID NO. 15 or CDR3 having 2 or 1 amino acid differences from SEQ ID NO. 15,

in some embodiments, CDR1 has the amino acid sequence of SEQ ID NO. 7, CDR2 has the amino acid sequence of SEQ ID NO. 11, and CDR3 has the amino acid sequence of SEQ ID NO. 15.

Non-limiting examples of such ISVD that specifically binds human IL-13 have one or more or all of the framework regions (and CDRs as defined in item B above) as shown in Table A-2 for construct 4B02/1, such as ISVD having the complete amino acid sequence of construct 4B02/1 (SEQ ID NO:3, see tables A-1 and A-2).

Furthermore, in one embodiment, the amino acid sequence of ISVD that specifically binds to human IL-13 can have greater than 90%, such as greater than 95% or greater than 99% sequence identity with SEQ ID NO 3, wherein the CDRs are optionally as defined in item B above. In some embodiments, the ISVD that binds IL-13 has the amino acid sequence of SEQ ID NO. 3.

When such an ISVD that binds IL-13 has a 2 or 1 amino acid difference in at least one CDR relative to the corresponding reference CDR sequence (item B above), in some embodiments, the ISVD has at least half, at least the same, or even a higher binding affinity for human IL-13 as compared to construct 4B02/1, wherein the binding affinity is measured using the same method (e.g., SPR).

C. ISVD that specifically binds to human IL-13 and comprises

i. CDR1 having the amino acid sequence of SEQ ID NO 9 or having a 2 or 1 amino acid difference from SEQ ID NO 9;

amino acid sequence having SEQ ID NO. 17 or CDR3 having 2 or 1 amino acid differences from SEQ ID NO. 17,

in some embodiments, CDR1 has the amino acid sequence of SEQ ID NO. 9, CDR2 has the amino acid sequence of SEQ ID NO. 13, and CDR3 has the amino acid sequence of SEQ ID NO. 17.

Non-limiting examples of such ISVD that specifically binds human IL-13 have one or more or all of the framework regions (and CDRs as defined in item C above) as shown in Table A-2 for construct 4B06/1, such as ISVD having the complete amino acid sequence of construct 4B06/1 (SEQ ID NO:5, see tables A-1 and A-2).

Furthermore, in one embodiment, the amino acid sequence of ISVD that specifically binds to human IL-13 can have greater than 90%, such as greater than 95% or greater than 99% sequence identity with SEQ ID NO 5, wherein the CDRs are optionally as defined in item C above. In some embodiments, the ISVD that binds IL-13 has the amino acid sequence of SEQ ID NO. 5.

When such an ISVD that specifically binds to IL-13 has a 2 or 1 amino acid difference in at least one CDR relative to the corresponding reference CDR sequence (item C above), the ISVD has at least half, at least the same, or even a higher binding affinity for human IL-13 compared to construct 4B06/1, wherein the binding affinity is measured using the same method (e.g., SPR).

In some embodiments, each ISVD as defined in items a to C above is comprised in a polypeptide of the present disclosure. In some embodiments, such a polypeptide of the disclosure comprising each ISVD as defined in items a to C above has at least half, at least the same, or even higher binding affinity for human OX40L and human IL-13 compared to a polypeptide consisting of the amino acids of SEQ ID No. 1, wherein the binding affinity is measured using the same method (e.g., SPR).

The SEQ ID NOs mentioned in the above items A to C are based on the CDR definitions according to the AbM definition (see Table A-2). Note that SEQ ID NOs defining the same CDRs according to the Kabat definition (see table a-2.1) can be used equally as well for items a to C above.

Thus, the specific ISVD that can use AbM definition to specifically bind to OX40L or IL-13 as described above in this disclosure can also be described using the Kabat definition as described in items A 'through C' below:

ISVD which specifically binds to human OX40L and comprises

i. CDR1 having the amino acid sequence of SEQ ID NO. 31 or having a 2 or 1 amino acid difference from SEQ ID NO. 31;

CDR2 having the amino acid sequence of SEQ ID No. 35 or having 2 or 1 amino acid differences from SEQ ID No. 35; and

in some embodiments, CDR1 has the amino acid sequence of SEQ ID NO. 31, CDR2 has the amino acid sequence of SEQ ID NO. 35, and CDR3 has the amino acid sequence of SEQ ID NO. 14.

Non-limiting examples of such ISVD that specifically binds to human OX40L have one or more or all of the framework regions (and CDRs as defined in item A' above) as shown in Table A-2.1 for construct 15B07AM, such as ISVD having the complete amino acid sequence of construct 15B07AM (SEQ ID NO:2, see tables A-1 and A-2.1).

B'. ISVD that specifically binds to human IL-13 and comprises

i. CDR1 having the amino acid sequence of SEQ ID NO. 32 or having a 2 or 1 amino acid difference from SEQ ID NO. 32;

CDR2 having the amino acid sequence of SEQ ID No. 36 or having 2 or 1 amino acid differences from SEQ ID No. 36; and

in some embodiments, CDR1 has the amino acid sequence of SEQ ID NO. 32, CDR2 has the amino acid sequence of SEQ ID NO. 36, and CDR3 has the amino acid sequence of SEQ ID NO. 15.

Non-limiting examples of such ISVD that specifically binds human IL-13 have one or more or all of the framework regions (and CDRs as defined in item B' above) as shown in Table A-2.1 for construct 4B02/1, such as ISVD having the complete amino acid sequence of construct 4B02/1 (SEQ ID NO:3, see tables A-1 and A-2.1).

C'. ISVD that specifically binds to human IL-13 and comprises

i. CDR1 having the amino acid sequence of SEQ ID No. 34 or having 2 or 1 amino acid differences from SEQ ID No. 34;

CDR2 having the amino acid sequence of SEQ ID No. 38 or having 2 or 1 amino acid differences from SEQ ID No. 38; and

in some embodiments, CDR1 has the amino acid sequence of SEQ ID NO. 34, CDR2 has the amino acid sequence of SEQ ID NO. 38, and CDR3 has the amino acid sequence of SEQ ID NO. 17.

Non-limiting examples of such ISVD that specifically binds human IL-13 have one or more or all of the framework regions (and CDRs as defined in the foregoing item C') as shown in Table A-2.1 for construct 4B06/1, such as ISVD having the complete amino acid sequence of construct 4B06/1 (SEQ ID NO:5, see tables A-1 and A-2.1).

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

Generally, for the purpose of determining the percentage of "sequence identity" between two amino acid sequences according to the calculation method outlined above, the amino acid sequence with the largest number of amino acid residues is taken as the "first" amino acid sequence and the other amino acid sequence is taken as the "second" amino acid sequence.

"amino acid differences" as used herein refers to deletions, insertions, or substitutions of a single amino acid residue relative to a reference sequence. In some embodiments, the amino acid difference is a substitution.

In some embodiments, the amino acid substitution is a conservative substitution. In some embodiments, such conservative substitutions are substitutions in which one amino acid in the following groups (a) - (e) is substituted with another amino acid residue in the same group: (a) small aliphatic, nonpolar or micropolarity residues: ala, ser, thr, pro and Gly; (b) Polar, negatively charged residues and (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) an aromatic residue: phe, tyr and Trp.

In some embodiments, the conservative substitutions are as follows: ala to Gly or Ser; arg becomes Lys; asn becomes Gln or His; asp becomes Glu; cys becomes Ser; gln becomes Asn; glu to Asp; gly to Ala or Pro; his becomes Asn or Gln; ile becomes Leu or Val; leu becomes Ile or Val; lys to Arg to Gln or Glu; met becomes Leu, tyr or Ile; phe to Met, leu or Tyr; ser becomes Thr; thr to Ser; trp becomes Tyr; tyr becomes Trp; and/or Phe to Val, ile or Leu.

5.2 specificity

The terms "specifically," "specifically bind," or "specifically bind" refer to the number of different target molecules (such as antigens) from the same organism that a particular binding unit (such as ISVD) can bind with sufficiently high affinity (see below). "specific," specifically binds, "or" specifically binds "are used interchangeably herein with" selectively, "" selectively binds, "or" selectively binds. According to some embodiments, the binding unit (e.g., ISVD) specifically binds to its designated target.

The specificity/selectivity of the binding unit can be determined based on affinity. Affinity refers to the strength or stability of molecular interactions. Affinity is typically given by KD or dissociation constant in moles/liter (or M). Affinity can also be expressed as an association constant KA, which is equal to 1/KD and has (mol/liter) ^-1 (or M) ^-1 ) Is a unit of (a).

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

Typically, the binding units used in this disclosure (e.g., ISVD) will be at 10 ^-5 To 10 ^-12 Molar/liter or less, e.g. 10 ^-7 To 10 ^-12 Molar/liter or less, more particularly such as 10 ^-8 To 10 ^-12 Dissociation constant (KD) in moles/liter (i.e. at 10 ⁵ To 10 ¹² Liter/mole or more, e.g. 10 ⁷ To 10 ¹² Liter/mole or higher, and more particularly such as 10 ⁸ To 10 ¹² The association constant (KA) in liters per mole) binds to its target (at room temperature).

Greater than 10 ^-4 Any KD value of mole/liter (or less than 10 ⁴ Any KA value of liters/mole) is generally considered to indicate non-specific binding.

KD for biological interactions believed to be specific (such as binding of immunoglobulin sequences to antigens) is typically at 10 ^-5 Molar/liter (10000 nM or 10. Mu.M) to 10 ^-12 Molar per liter (0.001 nM or 1 pM) or less.

Thus, specific/selective binding may mean that the binding unit (or polypeptide comprising it) is bound at 10 using the same measurement method (e.g.SPR) ^-5 To 10 ^-12 KD values of mole/liter or less bind to OX40L and/or IL-13 and are greater than 10 ^-4 The KD values of moles/liter bind to the relevant cytokines. Examples of OX 40L-related targets are human TRAIL, CD30L, CD L and RANKL. An example of an IL-13-related target is human IL-4. Thus, in one embodiment, at least one ISVD contained in the polypeptide is at 10 ^-5 To 10 ^-12 KD values of mole/liter or less bind to OX40L to greater than 10 ^-4 The KD value of mole/liter binds to TRAIL, CD30L, CD L and RANKL of the same species, and at least two ISVD contained in the polypeptide are 10 ^-5 To 10 ^-12 KD values of mole/liter or less bind IL-13 to greater than 10 ^-4 The molar KD value per liter is bound to IL-4 of the same species.

Thus, in some embodiments, the polypeptides of the disclosure have at least half, at least the same, or even higher binding affinity to human OX40L and human IL-13 as compared to a polypeptide consisting of the amino acids of SEQ ID NO. 1, wherein the binding affinity is measured using the same method (e.g., SPR).

Specific binding to a certain target from a certain species does not exclude that binding units may also specifically bind to similar targets from different species. For example, specific binding to human OX40L does not exclude that binding units or polypeptides comprising said binding units may also specifically bind to OX40L from cynomolgus monkey. Also, for example, specific binding to human IL-13 does not exclude that a binding unit or a polypeptide comprising said binding unit may also specifically bind to IL-13 from cynomolgus monkey ("cyno").

Specific binding of a binding unit to its designated target may be determined in any suitable manner known per se, including but not limited to Scatchard analysis and/or competitive binding assays, such as Radioimmunoassays (RIA), enzyme Immunoassays (EIA), and sandwich competition assays, as well as different variants thereof known per se in the art; as well as other techniques mentioned herein.

As will be clear to the skilled person, the dissociation constant may be an actual or apparent dissociation constant. The method for determining the dissociation constant will be clear to the skilled person and includes, for example, the techniques mentioned below. In this respect, it will also be clear that it may not be possible to measure more than 10 ^-4 Mol/liter or 10 ^-3 Moles/liter (e.g., 10 ^-2 Moles/liter). Optionally, it will also be clear to the skilled person that the association constant (KA) may be (actual or apparent) based by means of the relation [ kd=1/KA]To calculate (actual or apparent) dissociation constants.

The affinity of the molecular interaction between two molecules can be measured via different techniques known per se, 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 analysis of real-time biospecific interactions by detecting changes in protein concentration within a biosensor matrix, where one molecule is immobilized on a biosensor chip and another molecule is subjected to immobilized molecules under flow conditions, resulting in k _{Association with} 、k _{Dissociation of} Measured values, and thus K _D (or K) _A ) Values. For example, this may be done using well known techniquesSystem (BIAcore International AB, GE Healthcare, uppsala, sweden and Piscataway, N.J.). For further description, see Jonsson et al (1993, ann. Biol. Clin. 51:19-26), jonsson et al (1991Biotechniques 11:620-627), johnsson et al (1995, J. Mol. Recognit. 8:125-131), and Johnsson et al (1991, anal. Biochem. 198:268-277).

Another well-known biosensor technique to determine the affinity of biomolecular interactions is Biological Layer Interferometry (BLI) (see, e.g., abdiche et al 2008, anal. Biochem.377:209-217). As used herein, the term "bio-layer interferometry" or "BLI" refers to label-free optical techniques that analyze the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and a protein-immobilized layer (signal beam) on the biosensor tip. The change in the number of molecules bound to the biosensor tip will result in a shift in the interference pattern, reported as wavelength shift (nm), whose magnitude is a direct measure of the number of molecules bound to the surface of the biosensor tip. Since interactions can be measured in real time, association and dissociation rates and affinities can be determined. For example, BLI may use well known The system (ForteBio, division of Pall Life Sciences, gated lopak, usa).

Alternatively, it is possible to usePlatform (Sapidyne Instruments Inc, boysema, U.S.) measures affinity in a kinetic exclusion assay (KinExA) (see, e.g., drake et al 2004, anal. Biochem., 328:35-43). As used herein, the term "KinExA" refers to a solution-based method of measuring the true equilibrium binding affinity and kinetics of an unmodified molecule. The equilibrated solution of the antibody/antigen complex is passed through a column with beads pre-coated with antigen (or antibody) allowing free antibody (or antigen) to bind to the coated molecule. Detection of the thus captured antibody (or antigen) is accomplished with a fluorescently labeled protein that binds to the antibody (or antigen).

The immunoassay system provides a platform for automated biological analysis and rapid sample turnover (Fraley et al 2013,Bioanalysis 5:1765-74).

5.3 (in vivo) half-life extension

The polypeptide may further comprise one or more other groups, residues, moieties or binding units optionally linked via one or more peptide linkers, wherein the one or more other groups, residues, moieties or binding units provide the polypeptide with an increased (in vivo) half-life compared to the corresponding polypeptide without the one or more other groups, residues, moieties or binding units. By in vivo half-life extension is meant, for example, that the polypeptide has an increased half-life in a mammal (such as a human subject) after administration. Half-life may be expressed, for example, as t1/2 beta.

The type of group, residue, moiety or binding unit is generally not limited and may be selected, for example, from polyethylene glycol molecules, serum proteins or fragments thereof, binding units that may bind to serum proteins, fc moieties, and small proteins or peptides that may bind to serum proteins.

More specifically, the one or more other groups, residues, moieties or binding units providing the polypeptide with increased half-life may be selected from binding units that may bind to serum albumin (such as human serum albumin) or serum immunoglobulins (such as IgG). In some embodiments, the binding unit may bind to human serum albumin. In some embodiments, the binding unit is ISVD.

For example, WO 04/041685 (incorporated by reference in its entirety) describes binding to serum albumin (and in particular to human serum albumin)Which may be combined with other proteins (e.g. one or more other +.>) Ligation to increase half-life of the protein.

International application WO 06/122787, incorporated by reference in its entirety, describes a variety of antibodies directed against (human) serum albuminThese->Including what is known as Alb-1 (SEQ ID NO:52 of WO 06/122787 incorporated by reference in its entirety) and humanized variants thereof such as Alb-8 (SEQ ID NO:62 of WO 06/122787 incorporated by reference in its entirety) >Likewise, these can be used to extend the half-life of therapeutic proteins and polypeptides, as well as other therapeutic entities or moieties.

Furthermore, WO 2012/175400 (incorporated by reference in its entirety) describes a further improved form of Alb-1, referred to as Alb-23.

In one embodiment, the polypeptide comprises a serum albumin binding moiety selected from the group consisting of Alb-1, alb-3, alb-4, alb-5, alb-6, alb-7, alb-8, alb-9, alb-10 and Alb-23. In some embodiments, the polypeptide comprises Alb-8 or Alb-23, or variants thereof, as shown on pages 7-9 of WO 2012/175400, and albumin conjugates described in WO 2012/175741, WO 2015/173325, WO 2017/080850, WO 2017/085172, WO 2018/104444, WO 2018/134235, WO 2018/134234, each of which is incorporated herein by reference in its entirety. Some non-limiting examples of serum albumin binders are also shown in Table A-4. In some embodiments, the polypeptides of the disclosure comprise other components as described in item D:

D. ISVD binding to human serum albumin and comprising

CDR3 having the amino acid sequence of SEQ ID No. 16 or having 2 or 1 amino acid differences from SEQ ID No. 16;

in some embodiments, CDR1 has the amino acid sequence of SEQ ID NO. 8, CDR2 has the amino acid sequence of SEQ ID NO. 12, and CDR3 has the amino acid sequence of SEQ ID NO. 16.

Non-limiting examples of such ISVD binding to human serum albumin have one or more or all of the framework regions (and CDRs as defined in item D above) as shown for construct ALB23002 in Table A-2, such as ISVD with the complete amino acid sequence of construct ALB23002 (SEQ ID NO:4, see tables A-1 and A-2).

Item D can also be described as using the Kabat definition:

ISVD binding to human serum albumin and comprising

i. CDR1 having the amino acid sequence of SEQ ID NO. 33 or having a 2 or 1 amino acid difference from SEQ ID NO. 33;

CDR2 having the amino acid sequence of SEQ ID No. 37 or having 2 or 1 amino acid differences from SEQ ID No. 37; and

in some embodiments, CDR1 has the amino acid sequence of SEQ ID NO. 33, CDR2 has the amino acid sequence of SEQ ID NO. 37, and CDR3 has the amino acid sequence of SEQ ID NO. 16.

Non-limiting examples of such ISVD binding to human serum albumin have one or more or all of the framework regions (and CDRs as defined in item D' above) as shown for construct ALB23002 in Table A-2.1, such as ISVD with the complete amino acid sequence of construct ALB23002 (SEQ ID NO:4, see tables A-1 and A-2.1).

Furthermore, in one embodiment, the amino acid sequence of the ISVD that binds to human serum albumin can have greater than 90%, such as greater than 95% or greater than 99% sequence identity to SEQ ID No. 4, wherein the CDRs are optionally as defined in item D above. In some embodiments, the ISVD that binds human serum albumin has the amino acid sequence of SEQ ID NO. 4.

When such ISVD that binds to human serum albumin has a 2 or 1 amino acid difference in at least one CDR relative to the corresponding reference CDR sequence (item D above), the ISVD has at least half, at least the same, or even a higher binding affinity to human serum albumin than construct ALB23002, wherein the binding affinity is measured using the same method (e.g., SPR).

When such an ISVD that binds to human serum albumin has a C-terminal position, it exhibits a C-terminal alanine (A) or glycine (G) extension and may be selected from SEQ ID NOs 52, 53, 55, 57, 58, 59, 60, 61, 62 and 63 (see Table A-4 below). In one embodiment, the ISVD that binds human serum albumin has another position than the C-terminal position (i.e., is not the C-terminal ISVD of the polypeptides of this disclosure) and is selected from SEQ ID NOs: 4, 50, 51, 54, and 56 (see Table A-4 below).

5.4 nucleic acid molecules

Also provided is a nucleic acid molecule encoding a polypeptide of the disclosure.

A "nucleic acid molecule" (used interchangeably with "nucleic acid") is a chain of nucleotide monomers that are linked to one another via a phosphate backbone to form a nucleotide sequence. The nucleic acids may be used to transform/transfect a host cell or host organism, for example for expression and/or production of a polypeptide. Suitable hosts or host cells for production purposes will be apparent to the skilled person and may be, for example, any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism. Also included in the present disclosure are hosts or host cells comprising nucleic acids encoding polypeptides of the present disclosure.

The nucleic acid may be, for example, DNA, RNA or hybrids thereof, and may also comprise (e.g., chemically) modified nucleotides, such as PNA. It may be single-stranded or double-stranded DNA. For example, the nucleotide sequence of the present disclosure may be genomic DNA, cDNA.

The nucleic acids of the present disclosure may be prepared or obtained in a manner known per se and/or may be isolated from a suitable natural source. Nucleotide sequences encoding naturally occurring (poly) peptides may be subjected, for example, to site-directed mutagenesis in order to provide nucleic acid molecules encoding polypeptides having sequence variations. Also, as will be clear to the skilled person, several nucleotide sequences, such as at least one nucleotide sequence encoding a targeting moiety and e.g. a nucleic acid encoding one or more linkers, may also be joined together in a suitable manner for the preparation of a nucleic acid.

Techniques for producing nucleic acids will be apparent to the skilled artisan and may include, for example, but are not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and/or synthetic sequences (or two or more portions thereof), introducing a mutation that results in expression of the truncated expression product; introducing one or more restriction sites (e.g., to create cassettes and/or regions that may be readily digested and/or ligated using appropriate restriction enzymes), and/or introducing mutations via a PCR reaction using one or more "mismatched" primers.

5.5 vectors

Also provided is a vector comprising a nucleic acid molecule encoding a polypeptide of the disclosure. A vector as used herein is a vehicle suitable for carrying genetic material into a cell. Vectors include naked nucleic acids (e.g., plasmids or mRNA) or nucleic acids embedded in larger structures (e.g., liposomes or viral vectors).

Vectors typically comprise at least one nucleic acid, optionally linked to one or more regulatory elements (e.g., such as one or more suitable promoters, enhancers, terminators, etc.). The vector is an expression vector, i.e., a vector suitable for expressing the encoded polypeptide or construct under suitable conditions, e.g., when the vector is introduced into a (e.g., human) cell. For DNA-based vectors, it generally includes the presence of elements for transcription (e.g., promoters and polyA signals) and translation (e.g., kozak sequences).

In some embodiments, in the vector, the at least one nucleic acid and the regulatory element are "operably linked" to each other, which generally means that they are in a functional relationship with each other. For example, a promoter is considered "operably linked" to a coding sequence (where the coding sequence is understood to be "under the control of" the promoter ") if the promoter is capable of promoting or otherwise controlling/regulating transcription and/or expression of the coding sequence. Typically, when two nucleotide sequences are operably linked, they will be oriented identically and will typically also be in the same reading frame. They are also typically substantially continuous, although this may not be necessary.

In some embodiments, any regulatory elements of the vectors enable them to provide their intended biological function in the intended host cell or host organism.

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

5.6 compositions

The present disclosure also provides a composition comprising at least one polypeptide of the present disclosure, at least one nucleic acid molecule encoding a polypeptide of the present disclosure, or at least one vector comprising such a 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 one or more other pharmaceutically active polypeptides and/or compounds.

5.7 host organisms

The present disclosure also relates to host cells or host organisms comprising the polypeptides of the present disclosure, nucleic acids encoding the polypeptides of the present disclosure, and/or vectors comprising nucleic acid molecules encoding the polypeptides of the present disclosure.

Suitable host cells or host organisms will be apparent to the skilled person 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, E.coli (Escherichia coli) or Pichia pastoris (Pichia pastoris). In some embodiments, the host is pichia pastoris.

5.8 methods and uses of polypeptides

The present disclosure also provides a method for producing a polypeptide of the present disclosure. The method may comprise transforming/transfecting a host cell or host organism with a nucleic acid encoding the polypeptide, expressing the polypeptide in a host, optionally followed by one or more isolation and/or purification steps. Specifically, the method may include:

a) Expressing a nucleic acid sequence encoding the polypeptide in a suitable expression system (e.g., a suitable host cell or host organism or another expression system); optionally followed by:

b) Isolating and/or purifying the polypeptide.

Suitable hosts or host organisms for production purposes will be apparent to the skilled person and may be, for example, any suitable fungus, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism. Specific examples include HEK293 cells, CHO cells, e.coli or pichia pastoris. In some embodiments, the host is pichia pastoris.

The polypeptides of the present disclosure, nucleic acid molecules or vectors as described, or compositions comprising the polypeptides, nucleic acid molecules or vectors of the present disclosure, as described, or compositions comprising the polypeptides, can be used as medicaments.

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

Also provided are polypeptides of the disclosure, nucleic acid molecules or vectors as described, or compositions comprising polypeptides, nucleic acid molecules or vectors of the disclosure, for use in (prophylactic or therapeutic) treatment of autoimmune and/or inflammatory diseases and/or fibrotic diseases.

Also provided is a (prophylactic and/or therapeutic) method of treating an autoimmune and/or inflammatory and/or fibrotic disease, wherein the method comprises administering to a subject in need thereof a pharmaceutically active amount of a polypeptide of the present disclosure, a nucleic acid molecule or vector as described, or a composition comprising a polypeptide, nucleic acid molecule or vector of the present disclosure.

Also provided is the use of a polypeptide of the disclosure, a nucleic acid molecule or vector as described, or a composition comprising a polypeptide, nucleic acid molecule or vector of the disclosure, in the preparation of a pharmaceutical composition (e.g., a pharmaceutical composition for treating an autoimmune disease or an inflammatory disease or a fibrotic disease).

A "subject" as referred to in the context of the present disclosure may be any animal, such as a mammal. Between mammals, human and non-human mammals can be distinguished. The non-human animal may be, for example, a companion animal (e.g., a dog, cat), a livestock animal (e.g., a cow, horse, sheep, goat, or pig animal), or an animal commonly used for research purposes and/or for the production of antibodies (e.g., mice, rats, rabbits, cats, dogs, goats, sheep, horses, pigs, non-human primates (such as cynomolgus monkeys) or camelids (such as camels or alpacas).

In the context of prophylactic and/or therapeutic purposes, the subject may be any animal, and more particularly any mammal, such as a human subject.

The substance comprising the polypeptide, nucleic acid molecule and carrier, or composition may be administered to a subject by any suitable route of administration, for example by enteral (such as oral or rectal) or parenteral (such as epidermal, sublingual, buccal, nasal, intra-articular, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, transdermal or transmucosal) administration. Parenteral administration, such as intramuscular, subcutaneous or intradermal administration, may be used. In some embodiments, subcutaneous administration is used.

An effective amount of a polypeptide, a nucleic acid molecule or vector as described, or a composition comprising the polypeptide, nucleic acid molecule or vector, may be administered to a subject so as to provide a desired therapeutic result.

One or more doses may be administered. If more than one dose is administered, the doses may be administered at appropriate intervals so as to maximize the effect of the polypeptide, composition, nucleic acid molecule or vector. Table a-1: different monovalent V identified within tetravalent polypeptide F027100187 _HH The amino acid sequence of a building block ("ID" refers to SEQ ID NO as used herein)

Table a-2: the sequences of the CDRs and framework according to AbM numbering ("ID" refers to the given SEQ ID NO

Table a-2.1: the sequences of the CDRs and the frameworks according to Kabat numbering ("ID" refers to the given SEQ ID NO)

Table a-3: the amino acid sequence of the multivalent polypeptide of choice ("ID" refers to the given SEQ ID NO)

Table a-4: the serum albumin binding ISVD sequence ("ID" refers to SEQ ID NO as used herein

Table a-5: the linker sequence ("ID" refers to SEQ ID NO as used herein)

Example 6

6.1 example 1: multispecific ISVD construct generation

Polypeptide F027100187 (SEQ ID NO: 1) containing ISVD generated by data-driven bispecific engineering and formatting activities was identified, comprising anti-OX 40L building blocks (OX 40L01E07, OX40L01B11 and OX40L15B07, described in WO 2011073180 as SEQ ID NO:181, 180 and 179, respectively), anti-IL-13 building blocks (F0107003D 12, F0107009F07, F0107009G09, F0107004B02, F0107004B06 and F0107007C 10) and anti-HSA V _HH Building block ALB23002 (described as SEQ ID NO:10 in WO 2017085172). Different positions/orientations/valences of the building blocks and different linker lengths (9 GS and 20GS and 35 GS) were applied and proved to be critical for different parameters (potency, cross-reactivity, expression, etc.). Efficacy in this example refers to inhibition of in vitro IL-13 induced eosinophil chemokine release assay and inhibition of in vitro OX40L induced T cell co-stimulation as determined in examples 6 and 7, respectively.

Groups containing 123 constructs were transformed in pichia pastoris for small scale production. ISVD construct expression was induced by stepwise methanol addition. Clarified medium with secreted ISVD constructs was used as starting material for purification via protein a affinity chromatography followed by desalting. Purified samples were used for functional characterization and expression evaluation.

Depending on valency, linker length, ISVD building blocks used, and the relative positions of the ISVD building blocks, some constructs exhibit impaired potency and expression levels. In general, pentavalent ISVD constructs showed low expression levels, except for some ISVD with the divalent anti-OX 40L building block OX40L01E07 at the C-terminus (hereinafter "1E 07"). However, 1E07 located at the C-terminus showed insufficient potency for OX 40L. Lowering the valency by using monovalent OX40L arms improved expression levels, but again resulted in insufficient potency for OX 40L. Thus, specific compositions (valences, orientation of building blocks and use of linker lengths) were found to be important for potency and adequate expression levels.

To create an effective monovalent OX40L target arm for incorporation into a tetravalent multispecific ISVD construct, OX40L building blocks OX40L015B07 (hereinafter "15B 07") and OX40L001B11 (hereinafter "1B 11") were subjected to affinity maturation.

For each (V) _HH ) Building blocks, a pooled single site saturated library of all CDR positions was constructed for each CDR. Each single site saturation library was constructed using primers designed according to the 22c-trick method (Kille et al ACS Synth. Biol.,2013,2 (2), pages 83-92). Fixed human and cynomolgus monkey OX40L were subjected to dissociation rate screening based on surface plasmon resonance Spectroscopy (SPR) to identify individual mutations that lead to improved binding.

In a second step, a combinatorial library is constructed comprising the beneficial mutations identified in the first step. Dissociation rate screening was again performed on human and cynomolgus monkey OX40L to identify V with further improved binding _HH Variants. These variants were then purified for biophysical characterization of the affinity assay via SPR and functional characterization in PBMC activity assays (as described in example 7) to select final affinity matured variants. Table 1 lists the characteristics of affinity matured variants of ISVD OX40L015B07 and OX40L001B 11.

Table 1: summary of the characteristics of the final affinity matured variants 15B07AM and 01B11AM compared to the parental forms OX40L015B07 (15B 07) and OX40L001B11 (01B 11), respectively.

By using affinity matured versions of monovalent OX40L building blocks 15B07 and 1B11, an efficient and well-expressed tetravalent multispecific ISVD construct can be obtained.

The presence of affinity matured monovalent 15B07 building block (15B 07 AM) in the tetravalent multispecific ISVD construct provided 20-fold better efficacy in OX40L driven PBMC activity assays (as described in example 7) compared to the non-affinity matured counterpart (15B 07) (table 3).

In addition, the N-terminal position of the 15B07AM building block in the tetravalent multispecific ISVD construct is critical. A comparison of constructs F-027100172 and F-027100179 in Table 4 shows 10-fold better potency with the 15B07AM building block located at the N-terminal position compared to the C-terminal position.

The presence of the N-terminal 15B07AM building block in tetravalent multispecific ISVD of F-027100187 (SEQ ID NO: 1) is beneficial for CMC properties (i.e., expression and solubility) compared to pentavalent constructs that suffer from only low expression yields. As shown in Table 2, the three multispecific ISVD constructs F027100186 (SEQ ID NO: 99) (pentad), F027100187 (SEQ ID NO: 1) (tetravalent) and F027100188 (SEQ ID NO: 100) (pentad) exhibited very different initial CMC (chemical manufacturing and control) spectra. After 5L fermentation, the ISVD construct F027100187 (SEQ ID NO: 1) reached a titer of 4g/L, which is more than 2-fold that of the pentavalent ISVD constructs F027100186 (SEQ ID NO: 99) and F027100188 (SEQ ID NO: 100); and also exhibits superior solubility.

To leave sufficient space between the two IL13 building blocks to obtain optimal potency for IL-13, and to avoid the presence of long 35GS linkers in the F027100187 tetravalent multispecific ISVD construct, both IL13 building blocks are connected via a 9GS-ALB-9GS entity.

Finally, ISVD construct F027100187 was selected based on overall good potency for IL-13 and OX40L, and superior expression levels and CMC characteristics.

Table 2: expression yield and solubility of pentavalent ISVD F027100186 (SEQ ID NO: 99) and F027100188 (SEQ ID NO: 100) and tetravalent ISVD F027100187 (SEQ ID NO: 1). Alb=alb 23002, bb=building block.

Table 3: IC50 value for ISVD construct-mediated neutralization of human OX 40L-induced IL-2 release in PBMC assay

Table 4: IC50 value for ISVD construct-mediated neutralization of human OX 40L-induced IL-2 release in PBMC assay

6.2 example 2: binding affinity of multispecific ISVD constructs to OX40L, IL-13 and serum albumin

With equilibrium dissociation constant (K) _D ) The expressed affinity of F027100187 for human, cynomolgus monkey (cyno) and rhesus IL-13, for human and cynomolgus monkey OX40L and human, cynomolgus monkey and mouse serum albumin was quantified by affinity measurements in solution on Gyrolab xP Workstation (Gyros).

At K _D Under controlled measurement, serial dilutions of OX40L (in the range of 1.3. Mu.M-0.008 pM), IL-13 (in the range of 0.1. Mu.M-0.25 fM) or serum albumin (in the range of 100. Mu.M-3.2 pM) were mixed with fixed amounts of ISVD construct F027100187 (10 pM in the case of OX40L, 5pM in the case of IL-13, 100pM in the case of HSA and cynomolgus SA, and 30nM in the case of mouse SA) to allow interaction and incubation for 24 or 48 hours (in the case of OX40L and IL-13) or 2 hours (in the case of serum albumin) to reach equilibrium.

Serial dilutions of OX40L (ranging from 1.3 μm to 0.031 pM), IL-13 (ranging from 0.1 μm to 0.25 fM) or serum albumin (ranging from 100 μm to 3.2 pM) were mixed with fixed amounts of ISVD construct F027100187 (5 nM in the case of OX40L, 250pM in the case of IL-13, 30nM in the case of HSA and cynomolgus SA) under receptor controlled measurements to allow interactions and incubation for 24 or 48 hours (in the case of OX40L and IL-13) or 2 hours (in the case of serum albumin) to reach equilibrium.

Biotinylated human OX40L/IL-13/serum albumin was captured in the microstructure of the Gyrolab Bioaffy 1000CD, which contained a column of beads, and was used as a molecular probe to capture free F027100187 from the equilibration solution. A mixture of OX40L/IL 13/serum albumin and F027100187 (containing free OX 40L/IL-13/serum albumin, free F027100187, and OX40L/IL 13/serum albumin-F027100187 complex) was flowed through the beads and a small percentage of free F027100187 was captured, which was proportional to the free ISVD construct concentration. The fluorescently labeled anti-ISVD antibody ABH0086-Alexa647 was then injected to label any captured F027100187 and the change in fluorescence was measured after washing away excess fluorescent probe. Fitting of dilution series was done using Gyrolab Analysis software, where K was analyzed _D Curves of control and receptor control to determine K _D Values.

The results (Table 5) demonstrate that the multispecific ISVD construct binds human/cynomolgus monkey OX40L and human/cynomolgus monkey/rhesus monkey IL-13 with high affinity.

Table 5: f027100187 for human, rhesus and cynomolgus IL-13; human and cynomolgus monkey OX40L; and binding affinity of human, cynomolgus monkey and mouse serum albumin (cynomolgus monkey and rhesus monkey OX40L are identical in sequence and cynomolgus monkey and rhesus monkey albumin are identical in sequence)

6.3 example 3: binding of multispecific ISVD constructs to Membrane-bound OX40L

Flow cytometry of CHO-KI cells expressing human or cynomolgus monkey OX40L was used to demonstrate the binding of F027100187 to human and cynomolgus monkey membrane-bound OX 40L. Briefly, cells were fixed with 4% paraformaldehyde and 0.1% glutaraldehyde in PBS at 1X 10 ⁴ The individual cells/wells were seeded at density and incubated with ISVD F027100187 or a dilution series of the reference compound anti-hOX 40L mAb (designated comparator 3) from 100nM to 0.5pM for 48 hours at room temperature. Comparative 3 is a standard conventional monoclonal antibody directed against human OX40L, which is used as a reference in examples 1 to 12 described herein. Cells were washed 3 times, followed by anti-V _HH mAb (ABH 00119) was incubated together at 4 ℃ for 30min, washed again and incubated with goat anti-mouse PE or FITC labeled antibody for 30min at 4 ℃. The samples were washed and resuspended in FACS buffer (10% FBS and 0.05% sodium azide in D-PBS supplemented with 5nM TOPRO3). The cell suspension was then analyzed on an iQuescerener. EC50 values were calculated using GraphPad Prism. Table 6 shows the binding affinities of F0271000187 and anti-hOX 40L reference mAb comparative 3.

Table 6: f027100187 binding affinity to membrane-expressed human and cynomolgus monkey OX40L after 48h incubation compared to reference compound anti-hOX 40L mAb comparative 3.

6.4 example 4: multispecific ISVD constructs that selectively bind to OX40L and IL-13

Non-binding to OX40L and IL-13 related human cytokines was assessed via SPR (Proteon XPR 36). IL-4 was evaluated as an IL-13-associated cytokine. Human TRAIL, CD30L, CD L and RANKL were evaluated as OX 40L-related targets.

For this, cytokines were immobilized on a Proteon GLC sensor chip using amine coupling at 25 μg/mL for 600s, with EDC/NHS injection for 80 seconds for activation and 1M ethanolamine HCl injection for 150 seconds for deactivation (ProteOn Amine Coupling Kit, catalog No. 176-2410). The flow rate during activation and deactivation was set to 30 μl/min and the flow rate during ligand injection was set to 25 μl/min. The pH of the 10mM acetate fixation buffer was 6.0 for all cytokines, except for RANKL, where the pH was 5.0.

Next, 1. Mu. M F027100187 was injected for 2 minutes and dissociated for 600 seconds at a flow rate of 45. Mu.L/min. PBS (pH 7.4) +0.005% Tween 20 was used as running buffer. As positive controls, 100nM of alpha-hIL-4, alpha-hTRAIL, alpha-hCD 30L, alpha-hCD 40L Ab and alpha-hRANKL V were injected _HH (Nb). F027100187 and yangThe interaction between the sexual control and the immobilized target was measured by detecting the increase in refractive index due to the change in mass of the chip after binding.

All positive controls bound to the respective targets. No binding of ISVD construct F027100187 to human IL-4, TRAIL, CD30L, CD L and RANKL was detected.

6.5 example 5: simultaneous binding of multispecific ISVD to IL-13, OX40L and HSA

Using flow cytometry, it was determined whether ISVD construct F0271000187 could bind both recombinant soluble hIL-13 and cell membrane-bound hOX 40L. For this purpose, CHO-KI cells expressing human OX40L were used at 5X 10 ⁴ The density of individual cells/wells was seeded and incubated with 100nM ISVD construct F027100187 for 90 min at 4 ℃. The mixture was then incubated with a dilution series of biotinylated IL-13 starting at 500nM down to 7.6pM at 4℃in the presence of 30. Mu.M HSA for 30min. The cells were washed 3 times, followed by incubation with PE-labelled streptavidin at 4 ℃ for 30min, and washing again. The samples were washed and resuspended in FACS buffer (10% FBS and 0.05% sodium azide in D-PBS supplemented with 5nM TOPRO3). The cell suspension was then analyzed on an iQuescerener. Dose response curve (FIG. 1) demonstrates that ISVD construct F027100187 can bind both membrane bound hOX40L and soluble hIL-13 in the presence of HAS, while negative control V _HH IRR0096 is unable to bind as such.

6.6 example 6: in vitro inhibition of IL-13 induced eosinophil chemokine release by multispecific ISVD

Cell-based assays to study eosinophil chemokine release through a549 human lung cancer cells were used to study the functional activity of soluble IL-13 and inhibition of it by F027100187 from different species of interest (human, rhesus and cynomolgus).

For this, a549 suspension cells were cultured in Ham's F K supplemented with 10% FCS and seeded into 96-well plates at 400.000 cells/well. After 24 hours incubation, F027100187 or dilution series of reference compounds (anti-hIL-13 reference mAb comparator 1 and comparator 2) were added. Both of the comparisons 1 and 2 are standard conventional monoclonal antibodies to human IL-13, which are used as references in examples 1 to 12 described herein. After 20min incubation IL-13 (Sino Biological, catalog No. 10369-HNAC), cynomolgus monkey IL13 (Sino Biological, catalog No. 11057-CNAH) or cynomolgus monkey IL13 (R & D Systems, catalog No. 2674-RM-025) was added to a final concentration of 160pM after a further 24 h incubation in the presence of 30. Mu.M HSA heparin was added to enhance eosinophil chemokine expression. After a further 4 h incubation, eosinophil chemokine-3 secreted in the cell supernatant was quantified by using a human CCL 26/eosinophil chemokine-3 DuoSet ELISA (R & D Systems, DY 346).

F027100187 inhibited human, cynomolgus and rhesus IL-13 induced eosinophil chemokine-3 release in a concentration-dependent manner with IC 50's of 259pM (for human IL-13), 1940pM (for cynomolgus IL-13) and 858pM (for rhesus IL-13) (Table 7, FIG. 2).

Table 7: IC of F027100187 in comparison to reference compound anti-hIL-13 reference mAb comparator 1 and comparator 2 mediated neutralization of human, cynomolgus and rhesus IL-13 in eosinophil chemokine release assay ₅₀ Values.

6.7 example 7: in vitro inhibition of OX 40L-induced T cell co-stimulation by multispecific ISVD constructs

Cell-based assays (PBMC activity assays) to study OX 40L-induced T cell co-stimulation were used to study the functional activity of human and cynomolgus monkey OX40L and inhibition thereof by ISVD construct F027100187. The determination is performed by the following method: buffy coat derived PBMC (density 1X 10) ⁵ Individual cells/well) and OX 40L-overexpressing CHO-KI cells (density 1X 10) ⁴ Individual cells/well) were co-cultured in a clear 96-well plate. Dilution series of ISVD construct F027100187 or reference compound anti-hOX 40L mAb (referred to as comparator 3) Added to the co-culture and incubated at 37℃for 22 hours in the presence of 30. Mu.M HSA in a humidified incubator. The readings were made by evaluating the IL-2 levels in the cell supernatants using ELISA.

ISVD construct F027100187 inhibited human and cynomolgus monkey OX 40L-induced T cell activation in a concentration-dependent manner with IC50 of 1.9nM (for human OX 40L) and 14nM (for cynomolgus monkey OX 40L), which was comparable to reference compound anti-hOX 40L mAb comparator 3 (table 8, fig. 3). Table 8: IC50 values for ISVD construct F0271000187 compared to reference compound anti-hOX 40L mAb comparator 3 mediated neutralization of human and cynomolgus monkey OX40L in PBMC activity assay.

6.8 example 8: binding of multispecific ISVD constructs to pre-existing antibodies

The binding of pre-existing antibodies present in 96 serum samples from healthy volunteers to ISVD construct F027100187 was determined using ProteOn XPR36 (Bio-Rad Laboratories, inc.). PBS/Tween (phosphate buffered saline, pH 7.4,0.005% Tween 20) was used as running buffer and experiments were performed at 25 ℃.

The ISVD construct was captured on-chip via binding of ALB23002 building blocks to HSA immobilized on-chip. To immobilize HSA, ligand lanes of Proteon GLC sensor chip were activated with EDC/NHS (flow rate 30. Mu.l/min), and HSA was injected at 100. Mu.l/ml into Proteon acetate buffer pH 4.5 to achieve a immobilization level of about 2600 RU. After fixation, the surface was deactivated with ethanolamine HCI (flow rate 30. Mu.l/min).

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

Tetravalent ISVD construct F027100187, optimized for the reduction of pre-existing antibody binding by introducing mutations L11V and V89L and C-terminal alanine in each building block, showed a significant reduction in binding to pre-existing antibody compared to control, non-optimized pentavalent ISVD F027301186 (fig. 4).

6.9 example 9: inhibition of OX40L and IL-13 by multispecific ISVD construct F027100187 reduced IL-5 and CCL26 levels in the triple culture system:

to test for the physiological effect of OX40L blocking on T cell activation, PBMCs of healthy blood donors that were responsive to house dust mites were co-cultured with MRC5 (fibroblasts) and a549 (epithelial) cells. Mixing these cells provides additional activation, driving a type 2 immune response by inducing the production of IL-5 and IL-13. IL-13 triggers the production of CCL26 by local epithelial cells, resulting in inflammatory diseases mediated by type 2 immune responses, and the like. In addition, the recurrence of these cell types (recapitulates) is found in the tissues of interest (skin and lung). T cell responses were monitored 7 days after mixing the cells by measuring cytokines in the supernatant.

The method comprises the following steps:

7.5X10 in 500. Mu.l AIM V CTS medium with serum replacement CTS (assay medium) ⁴ MRC5 and 7.5X10 ⁴ Individual a549 cells were added to each well of the 24-well plate and incubated overnight. Then, 100 μl of ISVD construct F02710018, anti-OX 40L reference mAb comparative 3 or anti-hIL-13 reference mAb comparative 1 was added to the assay medium for 15min. Then, 1.2X10 ⁶ The thawed and resting allergic PBMC were added to 200. Mu.l assay medium200. Mu.l of low endotoxin house dust mites from the depleted cultures were then added. Cultures were incubated for 7 days and the culture supernatants were assayed in duplicate by Luminex assay for IL-5 and CCL 26. A summary of 4 donors is shown.

Results:

the overall results of the inhibition reactions of F027100187 and reference antibody, anti-hOX 40L mAb comparator 3 and reference anti-hIL-13 mAb comparator 1 are shown in tables 9 and 10 and fig. 5 and 6.

Taken together, these results demonstrate that ISVD F027100187 is equivalent to the anti-hOX 40L reference mAb (i.e., comparator 3) and nearly equivalent to the anti-hIL-13 reference mAb (i.e., comparator 1) in blocking the ability of both cytokines/chemokines (IL-5 and CCL 26) in a complex assay system comprising human PBMCs co-cultured with histocyte cells, highlighting its therapeutic potential for the treatment of type 2 inflammatory diseases (e.g., asthma and atopic dermatitis) as well as a broad range of immune disease indications.

Table 9: IL-5 average IC50 results.

Compounds of formula (I)	IL-5IC50(nM)
		F027100187	1
Comparative example 3	2.3
		Comparative 1	NA

Table 10: CCL26 averaged IC50 results.

Compounds of formula (I)	CCL26 IC50(pM)
		F027100187	40.7
Comparative 1	7.56
		Comparative example 3	NA

6.10 example 10: NSG humanized mouse model for evaluating F027100187 in vivo mediated target occupancy and pharmacodynamics

F027100187 targets both human OX40L and IL-13 and does not cross-react with murine orthologs. Thus, to evaluate the bioactivity of F027100187, a humanized model system of xenograft was used. Female NSG (NOD.Cg-Prkdcscid Il2rgtm1 Wjl/SzJ) was obtained from Jackson laboratories in Barbur, burmese, U.S.A.. These mice express human hematopoietic cytokines: stem Cell Factor (SCF), granulocyte/macrophage stimulating factor (GM-CSF), and interleukin-3 (IL-3), all of which are driven by human cytomegalovirus promoter/enhancer sequences. Triple transgenic mice constitutively produce the above cytokines, providing cell proliferation and survival signals, supporting stable transplantation of cd33+ myeloid lineages and several types of lymphocytes.

Briefly, the protocol followed for transplantation is as follows:

on study day 0, mice were transplanted with 200 μl Dulbecco phosphate buffered saline by Intravenous (IV) route5x 10 in saline (DPBS) ⁶ House dust mite sensitive Peripheral Blood Mononuclear Cells (PBMCs). Mice were challenged intranasally with 25 μg in 40 μl House Dust Mite (HDM) extract (Greerlab, catalog number XPB 70-X29) on days 1, 2, 3, 6, 7, 8, 9 and 10 of the study. Mice challenged with HDM received subcutaneous doses of vehicle or F27100187 (11.1, 3.72, 1.11 or 0.37 mg/kg) on days 1, 3, 6, 8, 10 and 13. On day 20, mice were anesthetized by isoflurane anesthesia. Blood was collected under isoflurane anesthesia via retroorbital bleeding. After blood collection and still under isoflurane anesthesia, mice were sacrificed by cervical dislocation. A portion of the lung was harvested and placed in culture for human cell phenotyping (flow cytometry). Plasma levels of human cytokines and chemokines from day 20 plasma samples were determined by Lumenix assessment (catalog number HSTCMAG28SPMX13, milliplex). Plasma levels of human IgE from day 20 plasma samples were determined by ELISA (catalog No. BMS2097, invitrogen).

The results of these experiments as shown in figures 7 and 8 demonstrate that F027100187 is able to significantly inhibit human T and B cell expansion in NSG mice. Figures 9 and 10 demonstrate that F027100187 is capable of significantly inhibiting the production of key types of cytokines (IL-2, IL-4, IL-5 and IL-10) and IgE. Overall, these results demonstrate the in vivo efficacy of F027100187.

Several key markers of type 2 allergic disease are increased in NSG-PBMC mouse models. The overall results of these experiments, as shown in figures 7-10, demonstrate that F27100187 is able to significantly inhibit key markers of type 2 allergic diseases, thus demonstrating the in vivo pharmacodynamic effects of F27100187 on human type 2 markers.

6.11 example 11: NSG-SGM3 humanized mouse model for evaluating F027100187 in vivo mediated target occupancy and pharmacodynamics

F027100187 targets both human OX40L and IL-13 and does not cross-react with murine orthologs. Thus, to evaluate the bioactivity of F027100187, a humanized model system of xenograft was used. Female NSG-SGM3 (NOD/SCID-IL 2 Rgamma-/-, NOD.Cg-PrkdcsccidIl 2 rgamma tm1 Wjl/SzJ) transplanted with human CD34+ cells was obtained from Jackson laboratories in Barbore, burma, U.S.A.. These mice express human hematopoietic cytokines: stem Cell Factor (SCF), granulocyte/macrophage stimulating factor (GM-CSF), and interleukin-3 (IL-3), all of which are driven by human cytomegalovirus promoter/enhancer sequences. Triple transgenic mice constitutively produce the above cytokines, providing cell proliferation and survival signals, supporting stable transplantation of cd33+ myeloid lineages and several types of lymphocytes. Animals were 80 to 100 days post-implantation. Briefly, the protocol followed for transplantation is as follows:

Jackson laboratories provide data from transplantation examinations by flow cytometry. Information from the transplantation examination was used to group mice. Mice received subcutaneous doses of vehicle or ISVD construct F27100187 on days 0 and 2. Mice were challenged intranasally with 7.5. Mu.g of 20. Mu.l human IL-33 (catalog No. 200-33-500UG, pepro Tech) on days 1, 2 and 3 of the study. On day 4, blood was collected under isoflurane anesthesia via retroorbital bleeding. After blood collection and still under isoflurane anesthesia, mice were sacrificed by cervical dislocation. A portion of the lung was harvested and placed in culture for human cell phenotyping (flow cytometry). Plasma levels of human cytokines and chemokines were determined by Lumenix assessment (catalog number HSTCMAG28SPMX13, milliplex). Plasma levels of human Il-13 from day 20 plasma samples were determined by ELISA (catalog number 88-7439-88, invitrogen).

The overall results of these experiments, as shown in fig. 11, demonstrate that F027100187 is able to significantly inhibit detectable levels of human IL-13 in plasma of humanized NSG-SGM3 mice, indicating target occupancy for human IL-13. In addition, F027100187 is capable of significantly inhibiting the key type 2 cytokines IL-5, TARC and mouse eosinophil chemokines. Thus, these results indicate that F027100187 is suitable for treating atopic dermatitis and/or asthma.

Example 12 allergic asthma model in young adult rhesus monkeys

30 male rhesus monkeys aged 2-4 years from the national primate research center (California National Primate Research Center, CNPRC) of california were selected based on the following: behavioral inhibition test, pulmonary function test of acetylmethylcholine reactivity (PFT), effective concentration 150% (EC 150) (< 3mg/ml of methacholine) and effective concentration 200% (EC 200) (< 8mg/ml of methacholine) values. All animals participating in this study were subjected to physical examination, complete Blood Count (CBC) and serum chemistry test suite.

All animals selected for this study underwent House Dust Mite (HDM) sensitization. Animals received a single subcutaneous injection of about 60ug of HDM extract (house dust mite (d. Pteronyssinus)) in 1mg alum once every two weeks (total volume per injection, greener B58a52, thermo 77161) for 28 weeks.

From week 12 of the study, nebulized HDM (8.5 ug house dust mites 1/ml, prepared from lyophilized house dust mites, greens) was administered via the nebulizer once every two weeks. Study animals were sedated with ketamine and dexmedetomidine and then placed in a semi-upright position on a child safety seat. The sedated animal is then fitted with a mask that covers both the nose and mouth. An oral plug is placed to ensure maximum aerosol passage into the trachea and lungs. A dose of atropine is administered to minimize saliva production normally caused by ketamine sedation, as excessive saliva can lead to premature termination of the procedure due to airway obstruction. Mask fit and head position are carefully adjusted without blocking the airway to prevent aerosol leakage. The HDM aerosol was applied through the mask for about 5-15 minutes. Heart rate and oxygen saturation were monitored continuously throughout the procedure. Following the procedure, sedation was reversed with an equal dose of atemezole.

Blood samples were collected at week 0 and then once a week starting at week 18. At weeks 20 and 29, serum blood samples were collected for pharmacokinetic analysis after test article administration. HDM intradermal injections and skin biopsies were performed on the shaved backs of each study animal. 100ul saline or HDM (1:1000W/V in 100ul saline) was injected intradermally at each site, 8 sites at each time point. HDM skin reactivity and skin biopsies were performed at weeks 19, 25 and 29. Biopsies were taken from the inter-scapular region using a 4mm drill according to the discretion of the veterinarian, and the site was closed with glue or sutures. Animals received ketoprofen (2-5 mg/kg, IM, 1-2 days after SID x biopsy) after each biopsy.

Bronchoalveolar lavage (BAL): the animals were placed in a supine position under sedation. The larynx was visualized using a laryngoscope and anesthetized with lidocaine. Bronchoscopes are placed in the sub Duan Zhi trachea. Manual instillation and aspiration of 2mg/kg Phosphate Buffered Saline (PBS). It was repeated twice.

Group allocation: groups of 6 animals were included in a rolling fashion in one of three groups according to PFT criteria (defined in the previous section). Eosinophil frequency/number obtained from BAL procedure during week 18 was used to assign animals to treatment or control (vehicle) groups.

Test article application: animals receive vehicle or test article by subcutaneous administration from week 20. The test article consisted of F027100187 administered once a week. The vehicle was also administered once a week.

Necropsy: animals will undergo necropsy immediately after the last PFT and BAL procedure at the end of week 30 or week 31. Euthanized by excess sodium pentobarbital. Blood will be collected and prepared for serum, plasma and PBMCs. The lungs were removed entirely and sections from each lobe were prepared for RNA, flow cytometry, and histology.

Plasma levels of IL-5 were determined by Simoa (catalog number 102860, quantix). Serum levels of IgE were determined by ELISA (catalog No. KA2450, abnova).

The results of these experiments, as shown in fig. 13, demonstrate that F027100187 is able to significantly inhibit inflammation of the lung (eosinophil density (histology), bal IL-5 and eosinophil percentages). Fig. 14 shows that F027100187 is able to significantly inhibit inflammation (histology) of the skin. Figure 15 shows that F027100187 is capable of significantly inhibiting the production of systemic IgE. Overall, these results demonstrate the in vivo efficacy of F027100187. These results further support that F27100187 is suitable for treating asthma.

7 industrial applicability

The polypeptides, nucleic acid molecules encoding the same, vectors comprising the nucleic acids and compositions described herein may be used, for example, to treat subjects suffering from inflammatory diseases.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the application is not entitled to antedate such publication by virtue of prior application.

While the application has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

Sequence listing

<110> Ablynx NV Ablynx Co., ltd

Sainofil (Sanofi)

<120> Polypeptides comprising immunoglobulin single variable domains targeting IL-13 and OX40L

<130> PAT20187-US-PSP

<160> 149

<170> patent In version 3.5

<210> 1

<211> 509

<212> PRT

<213> artificial sequence

<220>

<223> Nanobody sequence

<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 Arg Ser Ile Gly Arg Tyr Asp

20 25 30

Arg Met Gly Trp Tyr Arg His Arg Pro Gly Glu Pro Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn Tyr Gly Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Asn

85 90 95

Phe Asn Lys Tyr Gln Ile Ser Arg Asp Thr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val

115 120 125

Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu

130 135 140

Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr Arg Met

145 150 155 160

Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala

165 170 175

Leu Ser Gly Asp Gly Tyr Ser Thr Tyr Thr Ala Asn Ser Val Lys Gly

180 185 190

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln

195 200 205

Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Ala

210 215 220

Lys Leu Gln Tyr Val Ser Gly Trp Ser Tyr Asp Tyr Pro Tyr Trp Gly

225 230 235 240

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

245 250 255

Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro

260 265 270

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg

275 280 285

Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu

290 295 300

Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp

305 310 315 320

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

325 330 335

Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr

340 345 350

Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu

355 360 365

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val

370 375 380

Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu

385 390 395 400

Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Tyr Ala Met

405 410 415

Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser

420 425 430

Ile Thr Thr Gly Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val Lys Gly

435 440 445

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln

450 455 460

Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Asn

465 470 475 480

Val Pro Phe Gly Tyr Tyr Ser Glu His Phe Ser Gly Leu Ser Phe Asp

485 490 495

Tyr Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

500 505

<210> 2

<211> 117

<212> PRT

<213> artificial sequence

<220>

<223> Nanobody sequence

<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 Arg Ser Ile Gly Arg Tyr Asp

20 25 30

Arg Met Gly Trp Tyr Arg His Arg Pro Gly Glu Pro Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn Tyr Gly Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Asn

85 90 95

Phe Asn Lys Tyr Gln Ile Ser Arg Asp Thr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 3

<211> 123

<212> PRT

<213> artificial sequence

<220>

<223> Nanobody sequence

<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 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> 4

<211> 115

<212> PRT

<213> artificial sequence

<220>

<223> Nanobody sequence

<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 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> 5

<211> 127

<212> PRT

<213> artificial sequence

<220>

<223> Nanobody sequence

<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 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 Ala

115 120 125

<210> 6

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> CDR1

<400> 6

Arg Ser Ile Gly Arg Tyr Asp Arg Met Gly

1 5 10

<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 Arg Ser Phe Gly Met Ser

1 5 10

<210> 9

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> CDR1

<400> 9

Gly Phe Thr Phe Asn Asn Tyr Ala Met Lys

1 5 10

<210> 10

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> CDR2

<400> 10

Thr Ile Thr Gly Gly Ser Ser Ile Asn

1 5

<210> 11

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> CDR2

<400> 11

Ala Leu Ser Gly Asp Gly Tyr Ser Thr Tyr

1 5 10

<210> 12

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> CDR2

<400> 12

Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu

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> CDR3

<400> 14

Asn Lys Tyr Gln Ile Ser Arg Asp Thr

1 5

<210> 15

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> CDR3

<400> 15

Lys Leu Gln Tyr Val Ser Gly Trp Ser Tyr Asp Tyr Pro Tyr

1 5 10

<210> 16

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> CDR3

<400> 16

Gly Gly Ser Leu Ser Arg

1 5

<210> 17

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> CDR3

<400> 17

Val Pro Phe Gly Tyr Tyr Ser Glu His Phe Ser Gly Leu Ser Phe Asp

1 5 10 15

Tyr

<210> 18

<211> 25

<212> PRT

<213> artificial sequence

<220>

<223> FR1

<400> 18

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> 19

<211> 25

<212> PRT

<213> artificial sequence

<220>

<223> FR1

<400> 19

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> 20

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> FR2

<400> 20

Trp Tyr Arg His Arg Pro Gly Glu Pro Arg Glu Leu Val Ala

1 5 10

<210> 21

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> FR2

<400> 21

Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala

1 5 10

<210> 22

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> FR2

<400> 22

Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val Ser

1 5 10

<210> 23

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> FR2

<400> 23

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser

1 5 10

<210> 24

<211> 39

<212> PRT

<213> artificial sequence

<220>

<223> FR3

<400> 24

Tyr Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ile 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 Asn Phe

35

<210> 25

<211> 39

<212> PRT

<213> artificial sequence

<220>

<223> FR3

<400> 25

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> 26

<211> 39

<212> PRT

<213> artificial sequence

<220>

<223> FR3

<400> 26

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> 27

<211> 39

<212> PRT

<213> artificial sequence

<220>

<223> FR3

<400> 27

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> 28

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> FR4

<400> 28

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 29

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> FR4

<400> 29

Ser Ser Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 30

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> FR4

<400> 30

Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

1 5 10

<210> 31

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> CDR1

<400> 31

Arg Ser Ile Gly Arg Tyr Asp Arg Met Gly

1 5 10

<210> 32

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> CDR1

<400> 32

Ser Tyr Arg Met Gly

1 5

<210> 33

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> CDR1

<400> 33

Ser Phe Gly Met Ser

1 5

<210> 34

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> CDR1

<400> 34

Asn Tyr Ala Met Lys

1 5

<210> 35

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> CDR2

<400> 35

Thr Ile Thr Gly Gly Ser Ser Ile Asn Tyr Gly Asp Ser Val Lys Gly

1 5 10 15

<210> 36

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> CDR2

<400> 36

Ala Leu Ser Gly Asp Gly Tyr Ser Thr Tyr Thr Ala Asn Ser Val Lys

1 5 10 15

Gly

<210> 37

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> CDR2

<400> 37

Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 38

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> CDR2

<400> 38

Ser Ile Thr Thr Gly Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 39

<211> 25

<212> PRT

<213> artificial sequence

<220>

<223> FR1

<400> 39

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> 40

<211> 30

<212> PRT

<213> artificial sequence

<220>

<223> FR1

<400> 40

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 Arg Thr Phe Ser

20 25 30

<210> 41

<211> 30

<212> PRT

<213> artificial sequence

<220>

<223> FR1

<400> 41

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> 42

<211> 30

<212> PRT

<213> artificial sequence

<220>

<223> FR1

<400> 42

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> 43

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> FR2

<400> 43

Trp Tyr Arg His Arg Pro Gly Glu Pro Arg Glu Leu Val Ala

1 5 10

<210> 44

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> FR2

<400> 44

Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala

1 5 10

<210> 45

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> FR2

<400> 45

Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val Ser

1 5 10

<210> 46

<211> 32

<212> PRT

<213> artificial sequence

<220>

<223> FR3

<400> 46

Arg Phe Thr Ile Ser Ile 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 Asn Phe

20 25 30

<210> 47

<211> 32

<212> PRT

<213> artificial sequence

<220>

<223> FR3

<400> 47

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> 48

<211> 32

<212> PRT

<213> artificial sequence

<220>

<223> FR3

<400> 48

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> 49

<211> 32

<212> PRT

<213> artificial sequence

<220>

<223> FR3

<400> 49

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> 50

<211> 115

<212> PRT

<213> artificial sequence

<220>

<223> Nanobody sequence

<400> 50

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> 51

<211> 115

<212> PRT

<213> artificial sequence

<220>

<223> Nanobody sequence

<400> 51

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> 52

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> Nanobody sequence

<400> 52

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> 53

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> Nanobody sequence

<400> 53

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> 54

<211> 115

<212> PRT

<213> artificial sequence

<220>

<223> Nanobody sequence

<400> 54

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> 55

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> Nanobody sequence

<400> 55

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> 56

<211> 115

<212> PRT

<213> artificial sequence

<220>

<223> Nanobody sequence

<400> 56

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> 57

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> Nanobody sequence

<400> 57

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> 58

<211> 117

<212> PRT

<213> artificial sequence

<220>

<223> Nanobody sequence

<400> 58

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> 59

<211> 118

<212> PRT

<213> artificial sequence

<220>

<223> Nanobody sequence

<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 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> 60

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> Nanobody sequence

<400> 60

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> 61

<211> 117

<212> PRT

<213> artificial sequence

<220>

<223> Nanobody sequence

<400> 61

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> 62

<211> 118

<212> PRT

<213> artificial sequence

<220>

<223> Nanobody sequence

<400> 62

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> 63

<211> 116

<212> PRT

<213> artificial sequence

<220>

<223> Nanobody sequence

<400> 63

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> 64

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> linker sequence

<400> 64

Ala Ala Ala

1

<210> 65

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> linker sequence

<400> 65

Gly Gly Gly Gly Ser

1 5

<210> 66

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> linker sequence

<400> 66

Ser Gly Gly Ser Gly Gly Ser

1 5

<210> 67

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> linker sequence

<400> 67

Gly Gly Gly Gly Ser Gly Gly Ser

1 5

<210> 68

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> linker sequence

<400> 68

Gly Gly Gly Gly Ser Gly Gly Gly Ser

1 5

<210> 69

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> linker sequence

<400> 69

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 70

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> linker sequence

<400> 70

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 71

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> linker sequence

<400> 71

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Ser

<210> 72

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> linker sequence

<400> 72

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> 73

<211> 25

<212> PRT

<213> artificial sequence

<220>

<223> linker sequence

<400> 73

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> 74

<211> 30

<212> PRT

<213> artificial sequence

<220>

<223> linker sequence

<400> 74

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> 75

<211> 35

<212> PRT

<213> artificial sequence

<220>

<223> linker sequence

<400> 75

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> 76

<211> 40

<212> PRT

<213> artificial sequence

<220>

<223> linker sequence

<400> 76

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> 77

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> linker sequence

<400> 77

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

<210> 78

<211> 24

<212> PRT

<213> artificial sequence

<220>

<223> linker sequence

<400> 78

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> 79

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> linker sequence

<400> 79

Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala

1 5 10

<210> 80

<211> 62

<212> PRT

<213> artificial sequence

<220>

<223> linker sequence

<400> 80

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> 81

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> c terminal

<400> 81

Val Thr Val Ser Ser

1 5

<210> 82

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> c terminal

<400> 82

Val Lys Val Ser Ser

1 5

<210> 83

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> c terminal

<400> 83

Val Gln Val Ser Ser

1 5

<210> 84

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> c terminal

<400> 84

Val Thr Val Lys Ser

1 5

<210> 85

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> c terminal

<400> 85

Val Thr Val Gln Ser

1 5

<210> 86

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> c terminal

<400> 86

Val Lys Val Lys Ser

1 5

<210> 87

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> c terminal

<400> 87

Val Lys Val Gln Ser

1 5

<210> 88

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> c terminal

<400> 88

Val Gln Val Lys Ser

1 5

<210> 89

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> c terminal

<400> 89

Val Gln Val Gln Ser

1 5

<210> 90

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> c terminal

<400> 90

Val Thr Val Ser Ser Ala

1 5

<210> 91

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> c terminal

<400> 91

Val Lys Val Ser Ser Ala

1 5

<210> 92

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> c terminal

<400> 92

Val Gln Val Ser Ser Ala

1 5

<210> 93

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> c terminal

<400> 93

Val Thr Val Lys Ser Ala

1 5

<210> 94

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> c terminal

<400> 94

Val Thr Val Gln Ser Ala

1 5

<210> 95

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> c terminal

<400> 95

Val Lys Val Lys Ser Ala

1 5

<210> 96

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> c terminal

<400> 96

Val Lys Val Gln Ser Ala

1 5

<210> 97

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> c terminal

<400> 97

Val Gln Val Lys Ser Ala

1 5

<210> 98

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> c terminal

<400> 98

Val Gln Val Gln Ser Ala

1 5

<210> 99

<211> 635

<212> PRT

<213> artificial sequence

<220>

<223> Nanobody sequence

<400> 99

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 Arg Ser Ile Gly Arg Tyr Asp

20 25 30

Arg Met Gly Trp Tyr Arg His Arg Pro Gly Glu Pro Arg Glu Leu Val

35 40 45

Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn Tyr Gly Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Asn

85 90 95

Phe Asn Lys Tyr Gln Ile Ser Arg Asp Thr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val

115 120 125

Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu

130 135 140

Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr Arg Met

145 150 155 160

Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala

165 170 175

Leu Ser Gly Asp Gly Tyr Ser Thr Tyr Thr Ala Asn Ser Val Lys Gly

180 185 190

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln

195 200 205

Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Ala

210 215 220

Lys Leu Gln Tyr Val Ser Gly Trp Ser Tyr Asp Tyr Pro Tyr Trp Gly

225 230 235 240

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

245 250 255

Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro

260 265 270

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ile Gly Arg

275 280 285

Tyr Asp Arg Met Gly Trp Tyr Arg His Arg Pro Gly Glu Pro Arg Glu

290 295 300

Leu Val Ala Thr Ile Thr Gly Gly Ser Ser Ile Asn Tyr Gly Asp Ser

305 310 315 320

Val Lys Gly Arg Phe Thr Ile Ser Ile Asp Asn Ser Lys Asn Thr Val

325 330 335

Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr

340 345 350

Cys Asn Phe Asn Lys Tyr Gln Ile Ser Arg Asp Thr Trp Gly Gln Gly

355 360 365

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser

370 375 380

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

385 390 395 400

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Tyr

405 410 415

Ala Met Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

420 425 430

Ser Ser Ile Thr Thr Gly Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val

435 440 445

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

450 455 460

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

465 470 475 480

Ala Asn Val Pro Phe Gly Tyr Tyr Ser Glu His Phe Ser Gly Leu Ser

485 490 495

Phe Asp Tyr Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

500 505 510

Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly

515 520 525

Gly Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

530 535 540

Gly Phe Thr Phe Arg Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro

545 550 555 560

Gly Lys Gly Pro Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp

565 570 575

Thr Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

580 585 590

Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu

595 600 605

Asp Thr Ala Leu Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser

610 615 620

Ser Gln Gly Thr Leu Val Thr Val Ser Ser Ala

625 630 635

<210> 100

<211> 649

<212> PRT

<213> artificial sequence

<220>

<223> Nanobody sequence

<400> 100

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 Ile

20 25 30

Tyr Ala Lys Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe

35 40 45

Val Ala Ala Ile Ser Arg Ser Gly Arg Ser Thr Ser Tyr Ala Asp Ser

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val

65 70 75 80

Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr

85 90 95

Cys Ala Ala Val Gly Gly Ala Thr Thr Val Thr Ala Ser Glu Trp Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val

130 135 140

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg

145 150 155 160

Thr Phe Ser Ser Ile Tyr Ala Lys Gly Trp Phe Arg Gln Ala Pro Gly

165 170 175

Lys Glu Arg Glu Phe Val Ala Ala Ile Ser Arg Ser Gly Arg Ser Thr

180 185 190

Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

195 200 205

Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

210 215 220

Thr Ala Leu Tyr Tyr Cys Ala Ala Val Gly Gly Ala Thr Thr Val Thr

225 230 235 240

Ala Ser Glu Trp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

245 250 255

Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu

260 265 270

Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys

275 280 285

Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr Arg Met Gly Trp Phe Arg

290 295 300

Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Leu Ser Gly Asp

305 310 315 320

Gly Tyr Ser Thr Tyr Thr Ala Asn Ser Val Lys Gly Arg Phe Thr Ile

325 330 335

Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu

340 345 350

Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Ala Lys Leu Gln Tyr

355 360 365

Val Ser Gly Trp Ser Tyr Asp Tyr Pro Tyr Trp Gly Gln Gly Thr Leu

370 375 380

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val

385 390 395 400

Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu

405 410 415

Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe Gly Met

420 425 430

Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val Ser Ser

435 440 445

Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly

450 455 460

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln

465 470 475 480

Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Thr Ile

485 490 495

Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr Val Ser

500 505 510

Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu

515 520 525

Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys

530 535 540

Ala Ala Ser Gly Phe Thr Phe Asn Asn Tyr Ala Met Lys Trp Val Arg

545 550 555 560

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Thr Thr Gly

565 570 575

Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile

580 585 590

Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu

595 600 605

Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Asn Val Pro Phe Gly

610 615 620

Tyr Tyr Ser Glu His Phe Ser Gly Leu Ser Phe Asp Tyr Arg Gly Gln

625 630 635 640

Gly Thr Leu Val Thr Val Ser Ser Ala

645

<210> 101

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 101

Lys Glu Arg Glu

1

<210> 102

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 102

Lys Gln Arg Glu

1

<210> 103

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 103

Gly Leu Glu Trp

1

<210> 104

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 104

Lys Glu Arg Glu Leu

1 5

<210> 105

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 105

Lys Glu Arg Glu Phe

1 5

<210> 106

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 106

Lys Gln Arg Glu Leu

1 5

<210> 107

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 107

Lys Gln Arg Glu Phe

1 5

<210> 108

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 108

Lys Glu Arg Glu Gly

1 5

<210> 109

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 109

Lys Gln Arg Glu Trp

1 5

<210> 110

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 110

Lys Gln Arg Glu Gly

1 5

<210> 111

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 111

Thr Glu Arg Glu

1

<210> 112

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 112

Thr Glu Arg Glu Leu

1 5

<210> 113

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 113

Thr Gln Arg Glu

1

<210> 114

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 114

Thr Gln Arg Glu Leu

1 5

<210> 115

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 115

Lys Glu Cys Glu

1

<210> 116

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 116

Lys Glu Cys Glu Leu

1 5

<210> 117

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 117

Lys Glu Cys Glu Arg

1 5

<210> 118

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 118

Lys Gln Cys Glu

1

<210> 119

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 119

Lys Gln Cys Glu Leu

1 5

<210> 120

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 120

Arg Glu Arg Glu

1

<210> 121

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 121

Arg Glu Arg Glu Gly

1 5

<210> 122

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 122

Arg Gln Arg Glu

1

<210> 123

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 123

Arg Gln Arg Glu Leu

1 5

<210> 124

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 124

Arg Gln Arg Glu Phe

1 5

<210> 125

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 125

Arg Gln Arg Glu Trp

1 5

<210> 126

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 126

Gln Glu Arg Glu

1

<210> 127

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 127

Gln Glu Arg Glu Gly

1 5

<210> 128

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 128

Gln Gln Arg Glu

1

<210> 129

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 129

Gln Gln Arg Glu Trp

1 5

<210> 130

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 130

Gln Gln Arg Glu Leu

1 5

<210> 131

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 131

Gln Gln Arg Glu Phe

1 5

<210> 132

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 132

Lys Gly Arg Glu

1

<210> 133

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 133

Lys Gly Arg Glu Gly

1 5

<210> 134

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 134

Lys Asp Arg Glu

1

<210> 135

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 135

Lys Asp Arg Glu Val

1 5

<210> 136

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 136

Asp Glu Cys Lys Leu

1 5

<210> 137

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 137

Asn Val Cys Glu Leu

1 5

<210> 138

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 138

Gly Val Glu Trp

1

<210> 139

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 139

Glu Pro Glu Trp

1

<210> 140

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 140

Gly Leu Glu Arg

1

<210> 141

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 141

Asp Gln Glu Trp

1

<210> 142

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 142

Asp Leu Glu Trp

1

<210> 143

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 143

Gly Ile Glu Trp

1

<210> 144

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 144

Glu Leu Glu Trp

1

<210> 145

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 145

Gly Pro Glu Trp

1

<210> 146

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 146

Glu Trp Leu Pro

1

<210> 147

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 147

Gly Pro Glu Arg

1

<210> 148

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 148

Gly Leu Glu Arg

1

<210> 149

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> motif

<400> 149

Glu Leu Glu Trp

1

Claims

1. A polypeptide, a composition comprising the polypeptide, or a composition comprising a nucleic acid comprising a nucleotide sequence encoding the polypeptide, wherein the polypeptide comprises or consists of at least three Immunoglobulin Single Variable Domains (ISVD), wherein each of the ISVD comprises three complementarity determining regions (CDR 1 to CDR3, respectively) optionally linked via one or more peptide linkers; and wherein:

a) The first ISVD binds to OX40L and comprises

b) The second ISVD binds IL-13 and comprises

c) Third ISVD binds IL-13 and comprises

ix. CDR3 having the amino acid sequence of SEQ ID NO:17 or having a 2 or 1 amino acid difference from SEQ ID NO: 17.

2. The composition of claim 1, which is a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more other pharmaceutically active polypeptides and/or compounds.

3. The polypeptide or composition of claim 1 or 2, wherein:

4. A polypeptide or composition according to any one of claims 1 to 3, wherein:

5. The polypeptide or composition of any one of claims 1 to 4, wherein:

a) The first ISVD has an amino acid sequence of SEQ ID NO. 2;

c) The third ISVD has the amino acid sequence of SEQ ID NO. 5.

6. The polypeptide or composition of any one of claims 1 to 5, wherein the polypeptide further comprises one or more additional groups, residues, moieties or binding units optionally linked via one or more peptide linkers, wherein the one or more additional groups, residues, moieties or binding units provide the polypeptide with an increased half-life compared to a corresponding polypeptide without the one or more additional groups, residues, moieties or binding units.

7. The polypeptide or composition of claim 6 wherein the one or more other groups, residues, moieties or binding units that provide the polypeptide with increased half-life are selected from the group consisting of polyethylene glycol molecules, serum proteins or fragments thereof, binding units that bind to serum proteins, fc moieties, and small proteins or peptides that bind to serum proteins.

8. The polypeptide or composition of claim 6 or 7, wherein the one or more other groups, residues, moieties or binding units providing the polypeptide with increased half-life are selected from binding units that bind to serum albumin (such as human serum albumin) or serum immunoglobulin (such as IgG).

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

10. The polypeptide or composition of claim 9, wherein the ISVD that binds to human serum albumin comprises

11. The polypeptide or composition of claim 9 or 10, wherein the ISVD which binds to human serum albumin comprises CDR1 having the amino acid sequence of SEQ ID No. 8, CDR2 having the amino acid sequence of SEQ ID No. 12 and CDR3 having the amino acid sequence of SEQ ID No. 16.

12. The polypeptide or composition of any one of claims 9 to 11, wherein the amino acid sequence of the ISVD that binds to human serum albumin has greater than 90% sequence identity to SEQ ID No. 4.

13. The polypeptide or composition of any one of claims 9 to 12, wherein the ISVD that binds to human serum albumin has the amino acid sequence of SEQ ID No. 4.

14. The polypeptide or composition of any one of claims 1 to 13, wherein the amino acid sequence of the polypeptide has greater than 90% sequence identity to SEQ ID No. 1.

15. The polypeptide or composition of any one of claims 1 to 14, wherein the amino acid sequence of the polypeptide has the amino acid sequence of SEQ ID No. 1.

16. The polypeptide or composition according to any one of claims 1 to 15 for use as a medicament.

17. The polypeptide or composition according to any one of claims 1 to 15 for use in the treatment of an inflammatory disease, such as type 2 inflammatory disease.

18. The polypeptide or composition for the use according to claim 17, wherein the inflammatory disease type 2 is selected from asthma and/or atopic dermatitis.

19. A nucleic acid comprising a nucleotide sequence encoding the polypeptide of any one of claims 1 to 18.

20. A host or host cell comprising the nucleic acid of claim 19.

21. A method for producing a polypeptide according to any one of claims 1 to 18, the method comprising at least the steps of:

a) Expressing the nucleic acid of claim 19; optionally followed by:

b) Isolating and/or purifying the polypeptide.

22. A composition comprising at least one polypeptide according to any one of claims 1 to 18.

23. A composition comprising the nucleic acid of claim 19.

24. Use of a polypeptide according to any one of claims 1 to 18 or a composition according to any one of claims 22 in the manufacture of a pharmaceutical composition for the treatment of an autoimmune disease and/or an inflammatory disease such as inflammatory disease type 2.

25. The use of a polypeptide or composition according to claim 24, wherein the type 2 inflammatory disease is selected from asthma and atopic dermatitis.