Polypeptides containing immunoglobulin single variable domains that target TNFα and IL-23 - Patent application

Abstract

The present technology aims to provide a new type of drug. In particular, the present technology provides a polypeptide comprising at least three immunoglobulin single variable domains (ISVDs), wherein at least one ISVD binds to TNFα and at least two ISVDs bind to IL-23. The present technology also provides nucleic acids, vectors, and compositions.

Description

[Technical field]

[0001] 1 Field of this technology The present technology relates to a polypeptide that targets the p19 subunit of TNFα and IL-23.The present technology also relates to a nucleic acid molecule that encodes the polypeptide and a vector that contains the nucleic acid, as well as a composition that contains these polypeptides, nucleic acids, or vectors.The present technology further relates to these products for use in a method for treating a subject suffering from inflammatory bowel disease, psoriasis, psoriatic arthritis, or hidradenitis suppurativa.The present technology also relates to a method for producing these products. [Background technology]

[0002] 2 Technical background Autoimmune or inflammatory diseases are the result of immune response generated by the body against its own tissue. Autoimmune or inflammatory diseases are often chronic and can even be life-threatening. Among many others, autoimmune or inflammatory diseases include inflammatory bowel disease, such as Crohn's disease and ulcerative colitis, psoriasis, psoriatic arthritis and hidradenitis suppurativa. Inflammatory bowel disease, such as Crohn's disease and ulcerative colitis, is a chronic inflammatory disease with intestinal inflammation and associated epithelial damage. Other chronic autoimmune diseases, such as psoriasis, psoriatic arthritis and hidradenitis suppurativa, are characterized by red, dry, itchy or scaly patches of skin, painful inflammation of joints or inflamed and swollen lumps on the skin. It has been found that patients with psoriasis are more likely to have certain comorbidities, including diabetes and inflammatory bowel disease, such as Crohn's disease or ulcerative colitis, and cancer.

[0003] Interleukin 23 (IL-23) is an important cytokine in the activation of various immune cells during the induction of chronic inflammation. IL-23 is an upstream regulator of cytokines IL-6, IL-17, GM-CSF and IL-22, and is a heterodimer composed of a p19 subunit (IL-23 alpha subunit, sometimes referred to herein as IL-23p19) covalently linked to a p40 subunit (p40 subunit is shared with cytokine IL-12, also referred to as IL-12 beta subunit). Additionally, IL-23 plays an important role in regulating the inflammatory activity of innate lymphoid cells, as well as T cell inflammatory immune responses. IL-23 has been linked to inflammatory diseases, including inflammatory bowel disease and other autoimmune diseases.

[0004] Tumor necrosis factor alpha (TNFα) is produced primarily by monocytes and macrophages, but is also expressed by CD4 + and CD8 + It is a homotrimeric cytokine that is also known to be secreted by peripheral blood T lymphocytes. TNFα can exist in soluble form or as a transmembrane protein. The main role of TNFα is in the regulation of immune cells. TNFα acts as an endogenous pyrogen, and dysregulation of its production has been linked to various human diseases, including inflammatory bowel disease and other autoimmune diseases, such as psoriasis.

[0005] Treatments for inflammatory bowel disease currently approved by the FDA include anti-TNFα biologics (e.g., Simponi® [golimumab], Enbrel® [etanercept], Remicade® [infliximab], and Humira® [adalimumab]). However, current anti-TNFα treatments for inflammatory bowel disease are faced with a large percentage of patients who are non-responsive to currently available treatments, with loss of response to anti-TNFα treatment occurring in a high percentage of patients after 12 months of treatment.

[0006] For psoriasis and psoriatic arthritis, only a minority of patients are treated with biologics (including REMICADE® [infliximab] and HUMIRA® [adalimumab], as well as STELARA® [ustekinumab], which targets the shared p40 subunit of the cytokines IL-12 and IL-23). ​​Additional antibody treatments targeting IL-23 are in development, including guselkumab (Tremfya; approved in psoriasis and in phase 3 clinical trials for inflammatory bowel disease and psoriatic arthritis) and risankizumab (Skyrizi; approved in psoriasis and in phase 3 clinical trials for psoriatic arthritis and Crohn's disease). The class of p19-targeting anti-IL-23 antibodies may confer some disease-suppressive efficacy in psoriasis, but patients with different and / or additional autoimmune diseases do not necessarily benefit to the same extent from these treatments. For example, in psoriatic arthritis, treatment with anti-IL23 does not improve the response of inflamed joints.In inflammatory bowel disease, more than half of patients do not respond or lose response to TNF inhibitors.The same is true for hidradenitis suppurativa, for which the only approved treatment to date is HUMIRA (adalimumab), but only about 50% of patients respond.

[0007] Targeting of multiple disease factors can be achieved, for example, by the combined administration or combined use of two separate biological agents, such as antibodies that bind to different therapeutic targets.However, the combined administration or combined use of separate biological agents can be difficult from both practical and commercial perspectives.For example, two injections of separate products can result in a more inconvenient and more painful treatment regime for patients, negatively affecting compliance.For a single injection of two separate products, it can be difficult or impossible to provide a formulation that allows the required concentration and acceptable viscosity of both products with suitable stability.In addition, combined administration and combined formulation require the production of two separate drugs, which can increase overall costs.

[0008] Bispecific antibodies, capable of binding to two different antigens, have been suggested as one strategy to address such limitations associated with the co-administration or combined use of separate biological agents, such as antibodies.

[0009] Bispecific antibody constructs have been proposed in multiple formats. For example, bispecific antibody formats can involve chemical conjugation of two antibodies or fragments thereof (Non-Patent Document 1; Non-Patent Document 2). In one particular format, a single chain Fv (scFv) fragment that binds a specific antigen is linked to an IgG antibody that binds a separate antigen (e.g., Patent Document 1, which describes an anti-TNFα / anti-IL-23 IgG-scFv bispecific antibody). Patent Document 2 describes a heterodimeric IgG antibody in which one pair of heavy and light chain variable regions targets TNFα and the other pair of heavy and light chain variable regions targets IL-23.

[0010] Drawbacks of such bispecific antibody formats, however, include high viscosity at high concentrations, which makes e.g. subcutaneous administration difficult, and that each binding unit requires the interaction of two variable domains for specific and high affinity binding, with implications for polypeptide stability and efficiency of production.Such bispecific antibody formats can also potentially lead to chemistry, manufacturing and quality control (CMC) problems related to light chain mispairing or heavy chain mispairing. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] WO2016 / 073406 [Patent Document 2] WO2019 / 027780 [Non-patent literature]

[0012] [Non-Patent Document 1] Brennan, M et al., Science, 1985.229(4708): pp. 81-83 [Non-Patent Document 2] Glennie, MJ et al., J Immunol, 1987.139(7):2367-2375 Summary of the Invention [Means for solving the problem]

[0013] 3 Summary of this technology In some embodiments, the technology relates to polypeptides or constructs that specifically target TNFα and IL-23. Targeting TNFα and IL-23 simultaneously leads to increased efficiency in modulating inflammatory responses compared to monospecific anti-TNFα or anti-IL-23 polypeptides.

[0014] The above polypeptides shown to be highly potent against TNFα and IL-23 (e.g., human and cynomolgus monkey TNFα and IL-23) can be efficiently produced (e.g., in a microbial host, e.g., Pichia sp., e.g., P. pastoris) and exhibit low viscosity at high concentrations, which is advantageous and convenient for subcutaneous administration. Moreover, such polypeptides could be shown to have limited reactivity against 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). In other embodiments, such polypeptides exhibit a sufficiently long half-life in the subject to be treated to allow for convenient spacing of successive treatments.

[0015] The polypeptide of the present technology comprises or consists of at least three immunoglobulin single variable domains (ISVDs), where at least one ISVD specifically binds to TNFα and at least two ISVDs specifically bind to the p19 subunit of IL-23. In one embodiment, at least one ISVD that binds to TNFα specifically binds to human TNFα and at least two ISVDs that bind to IL-23 specifically bind to the p19 subunit of human IL-23.

[0016] In one embodiment, the polypeptide further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers, which provide the polypeptide with an increased half-life compared to a corresponding polypeptide without said one or more other groups, residues, moieties or binding units. For example, the binding unit can be an ISVD that binds to a (human) serum protein, such as human serum albumin.

[0017] Nucleic acid molecules capable of expressing the polypeptides of the present technology, vectors containing the nucleic acids, and compositions containing these polypeptides, nucleic acids or vectors are also provided.

[0018] The polypeptide of the present technology, a composition comprising the polypeptide, and a composition comprising a nucleic acid comprising a nucleotide sequence encoding the polypeptide can be used as a medicine. The polypeptide for use as a medicine comprises or consists of at least three immunoglobulin single variable domains (ISVDs), each of the ISVDs comprising three complementarity determining regions (CDR1 to CDR3, respectively), optionally linked via one or more peptidic linkers; a) The first ISVD is: i. CDR1, which is an amino acid sequence of SEQ ID NO:6 or an amino acid sequence having two or one amino acid difference from SEQ ID NO:6; ii. CDR2 having the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 10; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 14. Includes; b) The second ISVD is: iv. CDR1, which is an amino acid sequence of SEQ ID NO:7 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO:7; v. a CDR2 having the amino acid sequence of SEQ ID NO:11 or an amino acid sequence having two or one amino acid difference from SEQ ID NO:11; and vi. A CDR3 having the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 15 Includes; c) The third ISVD is: vii. CDR1, which is an amino acid sequence of SEQ ID NO:9 or an amino acid sequence having two or one amino acid difference from SEQ ID NO:9; viii. CDR2 having the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 13; and ix. CDR3 having the amino acid sequence of SEQ ID NO:17 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO:17 Including, The ISVDs are ordered starting from the N-terminus.

[0019] The composition of the present technology is for use as a medicine.The composition can be a pharmaceutical composition that further comprises at least one pharma-ceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally comprises one or more additional pharma-ceutically active polypeptides and / or compounds.

[0020] Such a polypeptide (optionally present in a composition or optionally encoded by a nucleic acid) comprises or consists of at least three immunoglobulin single variable domains (ISVDs), each of said ISVDs comprising three complementarity determining regions (CDR1 to CDR3, respectively), optionally linked via one or more peptidic linkers; a) The first ISVD is: i. CDR1, which is an amino acid sequence of SEQ ID NO:6 or an amino acid sequence having two or one amino acid difference from SEQ ID NO:6; ii. CDR2 having the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 10; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 14. Includes; b) The second ISVD is: iv. CDR1, which is an amino acid sequence of SEQ ID NO:7 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO:7; v. a CDR2 having the amino acid sequence of SEQ ID NO:11 or an amino acid sequence having two or one amino acid difference from SEQ ID NO:11; and vi. A CDR3 having the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 15 Includes; c) The third ISVD is: vii. CDR1, which is an amino acid sequence of SEQ ID NO:9 or an amino acid sequence having two or one amino acid difference from SEQ ID NO:9; viii. CDR2 having the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 13; and ix. CDR3 having the amino acid sequence of SEQ ID NO:17 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO:17 Including, The ISVDs are ordered starting from the N-terminus.

[0021] In one embodiment, the polypeptide specifically binds to TNFα and the p19 subunit of IL-23. In one embodiment, the polypeptide specifically binds to human TNFα and the p19 subunit of human IL-23. In one embodiment, the first ISVD present in the polypeptide specifically binds to TNFα, and the second and third ISVDs present in the polypeptide specifically bind to the p19 subunit of IL-23. In one embodiment, the first ISVD present in the polypeptide specifically binds to human TNFα, and the second and third ISVDs present in the polypeptide specifically bind to the p19 subunit of human IL-23.

[0022] The polypeptide of the present technology is: a) a first ISVD comprising 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) a second ISVD comprising 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 c) a third ISVD comprising a CDR1 having the amino acid sequence of SEQ ID NO: 9, a CDR2 having the amino acid sequence of SEQ ID NO: 13, and a CDR3 having the amino acid sequence of SEQ ID NO: 17; may include:

[0023] The polypeptide of the present technology is: a) a first ISVD having an amino acid sequence that comprises greater than 90% (e.g., 95%) sequence identity to SEQ ID NO:2; b) a second ISVD having an amino acid sequence that contains greater than 90% (e.g., 95%) sequence identity with SEQ ID NO:3; and c) a third ISVD having an amino acid sequence that has greater than 90% (e.g., 95%) sequence identity with SEQ ID NO:5; may include:

[0024] The polypeptide of the present technology is: a) a first ISVD comprising the amino acid sequence of SEQ ID NO:2; b) a second ISVD comprising the amino acid sequence of SEQ ID NO:3; and c) a third ISVD comprising the amino acid sequence of SEQ ID NO:5; may include:

[0025] The polypeptide of the present technology may further comprise one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers, which provide a polypeptide with an increased half-life compared to a corresponding polypeptide without the one or more other groups, residues, moieties or binding units. The one or more other groups, residues, moieties or binding units that provide a polypeptide with an increased half-life may be selected from the group consisting of polyethylene glycol molecules, serum proteins or fragments thereof, binding units that can bind to serum proteins, Fc portions, and small proteins or peptides that can bind to serum proteins. The binding units that provide a polypeptide with an increased half-life may be selected from the group consisting of binding units that can bind to serum albumin (e.g., human serum albumin) or serum immunoglobulins (e.g., IgG), such as human serum albumin.

[0026] The polypeptide of the present technology is: i. CDR1, which is an amino acid sequence of SEQ ID NO:8 or an amino acid sequence having two or one amino acid difference from SEQ ID NO:8; ii. CDR2 having the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 12; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 16. The antibody may comprise an ISVD that binds to human serum albumin, including

[0027] In one embodiment, the ISVD that binds human serum albumin comprises a CDR1 that is the amino acid sequence of SEQ ID NO: 8, a CDR2 that is the amino acid sequence of SEQ ID NO: 12, and a CDR3 that is the amino acid sequence of SEQ ID NO: 16. In one embodiment, the ISVD that binds human serum albumin comprises greater than 90% (e.g., 95%) sequence identity to SEQ ID NO: 4. In one embodiment, the ISVD that binds human serum albumin comprises or consists of the amino acid sequence of SEQ ID NO: 4.

[0028] The ISVD that binds human serum albumin can be located at any position in the polypeptide of the present technology (i.e., at the N-terminus, between the two building blocks, or at the C-terminus). In one embodiment, the ISVD that binds human serum albumin is located between the second and third ISVDs that specifically bind to the p19 subunit of IL-23.

[0029] The polypeptide of the present technology may comprise an amino acid sequence that comprises greater than 90% (e.g., 95%) sequence identity with SEQ ID NO: 1. In one embodiment, the polypeptide of the present technology comprises or consists of the amino acid sequence of SEQ ID NO: 1.

[0030] Nucleic acids comprising nucleotide sequences encoding the polypeptides of the present technology are also provided.

[0031] A host or host cell containing such nucleic acid is also provided.

[0032] Also provided is a method for producing a polypeptide of the present technology, the method comprising at least: a) expressing a nucleic acid encoding a polypeptide of the present technology in a suitable host cell or host organism or in another suitable expression system; optionally followed by: b) isolating and / or purifying the polypeptide Includes.

[0033] Also provided is a composition comprising at least one polypeptide of the present technology or a nucleic acid encoding the polypeptide of the present technology. The composition may be a pharmaceutical composition further comprising at least one pharma- ceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally one or more additional pharma- ceutically active polypeptides and / or compounds.

[0034] The polypeptides of the present technology can be used in the treatment. More particularly, the polypeptides of the present technology can be used in the treatment of autoimmune or inflammatory diseases, such as inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, psoriasis, psoriatic arthritis, and hidradenitis suppurativa.

[0035] Thus, the present technology also encompasses a method for treating autoimmune or inflammatory diseases. In some embodiments, the present technology encompasses a method for treating a disease selected from inflammatory bowel disease, such as Crohn's disease and ulcerative colitis, psoriasis, psoriatic arthritis, and hidradenitis suppurativa, comprising administering to a subject in need thereof a pharmacologic active amount of the polypeptide of the present technology, a nucleic acid encoding the polypeptide of the present technology, or a composition comprising the same.

[0036] Thus, the present technology also encompasses the use of the polypeptides of the present technology in the manufacture of pharmaceutical compositions for treating autoimmune or inflammatory diseases. In some embodiments, the present technology also encompasses the use of the polypeptides of the present technology in the manufacture of pharmaceutical compositions for treating diseases selected from inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, psoriasis, psoriatic arthritis, and hidradenitis suppurativa.

[0037] In particular, the present technology provides the following embodiments:

[0038] Embodiment 1. A polypeptide, a composition comprising said polypeptide, or a composition comprising a nucleic acid comprising a nucleotide sequence encoding said polypeptide, for use as a medicament, said polypeptide comprising or consisting of at least three immunoglobulin single variable domains (ISVDs), each of said ISVDs comprising three complementarity determining regions (CDR1 to CDR3, respectively), optionally linked via one or more peptidic linkers; a) The first ISVD is: x. CDR1, which is an amino acid sequence of SEQ ID NO:6 or an amino acid sequence having two or one amino acid difference from SEQ ID NO:6; xi. CDR2 having the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 10; and xii. A CDR3 having the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 14. Includes; b) The second ISVD is: xiii. CDR1, which is an amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 7; xiv. CDR2, which is an amino acid sequence of SEQ ID NO:11 or an amino acid sequence having two or one amino acid difference from SEQ ID NO:11; and xv. A CDR3 having the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 15 Includes; c) The third ISVD is: xvi. CDR1 having an amino acid sequence of SEQ ID NO:9 or an amino acid sequence having two or one amino acid difference from SEQ ID NO:9; xvii. CDR2 having the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 13; and xviii. CDR3 having the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 17 Including, The ISVD is in the order starting from the N-terminus: A polypeptide or composition for said use.

[0039] Embodiment 2. A composition for use according to embodiment 1, which is a pharmaceutical composition further comprising at least one pharma- ceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally one or more additional pharma- ceutically active polypeptides and / or compounds.

[0040] Embodiment 3. A polypeptide or composition for use according to embodiment 1 or 2, wherein said polypeptide specifically binds to TNFα and the p19 subunit of IL-23.

[0041] Embodiment 4. A polypeptide or composition for use according to any one of embodiments 1 to 3, wherein the polypeptide specifically binds to human TNFα and the p19 subunit of human IL-23.

[0042] Embodiment 5. A polypeptide or composition for use according to any one of embodiments 1 to 4, wherein the first ISVD specifically binds to TNFα and the second and third ISVDs specifically bind to the p19 subunit of IL-23.

[0043] Embodiment 6. A polypeptide or composition for use according to any one of embodiments 1 to 5, wherein the first ISVD specifically binds to human TNFα and the second and third ISVDs specifically bind to the p19 subunit of human IL-23.

[0044] In embodiment 7.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; c) the third ISVD comprises a CDR1 having an amino acid sequence of SEQ ID NO: 9, a CDR2 having an amino acid sequence of SEQ ID NO: 13, and a CDR3 having an amino acid sequence of SEQ ID NO: 17; A polypeptide or composition for use according to any one of embodiments 1 to 6.

[0045] In embodiment 8.a) the amino acid sequence of the first ISVD comprises greater than 90% (e.g., 95%) sequence identity with SEQ ID NO:2; b) the amino acid sequence of the second ISVD comprises greater than 90% (e.g., 95%) sequence identity with SEQ ID NO:3; c) the amino acid sequence of the third ISVD comprises greater than 90% (e.g., 95%) sequence identity with SEQ ID NO:5; A polypeptide or composition for use according to any one of embodiments 1 to 7.

[0046] In embodiment 9.a) the first ISVD comprises the amino acid sequence of SEQ ID NO:2; b) the second ISVD comprises the amino acid sequence of SEQ ID NO:3; c) the third ISVD comprises the amino acid sequence of SEQ ID NO:5; A polypeptide or composition for use according to any one of embodiments 1 to 8.

[0047] Embodiment 10. A polypeptide or composition for use according to any one of embodiments 1 to 9, wherein said polypeptide further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers, said one or more other groups, residues, moieties or binding units providing said polypeptide with an increased half-life compared to a corresponding polypeptide not having said one or more other groups, residues, moieties or binding units.

[0048] Embodiment 11. A polypeptide or composition for use according to embodiment 10, wherein said one or more other groups, residues, moieties or binding units providing said polypeptide with an increased half-life are selected from the group consisting of polyethylene glycol molecules, serum proteins or fragments thereof, binding units capable of binding to serum proteins, Fc moieties, and small proteins or peptides capable of binding to serum proteins.

[0049] Embodiment 12. A polypeptide or composition for use according to embodiment 10 or 11, wherein said binding unit providing said polypeptide with an increased half-life is selected from the group consisting of binding units capable of binding to serum albumin (e.g. human serum albumin) or serum immunoglobulin (e.g. IgG).

[0050] Embodiment 13. A polypeptide or composition for use according to embodiment 12, wherein said binding unit providing said polypeptide with an increased half-life is an ISVD capable of binding to human serum albumin.

[0051] Embodiment 14. The ISVD that binds to human serum albumin is i. CDR1, which is an amino acid sequence of SEQ ID NO:8 or an amino acid sequence having two or one amino acid difference from SEQ ID NO:8; ii. CDR2 having the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 12; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 16. 14. A polypeptide or composition for use according to embodiment 13, comprising:

[0052] Embodiment 15. A polypeptide or composition for use according to embodiment 13 or 14, wherein the ISVD that binds to human serum albumin comprises a CDR1 having the amino acid sequence of SEQ ID NO:8, a CDR2 having the amino acid sequence of SEQ ID NO:12 and a CDR3 having the amino acid sequence of SEQ ID NO:16.

[0053] Embodiment 16. A polypeptide or composition for use according to any one of embodiments 13 to 15, wherein the amino acid sequence of said ISVD that binds to human serum albumin comprises more than 90% (e.g. 95%) sequence identity with SEQ ID NO:4.

[0054] Embodiment 17. A polypeptide or composition for use according to any one of embodiments 13 to 16, wherein said ISVD that binds to human serum albumin comprises or consists of the amino acid sequence of SEQ ID NO: 4.

[0055] Embodiment 18. A polypeptide or composition for use according to any one of embodiments 1 to 17, wherein the amino acid sequence of the polypeptide comprises more than 90% (e.g. 95%) sequence identity with SEQ ID NO:1.

[0056] Embodiment 19. A polypeptide or composition for use according to any one of embodiments 1 to 18, wherein the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:1.

[0057] Embodiment 20. A polypeptide or composition according to any one of embodiments 1 to 19 for use in the treatment of an autoimmune or inflammatory disease, such as an inflammatory bowel disease, such as Crohn's disease and ulcerative colitis, psoriasis, psoriatic arthritis and hidradenitis suppurativa.

[0058] Embodiment 21. A polypeptide, comprising or consisting of at least three immunoglobulin single variable domains (ISVDs), each of said ISVDs comprising three complementarity determining regions (CDR1 to CDR3, respectively), optionally linked via one or more peptidic linkers; a) The first ISVD is: x. CDR1, which is an amino acid sequence of SEQ ID NO:6 or an amino acid sequence having two or one amino acid difference from SEQ ID NO:6; xi. CDR2 having the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 10; and xii. A CDR3 having the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 14. Includes; b) The second ISVD is: xiii. CDR1, which is an amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 7; xiv. CDR2, which is an amino acid sequence of SEQ ID NO:11 or an amino acid sequence having two or one amino acid difference from SEQ ID NO:11; and xv. A CDR3 having the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 15 Includes; c) The third ISVD is: xvi. CDR1 having an amino acid sequence of SEQ ID NO:9 or an amino acid sequence having two or one amino acid difference from SEQ ID NO:9; xvii. CDR2 having the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 13; and xviii. CDR3 having the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 17 Including, The ISVD is in the order starting from the N-terminus: The polypeptide.

[0059] Embodiment 22. A polypeptide according to embodiment 21, which specifically binds to the p19 subunit of TNFα and IL-23.

[0060] Embodiment 23. A polypeptide according to embodiment 21 or 22, which specifically binds to human TNFα and the p19 subunit of human IL-23.

[0061] Embodiment 24. A polypeptide according to any one of embodiments 21 to 23, wherein the first ISVD specifically binds to TNFα, and the second and third ISVDs specifically bind to the p19 subunit of IL-23.

[0062] Embodiment 25. A polypeptide according to any one of embodiments 21 to 24, wherein the first ISVD specifically binds to human TNFα, and the second and third ISVDs specifically bind to the p19 subunit of human IL-23.

[0063] Embodiment 26.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 CDR1 having the amino acid sequence of SEQ ID NO: 7, CDR2 having the amino acid sequence of SEQ ID NO: 11, and CDR3 having the amino acid sequence of SEQ ID NO: 15. Includes; c) the third ISVD comprises a CDR1 having an amino acid sequence of SEQ ID NO: 9, a CDR2 having an amino acid sequence of SEQ ID NO: 13, and a CDR3 having an amino acid sequence of SEQ ID NO: 17; A polypeptide according to any one of embodiments 21 to 25.

[0064] Embodiment 27.a) the amino acid sequence of the first ISVD comprises greater than 90% (e.g., 95%) sequence identity with SEQ ID NO:2; b) the amino acid sequence of the second ISVD comprises greater than 90% (e.g., 95%) sequence identity with SEQ ID NO:3; c) the amino acid sequence of the third ISVD comprises greater than 90% (e.g., 95%) sequence identity with SEQ ID NO:5; A polypeptide according to any one of embodiments 21 to 26.

[0065] Embodiment 28.a) the first ISVD comprises the amino acid sequence of SEQ ID NO:2; b) the second ISVD comprises the amino acid sequence of SEQ ID NO:3; c) the third ISVD comprises the amino acid sequence of SEQ ID NO:5; A polypeptide according to any one of embodiments 21 to 27.

[0066] Embodiment 29. A polypeptide according to any one of embodiments 21 to 28, wherein the polypeptide further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers, which provide the polypeptide with an increased half-life compared to a corresponding polypeptide not having said one or more other groups, residues, moieties or binding units.

[0067] Embodiment 30. The polypeptide of embodiment 29, wherein the one or more other groups, residues, moieties or binding units that provide the polypeptide with an increased half-life are selected from the group consisting of polyethylene glycol molecules, serum proteins or fragments thereof, binding units capable of binding to serum proteins, Fc moieties, and small proteins or peptides capable of binding to serum proteins.

[0068] Embodiment 31. The polypeptide of embodiment 29 or 30, wherein the one or more other groups, residues, moieties or binding units that provide said polypeptide with an increased half-life are selected from the group consisting of binding units capable of binding to serum albumin (e.g. human serum albumin) or serum immunoglobulin (e.g. IgG).

[0069] Embodiment 32. The polypeptide of embodiment 31, wherein the binding unit that provides the polypeptide with an increased half-life is an ISVD capable of binding to human serum albumin.

[0070] Embodiment 33. An ISVD that binds to human serum albumin is: i. CDR1, which is an amino acid sequence of SEQ ID NO:8 or an amino acid sequence having two or one amino acid difference from SEQ ID NO:8; ii. CDR2 having the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 12; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 16. 33. The polypeptide of embodiment 32, comprising:

[0071] Embodiment 34. The polypeptide of embodiment 32 or 33, wherein the ISVD that binds to human serum albumin comprises a CDR1 having the amino acid sequence of SEQ ID NO:8, a CDR2 having the amino acid sequence of SEQ ID NO:12, and a CDR3 having the amino acid sequence of SEQ ID NO:16.

[0072] Embodiment 35. A polypeptide described in any one of embodiments 32 to 34, wherein the amino acid sequence of the ISVD that binds to human serum albumin comprises greater than 90% (e.g., 95%) sequence identity with SEQ ID NO:4.

[0073] Embodiment 36. A polypeptide according to any one of embodiments 32 to 35, wherein said ISVD that binds to human serum albumin comprises or consists of the amino acid sequence of SEQ ID NO:4.

[0074] Embodiment 37. A polypeptide according to any one of embodiments 21 to 36, wherein the amino acid sequence of the polypeptide comprises more than 90% (e.g. 95%) sequence identity with SEQ ID NO:1.

[0075] Embodiment 38. A polypeptide according to any one of embodiments 21 to 37, comprising or consisting of the amino acid sequence of SEQ ID NO:1.

[0076] Embodiment 39. A nucleic acid comprising a nucleotide sequence encoding a polypeptide according to any one of embodiments 21 to 38.

[0077] Embodiment 40. A host or host cell comprising a nucleic acid according to embodiment 39.

[0078] Embodiment 41. A method for producing a polypeptide according to any one of embodiments 21 to 38, comprising at least: a) expressing the nucleic acid according to embodiment 39 in a suitable host cell or host organism or in another suitable expression system; optionally followed by: b) isolating and / or purifying the polypeptide according to any one of embodiments 21 to 38. The method comprising:

[0079] Embodiment 42. A composition comprising at least one polypeptide according to any one of embodiments 21 to 38, or a nucleic acid according to embodiment 39.

[0080] Embodiment 43. The composition according to embodiment 42, which is a pharmaceutical composition further comprising at least one pharma- ceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally one or more additional pharma- ceutically active polypeptides and / or compounds.

[0081] Embodiment 44. A method for treating an autoimmune or inflammatory disease, comprising administering to a subject in need thereof a pharma- ceutically active amount of a polypeptide according to any one of embodiments 21 to 38, or a composition according to embodiment 42 or 43.

[0082] Embodiment 45. The method of embodiment 44, wherein the autoimmune or inflammatory disease is selected from inflammatory bowel disease, e.g., Crohn's disease and ulcerative colitis, psoriasis, psoriatic arthritis, and hidradenitis suppurativa.

[0083] Embodiment 45. Use of a polypeptide according to any one of embodiments 21 to 38, or a composition according to embodiment 42 or 43, in the manufacture of a pharmaceutical composition for treating an autoimmune or inflammatory disease.

[0084] Embodiment 46. The use of a polypeptide or composition according to embodiment 45, wherein the autoimmune or inflammatory disease is selected from inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, psoriasis, psoriatic arthritis and hidradenitis suppurativa. [Brief description of the drawings]

[0085] 4. Brief Description of the Drawings [Figure 1] Sensorgram showing simultaneous binding of TNFα and IL-23 to F027500069 captured via human serum albumin (HSA). [Diagram 2] Inhibition of soluble human (A) and cynomolgus (B) TNFα by F027500069 and anti-hTNFα reference mAb in the Glo response™ HEK293_NFκB-NLucP reporter assay. IRR00096 is a negative control VHH. Data points are the overall mean (n=2) and error bars represent + / -SD. [Diagram 3] Inhibition of human (A) and cynomolgus (B) IL-23 using F027500069, anti-hIL-23 ref mAb1, and anti-hIL-23 ref mAb2 in the Glo response™ HEK293_human IL-23R / IL-12Rb1-Luc2P reporter assay. IRR00096 is a negative control VHH. Data points are overall means (n=2) and error bars represent + / -SD. [Figure 4] Box plot showing binding of pre-existing antibodies present in 96 human serum samples to F027500069 compared to the control F027301099. [Diagram 5] Box plot showing binding of pre-existing antibodies present in 96 human serum samples to F027500069, F027500093, F027500095 and F027500096 compared to control polypeptides F027301099 and F027301186. [Figure 6]Inhibition of polyarthritis in the Tg197 human TNF transgenic mouse model. Arthritis scores over time are plotted in different treatment groups (n=8 mice / group). Starting at 6 weeks of age, animals received two weekly intraperitoneal injections of the indicated compound. Mean weekly arthritis scores ± SEM are shown. Statistics are two-way ANOVA and Bonferroni multiple comparison test. [Figure 7] Bar graph of area under the curve of arthritis score over time in the Tg197 mouse model. Individual values ​​(symbols) and mean±SEM (bars) are shown. Statistics are one-way ANOVA and Bonferroni multiple comparison test. [Figure 8] Bar graph of paw histology scores of Tg197 mouse model. Individual values ​​(symbols) and mean±SEM (bars) are shown. Statistics are one-way ANOVA and Bonferroni multiple comparison test. [Figure 9] Inhibition of skin inflammation in the human IL-23 model. Bar graphs show ear skin swelling in different treatment groups (n=10 mice / group). Animals received daily intradermal injections of recombinant human IL-23 or PBS on days 1-4. Animals received intraperitoneal injections of the indicated compound on days 1 and 3. Shown are mean changes in ear skin thickness from baseline ± SEM on day 5. Statistics are ANOVA and Bonferroni multiple comparison test. [Figure 10] Bar graph of tissue IL-22 concentrations from skin biopsies on day 5 of the human IL-23-induced skin inflammation model. Individual values ​​(symbols) and mean ± SEM (bars) are shown. Statistics are ANOVA and Bonferroni multiple comparison test. [Figure 11] Inhibition of skin inflammation by F027500069 administered by both intraperitoneal and subcutaneous routes. Bar graphs show ear skin swelling in different treatment groups (n=8 mice / group). Animals received daily intradermal injections of recombinant human IL-23 or PBS on days 1-4. Intraperitoneal (IP) or subcutaneous (SC) injections of the indicated doses were administered on days 1 and 3. Mean change in ear skin thickness from baseline on day 5 ± SEM is shown. Statistics are ANOVA and Bonferroni multiple comparison test. [Figure 12]Bar graph of tissue IL-22 concentrations from skin biopsies on day 5 for F027500069 delivered by both intraperitoneal and subcutaneous routes. Individual values ​​(symbols) and mean±SEM (bars) are shown. Statistics are ANOVA and Bonferroni multiple comparison test. [Figure 13] Inhibition of arthritis scores in collagen antibody-induced arthritis (CAIA) model in human TNFα / TNFR1 knock-in mice. Shown are arthritis scores over time in different treatment groups (n=8 mice / group). Animals were given a single intraperitoneal injection of the indicated compound 6 hours after LPS injection on day 1. Shown are mean daily arthritis scores ± SEM. Statistics are two-way ANOVA and Bonferroni multiple comparison test. [Figure 14] Dual inhibition of IL-23 and TNFα induced inflammation in a human IL-23 skin injection model performed in human TNFα knock-in mice (n=4-10 mice / group). Animals received daily intradermal injections of recombinant human IL-23 or PBS on days 1-4. Intraperitoneal injections of the indicated compounds on days 1 and 3. Shown are mean changes in ear skin thickness from baseline ± SEM on day 5. Statistics are ANOVA and Bonferroni multiple comparison test. [Figure 15] Differential skin tissue gene expression in inflamed skin tissue following cytokine mono- or bispecific inhibition. Venn diagram of differentially expressed genes (DEGs, fold change >2 with p<0.001). 199 of the total 769 DEGs in F027500069 were specific to this treatment. [Figure 16] Schematic representation of ISVD construct F027500069, showing monovalent building blocks / ISVDs 6C11, 119A03 / 1, ALB23002, and 81A12 connected N- to C-terminus via a 9GS linker. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0086] 5 Detailed description of this technology The present technology aims to provide a new class of drugs for treating inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, psoriasis, psoriatic arthritis or hidradenitis suppurativa.

[0087] The inventors have surprisingly found that a polypeptide comprising at least three ISVDs, at least one of which specifically binds to TNFα and at least two of which specifically bind to the p19 subunit of IL-23, can be used for a more efficient treatment of autoimmune or inflammatory diseases compared to monospecific anti-TNFα or anti-IL-23p19 polypeptides.

[0088] In some embodiments, the polypeptide of the present technology exhibits high potency against TNFα and IL-23 (e.g., human or cynomolgus monkey TNFα and IL-23). ​​In some embodiments, the polypeptide of the present technology is efficiently produced (e.g., in a microbial host, such as Pichia sp., e.g., P. pastoris). In some embodiments, the polypeptide of the present technology has low viscosity at high concentration, which is advantageous and convenient for subcutaneous administration. Furthermore, in some embodiments, the polypeptide of the present technology has limited reactivity against pre-existing antibodies in the subject to be treated (i.e., antibodies present in the subject before the first treatment with the antibody construct). In other embodiments, such polypeptides exhibit a sufficiently long half-life to allow convenient spacing of successive treatments in the subject to be treated.

[0089] The polypeptides are at least bispecific, but can also be, for example, trispecific, tetraspecific, or pentaspecific. Further, the polypeptides are at least trivalent, but can also be, for example, tetravalent or pentavalent.

[0090] The terms "bispecific", "trispecific", "tetraspecific" or "pentaspecific" are all included in the term "multispecific" and refer to binding to two, three, four or five different target molecules, respectively. The terms "bivalent", "trivalent", "tetravalent" or "pentavalent" are all included in the term "multivalent" and refer to the presence of two, three, four or five binding units (e.g., ISVDs), respectively. For example, a polypeptide may be trispecific-tetravalent, e.g., a polypeptide comprises or consists of four ISVDs, one ISVD binds human TNFα, two ISVDs bind human IL-23 and one ISVD binds human serum albumin. Such a polypeptide may be simultaneously dual paratopic, e.g., when two ISVDs bind to two different epitopes on the p19 subunit of IL-23. The term "dual paratopic" refers to binding to two different parts (i.e., epitopes) of the same target molecule.

[0091] Terms such as "first ISVD", "second ISVD", "third ISVD", etc., as used herein, merely indicate the relative positions of the ISVDs relative to each other, with the numbering starting from the N-terminus of the polypeptide of the present technology. The "first ISVD" is therefore closer to the N-terminus than the "second ISVD", while the "second ISVD" is closer to the N-terminus than the "third ISVD", etc. Thus, the order of the ISVDs is opposite when considered from the C-terminus. Since the numbering is not absolute and merely indicates the relative positions of at least three ISVDs, it does not exclude that other binding units / building blocks, such as additional ISVDs that bind to TNFα or the p19 subunit of IL-23, or ISVDs that bind to another target, may be present in the polypeptide. Moreover, it does not exclude the possibility that other binding units / building blocks, such as ISVDs, may be located in between. For example, as described further below (see in particular Section 5.3 "Extended (In Vivo) Half-Life"), the polypeptide may further comprise another ISVD that binds to human serum albumin, which may even be located, for example, between the "second ISVD" and the "third ISVD".

[0092] In light of the above, the present technology provides a polypeptide comprising or consisting of at least three ISVDs, wherein at least one ISVD specifically binds to TNFα and at least two ISVDs specifically bind to the p19 subunit of IL-23.

[0093] At least two ISVDs that specifically bind to IL-23 bind to the p19 subunit of IL-23. At least two ISVDs that specifically bind to IL-23 may bind to different epitopes on the p19 subunit of IL-23. At least one of the ISVDs that specifically bind to IL-23 may have the ability to block the function of IL-23, for example, the ability to block the interaction between IL-23 and IL-23R and / or the ability to inhibit IL-23-induced release of IL-22.

[0094] The components of a polypeptide, such as ISVDs, can be linked to each other by one or more suitable linkers, such as peptidic linkers.

[0095] The use of linkers to connect two or more (poly)peptides is well known in the art. Exemplary peptidic linkers are shown in Table A-5. One frequently used class of peptidic linkers is known as "Gly-Ser" or "GS" linkers. These are linkers that consist essentially of glycine (G) and serine (S) residues and usually contain one or more repeats of a peptide motif, such as the GGGGS (SEQ ID NO: 47) motif (e.g., having the formula (Gly-Gly-Gly-Gly-Ser)n, where n can be 1, 2, 3, 4, 5, 6, 7 or more). Examples of such GS linkers that are used in some frequently are the 9GS linker (GGGGSGGGS, SEQ ID NO: 50), the 15GS linker (n=3; SEQ ID NO: 52) and the 35GS linker (n=7; SEQ ID NO: 57). See, e.g., Chen et al., Adv. Drug Deliv. Rev. 2013 Oct 15; 65(10): 1357-1369; and Klein et al., Protein Eng. Des. Sel. (2014) 27 (10): 325-330. In one embodiment of the polypeptides of the present technology, a 9GS linker is used to link components of the polypeptide together.

[0096] In one embodiment, an ISVD that specifically binds to TNFα is placed at the N-terminus of a polypeptide. The inventors have surprisingly found that such a configuration can increase the production yield of the polypeptide.

[0097] Also, in embodiments, one of the ISVDs that specifically binds IL-23 is located at the C-terminus of the polypeptide.

[0098] Thus, the polypeptide comprises or consists of, in order starting from the N-terminus of the polypeptide: an ISVD that specifically binds TNFα, a first ISVD that specifically binds IL-23 that can block a function of IL-23, an optional binding unit that provides a polypeptide with increased half-life as defined herein, and a second ISVD that specifically binds IL-23. In one embodiment, the binding unit that provides a polypeptide with increased half-life is an ISVD.

[0099] In one embodiment, the polypeptide comprises or consists of the following, in order starting from the N-terminus of the polypeptide: an ISVD that specifically binds TNFα, a linker, a first ISVD that specifically binds IL-23 that can block a function of IL-23, a linker, an ISVD that binds human serum albumin, a linker, and a second ISVD that specifically binds IL-23. In one embodiment, each linker is a 9GS linker.

[0100] Such configuration of the polypeptide may provide increased manufacturing yields as well as good CMC characteristics, including expression yield, viscosity and other biophysical properties.

[0101] In one embodiment, the polypeptide of the present technology exhibits reduced binding by pre-existing antibodies in human serum. To this end, in one embodiment, the polypeptide comprises a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering) in at least one ISVD. In one embodiment, the polypeptide comprises a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering) in each ISVD. In another embodiment, the polypeptide comprises an extension of 1 to 5 (naturally occurring) amino acids, such as a single alanine (A) extension, at the C-terminus of the C-terminal ISVD. The C-terminus of the ISVD is usually VTVSS (SEQ ID NO: 112). In another embodiment, the polypeptide comprises a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) in at least one ISVD. In another embodiment, the ISVDs comprise a lysine (K) or glutamine (Q) at position 112 (according to Kabat numbering) in at least one ISVD. In these embodiments, the C-terminus of the ISVD is VKVSS (SEQ ID NO:113), VQVSS (SEQ ID NO:114), VTVKS (SEQ ID NO:146), VTVQS (SEQ ID NO:147), VKVKS (SEQ ID NO:148), VKVQS (SEQ ID NO:149), VQVKS (SEQ ID NO:150), or VQVQS (SEQ ID NO:151), such that after the addition of a single alanine, the C-terminus of the polypeptide comprises, for example, the sequence VTVSSA (SEQ ID NO:115), VKVSSA (SEQ ID NO:116), VQVSSA (SEQ ID NO:117), VTVKSA (SEQ ID NO:152), VTVQSA (SEQ ID NO:153), VKVKSA (SEQ ID NO:154), VKVQSA (SEQ ID NO:155), VQVKSA (SEQ ID NO:156), or VQVQSA (SEQ ID NO:157). In one embodiment, the C-terminus comprises VKVSSA (SEQ ID NO:116).In another embodiment, the polypeptide comprises a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering) in each ISVD, optionally a lysine (K) or a glutamine (Q) at position 110 (according to Kabat numbering) in at least one ISVD, and comprises a stretch of 1 to 5 (naturally occurring) amino acids, such as a single alanine (A) stretch, at the C-terminus of the C-terminal ISVD, such that the C-terminus of the polypeptide comprises, for example, the sequence VTVSSA (SEQ ID NO: 115), VKVSSA (SEQ ID NO: 116) or VQVSSA (SEQ ID NO: 117), such as VKVSSA (SEQ ID NO: 116). For further information on this see, for example, WO2012 / 175741 and WO2015 / 173325.

[0102] In another embodiment, the polypeptide of the present technology comprises or consists of an amino acid sequence that comprises more than 90%, such as more than 95% or more than 99% sequence identity to SEQ ID NO:1, and the CDRs of the four ISVDs are as defined in items A to D (or A' to D', if the Kabat definitions are used) described in sections "5.1 Immunoglobulin Single Variable Domains" and "5.3 Extension of (In Vivo) Half-Life" below, respectively, in particular: an ISVD that specifically binds to TNFα, comprising 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; a first ISVD that specifically binds to the p19 subunit of IL-23, comprising 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; a second ISVD that specifically binds to the p19 subunit of IL-23, comprising a CDR1 having the amino acid sequence of SEQ ID NO:9, a CDR2 having the amino acid sequence of SEQ ID NO:13, and a CDR3 having the amino acid sequence of SEQ ID NO:17; the ISVD that binds to human serum albumin comprises a CDR1 having the amino acid sequence of SEQ ID NO:8, a CDR2 having the amino acid sequence of SEQ ID NO:12, and a CDR3 having the amino acid sequence of SEQ ID NO:16; Or alternatively, using the Kabat definition: an ISVD that specifically binds to TNFα, comprising a CDR1 having the amino acid sequence of SEQ ID NO: 122, a CDR2 having the amino acid sequence of SEQ ID NO: 130, and a CDR3 having the amino acid sequence of SEQ ID NO: 138; a first ISVD that specifically binds to the p19 subunit of IL-23, comprising a CDR1 having the amino acid sequence of SEQ ID NO: 123, a CDR2 having the amino acid sequence of SEQ ID NO: 131, and a CDR3 having the amino acid sequence of SEQ ID NO: 139; a second ISVD that specifically binds to the p19 subunit of IL-23, comprising a CDR1 that is the amino acid sequence of SEQ ID NO: 125, a CDR2 that is the amino acid sequence of SEQ ID NO: 133, and a CDR3 that is the amino acid sequence of SEQ ID NO: 141; The ISVD that binds to human serum albumin comprises a CDR1 having the amino acid sequence of SEQ ID NO: 124, a CDR2 having the amino acid sequence of SEQ ID NO: 132, and a CDR3 having the amino acid sequence of SEQ ID NO: 140.

[0103] In another embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 1. In another embodiment, the polypeptide consists of the amino acid sequence of SEQ ID NO:1.

[0104] In one embodiment, the polypeptide of the present technology has at least half the binding affinity, or at least the same binding affinity, for human TNFα and human IL-23 as a polypeptide consisting of the amino acid sequence of SEQ ID NO:1, where the binding affinity is measured using the same methods, e.g., surface plasmon resonance (SPR).

[0105] 5.1 Immunoglobulin Single Variable Domains The term "immunoglobulin single variable domain" (ISVD) is used interchangeably with "single variable domain" and defines an immunoglobulin molecule in which the antigen-binding site is present on and formed by a single immunoglobulin domain. This separates it from "conventional" immunoglobulins (e.g., monoclonal antibodies) or fragments thereof (e.g., Fab, Fab', F(ab')2, scFv, di-scFv) in which two immunoglobulin domains, in particular two variable domains, interact to form the antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (V H ) and the light chain variable domain (V L ) interact to form the antigen-binding site. In this case, V H and V L Both complementarity determining regions (CDRs) of the nucleotide sequence contribute to the antigen-binding site, i.e., all six CDRs are involved in forming the antigen-binding site.

[0106] In view of the above definitions, the antigen-binding domains of conventional four-chain antibodies (e.g., IgG, IgM, IgA, IgD or IgE molecules; known in the art), or of Fab fragments, F(ab')2 fragments, Fv fragments, e.g., disulfide-linked Fv or scFv fragments, or diabodies (all known in the art) derived from such conventional four-chain antibodies, are not typically considered to be ISVDs, and in these cases, binding to each epitope of an antigen does not typically occur by one single immunoglobulin domain, but by a pair of associated immunoglobulin domains, e.g., a light and heavy chain variable domain, i.e., the V of the immunoglobulin domains that together bind to the epitope of each antigen. H -V L It occurs in pairs.

[0107] In contrast, an ISVD has the ability to specifically bind to an epitope of an antigen without pairing with an additional immunoglobulin variable domain. The binding site of an ISVD consists of a single V H , a single V HH or a single V LIt is formed by domains.

[0108] Therefore, the ISVD is a sequence of a light chain variable domain (e.g., L sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., H Array or V HH sequence) or a suitable fragment thereof; provided that it has the ability to form a single antigen-binding unit; i.e., a functional antigen-binding unit consisting essentially of the ISVD, such that the single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit.

[0109] The ISVD can be, for example, a heavy chain ISVD, e.g., a camelized V H or humanized V HH Including, V H , V HH In one embodiment, it may be a camelized V H or humanized V HH Including, V HH The heavy chain ISVD can be derived from a traditional four chain antibody or a heavy chain antibody.

[0110] For example, an ISVD may be a single domain antibody (or a suitable amino acid sequence for use as a single domain antibody), a "dAb" or dAb (or a suitable amino acid sequence for use as a dAb) or Nanobody® (as defined herein, HH ); other single variable domains, or any suitable fragment of any one of these.

[0111] In particular, the ISVD is a Nanobody® (e.g., humanized V HH Or Camelification V H Including, V HH ) or a suitable fragment thereof. Nanobody®, Nanobodies® and Nanoclone® are registered trademarks.

[0112] V HH , V HH Antibody fragments, and V HH Antibodies, also known as "V HH The "V domain" was originally described as the antigen-binding immunoglobulin variable domain of a "heavy chain antibody"; i.e., an antibody lacking light chains. See Hamers-Casterman et al., Nature 363:446-448, 1993. HH The term "domain" is used herein to refer to H From the heavy chain variable domain present in conventional four-chain antibodies, referred to as the "V domain" and herein L These variable domains were chosen to distinguish them from the light chain variable domains present in conventional four-chain antibodies, referred to as "light chain variable domains." HH For further description, see the review article by Muyldermans (Reviews in Molecular Biotechnology 74:277-302, 2001).

[0113] Typically, the production of immunoglobulins involves immunization of laboratory animals, fusion of immunoglobulin-producing cells to generate hybridomas, and screening for the desired specificity. Alternatively, immunoglobulins can be produced by screening naive, immune, or synthetic libraries, for example by phage display.

[0114] The generation of immunoglobulin sequences, such as Nanobodies®, has been extensively described in various publications, among which WO94 / 04678, Hamers-Casterman et al., 1993 (Nature 363: 446-448, 1993) and Muyldermans et al., 2001 (Reviews in Molecular Biotechnology 74: 277-302, 2001) can be mentioned. In these methods, a camelid is immunized with a target antigen in order to induce an immune response against said target antigen. The repertoire of Nanobodies® resulting from said immunization is further screened for Nanobodies® that bind to the target antigen.

[0115] In these cases, the production of antibodies requires purified antigen for immunization and / or screening. The antigen can be purified from a natural source or during recombinant production.

[0116] Immunization and / or screening for immunoglobulin sequences can be carried out using peptide fragments of such antigens.

[0117] The technology may use immunoglobulin sequences of different origins, including mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences. The technology also includes fully human, humanized or chimeric sequences. For example, the technology includes camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized domain antibodies, such as camelized dAbs, as described in Ward et al. (Nature 341: 544, 1989) (see, for example, WO94 / 04678 and Davies and Riechmann, Febs Lett., 339: 285-290, 1994 and Prot. Eng., 9: 531-537, 1996). Furthermore, the technology also includes fused immunoglobulin sequences (one or more V domains), for example forming multivalent and / or multispecific constructs. HHFor multivalent and multispecific polypeptides containing domains and their production, see Conrath et al., J. Biol. Chem., vol. 276, 10: 7346-7350, 2001, but also e.g., WO 96 / 34103 and WO 99 / 23221), as well as immunoglobulin sequences containing tags or other functional moieties, e.g., toxins, labels, radiochemicals, etc., derivable from the immunoglobulin sequences of the present technology.

[0118] "Humanized V HH " is a naturally occurring V HH The amino acid sequence of the naturally occurring V HH One or more amino acid residues in the amino acid sequence of the sequence (particularly the framework sequence) may be replaced with a V H The present invention also includes amino acid sequences that have been "humanized" by replacing one or more of the amino acid residues occurring at the corresponding positions in the domain. This can be done in a manner known per se and will be clear to the skilled person, for example based on the further description herein and the prior art (e.g. WO2008 / 020079). Furthermore, such humanized V HH can be obtained in any suitable manner known per se, so that the naturally occurring V HH It should be noted that the present invention is not strictly limited to polypeptides obtained using a polypeptide containing the domain as a starting material.

[0119] "Camelization V H " is a naturally occurring V H The amino acid sequence of the naturally occurring V domain is "camelized", i.e., from a conventional four-chain antibody. H One or more amino acid residues in the amino acid sequence of the domain are HHThe term "camelized" includes amino acid sequences that have been "camelized" by replacing one or more of the amino acid residues occurring at the corresponding positions in the V domain. This can be done in a manner known per se and will be clear to the skilled person, for example on the basis of the further description herein and the prior art (e.g. WO2008 / 020079). Such "camelized" substitutions are usually H -V L The insertions are made at amino acid positions forming and / or present in the interface and / or at the so-called Camelidae hallmark residues as defined herein (see, for example, WO 94 / 04678 and Davies and Riechmann, 1994 and 1996, supra). H V, used as a starting material or starting point for generating or designing H The sequences are V from mammals H sequence, or human V H Array, e.g. V H 3 sequence. However, such camelized V H can be obtained in any suitable manner known per se, so that the naturally occurring V H It should be noted that the present invention is not strictly limited to polypeptides obtained using a polypeptide containing the domain as a starting material.

[0120] The structure of an ISVD sequence can be considered to be composed of four framework regions ("FR"), referred to in the art and herein as "framework region 1" ("FR1"); "framework region 2" ("FR2"); "framework region 3" ("FR3"); and "framework region 4" ("FR4"), respectively; these framework regions are flanked by three complementarity determining regions ("CDRs"), referred to in the art and herein as "complementarity determining region 1" ("CDR1"); "complementarity determining region 2" ("CDR2"); and "complementarity determining region 3" ("CDR3"), respectively.

[0121] As further described in WO08 / 020079, pages 58 and 59, paragraph q), the amino acid residues of the ISVD are similar to those of the VSVD from camelids in Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240(1-2): pages 185-195; see, e.g., FIG. 2 therein). HH As applied to the domain, the V given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91) H The domains can be numbered according to the common numbering system. H Domains and V HH It should be noted that, as is well known in the art for domains, the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by Kabat numbering. That is, 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 allowed by Kabat numbering. In general, this means that the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. H Domains and V HH The total number of amino acid residues in a domain is usually in the range of 110 to 120, often 112 to 115. However, it should be noted that shorter and longer sequences may also be suitable for the purposes described herein.

[0122] In this application, unless otherwise indicated, the CDR sequences were determined according to the AbM numbering as described in Kontermann and Dubel (eds., 2010, Antibody Engineering, Vol. 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pages 33-51). According to this method, FR1 comprises amino acid residues at positions 1-25, CDR1 comprises amino acid residues at positions 26-35, FR2 comprises amino acid residues at positions 36-49, CDR2 comprises amino acid residues at positions 50-58, FR3 comprises amino acid residues at positions 59-94, CDR3 comprises amino acid residues at positions 95-102, and FR4 comprises amino acid residues at positions 103-113.

[0123] The determination of the CDR regions can also be performed according to different methods: in the Kabat CDR determination, FR1 of the ISVD comprises amino acid residues at positions 1-30, CDR1 of the ISVD comprises amino acid residues at positions 31-35, FR2 of the ISVD comprises amino acid residues at positions 36-49, CDR2 of the ISVD comprises amino acid residues at positions 50-65, FR3 of the ISVD comprises amino acid residues at positions 66-94, CDR3 of the ISVD comprises amino acid residues at positions 95-102, and FR4 of the ISVD comprises amino acid residues at positions 103-113.

[0124] In such immunoglobulin sequences, the framework sequences may be any suitable framework sequences, and examples of suitable framework sequences will be clear to the skilled person, e.g. on the basis of standard handbooks and the further disclosure and prior art referred to herein.

[0125] The framework sequences are immunoglobulin framework sequences or suitable combinations of framework sequences derived from immunoglobulin framework sequences, for example by humanization or camelization. For example, the framework sequences may be those of the light chain variable domain (e.g., V L sequence) and / or a heavy chain variable domain (e.g., V HArray or V HH In one embodiment, the framework sequence is derived from the V HH and a framework sequence derived from a conventional V sequence, said framework sequences being optionally partially or fully humanized or camelized (as defined herein). H It is an array.

[0126] In particular, the framework sequences present in the ISVD sequences used in the present technology may contain one or more of the Hallmark residues (as defined herein), such that the ISVD sequence is a suitable component of a Nanobody®, e.g., a humanized VSVD. HH or Camelization V H Including, V HH Some non-limiting examples of suitable combinations of such framework sequences will become apparent from the further disclosure herein.

[0127] It is also possible to use any suitable fragment or combination of fragments as described generally herein for immunoglobulin sequences, e.g., a fragment containing one or more CDR sequences, preferably flanked by and / or linked through one or more framework sequences; e.g., these CDR and framework sequences are in the same order as they may occur in the full-length immunoglobulin sequence from which the fragment is derived.

[0128] However, it should be noted that the present technology is not limited with respect to the origin of the ISVD sequence or the origin of the nucleotide sequence used for its expression, nor with respect to the manner in which the ISVD sequence or the nucleotide sequence is generated or obtained or generated or obtained. Thus, the ISVD sequence may be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In a specific but non-limiting embodiment, the ISVD sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, including "humanized" immunoglobulin sequences (as defined herein) (e.g. partially or fully humanized mouse or rabbit immunoglobulin sequences, particularly partially or fully humanized VHV ... HH Immunoglobulin sequences that may be used include, but are not limited to, "camelized" immunoglobulin sequences (as defined herein), as well as immunoglobulin sequences obtained by techniques such as affinity maturation (e.g., starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combination of fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to those skilled in the art; or any suitable combination of any of the above.

[0129] Likewise, the nucleotide sequence may be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence, for example a sequence isolated by PCR from a suitable naturally occurring template, for example DNA or RNA isolated from a cell, a nucleotide sequence isolated from a library (in particular an expression library), a nucleotide sequence produced by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, for example mismatch PCR), a nucleotide sequence produced by PCR using overlapping primers, or a nucleotide sequence produced using techniques for DNA synthesis known per se.

[0130] As mentioned above, the ISVD may be a Nanobody® or a suitable fragment thereof. For a general description of Nanobodies®, see the further description below as well as the prior art referenced therein. In this regard, however, this description and the prior art are not intended to limit the scope of the invention to the so-called "V H 3 Classes of Nanobodies®, namely V H It should be noted that the present invention primarily describes Nanobodies with a high degree of sequence homology to three classes of human germline sequences, e.g. DP-47, DP-51 or DP-29. However, it should be noted that the present technology in its broadest sense can be used generally with any kind of Nanobody, e.g. the so-called "V H Nanobodies® belonging to the “4th class”, i.e. V H Four classes of Nanobodies® that have a high degree of sequence homology to human germline sequences, such as DP-78, are also used.

[0131] In general, Nanobodies® (especially the (partially) humanized V HH Sequence and camelization V H Contains arrays, V HH A Nanobody® may be characterized by the presence of one or more "hallmark residues" (again, as further described herein) in one or more of the framework sequences (as described herein). Thus, in general, a Nanobody® may have the (general) structure FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 where FR1-FR4 refer to framework regions 1-4, respectively, and CDR1-CDR3 refer to complementarity determining regions 1-3, respectively, and one or more of the hallmark residues are as further defined herein.

[0132] In particular, Nanobodies® have the (general) structure FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 where FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and the framework sequences are as further defined herein.

[0133] More particularly, Nanobodies® have the (general) structure FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively: One or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering may be selected from the Hallmark residues set out in Table A-6 below.

[0134] [Table 1-1] [Table 1-2]

[0135] The present technology employs ISVDs that can bind, inter alia, to TNFα or the p19 subunit of IL-23. In the context of the present technology, "binding to" a particular target molecule has its normal meaning in the art as understood in the context of antibodies and their respective antigens.

[0136] The polypeptides of the present technology may comprise one or more ISVDs that bind to TNFα and two or more ISVDs that bind to IL-23. For example, the polypeptide may comprise one ISVD that binds to TNFα and two ISVDs that bind to the p19 subunit of IL-23.

[0137] In some embodiments, at least one ISVD can functionally block its target molecule. For example, the ISVD can block the interaction between TNFα and TNFR (TNF receptor), or can block the interaction between IL-23 and IL-23R (IL-23 receptor). In the polypeptide of the present technology, at least one ISVD can functionally block IL-23, for example, by blocking the interaction between IL-23 and IL-23R and / or inhibiting IL-23-induced release of IL-22. Thus, in one embodiment, the polypeptide of the present technology comprises one ISVD that binds to TNFα and functionally blocks TNFα, and two ISVDs that bind to IL-23, one of which can functionally block IL-23.

[0138] The ISVDs used in the present technology form part of a polypeptide of the present technology that comprises or consists of at least three ISVDs, such that the polypeptide is capable of specifically binding to TNFα and IL-23.

[0139] Thus, the target molecules of at least three ISVDs as used in the polypeptide of the present technology are TNFα and IL-23. Examples are mammalian TNFα and IL-23. In addition to human TNFα (Uniprot accession P01375) and human IL-23 (p19 subunit, Uniprot accession for IL-23A: Q9NPF7), versions from other species are also applicable to the present technology, such as TNFα and IL-23 from mouse, rat, rabbit, cat, dog, goat, sheep, horse, pig, non-human primate, such as cynomolgus monkeys (sometimes referred to as "cyno" in this specification), or camelid, such as llama or alpaca.

[0140] Specific examples of ISVDs that specifically bind to the p19 subunit of TNFα or IL-23 that can be used in the present technology are as described in Sections A-C below:

[0141] A. Binds specifically to human TNFα i. CDR1, which is an amino acid sequence of SEQ ID NO:6 or an amino acid sequence having two or one amino acid difference from SEQ ID NO:6; ii. CDR2 having the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 10; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 14. ISVDs including:

[0142] In one embodiment, the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO:6, a CDR2 that is the amino acid sequence of SEQ ID NO:10, and a CDR3 that is the amino acid sequence of SEQ ID NO:14.

[0143] An example of such an ISVD that specifically binds to human TNFα has (in addition to the CDRs as defined in the preceding section A) one or more, or all, of the framework regions as indicated for construct 6C11 in Table A-2. In one embodiment, it is an ISVD that comprises or consists of the entire amino acid sequence of construct 6C11 (SEQ ID NO:2, see Tables A-1 and A-2).

[0144] In another embodiment, the amino acid sequence of the ISVD that specifically binds to human TNFα may have greater than 90%, such as greater than 95% or greater than 99% sequence identity to SEQ ID NO:2, and the CDRs are as defined in the preceding section A. In one embodiment, the ISVD that specifically binds to TNFα comprises or consists of the amino acid sequence of SEQ ID NO:2.

[0145] If such an ISVD that specifically binds to TNFα has two or one amino acid difference in at least one CDR compared to the corresponding reference CDR sequence (item A above), the ISVD has at least half the binding affinity, or at least the same binding affinity, for human TNFα compared to construct 6C11 (sequence number 2), and the binding affinity is measured using the same method, e.g., SPR.

[0146] B. Binds specifically to the p19 subunit of human IL-23; i. a CDR1 having an amino acid sequence of SEQ ID NO:7 or an amino acid sequence having two or one amino acid difference from SEQ ID NO:7; ii. CDR2 having the amino acid sequence of SEQ ID NO:11 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO:11; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 15. ISVDs including:

[0147] In one embodiment, the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO:7, a CDR2 that is the amino acid sequence of SEQ ID NO:11, and a CDR3 that is the amino acid sequence of SEQ ID NO:15.

[0148] An example of such an ISVD that specifically binds to human IL-23 has (in addition to the CDRs as defined in the preceding section B) one or more, or all, of the framework regions as indicated for construct 119A03 / 1 in Table A-2. In one embodiment, it is an ISVD that comprises or consists of the entire amino acid sequence of construct 119A03 / 1 (SEQ ID NO:3, see Tables A-1 and A-2).

[0149] Also, in another embodiment, the amino acid sequence of the ISVD that specifically binds to human IL-23 may have greater than 90% sequence identity, such as greater than 95% or greater than 99%, to SEQ ID NO:3, and the CDRs are as defined in the preceding section B. In one embodiment, the ISVD that binds to IL-23 comprises or consists of the amino acid sequence of SEQ ID NO:3.

[0150] If such an ISVD that binds to IL-23 has two or one amino acid difference in at least one CDR compared to the corresponding reference CDR sequence (item B above), the ISVD has at least half the binding affinity, or at least the same binding affinity, to human IL-23 compared to construct 119A03 / 1 (sequence number 3), and the binding affinity is measured using the same method, e.g., SPR.

[0151] In one embodiment, an ISVD comprising a CDR2 having two or one amino acid difference from SEQ ID NO: 11 (TIESGSRTN) does not have an E to N substitution at amino acid position 3 of the CDR2 sequence and / or does not have an N to Y substitution at amino acid position 9 of the CDR2 sequence. In another embodiment, such an ISVD does not have an E to N substitution at amino acid position 3 of the CDR2 sequence and does not have an N to Y substitution at amino acid position 9 of the CDR2 sequence. In such an embodiment of an ISVD comprising a CDR2 having two or one amino acid difference from SEQ ID NO: 11 (TIESGSRTN), E is maintained as the amino acid at position 3 and / or N is maintained as the amino acid at position 9 of said CDR2 sequence. In another embodiment, both E at amino acid position 3 and N at amino acid position 9 of said CDR2 sequence are maintained. Use of an ISVD that comprises an N at amino acid position 3 of the CDR2 sequence may result in reduced amino acid sequence stability during production of the polypeptide, e.g. due to deamination, compared to the same polypeptide that does not contain an N at that amino acid position, particularly compared to the same polypeptide that contains an E at that amino acid position. Use of an ISVD that comprises a Y at amino acid position 9 of the CDR2 sequence may result in increased protein aggregation of the polypeptide compared to the same polypeptide that does not contain a Y at that amino acid position, particularly compared to the same polypeptide that contains an N at that amino acid position.

[0152] C. Binds specifically to the p19 subunit of human IL-23; i. CDR1, which is an amino acid sequence of SEQ ID NO:9 or an amino acid sequence having two or one amino acid difference from SEQ ID NO:9; ii. CDR2 having the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 13; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 17. ISVDs including:

[0153] In one embodiment, the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO:9, a CDR2 that is the amino acid sequence of SEQ ID NO:13, and a CDR3 that is the amino acid sequence of SEQ ID NO:17.

[0154] An example of such an ISVD that specifically binds to human IL-23 has (in addition to the CDRs as defined in the preceding section C) one or more, or all, of the framework regions as indicated for construct 81A12 in Table A-2. In one embodiment, it is an ISVD that comprises or consists of the entire amino acid sequence of construct 81A12 (SEQ ID NO:5, see Tables A-1 and A-2).

[0155] Also, in another embodiment, the amino acid sequence of the ISVD that specifically binds to human IL-23 may have greater than 90% sequence identity, such as greater than 95% or greater than 99%, to SEQ ID NO:5, and the CDRs are as defined in the preceding section C. In one embodiment, the ISVD that binds to IL-23 comprises or consists of the amino acid sequence of SEQ ID NO:5.

[0156] If such an ISVD that specifically binds to IL-23 has two or one amino acid difference in at least one CDR compared to the corresponding reference CDR sequence (item C above), the ISVD has at least half the binding affinity, or at least the same binding affinity, to human IL-23 compared to construct 81A12 (SEQ ID NO:5), where the binding affinity is measured using the same method, e.g., SPR.

[0157] In embodiments, each of the ISVDs as defined above under items A-C is included in a polypeptide of the present technology. Such polypeptides of the present technology that include each of the ISVDs as defined above under items A-C have at least half the binding affinity, or at least the same binding affinity, for human TNFα and human IL-23 as a polypeptide consisting of the amino acid sequence of SEQ ID NO:1, where the binding affinity is measured using the same method, e.g., SPR.

[0158] The SEQ ID NOs referenced in the above sections A-C are based on the CDR definitions according to the AbM definition (see Table A-2). It is noted that SEQ ID NOs defining the same CDRs according to the Kabat definition (see Table A-2.1) can be used in the above sections A-C as well.

[0159] Thus, unique ISVDs that specifically bind to the p19 subunit of TNFα or IL-23 that can be used in the present technology as described above using the AbM definition can also be described using the Kabat definition as described in sections A'-C' below:

[0160] A'. Binds specifically to human TNFα; i. a CDR1 having an amino acid sequence of SEQ ID NO: 122 or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 122; ii. CDR2 having the amino acid sequence of SEQ ID NO: 130 or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 130; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 138. ISVDs including:

[0161] In one embodiment, the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO:122, a CDR2 that is the amino acid sequence of SEQ ID NO:130, and a CDR3 that is the amino acid sequence of SEQ ID NO:138.

[0162] An example of such an ISVD that specifically binds to human TNFα has (in addition to the CDRs as defined in the preceding section A') one or more, or all, of the framework regions as indicated for construct 6C11 in Table A-2.1. In one embodiment, it is an ISVD that comprises or consists of the entire amino acid sequence of construct 6C11 (SEQ ID NO:2, see Tables A-1 and A-2.1).

[0163] Also, in another embodiment, the amino acid sequence of the ISVD that specifically binds to human TNFα may have greater than 90% sequence identity, such as greater than 95% or greater than 99%, to SEQ ID NO:2, and the CDRs are as defined in the preceding section A'. In one embodiment, the ISVD that specifically binds to TNFα comprises or consists of the amino acid sequence of SEQ ID NO:2.

[0164] If such an ISVD that specifically binds to TNFα has two or one amino acid difference in at least one CDR compared to the corresponding reference CDR sequence (item A' above), the ISVD has at least half the binding affinity, or at least the same binding affinity, for human TNFα compared to construct 6C11 (sequence number 2), and the binding affinity is measured using the same method, e.g., SPR.

[0165] B'. Binds specifically to the p19 subunit of human IL-23; i. CDR1, which is an amino acid sequence of SEQ ID NO: 123, or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 123; ii. CDR2 having the amino acid sequence of SEQ ID NO: 131 or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 131; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 139 or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 139. ISVDs including:

[0166] In one embodiment, the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO:123, a CDR2 that is the amino acid sequence of SEQ ID NO:131, and a CDR3 that is the amino acid sequence of SEQ ID NO:139.

[0167] An example of such an ISVD that specifically binds to human IL-23 has (in addition to the CDRs as defined in the preceding section B') one or more, or all, of the framework regions as indicated for construct 119A03 / 1 in Table A-2.1. In one embodiment, it is an ISVD that comprises or consists of the entire amino acid sequence of construct 119A03 / 1 (SEQ ID NO:3, see Tables A-1 and A-2.1).

[0168] Also, in another embodiment, the amino acid sequence of the ISVD that specifically binds to human IL-23 may have greater than 90% sequence identity, such as greater than 95% or greater than 99%, to SEQ ID NO:3, and the CDRs are as defined in the preceding section B'. In one embodiment, the ISVD that binds to IL-23 comprises or consists of the amino acid sequence of SEQ ID NO:3.

[0169] If such an ISVD that binds to IL-23 has two or one amino acid difference in at least one CDR compared to the corresponding reference CDR sequence (item B' above), the ISVD has at least half the binding affinity, or at least the same binding affinity, to human IL-23 compared to construct 119A03 / 1 (sequence number 3), and the binding affinity is measured using the same method, e.g., SPR.

[0170] In one embodiment, an ISVD comprising a CDR2 comprising or consisting of an amino acid sequence having two or one amino acid difference from SEQ ID NO: 131 (TIESGSRTNYADSVKG) does not have an E to N substitution at amino acid position 3 and / or an N to Y substitution at amino acid position 9 of said CDR2 sequence. In another embodiment, such an ISVD does not have an E to N substitution at amino acid position 3 and does not have an N to Y substitution at amino acid position 9 of said CDR2 sequence. In such an embodiment of an ISVD comprising a CDR2 comprising or consisting of an amino acid sequence having two or one amino acid difference from SEQ ID NO: 131 (TIESGSRTNYADSVKG), E is maintained as the amino acid at position 3 and / or N is maintained as the amino acid at position 9 of said CDR2 sequence. In another embodiment, both E at amino acid position 3 and N at amino acid position 9 of said CDR2 sequence are maintained. Use of an ISVD that comprises an N at amino acid position 3 of the CDR2 sequence may result in reduced amino acid sequence stability during production of the polypeptide, e.g. due to deamination, compared to the same polypeptide that does not contain an N at said amino acid position, particularly compared to the same polypeptide that contains an E at said amino acid position. Use of an ISVD that comprises a Y at amino acid position 9 of the CDR2 sequence may result in increased protein aggregation of the polypeptide compared to the same polypeptide that does not contain a Y at said amino acid position, particularly compared to the same polypeptide that contains an N at said amino acid position.

[0171] C'. Binds specifically to the p19 subunit of human IL-23; i. CDR1, which is an amino acid sequence of SEQ ID NO: 125, or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 125; ii. CDR2 having the amino acid sequence of SEQ ID NO: 133 or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 133; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 141 or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 141. ISVDs including:

[0172] In one embodiment, the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO:125, a CDR2 that is the amino acid sequence of SEQ ID NO:133, and a CDR3 that is the amino acid sequence of SEQ ID NO:141.

[0173] An example of such an ISVD that specifically binds to human IL-23 has (in addition to the CDRs as defined in the preceding section C') one or more, or all, of the framework regions as indicated for construct 81A12 in Table A-2.1. In one embodiment, it is an ISVD that comprises or consists of the entire amino acid sequence of construct 81A12 (SEQ ID NO:5, see Tables A-1 and A-2.1).

[0174] Also, in another embodiment, the amino acid sequence of the ISVD that specifically binds to human IL-23 may have greater than 90% sequence identity, such as greater than 95% or greater than 99%, to SEQ ID NO:5, and the CDRs are as defined in the preceding section C'. In one embodiment, the ISVD that binds to IL-23 comprises or consists of the amino acid sequence of SEQ ID NO:5.

[0175] If such an ISVD that specifically binds to IL-23 has two or one amino acid difference in at least one CDR compared to the corresponding reference CDR sequence (item C' above), the ISVD has at least half the binding affinity, or at least the same binding affinity, to human IL-23 compared to construct 81A12 (SEQ ID NO:5), and the binding affinity is measured using the same method, e.g., SPR.

[0176] In embodiments, each of the ISVDs as defined under paragraphs A'-C' above is included in a polypeptide of the present technology. Such polypeptides of the present technology that include each of the ISVDs as defined under paragraphs A'-C' above have at least half the binding affinity, or at least the same binding affinity, for human TNFα and human IL-23 as a polypeptide consisting of the amino acid sequence of SEQ ID NO:1, where the binding affinity is measured using the same method, e.g., SPR.

[0177] The percentage of "sequence identity" between a first amino acid sequence and a second amino acid sequence can be calculated by dividing the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at corresponding positions in the second amino acid sequence by the total number of amino acid residues in the first amino acid sequence and multiplying by 100%, with each deletion, insertion, substitution or addition of amino acid residues in the second amino acid sequence compared to the first amino acid sequence being considered as a difference at a single amino acid residue (i.e. at a single position).

[0178] Typically, for purposes of determining the percentage of "sequence identity" between two amino acid sequences according to the calculation method outlined above, the amino acid sequence having the greatest number of amino acid residues is referred to as the "first" amino acid sequence and the other amino acid sequence is referred to as the "second" amino acid sequence.

[0179] "Amino acid difference" as used herein refers to a deletion, insertion or substitution of a single amino acid residue compared to a reference sequence. In one embodiment, an "amino acid difference" is a substitution.

[0180] In one embodiment, the amino acid substitution is a conservative substitution. Such a conservative substitution is a substitution in which one amino acid residue in the following groups (a) to (e) is replaced with another amino acid residue in the same group: (a) small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, non-polar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0181] In one embodiment, conservative substitutions are as follows: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln, or Glu; Met to Leu, Tyr or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile, or Leu.

[0182] 5.2 Specificity The terms "specificity", "specifically binds" or "specific binding" refer to the number of different target molecules, e.g., antigens, from the same organism to which a particular binding unit, e.g., an ISVD, can bind with sufficiently high affinity (see below). "Specificity", "specifically binds" or "specific binding" are used interchangeably herein with "selectivity", "selectively binds" or "selective binding". Binding units, e.g., ISVDs, specifically bind to their designated targets.

[0183] The specificity / selectivity of a binding unit can be determined based on affinity. Affinity describes the strength or stability of a molecular interaction. Affinity is commonly given by the KD, or dissociation constant, which has units of mol / liter (or M). Affinity can also be expressed as the association constant, KA, which is equal to 1 / KD, (mol / liter) -1 (or M -1 )

[0184] Affinity is a measure of the strength of binding between a binding site on a moiety and a target molecule: the lower the value of KD, the greater the binding strength between the target molecule and the targeting moiety.

[0185] Typically, the binding units used in the present technology, such as ISVDs, are 10 -5 ~10 -12 Moles / liter or less, 10 -7 ~10 -12 moles / liter or less, or 10 -8 ~10 -12 Dissociation constant (KD) in moles / liter (i.e. 10 5 ~10 12 Liters / mole or higher, 10 7 ~10 12 liters / mole or higher, or 10 8 ~10 12 They bind to their targets with an association constant (KA) in liters / mol.

[0186] 10 -4 Any K value higher than mol / liter (or 10 4 Any K A value lower than 1.0 liter / mol is generally considered to indicate nonspecific binding.

[0187] The KD for a biological interaction that is considered specific, such as the binding of an immunoglobulin sequence to an antigen, is typically greater than 10 -5 Mole / L (10000nM or 10μM) to 10 -12in the range of moles per liter (0.001 nM or 1 pM) or less.

[0188] Thus, specific / selective binding can be measured using the same measurement method, e.g., SPR, where the binding unit (or a polypeptide containing it) is 10 -5 ~10 -12 Binds to TNFα and / or IL-23 with a K value of 10 moles / liter or less -4 This may mean that the polypeptide binds to a related cytokine with a KD value higher than 10 moles per liter. An example of a related cytokine of IL-23 is IL-12, which shares a p40 subunit with IL-23. An example of a related cytokine of TNFα is the TNF superfamily members FASL, TNFβ, LIGHT, TL-1A, and RANKL. Therefore, in an embodiment of the present technology, at least one ISVD contained in the polypeptide is 10 -5 ~10 -12 Binds to (human) TNFα with a K value of 10 moles / liter or less -4 Binds to FASL, TNFβ, LIGHT, TL-1A, and RANKL of the same species with KD values ​​higher than 10 moles / liter, and at least two ISVDs contained in the polypeptide -5 ~10 -12 Binds to IL-23 with a K value of 10 moles / liter or less -4 It binds to IL-12 of the same species with a higher KD value than IL-12 per mole per liter.

[0189] As such, the polypeptides of the present technology have at least half the binding affinity, or at least the same binding affinity, for human TNFα and human IL-23 as a polypeptide consisting of the amino acid sequence of SEQ ID NO:1, where the binding affinity is measured using the same methods, e.g., SPR.

[0190] Specific binding to a particular target from a particular species does not exclude that the binding unit can also specifically bind to a similar target from a different species.For example, specific binding to human TNFα does not exclude that the binding unit or a polypeptide comprising it can also specifically bind to TNFα from cynomolgus monkeys.Similarly, specific binding to human IL-23, for example, does not exclude that the binding unit or a polypeptide comprising it can also specifically bind to IL-23 from cynomolgus monkeys ("cyno").

[0191] The specific binding of a binding unit to its designated target can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, as well as their different variants known per se in the art; as well as other techniques mentioned herein.

[0192] The dissociation constant may be an actual or apparent dissociation constant, as will be apparent to one of skill in the art. Methods for determining dissociation constants will be apparent to one of skill in the art, and include, for example, the techniques described below. In this regard, -4 moles / liter or 10 -3 Higher than moles / liter (e.g. 10 -2 It is also clear that it may not be possible to measure the dissociation constant (in moles / liter). In some cases, as will also be clear to those skilled in the art, the (actual or apparent) dissociation constant may be calculated based on the (actual or apparent) association constant (KA) according to the relationship [KD=1 / KA].

[0193] The affinity of a molecular interaction between two molecules can be measured via different techniques known per se, for example the well-known Surface Plasmon Resonance (SPR) biosensor technique (see for example Ober et al., 2001, Intern. Immunology 13:1551-1559). The term "surface plasmon resonance", as used herein, refers to an optical phenomenon that allows the analysis of real-time biomolecular specific interactions by detection of changes in protein concentration in a biosensor matrix, where one molecule is immobilized on a biosensor chip and the other molecule is passed under flow conditions over the immobilized molecule to measure k on , k off Measurements and therefore K D (or K A ) values ​​are obtained. This can be done, for example, using the well-known BIAcore® system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, NJ). For further explanation, see Jonsson et al. (1993, Ann. Biol. Clin. 51:19-26), Jonsson et al. (1991 Biotechniques 11:620-627), Johnsson et al. (1995, J. Mol. Recognit. 8:125-131), and Johnnson et al. (1991, Anal. Biochem. 198:268-277).

[0194] Another well-known biosensor technique for determining the affinity of biomolecular interactions is Biolayer Interferometry (BLI) (see, for example, Abdiche et al., 2008, Anal. Biochem. 377:209-217). The term "Biolayer Interferometry" or "BLI" as used herein refers to a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and a layer of immobilized proteins on a biosensor chip (signal beam). Changes in the number of molecules bound to the biosensor chip cause a shift in the interference pattern, reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the biosensor chip surface. Interactions can be measured in real time, so that association and dissociation rates as well as affinities can be determined. BLI can be performed, for example, using the well-known Octet® system (ForteBio, a division of Pall Life Sciences, Menlo Park, USA).

[0195] Alternatively, affinity can be measured in a Kinetic Exclusion Assay (KinExA) (see, e.g., Drake et al., 2004, Anal. Biochem., 328:35-43) using the KinExA® platform (Sapidyne Instruments Inc, Boise, USA). The term "KinExA" as used herein refers to a solution-based method for measuring the true equilibrium binding affinity and kinetics of unmodified molecules. An equilibrated solution of 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 achieved using a fluorescently labeled protein that binds to the antibody (or antigen).

[0196] The GYROLAB® Immunoassay System provides a platform for automated bioanalysis and rapid sample turnaround (Fraley et al., 2013, Bioanalysis 5:1765-74).

[0197] 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 peptidic linkers, which 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. The increased in vivo half-life means, for example, that the polypeptide has an increased half-life in a mammalian, for example, human subject, after administration. The half-life can be expressed, for example, as t1 / 2 beta.

[0198] The type of group, residue, moiety or binding unit is generally not restricted and can be selected, for example, from the group consisting of polyethylene glycol molecules, serum proteins or fragments thereof, binding units capable of binding to serum proteins, Fc portions, and small proteins or peptides capable of binding to serum proteins.

[0199] More particularly, the one or more other groups, residues, moieties or binding units that provide a polypeptide with increased half-life can be selected from the group consisting of binding units that can bind to serum albumin, such as human serum albumin, or serum immunoglobulin, such as IgG. In one embodiment, the one or more other groups, residues, moieties or binding units that provide a polypeptide with increased half-life are binding units that can bind to human serum albumin. In one embodiment, the binding unit is an ISVD.

[0200] For example, WO2004 / 041865 describes ISVDs that bind to serum albumin (particularly human serum albumin) that can be linked to other proteins (e.g. one or more other ISVDs that bind to a desired target) to increase the half-life of the protein.

[0201] International application WO2006 / 122787 describes a number of ISVDs for (human) serum albumin. These ISVDs include the ISVD called Alb-1 (SEQ ID NO: 52 in WO2006 / 122787) and its humanized variants, such as Alb-8 (SEQ ID NO: 62 in WO2006 / 122787). They can also be used to extend the half-life of therapeutic proteins and polypeptides and other therapeutic entities or moieties.

[0202] Furthermore, WO2012 / 175400 describes a further improved version of Alb-1, called Alb-23.

[0203] In one embodiment, the polypeptide comprises a serum albumin binding moiety selected from Alb-1, Alb-3, Alb-4, Alb-5, Alb-6, Alb-7, Alb-8, Alb-9, Alb-10 (WO2006 / 122787) and Alb-23. In one embodiment, the serum albumin binding moiety is Alb-8 or Alb-23 or a variant thereof, as set out on pages 7-9 of WO2012 / 175400. In one embodiment, the serum albumin binding moiety is selected from the albumin binders described in WO2012 / 175741, WO2015 / 173325, WO2017 / 080850, WO2017 / 085172, WO2018 / 104444, WO2018 / 134235, and WO2018 / 134234. Some serum albumin binders are also shown in Table A-4. In one embodiment, further components of the polypeptide of the present technology are as described in item D below:

[0204] D. binds to human serum albumin i. CDR1, which is an amino acid sequence of SEQ ID NO:8 or an amino acid sequence having two or one amino acid difference from SEQ ID NO:8; ii. CDR2 having the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having 2 or 1 amino acid difference from SEQ ID NO: 12; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 16. ISVDs including:

[0205] In one embodiment, the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO:8, a CDR2 that is the amino acid sequence of SEQ ID NO:12, and a CDR3 that is the amino acid sequence of SEQ ID NO:16.

[0206] An example of such an ISVD that binds to human serum albumin has (in addition to the CDRs as defined in the preceding section D) one or more, or all, of the framework regions as indicated for construct ALB23002 in Table A-2. In one embodiment, it is an ISVD that comprises or consists of the entire amino acid sequence of construct ALB23002 (SEQ ID NO:4, see Tables A-1 and A-2).

[0207] Item D can also be written using the Kabat definition as follows: D'. Binds to human serum albumin i. CDR1, which is an amino acid sequence of SEQ ID NO: 124, or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 124; ii. CDR2 having the amino acid sequence of SEQ ID NO: 132 or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 132; and iii. A CDR3 having the amino acid sequence of SEQ ID NO: 140 or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 140. ISVDs including:

[0208] In one embodiment, the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO:124, a CDR2 that is the amino acid sequence of SEQ ID NO:132, and a CDR3 that is the amino acid sequence of SEQ ID NO:140.

[0209] An example of such an ISVD that binds to human serum albumin has (in addition to the CDRs as defined in the preceding section D') one or more, or all, of the framework regions as indicated for construct ALB23002 in Table A-2.1. In one embodiment, it is an ISVD that comprises or consists of the entire amino acid sequence of construct ALB23002 (SEQ ID NO: 4, see Tables A-1 and A-2.1).

[0210] Also, in another embodiment, the amino acid sequence of the ISVD that binds human serum albumin may have greater than 90% sequence identity, such as greater than 95% or greater than 99%, to SEQ ID NO:4, and the CDRs are as defined in the preceding items D or D'. In one embodiment, the ISVD that binds human serum albumin comprises or consists of the amino acid sequence of SEQ ID NO:4.

[0211] If such an ISVD that binds to human serum albumin has two or one amino acid difference in at least one CDR compared to the corresponding reference CDR sequence (items D or D' above), the ISVD has at least half the binding affinity to human serum albumin compared to construct ALB23002 (sequence number 4), or at least the same binding affinity, where the binding affinity is measured using the same method, e.g., SPR.

[0212] In one embodiment, when such an ISVD that binds to human serum albumin has a C-terminal position, it exhibits a C-terminal extension, such as a C-terminal alanine (A) or glycine (G) extension. In one embodiment, such an ISVD is selected from SEQ ID NOs: 33, 34, 36, 38, 39, 40, 41, 42, 43, and 45 (see Table A-4 below). In another embodiment, the ISVD that binds to human serum albumin has a position other than the C-terminal position (i.e., it is not the C-terminal ISVD of the polypeptide of the present technology). In one embodiment, such an ISVD is selected from SEQ ID NOs: 4, 31, 32, 35, and 37 (see Table A-4 below).

[0213] 5.4 Nucleic acid molecules Nucleic acid molecules encoding the polypeptides of the present technology are also provided.

[0214] A "nucleic acid molecule" (used interchangeably with "nucleic acid") is a chain of nucleotide monomers linked together via a phosphate backbone to form a nucleotide sequence. Nucleic acids can be used for transformation / transfection of host cells or host organisms, e.g., for expression and / or production of polypeptides. Suitable hosts or host cells for production purposes will be apparent to the skilled artisan and may be, for example, any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism. Hosts or host cells comprising a nucleic acid encoding a polypeptide of the present technology are also encompassed by the present technology.

[0215] The nucleic acid may be, for example, DNA, RNA, or a hybrid thereof, and may also include (e.g., chemically) modified nucleotides such as PNA. It may be single-stranded or double-stranded. In one embodiment, it is in the form of double-stranded DNA. For example, the nucleotide sequence of the present technology may be genomic DNA, cDNA.

[0216] The nucleic acid of the present technology can be produced or obtained in a manner known per se and / or can be isolated from a suitable natural source.Nucleotide sequences encoding naturally occurring (poly)peptides can be subjected to, for example, site-directed mutagenesis, thereby providing nucleic acid molecules encoding polypeptides with sequence variations.Also, as will be clear to those skilled in the art, several nucleotide sequences, for example at least one nucleotide sequence encoding a targeting moiety and, for example, the nucleic acid encoding one or more linkers, can be linked in a suitable manner to produce nucleic acid.

[0217] Techniques for generating nucleic acids will be apparent to those of skill in the art and may include, but are not limited to, automated DNA synthesis; site-directed mutagenesis; the combination of two or more naturally occurring and / or synthetic sequences (or two or more portions thereof), the introduction of mutations that lead to the expression of truncated expression products; the introduction of one or more restriction sites (e.g., to create cassettes and / or regions that are easily digestible and / or ligatable using suitable restriction enzymes), and / or the introduction of mutations by PCR reactions using one or more "mismatched" primers.

[0218] 5.5 Vectors Also provided is a vector that comprises the nucleic acid molecule that codes for the polypeptide of the present technology.Vector, as used herein, is a suitable vehicle for carrying genetic material into cells.Vector includes naked nucleic acid, such as plasmid or mRNA, or nucleic acid embedded in larger structure, such as liposome or virus vector.

[0219] In some embodiments, a vector comprises at least one nucleic acid, optionally linked to one or more regulatory elements, such as one or more suitable promoters, enhancers, terminators, etc. In one embodiment, the vector is an expression vector, i.e. a vector suitable for expressing an encoded polypeptide or construct under suitable conditions, e.g., when the vector is introduced into a (e.g., human) cell. DNA-based vectors include the presence of elements for transcription (e.g., promoters and polyA signals) and translation (e.g., Kozak sequences).

[0220] In one embodiment, in a vector, the at least one nucleic acid and the regulatory element are "operably linked" to each other, which generally means that they are in a functional relationship with each other. For example, a promoter is considered to be "operably linked" to a coding sequence when it can initiate or otherwise control / regulate the transcription and / or expression of the coding sequence (in which case the coding sequence should be understood as being "under the control" of the promoter). Generally, when two nucleotide sequences are operably linked, they are in the same orientation and usually also in the same reading frame. They are also usually essentially contiguous, although this may not be required.

[0221] In one embodiment, any regulatory elements of the vector are such that they are capable of providing their intended biological function in the intended host cell or host organism.

[0222] For example, a promoter, enhancer or terminator should be "operable" in the intended host cell or host organism, by which is meant, for example, that said promoter should have the ability to initiate or otherwise control / regulate the transcription and / or expression of a nucleotide sequence, e.g., a coding sequence, to which it is operably linked.

[0223] 5.6 Composition The present technology also provides a composition comprising at least one polypeptide of the present technology, at least one nucleic acid molecule encoding the polypeptide of the present technology, or at least one vector comprising such a nucleic acid molecule.The composition may be a pharmaceutical composition.The composition may further comprise at least one pharma- ceutically acceptable carrier, diluent or excipient and / or adjuvant, and may optionally comprise one or more additional pharma- ceutically active polypeptides and / or compounds.

[0224] 5.7 Host organisms The present technology also relates to host cells or host organisms comprising a polypeptide of the present technology, a nucleic acid encoding a polypeptide of the present technology, and / or a vector comprising a nucleic acid molecule encoding a polypeptide of the present technology.

[0225] Suitable host cells or host organisms are clear to those skilled in the art, for example, any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism. Specific examples include HEK293 cells, CHO cells, Escherichia coli or Pichia pastoris. In one embodiment, the host is Pichia pastoris.

[0226] 5.8 Methods and Uses of Polypeptides The present technology also provides a method for producing the polypeptide of the present technology. The method may include transforming / transfecting a host cell or host organism with a nucleic acid encoding the polypeptide, expressing the polypeptide in the host, optionally followed by one or more isolation and / or purification steps. In particular, the method includes: a) expressing a nucleic acid sequence encoding the polypeptide in a suitable host cell or host organism or in another suitable expression system; optionally followed by: b) isolating and / or purifying the polypeptide may include:

[0227] Suitable host cells or host organisms for production purposes will be clear to the skilled artisan and may be, for example, any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism. Specific examples include HEK293 cells, CHO cells, E. coli or Pichia pastoris. In one embodiment, the host is Pichia pastoris.

[0228] The polypeptides of the present technology, the nucleic acid molecules or vectors as described, or compositions comprising the polypeptides, nucleic acid molecules or vectors of the present technology are useful as pharmaceuticals.

[0229] Thus, the present technology provides a polypeptide of the present technology, a nucleic acid molecule or vector as described, or a composition comprising the polypeptide, nucleic acid molecule or vector of the present technology for use as a medicament.

[0230] Also provided are polypeptides of the present technology, nucleic acid molecules or vectors as described, or compositions comprising the polypeptides, nucleic acid molecules or vectors of the present technology for use in treatment (prophylactic and / or therapeutic).

[0231] Also provided are polypeptides of the present technology, nucleic acid molecules or vectors as described, or compositions comprising the polypeptides, nucleic acid molecules or vectors of the present technology for use in the treatment (prophylactic and / or therapeutic) of autoimmune or inflammatory diseases.

[0232] Also provided are polypeptides of the present technology, nucleic acid molecules or vectors as described, or compositions comprising the polypeptides, nucleic acid molecules or vectors of the present technology for use in the treatment (prophylactic and / or therapeutic) of inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, psoriasis, psoriatic arthritis and hidradenitis suppurativa.

[0233] Further provided is a method of treating (prophylactic and / or therapeutic) an autoimmune or inflammatory disease, said method comprising administering to a subject in need thereof a pharma- ceutically active amount of a polypeptide of the present technology, a nucleic acid molecule or vector as described, or a composition comprising the polypeptide, nucleic acid molecule or vector of the present technology.

[0234] Further provided are methods of treating (prophylactic and / or therapeutic) inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, psoriasis, psoriatic arthritis and hidradenitis suppurativa, comprising administering to a subject in need thereof a pharma- ceutically active amount of a polypeptide of the present technology, a nucleic acid molecule or vector as described, or a composition comprising the polypeptide, nucleic acid molecule or vector of the present technology.

[0235] Further provided is the use of a polypeptide of the present technology, a nucleic acid molecule or vector as described, or a composition comprising the polypeptide, nucleic acid molecule or vector, in the manufacture of a pharmaceutical composition for treating an autoimmune or inflammatory disease.

[0236] There is further provided the use of a polypeptide of the present technology, a nucleic acid molecule or vector as described, or a composition comprising the polypeptide, nucleic acid molecule or vector, in the manufacture of a pharmaceutical composition for treating inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, psoriasis, psoriatic arthritis or hidradenitis suppurativa.

[0237] The inflammatory bowel disease may be, for example, Crohn's disease or ulcerative colitis.

[0238] "Subject" as referred to in the context of the present technology can be any animal. In one embodiment, the subject is a mammal. Among mammals, a distinction can be made between human and non-human mammals. Non-human animals can be, for example, companion animals (e.g., dogs, cats), livestock animals (e.g., bovine, equine, ovine, caprine, or porcine), or animals commonly used for research purposes and / or to produce antibodies (e.g., mice, rats, rabbits, cats, dogs, goats, sheep, horses, pigs, non-human primates, such as cynomolgus monkeys, or camelids, such as llamas or alpacas).

[0239] In the context of prophylactic and / or therapeutic purposes, the subject can be any animal, more particularly any mammal. In one embodiment, the subject is a human subject.

[0240] Substances or compositions, including polypeptides, nucleic acid molecules and vectors, can be administered to a subject by any suitable route of administration, for example enteral (e.g., oral or rectal) or parenteral (e.g., epicutaneous, sublingual, buccal, nasal, intraarticular, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, transdermal, or transmucosal) administration. In one embodiment, the substances are administered parenterally, for example intramuscular, subcutaneous, or intradermal administration. In one embodiment, subcutaneous administration is used.

[0241] An effective amount of a polypeptide, nucleic acid molecule or vector as described, or a composition comprising the polypeptide, nucleic acid molecule or vector, can be administered to a subject to provide the intended therapeutic result.

[0242] One or more doses can be administered. If more than one dose is administered, the doses can be administered at suitable intervals to maximize the effect of the polypeptide, composition, nucleic acid molecule or vector.

[0243] [Table 2]

[0244] [Table 3]

[0245] [Table 4]

[0246] [Table 5]

[0247] [Table 6-1] [Table 6-2]

[0248] [Table 7]

[0249] 6. Working Example 6.1 Example 1: Generation of polyspecific ISVD constructs Identification of the ISVD-containing polypeptide F027500069 (SEQ ID NO: 1) that binds to TNFα and IL-23 identifies anti-TNFα V HH Building blocks (TNF06C11 (WO2017 / 081320), TNF01C02 (WO2015 / 173325, SEQ ID NO: 327) and VHH#3E (WO2004 / 041862, SEQ ID NO: 4)), anti-IL-23p19 V HH Building blocks (23IL37D05, 23IL119A03 and 23IL81A12 (WO2009 / 068627)) and anti-HSA V HHThis resulted from a data-driven multispecific engineering and formatting campaign that included building blocks based on the building block ALB23002 (WO2017085172, SEQ ID NO: 10). Different positions / orientations of building blocks and different linker lengths (9GS vs. 35GS) were applied, which proved to be essential for different parameters (potency, cross-reactivity, expression, etc.). Potency in this context refers to the inhibition of IL-23-induced SIE promoter activation in vitro, in addition to the inhibition of TNFα-induced NFκB activation in vitro as assayed in Example 6 and the inhibition of IL-23-induced mIL-22 production ex vivo as assayed in Examples 7 and 8.

[0250] A panel of 38 constructs (Table 1) was transformed in Pichia pastoris for small-scale production. Induction of ISVD construct expression was performed by stepwise addition of methanol. Clarified medium into which the ISVD constructs were secreted 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.

[0251] Some constructs showed impaired potency depending on the linker length and the relative position of the ISVD building block. For example: the potency of bivalent VHH#3E against cynomolgus TNFα was strongly impaired when linked with a short 9GS linker. Another example is that the position of the anti-IL-23 ISVD building block, 37D05, in the multispecific construct was essential for maximum potency.

[0252] [Table 8-1] [Table 8-2]

[0253] The large panel was subsequently trimmed down to a panel of four multispecific constructs consisting of ISVD constructs F027500069, F027500093, F027500095 and F027500096, which were found to be potent against both targets (human and cynomolgus monkey) and contained the potential for high expression levels based on preliminary yield estimates.

[0254] A larger scale 2L production in Pichia pastoris was performed to determine the expression yields as well as to evaluate the biophysical properties and reactivity of existing antibodies. Table 2 shows that a specific orientation of the building blocks was required to obtain high expression levels in Pichia pastoris. The expression yields obtained from 5 ml cultures for the four formatted ISVD constructs with the same building blocks but different orientations and different linker lengths clearly indicate that ISVD 6C11 requires an N-terminal position for good expression. This was supported by 2L fermentations of F027500069 and F027500070, in which the ISVD construct with N-terminal 6C11 (F027500069) reached a titer of 6.4 g / L, which was 3.2-fold higher than F027500070 with C-terminal 6C11.

[0255] [Table 9]

[0256] Table 3 and Example 9 show that the reactivity of pre-existing antibodies is driven by the composition and valency of each ISVD construct.

[0257] [Table 10]

[0258] Finally, ISVD construct F027500069 was selected based on potency, reduced binding to existing antibodies, excellent expression levels and CMC characteristics, and low viscosity of 3.3 cP at a concentration of 100 mg / mL and 6.4 cP at 146 mg / mL in defined buffer.

[0259] 6.2 Example 2: Binding Affinities of Multispecific ISVD Constructs for TNFα, IL-23, and Serum Albumin The equilibrium dissociation constants (K D Affinities, expressed as affinity per unit time (A), were quantified by in-solution affinity measurements on a Gyrolab xP Workstation (Gyros).

[0260] K D Under control measurements, serial dilutions of TNFα or IL-23 (in the range of 1 μM to 0.1 pM) or serum albumin (in the range of 10 μM to 1 pM) and a fixed amount of F027500069 (50 pM for TNFα, 20 pM or 12.5 pM for IL-23, 1 nM for serum albumin) were mixed and allowed to interact and incubated for 24 or 48 h (for IL-23 and TNFα) or 2 h (for serum albumin) to reach equilibrium.

[0261] Under receptor-controlled measurements, serial dilutions of TNFα or IL-23 (in the range of 1 μM to 0.1 pM) or serum albumin (in the range of 10 μM to 1 pM) and a fixed amount of F027500069 (5 nM for TNFα, 1.25 nM for IL-23, and 50 nM for serum albumin) were mixed and allowed to interact and incubated for 24 or 48 h (for IL-23 and TNFα) or 2 h (for serum albumin) to reach equilibrium.

[0262] Biotinylated human TNFα / IL-23 / serum albumin was captured in a Gyrolab Bioaffy 1000 CD microstructure containing a column of beads and used as a molecular probe to capture free F027500069 from the equilibration solution. When a mixture of TNFα / IL-23 / serum albumin and F027500069 (containing free TNFα / IL-23 / serum albumin, free F027500069 and TNFα / IL-23 / serum albumin-F027500069 complex) was flowed through the beads, a small percentage of free F027500069 was captured, which is proportional to the free ISVD concentration. Fluorescently labeled anti-V HH Antibody was then injected to label any captured F027500069 and the change in fluorescence was determined after rinsing to remove excess fluorescent probe. Gyrolab Analysis software was used to fit the dilution series and determine the K D and receptor control curves were analyzed to determine K D value was determined.

[0263] The results (Table 4) demonstrate that the multispecific ISVD constructs bind with high affinity to human / cynomolgus IL-23 and human / cynomolgus TNFα.

[0264] [Table 11]

[0265] 6.3 Example 3: Multispecific ISVD constructs that bind membrane-bound TNFα Binding of F027500069 to membrane-bound TNFα was demonstrated using flow cytometry in human membrane TNFα-expressing HEK293H cells and in activated CD4+ cells isolated from PBMCs and stimulated with PMA and ionomycin (data shown for TNFα-expressing HEK293H cells). Briefly, cells were cultured at 1×10 4Cells were seeded at a density of 1000 cells / well and incubated with a dilution series of F027500069 starting at 100 nM down to 0.5 pM for 1 h at 4 °C. In parallel, cells were fixed with 4% paraformaldehyde and 0.1% glutaraldehyde in PBS before seeding (to increase detection of membrane-bound TNFα) and incubated with a dilution series of ISVD for 1 h at 4 °C or for 24 h at room temperature. Cells were washed three times and then stained with anti-V HH Cells were incubated with mAb for 30 min at 4°C, washed again and incubated with goat anti-mouse PE-labeled antibody for 30 min at 4°C. Samples were washed and resuspended in FACS buffer (D-PBS with 10% FBS and 0.05% sodium azide supplemented with 5 nM TOPRO3). Cell suspensions were then analyzed on an iQuescreener. EC50 values ​​were calculated using GraphPad Prism. The EC50 values ​​of F027500069 were in the same range for live and fixed cells after 1 h of incubation, but fixation of cells resulted in higher expression levels of TNFα on the membrane (Table 5). Equilibrium binding was reached after 24 h of incubation with a concomitant 6.6-fold improvement in EC50.

[0266] [Table 12]

[0267] 6.4 Example 4: Multispecific ISVD constructs that selectively bind TNFα and IL-23 Absence of binding to TNFα and IL-23 related human cytokines was assessed via SPR. hIL-12 was tested as it shares a p40 subunit with IL-23. TNF superfamily members human FASL, TNFβ, LIGHT, TL-1A, and RANKL were tested as related cytokines for TNFα.

[0268] Targets were immobilized using amine coupling at 10 μg / mL for 600 seconds, followed by a 420 second injection of EDC / NHS for activation and a 420 second injection of 1M ethanolamine HCl for deactivation (Sierra Sensors Amine Coupling Kit II, Cat. No. ACK-001-025). The flow rates for activation, deactivation and ligand injection were set at 10 μl / min. The pH of the 10 mM acetate immobilization buffer was selected by subtracting approximately 1.5 from the pI of each ligand.

[0269] Next, 1 μM of F027500069 was injected for 2 min and allowed to dissociate for 900 s at a flow rate of 45 μL / min. 1×HBS-EP+ pH 7.4 was used as the running buffer. As positive controls, 0.2 μM α-huIL-12 Ab and 0.5 μM α-huFASL Ab, 0.5 μM α-huTNFβ Ab, 0.5 μM α-huLIGHT Ab, 0.5 μM α-huTL-1A Ab, and 0.5 μM α-huRANKL V were used. HH Interaction between F027500069 and the positive control with the immobilized target was measured by detection of an increase in the refractory index that occurs as a result of a mass change on the chip upon binding.

[0270] All positive controls bound to their respective targets. No binding of F027500069 to human IL-12, FASL, TNFβ, LIGHT, TL-1A, or RANKL was detected.

[0271] 6.5 Example 5: Simultaneous Binding of Multispecific ISVD Constructs to hIL-23 and hTNFα Using a ProteOn XPR36 instrument, we determined whether F027500069 could bind hTNFα and hIL-23 simultaneously. To this end, HSA was immobilized on a GLC ProteOn sensor chip via amine coupling. 100 nM of F027500069 was injected over the HSA surface at 10 μl / min for 2 min to capture the ISVD via the ALB23002 building block. This was followed by injection of either 100 nM of hIL-23, hTNFα or hOX40L, or a mixture of 100 nM IL-23 + 100 nM TNFα, 100 nM IL-23 + 100 nM OX40L or 100 nM TNFα + 100 nM IL-13 at a flow rate of 10 μl / min for 2 min, followed by a 600 s dissociation step. The HSA surface was regenerated with a 2 min injection of HCl (100 mM) at 45 μl / min. The sensorgrams ( FIG. 1 ) demonstrate that F027500069 can simultaneously bind human IL-23 and human TNFα, as shown by the increase in response units: an increase of about 500 RU from TNFα alone, an increase of about 880 RU from IL-23 alone, and an increase of about 1300 RU for the IL-23 and TNFα mixture.

[0272] 6.6 Example 6: Inhibition of TNFα-induced NFkB activation in vitro by multispecific ISVD constructs HEK293_NFkB-NLucP cells are TNF receptor expressing cells stably transfected with a reporter construct encoding Nano luciferase under the control of an NFkB-dependent promoter. Incubation of cells with soluble human and cynomolgus TNFα resulted in NFκB-mediated Nano luciferase gene expression. Nano luciferase luminescence was measured using Nano-Glo luciferase substrate mixed with lysis buffer at a ratio of 1:50 added to the cells. Samples were mixed for 5 min on a shaker to obtain complete lysis.

[0273] Glo response™ HEK293_NFkB-NLucP cells were seeded at 20000 cells / well in regular growth medium in white tissue culture (TC) treated 96-well plates with clear bottom. A dilution series of F027500069 or a reference compound (anti-TNFα reference mAb) was added to 25 pM human or 70 pM cynomolgus TNFα and incubated with the cells for 5 hours at 37° C. in the presence of 30 μM HSA.

[0274] F027500069 inhibited human and cynomolgus TNFα-induced NFκB activation in a concentration-dependent manner with IC50 of 38.8 pM (human TNFα) and 128 pM (cynomolgus TNFα), comparable to the anti-TNFα reference mAb (Table 6, Figure 2).

[0275] [Table 13]

[0276] 6.7 Example 7: Inhibition of IL-23-induced mIL-22 production ex vivo by multispecific ISVD constructs Human (and cynomolgus) IL-23 stimulated mIL-17 and mIL-22 secretion from mouse splenocytes (Aggarwal et al., 2003, J. Biol. Chem. 278(3):1910-4). F027500069 was demonstrated to block IL-23-induced expression of mIL-22 ex vivo. Spleens of five C57BL / 6 mice were removed, splenocytes harvested, and single cell suspensions were prepared. Cells were cultured in the presence of 20 ng / ml recombinant mIL-2 and seeded at 400,000 cells / well in 96-well flat-bottom plates. Serial dilutions of F027500069 or reference compounds (anti-hIL-23 Reference mAb1 and anti-hIL-23 Reference mAb2) were preincubated with recombinant hIL-23 (36 pM) or recombinant IL-23 from cynomolgus monkeys (36 pM) in culture medium at room temperature for 30 min and then incubated with splenocytes in the presence of 30 μM HSA for an additional 3 days at 37° C. Supernatants were collected and levels of mIL-22 were measured using ELISA.

[0277] The results shown in Table 7 demonstrate that F027500069 inhibited hIL-23 and cynomolgus IL-23-induced mIL-22 production in a concentration-dependent manner with an IC50 of 43 pM (human IL-23) and 31 pM (cynomolgus IL-23). ​​The inhibition was more potent than that by the reference compounds anti-hIL-23 Ref mAb1 and anti-hIL-23 Ref mAb2.

[0278] 6.8 Example 8: Inhibition of IL-23-Induced SIE Promoter Activation In Vitro by Multispecific ISVD Constructs Glo response™ HEK293_human IL-23R / IL-12Rb1-Luc2P cells were stably transfected with a reporter construct containing the luciferase gene under the control of an SIE-responsive promoter. Additionally, these cells constitutively overexpress both subunits of the human IL-23 receptor, i.e., IL-12Rb1 and IL-23R. Upon induction of these cells with IL-23, the luciferase reporter protein is expressed, which is quantified based on its enzymatic activity by addition of the substrate, 5'-fluoroluciferin (Bio-Glo™ Luciferase Assay System).

[0279] Cells were cultured in regular growth medium and seeded at 15000 cells / well in 96-well white tissue culture treated plates with clear bottom. Serial dilutions of F027500069 or reference compounds (anti-hIL-23 ref mAb1 and anti-hIL-23 ref mAb2) were added to the cells, followed by recombinant hIL-23 (10 pM) or cynomolgus IL-23 (40 pM). Cells were incubated for 4 h 15 min at 37°C in the presence of 30 μM HSA. Bio-Glo was then added to the cells in each well and luciferase luminescence was measured. F027500069 inhibited human and cynomolgus IL-23-dependent signaling with IC50s of 250 pM and 323 pM, respectively (Table 7 and Figure 3).

[0280] [Table 14]

[0281] 6.9 Example 9: Multispecific ISVD Constructs that Bind to Existing Antibodies The reactivity of pre-existing antibodies of ISVD construct F027500069 was evaluated in normal human serum (n=96) using ProteOn XPR36 (Bio-Rad Laboratories, Inc.). PBS / Tween (phosphate-buffered saline, pH 7.4, 0.005% Tween20) was used as the running buffer and the experiment was performed at 25°C.

[0282] ISVDs were captured on the chip via binding of the ALB23002 building block to HSA immobilized on the chip. To immobilize HSA, the ligand lane of the ProteOn GLC sensor chip was activated with EDC / NHS (flow rate 30 μl / min) and HSA was injected at 100 μl / ml in ProteOn acetate buffer pH 4.5, resulting in an immobilization level of approximately 3200 RU. After immobilization, the surface was inactivated with ethanolamine HCI (flow rate 30 μl / min).

[0283] The ISVD construct was then injected over the HSA surface at 45 μl / min for 2 min, resulting in an ISVD capture level of approximately 800 RU. Samples containing pre-existing antibodies were centrifuged at 14,000 rpm for 2 min, and the supernatant was diluted 1:10 in PBS-Tween 20 (0.005%) before being injected at 45 μl / min for 2 min, followed by a 400 s dissociation step. After each cycle (i.e. before a new ISVD capture and blood sample injection step), the HSA surface was regenerated with a 2 min injection of HCI (100 mM) at 45 μl / min. Sensorgrams showing pre-existing antibody binding were obtained after 1) ISVD-HSA dissociation and 2) double matching by subtracting non-specific binding to the reference ligand lane. The binding level of pre-existing antibodies was determined by setting the reporting point at 125 s (5 s after the end of association). The percentage reduction in binding of the existing antibody was calculated relative to the binding level of the reference ISVD at 125 seconds.

[0284] The tetravalent ISVD construct F027500069, optimized for reduced binding of pre-existing antibodies by introduction of the mutations L11V and V89L in each building block and a C-terminal alanine, showed substantially lower binding to pre-existing antibodies compared to the control non-optimized tetravalent ISVD construct F027301099 (Table 8 and Figure 4).

[0285] [Table 15]

[0286] Binding of pre-existing antibodies depended on the valency and composition of the multispecific construct. Table 3 and Figure 5 demonstrate that construct F027500069 showed lower pre-existing antibody reactivity than constructs F027500095 and F027500096.

[0287] 6.10 Example 10: Evaluation of F027500069 in the human TNFα transgenic Tg197 polyarthritis model. F027500069 was profiled in the Tg197 mouse model of TNF-driven progressive polyarthritis (Keffer et al., 1991, EMBO J., 10:4025-4031). In these mice, a modified human TNFα gene was inserted as a transgene into the mice. The human gene was modified to make the transcribed mRNA more stable, leading to overexpression of TNFα in all four paws with 100% penetrance and spontaneous progressive arthritis. Signs and symptoms become visible at approximately 6 weeks of age and, if untreated, steadily increase, leading to significant moribundity and death from approximately 10 weeks of age onwards. Arthritis severity was assessed clinically by a scoring system as detailed below:

[0288] [Table 16]

[0289] The arthritis was sensitive to treatment with therapeutic agents directed at inhibiting human TNFα (Shealy et al., 2002, Arthritis Res. 4(5):R7).

[0290] For the purpose of establishing dose-dependent efficacy, different doses of F027500069 were administered in a therapeutic manner by intraperitoneal injection twice a week to 6-week-old animals (n=8 animals / group) with visible signs and symptoms of arthritis. Human IgG1 purified from human myeloma serum (BioXcell #BE0297) was used as a negative control and anti-hTNFα reference mAb was used as a positive control to suppress arthritis. F027500069 was administered at three different dose strengths, 1.3 mg / kg body weight, 4 mg / kg, and 13.5 mg / kg, respectively. Treatment continued until 11 weeks of age. Clinical arthritis scores were determined once a week. As shown in Figure 6, treatment with F027500069 resulted in a dose-dependent suppression of clinical arthritis scores over time.

[0291] Animals treated with human IgG1 negative control antibody had a mean arthritis score of 1.099 ± 0.1071 by week 11. Anti-hTNFα reference mAb completely inhibited arthritis progression with a mean score of 0.4844 ± 0.0594 by week 11. F027500069 reduced arthritis progression by week 11 with mean scores of 0.8047 ± 0.0929 (1.3 mg / kg), 0.7969 ± 0.0585 (4 mg / kg), and 0.6016 ± 0.0349 (13.5 mg / kg). Overall inhibition of arthritis was analyzed by area under the curve (AUC, Figure 7). All doses of F027500069 significantly inhibited arthritis progression comparable to anti-hTNFα reference mAb in the Tg197 arthritis model.

[0292] Upon completion of treatment, hind ankle joints were processed for histology and sections were assessed for structural signs of arthritis using the following scoring system:

[0293] [Table 17]

[0294] The results of histological scoring are depicted in Figure 8. Structural arthritis and joint destruction were significantly inhibited by the higher doses of F027500069.

[0295] In conclusion, the results demonstrate a dose-dependent suppression of arthritis signs and symptoms by F027500069 to a similar extent as the anti-TNFα reference mAb, as well as an inhibition of structural progression.

[0296] 6.11 Example 11: Evaluation of F027500069 in a human IL-23-induced skin inflammation model. Intradermal injection of recombinant IL-23 in mice led to acute skin inflammation with redness and swelling around the injection site. Histologically, features of psoriatic dermatitis were observed, such as epithelial thickening with keratinocyte hyperplasia, keratinization, and immune cell infiltration such as T cells and macrophages. At the molecular level, the transcriptomic changes in the model largely overlapped with those observed in human psoriatic lesional skin compared to normal skin (Gauld et al., 2018, Journal of Dermatological Science 92:45-53). Thus, the IL-23 skin inflammation model resembles a mechanistic model of psoriasis.

[0297] To investigate inhibition of IL-23-mediated inflammation, F027500069 was tested in a skin inflammation model adapted from Rizzo et al., 2011, J Immunol; 186:1495-1502. 1 μg of recombinant human IL-23 in a total volume of 20 μl was injected intradermally into the right ear of female C57BL / 6 mice on days 1, 2, 3, and 4. PBS was injected into the ear of one group of mice as a control. Thickening of the ear skin was measured by caliper every day. F027500069 and control compounds were administered on days 1 and 3 by intraperitoneal injection. On day 5, mice were sacrificed and skin punch biopsies were taken. Biopsies were homogenized in PBS supplemented with protease inhibitor cocktail, and downstream effector cytokine IL-22 levels were determined.

[0298] For the purpose of establishing dose-dependent efficacy, different doses of ISVD constructs were administered in a therapeutic manner: twice weekly intraperitoneal injection to 6-week-old animals (n=10 animals / group) with visible signs and symptoms of arthritis. Human IgG1 purified from human myeloma serum (BioXcell #BE0297) was used as a negative control and anti-hIL-23 reference mAb1 was used as a positive control to suppress skin inflammation. F027500069 was administered at four different dose strengths: 0.13 mg / kg body weight, 0.4 mg / kg, 1 mg / kg, and 4 mg / kg, respectively. As shown in Figure 9, treatment with F027500069 resulted in a dose-dependent suppression of skin swelling depicted as the change in ear thickness from baseline on day 5.

[0299] Skin biopsies were taken on day 5 and tissue homogenates were prepared. Mouse IL-22 levels were measured using the Mesoscale Discovery V-plex mouse IL-22 assay kit (#K152WVD, Figure 10). IL-23 administration led to measurable levels of IL-22, with all samples from PBS-injected skin below the limit of quantification (LLOQ) of the assay used. All doses of F027500069 as well as anti-hIL-23 reference mAb1 significantly suppressed IL-22 tissue levels (IL-22 levels were not determined for the 1 mg / kg dose group).

[0300] Additionally, the feasibility of subcutaneous administration was evaluated in another experiment in the IL-23-induced skin inflammation model. Two doses of F027500069 (0.1 mg / kg and 1 mg / kg) were administered as either intraperitoneal or subcutaneous injections on days 1 and 3 to obtain ear thickness changes (Figure 11). In this experiment, a dose of 1 mg / kg of unrelated V HH was used as a negative control (Nab control) and anti-hIL-23 reference mAb1 (IP) at a dose of 3 mg / kg was used as a positive control.

[0301] Additionally, tissue IL-22 levels were measured from skin biopsy homogenates on day 5 (Figure 12).

[0302] In conclusion, the results demonstrate the suppression of IL-23-induced skin inflammation in both skin thickening and tissue effector cytokine levels. Both intraperitoneal and subcutaneous routes of administration are feasible.

[0303] 6.12 Example 12: Evaluation of F027500069 in a collagen antibody-induced arthritis model in human TNFα / TNFR1 knock-in mice. F027500069 was profiled in a collagen antibody-induced arthritis (CAIA) model in proprietary mice in which both the TNFα and TNF receptor 1 (TNFR1) gene loci were replaced with the respective human loci.

[0304] Eight animals per group were injected on day 0 with 8 mg of a cocktail of monoclonal antibodies directed against cartilage collagen 2 (ArthritoMab, MDbioscience, CIA-MAB-2C). On day 1, the development of arthritis was induced by injection of 25 μg of bacterial lipopolysaccharide (LPS). Test compounds were administered once as a single injection, 6 hours after LPS injection. The evolution of signs and symptoms of arthritis was assessed daily up to day 7 based on the arthritis score as detailed below:

[0305] [Table 18]

[0306] F027500069 was administered at 1.3 mg / kg body weight and the anti-hTNFα reference mAb positive control was administered at 0.5 mg / kg. Figure 13 shows the evolution of arthritis signs and symptoms over time. A single prophylactic administration of either F027500069 or the anti-hTNFα reference mAb resulted in a complete inhibition of arthritis development.

[0307] 6.13 Example 13: Evaluation of F027500069 in a human IL-23 induced skin inflammation model in human TNFα knock-in mice. Since F027500069 binds and inhibits only human or primate targets (both IL-23 and TNFα), human IL-23-induced skin inflammation was recapitulated in TNFα-humanized mice. In this proprietary line, the entire genomic TNFα locus in mice was replaced with a human locus. The exon-intron structure and regulatory elements are conserved between mice and humans. Besides faithful expression, the functional ability of human TNFα to induce a response in mice was previously evaluated.

[0308] Intradermal injection of hIL-23 led to a modest increase in TNFα expression (own data and Gauld et al., 2018, Journal of Dermatological Science 92:45-53). Equimolar doses of F027500069 and the corresponding monospecific ISVD building blocks F027500101 (anti-TNFα) and F027500017 (anti-IL-23) were administered in this model to investigate the potential additive effect of dual targeting. The administered dose intensity was 3.6 nmol / kg, corresponding to approximately 0.1 mg / kg. This low dose was chosen to allow some residual free IL-23 and therefore downstream TNFα secretion. Additionally, anti-hIL-23 reference mAb1 was administered as a positive control in addition to the high dose of F027500069. Changes in ear thickness were normalized to high (Nab negative control) and low (no IL-23 injection) controls ((sample-low control) / (high control-low control)). Figure 14 illustrates the results. Monospecific TNFα inhibition with F027500101 at 3.6 nmol / kg did not inhibit skin swelling, whereas monospecific IL-23 inhibition with F027500017 at 3.6 nmol / kg had a moderate but significant effect. Dual targeting of both TNFα and IL-23 with F027500069 at 3.6 nmol / kg led to a numerically superior suppression of skin swelling.

[0309] Skin biopsies were taken on day 5 and mRNA was prepared using standard methods. mRNA was subjected to paired-end, bulk-based whole transcriptome sequencing on an Ilumina NovaSeq™ 6000 platform. Differentially expressed genes (DEGs, fold change >2 with p<0.001) compared to NAb control were analyzed for overlap between treatment groups. As shown in Figure 15, despite the large overlap of monospecific treatment groups with multispecific treatment groups, 199 of the 769 DEGs were still specific to F027500069 treatment. This indicates that dual targeting of both TNFα and IL-23 in this model led to unique molecular responses and possibly synergistic amelioration of human disease.

[0310] In conclusion, the results demonstrate that multispecific inhibition of both IL-23 and TNFα in IL-23-induced skin inflammation led to numerically additive effects on skin thickening and induced unique transcriptomic profiles. [Industrial Applicability]

[0311] 7 Industrial Applicability The polypeptides described herein, nucleic acid molecules encoding same, vectors and compositions comprising the nucleic acids can be used for example in the treatment of subjects suffering from inflammatory bowel disease, psoriasis, psoriatic arthritis or hidradenitis suppurativa.

Claims

1. 1. A polypeptide, a composition comprising said polypeptide, or a composition comprising a nucleic acid comprising a nucleotide sequence encoding said polypeptide, for use as a medicament, said polypeptide specifically binds to TNFα and the p19 subunit of IL-23, said polypeptide comprising or consisting of at least three immunoglobulin single variable domains (ISVDs), each of said ISVDs comprising three complementarity determining regions (CDR1 to CDR3, respectively), linked via one or more peptidic linkers; a) a first ISVD that specifically binds to TNFα, i. CDR1 which is the amino acid sequence of SEQ ID NO:6; ii. a CDR2 having the amino acid sequence of SEQ ID NO: 10; and iii. CDR3 having the amino acid sequence of SEQ ID NO: 14 Including; b) a second ISVD that specifically binds to the p19 subunit of IL-23; iv. CDR1 which is the amino acid sequence of SEQ ID NO:7; v. a CDR2 having the amino acid sequence of SEQ ID NO:11; and vi. CDR3 having the amino acid sequence of SEQ ID NO: 15 and c) A third ISVD that specifically binds to the p19 subunit of IL-23, vii. CDR1 which is the amino acid sequence of SEQ ID NO:9; viii. a CDR2 having the amino acid sequence of SEQ ID NO: 13; and ix. CDR3 having the amino acid sequence of SEQ ID NO: 17 Including; wherein the ISVDs are in order starting from the N-terminus: A polypeptide or composition for said use.

2. a) the amino acid sequence of the first ISVD comprises greater than 90% sequence identity with SEQ ID NO:2; b) the amino acid sequence of the second ISVD comprises greater than 90% sequence identity with SEQ ID NO:3; c) the amino acid sequence of the third ISVD comprises greater than 90% sequence identity with SEQ ID NO:5; A polypeptide or composition for use as claimed in claim 1.

3. a) said first ISVD consists of the amino acid sequence of SEQ ID NO:2; b) said second ISVD consists of the amino acid sequence of SEQ ID NO:3; c) the third ISVD consists of the amino acid sequence of SEQ ID NO:5; A polypeptide or composition for use according to claim 1 or 2.

4. The polypeptide is i. CDR1 which is the amino acid sequence of SEQ ID NO:8; ii. a CDR2 having the amino acid sequence of SEQ ID NO: 12; and iii. CDR3 having the amino acid sequence of SEQ ID NO: 16 Further comprising an ISVD that binds to human serum albumin comprising A polypeptide or composition for use according to any one of claims 1 to 3.

5. 5. A polypeptide or composition for use according to claim 4, wherein the amino acid sequence of the ISVD that binds to human serum albumin comprises greater than 90% sequence identity with SEQ ID NO:

4.

6. A polypeptide or composition for use according to any one of claims 1 to 5, wherein said polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:

1.

7. A polypeptide or composition for use according to any one of claims 1 to 6 for use in the treatment of an autoimmune or inflammatory disease.

8. The polypeptide or composition for use according to claim 7, wherein the autoimmune disease or inflammatory disease is selected from inflammatory bowel disease including Crohn's disease or ulcerative colitis, psoriasis, psoriatic arthritis and hidradenitis suppurativa.

9. A polypeptide comprising or consisting of at least three immunoglobulin single variable domains (ISVDs), each of said ISVDs comprising three complementarity determining regions (CDR1 to CDR3, respectively), linked via one or more peptidic linkers; a) a first ISVD that specifically binds to TNFα, i. CDR1 which is the amino acid sequence of SEQ ID NO:6; ii. a CDR2 having the amino acid sequence of SEQ ID NO: 10; and iii. CDR3 having the amino acid sequence of SEQ ID NO: 14 Including; b) a second ISVD that specifically binds to the p19 subunit of IL-23; iv. CDR1 which is the amino acid sequence of SEQ ID NO:7; v. a CDR2 having the amino acid sequence of SEQ ID NO:11; and vi. CDR3 having the amino acid sequence of SEQ ID NO: 15 and c) A third ISVD that specifically binds to the p19 subunit of IL-23, vii. CDR1 which is the amino acid sequence of SEQ ID NO:9; viii. a CDR2 having the amino acid sequence of SEQ ID NO: 13; and ix. CDR3 having the amino acid sequence of SEQ ID NO: 17 Including, The polypeptide.

10. a) the amino acid sequence of the first ISVD comprises greater than 90% sequence identity with SEQ ID NO:2; b) the amino acid sequence of the second ISVD comprises greater than 90% sequence identity with SEQ ID NO:3; c) the amino acid sequence of the third ISVD comprises greater than 90% sequence identity with SEQ ID NO:5; The polypeptide of claim 9.

11. a) said first ISVD consists of the amino acid sequence of SEQ ID NO:2; b) said second ISVD consists of the amino acid sequence of SEQ ID NO:3; c) the third ISVD consists of the amino acid sequence of SEQ ID NO:5; A polypeptide according to claim 9 or 10.

12. i. CDR1 which is the amino acid sequence of SEQ ID NO:8; ii. a CDR2 having the amino acid sequence of SEQ ID NO: 12; and iii. CDR3 having the amino acid sequence of SEQ ID NO: 16 Further comprising an ISVD that binds to human serum albumin comprising A polypeptide according to any one of claims 9 to 11.

13. The polypeptide of claim 12, wherein the amino acid sequence of the ISVD that binds to human serum albumin comprises greater than 90% sequence identity with SEQ ID NO:

4.

14. A polypeptide according to any one of claims 9 to 13, comprising or consisting of the amino acid sequence of SEQ ID NO:

1.

15. A nucleic acid comprising a nucleotide sequence encoding the polypeptide according to any one of claims 9 to 14.

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