Polypeptides

Abstract

The present invention provides, inter alia, a polypeptide comprising an immunoglobulin chain variable domain that binds to IL-23 wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4) wherein CDR1 comprises a sequence that shares 60% or more sequence identity with SEQ ID NO: 1, CDR2 comprises a sequence that shares 50% or more sequence identity with SEQ ID NO: 2, and CDR3 comprises a sequence that shares 50% or more sequence identity with SEQ ID NO: 3.

Description

[0001] This application is a divisional application of the Chinese patent application with an application date of June 19, 2020, application number 202080059154.1, and invention name “Polypeptide” (the corresponding PCT application with an application date of June 19, 2020 and application number PCT / GB2020 / 051495). Technical Field

[0002] The present invention relates to polypeptides comprising immunoglobulin chain variable domains (or ICVDs) that bind to interleukin-23 (IL-23), as well as constructs and pharmaceutical compositions comprising these polypeptides. The present invention also relates to nucleic acids encoding such polypeptides, methods for preparing such polypeptides, cDNAs and vectors comprising nucleic acids encoding such polypeptides, host cells expressing or capable of expressing such polypeptides, and uses of such polypeptides, pharmaceutical compositions or constructs. Background Art

[0003] IL-23 is a pleiotropic cytokine involved in regulating both physiology and pathology. IL-23 has a function associated with inflammation and immunomodulation and is considered to be important for the spread of chronic inflammation and associated pathology in autoimmune diseases, including inflammatory bowel disease (IBD, such as Crohn's disease (CD) and ulcerative colitis (UC) (Croxford et al., 2012; Teng et al., 2015: Furfaro et al., 2017)). In patients with IBD, the production of IL-23 increases at the site of intestinal inflammation. IL-23 causes inflammation by direct action on pathogenic T cells; IL-23 enhances the activation of IBD intraepithelial lymphocytes and NK cells and stimulates proinflammatory cytokines to produce (Eken et al., 2014) by innate lymphoid cells. IL-23 is a heterodimer cytokine within the IL-12 cytokine family. IL-23 and IL-12 share a common p40 subunit, which dimerizes with the ligand-specific IL-23p19 subunit (also known as p19, IL-23A, and interleukin-23 subunit α) to form IL-23 or with IL-12p35 to form IL-12. The results of preclinical studies in different models of inflammatory bowel disease clearly show that IL-23 promotes intestinal inflammation and pathology. Although structurally related to IL-12, studies on IL-23-specific neutralizing antibodies in these models reveal that IL-23 and IL-12 have different roles in mucosal and systemic immune responses, and that selective depletion of IL-23 can stop intestinal inflammation while avoiding systemic immune responses.

[0004] Anti-TNF monoclonal antibodies have transformed the treatment of Crohn's disease and ulcerative colitis. By neutralizing TNF activity, antibodies such as infliximab and adalimumab favor mucosal healing and induce long-term remission in many patients. However, approximately one-third of patients prescribed anti-TNF agents are primary non-responders. In primary responders, subsequent loss of response can vary between 10% and 50% per year (secondary non-response) (Colombel et al. 2007; Hanauer et al. 2002, 2006; Sandborn et al. 2007; Schreiber et al. 2007). Patients with primary non-response are unlikely to benefit from switching to a second anti-TNF agent; therefore, therapeutic strategies targeting other inflammatory pathways are needed.

[0005] The above suggests that IL-23 may be a therapeutic target for the treatment of IBD and also provides a rationale for local rather than systemic administration (McGovern and Powrie, 2007). In addition, this agent may provide effective treatment of IBD in settings where anti-TNF antibodies fail.

[0006] WO2007005955 and WO2007027714 disclose anti-IL-23p19 antibodies.

[0007] Recently, Bremelumab (AMG-139, a fully human IgG that selectively binds to the p19 subunit of IL-23) 2 The efficacy and safety of Bricupumab (monoclonal antibodies) have been studied in patients with active CD who are refractory to or intolerant of anti-TNFα therapy (Sandborn et al. 2018, Sands et al. 2017). The superiority of many polypeptides of the present invention relative to Bricupumab is demonstrated in Examples 2 and 5 below.

[0008] Another anti-IL-23 agent of the prior art is 37D5, which is an anti-IL-23p19 domain antibody (VHH) (Desmyter et al. 2017). The superiority of many polypeptides of the present invention relative to 37D5 is demonstrated in Example 5 below.

[0009] In at least some embodiments, the polypeptides of the present invention have one or more of the following advantages compared to the anti-IL-23 substances of the prior art:

[0010] (i) increased affinity for IL-23;

[0011] (ii) specificity for IL-23 increase;

[0012] (iii) increased neutralization capacity against IL-23;

[0013] (iv) increased specificity for IL-23 relative to IL-12;

[0014] (v) increased cross-reactivity with IL-23 from different species such as human and cynomolgus monkey;

[0015] (vi) reduced immunogenicity, for example, when administered to mice, cynomolgus monkeys or humans;

[0016] (vii) increased stability in the presence of proteases, for example (a) in the presence of proteases present in the small and / or large intestine and / or IBD inflammatory proteases such as trypsin, chymotrypsin, MMP3, MMP10, MMP12, other MMPs and cathepsins and / or (b) in the presence of proteases from intestinal commensal microflora and / or pathogenic bacteria, proteases actively secreted and / or released by lysis of microbial cells present in the small and / or large intestine;

[0017] (viii) Increased stability to protease degradation during production (e.g., resistance to yeast proteases)

[0018] (ix) increased suitability for oral administration;

[0019] (x) increased suitability for oral delivery to the intestinal tract and lamina propria following oral administration;

[0020] (xi) increased suitability for expression in heterologous hosts such as bacteria such as Escherichia coli or yeasts belonging to the genera Aspergillus, Saccharomyces, Kluyveromyces, Hansenula or Pichia, such as Saccharomyces cerevisiae or Pichia pastoris;

[0021] (xii) suitability and improved properties for use in medicine;

[0022] (xiii) suitability and improved properties for use in functional foods;

[0023] (xiv) improved tissue penetration, such as penetration of inflamed colonic mucosal epithelium and submucosal tissue to reach the submucosal lamina propria;

[0024] (xv) reduced immunogenicity in humans, e.g. due to increased sequence similarity to human immunoglobulins;

[0025] (xvi) increased suitability for formatting in multispecific formats;

[0026] (xvii) Binding to novel epitopes.

[0027] Advantages (i) to (xvii) above can potentially be achieved by the polypeptide of the present invention in a monovalent form or in a multivalent form such as a biheaded form (eg, a homobiheaded form or a heterobiheaded form). Summary of the invention

[0028] The inventors have produced unexpectedly advantageous polypeptides comprising immunoglobulin chain variable domains that bind to IL-23. These polypeptides are immunoglobulin chain variable domains ID-L253T, 10E2, 10G10 and polypeptides related to each of these immunoglobulin chain variable domains.

[0029] These polypeptides have been found to have unexpected advantages over the prior art anti-IL-23 agents Bremecumab and 37D5 (see in particular the Background section above and Examples 2 and 5 below).

[0030] These polypeptides benefit particularly from unexpectedly high efficacy. They are also able to cross-react with cynomolgus monkey IL-23 and remain stable when exposed to proteases in the small and large intestines. In one embodiment, these polypeptides have been further enhanced by engineering. These further enhanced polypeptides benefit from the above advantages, retaining their IL-23 neutralizing activity during passage through the intestine and further resisting degradation and / or inactivation by proteases in the intestine, such as digestibility, inflammatory and microbial proteases from, for example, mammalian species.

[0031] These polypeptides are expected to have particular use in the prevention or treatment of autoimmune diseases and or inflammatory diseases such as inflammatory bowel disease (eg, Crohn's disease or ulcerative colitis) or in the prevention or treatment of mucositis, particularly when administered orally.

[0032] Many polypeptides of the invention have been found to have superior properties compared to Bricumab, a fully human IgG2 monoclonal antibody that selectively binds to the p19 subunit of IL-23, which has been studied in patients with active CD who are refractory to or intolerant of anti-TNFα therapy. The superiority of many polypeptides of the invention relative to Bricumab is demonstrated in Examples 2 and 5 below.

[0033] It has been found that many polypeptides of the present invention have superior properties compared to 37D5, which is an anti-IL-23p19 domain antibody (VHH) (Desmyter et al. 2017). The superiority of many polypeptides of the present invention relative to 37D5 is demonstrated in Example 5 below.

[0034] In a specific embodiment, the inventors provide the above polypeptides in a 'double-headed' format as well as an anti-TNF-α polypeptide. The data provided herein show that a therapeutic approach combining gastrointestinal restricted antagonism of IL-23 and TNF-α achieves a greater degree of effectiveness in a greater proportion of patients with inflammatory bowel disease, for a longer period of time, than monotherapy directed against a single target.

[0035] In one aspect, the present invention provides a polypeptide comprising an immunoglobulin chain variable domain that binds to IL-23, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR1 comprises a sequence that shares 60% or greater sequence identity with SEQ ID NO:1, CDR2 comprises a sequence that shares 50% or greater sequence identity with SEQ ID NO:2, and CDR3 comprises a sequence that shares 50% or greater sequence identity with SEQ ID NO:3.

[0036] In another aspect, the present invention provides a polypeptide comprising an immunoglobulin chain variable domain that binds to IL-23, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR1 comprises a sequence that shares 60% or greater sequence identity with SEQ ID NO: 14, CDR2 comprises a sequence that shares 50% or greater sequence identity with SEQ ID NO: 15, and CDR3 comprises a sequence that shares 50% or greater sequence identity with SEQ ID NO: 16.

[0037] In another aspect, the present invention provides a polypeptide comprising an immunoglobulin chain variable domain that binds to IL-23, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR1 comprises a sequence that shares 60% or greater sequence identity with SEQ ID NO:22, CDR2 comprises a sequence that shares 50% or greater sequence identity with SEQ ID NO:23, and CDR3 comprises a sequence that shares 50% or greater sequence identity with SEQ ID NO:24.

[0038] In another aspect, the invention provides a construct comprising two or more identical polypeptides according to the invention.

[0039] In another aspect, the invention provides a construct comprising at least one polypeptide according to the invention and at least one different polypeptide, wherein the different polypeptide binds to TNF-α.

[0040] In another aspect, the invention provides a construct comprising at least one polypeptide according to the invention and at least one different polypeptide, wherein the different polypeptide binds to a target other than TNF-α.

[0041] Other aspects of the invention are disclosed elsewhere in this document. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 – Mean phosphorus intensity values ​​of isolated UC inflamed colonic mucosa tissue treated with ID-L210T

[0043] Figure 2 – Mean phosphorus intensity values ​​of isolated UC inflamed colonic mucosal tissue treated with ID-L210T (continued)

[0044] Figure 3 – Neutralization efficacy of FA1K (before and after trypsin cleavage) against TNF-α Figure 4 – Neutralization efficacy of FA1K (before and after trypsin cleavage) against IL-23

[0045] Figure 5 – Stability of FA1K parent monomers ID-38F and ID-L253T after 4 h incubation in human fecal supernatant

[0046] Figure 6 – Mean phosphorus intensity values ​​of isolated UC inflamed colonic mucosa tissues treated with ID-L210T and ID-38F

[0047] Figure 7 – Mean phosphorus intensity values ​​of isolated UC inflamed colonic mucosa tissue treated with ID-L210T and ID-38F (continued)

[0048] Sequence Description

[0049] SEQ ID NO:1 - Peptide sequence of ID-L253T CDR1

[0050] SEQ ID NO:2 - Peptide sequence of ID-L253T CDR2

[0051] SEQ ID NO:3 - Peptide sequence of ID-L253T CDR3

[0052] SEQ ID NO:4 - polypeptide sequence of ID-L253T FR1

[0053] SEQ ID NO:5 - polypeptide sequence of ID-L253T FR2

[0054] SEQ ID NO:6 - polypeptide sequence of ID-L253T FR3

[0055] SEQ ID NO:7 - polypeptide sequence of ID-L253T FR4

[0056] SEQ ID NO:8 - polypeptide sequence of ID-L253T

[0057] SEQ ID NO:9—Polypeptide sequence encoding ID-L253T (with stop codon)

[0058] SEQ ID NO:10 - polypeptide sequence encoding ID-L253T (without stop codon)

[0059] SEQ ID NO: 11-12G1 polypeptide sequence

[0060] SEQ ID NO: 12-1E2 polypeptide sequence

[0061] SEQ ID NO: 13 - polypeptide sequence of 10E2

[0062] SEQ ID NO:14-10E2 CDR1 polypeptide sequence

[0063] SEQ ID NO:15-10E2 CDR2 polypeptide sequence

[0064] SEQ ID NO:16-10E2 CDR3 polypeptide sequence

[0065] SEQ ID NO: 17-10E2 FR1 polypeptide sequence

[0066] SEQ ID NO: 18-10E2 FR2 polypeptide sequence

[0067] SEQ ID NO:19-10E2 FR3 polypeptide sequence

[0068] SEQ ID NO:20-10E2 FR4 polypeptide sequence

[0069] SEQ ID NO:21 - polypeptide sequence of 10G10

[0070] SEQ ID NO:22 - polypeptide sequence of 10G10 CDR1

[0071] SEQ ID NO:23 - polypeptide sequence of 10G10 CDR2

[0072] SEQ ID NO:24 - polypeptide sequence of 10G10 CDR3

[0073] SEQ ID NO:25 - polypeptide sequence of 10G10 FR1

[0074] SEQ ID NO:26 - polypeptide sequence of 10G10 FR2

[0075] SEQ ID NO:27 - polypeptide sequence of 10G10 FR3

[0076] SEQ ID NO:28 - polypeptide sequence of 10G10 FR4

[0077] SEQ ID NO:29 - polypeptide sequence of ID-L210T

[0078] SEQ ID NO:30 - polypeptide sequence of ID-L237T

[0079] SEQ ID NO:31 - polypeptide sequence of ID-L238T

[0080] SEQ ID NO:32 - polypeptide sequence of ID-L239T

[0081] SEQ ID NO:33 - polypeptide sequence of ID-L240T

[0082] SEQ ID NO:34 - polypeptide sequence of ID-L241T

[0083] SEQ ID NO:35 - polypeptide sequence of ID-L242T

[0084] SEQ ID NO:36 - polypeptide sequence of ID-L243T

[0085] SEQ ID NO:37 - polypeptide sequence of ID-L244T

[0086] SEQ ID NO:38 - polypeptide sequence of ID-L245T

[0087] SEQ ID NO:39 - polypeptide sequence of ID-L246T

[0088] SEQ ID NO:40 - polypeptide sequence of ID-L247T

[0089] SEQ ID NO:41 - polypeptide sequence of ID-L248T

[0090] SEQ ID NO:42 - polypeptide sequence of ID-L249T

[0091] SEQ ID NO:43 - polypeptide sequence of ID-L250T

[0092] SEQ ID NO:44 - polypeptide sequence of ID-L251T

[0093] SEQ ID NO:45 - polypeptide sequence of ID-L252T

[0094] SEQ ID NO:46 - polypeptide sequence of FA1K

[0095] SEQ ID NO:47 - polypeptide sequence of the ID-38 F arm in FA1K

[0096] SEQ ID NO:48 - polypeptide sequence of the ID-L253T arm in FA1K

[0097] SEQ ID NO:49 - polypeptide sequence of the unstable linker in FA1K

[0098] SEQ ID NO:50—Polynucleotide sequence encoding ID-L210T

[0099] SEQ ID NO:51—Polynucleotide sequence encoding ID-L237T

[0100] SEQ ID NO:52—Polynucleotide sequence encoding ID-L238T

[0101] SEQ ID NO:53—Polynucleotide sequence encoding ID-L239T

[0102] SEQ ID NO:54—Polynucleotide sequence encoding ID-L240T

[0103] SEQ ID NO:55—Polynucleotide sequence encoding ID-L241T

[0104] SEQ ID NO:56—Polynucleotide sequence encoding ID-L242T

[0105] SEQ ID NO:57—Polynucleotide sequence encoding ID-L243T

[0106] SEQ ID NO:58—Polynucleotide sequence encoding ID-L244T

[0107] SEQ ID NO:59—Polynucleotide sequence encoding ID-L245T

[0108] SEQ ID NO:60—Polynucleotide sequence encoding ID-L246T

[0109] SEQ ID NO:61—Polynucleotide sequence encoding ID-L247T

[0110] SEQ ID NO:62—Polynucleotide sequence encoding ID-L248T

[0111] SEQ ID NO:63—Polynucleotide sequence encoding ID-L249T

[0112] SEQ ID NO:64—Polynucleotide sequence encoding ID-L250T

[0113] SEQ ID NO:65—Polynucleotide sequence encoding ID-L251T

[0114] SEQ ID NO:66—Polynucleotide sequence encoding ID-L252T

[0115] SEQ ID NO:67 - polypeptide sequence of ID-38F

[0116] SEQ ID NO:68—Polynucleotide sequence of a 3' primer containing a SpeI site

[0117] SEQ ID NO:69-37D5 polypeptide sequence

[0118] SEQ ID NO:70—Brevicumab heavy chain

[0119] SEQ ID NO:71—Brevicumab light chain

[0120] SEQ ID NO:72 - polypeptide sequence of IL-23p19

[0121] SEQ ID NO:73 - polypeptide sequence of IL-23p40

[0122] SEQ ID NO:74—Protease-labile linker polypeptide sequence

[0123] SEQ ID NO:75—Polypeptide sequence of protease-labile linker

[0124] SEQ ID NO:76 - Polypeptide sequence with non-protease labile linker

[0125] SEQ ID NO:77 - Polypeptide sequence of a non-protease labile linker DETAILED DESCRIPTION

[0126] Polypeptides comprising antibodies and antibody fragments having VH and VHH

[0127] Conventional antibodies or immunoglobulins (Ig) are proteins comprising four polypeptide chains: two heavy (H) chains and two light (L) chains. Each chain is divided into a constant region and a variable domain. The heavy chain variable domain is abbreviated as VHC in this article, and the light (L) chain variable domain is abbreviated as VLC in this article. These domains, domains associated therewith, and domains derived therefrom are referred to herein as immunoglobulin chain variable domains. The VHC and VLC domains can be further subdivided into regions with high variability, referred to as "complementarity determining regions" ("CDRs"), interspersed with more conservative regions, referred to as "framework regions" ("FRs"). Framework regions and complementary determining regions have been accurately defined (Kabat et al. 1991). In conventional antibodies, each VHC and VLC is composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Conventional antibody tetramers of two heavy immunoglobulin chains and two light immunoglobulin chains are formed with heavy immunoglobulin chains and light immunoglobulin chains connected by, for example, disulfide bonds, and the heavy chains are similarly connected. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. The light chain constant region comprises one domain, CL. The variable domains of the heavy chain and the variable domains of the light chain are binding domains that interact with the antigen. The constant region of the antibody usually mediates the binding of the antibody to host tissues or factors, which include various immune system cells (e.g., effector cells) or the first component (C1q) of the classical complement system. The term antibody includes immunoglobulins of IgA, IgG, IgE, IgD, IgM types (and their subtypes), wherein the light chain of the immunoglobulin can be a κ type or a λ type. The overall structure of the immunoglobulin-γ (IgG) antibody assembled by two identical heavy (H) chains and two identical light (L) chain polypeptides is well established in mammals and is highly conserved (Padlan 1994).

[0128] An exception to conventional antibody structure is found in the serum of Camelidae. In addition to conventional antibodies, these serums have special IgG antibodies. These IgG antibodies (referred to as heavy chain antibodies (HCAb)) lack L chain polypeptides and lack the first constant domain (CH1). In its N-terminal region, the H chain of the homodimeric protein contains a dedicated immunoglobulin chain variable domain (referred to as VHH), which is used to associate with its cognate antigen (Muyldermans 2013, Hamers-Casterman et al. 1993, Muyldermans et al. 1994).

[0129] As used herein, an antigen binding fragment (or "antibody fragment" or "immunoglobulin fragment") refers to a portion of an antibody that specifically binds to IL-23 (e.g., a molecule in which one or more immunoglobulin chains are not full-length but specifically binds to IL-23). ​​Examples of binding fragments encompassed within antigen binding fragments include:

[0130] (i) Fab fragment (monovalent fragment consisting of VLC, VHC, CL and CH1 domains);

[0131] (ii) F(ab')2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region);

[0132] (iii) Fd fragment (consisting of VHC and CH1 domains);

[0133] (iv) Fv fragment (consisting of the VLC and VHC domains of a single arm of an antibody);

[0134] (v) scFv fragments (composed of the VLC and VHC domains joined using recombinant methods by a synthetic linker that enables the VLC and VHC domains to be made into a single protein chain, in which the VLC and VHC regions pair to form a monovalent molecule);

[0135] (vi) VH (the variable domain of the immunoglobulin chain consisting of the VHC domain (Ward et al. 1989);

[0136] (vii) VL (immunoglobulin chain variable domain consisting of the VLC domain);

[0137] (viii) V-NAR (immunoglobulin chain variable domain composed of the VHC domain of IgNAR from chondrichthyes (Roux et al. 1998 and Griffiths et al. 2013)

[0138] (ix) VHH.

[0139] The total number of amino acid residues in VHH or VH may be in the region of 110-130, suitably 115-125, and most suitably 121.

[0140] The immunoglobulin chain variable domains of the present invention can be obtained, for example, by preparing a nucleic acid encoding an immunoglobulin chain variable domain using a technique for nucleic acid synthesis, followed by expressing the nucleic acid thus obtained. According to a specific embodiment, the immunoglobulin chain variable domains of the present invention do not have an amino acid sequence that is completely identical (i.e., shares 100% sequence identity) to the amino acid sequence of a naturally occurring polypeptide (such as a VH or VHH domain of a naturally occurring antibody).

[0141] The examples provided herein relate to immunoglobulin chain variable domains that bind to IL-23 itself. However, the principles of the present invention disclosed herein are equally applicable to any polypeptide comprising immunoglobulin chain variable domains that bind to IL-23, such as antibodies and antibody fragments. For example, the anti-IL-23 immunoglobulin chain variable domains disclosed herein can be incorporated into polypeptides such as full-length antibodies. This method is demonstrated by McCoy et al. 2014, and McCoy et al. provide anti-HIV VHH engineered to be a fusion with a human Fc region (comprising hinge, CH2 and CH3 domains), expressed as a dimer construct.

[0142] Replacing at least one amino acid residue in the framework region of a non-human immunoglobulin variable domain with the corresponding residue from a human variable domain is humanization. Humanization of the variable domain can reduce immunogenicity in humans.

[0143] Suitably, the polypeptide of the present invention comprises an immunoglobulin chain variable domain. More suitably, the polypeptide of the present invention consists of an immunoglobulin chain variable domain, such as an immunoglobulin heavy chain variable domain. Suitably, the polypeptide of the present invention is an antibody or an antibody fragment. More suitably, the polypeptide of the present invention is an antibody fragment. Suitably, the antibody fragment is an immunoglobulin chain variable domain, such as VHH, VH or VL. Suitably, the antibody fragment is a VHH, VH, VL, V-NAR, scFv, Fab fragment or F(ab')2 fragment. Suitably, the antibody fragment is an immunoglobulin heavy chain variable domain. More suitably, the antibody fragment is a VHH or VH, and most suitably a VHH.

[0144] Specificity, affinity, avidity and cross-reactivity

[0145] Specificity refers to the number of different types of antigens or antigenic determinants to which a particular antigen binding polypeptide can bind. The specificity of an antigen binding polypeptide is the ability of the antigen binding polypeptide to recognize specific antigens as unique molecular entities and to distinguish them from one another.

[0146] Affinity, represented by the equilibrium constant (Kd) for the dissociation of the antigen from the antigen-binding polypeptide, is a measure of the strength of binding between an antigenic determinant and an antigen-binding site on the antigen-binding polypeptide: the smaller the value of Kd, the stronger the binding strength between the antigenic determinant and the antigen-binding polypeptide (alternatively, affinity can also be expressed as an affinity constant (Ka), which is 1 / Kd). Affinity can be determined by known methods depending on the specific antigen of interest. Suitably, affinity is measured using a dynamically switchable biosurface (e.g., See Knezevic et al. 2012) or determined by surface plasmon resonance.

[0147] Avidity is a measure of the binding between an antigen binding polypeptide and a related antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on an antigen binding polypeptide and the number of related binding sites present on the antigen binding polypeptide.

[0148] Suitably, the polypeptide of the invention will be present in 10 -6 M or smaller, preferably 10 -7 M or smaller, preferably 10 -8 M or smaller, preferably 10 -9 M or smaller, preferably 10 -10 M or smaller, preferably 10 -11 M or smaller, preferably 10 -12 M or smaller, preferably 10 -13 The IL-23 peptide binds to IL-23 with a dissociation constant (Kd) of M or less.

[0149] Less than 10 -6 Any Kd of M is considered to be indicative of binding. Specific binding of an antigen-binding polypeptide to an antigen or antigenic determinant can be determined in any suitable known manner, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competition assays, and different variants thereof known in the art. Suitably, the affinity of the polypeptide is determined by surface plasmon resonance.

[0150] In one embodiment, the affinity of the polypeptide is established at 25°C by coupling the α-p40 capture antibody, 10mM sodium acetate buffer in pH 5 to the sensor chip by amine. Then IL-23 is fixed on the chip by flowing at 2μg / mL or 0.5μg / mL for 60 seconds at 10μL / min. This allows the P19 subunit to bind to the polypeptide freely. Then, at pH 7.4, 0.01M HEPES, 0.15M NaCl, 0.05% polysorbate 20 and 3mM EDTA buffer, 0.0195 and 5nM (L253T) or 0.8 to 500nM (Brekumonab Fab preparation) in five different concentrations are added with 30μl / min for 300 seconds and stopped for 300 seconds. The anti-P40 bound is regenerated between cycles with 10μl / min for 60 seconds with 10mM glycine at pH2. This method is used in Example 5.2 below.

[0151] Anti-IL-23 polypeptides, polypeptides that interact with IL-23, or polypeptides directed against IL-23 are all effective polypeptides that bind to IL-23. The polypeptides of the present invention may bind to a linear or conformational epitope on IL-23.

[0152] Suitably, the polypeptide of the invention binds to human IL-23. More suitably, the polypeptide of the invention binds to both human and at least one additional primate IL-23 selected from baboon IL-23, marmoset IL-23, cynomolgus IL-23 and rhesus IL-23. More suitably, the polypeptide of the invention binds to both human and cynomolgus IL-23.

[0153] Suitably, the polypeptide of the invention neutralizes human IL-23. More suitably, the polypeptide of the invention neutralizes both human and at least one additional primate IL-23 selected from baboon IL-23, marmoset IL-23, cynomolgus IL-23 and rhesus IL-23. More suitably, the polypeptide of the invention neutralizes both human and cynomolgus IL-23.

[0154] Suitably, IL-23 is a polypeptide comprising SEQ ID NO: 72 (p19 subunit) and 73 (p40 subunit), more suitably, IL-23 is a polypeptide consisting of SEQ ID NO: 72 and 73. Suitably, IL-23p19 is a polypeptide comprising SEQ ID NO: 72. More suitably, IL-23p19 is a polypeptide consisting of SEQ ID NO: 72. The p19 and p40 subunits used in the following examples also each incorporate a single C-terminal 6xHis tag.

[0155] Polypeptides that are able to react with IL-23 from humans and IL-23 from another species, such as cynomolgus monkey IL-23 ("cross-react"), are advantageous because they allow for easier preclinical studies in animal models.

[0156] Suitably, the polypeptide of the present invention is directed against an epitope on IL-23 that is located in and / or forms part of one or more receptor binding sites of IL-23, such that upon binding to IL-23, the polypeptide of the present invention is capable of inhibiting or reducing IL-23 receptor cross-linking mediated by said IL-23 and / or signal transduction mediated by such receptor cross-linking.

[0157] The polypeptides of the invention bind to one or more epitopes on IL-23. In one aspect of the invention, a polypeptide is provided which binds to the same epitope on IL-23 as ID-L253T, 12G1, 1E2, 10E2 or 10G10, more suitably ID-L253T. As the polypeptides of the invention are expected to be specific for the p19 subunit of IL-23, references to "IL-23 throughout the description" may also be replaced with "p19" where appropriate.

[0158] Suitably, the polypeptides of the present invention are isolated. An "isolated" polypeptide is one that is removed from its original environment. For example, if a naturally occurring polypeptide of the present invention is separated from some or all of the coexisting materials in the natural system, the polypeptide is isolated.

[0159] Efficacy, Inhibition, and Neutralization

[0160] Efficacy is a measure of the activity of a therapeutic agent expressed in the amount required to produce an effect of a given intensity. Compared with a lower efficacy agent that causes a smaller response at low concentrations, a high efficacy agent causes a greater response at low concentrations. Efficacy is related to affinity and effectiveness. Effectiveness refers to the ability of a therapeutic agent to produce a biological response after being bound to a target ligand and the quantitative magnitude of this response. The term half-maximal effective concentration (EC50) refers to the concentration of a therapeutic agent that causes a response between half of the baseline and the maximum after a specified exposure time. Therapeutic agents can cause inhibition or stimulation. It is usually used as a measure of efficacy and is used as a measure of efficacy in this article.

[0161] Neutralizing polypeptides for the purposes of the present invention are polypeptides that bind to IL-23, thereby inhibiting the binding of IL-23 to its cognate receptor (IL-23R), as measured by an ELISA, such as the ELISA described in the Examples, Assessment Method A.

[0162] Suitably, the polypeptide of the present invention neutralizes human IL-23 in an IL-23-IL-23R neutralization ELISA (see Examples, Evaluation Method A) with an EC50 of 5 nM or less, such as 4 nM or less, such as 3 nM or less, such as 2 nM or less, such as 1.7 nM or less, such as 1.5 nM or less, such as 1.4 nM or less, such as 1.3 nM or less, such as 1.2 nM or less, such as 1.1 nM or less, such as 1.0 nM or less, such as 0.9 nM or less, such as 0.8 nM or less, such as 0.75 nM or less, such as 0.70 nM or less, such as 0.65 nM or less, such as 0.60 nM or less, such as 0.55 nM or less, such as 0.50 nM or less, such as 0.45 nM or less, such as 0.40 nM or less.

[0163] Peptide and polynucleotide sequences

[0164] For the purpose of comparing two closely related polypeptide sequences, the "% sequence identity" between a first polypeptide sequence and a second polypeptide sequence can be calculated using NCBI BLAST v2.0 (BLASTP) using the standard settings for polypeptide sequences. For the purpose of comparing two closely related polynucleotide sequences, the "% sequence identity" between a first nucleotide sequence and a second nucleotide sequence can be calculated using NCBI BLAST v2.0 (BLASTN) using the standard settings for nucleotide sequences. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the comparison. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a two-strand comparison as default values. For amino acid sequences, the BLASTP program uses a word length of 3 and an expectation (E) of 10 and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)) alignments (B) 50, expectation (E) 10, M = 5, N = -4, and comparison of both strands as defaults. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more suitably less than about 0.01, and most suitably less than about 0.001.

[0165] A polypeptide or polynucleotide sequence is said to be identical if it shares 100% sequence identity with the other polypeptide or polynucleotide sequence over its entire length. The residues in the sequence are numbered from left to right, i.e., from the N-terminus to the C-terminus of a polypeptide; from the 5' to the 3' end of a polynucleotide.

[0166] "Difference" between sequences refers to the insertion, deletion or substitution of a single amino acid residue in a position of the second sequence compared to the first sequence. Two polypeptide sequences may contain one, two or more such amino acid differences. Insertion, deletion or substitution in the second sequence that is otherwise identical to the first sequence (100% sequence identity) causes a reduction in sequence identity %. For example, if the length of the same sequence is 9 amino acid residues, one substitution in the second sequence produces 88.9% sequence identity. If the length of the same sequence is 17 amino acid residues, two substitutions in the second sequence produce 88.2% sequence identity. If the length of the same sequence is 7 amino acid residues, three substitutions in the second sequence produce 57.1% sequence identity. If the length of the first polypeptide sequence and the second polypeptide sequence is 9 amino acid residues and shares 6 identical residues, the first polypeptide sequence and the second polypeptide sequence share an identity greater than 66% (the first polypeptide sequence and the second polypeptide sequence share 66.7% identity). If the first polypeptide sequence and the second polypeptide sequence are 17 amino acid residues in length and share 16 identical residues, the first polypeptide sequence and the second polypeptide sequence share greater than 94% identity (the first polypeptide sequence and the second polypeptide sequence share 94.1% identity). If the first polypeptide sequence and the second polypeptide sequence are 7 amino acid residues in length and share 3 identical residues, the first polypeptide sequence and the second polypeptide sequence share greater than 42% identity (the first polypeptide sequence and the second polypeptide sequence share 42.9% identity).

[0167] Alternatively, for the purpose of comparing a first reference polypeptide sequence to a second comparison polypeptide sequence, the number of additions, substitutions and / or deletions made to the first sequence to produce the second sequence can be determined. An addition is the addition of an amino acid residue to the sequence of the first polypeptide (including additions at either end of the first polypeptide). A substitution is the replacement of an amino acid residue in the sequence of the first polypeptide with a different amino acid residue. A deletion is the deletion of an amino acid residue from the sequence of the first polypeptide (including deletions at either end of the first polypeptide).

[0168] For the purpose of comparing a first reference polynucleotide sequence to a second comparison polynucleotide sequence, the number of additions, substitutions and / or deletions made to the first sequence to produce the second sequence can be determined. An addition is the addition of a nucleotide residue to the sequence of the first polynucleotide (including additions at either end of the first polynucleotide). A substitution is the replacement of a nucleotide residue in the sequence of the first polynucleotide with a different nucleotide residue. A deletion is the deletion of a nucleotide residue from the sequence of the first polynucleotide (including deletions at either end of the first polynucleotide).

[0169] A "conservative" amino acid substitution is one in which an amino acid residue is replaced by another amino acid residue of similar chemical structure and is expected to have little effect on the function, activity or other biological properties of the polypeptide. Such conservative substitutions are suitably substitutions in which one amino acid within the following group is replaced by another amino acid residue from the same group:

[0170]

[0171]

[0172] Suitably, the hydrophobic amino acid residue is a non-polar amino acid. More suitably, the hydrophobic amino acid residue is selected from V, I, L, M, F, W or C.

[0173] As used herein, the numbering of polypeptide sequences and the definition of CDR and FR are as defined according to the Kabat system (Kabat et al. 1991). The "corresponding" amino acid residue between a first polypeptide sequence and a second polypeptide sequence is an amino acid residue in the first sequence that shares the same position with the amino acid residue in the second sequence according to the Kabat system, while the identity of the amino acid residue in the second sequence and the amino acid residue in the first sequence can be different. Suitably, if the framework and CDR are the same length according to the Kabat definition, the corresponding residues will share the same numbering (and letter). Alignment can be manually implemented or by using a known computer algorithm such as NCBI BLAST v2.0 (BLASTP or BLASTN) of sequence alignment using standard settings to realize.

[0174] Preferably, the polynucleotides used in the present invention are isolated. An "isolated" polynucleotide is a polynucleotide removed from its original environment. For example, if a naturally occurring polynucleotide is separated from some or all of the materials coexisting in a natural system, the polynucleotide is isolated. If, for example, a polynucleotide is cloned into a vector that is not a part of its natural environment or if it is contained in a cDNA, the polynucleotide is considered to be isolated.

[0175] In one aspect of the present invention, a polynucleotide is provided, which encodes a polypeptide of the present invention. Suitably, the polynucleotide comprises or consists of a sequence that shares 70% or greater, such as 80% or greater, such as 90% or greater, such as 95% or greater, such as 99% or greater sequence identity with SEQ ID NO:9, 10 or 50 to 66. More suitably, the polynucleotide comprises or consists of any one of SEQ ID NO:9, 10 or 50 to 66. In another aspect, a cDNA is provided, which comprises the polynucleotide.

[0176] In one aspect of the invention, a polynucleotide is provided which comprises or consists of a sequence that shares 70% or greater, such as 80% or greater, such as 90% or greater, such as 95% or greater, such as 99% or greater sequence identity with any of the portions of CDR1, CDR2 or CDR3 encoding the immunoglobulin chain variable domain encoded by SEQ ID NO:9, 10 or 50 to 66.

[0177] Suitably, the polypeptide sequence of the present invention contains at least one change relative to the native sequence. Suitably, the polynucleotide sequence of the present invention contains at least one change relative to the native sequence. Suitably, the polypeptide sequence or polynucleotide sequence is altered to increase the stability of the polypeptide or the encoded polypeptide to proteases present in the intestinal tract (e.g., trypsin and chymotrypsin).

[0178] Embodiments relating to the sequences of specific polypeptides of the invention are summarized below.

[0179] ID-L253T and related peptides

[0180] Peptide sequence of ID-L253T in Kabat form

[0181] The polypeptide sequence of ID-L253T (a particularly advantageous polypeptide of the present invention) is listed below in Kabat format. CDR1 (SEQ ID NO: 1) is labeled 'CDR-H1', CDR2 (SEQ ID NO: 2) is labeled 'CDR-H2' and CDR3 (SEQ ID NO: 3) is labeled 'CDR-H3'. The top row provides the Kabat number (with an 'H' prefix) of each polypeptide residue, the middle row provides the polypeptide residues and the bottom row provides the sequence number of each polypeptide residue.

[0182]

[0183]

[0184] CDRs of ID-L253T and related polypeptides

[0185] Suitably, the CDR1 of the polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 80% or greater sequence identity with SEQ ID NO:1.

[0186] Alternatively, the CDR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 2, more suitably no more than 1 addition compared to SEQ ID NO: 1. Suitably, the CDR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 2, more suitably no more than 1 substitution compared to SEQ ID NO: 1. Suitably, the CDR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 2, more suitably no more than 1 deletion compared to SEQ ID NO: 1.

[0187] Suitably, any residue of CDR1 which differs from its corresponding residue in SEQ ID NO: 1 is a conservative substitution relative to its corresponding residue.Suitably, CDR1 comprises, or more suitably consists of, SEQ ID NO: 1.

[0188] Suitably, the CDR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence sharing 55% or greater, more suitably 60% or greater, more suitably 70% or greater, more suitably 75% or greater, more suitably 80% or greater, more suitably 85% or greater, more suitably 90% or greater sequence identity with SEQ ID NO:2.

[0189] Alternatively, the CDR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition compared to SEQ ID NO: 2. Suitably, the CDR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution compared to SEQ ID NO: 2. Suitably, the CDR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion compared to SEQ ID NO: 2.

[0190] Suitably, any residue of CDR2 that differs from its corresponding residue in SEQ ID NO: 2 is a conservative substitution relative to its corresponding residue.

[0191] Suitably, the residue of CDR2 corresponding to residue number 9 of SEQ ID NO: 2 is D or H. Suitably, the residue of CDR2 corresponding to residue number 10 of SEQ ID NO: 2 is Y or D. Suitably, the residue of CDR2 corresponding to residue number 11 of SEQ ID NO: 2 is S, G, R or A (more suitably S, R or A, more suitably S or A). Suitably, the residue of CDR2 corresponding to residue number 14 of SEQ ID NO: 2 is V or A.

[0192] Suitably, the residue of CDR2 corresponding to residue number 9 of SEQ ID NO:2 is D or H; the residue of CDR2 corresponding to residue number 10 of SEQ ID NO:2 is Y or D; the residue of CDR2 corresponding to residue number 11 of SEQ ID NO:2 is S, G, R or A (more suitably S, R or A, more suitably S or A) and the residue of CDR2 corresponding to residue number 14 of SEQ ID NO:2 is V or A.

[0193] Suitably, CDR2 comprises, or more suitably consists of, SEQ ID NO:2.

[0194] Suitably, the CDR3 of the polypeptide of the invention comprises or more suitably consists of a sequence sharing 60% or greater, more suitably 65% ​​or greater, more suitably 75% or greater, more suitably 80% or greater, more suitably 90% or greater sequence identity with SEQ ID NO:3.

[0195] Alternatively, the CDR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 3, more suitably no more than 2, more suitably no more than 1 additions compared to SEQ ID NO: 3. Suitably, the CDR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 3, more suitably no more than 2, more suitably no more than 1 substitutions compared to SEQ ID NO: 3. Suitably, the CDR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 3, more suitably no more than 2, more suitably no more than 1 deletions compared to SEQ ID NO: 3. Suitably, any substitution is conservative relative to its corresponding residue in SEQ ID NO: 3.

[0196] Suitably, any residue of CDR3 that differs from its corresponding residue in SEQ ID NO: 3 is a conservative substitution relative to its corresponding residue.

[0197] Suitably, the residue of CDR3 corresponding to residue number 6 of SEQ ID NO: 3 is I or L.

[0198] Suitably, (a) the residue of CDR2 corresponding to residue number 9 of SEQ ID NO:2 is D or H; the residue of CDR2 corresponding to residue number 10 of SEQ ID NO:2 is Y or D; the residue of CDR2 corresponding to residue number 11 of SEQ ID NO:2 is S, G, R or A (more suitably S, R or A, more suitably S or A) and the residue of CDR2 corresponding to residue number 14 of SEQ ID NO:2 is V or A, and

[0199] (b) The residue of CDR3 corresponding to residue number 6 of SEQ ID NO: 3 is I or L.

[0200] FR of ID-L253T and related peptides

[0201] Suitably, FR1 of the polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 5%, 12%, 18%, 26%, 32%, 38%, 46%, 52%, 58%, 62%, 66%, 68%, 72%, 75%, 78%, 82%, 85%, 90%, 95% or more sequence identity with SEQ ID NO:4.

[0202] Alternatively, FR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 28, more suitably no more than 26, more suitably no more than 24, more suitably no more than 22, more suitably no more than 20, more suitably no more than 18, more suitably no more than 16, more suitably no more than 14, more suitably no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition compared to SEQ ID NO:4. Suitably, FR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 28, more suitably no more than 26, more suitably no more than 24, more suitably no more than 22, more suitably no more than 20, more suitably no more than 18, more suitably no more than 16, more suitably no more than 14, more suitably no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution compared to SEQ ID NO:4. Suitably, FR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 28, more suitably no more than 26, more suitably no more than 24, more suitably no more than 22, more suitably no more than 20, more suitably no more than 18, more suitably no more than 16, more suitably no more than 14, more suitably no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion compared to SEQ ID NO:4.

[0203] Suitably, any residue of FR1 that differs from its corresponding residue in SEQ ID NO: 4 is a conservative substitution relative to its corresponding residue.

[0204] Suitably, the residue of FR1 corresponding to residue number 1 of SEQ ID NO:4 is D or E. Suitably, the residue of FR1 corresponding to residue number 11 of SEQ ID NO:4 is Q or L. Suitably, the residue of FR1 corresponding to residue number 19 of SEQ ID NO:4 is S or R. Suitably, the residue of FR1 corresponding to residue number 23 of SEQ ID NO:4 is E or A. Suitably, the residue of FR1 corresponding to residue number 24 of SEQ ID NO:4 is S or A.

[0205] Suitably, the residue of FR1 corresponding to residue number 1 of SEQ ID NO:4 is D or E; the residue of FR1 corresponding to residue number 11 of SEQ ID NO:4 is Q or L; the residue of FR1 corresponding to residue number 19 of SEQ ID NO:4 is S or R; the residue of FR1 corresponding to residue number 23 of SEQ ID NO:4 is E or A and the residue of FR1 corresponding to residue number 24 of SEQ ID NO:4 is S or A.

[0206] Suitably, FR1 comprises, or more suitably consists of, SEQ ID NO:4.

[0207] Suitably, FR2 of a polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 10%, 15%, 25%, 30%, 40%, 45%, 55%, 60%, 70%, 75%, 85%, 90% or more sequence identity with SEQ ID NO:5.

[0208] Alternatively, the FR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition compared to SEQ ID NO: 5. Suitably, the FR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution compared to SEQ ID NO: 5. Suitably, FR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion compared to SEQ ID NO:5.

[0209] Suitably, any residue of FR2 that differs from its corresponding residue in SEQ ID NO: 5 is a conservative substitution relative to its corresponding residue.

[0210] Suitably, the residue of FR2 corresponding to residue number 2 of SEQ ID NO:5 is F or Y. Suitably, the residue of FR2 corresponding to residue number 5 of SEQ ID NO:5 is V or A. Suitably, the residue of FR2 corresponding to residue number 8 of SEQ ID NO:5 is K or H. Suitably, the residue of FR2 corresponding to residue number 9 of SEQ ID NO:5 is Q or E. Suitably, the residue of FR2 corresponding to residue number 10 of SEQ ID NO:5 is R or L. Suitably, the residue of FR2 corresponding to residue number 12 of SEQ ID NO:5 is L or F. Suitably, the residue of FR2 corresponding to residue number 13 of SEQ ID NO:5 is L or F. Suitably, the residue of FR2 corresponding to residue number 14 of SEQ ID NO:5 is L or F. Suitably, the residue of FR2 corresponding to residue number 15 of SEQ ID NO:5 is L or F. Suitably, the residue of FR2 corresponding to residue number 16 of SEQ ID NO:5 is L or F. Suitably, the residue of FR2 corresponding to residue number 17 of SEQ ID NO:5 is L or F. Suitably, the residue of FR2 corresponding to residue number 18 of SEQ ID NO:5 is L or F. Suitably, the residue of FR2 corresponding to residue number 19 of SEQ ID NO:5 is L or F. Suitably, the residue of FR2 corresponding to residue number 2 of SEQ ID NO:5 is F or Y. Suitably, the residue of FR2 corresponding to residue number 5 of SEQ ID NO:5 is V or A. Suitably, the residue of

[0211] Suitably, the residue of FR2 corresponding to residue number 2 of SEQ ID NO:5 is F or Y; the residue of FR2 corresponding to residue number 5 of SEQ ID NO:5 is V or A; the residue of FR2 corresponding to residue number 8 of SEQ ID NO:5 is K or H; the residue of FR2 corresponding to residue number 9 of SEQ ID NO:5 is Q or E; the residue of FR2 corresponding to residue number 10 of SEQ ID NO:5 is R or L and the residue of FR2 corresponding to residue number 12 of SEQ ID NO:5 is L or F.

[0212] Suitably, FR3 of a polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 8%, 15%, 20%, 26%, 32%, 40%, 45%, 52%, 58%, 65%, 70%, 76%, 80%, 82%, 90%, 85%, 92%, 95% or more sequence identity with SEQ ID NO:6.

[0213] Alternatively, the FR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 29, more suitably no more than 27, more suitably no more than 25, more suitably no more than 23, more suitably no more than 21, more suitably no more than 19, more suitably no more than 17, more suitably no more than 15, more suitably no more than 13, more suitably no more than 11, more suitably no more than 9, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition compared to SEQ ID NO:6. Suitably, the FR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 29, more suitably no more than 27, more suitably no more than 25, more suitably no more than 23, more suitably no more than 21, more suitably no more than 19, more suitably no more than 17, more suitably no more than 15, more suitably no more than 13, more suitably no more than 11, more suitably no more than 9, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution compared to SEQ ID NO:6. Suitably, the FR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 29, more suitably no more than 27, more suitably no more than 25, more suitably no more than 23, more suitably no more than 21, more suitably no more than 19, more suitably no more than 17, more suitably no more than 15, more suitably no more than 13, more suitably no more than 11, more suitably no more than 9, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion compared to SEQ ID NO:6.

[0214] Suitably, the residue of FR3 corresponding to residue number 4 of SEQ ID NO: 6 is I, L or M (more suitably I or M). Suitably, the residue of FR3 corresponding to residue number 13 of SEQ ID NO: 6 is L or V. Suitably, the residue of FR3 corresponding to residue number 14 of SEQ ID NO: 6 is Y or F. Suitably, the residue of FR3 corresponding to residue number 16 of SEQ ID NO: 6 is Q or E. Suitably, the residue of FR3 corresponding to residue number 18 of SEQ ID NO: 6 is N or D. Suitably, the residue of FR3 corresponding to residue number 20 of SEQ ID NO: 6 is L or V. Suitably, the residue of FR3 corresponding to residue number 22 of SEQ ID NO: 6 is P or S. Suitably, the residue of FR3 corresponding to residue number 25 of SEQ ID NO: 6 is T or A. Suitably, the residue of FR3 corresponding to residue number 27 of SEQ ID NO: 6 is V or R. Suitably, the residue of FR3 corresponding to residue number 31 of SEQ ID NO: 6 is A or N.

[0215] Suitably, the residue of FR3 corresponding to residue number 4 of SEQ ID NO:6 is I, L or M (more suitably I or M); the residue of FR3 corresponding to residue number 13 of SEQ ID NO:6 is L or V; the residue of FR3 corresponding to residue number 14 of SEQ ID NO:6 is Y or F; the residue of FR3 corresponding to residue number 16 of SEQ ID NO:6 is Q or E; the residue of FR3 corresponding to residue number 18 of SEQ ID NO:6 is N or D; the residue of FR3 corresponding to residue number 20 of SEQ ID NO:6 is L or V; the residue of FR3 corresponding to residue number 22 of SEQ ID NO:6 is P or S; the residue of FR3 corresponding to residue number 25 of SEQ ID NO:6 is T or A; the residue of FR3 corresponding to residue number 27 of SEQ ID NO:6 is V or R and the residue of FR3 corresponding to residue number 31 of SEQ ID NO:6 is A or N.

[0216] Suitably, any residue of FR3 which differs from its corresponding residue in SEQ ID NO: 6 is a conservative substitution relative to its corresponding residue. Suitably, FR3 comprises or more suitably consists of SEQ ID NO: 6.

[0217] Suitably, FR4 of a polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more sequence identity with SEQ ID NO:7.

[0218] Alternatively, the FR4 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition compared to SEQ ID NO: 7. Suitably, the FR4 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution compared to SEQ ID NO: 7. Suitably, FR4 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion compared to SEQ ID NO:7.

[0219] Suitably, any residue of FR4 which differs from its corresponding residue in SEQ ID NO: 7 is a conservative substitution relative to its corresponding residue. Suitably, FR4 comprises or more suitably consists of SEQ ID NO: 7.

[0220] Suitably, the residue of FR4 corresponding to residue number 6 of SEQ ID NO:7 is Q or R.

[0221] Full-length sequences of ID-L253T and related polypeptides

[0222] Suitably, a polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 50% or greater, more suitably 55% or greater, more suitably 60% or greater, more suitably 65% ​​or greater, more suitably 70% or greater, more suitably 75% or greater, more suitably 80% or greater, more suitably 85% or greater, more suitably 90% or greater, more suitably 95% or greater, more suitably 96% or greater, more suitably 97% or greater, more suitably 98% or greater, more suitably 99% or greater sequence identity with SEQ ID NO:8.

[0223] Alternatively, the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 20, more suitably no more than 15, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition compared to SEQ ID NO: 8. Suitably, the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 20, more suitably no more than 15, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution compared to SEQ ID NO: 8. Suitably, the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 20, more suitably no more than 15, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion compared to SEQ ID NO: 8.

[0224] Suitably, the N-terminus of the polypeptide is D. Suitably, the polypeptide comprises, or more suitably consists of, SEQ ID NO: 8.

[0225] 10E2 and related peptides

[0226] CDRs of 10E2 and related polypeptides

[0227] Suitably, the CDR1 of the polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 80% or greater sequence identity with SEQ ID NO:14.

[0228] Alternatively, the CDR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 2, more suitably no more than 1 addition compared to SEQ ID NO: 14. Suitably, the CDR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 2, more suitably no more than 1 substitution compared to SEQ ID NO: 14. Suitably, the CDR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 2, more suitably no more than 1 deletion compared to SEQ ID NO: 14.

[0229] Suitably, any residue of CDR1 which differs from its corresponding residue in SEQ ID NO: 14 is a conservative substitution relative to its corresponding residue. Suitably, CDR1 comprises or more suitably consists of SEQ ID NO: 14.

[0230] Suitably, the CDR2 of the polypeptide of the present invention comprises, or more suitably consists of, a sequence sharing 55% or greater, more suitably 60% or greater, more suitably 70% or greater, more suitably 75% or greater, more suitably 80% or greater, more suitably 85% or greater, more suitably 90% or greater sequence identity with SEQ ID NO: 15.

[0231] Alternatively, the CDR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition compared to SEQ ID NO: 15. Suitably, the CDR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution compared to SEQ ID NO: 15. Suitably, the CDR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion compared to SEQ ID NO: 15.

[0232] Suitably, any residue of CDR2 that differs from its corresponding residue in SEQ ID NO: 15 is a conservative substitution relative to its corresponding residue.

[0233] Suitably, CDR2 comprises or more suitably consists of SEQ ID NO: 15.

[0234] Suitably, the CDR3 of the polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 60% or greater, more suitably 65% ​​or greater, more suitably 75% or greater, more suitably 80% or greater, more suitably 90% or greater sequence identity with SEQ ID NO: 16.

[0235] Alternatively, the CDR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 3, more suitably no more than 2, more suitably no more than 1 additions compared to SEQ ID NO: 16. Suitably, the CDR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 3, more suitably no more than 2, more suitably no more than 1 substitutions compared to SEQ ID NO: 16. Suitably, the CDR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 3, more suitably no more than 2, more suitably no more than 1 deletions compared to SEQ ID NO: 16. Suitably, any substitution is conservative relative to its corresponding residue in SEQ ID NO: 16.

[0236] Suitably, any residue of CDR3 that differs from its corresponding residue in SEQ ID NO: 16 is a conservative substitution relative to its corresponding residue.

[0237] FR of 10E2 and related peptides

[0238] Suitably, FR1 of the polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 5%, 12%, 18%, 26%, 32%, 38%, 46%, 52%, 58%, 62%, 66%, 68%, 72%, 75%, 78%, 82%, 85%, 90%, 95% or more sequence identity with SEQ ID NO:17.

[0239] Alternatively, FR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 28, more suitably no more than 26, more suitably no more than 24, more suitably no more than 22, more suitably no more than 20, more suitably no more than 18, more suitably no more than 16, more suitably no more than 14, more suitably no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition compared to SEQ ID NO: 17. Suitably, FR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 28, more suitably no more than 26, more suitably no more than 24, more suitably no more than 22, more suitably no more than 20, more suitably no more than 18, more suitably no more than 16, more suitably no more than 14, more suitably no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution compared to SEQ ID NO: 17. Suitably, FR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 28, more suitably no more than 26, more suitably no more than 24, more suitably no more than 22, more suitably no more than 20, more suitably no more than 18, more suitably no more than 16, more suitably no more than 14, more suitably no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion compared to SEQ ID NO: 17.

[0240] Suitably, any residue of FR1 that differs from its corresponding residue in SEQ ID NO: 17 is a conservative substitution relative to its corresponding residue.

[0241] Suitably, FR1 comprises or more suitably consists of SEQ ID NO: 17.

[0242] Suitably, FR2 of a polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 10%, 15%, 25%, 30%, 40%, 45%, 55%, 60%, 70%, 75%, 85%, 90% or more sequence identity with SEQ ID NO: 18.

[0243] Alternatively, the FR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition compared to SEQ ID NO: 18. Suitably, the FR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution compared to SEQ ID NO: 18. Suitably, FR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion compared to SEQ ID NO: 18.

[0244] Suitably, any residue of FR2 that differs from its corresponding residue in SEQ ID NO: 18 is a conservative substitution relative to its corresponding residue.

[0245] Suitably, FR3 of a polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 8%, 15%, 20%, 26%, 32%, 40%, 45%, 52%, 58%, 65%, 70%, 76%, 80%, 82%, 90%, 85%, 92%, 95% or more sequence identity with SEQ ID NO: 19.

[0246] Alternatively, the FR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 29, more suitably no more than 27, more suitably no more than 25, more suitably no more than 23, more suitably no more than 21, more suitably no more than 19, more suitably no more than 17, more suitably no more than 15, more suitably no more than 13, more suitably no more than 11, more suitably no more than 9, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition compared to SEQ ID NO: 19. Suitably, the FR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 29, more suitably no more than 27, more suitably no more than 25, more suitably no more than 23, more suitably no more than 21, more suitably no more than 19, more suitably no more than 17, more suitably no more than 15, more suitably no more than 13, more suitably no more than 11, more suitably no more than 9, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution compared to SEQ ID NO: 19. Suitably, the FR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 29, more suitably no more than 27, more suitably no more than 25, more suitably no more than 23, more suitably no more than 21, more suitably no more than 19, more suitably no more than 17, more suitably no more than 15, more suitably no more than 13, more suitably no more than 11, more suitably no more than 9, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion compared to SEQ ID NO: 19.

[0247] Suitably, any residue of FR3 which differs from its corresponding residue in SEQ ID NO: 19 is a conservative substitution relative to its corresponding residue. Suitably, FR3 comprises or more suitably consists of SEQ ID NO: 19.

[0248] Suitably, FR4 of a polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more sequence identity with SEQ ID NO:20.

[0249] Alternatively, the FR4 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition compared to SEQ ID NO: 20. Suitably, the FR4 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution compared to SEQ ID NO: 20. Suitably, FR4 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion compared to SEQ ID NO: 20.

[0250] Suitably, any residue of FR4 which differs from its corresponding residue in SEQ ID NO: 20 is a conservative substitution relative to its corresponding residue. Suitably, FR4 comprises or more suitably consists of SEQ ID NO: 20.

[0251] Full-length sequences of 10E2 and related polypeptides

[0252] Suitably, a polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 50% or greater, more suitably 55% or greater, more suitably 60% or greater, more suitably 65% ​​or greater, more suitably 70% or greater, more suitably 75% or greater, more suitably 80% or greater, more suitably 85% or greater, more suitably 90% or greater, more suitably 95% or greater, more suitably 96% or greater, more suitably 97% or greater, more suitably 98% or greater, more suitably 99% or greater sequence identity with SEQ ID NO: 13.

[0253] Alternatively, the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 20, more suitably no more than 15, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition compared to SEQ ID NO: 13. Suitably, the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 20, more suitably no more than 15, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution compared to SEQ ID NO: 13. Suitably, the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 20, more suitably no more than 15, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion compared to SEQ ID NO: 13.

[0254] Suitably, the N-terminus of the polypeptide is D. Suitably, the polypeptide comprises, or more suitably consists of, SEQ ID NO: 13.

[0255] 10G10 and related peptides

[0256] CDRs of 10G10 and related peptides

[0257] Suitably, the CDR1 of the polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 80% or greater sequence identity with SEQ ID NO: 22.

[0258] Alternatively, the CDR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 2, more suitably no more than 1 addition compared to SEQ ID NO: 22. Suitably, the CDR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 2, more suitably no more than 1 substitution compared to SEQ ID NO: 22. Suitably, the CDR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 2, more suitably no more than 1 deletion compared to SEQ ID NO: 22.

[0259] Suitably, any residue of CDR1 which differs from its corresponding residue in SEQ ID NO: 22 is a conservative substitution relative to its corresponding residue. Suitably, CDR1 comprises or more suitably consists of SEQ ID NO: 22.

[0260] Suitably, the CDR2 of the polypeptide of the present invention comprises, or more suitably consists of, a sequence sharing 55% or greater, more suitably 60% or greater, more suitably 70% or greater, more suitably 75% or greater, more suitably 80% or greater, more suitably 85% or greater, more suitably 90% or greater sequence identity with SEQ ID NO:23.

[0261] Alternatively, the CDR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition compared to SEQ ID NO: 23. Suitably, the CDR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution compared to SEQ ID NO: 23. Suitably, the CDR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion compared to SEQ ID NO: 23.

[0262] Suitably, any residue of CDR2 that differs from its corresponding residue in SEQ ID NO: 23 is a conservative substitution relative to its corresponding residue.

[0263] Suitably, CDR2 comprises or more suitably consists of SEQ ID NO: 23.

[0264] Suitably, the CDR3 of the polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 60% or greater, more suitably 65% ​​or greater, more suitably 75% or greater, more suitably 80% or greater, more suitably 90% or greater sequence identity with SEQ ID NO:24.

[0265] Alternatively, the CDR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 3, more suitably no more than 2, more suitably no more than 1 additions compared to SEQ ID NO: 24. Suitably, the CDR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 3, more suitably no more than 2, more suitably no more than 1 substitutions compared to SEQ ID NO: 24. Suitably, the CDR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 3, more suitably no more than 2, more suitably no more than 1 deletions compared to SEQ ID NO: 24. Suitably, any substitution is conservative relative to its corresponding residue in SEQ ID NO: 24.

[0266] Suitably, any residue of CDR3 that differs from its corresponding residue in SEQ ID NO: 24 is a conservative substitution relative to its corresponding residue.

[0267] FRs of 10G10 and related peptides

[0268] Suitably, FR1 of the polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 5%, 12%, 18%, 26%, 32%, 38%, 46%, 52%, 58%, 62%, 66%, 68%, 72%, 75%, 78%, 82%, 85%, 90%, 95% or more sequence identity with SEQ ID NO:25.

[0269] Alternatively, FR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 28, more suitably no more than 26, more suitably no more than 24, more suitably no more than 22, more suitably no more than 20, more suitably no more than 18, more suitably no more than 16, more suitably no more than 14, more suitably no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition compared to SEQ ID NO: 25. Suitably, FR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 28, more suitably no more than 26, more suitably no more than 24, more suitably no more than 22, more suitably no more than 20, more suitably no more than 18, more suitably no more than 16, more suitably no more than 14, more suitably no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution compared to SEQ ID NO: 25. Suitably, FR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 28, more suitably no more than 26, more suitably no more than 24, more suitably no more than 22, more suitably no more than 20, more suitably no more than 18, more suitably no more than 16, more suitably no more than 14, more suitably no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion compared to SEQ ID NO: 25.

[0270] Suitably, any residue of FR1 that differs from its corresponding residue in SEQ ID NO: 25 is a conservative substitution relative to its corresponding residue.

[0271] Suitably, FR1 comprises or more suitably consists of SEQ ID NO:25.

[0272] Suitably, FR2 of a polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 10%, 15%, 25%, 30%, 40%, 45%, 55%, 60%, 70%, 75%, 85%, 90% or more sequence identity with SEQ ID NO:26.

[0273] Alternatively, the FR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition compared to SEQ ID NO: 26. Suitably, the FR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution compared to SEQ ID NO: 26. Suitably, FR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion compared to SEQ ID NO: 26.

[0274] Suitably, any residue of FR2 that differs from its corresponding residue in SEQ ID NO: 26 is a conservative substitution relative to its corresponding residue.

[0275] Suitably, FR3 of a polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 8%, 15%, 20%, 26%, 32%, 40%, 45%, 52%, 58%, 65%, 70%, 76%, 80%, 82%, 90%, 85%, 92%, 95% or more sequence identity with SEQ ID NO:27.

[0276] Alternatively, the FR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 29, more suitably no more than 27, more suitably no more than 25, more suitably no more than 23, more suitably no more than 21, more suitably no more than 19, more suitably no more than 17, more suitably no more than 15, more suitably no more than 13, more suitably no more than 11, more suitably no more than 9, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition compared to SEQ ID NO: 27. Suitably, the FR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 29, more suitably no more than 27, more suitably no more than 25, more suitably no more than 23, more suitably no more than 21, more suitably no more than 19, more suitably no more than 17, more suitably no more than 15, more suitably no more than 13, more suitably no more than 11, more suitably no more than 9, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution compared to SEQ ID NO: 27. Suitably, the FR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 29, more suitably no more than 27, more suitably no more than 25, more suitably no more than 23, more suitably no more than 21, more suitably no more than 19, more suitably no more than 17, more suitably no more than 15, more suitably no more than 13, more suitably no more than 11, more suitably no more than 9, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion compared to SEQ ID NO: 27.

[0277] Suitably, any residue of FR3 which differs from its corresponding residue in SEQ ID NO: 27 is a conservative substitution relative to its corresponding residue. Suitably, FR3 comprises or more suitably consists of SEQ ID NO: 27.

[0278] Suitably, FR4 of a polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more sequence identity with SEQ ID NO:28.

[0279] Alternatively, the FR4 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition compared to SEQ ID NO: 28. Suitably, the FR4 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution compared to SEQ ID NO: 28. Suitably, the FR4 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion compared to SEQ ID NO: 28.

[0280] Suitably, any residue of FR4 which differs from its corresponding residue in SEQ ID NO: 28 is a conservative substitution relative to its corresponding residue. Suitably, FR4 comprises or more suitably consists of SEQ ID NO: 28.

[0281] Full-length sequences of 10G10 and related polypeptides

[0282] Suitably, a polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 50% or greater, more suitably 55% or greater, more suitably 60% or greater, more suitably 65% ​​or greater, more suitably 70% or greater, more suitably 75% or greater, more suitably 80% or greater, more suitably 85% or greater, more suitably 90% or greater, more suitably 95% or greater, more suitably 96% or greater, more suitably 97% or greater, more suitably 98% or greater, more suitably 99% or greater sequence identity with SEQ ID NO: 21.

[0283] Alternatively, the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 20, more suitably no more than 15, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition compared to SEQ ID NO: 21. Suitably, the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 20, more suitably no more than 15, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution compared to SEQ ID NO: 21. Suitably, the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 20, more suitably no more than 15, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion compared to SEQ ID NO: 21.

[0284] Suitably, the N-terminus of the polypeptide is D. Suitably, the polypeptide comprises, or more suitably consists of, SEQ ID NO: 21.

[0285] Additional embodiments related to ID-L253T, 10E2, 10G10 and related polypeptides

[0286] In one embodiment, a polypeptide is provided which comprises an immunoglobulin chain variable domain that binds to IL-23, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein the complementarity determining regions (and more suitably framework regions) are selected from the complementarity determining regions (and framework regions) disclosed herein.

[0287] In one embodiment, a polypeptide is provided, comprising an immunoglobulin chain variable domain that binds to IL-23, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein:

[0288] CDR1 comprises a sequence selected from SEQ ID NO: 1, SEQ ID NO: 14 or SEQ ID NO: 22;

[0289] CDR2 comprises a sequence selected from SEQ ID NO: 2, SEQ ID NO: 15 or SEQ ID NO: 23;

[0290] CDR3 comprises a sequence selected from SEQ ID NO:3, SEQ ID NO:16 or SEQ ID NO:24.

[0291] Suitably, the polypeptide disclosed herein comprises or more suitably consists of a sequence sharing 70% or greater, more suitably 80% or greater, more suitably 90% or greater, more suitably 95% or greater, more suitably 98% or greater, more suitably 99% or greater, more suitably 99.5% or greater sequence identity with any one of SEQ ID No: 8, 11, 12, 13, 21 or 29-45.

[0292] In one embodiment, a polypeptide is provided which comprises or more suitably consists of a sequence sharing 70% or greater, more suitably 80% or greater, more suitably 90% or greater, more suitably 95% or greater, more suitably 98% or greater, more suitably 99% or greater, more suitably 99.5% or greater sequence identity with any one of SEQ ID No: 8, 11, 12, 13, 21 or 29-45.

[0293] Adapters and polymers

[0294] The construct according to the invention comprises a plurality of polypeptides and may therefore suitably be multivalent. Such a construct may comprise at least two identical polypeptides according to the invention. A construct consisting of two identical polypeptides according to the invention is a "homobivalent". In one aspect of the invention, a construct is provided comprising two or more identical polypeptides according to the invention.

[0295] Alternatively, the construct may comprise at least two polypeptides which are different but are still both polypeptides according to the invention ("homobihead").

[0296] Alternatively, such a construct may comprise (a) at least one polypeptide according to the invention and (b) at least one polypeptide which is not a polypeptide according to the invention, such as an antibody or antigen-binding fragment thereof (also a "heterobihead"). The at least one polypeptide of (b) may bind to IL-23 (e.g. via an epitope different from that of (a)), or alternatively may bind to a target other than IL-23. Suitably, the different polypeptides (b) bind to, for example: interleukins (such as IL-1, IL-1ra, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17 and IL-18), interleukin receptors (such as IL-6R and IL-7R), transcription factors (such as NF-kB), cytokines (such as TNF-α, IFN-γ, TGF-β and TSLP), transmembrane proteins (such as gp130 and CD3), surface glycoproteins (such as CD4, CD20, CD40), soluble proteins (such as CD40L), integrins (such as a4b7 and AlphaEbeta7), adhesion molecules (such as MAdCAM), chemokines (such as IP10 and CCL20), chemokine receptors (such as CCR2 and CCR9), inhibitory proteins (such as SMAD7), kinases (such as JAK3), G protein-coupled receptors (such as sphingosine-1-P receptor), other inflammatory mediators involved in human pathological processes or immune-related ligands. Therefore, different polypeptides (b) bind to, for example, IL-6R, IL-6, IL-12, IL-1-β, IL-17A, TNF-α or CD3; or other inflammatory mediators or immune-related ligands involved in human pathological processes. Most suitably, different polypeptides (b) bind to TNF-α, and more suitably, different polypeptides (b) bind to ID-38F.

[0297] Constructs can be multivalent and / or multispecific. Multivalent constructs (such as bivalent constructs) comprise two or more binding polypeptides, thus presenting two or more sites where connection to one or more antigens can occur. Examples of multivalent constructs can be homobiheaded or heterobiheaded. Multivalent constructs (such as bispecific constructs) comprise two or more different binding polypeptides, which present two or more sites where (a) connection to two or more different antigens can occur or (b) connection to two or more different epitopes on the same antigen can occur. Examples of multispecific constructs can be heterobiheaded. Multispecific constructs are multivalent.

[0298] Suitably, the polypeptide contained in the construct is an antibody fragment. More suitably, the polypeptide contained in the construct is selected from VHH, VH, VL, V-NAR, scFv, Fab fragment or F(ab')2 fragment. More suitably, the polypeptide contained in the construct is VH or VHH, most suitably VHH.

[0299] The polypeptides of the invention may be linked to each other directly (i.e., without the use of a linker) or via a linker. Suitably, the linker is a protease labile linker ('labile linker') or a non-protease labile linker. The linker is suitably a polypeptide and is selected so as to allow binding of the polypeptide to its epitope. If used for therapeutic purposes, the linker is suitably non-immunogenic in the subject to which the polypeptide is administered.

[0300] Suitably, the protease labile linker has the formula:

[0301] [-(G a S) x -B-(G b S) y -] z

[0302] in

[0303] a is 1 to 10;

[0304] b is 1 to 10;

[0305] x is 1 to 10;

[0306] y is 1 to 10;

[0307] z is 1 to 10 and

[0308] B is K or R (SEQ ID NO: 74).

[0309] And more appropriately:

[0310] [-(G 4 S) x -B-(G 4 S) y -] z

[0311] in

[0312] x is 1 to 10;

[0313] y is 1 to 10

[0314] z is 1 to 10 and

[0315] B is K or R.

[0316] More suitably, a is 2 to 5, b is 2 to 5, x is 1 to 3, y is 1 to 3, z is 1 and B is K.

[0317] More suitably, the protease labile linker has the formula -(G 4 S) 2 -K-(G 4 S) 2 -(SEQ ID NO:75).

[0318] Suitably, the polypeptides are all linked via non-protease labile linkers. Suitably, the non-protease labile linker has the formula (G 4 S) x , wherein x is 1 to 10 (SEQ ID NO: 76). Most suitably, x is 6 (SEQ ID NO: 77).

[0319] Therefore, a construct is provided, which comprises at least one polypeptide according to the invention and at least one different polypeptide, wherein the different polypeptide binds to TNF-α. Suitably, the polypeptide that binds to TNF-α is ID-38F or a variant thereof, such as a polypeptide that shares at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 99% sequence identity with ID-38F (SEQ ID NO: 67). More suitably, the polypeptide that binds to TNF-α is ID-38F. In one embodiment, this construct comprises a non-protease unstable linker, such as (G 4 S) 6 Alternatively, the construct comprises a protease-labile linker such as -(G 4 S) 2 -K-(G 4 S) 2 - Suitably, this construct shares at least 80%, such as at least 90%, such as at least 95% sequence identity with FAIK (SEQ ID NO: 46).

[0320] In embodiments where the construct comprises a non-protease labile linker, then suitably the construct as a whole (i.e. the binding polypeptide (which may be an immunoglobulin chain variable domain) and the non-protease labile linker) is substantially resistant to proteases such as trypsin and chymotrypsin. In embodiments where the construct comprises a protease labile linker, then suitably the polypeptide (i.e. the binding polypeptide, which may be an immunoglobulin chain variable domain) is substantially resistant to proteases such as trypsin and chymotrypsin, but the protease labile linker is susceptible to proteases such as trypsin and chymotrypsin.

[0321] Vector and host

[0322] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is connected. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which another DNA segment can be connected. Another type of vector is a viral vector, in which another DNA segment can be connected to the viral genome. Certain vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors with bacterial replication origins and free mammalian and yeast vectors). Other vectors (e.g., non-free mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and therefore replicated together with the host genome. In addition, certain vectors can guide the expression of genes operably connected thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA technology are generally in the form of plasmids. In the present specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used vector forms. However, the present invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses and adeno-associated viruses), which have the same function, as well as phage and phagemid systems. The present invention also relates to nucleotide sequences encoding polypeptide sequences or multivalent and / or multispecific constructs. As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell into which a recombinant expression vector has been introduced. Such terms are intended to refer not only to a specific subject cell, but also to the progeny of such a cell.

[0323] In one aspect of the invention, a vector is provided, comprising a polynucleotide encoding a polypeptide or construct of the present invention or a cDNA comprising the polynucleotide. In another aspect of the invention, a host cell transformed with the vector is provided, which is capable of expressing a polypeptide or construct of the present invention. Suitably, the host cell is a bacterium, such as Escherichia coli, a yeast belonging to the genus Aspergillus, Saccharomyces, Kluyveromyces, Hansenula or Pichia, such as Saccharomyces cerevisiae or Pichia pastoris.

[0324] stability

[0325] Suitably, the polypeptide or construct of the invention retains neutralizing capacity and / or efficacy upon oral delivery and after exposure to the intestinal tract (e.g., after exposure to proteases of the small and / or large intestine and / or IBD inflammatory proteases). Such proteases include enteropeptidases, trypsin, chymotrypsin, and irritable bowel inflammatory proteases (such as MMP3, MMP12, and cathepsins). Proteases of the small and / or large intestine or proteases produced in the small and / or large intestine include proteases derived from intestinal symbiotic microbial flora and / or pathogens, such as proteases wherein the proteases are cell membrane-attached proteases, excreted proteases, and proteases released by cell lysis). Most suitably, the proteases are trypsin and chymotrypsin.

[0326] Suitably, the intestinal tract is the intestinal tract of a dog, pig, human, cynomolgus monkey or mouse. More suitably, the intestinal tract is the intestinal tract of a human, cynomolgus monkey or mouse, more suitably mouse or human, most suitably human. The small intestine suitably consists of the duodenum, jejunum and ileum. The large intestine suitably consists of the cecum, colon, rectum and anal canal. In contrast to the gastrointestinal tract, the intestinal tract consists only of the small intestine and the large intestine.

[0327] A polypeptide or construct of the invention substantially retains neutralizing capacity when, following exposure to proteases and / or IBD inflammatory proteases present in the small and / or large intestine, suitably 10% or more, more suitably 20% or more, more suitably 30% or more, more suitably 40% or more, more suitably 50% or more, more suitably 60% or more, more suitably 70% or more, more suitably 80% or more, more suitably 90% or more, more suitably 95% or more, or most suitably 100% of the original neutralizing capacity of the polypeptide or construct of the invention is retained.

[0328] Suitably, the polypeptide or construct of the invention substantially retains neutralising ability after exposure to proteases present in the small intestine and / or large intestine and / or IBD inflammatory proteases for, e.g., at least 1 hour, more suitably at least 2 hours, more suitably at least 3 hours, more suitably at least 4 hours, more suitably at least 7 hours, more suitably at least 16 hours at 37°C.

[0329] 'Substantially retains neutralising ability' suitably means that the polypeptide or construct of the invention retains 10% or more, more suitably 20% or more, more suitably 30% or more, more suitably 40% or more, more suitably 50% or more, more suitably 60% or more, more suitably 70% or more, more suitably 80% or more, more suitably 90% or more of the neutralising ability of the polypeptide or construct of the invention.

[0330] After exposure to conditions of the intestine, more suitably the small or large intestine, more suitably a human faecal extract for 4 hours, suitably 10% or more, more suitably 20% or more, more suitably 30% or more, more suitably 40% or more, more suitably 50% or more, more suitably 60% or more, more suitably 70% or more of the neutralising capacity of the polypeptide or construct of the invention is retained.

[0331] After exposure to conditions of the intestine, more suitably the small or large intestine, more suitably a human faecal extract for 7 hours, suitably 10% or more, more suitably 20% or more, more suitably 30% or more, more suitably 40% or more, more suitably 50% or more, more suitably 60% or more, more suitably 70% or more of the neutralizing capacity of the polypeptide or construct of the invention is retained.

[0332] After exposure to conditions of the intestine, more suitably the small or large intestine, more suitably a human faecal extract for 16 hours, suitably 10% or more, more suitably 20% or more, more suitably 30% or more, more suitably 40% or more, more suitably 50% or more, more suitably 60% or more, more suitably 70% or more of the neutralising capacity of the polypeptide or construct of the invention is retained.

[0333] After exposure to conditions in the intestine, more suitably the small intestine or large intestine, more suitably mouse small intestine supernatant for 1 hour, suitably 10% or more, more suitably 20% or more, more suitably 30% or more, more suitably 40% or more, more suitably 50% or more, more suitably 60% or more, more suitably 70% or more of the neutralizing capacity of the polypeptide or construct of the invention is retained.

[0334] After 4 hours of exposure to conditions in the intestine, more suitably the small intestine or large intestine, more suitably mouse small intestine supernatant, suitably 10% or more, more suitably 20% or more, more suitably 30% or more, more suitably 40% or more, more suitably 50% or more, more suitably 60% or more, more suitably 70% or more of the neutralizing capacity of the polypeptide or construct of the invention is retained.

[0335] After exposure to the conditions of the intestine for 1, 2, 3, 4, 5, 6 or 7 hours, suitably 10% or more, more suitably 20% or more, more suitably 30% or more, more suitably 40% or more, more suitably 50% or more, more suitably 60% or more, more suitably 70% or more of the administered dose of the polypeptide or construct of the invention retains neutralizing capacity against IL-23 and is maintained in the feces (suitably excreted feces or feces removed from the intestine) of the mouse, cynomolgus monkey and / or human.

[0336] A polypeptide of the invention or a construct of the invention remains substantially intact when suitably 10% or more, more suitably 20% or more, more suitably 30% or more, more suitably 40% or more, more suitably 50% or more, more suitably 60% or more, more suitably 70% or more, more suitably 80% or more, more suitably 90% or more, more suitably 95% or more, more suitably 99% or more, most suitably 100% of the administered amount of the polypeptide of the invention or construct remains intact following exposure to proteases and / or IBD inflammatory proteases present in the small and / or large intestine.

[0337] Therapeutic Uses and Delivery

[0338] The therapeutically effective amount of the polypeptide, pharmaceutical composition or construct of the present invention is the amount of IL-23 in the subject that is effectively neutralized to a significant degree after a single or multiple doses are administered to the subject. The therapeutically effective amount can vary according to factors such as the disease state, age, sex and weight of the individual and the ability of the polypeptide, pharmaceutical composition or construct to elicit the desired response in the individual. The therapeutically effective amount is also the amount in which the therapeutic beneficial effect exceeds any toxic or deleterious effect of the polypeptide, pharmaceutical composition or construct of the present invention. The polypeptide or construct of the present invention can be incorporated into a pharmaceutical composition suitable for administration to a subject. The polypeptide or construct of the present invention can be in the form of a pharmaceutically acceptable salt.

[0339] The pharmaceutical composition of the present invention can be suitably formulated for oral, intramuscular, subcutaneous or intravenous delivery. The pharmaceutical composition of the present invention can be in various forms. These include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. Solid dosage forms are preferred. The polypeptide, pharmaceutical composition or construct of the present invention can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc.

[0340] Typically, the pharmaceutical composition comprises a polypeptide or construct of the present invention and a pharmaceutically acceptable diluent or carrier. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc. and combinations thereof. Pharmaceutically acceptable carriers may also include a small amount of auxiliary substances that enhance the shelf life or effectiveness of the polypeptide or construct of the present invention, such as a wetting agent or emulsifier, preservative or buffer. The pharmaceutical composition may include an anti-adherent, an adhesive, a coating, a disintegrant, a flavoring agent, a coloring agent, a lubricant, an adsorbent, a preservative, a sweetener, a lyophilizing excipient (including a lyophilizing protectant) or a compression aid.

[0341] Most suitably, the polypeptide, pharmaceutical composition or construct of the present invention are administered orally. The key issue of oral delivery is to ensure that enough polypeptides, pharmaceutical compositions or constructs arrive in the area of ​​the intestinal tract where they are needed. Factors that prevent the polypeptide, pharmaceutical composition or construct of the present invention from arriving in the area of ​​the intestinal tract where they are needed include the presence of proteases in the digestive secretions, which can degrade the polypeptide, pharmaceutical composition or construct of the present invention. Suitably, the polypeptide, pharmaceutical composition or construct of the present invention are basically stable due to the inherent properties of the polypeptide or construct itself in the presence of one or more of such proteases. Suitably, the polypeptide of the present invention or construct are lyophilized before being incorporated into the pharmaceutical composition.

[0342] The polypeptide of the present invention can also be provided with an enteric coating. An enteric coating is a polymer barrier applied to an oral drug that helps prevent the polypeptide from being affected by the low pH of the stomach. Materials used for enteric coatings include fatty acids, waxes, shellac, plastics, and plant fibers. Suitable enteric coating components include methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, cellulose acetate hydroxypropyl methyl succinate (hydroxypropyl methylcellulose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymers, sodium alginate, and stearic acid. Suitable enteric coatings include pH-dependent release polymers. These are polymers that are insoluble at the high acidic pH present in the stomach but dissolve rapidly at lower acidic pH. Therefore, suitably, the enteric coating is insoluble in the acidic gastric juice of the stomach (pH ~ 3), but dissolves in the higher pH environment present in the small intestine (pH above 6) or in the colon (pH above 7.0). The pH dependent release polymer is selected such that the polypeptide or construct of the invention is released approximately at the time that the dose reaches the small intestine.

[0343] The polypeptide, construct or pharmaceutical composition of the present invention can be formulated into a preparation for injection by dissolving, suspending or emulsifying them in an aqueous or non-aqueous solvent, such as a vegetable oil or other similar oil, a fatty acid glyceride, an ester of a higher fatty acid or propylene glycol; and if necessary, with conventional additives, such as a solubilizing agent, an isotonic agent, a suspending agent, an emulsifier, a stabilizer and a preservative. Acceptable carriers, excipients and / or stabilizers are nontoxic to the recipient at the dosage and concentration employed, and include buffers such as phosphoric acid, citric acid and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, metacresol, parachlorometacresol, methylparaben or propylparaben, benzalkonium chloride or a combination thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine amino acids, tyrosine, tryptophan, methionine, serine, proline, and combinations thereof; monosaccharides, diosmin, and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents, such as EDTA; sugars, such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbitol, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or nonionic surfactants, such as polysorbates, POE ethers, poloxamers, Triton-X, or polyethylene glycol.

[0344] The pharmaceutical compositions of the invention can be delivered topically to the skin (e.g., for the treatment of autoimmune diseases such as psoriasis or eczema). Such pharmaceutical compositions can suitably be in the form of creams, ointments, lotions, gels, foams, transdermal patches, powders, pastes or tinctures, and can suitably include vitamin D3 analogs (e.g., calcipotriol and maxacalcitol), steroids (e.g., fluticasone propionate, betamethasone valerate and clobetasol propionate), retinoic acid (e.g., tazarotene), coal tar and anthralin. Topical agents are often used in combination with each other (e.g., vitamin D3 and steroids) or with other agents such as salicylic acid. If the pharmaceutical composition of the invention is delivered topically for the treatment of psoriasis or eczema, suitably another substance considered effective in treating psoriasis or eczema may be included in the composition, such as a steroid, particularly a Class 4 or 5 steroid such as hydrocortisone (e.g. 1% hydrocortisone cream); cyclosporine or a similar macrolide agent or tretinoin.

[0345] For all modes of delivery, the polypeptide, pharmaceutical composition or construct of the present invention can be formulated in a buffer to stabilize the pH of the composition at a concentration between 5-50 g / l, or more suitably between 15-40 g / l, or more suitably between 25-30 g / l. Examples of suitable buffer components include physiological salts, such as sodium citrate and / or citric acid. Suitably, the buffer contains 100-200 mM, more suitably 125-175 mM of physiological salts, such as sodium chloride. Suitably, the buffer is selected to have a pKa close to the pH of the composition or the physiological pH of the patient.

[0346] Exemplary polypeptide or construct concentrations in the pharmaceutical composition can range from about 1 mg / mL to about 200 mg / ml, or from about 50 mg / mL to about 200 mg / mL, or from about 150 mg / mL to about 200 mg / mL.

[0347] Aqueous formulations of polypeptides, constructs or pharmaceutical compositions of the present invention can be prepared in a pH buffer solution, for example, in a range of about 4.0 to about 7.0, or about 5.0 to about 6.0, or alternatively about 5.5. Examples of suitable buffers include phosphate, histidine, citric acid, succinic acid, acetate buffers and other organic acid buffers. Depending on the desired tension of, for example, the buffer and formulation, the buffer concentration can be about 1 mM to about 100 mM, or about 5 mM to about 50 mM.

[0348] The tension of the pharmaceutical composition can be changed by including a tension regulator. Such tension regulators can be charged or uncharged chemical substances. Typical uncharged tension regulators include sugars or sugar alcohols or other polyols, preferably trehalose, sucrose, mannitol, glycerol, 1,2-propylene glycol, raffinose, sorbitol or lactitol (especially trehalose, mannitol, glycerol or 1,2-propylene glycol). Typical charged tension regulators include salts, such as sodium, potassium or calcium ions and chloride, sulfate, carbonate, sulfite, nitrate, lactate, succinate, acetate or maleate ions (especially sodium chloride or sodium sulfate); or amino acids such as arginine or histidine. Suitably, aqueous formulations are isotonic, although hypertonic or hypotonic solutions may be suitable. The term "isotonic" represents a solution having the same tension as a certain other solution to which it is compared, such as a physiological salt solution or serum. The tonicity agent may be used in an amount of about 5 mM to about 350 mM, for example in an amount of 1 mM to 500 nM. Suitably, at least one isotonicity agent is included in the composition.

[0349] Surfactant can also be added to the pharmaceutical composition to reduce the aggregation of the polypeptide or construct of preparation and / or to minimize the formation of particles in the preparation and / or to reduce adsorption. Exemplary surfactants include polyoxyethylene sorbitol fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (Poloxamer, Pluronic) and sodium lauryl sulfate (SDS). The example of suitable polyoxyethylene sorbitol-fatty acid esters is polysorbate 20 and polysorbate 80. The scope of the exemplary concentration of surfactant is from about 0.001% to about 10% w / v.

[0350] Lyoprotectants may also be added to protect the polypeptide or construct of the invention from destabilizing conditions during the freezing process. For example, known lyoprotectants include sugars (including glucose, sucrose, mannose, and trehalose); polyols (including mannitol, sorbitol, and glycerol); and amino acids (including alanine, glycine, and glutamic acid). The lyoprotectant may be included in an amount of about 10 mM to 500 mM.

[0351] The dosage range for administering the polypeptide of the present invention, the pharmaceutical composition of the present invention or the construct is those dosage ranges that produce the desired therapeutic effect. The required dosage range depends on the accurate nature of the polypeptide of the present invention, the pharmaceutical composition or the construct, the route of administration, the nature of the preparation, the age of the patient, the nature, degree or severity of the patient's condition, contraindications (if any) and the judgment of the attending physician. The variation of these dosage levels can be adjusted to optimize using standard empirical routines.

[0352] Suitably, the range of daily dosage of polypeptide of the present invention, pharmaceutical composition of the present invention or construct is 50ng-50mg / kg, such as 50ug-40mg / kg, such as 5-30mg / kg. Unit dosage can change from less than 100mg, but usually in the region of 250-2000mg / dose, it can be used daily or more frequently, for example 2,3 or 4 times / day or less frequently, for example once every two days or once a week, once every two weeks or once a month.

[0353] In one aspect of the present invention, there is provided a use of a polypeptide, pharmaceutical composition or construct of the present invention in the manufacture of a medicament for treating an autoimmune disease. In another aspect of the present invention, there is provided a method for treating an autoimmune disease, comprising administering a therapeutically effective amount of a polypeptide, pharmaceutical composition or construct of the present invention to a person in need thereof.

[0354] The word 'treatment' is intended to include prophylactic as well as therapeutic treatment. Treatment of a disease also includes treating exacerbations and also includes treating a patient in remission from disease symptoms to prevent recurrence of disease symptoms.

[0355] Combination therapy

[0356] The pharmaceutical compositions of the present invention may also include one or more active agents (e.g., active agents suitable for treating the diseases mentioned herein). It is within the scope of the present invention to use the pharmaceutical compositions of the present invention in therapeutic methods for treating autoimmune diseases as an addition to or in combination with other established therapies commonly used in treating autoimmune diseases.

[0357] For the treatment of IBD (such as Crohn's disease or ulcerative colitis), possible combinations include combinations with, for example, one or more active agents selected from the list comprising: 5-aminosalicylic acid or a prodrug thereof (such as sulfasalazine, olsalazide or bisalazide); corticosteroids (e.g., prednisolone, methylprednisolone or budesonide); immunosuppressants (e.g., cyclosporine, tacrolimus, methotrexate, azathioprine or 6-mercaptopurine); anti-TNF-α antibodies (e.g., infliximab, adalimumab, certolizumab pegol or golimumab); anti-IL12 / IL23 antibodies (e.g., ustekinumab); anti-IL6R antibodies or small molecule IL12 / IL23 inhibitors (e.g., apimod); anti-α -4-beta-7 antibody (e.g., vedolizumab); MAdCAM-1 blocker (e.g., PF-00547659); antibody against cell adhesion molecule α-4-integrin (e.g., natalizumab); antibody against IL2 receptor α subunit (e.g., daclizumab or basiliximab); JAK3 inhibitor (e.g., tofacitinib or R348); Syk inhibitor and its prodrug (e.g., fotantinib and R-406); phosphodiesterase-4 inhibitor (e.g., tetomilast); HMPL-004; probiotics; dersalazine; semamod / CPSI-2364; and protein kinase C inhibitor (e.g., AEB-071). The most suitable combination agent is infliximab, adalimumab, certolizumab pegol or golimumab.

[0358] Thus, another aspect of the invention provides a pharmaceutical composition of the invention in combination with one or more additional active agents, for example one or more of the active agents described above.

[0359] In another aspect of the invention, the polypeptide, pharmaceutical composition or construct and at least one active agent selected from the above list are administered sequentially, simultaneously or separately.

[0360] Similarly, another aspect of the present invention provides a combination comprising:

[0361] (A) a polypeptide, pharmaceutical composition or construct of the present invention; and

[0362] (B) one or more other active agents,

[0363] Wherein each of components (A) and (B) is mixed with a pharmaceutically acceptable adjuvant, diluent or carrier. In this aspect of the invention, the combination product can be a single (combination) formulation or a kit-of-parts. Therefore, this aspect of the invention encompasses a combined formulation comprising a polypeptide, pharmaceutical composition or construct of the invention and another therapeutic agent mixed with a pharmaceutically acceptable adjuvant, diluent or carrier.

[0364] The present invention also encompasses a kit comprising the following components:

[0365] (i) a polypeptide, pharmaceutical composition or construct of the present invention mixed with a pharmaceutically acceptable adjuvant, diluent or carrier; and

[0366] (ii) a formulation comprising one or more other active agents in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, wherein components (i) and (ii) are each provided in a form suitable for administration in combination with each other.

[0367] Therefore, component (i) of the kit of parts is the above component (A) mixed with a pharmaceutically acceptable adjuvant, diluent or carrier. Similarly, component (ii) is the above component (B) mixed with a pharmaceutically acceptable adjuvant, diluent or carrier. One or more other active agents (i.e., above component (B)) can be any of the agents mentioned in the above combination autoimmune diseases such as IBD (e.g., Crohn's disease and / or ulcerative colitis) treatment. If component (B) is more than one other active agent, these other active agents can be formulated with each other or with component (A) or they can be formulated separately. In one embodiment, component (B) is a kind of other therapeutic agent. In another embodiment, component (B) is two other therapeutic agents. The combination product (combination preparation or kit of parts) of this aspect of the present invention can be used for the treatment or prevention of autoimmune diseases (e.g., autoimmune diseases mentioned herein).

[0368] Suitably, the polypeptide, pharmaceutical composition or construct of the invention is for use as a medicament and more suitably for use in the treatment of autoimmune diseases and / or inflammatory diseases.

[0369] Autoimmune and / or inflammatory diseases

[0370] Autoimmune diseases occur when the immune system has adverse responses to normal body tissues. Autoimmune disorders can cause damage to body tissues, abnormal organ growth and / or changes in organ function. The disorder can only affect one organ or tissue type or can affect multiple organs and tissues. Organs and tissues that are usually affected by autoimmune disorders include blood components such as red blood cells, blood vessels, connective tissues, endocrine glands such as thyroid or pancreas, muscles, joints and skin. Inflammatory diseases are diseases characterized by inflammation. Many inflammatory diseases are autoimmune diseases and vice versa.

[0371] Autoimmune and / or inflammatory diseases of the GIT

[0372] Chronic inflammatory bowel disease (IBD) Crohn's disease and ulcerative colitis, which afflict children and adults, are examples of autoimmune and inflammatory diseases of the GIT (Hendrickson et al. 2002). Ulcerative colitis is defined as a condition in which the inflammatory response and morphological changes remain confined to the colon. The rectum is involved in 95% of patients. Inflammation is largely confined to the mucosa and includes variable severity of ulcers, edema and bleeding and continuous damage to the length of the colon (Hendrickson et al. 2002). Ulcerative colitis is usually manifested by the presence of mixed blood and mucus in the stool and lower abdominal cramps, which are most severe during defecation. Clinically, the presence of blood and mucosa in diarrhea distinguishes ulcerative colitis from irritable bowel syndrome in which blood is not present. Unlike ulcerative colitis, the presentation of Crohn's disease is usually less obvious, which leads to late diagnosis. Factors such as the location, extent and severity of the damage determine the extent of gastrointestinal symptoms. Patients with ileocolic damage usually have postprandial abdominal pain and right lower abdominal tenderness and occasional inflammatory masses.Symptoms associated with gastroduodenal Crohn's disease include early satiety, nausea, vomiting, epigastric pain or dysphagia.Perianal disease and anal tags, deep anal fissures and fistulas are common (Hendrickson et al. 2002).Other diseases of GIT include, for example, inflammatory disease mucositis (suitably, drug and radiation-induced mucositis).In mucositis, focus can occur anywhere from the mouth to the anus, and for the mouth and esophageal focus, a mouthwash or cream preparation containing variable domains can be used.For anal and rectal lesions, suppositories, creams or foams containing variable domains will be suitable for local application.Immunoglobulin chain variable domains will be removed from the lamina propria or other inflammatory sites via being adsorbed into the bloodstream at the inflammation site or via lymphatic removal and subsequently entering the bloodstream.Therefore, the domain arrives at the lungs via bloodstream and is removed via glomerular filtration in the kidney. There is therefore a good rationale for the domains to play a therapeutic role in diseases such as autoimmune diseases, type II diabetes and glomerulonephritis.

[0373] Suitably, the polypeptides, pharmaceutical compositions or constructs of the invention are used to treat autoimmune and / or inflammatory diseases of the GI (gastrointestinal) tract in which IL-23 contributes to the pathology of such diseases.

[0374] Suitably, the polypeptides, pharmaceutical compositions or constructs of the invention are used to treat autoimmune and / or inflammatory diseases of the GI tract selected from Crohn's disease, ulcerative colitis, irritable bowel disease, type II diabetes, glomerulonephritis, autoimmune hepatitis, Sjogren's syndrome, celiac disease and drug or radiation induced mucositis (more suitably Crohn's disease or ulcerative colitis, most suitably ulcerative colitis).

[0375] Oral delivery of immunoglobulin chain variable domains would ideally treat inflammatory diseases in which IL-23 contributes to at least a portion of the pathology, and the immunoglobulin chain variable domains can reach tissues in which IL-23 is biologically active.

[0376] Autoimmune and / or inflammatory diseases of the skin

[0377] Psoriasis is a debilitating dermatological autoimmune disease. Plaque psoriasis (the most common form of the disease) is characterized by red skin covered with silvery scales. Histologically, the condition is one of dedifferentiation and hyperproliferation of keratinocytes within psoriatic plaques with inflammatory cell infiltration (Ortonne, 1999). Psoriatic skin lesions are inflammatory, red, well-defined plaques of various shapes with characteristic silvery, shiny scales. The term psoriasis includes psoriasis and psoriasis symptoms including erythema, skin thickening / raised and scales.

[0378] Biologic agents used to treat psoriasis include anti-TNF-α therapies (such as monoclonal antibodies against TNF, e.g., adalimumab and infliximab, or TNF-α receptor fusion proteins, such as etanercept), humanized antibodies against CD11a (efalizumab), or agents that bind to CD2 such as afacept (thus blocking the CD2 LFA3 interaction). It should be noted that not all of the biologic agents listed here have been approved for the treatment of psoriasis.

[0379] The polypeptides of the invention may be incorporated into a cream / ointment or other topical vehicle for administration to inflammatory skin lesions where IL-23 contributes to the pathology of such lesions.

[0380] Suitably, the polypeptide, pharmaceutical composition or construct of the invention is for use in the treatment of an autoimmune and / or inflammatory disease of the skin selected from the group consisting of pemphigus, psoriasis, eczema and scleroderma.

[0381] Suitably, the polypeptide, pharmaceutical composition or construct is used to treat other autoimmune / inflammatory diseases in which IL-23 is responsible for part of the observed pathology.

[0382] Preparation method

[0383] The polypeptides of the present invention can be obtained and manipulated using, for example, the techniques disclosed in Green and Sambrook 2012 Molecular Cloning: A Laboratory Manual 4th edition Cold Spring Harbour Laboratory Press.

[0384] Monoclonal antibodies can be produced using hybridoma technology by fusing B cells that produce specific antibodies with myeloma (B cell cancer) cells that are selected for their ability to grow in tissue culture and the absence of antibody chain synthesis. and Milstein 1975 and Nelson et al. 2000 ).

[0385] Monoclonal antibodies directed against a defined antigen can be obtained, for example, by:

[0386] a) immortalizing lymphocytes obtained from the peripheral blood of an animal previously immunized with a defined antigen with immortalized cells, preferably myeloma cells, in order to form hybridomas,

[0387] b) Culturing the resulting immortalized cells (hybridomas) and recovering cells producing antibodies with the desired specificity.

[0388] Alternatively, there is no need to use hybridoma cells. Thus, monoclonal antibodies can be obtained by a method comprising the following steps:

[0389] a) cloning a DNA or cDNA sequence obtained from lymphocytes, especially peripheral blood lymphocytes, of an animal (suitably previously immunized with a defined antigen) into a vector, especially into a bacteriophage and more particularly a filamentous phage,

[0390] b) transforming prokaryotic cells with the above vector under conditions that allow the production of antibodies,

[0391] c) selecting the antibodies by subjecting the antibodies to antigen-affinity selection,

[0392] d) Recovering antibodies with the desired specificity.

[0393] Methods for immunizing camels, cloning the VHH repertoire of B cells circulating in the blood (Chomezynnski and Sacchi 1987) and isolating antigen-specific VHH from immune (Arbabi-Ghahroudi et al. 1997) and non-immune (Tanha et al. 2002) libraries using phage, yeast or ribosome display are known (WO92 / 01047, Nguyen et al. 2001 and Harmsen et al. 2007).

[0394] Antigen binding fragments of antibodies such as scFv and Fv fragments can be isolated and expressed in E. coli (Miethe et al. 2013, Skerra et al. 1988 and Ward et al. 1989).

[0395] The DNA or cDNA encoding the polypeptide may be mutated, which mutation is silent for the amino acid sequence of the polypeptide, but provides preferred codons for translation by a particular host. Preferred codons for translation of nucleic acids in, for example, Escherichia coli and Saccharomyces cerevisiae are known.

[0396] The sudden change of polypeptide can be realized for example by replacing, adding or lacking the nucleic acid encoding polypeptide.Replacement, adding or lacking the nucleic acid encoding polypeptide can be introduced by many methods, including for example fallibility PCR, reorganization, oligonucleotide directed mutagenesis, assembly PCR, PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, cycle overall mutagenesis, index overall mutagenesis, site-specific mutagenesis (Ling et al. 1997), gene reassembly, gene site saturation mutagenesis (GSSM), synthetic connection reassembly (SLR) or the combination of these methods.The modification, adding or lacking of nucleic acid can also be introduced by including the following method: reorganization, cycle sequence reorganization, thiophosphate modified DNA mutagenesis, template mutagenesis containing uracil, gap duplex mutagenesis (gapped duplex mutagenesis), point mismatch repair mutagenesis (point mismatch repair mutagenesis), repair-defective host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, overall mutagenesis, chimeric nucleic acid polymer generation or its combination.

[0397] Specifically, artificial gene synthesis can be used (Nambiar et al. 1984, Sakamar and Khorana 1988, Wells et al. 1985 and Grundstrom et al. 1985). The gene encoding the polypeptide of the present invention can be synthesized by, for example, solid phase DNA synthesis. The entire gene can be synthesized de novo without the need for precursor template DNA. In order to obtain the desired oligonucleotide, the building blocks are sequentially coupled to the growing oligonucleotide chain in the order required for the product sequence. After the chain assembly is completed, the product is released from the solid phase into the solution, deprotected and collected. The product can be separated by high performance liquid chromatography (HPLC) to obtain the desired high purity oligonucleotide (Verma and Eckstein 1998)

[0398] The expression of immunoglobulin chain variable domains such as VH and VHH can be achieved using suitable expression vectors such as prokaryotic cells such as bacteria, for example Escherichia coli (for example according to the protocol disclosed in WO94 / 04678, which is incorporated herein by reference and described in further detail below). The expression of immunoglobulin chain variable domains such as VH and VHH can also be achieved using eukaryotic cells such as insect cells, CHO cells, Vero cells or suitably yeast cells, such as yeast belonging to the genus Aspergillus, Saccharomyces, Kluyveromyces, Hansenula or Pichia. Suitably, Saccharomyces cerevisiae is used (for example, according to the protocol disclosed in WO94 / 025591, which is incorporated herein by reference and described in further detail below).

[0399] Specifically, VHH can be prepared using Escherichia coli according to the method disclosed in WO94 / 04678 by a method comprising the following steps:

[0400] a) cloning a DNA or cDNA sequence encoding a VHH (e.g. obtained from lymphocytes of camel or produced synthetically), optionally comprising a His-tag, in a Bluescript vector (Agilent Technologies),

[0401] b) after amplification, the cloned fragment was recovered using a 5' primer containing an XhoI site specific for VHH and a 3' primer containing a SpeI site having the sequence TC TTA ACT AGT GAG GAG ACG GTG ACC TG (SEQ ID NO: 68),

[0402] c) cloning of the fragment recovered in the phase in the Immuno PBS vector after digestion of the vector with XhoI and SpeI restriction enzymes (Huse et al. 1989),

[0403] d) transforming a host cell, especially Escherichia coli, by transfection using the recombinant Immuno PBS vector of step c,

[0404] e) Recovering the expression product of the VHH coding sequence, for example by affinity purification, such as by chromatography on a column using protein A, cation exchange or nickel-affinity resin (if the VHH comprises a His-tag).

[0405] Alternatively, immunoglobulin chain variable domains such as VH and VHH can be obtained by a method comprising the following steps:

[0406] a) obtaining a DNA or cDNA sequence encoding a VHH having a defined specific antigen binding site,

[0407] b) amplifying the obtained DNA or cDNA using a 5' primer containing a start codon and a Hindlll site and a 3' primer containing a stop codon and having an XhoI site,

[0408] c) recombining the amplified DNA or cDNA into the Hindlll (position 2650) and XhoI (position 4067) sites of plasmid pMM984 (Merchlinsky et al. 1983),

[0409] d) transfection of permissive cells, in particular NB-E cells (Faisst et al. 1995), with the recombinant plasmid,

[0410] e) recovering the product obtained.

[0411] Additionally, immunoglobulin chain variable domains such as VHH or VH can be produced using E. coli or S. cerevisiae according to the methods disclosed in Frenken et al. 2000 and WO99 / 23221 (incorporated herein by reference in their entirety), as follows:

[0412] After taking a blood sample from the immunized llama and enriching the lymphocyte population via Ficoll (neutral highly branched, high-quality hydrophilic polysaccharide that dissolves rapidly in aqueous solution - Pharmacia) discontinuous gradient centrifugation, isolating total RNA by acid guanidine thiocyanate extraction (Chomezynnski and Sacchi 1987), and first-strand cDNA synthesis (e.g., using a cDNA kit such as RPN 1266 (Amersham)), a DNA fragment encoding VHH and VH fragments and a portion of the short or long hinge region was amplified by PCR using specific primers described in detail on pages 22 and 23 of WO99 / 23221. After digestion of the PCR fragment with PstI and HindIII or BstEII, the DNA fragment with a length between about 300 and 450 bp was purified via agarose gel electrophoresis and ligated in the E. coli phagemid vector pUR4536 or the episomal S. cerevisiae expression vector pUR4548, respectively. pUR4536 was derived from pHEN (Hoogenboom et al. 1991) and contains lacI q pUR4548 was derived from pSY1 (Harmsen et al. 1993). The BstEII site in the leu2 gene was removed from this plasmid via PCR, and the cloning site between the SUC2 signal sequence and the terminator was replaced to facilitate the cloning of VH / VHH gene fragments. VH / VHH has a c-myc tag at the C-terminus for detection. Individual E. coli JM109 colonies were transferred to medium supplemented with 1% glucose and 100 mg L -1 After overnight growth (37°C), the cells were plated in 96-well microtiter plates containing 150 ml of 2TY medium containing 100 mg L -1 The plate is repeated in the 2TY culture medium of ampicillin and 0.1mMIPTG. After overnight incubation again and optionally freezing and thawing, the cells are centrifuged and precipitated, and the supernatant can be used in ELISA. Each Saccharomyces cerevisiae colony is transferred to a test tube containing a selective minimal culture medium (comprising 0.7% yeast nitrogen base, 2% glucose, supplemented with essential amino acids and alkali), and grown for 48h at 30°C. Subsequently, the culture is diluted ten times in YPGal culture medium (comprising 1% yeast extract, 2% bacterial peptone and 5% galactose). After growing for 24h and 48h, the cells are precipitated and the culture supernatant can be analyzed in ELISA. The absorbance (OD600) at 600nm is optionally measured.

[0413] Additionally, immunoglobulin chain variable domains such as VH / VHH can be produced using Saccharomyces cerevisiae using the following procedure:

[0414] Isolate the naturally occurring DNA sequence encoding VH / VHH or obtain a synthetically produced DNA sequence encoding VH / VHH, comprising a 5'-UTR, a signal sequence, a stop codon and flanked by Sad and HindIII sites (such synthetic sequences can be produced as outlined above or can be ordered, for example, from commercial suppliers such as Geneart (Life Technologies)).

[0415] The VH / VHH genes are transferred to the multicopy integration (MCI) vector pUR8569 or pUR8542 using restriction sites as described below. The DNA sequences encoding VHH, optionally contained in the shuttle vector, cassette or other synthetic gene construct and MCI vector, are cut with Sad and HindIII using the following: 25ul VHH DNA (Geneart plasmid or MCI vector), 1ul Sad, 1ul HindIII, 3ul of a suitable buffer for double enzyme digestion such as NEB buffer 1 (New England Biolabs), at 37°C overnight. 25ul of the digested VHH-encoding DNA and 25ul of the digested MCI vector are run on a 1.5% agarose gel with 1xTAE buffer and then gel extracted, for example, using a QIAquick gel extraction kit (Qiagen). The ligation of digested MCI vector and digested DNA encoding VH / VHH was set up as follows: 100 ng vector, 30 ng VHH gene, 1.5 ul 10× ligase buffer, 1 ul T4 DNA ligase and ddH 2 O. Ligation was then performed at 16°C overnight.

[0416] Next, transform the E. coli cells. For chemically competent XL-1 blue cells, 200ul of heat competent XL-1 blue cells were thawed and 5ul of ligation mixture was added on ice for about 30 minutes, followed by heat shock at 42°C for 90 seconds. Then 800ul of Luria-Bertani low salt medium supplemented with 2% glucose was added, and the cells were recovered at 37°C for 2 hours. The cells were plated on Luria-Bertani agar and ampicillin (100ug / ml) plates and kept overnight at 37°C. For electrocompetent TG1 E. coli cells, an electroporation cup was used. In the electroporation cup: 50ul of electrocompetent TG1 cells and 1ul of ligation mixture were thawed on ice for about 15 minutes. The cup was placed in a support and pulsed. 500ul of 2TY culture medium was added and the cells were recovered at 37°C for 30 minutes. 100ul of cells were plated on Luria-Bertani agar plates containing ampicillin (100ug / ml) and 2% glucose. The plates were kept at 37°C overnight.

[0417] After cloning the VH / VHH genes into E. coli as described in detail above, the linearized MCI vector can be used to transform S. cerevisiae. Prior to transformation, several steps are performed: (i) the DNA should be changed from circular to linear by digestion, otherwise the DNA cannot be integrated into the yeast genome, and (ii) the digested DNA should be cleared of impurities by ethanol precipitation. In addition, during the transformation process, the yeast cells are made semipermeable so that the DNA can pass through the membrane.

[0418] Preparation for yeast transformation: Midi-prep prepared from selected E. coli colonies expressing VH / VHH genes was digested with HpaI as described below. Prepare a 10 μl yeast culture medium containing 20 ng midi-prep, 5 μl HpaI, 10 μl of an appropriate buffer such as NEB4 buffer (BioLabs), and ddH 2 0.5 μl of 1% HO solution.

[0419] Cut the DNA with HpaI overnight at room temperature. Next, perform an ethanol precipitation (and set aside the 5ul sample from the HpaI digestion). Add 300ul of 100% ethanol to the 95ul HpaI digested midiprep, vortex and spin at full speed for 5 minutes. Carefully decant when a pellet is present, add 100ul of 70% ethanol, and then spin again at full speed for 5 minutes. Decant the sample again and keep at 50-60°C until the pellet is dry. Resuspend the pellet in 50ul ddH 2 O. Run 5ul on the gel next to 5ul of HpaI digested sample.

[0420] Yeast transformation: Prepare YNBglu plates. Use 10 g agar + 425 ml water (sterilized), 25 ml filtered 20× YNB (3.35 g YNB (yeast nitrogen base), in 25 ml sterilized HO. 2 0) and 50ml sterilized 20% glucose and poured into a petri dish. Select a yeast colony from the mother plate and grow overnight in 3ml YPD (yeast extract peptone glucose) at 30°C. Prepare about 600ml YPD on the second day and fill 3 flasks with 275ml, 225ml and 100ml YPD. Add 27.5ul yeast YPD culture to the first flask and mix lightly. Take 75ml from the first flask and place it in the second flask, mix lightly. Take 100ml from the second flask and place it in the third flask, mix lightly. Grow until an OD660 between 1 and 2 is reached. The flasks that reach this OD are distributed in 4 Falcon tubes, ±45ml in each. Spin at 4200rpm for 2 minutes. Discard the supernatant. The pellets in the two Falcon tubes are dissolved in 45ml H 2 O (reduce the number of tubes from 4 to 2). Spin at 4200 rpm for 2 minutes. Dissolve the pellet in 45 ml HO. 2 O (from 2 tubes to 1 tube). Spin at 4200rpm for 2 minutes. Dissolve the pellet lightly in 5ml lithium acetate (LiAc) (100mM) and spin for a few seconds. Carefully discard some of the LiAc, but keep more than half of the LiAc in the tube. Vortex the cells, boil the vector DNA for 5 minutes and quickly cool in ice water. Add to a 15ml tube containing the following: 240ul PEG, 50ul cells, 36uLiAc (1M), 25ul vector DNA, 45ul ethanol precipitated VH / VHH. Mix gently after each step (the blank sample is treated the same way, but there is no ethanol precipitated VH / VHH). Incubate at 30°C for 30 minutes, invert the tube slightly 3-4 times, and then heat shock at 42°C for 20-25 minutes. Spin at 6000rpm for a short time. Gently remove the supernatant and add 250ul ddH 2O and mix. All of them are streaked on YNBglu plates until the plates are dry and grown at 30°C for 4-5 days. Finally, YNBglu plates are prepared by the following: the plates are divided into 6 equal parts, each part is numbered 1 to 6, the largest colony is inoculated and number 1 is streaked. Repeat the above process for other colonies from 1 to 6 from large to small. Grow at 30°C for 3-4 days until colonies are produced. Glucose is used as a carbon source to grow VH / VHH colonies, and VH / VHH induction is performed by opening the galactose-7-promoter by adding 0.5% galactose. 3mL small-scale culture is carried out to test colonies, and the colonies showing the best expression of VH or VHH are selected. This colony is then used in purification.

[0421] Purification: VH / VHH are purified by cation exchange chromatography with a strong anion resin such as Capto S. On day 1, inoculate a selected yeast colony expressing VH / VHH in 5 ml YPD medium (YP medium + 2% glucose) and grow the cells in a 25 mL sealed sterilized tube at 30°C (shaking at 180 rpm). On day 2, dilute 5 ml of the overnight culture in 50 mL of freshly prepared YP medium + 2% glucose + 0.5% galactose and grow the cells in a 250 ml aerated baffled flask at 30°C for two nights (shaking at 180 rpm). On day 4, spin down the cells in a centrifuge at 4200 rpm for 20 min. Cation exchange chromatography using a strong anion resin: The pH of the supernatant containing the ligand is adjusted to 3.5. Wash with 50 mL ddH 2 0.75ml resin (+ / -0.5mL slurry) / 50mL supernatant was washed, followed by three washes with binding buffer. The washed resin was added to the supernatant and the suspension was incubated on a shaker at 4°C for 1.5 hours. The resin-bound VH / VHH was precipitated by centrifugation at 500g for 2 minutes and washed with washing buffer. The supernatant was decanted and the resin was resuspended with 10mL binding buffer. The filtrate was placed in a PD-10 column, the resin was poured into the column and the resin was precipitated for a while, and then the filtrate was added to the resin. Wait until all binding buffer runs through. VH / VHH was eluted with 6x 0.5ml elution buffer. The elution fractions were collected in eppendorf tubes. The protein concentration of 6 elution fractions was measured with Nanodrop. The fractions containing VHH were pooled and the solution was transferred to a 3,500Da cut-off dialysis membrane. The purified protein solution was dialyzed with 3L PBS at 4°C overnight. On day 5, the purified protein solution was dialyzed against 2 L of fresh PBS at 4 °C for another 2 h. Finally, the final concentration was calculated by BCA.

[0422] Although discussed in the context of VH / VHH, the techniques described above can also be applied to scFv, Fab, Fv and other antibody fragments if desired.

[0423] Multiple antigen binding fragments (suitably VH / VHH) can be fused by chemical cross-linking by reacting the amino acid residues with an organic derivatizing agent, such as described by Blattler et al. 1985. Alternatively, the antigen binding fragments can be genetically fused at the DNA level, i.e. the polynucleotide construct formed encodes a complete polypeptide construct comprising one or more antigen binding fragments. One way to join multiple antigen binding fragments via a genetic approach is to connect the antigen binding fragment coding sequences directly or via a peptide linker. For example, the carboxyl terminus of the first antigen binding fragment can be connected to the amino terminus of the next antigen binding fragment. This connection mode can be extended so that the antigen binding fragments are connected for the construction of tri-, tetra-, etc. functional constructs. Methods for generating multivalent (such as bivalent) VHH polypeptide constructs are disclosed in WO 96 / 34103 (incorporated herein in its entirety by reference).

[0424] Suitably, polypeptides of the invention (particularly VHHs of the invention) may be produced in fungi, such as yeast (e.g., Saccharomyces cerevisiae), according to the methods disclosed in WO02 / 48382, comprising growing the fungi on a medium comprising a carbon source, wherein 50-100 wt% of the carbon source is ethanol. Large-scale production of VHH fragments in Saccharomyces cerevisiae is described in Thomassen et al. 2002.

[0425] In one aspect of the present invention, there is provided a method for preparing a polypeptide or construct of the present invention, comprising the following steps:

[0426] i) cloning the polynucleotide of the present invention into a vector, such as a plasmid,

[0427] ii) transforming a cell capable of producing a polypeptide or construct of the invention, such as a bacterial cell or a yeast cell, with the vector under conditions allowing production of the polypeptide or construct,

[0428] iii) recovering the polypeptide or construct, such as by affinity chromatography.

[0429] The clauses listing further embodiments of the present invention are as follows:

[0430] 1. A polypeptide comprising an immunoglobulin chain variable domain that binds to IL-23, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR1 comprises a sequence that shares 60% or greater sequence identity with SEQ ID NO:1, CDR2 comprises a sequence that shares 50% or greater sequence identity with SEQ ID NO:2, and CDR3 comprises a sequence that shares 50% or greater sequence identity with SEQ ID NO:3.

[0431] 2. The polypeptide according to clause 1, wherein CDR3 comprises a sequence that shares 80% or greater sequence identity with SEQ ID NO:3.

[0432] 3. The polypeptide according to clause 2, wherein CDR3 consists of a sequence that shares 80% or greater sequence identity with SEQ ID NO:3.

[0433] 4. A polypeptide according to clause 2 or 3, wherein any residue of CDR3 that differs from its corresponding residue in SEQ ID NO: 3 is a conservative substitution relative to its corresponding residue.

[0434] 5. A polypeptide according to clause 2 or 3, wherein the residue of CDR3 corresponding to residue number 6 of SEQ ID NO: 3 is 1.

[0435] 6. A polypeptide according to clause 2 or 3, wherein the residue of CDR3 corresponding to residue number 6 of SEQ ID NO: 3 is L.

[0436] 7. A polypeptide according to clause 4, wherein the residue of CDR3 corresponding to residue number 6 of SEQ ID NO: 3 is I or L, and any other residue of CDR3 that differs from its corresponding residue in SEQ ID NO: 3 is a conservative substitution relative to its corresponding residue.

[0437] 8. The polypeptide according to clause 2, wherein CDR3 comprises SEQ ID NO: 3.

[0438] 9. The polypeptide according to clause 3, wherein CDR3 consists of SEQ ID NO: 3.

[0439] 10. The polypeptide according to any one of clauses 1 to 9, wherein CDR1 comprises a sequence sharing 80% or greater sequence identity with SEQ ID NO: 1.

[0440] 11. The polypeptide according to clause 10, wherein CDR1 consists of a sequence sharing 80% or greater sequence identity with SEQ ID NO: 1.

[0441] 12. A polypeptide according to any one of clauses 1 to 9, wherein any residue of CDR1 which differs from its corresponding residue in SEQ ID NO: 1 is a conservative substitution relative to its corresponding residue.

[0442] 13. The polypeptide according to clause 11, wherein CDR1 comprises SEQ ID NO: 1.

[0443] 14. The polypeptide according to clause 13, wherein CDR1 consists of SEQ ID NO: 1.

[0444] 15. A polypeptide according to any one of clauses 1 to 14, wherein CDR2 comprises a sequence that shares 55% or greater sequence identity, such as shares 60% or greater sequence identity, such as shares 70% or greater sequence identity, such as shares 75% or greater sequence identity, such as shares 80% or greater sequence identity, such as shares 85% or greater sequence identity, such as shares 90% or greater sequence identity with SEQ ID NO:2.

[0445] 16. A polypeptide according to clause 15, wherein CDR2 consists of a sequence that shares 55% or greater sequence identity, such as sharing 60% or greater sequence identity, such as sharing 70% or greater sequence identity, such as sharing 75% or greater sequence identity, such as sharing 80% or greater sequence identity, such as sharing 85% or greater sequence identity, such as sharing 90% or greater sequence identity with SEQ ID NO:2.

[0446] 17. A polypeptide according to any one of clauses 1 to 16, wherein the residue of CDR2 corresponding to residue number 9 of SEQ ID NO: 2 is D or H, and / or the residue of CDR2 corresponding to residue number 10 of SEQ ID NO: 2 is Y or D, and / or the residue of CDR2 corresponding to residue number 11 of SEQ ID NO: 2 is S, G, R or A (such as S, R or A, such as S or A) and / or the residue of CDR2 corresponding to residue number 14 of SEQ ID NO: 2 is V or A.

[0447] 18. A polypeptide according to any one of clauses 1 to 17, wherein any residue of CDR2 that differs from its corresponding residue in SEQ ID NO: 2 is a conservative substitution relative to its corresponding residue.

[0448] 19. The polypeptide according to clause 15, wherein CDR2 comprises SEQ ID NO: 2.

[0449] 20. The polypeptide according to clause 19, wherein CDR2 consists of SEQ ID NO: 2.

[0450] 21. A polypeptide according to any of clauses 1 to 20, wherein FR1 comprises a sequence that shares 5% or greater sequence identity, such as sharing 12% or greater sequence identity, such as sharing 18% or greater sequence identity, such as sharing 26% or greater sequence identity, such as sharing 32% or greater sequence identity, such as sharing 38% or greater sequence identity, such as sharing 46% or greater sequence identity, such as sharing 52% or greater sequence identity, such as sharing 58% or greater sequence identity, such as sharing 62% or greater sequence identity, such as sharing 66% or greater sequence identity, such as sharing 68% or greater sequence identity, such as sharing 72% or greater sequence identity, such as sharing 75% or greater sequence identity, such as sharing 78% or greater sequence identity, such as sharing 82% or greater sequence identity, such as sharing 85% or greater sequence identity, such as sharing 90% or greater sequence identity, such as sharing 95% or greater sequence identity with SEQ ID NO:4.

[0451] 22. A polypeptide according to clause 21, wherein FR1 consists of a sequence that shares 5% or greater sequence identity, such as sharing 12% or greater sequence identity, such as sharing 18% or greater sequence identity, such as sharing 26% or greater sequence identity, such as sharing 32% or greater sequence identity, such as sharing 38% or greater sequence identity, such as sharing 46% or greater sequence identity, such as sharing 52% or greater sequence identity, such as sharing 58% or greater sequence identity, such as sharing 62% or greater sequence identity, such as sharing 66% or greater sequence identity, such as sharing 68% or greater sequence identity, such as sharing 72% or greater sequence identity, such as sharing 75% or greater sequence identity, such as sharing 78% or greater sequence identity, such as sharing 82% or greater sequence identity, such as sharing 85% or greater sequence identity, such as sharing 90% or greater sequence identity, such as sharing 95% or greater sequence identity with SEQ ID NO:4.

[0452] 23. The polypeptide according to any one of clauses 1 to 22, wherein the residue of FR1 corresponding to residue number 1 of SEQ ID NO: 4 is D or E.

[0453] 24. The polypeptide according to clause 23, wherein the residue of FR1 corresponding to residue number 1 of SEQ ID NO: 4 is D.

[0454] 25. The polypeptide according to clause 24, wherein the residues of FR1 corresponding to residue numbers 1 to 5 of SEQ ID NO: 4 are DVQLV.

[0455] 26. A polypeptide according to any one of clauses 1 to 25, wherein any residue of FR1 which differs from its corresponding residue in SEQ ID NO: 4 is a conservative substitution relative to its corresponding residue.

[0456] 27. The polypeptide according to clause 21, wherein FR1 comprises SEQ ID NO:4.

[0457] 28. The polypeptide according to clause 27, wherein FR1 consists of SEQ ID NO:4.

[0458] 29. A polypeptide according to any one of clauses 1 to 28, wherein FR2 comprises a sequence that shares 10% or greater sequence identity, such as sharing 15% or greater sequence identity, such as sharing 25% or greater sequence identity, such as sharing 30% or greater sequence identity, such as sharing 40% or greater sequence identity, such as sharing 45% or greater sequence identity, such as sharing 55% or greater sequence identity, such as sharing 60% or greater sequence identity, such as sharing 70% or greater sequence identity, such as sharing 75% or greater sequence identity, such as sharing 85% or greater sequence identity, such as sharing 90% or greater sequence identity with SEQ ID NO:5.

[0459] 30. A polypeptide according to clause 29, wherein FR2 consists of a sequence that shares 10% or greater sequence identity, such as sharing 15% or greater sequence identity, such as sharing 25% or greater sequence identity, such as sharing 30% or greater sequence identity, such as sharing 40% or greater sequence identity, such as sharing 45% or greater sequence identity, such as sharing 55% or greater sequence identity, such as sharing 60% or greater sequence identity, such as sharing 70% or greater sequence identity, such as sharing 75% or greater sequence identity, such as sharing 85% or greater sequence identity, such as sharing 90% or greater sequence identity with SEQ ID NO:5.

[0460] 31. A polypeptide according to any one of clauses 1 to 30, wherein any residue of FR2 which differs from its corresponding residue in SEQ ID NO: 5 is a conservative substitution relative to its corresponding residue.

[0461] 32. The polypeptide according to clause 29, wherein FR2 comprises SEQ ID NO:5.

[0462] 33. The polypeptide according to clause 32, wherein FR2 consists of SEQ ID NO:5.

[0463] 34. A polypeptide according to any one of clauses 1 to 33, wherein FR3 comprises a sequence that shares 8% or greater sequence identity, such as sharing 15% or greater sequence identity, such as sharing 20% ​​or greater sequence identity, such as sharing 26% or greater sequence identity, such as sharing 32% or greater sequence identity, such as sharing 40% or greater sequence identity, such as sharing 45% or greater sequence identity, such as sharing 52% or greater sequence identity, such as sharing 58% or greater sequence identity, such as sharing 65% or greater sequence identity, such as sharing 70% or greater sequence identity, such as sharing 76% or greater sequence identity, such as sharing 80% or greater sequence identity, such as sharing 82% or greater sequence identity, such as sharing 85% or greater sequence identity, such as sharing 90% or greater sequence identity, such as sharing 92% or greater sequence identity, such as sharing 95% or greater sequence identity with SEQ ID NO:6.

[0464] 35. A polypeptide according to clause 34, wherein FR3 consists of a sequence that shares 8% or greater sequence identity, such as sharing 15% or greater sequence identity, such as sharing 20% ​​or greater sequence identity, such as sharing 26% or greater sequence identity, such as sharing 32% or greater sequence identity, such as sharing 40% or greater sequence identity, such as sharing 45% or greater sequence identity, such as sharing 52% or greater sequence identity, such as sharing 58% or greater sequence identity, such as sharing 65% or greater sequence identity, such as sharing 70% or greater sequence identity, such as sharing 76% or greater sequence identity, such as sharing 80% or greater sequence identity, such as sharing 82% or greater sequence identity, such as sharing 85% or greater sequence identity, such as sharing 90% or greater sequence identity, such as sharing 92% or greater sequence identity, such as sharing 95% or greater sequence identity with SEQ ID NO:6.

[0465] 36. A polypeptide according to any one of clauses 1 to 35, wherein any residue of FR3 which differs from its corresponding residue in SEQ ID NO: 6 is a conservative substitution relative to its corresponding residue.

[0466] 37. The polypeptide according to clause 34, wherein FR3 comprises SEQ ID NO:6.

[0467] 38. The polypeptide according to clause 37, wherein FR3 consists of SEQ ID NO: 6.

[0468] 39. A polypeptide according to any one of clauses 1 to 38, wherein FR4 comprises a sequence that shares 5% or greater sequence identity, such as sharing 10% or greater sequence identity, such as sharing 20% ​​or greater sequence identity, such as sharing 30% or greater sequence identity, such as sharing 40% or greater sequence identity, such as sharing 50% or greater sequence identity, such as sharing 60% or greater sequence identity, such as sharing 70% or greater sequence identity, such as sharing 80% or greater sequence identity, such as sharing 90% or greater sequence identity with SEQ ID NO:7.

[0469] 40. A polypeptide according to clause 39, wherein FR4 consists of a sequence that shares 5% or greater sequence identity, such as sharing 10% or greater sequence identity, such as sharing 20% ​​or greater sequence identity, such as sharing 30% or greater sequence identity, such as sharing 40% or greater sequence identity, such as sharing 50% or greater sequence identity, such as sharing 60% or greater sequence identity, such as sharing 70% or greater sequence identity, such as sharing 80% or greater sequence identity, such as sharing 90% or greater sequence identity with SEQ ID NO:7.

[0470] 41. A polypeptide according to any one of clauses 1 to 40, wherein any residue of FR4 which differs from its corresponding residue in SEQ ID NO: 7 is a conservative substitution relative to its corresponding residue.

[0471] 42. The polypeptide according to clause 39, wherein FR4 comprises SEQ ID NO:7.

[0472] 43. The polypeptide according to clause 42, wherein FR4 consists of SEQ ID NO:7.

[0473] 44. A polypeptide according to any one of clauses 1 to 43, comprising a sequence that shares 50% or greater sequence identity, such as sharing 55% or greater sequence identity, such as sharing 60% or greater sequence identity, such as sharing 65% or greater sequence identity, such as sharing 70% or greater sequence identity, such as sharing 75% or greater sequence identity, such as sharing 80% or greater sequence identity, such as sharing 85% or greater sequence identity, such as sharing 90% or greater sequence identity, such as sharing 95% or greater sequence identity, such as sharing 96% or greater sequence identity, such as sharing 97% or greater sequence identity, such as sharing 98% or greater sequence identity, such as sharing 99% or greater sequence identity with SEQ ID NO:8.

[0474] 45. A polypeptide according to clause 44, consisting of a sequence that shares 50% or greater sequence identity, such as sharing 55% or greater sequence identity, such as sharing 60% or greater sequence identity, such as sharing 65% or greater sequence identity, such as sharing 70% or greater sequence identity, such as sharing 75% or greater sequence identity, such as sharing 80% or greater sequence identity, such as sharing 85% or greater sequence identity, such as sharing 90% or greater sequence identity, such as sharing 95% or greater sequence identity, such as sharing 96% or greater sequence identity, such as sharing 97% or greater sequence identity, such as sharing 98% or greater sequence identity, such as sharing 99% or greater sequence identity with SEQ ID NO:8.

[0475] 46. ​​A polypeptide according to any one of clauses 1 to 45, wherein the N-terminus of the polypeptide is D.

[0476] 47. A polypeptide according to clause 44, comprising SEQ ID NO:8.

[0477] 48. A polypeptide according to clause 47 consisting of SEQ ID NO: 8.

[0478] 49. A polypeptide comprising an immunoglobulin chain variable domain that binds to IL-23, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein:

[0479] (a) CDR1 consists of a sequence that shares 60% or greater sequence identity with SEQ ID NO:1, CDR2 consists of a sequence that shares 70% or greater sequence identity with SEQ ID NO:2, and CDR3 consists of a sequence that shares 70% or greater sequence identity with SEQ ID NO:3;

[0480] (b) FR1 consists of a sequence that shares 70% or greater sequence identity with SEQ ID NO:4, FR2 consists of a sequence that shares 70% or greater sequence identity with SEQ ID NO:5, FR3 consists of a sequence that shares 70% or greater sequence identity with SEQ ID NO:6, and FR4 consists of a sequence that shares 70% or greater sequence identity with SEQ ID NO:7; and

[0481] (c) the polypeptide consists of a sequence that shares 70% or greater sequence identity with SEQ ID NO:8.

[0482] 50. A polypeptide comprising an immunoglobulin chain variable domain that binds to IL-23, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR1 comprises a sequence that shares 60% or greater sequence identity with SEQ ID NO: 14, CDR2 comprises a sequence that shares 50% or greater sequence identity with SEQ ID NO: 15, and CDR3 comprises a sequence that shares 50% or greater sequence identity with SEQ ID NO: 16.

[0483] 51. A polypeptide according to clause 50, wherein CDR3 comprises a sequence sharing 60% or greater sequence identity, such as 80% or greater sequence identity, with SEQ ID NO: 16.

[0484] 52. A polypeptide according to clause 51, wherein CDR3 consists of a sequence sharing 60% or greater sequence identity, such as 80% or greater sequence identity with SEQ ID NO: 16.

[0485] 53. A polypeptide according to any one of clauses 50 to 52, wherein any residue of CDR3 which differs from its corresponding residue in SEQ ID NO: 16 is a conservative substitution relative to its corresponding residue.

[0486] 54. The polypeptide according to clause 51, wherein CDR3 comprises SEQ ID NO: 16.

[0487] 55. The polypeptide according to clause 54, wherein CDR3 consists of SEQ ID NO: 16.

[0488] 56. The polypeptide according to any one of clauses 50 to 55, wherein CDR1 comprises a sequence that shares 80% or greater sequence identity with SEQ ID NO: 14.

[0489] 57. The polypeptide according to clause 56, wherein CDR1 consists of a sequence sharing 80% or greater sequence identity with SEQ ID NO: 14.

[0490] 58. A polypeptide according to any one of clauses 50 to 57, wherein any residue of CDR1 which differs from its corresponding residue in SEQ ID NO: 14 is a conservative substitution relative to its corresponding residue.

[0491] 59. The polypeptide according to clause 56, wherein CDR1 comprises SEQ ID NO:14.

[0492] 60. The polypeptide according to clause 59, wherein CDR1 consists of SEQ ID NO: 14.

[0493] 61. A polypeptide according to any one of clauses 50 to 60, wherein CDR2 comprises a sequence that shares 55% or greater sequence identity, such as shares 60% or greater sequence identity, such as shares 70% or greater sequence identity, such as shares 75% or greater sequence identity, such as shares 80% or greater sequence identity, such as shares 85% or greater sequence identity, such as shares 90% or greater sequence identity with SEQ ID NO: 15.

[0494] 62. A polypeptide according to clause 61, wherein CDR2 consists of a sequence that shares 60% or greater sequence identity, such as sharing 70% or greater sequence identity, such as sharing 75% or greater sequence identity, such as sharing 80% or greater sequence identity, such as sharing 85% or greater sequence identity, such as sharing 90% or greater sequence identity with SEQ ID NO: 15.

[0495] 63. A polypeptide according to any one of clauses 50 to 62, wherein any residue of CDR2 which differs from its corresponding residue in SEQ ID NO: 15 is a conservative substitution relative to its corresponding residue.

[0496] 64. The polypeptide according to clause 61, wherein CDR2 comprises SEQ ID NO:15.

[0497] 65. The polypeptide according to clause 64, wherein CDR2 consists of SEQ ID NO: 15.

[0498] 66. A polypeptide according to any one of clauses 50 to 65, wherein:

[0499] (a) FR1 comprises a sequence that shares 5% or greater sequence identity, such as sharing 12% or greater sequence identity, such as sharing 18% or greater sequence identity, such as sharing 26% or greater sequence identity, such as sharing 32% or greater sequence identity, such as sharing 38% or greater sequence identity, such as sharing 46% or greater sequence identity, such as sharing 52% or greater sequence identity, such as sharing 58% or greater sequence identity, such as sharing 62% or greater sequence identity, such as sharing 66% or greater sequence identity, such as sharing 68% or greater sequence identity, such as sharing 72% or greater sequence identity, such as sharing 75% or greater sequence identity, such as sharing 78% or greater sequence identity, such as sharing 82% or greater sequence identity, such as sharing 85% or greater sequence identity, such as sharing 90% or greater sequence identity, such as sharing 95% or greater sequence identity with SEQ ID NO: 17;

[0500] (b) FR2 comprises a sequence that shares 10% or greater sequence identity, such as sharing 15% or greater sequence identity, such as sharing 25% or greater sequence identity, such as sharing 30% or greater sequence identity, such as sharing 40% or greater sequence identity, such as sharing 45% or greater sequence identity, such as sharing 55% or greater sequence identity, such as sharing 60% or greater sequence identity, such as sharing 70% or greater sequence identity, such as sharing 75% or greater sequence identity, such as sharing 85% or greater sequence identity, such as sharing 90% or greater sequence identity with SEQ ID NO: 18;

[0501] (c) FR3 comprises a sequence that shares 8% or greater sequence identity, such as sharing 15% or greater sequence identity, such as sharing 20% ​​or greater sequence identity, such as sharing 26% or greater sequence identity, such as sharing 32% or greater sequence identity, such as sharing 40% or greater sequence identity, such as sharing 45% or greater sequence identity, such as sharing 52% or greater sequence identity, such as sharing 58% or greater sequence identity, such as sharing 65% or greater sequence identity, such as sharing 70% or greater sequence identity, such as sharing 76% or greater sequence identity, such as sharing 80% or greater sequence identity, such as sharing 82% or greater sequence identity, such as sharing 85% or greater sequence identity, such as sharing 90% or greater sequence identity, such as sharing 92% or greater sequence identity, such as sharing 95% or greater sequence identity with SEQ ID NO: 19; and / or

[0502] (d) FR4 comprises a sequence that shares 5% or greater sequence identity, such as sharing 10% or greater sequence identity, such as sharing 20% ​​or greater sequence identity, such as sharing 30% or greater sequence identity, such as sharing 40% or greater sequence identity, such as sharing 50% or greater sequence identity, such as sharing 60% or greater sequence identity, such as sharing 70% or greater sequence identity, such as sharing 80% or greater sequence identity, such as sharing 90% or greater sequence identity with SEQ ID NO:20.

[0503] 67. A polypeptide according to clause 66, wherein FR1 comprises SEQ ID NO: 17, and / or FR2 comprises SEQ ID NO: 18, and / or FR3 comprises SEQ ID NO: 19, and / or FR4 comprises SEQ ID NO: 20.

[0504] 68. A polypeptide according to any one of clauses 50 to 67, comprising a sequence that shares 50% or greater sequence identity, such as sharing 55% or greater sequence identity, such as sharing 60% or greater sequence identity, such as sharing 65% or greater sequence identity, such as sharing 70% or greater sequence identity, such as sharing 75% or greater sequence identity, such as sharing 80% or greater sequence identity, such as sharing 85% or greater sequence identity, such as sharing 90% or greater sequence identity, such as sharing 95% or greater sequence identity, such as sharing 96% or greater sequence identity, such as sharing 97% or greater sequence identity, such as sharing 98% or greater sequence identity, such as sharing 99% or greater sequence identity with SEQ ID NO: 13.

[0505] 69. A polypeptide according to clause 68 comprising or consisting of SEQ ID NO: 13.

[0506] 70. A polypeptide comprising an immunoglobulin chain variable domain that binds to IL-23, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR1 comprises a sequence that shares 60% or greater sequence identity with SEQ ID NO:22, CDR2 comprises a sequence that shares 50% or greater sequence identity with SEQ ID NO:23, and CDR3 comprises a sequence that shares 50% or greater sequence identity with SEQ ID NO:24.

[0507] 71. A polypeptide according to clause 70, wherein CDR3 comprises a sequence sharing 60% or greater sequence identity, such as 80% or greater sequence identity, with SEQ ID NO: 24.

[0508] 72. A polypeptide according to clause 71, wherein CDR3 consists of a sequence sharing 60% or greater sequence identity, such as 80% or greater sequence identity with SEQ ID NO: 24.

[0509] 73. A polypeptide according to any one of clauses 70 to 72, wherein any residue of CDR3 which differs from its corresponding residue in SEQ ID NO: 24 is a conservative substitution relative to its corresponding residue.

[0510] 74. The polypeptide according to clause 71, wherein CDR3 comprises SEQ ID NO:24.

[0511] 75. The polypeptide according to clause 74, wherein CDR3 consists of SEQ ID NO: 24.

[0512] 76. The polypeptide according to any one of clauses 70 to 75, wherein CDR1 comprises a sequence that shares 80% or greater sequence identity with SEQ ID NO: 22.

[0513] 77. The polypeptide according to clause 76, wherein CDR1 consists of a sequence sharing 80% or greater sequence identity with SEQ ID NO:22.

[0514] 78. A polypeptide according to any one of clauses 70 to 77, wherein any residue of CDR1 which differs from its corresponding residue in SEQ ID NO: 22 is a conservative substitution relative to its corresponding residue.

[0515] 79. The polypeptide according to clause 76, wherein CDR1 comprises SEQ ID NO:22.

[0516] 80. The polypeptide according to clause 79, wherein CDR1 consists of SEQ ID NO:22.

[0517] 81. A polypeptide according to any one of clauses 70 to 80, wherein CDR2 comprises a sequence that shares 55% or greater sequence identity, such as shares 60% or greater sequence identity, such as shares 70% or greater sequence identity, such as shares 75% or greater sequence identity, such as shares 80% or greater sequence identity, such as shares 85% or greater sequence identity, such as shares 90% or greater sequence identity with SEQ ID NO:23.

[0518] 82. A polypeptide according to clause 81, wherein CDR2 consists of a sequence that shares 60% or greater sequence identity, such as sharing 70% or greater sequence identity, such as sharing 75% or greater sequence identity, such as sharing 80% or greater sequence identity, such as sharing 85% or greater sequence identity, such as sharing 90% or greater sequence identity with SEQ ID NO:23.

[0519] 83. A polypeptide according to any one of clauses 70 to 82, wherein any residue of CDR2 that differs from its corresponding residue in SEQ ID NO: 23 is a conservative substitution relative to its corresponding residue.

[0520] 84. The polypeptide according to clause 81, wherein CDR2 comprises SEQ ID NO:23.

[0521] 85. The polypeptide according to clause 84, wherein CDR2 consists of SEQ ID NO:23.

[0522] 86. A polypeptide according to any one of clauses 70 to 85, wherein:

[0523] (a) FR1 comprises a sequence that shares 5% or greater sequence identity, such as sharing 12% or greater sequence identity, such as sharing 18% or greater sequence identity, such as sharing 26% or greater sequence identity, such as sharing 32% or greater sequence identity, such as sharing 38% or greater sequence identity, such as sharing 46% or greater sequence identity, such as sharing 52% or greater sequence identity, such as sharing 58% or greater sequence identity, such as sharing 62% or greater sequence identity, such as sharing 66% or greater sequence identity, such as sharing 68% or greater sequence identity, such as sharing 72% or greater sequence identity, such as sharing 75% or greater sequence identity, such as sharing 78% or greater sequence identity, such as sharing 82% or greater sequence identity, such as sharing 85% or greater sequence identity, such as sharing 90% or greater sequence identity, such as sharing 95% or greater sequence identity with SEQ ID NO:25;

[0524] (b) FR2 comprises a sequence that shares 10% or greater sequence identity, such as sharing 15% or greater sequence identity, such as sharing 25% or greater sequence identity, such as sharing 30% or greater sequence identity, such as sharing 40% or greater sequence identity, such as sharing 45% or greater sequence identity, such as sharing 55% or greater sequence identity, such as sharing 60% or greater sequence identity, such as sharing 70% or greater sequence identity, such as sharing 75% or greater sequence identity, such as sharing 85% or greater sequence identity, such as sharing 90% or greater sequence identity with SEQ ID NO: 26;

[0525] (c) FR3 comprises a sequence that shares 8% or greater sequence identity, such as sharing 15% or greater sequence identity, such as sharing 20% ​​or greater sequence identity, such as sharing 26% or greater sequence identity, such as sharing 32% or greater sequence identity, such as sharing 40% or greater sequence identity, such as sharing 45% or greater sequence identity, such as sharing 52% or greater sequence identity, such as sharing 58% or greater sequence identity, such as sharing 65% or greater sequence identity, such as sharing 70% or greater sequence identity, such as sharing 76% or greater sequence identity, such as sharing 80% or greater sequence identity, such as sharing 82% or greater sequence identity, such as sharing 85% or greater sequence identity, such as sharing 90% or greater sequence identity, such as sharing 92% or greater sequence identity, such as sharing 95% or greater sequence identity with SEQ ID NO:27; and / or

[0526] (d) FR4 comprises a sequence that shares 5% or greater sequence identity, such as sharing 10% or greater sequence identity, such as sharing 20% ​​or greater sequence identity, such as sharing 30% or greater sequence identity, such as sharing 40% or greater sequence identity, such as sharing 50% or greater sequence identity, such as sharing 60% or greater sequence identity, such as sharing 70% or greater sequence identity, such as sharing 80% or greater sequence identity, such as sharing 90% or greater sequence identity with SEQ ID NO:28.

[0527] 87. A polypeptide according to clause 86, wherein FR1 comprises SEQ ID NO:25, and / or FR2 comprises SEQ ID NO:26, and / or FR3 comprises SEQ ID NO:27, and / or FR4 comprises SEQ ID NO:28.

[0528] 88. A polypeptide according to any one of clauses 70 to 87, comprising a sequence that shares 50% or greater sequence identity, such as sharing 55% or greater sequence identity, such as sharing 60% or greater sequence identity, such as sharing 65% or greater sequence identity, such as sharing 70% or greater sequence identity, such as sharing 75% or greater sequence identity, such as sharing 80% or greater sequence identity, such as sharing 85% or greater sequence identity, such as sharing 90% or greater sequence identity, such as sharing 95% or greater sequence identity, such as sharing 96% or greater sequence identity, such as sharing 97% or greater sequence identity, such as sharing 98% or greater sequence identity, such as sharing 99% or greater sequence identity with SEQ ID NO:21.

[0529] 89. A polypeptide according to clause 88 comprising or consisting of SEQ ID NO: 21.

[0530] 90. The polypeptide according to any one of clauses 1 to 89, wherein the polypeptide is an antibody.

[0531] 91. The polypeptide according to clause 90, wherein the polypeptide is an antibody fragment.

[0532] 92. A polypeptide according to clause 91 which is selected from the group consisting of VHH, VH, VL, V-NAR, Fab fragment and F(ab')2 fragment.

[0533] 93. A polypeptide according to clause 92, wherein the polypeptide is a VHH.

[0534] 94. A polypeptide according to clause 92, wherein the polypeptide is VH.

[0535] 95. A construct comprising two or more identical polypeptides according to any one of clauses 1 to 94.

[0536] 96. A construct comprising at least one polypeptide according to any one of clauses 1 to 94 and at least one different polypeptide, wherein the different polypeptide binds to TNF-α.

[0537] 97. A construct comprising at least one polypeptide according to any one of clauses 1 to 94 and at least one different polypeptide, wherein the different polypeptide binds to a target other than TNF-α.

[0538] 98. A construct according to any one of clauses 95 to 97, wherein the polypeptides are linked via at least one protease labile linker.

[0539] 99. The construct according to clause 98, wherein the protease-labile linker has the formula:

[0540] [-(G a S) x -B-(G b S) y -] z

[0541] in

[0542] a is 1 to 10;

[0543] b is 1 to 10;

[0544] x is 1 to 10;

[0545] y is 1 to 10;

[0546] z is 1 to 10 and

[0547] B is K or R.

[0548] 100. A construct according to clause 99 wherein a is 2 to 5, b is 2 to 5, x is 1 to 3, y is 1 to 3, z is 1 and B is K.

[0549] 101. A construct according to clause 100, wherein the construct comprises or more suitably consists of SEQ ID NO: 46.

[0550] 102. A construct according to any one of clauses 95 to 97, wherein the polypeptides are all linked by non-protease labile linkers.

[0551] 103. A construct according to clause 102, wherein the non-protease labile linker has the formula

[0552] (G4 S) x

[0553] in

[0554] x is 1 to 10.

[0555] 104. The construct according to clause 103, wherein x is 6.

[0556] 105. A polypeptide or construct according to any one of clauses 1 to 104, which neutralizes human IL-23 in an IL-23-IL-23R neutralization ELISA (Assessment Method A) with an EC50 of 5 nM or less, such as 4 nM or less, such as 3 nM or less, such as 2 nM or less, such as 1.7 nM or less, such as 1.5 nM or less, such as 1.4 nM or less, such as 1.3 nM or less, such as 1.2 nM or less, such as 1.1 nM or less, such as 1.0nM or less, such as 0.9nM or less, such as 0.8nM or less, such as 0.75nM or less, such as 0.70nM or less, such as 0.65nM or less, such as 0.60nM or less, such as 0.55nM or less, such as 0.50nM or less, such as 0.45nM or less, such as 0.40nM or less, such as 0.35nM or less, such as 0.30nM or less, such as 0.25nM or less, such as 0.20nM or less.

[0557] 106. A polypeptide or construct according to any one of clauses 1 to 105 which is substantially resistant to one or more proteases.

[0558] 107. A polypeptide or construct according to clause 106, wherein the one or more proteases are present in the stomach or the small intestine or the large intestine.

[0559] 108. A polypeptide or construct according to clause 107, wherein the one or more proteases are present in the small intestine.

[0560] 109. A polypeptide or construct according to clause 106, wherein the one or more proteases are selected from enteropeptidase, trypsin, chymotrypsin and inflammatory bowel disease inflammatory protease.

[0561] 110. A polypeptide or construct according to clause 109, wherein the one or more proteases are selected from trypsin, chymotrypsin and inflammatory bowel disease inflammatory protease.

[0562] 111. A polypeptide or construct according to clause 109 or 110, wherein the inflammatory bowel disease inflammatory protease is one or more proteases selected from the group consisting of MMP3, MMP12 and cathepsins.

[0563] 112. A polypeptide or construct according to clause 110, wherein the proteases are trypsin and chymotrypsin.

[0564] 113. A pharmaceutical composition comprising a polypeptide or construct according to any one of clauses 1 to 112 and one or more pharmaceutically acceptable diluents or carriers.

[0565] 114. A pharmaceutical composition according to clause 113, wherein the composition is presented in an enteric-coated form.

[0566] 115. A pharmaceutical composition according to clause 113 or 114, comprising at least one additional active agent.

[0567] 116. A pharmaceutical composition according to clause 115, wherein the at least one additional active agent is selected from 5-aminosalicylic acid or a prodrug thereof (such as sulfasalazine, olsalazide or bisalazide); a corticosteroid (e.g., prednisolone, methylprednisolone or budesonide); an immunosuppressant (e.g., cyclosporine, tacrolimus, methotrexate, azathioprine or 6-mercaptopurine); an anti-TNF-α antibody (e.g., infliximab, adalimumab, certolizumab pegol or golimumab); an anti-IL12 / IL23 antibody (e.g., ustekinumab); an anti-IL6R antibody or a small molecule IL12 / IL23 inhibitor (e.g., apimod); an anti α-4-β-7 antibodies (e.g., vedolizumab); MAdCAM-1 blockers (e.g., PF-00547659); antibodies against the cell adhesion molecule α-4-integrin (e.g., natalizumab); antibodies against the α subunit of the IL2 receptor (e.g., daclizumab or basiliximab); JAK3 inhibitors (e.g., tofacitinib or R348); Syk inhibitors and their prodrugs (e.g., fostamitinib and R-406); phosphodiesterase-4 inhibitors (e.g., tetomilast); HMPL-004; probiotics; desalazine; semamod / CPSI-2364; and protein kinase C inhibitors (e.g., AEB-071).

[0568] 117. The pharmaceutical composition according to clause 116, wherein the at least one additional active agent is 5-aminosalicylic acid.

[0569] 118. A polypeptide, pharmaceutical composition or construct according to any one of clauses 1 to 117 for use as a medicament.

[0570] 119. A polypeptide, pharmaceutical composition or construct according to clause 118 for use in treating an autoimmune disease and / or an inflammatory disease.

[0571] 120. A polypeptide, pharmaceutical composition or construct according to clause 119, wherein the autoimmune disease and / or inflammatory disease is selected from Crohn's disease, ulcerative colitis, irritable bowel disease, type II diabetes, glomerulonephritis, autoimmune hepatitis, Sjögren's syndrome, celiac disease, drug- or radiation-induced mucositis, pemphigus, psoriasis, eczema and scleroderma.

[0572] 121. A polypeptide, pharmaceutical composition or construct for use according to clause 120, wherein the autoimmune disease and / or inflammatory disease is Crohn's disease.

[0573] 122. A polypeptide, pharmaceutical composition or construct for use according to any one of clauses 118 to 121 for oral administration.

[0574] 123. A polypeptide, pharmaceutical composition or construct for use according to any one of clauses 118 to 119 for topical administration to the skin.

[0575] 124. Use of a polypeptide, pharmaceutical composition or construct according to any one of clauses 1 to 117 in the manufacture of a medicament for treating an autoimmune disease and / or an inflammatory disease.

[0576] 125. Use of a polypeptide, pharmaceutical composition or construct according to clause 124, wherein the autoimmune and / or inflammatory disease is selected from Crohn's disease, ulcerative colitis, irritable bowel disease, type II diabetes, glomerulonephritis, autoimmune hepatitis, Sjögren's syndrome, celiac disease, drug- or radiation-induced mucositis, pemphigus, psoriasis, eczema and scleroderma.

[0577] 126. The use according to clause 125, wherein the autoimmune disease and / or inflammatory disease is Crohn's disease.

[0578] 127. The use according to any one of clauses 124 to 126, wherein the medicament is administered orally.

[0579] 128. The use according to clause 124, wherein the medicament is administered topically to the skin.

[0580] 129. A method of treating an autoimmune disease and / or an inflammatory disease comprising administering to a human in need thereof a therapeutically effective amount of a polypeptide, pharmaceutical composition or construct according to any one of clauses 1 to 117.

[0581] 130. A method for treating an autoimmune disease and / or an inflammatory disease according to clause 129, wherein the autoimmune disease and / or inflammatory disease is selected from Crohn's disease, ulcerative colitis, irritable bowel syndrome, type II diabetes, glomerulonephritis, autoimmune hepatitis, Sjögren's syndrome, celiac disease, drug- or radiation-induced mucositis, pemphigus, psoriasis, eczema and scleroderma.

[0582] 131. A method of treating an autoimmune disease according to clause 130, wherein the autoimmune disease is Crohn's disease.

[0583] 132. A method of treating an autoimmune disease according to any one of clauses 129 to 131, wherein the polypeptide, pharmaceutical composition or construct is administered orally.

[0584] 133. A method of treating an autoimmune disease according to clause 129, wherein the polypeptide, pharmaceutical composition or construct is administered topically to the skin.

[0585] 134. A polypeptide, pharmaceutical composition, construct, use or method according to any one of clauses 118 to 133, wherein the polypeptide, pharmaceutical composition or construct is administered sequentially, simultaneously or separately with at least one active agent selected from the following: 5-aminosalicylic acid or a prodrug thereof (such as sulfasalazine, olsalazide or bisalazide); corticosteroids (e.g., prednisolone, methylprednisolone or budesonide); immunosuppressants (e.g., cyclosporine, tacrolimus, methotrexate, azathioprine or 6-mercaptopurine); anti-TNF-α antibodies (e.g., infliximab, adalimumab, certolizumab pegol or golimumab); anti-IL12 / IL23 antibodies (e.g., ustekinumab); anti-IL6R antibodies or small molecules IL12 / IL23 inhibitors (e.g., apimod); anti-α-4-β-7 antibodies (e.g., vedolizumab); MAdCAM-1 blockers (e.g., PF-00547659); antibodies against the cell adhesion molecule α-4-integrin (e.g., natalizumab); antibodies against the α subunit of the IL2 receptor (e.g., daclizumab or basiliximab); JAK3 inhibitors (e.g., tofacitinib or R348); Syk inhibitors and their prodrugs (e.g., fotantinib and R-406); phosphodiesterase-4 inhibitors (e.g., tetomilast); HMPL-004; probiotics; desalazine; semamimod / CPSI-2364; and protein kinase C inhibitors (e.g., AEB-071).

[0586] 135. The polypeptide, pharmaceutical composition, construct or method according to clause 134, wherein the polypeptide, pharmaceutical composition or construct is administered sequentially, simultaneously or separately with infliximab, adalimumab, certolizumab pegol or golimumab.

[0587] 136. A polynucleotide comprising or consisting of a sequence that shares 70% or greater, such as 80% or greater, such as 90% or greater, such as 95% or greater, such as 99% or greater sequence identity with any of the portions of CDR1, CDR2 or CDR3 encoding the encoded immunoglobulin chain variable domain of SEQ ID NO:10.

[0588] 137. A polynucleotide encoding a polypeptide or construct according to any one of clauses 1 to 135.

[0589] 138. A polynucleotide according to clause 137, wherein the polynucleotide comprises or consists of a sequence that shares 70% or greater, such as 80% or greater, such as 90% or greater, such as 95% or greater, such as 99% or greater sequence identity with SEQ ID NO: 10.

[0590] 139. The polynucleotide according to clause 138, wherein the polynucleotide comprises or consists of SEQ ID NO: 10.

[0591] 140. A cDNA comprising the polynucleotide according to any one of clauses 136 to 139.

[0592] 141. A vector comprising the polynucleotide or cDNA according to any one of clauses 136 to 140.

[0593] 142. A host cell transformed with a vector according to clause 141 and capable of expressing a polypeptide or construct according to any one of clauses 1 to 112.

[0594] 143. A host cell transformed with a vector according to clause 142, wherein the host cell is a yeast cell, such as Saccharomyces cerevisiae or Pichia pastoris.

[0595] 144. A host cell transformed with a vector according to clause 142, wherein the host cell is a cell, such as Escherichia coli.

[0596] 145. A method for preparing a polypeptide or construct according to any one of clauses 1 to 112, comprising the following steps:

[0597] i) cloning a polynucleotide according to any one of clauses 136 to 139 into a vector, such as a plasmid

[0598] ii) transforming a cell, such as a bacterial cell or a yeast cell, capable of producing a polypeptide or construct according to any one of clauses 1 to 112 with the vector under conditions that allow production of the polypeptide or construct,

[0599] iii) recovering the polypeptide or construct, such as by affinity chromatography.

[0600] Additional clauses listing additional embodiments of the invention are as follows:

[0601] 1. A polypeptide comprising an immunoglobulin chain variable domain that binds to IL-23, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR1 comprises a sequence that shares 60% or greater sequence identity with SEQ ID NO:1, CDR2 comprises a sequence that shares 50% or greater sequence identity with SEQ ID NO:2, and CDR3 comprises a sequence that shares 50% or greater sequence identity with SEQ ID NO:3.

[0602] 2. A polypeptide according to clause 1, wherein CDR1 comprises a sequence that shares 80% or greater sequence identity with SEQ ID NO: 1, CDR2 comprises a sequence that shares 80% or greater sequence identity with SEQ ID NO: 2, and CDR3 comprises a sequence that shares 80% or greater sequence identity with SEQ ID NO: 3.

[0603] 3. The polypeptide according to clause 3, wherein CDR1 consists of SEQ ID NO: 1, CDR2 consists of SEQ ID NO: 2, and CDR3 consists of SEQ ID NO: 3.

[0604] 4. A polypeptide according to any one of clauses 1 to 3, wherein FR1 comprises a sequence that shares 80% or greater sequence identity with SEQ ID NO:4, FR2 comprises a sequence that shares 80% or greater sequence identity with SEQ ID NO:5, FR3 comprises a sequence that shares 80% or greater sequence identity with SEQ ID NO:6, and FR4 comprises a sequence that shares 80% or greater sequence identity with SEQ ID NO:7.

[0605] 5. A polypeptide according to any one of clauses 1 to 4, comprising a sequence sharing 70% or greater sequence identity with SEQ ID NO: 8.

[0606] 6. The polypeptide according to any one of clauses 1 to 5, wherein the polypeptide is an antibody or a fragment thereof.

[0607] 7. The polypeptide according to clause 6, wherein the polypeptide is VH or VHH.

[0608] 8. A construct comprising at least one polypeptide according to any one of clauses 1 to 7 and at least one different polypeptide, wherein the different polypeptide binds to TNF-α.

[0609] 9. A construct according to clause 8, wherein the polypeptides are linked via at least one protease-labile linker.

[0610] 10. A polypeptide or construct according to any one of clauses 1 to 9, wherein the polypeptide or construct neutralises human IL-23 with an EC50 of 2 nM or less in an IL-23-IL-23R neutralisation ELISA (Assessment Method A).

[0611] 11. A polypeptide or construct according to any one of clauses 1 to 10, wherein the polypeptide or construct is substantially resistant to one or more proteases present in the small intestine.

[0612] 12. The polypeptide or construct according to clause 11, wherein the proteases are trypsin and chymotrypsin.

[0613] 13. A polypeptide, pharmaceutical composition or construct according to any one of clauses 1 to 12 for use in the treatment of an autoimmune disease and / or an inflammatory disease.

[0614] 14. The polypeptide, pharmaceutical composition or construct for use according to clause 13, wherein the polypeptide, pharmaceutical composition or construct for use is administered orally.

[0615] 15. A polynucleotide encoding a polypeptide or construct according to any one of clauses 1 to 14.

[0616] The present invention provides embodiments including but not limited to the following:

[0617] 1. A polypeptide comprising an immunoglobulin chain variable domain that binds to IL-23, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR1 comprises a sequence that shares 60% or greater sequence identity with SEQ ID NO:1, CDR2 comprises a sequence that shares 50% or greater sequence identity with SEQ ID NO:2, and CDR3 comprises a sequence that shares 50% or greater sequence identity with SEQ ID NO:3.

[0618] 2. The polypeptide according to embodiment 1, wherein CDR1 comprises or consists of a sequence that shares 80% or greater sequence identity with SEQ ID NO: 1.

[0619] 3. A polypeptide according to embodiment 1 or 2, wherein CDR2 comprises or consists of a sequence that shares 80% or greater sequence identity with SEQ ID NO:2.

[0620] 4. The polypeptide according to any one of embodiments 1 to 3, wherein CDR3 comprises or consists of a sequence sharing 80% or greater sequence identity with SEQ ID NO:3.

[0621] 5. The polypeptide of embodiment 1, wherein CDR1 comprises or consists of SEQ ID NO: 1, CDR2 comprises or consists of SEQ ID NO: 2, and CDR3 comprises or consists of SEQ ID NO: 3.

[0622] 6. A polypeptide according to any one of embodiments 1 to 5, which comprises or consists of a sequence that shares 50% or greater sequence identity, such as 55% or greater sequence identity, such as 60% or greater sequence identity, such as 65% or greater sequence identity, such as 70% or greater sequence identity, such as 75% or greater sequence identity, such as 80% or greater sequence identity, such as 85% or greater sequence identity, such as 90% or greater sequence identity, such as 95% or greater sequence identity, such as 96% or greater sequence identity, such as 97% or greater sequence identity, such as 98% or greater sequence identity, such as 99% or greater sequence identity with SEQ ID NO:8.

[0623] 7. The polypeptide according to embodiment 6, comprising SEQ ID NO:8.

[0624] 8. The polypeptide according to embodiment 7, which consists of SEQ ID NO:8.

[0625] 9. The polypeptide of any one of embodiments 1 to 8, wherein the polypeptide is an antibody or an antibody fragment.

[0626] 10. The polypeptide of any one of embodiments 1 to 9, wherein the polypeptide consists of an immunoglobulin chain variable domain.

[0627] 11. A polypeptide according to any one of embodiments 1 to 10, wherein the immunoglobulin chain variable domain is VHH, VH or VL.

[0628] 12. A polypeptide according to embodiment 11, wherein the immunoglobulin chain variable domain is VHH or VH.

[0629] 13. A polypeptide according to embodiment 12, wherein the immunoglobulin chain variable domain is VHH.

[0630] 14. A construct comprising at least one polypeptide according to any one of embodiments 1 to 13 and at least one different polypeptide, wherein the different polypeptide binds to TNF-α.

[0631] 15. The construct of embodiment 14, wherein the different polypeptide comprises a sequence that shares at least 80% or greater sequence identity with SEQ ID NO:67.

[0632] 16. A construct according to embodiment 14 or 15, wherein the polypeptides are connected by at least one protease-labile linker comprising or consisting of SEQ ID NO: 74.

[0633] 17. A construct according to embodiment 16, wherein the construct comprises or consists of SEQ ID NO:46.

[0634] 18. A polypeptide or construct according to any one of embodiments 1 to 17, which neutralizes human IL-23 in evaluation method A with an EC50 of 5 nM or less, such as 4 nM or less, such as 3 nM or less, such as 2 nM or less, such as 1.7 nM or less, such as 1.5 nM or less, such as 1.4 nM or less, such as 1.3 nM or less, such as 1.2 nM or less, such as 1.1 nM or less, such as 1.0 nM or less, such as 0.9nM or less, such as 0.8nM or less, such as 0.75nM or less, such as 0.70nM or less, such as 0.65nM or less, such as 0.60nM or less, such as 0.55nM or less, such as 0.50nM or less, such as 0.45nM or less, such as 0.40nM or less, such as 0.35nM or less, such as 0.30nM or less, such as 0.25nM or less, such as 0.20nM or less.

[0635] 19. The polypeptide or construct according to any one of embodiments 1 to 18, wherein the polypeptide or construct is expressed in 10 -6 M or smaller, such as 10 -7 M or smaller, such as 10 -8 M or smaller, such as 10 -9 M or smaller, such as 10 -10 M or smaller, such as 10 -11 M or smaller, such as 10 -12 M or smaller, such as 10 -13 M or less binds to IL-23.

[0636] 20. The polypeptide or construct of any one of embodiments 1 to 19, wherein the polypeptide or polypeptides are substantially resistant to trypsin and chymotrypsin.

[0637] 21. A pharmaceutical composition comprising the polypeptide or construct according to any one of embodiments 1 to 20 and one or more pharmaceutically acceptable diluents or carriers.

[0638] 22. The pharmaceutical composition of embodiment 21, comprising at least one additional active agent.

[0639] 23. A polypeptide, pharmaceutical composition or construct according to any one of embodiments 1 to 22 for use as a medicament.

[0640] 24. A polypeptide, pharmaceutical composition or construct according to embodiment 23, for use in the treatment of autoimmune diseases and / or inflammatory diseases.

[0641] 25. A method for treating an autoimmune disease and / or an inflammatory disease, comprising administering to a person in need thereof a therapeutically effective amount of the polypeptide, pharmaceutical composition or construct according to any one of embodiments 1 to 22.

[0642] 26. Use of a polypeptide, pharmaceutical composition or construct according to any one of embodiments 1 to 22 in the manufacture of a medicament for treating autoimmune diseases and / or inflammatory diseases.

[0643] 27. A polypeptide for use, a pharmaceutical composition or construct, a method or a use according to any one of embodiments 23 to 26, wherein the polypeptide, pharmaceutical composition or construct is administered orally.

[0644] 28. The polypeptide for use, pharmaceutical composition or construct, method or use according to any one of embodiments 24 to 27, wherein the autoimmune disease and / or inflammatory disease is Crohn's disease or ulcerative colitis.

[0645] 29. A polynucleotide encoding a polypeptide or construct according to any one of embodiments 1 to 20.

[0646] 30. A polynucleotide according to embodiment 29, wherein the polynucleotide comprises or consists of a sequence that shares 70% or greater, such as 80% or greater, such as 90% or greater, such as 95% or greater, such as 99% or greater sequence identity with SEQ ID NO: 10.

[0647] 31. The polynucleotide of embodiment 30, wherein the polynucleotide comprises or consists of SEQ ID NO: 10.

[0648] The invention will now be further described by way of the following non-limiting examples.

[0649] Example

[0650] Evaluation Methods Used in Examples

[0651] Evaluation Method A: IL-23-IL-23R Neutralization ELISA

[0652] Maxisorp 96-well plates were coated with 50 μl / well 0.3 μg / ml IL-23R-Fc overnight and then blocked with 4% milk, 1% BSA. ICVD was serially diluted in 4% milk, 1% BSA and mixed 1:1 with 40ng / ml recombinant human IL-23. ICVD mixed with IL-23 was then added to the IL-23R coated plate. BAF219 anti-p40 biotinylated pAb (R&Dsystems) was used, followed by detection of bound IL-23 with exo-avidin-HRP. This allowed calculation of IL-23 neutralization activity in the sample.

[0653] Evaluation Method B: IL-23-IL-23R Neutralization ELISA Using High Salt Buffer

[0654] A high salt IL-23-IL-23R ELISA was used to measure anti-IL-23 ICVD concentrations in fecal extracts. This ELISA was identical to the EILSA above (Assessment Method A), except that the buffer used for fecal supernatant dilution and IL-23 preparation was 1% BSA, 4% milk in PBS containing 0.6 M NaCl, 0.05% Tween 20, and protease inhibitors.

[0655] Evaluation Method C: Mouse Splenocyte Assay

[0656] Splenocytes were isolated from mouse spleens and cultured at 4 × 10 5 Cells / 50 μL / well were placed in a 96-well round-bottom microplate in medium containing 20 ng / mL mouse IL-2 (2x assay concentration). The highest concentration of ICVD was prepared at 300 nM (2x) in medium containing 10 ng / mL THP-1-derived nhIL-23 (2x assay concentration), and subsequent 2.6-fold dilutions were prepared directly in medium containing nhIL-23 (2x). 50 μL of each ICVD mixture (2x) was then transferred to the cells (50 μL) to obtain the final 1x assay concentration of ICVD, hIL-23, and mIL-2. For mouse IL-17 stimulation and secretion in the culture supernatant, 'mIL-2 alone' and 'mIL-2+hIL-23' were used as negative and positive controls, respectively. Incubate at 37°C, 5% CO 2After incubation for 3 days at 4 °C, the plates were spun at 2000 rpm for 2 minutes and 50 μL of culture supernatant was recovered from each well. The mIL-17 level in the culture supernatant was measured using IL-17 binding ELISA, and neutralization of hIL-23 was determined.

[0657] Example 1: Immunization of ICVD in E. coli, phage library selection, master plate screening and production

[0658] Example 1.1: Immunization and phage library construction

[0659] Llamas were immunized with soluble human recombinant IL-23 and good titers of serum antibodies were obtained in both animals. RNA was isolated from leukocytes collected from each llama after several booster immunizations with IL-23.

[0660] Seven separate phage display libraries were generated using peripheral blood mononuclear cells (PBMCs) collected from each llama at the end of each immunization phase. Standard reagents and protocols were used to construct the ICVD phage display library and to express the primary selection of phages of ICVD with IL-23 binding activity. Typically, total RNA was extracted from peripheral blood lymphocytes isolated from each immunization llama. RNA was then used to generate cDNA, and PCR was performed to specifically amplify the variable region of the antibody with only the ICVD heavy chain. The cDNA fragment encoding the ICVD spectrum was cloned into a phagemid vector, and the library was introduced into Escherichia coli. Phage libraries were produced by culturing Escherichia coli with helper phage and precipitating the resulting ICVD display library. The number of each library was determined by titration and infection with logarithmic phase Escherichia coli strain TG1 with different dilutions. It is estimated that each library contains 6x10 7 5x10 8 ICVD sequences between .

[0661] Example 1.2: Library selection of phages with human IL-23 binding activity: propagation and production of periplasmic extracts become

[0662] Phage library selection strategy is set to enrich the ICVD that specifically binds to the IL-23p19 subunit. Phage is selected by panning hIL-23 or biotinylated hIL-23 in the presence of soluble hIL-12. The bound ICVD is washed using different methods and removed from the plate using the specific elution of IL-23R or the total elution of TEA. Selection and elution conditions are established to separate the ICVD that binds to the epitope of p19 present in the active soluble form of IL-23 with high affinity and interferes with the binding of IL-23 to IL-23 receptors (IL-23R).

[0663] Use the phage present in the eluent from selection to infect Escherichia coli TG1 cells. Randomly select colonies to 12 96-well master plates and breed to generate colony cultures. Crack the outer membrane of bacterial cells to release the periplasmic fraction (also referred to as "peri" in this article) containing ICVD by freezing-thawing. Remove cell debris by centrifugation and transfer supernatant to fresh 96-well plates to assess ICVD activity.

[0664] Example 1.3: Screening of periplasmic extracts

[0665] To identify ICVDs specific for inhibiting IL-23 signaling, periplasmic extracts were tested for their ability to disrupt three interactions: 1) IL-23 binding to IL-23R, 2) IL-23 binding to IL-12Rβ1, and 3) IL-12 binding to IL-12Rβ1. Each of these interactions was tested by ELISA.

[0666] A three-plate-ELISA format was developed in which periplasmic extracts containing ICVDs were mixed with the appropriate cytokines, after which the mixture was applied to Maxisorp plates coated with the appropriate cytokine receptors. This allowed capture of the cytokines in the absence of interfering cytokine-receptor binding by the ICVDs, which in all cases could be detected by biotinylated anti-p40 antibodies and subsequent exo-avidin-HRP. The neutralizing activity of each ICVD was measured as the ability to reduce the binding of the cytokine to its receptor relative to an "irrelevant" VHH control. Promising ICVD clones were selected for production in E. coli and further evaluation.

[0667] Example 1.4: Production of selected clones in E. coli

[0668] The DNA sequence of the selected ICVD was recloned into the vector pMEK222 (thereby introducing C-terminal FLAG and 6xHis tags) for production in E. coli, and the ICVD affinity was purified on Talon resin via the 6xHis tag for more detailed evaluation studies. ICVD that could not be well expressed by E. coli was excluded from further analysis.

[0669] Example 2: Efficacy of purified primary clones

[0670] The IL-23-IL-23R neutralization activity of purified ICVD was evaluated in an IL-23-IL-23R neutralization ELISA (Assessment Method A) to determine the efficacy against human IL-23. ICVDs found to have higher efficacy than the comparative example monoclonal antibody Bremekumab (heavy chain SEQ ID NO: 70, light chain SEQ ID NO: 71) are listed in the table below.

[0671]

[0672] The ELISA results showed that the L family clones (12G1 and 1E2) were significantly potent, not only showing higher efficacy in the IL-23-IL-23R ELISA compared to the comparative example anti-IL-23 antibody Bricupumab, but even showing subnanomolar efficacy. Clones 10E2 and 10G10 also showed higher efficacy than the Bricupumab comparative example in this assay.

[0673] 10E2, 12G1 and 1E2 were also tested in the cynomolgus monkey IL-23-IL-23R neutralization ELISA, and all showed high neutralization efficacy in this assay (data not shown), similar to those shown in the standard IL-23-IL-23R ELISA. It was also determined that these clones were unable to neutralize the interaction of IL-23 with IL-12R or IL-12 with IL-12R. Although not wishing to be bound by theory, the inventors believe that these clones are specific for the p19 subunit of IL-23.

[0674] Example 3: Intrinsic protease stability of purified primary clones

[0675] Orally administered ICVD may be susceptible to proteolytic digestion during passage through the small and large intestines. To further investigate resistance to intestinal proteases, the efficacy of purified ICVD was tested after incubation in the presence of both mouse small intestinal supernatant and supernatant prepared from pooled human fecal samples.

[0676] Mouse small intestinal supernatant: The contents of the small intestine from seven C57BL / 6 male mice were removed with 0.9% saline, combined, homogenized and centrifuged. The resulting supernatant was removed, aliquoted and frozen.

[0677] Human fecal supernatant: Fecal samples from five individuals were converted to slurries by adding 1x PBS. The slurries were then pooled, centrifuged and the supernatant removed, aliquoted and stored at -80°C. This process removed the fecal matrix, including any cellular material.

[0678] After incubation of ICVD at 37°C for different time periods, the anti-IL-23 activity of the "digested" ICVD samples was determined using an IL-23-IL-23R neutralization ELISA (Assessment Method B), and the percentage of activity maintained from the "undigested" ICVD to the "digested" ICVD was calculated. The results are provided in the table below.

[0679]

[0680] ICVDs showed significant intrinsic stability towards proteases present in mouse small intestinal material and human fecal material. L-family ICVD 12G1 was selected for further optimization based on its efficacy and intrinsic protease resistance.

[0681] Example 4: Generation and characterization of optimized variants of 12G1

[0682] L family ICVD 12G1 undergoes sequence modification to generate multiple variant ICVDs. These variant ICVDs were tested in the efficacy and protease stability assays described in detail above. Variant ICVDs, their mutations relative to ID-L253T, and their performance in these assays are described in detail in the following table (wherein EC50 values ​​are provided in nM, 'mouse spleen cells' refer to data obtained using the above evaluation method C, and relative to Kabat numbering, residues are numbered using N-terminal to C-terminal numbering).

[0683] It should be noted that generally variants maintain high efficacy and protease stability in ICVD. It can therefore be expected that variants of ID-L253T generally maintain high efficacy and protease stability.

[0684] Among these variant ICVDs, ID-L253T was ultimately selected for further characterization. 12G1 was varied from ID-L253T by the following mutations: E1D, L11Q, R19S, A23E, A24S, Y37F, A60S, M69I, V78L, F79Y, E81Q, D83N, V85L, A90T, N96A, L103I, and R116Q (numbering using N-terminus to C-terminus relative to Kabat numbering).

[0685]

[0686] Example 5: Efficacy, Binding Affinity and Protease Stability of ID-L253T Compared to Comparative Examples Bremecumab and 37D5

[0687] Bremelumab is a fully human IgG that selectively binds to the p19 subunit of IL-23. 2 Monoclonal antibody. 37D5 is a prior art domain antibody (VHH) that selectively binds to the p19 subunit of IL-23 (Desmyter et al. 2017, SEQ ID NO: 69). The efficacy, binding affinity, and protease stability of ID-L253T were evaluated relative to these prior art anti-IL-23 agents.

[0688] Example 5.1: Efficacy of ID-L253T compared to Bremelumab

[0689] The efficacy of ID-L253T and clinical comparator Bricumab were tested side by side in the IL-23-IL-23R inhibition ELISA (Assessment Method A). The results are summarized in the table below.

[0690]

[0691] As expected, based on the efficacy of the origin ICVD 12G1 and ID-L253T variants described above, ID-L253T showed higher efficacy than Bremelumab in the IL-23-IL-23R ELISA.

[0692] Example 5.2: Affinity of ID-L253T compared to Brevicumab Fab

[0693] The binding kinetics of ID-L253T were compared for Blake monoclonal antibody Fab (prepared using a commercially available Fab preparation kit) and evaluated in Biacore research. Anti-p40 mAb was fixed to the Biacore sensor plate and loaded with recombinant human IL-23. This allows the p19 subunit (Blake monoclonal antibody and ID-L253T are both specific to the p19 subunit) to interact with the anti-IL-23 ID-253T ICVD and Blake monoclonal antibody Fab fragments through the p40 subunit tethering IL-23 of IL-23, which then flows through the chip to detect binding. ID-L253T has an average K of 32pM D , while the Bremelumab Fab formulation had a much higher mean K of 1200 pM D , thus showing that Bremelumab Fab has much lower binding affinity than ID-L253T. These data are summarized in the table below.

[0694]

[0695]

[0696] While not wishing to be bound by theory, based on the data presented above, the inventors anticipate that ID-L253T binds to the pl9 subunit of IL-23.

[0697] Example 5.3: Protease stability of ID-L253T compared to 37D5

[0698] The stability of ID-L253T in mouse small intestine material is tested and compared with the stability of comparative example 37D5. The anti-IL-23 activity of "digested" ICVD sample is measured using IL-23-IL-23R neutralization ELISA (assessment method B), and the percentage of activity maintained from "undigested" ICVD to "digested" ICVD is calculated. These data are summarized in the table below.

[0699]

[0700] It is clear from these data that no ELISA signal could be established relative to 37D5 after 2 hours of digestion in mouse small intestine material, whereas after an incubation period twice as long, ID-L253T achieved 51% survival. ID-L253T is therefore significantly more stable in this digestible matrix than the comparative example 37D5. It should be noted that the data provided here for ID-L253T correspond to the same assay as described in detail in Example 4 above, and that the data provided here for 37D5 were generated in separate assays in different cases.

[0701] Example 6: ID-L253T specificity for human IL-23

[0702] Example 6.1: Cross-reactivity with IL-23 from toxicological species

[0703] Cynomolgus IL-23-IL-23R neutralization ELISA was performed. To this end, Maxisorp 96-well plates were coated with 50 μl / well 0.5 μg / ml cynomolgus IL-23R-Fc overnight and then blocked with 4% milk, 1% BSA. ICVD was serially diluted in 4% milk, 1% BSA and mixed 1:1 with 40ng / ml recombinant cynomolgus IL-23 (cIL-23), then incubated for 30 minutes to allow binding before being added to the cIL-23R coated plate. BAF219 anti-p40 pAb was used, and then the bound cIL-23 was detected with exo-avidin-HRP, and the neutralization level of the binding of cIL-23 to cIL-23R by ICVD was determined.

[0704] ID-L253T was active in the cynomolgus monkey IL-23-IL-23R neutralization assay (data not shown), making the cynomolgus monkey an appropriate toxicology species for any preclinical development studies.

[0705] Example 6.2: Specificity for non-target cytokines

[0706] The selectivity of ID-L253T for IL-23 related and non-related cytokines was tested in binding or inhibition ELISA. IL-12 shares the p40 subunit with IL-23 and is therefore the most closely related cytokine in humans. Human and rhesus IL-12 and another member of the IL-12 cytokine family, IL-27, were tested in this assay. Other important but non-related inflammatory cytokines TNFα, IL-6 and IFNγ were also tested. ID-L253T did not show interaction with the tested cytokines. This indicates that the binding of ID-L253T to off-target molecules is very unlikely in humans and non-human primates.

[0707] Example 7: Further protease stability studies on ID-L253T - tolerance to gastrointestinal matrix metalloproteinases and mouse gastrointestinal transit

[0708] Example 7.1: Resistance to Gastrointestinal Matrix Metalloproteinases

[0709] Levels of activated matrix metalloproteinases (MMPs) are increased in the inflamed mucosa of patients with inflammatory bowel disease. These MMPs are able to digest natural human IgG and therapeutic agents containing human IgG scaffolds (Biancheri et al., 2015). In the case of the anti-TNFα therapy etanercept, this digestion resulted in a significant reduction in TNFα neutralization efficacy. To confirm that ID-L253T is tolerant to MMPs, ID-L253T was incubated for approximately 20 hours in the presence of activated recombinant human MMP3 and MMP12. ID-L253T showed complete survival over the course of approximately 20 hours, while etanercept was degraded by the same enzyme preparation.

[0710] ID-L253T completely resists digestion by MMP3 and MMP12 over the course of approximately 20 hours at 37°C. This indicates that ID-L253T should retain high stability in the inflammatory environment of the IBD intestine where MMP levels are elevated. This finding, along with the above findings on general protease resistance, suggests that the polypeptides of the present invention have great potential as oral therapies for IBD.

[0711] Example 7.2: Gastrointestinal transit and survival in mice

[0712] The results of the in vitro studies described above show that ID-L253T tolerates inactivation caused by proteases present in supernatant extracts prepared from mouse small intestinal contents. According to the author's experience, mouse small intestinal supernatants are more proteolytically active than small intestinal supernatants obtained from humans or pigs. Studies were conducted to study the stability of ID-L253T during passage through the gastrointestinal system of mice. ID-L253T (see WO2016156465, SEQ ID NO:8 and Example 8 therein) was prepared with anti-TNF-αICVD ID-38F in milk and bicarbonate mixtures. It has been previously determined that ID-38F is stable during mouse gastrointestinal transit and therefore serves as a positive control here. After ICVD was administered to four mice by oral gavage, the concentration of ICVD in fecal pellets collected between 0h-3h and 3h-6h was measured.

[0713] ID-L253T was detected at both 0-3h and 3-6h after dosing at levels comparable to those of ID-38F in the same mouse. The results showed that ID-L253T survived as well or better than ID-38F in mice, reaching a maximum recovery concentration of over 15 μM in the supernatant from fecal pellets collected 3-6h after dosing.

[0714] Example 8: Evaluation of neutralizing activity of ID-L210T in human IBD tissues

[0715] use et al. (2014) and Crowe et al. (2018) to investigate the IL-23 neutralizing activity of ID-L210T (a variant of ID-L253T lacking only E1D, R19S and R92V) in ex vivo cultures of inflamed colonic mucosal tissue.

[0716] This model was used to test the inhibitory effect of ID-L210T for the elevated levels of signal transduction phosphoproteins present in IBD diseases under pathophysiological conditions. The study was performed using biopsy tissue samples from four patients with active UC. After incubation for 24 h with ID-L210T (at 150 nM concentration) or an isotype control ICVD (ID-2A, non-related anti-Clostridium difficile toxin ICVD, at 225 nM concentration) that is not bound to IL-23, the phosphoprotein levels of biopsy explants were analyzed using the proteome analyzer human phosphokinase array technology.

[0717] The mean phosphorintensity values ​​for all patients for each protein tested were Figure 1-2 The phosphorylation levels of most proteins on the array were reduced after treatment with ID-L210T.

[0718] The effect of treatment on the total protein phosphorylation signal detected for each biopsy (∑ n＝39 Following treatment with ID-L210T, total phosphorylation levels measured in biopsies from all UC patients were inhibited (20%-52%; mean 32% n=4).

[0719] ID-L253T differs from ID-L210T by only three amino acids, shows similar IL-23-IL-23R inhibitory efficacy, and is resistant to in vitro digestion and transport through mice. It is therefore expected that these findings on ID-L210T are substantially applicable to ID-L253T.

[0720] Example 9: Yeast productivity in fermentation culture

[0721] The Saccharomyces cerevisiae production strain expressing ID-L253T was inoculated into a 5 liter fermentation. Production levels were assessed by SDS-PAGE with Coomassie staining, and bioactivity was assessed by IL-23-IL-23R neutralization ELISA. Results showed clear bands in the end of fermentation (EoF) supernatant at the correct molecular weight with very few contaminants. The yield of ID-L253T was measured to be 0.188 g / L.

[0722] The productivity of ID-L253T in Pichia was also tested using standard yeast fermentation techniques. The production level was assessed by SDS-PAGE with Coomassie staining, and the results showed a clear band in the EoF supernatant at the correct molecular weight. The yield of ID-L253T from this fermentation was measured to be 1.3 g / L. Full neutralization activity was demonstrated by IL-23-IL-23R neutralization ELISA.

[0723] Example 10: Generation and characterization of the heterobiheaded anti-IL-23 / anti-TNF-α construct FA1K Example 10.1: FA1K produce

[0724] A heterobi-headed construct comprising anti-IL-23 ICVD ID-L253T and anti-TNF-α ICVD ID-38F (WO2016156465, SEQ ID NO: 8 therein and SEQ ID NO: 67 in this disclosure) (referred to herein as 'FA1K') was generated. 4 S) 4 A linker (SEQ ID NO: 49) separates the ICVD in this construct to create a trypsin cleavable site of the type disclosed in WO2016156466 (ID-38F-(G 4 S) 2 -K-(G 4 S) 2 -ID-L253T). The polypeptide sequence of the ID-38F arm used in FAlK (SEQ ID NO:47) was identical to the polypeptide sequence of ID-38F used above, and the polypeptide sequence of the ID-L253T arm used in FAlK (SEQ ID NO:48) was identical to the polypeptide sequence used above for ID-L253T but with a D1E substitution.

[0725] FA1K was cloned into the vector pUR9013 on a SacI / HindIII fragment to facilitate the transfer of vwkgal1 -The expression strain was stably integrated into the chromosome of Saccharomyces cerevisiae in multiple copies. Using this integration and expression system, FA1K was under the control of a galactose-inducible promoter in Saccharomyces cerevisiae and achieved dual-headed secretion via the yeast mating factor α signal sequence. In addition, FA1K with the same signal sequence was cloned into the chromosome of Pichia pastoris under the control of the methanol-inducible pAOX1 promoter. The expression of FA1K from Saccharomyces cerevisiae was evaluated in 50 mL of induction medium.

[0726] The supernatants from these shake flasks were purified. Unless otherwise stated, this purified preparation of FAlK was subsequently used for the experiments described below.

[0727] FAlK was also cloned into Pichia pastoris and showed excellent expression at 50 mL scale.

[0728] Example 10.2: In vitro characterization of FA1K

[0729] Incubation of FAlK with trypsin at 37°C resulted in rapid separation of the ID-L253T and ID-38F monomer arms. FAlK is therefore well formatted for rapid release of both monomer arms upon exposure to trypsin in the human small intestine.

[0730] The neutralization efficacy of FA1K (before and after trypsin cleavage) against TNF-α was investigated (see Figure 3 , where 'FA1K' refers to FA1K before trypsin treatment, and 'FA1K trypsin' refers to FA1K after trypsin treatment).

[0731] Biotinylated Xiu Mei Le (adalimumab) competition ELISA is carried out, which measures the competition for the epitope on TNF-α. When competition occurs, the epitope is occupied by unlabeled polypeptide, and less biotinylated Xiu Mei Le is bound to TNF α, thereby causing the determination signal to decrease. Human TNF α ELISA plates are coated with 100ng / mL in 250μg / mL bovine serum albumin (BSA) in phosphate buffered saline (1x PBS), and are blocked with 1% BSA in 1x PBS. For all experiments on polypeptides pretreated with trypsin, 1% BSA is used to add 0.1mM PMSF as a determination diluent. In other cases, 1% BSA is used as a determination diluent. Biotinylated adalimumab (LGC) is mixed with all standards and samples 1: 1 to obtain a final concentration of 2nM biotinylated adalimumab, and then the mixture is added to the plate. Bound biotinylated adalimumab was detected using exo-avidin-horseradish peroxidase (Sigma E2886) and visualized using TMB microwell substrate (KPL 50–76-00), followed by 0.5 M H 2 SO4 Stopping and reading at 450 nm, it was found that FAIK (before and after trypsin treatment) was essentially as potent as ID-38F in this competition ELISA.

[0732] The neutralization efficacy of FA1K (before and after trypsin cleavage) against IL-23 was investigated (see Figure 4 , wherein 'FA1K' refers to FA1K before trypsin treatment and 'FA1K trypsin' refers to FA1K after trypsin treatment). FA1K (before and after trypsin treatment) is at least as potent as ID-L253T against IL-23.

[0733] The inclusion of ID-L253T at the C-terminal position of FA1K, resulting in an N-terminal extension of the ID-L253T arm compared to the free ID-L253T monomer, also appears to provide benefits for IL-23 neutralization.

[0734] It was also confirmed by ELISA that FAIK produced and purified by expression in Pichia pastoris was fully potent against TNF-α and IL-23 (data not shown).

[0735] It was shown that after 4 h of incubation in human fecal supernatant, the two monomer arms of FA1K retained the favorable stability characteristics of the parent monomers ID-38F and ID-L253T (see Figure 5 , where the survival % of (a) ID-38F, (b) ID-38F in the form of a FAlK monomer arm, (c) ID-L253T, and (d) ID-L253T in the form of a FAlK monomer arm) are given from left to right.

[0736] Taken together, the above findings demonstrate that FA1K is a suitable form for delivering high concentrations of each of these monomers as a dual therapy. FA1K retains the favorable efficacy and protease stability characteristics of the parent monomer. Due to the labile linker form, exposure of FA1K to trypsin results in rapid release of the two monomer arms, enabling independent binding to their respective targets without interference from the other arm.

[0737] Example 11: Ex vivo characterization of anti-IL-23 (ID-L210T) and anti-TNF-α (ID-38F) combination administration to human IBD tissue

[0738] It has been demonstrated above and in other publications that ICVDs with TNF-α neutralizing activity (ID-38F) and IL-23 neutralizing activity (ID-L210T) can inhibit the phosphorylation of trypsin kinase receptors and signaling proteins that are increased in inflamed intestinal tissue samples taken from patients diagnosed with IBD (see Example 8 above and WO2016156465, Example 9 therein).

[0739] New studies were conducted to investigate the effect of combining ID-38F with L210T on the levels of phosphorylated biomarkers in ex vivo cultures of inflamed colonic mucosal tissue obtained from patients with UC. ID-L210T differs from ID-L253T by only three amino acids and exhibits similar IL-23-IL-23R inhibitory efficacy and tolerance to in vitro digestion.

[0740] The effects of individual ICVDs were compared to a mixture of two ICVDs and to an isotype control ICVD (ID-2A, an unrelated anti-C. difficile toxin ICVD) to assess what effects might be achieved by combining different anti-cytokine mechanisms.

[0741] Biopsies from each of four UC patients were incubated with different antibodies (control ID-2A, ID-38F, ID-L210T, or ID-38F+ID-L210T) for 24 hours, and after treatment, tissue lysates were analyzed on the phosphoprotein antibody array. The average signal intensity of the 45 phosphoproteins detected on each array (4 arrays / treatment) / patient was Figure 6-7 (where L210T refers to ID-L210T). The inhibitory effect of the different antibody treatments was demonstrated by the shift from significantly high levels of phosphorylation in biopsies treated with the control ICVD ID2A to relatively low phosphor intensity values ​​in biopsies treated with anti-TNF-α or anti-IL-23 ICVD or a combination of both.

[0742] The data presented herein show that a therapeutic approach combining GI-restricted antagonism of TNF-α and IL-23 achieves a greater degree of effectiveness, for a longer period of time, in a greater proportion of patients with inflammatory bowel disease than monotherapy directed at a single target.

[0743] Other aspects

[0744] All references, including patents and patent applications, cited in this application are incorporated herein by reference to the maximum extent possible.

[0745] Throughout this specification, unless the context requires otherwise, the word 'comprise' and variations such as 'comprises' and 'comprising' will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not the exclusion of any other integer, step, any other group of integers or any other group of steps.

[0746] The application of which this description and claims form a part may be used as a basis for claiming priority on any subsequent application. The claims of this subsequent application may be directed to any feature or combination of features described herein. They may be in the form of product, composition, method or use claims, and may include, for example, but not limited to, the following claims.

[0747] References

[0748] The following references are incorporated herein by reference in their entirety.

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Claims

1. A polypeptide comprising an immunoglobulin chain variable domain that binds to IL-23, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR1 comprises a sequence that shares 60% or greater sequence identity with SEQ ID NO:1, CDR2 comprises a sequence that shares 50% or greater sequence identity with SEQ ID NO:2, and CDR3 comprises a sequence that shares 50% or greater sequence identity with SEQ ID NO:

3. 2 . The polypeptide according to claim 1 , wherein CDR1 comprises or consists of a sequence that shares 80% or greater sequence identity with SEQ ID NO: 1 .

3. The polypeptide according to claim 1 or 2, wherein CDR2 comprises or consists of a sequence sharing 80% or greater sequence identity with SEQ ID NO:

2. 4 . The polypeptide according to claim 1 , wherein CDR3 comprises or consists of a sequence sharing 80% or greater sequence identity with SEQ ID NO:

3.

5. The polypeptide according to claim 1, wherein CDR1 comprises or consists of SEQ ID NO: 1, CDR2 comprises or consists of SEQ ID NO: 2, and CDR3 comprises or consists of SEQ ID NO:

3.

6. A polypeptide according to any one of claims 1 to 5, comprising or consisting of a sequence sharing 50% or greater sequence identity, such as sharing 55% or greater sequence identity, such as sharing 60% or greater sequence identity, such as sharing 65% or greater sequence identity, such as sharing 70% or greater sequence identity, such as sharing 75% or greater sequence identity, such as sharing 80% or greater sequence identity, such as sharing 85% or greater sequence identity, such as sharing 90% or greater sequence identity, such as sharing 95% or greater sequence identity, such as sharing 96% or greater sequence identity, such as sharing 97% or greater sequence identity, such as sharing 98% or greater sequence identity, such as sharing 99% or greater sequence identity with SEQ ID NO:

8.

7. A construct comprising at least one polypeptide according to any one of claims 1 to 6 and at least one different polypeptide, wherein the different polypeptide binds to TNF-α.

8. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 6 or a construct according to claim 7 and one or more pharmaceutically acceptable diluents or carriers.

9. A method for treating an autoimmune disease and / or an inflammatory disease, comprising administering a therapeutically effective amount of a polypeptide according to any one of claims 1 to 6, a pharmaceutical composition according to claim 8 or a construct according to claim 7 to a person in need thereof.

10. A polynucleotide encoding a polypeptide according to any one of claims 1 to 6 or a construct according to claim 7.

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