polypeptides

Abstract

To provide a polypeptide comprising an immunoglobulin chain variable domain which binds to IL-23.SOLUTION: The immunoglobulin chain variable domain binding to IL-23 comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR1 comprises a sequence sharing 60% or greater sequence identity with a specific amino acid sequence, CDR2 comprises a sequence sharing 50% or greater sequence identity with a specific amino acid sequence, and CDR3 comprises a sequence sharing 50% or greater sequence identity with a specific amino acid sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to polypeptides comprising an immunoglobulin chain variable domain (or ICVD) that binds interleukin-23 (IL-23), as well as to constructs and pharmaceutical compositions comprising these polypeptides. The invention further relates to nucleic acids encoding said polypeptides, methods for preparing said polypeptides, cDNAs and vectors comprising nucleic acids encoding said polypeptides, host cells that express or are capable of expressing said polypeptides, and uses of said polypeptides, pharmaceutical compositions, or constructs. [Background technology]

[0002] IL-23 is a pleiotropic cytokine involved in regulating both physiological and pathological functions. IL-23 has functions related to inflammation and immune regulation and is thought to be important in the prevalence of chronic inflammation and associated pathology in autoimmune diseases, including inflammatory bowel disease (IBD, e.g., Crohn's disease (CD) and ulcerative colitis (UC)) (Croxford et al., 2012; Teng et al., 2015; Furfaro et al., 2017). In IBD patients, IL-23 production is increased at sites of intestinal inflammation. IL-23 orchestrates inflammation through its direct effect on pathogenic T cells, enhances the activation of IBD intraepithelial lymphocytes and NK cells, and stimulates the production of pro-inflammatory cytokines by innate lymphoid cells (Eken et al., 2014). IL-23 is a heterodimeric 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. Preclinical studies in various models of inflammatory bowel disease clearly demonstrate that IL-23 promotes intestinal inflammation and pathology. Although structurally related to IL-12, studies using IL-23-specific neutralizing antibodies in these models have revealed that IL-23 and IL-12 have diverse roles in mucosal and systemic immune responses and that selective depletion of IL-23 can suppress intestinal inflammation while sparing systemic immune responses.

[0003] Anti-TNF monoclonal antibodies have revolutionized the treatment of Crohn's disease and ulcerative colitis. By neutralizing TNF activity, antibodies such as infliximab and adalimumab promote mucosal healing and induce long-term remission in many patients. However, approximately one-third of patients prescribed anti-TNF agents are primary non-responders. Among primary responders, subsequent loss of response can vary from 10 to 50% per year (secondary non-response) (Colombel et al. 2007; Hanauer et al. 2002, 2006; Sandborn et al. 2007; Schreiber et al. 2007). Primary non-responders are unlikely to benefit from switching to a second-line anti-TNF agent; therefore, therapeutic strategies targeting other inflammatory pathways are needed.

[0004] These findings suggest that IL-23 may be a therapeutic target for IBD, providing a rationale for local rather than systemic administration (McGovern and Powrie, 2007). Furthermore, such agents may provide an effective treatment for IBD in situations where anti-TNF antibodies have failed.

[0005] WO2007005955 and WO2007027714 disclose anti-IL-23p19 antibodies.

[0006] Recently, the efficacy and safety of brazikumab (AMG-139), a fully human IgG2 monoclonal antibody that selectively binds to the p19 subunit of IL-23, has been investigated in patients with active CD who have failed or are intolerant to anti-TNFα treatment (Sandborn et al. 2018, Sands et al. 2017). The superiority of many of the polypeptides of the present invention over brazikumab is demonstrated in Examples 2 and 5 below.

[0007] A further anti-IL-23 agent of the prior art is 37D5, an anti-IL-23p19 domain antibody (VHH) (Desmyter et al. 2017). The superiority of many of the polypeptides of the invention over 37D5 is demonstrated in Example 5 below.

[0008] Polypeptides of the present invention, in at least some embodiments, may have one or more of the following advantages over prior art anti-IL-23 agents. (i) increased affinity for IL-23; (ii) increased specificity for IL-23; (iii) increased neutralizing ability against IL-23; (iv) increased specificity for IL-23 over IL-12; (v) increased cross-reactivity with IL-23 from different species, such as humans and cynomolgus monkeys; (vi) reduced immunogenicity when administered to, for example, mice, cynomolgus monkeys, or humans; (vii) increased stability in the presence of proteases, for example, (a) proteases found in the small intestine and / or large intestine and / or IBD inflammatory proteases, e.g., trypsin, chymotrypsin, MMP3, MMP10, MMP12, other MMPs, and cathepsins, and / or (b) proteases from gut commensal flora and / or pathogenic bacteria that are actively secreted and / or released by lysis of microbial cells found in the small intestine and / or large intestine; (viii) increased stability against protease degradation during production (e.g., resistance to yeast proteases); (ix) increased suitability for oral administration; (x) increased suitability for local delivery to the intestinal tract and lamina propria after oral administration; (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, e.g., Saccharomyces cerevisiae or Pichia pastoris; (xii) suitability and improved properties for use in pharmaceuticals; (xiii) suitability and improved properties for use in functional foods; (xiv) enhanced tissue permeability, including penetration of inflamed colonic mucosal epithelium and submucosa to access the submucosal lamina propria; (xv) reduced immunogenicity in humans, for example, due to increased sequence similarity to human immunoglobulins; (xvi) increased compatibility of formatting in multispecific formats; (xvii) binding to a novel epitope;

[0009] The above advantages (i) to (xvii) can potentially be achieved by the polypeptides of the invention in a monovalent format or in a multivalent format, such as a bihead format (e.g., a homobihead or heterobihead format). Summary of the Invention

[0010] The present inventors have produced surprisingly advantageous polypeptides comprising immunoglobulin chain variable domains that bind to IL-23, such as the immunoglobulin chain variable domains ID-L253T, 10E2, and 10G10, and polypeptides related to each of these immunoglobulin chain variable domains.

[0011] These polypeptides have been found to have unexpected advantages over the prior art anti-IL-23 agents brazikumab and 37D5 (see especially the Background section above and Examples 2 and 5 below).

[0012] These polypeptides in particular benefit from surprisingly high potency. They are also able to cross-react with cynomolgus IL-23 and remain stable when exposed to small and large intestinal proteases. In one embodiment, these polypeptides have been further enhanced by engineering. These further enhanced polypeptides benefit from the above advantages, retain IL-23 neutralizing activity during passage through the intestinal tract, and further resist degradation and / or inactivation by intestinal proteases, e.g., digestive proteases, inflammatory proteases, and microbial proteases, e.g., from mammalian species.

[0013] These polypeptides may be expected to be useful, inter alia, when administered orally, in the prevention or treatment of autoimmune and / or inflammatory diseases, such as inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), or in the prevention or treatment of mucositis.

[0014] Many of the polypeptides of the present invention have been shown to have superior properties compared to brazikumab, and a fully human IgG2 monoclonal antibody that selectively binds to the p19 subunit of IL-23 has been investigated in patients with active CD who have failed or are intolerant to anti-TNFα therapy. The superiority of many of the polypeptides of the present invention over brazikumab is demonstrated in Examples 2 and 5 below.

[0015] Many of the polypeptides of the present invention have also been shown to have superior properties compared to the anti-IL-23p19 domain antibody (VHH), 37D5 (Desmyter et al. 2017). The superiority of many of the polypeptides of the present invention over 37D5 is demonstrated in Example 5 below.

[0016] In certain embodiments, the inventors have provided the above polypeptides in a "bihead" format together with an anti-TNF-α polypeptide. The data provided herein demonstrate that a therapeutic approach combining the gut-restricted antagonism of IL-23 and TNF-α can achieve a greater degree of efficacy, for a longer duration, in a higher proportion of patients with inflammatory bowel disease than monotherapy directed against either target alone.

[0017] In one embodiment, 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 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.

[0018] In a further 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 more sequence identity with SEQ ID NO: 14, CDR2 comprises a sequence that shares 50% or more sequence identity with SEQ ID NO: 15, and CDR3 comprises a sequence that shares 50% or more sequence identity with SEQ ID NO: 16.

[0019] In a further 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 more sequence identity with SEQ ID NO: 22, CDR2 comprises a sequence that shares 50% or more sequence identity with SEQ ID NO: 23, and CDR3 comprises a sequence that shares 50% or more sequence identity with SEQ ID NO: 24.

[0020] In a further aspect, the present invention provides a construct comprising two or more identical polypeptides according to the invention.

[0021] In a further aspect, the present 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.

[0022] In a further aspect, the present 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.

[0023] Further aspects of the invention are disclosed elsewhere herein. [Brief explanation of the drawings]

[0024] [Figure 1] Mean phospho-intensity values ​​of ex vivo UC inflamed colonic mucosal tissues treated with ID-L210T. [Figure 2] Mean phosphorus-intensity values ​​of ex vivo UC inflamed colonic mucosal tissues treated with ID-L210T (cont.). [Figure 3] Neutralizing potency of FA1K (before and after trypsin cleavage) against TNF-α. [Figure 4] Neutralizing potency of FA1K (before and after trypsin cleavage) against IL-23. [Figure 5] Stability of FA1K parent monomers ID-38F and ID-L253T after 4 hours of incubation in human fecal supernatant. [Figure 6] Mean phosphorus-intensity values ​​of ex vivo UC inflamed colonic mucosal tissues treated with ID-L210T and ID-38F. [Figure 7] Mean phosphorus-intensity values ​​of ex vivo UC inflamed colonic mucosal tissues treated with ID-L210T and ID-38F (cont.).

[0025] Sequence Listing SEQ ID NO:1-ID-L253T CDR1 polypeptide sequence, SEQ ID NO:2-ID-L253T CDR2 polypeptide sequence, SEQ ID NO:3-ID-L253T CDR3 polypeptide sequence, The polypeptide sequence of SEQ ID NO: 4-ID-L253T FR1, The polypeptide sequence of SEQ ID NO: 5-ID-L253T FR2, The polypeptide sequence of SEQ ID NO: 6-ID-L253T FR3, The polypeptide sequence of SEQ ID NO: 7-ID-L253T FR4, The polypeptide sequence of SEQ ID NO: 8-ID-L253T, SEQ ID NO: 9-ID-L253T encoding polynucleotide sequence (including stop codon); SEQ ID NO: 10-ID-Polynucleotide sequence encoding L253T (without stop codon), Polypeptide sequences of SEQ ID NOs: 11-12G1; SEQ ID NO: 12-1E2 polypeptide sequence, Polypeptide sequences of SEQ ID NOs: 13-10E2, SEQ ID NO: 14-10E2 Polypeptide sequence of CDR1, SEQ ID NO: 15-10E2 Polypeptide sequence of CDR2, SEQ ID NO: 16-10E2 CDR3 polypeptide sequence, SEQ ID NO: 17-10E2 FR1 polypeptide sequence, SEQ ID NO: 18-10E2 FR2 polypeptide sequence, SEQ ID NO: 19-10E2 FR3 polypeptide sequence, SEQ ID NO: 20-10E2 FR4 polypeptide sequence, SEQ ID NO: 21-10G10 polypeptide sequence; SEQ ID NO: 22-10G10 Polypeptide sequence of CDR1, SEQ ID NO: 23-10G10 CDR2 polypeptide sequence, SEQ ID NO: 24-10G10 CDR3 polypeptide sequence, SEQ ID NO: 25 - Polypeptide sequence of 10G10 FR1; SEQ ID NO: 26 - Polypeptide sequence of 10G10 FR2, SEQ ID NO: 27-10G10 FR3 polypeptide sequence; SEQ ID NO: 28-10G10 FR4 polypeptide sequence; The polypeptide sequence of SEQ ID NO: 29-ID-L210T, The polypeptide sequence of SEQ ID NO: 30-ID-L237T, The polypeptide sequence of SEQ ID NO: 31-ID-L238T, The polypeptide sequence of SEQ ID NO: 32-ID-L239T, The polypeptide sequence of SEQ ID NO: 33-ID-L240T, The polypeptide sequence of SEQ ID NO: 34-ID-L241T, The polypeptide sequence of SEQ ID NO: 35-ID-L242T, The polypeptide sequence of SEQ ID NO: 36-ID-L243T, The polypeptide sequence of SEQ ID NO: 37-ID-L244T, The polypeptide sequence of SEQ ID NO: 38-ID-L245T, The polypeptide sequence of SEQ ID NO: 39-ID-L246T, The polypeptide sequence of SEQ ID NO: 40-ID-L247T, The polypeptide sequence of SEQ ID NO: 41-ID-L248T, The polypeptide sequence of SEQ ID NO: 42-ID-L249T, The polypeptide sequence of SEQ ID NO: 43-ID-L250T, The polypeptide sequence of SEQ ID NO: 44-ID-L251T, The polypeptide sequence of SEQ ID NO: 45-ID-L252T, SEQ ID NO: 46 - Polypeptide sequence of FA1K, SEQ ID NO: 47-Polypeptide sequence of the ID-38F arm in FA1K; SEQ ID NO: 48-Polypeptide sequence of the ID-L253T arm in FA1K; SEQ ID NO: 49-The polypeptide sequence of the labile linker in FA1K; A polynucleotide sequence encoding SEQ ID NO: 50-ID-L210T, A polynucleotide sequence encoding SEQ ID NO: 51-ID-L237T, A polynucleotide sequence encoding SEQ ID NO: 52-ID-L238T, SEQ ID NO: 53-ID-L239T encoding polynucleotide sequence; SEQ ID NO: 54-ID-L240T, a polynucleotide sequence encoding SEQ ID NO: 55-ID-L241T encoding polynucleotide sequence; A polynucleotide sequence encoding SEQ ID NO: 56-ID-L242T, SEQ ID NO: 57-ID-L243T, a polynucleotide sequence encoding A polynucleotide sequence encoding SEQ ID NO: 58-ID-L244T, A polynucleotide sequence encoding SEQ ID NO: 59-ID-L245T, A polynucleotide sequence encoding SEQ ID NO: 60-ID-L246T, A polynucleotide sequence encoding SEQ ID NO: 61-ID-L247T, A polynucleotide sequence encoding SEQ ID NO: 62-ID-L248T, A polynucleotide sequence encoding SEQ ID NO: 63-ID-L249T, A polynucleotide sequence encoding SEQ ID NO: 64-ID-L250T, SEQ ID NO: 65-ID-L251T encoding polynucleotide sequence; A polynucleotide sequence encoding SEQ ID NO: 66-ID-L252T, The polypeptide sequence of SEQ ID NO: 67-ID-38F, SEQ ID NO:68—Polynucleotide sequence of 3′ primer containing SpeI site. Polypeptide sequences of SEQ ID NOs: 69-37D5; SEQ ID NO:70 - Brazikumab heavy chain, SEQ ID NO:71 - Brazikumab light chain, SEQ ID NO: 72—Polypeptide sequence of IL-23p19; SEQ ID NO: 73—Polypeptide sequence of IL-23p40; SEQ ID NO:74 - Polypeptide sequence of protease labile linker formula SEQ ID NO:75—Polypeptide sequence of protease labile linker, SEQ ID NO:76—Polypeptide sequence of non-protease labile linker formula SEQ ID NO:77—Polypeptide sequence of non-protease labile linker. DETAILED DESCRIPTION OF THE INVENTION

[0026] Polypeptides including antibodies and antibody fragments containing VH and VHH A conventional antibody or immunoglobulin (Ig) is a protein containing four polypeptide chains: two heavy (H) chains and two light (L) chains. Each chain is divided into a constant region and a variable region. The heavy chain variable domain is abbreviated herein as VHC, and the light chain variable domain is abbreviated herein as VLC. These domains, their related domains, and domains derived from them are referred to herein as immunoglobulin chain variable domains. The VHC and VLC domains can be further subdivided into regions of hypervariability called "complementarity-determining regions" ("CDRs"), interspersed with more conserved regions called "framework regions" ("FRs"). The framework regions and complementarity-determining regions have been precisely defined (Kabat et al., 1991). In conventional antibodies, the VHC and VLC each consist of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A conventional antibody tetramer, for example, consists of two immunoglobulin heavy chains and two immunoglobulin light chains interconnected by disulfide bonds, with the immunoglobulin heavy chains being similarity-connected. The heavy chain constant region contains three domains, CH1, CH2, and CH3. The light chain constant region consists of one domain, CL. The heavy chain variable domain and the light chain variable domain are binding domains that interact with antigens. The antibody constant region typically mediates antibody binding to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The term antibody includes immunoglobulins of types IgA, IgG, IgE, IgD, and IgM (and their subtypes), and the immunoglobulin light chains may be kappa or lambda types. The overall structure of immunoglobulin-γ (IgG) antibodies, assembled from two identical heavy (H) chain and two identical light (L) chain polypeptides, is well established and highly conserved in mammals (Padlan 1994).

[0027] An exception to the conventional antibody structure is found in camelid sera. In addition to conventional antibodies, these sera contain special IgG antibodies. These IgG antibodies, known as heavy-chain antibodies (HCAbs), lack the light-chain polypeptide and the first constant domain (CH1). In their N-terminal regions, the heavy chains of the homodimeric proteins contain dedicated immunoglobulin chain variable domains, termed VHHs, which are responsible for associating with their cognate antigens (Muyldermans 2013, Hamers-Casterman et al. 1993, Muyldermans et al. 1994).

[0028] An antigen-binding fragment (or "antibody fragment" or "immunoglobulin fragment") as used herein means 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 which specifically binds to IL-23). ​​Examples of binding fragments encompassed within the term antigen-binding fragment include the following: (i) FAb fragments (monovalent fragments consisting of the VLC, VHC, CL, and CH1 domains); (ii) F(ab')2 fragment (a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region); (iii) Fd fragment (consisting of the VHC and CH1 domains); (iv) Fv fragments (consisting of the VLC and VHC domains of a single arm of an antibody); (v) scFv fragments (consisting of VLC and VHC domains recombinantly linked by a synthetic linker, which allows the VLC and VHC domains to form a single protein chain, and the VLC and VHC regions pair to form a monovalent molecule); (vi) VH (immunoglobulin chain variable domain consisting of VHC domains (Ward et al. 1989) (vii) VL (immunoglobulin chain variable domain consisting of VLC domain), (viii) V-NAR (immunoglobulin chain variable domain consisting of the VHC domain from cartilaginous fish IgNAR (Roux et al. 1998 and Griffiths et al. 2013)); (ix) VHH.

[0029] The total number of amino acid residues in VHH or VH may be in the range of 110 to 130, preferably 115 to 125, and most preferably 121.

[0030] The immunoglobulin chain variable domains of the invention can be obtained, for example, by preparing a nucleic acid encoding the immunoglobulin chain variable domain using techniques for nucleic acid synthesis, and then expressing the nucleic acid thus obtained. According to certain embodiments, the immunoglobulin chain variable domains of the invention do not have an amino acid sequence that is exactly the same as (i.e., shares 100% sequence identity with) the amino acid sequence of a naturally occurring polypeptide, such as the VH domain or VHH domain of a naturally occurring antibody.

[0031] The examples provided herein relate to the immunoglobulin chain variable domain itself that binds to IL-23. However, the principles of the invention disclosed herein are equally applicable to any polypeptide containing an immunoglobulin chain variable domain that binds to IL-23, such as antibodies and antibody fragments. For example, the anti-IL-23 immunoglobulin chain variable domains disclosed herein may be incorporated into polypeptides such as full-length antibodies. Such an approach is demonstrated by McCoy et al. (2014), who provide an anti-HIV VHH designed as a fusion with a human Fc region (including the hinge, CH2, and CH3 domains) that is expressed as a dimeric construct.

[0032] Humanization is the replacement of 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. Humanization of variable domains can reduce immunogenicity in humans.

[0033] Preferably, the polypeptide of the present invention comprises an immunoglobulin chain variable domain. More preferably, the polypeptide of the present invention consists of an immunoglobulin chain variable domain such as an immunoglobulin heavy chain variable domain. Preferably, the polypeptide of the present invention is an antibody or an antibody fragment. More preferably, the polypeptide of the present invention is an antibody fragment. Preferably, the antibody fragment is an immunoglobulin variable domain such as a VHH, VH, or VL. Preferably, the antibody fragment is a VHH, VH, VL, V-NAR, scFv, FAb fragment, or F(ab')2 fragment. Preferably, the antibody fragment is an immunoglobulin heavy chain variable domain. More preferably, the antibody fragment is a VHH or VH, and most preferably a VHH.

[0034] Specificity, affinity, avidity, and cross-reactivity 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 a particular antigen as a unique molecular entity and distinguish it from other antigens.

[0035] Affinity, expressed as the equilibrium constant (Kd) for the dissociation of an antigen from an antigen-binding polypeptide, is an indicator of the binding strength between an antigenic determinant and an antigen-binding site on the antigen-binding polypeptide: the smaller the Kd value, the stronger the binding strength between the antigenic determinant and the antigen-binding polypeptide (alternatively, affinity can also be expressed as the affinity constant (Ka), which is 1 / Kd). Affinity can be determined by known methods depending on the particular antigen of interest. Preferably, affinity is determined using dynamically switchable biosurfaces (e.g., "switchSENSER", see Knezevic et al., 2012) or surface plasmon resonance.

[0036] Avidity is a measure of the strength of the bond between an antigen-binding polypeptide and an appropriate antigen. Avidity is related to both the affinity between an antigenic determinant on the antigen-binding polypeptide and its antigen-binding site, and the number of appropriate binding sites present on the antigen-binding polypeptide.

[0037] Preferably, the polypeptide of the present invention is -6 M or less, more preferably 10 -7 M or less, more preferably 10 -8 M or less, more preferably 10 -9 M or less, more preferably 10 -10 M or less, more preferably 10 -11 M or less, more preferably 10 -12 M or less, more preferably 10 -13 It binds to IL-23 with a dissociation constant (Kd) of less than M.

[0038] 10 -6 A Kd value less than M is considered to indicate binding. Specific binding of an antigen-binding polypeptide to an antigen or antigenic determinant can be determined by any suitable known method, including, for example, Scatchard analysis and / or competitive binding assays such as radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competition assays, and their different variants known in the art. Preferably, the affinity of a polypeptide is determined by surface plasmon resonance.

[0039] In one embodiment, the affinity of the polypeptide is established by amine coupling an α-p40 capture antibody to a sensor chip in 10 mM sodium acetate buffer at pH 5 at 25°C. IL-23 is then immobilized on the chip by flowing at 2 μg / mL or 0.5 μg / mL at 10 μL / min for 60 seconds, allowing the P19 subunit to freely bind to the polypeptide. Test polypeptides are then injected at five different concentrations, ranging from 0.0195 to 5 nM (L253T) or 0.8 to 500 nM (brazikumab Fab formulation), in a pH 7.4 buffer containing 0.01 M HEPES, 0.15 M NaCl, 0.05% polysorbate 20, and 3 mM EDTA, at 30 μL / min for 300 seconds on and 300 seconds off. Bound anti-P40 is regenerated during cycling with 10 mM glycine pH 2 for 60 seconds at 10 μl / min. This methodology was used in Example 5.2 below.

[0040] 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 can bind to a linear or conformational epitope on IL-23.

[0041] Preferably, the polypeptides of the present invention bind to human IL-23. More preferably, the polypeptides of the present invention bind to both human IL-23 and IL-23 of at least one additional primate selected from the group consisting of baboon IL-23, marmoset IL-23, cynomolgus IL-23, and rhesus IL-23. More preferably, the polypeptides of the present invention bind to both human IL-23 and cynomolgus IL-23.

[0042] Preferably, the polypeptides of the present invention neutralize human IL-23. More preferably, the polypeptides of the present invention neutralize both human IL-23 and IL-23 of at least one additional primate selected from the group consisting of baboon IL-23, marmoset IL-23, cynomolgus IL-23, and rhesus IL-23. More preferably, the polypeptides of the present invention neutralize both human IL-23 and cynomolgus IL-23.

[0043] Preferably, IL-23 is a polypeptide comprising SEQ ID NO: 72 (p19 subunit) and 73 (p40 subunit), more preferably, IL-23 is a polypeptide consisting of SEQ ID NO: 72 and 73. Preferably, IL-23p19 is a polypeptide comprising SEQ ID NO: 72. More preferably, 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.

[0044] Polypeptides that are capable of reacting with IL-23 from humans and with IL-23 from another species, for example, cynomolgus IL-23 ("cross-reactive"), are advantageous because they allow preclinical studies to be more easily performed in animal models.

[0045] Suitably, the polypeptides of the invention are directed against an epitope on IL-23 that is present in and / or forms part of the receptor binding site of IL-23, and upon binding to IL-23, the polypeptides of the invention are capable of inhibiting or reducing said IL-23-mediated IL-23 receptor cross-linking and / or signal transduction mediated by said receptor cross-linking.

[0046] The polypeptides of the present invention bind to one or more epitopes on IL-23. In one embodiment of the present invention, there are provided polypeptides that bind to the same epitope on IL-23 as ID-L253T, 12G1, 1E2, 10E2, or 10G10, more preferably ID-L253T. Because the polypeptides of the present invention are expected to be specific for the p19 subunit of IL-23, references to "IL-23" throughout this specification can be replaced with "p19" where appropriate.

[0047] Preferably, the polypeptides of the invention are isolated. An "isolated" polypeptide is one that has been removed from its original environment. For example, a naturally occurring polypeptide of the invention is isolated if it is separated from some or all of the coexisting materials in the natural system.

[0048] Potency, Inhibition, and Neutralization Potency is a measure of a therapeutic agent's activity in terms of the amount required to produce an effect of a given magnitude. A highly potent agent will produce a larger response at a lower concentration, compared to a less potent agent, which will produce a smaller response at a lower concentration. Potency is a function of affinity and efficacy. Efficacy refers to the ability of a therapeutic agent to produce a biological response upon binding to a target ligand and the quantitative magnitude of this response. The term maximal effective concentration (EC50) refers to the concentration of a therapeutic agent that produces a response halfway between the baseline and maximal concentrations after a specified exposure time. A therapeutic agent may produce either inhibition or stimulation. This is commonly, and is used herein, as an indication of efficacy.

[0049] For the purposes of the present invention, a neutralizing polypeptide is a polypeptide that binds to IL-23 and inhibits the binding of IL-23 to its cognate receptor (IL-23R), as measured by ELISA or the like, as described in Evaluation Method A in the Examples.

[0050] Suitably, the polypeptide of the present invention neutralises human IL-23 in IL-23-IL-23R neutralisation ELISA (see Evaluation method A in the Examples) with an EC50 of 5 nM or less, for example, 4 nM or less, for example, 3 nM or less, for example, 2 nM or less, for example, 1.7 nM or less, for example, 1.5 nM or less, for example, 1.4 nM or less, for example, 1.3 nM or less, for example, 1.2 nM or less, for example, 1.1 nM or less, for example, 1.0 nM or less, for example, 0.9 nM or less, for example, 0.8 nM or less, for example, 0.75 nM or less, for example, 0.70 nM or less, for example, 0.65 nM or less, for example, 0.60 nM or less, for example, 0.55 nM or less, for example, 0.50 nM or less, for example, 0.45 nM or less, for example, 0.40 nM or less.

[0051] Polypeptide and Polynucleotide Sequences 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 with standard settings for polypeptide sequences (BLASTP). 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 with NCBI BLAST v2.0 with standard settings for nucleotide sequences (BLASTN). The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as default a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as default a word length of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) with an alignment (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & 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 preferably less than about 0.01, and most preferably less than about 0.001.

[0052] A polypeptide or polynucleotide sequence is said to be identical or similar to another polypeptide or polynucleotide sequence if they share 100% sequence identity over their entire length. Residues in a sequence are numbered from left to right, i.e., from N-terminus to C-terminus for polypeptides and from 5' to 3' for polynucleotides.

[0053] A "difference" between sequences refers to the insertion, deletion, or substitution of a single amino acid residue at a position in the second sequence compared to the first sequence. Two polypeptide sequences can contain one, two, or more such amino acid differences. Insertions, deletions, or substitutions in a second sequence that is otherwise identical to the first sequence (100% sequence identity) result in a decrease in the percentage of sequence identity. For example, if an identical sequence is 9 amino acid residues long, one substitution in the second sequence will result in 88.9% sequence identity. If an identical sequence is 17 amino acid residues long, two substitutions in the second sequence will result in 88.2% sequence identity. If an identical sequence is 7 amino acid residues long, three substitutions in the second sequence will result in 57.1% sequence identity. If the first and second polypeptide sequences are 9 amino acid residues in length and share 6 identical residues, the first and second polypeptide sequences share greater than 66% identity (the first and second polypeptide sequences share 66.7% identity). If the first and second polypeptide sequences are 17 amino acid residues in length and share 16 identical residues, the first and second polypeptide sequences share greater than 94% identity (the first and second polypeptide sequences share 94.1% identity). If the first and second polypeptide sequences are 7 amino acid residues in length and share 3 identical residues, the first and second polypeptide sequences share greater than 42% identity (the first and second polypeptide sequences share 42.9% identity).

[0054] Alternatively, for the purpose of comparing a first reference polypeptide sequence with a second comparison polypeptide sequence, the number of additions, substitutions, and / or deletions made to the first sequence to generate the second sequence can be determined. An addition is the addition of one 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 one amino acid residue in the sequence of the first polypeptide with a different amino acid residue. A deletion is the removal of one amino acid residue from the sequence of the first polypeptide (including deletions at either end of the first polypeptide).

[0055] For purposes of comparing a first reference polynucleotide sequence with a second comparison polynucleotide sequence, the number of additions, substitutions, and / or deletions made to the first sequence to generate the second sequence can also be determined. An addition is the addition of a single 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 single nucleotide residue in the sequence of the first polynucleotide with a different nucleotide residue. A deletion is the removal of a single nucleotide residue from the sequence of the first polynucleotide (including deletions at either end of the first polynucleotide).

[0056] "Conservative" amino acid substitutions are those in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and are expected to have little effect on the function, activity, or other biological properties of the polypeptide. Such conservative substitutions are preferably those in which one amino acid residue within the following group is replaced with another amino acid residue from the same group:

[0057] [Table 1]

[0058] Preferably, the hydrophobic amino acid residue is a non-polar amino acid. More preferably, the hydrophobic amino acid residue is selected from V, I, L, M, F, W, or C.

[0059] As used herein, the numbering of polypeptide sequences and the definitions of CDRs and FRs are as defined according to the Kabat system (Kabat et al., 1991). A "corresponding" amino acid residue between a first and a second polypeptide sequence is an amino acid residue in the first sequence that shares the same position as an amino acid residue in the second sequence according to the Kabat system, although the amino acid residue in the second sequence may have a different identity from the first sequence. If the framework and CDRs are the same length according to the Kabat definition, then corresponding residues will share the same number (and letter) as appropriate. Alignment can be accomplished manually or using known computer algorithms for sequence alignment, such as, for example, NCBI BLAST v2.0 (BLASTP or BLASTN) using standard settings.

[0060] Preferably, the polynucleotide used in the present invention is isolated. An "isolated" polynucleotide is one that has been removed from its original environment. For example, a naturally occurring polynucleotide is isolated when it is separated from some or all of the coexisting materials in the natural system. A polynucleotide is considered to be isolated, for example, when it is cloned into a vector that is not part of its natural environment or when it is not contained in a cDNA.

[0061] In one embodiment of the present invention, a polynucleotide encoding a polypeptide of the present invention is provided. Preferably, the polynucleotide comprises or consists of a sequence sharing 70% or more, e.g., 80% or more, e.g., 90% or more, e.g., 95% or more, e.g., 99% or more, sequence identity with SEQ ID NO: 9, 10, or 50 to 66. More preferably, the polynucleotide comprises or consists of any one of SEQ ID NO: 9, 10, or 50 to 66. In a further embodiment, a cDNA comprising the polynucleotide is provided.

[0062] In one aspect of the present invention, there is provided a polynucleotide comprising or consisting of a sequence that encodes CDR1, CDR2, or CDR3 of an encoded immunoglobulin chain variable domain and shares 70% or more, for example, 80% or more, for example, 90% or more, for example, 95% or more, for example, 99% or more sequence identity with any one of SEQ ID NOs: 9, 10, or a portion of SEQ ID NOs: 50 to 66.

[0063] Preferably, the polypeptide sequences of the present invention comprise at least one modification relative to the native sequence. Preferably, the polynucleotide sequences of the present invention comprise at least one modification relative to the native sequence. Preferably, the modification to the polypeptide or polynucleotide sequence is made to increase the stability of the polypeptide or encoded polypeptide against proteases present in the intestinal tract (e.g., trypsin and chymotrypsin).

[0064] Specific polypeptide sequence embodiments of the invention are outlined below.

[0065] ID-L253T and related polypeptides Polypeptide sequence of ID-L253T in Kabat format The polypeptide sequence of ID-L253T, a particularly advantageous polypeptide of the invention, is set forth 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 line provides the Kabat numbering (with an "H" prefix) of each polypeptide residue, the middle line provides the polypeptide residues, and the bottom line provides the consecutive numbering of each polypeptide residue.

[0066] [Table 2]

[0067] CDRs of ID-L253T and related polypeptides Preferably, CDR1 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence sharing 80% or more sequence identity with SEQ ID NO:1.

[0068] Alternatively, CDR1 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has two or less, more preferably one or less additions compared to SEQ ID NO: 1. Preferably, CDR1 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has two or less, more preferably one or less substitutions compared to SEQ ID NO: 1. Preferably, CDR1 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has two or less, more preferably one or less deletions compared to SEQ ID NO: 1.

[0069] Preferably, any residues in CDR1 that differ from their corresponding residues in SEQ ID NO: 1 are conservative substitutions compared to their corresponding residues. Preferably, CDR1 comprises, or more preferably consists of, SEQ ID NO: 1.

[0070] Preferably, CDR2 of the polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 55% or more, more preferably 60% or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more sequence identity with SEQ ID NO:2.

[0071] Alternatively, CDR2 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer additions compared to SEQ ID NO: 2. Preferably, CDR2 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer substitutions compared to SEQ ID NO: 2. Preferably, CDR2 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer deletions compared to SEQ ID NO: 2.

[0072] Suitably, any residues in CDR2 that differ from their corresponding residues in SEQ ID NO:2 are conservative substitutions compared with their corresponding residues.

[0073] Preferably, the residue in CDR2 corresponding to residue number 9 of SEQ ID NO:2 is D or H. Preferably, the residue in CDR2 corresponding to residue number 10 of SEQ ID NO:2 is Y or D. Preferably, the residue in CDR2 corresponding to residue number 11 of SEQ ID NO:2 is S, G, R, or A (more preferably S, R, or A, more preferably S or A). Preferably, the residue in CDR2 corresponding to residue number 14 of SEQ ID NO:2 is V or A.

[0074] Preferably, the residue in CDR2 corresponding to residue number 9 of SEQ ID NO:2 is D or H, the residue in CDR2 corresponding to residue number 10 of SEQ ID NO:2 is Y or D, the residue in CDR2 corresponding to residue number 11 of SEQ ID NO:2 is S, G, R, or A (more preferably S, R, or A, more preferably S or A), and the residue in CDR2 corresponding to residue number 14 of SEQ ID NO:2 is V or A.

[0075] Preferably, CDR2 comprises, or more preferably consists of, SEQ ID NO:2.

[0076] Preferably, the CDR3 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence sharing 60% or more, more preferably 65% ​​or more, more preferably 75% or more, more preferably 80% or more, more preferably 90% or more sequence identity with SEQ ID NO: 3.

[0077] Alternatively, the CDR3 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has no more than 3, more preferably no more than 2, more preferably no more than 1 addition compared to SEQ ID NO: 3. Preferably, the CDR3 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has no more than 3, more preferably no more than 2, more preferably no more than 1 substitution compared to SEQ ID NO: 3. Preferably, the CDR3 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has no more than 3, more preferably no more than 2, more preferably no more than 1 deletion compared to SEQ ID NO: 3. Preferably, any substitutions are conservative to their corresponding residues in SEQ ID NO: 3.

[0078] Suitably, any residues in CDR3 that differ from their corresponding residues in SEQ ID NO: 3 are conservative substitutions compared with their corresponding residues.

[0079] Preferably, the residue in CDR3 corresponding to residue number 6 of SEQ ID NO:3 is I or L.

[0080] Preferably, (a) the residue in CDR2 corresponding to residue number 9 of SEQ ID NO:2 is D or H, the residue in CDR2 corresponding to residue number 10 of SEQ ID NO:2 is Y or D, the residue in CDR2 corresponding to residue number 11 of SEQ ID NO:2 is S, G, R, or A (more preferably S, R, or A, more preferably S or A), and the residue in CDR2 corresponding to residue number 14 of SEQ ID NO:2 is V or A; and (b) the residue in CDR3 corresponding to residue number 6 of SEQ ID NO:3 is I or L.

[0081] FR of ID-L253T and related polypeptides Preferably, FR1 of the polypeptide of the present invention comprises, or more preferably 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.

[0082] Alternatively, FR1 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having an addition of 28 or less, more preferably 26 or less, more preferably 24 or less, more preferably 22 or less, more preferably 20 or less, more preferably 18 or less, more preferably 16 or less, more preferably 14 or less, more preferably 13 or less, more preferably 12 or less, more preferably 11 or less, more preferably 10 or less, more preferably 9 or less, more preferably 8 or less, more preferably 7 or less, more preferably 6 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and more preferably 1 or less compared to SEQ ID NO: 4. Preferably, FR1 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 28 or less, more preferably 26 or less, more preferably 24 or less, more preferably 22 or less, more preferably 20 or less, more preferably 18 or less, more preferably 16 or less, more preferably 14 or less, more preferably 13 or less, more preferably 12 or less, more preferably 11 or less, more preferably 10 or less, more preferably 9 or less, more preferably 8 or less, more preferably 7 or less, more preferably 6 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and more preferably 1 or less substitutions compared to SEQ ID NO: 4. Preferably, FR1 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 28 or less, more preferably 26 or less, more preferably 24 or less, more preferably 22 or less, more preferably 20 or less, more preferably 18 or less, more preferably 16 or less, more preferably 14 or less, more preferably 13 or less, more preferably 12 or less, more preferably 11 or less, more preferably 10 or less, more preferably 9 or less, more preferably 8 or less, more preferably 7 or less, more preferably 6 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and more preferably 1 or less deletions compared to SEQ ID NO: 4.

[0083] Suitably, any residues in FR1 that differ from their corresponding residues in SEQ ID NO: 4 are conservative substitutions compared to their corresponding residues.

[0084] Preferably, the residue in FR1 corresponding to residue number 1 of SEQ ID NO:4 is D or E. Preferably, the residue in FR1 corresponding to residue number 11 of SEQ ID NO:4 is Q or L. Preferably, the residue in FR1 corresponding to residue number 19 of SEQ ID NO:4 is S or R. Preferably, the residue in FR1 corresponding to residue number 23 of SEQ ID NO:4 is E or A. Preferably, the residue in FR1 corresponding to residue number 24 of SEQ ID NO:4 is S or A.

[0085] Preferably, the residue in FR1 corresponding to residue number 1 of SEQ ID NO:4 is D or E, the residue in FR1 corresponding to residue number 11 of SEQ ID NO:4 is Q or L, the residue in FR1 corresponding to residue number 19 of SEQ ID NO:4 is S or R, the residue in FR1 corresponding to residue number 23 of SEQ ID NO:4 is E or A, and the residue in FR1 corresponding to residue number 24 of SEQ ID NO:4 is S or A.

[0086] Preferably, FR1 comprises, or more preferably consists of, SEQ ID NO:4.

[0087] Preferably, FR2 of the polypeptide of the present invention comprises, or more preferably 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.

[0088] Alternatively, FR2 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 13 or fewer, more preferably 12 or fewer, more preferably 11 or fewer, more preferably 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer additions compared to SEQ ID NO: 5. Preferably, FR2 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 13 or fewer, more preferably 12 or fewer, more preferably 11 or fewer, more preferably 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer substitutions compared to SEQ ID NO: 5. Preferably, FR2 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 13 or fewer, more preferably 12 or fewer, more preferably 11 or fewer, more preferably 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer deletions compared to SEQ ID NO: 5.

[0089] Suitably, any residues in FR2 that differ from their corresponding residues in SEQ ID NO:5 are conservative substitutions compared to their corresponding residues.

[0090] Preferably, the residue in FR2 corresponding to residue number 2 of SEQ ID NO:5 is F or Y. Preferably, the residue in FR2 corresponding to residue number 5 of SEQ ID NO:5 is V or A. Preferably, the residue in FR2 corresponding to residue number 8 of SEQ ID NO:5 is K or H. Preferably, the residue in FR2 corresponding to residue number 9 of SEQ ID NO:5 is Q or E. Preferably, the residue in FR2 corresponding to residue number 10 of SEQ ID NO:5 is R or L. Preferably, the residue in FR2 corresponding to residue number 12 of SEQ ID NO:5 is L or F.

[0091] Preferably, the residue in FR2 corresponding to residue number 2 of SEQ ID NO:5 is F or Y, the residue in FR2 corresponding to residue number 5 of SEQ ID NO:5 is V or A, the residue in FR2 corresponding to residue number 8 of SEQ ID NO:5 is K or H, the residue in FR2 corresponding to residue number 9 of SEQ ID NO:5 is Q or E, the residue in FR2 corresponding to residue number 10 of SEQ ID NO:5 is R or L, and the residue in FR2 corresponding to residue number 12 of SEQ ID NO:12 is L or F.

[0092] Preferably, FR3 of a polypeptide of the present invention comprises, or more preferably consists of, a sequence sharing 8%, 15%, 20%, 26%, 32%, 40%, 45%, 52%, 58%, 65%, 70%, 76%, 80%, 82%, 85%, 90%, 92%, 95% or more sequence identity with SEQ ID NO:6.

[0093] Alternatively, FR3 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having an addition of 29 or less, more preferably 27 or less, more preferably 25 or less, more preferably 23 or less, more preferably 21 or less, more preferably 19 or less, more preferably 17 or less, more preferably 15 or less, more preferably 13 or less, more preferably 11 or less, more preferably 9 or less, more preferably 7 or less, more preferably 6 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and more preferably 1 or less compared to SEQ ID NO: 6. Preferably, FR3 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 29 or less, more preferably 27 or less, more preferably 25 or less, more preferably 23 or less, more preferably 21 or less, more preferably 19 or less, more preferably 17 or less, more preferably 15 or less, more preferably 13 or less, more preferably 11 or less, more preferably 9 or less, more preferably 7 or less, more preferably 6 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and more preferably 1 or less substitutions compared to SEQ ID NO: 6. Preferably, FR3 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 29 or less, more preferably 27 or less, more preferably 25 or less, more preferably 23 or less, more preferably 21 or less, more preferably 19 or less, more preferably 17 or less, more preferably 15 or less, more preferably 13 or less, more preferably 11 or less, more preferably 9 or less, more preferably 7 or less, more preferably 6 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and more preferably 1 or less deletions compared to SEQ ID NO: 6.

[0094] Preferably, the residue in FR3 corresponding to residue number 4 of SEQ ID NO:6 is I, L, or M (more preferably I or M). Preferably, the residue in FR3 corresponding to residue number 13 of SEQ ID NO:6 is L or V. Preferably, the residue in FR3 corresponding to residue number 14 of SEQ ID NO:6 is Y or F. Preferably, the residue in FR3 corresponding to residue number 16 of SEQ ID NO:6 is Q or E. Preferably, the residue in FR3 corresponding to residue number 18 of SEQ ID NO:6 is N or D. Preferably, the residue in FR3 corresponding to residue number 20 of SEQ ID NO:6 is L or V. Preferably, the residue in FR3 corresponding to residue number 22 of SEQ ID NO:6 is P or S. Preferably, the residue in FR3 corresponding to residue number 25 of SEQ ID NO:6 is T or A. Preferably, the residue in FR3 corresponding to residue number 27 of SEQ ID NO:6 is V or R. Preferably, the residue in FR3 corresponding to residue number 31 of SEQ ID NO:6 is A or N.

[0095] Preferably, the residue in FR3 corresponding to residue number 4 of SEQ ID NO:6 is I, L, or M (more suitably I or M), the residue in FR3 corresponding to residue number 13 of SEQ ID NO:6 is L or V, the residue in FR3 corresponding to residue number 14 of SEQ ID NO:6 is Y or F, the residue in FR3 corresponding to residue number 16 of SEQ ID NO:6 is Q or E, the residue in FR3 corresponding to residue number 18 of SEQ ID NO:6 is N or D, the residue in FR3 corresponding to residue number 20 of SEQ ID NO:6 is L or V, the residue in FR3 corresponding to residue number 22 of SEQ ID NO:6 is P or S, the residue in FR3 corresponding to residue number 25 of SEQ ID NO:6 is T or A, the residue in FR3 corresponding to residue number 27 of SEQ ID NO:6 is V or R, and the residue in FR3 corresponding to residue number 31 of SEQ ID NO:6 is A or N.

[0096] Preferably, any residues in FR3 that differ from their corresponding residues in SEQ ID NO: 6 are conservative substitutions compared to their corresponding residues. Preferably, FR3 comprises, or more preferably consists of, SEQ ID NO: 6.

[0097] Preferably, FR4 of a polypeptide of the present invention comprises, or more preferably consists of, a sequence sharing 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more sequence identity with SEQ ID NO:7.

[0098] Alternatively, FR4 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer additions compared to SEQ ID NO: 7. Preferably, FR4 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer substitutions compared to SEQ ID NO: 7. Preferably, FR4 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer deletions compared to SEQ ID NO: 7.

[0099] Preferably, any residues in FR4 that differ from their corresponding residues in SEQ ID NO: 7 are conservative substitutions compared to their corresponding residues. Preferably, FR4 comprises, or more preferably consists of, SEQ ID NO: 6.

[0100] Preferably, the residue in FR4 corresponding to residue number 6 of SEQ ID NO:7 is Q or R.

[0101] Full-length sequences of ID-L253T and related polypeptides Preferably, the polypeptide of the present invention comprises, or more preferably consists of, a sequence sharing 50% or more, more preferably 55% or more, more preferably 60% or more, more preferably 65% ​​or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, and more preferably 99% or more sequence identity with SEQ ID NO:8.

[0102] Alternatively, the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has 20 or fewer, more preferably 15 or fewer, more preferably 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer additions compared to SEQ ID NO: 8. Preferably, the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has 20 or fewer, more preferably 15 or fewer, more preferably 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer substitutions compared to SEQ ID NO: 8. Preferably, the polypeptide of the present invention comprises, or more preferably consists of, a sequence having no more than 20 deletions compared to SEQ ID NO: 8, more preferably no more than 15 deletions, more preferably no more than 10 deletions, more preferably no more than 9 deletions, more preferably no more than 8 deletions, more preferably no more than 7 deletions, more preferably no more than 6 deletions, more preferably no more than 5 deletions, more preferably no more than 4 deletions, more preferably no more than 3 deletions, more preferably no more than 2 deletions, more preferably no more than 1 deletion compared to SEQ ID NO: 8.

[0103] Preferably, the N-terminus of the polypeptide is D. Preferably, the polypeptide comprises, or more preferably consists of, SEQ ID NO:8.

[0104] 10E2 and related polypeptides CDRs of 10E2 and related polypeptides Preferably, CDR1 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence sharing 80% or more sequence identity with SEQ ID NO: 14.

[0105] Alternatively, CDR1 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has not more than two, more preferably not more than one addition compared to SEQ ID NO: 14. Preferably, CDR1 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has not more than two, more preferably not more than one substitution compared to SEQ ID NO: 14. Preferably, CDR1 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has not more than two, more preferably not more than one deletion compared to SEQ ID NO: 14.

[0106] Preferably, any residues in CDR1 that differ from their corresponding residues in SEQ ID NO: 14 are conservative substitutions compared to their corresponding residues. Preferably, CDR1 comprises, or more preferably consists of, SEQ ID NO: 14.

[0107] Preferably, CDR2 of the polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 55% or more, more preferably 60% or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more sequence identity with SEQ ID NO: 15.

[0108] Alternatively, CDR2 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer additions compared to SEQ ID NO: 15. Preferably, CDR2 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer substitutions compared to SEQ ID NO: 15. Preferably, CDR2 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer deletions compared to SEQ ID NO: 15.

[0109] Suitably, any residues in CDR2 that differ from their corresponding residues in SEQ ID NO: 15 are conservative substitutions compared with their corresponding residues.

[0110] Preferably, CDR2 comprises, or more preferably consists of, SEQ ID NO:15.

[0111] Preferably, the CDR3 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence sharing 60% or more, more preferably 65% ​​or more, more preferably 75% or more, more preferably 80% or more, more preferably 90% or more sequence identity with SEQ ID NO: 16.

[0112] Alternatively, CDR3 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has no more than 3, more preferably no more than 2, more preferably no more than 1 addition compared to SEQ ID NO: 16. Preferably, CDR3 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has no more than 3, more preferably no more than 2, more preferably no more than 1 substitution compared to SEQ ID NO: 16. Preferably, CDR3 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has no more than 3, more preferably no more than 2, more preferably no more than 1 deletion compared to SEQ ID NO: 16. Preferably, any substitutions are conservative to their corresponding residues in SEQ ID NO: 16.

[0113] Suitably, any residues in CDR3 that differ from their corresponding residues in SEQ ID NO: 16 are conservative substitutions compared with their corresponding residues.

[0114] FR of 10E2 and related polypeptides Preferably, FR1 of the polypeptide of the present invention comprises, or more preferably 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.

[0115] Alternatively, FR1 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having an addition of 28 or less, more preferably 26 or less, more preferably 24 or less, more preferably 22 or less, more preferably 20 or less, more preferably 18 or less, more preferably 16 or less, more preferably 14 or less, more preferably 13 or less, more preferably 12 or less, more preferably 11 or less, more preferably 10 or less, more preferably 9 or less, more preferably 8 or less, more preferably 7 or less, more preferably 6 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and more preferably 1 or less compared to SEQ ID NO: 17. Preferably, FR1 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 28 or less, more preferably 26 or less, more preferably 24 or less, more preferably 22 or less, more preferably 20 or less, more preferably 18 or less, more preferably 16 or less, more preferably 14 or less, more preferably 13 or less, more preferably 12 or less, more preferably 11 or less, more preferably 10 or less, more preferably 9 or less, more preferably 8 or less, more preferably 7 or less, more preferably 6 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and more preferably 1 or less substitutions compared to SEQ ID NO: 17. Preferably, FR1 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 28 or less, more preferably 26 or less, more preferably 24 or less, more preferably 22 or less, more preferably 20 or less, more preferably 18 or less, more preferably 16 or less, more preferably 14 or less, more preferably 13 or less, more preferably 12 or less, more preferably 11 or less, more preferably 10 or less, more preferably 9 or less, more preferably 8 or less, more preferably 7 or less, more preferably 6 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and more preferably 1 or less deletions compared to SEQ ID NO: 17.

[0116] Suitably, any residues in FR1 that differ from their corresponding residues in SEQ ID NO: 17 are conservative substitutions compared to their corresponding residues.

[0117] Preferably, FR1 comprises, or more preferably consists of, SEQ ID NO:17.

[0118] Preferably, FR2 of the polypeptide of the present invention comprises, or more preferably 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.

[0119] Alternatively, FR2 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 13 or fewer, more preferably 12 or fewer, more preferably 11 or fewer, more preferably 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer additions compared to SEQ ID NO: 18. Preferably, FR2 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 13 or fewer, more preferably 12 or fewer, more preferably 11 or fewer, more preferably 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer substitutions compared to SEQ ID NO: 18. Preferably, FR2 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 13 or fewer, more preferably 12 or fewer, more preferably 11 or fewer, more preferably 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer deletions compared to SEQ ID NO: 18.

[0120] Suitably, any residues in FR2 that differ from their corresponding residues in SEQ ID NO: 18 are conservative substitutions compared to their corresponding residues.

[0121] Preferably, FR3 of a polypeptide of the invention comprises, or more preferably consists of, a sequence sharing 8%, 15%, 20%, 26%, 32%, 40%, 45%, 52%, 58%, 65%, 70%, 76%, 80%, 82%, 85%, 90%, 92%, 95% or more sequence identity with SEQ ID NO: 19.

[0122] Alternatively, FR3 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having an addition of 29 or less, more preferably 27 or less, more preferably 25 or less, more preferably 23 or less, more preferably 21 or less, more preferably 19 or less, more preferably 17 or less, more preferably 15 or less, more preferably 13 or less, more preferably 11 or less, more preferably 9 or less, more preferably 7 or less, more preferably 6 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and more preferably 1 or less compared to SEQ ID NO: 19. Preferably, FR3 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 29 or less, more preferably 27 or less, more preferably 25 or less, more preferably 23 or less, more preferably 21 or less, more preferably 19 or less, more preferably 17 or less, more preferably 15 or less, more preferably 13 or less, more preferably 11 or less, more preferably 9 or less, more preferably 7 or less, more preferably 6 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and more preferably 1 or less substitutions compared to SEQ ID NO: 19. Preferably, FR3 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 29 or less, more preferably 27 or less, more preferably 25 or less, more preferably 23 or less, more preferably 21 or less, more preferably 19 or less, more preferably 17 or less, more preferably 15 or less, more preferably 13 or less, more preferably 11 or less, more preferably 9 or less, more preferably 7 or less, more preferably 6 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and more preferably 1 or less deletions compared to SEQ ID NO: 19.

[0123] Preferably, any residues in FR3 that differ from their corresponding residues in SEQ ID NO: 19 are conservative substitutions compared to their corresponding residues. Preferably, FR3 comprises, or more preferably consists of, SEQ ID NO: 19.

[0124] Preferably, FR4 of a polypeptide of the present invention comprises, or more preferably consists of, a sequence sharing 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more sequence identity with SEQ ID NO:20.

[0125] Alternatively, FR4 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer additions compared to SEQ ID NO: 20. Preferably, FR4 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer substitutions compared to SEQ ID NO: 20. Preferably, FR4 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer deletions compared to SEQ ID NO: 20.

[0126] Preferably, any residues in FR4 that differ from their corresponding residues in SEQ ID NO: 20 are conservative substitutions compared to their corresponding residues. Preferably, FR4 comprises, or more preferably consists of, SEQ ID NO: 20.

[0127] Full-length sequences of 10E2 and related polypeptides Preferably, the polypeptide of the present invention comprises, or more preferably consists of, a sequence sharing 50% or more, more preferably 55% or more, more preferably 60% or more, more preferably 65% ​​or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, and more preferably 99% or more sequence identity with SEQ ID NO: 13.

[0128] Alternatively, the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has 20 or fewer, more preferably 15 or fewer, more preferably 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer additions compared to SEQ ID NO: 13. Preferably, the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has 20 or fewer, more preferably 15 or fewer, more preferably 10 or fewer, more preferably 9 or fewer, more preferably 13 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer substitutions compared to SEQ ID NO: 13. Preferably, the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has no more than 20 deletions compared to SEQ ID NO: 13, more preferably no more than 15 deletions, more preferably no more than 10 deletions, more preferably no more than 9 deletions, more preferably no more than 8 deletions, more preferably no more than 7 deletions, more preferably no more than 6 deletions, more preferably no more than 5 deletions, more preferably no more than 4 deletions, more preferably no more than 3 deletions, more preferably no more than 2 deletions, more preferably no more than 1 deletion compared to SEQ ID NO: 13, or more preferably no more than 15 deletions, more preferably no more than 10 deletions, more preferably no more than 9 deletions, more preferably no more than 8 deletions, more preferably no more than 7 deletions, more preferably no more than 6 deletions, more preferably no more than 5 deletions, more preferably no more than 4 deletions, more preferably no more than 3 deletions, more preferably no more than 2 deletions, more preferably no more than 1 deletion.

[0129] Preferably, the N-terminus of the polypeptide is D. Preferably, the polypeptide comprises, or more preferably consists of, SEQ ID NO:13.

[0130] 10G10 and related polypeptides CDRs of 10G10 and related polypeptides Preferably, CDR1 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence sharing 80% or more sequence identity with SEQ ID NO: 22.

[0131] Alternatively, CDR1 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has not more than two, more preferably not more than one addition compared to SEQ ID NO: 22. Preferably, CDR1 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has not more than two, more preferably not more than one substitution compared to SEQ ID NO: 22. Preferably, CDR1 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has not more than two, more preferably not more than one deletion compared to SEQ ID NO: 22.

[0132] Preferably, any residues in CDR1 that differ from their corresponding residues in SEQ ID NO: 22 are conservative substitutions compared to their corresponding residues. Preferably, CDR1 comprises, or more preferably consists of, SEQ ID NO: 22.

[0133] Preferably, CDR2 of the polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 55% or more, more preferably 60% or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more sequence identity with SEQ ID NO: 23.

[0134] Alternatively, CDR2 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer additions compared to SEQ ID NO: 23. Preferably, CDR2 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer substitutions compared to SEQ ID NO: 23. Preferably, CDR2 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer deletions compared to SEQ ID NO: 23.

[0135] Suitably, any residues in CDR2 that differ from their corresponding residues in SEQ ID NO: 23 are conservative substitutions compared with their corresponding residues.

[0136] Preferably, CDR2 comprises, or more preferably consists of, SEQ ID NO:23.

[0137] Preferably, the CDR3 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence sharing 60% or more, more preferably 65% ​​or more, more preferably 75% or more, more preferably 80% or more, more preferably 90% or more sequence identity with SEQ ID NO: 24.

[0138] Alternatively, CDR3 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has no more than 3, more preferably no more than 2, more preferably no more than 1 addition compared to SEQ ID NO: 24. Preferably, CDR3 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has no more than 3, more preferably no more than 2, more preferably no more than 1 substitution compared to SEQ ID NO: 24. Preferably, CDR3 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has no more than 3, more preferably no more than 2, more preferably no more than 1 deletion compared to SEQ ID NO: 24. Preferably, any substitutions are conservative to their corresponding residues in SEQ ID NO: 24.

[0139] Suitably, any residues in CDR3 that differ from their corresponding residues in SEQ ID NO: 24 are conservative substitutions compared with their corresponding residues.

[0140] FR of 10G10 and related polypeptides Preferably, FR1 of the polypeptide of the present invention comprises, or more preferably 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.

[0141] Alternatively, FR1 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having an addition of 28 or less, more preferably 26 or less, more preferably 24 or less, more preferably 22 or less, more preferably 20 or less, more preferably 18 or less, more preferably 16 or less, more preferably 14 or less, more preferably 13 or less, more preferably 12 or less, more preferably 11 or less, more preferably 10 or less, more preferably 9 or less, more preferably 8 or less, more preferably 7 or less, more preferably 6 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and more preferably 1 or less compared to SEQ ID NO: 25. Preferably, FR1 of the polypeptide of the present invention comprises or, more preferably, consists of a sequence having 28 or less, more preferably 26 or less, more preferably 24 or less, more preferably 22 or less, more preferably 20 or less, more preferably 18 or less, more preferably 16 or less, more preferably 14 or less, more preferably 13 or less, more preferably 12 or less, more preferably 11 or less, more preferably 10 or less, more preferably 9 or less, more preferably 8 or less, more preferably 7 or less, more preferably 6 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and more preferably 1 or less substitutions compared to SEQ ID NO: 25. Preferably, FR1 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 28 or less, more preferably 26 or less, more preferably 24 or less, more preferably 22 or less, more preferably 20 or less, more preferably 18 or less, more preferably 16 or less, more preferably 14 or less, more preferably 13 or less, more preferably 12 or less, more preferably 11 or less, more preferably 10 or less, more preferably 9 or less, more preferably 8 or less, more preferably 7 or less, more preferably 6 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and more preferably 1 or less deletions compared to SEQ ID NO: 25.

[0142] Suitably, any residues in FR1 that differ from their corresponding residues in SEQ ID NO: 25 are conservative substitutions compared to their corresponding residues.

[0143] Preferably, FR1 comprises, or more preferably consists of, SEQ ID NO:25.

[0144] Preferably, FR2 of the polypeptide of the present invention comprises, or more preferably 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.

[0145] Alternatively, FR2 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 13 or fewer, more preferably 12 or fewer, more preferably 11 or fewer, more preferably 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer additions compared to SEQ ID NO: 26. Preferably, FR2 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 13 or fewer, more preferably 12 or fewer, more preferably 11 or fewer, more preferably 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer substitutions compared to SEQ ID NO: 26. Preferably, FR2 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 13 or fewer, more preferably 12 or fewer, more preferably 11 or fewer, more preferably 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer deletions compared to SEQ ID NO: 26.

[0146] Suitably, any residues in FR2 that differ from their corresponding residues in SEQ ID NO: 26 are conservative substitutions compared to their corresponding residues.

[0147] Preferably, FR3 of a polypeptide of the present invention comprises, or more preferably consists of, a sequence sharing 8%, 15%, 20%, 26%, 32%, 40%, 45%, 52%, 58%, 65%, 70%, 76%, 80%, 82%, 85%, 90%, 92%, 95% or more sequence identity with SEQ ID NO:27.

[0148] Alternatively, FR3 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having an addition of 29 or less, more preferably 27 or less, more preferably 25 or less, more preferably 23 or less, more preferably 21 or less, more preferably 19 or less, more preferably 17 or less, more preferably 15 or less, more preferably 13 or less, more preferably 11 or less, more preferably 9 or less, more preferably 7 or less, more preferably 6 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and more preferably 1 or less compared to SEQ ID NO: 27. Preferably, FR3 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 29 or less, more preferably 27 or less, more preferably 25 or less, more preferably 23 or less, more preferably 21 or less, more preferably 19 or less, more preferably 17 or less, more preferably 15 or less, more preferably 13 or less, more preferably 11 or less, more preferably 9 or less, more preferably 7 or less, more preferably 6 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and more preferably 1 or less substitutions compared to SEQ ID NO: 27. Preferably, FR3 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 29 or less, more preferably 27 or less, more preferably 25 or less, more preferably 23 or less, more preferably 21 or less, more preferably 19 or less, more preferably 17 or less, more preferably 15 or less, more preferably 13 or less, more preferably 11 or less, more preferably 9 or less, more preferably 7 or less, more preferably 6 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and more preferably 1 or less deletions compared to SEQ ID NO: 27.

[0149] Preferably, any residues in FR3 that differ from their corresponding residues in SEQ ID NO: 27 are conservative substitutions compared to their corresponding residues. Preferably, FR3 comprises, or more preferably consists of, SEQ ID NO: 27.

[0150] Preferably, FR4 of a polypeptide of the present invention comprises, or more preferably consists of, a sequence sharing 5%, 10%, 28%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more sequence identity with SEQ ID NO:28.

[0151] Alternatively, FR4 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer additions compared to SEQ ID NO: 28. Preferably, FR4 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer substitutions compared to SEQ ID NO: 28. Preferably, FR4 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer deletions compared to SEQ ID NO: 28.

[0152] Preferably, any residues in FR4 that differ from their corresponding residues in SEQ ID NO: 28 are conservative substitutions compared to their corresponding residues. Preferably, FR4 comprises, or more preferably consists of, SEQ ID NO: 28.

[0153] Full-length sequences of 10G10 and related polypeptides Preferably, the polypeptide of the present invention comprises, or more preferably consists of, a sequence sharing 50% or more, more preferably 55% or more, more preferably 60% or more, more preferably 65% ​​or more, more preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, and more preferably 99% or more sequence identity with SEQ ID NO: 21.

[0154] Alternatively, the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has 20 or fewer, more preferably 15 or fewer, more preferably 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer additions compared to SEQ ID NO: 21. Preferably, the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has 20 or fewer, more preferably 15 or fewer, more preferably 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer substitutions compared to SEQ ID NO: 21. Preferably, the polypeptide of the present invention comprises, or more preferably consists of, a sequence having no more than 20 deletions compared to SEQ ID NO: 21, more preferably no more than 15 deletions, more preferably no more than 10 deletions, more preferably no more than 9 deletions, more preferably no more than 8 deletions, more preferably no more than 7 deletions, more preferably no more than 6 deletions, more preferably no more than 5 deletions, more preferably no more than 4 deletions, more preferably no more than 3 deletions, more preferably no more than 2 deletions, more preferably no more than 1 deletion compared to SEQ ID NO: 21.

[0155] Preferably, the N-terminus of the polypeptide is D. Preferably, the polypeptide comprises, or more preferably consists of, SEQ ID NO:21.

[0156] Further embodiments relating to ID-L253T, 10E2, 10G10, and related polypeptides 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), and the complementarity determining regions (and more preferably the framework regions) are selected from the complementarity determining regions (and framework regions) disclosed herein.

[0157] 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: CDR1 comprises a sequence selected from SEQ ID NO: 1, SEQ ID NO: 14, or SEQ ID NO: 22; CDR2 comprises a sequence selected from SEQ ID NO: 2, SEQ ID NO: 15, or SEQ ID NO: 23; The CDR3 comprises a sequence selected from SEQ ID NO:3, SEQ ID NO:16, or SEQ ID NO:24.

[0158] Preferably, the polypeptides disclosed in the present invention comprise, or more suitably consist of, a sequence sharing 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 98% or more, more preferably 99% or more, more preferably 99.5% or more sequence identity with SEQ ID NO: 8, 11, 12, 13, 21, or 29-45.

[0159] In one embodiment, a polypeptide is provided comprising, or more preferably consisting of, a sequence sharing 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 98% or more, more preferably 99% or more, and more preferably 99.5% or more sequence identity with SEQ ID NO: 8, 11, 12, 13, 21, or 29-45.

[0160] Linkers and multimers Constructs of the present invention may comprise multiple polypeptides and may be preferably multivalent. Such constructs may comprise at least two identical polypeptides of the present invention. A construct consisting of two identical polypeptides of the present invention is a "homobihead." In one aspect of the present invention, constructs are provided that comprise two or more identical polypeptides of the present invention.

[0161] Alternatively, the construct may comprise at least two polypeptides that are different but are both polypeptides according to the invention (a "heterobihead").

[0162] Alternatively, such a construct may comprise (a) at least one polypeptide of the invention and (b) at least one polypeptide, such as an antibody or antigen-binding fragment thereof, that is not a polypeptide of the invention (also a "heterobihead"). The at least one polypeptide in (b) may bind to IL-23 (e.g., via a different epitope than that of (a)), or alternatively may bind to a target other than IL-23. Preferably, the different polypeptide (b) is, for example, an interleukin (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), an interleukin receptor (such as IL-6R and IL-7R), a transcription factor (such as NF-kB), a cytokine (such as TNF-α, IFN-γ, TGF-β, and TSLP), a transmembrane protein (such as gp130 and CD3), a surface glycoprotein (such as CD4), or a polypeptide of interest (such as CD5). The different polypeptide (b) binds to, for example, IL-6R, IL-6, IL-12, IL-1-β, IL-17A, TNF-α, or CD3, or to other inflammatory mediators or immunologically relevant ligands involved in human pathological processes. Most preferably, the different polypeptide (b) binds to TNF-α, and more preferably, the different polypeptide (b) is ID-38F.

[0163] The construct may be multivalent and / or multispecific. A multivalent construct (such as a bivalent construct) comprises two or more binding polypeptides and therefore presents two or more sites at which binding to one or more antigens can occur. Examples of multivalent constructs may be homobiheads or heterobiheads. A multispecific construct (e.g., a bispecific construct) comprises two or more different binding polypeptides that present two or more sites at which (a) binding to two or more different antigens can occur, or (b) binding to two or more different epitopes on the same antigen can occur. A multispecific construct may be a heterobihead. A multispecific construct is multivalent.

[0164] Preferably, the polypeptide contained within the construct is an antibody fragment. More preferably, the polypeptide contained within the construct is selected from the list consisting of VHH, VH, VL, V-NAR, scFv, FAb fragment, or F(ab')2 fragment. More preferably, the polypeptide contained within the construct is VH or VHH, and most preferably, VHH.

[0165] The polypeptides of the present invention can be linked to each other directly (i.e., without the use of a linker) or via a linker. Preferably, the linker is a protease-labile linker or a non-protease-labile linker. The linker is preferably a polypeptide and is selected to allow binding of the polypeptide to its epitope. When used for therapeutic purposes, the linker is preferably non-immunogenic in the subject to which the polypeptide is administered.

[0166] Preferably, the protease labile linker is [-(G a S) x -B-(G b S) y -] z format, (In the formula, a is 1 to 10, b is 1 to 10; x is 1 to 10, y is 1 to 10, z is 1 to 10, and B is K or R (SEQ ID NO: 74); and, more preferably, [-(G4S) x -B-(G4S) y -] z It is a format (In the formula, x is 1 to 10, y is 1 to 10, z is 1 to 10, and B is K or R).

[0167] More preferably, 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.

[0168] More preferably, the protease labile linker is in the format -(G4S)2-K-(G4S)2- (SEQ ID NO: 75).

[0169] Preferably, the polypeptides are all connected by non-protease labile linkers. Preferably, the non-protease labile linkers are (G4S) x where x is 1 to 10 (SEQ ID NO: 76). Most preferably, x is 6 (SEQ ID NO: 77).

[0170] Thus, there is provided a construct comprising at least one polypeptide of the present invention and at least one different polypeptide, wherein the different polypeptide binds to TNF-α. Preferably, the TNF-α-binding polypeptide is ID-38F or a variant thereof, such as a polypeptide sharing 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, for example at least 99% sequence identity, with ID-38F (SEQ ID NO: 67). More preferably, the TNF-α-binding polypeptide is ID-38F. In one embodiment, the construct comprises a non-protease labile linker such as (G4S)6. Alternatively, the construct comprises a protease labile linker such as -(G4S)2-K-(G4S)2-. Preferably, the construct shares at least 80%, such as at least 90%, for example at least 95% sequence identity with FA1K (SEQ ID NO: 46).

[0171] In embodiments in which the construct comprises a non-protease labile linker, preferably the entire construct (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 in which the construct comprises a protease labile linker, preferably 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 labile to proteases, such as trypsin or chymotrypsin.

[0172] Vectors and hosts The term "vector," as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian and yeast vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of a host cell upon introduction into the host cell, thereby being replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "recombinant vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably, as the plasmid is the most commonly used form of vector. However, the invention is intended to include other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), as well as bacteriophage and phagemid systems, which serve equivalent functions. The present invention further relates to nucleotide sequences encoding the 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 the particular subject cell but also to the progeny of such a cell.

[0173] One aspect of the present invention provides a vector containing a polynucleotide encoding a polypeptide or construct of the present invention, or a cDNA containing the polynucleotide. A further aspect of the present invention provides a host cell transformed with the vector, capable of expressing the polypeptide or construct of the present invention. Preferably, the host cell is a bacterium such as Escherichia coli, or a yeast belonging to the genera Aspergillus, Saccharomyces, Kluyveromyces, Hansenula, or Pichia, such as Saccharomyces cerevisiae or Pichia pastoris.

[0174] stability Preferably, the polypeptides or constructs of the present invention substantially retain their neutralizing ability and / or efficacy when orally administered and after exposure to the intestinal tract (e.g., after exposure to small intestinal and / or large intestinal proteases and / or IBD inflammatory proteases). Such proteases include enteropeptidase, trypsin, chymotrypsin, and irritable bowel disease inflammatory proteases (e.g., MMP3, MMP12, and cathepsins). The small intestinal and / or large intestinal proteases or proteases produced therein include proteases supplied by intestinal commensal flora and / or pathogenic bacteria, e.g., cell membrane-associated proteases, excretory proteases, and proteases released during cell lysis. Most preferably, the proteases are trypsin and chymotrypsin.

[0175] Preferably, the intestinal tract is that of a dog, pig, human, cynomolgus monkey, or mouse. More suitably, the intestinal tract is that of a human, cynomolgus monkey, or mouse, more preferably a mouse or human, and most preferably a human. The small intestine preferably comprises the duodenum, jejunum, and ileum. The large intestine preferably comprises the cecum, colon, rectum, and anal canal. The gastrointestinal tract, in contrast to the gastrointestinal tract, comprises only the small intestine and large intestine.

[0176] A polypeptide or construct of the invention substantially retains its neutralizing ability if preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, or most preferably at least 100% of the original neutralizing ability of the polypeptide or construct of the invention is retained after exposure to proteases present in the small intestine and / or large intestine and / or IBD inflammatory proteases.

[0177] Preferably, the polypeptides or constructs of the invention substantially retain their neutralizing ability after exposure to small intestinal and / or large intestinal and / or IBD inflammatory proteases, for example at 37°C for up to at least 1 hour, more preferably up to at least 2 hours, more preferably up to at least 3 hours, more preferably up to at least 4 hours, more preferably up to at least 7 hours, more preferably up to at least 16 hours.

[0178] "Substantially retains neutralizing ability" preferably means that the polypeptide or construct of the present invention retains at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, and more preferably at least 90% of the neutralizing ability of the polypeptide or construct of the present invention.

[0179] Preferably, at least 10% of the neutralising capacity of the polypeptide or construct of the invention is retained after 4 hours of exposure to the conditions of the intestinal tract, more preferably the small intestine or large intestine, more preferably a human faecal extract, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%.

[0180] Preferably, at least 10% of the neutralising capacity of the polypeptide or construct of the invention is retained after 7 hours of exposure to the conditions of the intestinal tract, more preferably the small intestine or large intestine, more preferably a human faecal extract, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%.

[0181] Preferably, at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70% of the neutralising capacity of the polypeptide or construct of the invention is retained after 16 hours of exposure to the conditions of the intestinal tract, more preferably the small intestine or large intestine, more preferably a human faecal extract.

[0182] Preferably, at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, and more preferably at least 70% of the neutralizing capacity of the polypeptide or construct of the present invention is retained after 1 hour of exposure to the conditions of the intestinal tract, more preferably the small intestine or large intestine, more preferably mouse small intestinal supernatant.

[0183] Preferably, at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70% of the neutralizing capacity of the polypeptide or construct of the present invention is retained after 4 hours of exposure to the conditions of the intestinal tract, more preferably the small intestine or large intestine, more preferably mouse small intestinal supernatant.

[0184] Preferably, 10% or more, more preferably 20% or more, more preferably 30% or more, more preferably 40% or more, more preferably 50% or more, more preferably 60% or more, and more preferably 70% or more of an administered dose of a polypeptide or construct of the invention retains neutralising capacity against IL-23 and remains in the faeces (preferably voided faeces or faeces removed from the intestinal tract) of mice, cynomolgus monkeys and / or humans after 1, 2, 3, 4, 5, 6 or 7 hours of exposure to intestinal conditions.

[0185] A polypeptide of the invention or a construct of the invention remains substantially intact when preferably 10% or more, more preferably 20% or more, more preferably 30% or more, more preferably 40% or more, more preferably 50% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 99% or more, and most preferably 100% of an administered amount of the polypeptide of the invention or construct of the invention remains intact after exposure to proteases present in the small intestine and / or large intestine and / or IBD inflammatory proteases.

[0186] Therapeutic Uses and Delivery A therapeutically effective amount of a polypeptide, pharmaceutical composition, or construct of the present invention is an amount effective to neutralize IL-23 to a significant extent in a subject by single or multiple administration to the subject. The therapeutically effective amount may vary according to factors such as the individual's disease state, age, sex, and weight, as well as the ability of the polypeptide, pharmaceutical composition, or construct to elicit a desired response in the individual. A therapeutically effective amount is an amount such that any toxic or harmful effects of the polypeptide, pharmaceutical composition, or construct of the present invention are outweighed by the therapeutically beneficial effects. 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.

[0187] The pharmaceutical compositions of the present invention may be suitably formulated for oral, intramuscular, subcutaneous, or intravenous delivery. The pharmaceutical compositions of the present invention may be in various forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injection and injectable solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. Solid dosage forms are preferred. The polypeptides, pharmaceutical compositions, or constructs of the present invention may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc.

[0188] Typically, pharmaceutical compositions comprise 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 further contain minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the polypeptide or construct of the present invention. Pharmaceutical compositions may also include antiadherents, binders, coatings, disintegrants, flavors, colorants, lubricants, adsorbents, preservatives, sweeteners, lyophilization excipients (including lyoprotectants), or compression aids.

[0189] Most preferably, the polypeptide, pharmaceutical composition, or construct of the present invention is administered orally. A key issue with oral administration is ensuring that sufficient polypeptide, pharmaceutical composition, or construct reaches the region of the intestinal tract where it is needed. Factors that may prevent the polypeptide, pharmaceutical composition, or construct of the present invention from reaching the region of the intestinal tract where it is needed include the presence of proteases in digestive secretions that can degrade the polypeptide, pharmaceutical composition, or construct of the present invention. Preferably, the polypeptide, pharmaceutical composition, or construct of the present invention is substantially stable in the presence of one or more such proteases due to the inherent properties of the polypeptide or construct itself. Preferably, the polypeptide or construct of the present invention is lyophilized before being incorporated into a pharmaceutical composition.

[0190] The polypeptide of the present invention may be provided with an enteric coating. An enteric coating is a polymer barrier applied to oral medications that helps protect the polypeptide from the low pH of the stomach. Materials used for enteric coatings include fatty acids, waxes, shellac, plastics, and vegetable fibers. Suitable enteric coating components include methyl acrylate-methacrylic acid copolymer, cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymer, sodium alginate, and stearic acid. Suitable enteric coating agents include pH-dependent release polymers. These are polymers that are insoluble at the strongly acidic pH found in the stomach but rapidly dissolve at weakly acidic pH. Thus, preferably, the enteric coating will not dissolve in the acidic juices of the stomach (pH ∼ 3), but will dissolve in the high pH environments present in the small intestine (pH ≥ 6) or colon (pH ≥ 7.0). The pH-dependent release polymer is selected so that the polypeptide or construct of the invention is released around the time the administered dose reaches the small intestine.

[0191] The polypeptides, constructs, or pharmaceutical compositions of the present invention can be formulated into preparations for injection by dissolving, suspending, or emulsifying in aqueous or non-aqueous solvents, such as vegetable oils or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids, or propylene glycol, and, if desired, using conventional additives such as solubilizers, isotonicity agents, suspending agents, emulsifiers, stabilizers, and preservatives. Acceptable carriers, excipients, and / or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers, e.g., phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid, glutathione, cysteine, methionine, and citric acid; preservatives (ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methylparaben or propylparaben, benzalkonium chloride, or combinations thereof); arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tripeptide, riboflavin ... 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, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or non-ionic surfactants such as polysorbates, POE ethers, poloxamers, Triton-X, or polyethylene glycol.

[0192] The pharmaceutical composition of the present invention may be delivered topically to the skin (for example, for use in the treatment of autoimmune diseases such as psoriasis or eczema).Such pharmaceutical compositions may be suitably in the form of creams, ointments, lotions, gels, foams, transdermal patches, powders, pastes, or tinctures, and may suitably contain vitamin D3 analogs (for example, calcipotriol and maxacalcitol), steroids (for example, fluticasone propionate, betamethasone valerate, and clobetasol propionate), retinoids (for example, tazarotene), coal tar, and dithranol.Topical drugs are often used in combination with each other (for example, vitamin D3 and steroids) or in combination with additional agents such as salicylic acid. When the pharmaceutical composition of the invention is delivered topically for the treatment of psoriasis or eczema, additional substances believed to be effective in treating psoriasis or eczema may suitably be included in the composition, such as steroids, particularly Class 4 or Class 5 steroids, such as hydrocortisone (e.g., 1% hydrocortisone cream), cyclosporine, or similar macrolides, or retinoids.

[0193] For all delivery modes, the polypeptides, pharmaceutical compositions, or constructs of the present invention may be formulated in a buffer to stabilize the pH of the composition at a concentration between 5 and 50, or more preferably between 15 and 40, or even more preferably between 25 and 30 g / liter. Examples of suitable buffer components include physiological salts such as sodium citrate and / or citric acid. Preferably, the buffer contains 100 to 200, more preferably 125 to 175 mM of physiological salt, e.g., sodium chloride. Preferably, the buffer is selected to have a pKa close to the pH of the composition or the physiological pH of the patient.

[0194] Exemplary polypeptide or construct concentrations in pharmaceutical compositions 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.

[0195] Aqueous formulations of the polypeptides, constructs, or pharmaceutical compositions of the present invention can be prepared in pH buffer solutions, for example, at a pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively at about 5.5. Examples of suitable buffers include phosphate buffer, histidine buffer, citrate buffer, succinate buffer, acetate buffer, and other organic acid buffers. The buffer concentration can be, for example, from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending on the buffer and the desired tonicity of the formulation.

[0196] The tonicity of a pharmaceutical composition can be altered by including a tonicity modifier. Such tonicity modifiers can be charged or uncharged chemical species. Representative uncharged tonicity modifiers include sugars or sugar alcohols or other polyols, preferably trehalose, sucrose, mannitol, glycerol, 1,2-propanediol, raffinose, sorbitol, or lactitol (particularly trehalose, mannitol, glycerol, or 1,2-propanediol). Representative charged tonicity modifiers include salts such as combinations of sodium, potassium, or calcium ions with chloride, sulfate, carbonate, sulfite, nitrate, lactate, succinate, acetate, or maleate ions (particularly sodium chloride or sodium sulfate); or amino acids such as arginine or histidine. Preferably, aqueous formulations are isotonic, although hypertonic or hypotonic solutions may be appropriate. The term "isotonic" refers to a solution that has the same tonicity as some other solution to which it is being compared, such as physiological saline or serum. The tonicity adjusting 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. Preferably, at least one tonicity adjusting agent is included in the composition.

[0197] Surfactants can also be added to pharmaceutical compositions to reduce aggregation of the formulated polypeptide or construct, minimize the formation of particulates in the formulation, and / or reduce adsorption. Exemplary surfactants include polyoxyethylene sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (poloxamers, Pluronic®), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylene sorbitan fatty acid esters include polysorbate 20 and polysorbate 80. Exemplary concentrations of surfactants can range from about 0.001% w / v to about 10% w / v.

[0198] Lyoprotectants can also be added to protect the polypeptides or constructs of the present invention from destabilizing conditions during the lyophilization 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). Lyoprotectants can be included in amounts of about 10 mM to 500 mM.

[0199] Dosage ranges for administration of the polypeptides, pharmaceutical compositions, or constructs of the invention are those to produce the desired therapeutic effect. The required dosage range depends on the exact nature of the polypeptides, pharmaceutical compositions, or constructs of the invention, the route of administration, the nature of the formulation, the patient's age, the nature, extent, or severity of the patient's disease, contraindications (if any), and the judgment of the attending physician. Variations in these dosage levels can be adjusted using standard empirical methods for optimization.

[0200] Suitable daily dosages of the polypeptides, pharmaceutical compositions, or constructs of the invention range from 50 ng to 50 mg per kg, for example, 50 ug to 40 mg per kg of body weight, for example, 5 to 30 mg per kg. Unit dosages can vary from less than 100 mg, but are typically in the range of 250 to 2000 mg per administration, and unit dosages may be administered daily or more frequently, for example, 2, 3, or 4 times per day, or less frequently, for example, every other day, or once a week, once every two weeks, or once a month.

[0201] In one aspect of the invention there is provided the use of a polypeptide, pharmaceutical composition or construct of the invention in the manufacture of a medicament for the treatment of an autoimmune disease. In a further aspect of the invention there is provided a method of treating an autoimmune disease comprising administering to a person in need thereof a therapeutically effective amount of a polypeptide, pharmaceutical composition or construct of the invention.

[0202] The term "treatment" is intended to encompass prophylaxis as well as therapeutic treatment. Treatment of a disease includes treatment of its exacerbation, and also includes treatment of patients in remission from disease symptoms to prevent recurrence of disease symptoms.

[0203] Combination therapy The pharmaceutical compositions of the invention may also contain one or more active agents (e.g., active agents suitable for treating a disease described herein). It is within the scope of the invention to use the pharmaceutical compositions of the invention in therapeutic methods for the treatment of autoimmune diseases as an adjunct to, or in combination with, other established therapies commonly used in the treatment of autoimmune diseases.

[0204] For the treatment of IBD (such as Crohn's disease or ulcerative colitis), potential combinations include, for example, 5-aminosalicylic acid or its prodrugs (such as sulfasalazine, olsalazine, or bisalazide); corticosteroids (such as prednisolone, methylprednisolone, or budesonide); immunosuppressants (such as cyclosporine, tacrolimus, methotrexate, azathioprine, or 6-mercaptopurine); anti-TNF-α antibodies (such as infliximab, adalimumab, certolizumab pegol, or golimumab); anti-IL12 / IL23 antibodies (such as ustekinumab); anti-IL6R antibodies or small molecule IL12 / IL23 inhibitors (such as apilimod); anti-alpha-4-beta-7 antibodies. (e.g., vedolizumab); MAdCAM-1 blockers (e.g., PF-00547659); antibodies against the cell adhesion molecule alpha-4-integrin (e.g., natalizumab); antibodies against the IL2 receptor alpha subunit (e.g., daclizumab or basiliximab); JAK3 inhibitors (e.g., tofacitinib or R348); Syk inhibitors and their prodrugs (e.g., fostamatinib and R-406); phosphodiesterase-4 inhibitors (e.g., tetomilast); HMPL-004; probiotics; delsalazine; semapimod / CPSI-2364; and protein kinase C inhibitors (e.g., AEB-071). The most suitable combination agents are infliximab, adalimumab, certolizumab pego, or golimumab.

[0205] Accordingly, another aspect of the present invention provides a pharmaceutical composition of the present invention in combination with one or more additional active agents, such as one or more of the active agents described above.

[0206] In a further embodiment of the invention, the polypeptide, pharmaceutical composition or construct is administered sequentially, simultaneously or separately with at least one active agent selected from the list above.

[0207] Similarly, another aspect of the present invention is (A) a polypeptide, pharmaceutical composition, or construct of the present invention, and (B) one or more other active agents

[0013] The present invention also provides a combination product comprising: a polypeptide, pharmaceutical composition, or construct of the present invention and another therapeutic agent, each of which is formulated in admixture with a pharmaceutically acceptable adjuvant, diluent, or carrier; wherein each of components (A) and (B) is formulated in admixture with a pharmaceutically acceptable adjuvant, diluent, or carrier; in this aspect of the invention, the combination product can be either a single (combined) formulation or a kit-of-parts; thus, this aspect of the invention encompasses a combination formulation comprising a polypeptide, pharmaceutical composition, or construct of the present invention and another therapeutic agent, each of which is admixed with a pharmaceutically acceptable adjuvant, diluent, or carrier;

[0208] The present invention provides (i) a polypeptide, pharmaceutical composition, or construct of the invention mixed with a pharmaceutically acceptable adjuvant, diluent, or carrier; (ii) A formulation containing one or more other active agents mixed with a pharmaceutically acceptable adjuvant, diluent, or carrier. The present invention also encompasses a kit-of-parts comprising the components (i) and (ii), each of which is provided in a form suitable for administration in combination with the other.

[0209] Thus, component (i) of the kit-of-parts is component (A) described above mixed with a pharmaceutically acceptable adjuvant, diluent, or carrier. Similarly, component (ii) is component (B) described above mixed with a pharmaceutically acceptable adjuvant, diluent, or carrier. The one or more other active agents (i.e., component (B) described above) can be any of the agents described above for treating an autoimmune disease, such as IBD (e.g., Crohn's disease and / or ulcerative colitis). When component (B) is more than one active agent, these additional active agents can be formulated with each other, with component (A), or separately. In one embodiment, component (B) is one other therapeutic agent. In another embodiment, component (B) is two other therapeutic agents. The combination product of this aspect of the invention (either the combined formulation or the kit-of-parts) can be used in the treatment or prevention of an autoimmune disease (e.g., an autoimmune disease described herein).

[0210] The polypeptide, pharmaceutical composition or construct of the present invention is suitable for use as a medicament, more particularly for use in the treatment of autoimmune and / or inflammatory diseases.

[0211] Autoimmune and / or inflammatory diseases Autoimmune diseases occur when the immune system reacts adversely to normal body tissues. Autoimmune diseases can result in tissue damage, abnormal organ growth, and / or altered organ function. Damage can affect only one organ or tissue type, or multiple organs and tissues. Organs and tissues commonly affected by autoimmune diseases include blood components, such as red blood cells, blood vessels, connective tissue, endocrine glands, such as the thyroid or pancreas, muscles, joints, and skin. Inflammatory diseases are diseases characterized by inflammation. Many inflammatory diseases are autoimmune diseases, and vice versa.

[0212] Autoimmune and / or inflammatory diseases of the GIT The chronic inflammatory bowel diseases (IBDs) Crohn's disease and ulcerative colitis, which afflict both children and adults, are examples of autoimmune and inflammatory disorders of the gastrointestinal tract (GIT) (Hendrickson et al., 2002). Ulcerative colitis is defined as a condition in which the inflammatory response and morphologic changes remain localized to the colon. The rectum is involved in 95% of patients. The inflammation is largely confined to the mucosa and consists of a contiguous lesion of variable severity, with ulcers, edema, and bleeding along the length of the colon (Hendrickson et al., 2002). Ulcerative colitis is usually manifested by the presence of blood and mucus in the stool, along with the most severe lower abdominal cramps during bowel passage. Clinically, the presence of diarrhea with blood and mucus distinguishes ulcerative colitis from irritable bowel syndrome, in which blood is not present. Unlike ulcerative colitis, the symptoms of Crohn's disease are usually subtle, leading to delayed diagnosis. Factors such as the location, extent, and severity of the lesion determine the range of gastrointestinal symptoms. Patients with ileocolonic involvement usually have postprandial abdominal pain, accompanied by right lower quadrant tenderness and, occasionally, an inflammatory mass. Symptoms associated with gastroduodenal Crohn's disease include early satiety, nausea, vomiting, epigastric pain, or dysphagia. Perianal disease, with anal tags, deep anal fissures, and fistulas, is common (Hendrickson et al., 2002). Other diseases of the GIT include, for example, the inflammatory disease mucositis (e.g., drug-induced mucositis and radiation-induced mucositis). In mucositis, lesions can occur anywhere from the mouth to the anus. For lesions of the mouth and esophagus, mouthwash or cream preparations containing the variable domain can be used. For lesions of the anus and rectum, suppositories, creams, or foams containing the variable domain are suitable for topical application. Immunoglobulin chain variable domains are cleared from the lamina propria or other inflammatory sites via absorption into the bloodstream at the site of inflammation or via lymphatic clearance followed by entry into the bloodstream, thus reaching the liver via the bloodstream and being cleared by glomerular filtration in the kidney.Therefore, there is ample rationale that the domain may function therapeutically in diseases such as autoimmune hepatitis, type II diabetes, and glomerulonephritis.

[0213] Suitably, the polypeptide, pharmaceutical composition or construct of the invention is used in the treatment of autoimmune and / or inflammatory diseases of the GI (gastrointestinal) tract in which IL-23 contributes to the pathology of the disease.

[0214] Preferably, the polypeptide, pharmaceutical composition or construct of the present invention is used in the treatment of an autoimmune and / or inflammatory disease of the GI tract selected from the list consisting of Crohn's disease, ulcerative colitis, irritable bowel disease, type II diabetes, glomerulonephritis, autoimmune hepatitis, Sjogren's syndrome, celiac disease, and drug-induced or radiation-induced mucositis (more preferably Crohn's disease or ulcerative colitis, most preferably ulcerative colitis).

[0215] Oral delivery of immunoglobulin chain variable domains ideally treats inflammatory diseases in which IL-23 contributes to at least some of the pathology and in which the immunoglobulin chain variable domains can reach tissues in which IL-23 is biologically active.

[0216] Autoimmune and / or inflammatory diseases of the skin Psoriasis is a debilitating, autoimmune dermatological disease. Plaque psoriasis is the most common form of the disease and is characterized by red skin covered with silvery scales. Histologically, the picture is one of disordered differentiation and hyperproliferation of keratinocytes within psoriatic plaques accompanied by inflammatory cell infiltration (Ortonne, 1999). Psoriatic skin lesions are inflammatory, red, sharply demarcated plaques of various shapes accompanied by characteristic silvery, shiny scales. The term psoriasis includes psoriasis and its symptoms, including erythema, thickened / protuberant skin, and scales.

[0217] Biologics useful in treating psoriasis include anti-TNFα therapeutics (monoclonal antibodies against TNF, such as adalimumab and infliximab, or TNFα receptor fusion proteins, such as etanercept), humanized antibodies to CD11a (efalizumab), or agents that bind to CD2 (thereby blocking the CD2 LFA3 interaction), such as alefacept. Note that not all of the biologics presented herein are approved for use in treating psoriasis.

[0218] The polypeptides of the present invention may be incorporated into creams / ointments or other topical carriers for administration to inflammatory skin lesions where IL-23 contributes to the pathology of such lesions.

[0219] Suitably, the polypeptide, pharmaceutical composition or construct of the present invention is used in the treatment of an autoimmune and / or inflammatory disease of the skin selected from the list consisting of pemphigus, psoriasis, eczema and scleroderma.

[0220] Suitably, the polypeptide, pharmaceutical composition or construct is used in the treatment of other autoimmune / inflammatory diseases in which IL-23 is responsible for a proportion of the observed pathology.

[0221] Preparation method Polypeptides of the present invention can be obtained or engineered using, for example, the techniques disclosed in Green and Sambrook 2012 Molecular Cloning: A Laboratory Manual 4th Edition Cold Spring Harbour Laboratory Press.

[0222] Monoclonal antibodies can be produced using hybridoma technology by fusing myeloma (B-cell cancer) cells, selected for their ability to grow in tissue culture and the absence of antibody chain synthesis, with B-cells that produce the specific antibody.

[0223] Monoclonal antibodies directed against the determined antigens can be e.g. a) immortalizing lymphocytes obtained from the peripheral blood of an animal previously immunized with a determined antigen, with immortal cells, and preferably with bone marrow cells, to form hybridomas; b) The resulting immortalized cells (hybridomas) are cultured, and the cells that produce antibodies with the desired specificity are recovered.

[0224] Alternatively, the use of hybridoma cells is not required. Thus, monoclonal antibodies can be a) cloning into a vector, in particular a phage, more particularly a filamentous bacteriophage, a DNA or cDNA sequence obtained from lymphocytes, in particular peripheral blood lymphocytes, of an animal (preferably previously immunized with a determined antigen); b) transforming a prokaryotic cell with said vector under conditions that allow the production of the antibody; c) selecting antibodies by subjecting them to antigen-affinity selection; d) recovering antibodies with the desired specificity; It can be obtained by a process comprising:

[0225] Methods are known for immunizing camelids, cloning the VHH repertoire of circulating B cells (Chomezynski and Sacchi 1987), and isolating antigen-specific VHHs from immune (Arbabi-Ghahroudi et al. 1997) and non-immune (Tanha et al. 2002) libraries using phage, yeast, or ribosome display (WO92 / 01047, Nguyen et al. 2001, and Harmsen et al. 2007).

[0226] Antigen-binding fragments of antibodies, such as scFv and Fv fragments, have been isolated and expressed in E. coli (Miethe et al. 2013; Skerra et al. 1988; Ward et al. 1989).

[0227] Mutations can be made in the DNA or cDNA that are silent with respect to the amino acid sequence of the polypeptide but that encode the polypeptide by providing codons preferred for translation in a particular host. For example, preferred codons for translation of nucleic acids in E. coli and S. cerevisiae are known.

[0228] Mutation of a polypeptide can be achieved, for example, by substitution, addition, or deletion to the nucleic acid encoding the polypeptide. Substitutions, additions, or deletions to the nucleic acid encoding the polypeptide can be introduced by many methods, including, for example, error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, iterative ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis (Ling et al. 1997), gene rearrangement, gene site saturation mutagenesis (GSSM), synthetic ligation rearrangement (SLR), or a combination of these methods. Modifications, additions, or deletions to nucleic acids can also be introduced by methods including recombination, repeat sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, ensemble mutagenesis, chimeric nucleic acid multimer generation, or a combination thereof.

[0229] In particular, artificial gene synthesis may be used (Nambiar et al. 1984, Sakamar and Khorana 1988, Well et al. 1985, and Grundstrom et al. 1985). Genes encoding the polypeptides of the invention can be synthetically produced, for example, by solid-phase DNA synthesis. The entire gene can be synthesized de novo, without the need for precursor template DNA. To obtain the desired oligonucleotide, building blocks are sequentially coupled to a growing oligonucleotide chain in the order required by the product sequence. Once chain assembly is complete, the product is released from the solid phase into solution, deprotected, and collected. The product can be isolated by high-performance liquid chromatography (HPLC) to obtain the desired oligonucleotide in high purity (Verma and Eckstein 1998).

[0230] Expression of immunoglobulin chain variable domains, such as VH and VHH, can be achieved using suitable expression vectors, such as bacteria, e.g., prokaryotic cells, such as E. coli (for example, according to the protocols disclosed in WO94 / 04678, which is incorporated herein by reference and described in further detail below). Expression of immunoglobulin chain variable domains, such as VH and VHH, can also be achieved using eukaryotic cells, e.g., insect cells, CHO cells, Vero cells, or suitable yeast strains, such as those belonging to the genera Aspergillus, Saccharomyces, Kluyveromyces, Hansenula, or Pichia. Preferably, S. cerevisiae is used (for example, according to the protocols disclosed in WO94 / 025591, which is incorporated herein by reference and described in further detail below).

[0231] Specifically, VHHs: a) cloning a DNA or cDNA sequence encoding a VHH (e.g. obtained from camelid lymphocytes or produced synthetically), optionally containing a His-tag, into a Bluescript vector (Agilent Technologies); b) recovering the cloned fragment after amplification using a 5' primer specific for the VHH containing a XhoI site and a 3' primer containing a SpeI site with the sequence TC TTA ACT AGT GAG GAG ACG GTG ACC TG (SEQ ID NO: 68); c) cloning the recovered fragment in phase in the Immuno PBS vector (Huse et al. 1989) after digestion of the vector with the restriction enzymes XhoI and SpeI; d) transforming a host cell, in particular E. coli, by transfection with the recombinant Immuno PBS vector of step c; e) recovering the expression product of the VHH coding sequence by affinity purification, such as by Protein A, cation exchange, or, if the VHH contains a His tag, by chromatography on a column using a nickel affinity resin, using E. coli cells according to the method disclosed in WO94 / 04678.

[0232] Alternatively, immunoglobulin chain variable domains such as VH and VHH can be a) obtaining a DNA or cDNA sequence encoding a VHH having a determined specific antigen-binding site; b) amplifying the resulting DNA or cDNA using a 5' primer containing a start codon and a Hindlll site and a 3' primer containing a stop codon with a XhoI site; c) recombination of the amplified DNA or cDNA into the Hindlll (position 2650) and XhoI (position 4067) sites of the plasmid pMM984 (Merchlinsky et al. 1983); d) transfecting permissive cells, in particular NB-E cells (Faisst et al. 1995), with the recombinant plasmid; e) recovering the resulting product.

[0233] Furthermore, immunoglobulin chain variable domains such as VHH or VH can be produced according to the methods disclosed in Frenken et al., 2000 and WO99 / 23221 (incorporated herein by reference in their entireties) using E. coli or S. cerevisiae as follows.

[0234] Blood samples were collected from the immunized llamas, and the lymphocyte population was enriched by discontinuous centrifugation on a Ficoll (a neutral, highly branched, high-mass, hydrophilic polysaccharide that dissolves readily in aqueous solution - Pharmacia) gradient. Total RNA was isolated by acid guanidium thiocyanate extraction (Chomezynski and Sacchi 1987). After first-strand cDNA synthesis (using a cDNA kit such as RPN 1266 Amersham), DNA fragments encoding the VHH and VH fragments and the short or part of the long hinge region were amplified by PCR using specific primers as detailed on pages 22 and 23 of WO99 / 23221. Upon digestion of the PCR fragments with PstI and HindIII or BstEII, DNA fragments of approximately 300-450 bp in length are purified by agarose gel electrophoresis and ligated into the E. coli phagemid vector pUR4536 or the episomal S. cerevisiae expression vector pUR4548, respectively. pUR4536 is derived from pHEN (Hoogenboom et al., 1991) and is lacI-amplicon-converted to allow cloning of the llama VHH and VH genes. q pUR4548 is derived from pSY1 (Harmsen et al., 1993). The BstEII site in the leu2 gene was removed from this plasmid by PCR, and the cloning site between the SUC2 signal sequence and the terminator was replaced to facilitate the cloning of VH / VHH gene fragments. The VH / VHH have a c-myc tag at the C-terminus for detection. Individual E. coli JM109 colonies were cultured in 1% glucose and 100 mg L-1 After overnight growth (37°C), the plates were transferred to 96-well microtiter plates containing 150 ml of 2TY medium supplemented with ampicillin. -1 Duplicate cultures are grown in 2TY medium containing ampicillin and 0.1 mM IPTG. After a further overnight incubation, optionally freezing and thawing, the cells are centrifuged and pelleted, and the supernatant can be used in an ELISA. Individual S. cerevisiae colonies are transferred to test tubes containing selective minimal medium (0.7% yeast nitrogen base, 2% glucose, supplemented with essential amino acids and bases) and grown for 48 hours at 30°C. The cultures are then diluted 10-fold in YPGal medium (containing 1% yeast extract, 2% bacto peptone, and 5% galactose). After 24 and 48 hours of growth, the cells are pelleted, and the culture supernatant can be analyzed by ELISA. The optical density at 600 nm (OD600) is optionally measured.

[0235] Furthermore, immunoglobulin chain variable domains such as VH / VHH can be produced using S. cerevisiae using procedures such as the following.

[0236] Isolate a naturally occurring DNA sequence encoding a VH / VHH, or obtain a synthetically produced DNA sequence encoding a VH / VHH, including the 5'-UTR, signal sequence, stop codon, and flanked by SacI and HindIII sites (such synthetic sequences can be produced as outlined above or may be ordered from a commercial supplier, e.g., Geneart (Life Technologies)).

[0237] To transfer VH / VHH genes into the multicopy integration (MCI) vector pUR8569 or pUR8542, restriction sites are used as follows: Using 25 μl of VHH DNA (Geneart plasmid or MCI vector), 1 μl of SacI, 1 μl of HindIII, and 3 μl of a buffer suitable for double digestion, such as NEB buffer 1 (New England Biolabs), the DNA sequence encoding the VHH, optionally contained within a shuttle vector, cassette, or other synthetic gene construct, and the MCI vector with SacI and HindIII are digested overnight at 37°C. 25 μl of the digested DNA encoding the VHH and 25 μl of the digested MCI vector are run on a 1.5% agarose gel containing 1x TAE buffer, followed by gel extraction using, for example, a QIAquick Gel Extraction Kit (Qiagen). Ligation of the digested MCI vector and the digested DNA encoding VH / VHH is set up as follows: 100 ng of vector, 30 ng of VHH gene, 1.5 ul of 10x ligase buffer, 1 ul of T4 DNA ligase, and ddH2O. Ligation is then carried out overnight at 16°C.

[0238] Next, transform the E. coli cells. For chemically competent XL-1 Blue cells, thaw 200 μl of heat-competent XL-1 Blue cells and add 5 μl of ligation mix on ice for approximately 30 minutes, followed by a 90-second heat shock at 42°C. Then, add 800 μl of Luria-Bertani low-salt medium supplemented with 2% glucose, and allow the cells to recover at 37°C for 2 hours. Plate the cells on Luria-Bertani agar and ampicillin (100 μg / ml) plates and maintain them at 37°C overnight. For electrocompetent TG1 E. coli cells, use an electroporation cuvette. In the electroporation cuvette: thaw 50 μl of electrocompetent TG1 cells and 1 μl of ligation mix on ice for approximately 15 minutes. Place the cuvette in the holder and pulse. Add 500 μl of 2TY medium and allow the cells to recover at 37°C for 30 minutes. Plate 100 ul of cells onto Luria-Bertani agar plates containing ampicillin (100 ug / ml) and 2% glucose. Keep plates at 37°C overnight.

[0239] After cloning the VH / VHH genes into E. coli as detailed above, S. cerevisiae can be transformed with the linearized MCI vector. Before transformation can occur, several steps are performed: (i) the DNA must be converted from circular to linear by digestion, otherwise it cannot integrate into the yeast genome, and (ii) the digested DNA must be purified by ethanol precipitation. Furthermore, during the conversion process, yeast cells become semipermeable, allowing DNA to pass through the cell membrane.

[0240] Preparation for yeast transformation: Perform HpaI digestion of midi-preps prepared from selected E. coli colonies expressing VH / VHH genes as follows: Prepare 100 ul of a solution containing 20 ng of midi-prep, 5 ul of HpaI, 10 ul of an appropriate buffer such as NEB4 buffer (BioLabs), and ddH2O.

[0241] Digest the HpaI-digested DNA overnight at room temperature. Next, perform an ethanol precipitation (and set aside a 5 ul sample from the HpaI digestion). Add 300 ul of 100% ethanol to the 95 ul HpaI-digested midiprep, vortex, and spin at full speed for 5 minutes. If a pellet is present, carefully decant it and add 100 ul of 70% ethanol, then spin at full speed again for 5 minutes. Decant the sample again and keep at 50-60°C until the pellet is dry. Resuspend the pellet in 50 ul of ddH2O. Run 5 ul on a gel alongside the 5 ul HpaI-digested sample.

[0242] Yeast Transformation: Prepare YNBglu plates. Use 10 g agar + 425 ml water (sterilized), 25 ml filtered 20x YNB (3.35 g YNB (Yeast Nitrogen Base) in 25 ml sterile HO), and 50 ml sterile 20% glucose and pour into Petri dishes. Pick one yeast colony from the master plate and grow in 3 ml YPD (Yeast Extract Peptone Dextrose) overnight at 30°C. The next day, prepare approximately 600 ml of YPD and use this to fill three flasks with 275 ml, 225 ml, and 100 ml of YPD. Add 27.5 ul of the yeast YPD culture to the first flask and mix gently. Take 75 ml from the first flask and add to the second flask and mix gently. Take 100 ml from the second flask and place it in a third flask, mixing gently. Grow until an OD660 of 1-2 is reached. Once this OD is reached, split the flask into four Falcon tubes, each containing 45 ml. Spin at 4200 rpm for 2 minutes. Discard the supernatant. Dissolve the pellet in two Falcon tubes containing 45 ml of HO (reducing the number of tubes from four to two). Spin at 4200 rpm for 2 minutes. Dissolve the pellet in 45 ml of HO (reducing the number of tubes from two to one). Spin at 4200 rpm for 2 minutes. Gently dissolve the pellet in 5 ml of 100 mM lithium acetate (LiAc) and spin for a few seconds. Carefully discard some of the LiAc, retaining more than half of the LiAc in the tube. Vortex the cells and boil the carrier DNA for 5 minutes, then quickly cool in ice water. Add 240 μl of PEG, 50 μl of cells, 36 μl of LiAc (1M), 25 μl of carrier DNA, and 45 μl of ethanol-precipitated VH / VHH to a 15 ml tube. Mix gently after each step. (Treat a blank sample similarly, but without the ethanol-precipitated VH / VHH.) Incubate at 30°C for 30 minutes, gently invert 3-4 times, and then heat shock at 42°C for 20-25 minutes. Spin briefly at a maximum of 6000 rpm. Gently remove the supernatant and add 250 μl of ddH2O and mix.Streak all of the mixture onto a YNBglu plate until the plate is dry and grow at 30°C for 4-5 days. Finally, prepare YNBglu by dividing the plate into 6 equal parts, numbering the parts 1 to 6. Inoculate the largest colony and streak number 1. Repeat this process for the other colonies, starting from largest to smallest, starting with 1 to 6. Grow the colonies at 30°C for 3-4 days until colonies are produced. VH / VHH clones are grown using glucose as a carbon source, and VH / VHH expression is induced by adding 0.5% galactose to activate the galactose-7 promoter. Test the colonies by growing 3 mL small-scale cultures and select those that show the best expression of VH or VHH. These colonies are then used for purification.

[0243] Purification: VH / VHH are purified by cation exchange chromatography using a strong anionic resin (such as Capto S). On day 1, 5 ml of YPD medium (YP medium + 2% glucose) is inoculated with a selected yeast colony expressing the VH / VHH, and the cells are grown overnight at 30°C (shaking at 180 rpm) in a 25 ml sealed sterile tube. On day 2, 5 ml of the overnight culture is diluted into 50 ml of freshly prepared YP medium + 2% glucose + 0.5% galactose, and the cells are grown for two nights at 30°C (shaking at 180 rpm) in a 250 ml aerated, conditioned flask. On day 4, the cells are spun down in a centrifuge at 4200 rpm for 20 minutes to pellet the cells. Cation exchange purification step using a strong anionic resin: The pH of the ligand-containing supernatant is adjusted to 3.5. Wash 0.75 ml of resin (+ / - 0.5 ml slurry) per 50 ml of supernatant with 50 ml of ddH2O, then wash three times with binding buffer. Add the washed resin to the supernatant and incubate the suspension on a shaker at 4°C for 1.5 hours. Pellet the VH / VHH bound to the resin by centrifugation at 500 g for 2 minutes and wash it with wash buffer. Decant the supernatant and resuspend the resin in 10 ml of binding buffer. Place a filter in a PD-10 column, pour the resin into the column, allow the resin to settle for a while, and then add the filter on top of the resin. Wait until all the binding buffer has drained out. Elute the VH / VHH with 6 x 0.5 ml of elution buffer. Collect the eluate fractions in Eppendorf tubes. Measure the protein concentration of the six eluted fractions using a Nanodrop. Pool the fractions containing VHH and transfer the solution to a dialysis membrane with a 3,500 Da cutoff. Dialyze the purified protein solution against 3 L of PBS overnight at 4°C. On day 5, dialyze the purified protein solution against 2 L of fresh PBS for an additional 2 hours at 4°C. Finally, calculate the final concentration by BCA. Although described for VH / VHH, the techniques described above may also be used for scFv, Fab, Fv, and other antibody fragments, as appropriate.

[0244] Multiple antigen-binding fragments (preferably VH / VHH) can be fused by chemical crosslinking by reacting amino acid residues with organic derivatizing agents, such as those described by Blattler et al., 1985. Alternatively, antigen-binding fragments can be fused genetically at the DNA level, i.e., a polynucleotide construct encoding a complete polypeptide construct containing one or more antigen-binding fragments can be formed. One method for joining multiple antigen-binding fragments via a genetic pathway is by linking the sequences encoding the antigen-binding fragments directly or via a peptide linker. For example, the carboxy terminus of a first antigen-binding fragment can be linked to the amino terminus of the next antigen-binding fragment. This linking mode can be extended to link antigen-binding fragments for the construction of tri-, tetra-, etc. functional constructs. Methods for producing multivalent (e.g., bivalent) VHH polypeptide constructs are disclosed in WO 96 / 34103, incorporated herein by reference in its entirety.

[0245] Suitably, the polypeptides of the invention (in particular the VHHs of the invention) can be produced in fungi such as yeast (e.g., S. cerevisiae) according to the method disclosed in WO 02 / 48382, which involves growing the fungus on a medium containing a carbon source, wherein 50-100 wt% of the carbon source is ethanol. Large-scale production of VHH fragments in S. cerevisiae is described in Thomassen et al. (2002).

[0246] In one aspect of the invention there is provided a process for the preparation of a polypeptide or construct of the invention, said process comprising: i) cloning the polynucleotide of the present invention into a vector such as a plasmid; ii) transforming a cell, such as a bacterial or yeast cell, capable of producing the polypeptide or construct of the invention with said vector under conditions allowing the production of the polypeptide or construct; iii) recovering the polypeptide or construct, such as by affinity chromatography.

[0247] Further embodiments of the present invention are set forth below. 1. A polypeptide comprising an immunoglobulin chain variable domain that binds to IL-23, wherein said 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. 2. The polypeptide of Item 1, wherein CDR3 comprises a sequence that shares 80% or more sequence identity with SEQ ID NO:3. 3. The polypeptide according to Item 2, wherein CDR3 consists of a sequence that shares 80% or more sequence identity with SEQ ID NO:3. 4. The polypeptide of either paragraph 2 or 3, wherein any residues in CDR3 that differ from their corresponding residues in SEQ ID NO: 3 are conservative substitutions for their corresponding residues. 5. The polypeptide of either item 2 or 3, wherein the residue in CDR3 corresponding to residue number 6 in SEQ ID NO:3 is I. 6. The polypeptide of either item 2 or 3, wherein the residue in CDR3 corresponding to residue number 6 in SEQ ID NO:3 is L. 7. The polypeptide of paragraph 4, wherein the residue in CDR3 corresponding to residue number 6 of SEQ ID NO: 3 is I or L, and any other residue in CDR3 that differs from those corresponding residues in SEQ ID NO: 3 is a conservative substitution for those corresponding residues. 8. The polypeptide of item 2, wherein CDR3 comprises SEQ ID NO:3. 9. The polypeptide of item 3, wherein CDR3 consists of SEQ ID NO:3. 10. The polypeptide of any one of paragraphs 1-9, wherein CDR1 comprises a sequence that shares 80% or more sequence identity with SEQ ID NO:1. 11. The polypeptide according to item 10, wherein CDR1 consists of a sequence that shares 80% or more sequence identity with SEQ ID NO:1. 12. The polypeptide of any one of paragraphs 1-9, wherein any residues in CDR1 that differ from their corresponding residues in SEQ ID NO: 1 are conservative substitutions for their corresponding residues. 13. The polypeptide of clause 11, wherein CDR1 comprises SEQ ID NO:1. 14. The polypeptide of clause 13, wherein CDR1 consists of SEQ ID NO:1. 15. The polypeptide of any one of clauses 1-14, wherein CDR2 comprises a sequence that shares 55% or more sequence identity with SEQ ID NO:2, such as sharing 60% or more sequence identity, for example sharing 70% or more sequence identity, for example sharing 75% or more sequence identity, for example sharing 80% or more sequence identity, for example sharing 85% or more sequence identity, for example sharing 90% or more sequence identity. 16. The polypeptide of clause 15, wherein CDR2 consists of a sequence that shares 55% or more sequence identity with SEQ ID NO:2, such as sharing 60% or more sequence identity, for example, sharing 70% or more sequence identity, for example, sharing 75% or more sequence identity, for example, sharing 80% or more sequence identity, for example, sharing 85% or more sequence identity, for example, sharing 90% or more sequence identity. 17. The polypeptide of any one of paragraphs 1-16, wherein the residue in CDR2 corresponding to residue number 9 of SEQ ID NO:2 is D or H, and / or the residue in CDR2 corresponding to residue number 10 of SEQ ID NO:2 is Y or D, and / or the residue in CDR2 corresponding to residue number 11 of SEQ ID NO:2 is S, G, R, or A (e.g., S, R, or A, e.g., S or A), and / or the residue in CDR2 corresponding to residue number 14 of SEQ ID NO:2 is V or A. 18. The polypeptide of any one of paragraphs 1-17, wherein any residues in CDR2 that differ from their corresponding residues in SEQ ID NO:2 are conservative substitutions for their corresponding residues. 19. The polypeptide of clause 15, wherein CDR2 comprises SEQ ID NO:2. 20. The polypeptide of clause 19, wherein CDR2 consists of SEQ ID NO:2. 21. The polypeptide of any one of clauses 1-20, wherein FR1 comprises a sequence that shares 5% or more sequence identity with SEQ ID NO:4, such as sharing 12% or more sequence identity, for example sharing 18% or more sequence identity, for example sharing 26% or more sequence identity, for example sharing 32% or more sequence identity, for example sharing 38% or more sequence identity, for example sharing 46% or more sequence identity, for example sharing 52% or more sequence identity, for example sharing 58% or more sequence identity, such as sharing 62% or more sequence identity, for example sharing 66% or more sequence identity, for example sharing 68% or more sequence identity, for example sharing 72% or more sequence identity, such as sharing 75% or more sequence identity, for example sharing 78% or more sequence identity, for example sharing 82% or more sequence identity, such as sharing 85% or more sequence identity, for example sharing 90% or more sequence identity, for example sharing 95% or more sequence identity. 22. The polypeptide of clause 21, wherein FR1 consists of a sequence that shares 5% or more sequence identity with SEQ ID NO:4, such as sharing 12% or more sequence identity, for example, sharing 18% or more sequence identity, for example, sharing 26% or more sequence identity, for example, sharing 32% or more sequence identity, for example, sharing 38% or more sequence identity, for example, sharing 46% or more sequence identity, for example, sharing 52% or more sequence identity, for example, sharing 58% or more sequence identity, for example, sharing 62% or more sequence identity, for example, sharing 66% or more sequence identity, for example, sharing 68% or more sequence identity, for example, sharing 72% or more sequence identity, for example, sharing 75% or more sequence identity, for example, sharing 78% or more sequence identity, for example, sharing 82% or more sequence identity, for example, sharing 85% or more sequence identity, for example, sharing 90% or more sequence identity, for example, sharing 95% or more sequence identity. 23. The polypeptide of any one of paragraphs 1-22, wherein the residue in FR1 corresponding to residue number 1 of SEQ ID NO:4 is D or E. 24. The polypeptide of paragraph 23, wherein the residue in FR1 corresponding to residue number 1 in SEQ ID NO:4 is D. 25. The polypeptide of item 24, wherein the residues in FR1 corresponding to residues 1 to 5 in SEQ ID NO: 4 are DVQLV. 26. The polypeptide of any one of paragraphs 1-25, wherein any residues in FR1 that differ from their corresponding residues in SEQ ID NO: 4 are conservative substitutions for their corresponding residues. 27. The polypeptide of clause 21, wherein FR1 comprises SEQ ID NO:4. 28. The polypeptide of paragraph 27, wherein FR1 consists of SEQ ID NO:4. 29. The polypeptide of any one of clauses 1-28, wherein FR2 comprises a sequence that shares 10% or more sequence identity with SEQ ID NO:5, such as sharing 15% or more sequence identity, for example sharing 25% or more sequence identity, for example sharing 30% or more sequence identity, for example sharing 40% or more sequence identity, for example sharing 45% or more sequence identity, for example sharing 55% or more sequence identity, for example sharing 60% or more sequence identity, such as sharing 70% or more sequence identity, for example sharing 75% or more sequence identity, for example sharing 85% or more sequence identity, for example sharing 90% or more sequence identity. 30. The polypeptide of clause 29, wherein FR2 consists of a sequence that shares 10% or more sequence identity with SEQ ID NO: 5, such as sharing 15% or more sequence identity, for example sharing 25% or more sequence identity, for example sharing 30% or more sequence identity, for example sharing 40% or more sequence identity, for example sharing 45% or more sequence identity, for example sharing 55% or more sequence identity, such as sharing 60% or more sequence identity, for example sharing 70% or more sequence identity, for example sharing 75% or more sequence identity, for example sharing 85% or more sequence identity, for example sharing 90% or more sequence identity. 31. The polypeptide of any one of paragraphs 1-30, wherein any residues in FR2 that differ from their corresponding residues in SEQ ID NO:5 are conservative substitutions for their corresponding residues. 32. The polypeptide of paragraph 29, wherein FR2 comprises SEQ ID NO:5. 33. The polypeptide of paragraph 32, wherein FR2 consists of SEQ ID NO:5. 34. The polypeptide of any one of clauses 1-33, wherein FR3 comprises a sequence that shares 8% or more sequence identity with SEQ ID NO: 6, such as sharing 15% or more sequence identity, for example sharing 20% ​​or more sequence identity, for example sharing 26% or more sequence identity, for example sharing 32% or more sequence identity, such as sharing 40% or more sequence identity, for example sharing 45% or more sequence identity, for example sharing 52% or more sequence identity, such as sharing 58% or more sequence identity, for example sharing 65% or more sequence identity, such as sharing 70% or more sequence identity, for example sharing 76% or more sequence identity, for example sharing 80% or more sequence identity, such as sharing 82% or more sequence identity, for example sharing 85% or more sequence identity, for example sharing 90% or more sequence identity, for example sharing 92% or more sequence identity, for example sharing 95% or more sequence identity. 35. The polypeptide of clause 34, wherein FR3 consists of a sequence that shares 8% or more sequence identity with SEQ ID NO: 6, such as sharing 15% or more sequence identity, for example sharing 20% ​​or more sequence identity, for example sharing 26% or more sequence identity, for example sharing 32% or more sequence identity, such as sharing 40% or more sequence identity, for example sharing 45% or more sequence identity, for example sharing 52% or more sequence identity, such as sharing 58% or more sequence identity, for example sharing 65% or more sequence identity, such as sharing 70% or more sequence identity, for example sharing 76% or more sequence identity, for example sharing 80% or more sequence identity, such as sharing 82% or more sequence identity, for example sharing 85% or more sequence identity, for example sharing 90% or more sequence identity, such as sharing 92% or more sequence identity, for example sharing 95% or more sequence identity. 36. The polypeptide of any one of paragraphs 1-35, wherein any residues in FR3 that differ from their corresponding residues in SEQ ID NO: 6 are conservative substitutions for their corresponding residues. 37. The polypeptide of clause 34, wherein FR3 comprises SEQ ID NO:6. 38. The polypeptide of paragraph 37, wherein FR3 consists of SEQ ID NO:6. 39. The polypeptide of any one of clauses 1-38, wherein FR4 comprises a sequence that shares 5% or more sequence identity with SEQ ID NO: 7, such as sharing 10% or more sequence identity, for example sharing 20% ​​or more sequence identity, for example sharing 30% or more sequence identity, such as sharing 40% or more sequence identity, for example sharing 50% or more sequence identity, for example sharing 60% or more sequence identity, such as sharing 70% or more sequence identity, for example sharing 80% or more sequence identity, for example sharing 90% or more sequence identity. 40. The polypeptide of clause 39, wherein FR4 consists of a sequence that shares 5% or more sequence identity with SEQ ID NO: 7, such as sharing 10% or more sequence identity, for example sharing 20% ​​or more sequence identity, for example sharing 30% or more sequence identity, such as sharing 40% or more sequence identity, for example sharing 50% or more sequence identity, for example sharing 60% or more sequence identity, such as sharing 70% or more sequence identity, for example sharing 80% or more sequence identity, for example sharing 90% or more sequence identity. 41. The polypeptide of any one of paragraphs 1-40, wherein any residues in FR4 that differ from their corresponding residues in SEQ ID NO: 7 are conservative substitutions for their corresponding residues. 42. The polypeptide of clause 39, wherein FR4 comprises SEQ ID NO:7. 43. The polypeptide of paragraph 42, wherein FR4 consists of SEQ ID NO:7. 44. The polypeptide of any one of clauses 1-43, comprising a sequence that shares 50% or more sequence identity with SEQ ID NO: 8, such as sharing 55% or more sequence identity, for example sharing 60% or more sequence identity, for example sharing 65% or more sequence identity, for example sharing 70% or more sequence identity, for example sharing 75% or more sequence identity, for example sharing 80% or more sequence identity, for example sharing 85% or more sequence identity, for example sharing 90% or more sequence identity, for example sharing 95% or more sequence identity, such as sharing 96% or more sequence identity, for example sharing 97% or more sequence identity, for example sharing 98% or more sequence identity, for example sharing 99% or more sequence identity. 45. The polypeptide of clause 44, wherein the polypeptide consists of a sequence that shares 50% or more sequence identity with SEQ ID NO: 8, such as sharing 55% or more sequence identity, for example, sharing 60% or more sequence identity, for example, sharing 65% or more sequence identity, for example, sharing 70% or more sequence identity, for example, sharing 75% or more sequence identity, for example, sharing 80% or more sequence identity, for example, sharing 85% or more sequence identity, for example, sharing 90% or more sequence identity, for example, sharing 95% or more sequence identity, such as sharing 96% or more sequence identity, for example, sharing 97% or more sequence identity, for example, sharing 98% or more sequence identity, for example, sharing 99% or more sequence identity. 46. ​​The polypeptide of any one of paragraphs 1-45, wherein the N-terminus of the polypeptide is D. 47. The polypeptide of paragraph 44, comprising SEQ ID NO:8. 48. The polypeptide of paragraph 47, consisting of SEQ ID NO:8. 49. A polypeptide comprising an immunoglobulin chain variable domain that binds IL-23, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein: (a) CDR1 consists of a sequence that shares 60% or more sequence identity with SEQ ID NO: 1, CDR2 consists of a sequence that shares 70% or more sequence identity with SEQ ID NO: 2, and CDR3 consists of a sequence that shares 70% or more sequence identity with SEQ ID NO: 3; (b) FR1 consists of a sequence that shares 70% or more sequence identity with SEQ ID NO:4, FR2 consists of a sequence that shares 70% or more sequence identity with SEQ ID NO:5, FR3 consists of a sequence that shares 70% or more sequence identity with SEQ ID NO:6, and FR4 consists of a sequence that shares 70% or more sequence identity with SEQ ID NO:7, and (c) The polypeptide consists of a sequence that shares 70% or more sequence identity with SEQ ID NO:8. 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 more sequence identity with SEQ ID NO: 14, CDR2 comprises a sequence that shares 50% or more sequence identity with SEQ ID NO: 15, and CDR3 comprises a sequence that shares 50% or more sequence identity with SEQ ID NO: 16. 51. The polypeptide of clause 50, wherein the CDR3 comprises a sequence that shares 60% or more sequence identity, such as 80% or more sequence identity, with SEQ ID NO: 16. 52. The polypeptide of clause 51, wherein CDR3 consists of a sequence sharing 60% or more sequence identity, such as 80% or more sequence identity, with SEQ ID NO: 16. 53. The polypeptide of any one of paragraphs 50-52, wherein any residues in the CDR3 that differ from their corresponding residues in SEQ ID NO: 16 are conservative substitutions for their corresponding residues. 54. The polypeptide of clause 51, wherein CDR3 comprises SEQ ID NO:16. 55. The polypeptide of clause 54, wherein CDR3 consists of SEQ ID NO:16. 56. The polypeptide of any one of clauses 50-55, wherein CDR1 comprises a sequence that shares 80% or more sequence identity with SEQ ID NO: 14. 57. The polypeptide of clause 56, wherein CDR1 consists of a sequence that shares 80% or more sequence identity with SEQ ID NO: 14. 58. The polypeptide of any one of paragraphs 50-57, wherein any residues in CDR1 that differ from their corresponding residues in SEQ ID NO: 14 are conservative substitutions for their corresponding residues. 59. The polypeptide of clause 56, wherein CDR1 comprises SEQ ID NO:14. 60. The polypeptide of clause 59, wherein CDR1 consists of SEQ ID NO:14. 61. The polypeptide of any one of clauses 50-60, wherein CDR2 comprises a sequence that shares 55% or more sequence identity with SEQ ID NO: 15, such as sharing 60% or more sequence identity, for example sharing 70% or more sequence identity, for example sharing 75% or more sequence identity, such as sharing 80% or more sequence identity, for example sharing 85% or more sequence identity, for example sharing 90% or more sequence identity. 62. The polypeptide of clause 61, wherein CDR2 consists of a sequence that shares 60% or more sequence identity with SEQ ID NO: 15, such as sharing 70% or more sequence identity, for example sharing 75% or more sequence identity, for example sharing 80% or more sequence identity, for example sharing 85% or more sequence identity, for example sharing 90% or more sequence identity. 63. The polypeptide of any one of paragraphs 50-62, wherein any residues in CDR2 that differ from their corresponding residues in SEQ ID NO: 15 are conservative substitutions for their corresponding residues. 64. The polypeptide of clause 61, wherein CDR2 comprises SEQ ID NO:15. 65. The polypeptide of clause 64, wherein CDR2 consists of SEQ ID NO: 15. 66. (a) FR1 comprises a sequence that shares 5% or more sequence identity with SEQ ID NO: 17, such as sharing 12% or more sequence identity, for example sharing 18% or more sequence identity, for example sharing 26% or more sequence identity, for example sharing 32% or more sequence identity, for example sharing 38% or more sequence identity, for example sharing 46% or more sequence identity, for example sharing 52% or more sequence identity, for example sharing 58% or more sequence identity, for example sharing 62% or more sequence identity, such as sharing 66% or more sequence identity, for example sharing 68% or more sequence identity, for example sharing 72% or more sequence identity, for example sharing 75% or more sequence identity, for example sharing 78% or more sequence identity, for example sharing 82% or more sequence identity, such as sharing 85% or more sequence identity, for example sharing 90% or more sequence identity, for example sharing 95% or more sequence identity. (b) FR2 comprises a sequence that shares 10% or more sequence identity with SEQ ID NO: 18, such as sharing 15% or more sequence identity, for example, sharing 25% or more sequence identity, for example, sharing 30% or more sequence identity, such as sharing 40% or more sequence identity, for example, sharing 45% or more sequence identity, for example, sharing 55% or more sequence identity, such as sharing 60% or more sequence identity, for example, sharing 70% or more sequence identity, for example, sharing 75% or more sequence identity, for example, sharing 85% or more sequence identity, for example, sharing 90% or more sequence identity; (c) FR3 comprises a sequence sharing 8% or more sequence identity with SEQ ID NO: 19, such as sharing 15% or more sequence identity, for example sharing 20% ​​or more sequence identity, for example sharing 26% or more sequence identity, such as sharing 32% or more sequence identity, for example sharing 40% or more sequence identity, for example sharing 45% or more sequence identity, for example sharing 52% or more sequence identity, such as sharing 58% or more sequence identity, for example sharing 65% or more sequence identity, such as sharing 70% or more sequence identity, for example sharing 76% or more sequence identity, for example sharing 80% or more sequence identity, such as sharing 82% or more sequence identity, for example sharing 85% or more sequence identity, for example sharing 90% or more sequence identity, such as sharing 92% or more sequence identity, for example sharing 95% or more sequence identity; and / or (d) the polypeptide of any one of paragraphs 50-65, wherein FR4 comprises a sequence that shares 5% or more sequence identity with SEQ ID NO: 20, such as sharing 10% or more sequence identity, for example, sharing 20% ​​or more sequence identity, for example, sharing 30% or more sequence identity, for example, sharing 40% or more sequence identity, for example, sharing 50% or more sequence identity, such as sharing 60% or more sequence identity, for example, sharing 70% or more sequence identity, for example, sharing 80% or more sequence identity, for example, sharing 90% or more sequence identity. 67. The polypeptide of 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. 68. The polypeptide of any one of clauses 50-67, comprising a sequence that shares 50% or more sequence identity with SEQ ID NO: 13, such as sharing 55% or more sequence identity, for example sharing 60% or more sequence identity, for example sharing 65% or more sequence identity, such as sharing 70% or more sequence identity, for example sharing 75% or more sequence identity, for example sharing 80% or more sequence identity, such as sharing 85% or more sequence identity, for example sharing 90% or more sequence identity, such as sharing 95% or more sequence identity, for example sharing 96% or more sequence identity, for example sharing 97% or more sequence identity, such as sharing 98% or more sequence identity, for example sharing 99% or more sequence identity. 69. The polypeptide of paragraph 68, comprising or consisting of SEQ ID NO:13. 70. A polypeptide comprising an immunoglobulin chain variable domain that binds to L-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: 22, CDR2 comprises a sequence that shares 50% or more sequence identity with SEQ ID NO: 23, and CDR3 comprises a sequence that shares 50% or more sequence identity with SEQ ID NO: 24. 71. The polypeptide of clause 70, wherein the CDR3 comprises a sequence that shares 60% or more sequence identity, such as 80% or more sequence identity, with SEQ ID NO: 24. 72. The polypeptide of clause 71, wherein CDR3 consists of a sequence sharing 60% or more sequence identity, such as 80% or more sequence identity, with SEQ ID NO: 24. 73. The polypeptide of any one of paragraphs 70-72, wherein any residues in the CDR3 that differ from their corresponding residues in SEQ ID NO: 24 are conservative substitutions for their corresponding residues. 74. The polypeptide of clause 71, wherein CDR3 comprises SEQ ID NO:24. 75. The polypeptide of clause 74, wherein CDR3 consists of SEQ ID NO:24. 76. The polypeptide of any one of paragraphs 70-75, wherein CDR1 comprises a sequence sharing 80% or more sequence identity with SEQ ID NO: 22. 77. The polypeptide of clause 76, wherein CDR1 consists of a sequence that shares 80% or more sequence identity with SEQ ID NO: 22. 78. The polypeptide of any one of paragraphs 70-77, wherein any residues in CDR1 that differ from their corresponding residues in SEQ ID NO: 22 are conservative substitutions for their corresponding residues. 79. The polypeptide of clause 76, wherein CDR1 comprises SEQ ID NO:22. 80. The polypeptide of clause 79, wherein CDR1 consists of SEQ ID NO:22. 81. The polypeptide of any one of clauses 70-80, wherein CDR2 comprises a sequence that shares 55% or more sequence identity with SEQ ID NO: 23, such as sharing 60% or more sequence identity, for example sharing 70% or more sequence identity, such as sharing 75% or more sequence identity, for example sharing 80% or more sequence identity, such as sharing 85% or more sequence identity, for example sharing 90% or more sequence identity. 82. The polypeptide of clause 81, wherein CDR2 consists of a sequence that shares 60% or more sequence identity with SEQ ID NO: 23, such as sharing 70% or more sequence identity, for example sharing 75% or more sequence identity, for example sharing 80% or more sequence identity, for example sharing 85% or more sequence identity, for example sharing 90% or more sequence identity. 83. The polypeptide of any one of paragraphs 70-82, wherein any residues in CDR2 that differ from their corresponding residues in SEQ ID NO: 23 are conservative substitutions for their corresponding residues. 84. The polypeptide of clause 81, wherein CDR2 comprises SEQ ID NO:23. 85. The polypeptide of clause 84, wherein CDR2 consists of SEQ ID NO:23. 86. (a) FR1 comprises a sequence that shares 5% or more sequence identity with SEQ ID NO: 25, such as sharing 12% or more sequence identity, for example sharing 18% or more sequence identity, for example sharing 26% or more sequence identity, for example sharing 32% or more sequence identity, for example sharing 38% or more sequence identity, for example sharing 46% or more sequence identity, for example sharing 52% or more sequence identity, for example sharing 58% or more sequence identity, for example sharing 62% or more sequence identity, for example sharing 66% or more sequence identity, for example sharing 68% or more sequence identity, for example sharing 72% or more sequence identity, for example sharing 75% or more sequence identity, for example sharing 78% or more sequence identity, for example sharing 82% or more sequence identity, for example sharing 85% or more sequence identity, for example sharing 90% or more sequence identity, for example sharing 95% or more sequence identity. (b) FR2 comprises a sequence that shares 10% or more sequence identity with SEQ ID NO: 26, such as sharing 15% or more sequence identity, for example, sharing 25% or more sequence identity, for example, sharing 30% or more sequence identity, such as sharing 40% or more sequence identity, for example, sharing 45% or more sequence identity, for example, sharing 55% or more sequence identity, such as sharing 60% or more sequence identity, for example, sharing 70% or more sequence identity, for example, sharing 75% or more sequence identity, for example, sharing 85% or more sequence identity, for example, sharing 90% or more sequence identity; (c) FR3 comprises a sequence which shares 8% or more sequence identity, such as sharing 15% or more sequence identity, for example sharing 20% ​​or more sequence identity, for example sharing 26% or more sequence identity, such as sharing 32% or more sequence identity, for example sharing 40% or more sequence identity, for example sharing 45% or more sequence identity, such as sharing 52% or more sequence identity, for example sharing 58% or more sequence identity, such as sharing 65% or more sequence identity, for example sharing 70% or more sequence identity, for example sharing 76% or more sequence identity, such as sharing 80% or more sequence identity, for example sharing 82% or more sequence identity, for example sharing 85% or more sequence identity, such as sharing 90% or more sequence identity, for example sharing 92% or more sequence identity, for example sharing 95% or more sequence identity, with SEQ ID NO: 27; and / or (d) the polypeptide of any one of paragraphs 70-85, wherein FR4 comprises a sequence that shares 5% or more sequence identity, such as sharing 10% or more sequence identity, for example, sharing 20% ​​or more sequence identity, for example, sharing 30% or more sequence identity, for example, sharing 40% or more sequence identity, for example, sharing 50% or more sequence identity, for example, sharing 60% or more sequence identity, for example, sharing 70% or more sequence identity, for example, sharing 80% or more sequence identity, for example, sharing 90% or more sequence identity, with SEQ ID NO: 28. 87. The polypeptide of paragraph 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. 88. The polypeptide of any one of clauses 70-87, comprising a sequence that shares 50% or more sequence identity with SEQ ID NO: 21, such as sharing 55% or more sequence identity, for example sharing 60% or more sequence identity, for example sharing 65% or more sequence identity, such as sharing 70% or more sequence identity, for example sharing 75% or more sequence identity, for example sharing 80% or more sequence identity, such as sharing 85% or more sequence identity, for example sharing 90% or more sequence identity, such as sharing 95% or more sequence identity, for example sharing 96% or more sequence identity, for example sharing 97% or more sequence identity, such as sharing 98% or more sequence identity, for example sharing 99% or more sequence identity. 89. The polypeptide of paragraph 88, comprising or consisting of SEQ ID NO:21. 90. The polypeptide of any one of paragraphs 1-89, wherein the polypeptide is an antibody. 91. The polypeptide of paragraph 90, wherein the polypeptide is an antibody fragment. 92. The polypeptide of clause 91, selected from the list consisting of VHH, VH, VL, V-NAR, Fab fragment, and F(ab')2 fragment. 93. The polypeptide of paragraph 92, wherein the polypeptide is a VHH. 94. The polypeptide of clause 92, wherein the polypeptide is a VH. 95. A construct comprising two or more identical polypeptides according to any one of paragraphs 1-94. 96. A construct comprising at least one polypeptide according to any one of paragraphs 1-94 and at least one different polypeptide, wherein said different polypeptide binds to TNFα. 97. A construct comprising at least one polypeptide according to any one of paragraphs 1-94 and at least one different polypeptide, wherein said different polypeptide binds to a target other than TNFα. 98. The construct of any one of paragraphs 95-97, wherein the polypeptides are connected by at least one protease-labile linker. 99. The protease-labile linker has the following format: [-(G a S) x -B-(G b S) y -] z It is of where: a is 1 to 10, b is 1 to 10; x is 1 to 10, y is 1 to 10, z is 1 to 10, and 99. The construct of paragraph 98, wherein B is K or R. 100. The construct of paragraph 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. 101. The construct of paragraph 100, wherein the construct comprises, or more preferably consists of, SEQ ID NO:46. 102. The construct of any one of paragraphs 95-97, wherein all of the polypeptides are connected by non-protease labile linkers. 103. Non-protease labile linkers are of the following format: (G4S) x It is of where: 103. The construct of paragraph 102, wherein x is 1 to 10. 104. The construct of paragraph 103, wherein x is 6. 105.5nM or less, for example, 4nM or less, for example, 3nM or less, for example, 2nM or less, for example, 1.7nM or less, for example, 1.5nM or less, for example, 1.4nM or less, for example, 1.3nM or less, for example, 1.2nM or less, for example, 1.1nM or less, for example, 1.0nM or less, for example, 0.9nM or less, for example, 0.8nM or less, for example, 0.75nM or less, for example, 0.70nM or less, for example, 0.65nM or less, for example, The polypeptide or construct of any one of paragraphs 1-104, which neutralizes human IL-23 in IL-23-IL-23R neutralization ELISA (assessment method A) with an EC50 of 0.60 nM or less, for example, 0.55 nM or less, for example, 0.50 nM or less, for example, 0.45 nM or less, for example, 0.40 nM or less, for example, 0.35 nM or less, for example, 0.30 nM or less, for example, 0.25 nM or less, for example, 0.20 nM or less. 106. The polypeptide or construct of any one of paragraphs 1-105, which is substantially resistant to one or more proteases. 107. The polypeptide or construct of paragraph 106, wherein the one or more proteases are present in the stomach, or in the small intestine or large intestine. 108. The polypeptide or construct of paragraph 107, wherein the one or more proteases are present in the small intestine. 109. The polypeptide or construct of paragraph 106, wherein the one or more proteases are selected from the group consisting of enteropeptidase, trypsin, chymotrypsin, and inflammatory bowel disease proteases. 110. The polypeptide or construct of paragraph 109, wherein the one or more proteases are selected from the group consisting of trypsin, chymotrypsin, and inflammatory bowel disease proteases. 111. The polypeptide or construct of either of paragraphs 109 or 110, wherein the inflammatory bowel disease protease is one or more proteases selected from the group consisting of MMP3, MMP12, and cathepsins. 112. The polypeptide or construct of paragraph 110, wherein the proteases are trypsin and chymotrypsin. 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. 114. The pharmaceutical composition according to item 113, wherein the pharmaceutical composition is presented in an enterically coated form. 115. A pharmaceutical composition according to any of paragraphs 113 or 114, comprising at least one further active agent. 116. The at least one additional active agent is a 5-aminosalicylic acid or a prodrug thereof (such as sulfasalazine, olsalazine, or bisalazide); a corticosteroid (e.g., prednisolone, methylprednisolone, or budesonide); an immunosuppressant (e.g., cyclosporine, tacrolimus, methotrexate, azathioprine, or 6-mercaptopurine); an anti-TNFα 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., apilimod); an anti-alpha-4-beta-7 antibody (e.g., vedolizumab 116. The pharmaceutical composition of claim 115, wherein the compound is selected from the list comprising: an antibody against the cell adhesion molecule alpha-4-integrin (e.g., natalizumab); an antibody against the IL2 receptor alpha subunit (e.g., daclizumab or basiliximab); a JAK3 inhibitor (e.g., tofacitinib or R348); a Syk inhibitor and its prodrug (e.g., fostamatinib and R-406); a phosphodiesterase-4 inhibitor (e.g., tetomilast); HMPL-004; probiotics; delsalazine; semapimod / CPSI-2364; and a protein kinase C inhibitor (e.g., AEB-071). 117. The pharmaceutical composition of paragraph 116, wherein at least one additional active agent is 5-aminosalicylic acid. 118. A polypeptide, pharmaceutical composition, or construct according to any one of paragraphs 1 to 117, for use as a medicament. 119. The polypeptide, pharmaceutical composition, or construct according to paragraph 118, for use in the treatment of autoimmune and / or inflammatory diseases. 120. The polypeptide, pharmaceutical composition or construct according to paragraph 119, wherein the autoimmune and / or inflammatory disease is selected from the list consisting of Crohn's disease, ulcerative colitis, irritable bowel disease, diabetes mellitus type II, glomerulonephritis, autoimmune hepatitis, Sjogren's syndrome, celiac disease, drug-induced or radiation-induced mucositis, pemphigus, psoriasis, eczema and scleroderma. 121. The polypeptide, pharmaceutical composition, or construct according to paragraph 120, wherein the autoimmune and / or inflammatory disease is Crohn's disease. 122. A polypeptide, pharmaceutical composition, or construct according to any one of paragraphs 118 to 121, which is administered orally. 123. A polypeptide, pharmaceutical composition, or construct according to any one of paragraphs 118 to 119, which is administered topically to the skin. 124. Use of a polypeptide, pharmaceutical composition, or construct according to any one of paragraphs 1 to 117 in the manufacture of a medicament for the treatment of an autoimmune and / or inflammatory disease. 125. Use of a polypeptide, pharmaceutical composition or construct according to any one of clauses 124, wherein the autoimmune and / or inflammatory disease is selected from the list consisting of Crohn's disease, ulcerative colitis, irritable bowel disease, diabetes mellitus type II, glomerulonephritis, autoimmune hepatitis, Sjogren's syndrome, celiac disease, drug-induced or radiation-induced mucositis, pemphigus, psoriasis, eczema and scleroderma. 126. The use according to paragraph 125, wherein the autoimmune and / or inflammatory disease is Crohn's disease. 127. The use of any one of paragraphs 124 to 126, wherein the agent is administered orally. 128. The use according to paragraph 124, wherein the agent is administered topically to the skin. 129. A method for treating an autoimmune and / or inflammatory disease, comprising administering to a person in need thereof a therapeutically effective amount of a polypeptide, pharmaceutical composition, or construct described in any one of paragraphs 1 to 117. 130. The method of treating an autoimmune and / or inflammatory disease according to paragraph 129, wherein the autoimmune and / or inflammatory disease is selected from the list consisting of Crohn's disease, ulcerative colitis, irritable bowel syndrome, diabetes mellitus type II, glomerulonephritis, autoimmune hepatitis, Sjogren's syndrome, celiac disease, drug-induced or radiation-induced mucositis, pemphigus, psoriasis, eczema, and scleroderma. 131. The method of treating an autoimmune disease according to paragraph 130, wherein the autoimmune disease is Crohn's disease. 132. The method for treating an autoimmune disease according to any one of paragraphs 129 to 131, wherein the polypeptide, pharmaceutical composition, or construct is administered orally. 133. The method of treating an autoimmune disease according to paragraph 129, wherein the polypeptide, pharmaceutical composition, or construct is administered topically to the skin. 134. The polypeptide, pharmaceutical composition, or construct may be administered with or without a 5-aminosalicylic acid or a prodrug thereof (sulfasalazine, olsalazine, or bisalazide); a corticosteroid (e.g., prednisolone, methylprednisolone, or budesonide); an immunosuppressant (e.g., cyclosporine, tacrolimus, methotrexate, azathioprine, or 6-mercaptopurine); an anti-TNFα 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., apilimod); an anti-alpha-4-beta-7 antibody (e.g., vedolizumab); an MAdCAM-1 blocker (e.g., PF-00547659); a cell adhesion molecule 134. The polypeptide, pharmaceutical composition, construct, use, or method of any one of paragraphs 118 to 133, wherein the polypeptide, pharmaceutical composition, construct, use, or method is administered sequentially, simultaneously, or separately with at least one active agent selected from the list comprising: an antibody against alpha-4-integrin (e.g., natalizumab); an antibody against the IL2 receptor alpha subunit (e.g., daclizumab or basiliximab); a JAK3 inhibitor (e.g., tofacitinib or R348); a Syk inhibitor and its prodrug (e.g., fostamatinib and R-406); a phosphodiesterase-4 inhibitor (e.g., tetomilast); HMPL-004; a probiotic; delsalazine; semapimod / CPSI-2364; and a protein kinase C inhibitor (e.g., AEB-071). 135. The polypeptide, pharmaceutical composition, construct or method according to paragraph 134, wherein the polypeptide, pharmaceutical composition or construct is administered sequentially, simultaneously or separately with infliximab, adalimumab, certolizumab pegol or golimumab. 136. A polynucleotide comprising or consisting of a sequence sharing 70% or more, such as 80% or more, for example 90% or more, such as 95% or more, for example 99% or more sequence identity with any one of the portions of SEQ ID NO: 10 encoding CDR1, CDR2, or CDR3 of the encoded immunoglobulin chain variable domain. 137. A polynucleotide encoding a polypeptide or construct according to any one of paragraphs 1 to 135. 138. The polynucleotide of paragraph 137, wherein the polynucleotide comprises or consists of a sequence that shares 70% or more, such as 80% or more, for example 90% or more, such as 95% or more, for example 99% or more sequence identity with SEQ ID NO: 10. 139. The polynucleotide of paragraph 138, wherein the polynucleotide comprises or consists of SEQ ID NO:10. 140. A cDNA comprising the polynucleotide according to any one of paragraphs 136 to 139. 141. A vector comprising the polynucleotide or cDNA according to any one of paragraphs 136 to 140. 142. A host cell transformed with a vector according to paragraph 141 and capable of expressing a polypeptide or construct according to any one of paragraphs 1 to 112. 143. A host cell transformed with the vector of paragraph 142, wherein the host cell is a yeast cell such as S. cerevisiae or P. pastoris. 144. A host cell transformed with a vector according to paragraph 142, wherein the host cell is a bacterial cell such as E. coli. 145. A process for preparing a polypeptide or construct according to any one of clauses 1 to 112, said process comprising: i) cloning the polynucleotide according to any one of items 136 to 139 into a vector such as a plasmid; ii) transforming a cell, such as a bacterial cell or a yeast cell, capable of producing the polypeptide or construct according to any one of items 1 to 112 with said vector under conditions that allow the production of said polypeptide or construct; iii) recovering the polypeptide or construct, such as by affinity chromatography; The process includes:

[0248] Further sections describing further embodiments of the present invention follow. 1. A polypeptide comprising an immunoglobulin chain variable domain that binds to IL-23, wherein said 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. 2. The polypeptide of paragraph 1, wherein CDR1 comprises a sequence that shares 80% or more sequence identity with SEQ ID NO: 1, CDR2 comprises a sequence that shares 80% or more sequence identity with SEQ ID NO: 2, and CDR3 comprises a sequence that shares 80% or more sequence identity with SEQ ID NO: 3. 3. The polypeptide of paragraph 3, wherein CDR1 consists of SEQ ID NO: 1, CDR2 consists of SEQ ID NO: 2, and CDR3 consists of SEQ ID NO: 3. 4. The polypeptide of any one of items 1-3, wherein FR1 comprises a sequence that shares 80% or more sequence identity with SEQ ID NO:4, FR2 comprises a sequence that shares 80% or more sequence identity with SEQ ID NO:5, FR3 comprises a sequence that shares 80% or more sequence identity with SEQ ID NO:6, and FR4 comprises a sequence that shares 80% or more sequence identity with SEQ ID NO:7. 5. The polypeptide according to any one of Items 1 to 4, comprising a sequence that shares 70% or more sequence identity with SEQ ID NO:8. 6. The polypeptide according to any one of items 1 to 5, wherein the polypeptide is an antibody or a fragment thereof. 7. The polypeptide according to item 6, wherein the polypeptide is a VH or VHH. 8. A construct comprising at least one polypeptide according to any one of paragraphs 1 to 7 and at least one different polypeptide, wherein said different polypeptide binds to TNF-α. 9. The construct of paragraph 8, wherein the polypeptides are connected by at least one protease-labile linker. 10. The polypeptide or construct according to any one of paragraphs 1 to 9, wherein the polypeptide or construct neutralizes human IL-23 in an IL-23-IL-23R neutralization ELISA (evaluation method A) with an EC50 of 2 nM or less. 11. The polypeptide or construct of any one of paragraphs 1-10, wherein the polypeptide or construct is substantially resistant to one or more proteases present in the small intestine. 12. The polypeptide or construct of paragraph 11, wherein the proteases are trypsin and chymotrypsin. 13. A polypeptide, pharmaceutical composition, or construct according to any one of paragraphs 1 to 12, for use in the treatment of an autoimmune disease and / or an inflammatory disease. 14. The polypeptide, pharmaceutical composition, or construct according to item 13, wherein the polypeptide, pharmaceutical composition, or construct is administered orally. 15. A polynucleotide encoding the polypeptide or construct according to any one of items 1 to 14. The present invention will now be further illustrated by the following non-limiting examples. [Example]

[0249] Evaluation methods used in the examples Evaluation method A: IL-23-IL-23R neutralization ELISA Maxisorp 96-well plates were coated overnight with 50 μl / well of 0.3 μg / ml IL-23R-Fc and then blocked with 4% milk, 1% BSA. ICVD was serially diluted in 4% milk, 1% BSA and mixed 1:1 with 40 ng / ml recombinant human IL-23. The IL-23-mixed ICVD was then added to the IL-23R-coated plates. Bound IL-23 was detected with BAF219 anti-p40 biotinylated pAb (R&D systems) followed by Extravidin-HRP. This allowed for the calculation of IL-23 neutralizing activity in the samples.

[0250] Evaluation method B: IL-23-IL-23R neutralization ELISA using high salt buffer To measure anti-IL-23 ICVD concentrations in fecal extracts, a high salt IL-23-IL-23R ELISA was used, which was identical to the above ELISA (Assessment Method A) except that the buffer used for dilutions of fecal supernatants and IL-23 preparations was 1% BSA, 4% milk in PBS containing 0.6 M NaCl, 0.05% Tween 20, and protease inhibitors.

[0251] Evaluation method C: Mouse splenocyte assay Splenocytes were isolated from mouse spleens and cultured at 4x10 in medium containing 20ng / mL mouse IL-2 (2x assay concentration). 5Cells were plated in a 96-well round-bottom microplate at 50 μL per well. The top concentration of ICVD was prepared at 300 nM (2x) in medium containing 10 ng / mL THP-1-derived nhIL-23 (2x assay concentration). A 2.6-fold dilution was then made directly in medium containing nhIL-23 (2x). 50 μL of each ICVD mix (2x) was then transferred onto cells (50 μL) to obtain a final 1x assay concentration of ICVD, hIL-23, and mIL-2. For mouse IL-17 stimulation and secretion in the culture supernatant, "mIL-2 only" and "mIL-2 + hIL-23" were used as negative and positive controls, respectively. After 3 days of incubation at 37°C and 5% CO2, the plate was spun at 2000 rpm for 2 minutes, and 50 μL of culture supernatant was collected from each well. An IL-17 binding ELISA was used to measure the levels of mIL-17 in culture supernatants and determine the neutralization of hIL-23.

[0252] Example 1: Immunization, phage library selection, master plate screening, and production of ICVD in E. coli Example 1.1: Exemption and phage library construction 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 of IL-23.

[0253] Peripheral blood mononuclear cells (PBMCs) collected from each llama at the end of each immunization phase were used to generate seven separate phage display libraries. Construction of the ICVD phage display libraries and primary selection of phage expressing ICVD with IL-23 binding activity were performed using standard reagents and protocols. Generally, total RNA was extracted from peripheral blood lymphocytes isolated from each immunized llama. The RNA was then used to generate cDNA, and PCR was performed to specifically amplify the variable region of the ICVD heavy chain-only antibody. cDNA fragments encoding the ICVD repertoire were cloned into a phagemid vector, and the library was introduced into E. coli. Phage libraries were produced by culturing E. coli with helper phage and precipitating the resulting ICVD-displaying phage. The number of each library was determined by titration and infection of log-phase E. coli strain TG1 at various dilutions. Libraries consisted of 6x10 7 ~5x10 8 It was predicted that the ICVD sequence between

[0254] Example 1.2: Library selection for phage with human IL-23 binding activity: growth and generation of periplasmic extracts A phage library selection strategy was designed to enrich for ICVDs that specifically bind to the IL-23p19 subunit. Phages were selected by panning on either hIL-23 or biotinylated hIL-23 in the presence of soluble hIL-12. Bound ICVDs were washed using different methods and removed from the plate using either specific elution with IL-23R or total elution with TEA. Selection and elution conditions were established to isolate ICVDs that exist in the active, soluble form of IL-23 and bind with high affinity to an epitope on p19 that interferes with IL-23 binding to the IL-23 receptor (IL-23R).

[0255] Phage present in the eluate from the selection were used to infect E. coli TG1 cells. Colonies were randomly picked into twelve 96-well master plates and expanded to generate clonal cultures. The bacterial outer cell membrane was lysed by freeze-thawing to release the periplasmic fraction (also referred to herein as "peri"), which contains ICVD. Cell debris was removed by centrifugation, and the supernatant was transferred to a fresh 96-well plate for evaluation of ICVD properties.

[0256] Example 1.3: Screening of periplasmic extracts To identify ICVDs specific for inhibiting IL-23 signaling, periplasmic extracts were tested for their ability to interfere with three interactions: 1) IL-23 binding to IL-23R, 2) IL-23 binding to IL-12Rβ1, and 3) IL-12 binding to IL-12Rβ1. These interactions were tested by individual ELISA.

[0257] Three plate-ELISA formats were developed, in which periplasmic extracts containing ICVDs were mixed with the appropriate cytokine and then the mixture was applied to Maxisorp plates coated with the appropriate cytokine receptor. This allowed for capture of the cytokine without interference with cytokine receptor binding by ICVD, which in all cases could be detected with a biotinylated anti-p40 antibody followed by Extravidin-HRP. The neutralizing activity of each ICVD was measured as its ability to reduce cytokine binding to its receptor compared to an "irrelevant" VHH control. Promising ICVD clones were selected for production in E. coli and further evaluation.

[0258] Example 1.4: Production of selected clones in E. coli The DNA sequences of selected ICVDs were recloned into the vector pMEK222 (thus introducing a C-terminal FLAG and 6xHis tag) for production in E. coli, and ICVD affinities were purified on Talon resin via the 6xHis tag for more detailed characterization. ICVDs that could not be successfully expressed from E. coli were excluded from further analysis.

[0259] Example 2: Potency of purified primary clones To assess potency against human IL-23, the IL-23-IL-23R neutralizing activity of purified ICVDs was assessed by IL-23-IL-23R neutralization ELISA (Assessment Method A). ICVDs found to have higher potency than the comparative monoclonal antibody brazikumab (heavy chain SEQ ID NO: 70, light chain SEQ ID NO: 71) are shown in the table below.

[0260] [Table 3]

[0261] ELISA results showed that the family L clones (12G1 and 1E2) were significantly more potent than the comparative anti-IL-23 antibody brazikumab in the IL-23-IL-23R ELISA, and even demonstrated subnanomolar potency. Clones 10E2 and 10G10 also demonstrated greater potency in this assay than the brazikumab comparative.

[0262] 10E2, 12G1, and 1E2 were also tested in a cynomolgus monkey IL-23-IL-23R neutralization ELISA, and all demonstrated high neutralization potency in this assay (data not shown), similar to that demonstrated in the standard IL-23-IL-23R ELISA. It was also demonstrated that these clones were unable to neutralize the interaction of IL-23 with IL-12R or the interaction of IL-12 with IL-12R. Without wishing to be bound by theory, the inventors believe that these clones are specific for the p19 subunit of IL-23.

[0263] Example 3: Stability of endogenous proteases of purified primary clones Orally administered ICVD is likely subject to proteolytic digestion during passage through the small and large intestine. To further investigate its resistance to intestinal proteases, we tested the potency (retention) of purified ICVD after incubation in the presence of both mouse small intestinal supernatant and supernatant prepared from pooled human fecal samples.

[0264] Mouse small intestinal supernatant: Small intestinal contents from seven C57BL / 6 male mice were removed in 0.9% saline, combined, homogenized, and centrifuged. The resulting supernatant was removed, aliquoted, and frozen.

[0265] Human fecal supernatant: Fecal samples from five humans were slurried 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.

[0266] After incubating the ICVDs at 37°C for various periods of time, the anti-IL-23 activity of the "digested" ICVD samples was analyzed using the IL-23-IL-23R neutralization ELISA (Assessment Method B), and the percentage of activity retained from "undigested" to "digested" ICVD was calculated. The results are shown in the table below.

[0267] [Table 4]

[0268] ICVDs demonstrated substantial intrinsic stability against proteases present in mouse small intestinal material and human fecal material. Family L ICVD 12G1 was selected for further optimization based on its potency and intrinsic protease resistance.

[0269] Example 4: Production and characterization of optimized variants of 12G1 Family L ICVD 12G1 was subjected to sequence modifications to generate multiple mutant ICVDs. These mutant ICVDs were tested in the potency and protease stability assays detailed above. The mutant ICVDs, their mutation to ID-L253T, and their performance in these assays are detailed in the table below (where EC50 values ​​are given in nM, "mouse splenocytes" refers to data obtained using evaluation method C above, and residues are numbered using N- to C-terminal numbering, as opposed to Kabat numbering).

[0270] It was observed that high potency and protease stability were generally maintained in the mutant ICVDs, and therefore it can be expected that mutants of ID-L253T will generally substantially maintain high potency and protease stability.

[0271] Of these mutant ICVDs, ID-L253T was ultimately selected and carried forward for further characterization. 12G1 differs 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 from N-terminus to C-terminus, as opposed to Kabat numbering).

[0272] [Table 5]

[0273] Example 5: Potency, Binding Affinity, and Protease Stability of ID-L253T Compared to Comparative Examples Brazikumab and 37D5 Brazikumab is a prior art fully human IgG2 monoclonal antibody that selectively binds to the p19 subunit of IL-23. 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 potency, binding affinity, and protease stability of ID-L253T were evaluated in comparison to these prior art anti-IL-23 agents.

[0274] Example 5.1: Efficacy of ID-L253T compared to brazikumab ID-L253T and the clinical comparator brazikumab were comparatively tested for efficacy in the IL-23-IL-23R inhibition ELISA (Assessment Method A), and the results are summarized in the table below.

[0275] [Table 6]

[0276] As expected based on the potency of the precursor ICVD 12G1 and the variants of ID-L253T described above, ID-L253T showed greater potency than brazikumab in the IL-23-IL-23R ELISA.

[0277] Example 5.2: Affinity of ID-L253T compared to brazikumab Fab The binding kinetics of ID-L253T was compared to brazikumab Fab (prepared using a commercially available Fab preparation kit) and evaluated in Biacore experiments. Anti-p40 mAb was immobilized on a Biacore sensor plate and loaded with recombinant human IL-23. Tethering of IL-23 by its p40 subunit allows the p19 subunit (specific for both brazikumab and ID-L253T) to interact with the anti-IL-23 ID-253T ICVD and brazikumab FAb fragments, which are then flowed over the chip to detect binding. ID-L253T exhibited an average K of 32 pM. D whereas the brazikumab Fab preparation has a much higher average K of 1200 pM.D These data are summarized in the table below.

[0278] [Table 7]

[0279] Without wishing to be bound by theory, based on the data provided above, the inventors predict that ID-L253T binds to the p19 subunit of IL-23.

[0280] Example 5.3: Protease Stability of ID-L253T Compared to 37D5 The stability of ID-L253T in mouse small intestinal material was tested and compared to that of comparative example 37D5. The anti-IL-23 activity of the "digested" ICVD samples was analyzed using the IL-23-IL-23R neutral ELISA (Assessment Method B), and the percentage of activity retained from "undigested" to "digested" ICVD was calculated. These data are summarized in the table below.

[0281] [Table 8]

[0282] These data reveal that for 37D5, no ELISA signal was established after 2 hours of digestion with mouse small intestine material, whereas ID-L253T achieved 51% viability after twice the incubation period. Thus, ID-L253T is significantly more stable in this digestion matrix than comparative example 37D5. Note that the data provided here for ID-L253T correspond to the same assay detailed above in Example 4, while the data provided here for 37D5 were generated in a separate assay on a different occasion.

[0283] Example 6: ID-L253T specificity for human IL-23 Example 6.1: Cross-reactivity with IL-23 from toxicological species A cynomolgus IL-23-IL-23R neutralization ELISA was performed. To do so, Maxisorp 96-well plates were coated overnight with 50 μl / well of 0.5 μg / ml cynomolgus IL-23R-Fc and then blocked with 4% milk, 1% BSA. ICVD was serially diluted in 4% milk, 1% BSA and mixed 1:1 with 40 ng / ml recombinant cynomolgus IL-23 (cIL-23). ​​The mixture was then incubated for 30 minutes to allow binding before being added to the cIL-23R-coated plates. Bound cIL-23 was detected with BAF219 anti-p40 pAb followed by Extravidin-HRP to determine the level of ICVD neutralization of cIL-23 binding to cIL-23R.

[0284] ID-L253T was active in the cynomolgus monkey IL-23-IL-23R neutralization assay (data not shown), making the cynomolgus monkey a suitable toxicology species for any preclinical development studies.

[0285] Example 6.2: Specificity for non-target cytokines ID-L253T was tested for selectivity for IL-23-related or unrelated cytokines in either binding or inhibition ELISAs. IL-12 shares the p40 subunit with IL-23 and is therefore the most closely related cytokine in humans. Human and rhesus IL-12, along with an additional member of the IL-12 cytokine family (IL-27), were tested in this assay. Important but unrelated additional proinflammatory cytokines, TNFα, IL-6, and IFNγ, were also tested. ID-L253T showed no interaction with the cytokines tested, indicating that binding of ID-L253T to off-target molecules is highly unlikely in humans and nonhuman primates.

[0286] Example 7: Further protease stability studies on ID-L253T - resistance to gastrointestinal matrix metalloproteinases and mouse gastrointestinal transit Example 7.1: Resistance to gastrointestinal matrix metalloproteinases Levels of activated matrix metalloproteinases (MMPs) are elevated in the inflamed mucosa of patients with inflammatory bowel disease. These MMPs can digest native human IgG and therapeutic agents containing a human IgG scaffold (Biancheri et al., 2015). In the case of the anti-TNFα treatment etanercept, this digestion results in a significant decrease in TNFα neutralization efficacy. To confirm that ID-L253T is resistant 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, whereas etanercept was degraded by the same enzyme preparation.

[0287] ID-L253T is completely resistant to digestion by MMP3 and MMP12 for approximately 20 hours at 37°C. This indicates that ID-L253T should retain high stability in the inflamed environment of the IBD intestine when MMP levels are elevated. This finding, together with the above findings regarding general protease resistance, suggests that the polypeptides of the present invention have great potential as oral treatments for IBD.

[0288] Example 7.2: Mouse Gastrointestinal Transit and Survival The results of the in vitro studies described above demonstrated that ID-L253T is resistant to inactivation by proteases present in supernatant extracts prepared from mouse small intestinal contents. In the authors' experience, mouse small intestinal supernatants are significantly more proteolytically active than small intestinal supernatants obtained from humans or pigs. Studies were conducted to investigate the stability of ID-L253T during transit through the mouse gastrointestinal system. ID-L253T was formulated with the anti-TNF-α ICVD ID-38F (see WO2016156465, SEQ ID NO: 8, and Example 8) in a mixture of milk and bicarbonate. ID-38F has previously been established to be stable during transit through the mouse gastrointestinal tract and therefore serves as a positive control here. Four mice were co-administered with ICVD by oral gavage, and the concentrations of ICVD were measured in fecal pellets collected between 0 and 3 hours and between 3 and 6 hours.

[0289] ID-L253T was detected at levels comparable to those of ID-38F in the same mice at both 0-3 and 3-6 hours post-dose. Results showed that ID-L253T survived as well as or better than ID-38F in mice, reaching a maximum recovery concentration of >15 μM in the supernatant of fecal pellets collected 3-6 hours post-dose.

[0290] Example 8: Evaluation of ID-L210T neutralizing activity in human IBD tissues The IL-23 neutralizing activity of ID-L210T, a variant of ID-L253T lacking only the E1D, R19S, and R92V mutations, was investigated in ex vivo cultures of inflamed colonic mucosal tissue using the assay system described by Vossenkamper et al. (2014) and Crowe et al. (2018).

[0291] Using this model, we tested the inhibitory effect of ID-L210T on elevated levels of signaling phosphoproteins present in IBD disease tissue under pathophysiological conditions. Biopsy tissue samples from four patients with active UC were used. After 24 hours of incubation with either ID-L210T (at a concentration of 150 nM) or an isotype control ICVD (ID-2A, an unrelated anti-C. difficile toxin ICVD, at a concentration of 225 nM) that does not bind IL-23, biopsy explants were analyzed for phosphoprotein levels using proteome profiler human phosphokinase array technology.

[0292] The mean phosphorintensity values ​​across all patients for the various proteins tested are shown in Figures 1-2. The phosphorylation levels of most proteins on the array decreased after treatment with ID-L210T.

[0293] The total protein phosphorylation signal detected for each biopsy (Σ n=39 The effect of treatment on total phosphorylation levels (phosphoproteins) was also assessed. After treatment with ID-L210T, total phosphorylation levels measured in biopsies from all UC patients were inhibited by (20-52%; mean 32%, n=4).

[0294] ID-L253T differs from ID-L210T by only three amino acids and exhibits similar IL-23-IL-23R inhibitory potency, resistance to in vitro digestion, and passage in mice. Therefore, it is expected that these findings regarding ID-L210T will be substantially applicable to ID-L253T.

[0295] Example 9: Yeast productivity in fermentation cultures A 5-liter fermentation was inoculated with an S. cerevisiae production strain expressing ID-L253T. Production levels were assessed by SDS-PAGE with Coomassie staining, and biological activity was assessed by IL-23-IL-23R neutralization ELISA. Results showed a clean band at the appropriate molecular weight in the end-of-fermentation (EoF) supernatant with very few contaminants. The yield of ID-L253T was determined to be 0.188 g / L.

[0296] ID-L253T was also tested for production in P. pastoris using standard yeast fermentation techniques. Production levels were assessed by SDS-PAGE with Coomassie staining, which showed a clean band in the EoF supernatant at the appropriate molecular weight. The yield of ID-L253T from this fermentation was determined to be 1.3 g / L. Full neutralizing activity was demonstrated by IL-23-IL-23R neutralizing ELISA.

[0297] Example 10: Production and characterization of the heterobihead anti-IL-23 / anti-TNF-α construct FA1K Example 10.1: Production of FA1K A heterobihead construct (WO2016156465, SEQ ID NO: 8 therein, and SEQ ID NO: 67 in this disclosure) containing anti-IL-23 ICVD ID-L253T and anti-TNF-α ICVD ID-38F was generated (referred to herein as "FA1K"). The ICVDs in this construct were separated by a flexible, non-immunogenic (G4S)4 linker with a central lysine residue (SEQ ID NO: 49), creating a trypsin cleavage site of the type disclosed in WO2016156466 (ID-38F-(G4S)2K-(G4S)2-ID-L253T). The polypeptide sequence of the ID-38F group used in FA1K was identical to that of the ID-38F group used above (SEQ ID NO: 47), and the polypeptide sequence of the ID-L253T group used in FA1K was identical to that used above for ID-L253T, except for the D1E substitution (SEQ ID NO: 48).

[0298] FA1K was cloned on a SacI / HindIII fragment into vector pUR9013 and transformed into S. cerevisiae vwkgal1 - This facilitates stable multicopy integration into the chromosome of the expression strain. Using this integration and expression system, FA1K was under the control of a galactose-inducible promoter in S. cerevisiae, and bihead secretion was achieved via the yeast mating factor alpha signal sequence. In addition, FA1K with the same signal sequence was cloned into the chromosome of P. pastoris under the control of the methanol-inducible pAOX1 promoter. Expression of FA1K from S. cerevisiae was assessed in 50 mL induced cultures.

[0299] The supernatant from these shake flasks was purified, and this purified preparation of FA1K was then used in the experiments described below unless otherwise stated.

[0300] FA1K was cloned into P. pastoris and showed excellent expression at a 50 mL scale.

[0301] Example 10.2: In vitro characterization of FA1K Incubation of FA1K with trypsin at 37°C resulted in rapid separation of the ID-L253T and ID-38F monomer populations. Thus, FA1K is appropriately formatted for rapid release of both monomer populations upon exposure to trypsin in the human small intestine.

[0302] The neutralizing efficacy of FA1K against TNFα (before and after trypsin cleavage) was examined (see Figure 3, where "FA1K" refers to FA1K before trypsin treatment and "FA1K trypsin" refers to FA1K after trypsin treatment).

[0303] A biotinylated humira (adalimumab) competition ELISA was performed to measure competition for an epitope on TNF-α. If competition occurs, the epitope will be occupied by unlabeled polypeptide, resulting in fewer biotinylated humira binding to TNFα, resulting in a reduced assay signal. ELISA plates were coated with 100 ng / mL human TNFα in 250 μg / mL bovine serum albumin (BSA) in phosphate-buffered saline (1x PBS) and blocked with 1% BSA in 1x PBS. 1% BSA and 0.1 mM PMSF were used as the assay diluent for all experiments involving polypeptides with pre-trypsin treatment. Otherwise, 1% BSA was used as the assay diluent. Biotinylated adalimumab (LGC) was mixed 1:1 with all standards and samples to obtain a final concentration of 2 nM biotinylated adalimumab, and the mixture was then added to the plate. Bound biotinylated adalimumab was detected using ExtrAvidin horseradish peroxidase (Sigma E2886), visualized using TMB Microwell Substrate (KPL 50-76-00), stopped with 0.5 M H2SO4, and read at 450 nm in FA1K (pre-trypsin and post-trypsin) and found to be virtually as potent as ID-38F in this competitive ELISA.

[0304] The neutralization potency of FA1K (before and after trypsin cleavage) against IL-23 was examined (see Figure 4, where "FA1K" refers to FA1K before trypsin treatment and "FA1K trypsin" refers to FA1K after trypsin treatment). FA1K (before and after trypsin treatment) was at least as potent against IL-23 as ID-L253T.

[0305] The inclusion of ID-L253T at the C-terminal position of FA1K also appears to confer an advantage for IL-23 neutralization, as the ID-L253T population has an N-terminal extension compared to the free ID-L253T monomer.

[0306] FA1K produced and purified from expression in P. pastoris was also confirmed by ELISA to be fully potent against both TNF-α and IL-23 (data not shown). Both FA1K monomers were shown to retain the favorable stability properties of the parent monomers ID-38F and ID-L253T after 4 hours of incubation in human fecal supernatant (see Figure 5, which shows, from left to right, the % survival of (a) ID-38F, (b) the FA1K monomer form of ID-38F, (c) ID-L253T, and (d) the FA1K monomer form of ID-L253T).

[0307] Taken together, these findings demonstrate that FA1K is a suitable format for delivering high concentrations of each of these monomers as a dual therapy. FA1K retains the favorable potency and protease stability characteristics of the parent monomers. Due to the labile linker format, exposure of FA1K to trypsin results in the rapid release of both monomer groups, allowing them to bind to their respective targets independently without interference from the other groups.

[0308] Example 11: Ex vivo characterization of combined administration of anti-IL-23 (ID-L210T) and anti-TNF-α (ID-38F) to human IBD tissue It has been shown above and in other publications that ICVDs with neutralizing activity for TNF-α (ID-38F) and IL-23 (ID-L210T) can suppress the phosphorylation of tyrosine 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).

[0309] New studies were conducted to examine the combined effects of L210T and ID-38F on phosphoprotein biomarker levels 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 displays similar IL-23-IL-23R inhibitory potency and resistance to in vitro digestion.

[0310] To assess what effects could be achieved by combining different anti-cytokine mechanisms, the effects of individual ICVDs were compared with a mixture of two ICVDs and an isotype control ICVD (ID-2A, an unrelated anti- C. difficile toxin ICVD).

[0311] Biopsies from each of four UC patients were incubated with various antibodies (control ID-2A, ID-38F, ID-L210T, or ID-38F + ID-L210T) for 24 hours. After treatment, tissue lysates were analyzed on phosphoprotein antibody arrays. Mean signal intensity data per patient for 45 phosphoproteins detected on individual arrays (four arrays per treatment) are presented in Figures 6-7 (in the figures, L210T refers to ID-L210T). The inhibitory effects of the various antibody treatments are demonstrated by a shift from predominantly high levels of phosphorylation in biopsies treated with control ICVD ID2A to relatively low phosphointensity values ​​in biopsies treated with anti-TNF-α or anti-IL-23 ICVD, or a combination of the two.

[0312] The data provided herein demonstrate that a therapeutic approach combining GI restricted antagonism of TNF-α and IL-23 can achieve a greater degree of efficacy, over a longer duration, in a higher proportion of inflammatory bowel disease patients compared to monotherapy directed against either target alone.

[0313] Miscellaneous All documents referred to in this application, including patents and patent applications, are incorporated herein by reference to the fullest extent possible.

[0314] Throughout this specification, unless the context requires otherwise, it will be appreciated that the word "comprises" and variations such as "comprises" and "comprising" imply the inclusion of a stated integer, step, group of integers, or group of steps, but do not exclude other integers, steps, groups of integers, or groups of steps.

[0315] This specification and claims of the application of which it forms a part may be used as a basis for priority in respect of any subsequent application. The claims of such subsequent application may be directed to any feature or combination of features described herein. They may take the form of product, composition, process, or use claims and may include, by way of example and without limitation, the following claims:

[0316] References The following references are incorporated herein by reference in their entirety: ·Arbabi-Ghahroudi et al FEBS Lett 1997 414:521-526 ·Biancheri et al 2015 Gastroenterology 149(6):1564-1574 ·Blattler et al Biochemistry 1985 24:1517-1524 ·Chomezynnski and Sacchi Anal Biochem 1987 162:156-159 ·Colombel et al Gastroenterology 2007132:52-65 ·Crowe et al 2018 Sci.Rep. 8:1-13 ·Croxford et al Eur J Immunol.2012 42:2263-2273 ·Desmyter et al. Front Immunol 2017 8(884):1-10 ·Eken et al Inflamm Bowel Dis. 2014 20:587-595 ·Faisst et al J Virol 1995 69:4538-4543 ·Frenken et al J Biotech 2000 78:11-21 ·Furfaro et al Expert Rev Clin Immunol.2017 13:1-11 ·Green and Sambrook Molecular Cloning: A Laboratory Manual 2012 4th Edition Cold Spring Harbor Laboratory Press ·Griffiths et al Antibodies 2013 2:66-81 ·Grundstrom et al Nucl. Acids Res 1985 13:3305-3316 ·Hamers-Casterman et al Nature 1993 363(6428):446-448 ·Hanauer et al Lancet 2002 359:1541-1549 ·Hanauer et al Gastroenterology 2006 130:323-333 ·Harmsen et al Gene 1993 125:115-123 ·Harmsen et al Appl Microbiol Biotechnol 2007 77(1):13-22) ·Hendrickson et al Clin Microbiol Rev 2002 15(1):79-94 ·Hoogenboom et al Nucl Acid Res 1991 19:4133-4137 ·Huse et al Science 1989 246 (4935):1275-1281 ·Kabat et al Sequences of Proteins of Immunological Interest,Sequences of Proteins of Immunological Interest, Fifth Edition U.S. Department of Health and Human Services, 1991 NIH Publication Number 91-3242 ·Knezevic et al 2012 Journal of the American Chemical Society 134(37):15225-15228 ·Kohler and Milstein Nature 1975 256:495-497 ·Ling et al Anal Biochem 1997 254(2):157-178 ·McCoy et al Retrovirology 2014 11:83 ·McGovern and Powrie Gut 2007 56:1333-1336 ·Merchlinsky et al J. Virol.1983 47:227-232 ·Miethe et al J Biotech 2013 163(2):105-111 ·Muyldermans et al Protein Eng 1994 7(9):1129-1135 ·Muyldermans Annu Rev Biochem 2013 82:775-797 ·Nambiar et al Science 1984 223:1299-1301 ·Nelson et al Molecular Pathology 2000 53(3):111-117 ·Nguyen et al Adv Immunol 2001 79:261-296 ·Ortonne, Brit J Dermatol 1999 140(suppl 54):1-7 ·Padlan Mol Immunol 1994 31:169-217 ·Roux et al Proc Natl Acad Sci USA 1998 95:11804-11809 ·Sandborn et al N Engl J Med.2007 357:228-238 ·Sakamar and Khorana Nucl.Acids Res 1988 14:6361-6372 ·Schreiber et al N Engl J Med.2007 357:239-250 ·Skerra et al Science 1988 240(4855):1038-1041 ·Tanha et al J Immunol Methods 2002 263:97-109 ·Teng et al Nat Med. 2015 21:719-729 ·Thomassen et al Enzyme and Micro Tech 2002 30:273-278 ·Verma and Eckstein Annu Rev Biochem 1998 67:99-134 ·Vossenkamper et al 2014 Gastroenterology 147(1):172-183 ·Ward et al Nature 1989 341:544-546 ·Wells et al Gene 1985 34:315-323

Claims

1. A polypeptide comprising an immunoglobulin chain variable domain that binds to IL-23, 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.

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

1.

3. 3. The polypeptide of claim 1 or 2, wherein CDR2 comprises or consists of a sequence that shares 80% or more sequence identity with SEQ ID NO:

2.

4. The polypeptide of any one of claims 1 to 3, wherein the CDR3 comprises or consists of a sequence that shares 80% or more sequence identity with SEQ ID NO:

3.

5. 2. The polypeptide of 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. 6. The polypeptide of any one of claims 1 to 5, comprising or consisting of a sequence which shares 50% or more sequence identity with SEQ ID NO:8, such as sharing 55% or more sequence identity, for example sharing 60% or more sequence identity, such as sharing 65% or more sequence identity, for example sharing 70% or more sequence identity, such as sharing 75% or more sequence identity, for example sharing 80% or more sequence identity, such as sharing 85% or more sequence identity, for example sharing 90% or more sequence identity, such as sharing 95% or more sequence identity, for example sharing 96% or more sequence identity, such as sharing 97% or more sequence identity, for example sharing 98% or more sequence identity, such as sharing 99% or more sequence identity.

7. The polypeptide of claim 6 comprising SEQ ID NO:

8.

8. The polypeptide of claim 7 consisting of SEQ ID NO:

8.

9. The polypeptide of any one of claims 1 to 8, wherein the polypeptide is an antibody or an antibody fragment.

10. The polypeptide of any one of claims 1 to 9, wherein the polypeptide consists of an immunoglobulin chain variable domain.

11. The polypeptide of any one of claims 1 to 10, wherein the immunoglobulin chain variable domain is VHH, VH, or VL.

12. The polypeptide of claim 11, wherein the immunoglobulin chain variable domain is a VHH or VH.

13. The polypeptide of claim 12, wherein the immunoglobulin chain variable domain is a VHH.

14. A construct comprising at least one polypeptide according to any one of claims 1 to 13 and at least one different polypeptide, said different polypeptide binding to TNF-α.

15. The construct of claim 14 , wherein the different polypeptide comprises a sequence that shares at least 80% sequence identity with SEQ ID NO:

67.

16. 16. The construct of claim 14 or 15, wherein the polypeptides are connected by at least one protease-labile linker comprising or consisting of SEQ ID NO:

74.

17. 17. The construct of claim 16, wherein the construct comprises or consists of SEQ ID NO:

46.

18. 5 nM or less, for example, 4 nM or less, for example, 3 nM or less, for example, 2 nM or less, for example, 1.7 nM or less, for example, 1.5 nM or less, for example, 1.4 nM or less, for example, 1.3 nM or less, for example, 1.2 nM or less, for example, 1.1 nM or less, for example, 1.0 nM or less, for example, 0.9 nM or less, for example, 0.8 nM or less, for example, 0.75 nM or less, for example, 0.70 nM or less, for example, 0.65 nM 18. The polypeptide or construct of any one of claims 1 to 17, which neutralizes human IL-23 in evaluation method A with an EC50 of 0.5 nM or less, for example 0.60 nM or less, for example 0.55 nM or less, for example 0.50 nM or less, for example 0.45 nM or less, for example 0.40 nM or less, for example 0.35 nM or less, for example 0.30 nM or less, for example 0.25 nM or less, for example 0.20 nM or less.

19. The polypeptide or construct may be －６ M or less, for example, 10 －７ M or less, for example, 10 －８ M or less, for example, 10 －９ M or less, for example, 10 －１０ M or less, for example, 10 －１１ M or less, for example, 10 －１２ M or less, for example, 10 －１３ 19. A polypeptide or construct according to any one of claims 1 to 18, which binds to IL-23 with a Kd of M or less.

20. 20. The polypeptide or construct of any one of claims 1-19, wherein the polypeptide is substantially resistant to trypsin and chymotrypsin.

21. A pharmaceutical composition comprising a polypeptide or construct according to any one of claims 1 to 20 and one or more pharmaceutically acceptable excipients or carriers.

22. 22. The pharmaceutical composition of claim 21, comprising at least one additional active agent.

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

24. 24. A polypeptide, pharmaceutical composition or construct according to claim 23 for use in the treatment of an autoimmune and / or inflammatory disease.

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

26. Use of a polypeptide, pharmaceutical composition or construct according to any one of claims 1 to 22 in the manufacture of a medicament for the treatment of autoimmune and / or inflammatory diseases.

27. The polypeptide, pharmaceutical composition, or construct, method, or use according to any one of claims 23 to 26, wherein the polypeptide, pharmaceutical composition, or construct is administered orally.

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

29. A polynucleotide encoding a polypeptide or construct according to any one of claims 1 to 20.

30. 30. The polynucleotide of claim 29, wherein the polynucleotide comprises or consists of a sequence sharing 70% or more, such as 80% or more, for example 90% or more, such as 95% or more, for example 99% or more sequence identity with SEQ ID NO:

10.

31. 31. The polynucleotide of claim 30, wherein the polynucleotide comprises or consists of SEQ ID NO:10.

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