Peptides

Abstract

The present invention particularly provides polypeptides capable of inhibiting the binding of IL-7 and / or L-TSLP to IL-7R (IL-7R), as well as constructs and pharmaceutical compositions comprising these polypeptides.

Description

Technical Field

[0001] The present invention relates to polypeptides capable of inhibiting the binding of IL-7 and / or L-TSLP to IL-7R, as well as constructs and pharmaceutical compositions comprising these polypeptides. The present invention also relates to nucleic acids encoding such polypeptides, methods for preparing such polypeptides, cDNAs and vectors comprising nucleic acids encoding such polypeptides, host cells expressing or capable of expressing such polypeptides, and uses of such polypeptides. Background Art

[0002] Autoimmune diseases of the gastrointestinal tract

[0003] Autoimmune diseases of the gastrointestinal tract include inflammatory bowel diseases such as Crohn's disease (CD) and other autoimmune diseases such as eosinophilic esophagitis (EoE). Crohn's disease (also known as Crohn's syndrome) and regional enteritis cause a variety of symptoms. It mainly causes abdominal pain, diarrhea, vomiting and / or weight loss, but also causes complications outside the gastrointestinal tract (GIT), such as anemia, rash, arthritis, eye inflammation, fatigue and lack of concentration (Baumgart et al. 2012). Crohn's disease is currently an incurable lifelong gastrointestinal disease that is difficult to control with conventional therapy. EoE is a chronic disease defined by significant pathological eosinophil infiltration confined to the esophagus, which causes esophageal dysfunction and fibrosis if not treated. Esophageal fibrosis and esophageal stenosis are known EoE complications.

[0004] Antibody-based therapeutic agents have significant potential as effective treatments for autoimmune diseases such as IBD and EoE because they have high specificity and low inherent toxicity for their targets. Three anti-TNF-α antibodies, infliximab (trade name Remicade), adalimumab (trade name Humira) and certolizumab pegol (or 'certolizumab pegol', trade name Cimzia) are used clinically to treat Crohn's disease; however, these antibodies are generally considered unsuitable for oral therapeutic administration due to their inherent instability and susceptibility to proteolytic degradation by inflammatory proteases and intestinal microbial flora present at pathological sites in the digestive system and intestines. Therefore, these agents must be administered by intravenous infusion or subcutaneous injection, which requires professional training in order to correctly and safely use a subcutaneous syringe or needle. These agents also require sterile equipment, liquid formulations of therapeutic polypeptides, vial packaging of the polypeptide in a sterile and stable form, and a suitable site on the subject for inserting a needle. Subjects often experience psychological stress before receiving the injection and experience pain while receiving the injection. Long-term treatment with these systemic anti-TNF-α antibodies has an increased risk of serious infection and cancer. Combined with high production costs, these factors currently limit the use of these agents for patients with more serious diseases.

[0005] Several small molecule anti-inflammatory drugs and immunosuppressive drugs are also currently used for clinical development of Crohn's disease (Danese 2012, Shealy et al. 2010 and Vetter & Neurath 2017). Although these drugs are administered orally, many will be absorbed systemically after administration, and therefore may have systemic immunosuppressive effects that are unrelated to the effects on gastrointestinal lesions. In addition, because small molecules lack the specificity of antibodies, significant off-target side effects are maintained at high risk.

[0006] The ability to deliver oral therapeutics that are highly selective for targets associated with autoimmune diseases of the gastrointestinal tract but have exposure and activity restricted to the gastrointestinal tract, combined with significant safety improvements due to reduced systemic exposure, could provide efficacy similar to that of injectable antibodies.

[0007] Interleukin-7 (IL-7) and interleukin-7 receptor (IL-7R)

[0008] Interleukin-7 (IL-7) is a member of the cytokine family including IL-2, IL-4, IL-7, IL-15 and IL-21. IL-7 is constitutively produced by non-hematopoietic stromal cells and epithelial cells in lymphoid organs, intestines, skin and liver, and is necessary for the development of T lymphocytes in the thymus and for the survival and homeostasis regulation of peripheral blood T cells (Fry and Mackall, 2002; Fry and Mackall 2005). In the intestinal mucosa, IL-7 also regulates the phenotypically and functionally different populations of innate lymphoid cells, which are important for the initial initiation of immune responses to pathological microorganisms, and also regulate CD4+ lymphoid tissue induction (LTi) cells, which can promote lymphoid tissue organogenesis and some dendritic cell populations (Goldberg et al. 2015; Peters et al. 2015). In addition, IL-7 induces the proliferation of naive and memory T cells and enhances effector T cell responses, preferably helper T cell 1 (Th1) and Th17 responses (Dooms, 2013). The functional effects of IL-7 on T cells make IL-7 a key enhancer of protective immunity as well as autoimmunity and inflammation.

[0009] The effect of IL-7 on different target cells is mediated by IL-7R (a heterodimeric complex comprising IL-7Rα subunits (CD127) and common cytokine receptor γ chain (γc) (CD132). Full-length human IL-7Rα consists of an extracellular domain of 219 residues, a transmembrane domain of 25 residues, and an intracellular domain of 195 residues. It is believed that IL-7-induced receptor activation involves the initial interaction of IL-7 with IL-7Rα to form a complex, which then recruits γc to form an activated receptor signaling complex. The association of the two receptor subunits performed by IL-7 activates intracellular phosphorylation events through JAK / Stat, PI3 / Akt, and SRC signaling cascades (Walsh, 2012).

[0010] IL-7Rα exists not only in a cell membrane-bound form, but also as a soluble form (sIL-7Rα). sIL-7R is generated by shedding of membrane-bound receptors and also by alternative splicing (of IL-7Rα exon 6) that results in a protein lacking a transmembrane domain; sIL-7Rα present in human plasma is primarily derived from alternative splicing (Rose et al. 2009). Four common haplotypes of IL-7Rα have been identified (Teutsch et al. 2003). Two haplotypes are associated with altered expression and production of soluble forms of the receptor and susceptibility to autoimmune diseases including multiple sclerosis and type 1 diabetes (Hafler et al. 2007).

[0011] In addition to the role played by IL-7 / IL-7Rα in human T cell development and homeostasis, preclinical studies have demonstrated the involvement of the IL-7 / IL-7Rα pathway in animal models of various autoimmune and inflammatory diseases. These studies have identified additional IL-7-dependent mechanisms associated with disease pathology and have highlighted the IL-7 / IL-7Rα interaction as a possible target for the treatment of patients with related autoimmune and chronic inflammatory conditions.

[0012] Preclinical studies have demonstrated that short-term systemic administration of IL-7Rα blocking antibodies provides effective treatment in models of autoimmune disease and gastrointestinal inflammation. In the IBD model, the major mechanism of efficacy following IL-7R antagonist treatment appears to involve localized loss of functional inhibition of colitisogenic T cells (IL-7R+ effector / memory T cells), which express moderate to high levels of IL-7Rα (CD127) and can be activated in the inflamed intestine due to increased IL-7 production by stromal and epithelial cells. In the healthy mucosa, the gut-resident FOXP3, which is expected to express very low levels of IL-7R, is expressed in the gut. + CD25 + Regulatory T cells (Treg cells) suppress dysregulated CD4+ T responses to commensal bacteria. However, Heninger et al. 2012 showed that in an IL-7-rich environment, FOXP3 + CD25 + The ability of Tregs to suppress conventional T cell proliferation is abolished. In gastrointestinal diseases, blockade of IL-7 / IL-7R signaling can therefore help control T cell-mediated inflammation both by inhibiting the activation of effector T cells and by restoring the suppressive function of regulatory T cells. Evidence from murine IBD models and ex vivo human tissue studies also suggests that IL-7 / IL-7Rα-dependent activation of innate immune cells, including innate lymphoid cells (ILCs), contributes to processes involved in gastrointestinal inflammatory diseases.

[0013] Thymic stromal lymphopoietin (TSLP) and IL-7R

[0014] TSLP is a cytokine produced by epithelial cells in the skin, lungs, intestines and eye tissues, which appears to be involved in regulating the inflammatory process at the body's mucosal surfaces. TSLP stimulates dendritic cells (DCs) and innate lymphoid cells (ILCs) to induce the secretion of Th2 cytokines (IL-4, IL-5 and IL-13) and promote the development of Th2-type inflammation. TSLP is now considered to be the cause of the occurrence of some allergic conditions including atopic dermatitis and rhinitis, and also promotes intestinal conditions, including eosinophilic esophagitis (EoE) and ulcerative colitis (UC). Paradoxically, TSLP is also reported to be important for maintaining immune homeostasis and mucosal protection in the gastrointestinal tract. Recently, the discovery that TSLP can be expressed as two different isoforms provides a biological explanation for the significantly different activities of this cytokine (Fornasa et al. 2015; Tsilingiri et al. 2017). Molecular studies have also shown that the TSLP gene can produce two coding RNAs regulated by two different promoter regions. One of the transcripts encodes a long isoform of TSLP of 159 aa (L-TSLP) (UNIPROT entry Q969D9, SEQ ID NO: 62), and the second transcript encodes a short form of TSLP (S-TSLP) (UNIPROT entry Q969D9-2, SEQ ID NO: 63) that contains the C-terminal 63 aa of L-TSLP. L-TSLP acts on target cells via a receptor complex that includes a TSLP-specific receptor chain (TSLPR) and the IL-7Rα chain. Recently, structural studies have shown that the interaction of IL-7 and L-TSLP with the IL-7Rα chain of the TSLP-receptor complex involves a common IL-7Rα binding site (Verstraete et al. 2017).

[0015] S-TSLP does not bind to TSLPR and it cannot inhibit the binding of L-TSLP to this receptor. To the best of the authors' knowledge, no specific receptor for S-TSLP has been identified so far.

[0016] Importantly, it has been shown that S-TSLP is preferentially expressed by healthy skin and expressed by epithelial cells and lamina propria cells in healthy intestinal mucosal tissue. S-TSLP has anti-inflammatory activity; in vitro S-TSLP inhibits the production of proinflammatory cytokines by monocyte-derived DCs, and helps to regulate CD103+DCs to a tolerant phenotype. Therefore, it seems that S-TSLP produced by the intestinal epithelium can affect the following immune cells, including dendritic cells and lymphocytes, and is conducive to tolerance and regulatory responses in healthy people. S-TSLP also exhibits potent antimicrobial (bacterial and fungal) activity, and this can be important for preventing microbial invasion of mucosal epithelium (Bjerkan et al. 2016). S-TSLP expression in healthy tissues is constitutive, but can be upregulated by vitamin D3 and PPARγ agonists, or downregulated by proinflammatory pathogens. L-TSLP does not exist in healthy tissues, but is expressed in response to proinflammatory stimuli and plays a key role in promoting inflammation associated with Th2 cytokines by activating the effector functions of DCs and Th2 cells. Naive CD4+ T cells exposed to L-TSLP activated DCs undergo proliferation and differentiation into Th2 lymphocytes. L-TSLP is also able to stimulate Th2 innate immune responses through the activation of basophils, ILCs (ILC2s), and eosinophils.

[0017] Recent studies have shown that the expression pattern of two TSLP isoforms changes significantly relative to homeostasis in intestinal diseases including inflammatory bowel disease and eosinophilic esophagitis (EoE). Rimoldi et al. 2005 reported that TSLP (in this case, it can be S-TSLP specifically, consistent with the findings of Fornasa et al. 2015) is constitutively expressed by primary epithelial cells isolated from healthy colon tissue. However, it was found that TSLP expression was undetectable in epithelial cells from 6 / 9 patients with CD. CD epithelial cells cannot produce S-TSLP in diseased mucosa, which will lead to the failure of the mechanism that usually helps maintain intestinal homeostasis by generating a non-inflammatory environment. The defect of this mechanism can induce unwanted Th1 inflammatory response, thereby promoting the development of CD. Compared to the lamina propria Th1 cells that dominate in Crohn's disease, it is shown that the lamina propria T cells from patients with ulcerative colitis produce Th2-type cytokines IL-5 and IL-13, and these cells only show low levels of IL-4 production, which indicates that they do not show all the characteristics of classical Th2 cells. Functionally, IL-13 has been shown to promote fibrosis and lead to changes in tight junction function and apoptosis of intestinal epithelial cells, driving mucosal ulcers. Recently, Fornasa et al. 2015 reported that TSLP expression detected with an L-TSLP-specific antibody was significantly upregulated in intestinal tissue from patients with ulcerative colitis compared to levels detected in healthy colonic mucosa. Because TSLP-activated dendritic cells (DCs) can induce naive CD4+ T cells to differentiate into inflammatory Th2 cells that produce IL-5, IL-13, and TNF (Liu 2006), inhibition of this upstream cytokine acting at the epithelial cell-dendritic cell interface may prove to be an effective strategy for treating mucosal inflammation in patients with ulcerative colitis.

[0018] Epidemiological data support the involvement of atopic mechanisms and genetic factors in the development of EoE. Importantly, results from genome-wide association studies identified TSLP and its receptor TSLPR as candidate genes in the pathogenesis of EoE (Cianferoni and Spergel, 2015). TSLP expression is increased in esophageal biopsy specimens of patients with EoE and localized to stratified squamous epithelial tissue by immunohistochemical staining (Noti et al. 2013). L-TSLP appears to be a major upstream driver of disease pathology because it strongly promotes the production of cytokines (including CCL-26 / eotaxin-3, IL-4, IL-5, IL-9, and IL-13) and profibrotic factors that contribute to inflammation and tissue remodeling by Th2 cells, basophils, eosinophils, and mast cells.

[0019] In EoE, TSLP is thought to act as an upstream epithelial 'master switch' at the onset of the inflammatory cascade, and therefore, antagonism of this cytokine may allow the inflammatory cascade to be shut down further upstream than previous targeted anti-cytokine therapies. Positive results from a recent trial using tezepelumab (AMG 157), a first-in-class TSLP antagonist mAb, in patients with severe asthma provide support for this concept (Corren et al. 2017).

[0020] From the above, it will be appreciated that there is an unmet need for more effective therapies for inflammatory and / or autoimmune diseases such as IBD and EoE. Agents capable of inhibiting the binding of IL-7 and / or L-TSLP to IL-7R, particularly agents that can be administered orally, may represent such a therapy and are therefore highly desirable.

[0021] WO2013056984, WO2015189302, WO2011094259 and WO2011104687 disclose antibodies against IL-7R. Summary of the invention

[0022] The present inventors have produced polypeptides capable of inhibiting the binding of IL-7 and / or L-TSLP to IL-7R. These polypeptides bind to IL-7Rα. These polypeptides benefit particularly from unexpectedly high efficacy. They are able to cross-react with cynomolgus monkey IL-7Rα and remain stable when exposed to proteases in the small and large intestines. In one embodiment, these polypeptides have been further enhanced through engineering. These further enhanced polypeptides comprise sequences that have been humanized but still substantially maintain the above advantages.

[0023] It has been shown that in some embodiments, the polypeptides of the invention bind IL-7R with high affinity in Biacore studies and are potent inhibitors of the interaction of IL-7R with IL-7 and the IL-7-related cytokine L-TSLP in ELISA. In cell-based functional assays, certain polypeptides of the invention inhibit the biological effects of both cytokines (IL-7-induced Stat5 phosphorylation; L-TSLP-induced TARC production) with efficacy similar to that of clinical anti-IL-7R mAb829 (also referred to as "GSK2618960", an anti-IL-7Rα monoclonal antibody disclosed in Ellis et al. 2019).

[0024] Computer modeling techniques indicate that the dual antagonistic activity of certain polypeptides of the invention is due to the binding of the polypeptides to an epitope of IL-7R that also constitutes a shared binding site for both cytokines. In specificity studies, certain polypeptides of the invention did not show binding activity to other human IL-7R family or other cytokine receptors. In cross-species specificity assays, certain polypeptides of the invention did not bind to mouse IL-7R, but bound to cynomolgus monkey and human IL-7R with similar efficacy; therefore, cynomolgus monkeys may be a suitable species for preclinical development studies.

[0025] In ex vivo cultures of inflamed ulcerative colitis mucosal tissue, exemplary polypeptides of the invention inhibit phosphorylation of signaling proteins and production of cytokines and chemokines associated with proinflammatory and immunomodulatory pathways. The results demonstrate that antagonizing mucosal IL-7R+ve T cells by such polypeptides can inhibit inflammatory processes at least as effectively as clinical anti-IL-7R mAb829 in a model closely related to the disease setting, providing confidence that the polypeptides of the invention may be effective in patients suffering from ulcerative colitis in particular. Oral administration of certain polypeptides of the invention in vivo in normal mice showed high levels of colonic luminal exposure (micromolar), demonstrating tolerance to digestion during passage through the entire GI system.

[0026] Thus, these polypeptides are expected to have particular use in the prevention or treatment of autoimmune and or inflammatory diseases such as inflammatory bowel disease (eg, Crohn's disease or ulcerative colitis) or eosinophilic esophagitis, particularly when administered orally.

[0027] The present invention provides a polypeptide capable of inhibiting the binding of IL-7 and / or L-TSLP to IL-7R. The present invention also provides constructs and pharmaceutical compositions comprising these polypeptides. Also provided are nucleic acids encoding such polypeptides, methods for preparing such polypeptides, cDNAs and vectors comprising nucleic acids encoding such polypeptides, host cells capable of expressing such polypeptides, and uses of such polypeptides.

[0028] For the avoidance of doubt regarding the above terms 'and / or', 'polypeptides capable of inhibiting the binding of IL-7 to IL-7R' encompass polypeptides capable of inhibiting the binding of IL-7 and L-TSLP to IL-7R. Similarly, 'polypeptides capable of inhibiting the binding of L-TSLP to IL-7R' encompass polypeptides capable of inhibiting the binding of IL-7 and L-TSLP to IL-7R.

[0029] In at least some embodiments, the polypeptides of the present invention have one or more of the following advantages compared to prior art substances capable of inhibiting the binding of IL-7 and / or L-TSLP to IL-7R:

[0030] (i) increased affinity for IL-7Rα;

[0031] (ii) specificity for IL-7Rα increase;

[0032] (iii) increased neutralization capacity against IL-7 or L-TSLP bound to IL-7R;

[0033] (iv) inhibiting the binding of both IL-7 and L-TSLP to IL-7R;

[0034] (v) increased cross-reactivity with IL-7Rα from different species such as human and cynomolgus monkey;

[0035] (vi) reduced immunogenicity, for example, when administered to mice, cynomolgus monkeys or humans;

[0036] (vii) increased stability in the presence of proteases, for example (a) in the presence of proteases present in the small and / or large intestine and / or IBD inflammatory proteases such as trypsin, chymotrypsin, MMP3, MMP12, other MMPs and cathepsins and / or (b) in the presence of proteases from intestinal commensal microflora and / or pathogenic bacteria, proteases actively secreted and / or released by lysis of microbial cells present in the small and / or large intestine;

[0037] (viii) increased stability to protease degradation during production (e.g., resistance to yeast proteases);

[0038] (ix) increased suitability for oral administration;

[0039] (x) Increased suitability for topical delivery to the intestinal tract and lamina propria following oral administration;

[0040] (xi) increased suitability for topical delivery to the esophagus following oral administration;

[0041] (xii) increased suitability for expression in heterologous hosts such as bacteria such as Escherichia coli or yeasts belonging to the genera Aspergillus, Saccharomyces, Kluyveromyces, Hansenula or Pichia, such as Saccharomyces cerevisiae or Pichia pastoris;

[0042] (xiii) suitability and improved properties for use in medicine;

[0043] (xiv) suitability and improved properties for use in functional foods;

[0044] (xv) improved tissue penetration, such as penetration of inflamed colonic mucosal epithelium and submucosal tissue to reach the submucosal lamina propria;

[0045] (xvi) increased suitability for formatting in multispecific formats;

[0046] (xvii) Binding to novel epitopes.

[0047] Advantages (i) to (xvii) above can potentially be achieved by the polypeptide of the present invention in a monovalent form or in a multivalent form such as a biheaded form (eg, a homobiheaded form or a heterobiheaded form).

[0048] Since the polypeptides of the present invention specifically bind to the IL-7Rα subunit of IL-7R, references to “IL-7R” in the above points (and throughout the description) may also be replaced with “IL-7Rα” where appropriate. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 - Inhibition of IL-7-induced pSTAT-5 in human lymphocytes

[0050] Figure 2 - Inhibition of TSLP-induced TARC secretion in human monocytes

[0051] Figure 3 - Model structure of V7R-2E9 bound to IL-7Rα

[0052] Figure 4 - A62U, A59U and mAb829 inhibition of IL-7-induced pSTAT5 in human lymphocytes

[0053] Figure 5 - Cross-reactivity of ID-A40U with species IL-7Rα

[0054] Figure 6 - Cross-reactivity of ID-A59U and ID-A62U with human and cynomolgus monkey IL-7Rα

[0055] Figure 7 -Specificity of ID-A40U for human IL-7Rα

[0056] Figure 8 - Percent survival of V7R-2E9, ID-A24U, and ID-A40U in digestion matrix Fig. 9 -Percent stability of ID-A62U and ID-A41U in human fecal supernatant

[0057] Fig.10 -MMP digestion of etanercept, mAb829, and A40U-F / H

[0058] Fig.11 - Expected concentration in undiluted fecal supernatant (ID-A24U vs. ID-A40U vs. ID-38F)

[0059] Fig.12 - Expected concentration in undiluted supernatant of GIT (ID-A24U vs. ID-A40U vs. ID-38F)

[0060] Fig.13 - Expected concentration in undiluted fecal supernatant (ID-A40U relative to ID-38F)

[0061] Fig.14 - Expected concentration in undiluted GIT supernatant (ID-A40U vs. ID-38F)

[0062] Figure 15-18 -Protein phosphorylation profile of human IBD tissues

[0063] Fig.19 -Total phosphorylation level

[0064] Sequence Description

[0065] SEQ ID NO: 1-ID-A62U CDR1 polypeptide sequence

[0066] SEQ ID NO: 2-ID-A62U CDR2 polypeptide sequence

[0067] SEQ ID NO: 3-ID-A62U CDR3 polypeptide sequence

[0068] SEQ ID NO: 4-ID-A62U FR1 polypeptide sequence

[0069] SEQ ID NO: 5-ID-A62U FR2 polypeptide sequence

[0070] SEQ ID NO:6-ID-A62U FR3 polypeptide sequence

[0071] SEQ ID NO: 7-ID-A62U FR4 polypeptide sequence

[0072] SEQ ID NO: 8-ID-A62U polypeptide sequence

[0073] SEQ ID NO: 9-V7R-2B6 polypeptide sequence

[0074] SEQ ID NO: 10-V7R-2E5 polypeptide sequence

[0075] SEQ ID NO: 11-V7R-2E9 polypeptide sequence

[0076] SEQ ID NO: 12-V7R-2F6 polypeptide sequence

[0077] SEQ ID NO: 13 - Polypeptide sequence of V7R-3B5

[0078] SEQ ID NO: 14-V7R-4F6 polypeptide sequence

[0079] SEQ ID NO: 15-V7R-6C12 polypeptide sequence

[0080] SEQ ID NO: 16-ID-A2U polypeptide sequence

[0081] SEQ ID NO: 17-ID-A3U polypeptide sequence

[0082] SEQ ID NO: 18-ID-A4U polypeptide sequence

[0083] SEQ ID NO: 19-ID-A5U polypeptide sequence

[0084] SEQ ID NO: 20-ID-A6U polypeptide sequence

[0085] SEQ ID NO: 21-ID-A7U polypeptide sequence

[0086] SEQ ID NO: 22-ID-A8U polypeptide sequence

[0087] SEQ ID NO: 23-ID-A9U polypeptide sequence

[0088] SEQ ID NO: 24-ID-A10U polypeptide sequence

[0089] SEQ ID NO: 25-ID-A11U polypeptide sequence

[0090] SEQ ID NO: 26-ID-A12U polypeptide sequence

[0091] SEQ ID NO: 27-ID-A13U polypeptide sequence

[0092] SEQ ID NO: 28-ID-A14U polypeptide sequence

[0093] SEQ ID NO: 29-ID-A15U polypeptide sequence

[0094] SEQ ID NO: 30-ID-A16U polypeptide sequence

[0095] SEQ ID NO: 31-ID-A17U polypeptide sequence

[0096] SEQ ID NO: 32-ID-A18U polypeptide sequence

[0097] SEQ ID NO: 33-ID-A19U polypeptide sequence

[0098] SEQ ID NO: 34-ID-A20U polypeptide sequence

[0099] SEQ ID NO: 35-ID-A21U polypeptide sequence

[0100] SEQ ID NO: 36-ID-A23U polypeptide sequence

[0101] SEQ ID NO: 37-ID-A24U polypeptide sequence

[0102] SEQ ID NO: 38-ID-A25U polypeptide sequence

[0103] SEQ ID NO: 39-ID-A26U polypeptide sequence

[0104] SEQ ID NO: 40-ID-A27U polypeptide sequence

[0105] SEQ ID NO: 41-ID-A28U polypeptide sequence

[0106] SEQ ID NO: 42-ID-A29U polypeptide sequence

[0107] SEQ ID NO: 43-ID-A30U polypeptide sequence

[0108] SEQ ID NO: 44-ID-A31U polypeptide sequence

[0109] SEQ ID NO: 45-ID-A32U polypeptide sequence

[0110] SEQ ID NO: 46-ID-A33U polypeptide sequence

[0111] SEQ ID NO: 47-ID-A34U polypeptide sequence

[0112] SEQ ID NO: 48-ID-A35U polypeptide sequence

[0113] SEQ ID NO: 49-ID-A36U polypeptide sequence

[0114] SEQ ID NO: 50-ID-A37U polypeptide sequence

[0115] SEQ ID NO: 51-ID-A38U polypeptide sequence

[0116] SEQ ID NO: 52-ID-A39U polypeptide sequence

[0117] SEQ ID NO: 53-ID-A40U polypeptide sequence

[0118] SEQ ID NO: 54-ID-A43U polypeptide sequence

[0119] SEQ ID NO: polypeptide sequence of 55-ID-A50U

[0120] SEQ ID NO: polypeptide sequence of 56-ID-A52U

[0121] SEQ ID NO: 57-ID-A53U polypeptide sequence

[0122] SEQ ID NO: 58-ID-A54U polypeptide sequence

[0123] SEQ ID NO: 59-ID-A55U polypeptide sequence

[0124] SEQ ID NO:60-ID-A57U polypeptide sequence

[0125] SEQ ID NO:61-ID-A59U polypeptide sequence

[0126] SEQ ID NO:62 - Polypeptide sequence of L-TSLP

[0127] SEQ ID NO:63-S-TSLP polypeptide sequence

[0128] SEQ ID NO:64—Full-length human common gamma chain receptor polypeptide sequence

[0129] SEQ ID NO:65—Polypeptide sequence of full-length human IL-7Rα

[0130] SEQ ID NO: 66—Full-length cynomolgus monkey IL-7Rα polypeptide sequence

[0131] SEQ ID NO: 67 - Polypeptide sequence of the extracellular domain of cynomolgus monkey IL-7Rα

[0132] SEQ ID NO:68—Polypeptide sequence of human IL-7Rα extracellular domain

[0133] SEQ ID NO:69—Polynucleotide sequence encoding ID-A59U

[0134] SEQ ID NO:70—Polynucleotide sequence encoding ID-A62U

[0135] SEQ ID NO: 71 - polypeptide sequence of V7R-2B6 CDR1

[0136] SEQ ID NO: 72-V7R-2E9 CDR2 polypeptide sequence

[0137] SEQ ID NO: 73 - polypeptide sequence of V7R-2F6 CDR2

[0138] SEQ ID NO: 74-V7R-4F6 CDR2 polypeptide sequence

[0139] SEQ ID NO: 75-V7R-2B6 CDR2 polypeptide sequence

[0140] SEQ ID NO: 76-ID-A14U CDR2 polypeptide sequence

[0141] SEQ ID NO: 77-V7R-6C12 CDR3 polypeptide sequence

[0142] SEQ ID NO: 78-V7R-2B6 CDR3 polypeptide sequence

[0143] SEQ ID NO:79—Polypeptide sequence that suitably occupies residues 9-14 of ID-A62U FR2 (SEQ ID NO:5)

[0144] SEQ ID NO:80—Polypeptide sequence that suitably does not occupy residues 9-14 of ID-A62U FR2 (SEQ ID NO:5)

[0145] SEQ ID NO:81 - Polynucleotide sequence of a 3' primer containing a SpeI site

[0146] SEQ ID NO:82 - Polypeptide sequence of CDR1 with optional conservative substitution at residue 1 of SEQ ID NO:1

[0147] SEQ ID NO:83 - Polypeptide sequence of CDR2 with optional conservative substitutions at residues 2, 3, 7, 12 and 16 of SEQ ID NO:2

[0148] SEQ ID NO:84 - Polypeptide sequence of CDR3 with optional conservative substitutions at residues 3 and 9 of SEQ ID NO:3

[0149] SEQ ID NO: 85 - Polypeptide sequence of protease-labile linker formula 1

[0150] SEQ ID NO: 86 - Polypeptide sequence of protease-labile linker formula 2

[0151] SEQ ID NO: 87 - Polypeptide sequence of preferred variants of protease-labile linker formulas 1 and 2

[0152] SEQ ID NO: 88 - Polypeptide sequence of non-protease labile linker formula 3

[0153] SEQ ID NO: 89 - Preferred polypeptide sequence of a non-protease labile linker DETAILED DESCRIPTION

[0154] Polypeptides such as antibodies and antibody fragments comprising immunoglobulin chain variable domains (ICVD) such as VH and VHH

[0155] A polypeptide is an organic polymer composed of a plurality of amino acid residues bonded together in a chain. As used herein, 'polypeptide' is used interchangeably with 'protein' and 'peptide'. on Rate and / or k off A polypeptide is referred to as a binding polypeptide when it binds to one or more stretches of amino acid residues of an epitope on a target (eg, a binding rate, as further described herein).

[0156] Binding polypeptides include polypeptides such as DARPins (Binz et al. 2003), Affimers TM (Johnson et al. 2012), Fynomers TM (Grabulovski et al. 2007), Centyrins (Goldberg et al. 2016), Affitins (e.g. , Krehenbrink et al. 2008), cyclic peptides, antibodies and antibody fragments. Binding polypeptides also include polypeptides such as: Affibodies (Nygren 2008), Affilins (Ebersbach et al. 2007), Alphabodies (Desmet et al. 2014), Anticalins (Skerra et al. 2008), high affinity multimers (Silverman et al. 2005), Kunitz domain peptides (Nixon and Wood 2006), Monobodies (Koide and Koide 2007), nanoCLAMPs (Suderman et al. 2017), Adnectins (Lipovsek 2011) and bicyclic peptides.

[0157] Conventional antibodies or immunoglobulins (Ig) are proteins comprising four polypeptide chains: two heavy (H) chains and two light (L) chains. Each chain is divided into a constant region and a variable domain. The heavy chain variable domain is abbreviated as VHC in this article, and the light (L) chain variable domain is abbreviated as VLC in this article. These domains, domains associated therewith, and domains derived therefrom are referred to herein as immunoglobulin chain variable domains. The VHC and VLC domains can be further subdivided into regions with high variability, referred to as "complementarity determining regions" ("CDRs"), interspersed with more conservative regions, referred to as "framework regions" ("FRs"). Framework regions and complementary determining regions have been accurately defined (Kabat et al. 1991). In conventional antibodies, each VHC and VLC is composed of three CDRs and four FRs, arranged from amino terminal to carboxyl terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Conventional antibody tetramers of two heavy immunoglobulin chains and two light immunoglobulin chains are formed with heavy immunoglobulin chains and light immunoglobulin chains connected by, for example, disulfide bonds, and the heavy chains are similarly connected. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. The light chain constant region comprises one domain, CL. The variable domains of the heavy chain and the variable domains of the light chain are binding domains that interact with the antigen. The constant region of the antibody usually mediates the binding of the antibody to host tissues or factors, which include various immune system cells (e.g., effector cells) or the first component (C1q) of the classical complement system. The term antibody includes immunoglobulins of IgA, IgG, IgE, IgD, IgM types (and their subtypes), wherein the light chain of the immunoglobulin can be a kappa type or a lambda type. The overall structure of the immunoglobulin-γ (IgG) antibody assembled by two identical heavy (H) chains and two identical light (L) chain polypeptides is well established in mammals and is highly conserved (Padlan 1994).

[0158] An exception to conventional antibody structure is found in the serum of camelids. In addition to conventional antibodies, these sera possess special IgG antibodies. These IgG antibodies (referred to as heavy chain antibodies (HCAb)) lack L chain polypeptides and lack the first constant domain (CH1). In its N-terminal region, the H chain of the homodimeric protein contains a dedicated immunoglobulin chain variable domain (referred to as VHH), which is used to associate with its cognate antigen (Muyldermans 2013, Hamers-Casterman et al. 1993, Muyldermans et al. 1994).

[0159] As used herein, an antigen-binding fragment (or "antibody fragment" or "immunoglobulin fragment") refers to a portion of an antibody that specifically binds to IL-7Rα (e.g., a molecule in which one or more immunoglobulin chains are not full-length but specifically binds to IL-7Rα). Examples of binding fragments encompassed within antigen-binding fragments include:

[0160] (i) Fab fragment (monovalent fragment consisting of VLC, VHC, CL and CH1 domains);

[0161] (ii) F(ab′)2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region);

[0162] (iii) Fd fragment (consisting of VHC and CH1 domains);

[0163] (iv) Fv fragment (consisting of the VLC and VHC domains of a single arm of an antibody);

[0164] (v) scFv fragments (composed of the VLC and VHC domains joined using recombinant methods by a synthetic linker that enables the VLC and VHC domains to be made into a single protein chain, in which the VLC and VHC regions pair to form a monovalent molecule);

[0165] (vi) VH (the variable domain of the immunoglobulin chain consisting of the VHC domain (Ward et al. 1989);

[0166] (vii) VL (immunoglobulin chain variable domain consisting of the VLC domain);

[0167] (viii) V-NAR (immunoglobulin chain variable domain composed of the VHC domain of IgNAR from chondrichthyes (Roux et al. 1998 and Griffiths et al. 2013)

[0168] (ix) VHH.

[0169] The total number of amino acid residues in VHH or VH may be in the region of 110-130, suitably 115-120, and most suitably 118.

[0170] The immunoglobulin chain variable domains of the present invention can be obtained, for example, by preparing a nucleic acid encoding an immunoglobulin chain variable domain using a technique for nucleic acid synthesis, followed by expressing the nucleic acid thus obtained. According to a specific embodiment, the immunoglobulin chain variable domains of the present invention do not have an amino acid sequence that is completely identical (i.e., shares 100% sequence identity) to the amino acid sequence of a naturally occurring polypeptide (such as a VH or VHH domain of a naturally occurring antibody).

[0171] The examples provided herein relate to immunoglobulin chain variable domains that bind to IL-7Rα themselves. However, the principles of the invention disclosed herein are equally applicable to any IL-7Rα binding polypeptides, such as antibodies and antibody fragments. For example, the anti-IL-7Rα immunoglobulin chain variable domains disclosed herein can be incorporated into polypeptides such as full-length antibodies. This approach was demonstrated by McCoy et al. 2014, who provided anti-HIV VHHs constructed as fusions with human Fc regions (including hinge, CH2 and CH3 domains) expressed as dimer constructs.

[0172] Replacing at least one amino acid residue in the framework region of a non-human immunoglobulin chain variable domain with the corresponding residue from a human immunoglobulin chain variable domain is humanization. Humanization of the variable domain can reduce immunogenicity in humans.

[0173] Suitably, the polypeptide of the present invention comprises an immunoglobulin chain variable domain. More suitably, the polypeptide of the present invention consists of an immunoglobulin chain variable domain, such as an immunoglobulin heavy chain variable domain. Suitably, the polypeptide of the present invention is an antibody or an antibody fragment. More suitably, the polypeptide of the present invention is an antibody fragment. Suitably, the antibody fragment is an immunoglobulin chain variable domain, such as VHH, VH or VL. Suitably, the antibody fragment is a VHH, VH, VL, V-NAR, scFv, Fab fragment or F(ab')2 fragment. Suitably, the antibody fragment is an immunoglobulin heavy chain variable domain. More suitably, the antibody fragment is a VHH or VH, and most suitably a VHH.

[0174] Specificity, affinity, avidity and cross-reactivity

[0175] Specificity refers to the number of different types of antigens or antigenic determinants to which a particular antigen binding polypeptide can bind. The specificity of an antigen binding polypeptide is the ability of the antigen binding polypeptide to recognize specific antigens as unique molecular entities and to distinguish them from one another.

[0176] Affinity, represented by the equilibrium constant (Kd) for the dissociation of the target from the binding polypeptide, is a measure of the strength of binding between the target and the binding site on the binding polypeptide: the smaller the value of Kd, the stronger the binding strength between the target and the binding polypeptide (alternatively, affinity can also be expressed as an affinity constant (Ka), which is 1 / Kd). Affinity can be determined by known methods depending on the specific antigen of interest. Suitably, affinity is determined using a dynamically switchable biosurface (e.g., " ”, see Knezevic et al. 2012) or determined by surface plasmon resonance.

[0177] Avidity is a measure of the binding between an antigen binding polypeptide and a related antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on an antigen binding polypeptide and the number of related binding sites present on the antigen binding polypeptide.

[0178] Suitably, the polypeptide of the present invention is present in an amount of 10 -7 M or smaller, preferably 10 -8 M or smaller, preferably 10 -9 M or less and more preferably 10 -10 The IL-7Rα protein binds to IL-7Rα with an equilibrium dissociation constant (Kd) of M or less.

[0179] Suitably, the polypeptide of the invention binds to IL-7Rα with an equilibrium dissociation constant lower than that of mAb829 in the same assay. Suitably, the polypeptide of the invention binds to IL-7Rα with an equilibrium dissociation constant of 5.67×10 -10 M or lower, preferably lower than 5.67x10 -10 The equilibrium dissociation constant of mAb829 is 1.377 M and binds to IL-7Rα. mAb829 is also known as “GSK2618960”, an anti-IL-7Rα monoclonal antibody disclosed in Ellis et al. 2019.

[0180] In one embodiment, the affinity of the polypeptide of the invention is established by direct coating on a Biacore (or equivalent) sensor plate or by fusion to Fc and capture with anti-human IgG Fc, wherein the polypeptide flows through the plate to detect binding. Suitably, Biacore T200 is used in HBS-EP+ (GE Healthcare) running buffer at 30 ul / min at 25°C.

[0181] Anti-IL-7Rα polypeptides, IL-7Rα binding polypeptides, polypeptides that interact with IL-7Rα, or polypeptides directed against IL-7Rα are all effective polypeptides that bind to IL-7Rα. The polypeptides of the present invention may bind to a linear or conformational epitope on IL-7Rα.

[0182] Suitably, the polypeptide of the invention binds to human IL-7Rα. More suitably, the polypeptide of the invention binds to both human and at least one additional primate IL-7Rα selected from baboon IL-7Rα, marmoset IL-7Rα, cynomolgus IL-7Rα and rhesus IL-7Rα. Most suitably, the polypeptide of the invention binds to both human and cynomolgus IL-7Rα.

[0183] Suitably, the polypeptides of the invention neutralize the binding of human IL-7 and / or human L-TSLP to human IL-7R. More suitably, the polypeptides of the invention neutralize the binding of human IL-7 and / or human L-TSLP to both human and at least one additional primate IL-7R selected from baboon IL-7R, marmoset IL-7R, cynomolgus monkey IL-7R and rhesus monkey IL-7R. Most suitably, the polypeptides of the invention neutralize the binding of human IL-7 and human L-TSLP to human IL-7R.

[0184] Suitably, the polypeptides of the invention bind to IL-7Rα (e.g., human IL-7Rα, SEQ ID NO: 65 and / or cynomolgus monkey IL-7Rα, SEQ ID NO: 66) or to the γ chain receptor (e.g., human common γ chain receptor, SEQ ID NO: 64). More suitably, the polypeptides of the invention bind to IL-7Rα, most suitably human IL-7Rα. More specifically, the polypeptides of the invention bind to the extracellular region of IL-7Rα (SEQ ID NOs: 67 and 68, the extracellular regions of cynomolgus monkey and human IL-7Rα, respectively), i.e., the polypeptide sequence of IL-7Rα lacking the transmembrane helices and the cytoplasmic domain.

[0185] Suitably, IL-7Rα is a polypeptide comprising SEQ ID NO: 65 or SEQ ID NO: 66, more suitably, IL-7Rα is a polypeptide consisting of SEQ ID NO: 65 or SEQ ID NO: 66. More suitably, IL-7Rα is a polypeptide comprising SEQ ID NO: 65, more suitably, IL-7Rα is a polypeptide consisting of SEQ ID NO: 65. The polypeptide sequence of mature full-length human IL-7Rα is also available under UniProt entry P16871.

[0186] Polypeptides that are able to react with IL-7Rα from humans and IL-7Rα from another species, such as cynomolgus monkey IL-7Rα ("cross-react"), are advantageous because they allow for easier preclinical studies in animal models.

[0187] Suitably, the polypeptide of the present invention is directed against an epitope on IL-7Rα that is located in and / or forms part of one or more receptor binding sites of IL-7 and / or L-TSLP, such that the polypeptide of the present invention is capable of inhibiting or reducing IL-7 and / or L-TSLP signaling after binding to IL-7Rα.

[0188] The polypeptides of the present invention bind to one or more epitopes on IL-7Rα. In one aspect of the present invention, a polypeptide is provided that binds to the same epitope on IL-7Rα as V7R-2E5, V7R-2E9, V7R-2F6, V7R-6C12, V7R-2B6, V7R-3B5 or V7R-4F6.

[0189] Suitably, the polypeptides of the present invention are isolated. An "isolated" polypeptide is one that is removed from its original environment. For example, if a naturally occurring polypeptide of the present invention is separated from some or all of the coexisting materials in the natural system, the polypeptide is isolated.

[0190] Efficacy, Inhibition, and Neutralization

[0191] Efficacy is a measure of the activity of a therapeutic agent expressed in the amount required to produce an effect of a given intensity. Compared with a lower efficacy agent that causes a smaller response at low concentrations, a high efficacy agent causes a greater response at low concentrations. Efficacy is related to affinity and effectiveness. Effectiveness refers to the ability of a therapeutic agent to produce a biological response after being bound to a target ligand and the quantitative magnitude of this response. The term half-maximal effective concentration (EC50) refers to the concentration of a therapeutic agent that causes a response between half of the baseline and the maximum after a specified exposure time. Therapeutic agents can cause inhibition or stimulation. It is usually used as a measure of efficacy and is used as a measure of efficacy in this article.

[0192] Neutralizing polypeptides for the purposes of the present invention are polypeptides that bind to IL-7Rα, thereby inhibiting the binding of IL-7R to IL-7 and / or L-TSLP, as measured by ELISA. A specific ELISA method suitable for determining the level of inhibition in this context is described in detail in Example 3 below.

[0193] Suitably, the polypeptide of the present invention neutralizes the binding of IL-7R to IL-7 with an EC50 of 2.00 nM or less, such as 1.50 nM or less, such as 1.00 nM or less, such as 0.90 nM or less, such as 0.80 nM or less, such as 0.70 nM or less, such as 0.65 nM or less, such as 0.60 nM or less, such as 0.55 nM or less, such as 0.50 nM or less, such as 0.45 nM or less, such as 0.4 nM or less, such as 0.35 nM or less, such as 0.30 nM or less.

[0194] Suitably, the EC50 is determined using the IL-7 / IL-7R neutralisation ELISA described in detail in Example 3 below.

[0195] Peptide and polynucleotide sequences

[0196] For the purpose of comparing two closely related polypeptide sequences, the "% sequence identity" between a first polypeptide sequence and a second polypeptide sequence can be calculated using NCBI BLAST v2.0 (BLASTP) using the standard settings for polypeptide sequences. For the purpose of comparing two closely related polynucleotide sequences, the "% sequence identity" between a first nucleotide sequence and a second nucleotide sequence can be calculated using NCBI BLAST v2.0 (BLASTN) using the standard settings for nucleotide sequences. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the comparison. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a two-strand comparison as default values. For amino acid sequences, the BLASTP program uses a wordlength of 3 and an expectation (E) of 10 and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) 50, expectation (E) 10, M=5, N=-4, and comparison of both strands as defaults. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more suitably less than about 0.01, and most suitably less than about 0.001.

[0197] A polypeptide or polynucleotide sequence is said to be identical if it shares 100% sequence identity with the other polypeptide or polynucleotide sequence over its entire length. The residues in the sequence are numbered from left to right, i.e., from the N-terminus to the C-terminus of a polypeptide; from the 5' to the 3' end of a polynucleotide.

[0198] "Difference" between sequences refers to the insertion, deletion or substitution of a single amino acid residue in a position of the second sequence compared to the first sequence. Two polypeptide sequences may contain one, two or more such amino acid differences. Insertion, deletion or substitution in the second sequence that is otherwise identical to the first sequence (100% sequence identity) causes a reduction in sequence identity %. For example, if the length of the same sequence is 9 amino acid residues, one substitution in the second sequence produces 88.9% sequence identity. If the length of the same sequence is 17 amino acid residues, two substitutions in the second sequence produce 88.2% sequence identity. If the length of the same sequence is 7 amino acid residues, three substitutions in the second sequence produce 57.1% sequence identity. If the length of the first polypeptide sequence and the second polypeptide sequence is 9 amino acid residues and shares 6 identical residues, the first polypeptide sequence and the second polypeptide sequence share an identity greater than 66% (the first polypeptide sequence and the second polypeptide sequence share 66.7% identity). If the first polypeptide sequence and the second polypeptide sequence are 17 amino acid residues in length and share 16 identical residues, the first polypeptide sequence and the second polypeptide sequence share greater than 94% identity (the first polypeptide sequence and the second polypeptide sequence share 94.1% identity). If the first polypeptide sequence and the second polypeptide sequence are 7 amino acid residues in length and share 3 identical residues, the first polypeptide sequence and the second polypeptide sequence share greater than 42% identity (the first polypeptide sequence and the second polypeptide sequence share 42.9% identity).

[0199] Alternatively, for the purpose of comparing a first reference polypeptide sequence to a second comparison polypeptide sequence, the number of additions, substitutions and / or deletions made to the first sequence to produce the second sequence can be determined. An addition is the addition of an amino acid residue to the sequence of the first polypeptide (including additions at either end of the first polypeptide). A substitution is the replacement of an amino acid residue in the sequence of the first polypeptide with a different amino acid residue. A deletion is the deletion of an amino acid residue from the sequence of the first polypeptide (including deletions at either end of the first polypeptide).

[0200] For the purpose of comparing a first reference polynucleotide sequence to a second comparison polynucleotide sequence, the number of additions, substitutions and / or deletions made to the first sequence to produce the second sequence can be determined. An addition is the addition of a nucleotide residue to the sequence of the first polynucleotide (including additions at either end of the first polynucleotide). A substitution is the replacement of a nucleotide residue in the sequence of the first polynucleotide with a different nucleotide residue. A deletion is the deletion of a nucleotide residue from the sequence of the first polynucleotide (including deletions at either end of the first polynucleotide).

[0201] A "conservative" amino acid substitution is one in which an amino acid residue is replaced by another amino acid residue of similar chemical structure and is expected to have little effect on the function, activity or other biological properties of the polypeptide. Such conservative substitutions are suitably substitutions in which one amino acid within the following group is replaced by another amino acid residue from the same group:

[0202]

[0203] Suitably, the hydrophobic amino acid residue is a non-polar amino acid. More suitably, the hydrophobic amino acid residue is selected from V, I, L, M, F, W or C.

[0204] As used herein, the numbering of polypeptide sequences and the definition of CDR and FR are as defined according to the Kabat system (Kabat et al. 1991). The "corresponding" amino acid residue between a first polypeptide sequence and a second polypeptide sequence is an amino acid residue in the first sequence that shares the same position with the amino acid residue in the second sequence according to the Kabat system, while the identity of the amino acid residue in the second sequence and the amino acid residue in the first sequence can be different. Suitably, if the framework and CDR are the same length according to the Kabat definition, the corresponding residues will share the same numbering (and letter). Alignment can be manually implemented or by using a known computer algorithm such as NCBI BLAST v2.0 (BLASTP or BLASTN) of sequence alignment using standard settings to realize.

[0205]

[0206]

[0207]

[0208] Preferably, the polynucleotides used in the present invention are isolated. An "isolated" polynucleotide is a polynucleotide removed from its original environment. For example, if a naturally occurring polynucleotide is separated from some or all of the materials coexisting in a natural system, the polynucleotide is isolated. If, for example, a polynucleotide is cloned into a vector that is not a part of its natural environment or if it is contained in a cDNA, the polynucleotide is considered to be isolated.

[0209] In one aspect of the present invention, a polynucleotide is provided, which encodes a polypeptide or construct of the present invention. Suitably, the polynucleotide comprises or consists of a sequence that shares 70% or greater, such as 80% or greater, such as 90% or greater, such as 95% or greater, such as 99% or greater sequence identity with SEQ ID NO: 69 or 70, most suitably SEQ ID NO: 70. More suitably, the polypeptide comprises or consists of (most suitably consists of) SEQ ID NO: 69 or 70, most suitably SEQ ID NO: 70. In another aspect, a cDNA is provided, which comprises the polynucleotide.

[0210] In one aspect of the invention, a polynucleotide is provided which comprises or consists of a sequence that shares 70% or greater, such as 80% or greater, such as 90% or greater, such as 95% or greater, such as 99% or greater sequence identity with any of the portions of CDR1, CDR2 or CDR3 encoding the encoded immunoglobulin chain variable domain of SEQ ID NO: 69 or 70.

[0211] Suitably, the polypeptide sequence of the present invention contains at least one change relative to the native sequence. Suitably, the polynucleotide sequence of the present invention contains at least one change relative to the native sequence. Suitably, the polypeptide sequence or polynucleotide sequence is altered to increase the stability of the polypeptide or the encoded polypeptide to proteases present in the intestinal tract (e.g., trypsin and chymotrypsin).

[0212] Possible features of the CDRs and frameworks of the polypeptides of the invention are described below.

[0213] CDR1

[0214] Suitably, the CDR1 of the polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 80% or greater sequence identity with SEQ ID NO:1.

[0215] Alternatively, the CDR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 2, more suitably no more than 1 addition compared to SEQ ID NO: 1. Suitably, the CDR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 2, more suitably no more than 1 substitution compared to SEQ ID NO: 1. Suitably, the CDR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 2, more suitably no more than 1 deletion compared to SEQ ID NO: 1.

[0216] Suitably, any residue of CDR1 that differs from its corresponding residue in SEQ ID NO: 1 is a conservative substitution relative to its corresponding residue.

[0217] Suitably, the residues of CDR1 have the following identity (SEQ ID NO: 82):

[0218] 1 2 3 4 5 S / D D A M G

[0219] Suitably, CDR1 comprises or consists of SEQ ID NO: 1 or SEQ ID NO: 71. More suitably, CDR1 comprises or more suitably consists of SEQ ID NO: 1.

[0220] CDR2

[0221] Suitably, the CDR2 of the polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 65% or greater, 75% or greater, 80% or greater, 85% or greater or 90% or greater sequence identity with SEQ ID NO:2.

[0222] Alternatively, the CDR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition compared to SEQ ID NO: 2. Suitably, the CDR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution compared to SEQ ID NO: 2. Suitably, the CDR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion compared to SEQ ID NO: 2.

[0223] Suitably, any residue of CDR2 that differs from its corresponding residue in SEQ ID NO: 2 is a conservative substitution relative to its corresponding residue.

[0224] Suitably, the residues of CDR2 have the following identity (SEQ ID NO: 83):

[0225] 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 A I / T G / N W S G A / T V T H Y S / G D S V Q / K G

[0226] Suitably, the residue of CDR2 corresponding to residue number 16 of SEQ ID NO: 2 is Q or K, most suitably K. Suitably, CDR2 comprises or consists of SEQ ID NO: 2, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75 or SEQ ID NO: 76. More suitably, CDR2 comprises or more suitably consists of SEQ ID NO: 2.

[0227] CDR3

[0228] Suitably, the CDR3 of the polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 60% or greater, 70% or greater or 80% or greater sequence identity with SEQ ID NO:3.

[0229] Alternatively, the CDR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 3, more suitably no more than 2, more suitably no more than 1 additions compared to SEQ ID NO: 3. Suitably, the CDR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 3, more suitably no more than 2, more suitably no more than 1 substitutions compared to SEQ ID NO: 3. Suitably, the CDR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 3, more suitably no more than 2, more suitably no more than 1 deletions compared to SEQ ID NO: 3. Suitably, any substitution is conservative relative to its corresponding residue in SEQ ID NO: 3.

[0230] Suitably, any residue of CDR3 that differs from its corresponding residue in SEQ ID NO: 3 is a conservative substitution relative to its corresponding residue.

[0231] Suitably, the residues of CDR3 have the following identity (SEQ ID NO: 84):

[0232] 1 2 3 4 5 6 7 8 9 D Y D / V T D V W Q Y / H

[0233] Suitably, the sequence of CDR3 comprises or consists of SEQ ID NO: 3, SEQ ID NO: 77 or SEQ ID NO: 78. More suitably, CDR3 comprises or more suitably consists of SEQ ID NO: 3.

[0234] Specific CDR

[0235] Some particularly suitable CDR sequences are shown in the table below. Suitably, the CDR1 of the polypeptide of the present invention is one of the CDR1 sequences listed below. Suitably, the CDR2 of the polypeptide of the present invention is one of the CDR2 sequences listed below. Suitably, the CDR3 of the polypeptide of the present invention is one of the CDR3 sequences listed below. Suitably, the polypeptide of the present invention comprises a combination of two or more suitably three of the CDR sequences listed below.

[0236] Specific CDRs of the polypeptides of the invention are provided below.'Example' represents an exemplary ICVD comprising that CDR and the corresponding sequence identifier number of that sequence.

[0237] SEQ ID NO CDR1 Examples 1 SDAMG ID-A62U 71 DDAMG V7R-2B6

[0238] SEQ ID NO CDR2 Examples 72 AIGWSGAVTHYSDSVQG V7R-2E9 73 AIGWSGTVTHYSDSVQG V7R-2F6 2 AIGWSGAVTHYSDSVKG ID-A62U 74 ATGWSGAVTHYSDSVKG V7R-4F6 75 AINWSGAVTHYGDSVKG V7R-2B6 76 AIGWSGAVTHYSDSVKG ID-A14U

[0239] SEQ ID NO CDR3 Examples 3 DYDTDVWQ ID-A62U 77 DYVTDVW V7R-6C12 78 DYNAMIC V7R-2B6

[0240] FR1

[0241] Suitably, FR1 of the polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 5%, 12%, 18%, 26%, 32%, 38%, 46%, 52%, 58%, 62%, 66%, 68%, 72%, 75%, 78%, 82%, 85%, 90%, 95% or more sequence identity with SEQ ID NO:4.

[0242] Alternatively, FR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 28, more suitably no more than 26, more suitably no more than 24, more suitably no more than 22, more suitably no more than 20, more suitably no more than 18, more suitably no more than 16, more suitably no more than 14, more suitably no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition compared to SEQ ID NO:4. Suitably, FR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 28, more suitably no more than 26, more suitably no more than 24, more suitably no more than 22, more suitably no more than 20, more suitably no more than 18, more suitably no more than 16, more suitably no more than 14, more suitably no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution compared to SEQ ID NO:4. Suitably, FR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 28, more suitably no more than 26, more suitably no more than 24, more suitably no more than 22, more suitably no more than 20, more suitably no more than 18, more suitably no more than 16, more suitably no more than 14, more suitably no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion compared to SEQ ID NO:4.

[0243] Suitably, any residue of FR1 that differs from its corresponding residue in SEQ ID NO: 4 is a conservative substitution relative to its corresponding residue. Suitably, the residue of FR1 corresponding to residue number 1 of SEQ ID NO: 4 is D or E, most suitably D. Suitably, the residues of FR1 corresponding to residue numbers 1 to 5 of SEQ ID NO: 4 are DVQLV. Suitably, FR1 comprises or more suitably consists of SEQ ID NO: 4. Suitably, the residue of FR1 corresponding to residue number 24 of SEQ ID NO: 4 is S.

[0244] FR2

[0245] Suitably, FR2 of a polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 10%, 15%, 25%, 30%, 40%, 45%, 55%, 60%, 70%, 75%, 85%, 90% or more sequence identity with SEQ ID NO:5.

[0246] Alternatively, the FR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition compared to SEQ ID NO: 5. Suitably, the FR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution compared to SEQ ID NO: 5. Suitably, FR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion compared to SEQ ID NO:5.

[0247] Suitably, any residue of FR2 that differs from its corresponding residue in SEQ ID NO:5 is a conservative substitution relative to its corresponding residue. Suitably, the residue of FR2 corresponding to residue number 10 of SEQ ID NO:5 is R or L, most suitably L. Suitably, the residues of FR2 corresponding to residue numbers 8 to 11 of SEQ ID NO:5 are KEXE, wherein X is R or L, most suitably L. Alternatively, the residues of FR2 corresponding to residue numbers 9 to 12 of SEQ ID NO:5 are GLEW. Suitably, FR2 comprises or more suitably consists of SEQ ID NO:5. Suitably, the residue of FR2 corresponding to residue number 2 of SEQ ID NO:5 is F, and most suitably, in addition, the residue of FR2 corresponding to residue number 14 of SEQ ID NO:5 is A. Suitably, the residues of FR2 corresponding to residues 9-14 of SEQ ID NO:5 are ELEFLA (SEQ ID NO:79). Suitably, the residues of FR2 corresponding to residues 9-14 of SEQ ID NO:5 are not GLEWVS (SEQ ID NO:80). Suitably, the residue of FR2 corresponding to residue number 9 of SEQ ID NO:5 is not G. More suitably, the residue of FR2 corresponding to residue number 9 of SEQ ID NO:5 is E.

[0248] FR3

[0249] Suitably, FR3 of a polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 8%, 15%, 20%, 26%, 32%, 40%, 45%, 52%, 58%, 65%, 70%, 76%, 80%, 82%, 90%, 85%, 92%, 95% or more sequence identity with SEQ ID NO:6.

[0250] Alternatively, the FR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 29, more suitably no more than 27, more suitably no more than 25, more suitably no more than 23, more suitably no more than 21, more suitably no more than 19, more suitably no more than 17, more suitably no more than 15, more suitably no more than 13, more suitably no more than 11, more suitably no more than 9, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition compared to SEQ ID NO:6. Suitably, the FR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 29, more suitably no more than 27, more suitably no more than 25, more suitably no more than 23, more suitably no more than 21, more suitably no more than 19, more suitably no more than 17, more suitably no more than 15, more suitably no more than 13, more suitably no more than 11, more suitably no more than 9, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution compared to SEQ ID NO:6. Suitably, the FR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 29, more suitably no more than 27, more suitably no more than 25, more suitably no more than 23, more suitably no more than 21, more suitably no more than 19, more suitably no more than 17, more suitably no more than 15, more suitably no more than 13, more suitably no more than 11, more suitably no more than 9, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion compared to SEQ ID NO:6.

[0251] Suitably, any residue of FR3 that differs from its corresponding residue in SEQ ID NO: 6 is a conservative substitution relative to its corresponding residue. Suitably, FR3 comprises or more suitably consists of SEQ ID NO: 6. Suitably, residues of FR3 corresponding to residue numbers 18, 19 to 20 of SEQ ID NO: 6 are NSL. Suitably, residue of FR3 corresponding to residue number 21 of SEQ ID NO: 6 is R. Suitably, residue of FR3 corresponding to residue number 22 of SEQ ID NO: 6 is A.

[0252] FR4

[0253] Suitably, FR4 of a polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more sequence identity with SEQ ID NO:7.

[0254] Alternatively, the FR4 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition compared to SEQ ID NO: 7. Suitably, the FR4 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution compared to SEQ ID NO: 7. Suitably, FR4 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion compared to SEQ ID NO:7.

[0255] Suitably, any residue of FR4 which differs from its corresponding residue in SEQ ID NO: 7 is a conservative substitution relative to its corresponding residue. Suitably, FR4 comprises or more suitably consists of SEQ ID NO: 7.

[0256] Complete Peptide

[0257] Suitably, a polypeptide of the invention comprises, or more suitably consists of, a sequence sharing 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:8.

[0258] Alternatively, the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 20, more suitably no more than 15, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition compared to SEQ ID NO: 8. Suitably, the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 20, more suitably no more than 15, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution compared to SEQ ID NO: 8. Suitably, the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 20, more suitably no more than 15, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion compared to SEQ ID NO: 8.

[0259] Suitably, the N-terminus of the polypeptide is D. Suitably, the polypeptide comprises, or more suitably consists of, SEQ ID NO: 8.

[0260] Framework Implementation Plan

[0261] In one aspect of the present invention, a polypeptide is provided, which comprises four framework regions (FR1-FR4), wherein each framework region is a variant of the corresponding framework region of ID-A62U (i.e., SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7). Suitably, each variant framework region shares at least 50%, more suitably at least 60%, more suitably at least 70%, more suitably at least 80% or more suitably at least 90% identity with its corresponding framework region in ID-A62U. More suitably, the variant framework region comprises or more suitably consists of the corresponding framework region of ID-A62U. Suitably, the polypeptide comprising four framework regions is an antibody or an antibody fragment. Suitably, the antibody fragment is a VHH, VH, VL, V-NAR, scFv, Fab fragment or F(ab')2 fragment. More suitably, the antibody fragment is a VHH or VH, and most suitably a VHH.

[0262] Epitope

[0263] Example 5 details epitope modeling work performed on the polypeptide V7R-2E9 of the invention. This work indicated that V7R-2E9 binds to the following residues of IL-7Rα. Residues covering particularly significant regions at the interface are highlighted in bold. Residue numbering corresponds to SEQ ID NO: 65.

[0264]

[0265] Thus, in one aspect of the invention, a polypeptide is provided which binds to an epitope on IL-7Rα (SEQ ID NO: 65), the epitope comprising at least one residue of IL-7Rα selected from Glu27, Ser31, Leu57, Val58, Glu59, Lys77, Lys78, Phe79, Leu80, Leu81, Ile82, Thr104, Lys137, Lys138, Tyr139, Lys141, His191, Tyr192 and Phe193. Suitably, the polypeptide binds to an epitope on IL-7Rα comprising at least 8, more suitably at least 15, more suitably at least all of the residues of IL-7Rα selected from this list.

[0266] It can be noted that certain residues of IL-7Rα overlap particularly significant regions at the interface with V7R-2E9. Therefore, in another aspect of the present invention, a polypeptide is provided that binds to an epitope on IL-7Rα (SEQ ID NO: 65), the epitope comprising at least one residue of IL-7Rα selected from the group consisting of Ser31, Val58, Phe79, Leu80, Leu81, Ile82, Lys138, Tyr139 and Phe193. Suitably, the polypeptide binds to an epitope on IL-7Rα comprising at least Val58, Leu80 and Lys138 of IL-7Rα. More suitably, Val58, Leu80, Lys138, Ile82 and Tyr139 of IL-7Rα. More suitably, Val58, Leu80, Lys138, Ile82, Tyr139, Leu81 and Phe79 of IL-7Rα. Suitably, the polypeptide binds to an epitope on IL-7Rα comprising at least 5, more suitably at least 7, more suitably all residues of IL-7Rα selected from the group consisting of Ser31, Val58, Phe79, Leu80, Leu81, Ile82, Lys138, Tyr139 and Phe193.

[0267] Suitably, 'binding to an epitope' in this context may be defined as the relevant residues of IL-7Rα that constitute the epitope when bound by a polypeptide having at least the BSA cited in the table above for the relevant residues.

[0268] Adapters and polymers

[0269] The construct according to the invention comprises a plurality of polypeptides and may therefore suitably be multivalent. Such a construct may comprise at least two identical polypeptides according to the invention. A construct consisting of two identical polypeptides according to the invention is a "homobivalent". In one aspect of the invention, a construct is provided comprising two or more identical polypeptides according to the invention.

[0270] Alternatively, the construct may comprise at least two polypeptides which are different but are still both polypeptides according to the invention ("homobihead").

[0271] Alternatively, such a construct may comprise (a) at least one polypeptide according to the invention and (b) at least one polypeptide which is not a polypeptide according to the invention, such as an antibody or antigen-binding fragment thereof (also a "heterobihead"). The at least one polypeptide of (b) may bind to IL-7R (e.g. via an epitope different from that of (a)), or alternatively may bind to a target other than IL-7R. Suitably, the different polypeptides (b) bind to, for example: interleukins (such as IL-1, IL-1ra, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17 and IL-18), interleukin receptors (such as IL-6R), transcription factors (such as NF-kB), cytokines (such as TNF-α, IFN-γ, TGF-β), transmembrane proteins (such as gp130 and CD3), epitopes. Glycoproteins (such as CD4, CD20, CD40), soluble proteins (such as CD40L), integrins (such as a4b7 and AlphaEbeta7), adhesion molecules (such as MAdCAM), chemokines (such as IP10 and CCL20), chemokine receptors (such as CCR2 and CCR9), inhibitory proteins (such as SMAD7), kinases (such as JAK3), G protein-coupled receptors (such as sphingosine-1-P receptor), other inflammatory mediators involved in human pathological processes or immune-related ligands. Thus, different polypeptides (b) bind to, for example, IL-6R, IL-6, IL-12, IL-1-p, IL-17A, TNF-α or CD3; or other inflammatory mediators involved in human pathological processes or immune-related ligands.

[0272] Constructs can be multivalent and / or multispecific. Multivalent constructs (such as bivalent constructs) comprise two or more binding polypeptides, thus presenting two or more sites where connection to one or more antigens can occur. Examples of multivalent constructs can be homobiheaded or heterobiheaded. Multivalent constructs (such as bispecific constructs) comprise two or more different binding polypeptides, which present two or more sites where (a) connection to two or more different antigens can occur or (b) connection to two or more different epitopes on the same antigen can occur. Examples of multispecific constructs can be heterobiheaded. Multispecific constructs are multivalent.

[0273] Suitably, the polypeptide contained in the construct is an antibody fragment. More suitably, the polypeptide contained in the construct is selected from VHH, VH, VL, V-NAR, scFv, Fab fragment or F(ab')2 fragment. More suitably, the polypeptide contained in the construct is VH or VHH, most suitably VHH.

[0274] Suitably, the polypeptide included in the construct is selected from ICVD (such as VHH, VH, VL), V-NAR, scFv, Fab fragment or F(ab')2 fragment. More suitably, the polypeptide included in the construct is ICVD, more suitably, the polypeptide included in the construct is VH or VHH, most suitably VHH.

[0275] The polypeptides of the invention may be linked to each other directly (i.e., without the use of a linker) or via a linker. Suitably, the linker is a protease labile linker or a non-protease labile linker. The linker is suitably a polypeptide and is selected so as to allow binding of the polypeptide to its epitope. If used for therapeutic purposes, the linker is suitably non-immunogenic in the subject to which the polypeptide is administered. Suitably, the polypeptides are all linked by a non-protease labile linker. Suitably, the protease labile linker has the formula [-(G a S) x -BJB'-(G a S) y -] z , wherein J is lysine or arginine, B is 0 to 5 amino acid residues selected from the group consisting of R, H, N, Q, S, T, Y, G, A, V, L, W, P, M, C, F, K or I, B' is 0 to 5 amino acid residues selected from the group consisting of R, H, N, Q, S, T, Y, G, A, V, L, W, M, C, F, K or I, a is 1 to 10, x is 1 to 10; y is 1 to 10 and z is 1 to 10 (SEQ ID NO: 85). Suitably, a is 4. Most suitably, a is 4, J is lysine, B is 0, x is 1, y is 1 and z is 1. Alternatively, the protease labile linker has the formula -(G 4 S) x -K-(G4 S) y -, wherein x and y are each independently 1 to 5 (SEQ ID NO: 86), more preferably -(G 4 S) 2 -K-(G 4 S) 2 - (SEQ ID NO: 87).

[0276] Suitably, the non-protease labile linker has the formula (G 4 S) x (SEQ ID NO: 88). More suitably, x is 1 to 10, more suitably x is 4 to 8, more suitably x is 4, 6 or 8. Most suitably, x is 6 (SEQ ID NO: 89).

[0277] Vector and host

[0278] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is connected. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which another DNA segment can be connected. Another type of vector is a viral vector, in which another DNA segment can be connected to the viral genome. Certain vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors with bacterial replication origins and free mammalian and yeast vectors). Other vectors (e.g., non-free mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and therefore replicated together with the host genome. In addition, certain vectors can guide the expression of genes operably connected thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA technology are generally in the form of plasmids. In the present specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used vector forms. However, the present invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses and adeno-associated viruses), which have the same function, as well as phage and phagemid systems. The present invention also relates to nucleotide sequences encoding polypeptide sequences or multivalent and / or multispecific constructs. As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell into which a recombinant expression vector has been introduced. Such terms are intended to refer not only to a specific subject cell, but also to the progeny of such a cell.

[0279] In one aspect of the invention, a vector is provided, comprising a polynucleotide encoding a polypeptide or construct of the present invention or a cDNA comprising the polynucleotide. In another aspect of the invention, a host cell transformed with the vector is provided, which is capable of expressing a polypeptide or construct of the present invention. Suitably, the host cell is a bacterium, such as Escherichia coli, a yeast belonging to the genus Aspergillus, Saccharomyces, Kluyveromyces, Hansenula or Pichia, such as Saccharomyces cerevisiae or Pichia pastoris.

[0280] Autoimmune and / or inflammatory diseases

[0281] Autoimmune diseases occur when the immune system has adverse responses to normal body tissues. Autoimmune disorders can cause damage to body tissues, abnormal organ growth and / or changes in organ function. The disorder can only affect one organ or tissue type or can affect multiple organs and tissues. Organs and tissues that are usually affected by autoimmune disorders include blood components such as red blood cells, blood vessels, connective tissues, endocrine glands such as thyroid or pancreas, muscles, joints and skin. Inflammatory diseases are diseases characterized by inflammation. Many inflammatory diseases are autoimmune diseases and vice versa.

[0282] Suitably, the polypeptide, pharmaceutical composition or construct of the invention is for use as a medicament and more suitably for use in the treatment of autoimmune diseases and / or inflammatory diseases.

[0283] The polypeptides of the invention (suitably when delivered orally) are ideal for treating inflammatory diseases where IL-7 and / or L-TSLP contribute to at least part of the pathology and the polypeptides can reach tissues where IL-7 and / or L-TSLP are bioactive.

[0284] The polypeptide of the present invention binds to the receptor (IL-7R). It can therefore destroy yet undiscovered cytokines or other binding partners of IL-7R that may be involved in the disease.

[0285] Inhibition of IL-7 and L-TSLP binding to IL-7R

[0286] Evidence that different isoforms of TSLP are differentially expressed and act via different signaling pathways suggests that it may be possible to selectively target the disease-associated activities of L-TSLP rather than the physiologically beneficial effects of the short isoform of this cytokine. The polypeptides of the invention inhibit the binding of IL-7 to IL-7R. Information from computer simulations of the interaction of the V7R-2E9 molecule with IL-7Rα and the recently published structure of the TSLP:TSLPR:IL-7R complex (Verstraete et al. 2017) strongly suggests that V7R-2E9 and other polypeptides of the invention inhibit the binding of both IL-7 and L-TSLP to IL-7Rα. The ability of V7R-2E9 to potently block the receptor binding and cell-based biological activities of both IL-7 (Il-7-induced STAT5 phosphorylation) and L-TSLP (TSLP-induced TARC secretion) has now been confirmed. Because V7R-2E9 binds to IL-7Rα but not TSLP, V7R-2E9 and related ICVDs are expected to block only the pro-inflammatory activity of L-TSLP, and the important mucosal homeostatic function of S-TSLP is not affected.

[0287] Inflammatory bowel disease (IBD)

[0288] The chronic inflammatory bowel diseases Crohn's disease and ulcerative colitis that afflict children and adults are examples of autoimmune and inflammatory diseases of the GIT (Hendrickson et al. 2002). Ulcerative colitis is defined as a condition in which the inflammatory response and morphological changes remain confined to the colon. The rectum is involved in 95% of patients. Inflammation is largely confined to the mucosa and includes variable severity of ulcers, edema and bleeding and continuous damage to the length of the colon (Hendrickson et al. 2002). Ulcerative colitis is usually manifested by the presence of mixed blood and mucus in the stool and lower abdominal cramps, which are most severe during defecation. Clinically, the presence of blood and mucosa in diarrhea distinguishes ulcerative colitis from irritable bowel syndrome in which blood is not present. Unlike ulcerative colitis, the presentation of Crohn's disease is usually less obvious, which leads to late diagnosis. Factors such as the location, extent and severity of the damage determine the extent of gastrointestinal symptoms. Patients with ileocolic damage usually have postprandial abdominal pain and right lower abdominal tenderness and occasional inflammatory masses. Symptoms associated with gastroduodenal Crohn's disease include early satiety, nausea, vomiting, epigastric pain or dysphagia. Perianal disease as well as anal tags, deep anal fissures and fistulas are common (Hendrickson et al. 2002).

[0289] Suitably, the polypeptides, pharmaceutical compositions or constructs of the invention are used to treat autoimmune and / or inflammatory diseases of the GI tract in which IL-7 and / or L-TSLP contribute to the pathology of such diseases.

[0290] Suitably, the polypeptides, pharmaceutical compositions or constructs of the invention are used to treat autoimmune and / or inflammatory diseases of the GI tract selected from Crohn's disease, ulcerative colitis, irritable bowel disease, type II diabetes, glomerulonephritis, autoimmune hepatitis, Sjogren's syndrome, celiac disease and drug- or radiation-induced mucositis (more suitably Crohn's disease or ulcerative colitis, most suitably ulcerative colitis).

[0291] Eosinophilic Esophagitis (EoE)

[0292] Eosinophilic esophagitis (EoE, also spelled eosinophilic oesophagitis and also known as allergic esophagitis) is an allergic inflammatory condition of the esophagus involving eosinophils (a type of white blood cell). Symptoms are difficulty swallowing, food impaction, vomiting, and heartburn.

[0293] Suitably, the polypeptide, pharmaceutical composition or construct of the invention is used to treat eosinophilic esophagitis. More suitably, the polypeptide, pharmaceutical composition or construct is used to treat eosinophilic esophagitis and is administered orally.

[0294] Other autoimmune / inflammatory diseases

[0295] Other GIT diseases that may be treated, for example, via oral administration of a polypeptide of the invention include, for example, inflammatory diseases mucositis (suitably, drug- and radiation-induced mucositis), asthma, idiopathic pulmonary fibrosis, atopic dermatitis, allergic conjunctivitis, allergic rhinitis, Netherton syndrome, food allergies, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis (ABPA), allergic fungal sinusitis, cancer, COPD, scarring, chronic rhinosinusitis (CRS), nasal polyposis, chronic eosinophilic pneumonia, eosinophilic bronchitis, celiac disease, and Churg-Strauss syndrome.

[0296] In mucositis, focus can occur anywhere from the oral cavity to the anus, and for oral cavity and esophageal lesions, mouthwash or cream preparations containing variable domains can be used.For anus and rectal lesions, suppositories, creams or foams containing variable domains will be suitable for local application.Immunoglobulin chain variable domains will be removed from lamina propria or other inflammatory sites via being adsorbed in the blood stream at the inflammation site or removing via lymph and subsequently entering the blood stream.Therefore, domains arrive at the lungs via blood stream and remove via the glomerular filtration in the kidney.Therefore, domains play a therapeutic role in diseases such as autoimmune diseases, type II diabetes and glomerulonephritis and have good basic principles.

[0297] In one embodiment, the polypeptide or construct of the invention is for use in treating or preventing atopic dermatitis, suitably by topical delivery to and / or through the skin, suitably in the form of a cream, nanoparticles, ointment or hydrogel.

[0298] Suitably, the polypeptides, pharmaceutical compositions or constructs are used to treat other autoimmune / inflammatory diseases in which IL-7 and / or L-TSLP are responsible for part of the observed pathology.

[0299] Therapeutic Uses and Delivery

[0300] A therapeutically effective amount of a polypeptide, pharmaceutical composition or construct of the invention is an amount that is effective to inhibit the binding of IL-7 and / or L-TSLP to IL-7R in a subject to a significant degree after single or multiple doses are administered to a subject. The therapeutically effective amount may vary depending on factors such as the disease state, age, sex and weight of the individual and the ability of the polypeptide, pharmaceutical composition or construct to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or deleterious effects of the polypeptide, pharmaceutical composition or construct of the invention. The polypeptide or construct of the invention may be incorporated into a pharmaceutical composition suitable for administration to a subject. The polypeptide or construct of the invention may be in the form of a pharmaceutically acceptable salt.

[0301] The pharmaceutical composition of the present invention can be suitably formulated for oral, intramuscular, subcutaneous or intravenous delivery. The pharmaceutical composition of the present invention can be in various forms. These include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. Solid dosage forms are preferred. The polypeptide, pharmaceutical composition or construct of the present invention can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc. For the treatment of eosinophilic esophagitis, it is particularly preferred to deliver in the form of lozenges. For the treatment of atopic dermatitis, it is particularly preferred to deliver in the form of creams.

[0302] Typically, the pharmaceutical composition comprises a polypeptide or construct of the present invention and a pharmaceutically acceptable diluent or carrier. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc. and combinations thereof. Pharmaceutically acceptable carriers may also include a small amount of auxiliary substances that enhance the shelf life or effectiveness of the polypeptide or construct of the present invention, such as a wetting agent or emulsifier, preservative or buffer. The pharmaceutical composition may include an anti-adherent, an adhesive, a coating, a disintegrant, a flavoring agent, a coloring agent, a lubricant, an adsorbent, a preservative, a sweetener, a lyophilizing excipient (including a lyophilizing protectant) or a compression aid.

[0303] In patients with EoE and UC, it is believed that the inflamed intestinal mucosal epithelial barrier is impaired and will therefore facilitate the penetration of orally administered polypeptides of the present invention into the underlying mucosal tissue, thereby potentially inhibiting both IL-7 and L-TSLP activity in target tissues at the site of inflammation. Oral administration of the polypeptides of the present invention should limit systemic inhibition of IL-7 and L-TSLP activity, thereby reducing the risk of general immunosuppression associated with conventional IL-7 and L-TSLP antibodies given by injection. It is possible that short-term anti-IL-7R antibody treatment, such as occurs in animal models, provides a clinical effect (remission) for an extended period of time due to the depletion of pathogenic T cells. However, repeated systemic administration of existing IL-7Rα blocking antibodies to patients may result in inhibition of thymic T cell development and depletion of T cells in the periphery, resulting in significant systemic immunosuppression. Inflammatory bowel disease (Crohn's disease and ulcerative colitis) and EoE are primarily localized in the gastrointestinal tract; therefore, oral administration of the polypeptides of the present invention to the inflamed intestinal mucosa provides the potential to achieve a localized effect with limited systemic exposure, thereby reducing the risk of immunosuppression in non-diseased tissues.

[0304] Thus, suitably, the polypeptides, pharmaceutical compositions or constructs of the invention are administered orally. The polypeptides, pharmaceutical compositions or constructs may be delivered orally (such as for the treatment of EoE) to the oral cavity, pharynx and esophagus (more suitably, esophagus), or may be delivered orally (such as for the treatment of IBD) to the duodenum, jejunum, ileum, cecum, colon, rectum and / or anal canal.

[0305] The key issue of oral delivery is to ensure that enough polypeptides, pharmaceutical compositions or constructs reach the area of ​​the intestinal tract where it is needed. Factors that prevent the polypeptides, pharmaceutical compositions or constructs of the present invention from reaching the area of ​​the intestinal tract where it is needed include the presence of proteases in the digestive secretions, which can degrade the polypeptides, pharmaceutical compositions or constructs of the present invention. Suitably, the polypeptides, pharmaceutical compositions or constructs of the present invention are basically stable due to the inherent properties of the polypeptides or constructs themselves in the presence of one or more of such proteases. Suitably, the polypeptides of the present invention or constructs are lyophilized before being incorporated into the pharmaceutical composition.

[0306] The polypeptide of the present invention can also be provided with an enteric coating. An enteric coating is a polymer barrier applied to an oral drug that helps prevent the polypeptide from being affected by the low pH of the stomach. Materials used for enteric coatings include fatty acids, waxes, shellac, plastics, and plant fibers. Suitable enteric coating components include methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, cellulose acetate hydroxypropyl methyl succinate (hydroxypropyl methylcellulose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymers, sodium alginate, and stearic acid. Suitable enteric coatings include pH-dependent release polymers. These are polymers that are insoluble at the high acidic pH present in the stomach but dissolve rapidly at lower acidic pH. Therefore, suitably, the enteric coating is insoluble in the acidic gastric juice of the stomach (pH ~ 3), but dissolves in the higher pH environment present in the small intestine (pH above 6) or in the colon (pH above 7.0). The pH dependent release polymer is selected such that the polypeptide or construct of the invention is released approximately at the time that the dose reaches the small intestine.

[0307] If administered orally to treat IBD, the polypeptides of the invention are suitably provided with an enteric coating.If administered orally to treat EoE, the polypeptides of the invention are suitably provided in the form of a compressed tablet.

[0308] The polypeptide, construct or pharmaceutical composition of the present invention can be delivered topically. Such pharmaceutical compositions can be suitably in the form of creams, ointments, lotions, gels, foams, transdermal patches, powders, pastes or tinctures, and can suitably include vitamin D3 analogs (e.g., calcipotriol and maxacalcitol), steroids (e.g., fluticasone propionate, betamethasone valerate and clobetasol propionate), retinoic acid (e.g., tazarotene), coal tar and anthralin. Topical agents are often combined with each other (e.g., vitamin D3 and steroids) or with other agents such as salicylic acid.

[0309] The polypeptide, construct or pharmaceutical composition of the present invention can be formulated into a preparation for injection by dissolving, suspending or emulsifying them in an aqueous or non-aqueous solvent, such as a vegetable oil or other similar oil, a fatty acid glyceride, an ester of a higher fatty acid or propylene glycol; and if necessary, with conventional additives, such as a solubilizing agent, an isotonic agent, a suspending agent, an emulsifier, a stabilizer and a preservative. Acceptable carriers, excipients and / or stabilizers are nontoxic to the recipient at the dosage and concentration employed, and include buffers such as phosphoric acid, citric acid and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, metacresol, parachlorometacresol, methylparaben or propylparaben, benzalkonium chloride or a combination thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine amino acids, tyrosine, tryptophan, methionine, serine, proline, and combinations thereof; monosaccharides, diosmin, and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents, such as EDTA; sugars, such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbitol, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or nonionic surfactants, such as polysorbates, POE ethers, poloxamers, Triton-X, or polyethylene glycol.

[0310] For all modes of delivery, the polypeptide, pharmaceutical composition or construct of the present invention can be formulated in a buffer to stabilize the pH of the composition at a concentration between 5-50 g / l, or more suitably between 15-40 g / l, or more suitably between 25-30 g / l. Examples of suitable buffer components include physiological salts, such as sodium citrate and / or citric acid. Suitably, the buffer contains 100-200 mM, more suitably 125-175 mM of physiological salts, such as sodium chloride. Suitably, the buffer is selected to have a pKa close to the pH of the composition or the physiological pH of the patient.

[0311] Exemplary polypeptide or construct concentrations in the pharmaceutical composition can range from about 1 mg / mL to about 200 mg / ml, or from about 50 mg / mL to about 200 mg / mL, or from about 150 mg / mL to about 200 mg / mL.

[0312] Aqueous formulations of polypeptides, constructs or pharmaceutical compositions of the present invention can be prepared in a pH buffer solution, for example, in a range of about 4.0 to about 7.0, or about 5.0 to about 6.0, or alternatively about 5.5. Examples of suitable buffers include phosphate, histidine, citric acid, succinic acid, acetate buffers and other organic acid buffers. Depending on the desired tension of, for example, the buffer and formulation, the buffer concentration can be about 1 mM to about 100 mM, or about 5 mM to about 50 mM.

[0313] The tension of the pharmaceutical composition can be changed by including a tension regulator. Such tension regulators can be charged or uncharged chemical substances. Typical uncharged tension regulators include sugars or sugar alcohols or other polyols, preferably trehalose, sucrose, mannitol, glycerol, 1,2-propylene glycol, raffinose, sorbitol or lactitol (especially trehalose, mannitol, glycerol or 1,2-propylene glycol). Typical charged tension regulators include salts, such as sodium, potassium or calcium ions and chloride, sulfate, carbonate, sulfite, nitrate, lactate, succinate, acetate or maleate ions (especially sodium chloride or sodium sulfate); or amino acids such as arginine or histidine. Suitably, aqueous formulations are isotonic, although hypertonic or hypotonic solutions may be suitable. The term "isotonic" represents a solution having the same tension as a certain other solution to which it is compared, such as a physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, for example in an amount of 1 mM to 500 nM. Suitably, at least one isotonicity agent is included in the composition.

[0314] Surfactant can also be added to the pharmaceutical composition to reduce the aggregation of the polypeptide or construct of preparation and / or to minimize the formation of particles in the preparation and / or to reduce adsorption. Exemplary surfactants include polyoxyethylene sorbitol fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (Poloxamer, Pluronic) and sodium lauryl sulfate (SDS). The example of suitable polyoxyethylene sorbitol-fatty acid esters is polysorbate 20 and polysorbate 80. The scope of the exemplary concentration of surfactant is from about 0.001% to about 10% w / v.

[0315] Lyoprotectants may also be added to protect the polypeptide or construct of the invention from destabilizing conditions during the freezing process. For example, known lyoprotectants include sugars (including glucose, sucrose, mannose, and trehalose); polyols (including mannitol, sorbitol, and glycerol); and amino acids (including alanine, glycine, and glutamic acid). The lyoprotectant may be included in an amount of about 10 mM to 500 mM.

[0316] The dosage range for administering the polypeptide of the present invention, the pharmaceutical composition of the present invention or the construct is those dosage ranges that produce the desired therapeutic effect. The required dosage range depends on the accurate nature of the polypeptide of the present invention, the pharmaceutical composition or the construct, the route of administration, the nature of the preparation, the age of the patient, the nature, degree or severity of the patient's condition, contraindications (if any) and the judgment of the attending physician. The variation of these dosage levels can be adjusted to optimize using standard empirical routines.

[0317] Suitably, the range of daily dosage of polypeptide of the present invention, pharmaceutical composition of the present invention or construct is 50ng-50mg / kg, such as 50ug-40mg / kg, such as 5-30mg / kg. Unit dosage can change from less than 100mg, but usually in the region of 250-2000mg / dose, it can be used daily or more frequently, for example 2,3 or 4 times / day or less frequently, for example once every two days or once a week, once every two weeks or once a month.

[0318] In one aspect of the present invention, there is provided a use of a polypeptide, pharmaceutical composition or construct of the present invention in the manufacture of a medicament for treating an autoimmune disease. In another aspect of the present invention, there is provided a method for treating an autoimmune disease, comprising administering a therapeutically effective amount of a polypeptide, pharmaceutical composition or construct of the present invention to a person in need thereof.

[0319] In one aspect of the present invention, there is provided a use of a polypeptide, pharmaceutical composition or construct of the present invention in the manufacture of a medicament for treating an autoimmune disease / inflammatory disease. In another aspect of the present invention, there is provided a method for treating an autoimmune disease / inflammatory disease, comprising administering a therapeutically effective amount of a polypeptide, pharmaceutical composition or construct of the present invention to a person in need thereof.

[0320] The word 'treatment' is intended to include prophylactic as well as therapeutic treatment. Treatment of a disease also includes treating exacerbations and also includes treating a patient in remission from disease symptoms to prevent recurrence of disease symptoms.

[0321] Combination therapy

[0322] The pharmaceutical compositions of the present invention may also include one or more active agents (e.g., active agents suitable for treating the diseases mentioned herein). It is within the scope of the present invention to use the pharmaceutical compositions of the present invention in therapeutic methods for treating autoimmune diseases as an addition to or in combination with other established therapies commonly used in treating autoimmune diseases.

[0323] For the treatment of IBD (such as Crohn's disease or ulcerative colitis), possible combinations include combinations with, for example, one or more active agents selected from the list comprising: 5-aminosalicylic acid or a prodrug thereof (such as sulfasalazine, olsalazide or bisalazide); corticosteroids (e.g., prednisolone, methylprednisolone or budesonide); immunosuppressants (e.g., cyclosporine, tacrolimus, methotrexate, azathioprine or 6-mercaptopurine); anti-TNF-α antibodies (e.g., infliximab, adalimumab, certolizumab pegol or golimumab); anti-IL12 / IL23 antibodies (e.g., ustekinumab); anti-IL6R antibodies or small molecule IL12 / IL23 inhibitors (e.g., apimod); anti-α-4-β-7 antibodies (e.g., vedolizumab); MAdCAM-1 blockers (e.g., PF-00547659); antibodies against the cell adhesion molecule α-4-integrin (e.g., natalizumab); antibodies against the α subunit of the IL2 receptor (e.g., daclizumab or basiliximab); JAK3 inhibitors (e.g., tofacitinib or R348); Syk inhibitors and their prodrugs (e.g., fotantinib and R-406); phosphodiesterase-4 inhibitors (e.g., tetomilast); HMPL-004; probiotics; desalazone; semamod / CPSI-2364; and protein kinase C inhibitors (e.g., AEB-071). The most suitable combination agent is infliximab, adalimumab, certolizumab pegol or golimumab.

[0324] Thus, another aspect of the invention provides a pharmaceutical composition of the invention in combination with one or more additional active agents, for example one or more of the active agents described above.

[0325] In another aspect of the invention, the polypeptide, pharmaceutical composition or construct and at least one active agent selected from the above list are administered sequentially, simultaneously or separately.

[0326] Similarly, another aspect of the present invention provides a combination comprising:

[0327] (A) a polypeptide, pharmaceutical composition or construct of the present invention; and

[0328] (B) one or more other active agents,

[0329] Wherein each of components (A) and (B) is mixed with a pharmaceutically acceptable adjuvant, diluent or carrier. In this aspect of the invention, the combination product can be a single (combination) formulation or a kit-of-parts. Therefore, this aspect of the invention encompasses a combined formulation comprising a polypeptide, pharmaceutical composition or construct of the invention and another therapeutic agent mixed with a pharmaceutically acceptable adjuvant, diluent or carrier.

[0330] The present invention also encompasses a kit comprising the following components:

[0331] (i) a polypeptide, pharmaceutical composition or construct of the present invention mixed with a pharmaceutically acceptable adjuvant, diluent or carrier; and

[0332] (ii) a formulation comprising one or more other active agents in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, wherein components (i) and (ii) are each provided in a form suitable for administration in combination with each other.

[0333] Therefore, component (i) of the kit of parts is the above component (A) mixed with a pharmaceutically acceptable adjuvant, diluent or carrier. Similarly, component (ii) is the above component (B) mixed with a pharmaceutically acceptable adjuvant, diluent or carrier. One or more other active agents (i.e., above component (B)) can be any of the agents mentioned in the above combination autoimmune diseases such as IBD (e.g., Crohn's disease and / or ulcerative colitis) treatment. If component (B) is more than one other active agent, these other active agents can be formulated with each other or with component (A) or they can be formulated separately. In one embodiment, component (B) is a kind of other therapeutic agent. In another embodiment, component (B) is two other therapeutic agents. The combination product (combination preparation or kit of parts) of this aspect of the present invention can be used for the treatment or prevention of autoimmune diseases (e.g., autoimmune diseases mentioned herein).

[0334] stability

[0335] In one embodiment, the polypeptide or construct of the invention is delivered orally.Thus, the polypeptide or construct of the invention suitably substantially retains neutralizing capacity and / or efficacy when delivered orally.

[0336] Suitably, the polypeptide or construct of the invention retains neutralizing capacity and / or efficacy upon oral delivery and after exposure to the intestinal tract (e.g., after exposure to proteases of the small and / or large intestine and / or IBD inflammatory proteases). Such proteases include enteropeptidases, trypsin, chymotrypsin, and irritable bowel inflammatory proteases (such as MMP3, MMP12, and cathepsins). Proteases of the small and / or large intestine or proteases produced in the small and / or large intestine include proteases derived from intestinal symbiotic microbial flora and / or pathogens, such as proteases wherein the proteases are cell membrane-attached proteases, excreted proteases, and proteases released by cell lysis). Most suitably, the proteases are trypsin and chymotrypsin.

[0337] Suitably, the intestinal tract is the intestinal tract of a dog, pig, human, cynomolgus monkey or mouse. More suitably, the intestinal tract is the intestinal tract of a human, cynomolgus monkey or mouse, most suitably a human. The small intestine suitably consists of the duodenum, jejunum and ileum. The large intestine suitably consists of the cecum, colon, rectum and anal canal. In contrast to the gastrointestinal tract, the intestinal tract consists only of the small intestine and the large intestine. In one embodiment, the polypeptide or construct of the invention is substantially resistant to proteases of the intestinal tract, most suitably the human intestinal tract.

[0338] Stability in the oral cavity, pharynx, and esophagus

[0339] The oral cavity, pharynx and esophagus are in front of the stomach in the gastrointestinal tract. Suitably, the polypeptide or construct of the invention substantially retains neutralizing capacity and / or efficacy upon oral delivery and after exposure to the oral cavity, pharynx and esophagus (e.g., after exposure to proteases of the oral cavity, pharynx and esophagus). Proteases of the oral cavity, pharynx and esophagus include proteases derived from commensal microbial flora and / or pathogenic bacteria, such as where the proteases are cell membrane attached proteases, excreted proteases and proteases released by cell lysis).

[0340] Suitably, the oral cavity, pharynx and esophagus are those of dog, pig, human, cynomolgus monkey or mouse. More suitably, the oral cavity, pharynx and esophagus are those of human, cynomolgus monkey or mouse, most suitably human.

[0341] A polypeptide or construct of the invention substantially retains neutralizing capacity when, following exposure to proteases and / or IBD inflammatory proteases present in the small and / or large intestine, suitably 10% or more, more suitably 20% or more, more suitably 30% or more, more suitably 40% or more, more suitably 50% or more, more suitably 60% or more, more suitably 70% or more, more suitably 80% or more, more suitably 90% or more, more suitably 95% or more, or most suitably 100% of the original neutralizing capacity of the polypeptide or construct of the invention is retained.

[0342] Suitably, the polypeptide or construct of the invention substantially retains the neutralising ability after exposure to proteases present in the small and / or large intestine and / or IBD inflammatory proteases for, e.g., at least 2 hours, more suitably at least 3 hours, more suitably at least 4 hours, at least 5 hours, more suitably at least 5.5 hours, more suitably at least 6 hours, more suitably at least 6.5 hours, more suitably at least 7 hours, more suitably at least 7.5 hours, more suitably at least 10 hours, more suitably at least 13 hours or more suitably at least 16 hours at 37°C.

[0343] After at least 2 hours, more suitably at least 3 hours, more suitably at least 4 hours, more suitably at least 5 hours, more suitably at least 6 hours, more suitably at least 7 hours, more suitably at least 9 hours, more suitably at least 11 hours, more suitably at least 13 hours or more suitably at least 16 hours of exposure to the intestinal tract, more suitably the small intestine or large intestine, more suitably a human fecal extract, suitably 10% or more, more suitably 20% or more, more suitably 30% or more, more suitably 40% or more, more suitably 50% or more, more suitably 60% or more, more suitably 70% or more, more suitably 80% or more, more suitably 90% or more or more suitably 95% of the neutralizing capacity of the polypeptide or construct of the invention is retained.

[0344] After exposure to mouse small intestinal supernatant for suitably at least 1 hour, more suitably at least 2 hours, more suitably at least 3 hours, more suitably at least 4 hours, more suitably at least 5 hours or more suitably at least 6 hours, suitably 10% or more, more suitably 20% or more, more suitably 30% or more, more suitably 40% or more, more suitably 50% or more, more suitably 60% or more, more suitably 70% or more, more suitably 80% or more, more suitably 90% or more or more suitably 95% or more of the neutralizing capacity of the polypeptide or construct of the invention is retained.

[0345] Suitably 10% or more, more suitably 20% or more, more suitably 30% or more, more suitably 40% or more, more suitably 50% or more, more suitably 60% or more, more suitably 70% or more of the administered dose of the polypeptide or construct of the invention retains neutralizing capacity against IL-7 and / or L-TSLP and is maintained in the feces (suitably excreted feces or feces removed from the intestine) of mice, cynomolgus monkeys and / or humans at least 2 hours, more suitably at least 3 hours, more suitably at least 4 hours, more suitably at least 5 hours, more suitably at least 6 hours, more suitably at least 7 hours, more suitably at least 9 hours, more suitably at least 11 hours, more suitably at least 13 hours or more suitably at least 16 hours after administration.

[0346] A polypeptide of the invention or a construct of the invention remains substantially intact when suitably 10% or more, more suitably 20% or more, more suitably 30% or more, more suitably 40% or more, more suitably 50% or more, more suitably 60% or more, more suitably 70% or more, more suitably 80% or more, more suitably 90% or more, more suitably 95% or more, more suitably 99% or more, most suitably 100% of the administered amount of the polypeptide of the invention or construct remains intact following exposure to proteases and / or IBD inflammatory proteases present in the small and / or large intestine.

[0347] 'Stability' and 'survival rate' such as 'stability %' and 'survival %' are used interchangeably herein. "Substantially retaining neutralizing ability" and "substantially tolerating" are used interchangeably herein.

[0348] In one embodiment of the present invention, based on its stability in human fecal supernatant digest, a polypeptide is provided, which comprises or more suitably consists of the polypeptide sequence of any one of the ICVDs cited below:

[0349] V7R-2E9, ID-A59U, ID-A2U, ID-A9U, ID-A10U, ID-A11U, ID-A12U, ID-A14U, ID-A15U, ID-A16U, ID-A17U, ID-A18A, ID-A19U, ID -A20U, ID-A21U, ID-A24U, ID-A43U, ID-A25U, ID-A30U, ID-A31U, ID-A50U, ID-A33U, ID-A52U, ID-A34U, ID-A53U, ID-A35U, ID -A54U, ID-A36U, ID-A55U, ID-A37U, ID-A38U, ID-A57U, ID-A39U, ID-A40U, V7R-2F6, V7R-6C12, V7R-2B6, V7R-3B5, V7R-4F6, V 7R-2E5, ID-A3U, ID-A4U, ID-A5U, ID-A6U, ID-A7U, ID-A8U, ID-A13U, ID-A23U, ID-A26U, ID-A27U, ID-A28U, ID-A29U and ID-A32U.

[0350] A polypeptide is also provided, comprising three complementary determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR1 comprises the CDR1 sequence of any one of the above ICVDs or is more suitably composed thereof, CDR2 comprises the CDR2 sequence of any one of the above ICVDs or is more suitably composed thereof, and CDR3 comprises the CDR3 sequence of any one of the above ICVDs or is more suitably composed thereof. Most suitably, the polypeptide comprises all three CDRs from the above single ICVD.

[0351] Based on its stability in human fecal supernatant digest, a polypeptide is more suitably provided, which comprises or more suitably consists of any ICVD polypeptide sequence cited below:

[0352] V7R-2E9, ID-A59U, ID-A2U, ID-A9U, ID-A10U, ID-A11U, ID-A12U, ID-A14U, ID-A15U, ID-A16U, I D-A17U, ID-A18A, ID-A19U, ID-A20U, ID-A21U, ID-A24U, ID-A43U, ID-A25U, ID-A30U, ID-A31U, I D-A50U, ID-A33U, ID-A52U, ID-A34U, ID-A53U, ID-A35U, ID-A54U, ID-A36U, ID-A55U, ID-A37U, I D-A38U, ID-A57U, ID-A39U, ID-A40U, V7R-2F6, V7R-6C12, V7R-2B6, V7R-3B5, V7R-4F6 and V7R-2E5.

[0353] A polypeptide is also provided, comprising three complementary determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR1 comprises the CDR1 sequence of any one of the above ICVDs or is more suitably composed thereof, CDR2 comprises the CDR2 sequence of any one of the above ICVDs or is more suitably composed thereof, and CDR3 comprises the CDR3 sequence of any one of the above ICVDs or is more suitably composed thereof. Most suitably, the polypeptide comprises all three CDRs from the above single ICVD.

[0354] Based on its stability in human fecal supernatant digest, a polypeptide is more suitably provided, which comprises or more suitably consists of any ICVD polypeptide sequence cited below:

[0355] V7R-2E9, ID-A59U, ID-A2U, ID-A9U, ID-A10U, ID-A11U, ID-A12U, ID-A14U, ID-A1 5U, ID-A16U, ID-A17U, ID-A18A, ID-A19U, ID-A20U, ID-A21U, ID-A24U, ID-A43U, I D-A25U, ID-A30U, ID-A31U, ID-A50U, ID-A33U, ID-A52U, ID-A34U, ID-A53U, ID-A 35U, ID-A54U, ID-A36U, ID-A55U, ID-A37U, ID-A38U, ID-A57U, ID-A39U, ID-A40U.

[0356] A polypeptide is also provided, comprising three complementary determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR1 comprises the CDR1 sequence of any one of the above ICVDs or is more suitably composed thereof, CDR2 comprises the CDR2 sequence of any one of the above ICVDs or is more suitably composed thereof, and CDR3 comprises the CDR3 sequence of any one of the above ICVDs or is more suitably composed thereof. Most suitably, the polypeptide comprises all three CDRs from the above single ICVD.

[0357] Preparation method

[0358] The polypeptides of the present invention can be obtained and manipulated using, for example, the techniques disclosed in Green and Sambrook 2012 Molecular Cloning: A Laboratory Manual 4th edition Cold Spring Harbour Laboratory Press.

[0359] Monoclonal antibodies can be produced using hybridoma technology by fusing B cells that produce specific antibodies with myeloma (B cell cancer) cells that are selected for their ability to grow in tissue culture and the absence of antibody chain synthesis. and Milstein 1975 and Nelson et al. 2000 ).

[0360] Monoclonal antibodies directed against a defined antigen can be obtained, for example, by:

[0361] a) immortalizing lymphocytes obtained from the peripheral blood of an animal previously immunized with a defined antigen with immortalized cells, preferably myeloma cells, in order to form hybridomas,

[0362] b) Culturing the resulting immortalized cells (hybridomas) and recovering cells producing antibodies with the desired specificity.

[0363] Alternatively, there is no need to use hybridoma cells. Thus, monoclonal antibodies can be obtained by a method comprising the following steps:

[0364] a) cloning a DNA or cDNA sequence obtained from lymphocytes, especially peripheral blood lymphocytes, of an animal (suitably previously immunized with a defined antigen) into a vector, especially into a bacteriophage and more particularly a filamentous phage,

[0365] b) transforming prokaryotic cells with the above vector under conditions that allow the production of antibodies,

[0366] c) selecting the antibodies by subjecting the antibodies to antigen-affinity selection,

[0367] d) Recovering antibodies with the desired specificity.

[0368] Methods for immunizing camels, cloning the VHH repertoire of B cells circulating in the blood (Chomezynnski and Sacchi 1987) and isolating antigen-specific VHH from immune (Arbabi-Ghahroudi et al. 1997) and non-immune (Tanha et al. 2002) libraries using phage, yeast or ribosome display are known (WO92 / 01047, Nguyen et al. 2001 and Harmsen et al. 2007).

[0369] Antigen binding fragments of antibodies such as scFv and Fv fragments can be isolated and expressed in E. coli (Miethe et al. 2013, Skerra et al. 1988 and Ward et al. 1989).

[0370] The DNA or cDNA encoding the polypeptide may be mutated, which mutation is silent for the amino acid sequence of the polypeptide, but provides preferred codons for translation by a particular host. Preferred codons for translation of nucleic acids in, for example, Escherichia coli and Saccharomyces cerevisiae are known.

[0371] The sudden change of polypeptide can be realized for example by replacing, adding or lacking the nucleic acid encoding polypeptide.Replacement, adding or lacking the nucleic acid encoding polypeptide can be introduced by many methods, including for example fallibility PCR, reorganization, oligonucleotide directed mutagenesis, assembly PCR, PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, cycle overall mutagenesis, index overall mutagenesis, site-specific mutagenesis (Ling et al. 1997), gene reassembly, gene site saturation mutagenesis (GSSM), synthetic connection reassembly (SLR) or the combination of these methods.The modification, adding or lacking of nucleic acid can also be introduced by including the following method: reorganization, cycle sequence reorganization, thiophosphate modified DNA mutagenesis, template mutagenesis containing uracil, gap duplex mutagenesis (gapped duplex mutagenesis), point mismatch repair mutagenesis (point mismatch repair mutagenesis), repair-defective host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, overall mutagenesis, chimeric nucleic acid polymer generation or its combination.

[0372] Specifically, artificial gene synthesis can be used (Nambiar et al. 1984, Sakamar and Khorana 1988, Wells et al. 1985 and Grundstrom et al. 1985). The gene encoding the polypeptide of the present invention can be synthetically produced by, for example, solid phase DNA synthesis. The entire gene can be synthesized de novo without the need for precursor template DNA. In order to obtain the desired oligonucleotide, the building blocks are sequentially coupled to the growing oligonucleotide chain in the order required for the product sequence. After the chain assembly is completed, the product is released from the solid phase into the solution, deprotected and collected. The product can be separated by high performance liquid chromatography (HPLC) to obtain the desired high purity oligonucleotide (Verma and Eckstein 1998)

[0373] The expression of immunoglobulin chain variable domains such as VH and VHH can be achieved using suitable expression vectors such as prokaryotic cells such as bacteria, for example Escherichia coli (for example according to the protocol disclosed in WO94 / 04678, which is incorporated herein by reference and described in further detail below). The expression of immunoglobulin chain variable domains such as VH and VHH can also be achieved using eukaryotic cells such as insect cells, CHO cells, Vero cells or suitably yeast cells, such as yeast belonging to the genus Aspergillus, Saccharomyces, Kluyveromyces, Hansenula or Pichia. Suitably, Saccharomyces cerevisiae is used (for example, according to the protocol disclosed in WO94 / 025591, which is incorporated herein by reference and described in further detail below).

[0374] Specifically, VHH can be prepared using Escherichia coli according to the method disclosed in WO94 / 04678 by a method comprising the following steps:

[0375] a) cloning a DNA or cDNA sequence encoding a VHH (e.g. obtained from lymphocytes of camel or produced synthetically), optionally comprising a His-tag, in a Bluescript vector (Agilent Technologies),

[0376] b) After amplification, the cloned fragment was recovered using a 5' primer containing an XhoI site specific for VHH and a 3' primer containing a SpeI site with the following sequence,

[0377] TC TTA ACT AGT GAG GAG ACG GTG ACC TG (SEQ ID NO: 81),

[0378] c) cloning of the fragment recovered in the phase in the Immuno PBS vector after digestion of the vector with XhoI and SpeI restriction enzymes (Huse et al. 1989),

[0379] d) transforming a host cell, especially Escherichia coli, by transfection using the recombinant Immuno PBS vector of step c,

[0380] e) Recovering the expression product of the VHH coding sequence, for example by affinity purification, such as by chromatography on a column using protein A, cation exchange or nickel-affinity resin (if the VHH comprises a His-tag).

[0381] Alternatively, immunoglobulin chain variable domains such as VH and VHH can be obtained by a method comprising the following steps:

[0382] a) obtaining a DNA or cDNA sequence encoding a VHH having a defined specific antigen binding site,

[0383] b) amplifying the obtained DNA or cDNA using a 5' primer containing a start codon and a Hindlll site and a 3' primer containing a stop codon and having an XhoI site,

[0384] c) recombining the amplified DNA or cDNA into the Hindlll (position 2650) and XhoI (position 4067) sites of plasmid pMM984 (Merchlinsky et al. 1983),

[0385] d) transfecting permissive cells, in particular NB-E cells (Faisst et al. 1995), with the recombinant plasmid, and e) recovering the product obtained.

[0386] Additionally, immunoglobulin chain variable domains such as VHH or VH can be produced using E. coli or S. cerevisiae according to the methods disclosed in Frenken et al. 2000 and WO99 / 23221 (incorporated herein by reference in their entirety), as follows:

[0387] After taking a blood sample from the immunized llama and enriching the lymphocyte population via Ficoll (neutral highly branched, high-quality hydrophilic polysaccharide that dissolves rapidly in aqueous solution - Pharmacia) discontinuous gradient centrifugation, isolating total RNA by acid guanidine thiocyanate extraction (Chomezynnski and Sacchi 1987), and first-strand cDNA synthesis (e.g., using a cDNA kit such as RPN 1266 (Amersham)), a DNA fragment encoding VHH and VH fragments and a portion of the short or long hinge region was amplified by PCR using specific primers described in detail on pages 22 and 23 of WO99 / 23221. After digestion of the PCR fragment with PstI and HindIII or BstEII, the DNA fragment with a length between about 300 and 450 bp was purified via agarose gel electrophoresis and ligated in the E. coli phagemid vector pUR4536 or the episomal S. cerevisiae expression vector pUR4548, respectively. pUR4536 is derived from pHEN (Hoogenboom et al. 1991) and contains the lacIq gene and unique restriction sites to allow cloning of llama VHH and VH genes. pUR4548 is derived from pSY1 (Harmsen et al. 1993). The BstEII site in the leu2 gene was removed from this plasmid via PCR, and the cloning site between the SUC2 signal sequence and the terminator was replaced to facilitate cloning of VH / VHH gene fragments. VH / VHH has a c-myc tag at the C-terminus for detection. Individual E. coli JM109 colonies were transferred to medium supplemented with 1% glucose and 100 mg L -1 In a 96-well microtiter plate of 150ml 2TY culture medium of ampicillin. After overnight growth (37°C), repeat the plate in a 2TY culture medium containing 100mg L-1 ampicillin and 0.1mM IPTG. After overnight incubation again and optionally freezing and thawing, the cells are centrifuged and precipitated, and the supernatant can be used in ELISA. Each Saccharomyces cerevisiae colony is transferred to a test tube containing a selective minimal culture medium (comprising 0.7% yeast nitrogen base, 2% glucose, supplemented with essential amino acids and alkali), and grown at 30°C for 48h. Subsequently, the culture is diluted ten times in YPGal culture medium (comprising 1% yeast extract, 2% bacterial peptone and 5% galactose). After growing for 24h and 48h, the cells are precipitated and the culture supernatant can be analyzed in ELISA. The absorbance (OD600) at 600nm is optionally measured.

[0388] Additionally, immunoglobulin chain variable domains such as VH / VHH can be produced using Saccharomyces cerevisiae using the following procedure:

[0389] Isolate the naturally occurring DNA sequence encoding VH / VHH or obtain a synthetically produced DNA sequence encoding VH / VHH, comprising a 5'-UTR, a signal sequence, a stop codon and flanked by Sad and HindIII sites (such synthetic sequences can be produced as outlined above or can be ordered, for example, from commercial suppliers such as Geneart (Life Technologies)).

[0390] The VH / VHH genes are transferred to the multicopy integration (MCI) vector pUR8569 or pUR8542 using restriction sites as described below. The DNA sequences encoding VHH, optionally contained in the shuttle vector, cassette or other synthetic gene construct and MCI vector, are cut with Sad and HindIII using the following: 25ul VHH DNA (Geneart plasmid or MCI vector), 1ul Sad, 1ul HindIII, 3ul of a suitable buffer for double enzyme digestion such as NEB buffer 1 (New England Biolabs), at 37°C overnight. 25ul of the digested VHH-encoding DNA and 25ul of the digested MCI vector are run on a 1.5% agarose gel with 1xTAE buffer and then gel extracted, for example, using a QIAquick gel extraction kit (Qiagen). The ligation of digested MCI vector and digested DNA encoding VH / VHH was set up as follows: 100 ng vector, 30 ng VHH gene, 1.5 ul 10× ligase buffer, 1 ul T4 DNA ligase and ddH 2 O. Ligation was then performed at 16°C overnight.

[0391] Next, transform the E. coli cells. For chemically competent XL-1 blue cells, 200ul of heat competent XL-1 blue cells were thawed and 5ul of ligation mixture was added on ice for about 30 minutes, followed by heat shock at 42°C for 90 seconds. Then 800ul of Luria-Bertani low salt medium supplemented with 2% glucose was added, and the cells were recovered at 37°C for 2 hours. The cells were plated on Luria-Bertani agar and ampicillin (100ug / ml) plates and kept overnight at 37°C. For electrocompetent TG1 E. coli cells, an electroporation cup was used. In the electroporation cup: 50ul of electrocompetent TG1 cells and 1ul of ligation mixture were thawed on ice for about 15 minutes. The cup was placed in a support and pulsed. 500ul of 2TY culture medium was added and the cells were recovered at 37°C for 30 minutes. 100ul of cells were plated on Luria-Bertani agar plates containing ampicillin (100ug / ml) and 2% glucose. The plates were kept at 37°C overnight.

[0392] After cloning the VH / VHH genes into E. coli as described in detail above, the linearized MCI vector can be used to transform S. cerevisiae. Prior to transformation, several steps are performed: (i) the DNA should be changed from circular to linear by digestion, otherwise the DNA cannot be integrated into the yeast genome, and (ii) the digested DNA should be cleared of impurities by ethanol precipitation. In addition, during the transformation process, the yeast cells are made semipermeable so that the DNA can pass through the membrane.

[0393] Preparation for yeast transformation: Midi-prep prepared from selected E. coli colonies expressing VH / VHH genes was digested with HpaI as described below. Prepare a 10 μl yeast culture medium containing 20 ng midi-prep, 5 μl HpaI, 10 μl of an appropriate buffer such as NEB4 buffer (BioLabs), and ddH 2 0.5 μl of 1% HO solution.

[0394] Cut the DNA with HpaI overnight at room temperature. Next, perform an ethanol precipitation (and set aside the 5ul sample from the HpaI digestion). Add 300ul of 100% ethanol to the 95ul HpaI digested midiprep, vortex and spin at full speed for 5 minutes. Carefully decant when a pellet is present, add 100ul of 70% ethanol, and then spin again at full speed for 5 minutes. Decant the sample again and keep at 50-60°C until the pellet is dry. Resuspend the pellet in 50ul ddH 2 O. Run 5ul on the gel next to 5ul of HpaI digested sample.

[0395] Yeast transformation: Prepare YNBglu plates. Use 10 g agar + 425 ml water (sterilized), 25 ml filtered 20× YNB (3.35 g YNB (yeast nitrogen base), in 25 ml sterilized HO. 20) and 50ml sterilized 20% glucose and poured into a petri dish. Pick a yeast colony from the mother plate and grow it overnight in 3ml YSD (yeast extract powder glucose) at 30°C. Prepare about 600ml YSD on the second day and fill 3 flasks with 275ml, 225ml and 100ml YSD. Add 27.5ul yeast YSD culture to the first flask and mix lightly. Take 75ml from the first flask and place it in the second flask, mix lightly. Take 100ml from the second flask and place it in the third flask, mix lightly. Grow until an OD660 between 1 and 2 is reached. The flasks that reach this OD are distributed in 4 Falcon tubes, each with ±45ml. Spin at 4200rpm for 2 minutes. Discard the supernatant. The pellets in the two Falcon tubes are dissolved in 45ml H 2 O (reduce the number of tubes from 4 to 2). Spin at 4200 rpm for 2 minutes. Dissolve the pellet in 45 ml HO. 2 O (from 2 tubes to 1 tube). Spin at 4200rpm for 2 minutes. Dissolve the pellet lightly in 5ml lithium acetate (LiAc) (100mM) and spin for a few seconds. Carefully discard some of the LiAc, but keep more than half of the LiAc in the tube. Vortex the cells, boil the vector DNA for 5 minutes and quickly cool in ice water. Add to a 15ml tube containing the following: 240ul PEG, 50ul cells, 36uLiAc (1M), 25ul vector DNA, 45ul ethanol precipitated VH / VHH. Mix gently after each step (the blank sample is treated the same way, but there is no ethanol precipitated VH / VHH). Incubate at 30°C for 30 minutes, invert the tube slightly 3-4 times, and then heat shock at 42°C for 20-25 minutes. Spin at 6000rpm for a short time. Gently remove the supernatant and add 250ul ddH 2 O and mix. All of them are streaked on YNBglu plates until the plates are dry and grown at 30°C for 4-5 days. Finally, YNBglu plates are prepared by the following: the plates are divided into 6 equal parts, each part is numbered 1 to 6, the largest colony is inoculated and number 1 is streaked. Repeat the above process for other colonies from 1 to 6 from large to small. Grow at 30°C for 3-4 days until colonies are produced. Glucose is used as a carbon source to grow VH / VHH colonies, and VH / VHH induction is performed by opening the galactose-7-promoter by adding 0.5% galactose. 3mL small-scale culture is carried out to test colonies, and the colonies showing the best expression of VH or VHH are selected. This colony is then used in purification.

[0396] Purification: VH / VHH are purified by cation exchange chromatography with a strong anion resin such as Capto S. On day 1, inoculate a selected yeast colony expressing VH / VHH in 5 ml YSD medium (YS medium + 2% glucose) and grow the cells in a 25 mL sealed sterilized tube at 30°C (shaking at 180 rpm). On day 2, dilute 5 ml of the overnight culture in 50 mL of freshly prepared YS medium + 2% glucose + 0.5% galactose and grow the cells in a 250 ml aerated baffled flask at 30°C for two nights (shaking at 180 rpm). On day 4, spin down the cells at 4200 rpm in a centrifuge for 20 min. Cation exchange chromatography using a strong anion resin: The pH of the supernatant containing the ligand is adjusted to 3.5. Wash with 50 mL ddH 2 O wash 0.75ml resin (+ / -0.5mL slurry) / 50mL supernatant, then wash three times with binding buffer. The washed resin is added to the supernatant and the suspension is incubated on a shaker at 4°C for 1.5 hours. The resin-bound VH / VHH is precipitated by centrifugation at 500g for 2 minutes and washed with washing buffer. The supernatant is decanted and the resin is resuspended with 10mL binding buffer. The filtrate is placed in a PD-10 column, the resin is poured into the column and the resin is precipitated for a while, and then the filtrate is added to the resin. Wait until all the binding buffer runs through. Elute VH / VHH with 6x0.5ml elution buffer. Collect the elution fractions in an eppendorf tube. Measure the protein concentration of 6 elution fractions with Nanodrop. Pool the fractions containing VHH and transfer the solution to a 3,500Da cut-off dialysis membrane. Dialyze the purified protein solution with 3L PBS at 4°C overnight. On day 5, the purified protein solution was dialyzed against 2 L of fresh PBS at 4 °C for another 2 h. Finally, the final concentration was calculated by BCA.

[0397] Although discussed in the context of VH / VHH, the techniques described above can also be applied to scFv, Fab, Fv and other antibody fragments if desired.

[0398] Multiple antigen binding fragments (suitably VH / VHH) can be fused by chemical cross-linking by reacting the amino acid residues with an organic derivatizing agent, such as described by Blattler et al. 1985. Alternatively, the antigen binding fragments can be genetically fused at the DNA level, i.e. the polynucleotide construct formed encodes a complete polypeptide construct comprising one or more antigen binding fragments. One way to join multiple antigen binding fragments via a genetic approach is to connect the antigen binding fragment coding sequences directly or via a peptide linker. For example, the carboxyl terminus of the first antigen binding fragment can be connected to the amino terminus of the next antigen binding fragment. This connection mode can be extended so that the antigen binding fragments are connected for the construction of tri-, tetra-, etc. functional constructs. Methods for generating multivalent (such as bivalent) VHH polypeptide constructs are disclosed in WO 96 / 34103 (incorporated herein in its entirety by reference).

[0399] Suitably, polypeptides of the invention (particularly VHHs of the invention) may be produced in fungi, such as yeast (e.g., Saccharomyces cerevisiae), according to the methods disclosed in WO02 / 48382, comprising growing the fungi on a medium comprising a carbon source, wherein 50-100 wt% of the carbon source is ethanol. Large-scale production of VHH fragments in Saccharomyces cerevisiae is described in Thomassen et al. 2002.

[0400] In one aspect of the present invention, there is provided a method for preparing a polypeptide or construct of the present invention, comprising the following steps:

[0401] i) cloning the polynucleotide of the present invention into a vector, such as a plasmid,

[0402] ii) transforming a cell capable of producing a polypeptide or construct of the invention, such as a bacterial cell or a yeast cell, with the vector under conditions allowing production of the polypeptide or construct,

[0403] iii) recovering the polypeptide or construct, such as by affinity chromatography.

[0404] The clauses listing further embodiments of the present invention are as follows:

[0405] Terms

[0406] 1. A polypeptide capable of inhibiting the binding of IL-7 and / or L-TSLP to IL-7R.

[0407] 2. The polypeptide according to clause 1, wherein the polypeptide is capable of inhibiting the binding of IL-7 to IL-7R.

[0408] 3. The polypeptide according to clause 1, wherein the polypeptide is capable of inhibiting the binding of L-TSLP to IL-7R.

[0409] 4. A polypeptide according to any one of clauses 1 to 3, wherein the polypeptide is capable of inhibiting the binding of IL-7 to IL-7R and the binding of L-TSLP to IL-7R.

[0410] 5. The polypeptide according to any one of clauses 1 to 4, wherein the polypeptide binds to IL-7Rα.

[0411] 6. A polypeptide according to any one of clauses 1 to 5, wherein the polypeptide comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR1 comprises a sequence that shares 60% or greater sequence identity with SEQ ID NO: 1, CDR2 comprises a sequence that shares 60% or greater sequence identity with SEQ ID NO: 2, and CDR3 comprises a sequence that shares 60% or greater sequence identity with SEQ ID NO: 3.

[0412] 7. A polypeptide according to clause 6, wherein CDR1 comprises a sequence that shares 80% or greater sequence identity with SEQ ID NO: 1, CDR2 comprises a sequence that shares 80% or greater sequence identity with SEQ ID NO: 2, and CDR3 comprises a sequence that shares 80% or greater sequence identity with SEQ ID NO: 3.

[0413] 8. A polypeptide according to clause 7, wherein CDR1 comprises SEQ ID NO: 1 or SEQ ID NO: 71; CDR2 comprises SEQ ID NO: 2, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75 or SEQ ID NO: 76, and CDR3 comprises SEQ ID NO: 3, SEQ ID NO: 77 or SEQ ID NO: 78.

[0414] 9. The polypeptide according to clause 8, wherein the polypeptide comprises SEQ ID NO: 8.

[0415] 10. The polypeptide according to any one of clauses 1 to 9, wherein the polypeptide is an antibody or an antibody fragment.

[0416] 11. A polypeptide according to any one of clauses 1 to 10, wherein the polypeptide neutralises the binding of IL-7R to IL-7 with an EC50 of 2 nM or less.

[0417] 12. A polypeptide according to any one of clauses 1 to 11, wherein the polypeptide is substantially resistant to proteases of the human intestinal tract.

[0418] 13. A polypeptide according to any one of clauses 1 to 12 for use as a medicament.

[0419] 14. A polypeptide according to clause 13 for use in the treatment of autoimmune diseases and / or inflammatory diseases.

[0420] 15. A polypeptide for use according to clause 13 or 14, wherein the polypeptide is for oral administration.

[0421] The invention will now be further described by way of the following non-limiting examples.

[0422] Example

[0423] Example 1: Immunization and phage library construction

[0424] Two llamas were each immunized with soluble human recombinant IL-7Rα. Blood was collected from the two llamas at different time points during the immunization, and IL-7Rα binding and neutralization were tested to monitor the development of an immune response to IL-7Rα. Analysis showed that only one llama developed good anti-IL-7Rα antibody titers, while the other llama failed to respond to IL-7Rα immunization. RNA isolated from leukocytes collected from the responding llamas at the end of immunization was used to generate twelve separate phage display libraries.

[0425] Example 2: Selection of libraries with human IL-7Rα binding activity

[0426] The library selection strategy was developed to separate ICVDs that bind to epitopes present on the extracellular domain of the IL-7Rα subunit, including ICVDs that interfere with the binding of IL-7 to IL-7R. Several methods were used to selectively enrich phages that display ICVDs with IL-7Rα binding characteristics and other desired properties, including high binding affinity and tolerance to intestinal proteases. Phages present in the eluates selected from different libraries were used to infect Escherichia coli, and individual colonies were picked into mother plates and propagated to generate colony cultures. Periplasmic supernatants containing selected monoclonal ICVDs were used for primary evaluation studies to identify those with desired characteristics.

[0427] From a total of 630 library selected colonies from the eight original master plates picked for screening, a final set of seven primary clones were selected for production in E. coli and affinity purified for ICVD for more detailed evaluation studies.

[0428] The DNA sequences of the 7 primary clones (V7R-2E5, V7R-2E9, V7R-2F6, V7R-6C12, V7R-2B6, V7R-3B5 and V7R-4F6) isolated above were recloned into the vector pMEK222 (thereby introducing C-terminal FLAG and 6xHis tags) for production in E. coli, followed by affinity purification for more detailed evaluation studies. The polypeptide sequences of these ICVDs excluding FLAG and His tags are shown in the alignment provided in the section entitled "Polypeptide and Polynucleotide Sequences" above. The clinical anti-IL-7R antibody mAb829 (a 150 kDa antibody containing heavy and light chains, also referred to as "GSK2618960", as disclosed in Ellis et al. 2019) was produced and used as a comparator in a number of the following examples.

[0429] A key objective of the following examples was to identify those ICVD clones that possessed the ability to inhibit IL-7 binding to IL-7R and possessed a degree of inherent resistance to inactivation by intestinal proteases.

[0430] Example 3: Efficacy and protease resistance of primary clones

[0431] effect

[0432] The efficacy of seven primary clones was evaluated using IL-7 / IL-7R neutralization ELISA.

[0433] Primary clones were subcloned from phagemids into pMEK222 plasmid for addition of a C-terminal FLAG-6xHis tag and expression in E. coli. These ICVDs were expressed by E. coli TG1 and purified via the 6xHis tag.

[0434] Start at 300nM and use 3.2 dilution factors, prepare clones of 7 dilution series in 1% BSA (under 2x determination concentration).Use mAb829 contrast antibody as positive control in ELISA, concentration range is between 10nM and 0.088nM (2x determination concentration).Prepare enough volume in triplicate for each clone dilution, prepare enough volume in 2 triplicates (2 plates) for mAb829 simultaneously.Each ICVD (or mAb829) dilution of 85 μ L (or 170 μ L) is mixed with 10ng / mL IL-7 (2x determination concentration) of 85 μ L (or 170 μ L).85 μ L of IL-7 is mixed with 85 μ L of blocking buffer, to have IL-7 (1x) complete binding signal in each plate.Also add separate blocking buffer in each plate as blank. Bound IL-7 was then measured using biotinylated anti-hIL-7 followed by exo-avidin-HRP. The TMB reaction was stopped after 30 minutes.

[0435] A using ELISA signal blank correction 450 Data and 'log(inhibitor) vs. response - variable slope (four parameters)' curves were fitted and EC 50 To generate EC in Graphpad Prism 50 These values ​​are shown in Table 1 below. It was shown that all ICVDs were as effective as the comparator mAb829 in inhibiting the binding of hIL-7 to hIL-7R, and most of them were slightly more potent than mAb829.

[0436] Protease resistance

[0437] Seven purified ICVDs were incubated in the presence of mouse small intestinal supernatant ("mouse SI") and supernatant prepared from pooled human fecal samples ("HFP").

[0438] All ICVD stock dilutions were prepared at 250 g / mL in 1x PBS (30 L final volume) containing 1% BSA. Then digestion mixed reactions were prepared for each ICVD in PCR strips, with a final volume of 60 L and a final concentration of 20 g / mL (55.2 L of digestion matrix + 4.8 L of ICVD, 250 g / mL). 4.8 L of 1x PBS was used for non-ICVD controls. Subsequently, 25 L of each digestion reaction was transferred to (i) a new PCR tube containing cold stop buffer (these were used as T=0 time points) and frozen at -80 ° C; and (ii) a new empty PCR tube and incubated at 37 ° C in a PCR thermal cycler for digestion (digested and non-ICVD control samples). Samples digested in mouse SI were taken out from the thermal cycler after 2 h, and samples digested in HFP were taken out after 4 h. Samples were stopped immediately with 25 L of cold stop buffer and frozen at -80 ° C until analysis.

[0439] The digested samples were tested in the ELISA described in detail above in 'Efficacy' using a dilution factor of 1.8 (for digested samples) or 2.1 (for undigested samples) with subsequent 6 serial dilutions at a final starting dilution of 1:100 and assayed in triplicate wells in each plate. The undigested samples (0 h) were considered as the standard curve, since theoretically no protease digestion occurred in these samples (since they were treated on ice and stop buffer was added immediately followed by the addition of the digestion matrix).

[0440] If digestion had occurred in the 'digested' sample, the curve would be expected to be shifted to the left (compared to T=0 h). The greater the shift, the more unstable the ICVD. "% Survival" indicates the level of activity retained after digestion.

[0441] All clones showed a high level of resistance to proteolytic degradation in both matrices, and V7R-2E9 was identified as the most protease resistant ICVD, showing complete resistance to pooled human feces digestion for up to 4 hours.

[0442] These results are summarized in Table 1 below.

[0443] Table 1

[0444]

[0445] NT = Not Tested

[0446] SI = small intestinal supernatant

[0447] The polypeptides of the invention all show unexpectedly high efficacy and survival in digestive matrices.It can be noted that, although the ICVDs are clearly related, the CDRs and frameworks of the polypeptides differ from each other by multiple residues (see the alignment provided in "Polypeptide and Polynucleotide Sequences" above).

[0448] Example 4: Additional efficacy assays for V7R-2E9 and comparator mAb829

[0449] IL-7 / IL-7R Neutralization ELISA

[0450] The IL-7 / IL-7R neutralization ELISA described above in Example 3 was again performed on V7R-2E9 and the clinical anti-IL-7R comparator antibody mAb829. The results are shown in Table 2 below.

[0451] TSLP / TSLPR / IL-7R ELISA

[0452] The ability of V7R-2E9 to neutralize the binding of L-TSLP / TSLP-R complex to IL-7Rα was tested. A 96-well plate was coated with 0.25 μg / mL recombinant human IL-7Rα-His6-Fc+5 μg / mL BSA and then blocked. V7R-2E9 was serially diluted and mixed with recombinant human L-TSLP (15 ng / mL final concentration) and human TSLP-R (20 ng / mL final concentration) 1:1:1. The mixture was then incubated for 30 minutes to allow binding before being added to the IL-7Rα-coated plate. After a 2-hour incubation, 50 μL / well 0.3 μg / mL biotinylated rabbit anti-hTSLP antibody was used, and then 50 μL / well 1 / 2000 avidin-HRP was used to detect the bound L-TSLP, and GraphPad Prism was used to determine the level of neutralization of the binding of the L-TSLP / TSLP-R complex to IL-7Rα by ICVD. The results are shown in Table 2 below.

[0453] IL-7-induced pSTAT5 in hPBMCs

[0454] The ability of V7R-2E9 to inhibit IL-7 binding to IL-7Rα and prevent STAT5 phosphorylation was tested in vitro in human lymphocytes. Human peripheral blood mononuclear cells (PBMCs) respond to exogenous IL-7 by stimulating intracellular STAT5 phosphorylation through IL-7R signaling, but this response can be abolished by IL-7Rα-specific ICVD that prevents IL-7 / IL-7R binding.

[0455] Lymphocyte enriched populations were isolated from human buffy coat and stored in 90% FBS 10% DMSO in liquid nitrogen. Cells were thawed and placed in complete RPMI-1640 for recovery. After recovery, cells were plated at 100 μl of 2.5×10 5 Cells / well were plated in a round-bottom 96-well plate and starved for 1 h in complete RPMI without FBS. After starvation, the desired ICVD concentrate was added to each well (50 μL / well) and the plate was incubated at room temperature for 15 min. 50 μL / well of IL-7 was then added to each well and the plate was incubated at 37°C with 5% CO 2 The cells were incubated for 15 min at 4 °C for 20 min. The reaction was stopped by cooling the plate rapidly on ice, followed by centrifugation and removal of the supernatant. The cells were then processed for fixation, permeabilization and pSTAT5 intracellular staining. The cells were incubated for 20 min on ice with 100 μL / well Cytofix / Cytoperm solution (BD Bioscience #554722), washed twice with 150 μl / well 1x Perm / wash buffer (BD Bioscience #554723), incubated for 30 min on ice with 200 μL / well Perm buffer III (BD Bioscience #558050) and washed twice with 150 μL / well 1x PBS 2% BSA (FACS buffer). The cells were then stained with 25 μL / well pSTAT5 antibody ([47 / Stat5(pY694)](A488)(BD Bioscience#612598)) or isotype control mouse IgG1 ([B11 / 6](FITC)(Abcam#ab91356)) for 1 h at room temperature. The reaction was stopped by adding 150 μL / well FACS buffer. After 1 wash / centrifugation step, the cells were finally resuspended in 200 μL / well FACS buffer and data were acquired in a CytoFlex flow cytometer (BeckmanCoulter). Data analysis was performed using FlowJo software. The results are shown in Table 2 below.

[0456] TSLP-induced TARC secretion in human monocytes

[0457] V7R-2E9 was also tested in a human monocyte cell assay described in detail to confirm IL-7R neutralizing activity. Due to the engagement of TSLP / TSLP-R with cell surface IL-7R, human monocytes exhibited a TARC secretion response in culture medium after stimulation with TSLP. The ability of anti-IL-7Rα ICVD to inhibit the binding of TSLP / TSLP-R to IL-7R and prevent TARC secretion was tested in vitro in human monocytes.

[0458] Mononuclear cells were isolated from human buffy coat and 1×10 5 100 cells / well were plated in complete RPMI in a flat-bottom 96-well plate. To increase the purity of monocytes, the plated cells were incubated at 37°C with 5% CO. 2 The cells were incubated at 37°C for 2 h and then non-adherent cells were removed by aspirating the medium in each well followed by gentle 2x washing with warm cRPMI. 2 The cells were incubated with the desired concentration of ICVD for 24 h at 4 °C (diluted in complete RPMI, final volume 100 μL / well). After 24 h incubation, 75 μL of supernatant / well were collected and stored at -80°C for further analysis of secreted TARC.

[0459] The neutralization level of human TSLP by the anti-IL-7Rα agent was tested. A 96-well plate was coated with 50 μL / well anti-TARC antibody and then blocked. The human TARC standard was serially diluted in the assay diluent, and then 50 μL / well of the standard was added to the anti-TARC coated plate together with 50 μL / well of the recovered culture supernatant. Anti-human TARC polyclonal antibody was biotinylated, and then bound human TARC was detected with avidin-HRP. The neutralization level of human TSLP by the agent was determined. The results are shown in Table 2 below.

[0460] Table 2

[0461]

[0462] In conclusion, V7R-2E9 was shown to inhibit the binding of IL-7 and L-TSLP to IL-7R in both ELISA and cellular assays with similar efficacy as the comparator clinical anti-IL-7R antibody mAb829.

[0463] Example 5: Epitope Modeling

[0464] The computer builds the model structure and epitope of V7R-2E9. The protein data bank (PDB) '4ybq' file is used as a master to develop this model. 4ybq is the heavy chain of the FV that is bound to rat GLUT5 facilitated glucose transporter member 5. In terms of CDR3 loop length and expected conformation, the 4ybq template is particularly similar to V7R-2E9. PDB entry '3di3' is a high-resolution structure of the IL-7 that is bound to IL-7Rα. From this structure, IL-7 is simply missing to provide a target receptor for docking with the predicted V7R-2E9 structure.

[0465] Because the HEX 8.0 docking method employed works in a localized manner on the surface of each domain, multiple target regions were selected on the IL-7Rα structure and run in parallel. The best solution for V7R-2E9 represented a cluster of 46 solutions, which had a very high energy score of -894 after energy minimization (DARS force field) and no 'humps' (atomic positions that do not allow access). Figure 3 A ribbon schematic showing the position of docked V7R-2E9 on the IL-7Rα target. V7R-2E9 is located at the interface between the N-terminal domain and the C-terminal domain.

[0466] The epitope residues of IL-7Rα that contact V7R-2E9 are listed in Table 3. Residues covering particularly significant regions at the interface are highlighted in bold (refer to SEQ ID NO: 65).

[0467] Table 3

[0468]

[0469]

[0470] Example 6: Optimization to reduce immunogenicity

[0471] The amino acid sequence of V7R-2E9 is aligned with the human VH3 germline antibody sequence, and potential humanization changes are identified. The set of 18 single mutations at the end of framework 2 / CDR2 and 2 changes combined is introduced into the V7R-2E9 parent ICVD sequence, and mutants are produced from Escherichia coli. The effect of ICVD is initially tested in IL-7 / IL-7R and ELISA. The clone shows IL-7R neutralization activity similar to or larger than V7R-2E9, thereby indicating that the mutation introduced into the parent ICVD does not have a deleterious effect for antigen combination. Then all clones are digested in human feces supernatant material for 16 hours to measure its relative survival rate. (Table 4).

[0472] Table 4

[0473]

[0474]

[0475] Mutations that retained high efficacy in the IL-7 / IL-7Rα-His6-Fc ELISA and maintained similar resistance to human fecal pools were then combined to generate 19 humanized ICVDs (Table 5).

[0476] Table 5

[0477]

[0478]

[0479] Among these 19 humanized ICVDs, Table 6 summarizes the most favorable humanized ICVDs that maintained efficacy and tolerance to both human feces and mouse small intestinal proteases.

[0480] Table 6

[0481]

[0482]

[0483] A particularly noteworthy ICVD is ID-A62U, which contains E1D (for yeast expression to avoid the possibility of generating a product with a cyclized N-terminal glutamate) and humanized mutations R45L, Q65K, K87R, and S88A. Another particularly noteworthy ICVD is ID-A59U, which contains E1D, Q65K, K87R, and S88A. ID-A59U has a pI of 5.1 and a molecular weight of 12.966 kDa (pI and molecular weight calculated using CLC Sequence Viewer).

[0484] Example 7: Efficacy assays of humanized V7R-2E9 variants

[0485] The inhibitory efficacy and effectiveness (maximum inhibition) of ID-A40U (produced in E. coli) were confirmed in vitro in human PBMCs in an IL-7 / IL-7R neutralization ELISA and an IL-7-induced STAT5 phosphorylation assay. The ELISA signal blank-corrected A450 data and 'log(inhibitor) vs. response - variable slope (four parameters)' curves were fitted and EC was generated. 50 To generate EC in Graphpad Prism 50 value.

[0486] The results and comparisons are shown in Table 7a.

[0487] Table 7a

[0488]

[0489] In separate experiments, these same assays were performed on ID-A62U (produced in S. cerevisiae) and controls. The results are shown in Tables 7b and Figure 4 Shown in.

[0490] Table 7b

[0491]

[0492]

[0493] The ability of ID-A62U and mAb829 to neutralize the binding of the L-TSLP / TSLP-R complex to IL-7Rα was tested in the manner described above in Example 4. The results are shown in Table 7c below. The data set was subtracted prior to plotting, which was normalized to the highest concentration of the antibody tested (to overcome the high level of background on the plate).

[0494] Table 7c

[0495]

[0496] In summary, ID-A62U and ID-A40U were demonstrated to have comparable or greater efficacy than the comparator clinical anti-IL-7R antibody mAb829.

[0497] Example 8: Biacore estimation of ICVD-IL-7R binding affinity

[0498] The binding kinetics of ID-A40U were compared with mAb829 clinical antibody in Biacore studies. 6 -Fc was directly coated on the Biacore sensor plate (for mAb829 analysis) or captured by anti-human IgG Fc (for ICVD analysis), and ICVD / Ab was flowed through the plate to detect binding. ID-A40U has 7.8x10 -11 M, and mAb829 had a KD of 5.67x10 -10 The slightly lower affinity (K D ). The results indicated that ID-A40U showed strong binding to the antigen.

[0499] Example 9: Cross-reactivity with IL-7Rα from toxicological species

[0500] The cross-reactivity of ID-A40U, ID-A59U and ID-A62U with IL-7Rα from toxicological species was investigated.

[0501] The 96-well plate was incubated with 0.5 μg / mL recombinant human IL-7Rα His 6-Fc+5μg / mL BSA coating and then blocking. 0.5nM ICVD was mixed with the selected test compound serially diluted in 1% BSA 1:1, then incubated for 30 minutes to allow binding, and then added to the IL-7Rα coated plate. After 2 hours of incubation, the bound ICVD was detected with 50uL / well 1 / 20000 anti-FLAG-HRP goat antibody (GeneTex, GTX21238), and the neutralization level of the test compound on the ICVD-IL-7Rα binding was determined.

[0502] Murine IL-7Rα was found not to interfere with ICVD / IL-7Rα binding in this assay, indicating that mice or rats are not appropriate species for toxicology studies. However, cynomolgus monkey IL-7Rα interfered with the binding of these ICVDs to human IL-7Rα ( Figure 5 and Figure 6 ), making the cynomolgus monkey a suitable toxicology species for these and related ICVDs.

[0503] Example 10: Specificity for non-target cytokines

[0504] The selectivity of ID-A40U against proteins associated with human IL-7Rα was tested essentially in the manner described in Example 9 above. Human IL-12Rβ1 and human IL-12Rβ2 were found to be the most closely related human proteins to IL-7Rα identified using the NCBI BLASTp tool, with 29% and 30% sequence identity to IL-7Rα, respectively. In a competitive IL-7R binding ELISA assay, hIL-12Rβ1 and selected receptors within the IL-7R family (IL-2R, IL-21R and IL-9R) or unrelated receptors (TNFR-2 and IL-6R) were tested for their ability to inhibit the binding of ID-A40U to human IL-7Rα immobilized on the plate. None of human IL-12Rβ1, IL-2R, IL-21R, IL-9R, TNFR-2 or IL-6R interfered with the binding of ID-A40U to IL-7Rα, while the addition of increasing amounts of human IL-7Rα produced a dose-dependent curve ( Figure 7 ). This indicates that binding of ID-A40U ICVD to off-target molecules is very unlikely in humans.

[0505] Example 11: Tolerance of Gastrointestinal Extracts

[0506] In vitro incubation in intestinal supernatants can predict the stability of ICVD in the intestines of cynomolgus monkeys and humans. The activity of the major intestinal proteases trypsin and chymotrypsin is conserved in mammalian species, while the proteases present in the large intestine may be produced by host species-specific intestinal microbial flora. In order to generate test matrices reflecting these two environments, pooled mouse small intestinal supernatants and pooled fecal supernatants were prepared. These two matrices are highly digestible for non-selected non-engineered ICVD.

[0507] ICVD ID-38F has previously been shown to have high stability in these matrices (see WO2016 / 156465), and this property predicts that ID-38F will have high stability during transit through the intestine.

[0508] V7R-2E9, ID-A24U and ID-A40U were tested for survival in gastrointestinal extracts from both murine and human sources. ICVD was incubated with mouse small intestinal supernatant for 6 hours at 37°C and with human fecal supernatant for 16 hours. Survival was measured by IL-7 / IL-7R neutralization ELISA. All constructs showed good survival in all digestible matrices tested ( Figure 8 , where "SI" = mouse small intestinal fluid, and "HF" = human fecal supernatant). ID-A40U contains the same functional mutation as the yeast-produced ID-A59U.

[0509] In a separate experiment, ID-A62U and ID-A41U (unstable comparator ICVD) were tested for survival in the same human fecal supernatant assay. ID-A62U demonstrated approximately 100% survival ( ) compared to approximately 40% survival for ID-A41U. Fig. 9 ).

[0510] These are rigorous tests involving extended incubation periods. Therefore, it is expected that any of these ICVDs survive very well in the gastrointestinal environment.

[0511] Example 12: Resistance to Gastrointestinal Matrix Metalloproteinases

[0512] Levels of activated matrix metalloproteinases (MMPs) are increased in the inflamed mucosa of patients with intestinal diseases. These MMPs are able to digest native human IgG and therapeutic agents containing human IgG scaffolds (Biancheri et al. 2015). In the case of the anti-TNFα therapy etanercept, this digestion resulted in a significant reduction in TNFα neutralization efficacy. To confirm that ID-A40U is resistant to MMPs, protein immunoblotting was performed to detect ID-A40U as well as mAb829 and Enbrel after incubation for 22 hours at 37°C in the presence of human MMP3, MMP12, or TCNB buffer. Enbrel and mAb829 were detected by peroxidase-conjugated anti-human IgG specific for the γ chain. ID-A40U and TCNB buffer only controls were detected with pAb1219 primary rabbit α-ICVD and secondary HRP-conjugated pAb SwineαRabbit.

[0513] After incubation, ID-A40U was not digested by MMPs, as measured by western blot ( Fig.10 ), thus showing a band with a molecular weight corresponding to the expected full-length ICVD (lacking the Flag-His6 tag, which is cleaved during digestion). However, after the same incubation time, MMP3 and MMP12 digested full-length etanercept and mAb829 to smaller fragments. After MMP incubation, ID-A40U was shown to be fully potent in neutralizing IL-7R, as measured using the IL7 / IL-7R functional ELISA. Fig.10 The 'F / H' in the figure represents the presence of FLAG / His tag.

[0514] Example 13: Transport and survival in the mouse gastrointestinal tract

[0515] The results of the in vitro studies described above showed that the optimized V7R-2E9 derivatives were resistant to inactivation by proteases present in supernatant extracts prepared from mouse small intestinal contents.Subsequent studies were conducted to investigate the stability of ID-A24U and ID-A40U during passage through the gastrointestinal system of mice.

[0516] ID-A24U and ID-A40U were formulated with ID-38F (anti-TNFα ICVD, see WO2016 / 156465) in a milk and bicarbonate mixture to prevent denaturation at low pH and digestion by pepsin in the stomach. After ICVD was administered to mice by oral gavage, the concentration of ICVD in the stomach, small intestine, cecum, and colon was determined at 6 hours after administration. In addition, the ICVD concentration of fecal pellets collected at hourly intervals was measured.

[0517] Feces collected from all mice between 4 and 6 h after dosing were measured for ID-A24U and ID-A40U, with ID-A40U also measured from 2 mice at the 3 h time point ( Fig.11 ). In contrast, ID-A24U was measured in feces collected from mouse 6 (M6) during the first 3 hours, indicating that transit was particularly rapid in this mouse compared to other mice.

[0518] These results show that both ID-A24U and ID-A40U survive after transport through the mouse GI tract. In summary, the transport times of ID-A24U and ID-A40U look similar, with 1 mouse (M6 and M12) in each group being an exception. At the time of slaughter (6h), ID-A24U and ID-A40U were present in the cecum (CAE) and colon (COL) of all mice, and a fairly high amount was still measured in the stomach (STO) and small intestine (SI). Based on the concentration calculated by taking into account the dilution factor for slurry preparation, the expected concentrations of ID-A24U and ID-A40U at the time of slaughter were between 22.7 μM-140 μM and between 12 μM-22.5 μM, respectively ( Fig.11 The expected concentrations of ID-A24U in the cecum and colon are between 2.9μM-8μM and 9.7μM-16.5μM, respectively. The expected concentrations of ID-A40U in the cecum and colon are between 0.9μM-1.2μM and 0.8μM-6.2μM, respectively. Fig.12 ).

[0519] ID-38F, used as a control in this study, was measured at high levels in all feces and lower GIT samples, as previously observed. The expected concentration of ID-38F at the 6h time point varied in 6 mice, between 0.04μM-2.1μM in the cecum, between 0.33μM-5.1μM in the colon, and between 4.9μM-48μM in the feces. The experiment was repeated for ID-A40U and ID-38F. Similar results were obtained ( Fig.13 and Fig.14 ).

[0520] In summary, these results indicate that ID-A24U and ID-A40U survive well and similarly to ID-38F when transported through mice, thereby delivering high concentrations of active ICVD to the cecum and colon. This may reflect the relative stability of each ICVD in these intestinal compartments observed previously in vitro. It was shown that ID-A24U and ID-A40U were stable in the mouse small intestine. It is therefore expected that these and related ICVDs will have high stability in the human GI tract.

[0521] Example 14: Human IBD tissue study

[0522] Studies were conducted to investigate the activity of V7R-2E9 in a human ex vivo model that recapitulates the tissue environment of inflammatory bowel disease. Using tissues taken from four patients with active ulcerative colitis, V7R-2E9 and controls (ID-2A, anti-Clostridium difficile toxin ICVD; mAb829 and IgG1k (non-IL-7R binding purified IgG1k isotype control recombinant antibody) were tested in ex vivo culture for their effects on tissue phosphoprotein levels and the production of inflammatory cytokines.

[0523] Pathscan Phosphoprotein Array ( Figure 15-18 ) showed that V7R-2E9 treatment inhibited phosphorylation of a substantial portion of the 39 proteins detected on the array in biopsies from three of four UC patients when compared to the corresponding ID-2A treated biopsies. mAb829 also inhibited protein phosphorylation levels in biopsies from the same three UC patients, and the inhibition patterns obtained appeared similar to those achieved with V7R-2E9. Total phosphorylation intensity values ​​for each biopsy were calculated from the intensities of all 39 phosphoproteins on the array. Fig.19 Results presented in show that V7R-2E9 and mAb829 inhibited total phosphorylation levels in biopsies from three responsive UC patients, but had little or no effect on total phosphorylation in biopsies from patient UC2700. This patient had active disease while receiving azathioprine (T cell inhibitor) as part of his medication, so resistance to T cell directed therapy is a possible explanation for the lack of response to antibodies targeting IL-7R mediated T cell activation (V7R-2E9 and mAb829).

[0524] Analysis of culture media from biopsies of patients UC2698, UC2701, and UC2703 showed that V7R-2E9 treatment also inhibited the production of several cytokines, including IL-1β, IL-6, IL-8, and TNFα, but had no effect on the production of the anti-inflammatory cytokine IL-10 (data not shown). Consistent with the results of the phosphoprotein analysis, V7R-2E9 did not inhibit the production of pro-inflammatory cytokines in biopsy cultures of patient UC2700.

[0525] In summary, antagonism of IL-7R in UC biopsies by V7R-2E9 inhibits phosphorylation of signaling proteins and production of cytokines and chemokines associated with proinflammatory and immunomodulatory pathways. The results show that antagonism of mucosal IL-7R+ve T cells by V7R-2E9 (and mAb829) can inhibit inflammatory processes in a model that is closely related to the disease environment.

[0526] Example 15: Yeast productivity in fermentation culture

[0527] Saccharomyces cerevisiae expressing ID-A59U was inoculated into a fermentation of 5 liters of ethanol feed. The ID-A59U concentration of the end-of-fermentation (EoF) liquid culture supernatant was analyzed by SDS-PAGE and IL-7 / IL-7Rα-His6-Fc functional ELISA. SDS-PAGE indicated a high yield of ≤2g / L, and functional ELISA confirmed that ID-A59U was fully active in EoF and the final yield was at least 1.5g / L. Saccharomyces cerevisiae expressing ID-A62U was inoculated into a 50mL shaking flask expression system. A high yield of ICVD was obtained.

[0528] Conclusions from the above examples

[0529] Polypeptides that benefit from high efficacy in a neutralizing cell assay system measuring IL-7Rα activity were identified, including inhibition of IL-7 and / or L-TSLP binding to IL-7Rα. These polypeptides also benefit from high stability in the small intestine and / or human fecal supernatant in some cases. A humanized derivative of a specific polypeptide, V7R-2E9, was produced that substantially retains efficacy or benefits from increased efficacy compared to unmodified V7R-2E9 - while also retaining tolerance to intestinal proteases and the ability to be effectively produced in Saccharomyces cerevisiae. The most favorable combination for mutations (R45L, Q65K, K87R, S88A) of V7R-2E9 (including E1D yeast production mutations) is embodied by ID-A62U.

[0530] Other aspects

[0531] All references, including patents and patent applications, cited in this application are incorporated herein by reference to the maximum extent possible.

[0532] Throughout this specification and the following claims, unless the context requires otherwise, the word 'comprise' and variations such as 'comprises' and 'comprising' will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not the exclusion of any other integer, step, any other group of integers or any other group of steps.

[0533] The application of which this description and claims form a part may be used as a basis for claiming priority on any subsequent application. The claims of this subsequent application may be directed to any feature or combination of features described herein. They may be in the form of product, composition, method or use claims, and may include, for example, but not limited to, the following claims.

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[0609] Wells et al Gene 1985 34:315-323 Sequence Listing <110> wesquald gmbh <120> Peptides <130> VHS-P2585PCT <160> 89 <170> PatentIn Version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial sequence <220> <223> synthetic sequence <400> 1 Ser Asp Ala Met Gly 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 2 Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 3 Asp Tyr Asp Thr Asp Val Trp Gln Tyr 1 5 <210> 4 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 4 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ser Ile Ser Thr Phe Ser 20 25 30 <210> 5 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 5 Trp Phe Arg Gln Ala Pro Gly Lys Glu Leu Glu Phe Leu Ala 1 5 10 <210> 6 <211> 32 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 6 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Arg Tyr Tyr Cys Ala Glu 20 25 30 <210> 7 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 7 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 8 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 8 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Leu Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 9 <211> 118 <212> PRT <213> Llama <400> 9 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ala Arg Thr Phe Ser Asp Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Ala Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Asn Trp Ser Gly Ala Val Thr His Tyr Gly Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln His Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 10 <211> 118 <212> PRT <213> Llama <400> 10 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 11 <211> 118 <212> PRT <213> Llama <400> 11 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 12 <211> 118 <212> PRT <213> Llama <400> 12 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Gly Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Thr Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 13 <211> 118 <212> PRT <213> Llama <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Val Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 14 <211> 118 <212> PRT <213> Llama <400> 14 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Gly Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Thr Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Thr Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 15 <211> 118 <212> PRT <213> Llama <400> 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Val Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 16 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 16 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 17 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 17 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 18 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 18 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 19 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 19 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Gly Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 20 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 20 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 21 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 21 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 22 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 22 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 23 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 23 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 24 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 24 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Leu Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 25 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 25 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 26 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 26 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 27 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 27 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ser Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 28 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 28 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 29 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 29 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 30 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 30 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 31 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 31 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 32 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 32 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 33 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 33 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 34 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 34 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Val Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 35 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 35 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 36 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 36 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 37 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 37 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Leu Glu Trp Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 38 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 38 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Trp Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 39 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 39 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 40 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 40 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 41 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 41 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 42 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 42 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 43 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 43 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 44 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 44 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 45 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 45 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 46 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 46 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 47 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 47 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 48 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 48 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 49 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 49 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 50 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 50 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 51 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 51 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 52 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 52 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 53 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 53 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 54 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 54 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Leu Glu Trp Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 55 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 55 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 56 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 56 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 57 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 57 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 58 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 58 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 59 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 59 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 60 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 60 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 61 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 61 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 62 <211> 159 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 62 Met Phe Pro Phe Ala Leu Leu Tyr Val Leu Ser Val Ser Phe Arg Lys 1 5 10 15 Ile Phe Ile Leu Gln Leu Val Gly Leu Val Leu Thr Tyr Asp Phe Thr 20 25 30 Asn Cys Asp Phe Glu Lys Ile Lys Ala Ala Tyr Leu Ser Thr Ile Ser 35 40 45 Lys Asp Leu Ile Thr Tyr Met Ser Gly Thr Lys Ser Thr Glu Phe Asn 50 55 60 Asn Thr Val Ser Cys Ser Asn Arg Pro His Cys Leu Thr Glu Ile Gln 65 70 75 80 Ser Leu Thr Phe Asn Pro Thr Ala Gly Cys Ala Ser Leu Ala Lys Glu 85 90 95 Met Phe Ala Met Lys Thr Lys Ala Ala Leu Ala Ile Trp Cys Pro Gly 100 105 110 Tyr Ser Glu Thr Gln Ile Asn Ala Thr Gln Ala Met Lys Lys Arg Arg 115 120 125 Lys Arg Lys Val Thr Thr Asn Lys Cys Leu Glu Gln Val Ser Gln Leu 130 135 140 Gln Gly Leu Trp Arg Arg Phe Asn Arg Pro Leu Leu Lys Gln Gln 145 150 155 <210> 63 <211> 63 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 63 Met Phe Ala Met Lys Thr Lys Ala Ala Leu Ala Ile Trp Cys Pro Gly 1 5 10 15 Tyr Ser Glu Thr Gln Ile Asn Ala Thr Gln Ala Met Lys Lys Arg Arg 20 25 30 Lys Arg Lys Val Thr Thr Asn Lys Cys Leu Glu Gln Val Ser Gln Leu 35 40 45 Gln Gly Leu Trp Arg Arg Phe Asn Arg Pro Leu Leu Lys Gln Gln 50 55 60 <210> 64 <211> 369 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 64 Met Leu Lys Pro Ser Leu Pro Phe Thr Ser Leu Leu Phe Leu Gln Leu 1 5 10 15 Pro Leu Leu Gly Val Gly Leu Asn Thr Thr Ile Leu Thr Pro Asn Gly 20 25 30 Asn Glu Asp Thr Thr Ala Asp Phe Phe Leu Thr Thr Met Pro Thr Asp 35 40 45 Ser Leu Ser Val Ser Thr Leu Pro Leu Pro Glu Val Gln Cys Phe Val 50 55 60 Phe Asn Val Glu Tyr Met Asn Cys Thr Trp Asn Ser Ser Ser Glu Pro 65 70 75 80 Gln Pro Thr Asn Leu Thr Leu His Tyr Trp Tyr Lys Asn Ser Asp Asn 85 90 95 Asp Lys Val Gln Lys Cys Ser His Tyr Leu Phe Ser Glu Glu Ile Thr 100 105 110 Ser Gly Cys Gln Leu Gln Lys Lys Glu Ile His Leu Tyr Gln Thr Phe 115 120 125 Val Val Gln Leu Gln Asp Pro Arg Glu Pro Arg Arg Gln Ala Thr Gln 130 135 140 Met Leu Lys Leu Gln Asn Leu Val Ile Pro Trp Ala Pro Glu Asn Leu 145 150 155 160 Thr Leu His Lys Leu Ser Glu Ser Gln Leu Glu Leu Asn Trp Asn Asn 165 170 175 Arg Phe Leu Asn His Cys Leu Glu His Leu Val Gln Tyr Arg Thr Asp 180 185 190 Trp Asp His Ser Trp Thr Glu Gln Ser Val Asp Tyr Arg His Lys Phe 195 200 205 Ser Leu Pro Ser Val Asp Gly Gln Lys Arg Tyr Thr Phe Arg Val Arg 210 215 220 Ser Arg Phe Asn Pro Leu Cys Gly Ser Ala Gln His Trp Ser Glu Trp 225 230 235 240 Ser His Pro Ile His Trp Gly Ser Asn Thr Ser Lys Glu Asn Pro Phe 245 250 255 Leu Phe Ala Leu Glu Ala Val Val Ile Ser Val Gly Ser Met Gly Leu 260 265 270 Ile Ile Ser Leu Leu Cys Val Tyr Phe Trp Leu Glu Arg Thr Met Pro 275 280 285 Arg Ile Pro Thr Leu Lys Asn Leu Glu Asp Leu Val Thr Glu Tyr His 290 295 300 Gly Asn Phe Ser Ala Trp Ser Gly Val Ser Lys Gly Leu Ala Glu Ser 305 310 315 320 Leu Gln Pro Asp Tyr Ser Glu Arg Leu Cys Leu Val Ser Glu Ile Pro 325 330 335 Pro Lys Gly Gly Ala Leu Gly Glu Gly Pro Gly Ala Ser Pro Cys Asn 340 345 350 Gln His Ser Pro Tyr Trp Ala Pro Pro Cys Tyr Thr Leu Lys Pro Glu 355 360 365 Thr <210> 65 <211> 439 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 65 Glu Ser Gly Tyr Ala Gln Asn Gly Asp Leu Glu Asp Ala Glu Leu Asp 1 5 10 15 Asp Tyr Ser Phe Ser Cys Tyr Ser Gln Leu Glu Val Asn Gly Ser Gln 20 25 30 His Ser Leu Thr Cys Ala Phe Glu Asp Pro Asp Val Asn Thr Thr Asn 35 40 45 Leu Glu Phe Glu Ile Cys Gly Ala Leu Val Glu Val Lys Cys Leu Asn 50 55 60 Phe Arg Lys Leu Gln Glu Ile Tyr Phe Ile Glu Thr Lys Lys Phe Leu 65 70 75 80 Leu Ile Gly Lys Ser Asn Ile Cys Val Lys Val Gly Glu Lys Ser Leu 85 90 95 Thr Cys Lys Lys Ile Asp Leu Thr Thr Ile Val Lys Pro Glu Ala Pro 100 105 110 Phe Asp Leu Ser Val Ile Tyr Arg Glu Gly Ala Asn Asp Phe Val Val 115 120 125 Thr Phe Asn Thr Ser His Leu Gln Lys Lys Tyr Val Lys Val Leu Met 130 135 140 His Asp Val Ala Tyr Arg Gln Glu Lys Asp Glu Asn Lys Trp Thr His 145 150 155 160 Val Asn Leu Ser Ser Thr Lys Leu Thr Leu Leu Gln Arg Lys Leu Gln 165 170 175 Pro Ala Ala Met Tyr Glu Ile Lys Val Arg Ser Ile Pro Asp His Tyr 180 185 190 Phe Lys Gly Phe Trp Ser Glu Trp Ser Pro Ser Tyr Tyr Phe Arg Thr 195 200 205 Pro Glu Ile Asn Asn Ser Ser Gly Glu Met Asp Pro Ile Leu Leu Thr 210 215 220 Ile Ser Ile Leu Ser Phe Phe Ser Val Ala Leu Leu Val Ile Leu Ala 225 230 235 240 Cys Val Leu Trp Lys Lys Arg Ile Lys Pro Ile Val Trp Pro Ser Leu 245 250 255 Pro Asp His Lys Lys Thr Leu Glu His Leu Cys Lys Lys Pro Arg Lys 260 265 270 Asn Leu Asn Val Ser Phe Asn Pro Glu Ser Phe Leu Asp Cys Gln Ile 275 280 285 His Arg Val Asp Asp Ile Gln Ala Arg Asp Glu Val Glu Gly Phe Leu 290 295 300 Gln Asp Thr Phe Pro Gln Gln Leu Glu Glu Ser Glu Lys Gln Arg Leu 305 310 315 320 Gly Gly Asp Val Gln Ser Pro Asn Cys Pro Ser Glu Asp Val Val Ile 325 330 335 Thr Pro Glu Ser Phe Gly Arg Asp Ser Ser Leu Thr Cys Leu Ala Gly 340 345 350 Asn Val Ser Ala Cys Asp Ala Pro Ile Leu Ser Ser Ser Arg Ser Leu 355 360 365 Asp Cys Arg Glu Ser Gly Lys Asn Gly Pro His Val Tyr Gln Asp Leu 370 375 380 Leu Leu Ser Leu Gly Thr Thr Asn Ser Thr Leu Pro Pro Pro Phe Ser 385 390 395 400 Leu Gln Ser Gly Ile Leu Thr Leu Asn Pro Val Ala Gln Gly Gln Pro 405 410 415 Ile Leu Thr Ser Leu Gly Ser Asn Gln Glu Glu Ala Tyr Val Thr Met 420 425 430 Ser Ser Phe Tyr Gln Asn Gln 435 <210> 66 <211> 439 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 66 Glu Ser Gly Tyr Ala Gln Asn Gly Asp Leu Glu Asp Ala Glu Leu Asp 1 5 10 15 Asp Tyr Ser Phe Ser Cys Tyr Ser Gln Leu Glu Val Asn Gly Ser Gln 20 25 30 His Ser Leu Thr Cys Ala Phe Glu Asp Pro Asp Val Asn Thr Thr Asn 35 40 45 Leu Glu Phe Glu Ile Cys Gly Ala Leu Val Glu Val Lys Cys Leu Ser 50 55 60 Phe Arg Lys Leu Gln Glu Ile Tyr Phe Ile Glu Thr Lys Lys Phe Leu 65 70 75 80 Leu Ile Gly Lys Ser Asn Ile Cys Val Lys Val Gly Gly Lys Ser Leu 85 90 95 Thr Cys Lys Lys Ile Asp Leu Thr Thr Ile Val Lys Pro Glu Ala Pro 100 105 110 Phe Asp Leu Ser Val Ile Tyr Arg Glu Gly Ala Asn Asp Phe Val Val 115 120 125 Thr Phe Asn Thr Ser His Leu Gln Lys Lys Tyr Val Lys Val Leu Met 130 135 140 His Asp Val Ala Tyr Arg Gln Glu Lys Asp Glu Asn Lys Trp Met His 145 150 155 160 Val Asn Leu Ser Ser Thr Lys Leu Thr Leu Leu Gln Arg Asn Leu Gln 165 170 175 Pro Glu Ala Met Tyr Glu Ile Lys Val Arg Ser Ile Pro Asp His Tyr 180 185 190 Phe Lys Gly Phe Trp Ser Glu Trp Ser Pro Ser Tyr Tyr Phe Arg Thr 195 200 205 Pro Glu Ile Asn Asn Ser Pro Gly Glu Met Asp Pro Ile Leu Leu Thr 210 215 220 Ile Ser Leu Leu Ser Phe Phe Ser Val Ala Leu Leu Val Ile Leu Ala 225 230 235 240 Cys Val Leu Trp Lys Lys Arg Ile Lys Pro Ile Val Trp Pro Ser Leu 245 250 255 Pro Asp His Lys Lys Thr Leu Glu His Leu Cys Lys Lys Pro Arg Lys 260 265 270 Asn Leu Asn Val Ser Phe Asn Pro Glu Ser Phe Leu Asp Cys Gln Ile 275 280 285 His Arg Val Asp Asp Ile Gln Ala Arg Asp Glu Val Glu Gly Phe Leu 290 295 300 Gln Asp Thr Phe Pro Gln Gln Leu Glu Glu Ser Lys Lys Gln Arg Leu 305 310 315 320 Gly Gly Asp Val Gln Ser Pro Ser Cys Pro Ser Glu Asp Val Val Ile 325 330 335 Thr Pro Glu Ser Phe Glu Arg Asp Ser Ser Leu Arg Cys Leu Ala Gly 340 345 350 Asn Val Ser Ala Cys Asp Ala Pro Ile Leu Ser Ser Ser Arg Ser Leu 355 360 365 Asp Cys Arg Glu Ser Gly Lys Asn Gly Pro His Val Tyr Gln Asp Leu 370 375 380 Leu Leu Ser Leu Gly Thr Thr Asn Ser Thr Leu Pro Pro Pro Phe Ser 385 390 395 400 Leu Gln Ser Gly Ile Leu Thr Leu Asn Pro Val Ala Gln Gly Gln Pro 405 410 415 Ile Leu Thr Ser Leu Gly Ser Asn Gln Glu Glu Ala Tyr Val Thr Met 420 425 430 Ser Ser Phe Tyr Gln Asn Gln 435 <210> 67 <211> 220 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 67 Glu Ser Gly Tyr Ala Gln Asn Gly Asp Leu Glu Asp Ala Glu Leu Asp 1 5 10 15 Asp Tyr Ser Phe Ser Cys Tyr Ser Gln Leu Glu Val Asn Gly Ser Gln 20 25 30 His Ser Leu Thr Cys Ala Phe Glu Asp Pro Asp Val Asn Thr Thr Asn 35 40 45 Leu Glu Phe Glu Ile Cys Gly Ala Leu Val Glu Val Lys Cys Leu Ser 50 55 60 Phe Arg Lys Leu Gln Glu Ile Tyr Phe Ile Glu Thr Lys Lys Phe Leu 65 70 75 80 Leu Ile Gly Lys Ser Asn Ile Cys Val Lys Val Gly Gly Lys Ser Leu 85 90 95 Thr Cys Lys Lys Ile Asp Leu Thr Thr Ile Val Lys Pro Glu Ala Pro 100 105 110 Phe Asp Leu Ser Val Ile Tyr Arg Glu Gly Ala Asn Asp Phe Val Val 115 120 125 Thr Phe Asn Thr Ser His Leu Gln Lys Lys Tyr Val Lys Val Leu Met 130 135 140 His Asp Val Ala Tyr Arg Gln Glu Lys Asp Glu Asn Lys Trp Met His 145 150 155 160 Val Asn Leu Ser Ser Thr Lys Leu Thr Leu Leu Gln Arg Asn Leu Gln 165 170 175 Pro Glu Ala Met Tyr Glu Ile Lys Val Arg Ser Ile Pro Asp His Tyr 180 185 190 Phe Lys Gly Phe Trp Ser Glu Trp Ser Pro Ser Tyr Tyr Phe Arg Thr 195 200 205 Pro Glu Ile Asn Asn Ser Pro Gly Glu Met Asp Pro 210 215 220 <210> 68 <211> 219 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 68 Glu Ser Gly Tyr Ala Gln Asn Gly Asp Leu Glu Asp Ala Glu Leu Asp 1 5 10 15 Asp Tyr Ser Phe Ser Cys Tyr Ser Gln Leu Glu Val Asn Gly Ser Gln 20 25 30 His Ser Leu Thr Cys Ala Phe Glu Asp Pro Asp Val Asn Thr Thr Asn 35 40 45 Leu Glu Phe Glu Ile Cys Gly Ala Leu Val Glu Val Lys Cys Leu Asn 50 55 60 Phe Arg Lys Leu Gln Glu Ile Tyr Phe Ile Glu Thr Lys Lys Phe Leu 65 70 75 80 Leu Ile Gly Lys Ser Asn Ile Cys Val Lys Val Gly Glu Lys Ser Leu 85 90 95 Thr Cys Lys Lys Ile Asp Leu Thr Thr Ile Val Lys Pro Glu Ala Pro 100 105 110 Phe Asp Leu Ser Val Ile Tyr Arg Glu Gly Ala Asn Asp Phe Val Val 115 120 125 Thr Phe Asn Thr Ser His Leu Gln Lys Lys Tyr Val Lys Val Leu Met 130 135 140 His Asp Val Ala Tyr Arg Gln Glu Lys Asp Glu Asn Lys Trp Thr His 145 150 155 160 Val Asn Leu Ser Ser Thr Lys Leu Thr Leu Leu Gln Arg Lys Leu Gln 165 170 175 Pro Ala Ala Met Tyr Glu Ile Lys Val Arg Ser Ile Pro Asp His Tyr 180 185 190 Phe Lys Gly Phe Trp Ser Glu Trp Ser Pro Ser Tyr Tyr Phe Arg Thr 195 200 205 Pro Glu Ile Asn Asn Ser Ser Gly Glu Met Asp 210 215 <210> 69 <211> 355 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence <400> 69 gatgttcaat tggttgaatc tggtggtggt ttggttcaag ccggtggttc tttgagattg 60 tcttgtgaat cttctatctc caccttctca tctgatgcta tgggttggtt tagacaagct 120 ccaggtaaag aaagagaatt tttggctgct attggttgga gtggtgctgt tactcattat 180 tccgattctg ttaaaggtcg tttcaccatt tctagagata acgctaagaa caccgtctac 240 ttgcaaatga actctttgag agctgaagat accggtagat attactgcgc tgaagattac 300 gatactgatg tttggcaata ttggggtcaa ggtactcaag ttactgtctc ctcat 355 <210> 70 <211> 355 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence <400> 70 gatgttcaat tggttgaatc tggtggtggt ttggttcaag ccggtggttc tttgagattg 60 tcttgtgaat cttctatctc caccttctca tctgatgcta tgggttggtt tagacaagct 120 ccaggtaaag aattggaatt tttggctgct attggttgga gtggtgctgt tactcattat 180 tccgattctg ttaaaggtcg tttcaccatt tctagagata acgctaagaa caccgtctac 240 ttgcaaatga actctttgag agctgaagat accggtagat attactgcgc tgaagattac 300 gatactgatg tttggcaata ttggggtcaa ggtactcaag ttactgtctc ctcat 355 <210> 71 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 71 Asp Asp Ala Met Gly 1 5 <210> 72 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 72 Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val Gln 1 5 10 15 Gly <210> 73 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 73 Ala Ile Gly Trp Ser Gly Thr Val Thr His Tyr Ser Asp Ser Val Gln 1 5 10 15 Gly <210> 74 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 74 Ala Thr Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val Lys 1 5 10 15 Gly <210> 75 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 75 Ala Ile Asn Trp Ser Gly Ala Val Thr His Tyr Gly Asp Ser Val Lys 1 5 10 15 Gly <210> 76 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 76 Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val Lys 1 5 10 15 Gly <210> 77 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 77 Asp Tyr Val Thr Asp Val Trp Gln Tyr 1 5 <210> 78 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 78 Asp Tyr Asp Thr Asp Val Trp Gln His 1 5 <210> 79 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 79 Glu Leu Glu Phe Leu Ala 1 5 <210> 80 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 80 Gly Leu Glu Trp Val Ser 1 5 <210> 81 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence <400> 81 tcttaactag tgaggagacg gtgacctg 28 <210> 82 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <220> <221> MISC_Features <222> (1) <223> Xaa is Ser or Asp <400> 82 Xaa Asp Ala Met Gly 1 5 <210> 83 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <220> <221> MISC_Features <222> (2)..(2) <223> Xaa is Ile or Thr <220> <221> MISC_Features <222> (3) <223> Xaa is Gly or Asn <220> <221> MISC_Features <222> (7) <223> Xaa is Ala or Thr <220> <221> MISC_Features <222> (12)..(12) <223> Xaa is Ser or Gly <220> <221> MISC_Features <222> (16) <223> Xaa is Gln or Lys <400> 83 Ala Xaa Xaa Trp Ser Gly Xaa Val Thr His Tyr Xaa Asp Ser Val Xaa 1 5 10 15 Gly <210> 84 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <220> <221> MISC_Features <222> (3) <223> Xaa is Asp or Val <220> <221> MISC_Features <222> (9)..(9) <223> Xaa is Tyr or His <400> 84 Asp Tyr Xaa Thr Asp Val Trp Gln Xaa 1 5 <210> 85 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <220> <221> Variants <222> (1)..(11) <223> The residue may be present 1 to 10 times <220> <221> MISC_Features <222> (1)..(25) <223> The residue may be present 1 to 10 times <220> <221> Variants <222> (2)..(10) <223> The Gly residues may be absent, present in some or all <220> <221> MISC_Features <222> (12)..(12) <223> Xaa is selected from Arg, His, Asp, Gln, Ser, Thr, Tyr, Gly, Ala, Val, Leu, Trp, Pro, Met, Cys, Phe, 0 to 5 amino acid residues of Lys or Ile <220> <221> MISC_Features <222> (13) <223> Xaa is Lys or Arg <220> <221> MISC_Features <222> (14)..(14) <223> Xaa is selected from Arg, His, Asp, Gln, Ser, Thr, Tyr, Gly, Ala, Val, Leu, Trp, Pro, Met, Cys, Phe, 0 to 5 amino acid residues of Lys or Ile <220> <221> MISC_Features <222> (15)..(25) <223> The residue may be present 1 to 10 times <220> <221> MISC_Features <222> (16)..(24) <223> The Gly residues may be absent, present in some or all <400> 85 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Xaa Xaa Xaa Gly Gly 1 5 10 15 Gly Gly Gly Gly Gly Gly Gly Gly Ser 20 25 <210> 86 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <220> <221> Variants <222> (1)..(5) <223> The residue may be present 1 to 5 times <220> <221> Variants <222> (7)..(11) <223> The residue may be present 1 to 5 times <400> 86 Gly Gly Gly Gly Ser Lys Gly Gly Gly Gly Ser 1 5 10 <210> 87 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 87 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Gly Gly Gly Gly Ser 1 5 10 15 Gly Gly Gly Gly Ser 20 <210> 88 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <220> <221> Variants <222> (1)..(5) <223> The residue may be present 1 to 10 times <400> 88 Gly Gly Gly Gly Ser 1 5 <210> 89 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 89 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 30

Claims

1. A polypeptide capable of inhibiting the binding of IL-7 and / or L-TSLP to IL-7R, wherein the polypeptide comprises a VHH that binds to IL-7Rα, wherein the VHH comprises three complementarity determining regions CDR1-CDR3 and four framework regions FR1-FR4, wherein CDR1 consists of the sequence of SEQ ID NO: 1, CDR2 consists of the sequence of SEQ ID NO: 2, and CDR3 consists of the sequence of SEQ ID NO:

3.

2. The polypeptide of claim 1, wherein the polypeptide comprises SEQ ID NO:

8.

3. The polypeptide according to claim 2, wherein the polypeptide consists of SEQ ID NO:

8.

4. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 3 and one or more pharmaceutically acceptable diluents or carriers.

5. The pharmaceutical composition according to claim 4, comprising at least one additional active agent.

6. Use of the polypeptide according to any one of claims 1 to 3 in the manufacture of a medicament for treating autoimmune diseases and / or inflammatory diseases.

7. Use of the pharmaceutical composition according to claim 4 or 5 in the manufacture of a medicament for treating autoimmune diseases and / or inflammatory diseases.

8. The use according to claim 6 or 7, wherein the autoimmune disease and / or inflammatory disease is Crohn's disease or ulcerative colitis.

9. The use according to claim 6 or 7, wherein the autoimmune disease and / or inflammatory disease is atopic dermatitis.

10. The use according to claim 6 or 7, wherein the autoimmune disease and / or inflammatory disease is asthma.

11. A polynucleotide encoding the polypeptide according to any one of claims 1 to 3.

Images