polypeptides

Abstract

To provide an immunoglobulin heavy chain variable domain that binds to IL-7R.SOLUTION: Disclosed is an immunoglobulin heavy chain variable domain that binds to IL-7R, comprising complementarity determining region 1 comprising an amino acid sequence X1DAMG, complementarity determining region 2 comprising an amino acid sequence AX2X3WSGX4VTHYX5DSVX6G, and complementarity determining region 3 comprising an amino acid sequence DYX7TDVWQX8, where the immunoglobulin heavy chain variable domain inhibits binding of IL-7 to IL-7R or binding of L-TSLP to IL-7R and is VHH, where X1 is S or D, X2 is I or T, X3 is G or N, X4 is A or T, X5 is S or G, X6 is Q or K, X7 is V or D, X8 is Y or H.SELECTED DRAWING: None

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, constructs and pharmaceutical compositions comprising these polypeptides, nucleic acids encoding the polypeptides, methods for preparing the polypeptides, cDNAs and vectors comprising nucleic acids encoding the polypeptides, host cells expressing or capable of expressing the polypeptides, and uses of the polypeptides. [Background technology]

[0002] Autoimmune diseases of the gastrointestinal tract 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 or regional enteritis, causes a variety of symptoms. Crohn's disease primarily causes abdominal pain, diarrhea, vomiting, and / or weight loss, but can also cause non-gastrointestinal (GIT) complications such as anemia, skin rash, arthritis, eye inflammation, fatigue, and difficulty concentrating (Baumgart et al., 2012). Crohn's disease is a lifelong, intractable gastrointestinal disorder that is currently difficult to control with conventional treatments. EoE is a chronic condition defined by significant pathological eosinophil infiltration limited to the esophagus, which causes esophageal dysfunction and, if untreated, fibrosis. Esophageal fibrosis and esophageal strictures are known complications of EoE.

[0003] Antibody-based therapeutics have great potential as effective treatments for autoimmune diseases such as IBD and EoE due to their high target specificity and low inherent toxicity. Three anti-TNFα antibodies, infliximab (trade name Remicade), adalimumab (trade name Humira), and certolizumab (also known as "certolizumab pegol," trade name Cimzia), are clinically used to treat Crohn's disease. However, these antibodies are generally considered unsuitable for oral administration due to their inherent instability and susceptibility to proteolysis by inflammatory proteases present in the digestive system, lesions in the gastrointestinal tract, and intestinal microflora. Therefore, these agents must be administered intravenously or subcutaneously, requiring expert training for the accurate and safe use of hypodermic syringes or needles. These agents require sterile equipment, a liquid formulation of the therapeutic polypeptide, a vial containing the polypeptide in a sterile and stable form, and an appropriate site for needle penetration. "TNFα therapy is a promising treatment option for patients with severe rheumatoid arthritis, including rheumatoid arthritis, as well as rheumatoid arthritis. Patients typically experience psychological stress before receiving the injection and pain during the injection. Long-term treatment with systemic anti-TNFα antibodies is associated with an increased risk of serious infections and cancer. Furthermore, high manufacturing costs limit its use to more severely ill patients."

[0004] Several small molecule anti-inflammatory and immunosuppressive drugs are currently in clinical development for Crohn's disease (Danese 2012, Shealy et al. 2010, and Vetter & Neurath 2017). Although these drugs are administered orally, many are absorbed systemically after administration and may have systemic immunosuppressive effects unrelated to their effects on gastrointestinal lesions. Furthermore, because small molecules lack the specificity of antibodies, the risk of significant off-target side effects remains high.

[0005] The ability to deliver oral therapeutics with high selectivity for targets relevant to gastrointestinal autoimmune diseases, yet with exposure and activity limited to the gastrointestinal tract, combined with significantly improved safety due to reduced systemic exposure, can confer efficacy similar to injectable antibodies.

[0006] Interleukin-7 (IL-7) and interleukin-7 receptor (IL-7R) Interleukin-7 (IL-7) is a member of a family of cytokines that includes IL-2, IL-4, IL-7, IL-15, and IL-21. IL-7 is constitutively produced by non-hematopoietic stem cells and epithelial cells in lymphoid organs, the intestine, the skin, and the liver and is essential for T lymphocyte development in the thymus and for the survival and homeostatic control of peripheral T cells (Fry and Mackall, 2002; Fry and Mackall, 2005). In the intestinal mucosa, IL-7 further regulates phenotypically and functionally distinct populations of innate lymphoid cells important for the initial priming of immune responses to pathogenic microbial challenges, as well as CD4+ lymphoid tissue inducer (LTi) cells, which have the ability to promote lymphoid tissue organogenesis and several dendritic cell populations (Goldberg et al., 2015; Peters et al., 2015). Furthermore, IL-7 induces proliferation of naive and memory T cells and enhances effector T cell responses, preferentially T helper 1 (Th1) and Th17 responses (Dooms, 2013). The functional effects of IL-7 on T cells make it an important enhancer of protective immunity as well as autoimmunity and inflammation.

[0007] The effects of IL-7 on different target cells are mediated through the IL-7R, a heterodimeric complex containing the IL-7Rα subunit (CD127) and the common cytokine receptor γ chain (γc) (CD132). Full-length human IL-7Rα consists of a 219-residue extracellular domain, a 25-residue transmembrane domain, and a 195-residue intracellular domain. IL-7-induced receptor activation is thought to occur when IL-7 first interacts with IL-7Rα to form a complex, which then recruits γc to form the activated receptor signaling complex. IL-7-mediated association of the two receptor subunits activates intracellular phosphorylation events via the JAK / Stat, PI3 / Akt, and SRC signaling cascades (Walsh, 2012).

[0008] IL-7Rα is available not only in a cell membrane-bound format but also as a soluble form (sIL-7Rα). sIL-7R is generated by shedding of the membrane-bound receptor and is produced by alternative splicing (of IL-7Rα exon 6) resulting in a protein lacking the transmembrane domain; sIL-7Rα present in human plasma is primarily derived from alternative splicing (Rose et al., 2009). Four common haplotypes of the IL-7Rα gene have been identified (Teutsch et al., 2003). Two haplotypes are associated with altered expression and production of the soluble receptor and susceptibility to autoimmune diseases, including multiple sclerosis and type 1 diabetes (Hafler et al., 2007).

[0009] In addition to the role of 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 different autoimmune and inflammatory diseases. These studies have identified additional IL-7-dependent mechanisms associated with disease pathology and highlighted the IL-7 / IL-7Rα interaction as a potential target for the treatment of patients with related autoimmune and chronic inflammatory diseases.

[0010] 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 IBD models, the primary mechanism of efficacy after IL-7R-antagonist treatment is thought to involve local depletion or functional suppression of intestinal inflammation-inducing T cells (IL-7R+ effector / memory T cells) that express intermediate to high levels of IL-7Rα (CD127) and can be activated in the inflamed intestine by increased IL-7 production by stromal and epithelial cells. In healthy mucosa, gut-resident FOXP3 expresses very low levels of IL-7R. + CD25 +Regulatory T cells (Treg cells) are expected to suppress dysregulated CD4+ T responses to commensal bacteria. However, Heninger et al. (2012) reported that in an IL-7-rich environment, FOXP3 + CD25 + It has been suggested that the ability of Tregs to suppress the proliferation of conventional T cells is impaired. Therefore, in gastrointestinal diseases, blockade of IL-7 / IL-7R signaling can help control T cell-mediated inflammation by inhibiting the activation of effector T cells and restoring the suppressive function of regulatory T cells. Evidence from mouse 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.

[0011] Thymic stromal lymphopoietin (TSLP) and IL-7R TSLP is a cytokine produced by epithelial cells in the skin, lung, intestine, and eye tissues that is thought to be involved in regulating inflammatory processes at mucosal surfaces in the body. TSLP stimulates dendritic cells (DCs) and innate lymphoid cells (ILCs) to secrete Th2 cytokines (IL-4, IL-5, and IL-13), promoting the development of Th2-type inflammation. TSLP is thought to underlie the development of several allergic diseases, such as atopic dermatitis and rhinitis, and also promotes intestinal diseases, including eosinophilic esophagitis (EoE) and ulcerative colitis (UC). Paradoxically, TSLP has also been reported to be important for maintaining immune homeostasis and mucosal protection in the gastrointestinal tract. Recently, it has been discovered that TSLP can be expressed as two distinct isoforms, providing a biological explanation for the seemingly contrasting activities of this cytokine (Fornasa et al., 2015; Tsilingiri et al., 2017). Molecular biological studies have shown that the TSLP gene can give rise to two coding RNAs controlled by two distinct promoter regions. One transcript encodes the 159-aa long isoform of TSLP (L-TSLP) (UNIPROT entry Q969D9, SEQ ID NO: 62), while the other transcript encodes the short form of TSLP (S-TSLP) encompassing the C-terminal 63 aa of L-TSLP (UNIPROT entry Q969D9-2, SEQ ID NO: 63). L-TSLP acts on target cells through a receptor complex containing the TSLP-specific receptor chain (TSLPR) and the IL-7Rα chain. Recent 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).

[0012] S-TSLP does not bind to the TSLPR and cannot inhibit the binding of L-TSLP to this receptor. To the authors' knowledge, the specific receptor for S-TSLP has not been identified to date.

[0013] Importantly, S-TSLP has been shown to be preferentially expressed by healthy skin and by epithelial and lamina propria cells in healthy intestinal mucosal tissues. S-TSLP has anti-inflammatory activity, and in vitro, S-TSLP inhibits pro-inflammatory cytokine production by monocyte-derived DCs and contributes to conditioning CD103+ DCs toward a tolerogenic phenotype. Thus, S-TSLP produced by the intestinal epithelium appears to influence basal immune cells, including dendritic cells and lymphocytes, and promote tolerogenic and regulatory responses in health. S-TSLP also exhibits potent antibacterial (bacterial and fungal) activity, which may be important for protecting mucosal epithelia against microbial invasion (Bjerkan et al., 2016). S-TSLP expression in healthy tissues is constitutive but can be upregulated by vitamin D3 and PPARγ agonists and downregulated by pro-inflammatory pathogenic bacteria. L-TSLP is not present in healthy tissues but is expressed in response to proinflammatory stimuli and plays a key role in promoting Th2 cytokine-associated inflammation 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 can also stimulate Th2 innate immune responses through activation of basophils, ILCs (ILC2s), and eosinophils.

[0014] Recent studies have revealed that the expression patterns of both TSLP isoforms are dramatically altered from their steady-state state in intestinal diseases, including inflammatory bowel disease and eosinophilic esophagitis (EoE). Rimoldi et al. (2005) reported that TSLP (likely specifically S-TSLP in this case, similar to the findings of Fornasa et al. (2015)) was constitutively expressed by primary epithelial cells isolated from healthy colonic tissue. However, TSLP expression was found to be undetectable in epithelial cells from 6 / 9 patients with CD. The inability of CD epithelial cells to produce S-TSLP in the diseased mucosa disrupts a mechanism that normally helps maintain intestinal homeostasis by creating a non-inflammatory environment. Defects in this mechanism may induce unwanted Th1 inflammatory responses and contribute to the development of CD. In contrast to the lamina propria Th1 cells that predominate in Crohn's disease, T cells from patients with ulcerative colitis have been shown to produce the Th2 cytokines IL-5 and IL-13, but these cells only produce low levels of IL-4, suggesting that they do not display all the characteristics of classical Th2 cells. Functionally, IL-13 has been shown to promote fibrosis and induce changes in tight junction function and apoptosis in intestinal epithelial cells, thereby inducing mucosal ulcers. Recently, Fornasa et al. (2015) reported that TSLP expression, detected with an L-TSLP-specific antibody, was significantly elevated in the intestinal tissue of patients with ulcerative colitis compared with levels detected in healthy colonic mucosa. Because TSLP-activated dendritic cells (DCs) can induce the differentiation of naive CD4+ T cells into IL-5-, IL-13-, and TNF-producing proinflammatory Th2 cells (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.

[0015] Epidemiological data support the involvement of atopic mechanisms and genetic factors in the development of EoE. Importantly, genome-wide association studies have identified TSLP and its receptor, TSLPR, as candidate genes for EoE pathogenesis (Cianferoni and Spergel, 2015). TSLP expression is increased in esophageal biopsy specimens from EoE patients and has been localized to stratified squamous epithelial cells by immunohistochemical staining (Noti et al., 2013). L-TSLP is thought to be an upstream driver of disease pathology because it potently promotes the production of cytokines (including CCL-26 / eotaxin-3, IL-4, IL-5, IL-9, and IL-13) and profibrotic factors from Th2 cells, basophils, eosinophils, and mast cells, which contribute to inflammation and tissue remodeling.

[0016] In EoE, TSLP is thought to function as an upstream epithelial "master-switch" immediately after the initiation of the inflammatory cascade, and as a result, antagonizing this cytokine may halt the inflammatory cascade further upstream than previous targeted anti-cytokine therapies. This concept is supported by the positive results obtained in a recent trial of tezepelumab (AMG 157), a first-in-class TSLP antagonist mAb, in patients with severe asthma (Corren et al., 2017).

[0017] In light of the above, it will be appreciated that there is an unmet need for more effective treatments 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 if orally administrable, could represent such a treatment and would therefore be considered highly desirable.

[0018] WO2013056984, WO2015189302, WO2011094259, and WO2011104687 disclose antibodies directed against IL-7R. Summary of the Invention

[0019] The present inventors have generated 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 in particular from surprisingly high potency. They are capable of cross-reacting with cynomolgus monkey IL-7Rα and remain stable when exposed to small and large intestinal proteases.

[0020] In one embodiment, these polypeptides are further enhanced by engineering, and these further enhanced polypeptides comprise sequences that are humanized, yet still substantially maintain the advantages described above.

[0021] In some embodiments, polypeptides of the invention bind to IL-7R with high affinity in Biacore studies and are shown to be potent inhibitors of IL-7R interaction with both IL-7 and the IL-7-related cytokine L-TSLP in ELISA. In functional, cell-based assays, certain polypeptides of the invention inhibit the biological effects of both cytokines (IL-7-induced Stat5 phosphorylation, L-TSLP-induced TARC production) with potency similar to that of the clinical anti-IL-7R mAb 829 (also known as "GSK2618960," an anti-IL-7Rα monoclonal antibody disclosed in Ellis et al., 2019).

[0022] In silico modeling suggests that the dual antagonistic activity of certain polypeptides of the present invention is due to the polypeptide's binding to an epitope on IL-7R that also constitutes a shared binding site for both cytokines. Specificity tests showed that certain polypeptides of the present invention did not exhibit any binding activity to other members of the human IL-7R family or other cytokine receptors. In cross-species specificity assays, certain polypeptides of the present invention did not bind to mouse IL-7R, but bound to cynomolgus monkey and human IL-7R with similar potency. Therefore, cynomolgus monkeys are considered a suitable species for preclinical development studies.

[0023] In ex vivo cultures of inflamed ulcerative colitis mucosal tissue, exemplary polypeptides of the invention were shown to inhibit the phosphorylation of signaling proteins and the production of cytokines and chemokines associated with pro-inflammatory and immunoregulatory pathways. The results demonstrate that antagonism of mucosal IL-7R+ve T cells by the polypeptides can inhibit the inflammatory process at least as effectively as clinical anti-IL-7R mAb 829 in a model closely related to the disease environment, raising the possibility that the polypeptides of the invention may be particularly effective in patients with ulcerative colitis. In vivo, oral administration of certain polypeptides of the invention to normal mice demonstrated high levels of colonic luminal exposure (micromolar), indicating resistance to digestion during passage through the entire digestive system.

[0024] Thus, these polypeptides may be expected to be useful, inter alia, when administered orally, in the prevention or treatment of autoimmune and / or inflammatory diseases, such as inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis) or eosinophilic esophagitis.

[0025] The present invention provides polypeptides 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 the polypeptides, methods for preparing the polypeptides, cDNAs and vectors containing nucleic acids encoding the polypeptides, host cells capable of expressing the polypeptides, and uses of the polypeptides.

[0026] For the avoidance of doubt about the above term "and / or," "a polypeptide capable of inhibiting the binding of IL-7 to IL-7R" encompasses polypeptides capable of inhibiting the binding of IL-7 and L-TSLP to IL-7R. Similarly, "a polypeptide capable of inhibiting the binding of L-TSLP to IL-7R" encompasses polypeptides capable of inhibiting the binding of IL-7 and L-TSLP to IL-7R.

[0027] The polypeptides of the invention, in at least some embodiments, may have one or more of the following advantages over prior art substances capable of inhibiting the binding of IL-7 and / or L-TSLP to IL-7R: (i) increased affinity for IL-7Rα; (ii) increased specificity for IL-7Rα; (iii) increased neutralizing ability of IL-7 or L-TSLP against binding to IL-7R; (iv) inhibiting the binding of both IL-7 and L-TSLP to IL-7R; (v) increased cross-reactivity with IL-7Rα from different species, such as humans and cynomolgus monkeys; (vi) reduced immunogenicity when administered to, for example, mice, cynomolgus monkeys, or humans; (vii) increased stability in the presence of proteases, for example, (a) proteases found in the small intestine and / or large intestine, and / or IBD inflammatory proteases, such as trypsin, chymotrypsin, MMP3, MMP10, MMP12, other MMPs, and cathepsins, and (b) proteases from gut commensal flora and / or pathogenic bacteria that are actively secreted and / or released by lysis of microbial cells found in the small intestine and / or large intestine; (viii) increased stability against protease degradation during production (e.g., resistance to yeast proteases); (ix) increased suitability for oral administration; (x) increased suitability for local delivery to the intestinal tract and lamina propria after oral administration; (xi) increased suitability for local delivery to the esophagus after oral administration; (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, e.g., Saccharomyces cerevisiae or Pichia pastoris; (xiii) suitability and improved properties for use in pharmaceuticals; (xiv) suitability and improved properties for use in functional foods; (xv) enhanced tissue permeability, including penetration of inflamed colonic mucosal epithelium and submucosa to access the submucosal lamina propria; (xvi) increased compatibility of formatting in multispecific formats; (xvii) binding to a novel epitope;

[0028] The above advantages (i) to (xvii) can potentially be achieved by the polypeptides of the invention in a monovalent format or in a multivalent format, such as a bihead format (e.g., a homobihead or heterobihead format).

[0029] References to "IL-7R" above (and throughout this specification) may also be replaced with "IL-7Rα" where appropriate, since the polypeptides of the present invention specifically bind to the IL-7Rα subunit of IL-7R. [Brief explanation of the drawings]

[0030] [Figure 1] Inhibition of IL-7-induced pSTAT-5 in human lymphocytes. [Figure 2] Inhibition of TSLP-induced TARC secretion in human monocytes. [Figure 3] Model structure of V7R-2E9 bound to IL-7Rα. [Figure 4] A62U, A59U, and mAb829 inhibition of IL-7-induced pSTAT5 in human lymphocytes. [Figure 5] Cross-reactivity of ID-A40U with IL-7Rα species [Figure 6] Cross-reactivity of ID-A59U and ID-A62U with human and cynomolgus monkey IL-7Rα [Figure 7] Specificity of ID-A40U for human IL-7Rα [Figure 8] Percent survival of V7R-2E9, ID-A24U, and ID-A40U in the gut matrix [Figure 9] Percent stability of ID-A62U and ID-A41U in human fecal supernatant [Figure 10] Digestion of etanercept, mAb829, and A40U-F / H by MMP [Figure 11] Expected concentrations in undiluted fecal supernatant (ID-A24U vs. ID-A40U vs. ID-38F) [Figure 12] Expected concentrations in undiluted GI supernatant (ID-A24U vs. ID-A40U vs. ID-38F) [Figure 13] Expected concentrations in undiluted fecal supernatant (ID-A40U vs. ID-38F) [Figure 14]Expected concentrations in undiluted gastrointestinal supernatant (ID-A40U vs. ID-38F) [Figure 15] Phosphorylation profile of human IBD tissue proteins [Figure 16] Phosphorylation profile of human IBD tissue proteins [Figure 17] Phosphorylation profile of human IBD tissue proteins [Figure 18] Phosphorylation profile of human IBD tissue proteins [Figure 19] Total phosphorylation level

[0031] Sequence Listing SEQ ID NO: 1-ID-A62U CDR1 polypeptide sequence, SEQ ID NO: 2-ID-A62U CDR2 polypeptide sequence, SEQ ID NO:3-ID-A62U CDR3 polypeptide sequence, SEQ ID NO: 4-ID-A62U FR1 polypeptide sequence, The polypeptide sequence of SEQ ID NO: 5-ID-A62U FR2, The polypeptide sequence of SEQ ID NO: 6-ID-A62U FR3, The polypeptide sequence of SEQ ID NO: 7-ID-A62U FR4, The polypeptide sequence of SEQ ID NO: 8-ID-A62U, SEQ ID NO: 9 - Polypeptide sequence of V7R-2B6; SEQ ID NO: 10 - Polypeptide sequence of V7R-2E5; SEQ ID NO: 11 - Polypeptide sequence of V7R-2E9; SEQ ID NO: 12 - Polypeptide sequence of V7R-2F6; SEQ ID NO: 13 - Polypeptide sequence of V7R-3B5; SEQ ID NO: 14 - Polypeptide sequence of V7R-4F6; SEQ ID NO: 15-V7R-6C12 polypeptide sequence; Polypeptide sequence of SEQ ID NO: 16-ID-A2U, The polypeptide sequence of SEQ ID NO: 17-ID-A3U, Polypeptide sequence of SEQ ID NO: 18-ID-A4U, The polypeptide sequence of SEQ ID NO: 19-ID-A5U, The polypeptide sequence of SEQ ID NO: 20-ID-A6U, The polypeptide sequence of SEQ ID NO: 21-ID-A7U, The polypeptide sequence of SEQ ID NO: 22-ID-A8U, The polypeptide sequence of SEQ ID NO: 23-ID-A9U, The polypeptide sequence of SEQ ID NO: 24-ID-A10U, The polypeptide sequence of SEQ ID NO: 25-ID-A11U, The polypeptide sequence of SEQ ID NO: 26-ID-A12U, The polypeptide sequence of SEQ ID NO: 27-ID-A13U, The polypeptide sequence of SEQ ID NO: 28-ID-A14U, The polypeptide sequence of SEQ ID NO: 29-ID-A15U, The polypeptide sequence of SEQ ID NO: 30-ID-A16U, The polypeptide sequence of SEQ ID NO: 31-ID-A17U, The polypeptide sequence of SEQ ID NO: 32-ID-A18U, The polypeptide sequence of SEQ ID NO: 33-ID-A19U, The polypeptide sequence of SEQ ID NO: 34-ID-A20U, The polypeptide sequence of SEQ ID NO: 35-ID-A21U, The polypeptide sequence of SEQ ID NO: 36-ID-A23U, The polypeptide sequence of SEQ ID NO: 37-ID-A24U, The polypeptide sequence of SEQ ID NO: 38-ID-A25U, The polypeptide sequence of SEQ ID NO: 39-ID-A26U, The polypeptide sequence of SEQ ID NO: 40-ID-A27U, The polypeptide sequence of SEQ ID NO: 41-ID-A28U, The polypeptide sequence of SEQ ID NO: 42-ID-A29U, The polypeptide sequence of SEQ ID NO: 43-ID-A30U, The polypeptide sequence of SEQ ID NO: 44-ID-A31U, The polypeptide sequence of SEQ ID NO: 45-ID-A32U, The polypeptide sequence of SEQ ID NO: 46-ID-A33U, The polypeptide sequence of SEQ ID NO: 47-ID-A34U, The polypeptide sequence of SEQ ID NO: 48-ID-A35U, The polypeptide sequence of SEQ ID NO: 49-ID-A36U, The polypeptide sequence of SEQ ID NO: 50-ID-A37U, The polypeptide sequence of SEQ ID NO: 51-ID-A38U, The polypeptide sequence of SEQ ID NO: 52-ID-A39U, The polypeptide sequence of SEQ ID NO: 53-ID-A40U, The polypeptide sequence of SEQ ID NO: 54-ID-A43U, The polypeptide sequence of SEQ ID NO: 55-ID-A50U, The polypeptide sequence of SEQ ID NO: 56-ID-A52U, The polypeptide sequence of SEQ ID NO: 57-ID-A53U, The polypeptide sequence of SEQ ID NO: 58-ID-A54U, The polypeptide sequence of SEQ ID NO: 59-ID-A55U, The polypeptide sequence of SEQ ID NO: 60-ID-A57U, The polypeptide sequence of SEQ ID NO: 61-ID-A59U, SEQ ID NO: 62 - Polypeptide sequence of L-TSLP. SEQ ID NO: 63 - Polypeptide sequence of S-TSLP. SEQ ID NO: 64—Full-length human common gamma-chain receptor polypeptide sequence; SEQ ID NO: 65—Polypeptide sequence of full-length human IL-7Rα; SEQ ID NO: 66—Polypeptide sequence of full-length cynomolgus monkey IL-7Rα; SEQ ID NO:67—Polypeptide sequence of cynomolgus monkey IL-7Rα extracellular domain. SEQ ID NO: 68—Polypeptide sequence of human IL-7Rα extracellular domain. A polynucleotide sequence encoding SEQ ID NO: 69-ID-A59U, A polynucleotide sequence encoding SEQ ID NO: 70-ID-A62U, SEQ ID NO: 71 - Polypeptide sequence of V7R-2B6 CDR1, SEQ ID NO: 72 - Polypeptide sequence of V7R-2E9 CDR2, SEQ ID NO: 73 - V7R-2F6 CDR2 polypeptide sequence, SEQ ID NO: 74 - V7R-4F6 CDR2 polypeptide sequence, SEQ ID NO: 75 - Polypeptide sequence of V7R-2B6 CDR2, SEQ ID NO: 76-ID-A14U CDR2 polypeptide sequence, SEQ ID NO: 77-V7R-6C12 CDR3 polypeptide sequence, SEQ ID NO: 78 - Polypeptide sequence of V7R-2B6 CDR3, SEQ ID NO: 79-ID-A62U A polypeptide sequence preferably occupying residues 9-14 of FR2 (SEQ ID NO: 5), SEQ ID NO: 80-ID-A62U A polypeptide sequence that does not preferably occupy residues 9-14 of FR2 (SEQ ID NO: 5), SEQ ID NO:81—Polynucleotide sequence of 3′ primer containing SpeI site. SEQ ID NO:82 - Polypeptide sequence of CDR1 with optional conservative substitutions at residue 1 of SEQ ID NO:1; SEQ ID NO:83—Polypeptide sequence of CDR2 with any conservative substitutions at residues 2, 3, 7, 12, and 16 of SEQ ID NO:2; SEQ ID NO:84—Polypeptide sequence of CDR3 with optional conservative substitutions at residues 3 and 9 of SEQ ID NO:3; SEQ ID NO:85 - Polypeptide sequence of protease labile linker formula 1, SEQ ID NO:86 - Polypeptide sequence of protease labile linker formula 2, SEQ ID NO:87 - Polypeptide sequence of a preferred variant of protease labile linker formula 1 and 2, SEQ ID NO:88—Polypeptide sequence of non-protease labile linker formula 3, SEQ ID NO:89—Polypeptide sequence of a preferred non-protease labile linker. DETAILED DESCRIPTION OF THE INVENTION

[0032] Polypeptides such as antibodies and antibody fragments that contain immunoglobulin chain variable domains (ICVDs), such as VH and VHH

[0033] A polypeptide is an organic polymer consisting of a number of amino acid residues linked in a chain. As used herein, "polypeptide" is used interchangeably with "protein" and "peptide." A polypeptide binds to an epitope on a target with affinity (preferably, a Kd value, Ka value, k, as further described herein). on -rate, and / or k off A polypeptide is said to be a binding polypeptide if it contains a stretch of one or more amino acid residues forming a binding site to which the polypeptide can bind via a binding site (expressed as a -rate).

[0034] Binding polypeptides include polypeptides such as DARPins (Binz et al. 2003), Affimers™ (Johnson et al. 2012), Fynomers™ (Grabulovski et al. 2007), Centyrins (Goldberg et al. 2016), Affitins (e.g., Nanofitins®, Krehenbrink et al. 2008), circulating peptides, antibodies, and antibody fragments. Binding polypeptides include Affibodies (Nygren 2008), Affilins (Ebersbach et al. 2007), Alphabodies (Desmet et al. 2014), Anticalins (Skerra et al. 2008), Avimers (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.

[0035] A conventional antibody or immunoglobulin (Ig) is a protein containing four polypeptide chains: two heavy (H) chains and two light (L) chains. Each chain is divided into a constant region and a variable region. The heavy chain variable domain is abbreviated herein as VHC, and the light chain variable domain is abbreviated herein as VLC. These domains, their related domains, and domains derived from them are referred to herein as immunoglobulin chain variable domains. The VHC and VLC domains can be further subdivided into regions of hypervariability called "complementarity-determining regions" ("CDRs"), interspersed with more conserved regions called "framework regions" ("FRs"). The framework regions and complementarity-determining regions have been precisely defined (Kabat et al., 1991). In conventional antibodies, the VHC and VLC each consist of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A conventional antibody tetramer, for example, consists of two immunoglobulin heavy chains and two immunoglobulin light chains interconnected by disulfide bonds, with the immunoglobulin heavy chains being similarity-connected. The heavy chain constant region contains three domains, CH1, CH2, and CH3. The light chain constant region consists of one domain, CL. The heavy chain variable domain and the light chain variable domain are binding domains that interact with antigens. The antibody constant region typically mediates antibody binding to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The term antibody includes immunoglobulins of types IgA, IgG, IgE, IgD, and IgM (and their subtypes), and the immunoglobulin light chains may be kappa or lambda types. The overall structure of immunoglobulin-γ (IgG) antibodies, assembled from two identical heavy (H) chain and two identical light (L) chain polypeptides, is well established and highly conserved in mammals (Padlan 1994).

[0036] An exception to the conventional antibody structure is found in camelid sera. In addition to conventional antibodies, these sera contain special IgG antibodies. These IgG antibodies, known as heavy-chain antibodies (HCAbs), lack the light-chain polypeptide and the first constant domain (CH1). In their N-terminal regions, the heavy chains of the homodimeric proteins contain dedicated immunoglobulin chain variable domains, termed VHHs, which are responsible for associating with their cognate antigens (Muyldermans 2013, Hamers-Casterman et al. 1993, Muyldermans et al. 1994).

[0037] 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 which specifically binds to IL-7Rα). Examples of binding fragments encompassed within the term antigen-binding fragment include the following: (i) FAb fragments (monovalent fragments consisting of the VLC, VHC, CL, and CH1 domains); (ii) F(ab')2 fragment (a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region); (iii) Fd fragment (consisting of the VHC and CH1 domains); (iv) Fv fragments (consisting of the VLC and VHC domains of a single arm of an antibody); (v) scFv fragments (consisting of VLC and VHC domains recombinantly linked by a synthetic linker, which allows the VLC and VHC domains to form a single protein chain, and the VLC and VHC regions pair to form a monovalent molecule); (vi) VH (immunoglobulin chain variable domain consisting of VHC domains (Ward et al. 1989) (vii) VL (immunoglobulin chain variable domain consisting of VLC domain), (viii) V-NAR (immunoglobulin chain variable domain consisting of the VHC domain from cartilaginous fish IgNAR (Roux et al. 1998 and Griffiths et al. 2013)); (ix) VHH.

[0038] The total number of amino acid residues in VHH or VH may be in the range of 110 to 130, preferably 115 to 120, and most preferably 118.

[0039] The immunoglobulin chain variable domains of the invention can be obtained, for example, by preparing a nucleic acid encoding the immunoglobulin chain variable domain using techniques for nucleic acid synthesis, and then expressing the nucleic acid thus obtained. According to certain embodiments, the immunoglobulin chain variable domains of the invention do not have an amino acid sequence that is exactly the same as (i.e., shares 100% sequence identity with) the amino acid sequence of a naturally occurring polypeptide, such as the VH domain or VHH domain of a naturally occurring antibody.

[0040] 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 polypeptide, such as antibodies and antibody fragments. For example, the anti-IL-7Rα immunoglobulin chain variable domains disclosed herein may be incorporated into polypeptides such as full-length antibodies. Such an approach is demonstrated by McCoy et al. (2014), who provide anti-HIV VHHs engineered as fusions with a human Fc region (including the hinge, CH2, and CH3 domains) expressed as dimeric constructs.

[0041] Humanization involves replacing at least one amino acid residue in a framework region of a non-human immunoglobulin chain variable domain with the corresponding residue from a human immunoglobulin chain variable domain. Humanization of variable domains can reduce immunogenicity in humans.

[0042] Preferably, the polypeptide of the present invention comprises an immunoglobulin chain variable domain. More preferably, the polypeptide of the present invention consists of an immunoglobulin chain variable domain such as an immunoglobulin heavy chain variable domain. Preferably, the polypeptide of the present invention is an antibody or an antibody fragment. More preferably, the polypeptide of the present invention is an antibody fragment. Preferably, the antibody fragment is an immunoglobulin variable domain such as a VHH, VH, or VL. Preferably, the antibody fragment is a VHH, VH, VL, V-NAR, scFv, FAb fragment, or F(ab')2 fragment. Preferably, the antibody fragment is an immunoglobulin heavy chain variable domain. More preferably, the antibody fragment is a VHH or VH, and most preferably a VHH.

[0043] Specificity, affinity, avidity, and cross-reactivity Specificity refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding polypeptide can bind. The specificity of an antigen-binding polypeptide is the ability of the antigen-binding polypeptide to recognize a particular antigen as a unique molecular entity and distinguish it from other antigens.

[0044] Affinity, expressed as the equilibrium constant (Kd) for the dissociation of a target from a binding polypeptide, is an indicator of the binding strength between a target and a binding site on the binding polypeptide; the smaller the Kd value, the stronger the binding force 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. Preferably, affinity is determined using a dynamically switchable biosurface (e.g., "switchSENSER", see Knezevic et al., 2012) or surface plasmon resonance.

[0045] Avidity is a measure of the strength of the bond between an antigen-binding polypeptide and an appropriate antigen. Avidity is related to both the affinity between an antigenic determinant on the antigen-binding polypeptide and its antigen-binding site, and the number of appropriate binding sites present on the antigen-binding polypeptide.

[0046] Preferably, the polypeptide of the present invention is -7 M or less, more preferably 10 -8 M or less, more preferably 10 -9 M or less, and more preferably 10 -10 It binds to IL-7Rα with an equilibrium dissociation constant (Kd) of less than M.

[0047] Preferably, the polypeptides of the present invention bind to IL-7Rα with an equilibrium dissociation constant that is lower than the equilibrium dissociation constant of mAb 829 in the same assay. -10 M or less, more preferably 5.67×10 -10 It binds to IL-7Rα with an equilibrium dissociation constant lower than M. mAb829 is also known as "GSK2618960," an anti-IL-7Rα monoclonal antibody disclosed in Ellis et al. (2019).

[0048] In one embodiment, the affinity of a polypeptide of the invention is established by direct coating onto a Biacore (or equivalent) sensor plate, or by fusion to Fc and capture with anti-human IgG Fc, and the polypeptide is flowed across the plate to detect binding. Suitably, a Biacore T200 plate is used at 30 ul / min in HBS-EP+ (GE Healthcare) running buffer at 25°C.

[0049] 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 can bind to a linear or conformational epitope on IL-7Rα.

[0050] Preferably, the polypeptides of the present invention bind to human IL-7Rα. More preferably, the polypeptides of the present invention bind to both human IL-7Rα and IL-7Rα of at least one additional primate selected from the group consisting of baboon IL-7Rα, marmoset IL-7Rα, cynomolgus IL-7Rα, and rhesus IL-7Rα. Even more preferably, the polypeptides of the present invention bind to both human IL-7Rα and cynomolgus IL-7Rα.

[0051] Preferably, the polypeptides of the present invention neutralize the binding of human IL-7 and / or human L-TSLP to the human IL-7R. More preferably, the polypeptides of the present invention neutralize the binding of human IL-7 and / or human L-TSLP to both the human IL-7R and the IL-7R of at least one additional primate selected from the group consisting of baboon IL-7R, marmoset IL-7R, cynomolgus IL-7R, and rhesus IL-7R. Most preferably, the polypeptides of the present invention neutralize the binding of human IL-7 and human L-TSLP to the human IL-7R.

[0052] Preferably, the polypeptides of the present 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 a gamma chain receptor (e.g., human common gamma chain receptor, SEQ ID NO: 64). More preferably, the polypeptides of the present invention bind to IL-7Rα, most preferably human IL-7Rα. More specifically, the polypeptides of the present 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 cytoplasmic domain.

[0053] Preferably, IL-7Rα is a polypeptide comprising SEQ ID NO: 65 or SEQ ID NO: 66, more preferably IL-7Rα is a polypeptide consisting of SEQ ID NO: 65 or SEQ ID NO: 66. More preferably, IL-7Rα is a polypeptide comprising SEQ ID NO: 65, more preferably 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.

[0054] Polypeptides that are capable of reacting with IL-7Rα from humans and with IL-7Rα from another species, e.g., cynomolgus IL-7Rα ("cross-reactive"), are advantageous because they allow preclinical studies to be more easily performed in animal models.

[0055] Suitably, the polypeptides of the invention are directed against an epitope on IL-7Rα that is located in and / or forms part of the receptor binding site for IL-7 and / or L-TSLP, and upon binding to IL-7Rα, the polypeptides of the invention are capable of inhibiting or reducing IL-7 and / or L-TSLP signaling.

[0056] The polypeptides of the present invention bind to one or more epitopes on IL-7Rα. In one embodiment, the present invention provides polypeptides that bind to the same epitope on IL-7Rα as V7R-2E5, V7R-2E9, V7R-2F6, V7R-6C12, V7R-2B6, V7R-3B5, or V7R-4F6.

[0057] Preferably, the polypeptides of the invention are isolated. An "isolated" polypeptide is one that has been removed from its original environment. For example, a naturally occurring polypeptide of the invention is isolated if it is separated from some or all of the coexisting materials in the natural system.

[0058] Potency, Inhibition, and Neutralization Potency is a measure of a therapeutic agent's activity in terms of the amount required to produce an effect of a given magnitude. A highly potent agent will produce a larger response at a lower concentration, compared to a less potent agent, which will produce a smaller response at a lower concentration. Potency is a function of affinity and efficacy. Efficacy refers to the ability of a therapeutic agent to produce a biological response upon binding to a target ligand and the quantitative magnitude of this response. The term maximal effective concentration (EC50) refers to the concentration of a therapeutic agent that produces a response halfway between the baseline and maximal concentrations after a specified exposure time. A therapeutic agent may produce either inhibition or stimulation. This is commonly, and is used herein, as an indication of efficacy.

[0059] For purposes of the present invention, a neutralizing polypeptide is a polypeptide that binds to IL-7Rα and inhibits the binding of IL-7 and / or L-TSLP to IL-7R as measured by ELISA. A particular ELISA method suitable for determining the level of inhibition in this context is detailed in Example 3 below.

[0060] Suitably, the polypeptide of the present invention neutralises the binding of IL-7 to IL-7R with an EC50 of 2.00 nM or less, such as 1.50 nM or less, for example, 1.00 nM or less, for example, 0.90 nM or less, such as 0.80 nM or less, for example, 0.70 nM or less, for example, 0.65 nM or less, such as 0.60 nM or less, for example, 0.55 nM or less, such as 0.50 nM or less, for example, 0.45 nM or less, for example, 0.4 nM or less, such as 0.35 nM or less, for example, 0.30 nM or less.

[0061] Suitably, the EC50 is established using the IL-7 / IL-7R neutralisation ELISA detailed in Example 3 below.

[0062] Polypeptide and Polynucleotide Sequences For the purpose of comparing two closely related polypeptide sequences, the "% sequence identity" between a first polypeptide sequence and a second polypeptide sequence can be calculated using NCBI BLAST v2.0 with standard settings for polypeptide sequences (BLASTP). For the purpose of comparing two closely related polynucleotide sequences, the "% sequence identity" between a first nucleotide sequence and a second nucleotide sequence can be calculated with NCBI BLAST v2.0 with standard settings for nucleotide sequences (BLASTN). The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as default a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as default a word length of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) with an alignment (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0063] A polypeptide or polynucleotide sequence is said to be identical or similar to another polypeptide or polynucleotide sequence if they share 100% sequence identity over their entire length. Residues in a sequence are numbered from left to right, i.e., from N-terminus to C-terminus for polypeptides and from 5' to 3' for polynucleotides.

[0064] A "difference" between sequences refers to the insertion, deletion, or substitution of a single amino acid residue at a position in the second sequence compared to the first sequence. Two polypeptide sequences can contain one, two, or more such amino acid differences. Insertions, deletions, or substitutions in a second sequence that is otherwise identical to the first sequence (100% sequence identity) result in a decrease in the percentage of sequence identity. For example, if an identical sequence is 9 amino acid residues long, one substitution in the second sequence will result in 88.9% sequence identity. If an identical sequence is 17 amino acid residues long, two substitutions in the second sequence will result in 88.2% sequence identity. If an identical sequence is 7 amino acid residues long, three substitutions in the second sequence will result in 57.1% sequence identity. If the first and second polypeptide sequences are 9 amino acid residues in length and share 6 identical residues, the first and second polypeptide sequences share greater than 66% identity (the first and second polypeptide sequences share 66.7% identity). If the first and second polypeptide sequences are 17 amino acid residues in length and share 16 identical residues, the first and second polypeptide sequences share greater than 94% identity (the first and second polypeptide sequences share 94.1% identity). If the first and second polypeptide sequences are 7 amino acid residues in length and share 3 identical residues, the first and second polypeptide sequences share greater than 42% identity (the first and second polypeptide sequences share 42.9% identity).

[0065] Alternatively, for the purpose of comparing a first reference polypeptide sequence with a second comparison polypeptide sequence, the number of additions, substitutions, and / or deletions made to the first sequence to generate the second sequence can be determined. An addition is the addition of one amino acid residue to the sequence of the first polypeptide (including additions at either end of the first polypeptide). A substitution is the replacement of one amino acid residue in the sequence of the first polypeptide with a different amino acid residue. A deletion is the removal of one amino acid residue from the sequence of the first polypeptide (including deletions at either end of the first polypeptide).

[0066] For purposes of comparing a first reference polynucleotide sequence with a second comparison polynucleotide sequence, the number of additions, substitutions, and / or deletions made to the first sequence to generate the second sequence can also be determined. An addition is the addition of a single nucleotide residue to the sequence of the first polynucleotide (including additions at either end of the first polynucleotide). A substitution is the replacement of a single nucleotide residue in the sequence of the first polynucleotide with a different nucleotide residue. A deletion is the removal of a single nucleotide residue from the sequence of the first polynucleotide (including deletions at either end of the first polynucleotide).

[0067] "Conservative" amino acid substitutions are those in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and are expected to have little effect on the function, activity, or other biological properties of the polypeptide. Such conservative substitutions are preferably those in which one amino acid residue within the following group is replaced with another amino acid residue from the same group:

[0068] [Table 1]

[0069] Preferably, the hydrophobic amino acid residue is a non-polar amino acid. More preferably, the hydrophobic amino acid residue is selected from V, I, L, M, F, W, or C.

[0070] As used herein, the numbering of polypeptide sequences and the definitions of CDRs and FRs are as defined according to the Kabat system (Kabat et al., 1991). A "corresponding" amino acid residue between a first and a second polypeptide sequence is an amino acid residue in the first sequence that shares the same position as an amino acid residue in the second sequence according to the Kabat system, although the amino acid residue in the second sequence may have a different identity from the first sequence. If the framework and CDRs are the same length according to the Kabat definition, then corresponding residues will share the same number (and letter) as appropriate. Alignment can be accomplished manually or using known computer algorithms for sequence alignment, such as, for example, NCBI BLAST v2.0 (BLASTP or BLASTN) using standard settings.

[0071] The polypeptide sequence of ID-A62U, a polypeptide of the invention, is set forth below in Kabat format.

[0072] [Table 2]

[0073] The polypeptide sequence of a further polypeptide of the invention, ID-A59U, is set forth below in Kabat format.

[0074] [Table 3]

[0075] The polypeptide sequence of a further polypeptide of the invention, V7R-2E9, is set forth below in Kabat format.

[0076] [Table 4]

[0077] Residue numbering from the N-terminus to the C-terminus is given at the bottom. Kabat numbering includes the "H" prefix and is provided in the second row. CDR1, CDR2, and CDR3 are labeled as "CDR-H1," "CDR-H2," and "CDR-H3," respectively.

[0078] The polypeptide sequences of additional polypeptides of the present invention (discussed in Examples 2 and 3) are aligned below (note that V7R-6C12 is referred to below as IL-7R-6C12).

[0079] [Table 5]

[0080] Preferably, the polynucleotide used in the present invention is isolated. An "isolated" polynucleotide is one that has been removed from its original environment. For example, a naturally occurring polynucleotide is isolated when it is separated from some or all of the coexisting materials in the natural system. A polynucleotide is considered to be isolated, for example, when it is cloned into a vector that is not part of its natural environment or when it is not contained in a cDNA.

[0081] In one aspect of the present invention, there is provided a polynucleotide encoding a polypeptide or construct of the present invention. Preferably, the polynucleotide comprises or consists of a sequence sharing 70% or more, such as 80% or more, such as 90% or more, such as 95% or more, for example 99% or more, sequence identity with SEQ ID NO: 69 or 70, most preferably SEQ ID NO: 70. More preferably, the polynucleotide comprises or (most preferably) consists of either SEQ ID NO: 69 or 70, most preferably SEQ ID NO: 70. In a further aspect, there is provided a cDNA comprising said polynucleotide.

[0082] In one aspect of the invention there is provided a polynucleotide comprising or consisting of a sequence encoding CDR1, CDR2 or CDR3 of the encoded immunoglobulin chain variable domain, which sequence shares 70% or more, such as 80% or more, for example 90% or more, such as 95% or more, for example 99% or more sequence identity with any one of a portion of any of SEQ ID NOs: 69 or 70.

[0083] Preferably, the polypeptide sequences of the present invention comprise at least one modification relative to the native sequence. Preferably, the polynucleotide sequences of the present invention comprise at least one modification relative to the native sequence. Preferably, the modification to the polypeptide or polynucleotide sequence is made to increase the stability of the polypeptide or encoded polypeptide against proteases present in the intestinal tract (e.g., trypsin and chymotrypsin).

[0084] Potential features of the CDRs and frameworks of the polypeptides of the invention are described below.

[0085] CDR1 Preferably, CDR1 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence sharing 80% or more sequence identity with SEQ ID NO:1.

[0086] Alternatively, CDR1 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has two or less, more preferably one or less additions compared to SEQ ID NO: 1. Preferably, CDR1 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has two or less, more preferably one or less substitutions compared to SEQ ID NO: 1. Preferably, CDR1 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has two or less, more preferably one or less deletions compared to SEQ ID NO: 1.

[0087] Suitably, any residues in CDR1 that differ from their corresponding residues in SEQ ID NO: 1 are conservative substitutions compared with their corresponding residues.

[0088] Suitably, the residues of CDR1 have the following identity (SEQ ID NO: 82):

[0089] [Table 6]

[0090] Preferably, CDR1 comprises or consists of SEQ ID NO: 1 or SEQ ID NO: 71. More preferably, CDR1 comprises, or more preferably consists of SEQ ID NO: 1.

[0091] CDR2 Preferably, CDR2 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence sharing 65% or more, 75% or more, 80% or more, 85% or more, or 90% or more sequence identity with SEQ ID NO:2.

[0092] Alternatively, CDR2 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer additions compared to SEQ ID NO: 2. Preferably, CDR2 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer substitutions compared to SEQ ID NO: 2. Preferably, CDR2 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer deletions compared to SEQ ID NO: 2.

[0093] Suitably, any residues in CDR2 that differ from their corresponding residues in SEQ ID NO:2 are conservative substitutions compared with their corresponding residues.

[0094] Suitably, the residues of CDR2 have the following identity (SEQ ID NO: 83):

[0095] [Table 7]

[0096] Preferably, the residue in CDR2 corresponding to residue number 16 of SEQ ID NO:2 is Q or K, most preferably K. Preferably, 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 preferably, CDR2 comprises, or more preferably consists of, SEQ ID NO:2.

[0097] CDR3 Preferably, the CDR3 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence sharing 60% or more, 70% or more, or 80% or more sequence identity with SEQ ID NO:3.

[0098] Alternatively, the CDR3 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has no more than 3, more preferably no more than 2, more preferably no more than 1 addition compared to SEQ ID NO: 3. Preferably, the CDR3 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has no more than 3, more preferably no more than 2, more preferably no more than 1 substitution compared to SEQ ID NO: 3. Preferably, the CDR3 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has no more than 3, more preferably no more than 2, more preferably no more than 1 deletion compared to SEQ ID NO: 3. Preferably, any substitutions are conservative to their corresponding residues in SEQ ID NO: 3.

[0099] Suitably, any residues in CDR3 that differ from their corresponding residues in SEQ ID NO: 3 are conservative substitutions compared with their corresponding residues.

[0100] Suitably, the residues of CDR3 have the following identity (SEQ ID NO: 84):

[0101] [Table 8]

[0102] Preferably, the sequence of CDR3 comprises or consists of SEQ ID NO: 3, SEQ ID NO: 77, or SEQ ID NO: 78. More preferably, CDR3 comprises, or more preferably consists of, SEQ ID NO: 3.

[0103] Special CDR Some particularly suitable CDR sequences are shown in the table below. Preferably, the CDR1 of the polypeptide of the invention is one of the CDR1 sequences shown below. Preferably, the CDR2 of the polypeptide of the invention is one of the CDR2 sequences shown below. Preferably, the CDR3 of the polypeptide of the invention is one of the CDR3 sequences shown below. Preferably, the polypeptide of the invention comprises a combination of two, or more preferably three, of the CDR sequences shown below.

[0104] Particular CDRs of the polypeptides of the invention are provided below. "Example" indicates an exemplary CDR and its corresponding sequence identification number.

[0105] [Table 9]

[0106] FR1 Preferably, FR1 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence sharing 5%, 12%, 18%, 26%, 32%, 38%, 46%, 52%, 58%, 62%, 66%, 68%, 72%, 75%, 78%, 82%, 85%, 90%, 95% or more sequence identity with SEQ ID NO:4.

[0107] Alternatively, FR1 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having an addition of 28 or less, more preferably 26 or less, more preferably 24 or less, more preferably 22 or less, more preferably 20 or less, more preferably 18 or less, more preferably 16 or less, more preferably 14 or less, more preferably 13 or less, more preferably 12 or less, more preferably 11 or less, more preferably 10 or less, more preferably 9 or less, more preferably 8 or less, more preferably 7 or less, more preferably 6 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and more preferably 1 or less compared to SEQ ID NO: 4. Preferably, FR1 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 28 or less, more preferably 26 or less, more preferably 24 or less, more preferably 22 or less, more preferably 20 or less, more preferably 18 or less, more preferably 16 or less, more preferably 14 or less, more preferably 13 or less, more preferably 12 or less, more preferably 11 or less, more preferably 10 or less, more preferably 9 or less, more preferably 8 or less, more preferably 7 or less, more preferably 6 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and more preferably 1 or less substitutions compared to SEQ ID NO: 4. Preferably, FR1 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 28 or less, more preferably 26 or less, more preferably 24 or less, more preferably 22 or less, more preferably 20 or less, more preferably 18 or less, more preferably 16 or less, more preferably 14 or less, more preferably 13 or less, more preferably 12 or less, more preferably 11 or less, more preferably 10 or less, more preferably 9 or less, more preferably 8 or less, more preferably 7 or less, more preferably 6 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and more preferably 1 or less deletions compared to SEQ ID NO: 4.

[0108] Preferably, any residues in FR1 that differ from their corresponding residues in SEQ ID NO: 4 are conservative substitutions compared to their corresponding residues. Preferably, the residue in FR1 corresponding to residue number 1 in SEQ ID NO: 4 is D or E, most preferably D. Preferably, the residues in FR1 corresponding to residue numbers 1-5 in SEQ ID NO: 4 are DVQLV. Preferably, FR1 comprises, or more preferably consists of, SEQ ID NO: 4. Preferably, the residue in FR1 corresponding to residue number 24 in SEQ ID NO: 4 is S.

[0109] FR2 Preferably, FR2 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence sharing 10%, 15%, 25%, 30%, 40%, 45%, 55%, 60%, 70%, 75%, 85%, 90% or more sequence identity with SEQ ID NO:5.

[0110] Alternatively, FR2 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 13 or fewer, more preferably 12 or fewer, more preferably 11 or fewer, more preferably 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer additions compared to SEQ ID NO: 5. Preferably, FR2 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 13 or fewer, more preferably 12 or fewer, more preferably 11 or fewer, more preferably 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer substitutions compared to SEQ ID NO: 5. Preferably, FR2 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 13 or fewer, more preferably 12 or fewer, more preferably 11 or fewer, more preferably 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer deletions compared to SEQ ID NO: 5.

[0111] Preferably, any residues in FR2 that differ from their corresponding residues in SEQ ID NO:5 are conservative substitutions compared to their corresponding residues. Preferably, the residue in FR2 corresponding to residue 10 of SEQ ID NO:5 is R or L, most preferably L. Preferably, the residues in FR2 corresponding to residues 8-11 of SEQ ID NO:5 are KEXE, where X is R or L, most preferably L. Alternatively, the residues in FR2 corresponding to residues 9-12 of SEQ ID NO:5 are GLEW. Preferably, FR2 comprises, or more preferably consists of, SEQ ID NO:5. Preferably, the residue in FR2 corresponding to residue 2 of SEQ ID NO:5 is F, and more preferably, the residue in FR2 corresponding to residue 14 of SEQ ID NO:5 is A. Preferably, the residues in FR2 corresponding to residues 9-14 of SEQ ID NO:5 are ELEFLA (SEQ ID NO:79). Preferably, the residues in FR2 corresponding to residues 9-14 of SEQ ID NO:5 are not GLEWVS (SEQ ID NO:80). Preferably, the residue in FR2 corresponding to residue number 9 of SEQ ID NO:5 is not G. More preferably, the residue in FR2 corresponding to residue number 9 of SEQ ID NO:5 is E.

[0112] FR3 Preferably, FR3 of a polypeptide of the present invention comprises, or more preferably consists of, a sequence sharing 8%, 15%, 20%, 26%, 32%, 40%, 45%, 52%, 58%, 65%, 70%, 76%, 80%, 82%, 85%, 90%, 92%, 95% or more sequence identity with SEQ ID NO:6.

[0113] Alternatively, FR3 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having an addition of 29 or less, more preferably 27 or less, more preferably 25 or less, more preferably 23 or less, more preferably 21 or less, more preferably 19 or less, more preferably 17 or less, more preferably 15 or less, more preferably 13 or less, more preferably 11 or less, more preferably 9 or less, more preferably 7 or less, more preferably 6 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and more preferably 1 or less compared to SEQ ID NO: 6. Preferably, FR3 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 29 or less, more preferably 27 or less, more preferably 25 or less, more preferably 23 or less, more preferably 21 or less, more preferably 19 or less, more preferably 17 or less, more preferably 15 or less, more preferably 13 or less, more preferably 11 or less, more preferably 9 or less, more preferably 7 or less, more preferably 6 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and more preferably 1 or less substitutions compared to SEQ ID NO: 6. Preferably, FR3 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 29 or less, more preferably 27 or less, more preferably 25 or less, more preferably 23 or less, more preferably 21 or less, more preferably 19 or less, more preferably 17 or less, more preferably 15 or less, more preferably 13 or less, more preferably 11 or less, more preferably 9 or less, more preferably 7 or less, more preferably 6 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and more preferably 1 or less deletions compared to SEQ ID NO: 6.

[0114] Preferably, any residues in FR3 that differ from their corresponding residues in SEQ ID NO: 6 are conservative substitutions compared to their corresponding residues. Preferably, FR3 comprises, or more preferably consists of, SEQ ID NO: 6. Preferably, residues in FR3 corresponding to residues 18, 19, and 20 of SEQ ID NO: 6 are NSL. Preferably, the residue in FR3 corresponding to residue 21 of SEQ ID NO: 6 is R. Preferably, the residue in FR3 corresponding to residue 22 of SEQ ID NO: 6 is A.

[0115] FR4 Preferably, FR4 of a polypeptide of the present invention comprises, or more preferably consists of, a sequence sharing 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more sequence identity with SEQ ID NO:7.

[0116] Alternatively, FR4 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer additions compared to SEQ ID NO: 7. Preferably, FR4 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer substitutions compared to SEQ ID NO: 7. Preferably, FR4 of the polypeptide of the present invention comprises, or more preferably consists of, a sequence having 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer deletions compared to SEQ ID NO: 7.

[0117] Preferably, any residues in FR4 that differ from their corresponding residues in SEQ ID NO: 7 are conservative substitutions compared to their corresponding residues. Preferably, FR3 comprises, or more preferably consists of, SEQ ID NO: 6.

[0118] Total Polypeptide Preferably, a polypeptide of the invention comprises, or more preferably 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.

[0119] Alternatively, the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has 20 or fewer, more preferably 15 or fewer, more preferably 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer additions compared to SEQ ID NO: 8. Preferably, the polypeptide of the present invention comprises, or more preferably consists of, a sequence which has 20 or fewer, more preferably 15 or fewer, more preferably 10 or fewer, more preferably 9 or fewer, more preferably 8 or fewer, more preferably 7 or fewer, more preferably 6 or fewer, more preferably 5 or fewer, more preferably 4 or fewer, more preferably 3 or fewer, more preferably 2 or fewer, and more preferably 1 or fewer substitutions compared to SEQ ID NO: 8. Preferably, the polypeptide of the present invention comprises, or more preferably consists of, a sequence having no more than 20 deletions compared to SEQ ID NO: 8, more preferably no more than 15 deletions, more preferably no more than 10 deletions, more preferably no more than 9 deletions, more preferably no more than 8 deletions, more preferably no more than 7 deletions, more preferably no more than 6 deletions, more preferably no more than 5 deletions, more preferably no more than 4 deletions, more preferably no more than 3 deletions, more preferably no more than 2 deletions, more preferably no more than 1 deletion compared to SEQ ID NO: 8.

[0120] Preferably, the N-terminus of the polypeptide is D. Preferably, the polypeptide comprises, or more preferably consists of, SEQ ID NO:8.

[0121] Framework embodiment In one embodiment of the present invention, a polypeptide is provided comprising four framework regions (FR1 to FR4), each of which 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). Preferably, each variant framework region shares at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, or even more preferably at least 90% identity with its corresponding framework region of ID-A62U. More preferably, the variant framework region comprises, or more preferably consists of, the corresponding framework region of ID-A62U. Preferably, the polypeptide comprising the four framework regions is an antibody or antibody fragment. Preferably, the antibody fragment is a VHH, VH, VL, V-NAR, scFv, FAb fragment, or F(ab')2 fragment. More preferably, the antibody fragment is a VHH or VH, and most preferably a VHH.

[0122] epitope Example 5 details the epitope modeling work performed on V7R-2E9, a polypeptide of the invention. This work shows that V7R-2E9 binds to the following residues of IL-7Rα. Residues that fill regions of particular importance to the interface are highlighted in bold. Residue numbering corresponds to SEQ ID NO: 65.

[0123] [Table 10]

[0124] Thus, one embodiment of the present invention provides a polypeptide that binds to an epitope on IL-7Rα comprising at least one residue of IL-7Rα (SEQ ID NO: 65) selected from the list consisting of Glu27, Ser31, Leu57, Val58, Glu59, Lys77, Lys78, Phe79, Leu80, Leu81, Ile82, Thr104, Lys137, Lys138, Tyr139, Lys141, His191, Tyr192, and Phe193. Preferably, the polypeptide binds to an epitope on IL-7Rα comprising at least 8, more preferably at least 15, and even more preferably at least all of the residues of IL-7Rα selected from this list.

[0125] It may be noted that certain residues of IL-7Rα fill a region that is particularly important in the interface with V7R-2E9. Accordingly, a further aspect of the present invention provides a polypeptide that binds to an epitope on IL-7Rα (SEQ ID NO: 65) that contains at least one residue of IL-7Rα selected from the list consisting of Ser31, Val58, Phe79, Leu80, Leu81, Ile82, Lys138, Tyr139, and Phe193. Preferably, the polypeptide binds to an epitope on IL-7Rα that contains at least Val58, Leu80, and Lys138 of IL-7Rα. More preferably, it binds to Val58, Leu80, Lys138, Ile82, and Tyr139 of IL-7Rα. More preferably, the epitopes are Val58, Leu80, Lys138, Ile82, Tyr139, Leu81, and Phe79 of IL-7Rα. Preferably, the polypeptide binds to an epitope on IL-7Rα that includes at least five, more preferably at least seven, and more preferably all residues of IL-7Rα selected from Ser31, Val58, Phe79, Leu80, Leu81, Ile82, Lys138, Tyr139, and Phe193.

[0126] Suitably, "binds to an epitope" in this context can be defined as, upon polypeptide binding, the relevant residues of IL-7Rα that constitute the epitope have at least the BSA set forth in the table above for the relevant residues.

[0127] Linkers and multimers Constructs of the present invention may comprise multiple polypeptides and may be preferably multivalent. Such constructs may comprise at least two identical polypeptides of the present invention. A construct consisting of two identical polypeptides of the present invention is a "homobihead." In one aspect of the present invention, constructs are provided that comprise two or more identical polypeptides of the present invention.

[0128] Alternatively, the construct may comprise at least two polypeptides that are different but are both polypeptides according to the invention (a "heterobihead").

[0129] Alternatively, such a construct may comprise (a) at least one polypeptide of the present invention and (b) at least one polypeptide, such as an antibody or antigen-binding fragment thereof, that is not a polypeptide of the present invention (also a "heterobihead"). The at least one polypeptide in (b) may bind to IL-7R (e.g., via a different epitope than that of (a)), or alternatively may bind to a target other than IL-7R. Preferably, the different polypeptide (b) is, for example, an interleukin (such as IL-1, IL-1ra, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, and -18), an interleukin receptor (such as IL-6R), a transcription factor (such as NF-kB), a cytokine (such as TNF-α, IFN-γ, TGF-β), a transmembrane protein (such as gp130 and CD3), a surface glycoprotein (such as CD4, CD20, CD50), a phospholipid (such as phospholipid ... 40), 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 or immunologically relevant ligands involved in human pathological processes. Thus, different polypeptides (b) may bind, for example, to IL-6R, IL-6, IL-12, IL-1-β, IL-17A, TNF-α, or CD3, or other inflammatory mediators or immunologically relevant ligands involved in human pathological processes.

[0130] The construct may be multivalent and / or multispecific. A multivalent construct (such as a bivalent construct) comprises two or more binding polypeptides and therefore presents two or more sites at which binding to one or more antigens can occur. Examples of multivalent constructs may be homobiheads or heterobiheads. A multispecific construct (e.g., a bispecific construct) comprises two or more different binding polypeptides that present two or more sites at which (a) binding to two or more different antigens can occur, or (b) binding to two or more different epitopes on the same antigen can occur. A multispecific construct may be a heterobihead. A multispecific construct is multivalent.

[0131] Preferably, the polypeptide contained within the construct is an antibody fragment. More preferably, the polypeptide contained within the construct is selected from the list consisting of VHH, VH, VL, V-NAR, scFv, FAb fragment, or F(ab')2 fragment. More preferably, the polypeptide contained within the construct is VH or VHH, and most preferably, VHH.

[0132] Preferably, the polypeptide comprised within the construct is selected from the list consisting of an ICVD (e.g., VHH, VH, VL), a V-NAR, an scFv, a FAb fragment, or a F(ab')2 fragment. More preferably, the polypeptide comprised within the construct is an ICVD, more preferably, the polypeptide comprised within the construct is a VH or VHH, and most preferably, a VHH.

[0133] The polypeptides of the present invention can be linked to each other directly (i.e., without the use of a linker) or via a linker. Preferably, the linker is a protease-labile linker or a non-protease-labile linker. The linker is preferably a polypeptide and is selected to allow binding of the polypeptide to its epitope. When used for therapeutic purposes, the linker is preferably non-immunogenic in the subject to which the polypeptide is administered. Preferably, all polypeptides are connected by non-protease-labile linkers. Preferably, the protease-labile linker is [-(Ga S) x -BJB'-(G a S) y -] z wherein J is lysine or arginine, B is 0-5 amino acid residues selected from R, H, N, Q, S, T, Y, G, A, V, L, W, P, M, C, F, K, or I, B' is 0-5 amino acid residues selected from R, H, N, Q, S, T, Y, G, A, V, L, W, M, C, F, K, or I, a is 1-10, x is 1-10, y is 1-10, and z is 1-10 (SEQ ID NO: 85). Preferably, a is 4. Most preferably, a is 4, J is lysine, B is 0, x is 1, y is 1, and z is 1. Alternatively, the protease labile linker is -(GS) x -K-(G4S) y -, where x and y are each independently 1 to 5 (SEQ ID NO: 86), more preferably -(G4S)2-K-(G4S)2- (SEQ ID NO: 87).

[0134] Preferably, the non-protease labile linker is (G4S) x (SEQ ID NO: 88). More preferably, x is 1 to 10, more preferably, x is 4 to 8, and more preferably, x is 4, 6, or 8. Most preferably, x is 6 (SEQ ID NO: 89).

[0135] Vectors and hosts The term "vector," as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian and yeast vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of a host cell upon introduction into the host cell, thereby being replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "recombinant vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably, as the plasmid is the most commonly used form of vector. However, the invention is intended to include other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), as well as bacteriophage and phagemid systems, which serve equivalent functions. The present invention further relates to nucleotide sequences encoding the polypeptide sequences or multivalent and / or multispecific constructs. As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell into which a recombinant expression vector has been introduced. Such terms are intended to refer not only to the particular subject cell but also to the progeny of such a cell.

[0136] In one aspect of the present invention, a vector containing a polynucleotide encoding a polypeptide or construct of the present invention, or a cDNA containing the polynucleotide, is provided. In a further aspect of the present invention, a host cell transformed with the vector and capable of expressing the polypeptide or construct of the present invention is provided. Preferably, the host cell is a bacterium such as Escherichia coli, or a yeast belonging to the genera Aspergillus, Saccharomyces, Kluyveromyces, Hansenula, or Pichia, such as Saccharomyces cerevisiae or Pichia pastoris.

[0137] Autoimmune and / or inflammatory diseases Autoimmune diseases occur when the immune system reacts adversely to normal body tissues. Autoimmune diseases can result in tissue damage, abnormal organ growth, and / or altered organ function. Damage can affect only one organ or tissue type, or multiple organs and tissues. Organs and tissues commonly affected by autoimmune diseases include blood components, such as red blood cells, blood vessels, connective tissue, endocrine glands, such as the thyroid or pancreas, muscles, joints, and skin. Inflammatory diseases are diseases characterized by inflammation. Many inflammatory diseases are autoimmune diseases, and vice versa.

[0138] The polypeptide, pharmaceutical composition or construct of the present invention is suitable for use as a medicament, more particularly for use in the treatment of autoimmune and / or inflammatory diseases.

[0139] The polypeptides of the invention (preferably when delivered orally) are ideal for treating inflammatory diseases in which IL-7 and / or L-TSLP contribute to at least some proportion of the pathology, and the polypeptides have access to tissues in which IL-7 and / or L-TSLP are biologically active.

[0140] The polypeptide of the present invention binds to the receptor (IL-7R), and therefore may also interfere with as yet undiscovered cytokines or other binding partners of IL-7R that may be involved in disease.

[0141] Inhibition of IL-7 and L-TSLP-bound IL-7R Evidence that various TSLP isoforms are differentially expressed and act through different signaling pathways suggests that disease-related activities of L-TSLP may be selectively targeted rather than the physiologically beneficial effects of the shorter isoforms of this cytokine. The polypeptides of the present invention inhibit IL-7 binding to IL-7R. In silico modeling of the interaction of the V7R-2E9 molecule with IL-7Rα and information from the recently published structure of the TSLP:TSLPR:IL-7R complex (Verstraete et al., 2017) strongly suggest that V7R-2E9 and other polypeptides of the present 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) was confirmed. Because V7R-2E9 binds to IL-7Rα but not TSLP, it is expected that V7R-2E9 and related ICVDs will block only the pro-inflammatory activity of L-TSLP and that the important mucosal homeostatic functions of S-TSLP will be unaffected.

[0142] Inflammatory bowel disease (IBD) Chronic inflammatory bowel disease, Crohn's disease, and ulcerative colitis, which afflict both children and adults, are examples of autoimmune and inflammatory disorders of the gastrointestinal tract (Hendrickson et al., 2002). Ulcerative colitis is defined as a condition in which the inflammatory response and morphologic changes remain localized to the colon. The rectum is involved in 95% of patients. The inflammation is largely limited to the mucosa and consists of a contiguous lesion of variable severity, with ulcers, edema, and bleeding along the length of the colon (Hendrickson et al., 2002). Ulcerative colitis is usually manifested by the presence of blood and mucus in the stool, along with the most severe lower abdominal cramps during bowel passage. Clinically, the presence of diarrhea with blood and mucus distinguishes ulcerative colitis from irritable bowel syndrome, in which blood is not present. Unlike ulcerative colitis, the symptoms of Crohn's disease are usually subtle, leading to delayed diagnosis. Factors such as the location, extent, and severity of the involvement determine the spectrum of gastrointestinal symptoms. Patients with ileocolonic involvement usually have postprandial abdominal pain, accompanied by right lower quadrant tenderness and occasionally an inflammatory mass. Symptoms associated with gastroduodenal Crohn's disease include early satiety, nausea, vomiting, epigastric pain, or dysphagia. Perianal disease, with anal tags, deep anal fissures, and fistulas, is common (Hendrickson et al. 2002).

[0143] Suitably, the polypeptides, pharmaceutical compositions or constructs of the invention are used in the treatment of autoimmune and / or inflammatory diseases of the GI (gastrointestinal) tract in which IL-7 and / or L-TSLP contribute to the pathology of the disease.

[0144] Preferably, the polypeptide, pharmaceutical composition or construct of the present invention is used in the treatment of an autoimmune and / or inflammatory disease of the GI tract selected from the list consisting of Crohn's disease, ulcerative colitis, irritable bowel disease, type II diabetes, glomerulonephritis, autoimmune hepatitis, Sjogren's syndrome, celiac disease, and drug-induced or radiation-induced mucositis (more preferably Crohn's disease or ulcerative colitis, most preferably ulcerative colitis).

[0145] Eosinophilic esophagitis (EoE) Eosinophilic esophagitis (EoE, also spelled eosinophilic esophagitis and known as allergic esophagitis) is an allergic inflammatory disease of the esophagus involving eosinophils, a type of white blood cell. Symptoms include dysphagia, food impaction, vomiting, and heartburn. Preferably, the polypeptide, pharmaceutical composition, or construct of the present invention is used to treat eosinophilic esophagitis. More preferably, the polypeptide, pharmaceutical composition, or construct used to treat eosinophilic esophagitis is administered orally.

[0146] Other autoimmune / inflammatory diseases For example, other diseases of the GIT that can be treated by oral administration of the polypeptides of the present invention include, for example, the inflammatory disease mucositis (preferably drug-induced 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, keloids, chronic rhinosinusitis (CRS), nasal polyps, chronic eosinophilic pneumonia, eosinophilic bronchitis, celiac disease, and Churg-Strauss syndrome.

[0147] In mucositis, lesions can occur anywhere from the mouth to the anus. For lesions of the mouth and esophagus, mouthwash or cream preparations containing variable domains can be used. For lesions of the anus and rectum, suppositories, creams, or foams containing variable domains are suitable for topical application. Immunoglobulin chain variable domains are removed from the lamina propria or other inflammatory sites via absorption into the bloodstream at the site of inflammation or via lymphatic clearance and subsequent entry into the bloodstream. Thus, the domains reach the liver via the bloodstream and are removed by glomerular filtration in the kidney. Therefore, there is ample rationale for the therapeutic role of domains in diseases such as autoimmune hepatitis, type II diabetes, and glomerulonephritis.

[0148] In one embodiment, the polypeptide or construct of the invention is used in the treatment or prevention of atopic dermatitis, preferably in the form of a cream, nanoparticles, ointment or hydrogel, preferably by topical delivery to and / or through the skin.

[0149] Preferably, the polypeptide, pharmaceutical composition, or construct is used in the treatment of other autoimmune / inflammatory diseases in which IL-7 and / or L-TSLP are responsible for a proportion of the observed pathology.

[0150] Therapeutic Uses and Delivery A therapeutically effective amount of a polypeptide, pharmaceutical composition, or construct of the present invention is an amount effective to inhibit IL-7 and / or L-TSLP from binding to IL-7R to a significant extent in a subject upon single or multiple administration to the subject. A therapeutically effective amount may vary depending on factors such as the disease state, the 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 detrimental effects of the polypeptide, pharmaceutical composition, or construct of the present invention. The polypeptide or construct of the present invention may be incorporated into a pharmaceutical composition suitable for administration to a subject. The polypeptide or construct of the present invention may be in the form of a pharmaceutically acceptable salt.

[0151] The pharmaceutical compositions of the present invention may be formulated for oral, intramuscular, subcutaneous, or intravenous delivery. The pharmaceutical compositions of the present invention may be in various forms. These forms include liquid, semi-solid, 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 polypeptides, pharmaceutical compositions, or constructs of the present invention may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. For the treatment of eosinophilic esophagitis, delivery in the form of a lozenge is particularly preferred. For the treatment of atopic dermatitis, delivery in the form of a cream is particularly preferred.

[0152] Typically, pharmaceutical compositions comprise a polypeptide or construct of the present invention and a pharmaceutically acceptable diluent or carrier. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, etc., as well as combinations thereof. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the polypeptide or construct of the present invention. Pharmaceutical compositions may also comprise antiadherents, binders, coatings, disintegrants, flavors, colorants, lubricants, adsorbents, preservatives, sweeteners, freeze dry excipients (including lyoprotectants), or compression aids.

[0153] In patients with EoE and UC, the epithelial barrier of the inflamed intestinal mucosa is thought to be compromised, thereby facilitating penetration of orally administered polypeptides of the present invention into the underlying mucosal tissue, potentially resulting in the inhibition of 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 and reduce the risk of immunosuppression typically associated with conventional IL-7 and TSLP antibodies administered by injection. Short-term anti-IL-7R antibody treatment, such as that performed in animal models, can result in long-term clinical responses (remission) due to the depletion of pathogenic T cells. However, repeated systemic administration of existing IL-7Rα-blocking antibodies to patients is likely to result in significant systemic immunosuppression due to the inhibition of thymic T cell development and the depletion of peripheral T cells. Inflammatory bowel diseases (Crohn's disease and ulcerative colitis) and EoE are largely confined to the gastrointestinal tract, and consequently, oral administration of the polypeptides of the invention to the inflamed intestinal mucosa offers the potential to achieve a local effect with limited systemic exposure, thereby reducing the risk of immunosuppression in tissues not affected by the disease.

[0154] Thus, the polypeptides, pharmaceutical compositions, or constructs of the present invention are preferably administered orally. They may be delivered orally to the buccal cavity, pharynx, and esophagus (more preferably the esophagus) (such as for the treatment of EoE), or to the duodenum, jejunum, ileum, cecum, colon, rectum, and / or anal canal (such as for the treatment of IBD).

[0155] A key issue with oral delivery is ensuring that sufficient polypeptide, pharmaceutical composition, or construct reaches the region of the gastrointestinal tract where it is needed. Factors that prevent the polypeptide, pharmaceutical composition, or construct of the present invention from reaching the region of the gastrointestinal tract where it is needed include the presence of proteases in digestive secretions that can degrade the polypeptide, pharmaceutical composition, or construct of the present invention. Preferably, the polypeptide, pharmaceutical composition, or construct of the present invention is substantially stable in the presence of one or more such proteases due to the inherent properties of the polypeptide or its construct. Preferably, the polypeptide or construct of the present invention is lyophilized before being incorporated into a pharmaceutical composition.

[0156] The polypeptides of the present invention can also be provided with an enteric coating. An enteric coating is a polymeric barrier applied to oral dosage forms that helps protect the polypeptide from the low pH of the stomach. Materials used in enteric coatings include fatty acids, waxes, shellac, plastics, and vegetable fibers. Suitable enteric coating components include methyl acrylate-methacrylic acid copolymer, cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymer, sodium alginate, and stearic acid. Suitable enteric coatings include pH-dependent release polymers. These are polymers that are insoluble at the strongly acidic pH found in the stomach but rapidly dissolve at weakly acidic pH. Thus, preferably, the enteric coating does not dissolve in the acidic secretions of the stomach (pH ∼3) but dissolves in the higher pH environments present in the small intestine (pH above 6) or colon (pH above 7.0). The pH-dependent release polymer is selected so that the polypeptide or construct of the present invention is released at approximately the time that the administered dose reaches the small intestine.

[0157] When administered orally for the treatment of IBD, the polypeptides of the invention are preferably provided with an enteric coating. When administered orally for the treatment of EoE, the polypeptides of the invention are preferably provided in the form of compressed lozenges.

[0158] The polypeptides, constructs, or pharmaceutical compositions of the present invention are delivered topically. Such pharmaceutical compositions may be suitably in the form of creams, ointments, lotions, gels, foams, transdermal patches, powders, pastes, or tinctures, and may suitably contain vitamin D3 analogs (e.g., calcipotriol and maxacalcitol), steroids (e.g., fluticasone propionate, betamethasone valerate, and clobetasol propionate), retinoids (e.g., tazarotene), coal tar, and dithranol. Topical agents are often used in combination with each other (e.g., vitamin D3 and steroids) or with additional agents such as salicylic acid.

[0159] The polypeptides, constructs, or pharmaceutical compositions of the present invention can be formulated into preparations for injection by dissolving, suspending, or emulsifying in aqueous or non-aqueous solvents, such as vegetable oils or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids, or propylene glycol, and, if desired, using conventional additives such as solubilizers, isotonicity agents, suspending agents, emulsifiers, stabilizers, and preservatives. Acceptable carriers, excipients, and / or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers, e.g., phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid, glutathione, cysteine, methionine, and citric acid; preservatives (ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methylparaben or propylparaben, benzalkonium chloride, or combinations thereof); arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tripeptide, riboflavin ... low molecular weight (less than about 10 residues) polypeptides; proteins such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or non-ionic surfactants such as polysorbates, POE ethers, poloxamers, Triton-X, or polyethylene glycol.

[0160] For all delivery modes, the polypeptides, pharmaceutical compositions, or constructs of the present invention may be formulated in a buffer to stabilize the pH of the composition at a concentration between 5 and 50, or more preferably between 15 and 40, or even more preferably between 25 and 30 g / liter. Examples of suitable buffer components include physiological salts such as sodium citrate and / or citric acid. Preferably, the buffer contains 100 to 200, more preferably 125 to 175 mM of physiological salt, e.g., sodium chloride. Preferably, the buffer is selected to have a pKa close to the pH of the composition or the physiological pH of the patient.

[0161] Exemplary polypeptide or construct concentrations in pharmaceutical compositions can range from about 1 mg / mL to about 200 mg / mL, or from about 50 mg / mL to about 200 mg / ml, or from about 150 mg / mL to about 200 mg / ml.

[0162] Aqueous formulations of the polypeptides, constructs, or pharmaceutical compositions of the present invention can be prepared in pH buffer solutions, for example, at a pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively at about 5.5. Examples of suitable buffers include phosphate buffer, histidine buffer, citrate buffer, succinate buffer, acetate buffer, and other organic acid buffers. The buffer concentration can be, for example, from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending on the buffer and the desired tonicity of the formulation.

[0163] The tonicity of a pharmaceutical composition can be altered by including a tonicity modifier. Such tonicity modifiers can be charged or uncharged chemical species. Representative uncharged tonicity modifiers include sugars or sugar alcohols or other polyols, preferably trehalose, sucrose, mannitol, glycerol, 1,2-propanediol, raffinose, sorbitol, or lactitol (particularly trehalose, mannitol, glycerol, or 1,2-propanediol). Representative charged tonicity modifiers include salts such as combinations of sodium, potassium, or calcium ions with chloride, sulfate, carbonate, sulfite, nitrate, lactate, succinate, acetate, or maleate ions (particularly sodium chloride or sodium sulfate); or amino acids such as arginine or histidine. Preferably, aqueous formulations are isotonic, although hypertonic or hypotonic solutions may be appropriate. The term "isotonic" refers to a solution that has the same tonicity as some other solution to which it is being compared, such as physiological saline or serum. The tonicity adjusting agent may be used in an amount of about 5 mM to about 350 mM, for example, in an amount of 1 mM to 500 nM. Preferably, at least one tonicity adjusting agent is included in the composition.

[0164] Surfactants can also be added to pharmaceutical compositions to reduce aggregation of the formulated polypeptide or construct, minimize the formation of particulates in the formulation, and / or reduce adsorption. Exemplary surfactants include polyoxyethylene sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (poloxamers, Pluronic®), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylene sorbitan fatty acid esters include polysorbate 20 and polysorbate 80. Exemplary concentrations of surfactants can range from about 0.001% w / v to about 10% w / v.

[0165] Lyoprotectants can also be added to protect the polypeptides or constructs of the present invention from destabilizing conditions during the lyophilization process. For example, known lyoprotectants include sugars (including glucose, sucrose, mannose, and trehalose); polyols (including mannitol, sorbitol, and glycerol); and amino acids (including alanine, glycine, and glutamic acid). Lyoprotectants can be included in amounts of about 10 mM to 500 mM.

[0166] Dosage ranges for administration of the polypeptides, pharmaceutical compositions, or constructs of the invention are those to produce the desired therapeutic effect. The required dosage range depends on the exact nature of the polypeptides, pharmaceutical compositions, or constructs of the invention, the route of administration, the nature of the formulation, the patient's age, the nature, extent, or severity of the patient's disease, contraindications (if any), and the judgment of the attending physician. Variations in these dosage levels can be adjusted using standard empirical methods for optimization.

[0167] Suitable daily dosages of the polypeptides, pharmaceutical compositions, or constructs of the invention range from 50 ng to 50 mg per kg, for example, 50 ug to 40 mg per kg of body weight, for example, 5 to 30 mg per kg. Unit dosages can vary from less than 100 mg, but are typically in the range of 250 to 2000 mg per administration, and unit dosages may be administered daily or more frequently, for example, 2, 3, or 4 times per day, or less frequently, for example, every other day, or once a week, once every two weeks, or once a month.

[0168] In one aspect of the invention there is provided the use of a polypeptide, pharmaceutical composition or construct of the invention in the manufacture of a medicament for the treatment of an autoimmune disease. In a further aspect of the invention there is provided a method of treating an autoimmune disease comprising administering to a person in need thereof a therapeutically effective amount of a polypeptide, pharmaceutical composition or construct of the invention.

[0169] In one aspect of the invention there is provided the use of a polypeptide, pharmaceutical composition or construct of the invention in the manufacture of a medicament for the treatment of an autoimmune and / or inflammatory disease. In a further aspect of the invention there is provided a method of treating an autoimmune and / or inflammatory disease comprising administering to a person in need thereof a therapeutically effective amount of a polypeptide, pharmaceutical composition or construct of the invention.

[0170] The term "treatment" is intended to encompass prophylaxis as well as therapeutic treatment. Treatment of a disease includes treatment of its exacerbation, and also includes treatment of patients in remission from disease symptoms to prevent recurrence of disease symptoms.

[0171] Combination therapy The pharmaceutical compositions of the invention may also contain one or more active agents (e.g., active agents suitable for treating a disease described herein). It is within the scope of the invention to use the pharmaceutical compositions of the invention in therapeutic methods for the treatment of autoimmune diseases as an adjunct to, or in combination with, other established therapies commonly used in the treatment of autoimmune diseases.

[0172] For the treatment of IBD (such as Crohn's disease or ulcerative colitis), potential combinations include, for example, 5-aminosalicylic acid or its prodrugs (such as sulfasalazine, olsalazine, or bisalazide); corticosteroids (such as prednisolone, methylprednisolone, or budesonide); immunosuppressants (such as cyclosporine, tacrolimus, methotrexate, azathioprine, or 6-mercaptopurine); anti-TNF-α antibodies (such as infliximab, adalimumab, certolizumab pegol, or golimumab); anti-IL12 / IL23 antibodies (such as ustekinumab); anti-IL6R antibodies or small molecule IL12 / IL23 inhibitors (such as apilimod); anti-alpha-4-beta-7 antibodies. (e.g., vedolizumab); MAdCAM-1 blockers (e.g., PF-00547659); antibodies against the cell adhesion molecule alpha-4-integrin (e.g., natalizumab); antibodies against the IL2 receptor alpha subunit (e.g., daclizumab or basiliximab); JAK3 inhibitors (e.g., tofacitinib or R348); Syk inhibitors and their prodrugs (e.g., fostamatinib and R-406); phosphodiesterase-4 inhibitors (e.g., tetomilast); HMPL-004; probiotics; delsalazine; semapimod / CPSI-2364; and protein kinase C inhibitors (e.g., AEB-071). The most suitable combination agents are infliximab, adalimumab, certolizumab pego, or golimumab.

[0173] Accordingly, another aspect of the present invention provides a pharmaceutical composition of the present invention in combination with one or more additional active agents, such as one or more of the active agents described above.

[0174] In a further embodiment of the invention, the polypeptide, pharmaceutical composition or construct is administered sequentially, simultaneously or separately with at least one active agent selected from the list above.

[0175] Similarly, another aspect of the present invention is (A) a polypeptide, pharmaceutical composition, or construct of the present invention, and (B) one or more other active agents wherein each of components (A) and (B) is formulated in admixture with a pharmaceutically acceptable adjuvant, diluent, or carrier. In this aspect of the invention, the combination product can be either a single (combined) formulation or a kit-of-parts. Thus, this aspect of the invention encompasses combination formulations comprising a polypeptide, pharmaceutical composition, or construct of the invention and another therapeutic agent, admixed with a pharmaceutically acceptable adjuvant, diluent, or carrier.

[0176] The present invention provides (i) a polypeptide, pharmaceutical composition, or construct of the present invention, mixed with a pharmaceutically acceptable adjuvant, diluent, or carrier; and (ii) A formulation containing one or more other active agents mixed with a pharmaceutically acceptable adjuvant, diluent, or carrier. The present invention also encompasses a kit-of-parts comprising the components (i) and (ii), each of which is provided in a form suitable for administration in combination with the other.

[0177] Thus, component (i) of the kit-of-parts is component (A) described above mixed with a pharmaceutically acceptable adjuvant, diluent, or carrier. Similarly, component (ii) is component (B) described above mixed with a pharmaceutically acceptable adjuvant, diluent, or carrier. The one or more other active agents (i.e., component (B) described above) can be any of the agents described above for treating an autoimmune disease, such as IBD (e.g., Crohn's disease and / or ulcerative colitis). When component (B) is more than one active agent, these additional active agents can be formulated with each other, with component (A), or separately. In one embodiment, component (B) is one other therapeutic agent. In another embodiment, component (B) is two other therapeutic agents. The combination product of this aspect of the invention (either the combined formulation or the kit-of-parts) can be used in the treatment or prevention of an autoimmune disease (e.g., an autoimmune disease described herein).

[0178] stability In one embodiment, the polypeptides or constructs of the invention are delivered orally. Thus, the polypeptides or constructs of the invention suitably substantially retain their neutralizing ability and / or potency when delivered orally.

[0179] Preferably, the polypeptides or constructs of the present invention substantially retain their neutralizing ability and / or efficacy when orally administered and after exposure to the intestinal tract (e.g., after exposure to small intestinal and / or large intestinal proteases and / or IBD inflammatory proteases). Such proteases include enteropeptidase, trypsin, chymotrypsin, and irritable bowel disease inflammatory proteases (e.g., MMP3, MMP12, and cathepsins). The small intestinal and / or large intestinal proteases or proteases produced therein include proteases supplied by intestinal commensal flora and / or pathogenic bacteria, e.g., cell membrane-associated proteases, excretory proteases, and proteases released during cell lysis. Most preferably, the proteases are trypsin and chymotrypsin.

[0180] Preferably, the intestinal tract is that of a dog, pig, human, cynomolgus monkey, or mouse. More preferably, the intestinal tract is that of a human, cynomolgus monkey, or mouse, and most preferably that of a human. The small intestine preferably consists of the duodenum, jejunum, and ileum. The large intestine preferably consists of the cecum, colon, rectum, and anal canal. The gastrointestinal tract, in contrast to the gastrointestinal tract, consists only of the small intestine and large intestine. In one embodiment, the polypeptide or construct of the present invention is substantially resistant to proteases in the gastrointestinal tract, most preferably the human gastrointestinal tract.

[0181] Stability in the buccal cavity, pharynx, and esophagus The buccal cavity, pharynx, and esophagus precede the stomach in the gastrointestinal tract. Preferably, the polypeptide or construct of the present invention substantially retains neutralizing ability and / or potency when delivered orally and after exposure to the buccal cavity, pharynx, and esophagus (e.g., after exposure to buccal cavity, pharynx, and esophagus proteases). Proteases in the buccal cavity, pharynx, and esophagus include proteases sourced from commensal flora and / or pathogenic bacteria, e.g., proteases such as cell membrane-associated proteases, excretory proteases, and proteases released upon cell lysis.

[0182] Preferably, the buccal cavity, pharynx, and esophagus are from a dog, pig, human, cynomolgus monkey, or mouse. More preferably, the buccal cavity, pharynx, and esophagus are from a human, cynomolgus monkey, or mouse, and most preferably from a human.

[0183] A polypeptide or construct of the invention substantially retains its neutralizing ability if, preferably, 10% or more, more preferably 20% or more, more preferably 30% or more, more preferably 40% or more, more preferably 50% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, or most preferably 100% of the original neutralizing ability of the polypeptide or construct of the invention is retained after exposure to proteases present in the small intestine and / or large intestine and / or IBD inflammatory proteases.

[0184] Preferably, the polypeptides or constructs of the invention substantially retain their neutralizing ability after exposure to proteases present in the small intestine and / or large intestine and / or IBD inflammatory proteases, e.g., at 37°C, for up to at least 2 hours, more preferably up to at least 3 hours, more preferably up to at least 4 hours, more preferably up to at least 5 hours, more preferably up to at least 5.5 hours, more preferably up to at least 6 hours, more preferably up to at least 6.5 hours, more preferably up to at least 7 hours, more preferably up to at least 7.5 hours, more preferably up to at least 10 hours, more preferably up to at least 13 hours, or more preferably up to at least 16 hours.

[0185] Preferably, at least 10% of the neutralising capacity of the polypeptide or construct of the invention is retained after at least 2 hours, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95% of the neutralising capacity of the polypeptide or construct of the invention is retained after at least 2 hours, more preferably at least 3 hours, more preferably at least 4 hours, more preferably at least 5 hours, more preferably at least 6 hours, more preferably at least 7 hours, more preferably at least 9 hours, more preferably at least 11 hours, more preferably at least 13 hours, more preferably at least 16 hours of exposure to intestinal conditions, more preferably the small intestine or large intestine, more preferably a human faecal extract.

[0186] Preferably, 10% or more, more preferably 20% or more, more preferably 30% or more, more preferably 40% or more, more preferably 50% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more of the neutralising capacity of the polypeptide or construct of the invention is retained after exposure to mouse small intestinal supernatant for preferably at least 1 hour, more preferably at least 2 hours, more preferably at least 3 hours, more preferably at least 4 hours, more preferably at least 5 hours, or more preferably at least 6 hours.

[0187] Preferably, 10% or more, more preferably 20% or more, more preferably 30% or more, more preferably 40% or more, more preferably 50% or more, more preferably 60% or more, and more preferably 70% or more of an administered amount of a polypeptide or construct of the invention retains its ability to neutralize IL-7 and / or L-TSLP and remains in the feces (preferably voided feces or feces removed from the intestinal tract) of mice, cynomolgus monkeys, and / or humans at least 2 hours, more preferably at least 3 hours, more preferably at least 4 hours, more preferably at least 5 hours, more preferably at least 6 hours, more preferably at least 7 hours, more preferably at least 9 hours, more preferably at least 11 hours, more preferably at least 13 hours, or more preferably at least 16 hours after administration.

[0188] A polypeptide of the invention or a construct of the invention remains substantially intact when preferably 10% or more, more preferably 20% or more, more preferably 30% or more, more preferably 40% or more, more preferably 50% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 99% or more, and most preferably 100% of an administered amount of the polypeptide of the invention or construct of the invention remains intact after exposure to proteases present in the small intestine and / or large intestine and / or IBD inflammatory proteases.

[0189] "Stability" and "survival," e.g., "% stability" and "% survival," are used interchangeably herein. "Substantially retain neutralizing ability" and "substantially resistant" are used interchangeably herein.

[0190] In one embodiment of the present invention, there is provided a polypeptide comprising, or more preferably consisting of, the polypeptide sequence of any one of the ICVDs described below, based on their stability in digests of human fecal supernatant.

[0191] 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-A 54U, ID-A36U, ID-A55U, ID-A37U, ID-A38U, ID-A57U, ID-A39U, ID-A40U, V7R-2F6, V7R-6C12, V7R-2B6, V7R-3B5, V7R-4F6, V7R- 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.

[0192] Also provided is a polypeptide comprising three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR1 comprises, or more preferably consists of, the CDR1 sequence of any of the above ICVDs, CDR2 comprises, or more preferably consists of, the CDR2 sequence of any of the above ICVDs, and CDR3 comprises, or more preferably consists of, the CDR3 sequence of any of the above ICVDs. Most preferably, the polypeptide comprises all three CDRs from one of the above ICVDs.

[0193] Based on their stability in digests of human fecal supernatant, more preferably, polypeptides are provided which comprise, or more preferably consist of, the polypeptide sequence of any one of the ICVDs described below.

[0194] 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, and V7R-2E5.

[0195] Also provided is a polypeptide comprising three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR1 comprises, or more preferably consists of, the CDR1 sequence of any of the above ICVDs, CDR2 comprises, or more preferably consists of, the CDR2 sequence of any of the above ICVDs, and CDR3 comprises, or more preferably consists of, the CDR3 sequence of any of the above ICVDs. Most preferably, the polypeptide comprises all three CDRs from one of the above ICVDs.

[0196] Based on their stability in digests of human fecal supernatant, more preferably, polypeptides are provided which comprise, or more preferably consist of, the polypeptide sequence of any one of the ICVDs described below.

[0197] 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.

[0198] Also provided is a polypeptide comprising three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR1 comprises, or more preferably consists of, the CDR1 sequence of any of the above ICVDs, CDR2 comprises, or more preferably consists of, the CDR2 sequence of any of the above ICVDs, and CDR3 comprises, or more preferably consists of, the CDR3 sequence of any of the above ICVDs. Most preferably, the polypeptide comprises all three CDRs from one of the above ICVDs.

[0199] Preparation method Polypeptides of the present invention can be obtained or engineered using, for example, the techniques disclosed in Green and Sambrook 2012 Molecular Cloning: A Laboratory Manual 4th Edition Cold Spring Harbour Laboratory Press.

[0200] Monoclonal antibodies can be produced using hybridoma technology by fusing myeloma (B-cell cancer) cells, selected for their ability to grow in tissue culture and the absence of antibody chain synthesis, with B cells that produce the specific antibody (Kohler and Milstein 1975, and Nelson et al. 2000).

[0201] Monoclonal antibodies directed against the determined antigens can be e.g. a) immortalizing lymphocytes obtained from the peripheral blood of an animal previously immunized with a determined antigen, with immortal cells, and preferably with bone marrow cells, to form hybridomas; b) culturing the formed immortalized cells (hybridomas) and recovering cells that produce antibodies with the desired specificity; can be obtained by

[0202] Alternatively, the use of hybridoma cells is not required. Thus, monoclonal antibodies can be a) cloning into a vector, in particular a phage, more particularly a filamentous bacteriophage, a DNA or cDNA sequence obtained from lymphocytes, in particular peripheral blood lymphocytes, of an animal (preferably previously immunized with a determined antigen); b) transforming a prokaryotic cell with said vector under conditions that allow the production of the antibody; c) selecting antibodies by subjecting them to antigen-affinity selection; d) recovering antibodies with the desired specificity; It can be obtained by a process comprising:

[0203] Methods are known for immunizing camelids, cloning the VHH repertoire of circulating B cells (Chomezynski and Sacchi 1987), and isolating antigen-specific VHHs from immune (Arbabi-Ghahroudi et al. 1997) and non-immune (Tanha et al. 2002) libraries using phage, yeast, or ribosome display (WO92 / 01047, Nguyen et al. 2001, and Harmsen et al. 2007).

[0204] Antigen-binding fragments of antibodies, such as scFv and Fv fragments, have been isolated and expressed in E. coli (Miethe et al. 2013; Skerra et al. 1988; Ward et al. 1989).

[0205] Mutations can be made in the DNA or cDNA that are silent with respect to the amino acid sequence of the polypeptide but that encode the polypeptide by providing codons preferred for translation in a particular host. For example, preferred codons for translation of nucleic acids in E. coli and S. cerevisiae are known.

[0206] Mutation of a polypeptide can be achieved, for example, by substitution, addition, or deletion to the nucleic acid encoding the polypeptide. Substitutions, additions, or deletions to the nucleic acid encoding the polypeptide can be introduced by many methods, including, for example, error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, iterative ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis (Ling et al. 1997), gene rearrangement, gene site saturation mutagenesis (GSSM), synthetic ligation rearrangement (SLR), or a combination of these methods. Modifications, additions, or deletions to nucleic acids can also be introduced by methods including recombination, repeat sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, ensemble mutagenesis, chimeric nucleic acid multimer generation, or a combination thereof.

[0207] In particular, artificial gene synthesis may be used (Nambiar et al. 1984, Sakamar and Khorana 1988, Well et al. 1985, and Grundstrom et al. 1985). Genes encoding the polypeptides of the invention can be synthetically produced, for example, by solid-phase DNA synthesis. The entire gene can be synthesized de novo, without the need for precursor template DNA. To obtain the desired oligonucleotide, building blocks are sequentially coupled to the growing oligonucleotide chain in the order required by the product sequence. Once chain assembly is complete, the product is released from the solid phase into solution, deprotected, and collected. The product can be isolated by high-performance liquid chromatography (HPLC) to obtain the desired oligonucleotide in high purity (Verma and Eckstein 1998).

[0208] Expression of immunoglobulin chain variable domains, such as VH and VHH, can be achieved using suitable expression vectors, such as bacteria, e.g., prokaryotic cells, such as E. coli (for example, according to the protocols disclosed in WO94 / 04678, which is incorporated herein by reference and described in further detail below). Expression of immunoglobulin chain variable domains, such as VH and VHH, can also be achieved using eukaryotic cells, e.g., insect cells, CHO cells, Vero cells, or suitable yeast strains, such as those belonging to the genera Aspergillus, Saccharomyces, Kluyveromyces, Hansenula, or Pichia. Preferably, S. cerevisiae is used (for example, according to the protocols disclosed in WO94 / 025591, which is incorporated herein by reference and described in further detail below).

[0209] Specifically, VHHs: a) cloning a DNA or cDNA sequence encoding a VHH (e.g. obtained from camelid lymphocytes or produced synthetically), optionally containing a His-tag, into a Bluescript vector (Agilent Technologies); b) recovering the cloned fragment after amplification using a 5' primer specific for the VHH containing a XhoI site and a 3' primer containing a SpeI site with the sequence TC TTA ACT AGT GAG GAG ACG GTG ACC TG (SEQ ID NO: 81); c) cloning the recovered fragment in phase into the Immuno PBS vector (Huse et al. 1989) after digestion of the vector with the restriction enzymes XhoI and SpeI; d) transforming a host cell, in particular E. coli, by transfection with the recombinant Immuno PBS vector of step c; e) recovering the expression product of the VHH coding sequence by affinity purification, for example by Protein A, cation exchange, or, if the VHH contains a His tag, by chromatography on a column using a nickel affinity resin. It can be prepared according to the method disclosed in WO94 / 04678 using E. coli cells by a process comprising:

[0210] Alternatively, immunoglobulin chain variable domains such as VH and VHH can be a) obtaining a DNA or cDNA sequence encoding a VHH having a determined specific antigen-binding site; b) amplifying the resulting DNA or cDNA using a 5' primer containing a start codon and a Hindlll site and a 3' primer containing a stop codon with a XhoI site; c) recombination of the amplified DNA or cDNA into the Hindlll (position 2650) and XhoI (position 4067) sites of the plasmid pMM984 (Merchlinsky et al. 1983); d) transfecting permissive cells, in particular NB-E cells (Faisst et al. 1995), with the recombinant plasmid; e) recovering the resulting product It can be obtained by a process comprising:

[0211] Additionally, immunoglobulin chain variable domains such as VHH or VH can be produced according to the methods disclosed in Frenken et al. 2000 and WO99 / 23221 (incorporated herein by reference in their entireties) using E. coli or S. cerevisiae as follows.

[0212] Blood samples were collected from the immunized llamas, and the lymphocyte population was enriched by discontinuous centrifugation on a Ficoll (a neutral, highly branched, high-mass, hydrophilic polysaccharide that dissolves readily in aqueous solution - Pharmacia) gradient. Total RNA was isolated by acid guanidium thiocyanate extraction (Chomezynski and Sacchi 1987). After first-strand cDNA synthesis (using a cDNA kit such as RPN 1266 Amersham), DNA fragments encoding the VHH and VH fragments and the short or part of the long hinge region were amplified by PCR using specific primers as detailed on pages 22 and 23 of WO99 / 23221. Upon digestion of the PCR fragments with PstI and HindIII or BstEII, DNA fragments of approximately 300–450 bp in length were purified by agarose gel electrophoresis and ligated into the E. coli phagemid vector pUR4536 or the episomal S. cerevisiae expression vector pUR4548, respectively. pUR4536 is derived from pHEN (Hoogenboom et al. 1991) and is modified with lacI to allow cloning of the llama VHH and VH genes. q pUR4548 is derived from pSY1 (Harmsen et al. 1993). The BstEII site in the leu2 gene was removed from this plasmid by PCR, and the cloning site between the SUC2 signal sequence and the terminator was replaced to facilitate the cloning of VH / VHH gene fragments. The VH / VHH have a c-myc tag at the C-terminus for detection. Individual E. coli JM109 colonies were cultured in 1% glucose and 100 mg L-1 After overnight growth (37°C), the plates were transferred to 96-well microtiter plates containing 150 ml of 2TY medium supplemented with ampicillin. -1 Duplicate cultures are grown in 2TY medium containing ampicillin and 0.1 mM IPTG. After a further overnight incubation, optionally freezing and thawing, the cells are centrifuged and pelleted, and the supernatant can be used in an ELISA. Individual S. cerevisiae colonies are transferred to test tubes containing selective minimal medium (0.7% yeast nitrogen base, 2% glucose, supplemented with essential amino acids and bases) and grown at 30°C for 48 hours. The cultures are then diluted 10-fold in YPGal medium (containing 1% yeast extract, 2% bacto peptone, and 5% galactose). After 24 and 48 hours of growth, the cells are pelleted, and the culture supernatant can be analyzed by ELISA. The optical density at 600 nm (OD600) is optionally measured.

[0213] Furthermore, immunoglobulin chain variable domains such as VH / VHH can be produced using S. cerevisiae using procedures such as the following.

[0214] Isolate a naturally occurring DNA sequence encoding a VH / VHH, or obtain a synthetically produced DNA sequence encoding a VH / VHH, including the 5'-UTR, signal sequence, stop codon, and flanked by SacI and HindIII sites (such synthetic sequences can be produced as outlined above or may be ordered from a commercial supplier, e.g., Geneart (Life Technologies)).

[0215] To transfer VH / VHH genes into the multicopy integration (MCI) vector pUR8569 or pUR8542, restriction sites are used as follows: Using 25 μl of VHH DNA (Geneart plasmid or MCI vector), 1 μl of SacI, 1 μl of HindIII, and 3 μl of a buffer suitable for double digestion, such as NEB buffer 1 (New England Biolabs), the DNA sequence encoding the VHH, optionally contained within a shuttle vector, cassette, or other synthetic gene construct, and the MCI vector with SacI and HindIII are digested overnight at 37°C. 25 μl of the digested DNA encoding the VHH and 25 μl of the digested MCI vector are run on a 1.5% agarose gel containing 1×TAE buffer, followed by gel extraction using, for example, a QIAquick Gel Extraction Kit (Qiagen). Ligation of the digested MCI vector and the digested DNA encoding VH / VHH is set up as follows: 100 ng of vector, 30 ng of VHH gene, 1.5 ul of 10x ligase buffer, 1 ul of T4 DNA ligase, and ddH2O. Ligation is then carried out overnight at 16°C.

[0216] Next, transform the E. coli cells. For chemically competent XL-1 Blue cells, thaw 200 μl of heat-competent XL-1 Blue cells and add 5 μl of ligation mix on ice for approximately 30 minutes, followed by a 90-second heat shock at 42°C. Then, add 800 μl of Luria-Bertani low-salt medium supplemented with 2% glucose, and allow the cells to recover at 37°C for 2 hours. Plate the cells on Luria-Bertani agar and ampicillin (100 μg / ml) plates and maintain them at 37°C overnight. For electrocompetent TG1 E. coli cells, use an electroporation cuvette. Thaw 50 μl of electrocompetent TG1 cells and 1 μl of ligation mix in an electroporation cuvette on ice for approximately 15 minutes. Place the cuvette in the holder and pulse. Add 500 μl of 2TY medium, and allow the cells to recover at 37°C for 30 minutes. Plate 100 ul of cells onto Luria-Bertani agar plates containing ampicillin (100 ug / ml) and 2% glucose. Keep plates at 37°C overnight.

[0217] After cloning the VH / VHH genes into E. coli as detailed above, S. cerevisiae can be transformed with the linearized MCI vector. Before transformation can be performed, several steps must be performed: (i) the DNA must be converted from circular to linear by digestion, otherwise it cannot integrate into the yeast genome, and (ii) the digested DNA must be purified by ethanol precipitation. Furthermore, during the conversion process, yeast cells become semipermeable, allowing DNA to pass through the cell membrane.

[0218] Preparation for yeast transformation: Perform HpaI digestion of midi-preps prepared from selected E. coli colonies expressing VH / VHH genes as follows: Prepare 100 ul of a solution containing 20 ng of midi-prep, 5 ul of HpaI, 10 ul of an appropriate buffer such as NEB4 buffer (BioLabs), and ddH2O.

[0219] Digest the HpaI-digested DNA overnight at room temperature. Next, perform an ethanol precipitation (and set aside a 5 ul sample from the HpaI digestion). Add 300 ul of 100% ethanol to the 95 ul HpaI-digested midiprep, vortex, and spin at full speed for 5 minutes. If a pellet is present, carefully decant it and add 100 ul of 70% ethanol, then spin at full speed again for 5 minutes. Decant the sample again and keep at 50-60°C until the pellet is dry. Resuspend the pellet in 50 ul of ddH2O. Run 5 ul on a gel alongside the 5 ul HpaI-digested sample.

[0220] Yeast Transformation: Prepare YNBglu plates. Use 10 g agar + 425 ml water (sterilized), 25 ml filtered 20x YNB (3.35 g YNB (Yeast Nitrogen Base) in 25 ml sterile HO), and 50 ml sterile 20% glucose and pour into Petri dishes. Pick one yeast colony from the master plate and grow overnight at 30°C in 3 ml YSD (Yeast Extract Soytone Dextrose). The next day, prepare approximately 600 ml of YSD and use this to fill three flasks with 275 ml, 225 ml, and 100 ml of YSD. Add 27.5 ul of yeast YSD culture to the first flask and mix gently. Take 75 ml from the first flask and add to the second flask and mix gently. Take 100 ml from the second flask and place it in a third flask, mixing gently. Grow until an OD660 of 1-2 is reached. Once this OD is reached, split the flask into four Falcon tubes, each containing 45 ml. Spin at 4200 rpm for 2 minutes. Discard the supernatant. Dissolve the pellet in two Falcon tubes containing 45 ml of HO (reducing the number of tubes from four to two). Spin at 4200 rpm for 2 minutes. Dissolve the pellet in 45 ml of HO (reducing the number of tubes from two to one). Spin at 4200 rpm for 2 minutes. Gently dissolve the pellet in 5 ml of 100 mM lithium acetate (LiAc) and spin for a few seconds. Carefully discard some of the LiAc, retaining more than half of the LiAc in the tube. Vortex the cells and boil the carrier DNA for 5 minutes, then quickly cool in ice water. Add 240 μl of PEG, 50 μl of cells, 36 μl of LiAc (1M), 25 μl of carrier DNA, and 45 μl of ethanol-precipitated VH / VHH to a 15 ml tube. Mix gently after each step. (Treat a blank sample similarly, but without the ethanol-precipitated VH / VHH.) Incubate at 30°C for 30 minutes, gently invert 3-4 times, and then heat shock at 42°C for 20-25 minutes. Spin briefly at a maximum of 6000 rpm. Gently remove the supernatant and add 250 μl of ddH2O and mix.Streak all of the mixture onto a YNBglu plate until the plate is dry and grow at 30°C for 4-5 days. Finally, prepare YNBglu by dividing the plate into 6 equal parts, numbering the parts 1 to 6. Inoculate the largest colony and streak number 1. Repeat this process for the other colonies, starting from largest to smallest, starting with 1 to 6. Grow the mixture at 30°C for 3-4 days until colonies are produced. VH / VHH clones are grown using glucose as a carbon source, and VH / VHH expression is induced by adding 0.5% galactose to activate the galactose-7 promoter. Test the colonies by culturing 3 mL of small-scale cultures and select those that show the best expression of VH or VHH. These colonies are then used for purification.

[0221] Purification: VH / VHH are purified by cation exchange chromatography using a strong anionic resin (e.g., Capto S). On day 1, 5 ml of YSD medium (YS medium + 2% glucose) is inoculated with a selected yeast colony expressing the VH / VHH, and the cells are grown overnight at 30°C (shaking at 180 rpm) in a 25 ml sealed sterile tube. On day 2, 5 ml of the overnight culture is diluted into 50 ml of freshly prepared YS medium + 2% glucose + 0.5% galactose, and the cells are grown for two nights at 30°C (shaking at 180 rpm) in a 250 ml aerated, conditioned flask. On day 4, the cells are spun in a centrifuge at 4200 rpm for 20 minutes. Cation exchange purification step using a strong anionic resin: The pH of the ligand-containing supernatant is adjusted to 3.5. Wash 0.75 ml of resin (+ / - 0.5 ml slurry) per 50 ml of supernatant with 50 ml of ddH2O, then wash three times with binding buffer. Add the washed resin to the supernatant and incubate the suspension on a shaker at 4°C for 1.5 hours. Pellet the VH / VHH bound to the resin by centrifugation at 500 g for 2 minutes and wash it with wash buffer. Decant the supernatant and resuspend the resin in 10 ml of binding buffer. Place a filter in a PD-10 column, pour the resin into the column, allow the resin to settle for a while, and then add the filter on top of the resin. Wait until all the binding buffer has drained out. Elute the VH / VHH with 6 x 0.5 ml of elution buffer. Collect the eluate fractions in Eppendorf tubes. Measure the protein concentration of the six eluted fractions using a Nanodrop. Pool the fractions containing VHH and transfer the solution to a dialysis membrane with a 3,500 Da cutoff. Dialyze the purified protein solution against 3 L of PBS overnight at 4 °C. On day 5, dialyze the purified protein solution against 2 L of fresh PBS for an additional 2 h at 4 °C. Finally, calculate the final concentration by BCA.

[0222] Although described with respect to VH / VHH, the techniques described above may also be used for scFv, Fab, Fv, and other antibody fragments, as appropriate. Multiple antigen-binding fragments (preferably VH / VHH) can be fused by chemical crosslinking by reacting amino acid residues with organic derivatizing agents, such as those described by Blattler et al. (1985). Alternatively, antigen-binding fragments can be fused genetically at the DNA level, i.e., a polynucleotide construct encoding a complete polypeptide construct containing one or more antigen-binding fragments can be formed. One method for joining multiple antigen-binding fragments via the genetic pathway is by linking the sequences encoding the antigen-binding fragments directly or via a peptide linker. For example, the carboxy terminus of a first antigen-binding fragment can be linked to the amino terminus of the next antigen-binding fragment. This linking mode can be extended to link antigen-binding fragments for the construction of tri-, tetra-, etc. functional constructs. Methods for producing multivalent (such as bivalent) VHH polypeptide constructs are disclosed in WO96 / 34103, which is incorporated herein by reference in its entirety.

[0223] Suitably, the polypeptides of the invention (in particular the VHHs of the invention) can be produced in fungi such as yeast (e.g., S. cerevisiae) according to the method disclosed in WO 02 / 48382, which involves growing the fungus on a medium containing a carbon source, wherein 50-100 wt% of the carbon source is ethanol. Large-scale production of VHH fragments in S. cerevisiae is described in Thomassen et al. (2002).

[0224] In one aspect of the invention there is provided a process for the preparation of a polypeptide or construct of the invention, said process comprising: i) cloning the polynucleotide of the present invention into a vector such as a plasmid; ii) transforming a cell, such as a bacterial or yeast cell, capable of producing the polypeptide or construct of the invention with said vector under conditions allowing the production of the polypeptide or construct; iii) recovering the polypeptide or construct, such as by affinity chromatography.

[0225] Further embodiments of the present invention are set forth below.

[0226] term 1. A polypeptide capable of inhibiting the binding of IL-7 and / or L-TSLP to IL-7R. 2. The polypeptide according to item 1, wherein the polypeptide is capable of inhibiting the binding of IL-7 to IL-7R. 3. The polypeptide according to item 1, wherein the polypeptide is capable of inhibiting the binding of L-TSLP to IL-7R. 4. The polypeptide according to any one of items 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. 5. The polypeptide according to any one of items 1 to 4, wherein the polypeptide binds to IL-7Rα. 6. The polypeptide of any one of items 1-5, 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 more sequence identity with SEQ ID NO: 1, CDR2 comprises a sequence that shares 60% or more sequence identity with SEQ ID NO: 2, and CDR3 comprises a sequence that shares 60% or more sequence identity with SEQ ID NO: 3. 7. The polypeptide of paragraph 6, wherein CDR1 comprises a sequence that shares 80% or more sequence identity with SEQ ID NO: 1, CDR2 comprises a sequence that shares 80% or more sequence identity with SEQ ID NO: 2, and CDR3 comprises a sequence that shares 80% or more sequence identity with SEQ ID NO: 3. 8. The polypeptide of 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. 9. The polypeptide of paragraph 8, wherein the polypeptide comprises SEQ ID NO:8. 10. The polypeptide of any one of paragraphs 1-9, wherein the polypeptide is an antibody or antibody fragment. 11. The polypeptide according to any one of paragraphs 1 to 10, wherein the polypeptide neutralizes IL-7R binding to IL-7 with an EC50 of 2 nM or less. 12. The polypeptide of any one of paragraphs 1-11, wherein the polypeptide is substantially resistant to proteases of the human gastrointestinal tract. 13. The polypeptide according to any one of items 1 to 12, which is used as a drug. 14. The polypeptide according to paragraph 13, for use in the treatment of autoimmune and / or inflammatory diseases. 15. The polypeptide of any one of paragraphs 13 or 14, wherein the polypeptide is administered orally. The invention will now be further illustrated by the following non-limiting examples. [Example]

[0227] Example 1: Exemption and phage library construction Two llamas were each immunized with soluble human recombinant IL-7Rα. Blood samples were collected from both llamas at various times during the immunization and tested for IL-7Rα binding and neutralization to monitor the development of an immune response to IL-7Rα. Analysis showed that only one llama developed significant anti-IL-7Rα antibody titers, while the other llama failed to respond to IL-7Rα immunization. At the end of the immunization, RNA isolated from leukocytes collected from the responsive llama was used to generate 12 distinct phage display libraries.

[0228] Example 2: Library selection for phages with human IL-7Rα binding activity A library selection strategy was developed to isolate ICVDs that bind to epitopes present on the extracellular domain of the IL-7Rα subunit, including ICVDs that interfere with IL-7 binding to the IL-7R. Several methods were used to selectively enrich for phage exhibiting ICVDs with IL-7Rα binding properties and other desirable properties, including high binding affinity and resistance to intestinal proteases. Phage present in the eluates from various library selections were used to infect E. coli, and individual colonies were picked and expanded onto master plates to generate clonal cultures. Periplasmic supernatants containing selected monoclonal ICVDs were used in primary characterization studies to identify those with the desired properties.

[0229] From a total of 630 library-selected clones picked onto the original eight master plates screened, a final set of seven primary clones was selected for production in E. coli, ICVD affinity purified, and further detailed characterization studies.

[0230] The DNA sequences of the seven primary clones isolated above (V7R-2E5, V7R-2E9, V7R-2F6, V7R-6C12, V7R-2B6, V7R-3B5, and V7R-4F6) were recloned into the vector pMEK222 for production in E. coli (thus introducing C-terminal FLAG and 6xHis tags), followed by affinity purification and further detailed characterization. The polypeptide sequences of these ICVDs, excluding the FLAG and His tags, are shown in the alignment provided above in the section entitled "Polypeptide and Polynucleotide Sequences." The clinical anti-IL-7R antibody mAb829 (a 150 kDa antibody comprising heavy and light chains, also known as "GSK2618960" as disclosed in Ellis et al. 2019) was produced and used as a control in many of the following examples.

[0231] An important goal of the following examples was to identify those ICVD clones that have the ability to inhibit IL-7 binding to IL-7R and have some degree of intrinsic resistance to inactivation by small intestinal proteases.

[0232] Example 3: Potency and protease resistance of primary clones efficacy The potency of seven primary clones was assessed using an IL-7 / IL-7R neutralization ELISA.

[0233] The primary clones were subcloned from the phagemid into the pMEK222 plasmid, tagged with a C-terminal FLAG-6xHis tag, and expressed in E. coli. These ICVDs were expressed from E. coli TG1 and purified via the 6xHis tag.

[0234] Seven-point dilution series of clones were prepared in 1% BSA (2x assay concentration) starting at 300 nM and using a dilution factor of 3.2. The mAb829 control antibody was used as a positive control in the ELISA at concentrations ranging from 10 nM to 0.088 nM (2x assay concentration). Sufficient volumes for each clone dilution were prepared for triplicates, and for mAb829, sufficient volumes for two triplicates (two plates). 85 μL (or 170 μL) of each ICVD (or mAb829) dilution was mixed with 85 μL (or 170 μL) of 10 ng / mL IL-7 (2x assay concentration). 85 μL of IL-7 was mixed with 85 μL of blocking buffer to achieve a full IL-7 (1x) binding signal in each plate. Blocking buffer alone was added to each plate as a blank. Bound IL-7 was then measured using biotinylated anti-hIL-7 followed by Extravidin-HRP. The TMB reaction was stopped after 30 min.

[0235] A corrected for ELISA signal blank 450 EC2 analysis was performed in Graphpad Prism using the data and "Log (inhibitor) vs. response -- variable slope (4 parameters)". 50 Generate values ​​and fit the curve to EC50 These values ​​are shown in Table 1 below. All ICVDs were shown to be as effective as, and slightly more effective than, the control mAb 829 in inhibiting hIL-7 binding to hIL-7R.

[0236] Protease resistance The seven purified ICVDs were incubated in the presence of mouse small intestinal supernatant ("mouse SI") and supernatant prepared from pooled human fecal samples ("HFP").

[0237] All ICVD stock dilutions were prepared in 1x PBS containing 1% BSA at 250 μg / mL (30 μL final volume). Digestion mixture reactions were then prepared for each ICVD in a PCR strip in a final volume of 60 μL, with the ICVD at a final concentration of 20 μg / mL (55.2 μL digestion matrix at 250 μg / mL + 4.8 μL ICVD). For non-ICVD controls, 4.8 μL of 1x PBS was used instead. 25 μL of each digestion reaction was then transferred (i) to a new PCR tube containing cold stop buffer (used as the T=0 time point) and frozen at -80°C; and (ii) to a new empty PCR tube and incubated in a PCR thermocycler for digestion at 37°C (digested non-ICVD control samples). The digested samples in mouse SI were removed from the thermocycler after 2 hours, and the digested samples in HFP were removed after 4 hours. Samples were immediately stopped with 25 μL of cold stop buffer and frozen at −80° C. until analysis.

[0238] Digested samples were tested in the ELISA detailed above under "Potency" at a final starting dilution of 1:100, followed by six serial dilutions using dilution factors of 1.8 (for digested samples) or 2.1 (for undigested samples) and analyzed in triplicate wells on each plate. Undigested samples (time 0) serve as the standard curve, as no protease digestion should theoretically occur in these samples (they are processed on ice and stop buffer is added immediately after addition of digestion matrix).

[0239] If digestion occurs in the "digested" sample, there is expected to be a shift of the curve to the left (compared to T=0 hours). The greater the shift, the more unstable the ICVD. "% Survival" represents the level of activity retained after digestion.

[0240] All clones displayed high levels of resistance to proteolysis in both matrices, identifying V7R-2E9 as the most protease-resistant ICVD, which exhibited complete resistance to pooled digestion of human feces for up to 4 hours.

[0241] These results are summarized in Table 1 below.

[0242] [Table 11]

[0243] All of these polypeptides of the invention showed surprisingly high potency and survival rates in the digestion matrix. It should be noted that although these ICVDs are clearly related, the CDRs and frameworks of these polypeptides differ from each other by many residues (see alignments provided above under "Polypeptide and Polynucleotide Sequences").

[0244] Example 4: Further potency assays performed on V7R-2E9 and control mAb829 IL-7 / IL-7R neutralization ELISA The IL-7 / IL-7R neutralization ELISA described above in Example 3 was again performed on V7R-2E9 and the clinical anti-IL-7R control antibody, mAb 829. The results are shown in Table 2 below.

[0245] TSLP / TSLPR / IL-7R ELISA The ability of V7R-2E9 to neutralize the binding of the L-TSLP / TSLP-R complex to IL-7Rα was tested. 96-well plates were 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 1:1:1 with recombinant human L-TSLP (final concentration of 15 ng / mL) and human TSLP-R (final concentration of 20 ng / mL). 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, bound L-TSLP was detected with 50 μL / well of 0.3 μg / mL biotinylated rabbit anti-hTSLP antibody, followed by 50 μL / well of 1 / 2000 Extravidin-HRP. The level of neutralization of L-TSLP / TSLP-R complex binding to IL-7Rα by ICVD was determined using GraphPad Prism. The results are shown in Table 2 below.

[0246] IL-7-induced pSTAT5 in hPBMCs The ability of V7R-2E9 to inhibit IL-7 binding to IL-7Rα and interfere with STAT5 phosphorylation was tested in vitro in human lymphocytes. These human peripheral blood mononuclear cells (PBMCs) respond to exogenous IL-7 by stimulating intracellular STAT5 phosphorylation via IL-7R signaling, but this response can be abrogated by an IL-7Rα-specific ICVD that prevents IL-7 / IL-7R binding.

[0247] Lymphocyte-enriched populations were isolated from human buffy coats and stored in 90% FBS 10% DMSO in liquid nitrogen. Cells were thawed and quiescent for recovery in complete RPMI-1640. After recovery, cells were cultured in 100 μl of 2.5 × 10 5Cells were plated at 1000 cells / well in a round-bottom 96-well plate and starved for 1 hour in complete RPMI without FBS. After starvation, the desired ICVD concentration was added to each well (50 μL / well), and the plate was incubated at room temperature for 15 minutes. IL-7 (50 μL / well) was then added to each well, and the plate was incubated at 37°C, 5% CO2 for 15 minutes. The reaction was stopped by quickly chilling the plate on ice, followed by centrifugation and removal of the supernatant. Cells were then processed for fixation, permeabilization, and intracellular staining of pSTAT5. Cells were incubated on ice for 20 minutes with 100 μL / well of Cytofix / Cytoperm solution (BD Bioscience #554722), washed twice with 150 μL / well of 1x Perm / Wash buffer (BD Bioscience #554723), incubated on ice for 30 minutes with 200 μL / well of Perm buffer III (BD Bioscience #558050), and washed twice with 150 μL / well of 1x PBS 2% BSA (FACS buffer). Cells were then stained with 25 μL / well of pSTAT5 antibody ([47 / Stat5(pY694)](A488) (BD Bioscience #612598)) or isotype control mouse IgG1 ([B11 / 6](FITC) (Abcam #ab91356)) for 1 hour at room temperature. The reaction was stopped by adding 150 μL / well of FACS buffer. After one washing / centrifugation step, the cells were finally resuspended in 200 μL / well of FACS buffer and data were acquired on a CytoFlex flow cytometer (Beckman Coulter). Data analysis was performed using FlowJo software. The results are shown in Table 2 below.

[0248] TSLP-induced TARC secretion in human monocytes V7R-2E9 was also tested to confirm IL-7R neutralizing activity in a human monocyte cell assay, as detailed below. Human monocytes exhibit a TARC secretion response in culture following stimulation with TSLP as a result of binding of cell surface IL-7R to TSLP / TSLP-R. The ability of anti-IL-7Rα ICVD to inhibit TSLP / TSLP-R binding to IL-7R and prevent TARC secretion was tested in vitro in human monocytes.

[0249] Monocytes were isolated from human buffy coats and plated in flat-bottom 96-well plates (1 × 10 5 Monocytes were plated in 100 μL / well containing monocytes. To enhance monocyte purity, plated cells were rested for 2 hours at 37°C, 5% CO2, after which nonadherent cells were removed by aspirating the medium in each well and gently washing twice with warm cRPMI. Cells were incubated with the desired concentration of ICV in the presence of TSLP for 24 hours at 37°C, 5% CO2 (diluted in complete RPMI, final volume of 100 μL / well). After 24 hours of incubation, 75 μL of supernatant per well was collected and stored at -80°C for further analysis of secreted TARC.

[0250] The neutralization level of human TSLP by anti-IL-7Rα agents was tested. A 96-well plate was coated with 50 μL / well of anti-human TARC antibody and then blocked. Human TARC standards were serially diluted in assay diluent, and then 50 μL / well of the standards, along with 50 μL / well of harvested culture supernatant, were added to the anti-TARC-coated plate. Bound human TARC was detected with biotinylated anti-human TARC polyclonal antibody followed by avidin-HRP. The neutralization level of human TSLP by the agents was determined. The results are shown in Table 2 below.

[0251] [Table 12]

[0252] In summary, V7R-2E9 was shown to inhibit IL-7 and L-TSLP binding to IL-7R in both ELISA and cell-based assays with potency similar to that of the control clinical anti-IL-7R antibody mAb829.

[0253] Example 5: Epitope modeling A model structure and epitope for V7R-2E9 were established in silico. This model was developed using the Protein Data Bank (PDB) file "4ybq" as a template. 4ybq is the heavy chain of FV bound to rat GLUT5-facilitated glucose transporter member 5. The 4ybq template is particularly similar to V7R-2E9 in terms of CDR3 loop length and predicted conformation. PDB entry "3di3" is a high-resolution structure of IL-7 bound to IL-7Rα. Simply removing IL-7 from this structure provided a target receptor for docking with the predicted V7R-2E9 structure.

[0254] Because the HEX 8.0 docking method used operates locally across the surface of each domain, many target regions were selected and run in parallel on the IL-7Rα structure. The best solution for V7R-2E9 was representative of a cluster of 46 solutions, had a very high energy score of -894 (DARS force field), and had no "bumps" (unacceptably close atom positions) after energy minimization. Figure 3 shows a ribbon diagram of the position of docked V7R-2E9 on the IL-7Rα target. V7R-2E9 is located at the interface between the N- and C-terminal domains.

[0255] Table 3 shows the epitope residues of IL-7Rα that contact V7R-2E9. Residues that fill particularly important areas in the interface are highlighted in bold (by reference to SEQ ID NO: 65).

[0256] [Table 13]

[0257] Example 6: Optimization to reduce immunogenicity The amino acid sequence of V7R-2E9 was aligned with the human VH3 germline antibody sequence to identify potential humanization changes. Eighteen selected single mutations and two combinations of changes at the end of framework 2 / beginning of CDR2 were introduced into the V7R-2E9 parent ICVD sequence, and mutants were generated in E. coli. The ICVDs were first tested for potency in an IL-7 / IL-7R neutralization ELISA. The clones exhibited IL-7R neutralization activity comparable to or greater than that of V7R-2E9, indicating that none of the mutations introduced into the parent ICVD had a deleterious effect on antigen binding. All clones were then digested in human fecal supernatant for 16 hours, and their relative survival was measured (Table 4).

[0258] [Table 14]

[0259] Mutations that retained high potency in the IL-7 / IL-7Rα-His6-Fc ELISA and maintained resistance similar to the human fecal pool were then combined to generate 19 humanized ICVDs (Table 5).

[0260] [Table 15]

[0261] Table 6 summarizes the most advantageous humanized ICVDs among these 19 that maintained potency and resistance to both human fecal proteases and mouse small intestinal proteases.

[0262] [Table 16]

[0263] 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 were calculated using the CLC Sequence Viewer).

[0264] Example 7: Potency assays performed on humanized V7R-2E9 variants The inhibitory potency and efficacy (maximal inhibition) of ID-A40U (produced in E. coli) was confirmed in vitro in an IL-7 / IL-7R neutralization ELISA and in an IL-7-induced STAT5 phosphorylation assay in human PBMCs. 50 Values ​​are expressed as ELISA signal blank-corrected A 450 Using the data and "Log (inhibition) vs. response -- variable slope (four parameters)", a curve was generated and fitted in Graphpad Prism to obtain the EC 50 was generated.

[0265] The results are shown in Table 7a along with the control drug.

[0266] [Table 17]

[0267] In a separate experiment, these same assays were performed on ID-A62U (produced in S. cerevisiae) along with a control drug, the results of which are shown in Table 7b and Figure 4.

[0268] [Table 18]

[0269] The ability of ID-A62U to neutralize binding of the L-TSLP / TSLP-R complex to IL-7Rα was tested together with mAb829 using the method described above in Example 4. The results are shown in Table 7c below. Prior to display, the data sets were subtracted and normalized to the highest concentration of antibody tested (to overcome high levels of background on the plate).

[0270] [Table 19]

[0271] In summary, ID-A62U and ID-A40U were shown to have potency comparable to or greater than that of the control clinical anti-IL-7R antibody mAb829.

[0272] Example 8: Biacore estimation of ICVD-IL-7R binding affinity The binding kinetics of ID-A40U was compared to that of the mAb829 clinical antibody in Biacore experiments. IL-7Rα-His6-Fc was directly coated onto a Biacore sensor plate (for mAb829 analysis) and captured with anti-human IgG Fc (for ICVD analysis). Binding was detected by flowing ICVD / Ab over the plate. ID-A40U was 7.8 x 10 -11 Affinity of M (K D ), while mAb829 has a slightly lower 5.67 × 10 -10 Affinity of M (K D The results show that ID-A40U demonstrates strong binding to the antigen.

[0273] Example 9: Cross-reactivity with IL-7Rα from toxicological species The cross-reactivity of ID-A40U, ID-A59U, and ID-A62U to bind to IL-7Rα from toxicological species was examined.

[0274] 96-well plates were coated with 0.5 μg / mL recombinant human IL-7Rα His6-Fc + 5 μg / mL BSA and then blocked. 0.5 nM ICVD was mixed 1:1 with serially diluted selected test compounds in 1% BSA and then incubated for 30 minutes to allow binding before being added to the IL-7Rα-coated plates. After a 2-hour incubation, bound ICVD was detected with 50 μL / well of 1 / 20,000 anti-FLAG-HRP goat antibody (GeneTex, GTX21238) to determine the neutral level of ICVD-IL-7Rα binding by the test compound.

[0275] In this assay, mouse IL-7Rα was found not to interfere with ICVD / IL-7Rα binding, indicating that mice or rats are inappropriate species for toxicological testing. However, cynomolgus IL-7Rα interfered with these ICVD binding to human IL-7Rα (Figures 5 and 6), making cynomolgus monkeys an appropriate toxicological species for these and related ICVDs.

[0276] Example 10: Specificity for non-target cytokines ID-A40U was tested for selectivity for proteins that associate with human IL-7Rα substantially as described above in Example 9. Human IL-12Rβ1 and human IL-12Rβ2 were found to be the human proteins most closely related to IL-7Rα, identified using the NCBI BLASTp tool, with 29% and 30% sequence identity with IL-7Rα, respectively. In a competitive IL-7R binding ELISA assay, hIL-12Rβ1 and a selection of 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 a plate. Neither human IL-12Rβ1, IL-2R, IL-21R, IL-9R, TNFR-2, nor IL-6R interfered with ID-A40U binding to IL-7Rα, but the addition of increasing amounts of human IL-7Rα generated a dose-dependent curve (Figure 7), indicating that binding of ID-A40U ICVD to off-target molecules is highly unlikely in humans.

[0277] Example 11: Resistance to gastrointestinal extracts Ex vivo incubation in intestinal supernatant can predict ICVD stability in the gastrointestinal tract of cynomolgus monkeys and humans. While the activities of the major small intestinal proteases, trypsin and chymotrypsin, are conserved across mammalian species, proteases present in the large intestine are likely produced by the host's species-specific gut microbiota. To generate test matrices that reflect these two environments, pooled mouse small intestinal supernatant and pooled fecal supernatant were prepared. Both of these matrices are highly digestible to unselected, unmanipulated ICVD.

[0278] ICVD ID-38F has high stability in these matrices (see WO2016 / 156465), and this property predicts high stability of ID-38F during its passage through the intestine.

[0279] V7R-2E9, ID-A24U, and ID-A40U were tested for their survival in gastrointestinal extracts from both mouse and human sources. ICVDs were incubated in mouse small intestinal supernatant for 6 hours at 37°C and in human fecal supernatant for 16 hours. Survival was measured by IL-7 / IL-7R neutralization ELISA. All constructs showed good survival in all digestive matrices tested (Figure 8, where "SI" = mouse small intestinal fluid and "HF" = human fecal supernatant). ID-A40U contains the same functional mutations as ID-A59U produced in yeast.

[0280] In a separate experiment, ID-A62U was tested for viability alongside ID-A41U (a labile control drug, ICVD) in the same human fecal supernatant assay. ID-A62U showed approximately 100% survival compared to approximately 40% survival for ID-A41U (Figure 9).

[0281] These were rigorous tests involving long incubation times, and therefore, both of these ICVDs are expected to survive very well in the gastrointestinal environment.

[0282] Example 12: Resistance to gastrointestinal matrix metalloproteinases Levels of activated matrix metalloproteinases (MMPs) are elevated in the inflamed mucosa of patients with intestinal disease. These MMPs can digest native human IgG and therapeutic agents, including those with a human IgG scaffold (Biancheri et al. 2015). In the case of the anti-TNFα treatment etanercept, this digestion results in a significant decrease in TNFα neutralization potency. To confirm that ID-A40U is resistant to MMPs, mAb829 was detected by Western blotting with ID-A40U and Enbrel in the presence of human MMP3, MMP12, or TCNB buffer at 37°C for 22 hours. Enbrel and mAb829 were detected with anti-human IgG conjugated to a gamma chain-specific peroxidase. ID-A40U and TCNB buffer-only controls were detected with pAb 1219, and primary rabbit α-ICVD and secondary HRP-conjugated pAb SwineαRabbit.

[0283] After incubation, ID-A40U was not digested by MMPs (Figure 10) as determined by Western blotting, showing a band with a molecular weight corresponding to the expected full-length ICVD (lacking the Flag-His6 tag that is cleaved during digestion). However, after the same incubation time, MMP3 and MMP12 digested full-length etanercept and mAb829 into smaller fragments. After MMP incubation, ID-A40U was shown to be fully potent at neutralizing IL-7R, as determined using an IL7 / IL-7R functional ELISA. "F / H" in Figure 10 indicates the presence of the FLAG / His tag.

[0284] Example 13: Transit and survival through the mouse gastrointestinal tract The results of the in vitro studies described above demonstrated that the optimized V7R-2E9 derivatives were resistant to inactivation by proteases present in supernatant extracts prepared from the contents of mouse small intestine. Subsequent studies were performed to examine the stability of ID-A24U and ID-A40U during passage through the mouse gastrointestinal system.

[0285] Both ID-A24U and ID-A40U were formulated with ID-38F (anti-TNFα ICVD, see WO2016 / 156465) in a milk and bicarbonate mixture to protect against denaturation at low pH and digestion by pepsin in the stomach. After administering ICVD to mice by oral gavage, concentrations of ICVD in the stomach, small intestine, cecum, and colon were determined 6 hours after administration. In addition, ICVD concentrations were measured in fecal pellets collected at hourly intervals.

[0286] ID-A24U and ID-A40U were measured in feces collected from all mice 4–6 h after administration, and ID-A40U was also measured at 3 h from two mice (Figure 11). Instead, ID-A24U was measured in feces collected from mouse 6 (M6) during the first 3 h, indicating that transit was particularly rapid in this mouse compared to the other mice.

[0287] These results suggest that both ID-A24U and ID-A40U can survive passage through the mouse GI tract. Overall, the transit times of ID-A24U and ID-A40U appear to be very similar, with the exception of one mouse (M6 and M12) within each group. At the time of sorting (6 h), most of ID-A24U and ID-A40U were present in the cecum (CAE) and colon (COL) of all mice, with moderately high amounts still measured in the stomach (STO) and small intestine (SI). Based on calculated concentrations taking into account the dilution factor used in the slurry preparation, the expected concentrations of ID-A24U and ID-A40U at the time of sorting were between 22.7 μM and 140 μM and between 12 μM and 22.5 μM in feces, respectively (Figure 11). The predicted concentrations of ID-A24U in the cecum and colon were between 2.9 μM and 8 μM and between 9.7 μM and 16.5 μM, respectively. The predicted concentrations of ID-A40U in the cecum and colon were between 0.9 μM and 1.2 μM and between 0.8 μM and 6.2 μM, respectively (Figure 12).

[0288] ID-38F, used as a control in this study, was measured at high levels in all fecal and lower GIT samples, as previously observed. The predicted concentrations of ID-38F varied between 0.04 μM and 2.1 μM in the cecum, between 0.33 μM and 5.1 μM in the colon, and between 4.9 μM and 48 μM in the feces at 6 hours in six mice. Repeat experiments were performed with ID-A40U and ID-38F. Similar results were obtained (Figures 13 and 14).

[0289] Overall, these results suggest that ID-A24U and ID-A40U survive well and similarly to ID-38F during passage through mice, delivering high concentrations of active ICVD to the cecum and colon. This may reflect the relative stability of each ICVD in these intestinal compartments, as previously observed in vitro. ID-A24U and ID-A40U have been shown to be stable in the mouse small intestine. Therefore, it is expected that these and related ICVDs will have high stability in the human GI tract.

[0290] Example 14: Human IBD tissue studies Studies were conducted to examine the activity of V7R-2E9 in a human ex vivo model that mimics the inflammatory bowel disease tissue environment. V7R-2E9 and controls (ID-2A, an anti-C. difficile toxin ICVD; mAb 829; and IgG1k (a non-IL-7R-binding purified human IgG1k isotype control recombinant antibody) were tested in ex vivo cultures for their effects on tissue phosphoprotein levels and inflammatory cytokine production using tissue from four patients with active ulcerative colitis.

[0291] Analysis of tissue lysates on the Pathscan phosphoprotein array (Figures 15-18) showed that in biopsies from three of the four UC patients, V7R-2E9 treatment inhibited phosphorylation of a substantial proportion of the 39 proteins detected on the array compared to the corresponding ID-2A-treated biopsies. mAb829 also inhibited protein phosphorylation levels in biopsies from the same three UC patients, and the pattern of inhibition obtained appears similar to that achieved with V7R-2E9. The sum of the phosphorylation intensity values ​​for each biopsy was calculated from the intensities of all 39 phosphoproteins on the array. The results, presented in Figure 19, show that V7R-2E9 and mAb829 inhibited total phosphorylation levels in biopsies from three reactive UC patients but had little or no effect on total phosphorylation in the biopsy from patient UC2700. This patient presented with active disease while receiving azathioprine (a T-cell inhibitor) as part of those medications, and therefore resistance to T-cell-directed therapy may explain the lack of response to antibodies targeting IL-7R-mediated T-cell activation (V7R-2E9 and mAb829).

[0292] 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 phosphoprotein analysis, V7R-2E9 did not inhibit the production of pro-inflammatory cytokines in cultures from the biopsy of patient UC2700.

[0293] In conclusion, antagonism of IL-7R in UC biopsies with V7R-2E9 inhibited the phosphorylation of signaling proteins and the production of cytokines and chemokines associated with proinflammatory and immunoregulatory pathways. The results demonstrate that antagonism of mucosal IL-7R+ve T cells with V7R-2E9 (and mAb829) can inhibit inflammatory processes in a model closely related to the disease environment.

[0294] Example 15: Yeast productivity in fermentation cultures S. cerevisiae expressing ID-A59U was inoculated into a 5-liter ethanol-fed fermentation. Broth supernatants at the end of fermentation (EoF) were analyzed for ID-A59U concentration by SDS-PAGE and IL-7 / IL-7Rα-His6-Fc functional ELISA. SDS-PAGE indicated a high yield of ≤2 g / L, and functional ELISA confirmed that ID-A59U was fully active at EoF, with a final yield of at least 1.5 g / L. S. cerevisiae expressing ID-A62U was inoculated into a 50 mL shake flask expression system. High yields of ICVD were obtained.

[0295] Conclusions from the above examples Polypeptides have been identified that benefit from high potency in cellular assay systems measuring neutralization of IL-7Rα activity, including inhibition of IL-7 and / or L-TSLP binding to IL-7Rα. These polypeptides also, in some cases, benefit from high stability in the small intestine and / or human fecal supernatants. Humanized derivatives of one particular polypeptide (V7R-2E9) have been produced that substantially retain or benefit from increased potency compared to unmodified V7R-2E9, while also retaining resistance to intestinal proteases and the ability to be effectively produced in S. cerevisiae. The most preferred combination of mutations in V7R-2E9 (R45L, Q65K, K87R, S88A), including the E1D yeast production mutation, is embodied by ID-A62U.

[0296] Miscellaneous All documents referred to in this application, including patents and patent applications, are incorporated herein by reference to the fullest extent possible.

[0297] Throughout this specification and the claims that follow, unless the context requires otherwise, it is recognized that the word "comprises" and variations such as "comprises" and "comprising" imply the inclusion of a stated integer, step, group of integers, or group of steps, but not the exclusion of other integers, steps, groups of integers, or groups of steps.

[0298] This specification and claims of the application of which it forms a part may be used as a basis for priority in respect of any subsequent application. The claims of such subsequent application may be directed to any feature or combination of features described herein. They may take the form of product, composition, process, or use claims and may include, by way of example and without limitation, the following claims:

[0299] References The following references are incorporated herein by reference in their entirety: Arbabi-Ghahroudi et al FEBS Lett 1997 414:521-526 Baumgart et al The Lancet 2012 380(9853):1590-605 Bjerkan et al Pharmaceuticals (Basel).2016 9(3). pii: E41 Blattler et al Biochemistry 1985 24:1517-1524 Chomezynnski and Sacchi Anal Biochem 1987 162:156-159 Cianferoni and Spergel Curr Allergy Asthma Rep. 2015 15(9):58 Corren et al N Engl J Med.2017 377(10):936-946 Crawley et al J Immunol.2010 184(9):4679-4687 Danese Gut 2012 61:918-932 Desmet et al Nature Communications 2014 5:5237 Dooms J Autoimmun. 2013 45:40-48 Ebersbach et al. J. Mol. Biol. 2007 372 (1):172-185 Ellis et al Br J Clin Pharmacol. 2019 85(2):304-315 Faisst et al J Virol 1995 69:4538-4543 Fornasa et al J Allergy Clin Immunol.2015 136(2):413-422 Frenken et al J Biotech 2000 78:11-21 Fry & Mackall Blood 2002 99(11):3892-3904 Fry & Mackall J Immunol.2005 174(11):6571-6576 Goldberg et al Nat Rev Gastroenterol Hepatol 2015 (5):271-283 Goldberg et al Protein Eng Des Sel.2016 29(12):563-572 Green and Sambrook Molecular Cloning:A Laboratory Manual 2012 4th Edition Cold Spring Harbour Laboratory Press Griffiths et al Antibodies 2013 2:66-81 Grundstrom et al Nucl.Acids Res 1985 13:3305-3316 Hafler et al N Engl J Med.2007 357(9):851-862 Hamers-Casterman et al Nature 1993 363(6428):446-448 Harmsen et al Gene 1993 125:115-123 Harmsen et al Appl Microbiol Biotechnol 2007 77(1):13-22) Hendrickson et al Clin Microbiol Rev 2002 15(1):79-94 Heninger et al J Immunol.2012 189(12):5649-5658 Hoogenboom et al Nucl Acid Res 1991 19:4133-4137 Huse et al Science 1989 246 (4935):1275-1281 Johnson et al Anal.Chem. 2012 84(15):6553-6560 Kabat et al Sequences of Proteins of Immunological Interest, Fifth Edition U.S. Department of Health and Human Services, 1991 NIH Publication Number 91-3242 Kohler and Milstein Nature 1975 256:495-497 Koide and Koide Methods Mol.Biol. 2007 352: 95-109 Krehenbrink et al J. Mol.Biol. 2008 383 (5):1058-1068 Ling et al Anal Biochem 1997 254(2):157-178 Lipovsek Protein Eng Des Sel. 2011 24(1-2):3-9 Liu J Exp Med 2006 203(2):269-273 Liu et al Nat Med.2010 16(2):191-197 (retraction in: Nat Med. 2013 19(12):1673) McCoy et al Retrovirology 2014 11:83 Merchlinsky et al J. Virol.1983 47:227-232 Miethe et al J Biotech 2013 163(2):105-111 Muyldermans et al Protein Eng 1994 7(9):1129-1135 Muyldermans Annu Rev Biochem 2013 82:775-797 Nambiar et al Science 1984 223:1299-1301 Nelson et al Molecular Pathology 2000 53(3):111-117 Nguyen et al Adv Immunol 2001 79:261-296 Nixon and Wood Curr Opin Drug Discov Devel.2006 9(2):261-268 Noti et al Nat Med. 2013 19(8):1005-1013 Nygren FEBS J. 2008 275(11):2668-2676 Padlan Mol Immunol 1994 31:169-217 Peters et al Immunol Lett.2015 172:124-131 Rimoldi et al Nat Immunol. 2005 6(5):507-514 Rose et al J Immunol.2009 182(12):7389–7397 Roux et al Proc Natl Acad Sci USA 1998 95:11804–11809 Sandborn et al N Engl J Med.2007 357:228–238 Sakamar and Khorana Nucl.Acids Res 1988 14:6361–6372 Shealy et al mAbs 2010 2:428–439 Silverman et al Nat.Biotechnol. 2005 23(12):1556–1561 Skerra et al Science 1988 240(4855):1038–1041 Skerra et al FEBS J. 2008 275(11):2677–83 Suderman Protein Expression and Purification 2017 134:114–124 Tanha et al J Immunol Methods 2002 263:97–109 Teutsch et al Eur J Hum Genet.2003 11(7):509-515 Thomassen et al Enzyme and Micro Tech 2002 30:273-278 Tsilingiri et al Cell Mol Gastroenterol Hepatol. 2017 3(2):174-182 Verma and Eckstein Annu Rev Biochem 1998 67:99–134 Verstraete et al Nat Commun. 2017 8:14937. doi: 10.1038 / ncomms14937 Vetter & Neurath Therap Adv Gastroenterol. 2017 10(10):773-790 Walsh Immunol Rev. 2012 250(1):303-316 Ward et al Nature 1989 341:544-546 Wells et al Gene 1985 34:315-323

Claims

1. A polypeptide capable of inhibiting the binding of IL-7 and / or L-TSLP to IL-7R, said polypeptide comprising an immunoglobulin chain variable domain that binds to IL-7Rα, said immunoglobulin chain variable domain comprising three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR1 comprises a sequence that shares 60% or more sequence identity with SEQ ID NO:1, CDR2 comprises a sequence that shares 60% or more sequence identity with SEQ ID NO:2, and CDR3 comprises a sequence that shares 60% or more sequence identity with SEQ ID NO:

3.

2. The polypeptide of claim 1, wherein the polypeptide is capable of inhibiting the binding of IL-7 to IL-7R.

3. The polypeptide of claim 1, wherein the polypeptide is capable of inhibiting the binding of L-TSLP to IL-7R.

4. The polypeptide according to any one of claims 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.

5. 5. The polypeptide of any one of claims 1 to 4, wherein CDR1 comprises or consists of a sequence that shares 80% or more sequence identity with SEQ ID NO: 1, CDR2 comprises or consists of a sequence that shares 80% or more sequence identity with SEQ ID NO: 2, and CDR3 comprises or consists of a sequence that shares 80% or more sequence identity with SEQ ID NO:

3.

6. 6. The polypeptide of claim 5, wherein CDR1 comprises or consists of SEQ ID NO: 82, CDR2 comprises or consists of SEQ ID NO: 83, and CDR3 comprises or consists of SEQ ID NO:

84.

7. 7. The polypeptide of claim 6, wherein CDR1 comprises or consists of SEQ ID NO:1 or SEQ ID NO:71, 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, and CDR3 comprises or consists of SEQ ID NO:3, SEQ ID NO:77, or SEQ ID NO:

78.

8. 8. The polypeptide of claim 7, wherein CDR1 comprises or consists of SEQ ID NO: 1, CDR2 comprises or consists of SEQ ID NO: 2, and CDR3 comprises or consists of SEQ ID NO:

3.

9. 9. The polypeptide of any one of claims 1-8, wherein the polypeptide comprises or consists of a sequence sharing 50% or more sequence identity with SEQ ID NO:8, such as sharing 55% or more sequence identity, for example sharing 60% or more sequence identity, such as sharing 65% or more sequence identity, for example sharing 70% or more sequence identity, for example sharing 75% or more sequence identity, such as sharing 80% or more sequence identity, for example sharing 85% or more sequence identity, such as sharing 90% or more sequence identity, for example sharing 95% or more sequence identity, such as sharing 96% or more sequence identity, for example sharing 97% or more sequence identity, such as sharing 98% or more sequence identity, for example sharing 99% or more sequence identity.

10. The polypeptide of claim 9, wherein the polypeptide comprises SEQ ID NO:

8.

11. The polypeptide of claim 10, wherein the polypeptide consists of SEQ ID NO:

8.

12. 12. The polypeptide of any one of claims 1 to 11, wherein the polypeptide binds to an epitope on IL-7Rα that includes at least one residue of IL-7Rα selected from the list consisting of Glu27, Ser31, Leu57, Val58, Glu59, Lys77, Lys78, Phe79, Leu80, Leu81, Ile82, Thr104, Lys137, Lys138, Tyr139, Lys141, His191, Tyr192, and Phe193.

13. 1. A polypeptide that binds to an epitope on IL-7Rα, wherein the epitope comprises at least one residue of IL-7Rα selected from the list consisting of Glu27, Ser31, Leu57, Val58, Glu59, Lys77, Lys78, Phe79, Leu80, Leu81, Ile82, Thr104, Lys137, Lys138, Tyr139, Lys141, His191, Tyr192, and Phe193.

14. The polypeptide of any one of claims 1 to 13, wherein the polypeptide is an antibody or an antibody fragment.

15. The polypeptide of any one of claims 1-14, wherein the immunoglobulin chain variable domain is VHH, VH, or VL.

16. The polypeptide of claim 15, wherein the immunoglobulin chain variable domain is a VHH or VH.

17. A construct comprising at least one polypeptide according to any one of claims 1 to 16 and at least one different polypeptide.

18. 18. The polypeptide or construct of any one of claims 1 to 17, wherein said polypeptide neutralises the binding of IL-7 to IL-7R with an EC50 of 2.00nM or less, such as 1.50nM or less, for example 1.00nM or less, for example 0.90nM or less, such as 0.80nM or less, for example 0.70nM or less, such as 0.65nM or less, for example 0.60nM or less, such as 0.55nM or less, for example 0.50nM or less, such as 0.45nM or less, for example 0.4nM or less, such as 0.35nM or less, for example 0.30nM or less.

19. The polypeptide is －７ M or less, for example, more preferably 10 －８ M or less, for example, 10 －９ M or less, for example, 10 －１０ 19. The polypeptide or construct of any one of claims 1 to 18, which binds to IL-7Rα with an equilibrium dissociation constant (Kd) of M or less.

20. 20. A polypeptide or construct according to any one of claims 1 to 19, which is substantially resistant to trypsin and chymotrypsin.

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

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

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

24. 24. The polypeptide, pharmaceutical composition or construct of claim 23, wherein the polypeptide, pharmaceutical composition or construct is for use in the treatment of an autoimmune disease and / or an inflammatory disease.

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

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

27. The polypeptide, pharmaceutical composition, construct, method or use according to any one of claims 24 to 26, wherein the autoimmune and / or inflammatory disease is Crohn's disease or ulcerative colitis.

28. The polypeptide, pharmaceutical composition, construct, method or use according to any one of claims 24 to 26, wherein the autoimmune disease and / or inflammatory disease is atopic dermatitis.

29. 29. The polypeptide, pharmaceutical composition, construct, method or use according to any one of claims 23 to 28, wherein the polypeptide, pharmaceutical composition or construct is for oral administration.

30. 29. The polypeptide, pharmaceutical composition, construct, method or use of any one of claims 23 to 28, wherein the polypeptide, pharmaceutical composition or construct is for use in topical administration.

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

32. 32. The polynucleotide of claim 31 , wherein the polynucleotide comprises or consists of SEQ ID NO:70.

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