Composition

Abstract

Among other aspects, compositions comprising a TNF-α binding polypeptide and an IL-7R binding polypeptide are provided.

Description

Field of the Invention

[0001] The present invention relates to compositions and constructs comprising TNF-α binding polypeptides and IL-7R binding polypeptides. The present invention also relates to nucleic acids encoding such constructs, methods for preparing such compositions and constructs, cDNAs and vectors comprising nucleic acids encoding such constructs, host cells expressing or capable of expressing such constructs, and uses of such compositions and constructs. Background of the Invention

[0003] Tumor necrosis factor-α is a homotrimeric proinflammatory cytokine associated with systemic inflammation, which exists in both soluble and membrane-bound forms. TNF-α is secreted primarily by monocytes and macrophages, but is also secreted by tumor cell lines as well as CD4+ and CD8+ peripheral blood T lymphocytes and some cultured T- and B-cell lines. TNF-α is associated with inflammatory diseases, autoimmune diseases, viral, bacterial and parasitic infections, malignancies and / or neurodegenerative diseases, and is a target for specific biological therapies for autoimmune / autoinflammatory diseases such as rheumatoid arthritis and Crohn's disease (CD).

[0004] Interleukin-7 (IL-7) is produced constitutively by non-hematopoietic stromal cells and epithelial cells, and is necessary for the development of T lymphocytes in the thymus and for the survival and homeostasis regulation of peripheral blood T cells. In the intestinal mucosa, IL-7 also regulates different populations of innate lymphoid cells in terms of phenotype and function, which are important for the initial initiation of immune responses to pathogenic microorganisms, and also regulates CD4+ lymphoid tissue induction (LTi) cells, which can promote lymphoid tissue organogenesis and some dendritic cell populations. The functional effects of IL-7 on T cells make IL-7 a key enhancer of protective immunity, autoimmunity and inflammation. The effect of IL-7 on different target cells is mediated by the heterodimer complex IL-7R (which includes IL-7Rα subunits (CD127) and common cytokine receptor γ chain (γc) (CD132). IL-7Rα is available not only in the form of cell membrane binding, but also in a soluble form (sIL-7Rα). In addition to the role of IL-7 / IL-7Rα in human T cell development and homeostasis, preclinical studies have implicated the IL-7 / IL-7Rα pathway in animal models of different autoimmune and inflammatory diseases.

[0005] TSLP is a cytokine that appears to be involved in regulating the inflammatory process at the body's mucosal surfaces. TSLP stimulates dendritic cells (DCs) and innate lymphoid cells (ILCs) to induce the secretion of Th2 cytokines (IL-4, IL-5, and IL-13) and promote the development of Th2-type inflammation. TSLP is now believed to be the cause of the onset of some allergic conditions including atopic dermatitis and rhinitis, and also promotes intestinal conditions, including eosinophilic esophagitis (EoE) and ulcerative colitis (UC). Paradoxically, TSLP is also reported to be important for maintaining immune homeostasis and mucosal protection in the gastrointestinal tract. Recently, the discovery that TSLP can be expressed as two different isoforms provides a biological explanation for the significantly different activities of this cytokine (Fornasa et al. 2015; Tsilingiri et al. 2017). Molecular studies have also shown that the TSLP gene can produce two coding RNAs regulated by two different promoter regions. One of the transcripts encodes a long isoform of TSLP of 159 aa (L-TSLP) (UNIPROT entry Q969D9, SEQ ID NO: 22), and the second transcript encodes a short form of TSLP (S-TSLP) comprising the C-terminal 63 aa of L-TSLP (UNIPROT entry Q969D9-2, SEQ ID NO: 23). L-TSLP acts on target cells via a receptor complex comprising a TSLP-specific receptor chain (TSLPR) and the IL-7Rα chain. Recently, structural studies have shown that the interaction of IL-7 and L-TSLP with the IL-7Rα chain of the TSLP-receptor complex involves a common IL-7Rα binding site (Verstraete et al. 2017). S-TSLP does not bind to TSLPR, and it cannot inhibit the binding of L-TSLP to this receptor. To the best of the authors' knowledge, no specific receptor for S-TSLP has been identified so far. Importantly, S-TSLP has been shown to be preferentially expressed by healthy skin and by epithelial and lamina propria cells in healthy intestinal mucosal tissue. S-TSLP has anti-inflammatory activity; in vitro S-TSLP inhibits the production of pro-inflammatory cytokines by monocyte-derived DCs and helps modulate CD103+ DCs to a tolerogenic phenotype.

[0006] Therefore, TNF-α, IL-7, and L-TSLP are all cytokines that regulate cell types and pathways involved in the development and maintenance of intestinal inflammation in IBD. Anti-TNF-α antibodies have changed the treatment of Crohn's disease and ulcerative colitis. However, approximately one-third of patients using anti-TNF-α drugs are initial non-responders. Among initial responders, subsequent loss of response may vary between 10% and 50% per year (secondary non-response). Patients who are initially non-responsive are unlikely to benefit from switching to a second anti-TNF-α drug; therefore, new effective therapies are needed to better address the unmet clinical needs of these patients. Currently, the efficacy of IL-7R blocking antibodies has not been tested in patients with intestinal inflammatory diseases. However, preclinical studies have shown that short-term systemic administration of IL-7R blocking antibodies may be an effective treatment in models of gastrointestinal inflammation. In murine IBD models, the primary mechanism of efficacy following treatment with IL-7R antagonists involves local depletion or functional inhibition of pathogenic T cells (IL-7R+ effector / memory T cells) that express moderate to high levels of IL-7Rα and are activated due to increased production of IL-7 in inflamed intestinal tissue. Inhibition of the IL-7 / IL-7R pathway represents a novel strategy to address proinflammatory T cells through a mechanism distinct from that involved in the action of TNF-α neutralizing antibodies. Therefore, compositions or constructs that successfully combine the effects of anti-TNF-α and anti-IL-7R antibodies have the potential to improve efficacy and / or have efficacy in a broader group of IBD patients.

[0007] WO2004041862, WO2006122786 and Coppieters et al. 2006 (incorporated herein in their entirety by reference) disclose single domain antibodies against TNF-α and related aspects. WO2013056984, WO2015189302, WO2011094259 and WO2011104687 (incorporated herein in their entirety by reference) disclose antibodies against IL-7R and related aspects.

[0008] Compared with the materials of the prior art, the compositions or constructs of the present invention may have one or more of the following advantages in at least some embodiments. These advantages may be achieved by each component polypeptide in the compositions of the present invention itself, or alternatively the combination of polypeptides in the compositions of the present invention may result in the addition or even synergistic effect of one or more of the following advantages.

[0009] (a) increasing affinity and / or avidity for TNF-α and / or IL-7R;

[0010] (b) increased neutralization capacity for TNF-α and / or IL-7R;

[0011] (c) increase the inhibition of phosphorylation of signal transduction proteins;

[0012] (d) increased inhibition of cytokine production;

[0013] (e) reduced immunogenicity, e.g., when administered to mice, cynomolgus monkeys or humans;

[0014] (f) increased stability in the presence of proteases, e.g., (a) proteases present in the small and / or large intestine and / or IBD inflammatory proteases such as trypsin, chymotrypsin, enterokinase, MMP3, MMP10, MMP12, other MMPs and cathepsins and / or (b) proteases such as cell membrane attached proteases, secreted proteases and proteases released from cell lysis of intestinal commensal microflora and / or pathogenic bacteria present in the small and / or large intestine;

[0015] (g) increased stability to protease degradation during production (e.g., resistance to yeast proteases);

[0016] (h) increased suitability for oral administration;

[0017] (i) Increased suitability for topical delivery to the intestinal tract and lamina propria following oral administration;

[0018] (j) increased suitability for expression in heterologous hosts such as bacteria (such as Escherichia coli) or yeast (such as Saccharomyces cerevisiae or Pichia pastoris);

[0019] (k) suitability and improved properties for use in medicine;

[0020] (l) suitability and improved properties for use in functional foods;

[0021] (m) improved tissue penetration, such as penetration of inflamed colonic mucosal epithelium and submucosal tissue to reach the submucosal lamina propria;

[0022] (n) Reduced immunogenicity in humans, for example due to increased sequence similarity to human immunoglobulins:

[0023] (o) more effectively preventing or treating autoimmune and / or inflammatory diseases, including ameliorating symptoms thereof, particularly including inflammatory bowel disease and / or mucositis, particularly when administered orally; and

[0024] (p) Binding to novel epitopes. Summary of the invention

[0025] The present inventors have provided unexpectedly effective compositions and constructs comprising a TNF-α binding polypeptide and an IL-7R binding polypeptide. In particular, it has been found that providing a TNF-α binding polypeptide and an IL-7R binding polypeptide in a single composition may be more effective than providing each binding polypeptide separately. In some embodiments, the combination of these polypeptides in the compositions of the invention may have an additive effect, and in other embodiments, the combination of these polypeptides in the compositions of the invention may have a synergistic effect.

[0026] Based on the findings disclosed herein using intestinal mucosal tissue cultured in vitro from patients with inflammatory bowel disease (Examples 4 and 5), it can be expected that these compositions have special utility in preventing or treating autoimmune diseases and / or inflammatory diseases. More specifically, the findings disclosed herein show that these compositions have special utility in preventing or treating inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis, or colitis induced by checkpoint inhibitors) or preventing or treating mucositis or esophagitis, especially when administered orally.

[0027] It is expected that the same benefits can be obtained by using a TNF-α binding polypeptide together with an IL-7R binding polypeptide in the treatment or prevention of a disease, wherein the polypeptides are not combined in the same composition. Similarly, it is expected that the same benefits can be obtained by using an IL-7R binding polypeptide together with a TNF-α binding polypeptide in the treatment or prevention of a disease, wherein the polypeptides are not combined in the same composition.

[0028] The present invention provides a composition comprising a TNF-α binding polypeptide and an IL-7R binding polypeptide.

[0029] Also provided are constructs comprising a TNF-α binding polypeptide and an IL-7R binding polypeptide, and polynucleotides encoding the constructs.

[0030] Also provided are TNF-α binding polypeptides and IL-7R binding polypeptides for treating or preventing autoimmune diseases and / or inflammatory diseases; and IL-7R binding polypeptides and TNF-α binding polypeptides for treating or preventing autoimmune diseases and / or inflammatory diseases.

[0031] Specific TNF-α binding polypeptides and some of the data disclosed herein are also disclosed in PCT Application No. WO / 2016 / 156465 (incorporated herein by reference in its entirety, particularly with respect to that application relating to TNF-α binding polypeptide ID-38F). BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 -% survival of V7R-2E9 and ID-A40U in gastrointestinal extracts

[0033] Figure 2 - Survival % of ID-A62U and ID-A41U in human fecal supernatant

[0034] Figure 3 - Effect of combined ID-38F and ID-A62U on phosphorylation (phosphoprotein p38α to Lck) in UC biopsies

[0035] Figure 4 - Effect of combined ID-38F and ID-A62U on phosphorylation (phosphoproteins STAT5a to hsp60) in UC biopsies

[0036] Figure 5 - Total phosphorylation intensity values ​​of UC2747, UC2748, UC2749 and UC2750 measured on biopsies from patients after treatment with individual and combination agents

[0037] Figure 6 - Mean cytokine production measured in biopsies from all patients following treatment with individual and combination agents (IL-1β and IL-6)

[0038] Figure 7 - Mean cytokine production measured in biopsies from all patients following treatment with individual and combined agents (IL-8 and TNF-α)

[0039] Figure 8 - Mean cytokine production measured in biopsies from all patients following treatment with individual and combination agents (IL-17A and IL-10)

[0040] Fig. 9 - SDS PAGE analysis of ID-38F and ID-A62U released from the double-headed construct FU3K before and after trypsin treatment

[0041] Fig.10 - FU3K and ID-A62U potency by using IL-7 / IL-7R ELISA before and after trypsin treatment

[0042] Fig.11 -FU3K and ID-38F potency by competition ELISA using biotinylated Humira before and after trypsin treatment

[0043] Fig.12 - Survival % of (a) ID-A62U, (b) ID-38F, (c) ID-A62U and ID-38F after release from FU3K and (d) ID-A41U in human fecal supernatant

[0044] Sequence Description

[0045] SEQ ID NO: 1-CDR1 polypeptide sequence of ID-38F

[0046] SEQ ID NO: 2-CDR2 polypeptide sequence of ID-38F

[0047] SEQ ID NO: 3-CDR3 polypeptide sequence of ID-38F

[0048] SEQ ID NO: FR1 polypeptide sequence of 4-ID-38F

[0049] SEQ ID NO: FR2 polypeptide sequence of 5-ID-38F

[0050] SEQ ID NO: FR3 polypeptide sequence of 6-ID-38F

[0051] SEQ ID NO: FR4 polypeptide sequence of 7-ID-38F

[0052] SEQ ID NO: 8-ID-38F polypeptide sequence

[0053] SEQ ID NO: CDR1 polypeptide sequence of 9-ID-A62U

[0054] SEQ ID NO: 1CDR2 polypeptide sequence of O-ID-A62U

[0055] SEQ ID NO: CDR3 polypeptide sequence of 11-ID-A62U

[0056] SEQ ID NO: FR1 polypeptide sequence of 12-ID-A62U

[0057] SEQ ID NO: FR2 polypeptide sequence of 13-ID-A62U

[0058] SEQ ID NO: 14-ID-A62U FR3 polypeptide sequence

[0059] SEQ ID NO: FR4 polypeptide sequence of 15-ID-A62U

[0060] SEQ ID NO: 16-ID-A62U polypeptide sequence

[0061] SEQ ID NO: 17 - Polynucleotide sequence encoding ID-38F (including two stop codons 1

[0062] SEQ ID NO: 18—Polynucleotide sequence encoding ID-A62U (including two stop codons)

[0063] SEQ ID NO: 19 - polypeptide sequence of enterokinase cleavage site

[0064] SEQ ID NO:20—Polypeptide sequence comprising an enterokinase cleavage site

[0065] SEQ ID NO: 21 - Polypeptide sequence of the specific labile linker used in FU3K

[0066] SEQ ID NO: 22-L-TSLP polypeptide sequence

[0067] SEQ ID NO: 23-S-TSLP polypeptide sequence

[0068] SEQ ID NO: 24-ID-A40U polypeptide sequence

[0069] SEQ ID NO: 25-V7R-2E9 polypeptide sequence

[0070] SEQ ID NO: 26 - FU3K double-headed polypeptide sequence

[0071] SEQ ID NO: 27 - Polynucleotide sequence encoding FU3K double head

[0072] SEQ ID NO:28 - Polypeptide sequence of CDR1 with optional conservative substitution at residue 1 of SEQ ID NO:9

[0073] SEQ ID NO:29 - Polypeptide sequence of CDR2 with optional conservative substitutions at residues 2, 3, 7, 12 and 16 of SEQ ID NO:10

[0074] SEQ ID NO:30 - Polypeptide sequence of CDR3 with optional conservative substitutions at residues 3 and 9 of SEQ ID NO:11

[0075] SEQ ID NO:31 - Polypeptide sequence in the form of a non-protease-labile peptide linker

[0076] SEQ ID NO: 32 - Polypeptide sequence in the form of a trypsin-labile peptide linker

[0077] SEQ ID NO: 33 - Polypeptide sequence in the form of a trypsin-labile peptide linker

[0078] SEQ ID NO: 34 - Polypeptide sequence in the form of a peptide linker labile to a second trypsin

[0079] SEQ ID NO:35 - Polypeptide sequence of an exemplary trypsin-labile peptide linker

[0080] SEQ ID NO:36 - Polypeptide sequence of an exemplary trypsin-labile peptide linker

[0081] SEQ ID NO: 37 - Polypeptide sequence of an exemplary trypsin-labile peptide linker

[0082] SEQ ID NO: 38 - Polypeptide sequence of an exemplary trypsin-labile peptide linker

[0083] SEQ ID NO: 39 - Polypeptide sequence of an exemplary trypsin-labile peptide linker

[0084] SEQ ID NO:40 - Polypeptide sequence of an exemplary trypsin-labile peptide linker

[0085] SEQ ID NO:41 - Polypeptide sequence of an exemplary trypsin-labile peptide linker

[0086] SEQ ID NO: 42 - Polypeptide sequence in the form of a third trypsin-labile peptide linker

[0087] SEQ ID NO:43 - Polypeptide sequence in the form of a chymotrypsin-labile peptide linker DETAILED DESCRIPTION

[0088] Polypeptides such as antibodies and antibody fragments comprising immunoglobulin chain variable domains (ICVD) such as VH and VHH

[0089] A polypeptide is an organic polymer composed of multiple amino acid residues bonded together in a chain. As used herein, 'polypeptide' is used interchangeably with 'protein' and 'peptide'. on Rate and / or k off When a polypeptide contains one or more stretches of amino acid residues that bind to an epitope on a target (eg, a binding rate, as further described herein), the polypeptide is referred to as a binding polypeptide.

[0090] Binding polypeptides include polypeptides such as DARPins (Binz et al. 2003), Affimers TM (Johnson et al. 2012), Fynomers TM (Grabulovski et al. 2007), Centyrins (Goldberg et al. 2016), Affitins (e.g. Krehenbrink et al. 2008), cyclic peptides, antibodies and antibody fragments. Binding polypeptides also include polypeptides such as: Affibodies (Nygren 2008), Affilins (Ebersbach et al. 2007), Alphabodies (Desmet et al. 2014), Anticalins (Skerra et al. 2008), Avimers (Silverman et al. 2005), Kunitz domain peptides (Nixon and Wood 2006), Monobodies (Koide and Koide 2007), nanoCLAMPs (Suderman et al. 20171, Adnectins (Lipovsek 2011) and bicyclic peptides.

[0091] Conventional antibodies or immunoglobulins (Ig) are proteins comprising four polypeptide chains: two heavy (H) chains and two light (L) chains. Each chain is divided into a constant region and a variable domain. The heavy chain variable domain is abbreviated as VHC in this article, and the light (L) chain variable domain is abbreviated as VLC in this article. These domains, domains associated therewith, and domains derived therefrom are referred to herein as immunoglobulin chain variable domains ('ICVD').

[0092] The VHC and VLC domains can be further subdivided into regions of high variability, called "complementarity determining regions" ("CDRs"), interspersed with more conservative regions, called "framework regions" ("FRs"). Framework regions and complementary determining regions have been precisely defined (Kabat et al. 1991 Sequences of Proteins of Immunological Interest, 5th Edition, US Department of Health and Human Services, NIH Publication Number 91-3242, incorporated herein by reference in its entirety). In conventional antibodies, each VHC and VLC consists of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Conventional antibody tetramers of two immunoglobulin heavy chains and two immunoglobulin light chains are formed with immunoglobulin heavy chains and immunoglobulin light chains interconnected by, for example, disulfide bonds, and the heavy chains are similarly connected. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. The light chain constant region consists of one domain, CL.

[0093] The variable domains of the heavy chain and the variable domains of the light chain are binding domains that interact with the antigen. The constant region of the antibody usually mediates the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) or the first component (C1q) of the classical complement system. The term antibody includes immunoglobulins of the IgA, IgG, IgE, IgD, IgM types (and subtypes thereof), wherein the light chain of the immunoglobulin may be of the κ type or the λ type. The overall structure of the immunoglobulin-γ (IgG) antibody assembled by two identical heavy (H) chains and two identical light (L) chain polypeptides is well defined in mammals and is highly conserved (Padlan 1994 Mol Immunol 31: 169-217).

[0094] An exception to conventional antibody structure is present in the serum of Camelidae. In addition to conventional antibodies, these sera have special IgG antibodies. These IgG antibodies (referred to as heavy chain antibodies (HCAb)) lack L chain polypeptides and lack the first constant domain (CH1). In its N-terminal region, the H chain of the homodimeric protein contains a dedicated immunoglobulin chain variable domain (referred to as VHH), which is used to associate with its cognate antigen (Muyldermans et al. 2013, Hamers-Casterman et al. 1993, Muyldermans et al. 1994, incorporated herein by reference in their entirety).

[0095] As used herein, an antibody fragment refers to a portion of an antibody that specifically binds to a target (e.g., a molecule in which one or more immunoglobulin chains are not full-length but that specifically binds to a target). Examples of binding fragments encompassed within the term antibody fragment (or "antigen binding fragment") include:

[0096] (i) Fab fragment (monovalent fragment consisting of VLC, VHC, CL and CH1 domains);

[0097] (ii) F(ab')2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region);

[0098] (iii) Fd fragment (consisting of VHC and CH1 domains);

[0099] (iv) Fv fragment (consisting of the VLC and VHC domains of a single arm of an antibody);

[0100] (v) scFv fragments (composed of the VLC and VHC domains joined using recombinant methods by a synthetic linker that enables the domains to be made into a single protein chain in which the VLC and VHC regions pair to form a monovalent molecule);

[0101] (vi) VH (immunoglobulin chain variable domain consisting of the VHC domain from a conventional 4-chain immunoglobulin (Ward et al. 1989);

[0102] (vii) VL (immunoglobulin chain variable domain consisting of the VLC domain);

[0103] (viii) V-NAR (immunoglobulin chain variable domain consisting of the VHC domain from the chondrichthyes IgNAR (Roux et al. 1998 and Griffiths et al. 2013, incorporated herein by reference in their entirety)

[0104] (ix) VHH.

[0105] The total number of amino acid residues in VHH or VH may be in the region of 110-140, more suitably 110-120.

[0106] The examples provided herein relate to compositions comprising immunoglobulin chain variable domains that bind to TNF-α and immunoglobulin chain variable domains that bind to IL-7R. However, the principles of the invention disclosed herein are equally applicable to any composition comprising a polypeptide that binds to TNF-α and a polypeptide that binds to IL-7R (such as antibodies and antibody fragments). For example, immunoglobulin chain variable domains can be incorporated into polypeptides such as full-length antibodies. This method is demonstrated by McCoy et al. 2014, which provides anti-HIV VHH constructed as a fusion with a human Fc region (comprising hinge, CH2 and CH3 domains), expressed as a dimer construct.

[0107] Suitably, the polypeptide of the present invention comprises an immunoglobulin chain variable domain. More suitably, the polypeptide of the present invention consists of an immunoglobulin chain variable domain. Suitably, the polypeptide of the present invention is an antibody or an antibody fragment. More suitably, the polypeptide of the present invention is an antibody fragment. Suitably, the antibody fragment is a VHH, VH, VL, V-NAR, scFv, Fab fragment or F(ab')2 fragment. Suitably, the antibody fragment is an immunoglobulin chain variable domain (e.g., VHH, VH or VL). Suitably, the antibody fragment is an immunoglobulin heavy chain variable domain. More suitably, the antibody fragment is a VHH or VH, most suitably a VHH.

[0108] Specificity, affinity, avidity, potency, inhibition, and neutralization

[0109] Specificity refers to the number of different targets (eg, antigens or antigenic determinants) to which a particular binding polypeptide can bind. The specificity of a binding polypeptide is the ability of a binding polypeptide to recognize specific targets as unique molecular entities and to distinguish them from one another.

[0110] Affinity, represented by the equilibrium constant (Kd) for the dissociation of the target from the binding polypeptide, is a measure of the strength of binding between the target and the binding site on the binding polypeptide: the smaller the value of Kd, the stronger the binding strength between the target and the binding polypeptide (alternatively, affinity can also be expressed as an affinity constant (Ka), which is 1 / Kd). Affinity can be determined by known methods depending on the target specific antigen. Suitably, affinity is measured using a dynamically switchable biosurface (e.g., See Knezevic et al. 2012) or determined by surface plasmon resonance.

[0111] Avidity is a measure of the strength of binding between a binding polypeptide and a relevant target. Avidity is related to both the affinity between the target and its binding site on the binding polypeptide and the number of relevant binding sites present on the binding polypeptide.

[0112] Any value less than 10 -6 The Kd value of M is considered binding. Specific binding of a binding polypeptide to a target (e.g., an antigen or antigenic determinant) can be determined in any suitable known manner, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competition assays, and different variations thereof known in the art.

[0113] Efficacy is a measure of the activity of a therapeutic agent (e.g., a binding polypeptide) expressed in the amount required to produce an effect of a given intensity. A high efficacy agent causes a greater response at low concentrations compared to a lower efficacy agent that causes a smaller response at low concentrations. Efficacy varies with affinity and effectiveness. Effectiveness refers to the ability of a therapeutic agent to produce a biological response when bound to a target and the quantitative magnitude of this response. The term half-maximal effective concentration (EC50) refers to the concentration of a therapeutic agent that causes a response halfway between the baseline and the maximum after a specified exposure time. The therapeutic agent may cause inhibition (which may be specifically referred to as half-maximal inhibitory concentration, "IC50") or stimulation. EC50 and IC50 are commonly used and are used as a measure of efficacy herein. For TNF-α binding polypeptides and IL-7R binding polypeptides, EC50 and IC50 are used interchangeably herein because both binding polypeptides result in inhibition of the target.

[0114] Specific assays suitable for determining the potency of TNF-α binding polypeptides and IL-7R binding polypeptides are described in detail below under the headings "TNF-α binding polypeptides" and "IL-7R binding polypeptides," respectively.

[0115] Peptide and polynucleotide sequences

[0116] For the purpose of comparing two closely related polypeptide sequences, the "% sequence identity" between a first polypeptide sequence and a second polypeptide sequence can be calculated using NCBI BLAST v2.0 (BLASTP) using standard settings for polypeptide sequences. For the purpose of comparing two closely related polynucleotide sequences, the "% sequence identity" between a first nucleotide sequence and a second nucleotide sequence can be calculated using NCBI BLAST v2.0 (BLASTN) using standard settings for nucleotide sequences.

[0117] A polypeptide or polynucleotide sequence is said to be identical or equivalent if it shares 100% sequence identity with the other polypeptide or polynucleotide sequence over its entire length. The residues in the sequence are numbered from left to right, i.e., from the N-terminus to the C-terminus of a polypeptide; from the 5' end to the 3' end of a polynucleotide.

[0118] "Difference" between sequences refers to the insertion, deletion or replacement of a single amino acid residue in a position of the second sequence compared to the first sequence. Two polypeptide sequences may contain one, two or more such amino acid differences. An insertion, deletion or replacement in a second sequence that is otherwise identical to the first sequence (100% sequence identity) causes a reduction in sequence identity %. For example, if the length of the same sequence is 9 amino acid residues, one replacement in the second sequence produces 88.9% sequence identity. If the length of the same sequence is 17 amino acid residues, two replacements in the second sequence produce 88.2% sequence identity. If the length of the same sequence is 7 amino acid residues, three replacements in the second sequence produce 57.1% sequence identity. If the length of the first polypeptide sequence and the second polypeptide sequence is 9 amino acid residues and shares 6 identical residues, the first polypeptide sequence and the second polypeptide sequence share an identity greater than 66% (the first polypeptide sequence and the second polypeptide sequence share 66.7% identity). If the first polypeptide sequence and the second polypeptide sequence are 17 amino acid residues in length and share 16 identical residues, the first polypeptide sequence and the second polypeptide sequence share greater than 94% identity (the first polypeptide sequence and the second polypeptide sequence share 94.1% identity). If the first polypeptide sequence and the second polypeptide sequence are 7 amino acid residues in length and share 3 identical residues, the first polypeptide sequence and the second polypeptide sequence share greater than 42% identity (the first polypeptide sequence and the second polypeptide sequence share 42.9% identity).

[0119] Alternatively, for the purpose of comparing a first reference polypeptide sequence to a second comparison polypeptide sequence, the number of additions, substitutions and / or deletions made to the first sequence to produce the second sequence can be determined. An addition is the addition of an amino acid residue to the sequence of the first polypeptide (including additions at either end of the first polypeptide). A substitution is the replacement of an amino acid residue in the sequence of the first polypeptide with a different amino acid residue. A deletion is the deletion of an amino acid residue from the sequence of the first polypeptide (including deletions at either end of the first polypeptide).

[0120] A "conservative" amino acid substitution is one in which an amino acid residue is replaced by another amino acid residue of similar chemical structure and is expected to have little effect on the function, activity or other biological properties of the polypeptide. Such conservative substitutions are suitably substitutions in which one amino acid within the following group is replaced by another amino acid residue from the same group:

[0121]

[0122] Suitably, the hydrophobic amino acid residue is a non-polar amino acid. More suitably, the hydrophobic amino acid residue is selected from V, I, L, M, F, W or C.

[0123] As used herein, the numbering of polypeptide sequences and the definition of CDRs and FRs are as defined according to the Kabat system (Kabat et al. 1991 Sequences of Proteins of Immunological Interest, 5th Edition, US Department of Health and Human Services. NIH Publication Number 91-3242, incorporated herein by reference in its entirety). "Corresponding" amino acid residues between a first polypeptide sequence and a second polypeptide sequence are amino acid residues in the first sequence that share the same position as the amino acid residues in the second sequence according to the Kabat system, while the identity of the amino acid residues in the second sequence and the amino acid residues in the first sequence may be different. Suitably, if the framework and CDR are the same length according to the Kabat definition, the corresponding residues will share the same numbering (and letter). Alignment can be achieved manually or by using known computer algorithms such as NCBI BLAST v2.0 (BLASTP or BLASTN) for sequence alignment using standard settings.

[0124] The Kabat numbering system is applied below to specific TNF-α binding polypeptides and IL-7R binding polypeptides used in the Examples provided herein.

[0125]

[0126]

[0127]

[0128]

[0129] The numbers with the prefix "H" in the above ID-38F figure are Kabat numbers, and the numbers above the amino acid sequence are consecutive amino acid numbers from the N-terminus to the C-terminus. The residues of each CDR or FR can also be numbered from the N-terminus to the C-terminus of the CDR or FR.

[0130] ID-38F is encoded by the polynucleotide sequence of SEQ ID NO: 17. ID-A62U is encoded by the polynucleotide sequence of SEQ ID NO: 18. Suitably, the polynucleotides used in the present invention are isolated. An "isolated" polynucleotide is a polynucleotide removed from its original environment. For example, if a naturally occurring polynucleotide is separated from some or all of the coexisting materials in the natural system, the polynucleotide is isolated. If, for example, a polynucleotide is cloned into a vector that is not part of its natural environment or if it is contained within a cDNA, the polynucleotide is considered isolated.

[0131] TNF-α Binding Peptide

[0132] Features

[0133] Anti-TNF-α polypeptides, polypeptides that interact with TNF-α, or polypeptides that antagonize TNF-α are all effectively TNF-α binding polypeptides. TNF-α binding polypeptides can bind to linear or conformational epitopes on TNF-α. Suitably, TNF-α binding polypeptides bind to human TNF-α.

[0134] Suitably, the TNF-α binding polypeptide is present in an amount of 10 -6 M or smaller, more preferably 10 -7 M or smaller, more preferably 10 -8 M or smaller, more preferably 10 -9 It binds to TNF-α with an equilibrium dissociation constant (Kd) of M or less.

[0135] Suitably, the affinity of the TNF-α binding polypeptide is determined by using a dynamically switchable biosurface (e.g. See Knezevic et al. 2012) to determine.

[0136] In one embodiment, the affinity of the TNF-α binding polypeptide is established at 25°C by fusing the TNF-α binding polypeptide at its C-terminus to a single-stranded DNA and coupling the DNA-polypeptide fusion to a gold electrode (coated in a fluorescently labeled complementary strand of the single-stranded DNA), then exposing the chip-bound TNF-α binding polypeptide to a 10 kHz current and five different concentrations of human TNF-α ranging from 50 pM to 4.5 nM for 600 seconds and observing the dissociation by time-resolved fluorescence for 8 hours.

[0137] Suitably, the TNF-α binding polypeptide neutralizes TNF-α. TNF-α neutralizing polypeptide is a polypeptide that protects cells from the effects of TNF-α by, for example, inhibiting the biological effects of TNF-α. Typically, anti-TNF-α therapeutic antibody products have used the L929 mouse cell line with a cell death endpoint as a neutralization assay (Humphreys and Wilson 1999). L929 assays can be performed to determine the ability of the TNF-α binding polypeptide to neutralize the cytotoxic effects of TNF-α by determining the half-maximal effective concentration (EC50) of the TNF-α binding polypeptide. A detailed protocol for L929 assays is provided below.

[0138] L929 assay

[0139] L929 cells (10000 cells / well) were cultured for 24 hours with dilutions of purified polypeptides in the presence of soluble TNF-α (500 pg / ml) and actinomycin (0.75 ug / mL). At the end of the experiment, cytotoxicity was determined by using resazurin. The inhibition of soluble human TNF-induced cytotoxicity of mouse L929 cells was tested to determine the TNF-α neutralizing activity of each polypeptide bound to human TNF-α.

[0140] Material

[0141] -L929 cells (10,000 cells / well)

[0142] -Sterile polypropylene 96-well plates

[0143] -DMEM

[0144] -Human TNF-α concentration: 500pg / ml

[0145] -Actinomycin D concentration: 0.75ug / mL

[0146] -Purified test polypeptide

[0147] - Dilution range of purified polypeptide (for example): 300 nM-5 pM (1:3 dilution)

[0148] - Human TNF-α dose response curve: 10ng / mL-0.5pg / mL

[0149] Incubation time: 22h

[0150] -Resazurin Cell Viability Reagent

[0151] method

[0152] 10000 cells / well in 100ul were seeded in DMEM complete medium in 96-well microplates on day 0 and stored overnight at 37 degrees Celsius and 5% CO2. On day 1, a serial dilution of 1:3 (in DMEM+Act.D+TNF) was set up for each purified variable domain starting from a top concentration of 300nM (volume sufficient for three replicates for each point).

[0153] The following controls were added to the plate:

[0154] 1. DMEM complete + 0.75ug / mL actinomycin D

[0155] 2. DMEM complete + 0.75ug / mL actinomycin D + 0.5ng / mL h-TNF-α

[0156] 3. DMEM complete + 0.01% Triton (only in plates containing TNF-α dose response)

[0157] 4. DMEM complete (only in plates containing TNF-α dose response)

[0158] The medium was removed from each well of the microplate and the cells were incubated with 100ul of each peptide dilution or 100ul of different controls. After incubation for 22 hours at 37 degrees Celsius and 5% CO2, 10ul of Resazurin was added to each well and the cells were incubated at 37 degrees Celsius for 2 hours. Subsequently, 50ul of 3% SDS was added to each well. The plate was then read using a fluorescent plate reader Ex544nm / Em590nm.

[0159] Suitably, the TNF-α binding polypeptide neutralizes human TNF-α cytotoxicity in the L929 assay with an EC50 of 100 nM or less, such as 50 nM or less, such as 10 nM or less, such as 5 mM or less, such as 1 nM or less, such as 0.9 nM or less, such as 0.8 nM or less, such as 0.7 nM or less, such as 0.6 nM or less, such as 0.5 nM or less, such as 0.4 nM or less, such as 0.3 nM or less, such as 0.2 nM or less, such as 0.15 nM or less.

[0160] The neutralizing ability of the TNF-α binding polypeptide can also be determined by ELISA. Suitably, the TNF-α binding polypeptide inhibits the binding of human TNF-α to TNFR1 in an ELISA assay with an EC50 of 30 nM or less, more suitably 10 nM or less, more suitably 3 nM or less, more suitably 1 nM or less, more suitably 0.6 nM or less, more suitably 0.5 nM or less, more suitably 0.4 nM or less.

[0161] Alternatively or additionally, the TNF-α binding polypeptide inhibits the binding of human TNF-α to TNFR2 in an ELISA assay with an EC50 of 2 nM or less, more suitably 1 nM or less, more suitably 0.9 nM or less, more suitably 0.8 nM or less, more suitably 0.7 nM or less, more suitably 0.6 nM or less, more suitably 0.5 nM or less, more suitably 0.4 nM or less.

[0162] Suitably, the ELISA is performed as described in Example 2 of WO2018 / 060453.

[0163] In one embodiment, the affinity of the TNF-α binding polypeptide is determined by direct coating on a Biacore (or equivalent) sensor plate, wherein the polypeptide flows through the plate to detect binding. Suitably, Biacore T200 plates are used at 25° C. at 30 ul / min in HBS-EP+ (GE Healthcare) running buffer.

[0164] Structural characteristics

[0165] Suitably, the TNF-α binding polypeptide is a polypeptide comprising an antibody fragment. The polypeptide may be an antibody. Suitably, the antibody fragment is selected from: V-NAR, scFv, Fab fragment, F(ab')2 fragment or immunoglobulin chain variable domain, such as VL, VHH and VH. More suitably, the antibody fragment is an immunoglobulin chain variable domain, more suitably VHH or VH, most suitably VHH.

[0166] Suitably, the TNF-α binding polypeptide comprises three CDRs (CDR1, CDR2 and CDR3).

[0167] Suitably, the CDR1 of the TNF-α binding polypeptide comprises or more suitably consists of a sequence having 20% ​​or greater, more suitably 40% or greater, more suitably 60% or greater, more suitably 80% or greater sequence identity to SEQ ID NO:1.

[0168] Alternatively, the CDR1 of the TNF-α binding polypeptide comprises or more suitably consists of a sequence having no more than 3, more suitably no more than 2, more suitably no more than 1 additions, substitutions and / or deletions compared to SEQ ID NO:1.

[0169] Suitably, any residues in CDR1 of the TNF-alpha binding polypeptide which differ from their corresponding residues in SEQ ID NO: 1 are conservative substitutions relative to their corresponding residues. Suitably, CDR1 comprises or more suitably consists of SEQ ID NO: 1.

[0170] Suitably, the CDR2 of the TNF-α binding polypeptide comprises or more suitably consists of a sequence having 20% ​​or greater, more suitably 30% or greater, more suitably 40% or greater, more suitably 50% or greater, more suitably 55% or greater, more suitably 60% or greater, more suitably 70% or greater, more suitably 75% or greater, more suitably 80% or greater, more suitably 85% or greater, more suitably 90% or greater sequence identity to SEQ ID NO:2.

[0171] Alternatively, the CDR2 of the TNF-α binding polypeptide comprises or more suitably consists of a sequence having no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition, substitution and / or deletion compared to SEQ ID NO: 2.

[0172] Suitably, any residue of the CDR2 of the TNF-α binding polypeptide that differs from its corresponding residue in SEQ ID NO: 2 is a conservative substitution relative to its corresponding residue. Suitably, the residue of the CDR2 of the TNF-α binding polypeptide corresponding to residue number 10 of SEQ ID NO: 2 is R, H, D, E, N, Q, S, T, Y, G, A, V, L, W, P, M, C, F or I (most suitably H). Suitably, the CDR2 of the TNF-α binding polypeptide comprises or more suitably consists of SEQ ID NO: 2.

[0173] Suitably, the CDR3 of the TNF-α binding polypeptide comprises, or more suitably consists of, a sequence having 30%, more suitably 50%, more suitably 60%, more suitably 80% or greater sequence identity to SEQ ID NO:3.

[0174] Alternatively, the CDR3 of the TNF-α binding polypeptide comprises or more suitably consists of a sequence having no more than 3, more suitably no more than 2, more suitably no more than 1 additions, substitutions and / or deletions compared to SEQ ID NO:3.

[0175] Suitably, any residues in the CDR3 of the TNF-α binding polypeptide that differ from their corresponding residues in SEQ ID NO: 3 are conservative substitutions relative to their corresponding residues. Suitably, the residue of the CDR3 of the TNF-α binding polypeptide corresponding to residue number 3 of SEQ ID NO: 3 is R, H, D, E, N, O, S, T, Y, G, A, V, L, W, P, M, C, F or I; or suitably is R, H, D, E, N, O, S, T, Y, G, V, L, W, P, M, C, F or I (most suitably H). Suitably, the residue of the CDR3 of the TNF-α binding polypeptide corresponding to residue number 3 of SEQ ID NO: 3 is H and any other residues in the CDR3 of the TNF-α binding polypeptide that differ from their corresponding residues in SEQ ID NO: 3 are conservative substitutions relative to their corresponding residues. Suitably, the CDR3 of the TNF-α binding polypeptide comprises or more suitably consists of SEQ ID NO: 3.

[0176] Alternatively, the CDR3 of the TNF-α binding polypeptide comprises or more suitably consists of a sequence having 30%, such as 50%, such as 60%, such as 80% or greater sequence identity to SEQ ID NO: 3, and residue number 3 of CDR3 is R, D, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y or V (suitably H or a conservative substitution of H; more suitably H). Alternatively, residue number 3 of CDR3 is H or a conservative substitution of H (most suitably H), and any other residues of CDR3 that differ from their corresponding residues in SEQ ID NO: 3 are conservative substitutions.

[0177] Suitably, residue 1 of CDR1 of the TNF-α binding polypeptide is S, V or N; residues 2 to 4 are HWM and residue 5 is Y or C. Suitably, residues 1 to 9 of CDR2 of the TNF-α binding polypeptide are EINTNGLIT; residue 10 is H, K, S or N; residue 11 is Y, residue 12 is G, V, I or A; residue 13 is D; residue 14 is S or F; residue 15 is V or T; residue 16 is H, K, R or G and residue 17 is G. Suitably, residue 1 of CDR3 of the TNF-α binding polypeptide is N; residue 2 is Q or E; residue 3 is H, K, M or R and residues 4 to 6 are GLN.

[0178] Suitably, the TNF-α binding polypeptide comprises three CDRs (CDR1, CDR2 and CDR3) and four framework regions (FR1-FR4).

[0179] Suitably, FR1 of the TNF-α binding polypeptide comprises, or more suitably consists of, a sequence that shares 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.

[0180] Suitably, any residues in FR1 of the TNF-α binding polypeptide that differ from their corresponding residues in SEQ ID NO: 4 are conservative substitutions relative to their corresponding residues. Suitably, the residue of FR1 of the TNF-α binding polypeptide corresponding to residue number 1 of SEQ ID NO: 4 is G, A, V, L, I, F, P, S, T, Y, C, M, K, R, H, W, D, E or N (more suitably D or E, most suitably D). Suitably, the residue of FR1 of the TNF-α binding polypeptide corresponding to residue number 5 of SEQ ID NO: 4 is G, A, V, L, I, F, P, S, T, Y, C, M, K, R, H, W, D, E or N (suitably V). Suitably, the residue of FR1 of the TNF-α binding polypeptide corresponding to residue numbers 1 to 5 of SEQ ID NO: 4 is DVQLV. Suitably, the residues of FR1 of the TNF-α binding polypeptide corresponding to residue number 20 and / or 24 of SEQ ID NO: 4 are hydrophobic amino acids (most suitably L or A, respectively). Suitably, the residue of FR1 of the TNF-α binding polypeptide corresponding to residue number 29 of SEQ ID NO: 4 is F. Suitably, FR1 of the TNF-α binding polypeptide comprises or more suitably consists of SEQ ID NO: 4.

[0181] Suitably, FR2 of the TNF-α binding polypeptide comprises, or more suitably consists of, a sequence having 10%, 15%, 25%, 30%, 40%, 45%, 55%, 60%, 70%, 75%, 85% or 90% or greater sequence identity to SEQ ID NO:5.

[0182] Suitably, any residues in FR2 of the TNF-α binding polypeptide that differ from their corresponding residues in SEQ ID NO: 5 are conservative substitutions relative to their corresponding residues. Suitably, the residues of FR2 of the TNF-α binding polypeptide corresponding to residue numbers 8 to 11 of SEQ ID NO: 5 are KEXE, wherein X is R or L. Alternatively, the residues of FR2 of the TNF-α binding polypeptide corresponding to residue numbers 9 to 12 of SEQ ID NO: 5 are GLEW. Suitably, the FR2 of the TNF-α binding polypeptide comprises or more suitably consists of SEQ ID NO: 5.

[0183] Suitably, FR3 of the TNF-α binding polypeptide comprises, or more suitably consists of, a sequence having 8%, 15%, 20%, 26%, 32%, 40%, 45%, 52%, 58%, 65%, 70%, 76%, 80%, 82%, 85%, 90%, 92% or 95% or greater sequence identity to SEQ ID NO:6.

[0184] Suitably, the residue of FR3 of the TNF-α binding polypeptide corresponding to residue number 26 of SEQ ID NO: 6 is a hydrophobic amino acid (suitably A). Suitably, any residues in FR3 of the TNF-α binding polypeptide that differ from their corresponding residues in SEQ ID NO: 6 are conservative substitutions relative to their corresponding residues. Suitably, FR3 of the TNF-α binding polypeptide comprises or more suitably consists of SEQ ID NO: 6.

[0185] Suitably, FR4 of the TNF-α binding polypeptide comprises, or more suitably consists of, a sequence having 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or greater sequence identity to SEQ ID NO:7.

[0186] Suitably, any residues in FR4 of the TNF-α binding polypeptide that differ from their corresponding residues in SEQ ID NO: 7 are conservative substitutions relative to their corresponding residues. Suitably, FR4 of the TNF-α binding polypeptide comprises or more suitably consists of SEQ ID NO: 7.

[0187] Suitably, the TNF-α binding polypeptide comprises or more suitably consists of a sequence having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or greater sequence identity to SEQ ID NO: 8. Suitably, the N-terminus of the TNF-α binding polypeptide is D. Suitably, the TNF-α binding polypeptide comprises or more suitably consists of SEQ ID NO: 8.

[0188] According to a specific embodiment, the TNF-α binding polypeptide has an amino acid sequence that is not completely identical (ie, has 100% sequence identity) to the amino acid sequence of a naturally occurring polypeptide.

[0189] IL-7R binding peptide

[0190] Features

[0191] Anti-IL-7R polypeptides, polypeptides that interact with IL-7R, or polypeptides that antagonize IL-7R are all effectively IL-7R binding polypeptides. IL-7R binding polypeptides may bind to a linear or conformational epitope on IL-7R. Preferably, the IL-7R binding polypeptide binds to IL-7Rα.

[0192] Suitably, the IL-7R binding polypeptide will bind to human IL-7R. Suitably, the IL-7R binding polypeptide binds to both soluble and membrane-bound IL-7R.

[0193] Suitably, the polypeptides of the invention will neutralise IL-7 and / or human IL-7R which binds L-TSLP.

[0194] Suitably, the polypeptides of the invention neutralize the binding of human IL-7 and / or human L-TSLP to human IL-7R. More suitably, the polypeptides of the invention neutralize the binding of human IL-7 and / or human L-TSLP to both human and at least one additional primate IL-7R selected from the group consisting of: baboon IL-7R, marmoset IL-7R, cynomolgus monkey IL-7R and rhesus monkey IL-7R. Most suitably, the polypeptides of the invention neutralize the binding of human IL-7 and human L-TSLP to human IL-7R.

[0195] Suitably, the IL-7R binding polypeptide is directed against an epitope on IL-7R that is located in and / or forms part of the IL-7 binding site(s) of IL-7R, such that the polypeptide, when binding to IL-7R, results in inhibition or reduction of signalling mediated by IL-7R.

[0196] IL-7R binding polypeptides are neutralizing polypeptides for the purposes of the present invention provided that the polypeptide binds to IL-7R (suitably IL-7Rα) and inhibits the binding of IL-7R to IL-7 and / or L-TSLP as measured by ELISA. Example 1 below details a specific ELISA method suitable for determining the level of inhibition herein.

[0197] Suitably, the IL-7R binding polypeptide neutralizes IL-7R binding to IL-7 with an EC50 of 2.00 nM or less, such as 1.50 nM or less, such as 1.00 nM or less, such as 0.90 nM or less, such as 0.80 nM or less, such as 0.70 nM or less, such as 0.65 nM or less, such as 0.60 nM or less, such as 0.55 nM or less, such as 0.50 nM or less, such as 0.45 nM or less, such as 0.4 nM or less, such as 0.35 nM or less, such as 0.30 nM or less. Suitably, the EC50 is determined by using the IL-7 / IL-7R neutralization ELISA described in detail in Example 1 below.

[0198] Suitably, the IL-7R binding polypeptide neutralizes IL-7R-dependent, IL-7-induced STAT5 phosphorylation in human lymphocytes with an EC50 of 100 nM or less, such as 50 nM or less, such as 25 nM or less, such as 10 nM or less, such as 8 nM or less, such as 6 nM or less, such as 5 nM or less, such as 4 nM or less, such as 3 nM or less, such as 2 nM or less, such as 1.5 nM or less, such as 1 nM or less. Suitably, the EC50 is determined by using the IL-7-induced STAT5 phosphorylation assay detailed in Example 1 below.

[0199] Suitably, the affinity of the IL-7R binding polypeptide is determined by surface plasmon resonance.

[0200] Suitably, the IL-7R binding polypeptide is present at 10 -7 M or smaller, more preferably 10 -8 M or smaller, more preferably 10 -9 M or smaller, more preferably 10 -10 The IL-7R binds to IL-7R with an equilibrium dissociation constant (Kd) of M or less.

[0201] In one embodiment, the affinity of the IL-7R binding polypeptide is determined by direct coating onto a Biacore (or equivalent) sensor plate, wherein the polypeptide is flowed through the plate to detect binding. Suitably, Biacore T200 plates are used at 25°C at 30ul / min in HBS-EP+ (GE Healthcare) running buffer.

[0202] Structural characteristics

[0203] Suitably, the IL-7R binding polypeptide is a polypeptide comprising an antibody fragment. The polypeptide may be an antibody. Suitably, the antibody fragment is selected from: V-NAR, scFv, Fab fragment, F(ab')2 fragment or immunoglobulin chain variable domain, such as VL, VHH and VH. More suitably, the antibody fragment is an immunoglobulin chain variable domain, more suitably VHH or VH, most suitably VHH.

[0204] Suitably, the IL-7R binding polypeptide comprises three CDRs (CDR1, CDR2 and CDR3).

[0205] Suitably, the CDR1 of the IL-7R binding polypeptide comprises, or more suitably consists of, a sequence having 20%, 40%, 60% or 80% or greater sequence identity to SEQ ID NO:9.

[0206] Alternatively, the CDR1 of the polypeptide of the IL-7R binding polypeptide comprises or more suitably consists of a sequence having no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition, substitution and / or deletion compared to SEQ ID NO:9.

[0207] Suitably, any residues in the CDR1 of the IL-7R binding polypeptide that differ from their corresponding residues in SEQ ID NO: 9 are conservative substitutions relative to their corresponding residues. Suitably, the CDR1 of the IL-7R binding polypeptide comprises or more suitably consists of SEQ ID NO: 9.

[0208] Suitably, the residues of CDR1 have the following characteristics (SEQ ID NO: 28):

[0209] 1 2 3 4 5 S / D D A M G

[0210] More suitably, CDR1 comprises or more suitably consists of SEQ ID NO:9.

[0211] Suitably, the CDR2 of the IL-7R binding polypeptide comprises, or more suitably consists of, a sequence having 20% ​​or greater, more suitably 30% or greater, more suitably 40% or greater, more suitably 50% or greater, more suitably 55% or greater, more suitably 60% or greater, more suitably 65% ​​or greater, more suitably 70% or greater, more suitably 75% or greater, more suitably 80% or greater, more suitably 85% or greater, more suitably 90% or greater sequence identity to SEQ ID NO:10.

[0212] Alternatively, the CDR2 of the IL-7R binding polypeptide comprises or more suitably consists of a sequence having no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition, substitution and / or deletion compared to SEQ ID NO:10.

[0213] Suitably, any residues in the CDR2 of the IL-7R binding polypeptide that differ from their corresponding residues in SEQ ID NO: 10 are conservative substitutions relative to their corresponding residues. Suitably, the CDR2 of the IL-7R binding polypeptide comprises or more suitably consists of SEQ ID NO: 10.

[0214] Suitably, the residues of CDR2 have the following characteristics (SEQ ID NO: 29):

[0215] 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 A I / T G / N W S G A / T V T H Y S / G D S V Q / K G

[0216] Suitably, the residue of CDR2 corresponding to residue number 16 of SEQ ID NO: 10 is Q or K, most suitably K. Suitably, CDR2 comprises or consists of SEQ ID NO: 10. More suitably, CDR2 comprises or more suitably consists of SEQ ID NO: 10.

[0217] Suitably, the CDR3 of the IL-7R binding polypeptide comprises, or more suitably consists of, a sequence having 20% ​​or greater, more suitably 30% or greater, more suitably 40% or greater, more suitably 50% or greater, more suitably 55% or greater, more suitably 60% or greater, more suitably 65% ​​or greater, more suitably 70% or greater, more suitably 75% or greater, more suitably 80% or greater, more suitably 85% or greater, more suitably 90% or greater sequence identity to SEQ ID NO: 11.

[0218] Alternatively, the CDR3 of the IL-7R binding polypeptide comprises or more suitably consists of a sequence having no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition, substitution and / or deletion compared to SEQ ID NO: 11.

[0219] Suitably, any residues in the CDR3 of the IL-7R binding polypeptide that differ from their corresponding residues in SEQ ID NO: 11 are conservative substitutions relative to their corresponding residues.

[0220] Suitably, the residues of CDR3 have the following characteristics (SEQ ID NO: 30):

[0221] 1 2 3 4 5 6 7 8 9 D Y D / V T D V W Q Y / H

[0222] Suitably, the CDR3 of the IL-7R binding polypeptide comprises or more suitably consists of SEQ ID NO:11.

[0223] Suitably, the IL-7R binding polypeptide comprises three CDRs (CDR1, CDR2 and CDR3) and four framework regions (FR1-FR4).

[0224] Suitably, FR1 of the IL-7R binding polypeptide comprises, or more suitably consists of, a sequence that shares 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:12.

[0225] Suitably, any residue of FR1 that differs from its corresponding residue in SEQ ID NO: 12 is a conservative substitution relative to its corresponding residue. Suitably, the residue of FR1 corresponding to residue number 1 of SEQ ID NO: 12 is D or E, most suitably D. Suitably, the residue of FR1 corresponding to residue numbers 1 to 5 of SEQ ID NO: 12 is DVQLV. Suitably, FR1 comprises or more suitably consists of SEQ ID NO: 12. Suitably, the residue of FR1 corresponding to residue number 24 of SEQ ID NO: 12 is S.

[0226] Suitably, FR2 of the IL-7R binding polypeptide comprises, or more suitably consists of, a sequence having 10%, 15%, 25%, 30%, 40%, 45%, 55%, 60%, 70%, 75%, 85% or 90% or greater sequence identity to SEQ ID NO: 13.

[0227] Suitably, any residues of FR2 that differ from their corresponding residues in SEQ ID NO: 13 are conservative substitutions relative to their corresponding residues. Suitably, the residue of FR2 corresponding to residue number 10 of SEQ ID NO: 13 is R or L, more suitably L. Suitably, the residues of FR2 corresponding to residue numbers 8 to 11 of SEQ ID NO: 13 are KEXE, wherein X is R or L, more suitably L. Alternatively, the residues of FR2 corresponding to residue numbers 9 to 12 of SEQ ID NO: 13 are GLEW. Suitably, FR2 comprises or more suitably consists of SEQ ID NO: 13. Suitably, the residue of FR2 corresponding to residue number 2 of SEQ ID NO: 13 is F, more suitably, furthermore, the residue of FR2 corresponding to residue number 14 of SEQ ID NO: 13 is A. Suitably, the residues of FR2 corresponding to residues 9-14 of SEQ ID NO: 13 are ELEFLA (SEQ ID NO: 79). Suitably, the residue of FR2 corresponding to residues 9-14 of SEQ ID NO: 13 is not GLEWVS (SEQ ID NO: 80). Suitably, the residue of FR2 corresponding to residue number 9 of SEQ ID NO: 13 is not G. More suitably, the residue of FR2 corresponding to residue number 9 of SEQ ID NO: 13 is E.

[0228] Suitably, FR3 of the IL-7R binding polypeptide comprises, or more suitably consists of, a sequence having 8%, 15%, 20%, 26%, 32%, 40%, 45%, 52%, 58%, 65%, 70%, 76%, 80%, 82%, 85%, 90%, 92% or 95% or greater sequence identity to SEQ ID NO: 14.

[0229] Suitably, any residues of FR3 that differ from their corresponding residues in SEQ ID NO: 14 are conservative substitutions relative to their corresponding residues. Suitably, FR3 comprises or more suitably consists of SEQ ID NO: 14. Suitably, the residues of FR3 corresponding to residue numbers 18, 19 and 20 of SEQ ID NO: 14 are NSL. Suitably, the residue of FR3 corresponding to residue number 21 of SEQ ID NO: 14 is R. Suitably, the residue of FR3 corresponding to residue number 22 of SEQ ID NO: 14 is A.

[0230] Suitably, FR4 of the IL-7R binding polypeptide comprises, or more suitably consists of, a sequence having 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or greater sequence identity to SEQ ID NO: 15.

[0231] Suitably, any residues in FR4 of the IL-7R binding polypeptide that differ from their corresponding residues in SEQ ID NO: 15 are conservative substitutions relative to their corresponding residues. Suitably, FR4 of the IL-7R binding polypeptide comprises or more suitably consists of SEQ ID NO: 15.

[0232] Suitably, the IL-7R binding polypeptide comprises, or more suitably consists of, a sequence having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or greater sequence identity to SEQ ID NO: 16.

[0233] Suitably, the IL-7R binding polypeptide comprises or more suitably consists of SEQ ID NO:16.

[0234] According to a specific embodiment, the IL-7R binding polypeptide has an amino acid sequence that is not completely identical (ie, has 100% sequence identity) to the amino acid sequence of a naturally occurring polypeptide.

[0235] The structure and function of a specific combination of TNF-α binding polypeptide and IL-7R binding polypeptide are unique

[0236] Suitably, the TNF-α binding polypeptide and the IL-7R binding polypeptide are both immunoglobulin chain variable domains, wherein:

[0237] (a) the CDR1 of the TNF-α binding polypeptide comprises a sequence having 40% or greater sequence identity to SEQ ID NO: 1;

[0238] (b) the CDR2 of the TNF-α binding polypeptide comprises a sequence having 40% or greater sequence identity to SEQ ID NO:2;

[0239] (c) the CDR3 of the TNF-α binding polypeptide comprises a sequence having 50% or greater sequence identity to SEQ ID NO:3;

[0240] (d) CDR1 of the IL-7R binding polypeptide comprises a sequence having 40% or greater sequence identity to SEQ ID NO:9;

[0241] (e) the CDR2 of the IL-7R binding polypeptide comprises a sequence having 40% or greater sequence identity to SEQ ID NO: 10;

[0242] (f) the CDR3 of the IL-7R binding polypeptide comprises a sequence having 50% or greater sequence identity to SEQ ID NO: 11,

[0243] More suitably, wherein:

[0244] (a) the CDR1 of the TNF-α binding polypeptide comprises a sequence having 60% or greater sequence identity to SEQ ID NO: 1;

[0245] (b) the CDR2 of the TNF-α binding polypeptide comprises a sequence having 50% or greater sequence identity to SEQ ID NO: 2;

[0246] (c) the CDR3 of the TNF-α binding polypeptide comprises a sequence having 60% or greater sequence identity to SEQ ID NO:3;

[0247] (d) CDR1 of the IL-7R binding polypeptide comprises a sequence having 60% or greater sequence identity to SEQ ID NO:9;

[0248] (e) the CDR2 of the IL-7R binding polypeptide comprises a sequence having 50% or greater sequence identity to SEQ ID NO: 10;

[0249] (f) the CDR3 of the IL-7R binding polypeptide comprises a sequence having 60% or greater sequence identity to SEQ ID NO: 11,

[0250] More suitably, wherein:

[0251] (a) the CDR1 of the TNF-α binding polypeptide comprises a sequence having 60% or greater sequence identity to SEQ ID NO: 1;

[0252] (b) the CDR2 of the TNF-α binding polypeptide comprises a sequence having 70% or greater sequence identity to SEQ ID NO:2;

[0253] (c) the CDR3 of the TNF-α binding polypeptide comprises a sequence having 65% or greater sequence identity to SEQ ID NO:3;

[0254] (d) CDR1 of the IL-7R binding polypeptide comprises a sequence having 60% or greater sequence identity to SEQ ID NO:9;

[0255] (e) the CDR2 of the IL-7R binding polypeptide comprises a sequence having 70% or greater sequence identity to SEQ ID NO: 10;

[0256] (f) the CDR3 of the IL-7R binding polypeptide comprises a sequence having 65% or greater sequence identity to SEQ ID NO: 11,

[0257] More suitably, wherein:

[0258] (a) the CDR1 of the TNF-α binding polypeptide comprises a sequence having 80% or greater sequence identity to SEQ ID NO: 1;

[0259] (b) the CDR2 of the TNF-α binding polypeptide comprises a sequence having 85% or greater sequence identity to SEQ ID NO:2;

[0260] (c) the CDR3 of the TNF-α binding polypeptide comprises a sequence having 80% or greater sequence identity to SEQ ID NO:3;

[0261] (d) CDR1 of the IL-7R binding polypeptide comprises a sequence having 80% or greater sequence identity to SEQ ID NO:9;

[0262] (e) the CDR2 of the IL-7R binding polypeptide comprises a sequence having 85% or greater sequence identity to SEQ ID NO: 10;

[0263] (f) the CDR3 of the IL-7R binding polypeptide comprises a sequence having 80% or greater sequence identity to SEQ ID NO: 11,

[0264] and in each case, wherein most suitably, the TNF-α binding polypeptide neutralizes human TNF-α cytotoxicity in the L929 assay with an EC50 of 100 nM or less, and the IL-7R binding polypeptide neutralizes IL-7R-dependent, IL-7-induced STAT5 phosphorylation in human lymphocytes with an EC50 of 100 nM or less; and / or wherein the TNF-α binding polypeptide neutralizes human TNF-α cytotoxicity in the L929 assay with an EC50 of 100 nM or less -7 The IL-7R binding polypeptide binds to TNF-α with a Kd of 10 M or less. -7 The polypeptides described above bind to IL-7R with a Kd of 1 M or less. Most suitably, the polypeptides described above are provided as double-headed constructs for use in treating Crohn's disease or ulcerative colitis by oral administration.

[0265] Suitably, the TNF-α binding polypeptide and the IL-7R binding polypeptide are both immunoglobulin chain variable domains of the same type, such as VH, VHH or VL, more suitably VH or VHH.

[0266] Forms of TNF-α Binding Peptide and IL-7R Binding Peptide

[0267] The TNF-α binding polypeptide and the IL-7R binding polypeptide may or may not be linked.

[0268] In one embodiment, the TNF-α binding polypeptide and the IL-7R binding polypeptide may be present in the composition of the invention independently of each other (i.e., the TNF-α binding polypeptide and the IL-7R binding polypeptide are not linked to each other). In a specific embodiment, the composition comprises (a) ID-38F (SEQ ID NO: 8) or a polypeptide having at least 75%, more preferably at least 85% identity thereto, and (b) ID-A62U (SEQ ID NO: 16) or a polypeptide having at least 75%, more preferably at least 85% identity thereto.

[0269] In a further embodiment, the TNF-α binding polypeptide and the IL-7R binding polypeptide contained in the composition of the invention are linked to each other (thereby forming a single construct). This format may be convenient for recombinant expression purposes. Therefore, such constructs of the invention are multimeric and multivalent. If no additional polypeptides are included in the construct, then such constructs may be referred to as "heterobiheaded". Multivalent constructs contain two or more binding polypeptides, thereby providing two or more sites at which linkage to the antigen can occur (suitably before or after cleavage of an unstable peptide linker, provided that such a linker is present in the construct - see "Linkers" below).

[0270] Suitably, the molecular weight of each polypeptide in the construct is no more than 300 kDa, such as 250 kDa, such as 200 kDa, such as 180 kDa, such as 160 kDa, such as 140 kDa, such as 120 kDa, such as 100 kDa, such as 80 kDa, such as 60 kDa, such as 40 kDa, such as 30 kDa, such as 20 kDa, such as 15 kDa.

[0271] Connectors

[0272] The TNF-α binding polypeptide and the IL-7R binding polypeptide, if connected, can be connected to each other directly (i.e., without the use of a linker) or through a linker. A linker is suitably used. The linker can be a linker that is unstable to proteases or a non-protease unstable linker. The linker is suitably a peptide, suitably selected to allow the polypeptide to bind to its epitope. Suitably, the linker is flexible, for example flexible enough to allow two binding polypeptides to bind to their targets simultaneously. If used for therapeutic purposes, the linker is suitably non-immunogenic in the subject to which the polypeptide is administered. Suitably, the polypeptide and linker are expressed as a single contiguous polypeptide construct.

[0273] Suitably, the length of the peptide linker is at least 5, such as at least 7, such as at least 10, such as at least 13, such as at least 16, such as at least 19, such as at least 20, such as at least 21 residues. Suitably, the length of the peptide linker is not more than 40, such as not more than 35, such as not more than 30, such as not more than 25 residues.

[0274] Suitably, the peptide linker is composed of any amino acid selected from the following list: arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and tryptophan. Proline may be a suboptimal amino acid for inclusion in a linker, so the peptide linker is more suitably composed of any amino acid selected from the following list: arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and tryptophan.

[0275] The TNF-α binding polypeptide and the IL-7R binding polypeptide, if linked, can be in the orientation of anti-TNF-α-linker-anti-IL-7R (from N-terminus to C-terminus) or can be in the orientation of anti-IL-7R-linker-anti-TNF-α. Most suitably, their orientation is anti-TNF-α-linker-anti-IL-7R.

[0276] The composition of the present invention comprising a TNF-α binding polypeptide and an IL-7R binding polypeptide connected by a peptide linker can be obtained, for example, by preparing nucleic acids encoding the two binding polypeptides and the peptide linker using nucleic acid synthesis technology, and then expressing the nucleic acid thus obtained (as further described in detail under the heading "Preparation method" below).

[0277] Non-protease labile linker

[0278] Suitably, the non-protease labile linker is a peptide and does not comprise a cleavage site for a protease.

[0279] Suitably, the non-protease labile linker is of the form -(G a S b ) x - wherein a is 1 to 10, b is 1 to 5, and x is 1 to 15 (SEQ ID NO: 31). More suitably, a is 1 to 5, b is 1 to 2 and x is 1 to 10. More suitably a is 4, b is 1 and x is 1 to 8 (SEQ ID NO: 32).

[0280] Protease-labile linkers

[0281] Protease-labile linkers are more suitable than non-protease-labile linkers because protease-labile linkers allow the constituent monomers to be released and freely bind to their targets upon cleavage. Protease-labile linkers (or "labile peptide linkers") are peptides and contain at least one protease cleavage site. Including protease-labile linkers in the constructs of the present invention allows, for example, to conveniently generate constructs in heterobi-headed form, which are then cleaved into separate binding polypeptides after administration.

[0282] In one embodiment of the invention, the labile peptide linker can be engineered to resist protease cleavage to a desired degree and / or to be cleaved only when exposed to a specific region of the intestinal tract. For example, if the construct is recombinantly produced in a host such as yeast, a trypsin-like protease produced by the yeast can cleave the recombinant construct product. This can result in difficulty in purification and cause regulatory, clinical and commercial complications.

[0283] This can be achieved by incorporating shielding residues into unstable peptide linkers flanking (one or more) unstable sites. Shielding residues are located on the flanks of (one or more) unstable sites of unstable peptide linkers and reduce their instability. Cutting resistance can also be increased by positioning (one or more) unstable sites closer to unstable peptide linkers or at the periphery of unstable peptide linkers. This concept is referred to as "protected unstable sites" and provides controlled instability.

[0284] Alternatively, the unstable peptide linker can be engineered to be highly unstable to intestinal protease cleavage, thereby rapidly releasing the constituent polypeptides from the construct after oral administration. This is achieved by incorporating one or more unstable sites into the unstable peptide linker to expose the unstable site to proteolysis, for example, by positioning (one or more) unstable sites substantially in the center of the unstable peptide linker and / or making the unstable site substantially unshielded by flanking residues. This concept is referred to as "unshielded unstable sites".

[0285] It is expected that incorporating a P residue into the labile peptide linker of the construct of the invention immediately following an R or K residue will substantially prevent cleavage of the labile peptide linker. Suitably, the labile peptide linker does not comprise any P residues.

[0286] Trypsin-labile sites

[0287] Suitably, the unstable peptide linker comprises a cleavage site for trypsin or a trypsin-like protease. Suitably, the unstable peptide linker comprises at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10 K residues. Suitably, the unstable peptide linker comprises at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10 R residues. Preferably, the cleavage site is one or more K residues.

[0288] Suitably, the protease-labile linker comprises or more suitably consists of: a linker of the form [-(G a S b ) v -(G c S d ) w -B t -(G e S f ) x -(G g S h ) y ] z , wherein B is lysine or arginine; t is 1 to 5; a, c, e and g are each independently 0 to 10; b, d, f and h are each independently 0 to 5; v, w, x and y are each independently 0 to 10, and z is 1 to 5 (SEQ ID NO: 33). More suitably, B is lysine or arginine; t is 1 to 2; a, c, e and g are each independently 1 to 5; b, d, f and h are each independently 1 to 3; v, w, x and y are each independently 1 to 3, and z is 1 to 3, more suitably B is lysine or arginine; t is 1; a, c, e and g are each independently 2 to 4; b, d, f and h are each independently 1 to 2; v, w, x and y are each independently 1 to 2, and z is 1.

[0289] A particularly advantageous linker of this form is selected from -(G4S) x -K-(G4S) y -, wherein x and y are each independently 1 to 5 (SEQ ID NO: 34), more suitably -(G4S)2-K-(G4S)2- (ie, -GGGGSGGGGSKGGGGSGGGGS- (SEQ ID NO: 21).

[0290] Suitably, the labile peptide linker comprises or more suitably consists of: -B-(G a S) x -B'-form polypeptide sequence

[0291] in

[0292] a is 1 to 10:

[0293] x is 1 to 10:

[0294] B is K or R and

[0295] B' is K or R (SEQ ID NO: 35).

[0296] In one embodiment, a is 2 to 5, more suitably a is 4. In a further embodiment, x is 1 to 5. More suitably, x is 2. B may be present or absent. B' may be present or absent. Suitably, B is K. Suitably, B' is K.

[0297] Particularly advantageous linkers of this form are selected from the list of -K-(G4S)2-K-(SEQ ID NO: 36), -(G4S)2-K-(SEQID NO: 37), -K-(G4S)2-(SEQ ID NO: 38), -R-(G4S)2-R-(SEQ ID NO: 39), -(G4S)2-R-(SEQ ID NO: 40) and -R-(G4S)2-(SEQ ID NO: 41).

[0298] Suitably, the labile peptide linker comprises or more suitably consists of: -B-(G a S) x -B'-(G b S) y -B"-form polypeptide sequence

[0299] in

[0300] a is 1 to 10:

[0301] b is 1 to 10:

[0302] x is 1 to 10:

[0303] y is 1 to 10:

[0304] B is K or R:

[0305] B' is K or R and

[0306] B" is K or R (SEQ ID NO: 42).

[0307] In one embodiment, a is 2 to 5, more suitably a is 4. In one embodiment, b is 2 to 5, more suitably b is 4. In a further embodiment, x is 1 to 5. More suitably x is 2. In a further embodiment, y is 1 to 5. More suitably y is 2. Suitably, B is K. Suitably, B' is K. Suitably, B" is K.

[0308] Chymotrypsin-labile site

[0309] Alternatively or in addition to the site that is unstable to trypsin, the unstable peptide linker of the construct may also comprise a cleavage site for chymotrypsin or chymotrypsin-like protease. Suitably, the unstable peptide linker comprises at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10 residues, the residues being selected from the list of W, F, Y, L and M; more suitably selected from W, F and Y. Suitably, the unstable peptide linker consists of residues selected from the list of S, G, W, F, Y, L and M (e.g., S, G, W, F and Y).

[0310] Suitably, the protease-labile linker comprises or more suitably consists of: a linker of the form [-(G a S b ) v -(G c S d ) w -J t -(G e S f ) x -(G g S h ) y ] z , wherein J is W, F, Y, L or M; t is 1 to 5; a, c, e and g are each independently 0 to 10; b, d, f and h are each independently 0 to 5; v, w, x and y are each independently 0 to 10, and z is 1 to 5 (SEQ ID NO: 43). More suitably, J is W, F, Y, L or M; t is 1 to 2; a, c, e and g are each independently 1 to 5; b, d, f and h are each independently 1 to 3; v, w, x and y are each independently 1 to 3, and z is 1 to 3, more suitably J is W, F, Y, L or M; t is 1; a, c, e and g are each independently 2 to 4; b, d, f and h are each independently 1 to 2; v, w, x and y are each independently 1 to 2, and z is 1.

[0311] Enterokinase-labile site

[0312] Alternatively or in addition to the trypsin and / or chymotrypsin labile site, the labile peptide linker of the construct may also comprise an enterokinase cleavage site. Suitably, the labile peptide linker comprises the sequence DDDDK (SEQ ID NO: 19), for example a sequence comprising -G4S-DDDDK-G4S- (SEQ ID NO: 20) or consisting thereof.

[0313] MMP-labile sites

[0314] In one embodiment, the labile peptide linker of the construct may comprise a cleavage site for MMP3, MMP10 or MMP12.

[0315] Other unstable sites

[0316] In one embodiment, the labile peptide linker of the construct may comprise a cleavage site for other inflammatory or microbial proteases, where the cleavage site is known.

[0317] Stability of polypeptides in constructs

[0318] In embodiments where the construct comprises a non-protease labile linker, then suitably the construct as a whole (i.e. the binding polypeptide (which may be an immunoglobulin chain variable domain) and the non-protease labile linker) is substantially resistant to proteases such as trypsin and chymotrypsin. In embodiments where the construct comprises a protease labile linker, then suitably the polypeptide (i.e. the binding polypeptide, which may be an immunoglobulin chain variable domain) is substantially resistant to proteases such as trypsin and chymotrypsin, but the protease labile linker is labile to proteases such as trypsin or chymotrypsin.

[0319] Stability of unstable peptide linkers to expression hosts

[0320] Various organisms can be used to express recombinant polypeptides. Commonly used expression organisms include yeast, molds and mammalian cells. However, many of these expression organisms also produce proteases, such as trypsin-like proteases, which can cut the expressed recombinant polypeptide. If the expressed polypeptide contains a peptide linker that is unstable to one or more proteases present in the intestine, the peptide linker may also be undesirably unstable to the proteases produced by the expression organism, thereby preventing the effective expression of the complete polypeptide.

[0321] It is advantageous that the unstable peptide linker is substantially stable to the enzyme produced by the recombinant host used to produce the construct. Suitably, the unstable peptide linker is substantially resistant to the protease produced by the recombinant host, and the recombinant host is, for example, a bacterium, such as Escherichia coli or a yeast or mold such as belonging to Aspergillus, Saccharomyces, Kluyveromyces, Hansenula or Pichia; for example, Saccharomyces cerevisiae or Pichia pastoris.

[0322] Suitably, the recombinant host is a yeast or a mold. Suitably, yeast belongs to Saccharomyces, Kluyveromyces, Hansenula, Pichia, Candida or Torulopsis. Suitably, mold belongs to Aspergillus, Acremonium, Alternaria, Chrysosporium, Cladosporium, Dictyostelium, Fusarium, Mucor, Penicillium, Rhizopus, Stachybotrys, Trichoderma and Trichophyton.

[0323] Gastrointestinal and digestive enzymes

[0324] The gastrointestinal tract (GIT) is an organ system responsible for consuming and digesting food, absorbing nutrients, and excreting waste. In humans and other mammals, the gastrointestinal tract consists of the esophagus, stomach, small intestine (duodenum, jejunum, and ileum), and large intestine (cecum, colon, rectum, and anal canal). Various pathogens may colonize and various diseases may arise in different regions of the GIT. The intestinal tract (as opposed to the gastrointestinal tract) consists of the small intestine and the large intestine.

[0325] Different parts of the gastrointestinal tract each contain a complex mixture of digestive enzymes. Proteases are involved in the digestion of polypeptide chains into shorter fragments by cleaving the peptide bonds connecting the amino acid residues (proteolysis). Some detach the terminal amino acid from the protein chain (exopeptidases), others attack the internal peptide bonds of the protein (endopeptidases). Proteolysis can be highly promiscuous, so that a wide range of protein substrates are hydrolyzed. This is the case for proteases that cleave the large amounts of ingested polypeptides in the intestine into smaller polypeptide fragments.

[0326] Many proteases usually bind to a single amino acid (labile site) on the substrate and are therefore specific only for that residue. Proteases present in the intestine include trypsin, trypsin-like proteases, chymotrypsin, chymotrypsin-like proteases, carboxypeptidases, elastases, aminopeptidases, carboxypeptidases, and enteropeptidases. Trypsin-like proteases cleave peptide bonds following lysine or arginine residues. Chymotrypsin-like proteases cleave peptide bonds following hydrophobic residues such as tyrosine, phenylalanine, tryptophan, leucine, and methionine. In particular, tyrosine, phenylalanine, and tryptophan.

[0327] Particularly in the case of oral drugs, it may be desirable that the conjugated polypeptide is substantially resistant to one or more (eg, all) intestinal proteases, and that the labile peptide linker (if used) is labile to one or more (eg, all) intestinal proteases.

[0328] Suitably, the polypeptide in the construct is substantially resistant to one or more proteases, and suitably, the labile peptide linker (if present) is labile to one or more proteases, wherein the one or more proteases are present in the small intestine or large intestine, more suitably in the jejunum, ileum and / or cecum. The substantially resistant polypeptide substantially retains neutralizing capacity and / or potency when exposed to one or more proteases.

[0329] Such proteases include proteases derived from gastrointestinal symbiotic microflora or pathogenic bacteria, such as proteases wherein the proteases are cell membrane attached proteases, secreted proteases, and proteases released during cell lysis. Such proteases may also include IBD inflammatory proteases, such as MMP3, MMP10, and MMP12. Suitably, one or more proteases are serine proteases. Suitably, one or more proteases are selected from enteropeptidase, trypsin, trypsin-like proteases, chymotrypsin, and chymotrypsin-like proteases.

[0330] Suitably, the polypeptide substantially retains neutralizing capacity and / or potency when delivered orally and after exposure to the intestinal tract (e.g., after exposure to proteases of the small and / or large intestine and / or IBD inflammatory proteases. Proteases of the small and / or large intestine or proteases produced in the small and / or large intestine include proteases derived from intestinal commensal microflora and / or pathogenic bacteria, e.g., wherein the proteases are cell membrane attached proteases, excreted proteases, and proteases released by cell lysis). Most suitably, the proteases are trypsin and chymotrypsin.

[0331] Suitably, the intestinal tract is dog, pig, rat, guinea pig, hamster, rabbit, human, cynomolgus monkey or mouse. The small intestine suitably consists of the duodenum, jejunum and ileum. The large intestine suitably consists of the cecum, colon, rectum and anal canal.

[0332] When suitably at least 10%, more suitably 20%, more suitably 30%, more suitably 40%, more suitably 50%, more suitably 60%, more suitably 70%, more suitably 80%, more suitably 90%, more suitably 95%, more suitably 100% of the original neutralizing capacity of the polypeptide or construct is retained after exposure to proteases and / or IBD inflammatory proteases present in the small intestine and / or large intestine; after exposure to a given period of time at a given temperature, the polypeptide substantially retains neutralizing capacity. This concept is referred to herein as "% survival".

[0333] Suitably, the polypeptide retains substantial neutralising capacity after exposure to proteases present in the small and / or large intestine and / or IBD inflammatory proteases for, e.g., up to at least 2, more suitably up to at least 3, more suitably up to at least 4, more suitably up to at least 5, more suitably up to at least 5.5, more suitably up to at least 6, more suitably up to at least 12, more suitably up to at least 14 or more suitably up to at least 16 hours at 37 degrees Celsius.

[0334] When suitably at least 10% of the original neutralizing capacity of the polypeptide or construct, more suitably at least 20%, more suitably at least 30%, more suitably at least 40%, more suitably at least 50%, more suitably at least 60%, more suitably at least 70%, more suitably at least 80%, more suitably at least 90%, more suitably at least 95%, more suitably at least 100% is retained after exposure to human fecal supernatant or mouse small intestinal fluid; after exposure to a given time period at a given temperature, the polypeptide substantially retains neutralizing capacity. This concept is referred to herein as "% survival".

[0335] Suitably, the polypeptide retains substantial neutralizing capacity after exposure to human fecal supernatant or mouse small intestinal fluid at 37 degrees Celsius, for example up to at least 2, more suitably up to at least 3, more suitably up to at least 4, more suitably up to at least 5, more suitably up to at least 5.5, more suitably up to at least 6, more suitably up to at least 12, more suitably up to at least 14 or more suitably up to at least 16 hours.

[0336] Autoimmune and / or inflammatory diseases

[0337] The compositions and constructs provided by the invention are particularly useful for preventing or treating autoimmune diseases and / or inflammatory diseases. Suitably, the autoimmune diseases and / or inflammatory diseases are inflammatory bowel disease and / or mucositis, most suitably inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis, or colitis induced by a checkpoint inhibitor, most suitably ulcerative colitis or Crohn's disease).

[0338] Autoimmune diseases occur when the immune system has adverse responses to normal body tissues. Autoimmune disorders may result in damage to body tissues, abnormal organ growth and / or changes in organ function. The disorder may only affect one organ or tissue type or may affect multiple organs and tissues. Organs and tissues commonly affected by autoimmune disorders include blood components such as red blood cells, blood vessels, connective tissue, endocrine glands such as thyroid or pancreas, muscles, joints and skin. Inflammatory diseases are diseases characterized by inflammation. Many inflammatory diseases are autoimmune diseases and vice versa.

[0339] GIT autoimmune and / or inflammatory diseases

[0340] Chronic inflammatory bowel disease (IBD) Crohn's disease and ulcerative colitis, which affect children and adults, are examples of autoimmune and inflammatory diseases of the GIT (gastrointestinal tract) (Hendrickson et al. 2002, incorporated herein by reference in its entirety). Ulcerative colitis is defined as a condition in which the inflammatory response and morphological changes remain confined to the colon. The rectum is involved in 95% of patients. Inflammation is largely confined to the mucosa and includes variable severity of ulcers, edema and bleeding and continuous damage to the length of the colon (Hendrickson et al. 2002, incorporated herein by reference in its entirety). Ulcerative colitis is usually manifested by the presence of mixed blood and mucus in the stool and lower abdominal cramps, which are most severe during defecation. Clinically, the presence of blood and mucosa in diarrhea distinguishes ulcerative colitis from irritable bowel syndrome in which blood is not present. Unlike ulcerative colitis, the presentation of Crohn's disease is usually not obvious, which leads to late diagnosis. Factors such as the location, extent and severity of the damage determine the extent of gastrointestinal symptoms. Patients with ileocolic lesions typically have postprandial abdominal pain and right lower quadrant tenderness and occasional inflammatory masses. Symptoms associated with gastroduodenal Crohn's disease include early satiety, nausea, vomiting, epigastric pain or dysphagia. Perianal disease as well as anal tags, deep anal fissures and fistulas are common (Hendrickson et al. 2002, incorporated herein by reference in its entirety).

[0341] Other diseases of the GIT include, for example, inflammatory diseases mucositis (appropriate drugs and radiation-induced mucositis), in which inflammatory lesions are present in the mucosa and destroy epithelial tight junctions. In mucositis, lesions may occur anywhere from the oral cavity to the anus, and for oral and esophageal lesions, mouthwashes or cream preparations containing the composition can be used. For anal and rectal lesions, suppositories, creams or foams containing the composition will be suitable for topical application. The composition will be removed from the lamina propria or other inflammatory sites by being absorbed into the bloodstream at the site of inflammation or by lymphatic clearance and subsequent entry into the bloodstream. Therefore, the composition will reach the liver by bloodstream and be removed by glomerular filtration in the kidney. Therefore, it is well-founded that the composition of the present invention will play a therapeutic role in diseases such as autoimmune hepatitis, type II diabetes and glomerulonephritis.

[0342] Suitably, the composition of the invention is used to treat or prevent autoimmune and / or inflammatory diseases of the GIT.

[0343] Suitably, the composition of the invention is for use in the treatment of an autoimmune and / or inflammatory disease of the GIT selected from the group consisting of Crohn's disease, ulcerative colitis, irritable bowel disease, type II diabetes, glomerulonephritis, autoimmune hepatitis, Sjogren's syndrome, celiac disease and drug or radiation-induced mucositis, esophagitis and checkpoint inhibitor-induced colitis (more suitably Crohn's disease, ulcerative colitis or checkpoint inhibitor-induced colitis, most suitably ulcerative colitis or Crohn's disease).

[0344] Oral delivery of the compositions would ideally treat inflammatory diseases in which TNF-α (or TNF-α receptor) and / or IL-7R (or IL-7 or L-TSLP) contribute at least a portion of the pathology and, more preferably, in which the immunoglobulin chain variable domains can access tissues where these cytokines are biologically active.

[0345] Autoimmune and / or inflammatory diseases of the skin

[0346] Psoriasis is a debilitating autoimmune skin disease. Plaque psoriasis is the most common form of the disease and is characterized by red skin covered with silvery scales. Histologically, the condition is one of disordered differentiation and overproliferation of keratinocytes within the psoriatic plaques, accompanied by inflammatory cell infiltration (Ortonne, 1999). Psoriatic lesions are inflammatory, red, well-defined plaques of various shapes with characteristic silvery, shiny scales. The term psoriasis includes both psoriasis and the symptoms of psoriasis, including erythema, thickening / raising of the skin, and scaling.

[0347] Biologic agents used to treat psoriasis include anti-TNF-α therapy (e.g., monoclonal antibodies against TNF, such as adalimumab and infliximab, or TNF-α receptor fusion proteins, such as etanercept), humanized antibodies against CD11a (efalizumab), or agents that bind to CD2, such as alefacept (thus blocking the CD2 LFA3 interaction). It should be noted that not all of the biologic agents listed here have been approved for the treatment of psoriasis.

[0348] The compositions of the invention may be incorporated into a cream / ointment or other topical vehicle for administration to inflammatory skin lesions where TNF-α (or TNF-α receptor) and / or IL-7R (or IL-7 or L-TSLP) contribute to the pathology of such lesions.

[0349] Suitably, the composition of the present invention is for use in treating an autoimmune and / or inflammatory disease of the skin selected from pemphigus, psoriasis, eczema and scleroderma.

[0350] In one embodiment, the polypeptide or construct of the invention is for use in treating or preventing atopic dermatitis, suitably by topical delivery to and / or through the skin, suitably in the form of a cream, nanoparticles, ointment or hydrogel.

[0351] Therapeutic Uses and Delivery

[0352] Suitably, the compositions of the present invention are used as medicines, suitably by oral administration, suitably for treating or preventing GIT diseases and / or treating or preventing diseases such as autoimmune diseases and / or inflammatory diseases, such as inflammatory bowel disease. Suitably, the constructs of the present invention are used as medicines, suitably by oral administration. The compositions of the present invention may also be used for treating or preventing other medical conditions, such as metabolic disorders, such as obesity, by oral administration. In one embodiment, the compositions of the present invention are intended to have a local effect in the intestinal tract. In one embodiment, the compositions of the present invention are not used for treating or preventing diseases by being delivered into the circulation in a therapeutically effective amount.

[0353] In one aspect of the invention, there is provided a method of treating an autoimmune disease and / or an inflammatory disease, the method comprising administering to a person in need thereof a therapeutically effective amount of a composition of the invention. Suitably, the autoimmune disease and / or inflammatory disease is inflammatory bowel disease and / or mucositis.

[0354] The anti-TNF-α binding polypeptide and the anti-IL-7R binding polypeptide may be co-formulated in the composition of the present invention, or may be formulated separately and administered separately, sequentially or simultaneously.

[0355] The TNF-α binding polypeptide and the IL-7R binding polypeptide can be administered by the same route or by different routes. For example, the TNF-α binding polypeptide can be administered orally, while the IL-7R binding polypeptide can be administered rectally.

[0356] In one aspect of the invention, a TNF-α binding polypeptide is provided for use with an IL-7R binding polypeptide in treating or preventing autoimmune diseases and / or inflammatory diseases. In another aspect, an IL-7R binding polypeptide is provided for use with a TNF-α binding polypeptide in treating or preventing autoimmune diseases and / or inflammatory diseases.

[0357] In another aspect of the present invention, a method for treating an autoimmune disease and / or an inflammatory disease is provided, the method comprising administering a TNF-α binding polypeptide and an IL-7R binding polypeptide to a person in need thereof. A method for treating an autoimmune disease and / or an inflammatory disease is also provided, the method comprising administering an IL-7R binding polypeptide and a TNF-α binding polypeptide to a person in need thereof.

[0358] In another aspect of the present invention, a composition is provided, comprising a TNF-α binding polypeptide and an IL-7R binding polypeptide for use in treating or preventing inflammatory bowel disease and / or mucositis.

[0359] In another aspect of the present invention, a composition is provided, comprising a TNF-α binding polypeptide and an IL-7R binding polypeptide for treating or preventing inflammatory bowel disease and / or mucositis, wherein the composition is administered orally.

[0360] In another aspect of the present invention, there is provided use of a composition comprising a TNF-α binding polypeptide and an IL-7R binding polypeptide in the preparation of a medicament for treating or preventing an autoimmune disease and / or an inflammatory disease (suitably inflammatory bowel disease and / or mucositis).

[0361] In another aspect of the present invention, there is provided the use of a composition comprising a TNF-α binding polypeptide and an IL-7R binding polypeptide in the preparation of a medicament for treating or preventing an autoimmune disease and / or an inflammatory disease (suitably inflammatory bowel disease and / or mucositis) by oral administration.

[0362] In another aspect of the present invention, a method of treating or preventing inflammatory bowel disease and / or mucositis is provided, the method comprising administering a composition comprising a TNF-α binding polypeptide and an IL-7R binding polypeptide to a human in need thereof.

[0363] In another aspect of the present invention, a method of treating or preventing inflammatory bowel disease and / or mucositis is provided, the method comprising orally administering a composition comprising a TNF-α binding polypeptide and an IL-7R binding polypeptide to a human in need thereof.

[0364] In the above embodiments, the inflammatory disease is suitably mucositis or esophagitis, and the inflammatory bowel disease is suitably Crohn's disease, ulcerative colitis or colitis induced by a checkpoint inhibitor, most suitably ulcerative colitis or Crohn's disease. In addition, in the above embodiments, the TNF-α binding polypeptide is suitably a polypeptide comprising an antibody fragment, and / or the IL-7R binding polypeptide is suitably a polypeptide comprising an antibody fragment. More suitably, both TNF-α binding polypeptide and IL-7R binding polypeptide are polypeptides comprising an antibody fragment. More suitably, both polypeptides are ICVD.

[0365] Administration of one binding polypeptide "together" with another binding polypeptide means that the therapeutic windows of each binding polypeptide overlap each other. This means, for example, that a therapeutically effective amount of each binding polypeptide is present in the subject at the same time. In a specific embodiment, a therapeutically effective amount of each binding polypeptide is present at the site in need of treatment at the same time.

[0366] The therapeutically effective amount of the composition of the present invention is an effective amount that neutralizes the biological effect of the selected target in the subject to a significant extent when administered to the subject in a single or multiple doses. The therapeutically effective amount can vary according to various factors, such as the disease state, age, sex and body weight of the individual, and the ability of the construct to elicit a desired response in the individual. The therapeutically effective amount is also the amount in which the therapeutically beneficial effects of the construct outweigh any toxic or adverse effects thereof. The composition of the present invention can be incorporated into a pharmaceutical composition suitable for oral administration to a subject.

[0367] The compositions of the present invention can be formulated for oral delivery. The compositions of the present invention can be in various forms. These include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions, dispersions or suspensions, tablets, pills and powders. Solid dosage forms are preferred. Suitably, the compositions of the present invention are provided in the form of tablets, more suitably in the form of mini-tablets. Suitably, the constructs of the present invention are provided in the form of tablets, more suitably in the form of mini-tablets, for the treatment of ulcerative colitis or Crohn's disease. More suitably, the constructs of the present invention are provided in the form of mini-tablets for oral administration with an enteric coating.

[0368] 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.

[0369] The composition may comprise a pharmaceutically acceptable excipient, and may be suitably used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

[0370] Typically, the composition comprises a pharmaceutically acceptable excipient such as a carrier to form a pharmaceutical composition. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc. and combinations thereof. Pharmaceutically acceptable carriers may also include a small amount of auxiliary substances that enhance the shelf life or effectiveness of the polypeptide or construct of the present invention, such as a wetting agent or emulsifier, preservative or buffer. The pharmaceutical composition may include an anti-adherent, an adhesive, a coating, a disintegrant, a flavoring agent, a coloring agent, a lubricant, an adsorbent, a preservative, a sweetener, a lyophilizing excipient (including a lyophilizing protectant) or a compression aid. Suitably, the polypeptide in the composition of the present invention is lyophilized before being incorporated into the pharmaceutical composition.

[0371] The composition of the present invention may also be provided with an enteric coating. An enteric coating is a polymer barrier applied to an oral drug that helps prevent polypeptides from being affected by the low pH of the stomach. Materials used for enteric coatings include fatty acids, waxes, shellac, plastics, and plant fibers. Suitable enteric coating components include methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate (hydroxypropyl methylcellulose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymers, sodium alginate, and stearic acid. Suitable enteric coatings include pH-dependent release polymers. These are polymers that are insoluble at the high acid pH present in the stomach but rapidly dissolve at a lower acid pH. Thus, suitably, the enteric coating is insoluble in the acidic gastric juice of the stomach (pH ~ 3), but dissolves in the higher pH environment present in the small intestine (pH above 6) or in the colon (pH above 7.0). The pH-dependent release polymer is selected so that the composition of the present invention is released approximately at the time when the dose reaches the target area of ​​the intestine. .

[0372] The pharmaceutical composition of the present invention can be formulated in a buffer so that the pH of the composition is stabilized at a concentration between 5-50 g / l, or more suitably between 15-40 g / l, or more suitably between 25-30 g / l. Examples of suitable buffer components include physiological salts, such as sodium citrate and / or citric acid. Suitably, the buffer contains 100-200 mM, more suitably 125-175 mM of physiological salts, such as sodium chloride. Suitably, the buffer is selected to have a pKa close to the pH of the composition or the physiological pH of the patient.

[0373] Exemplary concentrations of the polypeptide in the pharmaceutical composition can range from about 1 mg / mL to about 200 mg / ml, or from about 50 mg / mL to about 200 mg / mL, or from about 150 mg / mL to about 200 mg / mL.

[0374] The aqueous formulation of the pharmaceutical composition of the present invention can be prepared in a pH buffer solution, for example, in a range of about 4.0 to about 7.0, or about 5.0 to about 6.0, or alternatively about 5.5 pH. Examples of suitable buffers include phosphoric acid, histidine, citric acid, succinic acid, acetate buffers, and other organic acid buffers. Depending on the desired tension of, for example, the buffer and the formulation, the buffer concentration can be about 1 mM to about 100 mM, or about 5 mM to about 50 mM.

[0375] The tension of the pharmaceutical composition can be changed by including a tension regulator. Such tension regulators can be charged or uncharged chemical substances. Typical uncharged tension regulators include sugars or sugar alcohols or other polyols, preferably trehalose, sucrose, mannitol, glycerol, 1,2-propylene glycol, raffinose, sorbitol or lactitol (especially trehalose, mannitol, glycerol or 1,2-propylene glycol). Typical charged tension regulators include salts, such as sodium ions, potassium ions or calcium ions and chloride ions, sulfate ions, carbonate ions, sulfite ions, nitrate ions, lactate ions, succinate ions, acetate ions or maleate ions (especially sodium chloride or sodium sulfate); or amino acids such as arginine or histidine. Suitably, aqueous formulations are isotonic, but hypertonic or hypotonic solutions may be suitable. The term "isotonic" represents a solution having the same tension as a certain other solution to which it is compared, such as a physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, for example in an amount of 1 mM to 500 nM. Suitably, at least one isotonicity agent is included in the composition.

[0376] Surfactant can also be added to the pharmaceutical composition to reduce the aggregation of the constructed body and / or to minimize the formation of particles in the formulation and / or to 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 (Pluronic)) and sodium lauryl sulfate (SDS). The example of suitable polyoxyethylene sorbitan-fatty acid esters is polysorbate 20 and polysorbate 80. The scope of the exemplary concentration of surfactant can be from about 0.001% to about 10% w / v.

[0377] Lyoprotectants may also be added to protect the polypeptide from destabilizing conditions during the freezing process. For example, known lyoprotectants include sugars (including glucose, sucrose, mannose, and trehalose); polyols (including mannitol, sorbitol, and glycerol); and amino acids (including alanine, glycine, and glutamic acid). The lyoprotectant may be included in an amount of about 10 mM to 500 mM.

[0378] The dosage range for using the pharmaceutical composition of the present invention is those dosage ranges that produce the desired therapeutic effect. The required dosage range depends on the accurate nature of the pharmaceutical composition or construct, the target area of ​​the intestinal tract, the nature of the preparation, the age of the patient, the nature, degree or severity of the patient's condition, contraindications (if any) and the judgment of the attending physician. The variation of these dosage levels can be adjusted to optimize using standard empirical routines.

[0379] Suitably, the scope of the daily dosage of the pharmaceutical composition of the present invention is 50ng-50mg / kg body weight, such as 50ug-40mg / kg body weight, such as 5-30mg / kg body weight. Unit dose can change from less than 100mg, but usually in the region of 250-2000mg / agent, it can be used daily or more frequently, for example 2,3 or 4 times / day or less frequently, for example once every two days or once a week.

[0380] The TNF-α binding polypeptide and the IL-7R binding polypeptide, when present in the composition of the present invention and when not linked to each other, may be present in a molar ratio of TNF-α binding polypeptide to IL-7R binding polypeptide of 20:1 to 1:20, such as 15:1 to 1:15, more suitably 10:1 to 1:10, more suitably 5:1 to 1:5, more suitably 3:1 to 1:3, more suitably 2:1 to 1:2, more suitably 1.5:1 to 1:1.5, and most suitably about 1:1.

[0381] Treatment of a disease also includes treating its progression, ameliorating its symptoms and also includes treating patients in remission from disease symptoms to prevent recurrence of disease symptoms.

[0382] Combination therapy

[0383] The pharmaceutical compositions of the present invention may also comprise one or more active agents (e.g., active agents suitable for treating diseases such as those mentioned herein). It is within the scope of the present invention to use the pharmaceutical compositions of the present invention in therapeutic methods for treating autoimmune diseases and / or inflammatory diseases as an adjunct to or in combination with other established therapies commonly used in treating bacterial diseases, autoimmune diseases and / or inflammatory diseases.

[0384] For the treatment of inflammatory bowel diseases (such as Crohn's disease or ulcerative colitis), possible combinations include combinations with, for example, one or more active agents selected from the list comprising: 5-aminosalicylic acid or a prodrug thereof (such as sulfasalazine, olsalazide or bisalazide); corticosteroids (e.g., prednisolone, methylprednisolone or budesonide); immunosuppressants (e.g., cyclosporine, tacrolimus, methotrexate, azathioprine or 6-mercaptopurine); anti-TNF-α antibodies (e.g., infliximab, adalimumab, certolizumab pegol or golimumab); anti-IL12 / IL23 antibodies (e.g., ustekinumab); anti-IL-23p19 specific antibodies (e.g., brazikumab, risankizumab or mirikizumab), Anti-IL6R antibodies or small molecule IL12 / IL23 inhibitors (e.g., apimod); anti-oncostatin M antibodies, anti-α-4-β-7 antibodies (e.g., vedolizumab); MAdCAM-1 blockers (e.g., PF-00547659); antibodies against the cell adhesion molecule α-4-integrin (e.g., natalizumab); antibodies against the α subunit of the IL2 receptor (e.g., daclizumab or basiliximab); JAK1 Inhibitors (e.g., filgotinib or upadacitinib); JAK3 inhibitors (e.g., tofacitinib or R348); Syk inhibitors and their prodrugs (e.g., fotantinib and R-406); phosphodiesterase-4 inhibitors (e.g., tetomilast); HMPL-004; probiotics; dersalazine; semamod / CPSI-2364; and protein kinase C inhibitors (e.g., AEB-071). The most suitable combination agent is a JAK1 inhibitor (e.g., filgotinib or upadacitinib); a JAK3 inhibitor (e.g., tofacitinib or R348), an anti-IL12 / IL23 antibody (e.g., ustekinumab); an anti-IL-23p19 specific antibody (e.g., brevitzumab, risankizumab or migizumab) or an anti-α-4-β-7 antibody (e.g., vedolizumab).

[0385] Therefore, another aspect of the present invention provides the pharmaceutical composition of the present invention combined with one or more other activating agents (for example one or more activating agents described above).In another aspect of the present invention, pharmaceutical composition or construct are used sequentially, simultaneously or individually with at least one activating agent selected from the above list.

[0386] Similarly, another aspect of the present invention provides a combination product comprising:

[0387] (A) a pharmaceutical composition of the present invention; and

[0388] (B) one or more other active agents,

[0389] Wherein each of component (A) and component (B) is mixed and formulated with a pharmaceutically acceptable adjuvant, diluent or carrier. In this aspect of the invention, the combination product can be a single (combination) formulation or a kit-of-parts. Therefore, this aspect of the invention encompasses a combined formulation comprising a pharmaceutical composition or construct of the invention and another therapeutic agent mixed with a pharmaceutically acceptable adjuvant, diluent or carrier.

[0390] The present invention also encompasses a kit of parts comprising the following components:

[0391] (i) a pharmaceutical composition of the present invention mixed with a pharmaceutically acceptable adjuvant, diluent or carrier; and

[0392] (ii) a formulation comprising one or more other active agents in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, wherein the components (i) and (ii) are each provided in a form suitable for administration in combination with each other.

[0393] Therefore, component (i) of the kit of components is the above component (A) mixed with a pharmaceutically acceptable adjuvant, diluent or carrier. Similarly, component (ii) is the above component (B) mixed with a pharmaceutically acceptable adjuvant, diluent or carrier. One or more other active agents (i.e., above component (B)) can be any of the agents mentioned above for the treatment of bacterial infection such as Clostridium difficile infection, autoimmune disease and / or inflammatory disease such as IBD (e.g., Crohn's disease and / or ulcerative colitis). If component (B) is more than one other active agent, these other active agents can be formulated with each other or with component (A) or they can be formulated separately. In one embodiment, component (B) is a kind of other therapeutic agent. In another embodiment, component (B) is two other therapeutic agents. The combination product (combination preparation or kit of components) of this aspect of the present invention can be used for the treatment or prevention of autoimmune diseases (e.g., autoimmune diseases mentioned herein).

[0394] Vector and host

[0395] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is connected. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which another DNA segment can be connected. Another type of vector is a viral vector, in which another DNA segment can be connected to a viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors with bacterial replication origins and free mammalian and yeast vectors). Other vectors (e.g., non-free mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, and therefore replicated together with the host genome. In addition, some vectors can guide the expression of genes operably connected thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). Generally, expression vectors useful in recombinant DNA technology are generally in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used vector forms. However, the present invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which have the same functions, as well as phage and phagemid systems. The present invention also relates to nucleotide sequences encoding the polypeptides in the compositions of the present invention. As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell into which a recombinant expression vector has been introduced. Such terms are intended to refer not only to a specific subject cell, but also to the progeny of such a cell.

[0396] In one aspect of the present invention, a vector comprising a polynucleotide encoding a construct of the present invention or a cDNA comprising the polynucleotide is provided. In another aspect of the present invention, a host cell transformed with the vector is provided, which is capable of expressing the construct of the present invention. Suitably, the host cell is a yeast, such as a yeast belonging to the genus Aspergillus, Saccharomyces, Kluyveromyces, Hansenula or Pichia, such as Saccharomyces cerevisiae, Escherichia coli or Pichia pastoris.

[0397] Preparation method

[0398] Polypeptides and constructs comprising polypeptides can be obtained and manipulated using techniques disclosed in, for example, Green and Sambrook 2012.

[0399] Monoclonal antibodies can be produced using hybridoma technology by fusing B cells that produce specific antibodies with myeloma (B cell cancer) cells that are selected for their ability to grow in tissue culture and the absence of antibody chain synthesis. et al., 1975 and Nelson et al., 2000, incorporated herein by reference in their entirety).

[0400] Monoclonal antibodies against a defined antigen can be obtained, for example, by:

[0401] a) immortalizing lymphocytes obtained from the peripheral blood of an animal previously immunized with a defined antigen with immortalized cells, preferably myeloma cells, in order to form hybridomas,

[0402] b) Culturing the resulting immortalized cells (hybridomas) and recovering cells producing antibodies with the desired specificity.

[0403] Alternatively, there is no need to use hybridoma cells. Thus, monoclonal antibodies can be obtained by a method comprising the following steps:

[0404] a) cloning a DNA or cDNA sequence obtained from lymphocytes, especially peripheral blood lymphocytes, of an animal (suitably previously immunized with a defined antigen) into a vector, especially into a bacteriophage and more particularly a filamentous phage,

[0405] b) transforming prokaryotic cells with the above vector under conditions that allow the production of antibodies,

[0406] c) selecting antibodies by subjecting them to antigen-affinity selection,

[0407] d) Recovering antibodies with the desired specificity.

[0408] Methods for immunizing camels, cloning VHH repertoires of B cells circulating in the blood (Chomezynnski et al., 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 are known (WO92 / 01047, Nguyen et al. 2001 and Harmsen et al. 2007). These references are incorporated herein by reference in their entirety.

[0409] Antibody fragments such as scFv and Fv fragments can be isolated and expressed in E. coli (Miethe et al. 2013, Skerra et al. 1988 and Ward et al. 1989, herein incorporated by reference in their entireties).

[0410] Mutations may be made to the DNA or cDNA encoding the polypeptide which are silent for the amino acid sequence of the polypeptide but provide preferred codons for translation in a particular host. Preferred codons for translation of nucleic acids in, for example, Escherichia coli, Pichia pastoris and Saccharomyces cerevisiae are known.

[0411] The sudden change of polypeptide can be realized for example by replacing, adding or lacking the nucleic acid of coded polypeptide.Replacement, adding or lacking the nucleic acid of coded polypeptide can be introduced by many methods, including for example fallibility PCR, mixed, oligonucleotide directed mutagenesis, assembly PCR, PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis (recursiveensemble mutagenesis), exponential ensemble mutagenesis (exponential ensemble mutagenesis), site-specific mutagenesis (Ling et al. 1997, incorporated herein by reference in its entirety), gene reassembly, gene site saturation mutagenesis (GSSM), synthetic connection reassembly (SLR) or the combination of these methods. Modifications, additions or deletions to the nucleic acid can also be introduced by methods including recombination, recursive sequence recombination, phosphorothioate-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.

[0412] Specifically, artificial gene synthesis can be used (Nambiar et al. 1984, Sakamar et al., 1988, Wells et al. 1985 and Grundstrom et al. 1985, incorporated herein by reference in their entirety). Genes encoding polypeptides can be synthesized by, for example, solid phase DNA synthesis. The entire gene can be synthesized from scratch without the need for precursor template DNA. In order to obtain the desired oligonucleotides, the building blocks are sequentially coupled to the growing oligonucleotide chain in the order required for the product sequence. Upon completion of chain assembly, the product is released from the solid phase to the solution, deprotected and collected. The product can be separated by high performance liquid chromatography (HPLC) to obtain the desired high purity oligonucleotides (Verma et al., 1998)

[0413] The expression of immunoglobulin chain variable domains such as VH and VHH can be achieved using suitable expression vectors such as prokaryotic cells such as bacteria, for example Escherichia coli (for example according to the protocol disclosed in WO94 / 04678, which is incorporated herein by reference and described in further detail below). The expression of immunoglobulin chain variable domains such as VH and VHH can also be achieved using eukaryotic cells such as insect cells, CHO cells, Vero cells or suitably yeast cells, such as yeast belonging to the genus Aspergillus, Saccharomyces, Kluyveromyces, Hansenula or Pichia. Suitably, Saccharomyces cerevisiae is used (for example, according to the protocol disclosed in WO94 / 025591, which is incorporated herein by reference and described in further detail below).

[0414] Specifically, VHH can be prepared using Escherichia coli according to the method disclosed in WO94 / 04678 by a method comprising the following steps:

[0415] a) cloning a DNA or cDNA sequence encoding a VHH (e.g. obtained from lymphocytes of camelids or produced synthetically), optionally comprising a His-tag, in a Bluescript vector (Agilent Technologies),

[0416] b) Recovering the cloned fragment after amplification using a 5' primer specific for the VHH containing an XhoI site and a 3' primer containing an SpeI site having the sequence,

[0417] TC TTA ACT AGT GAG GAG ACG GTG ACC TG (SEQ ID NO: 13),

[0418] c) cloning the fragment recovered in the phase in the Immuno PBS vector after digestion of the vector with XhoI and SpeI restriction enzymes (Huse et al. 1989, herein incorporated by reference in its entirety),

[0419] d) transforming a host cell, especially Escherichia coli, by transfection with the recombinant Immuno PBS vector of step c,

[0420] e) Recovering the expression product of the VHH coding sequence, for example by affinity purification, such as by chromatography on a column using protein A, cation exchange or nickel-affinity resin (if the VHH comprises a His-tag).

[0421] Alternatively, immunoglobulin chain variable domains such as VH and VHH can be obtained by a method comprising the following steps:

[0422] a) obtaining a DNA or cDNA sequence encoding a VHH having a defined specific antigen binding site,

[0423] b) amplifying the obtained DNA or cDNA using a 5' primer containing a start codon and a Hindlll site and a 3' primer containing a stop codon and having an XhoI site,

[0424] c) recombining the amplified DNA or cDNA into the Hindlll (position 2650) and XhoI (position 4067) sites of plasmid pMM984 (Merchlinsky et al. 1983, incorporated herein by reference in its entirety),

[0425] d) transfecting permissive cells, in particular NB-E cells (Faisst et al. 1995, herein incorporated by reference in its entirety), with the recombinant plasmid,

[0426] e) recovering the product obtained.

[0427] Additionally, immunoglobulin chain variable domains such as VHH or VH can be produced using E. coli or S. cerevisiae according to the methods disclosed in Frenken et al. 2000 and WO99 / 23221 (incorporated herein by reference in their entirety), as follows:

[0428] After taking a blood sample from the immunized llama and enriching the lymphocyte population via Ficoll (neutral highly branched, high-quality hydrophilic polysaccharide that dissolves rapidly in aqueous solution - Pharmacia) discontinuous gradient centrifugation, isolating total RNA by acid guanidine thiocyanate extraction (Chomezynnski et al., 1987) and first-strand cDNA synthesis (e.g., using a cDNA kit such as RPN 1266 (Amersham)), a DNA fragment encoding a portion of the VHH and VH fragments and the short or long hinge region is amplified by PCR using specific primers described in detail on pages 22 and 23 of WO99 / 23221. After digestion of the PCR fragment with PstI and HindIII or BstEII, the DNA fragment having a length between about 300 and 450 bp is purified via agarose gel electrophoresis and ligated in the E. coli phagemid vector pUR4536 or the episomal S. cerevisiae expression vector pUR4548, respectively. pUR4536 is derived from pHEN (Hoogenboom et al. 1991, incorporated herein by reference in its entirety) and contains lacI qpUR4548 was derived from pSY1 (Harmsen et al. 1993, incorporated herein by reference in its entirety). The BstEII site in the leu2 gene was removed from this plasmid via PCR, and the cloning site between the SUC2 signal sequence and the terminator was replaced to facilitate cloning of VH / VHH gene segments. VH / VHH had a c-myc tag at the C-terminus for detection. Individual E. coli JM109 colonies were transferred to medium supplemented with 1% glucose and 100 mg L -1 After overnight growth (37°C), the cells were plated in 96-well microtiter plates containing 150 ml 2TY medium containing 100 mg L -1 Replicate plates in 2TY medium of ampicillin and 0.1mM IPTG. After overnight incubation again and optionally freezing and thawing, the cells are centrifuged and granulated, and the supernatant can be used in ELISA. Each Saccharomyces cerevisiae colony is transferred to a test tube containing a selective minimal medium (comprising 0.7% yeast nitrogen base, 2% glucose, supplemented with essential amino acids and bases), and grown for 48h at 30°C. Subsequently, the culture is diluted ten times in YPGal medium (comprising 1% yeast extract, 2% bacterial peptone and 5% galactose). After growing for 24h and 48h, the cells are granulated and the culture supernatant can be analyzed in ELISA. The absorbance (OD600) at 600nm is optionally measured.

[0429] Additionally, immunoglobulin chain variable domains such as VH / VHH can be produced using Saccharomyces cerevisiae or Pichia pastoris using the following procedure:

[0430] Isolate the naturally occurring DNA sequence encoding VH / VHH or obtain a synthetically produced DNA sequence encoding VH / VHH, comprising a 5'-UTR, a signal sequence, a stop codon and flanked by Sad and HindIII sites (such synthetic sequences can be produced as outlined above or can be ordered, for example, from commercial suppliers such as Geneart (Life Technologies)).

[0431] The VH / VHH genes are transferred to the multicopy integration (MCI) vector pUR8569 or pUR8542 using restriction sites as described below. The DNA sequences encoding VHH, optionally contained in the shuttle vector, cassette or other synthetic gene construct and MCI vector, are cut with Sad and HindIII using the following: 25ul VHH DNA (Geneart plasmid or MCI vector), 1ul Sad, 1ul HindIII, 3ul of a suitable buffer for double digestion such as NEB buffer 1 (New England Biolabs), overnight at 37°C. 25ul of digested DNA encoding VHH and 25ul of digested MCI vector are run on a 1.5% agarose gel with 1xTAE buffer and then gel extracted, for example, using a QIAquick gel extraction kit (Qiagen). The ligation of the digested MCI vector and the digested DNA encoding VH / VHH was set up as follows: 100 ng vector, 30 ng VHH gene, 1.5 ul 10× ligase buffer, 1 ul T4 DNA ligase and ddH 2 O. The ligation was then performed at 16° C. overnight.

[0432] Next, transform the E. coli cells. For chemically competent XL-1 blue cells, 200ul of heat competent XL-1 blue cells were thawed and 5ul of ligation mixture was added on ice for about 30 minutes, followed by heat shock at 42°C for 90 seconds. Then 800ul of Luria-Bertani low salt medium supplemented with 2% glucose was added, and the cells were recovered at 37°C for 2 hours. The cells were plated on Luria-Bertani agar and ampicillin (100ug / ml) plates and kept overnight at 37°C. For electrocompetent TG1 E. coli cells, an electroporation cup was used. In the electroporation cup: 50ul of electrocompetent TG1 cells and 1ul of ligation mixture were thawed on ice for about 15 minutes. The cup was placed in a support and pulsed. 500ul of 2TY medium was added and the cells were recovered at 37°C for 30 minutes. 100ul of cells were plated on Luria-Bertani agar plates containing ampicillin (100ug / ml) and 2% glucose. The plates were kept at 37°C overnight.

[0433] After cloning the VH / VHH genes into E. coli as described in detail above, the linearized MCI vector can be used to transform Saccharomyces cerevisiae or Pichia pastoris. Before transformation, several steps are performed: (i) the DNA should be changed from circular to linear by digestion, otherwise the DNA cannot be integrated into the yeast genome, and (ii) the digested DNA should be cleared of impurities by ethanol precipitation. In addition, during the transformation process, the yeast cells are made semipermeable so that the DNA can pass through the membrane.

[0434] Preparation for yeast transformation: HpaI digestion of midi-prep prepared from selected E. coli colonies expressing VH / VHH genes was performed as follows. Prepare a 100 ul solution containing 20 ng midi-prep, 5 ul HpaI, 10 ul of an appropriate buffer such as NEB4 buffer (BioLabs) and ddH2O.

[0435] Cut DNA with HpaI at room temperature overnight. Next, perform ethanol precipitation (and place the 5ul sample from HpaI digestion on one side). Add 300ul 100% ethanol to the midiprep of 95ul HpaI digestion, vortex and spin at full speed for 5 minutes. Carefully decant when there is a pellet, add 100ul 70% ethanol, and then spin again at full speed for 5 minutes. Decant the sample again and keep at 50-60°C until the pellet is dry. Resuspend the pellet in 50ul ddH2O. Run 5ul on the gel next to the sample of 5ul HpaI digestion.

[0436] Yeast transformation: Prepare YNBglu plates. Use 10g agar + 425ml water (sterilized), 25ml filtered 20×YNB (3.35g YNB (yeast nitrogen base) in 25ml sterilized H2O) and 50ml sterile 20% glucose and pour into a petri dish. Pick up a yeast colony from the mother plate and grow overnight in 3ml YPD (yeast extract peptone dextrose) at 30°C. Prepare about 600ml YPD on the second day and fill 3 flasks with 275ml, 225ml and 100ml YPD. Add 27.5ul yeast YPD culture to the first flask and mix gently. Take 75ml from the first flask and place it in the second flask, mix gently. Take 100ml from the second flask and place it in the third flask, mix gently. Grow until an OD660 between 1 and 2 is reached. Split the flasks that reach this OD on 4 Falcon tubes, ±45ml in each. Spin at 4200rpm for 2 minutes. Discard the supernatant. Dissolve the pellets in two Falcon tubes in 45ml H2O (reduce the number of tubes from 4 to 2). Spin at 4200rpm for 2 minutes. Dissolve the pellets in 45ml H2O (from 2 tubes to 1 tube). Spin at 4200rpm for 2 minutes. Dissolve the pellets slightly in 5ml lithium acetate (LiAc) (100mM) and spin for a few seconds. Carefully discard some LiAc, but keep more than half of the LiAc in the tube. Vortex the cells, boil the vector DNA for 5 minutes and cool quickly in ice water. Add to a 15ml tube containing the following: 240ul PEG, 50ul cells, 36uLiAc (1M), 25ul vector DNA, 45ul ethanol-precipitated VH / VHH. Mix gently after each step (blank samples are treated the same way, but there is no ethanol-precipitated VH / VHH). Incubate at 30°C for 30 minutes, invert the tube slightly 3-4 times, and then heat shock at 42°C for 20-25 minutes. Rotate for a short time at up to 6000rpm. Gently remove the supernatant and add 250ul ddH2O and mix. On the YNBglu plate, all of them are streaked until the plate is dry, and grow for 4-5 days at 30°C. Finally, the YNBglu plate is prepared by the following: the plate is divided into 6 equal parts, each part is numbered 1 to 6, the largest colony is inoculated and No. 1 is streaked. Repeat the above process for 1 to 6 other colonies from large to small. Grow up at 30°C for 3-4 days until colonies are produced. Glucose is used as a carbon source to grow VH / VHH colonies, and galactose-7-promoter is opened by adding 0.5% galactose to induce VH / VHH expression. 3mL small-scale culture is carried out to test colonies, and the colonies showing the best expression of VH or VHH are selected. This colony was then used in the purification.

[0437] Purification: VH / VHH are purified by cation exchange chromatography with a strong anion resin such as Capto S. On day 1, a selected yeast colony expressing VH / VHH is inoculated in 5 ml YPD medium (YP medium + 2% glucose) and the cells are grown overnight in a 25 mL sealed sterile tube at 30°C (shaking at 180 rpm). On day 2, 5 ml of the overnight culture is diluted in 50 mL of freshly prepared YP medium + 2% glucose + 0.5% galactose and the cells are grown in a 250 ml aerated baffled flask at 30°C for two nights (shaking at 180 rpm). On day 4, the cells are spun down at 4200 rpm in a centrifuge for 20 min. Cation exchange purification step using a strong anion resin: The pH of the supernatant containing the ligand is adjusted to 3.5. 0.75ml resin (+ / -0.5mL slurry) / 50mL supernatant was washed with 50mL ddH2O, followed by three washes with binding buffer. The washed resin was added to the supernatant and the suspension was incubated on a shaker at 4°C for 1.5 hours. The resin-bound VH / VHH was pelletized by centrifugation at 500g for 2 minutes and washed with washing buffer. The supernatant was decanted and the resin was resuspended with 10mL binding buffer. The filter was placed in a PD-10 column, the resin was poured into the column and the resin was settled for a while, and then the filter was added to the resin. Wait until all binding buffer runs through. VH / VHH was eluted with 6x0.5ml elution buffer. Elution fractions were collected in eppendorf tubes. The protein concentration of 6 elution fractions was measured with Nanodrop. The fractions containing VHH were pooled and the solution was transferred to a 3,500Da cut-off dialysis membrane. The purified protein solution was dialyzed against 3L PBS overnight at 4°C. On day 5, the purified protein solution was dialyzed against 2 L of fresh PBS at 4 °C for another 2 h. Finally, the final concentration was calculated by BCA.

[0438] Although discussed in the context of VH / VHH, the techniques described above can also be applied to scFv, Fab, Fv and other antibody fragments if desired.

[0439] Multiple antigen binding fragments (suitably VH / VHH) can be fused by chemical cross-linking by reacting amino acid residues with an organic derivatizing agent, such as described by Blattler et al. 1985 (incorporated herein by reference in its entirety). Alternatively, the antigen binding fragments can be genetically fused at the DNA level, i.e., a polynucleotide construct is formed that encodes a complete polypeptide construct comprising one or more antigen binding fragments. One way to join multiple antigen binding fragments via a genetic approach is to connect the sequences encoding the antigen binding fragments directly or via a peptide linker. For example, the carboxyl terminus of the first antigen binding fragment can be connected to the amino terminus of the next antigen binding fragment. This connection mode can be extended so that antigen binding fragments are connected for the construction of trifunctional, tetrafunctional constructs, etc. Methods for generating multivalent (such as bivalent) VHH polypeptide constructs are disclosed in WO 96 / 34103 (incorporated herein by reference in its entirety).

[0440] Suitably, the polypeptide may be produced in a fungus, such as yeast (e.g., Saccharomyces cerevisiae or Pichia pastoris), according to the method disclosed in WO02 / 48382, comprising growing the fungus on a medium comprising a carbon source, wherein 50-100 wt % of the carbon source is ethanol. Large-scale production of VHH fragments in Saccharomyces cerevisiae is described in Thomassen et al. 2002 (incorporated herein in its entirety by reference).

[0441] In one aspect of the present invention, a method for preparing a composition of the present invention is provided, the method comprising the steps of expressing a polynucleotide encoding a TNF-α binding polypeptide using a suitable host, and expressing a polynucleotide encoding an IL-7R binding polypeptide in a suitable host and adding both polypeptides to the composition. In another aspect of the present invention, a method for preparing a construct of the present invention is provided, the method comprising the step of expressing a polynucleotide encoding a construct of the present invention using a suitable host.

[0442] Other embodiments of the invention are set forth in the following clauses.

[0443] Terms

[0444] 1. A composition comprising a TNF-α binding polypeptide and an IL-7R binding polypeptide.

[0445] 2. A composition according to clause 1, wherein:

[0446] (a) the TNF-α binding polypeptide comprises three complementarity determining regions (CDR1-CDR3), wherein CDR1 comprises a sequence having 60% or greater sequence identity to SEQ ID NO: 1, CDR2 comprises a sequence having 50% or greater sequence identity to SEQ ID NO: 2, and CDR3 comprises a sequence having 60% or greater sequence identity to SEQ ID NO: 3; and

[0447] (b) the IL-7R binding polypeptide comprises three complementarity determining regions (CDR1-CDR3), wherein CDR1 comprises a sequence having 60% or greater sequence identity to SEQ ID NO:9, CDR2 comprises a sequence having 50% or greater sequence identity to SEQ ID NO:10 and CDR3 comprises a sequence having 60% or greater sequence identity to SEQ ID NO:11.

[0448] 3. A composition according to clause 1 or 2, wherein the polypeptide comprising the antibody fragment is an immunoglobulin chain variable domain.

[0449] 4. A composition according to any one of clauses 1 to 3, wherein the TNF-α binding polypeptide is present in an amount of 10 -7 M or less Kd binds to TNF-α, and the IL-7R binding polypeptide binds to TNF-α with a Kd of 10 -7 The IL-7R binding protein was expressed in 4% IL-7R and had a Kd of M or less.

[0450] 5. A composition according to any one of clauses 1 to 4, wherein the TNF-α binding polypeptide neutralizes human TNF-α cytotoxicity in the L929 assay with an EC50 of 100 nM or less, and the IL-7R binding polypeptide neutralizes IL-7R-dependent, IL-7-induced STAT5 phosphorylation in human lymphocytes with an EC50 of 100 nM or less.

[0451] 6. The composition of any one of clauses 1 to 5, wherein the TNF-α binding polypeptide and the IL-7R binding polypeptide are linked.

[0452] 7. The composition according to clause 6, wherein the TNF-α binding polypeptide and the IL-7R binding polypeptide are linked by a protease-labile peptide linker.

[0453] 8. A composition according to clause 7, wherein the protease-labile peptide linker comprises K and / or R residues.

[0454] 9. A composition according to any one of clauses 1 to 8, wherein the polypeptide is substantially resistant to one or more proteases present in the small intestine or large intestine.

[0455] 10. A pharmaceutical composition comprising the composition according to any one of clauses 1 to 9 and a pharmaceutically acceptable excipient.

[0456] 11. A composition according to any one of clauses 1 to 10, wherein the composition comprises an enteric coating.

[0457] 12. The composition according to any one of clauses 1 to 11, wherein the composition is suitable for oral administration.

[0458] 13. A composition according to any one of clauses 1 to 12 for use in the treatment or prevention of an autoimmune disease and / or an inflammatory disease.

[0459] 14. A TNF-α binding polypeptide for use together with an IL-7R binding polypeptide in treating or preventing autoimmune diseases and / or inflammatory diseases.

[0460] 15. An IL-7R binding polypeptide for use together with a TNF-α binding polypeptide in treating or preventing autoimmune diseases and / or inflammatory diseases.

[0461] The invention will now be further described by way of the following non-limiting examples.

[0462] Example

[0463] Examples 1 to 3 provide information on the properties of specific IL-7R binding polypeptides. Examples 4 and 5 provide information on experiments involving IL-7R binding polypeptides and TNF-α binding polypeptides used in combination. Example 6 details the production of constructs comprising IL-7R binding polypeptides and TNF-α binding polypeptides.

[0464] Example 1: Potency of IL-7R Binding Peptides ID-A40U, V7R-2E9 and ID-A62U Compared to IL-7R Binding Peptides of the Prior Art

[0465] The inhibitory potency and efficacy (maximum inhibition) of IL-7R binding polypeptides ID-A40U (SEQ ID NO: 24) and V7R-2E9 (SEQ ID NO: 25) produced in Escherichia coli were determined in vitro in L-7 / IL-7R neutralization ELISA and compared with the clinical anti-IL-7R antibody mAb829 (also known as "GSK2618960", an anti-IL-7Rα monoclonal antibody disclosed in Ellis et al. 2019).

[0466] Start at 300nM and use 3.2 dilution factors, prepare 7-point dilution series of ICVD in 1% BSA (under 2x determination concentration).Use mAb829 contrast antibody as positive control in ELISA, concentration range is between 10nM and 0.088nM (2x determination concentration).Prepare enough volume in triplicate for each ICVD dilution, prepare enough volume in 2 triplicates (2 plates) for mAb829 simultaneously.Each ICVD (or mAb829) dilution of 85 μ L (or 170 μ L) is mixed with 10ng / mL IL-7 (2x determination concentration) of 85 μ L (or 170 μ L).85 μ L of IL-7 is mixed with 85 μ L of blocking buffer, to have IL-7 (1x) complete binding signal in each plate.Also add separate blocking buffer in each plate as blank. Bound IL-7 was then measured using biotinylated anti-hIL-7 followed by Extravidin-HRP. The TMB reaction was stopped after 30 minutes.

[0467] A using ELISA signal blank correction 450 The data were fitted with a 'log(inhibitor) vs. response - variable slope (four parameters)' curve and the EC 50 To generate EC in Graphpad Prism 50 value.

[0468] In addition, the ability of ID-A40U and V7R-2E9 to inhibit IL-7 binding to IL-7Rα and block STAT5 phosphorylation was tested in human lymphocytes in vitro. 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 abolished by IL-7Rα-specific ICVD that prevents IL-7 / IL-7R binding.

[0469] Lymphocyte enriched populations were isolated from human buffy coats and stored in 90% FBS 10% DMSO in liquid nitrogen. Cells were thawed and allowed to recover in complete RPMI-1640. After recovery, cells were plated at 100 μl of 2.5 x 10 5Cells / well were plated in round-bottom 96-well plates and starved for 1 h in complete RPMI without FBS. After starvation, the desired concentration of ICVD was added to each well (50 μL / well), and the plate was incubated for 15 min at room temperature. Then 50 μL / well of IL-7 was added to each well, and the plate was incubated for 15 min at 37°C 5% CO2. The reaction was stopped by rapidly cooling the plate on ice, followed by centrifugation and removal of the supernatant. The cells were then processed for fixation, permeabilization, and pSTAT5 intracellular staining. Cells were incubated on ice with 100 μL / well Cytofix / Cytoperm solution (BD Bioscience #554722) for 20 min, washed twice with 150 μL / well 1x Perm / wash buffer (BD Bioscience #554723), incubated on ice with 200 μL / well Perm buffer III (BD Bioscience #558050) for 30 min and washed twice with 150 μL / well 1x PBS 2% BSA (FACS buffer). Cells were then stained with 25 μL / well pSTAT5 antibody ([47 / Stat5 (pY694)] (A488) (BD Bioscience #612598)) or isotype control mouse IgG1 ([B11 / 6] (FITC) (Abcam #ab91356)) for 1 h at room temperature. The reaction was stopped by adding 150 μL / well FACS buffer. After 1 wash / centrifugation step, cells were finally resuspended in 200 μL / well of FACS buffer and data were acquired in a CytoFlex flow cytometer (Beckman Coulter). Data analysis was performed using FlowJo software.

[0470] The results of these experiments are shown in Table 1a along with the comparatives.

[0471] Table 1a

[0472]

[0473] In a separate experiment, the same IL-7 / IL-7R neutralization ELISA was performed on ID-A62U (SEQ ID NO: 16) (produced in S. cerevisiae) along with comparators. The results are shown in Table 1b. ID-A62U contains E1D and R45L mutations relative to ID-A40U.

[0474] Table 1b

[0475]

[0476] The ability of V7R-2E9 to neutralize the binding of the L-TSLP / TSLP-R complex to IL-7Rα was tested. A 96-well plate was coated with 0.25 μg / mL recombinant human IL-7Rα-His6-Fc+5 μg / mL BSA and then blocked. V7R-2E9 was serially diluted and mixed 1:1:1 with recombinant human L-TSLP (15 ng / mL final concentration) and human TSLP-R (20 ng / mL final concentration). The mixture was then incubated for 30 minutes to allow binding before being added to the IL-7Rα-coated plate. After a 2-hour incubation, 50 μL / well 0.3 μg / mL biotinylated rabbit anti-hTSLP antibody was used, and then 50 μL / well 1 / 2000 Extravidin-HRP was used to detect the bound L-TSLP, and the level of neutralization of the binding of the L-TSLP / TSLP-R complex to IL-7Rα by ICVD was determined using GraphPad Prism. The results are shown in Table 1c below.

[0477] Table 1c

[0478]

[0479] In summary, ID-A62U and ID-A40U exhibited comparable or greater potency than the comparator clinical anti-IL-7R antibody mAb829. Notably, both ID-A40U and ID-A62U, which belong to the same ICVD family, maintained high potency even though they do not have the same sequence (ID-A62U contains E1D and R45L mutations relative to ID-A40U) and are produced using different organisms.

[0480] Example 2: Biacore estimation of ICVD-IL-7R binding affinity

[0481] The binding kinetics of ID-A40U were compared to the mAb829 clinical antibody in a Biacore study. IL-7Rα-His6-Fc was directly coated on the Biacore sensor plate (for mAb829 analysis) or captured by anti-human IgG Fc (for ICVD analysis), and ICVD / Ab was flowed through the plate to detect binding. ID-A40U has a 7.8x10 -11 The affinity of M (K D ), and mAb829 had 5.67x10 -10 The slightly lower affinity (K D ). The results indicated that ID-A40U showed strong binding to the antigen. ID-A62U contained E1D and R45L mutations relative to ID-A40U.

[0482] Example 3: Tolerance to Gastrointestinal Extracts

[0483] In vitro incubation in intestinal supernatant can predict the stability of ICVD in the intestinal tract of cynomolgus monkey and human.The activity of major small intestinal protease trypsin and chymotrypsin is conserved in mammalian species, and the protease present in the large intestine may be produced by host species-specific intestinal microflora.In order to generate the test matrix reflecting these two environments, the mouse small intestinal supernatant and the fecal supernatant collected are prepared.These two matrices are highly digestible for non-selected non-engineered ICVD.

[0484] The anti-TNF-α ICVD ID-38F has been previously shown to have high stability in these matrices (see WO2016 / 156465), and this property predicts that ID-38F will have high stability during transit through the intestine.

[0485] V7R-2E9 and ID-A40U were tested for survival in gastrointestinal extracts from both mice and humans. ICVD was incubated with mouse small intestinal supernatant for 6 hours at 37°C and with human fecal supernatant for 16 hours. Survival was measured by the IL-7 / IL-7R neutralization ELISA detailed above in Example 1. Both constructs showed good survival in the digestible matrices tested ( Figure 1 , where "SI" = mouse small intestinal fluid, and "HF" = human fecal supernatant).

[0486] In a separate experiment, ID-A62U and ID-A41U (unstable comparator ICVD) were tested for survival in the same human fecal supernatant assay. ID-A62U demonstrated approximately 100% survival ( ) compared to approximately 40% survival for ID-A41U. Figure 2 , ID-A62U is marked as 'A62U' and ID-A41U is marked as 'A41U').

[0487] These are rigorous tests involving extended incubation periods. Therefore, it is expected that any of these ICVDs survive very well in the gastrointestinal environment.

[0488] Example 4: Study on the inhibitory effects of single and combined administration of TNF-α binding polypeptide and IL-7R binding polypeptide on phosphorylation of signal transduction proteins in ex vivo cultured IBD tissues

[0489] A study was conducted to investigate the inhibitory effects of TNF-α binding polypeptides and IL-7R binding polypeptides on the levels of phosphoprotein biomarkers and spontaneous production of inflammatory cytokines in ex vivo cultures of inflamed colonic mucosal tissue obtained from UC patients. The polypeptides tested were all ICVDs. The ICVDs were ID-A62U (the IL-7R-binding ICVD discussed above in Examples 1-3) and ID-38F (the TNF-α-binding ICVD disclosed in WO2016 / 156465). The effects of the individual ICVDs were compared with a mixture of the two ICVDs and a negative control ICVD ("ID-2A", an unrelated ICVD that does not bind to any target) to evaluate the effects of combining different anti-cytokine mechanisms.

[0490] Biopsies from four different UC patients were incubated with different ICVDs (control ID-2A 50 nM, ID-38F 50 nM, ID-A62U 50 nM, or ID-38F + ID-A62U, 50 nM each) for 24 h, and after treatment, tissue lysates were analyzed on a phosphoprotein antibody array. The resulting histogram ( Figure 3-4 ) provides a visual representation of the combined phosphorylation intensity data obtained for each biopsy (4 / treatment). The inhibitory effect of different ICVD treatments is shown as a change from the main high phosphorylation intensity value of the biopsy treated with control ICVD ID-2A to the relatively low phosphorylation intensity value of the biopsy treated with anti-TNF-α or anti-IL-7R ICVD or a combination of the two. These average results show that the overall inhibitory effect of ID-A62U and ID-38F on tissue phosphorylation levels is consistent, wherein the effect is greater when two ICVD combinations. It is not clear which phosphoprotein may be more important in this analysis, so the overall effect of treatment on protein phosphorylation in each biopsy is evaluated.

[0491] Figure 5 The results shown in show the total phosphorylation intensity values ​​measured for four biopsies per patient. For each biopsy, the total phosphorylation level was calculated by adding together the intensity values ​​measured for all 45 proteins on the array. For each patient, the total phosphorylation level measured for each treatment is shown. Three patients (UC2748, UC2749 and UC2750) showed significant inhibition (about 50% when used in combination), while the fourth UC2747 also showed significant inhibition, but the response was generally poor.

[0492] The combined inhibitory effect of ID-38F plus ID-A62U treatment exceeded that achieved by ICVD alone and resulted in near maximal inhibition of most phosphoproteins. Due to the unexpectedly high levels of inhibition observed in this experiment, definitive evidence of a synergistic effect of the combined agents in UC tissues needs to be explored further. However, the evidence of significant inhibition of inflammatory biomarkers in UC tissues by both the IL-7R binding polypeptide and the TNF-α binding polypeptide is encouraging and suggests good potential for observable synergy when lower concentrations of the polypeptides are combined.

[0493] Overall, these results demonstrate at least additive and potentially synergistic effects of co-administered anti-TNF-α and anti-IL-7R polypeptides.

[0494] Example 5: Study on the inhibitory effects of TNF-α binding polypeptide and IL-7R binding polypeptide alone and in combination on the production of cytokines in ex vivo cultures of IBD tissues

[0495] Figure 6-8 The effects of different treatments on the average spontaneous cytokine production in the biopsies discussed in Example 4 above are shown. Treatment with either ID-38F or ID-A62U alone inhibited the production of IL-8, TNFα, and IL-17F, but had little effect on other cytokines. However, when ID-38F and ID-A62U were used in combination, the inhibitory effects on IL-1β, IL-6, IL-8, IL-17A, and IL-33 were significantly greater than those seen with ICVD alone. The inhibitory effects of the combination on TNFα and IL-17F production were no greater than those seen with ICVD alone as described above.

[0496] In conclusion, the results obtained in three patients in this study indicate that treatment with either ID-38F or ID-A62U alone inhibited the production of some proinflammatory cytokines (IL-8, TNFα, IL-17F). The inhibitory effect on other cytokines was small / partial, which may reflect the submaximal concentrations of ICVDs selected for this study. Importantly, when both ICVDs were used in combination, the inhibitory effect on the production of most cytokines, including IL-1β, IL-6, IL-8, IL-17A, and IL-33, was increased.

[0497] Overall, these results demonstrate at least additive and potentially synergistic effects of co-administered anti-TNF-α and anti-IL-7R polypeptides.

[0498] Example 6: Generation, cleavage and testing of heterobiheads comprising a TNF-α binding polypeptide and an IL-7R binding polypeptide connected by a labile linker

[0499] The combination of ID-38F and ID-A62U separated by a flexible (G4S)2 linker (SEQ ID NO: 21) with a central lysine (K) residue to create a trypsin cleavable site resulted in a double-headed construct named "FU3K".

[0500] FU3K is cloned into the vector pUR9013 on the SacI / HindIII fragment to promote stable multi-copy integration into the chromosome of the Saccharomyces cerevisiae expression strain. This is achieved according to standard cloning procedures. Using this integration and expression system, FU3K is under the control of the galactose inducible promoter and realizes double-headed secretion through the yeast mating factor α signal sequence. The expression of FU3K from the yeast chromosome was evaluated in 50mL induced cultures. Full-length FU3K is expressed well in small-scale yeast cultures.

[0501] Incubation of FU3K with trypsin at 37°C resulted in rapid separation of the ID-38F and ID-A62U monomer arms. Analysis by SDS PAGE ( Fig. 9 , where lanes are time (min), St = standard (no trypsin added), L = molecular weight marker, equal volume loaded in each lane). Bands corresponding to the uncleaved FU3K double head (about 27 kDa) and the cleaved monomer (about 13.5 kDa) are clearly visible. This confirms that FU3K is in a well-formed state and rapidly releases the two monomer arms upon exposure to trypsin in the human small intestine or microbial trypsin-like proteases in the colon.

[0502] The IL-7 / IL-7R ELISA was performed using the method described in Example 1 above. Before digestion, FU3K had the same potency against IL-7R as ID-A62U, and after digestion, FU3K maintained high (subnanomolar) potency against IL-7R ( Fig.10 , ID-A62U is marked as "A62U").

[0503] Biotinylated Humira competition ELISA was performed to measure the competition of overlapping epitopes of ID-38F and Humira to TNFα. The ELISA plate was coated with 100ng / mL human TNFα in 250μg / mL bovine serum albumin (BSA) in phosphate buffered saline (1x PBS) and blocked with 1% BSA in 1x PBS. Biotinylated adalimumab was mixed with all standards and samples at 1:1, and a final concentration of 2nM biotinylated adalimumab was given before the mixture was added to the plate. ExtrAvidin-horseradish peroxidase was used to detect the bound biotinylated adalimumab, and TMB Microwell substrate was used for visualization, then 0.5M H2SO4 was terminated and read at 450nm.

[0504] In this competition ELISA, FU3K was found to be as effective as ID-38F before and after digestion ( Fig.11 ).

[0505] The two monomer arms of FU3K showed retention of the favorable stability characteristics of the parent monomers ID-38F and ID-A62U after incubation in human fecal supernatant for 4 hours (performed as described in Example 2 above). Fig.12 ).

[0506] These data suggest that FU3K is a suitable format to deliver high concentrations of each of these monomers directly to the human colon as a dual therapy.

[0507] Other aspects

[0508] All references, including patents and patent applications, cited in this application are incorporated herein by reference to the maximum extent possible.

[0509] Throughout this specification and the following claims, unless the context requires otherwise, the word 'comprise' and variations such as 'comprises' and 'comprising' will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not the exclusion of any other integer, step, any other group of integers or any other group of steps.

[0510] The application of which this description and claims form a part may be used as a basis for priority with respect to any subsequent application. The claims of such subsequent applications may be directed to any feature or combination of features described herein. They may be in the form of product, composition, method or use claims, and may include, for example, but not limited to, the following claims.

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[0565] Verstraete et al., Nat Commun. 2017 8: 14937. doi: 10.1038 / ncomms14937

[0566] Ward et al., Nature 1989 341: 544-546 Sequence Listing <110> wesquald gmbh <120> Composition <130> VHS-P2586PCT <160> 43 <170> PatentIn Version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 1 Ser His Trp Met Tyr 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 2 Glu Ile Asn Thr Asn Gly Leu Ile Thr His Tyr Gly Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 3 Asn Gln His Gly Leu Asn 1 5 <210> 4 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 4 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser 20 25 30 <210> 5 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 5 Trp Val Arg Gln Ala Pro Gly Lys Glu Leu Glu Trp Leu Ser 1 5 10 <210> 6 <211> 32 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 6 Arg Phe Thr Val Ser Arg Asn Asn Ala Ala Asn Lys Met Tyr Leu Glu 1 5 10 15 Leu Thr Arg Leu Glu Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Arg 20 25 30 <210> 7 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 7 Lys Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 8 <211> 115 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 8 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Ser His 20 25 30 Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Glu Leu Glu Trp Leu 35 40 45 Ser Glu Ile Asn Thr Asn Gly Leu Ile Thr His Tyr Gly Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asn Asn Ala Ala Asn Lys Met Tyr 65 70 75 80 Leu Glu Leu Thr Arg Leu Glu Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Arg Asn Gln His Gly Leu Asn Lys Gly Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115 <210> 9 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 9 Ser Asp Ala Met Gly 1 5 <210> 10 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 10 Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val Lys 1 5 10 15 Gly <210> 11 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 11 Asp Tyr Asp Thr Asp Val Trp Gln Tyr 1 5 <210> 12 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 12 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ser Ile Ser Thr Phe Ser 20 25 30 <210> 13 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 13 Trp Phe Arg Gln Ala Pro Gly Lys Glu Leu Glu Phe Leu Ala 1 5 10 <210> 14 <211> 32 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 14 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Arg Tyr Tyr Cys Ala Glu 20 25 30 <210> 15 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 15 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 16 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 16 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Leu Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 17 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 17 gatgttcaat tggttgaatc tggtggtggt ttggttcaac caggtggttc tttgaaattg 60 tcttgtgctg cttctggttt cgatttctct tctcattgga tgtactgggt tagacaagct 120 ccaggtaaag aattggaatg gttgtctgaa atcaacacca acggtttgat tacccattat 180 ggtgattctg tcaagggtag attcactgtc tctagaaaca atgctgctaa caagatgtac 240 ttggaattga ccagattgga accagaagat actgccttgt attactgcgc tagaaatcaa 300 catggtttga acaaaggtca aggtactcaa gttaccgttt cctcataatg a 351 <210> 18 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 18 gatgttcaat tggttgaatc tggtggtggt ttggttcaag ccggtggttc tttgagattg 60 tcttgtgaat cttctatctc caccttctca tctgatgcta tgggttggtt tagacaagct 120 ccaggtaaag aattggaatt tttggctgct attggttgga gtggtgctgt tactcattat 180 tccgattctg ttaaaggtcg tttcaccatt tctagagata acgctaagaa caccgtctac 240 ttgcaaatga actctttgag agctgaagat accggtagat attactgcgc tgaagattac 300 gatactgatg tttggcaata ttggggtcaa ggtactcaag ttactgtctc ctcatgataa 360 <210> 19 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 19 Asp Asp Asp Asp Lys 1 5 <210> 20 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 20 Gly Gly Gly Gly Ser Asp Asp Asp Asp Lys Gly Gly Gly Gly Ser 1 5 10 15 <210> 21 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 21 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Gly Gly Gly Gly Ser 1 5 10 15 Gly Gly Gly Gly Ser 20 <210> 22 <211> 159 <212> PRT <213> Homo sapiens <400> 22 Met Phe Pro Phe Ala Leu Leu Tyr Val Leu Ser Val Ser Phe Arg Lys 1 5 10 15 Ile Phe Ile Leu Gln Leu Val Gly Leu Val Leu Thr Tyr Asp Phe Thr 20 25 30 Asn Cys Asp Phe Glu Lys Ile Lys Ala Ala Tyr Leu Ser Thr Ile Ser 35 40 45 Lys Asp Leu Ile Thr Tyr Met Ser Gly Thr Lys Ser Thr Glu Phe Asn 50 55 60 Asn Thr Val Ser Cys Ser Asn Arg Pro His Cys Leu Thr Glu Ile Gln 65 70 75 80 Ser Leu Thr Phe Asn Pro Thr Ala Gly Cys Ala Ser Leu Ala Lys Glu 85 90 95 Met Phe Ala Met Lys Thr Lys Ala Ala Leu Ala Ile Trp Cys Pro Gly 100 105 110 Tyr Ser Glu Thr Gln Ile Asn Ala Thr Gln Ala Met Lys Lys Arg Arg 115 120 125 Lys Arg Lys Val Thr Thr Asn Lys Cys Leu Glu Gln Val Ser Gln Leu 130 135 140 Gln Gly Leu Trp Arg Arg Phe Asn Arg Pro Leu Leu Lys Gln Gln 145 150 155 <210> 23 <211> 63 <212> PRT <213> Homo sapiens <400> 23 Met Phe Ala Met Lys Thr Lys Ala Ala Leu Ala Ile Trp Cys Pro Gly 1 5 10 15 Tyr Ser Glu Thr Gln Ile Asn Ala Thr Gln Ala Met Lys Lys Arg Arg 20 25 30 Lys Arg Lys Val Thr Thr Asn Lys Cys Leu Glu Gln Val Ser Gln Leu 35 40 45 Gln Gly Leu Trp Arg Arg Phe Asn Arg Pro Leu Leu Lys Gln Gln 50 55 60 <210> 24 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 24 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 25 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 25 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ser Ser Ile Ser Thr Phe Ser Ser Asp 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45 Ala Ala Ile Gly Trp Ser Gly Ala Val Thr His Tyr Ser Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Arg Tyr Tyr Cys 85 90 95 Ala Glu Asp Tyr Asp Thr Asp Val Trp Gln Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 26 <211> 254 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 26 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Ser His 20 25 30 Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Glu Leu Glu Trp Leu 35 40 45 Ser Glu Ile Asn Thr Asn Gly Leu Ile Thr His Tyr Gly Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asn Asn Ala Ala Asn Lys Met Tyr 65 70 75 80 Leu Glu Leu Thr Arg Leu Glu Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Arg Asn Gln His Gly Leu Asn Lys Gly Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly 130 135 140 Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Glu Ser 145 150 155 160 Ser Ile Ser Thr Phe Ser Ser Asp Ala Met Gly Trp Phe Arg Gln Ala 165 170 175 Pro Gly Lys Glu Leu Glu Phe Leu Ala Ala Ile Gly Trp Ser Gly Ala 180 185 190 Val Thr His Tyr Ser Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 195 200 205 Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala 210 215 220 Glu Asp Thr Gly Arg Tyr Tyr Cys Ala Glu Asp Tyr Asp Thr Asp Val 225 230 235 240 Trp Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 245 250 <210> 27 <211> 768 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence <400> 27 gatgttcaat tggttgaatc tggtggtggt ttggttcaac caggtggttc tttgaaattg 60 tcttgtgctg cttctggttt cgatttctct tctcattgga tgtactgggt tagacaagct 120 ccaggtaaag aattggaatg gttgtctgaa atcaacacca acggtttgat tacccattat 180 ggtgattctg tcaagggtag attcactgtc tctagaaaca atgctgctaa caagatgtac 240 ttggaattga ccagattgga accagaagat actgccttgt attactgcgc tagaaatcag 300 catggtttga acaaaggtca aggtactcaa gttactgttt cttctggtgg aggcggttca 360 ggcggaggtg gctctaaggg cggtggcgga agtggtggag gcggttcaga agttcaatta 420 gttgagagtg gtggcggttt agtacaagct ggtggttcat taagattgtc ctgcgaatct 480 tctatctcca ccttttcatc tgatgctatg ggttggttca gacaagcacc tggaaaagag 540 ttagaattct tggctgctat tggttggagt ggtgctgtta ctcattactc tgattcagtt 600 aagggtcgtt tcaccatctc aagagataat gctaagaaca ccgtctactt gcagatgaat 660 tctttgagag ctgaagatac aggtaggtac tattgtgctg aagattacga tactgatgtc 720 tggcaatatt ggggacaagg cacccaagtt acagttagtt cttaatga 768 <210> 28 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <220> <221> MISC_FEATURE <222> (1)..(1) <223> Xaa is Ser or Asp <400> 28 Xaa Asp Ala Met Gly 1 5 <210> 29 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <220> <221> MISC_FEATURE <222> (2) <223> Xaa is Ile or Thr <220> <221> MISC_FEATURE <222> (3) <223> Xaa is Gly or Asn <220> <221> MISC_FEATURE <222> (7) <223> Xaa is Ala or Thr <220> <221> MISC_FEATURE <222> (12)..(12) <223> Xaa is Ser or Gly <220> <221> MISC_FEATURE <222> (16) <223> Xaa is Gln or Lys <400> 29 Ala Xaa Xaa Trp Ser Gly Xaa Val Thr His Tyr Xaa Asp Ser Val Xaa 1 5 10 15 Gly <210> 30 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <220> <221> MISC_FEATURE <222> (3) <223> Xaa is Asp or Val <220> <221> MISC_FEATURE <222> (9)..(9) <223> Xaa is Tyr or His <400> 30 Asp Tyr Xaa Thr Asp Val Trp Gln Xaa 1 5 <210> 31 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <220> <221> Variants <222> (1)..(15) <223> The residue may occur 1 to 15 times <220> <221> Variants <222> (2)..(10) <223> The Gly residues may not be present, or some or all of the Gly residues may be present. <220> <221> Variants <222> (12)..(15) <223> The Ser residues may not be present, or some or all of the Ser residues may be present. <400> 31 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Ser Ser Ser Ser 1 5 10 15 <210> 32 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <220> <221> Variants <222> (1)..(5) <223> The residue may occur 1 to 8 times <400> 32 Gly Gly Gly Gly Ser 1 5 <210> 33 <211> 65 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <220> <221> Variants <222> (1)..(65) <223> The residue may occur 1 to 5 times <220> <221> Variants <222> (1)..(15) <223> The residue may occur 0 to 10 times <220> <221> Variants <222> (2)..(10) <223> The Gly residues may not be present, or some or all of the Gly residues may be present. <220> <221> Variants <222> (12)..(15) <223> The Ser residues may not be present, or some or all of the Ser residues may be present. <220> <221> Variants <222> (16)..(30) <223> The residue may occur 0 to 10 times <220> <221> Variants <222> (17)..(25) <223> The Gly residues may not be present, or some or all of the Gly residues may be present. <220> <221> Variants <222> (27)..(30) <223> The Ser residues may not be present, or some or all of the Ser residues may be present. <220> <221> MISC_FEATURE <222> (31)..(35) <223> Xaa is Lys or Arg <220> <221> Variants <222> (32)..(35) <223> The Xaa residues may not be present, or some or all of the Xaa residues may be present. <220> <221> Variants <222> (36)..(50) <223> The residue may occur 0 to 10 times <220> <221> Variants <222> (37)..(45) <223> The Gly residues may not be present, or some or all of the Gly residues may be present. <220> <221> Variants <222> (47)..(50) <223> The Ser residues may not be present, or some or all of the Ser residues may be present. <220> <221> Variants <222> (51)..(65) <223> The residue may occur 0 to 10 times <220> <221> Variants <222> (52)..(60) <223> The Gly residues may not be present, or some or all of the Gly residues may be present. <220> <221> Variants <222> (62)..(65) <223> The Ser residues may not be present, or some or all of the Ser residues may be present. <400> 33 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Ser Ser Ser Ser Gly 1 5 10 15 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Ser Ser Ser Ser Xaa Xaa 20 25 30 Xaa Xaa Xaa Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Ser Ser 35 40 45 Ser Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Ser Ser Ser 50 55 60 Ser 65 <210> 34 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <220> <221> Variants <222> (1)..(5) <223> The residue may occur 1 to 5 times <220> <221> Variants <222> (7)..(11) <223> The residue may occur 1 to 5 times <400> 34 Gly Gly Gly Gly Ser Lys Gly Gly Gly Gly Ser 1 5 10 <210> 35 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <220> <221> MISC_FEATURE <222> (1) <223> Xaa is Lys or Arg <220> <221> Variants <222> (2)..(12) <223> The residue may occur 1 to 10 times <220> <221> Variants <222> (3)..(11) <223> The Gly residues may not be present, or some or all of the Gly residues may be present. <220> <221> MISC_FEATURE <222> (13) <223> Xaa is Lys or Arg <400> 35 Xaa Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Xaa 1 5 10 <210> 36 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 36 Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys 1 5 10 <210> 37 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 37 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys 1 5 10 <210> 38 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 38 Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 39 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 39 Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg 1 5 10 <210> 40 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 40 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg 1 5 10 <210> 41 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <400> 41 Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 42 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <220> <221> MISC_FEATURE <222> (1) <223> Xaa is Lys or Arg <220> <221> Variants <222> (2)..(12) <223> The residue may occur 1 to 10 times <220> <221> Variants <222> (3)..(11) <223> The Gly residues may not be present, or some or all of the Gly residues may be present. <220> <221> MISC_FEATURE <222> (13) <223> Xaa is Lys or Arg <220> <221> Variants <222> (14)..(24) <223> The residue may occur 1 to 10 times <220> <221> Variants <222> (15)..(23) <223> The Gly residues may not be present, or some or all of the Gly residues may be present. <220> <221> MISC_FEATURE <222> (25)..(25) <223> Xaa is Lys or Arg <400> 42 Xaa Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Xaa Gly Gly Gly 1 5 10 15 Gly Gly Gly Gly Gly Gly Gly Ser Xaa 20 25 <210> 43 <211> 61 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence <220> <221> Variants <222> (1)..(61) <223> The residue may occur 1 to 5 times <220> <221> Variants <222> (1)..(15) <223> The residue may occur 0 to 10 times <220> <221> Variants <222> (2)..(10) <223> The Gly residues may not be present, or some or all of the Gly residues may be present. <220> <221> Variants <222> (12)..(15) <223> The Ser residues may not be present, or some or all of the Ser residues may be present. <220> <221> Variants <222> (16)..(30) <223> The residue may occur 0 to 10 times <220> <221> Variants <222> (17)..(25) <223> The Gly residues may not be present, or some or all of the Gly residues may be present. <220> <221> Variants <222> (27)..(30) <223> The Ser residues may not be present, or some or all of the Ser residues may be present. <220> <221> MISC_FEATURE <222> (31)..(31) <223> Xaa is 1 to 5 residues selected from Trp, Phe, Tyr, Leu and Met <220> <221> Variants <222> (32)..(46) <223> The residue may occur 0 to 10 times <220> <221> Variants <222> (33)..(41) <223> The Gly residues may not be present, or some or all of the Gly residues may be present. <220> <221> Variants <222> (43)..(46) <223> The Ser residues may not be present, or some or all of the Ser residues may be present. <220> <221> Variants <222> (47)..(61) <223> The residue may occur 0 to 10 times <220> <221> Variants <222> (48)..(56) <223> The Gly residues may not be present, or some or all of the Gly residues may be present. <220> <221> Variants <222> (58)..(61) <223> The Ser residues may not be present, or some or all of the Ser residues may be present. <400> 43 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Ser Ser Ser Ser Gly 1 5 10 15 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Ser Ser Ser Ser Xaa Gly 20 25 30 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Ser Ser Ser Ser Gly Gly 35 40 45 Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Ser Ser Ser Ser 50 55 60

Claims

1. A composition comprising a TNF-α binding polypeptide and an IL-7R binding polypeptide, wherein: (a) the IL-7R binding polypeptide comprises SEQ ID NO: 16; and (b) the TNF-α binding polypeptide comprises SEQ ID NO: 8, wherein said polypeptides each comprise or consist of a VHH.

2. The composition according to claim 1, wherein the TNF-α binding polypeptide is present in an amount of 10 -7 M or less K binding to TNF-α, and the IL-7R binding polypeptide binds to TNF-α with a K of 10 -7 The IL-7R binding protein was expressed in 4% IL-7R and had a Kd of M or less.

3. The composition of claim 1 or 2, wherein the TNF-α binding polypeptide neutralizes human TNF-α cytotoxicity in the L929 assay with an EC50 of 100 nM or less, and the IL-7R binding polypeptide neutralizes IL-7R-dependent, IL-7-induced STAT5 phosphorylation in human lymphocytes with an EC50 of 100 nM or less.

4. The composition according to claim 1, wherein (a) the TNF-α binding polypeptide consists of SEQ ID NO: 8, and (b) the IL-7R binding polypeptide consists of SEQ ID NO:

16.

5. The composition of claim 1, wherein the TNF-α binding polypeptide and the IL-7R binding polypeptide are linked. 6 . The composition of claim 5 , wherein the TNF-α binding polypeptide and the IL-7R binding polypeptide are linked via a protease-labile peptide linker.

7. The composition of claim 6, wherein the protease-labile peptide linker comprises K and / or R residues.

8. The composition of claim 7, wherein the protease-labile linker comprises -(G4S) x -K-(G4S) y -sequence (SEQ ID NO: 34) or consisting thereof, wherein x and y are each independently 1 to 5.

9. The composition of claim 8, wherein the protease-labile linker comprises or consists of SEQ ID NO:

21.

10. The composition of claim 1, wherein the polypeptide is resistant to one or more proteases present in the small intestine or large intestine.

11. The composition of claim 10, wherein the proteases are trypsin and chymotrypsin.

12. A pharmaceutical composition comprising the composition according to any one of claims 1 to 11 and a pharmaceutically acceptable excipient.

13. Use of the composition according to any one of claims 1 to 11 in the preparation of a medicament for treating or preventing autoimmune diseases and / or inflammatory diseases.

14. Use according to claim 13, wherein the autoimmune disease and / or inflammatory disease is inflammatory bowel disease and / or mucositis.

15. The use according to claim 13, wherein the autoimmune disease and / or inflammatory disease is atopic dermatitis.

16. The use according to claim 13, wherein the composition is for oral administration.

17. The use according to claim 13, wherein the composition is for topical administration.

18. An IL-7R binding polypeptide for use in the preparation of a medicament for treating or preventing autoimmune diseases and / or inflammatory diseases together with a TNF-α binding polypeptide, wherein the IL-7R binding polypeptide comprises SEQ ID NO: 16, and the TNF-α binding polypeptide comprises SEQ ID NO:

8.

19. A polynucleotide encoding the composition according to any one of claims 1 to 11.

20. The polynucleotide according to claim 19, wherein: (a) the polynucleotide comprises a nucleic acid sequence having 80% or greater identity to SEQ ID NO: 17; (b) the polynucleotide comprises a nucleic acid sequence having 80% or greater identity to SEQ ID NO: 18; or (c) the polynucleotide comprises a nucleic acid sequence having 80% or higher identity with SEQ ID NO:

27.

21. A vector comprising the polynucleotide according to claim 19.

22. A host cell comprising the polynucleotide according to claim 19.

23. A method for preparing a composition according to any one of claims 1 to 11, wherein the method comprises providing a polynucleotide encoding the polypeptide into a host cell capable of expressing the polypeptide, producing the polypeptide in the host cell, and recovering the polypeptide from the host cell.

Images