US20180037639A1 - Peptide construct having a protease-cleavable linker - Google Patents

Peptide construct having a protease-cleavable linker Download PDF

Info

Publication number
US20180037639A1
US20180037639A1 US15/717,174 US201715717174A US2018037639A1 US 20180037639 A1 US20180037639 A1 US 20180037639A1 US 201715717174 A US201715717174 A US 201715717174A US 2018037639 A1 US2018037639 A1 US 2018037639A1
Authority
US
United States
Prior art keywords
construct
peptide linker
suitably
labile
chain variable
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US15/717,174
Other languages
English (en)
Inventor
Scott Crowe
Mike West
Kevin Roberts
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
VHsquared Ltd
Original Assignee
VHsquared Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by VHsquared Ltd filed Critical VHsquared Ltd
Publication of US20180037639A1 publication Critical patent/US20180037639A1/en
Assigned to VHSQUARED LIMITED reassignment VHSQUARED LIMITED CONFIRMATORY ASSIGNMENT Assignors: CROWE, SCOTT, ROBERTS, KEVIN, WEST, MIKE
Priority to US17/196,498 priority Critical patent/US12234279B2/en
Priority to US19/025,399 priority patent/US20250388655A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Gram-positive bacteria
    • C07K16/1282—Clostridium (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • the present invention relates to constructs suitable for oral administration comprising polypeptides connected by a labile peptide linker as well as to pharmaceutical compositions comprising such constructs.
  • the present invention also relates to methods for preparing such constructs, methods which assay the lability of such constructs, methods which utilise such constructs, nucleic acids encoding such constructs, cDNA and vectors comprising nucleic acids encoding such constructs, host cells expressing or capable of expressing such constructs and to uses of such constructs or pharmaceutical compositions.
  • Constructs comprising two or more polypeptides are a class of biomolecules with multi-functional properties. By genetically fusing two or more polypeptides together, the resultant construct may obtain many distinct functions derived from each component polypeptide. Such constructs have been utilised in biological research for many purposes such as protein purification and imaging. Such constructs have also become an important category of biopharmaceuticals. An effective construct requires a suitable linker. Direct fusion of polypeptides without a linker may lead to many undesirable outcomes including misfolding of the fused polypeptides, low yield in polypeptide production or impaired bioactivity. Therefore, the selection or rational design of a linker to join polypeptides is an important area in recombinant polypeptide technology.
  • linkers which are relatively stable for the purposes of in vivo delivery or recombinant production. Stable linkers covalently join functional domains together to act as one molecule throughout the in vivo or recombinant production processes.
  • a stable linkage between polypeptides may provide many advantages such as a prolonged plasma half-life and resistance to cleavage by host organism proteases.
  • stable linkers also have several potential drawbacks including steric hindrance between polypeptides, decreased bioactivity, and altered biodistribution (Chen et al. 2013 Adv Drug Deliv Rev. 65(10):1357-1369). It would be advantageous therefore to release free polypeptides from a construct in vivo and thereby potentially reduce steric hindrance, improve bioactivity or achieve independent functions of individual polypeptides.
  • such release would ideally take place in the intestinal tract after oral administration. It may be preferable that such release takes place only in one or a number of specific locations within the intestinal tract.
  • Constructs of the present invention may, in at least some embodiments, have one or more of the following advantages compared to substances of the prior art:
  • the present inventors have produced surprisingly advantageous constructs suitable for oral administration comprising a first polypeptide and a second polypeptide connected by a labile peptide linker. These constructs are particularly advantageous due to their convenience of production and their ability to release free component polypeptides within the intestinal tract.
  • the free component polypeptides may be released in one or more specific regions of the intestinal tract.
  • these constructs have particular utility in the prevention or treatment of diseases of the GIT such as autoimmune and/or inflammatory disease such as inflammatory bowel disease, or in the prevention or treatment of infection from an intestinal tract resident pathogenic microbe.
  • diseases of the GIT such as autoimmune and/or inflammatory disease such as inflammatory bowel disease
  • the present invention provides a construct suitable for oral administration comprising a first polypeptide and a second polypeptide connected by a labile peptide linker, wherein the labile peptide linker is labile to one or more proteases present in the intestinal tract and wherein the first and second polypeptides are substantially resistant to said one or more proteases. Also provided are related compositions, methods and nucleic acids relating to the inventive construct.
  • FIG. 1 Stable PAGE gel demonstrating non-lability of ID3A using the Trypsin Protease Assay
  • FIG. 2 Stable PAGE gel demonstrating lability of ID25A and ID26A using the Trypsin Protease Assay
  • FIG. 3 Stable PAGE gel demonstrating lability of ID27A and ID28A using the Trypsin Protease Assay
  • FIG. 4 Stable PAGE gel demonstrating lability of ID3A, ID25A, ID26A, ID27A and ID28A using the Faecal Protease Assay
  • FIG. 5 Stable PAGE gel demonstrating lability of ID55F, ID56F, ID57F, ID58F, ID59F and ID60F using the Faecal Protease Assay
  • FIG. 6 Sttained PAGE gels demonstrating storage stability of ID3A and ID25A
  • FIG. 7 Sttained PAGE gel demonstrating storage stability of ID26A, ID27A and ID28A
  • FIG. 8 Sttained PAGE gel demonstrating storage stability of ID4A
  • the construct of the invention comprises a labile peptide linker, which connects the first and second polypeptides.
  • the labile peptide linker can be engineered such that it resists cleavage by proteases to a desired extent and/or is only cleaved upon exposure to a specific area of the intestinal tract.
  • a construct is recombinantly produced in a host such as yeast
  • trypsin-like proteases produced by the yeast may cleave the recombinant construct product. This may result in difficulties in purification and cause regulatory, clinical and commercial complications.
  • the first polypeptide is a toxin
  • the second polypeptide of the construct may act to ‘quench’ the effects of the toxin until it is released at a suitable, target location.
  • shielding residues into the labile peptide linker flanking the labile site(s). Shielding residues flank the labile site(s) of the labile peptide linker and reduce the lability thereof. Cleavage resistance can also be increased by positioning the labile site(s) closer to or at the periphery of the labile peptide linker. This concept is referred to as a “shielded labile site” and provides controlled lability.
  • the labile peptide linker can be engineered such that it is highly labile to cleavage by intestinal tract proteases, thereby quickly releasing the constituent first and second polypeptides of the construct after oral administration.
  • This is achieved by incorporating one or more labile sites into the labile peptide linker such that the labile site is exposed for proteolysis, for example by positioning the labile site(s) substantially centrally in the labile peptide linker and/or by the labile site not being shielded substantially by flanking residues. This concept is referred to as a “non-shielded labile site”.
  • the labile peptide linker has a length of 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 labile peptide linker has a length of no greater than 40, such as no greater than 35, such as no greater than 30, such as no greater than 25, such as no greater than 20, such as no greater than 15 residues.
  • incorporation of a P residue into the labile peptide linker of the construct of the invention is expected to substantially prevent cleavage of the labile peptide linker.
  • the labile peptide linker does not comprise any P residues.
  • the labile peptide linker of the construct of the invention comprises a cleavage site for trypsin or a trypsin-like protease.
  • the labile 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.
  • the labile 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.
  • the cleavage site(s) is/are one or more K residue(s).
  • Shielding residues in the case of a trypsin or trypsin-like protease labile site may be D or E.
  • the labile peptide linker comprises one or more shielding residues selected from the list consisting of D or E.
  • K or R residues comprised within the labile peptide linker have at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5 shielding residues on their N-terminal side, wherein the shielding residues are selected from the list consisting of: D and E.
  • K or R residues comprised within the labile peptide linker have 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 5, 2 to 4, 2 to 3, 3 to 5, 3 to 4, 4 to 5 shielding residues on their N-terminal side, wherein the shielding residues are selected from the list consisting of: D and E.
  • K or R residues comprised within the labile peptide linker have and at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5 shielding residues on their C-terminal side, wherein the shielding residues are selected from the list consisting of: D and E.
  • K or R residues comprised within the labile peptide linker have 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 5, 2 to 4, 2 to 3, 3 to 5, 3 to 4, 4 to 5 shielding residues on their C-terminal side, wherein the shielding residues are selected from the list consisting of: D and E.
  • K and R residues have at least one shielding residue adjacent to them, suitably followed by one or more further contiguous shielding residues.
  • the shielding residues occur on one or both sides of one or more of the K or R residues.
  • the labile peptide linker comprises or more suitably consists of a polypeptide sequence of the format:
  • a is 2 to 5, more suitably a is 4.
  • Suitably b is 2 to 5, more suitably b is 4.
  • Suitably x is 1 to 5, more suitably x is 1.
  • Suitably y is 1 to 5, more suitably y is 1.
  • Suitably z is 1 to 3, more suitably z is 1.
  • B is K.
  • U if present, is D.
  • U′ if present, is D.
  • c is 1 and d is 1.
  • c is 0 and d is 0.
  • c is 4 and d is 0.
  • both U and U′ are each individually D and c and d are both 1.
  • the labile peptide linker comprises or more suitably consists of a polypeptide sequence of the format:
  • 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 is K.
  • B′ is K.
  • the labile peptide linker comprises or more suitably consists of a polypeptide sequence of the format:
  • a is 2 to 5, more suitably a is 4.
  • b is 2 to 5, more suitably b is 4.
  • x is 1 to 5. More suitably, x is 2.
  • y is 1 to 5. More suitably, y is 2.
  • B is K.
  • B′ is K.
  • B′′ is K.
  • the labile peptide linker of the construct of the invention comprises a cleavage site for trypsin or a trypsin-like protease.
  • the labile 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.
  • the labile 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.
  • the cleavage site(s) is/are one or more K residue(s).
  • K or R residues comprised within the labile peptide linker have at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5 non-shielding residues on their N-terminal side wherein the non-shielding residues are selected from the list consisting of: C, A, S, N, G, L, I, V, T, M, F, Y, H, W and Q; more suitably A, G, L, I, V, M, S and T; more suitably A, G, L, I, V and S; more suitably G and S.
  • K or R residues comprised within the labile peptide linker have 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 5, 2 to 4, 2 to 3, 3 to 5, 3 to 4, 4 to 5 non-shielding residues on their N-terminal side, wherein the shielding residues are selected from the list consisting of: C, A, S, N, G, L, I, V, T, M, F, Y, H, W and Q; more suitably A, G, L, I, V, M, S and T; more suitably A, G, L, I, V and S; more suitably G and S.
  • K or R residues comprised within the labile peptide linker have at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5 non-shielding residues on their C-terminal side, wherein the non-shielding residues are selected from the list consisting of: C, A, S, N, G, L, I, V, T, M, F, Y, H, W and Q; more suitably A, G, L, I, V, M, S and T; more suitably A, G, L, I, V and S; more suitably G and S.
  • K or R residues comprised within the labile peptide linker have 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 5, 2 to 4, 2 to 3, 3 to 5, 3 to 4, 4 to 5 non-shielding residues on their C-terminal side, wherein the shielding residues are selected from the list consisting of: C, A, S, N, G, L, I, V, T, M, F, Y, H, W and Q; more suitably A, G, L, I, V, M, S and T; more suitably A, G, L, I, V and S; more suitably G and S.
  • K and R residues have at least one non-shielding residue adjacent to them, suitably followed by one or more further contiguous non-shielding residues.
  • the non-shielding residues occur on one or both sides of one or more of the K or R residues.
  • the labile peptide linker does not comprise any D or E residues.
  • the labile peptide linker consists of residues selected from the list consisting of C, A, S, N, G, L, I, V, T, M, F, Y, H, K, R, W and Q; more suitably A, G, L, I, V, M, S, T, K and R residues; more suitably S, G, K and R residues.
  • the labile peptide linker comprises or more suitably consists of a polypeptide sequence of the format:
  • a is 2 to 5, more suitably a is 4.
  • b is 2 to 5, more suitably b is 4.
  • x is 1 to 5, more suitably x is 1.
  • y is 1 to 5, more suitably y is 1.
  • z is 1 to 3, more suitably z is 1.
  • B is K.
  • the labile peptide linker comprises or more suitably consists of a polypeptide sequence of the format:
  • x is 1 to 5, more suitably x is 1.
  • y is 1 to 5, more suitably y is 1.
  • z is 1 to 3, more suitably, z is 1.
  • B is K.
  • the labile peptide linker of the construct of the invention comprises a cleavage site for chymotrypsin or a chymotrypsin-like protease.
  • the labile 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 selected from the list consisting of W, F, Y, L and M; more suitably W, F and Y.
  • the labile peptide linker consists of residues selected from the list consisting of S, G, W, F, Y, L and M; such as S, G, W, F and Y.
  • the labile peptide linker comprises or more suitably consists of a polypeptide sequence of the format:
  • a is 2 to 5, in a further embodiment, b is 2 to 5, in a further embodiment x is 1 to 5, in a further embodiment, y is 1 to 5, in a further embodiment z is 1 to 3.
  • x is 1, y is 1 and z is 1.
  • the labile peptide linker comprises or more suitably consists of a polypeptide sequence of the format:
  • x is 1 to 5, in a further embodiment, y is 1 to 5, in a further embodiment z is 1 to 3.
  • x is 1, y is 1 and z is 1.
  • the labile peptide linker of the construct of the invention comprises a cleavage site for MMP3, MMP10 or MMP12.
  • the lability of a construct, and therefore the utility of a construct as a controlled-lability or highly labile construct can be assayed using the Faecal Protease Assay, the Trypsin Protease Assay and/or the Chymotrypsin Protease Assay defined in Examples 1 to 3.
  • At least 20%, such as at least 40%, such as at least 60%, such as at least 80%, such as at least 90%, such as at least 100% by mass of the construct remains uncleaved after at least 10, such as at least 20, such as at least 30, such as at least 40, such as at least 50, such as at least 60, such as at least 70, such as at least 80, such as at least 90, such as at least 100, such as at least 110, such as at least 120, such as at least 130, such as at least 140, such as at least 150, such as at least 160 minutes after mixing in the Faecal Protease Assay and/or Trypsin Protease Assay and/or Chymotrypsin Protease Assay.
  • At least 60%, such as at least 80%, such as at least 90%, such as at least 100% of the construct is cleaved after no more than 5, such as no more than 4, such as no more than 3, such as no more than 2, such as no more than 1 minute, after mixing in the Faecal Protease Assay and/or Trypsin Protease Assay and/or Chymotrypsin Protease Assay.
  • a method of assaying the lability of a construct of the invention comprising the steps of: (a) incubating the construct in a solution comprising trypsin, a solution comprising chymotrypsin, faecal supernatant, small intestinal fluid or a solution comprising enteropeptidase (such as by performing the Trypsin Protease Assay, the Chymotrypsin Protease Assay or the Faecal Protease Assay) then (b) ascertaining the proportion of cleaved construct after one or more periods of incubation.
  • a method of delivering a monomeric antibody or a monomeric antigen binding fragment thereof to a targeted region of the intestinal tract comprising the steps of: (a) performing the method of assaying construct lability described above, (b) selecting a construct with an appropriate level of lability for the targeted region of the intestinal tract, (c) producing the selected construct with an enterically coated packaging then (d) administering the packaged selected construct to a subject.
  • a method of preparing a product comprising a construct of the invention which has been selected, the method comprising adding the selected construct into the product, wherein the selected construct is selected and produced by a method comprising the steps of: (a) performing the method of assaying construct lability described above then (b) selecting a construct with an appropriate level of lability for the targeted region of the intestinal tract.
  • Various organisms may be used to express recombinant polypeptides.
  • Commonly used expression organisms include yeast, mould and mammalian cells.
  • many of these expression organisms also produce proteases, such as trypsin-like proteases, which may cleave the expressed recombinant polypeptide.
  • proteases such as trypsin-like proteases
  • the expressed polypeptide incorporates a peptide linker which is labile to one or more proteases present in the intestinal tract, then this peptide linker may undesirably also be labile to proteases produced by the expression organism, thus preventing effective expression of intact polypeptide.
  • the labile peptide linker is substantially non-labile to enzymes produced by the recombinant host used to produce the construct.
  • the labile peptide linker is substantially resistant to proteases produced by a recombinant host such as bacteria such as E. coli or such as a yeast or mould belonging to the genera Aspergillus, Saccharomyces, Kluyveromyces, Hansenula or Pichia ; such as Saccharomyces cerevisiae or Pichia pastoris .
  • the recombinant host is a yeast.
  • the recombinant host is a mould.
  • the yeast belongs to the genera Saccharomyces, Kluyveromyces, Hansenula or Pichia . Further examples of yeasts are those belonging to the genera Candida and Torulopsis .
  • the mould belongs to the genus Aspergillus . Further examples of moulds are those belonging to the genera Acremonium, Alternaria, Chrysosporium, Cladosporium, Dictyostelium, Fusarium, Mucor, Penicillium, Rhizopus, Stachybotrys, Trichoderma and Trichophyton .
  • the labile peptide linker is substantially resistant to proteases produced by a recombinant host for at least 2, such as at least 4, such as at least 9, such as at least 14, such as at least 60 days' storage at 4 degrees C.
  • a construct for use in the treatment by oral administration of a disease of the intestinal tract with a first immunoglobulin chain variable domain and a second immunoglobulin chain variable domain wherein the construct comprises the first immunoglobulin chain variable domain and the second immunoglobulin chain variable domain connected by a labile peptide linker, wherein:
  • a disease of the intestinal tract with a first immunoglobulin chain variable domain and a second immunoglobulin chain variable domain from a construct, wherein the construct comprises the first immunoglobulin chain variable domain and the second immunoglobulin chain variable domain connected by a labile peptide linker, wherein:
  • a method of delivering a first immunoglobulin chain variable domain and a second immunoglobulin chain variable domain to the intestinal tract comprising producing in yeast and then orally administering a construct comprising the first immunoglobulin chain variable domain and the second immunoglobulin chain variable domain connected by a labile peptide linker, wherein:
  • the step of producing the construct in yeast is performed by providing a host yeast cell which is capable of expressing the construct of the invention, transformed with a vector, wherein the vector comprises a polynucleotide encoding the construct of the invention and wherein the host yeast cell is exposed to conditions suitable for expression of the construct of the invention.
  • first immunoglobulin chain variable domain and the second immunoglobulin chain variable domain are substantially resistant to yeast proteases.
  • the methods outlined above further comprise the step of purifying the construct.
  • the labile peptide linker is cleaved in the intestinal tract by the one or more proteases present in the intestinal tract.
  • constructs for use and methods described above relate to the production of a construct in yeast. However, these constructs for use and methods are also equally applicable to production of a construct in any expression organism which produces proteases which may cleave a peptide linker, such as mammalian cells or moulds (such as moulds from any one or more of the genera Acremonium, Alternaria, Aspergillus, Chrysosporium, Cladosporium, Dictyostelium, Fusarium, Mucor, Penicillium, Rhizopus, Stachybotrys, Trichoderma and Trichophyton ).
  • mammalian cells or moulds such as moulds from any one or more of the genera Acremonium, Alternaria, Aspergillus, Chrysosporium, Cladosporium, Dictyostelium, Fusarium, Mucor, Penicillium, Rhizopus, Stachybotrys, Trichoderma and Trichophyton ).
  • a Labile Peptide Linker which is Labile to One or More Proteases Present in the Intestinal Tract
  • Examples 5 and 6 below provide details on specific constructs of the invention which comprise labile peptide linkers which are labile to one or more proteases present in the intestinal tract.
  • the labile peptide linker is labile to one or more proteases present in the intestinal tract such that greater than 50%, such as greater than 60%, such as greater than 70%, such as greater than 80%, such as greater than 85%, such as greater than 90% by mass of the construct is cleaved into first and second immunoglobulin chain variable domains after 160 minutes, more suitably after 105 minutes, more suitably after 60 minutes, more suitably after 25 minutes, more suitably after 10 minutes after mixing in the Trypsin Protease Assay.
  • Example 7 provides details on specific constructs of the invention which comprise labile peptide linkers which are stable to yeast proteases.
  • Example 9 provides details on a specific construct (ID4A) which is not stable to yeast proteases.
  • the stability of a linker to yeast proteases may be assessed using the Yeast Expression Protocol outlined as follows.
  • polypeptides such as constructs comprising ICVDs or monomeric ICVDs
  • induction in a suitable growth medium results in polypeptide expression and secretion into the extracellular supernatant.
  • the production strain used for expression and manufacture of polypeptides is a derivative of the CEN.PK series from the EUROSCARF collection (EUROpean Saccharomyces Cerevisiae ARchive for Functional analysis).
  • the genotype of the CEN.PK strain is: MATa/MAT ⁇ ura3-52/ura3-52; trp1-289/trp1-289; leu2-3,112/leu2-3,112; his3 ⁇ 1/his3 ⁇ 1; MAL2-8C/MAL2-8C; SUC2/SUC2.
  • This strain is then further modified to inhibit the ability to grow on galactose by deletion of the galactokinase gene (gal1::URA3). This is the final strain used for transformation by the polypeptide expression constructs.
  • Monomeric or multimeric DNA constructs in which polypeptides (such as constructs comprising ICVDs or monomeric ICVDs) joined by protein linkers, are cloned into a suitable multi-copy chromosomal integration vector (Lopes et al. 1991. Gene. 105, 83-90), generating an integration cassette.
  • the integration cassette may include an inducible promoter (for example pGal7, Nogi & Fukasawa (1983). Nucleic Acids Res. 11(24):8555-68), the polypeptide encoding region, a signal sequence that encodes secretion into the extracellular supernatant in yeast (Hashimoto et al. 1998.
  • polypeptide production may be achieved from a multi-copy episomal vector based on the yeast 2 ⁇ M plasmid with a similar expression cassette, without the need for chromosomal integration.
  • a colony of the resulting yeast strain is inoculated into 5 mL of yeast soytone broth supplemented with 2% glucose and grown overnight at 30° C. (150-200 rpm).
  • 50 mL of yeast soytone broth containing 2% glucose and 0.5% galactose (for induction) in a 500 mL Erlenmeyer flask is inoculated with the entire 5 mL overnight culture.
  • the resulting induction culture is incubated at 30° C., 200 rpm for 3 days.
  • the culture is then spun down at 4200 rpm for 20 min in a swing rotor centrifuge to remove yeast cells.
  • the supernatant is then filtered through 0.45 ⁇ m and 0.2 ⁇ m filters in series.
  • the polypeptide may then optionally undergo a storage period at 4° C. in the yeast supernatant under sterile conditions.
  • a construct of the invention is stable to yeast proteases such that no more than 10%, more suitably no more than 5%, more suitably no more than 1% by mass of the construct is cleaved into first and second immunoglobulin chain variable domains after producing the construct using the Yeast Expression Protocol (i.e. with no subsequent storage period) and then optionally storing for 2 days, more suitably 4 days or more suitably 9 days. “0 days storage” as used herein refers to no subsequent storage period.
  • Yeast-produced polypeptides may be distinct from those produced using alternative expression organisms such as bacteria, in that yeast-produced polypeptides may comprise post translational modifications such as glycosylation.
  • Examples 5 and 6 below provide details on specific constructs of the invention which comprise first and second immunoglobulin chain variable domains, wherein the first and second immunoglobulin chain variable domains are substantially resistant to one or more proteases present in the intestinal tract.
  • the first immunoglobulin chain variable domain and the second immunoglobulin chain variable domain are substantially resistant to one or more proteases present in the intestinal tract such that at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as about 100% by mass of the first immunoglobulin chain variable domain and at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as about 100% by mass of the second immunoglobulin chain variable domain remain uncleaved after 10 minutes, more suitably after 25 minutes, more suitably after 60 minutes, more suitably after 105 minutes, more suitably after 160 minutes after mixing in the Trypsin Protease Assay.
  • Example 7 provides details on specific constructs of the invention which comprise immunoglobulin chain variable domains, wherein the immunoglobulin chain variable domains are stable to yeast proteases.
  • the first immunoglobulin chain variable domain and the second immunoglobulin chain variable domain are substantially resistant to yeast proteases such that no more than 10%, more suitably no more than 5%, more suitably no more than 1% by mass of the first or second immunoglobulin chain variable domain are cleaved after producing the first or second immunoglobulin chain variable domain using the Yeast Expression Protocol and then optionally storing for 2 days, more suitably 4 days or more suitably 9 days.
  • the cleaved mass of the construct and/or the uncleaved mass of the first and second immunoglobulin chain variable domains and/or the cleaved mass of the first and second immunoglobulin chain variable domains are assessed by gel electrophoresis followed by visual inspection of the gel. More suitably, assessment is by quantitative gel electrophoresis. Alternatively, assessment is by gel electrophoresis followed by mass spectrometry.
  • Bands on a gel corresponding to cleaved construct and uncleaved immunoglobulin chain variable domain may be identified as those bands having approximately half the mass of the construct (i.e. the mass of one constituent immunoglobulin chain variable domain), by comparison with molecular weight marker. Bands on a gel corresponding to cleaved immunoglobulin chain variable domains are identifiable by having a molecular weight lower than that of the whole constituent immunoglobulin chain variable domain, by comparison to molecular weight marker bands.
  • the labile peptide linker of the construct of the invention does not comprise a polypeptide disclosed in WO2009/021754. More specifically, in one embodiment the labile peptide linker of the construct of the invention does not comprise the sequence GGGGSDDDDKGGGGS (SEQ ID NO: 4).
  • Polypeptides are organic polymers consisting of a number of amino acid residues bonded together in a chain.
  • polypeptide is used interchangeably with ‘protein’ and ‘peptide’.
  • Polypeptides are said to be antigen-binding when they contain one or more stretches of amino acid residues which form an antigen-binding site, capable of binding to an epitope on a target antigen with an affinity (suitably expressed as a Kd value, a Ka value, a k on -rate and/or a k off -rate, as further described herein).
  • Antigen-binding polypeptides include polypeptides such as antibodies, antibody fragments and antigen-binding fragments.
  • a polypeptide may comprise a region which is capable of binding a target with high affinity (suitably expressed as a Kd value, a Ka value, a k on -rate and/or a k off -rate, as further described herein).
  • a target with high affinity suitable for binding a target with high affinity
  • Such polypeptides include DARPins (Binz et al. Journal of Molecular Biology 332(2):489-503), AffimersTM, FynomersTM, Centyrins, Nanofitins® and cyclic peptides.
  • a conventional antibody or immunoglobulin (Ig) is a protein 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 domains are abbreviated herein as VHC, and the light (L) chain variable domains are abbreviated herein as VLC. These domains, domains related thereto and domains derived therefrom, are referred to herein as immunoglobulin chain variable domains.
  • the VHC and VLC domains can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (“CDRs”), interspersed with regions that are more conserved, termed “framework regions” (“FRs”).
  • CDRs complementarity determining regions
  • each VHC and VLC is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the conventional antibody tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains is formed with the heavy and the light immunoglobulin chains inter-connected by e.g. disulfide bonds, and the heavy chains similarity connected.
  • the heavy chain constant region includes three domains, CH1, CH2 and CH3.
  • the light chain constant region is comprised of one domain, CL.
  • the variable domain of the heavy chains and the variable domain of the light chains are binding domains that interact with an antigen.
  • the constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g. effector cells) and the first component (C1q) of the classical complement system.
  • the term antibody includes immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof), wherein the light chains of the immunoglobulin may be kappa or lambda types.
  • immunoglobulin-gamma (IgG) antibodies assembled from two identical heavy (H)-chain and two identical light (L)-chain polypeptides is well established and highly conserved in mammals (Padlan 1994 Mol Immunol 31:169-217).
  • HCAbs heavy-chain antibodies
  • the H chain of the homodimeric protein contains a dedicated immunoglobulin chain variable domain, referred to as the VHH, which serves to associate with its cognate antigen (Muyldermans 2013 Annu Rev Biochem 82:775-797, Hamers-Casterman et al 1993 Nature 363(6428):446-448, Muyldermans et al 1994 Protein Eng 7(9):1129-1135, herein incorporated by reference in their entirety).
  • VHH immunoglobulin chain variable domain
  • an antigen-binding 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 is not full length, but which specifically binds to a target).
  • a target e.g. a molecule in which one or more immunoglobulin chains is not full length, but which specifically binds to a target.
  • binding fragments encompassed within the term antigen-binding fragment include:
  • the total number of amino acid residues in a VHH or VH may be in the region of 110-130, is suitably 112-120, and is most suitably 115.
  • Constructs of the invention comprising polypeptides and labile peptide linkers may for example be obtained by preparing a nucleic acid encoding two or more polypeptides and a labile peptide linker using techniques for nucleic acid synthesis, followed by expression of the nucleic acid thus obtained (as detailed further herein).
  • a construct according to the invention does not have an amino acid sequence which is exactly the same as (i.e. shares 100% sequence identity with) the amino acid sequence of a naturally occurring polypeptide.
  • the first and/or second polypeptide of the construct of the invention is an antigen-binding polypeptide
  • the first and/or second antigen-binding polypeptide is an immunoglobulin chain variable domain, an antibody or an antigen-binding fragment thereof.
  • the antigen-binding fragment is selected from the group consisting of: VLs, V-NARs, scFvs, Fab fragments, F(ab′)2 fragments or immunoglobulin chain variable domains such as VHHs and VHs.
  • Specificity refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding polypeptide can bind.
  • the specificity of an antigen-binding polypeptide is the ability of the antigen-binding polypeptide to recognise a particular antigen as a unique molecular entity and distinguish it from another.
  • Affinity represented by the equilibrium constant for the dissociation of an antigen with an antigen-binding polypeptide (Kd) is a measure of the binding strength between an antigenic determinant and an antigen-binding site on an antigen-binding polypeptide: the lesser the value of the Kd, the stronger the binding strength between an antigenic determinant and the antigen-binding polypeptide (alternatively, the affinity can also be expressed as the affinity constant (Ka), which is 1/Kd). Affinity can be determined by known methods, depending on the specific antigen of interest.
  • Avidity is the measure of the strength of binding between an antigen-binding polypeptide and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen-binding site on the antigen-binding polypeptide and the number of pertinent binding sites present on the antigen-binding polypeptide.
  • antigen-binding polypeptides bind with a dissociation constant (Kd) of 10 ⁇ 6 to 10 ⁇ 12 M, more suitably 10 ⁇ 7 to 10 ⁇ 12 M, more suitably 10 ⁇ 8 to 10 ⁇ 12 M and more suitably 10 ⁇ 9 to 10 ⁇ 12 M.
  • Kd dissociation constant
  • any Kd value less than 10 ⁇ 6 is considered to indicate binding.
  • Specific binding of an antigen-binding polypeptide to an antigen or antigenic determinant can be determined in any suitable known manner, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known in the art.
  • the construct of the invention, the labile peptide linker and the first and second polypeptides comprised within the construct all comprise amino acid residues.
  • first and second polypeptides each comprise or more suitably consists of a sequence selected from the group consisting of SEQ ID NO: 18 and SEQ ID NO: 19
  • the construct comprises or more suitably consists of a sequence selected from the group consisting of SEQ ID NOs: 7 to 17.
  • the construct comprises or more suitably consists of a sequence selected from the group consisting of SEQ ID NOs: 8 to 11 and 13 to 16.
  • the labile peptide linker comprises or more suitably consists of a sequence selected from the group consisting of SEQ ID NOs: 1 to 6.
  • the labile peptide linker comprises or more suitably consists of a sequence selected from the group consisting of SEQ ID NOs: 2 to 5.
  • amino acid residue in regions of the construct other than the labile peptide linker can be replaced with another amino acid residue of similar chemical structure and which is expected to have little influence on the function, activity or other biological properties of the polypeptide.
  • Such a substitution is a “conservative” substitution
  • conservative substitutions suitably are substitutions in which one amino acid within the following groups is substituted by another amino acid residue from within the same group:
  • the construct excluding the labile peptide linker, may suitably include one or more conservative substitutions.
  • nucleic acid encoding the construct of the invention.
  • a construct according to the invention comprises a first polypeptide and a second polypeptide.
  • the inventive construct is therefore multimeric and may suitably be multivalent.
  • Such a construct may comprise a first polypeptide and a second polypeptide which are identical.
  • a construct consisting of two identical polypeptides is a “homobihead”.
  • a construct comprising two identical polypeptides.
  • a construct may consist of a first polypeptide and a second polypeptide which are different from one another (a “heterobihead”).
  • Constructs can be multivalent and/or multispecific.
  • a multivalent construct (such as a bivalent construct) comprises two or more binding polypeptides and therefore provides two or more sites at which attachment to antigens can occur suitably before or after cleavage of the labile peptide linker.
  • An example of a multivalent construct could be a homobihead or a heterobihead.
  • a multispecific construct such as a bispecific construct comprises two different binding polypeptides which present two sites at which either (a) attachment to two different antigens can occur or (b) attachment to two different epitopes on the same antigen can occur, suitably before or after cleavage of the labile peptide linker.
  • An example of a multispecific construct could be a heterobihead.
  • a multispecific construct is multivalent.
  • a construct of the invention may comprise an additional third polypeptide (connected to the first polypeptide by a peptide linker) and may also comprise or consist of an additional fourth polypeptide (connected to the second polypeptide by a peptide linker), wherein the third and fourth polypeptides are substantially resistant to the one or more proteases present in the intestinal tract and are as defined herein in respect of the first and second polypeptides.
  • a construct of the invention consisting of four polypeptides is known as a ‘quadrahead’.
  • the peptide linkers are substantially resistant to the one or more proteases present in the intestinal tract or alternatively the peptide linkers are labile peptide linkers as defined herein.
  • the first, second, third and/or fourth polypeptide has a molecular weight of no greater 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.
  • the gastrointestinal tract is an organ system responsible for consuming and digesting foodstuffs, absorbing nutrients, and expelling waste.
  • the GIT consists of the oesophagus, stomach, small intestine (duodenum, jejunum and ileum) and large intestine (cecum, colon, rectum and anal canal).
  • Various pathogens may colonise and various diseases may manifest in different areas of the GIT.
  • the intestinal tract (as opposed to the gastrointestinal tract) consists of the small and large intestine.
  • proteases include proteases, lipases, amylases and nucleases.
  • proteases include serine proteases, threonine proteases, cysteine proteases, aspartate proteases, glutamic acid proteases and metalloproteases.
  • proteolysis Some detach the terminal amino acids from the protein chain (exopeptidases), others attack internal peptide bonds of a protein (endopeptidases).
  • Proteolysis can be highly promiscuous such that a wide range of protein substrates are hydrolysed. This is the case for proteases which cleave the wide array of ingested polypeptides in the intestinal tract into smaller polypeptide fragments.
  • proteases typically bind to a single amino acid (a labile site) on the substrate and so only have specificity for that residue.
  • the proteases present in the intestinal tract include trypsin, trypsin-like proteases, chymotrypsin, chymotrypsin-like proteases, carboxypeptidase, elastase, aminopeptidase, carboxypeptidase and enteropeptidase. 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. Particularly tyrosine, phenylalanine and tryptophan.
  • the labile peptide linker is labile to one or more proteases present in the intestinal tract and wherein the first and second polypeptides are substantially resistant to said one or more proteases, wherein said one or more proteases are present in the small or large intestine, more suitably the jejunum, the ileum and/or the cecum.
  • the one or more proteases are serine proteases.
  • the one or more proteases are selected from the group consisting of enteropeptidase, trypsin, trypsin-like proteases, chymotrypsin and chymotrypsin-like proteases.
  • the first and second polypeptides of the construct of the invention are substantially resistant to all proteases present in the intestinal tract.
  • proteases include proteases sourced from gastrointestinal tract commensal microflora or pathogenic bacteria, for example wherein the proteases are cell membrane-attached proteases, secreted proteases and proteases released on cell lysis.
  • the intestinal tract is a mammalian intestinal tract, such as a human, simian, murine, bovine, ovine or porcine intestinal tract.
  • Diseases of the GIT refer to diseases involving the gastrointestinal tract, namely the oesophagus, stomach, small intestine (duodenum, jejunum and ileum) and large intestine (cecum, colon, rectum and anal canal).
  • the construct of the invention may be used in the treatment or prevention of such diseases. Exemplary diseases of the GIT are described below.
  • Autoimmune diseases develop when the immune system responds adversely 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 affect only 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 tissues, endocrine glands such as the thyroid or pancreas, muscles, joints and skin. An inflammatory disease is a disease characterised by inflammation. Many inflammatory diseases are autoimmune diseases and vice-versa.
  • Ulcerative colitis is usually manifested by the presence of blood and mucus mixed with stool, along with lower abdominal cramping which is most severe during the passage of bowel movements.
  • the presence of diarrhoea with blood and mucus differentiates ulcerative colitis from irritable bowel syndrome, in which blood is absent.
  • the presentation of Crohn's disease is usually subtle, which leads to a later diagnosis.
  • Factors such as the location, extent, and severity of involvement determine the extent of gastrointestinal symptoms. Patients who have ileocolonic involvement usually have postprandial abdominal pain, with tenderness in the right lower quadrant and an occasional inflammatory mass.
  • the pharmaceutical composition or construct of the invention is for use in the treatment of an autoimmune and/or inflammatory disease of the GIT selected from the list consisting of Crohn's disease, ulcerative colitis, irritable bowel syndrome, diabetes type II, glomerulonephritis, autoimmune hepatitis, Sjogren's syndrome, celiac disease and drug- or radiation-induced mucositis (most suitably Crohn's disease).
  • an autoimmune and/or inflammatory disease of the GIT selected from the list consisting of Crohn's disease, ulcerative colitis, irritable bowel syndrome, diabetes type II, glomerulonephritis, autoimmune hepatitis, Sjogren's syndrome, celiac disease and drug- or radiation-induced mucositis (most suitably Crohn's disease).
  • the construct of the invention may be used for the treatment or prevention of bacterial infection including infection by common bacterial GIT pathogens including Escherichia coli, Salmonella, Campylobacter, Vibrio cholerae, Shigella, Clostridium perfringens, Clostridium difficile, Bacillus cereus, Vibrio parahaemolyticus, Yersinia enerocolitica .
  • the construct of the invention may be used for the treatment or prevention of viral infection including common viral GIT pathogens which include rotavirus, norovirus and small round viruses.
  • the construct of the invention is for use in the treatment or prevention of nosocomial infection.
  • the construct of the invention is for use in the treatment or prevention of C. difficile infection.
  • the first and/or second polypeptide of the construct of the invention binds to a target accessible via the intestinal tract such as a target within the intestinal tract.
  • a target is a deleterious agent originating from an intestinal tract resident pathogenic microbe.
  • the target is selected from the group consisting of: an interleukin (such as IL-1, IL-1ra, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18 and IL-23), an interleukin receptor (such as IL-6R and IL-7R), a transcription factor (such as NF-kB), a cytokine (such as TNF-alpha, IFN-gamma TGF-beta and TSLP), a transmembrane protein (such as gp130 and CD3), a surface glycoprotein (such as CD4, CD20, CD40), a soluble protein (such as CD40L), an integrin (such as a4b7 and AlphaEbeta7), an adhesion molecule (such as MAdCAM), a chemokine (such as IP10 and CCL20), a chemokine (such
  • the target is selected from the group consisting of: TNF-alpha, C. difficile toxin A, C. difficile toxin B, CD3 and IL-6R; more suitably TNF-alpha or C. difficile toxin A.
  • the target of the first and second polypeptides are identical or different.
  • the first and/or second polypeptide of the construct of the invention does not bind to an interleukin (such as IL-1, IL-1ra, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18 and IL-23).
  • an interleukin such as IL-1, IL-1ra, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18 and IL-23).
  • first and/or second polypeptide of the construct of the invention does not bind to an interleukin receptor (such as IL-6R and IL-7R).
  • first and/or second polypeptide of the construct of the invention does not bind to a transcription factor (such as NF-kB).
  • first and/or second polypeptide of the construct of the invention does not bind to a cytokine (such as TNF-alpha, IFN-gamma TGF-beta and TSLP).
  • a cytokine such as TNF-alpha, IFN-gamma TGF-beta and TSLP.
  • first and/or second polypeptide of the construct of the invention does not bind to a transmembrane protein (such as gp130 and CD3).
  • first and/or second polypeptide of the construct of the invention does not bind to a surface glycoprotein (such as CD4, CD20 and CD40).
  • first and/or second polypeptide of the construct of the invention does not bind to a soluble protein (such as CD40L).
  • first and/or second polypeptide of the construct of the invention does not bind to an integrin (such as a4b7 and AlphaEbeta7).
  • first and/or second polypeptide of the construct of the invention does not bind to an adhesion molecule (such as MAdCAM)
  • first and/or second polypeptide of the construct of the invention does not bind to a chemokine (such as IP10 and CCL20).
  • first and/or second polypeptide of the construct of the invention does not bind to a chemokine receptor (such as CCR2 and CCR9).
  • first and/or second polypeptide of the construct of the invention does not bind to an inhibitory protein (SMAD7).
  • SAD7 an inhibitory protein
  • first and/or second polypeptide of the construct of the invention does not bind to a kinase (such as JAK3).
  • first and/or second polypeptide of the construct of the invention does not bind to a G protein-coupled receptor (such as sphingosine-1-P receptor).
  • first and/or second polypeptide of the construct of the invention does not bind to a toxin (such as C. difficile toxin A and C. difficile toxin B).
  • the construct or composition of the invention is for use as a medicament, suitably administered by oral administration, suitably for use in the treatment or prevention of diseases of the GIT (see intra).
  • the construct or composition of the invention may also be used in the treatment or prevention of other medical conditions by oral administration such as metabolic disorders, such as obesity.
  • the construct or composition of the invention is intended to have local effect in the intestinal tract.
  • the construct or composition of the invention is not for use in the treatment or prevention of diseases by delivery into the circulation in therapeutically effective quantities.
  • a method of treating autoimmune disease or C. difficile infection comprising administering to a person in need thereof a therapeutically effective amount of the inventive construct or composition.
  • a therapeutically effective amount of a construct of the invention is an amount which is effective, upon single or multiple dose administration to a subject, in neutralising the biological effects of a chosen target to a significant extent in a subject.
  • a therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the construct to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the construct are outweighed by the therapeutically beneficial effects.
  • the construct of the invention can be incorporated into pharmaceutical compositions suitable for oral administration to a subject.
  • the construct of the invention can be in the form of a pharmaceutically acceptable salt.
  • a method of treating a disease with a monomeric antibody or a monomeric antigen binding fragment thereof comprising administering to a subject the inventive construct.
  • a method of treating a disease with two or more monomeric antibodies or monomeric antigen binding fragments thereof comprising administering to a subject the inventive construct.
  • a pharmaceutical composition of the invention is formulated for oral delivery.
  • the pharmaceutical compositions of the invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions, dispersions or suspensions, tablets, pills and powders. Solid dosage forms are preferred.
  • the pharmaceutical composition may comprise a pharmaceutically acceptable excipient, and suitably may be used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • the pharmaceutical composition comprises a construct of the invention and a pharmaceutically acceptable excipient such as a carrier.
  • a pharmaceutically acceptable carrier include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
  • Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the polypeptide or construct of the invention.
  • compositions may include antiadherents, binders, coatings, disintegrants, flavours, colours, lubricants, sorbents, preservatives, sweeteners, freeze dry excipients (including lyoprotectants) or compression aids.
  • the construct of the invention is lyophilised before being incorporated into a pharmaceutical composition.
  • the first and second polypeptides of the construct are substantially resistant to all proteases present in the intestinal tract by virtue of the inherent properties of the polypeptides or construct itself.
  • a polypeptide of the invention may also be provided with an enteric coating.
  • An enteric coating is a polymer barrier applied on oral medication which protects the polypeptide from 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, hydroxy propyl methyl cellulose phthalate, hydroxy propyl methyl cellulose acetate succinate (hypromellose 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 which are insoluble at the highly acidic pH found in the stomach, but which dissolve rapidly at a less acidic pH. Thus, suitably, the enteric coating will not dissolve in the acidic juices of the stomach (pH ⁇ 3), but will do so in the higher pH environment present in the small intestine (pH above 6) or in the colon (pH above 7.0).
  • the pH-dependent release polymer is selected such that the construct of the invention will be released at about the time that the dosage reaches the target region of the intestinal tract.
  • the pharmaceutical composition or construct of the invention may be formulated in a buffer, in order to stabilise the pH of the composition, at a concentration between 5-50, or more suitably 15-40 or more suitably 25-30 g/litre.
  • suitable buffer components include physiological salts such as sodium citrate and/or citric acid.
  • physiological salts such as sodium citrate and/or citric acid.
  • buffers contain 100-200, more suitably 125-175 mM physiological salts such as sodium chloride.
  • the buffer is selected to have a pKa close to the pH of the composition or the physiological pH of the patient.
  • Exemplary construct concentrations in a pharmaceutical composition may 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.
  • An aqueous formulation of the construct or pharmaceutical composition of the invention may be prepared in a pH-buffered solution, e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5.
  • suitable buffers include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers.
  • the buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, for example, on the buffer and the desired tonicity of the formulation.
  • the tonicity of the pharmaceutical composition may be altered by including a tonicity modifier.
  • Such tonicity modifiers can be charged or uncharged chemical species.
  • Typical uncharged tonicity modifiers include sugars or sugar alcohols or other polyols, preferably trehalose, sucrose, mannitol, glycerol, 1,2-propanediol, raffinose, sorbitol or lactitol (especially trehalose, mannitol, glycerol or 1,2-propanediol).
  • Typical charged tonicity modifiers include salts such as a combination of sodium, potassium or calcium ions, with chloride, sulfate, carbonate, sulfite, nitrate, lactate, succinate, acetate or maleate ions (especially sodium chloride or sodium sulphate); or amino acids such as arginine or histidine.
  • the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable.
  • the term “isotonic” denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum.
  • Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 1 mM to 500 nM.
  • at least one isotonic agent is included in the composition.
  • a surfactant may also be added to the pharmaceutical composition to reduce aggregation of the formulated construct and/or minimize the formation of particulates in the formulation and/or reduce adsorption.
  • exemplary surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS).
  • suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, and polysorbate 80.
  • Exemplary concentrations of surfactant may range from about 0.001% to about 10% w/v.
  • a lyoprotectant may also be added in order to protect the construct of the invention against destabilizing conditions during the lyophilization process.
  • known lyoprotectants include sugars (including glucose, sucrose, mannose and trehalose); polyols (including mannitol, sorbitol and glycerol); and amino acids (including alanine, glycine and glutamic acid). Lyoprotectants can be included in an amount of about 10 mM to 500 mM.
  • the dosage ranges for administration of the pharmaceutical composition or construct of the invention are those to produce the desired therapeutic effect.
  • the dosage range required depends on the precise nature of the pharmaceutical composition or construct, the target region of the intestinal tract, the nature of the formulation, the age of the patient, the nature, extent or severity of the patients condition, contraindications, if any, and the judgement of the attending physician. Variations in these dosage levels can be adjusted using standard empirical routines for optimisation.
  • Suitable daily dosages of pharmaceutical composition or construct of the invention are in the range of 50 ng-50 mg per kg, such as 50 ug-40 mg per kg, such as 5-30 mg per kg of body weight.
  • the unit dosage can vary from less than 100 mg, but typically will be in the region of 250-2000 mg per dose, which may be administered daily or more frequently, for example 2, 3 or 4 times per day or less frequently for example every other day or once per week.
  • Treatment of diseases also embraces treatment of exacerbations thereof and also embraces treatment of patients in remission from disease symptoms to prevent relapse of disease symptoms.
  • a pharmaceutical composition of the 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 invention to use the pharmaceutical composition of the invention in therapeutic methods for the treatment of bacterial infection, autoimmune and/or inflammatory diseases as an adjunct to, or in conjunction with, other established therapies normally used in the treatment of bacterial, autoimmune and/or inflammatory diseases.
  • active agents e.g. active agents suitable for treating diseases such as those mentioned herein.
  • irritable bowel disease such as Crohn's disease or ulcerative colitis
  • active agents selected from the list comprising: 5-aminosalicylic acid, or a prodrug thereof (such as sulfasalazine, olsalazine or bisalazide); corticosteroids (e.g. prednisolone, methylprednisolone, or budesonide); immunosuppressants (e.g.
  • anti-TNF-alpha antibodies e.g., infliximab, adalimumab, certolizumab pegol or golimumab
  • anti-IL12/1L23 antibodies e.g., ustekinumab
  • anti-IL6R antibodies or small molecule IL12/1L23 inhibitors e.g., apilimod
  • Anti-alpha-4-beta-7 antibodies e.g., vedolizumab
  • MAdCAM-1 blockers e.g., PF-00547659
  • antibodies against the cell adhesion molecule alpha-4-integrin e.g., natalizumab
  • antibodies against the IL2 receptor alpha subunit e.g., daclizumab or basiliximab
  • JAK3 inhibitors e.g., tofacitinib or R348
  • Syk inhibitors e.g., tofacitinib or R348
  • possible combinations include combinations with, for example, one or more active agents selected from the list comprising C. difficile toxoid vaccine, ampicillin, amoxicillin, vancomycin, metronidazole, fidaxomicin, linezolid, nitazoxanide, rifaximin, ramoplanin, difimicin, clindamycin, cephalosporins (such as second and third generation cephalosporins), fluoroquinolones (such as gatifloxacin or moxifloxacin), macrolides (such as erythromycin, clarithromycin, azithromycin), penicillins, aminoglycosides, trimethoprim-sulfamethoxazole, chloramphenicol, tetracycline, imipenem, meropenem, antibacterial agents, bactericides, or bacteriostats. Possible combinations also include combinations with one or more active agents which are
  • compositions of the invention in combination with one or more further active agents, for example one or more active agents described above.
  • pharmaceutical composition or construct is administered sequentially, simultaneously or separately with at least one active agent selected from the list above.
  • the invention also encompasses a kit of parts comprising components:
  • component (i) of the kit of parts is thus component (A) above in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.
  • component (ii) is component (B) above in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.
  • the one or more other active agents i.e. component (B) above
  • component (B) is more than one further active agent, these further active agents can be formulated with each other or formulated with component (A) or they may be formulated separately. In one embodiment component (B) is one other therapeutic agent. In another embodiment component (B) is two other therapeutic agents.
  • the combination product (either a combined preparation or kit-of-parts) of this aspect of the invention may be used in the treatment or prevention of an autoimmune disease (e.g. the autoimmune diseases mentioned herein).
  • vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • a plasmid which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • a viral vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian and yeast vectors). Other vectors (e.g.
  • non-episomal mammalian vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”).
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector may be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g. replication defective retroviruses.
  • recombinant host cell (or simply “host cell”), as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. Such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell.
  • a vector comprising the polynucleotide encoding the construct of the invention or cDNA comprising said polynucleotide.
  • a host cell transformed with said vector, which is capable of expressing the construct of the invention.
  • the host cell is a yeast such as a yeast belonging to the genera Aspergillus, Saccharomyces, Kluyveromyces, Hansenula or Pichia , such as Saccharomyces cerevisiae, Escherchia coli or Pichia pastoris.
  • the labile peptide linker is cleaved by proteases present in the intestinal tract, but if the labile peptide linker is also substantially resistant to proteases of the host organism in which the construct is produced. Therefore, in one embodiment of the invention, the labile peptide linker is substantially resistant to proteases of the host organism in which the construct is produced.
  • artificial gene synthesis may be used to produce a construct according to the invention (Nambiar et al 1984 Science 223:1299-1301, Sakamar and Khorana 1988 Nucl. Acids Res 14:6361-6372, Wells et al 1985 Gene 34:315-323 and Grundstrom et al 1985 Nucl. Acids Res 13:3305-3316, herein incorporated by reference in their entirety).
  • a gene encoding a construct of the invention can be synthetically produced by, for example, solid-phase DNA synthesis. Entire genes may be synthesized de novo, without the need for precursor template DNA.
  • the building blocks are sequentially coupled to the growing oligonucleotide chain in the order required by the sequence of the product.
  • the product is released from the solid phase to solution, deprotected, and collected. Products can be isolated by high-performance liquid chromatography (HPLC) to obtain the desired oligonucleotides in high purity (Verma and Eckstein 1998 Annu Rev Biochem 67:99-134).
  • the constructs of the invention may be fused genetically at the DNA level i.e. a polynucleotide construct which encodes the complete polypeptide construct comprising one or more polypeptides such as antigen-binding polypeptides.
  • One way of joining multiple polypeptides via the genetic route is by linking the polypeptide coding sequences via a labile peptide linker coding sequence.
  • the carboxy-terminal end of the first polypeptide may be linked to the amino-terminal end of the next polypeptide via a labile peptide linker coding sequence.
  • This linking mode can be extended in order to link polypeptides for the construction of tri-, tetra-, etc. functional constructs.
  • a method for producing multivalent (such as bivalent) VHH polypeptide constructs is disclosed in WO96/34103 (herein incorporated by reference in its entirety).
  • Mutations can be made to the DNA or cDNA that encode constructs which are silent as to the amino acid sequence of the polypeptide, but which provide preferred codons for translation in a particular host.
  • the preferred codons for translation of a nucleic acid in, e.g., E. coli and S. cerevisiae are known.
  • Mutation of constructs can be achieved for example by substitutions, additions or deletions to a nucleic acid encoding the construct.
  • the substitutions, additions or deletions to a nucleic acid encoding the construct can be introduced by many methods, including for example error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis (Ling et al 1997 Anal Biochem 254(2):157-178, herein incorporated by reference in its entirety), gene reassembly, Gene Site Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR) or a combination of these methods.
  • GSSM Gene Site Saturation Mutagenesis
  • SLR synthetic ligation reassembly
  • the modifications, additions or deletions to a nucleic acid can also be introduced by a method comprising recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, ensemble mutagenesis, chimeric nucleic acid multimer creation, or a combination thereof.
  • constructs comprising immunoglobulin chain variable domains such as VHs and VHHs can be achieved using a suitable expression vector such as a prokaryotic cell such as bacteria, for example E. coli (for example according to the protocols disclosed in WO94/04678 and WO96/34103, which are incorporated herein by reference).
  • a suitable expression vector such as a prokaryotic cell such as bacteria, for example E. coli (for example according to the protocols disclosed in WO94/04678 and WO96/34103, which are incorporated herein by reference).
  • Expression of immunoglobulin chain variable domains such as VHs and VHHs can also be achieved using eukaryotic cells, for example insect cells, CHO cells, Vero cells or suitably yeast cells such as yeasts belonging to the genera Aspergillus, Saccharomyces, Kluyveromyces, Hansenula or Pichia .
  • S. cerevisiae is used (for example according to the protocols disclosed in WO94/025591,
  • the construct of the invention can be produced in a fungus such as a yeast (for example, S. cerevisiae ) comprising growth of the fungus on a medium comprising a carbon source wherein 50-100 wt % of said carbon source is ethanol, according to the methods disclosed in WO02/48382.
  • a yeast for example, S. cerevisiae
  • a carbon source wherein 50-100 wt % of said carbon source is ethanol
  • Trypsin Protease Assay was developed. This assay is performed as follows.
  • the beads are washed 3 times with water (250 ⁇ l beads+1.25 ml water) followed by washing 5 times with Trypsin buffer (TRYP buffer; 1 mM Tris-HCl, 20 mM CaCl2 [pH 8.0]). Finally, the resin is resuspended in TRYP buffer as a 50% (v/v) suspension.
  • the Trypsin Protease Assay protocol can be used wherein Trypsin beads are substituted with Chymotrypsin beads.
  • Faecal Extract Protease Assay was developed. Faecal extract is a physiologically relevant matrix in particular for polypeptides targeted to the large intestine. This assay is performed as follows.
  • Human faecal samples from multiple individuals are turned into slurries with addition of 1 ⁇ PBS at a ratio of 1, 2 or 3 mLs 1 ⁇ PBS per gram of faeces.
  • the slurries are then pooled (such that one pool represents the combined protease output from the faeces of multiple individuals), centrifuged and the supernatants removed, aliquoted and stored at ⁇ 70 degrees C. This process removes the faecal matrix, including any cellular material.
  • constructs are incubated at a concentration of 160.16 ⁇ g/ml at 37° C. in pooled human faecal supernatant for 60 mins in the absence of BSA carrier. Aliquots are taken after time intervals such as 0, 10, 30 and 60 minutes and are mixed 1:1 in Protease Stop Solution (1 ⁇ PBS+2 ⁇ protease inhibitor cocktail (Sigma)+1% PMSF) and immediately frozen at ⁇ 80° C.
  • samples are defrosted and 15 ⁇ l of each sample is mixed with 5 ⁇ l 4 ⁇ loading dye, boiled for 10 mins and 15 ⁇ l is loaded per lane on a polyacrylamide gel (such as NuPAGE 10% acrylamide Bis-Tris gel).
  • Gels are run in SDS-MES buffer at 200 V for 35 mins. Gels are fixed in 40% methanol, 7% acetic acid for 30 mins and stained in colloidal Coomassie Brilliant Blue stain overnight. Gels were destained in water before imaging (such as using ImageQuant LAS4000 with 7 secs exposure).
  • the quantity of intact constructs relative to cleaved constituent polypeptides can be assessed by comparing the corresponding higher and lower molecular weight bands in each time point lane.
  • the anti-TcdA constructs were homobiheads of ICVD ID1A, separated by different linkers.
  • the anti-TNF-alpha constructs were homobiheads of ICVD ID5F, encoded by polynucleotides which also included Flag-6His tags.
  • ID3A, ID4A and ID25A to ID28A were expressed from S. cerevisiae . These constructs do not carry any protein tags at either terminus.
  • ID25A to ID28A containing the labile peptide linkers, were generated by overlap PCR using ID1A encoding DNA as a template and cloned into the SacI/HindIII sites of shuttle vector pUR4547 (BAC) capable of replication in E. coli and S. cerevisiae . Expression, and full secretion into the supernatant, from S. cerevisiae was achieved from the shuttle vector via galactose induction. Finally, the constructs were purified using Capto S cation exchange resin (GE) and dialysed into 1 ⁇ PBS.
  • GE Capto S cation exchange resin
  • ID55F to ID60F were cloned directly into the SfiI/BsteII sites of vector pMEK222 (QVQ).
  • ID55F to ID60F carry C-terminal Flag-6His tags.
  • the resulting plasmids were transformed into E. coli BL 21 DE3 (Novagen) for expression and transport to the periplasm. Following overnight growth in Autoinduction medium Terrific broth (Formedium), cells were pelleted, frozen at ⁇ 80 degrees C. and re-suspended in 1/10 the original volume of 1 ⁇ PBS to disrupt the outer membrane.
  • ID55F to ID60F were purified from the supernatant by AKTA Prime FPLC using a Ni-NTA column to bind the His tags, followed by imidazole elution and dialysis into 1 ⁇ PBS.
  • Example 4 The lability of the constructs of Example 4 targeting TcdA were assayed using The Trypsin Protease Assay described in Example 1.
  • ID3A remained substantially intact over all time periods tested ( FIG. 1 ).
  • ID25A FIG. 2
  • the shielding D residues in ID26A FIG. 2
  • the labile residues situated at either terminus of the ID28A linker FIG. 3
  • the additional N-side shielding D residues in ID27A FIG. 3
  • the constructs in order of least to most labile were ID3A>ID28A>ID27A>ID26A>ID25A.
  • ID3A did not comprise a labile peptide linker and is not a construct according to the invention.
  • the first immunoglobulin chain variable domain and the second immunoglobulin chain variable domain in these constructs are themselves substantially resistant to one or more proteases present in the intestinal tract. It can be seen that there are no significant bands visible which correspond to fragments of ICVD monomers (the intact monomers running at a molecular weight of approximately 18 kDa) and thus at least 70% by mass of the immunoglobulin chain variable domains have clearly remained uncleaved after all time points tested including 160 minutes after mixing in the Trypsin Protease Assay.
  • Example 4 The lability of the constructs of Example 4 targeting TcdA and targeting TNF-alpha were assayed using The Faecal Protease Assay described in Example 3. The results of the assay are shown in SDS-PAGE gels in FIG. 4 (anti-TcdA constructs) and FIG. 5 (anti-TNF-alpha constructs). Lanes are labelled according to the construct tested and the period of incubation in minutes. ‘X’ indicates lane containing no ICVD (only faecal extract). In FIG. 5 , ‘B’ indicates bihead and ‘M’ indicates cleaved monomers.
  • ID3A remained substantially intact over all time periods tested.
  • ID25A was completely cleaved very quickly after between 0 and 10 minutes of incubation.
  • the shielding D residues in ID26A, the additional N-side shielding D residues in ID27A and the labile residues situated at either terminus of the ID28A linker all resulted in slower cleavage relative to ID25A. Digestion of ID26A, ID27A and ID28A was still ongoing after 30 minutes of incubation.
  • ID55F remained substantially intact over all time periods tested.
  • ID56F underwent instant total cleavage.
  • ID57F was completely cleaved very quickly after between 0 and 10 minutes of incubation.
  • the shielding D residues in ID58F, the labile residues situated at either terminus of the ID60F linker and the additional N-side shielding D restudies in ID59F all resulted in slower cleavage relative to ID57F. Digestion of ID58F, ID59F and ID60F was still ongoing after 60 minutes of incubation.
  • the anti-TcdA constructs (ID3A, ID25A, ID25A, ID27A and ID28A) were tested for their stability over time during storage. Samples were taken after 0, 2, 4 or 9 days of refrigeration at 4° C. ( FIGS. 6 and 7 ). It can be seen that the SDS-PAGE gels contain bands corresponding to uncleaved bihead constructs and do not contain bands for cleaved monomers. Accordingly, the constructs are stable for at least 9 days under these conditions.
  • the constituent immunoglobulin chain variable domains are substantially resistant to yeast proteases because no significant bands running at a lower molecular weight than that of a single ICVD monomer (running at approximately 18 kDa) are visible in FIG. 6 or 7 . Accordingly, it is clear that no more than 10% by mass of the first or second immunoglobulin chain variable domain is cleaved after producing the first or second immunoglobulin chain variable domain using the Yeast Expression Protocol (with 0 days storage) and also after a storage period of 2, 4 or 9 days.
  • Homobihead or heterobihead constructs containing a range of different linker sequences containing chymotrypsin-labile sites may be designed and expressed in a suitable host such as S. cerevisiae , using the methods analogous to those detailed in Example 4 above.
  • a suitable construct is provided in Table 4.
  • ID4A Anti-TcdA construct ID4A was expressed in S.cerevisiae using The Yeast Expression Protocol. ID4A is a homobihead of ICVD ID1A and does not carry any protein tag at either terminus. The sequence of the linker is given in Table 1 above.
  • ID4A was tested for its stability over time during production and storage. Samples were taken after 0, 2, 4 or 9 days of refrigeration at 4° C. ( FIG. 8 ). It can be seen that every lane of the SDS-PAGE gel contains bands corresponding to cleaved monomers, all running at an approximate molecular weight of 10 kDa and no lane clearly contains a band corresponding to uncleaved bihead. It appears that the linker used in ID4A was cleaved by yeast proteases during production.
  • ID4A is cleaved into first and second immunoglobulin chain variable domains after producing the construct using the Yeast Expression Protocol (with 0 days storage).
  • Constructs of the invention are stable to yeast proteases and therefore ID4A is not a construct of the invention.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Rheumatology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Pain & Pain Management (AREA)
  • Transplantation (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicinal Preparation (AREA)
US15/717,174 2015-03-31 2017-09-27 Peptide construct having a protease-cleavable linker Abandoned US20180037639A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US17/196,498 US12234279B2 (en) 2015-03-31 2021-03-09 Peptide construct having a protease-cleavable linker
US19/025,399 US20250388655A1 (en) 2015-03-31 2025-01-16 Peptide construct having a protease-cleavable linker

Applications Claiming Priority (9)

Application Number Priority Date Filing Date Title
EP15162112.5 2015-03-31
EP15162114.1 2015-03-31
EP15162115.8 2015-03-31
EP15162115 2015-03-31
EP15162112 2015-03-31
EP15162114 2015-03-31
EP16152320 2016-01-21
EP16152320.4 2016-01-21
PCT/EP2016/057022 WO2016156466A1 (en) 2015-03-31 2016-03-31 Peptide construct having a protease-cleavable linker

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2016/057022 Continuation WO2016156466A1 (en) 2015-03-31 2016-03-31 Peptide construct having a protease-cleavable linker

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US17/196,498 Continuation US12234279B2 (en) 2015-03-31 2021-03-09 Peptide construct having a protease-cleavable linker

Publications (1)

Publication Number Publication Date
US20180037639A1 true US20180037639A1 (en) 2018-02-08

Family

ID=55661404

Family Applications (3)

Application Number Title Priority Date Filing Date
US15/717,174 Abandoned US20180037639A1 (en) 2015-03-31 2017-09-27 Peptide construct having a protease-cleavable linker
US17/196,498 Active 2037-03-20 US12234279B2 (en) 2015-03-31 2021-03-09 Peptide construct having a protease-cleavable linker
US19/025,399 Pending US20250388655A1 (en) 2015-03-31 2025-01-16 Peptide construct having a protease-cleavable linker

Family Applications After (2)

Application Number Title Priority Date Filing Date
US17/196,498 Active 2037-03-20 US12234279B2 (en) 2015-03-31 2021-03-09 Peptide construct having a protease-cleavable linker
US19/025,399 Pending US20250388655A1 (en) 2015-03-31 2025-01-16 Peptide construct having a protease-cleavable linker

Country Status (11)

Country Link
US (3) US20180037639A1 (https=)
EP (1) EP3277317A1 (https=)
JP (2) JP7064670B2 (https=)
KR (1) KR102789821B1 (https=)
CN (1) CN107427577A (https=)
AU (2) AU2016239949A1 (https=)
CA (1) CA2981098A1 (https=)
HK (1) HK1250011A1 (https=)
IL (1) IL254589B2 (https=)
SG (1) SG11201706840WA (https=)
WO (1) WO2016156466A1 (https=)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10633438B2 (en) 2015-03-31 2020-04-28 Vhsquared Limited Polypeptides
WO2020191361A2 (en) 2019-03-21 2020-09-24 Codiak Biosciences, Inc. Extracellular vesicles for vaccine delivery
WO2020215010A1 (en) 2019-04-17 2020-10-22 Codiak Biosciences, Inc. Compositions of exosomes and aav
WO2021003445A1 (en) 2019-07-03 2021-01-07 Codiak Biosciences, Inc. Extracellular vesicles targeting t cells and uses thereof
US11623952B2 (en) 2019-06-21 2023-04-11 Sorriso Pharmaceuticals, Inc. IL-23 and TNF-alpha binding bi-specific heavy chain polypeptides
US11667719B2 (en) 2019-06-21 2023-06-06 Sorriso Pharmaceuticals, Inc. VHH immunoglobulin chain variable domain that binds to IL-7R and methods of use thereof for treating autoimmune and/or inflammatory diseases
US12173054B2 (en) 2015-03-31 2024-12-24 Sorriso Pharmaceuticals, Inc. Polypeptides
US12234279B2 (en) 2015-03-31 2025-02-25 Sorriso Pharmaceuticals, Inc. Peptide construct having a protease-cleavable linker
US12570736B2 (en) 2019-06-21 2026-03-10 Sorriso Pharmaceuticals, Inc. Compositions

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9134766B2 (en) 2009-10-20 2015-09-15 Hewlett-Packard Development Company, L.P. Keyboard hold down mechanism
GB201709222D0 (en) * 2017-06-09 2017-07-26 Univ Manchester C3b Inactivating Polypeptide
GB201800620D0 (en) 2018-01-15 2018-02-28 Univ Manchester C3b Binding Polypeptide
WO2020144164A1 (en) 2019-01-07 2020-07-16 Bactolife Aps Pathogen binding proteins

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110229476A1 (en) * 2007-08-15 2011-09-22 Bayer Schering Pharma Aktiengesellschaft Monospecific and multispecific antibodies and method of use
US8399188B2 (en) * 2006-09-28 2013-03-19 Illumina, Inc. Compositions and methods for nucleotide sequencing
US20140186365A1 (en) * 2013-01-03 2014-07-03 Institute For Cancer Research D/B/A The Research Institute Of Fox Chase Cancer Center Antibodies that specifically bind to serum albumin without interfering with albumin's capability to interact with the fcrn
US20140377287A1 (en) * 2002-03-01 2014-12-25 Immunomedics, Inc. Anti-trop-2 antibody-drug conjugates and uses thereof

Family Cites Families (151)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3300077A (en) 1965-02-18 1967-01-24 Clinch Tite Corp Pallet enclosure
JP3443119B2 (ja) 1989-08-07 2003-09-02 ペプテック リミテッド 腫瘍壊死因子結合リガンド
GB9015198D0 (en) 1990-07-10 1990-08-29 Brien Caroline J O Binding substance
DK0590060T3 (da) 1991-06-21 1998-05-11 Univ Cincinnati Oralt indgivelige terapeutiske proteiner samt fremgangsmåde til fremstilling
DK1621554T4 (da) 1992-08-21 2012-12-17 Univ Bruxelles Immunoglobuliner blottet for lette kæder
ES2162863T3 (es) 1993-04-29 2002-01-16 Unilever Nv Produccion de anticuerpos o fragmentos (funcionalizados) de los mismos derivados de inmunoglobulinas de cadena pesada de camelidae.
US5512459A (en) * 1993-07-20 1996-04-30 Bionebraska, Inc. Enzymatic method for modification or recombinant polypeptides
DE69432309D1 (de) 1993-09-20 2003-04-24 Anadis Ltd Verfahren zur herstellung von immunoglobulinen aus colostrum und deren verwendung in pharmazeutischen zusammensetzungen
EP0739981A1 (en) * 1995-04-25 1996-10-30 Vrije Universiteit Brussel Variable fragments of immunoglobulins - use for therapeutic or veterinary purposes
DK1027439T3 (da) 1997-10-27 2010-05-10 Bac Ip Bv Multivalente antigenbindende proteiner
EP0954978B1 (en) 1998-03-12 2011-11-30 VHsquared Limited New products comprising inactivated yeasts or moulds provided with active antibodies
ES2299244T3 (es) * 1998-05-13 2008-05-16 Domantis Limited Sistema de exposicion de fagos para la seleccion de proteinas correctamente plegadas.
AUPP702498A0 (en) 1998-11-09 1998-12-03 Silverbrook Research Pty Ltd Image creation method and apparatus (ART77)
GB9909301D0 (en) 1999-04-22 1999-06-16 Kci Medical Ltd Wound treatment apparatus employing reduced pressure
UA81743C2 (uk) 2000-08-07 2008-02-11 Центокор, Инк. МОНОКЛОНАЛЬНЕ АНТИТІЛО ЛЮДИНИ, ЩО СПЕЦИФІЧНО ЗВ'ЯЗУЄТЬСЯ З ФАКТОРОМ НЕКРОЗУ ПУХЛИН АЛЬФА (ФНПα), ФАРМАЦЕВТИЧНА КОМПОЗИЦІЯ, ЩО ЙОГО МІСТИТЬ, ТА СПОСІБ ЛІКУВАННЯ РЕВМАТОЇДНОГО АРТРИТУ
CZ303983B6 (cs) 2000-12-13 2013-07-31 Unilever N. V. Zpusob výroby heterologního proteinu
CA2450793A1 (en) 2001-06-20 2002-12-27 Prochon Biotech Ltd. Antibodies that block receptor protein tyrosine kinase activation, methods of screening for and uses thereof
TWI334439B (en) 2001-08-01 2010-12-11 Centocor Inc Anti-tnf antibodies, compositions, methods and uses
JP2005289809A (ja) 2001-10-24 2005-10-20 Vlaams Interuniversitair Inst Voor Biotechnologie Vzw (Vib Vzw) 突然変異重鎖抗体
US8658773B2 (en) 2011-05-02 2014-02-25 Immunomedics, Inc. Ultrafiltration concentration of allotype selected antibodies for small-volume administration
WO2004009776A2 (en) 2002-07-19 2004-01-29 Abbott Biotechnology Ltd. TREATMENT OF TNFα RELATED DISORDERS
MY150740A (en) 2002-10-24 2014-02-28 Abbvie Biotechnology Ltd Low dose methods for treating disorders in which tnf? activity is detrimental
EP1558645B1 (en) 2002-11-08 2011-07-27 Ablynx N.V. Stabilized single domain antibodies in pharmaceutical composition adapted for inhalation
US20060034845A1 (en) 2002-11-08 2006-02-16 Karen Silence Single domain antibodies directed against tumor necrosis factor alpha and uses therefor
WO2004041863A2 (en) 2002-11-08 2004-05-21 Ablynx N.V. Single domain antibodies directed against interferon- gamma and uses therefor
US20060034833A1 (en) 2002-11-08 2006-02-16 Els Beirnaert Single domain antibodies directed against interferron-gamma and uses therefor
EP1900753B1 (en) 2002-11-08 2017-08-09 Ablynx N.V. Method of administering therapeutic polypeptides, and polypeptides therefor
CA2817265A1 (en) 2003-04-11 2004-10-28 Medimmune, Llc Recombinant il-9 antibodies and uses thereof
US7758859B2 (en) 2003-08-01 2010-07-20 Genentech, Inc. Anti-VEGF antibodies
US8007827B2 (en) 2004-04-02 2011-08-30 Impax Laboratories, Inc. Pharmaceutical dosage forms having immediate release and/or controlled release properties
TW201705980A (zh) 2004-04-09 2017-02-16 艾伯維生物技術有限責任公司 用於治療ＴＮＦα相關失調症之多重可變劑量療法
CN1985064B (zh) 2004-07-14 2011-08-24 尤利乌斯·布卢姆有限公司 可旋转支承的操纵臂的操纵机构
BRPI0516681A (pt) 2004-11-25 2008-09-16 Unilever Nv sistema de entrega de anticorpos ao trato gastrointestinal, vetor de expressão, microorganismos transformados, método de entrega de anticorpos, imunoglobulinas de cadeia pesada e seu uso, produto alimentìcio e seu método de fabricação e utensìlio de distribuição
US7234616B2 (en) 2004-12-27 2007-06-26 Bel-Art Products, Inc. Friction arrangement for hosiery donning aid
WO2006071877A2 (en) 2004-12-27 2006-07-06 Progenics Pharmaceuticals (Nevada), Inc. Orally deliverable and anti-toxin antibodies and methods for making and using them
US8188223B2 (en) 2005-05-18 2012-05-29 Ablynx N.V. Serum albumin binding proteins
BRPI0611901A2 (pt) 2005-06-14 2012-08-28 Amgen, Inc composição, liofilizado, kit, e, processo para preparar uma composição
US20070042399A1 (en) * 2005-06-23 2007-02-22 Imperial College Innovations Limited Biological materials and uses thereof
SI2452694T1 (sl) 2005-06-30 2019-05-31 Janssen Biotech, Inc. Protitelesa proti-IL-23, sestavki in postopki uporabe
ES2626610T3 (es) 2005-08-30 2017-07-25 Intrexon Actobiotics Nv Bacterias de ácido láctico productoras del anti-TNF alfa para el tratamiento de la enfermedad inflamatoria intestinal
PT1931710T (pt) 2005-08-31 2017-03-28 Merck Sharp & Dohme Anticorpos anti-il-23 manipulados
WO2007048022A2 (en) * 2005-10-21 2007-04-26 Alexion Pharmaceuticals, Inc. Antibody-polypeptide fusion proteins and methods for producing and using same
AU2006326867A1 (en) 2005-12-20 2007-06-28 Cephalon Australia Pty Ltd Chimeric antibodies with part New World primate binding regions
US20110059101A9 (en) 2006-03-13 2011-03-10 Ablynx N.V. Amino Acid Sequences Directed Against Il-6 And Polypetides Comprising The Same For The Treatment Of Diseases And Disorders Associated With Il-6 Mediated Signalling
US20080026820A1 (en) 2006-07-05 2008-01-31 Aruze Gaming America, Inc. Slot machine and playing method thereof
US20080031770A1 (en) 2006-08-02 2008-02-07 Douglas Heselton Apparatus and method for using ozone as a disinfectant
WO2008020079A1 (en) 2006-08-18 2008-02-21 Ablynx N.V. Amino acid sequences directed against il-6r and polypeptides comprising the same for the treatment of deseases and disorders associated with il-6-mediated signalling
MX2009002756A (es) 2006-09-12 2009-05-25 Cosmo Technologies Ltd Composiciones farmaceuticas para la administracion oral o rectal de sustancias proteinicas.
US20100129379A1 (en) 2006-09-25 2010-05-27 John Carpenter Stabilized antibody formulations and uses thereof
CA2667466A1 (en) 2006-10-27 2008-05-02 Ablynx N.V. Intranasal delivery of polypeptides and proteins
TW200823720A (en) 2006-11-27 2008-06-01 Asustek Comp Inc Portable computer
US20080145420A1 (en) 2006-12-13 2008-06-19 Simon Michael R HUMAN SECRETORY IgA FOR THE TREATMENT OF CLOSTRIDIUM DIFFICILE ASSOCIATED DISEASES
EP2102244A2 (en) 2006-12-19 2009-09-23 Ablynx N.V. Amino acid sequences directed against a metalloproteinase from the adam family and polypeptides comprising the same for the treatment of adam-related diseases and disorders
WO2008075356A2 (en) 2006-12-19 2008-06-26 Shay Bushinsky Interactive broadcast system and method
TWI317699B (en) 2006-12-25 2009-12-01 Ind Tech Res Inst Device and method for removing residues
US7459949B2 (en) 2007-01-30 2008-12-02 Mosaid Technologies Incorporated Phase detector circuit and method therefor
US20160031971A9 (en) 2007-02-20 2016-02-04 Tufts University Methods and compositions with a recombinant neutralizing binding protein for treating toxin exposure
US8246950B2 (en) 2007-02-20 2012-08-21 Aptalis Pharma Limited Stable digestive enzyme compositions
EP2121757A2 (en) 2007-02-21 2009-11-25 Ablynx N.V. Amino acid sequences directed against vascular endothelial growth factor and polypeptides comprising the same for the treatment of conditions and diseases characterized by excessive and/or pathological angiogenesis or neovascularization
ES2393814T3 (es) 2007-04-04 2012-12-28 Sigmoid Pharma Limited Una composición farmacéutica oral
US7807168B2 (en) 2007-04-10 2010-10-05 Vaccinex, Inc. Selection of human TNFα specific antibodies
US20080260820A1 (en) 2007-04-19 2008-10-23 Gilles Borrelly Oral dosage formulations of protease-resistant polypeptides
TW201531484A (zh) 2007-05-21 2015-08-16 Alder Biopharmaceuticals Inc 抗ＴＮＦ-α之抗體及其用途
GB0724331D0 (en) 2007-12-13 2008-01-23 Domantis Ltd Compositions for pulmonary delivery
TWM333751U (en) 2007-09-07 2008-06-01 Jarllytec Co Ltd Supporting-type pivot structure
US20100239682A1 (en) 2007-09-17 2010-09-23 Antoine Andremont Colonic delivery of antimicrobial agents
EP2725035A1 (en) 2007-10-02 2014-04-30 Avaxia Biologics, Inc. Antibody therapy for use in the digestive tract
US20090149433A1 (en) 2007-11-13 2009-06-11 Meritage Pharma, Inc. Compositions for the treatment of inflammation of the gastrointestinal tract
EP2650311A3 (en) 2007-11-27 2014-06-04 Ablynx N.V. Amino acid sequences directed against heterodimeric cytokines and/or their receptors and polypeptides comprising the same
EA019181B1 (ru) 2008-02-07 2014-01-30 Тотал Петрокемикалс Рисерч Фелюй Дегидратация спиртов в присутствии инертного компонента
KR102057826B1 (ko) 2008-04-11 2019-12-20 추가이 세이야쿠 가부시키가이샤 복수 분자의 항원에 반복 결합하는 항원 결합 분자
LT2285408T (lt) 2008-06-05 2019-01-25 Ablynx N.V. Aminorūgščių sekos, nukreiptos prieš viruso apvalkalo baltymus, ir tokias sekas turintys polipeptidai, skirti virusinių ligų gydymui
US20110112229A1 (en) 2008-08-14 2011-05-12 Nippon Paper Chemicals Co., Ltd. Dispersion resin composition, and paint composition, ink composition, adhesive composition and primer composition, containing the dispersion resin composition
GB0815264D0 (en) * 2008-08-21 2008-09-24 Syntaxin Ltd Non-cytotoxic proteins
WO2010045506A2 (en) * 2008-10-16 2010-04-22 Kathleen Cogan Farinas Sustained drug delivery system
WO2010043424A1 (en) 2008-10-17 2010-04-22 Friedrich Streffer A method for digesting a biomass comprising lignin together with cellulose and/or hemicellulose
BRPI0919879A2 (pt) 2008-10-29 2016-02-16 Wyeth Llc métodos para purificação de moléculas de ligação a antígeno de domínio único
CA2738243C (en) 2008-10-29 2020-09-29 Wyeth Llc Formulations of single domain antigen binding molecules
US8697654B2 (en) * 2008-12-18 2014-04-15 E I Du Pont De Nemours And Company Peptide linkers for effective multivalent peptide binding
WO2010085643A1 (en) 2009-01-22 2010-07-29 University Of Miami Targeting il-7 signaling as a therapy for multiple sclerosis and other il-7 signaling dependent disorders
NZ595461A (en) 2009-04-10 2013-01-25 Ablynx Nv Improved amino acid sequences directed against il-6r and polypeptides comprising the same for the treatment of il-6r related diseases and disorders
US9056050B2 (en) 2009-04-13 2015-06-16 Curemark Llc Enzyme delivery systems and methods of preparation and use
CN101964403A (zh) 2009-07-23 2011-02-02 比克国际(天津)有限公司 一种电池组的封装方法及电池组
CN102090373A (zh) 2009-12-11 2011-06-15 中国医学科学院血液病医院（血液学研究所） 模拟人克隆氏病的小鼠结肠炎模型及其制备方法和用途
EP3603624A1 (en) 2009-12-24 2020-02-05 Rani Therapeutics, LLC Therapeutic agent preparations for delivery into a lumen of the intestinal tract using a swallowable drug delivery device
CL2010000019A1 (es) 2010-01-11 2010-06-11 Univ Chile Anticuerpo monoclonal contra el tnf humano y sus fragmentos, secuencias nucleotidicas que lo codifican, vector de expresion y celulas que las contienen, composiciones y kit que comprenden el anticuerpo, uso de los mismos y metodo para su obtencion.
PE20170687A1 (es) 2010-01-28 2017-06-13 Glaxo Group Ltd Proteinas de enlace a cd127
EP2533814A2 (en) 2010-02-11 2012-12-19 Ablynx N.V. Delivery of immunoglobulin variable domains and constructs thereof
AR080291A1 (es) 2010-02-24 2012-03-28 Rinat Neuroscience Corp Anticuerpos antagonistas anti receptor de il-7 y procedimientos
WO2011139629A2 (en) * 2010-04-26 2011-11-10 Biogen Idec Ma Inc. Light targeting molecules and uses thereof
WO2011135026A1 (en) 2010-04-30 2011-11-03 Ablynx Nv Amino acid sequences of nanobodies directed against p19 subunit of the heterodimeric cytokine il-23
WO2011135040A1 (en) * 2010-04-30 2011-11-03 F. Hoffmann-La Roche Ag Fluorescent antibody fusion protein, its production and use
EP2567433B1 (en) 2010-05-04 2016-06-29 Johnson Controls Technology Company Variable speed drive
CN103119062A (zh) 2010-07-16 2013-05-22 埃博灵克斯股份有限公司 修饰的单结构域抗原结合分子及其应用
EP2632949B1 (en) * 2010-10-25 2017-09-13 National Research Council of Canada Clostridium difficile-specific antibodies and uses thereof
US9460623B2 (en) 2010-11-22 2016-10-04 International Business Machines Corporation Parking management
AR084210A1 (es) 2010-12-08 2013-05-02 Abbott Lab PROTEINAS DE UNION AL TNF-a
US8566574B2 (en) 2010-12-09 2013-10-22 International Business Machines Corporation Secure encrypted boot with simplified firmware update
US8634563B2 (en) 2010-12-17 2014-01-21 Microsoft Corporation Attribute based encryption using lattices
AU2012234282B2 (en) 2011-03-28 2015-07-16 Ablynx Nv Method for producing solid formulations comprising immunoglobulin single variable domains
MX361242B (es) 2011-03-30 2018-11-30 Ablynx Nv Anticuerpos de dominio sencillo contra tnf-alfa y usos de los mismos.
MX2018012383A (es) 2011-03-30 2023-03-07 Chugai Pharmaceutical Co Ltd Método para alterar la retención en plasma e inmunogenicidad de la molécula de union al antígeno.
US9527925B2 (en) 2011-04-01 2016-12-27 Boehringer Ingelheim International Gmbh Bispecific binding molecules binding to VEGF and ANG2
SG10201805064SA (en) 2011-06-23 2018-07-30 Ablynx Nv Techniques for predicting, detecting and reducing aspecific protein interference in assays involving immunoglobulin single variable domains
PE20141522A1 (es) 2011-08-17 2014-11-17 Glaxo Group Ltd Proteinas y peptidos modificados
EP2583980A1 (en) 2011-10-19 2013-04-24 Effimune Antibodies directed against the alpha chain of IL7 receptor - their use for the preparation of drug candidates
KR20140084201A (ko) * 2011-10-19 2014-07-04 알렉시온 파마 홀딩 알칼리성 포스파타제 및/또는 나트륨이뇨 펩티드를 포함하는 조성물 및 그의 사용 방법
GB2504139B (en) 2012-07-20 2014-12-31 Argen X Bv Antibodies to highly conserved targets produced by the immunisation of Camelidae species
DE102011121238A1 (de) 2011-12-14 2013-06-20 Universitätsklinikum Hamburg-Eppendorf Einzeldomänen-antikörper gegen clostridium difficile toxine
DE102011121237A1 (de) 2011-12-14 2013-06-20 Universitätsklinikum Hamburg-Eppendorf Einzeldomänen-Antikörper gegen Clostridium difficile Toxin CDTa
US9879093B2 (en) 2011-12-20 2018-01-30 Adaerata, Limited Partnershp Single domain antibodies as inhibitors of PCSK9
US8874636B2 (en) 2012-01-03 2014-10-28 Teletech Holdings, Inc. Method for providing support services using consumer selected specialist and specialist ratings
SI2831111T1 (sl) * 2012-03-30 2019-06-28 Boehringer Ingelheim International Gmbh Ang2-vezavne molekule
EP2849723B1 (en) 2012-05-18 2018-05-02 Genentech, Inc. High-concentration monoclonal antibody formulations
CA2875783C (en) 2012-06-06 2018-12-11 Zoetis Llc Caninized anti-ngf antibodies and methods thereof
WO2014004746A2 (en) 2012-06-26 2014-01-03 Harvard Bioscience, Inc. Methods and compositions for promoting the structural integrity of scaffolds for tissue engineering
JP2014026709A (ja) 2012-07-30 2014-02-06 International Business Maschines Corporation ライブラリ・ストリング間でのカートリッジの移動方法、システム、及びプログラム
US9540449B2 (en) 2012-08-13 2017-01-10 Regeneron Pharmaceuticals, Inc. Anti-PCSK9 antibodies with pH-dependent binding characteristics
BR112015003851A2 (pt) 2012-08-21 2017-08-08 Glaxo Group Ltd composições compreendendo um domínio variável único e mesilato de camostato (cm)
CA2887682A1 (en) 2012-10-09 2014-04-17 Biogen Idec Ma Inc. Combination therapies and uses for treatment of demyelinating disorders
KR101911438B1 (ko) 2012-10-31 2018-10-24 삼성전자주식회사 이중 특이 항원 결합 단백질 복합체 및 이중 특이 항체의 제조 방법
CN113005091A (zh) 2013-02-06 2021-06-22 细胞基因公司 具有改进特异性的修饰的t淋巴细胞
AU2014229282B2 (en) 2013-03-15 2017-02-02 Glaxosmithkline Intellectual Property (No.2) Limited Low concentration antibody formulations
WO2015009996A1 (en) 2013-07-19 2015-01-22 The Trustees Of The University Of Pennsylvania Compositions and methods for treating eosinophilic esophagitis
US20150058173A1 (en) 2013-08-20 2015-02-26 Sears Brands, Llc Sales promotion using product comparison
US20160228371A1 (en) 2013-10-18 2016-08-11 Abbvie Inc. Stable solid units and methods of making the same
US9416197B2 (en) 2013-11-01 2016-08-16 Ibc Pharmaceuticals, Inc. Bispecific antibodies that neutralize both TNF-α and IL-6: novel therapeutic agent for autoimmune disease
WO2015100409A2 (en) * 2013-12-26 2015-07-02 Tufts University Methods, compositions and kits for treating a subject using a recombinant neutralizing binding protein
JP6832707B2 (ja) 2014-03-26 2021-02-24 セル メディカ スイッツァランド アーゲー ＴＮＦαに対する結合メンバー
US10220076B2 (en) 2014-05-15 2019-03-05 Incube Labs, Llc Pharmaceutical compositions and methods for fabrication of solid masses comprising glucose regulating proteins
EP2955196A1 (en) 2014-06-10 2015-12-16 Effimune Antibodies directed against CD127
EA202193002A2 (ru) 2014-09-03 2022-03-31 Бёрингер Ингельхайм Интернациональ Гмбх Соединение, нацеленное на ил-23a и фно-альфа, и его применение
US20160115247A1 (en) 2014-10-23 2016-04-28 Singh Biotechnology, Llc Single domain antibodies directed against intracellular antigens
EP3237000A1 (en) 2014-12-23 2017-11-01 Pfizer Inc Stable aqueous antibody formulation for anti tnf alpha antibodies
US20170260266A1 (en) 2015-03-11 2017-09-14 Glaxosmithkline Intellectual Property Development Limited Tslp binding proteins
EP3277706A1 (en) 2015-03-31 2018-02-07 VHsquared Limited Polypeptide comprising an immunoglobulin chain variable domain which binds to clostridium difficile toxin b
SG11201706840WA (en) 2015-03-31 2017-09-28 Vhsquared Ltd Peptide construct having a protease-cleavable linker
WO2016156468A1 (en) 2015-03-31 2016-10-06 Vhsquared Limited Polypeptides
CA3255406A1 (en) 2015-03-31 2025-07-03 Sorriso Pharmaceuticals, Inc. Tnf-alpha binding polypeptides
WO2016156474A1 (en) 2015-03-31 2016-10-06 Vhsquared Limited Polypeptide comprising an immunoglobulin chain variable domain which binds to clostridium difficile toxin a
EP3078675A1 (en) 2015-04-10 2016-10-12 Ares Trading S.A. Induction dosing regimen for the treatment of tnf alpha mediated disorders
GB201510758D0 (en) 2015-06-18 2015-08-05 Ucb Biopharma Sprl Novel TNFa structure for use in therapy
EP3405491A1 (en) 2016-01-21 2018-11-28 VHsquared Limited Polypeptides
SG11201807402PA (en) 2016-03-31 2018-09-27 Vhsquared Ltd Compositions
EP3519438A1 (en) * 2016-09-30 2019-08-07 VHsquared Limited Compositions
JP6986559B2 (ja) 2016-12-09 2022-01-05 オセ イムノセラピューティクス Ｃｄ１２７に対して方向付けられた抗体及びポリペプチド
WO2018225063A1 (en) 2017-06-04 2018-12-13 Rappaport Family Institute For Research In The Medical Sciences Method of predicting personalized response to cancer treatment with immune checkpoint inhibitors and kits therefor
AR112341A1 (es) 2017-08-02 2019-10-16 Lilly Co Eli ANTICUERPOS BIESPECÍFICOS ANTI-TNF- / ANTI-IL-23 DE IgG
KR20220063148A (ko) 2019-06-21 2022-05-17 소리소 파마슈티컬스 인크. 조성물
CA3144567A1 (en) 2019-06-21 2020-12-24 Scott Crowe Polypeptides
US12559552B2 (en) * 2019-06-21 2026-02-24 Sorriso Pharmaceuticals, Inc. IL-23 and TNF-alpha binding bi-specific heavy chain polypeptides

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140377287A1 (en) * 2002-03-01 2014-12-25 Immunomedics, Inc. Anti-trop-2 antibody-drug conjugates and uses thereof
US8399188B2 (en) * 2006-09-28 2013-03-19 Illumina, Inc. Compositions and methods for nucleotide sequencing
US20110229476A1 (en) * 2007-08-15 2011-09-22 Bayer Schering Pharma Aktiengesellschaft Monospecific and multispecific antibodies and method of use
US20140186365A1 (en) * 2013-01-03 2014-07-03 Institute For Cancer Research D/B/A The Research Institute Of Fox Chase Cancer Center Antibodies that specifically bind to serum albumin without interfering with albumin's capability to interact with the fcrn

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10633438B2 (en) 2015-03-31 2020-04-28 Vhsquared Limited Polypeptides
US12173054B2 (en) 2015-03-31 2024-12-24 Sorriso Pharmaceuticals, Inc. Polypeptides
US12234279B2 (en) 2015-03-31 2025-02-25 Sorriso Pharmaceuticals, Inc. Peptide construct having a protease-cleavable linker
WO2020191361A2 (en) 2019-03-21 2020-09-24 Codiak Biosciences, Inc. Extracellular vesicles for vaccine delivery
US12502427B2 (en) 2019-03-21 2025-12-23 Lonza Sales Ag Extracellular vesicles for vaccine delivery
WO2020215010A1 (en) 2019-04-17 2020-10-22 Codiak Biosciences, Inc. Compositions of exosomes and aav
US11623952B2 (en) 2019-06-21 2023-04-11 Sorriso Pharmaceuticals, Inc. IL-23 and TNF-alpha binding bi-specific heavy chain polypeptides
US11667719B2 (en) 2019-06-21 2023-06-06 Sorriso Pharmaceuticals, Inc. VHH immunoglobulin chain variable domain that binds to IL-7R and methods of use thereof for treating autoimmune and/or inflammatory diseases
US12247079B2 (en) 2019-06-21 2025-03-11 Sorriso Pharmaceuticals, Inc. Polynucleotides encoding, and method of making, a polypeptide comprising a VHH which binds interleukin-receptor (IL-7R)
US12559552B2 (en) 2019-06-21 2026-02-24 Sorriso Pharmaceuticals, Inc. IL-23 and TNF-alpha binding bi-specific heavy chain polypeptides
US12570736B2 (en) 2019-06-21 2026-03-10 Sorriso Pharmaceuticals, Inc. Compositions
WO2021003445A1 (en) 2019-07-03 2021-01-07 Codiak Biosciences, Inc. Extracellular vesicles targeting t cells and uses thereof

Also Published As

Publication number Publication date
IL254589B2 (en) 2023-11-01
AU2022201410A1 (en) 2022-03-24
AU2016239949A1 (en) 2017-09-07
CA2981098A1 (en) 2016-10-06
CN107427577A (zh) 2017-12-01
WO2016156466A1 (en) 2016-10-06
AU2022201410B2 (en) 2025-04-10
SG11201706840WA (en) 2017-09-28
US20210198345A1 (en) 2021-07-01
KR102789821B1 (ko) 2025-04-02
JP2022081634A (ja) 2022-05-31
IL254589A0 (en) 2017-11-30
US20250388655A1 (en) 2025-12-25
US12234279B2 (en) 2025-02-25
HK1250011A1 (zh) 2018-11-23
JP2018515596A (ja) 2018-06-14
IL254589B1 (en) 2023-07-01
EP3277317A1 (en) 2018-02-07
KR20170132154A (ko) 2017-12-01
JP7064670B2 (ja) 2022-05-11

Similar Documents

Publication Publication Date Title
US12234279B2 (en) Peptide construct having a protease-cleavable linker
US20250277043A1 (en) Polynucleotides encoding, and method of making, a polypeptide comprising a vhh which binds interleukin- receptor (il-7r)
US11623952B2 (en) IL-23 and TNF-alpha binding bi-specific heavy chain polypeptides
US12173054B2 (en) Polypeptides
JP7702363B2 (ja) 組成物
JP2018514226A (ja) ポリペプチド
HK40070559A (en) Compositions
HK40070559B (zh) 组合物
HK40070554A (en) Polypeptides
HK40070554B (zh) 多肽

Legal Events

Date Code Title Description
AS Assignment

Owner name: VHSQUARED LIMITED, UNITED KINGDOM

Free format text: CONFIRMATORY ASSIGNMENT;ASSIGNORS:CROWE, SCOTT;WEST, MIKE;ROBERTS, KEVIN;REEL/FRAME:048961/0196

Effective date: 20160816

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION