WO2023097363A1 - Protéines de liaison améliorées et leurs utilisations - Google Patents

Protéines de liaison améliorées et leurs utilisations Download PDF

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Publication number
WO2023097363A1
WO2023097363A1 PCT/AU2022/051430 AU2022051430W WO2023097363A1 WO 2023097363 A1 WO2023097363 A1 WO 2023097363A1 AU 2022051430 W AU2022051430 W AU 2022051430W WO 2023097363 A1 WO2023097363 A1 WO 2023097363A1
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npy
pyy
seq
binding protein
set forth
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PCT/AU2022/051430
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English (en)
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Daniel Christ
Peter Schofield
David Langley
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Garvan Institute Of Medical Research
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Priority claimed from AU2021903864A external-priority patent/AU2021903864A0/en
Application filed by Garvan Institute Of Medical Research filed Critical Garvan Institute Of Medical Research
Publication of WO2023097363A1 publication Critical patent/WO2023097363A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/02—Drugs for disorders of the endocrine system of the hypothalamic hormones, e.g. TRH, GnRH, CRH, GRH, somatostatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/06—Anabolic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • 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
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present disclosure relates to reagents and methods for treating cancer and weight loss.
  • the present disclosure relates to binding proteins, such as antibodies, which bind to neuropeptide Y (NPY) and peptide YY (PYY) and thereby inhibit NPY and PYY signalling, as well as the use of those binding proteins for treatment of cancer and weight loss.
  • binding proteins such as antibodies, which bind to neuropeptide Y (NPY) and peptide YY (PYY) and thereby inhibit NPY and PYY signalling, as well as the use of those binding proteins for treatment of cancer and weight loss.
  • cancer According to the World Health Organization (WHO), cancer is a leading cause of human death. Worldwide, cancer accounted for at least 7.6 million deaths in 2008, which was about 13% of all deaths that year. The most common forms of cancer are lung cancer (1.3 million deaths in 2008), stomach cancer (803,000 deaths in 2008), colorectal cancer (639,000 deaths in 2008), liver cancer (610,000 deaths in 2008), and breast cancer (519,000 deaths in 2008) Deaths from cancer continue to rise each year and WHO predicts in excess of 11 million cancer caused deaths by 2030. The National Cancer Institute estimates that the cost of cancer care in USA in 2010 alone was US$124.5 billion dollars.
  • cancer is caused by uncontrolled proliferation of cells in tissue of a subject. These cells can invade nearby tissues and, ultimately, can spread to more distant part of the body.
  • Exemplary cancer therapies will have reduced toxicity, e.g., compared to some forms of chemotherapy and/or radiation therapy.
  • the present disclosure an isolated neuropeptide Y (NPY) and peptide YY (PYY)- binding protein comprising (i) a heavy chain variable region (VH) comprising the complementarity determining regions (CDRs) of a VH comprising a sequence set forth in SEQ ID NO: 36; and (ii) a light chain variable region (VL) comprising the CDRs of a VL comprising a sequence set forth in SEQ ID NO: 37, wherein the VH and VL bind to form a Fv that specifically binds to NPY and PYY.
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • VL light chain variable region
  • the VH of the NPY and PYY-binding protein comprises a CDR1 set forth in SEQ ID NO: 42, a CDR2 set forth in SEQ ID NO: 43 and a CDR3 set forth in SEQ ID NO: 44
  • the VL of the NPY and PYY-binding protein comprises a CDR1 set forth in SEQ ID NO: 45, a CDR2 set forth in SEQ ID NO: 46 and a CDR3 set forth in SEQ ID NO: 47.
  • the VH of the NPY and PYY-binding protein comprises a sequence which is at least 90% identical to the sequence set forth in SEQ ID NO: 36, provided that the VH comprises a CDR1 set forth in SEQ ID NO: 42, a CDR2 set forth in SEQ ID NO: 43 and a CDR3 set forth in SEQ ID NO: 44, and the VL of the NPY and PYY-binding protein comprises a sequence which is at least 90% identical to the sequence set forth in SEQ ID NO: 37, provided that the VL comprises a CDR1 set forth in SEQ ID NO: 45, a CDR2 set forth in SEQ ID NO: 46 and a CDR3 set forth in SEQ ID NO: 47.
  • the VH of the NPY and PYY-binding protein comprises a sequence which is at least 95% identical to the sequence set forth in SEQ ID NO: 36, provided that the VH comprises a CDR1 set forth in SEQ ID NO: 42, a CDR2 set forth in SEQ ID NO: 43 and a CDR3 set forth in SEQ ID NO: 44, and the VL of the NPY and PYY-binding protein comprises a sequence which is at least 95% identical to the sequence set forth in SEQ ID NO: 37, provided that the VL comprises a CDR1 set forth in SEQ ID NO: 45, a CDR2 set forth in SEQ ID NO: 46 and a CDR3 set forth in SEQ ID NO: 47.
  • the VH of the NPY and PYY-binding protein comprises a sequence which is at least 96% (or at least 97% or at least 98% or at least 99%) identical to the sequence set forth in SEQ ID NO: 36, provided that the VH comprises a CDR1 set forth in SEQ ID NO: 42, a CDR2 set forth in SEQ ID NO: 43 and a CDR3 set forth in SEQ ID NO: 44, and the VL of the NPY and PYY-binding protein comprises a sequence which is at least 96% (or at least 97% or at least 98% or at least 99%) identical to the sequence set forth in SEQ ID NO: 37, provided that the VL comprises a CDR1 set forth in SEQ ID NO: 45, a CDR2 set forth in SEQ ID NO: 46 and a CDR3 set forth in SEQ ID NO: 47.
  • the NPY and PYY-binding protein comprises a VH comprising the sequence set forth in SEQ ID NO: 36 and/or a VL comprising the sequence set forth in SEQ ID NO: 37.
  • the NPY and PYY-binding protein may comprise a VH comprising the sequence set forth in SEQ ID NO: 36 and a VL comprising the sequence set forth in SEQ ID NO: 37.
  • the present disclosure also provides an isolated or recombinant neuropeptide Y (NPY) and peptide YY (PYY)-binding protein comprising a heavy chain variable region (VH) comprising a sequence set forth in SEQ ID NO: 36 and a light chain variable region (VL) comprising a sequence set forth in SEQ ID NO: 37, or a chimeric, deimmunized, CDR grafted, humanized or synhumanized form thereof, wherein the VH and VL bind to form a Fv that specifically binds to NPY and PYY.
  • VH heavy chain variable region
  • VL light chain variable region
  • the NPY and PYY-binding protein may neutralize NPY and PYY-mediated signalling in a cell.
  • the NPY and PYY-mediated signalling in a cell is ERK phosphorylation.
  • the NPY and PYY-binding protein binds NPY and/or PYY with a KD of 1X10' 8 M or less.
  • the NPY and PYY-binding protein may bind NPY and/or PYY with a KD of 5xl0' 9 M or less.
  • the NPY and PYY-binding protein binds NPY and/or PYY with a KD of 2xlO' 9 M or less.
  • the KD is determined using a biosensor, e.g., Biacore or Octet.
  • the NPY and PYY-binding protein binds to PYY with greater affinity than it binds to NPY.
  • the NPY and PYY-binding protein binds to PYY with a KD of 9x10" 10 M or less.
  • the NPY and PYY-binding protein does not significantly or detectably bind to pancreatic polypeptide (PP).
  • PP pancreatic polypeptide
  • the binding to PP is not detectable when assessed using an enzyme linked immunosorbent assay (ELISA).
  • ELISA enzyme linked immunosorbent assay
  • the NPY and PYY-binding protein comprises a variable domain which competitively inhibits binding of an antibody comprising a VH comprising a sequence set forth in SEQ ID NO: 36 and a VL comprising a sequence set forth in SEQ ID NO: 37.
  • the VH and the VL of the NPY and PYY-binding protein of the disclosure are in a single polypeptide chain.
  • the NPY and PYY-binding protein may be provided as:
  • a dimeric scFv (di-scFv); or (iii) at least one of (i) and/or (ii) linked to a Fc or a heavy chain constant domain (CH) 2 and/or CH3.
  • the binding protein may comprise a sequence set forth in SEQ ID NO: 31.
  • the VH and the VL of the NPY and PYY-binding protein of the disclosure are in separate polypeptide chains.
  • the NPY and PYY-binding protein may be provided as:
  • the binding protein may comprise a Fab heavy chain sequence set forth in SEQ ID NO: 48 and a Fab kappa light chain sequence set forth in SEQ ID NO: 49.
  • the NPY and PYY-binding protein is an antibody.
  • the present disclosure also provides an isolated or recombinant nucleic acid encoding the NPY and PYY-binding protein of the present disclosure.
  • the disclosure is not limited to the specific exemplified nucleic acids described herein, but also encompasses any nucleic acid that encodes a NPY and PYY-binding protein of the disclosure as a result of degeneracy of the genetic code.
  • the nucleic acid may be codon optimized for expression in a particular cell type.
  • such a nucleic acid is included in an expression construct in which the nucleic acid is operably- linked to a promoter.
  • an expression construct can be in a vector, e.g., a plasmid.
  • the expression construct may comprise a promoter linked to a nucleic acid encoding that polypeptide chain.
  • an expression construct of the disclosure comprises a nucleic acid encoding one of the polypeptides (e.g., comprising a VH) operably linked to a promoter and a nucleic acid encoding another of the polypeptides (e.g., comprising a VL) operably linked to a promoter.
  • the expression construct is a bicistronic expression construct, e.g., comprising the following operably linked components in 5’ to 3’ order:
  • the first polypeptide comprises a VH and the second polypeptide comprises a VL, or the first polypeptide comprises a VL and the second polypeptide comprises a VH.
  • the present disclosure also contemplates separate expression constructs one of which encodes a first polypeptide (e.g., comprising a VH) and another of which encodes a second polypeptide (e.g., comprising a VL).
  • a composition comprising:
  • a first expression construct comprising a nucleic acid encoding a polypeptide (e.g., comprising a VH operably linked to a promoter);
  • a second expression construct comprising a nucleic acid encoding a polypeptide (e.g., comprising a VL operably linked to a promoter), wherein the first and second polypeptides associate to form a NPY and PYY-binding protein of the present disclosure.
  • a polypeptide e.g., comprising a VL operably linked to a promoter
  • the present disclosure also provides an isolated cell expressing a NPY and PYY- binding protein of the disclosure or a recombinant cell genetically-modified to express a NPY and PYY-binding protein of the disclosure.
  • the cell comprises the expression construct of the disclosure or:
  • a first expression construct comprising a nucleic acid encoding a polypeptide (e.g., comprising a VH) operably linked to a promoter;
  • a second expression construct comprising a nucleic acid encoding a polypeptide (e.g., comprising a VL) operably linked to a promoter, wherein the first and second polypeptides associate to form a NPY and PYY-binding protein of the present disclosure.
  • a polypeptide e.g., comprising a VL
  • Examples of cells of the present disclosure include bacterial cells, yeast cells, insect cells or mammalian cells. Exemplary cells are mammalian.
  • the present disclosure additionally provides methods for producing a NPY and PYY- binding protein of the disclosure.
  • a method involves maintaining the expression construct(s) of the disclosure under conditions sufficient for the NPY and PYY- binding protein to be produced.
  • a method for producing a NPY and PYY-binding protein of the disclosure comprises culturing the cell of the disclosure under conditions sufficient for the protein to be produced and, optionally, secreted.
  • the method for producing a NPY and PYY-binding protein of the disclosure additionally comprises isolating the protein.
  • the present disclosure also provides a composition comprising the NPY and PYY- binding protein, nucleic acid, expression construct or cell of the present disclosure and a suitable carrier.
  • the composition comprises the NPY and PYY-binding protein of the present disclosure and a suitable carrier.
  • the carrier is pharmaceutically acceptable, e.g., the composition is a pharmaceutical composition.
  • the present disclosure additionally provides a method for treating or preventing a NPY and/or PYY-mediated condition, the method comprising administering to a subject the NPY and PYY-binding protein of the disclosure or the composition of the disclosure.
  • the present disclosure additionally provides a NPY and PYY-binding protein of the disclosure or the composition of the disclosure for use in treating or preventing a NPY and/or PYY-mediated condition.
  • the NPY and/or PYY-mediated condition is weight loss, e.g., anorexia or a wasting condition, such as, cachexia, pre-cachexia or sarcopenia (e.g., wasting associated with aging).
  • weight loss e.g., anorexia or a wasting condition, such as, cachexia, pre-cachexia or sarcopenia (e.g., wasting associated with aging).
  • the present disclosure additionally provides a method of treating or preventing cancer in a subject, the method comprising administering to a subject the NPY and PYY-binding protein of the disclosure or the composition of the disclosure.
  • preventing cancer includes preventing metastasis or recurrence of cancer in a subject.
  • the cancer may be selected from the group consisting of an adenocarcinoma, a squamous cell carcinoma, a digestive/gastrointestinal cancer, an eye cancer, a musculoskeletal cancer, a breast cancer, neuroblastoma, a genitourinary cancer, a germ cell cancer, a head and neck cancer, a hematologic/blood cancer, a respiratory cancer, a skin cancer, an AIDS -related malignancy or a genealogic cancer.
  • the cancer expresses a NPY receptor responsive to NPY and PYY.
  • administering the NPY and PYY-binding protein inhibits NPY and PYY signalling in cells of the subject.
  • the cancer expresses a Y1 receptor and/or a Y2 receptor and/or a Y5 receptor.
  • the method of the disclosure comprises administering an amount of the compound sufficient to induce or enhance an immune response against the cancer in the subject.
  • the immune response is a T cell response.
  • the present disclosure also contemplates administering a further compound to treat the cancer (e.g., chemotherapy) or exposing the subject to radiation therapy.
  • a further compound to treat the cancer (e.g., chemotherapy) or exposing the subject to radiation therapy.
  • the subject additionally suffers from a wasting condition, e.g., cancer cachexia, and the method additionally treats the wasting condition.
  • a wasting condition e.g., cancer cachexia
  • methods described herein for treating cancer shall be taken to apply mutatis mutandis to treatment of cancer accompanied by a wasting condition, such as cachexia.
  • the method of the present disclosure additionally comprises administering a further compound to treat the cancer or exposing the subject to radiation therapy.
  • the present disclosure also provides a method for inducing or enhancing an immune response in a subject, the method comprising administering to the subject the NPY and PYY- binding protein of the disclosure or the composition of the disclosure to thereby inhibit NPY and PYY signalling in cells of the subject.
  • the subject suffers from cancer and the immune response is against the cancer or a cell thereof.
  • FIG. 1 Graphical representations showing sequences of (A) heavy and (B) light chain variable regions of antibodies. Boxed regions contain CDRs (as indicated) as defined by the Kabat numbering system and the enhanced Chothia numbering system. CDRs defined by the Kabat numbering system are shown in bold. CDRs defined by the enhanced Chothia numbering system are underlined.
  • Figure 2 Schematic of the diversity introduced into CDR-H1, H3, L2 and L3 of h5E12 IGHV1-69/DPK9 scFv by degenerate codon oligonucleotides and kunkel mutagenesis to generate the first affinity maturation library of humanised 5E12.
  • FIG. 3 Identification of clones with improved apparent off-rate from the first h5E12 scFv affinity maturation library.
  • Clones from the libraries based on h5E12 IGV1-46/DPK9 and h5E12 IGHV1-69/DPK9 were selected through multiple rounds of phage display against biotinylated hPYY and streptavidin dynabeads. Soluble scFv supernatants from selected clones were screened for binding off-rate against biotinylated hPYY on streptavidin biosensors by Bio layer Interferometry (BLItz, Fortebio). Clones TA-F6, TA-A7, TA-D5 and SA-1B3 all demonstrated improvements in off-rate (lower kd).
  • FIG. 1 Amino acid sequence alignment of different h5E12 scFv affinity maturation variants. CDRs of the VH and VL domains are highlighted.
  • Figure 5 Amino acid sequence alignment of PSE2 VH and VL domains with a) humanised 5E12 VH domains and b) humanised 5E12 VL domains used to generate humanised scFv library by SOE PCR.
  • FIG. 6 Identification of clones with improved apparent off-rate from the humanised 5E12 scFv SOE PCR library containing 6 different humanised 5E12 VH variants and 4 different humanised 5E12 VL variants. Soluble scFv supernatants generated from picked colonies were screened for binding off-rate against biotinylated hPYY on streptavidin biosensors by Biolayer Interferometry (BLItz, Fortebio). Clones 1A5 and 1D8 all demonstrated improvements in off- rate (lower ka).
  • Figure 7 Amino acid sequence alignment of scFv variants designed to combine different combinations of optimal framework and improved off-rate CDR mutations observed in affinity maturation library.
  • Figure 8 Blitz analysis of h5E12 scFv variants with combined mutations.
  • Clone 4A which combined CDR-H1 N31D mutation and CDR-L2 mutations from clone TA-A7 with the CDR-L3 mutation of clone TA-D5 and the optimal VL framework of clone 1A5 had the best observed improvements in affinity.
  • Figure 9 Schematic of the diversity introduced into template variant 4A by kunkel mutagenesis for the second affinity maturation library.
  • FIG. 14 Structural soluble hPYY (SEQUENCE 6) in complex with a Fab comprising of h5E12 Fab DP47 heavy chain (SEQUENCE 38) and 4A3B2 kappa light chain (SEQUENCE 37), as solved by X-ray crystallography. Residues highlighted that differ between h5E12 IGHV1-69/DPK9 and fully matured clone 4A3B2 (Blue are framework residues, Red are CDR- H1 residues, Green are CDH-L2 residues and Grey are CDR-L3 residues).
  • FIG. 15 Structural soluble NPY (SEQUENCE 5) in complex with a Fab comprising of h5E12 IGHV1-69 Fab heavy chain (SEQUENCE 34) and 4A3B2 kappa light chain (SEQUENCE 37), as solved by X-ray crystallography. Residues highlighted that differ between h5E12 IGHV1-69/DPK9 and fully matured clone 4A3B2 (Blue are framework residues, Red are CDR-H1 residues, Green are CDH-L2 residues and Grey are CDR-L3 residues). KEY TO THE SEQUENCE LISTING
  • SEQ ID NO:1 Amino acid sequence for human NPY.
  • SEQ ID NO:2 Amino acid sequence for human PYY.
  • SEQ ID NO:3 Amino acid sequence for immunising peptide designated NPY20-36NH2.
  • SEQ ID NO:8 Amino acid sequence for TA-A7 scFv.
  • SEQ ID NO: 10 Amino acid sequence for SA3-1B5 scFv.
  • SEQ ID NO: 11 Amino acid sequence for heavy chain variable region of h5E12 DP47.
  • SEQ ID NO: 12 Amino acid sequence for heavy chain variable region of h5E12 IGHV1-46.
  • SEQ ID NO: 13 Amino acid sequence for heavy chain variable region of h5E12 IGHV1-69.
  • SEQ ID NO: 14 Amino acid sequence for heavy chain variable region of h5E12 HV108.
  • SEQ ID NO: 15 Amino acid sequence for heavy chain variable region of h5E12 HV108a.
  • SEQ ID NO: 16 Amino acid sequence for heavy chain variable region of h5E12 HV302.
  • SEQ ID NO: 17 Amino acid sequence for light chain variable region of h5E12 DPK9.
  • SEQ ID NO: 18 Amino acid sequence for light chain variable region of h5E12 KV103.
  • SEQ ID NO:20 Amino acid sequence for light chain variable region of h5E12 KV119.
  • SEQ ID NO:32 Amino acid sequence for heavy chain of PSE2 Fab.
  • SEQ ID NO:33 Amino acid sequence for kappa light chain of PSE2 Fab.
  • SEQ ID NO:34 Amino acid sequence for heavy chain of h5E12 IGHV1 Fab.
  • SEQ ID NO:35 Amino acid sequence for kappa light chain of h5E12 DPK9 Fab.
  • SEQ ID NO:36 Amino acid sequence for heavy chain variable region of 4A3B2.
  • SEQ ID NO:37 Amino acid sequence for light chain variable region of 4A3B2.
  • SEQ ID NO:38 Amino acid sequence for heavy chain of h5E12 DP47 Fab.
  • SEQ ID NO:39 Amino acid sequence of CDR2 of light chain variable region for TA-F6.
  • SEQ ID NO:40 Amino acid sequence of CDR3 of light chain variable region for TA-D5.
  • SEQ ID NO:41 Amino acid sequence of CDR3 of light chain variable region for SA3-1B5.
  • SEQ ID NO:42 Amino acid sequence of CDR1 of heavy chain variable region for 4A3B2.
  • SEQ ID NO:44 Amino acid sequence of CDR3 of heavy chain variable region for 4A3B2.
  • SEQ ID NO:45 Amino acid sequence of CDR1 of light chain variable region for 4A3B2.
  • SEQ ID NO:46 Amino acid sequence of CDR2 of light chain variable region for 4A3B2.
  • SEQ ID NO:47 Amino acid sequence of CDR3 of light chain variable region for 4A3B2.
  • SEQ ID NO:48 Amino acid sequence for heavy chain of 4A3B2 Fab.
  • SEQ ID NO:49 Amino acid sequence for kappa light chain of 4A3B2 Fab.
  • SEQ ID NO:50 Amino acid sequence for heavy chain variable region of 4A3B2vl.
  • SEQ ID NO:51 Amino acid sequence of CDR1 of heavy chain variable region for 4A3B2vl.
  • SEQ ID NO:53 Amino acid sequence for heavy chain of 4A3B2vl Fab.
  • composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter.
  • amino acid sequence of human NPY is set forth in SEQ ID NO: 1. Additional sequences of human NPY are set out in Genbank Gene Accession No. 4852. In one example, the amino acid sequence of human NPY comprises a sequence set forth in SEQ ID NO: 1.
  • amino acid sequence of human PYY is set forth in SEQ ID NO: 2. Additional sequences of human PYY are set out in Genbank Gene Accession No. 5697. In one example, the amino acid sequence of human PYY comprises a sequence set forth in SEQ ID NO: 2.
  • isolated protein or “isolated polypeptide” is intended to mean a protein or polypeptide that by virtue of its origin or source of derivation is not associated with naturally- associated components that accompany it in its native state; is substantially free of other proteins from the same source.
  • a protein may be rendered substantially free of naturally associated components or substantially purified by isolation, using protein purification techniques known in the art.
  • substantially purified is meant the protein is substantially free of contaminating agents, e.g., at least about 70% or 75% or 80% or 85% or 90% or 95% or 96% or 97% or 98% or 99% free of contaminating agents.
  • recombinant shall be understood to mean the product of artificial genetic recombination. Accordingly, in the context of a recombinant protein comprising an antibody antigen binding domain, this term does not encompass an antibody naturally-occurring within a subject’s body that is the product of natural recombination that occurs during B cell maturation. However, if such an antibody is isolated, it is to be considered an isolated protein comprising an antibody antigen binding domain. Similarly, if nucleic acid encoding the protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein comprising an antibody antigen binding domain. A recombinant protein also encompasses a protein expressed by artificial recombinant means when it is within a cell, tissue or subject, e.g., in which it is expressed.
  • the term “binds” in reference to the interaction of a NPY and PYY- binding protein with an antigen means that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen.
  • a particular structure e.g., an antigenic determinant or epitope
  • an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody binds to epitope “A”, the presence of a molecule containing epitope “A” (or free, unlabelled “A”), in a reaction containing labelled “A” and the antibody, will reduce the amount of labelled “A” bound to the antibody.
  • the term “specifically binds” or “binds specifically” shall be taken to mean that a protein reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a recited protein or proteins (e.g., NPY and PYY) than it does with another protein (e.g., with PP).
  • a protein that specifically binds to PYY and NPY binds with greater affinity, avidity, more readily, and/or with greater duration than it binds PP.
  • the degree of greater affinity, avidity, readiness, and/or with duration will depend on the application of the protein.
  • the degree of specificity should be sufficiently high to permit quantification (where required).
  • the degree of specificity should be sufficient to provide a therapeutic/prophylactic effect without serious adverse effects resulting from cross-reactivity of the protein and/or without sufficient cross-reactivity to significantly reduce PP biological activity in a subject.
  • a protein that specifically binds to NPY and PYY does not significantly or detectably bind to PP. It is also to be understood by reading this definition that “specific binding” does not necessarily require exclusive binding, this is encompassed by the term “selective binding”. Generally, but not necessarily, reference to binding means specific binding.
  • “specific binding” of a NPY and PYY- binding protein of the disclosure to an antigen means that the protein binds to the antigen with a KD of 1X10' 8 M or less, such as 5xl0' 9 M or less, for example 1X10' 9 M or less, such as 9x10" 10 M or less.
  • the term “does not significantly bind” shall be understood to mean that the level of binding to an antigen or epitope of a protein of the present disclosure is not statistically significantly higher than background, e.g., the level of binding signal detected in the absence of the protein and/or in the presence of a negative control protein (e.g., an isotype control antibody) and/or the level of binding detected in the presence of a negative control antigen or epitope.
  • the level of binding is assessed by ELISA, e.g., indirect ELISA.
  • the term “does not detectably bind” shall be understood to mean that a protein, e.g., an antibody, binds to an antigen or epitope at a level of 30% or 25% or 20% or 15% or 10% or less than the signal obtained with human or mouse NPY or PYY.
  • a protein e.g., an antibody
  • the level of binding can be detected using ELISA, e.g., indirect ELISA or Biacore analysis in which the protein is immobilized and contacted with an antigen or epitope.
  • binds to an epitope means that an antibody binds to amino acids within the sequence of the recited epitope. This term does not mean that the antibody binds to each and every amino acid recited, only that one or more of the recited amino acids are necessary for antibody binding.
  • epitope (syn. “antigenic determinant”) shall be understood to mean a region of NPY and PYY to which a protein comprising an antibody variable region binds. This term is not necessarily limited to the specific residues or structure to which the protein makes contact. For example, this term includes the region spanning amino acids contacted by the protein and/or at least 5-10 or 2-5 or 1-3 amino acids outside of this region. In some examples, the epitope is a linear series amino acids. However, the epitope is not restricted to only amino acid side-chains.
  • a protein described herein binds to a peptide comprising an amidated terminus but does not significantly bind to the same sequence of amino acids with a carboxy terminus. Sequences contained within exemplary epitopes in NPY/PYY are described herein.
  • C-terminus in the context of an epitope or peptide or antigen will be understood to mean that the recited sequence is positioned such that the last amino acid in the recited sequence is the carboxy terminal amino acid in the epitope or peptide or antigen.
  • amino acid or X-amide (wherein “X” is an amino acid) shall be understood to mean carboxy group of an amino acid or on the C-terminus of a peptide is replaced with an amide group.
  • the amino acid or peptide is a-amidated.
  • NPY and PYY-binding protein shall be taken to include a single polypeptide chain (i.e., a series of contiguous amino acids linked by peptide bonds), or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex) capable of binding to NPY and PYY in the manner described and/or claimed herein.
  • the series of polypeptide chains can be covalently linked using a suitable chemical or a disulphide bond.
  • non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.
  • polypeptide or “polypeptide chain” will be understood from the foregoing paragraph to mean a series of contiguous amino acids linked by peptide bonds.
  • the term “antibody” includes a protein capable of specifically binding to one or a few closely related antigens (e.g., NPY and PYY) by virtue of an antigen binding domain contained within a Fv.
  • This term includes four chain antibodies (e.g., two light chains and two heavy chains), recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted antibodies, primatized antibodies, de-immunized antibodies, synhumanized antibodies, half antibodies, and bispecific antibodies).
  • An antibody generally comprises constant domains, which can be arranged into a constant region or constant fragment or fragment crystallizable (Fc).
  • Exemplary forms of antibodies comprise a four-chain structure as their basic unit.
  • Full-length antibodies comprise two heavy chains (-50-70 kDa) covalently linked and two light chains (-23 kDa each).
  • a light chain generally comprises a variable region (if present) and a constant domain and in mammals is either a K light chain or a X light chain.
  • a heavy chain generally comprises a variable region and one or two constant domain(s) linked by a hinge region to additional constant domain(s).
  • Heavy chains of mammals are of one of the following types a, 6, 8, y, or p.
  • Each light chain is also covalently linked to one of the heavy chains.
  • the two heavy chains and the heavy and light chains are held together by inter-chain disulfide bonds and by non-covalent interactions. The number of inter-chain disulfide bonds can vary among different types of antibodies.
  • Each chain has an N-terminal variable region (VH or VL wherein each are -110 amino acids in length) and one or more constant domains at the C- terminus.
  • the constant domain of the light chain (CL which is -110 amino acids in length) is aligned with and disulfide bonded to the first constant domain of the heavy chain (CHI which is 330-440 amino acids in length).
  • the light chain variable region is aligned with the variable region of the heavy chain.
  • the antibody heavy chain can comprise 2 or more additional CH domains (such as, CH2, CH3 and the like) and can comprise a hinge region between the CHI and CH2 constant domains.
  • Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGi, IgG2, IgGs, IgG 4 , IgAi and IgA2) or subclass.
  • the antibody is a murine (mouse or rat) antibody or a primate (such as, human) antibody.
  • the antibody is humanized, synhumanized, chimeric, CDR-grafted or deimmunized.
  • variable region refers to the portions of the light and/or heavy chains of an antibody as defined herein that is(are) capable of specifically binding to an antigen and, includes amino acid sequences of complementarity determining regions (CDRs); i.e., CDR1, CDR2, and CDR3, and framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • the variable region comprises three or four FRs (e.g., FR1, FR2, FR3 and optionally FR4) together with three CDRs.
  • VH refers to the variable region of the heavy chain.
  • VL refers to the variable region of the light chain.
  • CDRs complementarity determining regions
  • CDR1, CDR2, and CDR3 refers to the amino acid residues of an antibody variable region the presence of which are major contributors to specific antigen binding.
  • Each variable region domain typically has three CDR regions identified as CDR1, CDR2 and CDR3.
  • amino acid positions assigned to CDRs and FRs are defined according to Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as “the Kabat numbering system”.
  • amino acid positions assigned to CDRs and FRs are defined according to the Enhanced Chothia Numbering Scheme (http://www.bioinfo.org.uk/mdex.html). According to the numbering system of Kabat, VH FRS and CDRs are positioned as follows: residues 1-30 (FR1), 31-35 (CDR1), 36-49 (FR2), 50-65 (CDR2), 66-94 (FR3), 95-102 (CDR3) and 103- 113 (FR4).
  • VL FRS and CDRs are positioned as follows: residues 1-23 (FR1), 24-34 (CDR1), 35-49 (FR2), 50-56 (CDR2), 57-88 (FR3), 89-97 (CDR3) and 98-107 (FR4).
  • the present disclosure is not limited to FRs and CDRs as defined by the Kabat numbering system, but includes all numbering systems, including the canonical numbering system or of Chothia and Eesk J. MolBiol. 796:901-917, 1987; Chothia etal.
  • the CDRs are defined according to the Kabat numbering system.
  • heavy chain CDR2 according to the Kabat numbering system does not comprise the five C-terminal amino acids listed herein or any one or more of those amino acids are substituted with another naturally- occurring amino acid.
  • light chain CDR1 does not comprise the four N-terminal amino acids listed herein or any one or more of those amino acids are substituted with another naturally-occurring amino acid.
  • Padlan et al., FASEB J., 9: 133-139, 1995 established that the five C-terminal amino acids of heavy chain CDR2 and/or the four N-terminal amino acids of light chain CDR1 are not generally involved in antigen binding.
  • FRs Framework regions
  • the term “Fv” shall be taken to mean any protein, whether comprised of multiple polypeptides or a single polypeptide, in which a VL and a VH associate and form a complex capable of specifically binding to an antigen.
  • the VH and the VL which form the antigen binding domain can be in a single polypeptide chain or in different polypeptide chains.
  • an Fv of the disclosure (as well as any protein of the disclosure) may have multiple antigen binding domains which may or may not bind the same antigen. This term shall be understood to encompass fragments directly derived from an antibody as well as proteins corresponding to such a fragment produced using recombinant means.
  • the VH is not linked to a heavy chain constant domain (CH) 1 and/or the VL is not linked to a light chain constant domain (CL).
  • exemplary Fv containing polypeptides or proteins include a Fab fragment, a Fab’ fragment, a F(ab’) fragment, a scFv, a diabody, a triabody, a tetrabody or higher order complex, or any of the foregoing linked to a constant region or domain thereof, e.g., CH2 or CH3 domain, e.g., a minibody.
  • a “Fab fragment” consists of a monovalent antigenbinding fragment of an immunoglobulin, and can be produced by digestion of a whole antibody with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain or can be produced using recombinant means.
  • a “Fab' fragment” of an antibody can be obtained by treating a whole antibody with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain comprising a VH and a single constant domain. Two Fab' fragments are obtained per antibody treated in this manner.
  • a Fab’ fragment can also be produced by recombinant means.
  • a “F(ab')2 fragment” of an antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds, and is obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction.
  • a “Fab2” fragment is a recombinant fragment comprising two Fab fragments linked using, for example a leucine zipper or a CH3 domain.
  • a “single chain Fv” or “scFv” is a recombinant molecule containing the variable region fragment (Fv) of an antibody in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable, flexible polypeptide linker.
  • NPY and PYY- binding protein of the disclosure reduces or prevents binding of a recited antibody to NPY or PYY. This may be due to the protein (or variable region or Fv thereof) and antibody binding to the same or an overlapping epitope. It will be apparent from the foregoing that the protein need not completely inhibit binding of the antibody, rather it need only reduce binding by a statistically significant amount, for example, by at least about 10% or 20% or 30% or 40% or 50% or 60% or 70% or 80% or 90% or 95%. Methods for determining competitive inhibition of binding are known in the art and/or described herein. For example, the antibody is exposed to NPY or PYY either in the presence or absence of the protein.
  • the protein is considered to competitively inhibit binding of the antibody.
  • the competitive inhibition of binding is caused by the epitope bound by the protein on NPY or PYY overlapping with the antigen binding domain of the antibody.
  • “Overlapping” in the context of two epitopes shall be taken to mean that two epitopes share a sufficient number of amino acid residues to permit a protein (or antigen binding domain thereof) that binds to one epitope to competitively inhibit the binding of a protein (or antigen binding domain) that binds to the other epitope.
  • the “overlapping” epitopes share at least 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 20 amino acids.
  • neutralize shall be taken to mean that a NPY and PYY- binding protein is capable of reducing or preventing NPY and/or PYY-mediated activity in a cell. Methods for determining neutralization are known in the art and/or described herein.
  • condition refers to a disruption of or interference with normal function, and is not to be limited to any specific condition, and will include diseases or disorders.
  • NPY and/or PYY-associated condition refers to any condition that is caused by or associated with NPY and/or PYY.
  • the skilled artisan will be readily able to determine such conditions based on the disclosure herein.
  • the condition is anorexia, unintended weight loss or a wasting condition or cancer.
  • wasting disorder refers to a disorder which involves, results at least in part from, or includes loss of weight, muscle atrophy, fatigue, weakness in someone who is not actively trying to lose weight.
  • Wasting disorders are commonly characterized by inadvertent and/or uncontrolled (in the absence of medical intervention) loss of muscle and/or fat.
  • the term encompasses cachexia or other forms of wasting, e.g., denervation-induced wasting.
  • cachexia will be understood to refer to metabolic condition associated with an underlying (or another) condition, wherein cachexia is characterized by loss of body weight and loss of muscle with or without loss of fat mass. Cachexia is generally associated with increased protein catabolism due to underlying disease(s). Contributory factors to the onset of cachexia are anorexia and metabolic alterations (e.g., increased inflammatory status, increased muscle proteolysis and impaired carbohydrate, protein and lipid metabolism). A prominent clinical feature of cachexia is weight loss in adults (optionally, corrected for fluid retention) or growth failure in children (excluding endocrine disorders). Anorexia, inflammation, insulin resistance and increased muscle protein breakdown are frequently associated with cachexia.
  • Cachexia is distinct from starvation, primary depression, malabsorption and hyperthyroidism and is associated with increased morbidity. Cachexia can be associated with or result from (directly or indirectly) various underlying disorders including cancer, metabolic acidosis (from decreased protein synthesis and increased protein catabolism), certain infectious diseases (e.g. bacterial infections, including tuberculosis, AIDS), some autoimmune disorders, addiction to drugs such as amphetamines or cocaine, chronic alcoholism and/or cirrhosis of the liver, chronic inflammatory disorders, anorexia, neurological conditions and/or neurodegenerative disease.
  • cachexia is cancer cachexia (cachexia associated with cancer).
  • muscle wasting and/or unintended body weight loss associated with neurological conditions, immobility or impaired mobility due to various diseases such as neurodegenerative disease, multiple sclerosis, spinal cord injury, are included in the term.
  • Cachexia can be diagnosed based on one or more of the following:
  • pre-cachexia will be understood to mean a condition associated with an underlying condition (e.g., chronic condition) and characterized by unintentional weight loss of less than about 5% of a subject’s body weight; and a chronic or recurrent systemic inflammatory response.
  • an underlying condition e.g., chronic condition
  • unintended body weight loss refers to a condition where the subject is incapable of maintaining a healthy body weight or loses a considerable amount of body weight, without actually attempting to reduce body weight.
  • a body mass index of less than 18.5 or any another BMI range defined by a medical specialist is considered underweight.
  • the terms “preventing”, “prevent” or “prevention” include administering a compound to thereby stop or hinder the development of at least one symptom of a condition. This term also encompasses treatment of a subject in remission to prevent or hinder relapse.
  • the terms “treating”, “treat” or “treatment” include administering a compound to thereby reduce or eliminate at least one symptom of a specified condition.
  • the term “subject” shall be taken to mean any animal, such as, a mammal.
  • the mammal is a human or non-human primate.
  • the mammal is a human.
  • sample should be understood as a reference to any sample derived from a subject such as, but not limited to, a body fluid (e.g., blood or blood fraction such as serum or plasma, tears, urine, synovial fluid or cerebrospinal fluid), cellular material (e.g. tissue aspirate), tissue biopsy specimens or surgical specimens.
  • a body fluid e.g., blood or blood fraction such as serum or plasma, tears, urine, synovial fluid or cerebrospinal fluid
  • cellular material e.g. tissue aspirate
  • tissue biopsy specimens or surgical specimens e.g., tissue biopsy specimens or surgical specimens.
  • sample is any one or more of serum, plasma, peripheral blood mononuclear cells (PBMC), or a buffy coat fraction.
  • PBMC peripheral blood mononuclear cells
  • expression construct is to be taken in its broadest context and includes a nucleic acid comprising one or more promoter sequences operably linked with one or more nucleic acids as described herein.
  • an expression vector refers to a nucleic acid comprising an expression construct that is additionally capable of maintaining and or replicating nucleic acid in an expressible format.
  • an expression vector may comprise a plasmid, bacteriophage, phagemid, cosmid, virus sub-genomic or genomic fragment. Selection of appropriate vectors is within the knowledge of those having skill in the art.
  • promoter is to be taken in its broadest context and includes the transcriptional regulatory sequences of a genomic gene, including the TATA box or initiator element, which is required for accurate transcription initiation, with or without additional regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers and silencers) that alter expression of a nucleic acid, e.g., in response to a developmental and/or external stimulus, or in a tissue specific manner.
  • promoter is also used to describe a recombinant, synthetic or fusion nucleic acid, or derivative which confers, activates or enhances the expression of a nucleic acid to which it is operably linked.
  • Exemplary promoters can contain additional copies of one or more specific regulatory elements to further enhance expression and/or alter the spatial expression and/or temporal expression of said nucleic acid.
  • operably linked to means positioning a promoter relative to a nucleic acid such that expression of the nucleic acid is controlled by the promoter.
  • a promoter can be operably linked to numerous nucleic acids, e.g., through an internal ribosome entry site.
  • structurally related to glutamine is meant an amino acid that is considered similar to glutamine based on the BLOSUM62 matrix and/or PAM250 matix and includes glutamine. Proteins Comprising Antibody Variable Region(s)
  • Table 1 Sequences of exemplary NPY and PYY-binding proteins
  • a NPY and PYY-binding protein of the disclosure may comprise the CDRs of an NPY and PYY-binding protein presented in Table 1.
  • an NPY and PYY-binding protein of the disclosure comprises the CDRs of the binding protein designated 4A3B2 in Table 1.
  • the CDRs of the binding protein designated 4A3B2 are CDR-H1 (SEQ ID NO: 42), CDR-H2 (SEQ ID NO: 43), CDR-H3 (SEQ ID NO: 44), CDR-L1 (SEQ ID NO: 45), CDR-L2 (SEQ ID NO: 46) and CDR-L3 (SEQ ID NO: 47).
  • the NPY and PYY-binding protein may comprise a VH comprising an amino acid sequence which is at least 90% identical to the sequence set forth in SEQ ID NO: 36 provided that it comprises the CDRs of the VH set forth in SEQ ID NO: 36, and a VL comprising an amino acid sequence which is at least 90% identical to the sequence set forth in SEQ ID NO: 37 provided that it comprises the CDRs of the VL set forth in SEQ ID NO: 37.
  • the NPY and PYY-binding protein may comprise a VH comprising an amino acid sequence which is at least 95% identical to the sequence set forth in SEQ ID NO: 36 provided that it comprises the CDRs of the VH set forth in SEQ ID NO: 36, and a VL comprising an amino acid sequence which is at least 95% identical to the sequence set forth in SEQ ID NO: 37 provided that it comprises the CDRs of the VL set forth in SEQ ID NO: 37.
  • the NPY and PYY-binding protein may comprise a VH comprising an amino acid sequence which is at least 96% identical to the sequence set forth in SEQ ID NO: 36 provided that it comprises the CDRs of the VH set forth in SEQ ID NO: 36, and a VL comprising an amino acid sequence which is at least 96% identical to the sequence set forth in SEQ ID NO: 37 provided that it comprises the CDRs of the VL set forth in SEQ ID NO: 37.
  • the NPY and PYY-binding protein may comprise a VH comprising an amino acid sequence which is at least 97% identical to the sequence set forth in SEQ ID NO: 36 provided that it comprises the CDRs of the VH set forth in SEQ ID NO: 36, and a VL comprising an amino acid sequence which is at least 97% identical to the sequence set forth in SEQ ID NO: 37 provided that it comprises the CDRs of the VL set forth in SEQ ID NO: 37.
  • the NPY and PYY-binding protein may comprise a VH comprising an amino acid sequence which is at least 98% identical to the sequence set forth in SEQ ID NO: 36 provided that it comprises the CDRs of the VH set forth in SEQ ID NO: 36, and a VL comprising an amino acid sequence which is at least 98% identical to the sequence set forth in SEQ ID NO: 37 provided that it comprises the CDRs of the VL set forth in SEQ ID NO: 37.
  • the NPY and PYY-binding protein may comprise a VH comprising an amino acid sequence which is at least 99% identical to the sequence set forth in SEQ ID NO: 36 provided that it comprises the CDRs of the VH set forth in SEQ ID NO: 36, and a VL comprising an amino acid sequence which is at least 99% identical to the sequence set forth in SEQ ID NO: 37 provided that it comprises the CDRs of the VL set forth in SEQ ID NO: 37.
  • the NPY and PYY-binding protein comprises a VH comprising an amino acid sequence set forth in SEQ ID NO: 36 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 37.
  • the binding protein may comprise a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 48 and a kappa light chain comprising an amino acid sequence set forth in SEQ ID NO: 49.
  • the binding protein may comprise a variable region comprising an amino acid sequence which is at least 90% identical to the sequence set forth in SEQ ID NO: 31 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 31.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 95% identical to the sequence set forth in SEQ ID NO: 31 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 31.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 96% identical to the sequence set forth in SEQ ID NO: 31 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 31.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 97% identical to the sequence set forth in SEQ ID NO: 31 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 31.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 98% identical to the sequence set forth in SEQ ID NO: 31 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 31.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 99% identical to the sequence set forth in SEQ ID NO: 31 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 31.
  • the NPY and PYY-binding protein may comprise a variable region comprising the amino acid sequence set forth in SEQ ID NO: 31.
  • an NPY and PYY-binding protein of the disclosure comprises the CDRs of the binding protein designated 4A3B2vlvl in Table 1.
  • the CDRs of the binding protein designated 4A3B2vl are CDR-H1 (SEQ ID NO: 51), CDR-H2 (SEQ ID NO: 43), CDR-H3 (SEQ ID NO: 44), CDR-L1 (SEQ ID NO: 45), CDR-L2 (SEQ ID NO: 46) and CDR-L3 (SEQ ID NO: 47).
  • the NPY and PYY-binding protein may comprise a VH comprising an amino acid sequence which is at least 90% identical to the sequence set forth in SEQ ID NO: 50 provided that it comprises the CDRs of the VH set forth in SEQ ID NO: 50, and a VL comprising an amino acid sequence which is at least 90% identical to the sequence set forth in SEQ ID NO: 37 provided that it comprises the CDRs of the VL set forth in SEQ ID NO: 37.
  • the NPY and PYY-binding protein may comprise a VH comprising an amino acid sequence which is at least 95% identical to the sequence set forth in SEQ ID NO: 50 provided that it comprises the CDRs of the VH set forth in SEQ ID NO: 50, and a VL comprising an amino acid sequence which is at least 95% identical to the sequence set forth in SEQ ID NO: 37 provided that it comprises the CDRs of the VL set forth in SEQ ID NO: 37.
  • the NPY and PYY-binding protein may comprise a VH comprising an amino acid sequence which is at least 96% identical to the sequence set forth in SEQ ID NO: 50 provided that it comprises the CDRs of the VH set forth in SEQ ID NO: 50, and a VL comprising an amino acid sequence which is at least 96% identical to the sequence set forth in SEQ ID NO: 37 provided that it comprises the CDRs of the VL set forth in SEQ ID NO: 37.
  • the NPY and PYY- binding protein may comprise a VH comprising an amino acid sequence which is at least 97% identical to the sequence set forth in SEQ ID NO: 50 provided that it comprises the CDRs of the VH set forth in SEQ ID NO: 50, and a VL comprising an amino acid sequence which is at least 97% identical to the sequence set forth in SEQ ID NO: 37 provided that it comprises the CDRs of the VL set forth in SEQ ID NO: 37.
  • the NPY and PYY-binding protein may comprise a VH comprising an amino acid sequence which is at least 98% identical to the sequence set forth in SEQ ID NO: 50 provided that it comprises the CDRs of the VH set forth in SEQ ID NO: 50, and a VL comprising an amino acid sequence which is at least 98% identical to the sequence set forth in SEQ ID NO: 37 provided that it comprises the CDRs of the VL set forth in SEQ ID NO: 37.
  • the NPY and PYY-binding protein may comprise a VH comprising an amino acid sequence which is at least 99% identical to the sequence set forth in SEQ ID NO: 50 provided that it comprises the CDRs of the VH set forth in SEQ ID NO: 50, and a VL comprising an amino acid sequence which is at least 99% identical to the sequence set forth in SEQ ID NO: 37 provided that it comprises the CDRs of the VL set forth in SEQ ID NO: 37.
  • the NPY and PYY-binding protein comprises a VH comprising an amino acid sequence set forth in SEQ ID NO: 50 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 37.
  • the binding protein may comprise a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 53 and a kappa light chain comprising an amino acid sequence set forth in SEQ ID NO: 49.
  • the binding protein may comprise a variable region comprising an amino acid sequence which is at least 90% identical to the sequence set forth in SEQ ID NO: 52 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 52.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 95% identical to the sequence set forth in SEQ ID NO: 52 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 52.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 96% identical to the sequence set forth in SEQ ID NO: 52 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 52.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 97% identical to the sequence set forth in SEQ ID NO: 52 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 52.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 98% identical to the sequence set forth in SEQ ID NO: 52 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 52.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 99% identical to the sequence set forth in SEQ ID NO: 52 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 52.
  • the NPY and PYY-binding protein may comprise a variable region comprising the amino acid sequence set forth in SEQ ID NO: 52.
  • an NPY and PYY-binding protein of the disclosure may comprise the CDRs of the binding protein designated 1A5 in Table 1.
  • the NPY and PYY- binding protein may comprise a variable region comprising an amino acid sequence which is at least 90% identical to the sequence set forth in SEQ ID NO: 21 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 21.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 95% identical to the sequence set forth in SEQ ID NO: 21 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 21.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 96% identical to the sequence set forth in SEQ ID NO: 21 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 21.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 97% identical to the sequence set forth in SEQ ID NO: 21 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 21.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 98% identical to the sequence set forth in SEQ ID NO: 21 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 21.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 99% identical to the sequence set forth in SEQ ID NO: 21 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 21.
  • the NPY and PYY-binding protein may comprise a variable region comprising the amino acid sequence set forth in SEQ ID NO: 21.
  • an NPY and PYY-binding protein of the disclosure comprises the CDRs of the binding protein designated 1D8 in Table 1.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 90% identical to the sequence set forth in SEQ ID NO: 22 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 22.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 95% identical to the sequence set forth in SEQ ID NO: 22 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 22.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 96% identical to the sequence set forth in SEQ ID NO: 22 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 22.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 97% identical to the sequence set forth in SEQ ID NO: 22 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 22.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 98% identical to the sequence set forth in SEQ ID NO: 22 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 22.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 99% identical to the sequence set forth in SEQ ID NO: 22 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 22.
  • the NPY and PYY-binding protein may comprise a variable region comprising the amino acid sequence set forth in SEQ ID NO:
  • an NPY and PYY-binding protein of the disclosure comprises the CDRs of the binding protein designated 1A in Table 1.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 90% identical to the sequence set forth in SEQ ID NO: 23 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 23.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 95% identical to the sequence set forth in SEQ ID NO: 23 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 23.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 96% identical to the sequence set forth in SEQ ID NO: 23 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 23.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 97% identical to the sequence set forth in SEQ ID NO: 23 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 23.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 98% identical to the sequence set forth in SEQ ID NO: 23 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 23.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 99% identical to the sequence set forth in SEQ ID NO: 23 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 23.
  • the NPY and PYY-binding protein may comprise a variable region comprising the amino acid sequence set forth in SEQ ID NO:
  • an NPY and PYY-binding protein of the disclosure comprises the CDRs of the binding protein designated 2B in Table 1.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 90% identical to the sequence set forth in SEQ ID NO: 24 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 24.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 95% identical to the sequence set forth in SEQ ID NO: 24 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 24.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 96% identical to the sequence set forth in SEQ ID NO: 24 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 24.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 97% identical to the sequence set forth in SEQ ID NO: 24 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 24.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 98% identical to the sequence set forth in SEQ ID NO: 24 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 24.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 99% identical to the sequence set forth in SEQ ID NO: 24 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 24.
  • the NPY and PYY-binding protein may comprise a variable region comprising the amino acid sequence set forth in SEQ ID NO:
  • an NPY and PYY-binding protein of the disclosure comprises the CDRs of the binding protein designated 3B in Table 1.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 90% identical to the sequence set forth in SEQ ID NO: 25 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 25.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 95% identical to the sequence set forth in SEQ ID NO: 25 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 25.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 96% identical to the sequence set forth in SEQ ID NO: 25 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 25.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 97% identical to the sequence set forth in SEQ ID NO: 25 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 25.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 98% identical to the sequence set forth in SEQ ID NO: 25 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 25.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 99% identical to the sequence set forth in SEQ ID NO: 25 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 25.
  • the NPY and PYY-binding protein may comprise a variable region comprising the amino acid sequence set forth in SEQ ID NO:
  • an NPY and PYY-binding protein of the disclosure comprises the CDRs of the binding protein designated 4A in Table 1.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 90% identical to the sequence set forth in SEQ ID NO: 26 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 26.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 95% identical to the sequence set forth in SEQ ID NO: 26 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 26.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 96% identical to the sequence set forth in SEQ ID NO: 26 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 26.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 97% identical to the sequence set forth in SEQ ID NO: 26 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 26.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 98% identical to the sequence set forth in SEQ ID NO: 26 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 26.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 99% identical to the sequence set forth in SEQ ID NO: 26 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 26.
  • the NPY and PYY-binding protein may comprise a variable region comprising the amino acid sequence set forth in SEQ ID NO: 26.
  • an NPY and PYY-binding protein of the disclosure comprises the CDRs of the binding protein designated 5 A in Table 1.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 90% identical to the sequence set forth in SEQ ID NO: 27 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 27.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 95% identical to the sequence set forth in SEQ ID NO: 27 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 27.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 96% identical to the sequence set forth in SEQ ID NO: 27 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 27.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 97% identical to the sequence set forth in SEQ ID NO: 27 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 27.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 98% identical to the sequence set forth in SEQ ID NO: 27 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 27.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 99% identical to the sequence set forth in SEQ ID NO: 27 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 27.
  • the NPY and PYY-binding protein may comprise a variable region comprising the amino acid sequence set forth in SEQ ID NO:
  • an NPY and PYY-binding protein of the disclosure comprises the CDRs of the binding protein designated 6 A in Table 1.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 90% identical to the sequence set forth in SEQ ID NO: 28 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 28.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 95% identical to the sequence set forth in SEQ ID NO: 28 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 28.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 96% identical to the sequence set forth in SEQ ID NO: 28 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 28.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 97% identical to the sequence set forth in SEQ ID NO: 28 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 28.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 98% identical to the sequence set forth in SEQ ID NO: 28 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 28.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 99% identical to the sequence set forth in SEQ ID NO: 28 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 28.
  • the NPY and PYY-binding protein may comprise a variable region comprising the amino acid sequence set forth in SEQ ID NO:
  • an NPY and PYY-binding protein of the disclosure comprises the CDRs of the binding protein designated 7 A in Table 1.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 90% identical to the sequence set forth in SEQ ID NO: 29 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 29.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 95% identical to the sequence set forth in SEQ ID NO: 29 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 29.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 96% identical to the sequence set forth in SEQ ID NO: 29 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 29.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 97% identical to the sequence set forth in SEQ ID NO: 29 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 29.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 98% identical to the sequence set forth in SEQ ID NO: 29 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 29.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 99% identical to the sequence set forth in SEQ ID NO: 29 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 29.
  • the NPY and PYY-binding protein may comprise a variable region comprising the amino acid sequence set forth in SEQ ID NO:
  • an NPY and PYY-binding protein of the disclosure comprises the CDRs of the binding protein designated 8A in Table 1.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 90% identical to the sequence set forth in SEQ ID NO: 30 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 30.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 95% identical to the sequence set forth in SEQ ID NO: 30 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 30.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 96% identical to the sequence set forth in SEQ ID NO: 30 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 30.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 97% identical to the sequence set forth in SEQ ID NO: 30 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 30.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 98% identical to the sequence set forth in SEQ ID NO: 30 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 30.
  • the NPY and PYY-binding protein may comprise a variable region comprising an amino acid sequence which is at least 99% identical to the sequence set forth in SEQ ID NO: 30 provided that it comprises the CDRs of the sequence set forth in SEQ ID NO: 30.
  • the NPY and PYY-binding protein may comprise a variable region comprising the amino acid sequence set forth in SEQ ID NO:
  • Multi-specific binding proteins comprise a plurality of variable regions each capable of binding to a different protein or different epitope within a protein.
  • the multi- specific binding protein comprises a variable domain that binds to NPY and a variable domain that binds to PYY.
  • the multi- specific binding protein comprises a variable domain that binds to one or more forms of NPY, a variable domain that binds to one or more additional forms of NPY and a variable domain that binds to PYY.
  • the multi- specific binding protein comprises a variable domain that binds to one or more forms of PYY, a variable domain that binds to one or more additional forms of PYY and a variable domain that binds to NPY.
  • Exemplary multi-specific binding proteins include:
  • dual variable domain immunoglobulins such as those comprising at least two domain antibodies that each bind to different proteins, e.g., as described in W02007/024715;
  • An exemplary form of multipecific binding protein of the disclosure comprises a scFv that binds to NPY, a scFv that binds to PYY and an antibody Fc region or heavy chain constant region.
  • the components can be arranged as SCFVI-FC-SCFV2 or SCFVI-SCFV2-FC or SCFV2-SCFVI- Fc (where Fc can be an Fc region of an antibody or an antibody constant region and scFvi bind to NPY and scFv2 binds to PYY).
  • One or both of the scFv can be replaced with domain antibodies. Additional scFv (or domain antibodies) can be added (e.g., fused to an existing scFv (or domain antibody) or to a constant region) to increase the number of proteins bound by the binding protein).
  • Another exemplary form of a multispecific binding protein of the disclosure comprises scFv that binds to NPY fused to a light chain constant region and a scFv that binds to PYY fused to a heavy chain constant region.
  • One or both of the scFv can be replaced with domain antibodies. Additional scFv (or domain antibodies) can be added (e.g., fused to an existing scFv (or domain antibody) or to a constant region) to increase the number of proteins bound by the binding protein).
  • a further exemplary form of a multispecific binding protein of the disclosure is a multispecific diabody, triabody or tetrabody, (see, e.g., Mack et al., Proc. Natl. Acad. Sci., 92. : 7021-7025, 1995).
  • Exemplary sources of variable regions that bind NPY for producing multi- specific binding proteins include, for example, an antibody described in Walter et al., Peptides, 15: 607- 613, 1994 or as are commercially available, e.g., from Abeam, Thermo Scientific Pierce or Bachem.
  • Exemplary sources of variable regions that bind PYY (or specific forms thereof) for producing multi- specific binding proteins include, for example, as described in WO/2006/108234 or as are commercially available, e.g., from Pierce, Abeam or Abnova.
  • the present disclosure encompasses a NPY and PYY-binding protein comprising an antigen binding domain of an antibody and a constant region or Fc or a domain thereof, e.g., CH2 and/or CH3 domain.
  • a constant region or Fc or a domain thereof e.g., CH2 and/or CH3 domain.
  • Suitable constant regions and/or domains will be apparent to the skilled artisan and/or the sequences of such polypeptides are readily available from publicly available databases. Kabat et al also provide description of some suitable constant regions/domains.
  • Constant regions and/or domains thereof are useful for providing biological activities such as, dimerization, extended serum half-life (e.g., by binding to FcRn), antigen dependent cell cytotoxicity (ADCC), complement dependent cytotoxicity (CDC, antigen dependent cell phagocytosis (ADCP).
  • ADCC antigen dependent cell cytotoxicity
  • CDC complement dependent cytotoxicity
  • ADCP antigen dependent cell phagocytosis
  • NPY and PYY-binding proteins comprising mutant constant regions or domains, e.g., as described in US7217797; US7217798; or US20090041770 (having increased half-life) or US2005037000 (increased ADCC).
  • Neutralizing NPY and PYY-binding proteins of the present disclosure can comprise an IgG4 constant region or a stabilized IgG4 constant region.
  • stabilized IgG4 constant region will be understood to mean an IgG4 constant region that has been modified to reduce Fab arm exchange or the propensity to undergo Fab arm exchange or formation of a halfantibody or a propensity to form a half antibody.
  • Fab arm exchange refers to a type of protein modification for human IgG4, in which an IgG4 heavy chain and attached light chain (halfmolecule) is swapped for a heavy-light chain pair from another IgG4 molecule.
  • IgG4 molecules may acquire two distinct Fab arms recognizing two distinct antigens (resulting in bispecific molecules).
  • Fab arm exchange occurs naturally in vivo and can be induced in vitro by purified blood cells or reducing agents such as reduced glutathione.
  • a “half antibody” forms when an IgG4 antibody dissociates to form two molecules each containing a single heavy chain and a single light chain.
  • a stabilized IgG4 constant region comprises a proline at position 241 of the hinge region according to the system of Kabat (Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 1987 and/or 1991). This position corresponds to position 228 of the hinge region according to the EU numbering system (Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 2001 and Edelman et al., Proc. Natl. Acad. USA, 63, 78-85, 1969). In human IgG4, this residue is generally a serine.
  • the IgG4 hinge region comprises a sequence CPPC.
  • the “hinge region” is a proline-rich portion of an antibody heavy chain constant region that links the Fc and Fab regions that confers mobility on the two Fab arms of an antibody.
  • the hinge region includes cysteine residues which are involved in inter-heavy chain disulfide bonds. It is generally defined as stretching from Glu226 to Pro243 of human IgGl according to the numbering system of Kabat.
  • Hinge regions of other IgG isotypes may be aligned with the IgGl sequence by placing the first and last cysteine residues forming inter-heavy chain disulphide (S-S) bonds in the same positions (see for example W02010/080538).
  • S-S inter-heavy chain disulphide
  • the present disclosure also provides a NPY and PYY-binding protein, or a nucleic acid encoding same, having at least 90% identity to a sequence disclosed herein, provided that the NPY and PYY-binding protein retains the CDRs of the sequences disclosed herein.
  • the NPY and PYY-binding protein may have at least 90% identity to the sequence(s) of an antibody described in Table 1 provided that it retains the CDRs of the respective antibody described in Table 1.
  • a NPY and PYY-binding protein of the disclosure comprises sequence at least about 95% or 97% or 98% or 99% identical to the sequence(s) of an antibody described in Table 1 provided that it retains the CDRs of the respective antibody described in Table 1.
  • the present disclosure provide nucleic acids which encode an NPY and PYY-binding protein as described herein.
  • the query sequence is at least 50 residues in length, and the GAP analysis aligns the two sequences over a region of at least 50 residues. For example, the query sequence is at least 100 residues in length and the GAP analysis aligns the two sequences over a region of at least 100 residues. For example, the two sequences are aligned over their entire length.
  • the present disclosure also contemplates a nucleic acid that hybridizes under stringent hybridization conditions to a nucleic acid encoding a NPY and PYY-binding protein described herein.
  • a “moderate stringency” is defined herein as being a hybridization and/or washing carried out in 2 x SSC buffer, 0.1% (w/v) SDS at a temperature in the range 45°C to 65°C, or equivalent conditions.
  • a “high stringency” is defined herein as being a hybridization and/or wash carried out in 0.1 x SSC buffer, 0.1% (w/v) SDS, or lower salt concentration, and at a temperature of at least 65°C, or equivalent conditions.
  • Reference herein to a particular level of stringency encompasses equivalent conditions using wash/hybridization solutions other than SSC known to those skilled in the art.
  • methods for calculating the temperature at which the strands of a double stranded nucleic acid will dissociate also known as melting temperature, or Tm are known in the art.
  • Tm melting temperature
  • a temperature that is similar to (e.g., within 5°C or within 10°C) or equal to the Tm of a nucleic acid is considered to be high stringency.
  • Medium stringency is to be considered to be within 10°C to 20°C or 10°C to 15°C of the calculated Tm of the nucleic acid.
  • mutant forms of a NPY and PYY-binding protein of the disclosure comprising one or more conservative amino acid substitutions within the frame work region compared to a sequence set forth herein.
  • the NPY and PYY-binding protein comprises 10 or fewer, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 conservative amino acid substitutions within the framework region.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain and/or hydropathicity and/or hydrophilicity.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), /-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Hydropathic indices are described, for example in Kyte and Doolittle J. Mol. Biol., 157: 105- 132, 1982 and hydrophy
  • the present disclosure also contemplates non-conservative amino acid changes.
  • non-conservative amino acid changes are substitutions of charged amino acids with another charged amino acid and with neutral or positively charged amino acids.
  • the NPY and PYY-binding protein comprises 10 or fewer, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 nonconservative amino acid substitutions within the framework region compared to a sequence set forth herein.
  • Exemplary methods for producing mutant forms of a NPY and PYY-binding protein include:
  • a nucleic acid encoding the polypeptide into a mutator cell, e.g., XL-lRed,
  • DNA shuffling e.g., as disclosed in Stemmer, Nature 570:389-91, 1994;
  • Exemplary methods for determining biological activity of the mutant NPY and PYY- binding proteins of the disclosure will be apparent to the skilled artisan and/or described herein, e.g., antigen binding.
  • methods for determining antigen binding, competitive inhibition of binding, affinity, association, dissociation and therapeutic efficacy are described herein.
  • a nucleic acid encoding a NPY and PYY-binding protein of the disclosure is introduced into an expression construct, such that it is operably linked to a promoter to thereby facilitate its expression.
  • Methods for producing expression constructs e.g., cloning into expression constructs/vectors are known in the art and/or described in Ausubel et al (In: Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987), and (Sambrook et al (In: Molecular Cloning: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Third Edition 2001) and US7270969.
  • the NPY and PYY-binding protein of the disclosure is expressed in a bacterial cell.
  • Typical promoters suitable for expression in bacterial cells such as for example a bacterial cell selected from the group comprising E. coli, Staphylococcus sp, Corynebacterium sp., Salmonella sp., Bacillus sp., and Pseudomonas sp., include, but are not limited to a promoter such as lacz, Ipp, a temperature- sensitive (L or (R promoters, T7, T3, SP6 or semiartificial promoters such as the IPTG-inducible tac promoter or lacUV5 promoter.
  • L or (R promoters, T7, T3, SP6 or semiartificial promoters such as the IPTG-inducible tac promoter or lacUV5 promoter.
  • the NPY and PYY-binding protein is expressed in a yeast cell.
  • Typical promoters suitable for expression in yeast cells such as, Pichia pastoris, Saccharomyces cerevisiae and S. pombe, include, but are not limited to promoters from the following genes ADH1, GALI, GAL4, CUP1, PH05, nmt, RPR1, or TEFl.
  • the NPY and PYY-binding protein is expressed in an insect cell.
  • Typical promoters suitable for expression in insect cells, or in insects include, but are not limited to, the OPEI2 promoter, the insect actin promoter isolated from Bombyx muri, the Drosophila sp. dsh promoter (Marsh et al Hum. Mol. Genet. 9, 13-25, 2000).
  • a NPY and PYY-binding protein of the disclosure can also be expressed in plant cells.
  • Promoters for expressing peptides in plant cells include, but are not limited to, the Hordeum vulgare amylase gene promoter, the cauliflower mosaic virus 35S promoter, the nopaline synthase (NOS) gene promoter, and the auxin inducible plant promoters Pl and P2.
  • a NPY and PYY-binding protein of the disclosure is expressed in a mammalian cell or in a mammal.
  • Typical promoters suitable for expression in a mammalian cell include, for example a promoter selected from the group consisting of, retroviral LTR elements, the SV40 early promoter, the SV40 late promoter, the CMV IE (cytomegalovirus immediate early) promoter, the EFi promoter (from human elongation factor 1), the EM7 promoter, the UbC promoter (from human ubiquitin C).
  • useful mammalian host cell lines include monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (HEK-293 cells) ; baby hamster kidney cells (BHK); Chinese hamster ovary cells (CHO); African green monkey kidney cells (VERO-76); or myeloma cells (e.g., NS/0 or SP2/0 cells).
  • COS-7 monkey kidney CV1 line transformed by SV40
  • HEK-293 cells human embryonic kidney line
  • BHK baby hamster kidney cells
  • Chinese hamster ovary cells CHO
  • African green monkey kidney cells e.g., NS/0 or SP2/0 cells.
  • expression constructs/vectors include, for example, enhancers, transcriptional terminators, polyadenylation sequences, nucleic acids encoding selectable or detectable markers and origins of replication.
  • an expression construct is a bicistronic expression construct.
  • bicistronic is meant a single nucleic acid molecule that is capable of encoding two distinct polypeptides from different regions of the nucleic acid, for example, a single nucleic acid capable of encoding a VH containing polypeptide and a VL containing polypeptide as distinct polypeptides.
  • the regions encoding each distinct polypeptide are separated by an internal ribosome entry site (IRES) and the region 5’ of the IRES does not comprise a transcription termination sequence.
  • IRESs are described, for example, in US20090247455.
  • a suitable expression construct Following production of a suitable expression construct, it is introduced into a suitable cell using any method known in the art.
  • Exemplary methods include microinjection, transfection mediated by DEAE-dextran, transfection mediated by liposomes such as by using lipofectamine (Gibco, MD, USA) and/or cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation and microparticle bombardment such as by using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA) amongst others.
  • the cells used to produce the NPY and PYY-binding protein of this disclosure are then cultured under conditions known in the art to produce a NPY and PYY-binding protein of the disclosure.
  • Cell free expression systems are also contemplated by the present disclosure, e.g., the TNT T7 and TNT T3 systems (Promega), the pEXPl-DEST and pEXP2-DEST vectors (Invitrogen).
  • a NPY and PYY-binding protein of the disclosure is purified using a method known in the art. Such purification provides the protein of the disclosure substantially free of nonspecific protein, acids, lipids, carbohydrates, and the like.
  • the protein will be in a preparation wherein more than about 90% (e.g. 95%, 98% or 99%) of the protein in the preparation is a NPY and PYY-binding protein of the disclosure.
  • Standard methods of peptide purification are employed to obtain an isolated or recombinant NPY and PYY-binding protein of the disclosure, including but not limited to various high-pressure (or performance) liquid chromatography (HPLC) and non-HPLC polypeptide isolation protocols, such as size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, mixed mode chromatography, phase separation methods, electrophoretic separations, precipitation methods, salting in/out methods, immunochromatography, and/or other methods.
  • HPLC high-pressure liquid chromatography
  • non-HPLC polypeptide isolation protocols such as size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, mixed mode chromatography, phase separation methods, electrophoretic separations, precipitation methods, salting in/out methods, immunochromatography, and/or other methods.
  • affinity purification is useful for isolating a fusion protein comprising a label.
  • Methods for isolating a protein using affinity chromatography are known in the art and described, for example, in Scopes (In: Protein purification: principles and practice, Third Edition, Springer Verlag, 1994).
  • an antibody or compound that binds to the label in the case of a polyhistidine tag this may be, for example, nickel- NT A
  • a sample comprising a protein is then contacted to the immobilized antibody or compound for a time and under conditions sufficient for binding to occur. Following washing to remove any unbound or non- specifically bound protein, the protein is eluted.
  • protein A or protein G or modified forms thereof can be used for affinity purification.
  • Protein A is useful for isolating purified proteins comprising a human yl, y2, or y4 heavy chain Fc region.
  • Protein G is recommended for all mouse Fc isotypes and for human y3.
  • a NPY and PYY-binding protein of the present disclosure is conjugated to a compound.
  • the compound is selected from the group consisting of a radioisotope, a detectable label, a therapeutic compound, a colloid, a toxin, a nucleic acid, a peptide, a protein, a compound that increases the half life of the NPY and PYY-binding protein in a subject and mixtures thereof.
  • the other compound can be directly or indirectly bound to the NPY and PYY-binding protein (e.g., can comprise a linker in the case of indirect binding).
  • examples of compounds include, a radioisotope (e.g., iodine-131, yttrium-90 or indium-i l l), a detectable label (e.g., a fluorophore or a fluorescent nanocrystal), a therapeutic compound (e.g., a chemotherapeutic or an anti-inflammatory), a colloid (e.g., gold), a toxin (e.g., ricin or tetanus toxoid), a nucleic acid, a peptide (e.g., a serum albumin binding peptide), a protein (e.g., a protein comprising an antigen binding domain of an antibody or serum albumin), a compound that increases the half life of the NPY and PYY-binding protein in a
  • NPY and PYY-binding proteins comprising antibody binding domains of the present disclosure are readily screened for biological activity, e.g., as described below. Binding Assays
  • assay is an antigen binding assay, e.g., as described in Scopes (In: Protein purification: principles and practice, Third Edition, Springer Verlag, 1994).
  • a method generally involves labelling the NPY and PYY-binding protein and contacting it with immobilized antigen, i.e., NPY and PYY or a peptide comprising conserved region thereof. Following washing to remove non-specific bound protein, the amount of label and, as a consequence, bound protein is detected.
  • the NPY and PYY-binding protein can be immobilized and the antigen(s) labelled.
  • the assay can be performed with immobilized NPY and PYY-binding protein and labelled antigen.
  • surface plasmon resonance assays can be used e.g., such as the Biacore assays described in the examples herein.
  • a binding assay is performed with peptide comprising an epitope of NPY and PYY.
  • peptide comprising an epitope of NPY and PYY.
  • Such an assay is also readily adapted to identify NPY and PYY-binding proteins that do not detectably or significantly bind to PP or any other protein or peptide described herein.
  • the protein is contacted with labelled PP and, following washing to remove non- specifically bound PP, the level of labelled protein detected.
  • a cell expressing a Y receptor e.g., Y2 receptor or Y1 receptor or Y4 receptor or Y5 receptor
  • a region of the receptor required for NPY/PYY binding is immobilized on a surface and contacted with NPY and/or PYY and with a NPY and PYY- binding protein to be tested (in the case of controls, no test NPY and PYY-binding protein is added).
  • a reduced level of NPY/PYY bound to the cell or receptor in the presence of the NPY and PYY-binding protein compared to in the absence of the NPY and PYY-binding protein indicates that the NPY and PYY-binding protein inhibits binding of NPY/PYY to the receptor.
  • the assay can also be performed with labelled NPY/PYY to assist with detection.
  • NPY and PYY-binding protein are tested and the concentration at which 50% of the maximum inhibition of binding of NPY and/or PYY to a receptor by the NPY and PYY-binding protein is determined (this concentration is known as EC50).
  • NPY and PYY-binding protein are tested and the concentration at which 50% inhibition of binding of NPY and/or PYY to a receptor by the NPY and PYY-binding protein is determined (this concentration is known as IC50).
  • NPY and/or PYY signal e.g., Y 1 receptor or Y2 receptor or Y4 receptor or Y5 receptor
  • NPY and/or PYY signal e.g., Y 1 receptor or Y2 receptor or Y4 receptor or Y5 receptor
  • the level of NPY/PYY- induced signalling in the cells is then detected.
  • NPY/PYY signalling can be determined by detecting the amount of phosphorylated ERK.
  • a protein that reduces the amount of phosphorylated ERK in the cells in the presence of NPY/PYY is considered to neutralize NPY and/or PYY signalling.
  • Additional assays include, for example, contacting Ewing sarcoma cells (e.g., SK-N- MC) in the presence of NPY and/or PYY and a NPY and PYY-binding protein. If NPY/PYY signal through Y1 and/or Y5 receptors in the cells, cell death is induced. Accordingly, a reduction in cell death in the presence of the NPY and PYY-binding protein compared to in the absence of the protein indicates that the protein neutralizes NPY/PYY signalling.
  • Ewing sarcoma cells e.g., SK-N- MC
  • Another assay for assessing NPY/PYY activity involves culturing HEL cells in the presence of a NPY and PYY-binding protein and NPY and/or PYY and assessing intracellular calcium levels. Reduced intracellular calcium levels in the presence of the NPY and PYY- binding protein compared to in the absence of the protein indicates that the protein neutralizes NPY/PYY signalling.
  • NPY and PYY-binding proteins of the present disclosure can also be assessed for therapeutic efficacy in an animal model of a condition, e.g., a NPY/PYY-mediated condition.
  • a condition e.g., a NPY/PYY-mediated condition.
  • the NPY and PYY-binding protein is administered to a model of cancer, e.g., to a mouse to which cancer cells are administered. Animal survival, tumour growth, tumour metastasis and/or other measures of cancer progression can then be assessed.
  • the tumour cells can be administered after the protein for prophylactic studies or before the protein for therapeutic studies.
  • an immune response e.g., T cell response against the cancer cells is assessed, e.g., using an ELISPOT assay.
  • a NPY and PYY-binding protein of the disclosure is administered to an animal and its effect on food consumption and/or body mass/weight is assessed.
  • a NPY and PYY-binding protein that increases food consumption and/or body mass/weight is considered to neutralize NPY/PYY signalling.
  • a NPY and PYY-binding protein of the disclosure is administered to a subject, T cells isolated from the subject and stimulated (e.g., with an anti-CD3 antibody) and the proliferation of T cells assessed.
  • T cells isolated from the subject e.g., with an anti-CD3 antibody
  • a NPY and PYY-binding protein that increases the amount of T cell proliferation neutralizes NPY/PYY signalling.
  • the antibody of the disclosure is conjugated to a detectable label, e.g., a fluorescent label or a radioactive label.
  • a detectable label e.g., a fluorescent label or a radioactive label.
  • the labelled antibody and the test NPY and PYY-binding protein are then mixed and contacted with NPY and/or PYY or a peptide comprising an epitope thereof.
  • the level of labelled antibody is then determined and compared to the level determined when the labelled antibody is contacted with the NPY and/or PYY or the peptide comprising an epitope thereof in the absence of the NPY and PYY-binding protein.
  • the NPY and PYY-binding protein competitively inhibits binding of the antibody.
  • the NPY and PYY-binding protein is conjugated to a different label than the antibody. This permits detection of the level of binding of the NPY and PYY-binding protein to NPY/PYY or epitope bearing peptide.
  • the NPY and PYY-binding protein is permitted to bind to NPY/PYY or a peptide comprising an epitope thereof prior to contacting the NPY/PYY or peptide with an antibody described herein.
  • a reduction in the amount of bound antibody in the presence of the NPY and PYY-binding protein compared to in the absence of the NPY and PYY-binding protein indicates that the NPY and PYY-binding protein competitively inhibits binding of the antibody to NPY/PYY.
  • a reciprocal assay can also be performed using labelled NPY and PYY- binding protein and first allowing the antibody to bind to NPY/PYY or the peptide.
  • a reduced amount of labelled NPY and PYY-binding protein bound to NPY/PYY or the peptide in the presence of the antibody compared to in the absence of antibody indicates that the NPY and PYY-binding protein competitively inhibits binding of the antibody to NPY/PYY.
  • the epitope bound by a protein described herein is mapped.
  • Epitope mapping methods will be apparent to the skilled artisan. For example, a series of overlapping peptides spanning the NPY/PYY sequence or a region thereof comprising an epitope of interest, e.g., peptides comprising 10-15 amino acids are produced. The NPY and PYY-binding protein is then contacted to each peptide or a combination thereof and the peptide(s) to which it binds determined. This permits determination of peptide(s) comprising the epitope to which the NPY and PYY-binding protein binds.
  • amino acid residues within NPY/PYY are mutated, e.g., by alanine scanning mutagenesis, and mutations that reduce or prevent protein binding are determined. Any mutation that reduces or prevents binding of the NPY and PYY-binding protein is likely to be within the epitope bound by the protein.
  • a further method involves binding NPY/PYY or a region thereof to an immobilized NPY and PYY-binding protein of the present disclosure and digesting the resulting complex with proteases. Peptide that remains bound to the immobilized protein are then isolated and analyzed, e.g., using mass spectrometry, to determine their sequence.
  • a further method involves converting hydrogens in NPY/PYY or a region thereof to deuterium atoms and binding the resulting protein to an immobilized NPY and PYY-binding protein of the present disclosure.
  • the deuterium atoms are then converted back to hydrogen, the NPY/PYY or region thereof isolated, digested with enzymes and analyzed, e.g., using mass spectrometry to identify those regions comprising deuterium, which would have been protected from conversion to hydrogen by the binding of a NPY and PYY-binding protein described herein.
  • the dissociation rate constant (kd) or association rate constant (ka) or equilibrium binding constant (KD) of a NPY and PYY-binding protein for NPY/PYY or a peptide comprising an epitope thereof is determined.
  • These constants for a NPY and PYY- binding protein are in one example measured by a radiolabelled or fluorescently-labelled NPY/PYY binding assay. This assay equilibrates the protein with a minimal concentration of labelled NPY/PYY in the presence of a titration series of unlabelled NPY/PYY. Following washing to remove unbound NPY/PYY, the amount of label is determined.
  • Affinity measurements can be determined by standard methodology for antibody reactions, for example, immunoassays, surface plasmon resonance (SPR) (Rich and Myszka Curr. Opin. Biotechnol 11: :54, 2000; Englebienne Analyst. 123 1599, 1998), isothermal titration calorimetry (ITC) or other kinetic interaction assays known in the art.
  • SPR surface plasmon resonance
  • ITC isothermal titration calorimetry
  • the constants are measured by using surface plasmon resonance assays, e.g., using BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) with immobilized NPY/PYY or a region thereof (e.g., a peptide described herein).
  • surface plasmon resonance assays e.g., using BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) with immobilized NPY/PYY or a region thereof (e.g., a peptide described herein).
  • BIAcore surface plasmon resonance e.g., using BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) with immobilized NPY/PYY or a region thereof (e.g., a peptide described herein).
  • Exemplary SPR methods are described in US7229619.
  • NPY and PYY-binding proteins encompassed by the present disclosure have an improved half-life, e.g., are modified to extend their half-life compared to NPY and PYY- binding proteins that are unmodified.
  • Methods for determining a NPY and PYY-binding protein with an improved half-life will be apparent to the skilled person. For example, the ability of a NPY and PYY-binding protein to bind to a neonatal Fc receptor (FcRn) is assessed. In this regard, increased binding affinity for FcRn increased the serum half-life of the NPY and PYY-binding protein (see for example, Kim et al., E r J Immunol., 24:2429, 1994).
  • the half-life of a NPY and PYY-binding protein of the disclosure can also be measured by pharmacokinetic studies, e.g., according to the method described by Kim et al, Eur J of Immunol 24:542, 1994. According to this method radiolabelled NPY and PYY-binding protein is injected intravenously into mice and its plasma concentration is periodically measured as a function of time, for example at 3 minutes to 72 hours after the injection.
  • the clearance curve thus obtained should be biphasic, that is, an alpha phase and beta phase.
  • the clearance rate in beta-phase is calculated and compared with that of the wild type or unmodified NPY and PYY-binding protein.
  • Stability of a NPY and PYY-binding protein of the disclosure can be assessed by any of a variety of assays.
  • the NPY and PYY-binding protein is exposed to a condition, e.g., heat or acid or stored for a period of time (e.g., 1 month) at room temperature. Aggregation of the NPY and PYY-binding protein can then be assessed by determining turbidity (with an increase in turbidity following exposure to the condition indicating instability), size exclusion chromatography, non-reducing gel electrophoresis or a binding or neutralization study described herein.
  • compositions contemplated by the present disclosure comprise compounds that inhibit Y receptor(s) (e.g., a Y1 receptor and/or a Y2 receptor and/or a Y4 receptor and/or a Y5 receptor) or a combination of compounds one that inhibits NPY and one that inhibits PYY.
  • Y receptor(s) e.g., a Y1 receptor and/or a Y2 receptor and/or a Y4 receptor and/or a Y5 receptor
  • Formulation of a NPY and PYY-binding protein or nucleic acid encoding same or cell expressing same or compound to be administered will vary according to the route of administration and formulation (e.g., solution, emulsion, capsule) selected.
  • An appropriate pharmaceutical composition can be prepared in a physiologically acceptable carrier.
  • suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • aqueous carriers include water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution and glycine.
  • Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers (See, generally, Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980).
  • the compositions can optionally contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents and toxicity adjusting agents, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride and sodium lactate.
  • the NPY and PYY-binding protein or other compound described herein of this disclosure can be lyophilized for storage and reconstituted in a suitable carrier prior to use according to art-known lyophilization and reconstitution techniques.
  • the optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures well known to the skilled artisan, and will depend on the ultimate pharmaceutical formulation desired.
  • the dosage ranges for the administration of the NPY and PYY-binding protein or other compound of the disclosure are those large enough to produce the desired effect.
  • the composition comprises a therapeutically or prophylactically effective amount of the NPY and PYY-binding protein or nucleic acid encoding same or cell expressing same.
  • the term “effective amount” shall be taken to mean a sufficient quantity of the NPY and PYY-binding protein, nucleic acid or cells or other compound to induce/increase or inhibit/reduce/prevent signalling of NPY and PYY in a subject.
  • the skilled artisan will be aware that such an amount will vary depending on, for example, the compound/protein and/or the particular subject and/or the type or severity of a condition being treated. Accordingly, this term is not to be construed to limit the disclosure to a specific quantity of compound/protein.
  • the term “therapeutically effective amount” shall be taken to mean a sufficient quantity of NPY and PYY-binding protein, nucleic acid or cells or other compound to reduce or inhibit one or more symptoms of a condition, e.g., cancer or to induce or stimulate an immune response against cancer cells.
  • prophylactically effective amount shall be taken to mean a sufficient quantity of NPY and PYY-binding protein, nucleic acid or cells or other compound to prevent or inhibit or delay the onset of one or more detectable symptoms of a condition, e.g. cancer and/or to prevent recurrence or metastasis of cancer.
  • the dosage should not be so large as to cause adverse side effects, such as hyper viscosity syndromes, pulmonary edema, congestive heart failure, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any complication. Dosage can vary from about 0.1 mg/kg to about 300mg/kg, e.g., from about 0.2mg/kg to about 200mg/kg, such as, from about 0.5mg/kg to about 20mg/kg, in one or more dose administrations daily, for one or several days.
  • the NPY and PYY-binding protein is administered at a dosage of between about Img/kg to about 50mg/kg. In one example, the NPY and PYY-binding protein is administered at a dosage of between about 5mg/kg to about 30mg/kg. In one example, the NPY and PYY-binding protein is administered subcutaneously or intravenously.
  • the NPY and PYY-binding protein or other compound is administered at an initial (or loading) dose which is higher than subsequent (maintenance doses).
  • the NPY and PYY-binding protein or other compound is administered at an initial dose of between about lOmg/kg to about 50mg/kg.
  • the NPY and PYY-binding protein or other compound is then administered at a maintenance dose of between about Img/kg to about lOmg/kg.
  • the maintenance doses may be administered every 7-35 days, such as, every 14 or 21 or 28 days.
  • a dose escalation regime in which a NPY and PYY-binding protein or other compound is initially administered at a lower dose than used in subsequent doses.
  • This dosage regime is useful in the case of subject’s initially suffering adverse events
  • multiple doses in a week may be administered.
  • increasing doses may be administered.
  • NPY and PYY-binding proteins of the present disclosure can be administered to an individual by an appropriate route, either alone or in combination with (before, simultaneous with, or after) another drug or agent.
  • the NPY and PYY- binding protein of the present disclosure can also be used in combination with a chemotherapy compound, such as caboplatin, cisplatin, cyclophosphamide, docetaxal, doxorubicin, erlotinib, etoposide, fluorouracil, irinotecan, methotrexate, paclitaxel, topotecan, vincristine or vinblastine.
  • a chemotherapy compound such as caboplatin, cisplatin, cyclophosphamide, docetaxal, doxorubicin, erlotinib, etoposide, fluorouracil, irinotecan, methotrexate, paclitaxel, topotecan, vincristine or vinblastine
  • the chemotherapy compound is selected from the group consisting of methotrexate, 1-asparaginase, vincristine, doxorubicin, danorubicin, cytarabine, idarubicin, mitoxantrone, cyclophosphamide, fludarabine, chlorambucil and combinations thereof.
  • the NPY and PYY-binding protein of the present disclosure can also be used in combination with a biologic useful for treating a cancer, e.g., rituximab, trastuzumab, bevacizumab, alemtuzumab, panitumumab, or cetuximab.
  • a biologic useful for treating a cancer e.g., rituximab, trastuzumab, bevacizumab, alemtuzumab, panitumumab, or cetuximab.
  • NPY and PYY-binding protein of the present disclosure can also be used in combination with radiation therapy.
  • cancer examples include, but are not limited to, an adenocarcinoma, a squamous cell carcinoma, a digestive/gastrointestinal cancer, an endocrine cancer, an eye cancer, a musculoskeletal cancer, a breast cancer, a neurologic cancer, a genitourinary cancer, a germ cell cancer, a head and neck cancer, a hematologic/blood cancer, a respiratory cancer, a skin cancer, an AIDS -related malignancy or a gynelogic cancer.
  • An adenocarcinoma is a cancer of an epithelium that originates in glandular tissue.
  • exemplary adenocarcinomas include forms of colorectal cancer, lung cancer, cervical cancer, prostate cancer, urachus cancer, vulval cancer, breast cancer, esophageal cancer, pancreatic cancer and gastric cancer.
  • Digestive/gastrointestinal cancers include anal cancer; bile duct cancer; extrahepatic bile duct cancer; appendix cancer; carcinoid tumour, gastrointestinal cancer; colon cancer; colorectal cancer including childhood colorectal cancer; esophageal cancer including childhood esophageal cancer; gallbladder cancer; gastric (stomach) cancer including childhood gastric (stomach) cancer; hepatocellular (liver) cancer including childhood hepatocellular (liver) cancer; pancreatic cancer including childhood pancreatic cancer; sarcoma, rhabdomyosarcoma; rectal cancer; and small intestine cancer.
  • Endocrine cancers include islet cell carcinoma (endocrine pancreas); adrenocortical carcinoma including childhood adrenocortical carcinoma; gastrointestinal carcinoid tumour; parathyroid cancer; pheochromocytoma; pituitary tumour; thyroid cancer including childhood thyroid cancer; childhood multiple endocrine neoplasia syndrome; and childhood carcinoid tumour.
  • Eye cancers include intraocular melanoma; and retinoblastoma.
  • Musculoskeletal cancers include Ewing's family of tumours; osteosarcoma/malignant fibrous histiocytoma of the bone; rhabdomyosarcoma including childhood rhabdomyosarcoma; soft tissue sarcoma including childhood soft tissue sarcoma; clear cell sarcoma of tendon sheaths; and uterine sarcoma.
  • Neurologic cancers include childhood brain stem glioma; brain tumour; childhood cerebellar astrocytoma; childhood cerebral astrocytoma/malignant glioma; childhood ependymoma; childhood medulloblastoma; childhood pineal and supratentorial primitive neuroectodermal tumours; childhood visual pathway and hypothalamic glioma; other childhood brain cancers; adrenocortical carcinoma; central nervous system lymphoma, primary; childhood cerebellar astrocytoma; neuroblastoma; craniopharyngioma; spinal cord tumours; central nervous system atypical teratoid/rhabdoid tumour; central nervous system embryonal tumours; and supratentorial primitive neuroectodermal tumours including childhood and pituitary tumour.
  • Genitourinary cancers include bladder cancer including childhood bladder cancer; renal cell (kidney) cancer; ovarian cancer including childhood ovarian cancer; ovarian epithelial cancer; ovarian low malignant potential tumour; penile cancer; prostate cancer; renal cell cancer including childhood renal cell cancer; renal pelvis and ureter, transitional cell cancer; testicular cancer; urethral cancer; vaginal cancer; vulvar cancer; cervical cancer; Wilms tumour and other childhood kidney tumours; endometrial cancer; and gestational trophoblastic tumour.
  • Germ cell cancers include childhood extracranial germ cell tumour; extragonadal germ cell tumour; ovarian germ cell tumour; and testicular cancer.
  • Head and neck cancers include lip and oral cavity cancer; childhood oral cancer; hypopharyngeal cancer; laryngeal cancer including childhood laryngeal cancer; metastatic squamous neck cancer with occult primary; mouth cancer; nasal cavity and paranasal sinus cancer; nasopharyngeal cancer including childhood nasopharyngeal cancer; oropharyngeal cancer; parathyroid cancer; pharyngeal cancer; salivary gland cancer including childhood salivary gland cancer; throat cancer; and thyroid cancer.
  • Hematologic/blood cell cancers include leukemia (e.g., acute lymphoblastic leukemia in adults and children; acute myeloid leukemia, e.g., in adults and children; chronic lymphocytic leukemia; chronic myelogenous leukemia; and hairy cell leukemia); a lymphoma (e.g., AIDS- related lymphoma; cutaneous T-cell lymphoma; Hodgkin's lymphoma including Hodgkin's lymphoma in adults and children; Hodgkin's lymphoma during pregnancy; non-Hodgkin's lymphoma including non-Hodgkin's lymphoma in adults and children; non-Hodgkin's lymphoma during pregnancy; mycosis fungoides; Sezary syndrome; Waldenstrom's macroglobulinemia; and primary central nervous system lymphoma); and other hematologic cancers (e.g., chronic myeloproliferative disorders; multiple mye
  • Respiratory cancers include non- small cell lung cancer; small cell lung cancer; malignant mesothelioma including malignant mesothelioma in adults and children; malignant thymoma; childhood thymoma; thymic carcinoma; bronchial adenomas/carcinoids including childhood bronchial adenomas/carcinoids; pleuropulmonary blastoma.
  • Skin cancers include Kaposi's sarcoma; Merkel cell carcinoma; melanoma; basal cell carcinoma and childhood skin cancer.
  • the cancer expresses a Y receptor, such as, a Y1 receptor and/or a Y2 receptor and/or a Y4 receptor and/or a Y5 receptor.
  • a Y receptor such as, a Y1 receptor and/or a Y2 receptor and/or a Y4 receptor and/or a Y5 receptor.
  • the cancer does not express a Y1 receptor and/or a Y2 receptor and/or a Y4 receptor and/or a Y5 receptor.
  • the cancer does not proliferate or grow in response to PYY and/or NPY.
  • the cancer is melanoma.
  • the cancer is lung cancer.
  • the cancer is breast cancer. Treatment of Non-cancerous Conditions
  • the present disclosure also contemplates treating non-cancerous conditions.
  • the disclosure contemplates treating a NPY and/or PYY-mediated condition, such as, anorexia or a wasting condition (which can be associated with cancer).
  • the condition is a wasting condition, such as cachexia.
  • the wasting condition is associated with a condition, such as, cancer, metabolic acidosis, infectious diseases, diabetes, autoimmune immune deficiency syndrome (AIDS), autoimmune disorders, addiction to drugs, cirrhosis of the liver, chronic inflammatory disorders, anorexia, chronic heart failure, chronic kidney disease, osteoporosis, skeletal muscle disease, motor neuron disease, multiple sclerosis, muscle atrophy and neurodegenerative disease.
  • a condition such as, cancer, metabolic acidosis, infectious diseases, diabetes, autoimmune immune deficiency syndrome (AIDS), autoimmune disorders, addiction to drugs, cirrhosis of the liver, chronic inflammatory disorders, anorexia, chronic heart failure, chronic kidney disease, osteoporosis, skeletal muscle disease, motor neuron disease, multiple sclerosis, muscle atrophy and neurodegenerative disease.
  • the wasting condition is cachexia or sarcopenia (e.g., wasting associated with aging).
  • the cachexia is associated with cancer, infectious disease (e.g., tuberculosis or leprosy), AIDS, autoimmune disease (including rheumatoid arthritis or type 1 diabetes), cystic fibrosis, drug addiction, alcoholism or liver cirrhosis.
  • infectious disease e.g., tuberculosis or leprosy
  • AIDS e.g., rheumatoid arthritis or type 1 diabetes
  • cystic fibrosis e.g., cystic fibrosis
  • drug addiction e.g., alcoholism or liver cirrhosis.
  • the wasting disorder is cachexia associated with cancer.
  • Exemplary cancers are described supra.
  • the method additionally comprises identifying a subject suffering from cachexia.
  • a subject can be identified, for example, based on detection of unintentional weight loss following diagnosis of another condition (e.g., cancer).
  • the subject can lose at least 5% of their body weight following diagnosis of another condition (e.g., cancer) or within the previous 30 days.
  • the method additionally comprises monitoring the weight of the subject and if their weight decreases or does not stabilize or increase administering a further dose of the compound(s).
  • the following assays can be performed with an antibody against a Y receptor, e.g., a Y1 receptor or a Y2 receptor or a Y4 receptor or a Y5 receptor, e.g., an antibody conjugated to a detectable label. Detection of the Y receptor(s) with an assay described herein is useful for identifying a subject suitable for treatment by performing a method described herein.
  • a Y receptor e.g., a Y1 receptor or a Y2 receptor or a Y4 receptor or a Y5 receptor
  • An immunoassay is an exemplary assay format for diagnosing a condition in a subject or detecting a Y receptor in a sample.
  • the present disclosure contemplates any form of immunoassay, including Western blotting, enzyme-linked immunosorbent assay (ELISA), fluorescence-linked immunosorbent assay (FLISA), competition assay, radioimmunoassay, lateral flow immunoassay, flow-through immunoassay, electrochemiluminescent assay, nephelometric-based assays, turbidometric-based assay, and fluorescence activated cell sorting (FACS)-based assays.
  • ELISA enzyme-linked immunosorbent assay
  • FLISA fluorescence-linked immunosorbent assay
  • competition assay radioimmunoassay
  • lateral flow immunoassay lateral flow immunoassay
  • flow-through immunoassay electrochemiluminescent assay
  • nephelometric-based assays n
  • One form of a suitable immunoassay is, for example, an ELISA or FLISA.
  • such an assay involves immobilizing an anti-Y receptor antibody onto a solid matrix, such as, for example a polystyrene or polycarbonate microwell or dipstick, a membrane, or a glass support (e.g. a glass slide).
  • a test sample is then brought into direct contact with the antibody and Y receptor bearing cells in the sample are bound or captured.
  • a protein that binds to the Y receptor or cell at a distinct epitope is brought into direct contact with the captured cell.
  • This detector protein is generally labelled with a detectable reporter molecule, such as for example, an enzyme (e.g.
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • P-galactosidase in the case of an ELISA or a fluorophore in the case of a FLISA.
  • a second labelled protein can be used that binds to the detector protein. Following washing to remove any unbound protein the detectable reporter molecule is detected by the addition of a substrate in the case of an ELISA, such as for example hydrogen peroxide, TMB, or toluidine, or 5-bromo- 4-chloro-3-indol-beta-D-galaotopyranoside (x-gal).
  • the immobilized (capture) protein and the detector protein may be used in the opposite manner.
  • the level of the antigen in the sample is then determined using a standard curve that has been produced using known quantities of the marker or by comparison to a control sample.
  • the assays described above are readily modified to use chemiluminescence or electrochemiluminescence as the basis for detection.
  • an immunosorbent method based on the description supra using a radiolabel for detection, or a gold label (e.g. colloidal gold) for detection, or a liposome, for example, encapsulating NAD+ for detection or an acridinium linked immunosorbent assay.
  • a radiolabel for detection or a gold label (e.g. colloidal gold) for detection
  • a liposome for example, encapsulating NAD+ for detection or an acridinium linked immunosorbent assay.
  • a tumour sample is assessed by immunofluorescence or immunohistochemistry for Y receptor expression.
  • the present disclosure additionally comprises a kit comprising one or more of the following:
  • kits for therapeutic/prophylactic use can additionally comprise a pharmaceutically acceptable carrier.
  • kit of the disclosure is packaged with instructions for use in a method described herein according to any example.
  • the present disclosure includes the following non-limiting Examples.
  • Example 1 Development of humanised antibodies against NPY/PYY with improved properties
  • NPY is formed from a precursor that is enzymatically cleaved and has an amidated C-terminal amino acid. Therefore, the peptide synthesized for these studies comprised the sequence: N terminus- YYSALRHYINLITRDRY-C term-carrier (SEQ ID NO: 3) with an NH group on its C- terminus.
  • This peptide designated “NPY20-36NH2” (or “HBY” or “immunizing peptide”) was synthesized using standard techniques at Mimotopes (Melbourne, Australia). It was conjugated to KLH via gluteraldehyde. The peptide was freeze-dried, then resuspended in sterile water at 448 pM (1 mg/mL) prior to immunization.
  • mice were immunized with the KLH conjugated HBY peptide using the regime summarized in Table 3.
  • Peptide was prepared for immunization by diluting the HBY-KLH peptide to either 500 pg/mL (prime) or 250 pg/mL (boost) in sterile PBS and either 50% V/v Imject Freund's Complete Adjuvant (FCA) or 50% V/v Imject Freund's Incomplete Adjuvant (FIA) — (Thermo Scientific). Mice were immunized intra-peritoneally. For the final boost, HBY-KLH peptide was diluted in sterile PBS only. The peptide preparation was injected i.v. into mice via one of the tail veins.
  • FCA V/v Imject Freund's Complete Adjuvant
  • FIA V/v Imject Freund's Incomplete Adjuvant
  • ELISA was used to select hybridomas secreting antibody that bound to the HBY peptide.
  • Any hybridomas secreting antibodies that bound to HBY peptide were expanded. Expanded wells with ongoing ELISA reactivity to HBY peptide were selected for subcloning. Cells were then diluted and subcloned and again screened for secretion of antibodies reactive with HBY peptide.
  • hybridoma 5E12 having immunoreactivity to the immunizing peptide was selected.
  • a sub clone B7 of hybridoma 5E12 i.e., 5E12-B7 was also selected.
  • Ig2a/2c Ig2a/2c
  • a hybridoma cell line was also submitted to Syd Labs (USA) for sequencing of the VH and VL, regions.
  • Humanized 5E12 VH genes were designed by grafting the 5E12-B7 VH CDRS onto the VH sequences of human germlines IGHV1-46 and IGHV1-69.
  • a humanized 5E12 VL gene was designed by grafting the 5E12-B7 VL CDRS onto the VL sequence of human germlines DPK9.
  • the humanized 5E12-B7 scFv genes were cloned into pHENI vector, and transformed TGI was grown on selective media (TYE, 100 ug/mL ampicillin, 4% glucose).
  • ELISA plates were blocked with 5% skim milk/PB ST then incubated with phage culture supernatant which had also been blocked with 5% skim milk/1% Tween (final con centration). Binding of schv-phage to wells was detected with Peroxidase conjugated anti-M13 secondary antibody.
  • Affinity maturation libraries were generated by kunkel mutagenesis using humanised 5E12 scFv variants h5E12 IGHV1-46/DPK9 and h5E12 IGHV1-69/DPK9 as templates (SEQ ID NOs: 4, 5 and 6, respectively).
  • Humanised 5E12 scFv genes were cloned into phage display vector pHENl, which were then used to transform E. coli strain CJ236.
  • pHENl vectors were rescued from transformations using KM 13 helper phage in the presence of 0.25 .g/mL uridine (Sigma). Phage were purified by PEG precipitation and ssDNA pHENl vector was isolated fromphage using QIAprep Spin M13 Kit (Qiagen) according to the manufacturer’s instructions.
  • Oligonucleotides containing degenerate codons were designed with the ability to anneal to four of the CDR-encoding regions (i.e., Hl, H2, L2 and L3) within the h5E12 scFv templates in ssDNA samples of pHENl vectors. Oligonucleotides were ordered with a 5’ phosphorylated modification from Integrated DNA Technologies (IDT) or 5’ phosphorylated samples were generated by incubating oligos with T4 PNK enzyme.
  • IDTT Integrated DNA Technologies
  • Oligonucleotides were combined with ssDNA pHENl template in a 3:1 molar ratio in TM buffer (500 mM Tris, 100 mM MgCE, pH 7.5), and allowed to anneal by heating to 90°C for 2 min, 50°C for 3 min then 20°C for 5 min.
  • TM buffer 500 mM Tris, 100 mM MgCE, pH 7.5
  • Covalently closed circular DNA was generated from annealed samples by adding the following to the reaction: 5 pl of 10 mM ATP, 5 pl of 25 mM dNTPs, 7.5 pl of 100 mM DTT, 7.5 pl of T4 DNA ligase (6000 NEB units), and 1.5 pl of T7 DNA polymerase (15 units) and incubating at room temperature (23 °C) for at least 3 hr.
  • Kunkel reactions were purified and desalted using a QIAquick PCR purification kit (Qiagen) with elution into MQ water (pH 7-8), then used to transform electrocompetent E. coli strain TGI using an electroporator (e.g. Bio-Rad Gene Pulser). Transformed bacteria were selected by growing overnight on TYE agar plates containing Ampicillin and glucose. Lawns from agar plates were resuspended in 2YT media and stored as glycerol stocks at -80°C or phage display libraries were produced directly through overnight incubation after rescue with KM13 helper phage, followed by purification of phage using PEG precipitation. This resulted in libraries with a theoretical diversity of over 5 x 10 6 variants (Figure 2)
  • phage maturation libraries Multiple rounds of phage selection were carried out on the maturation libraries. Briefly, purified phage maturation libraries were blocked for Jackpot in PBS containing 4% w/v skim milk, 1% v/v Tween with constant shaking, then spiked with N-terminally biotinylated human PYY (hPYY, SEQ ID NO: 2) generated at Mimotopes Australia) and incubated with shaking for an additional hour. Phage/peptide suspensions were then incubated for 15min with streptavidin- conjugated magnetic dynabeads (Thermo Fisher). Non-binding phage were removed by repeated washing steps phosphate buffered saline containing 0.05% Tween-20 (PBS-T).
  • PBS-T phosphate buffered saline containing 0.05% Tween-20
  • Binding phage were collected from the beads by incubation O.lmg/mL trypsin, then used to infect log-phase TGI cultures in 2YT media. Phage- infected TGI were grown as lawns overnight on TYE plates containing 4% w/v glucose and lOOug/mL ampicillin (TYE/Glu/Amp), and titres were determined on separate plates using serial dilutions. Lawns were used to generate phage for subsequent rounds of selection to enrich higher affinity clones. Titration plates from selection rounds 3 onwards were used as a source for picking colonies, which were subsequently cultured and induced with IPTG to express soluble scFv in 800 pL 96- well culture plates.
  • CDR grafting from mouse 5E12 scFv PSE2 was performed onto a total of 6 human VH domains and 4 human VL domains, where human sequences were selected based on level of sequence homology with mouse scFv PSE2 (SEQ ID NOs: 11-20, Figure 5).
  • An scFv library containing all possible combinations of humanised 5E12 VH and VL (24 possible scFvs) was then generated by SOE PCR reaction. The PCR products were used to transform electrocompetent E. coli TGI, which were grown overnight on TYE plates containing glucose and ampicillin. Over 100 individual clones were picked from the library and induced with IPTG to express soluble scFv into culture supernatant.
  • streptavidin biosensors (ForteBio) were loaded off-line in 200 pL wells containing 20 pg/mL biotinylated human PYY (SEQ ID NO: 2) in PBS for 5 min. Biosensors were then transferred to other wells containing 200 pL scFv-free TGI dummy cultures. Sensors were then transferred to a BLItz instrument (Fortebio, USA), and run on a basic protocol using the BLItz Pro 1.2 software.
  • BLItz screening of the individual clones selected following maturation in Section 1.8 identified several clones with apparent improvements in binding off-rate, including clones TA- F6, TA-A7, TA-D5 (originating from the h5E12 IGHV1-69/DPK9 kunkel library) and SA3- 1B5 (h5E12 IGHV1-46/DPK9 kunkel library). Clone TA-F6 showing the most dramatic apparent improvement, followed by clone TA-A7 ( Figure 3, Table 4).
  • Clones with evidence of improved soluble scFv off-rate by BLItz were sequenced by using PCR with pHENl primers LMB3+ and G3seq6 to amplify the scFv genes from glycerol stocks.
  • PCR products were purified using QIAquick purification kit (QIAGEN) and sent to AGRF for Sanger sequencing, or were sent as unpurified samples in 96-well plates to Beckman Coulter sequencing services (USA).
  • samples of purified scFv were biotinylated using EZ-link PEG4 Biotinylation reagent (Thermo Fisher) according to manufacturer’s instructions. This allowed for “flipped format” BLI measurements of affinity, where scFv was fixed to the streptavidin biosensors and binding kinetics of soluble hPYY or NPY were measured.
  • SEQ ID NO: 40 CDR-L3 sequences of QHFMYPPFT
  • QHFMYPPFT SEQ ID NO: 41
  • Figure 4 Of the clones matured in Section 1.9 by SOE PCR which had an improved off-rate, sequencing indicated that both clones 1A5 and 1D8 (SEQ ID NOs: 21 and 22 respectively) retained the IGHV1-69 VH framework, but 1A5 was using the KV103 VL framework with 18 initial residues from DPK9: most likely from the SOE PCR reaction (Figure 4). Clone 1D8 was contained the entire KV103 VL framework. Three additional clones with improved BLItz off- rates were also shown to have
  • variant 4A a combination of clone TA-A7 CDR-H1 mutation N31D, TA-A7 CDR-L2 mutated sequence NAEDLSD (SEQ ID NO: 39), and TA-D5 mutated CDR-L3 sequence QHFMYPPFT (SEQ ID NO: 40) had the most dramatic improvement in binding off-rate to B-hPYY ( Figure 8, Table 6).
  • the combined-mutant humanised 5E12 scFv variant 4A (SEQ ID NO: 26) was then selected as a template for generating a second affinity maturation library by kunkel mutagenesis.
  • the second affinity maturation library was prepared using the method described in Section 1.7, with the exception that a different suite of oligonucleotides was used to introduce a higher level of diversity into CDR’s Hl, H3, LI, L2 and L3 than in the initial library ( Figure 9).
  • Phage selection against B-hPYY and BLItz off-rate screening following the methodologies described in Sections 1.8 and 1.10 for the first humanised affinity maturation library identified clone 4A3B2 with dramatically improved off-rate compared to controls ( Figure 10).
  • Fab constructs were designed for 5E12 scFv variants PSE2 (SEQ ID NO: 4), h5E12 IGHV1-69/DPK9 (SEQ ID NO: 6) and the most affinity matured variant 4A3B2 (SEQ ID NO: 31).
  • the Fab constructs were synthesized (Genscript) and cloned into mammalian expression vectors (pCDNA3 or pCEP4).
  • Fab heavy and light chain genes were co-transfected at a 1:1 ratio into HEK-293F or Expi-293 (Thermo Fisher) expression systems according to manufacturer’s instructions.
  • Fab was then purified from culture supernatants via a C-terminal His tag on the Fab heavy chain using TALON affinity purification resin (Takara Bio) according to manufacturer’s instructions, and expressed in the Expi-293 mammalian expression system.
  • o PSE2 Fab heavy chain SEQ ID NO: 32
  • o PSE2 Fab kappa light chain SEQ ID NO: 33
  • o h5E12 IGHV1-69/DPK9 Fab heavy chain SEQ ID NO: 34
  • o h5E12 IGHV1-69/DPK9 Fab kappa light chain SEQ ID NO: 35
  • o 4A3B2 Fab heavy chain SEQ ID NO: 48
  • 4A3B2 Fab kappa light chain SEQ ID NO: 49.
  • Fab samples of PSE2 (SEQ ID NOs: 32 and 33), h5E12 IGHV1-69/DPK9 (SEQ ID NOs: 34 and 35) and clone 4A3B2 (SEQ ID NOs: 48 and 49) were biotinylated 5:1 using EZ-link PEG4 biotinylation reagent (Thermo Fisher) and loaded onto BiOptix streptavidin (SAD) sensorchips. The sensorchips were then loaded into the 404pi SPR instrument and preconditioned using the manufacturer’s protocol. Approximately 2000 resonance units (RU) of biotinylated Fab variants were then bound as ligand to two experimental channels, leaving two reference channels with no Fab loaded.
  • EZ-link PEG4 biotinylation reagent Thermo Fisher
  • SAD BiOptix streptavidin
  • Crystallography was attempted with several Fab format variants of h5E12 in complex with either NPY or hPYY.
  • crystals were formed for hPYY (SEQ ID NO: 2) in complex with a Fab containing h5E12 DP47 Fab heavy chain (SEQ ID NO: 38) and h5E12 4A3B2 light chain (SEQ ID NO: 36).
  • crystals were formed of NPY (SEQ ID NO: 1) in complex with a Fab containing h5E12 IGHV1-69 Fab heavy chain (SEQ ID NO: 34) and 4A3B2 light chain (SEQ ID NO: 49). Structures were solved for these two complexes using X-ray crystallography ( Figures 14 and 15).

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Abstract

La présente invention concerne des réactifs et des procédés de traitement du cancer et de la perte de poids. En particulier, la présente invention concerne des protéines de liaison, telles que des anticorps, qui se lient au neuropeptide Y (NPY) et au peptide YY (PYY) et inhibent ainsi la signalisation NPY et PYY, ainsi que l'utilisation de ces protéines de liaison pour le traitement du cancer et de la perte de poids.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005077094A2 (fr) * 2004-02-11 2005-08-25 Amylin Pharmaceuticals, Inc. Motifs de la famille de polypeptides pancreatiques et polypeptides les renfermant
WO2005118786A2 (fr) * 2004-06-03 2005-12-15 Compugen Ltd. Variants d'epissure de peptide yy,de neuropeptide y, de peptide pancreatique y et d'amyline et leurs utilisation
WO2013078511A1 (fr) * 2011-12-02 2013-06-06 Garvan Institute Of Medical Research Anticorps anti-npy et anti-pyy et leurs utilisations
US20180117170A1 (en) * 2016-10-27 2018-05-03 Janssen Pharmaceutica Nv Antibody-coupled cyclic peptide tyrosine tyrosine compounds as modulators of neuropeptide y receptors

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005077094A2 (fr) * 2004-02-11 2005-08-25 Amylin Pharmaceuticals, Inc. Motifs de la famille de polypeptides pancreatiques et polypeptides les renfermant
WO2005118786A2 (fr) * 2004-06-03 2005-12-15 Compugen Ltd. Variants d'epissure de peptide yy,de neuropeptide y, de peptide pancreatique y et d'amyline et leurs utilisation
WO2013078511A1 (fr) * 2011-12-02 2013-06-06 Garvan Institute Of Medical Research Anticorps anti-npy et anti-pyy et leurs utilisations
US20180117170A1 (en) * 2016-10-27 2018-05-03 Janssen Pharmaceutica Nv Antibody-coupled cyclic peptide tyrosine tyrosine compounds as modulators of neuropeptide y receptors

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LANGLEY DAVID B.; SCHOFIELD PETER; JACKSON JENNY; HERZOG HERBERT; CHRIST DANIEL: "Crystal structures of human neuropeptide Y (NPY) and peptide YY (PYY)", NEUROPEPTIDES, vol. 92, 7 February 2022 (2022-02-07), GB , pages 1 - 7, XP086991223, ISSN: 0143-4179, DOI: 10.1016/j.npep.2022.102231 *
MAKARENKO IRINA G, MICHAEL M. MEGUID , LOUIS GATTO , CHUNG CHEN , MICHAEL V. UGRUMOV : "Decreased NPY innervation of the hypothalamic nuclei in rats with cancer anorexia", BRAIN RESEARCH, vol. 961, no. 1, 24 January 2003 (2003-01-24), pages 100 - 108, XP093071249, DOI: 10.1016/s0006-8993(02)03850-7 *
ROKOT NATASYA TRIVENA, ATAKA KOJI, IWAI HARUKI, SUZUKI HAJIME, TACHIBE HOMARE, KAIRUPAN TIMOTHY SEAN, CHENG KAI-CHUN, AMITANI HARU: "Antagonism for NPY signaling reverses cognitive behavior defects induced by activity-based anorexia in mice", PSYCHONEUROENDOCRINOLOGY, vol. 126, 1 April 2021 (2021-04-01), GB , pages 1 - 8, XP093071246, ISSN: 0306-4530, DOI: 10.1016/j.psyneuen.2021.105133 *

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