WO2012136790A9 - Compositions comprenant des protéines de fusion ou des conjugués ayant une demi-vie améliorée - Google Patents

Compositions comprenant des protéines de fusion ou des conjugués ayant une demi-vie améliorée Download PDF

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WO2012136790A9
WO2012136790A9 PCT/EP2012/056327 EP2012056327W WO2012136790A9 WO 2012136790 A9 WO2012136790 A9 WO 2012136790A9 EP 2012056327 W EP2012056327 W EP 2012056327W WO 2012136790 A9 WO2012136790 A9 WO 2012136790A9
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composition according
peptide
albudab
seq
dab
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WO2012136790A1 (fr
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Bruce J HAMILTON
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Glaxo Group Limited
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6843—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/57563—Vasoactive intestinal peptide [VIP]; Related peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the present invention relates to drug fusions and conjugates that have improved serum half lives. These fusions and conjugates comprise immunoglobulin (antibody) single variable domains and insulintropic and/or incretin and/or gut peptide molecules.
  • the invention further relates to uses, formulations, compositions and devices comprising such drug fusions and conjugates.
  • the invention also relates to compositions which comprise more than one insulintropic and/or incretin and/or gut peptide molecules present as part of a fusion or conjugate and to uses and formulations thereof.
  • incretin hormones such as Glucagon-like peptide 1
  • exendin for example exendin-4
  • other gut peptides such as PYY.
  • Glucagon-like peptide (GLP)-l is an incretin hormone with potent glucose-dependent insulinotropic and glucagonostatic actions, trophic effects on the pancreatic ⁇ cells, and inhibitory effects on gastrointestinal secretion and motility, which combine to lower plasma glucose and reduce glycemic excursions. Furthermore, via its ability to enhance satiety, GLP-1 reduces food intake, thereby limiting weight gain, and may even cause weight loss (Drucker (2002) Gastroenterology 122:531-544, Giorgiano et al. (2006) Diabetes Research and Clinical Practice 74: SI 52- 155), Holt (2002) Diabetes/Metabolism Research and Reviews 18:430-441. Taken together, these actions give GLP-1 a unique profile, considered highly desirable for an antidiabetic agent, particularly since the glucose dependency of its
  • GLP-1 is highly susceptible to enzymatic degradation in vivo, and cleavage by dipeptidyl peptidase IV (DPP-IV) is probably the most relevant, since this occurs rapidly and generates a noninsulinotropic metabolite (Metlein (1999) Regulatory Peptides 85:9-244).
  • DPP-IV dipeptidyl peptidase IV
  • 02/46227 discloses heterologous fusion proteins comprising a polypeptide (for example, albumin) fused to GLP-1 or analogues (the disclosure of these analogues is incorporated herein by reference as examples of GLP-1 analogues that can be used in the present invention).
  • a polypeptide for example, albumin
  • WO05/003296, WO03/060071, WO03/059934 disclose amino fusion protein wherein GLP-1 has fused with albumin to attempt to increase the half-life of the hormone.
  • Peptide YY is a short (36 amino acid) protein released by neuroendocrine cells in response to feeding. PYY concentration in the circulation increases postprandially and decreases on fasting. It exerts its action through NPY receptors, inhibiting gastric motility and increasing water and electrolyte absorption in the colon. It is secreted by the neuroendocrine cells in the ileum and colon in response to a meal, and has been shown to reduce appetite Ballantyne (2006) Obesity Surgery 16:651-658, Batterham (2003) New England Journal of Medicine 349:941-8, Boey et al. (2007) Peptides 28:390-395, and Karra et al. (2009) Journal of Physiology 587:19-25).
  • Exendin-4 is a hormone found in the saliva of the Gila monster it is an agonist of GLP- 1 and also has a very potent insulinotropic effects. In contrast to GLP-1, exendin-4 has a much longer in vivo half-life
  • GLP-1 human glucagon-like peptide- 1
  • Exendin-4 enhances glucose- dependent insulin secretion by the pancreatic beta-cell, suppresses inappropriately elevated glucagon secretion, and slows gastric emptying.
  • NMU peptide refers to the Neuromedin U peptide and this is a neuropeptide which is an insulinotropic peptide is found in the gut and brain of humans abd other mammals and has a variety of functions including effect on muscle contraction, blood pressure and on appetite (see for example Pharmacological Reviews June 2004, Vol 56, No 2, pp 231-248).
  • compositions comprising incretins and/or insulinotropic and/or gut peptide agents such as NMU, GLP-1 peptides, PYY, exendin, or other agents that have an insulinotropic and/or incretin effect /or anorexic effect and which can be used in medicine e.g. in the treatment and/or prevention of metabolic conditions such as diabetes and obesity.
  • incretins and/or insulinotropic and/or gut peptide agents such as NMU, GLP-1 peptides, PYY, exendin, or other agents that have an insulinotropic and/or incretin effect /or anorexic effect and which can be used in medicine e.g. in the treatment and/or prevention of metabolic conditions such as diabetes and obesity.
  • incretins/insulinotropic/gut peptide containing agents e.g. NMU, GLP-1, exendin -4, PYY
  • agents e.g. NMU, GLP-1, exendin -4, PYY
  • compositions which comprise (or consist of) a single molecule (e.g. a single fusion or conjugate) which comprises combinations of (i.e. two or more) molecules selected from incretins and/or insulinotropic agents and/or gut peptides, which are e.g. present as fusions (chemical or genetic) or as a conjugates; or alternatively (b) a composition which comprises two or more individual molecules wherein each individual molecule comprises one or more incretins and/or insulinotropic agents and/or gut peptides.
  • These compositions (a) and/or (b) can also comprise further proteins or polypeptides e.g. half life extending proteins or polypeptides or peptides e.g. which can bind to serum albumin for example human serum albumin e.g. a dAb e.g. a dAb which binds to serum albumin such as human serum albumin.
  • the present invention provides a composition which comprises (or consists of) a single fusion (chemical or genetic) or a single conjugate molecule, wherein said fusion or conjugate comprises or consists of (a) two or more molecules which are selected from: insulinotropic and/or incretin molecules and/or gut peptides, (e.g. Neuromedin U (NMU) peptide such as a NMU-8 peptide, a Peptide YY (PYY) peptide such as PYY 3-36 or PYY 13-36, exendin-4, a GLP e.g. a GLP-1 e.g.
  • NMU Neuromedin U
  • PYY Peptide YY
  • GLP GLP-1
  • the GLP-1 (7-37) A8G mutant which are present as a single fusion or conjugate with (b) a domain antibody (dAb) which binds specifically to serum albumin, (e.g. the DOM 7h-14 (Vk) domain antibody (dAb), (the amino acid sequence of DOM 7h-14 is shown in Figure 1(h): SEQ ID NO 8), or e.g. the DOM 7h-14 -lO(Vk) domain antibody (dAb), (the amino acid sequence of DOM 7h-14-10 is shown in Figure l(o): SEQ ID NO 15 , or e.g.
  • the DOM 7h-14 -lO(Vk) domain antibody (dAb) which has the R108C mutation
  • the amino acid sequence of DOM 7h-14-10 R108C is shown in Figure l(r) SEQ ID NO 18 or the DOM 7h-l 1
  • the amino acid sequence of DOM 7h-l 1 is shown in Figure 1 (x) or DOM 7H-1 1-15
  • the amino acid sequence of DOM 7h-l 1-15 is shown in Figure 1 (t).
  • the fusion or conjugate is not the 2xGLP-l (7-37) A8G DOM7h-14 dAb fusion (DAT0114, with the amino acid sequence is shown in Figure 1 (a): SEQ ID NO 1 ).
  • the incretin/insulinotropic/gut peptide molecules can be different incretin/insulinotropic/gut peptide molecules.
  • the dAb that binds serum albumin i.e. the albudabTM
  • composition e.g. the single fusion or conjugate
  • the present invention further provides a composition, which comprises (or consists of) two or more individual fusions or conjugates and wherein each individual fusion or conjugate comprises or consists of (a) one or more molecules selected from:
  • insulinotropic and/or incretin molecules and/or gut peptides e.g. a NMU peptide such as a NMU-8 peptide , PYY peptide, exendin-4, a GLP e.g. a GLP-1 e.g. the GLP-1 (7-37) A8G mutant
  • a domain antibody (dAb) which binds specifically to serum albumin (e.g. the DOM 7h-14 (Vk) domain antibody (dAb), (the amino acid sequence of DOM 7h-14 is shown in Figure 1(h): SEQ ID NO 8) or or e.g.
  • the DOM 7h- 14 -lO(Vk) domain antibody (dAb) (the amino acid sequence of DOM 7h-14-10 is shown in Figure l(o): SEQ ID NO 15 , or e.g. the DOM 7h-14 -lO(Vk) domain antibody (dAb) which has the R108C mutation (the amino acid sequence of DOM 7h-14-10 R108 C is shown in Figure l(r) SEQ ID NO 18) or the DOM 7h-l 1, the amino acid sequence of DOM 7h-l 1 is shown in Figure 1 (x), or DOM 7H-11-15, the amino acid sequence of DOM 7h- 11-15 is shown in Figure 1 (t).
  • dAb the amino acid sequence of DOM 7h-14-10 is shown in Figure l(o): SEQ ID NO 15 , or e.g. the DOM 7h-14 -lO(Vk) domain antibody (dAb) which has the R108C mutation (the amino acid sequence of DOM 7h-14-10 R108 C is shown in
  • composition which comprises (or consists of) two or more individual fusions or conjugates comprises one or more NMU peptides e.g. one or more NMU-8 peptides e.g. the NMU-8 peptide with the sequence shown in Figure 1 (w).
  • this composition can comprise one or more molecules selected from those in: Figures la-lg and Figures lm-ln, and also figure 3a-3b.
  • compositions comprising (or consisting of) two or more fusions or conjugates as described above can be a combined preparation for simultaneous, separate or sequential use in therapy, e.g to treat or prevent a metabolic disease such as hyperglycemia , impaired glucose tolerance, beta cell deficiency, diabetes (for example type 1 or type 2 diabetes or gestational diabetes) non-alcoholic steatotic liver disease, polycstic ovarian syndrome, hyperlipidemia or obesity or diseases characterised by overeating and/or modify energy expenditure.
  • the fusions or conjugates of the invention can display synergy (by synergy we mean that their effect when administered is more than the simple additive effect of each when administered singly) when administered together or sequentially e.g.
  • a metabolic disease such as hyperglycemia , impaired glucose tolerance, beta cell deficiency, diabetes (for example type 1 or type 2 diabetes or gestational diabetes) non-alcoholic steatotic live deisease, polycstic ovarian syndrome, hyperlipidemia or obesity or diseases characterised by overeating and/or modify energy expenditure,
  • a metabolic disease such as hyperglycemia , impaired glucose tolerance, beta cell deficiency, diabetes (for example type 1 or type 2 diabetes or gestational diabetes) non-alcoholic steatotic live deisease, polycstic ovarian syndrome, hyperlipidemia or obesity or diseases characterised by overeating and/or modify energy expenditure,
  • compositions according to the invention can be for example selected from:
  • the GLP, PYY, exendin or NMU peptides can be any of those described in WO 2006/059106.
  • the mutants, analogues or derivatives of these peptides can be those which retain incretin and/or insulinotropic activity.
  • the insulinotropic and/or incretin and/or gut peptide molecules when present as a fusion (or conjugate) with a dAb can be linked to either the N- terminal or C-terminal of the dAb or at points within the dAb sequence.
  • one or more incretin and/or insulinotropic and/or gut peptide molecules are present as a fusion (or conjugate) with the N terminal of the dAb and one or more incretin and/or insulinotropic and/or gut peptide molecules are also present as a fusion (or conjugate) with the C terminal of the dAb.
  • An amino acid or chemical linker may also optionally be present joining the insulinotropic and/or incretin and/or gut peptide molecules, e.g. exendin-4 and/or GLP-1, e.g. with the dAb.
  • the linker can be for example a helical linker e.g. the helical linker of sequence shown in Figure 1 (k): SEQ ID NO 11, or it may be a gly-ser linker e.g. with an amino acid sequence shown in Figure 1 (1): SEQ ID NO 12.
  • linker can be e.g. a PEG linker e.g. the PEG linker shown in Figure
  • the fusions (or conjugates) of the invention can comprise further molecules e.g. further peptides or polypeptides.
  • the invention provides a composition which comprises or consists of the following two individual molecules:
  • the line represents the linker which is covalently attached to the free C terminal cysteine of the Dom7h-14-10 (R108C) AlbudAb and the lysine at position 10 of the PYY sequence.
  • the amino acid sequence and structure of this peptide conjugate is as follows (and is also shown in Figure 3 a) :
  • the chemical linker has the following structure:
  • Dom7h-14-10 (R108C) albudab conjugated to PYY3-36 via a lysine and 4 repeat PEG linker (of structure shown in figure 3) can be present as a combined preparation for simultaneous, separate or sequential suitable for uses in therapy as described herein.
  • the invention provides a composition which comprises or consists of the following two individual molecules: (a) Exendin -4 AlbudAb (DAT 01 15) and
  • a Dom7h-14-10 (R108C) Albudab conjugated to a NMU-8 linear peptide via a reactive maleimide group linked covalently via a 4 repeat PEG linker The chemical structure below represents the4 repeat PEG linker which is covalently attached to the free C terminal cysteine of the Dom7h-14-10 (R108C) AlbudAb and the first residue of the NMU-8 (here: Tyrosine).
  • the amino acid sequence and structure of this peptide conjugate is as follows (and is also shown in Figure 3b):
  • the chemical linker has the following structure including the attachment sites for the dAb and the peptide:
  • DAT 0115 DAT 0115
  • NMU-8 albudab NMU-8 albudab above (and which is also shown in Figure 3b) can be present as a combined preparation for simultaneous, separate or sequential suitable for uses in therapy as described herein.
  • the invention provides a composition which comprises or consists of the following two individual molecules:
  • a Dom7h-14-10 (R108C) Albudab conjugated to a NMU-8 linear peptide via a reactive maleimide group linked covalently via a 4 repeat PEG linker The chemical structure below represents the 4 repeat PEG linker which is covalently attached to the free C terminal cysteine of the Dom7h-14-10 (R108C) AlbudAb and the first residue of the NMU-8 (here: Tyrosine).
  • the amino acid sequence and structure of this peptide conjugate is as follows (and is also shown in Figure 3b):
  • the PEG chemical linker in the above has the following structure including the attachment sites for the dAb and the peptide:
  • DAT 0120 and NMU-8 albudab above can be present as a combined preparation for simultaneous, separate or sequential suitable for uses in therapy as described herein.
  • NMU AlbudAb peptide conjugate which is:
  • a peptide conjugate which is:
  • a Dom7h-14-10 (R108C) Albudab conjugated to a NMU-8 linear peptide via a reactive maleimide group linked covalently via a 4 repeat PEG linker The chemical structure below represents the4 repeat PEG linker which is covalently attached to the free C terminal cysteine of the Dom7h-14-10 (R108C) AlbudAb and the first residue of the NMU-8 (here: Tyrosine).
  • the amino acid sequence and structure of this peptide conjugate is as follows (and is also shown in Figure 3 b):
  • the PEG chemical linker in the above has the following structure including the attachment sites for the dAb and the peptide: Cysio 8 -DOM7h-14-10
  • Dom 7h-14 is a human immunoglobulin single variable domain or dAb (Vk) that binds to serum albumin and its amino acid sequence is shown in Figure 1(h): SEQ ID NO 8.
  • Vk human immunoglobulin single variable domain or dAb
  • the CDR regions of Dom7h-14 dAb are underlined in the amino acid sequence shown in Figure 1(h): SEQ ID NO 8.
  • Dom 7h-14-10 is a human immunoglobulin single variable domain or dAb (Vk) that binds to serum albumin and its amino acid sequence is shown in Figure 1(h): SEQ ID NO 8.
  • the CDR regions of Dom7h-14-10 dAb are underlined in the amino acid sequence shown in Figure l(o): SEQ ID NO 15.
  • Dom 7h-14-10 with a R108C mutation is a human immunoglobulin single variable domain or dAb (Vk) that binds to serum albumin and its amino acid sequence is shown in Figure 1(R): SEQ ID NO 18.
  • the invention also provides a molecule which comprises or consists of the sequence of the molecule shown in Figure 16 a and/or Figure 16b.
  • compositions of the invention can also comprise an NMU-albudAb which comprises or consists of the sequence of the molecule shown in Figure 16 a and/or Figure 16b.
  • fusion refers to a fusion protein that comprises as one moiety a dAb that binds serum albumin and further moieties which are insulinotropic and/or incretin and/or gut peptide molecules.
  • the dAb that binds serum albumin and the insulinotropic and/or an incretin and/or gut peptide molecules can be present as discrete parts (moieties) of a single continuous polypeptide chain.
  • the dAb and incretin / insulinotropic/gut peptide moieties can be directly bonded to each other through a peptide bond or linked through a suitable amino acid, or peptide or polypeptide linker. Additional moieties e.g. peptides or polypeptides (e.g. third, fourth) and/or linker sequences, can be present as appropriate.
  • the dAb can be in an N- terminal location, C-terminal location or it can be internal, relative to the incretin / insulinotropic/gut peptide molecules.
  • the fusion protein contains one or more than one (e.g. one to about 20) dAb moieties.
  • conjugate refers to a composition comprising a dAb that binds serum albumin to which an insulinotropic / incretin/gut peptide molecule is covalently or noncovalently bonded.
  • the insulinotropic / incretin/gut peptide molecule can be covalently bonded to the dAb directly or indirectly through a suitable linker moiety.
  • the insulinotropic / incretin/gut peptide molecule can be bonded to the dAb at any suitable position, such as the amino-terminus, the carboxyl-terminus or through suitable amino acid side chains (e.g., the ⁇ amino group of lysine, or thiol group of cysteine) either naturally occurring or engineered.
  • the insulinotropic / incretin/gut peptide molecule can be noncovalently bonded to the dAb directly (e.g., electrostatic interaction, hydrophobic interaction) or indirectly (e.g., through noncovalent binding of complementary binding partners (e.g., biotin and avidin), wherein one partner is covalently bonded to insulinotropic / incretin molecule and the complementary binding partner is covalently bonded to the dAb).
  • the dAb can be in an N- terminal location, C-terminal location or it can be internal relative to the incretin /
  • the conjugate protein contains one or more than one (e.g. one to about 20) dAb moieties.
  • the invention also provides compositions comprising nucleic acids encoding the fusions described herein for example comprising nucleic acids shown in Figure 2.
  • host cells e.g. non-embryonic host cells e.g. prokaryotic or eukaryotic (such as mammalian) hosts cells such as E. coli or or yeast host cells that comprise these nucleic acids.
  • the invention further provides a method for producing a fusion of the present invention which method comprises maintaining a host cell such as those described above that comprises a recombinant nucleic acid and/or construct that encodes a fusion of the invention under conditions suitable for expression of said recombinant nucleic acid, whereby a fusion is produced.
  • the invention also provides pharmaceutical compositions comprising the
  • compositions of the invention are provided.
  • the invention further provides a composition of the invention for use in medicine , e.g. for use in the treatment of e.g. a metabolic disease or condition such as hyperglycemia , impaired glucose tolerance, beta cell deficiency, diabetes (for example type 1 or type 2 diabetes or gestational diabetes) non-alcoholic steatotic liver deisease, polycstic ovarian syndrome, hyperlipidemia or obesity or diseases characterised by overeating e.g. it can be used to suppress appetite or modify energy expenditure, and which comprises administering to said individual a therapeutically effective amount of a composition of the invention.
  • a metabolic disease or condition such as hyperglycemia , impaired glucose tolerance, beta cell deficiency, diabetes (for example type 1 or type 2 diabetes or gestational diabetes) non-alcoholic steatotic liver deisease, polycstic ovarian syndrome, hyperlipidemia or obesity or diseases characterised by overeating e.g. it can be used to suppress appetite or modify energy expenditure, and which comprises administering to said individual a therapeutically
  • the invention also provides a method for treating an individual having a disease or disorder, such as those described herein e.g. a metabolic disease or condition such as hyperglycemia , impaired glucose tolerance, beta cell deficiency, diabetes (for example type 1 or type 2 diabetes or gestational diabetes) ), non-alcoholic steatotic liver disease, polycstic ovarian syndrome, hyperlipidemia, or obesity or diseases characterised by overeating e.g. it can be used to suppress appetite appetite or modify energy expenditure, and which comprises administering to said individual a therapeutically effective amount of a composition of the invention.
  • a disease or disorder such as those described herein e.g. a metabolic disease or condition such as hyperglycemia , impaired glucose tolerance, beta cell deficiency, diabetes (for example type 1 or type 2 diabetes or gestational diabetes) ), non-alcoholic steatotic liver disease, polycstic ovarian syndrome, hyperlipidemia, or obesity or diseases characterised by overeating e.g. it can be used to suppress appetite appetite
  • metabolic dieases or conditions include, but are not limited to, insulin resistance, insulin deficiency, hyperinsulinemia, hyperglycemia, dyslipidemia, hyperlipidemia, hyperketonemia, hyperglucagonemia,hypertension, coronary artery disease, atherosclerosis, renal failure, neuropathy (e.g., autonomic neuropathy, parasympathetic neuropathy, and polyneuropathy), retinopathy, cataracts, metabolic disorders (e.g., insulin and/or glucose metabolic disorders), endocrine disorders, obesity, weight loss, liver disorders (e.g., liver disease, steatosis of the liver, cirrhosis of the liver, and disorders associated with liver transplant), and conditions associated with these diseases or disorders.
  • metabolic disorders e.g., insulin and/or glucose metabolic disorders
  • endocrine disorders e.g., obesity disease, steatosis of the liver, cirrhosis of the liver, and disorders associated with liver transplant
  • liver disorders e.g., liver disease, stea
  • conditions associated with diabetes include, but are not limited to, hyperglycemia, obesity, diabetic retinopathy, mononeuropathy, polyneuropathy, atherosclerosis, ulcers, heart disease, stroke, anemia, gangrene (e.g., of the feet and hands), impotence, infection, cataract, poor kidney function, malfunctioning of the autonomic nervous system, impaired white blood cell function, Carpal tunnel syndrome, Dupuytren's contracture, and diabetic ketoacidosis.
  • the invention also provides methods for treating or preventing diseases associated with elevated blood glucose comprising administering at least one dose of a composition e.g. a pharmaceutical composition, of the present invention to a patient or subject.
  • a composition e.g. a pharmaceutical composition
  • patient or subject When patient or subject are described in the application this can mean a human or non- human patient or subject.
  • the invention further relates to methods of regulating insulin responsiveness in a patient, as well as methods of increasing glucose uptake by a cell, and methods of regulating insulin sensitivity of a cell, using the conjugates or fusions of the invention. Also provided are methods of stimulating insulin synthesis and release, enhancing adipose, muscle or liver tissue sensitivity towards insulin uptake, stimulating glucose uptake, slowing digestive process, reducing appetite, modifying energy expenditure,or blocking the secretion of glucagon in a patient, comprising administering to said patient a composition of the invention e.g.
  • compositions comprising administering at least one dose of a composition e.g. a pharmaceutical
  • composition of the present invention.
  • compositions e.g. pharmaceutical compositions, of the invention may be administered alone or in combination with other molecules or moieties e.g. polypeptides, therapeutic proteins (e.g. Albiglutide which is two molecules of GLP-1 covalently linked to a molecule of human serum albumin) and/or molecules (e.g., insulin and/or other proteins (including antibodies), peptides, or small molecules that regulate insulin sensitivity, weight, heart disease, hypertension, neuropathy, cell metabolism, and/or glucose, insulin, or other hormone levels, in a patient).
  • the conjugates or fusions of the invention are administered in combination with insulin (or an insulin derivative, analog, fusion protein, or secretagogue).
  • the invention also provides compositions of the invention for use in the treatment of a disease or disorder, such as any of those mentioned above e.g. a metabolic disorder such as hyperglycemia , pancreatitis, diabetes (type 1 or 2 or gestational diabetes) or obesity or diseases characterized by gut hypermotility.
  • a metabolic disorder such as hyperglycemia , pancreatitis, diabetes (type 1 or 2 or gestational diabetes) or obesity or diseases characterized by gut hypermotility.
  • the invention also provides for use of a composition of the invention in the manufacture of a medicament for treatment of a disease or disorder, such as any of those mentioned above e.g. a metabolic disorder such as hyperglycemia , diabetes (type 1 or 2 or gestational diabetes) or obesity, pancreatitis, or diseases characterized by gut hypermotility.
  • the invention also relates to use of any of the compositions described herein for use in therapy, diagnosis or prophylaxis.
  • compositions of the invention can be further formatted to have a larger hydrodynamic size to further extend the half life, for example, by attachment of a PEG group, serum albumin, transferrin, transferrin receptor or at least the transferrin-binding portion thereof, an antibody Fc region, or by conjugation to an antibody domain.
  • the dAb that binds serum albumin can be formatted as a larger antigen-binding fragment of an antibody (e.g., formatted as a Fab, Fab', F(ab) 2 , F(ab') 2 , IgG, scFv).
  • a domain that comprises the CDRs of a dAb that binds specifically to serum albumin e.g. CDRs of Dom7h-14, or Dom 7h-14-10 or Dom 7h-14-10 R108C, that binds serum albumin
  • the CDRs can be grafted onto a suitable protein scaffold or skeleton, eg an affibody, an SpA scaffold, an LDL receptor class A domain or an EGF domain.
  • the disclosure as a whole is to be construed accordingly to provide disclosure of such domains in place of a dAb.
  • a dAb can be used which competes for binding (e.g. for binding to the same or overlapping epitope) with the dAb sequences described herein and/or the dAb can be one which has 70, 80, 90, 95,96,97, 98,99 % sequence identity or homology with a dAb sequence described herein.
  • the fusion or conjugate of the invention can be one which has 70, 80, 90, 95,96, 97, 98,99 % sequence identity or homologywith a sequence of a fusion or conjugate described herein.
  • the sequence of the incretin or insulinotropic molecule can be one which has 70, 80, 90, 95,96, 97, 98,99 % sequence identity or homologywith a sequence of a such a molecule described herein and which retains its activity as an in
  • the invention provides a composition according to the invention that comprises a dual-specific ligand or multi-specific ligand that comprises a first dAb according to the invention that binds serum albumin e.g. Dom7h-14, and a second dAb that has the same or a different binding specificity from the first dAb and optionally in the case of multi-specific ligands further dAbs.
  • the second dAb (or further dAbs) may optionally bind a different target e.g. FgFr lc, or CD5 target.
  • the dAb component can be any of the dAbs disclosed in WO 2008096158 or WO051 18642 the details of which are incorporated by reference herein or one which has 70, 80, 90, 95,96, 97, 98,99 % sequence identity or homologywith a dAb therein.
  • the invention provides the compositions of the invention for delivery by parenteral administration e.g. by subcutaneous, intramuscular or intravenous injection, inhalation, nasal delivery, transmucosal (e.g. sub-lingual) delivery, transcutaneous, transdermal, oral delivery, delivery to the GI tract of a patient, rectal delivery or ocular delivery.
  • the invention provides the use of the fusions or conjugates of the invention in the manufacture of a medicament for delivery by subcutaneous injection or intramuscular, transdermal delivery, inhalation, intravenous delivery, nasal delivery, transmucossal delivery, oral delivery, delivery to the GI tract of a patient, rectal delivery or ocular delivery.
  • the invention provides a method for delivery to a patient by by subcutaneous, intramuscular or intravenous injection, inhalation, nasal delivery, transmucosal (e.g. sub-lingual) delivery, transcutaneous, transdermal, oral delivery, delivery to the GI tract of a patient, rectal delivery or ocular delivery, wherein the method comprises administering to the patient a pharmaceutically effective amount of a fusion or conjugate of the invention.
  • the invention provides an oral, injectable, inhalable, nebulisable, topical or ocular formulation comprising a fusion or conjugate of the invention.
  • the formulation can be a tablet, pill, capsule, liquid or syrup or ointment.
  • the compositions can be administered orally e.g. as a drink, for example marketed as a weight loss drink for obesity treatment.
  • the invention provides a formulation for rectal delivery to a patient, the fomulation can be provided e.g. as a suppository.
  • a composition for parenteral administration of GLP-1 compounds may, for example, be prepared as described in WO 03/002136 (incorporated herein by reference).
  • a composition for nasal administration of certain peptides may, for example, be prepared as generally described in European Patent No. 272097 (to Novo Nordisk A/S) or in WO 93/18785 (all incorporated herein by reference).
  • subject or “individual” is defined herein to include animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, guinea pigs, rats, mice or other bovine, ovine, equine, canine, feline, rodent or murine species.
  • mammals including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, guinea pigs, rats, mice or other bovine, ovine, equine, canine, feline, rodent or murine species.
  • the invention also provides a kit for use in administering compositions according to the invention to a subject (e.g., human patient), comprising a composition of the invention, a drug delivery device and, optionally, instructions for use.
  • a composition of the invention can be provided as a formulation, such as a freeze dried formulation.
  • the drug delivery device is selected from the group consisting of a syringe, a pen injection device, an inhaler, an intranasal or ocular administration device (e.g., a mister, eye or nose dropper), and a needleless injection device.
  • compositions (e.g conjugates or fusions) of this invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use.
  • Any suitable lyophilization method e.g., spray drying, cake drying
  • reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of antibody activity loss and that use levels may have to be adjusted to compensate.
  • the invention provides a composition comprising a lyophilized (freeze dried) composition as described herein.
  • the lyophilized (freeze dried) composition loses no more than about 20%, or no more than about 25%, or no more than about 30%, or no more than about 35%, or no more than about 40%, or no more than about 45%, or no more than about 50% of its activity (e.g. , binding activity for serum albumin) when rehydrated.
  • Activity is the amount of composition required to produce the effect of the composition before it was lyophilized. For example, the amount of conjugate or fusion needed to achieve and maintain a desired serum concentration for a desired period of time.
  • the activity of the composition can be determined using any suitable method before lyophilization, and the activity can be determined using the same method after rehydration to determine amount of lost activity.
  • the invention also provides sustained release formulations comprising the
  • compositions of the invention can comprise the composition of the invention in combination with, e.g. hyaluronic acid, microspheres or liposomes and other pharmaceutically or pharmacalogically acceptable carriers, excipients and/or diluents.
  • sustained release formulations can in the form of for example suppositories.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a composition of the invention, and a pharmaceutically or physiologically acceptable carrier, excipient or diluent.
  • Figure 1 is an illustration of the amino acid sequences of (a) DAT0114 (SEQ ID NO 1), (b) DAT0115 (SEQ ID NO 2), (c) DAT0116 (SEQ ID NO 3), (d) DAT0117 (SEQ ID NO 4), (e) DAT0118 (SEQ ID NO 5), (f) DAT0119 (SEQ ID NO 6) (g) DAT0120 (SEQ ID NO 7) (h) Dom7h-14 (SEQ ID NO 8) (dAb) ( the CDRs are underlined), (i) GLP-1 7-37 A(8)G (SEQ ID NO 9), (j) exendin-4 (SEQ ID NO 10), (k) Helical linker (SEQ ID NO 11 ) (1) Gly-ser linker (SEQ ID NO 12), (m) Exendin 4, (G4S)3, linker DOM7h-14-10 fusion (DMS7139: SEQ ID NO 13), (n) Exendin 4, (G4S)3, linker DOM7
  • Figure 2 is an illustration of the nucleic acid sequences of: (a) DAT0114 (mammalian construct) (SEQ ID NO 20) , (b) DAT0115 (mammalian construct) (SEQ ID NO 21), (c) DAT0115 (optimized for E.coli construct) (SEQ ID NO 22), (d) DAT0116 (mammalian construct) (SEQ ID NO 23), (e) DAT0116 (optimized for E.coli construct) (SEQ ID NO 24), (f) DAT0117 (mammalian construct) (SEQ ID NO 25), (g) DAT0117 (optimized for E.coli construct) (SEQ ID NO 26), (h) DAT0118 (mammalian construct) (SEQ ID NO 27), (i) DAT0119 (mammalian construct) (SEQ ID NO 28), (j) DAT0120 (mammalian construct) (SEQ ID NO 29), (k) Dom7h-14 (SEQ ID
  • Figure 3a shows a peptide conjugate which is:
  • Dom7h-14-10 (R108C) albudab conjugated to PYY3-36 via a lysine and 4 repeat PEG linker
  • Figure 3b shows a peptide conjugate which is:
  • the 4 repeat PEG chemical linker is covalently attached to the free C terminal cysteine of the Dom7h-14-10 (R108C) AlbudAb and the first residue of the NMU-8 (here: Tyrosine)
  • Figure 4 shows change in body weight over time in DIO mice treated with peptide- albudabs.
  • Figure 5 shows change in food intake over time in DIO mice treated with peptide- albudabs.
  • Figure 6 shows body fat % in DIO mice treated with peptide- albudabs.
  • Figure 7 shows change in body fat and lean mass in DIO mice (baseline vs 15 days) in mice treated with peptide- albudabs.
  • Figure 8 shows measurements of endocrine analytes in DIO mice treated with peptide- albudabs.
  • Figure 9 shows changes in histopathology in the liver on DIO mice treated with combinations of peptide- albudabs and controls.
  • Figure 10 shows measurements of glycosylated Haemoglobin Ale in db/db mice treated with peptide-albudabs.
  • Figure 11 shows the change in % HbAlc (baseline vs day 16) in db/db mice treated with peptide- albudabs.
  • Figure 12 shows plasma insulin levels (at day 16) in db/db mice treated with peptide- albudabs.
  • Figure 13 shows shows change in body weight over time in db/db mice treated with peptide- albudabs.
  • Figure 14 shows change in food intake over time in db/db mice treated with peptide- albudabs.
  • Figure 15 shows the amino acid sequences of leaders: (a) ompA (E. coli derived) (SEQ ID NO 38), (b) ompA-AMA (artificial sequence) (SEQ ID NO 39), (c) ompA-AWA (artificial sequence) (SEQ ID NO 40), (d) ompT (E. coli derived) (SEQ ID NO 41), (e) ompT-AMA (artificial sequence) (SEQ ID NO 42), (f) GAS (S.
  • Figure 16 amino acid sequences of NMU containing peptides: (a) DOM7h-14-10 AlbudAb and NMU8 (from example 14) (Seq ID No. 57)
  • insulinotropic agent means a compound which is able to stimulate, or cause the stimulation of, the synthesis or expression of, or the activity of the hormone insulin.
  • insulinotropic agents include but are not limited to e.g. glucose, GIP, NMU, GLP, Exendin (e.g. exendin-4 and exendin-3), PYY and OXM.
  • cretin means a type of gastrointestinal hormone that causes an increase in the amount of insulin released when glucose levels are normal or particularly when they are elevated.
  • they include GLP-1 , GIP, OXM, VIP, and PP (pancreatic polypeptide).
  • Gut peptides are a class of peptides released from various cells in different parts of the gut that provide a signaling function, PYY is also an example of a gut peptide.
  • analogue as used herein referring to a polypeptide means a modified peptide wherein one or more amino acid residues of the peptide have been substituted by other amino acid residues and/or wherein one or more amino acid residues have been deleted from the peptide and/or wherein one or more amino acid residues have been deleted from the peptide and or wherein one or more amino acid residues have been added to the peptide.
  • Such addition or deletion of amino acid residues can take place at the N-terminal of the peptide and/or at the C-terminal of the peptide or they can be within the peptide.
  • GLP-1 A8G (7-37 amino acids) designates a GLP-1 analogue wherein the naturally occuring alanine at position 8 has been substituted with a glycine residue.
  • Formulae of peptide analogs and derivatives thereof are drawn using standard single letter abbreviation for amino acids used according to IUPAC-IUB
  • fragment when used in reference to a polypeptide, is a polypeptide having an amino acid sequence that is the same as part but not all of the amino acid sequence of the entire naturally occurring polypeptide. Fragments may be "free-standing" or comprised within a larger polypeptide of which they form a part or region as a single continuous region in a single larger polypeptide.
  • a fragment of naturally occurring GLP-1 would include amino acids 7 to 36 of naturally occurring amino acids 1 to 36.
  • fragments of a polypeptide may also be variants of the naturally occurring partial sequence. For instance, a fragment of GLP-1 comprising amino acids 7-30 of naturally occurring GLP-1 may also be a variant having amino acid substitutions within its partial sequence.
  • suitable insulinotropic agents of the invention include NMU pepides such as NMU-8 or a derivative, fragment or analague thereof GLP-1, GLP-1 derivatives, GLP-1 analogues, or a derivative of a GLP-1 analogue.
  • NMU refers to an insulinotropic Neuromedin U peptide.
  • the NMU can be NMU-8 or a fragment, mutant, analogue or derivative which is an insulinotropic agent.
  • GLP-1 as used herein means GLP-1 (7-37), GLP-1 (7-36) , GLP-1 (7-35), GLP-1 (7-38), GLP-1 (7-39), GLP-1 (7-40), GLP-1 (7-41), a GLP-1 analogue, a GLP-1 peptide , a GLP-1 derivative or mutant or fragment or a derivative of a GLP-1 analogue.
  • Such peptides, mutants, analogues and derivatives are insulinotropic agents.
  • the GLP-1 can be GLP-1 (7-37) A8G mutant with the amino acid sequence shown in Figure 1 (i): SEQ ID NO 9.
  • GLP-1 analogues are described in International Patent Application No. 90/11296 (The General Hospital Corporation) which relates to peptide fragments which comprise GLP-1 (7-36) and functional derivatives thereof and have an insulinotropic activity which exceeds the insulinotropic activity of GLP-1 (1-36) or GLP-1 (1- 37) and to their use as insulinotropic agents (incorporated herein by reference, particularly by way of examples of drugs for use in the present invention).
  • exendin-4 peptide as used herein means exendin-4 (1-39), an exendin-4 analogue, a fragment of exendin-4 peptide, an exendin-4 derivative or a derivative of an exendin-4 analogue. Such peptides, fragments, analogues and derivatives are insulinotropic agents.
  • the amino acid sequence of exendin-4 (1-39) is shown in Figure 1 (j): SEQ ID NO 10.
  • PYY refers to the Peptide YY which is a short (36 amino acid) protein released in response to feeding. PYY concentration in the circulation increases postprandially and decreases on fasting. Fragments (e.g. active fragments) of the PYY peptide are also useful for the present inventin e.g. 3-36, 13-36 as are PYY analogues and derivatives which are useful in the present invention.
  • peptide refers to about two to about 50 amino acids that are joined together via peptide bonds.
  • polypeptide refers to at least about 50 amino acids that are joined together by peptide bonds. Polypeptides generally comprise tertiary structure and fold into functional domains.
  • display system refers to a system in which a collection of polypeptides or peptides are accessible for selection based upon a desired characteristic, such as a physical, chemical or functional characteristic.
  • the display system can be a suitable repertoire of polypeptides or peptides (e.g. , in a solution, immobilized on a suitable support).
  • the display system can also be a system that employs a cellular expression system (e.g. , expression of a library of nucleic acids in, e.g., transformed, infected, transfected or transduced cells and display of the encoded polypeptides on the surface of the cells) or an acellular expression system (e.g., emulsion compartmentalization and display).
  • Exemplary display systems link the coding function of a nucleic acid and physical, chemical and/or functional characteristics of a polypeptide or peptide encoded by the nucleic acid.
  • polypeptides or peptides that have a desired physical, chemical and/or functional characteristic can be selected and a nucleic acid encoding the selected polypeptide or peptide can be readily isolated or recovered.
  • bacteriophage display phage display, for example phagemid display
  • ribosome display emulsion compartmentalization and display
  • yeast display puromycin display
  • bacterial display display on plasmid, covalent display and the like.
  • “functional” describes a polypeptide or peptide that has biological activity, such as specific binding activity.
  • the term “functional polypeptide” includes an antibody or antigen-binding fragment thereof that binds a target antigen through its antigen-binding site.
  • target ligand refers to a ligand which is specifically or selectively bound by a polypeptide or peptide.
  • a polypeptide is an antibody or antigen-binding fragment thereof
  • the target ligand can be any desired antigen or epitope. Binding to the target antigen is dependent upon the polypeptide or peptide being functional.
  • an antibody refers to IgG, IgM, IgA, IgD or IgE or a fragment (such as a Fab , F(ab')2, Fv, disulphide linked Fv, scFv, closed conformation multispecific antibody, disulphide-linked scFv, diabody) whether derived from any species naturally producing an antibody, or created by recombinant DNA technology; whether isolated from serum, B-cells, hybridomas, transfectomas, yeast or bacteria.
  • a fragment such as a Fab , F(ab')2, Fv, disulphide linked Fv, scFv, closed conformation multispecific antibody, disulphide-linked scFv, diabody
  • antibody format refers to any suitable polypeptide structure in which one or more antibody variable domains can be incorporated so as to confer binding specificity for antigen on the structure.
  • suitable antibody formats are known in the art, such as, chimeric antibodies, humanized antibodies, human antibodies, single chain antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy chains and/or light chains, antigen-binding fragments of any of the foregoing (e.g., a Fv fragment (e.g., single chain Fv (scFv), a disulfide bonded Fv), a Fab fragment, a Fab' fragment, a F(ab') 2 fragment), a single antibody variable domain (e.g., a dAb, H, V H H, VL>, and modified versions of any of the foregoing (e.g., modified by the covalent attachment of polyethylene glycol or other suitable polymer or a humanized V H H)-
  • a dAb e
  • An immunoglobulin single variable domain can be present in a format (e.g., homo- or hetero-multimer) with other variable regions or variable domains where the other regions or domains are not required for antigen binding by the single immunoglobulin variable domain ( . e. , where the immunoglobulin single variable domain binds antigen independently of the additional variable domains).
  • a “domain antibody” or “dAb” is the same as an "immunoglobulin single variable domain” as the term is used herein.
  • a “single immunoglobulin variable domain” is the same as an "immunoglobulin single variable domain” as the term is used herein.
  • a “single antibody variable domain” is the same as an
  • immunoglobulin single variable domain is in one embodiment a human antibody variable domain, but also includes single antibody variable domains from other species such as rodent (for example, as disclosed in WO 00/29004, the contents of which are incorporated herein by reference in their entirety), nurse shark and Camelid VHH dAbs.
  • rodent for example, as disclosed in WO 00/29004, the contents of which are incorporated herein by reference in their entirety
  • Camelid VHH are immunoglobulin single variable domain polypeptides that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally devoid of light chains.
  • the V H H may be humanized.
  • a “domain” is a folded protein structure which has tertiary structure independent of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins, and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain.
  • a “single antibody variable domain” is a folded polypeptide domain comprising sequences characteristic of antibody variable domains. It therefore includes complete antibody variable domains and modified variable domains, for example, in which one or more loops have been replaced by sequences which are not characteristic of antibody variable domains, or antibody variable domains which have been truncated or comprise N- or C-terminal extensions, as well as folded fragments of variable domains which retain at least the binding activity and specificity of the full-length domain.
  • library refers to a mixture of heterogeneous polypeptides or nucleic acids.
  • the library is composed of members, each of which has a single polypeptide or nucleic acid sequence.
  • library is synonymous with "repertoire.” Sequence differences between library members are responsible for the diversity present in the library.
  • the library may take the form of a simple mixture of polypeptides or nucleic acids, or may be in the form of organisms or cells, for example bacteria, viruses, animal or plant cells and the like, transformed with a library of nucleic acids. In one embodiment, each individual organism or cell contains only one or a limited number of library members.
  • the nucleic acids are incorporated into expression vectors, in order to allow expression of the polypeptides encoded by the nucleic acids.
  • a library may take the form of a population of host organisms, each organism containing one or more copies of an expression vector containing a single member of the library in nucleic acid form which can be expressed to produce its corresponding polypeptide member.
  • the population of host organisms has the potential to encode a large repertoire of diverse polypeptides.
  • dose refers to the quantity of fusion or conjugate
  • dose can refer to the quantity of fusion or conjugate administered to a subject over the course of one day (24 hours) (daily dose), two days, one week, two weeks, three weeks or , one month, two months, three months, or six or more months (e.g., by a single administration, or by two or more administrations).
  • the interval between doses can be any desired amount of time.
  • half-life refers to the time taken for the serum or plasma concentration of the fusion or conjugate to reduce by 50%, in vivo, for example due to degradation and/or clearance or sequestration by natural mechanisms.
  • the compositions of the invention are stabilized in vivo and their half-life increased by binding to serum albumin molecules e.g. human serum albumin (HSA) which resist degradation and/or clearance or sequestration.
  • serum albumin molecules e.g. human serum albumin (HSA) which resist degradation and/or clearance or sequestration.
  • serum albumin molecules are naturally occurring proteins which themselves have a long half-life in vivo.
  • the half-life of a molecule is increased if its functional activity persists, in vivo, for a longer period than a similar molecule which is not specific for the half-life increasing molecule.
  • a composition of the invention comprising a dAb specific for human serum albumin (HSA) and incretin and/or insulinotropic and/or gut peptide molecules such as GLP- 1 , PYY or exendin is compared with the same ligand wherein the specificity to HSA is not present, that is does not bind HSA but binds another molecule. For example, it may bind a third target on the cell.
  • the half-life is increased by 10%, 20%, 30%, 40%, 50% or more. Increases in the range of 2x, 3x, 4x, 5x, lOx, 20x, 30x, 40x, 50x or more of the half-life are possible.
  • hydrodynamic size refers to the apparent size of a molecule (e.g., a protein molecule, ligand) based on the diffusion of the molecule through an aqueous solution. The diffusion, or motion of a protein through solution can be processed to derive an apparent size of the protein, where the size is given by the "Stokes radius” or “hydrodynamic radius” of the protein particle.
  • the “hydrodynamic size” of a protein depends on both mass and shape (conformation), such that two proteins having the same molecular mass may have differing hydrodynamic sizes based on the overall conformation of the protein.
  • sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, 80%, 90%, 100% of the length of the reference sequence.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “homology” is equivalent to amino acid or nucleic acid “identity”
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • Amino acid and nucleotide sequence alignments and homology, similarity or identity, as defined herein may be prepared and determined using the algorithm BLAST 2 Sequences, using default parameters (Tatusova, T. A. et al, FEMS Microbiol Lett, 774: 187-188 (1999).
  • the invention relates to isolated and/or recombinant nucleic acids encoding the compositions e.g. fusions, of the invention that are described herein.
  • Nucleic acids referred to herein as "isolated” are nucleic acids which have been separated away from other material ⁇ e.g., other nucleic acids such as genomic DNA, cDNA and/or RNA) in its original environment ⁇ e.g., in cells or in a mixture of nucleic acids such as a library).
  • An isolated nucleic acid can be isolated as part of a vector ⁇ e.g. , a plasmid).
  • Nucleic acids referred to herein as "recombinant” are nucleic acids which have been produced by recombinant DNA methodology, including methods which rely upon artificial recombination, such as cloning into a vector or chromosome using, for example, restriction enzymes, homologous recombination, viruses and the like, and nucleic acids prepared using the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the invention also relates to a recombinant host cell e.g.mammalian or microbial, which comprises a (one or more) recombinant nucleic acid or expression construct comprising nucleic acid(s) encoding a composition e.g. fusion, of the invention as described herein.
  • a method of preparing a composition, e.g. fusion, of the invention as described herein comprising maintaining a recombinant host cell e.g.mammalian or microbial, of the invention under conditions appropriate for expression of the fusion polypeptide.
  • the method can further comprise the step of isolating or recovering the fusion, if desired.
  • a nucleic acid molecule i.e., one or more nucleic acid molecules
  • a composition of the invention e.g. a fusion polypeptide of the invention, or an expression construct (i.e., one or more constructs) comprising such nucleic acid molecule(s)
  • an expression construct i.e., one or more constructs comprising such nucleic acid molecule(s)
  • can be introduced into a suitable host cell to create a recombinant host cell using any method appropriate to the host cell selected e.g. , transformation, transfection, electroporation, infection
  • the nucleic acid molecule(s) are operably linked to one or more expression control elements (e.g. , in a vector, in a construct created by processes in the cell, integrated into the host cell genome).
  • the resulting recombinant host cell can be maintained under conditions suitable for expression (e.g. , in the presence of an inducer, in a suitable animal, in suitable culture media supplemented with appropriate salts, growth factors, antibiotics, nutritional supplements, etc.), whereby the encoded peptide or polypeptide is produced.
  • the encoded peptide or polypeptide can be isolated or recovered (e.g. , from the mammal, the animal, the host cell, medium, milk). This process encompasses expression in a host cell of a transgenic animal (see, e.g. , WO 92/03918, GenPharm
  • the peptide or fusion protein or conjugate can subsequently be further modified e.g. chemically or enzymatically either in the expression host, in the culture medium, during or after purification e.g. via amidation of the C terminus.
  • compositions, e.g. fusion polypeptides, of the invention described herein can also be produced in a suitable in vitro expression system, e.g. by chemical synthesis or by any other suitable method.
  • compositions e.g. fusions and conjugates of the invention generally bind serum albumin with high affinity.
  • the fusions or conjugates can bind human serum albumin with an affinity (KD; [as determined by surface plasmon resonance) of about 5 micromolar to about 100 pM , e.g. about 1 micromolar to about 100 pM e.g. 400-800nm e.g. about 600nm.
  • KD affinity
  • compositions e.g. fusions or conjugates, of the invention can be expressed in E. coli or in Pichia species (e.g., P. pastoris).
  • the fusion is secreted in a quantity of at least about 0.5 mg/L when expressed in E. coli or in Pichia species (e.g., P. pastoris); or in mammalian cell culture (e.g. CHO, or HEK 293 cells).
  • the fusions or conjugates described herein can be secretable when expressed in E. coli or in Pichia species or mammalian cells they can be produced using any suitable method, such as synthetic chemical methods or biological production methods that do not employ E. coli or Pichia species.
  • compositions of the invention are efficacious in animal models of such as those described in WO 2006 /059106 (e.g. at pages 104-105 of published WO 2006 /059106) or those described in the examples herein, when an effective amount is administered.
  • an effective amount is about 0.0001 mg/kg to about 10 mg/kg (e.g., about 0.001 mg/kg to about 10 mg/kg, e.g. about 0.001 mg/kg to about 1 mg/kg, e.g. about 0.01 mg/kg to about 1 mg/kg, e.g. about 0.01 mg/kg to about 0.1 mg/kg).
  • the models of disease are recognized by those skilled in the art as being predictive of therapeutic efficacy in humans.
  • compositions of the invention will be utilised in purified form together with pharmacologically or physiologically appropriate carriers.
  • these carriers can include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, any including saline and/or buffered media.
  • Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's.
  • Suitable physiologically-acceptable adjuvants, if necessary to keep a polypeptide complex in suspension may be chosen from thickeners such as carboxymethylcellulose,
  • polyvinylpyrrolidone polyvinylpyrrolidone, gelatin and alginates, sucrose, trehalose, sorbitol, detergents such as tween-20 or tween-80.
  • Intravenous vehicles include fluid and nutrient replenishers and electrolyte
  • replenishers such as those based on Ringer's dextrose.
  • Preservatives and other additives such as antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack
  • formulations can be used, including extended release formulations.
  • compositions according to the invention may be any of those commonly known to those of ordinary skill in the art.
  • drug fusions or conjugates of the invention can be administered to any patient in accordance with standard techniques.
  • the administration can be by any appropriate mode, including parenterally, intravenously, transmucosal delivery (e.g. sub-lingual), by subcutaneous injection, intramuscularly, intraperitoneally, orally, transdermally, transmucosally via the pulmonary route, via nasal delivery, GI delivery, rectal delivery, or ocular delivery or also, appropriately, by direct infusion with a catheter.
  • parenterally intravenously, transmucosal delivery (e.g. sub-lingual), by subcutaneous injection, intramuscularly, intraperitoneally, orally, transdermally, transmucosally via the pulmonary route, via nasal delivery, GI delivery, rectal delivery, or ocular delivery or also, appropriately, by direct infusion with a catheter.
  • the dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs,
  • Administration can be local or systemic as indicated.
  • compositions of this invention can be lyophilised for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins and art-known lyophilisation and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and
  • compositions containing the present fusions or conjugates may also be administered in similar or slightly lower dosages, to prevent, inhibit or delay onset of disease (e.g., to sustain remission or quiescence, or to prevent acute phase).
  • onset of disease e.g., to sustain remission or quiescence, or to prevent acute phase.
  • the skilled clinician will be able to determine the appropriate dosing interval to treat, suppress or prevent disease.
  • composition of the invention When a composition of the invention is administered to treat, suppress or prevent disease, it can be administered up to four times per day, once per day, twice weekly, once weekly, once every two weeks, once a month, or once every two months, once every three months, once every six months, or at a longer interval, at a dose of, for example about 0.0001 mg/kg to about 10 mg/kg (e.g., about 0.001 mg/kg to about 10 mg/kg e.g. about 0.001 mg/kg to about 1 mg/kg e.g. about 0.01 mg/kg to about 1 mg/kg, e.g. about 0.01 mg/kg to about 0.1 mg/kg).
  • a dose of, for example about 0.0001 mg/kg to about 10 mg/kg e.g., about 0.001 mg/kg to about 10 mg/kg e.g. about 0.001 mg/kg to about 1 mg/kg e.g. about 0.01 mg/kg to about 1 mg/kg, e.g. about 0.
  • Treatment or therapy performed using the compositions described herein is considered “effective” if one or more symptoms or signs are reduced or alleviated (e.g., by at least 10% or at least one point on a clinical assessment scale), relative to such symptoms present before treatment, or relative to such symptoms in an individual (human or model animal) not treated with such composition or other suitable control. Symptoms will obviously vary depending upon the precise nature of the disease or disorder targeted, but can be measured by an ordinarily skilled clinician or technician. Similarly, prophylaxis performed using a composition as described herein is
  • compositions of the present invention may be administered in conjunction with other therapeutic or active agents e.g. other polypeptides or peptides or small molecules.
  • these further agents can include various drugs, such as for example metformin, insulin, glitazones (e.g. rosaglitazone), immunosuppresives, immunostimulants.
  • the compositions of the invention can be administered and/ or formulated together with one or more additional therapeutic or active agents. When a composition of the invention is administered with an additional therapeutic agent, the fusion or conjugate can be administered before,
  • composition of the invention and the additional agent are administered in a manner that provides an overlap of therapeutic effect.
  • Increased half-life of the insulinotropic and/or incretin and/or gut peptide molecule e.g. the GLP-1, PYY or exendin ligand is useful in in vivo applications.
  • the invention solves this problem by providing increased half-life of the insulinotropic agent and/or incretin and/or gut peptide drug e.g. GLP and exendin, in vivo and consequently longer persistence times in the body of the functional activity of these molecules.
  • compositions of the invention can have dramatically prolonged in vivo serum or plasma half-life and/or increased AUC and/or increased mean residence time (MRT), as compared to insulinotropic and/or incretin and/or gut peptide molecule alone.
  • the activity of the insulinotropic and/or incretin and/or gut peptide molecule is generally not substantially altered in the composition of the invention (e.g., the conjugate, or the fusion).
  • some change in the activity of compositions of the invention compared to insulinotropic and/or incretin and/or gut peptide molecule alone is acceptable and is generally compensated for by the improved pharmacokinetic properties of the compositions of the invention.
  • compositions of the invention may bind the target with lower affinity than incretin/insuliotropic agent alone, but have about equivalent or superior efficacy in comparison to incretin/insuliotropic agent alone due to the improved pharmacokinetic properties (e.g., prolonged in vivo serum half-life, larger AUC) of the composition.
  • the compositions of the invention due to the increased half life of the compositions of the invention they can be administed less frequently than the insulinotropic agent and/or incretin and/or gut peptide drug alone e.g.
  • Half lives (t1 ⁇ 2 alpha and t1 ⁇ 2 beta) and AUC and MRT can be determined from a curve of plasma or serum concentration of ligand against time.
  • the WinNonlin analysis package (available from Pharsight Corp., Mountain View, CA94040, USA) can be used, for example, to model the curve.
  • a first phase the alpha phase
  • a second phase (beta phase) is the terminal phase when the ligand has been distributed and the serum concentration is decreasing as the ligand is cleared from the patient.
  • the t alpha half life is the half life of the first phase and the t beta half life is the half life of the second phase.
  • a non-compartmental fitting model that is well known in the art can also be used to determine half life.
  • the present invention provides a composition, comprising fusion(s) or conjugate(s) , according to the invention wherein the fusion or conjugate has an elimination half-life e.g. in human subjects, in the range of about 12 hours or more, e.g. about 12 hours to about 21 days, e.g. about 24 hours to about 21 days, e.g. about 2-8 days e.g. about 3-4 days.
  • compositions of the invention i.e. those comprising the fusions and conjugates described herein, provide several further advantages.
  • the Domain antibody component is very stable, is small relative to antibodies and other antigen-binding fragments of antibodies, can be produced in high yields by expression in E. coli or yeast (e.g., Pichia pastor is), or mammalian cells (e.g. CHO cells) and antigen-binding fragments of antibodies that bind serum albumin can be easily selected from libraries of human origin or from any desired species.
  • compositions of the invention that comprise the dAb that binds serum albumin can be produced more easily than therapeutics that are generally produced in mammalian cells (e.g. , human, humanized or chimeric antibodies) and dAbs that are not immunogenic can be used (e.g., a human dAb can be used for treating or diagnosing disease in humans).
  • mammalian cells e.g. , human, humanized or chimeric antibodies
  • dAbs that are not immunogenic can be used (e.g., a human dAb can be used for treating or diagnosing disease in humans).
  • the immunogenicity of the insulinotropic and/or incretin and/or gut peptide molecule(s) can be reduced when it is part of a drug composition that contains a dAb that binds serum albumin.
  • the invention provides a compositions which can be less immunogenic (than e.g. the insulinotropic and/or incretin and/or gut peptide molecules alone) or which can be substantially non-immunogenic in the context of a drug composition that contains a dAb that binds serum albumin .
  • such compositions can be administered to a subject repeatedly over time with minimal loss of efficacy due to the elaboration of anti-drug antibodies by the subject's immune system.
  • compositions described herein can have an enhanced safety profile and fewer side effects than the insulinotropic and/or incretin and/or gut peptide agents alone.
  • the fusions and conjugates of the invention have enhanced residence time in the vascular circulation.
  • compositions of the invention are substantially unable to cross the blood brain barrier and to accumulate in the central nervous system following systemic
  • compositions of the invention can be administered with greater safety and reduced side effects in comparison to the insulinotropic and/or incretin and/or gut peptide agent alone alone.
  • compositions of the invention can have reduced toxicity toward particular organs (e.g., kidney or liver) than drug alone.
  • Example 1 Expression of genetic fusions of GLP-1 (A8G) or Exendin-4 and DOM7h-14 AlbudAb:
  • the linkers were included as spacers to separate the GLP-1 or exendin 4 spatially from the dAb to prevent steric hinderence of the binding between the GLP-1 or exendin-4 and the GLP- 1 receptor.
  • the sequences of the constructs are shown in Figure 1 (A-G) SEQ ID NOS 1-7.
  • Endotoxin free DNA was prepared in E.coli using alkaline lysis (using the endotoxin free plasmid Giga kit, obtainable from Qiagen CA) and used to transfect HEK293E cells
  • GLP-1 and Exendin-4 AlbudAb fusions were analysed by surface plasmon resonance (Biacore AB obtainable from GE Healthcare) to obtain information on affinity.
  • the analysis was performed using a CM5 Biacore chip (carboxymethylated dextran matrix) that was coated with serum albumin. About 1000 resonance units (RUs) of each serum albumin to be tested
  • Flow cell 1 of the Biocore AB was an uncoated, blocked negative control
  • flow cell 2 was coated with Human serum albumin (HSA) (815 RUs)
  • flow cell 3 was coated with Rat serum albumin (RSA)(826RUs)
  • flow cell 4 was coated with Mouse serum albumin (MSA) (938 RUs).
  • HSA Human serum albumin
  • MSA Mouse serum albumin
  • a range of concentrations of the fusion molecule were prepared (in the range 16nM to 2 ⁇ ) by dilution into BIACORE HBS-EP buffer (0.01M HEPES, pH7.4, 0.15M NaCl, 3mM EDTA, 0.005% surfactant P20) and flowed across the BIACORE chip.
  • KD Affinity
  • GLP-1 and exendin-4 AlbudAb fusions are active in a GLP-1 receptor binding assay (GLP-1R BA): Fusions were buffer exchanged into lOOmM NaVl, 20mM citrate pH 6.2. Meanwhile,CHO 6CRE GLPIR cells (CHO Kl cells (obtainable from the American Type Tissue Collection, ATCC) stably transfected with 6 cAMP response element driving a luciferase reporter gene and also with the human GLP-1 receptor) were seeded at 2 x 10 5 cells/mL in suspension media. Suspension culture was maintained for 24 hours.
  • GLP-1R BA GLP-1 receptor binding assay
  • Example 4 Expression of DAT0115, DAT0116, DAT0117 and DAT0120 in HEK 293 mammalian tissue culture followed by purification by protein L affinity capture and ion exchange chromatography:
  • the aim of this experiment was to produce protein for in vivo and in vitro characterisation.
  • Protein was expressed in mammalian tissue culture in HEK 293E cells from the pTT-5 vector as described in the previously. Briefly, endotoxin free DNA was prepared and purified and used to transfect HEK293E cells. Protein expression was for 5 days at 30°C in a shaking incubator and cultures were spun down and supernatant (containing the protein of interest) harvested. Protein was purified from the supernatant by affinity capture on protein L agarose streamline affinity resin (resin GE Healthcare, protein L coupled in house). Resin was then washed with approximately 10 column volumes of PBS and then protein was eluted with approximately 5 column volumes of 0.1 M glycine pH2.0.
  • Protein in tris-glycine was buffer exchanged to 20mM acetate pH 5.0 prior to loading using the Akta onto 1 (or 2 in parallel) 6ml resource S columns (GE healthcare) pre-equilibrated in 20mM acetate pH 5.0. After washing with the same buffer, protein was eluted via a 0-0.75M or NaCl gradient in 20mM acetate pH5.0. Fractions of the correct size were then identified by SDS-PAGE electrophoresis and by mass spectrometry and were then combined to make the final protein sample.
  • Example 5 Production of the PYY (3-36) Dom7h-14-10 (R108C) AlbudAb peptide conjugate (which has the structure shown in figure 3) and which is: a Dom7h-14-10 (R108C) albudab conjugated to the PYY3-36 via a lysine and a 4 repeat PEG linker):
  • the Dom7h-14-10 (R108C) albudab was expressed and purified as described as follows in E.coli: The gene encoding the DOM7h-14-10 (R108C) was cloned into vector pET30. To enable cloning into expression vector, fusions were produced as assembly PCRs with Ndel restriction site on 5' followed by the PEL B leader sequence (amino acid sequence shown in Figure 15 (i) SEQ ID NO 46). Vector and assembly PCRs were digested with Ndel and BamHI restriction endonucleases followed by ligation of the insert into the vector using a Quick Ligation Kit (NEB). 2 microlitres of this ligation was used for transformation of Machl cells.
  • NEB Quick Ligation Kit
  • Protein L streamline (GE Healthcare, Cat.No. 28-4058-03, protein L coupled), and eluted from the Protein L using 0.1M glycine pH2.0, then neutralized by addition of l/5 th elution volume of 1M Tris, pH8.0.
  • the protein was then pH adjusted using 0.1M Citric Acid to pH5 and applied to a 30ml Source S column (GE Healthcare) equilibrated with 50mM Sodium Citrate, pH5. A gradient from 0-100 of 50mM Sodium Citrate, pH5, 1M NaCl was applied using the AktaXpress FPLC (GE healthcare) over 150ml. Fractions were analyzed on SDS-PAGE and those containing the purest product were pooled. The final protein was desalted into 50mM Sodium Phosphate, pH6.5, 5mM EDTA.
  • the Dom7h-14-10 (R108C) albudab was then linked to a PYY 3-36 amino acid molecule (but with a lysine at position 10 which can be derivatised with PEG linker) using the PEG linker shown in figure 3.
  • the PYY and the PEG were prepared by standard chemical synthesis.
  • the maleimide at the end of the PEG linker was then used to conjugate the PYY peptide to the free cysteine of the Dom7h-14-10 (R108C) albudab prepared as described above.
  • Dom7h-14-10 The free cysteine of Dom7h-14-10 (R108C) was reduced by addition of Dithiothreitol (DTT) to a final concentration of 5mM, incubated for 30 minutes and finally desalted into 50mM Sodium Phosphate, pH6.5, 5mM EDTA to remove the DTT. Maleimide activated peptide was then mixed with the protein at a 1 : 1 ratio and incubated to allow the conjugation to occur. Conjugate was purified from un-reacted Dom7h- 14- 10 (Rl 08C) by Ion Exchange
  • Example 6 Expression and purification of genetic fusions of Exendin-4 and DOM7h-14-10/ DOM7h-l 1-15 AlbudAb.
  • DMS7139 is a fusion of exendin-4 with DOM7h-14-10 (a domain antibody (dAb) that binds serum albumin, also known as an albudab)
  • DMS7143 is a fusion of exendin-4 with DOM 7h- 11- 15 (a domain antibody (dAb) that binds serum albumin, also known as an albudab) in E. coli with correctly processed N-terminii.
  • the fusion could then be tested for activity of the exendin-4 portion and of the ALbudAb portion in subsequent experiments.
  • Exendin-4 was cloned as a fusion with DOM7h-14-10 or DOM7h-l l-15, where exendin-4 peptide was at the 5' end of the construct and AlbudAb at the 3' end.
  • two constructs were made each including (Gly4Ser)3 linker between the exendin-4 peptide and the AlbudAb.
  • the linker was included as a spacer to separate the exendin 4 spatially from the dAb to prevent steric hindrance of the binding between the exendin-4 and the GLP-1 receptor.
  • the sequences of the constructs are shown in figures l(m) and l(n) .
  • OmpT AWA the amino acid sequence is shown in figure l(q) , SEQ ID NO 17) signal peptide and with BamHI site on 3' terminus.
  • OmpT AWA signal peptide has the last three codons changed from wildtype "TCTTTTGCC” to "GCTTGGGCC” which codes AWA instead of SFA. That change improves processing at the correct site by the signal peptidase of E. coli.
  • the sequence of the fusion starts straight after the peptidase cleavage site.
  • An Ncol digestion site has been introduced, which overlaps with the last codon of the signal peptide and two first amino acids of exendin-4 sequence. This change facilitates future subcloning as well as leading to production of the fusion with free N-terminal end of exendin- 4.
  • the modified pET12a expression vector comprising the changes listed above was given the name pDOM35.
  • Vector and assembly PCRs were digested with Ndel and BamHI restriction endonucleases followed by ligation of the insert into the vector using a Quick Ligation Kit (NEB). 2 microlitres of this ligation was used for transformation of MachI cells. After the recovery growth period, cells were plated on agar plates containing carbenicilin and incubatesd at 37°C overnight. Colonies were sequenced and those containing the correct sequence were used for plasmid propagation and isolation (Plasmid Mini Prep kit, Qiagen). BL21(DE3) cells were transformed with plasmid DNA and resulting colonies were used for inoculation of expression culture.
  • NEB Quick Ligation Kit
  • Expression was performed by inoculation of a 4 x 0.5 litre culture of TB Onex media (supplemented with Overnight ExpressTM autoinduction solutions), 1 droplet of antifoam (antifoam A204; Sigma) and 100 microgram per milliliter of carbenicillin. Culture was incubated for 3 nights at 30° C with agitation 250 rpm, and then the culture supernatant was clarified by centrifugation at 3700xg for 1 hour. The expressed protein was then purified from the clarified supernatant using protein L streamline (GE Healthcare, Cat.No. 28-4058-03, protein L coupled), and eluted from the Protein L using 0.1M glycine pH2.0, then neutralized using 0.1 volume of 1M Tris pH8.0.
  • protein L streamline GE Healthcare, Cat.No. 28-4058-03, protein L coupled
  • Example 7 Pharmacologic profile of the Exendin-4 AlbudAb (DAT 0115 made as described above) and PYY (3-36) AlbudAb fusion peptide (made as described in example 5 and with the structure shown in figure 3) in the melanophore functional bioassay.
  • the pharmacologic profile of the Exendin-4 AlbudAb (DAT 0115) and the PYY(3-36) AlbudAB (as described in example 5 and with the structure shown in figure 3) was determined in a melanophore functional bioassay using cells transfected with receptors of interest.
  • the bioassay was performed essentially as described in Jayawickreme et al. (2005) Current Protocols in Pharmacology 12.9.1-12.9.16.
  • Selectivity values range from several hundred to > 1000 fold, when comparing peptide activity for NPY2R to the other NPY receptors within the same species (calculated from Table 5).
  • Example 8 Exendin-albudab (DAT 0115) in combination with PYY-albudab (as described in example 5 and with the structure shown in figure 3) causes synergistic effects on multiple parameters in diet induced obese (DIO) mice:
  • DIO C57BL/6 mice Male diet induced obese (DIO) C57BL/6 mice (Taconic, Hudson, NY) and lean C57BL/6 mice (Taconic, Hudson, NY) were used for all experiments.
  • DIO C57BL/6 mice were group housed and fed a high fat diet (45% fat by kcal) by the vendor from the time of weaning.
  • DIO mice 40-50g body weight
  • age-matched controls were single-housed and maintained at constant temperature (approximately 22° C) with 12 hr light/dark cycle (lights on from 5:00 AM to 5:00 PM). Mice were given ad libitum access to food (Research Diets D 12451, 45% fat for DIO; Lab Diet 5001, 13.5% fat for lean) and water.
  • peptide-albudabs were either prepared fresh daily or were prepared once and frozen at -70 deg C in aliquots. For combination dosing, the drugs were mixed together so that only one injection would be required.
  • ED 80 combo were given a single dose of: the PYY-albudab at 1.0 mg/kg mixed with the exendin-4 -albudab (DAT 01 15) at 0.1 mg/kg
  • a three day vehicle lead in period was used before the start of drug with the first day being vehicle and the second two days being mock injections.
  • Baseline fat mass and lean mass measurements were taken 3-4 days before the start of drug and on day 15 using a QMR instrument (Echo Medical Systems, Houston, TX.)
  • Body weight measurements were taken every Monday, Wednesday, and Friday starting four days before the first drug dose, with the first measurement being used to randomize the animals.
  • Food hopper weights were measured every weekday starting 4-6 days before the first drug dose, allowing for the calculation of food intake. Animals that created excessive food spillage were removed prior to the beginning of the study. During the study, excess food was removed from the cage and added to the food hopper weights for increased accuracy.
  • the PYY-AlbudAb 1.0 mg/kg group showed about a 7.8% decrease from vehicle and the Exendin-4-AlbudAb 0.1 mg/kg group showed a 16.8% decrease from vehicle; addition of those two dose groups would have yielded a 24.6% decrease in body weight.
  • a 32.8% decrease for the Combo ED 80 group was observed which is a statistically significant increase over the predicted additivity data
  • Amylin levels in the Combo ED 80 group were significantly lower than the vehicle controls (250 pg/ml in plasma; p ⁇ 0.0 ⁇ ). Moreover, the Combo ED 80 amylin levels were approximately the same as the lean control levels (87pg/ml in plasma).
  • Ghrelin levels were elevated in the Exendin-4-AlbudAb monotherapy groups to a level approximately equal to the combination groups. This indicates that Exendin-4 activity alone is most likely responsible for the increased ghrelin exposure.
  • the Lean Control group represents the relative difference between lean animals and the DIO group. Values represent changes for all other groups because these groups were randomized from a single population prior to the beginning of the study.
  • the Combo ED 20 group displayed some significant improvements on glucose and total cholesterol, while showing trends towards improvements in triglycerides and alanine transaminase (ALT) levels (Table 5).
  • alanine transaminase is elevated in the vehicle control DIO mice but treatment with the Combo ED 80 decreased levels by 79% to the level of the lean controls.
  • Other significant improvements include HbAlc, total cholesterol, triglycerides, total bilirubin, creatinine, aspartate aminotransferase (AST), alanine transaminase (ALT) and total protein. All of these changes made the DIO serum chemistries more closely resemble the lean control chemistries and were considered beneficial.
  • Cytoplasmic lipid droplets in the liver confirmed by osmium stain, were marked in severity in the DIO vehicle-control mice, affecting most hepatocytes.
  • the cytoplasmic lipid droplets were substantially decreased (minimal to undetectable) in DIO mice given Combo ED 0 (see figure 9).
  • a similar change with lesser response magnitude than seen in Combo ED 80 livers was noted in DIO mice given Combo ED 20 , PYY- AlbudAb (1.0 mg kg), Exendin- 4-AlbudAb (0.1 mg/kg) and Exendin-4 (0.1 mg/kg).
  • PYY- AlbudAb 1.0 mg kg
  • Exendin- 4-AlbudAb 0.1 mg/kg
  • Exendin-4 0.1 mg/kg
  • Example 9 Effects of Exendin- AlbudAb (DAT 0115) and PYY-Albudab (as described in example 5 and with the structure shown in figure 3) combination on diabetes parameters in db/db mice:
  • mice Male db/db C57BL/6J mice (Jackson Labs, Bar Harbor, ME) were used for all experiments.
  • the db/db mice (10-12 weeks of age), and age-matched controls were shipped to GSK where they were single-housed and maintained at constant temperature (approximately 22°C) with 12 hr light/dark cycle (lights on from 5:00 AM to 5:00 PM). Mice were given ad libitum access to food (LabDiet 5K67, 16% fat for db/db and their controls) and water.
  • the peptide-AlbudAbs were prepared fresh daily.
  • the correct dosing concentration of the drug was obtained by diluting the master stock using a citrate vehicle buffer comprised of 100 mM NaCl, 20 mM citric acid, pH 6.2 (filter sterilized).
  • a citrate vehicle buffer comprised of 100 mM NaCl, 20 mM citric acid, pH 6.2 (filter sterilized).
  • the drugs were mixed together so that only one injection would be required.
  • Chronic Diabetes Efficacy Studies The db/db mice and age-matched lean controls were habituated in house 2 weeks before the start of the study. Animals were dosed every two days between 2-4 pm subcutaneously with a dose volume of 5 ml/kg over a period of 15 days. A three day vehicle lead in period was used before the start of drug with the first day being vehicle and the second two days being mock injections. Baseline fat mass and lean mass measurements were taken 3 days before the start of drug and on day 15 using a QMR instrument (Echo Medical Systems, Houston, TX.) Body weight measurements were taken every Monday, Wednesday, and Friday starting four days before the first drug dose.
  • the vehicle control animals increased %HbAlc during the 18 days of the study from an average of 7.14% at baseline to an average of 9.03% by day 16. This indicates substantial progression of the diabetic phenotype during that time period. See Figures 10 and 11. An inhibition of the progression of the diabetic phenotype was observed in multiple dose groups including the Combo ED 20 , the PYY-AlbudAb 1.0 mg/kg, and the Exendin-4-AlbudAb 0.1 mg/kg groups (p ⁇ 0.05 vs. vehicle increase). An absolute decrease in %HbAlc was only observed for the Combo ED 80 group (p ⁇ 0.01 vs. baseline).
  • the Combo ED 80 group dropped from 6.83% glycosylated HbAlc down to 5.16% glycosylated HbAlc. There was no longer a significant difference in glycosylated HbAl c between the lean non-diabetic controls and the Combo ED 0 (p ⁇ 0.01). Therefore, the diabetic (db/db) mice in the Combo ED 80 treatment group had a completely normal level of % glycosylated HbAlc and were nearly "normalized" back to normal lean control animals.
  • the Combo groups were analyzed in a similar manner.
  • the PYY-AlbudAb 0.1 mg/kg group and the Exendin-4-AlbudAb 0.01 mg/kg groups showed no significant changes from the vehicle control levels while in combination (Combo ED 20 ), there was a 0.89% decrease in glycosylated HbAlc.
  • the predicted additive decrease would be 1.96% for the PYY-AlbudAb 1.0 mg/kg and Exendin-4-AlbudAb 0.1 mg/kg groups.
  • a 3.57% decrease in glycosylated HbAlc was observed. This decrease is significantly greater than what was predicted by additivity of the monotherapy groups (p ⁇ 0.05).
  • plasma insulin levels reached 21307 pg/ml which was significantly higher than the vehicle control group at 9470 pg/ml in plasma (p ⁇ 0.05).
  • the ED 8 o Pair-fed Control group had plasma insulin levels of 4438 pg/ml which was significantly lower than the vehicle control levels (p ⁇ 0.01), most likely due to the weight loss.
  • the Combo ED 80 group was analyzed in a similar manner. At day 15, the PYY- AlbudAb 1.0 mg/kg group showed a 5.9% decrease from vehicle and the Exendin-4- AlbudAb 0.1 mg/kg group showed a 9.2% decrease from vehicle; addition of those two dose groups would have yielded a 15.1% decrease in body weight. In fact, a 26.2% decrease for the Combo EDgo group was observed, which is a statistically significant increase over the predicted additivity data (p ⁇ 0.05).
  • the Pair-fed Controls (pair-fed to Combo ED 80 group) demonstrated a 12.8%) loss in body weight that was comparable to the Combo ED 80 group (12.3% weight loss) over the same time period.
  • the Pair-fed Controls gained weight at about the same rate as the vehicle controls, while the Combo ED 80 group maintained their weight loss. This resulted in a net weight loss of 8.4% for the pair-fed group and 16.7% for the Combo ED 80 group (pO.01 vs. baseline for both groups).
  • This rebound effect and resulting differences in body weight at day 15 suggests that a difference in metabolism is emerging between the pair-fed group and the Combo ED 80 group after eight days that is attributable to the combination and not merely to effects on weight.
  • Example 10 Expression and chemical conjugation of Cys base AlbudAb and NMU8 peptide, respectively.
  • the Dom7h-14-10 (R108C) albudab can be expressed and purified as described as follows in E.coli:
  • the gene encoding the DOM7h-14-10 (R108C) can be cloned into vector pET30.
  • fusions can be produced as assembly PCRs with Ndel restriction site on 5' followed by the PEL B leader sequence (amino acid sequence shown in Figure 15 (i) SEQ ID NO 46).
  • Vector and assembly PCRs can be digested with Ndel and BamHI restriction endonucleases followed by ligation of the insert into the vector using a Quick Ligation Kit (NEB). 2 microlitres of this ligation can then be used for transformation of Machl cells.
  • NEB Quick Ligation Kit
  • cells can be plated on agar plates containing Kanamycin and incubated at 37°C overnight. Colonies can then be sequenced and those containing the correct sequence used for plasmid propagation and isolation (Plasmid Mini Prep kit, Qiagen).
  • Kanamycin. At A600 0.5-1 cells can be induced with IPTG to 50uM final concentration, and growth continued at 23 degC overnight. Then the culture supernatant can be clarified by centrifugation at 3700xg for 1 hour.
  • a Protein L Streamline column was equilibrated with PBS, washed with 6M Guanidine Hydrochloride and re-equilibrated into PBS. 1L of clarified E.coli supernatant containing Dom7h-14-10R108C was applied to the column. The column was washed with PBS, then 20mM Tris, pH7.4 and finally eluted with 0.1M Glycine. The elution pool was neutralised by the addition of l/5 th volume of Tris, pH8 and filter sterilised.
  • the sample was pH adjusted to pH5 with 0.1M Citric acid and applied to a 30ml Source S column, previously cleaned with 0.1M NaOH and equilibrated with 50mM Sodium Citrate, pH5. Post sample application a 0-100% gradient of 50mM Sodium Citrate, pH5, 1M NaCl was performed over 150ml, collecting fractions with an A280 >5mAus.
  • Fractions containing protein as determined by SDS-PAGE analysis were pooled and filter sterilised. The protein was concentrated, pooled and desalted into 50mM Sodium Phosphate, pH6.5, 5mM EDTA on a 1L custom packed desalting column which had been previously cleaned with 100ml of 1M NaOH.
  • the Dom7h-14-10 (R108C) Albudab so produced can then be linked to a NMU-8 amino acid molecule (but with the N-terminal tyrosine derivatised with PEG linker) using the PEG linker shown in the figure 3b.
  • the NMU-8 and the PEG are prepared by standard chemical synthesis.
  • the maleimide at the end of the PEG linker was used to conjugate the NMU peptide to the free cysteine of the Dom7h-14-10 (R108C) Albudab prepared as described above.
  • the albudab solution prepared above was diluted to 100ml (approx. lmg/ml) and mixed with the NMU-8 peptide at 1 :1 molar ratio.
  • a 5ml SP FF HiTrap column was sanitized using 0.1M NaOH. It was then equilibrated with 50mM Sodium Citrate, pH5 at 5ml/min and the conjugation reactions were diluted to between 1 in 2 to 1 in 5 with equilibration buffer. The pH adjusted protein was applied to the SP FF column and after sample application the column was washed with equilibration buffer. The column was then subjected to a 0-100% gradient of 50mM Sodium Citrate, pH5, 450mM NaCl over 450ml. Fractions were collected and analysed by SDS-PAGE and the samples were stored at 4 degrees.
  • the protein was concentrated, filter sterilized into 0.5ml aliquots in cryovials and frozen at -80 degrees.
  • Example 11 Single AlbudAb fusions were made with both Exendin-4 and peptide YY:
  • Proteins were removed from storage at 4 degrees and DAT0116R108C was concentrated in 2X20ml concentrators to 12.5ml. DTT was added to final concentration 5mM and samples were incubated for 15 minutes. Proteins were then desalted into 20mM Bis Tris, pH6.57, 5mM EDTA, 10% Glycerol. Desalted fractions were pooled and for the R108C derivatives 1/10 th volume (approx. 2mgs) was added to 50ml falcon tubes containing n-ethylmaleimide. The remaining pooled protein was added to various masses of PYY peptide (batch ' 190') in 50ml falcons. The samples were incubated rolling at room temperature for 30 minutes, spun for 10 minutes in a bench top centrifuge at 4,500rpm, analysed by SDS-PAGE and then stored overnight at 4 degrees.
  • Samples were diluted 1/5 with 50mM Sodium Acetate, pH4.5 and applied to 2X6ml Resource S columns (previously cleaned with 0.5M NaOH and equilibrated with dilution buffer) at 2.5ml/min. Post samples application the column was washed with dilution buffer and then subjected to a 0-100% gradient with 50mM Sodium Acetate, pH4.5, 1M NaCl. The column was then washed with 2XPBS and finally cleaned with 0.5M NaOH. The samples were either pooled and stored at 4 degrees over the weekend in Sodium Acetate, pH4.5, and containing NaCl or were stored as fractions at 4 degrees over the weekend in Sodium Acetate,and containing NaCl pH4.5.
  • the Sodium Acetate fractions and the 2XPBS fractions were concentrated separately in multiple 20ml centrifugal concentrators, analysed by SDS-PAGE, filter sterilized and dialysed against 2X2L Sodium Citrate, pH6, lOOmM NaCl.
  • the two blocked samples were dialysed overnight (using a 3.5kDa cut off) against 2L IXPBS in an attempt to remove n-Ethyl maleimide. They were then filter sterilized and stored at 4 degrees.
  • the 2XPBS fractions of DAT01 15V15C:190 PYY were concentrated in several 20ml centrifugal concentrators, filter sterilized and stored at 4 degrees.
  • the proteins were submitted for MS analysis. Due to slight contamination of the DATOl 15R108C: 190PYY and DATOl 16R108C: 190PYY with peptide these proteins and the corresponding Sodium Acetate fraction pools were reapplied to a Protein L column.
  • a lml Protein L column was equilibrated with 1XPBS and cleaned with 6M Guanidine HC1.
  • the column was re-equilibrated with 1XPBS at 2ml/min and the DATOl 15R108C: 190 PYY Sodium Acetate elution pool was applied.
  • Post application the column was washed with lOOmM Sodium Citrate, pH6 and finally eluted with lOOmM Citric acid with a pH of 2.6.
  • the column was re-equilibrated with lOOmM Sodium Citrate, pH6 and the 2XPBS elution pool was applied and purified in a similar manner.
  • the column was cleaned with 6M Guanidine HC1 and the process was repeated for the DATOl 16R108C: 190 PYY derivatives.
  • the proteins were concentrated to between 1-1.5ml and were dialysed into 1.6L 50mM Sodium Acetate, pH6, 1 OOmM NaCl overnight at room temperature. The following morning the proteins were withdrawn from the dialysis cassettes, the OD measured, 200ul concentrated to 20ul for SDS-PAGE analysis. Only BH120608-02 and BH120608-04 were considered clean enough to assay and so the remaining material was filter sterilized and submitted for assay and Biacore QC.
  • Example 12 Expression of DOM7h-14-10 AlbudAb and PYY genetic fusion:
  • PYY 3-36 with an additional glycine introduced at the C-terminal was cloned as a fusion with DOM7h- 14- 10 (a domain antibody (dAb) which binds serum albumin (albudab) with an amino acid sequence shown below) into the pET30a vector (obtainable from Novagen
  • the PYY was at the 3' end of the construct and the dAb at the 5' end.
  • a TVAAPS linker was also introduced between the dAb and PYY sequence; the linker was included as a spacer to separate the dAb spatially from the PYY to prevent steric hinderence of the binding between the PYY and the NP receptor. The sequence of this construct is shown below.
  • Plasmid DNA was prepared in E.coli using alkaline lysis (using a miniprep kit, obtainable from Qiagen CA) and used to transform BL21(DE3) cells (obtainable from Invitrogen). A singly colony was picked and grown overnight at 37 °C in 100 ml of TB media at and then used to inoculate a 1 L culture via a 1/100 dilution. This culture was grown until the OD reached 0.7, at which point protein expression was induced by the addition of IPTG to a final concentration of 70 ⁇ . The culture was grown overnight at 23 °C then harvested by centrifugation and the pellet was stored at -20 °C.
  • inclusion bodies were prepared by lysing the cells with Bugbuster mix (12.5ml lOx bugbuster (Merck), 112.5 ml PBS, 250 ⁇ lysonase (Merck) and 4 complete protease inhibitor tablets (Roche).
  • Bugbuster mix (12.5ml lOx bugbuster (Merck), 112.5 ml PBS, 250 ⁇ lysonase (Merck) and 4 complete protease inhibitor tablets (Roche).
  • a pellet derived from 500 ml culture was resuspended in 100 ml bugbuster mix and incubated at room temperature for 30 minutes with agitiation then centrifuged at 32000g for 20 minutes, and the supernatant was discarded.
  • the pellet was washed in 2 M urea in PBS then centrifuged at 32000 g for 15 minutes and the supernatant was discarded.
  • the pellet was then resuspended in 1/12.5 of the original culture volume of 8 M urea in buffer B (100 mM NaCl, 100 niM Tris-HCl pH 8.0, 5% glycerol), agitated at room temperature for 1 hour and then centrifuged at 16000 rpm for 15 minutes.
  • the supernatant (inclusion body prep) was stored at 4 °C.
  • refolding buffer 100 mM MES pH 6.0, 60 mM NaCl, 0.001% triton-XlOO
  • buffer Z 20 mM sodium citrate pH 5.0 + 1 M NaCl. Thereafter protein was buffer- exchanged into 20 mM sodium citrate pH 6.2 plus 100 mM NaCl, concentrated and stored at - 80 °C.
  • NMU genetic fusions can be prepared in the same manner as described above for PYY.
  • Example 13 PYY 13-36 albudab in combination with NMU- AlbudAb (made as described in example 10) effects multiple parameters in diet induced obese (DIO) mice:
  • PYY 13-36 albudab was nade in the same way as described previously for making the PYY3- 36 albudab except the peptide PYY comprised only amino acid residues 13-36 instead of 3-36.
  • Male diet induced obese (DIO) C57BL/6 mice (Taconic, Hudson, NY) were used for all experiments. DIO mice were single-housed and maintained at constant temperature and humidity (approximately 22°C and 50% respectively) with 12 hr light/dark cycle (lights on from 5:00 AM to 5:00 PM). Mice were given ad libitum access to food (Research Diets D 12451 , 45% fat for DIO) and water.
  • NMU+PYY 13-36 Combo were given a dose of: the NMU- AlbudAb at 0.48 mg/kg every other day and a daily dose of the PYY 13-36- AlbudAb at 0.37 mg/kg.
  • a one day vehicle lead in period was used before the start of drug.
  • Baseline fat mass and lean mass measurements were taken four days before the start of drug and 6 days after the start of drug using a QMR instrument (Echo Medical Systems, Houston, TX.)
  • Body weight measurements were taken every Monday, Wednesday, and Friday starting four days before the first drug dose, with the first measurement, along with the body composition data, being used to randomize the animals.
  • Food hopper weights were measured frequently starting four days before the first drug dose, allowing for the calculation of food intake. Animals that created excessive food spillage were removed prior to the beginning of the study. During the study, excess food was removed from the cage and added to the food hopper weights for increased accuracy.
  • Table 7 shows Weight loss (change in body weight) , in DIO C57BL6 mice treated with NMU-8 AlbudAb and PYY 13-36 AlbudAb. Table 7:
  • NMU8 with an additional glycine introduced at the C-terminus was cloned as a fusion with DOM7h-14-10 (a domain antibody (dAb) which binds serum albumin (albudab) with an amino acid sequence shown below) into the pTT5 vector (obtainable from CNRC, Canada).
  • the NMU8 was at the 3 ' end of the construct and the dAb at the 5' end.
  • a TVAAPS linker was also introduced between the dAb and NMU8 sequence; the linker was included as a spacer to separate the dAb spatially from the NMU8 to prevent steric hindrance of the binding between the NMU8 and the NMU receptor (AXOR-13 and AXOR-34). The sequence of this construct is shown below:
  • Endotoxin free plasmid DNA was prepared in E.coli using alkaline lysis (the endotoxin free plasmid Mega kit, obtainable from Qiagen CA) and used to transfect HEK293E cells
  • the concentrated sample was dialysed at 4°C, for a minimum of 4h, twice, against a 100X sample volume of 0.02M sodium citrate, 0.1M NaCl, pH6.2 buffer.
  • the dialysed protein sample was applied to a Superdex-75 column (GE Healthcare Life Sciences) pre-equilibrated in 0.02M sodium citrate, O. IM NaCl, pH6.2 buffer. Fractions of correct size were identified by SDS-PAGE electrophoresis and were combined to make the final protein sample. Pooled samples were concentrated by ultrafiltration. The identity of pooled samples was confirmed as predominantly full-length unmodified protein by intact mass analysis.
  • PAM monooxygenase
  • Purified 8 ⁇ DMS7625 was incubated at ambient temperature for 2h in lOOmM MES pH 6.0, 0.001% Triton X-100, 30mM NaCl, 1% Ethanol, 2.5 mM sodium ascorbate and ⁇ copper chloride containing 20 ⁇ g/ml bovine catalase and 80nM peptidylglycine alpha- amidating monooxygenase.
  • the amidation reaction was concentrated by ultrafiltration to approximately 1/10 th of the reaction volume then dialysed against a 400X sample volume of 0.02M sodium citrate, 0.1M NaCl, pH6.2 buffer.
  • DMS7641 Activity of chemical conjugation (DMS7641), genetic fusion (DMS7625) and C-terminus amidated genetic fusion (DMS7652) ofDom7h-14-10 andNMU8 in melanophore cell-based functional assay
  • Melanophore assays are a sensitive screening platform for quantifying 7TM receptor-ligand interactions (Jayawickreme, 2005).
  • the melanophores can rapidly switch their melanosomes between two states, aggregated or dispersed, depending on intracellular cAMP or
  • DAG diacylglycerol
  • the melanophore cell colour reflects cAMP or DAG levels because it controls the molecular motor(s) responsible for positioning pigment within the cell.
  • Pigment translocation in melanophores can easily be detected within a few minutes following the activation of effector molecules, thereby providing a fast, sensitive and versatile reporter technology.
  • Gs/Gq coupled receptors activate either phospholipase-C (PLC) or protein kinase- A (PKA) resulting in pigment dispersion and Gi/Go coupled receptors inhibit PKA resulting in pigment aggregation.
  • PLC phospholipase-C
  • PKA protein kinase- A
  • Gi/Go coupled receptors inhibit PKA resulting in pigment aggregation.
  • the coupling of GPCRs to translocate melanosomes in the melanophores is the most well characterized signalling pathway.
  • NMU receptors are Gq/11 -coupled receptor and therefore increase intracellular DAG leading to
  • Melanophore receptor binding cell-based functional assays were performed as detailed in the methods below. Melanophore cells were washed, trypsinized and re-suspended in 0.7x PBS at a concentration of 15xl0 6 cells per ml. An 800 ⁇ 1 volume of cells was gently mixed with 40 ⁇ g of receptor cDNA and incubated on ice for 20 minutes. Following incubation, 800 ⁇ 1 of cells/cDNA mix were pipetted into a cuvette and electroporated at 500V, 725 ⁇ and 725 ohms.
  • Cells were then transferred from the cuvette directly into a T75 flask containing RFM (regular frog media) and incubated overnight in an incubator (25 °C, 0% C0 2 ). The next day, cells were trypsinized, counted and added to 96 half well plates at a density of 18,000 cells per well. Plates were placed in a sealed container and incubated overnight (25 °C, 0% C0 2 ). The following day media was aspirated and 25 ⁇ MAB (Melanophore Assay Buffer) containing 1% DMSO was added to each well. Cells were incubated for one hour and basal absorbance was measured on a SLT Spectra plate reader at 620nm.
  • RFM regular frog media
  • a dilution series (12 point series using 3 fold dilution intervals in MAB) of each sample, or standard, was prepared and 25 ⁇ was added directly to each well.
  • lOnM melatonin and MAB standards were included and used to establish the assay system maximum and basal response levels, respectively.
  • plates were incubated for 2 hour and absorbance measured as before. Data was analyzed using Robosage (GSK Excel add-in) by calculating (1-Tf/Ti) where Ti is initial baseline read, and Tf is the response read.
  • NMU8 peptide concentration curves were included as a control to show the correct functioning of the assay as a readout of AXOR 13 and AXOR34 receptor activity.
  • the GLP 1 was at the 5' end of the construct, the NMU8 was at the 3' end and the dAb at the centre.
  • a helical linker ((KEAAA x 3, KELAA) x 2) was introduced between GLP-1 and the dAb and a TVAAPS linker was also introduced between the dAb and NMU8 sequence; the linkers were included as spacers to separate the dAb spatially from the GLP1 and NMU8 to prevent steric hindrance of the binding between the GLP1, or NMU8 and the GLP1, or NMU receptors, respectively.
  • Endotoxin free plasmid DNA was prepared in E.coli using alkaline lysis (the endotoxin free plasmid Mega kit, obtainable from Qiagen CA) and used to transfect HEK293E cells
  • Bound protein was eluted from the column with 10 column volumes of 0.1M glycine.HCl, 50% ethylene glycol, pH2.0, collected as 5 1 column volume fractions and a final 5 column volume fraction.
  • the pH of the eluted protein solution was titrated to pH5.0 with an approximate 1/4* volume of 1M Tris.HCl, pH8.0.
  • PAM monooxygenase
  • Purified 8 ⁇ DMS7625 was dialysed overnight against 50mM sodium citrate, pH5.0. The dialysed protein was concentrated by ultrafiltration then incubated at 8 ⁇ at ambient temperature for 2h in lOOmM MES pH 6.0, 0.001% Triton X-100, 30mM NaCl, 1% Ethanol, 2.5 mM sodium ascorbate and ⁇ copper chloride containing 20 ⁇ g/ml bovine catalase and 80nM peptidylglycine alpha-amidating monooxygenase.
  • Melanophore assays are a sensitive screening platform for quantifying 7TM receptor-ligand interactions.
  • the melanophores can rapidly switch their melanosomes between two states, aggregated or dispersed, depending on intracellular cAMP or diacylglycerol (DAG) levels.
  • DAG diacylglycerol
  • the melanophore cell colour reflects cAMP or DAG levels because it controls the molecular motor(s) responsible for positioning pigment within the cell. Pigment translocation in melanophores can easily be detected within a few minutes following the activation of effector molecules, thereby providing a fast, sensitive and versatile reporter technology.
  • Gs/Gq coupled receptors activate either phospholipase-C (PLC) or protein kinase-A (PKA) resulting in pigment dispersion and Gi/Go coupled receptors inhibit PKA resulting in pigment aggregation.
  • PLC phospholipase-C
  • PKA protein kinase-A
  • Gi/Go coupled receptors inhibit PKA resulting in pigment aggregation.
  • GLP1 receptors are Gs- coupled and therefore increase cAMP and NMU receptors are Gq/1 1 -coupled and therefore increase intracellular DAG each leading to melanosome dispersion.
  • Melanophore receptor binding cell-based functional assays were performed as detailed in the methods below. Melanophore cells were washed, trypsinized and re-suspended in 0.7x PBS at a concentration of 15xl0 6 cells per ml. An 800 ⁇ 1 volume of cells was gently mixed with 40 ⁇ g of receptor cDNA and incubated on ice for 20 minutes. Following incubation, 800 ⁇ 1 of cells/cDNA mix were pipetted into a cuvette and electroporated at 500V, 725 ⁇ and 725 ohms.
  • Cells were then transferred from the cuvette directly into a T75 flask containing RFM (regular frog media) and incubated overnight in an incubator (25°C, 0% C0 2 ). The next day, cells were trypsinized, counted and added to 96 half well plates at a density of 18,000 cells per well. Plates were placed in a sealed container and incubated overnight (25°C, 0% C0 2 ). The following day media was aspirated and 25 ⁇ MAB (Melanophore Assay Buffer) containing 1% DMSO was added to each well. Cells were incubated for one hour and basal absorbance was measured on a SLT Spectra plate reader at 620nm.
  • RFM regular frog media
  • a dilution series (12 point series using 3 fold dilution intervals in MAB) of each sample or standard was prepared and 25 ⁇ was added directly to each well.
  • lOnM melatonin and MAB standards were included and used to establish the assay system maximum and basal response levels, respectively.
  • plates were incubated for 2 hour and absorbance measured as before. Data was analyzed using Robosage (GSK Excel add- in) by calculating (1-Tf/Ti) where Ti is initial baseline read, and Tf is the response read.
  • NMU8 peptide concentration curves were included as a control to show the correct functioning of the assay as a readout of AXOR13 and AXOR34 receptor activity.

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Abstract

La présente invention concerne des protéines de fusion et des conjugués de médicaments, qui ont des demi-vies améliorées dans le sérum. Ces protéines de fusion et conjugués comprennent des domaines variables uniques d'immunoglobuline (anticorps) et des molécules de peptides insulinotropes et/ou d'incrétine et/ou intestinaux. L'invention concerne en outre des utilisations, formulations, compositions et dispositifs comprenant de telles protéines de fusion et de tels conjugués de médicaments. L'invention concerne également des compositions qui comprennent plus d'une molécule de peptide insulinotrope et/ou d'incrétine et/ou intestinal présente comme partie de protéine de fusion ou d'un conjugué et leurs utilisations et formulations.
PCT/EP2012/056327 2011-04-07 2012-04-05 Compositions comprenant des protéines de fusion ou des conjugués ayant une demi-vie améliorée WO2012136790A1 (fr)

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EP3810186B1 (fr) 2018-06-21 2022-09-28 Novo Nordisk A/S Nouveaux composés pour le traitement de l'obésité
WO2022117044A1 (fr) * 2020-12-03 2022-06-09 Sunshine Lake Pharma Co., Ltd. Polypeptide agoniste du récepteur double glp-1/gcg et sa protéine de fusion
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