Title of Invention

BISPECIFIC DOMAIN ANTIBODIES TARGETING SERUM ALBUMIN AND GLP-1 OR PYY

Abstract In the fields of diabetes and obesity, there is a need to modify GLP-I and other insulinotropic peptides to provide longer duration of action in vivo while maintaining their low toxicity and therapeutic advantages. The invention relates to drug fusions having improved serum half lives and have the formula: a-(X)n1-b-(Y)n2-c-(Z)n3-d or a-(Z)n3-b-(Y)n2-c-(X)n1-d, wherein X is an insulinotropic agent or an analogue thereof; Y is an immunoglobulin heavy chain variable domain (VH) that has binding specificity for serum albumin, or an immunoglobulin light chain variable domain (VL) that has binding specificity for serum albumin; Z is a polypeptide drug that has binding specificity for a second target; a, b, c and d are each independently a polypeptide comprising one to about 100 amino acid residues or absent; n1 and n2 are one to about 10; and n3 is zero to about 10.
Full Text WO 2006/059106 PCT/GB2005/004599
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BISPECIFIC DOMAIN ANTIBODIES TARGETING SERUM ALBUMIN AND GLP-1 OR PYY
This application claims the benefit of U.S. Provisional Patent Application No.
60/632,361, filed on December 2, 2004 and the benefit of GB Patent Application No.
0511019.2. The entire teachings of the above applications are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
Many drugs that possess activities that could be useful for therapeutic and/or
diagnostic purposes have limited value because they are rapidly eliminated from the
body when administered. For example, many polypeptides that have therapeutically
useful activities are rapidly cleared from the circulation via the kidney. Accordingly, a
large dose must be administered in order to achieve a desired therapeutic effect. A need
exists for improved therapeutic and diagnostic agents that have improved
pharmacokinetic properties. Polypeptides that bind serum albumin are known in the art.
(See, e.g., EP 0486525 Bl (Cemu Bioteknik AB); US 6,267,964 Bl (Nygren et al);
WO 04/001064 A2 (Dyax, Corp.); WO 02/076489 Al(Dyax, Corp.); WO 01/45746
(Genentech, Inc.).)
One such class of drugs that have a short half life in the body or systemic
circulation is the incretin hormones such as Glucagon-like peptide 1, or Peptide YY.
Glucagon-like peptide (GLP)-l is an incretin hormone with potent glucose-
dependent insulinotropic and glucagonostatic actions, trophic effects on the pancreatic β
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. Taken together, these actions give GLP-1 a unique profile,
considered highly desirable for an antidiabetic agent, particularly since the glucose
dependency of its antihyperglycemic effects should minimize any risk of severe
hypoglycemia. However, its pharmacokinetic/pharmacodynamic profile is such that
native GLP-1 is not therapeutically useful. Thus, while GLP-1 is most effective when

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administered continuously, single subcutaneous injections have short-lasting effects.
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. Strategies for harnessing GLP-1 's
therapeutic potential, based on an understanding of factors influencing its metabolic
stability and pharmacokinetic/pharmacodynamic profile, have therefore been the focus
of intense research.
Extensive work has been done to attempt to inhibit the peptidase or to modify
GLP-1 in such a way that its degradation is slowed down while still maintaining
biological activity. WO05/027978 discloses GLP-1 derivatives having a protracted
profile of action (and incorporated herein by reference as examples of GLP-1
derivatives and analogues that can be used in the present invention). WO 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).
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.
However, despite these efforts a long lasting active GLP-1 has not been
produced.
As such, particularly in the fields of diabetes and obesity, there is a tremendous
need for improved GLP-1 peptides or other agents that similarly have an insulinotropic
effect amenable to treatment for diabetes and obesity in particular. There is thus a need
to modify GLP-1 and other insulinotropic peptides to provide longer duration of action
in vivo while maintaining their low toxicity and therapeutic advantages.
SUMMARY OF THE INVENTION
The invention relates to drug fusions and drug conjugates that have improved
serum half lives. In one aspect, the drug fusion is a continuous polypeptide chain
having the formula:


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Y is an immunoglobulin heavy chain variable domain (VH) that has binding
specificity for serum albumin, or an immunoglobulin light chain variable domain (VL)
that has binding specificity for serum albumin;
Z is a polypeptide drug that has binding specificity for a second target;
a, b, c and d are each independently absent or one to about 100 amino acid
residues;
nl is one to about 10;
n2 is one to about 10; and
n3 is zero to about 10,
with the proviso that when nl and n2 are both one and n3 is zero, X does not
comprise an antibody chain or a fragment of an antibody chain.
In some embodiments, Y comprises an amino acid sequence selected from the
group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 13,
SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26,
or an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ
ID NO:17, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID
NO:22 and SEQ ID NO:23. In particular embodiments, X is GLP-1 or a GLP-1
analogue.
In another aspect, the drug fusion comprises a continuous polypeptide chain,
said chain comprising moieties X' and Y', wherein
X' is a polypeptide drug, with the proviso that X' does not comprise an antibody
chain or a fragment of an antibody chain; and
Y' is an immunoglobulin heavy chain variable domain (VH) that has binding
specificity for serum albumin, or an immunoglobulin light chain variable domain (VL)
that has binding specificity for serum albumin. In some embodiments, Y' comprises an
amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID
NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID
NO:24, SEQ ID NO:25 and SEQ ID NO:26, or an amino acid sequence selected from
the group consisting of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID
NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23. In
particular embodiments, X' is GLP-1 or a GLP-1 analogue.

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In another aspect, the invention is a drug conjugate comprising an
immunoglobulin heavy chain variable domain (VH) that has binding specificity for
serum albumin, or an immunoglobulin light chain variable domain (VL) that has binding
specificity for serum albumin; and a drug that is covalently bonded to said VH or VL. In
some embodiments, the immunoglobulin heavy chain variable domain comprises an
amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID
NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID
NO:24, SEQ ID NO:25 and SEQ ID NO:26, or an amino acid sequence selected from
the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID
NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23. In
particular embodiments, the drug is GLP-1 or a GLP-1 analogue.
The invention also provides recombinant nucleic acids and constructs that
encode the drug fusions described herein, and host cells that comprise the recombinant
nucleic acids and/or constructs. The invention further provides a method for producing
a drug fusion comprising maintaining a host cell that comprises a recombinant nucleic
acid and/or construct that encodes a drug fusion described herein under conditions
suitable for expression of said recombinant nucleic acid, whereby a drug fusion is
produced.
The invention also provides compositions (e.g., pharmaceutical compositions)
comprising a drug fusion or drug conjugate of the invention. The invention also
provides a method for treating an individual having a disease or disorder, such as those
described herein, comprising administering to said individual a therapeutically effective
amount of a drug conjugate or drug fusion of the invention. In some embodiments, the
disease or disorder is an inflammatory disease, such as arthritis (e.g., rheumatoid
arthritis). In a further embodiment, the disease or disorder is a metabolic disease such as
diabetes or obesity. The invention also provides for use of a drug conjugate or drug
fusion of the invention for the manufacture of a medicament for treatment of a disease
or disorder, such as an inflammatory disease (e.g., arthritis (e.g., rheumatoid arthritis)),
or diabetes or obesity. The invention also relates to use of a drug fusion or drug
conjugate as described herein for use in therapy, diagnosis or prophylaxis.
In another aspect, the invention is a noncovalent drug conjugate comprising an
immunoglobulin heavy chain variable domain (VH) that has binding specificity for

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serum albumin, or an immunoglobulin light chain variable domain (VL) that has
binding specificity for serum albumin, and a drug that is noncovalently bonded to said
VH or VL. In some embodiments, the immunoglobulin heavy chain variable domain
comprises an amino acid sequence selected from the group consisting of SEQ ID
NO: 10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID
NO: 15, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, or an amino acid sequence
selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18,
SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23.
In a further embodiment, the invention provides an inactivated version of
Dom7h-8, iDom7h-8, which does not bind to serum albumin which is used as a research
tool and is predictive of the active serum albumin binding Dom7h-8.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is an alignment of the amino acid sequences of three V/cs selected by
binding to mouse serum albumin (MSA). The aligned amino acid sequences are from
VKS designated MSA16, which is also referred to as DOM7m-16 (SEQ ID NO:1), MSA
12, which is also referred to as DOM7m-12 (SEQ ID NO:2), and MSA 26, which is also
referred to as DOM7m-26 (SEQ ID NO:3).
FIG. 1B is an alignment of the amino acid sequences of six VKS selected by
binding to rat serum albumin (RSA). The aligned amino acid sequences are from VKS
designated DOM7r-l (SEQ ID NO:4), DOM7r-3 (SEQ ID NO:5), DOM7r-4 (SEQ ID
NO:6), DOM7r-5 (SEQ ID NO:7), DOM7r-7 (SEQ ID NO:8), and DOM7r-8 (SEQ ID
NO:9).
FIG. 1C is an alignment of the amino acid sequences of six VKS selected by
binding to human serum albumin (HSA). The aligned amino acid sequences are from
VKS designated DOM7h-2 (SEQ ID NO: 10), DOM7h-3 (SEQ ID NO: 11), DOM7h-4
(SEQ ID NO:12), DOM7h-6 (SEQ ID NO:13), DOM7h-l (SEQ ID NO: 14), DOM7h-7
(SEQ ID NO: 15).
FIG. 1D is an alignment of the amino acid sequences of seven VHS selected by
binding to human serum albumin and a consensus sequence (SEQ ID NO:23). The
aligned sequences are from VKS designated DOM7h-22 (SEQ ID NO: 16), DOM7h-23
(SEQ ID NO:17), DOM7h-24 (SEQ ID NO:18), DOM7h-25 (SEQ ID NO: 19),

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DOM7h-26 (SEQ ID NO:20), DOM7h-21 (SEQ ID NO:21), and DOM7h-27 (SEQ ID
NO:22).
FIG. 1E is an alignment of the amino acid sequences of three V/cs selected by
binding to human serum albumin and rat serum albumin. The aligned amino acid
sequences are from V/cs designated DOM7h-8 (SEQ ID NO:24), DOM7r-13 (SEQ ID
NO:25), and DOM7r-14 (SEQ ID NO:26).
FIG. 2A and 2B are schematics maps of the vectors used to express the
MSA16IL-lra (also referred to as DOM7m-16/IL-lra) and IL-lraMSA16 (also referred
to as IL-lra/DOM7m-16) fusions, respectively.
FIG. 2C-2D is an illustration of the nucleotide sequence (SEQ ID NO:27)
encoding the IL-lraMSA16 fusion (also referred to as IL-lra/DOM7m-16)_and of the
amino acid sequence (SEQ ID NO:28) of the fusion.
FIG. 2E-2F is an illustration of the nucleotide sequence (SEQ ID NO:29)
encoding the MSA16IL-lra fusion (also referred to as DOM7m-16/IL-lra)_and of the
amino acid sequence (SEQ ID NO:30) of the fusion.
FIG. 2G-2H is an illustration of the nucleotide sequence (SEQ ID NO:31)
encoding the DummylL-lra fusion that did not bind serum albumin, and of the amino
acid sequence (SEQ ID NO:32) of the fusion.
FIG. 3 A is an illustration showing that IL-1 induces the production of IL-8 by
HeLa cells, and showing the mechanism by which IL-8 is detected in an ELISA assay.
FIG. 3B is a graph showing that IL-lra (♦), MSA16IL-lra (■) and IL-lraMSA16
(A) each inhibited IL-1-induced secretion of IL-8 by cultured MRC-5 cells. The
observed inhibition was dose dependant for IL-lra, MSA16IL-lra and IL-lraMSA16.
FIGS. 4A-4C are graphs showing that IL-lra (♦) MSA16IL-lra (■) both
inhibited IL-1-induced secretion of IL-8 by cultured MRC-5 cells in assays that
included no mouse serum albumin (4A), 5% mouse serum albumin (4B) or 10% mouse
serum albumin (4C). The observed inhibition was dose dependant for IL-lra and
MSA16IL-lra under all conditions tested.
FIG. 5 is a schematic presentation of the results of an ELISA demonstrating that
the MSA16ILl-ra fusion and the IL-lraMSA16 fusion both bound serum albumin, but
the dummylLl-ra fusion did not.

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FIGS. 6A-6C are sensograms and tables showing BIACORE affinity data for
clone DOM7h-l binding to human serum albumin (HSA) (6A), DOM7h-7 binding to
HSA (6B) and DOM7r-l binding to rat serum albumin (RSA).
FIG. 7 is a table showing the affinities of DOM7h-l, DOM7r-l, DOM7h-2,
DOM7r-3, DOM7h-7, DOM7h-8, DOM7r-8, DOM7r-13, DOM7r-14, DOM7m-16,
DOM7h-22, DOM7h-23, DOM7h-26, DOM7r-16, DOM7m-26, DOM7r-27 and
DOM7R-31 for the serum albumins that they bind.
FIG. 8A is an illustration of the nucleotide sequence (SEQ ID NO:33) of a
nucleic acid encoding human interleukin 1 receptor antagonist (IL-lra) deposited in
GenBank under accession number NM_173842. The nucleic acid has an open reading
frame starting at position 65.
FIG. 8B is an illustration of the amino acid sequence of human IL-lra (SEQ ID
NO:34) encoded by the nucleic acid shown in FIG. 8A (SEQ ID NO:33). The mature
protein consists of 152 amino acid residues (amino acid residues 26-177 of SEQ ID
NO:34).
FIG. 9 is a graph showing the concentration (ug/mL) of MSA binding dAb/HA
epitope tag fusion protein in mouse serum following a single intravenous (i.v.) injection
(dose was about 1.5 mg/kg) into CD1 strain male animals over time (days). Serum
concentration was determined by ELISA using goat anti-HA (Abeam, UK) capture and
protein L-HRP (Invitrogen, USA) detection reagents. Standard curves of known
concentrations of MSA binding dAb/HA fusion were set up in the presence of lx mouse
serum to ensure comparability with the test samples. Modelling with a 1 compartment
model (WinNonlin Software, Pharsight Corp., USA) showed the MSA binding dAb/HA
epitope tag fusion protein had a terminal phase tl/2 of 29.1 hours and an area under the
curve of 559 hr-μg/mL.
FIG. 10 is an illustration of the amino acid sequences of the amino acid
sequences of VKS selected by binding to rat serum albumin (RSA). The illustrated
sequences are from V/cs designated DOM7r-15 (SEQ ID NO:37), DOM7r-16 (SEQ ID
NO:38), DOM7r-17 (SEQ ID NO:39), DOM7r-18 (SEQ ID NO:40), DOM7r-19 (SEQ
IDNO:41).
FIG. 11A-11B is an illustration of the amino acid sequences of the amino acid
sequences of Vks that bind rat serum albumin (RSA). The illustrated sequences are

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from VKS designated DOM7r-20 (SEQ ID NO:42), DOM7r-21 (SEQ ID NO:43),
DOM7r-22 (SEQ ID NO:44), DOM7r-23 (SEQ ID NO:45), DOM7r-24 (SEQ ID
NO:46), DOM7r-25 (SEQ ID NO:47), DOM7r-26 (SEQ ID NO:48), DOM7r-27 (SEQ
ID NO:49), DOM7r-28 (SEQ ID NO:50), DOM7r-29 (SEQ ID NO.51), DOM7r-30
(SEQ ID NO:52), DOM7r-31 (SEQ ID NO:53), DOM7r-32 (SEQ ID NO:54), DOM7r-
33 (SEQ ID NO:55).
FIG. 12 is a graph showing the concentration (% initial dose) of DOM7m-16,
DOM7m-26 or a control dAb that does not bind MSA, each of which contained an HA
epitope tag, in mouse serum following a single intravenous (i.v.) injection (dose was
about 1.5 mg/kg) into CD1 strain male animals over time. Serum concentration was
determined by ELISA using goat anti-HA (Abeam, UK) capture and protein L-HRP
(Invitrogen, USA) detection reagents. Standard curves of known concentrations of
MSA binding dAb/HA fusion were set up in the presence of lx mouse serum to ensure
comparability with the test samples. Modelling with a 1 compartment model
(WinNonlin Software, Pharsight Corp., USA) showed control dAb had a terminal phase
tl/2o! of 20 minutes, while DOM7m-16, DOM7m-26 persisted in serum significantly
longer.
FIG. 13 is a graph showing that DOM7m-16/IL-lra was more effective than IL-
lra or ENBREL® (entarecept; Immunex Corporation) in treating arthritis in a mouse
collagen-induced arthritis (CIA) model. Arthritis was induced and, beginning on day
21, mice were treated with Dexamethasone at 0.4 mg/Kg (Steroid), DOM7m-16/IL-lra
at 1 mg/Kg (IL-lra/anti-SA lmg/kg) or 10 mg/Kg (IL-lra/anti-SA 10 mg/kg), IL-lra at
1 mg/Kg or 10 mg/Kg, ENBREL® (entarecept; Immunex Corporation) at 5 mg/Kg, or
saline. The results show that DOM7m-16/IL-lra was more effective than IL-lra or
ENBREL® (entarecept; Immunex Corporation) in this study. The response to EL-Ira
was dose dependent, as expected, and that the response to DOM7m-16/IL-lra was also
dose dependent. The average scores for treatment with DOM7m-16/IL-lra at 1 mg/Kg
were consistently lower than the average scores obtained by treatment with IL-lra at 10
mg/kg. The results indicate that treatment with DOM7m-16/IL-lra was 10 times more
effective than IL-lra in this study.
FIGS. 14A-14G illustrate the amino acid sequences of saporin polypeptides.
FIG. 14A illustrates the amino acid sequence of saporin-2 precursor deposited as

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Swissprot Accession Number P27559 (SEQ ID NO:60). The signal peptide is amino
acids 1-24 of SEQ ID NO:60. FIG. 14B illustrates the amino acid sequence of saporin-
3 deposited as Swissprot Accession Number P27560 (SEQ ID NO:61). FIG. 14C
illustrates the amino acid sequence of saporin-4 precursor deposited as Swissprot
Accession Number P27561 (SEQ ID NO:62). The signal peptide is amino acids 1-24 of
SEQ ID NO:62. FIG. 14D illustrates the amino acid sequence of saporin-5 deposited as
Swissprot Accession Number Q41389 (SEQ ID NO:63). FIG. 14E illustrates the amino
acid sequence of saporin-6 precursor deposited as Swissprot Accession Number P20656
(SEQ ID NO:64). The signal peptide is amino acids 1-24 of SEQ ID NO:64, and a
potential propeptide is amino acids 278-299 of SEQ ID NO:64. The mature polypeptide
is amino acids 25-277 of SEQ ID NO:64 (SEQ ID NO:65). FIG. 14F illustrates the
amino acid sequence of saporin-7 deposited as Swissprot Accession Number Q41391
(SEQ ID NO:66). FIG. 14G illustrates a consensus amino acid sequence encompassing
several variants and isoforms of saporin-6 (SEQ ID NO:67).
FIG. 15 illustrates the amino acid sequences of several Camelid VHHS that bind
mouse serum albumin that are disclosed in WO 2004/041862. Sequence A (SEQ ID
NO:72), Sequence B (SEQ ID NO:73), Sequence C (SEQ ID NO:74), Sequence D
(SEQ ID NO:75), Sequence E (SEQ ID NO:76), Sequence F (SEQ ID NO:77)5
Sequence G (SEQ ID NO:78), Sequence H (SEQ ID NO:79), Sequence I (SEQ ID
NO:80), Sequence J (SEQ ID NO:81), Sequence K (SEQ ID NO:82), Sequence L (SEQ
ID NO:83), Sequence M (SEQ ID NO:84), Sequence N (SEQ ID NO:85), Sequence O
(SEQ ID NO:86), Sequence P (SEQ ID NO:87), Sequence Q (SEQ ID NO:88).
FIG 16A is an illustration of the nucleotide sequence encoding the [Pro9]GLP-l-
Dom7h8 fusion (SEQ ID NO: 175) and of the amino acid sequence of the fusion (SEQ
ID NO:176).
FIG 16B is an illustration of the nucleotide sequence encoding the [Pro9]GLP-l-
PSS-Dom7h8 fusion (SEQ ID NO: 177) and of the amino acid sequence of the fusion
(SEQ ID NO: 178).
FIG 16C is an illustration of the nucleotide sequence encoding the [Pro9]GLP-l-
PSSGAP-Dom7h8 fusion (SEQ ID NO: 179) and of the amino acid sequence of the
fusion (SEQ ID NO: 180).

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FIG 17 is a graph showing that [Pro9]GLP-l-PSSGAP-Dom7h8 fusion (□) had
an equivalent dose dependent cell proliferation activity to GLP-1 control, (A), Exendin-
4 (▼). Basal zero control is shown (♦).
FIG 18 is a graph showing that that [Pro9]GLP-l-PSSGAP-Dom7h8 fusion (D)
had an equivalent dose dependent insulin release to GLP-1 control, (A), Exendin-4
(▼). Basal zero control is shown (♦).
FIG 19A-19C illustrates the amino acid sequence of Dom7h-8 PYY (3-36) (SEQ
ID NO:181), PYY (3-36) DOM7h-8 (SEQ ID NO:182) and [Pro9]GLP-l(3-37)-DOM
7h-8 PYY (3-36) (SEQ ID NO: 183) fusions respectively.
DETAILED DESCRIPTION OF THE INVENTION
Within this specification embodiments have been described in a way which
enables a clear and concise specification to be written, but it is intended and will be
appreciated that embodiments may be variously combined or separated without parting
from the invention.
As used herein, "drug" refers to any compound (e.g., small organic molecule,
nucleic acid, polypeptide) that can be administered to an individual to produce a
beneficial therapeutic or diagnostic effect though binding to and/or altering the function
of a biological target molecule in the individual. The target molecule can be an
endogenous target molecule encoded by the individual's genome (e.g., an enzyme,
receptor, growth factor, cytokine encoded by the individual's genome) or an exogenous
target molecule encoded by the genome of a pathogen (e.g., an enzyme encoded by the
genome of a virus, bacterium, fungus, nematode or other pathogen).
As used herein the term "drug basis" refers to activities of drug compositions
and drugs that are normalized based on the amount of drug (or drug moiety) used to
assess, measure or determine activity. Generally, the drug compositions of the
invention (e.g., drug conjugate, noncovalent drug conjugate, drug fusion) have a larger
molecular weight than the drug they contain. Thus, equivalent amounts of drug
composition and drug, by weight, will contain different amounts of drug on a molecular
or molar basis. For example, if a drug composition of the invention has a molecular
weight that is twice the molecular weight of the drug it comprises, activities can be
determined on a "drug basis" using 2 μg of drug composition and 1 jug of drug, because

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these quantities would contain the same amount of drug (as free drug or as part of the
drug composition). Activities can be normalized and expressed on a "drug basis" using
appropriate calculations, for example, by expressing activity on a per target binding site
basis or, for enzyme drugs, on a per active site basis.
As used herein, "drug composition" refers to a composition comprising a drug
that is covalently or noncovalently bonded to a polypeptide binding moiety, wherein the
polypeptide binding moiety contains a binding site (e.g., an antigen-binding site) that
has binding specificity for a polypeptide that enhances serum half-life in vivo. The drug
composition can be a conjugate wherein the drug is covalently or noncovalently bonded
to the polypeptide binding moiety. The drag can be covalently or noncovalently bonded
to the polypeptide binding moiety directly or indirectly (e.g., through a suitable linker
and/or noncovalent binding of complementary binding partners (e.g., biotin and
avidin)). When complementary binding partners are employed, one of the binding
partners can be covalently bonded to the drag directly or through a suitable linker
moiety, and the complementary binding partner can be covalently bonded to the
polypeptide binding moiety directly or through a suitable linker moiety. When the drug
is a polypeptide or peptide, the drug composition can be a fusion protein, wherein the
polypeptide or peptide drug and the polypeptide binding moiety are discrete parts
(moieties) of a continuous polypeptide chain.
As used herein "conjugate" refers to a composition comprising an antigen-
binding fragment of an antibody that binds serum albumin that is bonded to a drag.
Such conjugates include "drag conjugates," which comprise an antigen-binding
fragment of an antibody that binds serum albumin to which a drag is covalently bonded,
and "noncovlaent drag conjugates," which comprise an antigen-binding fragment of an
antibody that binds serum albumin to which a drag is noncovalently bonded.
As used herein, "drug conjugate" refers to a composition comprising an antigen-
binding fragment of an antibody that binds serum albumin to which a drug is covalently
bonded. The drag can be covalently bonded to the antigen-binding fragment directly or
indirectly through a suitable linker moiety. The drag can be bonded to the antigen-
binding fragment at any suitable position, such as the amino-terminus, the carboxyl-
terminus or through suitable amino acid side chains (e.g., the e amino group of lysine, or
thiol group of cysteine).

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As used herein, "noncovalent drug conjugate" refers to a composition
comprising an antigen-binding fragment of an antibody that binds serum albumin to
which a drug is noncovalently bonded. The drug can be noncovalently bonded to the
antigen-binding fragment 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 drug and
the complementary binding partner is covalently bonded to the antigen-binding
fragment). When complementary binding partners are employed, one of the binding
partners can be covalently bonded to the drug directly or through a suitable linker
moiety, and the complementary binding partner can be covalently bonded to the
antigen-binding fragment of an antibody that binds serum albumin directly or through a
suitable linker moiety.
As used herein, "drug fusion" refers to a fusion protein that comprises an
antigen-binding fragment of an antibody that binds serum albumin and a polypeptide
drug. The antigen-binding fragment of an antibody that binds serum albumin and the
polypeptide drug are present as discrete parts (moieties) of a single continuous
polypeptide chain.
The term "albumin binding residue" as used herein means a residue which binds
non-covalently to human serum albumin. The albumin binding residue attached to the
therapeutic polypeptide typically has an affinity below 10 uM to human serum albumin
and preferably below 1 pM. In on embodiment, a range of albumin binding residues are
known among linear and branched lipohophillic moieties containing 4-40 carbon atoms,
compounds with a cyclopentanophenanthrene skeleton, peptides having 10-30 amino
acid residues etc.
As used herein "interleukin 1 receptor antagonist" (IL-lra) refers to
naturally occurring or endogenous mammalian IL-lra proteins and to proteins having an
amino acid sequence which is the same as that of a naturally occurring or endogenous
corresponding mammalian EL-lra protein (e.g., recombinant proteins, synthetic proteins
(i.e., produced using the methods of synthetic organic chemistry)). Accordingly, as
defined herein, the term includes mature protein, polymorphic or allelic variants, and
other isoforms of a IL-lra (e.g., produced by alternative splicing or other cellular
processes), and modified or unmodified forms of the foregoing (e.g., lipidated,

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glycosylated, PEGylated). Naturally occurring or endogenous IL-lra include wild type
proteins such as mature IL-lra, polymorphic or allelic variants and other isoforms
which occur naturally in mammals (e.g., humans, non-human primates). Such proteins
can be recovered or isolated from a source which naturally produces IL-lra, for
example. These proteins and IL-lra proteins having the same amino acid sequence as a
naturally occurring or endogenous corresponding IL-lra, are referred to by the name of
the corresponding mammal. For example, where the corresponding mammal is a
human, the protein is designated as a human IL-lra.
"Functional variants" of IL-lra include functional fragments, functional
mutant proteins, and/or functional fusion proteins which can be produce using suitable
methods (e.g., mutagenesis (e.g., chemical mutagenesis, radiation mutagenesis),
recombinant DNA techniques). A "functional variant" antagonizes interleukin-1 type 1
receptor. Generally, fragments or portions of IL-lra include those having a deletion
and/or addition (i.e., one or more amino acid deletions and/or additions) of an amino
acid (i.e., one or more amino acids) relative to the mature IL-lra (such as N-terminal, C-
terminal or internal deletions). Fragments or portions in which only contiguous amino
acids have been deleted or in which non-contiguous amino acids have been deleted
relative to mature IL-lra are also envisioned. A functional variant of human IL-lra can
have at least about 80%, or at least about 85%, or at least about 90%, or at least about
95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least
about 99% amino acid sequence identity with the mature 152 amino acid form of human
IL-lra and antagonize human Interleukin-1 type 1 receptor. (See, Eisenberg et ah,
Nature 343:341-346 1990). The variant can comprise one or more additional amino
acids (e.g., comprise 153 or 154 or more amino acids). For example, the variant IL-lra
can have an amino acid sequence that consists of an amiiio-termmal methionine residue
followed by residues 26 to 177 of SEQ ID NO:33. (KINERET® (anakinra), Amgen).
As referred to herein, the term "about" is optional, but is preferably interpreted
to mean plus or minus 20%, more preferably plus or minus 10%, even more preferably
plus or minus 5%, even more preferably plus or minus 2%, most preferably plus or
minus 1%.
The term "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

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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. A simple system is used to describe analogues of GLP-1: For
example [Arg34] GLP-1 (7-37) Lys designates a GLP-1 analogue wherein the naturally
occurring lysine at position 34 has been substituted with arginine and a lysine residue
has been added to the C-terminal (position 38). Formulae of peptide analogs and
derivatives thereof are drawn using standard single letter abbreviation for amino acids
used according to IUPAC-IUB nomenclature.
The term "GLP-1 peptide" as used herein means GLP-1 (7-37) (SEQ ID No.
158) or GLP-1 (7-36) (SEQ ID No. 159), a GLP-1 analogue, a GLP-1 derivative or a
derivative of a GLP-1 analogue. Such peptides, analogues and derivatives are
insulinotropic agents.
The term "insulinotropic agent" as used herein 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. Known examples of insulinotropic agents include but are not
limited to glucose, GIP, GLP, Exendin, and OXM.
The term "incretin" as used herein 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. By way of example they include GLP-1, GIP, and
OXM.
The term "exendin-4 peptide" as used herein means exendin-4 (1-39), an
exendin-4 analogue, an exendin-4 derivative or a derivative of an exendin-4 analogue.
In one embodiment the exendin-4 peptide is an insulinotropic agent. Such peptides,
analogues and derivatives are insulinotropic agents.
The term "DPP-IV protected" as used herein referring to a polypeptide means a
polypeptide which has been modified (eg, chemically modified) in order to render said
compound resistant to the plasma peptidase dipeptidyl aminopeptidase-4 (DPP-IV). The
DPP-IV enzyme in plasma is known to be involved in the degradation of several peptide
hormones, e. g. GLP-1, GLP-2, etc. Thus, a considerable effort is being made to

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develop analogues and derivatives of the polypeptides susceptible to DPP-IV mediated
hydrolysis in order to reduce the rate and/or extent of degradation by DPP-IV.
As used herein "saporin" refers to a family of single-chain ribosome-
inactivating polypeptides produced by the plant Saponaria officinalis. (Stirpe, F., et al.,
Biochem. J. 216:617-625 (1983), Bagga, S. et al., J. Biol. Chem. 278:4813-4820
(2003).) Saporin polypeptides exist is several forms that differ in length and/or amino
acid sequence. (See, e.g., Id. and Barthelemy, I. et al., J. Biol. Chem. 268:6541-6548
(1993).) Saporin-6 is the most active form of saporin. (Bagga, S. et al., J. Biol. Chem.
278:4813-4820 (2003).) At least four naturally occurring isoforms of saporin-6 in
which the amino acid at position 48 of the mature polypeptide (SEQ ID NO:65) is Asp
or Glu, and the amino acid a position 91 of the mature polypeptide (SEQ ID NO:65) is
Arg of Lys have been described. (Barthelemy, I. et al., J. Biol. Chem. 268:6541-6548
(1993).) Additional forms of saporin-6 include polypeptide in which the amino acid at
position 99 of the mature polypeptide (SEQ ID NO:65) is Ser of Leu, the amino acid at
position 134 of the mature polypeptide (SEQ ID NO:65) is Gin or Lys, the amino acid
at position 147 of the mature polypeptide (SEQ ID NO:65) is Ser or Leu, the amino acid
at position 149 of the mature polypeptide (SEQ ID NO:65) is Ser or Phe, the amino acid
at position 162 of the mature polypeptide (SEQ ID NO:65) is Asp or Asn, the amino
acid at position 177 of the mature polypeptide (SEQ ID NO:65) is Ala or Val, the amino
acid at position 188 of the mature polypeptide (SEQ ID NO:65) is He or Thr, the amino
acid at position 196 of the mature polypeptide (SEQ ID NO:65) is Asn or Asp, the
amino acid at position 198 of the mature polypeptide (SEQ ID NO:65) is Glu or Asp,
the amino acid at position 231 of the mature polypeptide (SEQ ID NO:65) is Asn or Ser,
and polypeptides in which the amino acid at position 233 of the mature polypeptide
(SEQ ID NO:65) is Lys or Arg. (Id.) A consensus sequence encompassing these
isoforms and variants is presented in FIG. 14G (SEQ ID NO:67).
Accordingly, the term "saporin" includes precursor protein, mature polypeptide,
native protein, polymorphic or allelic variants, and other isoforms (e.g., produced by
alternative splicing or other cellular processes), and modified or unmodified forms of
the foregoing (e.g., lipidated, glycosylated, PEGylated). Naturally occurring or
endogenous saporin include wild type proteins such as mature saporin (e.g., mature
saporin-6), polymorphic or allelic variants and other isoforms which occur naturally in

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Saponaria officinalis. Such proteins can be recovered or isolated from Saponaria
officinalis using any suitable methods. "Functional variants" of saporin include
functional fragments, functional mutant proteins, and/or functional fusion proteins
which can be produce using suitable methods (e.g., mutagenesis (e.g., chemical
mutagenesis, radiation mutagenesis), recombinant DNA techniques). Generally,
fragments or portions of saporin (e.g., saporin-6) include those having a deletion and/or
addition (i.e., one or more amino acid deletions and/or additions) of an amino acid (i.e.,
one or more amino acids) relative to mature saporin (such as N-terminal, C-terminal or
internal deletions). Fragments or portions in which only contiguous amino acids have
been deleted or in which non-contiguous amino acids have been deleted relative to
mature saporin are also envisioned. A variety of active variants of saporin can be
prepared. For example, fusion proteins of saporin-6 that contain amino-terminal
extensions have been prepared and shown to retain full ribosome-inhibiting activity in
rabbit reticulocyte lysate assays. (Barthelemy, I. et al., J. Biol. Chem. 268:6541-6548
(1993).) Variants or saporin-6 is which an active site residue, Tyr72, Tyrl20, Glul76,
Arg 179 or Trp208 (amino acids 72, 120, 176, 179 or 208 of SEQ ID NO:65), was
replaced with alanine had reduced cytotoxic activity in in vitro assays. (Bagga, S. et al.,
J. Biol. Chem. 278:4813-4820 (2003).) Accordingly, if preparing additional functional
variants of saporin is desired, mutation, substitution, replacement, deletion or
modification of the active site residues should be avoided. Preferably, a functional
variant of saporin that contains fewer amino acids than naturally occurring mature
polypeptide includes at least the active site. For example, a variant of saporin-6 that
contains fewer amino acids than naturally occurring mature saporin-6 can include the
active site residues of mature saporin-6 (Tyr72, Tyrl20, Glul76, Arg 179 and Trp208
(amino acids 72,120,176, 179 and 208 of SEQ ID NO:65)), and be at least about 137
amino acids in length, at least about 150 amino acids in length, at least about 175 amino
acids in length, at least about 200 amino acids in length, at least about 225 amino acids
in length or at least about 250 amino acids in length.
A "functional variant" of saporin has ribosome-inactivating activity (e.g.,
rRNA N-Glycosidase activity) and/or cytotoxic activity. Such activity can readily be
assessed using any suitable method, such as inhibition of protein synthesis using the
well-known rabbit reticulocyte lysate assay or any of the well-known cytotoxicity

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assays that employ tumor cell lines. (See, e.g., Bagga, S. et al., J. Biol. Chem.
278:4813-4820 (2003) and Barthelemy, I. et al., J. Biol. Chem. 268:6541-6548 (1993).)
In some embodiments, a functional variant of saporin has at least about
80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least
about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at
least about 96%, or at least about 97%, or at least about 98%, or at least about 99%
ammo acid sequence identity with mature saporin-6 (SEQ ID NO:65).
The invention relates to compositions that comprise a drug and a polypeptide
binding moiety that contains an antigen-binding site that has binding specificity for a
polypeptide that enhances serum half-live in vivo. As described herein in detail with
respect to compositions that comprise and antigen-binding fragment of an antibody that
has binding specificity for serum albumin, the drug and the binding polypeptide can be
conjugated covalently or noncovalently. In some embodiments, the composition is a
fusion protein that comprises a polypeptide drug and a polypeptide binding moiety that
contains an antigen-binding site that has binding specificity for a polypeptide that
enhances serum half-live in vivo. In other embodiments, the composition comprises a
drug that is covalently or noncovalently bonded to a polypeptide binding moiety that
contains an antigen-binding site that has binding specificity for a polypeptide that
enhances serum half-live in vivo.
The invention relates to drug compositions that comprise a drug and a
polypeptide binding moiety that contains a binding site {e.g., an antigen-binding site)
that has binding specificity for a polypeptide that enhances serum half-life in vivo. As
described herein in detail with respect to drug compositions that comprise an antigen-
binding fragment of an antibody that has binding specificity for serum albumin, the drug
and the polypeptide binding moiety can be bonded to each other covalently or
noncovalently. hi some embodiments, the drug composition is a fusion protein that
comprises a polypeptide drug and a polypeptide binding moiety that contains an
antigen-binding site that has binding specificity for a polypeptide that enhances serum
half-life in vivo. In other embodiments, the drug composition comprises a drug that is
covalently or noncovalently bonded to a polypeptide binding moiety that contains an
antigen-binding site that has binding specificity for a polypeptide that enhances serum
half-life in vivo.

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Typically, a polypeptide that enhances serum half-life in vivo is a polypeptide
which occurs naturally in vivo and which resists degradation or removal by endogenous
mechanisms which remove unwanted material from the organism (e.g., human). For
example, a polypeptide that enhances serum half-life in vivo can be selected from
proteins from the extracellular matrix, proteins found in blood, proteins found at the
blood brain barrier or in neural tissue, proteins localized to the kidney, liver, lung, heart,
skin or bone, stress proteins, disease-specific proteins, or proteins involved in Fc
transport.
Suitable polyp eptides that enhance serum half-life in vivo include, for example,
transferrin receptor specific ligand-neuropharmaceutical agent fusion proteins (see U.S.
Patent No. 5,977,307, the teachings of which are incorporated herein by reference),
brain capillary endothelial cell receptor, transferrin, transferrin receptor (e.g., soluble
transferrin receptor), insulin, insulin-like growth factor 1 (IGF 1) receptor, insulin-like
growth factor 2 (IGF 2) receptor, insulin receptor, blood coagulation factor X, cd-
antitrypsin and HNF la Suitable polypeptides that enhance serum half-life also include
alpha-1 glycoprotein (orosomucoid; AAG), alpha-1 antichymotrypsin (ACT), alpha-1
microglobulin (protein HC; AIM), antithrombin III (AT III), apolipoprotein A-l (Apo
A-l), apolipoprotein B (Apo B), ceruloplasmin (Cp), complement component C3 (C3),
complement component C4 (C4), Cl esterase inhibitor (Cl INH), C-reactive protein
(CRP), ferritin (FER), hemopexin (HPX), lipoprotein(a) (Lp(a)), mannose-binding
protein (MBP), myoglobin (Myo), prealbumin (transthyretin; PAL), retinol-binding
protein (RBP), and rheumatoid factor (RF).
Suitable proteins from the extracellular matrix include, for example, collagens,
laminins, integrins and fibronectin. Collagens are the major proteins of the extracellular
matrix. About 15 types of collagen molecules are currently known, found in different
parts of the body, e.g. type I collagen (accounting for 90% of body collagen) found in
bone, skin, tendon, ligaments, cornea, internal organs or type II collagen found in
cartilage, vertebral disc, notochord, and vitreous humor of the eye.
Suitable proteins from the blood include, for example, plasma proteins (e.g.,
fibrin, ce-2 macroglobulin, serum albumin, fibrinogen (e.g., fibrinogen A, fibrinogen B),
serum amyloid protein A, haptoglobin, profilin, ubiquitin, uteroglobulin and a-2-
microglobulin), enzymes and enzyme inhibitors (e.g., plasminogen, lysozyme, cystatin

WO 2006/059106 PCT/GB2005/004599
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C, alpha- 1-antitrypsin and pancreatic trypsin inhibitor), proteins of the immune system,
such as immunoglobulin proteins (e.g., IgA, IgD, IgE, IgG, IgM, immunoglobulin light
chains (kappa/lambda)), transport proteins (e.g., retinol binding protein, a-1
microglobulin), defensins (e.g., beta-defensin 1, neutrophil defensin 1, neutrophil
defensin 2 and neutrophil defensin 3) and the like.
Suitable proteins found at the blood brain barrier or in neural tissue include, for
example, melanocortin receptor, myelin, ascorbate transporter and the like.
Suitable polypeptides that enhances serum half-life in vivo also include proteins
localized to the kidney (e.g., polycystin, type IV collagen, organic anion transporter Kl,
Heymann's antigen), proteins localized to the liver (e.g., alcohol dehydrogenase, G250),
proteins localized to the lung (e.g., secretory component, which binds IgA), proteins
localized to the heart (e.g., HSP 27, which is associated with dilated cardiomyopathy),
proteins localized to the skin (e.g., keratin), bone specific proteins such as morphogenic
proteins (BMPs), which are a subset of the transforming growth factor R superfamily of
proteins that demonstrate osteogenic activity (e.g., BMP-2, BMP-4, BMP-5, BMP-6,
BMP-7, BMP-8), tumor specific proteins (e.g., trophoblast antigen, herceptin receptor,
oestrogen receptor, cathepsins (e.g., cathepsin B, which can be found in liver and
spleen)).
Suitable disease-specific proteins include, for example, antigens expressed only
on activated T-cells, including LAG-3 (lymphocyte activation gene), osteoprotegerin
ligand (OPGL; see Nature 402, 304-309 (1999)), OX40 (a member of the TNF receptor
family, expressed on activated T cells and specifically up-regulated in human T cell
leukemia virus type-I (HTLV-I)-producing cells; see Immunol. 165 (l):263-70 (2000)).
Suitable disease-specific proteins also include, for example, metalloproteases
(associated with arthritis/cancers) including CG6512 Drosophila, human paraplegin,
human FtsH, human AFG3L2, murine ftsH; and angiogenic growth factors, including
acidic fibroblast growth factor (FGF-1), basic fibroblast growth factor (FGF-2), vascular
endothelial growth factor/vascular permeability factor (VEGF/VPF), transforming
growth factor-a (TGF-a), tumor necrosis factor-alpha (TNF-a), angiogenin, interleukin-
3 (IL-3), interleukin-8 (IL-8), platelet-derived endothelial growth factor (PD-ECGF),
placental growth factor (P1GF), midkine platelet-derived growth factor-BB (PDGF),
and fractalkine.

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Suitable polypeptides that enhance serum half-life in vivo also include stress
proteins such as heat shock proteins (HSPs). HSPs are normally found intracellularly.
When they are found extracellularly, it is an indicator that a cell has died and spilled out
its contents. This unprogrammed cell death (necrosis) occurs when as a result of trauma,
disease or injury, extracellular HSPs trigger a response from the immune system.
Binding to extracellular HSP can result in localizing the compositions of the invention
to a disease site.
Suitable proteins involved in Fc transport include, for example, Brambell
receptor (also known as FcRB). This Fc receptor has two functions, both of which are
potentially useful for delivery. The functions are (1) transport of IgG from mother to
child across the placenta (2) protection of IgG from degradation thereby prolonging its
serum half-life. It is thought that the receptor recycles IgG from endosomes. (See,
Holliger et al, Nat Biotechnol 15(7):632-6 (1997).)
The drug compositions of the invention can comprise any polypeptide binding
moiety that contains a binding site {e.g., an antigen-binding site) that has binding
specificity for a polypeptide that enhances serum half-life in vivo. Preferably, the
polypeptide binding moiety comprises at least 31, at least about 40, at least about 50, at
least about 60, at least about 70, at least about 80 amino acids, at least about 90 amino
acids, at least about 100 amino acids or at lease about 110 amino acids as a separate
molecular entity. Preferably, the polypeptide binding moiety binds a polypeptide that
enhances serum half-life in vivo with a KD of at least about 5 mM KD (KD=Koff
(kd)/Kon (ka)). In some embodiments, the polypeptide binding moiety binds a
polypeptide that enhances serum half-life in vivo with a KD of about 10 to about 100
nM, or about 100 nM to about 500 nM, or about 500 nM to about 5 mM, as determined
by surface plasmon resonance {e.g., using a BIACORE instrument). In particular
embodiments, the polypeptide binding moiety binds a polypeptide that enhances serum
half-life in vivo with a KD of about 50 nM, or about 70 nM, or about 100 nM, or about
150 nM or about 200 nM.
Preferably, the polypeptide binding moiety that contains a binding site {e.g., an
antigen-binding site) that has binding specificity for a polypeptide that enhances serum
half-life in vivo is not a prokaryotic or bacterial polypeptide or peptide. Preferably, the

WO 2006/059106 PCT/GB2005/004599
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polypeptide binding moiety is a eukaryotic, mammalian or human polypeptide or
peptide.
In certain embodiments, the polypeptide binding moiety that contains a binding
site (e.g., an antigen-binding site) that has binding specificity for a polypeptide that
enhances serum half-life in vivo is a folded protein domain. In other embodiments, the
polypeptide binding moiety has a molecular weight of at least about 4 KDa, at least
about 4.5 KDa, at least about 5 KDa, at least about 5.5 KDa, at least about 6 KDa, at
least about 6.5 KDa, at least about 7 KDa, at least about 7.5 KDa or at least about 8
KDa as a separate molecular entity.
Suitable polypeptide binding moieties that contain a binding site (e.g., an
antigen-binding site) that has binding specificity for a polypeptide that enhances serum
half-life in vivo can be identified using any suitable method, such as by screening
naturally occurring or non-naturally occurring polypeptides in a suitable adhesion assay.
As described herein, preferred polypeptide binding moieties that have an antigen-
binding site for a polypeptide that enhances serum half-life in vivo are antigen-binding
fragments of antibodies that have binding specificity for serum albumin. However,
antigen-binding fragments of antibodies that have binding specificity for other
polypeptides that enhance serum half-life in vivo can be used in the invention.
If desired, one or more of the complementarity determining regions (CDRs) of
an antibody or antigen-binding fragment thereof that binds a polypeptide that enhances
serum half-life in vivo can be formatted into a non-immunoglobulin structure that
retains the antigen-binding specificity of the antibody or antigen-binding fragment. The
drug compositions of the invention can comprise such a non-immunoglobulin binding
moiety. Such non-immunoglobulin binding moieties can be prepared using any
suitable method, for example natural bacterial receptors such as SpA have been used as
scaffolds for the grafting of CDRs to generate polypeptide binding moieties which
specifically bind an epitope. Details of this procedure are described in U.S. Patent
Application No. 5,831,012, the teachings of which are incorporated herein by reference.
Other suitable scaffolds include those based on fibronectin and affibodies. Details of
suitable procedures are described in WO 98/58965. Other suitable scaffolds include
lipocallin and CTLA4, as described in van den Beuken et al, J. Mol. Biol. 310:591-601
(2001), and scaffolds such as those described in WO 00/69907 (Medical Research

WO 2006/059106 PCT/GB2005/004599
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Council), which are based for example on the ring structure of bacterial GroEL or other
chaperone polypeptides.
In some embodiments, the drug composition of the invention comprises a non-
immunoglobulin binding moiety that has binding specificity for serum albumin, wherein
the non-immunoglobulin binding moiety comprises one, two or three of the CDRs of a
VH, VK or VRH described herein and a suitable scaffold, hi certain embodiments, the
non-immunoglobulin binding moiety comprises CDR3 but not CDR1 or CDR2 of a VH,
VK or VHH described herein and a suitable scaffold. In other embodiments, the non-
immunoglobulin binding moiety comprises CDR1 and CDR2, but not CDR3 of a VH,
VK or VHH described herein and a suitable scaffold. In other embodiments, the non-
immunoglobulin binding moiety comprises CDR1, CDR2 and CDR3 of a VH, VK or
VHH described herein and a suitable scaffold. In other embodiments, the drug
composition comprises only CDR3 of a VH, VK or VHH described herein and a drug.
The drug compositions of the invention can be prepared using suitable methods,
such as the methods described herein for preparation of drug fusions, drug conjugates
and noncovalent drug conjugates. Additionally, the drug compositions of the invention
have the advantages and the utilities that are described in detail herein with respect to
drug fusions, drug conjugates and noncovalent drug conjugates.
The invention provides drug compositions (e.g., drug conjugates, noncovalent
drug conjugates, drug fusions) that have improved pharmacokinetic properties (e.g.,
increase serum half-life) and other advantages in comparison to the drug alone
(unconjugated drug, unfused drug). The drug conjugates, noncovalent drug conjugates
and drug fusions comprise an antigen-binding fragment of an antibody that has binding
specificity for serum albumin and one or more desired drugs.
As described herein, drug compositions (e.g., drug conjugates, noncovalent drug
conjugates, drug fusions) of the invention can have dramatically prolonged in vivo
serum half-life and/or increased AUC, as compared to drug alone. In addition, the
activity of the drug is generally not substantially altered in the drug composition (e.g.,
drug conjugate, noncovalent drug conjugate, drag fusion). However, some change in
the activity of a drag composition compared to drug alone is acceptable and is generally
compensated for by the improved pharmacokinetic properties of the drag composition
(e.g., drag conjugate, noncovalent drag conjugate, drag fusion). For example, drag

WO 2006/059106 PCT/GB2005/004599
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compositions (e.g., drug conjugates, noncovalent drag conjugates, drug fusions) may
bind the drug target with lower affinity than drug alone, but have about equivalent or
superior efficacy in comparison to drug alone due to the improved pharmacokinetic
properties (e.g., prolonged in vivo serum half-life, larger AUC) of the drug composition.
In addition, lower amounts of drug compositions (e.g., drug conjugates, noncovalent
drug conjugates and drug fusions) can be administered to achieve the desired
therapeutic or diagnostic effect. Preferably the activity of the drug composition (e.g.,
drug conjugate, noncovalent drug conjugate, drug fusion) differs from that of the drug
alone by a factor of no more than about 100, or no more than about 50, or no more than
about 10, or no more than about 5, or no more than about 4, or no more than about 3, or
no more than about 2. For example, a drug can have a KD, Ki or neutralizing dose 50
(ND50) of 1 nM, and a drug composition (e.g., drug conjugate, noncovalent drag
conjugate, drug fusion) can have a KD, Ki or ND50 of about 2 nM, or about 3 nM, or
about 4 nM, or about 5 nM, or about 10 nM.
Preferably, the activity of the drag composition (e.g., drag conjugate,
noncovalent drag conjugate, drag fusion) is not substantially reduced as compared to
the activity of the drag. In certain embodiments, the activity of the drag composition is
reduced, relative to the activity of drug, by no more than about 10%, no more than about
9%, no more than about 8%, no more than about 7%, no more than about 6%, no more
than about 5%, no more than about 4%, no more than about 3%, no more than about
2%, no more than about 1% or is substantially unchanged. Alternatively stated, the
drag composition (e.g., drug conjugate, noncovalent drag conjugate, drag fusion)
retains at least about 90%, at least about 91%, at least about 92%, at least about 93%, at
least about 94%, at least about 95%, at least about 96%, at least about 97%, at least
about 98%, at least about 99% of the activity of the drag, or substantially the same
activity as the drag. Preferably, the activity of drag compositions (e.g., drag conjugate,
noncovalent drag conjugate, drag fusion) and drugs are determined and/or compared on
a "drug basis."
As described and shown herein, the drag compositions (e.g., drag conjugate,
noncovalent drag conjugate, drag fusion) of the invention can have greater activity (e.g.,
in vivo activity) than drag alone. For example, as shown in Example 6, DOM7m-16/IL-
lra was more effective in treating arthritis in a mouse model than IL-lra when these

WO 2006/059106 PCT/GB2005/004599
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agents were administered at the same dose by weight (10 mg/Kg or 1 mg/Kg).
DOM7m-16/IL-lra was more effective even though its molecular weight is
approximately twice the molecular weight of IL-lra. Thus, mice that received
DOM7m-16/IL-lra received only about half of the IL-lra (as a moiety in D0M7m-
16/ILl-ra) as mice that received IL-lra.
In certain embodiments, the drug composition (e.g., drug conjugate, noncovalent
drug conjugate, drug fusion) has greater activity (e.g., in vivo activity) than drug, for
example, the drug composition can have at least about 100%, at least about 150%, at
least about 200%, at least about 250%, at least about 300%, at least about 350%, at least
about 400%, at least about 450%, or at least about 500% of the activity of drug.
Preferably, the activity of drug compositions (e.g., drug conjugate, noncovalent drug
conjugate, drug fusion) and drugs are determined and/or compared on a "drug basis."
The activity of drug compositions (e.g., drug conjugate, noncovalent drug conjugate,
drug fusion) and drugs can be determined using a suitable in vitro or in vivo system. In
certain embodiments, a drug composition (e.g., drug conjugate, noncovalent drug
conjugate, drug fusion) has greater activity than the drug it comprises, as determined in
vivo. In.other embodiments, a drug composition (e.g., drug conjugate, noncovalent drug
conjugate, drug fusion) has greater activity than the drug it comprises, as determined in
vitro.
Drug compositions (e.g., drug conjugates, noncovalent drug conjugates, drug
fusions) that comprise a domain antibody (dAb) that has binding specificity for serum
albumin provide further advantages. Domain antibodies are very stable, are 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 pastoris), and as
described herein antigen-binding fragments of antibodies that bind serum albumin can
be easily selected from libraries of human origin or from any desired species.
Accordingly, drug compositions (e.g., drug conjugates, noncovalent drug conjugates,
drug fusions) that comprise a 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 imrnunogenic can be used
(e.g., a human dAb can be used for a drug fusion or drug conjugate for treating or
diagnosing disease in humans.)

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The immunogenicity of a drag can be reduced when the drag is part of a drag
composition (e.g., drag conjugate, noncovalent drag conjugate, drag fusion) that
contains a polypeptide binding moiety that binds serum albumin (e.g., an antigen-
binding fragment of an antibody that binds serum albumin). Accordingly, a drag can be
less immunogenic (than drag alone) or be substantially non-immunogenic in the context
of a drag composition that contains a polypeptide binding moiety that binds serum
albumin (e.g., drag conjugate, noncovalent drag conjugate, drag fusion). Thus, such
drug compositions (e.g., drag conjugates, noncovalent drag conjugates, drag fusions)
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.
Additionally, the drag compositions (e.g., drag conjugates, noncovalent drug
conjugates, drag fusions) described herein can have an enhanced safety profile and
fewer side effects than drag alone. For example, as a result of the serum albumin-
binding activity of the antigen-binding fragment of an antibody that has binding
specificity for serum albumin, the drug fusions and conjugates (drag conjugate,
noncovalent drag conjugate) have enhanced residence time in the vascular circulation.
Additionally, the conjugates and drag fusions are substantially unable to cross the blood
brain barrier and to accumulate in the central nervous system following systemic
administration (e.g., intravascular administration). Accordingly, conjugates (drag
conjugate, noncovalent drag conjugate) and drag fusions that contain a drug that has
neurological toxicity or undesirable psychotropic effects can be administered with
greater safety and reduced side effects in comparison to the drug alone. Similarly, the
conjugates (drag conjugate, noncovalent drug conjugate) and drag fusions can have
reduced toxicity toward particular organs (e.g., kidney or liver) than drag alone. The
conjugates and drug fusions described herein can also be used to sequester a drag or a
target that binds a drug (e.g, a toxin) in the vascular circulation, thereby decreasing the
effects of the drag or target on tissues (e.g., inhibiting the effects of a toxin).
Suitable methods for pharmacokinetic analysis and determination of in vivo half-
life are well known in the art. Such methods are described, for example, in Kenneth, A
et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists, and in
Peters et al, Pharmacokinetc analysis: A Practical Approach (1996). Reference is also
made to "Pharmacokinetics", M Gibaldi & D Perron, published by Marcel Dekker, 2nd

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Rev. edition (1982), which describes pharmacokinetic parameters such as t alpha and t
beta half-lives (t1/2 alpha, t1/2 beta) and area under curve (AUC).
Half-lives (t1/2 alpha and t1/2 beta) and AUC can be determined from a curve of
serum concentration of conjugate or fusion against time. The WinNonlin analysis
package (available from Pharsight Corp., Mountain View, CA 94040, USA) can be
used, for example, to model the curve. In a first phase (the alpha phase) the drug
composition {e.g., drug conjugate, noncovalent drag conjugate, drug fusion) is
undergoing mainly distribution in the patient, with some elimination. A second phase
(beta phase) is the tenninal phase when the drug composition {e.g., drag conjugate,
noncovalent drag conjugate, drag fusion) has been distributed and the serum
concentration is decreasing as the drag composition 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. Thus, the present invention provides a drag composition {e.g., drag
conjugate, noncovalent drag conjugate, drug fusion) or a composition comprising a drag
composition {e.g., drag conjugate, noncovalent drug conjugate, drag fusion) according
to the invention having a ta half-life in the range of 15 minutes or more. In one
embodiment, the lower end of the range is 30 minutes, 45 minutes, 1 hour, 2 hours, 3
hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12
hours. In addition, or alternatively, a drug composition {e.g., drag conjugate,
noncovalent drag conjugate, drag fusion) or composition according to the invention will
have a tee half-life in the range of up to and including 12 hours. In one embodiment, the
upper end of the range is 11, 10, 9, 8, 7, 6 or 5 hours. An example of a suitable range is
1 to 6 hours, 2 to 5 hours or 3 to 4 hours.
Advantageously, the present invention provides drug compositions {e.g., drug
conjugates, noncovalent drag conjugates, drug fusions) having a tj(3 half-life in the range
of 2.5 hours or more. In one embodiment, the lower end of the range is 3 hours, 4
hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours. In
some embodiments, the drag compositions {e.g., drag conjugates, noncovalent drag
conjugates, drag fusions) have a tβ half-life in the range of up to and including 21 days.
In one embodiment, the upper end of the range is 12 hours, 24 hours, 2 days, 3 days, 5
days, 10 days, 15 days or 20 days. In particular embodiments, a drag composition {e.g.,
drag conjugate, noncovalent drag conjugate, drug fusion) according to the invention

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will have a tβ half-life in the range 12 to 60 hours. In a further embodiment, it will be
in the range 12 to 48 hours. In a further embodiment still, it will be in the range 12 to
26 hours.
In addition, or alternatively to the above criteria, the present invention provides
drug compositions {e.g., drug conjugates, noncovalent drug conjugates, drug fusions)
having an AUC value(area under the curve) in the range of 0.01 mg.min/mL or more, or
1 mg.min/mL or more. In one embodiment, the lower end of the range is 0.01, 0.1, 1, 5,
10,15, 20, 30, 100, 200 or 300 mg.min/mL. In particular embodiments, the drug
composition {e.g., drug conjugate, noncovalent drug conjugate, drug fusion) has an
AUC in the range of up to 600 mg.min/mL. In one embodiment, the upper end of the
range is 500, 400, 300, 200, 150,100, 75 or 50 mg.min/mL. In other embodiments, the
drug composition {e.g., drug conjugate, noncovalent drug conjugate, drug fusion) has an
AUC in the range selected from the group consisting of the following: 15 to 150
mg.min/mL, 15 to 100 mg.min/mL, 15 to 75 mg.min/mL, 15 to 50 mg.min/mL, 0.01 to
50 mg.min/mL, 0.1 to 50 mg.min/mL, 1 to 50 mg.min/mL, 5 to 50 mg.min/mL, and 10
to 50 mg.min/mL.
The invention relates to drug compositions {e.g., drug conjugates, noncovalent
drug conjugates, drug fusions) that comprise a drug and a polypeptide binding moiety
that contains a binding site {e.g., an antigen-binding site) that has binding specificity for
a polypeptide that enhances serum half-life in vivo. In preferred embodiments of drug
compositions, the polypeptide binding moiety that contains a binding site {e.g., an
antigen-binding site) that has binding specificity for a polypeptide that enhances serum
half-life in vivo, has binding specificity for serum albumin.
In some embodiments, the drug composition comprises a drug that is covalently
bonded to a polypeptide binding moiety that contains a binding site (e.g., an antigen-
binding site) that has binding specificity for a polypeptide that enhances serum half-life
in vivo. In these embodiments, the drug can be covalently bonded to the polypeptide
binding domain at any suitable position, such as the amino-terminus, the carboxyl-
terminus or through suitable amino acid side chains {e.g., the s amino group of lysine or
thiol group of cysteine).
In other embodiments, the drug composition comprises a drug that is
noncovalently bonded to a polypeptide binding moiety that contains a binding site (e.g.,

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an antigen-binding site) that has binding specificity for a polypeptide that enhances
serum half-life in vivo, hi such embodiments, the drug can be noncovalently bonded to
the antigen-binding fragment directly (e.g., through 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 drug and the complementary binding partner is covalently bonded
to the antigen-binding fragment). When complementary binding partners are employed,
one of the binding partners can be covalently bonded to the drug directly or through a
suitable linker moiety, and the complementary binding partner can be covalently bonded
to the polypeptide binding domain directly or through a suitable linker moiety.
In other embodiments, the drug composition is a fusion protein that comprises a
polypeptide binding moiety that contains a binding site (e.g., an antigen-binding site)
that has binding specificity for a polypeptide that enhances serum half-life in vivo and a
polypeptide drug. The fusion proteins comprise a continuous polypeptide chain, said
chain comprising a polypeptide binding moiety that contains a binding site (e.g., an
antigen-binding site) that has binding specificity for a polypeptide that enhances serum
half-life in vivo as a first moiety, and a polypeptide drug as a second moiety, which are
present as discrete parts (moieties) of the polypeptide chain. The first and second
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., third,
fourth) and/or linker sequences can be present as appropriate. The first moiety can be in
an N-terminal location, C-terminal location or internal relative to the second moiety
(i.e., the polypeptide drag). In certain embodiments, the fusion protein comprises one
or more one or more polypeptide binding moieties that contain a binding site that has
binding specificity for a polypeptide that enhances serum half-life in vivo and one or
more polypeptide drug moieties. In these embodiments, the fusion protein can comprise
one to about ten (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) polypeptide drug moieties that can be
the same or different, and one to about twenty (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12,
13, 14, 15, 16, 17, 18 19 or 20) polypeptide binding moieties that contain a binding site
that has binding specificity for a polypeptide that enhances serum half-life in vivo that
can be the same or different.

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The polypeptide binding moieties that contain a binding site that has binding
specificity for a polypeptide that enhances serum half-life in vivo and polypeptide drag
moieties can be present in any desired location. For example, proceeding from the
amino terminus to the carboxyl terminus, the moieties can be present in the following
order: one or more polypeptide binding moieties, one or more polypeptide drug
moieties, one or more polypeptide binding moieties. In another example, proceeding
from the amino terminus to the carboxyl terminus, the moieties can be present in the
following order: one or more polypeptide binding moieties, one or more polypeptide
drag moieties, one or more polypeptide binding moieties, one or more polypeptide drag
moieties, one or more polypeptide binding moieties. As described herein, the
polypeptide binding moieties and polypeptide drag moieties can be directly bonded to
each other through a peptide bond, or linked through a suitable amino acid, or peptide or
polypeptide linker.
In certain embodiments, the fusion protein is a continuous polypeptide chain that
has the formula (amino-terminal to carboxy-terminal):

wherein X is a polypeptide drug;
P and Q are each independently a polypeptide binding moiety that contains a
binding site that has binding specificity for a polypeptide that enhances serum half-life
in vivo;
a, b, c and d are each independently absent or one to about 100 amino acid
residues;
nl, n2 and n3 represent the number of X, P or Q moieties present, respectively;
nl is one to about 10;
n2 is zero to about 10; and
n3 is zero to about 10,
with the proviso that both n2 and n3 are not zero; and
with the proviso that when nl and n2 are both one and n3 is zero, X does not
comprise an antibody chain or a fragment of an antibody chain.

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In some embodiments, n2 is one, two, three, four, five or six, and n3 is zero. In
other embodiments, n3 is one, two, three, four, five or six, and n2 is zero. In other
embodiments, nl, n2 and n3 are each one.
In certain embodiments, X does not comprises an antibody chain or a fragment
of an antibody chain.
In preferred embodiments, P and Q are each independently a polypeptide
binding moiety that has binding specificity for serum albumin.
In particularly preferred embodiments, the drug composition {e.g., drug
conjugate, noncovalent drag conjugate, drug fusion) comprises a polypeptide binding
moiety that contains a binding site {e.g., an antigen-binding site) that has binding
specificity for a polypeptide that enhances serum half-life in vivo, wherein the
polypeptide binding domain is an antigen-binding fragment of an antibody that has
binding specificity for serum albumin.
Antigen-binding Fragment of an Antibody that Binds Serum Albumin
The drug conjugates, noncovalent drag conjugates and drag fusions of the
invention comprise an {i.e., one or more) antigen-binding fragment of an antibody that
binds serum albumin. The antigen-binding fragment can have binding specificity for
serum albumin of an animal to which the drag conjugate or drag fusion will be
administered. Preferably, the antigen-binding fragment has binding specificity for
human serum albumin. However, veterinary applications are contemplated and the
antigen-binding fragment can have binding specificity for serum albumin from a desired
animal, for example serum albumin from dog, cat, horse, cow, chicken, sheep, pig, goat,
deer, mink, and the like. In some embodiments the antigen-binding fragment has
binding specificity for serum albumin from more than one species. For example, as
described herein, human dAbs that have binding specificity for rat serum albumin and
mouse serum albumin, and a dAb that has binding specificity for rat, mouse and human
serum albumin have been produced. (Table 1 and FIG. 7) Such dAbs provide the
advantage of allowing preclinical and clinical studies using the same drag conjugate or
drug fusion and obviate the need to conduct preclinical studies with a suitable surrogate
drag fusion or drug conjugate.

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Antigen-binding fragments suitable for use in the invention include, for
example, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments (including
single chain Fv (scFv) and disulfide bonded Fv), a single variable domain, and dAbs
(VH, VL). Such antigen-binding fragments can be produced using any suitable method,
such as by proteolysis of an antibody using pepsin, papain or other protease having the
requisite cleavage specificity, or using recombinant techniques. For example, Fv
fragments can be prepared by digesting an antibody with a suitable protease or using
recombinant DNA technology. For example, a nucleic acid can be prepared that
encodes a light chain variable region and heavy chain variable region that are connected
by a suitable peptide linker, such as a chain of two to about twenty Glycyl residues.
The nucleic acid can be introduced into a suitable host (e.g., E. coli) using any suitable
technique (e.g., transfection, transformation, infection), and the host can be maintained
under conditions suitable for expression of a single chain Fv fragment. A variety of
antigen-binding fragments of antibodies can be prepared using antibody genes in which
one or more stop codons have been introduced upstream of the natural stop site. For
example, an expression construct encoding a F(ab')2 portion of an immunoglobulin
heavy chain can be designed by introducing a translation stop codon at the 3' end of the
sequence encoding the hinge region of the heavy chain. The drug conjugates,
noncovalent drug conjugates and drug fusions of the invention can comprise the
individual heavy and light chains of antibodies that bind serum albumin or portions of
the individual chains that bind serum albumin (e.g., a single VH, VK or Vx).
Antibodies and antigen-binding fragments thereof which bind a desired serum
albumin (e.g., human serum albumin) can be selected from a suitable collection of
natural or artificial antibodies or raised against an appropriate immunogen in a suitable
host. For example, antibodies can be raised by immunizing a suitable host (e.g., mouse,
human antibody-transgenic mouse, rat, rabbit, chicken, goat, non-human primate (e.g.,
monkey)) with serum albumin (e.g., isolated or purified human serum albumin) or a
peptide of serum albumin (e.g., a peptide comprising at least about 8, 9,10,11,12,15,
20, 25, 30, 33, 35, 37, or 40 amino acid residues). Antibodies and antigen-binding
fragments that bind serum albumin can also be selected from a library of recombinant
antibodies or antigen-binding fragments, such as a phage display library. Such libraries
can contain antibodies or antigen-binding fragments of antibodies that contain natural or

WO 2006/059106 PCT/GB2005/004599
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artificial amino acid sequences. For example, the library can contain Fab fragments
which contain artificial CDRs (e.g., random amino acid sequences) and human
framework regions. (See, for example, U.S. Patent No. 6,300,064 (Knappik, et al.).) In
other examples, the library contains scFv fragments or dAbs (single VH, single VK or
single V\) with sequence diversity in one or more CDRs. (See, e.g., WO 99/20749
(Tomlinson and Winter), WO 03/002609 A2 (Winter et al), WO 2004/003019A2
(Winter et al.).)
Suitable antibodies and antigen-binding fragments thereof that bind serum
albumin include, for example, human antibodies and antigen-binding fragments thereof,
humanized antibodies and antigen-binding fragments thereof, chimeric antibodies and
antigen-binding fragments thereof, rodent (e.g., mouse, rat) antibodies and antigen-
binding fragments thereof, and Camelid antibodies and antigen-binding fragments
thereof. In certain embodiments, the drug conjugates, noncovalent drug conjugates and
drug fusions comprises a Camelid VHH that binds serum albumin. Camelid VHHS are
immunoglobulin single variable domain polypeptides which are derived from heavy
chain antibodies that are naturally devoid of light chains. Such antibodies occur in
Camelid species including camel, llama, alpaca, dromedary, and guanaco. VHH
molecules are about ten times smaller than IgG molecules, and as single polypeptides,
are very stable and resistant to extreme pH and temperature conditions. Suitable
Camelid VHH that bind serum albumin include those disclosed in WO 2004/041862
(Ablynx N.V.) and herein (FIG. 15 and SEQ ID NOS:77-88). In certain embodiments,
the Camelid VHH binds human serum albumin and comprises an amino acid sequence
that has at least about 80%, or at least about 85%, or at least about 90%, or at least about
95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least
about 99% amino acid sequence identity with SEQ ID NO: 72, SEQ ID NO:73, SEQ ID
NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID
NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID
NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, or SEQ ID NO:88. Amino
acid sequence identity is preferably determined using a suitable sequence alignment
algorithm and default parameters, such as BLAST P (Karlin and Altschul, Proc. Natl.
Acad. Sci. USA 57(6):2264-2268 (1990)).

WO 2006/059106 PCT/GB2005/004599
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Preparation of the immunizing antigen, and polyclonal and monoclonal antibody
production can be performed using any suitable technique. A variety of methods have
been described. (See, e.g., Kohler et al, Nature, 256: 495-497 (1975) and Eur. J.
Immunol. 6: 511-519 (1976); Milstein et al, Nature 266: 550-552 (1977); Koprowski et
al, U.S. Patent No. 4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A
Laboratory Manual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, NY);
Current Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer '94),
Ausubel, F.M. et al, Eds., (John Wiley & Sons: New York, NY), Chapter 11, (1991).)
Generally, where a monoclonal antibody is desired, a hybridoma is produced by fusing
suitable cells from an immortal cell line {e.g., a myeloma cell line such as SP2/0,
P3X63Ag8.653 or a heteromyeloma) with antibody-producing cells. Antibody-
producing cells can be obtained from the peripheral blood or, preferably the spleen or
lymph nodes, of humans, human-antibody transgenic animals or other suitable animals
immunized with the antigen of interest. Cells that produce antibodies of human origin
{e.g., a human antibody) can be produced using suitable methods, for example, fusion of
a human antibody-producing cell and a heteromyeloma or trioma, or immortalization of
an activated human B cell via infection with Epstein Barr virus. (See, e.g., U.S. Patent
No. 6,197,582 (Trakht); Niedbala et al, Hybridoma, 17:299-304 (1998); Zanella et al,
J Immunol Methods, 156:205-215 (1992); Gustafsson et al, Hum Antibodies
Hybridomas, 2:26-32 (1991).) The fused or immortalized antibody-producing cells
(hybridomas) can be isolated using selective culture conditions, and cloned by limiting
dilution. Cells which produce antibodies with the desired specificity can be identified
using a suitable assay {e.g., ELISA).
Antibodies also can be prepared directly {e.g., synthesized or cloned) from an
isolated antigen-specific antibody producing cell {e.g., a cell from the peripheral blood
or, preferably the spleen or lymph nodes determined to produce an antibody with
desired specificity), of humans, human-antibody transgenic animals or other suitable
animals immunized with the antigen of interest (see, e.g., U.S. Patent No. 5,627,052
(Schrader)).
When the drug conjugate, noncovalent drug conjugate or drug fusion is for
administration to a human, the antibody or antigen-binding fragment thereof that binds
serum albumin {e.g., human serum albumin) can be a human, humanized or chimeric

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antibody or an antigen-binding fragment of such an antibody. These types of antibodies
and antigen-binding fragments are less immunogenic or non-immunogenic in humans
and provide well-known advantages. For example, drug conjugates, noncovalent drug
conjugates or drug fusions that contain an antigen-binding fragment of a human,
humanized or chimeric antibody can be administered repeatedly to a human with less or
no loss of efficacy (compared with other fully immunogenic antibodies) due to
elaboration of human antibodies that bind to the drug conjugate or drug fusion. When
the drug conjugate, noncovalent drug conjugate or drug fusion is intended for veterinary
administration, analogous antibodies or antigen-binding fragments can be used. For
example, CDRs from a murine or human antibody can be grafted onto framework
regions from a desired animal, such as a horse or cow.
Human antibodies and nucleic acids encoding same can be obtained, for
example, from a human or from human-antibody transgenic animals. Human-antibody
transgenic animals (e.g., mice) are animals that are capable of producing a repertoire of
human antibodies, such as XENOMOUSE (Abgenix, Fremont, CA), HUMAB-
MOUSE, KHUN TC MOUSE or KM-MOUSE (MEDAREX, Princeton, NJ).
Generally, the genome of human-antibody transgenic animals has been altered to
include a transgene comprising DNA from a human immunoglobulin locus that can
undergo functional rearrangement. An endogenous immunoglobulin locus in a human-
antibody transgenic animal can be disrupted or deleted to eliminate the capacity of the
animal to produce antibodies encoded by an endogenous gene. Suitable methods for
producing human-antibody transgenic animals are well known in the art. (See, for
example, U.S. Pat. Nos. 5,939,598 and 6,075,181 (Kucherlapati et al), U.S. Pat. Nos.
5,569,825, 5,545,806, 5,625,126, 5,633,425, 5,661,016, and 5,789,650 (Lonberg et al.),
Jakobovits et al, Proc. Natl. Acad. Sci. USA, 90: 2551-2555 (1993), Jakobovits et al.,
Nature, 362: 255-258 (1993), Jakobovits et al. WO 98/50433, Jakobovits et al. WO
98/24893, Lonberg et al. WO 98/24884, Lonberg et al. WO 97/13852, Lonberg et al.
WO 94/25585, Lonberg et al. EP 0 814 259 A2, Lonberg et al. GB 2 272 440 A,
Lonberg et al, Nature 368:856-859 (1994), Lonberg et al, Int Rev Immunol 13(1):65-
93 (1995), Kucherlapati et al. WO 96/34096, Kucherlapati et al EP 0 463 151 Bl,
Kucherlapati et al. EP 0 710 719 Al, Surani et al. US. Pat. No. 5,545,807, Bruggemann
et al. WO 90/04036, Bruggemann et al EP 0 438 474 Bl, Taylor et al, Int. Immunol.

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6(4)579-591 (1994), Taylor et al, Nucleic Acids Research 20(23):6287-6295 (1992),
Green et al, Nature Genetics 7:13-21 (1994), Mendez et al, Nature Genetics 15:146-
156 (1997), Tuaillon et al, ProcNatl AcadSci USA 90(8):3720-3724 (1993) and
Fishwild et al, Nat Biotechnol 14(7):845-851 (1996), the teachings of each of the
foregoing are incorporated herein by reference in their entirety.)
Human-antibody transgenic animals can be immunized with a suitable antigen
{e.g., human serum albumin), and antibody producing cells can be isolated and fused to
form hybridomas using conventional methods. Hybridomas that produce human
antibodies having the desired characteristics (e.g., specificity, affinity) can be identified
using any suitable assay (e.g., ELISA) and, if desired, selected and subcloned using
suitable culture techniques.
Humanized antibodies and other CDR-grafted antibodies can be prepared using
any suitable method. The CDRs of a CDR-grafted antibody can be derived from a
suitable antibody which binds a serum albumin (referred to as a donor antibody). Other
sources of suitable CDRs include natural and artificial serum albumin-specific
antibodies obtained from human or nonhuman sources, such as rodent (e.g., mouse, rat,
rabbit), chicken, pig, goat, non-human primate (e.g., monkey) or a library.
The framework regions of a humanized antibody are preferably of human origin,
and can be derived from any human antibody variable region having sequence similarity
to the analogous or equivalent region (e.g., heavy chain variable region or light chain
variable region) of the antigen-binding region of the donor antibody. Other sources of
framework regions of human origin include human variable region consensus
sequences. (See, e.g., Kettleborough, C.A. et al, Protein Engineering 4:773-783
(1991); Carter et al, WO 94/04679; Kabat, E.A., et al, Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services,
U.S. Government Printing Office (1991)). Other types of CDR grafted antibodies can
contain framework regions of suitable origin, such as framework regions encoded by
germline antibody gene segments from horse, cow, dog, cat and the like.
Framework regions of human origin can include amino acid substitutions or
replacements, such as "back mutations" which replace an amino acid residue in the
framework region of human or animal origin with a residue from the corresponding
position of the donor antibody. One or more mutations in the framework region can be

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made, including deletions, insertions and substitutions of one or more amino acids.
Variants can be produced by a variety of suitable methods, including mutagenesis of
nonhuman donor or acceptor human chains. (See, e.g., U.S. Patent Nos. 5,693,762
(Queen et al.) and 5,859,205 (Adair et al), the entire teachings of which are
incorporated herein by reference.)
Constant regions of antibodies, antibody chains {e.g., heavy chain, light chain)
or fragments or portions thereof, if present, can be derived from any suitable source.
For example, constant regions of human, humanized and certain chimeric antibodies,
antibody chains (e.g., heavy chain, light chain) or fragments or portions thereof, if
present can be of human origin and can be derived from any suitable human antibody or
antibody chain. For example, a constant region of human origin or portion thereof can
be derived from a human K or X light chain, and/or a human y (e.g., yl, y2, y3, y4), fi, a
(e.g., al, od), 8 or e heavy chain, including allelic variants. In certain embodiments, the
antibody or antigen-binding fragment (e.g., antibody of human origin, human antibody)
can include amino acid substitutions or replacements that alter or tailor function (e.g.,
effector function). For example, a constant region of human origin (e.g., yl constant
region, y2 constant region) can be designed to reduce complement activation and/or Fc
receptor binding. (See, for example, U.S. Patent Nos. 5,648,260 (Winter et al.),
5,624,821 (Winter et al.) and 5,834,597 (Tso et al), the entire teachings of which are
incorporated herein by reference.) Preferably, the amino acid sequence of a constant
region of human origin that contains such amino acid substitutions or replacements is at
least about 95% identical over the full length to the amino acid sequence of the
unaltered constant region of human origin, more preferably at least about 99% identical
over the full length to the amino acid sequence of the unaltered constant region of
human origin.
Humanized antibodies, CDR grafted antibodies or antigen-binding fragments of
a humanized or CDR grafted antibody can be prepared using any suitable method.
Several such methods are well-known in the art. (See, e.g., U.S. Patent No. 5,225,539
(Winter), U.S. Patent No. 5,530,101 (Queen et al.)) The portions of a humanized or
CDR grafted antibody (e.g., CDRs, framework, constant region) can be obtained or
derived directly from suitable antibodies (e.g., by de novo synthesis of a portion), or
nucleic acids encoding an antibody or chain thereof having the desired property (e.g.,

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binds serum albumin) can be produced and expressed. To prepare a portion of a chain,
one or more stop codons can be introduced at the desired position. For example, nucleic
acid (e.g., DNA) sequences coding for humanized or CDR grafted variable regions can
be constructed using PCR mutagenesis methods to alter existing DNA sequences. (See,
e.g., Kamman, M., et al., Nucl. Acids Res. 17:5404 (1989).) PCR primers coding for the
new CDRs can be hybridized to a DNA template of a previously humanized variable
region which is based on the same, or a very similar, human variable region (Sato, K., et
al., Cancer Research 53:851-856 (1993)). If a similar DNA sequence is not available
for use as a template, a nucleic acid comprising a sequence encoding a variable region
sequence can be constructed from synthetic oligonucleotides (see e.g., Kolbinger, F.,
Protein Engineering 8:971-980 (1993)). A sequence encoding a signal peptide can also
be incorporated into the nucleic acid (e.g., on synthesis, upon insertion into a vector).
The natural signal peptide sequence from the acceptor antibody, a signal peptide
sequence from another antibody or other suitable sequence can be used (see, e.g.,
Kettleborough, C.A., Protein Engineering 4:773-783 (1991)). Using these methods or
other suitable methods, variants can be readily produced. In one embodiment, cloned
variable regions can be mutated, and sequences encoding variants with the desired
specificity can be selected (e.g., from a phage library; see, e.g., U.S. Patent No.
5,514,548 (Krebber et al.) and WO 93/06213 (Hoogenboom et al.)).
The antibody or antigen-binding fragment that binds serum albumin can be a
chimeric antibody or an antigen-binding fragment of a chimeric antibody. The chimeric
antibody or antigen-binding fragment thereof comprises a variable region from one
species (e.g., mouse) and at least a portion of a constant region from another species
(e.g., human). Chimeric antibodies and antigen-binding fragments of chimeric
antibodies can be prepared using any suitable method. Several suitable methods are
well-known in the art. (See, e.g., U.S. Patent No. 4,816,567 (Cabilly et al), U.S. Patent
No. 5,116,946 (Capon et al.))
A preferred method for obtaining antigen-binding fragments of antibodies that
bind serum albumin comprises selecting an antigen-binding fragment (e.g., scFvs,
dAbs) that has binding specificity for a desired serum albumin from a repertoire of
antigen-binding fragments. For example, as described herein dAbs that bind serum
albumin can be selected from a suitable phage display library. A number of suitable

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bacteriophage display libraries and selection methods (e.g., monovalent display and
multivalent display systems) have been described. (See, e.g., Griffiths et al, U.S.
Patent No. 6,555,313 Bl (incorporated herein by reference); Johnson etal., U.S. Patent
No. 5,733,743 (incorporated herein by reference); McCafferty et al, U.S. Patent No.
5,969,108 (incorporated herein by reference); Mulligan-Kehoe, U.S. Patent No.
5,702,892 (incorporated herein by reference); Winter, G. et al, Annu. Rev. Immunol.
12:433-455 (1994); Soumillion, P. etal, Appl. Biochem. Biotechnol 47(2-3):175-189
(1994); Castagnoli, L. etal, Comb. Chem. High Throughput Screen, 4(2):121-133
(2001); WO 99/20749 (Tomlinson and Winter); WO 03/002609 A2 (Winter et al); WO
2004/003019A2 (Winter et al).) The polypeptides displayed in a bacteriophage library
can be displayed on any suitable bacteriophage, such as a filamentous phage (e.g., fd,
Ml3, Fl), a lytic phage (e.g., T4, T7, lambda), or an RNA phage (e.g., MS2), for
example, and selected for binding to serum albumin (e.g., human serum albumin).
Generally, a library of phage that displays a repertoire of polypeptides as fusion
proteins with a suitable phage coat protein is used. Such a library can be produced
using any suitable methods, such as introducing a library of phage vectors or phagemid
vectors encoding the displayed antibodies or antigen-binding fragments thereof into
suitable host bacteria, and culturing the resulting bacteria to produce phage (e.g., using a
suitable helper phage or complementing plasmid if desired). The library of phage can
be recovered from such a culture using any suitable method, such as precipitation and
centrifugation.
The library can comprise a repertoire of antibodies or antigen-binding fragments
thereof that contains any desired amount of amino acid sequence diversity. For
example, the repertoire can contain antibodies or antigen-binding fragments thereof that
have amino acid sequences that correspond to naturally occurring antibodies from a
desired organism, and/or can contain one or more regions of random or randomized
amino acid sequences (e.g., CDR sequences). The antibodies or antigen-binding
fragments thereof in such a repertoire or library can comprise defined regions of random
or randomized amino acid sequence and regions of common amino acid sequence. In
certain embodiments, all or substantially all polypeptides in a repertoire are a desired
type of antigen-binding fragment of an antibody (e.g., human VH or human VL). For

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example, each polypeptide in the repertoire can contain a VH, a VL or an Fv (e.g., a
single chain Fv).
Amino acid sequence diversity can be introduced into any desired region of
antibodies or antigen-binding fragments thereof using any suitable method. For
example, amino acid sequence diversity can be introduced into a target region, such as a
complementarity determining region of an antibody variable domain, by preparing a
library of nucleic acids that encode the diversified antibodies or antigen-binding
fragments thereof using any suitable mutagenesis methods (e.g., low fidelity PCR,
oligonucleotide-mediated or site directed mutagenesis, diversification using NNK
codons) or any other suitable method. If desired, a region of the antibodies or antigen-
binding fragments thereof to be diversified can be randomized.
A suitable phage display library can be used to selected antibodies or antigen-
binding fragments of antibodies that bind serum albumin and have other beneficial
properties. For example, antibodies or antigen-binding fragments that resist aggregation
when unfolded can be selected. Aggregation is influenced by polypeptide concentration
and is thought to arise in many cases from partially folded or unfolded intermediates.
Factors and conditions that favour partially folded intermediates, such as elevated
temperature and high polypeptide concentration, promote irreversible aggregation.
(Fink, A.L., Folding & Design J:R1-R23 (1998).) For example, storing purified
polypeptides in concentrated form, such as a lyophilized preparation, frequently results
in irreversible aggregation of at least a portion of the polypeptides. Also, production of
a polypeptide by expression in biological systems, such as E. coli, often results in the
formation of inclusion bodies which contain aggregated polypeptides. Recovering
active polypeptides from inclusion bodies can be very difficult and require adding
additional steps, such as a refolding step, to a biological production system.
Antibodies and antigen-binding fragments that resist aggregation and unfold
reversibly when heated can be selected from a suitable phage display library.
Generally, a phage display library comprising a repertoire of displayed antibodies or
antigen-binding fragments thereof is heated to a temperature (Ts) at which at least a
portion of the displayed antibodies or antigen-binding fragments thereof are unfolded,
then cooled to a temperature (Tc) wherein Ts>Tc, whereby at least a portion of the
antibodies or antigen-binding fragments thereof have refolded and a portion of the

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polypeptides have aggregated. Then, antibodies or antigen-binding fragments thereof
that unfold reversibly and bind serum albumin are recovered at a temperature (Tr). The
recovered antibody or antigen-binding fragment thereof that unfolds reversibly has a
melting temperature (Tm), and preferably, the repertoire was heated to Ts, cooled to Tc
and the antibody or antigen-binding fragment thereof that unfolds reversibly was
isolated at Tr, such that Ts>Tm>Tc, and Ts>Tm>Tr. Generally, the phage display
library is heated to about 80°C and cooled to about room temperature or about 4°C
before selection. Antibodies or antigen-binding fragment thereof that unfold reversibly
and resist aggregation can also be designed or engineered by replacing certain amino
acid residue with residues that confer the ability to unfold reversibly. (See, WO
2004/101790 (Jespers et ah), and U.S. Provisional Patent Application Nos: 60/470,340
(filed on May 14, 2003) and 60/554,021 (filed on March 17, 2004) for detailed
discussion of methods for selecting and for designing or engineering antibodies or
antigen-binding fragments thereof that unfold reversibly. The teachings of WO
2004/101790 and both of the foregoing U.S. Provisional Patent Applications are
incorporated herein by reference.).
Antibodies or antigen-binding fragments thereof that unfold reversibly and resist
aggregation provide several advantages. For example, due to their resistance to
aggregation, antibodies or antigen-binding fragments thereof that unfold reversibly can
readily be produced in high yield as soluble proteins by expression using a suitable
biological production system, such as E. coli. In addition, antibodies or antigen-binding
fragments thereof that unfold reversibly can be formulated and/or stored at higher
concentrations than conventional polypeptides, and with less aggregation and loss of
activity. DOM7h-26 (SEQ ID NO:20) is a human VH that unfolds reversibly.
Preferably, the antibody or antigen-binding fragment thereof that binds serum
albumin comprises a variable domain (VH, V/C, V\) in which one or more of the
framework regions (FR) comprise (a) the amino acid sequence of a human framework
region, (b) at least 8 contiguous amino acids of the amino acid sequence of a human
framework region, or (c) an amino acid sequence encoded by a human germline
antibody gene segment, wherein said framework regions are as defined by Kabat. In
certain embodiments, the amino acid sequence of one or more of the framework regions
is the same as the amino acid sequence of a corresponding framework region encoded

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by a human germline antibody gene segment, or the amino acid sequences of one or
more of said framework regions collectively comprise up to 5 amino acid differences
relative to the amino acid sequence of said corresponding framework region encoded by
a human germline antibody gene segment.
In other embodiments, the amino acid sequences of FR1, FR2, FR3 and FR4 are
the same as the amino acid sequences of corresponding framework regions encoded by
a human germline antibody gene segment, or the amino acid sequences of FR1, FR2,
FR3 and FR4 collectively contain up to 10 amino acid differences relative to the amino
acid sequences of corresponding framework regions encoded by said human germline
antibody gene segments. In other embodiments, the amino acid sequence of said FR1,
FR2 and FR3 are the same as the amino acid sequences of corresponding framework
regions encoded by said human germline antibody gene segment.
In particular embodiments, the antigen binding fragment of an antibody that
binds serum albumin comprises an immunoglobulin variable domain (e.g., VH, VL)
based on a human germline sequence, and if desired can have one or more diversified
regions, such as the complementarity determining regions. Suitable human germline
sequence for VH include, for example, sequences encoded by the VH gene segments
DP4, DP7, DP8, DP9, DP10, DP31, DP33, DP45, DP46, DP47, DP49, DP50, DP51,
DP53, DP54, DP65, DP66, DP67, DP68 and DP69, and the JH segments JH1, JH2,
JH3, JH4, JH4b, JH5 and JH6. Suitable human germline sequence for VL include, for
example, sequences encoded by the VK gene segments DPK1, DPK2, DPK3, DPK4,
DPK5, DPK6, DPK7, DPK8, DPK9, DPK10, DPK12, DPK13, DPK15, DPK16,
DPK18, DPK19, DPK20, DPK21, DPK22, DPK23, DPK24, DPK25, DPK26 and DPK
28, and the JK segments 3K 1, J7C 2, J/c 3, JK 4 and J/c 5.
In certain embodiments, the drug conjugate, noncovalent drug conjugate or drug
fusion does not contain a mouse, rat and/or rabbit antibody that binds serum albumin or
antigen-binding fragment of such an antibody.
The antigen-binding fragment can bind serum albumin with any desired affinity,
on rate and off rate. The affinity (KD), on rate (Kon or ka) and off rate (Koff or kd or Kd)
can be selected to obtain a desired serum half-life for a particular drug. For example, it
may be desirable to obtain a maximal serum half-life for a drug that neutralizes an
inflammatory mediator of a chronic inflammatory disorder (e.g., a dAb that binds and

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neutralizes an inflammatory cytokine), while a shorter half-life may be desirable for a
drug that has some toxicity (e.g., a chemotherapeutic agent). Generally, a fast on rate
and a fast or moderate off rate for binding to serum albumin is preferred. Drug
conjugates and drug fusions that comprise an antigen-binding fragment with these
characteristics will quickly bind serum albumin after being administered, and will
dissociate and rebind serum albumin rapidly. These characteristics will reduce rapid
clearance of the drug (e.g., through the kidneys) but still provide efficient delivery and
access to the drug target.
The antigen-binding fragment that binds serum albumin (e.g., dAb) generally
binds with a KD of about 1 nM to about 500 piM. In some embodiments, the antigen-
binding fragment binds serum albumin with a KD (KD=KOff (kd)/Kon (ka)) of about 10
to about 100 nM, or about 100 nM to about 500 nM, or about 500 nM to about 5 mM, as
determined by surface plasmon resonance (e.g., using a BIACORE instrument). In
particular embodiments, the drug conjugate, noncovalent drug conjugate or drug fusion
comprises and antigen-binding fragment of an antibody (e.g., a dAb) that binds serum
albumin (e.g., human serum albumin) with a KD of about 50 nM, or about 70 nM, or
about 100 nM, or about 150 nM or about 200 nM. The improved pharmacokinetic
properties (e.g., prolonged tl/2/3, increased AUC) of drug conjugates, noncovalent drug
conjugates and drug fusions described herein may correlate with the affinity of the
antigen-binding fragment that binds serum albumin. Accordingly, drug conjugates,
noncovalent drug conjugates and drug fusions that have improved pharmacokinetic
properties can generally be prepared using an antigen-binding fragment that binds
serum albumin (e.g., human serum albumin) with high affinity (e.g., KD of about 500
nM or less, about 250 nM or less, about 100 nM or less, about 50 nM or less, about 10
nM or less, or about 1 nM or less, or about 100 pM or less).
Preferably, the drug that is conjugated or fused to the antigen-binding fragment
that binds serum albumin, binds to its target (the drug target) with an affinity (KD) that
is stronger than the affinity of the antigen-binding fragment for serum albumin and/or a
Koff (kd) that is faster that the Koff of the antigen binding fragment for serum albumin, as
measured by surface plasmon resonance (e.g., using a BIACORE instrument). For
example, the drug can bind its target with an affinity that is about 1 to about 100000, or
about 100 to about 100000, or about 1000 to about 100000, or about 10000 to about

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100000 times stronger than the affinity of antigen-binding fragment that binds SA for
SA. For example, the antigen-binding fragment of the antibody that binds SA can bind
with an affinity of about 10 μM, while the drug binds its target with an affinity of about
100 pM.
In particular embodiments, the antigen-binding fragment of an antibody that
binds serum albumin is a dAb that binds human serum albumin. For example, a VK dAb
having an amino acid sequence selected from the group consisting of SEQ ID NO: 10,
SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ
ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, or a VH dAb having an amino acid
sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ
ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ
ID NO:23. In other embodiments, the antigen-binding fragment of an antibody that
binds serum albumin is a dAb that binds human serum albumin and comprises the
CDRs of any of the foregoing amino acid sequences. In other embodiments, the
antigen-binding fragment of an antibody that binds serum albumin is a dAb that binds
human serum albumin and comprises an amino acid sequence that has at least about
80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least
about 96%, or at least about 97%, or at least about 98%, or at least about 99% amino
acid sequence identity with SEQ ID NO: 10, SEQIDNO:11, SEQIDNO:12, SEQ ID
NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:24, SEQ ID NO:25, SEQ ID
NO:26, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID
NO:20, SEQ ID NO:21, SEQ ID NO:22 or SEQ ID NO:23. Amino acid sequence
identity is preferably determined using a suitable sequence alignment algorithm and
default parameters, such as BLAST P (Karlin and Altschul, Proc. Natl Acad. Sd. USA
57(6):2264-2268 (1990)).
Drugs
Certain drug compositions of the invention {e.g., drug conjugates, noncovalent
drug conjugates) can comprise any drug {e.g., small organic molecule, nucleic acid,
polypeptide) that can be administered to an individual to produce a beneficial
therapeutic or diagnostic effect, for example, through binding to and/or altering the
function of a biological target molecule in the individual. Other drug compositions of

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the invention (e.g., drug fusions) can comprise a polypeptide or peptide drug. In
preferred embodiments of drug fusions, the drug does not comprise an antibody chain or
fragment of an antibody chain (e.g., VH, VK, Vλ). In specific embodiments, the drug is
selected from an insulinotropic agent, and incretin, a glucagon-like 1 peptide, a GLP-1
peptide, a GLP-1 analogue, a GLP-1 derivative, PYY, a PYY peptide, a PYY analogue,
a PYY derivative, Exendin-3, an Exendin-3 peptide, an Exendin-3 analogue, an
Exendin-3 derivative, Exendin-4, an Exendin-4 peptide, an Exendin-4 analogue, an
Exendin-4 derivative or a combination of two or more of these (eg, GLP-1 peptide and a
PYY peptide).
Suitable drugs for use in the invention include, for example, immunosuppressive
agents (e.g., cyclosporin A, rapamycin, FK506, prednisone), antiviral agents (acyclovir,
ganciclovir, indinavir), antibiotics (penicillin, mynocyclin, tetracycline), anti-
infiammatory agents (aspirin, ibuprofen, prednisone), cytotoxins or cytotoxic agents
(e.g., paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin
C, etoposide, tenoposide, vincristine, vinblastine, colcbicine, doxorubicin, daunorubicin,
dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-
dihydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol,
puromycm, and analogs or homologs of any of the foregoing agents. Suitable drugs
also include antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,
cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,
thioepachlorambucil, CC-1065, melphalan, carmustine (BSNU), lomustine (CCNU),
cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-
dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin
(formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly
actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), radionuclides (e.g.,
iodine-125, -126) yttrium (e.g., yttrium-90, -91) and praseodymium (e.g.,
praseodymium-144, -145), and protease inhibitors (e.g., inhibitors of matrix
metalloproteinases). Other suitable drugs are nucleic acids such as antisense nucleic
acids and RNAi. Calicheamicin is also suitable for use in the invention.
Suitable drugs also include analgesic agents, including narcotics (e.g., codeine,
nalmefene, naloxone, fentanyl, meperidine, morphine, tramadol, propoxyphene,
oxycodone, methadone, nalbuphine), nonsteroidal anti-inflammatory agents (e.g.,

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indomethacin, ketorolac, arthrotec, ibuprofen, naproxen, salicylate, celecoxib,
rofecoxib), acetaminophen, capsaicin, ziconotide and the like.
In certain embodiments, the drug is a polypeptide toxin, for example, a toxin
such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin. Other suitable
polypeptide drugs include antibodies or antigen-binding fragments (e.g., dAbs) of
antibodies, polypeptide agonists, activators, secretagogues, antagonists or inhibitors.
For example, the polypeptide or peptide drug can bind and agonise or antagonize a cell
surface protein, such as a CD antigen, cytokine receptor (e.g., interleukin receptor,
chemokine receptor), adhesion molecule or costimulatory molecule. For example, the
polypeptide drug can bind a cytokine, growth factors, cytokine receptor, growth factor
receptor and other target ligand, which include but are not limited to: ApoE, Apo-SAA,
BDNF, Cardiotrophin-1, CEA, CD40, CD40 Ligand, CD56, CD38, CD138, EGF, EGF
receptor, ENA-78, Eotaxin, Eotaxin-2, Exodus-2, FAPa, FGF-acidic, FGF-basic,
fibroblast growth factor-10, FLT3 ligand, Fractalkine (CX3C), GDNF, G-CSF, GM-
CSF, GF-pl, human serum albumin, insulin, IFN-y, IGF-I, IGF-II, IL-la, IL-lp, IL-1
receptor, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (72 a.a.), IL-8 (77 a.a.), IL-9, IL-10, IL-
11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF), Inhibin a, Inhibin p, IP-10,
keratinocyte growth factor-2 (KGF-2), KGF, Leptin, LIF, Lymphotactin, Mullerian
inhibitory substance, monocyte colony inhibitory factor, monocyte attractant protein,
M-CSF, MDC (67 a.a.), MDC (69 a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4,
MDC (67 a.a.), MDC (69 a.a.), MIG, MIP-loc, MIP-lp, MIP-3a, MIP-3P, MIP-4,
myeloid progenitor inhibitor factor-1 (MPIF-1), NAP-2, Neurturin, Nerve growth
factor, p-NGF, NT-3, NT-4, Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4,
RANTES, SDFla, SDFip, SCF, SCGF, stem cell factor (SCF), TARC, TGF-a, TGF-p,
TGF-p2, TGF-p3, tumour necrosis factor (TNF), TNF-a, TNF-p, TNF receptor I, TNF
receptor II, TNIL-1, TPO, VEGF, VEGF A, VEGF B, VEGF C, VEGF D, VEGF
receptor 1, VEGF receptor 2, VEGF receptor 3, GCP-2, GRO/MGSA, GRO-p, GRO-y,
HCC1, 1-309, HER 1, HER 2, HER 3 and HER 4. It will be appreciated that this list is
by no means exhaustive.
Suitable drugs also include hormones, including pituitary hormone (PTH),
adrenocorticotropic hormone (ACTH), renin, luteinizing hormone-releasing hormone
(LHRH), gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), follicle

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stimulating hormone (FSH), aldosterone, and the like. Suitable drugs also include
keratinocyte growth factor, interferons (e.g., IFN-a, IFN-B, IFN-7), erythropoietin
(EPO), proteases, elastases, LHRH analogs, agonists and antagonists, opioid receptor
agonists, such as kappa opioid receptor agonists (e.g., dynorphin A), calcitonin and
calcitonin analogs, antidiuretic hormone (vasopressin), oxytocin antagonists, vasoactive
intestinal peptide, thrombin inhibitors, von Willebrand factor, surfactants and snail
venom (e.g., ziconotide).
Suitable drugs also include peptides and polypeptides that have anti-cancer
activities (e.g., proliferation inhibiting, growth inhibiting, apoptosis inducing, metastasis
inhibiting, adhesion inhibiting, neovascularization inhibiting). Several such peptides
and polypeptides are known in the art. (See. e.g., Janin Y.L., Amino Acids, 25:1-40
(2003). The entire teaching of this reference, particularly the peptides and polypeptides
disclosed therein, are incorporated herein by reference.) The amino acid sequences of
several such peptides are presented in Table 8.
Other suitable drags include peptides and polypeptides that have anti-viral
activity. Several such peptides and polypeptides are known in the art, for example the
peptides and polypeptides disclosed in Giannecchini, et al, J Viro., 77(6):3724-33
(2003); Wang, J., etal, Clin Chem (2003); Hilleman, M.R., Vaccine, 21(32):4626-49
(2003); Tziveleka, L.A., et al, Curr Top Med Chem, 3(13): 1512-35 (2003); Poritz,
M.A., et al, Virology, 313(7):170-83 (2003); Oevermann, A., et al, Antiviral Res,
59(i):23-33 (2003); Cole, A.M. et al, Curr Pharm Des, 9(18): 1463-73 (2003); Pinon,
J.D., et al, Virol, 77(5):3281-90 (2003); Sia, S.K., et al, Proc Natl Acad Sci USA,
99(23): 14664-9 (2002); Bahbouhi, B., et al, Biochem J, 66(Pt 5):863-72 (2002); de
Soultrait, V.R., et al, JMolBiol, 18(7):45-58 (2002); Witherell, G., Curr Opin Investig
Drugs, 2(5):340-7 (2001); Ruff, M.R., et al., Antiviral Res, 52(l):63-75 (2001);
Bultmann, H., et al., J. Virol, 75(6):2634-45 (2001); Egal, M., et al., Int JAntimicrob
AGents, 13(/):57-60 (1999); and Robinson, W.E., Jr., JLeukoc Biol, 63(7):94-
100(1998). The entire teachings of these references, particularly the peptides and
polypeptides disclosed therein, are incorporated herein by reference. These peptides
and polypeptides are examples of drugs that can be used in the compositions, drug
fusions, drug conjugates, noncovalent drug conjugates of the present invention.

WO 2006/059106 PCT/GB2005/004599
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The polypeptide drag can also be a cytokine or growth factor or soluble portion
of a receptor (e.g., a cytokine receptor, growth factor receptor, hormone receptor) or
other polypeptide such as the polypeptides listed above. For example, suitable
polypeptide drags also include receptor (e.g., growth factor receptor, cytokine receptor,
hormone receptor) agonists and antagonists, such as interleukin 1 receptor antagonist
(Eisenberg et ah, Nature 343:341-346 (1990)), thrombopoietin receptor agonists (e.g.,
GW395058 (de Serres et al, Stem Cells 17:316-326 (1999)), melanocortin receptor
antagonists (e.g., MCR-4 antagonists (Cepoi et al., Brain Res. 1000:64-71 (2004)),
anginex, 6DBF7 (Mayo et ah, J. Bioh Chem. 278:45746-45752 (2003)), chemokine
mimetics (e.g., RANTES mimetics (Nardese et al, Nat. Struct. Bioh 8:611-615 (2001)),
growth hormone (e.g., human growth hormone), growth hormone analogs and growth
hormone secretagogues (e.g., CP-424,391 (MacAndrew et ah, Eur. J. Pharmacol.
432:195-202 (2001)), growth hormone releasing hormone mimetics (e.g., MK-677
(Chapman etah, J. Clin. Endocrinol. Metab. 82:3455-3463 (1997)), inhibitors of
cellular adhesion molecule interactions (e.g., LFA-l/ICAM-1, VLA-l/VCAM-1
(Yusuf-Makagiansar et ah, Med. Res. Rev. 22:146-167 (2002)), mimetics of interferon
(e.g., SYR6 (Sato etah, Biochem. J. 371(Pt.2):603-608 (2003), mimetics ofherceptin
(Nature Biotechnoh 18:137 (2000)), inhibitors of antigen presentation (Bolin et ah, J.
Med. Chem. 43:2135-2148 (2000)), GPIB/IIIA antagonists (e.g., FK633 (Aoki etah,
Thromb. Res. 81:439-450 (1996)), alphavbeta3 antagonists (e.g., SC56631 (Engleman et
ah, J. Clin. Invest. 99:2284-2292 (1997)), erythropoietin mimetics (e.g., EMP1
(Johnson et ah, Biochemistry 37:3699-3710 (1998)), opioid receptor antagonists (e.g.,
[(2S, 3R)-TMT1]DPDPE (Liao etah, J. Med. Chem. 41:4161-4:116 (1998)),
hematopoietic factors (e.g., erythropoietin (EPO), granulocyte colony stimulating factor
(GM-CSF)).
Additional suitable peptide and polypeptide drugs include peptide antagonists
that bind human type 1IL-1 receptor (e.g., AF 11377 (FEWTPGYWQPYALPL, SEQ
ID NO:56), AF11869 (FEWTPGYWQJYALPL, SEQ ID NO:57 (J = l-azetidine-2-
carboxylic acid), FEWTPGYWQJY (SEQ ID NO:58), FEWTPGWYQJY (SEQ ID
NO:59), FEWTPGWYQJYALPL (SEQ ID NO:60), or any of the foregoing sequences
optionally containing an acylated amino terminus and/or an aminated carboxyl terminus
(Akeson et al, J. Bioh Chem. 271:30517-305123 (1996)), peptide antagonists of TNF-

WO 2006/059106 PCT/GB2005/004599
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alpha-mediated cytotoxicity (e.g., those disclosed in Chirinos-Rojas et al, J. Immunol.
161:5621-5626 (1998)), peptide agonists of erythropoietin receptor (e.g., those
disclosed in McConnel et al, Biol. Chem. 379:1279-1286 (1998) or Wrighton et al,
Science 273:458-464 (1996)), glucagon-like peptide-1 (GLP-1, e.g., GLP-l(7-37), GLP-
l(7-36)amide and analogs thereof (see, e.g., Ritzel U. etal, J. Endocrinology 159:93-
102 (1998)), and interferons (e.g., INF-a, INF-B, INF-X). Additional suitable
polypeptide and peptide drugs include integrin inhibitors (e.g., RGD peptides, such as
H-Glu[cyclo(Arg-Gly-Asp-D-Phe-Lys)]2 (Janssen, MX., etal, Cancer Research
62:6146- 6151 (2002)), cyclo(Arg-Gly-Asp-D-Phe-Lys) (Kantlehner M., et al, Agnew.
Chem. Int. Ed. 38:560 (1999)), cyclo(Arg-Gly-Asp-D-Tyr-Lys) (Haubner, R., et al, J.
Nucl. Med. 42:326-336 (2001)), ribosome-inactivating proteins (RTPs) such as Saporin
(e.g., SEQ ID NO:67), matrix metalloproteinase inhibitors (e.g., U.S. Patent No.
5,616,605), and antiviral peptides and polypeptides, such as HIV fusion inhibitors
(e.g.,T-1249 and T-20 (FUZEON® (enfuvirtide); Trimeris Inc.), and soluble receptor
antagonists such as immunoadhesins (e.g., LFA3-Ig, CTLA4-Ig).
Antimicrobial polypeptide and peptide drugs are also suitable for use in the
invention. Examples of suitable antimicrobial polypeptide and peptide drugs include
adenoregulin, dermcidin-lL, cathelicidins (e.g., cathelicidin-like peptide, human LL-
37/hCAP-18), defensins, including a-defensins (e.g., human neutrophil peptide 1 (HNP-
1), HNP-2, HNP-3, FfNP-4, human defensin 5, human defensin 6), B-defensins (e.g.,
human J3-defensin-l, human J3-defensin-2), and 0-defensins (e.g., 9-defensin-l),
histatins (e.g., histatin 1, histatin 3, histatin 5), lactoferricin-derived peptide and related
peptides (see, TomitaM., et al, Ada Paediatr. Jpn. 36:585-591 (1994) and Strom,
M.B., etal Biochem Cell Biol 80:65-74 (2002)).
In a preferred embodiment of the invention the drugs are insulinotropic drugs.
Examples of suitable insulinotropic drugs include GLP-1, GLP-1 derivative, GLP-1
analogues or a derivative of a GLP-1 analogue. In addition they include Exedin-4,
Exedin-4 analogues and Exedin-4 derivatives and Exedin-3, Exedin-3 derivatives and
Exedin-3 analogues.
Other suitable drugs include Peptide YY (3-36) or analogues. Peptide YY (PYY)
is a 36-residue peptide amide isolated originally from porcine intestine, and localized in
the endocrine cells of the gastrointestinal tract and pancreas (Tatemoto, et al. Proc. Natl.

WO 2006/059106 PCT/GB2005/004599
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Acad. Sci. 79:2514, 1982). Peptide YY has N-terminal and C-terminal tyrosine amides;
accordingly, these two tyrosines give PYY its name (Y represents the amino acid
tyrosine in the peptide nomenclature). In addition PYY shares a number of central and
peripheral regulatory roles with its homologous peptide neuropeptide Y (NPY), which
was originally isolated from porcine brain (Tatemoto, Proc. Natl. Acad. Sci. 79:5485,
1982). In contrast with the cellular location of PYY, NPY is present in submucous and
myenteric neurons which innervate the mucosal and smooth muscle layers, respectively
(Ekblad et al. Neuroscience 20:169, 1987). Both PYY and NPY are believed to inhibit
gut motility and blood flow (Laburthe, Trends Endocrinol. Metab. 1:168,1990), and
they are also thought to attenuate basal (Cox et al. Br. J. Pharmacol. 101:247,1990) and
secretagogue-induced intestinal secretion in rats (Lundberg et al Proc. Natl. Acad. Sci
USA 79:4471, 1982), as well as stimulate net absorption (MacFadyen et al.
Neuropeptides 7:219, 1986). Taken together, these observations suggest that PYY and
NPY are released into the circulation after a meal (Adrian et al. Gastroenterology
89:1070, 1985; Balasubramaniam et al. Neuropeptides 14:209, 1989), and thus play a
physiological role in regulating intestinal secretion and absorption.
A high affinity PYY receptor system which exhibits a slightly higher affinity for
PYY than NPY has been characterized in rat intestinal epithelia (Laburthe et al.
Endocrinology 118:1910,1986) and shown to be negatively coupled to adenylate
cyclase (Servin et al. Endocrinology 124:692, 1989). Structure-activity studies using
several partial sequences have led to the identification of PYY(22-36) as the active site
for interacting with intestinal PYY receptors (Balsubramaniam et al. Pept. Res. 1:32,
1988).
In addition, PYY has been implicated in a number of physiological activities
including nutrient uptake (Bilcheik et al. Digestive Disease Week 506:623,1993), cell
proliferation (Laburthe, Trends Endocrinol. Metab. 1:168,1990; Voisin et al. J. Biol.
Chem, 1993), lipolysis (Valet et al, J. Clin. Invest. 291, 1990), and vasoconstriction
(Lundberg et al, Proc. Natl. Acad. Sci., USA 79: 4471,1982).
WO 03/057235 and WO 03/026591 disclose method for decreasing calorie
intake, food intake and appetite by the administration of PYY or an agonist and GLP-1.
These publications are incorporated herein by reference in their entirety, in particular to

WO 2006/059106 PCT/GB2005/004599
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provide examples of PYY and GLP-1 drugs and methods that can be used in the present
invention.
Further other drugs that are suitable for use in the invention include insulin,
Resistin, Leptin, MC3R/MC4R antagonist, AgRP antagonist, Apolipoprotein A-IV,
Enterostatin, Gastrin-Releasing Peptide (GRP), IGF1, BMP-9, IL-22, ReglV, interferon
alfa, INGAP peptide, somatostatin, amylin, neurulin, interferon beta, interferon hybrids,
adiponectin, endocannabinoids, C peptide, WNTlOb, Orexin-A, adrenocorticotrophin,
Enterostatin, Cholecystokinin, oxyntomodulin, Melanocyte Stimulating Hormones,
melanocortin, Melanin concentrating hormone, BB-2, NPY Y2 agonists, NPY Y5/Y1
antagonists, OXM, Gal-IR antagonists, MCH-1R antagonists, MC-3/4 agonists, BRS-3
agonists, pancreatic polypeptide, anti-Ghrelin antibody fragment, brain-derived
neurotrophic factor, human growth hormone, parathyroid hormone, follicle stimulating
hormone, Gastric inhibitory peptide or an analogue thereof.
Drug Fusions
The drug fusions of the invention are fusion proteins that comprise a continuous
polypeptide chain, said chain comprising an antigen-binding fragment of an antibody
that binds serum albumin as a first moiety, linked to a second moiety that is a
polypeptide drug. The first and second 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., third, fourth) and/or linker sequences can
be present as appropriate. The first moiety can be in an N-terminal location, C-terminal
location or internal relative to the second moiety (i.e., the polypeptide drug). In certain
embodiments, each moiety can be present in more than one copy. For example, the
drug fusion can comprise two or more first moieties each comprising an antigen-binding
fragment of an antibody that binds serum albumin (e.g., a VH that binds human serum
albumin and a VL that bind human serum albumin or two or more VHS or VLS that bind
human serum albumin).
In some embodiments the drug fusion is a continuous polypeptide chain that has
the formula:


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wherein X is a polypeptide drug that has binding specificity for a first target;
Y is a single chain antigen-binding fragment of an antibody that has binding
specificity for serum albumin;
Z is a polypeptide drug that has binding specificity for a second target;
a, b, c and d are each independently absent or one to about 100 amino acid
residues;
nl is one to about 10;
n2 is one to about 10; and
n3 is zero to about 10,
with the proviso that when nl and n2 are both one and n3 is zero, X does not
comprise an antibody chain or a fragment of an antibody chain.
In one embodiment, neither X nor Z comprises an antibody chain or a fragment
of an antibody chain. In one embodiment, nl is one, n3 is one and n2 is two, three,
four, five, six, seven, eight or nine. Preferably, Y is an immunoglobulin heavy chain
variable domain (VH) that has binding specificity for serum albumin, or an
immunoglobulin light chain variable domain (VL) that has binding specificity for serum
albumin. More preferably, Y is a dAb (e.g., a VH, VK or Vx) that binds human serum
albumin. In a particular embodiment, X or Z is human GLP-1 or a GLP-1 derivatives or
analogue thereof.
In certain embodiments, Y comprises an amino acid sequence selected from the
group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13,
SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26.
In other embodiments, Y comprises an amino acid sequence selected from the group
consisting of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID
NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23.
In other embodiments, the drug fusion comprises moieties X' and Y', wherein
X' is a polypeptide drug, with the proviso that X' does not comprise an antibody chain
or a fragment of an antibody chain; and Y' is a single chain antigen-binding fragment of
an antibody that has binding specificity for serum albumin. Preferably, Y' is an
immunoglobulin heavy chain variable domain (VH) that has binding specificity for
serum albumin, or an immunoglobulin light chain variable domain (VL) that has binding
specificity for serum albumin. More preferably, Y' is a dAb (e.g., a VH, Vk or Vλ) that

WO 2006/059106 PCT/GB2005/004599
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binds human serum albumin. X' can be located amino terminally to Y', or Y' can be
located amino terminally to X'. In some embodiments, X' and Y' are separated by an
amino acid, or by a peptide or polypeptide linker that comprises from two to about 100
amino acids. In a particular embodiment, X' is human GLP-1 or GLP-1 derivative or
analogues thereof.
In certain embodiments, Y' comprises an amino acid sequence selected from the
group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13,
SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26.
In other embodiments, Y' comprises an amino acid sequence selected from the group
consisting of SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO:19, SEQ ID
NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23.
In particular embodiments the drug fusion comprises a dAb that binds serum
albumin and human IL-lra (e.g., SEQ ID NO: 28). Preferably, the dAb binds human
serum albumin and comprises human framework regions.
In other embodiments, the drug fusion or drug conjugate comprises a functional
variant of human IL-lra that has at least about 80%, or at least about 85%, or at least
about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at
least about 98%, or at least about 99% amino acid sequence identity with the mature
152 amino acid form of human IL-lra and antagonizes human Interleukin-1 type 1
receptor. (See, Eisenberg et al, Nature 343:341-346 (1990).) The variant can comprise
one or more additional amino acids (e.g., comprise 153 or 154 or more amino acids).
The drug fusions of the invention can be produced using any suitable method. For
example, some embodiments can be produced by the insertion of a nucleic acid
encoding the drug fusion into a suitable expression vector. The resulting construct is
then introduced into a suitable host cell for expression. Upon expression, fusion protein
can be isolated or purified from a cell lysate or preferably from the culture media or
periplasm using any suitable method. (See e.g., Current Protocols in Molecular
Biology (Ausubel, F.M. et al, eds., Vol. 2, Suppl. 26, pp. 16.4.1-16.7.8 (1991)).
In a further embodiment the drug fusion or drug conjugate comprises an
insulinotropic agent, hi a preferred embodiment the drug fusion or drug conjugate
comprises GLP-1, or an analogue or peptide of GLP-1. hi a further preferred
embodiment, the drug fusion or drug conjugate comprises Ser8GLP-l (7-36) amide.

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In a further embodiment, the drug fusion or drag conjugate comprises a GLP-1
analogue having one or more of the following substitutions: Val or Pro .
Preferably, the GLP-1 analogue is Pro9GLP-1(7-36) or Pro9GLP-l(7-37).
Further the GLP-1 analogue or peptide may include any one of the following C-terminal
extensions: PSS, PSSGAP or PSSGAPPPS.
In another embodiment, the drug fusion or drag conjugate comprises a GLP-1
analogue comprising the sequence of Formula I
His7-Xaa8-Xaa9-Gly10-Xaa11-Phe12-Thr13-Xaa14-Asp15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-
Xaa21-Xaa22-Xaa23-Xaa24-Xaa25-Xaa26-Xaa27-Phe28-Ile29-Xaa30-Xaa31-Xaa32-Xaa33-
Xaa34-Xaa35-Xaa36-Xaa37-Xaa38-Xaa39-Xaa40-Xaa41-Xaa42-Xaa43-Xaa44-Xaa45
FormulaI-SEQIDNO:171
wherein:
Xaa at position 8 is Ala, Gly, Ser, Thr, Leu, He, Val, Glu, Asp, or Lys;
Xaa at position 9 is Glu, or Asp;
Xaa at position 11 is Thr, Ala, Gly, Ser, Leu, He, Val, Glu, Asp, or Lys;
Xaa at position 14 is Ser, Ala, Gly, Thr, Leu, He, Val, Glu, Asp, or Lys;
Xaa at position 16 is Val, Ala, Gly, Ser, Thr,. Leu, He, Tyr, Glu, Asp, Trp, or Lys;
Xaa at position 17 is Ser, Ala, Gly, Thr, Leu, He, Val, Glu, Asp, or Lys;
Xaa at position 18 is Ser, Ala, Gly, Thr, Leu, He, Val, Glu, Asp, Trp, Tyr, or Lys;
Xaa at position 19 is Tyr, Phe, Trp, Glu, Asp, Gin, or Lys; Xaa at position 20 is Leu,
Ala, Gly, Ser, Thr, lie, Val, Glu, Asp, Met, Trp, Tyr, or Lys;
Xaa at position 21 is Glu, Asp, or Lys;
Xaa at position 22 is Gly, Ala, Ser, Thr, Leu, He, Val, Glu, Asp, or Lys;
Xaa at position 23 is Gin, Asn, Arg, Glu, Asp, or Lys;
Xaa at position 24 is Ala, Gly, Ser, Thr, Leu, He, Val, Arg, Glu, Asp, or Lys;
Xaa at position 25 is Ala, Gly, Ser, Thr, Leu, lie, Val, Glu, Asp, or Lys;
Xaa at position 26 is Lys, Arg, Gin, Glu, Asp, or His;
Xaa at position 27 is Leu, Glu, Asp, or Lys;
Xaa at position 30 is Ala, Gly, Ser, Thr, Leu, He, Val, Glu, Asp, or Lys;
Xaa at position 31 is Trp, Phe, Tyr, Glu, Asp, or Lys;
Xaa at position 32 is Leu, Gly, Ala, Ser, Thr, He, Val, Glu, Asp, or Lys;
Xaa at position 33 is Val, Gly, Ala, Ser, Thr, Leu, He, Glu, Asp, or Lys;

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Xaa at position 34 is Asn, Lys, Arg, Glu, Asp, or His;
Xaa at position 35 is Gly, Ala, Ser, Thr, Leu, He, Val, Glu, Asp, or Lys;
Xaa at position 36 is Gly, Arg, Lys, Glu, Asp, or His;
Xaa at position 37 is Pro, Gly, Ala, Ser, Thr, Leu, He, Val, Glu, Asp, or Lys, or is
deleted;
Xaa at position 38 is Ser, Arg, Lys, Glu, Asp, or His, or is deleted;
Xaa at position 39 is Ser, Arg, Lys, Glu, Asp, or His, or is deleted;
Xaa at position 40 is Gly, Asp, Glu, or Lys, or is deleted; Xaa at position 41 is Ala, Phe,
Trp, Tyr, Glu, Asp, or Lys, or is deleted;
Xaa at position 42 is Ser, Pro, Lys, Glu, or Asp, or is deleted;
Xaa at position 43 is Ser, Pro, Glu, Asp, or Lys, or is deleted;
Xaa at position 44 is Gly, Pro, Glu, Asp, or Lys, or is deleted;
and Xaa at position 45 is Ala, Ser, Val, Glu, Asp, or Lys, or is deleted;
provided that when the amino acid at position 37,38,39, 40,41,42,43, or 44 is deleted,
then each amino acid downstream of that amino acid is also deleted.
In another embodiment the drug fusion or drug conjugate comprises a GLP-1
analogue that comprises the amino acid sequence of the Formula (II):
Xaa7-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Xaa16-Ser-Xaa18-Xaa19-Xaa20-Glu-Xaa22-
Xaa23-Ala-Xaa25-Xaa26-Xaa27-Phe-Ile-Xaa30-Trp-Leu-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-
Xaa38-Xaa39-Xaa40-Xaa41-Xaa42-Xaa43-Xaa44-Xaa45-Xaa46
Formula (II) - SEQ ID NO: 172
wherein Xaa7 is L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, (3-
hydroxy-histidine, homohistidine, Na-acetyl-histidine, a-fluoromethyl-histidine, a-
methyl-histidine, 3- pyridylalanine, 2-pyridylalanine or 4-pyridylalanine ;
Xaa8 is Ala, Gly, Val, Leu, IIe, Lys, Aib, (1-aminocyclopropyl) carboxylic acid, (1-
aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl) carboxylic acid, (1-
aminocyclohexyl) carboxylic acid, (1-aminocycloheptyl) carboxylic acid, or (1-
aminocyclooctyl) carboxylic acid;
Xaa16 is Val or Leu;
Xaa18 is Ser, Lys or Arg;

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Xaa19 is Tyr or Gin;
Xaa20 is Leu or Met;
Xaa22isGly, GluorAib;
Xaa23 is Gin, Glu, Lys or Arg;
Xaa25 is Ala or Val;
Xaa26 is Lys, Glu or Arg;
Xaa27 is Glu or Leu;
Xaa30 is Ala, Glu or Arg;
Xaa33 is Val or Lys;
Xaa34 is Lys, Glu, Asn or Arg;
Xaa35 is Gly or Aib;
Xaa36 is Arg, Gly or Lys;
Xaa37 is Gly, Ala, Glu, Pro, Lys, amide or is absent;
Xaa38 is Lys, Ser, amide or is absent.
Xaa39 is Ser, Lys, amide or is absent;
Xaa40 is Gly, amide or is absent;
Xaa41 is Ala, amide or is absent;
Xaa42 is Pro, amide or is absent;
Xaa43 is Pro, amide or is absent;
Xaa44 is Pro, amide or is absent;
Xaa45 is Ser, amide or is absent;
Xaa46 is amide or is absent; provided that if Xaa38, Xaa39, Xaa40, Xaa41, Xaa42, Xaa43,
Xaa44, Xaa45 or Xaa46 is absent then each amino acid residue downstream is also absent.
In another embodiment of the invention the drug fusion or drug conjugate comprises a
GLP-1 peptide comprising the amino acid sequence of formula (III):

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Xaa7-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Xaa18-Tyr-Leu-Glu-Xaa22-Xaa23-
Ala-Ala-Xaa26-Glu-Phe-lle-Xaa30-Trp-Leu-Val-Xaa34-Xaa35-Xaa36-Xaa37-Xaa38
Formula (III) - SEQ ID NO: 173
Wherein
Xaa7 is L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, B-hydroxy-
histidine, homohistidine, N'1-acetyl-histidine, a-fluoromethyl-histidine, a -methyl-
histidine, 3- pyridylalanine, 2-pyridylalanine or 4-pyridylalanine;
Xaa8 is Ala, Gly, Val, Leu, He, Lys, a-aminoisobutyric acid (Aib), (1-
aminocyclopropyl) carboxylic acid, (1- aminocyclobutyl) carboxylic acid, (1-
aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl) carboxylic acid, (1-
aminocycloheptyl) carboxylic acid, or (1-aminocyclooctyl) carboxylic acid;
Xaa18 is Ser, Lys or Arg;
Xaa22 is Gly, Glu or Aib;
Xaa23 is Gin, Glu, Lys or Arg;
Xaa26 is Lys, Glu or Arg;
Xaa30 is Ala, Glu or Arg;
Xaa34 is Lys, Glu or Arg;
Xaa35 is Gly or Aib;
Xaa36 is Arg or Lys;
Xaa37 is Gly, Ala, Glu or Lys;
Xaa38 is Lys, amide or is absent.
In yet another embodiment of the invention the GLP-1 peptide is selected from
the group consisting of: GLP-1 (7-35), GLP-1 (7-36), GLP-1 (7-36) -amide, GLP-1 (7-
37), GLP-1 (7-38), GLP-1 (7-39), GLP-1 (7- 40), GLP-1 (7-41) or an analogue or
peptide thereof.
In another embodiment of the invention the GLP-1 peptide is GLP-1 (A-B)
wherein A is an integer from 1 to 7 and B is an integer from 37 to 45 or an analogue

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thereof comprising one albumin binding residue attached via a hydrophilic spacer to the
C-terminal amino acid residue and, optionally, a second albumin binding residue
attached to one of the other amino acid residues.
In another embodiment the GLP-1 peptide comprises no more than fifteen amino
acid residues which have been exchanged, added or deleted as compared to GLP-1 (7-
37) or no more than ten amino acid residues which have been exchanged, added or
deleted as compared to GLP-1 (7-37).
In another embodiment the GLP-1 peptide comprises no more than six
(preferably, no more than 5, 4, 3, 2 or 1) amino acid residues which have been
exchanged, added or deleted as compared to GLP-1 (7-37).
In another embodiment the GLP-1 peptide comprises no more than 4 preferably,
no more than 3, 2 or 1) amino acid residues which are not encoded by the genetic code.
In another embodiment the GLP-1 peptide is a DPPIV protected GLP-1 peptide.
In another embodiment the insulinotropic agent is DPPIV stabilised.
In another embodiment the GLP-1 peptide comprises an ce-aminoisobutyric acid
(Aib) residue in position 8.
In another embodiment the amino acid residue in position 7 of said GLP-1
peptide is selected from the group consisting of D-histidine, desamino-histidine, 2-
amino-histidine, B-hydroxy-histidine, homohistidine, Na-acetyl-histidine, a-
fluoromethyl-histidine, a-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine and 4-
pyridylalanine.
In another embodiment the GLP-1 peptide is selected from the group consisting of:
Arg34GLP-l (7-37), Arg26'34Lys38GLP-l(7-38), Arg26'34Lys38GLP-l (7-38)-OH,
Lys36Arg26>34GLP-l (7-36), Aib8'22'35 GLP-1 (7-37), Aib8'35 GLP-1 (7-37), Aib8'22 GLP-1
(7-37), Aib8>22'35Arg26'34Lys38GLP-l (7-38), Aib8'35Arg26'34Lys38GLP-l(7-38),
Aib8'22Arg26'34Lys38GLP-l (7-38), Aib8>22'35Arg26>34Lys38GLP-l (7-38),
Aib8>35Arg26'34Lys38GLP-1 (7-3 8), Aib8'22'35Arg26Lys38GLP-1 (7-3 8),
Aib8'35Arg26Lys38GLP-l(7-38), Aib8>22Arg26Lys38GLP-l (7-38), Aib8'22'
35Arg34Lys38GLP-l (7-38), Aib8'35Arg34Lys38GLP-l (7-38), Aib8'22Arg34Lys38GLP-l (7-
38), Aib8'22>35Ala37Lys38GLP-l (7-38), Aib8'35Ala37Lys38GLP-l (7-38),
Aib8'22Ala37Lys38GLP-l (7-38), Aib8>22'35Lys37GLP-l (7-37), Aib8'35Lys37GLP-l (7-37),
Aib8Arg26'34Glu22'23'30Lys38GLP-l(7-38),Gly8Arg26'34Lys36GLP-l(7-37),
Aib8Arg26'34Lys38GLP-l (7-38), Aib8Lys38GLP-l (7-38), Gly8Arg26>34Lys38GLP-l(7-
38), GLP-1 (7-37)amide, GLP-1 (7-37) amide, Aib8Arg26'34Lys36GLP-l(7-37),
Arg26'34Lys36GLP-l(7-37), Gly8Arg26'34Lys36GLP-l(7-37), Aib8'35Lys37GLP-l (7-37)-
OH, Ala8Arg26'34Lys38GLP-l(7-38), Aib8'22'35Lys38GLP-l (7-38),
Aib8Arg26'34Lys36GLP-l (7-36), Gly8Arg26'34Lys36GLP-l(7-37)-OH,

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Aib8l22>35Lys37GLP-1 (7-37)-NH2, Aib8Arg34GLP-1 (7-37)-OH, Gly8Arg26'34Lys38GLP-
1(7-38), Arg34GLP-l(7-37)-OH,Gly8Glu22'23'30Arg18>26'34Lys38GLP-l(7-38),
imidazolylpropionicacid7Asp18Aib22'35Lys38GLP-l(7-38), imidazolyrpropionic
acid7Aib22'35Lys38GLP-1 (7-38), [3-(5-Imidazoyl)propionyl7Aib8Arg26:34Lys38GLP-1 (7-
38), and Aib8'22Lys37GLP-l (7-38).
In another embodiment the GLP-1 peptide is attached to a hydrophilic spacer via
the amino acid residue in position 23, 26, 34, 36 or 38 of the native GLP-1 or GLP-1
analogue.
In another embodiment the insulinotropic agent is Lys20exendin-4(l-39)-NH2.
In another embodiment the GLP-1 peptide is
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK-amide - SEQ
ID NO: 174.
In another embodiment the GLP-1 peptide is
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGX - SEQ ID NO: 175
wherein X = P or Y, or a fragment or an analogue thereof.
In another embodiment of the invention the GLP-1 peptide is Arg18, Leu20,
Gin34, Lys33 (Ne-(Y-aminobutyroyl(Na-hexadecanoyl))) Exendin-4-(7-45)-amide or
Arg33, Leu20, Gln34,Lys18 (Ne-(γ-aminobutyroyl(Na-hexadecanoyl))) Exendin-
4-(7-45)-amide.
Examples of insulinotropic agents which can be useful as GLP-1 analogues or
derivatives or GLP-1 like drugs according to the present invention are described in
International Patent Application No. WO 87/06941 (The General Hospital Corporation)
which relates to a peptide fragment which comprises GLP-1 (7-37) and functional
derivatives thereof and to its use as an insulinotropic agent (incorporated herein by
reference, particularly by way of examples of drugs for use in the present invention).
Further 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).

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International Patent Application No. WO 91/11457 (Buckley et al..) discloses analogues
of the active GLP-1 peptides 7-34,7-35, 7-36, and 7-37 which can also be useful as
GLP-1 drugs according to the present invention (incorporated herein by reference,
particularly by way of examples of drugs for use in the present invention).
Further Exendin-analogs that are useful for the present invention are described
in PCT patent publications WO 99/25728 (Beeley et al.), WO 99/25727 Beeley et al.),
WO 98/05351 (Young et al.), WO 99/40788 (Young et al.), WO 99/07404 (Beeley et
al), and WO 99/43708 (Knudsen et al) (all incorporated herein by reference, particularly
by way of examples of drugs for use in the present invention).
Suitable expression vectors can contain a number of components, for example,
an origin of replication, a selectable marker gene, one or more expression control
elements, such as a transcription control element (e.g., promoter, enhancer, terminator)
and/or one or more translation signals, a signal sequence or leader sequence, and the
like. Expression control elements and a signal sequence, if present, can be provided by
the vector or other source. For example, the transcriptional and/or translational control
sequences of a cloned nucleic acid encoding an antibody chain can be used to direct
expression.
A promoter can be provided for expression in a desired host cell. Promoters can
be constitutive or inducible. For example, a promoter can be operably linked to a
nucleic acid encoding an antibody, antibody chain or portion thereof, such that it directs
transcription of the nucleic acid. A variety of suitable promoters for procaryotic (e.g.,
lac, tac, T3, T7 promoters for E. coli) and eucaryotic (e.g., simian virus 40 early or late
promoter, Rous sarcoma virus long terminal repeat promoter, cytomegalovirus
promoter, adenovirus late promoter) hosts are available.
In addition, expression vectors typically comprise a selectable marker for
selection of host cells carrying the vector, and, in the case of a replicable expression
vector, an origin or replication. Genes encoding products which confer antibiotic or
drug resistance are common selectable markers and may be used in procaryotic (e.g.,
lactamase gene (ampicillin resistance), Tet gene for tetracycline resistance) and
eucaryotic cells (e.g., neomycin (G418 or geneticin), gpt (mycophenolic acid),
ampicillin, or hygromycin resistance genes). Dihydrofolate reductase marker genes
permit selection with methotrexate in a variety of hosts. Genes encoding the gene

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product of auxotrophic markers of the host (e.g., LEU2, URA3, HIS3) are often used as
selectable markers in yeast. Use of viral (e.g., baculovirus) or phage vectors, and
vectors which are capable of integrating into the genome of the host cell, such as
retroviral vectors, are also contemplated. Suitable expression vectors for expression in
mammalian cells and prokaryotic cells (E. coli), insect cells (Drosophila Schnieder S2
cells, Sf9) and yeast (P. methanolica, P. pastoris, S. cerevisiae) are well-known in the
art.
Recombinant host cells that express a drug fusion and a method of preparing a
drug fusion as described herein are provided. The recombinant host cell comprises a
recombinant nucleic acid encoding a drug fusion. Drug fusions can be produced by the
expression of a recombinant nucleic acid encoding the protein in a suitable host cell, or
using other suitable methods. For example, the expression constructs described herein
can be introduced into a suitable host cell, and the resulting cell can be maintained (e.g.,
in culture, in an animal) under conditions suitable for expression of the constructs.
Suitable host cells can be prokaryotic, including bacterial cells such as E. coli, B.
suhtilis and or other suitable bacteria, eucaryotic, such as fungal or yeast cells (e.g.,
Pichia pastoris, Aspergillus species, Saccharomyces cerevisiae, Schizosaccharomyces
pornbe, Neurospora crassa), or other lower eucaryotic cells, and cells of higher
eucaryotes such as those from insects (e.g., Sf9 insect cells (WO 94/26087 (O'Connor))
or mammals (e.g., COS cells, such as COS-1 (ATCC Accession No. CRL-1650) and
COS-7 (ATCC Accession No. CRL-1651), CHO (e.g., ATCC Accession No. CRL-
9096), 293 (ATCC Accession No. CRL-1573), HeLa (ATCC Accession No. CCL-2),
CV1 (ATCC Accession No. CCL-70), WOP (Dailey et al, J. Virol. 54:139-149 (1985)),
3T3, 293T (Pear et al, Proc. Natl. Acad. Sci. U.S.A., 90:8392-8396 (1993)), NSO cells,
SP2/0, HuT 78 cells, and the like (see, e.g., Ausubel, F.M. et al., eds. Current Protocols
in Molecular Biology, Greene Publishing Associates and John Wiley & Sons Inc.,
(1993)).
The invention also includes a method of producing a drug fusion, comprising
maintaining a recombinant host cell of the invention under conditions appropriate for
expression of a drug fusion. The method can further comprise the step of isolating or
recovering the drug fusion, if desired, hi another embodiment, the components of the

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drug fusion (e.g., dAb that binds human serum albumin and EL-Ira) are chemically
assembled to create a continuous polypeptide chain.
Conjugates
In another aspect, the invention provides conjugates comprising an antigen-
binding fragment of an antibody that binds serum albumin that is bonded to a drug.
Such conjugates include "drug conjugates," which comprise an antigen-binding
fragment of an antibody that binds serum albumin to which a drug is covalently bonded,
and "noncovlaent drug conjugates," which comprise an antigen-binding fragment of an
antibody that binds serum albumin to which a drug is noncovalently bonded.
Preferably, the conjugates are sufficiently stable so that the antigen-binding fragment of
an antibody that binds serum albumin and drug remain substantially bonded (either
covalently or noncovalently) to each other under in vivo conditions {e.g., when
administered to a human). Preferably, no more than about 20%, no more than about
15%, no more than about 10%, no more than about 9%, no more than about 8%, no
more than about 7%, no more than about 6%, no more than about 5%, no more than
about 4%, no more than about 3%, no more than about 2%, no more than about 1% or
substantially none of the conjugates dissociate or break down to release drug and
antigen-binding fragment under in vivo conditions. For example, stability under "in
vivo" conditions can be conveniently assessed by incubating drug conjugate or
noncovalent drug conjugate for 24 hours in serum {e.g., human serum) at 37°C. In one
example of such a method, equal amounts of a drug conjugate and the unconjugated
drug are diluted into two different vials of serum. Half of the contents of each vial is
immediately frozen at -20°C, and the other half incubated for 24 hours at 37°C. All four
samples can then be analyzed using any suitable method, such as SDS-PAGE and/or
Western blotting. Western blots can be probed using an antibody that binds the drug.
All drugs in the drug conjugate lanes will run at the size of the drug conjugate if there
was no dissociation. Many other suitable methods can be used to assess stability under
"in vivo" conditions, for example, by analyzing samples prepared as described above
using suitable analytic methods, such as chromatography (e.g., gel filtration, ion
exchange, and reverse phase), ELISA, mass spectroscopy and the like.

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Drug Conjugates
In another aspect, the invention provides a drug conjugate comprising an
antigen-binding fragment of an antibody that has binding specificity for serum albumin,
and a drug that is covalently bonded to said antigen-binding fragment, with the proviso
that the drug conjugate is not a single continuous polypeptide chain.
In some embodiments, the drug conjugate comprises an immunoglobulin heavy
chain variable domain (VH) that has binding specificity for serum albumin, or an
immunoglobulin light chain variable domain (VL) that has binding specificity for serum
albumin, and a drug that is covalently bonded to said VH or VL, with the proviso that the
drug conjugate is not a single continuous polypeptide chain. Preferably the drug
conjugate comprises a single VH that binds serum albumin or a single VL that binds
serum albumin. In certain embodiments, the drug conjugate comprises a Vk dAb that
binds human serum albumin and comprises an amino acid sequence selected from the
group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,
SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26.
In other embodiments, the drug conjugate comprises a VH dAb that binds human serum
albumin and comprises an amino acid sequence selected from the group consisting of
SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ
ID NO:21, SEQ ID NO:22 and SEQ ID NO:23.
The drug conjugates can comprise any desired drug and can be prepared using
any suitable methods. For example, the drug can be bonded to the antigen-binding
fragment of an antibody that binds serum albumin directly or indirectly through a
suitable linker moiety at one or more positions, such as the amino-terminus, the
carboxyl-terminus or through amino acid side chains. In one embodiment, the drug
conjugate comprises a dAb that binds human serum albumin and a polypeptide drug
(e.g., human IL-lra or a functional variant of human IL-lra), and the amino-terminus of
the polypeptide drug (e.g., human IL-lra or a functional variant of human IL-lra) is
bonded to the carboxyl-terminus of the dAb directly or through a suitable linker moiety.
In another embodiment, the drug conjugate comprises a dAb that binds human serum
albumin and an insulinotropic drug (e.g., GLP-lor a GLP-1 analogue) and the amino-
terminus of the insulinotropic drug is free (i.e. not coupled or bonded in the conjugate)
and the carboxyl terminus is bonded to the amino-terminus of the dAb directly or

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through a suitable linker moiety. In other embodiments, the drug conjugate comprises a
dAb that binds human serum albumin and two or more different drugs that are
covalently bonded to the dAb. For example, a first drug can be covalently bonded
(directly or indirectly) to the carboxyl terminus of the dAb and a second drug can be
covalently bonded (directly or indirectly) to the ammo-terminus or through a side chain
ammo group (e.g., e amino group of lysine). In a preferred embodiment the amino-
terminus of the insulinotropic drug (eg. GLP-lor a GLP-1 analogue) is free. Such drug
conjugates can be prepared using well-known methods of selective coupling. (See, e.g.,
Hermanson, G. T., Bioconjugate Techniques, Academic Press: San Diego, CA (1996).)
A variety of methods for conjugating drugs to an antigen-binding fragment of an
antibody that has binding specificity for serum albumin can be used. The particular
method selected will depend on the drag to be conjugated. If desired, linkers that
contain terminal functional groups can be used to link the antigen-binding fragment and
the drug. Generally, conjugation is accomplished by reacting a drug that contains a
reactive functional group (or is modified to contain a reactive functional group) with a
linker or directly with an antigen-binding fragment of an antibody that binds serum
albumin. Covalent bonds form by reacting a drug that contains (or is modified to
contain) a chemical moiety or functional group that can, under appropriate conditions,
react with a second chemical group thereby forming a covalent bond. If desired, a
suitable reactive chemical group can be added to the antigen-binding fragment or to a
linker using any suitable method. (See, e.g., Hermanson, G. T., Bioconjugate
Techniques, Academic Press: San Diego, CA (1996).) Many suitable reactive chemical
group combinations are known in the art, for example an amine group can react with an
electrophilic group such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo), N-
hydroxysuccinmiidyl ester (NHS), and the like. Thiols can react with maleimide,
iodoacetyl, acrylolyl, pyridyl disulfides, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol),
and the like. An aldehyde functional group can be coupled to amine- or hydrazide-
containing molecules, and an azide group can react with a trivalent phosphorous group
to form phosphoramidate or phosphorimide linkages. Suitable methods to introduce
activating groups into molecules are known in the art (see for example, Hermanson, G.
T., Bioconjugate Techniques, Academic Press: San Diego, CA (1996)).

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In some embodiments, the antigen-binding fragment of an antibody that has
binding specificity for serum albumin is bonded to a drug by reaction of two thiols to
form a disulfide bond. In other embodiments, the antigen-binding fragment of an
antibody that has binding specificity for serum albumin is bonded to a drug by reaction
of an isothiocyanate group and a primary amine to produce an isothiourea bond.
Suitable linker moieties can be linear or branched and include, for example,
tetraethylene glycol, C2-Ci2 alkylene, -NH-(CH2)P-NH- or -(CH2)P-NH- (wherein p is
one to twelve), -CH2-O-CH2-CH2-O-CH2-CH2-O-CH-NH-, a polypeptide chain
comprising one to about 100 (preferably one to about 12) amino acids and the like.
Noncovalent Drug Conjugates
Some noncovalent bonds (e.g., hydrogen bonds, van der Waals interactions) can
produce stable, highly specific intermolecular connections. For example, molecular
recognition interactions achieved through multiple noncovalent bonds between
complementary binding partners underlie many important biological interactions, such
as the binding of enzymes to their substrates, the recognition of antigens by antibodies,
the binding of ligands to their receptors, and stabilization of the three dimensional
structure of proteins and peptide. Accordingly, such weak noncovalent interactions
(e.g., hydrogen bonding, van Der Waals interactions, electrostatic interactions,
hydrophobic interactions and the like) can be utilized to bind a drug to the antigen-
binding fragment of an antibody that has binding specificity for serum albumin.
Preferably, the noncovalent bond linking the antigen-binding fragment and drug
be of sufficient strength that the antigen-binding fragment and drug remain substantially
bonded to each under in vivo conditions (e.g., when administered to a human).
Generally, the noncovalent bond linking the antigen-binding fragment and drug has a
strength of at least about l010M"1. In preferred embodiments, the strength of the
noncovalent bond is at least about l011M-1, at least about l012M-1, at least about 1013M"
1, at least about 1014M'J or at least about l015M"1. The interactions between biotin and
avidin and between biotin and streptavidin are known to be very efficient and stable
under many conditions, and as described herein noncovalent bonds between biotin and
avidin or between biotin and streptavidin can be used to prepare a noncovalent drug
conjugate of the invention.

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The noncovalent bond can be formed directly between the antigen-binding
fragment of an antibody that has a specificity for serum albumin and drug, or can be
formed between suitable complementary binding partners (e.g., biotin and avidin or
streptavidin) wherein one partner is covalently bonded to drug and the complementary
binding partner is covalently bonded to the antigen-binding fragment. When
complementary binding partners are employed, one of the binding partners can be
covalently bonded to the drug directly or through a suitable linker moiety, and the
complementary binding partner can be covalently bonded to the antigen-binding
fragement of an antibody that binds serum albumin directly or through a suitable linker
moiety.
Complementary binding partners are pairs of molecules that selectively bind to
each other. Many complementary binding partners are known in the art, for example,
antibody (or an antigen-binding fragment thereof) and its cognate antigen or epitope,
enzymes and their substrates, and receptors and their ligands. Preferred complementary
binding partners are biotin and avidin, and biotin and streptavidin.
Direct or indirect covalent bonding of a member of a complementary binding
pair to an antigen-binding fragment that has binding specificity for serum albumin or a
drug can be accomplished as described above, for example, by reacting a
complementary binding partner that contains a reactive functional group (or is modified
to contain a reactive functional group) with an antigen-binding fragment of an antibody
that binds serum albumin, with or without the use of a linker. The particular method
selected will depend on the compounds (e.g., drug, complementary binding partner,
antigen-binding fragment of an antibody that binds serum albumin) to be conjugated. If
desired, linkers (e.g., homobifunctional linkers, heterobifunctional linkers) that contain
terminal reactive functional groups can be used to link the antigen-binding fragment
and/or the drug to a complementary binding partner. In one embodiment, a
heterobifunctional linker that contains two distinct reactive moieties can be used. The
heterobifunctional linker can be selected so that one of the reactive moieties will react
with the antigen-binding fragment of an antibody that has binding specificity for serum
albumin or the drug, and the other reactive moiety will react with the complementary
binding partner. Any suitable linker (e.g., heterobifunctional linker) can be used and

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many such linkers are known in the art and available for commercial sources (e.g.,
Pierce Biotechnology, Inc., IL).
Compositions and Therapeutic and Diagnostic Methods
Compositions comprising drug compositions of the invention (e.g., drug
conjugates, noncovalent drug conjugates, drug fusions), including pharmaceutical or
physiological compositions (e.g., for human and/or veterinary administration) are
provided. Pharmaceutical or physiological compositions comprise one or more drug
compositions (e.g., drug conjugate, noncovalent drug conjugate, drug fusion), and a
pharmaceutically or physiologically acceptable carrier. Typically, these carriers include
aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline
and/or buffered media. Parenteral vehicles 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, gelatin
and alginates. 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 (1982) Remington's Pharmaceutical Sciences, 16th Edition).
The compositions can comprise a desired amount of drug composition (e.g.,
drug conjugate, noncovalent drug conjugate, drag fusion). For example the
compositions can comprise about 5% to about 99% drug conjugate, noncovalent drug
conjugate or drug fusion by weight. In particular embodiments, the composition can
comprise about 10% to about 99%, or about 20% to about 99%, or about 30% to about
99% or about 40% to about 99%, or about 50% to about 99%, or about 60% to about
99%, or about 70% to about 99%, or about 80% to about 99%, or about 90% to about
99%, or about 95% to about 99% drug composition (e.g., drug conjugate, noncovalent
drag conjugate, drag fusion), by weight. In one example, the composition is freeze
dried (lyophilized).
The drag compositions (e.g., drag conjugates, noncovalent drag conjugates, drag
fusions), described herein will typically find use in preventing, suppressing or treating
inflammatory states (e.g., acute and/or chronic inflammatory diseases), such as chronic

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obstructive pulmonary disease (e.g., chronic bronchitis, chronic obstructive bronchitis,
emphysema), allergic hypersensitivity, cancer, bacterial or viral infection, pneumonia,
such as bacterial pneumonia (e.g., Staphylococcal pneumonia)), autoimmune disorders
(which include, but are not limited to, Type I diabetes, multiple sclerosis, arthritis (e.g.,
osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis,
lupus arthritis, spondylarthropathy (e.g., ankylosing spondylitis)), systemic lupus
erythematosus, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis),
Behcet's syndrome and myasthenia gravis), endometriosis, psoriasis, abdominal
adhesions (e.g., post abdominal surgery), asthma, and septic shock. The drug
compositions (e.g., drug conjugates, noncovalent drug conjugates, drug fusions),
described herein can be used for preventing, suppressing or treating pain, such as
chronic or acute traumatic pain, chronic or acute neuropathic pain, acute or chronic
musculoskeletal pain, chronic or acute cancer pain and the like. The drug compositions
(e.g., drug conjugates, noncovalent drug conjugates, drug fusions), described herein can
also be administered for diagnostic purposes.
Cancers that can be prevented, suppressed or treated using the drug
compositions (e.g., drug conjugates, noncovalent drug conjugates, drug fusions),
described herein include lymphomas (e.g., B cell lymphoma, acute myeloid lymphoma,
Hodgkin's lymphoma, non-Hodgkin's lymphoma), myelomas (e.g., multiple myeloma),
lung cancer (e.g., small cell lung carcinoma, non-small cell lung carcinoma), colorectal
cancer, head and neck cancer, pancreatic cancer, liver cancer, stomach cancer, breast
cancer, ovarian cancer, bladder cancer, leukemias (e.g., acute myelogenous leukemia,
chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic
leukemia), adenocarcinomas, renal cancer, haematopoetic cancers (e.g., myelodysplastic
syndrome, myeloproliferative disorder (e.g., polycythemia vera, essential (or primary)
thrombocythemia, idiopathic myelofibrosis), and the like.
The drug compositions (e.g., drug conjugates, noncovalent drug conjugates, drug
fusions) described herein are also suitable for use in preventing, suppressing or treating
endometriosis, fibrosis, infertility, premature labour, erectile dysfunction, osteoporosis,
diabetes (e.g., type II diabetes), growth disorder, HIV infection, respiratory distress
syndrome, tumours and bedwetting.

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In a preferred embodiment the present invention relates to the use of a
compound according to the invention for the preparation of a medicament for the
treatment of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1
diabetes, obesity, hypertension, syndrome X, dyslipidemia, (J3-cell apoptosis, j3-ce) i
deficiency, myocardial infarction, inflammatory bowel syndrome, dyspepsia, cognitive
disorders, e. g. cognitive enhancing, neuroprotection, atherosclerosis, coronary heart
disease and other cardiovascular disorders. In specific embodiments for these
indications, the drug is selected from an insulinotropic agent, and incretin, a glucagon-
like 1 peptide, a GLP-1 peptide, a GLP-1 analogue, a GLP-1 derivative, PYY, a PYY
peptide, a PYY analogue, a PYY derivative, Exendin-3, an Exendin-3 peptide, an
Exendin-3 analogue, an Exendin-3 derivative, Exendin-4, an Exendin-4 peptide, an
Exendin-4 analogue, an Exendin-4 derivative or a combination of two or more of these
(eg, GLP-1 peptide and a PYY peptide).
In another embodiment the present invention relates to the use of a compound
according to the invention for the preparation of a medicament for the treatment of
small bowel syndrome, inflammatory bowel syndrome or Crohns disease. In specific
embodiments for these indications, the drug is selected from an insulinotropic agent,
and incretin, a glucagon-like 1 peptide, a GLP-1 peptide, a GLP-1 analogue, a GLP-1
derivative, PYY, a PYY peptide, a PYY analogue, a PYY derivative, Exendin-3, an
Exendin-3 peptide, an Exendin-3 analogue, an Exendin-3 derivative, Exendin-4, an
Exendin-4 peptide, an Exendin-4 analogue, an Exendin-4 derivative or a combination of
two or more of these (eg, GLP-1 peptide and a PYY peptide).
In another embodiment the present invention relates to the use of a compound
according to the invention for the preparation of a medicament for the treatment of
hyperglycemia, type 1 diabetes, type 2 diabetes or 6-cell deficiency. In specific
embodiments for these indications, the drug is selected from an insulinotropic agent,
and incretin, a glucagon-like 1 peptide, a GLP-1 peptide, a GLP-1 analogue, a GLP-1
derivative, PYY, a PYY peptide, a PYY analogue, a PYY derivative, Exendin-3, an
Exendin-3 peptide, an Exendin-3 analogue, an Exendin-3 derivative, Exendin-4, an
Exendin-4 peptide, an Exendin-4 analogue, an Exendin-4 derivative or a combination of
two or more of these (eg, GLP-1 peptide and a PYY peptide).

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The treatment with a compound according to the present invention may also be
combined with a second or more pharmacologically active substances which may or
may not be part of the drug conjugate or fusion. For example, an active selected from
antidiabetic agents, antiobesity agents, appetite regulating agents, antihypertensive
agents, agents for the treatment and/or prevention of complications resulting from or
associated with diabetes and agents for the treatment and/or prevention of complications
and disorders resulting from or associated with obesity. In the present context the
expression "antidiabetic agent" includes compounds for the treatment and/or
prophylaxis of insulin resistance and diseases wherein insulin resistance is the
pathophysiological mechanism.
Examples of these pharmacologically active substances are: Insulin, GLP-1
agonists, sulphonylureas (e. g. tolbutamide, glibenclamide, glipizide and gliclazide),
biguanides e. g. metformin, meglitinides, glucosidase inhibitors (e. g. acorbose),
glucagon antagonists, DPP-IV (dipeptidyl peptidase-IV) inhibitors, inhibitors of hepatic
enzymes involved in stimulation of gluconeogenesis and/or glycogenolysis, glucose
uptake modulators, thiazolidinediones such as troglitazone and ciglitazone, compounds
modifying the lipid metabolism such as antihyperlipidemic agents as HMG CoA
inhibitors (statins), compounds lowering food intake, RXR agonists and agents acting
on the ATP-dependent potassium channel of the (fl-cells, e. g. glibenclamide, glipizide,
gliclazide and repaglinide; Cholestyramine, colestipol, clofibrate, gemfibrozil,
lovastatin, pravastatin, simvastatin, probucol, dextrothyroxine, neteglinide, repaglinide ;
(B-blockers such as alprenolol, atenolol, timolol, pindolol, propranolol and metoprolol,
ACE (angiotensin converting enzyme) inhibitors such as benazepril, captopril, enalapril,
fosinopril, lisinopril, alatriopril, quinapril and ramipril, calcium channel blockers such
as nifedipine, felodipine, nicardipine, isradipine, nimodipine, diltiazem and verapamil,
and a-blockers such as doxazosin, urapidil, prazosin and terazosin; CART (cocaine
amphetamine regulated transcript) agonists, NPY (neuropeptide Y) antagonists, MC4
(melanocortin 4) agonists, orexin antagonists, TNF (tumor necrosis factor) agonists,
CRF (corticotropin releasing factor) agonists, CRF BP (corticotropin releasing factor
binding protein) antagonists, urocortin agonists, B3 agonists, MSH (melanocyte-
stimulating hormone) agonists, MCH (melanocyte-concentrating hormone) antagonists,
CCK (cholecystokinin) agonists, serotonin re-uptake inhibitors, serotonin and

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noradrenaline re-uptake inhibitors, mixed serotonin and noradrenergic compounds, 5HT
(serotonin) agonists, bombesin agonists, galanin antagonists, growth hormone, growth
hormone releasing compounds, TRH (thyreotropin releasing hormone) agonists, UCP 2
or 3 (uncoupling protein 2 or 3) modulators, leptin agonists, DA agonists (bromocriptin,
doprexin), lipase/amylase inhibitors, RXR (retinoid X receptor) modulators, TR B
agonists; histamine H3 antagonists.
Further insulin can be in the form of one of the following analogues: AspB28-
human insulin, LysB28, ProB29-human insulin, LysB3 GluB29-human insulin,
GlyA21, ArgB31, ArgB32-human insulin and des (B30) human insulin.
Further other active drugs include, human growth hormone or an analogue
thereof, parathyroid hormone or an analogue thereof, a growth factor such as platelet-
derived growth factor (PDGF), transforming growth factor a (TGF-a), transforming
growth factor-13 (TGF-J3), epidermal growth factor (EGF), vascular endothelial growth
factor (VEGF), a somatomedin such as insulin growth factor I (IGF-I), insulin growth
factor 11 (IFG-II), erythropoietin (EPO), thrombopoietin (TPO) or angiopoietin,
interferon, prourokinase, urokinase, tissue plasminogen activator (t-PA), plasminogen
activator inhibitor 1, plasminogen activator inhibitor 2, von Willebrandt factor, a
cytokine, e. g. an interleukin such as interleukin (IL) 1, IL-1 Ra, IL-2, IL-4, IL-5, IL-6,
IL-9, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-20 or IL-21, a colony
stimulating factor (CFS) such as GM-CSF, stem cell factor, a tumor necrosis factor such
as TNF- a, lymphotoxin-a, Iymphotoxin-B, CD40L, or CD30L, a protease inhibitor e. g.
aprotinin, human follicle stimulating hormone or an analogue thereof, an enzyme such
as superoxide dismutase, asparaginase, arginase, arginine deaminase, adenosine
deaminase, ribonuclease, catalase, uricase, bilirubin oxidase, trypsin, papain, alkaline
phosphatase, (3-glucoronidase, purine nucleoside phosphorylase or batroxobin, an
opioid, e. g. endorphins, enkephalins or non-natural opioids, a hormone or neuropeptide,
e. g. calcitonin, glucagon, gastrins, adrenocorticotropic hormone (ACTH),
cholecystokmins, lutenizing hormone, gonadotropin-releassing hormone, chorionic
gonadotropin, corticotrophin-releasing factor, vasopressin, oxytocin, antidiuretic
hormones, thyroid-stimulating hormone, thyrotropin- releasing hormone, relaxin,
prolactin, peptide YY, neuropeptide Y, pancreastic polypeptide, leptin, CART (cocaine
and amphetamine regulated transcript), a CART related peptide, perilipin,

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melanocortins (melanocyte-stimulating hormones) such as MC-4, melanin-
concentrating hormones, natriuretic peptides, adrenomedullin, endothelin, secretin,
amylin, vasoactive intestinal peptide (VIP), pituary adenylate cyclase activating
polypeptide (PACAP), bombesin, bombesin-like peptides, thymosin, heparin-binding
protein, soluble CD4, hypothalmic releasing factor, melanotonins and analogues
thereof.
The drug conjugate or drug fusion described herein can also be administered for
diagnostic purposes or as an imaging agent.
In the instant application, the term "prevention" involves administration of the
protective composition prior to the induction of the disease. "Suppression" refers to
administration of the composition after an inductive event, but prior to the clinical
appearance of the disease. "Treatment" involves administration of the protective
composition after disease symptoms become manifest.
Animal model systems which can be used to screen the effectiveness of drug
compositions (e.g., drug conjugates, noncovalent drug conjugates, drug fusions) in
protecting against or treating the disease are available. Methods for the testing of
systemic lupus erythematosus (SLE) in susceptible mice are known in the art (Knight et
al. (1978) J. Exp. Med., 147: 1653; Reinersten et al. (1978) NewEng. J. Med., 299:
515). Myasthenia Gravis (MG) is tested in SJL/J female mice by inducing the disease
with soluble AchR protein from another species (Lindstrom et al. (1988) Adv. Immunol.,
42: 233). Arthritis is induced in a susceptible strain of mice by injection of Type II
collagen (Stuart et al. (1984) Ann. Rev. Immunol., 42: 233). A model by which adjuvant
arthritis is induced in susceptible rats by injection of mycobacterial heat shock protein
has been described (Van Eden et al. (1988) Nature, 331: 171). Effectiveness for
treating osteoarthritis can be assessed in a murine model in which arthritis is induced by
intra-articular injection of collagenase (Blom, A.B. et al., Osteoarthritis Cartilage
12:627-635 (2004). Thyroiditis is induced in mice by administration of thyroglobulin as
described (Maron et al. (1980) J. Exp. Med., 152: 1115). Insulin dependent diabetes
mellitus (IDDM) occurs naturally or can be induced in certain strains of mice such as
those described by Kanasawa et al. (1984) Diabetologia, 27: 113. EAE in mouse and
rat serves as a model for MS in human. Li this model, the demyelinating disease is
induced by administration of myelin basic protein (see Paterson (1986) Textbook of

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Immunopathology, Mischer et al., eds., Grune and Stratton, New York, pp. 179-213;
McFarlin et al. (1973) Science, 179: 478: and Satoh et al. (1987) J. Immunol, 138:
179).
The drag compositions {e.g., drag conjugates, noncovalent drug conjugates, drag
fusions) of the present invention maybe used as separately administered compositions
or in conjunction with other agents. These can include various immunotherapeutic
drugs, such as cylcosporine, methotrexate, adriamycin or cisplatinum, immunotoxins
and the like. Pharmaceutical compositions can include "cocktails" of various cytotoxic
or other agents in conjunction with the drag composition {e.g., drag conjugate,
noncovalent drag conjugate, drag fusion) of the present invention, or combinations of
drug compositions {e.g., drag conjugates, noncovalent drug conjugates, drag fusions)
according to the present invention comprising different drugs.
The drag compositions {e.g., drag conjugates, noncovalent drag conjugates, drag
fusions) can be administered to any individual or subject in accordance with any
suitable techniques. A variety of routes of administration are possible including, for
example, oral, dietary, topical, transdermal, rectal, parenteral {e.g., intravenous,
intraarterial, intramuscular, subcutaneous, intradermal, intraperitoneal, intrathecal,
intraarticular injection), and inhalation {e.g., intrabronchial, intranasal or oral inhalation,
intranasal drops) routes of administration, depending on the drag composition and
disease or condition to be treated. Administration can be local or systemic as indicated.
The preferred mode of administration can vary depending upon the drag composition
{e.g., drag conjugate, noncovalent drag conjugate, drag fusion) chosen, and the
condition {e.g., disease) being treated. The dosage and frequency of administration will
depend on the age, sex and condition of the patient, concurrent administration of other
drugs, counterindications and other parameters to be taken into account by the clinician.
A therapeutically effective amount of a drag composition {e.g., drag conjugate,
noncovalent drug conjugate, drug fusion) is administered. A therapeutically effective
amount is an amount sufficient to achieve the desired therapeutic effect, under the
conditions of administration.
In a preferred embodiment of the invention pharmaceutical compositions
containing a GLP-1 drag or GLP-1 analogue or derivative according to the present
invention may be administered parenterally to patients in need of such a treatment.

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Parenteral administration may be performed by subcutaneous, intramuscular or
intravenous injection by means of a syringe, optionally a pen-like syringe. Alternatively,
parenteral administration can be performed by means of an infusion pump. A further
option is a composition which may be a powder or a liquid for the administration of the
GLP-1 drug or GLP-1 analogue or derivative in the form of a nasal or pulmonal spray.
As a still further option, the GLP-1 drug or GLP-1 analogue or derivative of the
invention can also be administered transdermally, e. g. from a patch, optionally an
iontophoretic patch, or transmucosally, e. g. bucally. In other embodiments the
compositions are administered orally, eg as a pill, capsule, drink (eg, marketed as a
weight-loss drink for obesity treatment).
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 described in European Patent No. 272097 (to Novo Nordisk A/S) or in WO
93/18785 (all incorporated herein by reference).
The term "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.
The drug composition (e.g., drug conjugate, noncovalent drug conjugate, drug
fusion) can be administered as a neutral compound or as a salt. Salts of compounds
(e.g., drug compositions, drug conjugates, noncovalent drug conjugates, drug fusions)
containing an amine or other basic group can be obtained, for example, by reacting with
a suitable organic or inorganic acid, such as hydrogen chloride, hydrogen bromide,
acetic acid, perchloric acid and the like. Compounds with a quaternary ammonium
group also contain a counteranion such as chloride, bromide, iodide, acetate, perchlorate
and the like. Salts of compounds containing a carboxylic acid or other acidic functional
group can be prepared by reacting with a suitable base, for example, a hydroxide base.
Salts of acidic functional groups contain a countercation such as sodium, potassium and
the like.
The invention also provides a kit for use in administering a drug composition
(e.g., drug conjugate, noncovalent drug conjugate, drug fusion) to a subject (e.g.,

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patient), comprising a drug composition (e.g., drug conjugate, noncovalent drug
conjugate, drug fusion), a drug delivery device and, optionally, instructions for use. The
drug composition (e.g., drug conjugate, noncovalent drug conjugate, drug fusion) can be
provided as a formulation, such as a freeze dried formulation. In certain embodiments,
the drag delivery device is selected from the group consisting of a syringe, an inhaler,
an intranasal or ocular administration device (e.g., a mister, eye or nose dropper), and a
needleless injection device.
The drug composition (e.g., drug conjugate, noncovalent drag conjugate, drag
fusion) 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)
and/or 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 (e.g., with conventional immunoglobulins, IgM antibodies tend to
have greater activity loss than IgG antibodies) and that use levels may have to be
adjusted to compensate. In a particular embodiment, the invention provides a
composition comprising a lyophilized (freeze dried) drug composition (e.g., drug
conjugate, noncovalent drag conjugate, drag fusion) as described herein. Preferably,
the lyophilized (freeze dried) drag composition (e.g., drag conjugate, noncovalent drug
conjugate, drug fusion) 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 drug composition (e.g.,
drug conjugate, noncovalent drug conjugate, drug fusion) required to produce the effect
of the drag composition before it was lyophilized. For example, the amount of drag
conjugate or drug fusion needed to achieve and maintain a desired serum concentration
for a desired period of time. The activity of the drag composition (e.g., drag conjugate,
noncovalent drag conjugate, drug fusion) 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.
Compositions containing the drug composition (e.g., drag conjugate,
noncovalent drug conjugate, drug fusion) or a cocktail thereof can be administered for
prophylactic and/or therapeutic treatments. In certain therapeutic applications, an

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amount sufficient to achieve the desired therapeutic or prophylactic effect, under the
conditions of administration, such as at least partial inhibition, suppression, modulation,
killing, or some other measurable parameter, of a population of selected cells is denned
as a "therapeutically-effective amount or dose." Amounts needed to achieve this dosage
will depend upon the severity of the disease and the general state of the patient's own
immune system and general health, but generally range from about 10 ug/kg to about 80
mg/kg, or about 0.005 to 5.0 mg of drug conjugate or drug fusion per kilogram of body
weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonly used. For example,
a drug composition (e.g., drug fusion, drug conjugate, noncovalent drug conjugate) of
the invention can be administered daily (e.g., up to four administrations per day), every
two days, every three days, twice weekly, once weekly, once every two weeks, once a
month, or once every two months, at a dose of, for example, about 10 l-ig/kg to about 80
mg/kg, about 100 ug/kg to about 80 mg/kg, about 1 mg/kg to about 80 mg/kg, about 1
mg/kg to about 70 mg/kg, about 1 mg/kg to about 60 mg/kg, about 1 mg/kg to about 50
mg/kg, about 1 mg/kg to about 40 mg/kg, about 1 mg/kg to about 30 mg/kg, about 1
mg/kg to about 20 mg/kg , about 1 mg/kg to about 10 mg/kg, about 10 ug/kg to about
10 mg/kg, about 10 ug/kg to about 5 mg/kg, about 10 ug/kg to about 2.5 mg/kg, about 1
mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg,
about 7 mg/kg, about 8 mg/kg, about 9 mg/kg or about 10 mg/kg.
For prophylactic applications, compositions containing the drug composition (e.g., drug
conjugate, noncovalent drug conjugate, drug fusion) or cocktails thereof may also be
administered in similar or slightly lower dosages. A composition containing a drug
composition (e.g., drug conjugate, noncovalent drug conjugate, drug fusion) according
to the present invention may be utilised in prophylactic and therapeutic settings to aid in
the alteration, inactivation, killing or removal of a select target cell population in a
mammal.
EXAMPLES
Interleukin 1 receptor antagonist (ILl-ra) is an antagonist that blocks the
biologic activity of IL-1 by competitively inhibiting IL-1 binding to the interleukin-1
type 1 receptor (IL-1R1). IL-1 production is induced in response to inflammatory
stimuli and mediates various physiologic responses including inflammatory and

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imrnunological responses. IL-1 has a range of activities including cartilage degredation
and stimulation of bone resorption. In rheumatoid arthritis patients, the amount of
locally produed IL-1 is elevated and the levels of naturally occurring ILl-ra are
insufficient to compete with these abnormally increased amounts. There are several
treatments avavilable for RA including disease modifying antirheumatic drugs
(DMARDS) such as methotrexate, and biologies such as KTNERET® (anakinra,
Amgen).
KINERET® (anakinra, Amgen) is a recombinant, nonglycosylated form of the
human interleukin-1 receptor antagonist which consists of 153 amino acids and has a
molecular weight of 17.3 kilodaltons. (The amino acid sequence of KINERET®
(anakinra, Amgen) corresponds to the 152 amino acids in naturally occurring IL-lra and
an additional N-terminal methionine.) KINERET® (anakinra, Amgen) is indicated for
the reduction in signs and symptoms of moderate to severe rheumatoid arthritis in
patients 18 years of age or older who have failed one or more DMARDs. Dosage is a
single use daily subcutaneous injection of l00mgs of drug. The T1./2 is 4-6 hours and
71% of patients develop injection site reactions in 14-28 days.
Here we demonstrate that linking a therapeutic polypeptide to a serum-albumin
binding dAb results in a compound which (i) has activity similar to the therapeutic
polypeptide alone and (ii) also binds serum albumin. Furthermore, the present invention
provides a method to create a long serum half-life version of the therapeutic
polypeptide. For example, we have linked a serum albumin binding dAb to ILl-ra
which results in a compound of longer serum half life than ILl-ra alone.
Example 1 Selection of domain antibodies that bind mouse, rat and human serum
albumin
This example explains a method for making a single domain antibody (dAb)
directed against serum albumin. Selection of dAbs against mouse serum albumin
(MSA), human serum albumin (HSA) and rat serum albumin (RSA) is described.
The dAbs against mouse serum albumin were selected as described in WO
2004/003019 A2. Three human phage display antibody libraries were used. Each
library was based on a single human framework for VH (V3-23/DP47 and JH4b) or VK

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(ol2/o2/DPK9 and Jkl) with side chain diversity encoded by NNK codons incorporated
in complementarity determining regions (CDR1, CDR2 and CDR3).
Library 1 (VH):
Diversity at positions: H30, H31, H33, H35, H50, H52, H52a, H53, H55, H56, H58,
H95, H97, H98.
Library size: 6.2 x 109
Library 2 (VH):
Diversity at positions: H30, H31, H33, H35, H50, H52, H52a, H53, H55, H56, H58,
H95, H97, H98, H99, HI 00, H100A, H100B.
Library size: 4.3 x 109
Library 3 (Vk):
Diversity at positions: L30, L31, L32, L34, L50, L53, L91, L92, L93, L94, L96
Library size: 2x 109
The VH and VK libraries had been preselected for binding to generic ligands protein A
and protein L respectively so that the majority of clones in the selected libraries were
functional. The sizes of the libraries shown above correspond to the sizes after
preselection.
Two rounds of selection were performed on serum albumin using each of the
libraries separately. For each selection, antigen was coated on immunotube (nunc) in 4
mL of PBS at a concentration of 100 fig/ml. In the first round of selection, each of the
three libraries was panned separately against HSA (Sigma) or MSA (Sigma). In the
second round of selection, phage from each of the six first round selections was panned
against (i) the same antigen again (eg 1st round MSA, 2nd round MSA) and (ii) against
the reciprocal antigen (eg 1st round MSA, 2nd round HSA) resulting in a total of twelve
2nd round selections. In each case, after the second round of selection 48 clones were
tested for binding to HSA and MSA. Soluble dAb fragments were produced as
described for scFv fragments by Harrison et al, Methods Enzymol. 1996; 267: 83-109
and standard ELISA protocol was followed (Hoogenboom et al. (1991) Nucleic Acids
Res., 19: 4133) except that 2% tween PBS was used as a blocking buffer and bound

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dAbs were detected with either protein L-HRP (Sigma) (for the VKS) and protein A-
HRP (Amersham Pharmacia Biotech) (for the VHS).
dAbs that gave a signal above background indicating binding to MSA, HSA or
both were tested in ELISA insoluble form for binding to plastic alone but all were
specific for serum albumin. Clones were then sequenced (see Table 1) revealing that 21
unique dAb sequences had been identified. The minimum similarity (at the amino acid
level) between the V/c dAb clones selected was 86.25% ((69/80) XI00; the result when
all the diversified residues are different, e.g., clones 24 and 34). The minimum
similarity between the VH dAb clones selected was 94 % ((127/136) X100).
Next, the serum albumin binding dAbs were tested for their ability to capture
biotinylated antigen from solution. ELISA protocol (as above) was followed except that
ELISA plate was coated with 1 jug/ml protein L (for the VK clones) and 1 jug/ml protein
A (for the VH clones). Soluble dAb was captured from solution as in the protocol and
detection was with biotinylated MSA or HSA and streptavidin HRP. The biotinylated
MSA and HSA had been prepared according to the manufacturer's instructions, with the
aim of achieving an average of 2 biotins per serum albumin molecule. Twenty four
clones were identified that captured biotinylated MSA from solution in the ELISA. Two
of these (clones 2 and 38 below) also captured biotinylated HSA. Next, the dAbs were
tested for their ability to bind MSA coated on a CM5 Biacore chip. Eight clones were
found that bound MSA on the Biacore.
dAbs against human serum albumin and rat serum albumin were selected as
previously described for the anti-MSA dAbs except for the following modifications to
the protocol: The phage library of synthetic VH domains was the libray 4G, which is
based on a human VH3 comprising the DP47 germline gene and the JH4 segment. The
diversity at the following specific positions was introduced by mutagenesis (using NNK
codons; numbering according to Kabat) in CDR1: 30, 31, 33, 35; in CDR2: 50, 52, 52a,
53, 55, 56; and in CDR3: 4-12 diversified residues: e.g. H95, H96, H97, and H98 in 4G
HI 1 and H95, H96, H97, H98, H99, H100, Hl00a, Hl00b, Hl00c, Hl00d, Hl00e and
Hl00f in 4G H19. The last three CDR3 residues are FDY so CDR3 lengths vary from 7-
15 residues. The library comprises >lxl010 individual clones.
A subset of the VH and Vk libraries had been preselected for binding to generic
ligands protein A and protein L respectively so that the majority of clones in the

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unselected libraries were functional. The sizes of the libraries shown above correspond
to the sizes after preselection.
Two rounds of selection were performed on rat and human serum albumin using
subsets of the VH and Vk libraries separately. For each selection, antigen was either (i)
coated on immunotube (nunc) in 4ml of PBS at a concentration of 100μg/ml, or (ii)
bitotinylated and then used for soluble selection followed by capture on streptavidin
beads (in the 1st round) and neutravidin beads (in the 2nd round). (See Table 1 for
details of the selection strategy used to isolate each clone.) In each case, after the
second round of selection 24 phage clones were tested for binding to HS A or RS A.
If a significant proportion of the clones in one of the selections were positive in
the phage ELISA, then DNA from this selection was cloned into an expression vector
for production of soluble dAb, and individual colonies were picked. Soluble dAb
fragments were produced as described for scFv fragments by Harrison et al (Methods
Enzymol. 1996;267:83-109) and standard ELISA protocol was followed (Hoogenboom
et al. (1991) Nucleic Acids Res., 19: 4133) except that 2% TWEEN PBS was used as a
blocking buffer and bound dAbs were detected with anti-myc-HRP . Clones that were
positive in ELISA were then screened for binding to MSA, RSA or HSA using a
BIACORE surface plasmon resonance instrument (Biacore AB). dAbs which bound to
MSA, RSA or HSA were further analysed. Clones were then sequenced and unique
dAb sequences identified.
Table 1. Selection protocols for dAbs that bind serum albumin

dAb Library Rl selection R2 selection Biacore binding
DOM7r-l 4GVK l0μg/ml tube RSA l0μg/ml tubeRSA RSA
DOM7r-3 4GVK l0μg/ml tube RSA 10μg/mltubeRSA RSA
DOM7r-4 4GVk l0μg/ml tube RSA 10μg/ml tubeRSA RSA, MSA
DOM7r-5 4GVk 10μg/ml tube RSA 10μg/ml tubeRSA RSA
DOM7r-7 4GVK 10μg/ml tube RSA 10μg/ml tube RSA, MSA

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tube tube
dAbs that bound serum albumin on a BIACORE chip (Biacore AB) were then
further analysed to obtain information on affinity. The analysis was performed using a
CM5 chip (carboxymethylated dextran matix) that was coated with serum albumin.
Flow cell 1 was an uncoated, blocked negative control, flow cell 2 was coated with
HSA, flow cell 3 was coated with RSA and flow cell 4 was coated with MSA. The
serum albumins were immobilised in acetate buffer pH 5.5 using the BIACORE coating
wizard which was programmed to aim for 500 resonance units (RUs) of coated material.
Each dAb of interest was expressed in the periplasm of E. coli on a 200 mL-500 mL
scale and purified from the supernatant using batch absorption to protein A-streamline
affinity resin (Amersham, UK) for the VHS and to protein L-agarose affinity resin
(Affitech, Norway) for the VKs followed by elution with glycine at pH 2.2 and buffer
exchange to PBS. A range of concentrations of dAb were prepared (in the range 5nM to
5/xM) by dilution into BIACORE HBS-EP buffer and flowed across the BIACORE
chip.
Affinity (KD) was calculated from the BIACORE traces by fitting on-rate and
off-rate curves to traces generated by concentrations of dAb in the region of the KD.
dAbs with a range of different affinities to serum albumin were identified. Included in
the range 10-100nM, were the affinities of DOM7h-8 for HSA, DOM7h-2 for HSA and
DOM7r-l for RSA. Included in the range l00nM to 500nM were the affinities of
DOM7h-7 for HSA, DOM7h-8 for RSA and DOM7h-26 for HSA. Included in the
range 500nM to 5μM were the affinities of DOM7h-23 for HSA and DOM7h-l for
HSA. Example traces are included in FIGS. 6A-6C.
Example 2. Formatting anti-serum albumin antibodies as a fusion with IL-1 receptor
antagonist (IL-lra)
This example describes a method for making a fusion protein comprising IL-lra
and a dAb that binds to serum albumin. Two fusions were made, one with the dAb N-
terminal of the IL-lra (MSA16ILl-ra) and one with the dAb C-terminal of the IL-lra
(ILl-raMSA 16). The sequences of the fusions and the vector are shown in FIG. 2C and

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2D. A control fusion that did not bind MSA was also produced, and its sequence is
shown in FIG. 2E.
KINERET (anakinra, Amgen) has a short half life of 4-6 hours, and the
recommended dosing regime calls for daily injections. This regime lead to injection site
reaction in 14-28 days in 71% of cases. Therefore a form of human IL-lra that has a
longer serum half life would be beneficially and could increase efficacy and reduce
dosing frequency. These are both desirable properties for a pharmaceutical.
Cloning
Briefly, two multiple cloning sites (MCSs) were designed as detailed below and
inserted into an expression vector with a T7 promotor. The restriction sites were
designed for the insertion of ILl-ra, dAb, GAS leader and linker. One (MCS 1+3)
encodes a protein with the dAb N terminal of the IL-lra and the other (MCS 2 + 4)
encode a protein with the dAb C terminal of the IL-lra.
Cloning site 1+3 for dAblLl-ra fusion
Ndel, stuffer, Sail, NotI, stuffer, Xhol, BamHI
gcgcatatgttagtgcgtcgacgtcaaaaggccatagcgggcggccgctgcaggtctcgagtgcgatggatcc
(SEQ ID NO:35)
Cloning site 2+4 for ILl-radAb fusion
Ndel, stuffer, StUI, Sad, stuffer, Sail, NotI, TAA TAA BamHI
gcgcatatgttaagcgaggccttctggagagagctcaggagtgtcgacggacatccagatgacccaggcggccgctaataa
ggatccaatgc (SEQ ID NO:36)
The GAS leader was then inserted into each vector by digesting the MCS using
the appropriate restriction enzymes and ligating annealed primers coding for the leader.
Next, linker DNA coding for the linker was inserted in a similar manner. DNA coding
for IL-lra was obtained by PCR (using primers designed to add the required restriction
sites) from a cDNA clone and inserted into a TOPO cloning vector. After confirming

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the correct sequence by nucleic acid sequencing, DNA coding for IL-lra was excised
from the TOPO vector and ligated into the vectors containing leader and linker. Lastly,
DNA coding for the dAb was excised from the dAb expression vector and inserted into
the vectors by Sall/NotI digest of insert (purified by gel purification) and vector.
Expression and purification
MSA16ILl-ra, ILl-raMSA16 and dummylL-lra were expressed in the
periplasm of E. coli and purified from the supernatant using batch absorbtion to protein
L-agarose affinity resin (Affitech, Norway) followed by elution with glycine at pH 2.2.
The purified dAbs were then analysed by SDS-PAGE gel electrophoresis followed by
coomassie staining. For one of the proteins (IL-lraMSA 16), > 90% of the protein was
of the expected size and therefore was analysed for activity without further purification.
The other proteins (MSA16ELl-ra and dummy IL-lra) were contaminated by a smaller
band and were therefore further purified by FPLC ion exchange chromatography on the
RESOURSEQ ion exchange column at pH 9. Protein was eluted using a linear salt
gradient form 0-500 mM NaCl. After analysis by SDS-PAGE gel electrophoresis,
fractions containing a protein of the expected size were combined yielding a combined
fraction of >90% purity. This protein was used for further analysis
Example 3. Determination of activity of dAb ILl-ra fusion in vitro
MRC-5 IL-8 assay
MSA16IL-lra fusions were tested for the ability to neutralise the induction of
IL-8 secretion by IL-1 in MRC-5 cells (ATCC Accession No. CCL-171; American
Type Culture Collection, Manassas, VA). The method is adapted from Akeson, L. et al
(1996) Journal of Biological Chemistry 271,30517-30523, which describes the
induction of IL-8 by IL-1 in HUVEC, MRC-5 cells were used instead of the HUVEC
cell line. Briefly, MRC-5 cells plated in microtitre plates were incubated overnight with
dAblL-lra fusion proteins or IL-lra control, and IL-1 (100 pg/mL). Post incubation the
supernatant was aspirated off the cells and IL-8 concentration measured via a sandwich
ELISA (R&D Systems).
The activity of IL-lra in the fusion proteins led to a reduction in IL-8 secretion.
The reduction of IL-8 secretion resulting from activity of the MSA16ILl-ra fusion and

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from activity of the IL-lraMSA16 fusion was compared to the reduction seen with the
IL-lra control (recombinant human IL-lra, R&D systems). The neutralizing dose 50
(ND50) of each of the tested proteins was determined and is presented in Table 2.
Table 2

Protein ND50
IL-lra 0.5 nM
MSA16EL-lra 2nM
IL-lraMSA16 8nM
The results demonstrate that IL-lra remained active as part of a fusion construct
with an anti-serum albumin dAb. The MSA16IL-lra protein was further studied to
assess its pharmacokinetics (PK study).
Serum Albumin, anti IL-lra sandwich ELISA
Three dAb/IL-lra fusions were tested for the ability to bind serum albumin and
simultaneously be detected by a monoclonal anti-ILlra antibody. The fusions tested
were MSA16IL-lra, IL-lraMSA16 and dummylL-lra. Briefly, ELISA plate was coated
overnight with mouse serum albumin at 10 μg/ml, washed 5 x with 0.05% Tween PBS
and then blocked for 1 hour with 4% Marvel PBS. After blocking, the plate was washed
5 x with 0.05% Tween PBS and then incubated for 1 hour with each dAb, Il-lra fusion
diluted in 4% MPBS. Each fusion was incubated at 1 μM concentration and at 7
sequential 4-fold dilutions (ie down to 60pM). After the incubation, plates were washed
5 x with 0.05% Tween PBS and then incubated for 1 hour with the manufacturers
recommended dilution of a rabbit polyclonal antibody (ab-2573) to human IL-1 receptor
antagonist (Abeam, UK) diluted in 4% MPBS. After this incubation, plates were
washed 5 x with 0.05% Tween PBS and then incubated for lh with a 1/2000 dilution of
secondary antibody (anti-rabbit IgG-HRP) diluted in 4% MPBS. Following incubation
with the secondary antibody, plates were washed 3 x with 0.05% Tween PBS and 2 x
with PBS and then developed with 50/il per well of TMB microwell peroxidase
substrate (KPL, MA) and the reaction stopped with 50 μl per well of HCL. Absorption
was read at 450 nM.

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Both the MSA16IL-lra and IL-lraMSA16 proteins were detected at more than 2
x background level at 1 /iM concentration in the sandwich ELISA. The MSA16IL-lra
protein was detected at 2 x background or higher at dilutions down to 3.9 nM, whereas
the IL-lraMSA16 protein was detected at 2 x background only down to 500 nM.
Binding of the MSA16IL-lra fusion to serum albumin was shown to be specific for
serum albumin as the control construct (dummylL-lra) did not bind serum albumin.
Example 4. Determination of serum half life of drug fusions in mouse PK studies.
A. Determination of the serum half-life in mouse of a MSA binding dAb/HA
epitope tag fusion protein.
The MSA binding dAb/HA epitope tag fusion protein was expressed in the
periplasm of E. coli and purified using batch absorption to protein L-agarose affinity
resin (Affitech, Norway) followed by elution with glycine at pH 2.2. Serum half life of
the fusion protein was determined in mouse following a single intravenous (i.v.)
injection at approx 1.5 mg/kg into CD1 strain male animals. Analysis of serum levels
was by ELISA using goat anti-HA (Abeam, UK) capture and protein L-HRP
(Invitrogen, USA) detection which was blocked with 4% Marvel. Washing was with
0.05% Tween-20, PBS. Standard curves of known concentrations of MSA binding
dAb/HA fusion were set up in the presence of lx mouse serum to ensure comparability
with the test samples. Modelling with a 1 compartment model (WinNonlin Software,
Pharsight Corp., USA) showed the MSA binding dAb/HA epitope tag fusion protein
had a terminal phase tl/2 of 29.1 hours and an area under the curve of 559 hr.ug/ml.
This demonstrates a large improvement over the predicted half life for a HA epitope tag
peptide alone which could be a short as only several minutes.
The results of this study using the HA epitope tag as a drug model, demonstrate
that the in vivo serum half life of a drug can be extended when the drug is prepared as a
drug fusion or drug conjugate with an antigen-binding fragment of (e.g., dAb) of an
antibody that binds serum albumin.
The in vivo half life in mice of the anti-MSA dAbs DOM7m-16 and D0M7m-
26, and a control dAb that does not bind MSA were also assessed. Again, DOM7m-16,
DOM7m-26 and the control dAb contained an HA epitope tag, which serves as a model

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for a drag (e.g., a peptide drag). In this study, the control dAb, that does not bind MSA,
had an in vivo half life of 20 minutes, whereas the in vivo half lives of DOM7m-16 and
DOM7m-26 were significantly extended. (FIG. 12) DOM7m-16 was found to have an
in vivo half life in mice of 29.5 hours in further studies.
In another study, the in vivo half life (t1/2 β) of DOM7h-8 which contained an
HA epitope tag was evaluated in mice. Modelling with a 2 compartment model
(WinNonlin Software, Pharsight Corp., USA) showed that DOM7h-8 had a tl/2/3 of
29.1 hours.
The results of each of these study using the HA epitope tag as a model for a drag
{e.g., a peptide drag), demonstrate that the in vivo serum half life of a drug can be
dramatically extended when the drag is prepared as a drag fusion or drag conjugate with
an antigen-binding fragment of (e.g., dAb) of an antibody that binds serum albumin.
B. Determination of the serum half-life in mouse of MSA binding dAb/IL-lra
fusion protein.
The MSA binding dAb/IL-lra fusion protein (MSA16IL-lra) was expressed in
the periplasm of E. coli and purified using batch absorption to protein L-agarose affinity
resin (Affitech, Norway) followed by elution with glycine at pH 2.2. Serum half life of
the MSA16IL-lra (DOM7m-16/IL-lra), an IL-lra fusion with a dAb that does not bind
MSA (Dummy dAb/IL-lra), and an anti-MSA dAb fused to the HA epitope tag
(DOM7m-16 HA tag) was determined in mice following a single i.v. injection at
approximately 1.5 mg/kg into CD1 strain male animals.
Analysis of serum levels was by Il-lra sandwich ELISA (R&D Systems, USA).
Standard curves of known concentrations of dAb/IL-lra fusion were set up in the
presence of lx mouse serum to ensure comparability with the test samples. Modelling
was performed using the WinNonlin pharmacokinetics software (Pharsight Corp.,
USA).
It was expected that the IL-lra fusion with the anti-MSA dAb would increase
the serum half-life considerably when compared with the control which was a fusion of
a non-MSA binding dAb with IL-lra. The control non-MSA binding dAb/IL-lra fusion
was predicted to have a short serum half-life.

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The results of the study are presented in Table 3, and show that the IL-lra fusion
with anti-MSA dAb (DOM7m-16/IL-lra had a serum half life that was about 10 times
longer than the IL-lra fusion with a dAb that does not bind MSA (Dummy dAb/IL-lra).
The results also revealed that there was a > 200 fold improvement (increase) in the area
under the concentration time curve for DOM7m-16/IL-lra (AUC: 267 hr.ug/ml) as
compared to dummy/IL-lra (AUC: 1.5 hr.ug/ml)
Table 3

Agent Serum Half Life
DOM7m-16/IL-lra 4.3 hours
dummy/IL-lra 0.4 hours
DOM7m-16HAtag 29 hours
The results of these studies demonstrate that the in vivo serum half life and AUC
of a drug can be significantly extended when the drug is prepared as a drug fusion or
drug conjugate with an antigen-binding fragment of (e.g., dAb) of an antibody that
binds serum albumin.
Example 5. Determination of the serum half-life in rats of RSA binding dAb/HA
epitope tag fusion proteins.
Anti-rat serum albumin dAbs were expressed with C-terminal HA tags in the
periplasm of E. coli and purified using batch absorption to protein L-agarose affinity
resin (Affitech, Norway) for Vk dAbs and batch absorption to protein A affinity resin
for VH dAbs, followed by elution with glycine at pH 2.2. In order to determine serum
half life, groups of 4 rats were given a single i.v. injection at 1.5 mg/Kg of DOM7r-27,
DOM7r-31, DOM7r-16, DOM7r-3 or a control dAb (HEL4) that binds an irrelevant
antigen. Serum samples were obtained by serial bleeds from a tail vein over a 7 day
period and analyzed by sandwich ELISA using goat anti-HA (Abeam, Cambridge UK)
coated on an ELISA plate, followed by detection with protein A-HRP (for the VH dAbs)
or protein L-HRP (for VK dAbs). Standard curves of known concentrations of dAb
were set up in the presence of lx rat serum to ensure comparability with the test

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samples. Modelling with a 2 compartment model (using WinNonlin pharmacokinetics
software (Pharsight Corp., USA)) was used to calculate 11/2/3 and area under the curve
(AUC) (Table 4).
Table 4

Agent Scaffold Affmtity (KD) for rat serumalbumin tl/2/β AUC(μ,g.hr/mL)
DOM7r-3 12 nM 13.7 hours 224
DOM7r-16 Vk 1 μM 34.4 hours 170
DOM7r-27 VH 250 nM 14.8 hours 78.9
DOM7r-31 VH 5βM 5.96 hours 71.2
The results of this rat study using the HA epitope tag as a model for a drug (e.g.,
apeptide drug), demonstrate that the in vivo serum half life of a drug can be
dramatically extended when the drug is prepared as a drug fusion or drug conjugate with
an antigen-binding fragment of (e.g., dAb) of an antibody that binds serum albumin.
Prediction of half life in humans.
The in vivo half life of a dAb, drug fusion or drug conjugate in humans can
estimated from half life data obtained in animals using allometric scaling. The log of
the in vivo half lifes determined in 3 animals is plotted against the log of the weight of
the animal. A line is drawn through the plotted points and the slope and y-intercept of
the line are used to calculate the in vivo half life in humas using the formula log Y =
log(a) + b log(W), in which Y is the in vivo half life in humans, log(a) is the y-intercept,
b is the slope, and W is the weight of a human. The line can be produced using in vivo
half life data obtain in animals that weigh about 35 grams (e.g., mice), about 260 grams
(e.g., rats) and about 2,710 grams. For this calculation, the weight of a human can be
considered to be 70,000 grams.

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Example 6. Efficacy of anti-SA dAb/IL-lra drag fusion in mouse collagen induced
arthritis model of rheumatoid arthritis.
Efficacy of the fusion DOM7m-16/IL-lra and efficacy of IL-lra in a recognized
mouse model of rheumatoid arthritis (type II collagen induced arthritis (CIA) in DBA/1
mice) was assessed. Throughout the study, mice were maintained in a test facility in
standard type 2 cages that were housed in a HEPA-filtered Scantainer at 20-24°C with a
12-hours light, 12-hours dark cycle. Food (Harlan-Teklad universal diet 2016) and UV
sterilized water were provided ad libitum. The mice were imported to the test facility at
least 7 days before the start the study to assure proper acclimitization.
DBA/1 mice at 7-8 weeks of age (obtained from Taconic M and B, Domholtveg,
Denmark) were injected once with an emulsion of Arthrogen-CIA adjuvant and
Arthrogen-CIA collagen (both MD biosciences) emulsified at a 1:1 ratio until the
emulsion was stable. The emulsion was considered to be stable when a drop of the
emulsion added to a beaker of water formed a solid clump. The mice were then injected
with the emulsion.
Twenty-one days after the emulsion was injected, the 20 animals with the most
advanced arthritic disease were eliminated from the study, and the remaining mice were
divided into groups of 10 animals (each group contained 5 males and 5 females). The
mice were treated as shown in Table 5, and all treatments were delivered at a
concentration calculated so that 10 ml/Kg were administered.
Table 5

Group Treatment
1 IL-lra, 1 mg/Kg (intrapertoneal (ip.) bolus)
2 IL-lra, 10 mg/Kg (ip. bolus)
3 DOM7m-16/IL-lra, 1 mg/Kg (ip. bolus)
4 DOM7m-16/IL-lra, 10 mg/Kg (ip. bolus)
5 ENBREL® (entarecept; Immunex Corporation), 5 mg/Kg (ip. bolus)
6 saline (negative control), 10 ml/Kg (ip. bolus)
7 Dexamethasone (positive control), 0.4 mg/Kg (subcutaneousinjection)

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Clinical scores for the severity of arthritis were recorded 3 times a week from
day 21 to day 49. Mice were euthanized at day 49. Individual mice were euthanized
earlier if they presented an arthritic score of 12 or more, or had serious problems
moving.
For clinical scoring, each limb was scored according to the criteria below and
the scores for all four limbs were added to produce the total score for the mouse. This
method resulted is a score of 0 to 16 for each mouse. Scoring critera were: 0 = normal;
1 = mild but definite redness and swelling of the ankle or wrist, or apparent redness and
swelling limited to individual digits, regardless of the number of affected digits; 2 =
moderate redness and swelling of ankle and wrist; 3 = severe redness and swelling of
the entire paw including digits; 4 = maximally inflamed limb with involvement of
multiple joints.
Group average arthritic scores were calculated for each treatment group on every
treatment day using clinical scores from individual mice. Any animals that had been
removed from the study for ethical reasons were allocated the maximum score of 16.
The group average arthritic scores were plotted against time (FIG. 13).
Statistical analysis of the group average arthritic scores on day 49 were
performed using the Wilcoxon test. This statistical analysis revealed that the two
groups treated with DOM7m-16/IL-lra (at 1 mg/Kg or 10 mg/Kg (Groups 3 and 4)) had
significantly improved arthtritic scores at day 49 (at the P significance levels respectively) when compared to the saline control group (Group 6).
In contrast, treatment with IL-lra at 1 mg/Kg (Group 1) did not result in statistically
significant improvement in the arthritic score at day 49, while treatment with IL-lra at
10 mg/Kg (Group 2) resulted in a significant improvement at the P level. Treatment with ENBREL® (entarecept; Immunex Corporation) (Group 5)
resulted in significant improvement in the arthric score at day 49 at the P significance level.
Treatment with DOM7m-16/IL-lra at the 10 mg/Kg dose (Group 4), was
effective at improving the arthtritic score at day 49 (significant at the P when compared to standard treatment with ENBREL® (entarecept; Immunex
Corporation) at 5mg/Kg (Group 5). In addition, treatment with DOM7m-16/IL-lra at
the lower lmg/Kg dose (Group 3), was more efficacious at improving the arthtritic

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score at day 49 than treatment with IL-lra alone at the same dosage (Group 1)
(significant at the P The results of the study show that at certain doses DOM7m-16/IL-lra was more
effective than IL-lra or ENBREL® (entarecept; Immunex Corporation) in this study.
The response to IL-lra was dose dependant, as expected, and the response to D0M7m-
16/TL-lra was also dose dependant. The average scores for treatment with D0M7m-
16/IL-lra at lmg/Kg were consistently lower than the average scores obtained by
treatment with IL-lra at 10 mg/kg. These plotted results (FIG. 13) indicate that
treatment with DOM7m-16/IL-lra was about 10 times more effective than IL-lra in this
study.
This superior efficacy of DOM7m-16/IL-lra was observed even though the
DOM7-16/IL-lra fusion protein contains about half the number of IL-1 receptor binding
epitopes as IL-lra on a weight basis (e.g., 1 mg of DOM7m-16/IL-lra (MW .31.2 kD)
contains about half the number of IL-1 receptor binding epitopes as 1 mg of IL-lra
(MW. 17.1 kD).
The results of this study demonstrate that a dAb that binds serum albumin can be
linked to IL-lra (a clinically proven therapy for RA) and that the resulting drug fusion
has both long serum half life properties (conferred by the dAb) and IL-1 receptor
binding properties (conferred by the IL-lra). Due to the serum residence time of the
drug fusion, the dose of DOM7-16/IL-lra that was effective for treating CIA was
dramatically reduced relative to IL-lra.
The results of this study demonstrate that in addition to the benefits of extended
half life and increased AUC, drugs prepared as drug fusions or drug conjugates with an
antigen-binding fragment of (e.g., dAb) of an antibody that binds serum albumin are
highly effective therapeutic agents that provide advantages over drug alone. For
example, as demonstrated in the mouse CIA model, a lower dose of drug fusion was
effective and inhibited the joint inflammation and joint damage caused by IL-1 over a
longer period of time in comparison to IL-lra alone, and provided greater protection
against disease progression.
Example 7. Anti-SA dAb/Saporin noncovalent drug conjugate

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The ribosome-inactivating protein Saporin (an anti-cancer drug) is highly stable
to denaturants and proteases and has been used as a targeted toxin to T lymphocytes. A
non-covalent drug conjugate was prepared by coupling Saporin to DOM7h-8 via a
biotin-streptavidin link. Results obtained with this non-covalent drug conjugate
demonstrates that the DOM7h-8 retains its serum albumin binding characteristics when
coupled to a drug.
A variant DOM7h-8 referred to as DOM7h-8cys, in which the C-terminal
arginine at position 108 (amino acid 108 of SEQ ID NO: 24) was replaced with a
cysteine residue was prepared by expression of a recombinant nucleic acid in HB2151
cells. The cells were grown and induced at 30°C in overnight expression autoinduction
TB readymix (Merck KGa, Germany) for 72 hours before recovery of the supernatant
by centrifugation. DOM7h-8cys was purified from the supernatant using affinity
capture on protein L-agarose. The resin was then washed with 10 column volumes of 2
x PBS and DOM7h-8cys was eluted with 0.1 M glycine pH2. Eluted DOM7h-8cys was
neutralised with 0.2 x volume of Tris pH8 and concentrated to lmg/ml (using a
CENTRICON 20 ml concentrator (Millipore Corp., MA).
Concentrated DOM7h-8cys was buffer exchanged to PBS using a NAP5
desalting column (GE Healthcare/Amersham Biosciences, NJ) and concentration
determined. The dAb was then biotinylated (via primary amines) using EZ-LINK sulfo-
NHS-LC-biotin (Pierce Biotechnology Inc., IL). The biotinylated dAb was mixed with
streptavidin-saporin in a 1:1 molar ratio.
In order to confirm that the dAb/saporin complex was formed, a sandwich
ELISA was used to detect intact complexes. Human serum albumin (HAS) was coated
onto half of the wells of an ELISA plate (Nunc, NY) overnight at 10 μg/ml in a volume
of 100 /il per well. After overnight incubation, the plate was washed 3 times with PBS,
0.05% Tween and then the whole plate was blocked for 2 hours with 2% PBS. After
blocking, the plate was washed 3 times with PBS, 0.05% Tween and then incubated for
1 hour with DOM7h-8/saporin non-covalent conjugate diluted to 0.5 μM. in 2% Tween
PBS. As controls on the same ELISA plate, uncoupled saporin at 0.5 μM and
uncoupled DOM7h8 at 0.5 μM were incubated in 2% Tween PBS. Additional controls
were the same three diluted proteins incubated on wells of the ELISA plate not coated
with HSA and blocked with 2% Tween. After the incubation, the plate was washed 3

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times with PBS, 0.05% Tween and then incubated for 1 hour with 1/2000 dilution of
goat anti-saporin polyclonal antibody (Advanced Therapeutic Systems) diluted in 2%
Tween PBS. After the incubation, the plate was washed 3 times with PBS, 0.05%
Tween and then incubated for 1 hour with the secondary detection antibody (of 1/2000
anti-goat Ig HRP conjugate). After the incubation, the plate was washed 3 times with
PBS, 0.05% Tween and once with PBS and tapped dry on paper. The ELISA was
developed with 100 μl 3, 3', 5, 5'-tetramethylbenzidine as substrate and the reaction
stopped with 50 μl 1M hydrochloric acid. The presence of non-covalent conjugates of
DOM7h-8 and saporin was confirmed by comparing the OD600 of the conjugate with
that of either of the unconjugated parts.
Table 6

DOM7h-8/Saporin DOM7h-8 alone Saporin alone
OD600(plate coated with HAS) 0.311 0.060 0.079
OD600(plate blocked with 2%Tween PBS) 0.078 0.068 0.075
The results of this study demonstrate that a drug can be conjugated to an
antigen-binding fragement of an antibody that binds serum albumin, and that the
conjugated antigen-binding fragment retains serum albumin-binding activity. In
addition, due to the stability and strength of the biotin-streptavidin interation, the results
show that covalently bonded and noncovalently bonded conjugates can be prepared that
retain the serum albumin-binding activity of the antigen-binding fragment of an
antibody that binds serum albumin.
Example 8. Anti-SA dAb/Fluorescein conjugate
Fluorescein isothiocyanate (FITC) can be cross linked with amino, sulfhydryl,
imidazoyl, tyrosyl or carbonyl groups on a protein. It has a molecular weight of 389 Da
which is comparable in size to many small molecule drugs. Results obtained with this
conjugate demonstrate that the anti-sa dAb maintains its serum albumin binding

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characteristics when coupled to a small chemical entity, and indicate that small
molecule drugs can be conjugated to anti-SA dAbs.
Concentrated DOM7h-8cys was prepared as described in Example 7. The
concentrated dAb was buffer exchanged to 50 mM Borate pH 8 (coupling buffer) using
a NAP5 desalting column (GE Healthcare/Amersham Biosciences, NJ) and then
concentrated to 2.3 mg/ml using a 2 ml CENTRICON concentrator (Millipore Corp.,
MA). The FITC (Pierce Biotechnology Inc.) was diluted to 10 mg/ml in dimethyl
formamide (DMF) according to the manufacturer's instructions and then mixed with the
dAb in coupling buffer at a molar ratio of 24:1 FITC:dAb. The reaction was allowed to
proceed for 30 minutes. At this point, excess unreacted FITC was removed from the
reaction using a PD10 desalting column (GE Healthcare/Amersham Biosciences, NJ)
that was pre-equilibrated with PBS, and the DOM7h-8cys/FITC conjugate was eluted
with PBS.
In order to confirm that the FITC/dAb coupling reaction was successful, a
sandwich ELISA was used to detect coupled dAb. Human serum albumin (HSA) was
coated onto half of the wells of an ELISA plate (Nunc, NY) overnight at 10 μg/ml in a
volume of 100 μl per well. After overnight incubation, the whole plate was washed 3
times with PBS, 0.05% Tween and then all the wells were blocked for 2 hours with 2%
Tween PBS. After blocking, the plate was washed 3 times with PBS, 0.05% Tween and
then incubated for 1 hour with DOM7h-8cys/FITC diluted to 1 μM in 2% Tween PBS.
As controls on the same ELISA plate, a control FITC coupled antibody at 1 ΜM and
uncoupled DOM7h-8 at 1 μM were incubated in 2% Tween PBS. Additional controls
were the same three diluted proteins incubated on wells of the ELISA plate not coated
with HSA and blocked with 2% Tween. After the incubation, the plate was washed 3
times with PBS, 0.05% Tween and then incubated for 1 hour with 1/500 dilution of rat
anti FITC antibody (Serotec) diluted in 2% Tween PBS. After the incubation, the plate
was washed 3 times with PBS, 0.05% Tween, and then incubated for 1 hour with the
secondary detection antibody diluted in 2% Tween PBS (1/5000 anti-rat Ig HRP
conjugate). After the incubation, the plate was washed 3 times with PBS, 0.05% Tween
and once with PBS and tapped dry on paper. The ELISA was developed with 100 ul
per well 3,3',5,5'-tetramethylbenzidine as substrate and the reaction stopped with 50 ul
per well 1M hydrochloric acid. The presence of conjugates of DOM7h-8 and FITC was

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confirmed by comparing the OD600 of the conjugate with that of either of the
unconjugated parts.
Table 7

DOM7h-8/FITC DOM7h-8 alone FITC coupled
antibody
(negative control)
OD600 0.380 0.042 0.049
(plate coated with
HSA)
OD600 0.041 0.041 0.045
(plate blocked with
2% Tween PBS)
Example 9. anti-SA dAb/peptide conjugates.
Many peptides have therapeutic effects. Model peptides with an N or C terminal
cysteine can be coupled to an anti-serum albumin dAb.
In this case, four different peptides will be used: peptide 1
YPYDVPDYAKKKKKECC (SEQ ID NO:68); peptide 2 CKKKKKKYPYDVPDYA
(SEQ ID NO:69); peptide 3 HHHHHHKKKKKKC (SEQ ID NO:70) and peptide 4:
CKKKKKKHHHHHH (SEQ ID NO:71). Peptides 1 and 2 include the sequence of the
hemagglutinin tag (HA tag) and peptides 3 and 4 include the sequence of the His tag.
Concentrated DOM7h-8cys will be prepared as described in Example 7.
The concentrated dAb will be reduced with 5 mM dithiothreitol and then buffer
exchanged to coupling buffer (20 mM BisTris pH 6.5, 5 mM EDTA, 10% glycerol)
using a NAP5 desalting column (GE Healthcare/Amersham Biosciences, NJ). Cysteins
will be blocked (to prevent the dAb dimerising with itself) using a final concentration of
5 mM dithiodipyridine which will be added to the dAb solution form a stock of 100 mM
dithiodipyridine in DMSO. The dAb and dithiodipyrdine will be left to couple for 20-
30 minutes. Unreacted dithiodipyridine will then be removed using a PD10 desalting

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column and the dAb will be eluted in coupling buffer (20 mM BisTris pH 6.5, 5 mM
EDTA, 10% glycerol). The resulting protein will then be frozen until required.
Peptides 1-4 will be individually dissolved in water at a concentration of 200
μM, will be reduced using 5 mM DTT and then will be desalted using a NAP5 desalting
column (GE Healthcare/Amersham Biosciences, NJ). Each peptide will then be added
to a solution of reduced and blocked dAb at a 20:1 ratio, for the peptide-dAb coupling
to occur. In order to confirm success of the peptide, dAb coupling reactions, a sandwich
ELISA will be used to detect anti-SA dAb/peptide conjugates.
Human serum albumin will be coated onto an ELISA plate (Nunc, NY)
overnight at 10 μg/ml in a volume of 100 μl per well. After overnight incubation, the
plate will be washed 3 times with PBS, 0.05% Tween and then will be blocked for 2
hours with 4% Marvel PBS. After blocking, the plate will be washed 3 times with PBS,
0.05% Tween and then will be incubated for 1 hour with DOM7h-8/peptide conjugates
diluted to 1 μM in 4% Marvel PBS. As controls on the same ELISA plate, uncoupled
peptide at 20 μM and uncoupled DOM7h-8 at 1 μM. will be incubated in 4% MPBS.
After the incubation, the plate will be washed 3 times with PBS, 0.05% Tween and then
will be incubated for 1 hour with 1/2000 dilution of goat anti-HA antibody (Abeam) for
peptides 1 and 2, and a 1/2000 dilution of Ni NTA-HRP (for peptides 3 and 4) diluted
in 4% Marvel PBS. After incubation, the plate will be washed 3 times with PBS, 0.05%
Tween and the wells with the goat anti HA antibody will be incubated for lh with
seconday anti-goat HRP antibody diluted 1/2000 in 4% MPBS (other wells were
blocked for lh). After the incubation, the plate will be washed 3 times with PBS, 0.05%
Tween and once with PBS and will then be tapped dry on paper. The ELISA will be
developed with 3, 3', 5, 5'-tetramethylbenzidine as substrate and the reaction will be
stopped with 1M hydrochloric acid. The presence of conjugates of DOM7h-8/peptide
conjugate will be confirmed by comparing the OD600 of the conjugate with that of
either of the unconjugated parts.

Table 8 Anticancer Peptides
PeptideCategory Peptide Sequence Action/Application
LH-RHAgonistis and p-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 Treatment of sexhormone dependent

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The potency of an insulinotropic agent can be determined by calculating the
EC50 value from the dose-response curve. Purified plasma membranes from a stable
transfected cell line, BHK467-12A (tk-tsl3), expressing the human GLP-1 receptor will
be stimulated with GLP-1 and peptide analogues, and the potency of cAMP production
will be measured using the AlphaScreen™ cAMP Assay Kit from Perkin Elmer Life
Sciences.
A stable transfected cell line will be prepared and a high expressing clone
selected for screening. The cells will then be grown at 5% C02 in DMEM, 5% FCS, 1%
Pen/Strep and 0. 5 mg/ml G418.
Cells at approximate 80% confluence will be washed 2X with PBS and
harvested with Versene, centrifuged 5 min at 1000 rpm and the supernatant removed.
The additional steps will be made on ice. The cell pellet will be homogenized by the
Ultrathurax for 20-30 sec. in 10 mi of Buffer 1 (20 mM Na-HEPES, 10 mM EDTA,
pH7.4), centrifuged 15 min at 20.000 rpm and the pellet resuspended in 10 ml of Buffer
2 (20 mM Na-HEPES, 0.1 mM EDTA, pH7.4). The suspension will be homogenized for
20-30 sec and centrifuged 15 min at 20.000 rpm. Suspension in Buffer 2,
homogenization and centrifugation will be repeated once and the membranes
resuspended in Buffer 2 and ready for further analysis or stored at-80°C.
The functional receptor assay will be carried out by measurering the peptide
induced cAMP production by The AlphaScreen Technology. The basic principle of The
AlphaScreen Technology is a competition between endogenous cAMP and exogenously
added biotin-cAMP. The capture of cAMP is achieved by using a specific antibody
conjugated to acceptor beads. Formed cAMP will be counted and measured with an
AlphaFusion Microplate Analyzer. The EC50 values will be calculated using the Graph-
Pad Prisme software.
Resistance of a peptide to degradation by dipeptidyl aminopeptidase FV can be
determined by the following degradation assay: Aliquots of the peptides will be
incubated at 37 °C with an aliquot of purified dipeptidyl aminopeptidase IV for 4-22
hours in an appropriate buffer at pH 7-8 (buffer not being albumin). Enzymatic
reactions will be terminated by the addition of trifluoroacetic acid, and the peptide
degradation products will be separated and quantified using HPLC or LC-MS analysis.
The mixtures will be applied onto a Zorbax 300SB-C18 (30 nm pores, 5 um particles)

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150 x 2.1 mm column and eluted at a flow rate of 0.5 ml/min with a linear gradient of
acetonitrile in 0. 1% trifluoroacetic acid (0%-100% acetonitrile over 30 min). Peptides
and their degradation products may be monitored by their absorbance at 214 nm
(peptide bonds) or 280 nm (aromatic amino acids), and will be quantified by integration
of their peak areas. The degradation pattern can be determined by using LC-MS where
MS spectra of the separated peak can be determined. Percentage intact/degraded
compound at a given time is used for estimation of the peptides DPPIV stability.
A peptide is defined as DPPFV stabilised when it is 10 times more stable than
the natural peptide based on percentage intact compound at a given time. Thus, a
DPPIV stabilised GLP-1 compound is at least 10 times more stable than GLP-1 (7-37).
Stimulation of adenylate cyclase
BRTN-BD11 cells will be seeded into 24-well plates (3xl05/well) and cultured
for 48 h before being preincubated in media supplemented with tritiated adenine (2mCi)
for 16 h. The cells will be washed twice with cold Hanks buffered saline (HBS) and test
solution (400ul; 37C) added. The cells will then be exposed to varying concentrations
(10-10-10-5 M) of GLP-1 compounds in HBS buffer, in the presence of lmM IBMX
and 5.6mM glucose (20 min; 37C). Following incubation, test solutions will be removed
and 300ul of lysis solution (5% TFA, 3% SDS, 5mM of unlabelled ATP, and 300 uM of
unlabelled cAMP) added. Dowex and alumina exchange resins will be used to separate
tritiated cAMP from tritiated adenine and ATP in the cell lysate, as described (Miguel
JC, et al. Biochem. Pharmacol. 2003, 65:283).
Insulin secretory responses can be measured in the pancreatic /3-cell BRIN-
BD11 cells. Cells will be seeded into 24-multiwell plates at a density of lxlO5/well, and
allowed to attach during overnight culture. Acute studies of insulin release will be
preceded by 40 min pre-incubation at 37C in 1.0 ml Krebs-Ringer bicarbonate buffer
(115mM NaCl, 4.7mM KC1, 1.28mM CaCl2-2H2O, 1.2mM KH2PO4, 1.2mM
MgSO4H2O, l0mM NaHCO, and 5 g/L bovine serum albumin, pH 7.4) supplemented
with l.lmM glucose. Test incubations will be performed at 37C in the presence of
5.6mM glucose with a range of concentrations of GLP-1 compounds (10-12-10-6 M).
After 20 min incubation, the buffer will be removed from each well and aliquots stored
at -20C for measurement of insulin.

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Glucose-lowering and insulin secretory activity in obese diabetic (ob/ob) mice
The in vivo biological activity of GLP-1 compounds can be assessed in 12-16
week old obese diabetic (ob/ob) mice. The animals will be housed individually in an air-
conditioned room at 22±2C with a 12 h light: 12 h dark cycle. Animals will be allowed
drinking water ad libitum and continuous access to standard rodent maintenance diet.
Mice will be fasted for 18 h and intraperitoneally administered 8 ml/kg body weight
with saline (9 g/L NaCl), glucose alone (18mM/kg bodyweight), or in combination with
a GLP-1 compound (25 nM/kg body weight). Blood samples will be collected into
chilled fluoride/heparin microcentrifuge tubes immediately prior to injection and at 15,
30, and 60 min post injection, and the plasma obtained stored at -20C.
Other Assays
Plasma glucose levels can be determined using an Analox glucose analyser
(Hammersmith, London, UK), which employs the glucose oxidase method (Stevens JF,
Clin. Chim. Acta 1971, 32:199). Insulin levels can be assayed by dextran-coated
charcoal radioimmunoassay (Flatt PR and Bailey CJ, Diabetologia 1981,20:573).
Incremental areas under plasma glucose and insulin curves (AUC) can be calculated
using GraphPad PRISM version 3.0 (Graphpad Software, San Diego, CA, USA).
The activity of GLP-1 compound can be part of the drug composition of the
present invention as long as the GLP-1 drug is able to bind and induce signaling through
the GLP-1 receptor. GLP-1 receptor binding and signal transduction can be assessed
using in vitro assays such as those described in Examples 2, 3, and 4 of EP 619,322 and
Examples 1, 2, and 3 of U. S. Patent No. 5,120,712, respectively (incorporated herein by
reference).
Pharmacokinetics studies can be performed as described in Example 7 of WO 02/46227
(incorporated herein by reference).
Half-life extension of GLP-1 derivatives after i. v. or s. c. administration.

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The half-life extension of GLP-1 analogues can be determined by monitoring the
concentration thereof in plasma after sc administration to healthy pigs. For comparison
the concentration in plasma of GLP-1 (7-37) (natural active of form GLP-1 and used as
a control) after sc. administration can be followed.
The test substances will be dissolved in a vehicle suitable for subcutaneous or
intravenous administration. The concentration will be adjusted so the dosing volume is
approximately 1 ml. The study will be performed in 12 male Gottingen minipigs from
Ellegaard Gottingen Minipigs ApS. An acclimatisation period of approximately 10 days
will be allowed before the animals entered the study. At start of the acclimatisation
period the minipigs will be about 5 months old and in the weight range of 8-10 kg.
The study will be conducted in a suitable animal room with a room temperature
set at 21-23°C and the relative humidity approximately 50%. The room will be
illuminated to give a cycle of 12 hours light and 12 hours darkness. Light will be from
06.00 to 18.00h. The animals will be housed in pens with straw as bedding, six together
in each pen. The animals will have free access to domestic quality drinking water during
the study, but will be fasted from approximately 16.00h the day before dosing until
approximately 12 hours after dosing. The animals will be weighed on arrival and on the
days of dosing.
The animals will receive a single intravenous or subcutaneous injection. The
subcutaneous injection will be given on the right side of the neck, approximately 5-7 cm
from the ear and 7-9 cm from the middle of the neck. The injections will be given with a
stopper on the needle, allowing 0.5 cm of the needle to be introduced. Each test
substance will be given to three animals. Each animal received a dose of 2 nmol/kg
body weight. Six animals will be dosed per week while the remaining six rested.
A full plasma concentration-time profile will be obtained from each animal.
Blood samples will be collected according to the following schedule: After intravenous
administration: Predose (0), 0.17 (10 minutes), 0.5,1,2, 4,6, 8,12, 24,48, 72,96, and 120
hours after injection. After subcutaneous administration: Predose (0), 0.5,1, 2, 4, 6, 8,
12, 24,48, 72,96, and 120 hours after injection.
At each sampling time, 2 ml of blood will be drawn from each animal. The
blood samples will be taken from a jugular vein. The blood samples will be collected
into test tubes containing a buffer for stabilisation in order to prevent enzymatic

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degradation of the GLP-1 analogues. Plasma will be immediately transferred to
Micronic-tubes. Approximately 200 ul plasma will be transferred to each Micronic-
tube. The plasma was stored at-20°C until assayed. The plasma samples will be assayed
for the content of GLP-1 analogues using an immunoassay.
The plasma concentration-time profiles will be analysed by a non-
compartmental pharmacokinetic analysis. The following pharmacokinetic parameters
will be calculated at each occasion: AUC, AUC/Dose, AUC% Extrap, Cmax, tmax, ,λ2, CL,
CL/f,Vz,Vz/f and MRT.
Compostions of the invention can also be tested in Danish Landrace pigs.
Pigs (50% Duroc, 25% Yorkshire, 25% Danish Landrace, app 40 kg) will be
fasted from the beginning of the experiment. To each pig 0.5 nmol of test composition
per kg body weight will be administered in a 50 pM isotonic solution (5 mM phosphate,
pH 7.4, 0.02% Tween®-20 (Merck), 45 mg/ml mannitol (pyrogen free, Novo Nordisk).
Blood samples will be drawn from a catheter in vena jugularis. 5 ml of the blood
samples will be poured into chilled glasses containing 175 ul of the following solution:
0.18 M EDTA, 15000 KIE/ml aprotinin (Novo Nordisk) and 0.30 mM Valine-
Pyrrolidide (Novo Nordisk), pH 7.4. Within 30 min, the samples will be centrifuged for
10 min at 5-6000*g. Temperature will be kept at 4°C. The supernatant will be pipetted
into different glasses and kept at minus 20°C until use.
The plasma concentrations of the peptides will be determined in a sandwich
ELIS A or by RIA using different mono-or polyclonal antibodies. Choice of antibodies
depends of the GLP-1 analogue. The time at which the peak concentration in plasma is
achieved varies within wide limits, depending on the particular GLP-1 analogue
selected.
General assay protocol for sandwich ELISA in 96-wells microtitre plate
Coating buffer (PBS): Phosphate buffered saline, pH7.2
Wash-buffer (PBS-wash): Phosphate buffered saline, 0.05 % v/v Tween 20, pH
7.2
Assay-buffer (BSA-buffer): Phosphate buffered saline, 10 g/1 Bovin Serum
Albumin (Fluka 05477), 0.05 % v/v Tween 20, pH 7.2

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Streptavidin-buffer: Phosphate buffered saline, 0.5 M NaCI, 0.05 % v/v Tween
20, pH 7. 2
Standard: Individual compounds in a plasma-matrix
A-TNP: Nonsense antibody
AMDEX: Streptavin-horseradish-peroxodase (Amersham RPN4401V)
TMB-substrate: 3, 3', 5, 5'tetramethylbenzidine ( The assay can be carried out as follows (volume/well):
1.) Coat with 100 ul catching antibody 5 ug/ml in PBS-buffer, incubate o/n, 4 °C, 5x
PBS-wash, blocked with last wash in minimum 30 minute, then empty the plate
2.) 20 ul sample + 100 ul biotinylated detecting antibody 1 ug/ml in BSA-buffer with
10 ug/ml A-TNP; incubate 2 h, room temperature, on a shaker; 5x PBS-wash, then
empty the plate.
3.) 100 ul AMDEX 1: 8000 in Streptavidin-buffer, incubate 45-60 minute, room
temperature, on a shaker; 5x PBS-wash, then empty the plate.
4.) 100 ulTMB-substrate, incubate at room temperature on a shaker; stop the reaction
with 100 ul 4 M H3PO4. Read the absorbance at 450 nm with 620 nm as reference. The
concentration in the samples can be calculated from standard curves.
General assay protocol for RIA.
DB-buffer: 80 mM phosphate buffer, 0.1 % Human serum albumin, 10 mM
EDTA, 0.6 mM thiomersal, pH 7.5.
FAM-buffer: 40 mM phosphate buffer, 0.1 % Human Serum Albumin, 0.6 mM
thiomersal, pH 7.5.
Charcoal: 40 mM phosphate buffer, 0.6 mM thiomersal, 16.7 % bovine plasma,
15 g/1 activated carbon, pH 7.5 (mix the suspension minimum 1 h before use at 4 °C)
Standard: Individual compounds in a plasma-matrix.
The assay will be carried out in minisorp tubes 12x75 mm (volumen/tube) as
follows:

DB- Sample Antibody FAM Tracer Charcoal H2O

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Mix and incubate 30 min at 4 °C. Centrifuge at 3000 rpm for 30 min and
immediately after, transfer supernatants to new tubes, close with stopper and count on
gamma-counter for 1 minute. The concentration in the samples will be calculated from
individual standard curves.
GLP-1 radio receptor assay (RRA):
The GLP-1 radio receptor assay is a radiometric-ligand binding assay using
LEADseeker imaging particles. The assay is composed of membrane fragments
containing the GLP-1 receptor, unlabeled GLP-1 analogues, human GLP-1 labelled with
125I and PS LEADseeker particles coated with wheat germ agglutinin (WGA). Cold and
125I-labelled GLP-1 will compete for the binding to the receptor. When the LEADseeker
particles are added they will bind to carbohydrates residues on the membrane fragments
via the WGA-residues. The proximity between the 125I-molecules and the LEADseeker
particles causes light emission from the particles. The LEADseeker will image the
emitted light and it will be reversibly correlated to the amount of GLP-1 analogue
present in the sample.
Reagents & Materials:
Pre treatment of animal plasma: Animal plasma will be heat treated for 4 hrs at
56°C and centrifuged at 10,000 rpm for 10 minutes. Afterwards, Val-Pyr (10 |iM) and
aprotenin (500 KIE/ml) will be added and stored at -18°C until use.

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GLP-1 analogues standards: GLP-1 analogues will be spiked into heat-treated
plasma to produce dilution lines ranging from approximately 1 uM to 1 pM.
GLP-1 RRA assay buffer: 25 mM Na-HEPES (pH 7.5), 2.5 mM CaCl2, 1 mM
MgCI2, 50 mM NaCl, 0.1% ovalbumin, 0.003% Tween 20, 0.005% bacitracin, 0.05%
NaN3.
GLP-1 receptor suspension: GLP-1 receptor membrane fragments will be
purified from baby hamster kidney (BHK) cells expressing the human pancreatic GLP-1
receptor. Stored at- 80°C until use.
WGA-coupled polystyrene LEADseeker imaging beads (RPNQ0260,
Amersham): The beads will be reconstituted with GLP-1 RRA assay buffer to a
concentration of 13.3 mg/ml. The GLP-1 receptor membrane suspension will then be
added and incubated cold (2-8°C) for at least 1 hr prior to use.
Materials
125I-GLP-1 (7-36) amide (Novo Nordisk A/S). Stored at -18°C until use.
Ethanol 99.9% vol (De Dansk Sprotfabrikker A/S). Stored at -18°C until use.
Multiscreen® Solvinert 0.45 urn hydrophobic PTFE plate (MSRPN0450,
Millipore Corp.).
Polypropylene 384-well plates (cat. No. 781075, Greiner Bio-One).
Appartus:
Horizontal plate mixture
Centrifuge with a standard swinging bucket microtitre plate rotor assembly.
UltrVap, Drydown Sample concentrator (Provair)
LEADseeker® Multimodality Imaging System (Amersham)
Procedure:
Sample Preparation: Mount the Multiscreen® Solvinert filter plate on a
chemical-comparable receiver plate (ie polypropylene plates) to collect the filtrate.
Add 150 μl ice-cold ethanol 99.9% into the empty wells of the MultiScreen
Solvinertfilter plate followed by 50 ul calibrator or plasma sample. Place the storage lid
on the filter plate and incubate 15 minutes at 18-22°C on a horizontal plate mixer.

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The assembled filter and receiver plate, with the lid, will be placed into a
standard swinging-bucket microtitre plate rotor assembly. The filtrate will be collected
in the empty wells of the receiver plate at 1500 rpm for 2 minutes. The filtrate will be
dried down using the UltraVap with heated N2 (40°C) for 15 miuntes. The dry material
will be reconstituted by adding 100 ul GLP-1 RRA assay buffer into each well. This
will be incubated for 5 minutes on a horizontal mixer.
GLP-1 radio receptor assay: Use the following pipetting scheme and white polystyrene
3 84-well plates:
1. 3 5 μl GLP-1 RRA assay buffer
2. 5μl reconstituted filtrate
3. l0μl 125I-GLP-l(7-36)amide. The stock solution was diluted in GLP-1 RRA
assay buffer to 20,000 cpm/well prior to use.
4. 15 μL GLP-1 receptor membrane fragments (0. 5 ug/well) pre-coated to
WGA- polystyrene LEADseeker imaging beads (0.2 mg/well).
The plates will be sealed and incubated over night at 18-22°C. The light
emission from each well will be detected by using the LEADseeker Multimodality
Imaging System for duration of 10 minutes.
Stimulation of cAMP formation in a cell line expressing the cloned human GLP-
1 receptor.
Purified plasma membranes from a stable transfected cell line, BHK467-12A
(tk-tsl3), expressing the human GLP-1 receptor will be stimulated with GLP-1 and
peptide analogues, and the potency of cAMP production will be measured using the
AlphaScreen™ cAMP Assay Kit from Perkin Elmer Life Sciences.
A stable transfected cell line will be prepared and a high expressing clone will
be selected for screening. The cells will be grown at 5% C02 in DMEM, 5% FCS, 1%
Pen/Strep and 0. 5 mg/ml G418.
Cells at approximate 80% confluence will be washed 2X with PBS and
harvested with Versene, centrifuged 5 min at 1000 rpm and the supernatant removed.
The additional steps will be all made on ice. The cell pellet will be homogenized by the
Ultrathurax for 20-30 sec. in 10 ml of Buffer 1 (20 mM Na-HEPES, 10 mM EDTA, pH

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7.4), centrifuged 15 min at 20,000 rpm and the pellet resuspended in 10 ml of Buffer 2
(20 mM Na-HEPES, 0.1 mM EDTA, pH 7.4). The suspension will be homogenized for
20-30 sec and centrifuged 15 min at 20.000 rpm. Suspension in Buffer 2,
homogenization and centrifugation will be repeated once and the membranes will be
resuspended in Buffer 2 and ready for further analysis or stored at -80°C. The functional
receptor assay will be carried out by measuring the peptide induced cAMP production
by The AlphaScreen Technology. The basic principle of The AlphaScreen Technology
is a competition between endogenous cAMP and exogenously added biotin-cAMP. The
capture of cAMP will be achieved by using a specific antibody conjugated to acceptor
beads. Formed cAMP will be counted and measured on an AlphaFusion Microplate
Analyzer. The EC50 values will be calculated using the Graph-Pad Prisme software.
Example 11. Bacterial expression of a genetic fusion of glucagon like peptide-1 and
iDom7h-8 using the GAS leader.
GLP-1 (7-37), with glutamate at position 9 replaced by proline ([Pro9] GLP-l(7-37)),
was cloned as a fusion with iDOM7h-8 (a P96E mutation by Kabat numbering in
CDR3) into the pET 12a vector with a GAS leader (see WO 05/093074). The GLP-1
glutamate to proline 9 replacement was in order to render the GLP-1 part of the fusion
resistant to degradation by dipeptidyl peptidase IV (DPPIV) cleavage (Brian D. Green
et al. (2003) Metabolic Stability, Receptor Binding, cAMP Generation, Insulin secretion
and Antihyperglycaemic Activity of Novel N-terminal Glu9-substituted Analogues of
Glucagon-like-peptide-1: Biol. Chem. (384) 1543-1555). In total, three constructs were
made, one with no linker, one with PSS amino acids between [Pro9]GLP-1 (7-37) and
iDOM7h-8 and one with PSSGAP amino acids between [Pro9]GLP-1 (7-37) and
iDOM7h-8 (shown in Figure 16 as Dom7h-8 the albumin binding form). Expression
was in BL21 DE3 Plys S cells at 30°C for 48 hours using overnight expression
autoinduction TB readymix (Novagen) before recovery of the supernatant by
centrifugation. [Pro9] GLP-l(7-37) iDom7h-8 fusion was purified from the supernatant
using affinity capture on protein L-agarose. The resin was then washed with 10 column
volumes of PBS and bound protein eluted with 0.1 M glycine pH2. [Pro9]GLP-l(7-37)-
iDom7h-8 fusion was then loaded in the glycine buffer, onto a cation exchange column

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(1 ml S-column, GE healthcare) that was pre-equilibrated with 20mM citrate buffer at
pH 6.2. Elution was with a 0 - 50% gradient of the same buffer supplemented with 1M
NaCl. Peaks were collected and the size of the proteins determined by SDS PAGE
electrophoresis. Peaks with protein of the expected size were pooled and buffer
exchanged to PBS. Identity of the protein was confirmed by mass spectrometry and N-
terminal sequencing.
Example 12. GLP-1 activity determination of [Pro9]GLP-l(7-37)-PSSGAP-iDOM7h-8
fusion
In order to confirm that the [Pro9]GLP-l(7-37)-PSSGAP-iDOM7h-8 fusion
demonstrated GLP-1 activity, the fusion was subjected to two different biological
assays. In the first assay, the RINm5f rat insulinoma cell line (developed in 1980 by
Gadzar et al from x-ray induced transplantable insulinoma of the rat) was incubated
with varying concentrations (l0pM to O.luM) of GLP-1 and the [Pro9] GLP-1 (7-3 7)-
PSSGAP-iDOM7h-8 fusion for 60 min. Additionally, a single point assay of Exendin-4,
a GLP-1 analogue resistant to degradation by dipeptidyl peptidase IV, and a single point
buffer only assay were added as controls. Although the RTNm5f rat cells respond poorly
to glucose, when exposed to nutrients or non-secretagogues, they display secretory
responses similar to beta cells. Therefore, the effects of the compounds on cell
proliferation were assessed by measuring the incorporated levels of 5-bromo-2'-
deoxyuridine (BrdU) during DNA synthesis in proliferating cells using the Cell
proliferation ELISA system (Amersham, Little Chalfont, UK) see Figure 17. Using
OD450 to measure DNA levels, there was a dose dependent increase in DNA level with
increasing levels of [Pro9]GLP-l(7-37)-PSSGAP-iDOM7h-8 fusion up to a
concentration of lOOnM of the fusion. GLP-1 also showed the expected dose dependent
response.
In the second assay, RINm5f cells were incubated with varying concentrations (lOpM to
O.lμM) of GLP-1 and the [Pro9]GLP-l(7-37)-PSSGAP-iDOM7h-8 fusion in 5.6mM
glucose for 60 min. Additionally, a single point assay of Exendin-4, a GLP-1 analogue
resistant to degradation by DPPIV and a single point Krebs-Ringer bicarbonate buffer

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(KRB) only assay were added as controls. Insulin secretion was assayed after incubation
for 60 min at 37°C using KRB buffer supplemented GLP-1, 3A23 or exendin-4. The
medium was collected, centrifuged and the supernatant assayed for insulin
concentration using radioimmunoassay. Insulin concentration was normalised to cell
number within each well. Insulin concentration (measured in ng/ml/hr) was then plotted
against compound concentration. There was a dose dependent increase in insulin release
at escalating doses of [Pro9]GLP-l(7-37)-PSSGAP-iDOM7h-8 fusion up to a fusion
concentration of lOnM (see Figure 18). This agrees well with published data on GLP-1
alone.
Example 13. Bacterial expression of a genetic fusion of glucagon like peptide-1 and
iDom7h-8 using the OMP-T leader.
The same 3 constructs described in Example 11 (one with no linker, one with PSS
amino acids between [Pro9]GLP-l(7-37) and iDOM7h-8 and one with PSSGAP amino
acids between [Pro9]GLP-l(7-37) and iDOM7h-8) were remade with the OMP-T leader.
For clarity, the order of the elements in the construct were OMP-T leader, [Pro9] GLP-1,
Linker (where appropriate) and the iDom7h-8. Expression was in BL21 DE3 Plys S
cells at 25°C for 4 hours in TB media induced with 0.5mM IPTG at OD 16 before
recovery of the cell pellet by centrifugation. Secreted proteins were then recovered by
periplasmic preparation. GLP-1 iDom7h-8 fusions were purified from the periplasmic
fraction using affinity capture on protein L-agarose. The resin was then washed with 10
column volumes of PBS and bound protein eluted with 0.1 M glycine pH2. [Pro9]GLP-
1(7-37) fusion was then loaded in the glycine buffer, onto a cation exchange column (1
ml S-column, GE healthcare) that was pre-equilibrated with 20mM citrate buffer at pH
6.2. Elution was with a 0 - 50% gradient of the same buffer supplemented with 1M
NaCl. In this case, washing the column with 20mM citrate buffer at pH 6.2 (0% NaCl)
led to flow through of the band of the expected size and so this was concentrated using a
5K vivaspin column (Vivascience).
Example 14. Pichia pastoris expression of a genetic fusion of glucagon like peptide-1
and iDom7h-8.

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The [Pro9]GLP-l-PSS-iDOM7h-8 fusion construct (as described in Figure 16b but using
iDom7h-8) will be cloned into the pPICzct vector both alone and with an N-terminal
EAEA extension and transformed into Pichia pastoris KM71h. Protein will be
expressed (i) using methanol induction over 4 days at 30°C and (ii) using methanol
induction over 2 days at 25°C. Supernatant will be recovered by centrifugation and
protein checked for size on an SDS PAGE gel.
It is expected that the fusions will have the correct size by SDS Page and be active in
the GLP-1 assay as described in Example 10 and in Example 12.
Example 15. E.coli expression of glucagon like peptide-1 and iDom7h-8 in BL21 DE3
inclusion bodies.
The same fusions as described in Example 11 will be cloned into the pET21 expression
vector (Novagen), which is designed for protein expression in the cytoplasm.
Optionally, a protease cleavage site will be included in the constructs between a
sacrificial N-terminus and the HAP... of the [Pro9]GLP-1 (7-37). This will enable the
protein to be digested to ensure a fully native N-terminus. Enzymes that could be used
for this include Factor Xa, thrombin or DPPI. Protein will then be expressed at high
levels in BL21(DE3) cells upon IPTG induction and will accumulate in intracellular
inclusion bodies. Inclusion bodies will be isolated from the BL21 cells and solublised in
guanidine HC1. Following reduction, inclusion bodies will be refolded in a redox
shuffling buffer system (Buchner, J., Pastan, I. and Brinkmann, U. (1992). A method for
increasing the yield of properly folded recombinant fusion proteins. Anal. Biochem.
205, 263-270. After refolding, the protein will be dialysed and concentrated in a 5K
vivaspin column (Vivascience) and purified by S-column (GE healthcare).
It is expected that the fusions will have the correct size by SDS Page and be active in
the GLP-1 assay as described in Example 10 and in Example 12.
Example 16. Mammalian expression of glucagon like peptide-1 and a Dom7h-8

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The [Pro9]GLP-l-PSS-DOM7h-8 fusion construct (as described in Figure 16b) will be
cloned into the PcDNA(-) vector using a murine secretory signal peptide to promote
secretion of the translated protein into the media, lmg of DNA will be prepared in
E.coli using alkaline lysis (mega prep kit, qiagen, CA) and transfected into 1.5L of
HEK293 cells grown in Dulbecco's modified Eagle's medium (Gibco) for transient
protein expression. Protein will be expressed by incubating the culture at 37° for 5 days
and supernatant (containing expressed protein) will be recovered by centrifugation.
[Pro9]GLP-l-PSS-DOM7h-8 fusion will be purified from the periplasmic fraction using
affinity capture on protein L-agarose. The resin will then be washed with 10 column
volumes of PBS and bound protein eluted with 0.1 M glycine pH2. Protein will then be
loaded in the glycine buffer, onto a cation exchange column (1 ml S-column, GE
healthcare) that is pre-equilibrated with 20mM citrate buffer at pH 6.2. Elution will be
with a 0 - 50% gradient of the same buffer supplemented with 1M NaCl. Protein of the
correct size on an SDS-PAGE gel will then be concentrated using a 5K vivaspin column
(Vivascience) and buffer exchanged into PBS for biological assay.
Example 17. E.coli expression of Peptide YY fused to a Dom7h-8.
Peptide YY (3-36) (PYY: amino acid sequence ID No. 167 and nucleci acid sequence
ID No. 168 KPEAPGEDASPEELNRYYASLRHYLNLVTRQRY) inhibits food intake
in humans and has a short half life in plasma (10-30min). It is released in response to a
meal and acts via the Y2R in the arcuate nucleus to physiologically regulate food intake.
PYY will be cloned into the pET GAS vector (WO05093074) abutting the DOM7h-8
(see Figures 20a and 20b which show the peptide C-terminal and N-terminal of the
DOM7h-8 respectively.) Expression will be in BL21 DE3 Plys S cells at 25 °C for 4
hours in TB media induced with 0.5mM IPTG at before recovery of the cell pellet by
centrifugation. Secreted proteins will then be recovered by periplasmic preparation.
PYY Dom7h-8 fusion will be purified from the periplasmic fraction using affinity
capture on protein L-agarose. The resin will then be washed with 10 column volumes of
PBS and bound protein eluted with 0.1 M glycine pH2 and purified further by ion
exchange. Purified protein will then be buffer exchanged to PBS by dialysis,

WO 2006/059106 PCT/GB2005/004599
- 115 -
concentrated in a 5K vivaspin column (Vivascience) and subjected to biological assay to
measure stimulation of cAMP release as described (Goumain et al. (2001) The Peptide
YY-Preferring Receptor Mediating Inhibition of Small Intestinal Secretion Is a
Peripheral Y2 Receptor: Pharmacological Evidence and Molecular Cloning: Molecular
pharmacology: 60 124-134). Briefly, Isolated intestinal crypt cells at 200uJg protein/ml
will be incubated under continuous agitation for 45min at 15°C in 0.5ml of phosphate-
buffered saline, pH 7.0, containing 1.4% (w/v) bovine serum albumin, 0.1% bacitracin,
and 0.2mM 3-isobutyl-l-methylxanthine(IBMX) as described (Servin et al. (1989):
Peptide-YY and neuropeptide-Y inhibit vasoactive intestinal peptide-stimulated
adenosine 3',5'-monophosphate production in rat small intestine: structural requirements
of peptides for interacting with PYY-preferring receptors. Endocrinology 124: 692-
700). PYY alone or PYY-Dom7h-8 fusion will be added together with a potent
physiological stimulant of cAMP production in enterocytes (e.g., VIP). The reaction will
be initiated by adding cells and stopped after 45min by adding 50|j,l of 11M perchloric
acid. After centrifugation for l0min at 4,000g-, the cAMP present in the supernatant will
be succinylated, and its concentration will be measured by radioimmunoassay as
described (Laburthe et al., (1982) Alpha-adrenergic inhibition of cyclic AMP
accumulation in epithelial cells isolated from rat small intestine. Biochim Biophys Ada
721: 101-108).
It is expected that the fusion be of the expected size and will show PYY activity
equivalent to the non-fusion controls.
Example 18. E. coli expression of a Dom7h-8 Peptide YY, GLP-1, fusion
A [Pro9]GLP-l(7-37)-DOM7h-8-PYY (see Figure 19c) fusion will be cloned into the
pET GAS vector and then expressed as described for the Dom7h-8 PYY described in
Example 17. After purification, the fusion will be assayed for the biological activity of
both PYY and GLP-1 following the assays described in Examples 17 and Example 12
respectively.
It is expected that the fusions will be of the expected size will show PYY and GLP-1
activity equivalent to the non-fusion controls.

WO 2006/059106 PCT/GB2005/004599
-116-
While this invention has been particularly shown and described with references
to preferred embodiments thereof, it will be understood by those skilled in the art that
various changes in form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.

WO 2006/059106 PCT/GB2005/004599
117
WE CLAIM:
1. A drug fusion having the formula:
a-(X)n1-b-(Y)n2-c-(Z)n3-d or a-(Z)n3-b-(Y)n2-c-(X)n1-d,
wherein
X is an insulintropic agent or an analogue thereof;
Y is an immunoglobulin heavy chain variable domain (VH) that has
binding specificity for serum albumin, or an immunoglobulin light chain
variable domain (VL that has binding specificity for serum albumin;
Z is a polypeptide drug that has binding specificity for a target;
a, b, c and d are independently a polypeptide comprising one to about
100 amino acid residues or absent;
n1 is one to about 10;
n2 is one to about 10; and
n3 is zero to about 10.
2. The fusion as claimed in claim 1, wherein the or each X is GLP-l(7-37), GLP-l(7-36)
amide, [Ser8]GLP-l(7-36)amide [Pro9]GLP(7-37) or an analogue thereof.
3. The fusion as claimed in any preceding claim, wherein nl and n3 are both one, and n2 is
two to about 10.
4. The fusion as claimed in any preceding claim, wherein the or each Y comprises an amino
acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID
NO:ll, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ
ID NO:24, SEQ ID NO:25 and SEQ ID NO:26.
5. The fusion as claimed in any one of claims 1 to 4, wherein the or each Y comprises an
amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ
ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,
SEQ ID NO:22 and SEQ ID NO:23.
6. A drug fusion comprising moieties X' and Y', wherein X' is GLP-1 or an
analogue thereof; and

Y' is an immunoglobulin heavy chain variable domain (VH) that has binding
specificity for serum albumin, or an immunoglobulin light chain variable
domain (VL) that has binding specificity for serum albumin.
7. The drug fusion as claimed in claim 6, wherein X' is located amino terminally to Y'.
8. The drug fusion as claimed in claim 6, wherein Y' is located amino terminally to X'.
9. The drug fusion as claimed in claim 6, 7 or 8, wherein said VH and VL have binding
specificity for human serum albumin.
10. The drug fusion as claimed in claim 6, wherein Y' comprises an amino acid sequence
selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID
NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:24, SEQ
ID NO:25 and SEQ ID NO:26.
11. The drug fusion as claimed in claim 6, wherein Y' comprises an amino acid sequence
selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID
NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and
SEQ ID NO:23.
12. A drug conjugate comprising an immunoglobulin heavy chain variable domain
(VH) that has binding specificity for serum albumin, or an immunoglobulin light
chain variable domain (VL) that has binding specificity for serum albumin; and
GLP-1 or an analogue thereof that is covalently bonded to said VH or VL.
13. The drug conjugate as claimed in claim 12, wherein the drug conjugate comprises a single
VH.
14. The drug conjugate as claimed in claim 12, wherein the drug conjugate comprises a single
VL.

15. The drug conjugate as claimed in claim 12, 13 or 14, wherein said GLP-1 or analogue is
covalently bonded to said VH or VL through a linker moiety.
16. The drug conjugate as claimed in any one of claims 12 to 15 comprising one or more
different drugs covalently bonded to said VH or VL.
17. The drug conjugate as claimed in any one of claims 12 to 16, wherein said immunoglobulin
heavy chain variable domain (VH) that has binding specificity for serum
albumin, or said immunoglobulin light chain variable domain (VL) that has
binding specificity for serum albumin comprises an amino acid sequence
selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID

NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO:24, SEQ
ID NO:25, SEQ ID NO:26, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO:18,
SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID
N0:23.
18. A recombinant nucleic acid encoding the drug fusion as claimed in claim 1 or claim 6.
19. A nucleic acid construct comprising the recombinant nucleic acid as claimed in claim 18.
20. A method for producing a drug fusion comprising maintaining a host cell
comprising the recombinant nucleic acid as claimed in claim 18 under conditions suitable
for expression of said recombinant nucleic acid, whereby a drug fusion is produced.
21. A pharmaceutical composition comprising a drug fusion as claimed in claim 1 or claim 6,
or the drug conjugate as claimed in claim 12 and a physiologically acceptable carrier.
22. A drug conjugate or fusion comprising an insulinotropic agent and an antibody
fragment that binds an antigen, wherein the antigen acts to increase the half-life
of the drug conjugate or fusion in vivo.
23. The drug conjugate or fusion as claimed in claim 22, wherein the drug is a drug fusion
protein comprising the insulinotropic agent peptide bonded to the antibody
fragment.
24. The drug as claimed in claim 23, wherein the insulinotropic agent is fused to the antibody
fragment via peptide linker moiety.
25. The drug as claimed in any one of claims 22 to 24, wherein the antigen is serum albumin.
26. The drug as claimed in any one of claims 22 to 25, wherein the insulinotropic agent is a
glucagon-like peptide.
27. The drug as claimed in any one of claims 22 to 26, wherein the insulinotropic agent is
selected from the group consisting of GLP-1, GLP-1 analogue, Exendin-3, an
Exendin-3 analogue, Exendin-4 and an Exendin-4 analogue.
28. The drug as claimed in any one of claims 1,6, 12 and 27, wherein the GLP-1 or analogue
comprises an amino acid sequence that is at least 80% homologous to a
sequence selected from the group consisting of SEQ ID N0:s 157 or 159.
29. The drug as claimed in any one of claims 1,6, 12 and 27, wherein the GLP-1 or analogue
comprises Gly10, Thr12, Asp14, Phe27 and Ile29.

30. The drug as claimed in any one of claims 28 and 29, wherein the GLP-1 analogue differs
from SEQ ID NO: 157 or SEQ ID NO: 159 by no more than 6 amino acids.
31. The drug as claimed in any one of claims 1, 6, 12 and 27 comprising a single variable
domain specific for serum albumin (SA) which has a dissociation constant (Kd)
of InM to 500uM for SA, as determined by surface plasmon resonance.
32. The drug as claimed in claim 31, wherein the SA-specific domain binds SA in a standard
ligand binding assay with an IC50 of 1nM to 500µM.
33. The drug as claimed in claim 22, comprising 2,3 or 4 insulinotropic agent (IA) moieties.
34. The drug as claimed in claim 33, comprising IA-IA'-AF or IA-(AF)„,-LA', wherein IA and
LA' are the same or different insulinotropic agents and AF is an antibody
fragment as recited in claim 22, and n equals 1,2, 3,4, or 5.
35. The drug as claimed in claim 34, comprising [GLP-1]-[AF]-[GLP-1] or [GLP-1]-[GLP-1]-
[AF].
36. The drug as claimed in any one of the preceding claim, comprising an anti-satiety agent.
37. The drug as claimed in claim 36, wherein the anti-satiety agent is selected from the group
consisting of PYY, a PYY analogue, PYY (3-36).
38. The drug as claimed in claim 36 or 37 comprising IA-(AF)n,-(IA')x, -[anti-satiety agent] or
[anti-satiety agent]- (IA')X, -(AF)n, - IA, wherein n equals 1, 2, 3, 4, or 5 and x
equals zero, 1, 2, 3, 4, or 5.
39. The drug as claimed in any preceding claim having at ½ alpha of between 1 and 6 hours.
40. The drug as claimed in any preceding claim having at ½ beta of between 12 and 60 hours.
41. The drug as claimed in any one of claims 1, 6, 12 and 22 comprising an agent
selected from the group consisting of insulin, Exendin-4, Exendin-3, PYY (3-36),
Resistin, Leptin, MC3R/MC4R antagonist, AgRP antagonist,
Apolipoprotein A-IV, Enterostatin, Gastrin-Releasing Peptide (GRP), IGF1,
BMP-9, IL-22, ReglV, interferon alfa, INGAP peptide, somatostatin, amylin,
neurulin, interferon beta, interferon hybrids, adiponectin, endocannabinoids, C
peptide, WNTlOb, Orexin-A, adrenocorticotrophin, Enterostatin,
Cholecystokinin, oxyntomodulin, Melanocyte Stimulating Hormones,
melanocortin, Melanin concentrating hormone, BB-2, NPY Y2 agonists, NPY
Y5/Y1 antagonists, OXM, Gal-IR antagonists, MCH-1R antagonists, MC-3/4
agonists, BRS-3 agonists, pancreatic polypeptide, anti-Ghrelin antibody

fragment, brain-derived neurotrophic factor, human growth hormone,
parathyroid hormone, follicle stimulating hormone, Gastric inhibitory peptide or
an analogue thereof.
42. A drug comprising GLP-1 or an analogue thereof and a protein moiety
comprising an antigen binding site, wherein the antigen binding site binds an
antigen which acts to increase the half-life of the drug conjugate in vivo, with the
proviso that the protein moiety is not a peptide having 10-30 amino acids.
43. A drug fusion comprising GLP-1 or an analogue thereof and a protein moiety
comprising an antigen binding site, wherein the antigen binding site binds an
antigen which acts to increase the half-life of the drug conjugate in vivo.
44. A drug as claimed in claim 42 or 43, wherein the GLP or analogue is GLP-1 (7-37), GLP-
1(7-36) amide, [Ser8] GLP-1 (7-36)amide, [Pro9]GLP-l(7-36), [Pro9]GLP-l(7-37)
or an analogue thereof.
45. A drug as claimed in claim 44, wherein the GLP analogue has a C terminal peptide selected
from the list consisting of PSS, PSSGAP or PSSGAPPPS.
46. A drug as claimed in claim 42, 43, 44 or 45, wherein said antigen is serum albumin.
47. A drug as claimed in any one of claims 42 to 46, comprising a single protein moiety.
48. A recombinant nucleic acid encoding the drug as claimed in any one of claims 42 to 47.
49. A nucleic acid construct comprising the recombinant nucleic acid as claimed in claim 48.
50. A method for producing a drug fusion comprising maintaining a host cell
comprising the recombinant nucleic acid as claimed in claim 48 under conditions suitable for
expression of said recombinant nucleic acid, whereby a drug fusion is produced.
51. A pharmaceutical composition comprising a drug as claimed in any one of claims 42 to 47
and a physiologically acceptable carrier.
52. The drug as claimed in any one of claims 1 to 17 and 22 to 47 for treatment and/or
prevention of diabetes.
53. The drug as claimed in any one of claims 1 to 17 and 22 to 47 for treatment and/or
prevention of obesity.
54. The drug as claimed in any one of claims 1 to 17 and 22 to 47 for reducing food intake by a patient.
ABSTRACT
BISPECIFIC DOMAIN ANTIBODIES TARGETING SERUM ALBUMIN
AND GLP-1 OR PYY
In the fields of diabetes and obesity, there is a need to modify GLP-I and other insulinotropic
peptides to provide longer duration of action in vivo while maintaining their low toxicity and
therapeutic advantages. The invention relates to drug fusions having improved serum half
lives and have the formula:
a-(X)n1-b-(Y)n2-c-(Z)n3-d or a-(Z)n3-b-(Y)n2-c-(X)n1-d,
wherein
X is an insulinotropic agent or an analogue thereof;
Y is an immunoglobulin heavy chain variable domain (VH) that has binding specificity for
serum albumin, or an immunoglobulin light chain variable domain (VL) that has binding
specificity for serum albumin;
Z is a polypeptide drug that has binding specificity for a second target;
a, b, c and d are each independently a polypeptide comprising one to about 100 amino acid
residues or absent;
n1 and n2 are one to about 10; and
n3 is zero to about 10.


Documents:

02409-kolnp-2007-abstract.pdf

02409-kolnp-2007-assignment.pdf

02409-kolnp-2007-claims.pdf

02409-kolnp-2007-correspondence others 1.1.pdf

02409-kolnp-2007-correspondence others.pdf

02409-kolnp-2007-description complete.pdf

02409-kolnp-2007-drawings.pdf

02409-kolnp-2007-form 1.pdf

02409-kolnp-2007-form 3.pdf

02409-kolnp-2007-form 5.pdf

02409-kolnp-2007-international publication.pdf

02409-kolnp-2007-international search report.pdf

02409-kolnp-2007-pct request form.pdf

02409-kolnp-2007-pct request-1.1.pdf

02409-kolnp-2007-priority document.pdf

02409-kolnp-2007-sequence listing.pdf

2409-KOLNP-2007-(04-01-2012)-ABSTRACT.pdf

2409-KOLNP-2007-(04-01-2012)-AMANDED CLAIMS.pdf

2409-KOLNP-2007-(04-01-2012)-CORRESPONDENCE.pdf

2409-KOLNP-2007-(04-01-2012)-DESCRIPTION (COMPLETE).pdf

2409-KOLNP-2007-(04-01-2012)-DRAWINGS.pdf

2409-KOLNP-2007-(04-01-2012)-FORM-1.pdf

2409-KOLNP-2007-(04-01-2012)-FORM-3.pdf

2409-KOLNP-2007-(04-01-2012)-OTHER PATENT DOCUMENT.pdf

2409-KOLNP-2007-(04-01-2012)-OTHERS.pdf

2409-KOLNP-2007-(04-01-2012)-PA-CERTIFIED COPIES.pdf

2409-KOLNP-2007-(05-03-2012)-EXAMINATION REPORT REPLY RECIEVED.pdf

2409-KOLNP-2007-(05-03-2012)-OTHER PATENT DOCUMENT.pdf

2409-KOLNP-2007-(05-03-2012)-PA.pdf

2409-KOLNP-2007-(20-04-2012)-CORRESPONDENCE.pdf

2409-KOLNP-2007-(25-05-2012)-CORRESPONDENCE.pdf

2409-KOLNP-2007-(25-05-2012)-FORM-1.pdf

2409-KOLNP-2007-(25-05-2012)-PA.pdf

2409-kolnp-2007-form 18.pdf


Patent Number 253794
Indian Patent Application Number 2409/KOLNP/2007
PG Journal Number 35/2012
Publication Date 31-Aug-2012
Grant Date 24-Aug-2012
Date of Filing 29-Jun-2007
Name of Patentee DOMANTIS LIMITED
Applicant Address 980 GREAT WEST ROAD, BENTFORD, MIDDLESEX TW8 9GS
Inventors:
# Inventor's Name Inventor's Address
1 JESPERS LAURENT S DOMANTIS LIMITED 315 CAMBRIDGE SCIENCE PARK, CAMBRIDGE CB4 0WG
2 HOLMES STEVE DOMANTIS LIMITED 315 CAMBRIDGE SCIENCE PARK, CAMBRIDGE CB4 0WG
3 TOMLINSON IAN M DOMANTIS LIMITED 315 CAMBRIDGE SCIENCE PARK, CAMBRIDGE CB4 0WG
4 HOLT LUCY J DOMANTIS LIMITED 315 CAMBRIDGE SCIENCE PARK, CAMBRIDGE CB4 0WG
PCT International Classification Number C07K 16/46,A61P 1/00
PCT International Application Number PCT/GB2005/004599
PCT International Filing date 2005-11-30
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/632361 2004-12-02 U.S.A.
2 0511019.2 2005-05-31 U.S.A.