Title of Invention

A HETEROLOGOUS FUSION PROTEIN

Abstract The invention provides active therapeutic peptides fused to specific IgG4-Fc derivatives. These fusion proteins have an increased half-life, reduced half antibody formation, and reduced effector activity, while not being immunogenic. The fusion proteins are useful in treating human diseases as well as a variety of other conditions or disorders.
Full Text FIELD OF THE INVENTION
The present invention relates to heterologous fusion proteins comprising an active
therapeutic peptide and a constant heavy chain (Fc) portion of an immunoglobulin that
have the effect of extending the in vivo half-life of the active therapeutic peptide. These
heterologous fusion proteins can be used to treat human diseases as well as a variety of
other conditions or disorders.
Many active therapeutic peptides show promise in clinical trials for the treatment
of various diseases. However, the usefulness of therapy involving these peptides has
been limited by the fact that many peptides are poorly active, rapidly cleared in vivo, or
have extremely short in vivo half-lives. Various approaches have been undertaken to
extend the elimination half-life of these peptides or reduce clearance of these peptides
from the body while maintaining biological activity. One approach involves fusing an
active therapeutic peptide to the constant heavy chain (Fc) portion of an immunoglobulin.
Immunoglobulins typically have long circulating half-lives in vivo. For example, IgG
molecules can have a half-life in humans of up to 23 days. The Fc portion of the
immunoglobulin is responsible, in part, for this in vivo stability. These heterologous
fusion proteins take advantage of the stability provided by the Fc portion of an
immunoglobulin while preserving the biological activity of the peptides.
Although this approach is feasible for peptide therapeutics (See WO 02/46227),
there is a general concern regarding half antibody formation, unwanted effector function,
glycosylation sites, and heterogeneity expression. The present invention seeks to
overcome these problems by identifying and substituting amino acids at various positions
in the Fc portion of the molecule that reduce half antibodies and lessen or eliminate
effector function. In addition, the present invention also provides identifying and
substituting amino acids at various positions in the Fc portion of the molecule that do not
have glycosylation sites and have reduced heterogeneity during expression. Furthermore,
it is desired that identifying and substituting amino acids at various positions in the Fc
portion of the molecule does not induce an immune response after repeated and prolonged
administration of the heterologous fusion protein.
Compounds of the present invention include heterologous fusion proteins
comprising an active therapeutic peptide fused to the Fc portion of an immunoglobulin
comprising the sequence of SEQ ID NO:l
XaarGlu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro-Cys-Pro-Ala-Pro-
Xaai6-Xaa17-Xaa18-Gly-Gly-Pro-Ser-Val-Phe-Leu-Phe-Pro-Pro-Lys-Pro-
Lys-Asp-Thr-Leu-Met-Ile-Ser-Arg-Thr-Pro-Glu-Val-Thr-Cys-Val-
Val-Val-Asp-Val-Ser-Gln-Glu-Asp-Pro-Glu-Val-Gln-Phe-Asn-Trp-
Tyr-Val-Asp-Gly-Val-Glu-Val-His-Asn-Ala-Lys-Thr-Lys-Pro-Arg-
Glu-Glu-Gln-Phe-Xaa80-Ser-Thr-Tyr-Arg-Val-Val-Ser-Val-Leu-Thr-
Val-Leu-His-Gln-Asp-Trp-Leu-Asn-Gly-Lys-Glu-Tyr-Lys-Cys-Lys-
Val-Ser-Asn-Lys-Gly-Leu-Pro-Ser-Ser-Ile-Glu-Lys-Thr-Ile-Ser-
Lys-Ala-Lys-Gly-Gln-Pro-Arg-Glu-Pro-Gln-Val-Tyr-Thr-Leu-Pro-
Pro-Ser-G ln-G lu-G lu-Met-Thr-Lys-Asn-Gln-Val-Ser-Leu-Thr-Cys-
Leu-Val-Lys-Gly-Phe-Tyr-Pro-Ser-Asp-Ile-Ala-Val-Glu-Trp-Glu-
Ser-Asn-Gly-Gln-Pro-Glu-Asn-Asn-Tyr-Lys-Thr-Thr-Pro-Pro-Val-
Leu-Asp-Ser-Asp-Gly-Ser-Phe-Phe-Leu-Tyr-Ser-Arg-Leu-Thr-Val-
Asp-Lys-Ser-Arg-Trp-Gln-Glu-Gty-Asn-Val-Phe-Ser-Cys-Ser-Val-
Met-His-Glu-Ala-Leu-His-Asn-His-Tyr-Thr-Gln-Lys-Ser-Leu-Ser-
Leu-Ser-Leu-Gly-Xaa23o (SEQ ID NO.T)
wherein:
Xaa at position 1 is Ala or absent;
Xaa at position 16 is Pro or Glu;
Xaa at position 17 is Phe, Val, or Ala;
Xaa at position 18 is Leu, Glu, or Ala;
Xaa at position 80 is Asn or Ala; and
Xaa at position 230 is Lys or is absent.
The peptide portion and the Fc portion of the present invention are fused directly
together or via a linker. An example of a linker is a G-rich peptide linker having the
sequence Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID
NO:2). Other examples of linkers include, but are not limited to, Gly-Ser-Gly-Gly-Gly-
Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (1.5L) (SEQ
ID NO:4);Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-
Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-GIy-Ser (2L) (SEQ ID N0:6); Asp-Ala-
Ala-Ala-Lys-Glu-Ala-Ala-Ala-Lys-Asp-Ala-Ala-Ala-Arg-Glu-Ala-Ala-Ala-Arg-Asp-
Ala-Ala-Ala-Lys (SEQ ID N0:7) and Asn-Val-Asp-His-Lys-Pro-Ser-Asn-Thr-Lys-Val-
Asp-Lys-Arg (SEQ ID NO:8).
The C-terminus of the peptide portion and the N-terminus of the Fc portion are
fused together. Alternatively, the N-terminus of the peptide portion and the C-terminus
of the Fc portion are fused together. Additionally, the C-terminus of the peptide portion
is fused to the N-terminus of the Fc portion and the N-terminus of another peptide
molecule is fused to the C-terminus of the Fc portion, resulting in a peptide-Fc-peptide
fusion protein.
The present invention also includes polynucleotides encoding the heterologous
fusion proteins of the present invention, as well as vectors and host cells comprising such
polynucleotides. Methods of treating patients suffering from human diseases as well as a
variety of other conditions or disorders comprising administering a heterologous fusion
protein are also encompassed by the present invention.
The heterologous fusion proteins of the present invention comprise an
active therapeutic peptide portion and an Fc portion. The Fc portion comprises
substitutions to the human IgG4 sequence that provide the heterologous fusion
protein with increase in vivo stability compared to the active therapeutic peptide
not fused to an Fc sequence.
The heterologous fusion proteins of the present invention contain an Fc
portion which is derived from human IgG4, but comprises one or more
substitutions compared to the wild-type human sequence. As used herein, the Fc
portion of an immunoglobulin has the meaning commonly given to the term in
the field of immunology. Specifically, this term refers to an antibody fragment
which does not contain the two antigen binding regions (the Fab fragments) from
the antibody. The Fc portion consists of the constant region of an antibody from
both heavy chains, which associate through non-covalent interactions and
disulfide bonds. The Fc portion can include the hinge regions and extend
through the CH2 and CH3 domains to the c-terminus of the antibody. The Fc
portion can further include one or more glycosylation sites.
There are five types of human immunoglobulins with different effector
functions and pharmacokinetic properties. IgG is the most stable of the five
types having a serum half-life in humans of about 23 days. There are four IgG
subclasses (G1, G2, G3, and G4) each of which has different biological functions
known as effector functions. These effector functions are generally mediated
through interaction with the Fc gamma receptor (FcyR) or by binding a
subcomponent of complement 1 (Clq) which recognizes and binds to the heavy
chain of Immunoglobulin G or Immunoglobulin M initiating the classical
complement pathway. Binding to FcyR can lead to antibody dependent cell
mediated cytolysis, whereas binding to complement factors can lead to
complement mediated cell lysis. In designing heterologous fusion proteins
wherein the Fc portion is being utilized solely for its ability to extend half-life, it
is important to minimize any effector function. Thus, the heterologous fusion
proteins of the present invention are derived from the human IgG4 Fc region
because of its reduced ability to bind FcyR and complement factors compared to
other IgG sub-types. IgG4, however, has been shown to deplete target cells in
humans [Issacs et al., (1996) Clin. Exp. Immunol. 106:427-433]. Because the
heterologous fusion proteins of the present invention target cells in various
organs in the body, using an IgG4 derived region in an heterologous fusion
protein could initiate an immune response against the cells through interaction of
the heterologous fusion protein with receptors present on the target cells. Thus,
the IgG4 Fc region which is part of the heterologous fusion proteins of the
present invention contains substitutions that eliminate effector function. The
IgG4 Fc portion of the heterologous fusion proteins of the present invention may
contain one or more of the following substitutions: substitution of proline for
glutamate at residue 233, alanine or valine for phenylalanine at residue 234 and
alanine or glutamate for leucine at residue 235 (EU numbering, Kabat, E.A. et al.
(1991) Sequences of Proteins of Immunological Interest, 5th Ed. U.S. Dept. of
Health and Human Services, Bethesda, MD, NIH Publication no. 91-3242).
These residues corresponds to positions 16, 17 and 18 in SEQ ID NO:l. Further,
removing the N-linked glycosylation site in the IgG4 Fc region by substituting
Ala for Asn at residue 297 (EU numbering) which corresponds to position 80 of
SEQ ID NO: 1 is another way to ensure that residual effector activity is
eliminated in the context of a heterologous fusion protein.
In addition, the IgG4 Fc portion of the heterologous fusion proteins of the
present invention contain a substitution that stabilizes heavy chain dimer
formation and prevents the formation of half-IgG4 Fc chains. The heterologous
fusion proteins of the present invention preferably exist as dimers joined together
by disulfide bonds and various non-covalent interactions. Wild-type IgG4
contains a Pro-Pro-Cys-Pro-Ser-Cys (SEQ ID NO:3) motif beginning at residue
224 (EU numbering). This motif in a single active therapeutic peptide-Fc chain
forms disulfide bonds with the corresponding motif in another active therapeutic
peptide-Fc chain. However, the presence of serine in the motif causes the
formation of single chain heterologous fusion proteins. The present invention
encompasses heterologous fusion proteins wherein the IgG4 sequence is further
modified such that serine at position at 228 (EU numbering) is substituted with
proline (amino acid residue 11 in SEQ ID NO:l).
The C-terminal lysine residue present in the native molecule may be
deleted in the IgG4 derivative Fc portion of the heterologous fusion proteins
discussed herein (position 230 of SEQ ID NO:l; deleted lysine referred to as des-
K). Heterologous fusion proteins expressed in some cell types (such as NSO
cells) wherein lysine is encoded by the C-terminal codon are heterogeneous in
that a portion of the molecules have lysine as the C-terminal amino acid and a
portion have lysine deleted. The deletion is due to protease action during
expression in some types of mammalian cells. Thus, to avoid this heterogeneity,
it is preferred that heterologous fusion expression constructs lack a C-terminal
codon for lysine.
It is preferred that the C-terminal amino acid of the active therapeutic
peptide portion is fused to the N-terminus of the IgG4 Fc analog portion via a
glycine-rich linker. The in vivo function and stability of the heterologous fusion
proteins of the present invention can be optimized by adding small peptide
linkers to prevent potentially unwanted domain interactions. Further, a glycine-
rich linker provides some structural flexibility such that the active therapeutic
peptide portion can interact productively with its receptor on target cells. These
linkers, however, can significantly increase the risk that the heterologous fusion
protein will be immunogenic in vivo. Thus, it is preferred that the length be no
longer than necessary to prevent unwanted domain interactions and/or optimize
biological activity and/or stability. The preferred glycine-rich linker comprises
the sequence: Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser
(SEQ ID NO:2). Although more copies of this linker may be used in the
heterologous fusion proteins of the present invention, it is preferred that a single
copy of thislinker be used to minimize the risk of immunogenicity associated
with prolonged and repeated administration.
An active therapeutic peptide can be, without limitation, an enzyme, an enzyme
inhibitor, an antigen, an antibody, a hormone, a factor involved in the control of
coagulation, an interferon, a cytokine, a growth factor and/or differentiation factor, a
factor involved in the genesis/resorption of bone tissues, a factor involved in cellular
motility or migration, a bactericidal or antifungal factor, a chemotactic factor, a cytostatic
factor, a plasma or interstitial adhesive molecule or extracellular matrices, or alternatively
any peptide sequence which is an antagonist or agonist of molecular and/or intercellular
interactions involved in the pathologies of the circulatory and interstitial compartments
and for example the formation of arterial and venous thrombi, cancerous metastases,
tumor angiogenesis, inflammatory shock, autoimmune diseases, bone and osteoarticular
pathologies and the like. Examples of active therapeutic peptides include, but are not
limited to, G-CSF, GM-CSF, eosinophil (EOS)-CSF, macrophage (M)-CSF, multi-CSF,
erythropoietin (EPO), IL-1, IL-2, IL-4, IL-6, IL-7 IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,
IL-18, c-kit ligand, fibroblast growth factor (FGF) 21, Stem-cell factor (SCF), mast cell
growth factor, erythroid potentiating activity (EPA), Lactoferrin (LF), H- subunit ferritin
(i.e., acidic isoferritin), prostaglandin (PG) El and E2, tumor necrosis factor (TNF)-a, -ß
(i.e. lymphotoxin), interferon (IFN)-a (lb, 2a and 2b), -ß, - ? and -?; transforming growth
factor (TGF)-ß, activin, inhibin, leukemic inhibitory factor, oncostatin M, macrophage
inflammatory protein (MIP) -1-a (i.e. Stem-cell inhibitor), macrophage inflammatory
protein (MIP) -ß, macrophage inflammatory protein (MIP)-2-a (i.e., GRO-ß), GRO-a.
MIP-2-ß (i.e., GRO-?), platelet factor-4, macrophage chemotactic and activating factor.
IP-10, Calcitonin, Growth hormone, PTH, TR6, BLyS, BLyS single chain antibody,
Resistin, Growth hormone releasing factor, VEGF-2, KGF-2, D- SLAM, KDI, TR2.
Glucagon-like Peptide-1 (GLP-1). Excndin 4, and neuropeptide pituitary adenylate
cyclase-activating polypeptide (PACAP), or one of its receptors PAC-1, VPAC-l or
VPAC-2, or active analogs, fragments, or derivatives of any of the before mentioned
peptides.
The nomenclature used herein to Tefer to specific heterologous fusion proteins is
defined as follows: L refers to a linker with the sequence Gly-Gly-Gly-Gly-Ser-Gly-Gly-
Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:2). The number immediately preceding
the L refers to the number of linkers separating the active therapeutic peptide portion
from the Fc portion. A linker specified as 1.5L refers to the sequence Gly-Ser-Gly-Gly-
GIy-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser(SEQ ID
NO:4). A linker specified as 2L refers to the sequence Gly-Gly-Gly-Gly-Ser-Gly-Gly-
Gly-Gly-Ser-Gly-GIy-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-
Gly-Gly-Ser (SEQ ID NO:6). IgG4 refers to an analog of the human IgG4 Fc sequence
specified as SEQ ID NO:l. Substitutions in the IgG4 Fc portion of the heterologous
fusion protein are indicated in parenthesis. The wild-type amino acid is specified by its
common abbreviation followed by the position number in the context of the entire IgG4
sequence using the EU numbering system followed by the amino acid being substituted at
that position specified by its common abbreviation.
Although the heterologous fusion proteins of the present invention can be made by
a variety of different methods, because of the size of the heterologous fusion protein,
recombinant methods are preferred. For purposes of the present invention, as disclosed
and claimed herein, the following general molecular biology terms and abbreviations are
defined below.
"Base pair" or "bp" as used herein refers to DNA or RNA. The abbreviations
A,C,G, and T correspond to the 5'-monophosphate forms of the deoxyribonucleosides
(deoxy)adenosine, (deoxy)cytidine, (deoxy)guanosine, and thymidine, respectively, when
they occur in DNA molecules. The abbreviations U,C,G, and A correspond to the 5'-
monophosphate forms of the ribonucleosides uridine, cytidine, guanosine, and adenosine,
respectively when they occur in RNA molecules, in double stranded DNA, base pair may
refer to a partnership of A with T or C with G. In a DNA/RNA, heteroduplex base pair
may refer to a partnership of A with U or C with G. (See the definition of
"complementary"', infra.)
"Digestion" or "Restriction" of DNA refers to the catalytic cleavage of the DNA
with a restriction enzyme that acts only at certain sequences in the DNA ("sequence-
specific endonucleases"). The various restriction enzymes used herein are commercially
available and their reaction conditions, cofactors, and other requirements were used as
would be known to one of ordinary skill in the art. Appropriate buffers and substrate
amounts for particular restriction enzymes are specified by the manufacturer or can be
readily found in the literature.
"Ligation" refers to the process of forming phosphodiester bonds between two
double stranded nucleic acid fragments. Unless otherwise provided, ligation may be
accomplished using known buffers and conditions with a DNA ligase, such as T4 DNA
ligase.
"Plasmid" refers to an extrachromosomal (usually) self-replicating genetic
element.
"Recombinant DNA cloning vector" as used herein refers to any autonomously
replicating agent, including, but not limited to, plasmids and phages, comprising a DNA
molecule to which one or more additional DNA segments can or have been added.
"Recombinant DNA expression vector" as used herein refers to any recombinant
DNA cloning vector in which a promoter to control transcription of the inserted DNA has
been incorporated.
"Transcription" refers to the process whereby information contained in a
nucleotide sequence of DNA is transferred to a complementary RNA sequence.
"Transfection" refers to the uptake of an expression vector by a host cell whether
or not any coding sequences are, in fact, expressed. Numerous methods of transfection
are known to the ordinarily skilled artisan, for example, calcium phosphate co-
precipitation, liposome transfection, and electroporation. Successful transfection is
generally recognized when any indication of the operation of this vector occurs within the
host cell.
"Transformation" refers to the introduction of DNA into an organism so that the
DNA is replicablc. either as an extrachromosomal element or by chromosomal
integration. Methods of transforming bacterial and eukaryotic hosts are well known in
the art, many of which methods, such as nuclear injection, protoplast fusion or by calcium
treatment using calcium chloride are summarized in J. Sambrook, et al, Molecuiar
Cloning: A Laboratory Manual, (1989). Generally, when introducing DNA into Yeast the
term transformation is used as opposed to the term transfection.
"Translation" as used herein refers to the process whereby the genetic information
of messenger RNA (mRNA) is used to specify and direct the synthesis of a polypeptide
chain.
"Vector" refers to a nucleic acid compound used for the transfection and/or
transformation of cells in gene manipulation bearing polynucleotide sequences
corresponding to appropriate protein molecules which, when combined with appropriate
control sequences, confers specific properties on the host cell to be transfected and/or
transformed. Plasmids, viruses, and bacteriophage are suitable vectors. Artificial vectors
are constructed by cutting and joining DNA molecules from different sources using
restriction enzymes and ligases. The term "vector" as used herein includes Recombinant
DNA cloning vectors and Recombinant DNA expression vectors.
"Complementary" or "Complementarity", as used herein, refers to pairs of bases
(purines and pyrimidines) that associate through hydrogen bonding in a double stranded
nucleic acid. The following base pairs are complementary: guanine and cytosine;
adenine and thymine; and adenine and uracil.
"Primer" refers to a nucleic acid fragment which functions as an initiating
substrate for enzymatic or synthetic elongation.
"Promoter" refers to a DNA sequence which directs transcription of DNA to
RNA.
"Probe" refers to a nucleic acid compound or a fragment, thereof, which
hybridizes with another nucleic acid compound.
"Leader sequence" refers to a sequence of amino acids which can be
enzymatically or chemically removed to produce the desired polypeptide of interest.
"Secretion signal sequence" refers to a sequence of amino acids generally present
at the N-terminal region of a larger polypeptide functioning to initiate association of that
polypeptide with the cell membrane compartments like endoplasmic reticulum and
secretion of that polypeptide through the plasma membrane.
Wild-type human IgG4 proteins can be obtained from a variety of sources. For
example, these proteins can be obtained from a cDNA library prepared from cells which
express the mRNA of interest at a detectable level. Libraries can be screened with probes
designed using the published DNA or protein sequence for the particular protein of
interest. For example, immunoglobulin light or heavy chain constant regions are
described in Adams, et al. (1980) Biochemistry 19:2711-2719; Goughet, et al. (1980)
Biochemistry 19:2702-2710; Dolby, et al. (1980) Proc. Natl. Acad. Sci. USA 77:6027-
6031; Riceetal. (1982) Proc. Natl. Acad. Sci. USA 79:7862-7862; Falkner, et al. (1982)
Nature 298:286-288; and Morrison, etal. (1984) Ann. Rev. Immunol. 2:239-256.
Screening a cDNA or genomic library with the selected probe may be conducted
using standard procedures, such as described in Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, NY (1989). An alternative
means to isolate a gene encoding an immunoglobulin protein is to use PCR methodology
[Sambrook et al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, NY (1995)]. PCR primers can be designed based on
published sequences.
Generally the full-length wild-type sequences cloned from a particular library can
serve as a template to create the IgG4 Fc analog fragments of the present invention that
retain the ability to confer a longer plasma half-life on the active therapeutic peptide that
is part of the heterologous fusion protein. The IgG4 Fc analog fragments can be
generated using PCR techniques with primers designed to hybridize to sequences
corresponding to the desired ends of the fragment. PCR primers can also be designed to
create restriction enzyme sites to facilitate cloning into expression vectors.
DNA encoding the active therapeutic peptides of the present invention can be
made by a variety of different methods including cloning methods like those described
above as well as chemically synthesized DNA. Chemical synthesis may be attractive
given the short length of the encoded peptide. The amino acid sequence for the active
therapeutic peptides are generally known and published [Lopez, et al. (1983) Proc. Natl.
Acad. Sci., USA 80:5485-5489; Bell, etal. (1983) Nature, 302:716-718; Heinrich, G., et
al. (1984) Endocrinol, 115:2176-2181; Ghiglione, M., etal. (1984) Diabetologia 27:599-
600].
The gene encoding a heterologous fusion protein can then be constructed by
ligating DNA encoding an active therapeutic protein in-frame to DNA encoding the IgG
Fc proteins described herein. The DNA encoding an active therapeutic protein and IgG4
Fc fragments can be mutated either before ligation or in the context of a cDNA encoding
an entire heterologous fusion protein. A variety of mutagenesis techniques are well
known in the art. The gene encoding the active therapeutic protein and the gene encoding
the IgG4 Fc analog protein can also be joined in-frame via DNA encoding a G-rich linker
peptide. An example of a DNA sequence encoding one of the heterologous fusion
proteins of the present invention, Gly8-Glu22-Gly36-GLP-l(7-37)-lL-IgG4 (S228P,
F234A, L235A, des K), is provided as SEQ ID NO:5:
CACGGCGAGGGCACCTTCACCTCCGACGTGTCCTCCTATCTCGAGGAGCAGG
CCGCCAAGGAATTCATCGCCTGGCTGGTGAAGGGCGGCGGCGGTGGTGGTGG
CTCCGGAGGCGGCGGCTCTGGTGGCGGTGGCAGCGCTGAGTCCAAATATGGT
CCCCCATGCCCACCCTGCCCAGCACCTGAGGCCGCCGGGGGACCATCAGTCTT
CCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGG
TCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAA
CTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAG
GAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA
GGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTC
CCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGC
CACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGT
CAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGT
GGGAAAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCT
GGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGC
AGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGC
ACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGT (SEQ ID NO:5)
Host cells are transfected or transformed with expression or cloning vectors described
herein for heterologous fusion protein production and cultured in conventional nutrient media
modified as appropriate for inducing promoters, selecting transformants, or amplifying the
genes encoding the desired sequences. The culture conditions, such as media, temperature.
pH and the like, can be selected by the skilled artisan without undue experimentation. In
general, principles, protocols, and practical techniques for maximizing the productivity of
cell cultures can be found in Mammalian Cell Biotechnology: A Practical Approach. M.
Butler, ed. (IRL Press, 1991) and Sambrook, et al., supra. Methods of transfection are
known to the ordinarily skilled artisan, for example, CaPO4 and electroporation. General
aspects of mammalian cell host system transformations have been described in U.S. Patent
No. 4,399,216. Transformations into yeast are typically carried out according to the method
of van Solingen et al., J Bad. 130(2): 946-7 (1977) and Hsiao et al., Proc. Natl. Acad. Sci.
USA 76(8): 3829-33 (1979). However, other methods for introducing DNA into cells, such
as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or
polycations, e.g., polybrene or polyomithine, may also be used. For various techniques for
transforming mammalian cells, see Keown, et al., Methods in Enzymology 185: 527-37
(1990) and Mansour, et al., Nature 336(6197): 348-52 (1988).
Suitable host cells for cloning or expressing the nucleic acid (e.g., DNA) in the
vectors herein include yeast or higher eukaryote cells.
Eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or
expression hosts for heterologous fusion protein vectors. Saccharomyces cerevisiae is a
commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces
pombe [Beach and Nurse, Nature 290: 140-3 (1981); EP 139,383 published 2 May 1995];
Muyveromyces hosts [U.S. Patent No. 4,943,529; Fleer, et al., Bio/Technology 9(10): 968-75
(1991)] such as, e.g., K lactis (MW98-8C, CBS683, CBS4574) [de Louvencourt et al., J.
Bacterial. 154(2): 737-42 (1983)]; K. fiagilis (ATCC 12,424), K. bulgaricus (ATCC 16,045),
K wickeramii (ATCC 24,178), K waltii (ATCC 56,500), K. drosophilarum (ATCC 36.906)
[Van den Berg et al., Bio/Technology 8(2): 135-9 (1990)]; K. thermotoierans, and K.
marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070) [Sreekrishna et al., J. Basic
Microbiol. 28(4): 265-78 (1988)]; Candid; Trichoderma reesia (EP 244,234); Neurospora
crassa [Case, et al., Proc. Natl. Acad Sci. USA 76(10): 5259-63 (1979)]; Schwanniomyces
such as Schwanniomyces occidentulis (EP 394,538 published 31 October 1990); and
filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357
published 10 January 1991), and Aspergillus hosts such as A. nidulans [Ballance et al.,
Biochem. Biophys. Res. Comm. 112(1): 284-9 (1983)]; Tilburn, et al, Gene 26(2-3): 205-21
(1983); Yelton, el al.. Proc. Natl. Acad. Sci. USA 81(5): 1470-4 (1984)] and A. niger [Kelly
and Hynes. EMBO J. 4(2): 475-9 (1985)]. Methylotropic yeasts are selected from the genera
consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and
Rhodotoruia. A list of specific species that are exemplary of this class of yeast may be found
in C. Antony, The Biochemistry of Methylotrophs 269 (1982).
Suitable host cells for the expression of the heterologous fusion proteins of the
present invention are derived from multicellular organisms. Examples of invertebrate cells
include insect cells such as Drosophila S2 and Spodoptera Sp, Spodoptera High5 as well as
plant cells. Examples of useful mammalian host cell lines include NSO myeloma cells,
Chinese hamster ovary (CHO), SP2, and COS cells. More specific examples include monkey
kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney
line [293 or 293 cells subcloned for growth in suspension culture, Graham, et al., J. Gen
Virol., 36(1): 59-74 (1977)]; Chinese hamster ovary cellsADHFR [CHO, Urlaub and Chasin,
Proc. Natl. Acad. Sci. USA, 77(7): 4216-20 (1980)]; mouse Sertoli cells [TM4, Mather, Biol.
Reprod. 23(l):243-52 (1980)]; human lung cells (W138. ATCC CCL 75); human liver cells
(Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCC CCL51). A
preferred cell line for production of the heterologous fusion proteins of the present invention
is the NSO myeloma cell line available from the European Collection of Cell Cultures
(ECACC, catalog #85110503) and described in Galfre, G. and Milstein, C. ((1981) Methods
in Enzymology 73(13):3-46; and Preparation of Monoclonal Antibodies: Strategies and
Procedures, Academic Press, N.Y., N.Y.).
The heterologous fusion proteins of the present invention may be recombinantly
produced directly, or as a protein having a signal sequence or other additional sequences
which create a specific cleavage site at the N-terminus of the mature heterologous fusion
protein. In general, the signal sequence may be a component of the vector, or it may be a
part of the heterologous fusion protein-encoding DNA that is inserted into the vector. For
yeast secretion the signal sequence may be, e.g., the yeast invertase leader, alpha factor
leader (including Saccharomyces and Kluyveromyces cc-factor leaders, the latter described
in U.S. Patent No. 5,010,182), or acid phosphatase leader, the C. albicans glucoamylase
leader (EP 362,179), or the signal described in WO 90/13646. In mammalian cell
expression, mammalian signal sequences may be used to direct secretion of the protein, such
as signal sequences from secreted polypeptides of the same or related species as well as viral
secretory leaders.
Both expression and cloning vectors contain a nucleic acid sequence that enables the
vector to replicate in one or more selected host cells. Expression and cloning vectors will
typically contain a selection gene, also termed a selectable marker. Typical selection genes
encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., neomycin,
methotrexate, or tetracycline, (b) complement autotrophic deficiencies, or (c) supply critical
nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for
Bacilli.
An example of suitable selectable markers for mammalian cells are those that enable
the identification of cells competent to take up the heterologous fusion protein-encoding
nucleic acid, such as DHFR or thymidine kinase. An appropriate host cell when wild-type
DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated
as described [Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77(7): 4216-20 (1980)]. A
suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7
[Stinchcomb, et al. Nature 282(5734): 39-43 (1979); Kingsman, et al., Gene 7(2): 141-52
(1979); Tschumper, et al., Gene 10(2): 157-66 (1980)]. The trpl gene provides a selection
marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example,
ATCC No. 44076 or PEPC1 [Jones, Genetics 85: 23-33 (1977)].
Expression and cloning vectors usually contain a promoter operably linked to the
heterologous fusion protein-encoding nucleic acid sequence to direct mRNA synthesis.
Promoters recognized by a variety of potential host cells are well known. Examples of
suitable promoting sequences for use with yeast hosts include the promoters for 3-
phosphoglycerate kinase [Hitzeman, et al., J. Biol. Chem. 255(24): 12073-80 (1980)] or other
glycolytic enzymes [Hess et al., J. Adv. Enzyme Reg. 7: 149 (1968); Holland, Biochemistry
17(23): 4900-7 (1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,
hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose
isomerase, and glucokinase. Other yeast promoters, which are inducible promoters having
the additional advantage of transcription controlled by growth conditions, are the promoter
regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative
enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate
dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable
vectors and promoters for use in yeast expression are further described in EP 73,657.
Transcription of heterologous fusion protein-encoding mRNA from vectors in mammalian
host cells may be controlled, for example, by promoters obtained from the genomes of
viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine
papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and
Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter
or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters
are compatible with the host cell systems.
Transcription of a polynucleotide encoding a heterologous fusion protein by higher
eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers
are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to
increase its transcription. Many enhancer sequences are now known from mammalian genes
(globin, elastase, albumin, a-ketoprotein, and insulin). Typically, however, one will use an
enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side
of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the
polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The
enhancer may be spliced into the vector at a position 5' or 3' to the heterologous fusion
protein coding sequence but is preferably located at a site 5' from the promoter.
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal,
human, or nucleated cells from other multicellular organisms) will also contain sequences
necessary for the termination of transcription and for stabilizing the mRNA. Such sequences
are commonly available from the 5' and occasionally 3' untranslated regions of eukaryotic or
viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as
polyadenylated fragments in the untranslated portion of the mRNA encoding the
heterologous fusion protein.
Various forms of a heterologous fusion protein may be recovered from culture
medium or from host cell lysates. If membrane-bound, it can be released from the membrane
using a suitable detergent solution (e.g., Triton-X 100) or by enzymatic cleavage. Cells
employed in expression of a heterologous fusion protein can be disrupted by various physical
or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell
lysing agents.
Once the heterologous fusion proteins of the present invention are expressed in the
appropriate host cell, the analogs can be isolated and purified. The following procedures are
exemplary of suitable purification procedures: fractionation on carboxymethyl cellulose; gel
filtration such as Sephadex G-75; anion exchange resin such as DEAE or Mono-Q; cation
exchange such as CM or Mono-S; metal chelating columns to bind epitope-tagged forms of
the polypeptide: reversed-phase HPLC; chromatofocusing; silica gel; ethanol precipitation;
and ammonium sulfate precipitation.
Various methods of protein purification may be employed and such methods are
known in the art and described, for example, in Deutscher, Methods in Enzymology 182:
83-9 (1990) and Scopes, Protein Purification: Principles and Practice, Springer-Verlag,
NY (1982). The purification step(s) selected will depend on the nature of the production
process used and the particular heterologous fusion protein produced. For example,
heterologous fusion proteins comprising an Fc fragment can be effectively purified using
a Protein A or Protein G affinity matrix. Low or high pH buffers can be used to elute the
heterologous fusion protein from the affinity matrix. Mild elution conditions will aid in
preventing irreversible denaturation of the heterologous fusion protein.
The heterologous fusion proteins of the present invention may be formulated with one
or more excipients. The heterologous fusion proteins of the present invention may be
combined with a pharmaceutically acceptable buffer, and the pH adjusted to provide
acceptable stability, and a pH acceptable for administration such as parenteral administration.
Optionally, one or more pharmaceutically-acceptable anti-microbial agents may be added.
Meta-cresol and phenol are preferred pharmaceutically-acceptable microbial agents. One or
more pharmaceutically-acceptable salts may be added to adjust the ionic strength or tonicity.
One or more excipients may be added to further adjust the isotonicity of the formulation.
Glycerin is an example of an isotonicity-adjusting excipient. Pharmaceutically acceptable
means suitable for administration to a human or other animal and thus, does not contain toxic
elements or undesirable contaminants and does not interfere with the activity of the active
compounds therein.
The heterologous fusion proteins of the present invention may be formulated as a
solution formulation or as a lyophilized powder that can be reconstituted with an appropriate
diluent. A lyophilized dosage form is one in which the heterologous fusion protein is stable,
with or without buffering capacity to maintain the pH of the solution over the intended in-use
shelf-life of the reconstituted product. It is preferable that the solution comprising the
heterologous fusion proteins discussed herein before lyphilization be substantially isotonic to
enable formation of isotonic solutions after reconstitution.
A pharmaceutically-acceptable salt form of the heterologous fusion proteins of the
present invention are within the scope of the invention. Acids commonly employed to form
acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydriodic
acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as P-toluenesulfonic
acid, methanesulfonic acid, oxalic acid, p-bromophenyl-sulfonic acid, carbonic acid, succinic
acid, citric acid, benzoic acid, acetic acid, and the like. Preferred acid addition salts are those
formed with mineral acids such as hydrochloric acid and hydrobromic acid.
Base addition salts include those derived from inorganic bases, such as
ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the
like. Such bases useful in preparing the salts of this invention thus include sodium
hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, and the
like.
The heterologous fusion proteins of the present invention have biological activity.
Biological activity refers to the ability of the heterologous fusion protein to bind to and
activate a receptor in vivo and elicit a response. A representative number of heterologous
fusion proteins were tested for in vitro as well as in vivo activity. Examples 1 and 2 provide
a representation of in vitro activity based on the ability of the heterologous fusion protein to
interact with and activate the human GLP-1 receptor. In both sets of experiments, HEK293
cells over-expressing the human GLP-1 receptor are used. Activation of the GLP-1 receptor
in these cells causes adenylyl cyclase activation which in turn induces expression of a
reporter gene driven by a cyclic AMP response element (CRE). Example 1 (table 1) provides
representative data wherein the reporter gene is beta lactamase, and example 2 (table 2)
provides representative data wherein the reporter gene is luciferase. Example 3 provides
representative data generated after administration of a heterologous fusion proteins of the
present invention to rats. Example 4 (table 6) provides representative data generated after
administration of a heterologous fusion proteins of the present invention to monkeys.
Example 5 (table 7) provides representative data of the assessment of the potential formation
of antibodies following repeat subcutanesous injections of a heterologous fusion protein.
Example 6 (table 8) provides representative data from a pharmacodynamic study following
an injection of a heterologous fusion protein to monkey. Example 7 (table 9) provides
representative data from a pharmacodynamic study following injections of three different
doses to rats. Example 8 (table 10) provides representative data generated after
administration of a different heterologous fusion proteins of the present invention to mice.
Together the representative data show that the heterologous fusion proteins are able to bind
to and activate their receptor, appear more potent than the active therapeutic peptide, are
active in vivo and have a longer half-life than the active therapeutic peptide, are not
immunogenic, and are dose responsive.
Administration of the heterologous fusion proteins may be via any route known to be
effective by the physician of ordinary skill. Peripheral parenteral is one such method.
Parenteral administration is commonly understood in the medical literature as the injection of
a dosage form into the body by a sterile syringe or some other mechanical device such as an
infusion pump. Peripheral parenteral routes can include intravenous, intramuscular,
subcutaneous, and intraperitoneal routes of administration.
The heterologous fusion proteins of the present invention may also be amenable to
administration by oral, rectal, nasal, or lower respiratory routes, which are non-parenteral
routes. Of these non-parenteral routes, the lower respiratory route and the oral route are
preferred.
The heterologous fusion proteins of the present invention can be used to treat a wide
variety of diseases and conditions.
An effective amount of the heterologous fusion proteins described herein is the
quantity which results in a desired therapeutic and/or prophylactic effect without causing
unacceptable side-effects when administered to a subject in need of the active therapeutic
peptide receptor stimulation. A "desired therapeutic effect" includes one or more of the
following: 1) an amelioration of the symptom(s) associated with the disease or condition;
2) a delay in the onset of symptoms associated with the disease or condition; 3) increased
longevity compared with the absence of the treatment; and 4) greater quality of life
compared with the absence of the treatment.
It is preferable that the heterologous fusion proteins of the present invention be
administered either once every two weeks or once a week. Depending on the disease
being treated, it may be necessary to administer the heterologous fusion protein more
frequently such as two to three time per week.
The present invention will now be described only by way of non-limiting example
with reference to the following Examples.
EXAMPLES
Example 1 - In vitro GLP-1 receptor activation assay
HEK-293 cells expressing the human GLP-1 receptor, using a CRE-BLAM
system, are seeded at 20,000 to 40,000 cells/well/100 µl DMEM medium with 10%FBS
into a poly-d-lysine coated 96 well black, clear-bottom plate. The day after seeding, the
medium is flicked off and 80 µl plasma-free DMEM medium is added. On the third day
after seeding, 20 µl of plasma-free DMEM medium with 0.5% BSA containing different
concentrations of various GLP-1-Fc heterologous fusion protein is added to each well to
generate a dose response curve. Generally, fourteen dilutions containing from 3
nanomolar to 30 nanomolar or heterologous GLP-1 Fc fusion protein are used to generate
a dose response curve from which EC50 values can be determined. After 5 hours of
incubation with the fusion protein, 20 µl of ß-lactamase substrate (CCF2/AM, PanVera
LLC) is added and incubation continued for 1 hour at which time fluorescence is
determined on a cytofluor. The assay is further described in Zlokarnik, et al. (1998),
Science, 278:84-88. Various GLP-l-Fc fusion proteins are tested and EC50 values are
represented in Table 1. The values are relative to values determined for Val8-GLP-1(7-
37)OH which is run as an internal control with every experiment.

Example 2 - In vitro GLP-1 receptor activation assay
HEK-293 cells stably expressing the human GLP-1 receptor, using a CRE-
Luciferase system, are seeded at 30,000 cells/well/80 µl low serum DMEM F12 medium
into 96 well plates. The day after seeding, 20 µl aliquots of test protein dissolved in 0.5%
BSA are mixed and incubated with the cells for 5 hours. Generally 12 dilutions
containing from 3 pM to 3 nM are prepared at a 5X concentration for each test protein
before addilion to the cells to generate a dose response curve from which EC50 values are
determined. After incubation, 100 µl of Luciferase reagent is added directly to each plate
and mixed gently for 2 minutes. Plates are placed in a Tri-lux luminometer and light
output resulting from luciferase expression is calculated. Various GLP-l-Fc fusion
proteins are tested and EC50 values are represented in Table 2. The values are relative to
values determined for Val8-GLP-l(7-37)OH which is run as an internal control with every
experiment. Because the heterologous fusion proteins tested below are dimers, values are
corrected taking into account a 2-fold difference in molarity.

Example 3 Intravenous Glucose Tolerance Test in Rats
The heterologous fusion protein, Gly8-Glu22-Gly36-GLP-l(7-37)-L-IgG4
(S228P,F234A,L235A), is evaluated in an intravenous glucose tolerance test (IVGTT) in
rats. At least four rats are included into each of three groups. Group I receives vehicle
(table 3), Group II receives 1.79 mg/kg of Gly8-Glu22-Gly36-GLP-l(7-37)-L-IgG4
(S228P,F234A,L235A) as a single subcutaneous injection (table 4), and Group III
receives 0.179 mg/kg of Gly8-Glu22-Gly36-GLP-l(7-37)-L-lgG4 (S228P,F234A,L235A)
as a single subcutaneous injection (table 5). Rats are subcutaneously injected the morning
of Day 1. Twenty-four hours following the first injection, 1 µL of glucose (D50) per
gram rat body weight is infused as a bolus. Blood samples are taken at 2, 4, 6, 10, 20, and
30 minutes following the bolus infusion of glucose.
Example 4 Pharmacokinetic Study Following a Single Subcutaneous Injection to
Cynomolgus Monkeys.
A study is performed to characterize the pharmacokinetics (PK) of the
heterologous fusion protein, Gly8-Glu22-Gly36-GLP-l(7-37)-L-IgG4
(S228P,F234A,L235A), when administered as a 0.1 mg/kg by subcutaneous (SC)
injection to male cynomolgus monkeys. R1A antibody is specific for the middle portion
of GLP. ELISA uses an N-terminus specific capture antibody and an Fc specific
detection antibody.
Resulting plasma concentrations from both the ELISA and the RLA are used to determine
the represented pharmacokinetic parameter values.
A representation of the resulting PK parameter values is summarized in table 6.
Single-dose SC PK from the RLA is associated with a mean Cmax of 446.7 ng/mL with a
corresponding Tmax of 17.3 hours. The mean elimination half-life is approximately 79.3
hours (3.3 days). The PK from the ELISA is associated with a mean Cmax of 292.2 ng/mL
with a corresponding Tmax of 16.7 hours. The mean elimination half-life is approximately
51.6 hours (2.2 days).
Example 5 Assessment of the potential formation of antibodies following repeat
subcutanesous injections.
Designated serum samples from cynomolgus monkeys are tested for the formation
of antibodies against Gly8-Glu22-Gly36-GLP-l(7-37)-L-IgG4 (S228P,F234A,L235A)
using a direct ELISA format. . Microtiter plates are coated with Gly8-Glu22-Gly36-GLP-
l(7-37)-L-IgG4 (S228P,F234A,L235A) at a 0.1 ng/mL concentration. Monkey serum
samples are diluted 50, 500,1000 and 5000 fold into blocking solution, and 0.05 mL
sample/well are incubated approximately one hour. Secondary antibody, Goat Fab'2>-Peroxidase (with 75% cross reactivity to human), is diluted 10,000 fold into block
and added at 0.05 mL/well and incubated approximately one hour. Color development
using tetramethylbenzidine (TMB) substrate is read at an optical density of 450nm -
630nm. Duplicate readings are averaged. A GLP-1 antibody was used as a positive
control and goat(H+L)-Peroxidase conjugate is the secondary used for detection.
Point serum samples are collected prior to dosing, at 24 hours following the second dose,
and 168 hours following the first and second SC dose for an evaluation of potential
immunogenicity. The presence of antibody titers to G8E22-CEX-L-hIgG4 is interpreted
by comparison to predose serum samples and positive control. A representation of the
results is presented in table 7.
Example 6 Pharmacodynamic Study Following a Single Subcutaneously Injection to
Cynomolgus Monkeys in the Fasting State and During a Graded Intravenous Glucose
Infusion.
In Phase 1 (Study Day 1) a subcutaneous injection of vehicle is administered. A
graded intravenous glucose (20% dextrose) infusion of 5, 10, and 25 mg/kg/min is then
administered immediately after the vehicle injection. In Phase 2 (Study Day 3), a
subcutaneous injection of a GLP-1 fusion protein (0.1 mg/kg) is administered. In Phase
3,a graded intravenous glucose infusion is performed approximately 96 hours following
the GLP-1 fusion injection.
Graded intravenous glucose infusion procedures are conducted in sedated
monkeys after a 16-hr overnight fast. For both intravenous glucose infusions, baseline
samples will be drawn every 10 min for 20 min to define baseline. A stepped-up glucose
infusion is initiated at +20 min at a rate of 5 mg/kg/min, followed by infusions of 10
mg/kg/min, and 25 mg/kg/min. Each infusion rate is administered for a period of 20
minutes. Blood samples are taken at 10 minute intervals for measurement of glucose,
insulin, and glucagon. Approximately 1.0 mL of blood is collected at -20, -10 min, 0 pre-
glucose infusions, and at 10, 20, 30, 40, 50, and 60 minutes following glucose infusion
for Phases 1 and 3.
A representation of the data are shown in table 8.
Example 7 Pharmacodynamic Study Following Single Subcutaneously Injections of Three
Different Doses to Rats in the Fasting State and During a Graded Intravenous Glucose
Infusion.
Chronically cannulated rats are assigned to either vehicle control (saline) or one of
3 treatment groups (GLP-1 fusion protein; 0.0179 mg/kg, 0.179 mg/kg, or 1.79 mg/kg).
The GLP-1 fusion protein and vehicle are administered via subcutaneous injection.
Twenty-four hours after treatment, overnight fasted (16h) rats are subjected to a graded
intravenous glucose infusion test. The graded glucose infusion test consists of a baseline
saline infusion period (20 min), followed by two 30 min glucose infusion phases at 5 and
15 mg/kg/min, respectively. Plasma samples are collected at -20, -10 min, 0 pre-glucose
infusions (baseline), and at 10, 20, 30, 40, 50, and 60 minutes.
Example 8 Pharmacokinetic analysis of FGF-21 Fusion Protein
FGF-21 fusion proteins are administered by intravenous (IV) or subcutaneous
(SC) routes at a dose of 0.4 mg/kg to CD-I mice. The animals are bled at various times
between 0 and 336 hours after dosing. Plasma is collected from each sample and
analyzed by radioimmunoassay. Pharmacokinetic parameters are calculated using model-
dependent (IV data) and independent (SC data) methods (WinNonlin Pro) and are
reported in table 10 below. By IV administration, the FGF-21-Fc fusion protein has an
elimination half-life of approximately 53.9 hours compared to an elimination half-life of
0.5 hours for native FGF-21. By SC administration the FGF-21-Fc fusion protein has an
elimination half-life of approximately 24 hours compared to an elimination half-life of
0.6 hours for native FGF-21. By both routes of administration the FGF-21-Fc fusion
protein demonstrates prolonged time action when compared to native FGF-21.
1. A heterologous fusion protein comprising an active therapeutic peptide
fused to the Fc portion of an immunoglobulin, the Fc portion comprising the
sequence of SEQ ID NO: 1
Xaa1-Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro-Cys-Pro-Ala-Pro-
Xaa16-Xaa17-Xaa18-Gly-Gly-Pro-Ser-Val-Phe-Leu-Phe-Pro-Pro-Lys-Pro-
Lys-Asp-Thr-Leu-Met-Ile-Ser-Arg-Thr-Pro-Glu-Val-Thr-Cys-Val-
Val-Val-Asp-Val-Ser-Gln-Glu-Asp-Pro-Glu-Val-Gln-Phe-Asn-Trp-
Tyr-Val-Asp-Gly-Val-Glu-Val-His-Asn-Ala-Lys-Thr-Lys-Pro-Arg-
Glu-Glu-Gln-Phe-Xaa80-Ser-Thr-Tyr-Arg-Val-Val-Ser-Val-Leu-Thr-
Val-Leu-His-Gln-Asp-Trp-Leu-Asn-Gly-Lys-Glu-Tyr-Lys-Cys-Lys-
Val-Ser-Asn-Lys-Gly-Leu-Pro-Ser-Ser-Ile-Glu-Lys-Thr-Ile-Ser-
Lys-Ala-Lys-Gly-Gln-Pro-Arg-Glu-Pro-Gln-Val-Tyr-Thr-Leu-Pro-
Pro-Ser-Gln-Glu-Glu-Met-Thr-Lys-Asn-Gln-Val-Ser-Leu-Thr-Cys-
Leu-Val-I.ys-Gly-Phe-Tyr-Pro-Ser-Asp-Ile-Ala-Val-Glu-Trp-Glu-
Ser-Asn-Gly-Gln-Pro-Glu-Asn-Asn-Tyr-Lys-Thr-Thr-Pro-Pro-Val-
Leu-Asp-Ser-Asp-Gly-Ser-Phe-Phe-Leu-Tyr-Ser-Arg-Leu-Thr-Val-
Asp-Lys-Ser-Arg-Trp-Gln-Glu-Gly-Asn-Val-Phe-Ser-Cys-Ser-Val-
Met-His-Glu-Ala-Leu-His-Asn-His-Tyr-Thr-Gln-Lys-Ser-Leu-Ser-
Leu-Ser-Leu-Gly-Xaa230 (SEQ ID NO : 1)
wherein:
Xaa at position 1 is Ala or absent;
Xaa at position 16 is Pro or Glu;
Xaa at position 17 is Val, or Ala;
Xaa at position 18 is Ala;
Xaa at position 80 is Asn or Ala; and
Xaa at position 230 is Lys or is absent.
2. The heterologous fusion protein as claimed in claim 1 wherein the C-terminal amino
acid of the active therapeutic peptide is fused to the N-terminal residue of the Fc
portion via a peptide linker comprising a sequence selected from:
a. Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID
NO:2);
b. Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-
Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:4);
c. Gly-Gly-Gly-Gly-Ser-GIy-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-
Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-GIy-Gly-Gly-Gly-Ser (SEQ ID NO:6);
d. Asp-Ala-Ala-Ala-Lys-Glu-Ala-Ala-Ala-Lys-Asp-Ala-Ala-Ala-Arg-Glu-
Ala-Ala-Ala-Arg-Asp-Ala-Ala-AIa-Lys (SEQ ID NO:7); and
e. Asn-Val-Asp-His-Lys-Pro-Ser-Asn-Thr-Lys-Val-Asp-Lys-Arg (SEQ ID NO: 8).
3. The heterologous fusion protein as claimed in claim 1 or claim 2 for use as a
medicament.

The invention provides active therapeutic peptides fused to specific IgG4-Fc derivatives.
These fusion proteins have an increased half-life, reduced half antibody formation, and
reduced effector activity, while not being immunogenic. The fusion proteins are useful in
treating human diseases as well as a variety of other conditions or disorders.

Documents:

2526-kolnp-2005-granted-abstract.pdf

2526-kolnp-2005-granted-assignment.pdf

2526-kolnp-2005-granted-claims.pdf

2526-kolnp-2005-granted-correspondence.pdf

2526-kolnp-2005-granted-description (complete).pdf

2526-kolnp-2005-granted-examination report.pdf

2526-kolnp-2005-granted-form 1.pdf

2526-kolnp-2005-granted-form 13.pdf

2526-kolnp-2005-granted-form 18.pdf

2526-kolnp-2005-granted-form 2.pdf

2526-kolnp-2005-granted-form 26.pdf

2526-kolnp-2005-granted-form 3.pdf

2526-kolnp-2005-granted-form 5.pdf

2526-kolnp-2005-granted-reply to examination report.pdf

2526-kolnp-2005-granted-specification.pdf


Patent Number 233877
Indian Patent Application Number 2526/KOLNP/2005
PG Journal Number 16/2009
Publication Date 17-Apr-2009
Grant Date 16-Apr-2009
Date of Filing 07-Dec-2005
Name of Patentee ELI LILLY AND COMPANY
Applicant Address LILLY CORPORATE CENTER, INDIANAPOLIS, IN
Inventors:
# Inventor's Name Inventor's Address
1 GLAESNER, WOLFANG 7512 FIELDSTONE COURT, INDIANAPOLIS, IN 46254
2 VICK, ANDREW, MARK 10736 GATEWAY DRIVE, FISHERS, IN 46038
3 MILLICAN, ROHN, LEE, JR. 8145 GRASSY MEADOW COURT, INDIANAPOLIS, IN 46259
4 TIAN, YU 13695 FLINTRIDGE PASS, CARMEL, IN 46033
5 TSCHANG, SHENG-HUNG, RAINBOW 4963 RILEY MEWS, CARMEL, IN 46033
PCT International Classification Number A61K 38/00
PCT International Application Number PCT/US2004/016611
PCT International Filing date 2004-06-10
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/477,880 2003-06-12 U.S.A.
2 60/570,908 2004-05-13 U.S.A.