U.S. patent application number 10/407078 was filed with the patent office on 2003-10-16 for use of transthyretin peptide/protein fusions to increase the serum half-life of pharmacologically active peptides/proteins.
Invention is credited to Walker, Kenneth, Xiong, Fei.
Application Number | 20030195154 10/407078 |
Document ID | / |
Family ID | 28674128 |
Filed Date | 2003-10-16 |
United States Patent
Application |
20030195154 |
Kind Code |
A1 |
Walker, Kenneth ; et
al. |
October 16, 2003 |
Use of transthyretin peptide/protein fusions to increase the serum
half-life of pharmacologically active peptides/proteins
Abstract
The present invention provides a means for increasing the serum
half-life of a selected biologically active agent by utilizing
transthyretin (TTR) as a fusion partner with a biologically active
agent. Specifically, the present invention provides substantially
homogenous preparations of TTR (or a TTR variant)-biologically
active agent fusions and PEG-TTR (PEG-TTR variant)-biologically
active agent fusions. As compared to the biologically active agent
alone, the TTR-biologically active agent fusion and/or
PEG-TTR-biologically active agent fusion has substantially
increased serum half-life.
Inventors: |
Walker, Kenneth; (Newbury
Park, CA) ; Xiong, Fei; (Thousand Oaks, CA) |
Correspondence
Address: |
AMGEN INCORPORATED
MAIL STOP 27-4-A
ONE AMGEN CENTER DRIVE
THOUSAND OAKS
CA
91320-1799
US
|
Family ID: |
28674128 |
Appl. No.: |
10/407078 |
Filed: |
April 3, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10407078 |
Apr 3, 2003 |
|
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10117109 |
Apr 4, 2002 |
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Current U.S.
Class: |
514/6.9 ;
514/7.8 |
Current CPC
Class: |
A61P 9/12 20180101; C07K
14/47 20130101; A61K 47/58 20170801; A61K 47/61 20170801; A61K
47/643 20170801; A61P 3/10 20180101; A61K 38/29 20130101; A61K
38/043 20130101; A61K 38/20 20130101; A61K 47/60 20170801; A61P
5/18 20180101; A61P 7/04 20180101; C07K 2319/31 20130101; A61K
38/10 20130101 |
Class at
Publication: |
514/12 |
International
Class: |
A61K 038/00 |
Claims
What is claimed is:
1. A method for increasing the serum half-life of a biologically
active agent comprising fusing the biologically active agent to
transthyretin (TTR) or a TTR variant.
2. The method of claim 1 where said TTR or TTR variant is
chemically modified with a chemical selected from the group
consisting of dextran, poly(n-vinyl pyurrolidone), polyethylene
glycols, propropylene glycol homopolymers, polypropylene
oxide/ethylene oxide co-polymers, polyoxyethylated polyols and
polyvinyl alcohols.
3. The method of claim 2 where said TTR or TTR variant is
chemically modified with polyethylene glycol.
4. The method of claim 3 wherein said polyethylene glycol has a
molecular weight of between about 1 kD and 100 kD.
5. The method of claim 4 wherein said polyethylene glycol has a
molecular weight of between about 5 kD and 30 kD.
6. The method of claim 1 wherein said TTR is encoded by the nucleic
acid of SEQ ID NO:2.
7. The method of claim 1 wherein the TTR variant is encoded by the
nucleic acid of SEQ ID NO:8.
8. The method of claim 1 wherein the biologically active agent is a
protein.
9. The method of claim 1 wherein the biologically active agent is a
peptide.
10. The method of claim 9 wherein the peptide is a TPO mimetic
peptide (TMP).
11. The method of claim 9 wherein the biologically active agent is
a Glucagon-like Peptide-1 (GLP-1).
12. A substantially homogenous preparation of a TTR-biologically
active agent fusion, optionally in a pharmaceutically acceptable
diluent, carrier or adjuvant.
13. A substantially homogenous preparation of a
PEG-TTR-biologically active agent fusion, optionally in a
pharmaceutically acceptable diluent, carrier or adjuvant.
14. The preparation of claim 13 wherein the biologically active
agent is a protein.
15. The preparation of claim 13 wherein the biologically active
agent is a peptide.
16. The preparation of claim 15 wherein the peptide is a TMP.
17. The preparation of claim 15 wherein the peptide is a GLP-1.
18. A substantially homogenous preparation of a TTR
variant-biologically active agent fusion, optionally in a
pharmaceutically acceptable diluent, carrier or adjuvant.
19. A substantially homogenous preparation of a PEG-TTR
variant-biologically active agent fusion, optionally in a
pharmaceutically acceptable diluent, carrier or adjuvant.
20. The preparation of claim 19 wherein the biologically active
agent is a protein.
21. The preparation of claim 19 wherein the biologically active
agent is a peptide.
22. The preparation of claim 21 wherein the peptide is a TMP.
23. The preparation of claim 21, wherein the peptide is GLP-1.
24. The preparation of any of claims 10-23 wherein the fusion
contains a linker peptide.
25. A process for preparing a substantially homogenous preparation
of a TTR-biologically active agent fusion comprising: (a) fusing
said TTR to a biologically active agent to provide a
TTR-biologically active agent fusion; and (b) isolating said
TTR-biologically active agent fusion.
26. A process for preparing a substantially homogenous preparation
of a TTR variant-biologically active agent fusion comprising: (a)
engineering a cysteine residue into a specific amino acid position
within the amino acid sequence of said TTR to provide a variant of
said TTR; (b) fusing said TTR variant to a biologically active
agent to provide a TTR variant-biologically active agent fusion;
and (c) isolating said TTR variant-biologically active agent
fusion.
27. A process for preparing a substantially homogenous preparation
of a PEG-TTR-biologically active agent fusion comprising: (a)
conjugating a polyethylene glycol to said TTR to provide a PEG-TTR;
(b) fusing said PEG-TTR to a biologically active agent to provide a
PEG-TTR-biologically active agent fusion; and (c) isolating said
PEG-TTR-biologically active agent fusion.
28. A process for preparing a substantially homogenous preparation
of a PEG-TTR variant-biologically active agent fusion comprising:
(a) engineering a cysteine residue into a specific amino acid
position within the amino acid sequence of said TTR to provide a
variant of said TTR; (b) conjugating a polyethylene glycol to said
TTR variant at said cysteine residue to provide a PEG-TTR variant;
(c) fusing said PEG-TTR variant to a biologically active agent to
provide a PEG-TTR-biologically active agent fusion; and (d)
isolating said PEG-TTR-biologically active agent fusion.
29. A method of treating thrombocytopenia comprising administering
a therapeutically effective dose of a preparation of claim 16.
30. A method of treating thrombocytopenia comprising administering
a therapeutically effective dose of a preparation of claim 22.
31. A method of treating non-insulin dependent diabetes comprising
administering a therapeutically effective dose of a preparation of
claim 17.
32. A method of treating non-insulin dependent diabetes comprising
administering a therapeutically effective dose of a preparation of
claim 23.
33. A fusion protein comprising a TTR protein fused to a
heterologous sequence.
34. A fusion protein of claim 33 wherein the heterologous sequence
is a TMP.
35. A fusion protein of claim 33 wherein the heterologous sequence
is a GLP-1.
36. A fusion protein of any one of claims 33, 34 or 35 further
comprising a linker sequence between the TTR protein and the
heterologous sequence.
37. A nucleic acid encoding the fusion protein of any one of claims
33, 34 or 35.
Description
[0001] This application is a Continuation in Part of U.S.
application Ser. No. 10/117,109, filed Apr. 4, 2002, which is
hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] Proteins, peptides and other drug molecules for therapeutic
use are currently available in suitable forms in adequate
quantities largely as a result of the advances in recombinant DNA
technologies. The availability of such peptides and proteins has
engendered advances in protein formulation and chemical
modification. Chemical modification of biologically active
peptides, proteins, oligonucleotides and other drugs for purposes
of extending the serum half-life of such bioactive agents has been
extensively studied. The ability to extend the serum half-life of
such agents allows for the therapeutic potential of the agent to be
realized without the need for high dosages and frequent
administration.
[0003] Chemical modification used to extend the half-lives of
proteins in vivo includes the chemical conjugation of a water
soluble polymer, such as polyethylene glycol (PEG), to the protein
of interest. A variety of approaches have been used to attach the
polyethylene glycol molecules to the protein (PEGylation). For
example, Royer (U.S. Pat. No. 4,002,531) states that reductive
alkylation was used for attachment of polyethylene glycol molecules
to an enzyme. Davis et al. (U.S. Pat. No. 4,179,337) disclose
PEG:protein conjugates involving, for example, enzymes and insulin.
Shaw (U.S. Pat. No. 4,904,584) disclose the modification of the
number of lysine residues in proteins for the attachment of
polyethylene glycol molecules via reactive amine groups. Hakimi et
al. (U.S. Pat. No. 5,834,594) disclose substantially
non-immunogenic water soluble PEG:protein conjugates, involving for
example, the proteins IL-2, interferon alpha, and IL-1ra. The
methods of Hakimi et al. involve the utilization of unique linkers
to connect the various free amino groups in the protein to PEG.
Kinstler et al. (U.S. Pat. Nos. 5,824,784 and 5,985,265) teach
methods allowing for selectively N-terminally chemically modified
proteins and analogs thereof, including G-CSF and consensus
interferon.
[0004] Other approaches designed to extend the serum half-life of
bioactive agents include: conjugation of the peptides to a large,
stable protein which is too large to be filtered through the
kidneys (e.g., serum albumin); G. D. Mao et al., Biomat., Art.
Cells, Art. Org. 17:229-244 (1989); use of low- and high-density
lipoproteins as transport vehicles and to increase serum half-life;
P. Chris de Smidt et al., Nuc. Acids. Res., 19(17):4695-4700
(1991); the use of the Fc region of immunoglobulins to produce
Fc-protein fusions; PCT WO 98/28427 (Mann et al, and references
cited therein); and the use of the Fc domain to increase in vivo
half-life of one or more biologically active peptides; PCT WO
00/24782 (Feige et al, and references cited therein).
[0005] Transthyretin (TTR) (formerly called prealbumin) is a 56 kDa
tetrameric serum protein that plays important physiological roles
as a transporter of thyroxine and retinol-binding protein; Hamilton
and Benson, Cell. Mol. Life Sci., 58:1491-1521 (2001), and
references cited therein. Blaney et al., in U.S. Pat. No.
5,714,142, describe the exploitation of TTR by endowing the drug to
be administered with functionality that allows it to bind
specifically to the protein. Specifically, Blaney et al.
demonstrate that covalent attachment of a peptide, protein,
nucleotide, oligonucleotide, oligosaccharide or other drug to a
transthyretin-selective ligand will reversibly bind the drug to TTR
and thereby increase the serum half-life of the agent based on the
affinity of the ligand for TTR. It is stated that the intrinsic
activity of the drug is not adversely affected and the resulting
drug-TTR ligand conjugate will still be small enough to be orally
absorbed.
SUMMARY OF THE INVENTION
[0006] It has been found, surprisingly and importantly, that TTR
(or a TTR variant), and in particular, a TTR or TTR variant which
has been chemically modified via conjugation to a water soluble
polymer, e.g., can be used as a fusion partner with a biologically
active agent to increase the serum half-life of the biologically
active agent. Accordingly, the present invention provides a means
for increasing the serum half-life of a selected biologically
active agent.
[0007] The present invention thus relates to substantially
homogenous preparations of TTR (or a TTR variant)-biologically
active agent fusions and PEG-TTR (PEG-TTR variant)-biologically
active agent fusions. As compared to the biologically active agent
alone, the TTR-biologically active agent fusion and/or
PEG-TTR-biologically active agent fusion has substantially
increased serum half-life.
[0008] The present invention further relates to TTR-biologically
active agent fusions and PEG-TTR-biologically active agent fusions,
in a pharmaceutically acceptable carrier, to provide a
pharmacologically active compound.
[0009] The present invention further relates to the preparation of
TTR variants. Specifically, TTR proteins are modified such that
cysteine residue(s) are engineered into the TTR protein sequence.
The TTR variants are recoverable in high yield and are then
chemically modified via conjugation of a water soluble polymer at
the cysteine residue to provide a chemically modified TTR variant
which can then be fused to a selected biologically active
agent.
[0010] The present invention further relates to processes for
preparing pharmacologically active compounds. For example, the
principal embodiment of the method for making the substantially
homogenous preparation of a PEG-TTR-peptide fusion comprises: (a)
engineering a cysteine residue into a specific amino acid position
within the amino acid sequence of said TTR to provide a variant of
said TTR; (b) conjugating a polyethylene glycol to said TTR variant
at said cysteine residue to provide a PEG-TTR; (c) fusing said
PEG-TTR to a peptide of interest to provide a PEG-TTR-peptide
fusion; and (d) isolating said PEG-TTR-peptide fusion.
[0011] The present invention also relates to methods of treatment
of individuals using the pharmacologically active compounds as
above.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is an SDS gel that depicts the purification of an E.
coli expressed, recombinant human transthyretin (TTR) variant
(C10A/G83C) with a Bradykinin peptide fused to the C-terminus of
TTR. Lane 1 contains Novex Mark 12 molecular weight standards, and
lanes 2-7 contain the following respectively: cell lysate,
post-heating supernatant, pool from Q-sepharose chromatography
step, pool from phenyl sepharose chromatography step, pool from
hydroxyapatite chromatography step, and pool from source Q
chromatography step.
[0013] FIG. 2 demonstrates by size exclusion chromatography that
fusion of peptides to the amino-terminus or carboxy-terminus of a
TTR variant, TTR(C10A/G83C), does not alter its oligomeric
structure. Solid line is TTR(C10A/G83C), dashed line is parathyroid
hormone (PTH) fused to the amino-terminus of TTR(C10A/G83C), and
the dotted line is Bradykinin fused to the carboxy-terminus of
TTR(C10A/G83C).
[0014] FIG. 3 demonstrates by size exclusion chromatography that
fusion of proteins to the amino-terminus or carboxy-terminus of a
TTR variant, TTR(C10A), does not alter its oligomeric structure.
Solid line is TTR(C10A), dashed line is IL-1-ra fused to the
carboxy-terminus of TTR(C10A), and the dotted line is IL-1-ra fused
to the amino-terminus of TTR(C10A).
[0015] FIG. 4 shows the binding observed using BIAcore of various
TPO-mimetic peptide (TMP) constructs to human MPL receptor:
.box-solid. Fc-TMP, .circle-solid. TMP(m)-TTR, .tangle-solidup.
TMP(m)-TTR-PEG5K, .tangle-soliddn. TMP(m)-TTR-PEG20K.
[0016] FIG. 5 shows that injection of TMP(m)-TTR-PEG5K induces
platelet formation in mice. The following symbols correspond to the
corresponding constructs: .box-solid. Carrier, .circle-solid.
Fc-TMP, .tangle-solidup. TTR-TMP, .tangle-soliddn. TMP(m)-TTR, and
.diamond-solid. TMP(m)-TTR-PEG5K.
[0017] FIG. 6 demonstrates by size exclusion chromatography that
native TTR and TTR(C10A) maintain a similar oligomeric
configuration (an apparent tetramer). Solid line is native TTR and
the dashed line is TTR(C10A).
[0018] FIG. 7 demonstrates by size exclusion chromatography that
conjugation of PEG to TTR increases its molecular size in a
predictable uniform manner. Solid lines indicate no PEG conjugated,
dashed lines indicate 5K PEG fused, and dotted lines indicate 20K
PEG fused. The following constructs were used: A)
TMP-TTR(C10A/A37C), B) TMP-TTR(C10A/D38C), C) TMP-TTR(C10A/A81C),
and D) TMP-TTR(C10A/G83C).
[0019] FIG. 8 is an SDS gel that depicts the extent of pegylation
of various TMP-TTR constructs involving TTR variants having a
non-native cysteine engineered in at one of four different
locations. Lane 1 contains Novex Mark 12 molecular weight
standards; lane 2 is unpegylated TMP-TTR(C10A/A37C); lanes 3-6 are
5K pegylated versions of TMP-TTR(C10A/A37C), TMP-TTR(C10A/D38C),
TMP-TTR(C10A/A81C), and TMP-TTR(C10A/G83C) respectively; lanes 7-10
are 20K pegylated versions of TMP-TTR(C10A/A37C),
TMP-TTR(C10A/D38C), TMP-TTR(C10A/A81C), and TMP-TTR(C10A/G83C),
respectively.
[0020] FIGS. 9A-C compare the competitive binding of Fc-TMP and
TMP-TTR to human MPL by BIAcore analysis. A) .box-solid. Fc-TMP,
.circle-solid. TMP-TTR(C10A/A37C), .tangle-solidup.
TMP-TTR(C10A/D38C), .tangle-soliddn. TMP-TTR(C10A/A81C),
.diamond-solid. TMP-TTR(C10A/G83C). B) .box-solid. Fc-TMP, 5K
pegylated versions of TMP-TTR(C10A/A37C) (.circle-solid.),
TMP-TTR(C10A/D38C) (.tangle-solidup.),
TMP-TTR(C10A/A81C)(.tangle-soliddn- .),
TMP-TTR(C10A/G83C)(.diamond-solid.). C) .box-solid. Fc-TMP, 20K
pegylated versions of TMP-TTR(C10A/A37C)(.circle-solid.),
TMP-TTR(C10A/D38C) (.tangle-soliddn.), TMP-TTR(C10A/A81C)
(.tangle-solidup.), TMP-TTR(C10A/G83C)(.diamond-solid.).
[0021] FIGS. 10A-C show that injection of TMP-TTR with PEG
conjugated to engineered cysteines induces platelet formation in
mice. A) .box-solid. TTR(C10A), .circle-solid. Fc-TMP,
.tangle-soliddn. TMP-TTR(C10A/A37C), .tangle-solidup.
TMP-TTR(C10A/D38C) (carboxamidomethylated), .diamond-solid.
TMP-TTR(C10A/A81C), TMP-TTR(C10A/G83C). B) .box-solid. TTR(C10A),
.circle-solid. Fc-TMP, 5K pegylated versions of TMP-TTR(C10A/A37C)
(.tangle-soliddn.), TMP-TTR(C10A/D38C)(.tangle-solidup- .),
TMP-TTR(C10A/A81C) (.diamond-solid.), TMP-TTR(C10A/G83C)(). C)
.box-solid. TTR(C10A), .circle-solid. Fc-TMP, 20K pegylated
versions of TMP-TTR(C10A/A37C) (.tangle-soliddn.),
TMP-TTR(C10A/D38C) (.tangle-solidup.),
TMP-TTR(C10A/A81C)(.diamond-solid.), TMP-TTR(C10A/G83C) ().
[0022] FIG. 11 shows that injection of PTH-TTR with PEG conjugated
to engineered cysteines induces ionized calcium release in mice.
The following symbols correspond to the corresponding constructs:
.box-solid. TTR(C10A), .circle-solid. PTH-Fc, .tangle-soliddn.
PTH-TTR, .tangle-solidup. PTH-TTR(C10A/K15A/A37C)
(carboxamidomethylated), .diamond-solid. 5K pegylated version of
PTH-TTR(C10A/K15A/A37C), 20K pegylated version of
PTH-TTR(C10A/K15A/A37C), z,901 PTH-TTR(C10A/K15A/G83C)
(carboxamidomethylated), 5K pegylated version of
PTH-TTR(C10A/K15A/G83C), and 20K pegylated version of
PTH-TTR(C10A/K15A/G83C).
[0023] FIG. 12 shows that injection of Glucagon-like Peptide 1
(GLP1)-TTR with PEG conjugated to engineered cysteines lowers blood
glucose levels in mice. The following symbols correspond to the
corresponding constructs: .box-solid. TTR(C10A), .circle-solid.
GLP1-Fc, .tangle-soliddn. GLPl-TTR(C10A/K15A/G83C) (PEG 5K), and
.tangle-solidup. GLP1-TTR(C10A/K15A/G83C) (PEG 20K).
[0024] FIG. 13 shows that injection of TMP-TTR conjugates with
fused CH2 domains increase serum platelet levels in mice. The
following symbols correspond to the corresponding constructs:
.box-solid. TTR(C10A), .circle-solid. Fc-TMP, A TMP-TTR(C10A)-CH2,
.tangle-solidup. TTR(C10A)-CH2-TMP, and .diamond-solid.
TMP-CH2-TTR(C10A).
[0025] FIG. 14 shows that injection of and carboxy-terminal fusions
of TMP with pegylated TTR increases blood platelet counts in mice.
The following symbols correspond to the corresponding constructs:
.box-solid. TTR(C10A), .circle-solid. Fc-TMP, .tangle-soliddn.
TTR(C10A/K15A/A37C)-TMP (PEG 20K), .tangle-solidup.
TTR(C10A/K15A/A81C)-TMP (PEG 20K), .diamond-solid.
TTR(C10A/K15A/G83C)-TMP (PEG 20K), TMP-TTR(C10A/K15A/A37C) (PEG
20K), TMP-TTR(C10A/K15A/A81C) (PEG 20K), TMP-TTR(C10A/K15A/G83C)
(PEG 20K).
[0026] FIGS. 15A-C show that injection of pegylated TMP-TTR fusions
containing a K15A alteration increases blood platelet counts in
mice. The following symbols correspond to the corresponding
constructs: A) .box-solid. TTR(C10A), .circle-solid. Fc-TMP,
.tangle-soliddn. TMP-TTR(C10A/K15A/A37C) (carboxyamidomethylated),
and .tangle-solidup. TMP-TTR(C10A/K15A/A81C)
(carboxyamidomethylated); B) .box-solid. TTR(C10A), .circle-solid.
Fc-TMP, .tangle-soliddn. TMP-TTR(C10A/K15A/A37C) (PEG 5K),
.tangle-solidup. TMP-TTR(C10A/K15A/A81C) (PEG 5K), and
.diamond-solid. TMP-TTR(C10A/K15A/G83C) (PEG 5K); C) .box-solid.
TTR(C10A), .circle-solid. Fc-TMP, .tangle-soliddn.
TMP-TTR(C10A/K15A/A37C) (PEG 20K), .tangle-solidup.
TMP-TTR(C10A/K15A/A81C) (PEG 20K), and .diamond-solid.
TMP-TTR(C10A/K15A/G83C) (PEG 20K).
DETAILED DESCRIPTION OF THE INVENTION
[0027] For purposes of describing the present invention, the
following terms are defined as set forth below.
[0028] The term "biologically active agent" refers to any chemical
material or compound useful for prophylactic, therapeutic or
diagnostic application. The term "pharmacologically active
compound" refers to a compound suitable for administration to a
mammalian, preferably a human individual, which induces a desired
local or systemic effect.
[0029] The terms "peptide", "polypeptide" and "protein" describe a
type of biologically active agents, and the terms are used
interchangeably herein to refer to a naturally occurring,
recombinantly produced or chemically synthesized polymer of amino
acids. The terms are intended to include peptide molecules
containing as few as 2 amino acids, chemically modified
polypeptides, consensus molecules, analogs, derivatives or
combinations thereof.
[0030] Any number of peptides may be used in conjunction with the
present invention. Of particular interest are peptides that mimic
the activity of erythropoietin (EPO), thrombopoietin (TPO),
Glucagon-like Peptide 1 (GLP-1), parathyroid hormone (PTH),
granulocyte-colony stimulating factor (G-CSF), granulocyte
macrophage-colony stimulating factor (GM-CSF), interleukin-1
receptor antagonist (IL-1ra), leptin, cytotoxic T-lymphocyte
antigen 4 (CTLA4), TNF-related apoptosis-inducing ligand (TRAIL),
tumor growth factor-alpha and beta (TGF-.alpha. and TGF-.beta.,
respectively), and growth hormones. The terms "-mimetic peptide"
and "-agonist peptide" refer to a peptide having biological
activity comparable to a protein (e.g., GLP-1, PTH, EPO, TPO,
G-CSF, etc.) that interacts with a protein of interest. These terms
further include peptides that indirectly mimic the activity of a
protein of interest, such as by potentiating the effects of the
natural ligand of the protein of interest. Thus, the term
"EPO-mimetic peptide" comprises any peptides that can be identified
or derived as having EPO-mimetic subject matter; see, for example,
Wrighton et al., Science, 273:458-63 (1996); and Naranda et al.,
Proc. Natl. Acad. Sci. USA 96:7569-74 (1999). Those of ordinary
skill in the art appreciate that each of these references enables
one to select different peptides than actually disclosed therein by
following the disclosed procedures with different peptide
libraries.
[0031] The term "TPO-mimetic peptide" (TMP) comprises peptides that
can be identified or derived as having TPO-mimetic subject matter;
see, for example, Cwirla et al., Science, 276:1696-9 (1997); U.S.
Pat. Nos. 5,869,451 and 5,932,946; and PCT WO 00/24782 (Liu et al,
and references cited therein), hereby incorporated by reference in
its entirety. Those of ordinary skill in the art appreciate that
each of these references enables one to select different peptides
than actually disclosed therein by following the disclosed
procedures with different peptide libraries.
[0032] The term "G-CSF-mimetic peptide" comprises any peptides that
can be identified as having G-CSF-mimetic subject matter; see, for
example, Paukovits et al., Hoppe-Seylers Z. Physiol. Chem.
365:303-11 (1984). Those of ordinary skill in the art appreciate
that each of these references enables one to select different
peptides than actually disclosed therein by following the disclosed
procedures with different peptide libraries.
[0033] The term "CTLA4-mimetic peptide" comprises any peptides that
can be identified or derived as described in Fukumoto et al.,
Nature Biotech. 16:267-70 (1998). Those of ordinary skill in the
art appreciate that each of these references enables one to select
different peptides than actually disclosed therein by following the
disclosed procedures with different peptide libraries.
[0034] Peptide antagonists are also of interest, particularly those
antagonistic to the activity of TNF, leptin, any of the
interleukins, and proteins involved in complement activation (e.g.,
C3b). The term "-antagonist peptide" or "inhibitor peptide" refers
to a peptide that blocks or in some way interferes with the
biological activity of the associated protein of interest, or has
biological activity comparable to a known antagonist or inhibitor
of the associated protein of interest. Thus, the term
"TNF-antagonist peptide" comprises peptides that can be identified
or derived as having TNF-antagonistic subject matter; see, foe
example, Takasaki et al., Nature Biotech., 15:1266-70 (1997). Those
of ordinary skill in the art appreciate that each of these
references enables one to select different peptides than actually
disclosed therein by following the disclosed procedures with
different peptide libraries.
[0035] The terms "IL-1 antagonist" and "IL-1ra-mimetic peptide"
comprises peptides that inhibit or down-regulate activation of the
IL-1 receptor by IL-1. IL-1 receptor activation results from
formation of a complex among IL-1, IL-1 receptor, and IL-1 receptor
accessory protein. IL-1 antagonist or IL-1ra-mimetic peptides bind
to IL-1, IL-1 receptor, or IL-1 receptor accessory protein and
obstruct complex formation among any two or three components of the
complex. Exemplary IL-1 antagonist or IL-1ra-mimetic peptides can
be identified or derived as described in U.S. Pat. Nos. 5,608,035,
5,786,331, 5,880,096. Those of ordinary skill in the art appreciate
that each of these references enables one to select different
peptides than actually disclosed therein by following the disclosed
procedures with different peptide libraries.
[0036] The term "VEGF-antagonist peptide" comprises peptides that
can be identified or derived as having VEGF-antagonistic subject
matter; see, for example, Fairbrother, Biochem., 37:17754-64
(1998). Those of ordinary skill in the art appreciate that each of
these references enables one to select different peptides than
actually disclosed therein by following the disclosed procedures
with different peptide libraries.
[0037] The term "MMP inhibitor peptide" comprises peptides that can
be identified or derived as having MMP inhibitory subject matter;
see, for example, Koivunen, Nature Biotech., 17:768-74 (1999).
Those of ordinary skill in the art appreciate that each of these
references enables one to select different peptides than actually
disclosed therein by following the disclosed procedures with
different peptide libraries.
[0038] Targeting peptides are also of interest, including
tumor-homing peptides, membrane-transporting peptides, and the
like.
[0039] Exemplary peptides may be randomly generated by various
techniques known in the art. For example, solid phase synthesis
techniques are well known in the art, and include those described
in Merrifield, Chem. Polypeptides, pp. 335-61 (Katsoyannis and
Panayotis eds.)(1973); Merrifield, J. Am. Chem. Soc., 85:2149
(1963); Davis et al., Biochem. Intl., 10:394-414 (1985); Stewart
and Young, Solid Phase Peptide Synthesis (1969); U.S. Pat. No.
3,941,763; Finn et al., The Proteins, 3rd ed., 2:105-253 (1976);
and Erickson et al., The Proteins, 3rd ed., 2:257-527 (1976). Solid
phase synthesis is the preferred technique of making individual
peptides since it is the most cost-effective method of making small
peptides.
[0040] Phage display is another useful method in generating
peptides for use in the present invention. It has been stated that
affinity selection from libraries of random peptides can be used to
identify peptide ligands for any site of any gene product; Dedman
et al., J. Biol. Chem., 268:23025-30 (1993). Phage display is
particularly well suited for identifying peptides that bind to such
proteins of interest as cell surface receptors or any proteins
having linear epitopes; Wilson et al., Can. J. Microbiol.,
44:313-29 (1998); Kay et al., Drug Disc. Today, 3:370-8 (1998).
Such proteins are extensively reviewed in Herz et al., J. Receptor
& Signal Transduction Res., 17(5):671-776 (1997), which is
hereby incorporated by reference.
[0041] The peptides may also be made in transformed host cells
using recombinant DNA techniques. To do so, a recombinant DNA
molecule coding for the peptide is prepared. Methods of preparing
such DNA and/or RNA molecules are well known in the art. For
instance, sequences coding for the peptides could be excised from
DNA using suitable restriction enzymes. The relevant sequences can
be created using the polymerase chain reaction (PCR) with the
inclusion of useful restriction sites for subsequent cloning.
Alternatively, the DNA/RNA molecule could be synthesized using
chemical synthesis techniques, such as the phosphoramidite method.
Also, a combination of these techniques could be used.
[0042] Additional biologically active agents contemplated for use
include recombinant or naturally occurring proteins, whether human
or animal, hormones, cytokines, hematopoietic factors, growth
factors, antiobesity factors, trophic factors, anti-inflammatory
factors, and enzymes. Such proteins would include but are not
limited to interferons (see, U.S. Pat. Nos. 5,372,808, 5,541,293
4,897,471, and 4,695,623 hereby incorporated by reference including
drawings), interleukins (see, U.S. Pat. No. 5,075,222, hereby
incorporated by reference including drawings), erythropoietins
(see, U.S. Pat. Nos. 4,703,008, 5,441,868, 5,618,698 5,547,933, and
5,621,080 hereby incorporated by reference including drawings),
granulocyte-colony stimulating factors (see, U.S. Pat. Nos.
4,810,643, 4,999,291, 5,581,476, 5,582,823, and PCT Publication No.
94/17185, hereby incorporated by reference including drawings),
stem cell factor (PCT Publication Nos. 91/05795, 92/17505 and
95/17206, hereby incorporated by reference including drawings),
NESP(PCT Publication No. US94/02957, hereby incorporated by
reference including drawings), osteoprotegerin (PCT Publication No.
97/23614, hereby incorporated by reference including drawings),
interleukin-1 receptor antagonist (IL-1ra)(PCT Publication Nos.
91/08285 and 92/16221) and leptin (OB protein) (PCT publication
Nos. 96/40912, 96/05309, 97/00128, 97/01010 and 97/06816 hereby
incorporated by reference including figures).
[0043] In addition, biologically active agents can also include but
are not limited to insulin, gastrin, prolactin, adrenocorticotropic
hormone (ACTH), thyroid stimulating hormone (TSH), luteinizing
hormone (LH), follicle stimulating hormone (FSH), human chorionic
gonadotropin (HCG), motilin, interferons (alpha, beta, gamma),
interleukins (IL-1 to IL-12), tumor necrosis factor (TNF), tumor
necrosis factor-binding protein (TNF-bp), brain derived
neurotrophic factor (BDNF), glial derived neurotrophic factor
(GDNF), neurotrophic factor 3 (NT3), fibroblast growth factors
(FGF), neurotrophic growth factor (NGF), bone growth factors such
as osteoprotegerin (OPG), insulin-like growth factors (IGFs),
macrophage colony stimulating factor (M-CSF), granulocyte
macrophage colony stimulating factor (GM-CSF), megakaryocyte
derived growth factor (MGDF), keratinocyte growth factor (KGF),
thrombopoietin, platelet-derived growth factor (PGDF), colony
simulating growth factors (CSFs), bone morphogenetic protein (BMP),
superoxide dismutase (SOD), tissue plasminogen activator (TPA),
urokinase, streptokinase and kallikrein.
[0044] Transthyretin (TTR) contemplated for use in the present
invention will have the DNA and amino acid sequences of TTR as
reported in Mita et al., Biochem. Biophys. Res. Commun.,
124(2):558-564 (1984). These sequences have been deposited in
Genbank as accession number K02091. The 127 amino acid TTR sequence
used herein does not include the signal sequence (amino acids 1-20)
of the K02091 sequence and is depicted below as SEQ ID NO:1.
1 GPTGTGESKCPLMVKVLDAVRGSPAINVAVHVFRKAADDTWEPFASGKTSESGEL SEQ ID
NO:1 HGLTTEEEFVEGIYKVEIDTKSYWKALGISPFHEHAEVVFTANDSGPRRYT- IAAL
LSPYSYSTTAVVTNPKE
[0045] The term "TTR variant" refers to a molecule or sequence that
is a modified form of a native TTR. For example, a native TTR
comprises sites that may be removed because they provide structural
features or biological activity that are not required for the
fusion molecules of the present invention. Thus, the term "TTR
variant" comprises a molecule or sequence that lacks one or more
native TTR sites or residues or that has had one or more native TTR
sites or residues replaced with a different amino acid or that has
had one or more residues added to the sequence. For purposes of an
example, a TTR variant wherein the Alanine residue at amino acid
sequence position 37 has been replaced with a Cysteine residue,
will be designated TTR variant (A37C); and a TTR variant wherein
both the Alanine residue at amino acid sequence position 37 and the
Glycine residue at amino acid sequence position 83 have both been
replaced with a Cysteine residue will be designated TTR variant
(A37C/G83C).
[0046] In one embodiment, a TTR or TTR variant fused to a
biologically active agent may be fused to a third protein or
protein fragment that further stabilizes the TTR-biologically
active agent fusion protein, and thereby increases the half-life of
the resulting fusion in serum. Examples of such additional proteins
or fragments thereof that can be used in such methods and
compositions include the Fc domain or CH2 domain of an
immunoglobulin, or any other protein domain that one of skill in
the art would recognize as having properties that would increase
protein stability (see, e.g., Example 29 below). Such protein
groups can be fused to the carboxy or amino terminus of the
TTR-biologically active agent fusion protein, or can be placed
between the TTR and the biologically active agent. It is
contemplated that linkers or spacers can be placed, as needed,
between each of the domains of the fusion protein to facilitate
their desired activity.
[0047] In another embodiment, the TTR or TTR variant of the
invention can be chemically crosslinked to the biologically active
agent. Cross-linking of proteins can be performed by using, for
example, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP)
according to established, published procedures. Additional
cross-linking agents are readily available and can be identified by
one of skill in the art. For details on the above procedure, see,
e.g., Karpovsky et al, J. Exp. Med. 160, 1686-1701 (1984); Perez et
al, Nature, 316, 354-356 (1985) or Titus et al, Journal of
Immunology, 139, 3153-3158 (1987).
[0048] In another embodiment, a molecule can be covalently linked
to the fusion protein such that stability and/or half-life in serum
are increased. For example, a preferred TTR or TTR variant may be
chemically modified using water soluble polymers such as
polyethylene glycol (PEG). The PEG group may be of any convenient
molecular weight and may be straight chain or branched. The average
molecular weight of the PEG will preferably range from about 2 kDa
to about 100 kDa, more preferably from about 5 kDa to about 50 kDa,
most preferably about 20 kDa.
[0049] The PEG groups will generally be attached to the compounds
of the invention via acylation, reductive alkylation, Michael
addition, thiol alkylation or other chemoselective
conjugation/ligation methods through a reactive group on the peg
moiety (e.g., an aldehyde, amino, ester, thiol, -haloacetyl,
maleimido or hydrazino group) to a reactive group on the target
compound (e.g., an aldehyde, amino, ester, thiol, haloacetyl,
maleimido or hydrazino group).
[0050] Other water soluble polymers used include copolymers of
ethylene glycol/propylene glycol, carboxymethylcellulose, polyvinyl
alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,
poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer,
polyaminoacids (either homopolymers or random copolymers), and
dextran.
[0051] A DNA molecule encoding the peptide of interest, protein of
interest, TTR or TTR variant can be prepared using well known
recombinant DNA technology methods such as those set forth in
Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. [1989])
and/or Ausubel et al., eds, Current Protocols in Molecular Biology,
Green Publishers Inc. and Wiley and Sons, NY (1994). A gene or cDNA
encoding the protein of interest or fragment thereof may be
obtained for example by screening a genomic or cDNA library with a
suitable probe. Suitable probes include, for example,
oligonucleotides, cDNA fragments, or genomic DNA fragments, that
are expected to have some homology to the gene encoding the protein
of interest, such that the probe will hybridize with the gene
encoding the protein of interest under selected hybridization
conditions. An alternate means of screening a DNA library is by
polymerase chain reaction "PCR" amplification of the gene encoding
the protein of interest. PCR is typically accomplished using
oligonucleotide "primers" which have a sequence that is believed to
have sufficient homology to the gene to be amplified such that at
least a sufficient portion of the primer will hybridize with the
gene.
[0052] Alternatively, a gene encoding the peptide of interest or
protein of interest may be prepared by chemical synthesis using
methods well known to the skilled artisan such as those described
by Engels et al., Angew. Chem. Intl. Ed., 28:716-734 (1989). These
methods include, inter alia, the phosphotriester, phosphoramidite,
and H-phosphonate methods for nucleic acid synthesis. A preferred
method for such chemical synthesis is polymer-supported synthesis
using standard phosphoramidite chemistry. Typically, the DNA
encoding the protein of interest will be several hundred
nucleotides in length. Nucleic acids larger than about 100
nucleotides can be synthesized as several fragments using these
methods. The fragments can then be ligated together to form a gene
coding for the full length protein of interest. Usually, the DNA
fragment encoding the amino terminus of the polypeptide will have
an ATG, which encodes a methionine residue. This methionine may or
may not be present on the mature form of the protein of interest.
The methionine can be removed inside the cell or during the process
of secretion. Preferred TTR polypeptides may include TTR with the
nucleic acid sequence altered to optimize expression in E. coli and
to introduce convenient restriction sites. A general discussion of
codon optimization for expression in E. coli is described in Kane,
Curr. Opin. Biotechnol., 6:494-500 (1995).
[0053] Once the genes encoding the protein of interest and the TTR
polypeptide have been obtained, they may be modified using standard
methods to create restriction endonuclease sites at the 5' and/or
3' ends. Creation of the restriction sites permits the genes to be
properly inserted into amplification and/or expression vectors.
Addition of restriction sites is typically accomplished using PCR,
where one primer of each PCR reaction typically contains, inter
alia, the nucleotide sequence of the desired restriction site.
[0054] The gene or cDNA encoding the peptide of interest, or
protein of interest can be inserted into an appropriate expression
vector for expression in a host cell. The vector is selected to be
functional in the particular host cell employed (i.e., the vector
is compatible with the host cell machinery such that amplification
and/or expression of the gene encoding the protein of interest can
occur).
[0055] Typically, the vectors used in any of the host cells will
contain a promoter (also referred to as a "5' flanking sequence")
and other regulatory elements as well such as an enhancer(s), an
origin of replication element, a transcriptional termination
element, a ribosome binding site element, a polylinker region for
inserting the nucleic acid encoding the polypeptide to be
expressed, and a selectable marker element. Each of these elements
is discussed below. Optionally, the vector may contain a "tag" DNA
sequence, i.e., an oligonucleotide sequence located at either the
5' or 3' end of the fusion DNA construct. The tag DNA encodes a
molecule such as hexaHis, c-myc, FLAG (Invitrogen, San Diego,
Calif.) or another small immunogenic sequence. When placed in the
proper reading frame, this tag will be expressed along with the
fusion protein, and can serve as an affinity tag for purification
of the fusion protein from the host cell. Optionally, the tag can
subsequently be removed from the purified fusion protein by various
means such as using a selected peptidase for example.
[0056] The promoter may be homologous (i.e., from the same species
and/or strain as the host cell), heterologous (i.e., from a species
other than the host cell species or strain), hybrid (i.e., a
combination of promoters from more than one source), synthetic, or
it may be the native protein of interest promoter. Further, the
promoter may be a constitutive or an inducible promoter. As such,
the source of the promoter may be any unicellular prokaryotic or
eukaryotic organism, any vertebrate or invertebrate organism, or
any plant, provided that the promoter is functional in, and can be
activated by, the host cell machinery.
[0057] The promoters useful in the vectors of this invention may be
obtained by any of several methods well known in the art.
Typically, promoters useful herein will have been previously
identified by mapping and/or by restriction endonuclease digestion
and can thus be isolated from the proper tissue source using the
appropriate restriction endonucleases. In some cases, the full
nucleotide sequence of the promoter may be known. Here, the
promoter may be synthesized using the methods described above for
nucleic acid synthesis or cloning.
[0058] Where all or only a portion of the promoter sequence is
known, the complete promoter may be obtained using PCR and/or by
screening a genomic library with suitable oligonucleotide and/or 5'
flanking sequence fragments from the same or another species.
[0059] Suitable promoters for practicing this invention are
inducible promoters such as the lux promoter, the lac promoter, the
arabinose promoter, the trp promoter, the tac promoter, the tna
promoter, synthetic lambda promoters (from bacteriophage lambda),
and the T5 or T7 promoters. Preferred promoters include the lux,
and lac promoters.
[0060] The origin of replication element is typically a part of
prokaryotic expression vectors whether purchased commercially or
constructed by the user. In some cases, amplification of the vector
to a certain copy number can be important for optimal expression of
the protein or polypeptide of interest. In other cases, a constant
copy number is preferred. In any case, a vector with an origin of
replication that fulfills the requirements can be readily selected
by the skilled artisan. If the vector of choice does not contain an
origin of replication site, one may be chemically synthesized based
on a known sequence, and ligated into the vector.
[0061] The transcription termination element is typically located
3' of the end of the fusion protein DNA construct, and serves to
terminate transcription of the RNA message coding for the fusion
polypeptide. Usually, the transcription termination element in
prokaryotic cells is a G-C rich fragment followed by a poly T
sequence. While the element is easily cloned from a library or even
purchased commercially as part of a vector, it can also be readily
synthesized using methods for nucleic acid synthesis such as those
described above.
[0062] Expression vectors typically contain a gene coding for a
selectable marker. This gene encodes a protein necessary for the
survival and growth of a host cell grown in a selective culture
medium. Typical selection marker genes encode proteins that (a)
confer resistance to antibiotics or other toxins, e.g., ampicillin,
tetracycline, chloramphenicol, or kanamycin for prokaryotic host
cells, (b) complement auxotrophic deficiencies of the cell; or (c)
supply critical nutrients not available from complex media.
Preferred selectable markers are the kanamycin resistance gene, the
ampicillin resistance gene, the chloramphenicol resistance gene,
and the tetracycline resistance gene.
[0063] The ribosome binding element, commonly called the
Shine-Dalgarno sequence in prokaryotes, is necessary for the
initiation of translation of mRNA. The element is typically located
3' to the promoter and 5' to the coding sequence of the fusion
protein DNA construct. The Shine-Dalgarno sequence is varied but is
typically a polypurine (i.e., having a high A-G content). Many
Shine-Dalgarno sequences have been identified, each of which can be
readily synthesized using methods set forth above and used in a
prokaryotic vector.
[0064] Where one or more of the elements set forth above are not
already present in the vector to be used, they may be individually
obtained and ligated into the vector. Methods used for obtaining
each of the elements are well known to the skilled artisan and are
comparable to the methods set forth above (i.e., synthesis of the
DNA, library screening, and the like).
[0065] Each element may be individually ligated into the vector by
cutting the vector with the appropriate restriction endonuclease(s)
such that the ends of the element to be ligated in and the ends of
the vector are compatible for ligation. In some cases, it may be
necessary to "blunt" the ends to be ligated together in order to
obtain a satisfactory ligation. Blunting can be accomplished by
first filling in "sticky ends" using an enzyme such as Klenow DNA
polymerase or T4 DNA polymerase in the presence of all four
nucleotides. This procedure is well known in the art and is
described for example in Sambrook et al., supra.
[0066] Alternatively, two or more of the elements to be inserted
into the vector may first be ligated together (if they are to be
positioned adjacent to each other) and then ligated into the
vector.
[0067] Another method for constructing the vector is to conduct all
ligations of the various elements simultaneously in one reaction
mixture. Here, many nonsense or nonfunctional vectors may be
generated due to improper ligation or insertion of the elements,
however the functional vector may be identified by expression of
the selectable marker. Proper sequence of the ligation product can
be confirmed by digestion with restriction endonucleases or by DNA
sequencing.
[0068] After the vector has been constructed and a fusion protein
DNA construct has been inserted into the proper site of the vector,
the completed vector may be inserted into a suitable host cell for
fusion protein expression.
[0069] Host cells suitable for the present invention are bacterial
cells. For example, the various strains of E. coli (e.g., HB101,
JM109, DH5.alpha., DH10, and MC1061) are well-known host cells for
use in preparing recombinant polypeptides. The choice of bacterial
strain is typically made so that the strain and the expression
vector to be used are compatible. Various strains of B. subtilis,
Pseudomonas spp., other Bacillus spp., Streptomyces spp., and the
like may also be employed in practicing this invention in
conjunction with appropriate expression vectors.
[0070] Insertion (also referred to as "transformation" or
"transfection") of the vector into the selected host cell may be
accomplished using such methods as calcium phosphate precipitation
or electroporation. The method selected will in part be a function
of the type of host cell to be used. These methods and other
suitable methods are well known to the skilled artisan, and are set
forth, for example, in Sambrook et al., supra.
[0071] The host cells containing the vector (i.e., transformed or
transfected host cells) may be cultured using one or more standard
media well known to the skilled artisan. The selected medium will
typically contain all nutrients necessary for the growth and
survival of the host cells. Suitable media for culturing E. coli
cells, are, for example, Luria broth ("LB"), YT broth, SOB, SOC,
and/or Terrific Broth ("TB").
[0072] There are several ways to prepare the DNA construct encoding
the fusion protein which comprises the TTR gene, the gene encoding
the peptide or protein of interest, and, optionally, a DNA molecule
encoding a linker peptide which is located between the two
genes.
[0073] In one procedure, the TTR gene and gene encoding the protein
of interest (the "fusion partner genes") can be ligated together in
either orientation (e.g., TTR gene at the 5' or 3' end of the
construct). Where a linker DNA molecule is to be included, it can
first be ligated to one of the fusion partner genes, and that
construct can then be ligated to the other fusion partner gene.
Ligations are typically accomplished using DNA ligase enzyme in
accordance with the manufacturer's instructions.
[0074] A separate procedure provides for first ligating one fusion
partner gene into the selected vector, after which the other fusion
partner gene can be ligated into the vector in a position that is
either 3' or 5' to the first fusion partner gene. Where a linker
DNA molecule is to be included, the linker DNA molecule may be
ligated to either fusion partner gene either before or after that
gene has been ligated into the vector.
[0075] The TTR-TMPs of the present invention can be used to treat
conditions generally known as those that involve an existing
megakaryocyte/platelet deficiency or an expected
megakaryocyte/platelet deficiency (e.g., because of planned surgery
or platelet donation). Such conditions will usually be the result
of a deficiency (temporary or permanent) of active Mp1 ligand in
vivo. The generic term for platelet deficiency is thrombocytopenia,
and hence the methods and compositions of the present invention are
generally available for treating thrombocytopenia in patients in
need thereof. Thrombocytopenia (platelet deficiencies) may be
present for various reasons, including chemotherapy and other
therapy with a variety of drugs, radiation therapy, surgery,
accidental blood loss, and other specific disease conditions.
[0076] Exemplary specific disease conditions that involve
thrombocytopenia and may be treated in accordance with this
invention are: aplastic anemia, idiopathic thrombocytopenia,
metastatic tumors which result in thrombocytopenia, systemic lupus
erythematosus, splenomegaly, Fanconi's syndrome, vitamin B12
deficiency, folic acid deficiency, May-Hegglin anomaly,
Wiskott-Aldrich syndrome, and paroxysmal nocturnal hemoglobinuria.
Also, certain treatments for AIDS result in thrombocytopenia (e.g.,
AZT). Certain wound healing disorders might also benefit from an
increase in platelet numbers.
[0077] With regard to anticipated platelet deficiencies, e.g., due
to future surgery, a compound of the present invention could be
administered several days to several hours prior to the need for
platelets. With regard to acute situations, e.g., accidental and
massive blood loss, a compound of this invention could be
administered along with blood or purified platelets.
[0078] The TMP compounds of this invention may also be useful in
stimulating certain cell types other than megakaryocytes if such
cells are found to express Mpl receptor. Conditions associated with
such cells that express the Mpl receptor, which are responsive to
stimulation by the Mpl ligand, are also within the scope of this
invention.
[0079] The TMP compounds of this invention may be used in any
situation in which production of platelets or platelet precursor
cells is desired, or in which stimulation of the c-Mpl receptor is
desired. Thus, for example, the compounds of this invention may be
used to treat any condition in a mammal wherein there is a need of
platelets, megakaryocytes, and the like. Such conditions are
described in detail in the following exemplary sources: WO95/26746;
WO95/21919; WO95/18858; WO95/21920 and are incorporated herein.
[0080] The TMP compounds of this invention may also be useful in
maintaining the viability or storage life of platelets and/or
megakaryocytes and related cells. Accordingly, it could be useful
to include an effective amount of one or more such compounds in a
composition containing such cells.
[0081] The therapeutic methods, compositions and compounds of the
present invention may also be employed, alone or in combination
with other cytokines, soluble Mpl receptor, hematopoietic factors,
interleukins, growth factors or antibodies in the treatment of
disease states characterized by other symptoms as well as platelet
deficiencies. It is anticipated that the inventive compound will
prove useful in treating some forms of thrombocytopenia in
combination with general stimulators of hematopoiesis, such as IL-3
or GM-CSF. Other megakaryocytic stimulatory factors, i.e., meg-CSF,
stem cell factor (SCF), leukemia inhibitory factor (LIF),
oncostatin M (OSM), or other molecules with megakaryocyte
stimulating activity may also be employed with Mpl ligand.
Additional exemplary cytokines or hematopoietic factors for such
co-administration include IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4,
IL-5, IL-6, IL-11, colony stimulating factor-1 (CSF-1), SCF,
GM-CSF, granulocyte colony stimulating factor (G-CSF), EPO,
interferon-alpha (IFN-alpha), consensus interferon, IFN-beta, or
IFN-gamma. It may further be useful to administer, either
simultaneously or sequentially, an effective amount of a soluble
mammalian Mpl receptor, which appears to have an effect of causing
megakaryocytes to fragment into platelets once the megakaryocytes
have reached mature form. Thus, administration of an inventive
compound (to enhance the number of mature megakaryocytes) followed
by administration of the soluble Mpl receptor (to inactivate the
ligand and allow the mature megakaryocytes to produce platelets) is
expected to be a particularly effective means of stimulating
platelet production. The appropriate dosage would be adjusted to
compensate for such additional components in the therapeutic
composition. Progress of the treated patient can be monitored by
conventional methods.
[0082] In non-insulin dependent diabetes mellitus (NIDDM), also
known as type 2 diabetic patients, the administration of
glucagon-like peptide-1 (GLP-1) has antidiabetic properties.
However, GLP-1 is rapidly degraded by dipeptidyl peptidase IV
(DPPIV) after its release in vivo. Thus, it is an advantage of the
present invention that a GLP-1 peptide or variant thereof can be
fused to a TTR polypeptide of the invention to stabilize GLP-1 and
increase its half life in vivo. Accordingly, in another embodiment
of the invention, a TTR-GLP1 fusion protein as described herein can
be used to treat conditions generally known to involve non-insulin
dependent diabetes mellitus (NIDDM), which is also known as type II
diabetes.
[0083] One of skill in the art will recognize that the sequence of
a GLP-1 peptide can be varied such that it retains its
insulinotropic effects. Particular examples of such variations
known in the art include, for example, GLP-1(7-34), (7-35), (7-36)
or (7-37), Gln.sup.9-GLP-1(7-37), D-Gln.sup.9-GLP-1(7-37), Thr
.sup.16-Lys.sup.18-GLP-1(7-37), and Lys.sup.18-GLP-1(7-37).
Additional examples of GLP-1 variants are described in U.S. Pat.
Nos. 5,118,666, 5,545,618, 5,977,071, and WO 02/46227 and in
Adelhorst et al., J. Biol. Chem. 269:6275 (1994), which are
incorporated by reference. Accordingly, any GLP-1 peptide can be
used to generate fusion proteins of the invention, as long as the
GLP-1 fusion protein is capable of binding and inducing a signal
through it's cognate receptor. Receptor binding and activation can
be measured by standard assays (U.S. Pat. No. 5,120,712).
[0084] The dose of fusion protein effective to normalize a
patient's blood glucose will depend on a number of factors among
which are included the subject's weight, age, severity of their
inability to regulate blood glucose, the route of administration,
the bioavailability, the pharmokinetic profile of the fusion
protein and the formulation as is discussed more fully below.
[0085] The therapeutic methods, compositions and compounds of the
present invention may also be employed, alone or in combination
with other diabetes treatments, including but not limited to
insulin, DPPIV-inhibitors and the like. The dosage of the GLP-1
fusion protein would be adjusted to compensate for such additional
components in the therapeutic composition. Progress of the treated
patient can be monitored by conventional methods, such as, for
example, the monitoring of blood glucose levels.
[0086] The present invention also provides pharmaceutical
compositions of the inventive compounds. Such pharmaceutical
compositions may be for administration for injection, or for oral,
nasal, transdermal or other forms of administration, including,
e.g., by intravenous, intradermal, intramuscular, intramammary,
intraperitoneal, intrathecal, intraocular, retrobulbar,
intrapulmonary (e.g., aerosolized drugs) or subcutaneous injection
(including depot administration for long term release); by
sublingual, anal, vaginal, or by surgical implantation, e.g.,
embedded under the splenic capsule, brain, or in the cornea. The
treatment may consist of a single dose or a plurality of doses over
a period of time. In general, comprehended by the invention are
pharmaceutical compositions comprising effective amounts of a
compound of the invention together with pharmaceutically acceptable
diluents, preservatives, stabilizers, solubilizers, emulsifiers,
adjuvants and/or carriers. Such compositions include diluents of
various buffer content (e.g., Tris-HCl, acetate, phosphate,
citrate, etc.), pH and ionic strength; additives such as detergents
and solubilizing agents (e.g., Tween 80, Polysorbate 80, etc.),
anti-oxidants (e.g., ascorbic acid, sodium metabisulfite),
preservatives (e.g., Thimersol, benzyl alcohol) and bulking
substances (e.g., lactose, mannitol); incorporation of the material
into particulate preparations of polymeric compounds such as
polylactic acid, polyglycolic acid, etc. or into liposomes.
Hyaluronic acid may also be used, and this may have the effect of
promoting sustained duration in the circulation. The pharmaceutical
compositions optionally may include still other pharmaceutically
acceptable liquid, semisolid, or solid diluents that serve as
pharmaceutical vehicles, excipients, or media, including but are
not limited to, polyoxyethylene sorbitan monolaurate, magnesium
stearate, methyl- and propylhydroxybenzoate, starches, sucrose,
dextrose, gum acacia, calcium phosphate, mineral oil, cocoa butter,
and oil of theobroma. Such compositions may influence the physical
state, stability, rate of in vivo release, and rate of in vivo
clearance of the present proteins and derivatives. See, e.g.,
Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack
Publishing Co., Easton, Pa. 18042) pages 1435-1712 which are herein
incorporated by reference. The compositions may be prepared in
liquid form, or may be in dried powder, such as lyophilized form.
Implantable sustained release formulations are also contemplated,
as are transdermal formulations.
[0087] Controlled release formulation may be desirable. The drug
could be incorporated into an inert matrix which permits release by
either diffusion or leaching mechanisms e.g., gums. Slowly
degenerating matrices may also be incorporated into the
formulation, e.g., alginates, polysaccharides. Another form of a
controlled release of this therapeutic is by a method based on the
Oros therapeutic system (Alza Corp.), i.e., the drug is enclosed in
a semipermeable membrane which allows water to enter and push drug
out through a single small opening due to osmotic effects. Some
enteric coatings also have a delayed release effect.
[0088] Also contemplated herein is pulmonary delivery of the
present protein (or derivatives thereof). The protein (or
derivative) is delivered to the lungs of a mammal while inhaling
and traverses across the lung epithelial lining to the blood
stream. (Other reports of this include Adjei et al., Pharmaceutical
Research 7:565-569 (1990); Adjei et al., International Journal of
Pharmaceutics 63:135-144 (1990)(leuprolide acetate); Braquet et
al., Journal of Cardiovascular Pharmacology 13 (suppl.5): s.143-146
(1989)(endothelin-1); Hubbard et al., Annals of Internal Medicine
3:206-212 (1989)(1-antitrypsin); Smith et al., J. Clin. Invest.
84:1145-1146 (1989)(1-proteinase); Oswein et al., "Aerosolization
of Proteins", Proceedings of Symposium on Respiratory Drug Delivery
II, Keystone, Colorado, March, 1990 (recombinant human growth
hormone); Debs et al., The Journal of Immunology 140:3482-3488
(1988)(interferon- and tumor necrosis factor ) and Platz et al.,
U.S. Pat. No. 5,284,656 (granulocyte colony stimulating
factor).
[0089] Contemplated for use in the practice of this invention are a
wide range of mechanical devices designed for pulmonary delivery of
therapeutic products, including but not limited to nebulizers,
metered dose inhalers, and powder inhalers, all of which are
familiar to those skilled in the art.
[0090] Some specific examples of commercially available devices
suitable for the practice of this invention are the Ultravent
nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the
Acorn II nebulizer, manufactured by Marquest Medical Products,
Englewood, Colorado; the Ventolin metered dose inhaler,
manufactured by Glaxo Inc., Research Triangle Park, North Carolina;
and the Spinhaler powder inhaler, manufactured by Fisons Corp.,
Bedford, Mass.
[0091] All such devices require the use of formulations suitable
for the dispensing of the inventive compound. Typically, each
formulation is specific to the type of device employed and may
involve the use of an appropriate propellant material, in addition
to diluents, adjuvants and/or carriers useful in therapy.
[0092] The inventive compound should most advantageously be
prepared in particulate form with an average particle size of less
than 10 .mu.m (or microns), most preferably 0.5 to 5 .mu.m, for
most effective delivery to the distal lung.
[0093] Carriers include carbohydrates such as trehalose, mannitol,
xylitol, sucrose, lactose, and sorbitol. Other ingredients for use
in formulations may include DPPC, DOPE, DSPC and DOPC. Natural or
synthetic surfactants may be used. Polyethylene glycol may be used
(even apart from its use in derivatizing the protein or analog).
Dextrans, such as cyclodextran, may be used. Bile salts and other
related enhancers may be used. Cellulose and cellulose derivatives
may be used. Amino acids may be used, such as use in a buffer
formulation.
[0094] The dosage regimen involved in a method for treating the
above-described conditions will be determined by the attending
physician, considering various factors which modify the action of
drugs, e.g. the age, condition, body weight, sex and diet of the
patient, the severity of any infection, time of administration and
other clinical factors. Generally, the dose should be in the range
of 0.1 .mu.g to 100 mg of the inventive compound per kilogram of
body weight per day, preferably 0.1 to 1000 .mu.g/kg; and more
preferably 0.1 to 150 .mu.g/kg, given in daily doses or in
equivalent doses at longer or shorter intervals, e.g., every other
day, twice weekly, weekly, or twice or three times daily.
[0095] The inventive compounds may be administered by an initial
bolus followed by a continuous infusion to maintain therapeutic
circulating levels of drug product. As another example, the
inventive compounds may be administered as a one-time dose. Those
of ordinary skill in the art will readily optimize effective
dosages and administration regimens as determined by good medical
practice and the clinical condition of the individual patient. The
frequency of dosing will depend on the pharmacokinetic parameters
of the agents and the route of administration. The optimal
pharmaceutical formulation will be determined by one skilled in the
art depending upon the route of administration and desired dosage.
See for example, Remington's Pharmaceutical Sciences, 18th Ed.
(1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712, the
disclosure of which is hereby incorporated by reference. Depending
on the route of administration, a suitable dose may be calculated
according to body weight, body surface area or organ size.
[0096] Appropriate dosages may be ascertained through use of
established assays for determining serum levels in conjunction with
appropriate dose-response data. The final dosage regimen will be
determined by the attending physician, considering various factors
which modify the action of drugs, e.g. the drug's specific
activity, the severity of the damage and the responsiveness of the
patient, the age, condition, body weight, sex and diet of the
patient, the severity of any infection, time of administration and
other clinical factors. As studies are conducted, further
information will emerge regarding the appropriate dosage levels and
duration of treatment for various diseases and conditions.
[0097] The following Examples are intended for illustration
purposes only, and should not be construed to limit the invention
in any way.
EXAMPLE 1
[0098] This example describes the preparation of DNA for native
recombinant human transthyretin (TTR) and the following TTR
variants: TTR(C10A), TTR(C10A/A37C), TTR(C10A/D38C),
TTR(C10A/A81C), TTR(C10A/G83C), and TTR(C10A/K15A/G83C).
[0099] The expression plasmid pAMG21 is available from the ATCC
under accession number 98113, which was deposited on Jul. 24, 1996
(see PCT WO 97/23614, published Jul. 3, 1997 for a description of
pAMG21). DNA sequence coding for TTR, TTR variants or TTR-peptide
fusions was placed under control of the LuxPR promoter in
pAMG21.
[0100] The bacterial host GM221 is an E.coli K-12 strain that has
been modified to contain both the temperature sensitive lambda
repressor c1857s7 in the early ebg region and the lacI.sup.Q
repressor in the late ebg region (68 minutes). The presence of
these two repressor genes allows the use of this host with a
variety of expression systems, however both of these repressors are
irrelevant to the expression from luxP.sub.R. The untransformed
host has no antibiotic resistances. The ribosomal binding site of
the cI857s7 gene has been modified to include an enhanced RBS. It
has been inserted into the ebg operon between nucleotide position
1170 and 1411 as numbered in Genbank accession number M64441 Gb_Ba
with deletion of the intervening ebg sequence. The construct was
delivered to the chromosome using a recombinant phage called
MMebg-cI857s7 enhanced RBS #4 into F'tet/393. After recombination
and resolution only the chromosomal insert described above remains
in the cell. It was renamed F'tet/GM101. F'tet/GM101 was then
modified by the delivery of a lacI.sup.Q construct into the ebg
operon between nucleotide position 2493 and 2937 as numbered in the
Genbank accession number M64441 Gb_Ba with the deletion of the
intervening ebg sequence. The construct was delivered to the
chromosome using a recombinant phage called AGebg-lacI.sup.Q #5
into F'tet/GM101. After recombination and resolution only the
chromosomal insert described above remains in the cell. It was
renamed F'tet/GM221. The F'tet episome was cured from the strain
using acridine orange at a concentration of 25 .mu.g/ml in LB. The
cured strain was identified as tetracyline sensitive and was stored
as GM221.
[0101] Oligonucleotides (1.0 nm each) were synthesized by
phosphoramidite method. Nucleotides were, in some cases, altered
for optimized expression in E. coli. These codon changes did not
result in changes in the amino acid sequence. Each of the
oligonucleotides utilized in this example are listed in Table
1.
[0102] PCR was performed with the Expand Long Polymerase according
to the manufacturer's protocol (Boehringer Mannheim). PCR products
were verified by agarose gel electrophoresis, purified and digested
with Nde1 and Xho1 (New England Biolabs). Expression vector pAMG21
was digested in the same manner and then treated with calf
intestinal phosphatase (Boehringer Mannheim). The vector and insert
were purified from an agarose gel, then mixed and ligated by T4 DNA
ligase (New England Biolabs). Ligation was done at 4.degree. C. for
2 hrs. Each ligation mixture was transformed by electroporation
into the host strain GM221 described above with a Biorad GenePulser
(Biorad Laboratories) using 2.5V, 25 uFD, and 200 ohms in a cuvette
with a gap length of about 2 mm. After electroporation, the cells
were allowed to recover in 1 ml of Luria broth (LB) for about one
hour at 37.degree. C. with gentle shaking. The entire
transformation mix was plated on LB agar containing 50 ug/ml
kanamycin. Colonies were screened for presence of the desired
molecular weight by PCR using oligonucleotides directed against
flanking vector sequence. The PCR products were evaluated by
agarose gel electrophoresis. Positive clones were further screened
for the ability to produce the recombinant protein product and
finally verified by nucleotide sequencing.
[0103] The DNA and amino acid sequences of TTR are known (Mita, S
et al., Biochem. Biophys. Res. Commun. 124 (2), 558-564 [1984]).
These sequences have been deposited in Genbank as accession number
K02091. The cDNA of native TTR excluding the signal peptide was
cloned from a cDNA library derived from human liver (Clontech).
Specifically, an oligonucleotide encoding eight codons of the TTR
5' (Oligo 2693-79) end and an oligonucleotide encoding seven codons
of TTR 3' end including a terminating codon (Oligo 2693-80) were
synthesized and used to amplify the full-length mature TTR with
Expand Long polymerase using human liver cDNA library as template.
The resulting PCR fragment was digested with NdeI and XhoI, gel
purified and ligated with NdeI/XhoI restricted expression vector
pAMG21. After 2 hours at 4.degree. C., the ligation mixture was
electroporated into GM221 cells. Single colonies were picked and
plasmid DNA was prepared and sequenced. One resulting plasmid
(strain #5316) was shown to have the correct DNA sequence of native
TTR (plus a methionine at the N-terminus) and was used for
expression. This DNA sequence is identified in SEQ ID NO:2.
[0104] Mutant TTR(C10A) was made by using oligonucleotide 2693-80
above and oligonucleotide 2820-88 (encompasses the first 11 codons
of native TTR in which the codon Cys at the tenth position was
changed to Ala). The PCR procedure and the process for selecting
the expression strain were similar to that described above. The
resulting strain (strain #5619) had the DNA sequence identified in
SEQ ID NO:3.
[0105] Plasmid 5619 was further modified by replacing the amino
acids at the following positions: A37, D38, A81 and G83, with the
amino acid Cysteine. As described below, each pair of the
complementary oligonucleotides harboring the desired mutations was
used in conjunction with TTR 5' and 3' primers described above in a
standard two-step PCR procedure designed for site-specific
mutagenesis. Each of the forward primers were used with a TTR 3'
primer and each of the reverse primers were used with a TTR 5'
primer in a 20-cycle PCR in which plasmid derived from strain 5619
was used as the template. The resulting PCR amplified 5' and 3'
fragments were mixed and used as the template for the second step
PCR to generate the full-length mutants. Subsequent cloning and
sequencing procedures were similar to those already described. The
following oligonucleotides were utilized: TTR(A37C) forward (Oligo
2823-91); TTR(A37C) reverse (Oligo 2823-92); TTR(D38C) forward
(Oligo 2823-93); TTR(D38C) reverse (Oligo 2823-94); TTR(A81C)
forward (Oligo 2823-95); TTR(A81C) reverse (Oligo 2823-96);
TTR(G83C) forward (Oligo 2823-97); TTR(G83C) reverse (Oligo
2823-98). The resulting E. coli strains containing the plasmids are
described as follows: TTR(C10A/A37C)(strain 5641) had the DNA
sequence identified in SEQ ID NO:4. TTR(C10A/D38C)(strain 5642) had
the DNA sequence identified in SEQ ID NO:5. TTR(C10A/A81C)(strain
5643) had the DNA sequence identified in SEQ ID NO:6.
TTR(C10A/G83C)(strain 5651) had the DNA sequence identified in SEQ
ID NO:7.
[0106] The Lys in the 15th position in strain 5651 was further
mutagenized to Ala using oligonucleotides 2953-67 and 2953-68 by a
procedure similar to that described for strains 5641, 5642, 5643
and 5651. The resulting strain, TTR(C10A/K15A/G83C)(strain 5895)
had the DNA sequence identified in SEQ ID NO:8.
2TABLE 1 SEQ ID Oligo Sequence Number 2693-79
GAGGAATAACATATGGGTCCAACTGGTACCGGTGAA 18 2693-80
CCGCGGATCCTCGAGATTATTCCTTGGGATTGGTGA 19 2820-88
GAGGAATAACATATGGGTCCAACTGGTACCGGTGAA 20 TCCAAGGCTCCT 2823-91
AGAAAGGCTTGTGATGACACCTGG 21 2823-92 CCAGGTGTCATCACAAGCCTTTCT 22
2823-93 AGAAAGGCTGCTTGTGACACCTGG 23 2823-94
CCAGGTGTCACAAGCAGCCTTTCT 24 2823-95 TACTGGAAGTGTCTTGGCATCTCC 25
2823-96 GGAGATGCCAAGACACTTCCAGTA 26 2823-97
AAGGCACTTTGCATCTCCCCATTC 27 2823-98 GAATGGGGAGATGCAAAGTGCCTT 28
2953-67 CTGATGGTCGCAGTTCTAGAT 29 2953-68 ATCTAGAACTGCGACCATCAG
30
EXAMPLE 2
[0107] This example describes the preparation of various TMP-TTR
fusions. Several fusion proteins containing TTR and a TMP were
prepared. Each of the oligonucleotides utilized in this example are
listed in Table 2.
[0108] A fragment containing the TMP was first amplified from a
strain harboring a plasmid encoding a full-length TMP-Fc fusion
(see PCT Publication No. 00/24770) using oligonucleotides 2743-96
which encodes the first 7 codons of the TMP plus a 12 nucleotide 5'
extension including a Nde1 site and 2743-97 which encodes the first
7 codons of native TTR and the last 7 codons of the TMP of
interest. The resulting PCR fragment was mixed with plasmid derived
from strain 5619 and the mixture was used as a template for
oligonucleotide primers 2743-96 and 2693-80 to amplify full-length
TMP-TTR. Similar procedures described above were used for cloning
and expression. The resulting strain, TMP-TTR (strain 5513) had the
DNA sequence identified in SEQ ID NO:9.
[0109] The TMP was then introduced to the N-terminus of strains
5641, 5642, 5643 and 5651, respectively. Plasmid 5513 was digested
with Xba1, the resulting Xba1/Xba1 insert containing the TMP and
the first 18 codons of TTR(C10A) was gel purified and ligated with
Xba1 restricted, phosphatase treated and gel purified vector
derived from 5641, 5642, 5643 and 5651. DNA sequencing was
performed to select the correct orientation for each fusion. The
resulting E. coli strains containing the plasmids are described as
follows: TMP-TTR(C10A/A37C)(strain 5704) had the DNA sequence
identified in SEQ ID NO:10. TMP-TTR(C10A/D38C)(strain 5705) had the
DNA sequence identified in SEQ ID NO:11. TMP-TTR(C10A/A81C)(strain
5706) had the DNA sequence identified in SEQ ID NO:12.
TMP-TTR(C10A/G83C)(strain 5707) had the DNA sequence identified in
SEQ ID NO:13.
3TABLE 2 SEQ ID Oligo Sequence Number 2743-96
GAGGAATAACATATGATCGAAGGTCCGACTCTGCGT 31 2743 97
TTCACCGGTACCAGTTGGACCTGCGCGTGCTGCAAG 32 CCATT
EXAMPLE 3
[0110] This example describes the preparation of PTH
(1-34)-TTR(C10A/K15A/G83C) fusion. Each of the oligonucleotides
utilized in this example are listed in Table 3.
[0111] Two new oligonucleotides, oligonucleotide 2694-01, which
encodes the first 7 codons of human PTH, and oligonucleotide
2694-03, which encodes the first 7 codons of TTR and amino acids
28-34 of PTH, were synthesized to make the fusion. Oligonucleotides
2694-01 and 2694-03 were used in a 20-cycle PCR procedure as
described above to amplify PTH (1-34) with the TTR linker. The
template for this reaction was a strain which harbors a plasmid
encoding a PTH1-34-Fc fusion (see PCT Publication No. 01/81415).
The resulting PCR mixture was combined with strain 5895 and used as
the template to amplify the full length PTH
(1-34)-TTR(C10A/K15A/G83C) using primers 2694-01 and 2693-80. After
sequence confirmation, the resulting expression strain containing
the new plasmid was designated PTH-TTR(C10A/K15A/G83C)(strain 5920)
and had the DNA sequence identified in SEQ ID NO:14.
4TABLE 3 SEQ ID Oligo Sequence Number 2694-01
GAGGAATAACATATGTCTGTTTCTGAAATCCAG 33 2694-03
TTCACCGGTACCAGTTGGACCAAAGTTATGAACGTC 34
EXAMPLE 4
[0112] This example describes the preparation of an
IL-1ra-TTR(C10A) fusion and a TTR(C10A)-GSGS-IL-1ra fusion. Each of
the oligonucleotides utilized in this example are listed in Table
4.
[0113] To prepare the IL-1ra-TTR(C10A) fusion, two
oligonucleotides, oligonucleotide 2823-13, which encodes the first
7 codons of the human protein IL-1ra, and oligonucleotide 2823-14,
which encodes the last 7 amino acids of IL-1ra and the first 7
amino acids of TTR, were synthesized. The plasmid derived from a
strain which expresses IL-1ra (see PCT Publication No. 91/08285)
was amplified using oligonucleotides 2823-13 and 2823-14. The
resulting PCR product was mixed with plasmid purified from strain
5619 and used as a template to amplify full-length
IL-1-ra-TTR(C10A) using oligonucleotide primers 2823-13 and
2693-80. The PCR product was cloned, sequenced and expressed as
described above. The resultant strain containing the new plasmid
was designated IL-1ra-TTR(C10A)(strain 5644) and had the DNA
sequence identified in SEQ ID NO:15.
[0114] To make TTR(C10A)-IL-1ra, the following two
oligonucleotides, oligonucleotide 2787-32, which encodes the last 7
amino acids of TTR, the first 7 amino acids of IL-1-ra between
which a GSGS linker was introduced, and oligonucleotide 2787-33,
which encodes the last 7 codons of IL-1-ra, were synthesized. These
two oligonucleotide primers were used to amplify plasmid 2693, and
the resulting PCR product was mixed with plasmid 5619, and together
these were used as a template to amplify full-length
TTR(C10A)-IL-1ra using primers 2787-33 and 2693-79. The PCR product
was cloned, sequenced and expressed as described above. The
resultant strain containing the new plasmid was designated
TTR(C10A)-IL-1ra (strain 5645) and had the DNA sequence identified
in SEQ ID NO:16.
5TABLE 4 SEQ ID Oligo Sequence Number 2823-13
GAGGAATAACATATGCGACCGTCCGGACGTAA 35 2823-14
TTCTACTTCCAGGAAGACGAAGGTCCAACTGGTACC 36 2787-32
GTCGTCACCAATCCCAAGGAAGGTAGTGGTAGCCGA 37 CCGTCCGGCCGTAAGAGC 2787-33
CCGCGGATCCTCGAGATTATTCGTCTTCCT- GGAAGT 38 AGAA
EXAMPLE 5
[0115] This example describes the preparation of
TTR(C10A/G83C)-Bradykinin- . Each of the oligonucleotides utilized
in this example are listed in Table 5.
[0116] Plasmid purified from strain 5651 was used for PCR with
oligonucleotide primer 2693-79 and oligonucleotide primer 2943-47,
which is a TTR 3' primer containing a PstI restriction site. This
PCR product was gel purified and restriction digested with NdeI and
PstI. The resulting DNA fragment was used in a ligation mixture
containing AMG21, digested with NdeI and XhoI, and the annealed
oligonucleotide linkers 2943-48, which encodes the GSGSG linker,
and oligonucleotide 2943-49, which encodes the Bradykinin
antagonist peptide KRPPGFSPL with PstI 5' and XhoI 3' overlapping
ends. GM121 was transformed with this ligation product and DNA was
purified from the kanamycin resistant colonies. The DNA sequence
was then confirmed in the resistant colonies. The confirmed strain
was grown at 30.degree. C. and induced for expression in a 10-liter
fermentation described below. The new strain was designated
TTR(C10A/G83C)-Bradykinin (strain 5914) and had the DNA sequence
identified in SEQ ID NO:17.
6TABLE 5 SEQ ID Oligo Sequence Number 2693-79
GAGGAATAACATATGGGTCCAACTGGTACCGGTGAA 39 2943-47
AATATACTGCAGTGGTGGAATAGGAG 40 2943-48
GTCGTCACCAATCCCAAGGAAGGATCAGGATCCGGAAAACGTCCGCCGGGTTTCTCCCCGCTGTAATC
41 2943-49 TCGAGATTACAGCGGGGAGAAACCCGGCGGACGTTTTCCGGATCCTGATCC-
TTCCTTGGGATTGGTGACGACTGCA 42
EXAMPLE 6
[0117] This example describes the recombinant expression of TTR and
the TTR fusion constructs in E. coli. Each of the newly constructed
TTR or TTR fusions were first examined for soluble expression at
temperatures ranging from 16.degree. C. to 37.degree. C. For this
purpose, cultures (25 ml) of GM221 expressing each of the TTR or
TTR fusions were grown in LB medium supplemented with 50 .mu.g/ml
kanamycin at 37.degree. C. until the optical density (OD) at 600 nm
reached 0.5 to 1.0. The cultures were then placed in shakers with
temperature settings at 16.degree. C., 20.degree. C., 25.degree.
C., 30.degree. C., 34.degree. C. and 37.degree. C., respectively.
The induction of gene product expression from the luxPR promoter
was achieved following the addition of the synthetic autoinducer
N-(3-oxohexanoyl)-DL-homoserine lactone to the culture media to a
final concentration of 20 ng/ml. After 6 hours, the bacterial
cultures were examined by microscopy for the presence of inclusion
bodies. Often soluble or partial soluble expression could be
achieved by growing the cultures at temperatures lower than
30.degree. C. for TTR and its fusions, and this temperature was
used for large-scale expression. In cases where soluble expression
could not be achieved, temperatures at which the level of
expression was at the highest were used for large-scale shakers or
fermentors.
[0118] Large-scale expression was normally done in 4 liter flasks.
Four to eight 4 liter shakers containing 1 liter of LB was
inoculated with overnight cultures of TTR or its fusion strains.
Expression was done essentially as described above. Cells were
collected by centrifugation.
[0119] The fermentation stage, employing aseptic technique, begins
with the inoculation from a seed culture of strains produced in a
shake flask containing 500 mL of sterilized Luria broth. When this
culture obtained the appropriate cell density (0.8-2 at 600 nm),
the contents were used to inoculate a 20 liter fermentor containing
10 liter of complex based growth medium. The fermentor is
maintained at 30.degree. C. and pH 7 with dissolved oxygen levels
kept at 30% saturation. When the cell density reached an optical
density of 10-12 OD units at 600 nm, at which point the culture was
induced by the addition of N-(3-oxo-hexanoyl) homoserine lactone.
At 6 hours post-induction the cells were harvested from the
fermentor by centrifugation.
EXAMPLE 7
[0120] This example describes the purification of
TTR(C10A/G83C)-Bradykini- n. About 193 g of E. coli paste from
clone 5914 stored at -80.degree. C. was defrosted in 1447 ml of 50
mm tris HCl, 5 mM EDTA, pH 8.0.50 tablets of Sigma protease
inhibitor cocktail 1-873-580 (Saint Louis, Mo.) was dissolved in
the cell suspension and the suspension was passed through a model
110-Y microfluidizer (Microfluidics, Newton, Mass.) twice at 12,000
PSI. The lysate (FIG. 1, Lane 2) was centrifuged at 11,325.times.g
for 50 min 4.degree. C. The supernatant was removed as the soluble
fraction. The soluble fraction was heated in a 65.degree. C. water
bath for 30 minutes in polypropylene bottles, at which time the
temperature of the contents was 63.degree. C. The soluble fraction
was centrifuged at 11,325.times.g for 50 minutes 40C. The
supernatant was removed as Heat Soluble (FIG. 1, Lane 3). The heat
soluble fraction was filtered through a 0.45 .mu.m cellulose
acetate filter with two prefilters and then loaded on to a 240 ml
Q-sepharose fast flow (5 cm ID) column (Amersham Pharmacia Biotech,
Piscataway, N.J.) at 20 ml/min equilibrated in Q-Buffer A (20 mM
tris HCl, 2.5 mM EDTA, pH 8.0) at room temperature (about
23.degree. C.). Column was washed with about 2300 ml Q-Buffer A at
20 ml/min. Q-column was eluted with a 15 column volume linear
gradient to 60% Q-Buffer B (20 mM tris HCl, 1 M NaCl, 2.5 mM EDTA,
pH 8.0) followed by a 2 column volume step to 100% Q-Buffer B.
Fractions containing the TTR fusion as determined by SDS-PAGE were
pooled into a single Q-pool (1150 ml) (FIG. 1, Lane 4) and 1.77 g
of DTT was added. The Q-pool was gently stirred for 30 min at room
temperature (about 23.degree. C.). To the Q-pool, 410 ml of 3.8 M
ammonium sulfate pH 7.0 was slowly added and the pH was lowered
from about 7.5 to 7.0 by slow addition of 1 M HCl. About one-half
of the Q-pool was then loaded on to an 84 ml phenyl sepharose high
performance column (2.6 cm ID) (Amersham Pharmacia Biotech) in
P-Buffer A (50 mM NaH.sub.2PO.sub.4, 1 M ammonium sulfate, pH 7.0)
at 10 ml/min. The column was washed with about 170 ml P-Buffer A
followed by three step elutions using 50%, 80%, and 100% P-Buffer B
(50 mM NaH.sub.2PO.sub.4, pH 7.0). The remaining half of the Q-pool
was then processed using the same protocol as the first half.
Fractions containing the TTR fusion as determined by SDS-PAGE were
pooled into a single P-pool (260 ml) (FIG. 1, Lane 5) and the
P-pool was dialyzed against 4 L of HA-Buffer A (10 mM
NaH.sub.2PO.sub.4, pH 7.0) for 2 hours at room temperature (about
23.degree. C.) using 20.4 mm diameter 8 kDa cutoff dialysis tubing
(Spectrum Laboratories Inc., Rancho Dominguez, Calif.). The
dialysis buffer was changed with a fresh 4 L of HA-Buffer A and
dialysis was continued for approximately an additional 15 hours.
The P-pool was removed from dialysis and 600 .mu.l of 1 M DTT was
added followed by incubation at room temperature (about 23.degree.
C.) for about 1 hour. P-pool was loaded on to a 105 ml (2.6 cm)
type 1 ceramic hydroxyapatite column (Bio-Rad Inc., Hercules,
Calif.) at 10 ml/min in HA-Buffer A. Column was washed with
approximately 210 ml HA-Buffer A at 10 ml/min followed by 4 steps
of 12.5%, 25%, 50%, and 100% HA-Buffer B (400 mM NaH.sub.2PO.sub.4,
pH 7.0). The flowthrough was pooled as HA-pool (340 ml) (FIG. 1,
Lane 6) and 524 mg of DTT was added followed by incubation at room
temperature (about 23.degree. C.) for 1 hour.
[0121] About one-half of the HA-pool was loaded on to a 47 ml
source 15Q (2.6 cm ID) column (Amersham Pharmacia Biotech) at 10
ml/min followed by a wash with about 250 ml Q-Buffer A. Column was
eluted with a 20 column volume linear gradient from 10% to 50%
Q-Buffer B followed a step of 2 column volumes of 100% Q-Buffer B.
The remaining half of the HA-Pool was then processed using the same
protocol as the first half. Fractions containing the TTR fusion as
determined by SDS-PAGE were pooled into a single Q2-pool (260 ml)
and concentrated to about 75 ml using a stirred cell with a 10 kDa
membrane. Q2-pool (FIG. 1, Lane 7) was then filtered through a 0.22
.mu.m cellulose acetate filter and the protein concentration was
determined to be 16.9 mg/ml using a calculated extinction
coefficient of 18,450 M.sup.-1 cm.sup.-1. The pyrogen level was
determined to be <1 EU/mg of protein using the Limulus
Ameboycyte Lysate assay (Associates of Cape Cod, Falmouth, Mass.).
The nucleic acid content was determined to be negligible, since the
ratio of the absorbance at 260 nm over 280 nm was determined to be
0.52.
EXAMPLE 8
[0122] This example demonstrates that fusing a peptide to either
the C-terminus or N-terminus of TTR(C10A/G83C)does not have a
significant impact on its oligomeric structure. TTR(C10A/G83C),
PTH-TTR(C10A/K15A/G83C), and TTR(C10A/G83C)-Bradykinin in 20 mM
tris pH 8.0 and about 250 mM NaCl were reduced with 9 mM DTT for
about 1 hour at room temperature (about 23.degree. C.). About 50
.mu.g of the reduced TTR was injected on to a Biosep-Sec-S 3000
column (7.8 mm ID.times.300 mm) (Phenomenex, Torrance, Calif.) in
SEC-Buffer (50 mM NaH.sub.2PO.sub.4, 500 mM NaCl, pH 6.7) at 1
ml/min. Bio-Rad molecular weight standards (151-1901) were used to
calibrate the column and calculate the approximate molecular size
of the injected samples. As can be seen in FIG. 2, TTR(C10A/G83C)
eluted at approximately 8.8 min corresponding to a molecular size
of 49 kDa, which is comparable to the calculated molecular weight
of the tetramer at 55 kDa. PTH-TTR(C10A/K15A/G83C) eluted at about
8.6 min corresponding to a molecular size of 67 kDa, which is close
to the calculated 71 kDa for the tetramer.
TTR(C10A/G83C)-Bradykinin eluted at about 8.7 min corresponding to
a molecular size of 57 kDa, which is also close to the calculated
60 kDa for the tetramer.
EXAMPLE 9
[0123] This example demonstrates that fusing a protein containing
disulfide bonds to either the C-terminus or N-terminus of TTR(C10A)
does not have a significant impact on its oligomeric structure.
About 50 .mu.g each of TTR(C10A), IL-1-ra-TTR(C10A), and
TTR(C10A)-IL-1-ra was injected on to a Biosep-Sec-S 3000 column
(7.8 mm ID.times.300 mm) (Phenomenex) in SEC-Buffer at 1 ml/min.
Bio-Rad molecular weight standards (151-1901) were used to
calibrate the column and calculate the approximate molecular weight
of the injected samples. As can be seen in FIG. 3, TTR(C10A) elutes
at approximately 8.8 min, which corresponds to a molecular size of
49 kDa which is comparable to the calculated molecular weight of
the tetramer at 55 kDa. The IL-1-ra-TTR(C10A) fusion eluted at
about 7.9 min corresponding to a molecular size of 188 kDa, which
is noticeably larger than that expected for the tetramer at 124
kDa. Similarly, TTR(C10A)-IL-1-ra eluted at about 7.9 min, again
corresponding to a molecular size of 188 kDa compared to the 124
kDa expected for the tetramer. These size discrepancies are likely
due to differences in the shape of the molecule, since size
exclusion chromatography is shape dependant and the standards are
calibrated for globular proteins.
EXAMPLE 10
[0124] This example compares the binding of a TMP sequence fused to
the carboxy-terminus of human immunoglobulin Fc (Fc-TMP) and
TMP(m)-TTR to soluble human myeloproliferative leukemia (MPL)
receptor. In addition, this example shows the effect of pegylation
of the native TTR cysteine on the binding of the TMP fusion to the
MPL receptor. The preparation of the pegylated TTR fusions is
described in detail in Example 13.
[0125] For this example, human MPL receptor was covalently bound to
a BIAcore CM5 chip at R.sub.L=1300 R.sub.U using the EDC/NHS
chemistry as per the manufacturer's instructions (BIAcore, Uppsula,
Sweden). All samples were passed over the chip at 50 .mu.l/min in
Dulbecco's PBS (Gibco BRL, Gaithersburg, Md.) with 0.1 mg/ml bovine
serum albumin and 0.005% P20 (polyoxyethylenesorbitan). The
equilibrium endpoint was taken 3 min post injection. As can be seen
in FIG. 4, Fc-TMP shows superior binding characteristics compared
to TMP(m)-TTR. Further, this figure demonstrates that pegylation of
the native TTR cysteine (Cys 10) interferes with the binding of TMP
to the MPL receptor. The binding of TMP(m)-TTR-PEG5K showed a
significantly repressed binding response compared to its
non-pegylated counterpart, and TMP(m)-TTR-PEG20K showed an even
more severe inhibition. This indicates that the presence of PEG on
cysteine 10 likely causes steric interference for binding of the
fused TMP to the MPL receptor, and larger PEGs produce more
interference.
EXAMPLE 11
[0126] This example shows the effect of injecting TMP(m)-TTR into
mice on blood platelet count. For this example 50 BDF1 mice
(Charles River Laboratories, Wilmington, Mass.) were split into 5
groups and injected (day 0) subcutaneously with either diluting
agent (Dulbecco's PBS with 0.1% bovine serum albumin) or diluting
agent with 50 .mu.g test protein per kg animal. Each group was
divided in half and bled (140 .mu.l) on alternate time points (day
0, 3, 5, 7, 11, 12, 14, and 17). Mice were anesthetized with
isoflurane prior to collection.
[0127] The collected blood was analyzed for a complete and
differential count using an ADVIA 120 automated blood analyzer with
murine software (Bayer Diagnostics, New York, N.Y.). As seen in
FIG. 5, Fc-TMP showed the greatest response with platelet count
peaking at 4.3.times.10.sup.12 platelets L.sup.-1 on day 5, which
is over 3.4 times baseline at 1.2.times.10.sup.12 platelets
L.sup.-1. TMP(m)-TTR-PEG 5K was a moderate responder peaking at
2.3.times.10.sup.12 platelets L.sup.-1 which is just under twice
the baseline level. The non-pegylated form of TMP(m)-TTR shows very
little response at 1.5.times.10.sup.12 platelets L.sup.-1 which is
only 20% over the baseline level. The non-pegylated form of
TMP(m)-TTR shows better binding in vitro than its pegylated
counterparts (FIG. 4), but it has poor performance in vivo compared
to TMP(m)-TTR-PEG 5K. This indicates that PEG is required to
improve the biological half-life of the TTR construct, and this
more than compensates for the reduced affinity for the
receptor.
EXAMPLE 12
[0128] This example demonstrates that mutation of cysteine 10 on
TTR to alanine TTR(C10A) does not have a significant impact on its
oligomeric structure. About 50 .mu.g each of TTR and TTR(C10A) was
injected on to a Biosep-Sec-S 3000 column (7.8 mm ID.times.300 mm)
(Phenomenex) in SEC-Buffer at 1 ml/min. Bio-Rad molecular weight
standards (151-1901) were used to calibrate the column and
calculate the approximate molecular size of the injected samples.
As can be seen in FIG. 6, TTR(C10A) elutes at approximately 8.8
min, which corresponds to a molecular size of 57 kDa which is
similar to the calculated molecular weight of the tetramer at 55
kDa. This data combined with the observation that both forms of TTR
are resistant to precipitation at 65.degree. C. (data not shown)
indicates that mutation of cysteine 10 to alanine does not have a
significant impact on the structure or stability of TTR.
EXAMPLE 13
[0129] This example demonstrates that mutation of alanine 37 to
cysteine TMP-TTR(C10A/A37C), aspartate 38 to cysteine
TMP-TTR(C10A/D38C), alanine 81 to cysteine TMP-TTR(C10A/A81C), or
glycine 83 to cysteine TMP-TTR(C10A/G83C) in a cysteine 10 to
alanine background does not have a significant impact on the
oligomeric structure of TTR. In addition, this example demonstrates
that pegylation of these mutant forms of TTR with a 5K or 20K PEG
produces two distinct species of TTR with significantly greater
molecular size than the unpegylated form. The pegylation of TTR was
carried out by first reducing about 8 ml of the TTR (7.28 mg/ml)
with 10 mM DTT for 30 minutes at 30.degree. C. in the presence of
50 mM tris HCl, pH 8.5. The reduced TTR was then desalted using a
26 ml SEPHADEX.TM. G25 medium column (2.6 cm ID) (Amersham
Pharmacia Biotech) at 2.5 ml/min in 20 mM tris HCl, pH 8.5. The
concentration was then determined by measuring the absorbance of
the reduced TTR at 280 nm and using the calculated extinction
coefficient (29,450 M.sup.-1 for TMP-TTR(C10A/A37C) (5.14 mg/ml).
One-half (4.6 ml) of the reduced sample was then immediately mixed
with 810 .mu.l of 5 mM methoxy-PEG-maleimide 5K (Shearwater
Corporation, Huntsville, Ala.) and the remaining half was mixed
with 1620 .mu.l 2.5 mM methoxy-PEG-maleimide 20K (Shearwater
Corporation). The reaction was allowed to proceed at 30.degree. C.
for 30 min and was quenched by the addition of 46 .mu.l 1 M DTT.
Each pegylated sample was then loaded on to a 5 ml HiTrap
Q-sepharose column at 2.5 ml/min and washed with several column
volumes of Q-Buffer A (20 mM tris HCl, pH 8.0) at 5 ml/min. The
columns were eluted with a linear gradient to 40% Q-Buffer B (20 mM
tris HCl, 1 M NaCl, pH 8.0) followed by a 2 column volume step to
100% Q-Buffer B. Peak fractions were pooled and the concentration
determined by measuring the absorbance of the pool at 280 nm. About
50 .mu.g of each sample was injected on to a Biosep-Sec-S 3000
column (7.8 mm ID.times.300 mm) (Phenomenex) in SEC-Buffer at 1
ml/min. Bio-Rad molecular weight standards (151-1901) were used to
calibrate the column and calculate the approximate molecular size
of the injected samples. As can be seen in FIG. 7, the apparent
molecular size of the 4 non-pegylated TMP-TTR constructs is between
40 and 45 kDa which is noticeably lower than the expected 70 kDa
tetramer. This retarded elution time is likely due to a slight
interaction of the TMP-TTR construct with the size exclusion resin,
which has been observed with several other TMP constructs (data not
shown). After conjugation with the 5K PEG, the apparent molecular
size increases to between 421 and 428 kDa (1.53-1.64 minutes more
advanced elution than the unpegylated counterparts), which is much
greater than the expected 90 kDa. The observation of an exaggerated
molecular weight of pegylated molecules on size exclusion
chromatography is frequently observed phenomenon (data not shown).
The 20K PEG constructs elute earlier than the largest calibration
standard (670 kDa) showing a 1.28-1.40 minutes more advanced
elution than their 5K pegylated counterparts. This data taken
together demonstrates that all 4 engineered mutant forms of TMP-TTR
can be pegylated drastically increasing their apparent molecular
size.
[0130] About 2 .mu.g of the pegylated TMP-TTR constructs were
analyzed by SDS-PAGE (FIG. 8). This figure demonstrates by gel
shift that most of the TMP-TTR monomers were modified by only one
methoxy-PEG-maleimide, and the reaction was nearly complete leaving
very little unmodified monomer.
EXAMPLE 14
[0131] This example demonstrates that Fc-TMP, TMP-TTR(C10A/A37C),
TMP-TTR(C10A/D38C), TMP-TTR(C10A/A81C), and TMP-TTR(C10A/G83C) have
similar affinities for binding human MPL receptor in vitro. For
this example, Fc-TMP was bound to a BIAcore protein G chip at high
density as per the manufacturer's instructions (BIAcore, Uppsula,
Sweden). Test proteins were preincubated with 5 nM MPL receptor in
Binding Buffer (Dulbecco's PBS (Gibco BRL, Gaithersburg, Md.) with
0.1 mg/ml bovine serum albumin and 0.005% P20
(polyoxyethylenesorbitan) for >2 hours at room temperature
(about 23.degree. C.). For non-pegylated proteins, 0.1 mg/ml
heparin was added to prevent non-specific binding. All samples were
then passed over the chip at 50 .mu.l/min in Binding Buffer. The
equilibrium endpoint was taken 3 min post injection. As can be seen
in FIG. 9, all TTR constructs showed similar affinity for the MPL
receptor with affinities ranging from 0.881 to 2.333 nm, while the
Fc-TMP construct had affinities ranging from 3.276 to 5.369 nm.
EXAMPLE 15
[0132] This example shows the effect of injecting pegylated TMP-TTR
constructs into mice on blood platelet count. For this example 170
BDF1 mice were split into 17 groups and injected (day 0)
subcutaneously with 50 .mu.g test protein per kg animal (TMP fusion
construct, Fc-TMP, or a TTR(C10A) control). Each group was divided
in half and bled (140 .mu.l) on alternate time points (day 0, 3, 5,
7, 10, 12, and 14). Mice were anesthetized with isoflurane prior to
collection.
[0133] The collected blood was analyzed for a complete and
differential count using an ADVIA 120 automated blood analyzer with
murine software (Bayer Diagnostics, New York, N.Y.). As seen in
FIG. 10A, Fc-TMP showed the greatest response with platelet count
rising to over 4.2.times.10.sup.12 platelets L.sup.-1 on day 5
which is 3 times baseline at 1.4.times.10.sup.12 platelets
L.sup.-1. All 4 of the non-pegylated TMP-TTR constructs preformed
better than the control, but not as well as Fc-TMP with platelet
counts between 1.8 and 2.9.times.10.sup.12 platelets L.sup.-1 on
day 5, which is between a 29% and 107% improvement over baseline.
As can be seen in FIG. 10B, addition of a 5K PEG group to the
engineered cysteine of all 4 TMP-TTR constructs substantially
improves efficacy with platelet counts between 3.7 and
4.4.times.10.sup.12 platelets L.sup.-1 (2.8 to 3.4 times
baseline).
[0134] Also as can be seen in FIG. 10C, conjugation of a 20K PEG to
TMP-TTR results in an additional, but less dramatic improvement in
efficacy with platelet counts between 4.2 and 4.6.times.10.sup.12
platelets L.sup.-1 (3.2 to 3.5 times baseline). Since all of the
TMP fusion constructs had similar binding affinities for MPL in
vitro, this difference is likely due to the effect of PEG
conjugation increasing the effective biological half-life of the
construct.
EXAMPLE 16
[0135] This example shows the effect of injecting pegylated PTH-TTR
constructs into mice on blood ionized calcium release. For this
example 60 male, BDF1, 4 week-old mice were split into 12 groups
and injected (day 0) subcutaneously with 8.91 mg test protein per
kg animal (PTH fusion construct, PTH-Fc, or a TTR(C10A) control).
Each group was bled (75 .mu.l) at time points 0, 24, 48, and 72
hours. Mice were anesthetized with isoflurane prior to
collection.
[0136] The collected blood was analyzed for ionized calcium using a
Ciba*Corning 634 Ca++/pH analyzer. As seen in FIG. 11, PTH-Fc,
PTH-TTR(C10A/K15A/A37C) (PEG 5K), PTH-TTR(C10A/K15A/A37C) (PEG
20K), PTH-TTR(C10A/K15A/G83C) (PEG 5K), and PTH-TTR(C10A/K15A/G83C)
(PEG 20K) showed the greatest response with ionized calcium levels
rising between 2.2 and 2.7 mmol per L at 24 hours post-injection,
which is 1.7 times baseline at 1.3 mmol per L. At 72 hours post
injection, the ionized calcium levels of all groups returned to
baseline, except PTH-TTR(C10A/K15A/A37C) (PEG 5K),
PTH-TTR(C10A/K15A/G83C) (PEG 5K), and PTH-TTR(C10A/K15A/G83C) (PEG
20K) treated groups that maintained elevated ionized calcium levels
between 1.8 and 1.9 mmol per L. The non-pegylated PTH-TTR
constructs were equivalent to or slightly better than the TTR(C10A)
control at raising serum ionized calcium levels.
EXAMPLE 17
[0137] This example describes the construction of a
PTH-TTR(C10A/K15A/A81C) containing plasmid. The Xba1/Xba1 fragment
of 5920 was ligated with the purified vector derived from digesting
plasmid 5643 (described in example 1) with Xba1. The E. coli strain
containing the resulting plasmid is described as 5933
PTH-TTRC10A/K15A/A81C.
7 ATGTCTGTTTCTGAAATCCAGCTGATGCATAACCTGGGTAAACATCTGAACTCTA SEQ ID
NO:43 TGGAACGTGTTGAATGGCTGCGTAAGAAACTGCAGGACGTTCATAACTTT- GGTCC
AACTGGTACCGGTGAATCCAAGGCTCCTCTGATGGTCGCAGTTCTAGATGCT- GTC
CGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGTGTTCAGAAAGGCTGCTGAT- G
ACACCTGGGAGCCATTTGCCTCTGGGAAAACCAGTGAGTCTGGAGAGCTGCATGG
GCTCACAACTGAGGAGGAATTTGTAGAAGGGATATACAAAGTGGAAATAGACACC
AAATCTTACTGGAAGTGTCTTGGCATCTCCCCATTCCATGAGCATGCAGAGGTGG
TATTCACAGCCAACGACTCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAG
CCCCTACTCCTATTCCACCACGGCTGTCGTCACCAATCCCAAGGAATAA:
EXAMPLE 18
[0138] This example describes the preparation of a
GLP-1-TTR(C10A/G83C) fusion and a GLP-1-TTR(C10A/K15A/G83C) fusion.
These constructs were cloned using plasmid pAMG21, which is
described in example 1. Each of the oligonucleotides utilized in
this example are listed in Table 6.
[0139] The bacterial host GM121 is an E. coli K-12 strain that has
been modified to contain the lacI.sup.Q repressor in the late ebg
region (68 minutes). The presence of this repressor gene allows the
use of this host with a variety of expression systems, however this
repressor is irrelevant to the expression from luxPR. The
untransformed host has no antibiotic resistances. Specifically,
F'tet/393 was modified by the delivery of a lacI.sup.Q construct
into the ebg operon between nucleotide position 2493 and 2937 as
numbered in the Genbank accession number M64441 Gb_Ba with the
deletion of the intervening ebg sequence. The construct was
delivered to the chromosome using a recombinant phage called
AGebg-lacI.sup.Q #5.
[0140] After recombination and resolution only the chromosomal
insert described above remains in the cell. It was renamed
F'tet/GM120. F'tet/GM120 was then mutated in the hsdR gene to
inactivate it. This was renamed F'tet/GM121. The F'tet episome was
cured from the strain, verified as tetracyline sensitive and was
stored as GM121 (ATCC #202174).
[0141] PCR was performed with Roche PCR Core Kit (Cat. No. 1 578
553) in 80 ul reactions containing 2-4 ul mini-prep plasmid DNA
template, 1 uM each oligonucleotide, 0.2 mM each oligonucleotide,
5% DMSO (Sigma), and 2U Taq DNA polymerase in order to amplify the
GLP-1 sequence and a linker. Reaction cycles were 94.degree. C. for
5 min followed by 35 cycles of [94.degree. C. for 20 sec,
45.degree. C. for 30 sec, 72.degree. C. for 1 min]. PCR products
were purified with QIAquick.RTM. PCR Purification Kit according to
the manufacturer's protocol (QIAGEN). PCR products and vectors were
then digested with NdeI and KpnI (New England Biolabs).
[0142] Digested DNA was purified from an agarose gel, then mixed
and ligated by T4 DNA ligase (New England Biolabs) for 1.5-2 hours
at room temperature. Each ligation mixture was transformed by
electroporation into the host strain GM121 described above with a
Biorad E. coli Pulser at 2.5 KV in a cuvette with a gap length of 2
mm. The cells were allowed to recover in 2 ml Terrific Broth (TB)
for about 3 hours at 37.degree. C. at 250 rpm. 70-100 .mu.l of the
recovery culture was plated on LB agar containing 40 ug/ml
kanamycin. DNA mini-preps were prepared and correct clones were
verified by nucleotide sequencing.
[0143] To prepare the GLP-1-TTR(C10A/G83C) fusion, two
oligonucleotides, oligonucleotide 1209-85, which binds the luxR
promoter region, and 3131-63, which encodes the last 12 amino acids
of the fusion linker and the first 8 amino acids of TTR, were
synthesized. A pAMG21 plasmid derived from a strain which expresses
a GLP-1 sequence with a N-terminal Met-Lys start followed by a
seven Histidine sequence for nickel column purification, an
Aspartic acid-Glutamic acid-Valine-Aspartic acid sequence for
cleavage before the first Histidine of GLP-1 by caspase, the
GLP-1(A2G) sequence, and a 27 amino acid fusion linker was
amplified using oligonucleotides 1209-85 and 3131-63. The PCR
product was cloned and sequenced as described above. The resultant
strain containing the new plasmid was designated
GLP-1-TTR(C10A/G83C) (strain 6298) and had the DNA sequence
identified in SEQ ID NO:47.
[0144] To prepare the GLP-1-TTR(C10A/K15A/G83C) fusion, two
oligonucleotides, oligonucleotide 3183-83, which contains and NdeI
site and encodes the purification and cleavage sequence described
above plus the first six amino acids of GLP-1(A2G), and 3183-84,
which encodes the last 6 amino acids of the fusion linker and the
first 8 amino acids of TTR, were synthesized.
[0145] A pAMG21 plasmid derived from a strain which expresses a
GLP-1 sequence with a N-terminal Met-Lys start followed by a seven
Histidine sequence for nickel column purification, an Aspartic
acid-Glutamic acid-Valine-Aspartic acid sequence for cleavage
before the first Histidine of GLP-1 by caspase, the GLP-1(A2G)
sequence, and a 25 amino acid fusion linker was amplified using
oligonucleotides 3183-83 and 3183-84. The PCR product was cloned
and sequenced as described above. The resultant strain containing
the new plasmid was designated GLP-1-TTR(C10A/K15A/G83C) (strain
6450) and had the DNA sequence identified in SEQ ID NO:48.
8TABLE 6 SEQ ID Oligo Sequence Number 1209-85
CGTACAGGTTTACGCAAGAAAATGG 44 3131-63
GGATTCACCGGTACCAGTTGGACCACCACCACCAC 45 CACCACCCGCACTGCCTGAACCAGAGC
3183-83 TGACTAAGCCATATGAAACATCATCACCATCACCAT 46
CATGACGAAGTTGATCACGGTGAAGGTACTTTCAC 3183-84
GGATTCACCGGTACCAGTTGGACCACCACCACCAC 47 CACCGCTAC
[0146]
9 ATGAAACATCATCACCATCACCATCATGACGAAGTTGATCACGGTGAAGGTACTT SEQ ID
NO:48 TCACTTCTGACGTTTCTTCTTATCTGGAAGGTCAGGCTGCTAAAGAATTC- ATCGC
TTGGCTGGTTAAAGGTCGTGGTGGTTCTGGTTCTGCTACTGGTGGTTCCGGC- TCC
ACCGCAAGCTCTGGTTCAGGCAGTGCGGGTGGTGGTGGTGGTGGTGGTCCAACT- G
GTACCGGTGAATCCAAGGCTCCTCTGATGGTCAAAGTTCTAGATGCTGTCCGAGG
CAGTCCTGCCATCAATGTGGCCGTGCATGTGTTCAGAAAGGCTGCTGATGACACC
TGGGAGCCATTTGCCTCTGGGAAAACCAGTGAGTCTGGAGAGCTGCATGGGCTCA
CAACTGAGGAGGAATTTGTAGAAGGGATATACAAAGTGGAAATAGACACCAAATC
TTACTGGAAGGCACTTTGCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTC
ACAGCCAACGACTCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCCT
ACTCCTATTCCACCACGGCTGTCGTCACCAATCCCAAGGAATAA
[0147]
10 ATGAAACATCATCACCATCACCATCATGACGAAGTTGATCACGGTGAAGGTACTT SEQ ID
NO:49 TCACTTCTGACGTTTCTTCTTATCTGGAAGGTCAGGCTGCTAAAGAATTC- ATCGC
TTGGCTGGTTAAAGGTCGTGGTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCT- GGT
GGTGGTGGTTCTGGCGGCGGTGGTAGCGGTGGTGGTGGTGGTGGTCCAACTGGT- A
CCGGTGAATCCAAGGCTCCTCTGATGGTCGCAGTTCTAGATGCTGTCCGAGGCAG
TCCTGCCATCAATGTGGCCGTGCATGTGTTCAGAAAGGCTGCTGATGACACCTGG
GAGCCATTTGCCTCTGGGAAAACCAGTGAGTCTGGAGAGCTGCATGGGCTCACAA
CTGAGGAGGAATTTGTAGAAGGGATATACAAAGTGGAAATAGACACCAAATCTTA
CTGGAAGGCACTTTGCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACA
GCCAACGACTCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCCTACT
CCTATTCCACCACGGCTGTCGTCACCAATCCCAAGGAATAA
EXAMPLE 19
[0148] This example describes the preparation of a GLP-1(A2G)-K-Fc
fusion. This construct was cloned using plasmid pAMG33*, which
differs from pAMG21 in that the lux protein and promoters are
replaced with lacI binding sites and an IPTG inducible promoter and
the ribosomal binding site sequence is shorter (the sequence
between the AatII and ClaI recognition sites is replaced with
AATTGTGAGCGGATAACAATTGAC
AAATGCTAAAATTCTTGATTAATTGTGAGCGGATAACAATTTATCGATTTGATTC
TAGAAGGAGGAATAA) and some of the sequence after the SacII
recognition site was deleted (leaving
11 ATAAATAAGTAACGATCCGGTCCAGTAATGACCTCAGAAC
TCCATCTGGATTTGTTCAGAACGCTCGGTTGCCGCCGGGCGTTTTTTATT
GGTGAGAATCGCAGCAACTTGTCGCGCCAATCGAGCCATGTCGTCGTCAA
CGACCCCCCATTCAAGAACAGCAAGCAGCATTGAGAACTTTGGAATCCAG
TCCCTCTTCCACCTGCTGACCG).
[0149] Each of the oligonucleotides utilized in this example are
listed in Table 7.
[0150] To prepare the GLP-1(A2G)-Fc fusion, two oligonucleotides,
oligonucleotide 2985-92, which contains and NdeI site and encodes
the purification and cleavage sequence described above plus the
first eight amino acids of GLP-1(A2G), and 2687-50, which encodes
the amino acids 18 through 23 of the Fc, were synthesized. A
pAMG33* plasmid derived from a strain which expresses a GLP-1(A2G)
sequence with a N-terminal Met start, a 27 amino acid linker, and
an Fc sequence was amplified using oligonucleotides 2985-92 and
2687-50. The PCR product was cloned and sequenced as described
above except the enzymes used were NdeI and EcoRI. A colony
screening step was included which verified the presence of insert
by PCR with oligonucleotides directed against upstream vector
sequence and the 5 His-Aspartic acid sequence which the insert
introduced. The resultant strain containing the new plasmid was
designated GLP-1(A2G)-K-Fc (strain 5945) and had the DNA sequence
identified in SEQ ID NO:51.
12TABLE 7 SEQ ID Oligo Sequence Number 2985-92
AGACCTGTACATATGAAACATCATCACCATCACCAT 50
CATGACGAAGTTGATCACGGTGAAGGTACTTTCAC TTCTG 2687-50
GGGGGAAGAGGAAAACTGAC 51
[0151]
13 ATGAAACATCATCACCATCACCATCATGACGAAGTTGATCACGGTGAAGGTACTT SEQ ID
NO: 52 TCACTTCTGACGTTTCTTCTTATCTGGAAGGTCAGGCTGCTAAAGAATT- CATCGC
TTGGCTGGTTAAAGGTCGTGGTGGTTCTGGTTCTGCTACTGGTGGTTCCGG- CTCC
ACCGCAAGCTCTGGTTCAGGCAGTGCGACTCATGGTGGTGGTGGTGGTGACAA- AA
CTCACACATGTCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTTTT
CCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTC
ACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGT
ACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA
CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTG
AATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCG
AGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCT
GCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTC
AAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG
AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCT
CTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCA
TGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCC
TGTCTCCGGGTAAA
EXAMPLE 20
[0152] This example describes the cloning of the CH2 domain of an
immunoglobulin molecule to the TTR(C10A) to generate
TMP-CH2-TTRC10A and TTRC10A-CH2-TMP.
[0153] The CH2 domain derived from TMP-Fc was linked to the
C-terminal end of TTR(C10A), i.e., strain 5619, by a two-step PCR
procedure. The CH2 domain (containing from 5' to 3': the last 7
codons of TTR, CH2 and a BamH1-XhoI linker) was first amplified by
the following oligos:
[0154] 2973-77:
14 (SEQ ID NO:53) GTC GTC ACC AAT CCC AAG GAA GGT TCT GGC TCC GGA
TCA GGG GGA CCG TCA GTT TTC CTC, and
[0155] 2973-78:
15 (SEQ ID NO:54) CCG CGG ATC CTC GAG ATT AGG ATC CAG AAC CCC CTT
TGG CTT TGG AGA TGG T.
[0156] This fragment was then fused to 5619 in a subsequent PCR by
oligos 2973-78 and
[0157] 2973-79:
16 (SEQ ID NO:55) GAG GAA TAA CAT ATG GGT CCA ACT GGT ACC GGT GAA
TCC AAG,
[0158] followed by Nde1 /XhoI digest and cloning into similarly
restricted pAMG21. The resulting plasmid is described as 6017
(TTRC10A-CH2):
17 ATGGGTCCAACTGGTACCGGTGAATCCAAGGCTCCTCTGATGGTCAAAGTTCTAG SEQ ID
NO:56 ATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGTGTTCAGA- AAGGC
TGCTGATGACACCTGGGAGCCATTTGCCTCTGGGAAAACCAGTGAGTCTGGA- GAG
CTGCATGGGCTCACAACTGAGGAGGAATTTGTAGAAGGGATATACAAAGTGGAA- A
TAGACACCAAATCTTACTGGAAGGCACTTGGCATCTCCCCATTCCATGAGCATGC
AGAGGTGGTATTCACAGCCAACGACTCCGGCCCCCGCCGCTACACCATTGCCGCC
CTGCTGAGCCCCTACTCCTATTCCACCACGGCTGTCGTCACCAATCCCAAGGAAG
GTTCTGGCTCCGGATCAGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAA
GGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTG
AGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGC
ATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGT
CAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGC
AAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCA
AAGGGGGTTCTGGATCCTAA:
[0159] The Xba1/Xba1 fragment of 6017 was replaced with the
corresponding fragment of 5704 as described above to construct
TMP-TTRC10A-CH2 (Strain 6024):
18 ATGATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCTGGCGGTGGTG SEQ ID
NO:57 GCGGAGGGGGTGGCATTGAGGGCCCAACCCTTCGCCAATGGCTTGCAGCA- CGCGC
AGGTCCAACTGGTACCGGTGAATCCAAGGCTCCTCTGATGGTCAAAGTTCTA- GAT
GCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGTGTTCAGAAAGGCT- G
CTGATGACACCTGGGAGCCATTTGCCTCTGGGAAAACCAGTGAGTCTGGAGAGCT
GCATGGGCTCACAACTGAGGAGGAATTTGTAGAAGGGATATACAAAGTGGAAATA
GACACCAAATCTTACTGGAAGGCACTTGGCATCTCCCCATTCCATGAGCATGCAG
AGGTGGTATTCACAGCCAACGACTCCGGCCCCCGCCGCTACACCATTGCCGCCCT
GCTGAGCCCCTACTCCTATTCCACCACGGCTGTCGTCACCAATCCCAAGGAAGGT
TCTGGCTCCGGATCAGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAAGG
ACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAG
CCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAT
AATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCA
GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAA
GGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAA
GGGGGTTCTGGATCCTAA:
[0160] Construction of TTRC10A-CH2-TMP was done as follows: the TMP
fragment containing a 5' BamHI linker and 3' XhoI linker was
amplified by oligos 2694-19 and
[0161] 2974-70:
19 (SEQ ID NO:58) GAG GAA TAA GGA TCC ATC GAA GGT CCG ACT CTG
CG
[0162] The amplified fragment was digested with BamH1 and Xho1 and
was subsequently ligated with similarly restricted 6017. The
resulting clone is described as strain 6104 (TTRC10A-CH2-TMP).
20 ATGGGTCCAACTGGTACCGGTGAATCCAAGGCTCCTCTGATGGTCAAAGTTCTAG SEQ ID
NO:59 ATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGTGTTCAGA- AAGGC
TGCTGATGACACCTGGGAGCCATTTGCCTCTGGGAAAACCAGTGAGTCTGGA- GAG
CTGCATGGGCTCACAACTGAGGAGGAATTTGTAGAAGGGATATACAAAGTGGAA- A
TAGACACCAAATCTTACTGGAAGGCACTTGGCATCTCCCCATTCCATGAGCATGC
AGAGGTGGTATTCACAGCCAACGACTCCGGCCCCCGCCGCTACACCATTGCCGCC
CTGCTGAGCCCCTACTCCTATTCCACCACGGCTGTCGTCACCAATCCCAAGGAAG
GTTCTGGCTCCGGATCAGGGGGACCGTCAGTTTTCCTCTTCCCCCCAAAACCCAA
GGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTG
AGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGC
ATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGT
CAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGC
AAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCA
AAGGGGGTTCTGGATCCATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCG
TGCTGGCGGTGGTGGCGGAGGGGGTGGCATTGAGGGCCCAACCCTTCGCCAATGG
CTTGCAGCACGCGCATAA:
[0163] Another configuration of this fusion was made as
TMP-CH2-TTR2. The CH2 domain derived from TMP-Fc was first linked
to N-terminus of TTRC10A by a two-step PCR. The CH2 domain
(containing from 5' to 3': a NdeI-BamHI linker, CH2 and the first 7
codons of TTR C10A) was first amplified by oligos
[0164] 2974-65:
21 (SEQ ID NO:60) TTC ACC GGT ACC AGT TGG ACC AGA ACC CCC TTT GGC
TTT GGA GAT GGT, and
[0165] 2974-66:
22 (SEQ ID NO:61) GAG GAA TAA CAT ATG GGA TCC GGT TCT GGG GGA CCG
TCA GTT TTC CTC.
[0166] This fragment was fused to 5619 in a subsequent PCR by
oligos 2974-66 and 2693-80 (example 1), followed by restriction
with NdeI/XhoI and cloning into similarly restricted pAMG21. The
resulting clone is described as 6016 (CH2-TTRC10A):
23 SEQ ID NO:62 ATGGGATCCGGTTCTGGGGGACCGTCAGTTTTCCTCTTCCCCC-
CAAAACC CAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTG- G
TGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGAC
GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAA
CAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGC
TGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCC
CCCATCGAGAAAACCATCTCCAAAGCCAAAGGGGGTTCTGGTCCAACTGG
TACCGGTGAATCCAAGGCTCCTCTGATGGTCAAAGTTCTAGATGCTGTCC
GAGGCAGTCCTGCCATCAATGTGGCCGTGCATGTGTTCAGAAAGGCTGCT
GATGACACCTGGGAGCCATTTGCCTCTGGGAAAACCAGTGAGTCTGGAGA
GCTGCATGGGCTCACAACTGAGGAGGAATTTGTAGAAGGGATATACAAAG
TGGAAATAGACACCAAATCTTACTGGAAGGCACTTGGCATCTCCCCATTC
CATGAGCATGCAGAGGTGGTATTCACAGCCAACGACTCCGGCCCCCGCCG
CTACACCATTGCCGCCCTGCTGAGCCCCTACTCCTATTCCACCACGGCTG
TCGTCACCAATCCCAAGGAATAA:
[0167] The TMP fragment containing a NdeI linker at 5' end and a
BamHI linker at 3' end was amplified by oligos
[0168] 2974-68:
24 (SEQ ID NO:63) GAG GAA TAA CAT ATG ATC GAA GGT CCG ACT CTG,
and
[0169] 2974-69:
25 (SEQ ID NO:64) TAA CAT ATG GGA TCC TGC GCG TGC TGC AAG CCA
TTG.
[0170] This fragment was then digested with NdeI/BamHI and ligated
with the vector which was similarly restricted, gel purified from
strain 6016. The resulting clone is described as 6110
(TMP-CH2-TTRC10A):
26 SEQ ID NO:65 ATGATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTG-
CTGGCGG TGGTGGCGGAGGGGGTGGCATTGAGGGCCCAACCCTTCGCCAATGGCTT- G
CAGCACGCGCAGGATCCGGTTCTGGGGGACCGTCAGTTTTCCTCTTCCCC
CCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATG
CGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGT
ACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG
CAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA
GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCC
TCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGGGTTCTGGT
CCAACTGGTACCGGTGAATCCAAGGCTCCTCTGATGGTCAAAGTTCTAGA
TGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGTGTTCAGAA
AGGCTGCTGATGACACCTGGGAGCCATTTGCCTCTGGGAAAACCAGTGAG
TCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGAATTTGTAGAAGGGAT
ATACAAAGTGGAAATAGACACCAAATCTTACTGGAAGGCACTTGGCATCT
CCCCATTCCATGAGCATGCAGAGGTGGTATTCACAGCCAACGACTCCGGC
CCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCCTACTCCTATTCCAC
CACGGCTGTCGTCACCAATCCCAAGGAATAA:
EXAMPLE 21
[0171] This example describes the construction of TTRC10A/K15A-TMP,
TTRC10A/K15A/A81C-TMP and TTRC10A/K15A/G83C-TMP.
[0172] TMP was also cloned at the C- termini of TTR and variants
thereof. The full length TMP containing at its N- terminal end a
5-amino acids linker (gsgsg) plus the last 7 amino acids of wt TTR
was amplified by the following set of oligonucleotides in a
standard PCR procedure.
[0173] 2694-18:
27 (SEQ ID NO:66) GTC GTC ACC AAT CCC AAG GAA GGT TCT GGT TCT GGT
ATC GAA, and
[0174] 2694-19:
28 (SEQ ID NO:67) CCG CGG ATC CTC GAG ATT ATG CGC GTG CTG CAA GCC
ATT G.
[0175] This PCR fragment was further linked to the 3' end of wt TTR
by a second PCR utilizing oligos 2694-19 and 2693-79 as described
in example 1. The resulting clone was sequence confirmed and is
described as strain 5365 (TTR-TMP):
29 SEQ ID NO:68 ATGGGTCCAACTGGTACCGGTGAATCCAAGTGTCCTCTGATGG-
TCAAAGT TCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGTG- T
TCAGAAAGGCTGCTGATGACACCTGGGAGCCATTTGCCTCTGGGAAAACC
AGTGAGTCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGAATTTGTAGA
AGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAAGGCACTTG
GCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAGCCAACGAC
TCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCCTACTCCTA
TTCCACCACGGCTGTCGTCACCAATCCCAAGGAAGGTTCTGGTTCTGGTA
TCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCTGGCGGTGGT
GGCGGAGGGGGTGGCATTGAGGGCCCAACCCTTCGCCAATGGCTTGCAGC ACGCGCATAA:
[0176] The Xba1/Xba1 fragment of 5365 was then replaced by the
corresponding Xba1/Xba1 fragment of strain 5895 to make strain 5921
(TTRC10A/K15A-TMP) as described above:
30 SEQ ID NO:69 ATGGGTCCAACTGGTACCGGTGAATCCAAGGCTCCTCTGATGG-
TCGCAGT TCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGTG- T
TCAGAAAGGCTGCTGATGACACCTGGGAGCCATTTGCCTCTGGGAAAACC
AGTGAGTCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGAATTTGTAGA
AGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAAGGCACTTG
GCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAGCCAACGAC
TCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCCTACTCCTA
TTCCACCACGGCTGTCGTCACCAATCCCAAGGAAGGTTCTGGTTCTGGTA
TCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCTGGCGGTGGT
GGCGGAGGGGGTGGCATTGAGGGCCCAACCCTTCGCCAATGGCTTGCAGC ACGCGCATAA:
[0177] Plasmid 5921 was subsequently modified by replacing the
amino acids at the following positions: A37, A81 and G83, with the
amino acid Cysteine as described in example 1, except that the TTR
3' oligo utilized with the mutation oligos (2693-80) in example 1
was replaced with 2694-19, resulting in Strain 5982, containing
TTRC10A/K15A/A37C-TMP (SEQ ID NO:70), Strain 5983 containing
TTRC10A/K15A/A81C-TMP (SEQ ID NO:71), and Strain 5984 containing
TTRC10A/K15A/G83C-TMP (SEQ ID NO:72).
31 SEQ ID NO:70: ATGGGTCCAACTGGTACCGGTGAATCCAAGGCTCCTCTGATG-
GTCGCAGT TCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGT- GT
TCAGAAAGGCTTGTGATGACACCTGGGAGCCATTTGCCTCTGGGAAAACC
AGTGAGTCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGAATTTGTAGA
AGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAAGGCACTTG
GCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAGCCAACGAC
TCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCCTACTCCTA
TTCCACCACGGCTGTCGTCACCAATCCCAAGGAAGGTTCTGGTTCTGGTA
TCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCTGGCGGTGGT
GGCGGAGGGGGTGGCATTGAGGGCCCAACCCTTCGCCAATGGCTTGCAGC ACGCGCATAA
[0178]
32 SEQ ID NO:71: ATGGGTCCAACTGGTACCGGTGAATCCAAGGCTCCTCTGATG-
GTCGCAGT TCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGT- GT
TCAGAAAGGCTGCTGATGACACCTGGGAGCCATTTGCCTCTGGGAAAACC
AGTGAGTCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGAATTTGTAGA
AGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAAGTGTCTTG
GCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAGCCAACGAC
TCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCCTACTCCTA
TTCCACCACGGCTGTCGTCACCAATCCCAAGGAAGGTTCTGGTTCTGGTA
TCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCTGGCGGTGGT
GGCGGAGGGGGTGGCATTGAGGGCCCAACCCTTCGCCAATGGCTTGCAGC ACGCGCATAA
[0179]
33 SEQ ID NO:72: ATGGGTCCAACTGGTACCGGTGAATCCAAGGCTCCTCTGATG-
GTCGCAGT TCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGT- GT
TCAGAAAGGCTGCTGATGACACCTGGGAGCCATTTGCCTCTGGGAAAACC
AGTGAGTCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGAATTTGTAGA
AGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAAGGCACTTT
GCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAGCCAACGAC
TCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCCTACTCCTA
TTCCACCACGGCTGTCGTCACCAATCCCAAGGAAGGTTCTGGTTCTGGTA
TCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCTGGCGGTGGT
GGCGGAGGGGGTGGCATTGAGGGCCCAACCCTTCGCCAGTGGCTTGCAGC ACGCGCATAA
EXAMPLE 22
[0180] This example describes the construction of
TMP-TTRC10A/K15A/A81C and TMP-TTRC10A/K15A/A37C. The Lys at 15th
position of TTR was mutagenized to Ala in strains 5704, 5706 and
5707 by the following methods. Plasmid 5513 was digested with
Nde1/Kpn1, the insert harboring TMP fragment and the first 6 amino
acids of TTR was purified and ligated with Nde1/Kpn1 restricted and
gel purified vector derived from strain 5895. The bacterial strain
containing the resulting plasmid is described as 5919
(TMP-TTRC10A/K15A/G83C). Plasmid 5919 was then digested with Xba1,
the resulting Xba1/Xba1 fragment containing TMP and the first 18
codons of TTR including the C10A and K15A mutations was gel
purified and ligated with Xba1 digested, phosphatase treated and
gel purified vectors derived from strain 5704 and 5706. The new
strains are described as 5918 (TMP-TTRC10A/K15A/A81C) and 6023
(TMP-TTRC10A/K15A/A37C).
34 TMP-TTRC10A/K15A/G83C: (SEQ ID NO:73)
ATGATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCTGGCGG
TGGTGGCGGAGGGGGTGGCATTGAGGGCCCAACCCTTCGCCAATGGCTTG
CAGCACGCGCAGGTCCAACTGGTACCGGTGAATCCAAGGCTCCTCTGATG
GTCGCAGTTCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGT
GCATGTGTTCAGAAAGGCTGCTGATGACACCTGGGAGCCATTTGCCTCTG
GGAAAACCAGTGAGTCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGAA
TTTGTAGAAGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAA
GGCACTTTGCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAG
CCAACGACTCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCC
TACTCCTATTCCACCACGGCTGTCGTCACCAATCCCAAGGAATAA
[0181]
35 TMP-TTRC10A/K15A/A81C: (SEQ ID NO:74)
ATGATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCTGGCGG
TGGTGGCGGAGGGGGTGGCATTGAGGGCCCAACCCTTCGCCAATGGCTTG
CAGCACGCGCAGGTCCAACTGGTACCGGTGAATCCAAGGCTCCTCTGATG
GTCGCAGTTCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGT
GCATGTGTTCAGAAAGGCTGCTGATGACACCTGGGAGCCATTTGCCTCTG
GGAAAACCAGTGAGTCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGAA
TTTGTAGAAGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAA
GTGTCTTGGCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAG
CCAACGACTCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCC
TACTCCTATTCCACCACGGCTGTCGTCACCAATCCCAAGGAATAA
[0182]
36 TMP-TTRC10A/K15A/A37C: (SEQ ID NO:75)
ATGATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCTGGCGG
TGGTGGCGGAGGGGGTGGCATTGAGGGCCCAACCCTTCGCCAATGGCTTG
CAGCACGCGCAGGTCCAACTGGTACCGGTGAATCCAAGGCTCCTCTGATG
GTCGCAGTTCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGT
GCATGTGTTCAGAAAGGCTTGTGATGACACCTGGGAGCCATTTGCCTCTG
GGAAAACCAGTGAGTCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGAA
TTTGTAGAAGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAA
GGCACTTGGCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAG
CCAACGACTCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCC
TACTCCTATTCCACCACGGCTGTCGTCACCAATCCCAAGGAATAA
EXAMPLE 23
[0183] This example describes the expression of GLP-1 fusions
proteins in E. coli. 25-100 ml of a saturated overnight culture was
used to inoculate 50 ml TB with 20 ug/ml kanamycin in a 250 ml
baffled flask and incubated at 37C, 250 rpm overnight. 10-35 ml of
these overnight cultures were used to inoculate 1L TB with 20 ug/ml
kanamycin in a 2L baffled flask and incubated at 37C, 250 rpm until
the optical density at 600 nm reached approximately 0.7. The
cultures were then induced to express recombinant protein by the
addition of: 1 ml of ethanol containing 30 ug/ml
N-(B-ketocaproyl)-DL-homoserine lactone (Sigma) in the case of
pAMG21, or IPTG to 0.1 mM in the case of pAMG33*. The incubation
was continued for an additional 2-4 hours and the cells were
collected by centrifugation.
EXAMPLE 24
[0184] This example describes the purification of
PTH-TTR(C10A/K15A/A81C). About 197 g of E. coli paste from clone
5933 stored at -80.degree. C. was defrosted in 1480 ml of 50 mM
tris HCl, 5 mM EDTA, pH 8.0. 60 tablets of Sigma protease inhibitor
cocktail 1-873-580 (Saint Louis, Mo.) was dissolved in the cell
suspension and the suspension was passed through a model 110-Y
microfluidizer (Microfluidics, Newton, Mass.) twice at 14,000 PSI.
The lysate was centrifuged at 11,325.times.g for 50 min 4.degree.
C. The supernatant was removed as the soluble fraction. The soluble
fraction was heated in a 65.degree. C. water bath for 30 minutes in
polypropylene bottles, at which time the temperature of the
contents was 63.degree. C. The soluble fraction was centrifuged at
11,325.times.g for 50 minutes 4.degree. C. The supernatant was
removed as Heat Soluble. The heat soluble fraction was filtered
through a 0.45 .mu.m cellulose acetate filter with two prefilters
and then loaded on to a 240 ml Q-sepharose fast flow (5 cm ID)
column (Amersham Pharmacia Biotech, Piscataway, N.J.) at 25 ml/min
equilibrated in Q-Buffer A (20 mM tris HCl, 2.5 mM EDTA, pH 8.0) at
room temperature (about 23.degree. C.). Column was washed with
about 2200 ml Q-Buffer A at 30 ml/min. Q-column was eluted with a
15 column volume linear gradient to 60% Q-Buffer B (20 mM tris HCl,
1 M NaCl, 2.5 mM EDTA, pH 8.0) followed by a 2 column volume step
to 100% Q-Buffer B. Fractions containing the TTR fusion as
determined by SDS-PAGE were pooled into a single Q-pool (1300 ml).
To the Q-pool, 464 ml of 3.8 M ammonium sulfate pH 7.2 was slowly
added. The solution was centrifuged at 11,325.times.g for 50 min
4.degree. C. The supernatant was removed as the ammonium sulfate
soluble fraction and discarded, and the pellet was resuspended in
450 ml 10 mM NaH.sub.2PO.sub.4, pH 7.0 by gentle agitation at room
temperature for about 30 min. The solution was centrifuged at
11,325.times.g for 50 min 4.degree. C. Supernatant was removed as
phosphate buffer soluble fraction and filtered through a 0.45 .mu.m
cellulose acetate filter. Added 240 .mu.l 1 M dithiothreitol to the
phosphate buffer soluble fraction and loaded on to a 105 ml (2.6
cm) type 1 ceramic hydroxyapatite column (Bio-Rad Inc., Hercules,
Calif.) at 10 ml/min in HA-Buffer A. Column was washed with
approximately 210 ml HA-Buffer A at 10 ml/min followed by 3 steps
of 25%, 50%, and 100% HA-Buffer B (400 mM NaH.sub.2PO.sub.4, pH
7.0). The fractions from the 50% elution were pooled as HA-pool
(725 ml) and filtered through a 0.22 .mu.m cellulose acetate
filter. 1.16 g of dithiothreitol was added to HA-Pool, and the pH
was raised to 8.0 using tris base followed by incubation at room
temperature for about 30 minutes. Diluted HA-pool with 750 ml water
and loaded on to a 50 ml source 15Q (2.6 cm ID) column (Amersham
Pharmacia Biotech) at 10 ml/min followed by a wash with about 250
ml Q-Buffer A. Column was eluted with a 20 column volume linear
gradient from 10% to 60% Q-Buffer B followed a step of 2 column
volumes of 100% Q-Buffer B. Fractions containing the TTR fusion as
determined by SDS-PAGE were pooled into a single Q2-pool (170 ml)
and filtered through a 0.22 .mu.m cellulose acetate filter. The
protein concentration was determined to be 3.7 mg/ml using a
calculated extinction coefficient of 23,950 M.sup.-1 cm.sup.-1. The
pyrogen level was determined to be <1 EU/mg of protein using the
Limulus Ameboycyte Lysate assay (Associates of Cape Cod, Falmouth,
Mass.). The nucleic acid content was determined to be negligible,
since the ratio of the absorbance at 260 nm over 280 nm was
determined to be 0.61.
EXAMPLE 25
[0185] This example describes the purification of
TMP-TTR(C10A/D38C). About 170 g of E. coli paste from clone 5705
stored at -80.degree. C. was defrosted in 1275 ml of 50 mM tris
HCl, 5 mM EDTA, pH 8.0. 50 tablets of Sigma protease inhibitor
cocktail 1-873-580 (Saint Louis, Mo.) was dissolved in the cell
suspension and the suspension was passed through a model 110-Y
microfluidizer (Microfluidics, Newton, Mass.) twice at 14,000 PSI.
The lysate was centrifuged at 11,325.times.g for 30 min 4.degree.
C. The supernatant was removed as the soluble fraction and
discarded. The pellets were resuspended in 1200 ml water using a
tissue grinder and 20 more Sigma protease inhibitor tablets were
added. The suspension was centrifuged at 11,325.times.g for 30 min
4.degree. C. The supernatant was filtered through a Whatman GF/A
filter and 2.1 g of dithiothreitol was added followed by incubation
at 7.degree. C. for 30 minutes. The reduced sample was loaded on to
a 240 ml Q-sepharose fast flow (5 cm ID) column (Amersham Pharmacia
Biotech, Piscataway, N.J.) at 30 ml/min equilibrated in Q-Buffer A
(20 mM tris HCl, 0.02% sodium azide, pH 8.0) at 7.degree. C. Column
was washed with about 1920 ml Q-Buffer A at 30 ml/min. Q-column was
eluted with 3 steps of 20%, 35%, and 100% Q-Buffer B (20 mM tris
HCl, 1 M NaCl, 0.02% sodium azide, pH 8.0). Added 13 ml 500 mM EDTA
pH 8.0 to the flowthrough from the Q-column and centrifuged for 30
min at 11,325 g at 4.degree. C. Supernatant was discarded, and the
pellet was resuspended in 700 ml 4 M urea, 20 mM tris HCl, pH 8.0.
The urea solublized pellet was then filtered through a Whatman GF/A
filter and loaded on to a 240 ml Q-sepharose fast flow (5 cm ID)
column (Amersham Pharmacia Biotech, Piscataway, N.J.) at 30 ml/min
equilibrated in Q-Buffer A (20 mM tris HCl, 0.02% sodium azide, pH
8.0) at 7.degree. C. Column was washed with about 1920 ml Q-Buffer
A at 30 ml/min. Q-column was eluted with 3 steps of 20%, 35%, and
100% Q-Buffer B (20 mM tris HCl, 1 M NaCl, 0.02% sodium azide, pH
8.0) at 15 ml/min. Fractions containing the 35% elution peak were
pooled, filtered through a 0.22 .mu.m cellulose acetate filter, and
0.5 g of dithiothreitol (10 mM final concentration) was added
followed by incubation for 30 min at 7.degree. C. The 35% Q-pool
was then loaded on to a 45 ml (2.6 cm) type 1 ceramic
hydroxyapatite column (Bio-Rad Inc., Hercules, Calif.) at 5 ml/min
in 20 mM tris HCl, 350 mM NaCl, pH 8.0 at 7.degree. C. Column was
washed with approximately 70 ml 20 mM tris HCl, 350 mM NaCl, pH 8.0
at 5 ml/min followed by 3 steps of 2.5%, 25%, and 100% HA-Buffer B
(400 mM NaH.sub.2PO.sub.4, pH 7.0). The fractions from the 2.5%
elution were pooled as HA-pool (80 ml) and filtered through a 0.22
.mu.m cellulose acetate filter. The protein concentration was
determined to be 6.8 mg/ml using a calculated extinction
coefficient of 29,450 M.sup.-1 cm.sup.-1. The pyrogen level was
determined to be <1 EU/mg of protein using the Limulus
Ameboycyte Lysate assay (Associates of Cape Cod, Falmouth, Mass.).
The nucleic acid content was determined to be negligible, since the
ratio of the absorbance at 260 nm over 280 nm was determined to be
0.54.
EXAMPLE 26
[0186] This example describes the refolding and purification of
TTR(C10A)-CH2-TMP. About 23 g of E. coli paste from clone 6104
stored at -80.degree. C. was defrosted in 200 ml of 50 mM tris HCl,
5 mM EDTA, pH 8.0. 10 tablets of Sigma protease inhibitor cocktail
1-873-580 (Saint Louis, Mo.) was dissolved in the cell suspension
and the suspension was passed through a microfluidizer
(Microfluidics, Newton, Mass.) twice at 12,000 PSI. The lysate was
centrifuged at 15,344.times.g for 50 min 4.degree. C. The
supernatant was removed as the soluble fraction and discarded. The
pellet was resuspended in 200 ml 50 mM tris HCl, 5 mM EDTA, pH 8.0
using a tissue grinder. The suspension was centrifuged at
15,344.times.g for 50 min 4.degree. C. The supernatant was removed
as the wash and discarded. The pellet was resuspended in 50 ml 50
mM tris HCl, 5 mM EDTA, pH 8.0 using a tissue grinder. The
suspension was centrifuged at 14,000.times.g for 10 min room
temperature. The supernatant was removed as the wash and discarded.
The pellets were dissolved in 50 ml 8 M guanidine HCl, 50 mM tris
HCl, pH 8.0 using a sonicator for about 1 min. Dissolved protein
was reduced for 30 min room temperature by adding 500 .mu.l 1 M
DTT. Reduced protein was centrifuged for 30 min at 20.degree. C. at
27,216 g. Supernatant was then added to 4 L 50 mM tris base, 160 mM
arginine base, 1 M urea, 1 mM cystamine, 4 mM cysteine, pH 9.5 at 2
ml/min and incubated about 16 hours 4.degree. C. Refolded protein
was then filtered through a Gellman SUPORCAP.RTM. 50 and then
concentrated to about 500 ml using a Pall Filtron 3 square foot
YM10 membrane tangential flow system followed by diafiltration
against 2 L 20 mM tris HCl pH 8.0. Concentrated protein was then
loaded on to a 45 ml source 15Q (2.6 cm ID) column (Amersham
Pharmacia Biotech) at 18 ml/min followed by a wash with about 150
ml Q-Buffer A (20 mM tris HCl pH 8.0). Column was eluted with a 20
column volume linear gradient from 0% to 60% Q-Buffer B followed a
step of 2 column volumes of 100% Q-Buffer B. Fractions containing
the TTR fusion as determined by SDS-PAGE were pooled into a single
Q-pool (29 ml). The Q-Pool was then concentrated to about 6.3 ml
using a Millipore CENTRIPREP.TM. 10 and then passed through a Pall
ACRODISC.RTM. MUSTANG.TM. E membrane filter at 1 ml/min. The
protein concentration was determined to be 10.5 mg/ml using a
calculated extinction coefficient of 46,410 M.sup.-1 cm.sup.-1. The
pyrogen level was determined to be <1 EU/mg of protein using the
Limulus Ameboycyte Lysate assay (Associates of Cape Cod, Falmouth,
Mass.). The nucleic acid content was determined to be negligible,
since the ratio of the absorbance at 260 nm over 280 nm was
determined to be 0.51.
EXAMPLE 27
[0187] This example describes the purification of GLP1-TTR
(C10A/K15A/G83C). About 30 g of E. coli paste from clone 6450
stored at -80.degree. C. was defrosted in 250 ml of 50 mM
NaH.sub.2PO.sub.4, pH 7.0. Cell suspension was passed through a
microfluidizer (Microfluidics, Newton, Mass.) twice at 12,000 PSI.
The lysate was centrifuged at 15,344.times.g for 50 min 4.degree.
C. The supernatant was discarded as the soluble fraction, and the
pellet was resuspended in 200 ml deoxycholate using a tissue
grinder. The suspension was centrifuged at 15,344.times.g for 50
min 4.degree. C. The supernatant was discarded as the wash, and the
pellet was resuspended in 200 ml water using a tissue grinder. The
suspension was centrifuged at 15,344.times.g for 50 min 4.degree.
C. The supernatant was discarded as the wash, and the pellet was
resuspended in 100 ml water using a tissue grinder. The suspension
was centrifuged at 27,216.times.g for 30 min room temperature. The
supernatant was discarded as the wash, and about 2/3 of the pellets
were dissolved in 75 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0
by agitation for about 15 min. The suspension was centrifuged at
27,216.times.g for 30 min room temperature, and the supernatant was
diluted with 18 ml water. Sample was then loaded on to a 50 ml
chelating sepharose fast flow column (Amersham Pharmacia Biotech,
Piscataway, N.J.), loaded with NiCl.sub.2, at 5 ml/min. After
washing with about 150 ml Ni-Buffer A (6 M guanidine HCl, 37.5 ml
tris HCl, pH 8.0) at 10 ml/min, eluted with two step of 10% and
100% Ni-Buffer B (6 M guanidine HCl, 37.5 mM tris HCl, 400 mM
imidazole, pH 8.0). Combined the peak containing the fusion
construct as Ni-Pool (40 ml) and determined the protein content to
be 6.4 mg/ml by observing the absorbance at 280 nm in 8 M guanidine
HCl using an extinction coefficient of 25,440 M.sup.-1. Added 800
.mu.l 500 mM EDTA pH 8.0 and removed 80 mg of protein for the
PEGylation reaction. Added 230 .mu.l 1 M DTT and incubated for 30
min at 30.degree. C. Loaded on to a 130 ml SEPHADEX.TM. G25 medium
column (2.6 cm ID) (Amersham Pharmacia Biotech, Piscataway, N.J.)
at 8 ml/min in 20 mM tris HCl, 6 M urea, pH 8.5. Pooled the protein
peak as determined by absorbance at 280 nm (22 ml) and determined
the concentration to be 3.2 mg/ml by observing the absorbance at
280 nm in 20 mM tris HCl, 6 M urea, pH 8.5 using an extinction
coefficient of 25,440 M.sup.-1. Reacted 45% of the buffer exchanged
material with 950 .mu.l of 5 mM methoxy-PEG-maleimide 5K
(Shearwater Corporation, Huntsville, Ala.) for 140 min at
30.degree. C. Added 100 .mu.l 1 M 2-mercaptoethanol to each
reaction to quench. Dialyzed reaction against 1 L 25 mM
NaH.sub.2PO.sub.4, 3 M urea, pH 7.25 using a Pierce 10 kDa
Slidealyzer for 2 hour room temperature. Changed the dialysis
buffer for 25 mM NaH.sub.2PO.sub.4, 10% sucrose, 2 mM EDTA, pH 7.25
and incubated for about 16 hours room temperature. Added 140 .mu.l
5% CHAPS and 7.28 .mu.l 2-mercaptoethanol and 0.475 ml of 3 mg/ml
caspase 3 followed by a 2 hour incubation at room temperature.
Reaction mixture was loaded on to a 5 ml HiTrap Q-sepharose HP
column (Amersham Pharmacia Biotech, Piscataway, N.J.) at 1 ml/min
in 20 mM tris HCl pH 8.0 followed by about a 15 ml wash in the same
buffer. Column was then developed at 5 ml/min using a linear
gradient to 60% 20 mM tris HCl, 1 M NaCl, pH 8.0 followed by a step
to 100% of the elution buffer. Fractions containing the TTR fusion
as determined by SDS-PAGE were pooled into a single Q-pool (9.5
ml). Concentrated Q-Pool to 3.2 ml using a Millipore CENTRIPREP.TM.
30 kDa and filtered through a Pall MUSTANGS E membrane at about 1
ml/min. Diluted Q-Pool to 6.5 ml with water and added 375 .mu.l
acetonitrile. Injected on to a Vydac Protein/Peptide 10.times.250
mm C.sub.4 column (Vydac, Hisperia, Calif.) in 95% RP-Buffer A
(0.1% trifluoroacetic acid) with 5% RP-Buffer B (95% acetonitrile,
0.1% trifluoroacetic acid) at 5 ml/min. Developed column running a
linear gradient to 100% RP-Buffer B. Concentrated protein peak to
about 3 ml using a Millipore CENTRIPREP.TM. 30 kDa and diluted to
15 ml using 20 mM tris HCl pH 8.0. Repeated buffer exchange 3 more
times then passed though a Pall MUSTANG.TM. E membrane at about 1
ml/min. The protein concentration was determined to be 7.7 mg/ml
using a calculated extinction coefficient of 25,440 M.sup.-1
cm.sup.-1. The pyrogen level was determined to be <1 EU/mg of
protein using the Limulus Ameboycyte Lysate assay (Associates of
Cape Cod, Falmouth, Mass). The nucleic acid content was determined
to be negligible, since the ratio of the absorbance at 260 nm over
280 nm was determined to be 0.54.
EXAMPLE 28
[0188] This example shows the effect of injecting pegylated
GLP1-TTR constructs into mice on blood glucose levels. For this
example 40 male, db/db, 9 week-old mice were split into 4 groups
and injected (hour 0) intraperitoneal with 7.4-16.6 mg test protein
per animal (538 pmol monomers for all groups) (5K pegylated
GLP1-TTR fusion construct 10 mg, 20K pegylated GLP1-TTR fusion
construct 10 mg, GLP1-Fc 16.6 mg, and a TTR(C10A) control 7.4 mg).
Each group was bled at time points 0(baseline measurement), 1, 4,
6, 12, 24, and 48 hours post injection. Food was withheld from the
mice for the first 6 hours of the experiment and replaced after the
bleed at the 6 hour time point.
[0189] Each collected drop of blood per time point was analyzed for
glucose content using a One Touch Profile glucose meter and the
results are depicted in FIG. 12.
EXAMPLE 29
[0190] This example shows the effect of injecting TMP-TTR
constructs with fused antibody CH2 domains into mice on blood
platelet count. For this example 50 female BDF1 mice were split
into 5 groups and injected (day 0) subcutaneously with 50 mg test
protein per kg animal (TMP fusion construct, Fc-TMP, or a TTR(C10A)
control). Each group was divided in half and bled (140 ml) on
alternate time points (day 0, 3, 5, 7, and 10). Mice were
anesthetized with isoflurane prior to collection.
[0191] The collected blood was analyzed for a complete and
differential count using an ADVIA 120 automated blood analyzer with
murine software (Bayer Diagnostics, New York, N.Y.). As seen in
FIG. 13, Fc-TMP showed the greatest response with platelet count
rising to over 4.2.times.10.sup.12 platelets L-1 on day 5 which is
3 times baseline at 1.4.times.10.sup.12 platelets L-1. All three of
the CH2 fused TMP-TTR constructs preformed better than the control,
but not as well as Fc-TMP with platelet counts between
2.3.times.1012 and 2.6.times.1012 platelets L-1 on day 5, which is
between a 64% and 86% improvement over baseline.
EXAMPLE 30
[0192] This example shows the effect of injecting pegylated TTR
constructs with TMP fused to the carboxy-terminus of pegylated TTR
into mice on blood platelet count. For this example 80 BDF1 mice
were split into 8 groups and injected (day 0) subcutaneously with
50 mg test protein per kg animal (TMP fusion constructs, Fc-TMP, or
a TTR(C10A) control). Each group was divided in half and bled (140
ml) on alternate time points (day 0, 3, 5, 7, 10, and 12). Mice
were anesthetized with isoflurane prior to collection.
[0193] The collected blood was analyzed for a complete and
differential count using an ADVIA 120 automated blood analyzer with
murine software (Bayer Diagnostics, New York, N.Y.). As seen in
FIG. 14, Fc-TMP and the three amino terminal (TMP-TTR) fusions
showed the greatest response with platelet count rising between
4.3.times.10.sup.12 and 4.6.times.10.sup.12 platelets L-1 on day 5
which is over three times baseline at 1.3.times.10.sup.12 platelets
L-1. All three of the carboxy terminal (TTR-TMP) constructs
performed better than the control.
EXAMPLE 31
[0194] This example shows the effect of injecting pegylated TTR-TMP
constructs containing a K15A alteration into mice on blood platelet
count. For this example 120 BDF1 mice were split into 12 groups and
injected (day 0) subcutaneously with 50 mg test protein per kg
animal (TMP fusion constructs, Fc-TMP, or a TTR(C10A) control)
(this study was split into two batches (PEG 20K in one and the PEG
5K and non-pegylated samples in the other) completed at separate
times with repeated controls). Each group was divided in half and
bled (140 ml) on alternate time points (day 0, 3, 5, 7, 10, and
12). Mice were anesthetized with isoflurane prior to
collection.
[0195] The collected blood was analyzed for a complete and
differential count using an ADVIA 120 automated blood analyzer with
murine software (Bayer Diagnostics, New York, N.Y.). As seen in
FIG. 15A, the two non-pegylated constructs outperformed the
baseline (1.3.times.10.sup.12 platelets L-1) with platelet
responses at day 5 rising between 1.8.times.10.sup.12 and
2.0.times.10.sup.12 platelets L-1. As seen in FIG. 15B, Fc-TMP and
the three 5K pegylated fusions showed equivalent responses at day 5
with platelet counts rising between 3.5.times.10.sup.12 and
4.4.times.10.sup.12 platelets L-1 which is at least 2.7 times
baseline (1.3.times.10.sup.12 platelets L-1). As seen in FIG. 15C,
Fc-TMP and the three 20K pegylated fusions showed equivalent
responses at day 5 with platelet count rising between
4.3.times.10.sup.12 and 4.6.times.10.sup.12 platelets L-1 which is
over three times baseline at 1.3.times.10.sup.12 platelets L-1.
[0196] In addition, the 20K pegylated TTR constructs appear to have
an improved sustained response with platelets at day 7 ranging from
3.7.times.10.sup.12 to 4.9.times.10.sup.12 platelets L-1 compared
to Fc-TMP at 3.1.times.10.sup.12 platelets L-1. This sustained
response is maintained at day 10 for the three 20K pegylated TTR
constructs with platelets ranging from 2.3.times.10.sup.12 to
3.1.times.10.sup.12 platelets L-1 compared to Fc-TMP at
2.0.times.10.sup.12 platelets L-1.
Sequence CWU 1
1
75 1 127 PRT Homo sapiens 1 Gly Pro Thr Gly Thr Gly Glu Ser Lys Cys
Pro Leu Met Val Lys Val 1 5 10 15 Leu Asp Ala Val Arg Gly Ser Pro
Ala Ile Asn Val Ala Val His Val 20 25 30 Phe Arg Lys Ala Ala Asp
Asp Thr Trp Glu Pro Phe Ala Ser Gly Lys 35 40 45 Thr Ser Glu Ser
Gly Glu Leu His Gly Leu Thr Thr Glu Glu Glu Phe 50 55 60 Val Glu
Gly Ile Tyr Lys Val Glu Ile Asp Thr Lys Ser Tyr Trp Lys 65 70 75 80
Ala Leu Gly Ile Ser Pro Phe His Glu His Ala Glu Val Val Phe Thr 85
90 95 Ala Asn Asp Ser Gly Pro Arg Arg Tyr Thr Ile Ala Ala Leu Leu
Ser 100 105 110 Pro Tyr Ser Tyr Ser Thr Thr Ala Val Val Thr Asn Pro
Lys Glu 115 120 125 2 387 DNA Homo sapiens 2 agtggtccaa ctggtaccgg
tgaatccaag tgtcctctga tggtcaaagt tctagatgct 60 gtccgaggca
gtcctgccat caatgtggcc gtgcatgtgt tcagaaaggc tgctgatgac 120
acctgggagc catttgcctc tgggaaaacc agtgagtctg gagagctgca tgggctcaca
180 actgaggagg aatttgtaga agggatatac aaagtggaaa tagacaccaa
atcttactgg 240 aaggcacttg gcatctcccc attccatgag catgcagagg
tggtattcac agccaacgac 300 tccggccccc gccgctacac cattgccgcc
ctgctgagcc cctactccta ttccaccacg 360 gctgtcgtca ccaatcccaa ggaataa
387 3 387 DNA Homo sapiens 3 atgggtccaa ctggtaccgg tgaatccaag
gctcctctga tggtcaaagt tctagatgct 60 gtccgaggca gtcctgccat
caatgtggcc gtgcatgtgt tcagaaaggc tgctgatgac 120 acctgggagc
catttgcctc tgggaaaacc agtgagtctg gagagctgca tgggctcaca 180
actgaggagg aatttgtaga agggatatac aaagtggaaa tagacaccaa atcttactgg
240 aaggcacttg gcatctcccc attccatgag catgcagagg tggtattcac
agccaacgac 300 tccggccccc gccgctacac cattgccgcc ctgctgagcc
cctactccta ttccaccacg 360 gctgtcgtca ccaatcccaa ggaataa 387 4 387
DNA Homo sapiens 4 atgggtccaa ctggtaccgg tgaatccaag gctcctctga
tggtcaaagt tctagatgct 60 gtccgaggca gtcctgccat caatgtggcc
gtgcatgtgt tcagaaaggc ttgtgatgac 120 acctgggagc catttgcctc
tgggaaaacc agtgagtctg gagagctgca tgggctcaca 180 actgaggagg
aatttgtaga agggatatac aaagtggaaa tagacaccaa atcttactgg 240
aaggcacttg gcatctcccc attccatgag catgcagagg tggtattcac agccaacgac
300 tccggccccc gccgctacac cattgccgcc ctgctgagcc cctactccta
ttccaccacg 360 gctgtcgtca ccaatcccaa ggaataa 387 5 387 DNA Homo
sapiens 5 atgggtccaa ctggtaccgg tgaatccaag gctcctctga tggtcaaagt
tctagatgct 60 gtccgaggca gtcctgccat caatgtggcc gtgcatgtgt
tcagaaaggc tgcttgtgac 120 acctgggagc catttgcctc tgggaaaacc
agtgagtctg gagagctgca tgggctcaca 180 actgaggagg aatttgtaga
agggatatac aaagtggaaa tagacaccaa atcttactgg 240 aaggcacttg
gcatctcccc attccatgag catgcagagg tggtattcac agccaacgac 300
tccggccccc gccgctacac cattgccgcc ctgctgagcc cctactccta ttccaccacg
360 gctgtcgtca ccaatcccaa ggaataa 387 6 387 DNA Homo sapiens 6
atgggtccaa ctggtaccgg tgaatccaag gctcctctga tggtcaaagt tctagatgct
60 gtccgaggca gtcctgccat caatgtggcc gtgcatgtgt tcagaaaggc
tgctgatgac 120 acctgggagc catttgcctc tgggaaaacc agtgagtctg
gagagctgca tgggctcaca 180 actgaggagg aatttgtaga agggatatac
aaagtggaaa tagacaccaa atcttactgg 240 aagtgtcttg gcatctcccc
attccatgag catgcagagg tggtattcac agccaacgac 300 tccggccccc
gccgctacac cattgccgcc ctgctgagcc cctactccta ttccaccacg 360
gctgtcgtca ccaatcccaa ggaataa 387 7 387 DNA Homo sapiens 7
atgggtccaa ctggtaccgg tgaatccaag gctcctctga tggtcaaagt tctagatgct
60 gtccgaggca gtcctgccat caatgtggcc gtgcatgtgt tcagaaaggc
tgctgatgac 120 acctgggagc catttgcctc tgggaaaacc agtgagtctg
gagagctgca tgggctcaca 180 actgaggagg aatttgtaga agggatatac
aaagtggaaa tagacaccaa atcttactgg 240 aaggcacttt gcatctcccc
attccatgag catgcagagg tggtattcac agccaacgac 300 tccggccccc
gccgctacac cattgccgcc ctgctgagcc cctactccta ttccaccacg 360
gctgtcgtca ccaatcccaa ggaataa 387 8 387 DNA Homo sapiens 8
atgggtccaa ctggtaccgg tgaatccaag gctcctctga tggtcgcagt tctagatgct
60 gtccgaggca gtcctgccat caatgtggcc gtgcatgtgt tcagaaaggc
tgctgatgac 120 acctgggagc catttgcctc tgggaaaacc agtgagtctg
gagagctgca tgggctcaca 180 actgaggagg aatttgtaga agggatatac
aaagtggaaa tagacaccaa atcttactgg 240 aaggcacttt gcatctcccc
attccatgag catgcagagg tggtattcac agccaacgac 300 tccggccccc
gccgctacac cattgccgcc ctgctgagcc cctactccta ttccaccacg 360
gctgtcgtca ccaatcccaa ggaataa 387 9 495 DNA Homo sapiens 9
atgatcgaag gtccgactct gcgtcagtgg ctggctgctc gtgctggcgg tggtggcgga
60 gggggtggca ttgagggccc aacccttcgc caatggcttg cagcacgcgc
aggtccaact 120 ggtaccggtg aatccaagtg tcctctgatg gtcaaagttc
tagatgctgt ccgaggcagt 180 cctgccatca atgtggccgt gcatgtgttc
agaaaggctg ctgatgacac ctgggagcca 240 tttgcctctg ggaaaaccag
tgagtctgga gagctgcatg ggctcacaac tgaggaggaa 300 tttgtagaag
ggatatacaa agtggaaata gacaccaaat cttactggaa ggcacttggc 360
atctccccat tccatgagca tgcagaggtg gtattcacag ccaacgactc cggcccccgc
420 cgctacacca ttgccgccct gctgagcccc tactcctatt ccaccacggc
tgtcgtcacc 480 aatcccaagg aataa 495 10 495 DNA Homo sapiens 10
atgatcgaag gtccgactct gcgtcagtgg ctggctgctc gtgctggcgg tggtggcgga
60 gggggtggca ttgagggccc aacccttcgc caatggcttg cagcacgcgc
aggtccaact 120 ggtaccggtg aatccaaggc tcctctgatg gtcaaagttc
tagatgctgt ccgaggcagt 180 cctgccatca atgtggccgt gcatgtgttc
agaaaggctt gtgatgacac ctgggagcca 240 tttgcctctg ggaaaaccag
tgagtctgga gagctgcatg ggctcacaac tgaggaggaa 300 tttgtagaag
ggatatacaa agtggaaata gacaccaaat cttactggaa ggcacttggc 360
atctccccat tccatgagca tgcagaggtg gtattcacag ccaacgactc cggcccccgc
420 cgctacacca ttgccgccct gctgagcccc tactcctatt ccaccacggc
tgtcgtcacc 480 aatcccaagg aataa 495 11 495 DNA Homo sapiens 11
atgatcgaag gtccgactct gcgtcagtgg ctggctgctc gtgctggcgg tggtggcgga
60 gggggtggca ttgagggccc aacccttcgc caatggcttg cagcacgcgc
aggtccaact 120 ggtaccggtg aatccaaggc tcctctgatg gtcaaagttc
tagatgctgt ccgaggcagt 180 cctgccatca atgtggccgt gcatgtgttc
agaaaggctg cttgtgacac ctgggagcca 240 tttgcctctg ggaaaaccag
tgagtctgga gagctgcatg ggctcacaac tgaggaggaa 300 tttgtagaag
ggatatacaa agtggaaata gacaccaaat cttactggaa ggcacttggc 360
atctccccat tccatgagca tgcagaggtg gtattcacag ccaacgactc cggcccccgc
420 cgctacacca ttgccgccct gctgagcccc tactcctatt ccaccacggc
tgtcgtcacc 480 aatcccaagg aataa 495 12 495 DNA Homo sapiens 12
atgatcgaag gtccgactct gcgtcagtgg ctggctgctc gtgctggcgg tggtggcgga
60 gggggtggca ttgagggccc aacccttcgc caatggcttg cagcacgcgc
aggtccaact 120 ggtaccggtg aatccaaggc tcctctgatg gtcaaagttc
tagatgctgt ccgaggcagt 180 cctgccatca atgtggccgt gcatgtgttc
agaaaggctg ctgatgacac ctgggagcca 240 tttgcctctg ggaaaaccag
tgagtctgga gagctgcatg ggctcacaac tgaggaggaa 300 tttgtagaag
ggatatacaa agtggaaata gacaccaaat cttactggaa gtgtcttggc 360
atctccccat tccatgagca tgcagaggtg gtattcacag ccaacgactc cggcccccgc
420 cgctacacca ttgccgccct gctgagcccc tactcctatt ccaccacggc
tgtcgtcacc 480 aatcccaagg aataa 495 13 495 DNA Homo sapiens 13
atgatcgaag gtccgactct gcgtcagtgg ctggctgctc gtgctggcgg tggtggcgga
60 gggggtggca ttgagggccc aacccttcgc caatggcttg cagcacgcgc
aggtccaact 120 ggtaccggtg aatccaaggc tcctctgatg gtcaaagttc
tagatgctgt ccgaggcagt 180 cctgccatca atgtggccgt gcatgtgttc
agaaaggctg ctgatgacac ctgggagcca 240 tttgcctctg ggaaaaccag
tgagtctgga gagctgcatg ggctcacaac tgaggaggaa 300 tttgtagaag
ggatatacaa agtggaaata gacaccaaat cttactggaa ggcactttgc 360
atctccccat tccatgagca tgcagaggtg gtattcacag ccaacgactc cggcccccgc
420 cgctacacca ttgccgccct gctgagcccc tactcctatt ccaccacggc
tgtcgtcacc 480 aatcccaagg aataa 495 14 489 DNA Homo sapiens 14
atgtctgttt ctgaaatcca gctgatgcat aacctgggta aacatctgaa ctctatggaa
60 cgtgttgaat ggctgcgtaa gaaactgcag gacgttcata actttggtcc
aactggtacc 120 ggtgaatcca aggctcctct gatggtcgca gttctagatg
ctgtccgagg cagtcctgcc 180 atcaatgtgg ccgtgcatgt gttcagaaag
gctgctgatg acacctggga gccatttgcc 240 tctgggaaaa ccagtgagtc
tggagagctg catgggctca caactgagga ggaatttgta 300 gaagggatat
acaaagtgga aatagacacc aaatcttact ggaagtgtct tggcatctcc 360
ccattccatg agcatgcaga ggtggtattc acagccaacg actccggccc ccgccgctac
420 accattgccg ccctgctgag cccctactcc tattccacca cggctgtcgt
caccaatccc 480 aaggaataa 489 15 843 DNA Homo sapiens 15 atgcgaccgt
ccggccgtaa gagctccaaa atgcaggctt tccgtatctg ggacgttaac 60
cagaaaacct tctacctgcg caacaaccag ctggttgctg gctacctgca gggtccgaac
120 gttaacctgg aagaaaaaat cgacgttgta ccgatcgaac cgcacgctct
gttcctgggt 180 atccacggtg gtaaaatgtg cctgagctgc gtgaaatctg
gtgacgaaac tcgtctgcag 240 ctggaagcag ttaacatcac tgacctgagc
gaaaaccgca aacaggacaa acgtttcgca 300 ttcatccgct ctgacagcgg
cccgaccacc agcttcgaat ctgctgcttg cccgggttgg 360 ttcctgtgca
ctgctatgga agctgaccag ccggtaagcc tgaccaacat gccggacgaa 420
ggcgtgatgg taaccaaatt ctacttccag gaagacgaag gtccaactgg taccggtgaa
480 tccaaggctc ctctgatggt caaagttcta gatgctgtcc gaggcagtcc
tgccatcaat 540 gtggccgtgc atgtgttcag aaaggctgct gatgacacct
gggagccatt tgcctctggg 600 aaaaccagtg agtctggaga gctgcatggg
ctcacaactg aggaggaatt tgtagaaggg 660 atatacaaag tggaaataga
caccaaatct tactggaagg cacttggcat ctccccattc 720 catgagcatg
cagaggtggt attcacagcc aacgactccg gcccccgccg ctacaccatt 780
gccgccctgc tgagccccta ctcctattcc accacggctg tcgtcaccaa tcccaaggaa
840 taa 843 16 855 DNA Homo sapiens 16 atgggtccaa ctggtaccgg
tgaatccaag gctcctctga tggtcaaagt tctagatgct 60 gtccgaggca
gtcctgccat caatgtggcc gtgcatgtgt tcagaaaggc tgctgatgac 120
acctgggagc catttgcctc tgggaaaacc agtgagtctg gagagctgca tgggctcaca
180 actgaggagg aatttgtaga agggatatac aaagtggaaa tagacaccaa
atcttactgg 240 aaggcacttg gcatctcccc attccatgag catgcagagg
tggtattcac agccaacgac 300 tccggccccc gccgctacac cattgccgcc
ctgctgagcc cctactccta ttccaccacg 360 gctgtcgtca ccaatcccaa
ggaaggtagt ggtagccgac cgtccggccg taagagctcc 420 aaaatgcagg
ctttccgtat ctgggacgtt aaccagaaaa ccttctacct gcgcaacaac 480
cagctggttg ctggctacct gcagggtccg aacgttaacc tggaagaaaa aatcgacgtt
540 gtaccgatcg aaccgcacgc tctgttcctg ggtatccacg gtggtaaaat
gtgcctgagc 600 tgcgtgaaat ctggtgacga aactcgtctg cagctggaag
cagttaacat cactgacctg 660 agcgaaaacc gcaaacagga caaacgtttc
gcattcatcc gctctgacag cggcccgacc 720 accagcttcg aatctgctgc
ttgcccgggt tggttcctgt gcactgctat ggaagctgac 780 cagccggtaa
gcctgaccaa catgccggac gaaggcgtga tggtaaccaa attctacttc 840
caggaagacg aataa 855 17 439 DNA Homo sapiens 17 catatgggtc
caactggtac cggtgaatcc aaggctcctc tgatggtcaa agttctagat 60
gctgtccgag gcagtcctgc catcaatgtg gccgtgcatg tgttcagaaa ggctgctgat
120 gacacctggg agccatttgc ctctgggaaa accagtgagt ctggagagct
gcatgggctc 180 acaactgagg aggaatttgt agaagggata tacaaagtgg
aaatagacac caaatcttac 240 tggaaggcac tttgcatctc cccattccat
gagcatgcag aggtggtatt cacagccaac 300 gactccggcc cccgccgcta
caccattgcc gccctgctga gcccctactc ctattccacc 360 actgcagtcg
tcaccaatcc caaggaagga tcaggatccg gaaaacgtcc gccgggtttc 420
tccccgctgt aatctcgag 439 18 36 DNA Homo sapiens 18 gaggaataac
atatgggtcc aactggtacc ggtgaa 36 19 36 DNA Homo sapiens 19
ccgcggatcc tcgagattat tccttgggat tggtga 36 20 48 DNA Homo sapiens
20 gaggaataac atatgggtcc aactggtacc ggtgaatcca aggctcct 48 21 24
DNA Homo sapiens 21 agaaaggctt gtgatgacac ctgg 24 22 24 DNA Homo
sapiens 22 ccaggtgtca tcacaagcct ttct 24 23 24 DNA Homo sapiens 23
agaaaggctg cttgtgacac ctgg 24 24 24 DNA Homo sapiens 24 ccaggtgtca
caagcagcct ttct 24 25 24 DNA Homo sapiens 25 tactggaagt gtcttggcat
ctcc 24 26 24 DNA Homo sapiens 26 ggagatgcca agacacttcc agta 24 27
24 DNA Homo sapiens 27 aaggcacttt gcatctcccc attc 24 28 24 DNA Homo
sapiens 28 gaatggggag atgcaaagtg cctt 24 29 21 DNA Homo sapiens 29
ctgatggtcg cagttctaga t 21 30 21 DNA Homo sapiens 30 atctagaact
gcgaccatca g 21 31 36 DNA Homo sapiens 31 gaggaataac atatgatcga
aggtccgact ctgcgt 36 32 41 DNA Homo sapiens 32 ttcaccggta
ccagttggac ctgcgcgtgc tgcaagccat t 41 33 33 DNA Homo sapiens 33
gaggaataac atatgtctgt ttctgaaatc cag 33 34 36 DNA Homo sapiens 34
ttcaccggta ccagttggac caaagttatg aacgtc 36 35 32 DNA Homo sapiens
35 gaggaataac atatgcgacc gtccggacgt aa 32 36 36 DNA Homo sapiens 36
ttctacttcc aggaagacga aggtccaact ggtacc 36 37 54 DNA Homo sapiens
37 gtcgtcacca atcccaagga aggtagtggt agccgaccgt ccggccgtaa gagc 54
38 40 DNA Homo sapiens 38 ccgcggatcc tcgagattat tcgtcttcct
ggaagtagaa 40 39 36 DNA Homo sapiens 39 gaggaataac atatgggtcc
aactggtacc ggtgaa 36 40 26 DNA Homo sapiens 40 aatatactgc
agtggtggaa taggag 26 41 68 DNA Homo sapiens 41 gtcgtcacca
atcccaagga aggatcagga tccggaaaac gtccgccggg tttctccccg 60 ctgtaatc
68 42 76 DNA Homo sapiens 42 tcgagattac agcggggaga aacccggcgg
acgttttccg gatcctgatc cttccttggg 60 attggtgacg actgca 76 43 489 DNA
Homo sapiens 43 atgtctgttt ctgaaatcca gctgatgcat aacctgggta
aacatctgaa ctctatggaa 60 cgtgttgaat ggctgcgtaa gaaactgcag
gacgttcata actttggtcc aactggtacc 120 ggtgaatcca aggctcctct
gatggtcgca gttctagatg ctgtccgagg cagtcctgcc 180 atcaatgtgg
ccgtgcatgt gttcagaaag gctgctgatg acacctggga gccatttgcc 240
tctgggaaaa ccagtgagtc tggagagctg catgggctca caactgagga ggaatttgta
300 gaagggatat acaaagtgga aatagacacc aaatcttact ggaagtgtct
tggcatctcc 360 ccattccatg agcatgcaga ggtggtattc acagccaacg
actccggccc ccgccgctac 420 accattgccg ccctgctgag cccctactcc
tattccacca cggctgtcgt caccaatccc 480 aaggaataa 489 44 25 DNA Homo
sapiens 44 cgtacaggtt tacgcaagaa aatgg 25 45 62 DNA Homo sapiens 45
ggattcaccg gtaccagttg gaccaccacc accaccacca cccgcactgc ctgaaccaga
60 gc 62 46 71 DNA Homo sapiens 46 tgactaagcc atatgaaaca tcatcaccat
caccatcatg acgaagttga tcacggtgaa 60 ggtactttca c 71 47 44 DNA Homo
sapiens 47 ggattcaccg gtaccagttg gaccaccacc accaccaccg ctac 44 48
594 DNA Homo sapiens 48 atgaaacatc atcaccatca ccatcatgac gaagttgatc
acggtgaagg tactttcact 60 tctgacgttt cttcttatct ggaaggtcag
gctgctaaag aattcatcgc ttggctggtt 120 aaaggtcgtg gtggttctgg
ttctgctact ggtggttccg gctccaccgc aagctctggt 180 tcaggcagtg
cgggtggtgg tggtggtggt ggtccaactg gtaccggtga atccaaggct 240
cctctgatgg tcaaagttct agatgctgtc cgaggcagtc ctgccatcaa tgtggccgtg
300 catgtgttca gaaaggctgc tgatgacacc tgggagccat ttgcctctgg
gaaaaccagt 360 gagtctggag agctgcatgg gctcacaact gaggaggaat
ttgtagaagg gatatacaaa 420 gtggaaatag acaccaaatc ttactggaag
gcactttgca tctccccatt ccatgagcat 480 gcagaggtgg tattcacagc
caacgactcc ggcccccgcc gctacaccat tgccgccctg 540 ctgagcccct
actcctattc caccacggct gtcgtcacca atcccaagga ataa 594 49 591 DNA
Homo sapiens 49 atgaaacatc atcaccatca ccatcatgac gaagttgatc
acggtgaagg tactttcact 60 tctgacgttt cttcttatct ggaaggtcag
gctgctaaag aattcatcgc ttggctggtt 120 aaaggtcgtg gtggtggtgg
tggttctggt ggtggtggtt ctggtggtgg tggttctggc 180 ggcggtggta
gcggtggtgg tggtggtggt ccaactggta ccggtgaatc caaggctcct 240
ctgatggtcg cagttctaga tgctgtccga ggcagtcctg ccatcaatgt ggccgtgcat
300 gtgttcagaa aggctgctga tgacacctgg gagccatttg cctctgggaa
aaccagtgag 360 tctggagagc tgcatgggct cacaactgag gaggaatttg
tagaagggat atacaaagtg 420 gaaatagaca ccaaatctta ctggaaggca
ctttgcatct ccccattcca tgagcatgca 480 gaggtggtat tcacagccaa
cgactccggc ccccgccgct acaccattgc cgccctgctg 540 agcccctact
cctattccac cacggctgtc gtcaccaatc ccaaggaata a 591 50 76 DNA Homo
sapiens 50 agacctgtac atatgaaaca tcatcaccat caccatcatg acgaagttga
tcacggtgaa 60 ggtactttca cttctg 76 51 20 DNA Homo sapiens 51
gggggaagag gaaaactgac 20 52 894 DNA Homo sapiens 52 atgaaacatc
atcaccatca ccatcatgac gaagttgatc acggtgaagg tactttcact 60
tctgacgttt cttcttatct ggaaggtcag gctgctaaag aattcatcgc ttggctggtt
120 aaaggtcgtg gtggttctgg ttctgctact ggtggttccg gctccaccgc
aagctctggt 180 tcaggcagtg cgactcatgg tggtggtggt ggtgacaaaa
ctcacacatg tccaccgtgc 240 ccagcacctg aactcctggg gggaccgtca
gttttcctct tccccccaaa acccaaggac 300 accctcatga tctcccggac
ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 360 gaccctgagg
tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 420
aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg
480 caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa
agccctccca 540 gcccccatcg agaaaaccat ctccaaagcc aaagggcagc
cccgagaacc acaggtgtac 600 accctgcccc catcccggga tgagctgacc
aagaaccagg tcagcctgac ctgcctggtc 660 aaaggcttct
atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac 720
aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag
780 ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc
cgtgatgcat 840 gaggctctgc acaaccacta cacgcagaag agcctctccc
tgtctccggg taaa 894 53 60 DNA Homo sapiens 53 gtcgtcacca atcccaagga
aggttctggc tccggatcag ggggaccgtc agttttcctc 60 54 52 DNA Homo
sapiens 54 ccgcggatcc tcgagattag gatccagaac cccctttggc tttggagatg
gt 52 55 42 DNA Homo sapiens 55 gaggaataac atatgggtcc aactggtacc
ggtgaatcca ag 42 56 735 DNA Homo sapiens 56 atgggtccaa ctggtaccgg
tgaatccaag gctcctctga tggtcaaagt tctagatgct 60 gtccgaggca
gtcctgccat caatgtggcc gtgcatgtgt tcagaaaggc tgctgatgac 120
acctgggagc catttgcctc tgggaaaacc agtgagtctg gagagctgca tgggctcaca
180 actgaggagg aatttgtaga agggatatac aaagtggaaa tagacaccaa
atcttactgg 240 aaggcacttg gcatctcccc attccatgag catgcagagg
tggtattcac agccaacgac 300 tccggccccc gccgctacac cattgccgcc
ctgctgagcc cctactccta ttccaccacg 360 gctgtcgtca ccaatcccaa
ggaaggttct ggctccggat cagggggacc gtcagttttc 420 ctcttccccc
caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 480
gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc
540 gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag
cacgtaccgt 600 gtggtcagcg tcctcaccgt cctgcaccag gactggctga
atggcaagga gtacaagtgc 660 aaggtctcca acaaagccct cccagccccc
atcgagaaaa ccatctccaa agccaaaggg 720 ggttctggat cctaa 735 57 843
DNA Homo sapiens 57 atgatcgaag gtccgactct gcgtcagtgg ctggctgctc
gtgctggcgg tggtggcgga 60 gggggtggca ttgagggccc aacccttcgc
caatggcttg cagcacgcgc aggtccaact 120 ggtaccggtg aatccaaggc
tcctctgatg gtcaaagttc tagatgctgt ccgaggcagt 180 cctgccatca
atgtggccgt gcatgtgttc agaaaggctg ctgatgacac ctgggagcca 240
tttgcctctg ggaaaaccag tgagtctgga gagctgcatg ggctcacaac tgaggaggaa
300 tttgtagaag ggatatacaa agtggaaata gacaccaaat cttactggaa
ggcacttggc 360 atctccccat tccatgagca tgcagaggtg gtattcacag
ccaacgactc cggcccccgc 420 cgctacacca ttgccgccct gctgagcccc
tactcctatt ccaccacggc tgtcgtcacc 480 aatcccaagg aaggttctgg
ctccggatca gggggaccgt cagttttcct cttcccccca 540 aaacccaagg
acaccctcat gatctcccgg acccctgagg tcacatgcgt ggtggtggac 600
gtgagccacg aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat
660 aatgccaaga caaagccgcg ggaggagcag tacaacagca cgtaccgtgt
ggtcagcgtc 720 ctcaccgtcc tgcaccagga ctggctgaat ggcaaggagt
acaagtgcaa ggtctccaac 780 aaagccctcc cagcccccat cgagaaaacc
atctccaaag ccaaaggggg ttctggatcc 840 taa 843 58 35 DNA Homo sapiens
58 gaggaataag gatccatcga aggtccgact ctgcg 35 59 843 DNA Homo
sapiens 59 atgggtccaa ctggtaccgg tgaatccaag gctcctctga tggtcaaagt
tctagatgct 60 gtccgaggca gtcctgccat caatgtggcc gtgcatgtgt
tcagaaaggc tgctgatgac 120 acctgggagc catttgcctc tgggaaaacc
agtgagtctg gagagctgca tgggctcaca 180 actgaggagg aatttgtaga
agggatatac aaagtggaaa tagacaccaa atcttactgg 240 aaggcacttg
gcatctcccc attccatgag catgcagagg tggtattcac agccaacgac 300
tccggccccc gccgctacac cattgccgcc ctgctgagcc cctactccta ttccaccacg
360 gctgtcgtca ccaatcccaa ggaaggttct ggctccggat cagggggacc
gtcagttttc 420 ctcttccccc caaaacccaa ggacaccctc atgatctccc
ggacccctga ggtcacatgc 480 gtggtggtgg acgtgagcca cgaagaccct
gaggtcaagt tcaactggta cgtggacggc 540 gtggaggtgc ataatgccaa
gacaaagccg cgggaggagc agtacaacag cacgtaccgt 600 gtggtcagcg
tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc 660
aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg
720 ggttctggat ccatcgaagg tccgactctg cgtcagtggc tggctgctcg
tgctggcggt 780 ggtggcggag ggggtggcat tgagggccca acccttcgcc
aatggcttgc agcacgcgca 840 taa 843 60 48 DNA Homo sapiens 60
ttcaccggta ccagttggac cagaaccccc tttggctttg gagatggt 48 61 48 DNA
Homo sapiens 61 gaggaataac atatgggatc cggttctggg ggaccgtcag
ttttcctc 48 62 723 DNA Homo sapiens 62 atgggatccg gttctggggg
accgtcagtt ttcctcttcc ccccaaaacc caaggacacc 60 ctcatgatct
cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 120
cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag
180 ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac
cgtcctgcac 240 caggactggc tgaatggcaa ggagtacaag tgcaaggtct
ccaacaaagc cctcccagcc 300 cccatcgaga aaaccatctc caaagccaaa
gggggttctg gtccaactgg taccggtgaa 360 tccaaggctc ctctgatggt
caaagttcta gatgctgtcc gaggcagtcc tgccatcaat 420 gtggccgtgc
atgtgttcag aaaggctgct gatgacacct gggagccatt tgcctctggg 480
aaaaccagtg agtctggaga gctgcatggg ctcacaactg aggaggaatt tgtagaaggg
540 atatacaaag tggaaataga caccaaatct tactggaagg cacttggcat
ctccccattc 600 catgagcatg cagaggtggt attcacagcc aacgactccg
gcccccgccg ctacaccatt 660 gccgccctgc tgagccccta ctcctattcc
accacggctg tcgtcaccaa tcccaaggaa 720 taa 723 63 33 DNA Homo sapiens
63 gaggaataac atatgatcga aggtccgact ctg 33 64 36 DNA Homo sapiens
64 taacatatgg gatcctgcgc gtgctgcaag ccattg 36 65 831 DNA Homo
sapiens 65 atgatcgaag gtccgactct gcgtcagtgg ctggctgctc gtgctggcgg
tggtggcgga 60 gggggtggca ttgagggccc aacccttcgc caatggcttg
cagcacgcgc aggatccggt 120 tctgggggac cgtcagtttt cctcttcccc
ccaaaaccca aggacaccct catgatctcc 180 cggacccctg aggtcacatg
cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 240 ttcaactggt
acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 300
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg
360 aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc
catcgagaaa 420 accatctcca aagccaaagg gggttctggt ccaactggta
ccggtgaatc caaggctcct 480 ctgatggtca aagttctaga tgctgtccga
ggcagtcctg ccatcaatgt ggccgtgcat 540 gtgttcagaa aggctgctga
tgacacctgg gagccatttg cctctgggaa aaccagtgag 600 tctggagagc
tgcatgggct cacaactgag gaggaatttg tagaagggat atacaaagtg 660
gaaatagaca ccaaatctta ctggaaggca cttggcatct ccccattcca tgagcatgca
720 gaggtggtat tcacagccaa cgactccggc ccccgccgct acaccattgc
cgccctgctg 780 agcccctact cctattccac cacggctgtc gtcaccaatc
ccaaggaata a 831 66 42 DNA Homo sapiens 66 gtcgtcacca atcccaagga
aggttctggt tctggtatcg aa 42 67 40 DNA Homo sapiens 67 ccgcggatcc
tcgagattat gcgcgtgctg caagccattg 40 68 510 DNA Homo sapiens 68
atgggtccaa ctggtaccgg tgaatccaag tgtcctctga tggtcaaagt tctagatgct
60 gtccgaggca gtcctgccat caatgtggcc gtgcatgtgt tcagaaaggc
tgctgatgac 120 acctgggagc catttgcctc tgggaaaacc agtgagtctg
gagagctgca tgggctcaca 180 actgaggagg aatttgtaga agggatatac
aaagtggaaa tagacaccaa atcttactgg 240 aaggcacttg gcatctcccc
attccatgag catgcagagg tggtattcac agccaacgac 300 tccggccccc
gccgctacac cattgccgcc ctgctgagcc cctactccta ttccaccacg 360
gctgtcgtca ccaatcccaa ggaaggttct ggttctggta tcgaaggtcc gactctgcgt
420 cagtggctgg ctgctcgtgc tggcggtggt ggcggagggg gtggcattga
gggcccaacc 480 cttcgccaat ggcttgcagc acgcgcataa 510 69 510 DNA Homo
sapiens 69 atgggtccaa ctggtaccgg tgaatccaag gctcctctga tggtcgcagt
tctagatgct 60 gtccgaggca gtcctgccat caatgtggcc gtgcatgtgt
tcagaaaggc tgctgatgac 120 acctgggagc catttgcctc tgggaaaacc
agtgagtctg gagagctgca tgggctcaca 180 actgaggagg aatttgtaga
agggatatac aaagtggaaa tagacaccaa atcttactgg 240 aaggcacttg
gcatctcccc attccatgag catgcagagg tggtattcac agccaacgac 300
tccggccccc gccgctacac cattgccgcc ctgctgagcc cctactccta ttccaccacg
360 gctgtcgtca ccaatcccaa ggaaggttct ggttctggta tcgaaggtcc
gactctgcgt 420 cagtggctgg ctgctcgtgc tggcggtggt ggcggagggg
gtggcattga gggcccaacc 480 cttcgccaat ggcttgcagc acgcgcataa 510 70
510 DNA Homo sapiens 70 atgggtccaa ctggtaccgg tgaatccaag gctcctctga
tggtcgcagt tctagatgct 60 gtccgaggca gtcctgccat caatgtggcc
gtgcatgtgt tcagaaaggc ttgtgatgac 120 acctgggagc catttgcctc
tgggaaaacc agtgagtctg gagagctgca tgggctcaca 180 actgaggagg
aatttgtaga agggatatac aaagtggaaa tagacaccaa atcttactgg 240
aaggcacttg gcatctcccc attccatgag catgcagagg tggtattcac agccaacgac
300 tccggccccc gccgctacac cattgccgcc ctgctgagcc cctactccta
ttccaccacg 360 gctgtcgtca ccaatcccaa ggaaggttct ggttctggta
tcgaaggtcc gactctgcgt 420 cagtggctgg ctgctcgtgc tggcggtggt
ggcggagggg gtggcattga gggcccaacc 480 cttcgccaat ggcttgcagc
acgcgcataa 510 71 510 DNA Homo sapiens 71 atgggtccaa ctggtaccgg
tgaatccaag gctcctctga tggtcgcagt tctagatgct 60 gtccgaggca
gtcctgccat caatgtggcc gtgcatgtgt tcagaaaggc tgctgatgac 120
acctgggagc catttgcctc tgggaaaacc agtgagtctg gagagctgca tgggctcaca
180 actgaggagg aatttgtaga agggatatac aaagtggaaa tagacaccaa
atcttactgg 240 aagtgtcttg gcatctcccc attccatgag catgcagagg
tggtattcac agccaacgac 300 tccggccccc gccgctacac cattgccgcc
ctgctgagcc cctactccta ttccaccacg 360 gctgtcgtca ccaatcccaa
ggaaggttct ggttctggta tcgaaggtcc gactctgcgt 420 cagtggctgg
ctgctcgtgc tggcggtggt ggcggagggg gtggcattga gggcccaacc 480
cttcgccaat ggcttgcagc acgcgcataa 510 72 510 DNA Homo sapiens 72
atgggtccaa ctggtaccgg tgaatccaag gctcctctga tggtcgcagt tctagatgct
60 gtccgaggca gtcctgccat caatgtggcc gtgcatgtgt tcagaaaggc
tgctgatgac 120 acctgggagc catttgcctc tgggaaaacc agtgagtctg
gagagctgca tgggctcaca 180 actgaggagg aatttgtaga agggatatac
aaagtggaaa tagacaccaa atcttactgg 240 aaggcacttt gcatctcccc
attccatgag catgcagagg tggtattcac agccaacgac 300 tccggccccc
gccgctacac cattgccgcc ctgctgagcc cctactccta ttccaccacg 360
gctgtcgtca ccaatcccaa ggaaggttct ggttctggta tcgaaggtcc gactctgcgt
420 cagtggctgg ctgctcgtgc tggcggtggt ggcggagggg gtggcattga
gggcccaacc 480 cttcgccagt ggcttgcagc acgcgcataa 510 73 495 DNA Homo
sapiens 73 atgatcgaag gtccgactct gcgtcagtgg ctggctgctc gtgctggcgg
tggtggcgga 60 gggggtggca ttgagggccc aacccttcgc caatggcttg
cagcacgcgc aggtccaact 120 ggtaccggtg aatccaaggc tcctctgatg
gtcgcagttc tagatgctgt ccgaggcagt 180 cctgccatca atgtggccgt
gcatgtgttc agaaaggctg ctgatgacac ctgggagcca 240 tttgcctctg
ggaaaaccag tgagtctgga gagctgcatg ggctcacaac tgaggaggaa 300
tttgtagaag ggatatacaa agtggaaata gacaccaaat cttactggaa ggcactttgc
360 atctccccat tccatgagca tgcagaggtg gtattcacag ccaacgactc
cggcccccgc 420 cgctacacca ttgccgccct gctgagcccc tactcctatt
ccaccacggc tgtcgtcacc 480 aatcccaagg aataa 495 74 495 DNA Homo
sapiens 74 atgatcgaag gtccgactct gcgtcagtgg ctggctgctc gtgctggcgg
tggtggcgga 60 gggggtggca ttgagggccc aacccttcgc caatggcttg
cagcacgcgc aggtccaact 120 ggtaccggtg aatccaaggc tcctctgatg
gtcgcagttc tagatgctgt ccgaggcagt 180 cctgccatca atgtggccgt
gcatgtgttc agaaaggctg ctgatgacac ctgggagcca 240 tttgcctctg
ggaaaaccag tgagtctgga gagctgcatg ggctcacaac tgaggaggaa 300
tttgtagaag ggatatacaa agtggaaata gacaccaaat cttactggaa gtgtcttggc
360 atctccccat tccatgagca tgcagaggtg gtattcacag ccaacgactc
cggcccccgc 420 cgctacacca ttgccgccct gctgagcccc tactcctatt
ccaccacggc tgtcgtcacc 480 aatcccaagg aataa 495 75 495 DNA Homo
sapiens 75 atgatcgaag gtccgactct gcgtcagtgg ctggctgctc gtgctggcgg
tggtggcgga 60 gggggtggca ttgagggccc aacccttcgc caatggcttg
cagcacgcgc aggtccaact 120 ggtaccggtg aatccaaggc tcctctgatg
gtcgcagttc tagatgctgt ccgaggcagt 180 cctgccatca atgtggccgt
gcatgtgttc agaaaggctt gtgatgacac ctgggagcca 240 tttgcctctg
ggaaaaccag tgagtctgga gagctgcatg ggctcacaac tgaggaggaa 300
tttgtagaag ggatatacaa agtggaaata gacaccaaat cttactggaa ggcacttggc
360 atctccccat tccatgagca tgcagaggtg gtattcacag ccaacgactc
cggcccccgc 420 cgctacacca ttgccgccct gctgagcccc tactcctatt
ccaccacggc tgtcgtcacc 480 aatcccaagg aataa 495
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