U.S. patent application number 11/036256 was filed with the patent office on 2006-02-02 for methods of producing peptides/proteins in plants and peptides/proteins produced thereby.
Invention is credited to Marcia J. Kieliszewski, Jianfeng Xu.
Application Number | 20060026719 11/036256 |
Document ID | / |
Family ID | 34812077 |
Filed Date | 2006-02-02 |
United States Patent
Application |
20060026719 |
Kind Code |
A1 |
Kieliszewski; Marcia J. ; et
al. |
February 2, 2006 |
Methods of producing peptides/proteins in plants and
peptides/proteins produced thereby
Abstract
Methods of increasing the yield in plant expression of
recombinant proteins comprising: engineering glycosylation sites
into cloned genes or cDNAs for proteins using codons that drive
post-translational modifications in plants; and engineering the
cloned genes or cDNAs to contain a plant secretory signal sequence
that targets the gene products (protein) for secretion. The methods
result in increased recombinant glycosylated protein yields.
Proteins produced according to these methods are disclosed.
Inventors: |
Kieliszewski; Marcia J.;
(Albany, OH) ; Xu; Jianfeng; (Athens, OH) |
Correspondence
Address: |
CALFEE HALTER & GRISWOLD, LLP
800 SUPERIOR AVENUE
SUITE 1400
CLEVELAND
OH
44114
US
|
Family ID: |
34812077 |
Appl. No.: |
11/036256 |
Filed: |
January 14, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60536486 |
Jan 14, 2004 |
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60582027 |
Jun 22, 2004 |
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60602562 |
Aug 18, 2004 |
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Current U.S.
Class: |
800/288 ;
435/419; 435/468; 530/370; 536/23.6 |
Current CPC
Class: |
A61P 5/00 20180101; A61P
37/08 20180101; A61P 25/00 20180101; A61P 25/20 20180101; C07K
14/61 20130101; A61P 25/02 20180101; A61P 21/04 20180101; A61P 3/00
20180101; A61P 37/04 20180101; C07K 14/415 20130101; A61P 3/10
20180101; C07K 14/56 20130101; A61P 9/10 20180101; A61P 17/02
20180101; A61P 27/02 20180101; A61P 35/00 20180101; A61P 21/00
20180101; C12N 15/8257 20130101; A61P 3/04 20180101; A61P 13/12
20180101; C12P 21/005 20130101; A61P 5/06 20180101; A61P 11/00
20180101; A61P 19/08 20180101; A61P 19/10 20180101; A61P 25/28
20180101; C12N 15/62 20130101; A61K 38/27 20130101; C07K 14/765
20130101; A61P 35/02 20180101; A61P 43/00 20180101; A61P 37/02
20180101; A61P 9/04 20180101 |
Class at
Publication: |
800/288 ;
530/370; 536/023.6; 435/419; 435/468 |
International
Class: |
A01H 1/00 20060101
A01H001/00; C07H 21/04 20060101 C07H021/04; C07K 14/42 20060101
C07K014/42; C12N 15/82 20060101 C12N015/82; C12N 5/04 20060101
C12N005/04 |
Claims
1. A nucleic acid construct for expression of at least one
biologically active protein in plants comprising: a) at least one
nucleic acid sequence encoding a glycosylation site and b) at least
one nucleic acid sequence encoding a biologically active
protein.
2. A plant-derived biologically active mammalian fusion
glycoprotein comprising: a) at least one glycomodule, covalently
linked to b) a mammalian biologically active protein.
3. The plant-derived biologically active mammalian fusion protein
according to claim 2, wherein the at least one glycomodule is
chosen from i) X-Hyp.sub.n (SEQ ID NO: 2) or X-Pro-Hyp.sub.n (SEQ
ID NO: 1), where n is from 2 to about 1000, ii) Hyp.sub.n-X (SEQ ID
NO: 156), where n is from 2 to about 1000, iii) (Hyp-X).sub.n (SEQ
ID NO: 157), where n is from 1 to about 1000, and iv) (X-Hyp).sub.n
(SEQ ID NO: 3), where n is from 1 to about 1000; wherein X is any
amino acid.
4. The plant-derived biologically active mammalian fusion protein
according to claim 3, wherein X is chosen from Lys, Ser, Ala, Thr,
Gly and Val for the glycomodules X-Hyp.sub.n, Hyp.sub.n-X,
(Hyp-X).sub.n, and (X-Hyp).sub.n.
5. The plant-derived biologically active mammalian fusion protein
according to claim 4, wherein X is chosen from Ser, Ala, Thr, and
Val.
6. The plant-derived biologically active mammalian fusion protein
according to claim 3, wherein the at least one glycomodule is
covalently linked at a location chosen from the N-terminus and the
C-terminus of the protein.
7. The plant-derived biologically active mammalian fusion protein
according to claim 3, wherein the at least one glycomodule is
within the interior of the biologically active mammalian
protein.
8. The plant-derived biologically active mammalian fusion protein
according to claim 2, wherein the biologically active mammalian
protein is chosen from growth hormone, growth hormone antagonists,
growth hormone releasing hormone, somatostatin, ghrelin, leptin,
prolactin, monocyte chemoattractant protein-1, interleukin-10,
pleiotropin, interleukin-7, interleukin-8, interferon omega,
interferon-Alpha 2, interferon gamma, interleukin-1, fibroblast
growth factor 6, IFG-1, insulin-like growth factor I, insulin,
erythropoietin, GMCSF, and any humanized monoclonal antibody
wherein the glycomodule comprises i) X-Hyp.sub.n (SEQ ID NO: 2),
X-Pro-Hyp.sub.n (SEQ ID NO: 1), or Hyp.sub.n-X (SEQ ID NO: 156)
where n is from 2 to about 1000, and ii) (X-Hyp).sub.n (SEQ ID NO:
3) or (Hyp-X).sub.n (SEQ ID NO: 157) where n is from 1 to about
1000; and wherein X is any amino acid.
9. The plant-derived biologically active mammalian fusion protein
according to claim 8, wherein X is chosen from Lys, Ser, Ala, Thr,
Gly and Val for the glycomodules X-Hyp.sub.n, Hyp.sub.n-X,
(Hyp-X).sub.n, and (X-Hyp).sub.n.
10. The plant-derived biologically active mammalian fusion protein
according to claim 9, wherein X is chosen from Ser, Ala, Thr, and
Val.
11. The plant-derived biologically active mammalian fusion protein
according to claim 8, wherein the biologically active mammalian
protein is a human protein.
12. The plant-derived biologically active mammalian fusion protein
according to claim 8, wherein the glycomodule comprises
(X-Hyp).sub.n or (Hyp-X).sub.n, wherein X is chosen from Lys, Ser,
Ala, Thr, Gly and Val.
13. The plant-derived biologically active mammalian fusion protein
according to claim 12, wherein X is chosen from Ser, Ala, Thr, and
Val.
14. The plant-derived biologically active mammalian fusion protein
according to claim 13, wherein the protein is human growth hormone,
and the glycomodule comprises (Ser-Hyp).sub.10 (SEQ ID NO: 4).
15. The plant-derived biologically active mammalian fusion protein
according to claim 2, wherein the fusion glycoprotein is covalently
linked to at least one carbohydrate molecule.
16. A method of increasing the aqueous solubility of a protein
molecule, comprising: preparing a nucleic acid sequence encoding:
a) at least one glycosylation site and b) at least one protein; and
expressing the nucleic acid construct as a fusion glycoprotein;
wherein carbohydrate component of the glycoprotein accounts for
greater than or equal to about 10% of the molecular weight of the
glycoprotein.
17. The method of increasing aqueous solubility of a protein
molecule according to claim 16, wherein carbohydrate component of
the glycoprotein accounts for greater than or equal to about 50% of
the molecular weight of the glycoprotein.
18. The method of increasing aqueous solubility of a protein
molecule according to claim 17, wherein carbohydrate component of
the glycoprotein accounts for greater than or equal to about 75% of
the molecular weight of the glycoprotein.
19. The method of increasing aqueous solubility of a protein
molecule according to claim 18, wherein carbohydrate component of
the glycoprotein accounts for greater than or equal to about 90% of
the molecular weight of the glycoprotein.
20. A method of producing a biologically active fusion protein,
comprising: expressing in a plant at least one nucleic acid
sequence encoding: a) at least one glycosylation site and b) at
least one biologically active protein, as a glycoprotein; wherein
the molecular weight of the glycoprotein is greater than or equal
to about 10 kD and wherein the carbohydrate component of the
glycoprotein accounts for greater than or equal to about 10% of the
molecular weight of the glycoprotein.
21. The method of producing a biologically active fusion protein
according to claim 20, wherein the at least one biologically active
protein is selected from insulin, insulin-like growth factor,
somatostatin, growth hormone releasing hormone, ghrelin, prolactin,
placental lactogen, growth hormone, and growth hormone
antagonist.
22. The method of producing a biologically active fusion protein
according to claim 20, wherein the molecular weight of the
glycoprotein is greater than or equal to about 35 kD.
23. The method of producing a biologically active fusion protein
according to claim 22, wherein the molecular weight of the
glycoprotein is greater than or equal to about 40 kD.
24. The method of producing a biologically active fusion protein
according to claim 23, wherein the molecular weight of the
glycoprotein is greater than or equal to about 45 kD.
25. The method of producing a biologically active fusion protein
according to claim 20, wherein the pharmacokinetic half-life of the
glycoprotein is greater than the pharmacokinetic half-life of a
corresponding wild-type protein.
26. The method of producing a biologically active fusion protein
molecule according to claim 20, wherein the at least one
glycosylation site is chosen from i) X-Pro.sub.n (SEQ ID NO: 158)
or Pro.sub.n-X (SEQ ID NO: 159), where n is from 6 to about 100,
and ii) (X-Pro).sub.n (SEQ ID NO: 160) or (Pro-X).sub.n (SEQ ID NO:
161), where n is from 6 to about 100; wherein X is any amino
acid.
27. The method of producing a biologically active fusion protein
molecule according to claim 26, wherein X is chosen from Lys, Ser,
Ala, Thr, Gly and Val.
28. The method of producing a biologically active fusion protein
according to claim 27, wherein X is chosen from Ser, Ala, Thr, and
Val.
29. The method of producing a biologically active fusion protein
according to claim 20, wherein the biologically active protein is
growth hormone and the glycoprotein comprises (Ser-Hyp).sub.10 (SEQ
ID NO: 4).
30. The method of producing a biologically active fusion protein
according to claim 29, wherein the (Ser-Hyp).sub.10 (SEQ ID NO: 4)
is covalently attached to the C-terminus of the growth hormone.
31. The method of producing a biologically active fusion protein
according to any of claims 29 or 30, wherein the growth hormone is
human growth hormone.
32. A method of increasing the yield in plant production of a
protein, comprising: preparing a nucleic acid construct comprising:
a) at least one signal peptide nucleic acid coding sequence, b) at
least one glycosylation site nucleic acid coding sequence, and c)
at least one protein nucleic acid coding sequence; and expressing
the nucleic acid construct as a glycoprotein.
33. The method according to claim 32, wherein the at least one
glycosylation site is chosen from i) X-Pro.sub.n (SEQ ID NO: 5) or
Pro.sub.n-X (SEQ ID NO: 96), where n is from 2 to about 1000, and
ii) (X-Pro).sub.n (SEQ ID NO: 11) or (Pro-X).sub.n (SEQ ID NO: 94),
where n is from 1 to about 1000; wherein X is any amino acid.
34. The method according to claim 33, wherein X is chosen from Gly,
Lys, Ser, Thr, Ala, and Val.
35. The method according to claim 34, wherein X is chosen from Ser,
Ala, Thr, and Val.
36. The method according to claim 32, wherein the nucleic acid
construct excludes a nucleic acid sequence coding for green
fluorescent protein.
37. A protein produced according to the method according to claim
36.
38. A method of preventing allergic immune response in a mammal
comprising performing at least one administration to the animal of
a plant-derived fusion glycoprotein comprising: a) at least one
glycomodule, covalently linked to b) a biologically active protein,
wherein the at least one glycomodule is chosen from X-Hyp.sub.n
(SEQ ID NO: 2) and X-Pro-Hyp.sub.n (SEQ ID NO: 1), where n is from
2 to about 1000, and wherein X is any amino acid.
39. The method of preventing allergic immune response according to
claim 38, wherein X is chosen from Lys, Ser, Ala, Thr, Gly and Val.
Description
[0001] This application claims priority to U.S. Provisional
Application Nos. 60/536,486, filed Jan. 14, 2004, and 60/582,027,
filed Jun. 22, 2004, and 60/602,562, filed Aug. 18, 2004, the
entire disclosure of each of which is incorporated by reference
herein.
[0002] The instant application contains a "lengthy" Sequence
Listing which has been submitted via CD-R in lieu of a printed
paper copy, and is hereby incorporated by reference in its
entirety. Said CD-R, recorded on May 16, 2005, are labeled CRF,
"Copy 1" and "Copy 2", respectively, and each contains only one
identical 976 KB file (27211413.APP).
DESCRIPTION OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to novel methods of producing
fusion peptides, polypeptides, and proteins in plants, the nucleic
acid constructs used in these methods, and the products produced
according to these methods. The methods generally involve
expressing the peptide, polypeptide, or protein as fusion proteins,
which are glycosylated by the plant. In some embodiments, a
plant-based signal peptide is expressed as part of the fusion
protein. According to the present invention, novel glycoproteins
are presented.
[0005] 2. Background of the Invention
[0006] Support of young growing plant tissues depends largely on
the turgidity of cells restrained by an elastic cell wall comprised
of three interpenetrating networks, namely, cellulosic-xyloglucan,
pectin, and hydroxyproline-rich glycoproteins (HRGPs). When these
networks are loosened, turgor drives cell extension. Significantly,
HRGPs have no animal homologs, thus emphasizing a plant-specific
function.
[0007] Quantitatively, most of the cell surface HRGPs (extensins)
form a covalently cross-linked cell wall network. Unlike extensins,
another set of HRGPs, arabinogalactan-proteins (AGPs) occur as
monomers that are hyperglycosylated by arabinogalactan
polysaccharides. AGPs are initially tethered to the plasma membrane
by a lipid anchor whose cleavage results in their movement from the
periplasm through the cell wall to the exterior. Although
implicated in diverse aspects of plant growth and development, the
precise functions of AGPs remain unclear.
SUMMARY OF THE INVENTION
[0008] The present invention provides novel methods of producing
glycoproteins in plants. The glycoproteins include a glycosylation
site element and a core protein element. In some embodiments, the
core protein element can be of mammalian (including human) origin,
and in some embodiments, the core protein element can be a
biologically active protein. In some cases, the protein can be an
FDA-approved recombinant protein that is used therapeutically, e.g.
recombinant human growth hormone ("hGH"). The glycosylation site is
an amino acid sequence that acts as a target for glycosylation by
the plant.
[0009] One feature of the present method is an increase in yield in
protein production. By including a glycosylation site(s) and a
signal peptide sequence in the expressed protein, recombinant
protein yield considerably increases in comparison to expression of
the same protein in plants without the glycosylation site and
signal peptide sequence.
[0010] Glycoproteins produced according to the method exhibit
additional advantages over their wild-type counterparts, including
increased solubility, increased resistance to proteolytic enzymes,
and increased stability. Another important feature includes
increased biological half-life as compared to wild-type
proteins.
[0011] Additional features and advantages of the invention will be
set forth in part in the description that follows, and in part will
be obvious from the description, or may be learned by practice of
the invention. The features and advantages of the invention will be
realized and attained by means of the elements and combinations
particularly pointed out in the appended claims.
[0012] The present invention provides nucleic acid constructs for
expression of at least one biologically active protein in plants
comprising: a) at least one nucleic acid sequence encoding a
glycosylation site utilized in plants and b) at least one nucleic
acid sequence encoding a biologically active protein.
[0013] The invention also provides plant-derived biologically
active fusion proteins comprising: a) at least one glycomodule
covalently linked to b) a biologically active protein. In some
embodiments, the at least one glycomodule comprises a glycosylation
site chosen from i) X-Pro-Hyp.sub.n (SEQ ID NO: 1), where n is from
2 to about 1000, ii) X-Hyp.sub.n (SEQ ID NO: 2), where n is from 2
to about 1000, and iii) (X-Hyp).sub.n (SEQ ID NO: 3), where n is
from 1 to about 1000; wherein X is chosen from Lys, Ser, Ala, Thr,
Gly, and Val, but is more preferably selected from Ser, Thr, Val,
and Ala. In some embodiments, the at least one glycomodule is
covalently linked at a location chosen from the N-terminus and/or
the C-terminus of the protein. In some embodiments, the at least
one glycomodule is within the interior of the biologically active
mammalian protein. While Lys, Ser, Thr, Val, Gly, and Ala, are
specifically identified above as corresponding to X, it is believed
that any amino acid can serve that purpose, and that the motif will
be glycosylated in plants.
[0014] The biologically active mammalian protein can be selected
from a group including growth hormone, growth hormone antagonists,
growth hormone releasing hormone, somatostatin, ghrelin, leptin,
prolactin, monocyte chemoattractant protein-1, interleukin-10,
pleiotropin, interleukin-7, interleukin-8, interferon omega,
interferon-Alpha 2a and 2b, interferon gamma, interleukin-1,
fibroblast growth factor 6, IFG-1, insulin-like growth factor I,
insulin, erythropoietin, GMCSF, and any humanized monoclonal
antibody or monoclonal antibody, wherein the glycomodule comprises
a glycosylation site chosen from i) X-Pro-Hyp.sub.n (SEQ ID NO: 1),
where n is from 2 to about 1000, ii) X-Hyp.sub.n (SEQ ID NO: 2),
where n is from 2 to about 1000, and iii) (X-Hyp).sub.n (SEQ ID NO:
3), where n is from 1 to about 1000; and wherein X is selected from
Lys, Ser, Ala, Thr, Gly, and Val, and is preferably Ser, Ala, Thr,
and Val. In some embodiments, the glycomodule comprises
(X-Hyp).sub.n (SEQ ID NO: 3), X is selected from Lys, Ser, Ala,
Thr, Gly and Val, more preferably Ser, Ala, Thr, and Val, and
n=1-1000. In some embodiments, the protein is human growth hormone,
and the glycomodule comprises (Ser-Hyp).sub.10 (SEQ ID NO: 4).
While Lys, Ser, Thr, Val, Gly, and Ala, are specifically identified
as corresponding to X, it is believed that any amino acid can serve
that purpose, and that the motif will be glycosylated in
plants.
[0015] In some embodiments, the plant-derived biologically active
mammalian fusion glycoproteins of the invention are covalently
linked to at least one carbohydrate molecule. In some embodiments,
the carbohydrate is an arabinogalactan moiety, and in some it is an
arabinosyl moiety.
[0016] The invention also provides methods of increasing the
aqueous solubility of a protein molecule, wherein one: prepares a
nucleic acid construct encoding a) at least one glycosylation site
and b) at least one peptide or protein; and expressing the nucleic
acid construct as a glycoprotein; wherein carbohydrate component of
the glycoprotein accounts for greater than or equal to about 10% of
the molecular weight of the glycoprotein. The carbohydrate
component of the glycoprotein can account for greater than or equal
to about 50%, about 75%, or about 90% of the molecular weight of
the glycoprotein.
[0017] The invention also provides methods of producing a
biologically active fusion glycoprotein, comprising: expressing in
a plant at least one nucleic acid construct comprising: a) at least
one nucleic acid sequence encoding a glycosylation site and b) at
least one nucleic acid sequence encoding a biologically active
protein, as a glycoprotein; wherein the molecular weight of the
glycoprotein is greater than or equal to about 10 kD and wherein
the carbohydrate component of the glycoprotein accounts for greater
than or equal to about 10% of the molecular weight of the
glycoprotein. In some embodiments, the molecular weight of the
glycoprotein is greater than or equal to about 35 kD, about 40 kD,
about 45 kD, about 50 kD, or about 55 kD. In some embodiments, the
pharmacokinetic half-life of the glycoprotein is greater than the
pharmacokinetic half-life of a corresponding wild-type protein. In
some embodiments, the at least one glycosylation site is chosen
from i) X-Pro.sub.n (SEQ ID NO: 5), where n is from 2 to about
1000, and ii) (X-Pro).sub.n (SEQ ID NO: 6), where n is from 2 to
about 1000; wherein X is any amino acid or is selected from Lys,
Ser, Ala, Thr, Gly and Val, or more preferably from Ser, Ala, Thr,
and Val. Of course, n can range from 4 to 200 or from 6 to 100 or
from 8 to 50 or from 10 to 25, or any number in between or any
combination thereof. In some embodiments, the biologically active
protein is human growth hormone and the glycoprotein comprises
(Ser-Hyp).sub.10 (SEQ ID NO: 4), and in some embodiments, the
(Ser-Hyp).sub.10 (SEQ ID NO: 4) is covalently attached to the
C-terminus of the human growth hormone protein.
[0018] The invention also provides injectable pharmaceutical
formulations comprising glycosylated human growth hormone, and
excluding additional excipients normally required for solvating or
increasing the solubility of proteins. In some embodiments, the
formulation excludes at least one excipient chosen from mannitol,
sorbitol, trehalose, glucose, glycine, leucine, trileucine,
histidine, and phospholipid. In some embodiments, the glycosylated
human growth hormone comprises a glycomodule chosen from i)
X-Pro-Hyp.sub.n (SEQ ID NO: 7), where n is from 2 to about 100, and
wherein X is any amino acid, or is chosen from Lys, Ser, Ala, Thr,
Gly and Val, or more preferably chosen from Ser, Ala, Thr, and Val,
ii) X-Hyp.sub.n (SEQ ID NO: 8), where n is from 2 to about 100, and
wherein X is any amino acid, or is chosen from Lys, Ser, Ala, Thr,
Gly and Val, or more preferably from Ser, Ala, Thr, and Val, and
iii) (X-Hyp).sub.n (SEQ ID NO: 9), where n is from 1 to about 100;
wherein X is any amino acid or is selected from Lys, Ser, Ala, Thr,
Gly and Val, or more preferably from Ser, Ala, Thr, and Val. The
glycosylated growth hormone can comprise X-Hyp.sub.n (SEQ ID NO:
10), where n is from 2 to about 20; wherein X is selected from Lys,
Ser, Ala, Thr, Gly and Val, or more preferably from Ser, Ala, Thr,
and Val.
[0019] The invention also provides lyophilized powder formulations
of glycosylated human growth hormone exhibiting a solubility of
greater than or equal to about 10 mg/ml, wherein the formulation
excludes additional excipients required for peptide solubility. In
some embodiments, the excipient is chosen from mannitol, sorbitol,
trehalose, glucose, glycine, leucine, trileucine, histidine, and
phospholipid.
[0020] The invention still further provides methods of increasing
the yield in plant production of a protein, comprising: preparing a
nucleic acid construct comprising: a) at least one nucleic acid
sequence encoding a secretory signal peptide, b) at least one
nucleic acid sequence encoding a glycosylation site, and c) at
least one nucleic acid sequence encoding a protein; and expressing
the nucleic acid construct as a glycoprotein in plants or plant
cell cultures. In some embodiments, the at least one HRGP
glycosylation site is chosen from i) X-Pro.sub.n (SEQ ID NO: 5),
where n is from 2 to about 1000, and ii) (X-Pro).sub.n (SEQ ID NO:
11), where n is from 1 to about 1000; wherein X is any amino acid,
or is chosen from Lys, Ser, Ala, Thr, Gly and Val, or more
preferably from Ser, Ala, Thr, and Val. The nucleic acid construct
can also include or exclude a nucleic acid sequence encoding green
fluorescent protein. The invention also provides proteins produced
according to these methods.
[0021] The invention also provides growth hormone molecules
covalently attached to an amino acid sequence comprising a
glycomodule, wherein the glycomodule is chosen from i)
X-Pro-Hyp.sub.n (SEQ ID NO: 7), where n is from 2 to about 100, ii)
X-Hyp.sub.n (SEQ ID NO: 8), where n is from 2 to about 100, and ii)
(X-Hyp).sub.n (SEQ ID NO: 9), where n is from 1 to about 100;
wherein X is chosen from Lys, Ser, Ala, Thr, Gly and Val, or more
preferably from Ser, Ala, Thr, and Val.
[0022] The invention also provides growth hormone antagonist
molecules covalently attached to an amino acid sequence comprising
a glycomodule, wherein the glycomodule is chosen from i)
X-Pro-Hyp.sub.n (SEQ ID NO: 7), where n is from 2 to about 100, ii)
X-Hyp.sub.n (SEQ ID NO: 8), where n is from 2 to about 100, and ii)
(X-Hyp).sub.n (SEQ ID NO: 9), where n is from 1 to about 100;
wherein X is chosen from Lys, Ser, Ala, Thr, Gly and Val, or more
preferably from Ser, Ala, Thr, and Val.
[0023] Also provided are methods of treating a patient suffering
from growth hormone deficiency or insufficiency comprising
administering a therapeutically effective amount of glycosylated
human growth hormone.
[0024] Also provided are methods of treating a patient suffering
from excess human growth hormone or growth hormone action
comprising administering a therapeutically effective amount of
glycosylated human growth hormone antagonist.
[0025] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the invention, as
claimed.
[0026] The accompanying drawings, which are incorporated in and
constitute a part of this specification, may illustrate embodiments
of the invention, and together with the description, serve to
explain the principles of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 shows oligonucleotide sets used to build (a)
[Gum].sub.n (n=8, 20)m (SEQ ID NOS 12 & 13), and (b) [HP].sub.m
(m=2, 4, 8) synthetic genes by mutual priming and extension (SEQ ID
NOS 14 & 15). The overlap is underlined. The restriction sites
are in bold italic.
[0028] FIG. 2 shows the DNA sequence of (a) [Gum].sub.3 (SEQ ID NOS
16 & 18; encoding SEQ ID NOS 17 & 19 respectively), (b)
[Gum].sub.8 and [Gum].sub.20 synthetic gene constructed in pUC18
plasmid between signal sequence (underlined) and GFP gene (SEQ ID
NOS 20 & 22 encoding SEQ ID NOS 21 & 23, respectively when
n=4: SEQ ID NOS 88 & 22 encoding SEQ ID NOS 89 & 23,
respectively when n=10). The restriction sites are in bold
italic.
[0029] FIG. 3 shows the DNA sequence of [HP].sub.4 and [HP].sub.8
synthetic gene constructed in pUC18 plasmid between signal sequence
(underlined) and GFP gene (SEQ ID NOS 24 & 28 encoding SEQ ID
NOS 25 & 29, respectively when n=4: SEQ ID NOS 26 & 28
encoding SEQ ID NOS 27 & 29, respectively when n=8). The
restriction sites are in bold italic.
[0030] FIG. 4 shows the DNA sequence of [Gum].sub.8[HP].sub.2 and
[Gum].sub.8[HP].sub.4 synthetic gene constructed in pUC18 plasmid
between signal sequence (underlined) and GFP gene (SEQ ID NOS 30
& 34 encoding SEQ ID NOS 31 & 35, respectively when n=2;
SEQ ID NOS 32 & 34 encoding SEQ ID NOS 33 & 35,
respectively when n=4). The restriction sites are in bold
italic.
[0031] FIG. 5 shows a schematic representation of the construction
of the hGH-(SP).sub.10-EGFP ((SP).sub.10 disclosed as SEQ ID NO:
51) gene cassette (Drimers disclosed as SEQ ID NOS 36 & 37;
vector disclosed as SEQ ID NO: 38).
[0032] FIG. 6 shows a schematic representation of the construction
of the hGH-(SP).sub.10 ((SP).sub.10 disclosed as SEQ ID NO: 51)
gene cassette (nucleotide sequence disclosed as SEQ ID NO: 39).
[0033] FIG. 7 shows a schematic representation of the construction
of the INF-(SP).sub.10 ((SP).sub.10 disclosed as SEQ ID NO: 51)
gene cassette (Drimers disclosed as SEQ ID NOS 40 & 41).
[0034] FIG. 8 shows a schematic representation of the construction
of the HSA-(SP).sub.10 ((SP).sub.10 disclosed as SEQ ID NO: 51)
gene cassette (primers disclosed as SEQ ID NOS 42 & 43).
[0035] FIG. 9 shows a schematic representation of the construction
of the DomainI-(SP).sub.10 ((SP).sub.10 disclosed as SEQ ID NO: 51)
gene cassette (primers disclosed as SEQ ID NOS 40 & 44).
[0036] FIG. 10A shows the gene construct for the expression of
human growth hormone (hGH) (SEQ ID NO: 45 encoding SEQ ID NO: 46)
with a (Ser-Hyp).sub.10 motif (SEQ ID NO: 4) attached. FIG. 10B
shows how the (SP).sub.10 ((SP).sub.10 disclosed as SEQ ID NO:
51)-gene was constructed by primer extension (SEQ ID NOS 47-49, 50
encoding 51, 47, 52-53, and 54 encoding 55, respectively in order
of appearance).
[0037] FIG. 11 shows the gene construct for the expression of human
growth hormone (hGH) connected to green fluorescent protein, with a
(Ser-Hyp).sub.10 motif (SEQ ID NO: 4) connecting the two (SEQ ID
NOS 56 & 58 encoding SEQ ID NOS: 57 & 59, respectively),
((SP).sub.10 disclosed as SEQ ID NO: 51).
[0038] FIG. 12 (A and B) shows the gene construct for the
expression of human serum albumin (HSA) with a (Ser-Hyp).sub.10
motif (SEQ ID NO: 4) attached (SEQ ID NO: 60 encoding SEQ ID NO:
61), ((SP).sub.10 disclosed as SEQ ID NO: 51).
[0039] FIG. 13 shows the gene construct for the expression of human
serum albumin domain I with a (Ser-Hyp).sub.10 motif (SEQ ID NO: 4)
attached (SEQ ID NO: 62 encoding SEQ ID NO: 63), ((SP).sub.10
disclosed as SEQ ID NO: 51).
[0040] FIG. 14 shows the gene construct for the expression of
interferon-alpha 2a (INF2a) with a (Ser-Hyp).sub.10 motif (SEQ ID
NO: 4) attached (SEQ ID NO: 64 encoding SEQ ID NO: 65),
((SP).sub.10 disclosed as SEQ ID NO: 51).
[0041] FIG. 15 shows the detection of hGH equivalents secreted into
the medium of ten lines of tobacco cells transformed with either
hGH-(SO).sub.10 ((SO).sub.10disclosed as SEQ ID NO: 4) and hGH.
[0042] FIG. 16 shows the time course of cell growth and production
of hGH equivalents in the culture medium of tobacco cells
transformed with hGH-(SP).sub.10((SP).sub.10 disclosed as SEQ ID
NO: 51).
[0043] FIG. 17 shows Western blot detection of hGH-(SO).sub.10
((SO).sub.10 disclosed as SEQ ID NO: 4) (left panel) and
hGH-(SO).sub.10-EGFP ((SO).sub.10 disclosed as SEQ ID NO: 4) in
culture medium.
[0044] FIG. 18 shows chromatographic profiles for the isolation of
hGH-(SO).sub.10 ((SO)10 disclosed as SEQ ID NO: 4) and
hGH-(SO).sub.10-EGFP ((SO).sub.10 disclosed as SEQ ID NO: 4) by
reversed-phase HPLC.
[0045] FIG. 19 shows the gene sequence of SS.sup.tob-hGH-(SP).sub.1
construct (SEQ ID NO: 66 encoding SEQ ID NO: 67). The restriction
sites are in bold italic.
[0046] FIG. 20 shows the gene sequence of SS.sup.tob-hGH-(SP).sub.2
((SP).sub.2 disclosed as SEQ ID NO: 90) construct (SEQ ID NO: 68
encoding SEQ ID NO: 69). The restriction sites are in bold
italic.
[0047] FIG. 21 shows the gene sequence of SS.sup.tob-hGH-(SP).sub.5
((SP).sub.5 disclosed as SEQ ID NO: 92) construct (SEQ ID NO: 70
encoding SEQ ID NO: 71). The restriction sites are in bold
italic.
[0048] FIG. 22 shows the gene sequence of
SS.sup.tob-hGH-(SP).sub.20 ((SP).sub.20 disclosed as SEQ ID NO: 93)
construct (SEQ ID NO: 72 encoding SEQ ID NO: 73). The restriction
sites are in bold italic.
[0049] FIG. 23 shows the gene sequence of
SS.sup.tob-(SP).sub.10-hGH-(SP).sub.10 ((SP).sub.10 disclosed as
SEQ ID NO: 51) construct (SEQ ID NO: 74 encoding SEQ ID NO: 75).
The restriction sites are in bold italic.
[0050] FIG. 24 shows the gene sequence of
SS.sup.tob-hGHA-(SP).sub.10 (SP).sub.10 disclosed as SEQ ID NO: 51)
construct (SEQ ID NO: 76 encoding SEQ ID NO: 77). The restriction
sites are in bold italic.
[0051] FIG. 25 shows the gene sequence of SS.sup.tob-INF-(SP).sub.5
((SP).sub.5 disclosed as SEQ ID NO: 92) construct (SEQ ID NO: 78
encoding SEQ ID NO: 79). The restriction sites are in bold
italic.
[0052] FIG. 26 shows the gene sequence of
SS.sup.tob-(SP).sub.5-INF-(SP).sub.5 ((SP).sub.5 disclosed as SEQ
ID NO: 92) construct (SEQ ID NO: 80 encoding SEQ ID NO: 81). The
restriction sites are in bold italic.
[0053] FIG. 27 shows the gene sequence of SS.sup.tob-(SP).sub.5-INF
((SP).sub.5 disclosed as SEQ ID NO: 92) construct (SEQ ID NO: 82
encoding SEQ ID NO: 83). The restriction sites are in bold
italic.
[0054] FIG. 28 shows the gene sequence of
SS.sup.tob-INF-(SP).sub.20 ((SP).sub.20 disclosed as SEQ ID NO: 93)
construct (SEQ ID NO: 84 encoding SEQ ID NO: 85). The restriction
sites are in bold italic.
[0055] FIG. 29 shows the gene sequence of
SS.sup.tob-(SP).sub.10-INF-(SP).sub.10 ((SP).sub.10 disclosed as
SEQ ID NO: 51) construct (SEQ ID NO: 86 encoding SEQ ID NO: 87).
The restriction sites are in bold italic.
[0056] FIG. 30 shows a binding curve for hGH-(SP).sub.10-EGFP
((SP).sub.10 disclosed as SEQ ID NO:51).
[0057] FIG. 31 shows a binding curve for commercially available
hGH.
[0058] FIG. 32 shows a binding curve for hGH-(SP).sub.10
((SP).sub.10 disclosed as SEQ ID NO: 51).
[0059] FIG. 33 shows serum concentration of commercially available
hGH and hGH-(SP).sub.10 ((SP).sub.10 disclosed as SEQ ID NO: 51)
following a single administration of each to mice.
[0060] FIG. 34 shows serum IGF-1 concentration following a single
administration to mice of commercially available hGH and
hGH-(SP).sub.10 ((SP).sub.10 disclosed as SEQ ID NO: 51).
[0061] FIG. 35 shows blood concentration of hGH equivalents
following a single administration of commercially available hGH and
hGH-(SP).sub.10 ((SP).sub.10 disclosed as SEQ ID NO: 51) to
mice.
[0062] FIG. 36 shows serum concentration of commercially available
hGH and hGH-(SP).sub.10 ((SP).sub.10 disclosed as SEQ ID NO: 51)
(and PBS controls) following two administrations per day for five
days to mice.
[0063] FIG. 37 shows serum IGF-1 concentration following
administration of commercially available hGH and hGH-(SP).sub.10
((SP).sub.10 disclosed as SEQ ID NO: 51) (and PBS control)
following two administrations per day for five days to mice.
[0064] FIG. 38 shows growth hormone levels following once daily
administration of a lower concentration (1 .mu.g/gm) of
commercially available hGH and hGH-(SP).sub.10 ((SP).sub.10
disclosed as SEQ ID NO: 51) (and PBS control) for five days.
[0065] FIG. 39 shows serum IGF-1 concentration following
administration of a lower concentration (1 .mu.g/gm) of
commercially available hGH and hGH-(SP).sub.10 ((SP).sub.10
disclosed as SEQ ID NO: 51) (and PBS control) following one
administration per day for five days.
[0066] FIG. 40 shows the increase in body mass over the course of a
two-week treatment with commercially available hGH and
hGH-(SP).sub.10 ((SP).sub.10 disclosed as SEQ ID NO: 51).
DESCRIPTION OF THE EMBODIMENTS
[0067] The connection between structure and function is one of the
profound lessons in biology. At least two aspects of
hydroxyproline-rich glycoprotein (HRGP) structural biology appear
to be of functional significance: glycosylation and covalent
cross-links. Because HRGPs tend to be extended repetitive modular
glycoproteins, research leading to this point has focused on
dissecting HRGP functional properties, module by module. Synthetic
genes were designed as analogs of each putative module. This
approach allowed for many discoveries: unraveling glycosylation
codes, structural elucidation of glyco-substituents, identification
of crosslink motifs, and the design of novel glycoproteins,
including improved biomedical products.
[0068] The present work extended, and expanded upon, the
Hyp-contiguity hypothesis originally proposed by Kieliszewski et
al. It has now been discovered that this O-Hyp glycosylation code
predicts the glycosylation sites and substituents of HRGPs. The
present disclosure applies this discovery in a number of ways.
[0069] Some embodiments are directed to methods for improving the
yield of protein production in plants. Some embodiments involve
modified proteins produced in accordance with the present
disclosure, which can exhibit improved properties overall, and
specific advantages in vivo, including extended biological
half-life and improved bioavailability.
[0070] The present invention will now be described by reference to
more detailed embodiments, with occasional reference to the
accompanying drawings. This invention may, however, be embodied in
different forms and should not be construed as limited to the
embodiments set forth herein. Rather, these embodiments are
provided so that this disclosure will be thorough and complete, and
will fully convey the scope of the invention to those skilled in
the art.
[0071] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. The
terminology used in the description of the invention herein is for
describing particular embodiments only and is not intended to be
limiting of the invention. As used in the description of the
invention and the appended claims, the singular forms "a," "an,"
and "the" are intended to include the plural forms as well, unless
the context clearly indicates otherwise. All publications, patent
applications, patents, and other references mentioned herein are
expressly incorporated by reference in their entirety.
[0072] Unless otherwise indicated, all numbers expressing
quantities of ingredients, reaction conditions, and so forth used
in the specification and claims are to be understood as being
modified in all instances by the term "about." Accordingly, unless
indicated to the contrary, the numerical parameters set forth in
the following specification and attached claims are approximations
that may vary depending upon the desired properties sought to be
obtained by the present invention. At the very least, and not as an
attempt to limit the application of the doctrine of equivalents to
the scope of the claims, each numerical parameter should be
construed in light of the number of significant digits and ordinary
rounding approaches.
[0073] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the invention are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. Any numerical value, however,
inherently contains certain errors necessarily resulting from the
standard deviation found in their respective testing measurements.
Every numerical range given throughout this specification will
include every narrower numerical range that falls within such
broader numerical range, as if such narrower numerical ranges were
all expressly written herein.
[0074] Throughout this disclosure, reference will be made to
compounds according to the invention. Reference to such compounds,
in the specification and claims, includes esters and salts of such
compounds. Thus, even if not explicitly recited, such esters and
salts are contemplated, and encompassed, by reference to the
compounds themselves.
[0075] Additionally, as used herein, "peptide," "polypeptide," and
"protein," can and will be used interchangeably.
"Peptide/polypeptide/protein" will occasionally be used to refer to
any of the three, but recitations of any of the three contemplate
the other two. That is, there is no intended limit on the size of
the amino acid polymer (peptide, polypeptide, or protein), that can
be expressed using the present invention. Additionally, the
recitation of "protein" is intended to encompass enzymes, hormone,
receptors, channels, intracellular signaling molecules, and
proteins with other functions. Multimeric proteins can also be made
in accordance with the present invention.
[0076] While the naturally occurring amino acids are discussed
throughout this disclosure, non-naturally occurring amino acids, or
modified amino acids, are also contemplated and within the scope of
the invention. In fact, as used herein, "amino acid" refers to
natural amino acids, non-naturally occurring amino acids, and amino
acid analogs, all in their D and L stereoisomers. Natural amino
acids include alanine (A), arginine (R), asparagine (N), aspartic
acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine
(G), histidine (H), isoleucine (I), leucine (L), lysine (K),
methionine (M), phenylalanine (F), proline (P), serine (S),
threonine (T), tryptophan (W), tyrosine (Y), valine (V),
hydroxyproline (O and/or Hyp), isodityrosine (IDT), and
di-isodityrosine (di-IDT). Hydroxyproline, isodityrosine, and
di-isodityrosine are formed post-translationally. Use of natural
amino acids, in particular the 20 genetically encoded amino acids,
is preferred.
[0077] Non-naturally occurring amino acids include, but are not
limited to azetidinecarboxylic acid, 2-aminoadipic acid,
3-aminoadipic acid, beta-alanine, aminopropionic acid,
2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid,
2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric
acid, 2-aminopimelic acid, 2,4 diaminoisobutyric acid, desmosine,
2,2'-diaminopimelic acid, 2,3-diaminopropionic acid,
N-ethylglycine, N-ethylasparagine, hydroxylysine,
allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline,
isodesmosine, allo-isoleucine, trileucine, N-methylglycine,
N-methylisoleucine, N-methylvaline, norvaline, norleucine,
ornithine, and pipecolic acid.
[0078] Additionally, while specific reference is made to discrete
peptides, polypeptides, and/or proteins, mutants or variants of
those peptides or proteins are specifically contemplated as well. A
"variant" as used herein, refers to a protein (or peptide or
polypeptide) whose amino acid sequence is similar to a reference
peptide/polypeptide/protein, but does not have 100% identity to the
respective peptide/polypeptide/protein sequence. A variant
peptide/polypeptide/protein has an altered sequence in which one or
more of the amino acids in the reference sequence is deleted or
substituted, or one or more amino acids are inserted into the
sequence of the reference amino acid sequence. A variant can have
any combination of deletions, substitutions, or insertions. As a
result of the alterations, a variant peptide/polypeptide/protein
can have an amino acid sequence which is at least about 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,
70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or higher
percent, identical to the reference sequence. Lower percent
identity is also acceptable, and can range to as low as 20%.
[0079] In order to determine whether a mutant polypeptide is
substantially identical with any vertebrate polypeptide, the mutant
polypeptide sequence can be aligned with the sequence of a first
reference vertebrate polypeptide. One method of alignment is by
BlastP, using the default setting for scoring matrix and gap
penalties. In one embodiment, the first reference vertebrate
polypeptide is the one for which such an alignment results in the
lowest E value, that is, the lowest probability that an alignment
with an alignment score as good or better would occur through
chance alone. Alternatively, it is the one for which such alignment
results in the highest percentage identity.
[0080] Substitutions can be conservative and/or nonconservative. In
conservative amino acid substitutions, the substituted amino acid
has similar structural and/or chemical properties with the
corresponding amino acid in the reference sequence. By way of
example, conservative substitutions (replacements) are defined as
exchanges within the groups set forth below:
[0081] I small aliphatic, nonpolar or slightly polar residues--Ala,
Ser, Thr (Pro, Gly)
[0082] II negatively charged residues and their amides--Asn Asp Glu
Gln
[0083] III positively charged residues--His Arg Lys
[0084] IV large aliphatic nonpolar residues--Met Leu Ile Val
(Cys)
[0085] V large aromatic residues--Phe Tyr Trp
[0086] Three residues are parenthesized because of their special
roles in protein architecture. Gly is the only residue without a
side chain and therefore imparts flexibility to the chain. Pro has
an unusual geometry which tightly constrains the chain. Cys can
participate in disulfide bonds, which hold proteins into a
particular folding; the four cysteines of bGH are highly conserved.
With conservative substitutions, even variants with low levels of
identity can exhibit very similar activities to the unmodified
peptide/polypeptide/protein.
[0087] It should be noted that "variants" in accordance with the
invention include peptides/polypeptides/proteins that have greater
than or fewer than the number of amino acids in the wild-type
version. With respect to growth hormone, for example, the wild-type
has a molecular weight of about 22 kDa, yet variants of 20 and 17
kDa also exist. These sorts of variants, which may or may not be
naturally occurring, are expressly contemplated. Growth hormone
antagonist, which has an approximate molecular weight of 22 kDa,
also can exist in 20 and 17 kDa forms, and these forms of growth
hormone antagonist are also expressly contemplated.
[0088] "Biologically active" substance refers to a substance, such
as any peptide, polypeptide, or protein, which causes an observable
change in the structure, function, or composition of a cell upon
uptake by the cell. In some embodiments, the substance is an animal
protein, in some embodiments a mammalian protein, and in some
embodiments human protein. Observable changes include, but are not
limited to, increased or decreased expression of one or more mRNAs,
increased or decreased expression of one or more proteins,
phosphorylation or dephosphorylation of a protein or other cell
component, inhibition or activation of an enzyme, inhibition or
activation of binding between members of a binding pair, an
increased or decreased rate of synthesis of a metabolite, increased
or decreased cell proliferation, and increase or decrease effect on
the outward phenotype of an organism and the like. For example,
administration of hGH to GH deficient children will ultimately
stimulate growth. Or administration of a human GH antagonist to
acromegalic individuals, will result in lower levels of IGF-1 and
clinical curing of the disorder. Fragments of biologically active
proteins, wherein the fragments retain biological activity, are
expressly contemplated.
[0089] It should also be noted that the present methods can be used
to produce fusion proteins in plants. The basic protein that is
modified in the fusion protein can be of any source, plant or
animal. Animal source proteins include mammalian and non-mammalian.
Of course, mammalian proteins include human proteins. Frequently
throughout this document, reference will be made to human forms of
proteins. It should be recognized that where reference is made to
human proteins, the same proteins are often also found in other
non-human mammals. These other non-human mammalian proteins are
expressly contemplated.
[0090] Glycosylation
[0091] The present invention generally involves expressing
glycoproteins in plants using a novel approach. The approach
generally involves genetically engineering nucleic acid sequences
coding for glycosylation sites into genes for non-HRGP
proteins/peptides/polypeptides using the codes that drive these
post-translational modifications in plants. The sequences for
glycosylation can be constructed as separate units attached to one
or the other end of the gene, to form fusion proteins. These genes
can also be engineered to code for plant signal peptide sequences
to target the gene products for secretion.
[0092] Glycosylation types include, but are not limited to,
arabinosylation and arabinogalactan-polysaccharide addition.
Arabinosylation generally involves the addition of short (e.g.,
generally about 1-5) arabinooligosaccharide (generally
L-arabinofuranosyl residues) chains.
Arabinogalactan-polysaccharides, on the other hand, are larger and
generally are formed from a core .beta.-1,3-D-galactan backbone
periodically decorated with 1,6-additions of small side chains of
D-galactose and L-arabinose and occasionally with other sugars such
as L-rhamnose and sugar acids such as D-glucuronic acid and its
4-o-methyl derivative. Arabinogalactan-polysaccharides can also
take the form of a core .beta.-1,6-D-galactan backbone periodically
decorated with 1,6-additions of small side chains of
arabinofuranosyl. Note that these adducts are added by a plant's
natural enzymatic systems to proteins/peptides/polypeptides that
include the target sites for glycosylation, i.e., the glycosylation
sites. There may be variation in the actual molecular structure of
the glycosylation that occurs. Basically, any sugar that can be
added by a plant cell, including but not limited to, The
oligosaccharide chains may include any sugar which can be provided
by the host cell, including, without limitation, Gal, GalNAc, Glc,
GlcNAc, and Fuc, can make up the oligosaccharide chain. It should
be noted that glycosylation can be achieved in vitro.
[0093] As used herein, the term "glycomodule" is meant to refer to
an amino acid sequence comprising at least one proline residue that
is hydroxylated and glycosylated. As used herein, the term
"glycosylation site" is meant to refer an amino acid sequence
comprising at least one proline residue that acts as a target site
of hydroxylation and subsequent glycosylation. Glycosylation
generally occurs following hydroxylation of the one or more of the
proline residues in the site. Thus, within glycosylation sites,
proline residues may be hydroxylated to form hydroxyprolines.
[0094] The two major types of glycosylation are achieved in
accordance with the present invention by the introduction of one or
more glycosylation sites into a peptide/polypeptide/protein.
Glycosylation is generally of two types: 1) arabinogalactan
glycomodules comprise clustered non-contiguous hydroxyproline (Hyp)
residues in which the Hyp residues are O-glycosylated with
arabinogalactan adducts (the structure of which is described
above); and 2) arabinosylation glycomodules comprise contiguous Hyp
residues in which some or all of the Hyp residues are
arabinosylated (O-glycosylated) with chains of arabinose about 1-5
residues long. See the following U.S. patents and published
applications for a more detailed discussion of target sites for
glycosylation, and the Hyp-contiguity theory: U.S. Pat. Nos.
6,548,642, 6,570,062, 6,639,050 and Application Nos. 2004/0009555
and 2004/0009557. The entire disclosure of each of these patents
and patent applications is incorporated herein by reference.
[0095] In particular, glycosylation sites can be introduced as
follows. For arabinogalactan glycomodules (where the glycosylation
sites are clustered non-contiguous Hyp residues), the genes will
encode variations of (Pro-X).sub.n (SEQ ID NO: 94) and
(X-Pro).sub.n (SEQ ID NO: 11), where n=1-1000, and
(X-Pro-X.sub.1-9), where X can be Lys, Ala, Ser, Thr, Gly or Val,
or more preferably Ser, Ala, Thr, or Val. In other embodiments, n
is greater than 2, 3, 5, 5, 6, 7, 8, 9, 10, 50, 100, or 500, or
less than 999, 998, 997, 996, 995, 994, 993, 992, 991, 990, 900,
800, 700, 600, or 500; n can range from any number to any number
between 1 and 1000. In some embodiments, n ranges from 1-100, or
from 1-75, or from 1-50, or from 2-25, or from 2-10, or from 2-6.
Many of the Pro residues in these sequences will be hydroxylated to
hydroxyproline (Hyp) and subsequently O-glycosylated with
arabinogalactan oligosaccharides or polysaccharides. It should be
noted that (X-Pro).sub.n or (Pro-X).sub.n repeats can be
interspersed with each other and with other amino acids, and that
such interspersed repeating groups are expressly contemplated.
While Lys, Ser, Thr, Val, Gly, and Ala, are specifically identified
as corresponding to X, it is believed that any amino acid can serve
that purpose, and that the motif will be glycosylated in plants. As
noted, X is more preferably selected from Ser, Ala, Thr, or
Val.
[0096] For arabinosylation glycomodules (where glycosylation sites
are contiguous Hyp residues), genes tailored for expression will
encode contiguous Pro residues (Pro).sub.n (SEQ ID NO: 95), where
n=2-1000. In other embodiments, n is greater than 3, 4, 5, 6, 7, 8,
9, 10, 50, 100, or 500, or less than 999, 998, 997, 996, 995, 994,
993, 992, 991, 990, 900, 800, 700, 600, or 500; n can range from
any number to any number between 2 and 1000. In some embodiments, n
ranges from 1-100, or from 1-75, or from 1-50, or from 2-25, or
from 2-10, or from 2-6. Most of the Pro residues in these sequences
will be hydroxylated to hydroxyproline and subsequently
O-glycosylated with arabinosides ranging in size from one to five
arabinose residues. It should be noted that (Pro).sub.n repeats can
be interspersed with other amino acids, and that such interspersed
repeating groups are expressly contemplated.
[0097] So as to avoid confusion, it is noted that reference to
nucleic acid constructs and genes reflects the fact that the
nucleotides will encode proline, not hydroxyproline. Thus, nucleic
acid constructs, genes, etc., will refer to Pro or P (in single
letter form). Reference to genes encoding repeating units might
look like: (SP).sub.10 (SEQ ID NO: 51), which refers to a nucleic
acid construct that codes for ten repeating units of Ser-Pro. To
differentiate peptides/polypeptides/proteins that have been
produced in plants, reference is made to hydroxyproline, or hyp, or
O (in single letter form). Thus, once the (SP).sub.10 (SEQ ID NO:
51) has been expressed in plants, it may be referred to as
(SO).sub.10 (SEQ ID NO: 4).
[0098] Any combination of glycomodules within a single glycoprotein
can also be made. That is, a glycoprotein can include
arabinosylation glycomodules and arabinogalactan glycomodules.
Thus, a single gene construct can include nucleic acid sequences
coding for one or more arabinosylation sites and/or one or more
arabinogalactan polysaccharide sites, which are hydroxylated and
glycosylated upon expression in a plant host.
[0099] The sites for glycosylation can be placed at either or both
termini of the peptide/polypeptide/protein, and/or in the interior
of the molecule if desired. For example, in a smaller molecule, the
N- or C-terminus might be modified by the addition of glycosylation
sites; in a larger molecule, an interior substitution might be more
desirable. Of course, smaller molecules can be modified on their
interiors and larger molecules modified on either or both
termini--the choice is left to the practitioner.
[0100] In the case of membrane-spanning or -anchored enzymes, a
construct can be prepared that modifies the N-terminus by replacing
the membrane-spanning or -anchoring domain (avoiding the intrinsic
tendency of glycosyltransferases, for example, to associate with
ER/Golgi membranes) with an N-terminal secretion signal sequence,
followed by the glycosylation sequence, such as, for example, a
short (Ser-Hyp).sub.n or (Ala-Hyp).sub.n repeat. (For example, some
enzymes, such as glycosyltransferases, can be modified by replacing
the N-terminal membrane-spanning sequence that often anchors the
enzymes to membranes, with a signal sequence and glycomodule,
allowing the glycosyltransferase to be glycosylated and secreted
rather than retained in the ER or Golgi membranes.) The transgenes
are designed to encode a signal sequence for secretion through the
endomembrane system. The strategy of using a secretion signal
sequence to target the entire molecule for secretion can be used in
any construct, and is not limited to the secretion of normally
membrane-tethered, -spanning, or -anchored proteins.
[0101] The addition of a glycosylation site and the subsequent
glycosylation, be it by arabinosylation and/or arabinogalactan
polysaccharide addition, can have a number of different effects. In
some instances, the glycosylation of the
peptide/polypeptide/protein will result in an increased yield and
secretion of the expressed product as compared to a
non-glycosylated product that is otherwise identical. That is,
adding at least one site for arabinosylation or arabinogalactan
polysaccharide addition can result in an increased secreted product
yield as compared to product expressed without the addition(s). The
yield can be increased by about 10%, 25%, 50%, 100%, 200%, 300%,
400%, 500%, or 1000%, or more.
[0102] Glycosylation can provide additional means for isolation of
a peptides/polypeptides/protein of interest. For example, by
introducing a glycosylation site into a protein's gene and
subsequently expressing the gene in plants, the product can be
isolated and/or separated by affinity chromatography or by use of a
lectin-based chromatography.
[0103] The addition of arabinooligosaccharides or arabinogalactan
polysaccharides can have effects on the physicochemical activity of
the peptides/polypeptides/proteins. The additions can increase the
molecular weight, change the isoelectric point, and change the
ability of the peptide/polypeptide/protein to modify the effects of
other media. For example, glycosylation can have the effect of
increasing a protein's ability to act as an emulsifier. Thus,
glycoproteins made in accordance with the present invention can be
used as emulsifiers. In some embodiments, glycoproteins of the
invention, which act as emulsifiers, are combined with emulsifiers
in pharmaceutical compositions, to improve the administration of
the glycoprotein or to improve the administration of another
biologically active substance.
[0104] Glycosylation can increase the molecular weight of a
peptide/polypeptide/protein. The glycosylation can account for 1%,
2%, 3%, 4%, 5%, 8%, 12%, 16%, 24%, 33%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, or even higher percent of the
total weight of the glycoprotein. Glycosylation can add 0.1, 0.2,
0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,
60, 70, 80, 90, or 100 kDa or more to a
peptide/polypeptide/protein. Generally, glycosylation can increase
the molecular weight by any percentage increment.
[0105] Glycosylation of a protein according to this invention can
render insoluble proteins soluble, and can increase the solubility
of already soluble proteins. Thus, in some embodiments,
peptides/polypeptides/proteins modified according to these methods
can be isolated or dissolved in water, where a wild-type protein
may require buffer solutions. In some embodiments of the invention,
the glycoproteins are more stable, in comparison to wild-type
proteins, which aggregate or form multimers if not treated
properly.
[0106] In particular, with regard to solubility of growth hormone
and growth hormone antagonists, solubility is increased over that
of the non-glycosylated versions. Increased solubility is observed
in the absence of other elements required for solubility in
non-glycosylated forms, such as buffers or other additives.
Solubility can be greater than or equal to about 10, 15, 20, 25,
30, 40, 50, or more mg/ml.
[0107] Glycosylation of peptides/polypeptides/proteins according to
the invention can have the desired effect of increasing resistance
to enzymatic degradation. While it is not entirely clear why this
occurs, it appears that the bulky carbohydrate substituents added
in accordance with the invention block or prevent access to the
sites of enzymatic degradation. Thus, where a peptidase may have
specificity for a particular terminus or for a particular amino
acid sequence, the glycosylation blocks, impedes, or hinders
peptidase access to those sites. This protective effect has a
number of real world utilities, including increasing shelf life,
reducing breakdown by microbes, and of increasing the likelihood of
gastrointestinal passage and thus, in some cases, allowing for oral
administration.
[0108] In some embodiments, modified peptides/polypeptides/proteins
of the invention that have been lyophilized can be dissolved with
ease, whereas the wild-type peptides/polypeptides/proteins are more
difficult to dissolve. This aspect of the invention is important
in, and leads to utility in, for example, reconstituting
lyophilized modified peptides/polypeptides/proteins of the
invention prior to injection, which can be, for example, IM, SC,
IV, or IP. Where wild-type peptides/polypeptides/proteins may be
difficult to solubilize, requiring buffers, salts, or other
solubilizing elements, which can cause burning or irritation on
injection, some modified peptides/polypeptides/proteins of the
invention can avoid those undesirable additives. Thus, in one
embodiment, for example, a modified human growth hormone is made in
accordance with the present invention, prepared, and packaged in
the absence of mannitol; a lyophilized powder or solution for
injection excludes mannitol. In one embodiment, a modified human
growth hormone is made in accordance with the present invention,
prepared, and packaged in the absence of added glycine; a
lyophilized powder or solution for injection excludes added
glycine. In one embodiment, a modified human growth hormone is made
in accordance with the present invention, prepared, and packaged in
the absence of added leucine; a lyophilized powder or solution for
injection excludes added leucine. In one embodiment, a modified
human growth hormone is made in accordance with the present
invention, prepared, and packaged in the absence of added
phospholipids; a lyophilized powder or solution for injection
excludes added phospholipids. In one embodiment, a modified human
growth hormone is made in accordance with the present invention,
prepared, and packaged in the absence of added trehalose; a
lyophilized powder or solution for injection excludes added
trehalose. In one embodiment, a modified human growth hormone is
made in accordance with the present invention, prepared, and
packaged in the absence of added histidine; a lyophilized powder or
solution for injection excludes added histidine. Indeed, modified
growth hormone formulations of the invention, for example, can
exclude any excipients normally required in other growth hormone
formulations.
[0109] These impacts on physicochemical properties can be achieved
without influencing biological activity. In some cases, however,
glycosylation imparts additional advantages.
[0110] Because of the increased solubility and ease of dissolution,
some modified peptides/polypeptides/proteins of the invention can
be delivered by inhalation to the lung for a pharmacological
effect. For example, a wild-type protein may be difficult to
dissolve without additives. On inhalation of the wild-type protein
in lyophilized powder form, dissolution in the membrane of the lung
is very slow, which a) slows the rate of uptake, b) allows for
phagocytosis of the particulate matter, and c) allows cilia to
carry the particulate matter up and out of the lung. A modified
peptide/polypeptide/protein of the invention, however, can dissolve
much more quickly, thereby increasing the rate of uptake,
decreasing the opportunity for phagocytosis, and preventing
expulsion through ciliary action. The net effect is the creation of
a drug that can be delivered by inhalation, where such delivery is
not feasible for the wild-type drug.
[0111] In some embodiments of peptides/polypeptides/proteins having
biological activity, the arabinosylation and/or arabinogalactan
polysaccharide addition can alter the biological activity.
Alteration in biological activity can be, for example,
pharmacodynamic, i.e., modifying the agonist and/or antagonist
activity of the peptide/polypeptide/protein. For example, a
modified agonist can exhibit antagonist activity; thus, an
antagonist can be made from an agonist. In other examples,
modifications result in an increase or decrease in receptor
affinity.
[0112] Alteration in biological activity can be, for example,
pharmacokinetic, i.e., modifing the absorption, distribution,
localization in tissues, biotransformation, and/or excretion of the
peptide/polypeptide/protein. For example, a glycosylated
peptide/polypeptide/protein can exhibit an increased
bioavailability or half-life, relative to the non-glycosylated
peptide/polypeptide/protein. Bioavailability or half-life can be
increased by about 10%, 25%, 50%, 100%, 200%, 300%, 400%, 500%, or
1000%, or more.
[0113] Bioavailability can be generally measured by the area under
the curve (AUC). The area under the curve is a plot of plasma
concentration of drug (not logarithm of the concentration) against
time after drug administration. The area can generally be
determined by the "trapezoidal rule," wherein the data points are
connected by straight line segments, perpendiculars are erected
from the abscissa to each data point, and the sum of the areas of
the triangles and trapezoids so constructed is computed. Area under
the curve can be calculated using any means known in the art for
calculating this value. In accordance with the invention, AUC can
be increased by about 10%, 25%, 50%, 100%, 200%, 300%, 400%, 500%,
or 1000%, or more.
[0114] An increase in bioavailability can also be reflected in an
increased peak plasma concentration (C.sub.max). In accordance with
the invention, peak plasma concentration can be increased by about
10%, 25%, 50%, 100%, 200%, 300%, 400%, 500%, or 1000%, or more.
[0115] Thus, biologically active proteins produced in accordance
with the present invention can have the advantage of exhibiting
extended half-life and/or bioavailability, and thus exhibiting an
increased or prolonged effect in the body. While it is not entirely
clear how or why this occurs, it may relate to the charge and
increased size imparted on the biological molecule by the
carbohydrate motifs of the invention.
[0116] Another effect of the glycosylation in accordance with this
invention is a lack of change in immunogenicity or antigenicity.
Thus, the immunogenicity or antigenicity of a
peptide/polypeptide/protein can be unchanged by producing it as a
glycoprotein in accordance with this invention. In some
embodiments, the immunogenicity or antigenicity is actually
decreased. In either case--no change or decrease--this is important
for vaccines or other parenterally introduced molecules that
exhibit a desirable biological effect but are hindered by their
immunogenicity/antigenicity. Specific examples include, but are not
limited to, the beta-amyloid peptide.
[0117] The reduced immunogenicity (or allergenicity) relative to a
base protein may result from antibodies' (in)ability to recognize
the core protein. However, it should also be noted that the
carbohydrate moieties can also be the epitope of an antibody, and
thus, can function as an immunogen or allergen. While it's unclear
what is necessary to cause antibodies to recognize those
carbohydrate moieties as foreign, it is believed that glycoproteins
manufactured in accordance with the present invention can serve as
sensitizing agents for allergy immunotherapy. That is,
glycoproteins made in accordance with the present invention can be
used for repeated injections with the desired long-term effect of
reducing an allergic response. In particular, arabinosylated
glycoproteins (including glycopeptides or even glycosylated amino
acids, such as a single hydroxyproline that has at least one
arabinose attached), which include the glycomodules X-Hyp.sub.n,
are believed to be useful in allergy immunotherapy.
[0118] Peptides/Polypeptides/Proteins
[0119] The peptides/polypeptides/proteins that can be modified in
accordance with the present invention can be from various
organisms, including but not limited to, humans and other mammals
and/or vertebrates, invertebrates, plants, sponges, bacteria,
fungi, algae, archebacteria, etc. Additionally, synthetic proteins
and peptides are expressly contemplated, as are derivatives and
analogs of any protein such as antagonists, peptide agonists or
antagonists, or antibodies.
[0120] The peptides/polypeptides/proteins can be large or small,
monomeric or multimeric, and have any type of utility. In some
embodiments, the peptides/polypeptides/proteins are small, such as
less than about 25 kDa. Through glycosylation according to this
invention, their molecular weight can be increased to 40 kDa or
higher.
[0121] In some embodiments, the peptides/polypeptides/proteins are
not post-translationally modified, except for disulfide bond
formation or N-linked glycosylation. In some embodiments, peptides
with many proline residues, which may be targets for hydroxylation
and subsequent Hyp-glycosylation, are avoided.
[0122] Peptides/polypeptides/proteins that can be expressed using
the present invention include, but are not limited to, those
molecules in the growth hormone superfamily, including but not
limited to, growth hormone, prolactin, placental lactogen, and
other interleukins. Other specific examples include, but are not
limited to, monocyte chemoattractant protein-1, interleukin-10,
pleiotropin, interleukin-7, interleukin-8, interferon omega,
interferon-Alpha 2a and 2b, interferon gamma, interleukin-1,
fibroblast growth factor 6, IGF-1, insulin-like growth factor I and
II, adrenocorticotropic hormone, beta-amyloid, amylin, atrial
natriuretic polypeptide (e.g., alpha), bombesin, bradykinin, brain
natriuretic peptide, calcitonin, calcitonin gene related peptide,
corticotropin releasing factor, dynorphin, endorphin, endothelin
(e.g., -1, -2, and -3), enkephalin, epidermal growth factor,
gastric inhibitory peptide, gastrin, gastrin releasing peptide,
growth hormone releasing hormone, HIV-1 envelope proteins,
katacalcin, luteinizing hormone-releasing hormone, neurokinins
(e.g., A and B), neuromedins (e.g., B and C), neuropeptide Y,
neurotensin, oxytocin, pancreatic polypeptide, pancreatic
polypeptide, pancreastin, pancreastatin, parathyroid hormone,
secretin, somatostatin, substance P, transforming growth factor
(e.g., alpha), vasoactive intestinal peptide, vasopressin,
vasotocin, glucagon and the glucagon-like peptides, erythropoietin,
granulocyte colony stimulating factor, PORF-1 and -2 (preoptic
regulatory factors), and PYY 3-36. Also included are any protein
growth factor, hormone, antibody, cytokine, oncoprotein (cancer
causing protein), lymphokine, or derivative thereof. Also included
are proteins involved in metabolic processes, including but not
limited to, insulin, ghrelin, leptin, adiponectin, resistin,
etc.
[0123] For example, the present invention can be used to express a
modified growth hormone. Growth hormone (GH) is secreted by the
pituitary gland. It is an approximately 22-kDa protein that
exhibits a variety of biological activities. Hyposecretion of
growth hormone results in dwarfism while hypersecretion results in
gigantism and/or acromegaly. A recombinant DNA construct can be
prepared that includes: the nucleic acids encoding hGH, nucleic
acids coding for a hydroxyproline glycosylation site, along with
nucleic acids coding for a plant signal sequence. The nucleic acids
coding for a hydroxyproline glycosylation site can code for
X-Pro.sub.n (SEQ ID NO: 5) (or Pro.sub.n-X (SEQ ID NO: 96)), where
X is Lys, Ser, Thr, Ala, Gly, Val or any amino acid, or more
preferably Ser, Ala, Thr, or Val, and n is from 2 to 1000; or the
nucleic acids can code for (X-Pro).sub.n (SEQ ID NO: 11) (or
(Pro-X).sub.n (SEQ ID NO: 94)), where X is any amino acid, such as
Lys, Ser, Thr, Ala, Gly, or Val, or more preferably Ser, Ala, Thr,
or Val, and n is from 1 to 1000. For bulky amino acids, the first
Pro in the XPPPPP (SEQ ID NO: 97) series may not be hydroxylated,
but the others will be. In one embodiment, for example, the nucleic
acids code for (Ser-Pro).sub.10 (SEQ ID NO: 51). In this
embodiment, hGH is expressed as a GH-(Ser-Pro).sub.10 (SEQ ID NO:
51) (modified on the N- or C-terminus); the Pro is hydroxylated by
the plant and then glycosylated with arabinogalactan chains. The
product is an hGH glycoprotein comprising (Ser-Hyp).sub.10 (SEQ ID
NO: 4). The glycoprotein exhibits the same activity as the
wild-type hGH, yet exhibits a significantly increased
pharmacokinetic half-life. (The production and testing of this
embodiment is described in more detail in Example 6, herein
below.)
[0124] HGH modified in accordance with the present invention can
produce a peak plasma concentration of greater than about 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 or more
hours, following a single subcutaneous (SC) injection. This is a
substantial increase over the half-life of wild-type growth
hormone, which exhibits a half-life of about 20-30 minutes.
[0125] In one embodiment, the nucleic acids encoding hGH are
engineered to create an hGH antagonist and the glycosylation site
is added at the C-terminal. For example, the Gly at position 119
(found in a variety of wild-type animal's growth hormone) or Gly
120 (of hGH) can be replaced with any amino acid other that alanine
and generate an antagonist. In one embodiment, Gly 120 of hGH is
replaced with Lys, which produces a human growth hormone
antagonist. Also, a (Ser-Hyp).sub.10 (SEQ ID NO: 4) motif is
attached at the C-terminal. The result is a glycoprotein that
exhibits hGH antagonist activity and increased half-life, as
compared to the half life of unglycosylated hGH antagonist is 20-30
minutes.
[0126] Of course, similar constructs can be created with a 20-kD
variant of growth hormone, with similar results. For example, the
Gly at position 104 (found in a variety of wild-type animal's
20-kDa growth hormone) or Gly 105 (of the 20-kDa human growth
hormone) can be replaced with any amino acid other that alanine and
generate an antagonist. In one embodiment, Gly 105 of hGH (20-kDa
form) is replaced with Lys, which produces an hGH antagonist. Also,
a (Ser-Hyp).sub.10 (SEQ ID NO: 4) motif can be attached at the
C-terminal.
[0127] In one embodiment, the nucleic acids coding for hGH are
engineered to insert the hydroxyproline glycosylation site in an
internal part of the protein. In the case of 22-kDa GH, for
example, the Gly normally at position 119 or 120 is deleted and
Ser-Pro-Pro-Pro-Pro (SEQ ID NO: 98) inserted in its place. With
this construct, the prolines will be hydroxylated and then
arabinosylated. The result will be an antagonist with increased
half-life.
[0128] The following more general description of is informative of
fusion peptides/proteins of the growth hormone superfamily that can
be made in accordance with this invention. In one embodiment of the
present invention, the fusion protein of the present invention
comprises a) at least one glycomodule, and b) a naturally occurring
vertebrate hormone belonging to the GH-PRL-PL superfamily, as
defined below. Vertebrate growth hormone, prolactin, or placental
lactogen are of particular interest.
[0129] In another embodiment of the present invention, the fusion
protein of the present invention comprises a) at least one
glycomodule, and b) a biologically active mutant polypeptide which
is substantially identical, but not completely identical, to a
naturally occurring vertebrate growth hormone, prolactin, or
placental lactogen.
[0130] The term "naturally occurring" presupposes the absence of
human intervention, i.e., the fact that a transgenic mouse has been
genetically engineered to produce a foreign protein does not mean
that the foreign protein in question occurs naturally in mice.
[0131] This mutant may be an agonist, that is, it possesses at
least one biological activity of a vertebrate growth hormone,
prolactin, or placental lactogen. It should be noted that a growth
hormone may be modified to become a better prolactin or placental
lactogen agonist, and vice versa. The mutant may be characterized
as a growth hormone mutant if, after alignments by BlastP, it has a
higher percentage identity with a vertebrate growth hormone than it
does with any known vertebrate prolactin or placental lactogen.
Prolactin and placental lactogen mutants are analogously
defined.
[0132] Alternatively, the mutant may be an antagonist of a
vertebrate growth hormone, prolactin, or placental lactogen. In
general, the contemplated antagonist is a receptor antagonist, that
is, a molecule that binds to the receptor but which substantially
fails to activate it, thereby antagonizing receptor activity via
the mechanism of competitive inhibition. The first identification
of GH mutants that encoded biologically active GH receptor
antagonists was in Kopchick et al., U.S. Pat. Nos. 5,350,836,
5,681,809, 5,958,879, 6,583,115, and 6,787,336, and in Chen et al.,
1991, "Functional antagonism between endogenous mouse growth
hormone (GH) and a GH analog results in dwarf transgenic mice",
Endocrinology 129:1402-1408, Chen et al., 1991, "Glycine 119 of
bovine growth hormone is critical for growth promoting activity"
Mol. Endocrinology 5:1845-1852, and Chen et al., 1991, "Mutations
in the third .alpha.-helix of bovine growth hormone dramatically
affect its intracellular distribution in vitro and growth
enhancement in transgenic mice", J. Biol. Chem. 266:2252-2258. All
of these references (hereinafter, "Kopchick, et al., supra") are
hereby incorporated by reference in their entirety.
[0133] In order to determine whether the mutant polypeptide is
substantially identical with any vertebrate hormone of the
GH-PRL_PL superfamily, the mutant polypeptide sequence can be
aligned with the sequence of a first reference vertebrate hormone
of that superfamily. One method of alignment is by BlastP, using
the default setting for scoring matrix and gap penalties. In one
embodiment, the first reference vertebrate hormone is the one for
which such an alignment results in the lowest E value, that is, the
lowest probability that an alignment with an alignment score as
good or better would occur through chance alone. Alternatively, it
is the one for which such alignment results in the highest
percentage identity.
[0134] In general, the mutant polypeptide agonist is considered
substantially identical to the reference vertebrate hormone if all
of the differences can be justified as being (1) conservative
substitutions of amino acids known to be preferentially exchanged
in families of homologous proteins, (2) non-conservative
substitutions of amino acid positions known or determinable (e.g.,
by virtue of alanine scanning mutagenesis) to be unlikely to result
in the loss of the relevant biological activity, or (3) variations
(substitutions, insertions, deletions) observed within the
GH-PRL-PL superfamily (or, more particularly, within the relevant
family). The mutant polypeptide antagonist will additionally differ
from the reference vertebrate hormone by virtue of one or more
receptor antagonizing mutations.
[0135] With regard to applying point (3) above to insertions and
deletions, it is necessary to align the mutant polypeptide with at
least two different reference hormones. This is done by pairwise
alignment of each reference hormone to the mutant polypeptide.
[0136] When two sequences are aligned to each other, the alignment
algorithm(s) may introduce gaps into one or both sequences. If
there is a length one gap in sequence A corresponding to position X
in sequence B, then we can say, equivalently, that (1) sequence A
differs from sequence B by virtue of the deletion of the amino acid
at position X in sequence B, or (2) sequence B differs from
sequence A by virtue of the insertion of the amino acid at position
X of sequence B, between the amino acids of sequence A which were
aligned with positions X-1 and X+1 of sequence B.
[0137] If alignment of the mutant sequence to the first reference
hormone creates a gap in the mutant sequence, then the mutant
sequence can be characterized as differing from the first reference
hormone by deletion of the amino acid at that position in the first
reference hormone, and such deletion is justified under clause (3)
if another reference hormone differs from the first reference
hormone in the same way.
[0138] Likewise, if the alignment of the mutant sequence to the
first reference hormone creates a gap in the reference sequence,
then the mutant sequence can be characterized as differing from the
first reference hormone by insertion of the amino acid aligned with
that gap, and such insertion is justified under clause (3) if
another reference hormone differs from the first reference hormone
in the same way.
[0139] The preferred vertebrate GH-derived GH receptor agonists of
the present invention are fusion proteins which comprise a
polypeptide sequence P for which the differences, if any, between
said amino acid sequence and the amino acid sequence of a first
reference vertebrate growth hormone, are independently selected
from the group consisting of [0140] (a) a substitution of a
conservative replacement amino acid for the corresponding first
reference vertebrate growth hormone residue; [0141] (b) a
substitution of a non-conservative replacement amino acid for the
corresponding first reference vertebrate growth hormone residue
where [0142] (i) another reference vertebrate growth hormone exists
for which the corresponding amino acid is a non-conservative
substitution for the corresponding first reference vertebrate
growth hormone residue, and/or [0143] (ii) the binding affinity of
a single substitution mutant of the first reference vertebrate
growth hormone, wherein said corresponding residue, which is not
alanine, is replaced by alanine, is at least 10% of the binding
affinity of the first vertebrate growth hormone for the vertebrate
growth hormone receptor to which the first vertebrate growth
hormone natively binds; [0144] (c) a deletion of one or more
residues found in said first reference vertebrate growth hormone
but deleted in another reference vertebrate growth hormone; [0145]
(d) insertion of one or more residues into said first reference
vertebrate growth hormone between adjacent amino acid positions of
said first reference vertebrate growth hormone, where another
reference vertebrate growth hormone exists which differs from said
first reference growth hormone by virtue of an insertion at the
same location of said first reference vertebrate growth hormone;
and [0146] (e) truncation of the first 1-8, 1-6, 1-4, or 1-3
residues and/or the last 1-8, 1-6, 1-4, or 1-3 residues found in
said first reference vertebrate growth hormone ("truncation" is
intended to refer to a deletion of residues at the N- or C-terminal
of the peptide); [0147] where the polypeptide sequence has at least
10% of the binding affinity of said first reference vertebrate
growth hormone for a vertebrate growth hormone receptor, preferably
one to which said first reference vertebrate growth hormone
natively binds, and [0148] where said fusion protein binds to and
thereby activates a vertebrate growth hormone receptor. We
characterize the fusion protein as "GH-derived" because the
polypeptide sequence P qualifies as a vertebrate GH or as a
vertebrate GH mutant as defined above.
[0149] A growth hormone natively binds a growth hormone receptor
found in the same species, i.e., human growth hormone natively
binds a human growth hormone receptor, bovine growth hormone, a
bovine GH receptor, and so forth.
[0150] Based on analyses of the frequencies of amino acid changes
between homologous proteins of different organisms, such as those
presented in Table 1-2 of Schulz and Schirmer, Principles of
Protein Structure and FIG. 3-9 of Creighton, Proteins, we define
conservative substitutions (replacements) as exchanges within the
groups set forth below:
[0151] I small aliphatic, nonpolar or slightly polar residues--Ala,
Ser, Thr (Pro, Gly)
[0152] II negatively charged residues and their amides--Asn Asp Glu
Gln
[0153] III positively charged residues--His Arg Lys
[0154] IV large aliphatic nonpolar residues--Met Leu Ile Val
(Cys)
[0155] V large aromatic residues--Phe Tyr Trp
[0156] Three residues are parenthesized because of their special
roles in protein architecture. Gly is the only residue without a
side chain and therefore imparts flexibility to the chain. Pro has
an unusual geometry which tightly constrains the chain. Cys can
participate in disulfide bonds, which hold proteins into a
particular folding; the four cysteines of bGH are highly
conserved.
[0157] Mutations which exchange I/II, or which exchange III/IV/V,
may be considered semi-conservative, which are a subset of
nonconservative mutations. Nonconservative mutations, which are not
characterized as semi-conservative may be characterized as
"strongly non-conservative." Semi-conservative mutations are
preferred over strongly non-conservative mutations.
[0158] For binding to the human growth hormone receptor, binding
affinity is determined by the method described in Cunningham and
Wells, "High-Resolution Mapping of hGH-Receptor Interactions by
Alanine Scanning Mutagenesis", Science 284: 1081 (1989), and thus
uses the hGHRbp as the target. For binding to the human prolactin
receptor, binding is determined by the method described in
WO92/03478, and thus uses the hPRLbp as the target. For binding to
nonhuman vertebrate hormone receptors, binding affinity is
determined by use, in order of preference, of the extracellular
binding domain of the receptor, the purified whole receptor, and an
unpurified source of the receptor (e.g., a membrane
preparation).
[0159] The receptor binding fusion protein preferably has growth
promoting activity in a vertebrate. Growth promoting (or
inhibitory) activity may be determined by the assays set forth in
Kopchick, et al., which involve transgenic expression of the GH
agonist or antagonist in mice. Or it may be determined by examining
the effect of pharmaceutical administration of the GH agonist or
antagonist to humans or nonhuman vertebrates.
[0160] Preferably, one or more of the following further conditions
apply: [0161] (1) the polypeptide sequence P is at least 50%, more
preferably at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90% or most
preferably at least 95% identical to said first reference
vertebrate growth hormone, [0162] (2) the conservative replacement
amino acids are highly conservative replacement amino acids, [0163]
(3) any deletion under clause (c) is of a residue which is not
located at a conserved residue position of the vertebrate growth
hormone family, and, more preferably is not a conserved residue
position of the mammalian growth hormone subfamily, [0164] (4) the
first reference vertebrate growth hormone is a mammalian growth
hormone, more preferably, a human or bovine growth hormone, [0165]
(5) any insertion under clause (d) is of a length such that another
reference vertebrate growth hormone exists which differs from said
first reference growth hormone by virtue of an equal length
insertion at the same location of said first reference vertebrate
growth hormone [0166] (6) the differences are limited are limited
to substitutions pursuant to clauses (a) and/or (b), [0167] (7) if
the first reference vertebrate growth hormone is a nonhuman growth
hormone, and the intended use is in binding or activating the human
growth hormone receptor, the differences increase the overall
identity to human growth hormone, [0168] (8) one or more of the
substitutions are selected from the group consisting of one or more
of the mutations characterizing the hGH mutants B2024 and/or B2036
as described below, [0169] (9) the polypeptide sequence P is at
least 50%, more preferably at least 55%, at least 60%, at least
65%, at least 70% at least 75%, at least 80%, at least 85%, at
least 90%, at least 95% or, if an agonist, most preferably 100%
similar to said first reference vertebrate growth hormone, or
[0170] (10) the polypeptide sequence P, when aligned to the first
reference vertebrate growth hormone by BlastP using the Blosum62
matrix and the gap penalties -11 for gap creation and -1 for each
gap extension, results in an alignment for which the E value is
less than e-10, more preferably less than e-20, e-30, e-40, e-50,
e-60, e-70, e-80, e-90 or most preferably e-100.
[0171] For purposes of condition (1), percentage identity is
calculated by the BlastP methodology, i.e., identities as a
percentage of the aligned overlap region including internal gaps.
For purposes of condition (2), highly conservative amino acid
replacements are as follows: Asp/Glu, Arg/His/Lys, Met/Leu/Ile/Val,
and Phe/Tyr/Trp. For purposes of condition (3), the conserved
residue positions are those which, when all vertebrate growth
hormones whose sequences are in a publicly available sequence
database as of the time of filing are aligned as taught herein, are
occupied only by amino acids belonging to the same conservative
substitution exchange group (I, II, III, IV or V) as defined above.
The unconserved residue positions are those which are occupied by
amino acids belonging to different exchange groups, and/or which
are unoccupied (i.e., deleted) in one or more of the vertebrate
growth hormones. The fully conserved residue positions of the
vertebrate growth hormone family are those residue positions are
occupied by the same amino acid in all of said vertebrate growth
hormones. Clause (c) does not permit deletion of a residue at one
of the fully conserved residue positions. One may analogously
define fully conserved, conserved, and unconserved residue
positions of the mammalian growth hormone family.
[0172] For purposes of condition (4), hGH is preferably the form of
hGH which corresponds to the mature portion (AAs 27-217) of the
sequence set forth in Swiss-Prot SOMA_HUMAN, P01241, isoform 1 (22
kDa), and bovine growth hormone is preferably the form of bovine
growth hormone which corresponds to the mature portion (AA 28-217)
of the sequence set forth in Swiss-Prot SOMA_BOVIN, P01246, per
Miller W. L., Martial J. A., Baxter J. D.; "Molecular cloning of
DNA complementary to bovine growth hormone mRNA."; J. Biol. Chem.
255:7521-7524(1980). These references are incorporated by reference
in their entirety. For purpose of condition (10), percentage
similarity is calculated by the BlastP methodology, i.e., positives
(aligned pairs with a positive score in the Blosum62 matrix) as a
percentage of the aligned overlap region including internal
gaps.
[0173] Vertebrate GH-derived GH receptor antagonists of the present
invention may be similarly defined, except that the polypeptide
sequence must additionally differ from the sequence of the
reference vertebrate growth hormone, e.g., at the position
corresponding to Gly 119 in bovine growth hormone or Gly 120 in
human growth hormone, in such manner as to impart GH receptor
antagonist (binds but does not activate) activity to the
polypeptide sequence and thereby to the fusion protein. Note that
bGH Gly119/hGH Gly 120 is presently believed to be a fully
conserved residue position in the vertebrate GH family. It has been
reported that an independent mutation, R77C, can result in growth
inhibition. See Takahashi Y, Kaji H, Okimura Y, Goji K, Abe H,
Chihara K., "Brief report: short stature caused by a mutant growth
hormone.", N Engl J Med. 1996 Feb. 15; 334(7):432-6.
[0174] Preferably, the GH receptor antagonist has growth inhibitory
activity. The compound is considered to be growth-inhibitory if the
growth of test animals of at least one vertebrate species which are
treated with the compound (or which have been genetically
engineered to express it themselves) is significantly (at a 0.95
confidence level) slower than the growth of control animals (the
term "significant" being used in its statistical sense). In some
embodiments, it is growth-inhibitory in a plurality of species, or
at least in humans and/or bovines.
[0175] Also,the GH antagonists may comprise an alpha helix
essentially corresponding to the third major alpha helix of the
first reference vertebrate growth hormone, and at least 50%
identical (more preferably at least 80% identical) therewith.
However, the mutations need not be limited to the third major alpha
helix.
[0176] The contemplated vertebrate GH antagonists include, in
particular, fusions in which the polypeptide P corresponds to the
hGH mutants B2024 and B2036 as defined in U.S. Pat. No. 5,849,535.
Note that B2024 and B2036 are both hGH mutants including, inter
alia, a G10K substitution. In addition, we contemplate GH
antagonists in which B2024 and B2036 are further mutated in
accordance, mutatis mutandis, with the principles set forth above,
i.e., in which B2024 or B2036 serves in place of a naturally
occurring GH such as HGH as the reference vertebrate GH.
[0177] In a like manner, one may define vertebrate prolactin
agonists and antagonists, and vertebrate placental lactogen
agonists and antagonists, which agonize or antagonize a vertebrate
prolactin receptor. One may also have mutants of a vertebrate
growth hormone, which agonize or antagonize the prolactin receptor
(with or without retention of activity against a growth hormone
receptor), and mutants of a vertebrate prolactin or placental
lactogen, which agonize or antagonize a vertebrate growth hormone
receptor (with or without retention of activity against a prolactin
receptor). In a like manner, one may define agonists and
antagonists that are hybrids, or are mutants of hybrids, of two or
more reference hormones of the vertebrate growth
hormone--prolactin--placental lactogen hormone superfamily, and
which retain at least 10% of at least one receptor binding activity
of at least one of the reference hormones.
[0178] There are several ways in which these hybrids can be
defined. In one embodiment, we simply permit the first reference
vertebrate growth hormone and the another reference vertebrate
growth hormone to be any vertebrate hormone which is a member of
the superfamily. In a second embodiment, the mutant is mostly
defined on the basis of one family, e.g., GH, but at a limited
number of positions, e.g., less than 10% or less than 5% of the
sequence P, it is permitted to choose from another family. In this
category is the Cunningham prolactin octomutant, infra, which binds
hGH. In a third embodiment, the hybrid is a segmented hybrid, such
as a dihybrid visualized as consisting of segments which are
alternately derived from (a) the vertebrate growth hormone family
or (b) the vertebrate prolactin family, starting with either. The
number of segments may be odd or even, e.g., 2, 3, 4, 5, 6, 7, 8, 9
or 10. Preferably, there are not more than ten segments. In a
GH-derived segment, the reference hormones are vertebrate GHs, and
in prolactin-derived segments, the reference hormones are
vertebrate prolactins. Preferably, each segment is at least ten
consecutive amino acids long. The segments may be unequal in
length. Cunningham, infra, describes several GH/prolactin hybrids
(or mutants thereof) which have three segments, of the format
(GH-derived)-(prolactin-derived)-(GH derived). In a like manner,
the segmented hybrid may be a GH/PL or PL/PRL dihybrid, or a
GH/PRL/PL trihybrid (in the last case, the rule is that adjacent
segments are derived from different families, whether GH, PRL or
PL).
[0179] Growth Hormone-Prolactin-Placental Lactogen Family
[0180] Growth hormones, placental lactogens, and prolactins are
homologous proteins, thought to have arisen from a common ancestral
molecule. Prolactins and growth hormones are believed to have
diverged about 400 million years ago, hence the presence of
distinct prolactins and growth hormones in fish. Placental
lactogens are only observed in mammals, and it has been
hypothesized that primate PLs evolved from the growth hormone
lineage and non-primate PLs from the prolactin one. The protein hCS
is thought to have evolved by gene duplication from hGH. There are
also somatolactins in fish, with sequences intermediate between
those of prolactin and GH.
[0181] The mature growth hormones, prolactins, and placental
lactogens are typically composed of 190-200 residues, with
molecular weights of 22,000-23,000 daltons. However, these sizes
are not required; e.g., mature flounder GH is not more than 173
residues long.
[0182] The amino acid sequences of these proteins are too similar
to have arisen by chance alone; a BlastP search, using mature hGH
as the query sequence, with the default scoring matrix (Blosum62)
and gap penalties (11 creation/1 extension), and no low complexity
filter, yields an E value of 1e-106, 9e-90 for the alignment with
human placental lactogen (prf 731144A), and 6e-11 for the alignment
with human prolactin (ref NP.sub.--000939.1).
[0183] Functional considerations also justify the definition of the
growth hormone-placental lactogen-prolactin superfamily. Even if
there is also a distinct placental lactogen receptor, see Freemark,
J. Clin. Investig., 83: 883-9 (1989), the effect of placental
lactogens on the prolactin receptor is significant. Classically,
the GH receptor is the specific receptor for GH, and the prolactin
(a.k.a. lactogen) receptor is the specific receptor for prolactin
and placental lactogen. However, primate GHs can bind to the
prolactin receptor with high affinity, and some non-human mammalian
placental lactogens can bind to the somatogen (GH) receptor.
Reference may also be made to the structural similarities of the GH
and prolactin receptor proteins. See Goffin, et al.,
"Sequence-Function Relationships Within the Expanding Family of
Prolactin, Growth Hormone, Placental Lactogen, and Related Proteins
in Mammals", Endocrine Revs., 17(4): 385-410 (1996); Nicoll, et
al., "Structural Features of Prolactins and Growth Hormones that
Can Be Related to Their Biological Properties", Endocrine Revs.,
7(2): 169-203 (1986).
[0184] For the purpose of the present application, the GH-PRL-PL
superfamily is composed of all proteins which, when aligned to hGH
(mature portion of ref NP.sub.--000506.2) by BlastP as set forth
above, yield an alignment for which the E value is less than (i.e.,
better than) e-06.
[0185] The growth hormones (GHs) are a family of vertebrate
proteins with about 191 amino acid residues, the number varying
from species to species. There are four cysteine residues, and two
disulfide bridges. See generally Harvey, et al., Growth Hormone
(CRC Press: 1995). The amino acid sequence of the growth hormones
isolated from various vertebrate species are highly conserved. In
the aforementioned BlastP search, the E value for alignments of
mature hGH with a few of the many other database GHs were as
follows (best alignment for each species cited): 1e-106 (Pan
troglodytes), 3e-97 (Caallithrix jacchus, common marmoset), 3e-68
(Balaenoptera borealis, fm whale; Delphinus delphis, common
dolphin; Hippopotamus amphibius), 4e-68 (Canis familiaris, dog; Sus
scrofa domestica, pig), 2e-67 (Mus musculus), 1e-66 (Rattus
norvegicus, Norwegian rat; Oryctolagus cuniculus, domestic rabbit;
Cavia porcellus, guinea pig), 2e-65 (Capra hircus, goat; Giraffa
camelopardalis, giraffe; Bos taurus, bovine); 3e-65 (Ovis aries,
domestic sheep); 4e-59 (Crocodulus novaeguineae), 4e-58 (Chelonia
mydas), 5e-58 (Gallus gallus, chicken); 2e-55 (Tarsius syrichta,
Philippine tarsier) (a relative high E value for a mammal) ; 1e-53
(Lepisosteus osseus, a bony fish); 8e-08 (Torpedo californica). The
best scoring somatolactin is sp P20362, E value of2e-18. The best
(lowest) E value is that which would be obtained if the query and
database sequence were identical (or if one comprised the other);
in a recent search in which the query sequence was the mature HGH,
the best E value was that for the alignment of the mature HGH with
the database HGH precursor (ref NP.sub.--000506.2): 1e-106.
[0186] If the E value for an alignment is low, the alignment score
must have been high relative to those which would occur by chance
alone. The alignment score for each alignment is calculated by
adding up the individual amino acid pair scores dictated by the
scoring matrix, and subtracting the appropriate gap penalties for
any gaps. The alignment algorithm introduces gaps only if they
result in a net improvement in the overall alignment score. In the
scoring matrix, identities tend to have the higher values, and
hence alignments with high alignment scores will also tend to be
characterized as having high percentage identities. However,
alignments ranked by alignment score will not necessarily have the
same order as if those same alignments were ranked by percentage
identity.
[0187] In BlastP, the percentage identity is calculated as being
the number of identities expressed as a percentage of the length of
the "overlap", the aligned region. This region begins and ends with
aligned amino acid pairs (not necessarily identical) and may
include one or more gaps in either or both sequences. A gap occurs
where one or more consecutive amino acids inside one sequence are
left unpaired with amino acids in the other sequence (this may be
symbolized by aligning each of them with a null symbol, such as a
hyphen, in that other sequence). The calculated length of the
overlap region is the sum of the number of aligned pairs and the
lengths of the gaps. If one sequence overhangs another, the
overhang is an end-gap, outside the overlap region, and does not
count in calculating the percentage identity.
[0188] The following are examples of the BlastP percentage identity
of human GH (ref NP.sub.--000506.2) with other members of the
GH-PRL-PL superfamily: human placental lactogen (85%, 161/189),
whale, dolphin and hippopotamus GH (67%, 130/192, 3/192 in gaps),
pig GH (67%, 130/193, 3/192 in gaps), mouse GH (65%, 126/192, 3/192
in gaps), bovine GH (66%, 127/192, 3/192 in gaps), crocodile GH
(59%, 113/190, 3/190 in gaps), chicken GH (57%, 110/190, 3/190 in
gaps), Syrian hamster GH (62%, 108/172, 2/172 in gaps), Lepisosteus
osseus GH (54%, 102/186, 3/186 in gaps), Japanese flounder (27%,
53/190, 8/190 in gaps), human prolactin (23%, 45/191, 12/191 in
gaps).
[0189] The overall percentage identity of bovine growth hormone
with other non-primate, mammalian growth hormones is very high:
porcine (92%), ovine (99%), and rat (87%). Watahiki, et al., J.
Biol. Chem., 264:312 (1989) compared the sequences of flounder,
yellowtail, tuna, salmon, chicken, rat, porcine, ovine, bovine and
human growth hormones. Watahiki's FIG. 3 identifies residues
conserved among the GHs and residues predicted to be important for
the manifestation of growth-promoting activity. He identified five
conserved domains which he labeled GD1-GD5. Mutations in these
conserved domains are more likely to affect activity.
[0190] The 3-dimensional structures of two GHs are known, and they
are quite similar. Porcine GH is a single domain protein arranged
as a four helix bundle with the helices in an antiparallel
(up-up-down-down) relationship. Its four helixes are made up of
residues 7-34, 75-87, 106-127 and 152-183. See Abdel-Meguid et al.,
Proc. Nat. Acad. Sci. USA 84: 6434 (1987). Human growth hormone
features a bundle of four major helices (9-34, 72-92, 106-128, and
155-184), connected by loops (35-71, 93-105 and 129-154). Loop 1
(between helices 1 and 2) comprises mini-helices at 38-47 and
64-70, and Loop 2 (between helices 2 and 3) one at 94-100.
Reference to helices 1-4 of hGH is a reference to the major
helices, not to the mini-helices. Helix 2 is kinked at Pro-89. See
DeVos, et al., Science, 255:306-312 (1992).
[0191] The other GHs are also believed to be four-helix proteins,
on the basis of secondary structure prediction methods, sequence
alignment, and knowledge of the 3-D structures of pGH and/or hGH.
For example, bovine growth hormone is 92% homologous at the amino
acid sequence level with porcine growth hormone, and bGH's
structure has been deduced by study of the two sequences and of the
structure of porcine growth hormone. Its four alpha helixes have
been reported to be assumed by amino acids 4-33, 66-80, 108-127 and
150-179. The third alpha helix of bGH is defined as amino acids
106-129. However, it will be noted that the ends of this helix have
a less marked alpha helical secondary structure than does the
central region, which is 109-126. The exact bounds of the third
alpha helix may differ for other GH's, depending on the alpha
helical tendencies of the "end" amino acids. The conformation is
reasonably consistent with the predictions made by Chen and
Sonenberg, Biochemistry, 16:2110 (1977) using the method of Chou
and Fasman, Biochemistry, 13:222 (1974) (AAs 10-34, 66-87, 111-127,
186-191). For preliminary work in determining the 3-D structure of
bGH, see Bell, et al., J. Biol. Chem., 260:8520-25 (1985).
[0192] Growth hormones can have considerable inter-species
cross-reactivity. In general, the trend is for "higher" growth
hormones to activate "lower" GH receptors, but not the reverse.
Human GH is active in nonhuman mammals, but nonhuman, nonprimate
GHs are generally inactive in humans. Bovine GH is active in the
horse (see De Kock, et al., J. Endocrinol., 171(1): 163-171
(2001)). Mammalian and bird GHs are active in fish, see Gill, et
al., Biotechnology, 3:643 (1985) reported that recombinant chicken
and bovine growth hormones accelerate growth in juvenile pacific
salmon. Mutation of a nonhuman GH, to increase its similarity to
human GH, will render it more likely to be active against the human
GH receptor. For studies of the structural origins of species
specificity in GH or its receptor, see Liu, et al., "Episodic
Evolution of Growth Hormone in Primates and Emergence of the
Species Specificity of Human Growth Hormone Receptor", Mol. Biology
& Evolution, 17: 945-53 (2001); Allan, et al., "Identification
of Novel Sites in the Ovine Growth Hormone Receptor Involved in
Binding Hormone and Conferring Species Specificity", Eur. J.
Biochem., 261(2): 555-62 (1999).
[0193] Human placental lactogen has an overall sequence identity
with hGH of 85%, but its binding to hGH bp is .about.2,000-fold
weaker. WO97/11178 at p. 100. For a comparison of placental
lactogens, see Forsyth, Exp. Clin. Endocrinol., 102(3): 244-51
(1994).
[0194] Human prolactin is a 199-residue (23 kDa protein), with 23%
identity (BlastP) to human GH. The 3-D structure of human prolactin
has been determined; as expected, it has four primary helices, with
an up-up-down-down topology, just as does human growth hormone.
There are also interesting differences. The first extended loop of
hPRL is missing the first of the two mini-helices found in the
comparable loop of hGH, while the second mini-helix deviates in
angle from its hGH counterpart. Both hPRL and hGH have a short loop
connecting the primary helices 2 and 3, but the loop is shorter in
hPRL, and there is no component mini-helix. Finally, the N-terminal
of hPRL is longer than that of hGH, and contains a short
disulfide-linked loop. See Keeler, et al., "The Tertiary Structure
and Backbone Dynamics of Human Prolactin", J. Molec. Biol., 328:
1105-221 (2003). In Keeler's FIG. 1, HGH Gly-120 is aligned with
hPRL Gly-129. G129X mutants of hPRL are known to exhibit prolactin
receptor antagonist activity, see below.
[0195] Growth Hormone (Somatotropic) Receptor
[0196] The hGH receptor belongs to a large family of receptors of
hematopoietic origin, which includes interleukin-3 and granulocyte
colony stimulating factor receptors. For purification and
characterization of a human growth hormone receptor, see Leung, et
al., Nature, 330:537-43 (1987).
[0197] The extracellular domain of the hGH receptor is designated
hGHbp. The affinity (Kd) of hGH for hGHbp was reported by
Cunningham et al. (1989) to be 0.34 nM. WO92/03478 reports the
affinity of hGH for the hGHbp in the presence of EDTA is such that
the Kd is 0.42 nM, while in the presence of ZnCl2 the affinity is
reduced (KD of 1.6 nM). It also reports that the affinity of hPRL
for the hGHbp is extremely low (KD>100,000 nM whether in
presence of EDTA or ZnCl2, see Table 1). The affinity of hPL for
hGHbp is very low (949.2 nM, Table 13), but not as low as that of
hRPL.
[0198] 3D Structure of GH: GH Receptor Complexes
[0199] The 3D structure of the hGH:hGHbp complex is also known (see
Wells and DeVos, Ann. Rev. Biophys. Biomol. Struct., 22: 329-51
(1993) and DeVos, et al., Science, 255:306 (1992)). These
researchers examined the complex of hGH and the extracellular
domain of its receptor (hGHR) by X-ray diffraction. The complex had
the form hGH (hGHR).sub.2; that is, the receptor dimerizes to
interact with hGH.
[0200] The first receptor-binding region ("site 1") of hGH is
concave and is formed mainly by residues on exposed faces of helix
4, but also by exposed residues of helix 1 and residues in the
region connecting helices 1 and 2. The second receptor-binding
region ("site 2") comprises the exposed sides of helices 1 and 3
and is relatively flat. The role of the helix 3 is shown best in
DeVos' FIG. 5; there is a significant decrease in solvent
accessibility around hGH E119 upon complex formation. GH
antagonists that are GH mutants with mutations corresponding to
bGH119X (or hGH120X) appear to interfere with receptor
dimerization.
[0201] The site 1 residues of hGH are H18, H21, Q22, F25, K41, Y42,
L45, Q46, P61, S62, N63, E66, R167, K168, D171, K172, I175, R178,
C182 and C189. The site 2 residues are T3, I4, L6, L9, N12, L15,
r16, R19, Q22, Y103, N109, D116, D119, G120 and T123. See Tables 4
and 5 of U.S. Pat. No. 5,506,107 for details on the nature of the
interactions between these residues and hGHbp.
[0202] According to the X-ray structure of the hgh(hGHbp)2 complex,
the two HGHbp's contact each other at Ser201. Consequently, an
hGHbp(S201C)-matrix can be used to test variants of hGH for binding
to site 1 alone. See WO97/11178.
[0203] Prolactin Receptor
[0204] The extracellular binding domain (AAs 1-211) of the
prolactin receptor is designated hPRLbp. It is about 32% identical
to hGHbp, see WO90/04788 p. 89. WO92/03478 initially reports (table
1) the affinity of hPRL for the hPRLbp in the presence of EDTA is
such that the Kd is 2.1 nM, while in the presence of ZnCl2 the
affinity is reduced (KD of 2.6 nM). However, in table 11 the
affinity of hPRL for hPRLbp without zinc is said to be 2.8 nM.
[0205] Human GH also binds the human prolactin receptor. (See
Boutin et al., Cell, 53: 69 (1988)). WO92/03478 reports the
affinity of hGH for the hPRLbp in the presence of EDTA is such that
the Kd is 270 nM, while in the presence of ZnCl2 the affinity is
substantially increased (KD of 0.033 nM, i.e., 33 pM). Increased
affinity is also observed for the single Ala substitution hGH
mutants H18A (370 to 4.5 nM), H21A (200 to 3 nM), E174A (360 to 12
nM) and D171A (ND to 0.037 nM).
[0206] The hGH binding epitope for the prolactin receptor is
composed of determinants in the middle of helix 1 (comprising
residues F25 and D26), a loop region (including I58 and R64), and
center portion of helix 4 (including K168m K172, E174, and F176).
See WO90/04788 p. 56. This patch overlaps, but is not identical to,
the hGH epitope for the hGH receptor. Binding affinities of various
hGH mutants for hPRLbp in presence of ZnCl2 are given in Tables
7-9. WO92/03478, p. 13, suggests that the binding of zinc to the
hGH:hPRLbp complex is mediated by hGH residues H18, H21 and
E174.
[0207] The affinity of hPL for hPRLbp in the presence of ZnCl2 is
50 pM. In the absence of zinc the hPL precipitated. The hPRLbp
affinities of hPL mutants D56E, M64R, E174A, M179I,
D56E/M64R/M179I, and V4I/D56E/M64R/M179I are given in Table 12 of
WO92/03478.
[0208] Hybrid Proteins and Homologue-Scanning Mutagenesis
[0209] Cunningham et al., Science 243: 1330-1336 (1989) used a
technique called homologue-scanning mutagenesis to identify
residues involved in the binding of hGH to hGHbp. In essence,
selected segments of the hGH polypeptide were replaced with the
corresponding segments (according to Cunningham's sequence
alignment) of a homologous hormone (pGH, hPL or hPRL). This in
effect created proteins which were hybrids of hGH and a homologous
hormone. It should be noted that Cunningham did not always replace
all residues of the target segment.
[0210] A comparison of the binding affinities of these mutants GHs
and wild-type hGH to a cloned liver hGH receptor led to the
conclusion that there were three discontinuous polypeptide
determinants in hGH involved in receptor binding. They were located
at the NH.sub.2 terminus, the COOH terminus, and within a loop
between amino acid residues 54 and 74. These putative binding
domains were further analyzed by an alanine-scanning mutagenesis
technique in which alanine residues were systematically substituted
throughout those regions (see below).
[0211] The mutations introduced into hGH by Cunningham are set
forth below: TABLE-US-00001 hGH hPRL bp bp Bind Bind Region Kd Kd
probed Mutant Name Mutations Introduced (nM) (nM) w + hGH none 0.34
2.3 A11-33 hPL(12-25) N12H, F25L 1.4 ND pGH(11-33) D11A, M14V,
H18Q, R19H, F25A, Q29K, 1.2 852 E33R hPRL(12-33) N12R, M14V, L15V,
R16L, R19Y, F25S, 3.6 ND D26E, Q29S, E30Q, E33K hPRL(12-19) N12R,
M14V, L15V, R16L, R19Y 5.8 3.2 hPRL(22-33) Q22N, F25S, D26E, Q29S,
E30Q, E33K 0.29 168 B46-82 hPL(46-52) Q46H, N47D, P48S, Q49E, L52F
2.5 4.4 pGH(48-52) P48A, T50A, S51A, L52F 0.94 2.0 C54-74
hPL(56-64) E56D, R64M 10 41 pGH(57-73) S57T, T60A, S62T, N63G,
R64K, E65D, 5.8 167 T67A, K70R, N72D, L73V hPRL(54-74) F54H, S55T,
E56S, I58L, P59A, S62E, 23 2.5 N63D, R64K, E66Q, T67A, K70M, S71N,
N72Q, L73K, E74D D88-104 hPRL(88-95) E88G, Q91Y, F92H, R94T, S95E
0.47 3.8 hPRL(97-104) F97R, A98G, N99M, S100Q, L101D, 0.53 12.1
V102A, Y103P, G104E E108-136 hPL(109-112) N109D, V110D, D112H 0.61
ND hPRL(111-129) Y111V, L113I, K115E, D116Q, E118K, 0.52 2.6 E119R,
G120L, Q122E, T123G, G126L, R127I, E129S hPRL(126-136) R127D,
L128V, E129H, D130P, G131E, 0.58 ND S132T, P133K, R134E, T135N
F164-190 pGH(164-190) Y164S, R167K, M170L, D171H, V173A, .gtoreq.34
ND F176Y, I179V, V180M, Q181K, S184R, i184f, G187S, G190A
PGH(167-181) R167K, D171H, I179V, Q181K 9.2 ND w+ hPRL none .sup.
10.sup.5 7.6 The first four columns are based on Cunningham et al.
(1989), and the last column on Table XVIII of WO90/04788. The hGHbp
data for w+ hPRL is also from WO90/04788. The data for w+ hGH
binding hGH bp is from Table III of WO94/04788.
[0212] First Ala Scanning Mutagenesis Study
[0213] Alanine scanning mutagenesis was first described by
Cunningham and Wells ("High-Resolution Mapping of hGH-Receptor
Interactions by Alanine Scanning Mutagenesis", Science 284: 1081
(1989)). In view of the results of homologue scanning mutagenesis,
their study was directed to residues 2-19, 54-75, and 167-191.
Amino acid residues at positions 10, 58, 64, 68, 172, 174, 175, and
176 of hGH were shown to be important for GH receptor binding.
However, none of the single Ala substitution mutant GHs tested were
reported to inhibit growth.
[0214] Based on the alanine scanning mutagenesis, preferred
replacement amino acids for hGH residues F10, F54, E56, I58, R64,
Q68, D171, K172, E174, T175, F176, R178, C182 and V185 are listed
in Table IV, p. 52, of WO90/04788. These residues are those for
which the alanine substitution resulted in a more than four-fold
effect on the Kd. Table V of the same reference listed the residues
for which the alanine substitution resulted in a less than two fold
effect, and Table VI those for which it had a favorable effect.
Table X sets forth suggested replacement AAs for hGH residues S43,
F44, H18, E65, L73, E186, S 188, F191, F97, A98, N99, S100 L101,
V102, Y103, G104, R19, Q22, D26, Q29, E30 and E33.
[0215] hGH174 Study
[0216] Since the mutation E174A resulted in a substantial increase
in hGH:hGHbp affinity, twelve alternative substitutions at this
site were tested for activity. Side chain size appeared to be the
major factor determining affinity. The optimal AA remained Ala
(0.075), followed by Ser (0.11), Gly (0.15), Gln (0.21), Asn
(0.26), Glu (wild type, 0.37), His (0.43), Lys (1.14), Leu (2.36)
and Tyr (2.9). There was no expression of E174D or E174R. See Table
6 of WO92/03478.
[0217] Second Ala Scanning Mutagenesis Study
[0218] Residues K41, Y42, L45 and Q46, which belong to the first
minihelix, were not evaluated in the first study, and hence were
studied subsequently. Kd values are given in Table 3 of U.S. Pat.
No. 5,534,617. WO97/11178 comments at p. 106 that "a starting point
for efficient optimization of affinity is a complete alanine scan
of the relevant interface."
[0219] Double Mutants
[0220] Several double mutants were prepared with the intent of
altering hGH/hPRL receptor preference. For wt hGH, binding is 2.3
nM to hPRLr and 0.34 to hGHr. For K168A/E174A, the values are 1950
and 0.09, and for K172A/F176A, they are .about.40,000 and 190.
These double mutants thus evidence increased preference for hGHr
over hPRLr. See WO90/04788.
[0221] Additivity of Single Substitution Effects
[0222] Table XXI OF WO90/04788 analyzes the additivity of the
effects of various single substitutions on binding to the hGH or
hPRL receptors. These effects are characterized as being
"strikingly additive."
[0223] Helix-4a Library
[0224] A combinatorial library of mutants was prepared in which
wild type hGH was randomized at residues K172, E174, F176 and R178.
These residues were targeted for random mutagenesis because they
all lie on or near the surface of hGH, contribute significantly to
receptor binding as shown by Ala scanning mutagenesis, lie within a
well defined structure occupying two turns on the same side of
helix 4, and are each substituted by at least one amino acid among
known evolutionary variants of hGH. See p. 32 of WO92/09690. The
mutants selected by competitive binding to hGHbp were KSYR (SEQ ID
NO: 99) (0.06 nM), RSFR (SEQ ID NO: 100) (0.10), RAYR (SEQ ID NO:
101) (0.13), KTYK (SEQ ID NO: 102) (0.16), RSYR (SEQ ID NO: 103)
(0.20), KAYR (SEQ ID NO: 104) (0.22), RFFR (SEQ ID NO: 105) (0.26),
KQYR (SEQ ID NO: 106) (0.33), KEFR (SEQ ID NO: 107) (wild type,
0.34), RTYH (SEQ ID NO: 108) (0.68), QRYR (SEQ ID NO: 109) (0.83),
KKYK (SEQ ID NO: 110) (1.1), RSFS (SEQ ID NO: 111) (1.1) and KSNR
(SEQ ID NO: 112) (3.1), with, e.g., "KSYR" (SEQ ID NO: 99) denoting
K172, S174, Y176 and R178. The tightest binding mutant (E174S,
F176Y) had an affinity about six-fold higher than wild-type hGH.
See table VII of WO92/09690.
[0225] For sequences of some non-selected mutants (thereby
illustrating the diversity of the library), see Table VI of U.S.
Pat. No. 5,780,279. These mutants should have lower hGHbp affinity
than the selected mutants, but are not necessarily entirely
non-binding.
[0226] Helix-4b Library
[0227] A combinatorial library of mutants was prepared in which the
mutant hGH (E174S, F176Y) was randomly mutated at R167, D171, T175
and I179. Table XI of WO92/09690 shows that N, K, S, D, T, E and A
were all accepted at 167 (wt=R); S, N and D at 171 (wt=D); T, A and
S at 175 (wt=T); and T, N, Q, I and L at 179 (wt=I).
[0228] Some mutations were over-represented among the selected
clones compared to the expected frequency of those mutations in the
library based on the codon (NNS) used to encode them. This
over-representation may be expressed in standard deviation units by
(observed frequency-expected frequency)/standard deviation. In the
56 clones sequenced, the over-represented mutations (with a score
of at least 2.0 standard deviation units) were R167N (25.6 sd),
R167K (4.1), D171S (14.1), D171 (4.8), D171N (4.1), T175 (29.1),
I179N (18.6), I179N (4.1). See Table 4 of U.S. Pat. No. 5,534,617.
The best library member was a pentamutant (R167D, D171S, E174S,
F176Y, I179T), with three new mutations relative to the two
mutation background, which bound hGH receptor about 8-fold better
than wild-type hGH.
[0229] Helix-1 Library
[0230] A combinatorial library of mutants was prepared in which
wild-type hGH was randomly mutated at F10, M14, H18 and H21. After
4 rounds of selection, a tetramutant (F10A, M14W, H18D, H21N) was
isolated which bound the receptor about 3-fold better (Kd 0.10 nM)
than wild-type hGH. In the 68 clones sequenced, the following amino
acids were over-represented at the mutated positions with a score
of at least 2.0 standard deviation units: F10A (12.0 sd), F10 (10.4
sd), F10H (6.2 sd), M14W (11.1), M14S (4.8), M14Y (2.7), M14N
(2.7), M14H (2.0), H18D (18.8), H18F (4.1), H18N (3.4), H21N
(20.2), and H21 (4.8). See Table 4 of U.S. Pat. No. 5,534,617. More
generally, Table VIII of WO92/09690 shows that H, A, Y, L, I, and F
were all accepted at position 10, G, W, T, N and S at 14; N, D, V,
I S, and F at 18, and N, H, G and L at 21.
[0231] Minihelix-1 Library
[0232] A combinatorial library of mutants was prepared in which
wild type hGH was mutated at minihelix-1 positions K41, Y42, L45
and Q46. Results are shown in Table 4 of U.S. Pat. No. 5,534,617.
Seventeen clones were sequenced. By the standard deviation
criterion there was a mild-preference (3.7 std. dev. units) for
K41R, a slight preference for Y42R (2.0 sd) or Y42Q (2.0 sd), a
strong preference for L45W (4.8 sd) or wild type L45 (4.5 sd), and
a stronger preference for Q46W (7.6). Also observed were K41F (2.0
sd), Q46F (2.0 sd) and Q46Y (2.0 sd). The best of the library
member was clone 835.A6 (41I, 42H, 45W, 46W), with a 4.5-fold
improved affinity over wild-type hGH. See Table 5 of U.S. Pat. No.
5,534,617.
[0233] Loop-A Library
[0234] A combinatorial library of mutants was prepared in which
wild-type hGH was randomly mutated at loop-A positions F54, E56,
I58 and R4. In the 26 clones sequenced, the over-represented
mutations (at least 2 sd) were F54P (14.1 sd), E56D (4.7), E56W
(4.7), E56Y (2.5), I58 (8.1), I58V (3.5) and R64K (22.8). The R64K
mutant, found in 81% of the clones, was previously known to by
itself cause a 3-fold improvement in affinity. The best of the
library members tested was the tetramutant (F54P, E56D, I58T,
R64K), which had a 5.6-fold greater affinity than wild type
hGH.
[0235] Combinatorial Library Use, Generally
[0236] WO97/11178 comments (p. 107) that ideally one should
randomize residues which contact each other in the same mutagenesis
step so that they are allowed to co-vary. While such covariation
allows the detection of non-additive multiple substitution effects,
most improvements were simple additive effects. See WO97/11178, p.
108.
[0237] Noncombinatorial Multiple Substitution Mutants
[0238] Various combinations of the following subcombinations of
multiple mutations were synthesized and tested as shown in Table 6
of U.S. Pat. No. 5,534,617: [0239] A=F10H, M14G, H18N, H21N [0240]
B=F10A, M14W, H18D, H21N (0.10) [0241] C=M14S, H18F, H21L (0.68)
[0242] D=R167N, D171S, E174S, F176Y, I179T (0.04) [0243] E=R167E,
D171S, E174S, F176Y (0.04) [0244] F=R167N, D171N, E174S, F176Y,
I179T (0.06) [0245] 852b=K41I, Y42H, L45W, Q46W, F54P, R64K
(0.0079)
[0246] Combinations of the Helix-1 variants A, B or C, with the
Helix-4b variants D, E or F, were prepared. The variant A, and
combinations AD, AE and AF, formed disulfide dimers and hence were
not pursued further. Variant C also formed a disulfide dimer, but
CD, CE and CF did not. It is unclear whether BE was prepared; no
reference to it is made.
[0247] The tested combinations, and their Kd values (nM), were BD
(0.01), CD (0.011), CE (0.014), BF (0.016), CF (0.021)and 852d
(BD+852b) (0.0009). Note that 852d differs by 15 substitutions from
wild-type hGH.
[0248] Joint Selection Combinatorial Library
[0249] Some attempt has been made to combinatorially explore
simultaneous helix-1 and helix-4 mutations. Mutating four residues
in helix-1 and 4 residues in helix 4 so as to systematically
explore all 20 possible AAs at each of these eight positions would
mean preparing a pool of 1.1e12 DNA sequences which by NNS
degeneracy encode 2.6e10 different polypeptides. Obtaining a random
phagemid library large enough (perhaps e13 transformants) to assure
representation of all variants was not feasible in 1991.
[0250] Consequently, a library was constructed by randomly ligating
selected DNA pools from the helix-1 and helix-4b library screens,
and nondegenerate DNA to complete the coding sequence, so as to
create a combined pool. There would be some amount of diversity in
each of the donor pools. The results are shown in Table XIII-A of
WO92/09690. See also Table 7 of WO97/11178.
[0251] Third Alpha Helix Mutants of Growth Hormones which Function
as GH Antagonists
[0252] Mutants of hGH and bGH which function as GH antagonists were
first identified in Kopchick et al. Kopchick et al. discovered that
mutation of Gly119 in bGH to Arg ("G119R"), Pro ("G119P"), Lys
("G119K"), Trp ("G119W") or Leu ("G119L"), or the homologous Gly120
in hGH to Arg or Trp, results in a mutein (mutant protein or
peptide fragment thereof) which has growth-inhibitory activity in
vertebrates, especially mammals.
[0253] Kopchick et al. discovered that the bGH mutants, when
expressed in transgenic mice, resulted in mice with a growth ratio
of between 0.57 and 1.0. The growth ratio of the mice was
negatively correlated with the serum level of the bGH analog, i.e.,
as the serum level of the bGH analog increased, the growth ratio of
the animals decreased. Also, these analogs, when expressed to
NIH-3T3-preadipocytes, did not result in stimulation of
preadipocytes differentiation, whereas native GH will promote this
differentiation. In fact, these analogs will antagonize the ability
of wild type GH to promote preadipocyte differentiation. Kopchick
et al. referred to these analogs as "functional antagonists."
[0254] Kopchick et al. also generated transgenic mice which express
either wild type hGH, hGH G120A, hGH G120R and hGH G120W. Mice
which express hGH G120A show a growth enhanced phenotype similar to
mice which express wild type hGH. In contrast, substitution of R or
W for G at position 120 in hGH, and subsequent expression in
transgenic mice, results in animals with a growth ratio between
0.73 and 0.96, and whose level of serum hGH is negatively
correlated with the growth phenotype; i.e., as the serum levels of
these hGH 120 analogs increase, the growth ratios decrease.
[0255] It has since been shown by Genentech researchers that the
G120R mutant of hGH binds to hGHbp, and that its affinity for
hGHbp(S237C) was Kd=1.6 nM, and for hGHbp (S201C) was Kd=2.7 nM. In
the same experiment, the KD for the binding of wild type hGH to
hGHbp (S201C) was 0.9 nM. It is important to note when hGh and bGH
are aligned according to commonly accepted principles of sequence
alignment, that the glycine residue in bGH at position 119 is
aligned with (i.e., corresponds to) the glycine residue in hGH at
position 120. They are both located in the central portion of the
third alpha helix.
[0256] The preferred growth-inhibitory mutants are characterized by
a modification of the surface topography of the third alpha helix.
In the third alpha helix of "wild-type" bovine growth hormone,
there is a surface cleft or depression beginning, at the
Aspartate-115, deepening at the Glycine-119, and ending with the
Alanine-122. All of the mutants discussed in the references cited
in this section, both those which retain the wild-type
growth-promoting activity and those which do not, are consistent
with the theory that growth-promoting activity requires the
presence of this cleft or depression and that, if the center of
this cleft is "filled in" by substitution of amino acids with
bulkier side chains, the mutein inhibits the growth of the
subject.
[0257] With respect to amino acid 119, glycine is both the smallest
amino acid residue and the one least favorable to alpha-helix
formation. Thus, it is believed that any other amino acid may be
substituted for it without destabilizing the alpha helix, while at
the same time filling in the aforementioned cleft. All of the G119
bGH substitutions tested resulted in a "small animal" phenotype.
These substitutions were arginine (a large, positively charged AA),
proline (a cyclic aliphatic AA), lysine (a large, positively
charged AA), tryptophan (a large aromatic AA) and leucine (a large,
nonpolar, aliphatic AA).
[0258] In hGH, the homologous glycine is at position 120.
Substitution of arginine or tryptophan resulted in an antagonist,
however, hGH G120A retained growth-promoting activity.
Consequently, it is presently believed that if antagonist activity
is desired, this glycine, which is conserved in all vertebrate GHs,
may be replaced by any amino acid other than alanine (the second
smallest amino acid), and more preferably by any amino acid which
is at least as large as proline (the smallest replacement amino
acid known to result in a "small" animal phenotype).
[0259] Modification of position 115 is suggested by Kopchick et
al.'s "cleft" theory. The aspartate at position 115 may be replaced
by a bulkier amino acid, which does not destroy the alpha helix.
Preferably, the replacement amino acid has a size greater than that
of aspartate. The amino acids histidine, methionine, isoleucine,
leucine, lysine, arginine, phenylalanine, tyrosine, and tryptophan
are substantially larger than aspartate. Of these, His, Met, Leu,
and Trp are more preferred because they combine the advantages of
bulk with a reasonably strong alphahelical propensity. Note,
however, that Glu is the strongest alpha-helix former of all of the
amino acids. The D115A mutant of bGH is not a GH antagonist, but
Alanine is smaller than Aspartic Acid, so this is not probative of
the value of replacing Asp115 with a bulkier amino acid.
[0260] It is possible to systematically screen for the effect of
all possible amino acid substitutions at the position corresponding
to bGH 119 alone, or at positions corresponding to bGH 115 and/or
119, too. It is possible that G119A will lead to a "small"
phenotype if coupled with other mutations, e.g., at 115 and 122.
Thus, one could screen a combinatorial library in which all library
members contain the mutation G119A, and positions 115 and 122 are
each varied thorough the 20 possible amino acids.
[0261] This approach may be extended, if desired, to other amino
acid positions in the third alpha helix. Amino acids which are
particularly preferred for screening are the six amino acids
spatially nearest bGH's Gly119, that is, Ala122, Leu123, Ile120,
Leu116, Asp115 and Glu118. Screening for the effects of all
possible mutations of position 119 and these six proximate
positions would require a library with 207 members. If such a
library cannot be prepared one could prepare 19 separate libraries,
each characterized by a particular bGH G119X background mutation,
and randomization of the six proximate positions (for 20.sup.6
different library members per library).
[0262] Besides the mutation at the position corresponding to bGH
119, which is deemed necessary to impart the desired
growth-inhibitory activity, additional mutations are possible which
will leave the growth-inhibitory activity or other antagonist
activity intact. These mutations may take the form of single or
multiple substitutions, deletions, or insertions, in nonessential
regions of the polypeptide. For example, it is possible to alter
another amino acid in the alpha helix if the substitution does not
destroy the alpha helix. Preferably, such alterations replace an
amino acid with one of similar size and polarity. It may be
advantageous to modify amino acids flanking the primary mutation
site 119 in order to increase the alpha-helical propensities of the
sequence, particularly if the mutation at 119 is one expected to
destabilize the helix.
[0263] The GH antagonist activity was manifested, not only in these
single substitution mutants, but in multiple substitution mutants.
The first such studied by Kopchick et al. was the bGH mutant
E117L/G119R/A122D, which inhibited growth in transgenic mice. Mouse
L cell secretion of the mutant protein was observed in the case of
the bGH mutants E117/G119R, E111L/G119W, E111L/G119W/L121R/M124K,
E111L/G119W/R125L, and E111L/G119W/L121R/M124K.
[0264] B2024and B2036 GHA Mutants
[0265] In view of the foregoing mutational analyses, two mutants of
hGH were singled out for special attention. The B2024 mutant is
characterized by the mutations H18A, Q22A, F25A, D26A, Q29A, E65A,
G120K, K168A, and E174A. The B2036 mutant is characterized by the
mutations H18D, H21N, G120K, R167N, K168A, D171S, K172R, E174S, and
I179T. In both cases, the boldfaced mutation imparts antagonist
activity and the other mutations improve "site 1" binding to the
hGH receptor. See WO 97/11178.
[0266] The B036 mutant may be compared with the 852d GH agonist
mutant described previously. The R64K mutation of 852d was omitted
to protect site 1 binding residues from PEGylation. Likewise, the
mutations K168A and K172R were added to B2036 to reduce the number
of site 1 PEGylation sites. Some of the mutations of 852d were
omitted from B2036 because they make only modest enhancements to
affinity, and their omission was considered likely to reduce
antigenicity in humans. The B2024 mutant carries this theme
further, omitting additional mutations. Both B2036 and B2024 could
be converted into agonists by reversing the G120 mutation.
[0267] In a cell-based assay of antagonist activity, non-PEGylated
B2036 had an IC50 of 0.19 ug/ml, while the IC50 for a PEGylated
form (PEG-4/5-B2036) of B2036 was 13.1 ug/ml. Later, it was shown
that another PEGylated form, PEG(20,000)-B2036, had an IC50 of 0.25
ug/ml. See WO97/11178 at p. 135. Both PEGylated and non-PEGylated
forms of B2036 have been shown to reduce IGF-1 levels in rhesus
monkeys. WO97/11178 at p. 136. (See, generally, Ross et al., JCE,
2001, vol 86, pages 1716-1723, for its discussion of PEGylated
growth hormones and their binding.)
[0268] Chemically Modified (Including PEGylated) GH Agonists and
Antagonists
[0269] In order to reduce immunogenicity and/or increase half-life,
a polyol can be conjugated to a GH agonist or antagonist at one or
more amino acid residues, e.g., lysine(s). See WO93/00109. Suitable
polyols include, but are not limited to, those substituted at one
or more hydroxyl positions with a chemical group, such as an alkyl
group having between one and four carbon atoms. Typically, the
polyol is a poly(alkylene)glycol, such as poly(ethylene)glycol
(PEG). The process of conjugating PEG to hGH (or a hGH mutant) is
called PEGylation, but the process is also applicable to
conjugation of other polyols. Preferably, the PEG has a molecular
weight of 500 to 30,000 daltons, with an average molecular weight
of 5,000 D being especially preferred.
[0270] Preferably, the process is such that two to seven, more
preferably four to six, molecules of PEG are conjugated to each
molecule of hGH (or mutant). The final composition may be
homogeneous, i.e., all molecules bear the same number of PEGs at
the same PEGylation sites, or heterogeneous, i.e., the number of
PEGs or the sites of attachment of the PEGs varies from conjugate
to conjugate.
[0271] Preferably, the reaction conditions are such that the
conjugation does not destroy site 1 binding activity. Also, if the
conjugate is to be used as a GH agonist, the conjugation should not
destroy site 2 binding activity. See generally WO97/11178. Note
that the G120K mutation contemplated above provides an additional
PEGylation site.
[0272] Prolactin Mutants
[0273] Based on the data set forth above, Cunningham, et al.,
Science, 247: 1461 (Mar. 11, 1990) designed a human prolactin
octamutant, which bound hGHbp (Kd of 2.1 nM) more than 10,000-fold
more strongly than does wild type human prolactin (Kd>40,000).
This hPRL octamutant bound hGHbp about one-sixth as strongly as
wild type hGH (Kd of 0.34 nM), yet has only 26% overall sequence
identity with hGH. The octamutant was characterized by the
mutations (hGH numbering, Cunningham hGH:hPRL alignment) H171D,
N175T, Y176F, K178R, E174A, E62S, D63N, and Q66E. The additional
mutation L179I did not alter the affinity. WO90/04788 suggests the
possibility of improving the binding further with the mutations
V14M and H185V, see P. 113.
[0274] Mutational Studies Inspired by the Comparison of hGH and
hPL
[0275] Within the three regions (hGH residues 4-14, 54-74, 171-185)
which were identified by Ala scanning mutagenesis as constituting
the hGHr binding epitope of hGH, hPL differs at only seven
positions from hGH, as follows: P2Q, I4V, N12H, R16Q, E56D, R64M,
and I179M, where, e.g., "P2Q" means that the proline at position 2
of hGH is replaced with Q in the corresponding AA of aligned hPL.
All of these seven positions were Ala-scanned in hGH, and four of
the Ala substitutions (14A, E56A, R64A, and I179A) resulted in a
two-fold or greater reduction in binding affinity.
[0276] The hGH single substitution mutant I179M reduced hGH
affinity by just 1.7 fold (as compared to 2.7 fold for I179A). The
R64A and R64M mutations both caused 20-fold reductions in affinity.
The hGH double mutant E56D/R64M evidenced a total reduction in
affinity of 30-fold.
[0277] Placental Lactogen Mutants
[0278] Wild type hPL binds hGHbp (S201 C) with an affinity (KD) of
1800 nM, while wild type hGH binds the same target with an affinity
of 1.4 nM. The mutant hPL (0274), characterized by the mutations
10Y, 14E, 18R, 21G, binds hGHbp (S201C) with an affinity of 1.1 nM,
i.e., superior to that of wild type hGH. See WO97/11178, Table 9 on
p. 101.
[0279] WO90/04788 p. 116 says that the double mutant D56E, M64R in
hPL substantially enhances its binding affinity for the hGH
receptor, and also suggests the additional modifications M179I and
V4I. The G120R variant of hPL inhibits hGH-stimulated growth of
FDC-P1 cells transfected with the hPRL receptor. The IC50 for
G120R-hPL is about 8-fold higher than for G120R-hGH. See Fuh &
Wells, J. Biol. Chem., 270: 13133 (1995).
[0280] Beyond the growth hormone superfamily of proteins, variants
of all of the peptides/polypeptides/proteins mentioned herein are
specifically contemplated. Thus, any of the amino acids at any
position can be modified by deletion/insertion/mutation. These
variations can be made in addition to, or as part of, the
glycosylation motif.
[0281] For Drug Delivery/Emulsification: Small hydrophobic or
amphipathic proteins are tagged with the desired motif to make drug
emulsifiers. Examples include but are not limited to, human serum
albumin, including its individual domains. Of course, hSA can be
made with glycomodules according to the invention, for any purpose
or use, not just for drug delivery/emulsification.
[0282] The following modified proteins are specifically
contemplated: 1) human growth hormone modified at the C- or
N-terminus with (Ser-Hyp).sub.n (SEQ ID NO: 113) where n is from
about 1 to about 20, or about 2 to about 18, or about 4 to about
16, or about 6 to about 14, or about 8 to about 12, or about 10; 2)
human prolactin modified at the C- or N-terminus with
(Ser-Hyp).sub.n (SEQ ID NO: 113) where n is from about 1 to about
20, or about 2 to about 18, or about 4 to about 16, or about 6 to
about 14, or about 8 to about 12, or about 10; 3) human placental
lactogen, modified at the C- or N-terminus with (Ser-Hyp).sub.n
(SEQ ID NO: 113) where n is from about 1 to about 20, or about 2 to
about 18, or about 4 to about 16, or about 6 to about 14, or about
8 to about 12, or about 10; 4) interferon-2-alpha, modified at the
C- or N-terminus with (Ser-Hyp).sub.n (SEQ ID NO: 113) where n is
from about 1 to about 20, or about 2 to about 18, or about 4 to
about 16, or about 6 to about 14, or about 8 to about 12, or about
10; and 5) insulin, modified at the C- or N-terminus with
(Ser-Hyp).sub.n (SEQ ID NO: 113) where n is from about 1 to about
20, or about 2 to about 18, or about 4 to about 16, or about 6 to
about 14, or about 8 to about 12, or about 10.
[0283] In some embodiments, N-terminal "insertions" are at the
N-terminus of the mature or circulatory form of the various
hormones. This placement may be desirable for proteins hormones
that are found in the blood stream, which are generated by way of
an amino terminal secretory peptide that is cleaved during the
secretory process.
[0284] In addition to the specific proteins set forth above,
antibodies, including monoclonal antibodies and humanized
monoclonal antibodies, can also be expressed in accordance with the
present invention. For example, glycosylated antibodies to growth
hormone or to the growth hormone receptor can be made in accordance
with the present invention.
[0285] Expression in Plants
[0286] There combinant genes are expressed in plant cells, such as
cell suspension cultured cells, including but not limited to, BY2
tobacco cells. Expression can also be achieved in a range of intact
plant hosts, and other organisms including but not limited to,
invertebrates, plants, sponges, bacteria, fungi, algae,
archebacteria.
[0287] In some embodiments, the expression
construct/plasmid/recombinant DNA comprises a promoter. It is not
intended that the present invention be limited to a particular
promoter. Any promoter sequence which is capable of directing
expression of an operably linked nucleic acid sequence encoding at
least a portion of nucleic acids of the present invention, is
contemplated to be within the scope of the invention. Promoters
include, but are not limited to, promoter sequences of bacterial,
viral and plant origins. Promoters of bacterial origin include, but
are not limited to, octopine synthase promoter, nopaline synthase
promoter, and other promoters derived from native Ti plasmids.
Viral promoters include, but are not limited to, 35S and 19S RNA
promoters of cauliflower mosaic virus (CaMV), and T-DNA promoters
from Agrobacterium. Plant promoters include, but are not limited
to, ribulose-1,3-bisphosphate carboxylase small subunit promoter,
maize ubiquitin promoters, phaseolin promoter, E8 promoter, and
Tob7 promoter.
[0288] The invention is not limited to the number of promoters used
to control expression of a nucleic acid sequence of interest. Any
number of promoters may be used so long as expression of the
nucleic acid sequence of interest is controlled in a desired
manner. Furthermore, the selection of a promoter may be governed by
the desirability that expression be over the whole plant, or
localized to selected tissues of the plant, e.g., root, leaves,
fruit, etc. For example, promoters active in flowers are known
(Benfy et al. (1990) Plant Cell 2:849-856).
[0289] Transformation of plant cells may be accomplished by a
variety of methods, examples of which are known in the art, and
include for example, particle mediated gene transfer (see, e.g.,
U.S. Pat. No. 5,584,807 hereby incorporated by reference);
infection with an Agrobacterium strain containing the foreign
DNA-for random integration (U.S. Pat. No. 4,940,838 hereby
incorporated by reference) or targeted integration (U.S. Pat. No.
5,501,967 hereby incorporated by reference) of the foreign DNA into
the plant cell genome; electroinjection (Nan et al. (1995) In
"Biotechnology in Agriculture and Forestry," Ed. Y. P. S. Bajaj,
Springer-Verlag Berlin Heidelberg, Vol 34:145-155; Griesbach (1992)
HortScience 27:620); fusion with liposomes, lysosomes, cells,
minicells, or other fusible lipid-surfaced bodies (Fraley et al.
(1982) Proc. Natl. Acad. Sci. USA 79:1859-1863; polyethylene glycol
(Krens et al. (1982) Nature 296:72-74); chemicals that increase
free DNA uptake; transformation using virus, and the like.
[0290] The terms "infecting" and "infection" with a bacterium refer
to co-incubation of a target biological sample, (e.g., cell,
tissue, etc.) with the bacterium under conditions such that nucleic
acid sequences contained within the bacterium are introduced into
one or more cells of the target biological sample.
[0291] The term "Agrobacterium" refers to a soil-borne,
Gram-negative, rod-shaped bacterium, which causes crown gall. The
term "Agrobacterium" includes, but is not limited to, the strains
Agrobacterium tumefaciens, (which typically causes crown gall in
infected plants), and Agrobacterium rhizogenes (which causes hairy
root disease in infected host plants). Infection of a plant cell
with Agrobacterium generally results in the production of opines
(e.g., nopaline, agropine, octopine, etc.) by the infected cell.
Thus, Agrobacterium strains which cause production of nopaline
(e.g., strain LBA4301, C58, A208) are referred to as
"nopaline-type" Agrobacteria; Agrobacterium strains which cause
production of octopine (e.g., strain LBA4404, Ach5, B6) are
referred to as "octopine-type" Agrobacteria; and Agrobacterium
strains which cause production of agropine (e.g., strain EHA105,
EHA101, A281) are referred to as "agropine-type" Agrobacteria.
[0292] The terms "bombarding," "bombardment," and "biolistic
bombardment" refer to the process of accelerating particles towards
a target biological sample (e.g., cell, tissue, etc.) to effect
wounding of the cell membrane of a cell in the target biological
sample and/or entry of the particles into the target biological
sample. Methods for biolistic bombardment are known in the art
(e.g., U.S. Pat. No. 5,584,807, the contents of which are herein
incorporated by reference), and are commercially available (e.g.,
the helium gas-driven microprojectile accelerator (PDS-1000/He)
(BioRad).
[0293] The term "microwounding" when made in reference to plant
tissue refers to the introduction of microscopic wounds in that
tissue. Microwounding may be achieved by, for example, particle, or
biolistic bombardment.
[0294] Plant cells can also be transformed according to the present
invention through chloroplast genetic engineering, a process that
is described in the art. Methods for chloroplast genetic
engineering can be performed as described, for example, in U.S.
Pat. No. 6,680,426, and in published U.S. Application Nos.
2003/0009783, 2003/0204864, 2003/0041353, 2002/0174453,
2002/0162135, the entire contents of each of which is incorporated
herein by reference.
[0295] A variety of host cells are contemplated for use in this
invention, including eukaryotic and prokaryotic cells. It is not
intended that the present invention be limited by the host cells
used for expression of the synthetic genes of the present
invention. Generally, the present invention is contemplated in
plants. As used herein, "plants" encompasses any organism that is
photoautotrophic, which includes blue-green algae. Also
specifically contemplated are green, red, and brown algae.
[0296] Plants that can be used as host cells include vascular and
non-vascular plants. Non-vascular plants include, but are not
limited to, Bryophytes, which further include but are not limited
to, mosses (Bryophyta), liverworts (Hepaticophyta), and hormworts
(Anthocerotophyta). Vascular plants include, but are not limited
to, lower (e.g., spore-dispersing) vascular plants, such as,
Lycophyta (club mosses), including Lycopodiae, Selaginellae, and
Isoetae, horsetails or equisetum (Sphenophyta), whisk ferns
(Psilotophyta), and ferns (Pterophyta).
[0297] Vascular plants include, but are not limited to, i) fossil
seed ferns (Pteridophyta), ii) gymnosperms (seed not protected by a
fruit), such as Cycadophyta (Cycads), Coniferophyta (Conifers, such
as pine, spruce, fir, hemlock, yew), Ginkgophyta (e.g., Ginkgo),
Gnetophyta (e.g., Gnetum, Ephedra, and Welwitschia), and iii)
angiosperms (flowering plants--seed protected by a fruit), which
includes Anthophyta, further comprising dicotyledons (dicots) and
monocotyledons (monocots). Specific plant host cells that can be
used in accordance with the invention include, but are not limited
to, legumes (e.g., soybeans) and solanaceous plants (e.g., tobacco,
tomato, etc.). Other cells contemplated to be within the scope of
this invention are green algae types, Chlamydomonas, Volvox, and
duckweed (Lemna).
[0298] The present invention is not limited by the nature of the
plant cells. All sources of plant tissue are contemplated. In one
embodiment, the plant tissue which is selected as a target for
transformation with vectors which are capable of expressing the
invention's sequences are capable of regenerating a plant. The term
"regeneration" as used herein, means growing a whole plant from a
plant cell, a group of plant cells, a plant part or a plant piece
(e.g., from seed, a protoplast, callus, protocorm-like body, or
tissue part). Such tissues include but are not limited to seeds.
Seeds of flowering plants consist of an embryo, a seed coat, and
stored food. When fully formed, the embryo generally consists of a
hypocotyl-root axis bearing either one or two cotyledons and an
apical meristem at the shoot apex and at the root apex. The
cotyledons of most dicotyledons are fleshy and contain the stored
food of the seed. In other dicotyledons and most monocotyledons,
food is stored in the endosperm and the cotyledons function to
absorb the simpler compounds resulting from the digestion of the
food.
[0299] Species from the following examples of genera of plants may
be regenerated from transformed protoplasts: Fragaria, Lotus,
Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum,
Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus,
Sinapis, Atropa, Capsicum, Hyoscyamus, Lycopersicon, Nicotiana,
Solanum, Petunia, Digitalis, Majorana, Ciohorium, Helianthus,
Lactuca, Bromus, Asparagus, Antirrhinum, Hererocallis, Nemesia,
Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio,
Salpiglossis, Cucumis, Browaalia, Glycine, Lolium, Zea, Triticum,
Sorghum, and Datura.
[0300] For regeneration of transgenic plants from transgenic
protoplasts, a suspension of transformed protoplasts or a petri
plate containing transformed explants is first provided. Callus
tissue is formed and shoots may be induced from callus and
subsequently rooted. Alternatively, somatic embryo formation can be
induced in the callus tissue. These somatic embryos germinate as
natural embryos to form plants. The culture media will generally
contain various amino acids and plant hormones, such as auxin and
cytokinins. It is also advantageous to add glutamic acid and
proline to the medium, especially for such species as corn and
alfalfa. Efficient regeneration will depend on the medium, on the
genotype, and on the history of the culture. These three variables
may be empirically controlled to result in reproducible
regeneration.
[0301] Plants may also be regenerated from cultured cells or
tissues. Dicotyledonous plants which have been shown capable of
regeneration from transformed individual cells to obtain transgenic
whole plants include, for example, apple (Malus pumila), blackberry
(Rubus), Blackberry/raspberry hybrid (Rubus), red raspberry
(Rubus), carrot (Daucus carota), cauliflower (Brassica oleracea),
celery (Apium graveolens), cucumber. (Cucumis sativus), eggplant
(Solanum melongena), lettuce (Lactuca sativa), potato (Solanum
tuberosum), rape (Brassica napus), wild soybean (Glycine
canescens), strawberry (Fragaria x ananassa), tomato (Lycopersicon
esculentum), walnut (Juglans regia), melon (Cucumis melo), grape
(Vitis vinifera), and mango (Mangifera indica). Monocotyledonous
plants which have been shown capable of regeneration from
transformed individual cells to obtain transgenic whole plants
include, for example, rice (Oryza sativa), rye (Secale cereale),
and maize.
[0302] In addition, regeneration of whole plants from cells (not
necessarily transformed) has also been observed in: apricot (Prunus
armeniaca), asparagus (Asparagus officinalis), banana (hybrid
Musa), bean (Phaseolus vulgaris), cherry (hybrid Prunus), grape
(Vitis vinifera), mango (Mangifera indica), melon (Cucumis melo),
ochra (Abelmoschus esculentus), onion (hybrid Allium), orange
(Citrus sinensis), papaya (Carrica papaya), peach (Prunus persica),
plum (Prunus domestica), pear (Pyrus communis), pineapple (Ananas
comosus), watermelon (Citrullus vulgaris), and wheat (Triticum
aestivum).
[0303] The regenerated plants are transferred to standard soil
conditions and cultivated in a conventional manner. After the
expression vector is stably incorporated into regenerated
transgenic plants, it can be transferred to other plants by
vegetative propagation or by sexual crossing. For example, in
vegetatively propagated crops, the mature transgenic plants are
propagated by the taking of cuttings or by tissue culture
techniques to produce multiple identical plants. In seed propagated
crops, the mature transgenic plants are self crossed to produce a
homozygous inbred plant which is capable of passing the transgene
to its progeny by Mendelian inheritance. The inbred plant produces
seed containing the nucleic acid sequence of interest. These seeds
can be grown to produce plants that would produce the desired
polypeptides. The inbred plants can also be used to develop new
hybrids by crossing the inbred plant with another inbred plant to
produce a hybrid.
[0304] It is not intended that the present invention be limited to
only certain types of plants. Both monocotyledons and dicotyledons
are contemplated. Monocotyledons include grasses, lilies, irises,
orchids, cattails, palms, Zea mays (such as corn), rice barley,
wheat and all grasses. Dicotyledons include almost all the familiar
trees and shrubs (other than confers) and many of the herbs
(non-woody plants).
[0305] Tomato cultures are one example of a recipient for
repetitive HRGP modules to be hydroxylated and glycosylated. The
cultures produce cell surface HRGPs in high yields easily eluted
from the cell surface of intact cells and they possess the required
posttranslational enzymes unique to plants--HRGP prolyl
hydroxylases, hydroxyproline O-glycosyltransferases and other
specific glycosyltransferases for building complex polysaccharide
side chains. Other recipients for the invention's sequences
include, but are not limited to, tobacco cultured cells and plants,
e.g., tobacco BY 2 (bright yellow 2).
[0306] In short, the present expression strategy can be used in
plants, such as intact monocots and dicots, gymnosperms, ferns,
bryophytes, cell suspension cultures, and algae, etc., to express
proteins from various organisms, such as humans and other mammals
and/or vertebrates, invertebrates, plants, sponges, bacteria,
fungi, algae, archebacteria, potentially any organism on this
planet.
[0307] Utilities
[0308] Depending on the particular peptide/polypeptide/protein
expressed, a variety of utilities for the product are contemplated.
If the expressed product includes green fluorescent protein, for
example, the product or cells containing the product can be used in
fluorescent screening assays. If the product is biologically
active, for example, the expressed product may be used as a
receptor antagonist or agonist, and may be used in vitro and in
vivo. In vitro utilities include, for example, use in screening
assays. In vivo utilities include, but are not limited to, use of
the compounds for treatment of humans or other animals, based on
the agonist or antagonist activities.
[0309] The term "treatment" as used herein with reference to a
disease is used broadly and is not limited to a method of curing
the disease. The term "treatment" includes any method that serves
to reduce one or more of the pathological effects or symptoms of a
disease or to reduce the rate of progression of one or more of such
pathological effects or symptoms.
[0310] While space limits a description of all of the utilities for
all of the peptides/polypeptides/proteins that can be made in
accordance with this invention, examples will be specifically
described with reference to growth hormone. The administration of
the growth hormone described herein can be used for: treating
growth hormone deficient humans or other animals, including dogs,
cats, pigs, cows, horses; reducing catabolic side effects of
glucocorticoids; treating osteoporosis; stimulating the immune
system; accelerating wound healing; accelerating bone fracture
repair; treating growth retardation; treating congestive heart
failure; treating acute or chronic renal failure or insufficiency;
treating physiological short stature, including growth hormone
deficient children; treating short stature associated with chronic
illness; treating obesity; treating growth retardation associated
with Prader-Willi syndrome and Turner's syndrome; treating
Metabolic syndrome (also known as Syndrome X); accelerating
recovery and reducing hospitalization of burn patients or following
major surgery; treating intrauterine growth retardation, skeletal
dysplasia, hypercortisonism and Cushings syndrome; replacing growth
hormone in stressed patients; treating osteochondrodysplasias,
Noonans syndrome, sleep disorders, Alzheimer's disease, delayed
wound healing, and psychosocial deprivation; treating pulmonary
dysfunction and ventilator dependency; attenuating protein
catabolic response after a major operation; treating malabsorption
syndromes, reducing cachexia and protein loss due to chronic
illness such as cancer or AIDS; accelerating weight gain and
protein accretion in patients on total parenteral nutrition;
treating hyperinsulinemia including nesidioblastosis; adjuvant
treatment for ovulation induction and to prevent and treat gastric
and duodenal ulcers; stimulating thymic development and preventing
age-related decline of thymic function; adjunctive therapy for
patients on chronic hemodialysis; treating immunosuppressed
patients and enhancing antibody response following vaccination;
improving muscle strength, increasing muscle mass, mobility,
maintenance of skin thickness, metabolic homeostasis, renal
homeostasis in the frail elderly; stimulating osteoblasts, bone
remodeling, and cartilage growth; treating neurological diseases
such as peripheral and drug induced neuropathy, Guillian-Barre
Syndrome, amyotrophic lateral sclerosis, multiple sclerosis,
cerebrovascular accidents and demyelinating diseases; and
stimulating wool growth in sheep.
[0311] In farm animals, growth hormone can be used for increasing
meat production in, for example, chickens, turkeys, sheep, pigs,
and cattle; stimulation of pre- and post-natal growth, enhanced
feed efficiency in animals raised for meat production, improved
carcass quality (increased muscle to fat ratio); increased milk
production in dairy cattle or in other mammalian species; improved
body composition; modification of other GH-dependent metabolic and
immunologic functions such as enhancing antibody response following
vaccination or improved developmental processes; and accelerate
growth and improve the protein-to-fat ratio in fish.
[0312] In companion animals, uses of growth hormone includes
stimulating thymic development and preventing age-related decline
of thymic function; preventing age-related decline of thymic
function; preventing age-related decline in cognition; accelerating
wound healing; accelerating bone fracture repair; stimulating
osteoblasts; bone remodeling and cartilage growth; attenuating
protein catabolic response after major surgery, accelerating
recovery from burn injuries and major surgeries such as
gastrointestinal surgery; stimulating the immune system and
enhancing antibody response following vaccination; treating
congestive heart failure, treating acute or chronic renal failure
or insufficiency, treating obesity; treating growth retardation,
skeletal dysplasia and osteochondrodysplasias; preventing catabolic
side effects of glucocorticoids; treating Cushing's syndrome;
treating malabsorption syndromes, reducing cachexia and protein
loss due to chronic illness such as cancer; accelerating weight
gain and protein accretion in animals receiving total parenteral
nutrition; providing adjuvant treatment for ovulation induction and
to prevent gastrointestinal ulcers; improving muscle mass, strength
and mobility; maintenance of skin thickness, and improving vital
organ function and metabolic homeostasis or in promoting growth of
small animals to larger animals.
[0313] With regard to growth hormone antagonists described herein,
diseases that may be treated are characterized by one or more of
the following criteria: elevated levels of growth hormone
production, elevated levels of growth hormone receptor production,
and elevated cellular response of receptors to growth hormone. The
term "elevated" as used herein is used with respect to the normal
levels of growth hormone production, growth hormone receptor
production, or growth hormone-mediated cellular response in a
tissue (or tissues) of a diseased person (or animal) as compared to
level in a normal individual. Diseases that may be treated with
growth hormone antagonists by the methods of the invention include,
but are not limited to, acromegaly, gigantism, cancer, diabetes,
vascular eye diseases (diabetic retinopathy, retinopathy of
prematurity, age-related macular degeneration, retinopathy of
sickle-cell anemia, etc.) as well as nephropathy and
glomerulosclerosis and in critically ill individuals in intensive
care unit of a hospital.
[0314] Cancers that may be treated by the invention include, but
are not limited to, cancers comprising tumor cells that express
growth hormone receptors. Cancers that maybe treated by the methods
of the invention include, but are not limited to: cardiac: sarcoma
(angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma),
myxoma, rhabdomyoma, fibroma, lipoma and teratoma; lung:
bronchogenic carcinoma (squamous cell, undifferentiated small cell,
undifferentiated large cell, adenocarcinoma), alveolar
(bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma,
chondromatous hamartoma, mesothelioma; gastrointestinal: esophagus
(squamous cell carcinoma, adenocarcinoma, leiomyosarcoma,
lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas
(ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma,
carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma,
carcinoid tumors, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma,
neurofibroma, fibroma), large bowel (adenocarcinoma, tubular
adenoma, villous adenoma, hamartoma, leiomyoma); genitourinary
tract: kidney (adenocarcinoma, Wilm's tumor (nephroblastoma),
lymphoma, leukemia), bladder and urethra (squamous cell carcinoma,
transitional cell carcinoma, adenocarcinoma), prostate
(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal
carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial
cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma);
liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma,
hepatoblastom, angiosarcoma, hepatocellular adenoma, hemangioma;
bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant
fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant
lymphoma (reticulum cell sarcoma), multiple myeloma, malignant
giant cell tumor, chordoma, osteochronfroma (osteocartilaginous
exostoses), benign chondroma, chondroblastoma, choridromyxofibroma,
osteoid osteoma and giant cell tumors; nervous system: skull
(osteoma, hemangioma, granuloma, xanthoma, osteitis deformans),
meninges (meningioma, meningiosarcoma, gliomatosis), brain
(astrocytoma, medulloblastoma, glioma, ependymoma, germinoma
[pinealoma], glioblastoma multiforme, oligodendroglioma,
schwannoma, retinoblastoma, congenital tumors), spinal cord
(neurofibroma, meningioma, glioma, sarcoma); gynecological: uterus
(endometrial carcinoma), cervix (cervical carcinoma, pre-tumor
cervical dysplasia), ovaries (ovarian carcinoma [serous
cystadenocarcinoma, mucinous cystadenocarcinoma, endometrioid
tumors, celioblastoma, clear cell carcinoma, unclassified
carcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell
tumors, dysgerminoma, malignant teratoma), vulva (squamous cell
carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma,
melanoma), vagina (clear cell carcinoma, squamous cell carcinoma,
botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes
(carcinoma); hematologic: blood (myeloid leukemia (acute and
chronic), acute lymphoblastic leukemia, chronic lymphocytic
leukemia, myeloproliferative diseases, multiple-myeloma,
myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's
lymphoma [malignant lymphoma]; skin: malignant melanoma, basal cell
carcinoma, squamous cell carcinoma, Kaposi's sarcoma, moles,
dysplastic nevi, lipoma, angioma, dermatofibroma, keloids,
psoriasis; and adrenal glands: neuroblastoma. Specifically
contemplated are uses in breast, colon, and prostate cancers, as
well as leukemias and lymphomas.
[0315] The growth hormone agonist or antagonist may be combined
with compatible, nontoxic pharmaceutical excipients and
administered. In the case of administration to nonhuman animals, it
may be preferable to incorporate the drug into the animal's feed,
possibly in a prepared combination of drug and nutritional material
ready for use by a farmer. Growth hormone or growth hormone
antagonists may be administered orally, rectally, transdermally, by
pulmonary infiltration, insufflation, or parenterally (including
intravenously, subcutaneously and intramuscularly) to humans, in
any suitable pharmaceutical dosage form. Polyethylene glycol
moieties can also be added to growth hormone or growth hormone
antagonists. In the case of treatment of retinopathy, it may be
administered directly onto or into the eye by means of a
conventional ocular pharmaceutical form.
[0316] An effective dosage and treatment protocol may be determined
by conventional means, starting with a low dose in laboratory
animals and then increasing the dosage while monitoring the
effects, and systematically varying the dosage regimen as well.
Generally, a clinical end point for GH action is measuring the
level of serum IGF-1. As GH goes up, so does IGF-1. As GH goes
down, so does IGF-1. So in conditions of GH deficiency, both GH and
IGF-1 are low. When one give recombinant GH to these individuals,
IGF-1 levels will rise. The clinician will attempt to keep IGF-1
level in age adjusted normal ranges. On the other hand, if one has
too much GH, then IGF-1 will be high. When one gives the GH
antagonist, IGF-1 levels will fall. The clinician will try to dose
the patient such that the IGF-1 level will return to normal,
age-adjusted levels. Numerous factors may be taken into
consideration by a clinician when determining an optimal dosage for
a given subject. Primary among these is the amount of growth
hormone normally secreted by the pituitary, which is on the order
of 0.5 mg/day for healthy adult humans. Additional factors include
the size of the patient, the age of the patient, the general
condition of the patient, the particular disease being treated, the
severity of the disease, the presence of other drugs in the
patient, the in vivo activity of the agonist or antagonist, and the
like. The trial dosages would be chosen after consideration of the
results of animal studies and the clinical literature with respect
to administration of growth hormones, and/or of somatostatin (a
growth hormone release inhibitor). It will be appreciated by the
person of ordinary skill in the art that information such as
binding constants and Ki derived from in vitro growth hormone
binding competition assays may also be used in calculating
dosages.
[0317] A typical human dose of a growth hormone antagonist would be
from about 0.1 mg/day to about 10 mg/day, or from about 0.5 mg/day
to about 2 mg/day, or about 1 mg/day. A typical human dose of a
growth hormone agonist would be from about 10 mg/day to about 80
mg/day, or from about 20 mg/day to about 40 mg/day, or about 30
mg/day. As noted above, the appropriate dose can be determined
empirically, by monitoring the IGF-1 level. For example, one gives
enough GH antagonist to return IGF-1 levels to normal.
[0318] It should be noted that the glycosylation of proteins
according to the invention can increase the molecular weight
significantly. Growth hormone (22 kDa) modified with
(Ser-Hyp).sub.10 (SEQ ID NO: 4), for example, exhibits a molecular
weight of over 45 kDa. Thus, the molecular weight can more than
double--yet activity remain the same. This should be taken into
account when determining dose and dose equivalence should be
considered on a molar basis.
[0319] The invention also provides pharmaceutical formulations for
use in the subject methods of treating disease. The formulations
can comprise at least one biologically active protein, such as, for
example, growth hormone agonist or antagonist, and can include a
pharmaceutically acceptable carrier. A variety of aqueous carriers
may be used, e.g., water, buffered water, 0.4% saline, 0.3%
glycine, and the like. The pharmaceutical formulations may also
comprise additional components that serve to-extend the shelf-life
of pharmaceutical formulations, including preservatives, protein
stabilizers, and the like. The formulations are preferably sterile
and free of particulate matter (for injectable forms). These
compositions may be sterilized by conventional, well-known
sterilization techniques. The compositions may contain
pharmaceutically acceptable auxiliary substances as required to
approximate physiological conditions such as pH adjusting and
buffering agents, toxicity adjusting agents and the like, e.g.,
sodium acetate, sodium chloride, potassium chloride, calcium
chloride, sodium lactate, etc. The formulations of the invention
may be adapted for various forms of administration, including
intramuscularly, subcutaneously, intravenously, intraocularly, and
the like. The subject formulations may also be formulated so as to
provide for the sustained release of growth hormone agonist or
antagonist. Additional details for methods for preparing
parenterally administrable compositions and adjustments necessary
for administration to subjects are described in more detail in, for
example, Remington's Pharmaceutical Science, which is incorporated
herein by reference.
[0320] Other utilities will be readily apparent to those of skill
in the art from reading this description.
EXAMPLES
Example 1
Expression of Gum Arabic Glycoprotein Analogs with Transgenic
Tobacco Cells
[0321] Gum arabic glycoprotein (GAGP), an arabinoglactan protein
(AGP), is the surface-active component accounting for gum arabic's
emulsification properties. This functional GAGP is a typical HRGP
that consists of four main carbohydrate moieties including
galactose, arabinose, rhanmose and glucuronic acid, and a small
proportion (10%, w/w) of Hyp-rich protein as an integral part of
the structure (Islam A. M., Phillips G. O., Sljivo A., Snowden M.
J. and William P. A. (1997), Food Hydrocolloids 11(4):493-505.).
The GAGP has already been isolated and well characterized. However,
the gene encoding GAGP has not yet been cloned so far, nor has been
the elucidation of the precise mechanism by which GAGP exhibits
emulsifying ability and unique properties. Recently, the dominant
amino acid sequence of GAGP polypeptide backbone was derived. It
contains a repetitive 19-residue consensus motif
SOOO(O/T/S)LSOSOTOTOO(O/L)GPH (SEQ ID NO: 114) (O: hydroxyproline)
(Goodrum L. J., Patel A., Leykam J. F. and Kieliszewski M. J.
(2000), Phytochem 54(1): 99-106). This provides the possibility to
express GAGP analogs in transgenic plant cells by use of the
synthetic gene technology. The genes encoding seven GAGP analogs
were designed and constructed. They include three types: a)
[Gum].sub.3, [Gum].sub.8 and [Gum].sub.20 are the genes that
encoded three, eight, and twenty repeats of GAGP consensus motif,
respectively; b) [HP].sub.4 and [HP].sub.8, which are the genes
encoding four and eight repeats of the GAGP hydrophobic peptide
[HP] that was also derived from the GAGP backbone polypeptide; and
c) [Gum].sub.8[HP].sub.2 and [Gum].sub.8[HP].sub.4 are those of the
combination of [Gum].sub.8 with two and four repeats of [HP]. These
synthetic analogs were expressed as fusion proteins with enhanced
green fluorescence protein (EGFP) in tobacco cells.
[0322] Materials and Methods
[0323] Gene Construction
[0324] All the gene cassettes constructed to express the GAGP
analogs have a "SS.sup.tob-[Synthetic gene]-EGFP" structure, in
which the synthetic gene encoding various GAGP analogs was inserted
between SS.sup.tob, which encodes the extensin signal sequence from
tobacco (De Loose, M., Gheysen, G., Tire, C., Gielen, J.,
Villarroel, R., Genetello, C., Van Montagu, M., Depicker, A. and
Inze, D. (1991), Gene, 99: 95-100), and the gene for EGFP.
[0325] 1) [Gum].sub.3, [Gum].sub.8 and [Gum].sub.20 Gene
Synthesis
[0326] The [Gum].sub.3 gene encoding three repeats of
SPSPTPTAPPGPHSPPPTL (SEQ ID NO: 115) was constructed by
head-to-tail polymerization of three sets of partially overlapping,
complementary oligonucleotide pairs including 5'-linker, internal
GAGP repeat and 3'-linker as described by Shpak et al (Shpak, E.,
Leykam, J. F., and Kieliszewski, M. J. (1999), Proceedings of the
National Academy of Sciences (USA), 96: 14736-14741).
[0327] The [Gum].sub.8 and [Gum].sub.20 were designed to encode 4
and 10 repeats of GPHSPPPPLSPSPTPSPPL-GPHSPPPTLSPSPTPTPPP (SEQ ID
NO: 116), which was designated [Gum].sub.2. It has slight
differences in alternating repeats, thus more closely resembles the
native GAGP. The [Gum].sub.2 gene was synthesized by primer
extension of two mutually priming oligonucleotides (FIG. 1a)
(Integrated DNA Technologies, Inc. Coralville, Iowa). The duplex
was placed into pUC18 plasmid as a HindIII/EcoRI fragment. The
construction of four and ten repeats of the synthetic gene involved
annealing compatible but non-regenerable restriction sites (XmaI
and BsrFI) of [Gum].sub.2 fragment to generate double number of
repeats (Lewis R. V., Hinman M., Kothakota S. and Fournier M.
(1996), Protein Expression Purif 7:400-406). By reiteration, such a
gene fragment could be geometrically multiplied to four and ten
repeats in length.
[0328] 2) [HP].sub.2, [HP].sub.4 and [HP].sub.8 Gene Synthesis
[0329] The [HP].sub.2, [HP].sub.4 and [HP].sub.8 genes were
designed to encode two, four and eight repeats of
TPLPTLTPLPAPTPPLLPH (SEQ ID NO: 117), as designated [HP].sub.1.
[HP].sub.1 was also synthesized by primer extension of two mutually
priming oligonucleotides (FIG. 1b) as above. The duplex was placed
into pUC18 plasmid as a HindIII/EcoRI fragment. The construction of
two ([HP].sub.2), four ([HP].sub.4) and eight ([HP].sub.8) repeats
of the synthetic gene involved annealing compatible but
non-regenerable restriction sites (BspEI and XmaI) of [HP].sub.1
fragment as described above.
[0330] 3) pUC-SS.sup.tob-[Gum].sub.n-EGFP (n=3, 8, 10) Plasmid
Construction
[0331] The plasmid pUC-SS.sup.tob-[Gum].sub.3-EGFP was constructed
according to Shpak et al. (Shpak, E., Leykam, J. F., and
Kieliszewski, M. J. (1999), Proceedings of the National Academy of
Sciences (USA), 96: 14736-14741) (FIG. 2a). The polymerized
[Gum].sub.8 and [Gum].sub.20 gene were subcloned into
pUC-SS.sup.tob-EGFP (Shpak, E., Leykam, J. F., and Kieliszewski, M.
J. (1999), Proceedings of the National Academy of Sciences (USA),
96: 14736-14741) as a BspEI/AgeI fragment between SS.sup.tob and
EGFP gene to generate the plasmid designated
pUC-SS.sup.tob-[Gum].sub.8-EGFP and
pUC-SS.sup.tob-[Gum].sub.20-EGFP (FIG. 2b).
[0332] 4) pUC-SS.sup.tob-[HP].sub.n-EGFP (n=4, 8) Plasmid
Construction
[0333] The polymerized [HP].sub.4 and [HP].sub.8 genes were
subcloned into pUC-SS.sup.tob-EGFP (Shpak, E., Leykam, J. F., and
Kieliszewski, M. J. (1999), Proceedings of the National Academy of
Sciences (USA), 96: 14736-14741) as a AgeI/NcoI fragment between
SS.sup.tob and EGFP gene to generate the plasmid designated
pUC-SS.sup.tob-[HP].sub.4-GFP and pUC-SS.sup.tob-[HP].sub.8-EGFP
(FIG. 3).
[0334] 5) pUC-SS.sup.tob[Gum].sub.8[HP].sub.n-EGFP (n=2, 4) Plasmid
Construction
[0335] The polymerized [HP].sub.2 and [HP].sub.4 gene were
sub-cloned into pUC-SS.sup.tob-[Gum].sub.8-EGFP as a AgeI/NcoI
fragment between [Gum].sub.8 and EGFP gene to generate the plasmid
designated pUC-SS.sup.tob-[Gum].sub.8 [HP].sub.2-EGFP and
pUC-SS.sup.tob-[Gum].sub.8 [HP].sub.4-EGFP (FIG. 4).
[0336] The DNA sequencing of all the genes constructed above was
performed in Department of Environmental and Plant Biology, Ohio
University.
[0337] Plant Transformation Vector Construction
[0338] The entire "SS.sup.tob-[Synthetic gene]-EGFP" construct was
then sub-cloned into plant vector pBI121 (Clontech, CA) as a
BamHI/SacI fragment in place of the .beta.-glucuronidase reporter
gene to generate plasmids pBI-SS.sup.tob-[Synthetic gene]-EGFP. The
expression of these synthetic genes was under the control of the
35S cauliflower mosaic virus (CaMV) promoter.
[0339] Plant Cell Transformation and Selection
[0340] Plasmid pBI121-SS.sup.tob-[Synthetic gene]-EGFP was
introduced into Agrobacterrium tumefaciens strain LBA4404 by the
freeze-thaw method (Holsters et al., 1978), then
suspension-cultured tobacco cells (Nicotiana tabacum, BY2) were
transformed with the Agrobacterium as described earlier (An, G.
(1985), Plant Physiol, 79:568-570) and selected on solid Schenk
& Hildebrandt (SH) medium (Schenk and Hildebrandt, 1972)
containing 0.4 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 200
mg/L kanamycin (Sigma) and 400 mg/L timentin (SmithKline Beecham,
Pa.). At least ten cell lines of each construct were chosen and
transferred into liquid SH medium comprised of the same components
as above, except excluding timentin. After 10 days of culture at
room temperature on an Innova gyrotary shaker (New Brunswick
Scientific, Edison, N.J.) rotating at 90 rpm, the culture medium of
each cell line was screened for target protein expression by
determining the green fluorescence intensity. The cell lines
producing the highest green fluorescence intensity of each
construct were selected for subcultures.
[0341] Isolation of GAGP Analog-EGFP Fusion Glycoprotein from
Medium
[0342] The culture medium, harvested after 12-14 days of culture,
was concentrated about 10-fold by rotorevaporation under 30.degree.
C. An aliquot of 100-200 ml of medium containing 2 M sodium
chloride was loaded onto a hydrophobic-interaction chromatography
(HIC) column (Phenyl-Sepharose 6 Fast Flow, 16.times.700 mm,
Amersharn Pharmacia Biotech, Piscataway, N.J.) equilibrated in 2 M
sodium chloride, and eluted with step-wise sodium chloride gradient
from 2M, 1M to distilled water. The green fluorescent fraction
eluted in distilled water was pooled, concentrated by
freeze-drying, and then fractionated with a Superose-12 gel
permeation chromatography (GPC) column (16.times.700 mm, Amersham
Pharmacia Biotech) equilibrated in 200 mM sodium phosphate buffer
(pH 7). The fluorescent fraction collected from the GPC column was
further purified with HPLC by injecting into a Hamilton PRP-1
semi-preparative column (10 .mu.m, 7.times.305 mm, Hamilton Co.,
Reno, Nev.) equilibrated with starting buffer A (0.1%
trifluoroacetic acid). Proteins were eluted with buffer B (0.1%
trifluoroacetic acid+80% acetonitrile, v/v) with a linear gradient
of 0-70% B in 100 min at a flow rate of 1.0 ml/min.
[0343] Removal of EGFP from Fusion Glycoprotein by Tryptic
Digestion
[0344] About 100 mg of fusion glycoprotein was heat-denatured in
boiling water for 2 min, cooled, then combined with an equal volume
of freshly prepared 2% (w/v) ammonium bicarbonate containing 10 mM
calcium chloride and 100 .mu.g trypsin. After overnight incubation
at room temperature, the sample was fractionated with Superose-12
GPC column and further purified with HPLC using the same method as
described above.
[0345] Emulsification Properties Characterization
[0346] Emulsion assays were carried out according to the method of
Pearce and Kinsella (Pearce K. N. and Kinsella J. E. (1978), J
Agric Food Chem 26(3):716-723) with some modifications. An emulsion
was prepared by sonicating 0.4 mL of orange oil and 0.6 mL of 0.5%
(w/v) protein solution (in 0.05M phosphate buffer, pH 6.5) in a
glass tube with a Sonic Dismembrator (Fisher Scientific) equipped
with a Microtip.RTM. probe. The amplitude was set at 4 and the
oil/water mixture was treated for 60 s and kept on ice the whole
time. A 100-.mu.l aliquot of the emulsion thus obtained was then
diluted serially with 0.1% SDS (sodium dodecyl sulfate) solution to
give a final dilution of 1/1500. The optical density of the 1/1500
dilution was then determined at 500 nm, which was defined as
emulsifying ability (EA). The remaining emulsion was stored
vertically in the glass tube for 2 hr at room temperature, and then
the optical density of the 1/1500 dilution was measured again. The
percentage optical density remaining after 2 hr of storage is
defined as emulsifying stability (ES).
[0347] Results
[0348] All of the GAGP analogs expressed by tobacco cells exhibited
lower emulsifying ability than the native GAGP. The order of
emulsifying ability of these GAGP analogs was
[HP].sub.8>[HP].sub.4>[Gum].sub.8[HP].sub.4>[Gum].sub.8[HP].sub.-
2>[Gum].sub.20>[Gum].sub.8>[Gum].sub.3. However, as shown
in Table 1, when the EGFP was attached to these synthetic GAGP
analogs, all the fusion proteins exhibited better emulsifying
ability than native GAGP. TABLE-US-00002 TABLE 1 The emulsification
properties of the recombinant GAGP Analogs Emulsifying ability
Emulsifying stability Constructs (EA) (ES) [Gum].sub.3 0.035 0
[Gum].sub.8 0.055 0 [Gum].sub.20 0.145 7.5% [HP].sub.4 0.523 44.2%
[HP].sub.8 0.589 53.1% [Gum].sub.8[HP].sub.2 0.181 18.2%
[Gum].sub.8[HP].sub.4 0.356 64.5% [Gum].sub.3-EGFP 1.223 94.5%
[Gum].sub.8-EGFP 1.034 91.7% [Gum].sub.20-EGFP 0.968 93.4%
[HP].sub.4-EGFP 1.445 81.2% [HP].sub.8-EGFP 1.334 83.4%
[Gum].sub.8[HP].sub.2-EGFP 0.954 90.8% [Gum].sub.8[HP].sub.4-EGFP
0.938 91.5% Control GAGP 0.784 93.7% EGFP 0.156 17.9%
Example 2
Increased Yield by Glycosylation
[0349] Some transgenic proteins expressed in plant cells generally
give very low yields, thus their expression in plant systems is
expensive, inefficient, and impractical. The present invention
includes new ways to increase the yields of transgenic proteins
produced in plant cells by producing the transgenic proteins as
fusion glycoproteins possessing at least one hydroxyproline-rich
glycoprotein (HRGP) glycomodule. This example employs some of the
techniques described in Example 1 above to create novel proteins
with glycomodules. By including these glycomodules, the yield of
protein expressed into the medium is increased.
[0350] Briefly, there are two general types of glycomodules: 1)
arabinogalactan glycomodules comprising clustered non-contiguous
hydroxyproline (Hyp) residues in which the Hyp residues are
O-glycosylated with arabinogalactan adducts (for example,
Xaa-Hyp-Xaa-Hyp-Xaa-Hyp repeats where Xaa is Ser or Ala, but can be
other amino acids like Thr or Val (or Lys or Gly). For example
[Ser-Hyp].sub.n or [Ala-Hyp].sub.n); and 2) arabinosylation
glycomodules comprising contiguous Hyp residues in which some or
all of the Hyp residues are arabinosylated with chains of
arabinooligosaccharides from about 1-5 residues long (for example,
Xaa-Hyp-Hyp-Hyp-Hyp.sub.n (SEQ ID NO: 118) modules, where Xaa can
be Ser or Ala or other amino acids, e.g.,
[Ser-Hyp-Hyp-Hyp-Hyp].sub.n (SEQ ID NO: 119) or
[Ser-Hyp-Hyp].sub.n).
[0351] Tailoring the Genes for Expression:
[0352] The transgenes can include a signal sequence for secretion
through the endomembrane system. For example, tobacco extensin
signal sequence: MASLFATFLVVLSLSLAQTTRSA (SEQ ID NO: 120) (Shpak,
E., Leykam, J. F., and Kieliszewski, M. J. (1999), Proceedings of
the National Academy of Sciences (USA), 96:14736-14741); Tomato
LeAGP-1 signal sequence: MDRKFVFLVSILCIVVASVTG (SEQ ID NO: 121) (Li
& Showalter, Li and Showalter, Plant Mol Biol. (1996) November;
32(4):641-52; Zhao Z D, Tan L, Showalter A M, Lamport D T,
Kieliszewski M J., Plant J. 2002 August; 31(4):431-44).
[0353] 1) Gene Construction
[0354] For these examples, the gene cassettes were constructed to
have following structures: ##STR1##
[0355] FIGS. 5, 6, 7, 8, and 9 show, respectively, schematics for
the construction of gene cassettes for hGH-(SP).sub.10-EGFP
((SP).sub.10 disclosed as SEQ ID NO: 51), hGH-(SP).sub.10
((SP).sub.10 disclosed as SEQ ID NO: 51), INF-(SP).sub.10
((SP).sub.10 disclosed as SEQ ID NO: 51), HAS (human serum
albumin)-(SP).sub.10 ((SP).sub.10 disclosed as SEQ ID NO: 51), and
DomainI (domain I of HSA-(SP).sub.10 ((SP).sub.10 disclosed as SEQ
ID NO: 51). FIG. 10A shows the genetic construct for the expression
of hGH-(SP).sub.10 ((SP).sub.10 disclosed as SEQ ID NO: 51); FIG.
10B shows how the construct was created by primer extension. FIGS.
11, 12 (A and B), 13, and 14, show, respectively, the genetic
constructs for the expression of hGH-(SP).sub.10-EGFP ((SP).sub.10
disclosed as SEQ ID NO: 51), HSA-(SP).sub.10 DomainI (of
HSA)-(SP).sub.10 ((SP).sub.10 disclosed as SEQ ID NO: 51), and
INF2a (interferon 2.alpha.)-(SP).sub.10 ((SP).sub.10 disclosed as
SEQ ID NO: 51).
[0356] Summary of Results
[0357] EGFP was expressed with an N-terminal signal sequence that
targeted EGFP for secretion. However, even with the signal sequence
attached, the average amounts secreted into the medium were so low
that they could not be quantified accurately.
[0358] In contrast, when EGFP was expressed as HRGP fusion proteins
of various types the yields increased dramatically. Tables 2 and 3
below give examples of different types of plants, proteins, and
constructs that gave increased yield. TABLE-US-00003 TABLE 2 Yield
examples of purified HRGP-EGFP fusion glycoproteins expressed in
tobacco BY2 cells (those also expressed in tomato or Arabidopsis
are noted) Purified Fusion Glycoprotein mg purified/L medium
collected ARABINOGALACTAN GLYCOMODULE ADDED (Ser-Hyp).sub.32-EGFP
(SEQ ID NO:122) 23 Shpak et al. (1999) (Ala-Hyp).sub.51-EGFP (SEQ
ID NO:123) 30 Tan et al. (2003) (Thr-Hyp).sub.99-EGFP (SEQ ID
NO:124) 10 Tan et al. (2003) (Val-Hyp).sub.10-EGFP (SEQ ID NO:125)
6 Tan et al. (2003) ARABINOSYLATION GLYCOMODULE ADDED
(Ser-Hyp-Hyp).sub.24-EGFP (SEQ ID NO:126) 10 Shpak et al. (2001)
(Ser-Hyp-Hyp-Hyp).sub.15-EGFP (SEQ ID NO:127) 36 Shpak et al.
(2001) (Ser-Hyp-Hyp-Hyp-Hyp).sub.18-EGFP (SEQ ID NO:128) 23 Shpak
et al. (2001) (YK).sub.20-EGFP.sup.a 3-27 Held et al 2004 Journal
of Biological Chemistry Vol 279: 55474-55482 (YK).sub.8-EGFP.sup.a
4-7 Held et al (YL).sub.8-EGFP.sup.a 6-23 Held et al (2004)
(FK).sub.9-EGFP.sup.a 0-3.3 Held et al, (2004) BOTH TYPES OF
GLYCOMODULE ADDED (Ala-Hyp).sub.4-(YK).sub.20-EGFP (SEQ ID NO:129)
111 unpublished (GAGP).sub.3-EGFP 8 Shpak et al (1999)
(Ala-Ala-Ser-Ser-Hyp-Hyp-Leu).sub.6-EGFP (SEQ ID NO:130) and
(Ala-Ala-Gly-Thr-Thr-Hyp-Hyp).sub.6-EGFP (SEQ ID NO:131) >50
unpublished (tobacco and tomato) EGFP-LeAGP-1.DELTA.GPI >50
unpublished (tobacco and Arabidopsis) .sup.a(YK).sub.20 and
(YK).sub.8 designate the sequences:
(Ser-Hyp.sub.4-Ser-Hyp-Ser-Hyp.sub.4-Tyr-Tyr-Tyr-Lys).sub.20(SEQ ID
NO:132) and
(Ser-Hyp.sub.4-Ser-Hyp-Ser-Hyp.sub.4-Tyr-Tyr-Tyr-Lys).sub.8 (SEQ ID
NO:133) respectively; (YL).sub.8 designates
(Ser-Hyp.sub.4-Ser-Hyp-Ser-Hyp.sub.4-Tyr-Tyr-Tyr-Leu).sub.8 (SEQ ID
NO:134); (FK).sub.8 designates
(Ser-Hyp.sub.4-Ser-Hyp-Ser-Hyp.sub.4-Phe-Phe-Phe-Lys).sub.8 (SEQ ID
NO:135).
[0359] TABLE-US-00004 TABLE 3 Yield of non-plant proteins expressed
as a secreted protein in Nicotiana tabacum suspension cultured
cells ((Ser-Hyp).sub.10 disclosed as SEQ ID NO: 4) HGH fusion
protein mg/L medium hGH-EGFP None detected unpublished
hGH-(Ser-Hyp).sub.10-EGFP 16-24 unpublished hGH None detected
unpublished hGH-(Ser-Hyp).sub.10 20-32 mg unpublished INF.alpha.2
None detected unpublished INF.alpha.2-(Ser-Hyp).sub.10 +
unpublished HSA + unpublished HSA-(Ser-Hyp).sub.10 + unpublished
HSADomI unpublished HSADomI-(Ser-Hyp).sub.10 + unpublished
[0360] Detailed Breakdown of Results
[0361] The results summarized above are taken from a number of
different studies, with different constructs and different proteins
expressed, and were selected as being representative of each
particular study. The following section breaks down the process of
expression, observed at various stages, focusing on the expression
of a) an hGH construct without a glycosylation module, and b) an
hGH construct having a glycosylation module. In some instances, the
expression of hGH-(SO).sub.10 ((SO).sub.10 disclosed as SEQ ID NO:
4) was compared to hGH-(SO).sub.10 -EGFP ((SO).sub.10 disclosed as
SEQ ID NO: 4), to observe how different peptide elements were
expressed.
[0362] FIG. 15 shows detection of hGH equivalents secreted into the
medium of tobacco cells transformed with hGH-(SO).sub.10
((SO).sub.10 disclosed as SEQ ID NO: 4) and hGH. Frame (A) shows a
dot blot assay of hGH equivalents occurring in one .mu.L of medium
from 10 cell lines transformed with either hGH-(SO).sub.10
((SO).sub.10 disclosed as SEQ ID NO: 4) (upper) or hGH (lower)
Frame (B) shows sandwich ELISA quantitation of the hGH equivalents
in the medium from the same two sets of ten cell lines. These
results demonstrate that attachment of a glycosylation module
significantly increases the secretion of expressed protein into the
medium.
[0363] FIG. 16 shows the time course of cell growth and hGH
equivalents in BY-2 tobacco cells transformed with hGH-(SO).sub.10
((SO).sub.10 disclosed as SEQ ID NO: 4). The tobacco cells were
grown in 250-mL Erlenmeyer flasks containing 100 mL medium. Three
flasks were withdrawn at 2-day intervals to measure the cell dry
weight and hGH equivalents in the medium. The cultured cells were
harvested by filtration on a sintered funnel, and the filtrate
(culture medium) collected for hGH assays; the cells were washed
three times with distilled water, then lyophilized for three days
before dry weight measurements. The hGH equivalents were measured
via sandwich ELISA assays.
[0364] The medium from transformed cells was harvested after 8-10
days of culture by filtration on a coarse sintered funnel and
supplemented with sodium chloride to a final concentration of 2 M.
Insoluble material was pelleted by centrifugation at 25,000.times.G
for 20 min at 4 C. The supernatant was fractionated by
hydrophobic-interaction chromatography (HIC) on a Phenyl-Sepharose
6 column (Phenyl-Sepharose 6 Fast Flow, 16 by 700 mm, Amersham
Pharmacia Biotech) equilibrated in 2 M sodium chloride. After the
medium was completely loaded onto the HIC column, the proteins were
eluted step-wise first with Tris buffer (25 mM, pH 8.5)/2M sodium
chloride, followed by Tris buffer (25 mM, pH 8.5)/0.8M sodium
chloride, and then the Tris buffer (25 mM, pH 8.5)/0.2N sodium
chloride. The flow rate was 1.0 ml/min, and the fractions were
monitored at 220 nm with a UV detector. Each eluted fraction was
assayed for the presence of hGH by dot blots and ELISA assays. The
Tris buffer (25 mM, pH 8.5)/0.2N NaCl fraction containing most of
the hGH-(SO).sub.10 fusion glycoprotein ((SO).sub.10 disclosed as
SEQ ID NO: 4 was concentrated by ultrafiltration at 4.degree. C.,
and either used for hGH binding and activity assays, or further
purification by reversed phase chromatography.
[0365] FIG. 18 shows the isolation of hGH-(SO).sub.10 ((SO).sub.10
disclosed as SEQ ID NO: 4, (A) and hGH-(SO).sub.10-EGFP
((SO).sub.10 disclosed as SEQ ID NO: 4) (B) by reversed-phase
chromatography on a Hamilton polymeric reversed phase-1 (PRP-1)
column equilibrated with buffer A (0.1% trifluoroacetic acid).
Proteins were eluted with buffer B (0.1% trifluoroacetic acid, 80%
acetonitrile, v/v) using a two step linear gradient of 0-30% B in
15 min, followed by 30%-70% B in 90 min at a flow rate of 0.5
ml/min. Absorbance was measured at 220 nm. The fusion protein
hGH-(SO).sub.10-EGFP ((SO).sub.10 disclosed as SEQ ID NO: 4) was
first fractionated by gel permeation chromatography on a
Superose-12 column before injection onto the PRP-1.
[0366] FIG. 17 shows Western blot detection of hGH-(SO).sub.10
((SO).sub.10 disclosed as SEQ ID NO: 4) (Left hand panel) and
hGH-(SO).sub.10-EGFP ((SO).sub.10 disclosed as SEQ ID NO: 4 (Right
hand panel) using anti-hGH antibodies. The gels were run after
fractionation of the culture medium using hydrophobic interaction
chromatography. Samples (10 .mu.g protein) were run on a 4-15%
SDS-PAGE, then transferred to a NitroBind membrane. Rabbit
polyclonal anti-hGH antibody diluted at 1:500 in TTBS buffer (100
mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.1% Tween 20) and alkaline
phosphatase-conjugated goat anti-rabbit IgG diluted at 1:1000 in
TTBS buffer were used as primary and secondary antibodies,
respectively. Lanes 1: molecular marker; Lanes 2, 3, 4:
hGH-(SO).sub.10 ((SO).sub.10 disclosed as SEQ ID NO: 4) (A) or
hGH-(SO).sub.10-EGFP ((SO).sub.10 disclosed as SEQ ID NO: 4) (B)
culture medium; Lanes 5: hGH standard (2 .mu.g).
[0367] The fuzzy bands at 50-75 kDa (A) or 75 to 100 kDa is typical
for arabinogalactan-proteins, which includes hGH-(SO).sub.10 and
hGH-(SO).sub.10-EGFP ((SO).sub.10 disclosed as SEQ ID NO: 4).
Enough O-Hyp arabinogalactans were added to bring the molecular
mass to .about.50 kDa. Carbohydrate not only creates sites of
microheterogeneity, but also interferes with SDS binding, which
produces the fuzziness seen in the gel. The band at >150 kDa in
(A) may be a contaminant. The band at .about.22 kDa in (A) is
probably hGH released from the hGH-(SO).sub.10 ((SO).sub.10
disclosed as SEQ ID NO: 4) fusion protein either during the
isolation process or on heat treatment in the pH 8 loading buffer.
We have observed that SOSO-rich constructs (SEQ ID NO: 136) (O=Hyp)
are somewhat labile when heated in base (pH 8) perhaps due to an
N-<O acyl shift, which is an issue around Ser residues. Rather
than heating the constructs before SDS PAGE, the proteins can be
incubated at room temperature for several hours in the loading
buffer (no heat), which appears to solve the problem. The band at
.about.25 kDa in (B) could be EGFP, hGH with some SO and glycan
attached, or some contamninant.
[0368] The presence of an EGFP element did not significantly change
the glycosylation profile of the expressed protein. As shown in
Table 4 below, galactose and arabinose comprised the major
monosaccharides in hGH(SO).sub.10 or hGH-(SO).sub.10-EGFP
((SO).sub.10 disclosed as SEQ ID NO: 4), with lesser amounts of
rhamnose and uronic acid. The sugar accounted for 55.5% of the dry
weight of hGH(SO).sub.10 ((SO).sub.10 disclosed as SEQ ID NO: 4),
and 46.5% of the dry weight of hGH-(SO).sub.10-EGFP fusion
glycoproteins ((SO).sub.10 disclosed as SEQ ID NO: 4).
TABLE-US-00005 TABLE 4 Glycosyl composition of hGH-(SO).sub.10 and
hGH- (SO).sub.10-EGFP ((SO).sub.10 disclosed as SEQ ID NO: 4)
Glycosyl hGH-(SO).sub.10 hGH-(SO).sub.10-EGFP residue Mol % (weight
% Mol % weight % Rha 7 3.9 8 3.7 Ara 32 15.2 28 11.0 Gal 43 25.1 49
24.2 GlcUA 18 11.3 14 7.6 Total 100 55.5 100 46.5
[0369] Table 5 shows the glycosylation profile of INF-(SO).sub.10
((SO).sub.10 disclosed as SEQ ID NO: 4), which was similar to that
of hGH-(SO).sub.10 ((SO).sub.10 disclosed as SEQ ID NO: 4).
TABLE-US-00006 TABLE 5 Glycosyl composition of INF-(SO).sub.10
((SO).sub.10 disclosed as SEQ ID NO: 4) INF-(SO).sub.10 Molar
percentage Weight percentage Glycosyl residue.sup.a (mol %) (wt %)
Rha 9 4.6 Ara 30 17.6 Gal 45 29.3 Uronic acids 16 12.3 Total 100
63.8
[0370] As predicted by the Hyp contiguity hypothesis (Shpak, E.,
Leykam, J. F., and Kieliszewski, M. J. (1999), Proceedings of the
National Academy of Sciences (USA), 96: 14736-14741; Shpak, E.,
Barbar, E., Leykam, J. F. & Kieliszewski, M. J. J. Biol. Chem.
276, 11272-11278 (2001)), both hGH-(SO).sub.10 and
hGH-(SO).sub.10-EGFP fusion glycoproteins ((SO).sub.10 disclosed as
SEQ ID NO: 4) contained only Hyp-polysaccharide (Table 6). The same
effect was observed in INF-(SO).sub.10 ((SO).sub.10 disclosed as
SEQ ID NO: 4) (Table 7). TABLE-US-00007 TABLE 6 Represented as
percent of total hydroxyproline Hyp glycoside Predicted
hGH-(SO).sub.10 hGH-(SO).sub.10-EGFP Hyp-PS 100 100 100 Hyp-Ara4 0
0 Hyp-Ara3 0 0 Hyp-Ara2 {close oversize brace} 0 0 0 Hyp-Ara1 0 0
NG-Hyp Trace Trace Hyp-PS, Hyp polysaccharide; Hyp-Ara.sub.n,
Hyp-arabinoside.sub.1-4; NG-Hyp, non-glycosylated Hyp ((SO).sub.10
disclosed as SEQ ID NO: 4)
[0371] TABLE-US-00008 TABLE 7 Hydroxyproline glycoside profiles of
INF-(SO).sub.10 ((SO).sub.10 disclosed as SEQ ID NO: 4) Molar
percentage of total hydroxyproline Hyp glycoside Predicted
INF-(SO).sub.10 Hyp-PS 100 100 Hyp-Ara4 0 Hyp-Ara3 0 Hyp-Ara2
{close oversize brace} 0 0 Hyp-Ara1 0 NG-Hyp Trace Hyp-PS, Hyp
polysaccharide; Hyp-Ara.sub.n, Hyp-arabinoside.sub.1-4; NG-Hyp,
non-glycosylated Hyp
[0372] Table 8 shows the glycosyl linkage analysis of
hGH-(SO).sub.10 ((SO).sub.10 disclosed as SEQ ID NO: 4).
TABLE-US-00009 TABLE 8 Glycosyl Linkage Mole Percent t-Rha (p) 6
t-Ara (f) 17 t-Ara (p) 6 1,4-Ara (p) 7 1,5-Ara (f) 8 1,2,3,5-Ara
(f) 1 t-Gal (p) 4 1,3-Gal (p) 10 1,6-Gal (p) 5 1,3,4-Gal (p) 1
1,3,6-Gal (p) 17 1,3,4,6-Gal (p) 1 1,2,3,4,6-Gal (p) 1 t-GlcA (p) 2
1,4-Glc(p) 10 1,4-GlcA (p) 4 Terminal residues 35 Branched residues
65
[0373] General Cloning Description
[0374] The gene cassettes were built to encode the glycosylation
site at either the N-terminus or C-terminus of the protein, and
were sub-cloned into pUC18-SS.sup.tob-EGFP (pUC18 vector encoding
the tobacco extensin signal sequence [SS.sup.tob] and EGFP). The
genes were sequenced and then subcloned into pB121 (Clontech) as
BamHI/SacI fragments in place of the .beta.-glucuronidase gene and
behind the Cauliflower Mosaic Virus 35S promoter.
[0375] Plant Transformation
[0376] The pBI121-derived plasmids containing the gene cassettes
were transferred into Agrobacterium tumefaciens strain LBA4404. The
transformation of tobacco cells followed methods described earlier
(An, G. (1985), Plant Physiol, 79:568-570; Shpak, E., Leykam, J.
F., and Kieliszewski, M. J. (1999), Proceedings of the National
Academy of Sciences (USA), 96: 14736-14741; Zhao Z D, Tan L,
Showalter A M, Lamport D T, Kieliszewski M J., Plant J. 2002
August; 31(4):431-44). The tomato cells were transformed with leaf
disk method (McCormick et al, 1986 Leaf disc transformation of
cultivated tomato (L. esculentum) using Agrobacterium tumefaciens
McCormick, S.; Niedermeyer, J.; Fry, J.; Barnason, A.; Horsch, R.;
Fraley, R. Plant Cell Reports 5: 81-84) The Arabidopsis cells were
transformed using the method of Forreiter et al. (Forreiter C,
Kirschner M, Nover L., Plant Cell. 1997 December;
9(12):2171-81).
[0377] Cell Cultures
[0378] All the transformed cells were cultured in SH medium (Schenk
and Hildebrandt, 1972) containing 34 g/L sucrose, 0.4 mg/L
2,4-dichlorophenoxyacetic acid (2,4-D) and 200 mg/L kanamycin
(Sigma). Flasks (250-ml or 1000-ml) were placed on gyrotary shakers
rotating at 90 rpm at room temperature. Media were collected after
10-20 days cultures for isolation of target proteins.
[0379] Glycoprotein Isolation
[0380] Glycoproteins were isolated from media using
hydrophobic-interaction chromatography (HIC) and reversed-phase
chromatography, as shown before (NOTE the following differences: 2
M NaCl/25 mM Tris pH 8.5 was used to equilibrate the HIC column;
and the column was eluted with a stepwise gradient of a second
buffer containing just 25 mM Tris pH 8.5. The hGH derivative eluted
in the gradient at 25 mM Tris/0.2 M) (Shpak, E., Barbar, E.,
Leykam, J. F. & Kieliszewski, M. J. J. Biol. Chem. 276,
11272-11278 (2001); Zhao Z D, Tan L, Showalter A M, Lamport D T,
Kieliszewski M J., Plant J. 2002 August; 31(4):431-44; Li L C,
Bedinger P A, Volk C, Jones A D, Cosgrove D J, Plant Physiol. 2003
August; 132(4):2073-85).
Example 3
Examples of Additional Human Growth Hormone Constructs
[0381] In addition to the hGH constructs described in Example 2
above, the following constructs have also been synthesized and
transformed into tobacco cells.
[0382] 1. hGH-(SO).sub.1
[0383] SS.sup.tob-hGH-(SP).sub.1 gene fragment was amplified with
PCR using pUC-SS.sup.tob-hGH-(SP).sub.10 ((SP).sub.10 disclosed as
SEQ ID NO: 51) as template and the following primer set:
TABLE-US-00010 (SEQ ID NO:139) 5'-AGAGGATCCGCAATGGGAAAAATGGC-3' and
(SEQ ID NO:138) 5'-TAAGTGTACAATCAGGGTGAGAAGCCGCAGCTG-3'
[0384] The resulting PCR fragment was then sub-cloned into
pUC-SS.sup.tob-EGFP as a BamHI/BsrGI fragment, replacing
SS.sup.tob-EGFP, to generate the plasmid designated
pUC-SS.sup.tob-hGH-(SP).sub.1 (FIG. 19).
[0385] 2. hGH-(SO).sub.2 ((SO).sub.2 Disclosed as SEQ ID NO:
136)
[0386] SS.sup.tob-hGH-(SP).sub.2 ((SP)2 disclosed as SEQ ID NO: 90)
gene fragment was amplified with PCR using
pUC-SS.sup.tob-hGH-(SP).sub.10 ((SP).sub.10 disclosed as SEQ ID NO:
51) as template and the following primer set: TABLE-US-00011 (SEQ
ID NO:139) 5'-AGAGGATCCGCAATGGGAAAAATGGC-3' and (SEQ ID NO:140)
5'-TAAGTGTACAATCATGGAGAGGGTGAGAAGCC-3'
[0387] The resulting PCR fragment was then sub-cloned into
pUC-SS.sup.tob-EGFP as a BamHI/BsrGI fragment, replacing
SS.sup.tob-EGFP, to generate the plasmid designated
pUC-SS.sup.tob-hGH-(SP).sub.2 ((SP).sub.2 disclosed as SEQ ID NO:
90) (FIG. 20).
[0388] 3. hGH-(SO).sub.5 ((SO).sub.5 Disclosed as SEQ ID NO:
143)
[0389] SS.sup.tob-hGH-(SP).sub.5 ((SP).sub.5 disclosed as SEQ ID
NO: 92) gene fragment was amplified with PCR using
pUC-SS.sup.tob-hGH-(SP).sub.10 ((SP).sub.10 disclosed as SEQ ID NO:
51) as template and the following primer set: TABLE-US-00012 (SEQ
ID NO:139) 5'-AGAGGATCCGCAATGGGAAAAATGGC-3' and (SEQ ID NO:141)
5'-TAAGTGTACAATCAAGGCGATGGGGAAGGGCTTGG-3'
[0390] The resulting PCR fragment was then sub-cloned into
pUC-SS.sup.tob-EGFP as a BamHI/BsrGI fragment, replacing
SS.sup.tob-EGFP, to generate the plasmid designated
pUC-SS.sup.tob-hGH-(SP).sub.5 ((SP).sub.5 disclosed as SEQ ID NO:
92) (FIG. 21).
[0391] 4. hGH-(SO).sub.20 ((SO).sub.20 Disclosed as SEQ ID NO:
144)
[0392] A NcoI restriction site was first introduced right after
SS.sup.tob-hGH-(SP).sub.10 gene fragment ((SP).sub.10 disclosed as
SEQ ID NO: 51) with PCR using pUC-S.sup.tob-hGH-(SP).sub.10
((SP).sub.10 disclosed as SEQ ID NO: 51) as template and the
following primer set: TABLE-US-00013 (SEQ ID NO:139)
5'-AGAGGATCCGCAATGGGAAAAATGGC-3' and (SEQ ID NO:142)
5'-ATAAGCCATGGTTGGGCTGGGAGAAGGGGATGG-3'
[0393] The resulting PCR fragment,
SS.sup.tob-hGH-(SP).sub.10.sup.NcoI ((SP).sub.10 disclosed as SEQ
ID NO: 51) was then sub-cloned into
pUC-SS.sup.tob-hGH.sup.NcoI-(SP).sub.10* ((SP).sub.10 disclosed as
SEQ ID NO: 51) as a BamHI/NcoI fragment, replacing
SS.sup.tob-hGH.sup.NcoI, to generate the plasmid designated
pUC-SS.sup.tob-hGH-(SP).sub.20 ((SP).sub.20 disclosed as SEQ ID NO:
93 (FIG. 22). The extra nucleotides introduced into this plasmid
for cloning purpose were then removed by site-directed mutagenesis
using the QuickChange Mutagenesis kit (Strategies, Calif.). (*:
pUC-SS.sup.tob-hGH.sup.NcoI-(SP).sub.10 ((SP).sub.10 disclosed as
SEQ ID NO: 51) is the preliminary pUC-SS.sup.tob-hGH-(SP).sub.10
plasmid ((SP).sub.10 disclosed as SEQ ID NO: 51) without subject to
site-directed mutation to remove the NcoI restriction site.)
[0394] 5. (SO).sub.10-hGH-(SO).sub.10 ((SO).sub.10 Disclosed as SEQ
ID NO: 4)
[0395] A (SP).sub.10 fragment ((SP).sub.10 disclosed as SEQ ID NO:
51) was first amplified with PCR using
pUC-SS.sup.tob-hGH-(SP).sub.10 ((SP).sub.10disclosed as SEQ ID NO:
51) as template and the following primer set: TABLE-US-00014 (SEQ
ID NO:145) 5'-TTATCCCGGGCCTCACCCTCTCCAAGCCCTTCC-3' and (SEQ ID
NO:146) 5'-TTATCCCGGGTGGGCTGGGAGAAGGGGATGG-3'
[0396] The resulting PCR fragment, .sup.XmaI(SP).sub.10.sup.XmaI
((SP).sub.10 disclosed as SEQ ID NO: 51 was sub-cloned into
pUC-SS.sup.tob-.sup.XmaIhGH-(SP).sub.10** ((SP).sub.10 disclosed as
SEQ ID NO: 51) at the XmaI site, inserting between SS.sup.tob and
hGH-.sup.XmaI(SP).sub.10 ((SP).sub.10 disclosed as SEQ ID NO: 51 to
generate the plasmid designated
pUC_S.sup.tob-(SP).sub.10-hGH-(SP).sub.10 ((SP).sub.10 disclosed as
SEQ ID NO: 51) (FIG. 23). The extra nucleotides introduced into
this plasmid for cloning purpose were then removed by site-directed
mutagenesis using the QuickChange Mutagenesis kit (Strategies, CA).
(**pUC-SS.sup.tob-.sup.XmaIhGH-(SP).sub.10 ((SP).sub.10 disclosed
as SEQ ID NO: 51) is the preliminary pUC-SS.sup.tob-hGH-(SP).sub.10
plasmid ((SP).sub.10 disclosed as SEQ ID NO: 51) without subject to
site-directed mutation to remove the XmaI restriction site.
[0397] 6. hGHA-(SO).sub.10 (hGRA: Human Growth Hormone Antagonist)
((SO).sub.10 Disclosed as SEQ ID NO: 4)
[0398] pUC-SS.sup.tob-hGHA-(SP).sub.10 ((SP).sub.10 disclosed as
SEQ ID NO: 51) (FIG. 24) was generated by site-directed mutagenesis
of plasmid pUC-SS.sup.tob-hGH-(SP).sub.10 ((SP).sub.10 disclosed as
SEQ ID NO: 51) (from encoding Gly.sup.120 to encoding Lys.sup.120)
using the following primer set: TABLE-US-00015 (SEQ ID NO:147)
5'-GGACCTAGAGGAAAAGATCCAAACGCTG-3' and (SEQ ID NO:148)
5'-CAGCGTTTGGATCTTTTCCTCTAGGTCC-3'.
Example 4
Additional Examples of Interferon Constructs
[0399] In addition to the interferon alpha2 construct
(INF-(SO).sub.10) ((SO).sub.10 disclosed as SEQ ID NO: 4) described
in Example 2 above, the following additional constructs were
made.
[0400] 1. INF-(SO).sub.5 ((SO).sub.5 Disclosed as SEQ ID NO:
1434)
[0401] SS.sup.tob-INF-(SP).sub.5 ((SP).sub.5 disclosed as SEQ ID
NO: 92) gene fragment was amplified with PCR using
pUC-SS.sup.tob-INF-(SP).sub.10 ((SP).sub.10 disclosed as SEQ ID NO:
51) as template and the following primer set: TABLE-US-00016 (SEQ
ID NO:139) 5'-AGAGGATCCGCAATGGGAAAAATGGC-3' and (SEQ ID NO:141)
5'-TAAGTGTACAATCAAGGCGATGGGGAAGGGCTTGG-3'
[0402] The resulting PCR fragment was sub-cloned into
pUC-SS.sup.tob-EGFP as a BamHI/BsrGI fragment, replacing
SS.sup.tob-EGFP, to generate the plasmid designated
pUC-SS.sup.tob-INF-(SP).sub.5 ((SP).sub.5 disclosed as SEQ ID NO:
92) (FIG. 25). This transformation was performed in Arabidopsis
thaliana cells.
[0403] 2. (SO).sub.5-INF-(SO).sub.5 ((SO).sub.5 Disclosed as SEQ ID
NO: 143)
[0404] SS.sup.tob-(SP).sub.5 ((SP).sub.5 disclosed as SEQ ID NO:
92) gene fragment was amplified with PCR using
pU_SS.sup.tob-(SP).sub.10-hGH-(SP).sub.10 as template ((SP).sub.10
disclosed as SEQ ID NO: 51) and the following primer set:
TABLE-US-00017 (SEQ ID NO:139) 5'-AGAGGATCCGCAATGGGAAAAATGGC-3' and
(SEQ ID NO:137) 5'-ATAAGGCCCGGGTAGGCGATGGGGAAGGGCTTG-3'
[0405] The resulting PCR fragment was sub-cloned into
pUC-SS.sup.tob-INF-(SP).sub.5 ((SP).sub.5 disclosed as SEQ ID NO:
92) as a BamHI/XmaI fragment, replacing SS.sup.tob, to generate the
plasmid designated pUC-SS.sup.tob-(SP).sub.5-INF-(SP).sub.5
((SP).sub.5 disclosed as SEQ ID NO: 92) (FIG. 26). The extra
nucleotides introduced into this plasmid for cloning purpose were
then removed by site-directed mutagenesis using the QuickChange
Mutagenesis kit (Strategies, CA). This transformation was performed
in Arabidopsis thaliana cells.
[0406] 3. (SO).sub.5-INF((SO).sub.5 Disclosed as SEQ ID NO:
143)
[0407] SS.sup.tob-(SP).sub.5 ((SP).sub.5 disclosed as SEQ ID NO:
92) gene fragment was amplified with PCR as above. The resulting
PCR fragment was sub-cloned into pUC-SS.sup.tob-INF as a BamHI/XmaI
fragment, replacing SS.sup.tob, to generate the plasmid designated
pUC-SS.sup.tob-(SP).sub.5-INF ((SP).sub.5 disclosed as SEQ ID NO:
92) (FIG. 27). The extra nucleotides introduced into this plasmid
for cloning purpose were then removed by site-directed mutagenesis
using the QuickChange Mutagenesis kit (Strategies, CA). This
transformation was performed in Arabidopsis thaliana cells.
[0408] 4. INF-(SO).sub.20-((SO).sub.20Disclosed as SEQ ID
NO:144)
[0409] A NcoI restriction site was first introduced right after
SS.sup.tob-INF-(SP).sub.10 ((SP).sub.10 disclosed as SEQ ID NO: 51)
gene fragment with PCR using pUC-SS.sup.tob-INF-(SP).sub.10
((SP).sub.10 disclosed as SEQ ID NO: 51) as template and the
following primer set: TABLE-US-00018 (SEQ ID NO:139)
5'-AGAGGATCCGCAATGGGAAAAATGGC-3' and (SEQ ID NO:142)
5'-ATAAGCCATGGTTGGGCTGGGAGAAGGGGATGG-3'
[0410] The resulting PCR fragment,
SS.sup.tobINF-(SP).sub.10.sup.NCoI ((Sp).sub.10 disclosed as SEQ ID
NO: 51) was then sub-cloned into
pUC-SS.sup.tob-hGH.sup.NcoI-(SP).sub.10 ((SP).sub.10 disclosed as
SEQ ID NO: 51) as a BamHI/NcoI fragment, replacing
SS.sup.tob-INF.sup.NcoI, to generate the plasmid designated
pUC-SS.sup.tob-INF-(SP).sub.20 ((SP).sub.20 disclosed as SEQ ID NO:
93) (FIG. 28). The extra nucleotides introduced into this plasmid
for cloning purpose were then removed by site-directed mutagenesis
using the QuickChange Mutagenesis kit (Strategies, CA). This
transformation was performed in tobacco cells.
[0411] 5. (SO).sub.10-INF(SO).sub.10 ((SO).sub.10 Disclosed as SEQ
ID NO: 4)
[0412] A SS.sup.tob-(SP).sub.10 ((SP).sub.10 disclosed as SEQ ID
NO: 51) fragment was generated by digestion of
pUC_SS.sup.tob-(SP).sub.10.sup.XmaI-hGH-(SP).sub.10*** ((SP).sub.10
disclosed as SEQ ID NO: 51) with BamHI/XmaI. This fragment was then
sub-cloned into pUC-SS.sup.tob-INF-(SP).sub.10 ((SP).sub.10
disclosed as SEQ ID NO: 51), replacing SS.sup.tob, to generate the
plasmid designated pUC_SS.sup.tob-(SP).sub.10-INF-(SP).sub.10
((SP).sub.10 disclosed as SEQ ID NO: 51) (FIG. 29). The extra
nucleotides introduced into this plasmid for cloning purpose were
then removed by site-directed mutagenesis using the QuickChange
Mutagenesis kit (Strategies, CA). (***:
pUS_SS.sup.tob-(SP).sub.10.sup.XmaI-hGH-(SP).sub.10 ((SP).sub.10
disclosed as SEQ ID NO: 51) is the preliminary
pUC_SS.sup.tob(SP).sub.10-hGH-(SP).sub.10 ((SP).sub.10 disclosed as
SEQ ID NO: 51) plasmid without subject to site-directed mutation to
remove the XmaI restriction site. This transformation was performed
in tobacco cells.
Example 5
Propehtic Examples of EGFP Fusion Proteins
[0413] Other EGFP fusion proteins that can be made in accordance
with the present invention include, but are not limited to,
(Ala-Hyp).sub.11-EGFP (peptide disclosed as SEQ ID NO: 149),
(Thr-Hyp).sub.11-EGFP (peptide disclosed as SEQ ID NO: 150),
(Thr-Hyp).sub.101-EGFP (peptide disclosed as SEQ ID NO: 151), and
(Val-Hyp).sub.11-EGFP (peptide disclosed as SEQ ID NO: 152). These
are just a few of the examples that are specifically contemplated.
The invention is hardly limited to these examples; essentially any
combination or number of X-Hyp repeats can be made (where X is an
amino acid and Hyp is hydroxyproline).
Example 6
Improving the Biological Characteristics of Peptides by
Glycosylation
[0414] Human growth hormone (hGH) is a polypeptide hormone secreted
by the pituitary gland and transported by the blood to target
tissues such as the liver, muscle, bone, and adipose. Human GH
induces metabolic changes in the target tissues, ultimately
stimulating the processes that result in body growth. Hyposecretion
of hGH results in dwarfism and hypersecretion results in gigantism
and acromegaly. Additionally, hGH influences the metabolism of
adipocytes and muscle cells and processes such as aging; hence, the
intense interest in manipulating hGH levels in blood and
tissues.
[0415] Despite these important utilities, native or recombinant GH
is generally unsuitable as a polypeptide drug because its small
size results in rapid kidney clearance and a very short circulating
half-life (.about.30 min). Thus, patients undergoing treatment for
dwarfism require too-frequent injections of hGH.
[0416] The attachment of polyethylene glycol (PEG) groups to lysine
residues in the polypeptide--a process called
PEGylation--dramatically improves the pharmacological properties of
hGH. PEGylation makes hGH more clinically effective by increasing
its molecular mass, thereby preventing renal filtration and slowing
clearance of hGH from the body; it also protects the polypeptide
from proteolysis and reduces immunogenicity.
[0417] PEGylation has some drawbacks however. The relatively
non-specific targeting of lysine residues dramatically reduces
receptor binding affinities, by as much as 1500-fold Furthermore,
the process of PEGylation is time-consuming and inconvenient, as it
requires purification of the derivatized polypeptide, greatly
increasing drug costs.
[0418] This Example describes work in which the inventors increased
the effective molecular weight of hGH and its corresponding
circulating stability by expressing it in plant cells as a
glycoprotein.
[0419] Materials and Methods
[0420] Construction of the plant transformation plasmid pBI
SS.sup.tob-hGH-(SP).sub.10 pBI121 ((SP).sub.10 disclosed as SEQ ID
NO: 51) is a plasmid commercially available from Clontech. A
derivative of it was made for this work.
[0421] Human growth hormone cDNA was produced by RT-PCR from the
total RNA extracted from mouse L-cells stably transfected with hGH
gene (Chen et al, 1994) using the following primer set:
5'-ACCCGGGCCTTCCCAACCATTCCCTTATCC-3' (SEQ ID NO: 153) and
5'-GATTCCATGGTGAAGCCACAGCTGCCCTCCAC-3' (SEQ ID NO: 91). The
resulting PCR fragment contained the open reading frame for hGH but
lacked its signal peptide. This fragment was cloned into
pUC-SS.sup.tob-EGFP as an XmaI/NcoI fragment between SS.sup.tob,
which encodes the extensin signal sequence (SS) from tobacco, and
the gene for enhanced green fluorescent protein (EGFP) to generate
the plasmid designated pUC-SS.sup.tob-hGH-EGFP. The synthetic gene
encoding ten repeats of the dipeptide Ser-Pro (SP).sub.10
((SP).sub.10 disclosed as SEQ ID NO: 51) was constructed by primer
extension of two mutually priming oligonucleotides (Integrated DNA
Technologies, Inc. Coralville, Iowa) (FIG. 10B)
[0422] The (SP).sub.10 gene ((SP).sub.10 disclosed as SEQ ID NO:
51) was subcloned into pUC-SS.sup.tob-hGH-EGFP as a NcoI and BsrGI
fragment, replacing EGFP to generate
pUC-SS.sup.tob-hGH-(SP).sub.10. ((SP).sub.10 disclosed as SEQ ID
NO: 51) The extra nucleotides introduced into the
SS.sup.tob-hGH-(SP).sub.10 ((SP).sub.10 disclosed as SEQ ID NO: 51)
Gene cassette for cloning purpose were then removed by
site-directed mutagenesis using the QuickChange Mutagenesis kit
(Strategies, CA). Sequencing of
SS.sup.tob-hGH-(SP).sub.10((SP).sub.10 disclosed as SEQ ID NO: 51)
was performed in Department of Environmental and Plant Biology,
Ohio University.
[0423] The entire SS.sup.tob-hGH-(SP).sub.10 ((SP).sub.10disclosed
as SEQ ID NO: 51) construct (FIG. 10A) was then cloned into plant
transformation vector pBI121 (Clontech, CA) as a BamHI and SacI
fragment in place of the .beta.-glucuronidase reporter gene to give
plasmid pBI-SS.sup.tob-hGH-(SP).sub.10 ((SP).sub.10 disclosed as
SEQ ID NO: 51). The expression of SS.sup.tob-hGH-(SP).sub.10
((SP).sub.10 disclosed as SEQ ID NO: 51) was under the control of
the 35S cauliflower mosaic virus promoter.
[0424] Plant Cell Transformation and Selection
[0425] Plasmid pBI-SS.sup.tob-hGH-(SP).sub.10 ((SP).sub.10
disclosed as SEQ ID NO: 51) was introduced into Agrobacterium
tumefaciens strain LBA4404 by the freeze-thaw method (Holsters et
al., 1978), then suspension-cultured tobacco cells (Nicotiana
tabacum, BY2) were transformed with the Agrobacterium as described
earlier (An, G. (1985) High efficiency transformation of cultured
tobacco cells. Plant Physiol, 79:568-570) and selected on solid
Schenk & Hildebrandt (SH) medium (Schenk and Hildebrandt,
(1972) Medium and techniques for induction and growth of
monocotyledonous and dicotyledonous plant cell cultures. Can J Bot,
50:199-204) containing 0.4 mg/L 2,4-dichlorophenoxyacetic acid
(2,4-D), 200 mg/L kanamycin (Sigma), and 400 mg/L TIMENTIN
(SmithKline Beecham, PA).
[0426] For production of the transgene product, cells were gown in
liquid SH medium comprised of the same components as above, except
excluding TIMENTIN. After 8 to 10 days of culture at room
temperature on an Innova gyrotary shaker (New Brunswick Scientific,
Edison, N.J.), rotating at 90 rpm, the culture medium for each cell
line was screened for hGH expression by dot blotting and ELISA
assay (see below). Three high-yield cell lines were chosen for
subculture under the conditions described above.
[0427] Isolation of the hGH-(SO).sub.10 Fusion Glycoprotein
((SO).sub.10 Disclosed as SEQ ID NO: 4)
[0428] The medium from transformed cells was harvested after 8-10
days of culture by filtration on a coarse sintered funnel and
supplemented with sodium chloride to a final concentration of 2 M.
Insoluble material was pelleted by centrifugation at 25,000.times.G
for 20 min at 4 C. The supernatant was fractionated by
hydrophobic-interaction chromatography (HIC) on a Phenyl-Sepharose
6 column (Phenyl-Sepharose 6 Fast Flow, 16 by 700 mm, Amersham
Pharmacia Biotech) equilibrated in 2 M sodium chloride. After the
medium was completely loaded onto the HIC column, the proteins were
eluted step-wise first with Tris buffer (25 mM, pH 8.5)/2M sodium
chloride, followed by Tris buffer (25 mM, pH 8.5)/0.8M sodium
chloride, and then the Tris buffer (25 mM, pH 8.5)/0.2N sodium
chloride. The flow rate was 1.0 ml/min, and the fractions were
monitored at 220 nm with a UV detector. Each elute fraction was
assayed for the presence of hGH by dot blots and ELISA assays. The
Tris buffer (25 mM, pH 8.5)/0.2N NaCl fraction containing most of
the hGH-(SO).sub.10 fusion glycoprotein ((SO).sub.10 disclosed as
SEQ ID NO: 4 was concentrated by ultrafiltration at 4.degree. C.,
and either used for hGH binding and activity assays, or further
purification by reversed phase chromatography.
[0429] Each eluate fraction was assayed for the presence of hGH by
dot blots and ELISA assays. The fraction from the HIC column, which
contained the fusion glycoprotein (designated hGH-(SO).sub.10
((SO).sub.10 disclosed as SEQ ID NO: 4)), was concentrated by
ultrafiltration at 4.degree. C. and either used for hGH binding and
activity assays The HIC hGH-(SO).sub.10 ((SO).sub.10 disclosed as
SEQ ID NO: 4) rich fraction further fractionated by reversed phase
chromatography on a Hamilton polymeric reversed phase-1 (PRP-1)
analytical column (4.1.times.150 mm, Hamilton Co., Reno, Nev.)
equilibrated with buffer A (0.1% trifluoroacetic acid). Proteins
were eluted with buffer B (0.1% trifluoroacetic acid, 80%
acetonitrile, v/v) using a two-step linear gradient of 0-30% B in
15 min, followed by 30%-70% B in 90 min at a flow rate of 0.5
ml/min. Absorbance was measured at 220 nm.
[0430] Western Blot Analysis
[0431] Samples (10-.mu.g) of hGH-(SO).sub.10 ((SO).sub.10 disclosed
as SEQ ID NO: 4) were mixed with an equal volume of 2.times.
reducing sample buffer and electrophoresed on a 4-12%
SDS-polyacrylamide gel (BioRad, CA), then transferred to a
NitroBind membrane (MSI, Westboro, Mass.) using a BioRad mini
Trans-Blot cell. Rabbit polyclonal anti-hGH antibody (Fitzgerald
Industries International, Concord, Mass.) diluted at 1:500 in TTBS
buffer (100 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20)
and alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma)
diluted at 1:1000 in TTBS buffer were used as primary and secondary
antibodies, respectively.
[0432] Quantification of hGH by ELISA
[0433] The concentration of hGH equivalents in the medium or in
column eluant was determined using a sandwich hGH ELISA kit (Roche
Molecular Biochemicals, Germany) according to manufacturer's
instructions.
[0434] Glycosyl Composition and Hydroxyproline Glycoside
Profiles
[0435] Neutral sugars were analyzed as alditol acetates derivatives
by gas chromatography using a Hewlett-Packard HP-5 column
(crosslinked 5% PH ME Siloxane, 30 m.times.0.32 mm.times.0.25
.mu.m) programmed from 130.degree. C. to 177.degree. C. at
1.2.degree. C./min. Data were captured by Hewlett-Packard
ChemStation software. One hundred pg of hGH-(SO).sub.10
((SO).sub.10 disclosed as SEQ ID NO: 4) was used for each analysis
with 50 nmol of myo-inositol as the internal standard. Uronic acids
were assayed by the colorimetric method based on reaction with
m-hydroxydiphenyl, with D-glucuronic acid as the standard.
[0436] Amino Acid Sequencing and Composition Assay
[0437] The N-terminal amino acid sequence of hGH-(SO).sub.10
((SO).sub.10 disclosed as SEQ ID NO: 4) was determined at the
Michigan State University Macromolecular Facility on a 477-A
Applied Biosystems Gas Sequencer. The hGH-(SO).sub.10 amino acid
composition ((SO).sub.10 disclosed as SEQ ID NO: 4) was determined
by reversed phase HPLC on a Beckman Gold System (Beckman
Instruments Inc., CA) after hydrochloric acid hydrolysis and
subsequent phenylisothiocyanate derivatization (Bergman, T,
Carlquist, M, Jomvall, H. (1986) Amino acid analysis by high
performance liquid chromatography of phenylthiocarbamyl
derivatives. In: Wittmann-liebold B, editor. Advanced Methods in
Protein Microsequence Analysis. Berlin: Springer-Verlag. p 45-55).
The Hyp content of samples was assayed colorimetrically as
described earlier (Kivirikko, K. I. and Liesmaa, M. (1959) A
colorimetric method for determination of hydroxyproline in tissue
hydrolysates. Scandinavian J Clin Lab, 11:128-131). TABLE-US-00019
TABLE 9 Amino acid composition and N-terminal sequence of
hGH-(SO).sub.10 ((SO).sub.10 disclosed as SEQ ID NO: 4) Composition
(mol %) Amino hGH(SO).sub.10 Acid hGH(SO).sub.10 cDNA
Predicted.sup.c Hyp 4.5 -- 4.7 Pro 6.8 8.5 3.8 Asx.sup.a 2.5 9.5
9.5 Glx.sup.b 14.6 12.8 12.8 Thr 3.9 5.2 5.2 Ser 14.8 13.7 13.7 Gly
5.6 3.8 3.8 Ala 7.4 3.3 3.3 Val 3.1 3.3 3.3 Met 1.8 0.5 0.5 Ile 3.1
3.3 3.3 Leu 9.7 12.3 12.3 Tyr 2.0 3.8 3.8 Phe 5.2 6.2 6.2 His 2.9
0.5 0.5 Lys 6.9 4.3 4.3 Arg 4.6 4.7 4.7 Cys 0.5 1.9 1.9 Trp nd 0.0
0.0 N-terminal sequence (main sequence) Phe-Pro-Thr-
Ile-Pro-Leu-Ser-Arg-Leu-Phe- Asp-Asn-Ala-Met-Leu . . . (SEQ ID NO:
154) (minor sequence) Ser-His-Asn- Asp-Asp-Ala-Leu-Leu-Lys-Asn-
Tyr-Gly-Leu-Leu-Tyr . . . (SEQ ID NO: 155) .sup.aAsx includes Asp
and Asn .sup.bGlx includes Glu and Gln .sup.cpredicted from the
designed peptide sequence of hGH(SO).sub.10 glycoprotein
((SO).sub.10 disclosed as SEQ ID NO: 4) and Hyp contiguity theory
(Shpak, E., Leykam, J. F., and Kieliszewski, M. J. (1999),
Proceedings of the National Academy of Sciences (USA), 96:
14736-14741; Shpak, E., Barbar, E., Leykam, J. F. &
Kieliszewski, M. J. J. Biol. Chem. 276, 11272-11278 (2001))
[0438] The major sequence above is the N-terminus of intact
hGH-(SP).sub.10 ((SP).sub.10 disclosed as SEQ ID NO: 51), the minor
sequence occurs after proteolytic cleavage at one labile site
(N150-S151) in the hGH domain of hGH-(SP).sub.10 ((SP).sub.10
disclosed as SEQ ID NO: 51). Analysis of hGH expressed as a
targeted protein in our BY-2 system showed it contained no Hyp,
suggesting that hGH in our fusion glycoproteins contains Hyp only
in the SO module. This amino acid composition indicates there are
9.5 Hyp residues in the 211 amino acid sequence.
[0439] Radioreceptor Binding Assays of hGH-(SO).sub.10 ((SO).sub.10
Disclosed as SEQ ID NO: 4)
[0440] Binding assays of hGH-(SP).sub.10((SP).sub.10 disclosed as
SEQ ID NO: 51), isolated with HIC (Hydrophobic Interaction
Chromatography), were performed using a monolayer cell surface
binding assay.
[0441] Briefly, growth hormone receptor (GHR)-expressing NIH 3T3-L1
cells were grown to confluence in 12-well cell culture plates. The
cells were depleted of serum overnight with plain DMEM. The cells
were rinsed twice with 1 ml PBS containing 0.1% BSA at room
temperature prior to the binding assay. Cells were incubated in the
presence of a constant amount of [.sup.125I]-hGH (Perkin Elmer)
with varying amounts of GH preparations in 1-mL reaction volumes
containing 0.1% BSA at room temperature for 2 hours on an orbital
shaker. Binding reaction was terminated by rinsing cells 3 times
with 1 mL of ice-cold PBS containing 0.1% BSA. Cells were
solubilized with 0.1N NaOH and neutralized with 0.1N HCl and cell
surface bound radioactivity was measured using a liquid
scintillation counter.
[0442] This binding assay was repeated with an hGH-(SP).sub.10-EGFP
((SP).sub.10 disclosed as SEQ ID NO: 51) construct. The results,
presented in FIG. 30, show that even with green fluorescent protein
attached, the modified hGH binds to the receptor with relatively
high affinity (EC.sub.50 of approximately 10 nM). The results also
show that the glycosylation motif can be interiorly situated; it is
not necessary that the glycosylation motif be on either
terminus.
[0443] The results for the commercially available hGH for
hGH-(SP).sub.10 ((SP).sub.10 disclosed as SEQ ID NO: 51) are
presented in FIGS. 31 and 32. The EC.sub.50 for hGH-(SP).sub.10
((SP).sub.10 disclosed as SEQ ID NO: 51) was 1 nM, consistent with
commercially available hGH binding of its receptor (FIG. 31).
[0444] In Vivo Effects of hGH-(SP).sub.10 ((SP).sub.10 Disclosed as
SEQ ID NO: 51)
[0445] In order to determine the pharmacological effect and rate of
clearance of the modified growth hormone, hGH-(SP).sub.10
((SP).sub.10 disclosed as SEQ ID NO: 51) and commercially available
hGH (Fitzgerald Industries International, Inc. 34 Junction Square
Drive, Concord, Mass. 01742-3049 USA) were tested in mice.
[0446] For these tests, 5 to 6 month old C57BL/6J mice were
injected intraperitoneally with GH samples prepared in PBS. Plasma
was assayed for levels of growth hormone and insulin-like growth
factor I ("IGF-1"; released by the body in response to growth
hormone). (The growth hormone ELISA kits and IGF-1 kits were
purchased from Diagnostic Systems Laboratories Inc.)
[0447] Test 1: Single injection of 2 .mu.g GH/g body weight. Plasma
was sampled at 1 and 4 days after injection. The results are shown
in FIGS. 33 (growth hormone concentration) and 34 (IGF-1
concentration). Clearly, the hGH-(SP).sub.10 ((SP).sub.10 disclosed
as SEQ ID NO: 51) exhibited a much higher concentration at the
one-day measurement and exhibited a dramatically increased
half-life and area under the curve. The IGF-1 levels show that the
biological effect of GH was both enhanced and extended.
[0448] In another test of hGH half-life, each group of mice (two)
was given a single dose of 30 .mu.g of hGH equivalent. Serum
samples (30 .mu.l) were taken over intervals extending to 48 hours
and analyzed for hGH concentration by ELISA. The results, shown in
FIG. 35, demonstrate a significant extension of plasma half-life by
glycosylation.
[0449] Test 2: 2 .mu.g GH/g body weight/day. The growth hormone
(modified and control) was administered daily as two injections, 12
hours apart, for 5 days. Plasma was sampled at 1, 4, 6, 8, 11, and
18 days after the first injection. The results are shown in FIGS.
36 and 37. FIG. 36 shows the serum concentration of growth hormone;
FIG. 37 shows the serum concentration of IGF-1.
[0450] Again, note how GH levels (FIG. 36) are insignificant at one
day with a commercial growth hormone preparation, whereas the
glycosylated form has a much higher concentration at day one. The
biological effect (shown in FIG. 37) for the commercially available
growth hormone essentially ceases less than 5 days after the last
administration (on day 5), whereas the glycosylated form continues
to produce measurable IGF-1 levels more than two weeks after the
last administration. These results suggest that this may be the
longest acting growth hormone ever developed.
[0451] Test 3: 1 .mu.g GH/g body weight/day. The GH was
administered in single daily injections for 5 days and plasma was
sampled at 1, 4, 7, 9, and 11 days after the first injection. The
results are shown in FIGS. 38 and 39. Even at this lower dose, a
significant difference is observed between the commercially
available growth hormone and the glycosylated form of the
invention.
[0452] Test 4: Effects of hGH-(SO).sub.10 ((SO).sub.10 disclosed as
SEQ ID NO: 4) on whole body growth.
[0453] Seven- to eight-week old mice were randomly divided into 3
treatment groups, i.e., control (n=3), hGH (n=3), and
hGH-(SO).sub.10 (n=4) ((SO).sub.10 disclosed as SEQ ID NO: 4). Mice
were caged in groups of two or three individuals. hGH (Fittzgerald,
Concord Mass.) and hGH-(SO).sub.10 ((SO).sub.10 disclosed as SEQ ID
NO: 4) were prepared at 100 .mu.g/mL in PBS. Mice were
intraperitoneally injected with a total dose of 1 .mu.g per gram of
body weight, twice daily at about 9 AM and 9 PM, for six days.
After a one-day intermission, dosage was increased to 2 .mu.g per
gram of body weight for an additional 7 days.
[0454] FIG. 40 shows the weight gain of the mice in the test.
Briefly, control mice gained average of 0.83 g over two-week
period. Mice receiving hGH gained an average of 2.13 g and mice
received hGH-(SO).sub.10 ((SO).sub.10 disclosed as SEQ ID NO: 4)
gained average of 2.15 g over the two-week period. Weight gain over
control mice was significant (p<0.05, ANOVA) for both hGH and
hGH-(SO).sub.10 ((SO).sub.10 disclosed as SEQ ID NO: 4) and there
was no significant difference between hGH and hGH-(SO).sub.10
((SO).sub.10 disclosed as SEQ ID NO: 4) treatments.
[0455] Immunogenicity Assay of hGH-(SO).sub.10 ((SO).sub.10
Disclosed as SEQ ID NO: 4)
[0456] hGH-(SO).sub.10 ((SO).sub.10 disclosed as SEQ ID NO: 4) was
injected into mice to test its immunogenicity as compared to
wild-type growth hormone.
[0457] Immunization regimen: Two female Balb/C mice (.about.6-7
weeks old) were bled and immunized four times at two-week
intervals. Each mouse received 50 .mu.g of hGH-(SP).sub.10
((SP).sub.10 disclosed as SEQ ID NO: 51) subcutaneously split
between 2 sites (right and left flank. 0.05 mL/site). Serum was
frozen at -20.degree. C. until assayed for antibody activity by
ELISA.
[0458] ELISA Protocol:
[0459] EIA plates (NINC polystyrene) are coated with 40 .mu.g/mL
immunogen (hGH-(SP).sub.10) ((SP).sub.10 disclosed as SEQ ID NO:
51) in carbonate-bicarbonate buffer, pH 9.0, 50 .mu.L/well, and
left overnight. An equal number of wells is coated with buffer
only, (SP).sub.10 ((SP).sub.10 disclosed as SEQ ID NO: 51) only (20
.mu.g/mL) or hGH only (20 .mu.g/mL). Immunogen is decanted and 200
.mu.L of PBS-5% BSA (+0.05% Tween-20) is added per well for two
hours at room temperature to block nonspecific binding.
[0460] BSA is decanted and to each well is added 50 .mu.L
(duplicate wells on both immunogen-coated and uncoated wells) of
PBS-1% BSA only or mouse serum dilutions in PBS-BSA. Pre-immune
serum and most recent serum sample is compared from same mouse on
each plate.
[0461] Incubation is performed for four hours at room temperature
or overnight at 4.degree. C. The wells are washed 4.times. in
PBS-Tween.
[0462] To each well is added 50 .mu.l of a 1:5000 dilution of
peroxidase-conjugated goat anti-mouse Ig (all isotypes) in PBS-BSA
for one hour at room temperature. Wells are washed four times.
[0463] The assay is developed by addition of 50 .mu.L/well of OPD
substrate in citrate-phosphate buffer, pH 6. The reaction is
stopped by addition of 50 .mu.l/well 12.5% sulfuric acid when good
contrast between background and samples is seen. The ELISA is read
at 490 nm.
[0464] Interpretation.
[0465] Both mice showed a strong antibody response to hGH after a
single injection of hGH. The Antibody levels rose with repeated
injection.
[0466] Both mice possessed marginally detectable antibody to
purified (SP).sub.10 ((SP).sub.10 disclosed as SEQ ID NO: 51) after
the 2.sup.nd and 3.sup.rd injections. The low response may be due
to poor detection as a result of low binding of the purified
(SP).sub.10 ((SP).sub.10 disclosed as SEQ ID NO: 51) antigen to the
ELISA plate (see below), which would reduce the reactions seen.
[0467] Both mice had an unexpected high antibody level to the
hGH-SP10 conjugate antigen even before immunization. Since
pre-immune serum did not react with purified (SP).sub.10
((SP).sub.10 disclosed as SEQ ID NO: 51) or hGH alone, this can be
explained by the mice having preformed anti-(SP).sub.10
((SP).sub.10 disclosed as SEQ ID NO: 51), a cross-reactive antibody
to some other antigen they have seen. It is possible that it is
only detected when conjugated to hGH because the hGH-(SP).sub.10
((SP).sub.10 disclosed as SEQ ID NO: 51) conjugate strongly
attached to the ELISA plate, allowing better detection of
anti-(SP).sub.10 ((SP).sub.10 disclosed as SEQ ID NO: 51). While
this is speculation, it is consistent with observations in other
mice where plant materials produced background responses without
immunization.
[0468] The OD values were higher to the hGH coated plates than to
the hGH-(SP).sub.10 ((SP).sub.10 disclosed as SEQ ID NO: 51) coated
plates, which may reflect a differential recognition or simply
different levels of the recognized determinants on the two plates.
TABLE-US-00020 TABLE 10 Time post- anti- anti-hGHSP10 immunization
Mouse# anti-SP10 (OD) hGH(OD) (OD) 1:100 serum dilution 0 weeks 1
0.05 0.26 0.97 2 0.05 0 0.87 2 weeks 1 0.05 1.79 1.3 2 0.05 1.52
1.1 4 weeks 1 0.14 2.48 2.06 2 0.1 2.27 1.87 6 weeks 1 0.28 2.33
2.15 2 0.23 2.29 2.1 1:500 serum dilution 0 weeks 1 0.04 0.11 0.41
2 0.03 0.04 0.36 2 weeks 1 0.03 1.16 0.55 2 0.02 0.83 0.46 4 weeks
1 0.1 2.29 1.82 2 0.06 1.99 1.44 6 weeks 1 0.16 2.23 1.98 2 0.15
2.15 1.86
[0469] Competition experiments were also performed using
hGH-(SO).sub.10-coated plates, anti-hGH-(SO).sub.10 antibodies
(1:10,000 serum dilution), and 100 .quadrature.g/ml (SO).sub.10
((SO).sub.10 disclosed as SEQ ID NO: 4) as competitive inhibitor of
the antibody binding. A 5% inhibition of the reaction was
observed.
[0470] In summary, the hGH fusion glycoprotein, designated
hGH-(SO).sub.10 ((SO).sub.10 disclosed as SEQ ID NO: 4), contained
at the C-terminus ten tandem repeats of the glycosylation site
Ser-Hyp (SO), which directed the addition of
rhamnoglucuronoarabinogalactan polysaccharides to each Hyp residue
and increased the molecular mass of hGH from 22 kDa to about 50 kDa
and the circulating half-life from minutes to several hours or even
days. The EC50 for hGH-(SO).sub.10 ((SO).sub.10 disclosed as SEQ ID
NO: 4) was 1 nM, consistent with wild type GH binding of its
receptor; furthermore hGH-(SO).sub.10 ((SO).sub.10 disclosed as SEQ
ID NO: 4) stimulated the phosphorylation of JAK 5 in cultured cells
and ultimately produced the same physiological response as wild
type hGH. Preliminary evaluation of the antigenicity of
hGH-(SO).sub.10 ((SO).sub.10 disclosed as SEQ ID NO: 4) injected
subcutaneously into mice indicates that it is not more immunogenic
than wild-type growth hormone.
[0471] Other embodiments of the invention will be apparent to those
skilled in the art from consideration of the specification and
practice of the invention disclosed herein. It is intended that the
specification and examples be considered as exemplary only, with a
true scope and spirit of the invention being indicated by the
following claims.
* * * * *