U.S. patent application number 13/009414 was filed with the patent office on 2011-06-09 for modified growth hormone.
This patent application is currently assigned to THE UNITED STATES OF AMERICA, REPRESENTED BY, DEPA RTMENT OF HEALTH. Invention is credited to Bruce J. Baum, Niamh Cawley, Yoke Peng Loh, Christopher R. Snell.
Application Number | 20110135614 13/009414 |
Document ID | / |
Family ID | 23117747 |
Filed Date | 2011-06-09 |
United States Patent
Application |
20110135614 |
Kind Code |
A1 |
Loh; Yoke Peng ; et
al. |
June 9, 2011 |
MODIFIED GROWTH HORMONE
Abstract
The invention provides, a nucleic acid molecule encoding a
growth hormone (GH) in which the RSP sorting signal has been
mutated, such that the GH can be constitutively secreted by the
nonregulated secretory pathway (NRSP) in a mammalian cell. The
invention also provides a nucleic acid molecule encoding a GH in
which the three-dimensional conformation of the RSP sorting signal
has been altered such that the GH can be constitutively secreted by
the NRSP in a mammalian cell.
Inventors: |
Loh; Yoke Peng; (Bethesda,
MD) ; Cawley; Niamh; (Bethesda, MD) ; Baum;
Bruce J.; (Bethesda, MD) ; Snell; Christopher R.;
(Norfolk, GB) |
Assignee: |
THE UNITED STATES OF AMERICA,
REPRESENTED BY, DEPA RTMENT OF HEALTH
BETHESDA
MD
|
Family ID: |
23117747 |
Appl. No.: |
13/009414 |
Filed: |
January 19, 2011 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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11850947 |
Sep 6, 2007 |
7888070 |
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13009414 |
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10477651 |
Nov 14, 2003 |
7271150 |
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PCT/US02/15172 |
May 14, 2002 |
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11850947 |
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60290836 |
May 14, 2001 |
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Current U.S.
Class: |
424/93.21 ;
514/44R; 530/397; 536/23.51 |
Current CPC
Class: |
A61P 43/00 20180101;
C07K 2319/055 20130101; A61K 38/00 20130101; C07K 2319/02 20130101;
C07K 14/61 20130101; C12N 2799/022 20130101; A61P 5/06
20180101 |
Class at
Publication: |
424/93.21 ;
514/44.R; 530/397; 536/23.51 |
International
Class: |
A61K 35/12 20060101
A61K035/12; A61K 48/00 20060101 A61K048/00; C07K 14/61 20060101
C07K014/61; C07H 21/00 20060101 C07H021/00; A61P 5/06 20060101
A61P005/06; C07H 1/00 20060101 C07H001/00; C07K 1/107 20060101
C07K001/107 |
Claims
1-35. (canceled)
36. A method of treating GHD in a mammal, which method comprises
administering to the mammal an isolated and purified nucleic acid
molecule encoding GH in which the RSP sorting signal has been
mutated such that the GH can be constitutively secreted by the
nonregulated secretory pathway (NRSP) in a mammalian cell or a
vector comprising the isolated and purified nucleic acid, which
expresses an effective amount of the encoded GH, whereupon the GHD
in the mammal is treated.
37. The method of claim 36, wherein the vector is administered to
the mammal by infusion via the main excretory ducts of the salivary
gland of the mammal.
38. The method of claim 36, wherein the isolated and purified
nucleic acid molecule encodes the amino acid sequence of SEQ ID NO:
2 in which the sorting signal comprises Glu 174, Leu 177, Val 185
and Glu 186 and one or more of the codons encoding the
aforementioned amino acids is mutated or a vector comprising the
isolated and purified nucleic acid, which expresses an effective
amount of the encoded GH, whereupon the GHD in the mammal is
treated.
39. The method of claim 38, wherein the vector is administered to
the mammal by infusion via the main excretory ducts of the salivary
gland of the mammal.
40. A method of treating GHD in a mammal, which method comprises
administering to the mammal an isolated and purified nucleic acid
molecule encoding GH in which the three-dimensional conformation of
the RSP sorting signal has been altered such that the GH can be
constitutively secreted by the NRSP in a mammalian cell or a vector
comprising the isolated and purified nucleic acid, which expresses
an effective amount of the encoded GH, whereupon the GHD in the
mammal is treated.
41. The method of claim 40, wherein the vector is administered to
the mammal by infusion via the main excretory ducts of the salivary
gland of the mammal.
42. A method of treating GHD in a mammal, which method comprises
administering to the mammal host cells which have been contacted ex
vivo with an isolated and purified nucleic acid molecule encoding
GH in which the RSP sorting signal has been mutated such that the
GH can be constitutively secreted by the nonregulated secretory
pathway (NRSP) in a mammalian cell or a vector comprising the
isolated and purified nucleic acid, which expresses an effective
amount of the encoded GH, whereupon the GHD in the mammal is
treated.
43. The method of claim 42, wherein the host cells are autologous
to the mammal.
44. The method of claim 43, wherein the host cells are derived from
a biopsy of secretory gland tissue.
45. The method of claim 44, wherein the secretory gland tissue is
salivary gland tissue.
46. The method of claim 42, wherein the isolated and purified
nucleic acid molecule of claim 36, which encodes the amino acid
sequence of SEQ ID NO: 2 in which the sorting signal comprises Glu
174, Leu 177, Val 185 and Glu 186 and one or more of the codons
encoding the aforementioned amino acids is mutated or a vector
comprising the isolated and purified nucleic acid, which expresses
an effective amount of the encoded GH, whereupon the GHD in the
mammal is treated.
47. The method of claim 46, wherein the host cells are autologous
to the mammal.
48. The method of claim 47, wherein the host cells are derived from
a biopsy of secretory gland tissue.
49. The method of claim 48, wherein the secretory gland tissue is
salivary gland tissue.
50. A method of treating GHD in a mammal, which method comprises
administering to the mammal host cells which have been contacted ex
vivo with an isolated and purified nucleic acid molecule encoding
GH in which the three-dimensional conformation of the RSP sorting
signal has been altered such that the GH can be constitutively
secreted by the NRSP in a mammalian cell or a vector comprising the
isolated and purified nucleic acid, which expresses an effective
amount of the encoded GH, whereupon the GHD in the mammal is
treated.
51. The method of claim 50, wherein the host cells are autologous
to the mammal.
52. The method of claim 51, wherein the host cells are derived from
a biopsy of secretory gland tissue.
53. The method of claim 52, wherein the secretory gland tissue is
salivary gland tissue.
54. A method making a GH in which the RSP sorting signal is
mutated, which method comprises mutating one or more amino acids in
the RSP sorting signal in GH, whereupon a GH in which the RSP
sorting signal is mutated is obtained.
55. A GH with a mutated sorting signal obtained in accordance with
claim 54.
56. A method making a GH in which the three-dimensional
conformation of the RSP sorting signal is altered, which method
comprises mutating one or more amino acids outside of the RSP
sorting signal so that the three-dimensional conformation of the
amino acids of the RSP sorting signal in GH is altered, whereupon a
GH with an RSP having an altered three-dimensional conformation is
obtained.
57. A GH with an RSP sorting signal having an altered
three-dimensional conformation obtained in accordance with claim
56.
58. A method of making a nucleic acid molecule encoding a GH that
can be constitutively secreted by the NRSP in a mammalian cell,
which method comprises mutating one or more codons encoding amino
acids in the RSP sorting signal in an isolated and purified nucleic
acid molecule encoding GH such that, upon expression in a mammalian
cell, the GH can be constitutively secreted by the NRSP in the
mammalian cell.
59. A nucleic acid molecule encoding a GH with a mutated RSP
sorting signal obtained in accordance with the method of claim
58.
60. A method of making a nucleic acid molecule encoding a GH that
can be constitutively secreted by the NRSP in a mammalian cell,
which method comprises mutating one or more codons encoding amino
acids outside of the RSP sorting signal in an isolated and purified
nucleic acid molecule encoding GH such that, the three-dimensional
conformation of the amino acids of the RSP sorting signal in GH is
altered and, upon expression in a mammalian cell, the GH can be
constitutively secreted by the NRSP in the mammalian cell.
61. A nucleic acid molecule encoding a GH with an RSP sorting
signal having an altered three-dimensional conformation obtained in
accordance with the method of claim 60.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention relates to modified growth hormone and
related compositions, nucleic acids, vectors, isolated host cells
comprising such vectors, and methods of manufacture and use.
BACKGROUND OF THE INVENTION
[0002] Growth hormone acts through binding to membrane receptors
that belong to the cytokine receptor superfamily (Finidori, Vitam.
Horm. 59: 71-97 (2000)). Ligand binding induces receptor
dimerization and activation of the receptor-associated kinase
JAK-2, resulting in phosphorylation of the kinase, the receptor and
many cellular proteins (Finidori (2000), supra). Activation by
growth hormone is very transient and several mechanisms are
involved in downregulation, including internalization and
degradation of the receptor and recruitment of phosphatases or
specific inhibitors of the JAK/Stat pathway, namely the SOCS
proteins (Finidori (2000), supra).
[0003] There are variant forms of human growth hormone (hGH) which
include a disulfide dimer, a glycosylated form (20 kD hGH) and two
pituitary peptides made up of portions of 22 kD hGH (Lewis et al.,
Endocr. J. 47 Suppl: S1-8 (March 2000)). The two pituitary peptides
(hGH (1-43) and hGH (44-191)) have, respectively,
insulin-potentiating and anti-insulin properties (Lewis et al.
(March 2000), supra). The smaller peptide may be useful in
decreasing the amount of exogenous insulin required by diabetics,
whereas the larger peptide may be involved in diabetic retinopathy
(Lewis et al. (March 2000), supra).
[0004] The increased availability of growth hormone (GH) in the
mid-1980s, due to advances in recombinant DNA technology, has
allowed research into the use of this hormone at physiological
dosage as replacement therapy for adults and children with GH
deficiency (GHD) (see, e.g., Carroll et al., Trends Endocrinol.
Metab. 11(6): 231-238 (August 2000)) and at pharmocological dosages
as a possible therapeutic agent for a number of disease states
(Murray et al., Expert Opin. Pharmacother. 1(5): 975-990 (July
2000); see, also, Wit, Endocr. Regul. 34(1): 28-32 (March 2000)).
Such disease states include frailty associated with ageing,
osteoporosis, morbid obesity, cardiac failure, major thermal
injury, hypoglycemic unawareness in diabetes mellitus (Sonksen et
al., U.S. Pat. No. 5,426,096, issued Jun. 20, 1995), various acute
and chronic catabolic conditions (Murray et al. (July 2000), supra;
see, also, Mehls et al., Growth Horm. IGF Res. 10 Suppl. B: S31-37
(April 2000)) and intoxication with poisonous substances that are
degraded in the liver by microsomal enzymes (Jorgensen, U.S. Pat.
No. 4,816,439, issued Mar. 28, 1989). In combination with DHEA, its
use has been proposed for regenerating an involuted thymus (Fahy,
International Patent Application WO 95/32991, published Dec. 7,
1995).
[0005] MAD in humans is currently treated by growth hormone
injection. The problem with such a treatment method is that
injections are required every day or couple of days (MacGillivray
et al., J. Clin. Endocrinol. Metab. 81(5):1806-1809 (May 1996)).
The present invention seeks to overcome such a problem. This and
other objects and advantages, as well as additional inventive
features, will become apparent from the detailed description
provided herein.
BRIEF DESCRIPTION OF THE INVENTION
[0006] The present invention provides an isolated and purified GH
in which the regulated secretory pathway (RSP) sorting signal has
been mutated as well as an isolated and purified GH in which the
three-dimensional conformation of the RSP sorting signal has been
altered. Also provided is a composition comprising an effective
amount of such an isolated and purified GH in an excipient.
[0007] Further provided is an isolated and purified nucleic acid
molecule encoding GH in which the RSP sorting signal has been
mutated such that the GH can be constitutively secreted by the
nonregulated secretory pathway (NRSP) in a mammalian cell as well
as an isolated and purified nucleic acid molecule encoding GH in
which the three-dimensional conformation of the RSP sorting signal
has been altered such that the GH can be constitutively secreted by
the NRSP in a mammalian cell. Still further provided are a vector
comprising such an isolated and purified nucleic acid molecule and
an isolated host cell comprising such a vector.
[0008] A method of treating GHD in a mammal is also provided. The
method comprises administering to the mammal the aforementioned
composition, nucleic acid or vector, wherein the nucleic acid or
vector expresses an effective amount of the encoded GH and
whereupon GHD in the mammal is treated.
[0009] Also provided are a method of making a GH in which the RSP
sorting signal is mutated and the GH so produced. The method
comprises mutating one or more amino acids in the RSP sorting
signal in GH, whereupon a GH in which the RSP sorting signal is
mutated is obtained.
[0010] Still also provided are a method of making a GH in which the
three-dimensional conformation of the RSP sorting signal is altered
and the GH so produced. The method comprises mutating one or more
amino acids outside of the RSP sorting signal so that the
three-dimensional conformation of the amino acids of the RSP
sorting signal in GH is altered, whereupon a GH with an RSP having
an altered three-dimensional conformation is obtained.
[0011] Similarly provided is a method of making a nucleic acid
molecule encoding a GH that can be constitutively secreted by the
NRSP in a mammalian cell and the nucleic acid molecule so produced.
The method comprises mutating one or more codons encoding amino
acids in the RSP sorting signal in an isolated and purified nucleic
acid molecule encoding GH such that, upon expression in a mammalian
cell, the GH can be constitutively secreted by the NRSP in a
mammalian cell.
[0012] Also similarly provided is a method of making a nucleic acid
molecule encoding a GH that can be constitutively secreted by the
NRSP in a mammalian cell and the nucleic acid molecule so produced.
The method comprises mutating one or more codons encoding amino
acids outside of the RSP sorting signal in an isolated and purified
nucleic acid molecule encoding GH such that the three-dimensional
conformation of the amino acids of the RSP sorting signal in GH is
altered and, upon expression in a mammalian cell, the GH can be
constitutively secreted by the NRSP in a mammalian cell.
BRIEF DESCRIPTION OF THE FIGURE
[0013] FIG. 1 sets forth nucleic acid (SEQ ID NO: 1) and amino acid
(SEQ ID NOS: 2 (lower case letters represent single letter
designations of amino acids in accordance with convention) and 3)
sequences with respect to GH and modified GH.
DETAILED DESCRIPTION OF THE INVENTION
[0014] The present invention provides an isolated and purified GH
in which the RSP sorting signal has been mutated. Any GH can be
modified in accordance with the present invention. Preferably, the
GH is mammalian. Particularly preferred is hGH. The amino acid
sequence of hGH is known (Genbank accession no. A 15072; see also
biosynthetic hGH of Dalboge et al., U.S. Pat. No. 5,633,352, issued
May 27, 1997, and U.S. Pat. No. 5,635,604, issued Jun. 3, 1997) and
is reproduced herein as SEQ ID NO: 2 (see FIG. 1). By "mutated" is
meant chemical modification, substitution, deletion or insertion.
Methods of chemical modification, substitution, deletion and
insertion are known in the art and include in vitro chemical
synthesis (e.g., Merrifield synthesis) of the desired mutant GH
(see, e.g., Barany et al., in The Peptides, Gross and Meienhofer,
eds. Academic Press: New York (1979), Vol. 2, pp. 3-254; and
Parkhurst et al., J. Immunol. 157: 2539-2548 (1996)). Substitution
is preferred. Preferably, the isolated and purified GH consists
essentially of the amino acid sequence of SEQ ID NO: 2 in which the
sorting signal comprises glutamic acid at amino acid position 174
(Glu 174), leucine at amino acid position 177 (Leu 177), valine at
amino acid position 185 (Val 185) and glutamic acid at amino acid
position 186 (Glu 186) and one or more of the aforementioned amino
acids is mutated. Preferably, each of Glu 174 and Glu 186 is
mutated, preferably by substitution with alanine.
[0015] Especially preferred is when the isolated and purified GH
consists essentially of the amino acid sequence of SEQ ID NO: 2 in
which the sorting signal comprises glutamic acid at amino acid
position 174 (Glu 174), leucine at amino acid position 177 (Leu
177), valine at amino acid position 185 (Val 185) and glutamic acid
at amino acid position 186 (Glu 186), each of Glu 174 and Glu 186
is substituted with alanine, and Phe 191 is substituted with
LLGILQISSTVAAARV (see SEQ ID NO: 3 in FIG. 1). Optionally, Leu 177
and/or Val 185 is/are mutated, such as by substitution.
[0016] Also provided is an isolated and purified GH in which the
three-dimensional conformation of the RSP sorting signal has been
altered. Preferably, the isolated and purified GH consists
essentially of the amino acid sequence of SEQ ID NO: 2 in which the
sorting signal comprises Glu 174, Leu 177, Val 185 and Glu 186 and
in which one or more amino acids outside of the RSP sorting signal
is mutated. Preferably, the cysteine at amino acid position 189
(Cys 189) is mutated, preferably by substitution with serine.
[0017] While the above-described mutations are preferred, other
mutations that either interfere with the charge of the acidic
residues or alter the three-dimensional conformation of the sorting
signal are possible. For example, a positively charged residue(s),
such as arginine or lysine, can be introduced close to or beside
either one or both of the glutamic acid residues in order to
neutralize the acidic charge of the neighboring glutamic acid
residue. Alternatively, a praline residue can be introduced near
the sorting signal so as to alter the three-dimensional
conformation of the sorting signal region.
[0018] In view of the above, the present invention also provides a
composition comprising an effective amount of an above-described
isolated and purified GH in an excipient, such as a vehicle,
adjuvant, carrier or diluent, which is desirably pharmaceutically
acceptable, as known in the art. See, for example, Pharmaceutics
and Pharmacy Practice, J. B. Lippincott Co., Philadelphia, Pa.,
Banker and Chalmers, eds. (1982), and ASHP Handbook on Injectable
Drugs, Toissel, 4.sup.th ed. (1986).
[0019] Such compositions can further comprise asparagine (Sorensen,
U.S. Pat. No. 5,851,992, issued Dec. 22, 1998). Injectable aqueous
formulations, such as those which comprise a buffer, nonionic
surfactants and neutral salts are known in the art (see, e.g.,
O'Connor et al., U.S. Pat. No. 5,763,394, issued Jun. 9, 1998; and
U.S. Pat. No. 5,981,485, issued Nov. 9, 1999). Sustained-release
compositions, such as those comprising GH complexed with a metal,
such as zinc, are described by Johnson et al. (U.S. Pat. No.
5,667,808, issued Sep. 16, 1997). Metal-complexed GH can be further
combined with a biocompatible polymer (see, e.g., Johnson et al.,
U.S. Pat. No. 5,654,010, issued Aug. 5, 1997; U.S. Pat. No.
5,891,478, issued Apr. 6, 1999; U.S. Pat. No. 6,051,259, issued
Apr. 18, 2000; and International Patent Application WO 96/40072).
Glycine and mannitol also can be used to stabilize GH for
parenterally administered formulations (see, e.g., Pikal et al.,
U.S. Pat. No. 5,612,315, issued Mar. 18, 1997; and Pearlman et al.,
U.S. Pat. No. 5,096,885, issued Mar. 17, 1992). Saccharose, alone
or in further combination with mannitol, can be used to stabilize
GH as a solid intimate mixture (see, e.g., Samaritani, U.S. Pat.
No. 5,898,030, issued Apr. 27, 1999). Injectable GH formulations
which comprise citrate can be stable for at least 12 months (see,
e.g., Castensson et al., U.S. Pat. No. 5,567,677, issued Oct. 22,
1996). Solubility of GH in an aqueous solution can be enhanced by
the presence of creatinine, an acetyl tryptophan salt and/or
nicotinamide (see, e.g., U.S. Pat. No. 6,013,773, issued Jan. 11,
2000).
[0020] Also in view of the above, the present invention provides an
isolated and purified nucleic acid molecule encoding GH in which
the RSP sorting signal has been mutated such that the hGH can be
constitutively secreted by the nonregulated secretory pathway
(NRSP) in a mammalian cell. Any nucleic acid molecule encoding a GH
can be modified in accordance with the present invention.
Preferably, the GH is mammalian. Particularly preferred is hGH. The
nucleotide sequence of hGH is known (Genbank accession no. A 15072)
and is reproduced herein as SEQ ID NO: 1 (see FIG. 1). Methods of
introducing mutations at the nucleic acid level are known in the
art and include the methods of Example 1, site-specific mutagenesis
(Carter et al., Nucl. Acids Res. 13: 4331 (1986); and Zoller et
al., Nucl. Acids Res. 10: 6487 (1987)), cassette mutagenesis (Wells
et al., Gene 34: 315 (1985)), restriction selection mutagenesis
(Wells et al., Philos. Trans. R. Soc. London SerA 317: 415 (1986))
and DNA synthesis of the mutated GH. When modifying the nucleic
acid so that a new amino acid is substituted for that which is
naturally occurring, the codon encoding the amino acid sequence to
be substituted may be any of the alternative codons known to code
for the particular amino acid (see, e.g. Lewin, GENES V, Oxford
University Press, page 172 (1994)). For example, when the desired
substitution is to result in the amino acid alanine, the codons
which could be used include GCT, GCC, GCA or GCG. Substitution is
preferred. Preferably, the isolated and purified nucleic acid
molecule encodes the amino acid sequence of SEQ ID NO: 2 in which
the sorting signal comprises Glu 174, Leu 177, Val 185 and Glu 186
and one or more of the codons encoding the aforementioned amino
acids is mutated. Preferably, each of the codons encoding Glu 174
and Glu 186 is mutated, preferably mutated to encode alanine.
[0021] Especially preferred is when the isolated and purified
nucleic acid molecule encodes the amino acid sequence of SEQ ID NO:
3. Optionally, the isolated and purified nucleic acid molecule
encodes a mutation of Leu 177 and/or Val 185, such as a
substitution.
[0022] Still also in view of the above, the present invention
provides an isolated and purified nucleic acid molecule encoding GH
in which the three-dimensional conformation of the RSP sorting
signal has been altered such that the GH can be constitutively
secreted by the NRSP in a mammalian cell. Preferably, the isolated
and purified nucleic acid molecule encodes the amino acid sequence
of SEQ ID NO: 2 in which the sorting signal comprises Glu 174, Len
177, Val 185 and Glu 186 and one or more of the codons encoding
amino acids outside of the sorting signal is mutated. Preferably,
the codon encoding Cys 189 is mutated, preferably mutated to encode
serine.
[0023] A vector comprising an above-described isolated and purified
nucleic acid molecule is also provided. Vectors and vector
construction are known in the art (see, e.g., Maniatis et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, N.Y. (1982)). Preferred vectors for use in the context
of the present invention include adenoviral vectors and
adeno-associated viral (AAV) vectors. AAV vectors have been
developed for a number of AAV serotypes, including AAV2 (see, e.g.,
Carter et al., U.S. Pat. No. 4,797,368, issued Jan. 10, 1989, and
U.S. Pat. No. 5,587,308, issued Dec. 24, 1996), AAV4 (see, e.g.,
Chiorini et al., International Patent Application WO 98/11244,
published Mar. 19, 1998) and AAV5 (see, e.g., Chiorini, et al. WO
99/61601. published Dec. 2, 1999). Other vectors which may be
useful include lentivirus-based vectors (see, e.g., D'Costa et al.,
J. Gen. Virol. 82(Pt 2): 425-434 (February 2001); Arya,
International Patent Application WO 00/40741, published Jul. 13,
2000; and Morgan et al., International Patent Application WO
98/13511, published Apr. 2, 1998) and hybrid or chimeric viral
vectors or vector systems comprising, for example, an adenoviral
backbone with lentiviral components (see, e.g., Zheng et al.,
Nature Biotechnology 18(2): 176-80 (February 2000); Curiel et al.,
International Patent Application WO 98/22143, published May 28,
1998; Ramsey et al., International Patent Application WO 98/46778,
published Oct. 22, 1998; and Ramsey et al., International Patent
Application WO 00/17376, published Mar. 30, 2000), or an adenoviral
backbone with AAV components (Fisher et al., Human Gene Therapy 7:
2079-2087 (1996)). While the promoter native to hGH can be used,
preferably a nonnative promoter is used. Examples of such nonnative
promoters include various constitutive and regulatable promoters.
Examples of regulatable promoters include inducible, repressible
and tissue-specific promoters. Specific examples include viral
promoters, preferable adenoviral promoters and AAV promoters, and a
promoter that is specific for expression in the salivary gland,
such as the promoter from the amylase gene. Preferably, the
promoter is an adenoviral promoter.
[0024] Accordingly, an isolated host cell comprising the
above-described vector is also provided. Any suitable host cell can
be used. Examples include prokaryotic host cells, such as E. coil,
in particular K12 strain 294 (American Type Culture Collection
(ATCC) No.
[0025] 31446), B, X1776 (ATCC No. 31537), c600, c600hfl, W3110
(ATCC No. 27,325), JM101, HB101, NM522, NM538 and NM539, Bacillus
subtilis, Salmonella typhimurium, Serratia marcescens, and
Pseudomonas. Eukaryotic host cells include, for example, yeast and
cells derived from a mammal, including human cell lines. Specific
examples of suitable eukaryotic host cells include VERO, HeLa, 3T3,
Chinese hamster ovary (CHO) cells, W138 BHK, COS-7 and MDCK.
Alternatively, cells from a human to be treated in accordance with
the methods described herein can be used as host cells. Methods of
introducing vectors into isolated host cells and the culture and
selection of transformed host cells in vitro are known in the art
and include the use of calcium chloride-mediated transformation,
transduction, conjugation, triparental mating, DEAE,
dextran-mediated transfection, infection, membrane fusion with
liposomes, high velocity bombardment with DNA-coated
microprojectiles, direct microinjection into single cells, and
electroporation (see, e.g., Sambrook et al., Molecular Biology: A
Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. (1989);
Davis et al., Basic Methods in Molecular Biology (1986), and
Neumann et al., EMBO J. 1: 841 (1982)).
[0026] The forms of the introduced vector can vary with the
rationale underlying the introduction of the vector into the host
cell. For example, the nucleic acid can be closed circular, nicked,
or linearized, depending on whether the vector is to be maintained
extragenomically (i.e., as an autonomously replicating vector),
integrated as a provirus or prophage, transiently transfected,
transiently infected as with use of a replication-deficient or
conditionally replicating virus or phage, or stably introduced into
the host genome through double or single crossover recombination
events.
[0027] In addition to the above, the present invention provides a
method of treating GHD in mammals, in particular a human. In one
embodiment, the method comprises administering to the mammal an
above-described composition, whereupon the GHD in the mammal is
treated. While any species of mammal can be used as the source of
the GH, desirably the GH is from the same species as the mammal
being treated. Any suitable route of administration can be used in
the context of this method, including local and systemic
administration, such as parenteral, i.e., subcutaneous,
intramuscular, intravenous, intraarterial and intraperitoneal
administration. Preferably, the composition is administered to the
mammal by subsutaneous injection to the mammal. In another
embodiment, the method comprises administering to the mammal an
above-described nucleic acid or vector that expresses an effective
amount of the encoded GH, whereupon the GHD in the mammal is
treated. As indicated above, while any species can be used as the
source of nucleic acid encoding GH, desirably the GH is from the
same species as the mammal being treated. While any suitable route
of administration can be used in the context of this method,
preferably, the vector is administered to the mammal in vivo, such
as by infusion via the main excretory ducts of the salivary gland
("salivary gland" includes glandulae salivariae majores (parotid,
sublingual and submandibular glands) and glandulae salivariae
minores of the tongue, lips, cheeks and palate (labial, buccal,
molar, palatine, lingual and anterior lingual glands)) of the
mammal (see, e.g., Example 4 and the references cited therein and
German et al., U.S. Pat. No. 5,885,971, issued Mar. 23, 1999).
Alternatively, an above-described nucleic acid or vector encoding
GH in accordance with the present invention can be contacted with
host cells ex vivo and subsequently administered to the mammal to
be treated. Preferably, the host cells are autologous cells, such
as biopsied secretory gland tissue, e.g., salivary gland tissue
(see, e.g., German et al., supra).
[0028] Generally, the effective amount of modified GH administered
parenterally per dose is in the range of about 1 .mu.g/kg body
weight/day to about 100 .mu.g/kg body weight/day. Usually, the
effective amount of modified GH administered parenterally per dose
is in the range of about 0.01 mg/kg body weight/day and 10 mg/kg
body weight/ day. Even more usually, the effective amount of
modified hGH administered parenterally per dose is in the range of
about 0.01 .mu.g/kg/day and 1 .mu.g/kg/day. If given continuously,
the modified GH is typically administered at a dose rate of about 1
.mu.g/kg body weight/hr to about 50 .mu.g/kg body weight/hr, such
as by one to four injections per day or by continuous subcutaneous
infusions. Administration can be repeated daily, three times per
week, every three days or once a month. Typically, administration
is repeated about once a day to every 2-3 days.
[0029] Desirably, an above-described vector that expresses an
effective amount of modified GH is administered. When an
above-described nucleic acid or vector is administered to the
salivary gland, from about 1 .mu.g to 200 mg, preferably from about
100 .mu.g to 100 mg, more preferably from about 500 .mu.g to 50 mg,
most preferably about 10 mg, of vector are administered. If the
vector is a viral vector, preferably a tissue concentration of
about 10.sup.2 to about 10.sup.12 viral particles per ml is
attained. Generally, the amount of vector necessary can be
extrapolated from animal models. For example, the amount of DNA to
be administered to a human is about 10-100 times the amount of DNA
to be administered to a rat. Use of an adequate vector, which is
preferably a viral vector, obviates the need for frequent repeat
administrations. When a vector is administered, the vector is
preferably administered once or up to about once per month.
[0030] The present inventive method of treatment can be used to
treat other conditions or disease states in addition to MD in which
the administration of hGH would be beneficial. For example, the
method can be used to treat frailty associated with ageing,
osteoporosis, morbid obesity, cardiac failure, major thermal
injury, hypoglycemic unawareness in diabetes mellitus (Sonksen et
al. (Jun. 20, 1995), supra), various acute and chronic catabolic
conditions (Murray et al. (July 2000), supra; Mehls et al. (April
2000), supra) and intoxication with poisonous substances that are
degraded in the liver by microsomal enzymes (Jorgensen (Mar. 28,
1989), supra).
[0031] A method of making a GH in which the RSP sorting signal is
mutated is also provided. The method comprises mutating one or more
amino acids in the RSP sorting signal in GH, whereupon a GH in
which the RSP sorting signal is mutated is obtained. As indicated
above, methods of mutating amino acids are known in the art.
Accordingly, a GH with a mutated RSP sorting signal obtained in
accordance with such a method is also provided. Further provided is
a method of making a GH in which the three-dimensional conformation
of the RSP sorting signal is altered. The method comprises mutating
one or more amino acids outside of the RSP sorting signal so that
the three-dimensional conformation of the amino acids of the RSP
sorting signal in GH is altered, whereupon a GH with an RSP having
an altered three-dimensional conformation is obtained. Methods of
mutating amino acids are known in the art as indicated above.
Accordingly, a GH with an RSP sorting signal having an altered
three-dimensional conformation obtained in accordance with such a
method is also provided.
[0032] Still further provided is a method of making a nucleic acid
molecule encoding a GH that can be constitutively secreted by the
NRSP in a mammalian cell. The method comprises mutating one or more
codons encoding amino acids in the RSP sorting signal in an
isolated and purified nucleic acid molecule encoding GH such that,
upon expression in a mammalian cell, the GH can be constitutively
secreted by the NRSP in the mammalian cell. As indicated above,
methods of introducing mutations at the nucleic acid level are
known in the art. Accordingly, a nucleic acid molecule encoding a
GH with a mutated RSP sorting signal obtained in accordance with
such a method is also provided.
[0033] Yet still further provided is a method of making a nucleic
acid molecule encoding a GH that can be constitutively secreted by
the NRSP in a mammalian cell. The method comprises mutating one or
more codons encoding amino acids outside of the RSP sorting signal
in an isolated and purified nucleic acid molecule encoding GH such
that the three-dimensional conformation of the amino acids of the
RSP sorting signal in GH is altered and, upon expression in a
mammalian cell, the GH can be constitutively secreted by the NRSP
in the mammalian cell. Methods of introducing mutations at the
nucleic acid level are known in the art as indicated above.
Accordingly, a nucleic acid molecule encoding a GH with an RSP
sorting signal having an altered three-dimensional conformation
obtained in accordance with such a method is also provided.
[0034] Whether or not a recombinantly produced GH is secreted by
the NRSP in a mammalian cell and has biological activity can be
determined in accordance with the methods set forth in the
Examples. An alternative method of determining the biological
activity of recombinantly produced GH is described in Zaslaysky,
U.S. Pat. No. 5,734,024, issued Mar. 31, 1998.
EXAMPLES
[0035] The following examples serve to illustrate further the
present invention and are not intended to limit its scope in any
way.
Example 1
[0036] This example demonstrates the existence of an RSP sorting
signal in hGH and describes the essential amino acid residues of
the RSP sorting signal motif and their mutation leading to
constitutive secretion of hGH.
[0037] Experimental data have evidenced the existence of RSP
sorting signals in proopiomelanocortin (POMC; see, e.g., Cool et
al., J. Biol. Chem. 270(15): 8723-8729 (Apr. 14, 1995)) and
chromogranin B. Thus, hGH was examined for the presence of an RSP
sorting signal.
[0038] Initially, the amino acid sequences of growth hormones from
multiple species were analyzed to identify evolutionarily conserved
amino acids. Since the RSP sorting signals of POMC and chromogranin
B included acidic residues, acidic residues in the conserved amino
acid sequences were identified.
[0039] The X-ray crystal structure of hGH (Brookhaven Protein
Database accession no. 1HGU) was then analyzed to determine if the
conserved amino acids were exposed on the surface of the molecule
and, therefore, accessible to the sorting receptor. Since NMR
structural data were available for POMC and the molecular distances
between the amino acids of the POMC sorting signal were known,
several exposed amino acid residues in hGH having similar molecular
distances to those of POMC were selected. The selected amino acid
residues were in a region of the hGH molecule that was not involved
in the binding of hGH to the physiological GH receptor so as to
maintain biological activity for physiological signal
transduction.
[0040] Molecular distances between amino acid residues of the
proposed RSP sorting signal in hGH as compared to the molecular
distances between amino acid residues of the RSP sorting signal in
POMC
TABLE-US-00001 POMC hGH Acidic residue 1 (Asp10 for POMC; 3.82 4.57
Glu174 for hGH) to hydrophobic residue 1 (Leu11 for POMC; Leu177
for hGH) Acidic residue 2 (Glu14 for POMC; 9.29 3.79 Glu186 for
hGH) to hydrophobic residue 2 (Leu18 for POMC; Leu185 for hGH)
Acidic residue 1 (Asp10 for POMC; 11.6 17.59 Glu174 for hGH) to
acidic residue 2 (Glu14 for POMC; Glu186 for hGH)
Molecular distances are in Angstroms measured between the alpha
carbons of each indicated amino acid. The data for growth hormone
were obtained from its X-ray crystal structure and the data for
POMC were obtained from its NMR structure.
[0041] The molecular distances between the amino acid residues of
hGH as compared to POMC were sufficiently variable so as to require
experimental proof of a sorting signal for hGH. The selected amino
acid residues were tested empirically for their ability to direct
hGH to the RSP by mutating them and assaying for secretion. Mutants
were generated as follows. The wild-type hGH was generated by
reverse-transcriptase polymerase chain reaction (RT-PCR) from a
human pituitary cDNA library (Clontech, Palo Alto, Calif.) using a
kit from Boehringer-Mannheim (Indianapolis, Ind.). The hGH cDNA was
directionally subcloned into a mammalian expression vector,
pcDNA3.1 (InVitrogen, Carlsbad, Calif.). The plasmid was used as
the template for mutagenesis.
[0042] Mutagenesis was performed using the Quick Change mutagenesis
kit (Stratagene, LaJolla, Calif.). Briefly, oligonucleotide primers
bearing the mutant nucleotide were used in a PCR reaction to
amplify the pcDNA3.1-hGH plasmid. The parental DNA was then
digested with the restriction endonuclease, Dpn 1. The remaining
amplified DNA was transformed into a special strain of E. coli
(from Stratagene) and cultured. Colonies were picked and the
plasmid DNA isolated from the colonies was sequenced to confirm
that they contained the mutations. For double mutants, a second
round of mutagenesis with new mutant primers was carried out using
the first mutant as the PCR template.
[0043] The two acidic residues that caused mis-sorting to the
constitutive pathway were Glu 174 and Glu 186 and, thus, were
determined to be an essential part of the sorting signal motif.
Mutation of these glutamic acid residues to alanines, thereby
removing the negative charges associated with these residues
without causing major structural changes in the loop structure of
the sorting signal motif resulted in mis-sorting to the
constitutive pathway (i.e., NRSP). It was also determined that
mutation of Cys 189 in the loop disrupted stability afforded by the
disulfide bridge between Cys 189 and Cys 182. Mutations of either
or both of the two cysteine residues can affect the
three-dimensional conformation of the loop structure of the sorting
signal motif by causing unfolding of the C-terminal loop, thereby
disrupting the alignment of the acidic residues necessary for
sorting via the RSP.
Example 2
[0044] This example describes a method of assaying mutant GH for
secretion.
[0045] The RSP in (neuro)-endocrine cells can be stimulated to
release the peptide hormones that are stored within the cells. The
mechanism for stimulating these cells is widely used. Typically,
this is done by depolarizing the plasma membrane by adding 50 mM
K.sup.+ to the medium in the presence of calcium. Alternatively,
specific chemicals or other proteins (or peptides) can be added to
the medium that bind to a receptor on the plasma membrane and cause
stimulated secretion via signal transduction. Primarily, a
depolarizing procedure based on the procedure described in Gorr et
al., Am. J. Physiol. 277: C121-131 (1999), was used.
[0046] Plasmids of pcDNA3.1-GH or pcDNA3.1-mutant-GH were
transiently transfected into PC12 or AtT20 cells, which are model
(neuro)-endocrine cell lines that contain both a regulated and a
constitutive secretory pathway. Forty-eight hours after
transfection with lipofectamine 2000 (Gibco BRL, Rockville, Md.),
the PC12 cells were pre-incubated twice in a basal buffer (129 mM
NaCl, 10 mM HEPES, 5 mM NaHCO.sub.3, 4.8 mM KCl, 2.8 mM Glucose,
1.2 mM KH.sub.2PO.sub.4, 1.2 mM MgCl.sub.2, and 1 mM CaCl.sub.2, pH
7.4) for 15 and 30 minutes, respectively. Afterwards, the cells
were incubated in 1 ml of fresh basal buffer for 2 hr. This buffer
(M1) was then saved for Western blot analysis and replaced with
either of the same volume of basal buffer or stimulation buffer
(same as basal buffer but with 79 mM NaCl, 50 mM KCl, 2 mM
BaCl.sub.2 and no CaCl.sub.2) and incubated for 10 min. This buffer
(M2.sup.- and M2.sup.+) was also saved for Western blot analysis.
Immediately upon collection of either of the basal or stimulation
buffers, they were centrifuged at 1000.times.g to remove cell
debris prior to being processed for Western blot analysis. The
cells were washed twice with PBS and then harvested in 1 ml of
lysis buffer (50 mM HEPES, 150 mM NaCl, 10 mM EDTA, 10 mM sodium
pyrophosphate, 100 mM NaF, 2 mM sodium orthovanadate, 1% Triton
X-100, Boehringer Mannheim complete mini protease cocktail, and 1
.mu.M pepstatin A, pH 7.5). A soluble cell extract (L) was obtained
from the lysate after three freeze-thaw cycles and centrifugation
at 13,000 rpm for 10 min. The levels of GH in the media and lysates
were detected by Western blot analysis and/or radioimmunoassay
(RIA).
Example 3
[0047] This example describes a method of assaying mutant GH for
bioactivity.
[0048] 32D-rGHR-IRS-1 are special cells that have been engineered
to express the rabbit growth hormone receptor (rGHR) and an insulin
response substrate (IRS) (Liang et al., Endocrinology 140:
1972-1983 (1999)). These cells were provided by Dr. Stuart J.
Frank, University of Alabama. These cells require GH to survive
through the action of the rabbit GH receptor. They normally get
sufficient GH to survive from fetal bovine serum, which is added to
the regular culture medium.
[0049] In order to test the bioactivity of the GH mutants, the
32D-rGHR-IRS-1 cells were starved of serum for 5 hr and then plated
into a 24-well plate. Control serum-free medium, GH standards
(purified protein), and expressed GH (wild-type or mutants in
conditioned medium) were separately added to the serum starved
cells and the cells were allowed to grow for 12-16 hr. The number
of viable cells remaining in the wells was then quantified by
trypan blue exclusion. Dead cells absorb the blue dye, whereas
viable cells actively exclude the dye. In the presence of either GH
standard or any GH expressed from the wild-type or mutant
constructs, the number of viable cells remained high. In the
presence of serum-free medium that contained no GH, the number of
viable cells was dramatically reduced. Thus, the mutant GH proteins
were biologically active. In addition, the mutants appeared to be
as biologically active as wild-type GH.
Example 4
[0050] This example describes a method of using an adenoviral
vector to transfer a gene encoding mutant GH to a salivary gland of
a mammal in vivo.
[0051] Adenoviral vectors were constructed in accordance with the
methods of Becker et al., Methods Cell. Biol. 43 Pt A: 161-189
(1994); Delporte et al., J. Biol. Chem. 271: 22070-22075 (1996);
and He et al., Gene Therapy 5: 537-541 (1998). "First generation"
recombinant adenoviruses (serotype 5, E1-deleted) were used The
adenoviral vectors encoding mutant GH were transferred to salivary
glands, such as parotids, submandibular glands and sublingual
glands, by retrograde infusion following cannulation of the main
excretory ducts (see, e.g., Mastrangeli et al., Am. J. Physiol.
266: G1146-01155 (1994); Baum et al. Ann. N.Y. Acad. Sci. 875:
294-300 (1999); Baccaglini et al., J. Gene. Med. 3: 82-90 (2001);
Wang et al., J. Dental Res. 79: 701-708 (1999); and O'Connell et
al., Cancer Gene Ther. 6: 505-513 (1999)).
[0052] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0053] All amino acid numbering herein is based on the mature
proteins. The amino acids are numbered consecutively from the
N-terminus to the C-terminus of the mature protein starting with
"1" in accordance with convention.
[0054] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. Recitation of ranges of values
herein are merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range, unless otherwise indicated herein, and each separate value
is incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0055] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Of course, variations of those preferred
embodiments will become apparent to those of ordinary skill in the
art upon reading the foregoing description. The inventors expect
skilled artisans to employ such variations as appropriate, and the
inventors intend for the invention to be practiced otherwise than
as specifically described herein. Accordingly, this invention
includes all modifications and equivalents of the subject matter
recited in the claims appended hereto as permitted by applicable
law. Moreover, any combination of the above-described elements in
all possible variations thereof is encompassed by the invention
unless otherwise indicated herein or otherwise clearly contradicted
by context.
Sequence CWU 1
1
31651DNAHomo sapiens 1atggctacag gctcccggac gtccctgctc ctggcttttg
gcctgctctg cctgccctgg 60cttcaagagg gcagtgcctt cccaaccatt cccttatcca
ggctttttga caacgctatg 120ctccgcgccc atcgtctgca ccagctggcc
tttgacacct accaggagtt tgaagaagcc 180tatatcccaa aggaacagaa
gtattcattc ctgcagaacc cccagacctc cctctgtttc 240tcagagtcta
ttccgacacc ctccaacagg gaggaaacac aacagaaatc caacctagag
300ctgctccgca tctccctgct gctcatccag tcgtggctgg agcccgtgca
gtccctcagg 360agtgtcttcg ccaacagcct ggtgtacggc gcctctgaca
gcaacgtcta tgacctccta 420aaggacctag aggaaggcat ccaaacgctg
atggggaggc tggaagatgg cagcccccgg 480actgggcaga tcttcaagca
gacctacagc aagttcgaca caaactcaca caacgatgac 540gcactactca
agaactacgg gctgctctac tgcttcagga aggacatgga caaggtcgag
600acattcctgc gcatcgtgca gtgccgctct gtggagggca gctgtggctt c
6512217PRTHomo sapiens 2Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu
Ala Phe Gly Leu Leu1 5 10 15Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala
Phe Pro Thr Ile Pro Leu 20 25 30Ser Arg Leu Phe Asp Asn Ala Met Leu
Arg Ala His Arg Leu His Gln 35 40 45Leu Ala Phe Asp Thr Tyr Gln Glu
Phe Glu Glu Ala Tyr Ile Pro Lys 50 55 60Glu Gln Lys Tyr Ser Phe Leu
Gln Asn Pro Gln Thr Ser Leu Cys Phe65 70 75 80Ser Glu Ser Ile Pro
Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys 85 90 95Ser Asn Leu Glu
Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp 100 105 110Leu Glu
Pro Val Gln Ser Leu Arg Ser Val Phe Ala Asn Ser Leu Val 115 120
125Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu
130 135 140Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly Ser
Pro Arg145 150 155 160Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys
Phe Asp Thr Asn Ser 165 170 175His Asn Asp Asp Ala Leu Leu Lys Asn
Tyr Gly Leu Leu Tyr Cys Phe 180 185 190Arg Lys Asp Met Asp Lys Val
Glu Thr Phe Leu Arg Ile Val Gln Cys 195 200 205Arg Ser Val Glu Gly
Ser Cys Gly Phe 210 2153232PRTArtificialSynthetic 3Met Ala Thr Gly
Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu1 5 10 15Cys Leu Pro
Trp Leu Gln Glu Gly Ser Ala Phe Pro Thr Ile Pro Leu 20 25 30Ser Arg
Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln 35 40 45Leu
Ala Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys 50 55
60Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe65
70 75 80Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln
Lys 85 90 95Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln
Ser Trp 100 105 110Leu Glu Pro Val Gln Ser Leu Arg Ser Val Phe Ala
Asn Ser Leu Val 115 120 125Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp
Leu Leu Lys Asp Leu Glu 130 135 140Glu Gly Ile Gln Thr Leu Met Gly
Arg Leu Glu Asp Gly Ser Pro Arg145 150 155 160Thr Gly Gln Ile Phe
Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser 165 170 175His Asn Asp
Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe 180 185 190Arg
Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys 195 200
205Arg Ser Val Glu Gly Ser Cys Gly Leu Leu Gly Ile Leu Gln Ile Ser
210 215 220Ser Thr Val Ala Ala Ala Arg Val225 230
* * * * *