U.S. patent application number 13/770606 was filed with the patent office on 2014-01-23 for follicle stimulating hormone superagonists.
This patent application is currently assigned to TROPHOGEN, INC.. The applicant listed for this patent is TROPHOGEN, INC.. Invention is credited to MARIUSZ W. SZKUDLINSKI, BRUCE D. WEINTRAUB.
Application Number | 20140024589 13/770606 |
Document ID | / |
Family ID | 34994354 |
Filed Date | 2014-01-23 |
United States Patent
Application |
20140024589 |
Kind Code |
A1 |
SZKUDLINSKI; MARIUSZ W. ; et
al. |
January 23, 2014 |
Follicle Stimulating Hormone Superagonists
Abstract
Modified VEGF proteins that inhibit VEGF-mediated activation or
proliferation of endothelial cells are disclosed. The analogs may
be used to inhibit VEGF-mediated activation of endothelial cells in
angiogenesis-associated diseases such as cancer, inflammatory
diseases, eye diseases, and skin disorders.
Inventors: |
SZKUDLINSKI; MARIUSZ W.;
(ROCKVILLE, MD) ; WEINTRAUB; BRUCE D.; (ROCKVILLE,
MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TROPHOGEN, INC. |
Rockville |
MD |
US |
|
|
Assignee: |
TROPHOGEN, INC.
ROCKVILLE
MD
|
Family ID: |
34994354 |
Appl. No.: |
13/770606 |
Filed: |
February 19, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13187956 |
Jul 21, 2011 |
8377879 |
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13770606 |
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10593466 |
Sep 19, 2006 |
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PCT/US05/08960 |
Mar 18, 2005 |
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13187956 |
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60554419 |
Mar 19, 2004 |
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Current U.S.
Class: |
514/9.9 |
Current CPC
Class: |
C07K 14/59 20130101;
A61P 5/00 20180101; A61K 38/24 20130101; A61P 15/08 20180101; A61K
38/24 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/9.9 |
International
Class: |
A61K 38/24 20060101
A61K038/24 |
Claims
1-137. (canceled)
138. A method of inducing superovulation in an animal, comprising:
administering an effective amount of a superactive follicle
stimulating hormone (FSH) to the animal, wherein said superactive
FSH comprises an alpha chain comprising basic amino acid
substitutions at positions 18, 20 and 24; and the alpha chain
further comprises an ANITV (SEQ ID NO: 3) N-terminal extension.
139. The method of claim 138, wherein superovulation is
characterized by an increase in oocyte number as compared to a like
animal receiving the same amount of recombinant FSH.
140. The method of claim 139, wherein the oocyte number increases
at least about 10% as a result of administration of said
superactive equine FSH at the maximally effective dose for oocyte
number.
141. The method of claim 138, wherein the alpha chain further
comprises a basic amino acid substitution at position 17.
142. The method of claim 138, wherein the basic amino acid is an
arginine, a lysine, or a histidine, or a modification thereof.
143. The method of claim 138, wherein the amino acid substitutions
at positions 18, 20 and 24 are arginine substitutions.
144. The method of claim 141, wherein the amino acid substitutions
at positions 17, 18, 20 and 24 are arginine substitutions.
145. The method of claim 138, wherein said ANTIV (SEQ ID NO: 3)
extension prolongs the in vivo half life of the superactive
FSH.
146. The method of claim 138, wherein the superactive FSH is a
bovine FSH further comprising a bovine beta subunit, and the animal
is a cow.
147. The method of claim 144, wherein the superactive FSH is a
bovine FSH further comprising a bovine beta subunit, and the animal
is a cow.
148. The method of claim 138, wherein the superactive FSH is an
equine FSH further comprising a equine beta subunit, and the animal
is a horse.
149. The method of claim 143, wherein the superactive FSH is an
equine FSH further comprising a equine beta subunit, and the animal
is a horse.
150. The method of claim 138, wherein the superactive FSH is an
ovine FSH further comprising a wild ovine beta subunit, and the
animal is a sheep.
151. The method of claim 145, wherein the superactive FSH is an
ovine FSH further comprising a wild ovine beta subunit, and the
animal is a sheep.
152. The method of claim 138, wherein the superactive FSH is a
porcine FSH further comprising a wild porcine beta subunit, and the
animal is a pig.
153. The method of claim 144, wherein the superactive FSH is a
porcine FSH further comprising a wild porcine beta subunit, and the
animal is a pig.
154. The method of claim 138, wherein said superactive FSH is
administered by injection.
155. The method of claim 138, wherein the injection is
intramuscular injection.
156. The method of claim 138, wherein said superactive FSH is
administered by ingestion.
Description
FIELD OF INVENTION
[0001] This invention relates generally to modified follicle
stimulating hormones (FSH) having superagonist activity, and the
use thereof in the treatment of conditions associated with
glycoprotein hormone activity. More specifically, this invention
relates to modified FSH molecules containing two or more amino acid
substitutions as compared to wild type FSH, wherein such modified
FSH molecules exhibit enhanced pharmacological properties as
compared to wild type FSH.
BACKGROUND OF INVENTION
[0002] Follitropin (follicle-stimulating hormone, FSH) and the
gonadotropins chorionic gonadotropin, (CG), lutropin (luteinizing
hormone, LH), and thyrotropin (thyroid-stimulating hormone, TSH)
comprise the family of glycoprotein hormones. Each hormone is a
heterodimer of two non-covalently linked subunits: alpha and beta.
Within the same species, the amino acid sequence of the
alpha-subunit is identical in all the hormones, whereas the
sequence of the beta-subunit is hormone specific (Pierce, J. G. and
Parsons, T. F. "Glycoprotein hormones: structure and function."
Ann. Rev. Biochem. 50:465-495 (1981)). The fact that the sequences
of the subunits are highly conserved from fish to mammals implies
that these hormones have evolved from a common ancestral protein
(Fontaine Y-A. and Burzawa-Gerard, E. "Esquisse de l' evolution des
hormones gonadotopes et thyreotropes des vertebres." Gen. Comp.
Endocrinol. 32:341-347 (1977)).
[0003] Recombinant follitropin has been used in certain therapies,
such as in the treatment of patients suffering from infertility
(Lathi and Milki, "Recombinant gonadotropins," Curr Womens Health
Rep. 1(2):157-63 (2001)). The hormone has been used in women to
induce ovulation, and also in men to induce spermatogenesis
(Bouloux et al., "Induction of spermatogenesis by recombinant
follicle-stimulating hormone (puregon) in hypogonadotropic
azoospermic men who failed to respond to human chorionic
gonadotropin alone," J. Androl. 24(4):604-11 (2003)), improve
disturbed sperm structures (Haidl et al., "Drug treatment of male
fertility disorders," Asian J. Androl. 2(2):81-5 (2000)), and treat
conditions associated with decreased levels of testosterone (see
U.S. Pat. Nos. 5,574,011 and 6,562,790, each incorporated by
reference). The response of women to exogenous FSH therapy, has
been shown to be variable, with some demonstrating a poor response
to a standard therapy protocol (requiring adjustment of the FSH
doses), and others demonstrating ovarian hyperstimulation syndrome
(Perez et al., "Ovarian response to follicle-stimulating hormone
(FSH) stimulation depends on the FSH receptor genotype," J Clin
Endocrinol Metab. 85(9):3365-9 (2000)). What is needed are modified
derivatives of FSH having increased activity, to facilitate
treatment of poor responders while permitting lower dose therapy
regimens of patients prone to ovarian hyperstimulation.
SUMMARY OF INVENTION
[0004] This invention encompasses modified FSH proteins and nucleic
acids encoding the same, wherein the in vivo and in vitro
bioactivities of the modified proteins are substantially increased
as compared to wild type FSH. In particular, the modified analogs
of the invention demonstrate surprisingly enhanced pharmacological
properties, including potency and Vmax (efficacy), as compared to
wild type FSH. Further, the modified analogs of the invention
provide dramatic increases in the quantity and quality of oocytes,
blastocysts and embryos of treated animals. The analogs of the
invention thus provide a long awaited solution for a wide spectrum
of patients suffering from infertility, including women
demonstrating a poor response following in vitro fertilization
(IVF), women who have been disqualified from IVF, women
demonstrating low numbers of FSH receptors and women with FSH
receptor mutations leading to infertility.
[0005] The modified FSH molecules of the invention contain at least
a modified .alpha.-subunit containing a combination of at least two
mutations in peripheral loops of FSH, which lead to a modified FSH
having increased potency over wild type FSH or modified proteins
comprising the specified mutations alone. Typically, the modified
FSH proteins of the invention demonstrate at least about a ten fold
increase in potency over wild type FSH, with preferred
.alpha.-subunit mutations comprising at least two basic amino acids
at positions corresponding to positions 13, 14, 16, 17, 20, 21, 22,
66, 68, 73, 74 and 81 of SEQ ID No. 1.
[0006] The modified FSH proteins of the invention may further
comprise a modified .beta.-subunit, particularly a modified
.beta.-subunit comprising at least one basic amino acid at a
position corresponding to any one of positions 2, 4, 14, 63, 64, 67
and 69 of SEQ ID No. 2. The modified FSH proteins of the invention
may also demonstrate an increased or decreased plasma half-life as
compared to wild type FSH or a decreased plasma half-life as
compared to wild type FSH. An increase in plasma half-life may be
facilitated by pegylation, by inclusion of a potential
glycosylation site or by other means.
[0007] The invention also includes methods of assisting
reproduction in a subject comprising administering an assisting
amount of the modified FSH of the invention, for instance in an in
vitro fertilization protocol or artificial insemination protocol or
other protocol in which ovulation or spermatogenesis is induced.
Also included are methods of diagnosing and treating conditions
associated with glycoprotein hormone activity in women, including
but not limited to ovulatory dysfunction, luteal phase defects,
time-limited conception, low FSH receptor expression in growing
follicles, low FSH receptor sensitivity, FSH receptor binding
and/or coupling deficiencies, pituitary failure or injury,
unexplained infertility and ovarian carcinoma. The modified FSH
proteins of the invention are particularly useful for treating
women prone to ovarian hyperstimulation, where analogs with the
longest half-life may be applied early in the cycle and those with
shorter half-life later in the cycle to prevent or reduce the
possibility of ovarian hyperstimulation syndrome (OHHS). Also
included are methods of diagnosing and treating conditions
associated with glycoprotein hormone activity in men, including but
not limited to male factor infertility, pituitary failure or
injury, male pattern baldness, testicular carcinoma and any
condition associated with deficient levels of testosterone
production.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIGS. 1A-E are graphs showing a comparison of the effect of
various single mutations on FSH bioactivity in vitro compared to
wild type (WT), as measured using transient transfection of
CHO-FSHR cells.
[0009] FIG. 2 is a graph showing the effect of the beta E4R
mutation on hFSH production in transfected CHO-FSHR cells.
[0010] FIGS. 3A and 3B are graphs showing a comparison of the
effect of various combined mutations on FSH bioactivity in vitro,
as measured using transient transfection of CHO-FSHR cells.
[0011] FIGS. 4A and B are graphs showing a comparison of
cross-reactivity of rat and human LHR to analog FSH and wild type
FSH. FIG. 4A shows cross-reactivity between FSH TR-4402 and rat
luteinizing hormone receptor. FIG. 4B shows that there is no
cross-reactivity between FSH TR-4402 and human luteinizing
hormone.
[0012] FIG. 5 is a diagram of the structure of FSH showing the
loops in the alpha and beta subunits.
[0013] FIGS. 6A and B are graphs showing cAMP production in CHO
cells in response to purified analog TR-4402 versus wild type FSH
and purified analog TR-4401 versus wild type FSH, respectively.
[0014] FIG. 7 is a graph showing cAMP production in KGN cells in
response to purified analog TR-4402 versus wild type FSH.
[0015] FIG. 8 is a graph showing cAMP production in GLHR-15 cells
in response to purified analog TR-4402 versus wild type FSH.
[0016] FIG. 9 is a graph showing follicle survival in the presence
of wild type hFSH (compound #3) and analog TR-4402 (compound #4),
observed during an in vitro follicle bioassay.
[0017] FIG. 10 is a graph showing antrum formation in the presence
of wild type hFSH (compound #3) and analog TR-4402 (compound #4),
observed during an in vitro follicle bioassay.
[0018] FIG. 11A is a graph showing mucification of COC in the
presence of wild type hFSH (compound #3) and analog TR-4402
(compound #4), observed during an in vitro follicle bioassay. FIG.
11B is a graph showing % oocyte release upon hCG stimulation in the
presence of wild type hFSH (compound #3) and analog TR-4402
(compound #4), observed during an in vitro follicle bioassay.
[0019] FIG. 12 is a graph showing oocyte nuclear maturation as
measured by PB extrusion in the presence of wild type hFSH
(compound #3) and analog TR-4402 (compound #4), observed during an
in vitro follicle bioassay.
[0020] FIGS. 13A and B are graphs showing progesterone production
in the presence of wild type hFSH (compound #3) (13A) and analog
TR-4402 (compound #4) (13B), observed during an in vitro follicle
bioassay.
[0021] FIGS. 14A-D show the results of a Steelman-Pohley Bioassay
performed using immature Sprague-Dawley Female Rats (Steelman and
Pohley, 1953). The graphs in FIGS. 14A, C and D show differences in
ovarian weight measured in response to TR-4402 as compared to wild
type (Follistim). The graph in FIG. 14B compares the serum levels
of TR-4402 and wild type FSH during the bioassay.
[0022] FIG. 15 is a graph showing the intra-ovarian estradiol
content of rats treated with wild type FSH (Follistim) as compared
to rats treated with the analog TR-4402.
[0023] FIG. 16 is a graph showing serum inhibin B levels in rats
after stimulation with corresponding doses of wild type FSH
(Follistim) and the analog TR-4402.
[0024] FIGS. 17 A and B are graphs showing the elimination and
absorption of FSH analogs TR-4901, TR-4401, and TR-4402 versus wild
type FSH.
[0025] FIG. 18 shows N-terminal extensions which can be used to
prolong half life of FSH analogs (SEQ. ID Nos. 3, 4, 5, 6, 7, 8, 9,
10, 11, and 12).
[0026] FIG. 19 is a graph showing cAMP production in CHO cells in
response to LA1-4402 (TR-4402 further modified to increase FSH
serum half-life) versus LA1 FSH (FSH modified to increase FSH serum
half-life), TR-4402, and wild type FSH.
[0027] FIG. 20 is a graph showing an increase in number of ovulated
oocytes produced in vivo in response to LA1-4402 (TR-4402 modified
to increase serum half-life) versus wild type FSH (Follistem) and
hCG only.
[0028] FIG. 21 is a graph showing the increase in total number of
oocytes produced in vivo after administration of TR 4401 versus
wild type FSH (Gonal F).
[0029] FIG. 22 is a graph showing the increase in fertilization
rate of oocytes after in vivo administration of TR 4401 versus wild
type FSH (Gonal F).
[0030] FIG. 23 is a graph showing the increase in blastocyst
formation rate after in vivo administration of TR 4401 versus wild
type FSH (Gonal F).
[0031] FIG. 24 is a graph showing the increase in total number of
embryos after in vivo administration of TR 4401 versus wild type
FSH (Gonal F).
DETAILED DESCRIPTION OF THE INVENTION
[0032] The present invention provides modified "superactive" FSH
molecules showing surprisingly enhanced potency as compared to wild
type FSH. Being "modified" means that, while the protein contains
an amino acid sequence which differs from the wild-type FSH, the
sequence has not been changed such that it is identical to the
known FSH sequence of another species. "Superactivity" may be
assessed according to a variety of parameters, including potency
and efficacy. "Potency" is a parameter of bioactivity that is
determined by measuring the half maximal response. Differences in
"potency" are determined by comparing the value of the FSH response
of the analog halfway between baseline and maximum (EC50) versus
that of wild type FSH. FSH responses may be measured in vitro using
purified proteins, or may be estimated following transient
transfection of a nucleic acid encoding the modified protein. FSH
responses may also be measured in vivo, i.e. in an animal
responsive to said FSH analog. Such responses encompass any known
cellular or biological and quantitative or qualitative response of
FSH binding to its receptor, i.e. cAMP production, synthesis of
proteins such as progesterone, fertilization rate, blastocyst
formation rate, embryo development per fertilized oocyte, etc.
[0033] "Efficacy" (Vmax) or maximum response is another parameter
of bioactivity. As discussed herein, parameters of bioactivity may
vary depending on receptor number and receptor coupling in the
assay cell line. In systems with lower receptor numbers or impaired
coupling, differences are more discernable in terms of Vmax
(efficacy). In systems where receptors are overexpressed,
differences in potency are more visible.
[0034] In vivo quantitative and qualitative parameters such as
quantity of oocytes, fertilization rate and blastocyst and embryo
formation rates may be measured at the maximally effective dose for
oocyte number. The maximally effective dose for oocyte number is
the optimal amount of superactive FSH for both oocyte quality and
quantity. The maximally effective dose for oocyte number is
dependent on an animal's weight and rate of metabolism. For
example, the maximally effective dose for a larger animal with a
slower rate of metabolism is greater than the maximally effective
dose for a smaller animal with a higher rate of metabolism. The
maximally effective dose is determined empirically for each
animal.
[0035] However, regardless of the system used, the modified
superactive FSH proteins of the invention demonstrate at least
about a 10 fold increase in potency more preferably at least about
a 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90
fold or even 100 fold increase in potency compared to wild type
FSH, or about a 10% increase in maximal efficacy, more preferably
at least a 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%
increase in maximal efficacy compared to wild type FSH. The
superactive analogs of the invention may also provide about a five
to ten fold increase in potency or 5% to 10% increase in maximal
efficacy as compared to wildtype FSH. Some of the modified proteins
of the invention demonstrate at least about a thirty to fifty fold
increase in potency or 30% to 50% increase in maximal efficacy as
compared to wild type. Thus, the modified FSH proteins of the
present invention are particularly useful for treating patients
with low receptor number or deficiencies in receptor response,
since the modified proteins of the invention maintain at least a 10
fold increase in potency or 10% increase in maximal efficacy even
in systems with low receptor number or response.
[0036] The rate of absorption of a modified superactive FSH may
result in increased or decreased duration of action. A modified FSH
analog with an increased rate of absorption and decreased duration
of action may be beneficial for hypersensitive patients at risk for
hyperstimulation syndrome. The rate of absorption is measured by
K.sub.a. The rate of elimination is measured by K.sub.e.
[0037] The modified FSH molecules of the invention include modified
proteins of species selected from the group consisting of human,
bovine, equine, porcine, ovine, murine, rat, rabbit, primate, fish,
etc. Fish FSH (also known as GTH-1) may be used in aquaculture,
i.e., in order to grow endangered or other fish species in
captivity. Other species of modified FSH find use in agriculture
breeding, and in the laboratory setting for testing the effects of
different combined mutations on various male and female
glycoprotein hormone-related conditions. The modified FSH molecules
of other species have substitutions at positions corresponding to
those in the modified human FSH molecules disclosed herein, which
may be identified using any alignment program, including but not
limited to DNASIS, ALIONment, SIM and GCG programs such as Gap,
BestFit, FrameAlign and Compare.
[0038] Modified human FSH molecules of the present invention
comprise at least a modified .alpha.-subunit, wherein the alpha
subunit comprises at least two basic amino acids such as those at
positions corresponding to positions 13, 14, 16, 17, 20, 21, 22,
66, 68, 73, 74 and 81 of wild type human FSH alpha (SEQ ID No. 1).
The modified proteins may also contain a modified beta subunit,
wherein the beta subunit comprises at least one basic amino acid at
positions corresponding to positions 2, 4, 14, 63, 64, 67 and 69 of
wild type human FSH beta (SEQ ID No. 2). The modified proteins of
the invention may also contain further substitutions, particularly
conservative substitutions that do not alter the enhanced
properties of the protein. Typically, however, such modified
proteins will contain less than five substitutions at positions
other than those listed above, and may exhibit complete amino acid
sequence identity with the corresponding wild-type FSH alpha and
beta subunits in positions other than the positions listed
above.
[0039] Basic amino acids comprise the amino acids lysine, arginine,
and histidine, and any other basic amino acid which may be a
modification to any of these three amino acids, synthetic basic
amino acids not normally found in nature, or any other amino acid
which is positively charged at a neutral pH. Preferred basic amino
acids, among others, are selected from the group consisting of
lysine and arginine.
[0040] Exemplary modified FSH molecules having two basic amino acid
substitutions include but are not limited to proteins with
substitutions at positions 14 and 66 of the .alpha.-subunit,
particularly E14R and N66R, positions 14 and 73 of the
.alpha.-subunit, particularly E14R and G73R positions 16 and 20 of
the .alpha.-subunit, particularly P16R and Q20R, and positions 20
and 21 of the .alpha.-subunit, particularly Q20R and P21R.
[0041] The modified FSH proteins of the invention may also have an
.alpha.-subunit comprising three basic amino acid substitutions at
positions selected from the group consisting of positions 13, 14,
16, 17, 20, 21, 22, 66, 68, 73, 74 and 81. Such modified proteins
include but are not limited to proteins with combined substitutions
at positions 16, 20 and 21, particularly P16R, Q20R and P21R,
positions 14, 20 and 73, particularly E14R, Q20R and G73R,
positions 66, 73 and 81, particularly N66K, G73K and A81K,
positions 14, 66 and 73, particularly E14R, N66R and G73R, and
positions 14, 21 and 73, particularly E14R, P21R and G73R.
[0042] The modified FSH proteins of the invention may also have an
.alpha.-subunit comprising four basic amino acid substitutions at
positions selected from the group consisting of positions 13, 14,
16, 17, 20, 21, 22, 66, 68, 73, 74 and 81. Such modified proteins
include but are not limited to proteins with combined substitutions
at positions 13, 14, 16 and 20, particularly the combination of
Q13R, E14R, P16R and Q20R, and the combination of Q13K, E14K, P16K
and Q20K.
[0043] The modified FSH proteins of the invention may also have an
.alpha.-subunit comprising five basic amino acid substitutions at
positions selected from the group consisting of positions 13, 14,
16, 17, 20, 21, 22, 66, 68, 73, 74 and 81. Such modified proteins
include but are not limited to proteins with combined substitutions
at positions 14, 20, 21, 66 and 73, particularly E14R, Q20R, P21R,
N66R and G73R, and positions 14, 16, 20, 66 and 73, particularly
E14R, P16R, Q20R, N66R and G73R.
[0044] The modified FSH proteins of the invention may also have an
.alpha.-subunit comprising six basic amino acid substitutions at
positions selected from the group consisting of positions 13, 14,
16, 17, 20, 21, 22, 66, 68, 73, 74 and 81. Such modified proteins
include but are not limited to proteins with combined substitutions
at positions 13, 14, 16, 20, 66 and 73, particularly Q13K, E14K,
P16K, Q20K, N66K and G73K, and positions 14, 16, 20, 21, 66 and 73,
particularly E14R, P16R, Q20R, P21R, N66R and G73R.
[0045] A particularly effective modified O-subunit of the invention
comprises a basic amino acid at a position corresponding to
position 4 of SEQ ID No. 2, and more particularly, E4R. This
substitution results in a unique increase in FSH potency and
expression level. The inventors have found that this mutation
results in 2-3 fold higher production of recombinant FSH when used
in combination with the other substitutions disclosed herein.
[0046] Design of FSH Superagonists
[0047] Superagonists encompassed by the present invention may be
designed by comparing the amino acid sequences of the alpha and
beta FSH of interest to that of other species to identify basic
residues in the proteins of FSH of other species. Such methods are
disclosed in U.S. Pat. No. 6,361,992, which is herein incorporated
by reference in its entirety. Consideration may also be given to
the relative biological activity of FSH from various species as to
which species to chose for comparison and substitution. Further,
homology modeling based on the structure of related glycoprotein
hormones is useful to identify surface-exposed amino acid
residues.
[0048] Accordingly, the present invention also provides a modified
FSH protein having increased potency over a wild-type FSH from the
same species, wherein the modified FSH comprises a basic amino acid
substituted at a position corresponding to the same amino acid
position in a FSH protein from another species having an increased
potency over the wild-type FSH protein. The glycoprotein being
modified to increase its potency can be from a non-human species.
For example, one can compare porcine FSH to bovine FSH, design
porcine FSH proteins with amino acid substitutions at positions
where the porcine and the bovine sequences are different, construct
porcine FSH proteins with the selected changes, and administer the
modified porcine FSH to porcine animals. Alternatively, the FSH
being modified can be bovine.
[0049] The present invention also provides a modified FSH having
increased potency over the wild-type FSH from the same species,
wherein the modified FSH comprises a basic amino acid substituted
at a position corresponding to the same amino acid position in a
different glycoprotein hormone from the same species having an
increased potency over the wild-type glycoprotein hormone. For
example, the beta subunits of human FSH and human chorionic
gonadotropin can be compared and amino acid substitutions to the
FSH beta subunit can be made based on any sequence divergence.
Naturally, only those changes which generally increase the potency
of the modified FSH are contemplated since the hormone receptor
specificity will still need to be retained.
[0050] To modify additional amino acid positions, glycoprotein
hormone sequences from human and non-humans can be aligned using
standard computer software programs such as DNASIS (Hitachi
Software Engineering Co. Ltd.) or any of the other alignment
programs listed above, including but not limited to ALIONment, SIM
and GCG programs such as Gap, BestFit, FrameAlign and Compare. The
amino acid residues that differ between the human and the non-human
glycoprotein hormone can then be substituted using one of the
above-mentioned techniques, and the resultant glycoprotein hormone
assayed for its potency using one of the herein-mentioned
assays.
[0051] The present invention also encompasses fragments of the
analogs described herein that have either superagonist or
antagonist activity. For example, fragments of the modified alpha
chains of the invention may be used either alone or in combination
with either a fragment or full length beta chain to create
superagonist compounds. Likewise, fragments of the modified beta
chains of the invention may be used either alone or in combination
with either a fragment or full length alpha chain to create
superagonist compounds. In some cases, fragments of the modified
FSH molecules of the invention may also be used as antagonists, for
instance, to limit the duration of activity of an FSH therapeutic
after it has been administered.
[0052] The present invention also encompasses single chain analogs
and chimeric proteins incorporating the mutated regions of the
analogs described herein. For instance, the present inventors have
found that incorporation of superpotency substitutions within the
alpha subunit of dual-activity gonadotropins results in a 3-5 fold
increase of both luteotropic and follitropic activities indicating
that the intrinsic activities of dual-activity gonadotropins can be
further enhanced by the combined substitutions of the present
invention. Construction of dual-activity gonadotropins is described
in U.S. Pat. No. 4,237,224, which is herein incorporated by
reference in its entirety.
[0053] Characterization of FSH Superagonists
[0054] The effect of the modification or modifications to the
wild-type FSH described herein can be ascertained in any number of
ways. For example, cyclic AMP (cAMP) production in cells
transfected with a nucleic acid encoding the modified glycoprotein
can be measured and compared to the cAMP production of similar
cells transfected with a nucleic acid encoding the wild-type
glycoprotein hormone. Alternatively, progesterone production in
cells transfected with the modified glycoprotein can be measured
and compared to the progesterone production of similar cells
transfected with the wild-type glycoprotein hormone. Alternatively,
the activity of a modified glycoprotein hormone can be determined
from receptor binding assays, from thymidine uptake assays, or from
T4 secretion assays. Specific examples of such assays for
determining the activity of modified glycoprotein hormones are set
forth in the Example section contained herein. One skilled in the
art can readily determine any appropriate assay to employ to
determine the activity of either a wild-type or a modified
glycoprotein hormone.
[0055] In one embodiment of the present invention, the modified
glycoprotein hormone has a potency which is increased over the
potency of the wild type glycoprotein hormone by at least about 10
fold. This increased potency can be assessed by any of the
techniques mentioned above and described in the Example contained
herein, or in any other appropriate assay as readily determined by
one skilled in the art. The increased potency does not have to be
consistent from assay to assay, or from cell line to cell line, as
these of course, will vary. The modified FSH molecules of the
invention may demonstrate an increase in potency of at least about
20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90
fold or even 100 fold over wild type using cell lines expressing
responsive FSH receptors at varying levels.
[0056] In another embodiment of the present invention, the modified
glycoprotein hormone has a maximal efficacy which is increased over
the maximal efficacy of the wild type glycoprotein hormone by at
least about 10%. This increased maximal efficacy can be assessed by
any of the techniques mentioned above and described in the Example
contained herein, or in any other appropriate assay as readily
determined by one skilled in the art. The increased maximal
efficacy does not have to be consistent from assay to assay, or
from cell line to cell line, as these of course, will vary. The
modified FSH molecules of the invention may demonstrate an increase
in maximal efficacy of at least about 10% fold, at least about 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, over wild type using
cell lines expressing responsive FSH receptors at varying
levels.
[0057] Other assays suitable for characterizing the analogs
described herein are described in PCT/US99/05908, which is herein
incorporated by reference in its entirety. For instance, various
immunoassays may be used including but not limited to competitive
and non-competitive assay systems using techniques such as
radioimmunoassays, ELISA, sandwich immunoassays, immunoradiometric
assays, gel diffusion precipitin reactions, immunodiffusion assays,
in situ immunoassays, western blots, precipitation reactions,
agglutination assays, complement fixation assays,
immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc.
[0058] Improvements in the quality and quantity of oocytes can be
assessed by in vitro and in vivo assays. It is common for
improvements in oocyte quantity and quality to be determined using
different end points of the in vitro fertilization process such as
oocyte formation, oocyte fertilization, and blastocyst formation.
In vitro fertilization experiments may follow a "superovulation
protocol" in which subjects are treated with a superactive FSH
analog according to the present invention, which leads to the
release and maturation of multiple oocytes. In in vitro
fertilization experiments, FSH (superactive FSH and recombinant
wild type FSH) may be administered with hCG to trigger ovulation. A
control animal may be used which receives only hCG or pregnant mare
serum gonadotropin (PMSG).
[0059] The quality of oocytes can be improved by increasing the
fertilization rate of oocytes in an animal. The fertilization rate
of a superactive follicle stimulating hormone can be determined in
vivo or in vitro by comparing the fertilization rate achieved with
a superactive FSH to the fertilization rate achieved with the same
amount of recombinant wild type FSH. A control animal may also be
used that receives hCG.
[0060] The rate of fertilization can be measured by the percent of
two-cell embryos which develop per total number of oocytes. If
fertilization takes place in vitro, two cell embryos can be counted
in fertilization dishes. In mice, two cell embryos develop
approximately twenty-four hours after fertilization.
[0061] The fertilization rate varies based on the amount of
superactive FSH administered. An animal may receive multiple does
of superactiveFSH. The rate of fertilization increases by at least
about 10 percent as a result of administration of superactive FSH
at the maximally effective dose for oocyte number. The rate of
fertilization may increase by at least about 20 percent, preferably
at least 30 percent, 40%, 50%, 60%, 70%, 80%, 90%, or 100% as a
result of administration of superactive FSH at the maximally
effective dose for oocyte number.
[0062] Superactive follicle stimulating hormone can improve the
quality of oocytes by improving the blastocyst formation rate per
fertilized oocyte. The rate of blastocyst formation can be measured
by determining the percentage of two-cell embryos which form
blastocysts. The rate of blastocyst formation increases whether the
blastocyst forms in vivo or in vitro. The blastocyst formation rate
is dependent on the amount of superactive follicle stimulating
hormone administered. The rate of blastocyst formation increases at
least about 10 percent as a result of administration of a
superactive follicle stimulating hormone at the maximally effective
dose for oocyte number. The rate of blastocyst formation may
increase at least about 20 percent, preferably at least 30%, 40%,
50%, 60%, 70%, 80%, 90%, or 100% as a result of administration of
superactive FSH at the maximally effective dose for oocyte
number.
[0063] Superactive follicle stimulating hormone can improve the
quality of oocytes by increasing the total number of embryos per
fertilized oocyte. The increase in total number of embryos per
fertilized oocyte increases whether fertilization occurs in vivo or
in vitro. The increase in total number of embryos per fertilized
oocyte is dependent on the amount of superactive follicle
stimulating hormone administered. The total number of embryos per
fertilized oocyte increases at least about 10 percent as a result
of administration of a superactive follicle stimulating hormone at
the maximally effective dose for oocyte number. The total number of
embryos per fertilized oocyte may increase by at least about 20
percent, preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or
100% as a result of administration of superactive FSH at the
maximally effective dose for oocyte number.
[0064] Superactive FSH can be used to improve the quality and
quantity of oocytes from animals, including but not limited to,
human, mouse, rat, primate, rabbit, pig, horse, sheep, and dog.
Preferably, a superactive FSH is administered to a human FSH.
[0065] FSH Analogs with Increased Serum Half-Life
[0066] The modified FSH proteins of the invention may also be
further modified such that the plasma half-life is increased as
compared to wild type FSH. For instance, the modified FSH proteins
of the invention may further comprise at least one sequence with a
potential glycosylation site including sequences comprising
N-glycosylation and/or O-glycosylation sites on either the alpha or
beta chain. Sequences providing potential glycosylation recognition
sites may be either an N-terminal or C-terminal extension on either
the alpha or beta chain. Exemplary modified proteins contain an
N-terminal extension on the .alpha. chain that is selected from the
group consisting of ANITV (SEQ ID No. 3) and ANITVNITV (SEQ ID No.
4). Other exemplary modified proteins contain a further
substitution in said .beta. chain, wherein said substitution is
selected from the group consisting of Y58N and V78N.
[0067] Increased half-life may also be provided by pegylation or
conjugation of other appropriate chemical groups or by constructing
fusion proteins having increased half life or any other method.
Such methods are known in the art, for instance as described in
U.S. Pat. No. 5,612,034, U.S. Pat. No. 6,225,449, and U.S. Pat. No.
6,555,660, each of which is incorporated by reference in its
entirety. Half-life may also be increased by increasing the number
of negatively charged residues within the molecule, for instance,
the number of glutamate and/or aspartate residues. Such alteration
may be accomplished by site directed mutagenesis, with preferred
alterations selected from the group consisting of alpha subunit
substitutions A85E and A85D, among others. Such alteration may also
be achieved via an insertion of an amino acid sequence containing
one or more negatively charged residues into said modified FSH,
including insertions selected from the group consisting of GEFT
(SEQ ID No. 5) and GEFTT (SEQ ID No. 6), among others. In one
embodiment, the insertion is in the alpha subunit, and is selected
from the group consisting of APD-GEFT-VQDC (SEQ ID No. 7) and
APD-GEFTT-QDC (SEQ ID No. 8), among others.
[0068] The half-life of a protein is a measurement of protein
stability and indicates the time necessary for a one-half reduction
in the concentration of the protein. The serum half-life of the
modified FSH molecules described herein may be determined by any
method suitable for measuring FSH levels in samples from a subject
over time, for example but not limited to, immunoassays using
anti-FSH antibodies to measure FSH levels in serum samples taken
over a period of time after administration of the modified FSH, or
by detection of labeled
[0069] FSH molecules, i.e., radiolabeled molecules, in samples
taken from a subject after administration of the labeled FSH.
[0070] Expression and/or Synthesis of the FSH Superagonists
[0071] The present invention also includes nucleic acids encoding
the modified FSH .alpha. and .beta. subunits of the invention, as
well as vectors and host cells for expressing the nucleic acids.
Appropriate promoters for the expression of nucleic acids in
different host cells are well known in the art, and are readily
interchanged depending on the vector-host system used for
expression. Exemplary vectors and host cells are described in U.S.
Pat. No. 6,361,992, which is herein incorporated by reference in
its entirety.
[0072] For instance, once a nucleic acid encoding a particular
glycoprotein hormone of interest, or a region of that nucleic acid,
is constructed, modified, or isolated, that nucleic acid can then
be cloned into an appropriate vector, which can direct the in vivo
or in vitro synthesis of that wild-type and/or modified
glycoprotein hormone. The vector is contemplated to have the
necessary functional elements that direct and regulate
transcription of the inserted gene, or hybrid gene. These
functional elements include, but are not limited to, a promoter,
regions upstream or downstream of the promoter, such as enhancers
that may regulate the transcriptional activity of the promoter, an
origin of replication, appropriate restriction sites to facilitate
cloning of inserts adjacent to the promoter, antibiotic resistance
genes or other markers which can serve to select for cells
containing the vector or the vector containing the insert, RNA
splice junctions, a transcription termination region, or any other
region which may serve to facilitate the expression of the inserted
gene or hybrid gene. (See generally, Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2nd ed. (1989)).
[0073] There are numerous E. coli (Escherichia coli) expression
vectors known to one of ordinary skill in the art which are useful
for the expression of the nucleic acid insert. Other microbial
hosts suitable for use include bacilli, such as Bacillus subtilis,
and other enterobacteriaceae, such as Salmonella, Serratia, and
various Pseudomonas species. In these prokaryotic hosts one can
also make expression vectors, which will typically contain
expression control sequences compatible with the host cell (e.g.,
an origin of replication). In addition, any number of a variety of
well-known promoters will be present, such as the lactose promoter
system, a tryptophan (Trp) promoter system, a beta-lactamase
promoter system, or a promoter system from phage lambda. The
promoters will typically control expression, optionally with an
operator sequence, and have ribosome binding site sequences for
example, for initiating and completing transcription and
translation. If necessary, an amino terminal methionine can be
provided by insertion of a Met codon 5' and in-frame with the
downstream nucleic acid insert. Also, the carboxy-terminal
extension of the nucleic acid insert can be removed using standard
oligonucleotide mutagenesis procedures.
[0074] Additionally, yeast expression can be used. There are
several advantages to yeast expression systems. First, evidence
exists that proteins produced in a yeast secretion systems exhibit
correct disulfide pairing. Second, post-translational glycosylation
is efficiently carried out by yeast secretory systems. The
Saccharomyces cerevisiae pre-pro-alpha-factor leader region
(encoded by the MF''-1 gene) is routinely used to direct protein
secretion from yeast. (Brake, et al., ".varies.-Factor-Directed
Synthesis and Secretion of Mature Foreign Proteins in Saccharomyces
cerevisiae." Proc. Nat. Acad. Sci., 81:4642-4646 (1984)). The
leader region of pre-pro-alpha-factor contains a signal peptide and
a pro-segment which includes a recognition sequence for a yeast
protease encoded by the KEX2 gene: this enzyme cleaves the
precursor protein on the carboxyl side of a Lys-Arg dipeptide
cleavage signal sequence. The FSH coding sequence can be fused
in-frame to the pre-pro-alpha-factor leader region. This construct
is then put under the control of a strong transcription promoter,
such as the alcohol dehydrogenase I promoter or a glycolytic
promoter. The nucleic acid coding sequence is followed by a
translation termination codon which is followed by transcription
termination signals. Alternatively, the nucleic acid coding
sequences can be fused to a second protein coding sequence, such as
Sj26 or beta.-galactosidase, which may be used to facilitate
purification of the fusion protein by affinity chromatography. The
insertion of protease cleavage sites to separate the components of
the fusion protein is applicable to constructs used for expression
in yeast. Efficient post-translational glycosolation and expression
of recombinant proteins can also be achieved in Baculovirus
systems.
[0075] Mammalian cells permit the expression of proteins in an
environment that favors important post-translational modifications
such as folding and cysteine pairing, addition of complex
carbohydrate structures, and secretion of active protein. Vectors
useful for the expression of active proteins in mammalian cells are
characterized by insertion of the protein coding sequence between a
strong viral promoter and a polyadenylation signal. The vectors can
contain genes conferring hygromycin resistance, gentamicin
resistance, or other genes or phenotypes suitable for use as
selectable markers, or methotrexate resistance for gene
amplification. The chimeric protein coding sequence can be
introduced into a Chinese hamster ovary (CHO) cell line using a
methotrexate resistance-encoding vector, or other cell lines using
suitable selection markers. Presence of the vector DNA in
transformed cells can be confirmed by Southern blot analysis.
Production of RNA corresponding to the insert coding sequence can
be confirmed by Northern blot analysis. A number of other suitable
host cell lines capable of secreting intact human proteins have
been developed in the art, and include the CHO cell lines, HeLa
cells, myeloma cell lines, Jurkat cells, etc. Expression vectors
for these cells can include expression control sequences, such as
an origin of replication, a promoter, an enhancer, and necessary
information processing sites, such as ribosome binding sites, RNA
splice sites, polyadenylation sites, and transcriptional terminator
sequences. Exemplary expression control sequences are promoters
derived from immunoglobulin genes, SV40, Adenovirus, Bovine
Papilloma Virus, etc. The vectors containing the nucleic acid
segments of interest can be transferred into the host cell by
well-known methods, which vary depending on the type of cellular
host. For example, calcium chloride transformation is commonly
utilized for prokaryotic cells, whereas calcium phosphate, DEAE
dextran, or lipofectin mediated transfection or electroporation may
be used for other cellular hosts.
[0076] Alternative vectors for the expression of genes in mammalian
cells, those similar to those developed for the expression of human
gamma-interferon, tissue plasminogen activator, clotting Factor
VIII, hepatitis B virus surface antigen, protease Nexinl, and
eosinophil major basic protein, can be employed. Further, the
vector can include CMV promoter sequences and a polyadenylation
signal available for expression of inserted nucleic acids in
mammalian cells (such as COS-7). Expression of the gene or hybrid
gene can be by either in vivo or in vitro. In vivo synthesis
comprises transforming prokaryotic or eukaryotic cells that can
serve as host cells for the vector. Alternatively, expression of
the gene can occur in an in vitro expression system. For example,
in vitro transcription systems are commercially available which are
routinely used to synthesize relatively large amounts of mRNA. In
such in vitro transcription systems, the nucleic acid encoding the
glycoprotein hormone would be cloned into an expression vector
adjacent to a transcription promoter. For example, the Bluescript
II cloning and expression vectors contain multiple cloning sites
which are flanked by strong prokaryotic transcription promoters.
(Stratagene Cloning Systems, La Jolla, Cailf.). Kits are available
which contain all the necessary reagents for in vitro synthesis of
an RNA from a DNA template such as the Bluescript vectors.
(Stratagene Cloning Systems, La Jolla, Cailf.). RNA produced in
vitro by a system such as this can then be translated in vitro to
produce the desired glycoprotein hormone. (Stratagene Cloning
Systems, La Jolla, Cailf.).
[0077] Another method of producing a glycoprotein hormone is to
link two peptides or polypeptides together by protein chemistry
techniques. For example, peptides or polypeptides can be chemically
synthesized using currently available laboratory equipment using
either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc
(tert-butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc.,
Foster City, Calif.). One skilled in the art can readily appreciate
that a peptide or polypeptide corresponding to a hybrid
glycoprotein hormone can be synthesized by standard chemical
reactions. For example, a peptide or polypeptide can be synthesized
and not cleaved from its synthesis resin whereas the other fragment
of a hybrid peptide can be synthesized and subsequently cleaved
from the resin, thereby exposing a terminal group which is
functionally blocked on the other fragment. By peptide condensation
reactions, these two fragments can be covalently joined via a
peptide bond at their carboxyl and amino termini, respectively, to
form a hybrid peptide. (Grant, G. A., "Synthetic Peptides: A User
Guide," W. H. Freeman and Co., N.Y. (1992) and Bodansky, M. and
Trost, B., Ed., "Principles of Peptide Synthesis," Springer-Verlag
Inc., N.Y. (1993)). Alternatively, the peptide or polypeptide can
by independently synthesized in vivo as described above. Once
isolated, these independent peptides or polypeptides may be linked
to form a glycoprotein hormone via similar peptide condensation
reactions. For example, enzymatic or chemical ligation of cloned or
synthetic peptide segments can allow relatively short peptide
fragments to be joined to produce larger peptide fragments,
polypeptides or whole protein domains (Abrahmsen, L., et al.,
Biochemistry, 30:4151 (1991); Dawson, et al., "Synthesis of
Proteins by Native Chemical Ligation," Science, 266:776-779
(1994)).
[0078] The invention also provides fragments of modified
glycoprotein hormones which have either superagonist or antagonist
activity. The polypeptide fragments of the present invention can be
recombinant proteins obtained by cloning nucleic acids encoding the
polypeptide in an expression system capable of producing the
polypeptide fragments thereof. For example, one can determine the
active domain of a modified FSH protein which, together with the
beta subunit, can interact with a glycoprotein hormone receptor and
cause a biological effect associated with the glycoprotein hormone.
In one example, amino acids found to not contribute to either the
activity or the binding specificity or affinity of the glycoprotein
hormone can be deleted without a loss in the respective
activity.
[0079] For example, amino or carboxy-terminal amino acids can be
sequentially removed from either the native or the modified
glycoprotein hormone and the respective activity tested in one of
many available assays described above. In another example, the
modified proteins of the invention may have a portion of either
amino terminal or carboxy terminal amino acids, or even an internal
region of the hormone, replaced with a polypeptide fragment or
other moiety, such as biotin, which can facilitate in the
purification of the modified glycoprotein hormone. For example, a
modified glycoprotein can be fused to a maltose binding protein,
through either peptide chemistry of cloning the respective nucleic
acids encoding the two polypeptide fragments into an expression
vector such that the expression of the coding region results in a
hybrid polypeptide. The hybrid polypeptide can be affinity purified
by passing it over an amylose affinity column, and the modified
glycoprotein can then be separated from the maltose binding region
by cleaving the hybrid polypeptide with the specific protease
factor Xa. (See, for example, New England Biolabs Product Catalog,
1996, pg. 164)
[0080] Active fragments of the FSH molecules of the invention can
also be synthesized directly or obtained by chemical or mechanical
disruption of larger glycoprotein hormone. An active fragment is
defined as an amino acid sequence of at least about 5 consecutive
amino acids derived from the naturally occurring amino acid
sequence, which has the relevant activity, e.g., binding or
regulatory activity. The fragments, whether attached to other
sequences or not, can also include insertions, deletions,
substitutions, or other selected modifications of particular
regions or specific amino acids residues, provided the activity of
the peptide is not significantly altered or impaired compared to
the modified glycoprotein hormone. These modifications can provide
for some additional property, such as to remove/add amino acids
capable of disulfide bonding, to increase its bio-longevity, etc.
In any case, the peptide must possess a bioactive property, such as
binding activity, regulation of binding at the binding domain, etc.
Functional or active regions of the glycoprotein hormone may be
identified by mutagenesis of a specific region of the hormone,
followed by expression and testing of the expressed polypeptide.
Such methods are readily apparent to a skilled practitioner in the
art and can include site-specific mutagenesis of the nucleic acid
encoding the receptor. (Zoller, M. J. et al.).
[0081] The present invention also encompasses fusion proteins and
chimeric proteins comprising the mutations described herein,
including for instance, fusions to the CTEP domain of LH or CG
proteins. Such a fusion protein may be made by ligating the
appropriate nucleic acid sequences encoding the desired amino acid
sequences to each other by methods known in the art, in the proper
coding frame, and expressing the fusion protein by any of the means
described above. Alternatively, such a fusion protein may be made
by protein synthesis techniques, for example, using a peptide
synthesizer. The single chain analogs and chimeric proteins of the
invention may incorporate a peptide linker between the alpha and
beta subunits, or between different portions of the chimeric
protein.
[0082] Methods of Treatment
[0083] The modified FSH superagonists of the present invention may
be used to treat any condition associated with glycoprotein hormone
activity. Conditions "associated with glycoprotein hormone
activity" are ones that are either completely or partially caused
by altered glycoprotein hormone responsiveness, or ones that
benefit from the administration of glycoprotein hormone. For
instance, such conditions include, but are not limited to ovulatory
dysfunction, luteal phase defects, unexplained infertility, male
factor infertility, time-limited conception, low FSH receptor
expression, low FSH receptor sensitivity, FSH receptor binding
deficiencies, FSH receptor coupling deficiencies, low testosterone
production, male pattern baldness, and pituitary failure or
injury.
[0084] In particular, the quantity and quality of oocytes can be
improved by administering a superactive FSH analog as described
herein to an animal. For example, as reported herein, Applicants
have surprisingly found that by administering a superactive FSH
containing a modified alpha-subunit with basic amino acids at
position 13, 14, 16 and 20, a dramatic increase in the quantity and
quality of oocytes is obtained. The effects of a superactive FSH on
oocyte quantity and quality may be further enhanced by increasing
the FSH serum half-life of the superactive FSH. The FSH serum
half-life can be increased by further modifying the superactive
FSH. Further modifications, including but not limited to those
previously described, can be used to increase FSH serum half-life.
For instance, an ANITV (SEQ ID No. 3) extension may be used to
prolong FSH serum half-life.
[0085] According to U.S. Pat. No. 5,574,011, herein incorporated by
reference in its entirety, FSH stimulates the gonads to produce
steroids, such as testosterone. Accordingly, the FSH analogs of the
invention could be used to treat any condition associated with low
steroid production, and particularly low testosterone production.
According to U.S. Pat. No. 6,562,790, herein incorporated by
reference, coronary artery blockage is treatable with testosterone.
Therefore, the analogs of the present invention may be used to
elevate testosterone levels in patients exhibiting coronary artery
disease.
[0086] The analogs of the present invention may also be used in
therapeutic regimens of assisted reproduction in either a male or
female subject comprising administering an assisting amount of the
modified FSH to the subject. In such methods, the analogs may be
administered alone or in combination with other therapeutics, for
instance, including but not limited to Clomiphene citrate, GnRH
(gonotropin releasing hormone) and LH (Luteinizing hormone). For
example, in a subject with isolated gonadotropin deficiency (IGD),
administration of modified FSH and LH may be administered to the
subject to restore normal gonadal function. It is widely known in
the art that glycoprotein hormones such as FSH and LH are integral
in female reproductive physiology, and these glycoprotein hormones
may be administered to a subject to overcome a number of
reproductive disorders and thereby assist reproduction.
[0087] The analogs of the invention are particularly useful for
treating women prone to ovarian hyperstimulation, for instance by
using analogs having different serum half-lives in a combined
regimen. Such methods may include (a) administering an assisting
amount of a first modified FSH according to the invention wherein
the plasma half-life of said first modified FSH is increased as
compared to wild type FSH, and (b) subsequently administering an
assisting amount of a second modified FSH according to the
invention wherein the plasma half-life of said second modified FSH
is decreased as compared to said first modified FSH. For instance,
analogs demonstrating decreased half-life as compared to wild-type
FSH, i.e. TR-4401, may be useful for treating women prone to
ovarian hyperstimulation.
[0088] A skilled practitioner in the art can readily determine the
effective amount of the glycoprotein hormone to administer and will
depend on factors such as weight, size, the severity of the
specific condition, and the type of subject itself. The
therapeutically effective amount can readily be determined by
routine optimization procedures. The present invention provides
glycoprotein hormones with increased potency relative to the
wild-type glycoprotein hormone. These modified glycoprotein
hormones will allow a skilled practitioner to administer a lower
dose of a modified glycoprotein hormone relative to the wild-type
glycoprotein hormones to achieve a similar therapeutic effect, or
alternatively, administer a dose of the modified glycoprotein
hormone similar to the dose of the wild-type glycoprotein hormone
to achieve an increased therapeutic effect.
[0089] Depending on whether the glycoprotein hormone is
administered orally, parenterally, or otherwise, the administration
of the prostaglandin can be in the form of solid, semi-solid, or
liquid dosage forms, such as, for example, tablets, pills,
capsules, powders, liquids, creams, and suspensions, or the like,
preferably in unit dosage form suitable for delivery of a precise
dosage. The glycoprotein hormone may include an effective amount of
the selected glycoprotein hormone in combination with a
pharmaceutically acceptable carrier and, in addition, may include
other medicinal agents, pharmaceutical agents, carriers, adjuvants,
diluents, etc. By "pharmaceutically acceptable" is meant a material
that is not biologically or otherwise undesirable, i.e., the
material may be administered to an individual along with the
selected glycoprotein hormone without causing unacceptable
biological effects or interacting in an unacceptable manner with
the glycoprotein hormone. Actual methods of preparing such dosage
forms are known, or will be apparent, to those skilled in this art;
for example, see Remington's Pharmaceutical Sciences, latest
edition (Mack Publishing Co., Easton, Pa.).
[0090] Genetic therapy is another approach for treating hormone
disorders with the modified glycoprotein hormones of the present
invention. In this approach, a gene encoding the modified
glycoprotein hormone can be introduced into a cell, such as a germ
line cell or a somatic cell, so that the gene is expressed in the
cell and subsequent generations of those cells are capable of
expressing the introduced gene. For example, a nucleic acid
encoding a modified FSH protein of the invention can be inserted
into an ovarian cell, or its precursor, to enhance ovulation.
Suitable vectors to deliver the coding sequence are well known in
the art. For example, the vector could be viral, such as
adenoviral, adenoassociated virus, retrovirus, or non-viral, such
as cationic liposomes.
[0091] The analogs of the present invention have an enhanced
activity over wild type protein and are therefore particularly
suitable for delivering agents to cells expressing glycoprotein
hormone receptors. Accordingly, the present invention further
provides a method of delivering an agent to a cell expressing a
glycoprotein receptor in a subject in need thereof using the
modified glycoprotein hormones of the invention. The method of
delivering an agent to a cell (i.e. targeted delivery) can employ
any suitable agent, depending on the nature of the subject's
illness or suspected illness. The agent can be a cytoprotective
compound, antibody, drug, sensitizer, biological response modifier,
radionuclide, toxin or combination thereof.
[0092] In certain embodiments, the methods of targeted delivery are
for the treatment of a subject with a disorder or suspected
disorder associated with abnormal glycoprotein receptor expression.
In certain embodiments, the methods of targeted delivery are for
the diagnosis or detection of a disorder associated with abnormal
glycoprotein receptor expression. In certain embodiments, the
methods of targeted delivery can be used in conjunction with other
therapies, diagnostic procedures or clinical modalities, including
radiation and/or surgery.
[0093] In one embodiment, the methods provide for targeted delivery
of an agent, wherein the agent is a cytoprotective compound.
Cytoprotective compounds are those compounds which act to protect
or decrease the incidence or severity of injury to a cell.
Commercially available cytoprotective compounds include mesna
(MESNEX.RTM., Bristol-Myers Squibb), amifostine (ETHYOL.RTM.,
Alza), dexrazoxane (ZINECARD.RTM., Pharmacia & Upjohn) and
leucovorin (multiple manufacturers).
[0094] In one embodiment, the agent can be any drug used to treat
various forms of cancer, such as, for example, natural or synthetic
estrogens, estrogen receptor modulators, progestins, androgens,
gonadotropin-releasing hormones, androgen inhibitors,
bisphosphonates, glucocorticoids, thyroid hormones, antithyroid
agents, iodine agents, bromocriptine, alkylating agents,
antimetabolites, antimitotic agents, epipodophyllotoxins,
antineoplastic antibiotics, antineoplastic hormones, platinum
coordination complex agents, anthracenediones, substituted ureas,
methylhydrazine derivatives, DNA topoisomerase inhibitors,
retinoids, porfimer, mitotane or combinations thereof.
[0095] In one embodiment, the agent can be any drug used to treat
cancers of the male or female reproductive systems (e.g.
endometrial cancer, uterine cancer, cervical cancer, breast cancer,
testicular cancer). In a preferred embodiment, the agent can be
clomiphene, finasteride, propylthiouracil, methimazole, bleomycin,
vincristine, vinblastine, cisplatin, mitomycin, ifosfamide,
cyclophosphamide, doxorubicin, paclitaxel, fluorouracil,
carboplatin, epirubicin, altretamine, vinorelbine, mitoxantrone,
prednisone or combinations thereof.
[0096] Drugs known to enhance the cytotoxic effect of certain
anti-cancer drugs and radiopharmaceuticals can also be used. Such
drugs are commonly referred to as sensitizers. Examples of
sensitizers which enhance the activity of various therapeutic drugs
(e.g., anti-cancer drugs) are buthionine sulfoximine and calcium
channel blockers such as verapamil, and diltiazem. (See, U.S. Pat.
No. 4,628,047 and Important Advances in Oncology 1986, DeVita, et
al., Eds., J. B. Lippincott Co., Philadelphia, pages 146-157
(1986), incorporated herein by reference in their entireties.)
Other sensitizers known in the art are metronidazole, misonidazole,
certain 2-sulfamyl-6-nitrobenzoic acid derivatives,
2,6-disubstituted derivatives of 3-nitropyrazine, and certain
isoindoledione compounds. (See, U.S. Pat. Nos. 4,647,588;
4,654,369; 4,609,659 and 4,494,547, incorporated herein by
reference in their entireties.)
[0097] In certain embodiments, the agent can be a biological
response modifier. Any biological response modifier can be used in
the scope of the invention. Examples of biological response
modifiers useful in the methods of the invention include, but are
not limited to interferon-.alpha., interferon-.beta.,
interferon-.gamma., tumor necrosis factor, lymphotoxin,
interleukin-1, interleukin-2, interleukin-3, interleukin-4,
interleukin-5, interleukin-6 or combinations thereof.
[0098] In certain embodiments, the agent can be an antibody. The
antibody can be a monoclonal or polyclonal antibody. In certain
embodiments, the antibodies can be humanized antibodies, chimeric
antibodies, or functional antibody fragments including, for
example, Fab1, Fab2, etc.
[0099] Examples of toxins which can be employed in the methods of
the invention are ricin, abrin, diphtheria toxin, Pseudomonas
exotoxin A, ribosomal inactivating proteins, and mycotoxins; e.g.,
trichothecenes. Trichothecenes are a species of mycotoxins produced
by soil fungi of the class fungi imperfecti or isolated from
Baccharus megapotamica (Bamburg, Proc. Molec. Subcell Bio. 1983,
8:41-110, Jarvis and Mazzola, Acc. Chem. Res. 1982, 15:338-395,
incorporated herein by reference in their entireties.)
Therapeutically effective modified toxins or fragments thereof,
such as those produced through genetic engineering or protein
engineering techniques, can be used.
[0100] Any means of coupling or linking an agent to a modified
glycoprotein hormone can be employed. For example a number of
different cleavable linkers have been described previously. See,
U.S. Pat. Nos. 4,618,492; 4,542,225; and 4,625,014, incorporated
herein by reference in their entireties. The mechanisms for release
of an agent from these linker groups include by irradiation of a
photolabile bond, and acid-catalyzed hydrolysis. U.S. Pat. No.
5,563,250, incorporated herein by reference in its entirety,
discloses immunoconjugates comprising linkers of specified chemical
structure, wherein the linkage is cleaved in vivo, releasing the
compound (radiopharmaceutical, drug, toxin, etc.) in its native
form. The linker is susceptible to cleavage at mildly acidic pH,
and is believed to be cleaved during transport into the cytoplasm
of a target cell, thereby releasing the biologically active
compound inside a target cell. U.S. Pat. No. 4,671,958,
incorporated herein by reference in its entirety, includes a
description of immunoconjugates comprising linkers which are
cleaved at the target site in vivo by the proteolytic enzymes of
the patient's complement system.
[0101] Other means of coupling or linking have been described. For
example, linker molecules are commercially available, such as those
available from Pierce Chemical Company, Rockford, Ill. (See the
Pierce 1986-87 General Catalog, pages 313-354, incorporated herein
by reference in its entirety.) Means for coupling to an antibody,
(See, for example, U.S. Pat. No. 4,671,958 and U.S. Pat. No.
4,659,839, incorporated herein by reference in their entireties)
and means of linking or coupling radionuclide metal chelates,
toxins and drugs to proteins are known. See, for example, European
Patent Application Publication No. 188,256; U.S. Pat. Nos.
4,671,958; 4,659,839, 4,414,148; 4,699,784; 4,680,338; 4,569,789;
and 4,590,071; Borlinghaus et al. Canc. Res. 47:4071-4075, Aug. 1,
1987, Foran, Best Pract. Res. Clin. Haematol. 2002, 15(3): 449-65
and Fotiou, et al., Eur. J. Gynaecol. Oncol. 1988, 9(4): 304-7
incorporated herein by reference in their entireties. In view of
the large number of methods that have been reported for coupling a
variety of radiodiagnostic compounds, radiopharmaceuticals, drugs,
toxins, and other agents to proteins, one skilled in the art will
be able to determine a suitable method for attaching a given agent
to a modified glycoprotein.
[0102] Methods of Imaging
[0103] The analogs of the present invention have an enhanced
activity over wild type protein and are therefore particularly
suitable for imaging cells expressing glycoprotein hormone
receptors. Accordingly, in one embodiment, the invention further
provides methods of imaging cells comprising a glycoprotein hormone
receptor using the modified glycoprotein hormones of the present
invention. The method of imaging and detecting the hormone can be
any method known to those of skill in the art. Commonly used
imaging methods include, for example, magnetic resonance imaging
(MRI), X-ray, computed tomography (CT), positron emission
tomography (PET), mammography and ultrasound.
[0104] Methods of imaging subjects using basic radiologic
techniques have been described, for example, "Textbook of Radiology
and Imaging," Sutton and Livingstone, 7th Edition, (2 Volume set),
Churchill Livingstone (Elsevier Sciences), London, 2002, "A Concise
Textbook of Radiology," Armstrong and Wastie (eds.) Arnold
Publishing (The Thomson Corporation), Scarborough, Ontario, Canada,
2001, "Walter & Miller's Textbook of Radiotherapy," Bomford and
Knuckler, 6th Edition, Churchill Livingstone (Elsevier Sciences),
London, 2001, incorporated herein by reference in their entireties.
See also, Bottomley, Comput. Radiol. 1984, 8(2): 57-77, Dixon,
Radiology 1984, 153(1):189-94, Daley and Cohen, Cancer Res. 1989,
49(4):770-9, Ellis, et al., Clin. Radiol. 2001, 56(9):691-9,
Paushter, et al., Med. Clin. North Am. 1984, 68(6):1393-421,
Blecher, Aust. Fam. Physician 1983 12(6):449-50, 452, Bragg, Cancer
1977, 40(1 Suppl):500-8, Moseley, Br. Med. J. (Clin. Res. Ed.)
1982, 284(6323):1141-4, Lentle and Aldrich, Lancet 1997,
350(9073):280-5, Weber, et al., Strahlenther Onkol. 1999,
75(8):356-73, Hanbidge, Can. J. Gastroenterol. 2002, 16(2):101-5,
Miles, Eur. Radiol. 2003, Suppl 5:M134-8, Prigent-Le Jeune, et al.,
Eur. J. Nucl. Med. Mol. Imaging. 2004, February 19 [Epub ahead of
print], DeSimone, et al., Gynecol. Oncol. 2003, 89(3):543-8 and
Goldenberg, et al., J. Clin. Oncol. 1987, 5(11):1827-35,
incorporated herein by reference in their entireties.
[0105] Any suitable means of imaging or detecting can be employed,
depending, inter alia, on the nature of the subject's disorder or
suspected disorder, the tissue to be imaged and whether functional
(physiologic) or structural (anatomic) images are desired. In some
embodiments, among others, the methods of imaging provide that
detecting an amount of a labeled modified glycoprotein hormone in a
subject or detecting increased levels of a modified glycoprotein
hormone in a subject indicates the presence of an autoimmune
disorder or a cancerous disorder selected from the group consisting
of ovarian cancer, uterine cancer, cervical cancer, endometrial
cancer, breast cancer, testicular cancer or pituitary tumor.
[0106] Imaging methods can be broadly categorized as those that
provide information regarding the structure or anatomy of a subject
or those that provide function or physiology of a subject.
Structural imaging provides the shape of a bone or tissue component
to determine if there are abnormal formations or destruction of
certain elements. Tumors or the presence of cancerous cells can
appear as structural changes. A newer type of structural imaging
provides the chemical composition of different parts of a tissue in
order to determine if there is ongoing injury or abnormal
biochemical processes (e.g. presence or growth of cancerous cells).
See, for example, Bonilha, et al., Med. Sci. Monit. 2004,
10(3):RA40-6, epub 2004 Mar. 1, Ballmaier, et al., Psychiatry Res.
2004, 15; 130(1):43-55, Ballmaier, et al., Biol. Psychiatry, 2004:
55(4):382-9, Cha, Magn. Reson. Imaging Clin. N. Am. 2003,
11(3):403-13 and Kopelman, et al., Hippocampus, 2003; 13(8):879-91,
incorporated herein by reference in their entireties.
[0107] Functional imaging is a relatively new technique which seeks
to ascertain whether particular tissues or organs are performing
particular functional tasks. This technique can capitalize on a
number of physiologic processes, including, for example, blood flow
and activity-associated with changes in blood flow (i.e. neoplastic
presence or growth) and monitoring responses to chemotherapy. See,
for example, Takeuchi, et al., J. Med. Invest. 2004, 51(1-2):59-62,
Otsuka, et al., J. Med. Invest. 2004, 51(1-2):14-9, Martincich, et
al., Breast Cancer Res. Treat. 2004, 83(1):67-76, Cohen and
Goadsby, Curr. Neurol. Neurosci. Rep. 2004, 4(2):105-10 and Lewis,
et al., Eur. J. Neurosci. 2004, 19(3):755-60, incorporated herein
by reference in their entireties.
[0108] In general, radiological methods such as, for example,
magnetic resonance imaging (MRI), X-ray, computed tomography (CT),
mammography and ultrasound provide structural or anatomic
information regarding a subject. Radiological methods such as, for
example, nuclear medicine, radionuclide imaging and positron
emission tomography (PET) provide functional or physiologic
information regarding a subject. Both structural and functional
imaging are within the scope of the present invention.
[0109] In one embodiment of the invention, the imaging methods
provide that the modified glycoprotein hormone is labeled (i.e. a
contrast agent is used). Any label or contrast agent can be used.
See, Minato, et al. J. Comput. Assist. Tomogr. 2004, 28(1):46-51,
Antoch, et al., JAMA 2003, 290(24):3199-206, Brinker, Rev.
Cardiovasc. Med. 2003; 4 Suppl 5:S19-27, el-Diasty, et al., J.
Urol. 2004, 171(1):31-4, Williams, et al., Int. J. Oral Maxillofac.
Surg. 2003, 32(6):651-2, Follen, et al., Cancer 2003, 98(9
Suppl):2028-38, Behrenbruch, et al., Med. Image Anal. 2003,
7(3):311-40, Knopp, et al., Mol. Cancer. Ther. 2003, 2(4):419-26,
incorporated herein by reference in their entireties. The label can
be any label known to those of skill in the art. In one embodiment,
the label can be a radiopaque label, radioactive label,
fluorescence label or paramagnetic label.
[0110] Radionuclides generally emit either beta (.beta.) or gamma
(.gamma.) radiation. 1131 emits about 90% .beta.-radiation and
about 10% .gamma.-particles has a physical half life of about 8
days. Tc99m emits .gamma.-radiation and has a half life of about 6
hours. Following administration of, for example, a Tc99m-labeled
protein, the biodistribution of the radionuclide can be detected by
scanning the patient with a gamma camera using known procedures.
Accumulations of Tc99m at the target site(s) is thus easily imaged.
See, Toohey, Radiographics. 2000; 20:533-546, Kostakoglu, et al.,
RadioGraphics 2003, 23:315-340, Saremi, et al., RadioGraphics 2002,
22:477-490, Intenzo, et al., RadioGraphics 2001, 21:957-964,
Ranger, RadioGraphics 1999, 19:481-502, Simpkin, RadioGraphics
1999, 19:155-167, Janoki and Kerekes, Acta Physiol. Hung. 1992,
79(2):183-96, Hoefnagel, Anticancer Drugs 1991, 2(2):107-32,
Hoefnagel, Eur. J. Nucl. Med. 1991, 18(6):408-31, Gatley, et al.,
Acta Radiol. Suppl. 1990, 374:7-11, Ott, Br. J. Radiol. 1989,
62(737):421-32, Andersen, Cerebrovasc. Brain Metab. Rev. 1989,
1(4):288-318 and Miraldi, Int. J. Radiat. Oncol. Biol. Phys. 1986,
12(7):1033-9, incorporated herein by reference in their
entireties.
[0111] In addition to I131 or Tc99m, any radioisotope known to
those of skill in the art can be employed in the methods of the
invention. Other radionuclides and chelates can include, for
example, Co57, Co58, Cr51, F18 FDG, Ga67, In111 chloride, In111
pentetate (DTPA), In111oxyquinoline (oxine), In111 Capromab
pendetide, In111 Imciroma pentetate, In111, pentetreotide, In111
satumomab pendetide, I 123, I125 iothalamate, I125 human serum
albumin (RISA), I131 iodohippurate, I131 iodomethylnorcholesterol
(NP-59), I131 metaiodobenzylguanidine (MIBG), Kr81m gas, P32
chromic phosphate, P32 sodium phosphate, Ru82, Sm 153 lexidronam
(Sm-153 EDTMP), Sr89, T1 201 and Xe133.
[0112] Diagnostic Assays
[0113] The present invention further provides for the detection of
analytes that interfere with the binding of the modified
glycoprotein hormones of the invention to a glycoprotein hormone
receptor. In one embodiment, the methods provide for the detection
of an analyte that interferes with the binding of a modified
glycoprotein hormone receptor in a biological sample, said method
comprising (I) contacting the sample with a modified glycoprotein
hormone according to the present invention and (ii) detecting a
signal wherein the presence or amount of the signal detected
indicates the presence or absence of an analyte that interferes
with the binding of a modified glycoprotein hormone to a
glycoprotein receptor.
[0114] In one embodiment, the method for the detection of an
analyte is a competitive binding assay. A competitive binding assay
is an assay based on the competition between a labeled and an
unlabelled ligand in the reaction with a receptor binding agent
(e.g. antibody, receptor, transport protein). IUPAC Compendium of
Chemical Terminology, 1997, 2nd edition, "Competitive Protein
Binding Assays" Odell and Daughaday, W.H. Lippincott, 1972 and
"Principles of Competitive Protein-binding Assays" Odell and
Franchimont, P. John Wiley & Sons Inc., 1983, incorporated
herein by reference in their entireties. See also, U.S. Pat. No.
6,537,1760, incorporated herein by reference in its entirety.
[0115] In certain embodiments, the signal is the presence or amount
of the modified glycoprotein hormone bound with the glycoprotein
receptor in the sample. In certain embodiments, the method employs
the detection of a secondary signal, such as, for example, the
detection of the presence or amount of cAMP or a steroid (e.g.
progesterone). In certain embodiments, the methods employ the use
of whole cells in the biological sample. In certain embodiments,
the methods employ only parts of cells, for example, cell
membranes.
[0116] In certain embodiments, the assay can be performed in
solution. In certain embodiments, one or more components of the
assay can be immobilized on a solid phase. Plastic surfaces,
microparticles, magnetic particles, filters, polymer gel materials
and other solid-phase substrates can be used as solid phases. See,
for example, 6,664,114; 6,589,798; 6,479,296 and 6,294,342,
incorporated herein by reference in their entireties. It is
possible to automate the methods of assay provided in the
invention.
[0117] Methods of Designing Glycoprotein Receptor Agonists and
Antagonists Using FSH Superagonists
[0118] The present invention also provides methods of designing new
receptor agonists and antagonists based on the interaction of the
FSH proteins of the invention and a cognate receptor. Such methods
involve predicting interactions of charge motifs in the FSH
proteins of the invention with complementary amino acid residues
within a cognate receptor. For instance, such a method may involve
comparing the differences in interaction in terms of binding and
bioactivity of FSH to receptors from evolutionary distant species,
e.g., human LH versus rat LH receptor, localizing charged amino
acids within extracellular domains and/or extracellular loops that
are present in only one of the two receptor sequences, performing
alanine scanning and charge reversal mutagenesis to further
validate given prediction, building a model of hormone-receptor
complex incorporating validated interactions, and designing new
hormone analogs and receptor antagonists using the model. New
hormone analogs include those that are predicted to bind to the
receptor using the model. New antagonists include those that are
designed from the domains and/or loop of the receptor protein that
are predicted to bind to the FSH analog using the model.
[0119] For example, one of the analogs of the invention (TR-4402,
comprising the substitutions alpha(E14R+Q20R+G73R)+betaE4R), has
been found to interact with the rat LH receptor (SEQ ID No. 23,
NCBI Accession No. NP.sub.--037110) at high concentrations, but not
the human LH receptor (SEQ ID No. 24, NCBI Accession No.
NP.sub.--000224, data not shown). Based on the difference in
specificity of TR-4402 at these receptors, Arg14, Arg20 and Arg73
should interact with negatively charged residues Asp and Glu in the
rat LH receptor. The negatively charged residues present in the rat
receptor but absent in the human receptor are Asp 312 and Glu 314
(based on the human LH receptor amino acid sequence with signal
peptide) (Ser and Lys, respectively, in the human LH receptor). The
corresponding residues in the human FSH receptor (SEQ ID No. 22,
NCBI Accession No. AAA52477) are Glu316, Asp317 and G1u319. This
cluster of acidic amino acids, therefore, is predicted to interact
with Arg14, Arg20 and Arg73 of the alpha subunit of TR-4402. This
information should allow for better modeling of glycoprotein
hormone interactions, and will contribute to the design of new
glycoprotein analogs, including peptide/protein antagonists
containing the sequence corresponding to 298-338 of the human FSH
receptor and including G1u300 and Asp302.
[0120] The following examples are provided to describe and
illustrate the present invention. As such, they should not be
construed to limit the scope of the invention. Those in the art
will well appreciate that many other embodiments also fall within
the scope of the invention, as it is described hereinabove and in
the claims.
EXAMPLES
Example 1
Production and Characterization of FSH Superagonists
[0121] Site Directed Mutagenesis. Site directed mutagenesis of
human alpha (SEQ ID No. 1) and FSH beta (SEQ ID No. 2) subunit cDNA
was performed using QuickChange Mutagenesis Kit from Stratagene.
Analogs were designed according to the methods described in U.S.
Pat. No. 6,361,992, herein incorporated by reference in its
entirety.
[0122] After subcloning into the expression vectors, the entire PCR
products of all constructs were sequenced to verify the mutations
and to rule out any undesired polymerase errors.
[0123] Transient Expression. Analogs were expressed transiently in
Chinese hamster ovary (CHO-K1) cells. Cells were transiently
co-transfected in 60 or 100 min culture dishes with wild-type or
mutant subunit cDNAs (alpha and FSH beta), using a transient
transfection protocol based on a liposome formulation
(LipofectAMiNE reagent, Gibco BRL). After recovery for 12 hours in
regular growth medium, transfected cells were cultured in CHO-serum
free medium (CHO-SFM, Gibco BRL) for 72 hours. Subsequently, the
conditioned media including control medium from mock transfections
using the expression plasmids without gene inserts, were harvested,
concentrated with Centriprep 10 concentrators (Amicon, Beverly,
Mass.) and stored at -70.degree. C. Analogs were quantitated with a
panel of different monoclonal and polyclonal antibodies recognizing
different FSH epitopes.
[0124] FSH Bioactivity Assay. The follitropic activity of the
analogs was assessed by their ability to induce cAMP production in
CHO cells expressing hFSH receptors. CHO cells stably expressing
the hFSH receptor were grown to confluence in 96-well tissue
culture plates. Subsequently, cells were incubated either in
salt-free conditions (2 h) or with physiological media (1 h) at
37.degree. C., 5% CO2 with serial dilutions of wild-type and mutant
FSH as well as control medium from mock transfections. The amount
of cAMP produced was determined by radioimmunoassay.
[0125] FSH mutations showing the highest bioactivity in vitro and
no adverse effects on FSH production were chosen for combination
strains. FIG. 1 includes graphs showing a comparison of the effect
of various single mutations on FSH bioactivity in vitro, as
measured using transient transfection of CHO-FSHR cells. Single
mutations showing the highest potency included basic substitutions
at alpha positions Q13, E14, V68, P21 and G73, and at beta position
E4. An arginine substitution at F18 resulted in a loss of
bioactivity. Beta E4R in particular resulted in enhanced FSH
production (see FIG. 2). The synergistic effect on bioactivity of
several combined substitutions is shown in FIG. 3.
[0126] In total, 26 single mutations in the alpha subunit and 23
single mutations in the beta subunit were tested, and the top
mutations in each subunit were selected to construct lead analogs
with combined substitutions. Table 1 below shows combined mutations
with a proven increase in bioactivity in vitro.
TABLE-US-00001 TABLE 1 Combined Substitutions Resulting in Enhanced
Potency Analog Substitutions 4201 alphaE14R + betaE4R 4202
alpha(E14R + N66R) + WT beta 4203 alpha(E14R + G73R) + WT beta 4204
alpha(P16R + Q20R) + WT beta 4205 alpha(Q20R + P21R) + WT beta 4301
alpha(E14R + Q20R + G73R) + WT beta 4302 alpha(E14R + P21R + G73R)
+ WT beta 4303 alpha(E14R + N66R + G73R) + WT beta 4304 alpha(E14R
+ N66R) + betaE4R 4305 alpha(E14R + G73R) + betaE4R 4306 alpha(P16R
+ Q20D + P21R) + WT beta 4307 alpha(P16R + Q20R + P21R) + WT beta
4308 alpha(N66K + G73K + A81K) + WT beta 4401 alpha(Q13R + E14R +
P16R + Q20R) + WT beta 4402 alpha(E14R + Q20R + G73R) + betaE4R
4403 alpha(E14R + P21R + G73R) + betaE4R 4404 alpha(E14R + N66R +
G73R) + betaE4R 4405 alpha(Q13K + E14K + P16K + Q20K) + WT beta
4501 alpha(E14R + Q20R + P21R + N66R + G73R) + WT beta 4601
alpha(Q13K + E14K + P16K + Q20K + N66K + G73K) + WT beta 4602
alpha(E14R + P16R + Q20R + P21R + N66R + G73R) + WT beta 4603
alpha(E14R + Q20R + P21R + N66R + G73R) + betaE4R 4701 alpha(E14R +
P16R + Q20R + P21R + N66R + G73R) + betaE4R 4901 [alpha(E14R + Q20R
+ G73R) + betaE4R] with N-terminal ANITV (SEQ ID No. 3) extension
in the alpha subunit 4910 [alpha(Q13R + E14R + P16R + Q20R) + WT
beta] with N- terminal ANITV (SEQ ID No. 3)extension in the alpha
subunit
[0127] As known in the art, a particular drug may exhibit different
efficiencies depending on the system used. See Kenakin, "Predicting
Therapeutic Value in the Lead Optimization Phase of Drug
Disclovery," Nature Rev. 2: 429-38 (2003). Therefore, the
bioactivity of the analogs of the invention was also tested using
rat granulosa cells expressing low quantities of FSH receptor
(GLHR-15 cells). As shown in the graphs in FIG. 4, the analogs of
the invention still resulted in significant cAMP responses, as
compared to wild type FSH and free alpha chain, which did not show
a significant dose dependent response.
Example 2
Purification and Characterization of Analog TR-4402
[0128] Analog TR-4402 was chosen for purification and further
characterization. TR-4402 is a FSH analog containing two mutations
in the alpha L1 loop (.alpha.E14R and .alpha.Q20R), one mutation in
the alpha L3 loop (.alpha.G73R), and one mutation in the beta L1
loop (.beta.E4R). See FIG. 5. The cell line producing TR-4402 was
established by co-transfection of a modified pED vector containing
the cDNA of the human alpha subunit and dihydrofolate reductase
(DHFR) gene, and a modified pIRES vector containing the cDNA of FSH
beta subunit, separated by an IRES sequence and the amplifiable
gene marker, adenosine deaminase (ADA), into CHO-DHFR(-) by
lipofectamine method. Transfected cells were cultured in selection
medium (ribonucleosides and deoxyribonucleosides deficient aMEM
with 10% dialyzed FBS). For stable production of hFSH analogs in a
CHO double deletion mutant (dhfr-/dhfr-), the CHO-DG44 cell line
was kindly provided by Dr. L. Chasin (Columbia University, New
York, N.Y.).
[0129] Clonal cell lines secreting FSH#4402 were cultured in
increasing concentrations of methotrexate (MTX) up to 2 .mu.M. DHFR
amplification is based on systematic increases of MTX in medium
without added nucleosides. Cells were qualified for next
amplification step after regaining their polygonal morphology (2-3
weeks). Since the concentration of MTX increased about 800.times.
(from 0.005 uM to about 4 .mu.M) the amplification process took
about 4 months. Clones with highest secretion level were also
subjected to a second treatment, involving the utilization of
deoxycoformycin, directed to amplify the ADA marker gene.
[0130] Single clones were tested for expression using FSH
immunoassay established for detection of FSH#4402. A stable cell
line (clone ID: H-2-3), transfected with pED-analog
a+pIRES-ADA-analog b (molar ratio of a:b=1:5, 5 mg total DNA), was
selected and propagated in alpha-minimum essential medium (a-MEM:
Cat #: 12561-056, Lot #1141509, with L-glutamine, without
ribonucleosides and deoxyribonucleosides); GIBCO, Grand Island,
N.Y.), supplemented with 10% dialyzed fetal bovine serum (Gibco,
Cat No: 26400-044) and 2 mM methotrexate (MTX; ICN, Cat. No:
102299, Aurora, Ohio).
[0131] Preparation of bioreactor inoculums. When >90% CHO-DG44
cells were confluent in the 500-cm2 T-flasks, the cells were
trypsinized, centrifuged at 400 g for 5 min at 4.degree. C. Total
cells (1.6 `109/500 ml of a-MEM culture medium) were inoculated
into the Celligen plus Bioreactor by feeding cell suspension in a 2
L-plastic bottle.
[0132] After amplification, the cell line producing the highest
amount of FSH#4402 was grown in multiple flasks. Cells were further
propagated using perfusion mode in Packed-Bed Bioreactor 3.5 L
Bioreactor with internal retention device (basket) and vertical
mixing system (Celligen Plus, New Brunswick Scientific, Edison,
NJ). Cells were trapped onto Fibracel disks located inside the
retention assembly. Dissolved oxygen was kept at 50% saturation.
Temperature was 37 C. Agitation was 100 rpm. The pH 7.2 was
maintained using a four-gas mixing system and automatic injection
of sodium bicarbonate. Perfusion was adjusted to keep glucose level
above 1.5 g/L and lactate below 1.5 g/L.
[0133] FBS weaning process and FSH-TR 4402 analog production in
Celligen plus bioreactor. CHO III A (Gibco, Formula No.: 97-0147DK,
Lot No.: 1147268) supplemented with hypoxanthine-thymidine
supplement (Gibco, 100'), penicillin-streptomycin (Gibco, 100'; Cat
#: 15140-122, Lot #: 1161387), glutamax-1 (Gibco, 100'; Cat #:
35050-061, Lot #: 1163550), 10% pluronic F-68 (Gibco, 100', Cat #:
24040-032, Lot #: 1153058), and 1% dialyzed fetal bovine serum
(Gibco) was used for serum weaning in the bioreactor. At day 17 of
the bioreactor operation, we changed CHO-III A culture medium to
CHO protein free, animal component-free medium (Sigma, Cat #:
C-8730 Lot No.: 122K8401) until day 23 (see SLIDE summarizing
bioreactor run). Medium from bioreactor was harvested, centrifuged,
filtered (0.45 .mu.m membrane) and concentrated using Millipore
concentrators.
[0134] Purification. TR-4402 was purified using immunoaffinity
(monoclonal Ab ME.112 from Maine Biotechnology Services, Inc.) and
hydrophobic interaction chromatography. Purity was assessed based
on SDS-PAGE (.about.85%).
[0135] Modified analog TR-4402 was characterized using in vitro
bioassays employing CHO, human granulosa-like tumor (KGN) and rat
granulosa (GLHR-15) cell lines expressing human FSH receptor and
total cAMP production as an end point (see FIGS. 6-8). Using
CHO-FSHR cells, TR-4402 showed a 30 fold increase in potency and
17% increase in Vmax as compared to Follistim (wild type FSH).
Using KGN-FSHR, TR-4402 also showed a 30 fold increase in potency
as compared to wild type FSH.
[0136] TR-4402 was also tested for binding to LH and TSH receptors
to confirm FSH analog specificity, and isoelectric focusing was
employed to confirm carbohydrate chain heterogeneity, i.e., the
presence of alpha and beta subunits (data not shown).
[0137] The effect of TR-4402 as compared to wild type FSH was also
tested on mouse follicles in vitro using EggCentris in vitro
follicle bioassay. This assay studies the growth and development of
early preantral follicles up to the ovulatory stage. The whole in
vitro, process closely mimics the physiology of in vivo
folliculogenesis. The culture system begins with isolation of a
homogenous class of mouse preantral follicles between 100 and 130
.mu.M in diameter. The follicules were individually plated and
cultured for 12 days with 1, 3 and 9 mIU/mL of wild type ("compound
3") and TR-4402 ("compound 4").
[0138] The follicle bioassay indicated that quality of the oocytes
is improved after exposure to TR-4402 in comparison to Follistim
(wild type) as shown by enhanced follicle survival (FIG. 9),
enhanced antrum formation (FIG. 10), enhanced mucification of COC
(FIG. 11), enhanced nuclear maturation (FIG. 12) and enhanced
progesterone production (FIG. 13). Such differences could be
related to the presence and anti-apoptotic action of FSH receptor
in the oocyte cell membrane (see, e.g., Meduri et al., J. Clin.
Endocrinol. Metab. 87(5): 2266-76), and indicate that the modified
superagonists of the invention may be used to improve the
performance of oocytes in patients seeking assisted reproduction
therapy.
Example 3
In Vivo Studies Using TR-4402
[0139] In vivo studies of TR4402 were performed using immature
21-day old Sprague-Dawley female rats. The FSH injection was
performed subcutaneously once a day for 3 days (0, 24 and 48 h). At
72 h blood samples were collected and autopsy was performed. The
weight of both ovaries was measured. FSH and inhibin B levels in
sera were determined using ICN-IRMA FSH immunoassay. Intra-ovarian
estradiol content was determined after homogenization of
ovaries--using CT 17beta-estradiol kit (ICN Pharmaceuticals,
Inc.).
[0140] An increase of ovarian weight has been previously correlated
with injected dose of FSH (Steelman and Pohley, 1953). FSH
stimulates follicle growth (granulosa cells proliferation,
hyperaemia, estradiol and inhibin B production). Statistically
significant differences in ovarian weight (FIGS. 14A, C and D),
intra-ovarian estradiol content (FIG. 15) and serum inhibin B
levels (FIG. 16) after stimulation with corresponding doses of
TR-4402 and Follistim were observed. Such advantage of TR-4402 over
wild type FSH in terms of ovarian weight, inhibin and estradiol
production was observed despite a 40-50% lower level of TR-4402
than Follistim remaining in sera at the end of each experiment (see
FIG. 14B).
[0141] Since studies in rodents are generally considered as good
indicators of clinical efficacy of FSH preparations in humans, it
is expected that TR-4402 should show considerable advantage over
Follistim for the treatment of human patients. Moreover, a
superactive FSH with faster clearance rate (such as TR-4402) should
have immediate applications at the second phase of IVF protocol and
result in decreased occurrence of ovarian hyperstimulation syndrome
(OHSS).
Example 4
In Vitro Fertilization, Embryo Development, and Live Births Studies
Comparing FSH Analogs to Wild Type FSH
[0142] Twenty-three day old B6D2F1 female mice (groups of 5)
received one subcutaneous injection of 10 IU of TR-4401, 10 IU
TR-4901, 10 IU wild type FSH (Follistim), or 20 IU wild type FSH
(Follistim) on day one of the experiment. An ovulatory dose of hCG
was administered by an intraperitoneal injection in at least one
animal as a control.
[0143] After seventy-two hours from the FSH injection, sperm and
oocytes were collected and fertilization occurred. Sperm was
collected from male B6D2 and CB6F1 mice greater than 2 months of
age. The male mice were sacrificed by cervical dislocation. An
incision was made in the lower area of the abdomen, and the
epididymis and vas deferens were dissected out and placed in a
sperm dish. The epididymis and vas deferens were cut 3 to 5 times,
and the sperm was gently squeezed out of the organs on to the sperm
dish. The sperm dish with sperm was placed in an incubator at
37.degree. C. and 5% CO2 and allowed to capacitate 30 to 90
minutes.
[0144] Oocytes were collected from the superovulated female mice
which had received TR-4401, TR-4901, or wild-type FSH (Follistim)
by sacrificing the female mice and dissecting out the oviducts. The
oviducts were placed in a drops of HTF medium and the ampulae were
torn to release egg clutches. The intact egg clutches were
transferred to fertilization dishes and counted. Table 2 provides
the count of oocytes per group of five mice. FSH analogs TR-4401
and TR-4901 produced more oocytes at the 10 IU dosage than
recombinant wild type FSH (Follistem) at the 10 IU and 20 IU
dosages.
[0145] After the oocytes were placed in the fertilization dishes,
aliquots of sperm (1.times.106 to 2.times.106 sperm/ml) were added
to each fertilization dish. The fertilization dishes were placed in
the incubator at 37.degree. C. and 5% CO2 for a minimum of four
hours to allow fertilization to occur. After the four hours of
incubation, the fertilized eggs were transferred from the
fertilization dishes to wash dishes where they were washed at least
two times in drops of 250 .mu.l of HTF medium to remove debris. The
oocytes were stored in HTF drops in the dishes in the 37.degree. C.
and 5% CO2 incubator overnight.
[0146] Twenty-four hours after fertilization, the cells were
removed from the incubator. Two cell embryos were counted (Table 2,
column titled "number of 2 cell embryos"), and the fertilization
rate was determined by the percentage of oocytes which developed
into two cell embryos (Table 2, column "% of 2-cell embryos"). The
number of resulting two cell embryos was greater for the groups of
mice treated with FSH analogs TR-4401 and TR-4901. The
fertilization rate for all groups (FSH analogs and recombinant wild
type FSH) was high.
[0147] The two-cell embryos were subsequently transferred to
cultured dishes for further development (Table 2, column titled
"number of 2-cell embryos remaining in culture") or implanted in
pseudopregnant females (Table 2, column titled "number of 2-cell
embryos transferred").
[0148] The embryos which remained in the culture dishes were
observed for blastocyst formation on the fourth day after
fertilization. The number of developing blastocysts is provided in
Table 2 in the column titled "number of developing blastocysts".
Table 2 provides both the total number of blastocysts and the
number of blastocysts which hatched.
[0149] The two cell embryos which were implanted for fertilization
were implanted in CD1 females between six and eight weeks old.
Sixty 2-cell embryos were implanted in each test group of three
mice with the exception of the TR-4401 group which had forty 2-cell
embryos implanted. The mice were anesthetized with a solution of
ketamine/zylazine by intraperitoneal injection. Once anesthetized,
each mouse was shaved, and a small (0.5 cm) incision was made
caudal of the rib cage and at the first one third of the flank of
the dorsal to ventral. Another incision was made in the body wall
to provide access to the abdominal cavity. Forceps were used to
grasp the ovarian fat pad and gently withdraw the ovary, oviduct,
and proximal end of the uterus through the body wall. The ovary and
oviduct were positioned on a cotton swab to create an angle on the
ovarian-oviductal junction. The infundibulum was identified under a
stereomicroscope, and two pairs of superfine forceps were used to
make a hole in the bursa. Embryos were transferred by pipetting a
minimal volume of M2 medium with the embryos into the infundibulum.
The organs were then relocated into the body wall and sutured with
one or two stitches. The skin incision was closed with one or two
wound clips. The mice were observed for daily. After ten days, the
recipient mice were checked for pregnancy. Table 2, column
"pregnancy from 2-cell embryo transfer" provides the number of
resulting pregnancies per test group. FSH analog TR-4901 produced
the most pregnancies.
[0150] A similar experiment comparing TR-4401 to recombinant wild
type FSH was performed using birth as an end-point. Female mice (3
mice/group) were injected with 1 IU hCG and 3 IU of pregnant mare
serum gonadotropin (PMSG) as a control, 11CT wild type FSH (Gonal
F), 3 IU wild type FSH (Gonal F), 1 IU TR-4401, or 3 IU TR-4401.
The mice were injected 48 hours later with an ovulatory dose of 5
ILT hCG. Twenty hours after the ovulatory dose, oocytes were
counted and in vitro fertilization was allowed to take place as
previously described. Subsequently, twenty 2-cell embryos were
implanted in pseudo-pregnant mothers. Table 3 provides the results
of this experiment. The test groups which received 1 IU or 3 IU of
TR-4401 achieved greater oocyte counts, higher rates of blastocyst
development, and higher birth rates compared to the control group
and test groups treated with recombinant wild type FSH.
TABLE-US-00002 TABLE 2 In vitro Fertilization and Embryo Transfer
Experiment Comparing TR-4401 and TR-4901 to Recombinant Wild-Type
FSH (Follistim) Test Article Oocytes Number of Number of 2-cell
Number of 2-cell Number of Pregnancy from Dose Level Count 2-cell %
of 2-cell embryos embryos remaining developing 2-cell embryo
(IU/mouse) (per group) embryos embryos transferred in culture
blastocysts transfer 10 IU Follistim 77 77 100% 60 (3 females) 12 6
total (3 hatched) 0 20 IU Follistim 165 161 98% 60 (3 females) 101
52 (37 hatched) 2 10 IU TR-4401 207 204 99% 40 (2 females) 164 75
(38 hatched) 1 10 IU TR-4901 376 369 98% 60 (3 females) 171 197
(126 hatched) 3
TABLE-US-00003 TABLE 3 In Vitro Fertilization, Embryo Development,
and Live Births Experiment Comparing TR-4401 to Recombinant Wild
Type FSH (Gonal-F) and PMSG (control) Test Article Oocytes Number
of Number of 2-cell Number of % of Dose Level Count 2-cell % of
2-cell embryos remaining developing developing Birth (IU/mouse)
(per group) embryos embryos in culture blastocysts blastocysts Rate
3 IU PMSG 58 51 88% 31 18 58% 0/20 1 IU Gonal F 26 26 100% 6 4 67%
0/20 3 IU Gonal F 21 21 100% 1 0 0% 0/20 1 IU TR-4401 78 59 76% 19
12 63% 5/40 3 IU TR4401 116 113 97% 53 38 72% 11/60
Example 5
Comparison of the Quantity and Quality of Oocytes from Mice Treated
with TR-4401 FSH Analog and Recombinant Wild Type FSH (Gonal F)
[0151] Oocytes from B6CBAF1 mice were quantitatively and
qualitatively assessed after in vivo treatment with a control or
various doses of recombinant wild type FSH or FSH analog TR-4401 as
described in Table 4. In vitro fertilization took place on day 1
(72 hours post treatment) according to the protocol previously
described.
TABLE-US-00004 TABLE 4 Treatment groups (3 mice/group) Treatment
(Day -2) hCG (Day 0) Control: 2.5 IU PMSG (Folligon) 5 IU hCG
(Chorulon) Control: hCG only 5 IU hCG 0.5 IU Recombinant FSH (Gonal
F) + 1 IU 5 IU hCG (Ovitrelle) hCG (Ovitrelle) 0.5 IU TR-4401 + 1
IU hCG (Ovitrelle) 5 IU hCG (Ovitrelle) 1 IU Recombinant FSH (Gonal
F) + 1 IU 5 IU hCG (Ovitrelle) hCG (Ovitrelle) 1 IU TR-4401 + 1 IU
hCG (Ovitrelle) 5 IU hCG (Ovitrelle) 3 IU Recombinant FSH (Gonal F)
+ 1 IU 5 IU hCG (Ovitrelle) hCG (Ovitrelle) 3 IU TR-4401 + 1 IU hCG
(Ovitrelle) 5 IU hCG (Ovitrelle)
[0152] Treatment with TR-4401 was found to significantly increase
the number of oocytes produced. FIG. 21 provides the total number
of oocytes per group at the time of sperm washing (immediately
prior to in vitro fertilization). The figure shows that TR-4401
produced more oocytes at all doses (0.5 IU, 1 IU, and 3 IU) than
the test groups treated with recombinant wild type follicle
stimulating hormone.
[0153] Treatment with TR-4401 increased the total number of embryos
resulting from in vitro fertilization. FIG. 24 provides the total
number of 2-cell embryos per group. The figure shows that TR-4401
produced more 2-cell embryos at all doses (0.5 IU, 1 IU, and 3 IU)
than the test groups treated with recombinant wild type FSH.
[0154] In a similar experiment, test groups received 3 doses of
0.5, 1, or 3 IU of TR-4401 or recombinant wild type FSH (Gonal F)
combined with 1 IU of hCG. A control group received 3 doses of 1 IU
hCG. On day 3, all groups were given one ovulatory dose of 15 IU
hCG. In vitro fertilization was performed on mice as previously
described. FIG. 22 shows that mice treated with the TR-4401 FSH
analog displayed higher fertilization rates at all doses (3.times.
(0.5, 1, 3 IU)+1 IU hCG) than mice treated with recombinant wild
type FSH (Gonal F) or the control. Furthermore, oocytes from mice
treated with the lowest dose of TR-4401 (3.times.0.5 TR-4401+1 IU
hCG) displayed a higher fertilization rate than those from mice
treated with higher doses of TR-4401. FIG. 23 shows that embryos
from mice treated with the TR-4401 FSH analog (all dosages)
displayed higher blastocyst formation rates than the embryos from
the test group treated with recombinant wild type FSH. Embryos from
mice treated with the lowest dose of TR-4401 (3.times.0.5 TR-4401+1
hCG) displayed a greater blastocyst formation rate than embryos
from mice treated with higher doses of TR-4401.
Example 6
Pharmokinetics Comparison of FSH Analogs
[0155] Pharmokinetics experiments were performed to determine the
rates of absorption and elimination of FSH analogs TR-4401,
TR-4402, and TR-4901 compared to recombinant wild type FSH. A FSH
Clearance Assay was performed to determine the amount of serum FSH
in mIU/ml over time for TR-4401, TR-4402, and TR-4901 compared to
recombinant wild type FSH. FIG. 17A provides the results of the
assay. The figure shows delayed clearance of FSH analog TR-4401
compared to TR-4402 and TR-4901. FSH analog TR-4402 exhibited a
reduced duration of action compared to the other analogs.
Similarly, FIG. 17 B shows the rate of elimination (ln [serum FSH
mIU/ml] over time) for FSH analogs TR-4401, TR-4402, and TR-4901
compared to recombinant wild type FSH. TR-4402 was eliminated at a
faster rate than the other analogs and recombinant wild type FSH
(Follistim). Table 5 provides the data from the pharmokinetics
experiment. The data confirms that the rate of elimination (Ke) and
the rate of absorption (Ka) were greatest for FSH analog TR-4402.
As expected, the serum half life (T1/2) was lowest for TR-4402
compared to the other analogs and the recombinant wild type
FSH.
TABLE-US-00005 TABLE 5 Pharmokinetics Data for FSH Analogs TR-4901,
TR-4401, and TR-4402 and Recombinant Wild Type FSH (Follistem)
TR-4901 TR-4401 TR-4402 Wild Type FSH Ke 0.09 0.076 0.124 0.099 Ka
0.5794 0.5654 1.55 0.3503 V 17.47 31.55 20.46 12.51 AUC (0-t) 9328
6328 6868 8768 (mIU * hour/ml) Tmax (hour) 3.8 4.1 1.8 5.0 Cmax
(mIU/ml) 609.6 348.1 588.6 728.8 T1/2 (hour) 7.7 9.1 5.6 7.0 AUC
9508.8 6570.9 6897.1 8804.5 (mIU * Hour/ml)
[0156] Pharmokinetics may have a dramatic effect on how a patient
reacts to a FSH analog. Hypersensitive patients at risk for
hyperstimulation syndrome may benefit from a FSH analog such as
TR-4402 which acts faster and for a shorter duration than the other
analogs. Other patients would likely benefit from a FSH analog such
as TR-4401 which demonstrates a prolonged pharmokinetics
action.
[0157] Additional pharmokinetics experiments were performed
comparing TR-4401 to recombinant wild type FSH (Gonal F). In one
experiment mice were injected with a single dose of recombinant
wild type FSH or TR-4401. Terminal blood levels were determined 68
hours following the injection at necropsy. Terminal blood FSH
values were at least 5-6 times higher with TR-4401 compared to
recombinant wild type FSH. Table 6 provides dosing and terminal
blood FSH data.
TABLE-US-00006 TABLE 6 Terminal Blood FSH Data for FSH Terminal
Blood FSH FSH Dosage (.mu.g) Values (mIU/ml) Recombinant Wild 2.22
.mu.g all animals less than 2.5 mIU/ml Type FSH (Gonal F) TR-4401
0.22 .mu.g all animals less than 2.5 mIU/ml TR-4401 2.2 .mu.g all
animals between 12-15 mIU/ml
Example 7
Modifications to Increase Serum Half Life of FSH Analogs
[0158] FHS analog TR-4402 was further modified by an N-terminal
extension as previously described to enhance serum half life.
Examples of further modifications which may enhance serum half life
for FHS analogs are provided in FIG. 18. In vitro cAMP stimulation
studies using CHO cells were conducted to compare N-terminal
modified TR-4402 (LA1-4402), "wild type" N-terminal modified FSH
(LA1-Wt), TR-4402, and recombinant wild type FSH (Follistem). FIG.
19 shows that the modifications extended the FSH serum half-life
for the FSH analog TR-4402 and recombinant wild type FHS.
[0159] An in vivo ovulation assay was conducted in hybrid B6D2F1
mice to compare N-terminal modified TR-4402 to recombinant wild
type FSH. The mice treated with N-terminal modified TR-4402
produced more oocytes than those treated with recombinant wild type
FSH or hCG (control) for doses 0.5 IU, 1.0 IU, 2.5 IU, 5.0 IU, and
10 IU. The results of the experiment are shown in FIG. 20.
[0160] All publications, patents and patent applications discussed
in this application are incorporated herein by reference. While in
the foregoing specification this invention has been described in
relation to certain preferred embodiments thereof, and many details
have been set forth for purposes of illustration, it will be
apparent to those skilled in the art that the invention is
susceptible to additional embodiments and that certain of the
details described herein may be varied considerably without
departing from the basic principles of the invention.
Sequence CWU 1
1
24192PRTHomo sapiens 1Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr
Leu Gln Glu Asn Pro 1 5 10 15 Phe Phe Ser Gln Pro Gly Ala Pro Ile
Leu Gln Cys Met Gly Cys Cys 20 25 30 Phe Ser Arg Ala Tyr Pro Thr
Pro Leu Arg Ser Lys Lys Thr Met Leu 35 40 45 Val Gln Lys Asn Val
Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser 50 55 60 Tyr Asn Arg
Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr 65 70 75 80 Ala
Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser 85 90 2111PRTHomo
sapiens 2Asn Ser Cys Glu Leu Thr Asn Ile Thr Ile Ala Ile Glu Lys
Glu Glu 1 5 10 15 Cys Arg Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys
Ala Gly Tyr Cys 20 25 30 Tyr Thr Arg Asp Leu Val Tyr Lys Asp Pro
Ala Arg Pro Lys Ile Gln 35 40 45 Lys Thr Cys Thr Phe Lys Glu Leu
Val Tyr Glu Thr Val Arg Val Pro 50 55 60 Gly Cys Ala His His Ala
Asp Ser Leu Tyr Thr Tyr Pro Val Ala Thr 65 70 75 80 Gln Cys His Cys
Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys Thr Val 85 90 95 Arg Gly
Leu Gly Pro Ser Tyr Cys Ser Phe Gly Glu Met Lys Glu 100 105 110
35PRTArtificial SequenceAmino terminal extension; potential
glycosylation recognition site 3Ala Asn Ile Thr Val 1 5
49PRTArtificial SequenceAmino terminal extension; potential
glycosylation recognition site 4Ala Asn Ile Thr Val Asn Ile Thr Val
1 5 54PRTArtificial SequenceNegatively charged amino acid insert to
modify protein half-life 5Gly Glu Phe Thr 1 65PRTArtificial
SequenceNegatively charged amino acid insert to modify protein
half-life 6Gly Glu Phe Thr Thr 1 5 711PRTArtificial SequenceFSH
segment with negatively charged amino acid insert to modify protein
half-life 7Ala Asp Pro Gly Glu Phe Thr Val Gln Asp Cys 1 5 10
811PRTArtificial SequenceFSH segment with negatively charged amino
acid insert to modify protein half-life 8Ala Asp Pro Gly Glu Phe
Thr Thr Gln Asp Cys 1 5 10 997PRTArtificial SequenceMutated FSH
alpha mature peptide sequence with N-terminal extension 9Ala Asn
Ile Thr Val Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr 1 5 10 15
Leu Gln Glu Asn Pro Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln 20
25 30 Cys Met Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg
Ser 35 40 45 Lys Lys Thr Met Leu Val Gln Lys Asn Val Thr Ser Glu
Ser Thr Cys 50 55 60 Cys Val Ala Lys Ser Tyr Asn Arg Val Thr Val
Met Gly Gly Phe Lys 65 70 75 80 Val Glu Asn His Thr Ala Cys His Cys
Ser Thr Cys Tyr Tyr His Lys 85 90 95 Ser 1097PRTArtificial
SequenceMutated FSH alpha mature peptide sequence with N-terminal
extension 10Ala Asn Ile Thr Val Ala Pro Asp Val Gln Asp Cys Pro Glu
Cys Thr 1 5 10 15 Leu Gln Arg Asn Pro Phe Phe Ser Arg Pro Gly Ala
Pro Ile Leu Gln 20 25 30 Cys Met Gly Cys Cys Phe Ser Arg Ala Tyr
Pro Thr Pro Leu Arg Ser 35 40 45 Lys Lys Thr Met Leu Val Gln Lys
Asn Val Thr Ser Glu Ser Thr Cys 50 55 60 Cys Val Ala Lys Ser Tyr
Asn Arg Val Thr Val Met Gly Arg Phe Lys 65 70 75 80 Val Glu Asn His
Thr Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys 85 90 95 Ser
11101PRTArtificial SequenceMutated FSH alpha mature peptide
sequence with N-terminal extension 11Ala Asn Ile Thr Val Asn Ile
Thr Val Ala Pro Asp Val Gln Asp Cys 1 5 10 15 Pro Glu Cys Thr Leu
Gln Glu Asn Pro Phe Phe Ser Gln Pro Gly Ala 20 25 30 Pro Ile Leu
Gln Cys Met Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr 35 40 45 Pro
Leu Arg Ser Lys Lys Thr Met Leu Val Gln Lys Asn Val Thr Ser 50 55
60 Glu Ser Thr Cys Cys Val Ala Lys Ser Tyr Asn Arg Val Thr Val Met
65 70 75 80 Gly Gly Phe Lys Val Glu Asn His Thr Ala Cys His Cys Ser
Thr Cys 85 90 95 Tyr Tyr His Lys Ser 100 12101PRTArtificial
SequenceMutated FSH alpha mature peptide sequence with N-terminal
extension 12Ala Asn Ile Thr Val Asn Ile Thr Val Ala Pro Asp Val Gln
Asp Cys 1 5 10 15 Pro Glu Cys Thr Leu Gln Arg Asn Pro Phe Phe Ser
Arg Pro Gly Ala 20 25 30 Pro Ile Leu Gln Cys Met Gly Cys Cys Phe
Ser Arg Ala Tyr Pro Thr 35 40 45 Pro Leu Arg Ser Lys Lys Thr Met
Leu Val Gln Lys Asn Val Thr Ser 50 55 60 Glu Ser Thr Cys Cys Val
Ala Lys Ser Tyr Asn Arg Val Thr Val Met 65 70 75 80 Gly Arg Phe Lys
Val Glu Asn His Thr Ala Cys His Cys Ser Thr Cys 85 90 95 Tyr Tyr
His Lys Ser 100 13111PRTArtificial SequenceMutated FSH beta mature
peptide sequence 13Asn Ser Cys Glu Leu Thr Asn Ile Thr Ile Ala Ile
Glu Lys Glu Glu 1 5 10 15 Cys Arg Phe Cys Ile Ser Ile Asn Thr Thr
Trp Cys Ala Gly Tyr Cys 20 25 30 Tyr Thr Arg Asp Leu Val Tyr Lys
Asp Pro Ala Arg Pro Lys Ile Gln 35 40 45 Lys Thr Cys Thr Phe Lys
Glu Leu Val Tyr Glu Thr Val Arg Val Pro 50 55 60 Gly Cys Ala His
His Ala Asp Ser Leu Tyr Thr Tyr Pro Asn Ala Thr 65 70 75 80 Gln Cys
His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys Thr Val 85 90 95
Arg Gly Leu Gly Pro Ser Tyr Cys Ser Phe Gly Glu Met Lys Glu 100 105
110 14111PRTArtificial SequenceMutated FSH beta mature peptide
sequence 14Asn Ser Cys Arg Leu Thr Asn Ile Thr Ile Ala Ile Glu Lys
Glu Glu 1 5 10 15 Cys Arg Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys
Ala Gly Tyr Cys 20 25 30 Tyr Thr Arg Asp Leu Val Tyr Lys Asp Pro
Ala Arg Pro Lys Ile Gln 35 40 45 Lys Thr Cys Thr Phe Lys Glu Leu
Val Tyr Glu Thr Val Arg Val Pro 50 55 60 Gly Cys Ala His His Ala
Asp Ser Leu Tyr Thr Tyr Pro Asn Ala Thr 65 70 75 80 Gln Cys His Cys
Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys Thr Val 85 90 95 Arg Gly
Leu Gly Pro Ser Tyr Cys Ser Phe Gly Glu Met Lys Glu 100 105 110
15111PRTArtificial SequenceMutated FSH beta mature peptide sequence
15Asn Ser Cys Glu Leu Thr Asn Ile Thr Ile Ala Ile Glu Lys Glu Glu 1
5 10 15 Cys Arg Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys Ala Gly Tyr
Cys 20 25 30 Tyr Thr Arg Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro
Lys Ile Gln 35 40 45 Lys Thr Cys Thr Phe Lys Glu Leu Val Asn Glu
Thr Val Arg Val Pro 50 55 60 Gly Cys Ala His His Ala Asp Ser Leu
Tyr Thr Tyr Pro Val Ala Thr 65 70 75 80 Gln Cys His Cys Gly Lys Cys
Asp Ser Asp Ser Thr Asp Cys Thr Val 85 90 95 Arg Gly Leu Gly Pro
Ser Tyr Cys Ser Phe Gly Glu Met Lys Glu 100 105 110
16111PRTArtificial SequenceMutated FSH beta mature peptide sequence
16Asn Ser Cys Arg Leu Thr Asn Ile Thr Ile Ala Ile Glu Lys Glu Glu 1
5 10 15 Cys Arg Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys Ala Gly Tyr
Cys 20 25 30 Tyr Thr Arg Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro
Lys Ile Gln 35 40 45 Lys Thr Cys Thr Phe Lys Glu Leu Val Asn Glu
Thr Val Arg Val Pro 50 55 60 Gly Cys Ala His His Ala Asp Ser Leu
Tyr Thr Tyr Pro Val Ala Thr 65 70 75 80 Gln Cys His Cys Gly Lys Cys
Asp Ser Asp Ser Thr Asp Cys Thr Val 85 90 95 Arg Gly Leu Gly Pro
Ser Tyr Cys Ser Phe Gly Glu Met Lys Glu 100 105 110 17121PRTHomo
sapiens 17Ser Arg Glu Pro Leu Arg Pro Trp Cys His Pro Ile Asn Ala
Ile Leu 1 5 10 15 Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr
Val Asn Thr Thr 20 25 30 Ile Cys Ala Gly Tyr Cys Pro Thr Met Met
Arg Val Leu Gln Ala Val 35 40 45 Leu Pro Pro Leu Pro Gln Val Val
Cys Thr Tyr Arg Asp Val Arg Phe 50 55 60 Glu Ser Ile Arg Leu Pro
Gly Cys Pro Arg Gly Val Asp Pro Val Val 65 70 75 80 Ser Phe Pro Val
Ala Leu Ser Cys Arg Cys Gly Pro Cys Arg Arg Ser 85 90 95 Thr Ser
Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp His 100 105 110
Pro Gln Leu Ser Gly Leu Leu Phe Leu 115 120 1824PRTHomo sapiens
18Met Asp Tyr Tyr Arg Lys Tyr Ala Ala Ile Phe Leu Val Thr Leu Ser 1
5 10 15 Val Phe Leu His Val Leu His Ser 20 1918PRTHomo sapiens
19Met Lys Thr Leu Gln Phe Phe Phe Leu Phe Cys Cys Trp Lys Ala Ile 1
5 10 15 Cys Cys 2020PRTHomo sapiens 20Met Glu Met Leu Gln Gly Leu
Leu Leu Leu Leu Leu Leu Ser Met Gly 1 5 10 15 Gly Ala Trp Ala 20
21692PRTRattus norvegicus 21Met Ala Leu Leu Leu Val Ser Leu Leu Ala
Phe Leu Gly Thr Gly Ser 1 5 10 15 Gly Cys His His Trp Leu Cys His
Cys Ser Asn Arg Val Phe Leu Cys 20 25 30 Gln Asp Ser Lys Val Thr
Glu Ile Pro Thr Asp Leu Pro Arg Asn Ala 35 40 45 Ile Glu Leu Arg
Phe Val Leu Thr Lys Leu Arg Val Ile Pro Lys Gly 50 55 60 Ser Phe
Ala Gly Phe Gly Asp Leu Glu Lys Ile Glu Ile Ser Gln Asn 65 70 75 80
Asp Val Leu Glu Val Ile Glu Ala Asp Val Phe Ser Asn Leu Pro Lys 85
90 95 Leu His Glu Ile Arg Ile Glu Lys Ala Asn Asn Leu Leu Tyr Ile
Asn 100 105 110 Pro Glu Ala Phe Gln Asn Leu Pro Ser Leu Arg Tyr Leu
Leu Ile Ser 115 120 125 Asn Thr Gly Ile Lys His Leu Pro Ala Val His
Lys Ile Gln Ser Leu 130 135 140 Gln Lys Val Leu Leu Asp Ile Gln Asp
Asn Ile Asn Ile His Ile Val 145 150 155 160 Ala Arg Asn Ser Phe Met
Gly Leu Ser Phe Glu Ser Val Ile Leu Trp 165 170 175 Leu Ser Lys Asn
Gly Ile Glu Glu Ile His Asn Cys Ala Phe Asn Gly 180 185 190 Thr Gln
Leu Asp Glu Leu Asn Leu Ser Asp Asn Asn Asn Leu Glu Glu 195 200 205
Leu Pro Asn Asp Val Phe Gln Gly Ala Ser Gly Pro Val Ile Leu Asp 210
215 220 Ile Ser Arg Thr Lys Val His Ser Leu Pro Asn His Gly Leu Glu
Asn 225 230 235 240 Leu Lys Lys Leu Arg Ala Arg Ser Thr Tyr Arg Leu
Lys Lys Leu Pro 245 250 255 Asn Leu Asp Lys Phe Val Thr Leu Met Glu
Ala Ser Leu Thr Tyr Pro 260 265 270 Ser His Cys Cys Ala Phe Ala Asn
Leu Lys Arg Gln Ile Ser Glu Leu 275 280 285 His Pro Ile Cys Asn Lys
Ser Ile Leu Arg Gln Asp Ile Asp Asp Met 290 295 300 Thr Gln Ile Gly
Asp Gln Arg Val Ser Leu Ile Asp Asp Glu Pro Ser 305 310 315 320 Tyr
Gly Lys Gly Ser Asp Met Met Tyr Asn Glu Phe Asp Tyr Asp Leu 325 330
335 Cys Asn Glu Val Val Asp Val Thr Cys Ser Pro Lys Pro Asp Ala Phe
340 345 350 Asn Pro Cys Glu Asp Ile Met Gly Tyr Asn Ile Leu Arg Val
Leu Ile 355 360 365 Trp Phe Ile Ser Ile Leu Ala Ile Thr Gly Asn Thr
Thr Val Leu Val 370 375 380 Val Leu Thr Thr Ser Gln Tyr Lys Leu Thr
Val Pro Arg Phe Leu Met 385 390 395 400 Cys Asn Leu Ala Phe Ala Asp
Leu Cys Ile Gly Ile Tyr Leu Leu Leu 405 410 415 Ile Ala Ser Val Asp
Ile His Thr Lys Ser Gln Tyr His Asn Tyr Ala 420 425 430 Ile Asp Trp
Gln Thr Gly Ala Gly Cys Asp Ala Ala Gly Phe Phe Thr 435 440 445 Val
Phe Ala Ser Glu Leu Ser Val Tyr Thr Leu Thr Ala Ile Thr Leu 450 455
460 Glu Arg Trp His Thr Ile Thr His Ala Met Gln Leu Glu Cys Lys Val
465 470 475 480 Gln Leu Arg His Ala Ala Ser Val Met Val Leu Gly Trp
Thr Phe Ala 485 490 495 Phe Ala Ala Ala Leu Phe Pro Ile Phe Gly Ile
Ser Ser Tyr Met Lys 500 505 510 Val Ser Ile Cys Leu Pro Met Asp Ile
Asp Ser Pro Leu Ser Gln Leu 515 520 525 Tyr Val Met Ala Leu Leu Val
Leu Asn Val Leu Ala Phe Val Val Ile 530 535 540 Cys Gly Cys Tyr Thr
His Ile Tyr Leu Thr Val Arg Asn Pro Thr Ile 545 550 555 560 Val Ser
Ser Ser Ser Asp Thr Lys Ile Ala Lys Arg Met Ala Thr Leu 565 570 575
Ile Phe Thr Asp Phe Leu Cys Met Ala Pro Ile Ser Phe Phe Ala Ile 580
585 590 Ser Ala Ser Leu Lys Val Pro Leu Ile Thr Val Ser Lys Ala Lys
Ile 595 600 605 Leu Leu Val Leu Phe Tyr Pro Ile Asn Ser Cys Ala Asn
Pro Phe Leu 610 615 620 Tyr Ala Ile Phe Thr Lys Asn Phe Arg Arg Asp
Phe Phe Ile Leu Leu 625 630 635 640 Ser Lys Phe Gly Cys Tyr Glu Met
Gln Ala Gln Ile Tyr Arg Thr Glu 645 650 655 Thr Ser Ser Ala Thr His
Asn Phe His Ala Arg Lys Ser His Cys Ser 660 665 670 Ser Ala Pro Arg
Val Thr Asn Ser Tyr Val Leu Val Pro Leu Asn His 675 680 685 Ser Ser
Gln Asn 690 22695PRTHomo sapiens 22Met Ala Leu Leu Leu Val Ser Leu
Leu Ala Phe Leu Ser Leu Gly Ser 1 5 10 15 Gly Cys His His Arg Ile
Cys His Cys Ser Asn Arg Val Phe Leu Cys 20 25 30 Gln Glu Ser Lys
Val Thr Glu Ile Pro Ser Asp Leu Pro Arg Asn Ala 35 40 45 Ile Glu
Leu Arg Phe Val Leu Thr Lys Leu Arg Val Ile Gln Lys Gly 50 55 60
Ala Phe Ser Gly Phe Gly Asp Leu Glu Lys Ile Glu Ile Ser Gln Asn 65
70 75 80 Asp Val Leu Glu Val Ile Glu Ala Asp Val Phe Ser Asn Leu
Pro Lys 85 90 95 Leu His Glu Ile Arg Ile Glu Lys Ala Asn Asn Leu
Leu Tyr Ile Thr 100 105 110 Pro Glu Ala Phe Gln Asn Leu Pro Asn Leu
Gln Tyr Leu Leu Ile Ser 115 120 125 Asn Thr Gly Ile Lys His Leu Pro
Asp Val His Lys Ile His Ser Leu 130 135 140 Gln Lys Val Leu Leu Asp
Ile Gln Asp Asn Ile Asn Ile
His Thr Ile 145 150 155 160 Glu Arg Asn Ser Phe Val Gly Leu Ser Phe
Glu Ser Val Ile Leu Trp 165 170 175 Leu Asn Lys Asn Gly Ile Gln Glu
Ile His Asn Cys Ala Phe Asn Gly 180 185 190 Thr Gln Leu Asp Ala Val
Asn Leu Ser Asp Asn Asn Asn Leu Glu Glu 195 200 205 Leu Pro Asn Asp
Val Phe His Gly Ala Ser Gly Pro Val Ile Leu Asp 210 215 220 Ile Ser
Arg Thr Arg Ile His Ser Leu Pro Ser Tyr Gly Leu Glu Asn 225 230 235
240 Leu Lys Lys Leu Arg Ala Arg Ser Thr Tyr Asn Leu Lys Lys Leu Pro
245 250 255 Thr Leu Glu Lys Leu Val Ala Leu Met Glu Ala Ser Leu Thr
Tyr Pro 260 265 270 Ser His Cys Cys Ala Phe Ala Asn Trp Arg Arg Gln
Ile Ser Glu Leu 275 280 285 His Pro Ile Cys Asn Lys Ser Ile Leu Arg
Gln Glu Val Asp Tyr Met 290 295 300 Thr Gln Ala Arg Gly Gln Arg Ser
Ser Leu Ala Glu Asp Asn Glu Ser 305 310 315 320 Ser Tyr Ser Arg Gly
Phe Asp Met Thr Tyr Thr Glu Phe Asp Tyr Asp 325 330 335 Leu Cys Asn
Glu Val Val Asp Val Thr Cys Ser Pro Lys Pro Asp Ala 340 345 350 Phe
Asn Pro Cys Glu Asp Ile Met Gly Tyr Asn Ile Leu Arg Val Leu 355 360
365 Ile Trp Phe Ile Ser Ile Leu Ala Ile Thr Gly Asn Ile Ile Val Leu
370 375 380 Val Ile Leu Thr Thr Ser Gln Tyr Lys Leu Thr Val Pro Arg
Phe Leu 385 390 395 400 Met Cys Asn Leu Ala Phe Ala Asp Leu Cys Ile
Gly Ile Tyr Leu Leu 405 410 415 Leu Ile Ala Ser Val Asp Ile His Thr
Lys Ser Gln Tyr His Asn Tyr 420 425 430 Ala Ile Asp Trp Gln Thr Gly
Ala Gly Cys Asp Ala Ala Gly Phe Phe 435 440 445 Thr Val Phe Ala Ser
Glu Leu Ser Val Tyr Thr Leu Thr Ala Ile Thr 450 455 460 Leu Glu Arg
Trp His Thr Ile Thr His Ala Met Gln Leu Asp Cys Lys 465 470 475 480
Val Gln Leu Arg His Ala Ala Ser Val Met Val Met Gly Trp Ile Phe 485
490 495 Ala Phe Ala Ala Ala Leu Phe Pro Ile Phe Gly Ile Ser Ser Tyr
Met 500 505 510 Lys Val Ser Ile Cys Leu Pro Met Asp Ile Asp Ser Pro
Leu Ser Gln 515 520 525 Leu Tyr Val Met Ser Leu Leu Val Leu Asn Val
Leu Ala Phe Val Val 530 535 540 Ile Cys Gly Cys Tyr Ile His Ile Tyr
Leu Thr Val Arg Asn Pro Asn 545 550 555 560 Ile Val Ser Ser Ser Ser
Asp Thr Arg Ile Ala Lys Arg Met Ala Met 565 570 575 Leu Ile Phe Thr
Asp Phe Leu Cys Met Ala Pro Ile Ser Phe Phe Ala 580 585 590 Ile Ser
Ala Ser Leu Lys Val Pro Leu Ile Thr Val Ser Lys Ala Lys 595 600 605
Ile Leu Leu Val Leu Phe His Pro Ile Asn Ser Cys Ala Asn Pro Phe 610
615 620 Leu Tyr Ala Ile Phe Thr Lys Asn Phe Arg Arg Asp Phe Phe Ile
Leu 625 630 635 640 Leu Ser Lys Cys Gly Cys Tyr Glu Met Gln Ala Gln
Ile Tyr Arg Thr 645 650 655 Glu Thr Ser Ser Thr Val His Asn Thr His
Pro Arg Asn Gly His Cys 660 665 670 Ser Ser Ala Pro Arg Val Thr Ser
Gly Ser Thr Tyr Ile Leu Val Pro 675 680 685 Leu Ser His Leu Ala Gln
Asn 690 695 23700PRTRattus sp. 23Met Gly Arg Arg Val Pro Ala Leu
Arg Gln Leu Leu Val Leu Ala Val 1 5 10 15 Leu Leu Leu Lys Pro Ser
Gln Leu Gln Ser Arg Glu Leu Ser Gly Ser 20 25 30 Arg Cys Pro Glu
Pro Cys Asp Cys Ala Pro Asp Gly Ala Leu Arg Cys 35 40 45 Pro Gly
Pro Arg Ala Gly Leu Ala Arg Leu Ser Leu Thr Tyr Leu Pro 50 55 60
Val Lys Val Ile Pro Ser Gln Ala Phe Arg Gly Leu Asn Glu Val Val 65
70 75 80 Lys Ile Glu Ile Ser Gln Ser Asp Ser Leu Glu Arg Ile Glu
Ala Asn 85 90 95 Ala Phe Asp Asn Leu Leu Asn Leu Ser Glu Leu Leu
Ile Gln Asn Thr 100 105 110 Lys Asn Leu Leu Tyr Ile Glu Pro Gly Ala
Phe Thr Asn Leu Pro Arg 115 120 125 Leu Lys Tyr Leu Ser Ile Cys Asn
Thr Gly Ile Arg Thr Leu Pro Asp 130 135 140 Val Thr Lys Ile Ser Ser
Ser Glu Phe Asn Phe Ile Leu Glu Ile Cys 145 150 155 160 Asp Asn Leu
His Ile Thr Thr Ile Pro Gly Asn Ala Phe Gln Gly Met 165 170 175 Asn
Asn Glu Ser Val Thr Leu Lys Leu Tyr Gly Asn Gly Phe Glu Glu 180 185
190 Val Gln Ser His Ala Phe Asn Gly Thr Thr Leu Ile Ser Leu Glu Leu
195 200 205 Lys Glu Asn Ile Tyr Leu Glu Lys Met His Ser Gly Ala Phe
Gln Gly 210 215 220 Ala Thr Gly Pro Ser Ile Leu Asp Ile Ser Ser Thr
Lys Leu Gln Ala 225 230 235 240 Leu Pro Ser His Gly Leu Glu Ser Ile
Gln Thr Leu Ile Ala Leu Ser 245 250 255 Ser Tyr Ser Leu Lys Thr Leu
Pro Ser Lys Glu Lys Phe Thr Ser Leu 260 265 270 Leu Val Ala Thr Leu
Thr Tyr Pro Ser His Cys Cys Ala Phe Arg Asn 275 280 285 Leu Pro Lys
Lys Glu Gln Asn Phe Ser Phe Ser Ile Phe Glu Asn Phe 290 295 300 Ser
Lys Gln Cys Glu Ser Thr Val Arg Lys Ala Asp Asn Glu Thr Leu 305 310
315 320 Tyr Ser Ala Ile Phe Glu Glu Asn Glu Leu Ser Gly Trp Asp Tyr
Asp 325 330 335 Tyr Gly Phe Cys Ser Pro Lys Thr Leu Gln Cys Ala Pro
Glu Pro Asp 340 345 350 Ala Phe Asn Pro Cys Glu Asp Ile Met Gly Tyr
Ala Phe Leu Arg Val 355 360 365 Leu Ile Trp Leu Ile Asn Ile Leu Ala
Ile Phe Gly Asn Leu Thr Val 370 375 380 Leu Phe Val Leu Leu Thr Ser
Arg Tyr Lys Leu Thr Val Pro Arg Phe 385 390 395 400 Leu Met Cys Asn
Leu Ser Phe Ala Asp Phe Cys Met Gly Leu Tyr Leu 405 410 415 Leu Leu
Ile Ala Ser Val Asp Ser Gln Thr Lys Gly Gln Tyr Tyr Asn 420 425 430
His Ala Ile Asp Trp Gln Thr Gly Ser Gly Cys Gly Ala Ala Gly Phe 435
440 445 Phe Thr Val Phe Ala Ser Glu Leu Ser Val Tyr Thr Leu Thr Val
Ile 450 455 460 Thr Leu Glu Arg Trp His Thr Ile Thr Tyr Ala Val Gln
Leu Asp Gln 465 470 475 480 Lys Leu Arg Leu Arg His Ala Ile Pro Ile
Met Leu Gly Gly Trp Leu 485 490 495 Phe Ser Thr Leu Ile Ala Thr Met
Pro Leu Val Gly Ile Ser Asn Tyr 500 505 510 Met Lys Val Ser Ile Cys
Leu Pro Met Asp Val Glu Ser Thr Leu Ser 515 520 525 Gln Val Tyr Ile
Leu Ser Ile Leu Ile Leu Asn Val Val Ala Phe Val 530 535 540 Val Ile
Cys Ala Cys Tyr Ile Arg Ile Tyr Phe Ala Val Gln Asn Pro 545 550 555
560 Glu Leu Thr Ala Pro Asn Lys Asp Thr Lys Ile Ala Lys Lys Met Ala
565 570 575 Ile Leu Ile Phe Thr Asp Phe Thr Cys Met Ala Pro Ile Ser
Phe Phe 580 585 590 Ala Ile Ser Ala Ala Phe Lys Val Pro Leu Ile Thr
Val Thr Asn Ser 595 600 605 Lys Ile Leu Leu Val Leu Phe Tyr Pro Val
Asn Ser Cys Ala Asn Pro 610 615 620 Phe Leu Tyr Ala Ile Phe Thr Lys
Ala Phe Gln Arg Asp Phe Leu Leu 625 630 635 640 Leu Leu Ser Arg Phe
Gly Cys Cys Lys Arg Arg Ala Glu Leu Tyr Arg 645 650 655 Arg Lys Glu
Phe Ser Ala Tyr Thr Ser Asn Cys Lys Asn Gly Phe Pro 660 665 670 Gly
Ala Ser Lys Pro Ser Gln Ala Thr Leu Lys Leu Ser Thr Val His 675 680
685 Cys Gln Gln Pro Ile Pro Pro Arg Ala Leu Thr His 690 695 700
24699PRTHomo sapiens 24Met Lys Gln Arg Phe Ser Ala Leu Gln Leu Leu
Lys Leu Leu Leu Leu 1 5 10 15 Leu Gln Pro Pro Leu Pro Arg Ala Leu
Arg Glu Ala Leu Cys Pro Glu 20 25 30 Pro Cys Asn Cys Val Pro Asp
Gly Ala Leu Arg Cys Pro Gly Pro Thr 35 40 45 Ala Gly Leu Thr Arg
Leu Ser Leu Ala Tyr Leu Pro Val Lys Val Ile 50 55 60 Pro Ser Gln
Ala Phe Arg Gly Leu Asn Glu Val Ile Lys Ile Glu Ile 65 70 75 80 Ser
Gln Ile Asp Ser Leu Glu Arg Ile Glu Ala Asn Ala Phe Asp Asn 85 90
95 Leu Leu Asn Leu Ser Glu Ile Leu Ile Gln Asn Thr Lys Asn Leu Arg
100 105 110 Tyr Ile Glu Pro Gly Ala Phe Ile Asn Leu Pro Gly Leu Lys
Tyr Leu 115 120 125 Ser Ile Cys Asn Thr Gly Ile Arg Lys Phe Pro Asp
Val Thr Lys Val 130 135 140 Phe Ser Ser Glu Ser Asn Phe Ile Leu Glu
Ile Cys Asp Asn Leu His 145 150 155 160 Ile Thr Thr Ile Pro Gly Asn
Ala Phe Gln Gly Met Asn Asn Glu Ser 165 170 175 Val Thr Leu Lys Leu
Tyr Gly Asn Gly Phe Glu Glu Val Gln Ser His 180 185 190 Ala Phe Asn
Gly Thr Thr Leu Thr Ser Leu Glu Leu Lys Glu Asn Val 195 200 205 His
Leu Glu Lys Met His Asn Gly Ala Phe Arg Gly Ala Thr Gly Pro 210 215
220 Lys Thr Leu Asp Ile Ser Ser Thr Lys Leu Gln Ala Leu Pro Ser Tyr
225 230 235 240 Gly Leu Glu Ser Ile Gln Arg Leu Ile Ala Thr Ser Ser
Tyr Ser Leu 245 250 255 Lys Lys Leu Pro Ser Arg Glu Thr Phe Val Asn
Leu Leu Glu Ala Thr 260 265 270 Leu Thr Tyr Pro Ser His Cys Cys Ala
Phe Arg Asn Leu Pro Thr Lys 275 280 285 Glu Gln Asn Phe Ser His Ser
Ile Ser Glu Asn Phe Ser Lys Gln Cys 290 295 300 Glu Ser Thr Val Arg
Lys Val Ser Asn Lys Thr Leu Tyr Ser Ser Met 305 310 315 320 Leu Ala
Glu Ser Glu Leu Ser Gly Trp Asp Tyr Glu Tyr Gly Phe Cys 325 330 335
Leu Pro Lys Thr Pro Arg Cys Ala Pro Glu Pro Asp Ala Phe Asn Pro 340
345 350 Cys Glu Asp Ile Met Gly Tyr Asp Phe Leu Arg Val Leu Ile Trp
Leu 355 360 365 Ile Asn Ile Leu Ala Ile Met Gly Asn Met Thr Val Leu
Phe Val Leu 370 375 380 Leu Thr Ser Arg Tyr Lys Leu Thr Val Pro Arg
Phe Leu Met Cys Asn 385 390 395 400 Leu Ser Phe Ala Asp Phe Cys Met
Gly Leu Tyr Leu Leu Leu Ile Ala 405 410 415 Ser Val Asp Ser Gln Thr
Lys Gly Gln Tyr Tyr Asn His Ala Ile Asp 420 425 430 Trp Gln Thr Gly
Ser Gly Cys Ser Thr Ala Gly Phe Phe Thr Val Phe 435 440 445 Ala Ser
Glu Leu Ser Val Tyr Thr Leu Thr Val Ile Thr Leu Glu Arg 450 455 460
Trp His Thr Ile Thr Tyr Ala Ile His Leu Asp Gln Lys Leu Arg Leu 465
470 475 480 Arg His Ala Ile Leu Ile Met Leu Gly Gly Trp Leu Phe Ser
Ser Leu 485 490 495 Ile Ala Met Leu Pro Leu Val Gly Val Ser Asn Tyr
Met Lys Val Ser 500 505 510 Ile Cys Phe Pro Met Asp Val Glu Thr Thr
Leu Ser Gln Val Tyr Ile 515 520 525 Leu Thr Ile Leu Ile Leu Asn Val
Val Ala Phe Phe Ile Ile Cys Ala 530 535 540 Cys Tyr Ile Lys Ile Tyr
Phe Ala Val Arg Asn Pro Glu Leu Met Ala 545 550 555 560 Thr Asn Lys
Asp Thr Lys Ile Ala Lys Lys Met Ala Ile Leu Ile Phe 565 570 575 Thr
Asp Phe Thr Cys Met Ala Pro Ile Ser Phe Phe Ala Ile Ser Ala 580 585
590 Ala Phe Lys Val Pro Leu Ile Thr Val Thr Asn Ser Lys Val Leu Leu
595 600 605 Val Leu Phe Tyr Pro Ile Asn Ser Cys Ala Asn Pro Phe Leu
Tyr Ala 610 615 620 Ile Phe Thr Lys Thr Phe Gln Arg Asp Phe Phe Leu
Leu Leu Ser Lys 625 630 635 640 Phe Gly Cys Cys Lys Arg Arg Ala Glu
Leu Tyr Arg Arg Lys Asp Phe 645 650 655 Ser Ala Tyr Thr Ser Asn Cys
Lys Asn Gly Phe Thr Gly Ser Asn Lys 660 665 670 Pro Ser Gln Ser Thr
Leu Lys Leu Ser Thr Leu His Cys Gln Gly Thr 675 680 685 Ala Leu Leu
Asp Lys Thr Arg Tyr Thr Glu Cys 690 695
* * * * *