U.S. patent application number 10/664801 was filed with the patent office on 2004-06-17 for method for down-regulating osteoprotegerin ligand activity.
This patent application is currently assigned to M&E Biotech A/S. Invention is credited to Haaning, Jesper, Halkier, Torben.
Application Number | 20040115199 10/664801 |
Document ID | / |
Family ID | 26065320 |
Filed Date | 2004-06-17 |
United States Patent
Application |
20040115199 |
Kind Code |
A1 |
Halkier, Torben ; et
al. |
June 17, 2004 |
Method for down-regulating osteoprotegerin ligand activity
Abstract
The invention provides a novel method for down-regulating the
biological activity of osteoprotegerin ligand (OPGL, TRANCE)
thereby rendering possible the treatment/amelioration of diseases
characterized by excessive loss of bone mass, e.g. osteoporosis.
Down-regulation is effected by inducing an immune response against
OPGL in an individual in need thereof. Immune responses can be
raised by classical immunization with immunogenic variants of OPGL
or by nucleic acid immunization where the nucleic acids encode the
OPGL variant. The invention also pertains to compositions,
polypeptides and nucleic acids useful in the invention, as well as
to vectors and transformed host cells useful in the preparation
thereof.
Inventors: |
Halkier, Torben; (Birkerod,
DK) ; Haaning, Jesper; (Birkerod, DK) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
M&E Biotech A/S
Horsholm
DK
|
Family ID: |
26065320 |
Appl. No.: |
10/664801 |
Filed: |
September 17, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10664801 |
Sep 17, 2003 |
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09396937 |
Sep 15, 1999 |
|
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|
6645500 |
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60102896 |
Oct 2, 1998 |
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Current U.S.
Class: |
424/145.1 ;
424/185.1 |
Current CPC
Class: |
C12N 2799/021 20130101;
Y02A 50/30 20180101; C07K 14/70575 20130101; A61K 39/00 20130101;
A61P 19/10 20180101; A61K 2039/51 20130101; C07K 2319/02
20130101 |
Class at
Publication: |
424/145.1 ;
424/185.1 |
International
Class: |
A61K 039/395; A61K
039/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 15, 1998 |
DK |
PA 1998 01164 |
Claims
1. A method for in vivo down-regulation of osteoprotegerin ligand
(OPGL) activity in an animal, including a human being, the method
comprising effecting presentation to the animal's immune system of
an immunogenically effective amount of at least one OPGL
polypeptide or subsequence thereof which has been formulated so
that immunization of the animal with the OPGL polypeptide or
subsequence thereof induces production of antibodies against the
OPGL polypeptide, and/or at least one OPGL analogue wherein is
introduced at least one modification in the OPGL amino acid
sequence which has as a result that immunization of the animal with
the analogue induces production of antibodies against the OPGL
polypeptide.
2. The method according to claim 1, wherein is presented an OPGL
analogue with at least one modification of the OPGL amino acid
sequence.
3. The method according to claim 2, wherein the modification has as
a result that a substantial fraction of OPGL B-cell epitopes are
preserved and that at least one foreign T helper lymphocyte epitope
(T.sub.H epitope) is introduced, and/or at least one first moiety
is introduced which effects targeting of the modified molecule to
an antigen presenting cell (APC) or a B-lymphocyte, and/or at least
one second moiety is introduced which stimulates the immune system,
and/or at least one third moiety is introduced which optimizes
presentation of the modified OPGL polypeptide to the immune
system.
4. The method according to claim 3, wherein the modification
includes introduction as side groups, by covalent or non-covalent
binding to suitable chemical groups in OPGL or a subsequence
thereof, of the foreign T.sub.H epitope and/or of the first and/or
of the second and/or of the third moiety.
5. The method according to claim 3 or 4, wherein the modification
includes amino acid substitution and/or deletion and/or insertion
and/or addition.
6. The method according to claim 5, wherein the modification
results in the provision of a fusion polypeptide.
7. The method according to claim 5 or 6, wherein introduction of
the amino acid substitution and/or deletion and/or insertion and/or
addition results in a substantial preservation of the overall
tertiary structure of OPGL.
8. The method according to any one of claims 2-7, wherein the
modification includes duplication of at least one OPGL B-cell
epitope and/or introduction of a hapten.
9. The method according to any one of claims 3-8, wherein the
foreign T-cell epitope is immunodominant in the animal.
10. The method according to any one of claims 3-9, wherein the
foreign T-cell epitope is promiscuous.
11. The method according to claim 10, wherein the at least one
foreign T-cell epitope is selected from a natural promiscuous
T-cell epitope and an artificial MHC-II binding peptide
sequence.
12. The method according to claim 11, wherein the natural T-cell
epitope is selected from a Tetanus toxoid epitope such as P2 or
P30, a diphtheria toxoid epitope, an influenza virus hemagluttinin
epitope, and a P. falciparum CS epitope.
13. The method according to any one of claims 3-12, wherein the
first moiety is a substantially specific binding partner for a
B-lymphocyte specific surface antigen or for an APC specific
surface antigen such as a hapten or a carbohydrate for which there
is a receptor on the B-lymphocyte or the APC.
14. The method according to any one of claims 3-13, wherein the
second moiety is selected from a cytokine, a hormone, and a
heat-shock protein.
15. The method according to claim 6, wherein the cytokine is
selected from, or is an effective part of, interferon .gamma.
(IFN-.gamma.), Flt3L, interleukin 1 (IL-1), interleukin 2 (IL-2),
interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 12 (IL-12),
interleukin 13 (IL-13), interleukin 15 (IL-15), and
granulocyte-macrophage colony stimulating factor (GM-CSF), and the
heat-shock protein is selected from, or is an effective part of,
HSP70, HSP90, HSC70, GRP94, and calreticulin (CRT).
16. The method according to any one of claims 3-15, wherein the
third moiety is of lipid nature, such as a palmitoyl group, a
myristyl group, a farnesyl group, a geranyl-geranyl group, a
GPI-anchor, and an N-acyl diglyceride group.
17. The method according to any one of the preceding claims,
wherein the OPGL polypeptide or subsequence thereof has been
modified in any one of positions 170-192, any one of positions
198-218, any one of positions 221-246, any one of positions
256-261, or in any one of positions 285-316, the amino acid
numbering conforming with that of any one of SEQ ID NOs: 4, 6, and
12, or wherein the OPGL polypeptide has been modified in any one of
positions 171-193, any one of positions 199-219, any one of
positions 222-247, any one of positions 257-262, or in any one of
positions 286-317, the amino acid numbering conforming with that of
SEQ ID NO: 2.
18. The method according to claim 17, wherein the modification
comprises a substitution of at least one amino acid sequence within
a position defined in claim 17 with an amino acid sequence of equal
or different length which contains a foreign T.sub.H epitope.
19. The method according to claim 18, wherein the amino acid
sequence containing the foreign T.sub.H epitope substitutes amino
acids 256-261 and/or 288-302 and/or 221-241 found in SEQ ID NO: 4
or amino acids 257-262 and/or 289-303 and/or 222-243 in SEQ ID NO:
2 or in a polypeptide where a cysteine corresponding to Cys-221 has
been substituted with Ser.
20. The method according to any one of the preceding claims,
wherein presentation to the immune system is effected by having at
least two copies of the OPGL polypeptide, the subsequence thereof
or the modified OPGL polypeptide covalently of non-covalently
linked to a carrier molecule capable of effecting presentation of
multiple copies of antigenic determinants.
21. The method according to any the preceding claims, wherein the
OPGL polypeptide, the subsequence thereof, or the modified OPGL
polypeptide has been formulated with an adjuvant which facilitates
breaking of autotolerance to autoantigens.
22. The method according to any one of the preceding claims,
wherein an effective amount of the OPGL polypeptide or the OPGL
analogue is administered to the animal via a route selected from
the parenteral route such as the intradermal, the subdermal, the
intracutaneous, the subcutaneous, and the intramuscular routes; the
peritoneal route; the oral route; the buccal route; the sublinqual
route; the epidural route; the spinal route; the anal route; and
the intracranial route.
23. The method according to claim 22, wherein the effective amount
is between 0.5 .mu.g and 2,000 .mu.g of the OPGL polypeptide, the
subsequence thereof or the analogue thereof.
24. The method according to claim 22 or 23, wherein the OPGL
polypeptide or analogue is contained in a virtual lymph node (VLN)
device.
25. The method according to any one of claims 1-21, wherein
presentation of modified OPGL to the immune system is effected by
introducing nucleic acid(s) encoding the modified OPGL into the
animal's cells and thereby obtaining in vivo expression by the
cells of the nucleic acid(s) introduced.
26. The method according to claim 25, wherein the nucleic acid(s)
introduced is/are selected from naked DNA, DNA formulated with
charged or uncharged lipids, DNA formulated in liposomes, DNA
included in a viral vector, DNA formulated with a
transfection-facilitating protein or polypeptide, DNA formulated
with a targeting protein or polypeptide, DNA formulated with
Calcium precipitating agents, DNA coupled to an inert carrier
molecule, DNA encapsulated in chitin or chitosan, and DNA
formulated with an adjuvant.
27. The method according to claim 27, wherein the nucleic acid(s)
is/are contained in a VLN device.
28. The method according to any one of claims 22-27, which includes
at least one administration/introduction per year, such as at least
2, at least 3, at least 4, at least 6, and at least 12
administrations/introduc- tions.
29. A method for treating and/or preventing and/or ameliorating
osteoporosis or other diseases and conditions characterized by
excess bone resorption, the method comprising down-regulating OPGL
activity according to the method of any one of claims 1-28 to such
an extent that the rate of bone resorption is significantly
decreased, such as a decrease of at least 3%, at least 7%, at least
9%, at least 11%, at least 13%, at least 15%, at least 17%, at
least 20%, and at least 30%.
30. An OPGL analogue which is derived from an animal OPGL
polypeptide wherein is introduced a modification which has as a
result that immunization of the animal with the analogue induces
production of antibodies against the OPGL polypeptide.
31. An OPGL analogue according to claim 30, wherein the
modification is as defined in any one of claims 1-19.
32. An immunogenic composition comprising an immunogenically
effective amount of an OPGL polypeptide autologous in an animal,
said OPGL polypeptide being formulated together with an
immunologically acceptable adjuvant so as to break the animal's
autotolerance towards the OPGL polypeptide, the composition further
comprising a pharmaceutically and immunologically acceptable
carrier and/or vehicle, or an immunogenically effective amount of
an OPGL analogue according to claim 30 or 31, the composition
further comprising a pharmaceutically and immunologically
acceptable carrier and/or vehicle and optionally an adjuvant.
33. A nucleic acid fragment which encodes an OPGL analogue
according to claim 30 or 31.
34. A vector carrying the nucleic acid fragment according to claim
33.
35. The vector according to claim 34 which is capable of autonomous
replication.
36. The vector according to claim 34 or 35 which is selected from
the group consisting of a plasmid, a phage, a cosmid, a
mini-chromosome, and a virus.
37. The vector according to any one of claims 34-36, comprising, in
the 5'.cndot..cndot.3' direction and in operable linkage, a
promoter for driving expression of the nucleic acid fragment
according to claim 33, optionally a nucleic acid sequence encoding
a leader peptide enabling secretion of or integration into the
membrane of the polypeptide fragment, the nucleic acid fragment
according to claim 33, and optionally a terminator.
38. The vector according to any one of claims 34-37 which, when
introduced into a host cell, is capable or incapable of being
integrated in the host cell genome.
39. The vector according to claim 37 or 38, wherein a promoter
drives expression in a eukaryotic cell and/or in a prokaryotic
cell.
40. A transformed cell carrying the vector of any one of claims
34-39.
41. The transformed cell according to claim 40 which is capable of
replicating the nucleic acid fragment according to claim 33.
42. The transformed cell according to claim 41, which is a
microorganism selected from a bacterium, a yeast, a protozoan, or a
cell derived from a multicellular organism selected from a fungus,
an insect cell such as an S.sub.2 or an SF cell, a plant cell, and
a mammalian cell.
43. The transformed cell according to any one of claims 40-42,
which expresses the nucleic acid fragment according to claim
33.
44. The transformed cell according to claim 43, which secretes or
carries on its surface, the OPGL analogue according to claim 30 or
31.
45. The method according to any one of claims 1-19, wherein
presentation to the immune system is effected by administering a
non-pathogenic microorganism or virus which is carrying a nucleic
acid fragment which encodes and expresses the OPGL polypeptide or
analogue.
46. A composition for inducing production of antibodies against
OPGL, the composition comprising a nucleic acid fragment according
to claim 33 or a vector according to any one of claims 34-39, and a
pharmaceutically and immunologically acceptable carrier and/or
vehicle and/or adjuvant.
47. A stable cell line which carries the vector according to any
one of claims 34-39 and which expresses the nucleic acid fragment
according to claim 33, and which optionally secretes or carries the
OPGL analogue according to claim 30 or 31 on its surface.
48. A method for the preparation of the cell according to any one
of claims 40-44, the method comprising transforming a host cell
with the nucleic acid fragment according to claim 33 or with the
vector according to any one of claims 34-39.
49. A method for the identification of a modified OPGL polypeptide
which is capable of inducing antibodies against unmodified OPGL in
an animal species where the unmodified OPGL polypeptide is a
self-protein, the method comprising preparing, by means of peptide
synthesis or genetic engineering techniques, a set of mutually
distinct modified OPGL polypeptides wherein amino acids have been
added to, inserted in, deleted from, or substituted into the amino
acid sequence of an OPGL polypeptide of the animal species thereby
giving rise to amino acid sequences in the set which comprise
T-cell epitopes which are foreign to the animal species, or
preparing a set of nucleic acid fragments encoding the set of
mutually distinct modified OPGL polypeptides, testing members of
the set of modified OPGL polypeptides or nucleic acid fragments for
their ability to induce production of antibodies by the animal
species against the unmodified OPGL, and identifying and optionally
isolating the member(s) of the set of modified OPGL polypeptides
which significantly induces antibody production against unmodified
OPGL in the species or identifying and optionally isolating the
polypeptide expression products encoded by members of the set of
nucleic acid fragments which significantly induces antibody
production against unmodified OPGL in the animal species.
50. A method for the preparation of an immunogenic composition
comprising at least one modified OPGL polypeptide which is capable
of inducing antibodies against unmodified OPGL in an animal species
where the unmodified OPGL polypeptide is a self-protein, the method
comprising preparing, by means of peptide synthesis or genetic
engineering techniques, a set of mutually distinct modified OPGL
polypeptides wherein amino acids have been added to, inserted in,
deleted from, or substituted into the amino acid sequence of an
OPGL polypeptide of the animal species thereby giving rise to amino
acid sequences in the set comprising T-cell epitopes which are
foreign to the animal, testing members of the set for their ability
to induce production of antibodies by the animal species against
the unmodified OPGL, and admixing the member(s) of the set which
significantly induces production of antibodies in the animal
species which are reactive with OPGL with a pharmaceutically and
immunologically acceptable carrier and/or vehicle, optionally in
combination with at least one pharmaceutically and immunologically
acceptable adjuvant.
51. The method according to claim 49 or 50, wherein preparation of
the members of the set comprises preparation of mutually distinct
nucleic acid sequences, each sequence being a nucleic acid sequence
according to claim 33, insertion of the nucleic acid sequences into
appropriate expression vectors, transformation of suitable host
cells with the vectors, and expression of the nucleic acid
sequences, optionally followed by isolation of the expression
products.
52. The method according to claim 51, wherein the preparation of
the nucleic acid sequences and/or the vectors is achieved by the
aid of a molecular amplification technique such as PCR or by the
aid of nucleic acid synthesis.
53. Use of OPGL or a subsequence thereof for the preparation of an
immunogenic composition comprising an adjuvant for down-regulating
OPGL activity in an animal.
54. Use of OPGL or a subsequence thereof for the preparation of an
immunogenic composition comprising an adjuvant for the treatment,
prophylaxis or amelioration of osteoporosis or other conditions
characterized by excessive bone resorption.
55. Use of an OPGL analogue for the preparation of an immunogenic
composition optionally comprising an adjuvant for down-regulating
OPGL activity in an animal.
56. Use of an OPGL analogue for the preparation of an immunogenic
composition optionally comprising an adjuvant for the treatment,
prophylaxis or amelioration of osteoporosis or other conditions
characterized by excessive bone resorption.
Description
[0001] This application is a divisional of co-pending U.S. patent
application Ser. No. 09/396,937 filed on Sep. 15, 1999, the entire
contents of which are hereby incorporated by reference and for
which priority is claimed under 35 U.S.C. .sctn.120; and this
application claims priority of U.S. Provisional Application No.
60/102,896 filed Oct. 2, 1998 and Danish Patent Application PA 1998
01164 filed in Denmark on Sep. 15, 1998 under 35. U.S.C.
.sctn.119.
FIELD OF THE INVENTION
[0002] The present invention relates to improvement in therapy and
prevention of osteoporosis and other diseases characterized by
continued loss of bone tissue. More specifically, the present
invention provides a method for down-regulating osteoprotegerin
ligand (OPGL) by enabling the production of antibodies against OPGL
in subjects suffering from or in danger of suffering from
osteoporosis. The invention also provides for methods of producing
modified OPGL useful in this method as well as for the modified
OPGL as such. Also encompassed by the present invention are nucleic
acid fragments encoding modified OPGL as well as vectors
incorporating these nucleic acid fragments and host cells and cell
lines transformed therewith. The invention also provides for a
method for the identification of OPGL analogues which are useful in
the method of the invention as well as for compositions comprising
modified OPGL or comprising nucleic acids encoding the OPGL
analogues.
[0003] Osteoporosis is a major and growing health problem
worldwide. It affects an estimated 75 million people in the United
States of America, Europe and Japan combined. Thus, it is the most
common systemic bone disorder in the industrialised part of the
world.
[0004] Osteoporosis affects one in four postmenopausal women and a
majority of the elderly, including a substantial number of men. The
cost of osteoporosis in the United States of America with 15
million affected people was estimated to be 3.8 billion USD
annually in 1984. This translates by extrapolation to a worldwide
cost of something in the order of at least 20 billion USD.
[0005] Osteoporosis is a systemic skeletal disease characterised by
low bone mass and micro-architectural deterioration of bone tissue,
with a consequent increase in bone fragility and susceptibility to
fractures. Although all bones are affected, fractures of the spine,
wrist and hip are typical and the most common. The risk of
developing osteoporosis increases with age and is higher in women
than in men. Its etiology appears to reside in the mechanisms
underlying an accentuation of the normal loss of bone mass, which
follows the menopause in women and occurs in all individuals with
advancing age.
[0006] Peak bone mass is achieved at about 35 years of age. After
reaching its peak, bone mass declines throughout life due to an
imbalance in remodelling. Bones lose both mineral and organic
matrix but retain their basic organisation.
[0007] Bone consists of a mineralized extracellular matrix composed
of a variety of proteins and proteoglycans; the principal component
being type I collagen. The mineral encrusting the extracellular
matrix is hydroxyapatite (Ca.sub.3 (PO.sub.4).sub.2Ca (OH).sub.2).
Bone is continuously modelled during growth and development and
remodelled throughout life in response to physical and chemical
signals.
[0008] The growth, development and maintenance of bone are highly
regulated processes, which at the cellular level involves the
co-ordinate regulation of bone-forming cells (osteoblasts) and
bone-resorbing cells (osteoclasts). The level of bone mass reflects
the balance of bone formation and resorption.
[0009] Osteoblasts arise from mesenchymal stem cells and produce
bone matrix during development, after bone injury, and during the
normal bone remodelling that occurs throughout life. Osteoclasts
differentiate from hemtopoietic precursors of the
monocyte-macrophage lineage and resorb bone matrix.
[0010] An imbalance of osteoblast and osteoclast functions can
result in the skeletal abnormalities characterized by increased
bone mass (osteopetrosis) or by decreased bone mass
(osteoporosis).
[0011] Studies of osteopetrosis in mutant mice have shown that
genetic defects in osteoclast development, maturation, and/or
activation lead to decreased bone resorption and uniformly result
in severe osteopetrosis (Marks, 1989). Nevertheless, relatively
little has so far been known about the soluble factors that act
physiologically to regulate osteoclast development.
[0012] Recently, however, two proteins that take part in this
regulation have been described and characterized (Simonet et al.,
1997; Lacey et al., 1998). These two proteins are osteoprotegerin
and osteoprotegerin ligand.
[0013] Osteoprotegerin is a novel secreted member of the tumour
necrosis factor receptor family. In vitro, osteoprotegerin blocks
osteoclastogenesis in a dose dependent manner. Transgenic mice
expressing osteoprotegerin exhibit a generalized increase in bone
density (osteopetrosis) associated with a decrease in osteoclasts.
Administration of recombinant osteoprotegerin produces similar
effects in normal mice and protects against ovariectomy-associated
bone loss in rats (Simonet et al., 1997). In addition,
osteoprotegerin-deficient mice (knock out mice) while normal at
birth develop early onset osteoporosis and arterial calcification
(Bucay et al., 1998). These observations strongly point to the
possibility that osteoprotegerin blocks the differentiation of
osteoclasts, the principal if not sole bone-resorbing cell type,
suggesting that it can act as a humoral regulator of bone
resorption. Osteoprotegerin is the subject matter of WO
97/23614.
[0014] It was hypothesized that osteoprotegerin may exert its
effect by binding to and neutralising a factor that stimulates
osteoclast development, thus inhibiting osteoclast maturation
(Simonet et al., 1997).
[0015] Osteoprotegerin ligand (OPGL) is a novel member of the
tumour necrosis factor family of cytokines that exists in both a
membrane-bound and a soluble form. OPGL binds to osteoprotegerin
with a binding affinity of 4 nM. In vitro, OPGL activates mature
osteoclasts and modulates osteoclast formation from bone marrow
precursors in the presence of CSF-1. It has also been demonstrated
that OPGL binds to the surface of osteoclast progenitors in
CSF-1-treated bone marrow. The receptor for OPGL on these
hematopoeitic progenitor cells is, however, unknown. Recombinant
soluble OPGL is a potent inducer of bone resorption in vivo (Lacey
et al., 1998).
[0016] Description of OPGL
[0017] OPGL is synthesised as a type II transmembrane protein
consisting of 317 amino acid residues (human, cf. SEQ ID NO: 2) or
316 amino acid residues (murine, cf. SEQ ID NOs: 4 and 6).
Alignment of the two amino acid sequences show that identical amino
acid residues are found at 87% of the homologous positions.
[0018] The OPGL amino acid sequence contains a short cytoplasmic
domain in the N-terminus followed by the putative transmembrane
region between amino acid residues 49 and 69. Based on its homology
to tumour necrosis factor alpha, the extracellular part of OPGL has
been suggested to be comprised by two domains: a stalk region
extending from amino acid residue 70 to 157, and the active ligand
moiety extending from amino acid residue 158 to the C-terminus.
[0019] The most closely related protein to OPGL appears to be the
apoptosis inducing cytokine TRAIL with less that 25% identical
amino acid residues. OPGL has also very recently been cloned in
other contexts and was called TRANCE (Wong et al., 1997, J. Biol.
Chem. 272: 25190-25194) and RANKL, respectively (Anderson et al.,
1997, Nature 390: 175-179. The protein is also known as osteoclast
differentiation factor (ODF).
[0020] Several N-terminal deletion variants of murine OPGL have
been expressed in E. coli and purified. These variants consisted of
amino acid residues 75-316, 128-316, 137-316, and 158-316,
respectively. The three shortest variants had similar .beta.-sheet
structure based on circular dichroism studies, and all were able to
bind to osteoprotegerin. More important, though, is that the three
variants were active in in vitro assays (Lacey et al., 1998).
[0021] The shortest variant was studied further. Like tumour
necrosis factor alpha, this variant OPGL exists as a trimer in
solution and forms 3:3 complexes when incubated with
osteoprotegerin. The binding affinity was found to be 4 nM. This
variant induces significant increases in blood ionized calcium
(hypercalcemia) in mice in vivo. Co-administration of
osteoprotegerin significantly reduced this hypercalcemic effect of
OPGL.
[0022] The longest variant (amino acid residues 75-316) of OPGL did
not bind to osteoprotegerin and it did not have any biological
activity.
[0023] At the time of construction of the N-terminal deletion
variants the natural cleavage site in OPGL was not known.
Expression of full-length OPGL in human 293 fibroblasts resulted in
soluble OPGL beginning at amino residue 139 in the murine protein
or at the homologous amino acid residue 140 in the human protein.
These expression studies also showed that soluble OPGL resulting
from expression in human cells is glycosylated. This is not
surprising as both murine and human OPGL contain three potential
N-glycosylation sites in the C-terminal ligand domain.
[0024] The concentrations of osteoprotegerin in blood and tissues
are not known but the protein has significant biological activity
at a concentration of 1 ng/ml.
[0025] Biological Activity of OPGL
[0026] OPGL is a potent osteoclast differentiation factor when
combined with CSF-1. Neither of these components alone are capable
of inducing osteoclast differentiation from progenitor cells.
[0027] OPGL is a potent activator of mature osteoclast. On its own,
OPGL activates mature osteoclasts to resorb bone. OPGL has not been
observed to act as an osteoclast growth factor or osteoclast
survival factor in these experiments.
[0028] The action of OPGL does not seem to be species restricted as
murine OPGL also induced osteoclast formation in cultures of human
peripheral blood mononuclear cells.
OBJECT OF THE INVENTION
[0029] The object of the present invention is to provide novel
therapies against conditions characterized by excess bone
resorption, such as osteoporosis. A further object is to develop an
autovaccine against OPGL, in order to obtain a novel treatment for
osteoporosis and for other pathological disorders involving excess
bone resorption.
SUMMARY OF THE INVENTION
[0030] We find that the above-referenced data suggests a
pathophysiological role of OPGL. The in vivo evidence is partially
circumstantial or indirect but is in our opinion convincing
especially in combination with the direct evidence.
[0031] Observing that injection into mice of the recombinant
C-terminal domain of OPGL results in severe hypercalcemia in our
opinion points directly to a pathophysiological role.
[0032] Indirect evidence comes from the osteoprotegerin-deficient
mice (knock out mice) that even though normal at birth develop
early onset osteoporosis. This shows that removing a protein that
binds OPGL and neutralises its effects leads to osteoporosis. We
conclude that the most likely reason for this is an increased
osteoclast maturation and activation caused by OPGL.
[0033] Two other pieces of indirect evidence are that both mice
transgenic for osteoprotegerin and mice injected with recombinant
osteoprotegerin develop osteopetrosis. This shows that unnatural
high levels of a protein that binds OPGL and neutralises its
effects leads to osteopetrosis. Here, we conclude that this has its
reasons in a decreased osteoclast maturation and activation caused
by neutralisation of OPGL.
[0034] We therefore suggest a model in which OPGL and
osteoprotegerin act as positive and negative regulators of
osteoclast development, respectively. In other words OPGL promotes
bone resorption while osteoprotegerin inhibits bone resorption.
[0035] Thus, in relation to osteoporosis OPGL could be thought of
as a "pathogenic agent" which promotes the bone resorption that in
the end leads to osteoporosis. Likewise osteoprotegerin can be
visualised as a "therapeutic agent" which counteracts the
"pathogenic agent" through neutralisation of its effects.
[0036] We hence propose to down-regulate osteoclast
differentiation/maturation/formation and osteoclast activation
through in vivo production of antibodies capable of neutralizing
OPGL, thereby providing a safe and efficient means for
treating/ameliorating and/or preventing osteoporosis and other
diseases characterized by an excess rate of bone resorption
compared to the rate of bone formation.
[0037] Thus, in its broadest and most general scope, the present
invention relates to a method for in vivo down-regulation of
osteoprotegerin ligand (OPGL) activity in an animal, including a
human being, the method comprising effecting presentation to the
animal's immune system of an immunologically effective amount
of
[0038] at least one OPGL polypeptide or subsequence thereof which
has been formulated so that immunization of the animal with the
OPGL polypeptide or subsequence thereof induces production of
antibodies against the OPGL polypeptide, and/or
[0039] at least one OPGL analogue wherein is introduced a
modification in the OPGL polypeptide which has as a result that
immunization of the animal with the analogue induces production of
antibodies against the OPGL polypeptide.
[0040] The most attractive aspect of this approach is that e.g.
osteoporosis can be controlled by periodic but not very frequent
immunizations, in contrast to a therapeutic approach which involves
frequent (e.g. daily) administration of osteoprotegerin or
molecules having a binding affinity to OPGL analogous therewith. It
is expected that 1-4 annual injections with an immunogenic
composition will be sufficient to obtain the desired effect,
whereas administration of osteoprotegerin or other inhibitors of
OPGL activity would require daily administrations.
[0041] The invention also relates to OPGL analogues as well as to
nucleic acid fragments encoding a subset of these. Also immunogenic
compositions comprising the analogues or the nucleic acid fragments
are part of the invention.
[0042] The invention also relates to a method of identifying
analogues of OPGL as well as a method for preparing composition
comprising the OPGL analogues.
[0043] Finally, the invention relates to a method treating
osteoporosis and other diseases characterized in excess bone
resorption, wherein is administered a non-OPGL molecule (typically
an antibody) which blocks the interaction between OPGL and its
receptor on osteoclast cells.
DETAILED DISCLOSURE OF THE INVENTION
[0044] Definitions
[0045] In the following a number of terms used in the present
specification and claims will be defined and explained in detail in
order to clarify the metes and bounds of the invention.
[0046] The terms "T-lymphocyte" and "T-cell" will be used
interchangeably for lymphocytes of thymic origin which are
responsible for various cell mediated immune responses as well as
for helper activity in the humoral immune response. Likewise, the
terms "B-lymphocyte" and "B-cell" will be used interchangeably for
antibody-producing lymphocytes.
[0047] An "OPGL polypeptide" is herein intended to denote
polypeptides having the amino acid sequence of the above-discussed
OPGL proteins derived from humans and mice (or truncates thereof
sharing a substantial amount of B-cell epitopes with intact OPGL),
but also polypeptides having the amino acid sequence identical to
analogues of these two proteins isolated from other species are
embraced by the term. Also unglycosylated forms of OPGL which are
prepared in prokaryotic system are included within the boundaries
of the term as are forms having varying glycosylation patterns due
to the use of e.g. yeasts or other non-mammalian eukaryotic
expression systems. It should, however, be noted that when using
the term "an OPGL polypeptide" it is intended that the polypeptide
in question is normally non-immunogenic when presented to the
animal to be treated. In other words, the OPGL polypeptide is a
self-protein or is an analogue of such a self-protein which will
not normally give rise to an immune response against OPGL of the
animal in question.
[0048] An "OPGL analogue" is an OPGL polypeptide which has been
subjected to changes in its primary structure. Such a change can
e.g. be in the form of fusion of an OPGL polypeptide to a suitable
fusion partner (i.e. a change in primary structure exclusively
involving C- and/or N-terminal additions of amino acid residues)
and/or it can be in the form of insertions and/or deletions and/or
substitutions in the OPGL polypeptide's amino acid sequence. Also
encompassed by the term are derivatized OPGL molecules, cf. the
discussion below of modifications of OPGL.
[0049] It should be noted that the use as a vaccine in a human of a
xeno-analogue (e.g. a canine or porcine analogue) of human OPGL can
be imagined to produce the desired immunity against OPGL. Such use
of an xeno-analogue for immunization is also considered part of the
invention.
[0050] The term "polypeptide" is in the present context intended to
mean both short peptides of from 2 to 10 to amino acid residues,
oligopeptides of from 11 to 100 amino acid residues, and
polypeptides of more than 100 amino acid residues. Furthermore, the
term is also intended to include proteins, i.e. functional
biomolecules comprising at least one polypeptide; when comprising
at least two polypeptides, these may form complexes, be covalently
linked, or may be non-covalently linked. The polypeptide(s) in a
protein can be glycosylated and/or lipidated and/or comprise
prosthetic groups.
[0051] The term subsequence means any consecutive stretch of at
least 3 amino acids or, when relevant, of at least 3 nucleotides,
derived directly from a naturally occurring OPGL amino acid
sequence or nucleic acid sequence, respectively.
[0052] The term "animal" is in the present context in general
intended to denote an animal species (preferably mammalian), such
as Homo sapiens, Canis domesticus, etc. and not just one single
animal. However, the term also denotes a population of such an
animal species, since it is important that the individuals
immunized according to the method of the invention all harbour
substantially the same OPGL allowing for immunization of the
animals with the same immunogen(s). If, for instance, genetic
variants of OPGL exists in different human population it may be
necessary to use different immunogens in these different
populations in order to be able to break the autotolerance towards
OPGL in each population. It will be clear to the skilled person
that an animal in the present context is a living being which has
an immune system. It is preferred that the animal is a vertebrate,
such as a mammal.
[0053] By the term "in vivo down-regulation of OPGL activity" is
herein meant reduction in the living organism of the number of
interactions between OPGL and its (unknown) receptor (or between
OPGL and other possible biologically important binding partners for
this molecule). The down-regulation can be obtained by means of
several mechanisms: Of these, simple interference with the active
site in OPGL by antibody binding is the most simple. However, it is
also within the scope of the present invention that the antibody
binding results in removal of OPGL by scavenger cells (such as
macrophages and other phagocytic cells).
[0054] The expression "effecting presentation . . . to the immune
system" is intended to denote that the animal's immune system is
subjected to an immunogenic challenge in a controlled manner. As
will appear from the disclosure below, such challenge of the immune
system can be effected in a number of ways of which the most
important are vaccination with polypeptide containing
"pharmaccines" (i.e. a vaccine which is administered to treat or
ameliorate ongoing disease) or nucleic acid "pharmaccine"
vaccination. The important result to achieve is that immune
competent cells in the animal are confronted with the antigen in an
immunologically effective manner, whereas the precise mode of
achieving this result is of less importance to the inventive idea
underlying the present invention.
[0055] The term "immunogenically effective amount" has its usual
meaning in the art, i.e. an amount of an immunogen which is capable
of inducing an immune response which significantly engages
pathogenic agents which share immunological features with the
immunogen.
[0056] When using the expression that the OPGL has been "modified"
is herein meant a chemical modification of the polypeptide which
constitutes the backbone of OPGL. Such a modification can e.g. be
derivatization (e.g. alkylation) of certain amino acid residues in
the OPGL sequence, but as will be appreciated from the disclosure
below, the preferred modifications comprise changes of the primary
structure of the OPGL amino acid sequence.
[0057] When discussing "autotolerance towards OPGL" it is
understood that since OPGL is a self-protein in the population to
be vaccinated, normal individuals in the population do not mount an
immune response against OPGL; it cannot be excluded, though, that
occasional individuals in an animal population might be able to
produce antibodies against native OPGL, e.g. as part of a
autoimmune disorder. At any rate, an animal will normally only be
autotolerant towards its own OPGL, but it cannot be excluded that
OPGL analogues derived from other animal species or from a
population having a different OPGL phenotype would also be
tolerated by said animal.
[0058] A "foreign T-cell epitope" (or: "foreign T-lymphocyte
epitope") is a peptide which is able to bind to an MHC molecule and
which stimulates T-cells in an animal species. Preferred foreign
T-cell epitopes in the invention are "promiscuous" epitopes, i.e.
epitopes which bind to a substantial fraction of a particular class
of MHC molecules in an animal species or population. Only a very
limited number of such promiscuous T-cell epitopes are known, and
they will be discussed in detail below. It should be noted that in
order for the immunogens which are used according to the present
invention to be effective in as large a fraction of an animal
population as possible, it may be necessary to 1) insert several
foreign T-cell epitopes in the same OPGL analogue or 2) prepare
several OPGL analogues wherein each analogue has a different
promiscuous epitope inserted. It should be noted also that the
concept of foreign T-cell epitopes also encompasses use of cryptic
T-cell epitopes, i.e. epitopes which are derived from a
self-protein and which only exerts immunogenic behaviour when
existing in isolated form without being part of the self-protein in
question.
[0059] A "foreign T helper lymphocyte epitope" (a foreign T.sub.H
epitope) is a foreign T cell epitope which binds an MHC Class Class
II molecule and can be presented on the surface of an antigen
presenting cell (APC) bound to the MHC Class II molecule.
[0060] A "functional part" of a (bio)molecule is in the present
context intended to mean the part of the molecule which is
responsible for at least one of the biochemical or physiological
effects exerted by the molecule. It is well-known in the art that
many enzymes and other effector molecules have an active site which
is responsible for the effects exerted by the molecule in question.
Other parts of the molecule may serve a stabilizing or solubility
enhancing purpose and can therefore be left out if these purposes
are not of relevance in the context of a certain embodiment of the
present invention. For instance it is possible to use certain
cytokines as a modifying moiety in OPGL (cf. the detailed
discussion below), and in such a case, the issue of stability may
be irrelevant since the coupling to OPGL provides the stability
necessary.
[0061] The term "adjuvant" has its usual meaning in the art of
vaccine technology, i.e. a substance or a composition of matter
which is 1) not in itself capable of mounting a specific immune
response against the immunogen of the vaccine, but which is 2)
nevertheless capable of enhancing the immune response against the
immunogen. Or, in other words, vaccination with the adjuvant alone
does not provide an immune response against the immunogen,
vaccination with the immunogen may or may not give rise to an
immune response against the immunogen, but the combined vaccination
with immunogen and adjuvant induces an immune response against the
immunogen which is stronger than that induced by the immunogen
alone.
[0062] "Targeting" of a molecule is in the present context intended
to denote the situation where a molecule upon introduction in
the-animal will appear preferentially in certain tissue(s) or will
be preferentially associated with certain cells or cell types. The
effect can be accomplished in a number of ways including
formulation of the molecule in composition facilitating targeting
or by introduction in the molecule of groups which facilitates
targeting. These issues will be discussed in detail below.
[0063] "Stimulation of the immune system" means that a substance or
composition of matter exhibits a general, non-specific
immunostimulatory effect. A number of adjuvants and putative
adjuvants (such as certain cytokines) share the ability to
stimulate the immune system. The result of using an
immunostimulating agent is an increased "alertness" of the immune
system meaning that simultaneous or subsequent immunization with an
immunogen induces a significantly more effective immune response
compared to isolated use of the immunogen
[0064] Preferred Embodiments of OPGL Activity Down-regulation
[0065] It is preferred that the OPGL polypeptide used as an
immunogen in the method of the invention is a modified molecule
wherein at least one change is present in the OPGL amino acid
sequence, since the chances of obtaining the all-important breaking
of autotolerance towards OPGL is greatly facilitated that way. It
should be noted that this does not exclude the possibility of using
such a modified OPGL in formulations which further facilitate the
breaking of autotolerance against OPGL, e.g. formulations
containing adjuvants.
[0066] It has been shown (in Dalum I et al., 1996, J. Immunol. 157:
4796-4804) that potentially self-reactive B-lymphocytes recognizing
self-proteins are physiologically present in normal individuals.
However, in order for these B-lymphocytes to be induced to actually
produce antibodies reactive with the relevant self-proteins,
assistance is needed from cytokine producing T-helper lymphocytes
(T.sub.H-cells or T.sub.H-lymphocytes). Normally this help is not
provided because T-lymphocytes in general do not recognize T-cell
epitopes derived from self-proteins when presented by antigen
presenting cells (APCs). However, by providing an element of
"foreignness" in a self-protein (i.e. by introducing an
immunologically significant modification), T-cells recognizing the
foreign element are activated upon recognizing the foreign epitope
on an APC (such as, initially, a mononuclear cell). Polyclonal
B-lymphocytes (which are also APCs) capable of recognising
self-epitopes on the modified self-protein also internalise the
antigen and subsequently presents the foreign T-cell epitope(s)
thereof, and the activated T-lymphocytes subsequently provide
cytokine help to these self-reactive polyclonal B-lymphocytes.
Since the antibodies produced by these polyclonal B-lymphocytes are
reactive with different epitopes on the modified polypeptide,
including those which are also present in the native polypeptide,
an antibody cross-reactive with the non-modified self-protein is
induced. In conclusion, the T-lymphocytes can be led to act as if
the population of polyclonal B-lymphocytes have recognised an
entirely foreign antigen, whereas in fact only the inserted
epitope(s) is/are foreign to the host. In this way, antibodies
capable of cross-reacting with non-modified self-antigens are
induced.
[0067] Several ways of modifying a peptide self-antigen in order to
obtain breaking of autotolerance are known in the art. Hence,
according to the invention, the modification can include that
[0068] at least one foreign T-cell epitope is introduced,
and/or
[0069] at least one first moiety is introduced which effects
targeting of the modified molecule to an antigen presenting cell
(APC), and/or
[0070] at least one second moiety is introduced which stimulates
the immune system, and/or
[0071] at least one third moiety is introduced which optimizes
presentation of the modified OPGL polypeptide to the immune
system.
[0072] However, all these modifications should be carried out while
maintaining a substantial fraction of the original B-lymphocyte
epitopes in OPGL, since the B-lymphocyte recognition of the native
molecule is thereby enhanced.
[0073] In one preferred embodiment, side groups (in the form of
foreign T-cell epitopes or the above-mentioned first, second and
third moieties) are covalently or non-covalently introduced. This
is to mean that stretches of amino acid residues derived from OPGL
are derivatized without altering the primary amino acid sequence,
or at least without introducing changes in the peptide bonds
between the individual amino acids in the chain.
[0074] An alternative, and preferred, embodiment utilises amino
acid substitution and/or deletion and/or insertion and/or addition
(which may be effected by recombinant means or by means of peptide
synthesis; modifications which involves longer stretches of amino
acids can give rise to fusion polypeptides). One especially
preferred version of this embodiment is the technique described in
WO 95/05849, which discloses a method for down-regulating
self-proteins by immunising with analogues of the self-proteins
wherein a number of amino acid sequence(s) has been substituted
with a corresponding number of amino acid sequence(s) which each
comprise a foreign immunodominant T-cell epitope, while at the same
time maintaining the overall tertiary structure of the self-protein
in the analogue. For the purposes of the present invention, it is
however sufficient if the modification (be it-an insertion,
addition, deletion or substitution) gives rise to a foreign T-cell
epitope and at the same time preserves a substantial number of the
B-cell epitopes in OPGL. However, in order to obtain maximum
efficacy of the immune response induced, it is preferred that the
overall tertiary structure of OPGL is maintained in the modified
molecule.
[0075] The following formula describes the OPGL constructs
generally covered by the invention:
(MOD.sub.1).sub.s1(OPGL.sub.e1).sub.n1(MOD.sub.2).sub.s2(OPGL.sub.e2).sub.-
n2 . . . (MOD.sub.x).sub.sx(OPGL.sub.ex).sub.nx (I)
[0076] where OPGL.sub.e1-OPGL.sub.ex are x B-cell epitope
containing subsequences of OPGL which independently are identical
or non-identical and which may contain or not contain foreign side
groups, x is an integer .cndot..cndot.3, n1-nx are x integers
.cndot..cndot.0 (at least one is .cndot..cndot.1),
MOD.sub.1-MOD.sub.x are x modifications introduced between the
preserved B-cell epitopes, and s.sub.1-s.sub.x are x integers
.cndot..cndot.0 (at least one is .cndot..cndot.1 if no side groups
are introduced in the OPGL.sub.e sequences). Thus, given the
general functional restraints on the immunogenicity of the
constructs, the invention allows for all kinds of permutations of
the original OPGL sequence, and all kinds of modifications therein.
Thus, included in the invention are modified OPGL obtained by
omission of parts of the OPGL sequence which e.g. exhibit adverse
effects in vivo or omission of parts which are normally
intracellular and thus could give rise to undesired immunological
reactions.
[0077] Maintenance of a substantial fraction of B-cell epitopes or
even the overall tertiary structure of a protein which is subjected
to modification as described herein can be achieved in several
ways. One is simply to prepare a polyclonal antiserum directed
against OPGL (e.g. an antiserum prepared in a rabbit) and
thereafter use this antiserum as a test reagent (e.g. in a
competitive ELISA) against the modified proteins which are
produced. Modified versions (analogues) which react to the same
extent with the antiserum as does OPGL must be regarded as having
the same overall tertiary structure as OPGL whereas analogues
exhibiting a limited (but still significant and specific)
reactivity with such an antiserum are regarded as having maintained
a substantial fraction of the original B-cell epitopes.
[0078] Alternatively, a selection of monoclonal antibodies reactive
with distinct epitopes on OPGL can be prepared and used as a test
panel. This approach has the advantage of allowing 1) an epitope
mapping of OPGL and 2) a mapping of the epitopes which are
maintained in the analogues prepared.
[0079] Of course, a third approach would be to resolve the
3-dimensional structure of OPGL or of a biologically active
truncate thereof (cf. above) and compare this to the resolved
three-dimensional structure of the analogues prepared.
Three-dimensional structure can be resolved by the aid of X-ray
diffraction studies and NMR-spectroscopy. Further information
relating to the tertiary structure can to some extent be obtained
from circular dichroism studies which have the advantage of merely
requiring the polypeptide in pure form (whereas X-ray diffraction
requires the provision of crystallized polypeptide and NMR requires
the provision of isotopic variants of the polypeptide) in order to
provide useful information about the tertiary structure of a given
molecule. However, ultimately X-ray diffraction and/or NMR are
necessary to obtain conclusive data since circular dichroism can
only provide indirect evidence of correct 3-dimensional structure
via information of secondary structure elements.
[0080] One preferred embodiment of the invention utilises multiple
presentations of B-lymphocyte epitopes of OPGL (i.e. formula I
wherein at least one B-cell epitope is present in two positions).
This effect can be achieved in various ways, e.g. by simply
preparing fusion polypeptides comprising the structure
(OPGL).sub.m, where m is an integer .cndot..cndot.2 and then
introduce the modifications discussed herein in at least one of the
OPGL sequences. It is preferred that the modifications introduced
includes at least one duplication of a B-lymphocyte epitope and/or
the introduction of a hapten.
[0081] As mentioned above, the introduction of a foreign T-cell
epitope can be accomplished by introduction of at least one amino
acid insertion, addition, deletion, or substitution. Of course, the
normal situation will be the introduction of more than one change
in the amino acid sequence (e.g. insertion of or substition by a
complete T-cell epitope) but the important goal to reach is that
the OPGL analogue, when processed by an antigen presenting cell
(APC), will give rise to such a foreign immunodominant T-cell
epitope being presented in context of an MCH Class II molecule on
the surface of the APC. Thus, if the OPGL amino acid sequence in
appropriate positions comprises a number of amino acid residues
which can also be found in a foreign T.sub.H epitope then the
introduction of a foreign T.sub.H epitope can be accomplished by
providing the remaining amino acids of the foreign epitope by means
of amino acid insertion, addition, deletion and substitution. In
other words, it is not necessary to introduce a complete T.sub.H
epitope by insertion or substitution in order to fulfill the
purpose of the present invention.
[0082] It is preferred that the number of amino acid insertions,
deletions, substitutions or additions is at least 2, such as 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 25
insertions, substitutions, additions or deletions. It is
furthermore preferred that the number of amino acid insertions,
substitutions, additions or deletions is not in excess of 150, such
as at most 100, at most 90, at most 80, and at most 70. It is
especially preferred that the number of substitutions, insertions,
deletions, or additions does not exceed 60, and in particular the
number should not exceed 50 or even 40. Most preferred is a number
of not more than 30. With respect to amino acid additions, it
should be noted that these, when the resulting construct is in the
form of a fusion polypeptide, is often considerably higher than
150.
[0083] Preferred embodiments of the invention includes modification
by introducing at least one foreign immunodominant T-cell epitope.
It will be understood that the question of immune dominance of a
T-cell epitope-depends on the animal species in question. As used
herein, the term "immunodominance" simply refers to epitopes which
in the vaccinated individual/population gives rise to a significant
immune response, but it is a well-known fact that a T-cell epitope
which is immunodominant in one individual/population is not
necessarily immunodominant in another individual of the same
species, even though it may be capable of binding MHC-II molecules
in the latter individual. Hence, for the purposes of the present
invention, an immune dominant T-cell epitope is a T-cell epitope
which will be effective in providing T-cell help when present in an
antigen. Typically, immune dominant T-cell epitopes has as an
inherent feature that they will substantially always be presented
bound to an MHC Class II molecule, irrespective of the polypeptide
wherein they appear.
[0084] Another important point is the issue of MHC restriction of
T-cell epitopes. In general, naturally occurring T-cell epitopes
are MHC restricted, i.e. a certain peptides constituting a T-cell
epitope will only bind effectively to a subset of MHC Class II
molecules. This in turn has the effect that in most cases the use
of one specific T-cell epitope will result in a vaccine component
which is only effective in a fraction of the population, and
depending on the size of that fraction, it can be necessary to
include more T-cell epitopes in the same molecule, or alternatively
prepare a multi-component vaccine wherein the components are OPGL
variants which are distinguished from each other by the nature of
the T-cell epitope introduced.
[0085] If the MHC restriction of the T-cells used is completely
unknown (for instance in a situation where the vaccinated animal
has a poorly defined MHC composition), the fraction of the
population covered by a specific vaccine composition can be
determined by means of the following formula 1 f population = 1 - i
= 1 n ( 1 - p i ) ( II )
[0086] where p.sub.i is the frequency in the population of
responders to the i.sup.th foreign T-cell epitope present in the
vaccine composition, and n is the total number of foreign T-cell
epitopes in the vaccine composition. Thus, a vaccine composition
containing 3 foreign T-cell epitopes having response frequencies in
the population of 0.8, 0.7, and 0.6, respectively, would give
1-0.2.times.0.3.times.0.4=0.976
[0087] i.e. 97.6 percent of the population will statistically mount
an MHC-II mediated response to the vaccine.
[0088] The above formula does not apply in situations where a more
or less precise MHC restriction pattern of the peptides used is
known. If, for instance a certain peptide only binds the human
MHC-II molecules encoded by HLA-DR alleles DR1, DR3, DR5, and DR7,
then the use of this peptide together with another peptide which
binds the remaining MHC-II molecules encoded by HLA-DR alleles will
accomplish 100% coverage in the population in question. Likewise,
if the second peptide only binds DR3 and DR5, the addition of this
peptide will not increase the coverage at all. If one bases the
calculation of population response purely on MHC restriction of
T-cell epitopes in the vaccine, the fraction of the population
covered by a specific vaccine composition can be determined by
means of the following formula: 2 f population = 1 - j = 1 3 ( 1 -
j ) 2 ( III )
[0089] wherein .PHI..sub.j is the sum of frequencies in the
population of allelic haplotypes encoding MHC molecules which bind
any one of the T-cell epitopes in the vaccine and which belong to
the j.sup.th of the 3 known HLA loci (DP, DR and DQ); in practice,
it is first determined which MHC molecules will recognize each
T-cell epitope in the vaccine and thereafter these are listed by
type (DP, DR and DQ)--then, the individual frequencies of the
different listed allelic haplotypes are summed for each type,
thereby yielding .PHI..sub.1, .PHI..sub.2, and .PHI..sub.3.
[0090] It may occur that the value p.sub.i in formula II exceeds
the corresponding theoretical value .PI..sub.i: 3 i = 1 - j = 1 3 (
1 - v j ) 2 ( IV )
[0091] wherein u.sub.j is the sum of frequencies in the population
of allelic haplotype encoding MHC molecules which bind the i.sup.th
T-cell epitope in the vaccine and which belong to the j.sup.th of
the 3 known HLA loci (DP, DR and DQ) . This means that in
1-.PI..sub.i of the population is a frequency of responders of
f.sub.residual.sub..sub.--.sub-
.i=(p.sub.i-.PI..sub.i)/(1-.PI..sub.i). Therefore, formula III can
be adjusted so as to yield formula V: 4 f population = 1 - j = 1 3
( 1 - j ) 2 + ( 1 - i = 1 n ( 1 - f residual_i ) ) ( V )
[0092] where the term 1-f.sub.residual-i is set to zero if
negative. It should be noted that formula V requires that all
epitopes have been haplotype mapped against identical sets of
haplotypes.
[0093] Therefore, when selecting T-cell epitopes to be introduced
in the OPGL analogue, it is important to include all knowledge of
the epitopes which is available: 1) The frequency of responders in
the population to each epitope, 2) MHC restriction data, and 3)
frequency -in the population of the relevant haplotypes.
[0094] There exist a number of naturally occurring "promiscuous"
T-cell epitopes which are active in a large proportion of
individuals of an animal species or an animal population and these
are preferably introduced in the vaccine thereby reducing the need
for a very large number of different OPGL analogues in the same
vaccine.
[0095] The promiscuous epitope can according to the invention be a
naturally occurring human T-cell epitope such as epitopes from
tetanus toxoid (e.g. the P2 and P30 epitopes), diphtheria toxoid,
Influenza virus hemagluttinin (HA), and P. falciparum CS
antigen.
[0096] Over the years a number of other promiscuous T-cell epitopes
have been identified. Especially peptides capable of binding a
large proportion of HLA-DR molecules encoded by the different
HLA-DR alleles have been identified and these are all possible
T-cell epitopes to be introduced in the OPGL analogues used
according to the present invention. Cf. also the epitopes discussed
in the following references which are hereby all incorporated by
reference herein: WO 98/23635 (Frazer I H et al., assigned to The
University of Queensland); Southwood S et. al, 1998, J. Immunol.
160: 3363-3373; Sinigaglia F et al., 1988, Nature 336: 778-780;
Chicz R M et al., 1993, J. Exp. Med 178: 27-47; Hammer J et al.,
1993, Cell 74: 197-203; and Falk K et al., 1994, Immunogenetics 39:
230-242. The latter reference also deals with HLA-DQ and -DP
ligands. All epitopes listed in these 5 references are relevant as
candidate natural epitopes to be used in the present invention, as
are epitopes which share common motifs with these.
[0097] Alternatively, the epitope can be any artificial T-cell
epitope which is capable of binding a large proportion of MHC Class
II molecues. In this context the pan DR epitope peptides ("PADRE")
described in WO 95/07707 and in the corresponding paper Alexander J
et al., 1994, Immunity 1: 751-761 (both disclosures are
incorporated by reference herein) are interesting candidates for
epitopes to be used according to the present invention. It should
be noted that the most effective PADRE peptides disclosed in these
papers carry D-amino acids in the C- and N-termini in order to
improve stability when administered. However, the present invention
primarily aims at incorporating the relevant epitopes as part of
the modified OPGL which should then subsequently be broken down
enzymatically inside the lysosomal compartment of APCs to allow
subsequent presentation in the context of an MHC-II molecule and
therefore it is not expedient to incorporate D-amino acids in the
epitopes used in the present invention.
[0098] One especially preferred PADRE peptide is the one having the
amino acid sequence AKFVAAWTLKAAA or an immunologically effective
subsequence thereof. This, and other epitopes having the same lack
of MHC restriction are preferred T-cell epitopes which should be
present in the OPGL analogues used in the inventive method. Such
super-promiscuous epitopes will allow for the most simple
embodiments of the invention wherein only one single modified OPGL
is presented to the vaccinated animal's immune system.
[0099] As mentioned above, the modification of OPGL can also
include the introduction of a first moiety which targets the
modified OPGL to an APC or a B-lymphocyte. For instance, the first
moiety can be a specific binding partner for a B-lymphocyte
specific surface antigen or for an APC specific surface antigen.
Many such specific surface antigens are known in the art. For
instance, the moiety can be a carbohydrate for which there is a
receptor on the B-lymphocyte or the APC (e.g. mannan or mannose).
Alternatively, the second moiety can be a hapten. Also an antibody
fragment which specifically recognizes a surface molecule on APCs
or lymphocytes can be used as a first moiety (the surface molecule
can e.g. be an FC.gamma. receptor of macrophages and monocytes,
such as FC.gamma.RI or, alternatively any other specific surface
marker such as CD40 or CTLA-4). It should be noted that all these
exemplary targeting molecules can be used as part of an adjuvant
also, cf. below.
[0100] As an alternative or supplement to targeting the modified
OPGL polypeptide to a certain cell type in order to achieve an
enhanced immune response, it is possible to increase the level of
responsiveness of the immune system by including the
above-mentioned second moiety which stimulates the immune system.
Typical examples of such second moieties are cytokines, and
heat-shock proteins or molecular chaperones, as well as effective
parts thereof.
[0101] Suitable cytokines to be used according to the invention are
those which will normally also function as adjuvants in a vaccine
composition, i.e. for instance interferon .gamma. (IFN-.gamma.),
interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 4 (IL-4),
interleukin 6 (IL-6), interleukin 12 (IL-12), interleukin 13
(IL-13), interleukin (IL-15), and granulocyte-macrophage colony
stimulating factor (GM-CSF); alternatively, the functional part of
the cytokine molecule may suffice as the second moiety. With
respect to the use of such cytokines as adjuvant substances, cf.
the discussion below.
[0102] According to the invention, suitable heat-shock proteins or
molecular chaperones used as the second moiety can be HSP70, HSP90,
HSC70, GRP94 (also known as gp96, cf. Wearsch PA et al. 1998,
Biochemistry 37: 5709-19), and CRT (calreticulin).
[0103] Alternatively, the second moiety can be a toxin, such as
listeriolycin (LLO), lipid A and heat-labile enterotoxin. Also, a
number of mycobacterial derivatives such as MDP (muramyl
dipeptide), CFA (complete Freund's adjuvant) and the trehalose
diesters TDM and TDE are interesting possibilities.
[0104] Also the possibility of introducing a third moiety which
enhances the presentation of the modified OPGL to the immune system
is an important embodiment of the invention. The art has shown
several examples of this principle. For instance, it is known that
the palmitoyl lipidation anchor in the Borrelia burgdorferi protein
OspA can be utilised so as to provide self-adjuvating polypeptides
(cf. e.g. WO 96/40718)--it seems that the lipidated proteins form
up micelle-like structures with a core consisting of the lipidation
anchor parts of the polypeptides and the remaining parts of the
molecule protruding therefrom, resulting in multiple presentations
of the antigenic determinants. Hence, the use of this and related
approaches using different lipidation anchors (e.g. a myristyl
group, a myristyl group, a farnesyl group, a geranyl-geranyl group,
a GPI-anchor, and an N-acyl diglyceride group) are preferred
embodiments of the invention, especially since the provision of
such a lipidation anchor in a recombinantly produced protein is
fairly straightforward and merely requires use of e.g. a naturally
occurring signal sequence as a fusion partner for the modified OPGL
polypeptide. Another possibility is use of the C3d fragment of
complement factor C3 or C3 itself (cf. Dempsey et al., 1996,
Science 271, 348-350 and Lou & Kohler, 1998, Nature
Biotechnology 16, 458-462).
[0105] An alternative embodiment of the invention which also
results in the preferred presentation of multiple (e.g. at least 2)
copies of the important epitopic regions of OPGL to the immune
system is the covalent coupling of OPGL, subsequence or variants
thereof to certain molecules. For instance, polymers can be used,
e.g. carbohydrates such as dextran, cf. e.g. Lees A et al., 1994,
Vaccine 12: 1160-1166; Lees A et al., 1990, J Immunol. 145:
3594-3600, but also mannose and mannan are useful alternative.
Integral membrane proteins from e.g. E. coli and other bacteria are
also useful conjugation partners. The traditional carrier molecules
such as keyhole limpet hemocyanin (KLH), tetanus toxoid, diphtheria
toxoid, and bovine serum albumin (BSA) are also preferred and
useful conjugation partners.
[0106] Certain areas of native OPGL seems to be most suited for
performing modifications. Because of OPGL's structural relationship
with TNF-.alpha. and other members of the tumour necrosis factor
family, it is predicted that introductions of T-cell epitopes or
other modifications in areas defined by positions 170-192, 198-218,
221-246, 256-261, or 285-316, (the amino acid numbering of SEQ ID
NOs: 4, 6, and 12) will be most likely to produce the desired
results. These positions refer to the murine OPGL--the
corresponding positions in the human molecule are 171-193, 199-219,
222-247, 257-262, and 286-317 (the amino acid numbering of SEQ ID
NO: 2).
[0107] Considerations underlying these chosen areas are a)
preservation of known and predicted B-cell epitopes, b)
preservation of tertiary structure etc. At any rate, as discussed
above, it is fairly easy to screen a set of modified OPGL molecules
which have all been subjected to introduction of a T-cell epitope
in different locations.
[0108] Since the most preferred embodiments of the present
invention involves down-regulation of human OPGL, it is
consequently preferred that the OPGL polypeptide discussed above is
a human OPGL polypeptide. In this embodiment, it is especially
preferred that the human OPGL polypeptide has been modified by
substituting at least one amino acid sequence in SEQ ID NO: 2 (or
in a polypeptide where Cys-221 in SEQ ID NO: 2 has been substituted
with serine) with at least one amino acid sequence of equal or
different length and containing a foreign T.sub.H epitope. The
substituted amino acid residues are selected from residues 257-262,
289-303 and 222-243 in SEQ ID NO: 2. The rationale behind such
constructs is discussed in detail in the examples.
[0109] Formulation of OPGL and Modified OPGL Polypeptides
[0110] When effecting presentation of the OPGL polypeptide or the
modified OPGL polypeptide to an animal's immune system by means of
administration thereof to the animal, the formulation of the
polypeptide follows the principles generally acknowledged in the
art.
[0111] Preparation of vaccines which contain peptide sequences as
active ingredients is generally well understood in the art, as
exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231;
4,599,230; 4,596,792; and 4,578,770, all incorporated herein by
reference. Typically, such vaccines are prepared as injectables
either as liquid solutions or suspensions; solid forms suitable for
solution in, or suspension in, liquid prior to injection may also
be prepared. The preparation may also be emulsified. The active
immunogenic ingredient-is often mixed with excipients which are
pharmaceutically acceptable and compatible with the active
ingredient. Suitable excipients are, for example, water, saline,
dextrose, glycerol, ethanol, or the like, and combinations thereof.
In addition, if desired, the vaccine may contain minor amounts of
auxiliary substances such as wetting or emulsifying agents, pH
buffering agents, or adjuvants which enhance the effectiveness of
the vaccines; cf. the detailed discussion of adjuvants below.
[0112] The vaccines are conventionally administered parenterally,
by injection, for example, either subcutaneously, intracutaneously,
intradermally, subdermally or intramuscularly. Additional
formulations which are suitable for other modes of administration
include suppositories and, in some cases, oral, buccal, sublinqual,
intraperitoneal, intravaginal, anal, epidural, spinal, and
intracranial formulations. For suppositories, traditional binders
and carriers may include, for example, polyalkalene glycols or
triglycerides; such suppositories may be formed from mixtures
containing the active ingredient in the range of 0.5% to 10%,
preferably 1-2%. Oral formulations include such normally employed
excipients as, for example, pharmaceutical grades of mannitol,
lactose, starch, magnesium stearate, sodium saccharine, cellulose,
magnesium carbonate, and the like. These compositions take the form
of solutions, suspensions, tablets, pills, capsules, sustained
release formulations or powders and contain 10-95% of active
ingredient, preferably 25-70%. For oral formulations, cholera toxin
is an interesting formulation partner (and also a possible
conjugation partner).
[0113] The polypeptides may be formulated into the vaccine as
neutral or salt forms. Pharmaceutically acceptable salts include
acid addition salts (formed with the free amino groups of the
peptide) and which are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids as
acetic, oxalic, tartaric, mandelic, and the like. Salts formed with
the free carboxyl groups may also be derived from inorganic bases
such as, for example, sodium, potassium, ammonium, calcium, or
ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the
like.
[0114] The vaccines are administered in a manner compatible with
the dosage formulation, and in such amount as will be
therapeutically effective and immunogenic. The quantity to be
administered depends on the subject to be treated, including, e.g.,
the capacity of the individual's immune system to mount an immune
response, and the degree of protection desired. Suitable dosage
ranges are of the order of several hundred micro-grams active
ingredient per vaccination with a preferred range from about 0.1
.mu.g to 2,000 .mu.g (even though higher amounts in the 1-10 mg
range are contemplated), such as in the range from about 0.5 .mu.g
to 1,000 .mu.g, preferably in the range from 1 .mu.g to 500 .mu.g
and especially in the range from about 10 .mu.g to 100 .mu.g.
Suitable regimens for initial administration and booster shots are
also variable but are typified by an initial administration
followed by subsequent inoculations or other administrations.
[0115] The manner of application may be varied widely. Any of the
conventional methods for administration of a vaccine are
applicable. These include oral application on a solid
physiologically acceptable base or in a physiologically acceptable
dispersion, parenterally, by injection or the like. The dosage of
the vaccine will depend on the route of administration and will
vary according to the age of the person to be vaccinated and the
formulation of the antigen.
[0116] Some of the polypeptides of the vaccine are sufficiently
immunogenic in a vaccine, but for some of the others the immune
response will be enhanced if the vaccine further comprises an
adjuvant substance.
[0117] Various methods of achieving adjuvant effect for the vaccine
are known. General principles and methods are detailed in "The
Theory and Practical Application of Adjuvants", 1995, Duncan E. S.
Stewart-Tull (ed.), John Wiley & Sons Ltd, ISBN 0-471-95170-6,
and also in "Vaccines: New Generationn Immunological Adjuvants",
1995, Gregoriadis G et al. (eds.), Plenum Press, New York, ISBN
0-306-45283-9, both of which are hereby incorporated by reference
herein.
[0118] It is especially preferred to use an adjuvant which can be
demonstrated to facilitate breaking of the autotolerance to
autoantigens; in fact, this is essential in cases where unmodified
OPGL is used as the active ingredient in the autovaccine.
Non-limiting examples of suitable adjuvants are selected from the
group consisting of an immune targeting adjuvant; an immune
modulating adjuvant such as a toxin, a cytokine, and a
mycobacterial derivative; an oil formulation; a polymer; a micelle
forming adjuvant; a saponin; an immunostimulating complex matrix
(ISCOM matrix); a particle; DDA; aluminium adjuvants; DNA
adjuvants; .gamma.-inulin; and an encapsulating adjuvant. In
general it should be noted that the disclosures above which relate
to compounds and agents useful as first, second and third moieties
in the analogues also refer mutatis mutandis to their use in the
adjuvant of a vaccine of the invention.
[0119] The application of adjuvants include use of agents such as
aluminum hydroxide or phosphate (alum), commonly used as 0.05 to
0.1 percent solution in buffered saline, admixture with synthetic
polymers of sugars (e.g. Carbopol.RTM.) used as 0.25 percent
solution, aggregation of the protein in the vaccine by heat
treatment with temperatures ranging between 70.degree. to
101.degree. C. for 30 second to 2 minute periods respectively and
also aggregation by-means of cross-linking agents are possible.
Aggregation by reactivation with pepsin treated antibodies (Fab
fragments) to albumin, mixture with bacterial cells such as C.
parvum or endotoxins or lipopolysaccharide components of
gram-negative bacteria, emulsion in physiologically acceptable oil
vehicles such as mannide mono-oleate (Aracel A) or emulsion with 20
percent solution of a perfluorocarbon (Fluosol-DA) used as a block
substitute may also be employed. Admixture with oils such as
squalene and IFA is also preferred.
[0120] According to the invention DDA (dimethyldioctadecylammonium
bromide) is an interesting candidate for an adjuvant as is DNA and
.gamma.-inulin, but also Freund's complete and incomplete adjuvants
as well as quillaja saponins such as QuilA and QS21 are interesting
as is RIBI. Further possibilities are monophosphoryl lipid A (MPL),
the above mentioned C3 and C3d, and muramyl dipeptide (MDP).
[0121] Liposome formulations are also known to confer adjuvant
effects, and therefore liposome adjuvants are preferred according
to the invention.
[0122] Also immunostimulating complex matrix type (ISCOM.RTM.
matrix) adjuvants are preferred choices according to the invention,
especially since it has been shown that this type of adjuvants are
capable of up-regulating MHC Class II expression by APCs. An
ISCOM.RTM. matrix consists of (optionally fractionated) saponins
(triterpenoids) from Quillaja saponaria, cholesterol, and
phospholipid. When admixed with the immunogenic protein, the
resulting particulate formulation is what is known as an ISCOM
particle where the saponin constitutes 60-70% w/w, the cholesterol
and phospholipid 10-15% w/w, and the protein 10-15% w/w. Details
relating to composition and use of immunostimulating complexes can
e.g. be found in the above-mentioned text-books dealing with
adjuvants, but also Morein B et al., 1995, Clin. Immunother. 3:
461-475 as well as Barr I G and Mitchell G F, 1996, Immunol. and
Cell Biol. 74: 8-25 (both incorporated by reference herein) provide
useful instructions for the preparation of complete
immunostimulating complexes.
[0123] Another highly interesting (and thus, preferred) possibility
of achieving adjuvant effect is to employ the technique described
in Gosselin et al., 1992 (which is hereby incorporated by reference
herein). In brief, the presentation of a relevant antigen such as
an antigen of the present invention can be enhanced by conjugating
the antigen to antibodies (or antigen binding antibody fragments)
against the Fc.gamma. receptors on monocytes/macrophages.
Especially conjugates between antigen and anti-Fc.gamma.RI have
been demonstrated to enhance immunogenicity for the purposes of
vaccination.
[0124] Other possibilities involve the use of the targeting and
immune modulating substances (i.a. cytokines) mentioned above as
candidates for the first and second moieties in the modified
versions of OPGL. In this connection, also synthetic inducers of
cytokines like poly I:C are possibilities.
[0125] Suitable mycobacterial derivatives are selected from the
group consisting of muramyl dipeptide, complete Freund's adjuvant,
RIBI, and a diester of trehalose such as TDM and TDE.
[0126] Suitable immune targeting adjuvants are selected from the
group consisting of CD40 ligand and CD40 antibodies or specifically
binding fragments thereof (cf. the discussion above), mannose, a
Fab fragment, and CTLA-4.
[0127] Suitable polymer adjuvants are selected from the group
consisting of a carbohydrate such as dextran, PEG, starch, mannan,
and mannose; a plastic polymer such as; and latex such as latex
beads.
[0128] Yet another interesting way of modulating an immune response
is to include the OPGL immunogen (optionally together with
adjuvants and pharmaceutically acceptable carriers and vehicles) in
a "virtual lymph node" (VLN) (a proprietary medical device
developed by ImmunoTherapy, Inc., 360 Lexington Avenue, New York,
N.Y. 10017-6501). The VLN (a thin tubular device) mimics the
structrue and function of a lymph node. Insertion of a VLN under
the skin creates a site of sterile inflammation with an upsurge of
cytokines and chemokines. T- and B-cells as well as APCs rapidly
respond to the danger signals, home to the inflamed site and
accumulate inside the porous matrix of the VLN. It has been shown
that the necessary antigen dose required to mount an immune
response to an antigen is reduced when using the VLN and that
immune protection conferred by vaccination using a VLN surpassed
conventional immunization using Ribi as an adjuvant. The technology
is i.a. described briefly in Gelber C et al., 1998, "Elicitation of
Robust Cellular and Humoral Immune Responses to Small Amounts of
Immunogens Using a Novel Medical Device Designated the Virtual
Lymph Node", in: "From the Laboratory to the Clinic, Book of
Abstracts, Oct. 12-15, 1998, Seascape Resort, Aptos, Calif.".
[0129] It is expected that the vaccine should be administered 1-6
times per year, such as 1, 2, 3, 4, 5, or 6 times a year to an
individual in need thereof. It has previously been shown that the
memory immunity induced by the use of the preferred autovaccines
according to the invention is not permanent, and therefore the
immune system needs to be periodically challenged with the OPGL or
modified OPGL polypeptides.
[0130] Due to genetic variation, different individuals may react
with immune responses of varying strength to the same polypeptide.
Therefore, the vaccine according to the invention may comprise
several different polypeptides in order to increase the immune
response, cf. also the discussion above concerning the choice of
foreign T-cell epitope introductions. The vaccine may comprise two
or more polypeptides, where all of the polypeptides are as defined
above.
[0131] The vaccine may consequently comprise 3-20 different
modified or unmodified polypeptides, such as 3-10 different
polypeptides.
[0132] Nucleic Acid Vaccination
[0133] As an alternative to classic administration of a
peptide-based vaccine, the technology of nucleic acid vaccination
(also known as "nucleic acid immunisation", "genetic immunisation",
and "gene immunisation") offers a number of attractive
features.
[0134] First, in contrast to the traditional vaccine approach,
nucleic acid vaccination does not require resource consuming
large-scale production of the immunogenic agent (e.g. in the form
of industrial scale fermentation of microorganisms producing
modified OPGL). Furthermore, there is no need to device
purification and refolding schemes for the immunogen. And finally,
since nucleic acid vaccination relies on the biochemical apparatus
of the vaccinated individual in order to produce the expression
product of the nucleic acid introduced, the optimum
posttranslational processing of the expression product is expected
to occur; this is especially important in the case of
autovaccination, since, as mentioned above, a significant fraction
of the original OPGL B-cell epitopes should be preserved in the
modified molecule, and since B-cell epitopes in principle can be
constituted by parts of any (bio)molecule (e.g. carbohydrate,
lipid, protein etc.). Therefore, native glycosylation and
lipidation patterns of the immunogen may very well be of importance
for the overall immunogenicity and this is best ensured by having
the host producing the immunogen.
[0135] Hence, a preferred embodiment of the invention comprises
effecting presentation of modified OPGL to the immune system by
introducing nucleic acid(s) encoding the modified OPGL into the
animal's cells and thereby obtaining in vivo expression by the
cells of the nucleic acid(s) introduced.
[0136] In this embodiment, the introduced nucleic acid is
preferably DNA which can be in the form of naked DNA, DNA
formulated with charged or uncharged lipids, DNA formulated in
liposomes, DNA included in a viral vector, DNA formulated with a
transfection-facilitating protein or polypeptide, DNA formulated
with a targeting protein or polypeptide, DNA formulated with
Calcium precipitating agents, DNA coupled to an inert carrier
molecule, DNA encapsulated in chitin or chitosan, and DNA
formulated with an adjuvant. In this context it is noted that
practically all considerations pertaining to the use of adjuvants
in traditional vaccine formulation apply for the formulation of DNA
vaccines. Hence, all disclosures herein which relate to use of
adjuvants in the context of polypeptide based vaccines apply
mutatis mutandis to their use in nucleic acid vaccination
technology.
[0137] As for routes of administration and administration schemes
of polypeptide based vaccines which have been detailed above, these
are also applicable for the nucleic acid vaccines of the invention
and all discussions above pertaining to routes of administration
and administration schemes for polypeptides apply mutatis mutandis
to nucleic acids. To this should be added that nucleic acid
vaccines can suitably be administered intraveneously and
intraarterially. Furthermore, it is well-known in the art that
nucleic acid vaccines can be administered by use of a so-called
gene gun, and hence also this and equivalent modes of
administration are regarded as part of the present invention.
Finally, also the use of a VLN in the administration of nucleic
acids has been reported to yield good results, and therefore this
particular mode of administration is particularly preferred.
[0138] Furthermore, the nucleic acid(s) used as an immunization
agent can contain regions encoding the 1.sup.st, 2.sup.nd and/or
3.sup.rd moieties, e.g. in the form of the immunomodulating
substances described above such as the cytokines discussed as
useful adjuvants. A preferred version of this embodiment
encompasses having the coding region for the analogue and the
coding region for the immunomodulator in different reading frames
or at least under the control of different promoters. Thereby it is
avoided that the analogue or epitope is produced as a fusion
partner to the immunomodulator. Alternatively, two distinct
nucleotide fragments can be used, but this is less preferred
because of the advantage of ensured co-expression when having both
coding regions included in the same molecule.
[0139] Accordingly, the invention also relates to a composition for
inducing production of antibodies against OPGL, the composition
comprising
[0140] a nucleic acid fragment or a vector of the invention (cf.
the discussion of vectors below), and
[0141] a pharmaceutically and immunologically acceptable vehicle
and/or carrier and/or adjuvant as discussed above.
[0142] Under normal circumstances, the OPGL variant-encoding
nucleic acid is introduced in the form of a vector wherein
expression is under control of a viral promoter. For more detailed
discussions of vectors according to the invention, cf. the
discussion below. Also, detailed disclosures relating to the
formulation and use of nucleic acid vaccines are available, cf.
Donnelly J J et al, 1997, Annu. Rev. Immunol. 15: 617-648 and
Donnelly J J et al., 1997, Life Sciences 60: 163-172. Both of these
references are incorporated by reference herein.
[0143] Live Vaccines
[0144] A third alternative for effecting presentation of modified
OPGL to the immune system is the use of live vaccine technology. In
live vaccination, presentation to the immune system is effected by
administering, to the animal, a non-pathogenic microorganism which
has been transformed with a nucleic acid fragment encoding a
modified OPGL or with a vector incorporating such a nucleic acid
fragment. The non-pathogenic microorganism can be any suitable
attenuated bacterial strain (attenuated by means of passaging or by
means of removal of pathogenic expression products by recombinant
DNA technology), e.g. Mycobacterium bovis BCG., non-pathogenic
Streptococcus spp., E. coli, Salmonella spp., Vibrio cholerae,
Shigella, etc. Reviews dealing with preparation of state-of-the-art
live vaccines can e.g. be found in Saliou P, 1995, Rev. Prat. 45:
1492-1496 and Walker P D, 1992, Vaccine 10: 977-990, both
incorporated by reference herein. For details about the nucleic
acid fragments and vectors used in such live vaccines, cf. the
discussion below.
[0145] As an alternative to bacterial live vaccines, the nucleic
acid fragment of the invention discussed below can be incorporated
in a non-virulent viral vaccine vector such as a vaccinia strain or
any other suitable pox virus.
[0146] Normally, the non-pathogenic microorganism or virus is
administered only once to the animal, but in certain cases it may
be necessary to administer the microorganism more than once in a
lifetime in order to maintain protective immunity. It is even
contemplated that immunization schemes as those detailed above for
polypeptide vaccination will be useful when using live or virus
vaccines.
[0147] Alternatively, live or virus vaccination is combined with
previous or subsequent polypeptide and/or nucleic acid vaccination.
For instance, it is possible to effect primary immunization with a
live or virus vaccine followed by subsequent booster immunizations
using the polypeptide or nucleic acid approach.
[0148] The microorganism or virus can be transformed with nucleic
acid(s) containing regions encoding the 1.sup.st, 2.sup.nd and/or
3.sup.rd moieties, e.g. in the form of the immunomodulating
substances described above such as the cytokines discussed as
useful adjuvants. A preferred version of this embodiment
encompasses having the coding region for the analogue and the
coding region for the immunomodulator in different reading frames
or at least under the control of different promoters. Thereby it is
avoided that the analogue or epitopes are produced as fusion
partners to the immunomodulator. Alternatively, two distinct
nucleotide fragments can be used as transforming agents. Of course,
having the 1.sup.st and/or 2.sup.nd and/or 3.sup.rd moieties in the
same reading frame can provide as an expression product, an
analogue of the invention, and such an embodiment is especially
preferred according to the present invention.
[0149] Use of the Method of the Invention in Disease Treatment
[0150] As will be appreciated from the discussions above, the
provision of the method of the invention allows for control of
diseases characterized by excessive loss of bone mass. In this
context, the disease osteoporosis is the key target for the
inventive method but also bone loss associated with complicated
bone fractures is a feasible target for treatment/amelioration.
Hence, an important embodiment of the method of the invention for
down-regulating OPGL activity comprises treating and/or preventing
and/or ameliorating osteoporosis or other conditions characterized
by excess bone resorption, the method comprising down-regulating
OPGL activity according to the method of the invention to such an
extent that the rate of bone resorption is significantly
decreased
[0151] In the present context such a significant decrease in bone
resorption is at least 3% compared to the pathological rate, but
higher percentages are contemplated, such as at least 5%, at least
7%, at least 9%, at least 11%, at least 13%, at least 15%, and at
least 17%, but even higher percentages are expected, such as at
least 20%, or even at least 30%. It is especially preferred that
the decrease in bone resorption results in an inversion of the
balance between bone formation and bone resorption, i.e. that the
rate of bone formation is brought to exceed the rate of bone
resorption. Of course, this imbalance should not be maintained
(since it would result in osteopetrosis), but by carefully
controlling the number and immunological impact of immunizations of
the individual in need thereof it is possible to obtain a balance
over time which results in a net conservation of bone mass.
Alternatively, if in an individual the method of the invention
cannot terminate bone loss, the method of the invention can
(optionally in combination with other known methods for reducing
bone loss in osteoporosis patients) be used to obtain a significant
reduction in bone loss, thereby prolonging the time where
sufficient bone mass is present in the individual.
[0152] Methods for measuring the rate of bone resorption and bone
formation are known in the art. It is by means of biochemical
assays possible to gauge the rate of bone resorption by measuring
the blood concentration of certain fragments of collagen type I
(cf. WO 93/15107 and WO 94/14844). Alternatively, the rate of bone
loss can be assessed by physical means; representative disclosures
in the art of methods for assessing bone mass by non-invasive,
physical methods can be found in WO 88/06862, WO 94/12855, WO
95/14431, and WO 97/00643.
[0153] Peptides, Polypeptides, and Compositions of the
Invention
[0154] As will be apparent from the above, the present invention is
based on the concept of immunising individuals against the OPGL
antigen in order to indirectly obtain a reduced osteoclast
activity. The preferred way of obtaining such an immunization is to
use modified versions of OPGL, thereby providing molecules which
have not previously been disclosed in the art.
[0155] It is believed that the modified OPGL molecules discussed
herein are inventive in their own right, and therefore an important
part of the invention pertains to an OPGL analogue which is derived
from an animal OPGL wherein is introduced a modification which has
as a result that immunization of the animal with the analogue
induces production of antibodies reacting specifically with the
unmodified OPGL polypeptide. Preferably, the nature of the
modification conforms with the types of modifications described
above when discussing various embodiments of the method of the
invention when using modified OPGL. Hence, any disclosure presented
herein pertaining to modified OPGL molecules are relevant for the
purpose of describing the OPGL analogues of the invention, and any
such disclosures apply mutatis mutandis to the description of these
analogues.
[0156] It should be noted that preferred modified OPGL molecules
comprises modifications which results in a polypeptide having a
sequence identity of at least 70% with OPGL or with a subsequence
thereof of at least 10 amino acids in length. Higher sequence
identities are preferred, e.g. at least 75% or even at least 80,
85, 90, or 95%. The sequence identity for proteins and nucleic
acids can be calculated as (N.sub.ref-N.sub.dif).mul-
tidot.100/N.sub.ref, wherein N.sub.dif is the total number of
non-identical residues in the two sequences when aligned and
wherein N.sub.ref is the number of residues in one of the
sequences. Hence, the DNA sequence AGTCAGTC will have a sequence
identity of 75% with the sequence AATCAATC (N.sub.dif=2 and
N.sub.ref=8).
[0157] The invention also pertains to compositions useful in
exercising the method of the invention. Hence, the invention also
relates to an immunogenic composition comprising an immunogenically
effective amount of an OPGL polypeptide which is a self-protein in
an animal, said OPGL polypeptide being formulated together with an
immunologically acceptable adjuvant so as to break the animal's
autotolerance towards the OPGL polypeptide, the composition further
comprising a pharmaceutically and immunologically acceptable
diluent and/or vehicle and/or carrier and/or excipient. In other
words, this part of the invention pertains to the formulations of
naturally occurring OPGL polypeptides which have been described in
connection with embodiments of the method of the invention.
[0158] The invention also relates to an immunogenic composition
comprising an immunologically effective amount of an OPGL analogue
defined above, said composition further comprising a
pharmaceutically and immunologically acceptable diluent and/or
vehicle and/or carrier and/or excipient and optionally an adjuvant.
In other words, this part of the invention concerns formulations of
modified OPGL, essentially as described above. The choice of
adjuvants, carriers, and vehicles is accordingly in line with what
has been discussed above when referring to formulation of modified
and unmodified OPGL for use in the inventive method for the
down-regulation of OPGL.
[0159] The polypeptides are prepared according to methods
well-known in the art. Longer polypeptides are normally prepared by
means of recombinant gene technology including introduction of a
nucleic acid sequence encoding the OPGL analogue into a suitable
vector, transformation of a suitable host cell with the vector,
expression of the nucleic acid sequence, recovery of the expression
product from the host cells or their culture supernatant, and
subseqeunt purification and optional further modification, e.g.
refolding or derivatization.
[0160] Shorter peptides are preferably prepared by means of the
well-known techniques of solid- or liquid-phase peptide synthesis.
However, recent advances in this technology has rendered possible
the production of full-length polypeptides and proteins by these
means, and therefore it is also within the scope of the present
invention to prepare the long constructs by synthetic means.
[0161] Nucleic Acid Fragments and Vectors of the Invention
[0162] It will be appreciated from the above disclosure that
modified OPGL polypeptides can be prepared by means of recombinant
gene technology but also by means of chemical synthesis or
semisynthesis; the latter two options are especially relevant when
the modification consists in coupling to protein carriers (such as
KLH, diphtheria toxoid, tetanus toxoid, and BSA) and
non-proteinaceous molecules such as carbohydrate polymers and of
course also when the modification comprises addition of side chains
or side groups to an -OPGL polypeptide-derived peptide chain.
[0163] For the purpose of recombinant gene technology, and of
course also for the purpose of nucleic acid immunization, nucleic
acid fragments encoding modified OPGL are important chemical
products. Hence, an important part of the invention pertains to a
nucleic acid fragment which encodes an OPGL analogue, i.e. an OPGL
derived polypeptide which either comprises the natural OPGL
sequence to which has been added or inserted a fusion partner or,
preferably an OPGL derived polypeptide wherein has been introduced
a foreign T-cell epitope by means of insertion and/or addition,
preferably by means of substitution and/or deletion. The nucleic
acid fragments of the invention are either DNA or RNA
fragments.
[0164] The nucleic acid fragments of the invention will normally be
inserted in suitable vectors to form cloning or expression vectors
carrying the nucleic acid fragments of the invention; such novel
vectors are also part of the invention. Details concerning the
construction of these vectors of the invention will be discussed in
context of transformed cells and microorganisms below. The vectors
can, depending on purpose and type of application, be in the form
of plasmids, phages, cosmids, mini-chromosomes, or virus, but also
naked DNA which is only expressed transiently in certain cells is
an important vector. Preferred cloning and expression vectors of
the invention are capable of autonomous replication, thereby
enabling high copy-numbers for the purposes of high-level
expression or high-level replication for subsequent cloning.
[0165] The general outline of a vector of the invention comprises
the following features in the 5'.cndot..cndot.3' direction and in
operable linkage: a promoter for driving expression of the nucleic
acid fragment of the invention, optionally a nucleic acid sequence
encoding a leader peptide enabling secretion (to the extracellular
phase or, where applicable, into the periplasma) of or integration
into the membrane of the polypeptide fragment, the nucleic acid
fragment of the invention, and optionally a nucleic acid sequence
encoding a terminator. When operating with expression vectors in
producer strains or cell-lines it is for the purposes of genetic
stability of the transformed cell preferred that the vector when
introduced into a host cell is integrated in the host cell genome.
In contrast, when working with vectors to be used for effecting in
vivo expression in an animal (i.e. when using the vector in DNA
vaccination) it is for security reasons preferred that the vector
is incapable of being integrated in the host cell genome;
typically, naked DNA or non-integrating viral vectors are used, the
choices of which are well-known to the person skilled in the
art
[0166] The vectors of the invention are used to transform host
cells to produce the modified OPGL polypeptide of the invention.
Such transformed cells, which are also part of the invention, can
be cultured cells or cell lines used for propagation of the nucleic
acid fragments and vectors of the invention, or used for
recombinant production of the modified OPGL polypeptides of the
invention. Alternatively, the transformed cells can be suitable
live vaccine strains wherein the nucleic acid fragment (one single
or multiple copies) have been inserted so as to effect secretion or
integration into the bacterial membrane or cell-wall of the
modified OPGL.
[0167] Preferred transformed cells of the invention are
microorganisms such as bacteria (such as the species Escherichia
[e.g. E. coli], Bacillus [e.g. Bacillus subtilis], Salmonella, or
Mycobacterium [preferably non-pathogenic, e.g. M. bovis BCG]),
yeasts (such as Saccharomyces cerevisiae), and protozoans.
Alternatively, the transformed cells are derived from a
multicellular organism such as a fungus, an insect cell, a plant
cell, or a mammalian cell. Most preferred are cells derived from a
human being, cf. the discussion of cell lines and vectors below.
Recent results have shown great promise in the use of a
commercially available Drosophila melanogaster cell line (the
Schneider 2 (S.sub.2)cell line and vector system available from
Invitrogen) for the recombinant production of polypeptides in
applicants' lab, and therefore this expression system is
particularly preferred.
[0168] For the purposes of cloning and/or optimized expression it
is preferred that the transformed cell is capable of replicating
the nucleic acid fragment of the invention. Cells expressing the
nucleic fragment are preferred useful embodiments of the invention;
they can be used for small-scale or large-scale preparation of the
modified OPGL or, in the case of non-pathogenic bacteria, as
vaccine constituents in a live vaccine.
[0169] When producing the modified OPGL of the invention by means
of transformed cells, it is convenient, although far from
essential, that the expression product is either exported out into
the culture medium or carried on the surface of the transformed
cell.
[0170] When an effective producer cell has been identified it is
preferred, on the basis thereof, to establish a stable cell line
which carries the vector of the invention and which expresses the
nucleic acid fragment encoding the modified OPGL. Preferably, this
stable cell line secretes or carries the OPGL analogue of the
invention, thereby facilitating purification thereof.
[0171] In general, plasmid vectors containing replicon and control
sequences which are derived from species compatible with the host
cell are used in connection with the hosts. The vector ordinarily
carries a replication site, as well as marking sequences which are
capable of providing phenotypic selection in transformed cells. For
example, E. coli is typically transformed using pBR322, a plasmid
derived from an E. coli species (see, e.g., Bolivar et al., 1977).
The pBR322 plasmid contains genes for ampicillin and tetracycline
resistance and thus provides easy means for identifying transformed
cells. The pBR plasmid, or other microbial plasmid or phage must
also contain, or be modified to contain, promoters which can be
used by the prokaryotic microorganism for expression.
[0172] Those promoters most commonly used in recombinant DNA
construction include the B-lactamase (penicillinase) and lactose
promoter systems (Chang et al., 1978; Itakura et al., 1977; Goeddel
et al., 1979) and a tryptophan (trp) promoter system (Goeddel et
al., 1979; EP-A-0 036 776). While these are the most commonly used,
other microbial promoters have been discovered and utilized, and
details concerning their nucleotide sequences have been published,
enabling a skilled worker to ligate them functionally with plasmid
vectors (Siebwenlist et al., 1980). Certain genes from prokaryotes
may be expressed efficiently in E. coli from their own promoter
sequences, precluding the need for addition of another promoter by
artificial means.
[0173] In addition to prokaryotes, eukaryotic microbes, such as
yeast cultures may also be used, and here the promoter should be
capable of driving expression. Saccharomyces cerevisiase, or common
baker's yeast is the most commonly used among eukaryotic
microorganisms, although a number of other strains are commonly
available. For expression in Saccharomyces, the plasmid YRp7, for
example, is commonly used (Stinchcomb et al., 1979; Kingsman et
al., 1979; Tschemper et al., 1980). This plasmid already contains
the trpl gene which provides a selection marker for a mutant strain
of yeast lacking the ability to grow in tryptophan for example ATCC
No. 44076 or PEP4-l (Jones, 1977). The presence of the trpl lesion
as a characteristic of the yeast host cell genome then provides an
effective environment for detecting transformation by growth in the
absence of tryptophan.
[0174] Suitable promoting sequences in yeast vectors include the
promoters for 3-phosphoglycerate kinase (Hitzman et al., 1980) or
other glycolytic enzymes (Hess et al., 1968; Holland et al., 1978),
such as enolase, glyceraldehyde-3-phosphate dehydrogenase,
hexokinase, pyruvate decarboxylase, phosphofructokinase,
glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate
kinase, triosephosphate isomerase, phosphoglucose isomerase, and
glucokinase. In constructing suitable expression plasmids, the
termination sequences associated with these genes are also ligated
into the expression vector 3' of the sequence desired to be
expressed to provide polyadenylation of the mRNA and
termination.
[0175] Other promoters, which have the additional advantage of
transcription controlled by growth conditions are the promoter
region for alcohol dehydrogenase 2, isocytochrome C, acid
phosphatase, degradative enzymes associated with nitrogen
metabolism, and the aforementioned glyceraldehyde-3-phosphate
dehydrogenase, and enzymes responsible for maltose and galactose
utilization. Any plasmid vector containing a yeast-compatible
promoter, origin of replication and termination sequences is
suitable.
[0176] In addition to microorganisms, cultures of cells derived
from multicellular organisms may also be used as hosts. In
principle, any such cell culture is workable, whether from
vertebrate or invertebrate culture. However, interest has been
greatest in vertebrate cells, and propagation of vertebrate in
culture (tissue culture) has become a routine procedure in recent
years (Tissue Culture, 1973). Examples of such useful host cell
lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell
lines, and W138, BHK, COS-7 293, Spodoptera frugiperda (SF) cells
(commercially available as complete expression systems from i.a.
Protein Sciences, 1000 Research Parkway, Meriden, Conn. 06450,
U.S.A. and from Invitrogen), and MDCK cell lines. In the present
invention, an especially preferred cell line is S.sub.2 available
from Invitrogen, PO Box 2312, 9704 CH Groningen, The
Netherlands.
[0177] Expression vectors for such cells ordinarily include (if
necessary) an origin of replication, a promoter located in front of
the gene to be expressed, along with any necessary ribosome binding
sites, RNA splice sites, polyadenylation site, and transcriptional
terminator sequences.
[0178] For use in mammalian cells, the control functions on the
expression vectors are often provided by viral material. For
example, commonly used promoters are derived from polyoma,
Adenovirus 2, and most frequently Simian Virus 40 (SV40). The early
and late promoters of SV40 virus are particularly useful because
both are obtained easily from the virus as a fragment which also
contains the SV40 viral origin of replication (Fiers et al., 1978).
Smaller or larger SV40 fragments may also be used, provided there
is included the approximately 250 bp sequence extending from the
HindIII site toward the BglI site located in the viral origin of
replication. Further, it is also possible, and often desirable, to
utilize promoter or control sequences normally associated with the
desired gene sequence, provided such control sequences are
compatible with the host cell systems.
[0179] An origin of replication may be provided either by
construction of the vector to include an exogenous origin, such as
may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV,
BPV) or may be provided by the host cell chromosomal replication
mechanism. If the vector is integrated into the host cell
chromosome, the latter is often sufficient.
[0180] Identification of Useful OPGL Analogues
[0181] It will be clear to the skilled person that not all possible
variants or modifications of native OPGL will have the ability to
elicit antibodies in an animal which are cross-reactive with the
native form. It is, however, not difficult to set up an effective
standard screen for modified OPGL molecules which fulfill the
minimum requirements for immunological reactivity discussed herein.
Hence, another part of the invention concerns a method for the
identification of a modified OPGL polypeptide which is capable of
inducing antibodies against unmodified OPGL in an animal species
where the unmodified OPGL polypeptide is a (non-immunogenic)
self-protein, the method comprising
[0182] preparing, by means of peptide synthesis or genetic
engineering techniques, a set of mutually distinct modified OPGL
polypeptides wherein amino acids have been added to, inserted in,
deleted from, or substituted into the amino acid sequence of an
OPGL polypeptide of the animal species thereby giving rise to amino
acid sequences in the set which comprise T-cell epitopes which are
foreign to the animal species, or preparing a set of nucleic acid
fragments encoding the set of mutually distinct modified OPGL
polypeptides,
[0183] testing members of the set of modified OPGL polypeptides or
nucleic acid fragments for their ability to induce production of
antibodies by the animal species against the unmodified OPGL,
and
[0184] identifying and optionally isolating the member(s) of the
set of modified OPGL polypeptides which significantly induces
antibody production against unmodified OPGL in the species or
identifying and optionally isolating the polypeptide expression
products encoded by members of the set of nucleic acid fragments
which significantly induces antibody production against unmodified
OPGL in the animal species.
[0185] In this context, the "set of mutually distinct modified OPGL
polypeptides" is a collection of non-identical modified OPGL
polypeptides which have e.g. been selected on the basis of the
criteria discussed above (e.g. in combination with studies of
circular dichroism, NMR spectra, and/or X-ray diffraction
patterns). The set may consist of only a few members but it is
contemplated that the set may contain several hundred members.
[0186] The test of members of the set can ultimately be performed
in vivo, but a number of in vitro tests can be applied which narrow
down the number of modified molecules which will serve the purpose
of the invention.
[0187] Since the goal of introducing the foreign T-cell epitopes is
to support the B-cell response by T-cell help, a prerequisite is
that T-cell proliferation is induced by the modified OPGL. T-cell
proliferation can be tested by standardized proliferation assays in
vitro. In short, a sample enriched for T-cells is obtained from a
subject and subsequently kept in culture. The cultured T-cells are
contacted with APCs of the subject which have previously taken up
the modified molecule and processed it to present its T-cell
epitopes. The proliferation of T-cells is monitored and compared to
a suitable control (e.g. T-cells in culture contacted with APCs
which have processed intact, native OPGL). Alternatively,
proliferation can be measured by determining the concentration of
relevant cytokines released by the T-cells in response to their
recognition of foreign T-cells.
[0188] Having rendered highly probable that at least one modified
OPGL of either type of set is capable of inducing antibody
production against OPGL, it is possible to prepare an immunogenic
composition comprising at least one modified OPGL polypeptide which
is capable of inducing antibodies against unmodified OPGL in an
animal species where the unmodified OPGL polypeptide is a
self-protein, the method comprising admixing the member(s) of the
set which significantly induces production of antibodies in the
animal species which are reactive with OPGL with a pharmaceutically
and immunologically acceptable carrier and/or vehicle and/or
diluent and/or excipient, optionally in combination with at least
one pharmaceutically and immunologically acceptable adjuvant.
[0189] The above aspects of the invention pertaining to test of
polypeptide sets are conveniently carried out by initially
preparing a number of mutually distinct nucleic acid sequences or
vectors of the invention, inserting these into appropriate
expression vectors, transforming suitable host cells with the
vectors, and expressing the nucleic acid sequences of the
invention. These steps can be followed by isolation of the
expression products. It is preferred that the nucleic acid
sequences and/or vectors are prepared by methods comprising
exercise of a molecular amplification technique such as PCR or by
means of nucleic acid synthesis.
[0190] Another part of the invention concerns a method for the
treatment, prophylaxis or amelioration of diseases characterized by
excess bone resorption in an animal, including a human being, the
method comprising administering, to the animal, an effective amount
of at least one substance different from osteoprotegerin which
blocks the stimulatory effect of OPGL on osteoclast activity. It is
presently believed that such an approach has never been suggested
in the art.
[0191] The preferred embodiment of this part of the invention
involves use of an OPGL-specific antibody (poly- or monoclonal) or
a specifically binding variant thereof as the substance blocking
the stimulatory effect of OPGL. It is preferred that the antibody
is an IgG or IgM molecule, or that the specifically binding varian
is derived from IgG or IgM. The specifically binding variant of the
antibody can conveniently be a Fab fragment, a F(ab').sub.2
fragment, a humanized monoclonal anti-body or fragment thereof, or
a di- or multimeric antibody fragment such as a diabody (a
bispecific and dimeric artificial antibody-derived molecule
produced by Cambridge Antibody Technology).
EXAMPLE
[0192] It has been decided to clone or synthesize cDNAs encoding
murine and human OPGL in the truncated version comprising amino
acid residues 158-316 in the murine case and residues 159-317 in
the human case (numbers correspond to the numbering in SEQ ID NOs:
2 and 4, respectively). As these truncated versions exhibit
biological activity, it is logical to direct the autoantibodies
against this part of OPGL. In addition, it makes the proteins
smaller and thus easier to handle.
[0193] A synthetic cDNA encoding the murine OPGL residues 158-316
has been synthesized removing sub-optimal Eschericia coli and
Pichia pastoris codons from the published sequence. Additionally,
an N-terminal Histidine tag, part of the cleavage site of the alpha
mating factor signal sequence from Sacharomyces cerevisiae, and
suitable restriction enzymes have been incorporated into the open
reading frame (cf. SEQ ID NO: 7).
[0194] This cDNA encoding wild type murine OPGL has been cloned
into a standard Eschericia coli expression vector (pTrc99a) using
BspHI and HindIII restriction enzymes and a standard cloning vector
(pBluescript KS+) using SacI and KpnI restriction enzymes (yielding
SEQ ID NO: 9).
[0195] Expression in Eschericia coli cells resulted in
approximately 30% recombinant OPGL of the total Eschericia coli
protein. The protein has been refolded and purified using the
following procedure:
[0196] 1. Cells are harvested by centrifugation.
[0197] 2. Cells are resuspended in phosphate buffered saline (PBS)
and recentrifuged.
[0198] 3. The supernatant is discarded and the cells are
resuspended in three volumes (100 mM
Tris[hydroxymethyl]aminomethane hydrochloride, 5 mM dithiotreitol
(DTT), 0.5 M NaCl, pH 8.0).
[0199] 4. The cells are added 8 .mu.l 50 mM PMSF and 80 .mu.l
lysozyme (10 mg/ml) per gram cell and incubated at room temperature
for 20 min.
[0200] 5. For each gram cell pellet, 4 mg deoxychloric acid is
added, and the suspension is incubated at 37.degree. C. until it
appears viscous.
[0201] 6. 20 .mu.l DNase (1 mg/ml) pr. gram cell weight is added,
and MgCl.sub.2 to 5 mM, and the suspension is incubated at room
temperature for 30 min.
[0202] 7. The suspension is sonicated on ice until the viscosity
disappears.
[0203] 8. After centrifugation (20000.times.g for 30 min) the
pellet is resuspended in H.sub.2O, recentrifuged and resuspended in
3 ml 1 M urea per gram cell weight.
[0204] 9. After centrifugation (20000.times.g for 30 min) the
pellet is resuspended in 1 M Guanidine hydrochloride, 100 mM
Tris[hydroxymethyl]aminomethane hydrochloride, pH 7.5.
[0205] 10. After centrifugation (20000.times.g for 30 min) the
pellet is resuspended in 6 M Guanidine hydrochloride, 20 mM
Tris[hydroxymethyl]amin- omethane hydrochloride, 5% ethanol, 1%
beta-mercaptoethanol, pH 8.0, and stirred at 4.degree. C.
overnight.
[0206] 11. After centrifugation (40000.times.g for 1-4 hours) the
supernatant is filtered and stored at -20.degree. C.
[0207] 12. The solubilized inclusion bodies are separated by gel
filtration chromatography using Superdex 200 material
(Pharmacia).
[0208] 13. The fractions containing the recombinant OPGL are pooled
and diluted to 0.1 mg/ml with 1,5M Guanidine hydrochloride, mM
Tris[hydroxymethyl]aminomethane hydrochloride, 1 mM DTT, pH
7.5.
[0209] 14. The material is dialyzed overnight at 4.degree. C.
against 10 volumes 1,5M Guanidine hydrochloride, 20 mM
Tris[hydroxymethyl]aminometha- ne hydrochloride, 1 mM DTT, pH
7.5
[0210] 15. The material is dialyzed overnight at 4.degree. C.
against volumes 1,0 M Guanidine hydrochloride, 20 mM
Tris[hydroxymethyl]aminometh- ane hydrochloride, 1 mM DTT, pH
7.5
[0211] 16. The material is dialyzed overnight at 4.degree. C.
against 10 volumes 0,5 M Guanidine hydrochloride, 20 mM
Tris[hydroxymethyl]aminometh- ane hydrochloride, 1 mM DTT, pH
7.5
[0212] 17. The material is dialyzed overnight at 4.degree. C.
against 10 volumes 20 mM Tris[hydroxymethyl]aminomethane
hydrochloride, 150 mM Arginine, 1 mM DTT, pH 7.5
[0213] 18. The material is dialyzed overnight at 4.degree. C.
against 10 volumes 20 mM Tris[hydroxymethyl]aminomethane
hydrochloride, 150 mM Arginine, pH 7.5
[0214] 19. The refolded material is freeze dried and stored at
-20.degree. C.
[0215] The efficiency of refolding using this procedure is
approximately 40%, and the purity in excess of 65%. The
purification procedure and refolding process are still subject to
further improvements. Immobilized refolding are under
investigation, and enzymatic removal of the Histidine-tag will e
performed essentially as described by Pedersen et al., 1999. The
nature of the recombinant protein has been characterized and
verified using SDS-PAGE, N-terminal sequencing, amino acid
analysis, and mass spectrometry.
[0216] A cysteine substitution mutant of the wild type murine OPGL
is under construction (wherein a cysteine corresponding to amino
acid residue in SEQ ID NO: 4 is substituted with serine; cf. SEQ ID
NOs: 11 and 12). This is done to eliminate potential stability
problems with the purified recombinant protein. This mutated OPGL
truncate will serve as basis for vaccine constructs in complete
analogy with the description below which sets out from the DNA
having SEQ ID NO: 9. Further, a corresponding Cys.cndot..cndot.Ser
mutant (where Cys-221 is substituted) of human OPGL will also be
produced for the same purposes.
[0217] The vaccine molecules are initially constructed by insertion
or in-substitution of either the P2 or P30 epitope from tetanus
toxoid at selected positions. Other suitable immunodominant T-cell
epitopes may be used at a later stage.
[0218] The selected positions for the introduction of variation are
chosen based on knowledge of existing or predicted B-cell epitopes
and predicted secondary structure elements of the native molecule,
as well as using alignments with the existing three dimensional
structures of TNF.alpha. (1a8m.pdb) and CD40 ligand (1aly.pdb) for
modelling the secondary and tertiary structure of the extracellular
part of OPGL. The introduction in the murine molecule will take
place in areas corresponding to amino acid residues 170-192,
198-218, 221-246, 256-261, and 285-316 (cf. the amino acid
numbering in SEQ ID NO: 4), whereas the introduction in the human
molecule will take place in areas corresponding to amino acid
residues 171-193, 199-219, 222-247, 257-262, and 286-317.
[0219] Four variants of murine OPGL have by now been constructed
and expressed recombinantly in Eschericia coli:
[0220] DNA Encoding mOPGL[158-316]_P30[256-261] with an N-terminal
Histidine Tag (SEQ ID NO: 13):
[0221] PCR of SEQ ID NO: 9 was performed using SEQ ID NOs: 22 and
25 as primers. The resulting PCR fragment was restriction digested
with SacII and KpnI and subsequently purified from an agarose gel.
A second PCR using SEQ ID NO: 9 as template was performed using
primer SEQ ID NO: 26 and a vector specific primer. The resulting
PCR fragment was restriction digested with KpnI and HindIII. Both
fragments were then ligated to SEQ ID NO: 9 in pBluescript KS+
restriction digested with SacII and HindIII. To correct a single
base mutation in this construct, PCR using the construct as
template was performed with primers SEQ ID NOs: 33 and 29. The
resulting PCR fragment was restriction digested with PstI+ EcoRI,
gel purified and subsequently ligated to the erroneous construct
digested with PstI and EcoRI. The verified construct (SEQ ID NO:
13) was then transferred to pTrc99a using BspHI and HindIII
restriction enzymes.
[0222] DNA Encoding mOPGL[158-316]_P2[256-261] with an N-terminal
Histidine Tag (SEQ ID NO: 15):
[0223] PCR was performed using primers SEQ ID NOs: 27 and 28
without template. The resulting PCR fragment was restriction
digested with PstI and EcoRI and subsequently purified from an
agarose gel. The resulting fragment was then ligated to SEQ ID NO:
9 in pBluescript KS+ restriction digested with SacII and HindIII.
The verified construct (SEQ ID NO: 15) was subsequently transferred
to pTrc99a using BspHI and HindIII restriction enzymes
[0224] DNA Encoding mOPGL[158-316]_P2[288-302] with an N-terminal
Histidine Tag (SEQ ID NO: 17):
[0225] PCR of SEQ ID NO: 9 was performed using primers SEQ ID NOs:
22 and 29. The resulting PCR fragment was restriction digested with
PstI and BstBI and subsequently purified from an agarose gel. A
second PCR using SEQ ID NO: 9 as template was performed using
primer SEQ ID NO: 30 and a vector specific primer. The resulting
PCR fragment was restriction digested with BstBI and KpnI and
subsequently gel purified. Both fragments were then ligated to SEQ
ID NO: 9 in pBluescript KS+ restriction digested with PstI and
KpnI. The verified construct (SEQ ID NO: 17) was then transferred
to pTrc99a using BspHI and HindIII restriction enzymes.
[0226] DNA Encoding mOPGL[158-316]_P30[221-241] with an N-terminal
Histidine Tag (SEQ ID NO: 19):
[0227] PCR of SEQ ID NO: 9 was performed using primers SEQ ID NOs:
22 and 23. The resulting PCR fragment was restriction digested with
SacII and KpnI and subsequently purified from an agarose gel. A
second PCR using SEQ ID NO: 9 as template was performed using
primer SEQ ID NOs: 24 and 31. The PCR fragment was restriction
digested with KpnI and EcoRI and subsequently gel purified. Both
fragments were then ligated to SEQ ID NO: 9 in pBluescript KS+
restriction digested with SacII and EcoRI. The verified construct
(SEQ ID NO: 19) was then transferred to pTrc99a using BspHI and
HindIII restriction enzymes.
[0228] Expression of these truncated variants of OPGL have taken
place in E. coli yielding over 20% of total protein for all
variants. A further development of the above-described purification
and refolding procedure for the wild type protein (SEQ ID NO: 9)
will be performed. This procedure will serve as a basis for the
development of optimal procedures for each of the variants.
Immobilized refolding of the variant proteins utilizing the
Histidine tag is another approach that is being pursued.
[0229] Alternatively, the variants can be directly transferred to
Pichia pastoris expression vectors using restriction enzymes, or
other yeast expression systems using PCR if glycosylation is
desired. It should be noted that the glycosylation is not needed
for biological activity in vivo of OPGL. It is also possible to
express the truncated OPGL in human 293 fibro blasts as reported in
Lacey et al. Expression in insect cells will also be considered
(e.g. the Schneider 2 (S.sub.2)cell line and vector system or the
Spodoptera frugiperda (SF) cell and vector systems, both available
from Invitrogen).
[0230] The purified variants will be used for antibody production
in rabbits for later use as detection tools as there exist no
commercially available antibodies. In addition, this material will
be a very valuable tool in the biological assays needed to evaluate
the autovaccine candidates. The preparation of the antibodies will
be performed using standard methods known in the art.
[0231] Selecting the best autovaccine candidate is based on
assessment of inhibitory activity in in vitro assays for osteoclast
maturation/activation or in in vivo animal models for osteoporosis.
Useful assay and model systems are described in the literature
(e.g. in Lacey et al., Fuller et al., and Simonet et al.).
[0232] The activity of the recombinant proteins will be analyzed by
intravenous injection of 100 .mu.l into un-anaesthetized male
Balb/C mice (0, 0.1, and 1.0 mg protein pr. kg mouse) and one hour
later withdrawal of 125 .mu.l blood from the major eye vein using
capillary tubes coated with calcium saturated heparin. The calcium
levels are measured using an ICA2 (Radiometer, Denmark). Purified,
and refolded recombinant murine OPGL (SEQ ID NO: 9) is reactive in
this assay, increasing the circulating levels of ionized calcium by
up to 10%.
[0233] The autovaccine candidates will e.g. be evaluated using
autovaccination and subsequent monitoring of their inhibition of
the release of ionized calcium to peripheral blood upon injection
of recombinant mOPGL into mice (as described by Burgess et
al.).
[0234] It should be noted that as an alternative to modified OPGL,
antiidiotypic antibodies directed against the idiotype of an
anti-OPGL antibody will also serve as useful immunogens within the
scope of the present invention. Likewise, the use of mimotypic
polypeptides which can be isolated in e.g. a phage display system
using anti-OPGL or osteoprotegerin as catching probe are also
considered as part of the immunogens of the invention.
List of References
[0235] 1. Bucay, N. et al. (1998), Genes Dev. 12, 1260-1268.
[0236] 2. Lacey, D. L. et al. (1998), Cell 93, 165-176.
[0237] 3. Marks, S. C., Jr. (1989), Am. J. Med. Genet. 34,
43-53.
[0238] 4. Simonet, W. S. et al. (1997), Cell 89, 309-319.
[0239] 5. Fuller, K. et al. (1998), J. Exp. Med. 188, 997-1001.
[0240] 6. Burgess, T. L. et al. (1999), J. Cell Biol. 145,
527-538.
[0241] 7. Pedersen J et al. (1999) Protein Expr. Purif. 15,
389-400.
Sequence CWU 1
1
35 1 2271 DNA Homo sapiens CDS (185)..(1138) 1 aagcttggta
ccgagctcgg atccactact cgacccacgc gtccgcgcgc cccaggagcc 60
aaagccgggc tccaagtcgg cgccccacgt cgaggctccg ccgcagcctc cggagttggc
120 cgcagacaag aaggggaggg agcgggagag ggaggagagc tccgaagcga
gagggccgag 180 cgcc atg cgc cgc gcc agc aga gac tac acc aag tac ctg
cgt ggc tcg 229 Met Arg Arg Ala Ser Arg Asp Tyr Thr Lys Tyr Leu Arg
Gly Ser 1 5 10 15 gag gag atg ggc ggc ggc ccc gga gcc ccg cac gag
ggc ccc ctg cac 277 Glu Glu Met Gly Gly Gly Pro Gly Ala Pro His Glu
Gly Pro Leu His 20 25 30 gcc ccg ccg ccg cct gcg ccg cac cag ccc
ccc gcc gcc tcc cgc tcc 325 Ala Pro Pro Pro Pro Ala Pro His Gln Pro
Pro Ala Ala Ser Arg Ser 35 40 45 atg ttc gtg gcc ctc ctg ggg ctg
ggg ctg ggc cag gtt gtc tgc agc 373 Met Phe Val Ala Leu Leu Gly Leu
Gly Leu Gly Gln Val Val Cys Ser 50 55 60 gtc gcc ctg ttc ttc tat
ttc aga gcg cag atg gat cct aat aga ata 421 Val Ala Leu Phe Phe Tyr
Phe Arg Ala Gln Met Asp Pro Asn Arg Ile 65 70 75 tca gaa gat ggc
act cac tgc att tat aga att ttg aga ctc cat gaa 469 Ser Glu Asp Gly
Thr His Cys Ile Tyr Arg Ile Leu Arg Leu His Glu 80 85 90 95 aat gca
gat ttt caa gac aca act ctg gag agt caa gat aca aaa tta 517 Asn Ala
Asp Phe Gln Asp Thr Thr Leu Glu Ser Gln Asp Thr Lys Leu 100 105 110
ata cct gat tca tgt agg aga att aaa cag gcc ttt caa gga gct gtg 565
Ile Pro Asp Ser Cys Arg Arg Ile Lys Gln Ala Phe Gln Gly Ala Val 115
120 125 caa aag gaa tta caa cat atc gtt gga tca cag cac atc aga gca
gag 613 Gln Lys Glu Leu Gln His Ile Val Gly Ser Gln His Ile Arg Ala
Glu 130 135 140 aaa gcg atg gtg gat ggc tca tgg tta gat ctg gcc aag
agg agc aag 661 Lys Ala Met Val Asp Gly Ser Trp Leu Asp Leu Ala Lys
Arg Ser Lys 145 150 155 ctt gaa gct cag cct ttt gct cat ctc act att
aat gcc acc gac atc 709 Leu Glu Ala Gln Pro Phe Ala His Leu Thr Ile
Asn Ala Thr Asp Ile 160 165 170 175 cca tct ggt tcc cat aaa gtg agt
ctg tcc tct tgg tac cat gat cgg 757 Pro Ser Gly Ser His Lys Val Ser
Leu Ser Ser Trp Tyr His Asp Arg 180 185 190 ggt tgg gcc aag atc tcc
aac atg act ttt agc aat gga aaa cta ata 805 Gly Trp Ala Lys Ile Ser
Asn Met Thr Phe Ser Asn Gly Lys Leu Ile 195 200 205 gtt aat cag gat
ggc ttt tat tac ctg tat gcc aac att tgc ttt cga 853 Val Asn Gln Asp
Gly Phe Tyr Tyr Leu Tyr Ala Asn Ile Cys Phe Arg 210 215 220 cat cat
gaa act tca gga gac cta gct aca gag tat ctt caa cta atg 901 His His
Glu Thr Ser Gly Asp Leu Ala Thr Glu Tyr Leu Gln Leu Met 225 230 235
gtg tac gtc act aaa acc agc atc aaa atc cca agt tct cat acc ctg 949
Val Tyr Val Thr Lys Thr Ser Ile Lys Ile Pro Ser Ser His Thr Leu 240
245 250 255 atg aaa gga gga agc acc aag tat tgg tca ggg aat tct gaa
ttc cat 997 Met Lys Gly Gly Ser Thr Lys Tyr Trp Ser Gly Asn Ser Glu
Phe His 260 265 270 ttt tat tcc ata aac gtt ggt gga ttt ttt aag tta
cgg tct gga gag 1045 Phe Tyr Ser Ile Asn Val Gly Gly Phe Phe Lys
Leu Arg Ser Gly Glu 275 280 285 gaa atc agc atc gag gtc tcc aac ccc
tcc tta ctg gat ccg gat cag 1093 Glu Ile Ser Ile Glu Val Ser Asn
Pro Ser Leu Leu Asp Pro Asp Gln 290 295 300 gat gca aca tac ttt ggg
gct ttt aaa gtt cga gat ata gat tga 1138 Asp Ala Thr Tyr Phe Gly
Ala Phe Lys Val Arg Asp Ile Asp 305 310 315 gccccagttt ttggagtgtt
atgtatttcc tggatgtttg gaaacatttt ttaaaacaag 1198 ccaagaaaga
tgtatatagg tgtgtgagac tactaagagg catggcccca acggtacacg 1258
actcagtatc catgctcttg accttgtaga gaacacgcgt atttacagcc agtgggagat
1318 gttagactca tggtgtgtta cacaatggtt tttaaatttt gtaatgaatt
cctagaatta 1378 aaccagattg gagcaattac gggttgacct tatgagaaac
tgcatgtggg ctatgggagg 1438 ggttggtccc tggtcatgtg ccccttcgca
gctgaagtgg agagggtgtc atctagcgca 1498 attgaaggat catctgaagg
ggcaaattct tttgaattgt tacatcatgc tggaacctgc 1558 aaaaaatact
ttttctaatg aggagagaaa atatatgtat ttttatataa tatctaaagt 1618
tatatttcag atgtaatgtt ttctttgcaa agtattgtaa attatatttg tgctatagta
1678 tttgattcaa aatatttaaa aatgtcttgc tgttgacata tttaatgttt
taaatgtaca 1738 gacatattta actggtgcac tttgtaaatt ccctggggaa
aacttgcagc taaggagggg 1798 aaaaaaatgt tgtttcctaa tatcaaatgc
agtatatttc ttcgttcttt ttaagttaat 1858 agattttttc agacttgtca
agcctgtgca aaaaaattaa aatggatgcc ttgaataata 1918 agcaggatgt
tggccaccag gtgcctttca aatttagaaa ctaattgact ttagaaagct 1978
gacattgcca aaaaggatac ataatgggcc actgaaatct gtcaagagta gttatataat
2038 tgttgaacag gtgtttttcc acaagtgccg caaattgtac cttttttttt
ttttcaaaat 2098 agaaaagtta ttagtggttt atcagcaaaa aagtccaatt
ttaatttagt aaatgttatc 2158 ttatactgta caataaaaac attgcctttg
aatgttaatt ttttggtaca aaaataaatt 2218 tatatgaaaa aaaaaaaaaa
agggcggccg ctctagaggg ccctattcta tag 2271 2 317 PRT Homo sapiens 2
Met Arg Arg Ala Ser Arg Asp Tyr Thr Lys Tyr Leu Arg Gly Ser Glu 1 5
10 15 Glu Met Gly Gly Gly Pro Gly Ala Pro His Glu Gly Pro Leu His
Ala 20 25 30 Pro Pro Pro Pro Ala Pro His Gln Pro Pro Ala Ala Ser
Arg Ser Met 35 40 45 Phe Val Ala Leu Leu Gly Leu Gly Leu Gly Gln
Val Val Cys Ser Val 50 55 60 Ala Leu Phe Phe Tyr Phe Arg Ala Gln
Met Asp Pro Asn Arg Ile Ser 65 70 75 80 Glu Asp Gly Thr His Cys Ile
Tyr Arg Ile Leu Arg Leu His Glu Asn 85 90 95 Ala Asp Phe Gln Asp
Thr Thr Leu Glu Ser Gln Asp Thr Lys Leu Ile 100 105 110 Pro Asp Ser
Cys Arg Arg Ile Lys Gln Ala Phe Gln Gly Ala Val Gln 115 120 125 Lys
Glu Leu Gln His Ile Val Gly Ser Gln His Ile Arg Ala Glu Lys 130 135
140 Ala Met Val Asp Gly Ser Trp Leu Asp Leu Ala Lys Arg Ser Lys Leu
145 150 155 160 Glu Ala Gln Pro Phe Ala His Leu Thr Ile Asn Ala Thr
Asp Ile Pro 165 170 175 Ser Gly Ser His Lys Val Ser Leu Ser Ser Trp
Tyr His Asp Arg Gly 180 185 190 Trp Ala Lys Ile Ser Asn Met Thr Phe
Ser Asn Gly Lys Leu Ile Val 195 200 205 Asn Gln Asp Gly Phe Tyr Tyr
Leu Tyr Ala Asn Ile Cys Phe Arg His 210 215 220 His Glu Thr Ser Gly
Asp Leu Ala Thr Glu Tyr Leu Gln Leu Met Val 225 230 235 240 Tyr Val
Thr Lys Thr Ser Ile Lys Ile Pro Ser Ser His Thr Leu Met 245 250 255
Lys Gly Gly Ser Thr Lys Tyr Trp Ser Gly Asn Ser Glu Phe His Phe 260
265 270 Tyr Ser Ile Asn Val Gly Gly Phe Phe Lys Leu Arg Ser Gly Glu
Glu 275 280 285 Ile Ser Ile Glu Val Ser Asn Pro Ser Leu Leu Asp Pro
Asp Gln Asp 290 295 300 Ala Thr Tyr Phe Gly Ala Phe Lys Val Arg Asp
Ile Asp 305 310 315 3 951 DNA Mus musculus CDS (1)..(951)
misc_feature (142)..(213) Transmembrane domain 3 atg cgc cgg gcc
agc cga gac tac ggc aag tac ctg cgc agc tcg gag 48 Met Arg Arg Ala
Ser Arg Asp Tyr Gly Lys Tyr Leu Arg Ser Ser Glu 1 5 10 15 gag atg
ggc agc ggc ccc ggc gtc cca cac gag ggt ccg ctg cac ccc 96 Glu Met
Gly Ser Gly Pro Gly Val Pro His Glu Gly Pro Leu His Pro 20 25 30
gcg cct tct gca ccg gct ccg gcg ccg cca ccc gcc gcc tcc cgc tcc 144
Ala Pro Ser Ala Pro Ala Pro Ala Pro Pro Pro Ala Ala Ser Arg Ser 35
40 45 atg ttc ctg gcc ctc ctg ggg ctg gga ctg ggc cag gtg gtc tgc
agc 192 Met Phe Leu Ala Leu Leu Gly Leu Gly Leu Gly Gln Val Val Cys
Ser 50 55 60 atc gct ctg ttc ctg tac ttt cga gcg cag atg gat cct
aac aga ata 240 Ile Ala Leu Phe Leu Tyr Phe Arg Ala Gln Met Asp Pro
Asn Arg Ile 65 70 75 80 tca gaa gac agc act cac tgc ttt tat aga atc
ctg aga ctc cat gaa 288 Ser Glu Asp Ser Thr His Cys Phe Tyr Arg Ile
Leu Arg Leu His Glu 85 90 95 aac gca ggt ttg cag gac tcg act ctg
gag agt gaa gac aca cta cct 336 Asn Ala Gly Leu Gln Asp Ser Thr Leu
Glu Ser Glu Asp Thr Leu Pro 100 105 110 gac tcc tgc agg agg atg aaa
caa gcc ttt cag ggg gcc gtg cag aag 384 Asp Ser Cys Arg Arg Met Lys
Gln Ala Phe Gln Gly Ala Val Gln Lys 115 120 125 gaa ctg caa cac att
gtg ggg cca cag cgc ttc tca gga gct cca gct 432 Glu Leu Gln His Ile
Val Gly Pro Gln Arg Phe Ser Gly Ala Pro Ala 130 135 140 atg atg gaa
ggc tca tgg ttg gat gtg gcc cag cga ggc aag cct gag 480 Met Met Glu
Gly Ser Trp Leu Asp Val Ala Gln Arg Gly Lys Pro Glu 145 150 155 160
gcc cag cca ttt gca cac ctc acc atc aat gct gcc agc atc cca tcg 528
Ala Gln Pro Phe Ala His Leu Thr Ile Asn Ala Ala Ser Ile Pro Ser 165
170 175 ggt tcc cat aaa gtc act ctg tcc tct tgg tac cac gat cga ggc
tgg 576 Gly Ser His Lys Val Thr Leu Ser Ser Trp Tyr His Asp Arg Gly
Trp 180 185 190 gcc aag atc tct aac atg acg tta agc aac gga aaa cta
agg gtt aac 624 Ala Lys Ile Ser Asn Met Thr Leu Ser Asn Gly Lys Leu
Arg Val Asn 195 200 205 caa gat ggc ttc tat tac ctg tac gcc aac att
tgc ttt cgg cat cat 672 Gln Asp Gly Phe Tyr Tyr Leu Tyr Ala Asn Ile
Cys Phe Arg His His 210 215 220 gaa aca tcg gga agc gta cct aca gac
tat ctt cag ctg atg gtg tat 720 Glu Thr Ser Gly Ser Val Pro Thr Asp
Tyr Leu Gln Leu Met Val Tyr 225 230 235 240 gtc gtt aaa acc agc atc
aaa atc cca agt tct cat aac ctg atg aaa 768 Val Val Lys Thr Ser Ile
Lys Ile Pro Ser Ser His Asn Leu Met Lys 245 250 255 gga ggg agc acg
aaa aac tgg tcg ggc aat tct gaa ttc cac ttt tat 816 Gly Gly Ser Thr
Lys Asn Trp Ser Gly Asn Ser Glu Phe His Phe Tyr 260 265 270 tcc ata
aat gtt ggg gga ttt ttc aag ctc cga gct ggt gaa gaa att 864 Ser Ile
Asn Val Gly Gly Phe Phe Lys Leu Arg Ala Gly Glu Glu Ile 275 280 285
agc att cag gtg tcc aac cct tcc ctg ctg gat ccg gat caa gat gcg 912
Ser Ile Gln Val Ser Asn Pro Ser Leu Leu Asp Pro Asp Gln Asp Ala 290
295 300 acg tac ttt ggg gct ttc aaa gtt cag gac ata gac tga 951 Thr
Tyr Phe Gly Ala Phe Lys Val Gln Asp Ile Asp 305 310 315 4 316 PRT
Mus musculus 4 Met Arg Arg Ala Ser Arg Asp Tyr Gly Lys Tyr Leu Arg
Ser Ser Glu 1 5 10 15 Glu Met Gly Ser Gly Pro Gly Val Pro His Glu
Gly Pro Leu His Pro 20 25 30 Ala Pro Ser Ala Pro Ala Pro Ala Pro
Pro Pro Ala Ala Ser Arg Ser 35 40 45 Met Phe Leu Ala Leu Leu Gly
Leu Gly Leu Gly Gln Val Val Cys Ser 50 55 60 Ile Ala Leu Phe Leu
Tyr Phe Arg Ala Gln Met Asp Pro Asn Arg Ile 65 70 75 80 Ser Glu Asp
Ser Thr His Cys Phe Tyr Arg Ile Leu Arg Leu His Glu 85 90 95 Asn
Ala Gly Leu Gln Asp Ser Thr Leu Glu Ser Glu Asp Thr Leu Pro 100 105
110 Asp Ser Cys Arg Arg Met Lys Gln Ala Phe Gln Gly Ala Val Gln Lys
115 120 125 Glu Leu Gln His Ile Val Gly Pro Gln Arg Phe Ser Gly Ala
Pro Ala 130 135 140 Met Met Glu Gly Ser Trp Leu Asp Val Ala Gln Arg
Gly Lys Pro Glu 145 150 155 160 Ala Gln Pro Phe Ala His Leu Thr Ile
Asn Ala Ala Ser Ile Pro Ser 165 170 175 Gly Ser His Lys Val Thr Leu
Ser Ser Trp Tyr His Asp Arg Gly Trp 180 185 190 Ala Lys Ile Ser Asn
Met Thr Leu Ser Asn Gly Lys Leu Arg Val Asn 195 200 205 Gln Asp Gly
Phe Tyr Tyr Leu Tyr Ala Asn Ile Cys Phe Arg His His 210 215 220 Glu
Thr Ser Gly Ser Val Pro Thr Asp Tyr Leu Gln Leu Met Val Tyr 225 230
235 240 Val Val Lys Thr Ser Ile Lys Ile Pro Ser Ser His Asn Leu Met
Lys 245 250 255 Gly Gly Ser Thr Lys Asn Trp Ser Gly Asn Ser Glu Phe
His Phe Tyr 260 265 270 Ser Ile Asn Val Gly Gly Phe Phe Lys Leu Arg
Ala Gly Glu Glu Ile 275 280 285 Ser Ile Gln Val Ser Asn Pro Ser Leu
Leu Asp Pro Asp Gln Asp Ala 290 295 300 Thr Tyr Phe Gly Ala Phe Lys
Val Gln Asp Ile Asp 305 310 315 5 2299 DNA Mus musculus CDS
(170)..(1120) 5 gagctcggat ccactactcg acccacgcgt ccgcccacgc
gtccggccag gacctctgtg 60 aaccggtcgg ggcgggggcc gcctggccgg
gagtctgctc ggcggtgggt ggccgaggaa 120 gggagagaac gatcgcggag
cagggcgccc gaactccggg cgccgcgcc atg cgc cgg 178 Met Arg Arg 1 gcc
agc cga gac tac ggc aag tac ctg cgc agc tcg gag gag atg ggc 226 Ala
Ser Arg Asp Tyr Gly Lys Tyr Leu Arg Ser Ser Glu Glu Met Gly 5 10 15
agc ggc ccc ggc gtc cca cac gag ggt ccg ctg cac ccc gcg cct tct 274
Ser Gly Pro Gly Val Pro His Glu Gly Pro Leu His Pro Ala Pro Ser 20
25 30 35 gca ccg gct ccg gcg ccg cca ccc gcc gcc tcc cgc tcc atg
ttc ctg 322 Ala Pro Ala Pro Ala Pro Pro Pro Ala Ala Ser Arg Ser Met
Phe Leu 40 45 50 gcc ctc ctg ggg ctg gga ctg ggc cag gtg gtc tgc
agc atc gct ctg 370 Ala Leu Leu Gly Leu Gly Leu Gly Gln Val Val Cys
Ser Ile Ala Leu 55 60 65 ttc ctg tac ttt cga gcg cag atg gat cct
aac aga ata tca gaa gac 418 Phe Leu Tyr Phe Arg Ala Gln Met Asp Pro
Asn Arg Ile Ser Glu Asp 70 75 80 agc act cac tgc ttt tat aga atc
ctg aga ctc cat gaa aac gca ggt 466 Ser Thr His Cys Phe Tyr Arg Ile
Leu Arg Leu His Glu Asn Ala Gly 85 90 95 ttg cag gac tcg act ctg
gag agt gaa gac aca cta cct gac tcc tgc 514 Leu Gln Asp Ser Thr Leu
Glu Ser Glu Asp Thr Leu Pro Asp Ser Cys 100 105 110 115 agg agg atg
aaa caa gcc ttt cag ggg gcc gtg cag aag gaa ctg caa 562 Arg Arg Met
Lys Gln Ala Phe Gln Gly Ala Val Gln Lys Glu Leu Gln 120 125 130 cac
att gtg ggg cca cag cgc ttc tca gga gct cca gct atg atg gaa 610 His
Ile Val Gly Pro Gln Arg Phe Ser Gly Ala Pro Ala Met Met Glu 135 140
145 ggc tca tgg ttg gat gtg gcc cag cga ggc aag cct gag gcc cag cca
658 Gly Ser Trp Leu Asp Val Ala Gln Arg Gly Lys Pro Glu Ala Gln Pro
150 155 160 ttt gca cac ctc acc atc aat gct gcc agc atc cca tcg ggt
tcc cat 706 Phe Ala His Leu Thr Ile Asn Ala Ala Ser Ile Pro Ser Gly
Ser His 165 170 175 aaa gtc act ctg tcc tct tgg tac cac gat cga ggc
tgg gcc aag atc 754 Lys Val Thr Leu Ser Ser Trp Tyr His Asp Arg Gly
Trp Ala Lys Ile 180 185 190 195 tct aac atg acg tta agc aac gga aaa
cta agg gtt aac caa gat ggc 802 Ser Asn Met Thr Leu Ser Asn Gly Lys
Leu Arg Val Asn Gln Asp Gly 200 205 210 ttc tat tac ctg tac gcc aac
att tgc ttt cgg cat cat gaa aca tcg 850 Phe Tyr Tyr Leu Tyr Ala Asn
Ile Cys Phe Arg His His Glu Thr Ser 215 220 225 gga agc gta cct aca
gac tat ctt cag ctg atg gtg tat gtc gtt aaa 898 Gly Ser Val Pro Thr
Asp Tyr Leu Gln Leu Met Val Tyr Val Val Lys 230 235 240 acc agc atc
aaa atc cca agt tct cat aac ctg atg aaa gga ggg agc 946 Thr Ser Ile
Lys Ile Pro Ser Ser His Asn Leu Met Lys Gly Gly Ser 245 250 255 acg
aaa aac tgg tcg ggc aat tct gaa ttc cac ttt tat tcc ata aat 994 Thr
Lys Asn Trp Ser Gly Asn Ser Glu Phe His Phe Tyr Ser Ile Asn 260 265
270 275 gtt ggg gga ttt ttc aag ctc cga gct ggt gaa gaa att agc att
cag 1042 Val Gly Gly Phe Phe Lys Leu Arg Ala Gly Glu Glu Ile Ser
Ile Gln 280 285 290 gtg tcc aac cct tcc ctg ctg gat ccg gat caa gat
gcg acg tac ttt 1090 Val Ser Asn Pro Ser Leu Leu Asp Pro Asp Gln
Asp Ala Thr Tyr Phe 295 300 305 ggg gct ttc aaa gtt cag gac ata gac
tga gactcatttc gtggaacatt 1140 Gly Ala Phe Lys Val Gln Asp
Ile Asp 310 315 agcatggatg tcctagatgt ttggaaactt cttaaaaaat
ggatgatgtc tatacatgtg 1200 taagactact aagagacatg gcccacggtg
tatgaaactc acagccctct ctcttgagcc 1260 tgtacaggtt gtgtatatgt
aaagtccata ggtgatgtta gattcatggt gattacacaa 1320 cggttttaca
attttgtaat gatttcctag aattgaacca gattgggaga ggtattccga 1380
tgcttatgaa aaacttacac gtgagctatg gaagggggtc acagtctctg ggtctaaccc
1440 ctggacatgt gccactgaga accttgaaat taagaggatg ccatgtcatt
gcaaagaaat 1500 gatagtgtga agggttaagt tcttttgaat tgttacattg
cgctgggacc tgcaaataag 1560 ttcttttttt ctaatgagga gagaaaaata
tatgtatttt tatataatgt ctaaagttat 1620 atttcaggtg taatgttttc
tgtgcaaagt tttgtaaatt atatttgtgc tatagtattt 1680 gattcaaaat
atttaaaaat gtctcactgt tgacatattt aatgttttaa atgtacagat 1740
gtatttaact ggtgcacttt gtaattcccc tgaaggtact cgtagctaag ggggcagaat
1800 actgtttctg gtgaccacat gtagtttatt tctttattct ttttaactta
atagagtctt 1860 cagacttgtc aaaactatgc aagcaaaata aataaataaa
aataaaatga ataccttgaa 1920 taataagtag gatgttggtc accaggtgcc
tttcaaattt agaagctaat tgactttagg 1980 agctgacata gccaaaaagg
atacataata ggctactgaa atctgtcagg agtatttatg 2040 caattattga
acaggtgtct ttttttacaa gagctacaaa ttgtaaattt tgtttctttt 2100
ttttcccata gaaaatgtac tatagtttat cagccaaaaa acaatccact ttttaattta
2160 gtgaaagtta ttttattata ctgtacaata aaagcattgt ctctgaatgt
taattttttg 2220 gtacaaaaaa taaatttgta cgaaaacctg aaaaaaaaaa
aaaaaaaggg cggccgctct 2280 agagggccct attctatag 2299 6 316 PRT Mus
musculus 6 Met Arg Arg Ala Ser Arg Asp Tyr Gly Lys Tyr Leu Arg Ser
Ser Glu 1 5 10 15 Glu Met Gly Ser Gly Pro Gly Val Pro His Glu Gly
Pro Leu His Pro 20 25 30 Ala Pro Ser Ala Pro Ala Pro Ala Pro Pro
Pro Ala Ala Ser Arg Ser 35 40 45 Met Phe Leu Ala Leu Leu Gly Leu
Gly Leu Gly Gln Val Val Cys Ser 50 55 60 Ile Ala Leu Phe Leu Tyr
Phe Arg Ala Gln Met Asp Pro Asn Arg Ile 65 70 75 80 Ser Glu Asp Ser
Thr His Cys Phe Tyr Arg Ile Leu Arg Leu His Glu 85 90 95 Asn Ala
Gly Leu Gln Asp Ser Thr Leu Glu Ser Glu Asp Thr Leu Pro 100 105 110
Asp Ser Cys Arg Arg Met Lys Gln Ala Phe Gln Gly Ala Val Gln Lys 115
120 125 Glu Leu Gln His Ile Val Gly Pro Gln Arg Phe Ser Gly Ala Pro
Ala 130 135 140 Met Met Glu Gly Ser Trp Leu Asp Val Ala Gln Arg Gly
Lys Pro Glu 145 150 155 160 Ala Gln Pro Phe Ala His Leu Thr Ile Asn
Ala Ala Ser Ile Pro Ser 165 170 175 Gly Ser His Lys Val Thr Leu Ser
Ser Trp Tyr His Asp Arg Gly Trp 180 185 190 Ala Lys Ile Ser Asn Met
Thr Leu Ser Asn Gly Lys Leu Arg Val Asn 195 200 205 Gln Asp Gly Phe
Tyr Tyr Leu Tyr Ala Asn Ile Cys Phe Arg His His 210 215 220 Glu Thr
Ser Gly Ser Val Pro Thr Asp Tyr Leu Gln Leu Met Val Tyr 225 230 235
240 Val Val Lys Thr Ser Ile Lys Ile Pro Ser Ser His Asn Leu Met Lys
245 250 255 Gly Gly Ser Thr Lys Asn Trp Ser Gly Asn Ser Glu Phe His
Phe Tyr 260 265 270 Ser Ile Asn Val Gly Gly Phe Phe Lys Leu Arg Ala
Gly Glu Glu Ile 275 280 285 Ser Ile Gln Val Ser Asn Pro Ser Leu Leu
Asp Pro Asp Gln Asp Ala 290 295 300 Thr Tyr Phe Gly Ala Phe Lys Val
Gln Asp Ile Asp 305 310 315 7 564 DNA Artificial Sequence CDS
(1)..(564) Description of Artificial Sequence Synthetic PCR product
with optimum codons for E. coli and P. pastoris expression 7 gag
ctc gga tcc ctc gag aaa aga gag gct gaa gct cat gtc atg aaa 48 Glu
Leu Gly Ser Leu Glu Lys Arg Glu Ala Glu Ala His Val Met Lys 1 5 10
15 cac caa cac caa cat caa cat caa cat caa cat caa aaa cct gaa gct
96 His Gln His Gln His Gln His Gln His Gln His Gln Lys Pro Glu Ala
20 25 30 cag cca ttc gct cat ctg acc atc aac gct gca tcg atc cct
tct ggt 144 Gln Pro Phe Ala His Leu Thr Ile Asn Ala Ala Ser Ile Pro
Ser Gly 35 40 45 tct cat aaa gtt acc ctg tct tct tgg tat cac gac
cgc ggt tgg gct 192 Ser His Lys Val Thr Leu Ser Ser Trp Tyr His Asp
Arg Gly Trp Ala 50 55 60 aaa atc tct aac atg acc ctg tct aac ggt
aaa ctg aga gtt aac cag 240 Lys Ile Ser Asn Met Thr Leu Ser Asn Gly
Lys Leu Arg Val Asn Gln 65 70 75 80 gac ggt ttc tac tac ctg tac gct
aac atc tgt ttc aga cat cac gaa 288 Asp Gly Phe Tyr Tyr Leu Tyr Ala
Asn Ile Cys Phe Arg His His Glu 85 90 95 acc tct ggt tct gtt cca
acc gac tac ctg cag ctg atg gtt tac gtt 336 Thr Ser Gly Ser Val Pro
Thr Asp Tyr Leu Gln Leu Met Val Tyr Val 100 105 110 gtt aaa acc tct
atc aaa atc cca tct tca cat aac ctg atg aaa ggt 384 Val Lys Thr Ser
Ile Lys Ile Pro Ser Ser His Asn Leu Met Lys Gly 115 120 125 ggt tct
acc aaa aac tgg tct ggt aac tct gaa ttc cat ttc tac tct 432 Gly Ser
Thr Lys Asn Trp Ser Gly Asn Ser Glu Phe His Phe Tyr Ser 130 135 140
atc aac gtt ggt ggt ttc ttc aaa ctg aga gct ggt gaa gaa atc tct 480
Ile Asn Val Gly Gly Phe Phe Lys Leu Arg Ala Gly Glu Glu Ile Ser 145
150 155 160 atc cag gtt tct aac cct tct ctg ctg gac cca gac cag gac
gct acc 528 Ile Gln Val Ser Asn Pro Ser Leu Leu Asp Pro Asp Gln Asp
Ala Thr 165 170 175 tac ttc ggg gcc ttc aaa gtt cag gac atc gac tag
564 Tyr Phe Gly Ala Phe Lys Val Gln Asp Ile Asp 180 185 8 187 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
PCR product with optimum codons for E. coli and P. pastoris
expression 8 Glu Leu Gly Ser Leu Glu Lys Arg Glu Ala Glu Ala His
Val Met Lys 1 5 10 15 His Gln His Gln His Gln His Gln His Gln His
Gln Lys Pro Glu Ala 20 25 30 Gln Pro Phe Ala His Leu Thr Ile Asn
Ala Ala Ser Ile Pro Ser Gly 35 40 45 Ser His Lys Val Thr Leu Ser
Ser Trp Tyr His Asp Arg Gly Trp Ala 50 55 60 Lys Ile Ser Asn Met
Thr Leu Ser Asn Gly Lys Leu Arg Val Asn Gln 65 70 75 80 Asp Gly Phe
Tyr Tyr Leu Tyr Ala Asn Ile Cys Phe Arg His His Glu 85 90 95 Thr
Ser Gly Ser Val Pro Thr Asp Tyr Leu Gln Leu Met Val Tyr Val 100 105
110 Val Lys Thr Ser Ile Lys Ile Pro Ser Ser His Asn Leu Met Lys Gly
115 120 125 Gly Ser Thr Lys Asn Trp Ser Gly Asn Ser Glu Phe His Phe
Tyr Ser 130 135 140 Ile Asn Val Gly Gly Phe Phe Lys Leu Arg Ala Gly
Glu Glu Ile Ser 145 150 155 160 Ile Gln Val Ser Asn Pro Ser Leu Leu
Asp Pro Asp Gln Asp Ala Thr 165 170 175 Tyr Phe Gly Ala Phe Lys Val
Gln Asp Ile Asp 180 185 9 519 DNA Artificial Sequence Description
of Artificial Sequence DNA encoding murine OPGL, residues 158-316,
fused to His tag 9 atg aaa cac caa cac caa cat caa cat caa cat caa
cat caa aaa cct 48 Met Lys His Gln His Gln His Gln His Gln His Gln
His Gln Lys Pro 1 5 10 15 gaa gct cag cca ttc gct cat ctg acc atc
aac gct gca tcg atc cct 96 Glu Ala Gln Pro Phe Ala His Leu Thr Ile
Asn Ala Ala Ser Ile Pro 20 25 30 tct ggt tct cat aaa gtt acc ctg
tct tct tgg tat cac gac cgc ggt 144 Ser Gly Ser His Lys Val Thr Leu
Ser Ser Trp Tyr His Asp Arg Gly 35 40 45 tgg gct aaa atc tct aac
atg acc ctg tct aac ggt aaa ctg aga gtt 192 Trp Ala Lys Ile Ser Asn
Met Thr Leu Ser Asn Gly Lys Leu Arg Val 50 55 60 aac cag gac ggt
ttc tac tac ctg tac gct aac atc tgt ttc aga cat 240 Asn Gln Asp Gly
Phe Tyr Tyr Leu Tyr Ala Asn Ile Cys Phe Arg His 65 70 75 80 cac gaa
acc tct ggt tct gtt cca acc gac tac ctg cag ctg atg gtt 288 His Glu
Thr Ser Gly Ser Val Pro Thr Asp Tyr Leu Gln Leu Met Val 85 90 95
tac gtt gtt aaa acc tct atc aaa atc cca tct tca cat aac ctg atg 336
Tyr Val Val Lys Thr Ser Ile Lys Ile Pro Ser Ser His Asn Leu Met 100
105 110 aaa ggt ggt tct acc aaa aac tgg tct ggt aac tct gaa ttc cat
ttc 384 Lys Gly Gly Ser Thr Lys Asn Trp Ser Gly Asn Ser Glu Phe His
Phe 115 120 125 tac tct atc aac gtt ggt ggt ttc ttc aaa ctg aga gct
ggt gaa gaa 432 Tyr Ser Ile Asn Val Gly Gly Phe Phe Lys Leu Arg Ala
Gly Glu Glu 130 135 140 atc tct atc cag gtt tct aac cct tct ctg ctg
gac cca gac cag gac 480 Ile Ser Ile Gln Val Ser Asn Pro Ser Leu Leu
Asp Pro Asp Gln Asp 145 150 155 160 gct acc tac ttc ggg gcc ttc aaa
gtt cag gac atc gac 519 Ala Thr Tyr Phe Gly Ala Phe Lys Val Gln Asp
Ile Asp 165 170 10 173 PRT Artificial Sequence Description of
Artificial Sequence DNA encoding murine OPGL, residues 158-316,
fused to His tag 10 Met Lys His Gln His Gln His Gln His Gln His Gln
His Gln Lys Pro 1 5 10 15 Glu Ala Gln Pro Phe Ala His Leu Thr Ile
Asn Ala Ala Ser Ile Pro 20 25 30 Ser Gly Ser His Lys Val Thr Leu
Ser Ser Trp Tyr His Asp Arg Gly 35 40 45 Trp Ala Lys Ile Ser Asn
Met Thr Leu Ser Asn Gly Lys Leu Arg Val 50 55 60 Asn Gln Asp Gly
Phe Tyr Tyr Leu Tyr Ala Asn Ile Cys Phe Arg His 65 70 75 80 His Glu
Thr Ser Gly Ser Val Pro Thr Asp Tyr Leu Gln Leu Met Val 85 90 95
Tyr Val Val Lys Thr Ser Ile Lys Ile Pro Ser Ser His Asn Leu Met 100
105 110 Lys Gly Gly Ser Thr Lys Asn Trp Ser Gly Asn Ser Glu Phe His
Phe 115 120 125 Tyr Ser Ile Asn Val Gly Gly Phe Phe Lys Leu Arg Ala
Gly Glu Glu 130 135 140 Ile Ser Ile Gln Val Ser Asn Pro Ser Leu Leu
Asp Pro Asp Gln Asp 145 150 155 160 Ala Thr Tyr Phe Gly Ala Phe Lys
Val Gln Asp Ile Asp 165 170 11 519 DNA Artificial Sequence
Description of Artificial Sequence Fusion of murine OPGL, residues
158-316 with C to S mutation, and His tag 11 atg aaa cac caa cac
caa cat caa cat caa cat caa cat caa aaa cct 48 Met Lys His Gln His
Gln His Gln His Gln His Gln His Gln Lys Pro 1 5 10 15 gaa gct cag
cca ttc gct cat ctg acc atc aac gct gca tcg atc cct 96 Glu Ala Gln
Pro Phe Ala His Leu Thr Ile Asn Ala Ala Ser Ile Pro 20 25 30 tct
ggt tct cat aaa gtt acc ctg tct tct tgg tat cac gac cgc ggt 144 Ser
Gly Ser His Lys Val Thr Leu Ser Ser Trp Tyr His Asp Arg Gly 35 40
45 tgg gct aaa atc tct aac atg acc ctg tct aac ggt aaa ctg aga gtt
192 Trp Ala Lys Ile Ser Asn Met Thr Leu Ser Asn Gly Lys Leu Arg Val
50 55 60 aac cag gac ggt ttc tac tac ctg tac gct aac atc tcc ttc
aga cat 240 Asn Gln Asp Gly Phe Tyr Tyr Leu Tyr Ala Asn Ile Ser Phe
Arg His 65 70 75 80 cac gaa acc tct ggt tct gtt cca acc gac tac ctg
cag ctg atg gtt 288 His Glu Thr Ser Gly Ser Val Pro Thr Asp Tyr Leu
Gln Leu Met Val 85 90 95 tac gtt gtt aaa acc tct atc aaa atc cca
tct tca cat aac ctg atg 336 Tyr Val Val Lys Thr Ser Ile Lys Ile Pro
Ser Ser His Asn Leu Met 100 105 110 aaa ggt ggt tct acc aaa aac tgg
tct ggt aac tct gaa ttc cat ttc 384 Lys Gly Gly Ser Thr Lys Asn Trp
Ser Gly Asn Ser Glu Phe His Phe 115 120 125 tac tct atc aac gtt ggt
ggt ttc ttc aaa ctg aga gct ggt gaa gaa 432 Tyr Ser Ile Asn Val Gly
Gly Phe Phe Lys Leu Arg Ala Gly Glu Glu 130 135 140 atc tct atc cag
gtt tct aac cct tct ctg ctg gac cca gac cag gac 480 Ile Ser Ile Gln
Val Ser Asn Pro Ser Leu Leu Asp Pro Asp Gln Asp 145 150 155 160 gct
acc tac ttc ggg gcc ttc aaa gtt cag gac atc gac 519 Ala Thr Tyr Phe
Gly Ala Phe Lys Val Gln Asp Ile Asp 165 170 12 173 PRT Artificial
Sequence Description of Artificial Sequence Fusion of murine OPGL,
residues 158-316 with C to S mutation, and His tag 12 Met Lys His
Gln His Gln His Gln His Gln His Gln His Gln Lys Pro 1 5 10 15 Glu
Ala Gln Pro Phe Ala His Leu Thr Ile Asn Ala Ala Ser Ile Pro 20 25
30 Ser Gly Ser His Lys Val Thr Leu Ser Ser Trp Tyr His Asp Arg Gly
35 40 45 Trp Ala Lys Ile Ser Asn Met Thr Leu Ser Asn Gly Lys Leu
Arg Val 50 55 60 Asn Gln Asp Gly Phe Tyr Tyr Leu Tyr Ala Asn Ile
Ser Phe Arg His 65 70 75 80 His Glu Thr Ser Gly Ser Val Pro Thr Asp
Tyr Leu Gln Leu Met Val 85 90 95 Tyr Val Val Lys Thr Ser Ile Lys
Ile Pro Ser Ser His Asn Leu Met 100 105 110 Lys Gly Gly Ser Thr Lys
Asn Trp Ser Gly Asn Ser Glu Phe His Phe 115 120 125 Tyr Ser Ile Asn
Val Gly Gly Phe Phe Lys Leu Arg Ala Gly Glu Glu 130 135 140 Ile Ser
Ile Gln Val Ser Asn Pro Ser Leu Leu Asp Pro Asp Gln Asp 145 150 155
160 Ala Thr Tyr Phe Gly Ala Phe Lys Val Gln Asp Ile Asp 165 170 13
564 DNA Artificial Sequence Description of Artificial Sequence
Fusion of murine OPGL, residues 158-316 modified by introduction of
tetanus toxoid P30 epitope, and His tag 13 atg aaa cac caa cac caa
cat caa cat caa cat caa cat caa aaa cct 48 Met Lys His Gln His Gln
His Gln His Gln His Gln His Gln Lys Pro 1 5 10 15 gaa gct cag cca
ttc gct cat ctg acc atc aac gct gca tcg atc cct 96 Glu Ala Gln Pro
Phe Ala His Leu Thr Ile Asn Ala Ala Ser Ile Pro 20 25 30 tct ggt
tct cat aaa gtt acc ctg tct tct tgg tat cac gac cgc ggt 144 Ser Gly
Ser His Lys Val Thr Leu Ser Ser Trp Tyr His Asp Arg Gly 35 40 45
tgg gct aaa atc tct aac atg acc ctg tct aac ggt aaa ctg aga gtt 192
Trp Ala Lys Ile Ser Asn Met Thr Leu Ser Asn Gly Lys Leu Arg Val 50
55 60 aac cag gac ggt ttc tac tac ctg tac gct aac atc tgt ttc aga
cat 240 Asn Gln Asp Gly Phe Tyr Tyr Leu Tyr Ala Asn Ile Cys Phe Arg
His 65 70 75 80 cac gaa acc tct ggt tct gtt cca acc gac tac ctg cag
ctg atg gtt 288 His Glu Thr Ser Gly Ser Val Pro Thr Asp Tyr Leu Gln
Leu Met Val 85 90 95 tac gtt gtt aaa acc tct atc aaa atc cca tct
tca cat aac ctg atg 336 Tyr Val Val Lys Thr Ser Ile Lys Ile Pro Ser
Ser His Asn Leu Met 100 105 110 ttc aac aac ttc acc gtt tct ttc tgg
ctg agg gta ccg aaa gtt tct 384 Phe Asn Asn Phe Thr Val Ser Phe Trp
Leu Arg Val Pro Lys Val Ser 115 120 125 gct tct cac ctg gaa aac tgg
tct ggt aac tct gaa ttc cat ttc tac 432 Ala Ser His Leu Glu Asn Trp
Ser Gly Asn Ser Glu Phe His Phe Tyr 130 135 140 tct atc aac gtt ggt
ggt ttc ttc aaa ctg aga gct ggt gaa gaa atc 480 Ser Ile Asn Val Gly
Gly Phe Phe Lys Leu Arg Ala Gly Glu Glu Ile 145 150 155 160 tct atc
cag gtt tct aac cct tct ctg ctg gac cca gac cag gac gct 528 Ser Ile
Gln Val Ser Asn Pro Ser Leu Leu Asp Pro Asp Gln Asp Ala 165 170 175
acc tac ttc ggg gcc ttc aaa gtt cag gac atc gac 564 Thr Tyr Phe Gly
Ala Phe Lys Val Gln Asp Ile Asp 180 185 14 188 PRT Artificial
Sequence Description of Artificial Sequence Fusion of murine OPGL,
residues 158-316 modified by introduction of tetanus toxoid P30
epitope, and His tag 14 Met Lys His Gln His Gln His Gln
His Gln His Gln His Gln Lys Pro 1 5 10 15 Glu Ala Gln Pro Phe Ala
His Leu Thr Ile Asn Ala Ala Ser Ile Pro 20 25 30 Ser Gly Ser His
Lys Val Thr Leu Ser Ser Trp Tyr His Asp Arg Gly 35 40 45 Trp Ala
Lys Ile Ser Asn Met Thr Leu Ser Asn Gly Lys Leu Arg Val 50 55 60
Asn Gln Asp Gly Phe Tyr Tyr Leu Tyr Ala Asn Ile Cys Phe Arg His 65
70 75 80 His Glu Thr Ser Gly Ser Val Pro Thr Asp Tyr Leu Gln Leu
Met Val 85 90 95 Tyr Val Val Lys Thr Ser Ile Lys Ile Pro Ser Ser
His Asn Leu Met 100 105 110 Phe Asn Asn Phe Thr Val Ser Phe Trp Leu
Arg Val Pro Lys Val Ser 115 120 125 Ala Ser His Leu Glu Asn Trp Ser
Gly Asn Ser Glu Phe His Phe Tyr 130 135 140 Ser Ile Asn Val Gly Gly
Phe Phe Lys Leu Arg Ala Gly Glu Glu Ile 145 150 155 160 Ser Ile Gln
Val Ser Asn Pro Ser Leu Leu Asp Pro Asp Gln Asp Ala 165 170 175 Thr
Tyr Phe Gly Ala Phe Lys Val Gln Asp Ile Asp 180 185 15 546 DNA
Artificial Sequence Description of Artificial Sequence Fusion
between murine OPGL, residues 158-316 with tetanus toxoid P2
epitope introduced, and His tag 15 atg aaa cac caa cac caa cat caa
cat caa cat caa cat caa aaa cct 48 Met Lys His Gln His Gln His Gln
His Gln His Gln His Gln Lys Pro 1 5 10 15 gaa gct cag cca ttc gct
cat ctg acc atc aac gct gca tcg atc cct 96 Glu Ala Gln Pro Phe Ala
His Leu Thr Ile Asn Ala Ala Ser Ile Pro 20 25 30 tct ggt tct cat
aaa gtt acc ctg tct tct tgg tat cac gac cgc ggt 144 Ser Gly Ser His
Lys Val Thr Leu Ser Ser Trp Tyr His Asp Arg Gly 35 40 45 tgg gct
aaa atc tct aac atg acc ctg tct aac ggt aaa ctg aga gtt 192 Trp Ala
Lys Ile Ser Asn Met Thr Leu Ser Asn Gly Lys Leu Arg Val 50 55 60
aac cag gac ggt ttc tac tac ctg tac gct aac atc tgt ttc aga cat 240
Asn Gln Asp Gly Phe Tyr Tyr Leu Tyr Ala Asn Ile Cys Phe Arg His 65
70 75 80 cac gaa acc tct ggt tct gtt cca acc gac tac ctg cag ctg
atg gtt 288 His Glu Thr Ser Gly Ser Val Pro Thr Asp Tyr Leu Gln Leu
Met Val 85 90 95 tac gtt gtt aaa acc cct atc aaa atc caa tct tca
cat aac ctg atg 336 Tyr Val Val Lys Thr Pro Ile Lys Ile Gln Ser Ser
His Asn Leu Met 100 105 110 cag tac atc aaa gct aat tcg aaa ttc atc
ggt atc acc gaa ctg aac 384 Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile
Gly Ile Thr Glu Leu Asn 115 120 125 tgg tct ggt aac tct gaa ttc cat
ttc tac tct atc aac gtt ggt ggt 432 Trp Ser Gly Asn Ser Glu Phe His
Phe Tyr Ser Ile Asn Val Gly Gly 130 135 140 ttc ttc aaa ctg aga gct
ggt gaa gaa atc tct atc cag gtt tct aac 480 Phe Phe Lys Leu Arg Ala
Gly Glu Glu Ile Ser Ile Gln Val Ser Asn 145 150 155 160 cct tct ctg
ctg gac cca gac cag gac gct acc tac ttc ggg gcc ttc 528 Pro Ser Leu
Leu Asp Pro Asp Gln Asp Ala Thr Tyr Phe Gly Ala Phe 165 170 175 aaa
gtt cag gac atc gac 546 Lys Val Gln Asp Ile Asp 180 16 182 PRT
Artificial Sequence Description of Artificial Sequence Fusion
between murine OPGL, residues 158-316 with tetanus toxoid P2
epitope introduced, and His tag 16 Met Lys His Gln His Gln His Gln
His Gln His Gln His Gln Lys Pro 1 5 10 15 Glu Ala Gln Pro Phe Ala
His Leu Thr Ile Asn Ala Ala Ser Ile Pro 20 25 30 Ser Gly Ser His
Lys Val Thr Leu Ser Ser Trp Tyr His Asp Arg Gly 35 40 45 Trp Ala
Lys Ile Ser Asn Met Thr Leu Ser Asn Gly Lys Leu Arg Val 50 55 60
Asn Gln Asp Gly Phe Tyr Tyr Leu Tyr Ala Asn Ile Cys Phe Arg His 65
70 75 80 His Glu Thr Ser Gly Ser Val Pro Thr Asp Tyr Leu Gln Leu
Met Val 85 90 95 Tyr Val Val Lys Thr Pro Ile Lys Ile Gln Ser Ser
His Asn Leu Met 100 105 110 Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile
Gly Ile Thr Glu Leu Asn 115 120 125 Trp Ser Gly Asn Ser Glu Phe His
Phe Tyr Ser Ile Asn Val Gly Gly 130 135 140 Phe Phe Lys Leu Arg Ala
Gly Glu Glu Ile Ser Ile Gln Val Ser Asn 145 150 155 160 Pro Ser Leu
Leu Asp Pro Asp Gln Asp Ala Thr Tyr Phe Gly Ala Phe 165 170 175 Lys
Val Gln Asp Ile Asp 180 17 519 DNA Artificial Sequence Description
of Artificial Sequence Fusion between murine OPGL, residues 158-316
with tetanus toxoidP2 epitope introduced, and His tag 17 atg aaa
cac caa cac caa cat caa cat caa cat caa cat caa aaa cct 48 Met Lys
His Gln His Gln His Gln His Gln His Gln His Gln Lys Pro 1 5 10 15
gaa gct cag cca ttc gct cat ctg acc atc aac gct gca tcg atc cct 96
Glu Ala Gln Pro Phe Ala His Leu Thr Ile Asn Ala Ala Ser Ile Pro 20
25 30 tct ggt tct cat aaa gtt acc ctg tct tct tgg tat cac gac cgc
ggt 144 Ser Gly Ser His Lys Val Thr Leu Ser Ser Trp Tyr His Asp Arg
Gly 35 40 45 tgg gct aaa atc tct aac atg acc ctg tct aac ggt aaa
ctg aga gtt 192 Trp Ala Lys Ile Ser Asn Met Thr Leu Ser Asn Gly Lys
Leu Arg Val 50 55 60 aac cag gac ggt ttc tac tac ctg tac gct aac
atc tgt ttc aga cat 240 Asn Gln Asp Gly Phe Tyr Tyr Leu Tyr Ala Asn
Ile Cys Phe Arg His 65 70 75 80 cac gaa acc tct ggt tct gtt cca acc
gac tac ctg cag ctg atg gtt 288 His Glu Thr Ser Gly Ser Val Pro Thr
Asp Tyr Leu Gln Leu Met Val 85 90 95 tac gtt gtt aaa acc tct atc
aaa atc cca tct tca cat aac ctg atg 336 Tyr Val Val Lys Thr Ser Ile
Lys Ile Pro Ser Ser His Asn Leu Met 100 105 110 aaa ggt ggt tct acc
aaa aac tgg tct ggt aac tct gaa ttc cat ttc 384 Lys Gly Gly Ser Thr
Lys Asn Trp Ser Gly Asn Ser Glu Phe His Phe 115 120 125 tac tct atc
aac gtt ggt ggt ttc ttc aaa ctg aga gct ggt gaa gaa 432 Tyr Ser Ile
Asn Val Gly Gly Phe Phe Lys Leu Arg Ala Gly Glu Glu 130 135 140 cag
tac atc aaa gct aat tcg aaa ttc atc ggt atc acc gaa ctg gac 480 Gln
Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Asp 145 150
155 160 gct acc tac ttc ggg gcc ttc aaa gtt cag gac atc gac 519 Ala
Thr Tyr Phe Gly Ala Phe Lys Val Gln Asp Ile Asp 165 170 18 173 PRT
Artificial Sequence Description of Artificial Sequence Fusion
between murine OPGL, residues 158-316 with tetanus toxoid P2
epitope introduced, and His tag 18 Met Lys His Gln His Gln His Gln
His Gln His Gln His Gln Lys Pro 1 5 10 15 Glu Ala Gln Pro Phe Ala
His Leu Thr Ile Asn Ala Ala Ser Ile Pro 20 25 30 Ser Gly Ser His
Lys Val Thr Leu Ser Ser Trp Tyr His Asp Arg Gly 35 40 45 Trp Ala
Lys Ile Ser Asn Met Thr Leu Ser Asn Gly Lys Leu Arg Val 50 55 60
Asn Gln Asp Gly Phe Tyr Tyr Leu Tyr Ala Asn Ile Cys Phe Arg His 65
70 75 80 His Glu Thr Ser Gly Ser Val Pro Thr Asp Tyr Leu Gln Leu
Met Val 85 90 95 Tyr Val Val Lys Thr Ser Ile Lys Ile Pro Ser Ser
His Asn Leu Met 100 105 110 Lys Gly Gly Ser Thr Lys Asn Trp Ser Gly
Asn Ser Glu Phe His Phe 115 120 125 Tyr Ser Ile Asn Val Gly Gly Phe
Phe Lys Leu Arg Ala Gly Glu Glu 130 135 140 Gln Tyr Ile Lys Ala Asn
Ser Lys Phe Ile Gly Ile Thr Glu Leu Asp 145 150 155 160 Ala Thr Tyr
Phe Gly Ala Phe Lys Val Gln Asp Ile Asp 165 170 19 519 DNA
Artificial Sequence Description of Artificial Sequence Fusion
betweenmurine OPGL, residues 158-316 with tetanus toxoid P30
epitope introduced, and His tag 19 atg aaa cac caa cac caa cat caa
cat caa cat caa cat caa aaa cct 48 Met Lys His Gln His Gln His Gln
His Gln His Gln His Gln Lys Pro 1 5 10 15 gaa gct cag cca ttc gct
cat ctg acc atc aac gct gca tcg atc cct 96 Glu Ala Gln Pro Phe Ala
His Leu Thr Ile Asn Ala Ala Ser Ile Pro 20 25 30 tct ggt tct cat
aaa gtt acc ctg tct tct tgg tat cac gac cgc ggt 144 Ser Gly Ser His
Lys Val Thr Leu Ser Ser Trp Tyr His Asp Arg Gly 35 40 45 tgg gct
aaa atc tct aac atg acc ctg tct aac ggt aaa ctg aga gtt 192 Trp Ala
Lys Ile Ser Asn Met Thr Leu Ser Asn Gly Lys Leu Arg Val 50 55 60
aac cag gac ggt ttc tac tac ctg tac gct aac atc tgt ttc aac aac 240
Asn Gln Asp Gly Phe Tyr Tyr Leu Tyr Ala Asn Ile Cys Phe Asn Asn 65
70 75 80 ttc acc gtt tct ttc tgg ctg agg gta ccg aaa gtt tct gct
tct cac 288 Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser Ala
Ser His 85 90 95 ctg gaa gtt aaa acc tct atc aaa atc cca tct tca
cat aac ctg atg 336 Leu Glu Val Lys Thr Ser Ile Lys Ile Pro Ser Ser
His Asn Leu Met 100 105 110 aaa ggt ggt tct acc aaa aac tgg tct ggt
aac tct gaa ttc cat ttc 384 Lys Gly Gly Ser Thr Lys Asn Trp Ser Gly
Asn Ser Glu Phe His Phe 115 120 125 tac tct atc aac gtt ggt ggt ttc
ttc aaa ctg aga gct ggt gaa gaa 432 Tyr Ser Ile Asn Val Gly Gly Phe
Phe Lys Leu Arg Ala Gly Glu Glu 130 135 140 atc tct atc cag gtt tct
aac cct tct ctg ctg gac cca gac cag gac 480 Ile Ser Ile Gln Val Ser
Asn Pro Ser Leu Leu Asp Pro Asp Gln Asp 145 150 155 160 gct acc tac
ttc ggg gcc ttc aaa gtt cag gac atc gac 519 Ala Thr Tyr Phe Gly Ala
Phe Lys Val Gln Asp Ile Asp 165 170 20 173 PRT Artificial Sequence
Description of Artificial Sequence Fusion between murine OPGL,
residues 158-316 with tetanus toxoidP30 epitope introduced, and His
tag 20 Met Lys His Gln His Gln His Gln His Gln His Gln His Gln Lys
Pro 1 5 10 15 Glu Ala Gln Pro Phe Ala His Leu Thr Ile Asn Ala Ala
Ser Ile Pro 20 25 30 Ser Gly Ser His Lys Val Thr Leu Ser Ser Trp
Tyr His Asp Arg Gly 35 40 45 Trp Ala Lys Ile Ser Asn Met Thr Leu
Ser Asn Gly Lys Leu Arg Val 50 55 60 Asn Gln Asp Gly Phe Tyr Tyr
Leu Tyr Ala Asn Ile Cys Phe Asn Asn 65 70 75 80 Phe Thr Val Ser Phe
Trp Leu Arg Val Pro Lys Val Ser Ala Ser His 85 90 95 Leu Glu Val
Lys Thr Ser Ile Lys Ile Pro Ser Ser His Asn Leu Met 100 105 110 Lys
Gly Gly Ser Thr Lys Asn Trp Ser Gly Asn Ser Glu Phe His Phe 115 120
125 Tyr Ser Ile Asn Val Gly Gly Phe Phe Lys Leu Arg Ala Gly Glu Glu
130 135 140 Ile Ser Ile Gln Val Ser Asn Pro Ser Leu Leu Asp Pro Asp
Gln Asp 145 150 155 160 Ala Thr Tyr Phe Gly Ala Phe Lys Val Gln Asp
Ile Asp 165 170 21 68 DNA Artificial Sequence Description of
Artificial Sequence Synthetic PCR primer 21 agctgcaggt agtcggttgg
aacagaacca gaggtttcgt gatgtctgaa acagatgtta 60 gcgtacag 68 22 24
DNA Artificial Sequence Description of Artificial Sequence
Synthetic PCR primer 22 ctcatctgac catcaacgct gcat 24 23 64 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
PCR primer 23 tttcggtacc ctcagccaga aagaaacggt gaagttgttg
aaacagatgt tagcgtacag 60 gtag 64 24 61 DNA Artificial Sequence
Description of Artificial Sequence Synthetic PCR primer 24
tgagggtacc gaaagtttct gcttctcacc tggaagttaa aacccctatc aaaatccaat
60 c 61 25 63 DNA Artificial Sequence Description of Artificial
Sequence Synthetic PCR primer 25 tttcggtacc ctcagccaga aagaaacggt
gaagttgttg aacatcaggt tatgtgaaga 60 ttg 63 26 62 DNA Artificial
Sequence Description of Artificial Sequence Synthetic PCR primer 26
tgagggtacc gaaagtttct gcttctcacc tggaaaactg gtctggtaac tctgaattcc
60 at 62 27 79 DNA Artificial Sequence Description of Artificial
Sequence Synthetic PCR primer 27 tacctgcagc tgatggttta cgttgttaaa
acccctatca aaatccaatc ttcacataac 60 ctgatgcagt acatcaaag 79 28 83
DNA Artificial Sequence Description of Artificial Sequence
Synthetic PCR primer 28 tggaattcag agttaccaga ccagttcagt tcggtgatac
cgatgaattt cgaattagct 60 ttgatgtact gcatcaggtt atg 83 29 49 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
PCR primer 29 gaatttcgaa ttagctttga tgtactgttc ttcaccagct ctcagtttg
49 30 53 DNA Artificial Sequence Description of Artificial Sequence
Synthetic PCR primer 30 gctaattcga aattcatcgg tatcaccgaa ctggacgcta
cctacttcgg ggc 53 31 26 DNA Artificial Sequence Description of
Artificial Sequence Synthetic PCR primer 31 cttactagtc gatgtcctga
actttg 26 32 74 DNA Artificial Sequence Description of Artificial
Sequence Synthetic PCR primer 32 agtggaattc agagttacca gaccagtttt
tggtagaacc acctttcatc aggttatgtg 60 aagatgggat tttg 74 33 65 DNA
Clostridium tetani 33 actacctgca gctgatggtt tacgttgtta aaacctctat
caaaatccca tcttcacata 60 acctg 65 34 15 PRT Clostridium tetani 34
Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu 1 5 10
15 35 21 PRT Clostridium tetani 35 Phe Asn Asn Phe Thr Val Ser Phe
Trp Leu Arg Val Pro Lys Val Ser 1 5 10 15 Ala Ser His Leu Glu
20
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