ZA200102131B - Method for down-regulating osteoprotegerin ligand activity. - Google Patents
Method for down-regulating osteoprotegerin ligand activity. Download PDFInfo
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- ZA200102131B ZA200102131B ZA200102131A ZA200102131A ZA200102131B ZA 200102131 B ZA200102131 B ZA 200102131B ZA 200102131 A ZA200102131 A ZA 200102131A ZA 200102131 A ZA200102131 A ZA 200102131A ZA 200102131 B ZA200102131 B ZA 200102131B
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Description
L
METHOD FOR DOWN—REGULATING OSTEOPROTEGERIN LIGAND ACTIVITY
The present invention relates to improvements 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 osteoprotege- rin ligand (OPGL) by enabling the production of antibodies against OPGL in subjects suffering from or in danger of suf- fering 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 identifica- tion 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.
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.
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 bil- lion USD annually in 1984. This translates by extrapolation to a worldwide cost of something in the order of at least 20 billion USD.
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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, 5 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 eticlogy 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.
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.
Bone consists of a mineralised 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;{(PO,), Ca (OH),). Bone is continuously modelled during growth and development and remodelled throughout life in response to physical and chemi- cal signals.
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.
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. Osteo- clasts differentiate from hematopoietic precursors of the monocyte-macrophage lineage and resorb bone matrix.
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An imbalance of osteoblast and osteoclast functions can result in the skeletal abnormalities characterised by increased bone mass (osteopetrosis) or by decreased bone mass (osteoporosis).
Studies of osteopetrosis in mutant mice have shown that ge- netic 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 deve- lopment.
Recently, however, two proteins that take part in this regula- tion have been described and characterized (Simonet et al., 1997; Lacey et al., 1998). These two proteins are osteoprotegerin and osteoprotegerin ligand.
Osteoprotedgerin is a novel secreted member of the tumour necrosis factor receptor family. In vitro, osteoprotegerin blocks osteoclastogenesis in a dose dependent manner. Trans- genic 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 osteo- clasts, 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
CoE Cy : Co
It was hypothesized that osteoprotegerin may exert its effect by binding to and neutralising a factor that stimulates osteo- clast development, thus inhibiting osteoclast maturation (Simonet et al., 1997).
QOsteoprotegerin ligand (OPGL) is a novel member of the tumouf necrosis factor family of cytokines that exists in both a membrane-bound and a soluble form. OPGL binds to osteoprote- gerin with a binding affinity of 4 nM. In vitro, OPGL acti- vates 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-l-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).
Description of OPGL
OPGL is synthesised as a type I1 transmembrane protein consis- ting 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 posi- tions.
The OPGL amino acid sequence contains a short cytoplasmic domain in the N-terminus followed by the putative transmem- brane region between amino acid residues 49 and 69. Based on its homology to tumour necrosis factor alpha, the extracellu- lar 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.
, PCT/DK99/00481
The most closely related protein to OPGL appears to be the apoptosis inducing cytokine TRAIL with less than 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 (i.e. Receptor Activator of Nuclear-factor Kappa B ligand), respectively (Anderson et al., 1997, Nature 390: 175-179. The protein is also known as osteoclast differentiation factor (ODF).
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 8-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 vitro assays (Lacey et al., 1998).
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.
The longest variant {amino acid residues 75-316) of OPGL did not bind to osteoprotegerin and it did not have any biological activity.
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
AMENDED SHEET c : . 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.
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.
Biological activity of OPGL
OPGL is a potent ostecclast differentiation factor when com- bined with CSF-1. Neither of these components alone are cap- able of inducing osteoclast differentiation from progenitor cells.
OPGL is a potent activator of mature osteoclast. On its own,
OPGL activates mature osteoclasts to resorb bone. COPGL has not been observed to act as an osteoclast growth factor or osteo- clast survival factor in these experiments.
The action of OPGL does not seem to be species restricted as murine OPGL alsc induced osteoclast formation in cultures of human peripheral blood mononuclear cells.
The object of the present invention is to provide novel thera- pies against conditions characterized by excess bone resorp- tion, 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 invol- ving excess bone resorption.
. . : .
We find that the above-referenced data suggests a pathophysiological role of OPGL. The in vivo evidence is par-— tially circumstantial or indirect but is in our opinion convincing especially in combination with the direct evidence.
Observing that injection into mice of the recombinant C-termi- nal domain of OPGL results in severe hypercalcemia in our opinion points directly to a pathophysiological role.
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 osteopo- rosis. We conclude that the most likely reason for this is an increased osteoclast maturation and activation caused by OPGL.
Two other pieces of indirect evidence are that both mice transgenic for osteoprotegerin and mice injected with recombi- nant osteoprotegerin develop osteopetrosis. This shows that unnatural high levels of a protein that binds OPGL and neu- tralises 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.
We therefore suggest a model in which OPGL and osteoprotegerin act as positive and negative regulators of osteoclast develop- ment, respectively. In other words OPGL promotes bone resorp- tion while osteoprotegerin inhibits bone resorption.
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.
We hence propose to down-regulate osteoclast differentia- tion/maturation/formation and osteoclast activation through in vivo production of antibodies capable of neutralizing OPGL, — thereby providing a safe and efficient means for treating/a- meliorating and/or preventing osteoporosis and other diseases characterized by an excess rate of bone resorption compared to the rate of bone formation.
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 - 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 a modification in the OPGL polypeptide which has as a re- sult that immunization of the animal with the analogue induces production of antibodies against the OPGL polypeptide.
The most attractive aspect of this approach is that e.g. osteoporosis can be controlled by periodic but not very fre- quent 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
. ' ' » N : the desired effect, whereas administration of osteoprotegerin or other inhibitors of OPGL activity would require daily administrations.
The invention also relates to OPGL analogues as well as to 5 nucleic acid fragments encoding a subset of these. Also — immunogenic compositions comprising the analogues or the nucleic acid fragments are part of the invention.
The invention also relates to a method of identifying ana- logues of OPGL as well as a method for preparing composition comprising the OPGL analogues.
Finally, the invention relates to a method treating osteoporo- sis and other diseases characterized in excess bone resorp- tion, wherein is administered a non-OPGL molecule (typically an antibody) which blocks the interaction between OPGL and its receptor on osteoclast cells.
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 inven- tion.
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. Like- wise, the terms “B-lymphocyte” and “B-cell” will be used interchangeably for antibody-producing lymphocytes.
An “OPGL polypeptide” is herein intended to denote polypep- tides 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 unglycosyla- ted 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-immuncgenic when presented to the animal to be treated. In other words, the OPGL polypeptide is a self-pro- tein 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.
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 polypep- tide’s amino acid sequence. Also encompassed by the term are derivatized OPGL molecules, cf. the discussion below of modi- fications of OPGL.
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.
The term “polypeptide” is in the present context intended to mean both short peptides of from 2 to 10 amino acid residues, oligopeptides of from 11 to 100 amino acid residues, and polypeptides of more than 100 amino acid residues. Further-— more, 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.
The term “subsequence” means any consecutive stretch of at least 3 amino acids or, when relevant, of at least 3 nucleo- tides, derived directly from a naturally occurring OPGL amino acid sequence or nucleic acid sequence, respectively.
The term “animal” is in the present context in general in- tended 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 indi- viduals 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.
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
Claims (104)
1. Use of at least one osteoprotegerin ligand (OPGL) polypeptide which is a full-length wild-type OPGL amino acid sequence or a subsequence thereof or nucleic acid(s} encoding the OPGL polypeptide or a non-pathogenic microorganism or virus which is carrying a nucleic acid fragment which encodes and expresses the OPGL polypeptide which has been formulated so that immunization of an animal with the OPGL polypeptide or subsequence thereof or the nucleic acid(s) encoding the OPGL polypeptide or the non- pathogenic microorganism or virus which is carrying the nucleic acid fragment which encodes and expresses the OPGL polypeptide induces production of antibodies against the OPGL polypeptide, in the manufacture of a preparation for in vivo down-regulation of OPGL activity in the animal, including a human being, by effecting presentation to the animal’s immune system of an immunogenically effective amount of said preparation, whereby the animal’s own OPGL is down-regulated due to binding thereof to the antibodies, OPGL being a protein which acts as an osteoclast differentiation factor and which has an amino acid sequence as set forth in SEQ ID NO: 2 for human OPGL and in SEQ ID NOs: 4 and 6 for murine OPGL.
2. Use of at least one OPGL analogue or nucleic acid(s) encoding the OPGL analogue or a non-pathogenic microorganism or virus which is carrying a nucleic acid fragment which encodes and expresses the OPGL analogue wherein is introduced at least one modification in the OPGL amino acid sequence which has as a result that immunization of an animal with the analogue or the nucleic acid(s) encoding the OPGL analogue or the non- pathogenic microorganism or virus which is carrying the nucleic acid fragment which encodes and expresses the OPGL analogue induces production of antibodies against the OPGL polypeptide, in the manufacture of a preparation AMENDED SHEET
PCT/DK99/00481 for in vivo down-regulation of OPGL activity in the animal, including a human being, by effecting presentation to the animal's immune system of an immunogenically effective amount of said preparation, whereby the animal’s own OPGL is down-regulated due to binding thereof to the antibodies, OPGL being a protein which acts as an osteoclast differentiation factor and which has an amino acid sequence as set forth in SEQ ID NO: 2 for human OPGL and in SEQ ID NOs: 4 and 6 for murine OPGL.
3. Use of at least one OPGL polypeptide which is a full-length wild-type OPGL amino acid sequence or a subsequence thereof or nucleic acid(s) encoding the OPGL polypeptide or a non-pathogenic microorganism or virus which is carrying a nucleic acid fragment which encodes and expresses the OPGL polypeptide which has been formulated so that immunization of an animal with the OPGL polypeptide or subsequence thereof or the nucleic acid(s) encoding the OPGL polypeptide or the non-pathogenic microorganism or virus which is carrying the nucleic acid fragment which encodes and expresses the OPGL polypeptide induces production of antibodies against the OPGL polypeptide, and at least one OPGL analogue or nucleic acid(s) encoding the OPGL analogue or a non-pathogenic microorganism or virus which is carrying a nucleic acid fragment which encodes and expresses the OPGL or 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 or the nucleic acid(s) encoding the OPGL analogue or the non-pathogenic microorganism or virus which is carrying the nucleic acid fragment which encodes and expresses the OPGL analogue induces production of antibodies against the OPGL polypeptide, in the manufacture of a preparation for in vivo down-regulation of OPGL activity in the animal, including a human being, by effecting presentation to the animal's immune system of an immunogenically effective amount of said preparation AMENDED SHEET
PCT/DK99/00481 whereby the animal's own OPGL is down-regulated due to binding thereof to the antibodies, OPGL being a protein which acts as an osteoclast differentiation factor and which has an amino acid sequence as set forth in SED ID NO: 2 for human OPGL and in SEQ ID NOs: 4 and 6 for murine OPGL. 4, Use according to claim 2 or claim 3, wherein is presented an OPGL analogue with at least one modification of the OPGL amino acid sequence.
5. Use according to claim 4, 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, 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.
6. Use according to claim 5, 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,, epitope and/or of the first and/or of the second and/or of the third moiety.
7. Use according to claim 5 or 6, wherein the modification includes amino acid substitution and/or deletion and/or insertion and/or addition.
8. Use according to claim 7, wherein the modification results in the provision of a fusion polypeptide. AMENDED SHEET
PCT/DK99/00481
9. Use according to claim 7 or 8, 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.
10. Use according to any one of claims 4 - 9, wherein the modification includes duplication of at least one OPGL B-cell epitope and/or introduction of a hapten.
11. Use according to any one of claims 5 - 10, wherein the foreign T-cell epitope is immunodominant in the animal.
12. Use according to any one of claims 5 - 11, wherein the foreign T-cell epitope is capable of binding to a large proportion of MHC class || molecules.
13. Use according to claim 12, wherein the at least one foreign T-cell epitope is selected from a natural T-cell epitope and an artificial MHC-II binding peptide sequence.
14. Use according to claim 13, 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.
15. Use according to any one of claims 5 - 14, wherein the first moiety is a 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.
16. Use according to any one of claims 5 - 15, wherein the second moiety is selected from a cytokine, a hormone, and a heat-shock protein. AMENDED SHEET
PCT/DK99/00481
17. Use according to claim 8, wherein the cytokine is selected from, or is an effective part of, interferon y (IFN-y, Fit3L, 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).
18. Use according to any one of claims 5 - 17, wherein the third moiety is of lipid nature, such as a palmitoyl group, a myristyl group, a farnesyl group, a geranyl-geranyl group, a GPl-anchor, and an N-acyl diglyceride group.
19. Use according to any one of the preceding claims insofar they are directly or indirectly dependent on claim 1 or claim 3, 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.
20. Use according to claim 19, wherein the modification comprises a substitution of at least one amino acid sequence within a position defined in claim 19 with an amino acid sequence of equal or different length which contains a foreign T, epitope.
21. Use according to claim 20, wherein the amino acid sequence containing the foreign T, 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 AMENDED SHEET
PCT/DK99/00481 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.
22. Use according to any one of the preceding claims insofar they are directly or indirectly dependent on claim 1 or claim 3, 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 or non-covalently linked to a carrier molecule capable of effecting presentation of multiple copies of antigenic determinants.
23. Use according to any of the preceding claims insofar they are directly or indirectly dependent on claim 1 or claim 3, 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.
24. Use 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.
25. Use according to claim 24, wherein the effective amount is between
0.5 ug and 2,000 ug of the OPGL polypeptide, the subsequence thereof or the analogue thereof.
26. Use according to claim 24 or 25, wherein the OPGL polypeptide or analogue is contained in a virtual lymph node (VLN) device. AMENDED SHEET
PCT/DK99/00481
27. Use according to any one of claims 1 - 23 insofar they are directly or indirectly dependent on claim 2 or claim 3, wherein presentation of modified OPGL to the immune system is effected by introducing said 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.
28. Use accordingto claim 27, wherein the nucleic acid(s) introduced is/are selected from naked DNA, DNA formulated with charged or uncharged lipids, DNA formulated in liposomes, DNA includedin 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.
29. Use according to claim 28, wherein the nucleic acid{s) is/are contained in a VLN device.
30. Use according to any one of claims 24 - 29, 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/introductions.
31. Use of a preparation as defined in any one of claims 1 - 30 in a method for preventing osteoporosis or other diseases and conditions characterized by excess bone resorption; the method comprising down-regulating OPGL activity as defined in 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 156%, at least 17%, at least 20%, and at least 30%.
32. An OPGL analogue which is derived from an animal OPGL polypeptide wherein is introduced a modification which has as a result that immunization AMENDED SHEET
PCT/DK99/00481 of the animal with the analogue induces production of antibodies against the OPGL polypeptide and wherein the modification is as defined in any one of claims 19 - 21 while a substantial fraction of the animal OPGL B-cell epitopes is preserved.
33. An OPGL analogue according to claim 32, wherein the modification is as defined in claim 21.
34. 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 32 or 33, the composition further comprising a pharmaceutically and immunologically acceptable carrier and/or vehicle and optionally an adjuvant.
35. A nucleic acid fragment which encodes an OPGL analogue according to claim 32 or 33.
36. A vector carrying the nucleic acid fragment according to claim 35.
37. The vector according to claim 36 which is capable of autonomous replication.
38. The vector according to claim 36 or 37 which is selected from the group consisting of a plasmid, a phage, a cosmid, a mini-chromosome, and a virus. AMENDED SHEET
PCT/DK99/00481
39. The vector according to any one of claims 36 - 38, comprising, in the 5'->3’ direction and in operable linkage, a promoter for driving expression of the nucleic acid fragment according to claim 35 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 35, and optionally a terminator.
40. The vector according to any one of claims 36 - 39 which, when introduced into a host cell, is capable or incapable of being integrated in the host cell genome.
41. The vector according to claim 39 or 40, wherein a promoter drives expression in a eukaryotic cell and/or in a prokaryotic cell.
42. A transformed cell carrying the vector of any one of claims 36 - 41.
43. The transformed cell according to claim 42 which is capable of replicating the nucleic acid fragment according to claim 35.
44. The transformed cell according to claim 43, 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 an S, or an SF cell, a plant cell, and a mammalian cell.
45. The transformed cell according to any one of claims 42 - 44, which expresses the nucleic acid fragment according to claim 35.
46. The transformed cell according to claim 45, which secretes or carries on its surface, the OPGL analogue according to claim 32 or 33. AMENDED SHEET
PCT/DK99/00481
47. Use according to any one of claims 1 - 21, wherein presentation to the immune system is effected by administering said non-pathogenic microorganism or virus which is carrying said nucleic acid fragment which encodes and expresses the OPGL polypeptide or analogue.
48. A composition for inducing production of antibodies against OPGL, the composition comprising - a nucleic acid fragment according to claim 35 or a vector according to any one of claims 36 - 41, and - a pharmaceutically and immunologically acceptable carrier and/or vehicle and/or adjuvant.
49. A stable cell line which carries the vector according to any one of claims 36 - 41 and which expresses the nucleic acid fragment according to claim 35, and which optionally secretes or carries the OPGL analogue according to claim 32 or 33 on its surface.
50. A method for the preparation of the cell according to any one of claims 42 - 46, the method comprising transforming a host cell with the nucleic acid fragment according to claim 35 or with the vector according to any one of claims 36 - 41.
51. 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 AMENDED SHEET
PCT/DK99/00481 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.
52. 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. AMENDED SHEET
PCT/DK99/00481
53. The method according to claim 51 or 52, 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 35, 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.
54. The method according to claim 53, 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.
55. 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.
56. 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.
57. Use of an OPGL analogue according to claim 32 or 33 for the preparation of an immunogenic composition optionally comprising an adjuvant for down-regulating OPGL activity in an animal.
58. Use of an OPGL analogue according to claim 32 or 33 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. AMENDED SHEET
PCT/DK99/00481
59. A substance or composition for use in a method for in vivo down- regulation of osteoprotegerin ligand {OPGL) activity in an animal, including a human being, said substance or composition comprising 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 said method comprising effecting presentation to the animal’s immune system of an immunogenically effective amount of said substance or composition, whereby the animal's own OPGL is down-regulated due to binding thereof to the antibodies, OPGL being a protein which acts as an osteoclast differentiation factor and which has an amino acid sequence as set forth in SEQ ID NO: 2 for human OPGL and in SEQ ID NOs: 4 and 6 for murine OPGL.
60. A substance or composition for use in a method for in vivo down- regulation of osteoprotegerin ligand (OPGL) activity in an animal, including a human being, said substance or composition comprising 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, and said method comprising effecting presentation to the animal's immune system of an immunogenically effective amount of said substance or composition, whereby the animal’s own OPGL is down-regulated due to binding thereof to the antibodies, OPGL being a protein which acts as an osteoclast differentiation factor and which has an amino acid sequence as set forth in SEQ ID NO: 2 for human OPGL and in SEQ ID NOs: 4 and 6 for murine OPGL.
61. A substance or composition for use in a method for /n vivo down- regulation of osteoprotegerin ligand (OPGL) activity in an animal, including a human being, said substance or composition comprising at least one OPGL AMENDED SHEET
PCT/DK99/00481 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 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, and said method comprising effecting presentation to the animal's immune system of an immunogenically effective amount of said substance or composition, whereby the animal's own OPGL is down-regulated due to binding thereof to the antibodies, OPGL being a protein which acts as an osteoclast differentiation factor and which has an amino acid sequence as set forth in SEQ ID NO: 2 for human OPGL and in SEQ ID NOs: 4 and 6 for murine OPGL.
62. A substance or composition for use in a method of treatment according to claim 60 or claim 61, wherein is presented an OPGL analogue with at least one modification of the OPGL amino acid sequence.
63. A substance or composition for use in a method of treatment according to claim 62, 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 epitoe (T, 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. AMENDED SHEET
PCT/DK99/00481
64. A substance or composition for use in a method of treatment according to claim 63, 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,, epitope and/or of the first and/or of the second and/or of the third moiety.
65. A substance or composition for use in a method of treatment according to claim 63 or 64, wherein the modification includes amino acid substitution and/or deletion and/or insertion and/or addition.
66. A substance or composition for use in a method of treatment according to claim 65, wherein the modification results in the provision of a fusion polypeptide.
67. A substance or composition for use in a method of treatment according to claim 65 or 66, 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.
68. A substance or composition for use in a method of treatment according to any one of claims 62-67, wherein the modification includes duplication of at least one OPGL B-cell epitope and/or introduction of a hapten.
69. A substance or composition for use in a method of treatment according to any one of claims 63-68, wherein the foreign T-cell epitope is immunodominant in the animal. 70 A substance or composition for use in a method of treatment according to any one of claims 63-69, wherein the foreign T-cell epitope is capable of binding to a large proportion of MHC Class ll molecules. AMENDED SHEET
PCT/DK99/00481
71. A substance or composition for use in a method of treatment according to claim 70, wherein the at least one foreign T-cell epitope is selected from a natural T-cell epitope and an artificial MHC-II binding peptide sequence.
72. A substance or composition for use in a method of treatment according to claim 71, 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.
73. A substance or composition for use in a method of treatment according to any one of claims 63-72, wherein the first moiety is a 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.
74. A substance or composition for use in a method of treatment according to any one of claims 63-73, wherein the second moiety is selected from a cytokine, a hormone, and a heat-shock protein.
75. A substance or composition for use in a method of treatment according to claim 66, wherein the cytokine is selected from, or is an effective part of, interferon y (IFN-y, FIt3L, 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).
76. A substance or composition for use in a method of treatment according to any one of claims 63-75, wherein the third moiety is of lipid nature, such as a palmitoyl group, a myristyl group, a farnesy! group, a geranyl-geranyl group, a GPl-anchor, and an N-acyl diglyceride group. AMENDED SHEET
PCT/DK99/00481
77. A substance or composition for use in a method of treatment according to any one of claims 59 to 76, insofar they are directly or indirectly dependent on claim 59 or claim 61, 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 2567-262, or in any one of positions 286-317, the amino acid numbering conforming with that of SEQ ID NO: 2.
78. A substance or composition for use in a method of treatment according to claim 77, wherein the modification comprises a substitution of at least one amino acid sequence within a position defined in claim 77, with an amino acid sequence of equal or different length which contains a foreign Ty epitope.
79. A substance or composition for use in a method of treatment according to claim 78, wherein the amino acid sequence containing the foreign Ty 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.
80. A substance or composition for use in a method of treatment according to any one of claims 59 to 79, insofar they are directly or indirectly dependent on claim 59 or claim 61, 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 or non- AMENDED SHEET
PCT/DK99/00481 covalently linked to a carrier molecule capable of effecting presentation of multiple copies of antigenic determinants.
81. A substance or composition for use in a method of treatment according to any one of claims 59 to 80 insofar they are directly or indirectly dependent on claim 59 or claim 61, 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.
82. A substance or composition for use in a method of treatment according to any one of claims 59 to 81, wherein an effective amount of the OPGL polypeptide or the OPGL analogue is administered to the animal via a route selected from the parenteralroute such as the intradermal, the subdermal, the intracutaneous, the subcutaneous, and the intramuscular routes; the peritoneal route; the oral route; the buccal route; the sublingual route; the epidural route; the spinal route; the anal route; and the intracranial route.
83. A substance or composition for use in a method of treatment according to claim 82, wherein the effective amount is between 0.5 ug and 2,000 ug of the OPGL polypeptide, the subsequence thereof or the analogue thereof.
84. A substance or composition for use in a method of treatment according to claim 82 or 83, wherein the OPGL polypeptide or analogue is contained in a virtual lymph node (VLN) device.
85. A substance or composition for use in a method of treatment according to any one of claims 58-81 insofar they are directly or indirectly dependent on claim 60 or claim 61, 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. AMENDED SHEET
PCT/DK99/00481
86. A substance or composition for use in a method of treatment according to claim 85, 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.
87. A substance or composition for use in a method of treatment according to claim 87, wherein the nucleic acid(s) is/are contained in a VLN device.
88. A substance or composition for use in a method of treatment according to any one of claims 82-87, 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/introductions.
89. A substance or composition for use in a method of treatment according to any one of claims 59-79, 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.
90. Use according to any one of claims 1 to 30, in the manufacture of a preparation for treating and/or preventing and/or ameliorating osteoporosis or other diseases and conditions characterized by excess bone resorption; by regulating OPGL activity 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%. AMENDED SHEET
PCT/DK99/00481
91. A substance or composition for use in a method for treating and/or preventing and/or ameliorating osteoporosis or other diseases and conditions characterized by excess bone resorption; which comprises down-regulating OPGL activity according to any one of claims 59 to 88 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%.
92. A substance or composition for down-regulating OPGL activity in an animal, said substance or composition comprising OPGL or a subsequence thereof, and an adjuvant, and said method comprising administering an immunogenic effective amount of said substance or composition.
93. A substance or composition for use in the treatment, prophylaxis or amelioration of osteoporosis or other conditions characterized by excessive bone resorption, said substance or composition comprising OPGL or a subsequence thereof and an adjuvant, and said method comprising administering an immunogenic effective amount of said substance or composition.
94. A substance or composition for down-regulating OPGL activity in an animal, said substance or composition comprising an OPGL analogue according to claim 32 or 33 and optionally an adjuvant, and said method comprising administering animmunogenic effective amount of said substance or composition.
95. A substance or composition for the treatment, prophylaxis or amelioration of osteoporosis or other conditions characterized by excessive bone resorption, said substance or composition comprising an OPGL analogue according to claim 32 or 33 and optionally and adjuvant, and said method AMENDED SHEET
PCT/DK99/00481 comprising administering an immunogenic effective amount of said substance or composition.
96. Use according to any one of claims 1, 2, 3, 55, 56, 57 or BS, substantially as herein described and illustrated.
97. An OPGL analogue according to claim 32, substantially as herein described and illustrated.
98. A nucleic acid fragment according to claim 35, substantially as herein described and illustrated.
99. A vector according to claim 36, substantially as herein described and illustrated.
100. A cell according to claim 42, substantially as herein described and illustrated.
101. A cell line according to claim 49, substantially as herein described and illustrated.
102. A method according to any one of claims 31, 50 to 52, substantially as herein described and illustrated.
103. A substance or composition for use in a method of treatment according to any one of claims 59, 60, 61 or 91 to 95, substantially as herein described and illustrated.
104. New use of an OPGL or subsequence thereof as defined in claim 1, and/or an OPGL analogue as defined in claim 2, claim 32 or claim 33; a new compound; a new nucleic acid fragment; a new vector; a new cell; a new cell AMENDED SHEET
PCT/DK99/00481 line; a new method for preventing osteoporosis, for the preparation of a cell, for the identification of a compound, or for the preparation of a composition; or a substance or composition for a new use in a method of treatment, substantially as herein described.
AMENDED SHEET _
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DKPA199801164 | 1998-09-15 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| ZA200102131B true ZA200102131B (en) | 2002-06-14 |
Family
ID=27675500
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ZA200102131A ZA200102131B (en) | 1998-09-15 | 2001-03-14 | Method for down-regulating osteoprotegerin ligand activity. |
Country Status (4)
| Country | Link |
|---|---|
| CN (1) | CN101260152A (en) |
| EA (1) | EA006940B1 (en) |
| RS (1) | RS49960B (en) |
| ZA (1) | ZA200102131B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019061297A1 (en) * | 2017-09-29 | 2019-04-04 | 苏州工业园区唯可达生物科技有限公司 | Cd4 helper t-cell epitope fusion peptide and vaccine thereof |
| CN110923266A (en) * | 2019-11-07 | 2020-03-27 | 苏州工业园区唯可达生物科技有限公司 | Recombinant virus vector, immune composition containing same and application |
-
1999
- 1999-09-13 CN CNA2007101927772A patent/CN101260152A/en active Pending
- 1999-09-13 EA EA200100356A patent/EA006940B1/en not_active IP Right Cessation
- 1999-09-13 RS YUP-197/01A patent/RS49960B/en unknown
-
2001
- 2001-03-14 ZA ZA200102131A patent/ZA200102131B/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| RS49960B (en) | 2008-09-29 |
| YU19701A (en) | 2005-06-10 |
| EA006940B1 (en) | 2006-06-30 |
| EA200100356A1 (en) | 2001-08-27 |
| CN101260152A (en) | 2008-09-10 |
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