WO1997004098A2 - MODIFIED HUMAN CHORIONIC (β-hCG) PROTEINS AND THEIR MEDICAL USE - Google Patents
MODIFIED HUMAN CHORIONIC (β-hCG) PROTEINS AND THEIR MEDICAL USE Download PDFInfo
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- WO1997004098A2 WO1997004098A2 PCT/GB1996/001717 GB9601717W WO9704098A2 WO 1997004098 A2 WO1997004098 A2 WO 1997004098A2 GB 9601717 W GB9601717 W GB 9601717W WO 9704098 A2 WO9704098 A2 WO 9704098A2
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- hcg
- modified
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- nucleic acid
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 34
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 32
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- 102000009151 Luteinizing Hormone Human genes 0.000 description 3
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- 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
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/827—Proteins from mammals or birds
-
- 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
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/827—Proteins from mammals or birds
- Y10S530/85—Reproductive organs or embryos
Definitions
- the present invention relates to substances, in particular to modified human chorionic gonadotropin ( ⁇ -hCG) proteins/genes, and their medical use, for example as immunological contraceptives having improved specificity and/or which in vivo avoid producing antibodies having undesirable cross-reactivity, for example with other natural hormones.
- ⁇ -hCG modified human chorionic gonadotropin
- hCG Shortly after fertilization of the ovum, the hormone hCG which at other times is essentially absent from the body, is produced and acts on the corpus luteum in the ovary to promote synthesis of progesterone. Progesterone is vital for the maintenance of the fertilized egg in the uterus and so the production of antibodies to neutralise the hCG will effectively prevent the pregnancy from proceeding. This strategy has been successfully employed to block fertility in baboons 1 and marmosets 4 and more recently in humans 3 .
- hCG itself is composed of two chains, ⁇ and ⁇ .
- the ⁇ -chain is common to other hormones (FSH, TSH and LH), which contribute to normal physiological function, so that autoantibodies made to this chain would be highly undesirable.
- the ⁇ -chain of hCG is far more specific, but a major problem still remains in that there is an 85% homology of ⁇ -hCG with the ⁇ -chain of luteinizing hormone
- the epitopes specific for ⁇ -hCG other than the C-terminus are discontinuous, i.e. the residues making up the epitope may be separate from each other in primary structure but are brought together by the protein folding.
- the contact residues forming these discontinuous epitopes are very difficult to identify and even if they could be, the "floppiness" of any synthetic peptide formed from these residues would make it a poor immunogen with respect to the generation of antibodies with high affinity.
- the present invention provides a substance which has the property of inducing a neutralising antibody response to ⁇ -hCG in vivo, said antibodies not substantially cross-reacting with LH, the substance comprising one or more of the conformational epitopes specific to native ⁇ -hCG, or functional equivalents or mimetics of these epitopes.
- the substance is a modified ⁇ -hCG protein having one or more conformational epitopes specific to native ⁇ -hCG, the protein being modified at one or more amino acid residues forming epitope (s) of native ⁇ -hCG that cross-react with LH, to reduce the cross-reactivity the ⁇ -hCG protein with LH, as defined by the ability of both proteins to react with the same antibody.
- the present invention also includes substances which are variants, derivatives, functional equivalents or mimetics of these above proteins.
- the substance includes two or more epitopes that are specific to native ⁇ -hCG. This helps to induce the production of antibodies specific for these epitopes, which will form complexes of the ⁇ -hCG with two or more antibody molecules, so helping to improve the in vivo neutralising activity caused by the substance.
- the modified amino acid residues are selected from the following residues of native ⁇ -hCG; Lys20, Glu21, Gly22, Pro24, Val25, Glu65, Arg68, Gly71, Arg74, Gly75 and/or Val79.
- residues of native ⁇ -hCG Lys20, Glu21, Gly22, Pro24, Val25, Glu65, Arg68, Gly71, Arg74, Gly75 and/or Val79.
- residues common to ⁇ -hCG and ⁇ -LH which lie on the outside of the protein molecule accessible to the aqueous solvent phase, which might potentially be immunogenic and give rise to cross-reacting antibodies. This would have to be established following immunisation with the mutant ⁇ -hCG and a similar further mutation procedure would then be required to abolish the epitopes reacting with these new antibodies.
- the rationale for selecting the residues to replace the native residues is set out below in more detail. Preferred modifications are set out in table 2.
- the present invention provides nucleic acid encoding the above proteins, vectors incorporating the nucleic acid and host cells transformed with the vectors.
- the present invention includes compositions comprising one or more of the above substances, in combination with a physiologically acceptable carrier.
- the compositions will be contraceptive compositions in a form suitable for immunisation.
- the substances, proteins or compositions described herein may prove useful in hCG-specific immunoassays and for applications where hCG is active, such as the inhibition of Kaposi sarcoma.
- the present invention provides a method of contraception, more strictly in this context contragestative, for a female mammal comprising immunising the female mammal with contraceptively effective amount of one or more of the substances.
- the present invention includes the use of the substances in the manufacture of a contraceptive composition.
- the immunogenicity of the substance may be enhanced by linking it to a carrier such as tetanus toxoid, or to appropriate sequences from such a carrier acting as T-helper epitopes.
- the substance may be engineered as a fusion protein with an appropriately immunogenic partner.
- Engineered DNA constructs containing nucleotide sequences encoding the substance together with, for example, additional sequences encoding T-helper epitopes or cytokine adjuvants, may be directly administered as a nucleic acid, preferably DNA, vaccine.
- the nucleic acid construct encoding the modified ⁇ -hCG protein can be used as a initial vaccine to prime an immune response. This initial response can then be boosted by subsequent injection of the modified ⁇ -hCG protein itself. Likewise, the modified ⁇ -hCG protein could be used first followed by the nucleic acid to boost the immune response.
- the designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a "lead" compound. This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, eg peptides are unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases, in the alimentary canal.
- Mimetic design, synthesis and testing is generally used to avoid randomly screening large number of molecules for a target property.
- the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this the design of the mimetic.
- a template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted.
- the template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound.
- the mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
- Figure 1 shows schematically a modified ⁇ -hCG protein in which an epitope which cross-reacts with LH is modified
- epitope deletion mutant which has lost epitope ( ⁇ ) cross-reacting with ⁇ -LH but still retains ⁇ - hCG specific epitope ( ⁇ ).
- Figure 2 shows the fluorescence of cell transfected with ⁇ - hCG mutant 6 determined by Facscan: (a) control antibody;
- Figure 3 shows the relative spatial distribution of the ⁇ - hCG epitope clusters
- Figure 4 shows the results of the binding of Mabs to different mutants.
- Figure 5 shows the amino acid substitutions made in ⁇ -hCG.
- mutant ⁇ -hCG has a size of 145 amino acid residues which include 12 cysteines that form 6 conserved di-sulphide bridges.
- the subunit is heavily glycosylated with N-linked carbohydrates at position Asnl3 and Asn30 in addition to four O-linked carbohydrates in the C-terminus at Ser121, 127, 132 and 138. To ensure correct folding of the recombinant molecules we opted for expression in mammalian cells.
- ⁇ -hCG is synthesized as a fusion protein with a C-terminal extension that consists of the 17 amino acids proximal to the membrane, the transmembrane and the cytoplasmic portion of the H2-D b molecule
- the expression level was determined with the hCG specific Mab OT3A on a Becton Dickinson Facscan as shown in Figure 2.
- the Mab OT3A which recognizes a linear epitope in the C-terminal extension of ⁇ -hCG can be used to quantitate the expression level of the wild type and mutant recombinant proteins following transient transfection in the COS7 cells. Berger et al 8 & 9 have previously used a panel of Mab to define
- the target residues for mutation were selected from the crystal structure of hCG to have side chains protruding from the surface of the molecule which could contribute to the antibody binding site.
- the substitutions were selected by comparing the same residues in the different members of the same family.
- the changes were designed, however, to introduce amino acids with sufficiently dissimilar properties in their side chains (e.g. charge, size, polarity) from the ⁇ -hCG residues, to disrupt any Mab binding in this region.
- Computer graphic model building of the mutant ⁇ -hCG molecules ensured that the side chains of the amino acid substitutions could be accommodated into the predicted structure without grossly altering the overall conformation.
- Table 2 (figure 5) summarizes the amino acid changes in fifteen of the mutants used in this study. Expression vector construct and production of mutants
- ⁇ -hCG cDNA Full length ⁇ -hCG cDNA was cloned from human placental third trimester RNA using RT-PCR and the sense cloning primer 5'ACCGGAATTCCAGGGGCTCCTGCTGTTG3' (corresponding to nucleotide (nt) -51 ⁇ -33) and the antisense cloning primer 5'TTGGTCGACTTGTGGGAGGATCGGGGTGTCC3 ' (nt 414 ⁇ 435) .
- the hCG cDNA was cloned into pCDM8 10 into which a DNA fragment from H2-Db containing the 17 membrane proximal amino acid residues, the transmembrane region and cytoplasmic tail had been inserted.
- This fragment was obtained using RT-PCR amplification using RNA from a spleen of a C57BL/10 mouse with the sense primer 5'GCGTTGGTCGACCATGAGGGGCTGCCTGAGCCC3' (nt 547 ⁇ 566) and an antisense primer 5'CACAGGAGAGACCTGAACACATCG3' (nt 809 ⁇ 832).
- the sequence of ⁇ -hCG is as published 11 .
- ⁇ -hCG or the mutants themselves, were used to generate mutants/further mutants.
- COS cells were transfected using a modified DEAE dextran- chloroquine method (based on Seed & Aruffo 13 ). Briefly, 1.5 ⁇ 10 cells were seeded into an 80cm 3 flask on the day before transfection. 6ml of the transfection mixture, ( 10% NuSerum (Becton Dickinson, Bedford MA) ;1-2 ⁇ g/ml supercoiled DNA (CsCl prepared or PEG prepared); 250 ⁇ g/ml DEAE dextran) was added to the washed monolayer and left in 37°C incubator for 60 minutes. Chloroquine was then added to a final concentration of 200 ⁇ M and the cells incubated for a further 120 minutes.
- a modified DEAE dextran- chloroquine method based on Seed & Aruffo 13 . Briefly, 1.5 ⁇ 10 cells were seeded into an 80cm 3 flask on the day before transfection. 6ml of the transfection mixture, ( 10% NuSerum (Becton Dickinson,
- the transfection mixture was then removed, the monolayer washed with PBS and 3ml 10%DMSO (in PBS) added for 2 minutes. The cells were washed again and complete medium added. The cells were split 1:1 24 hours later and harvested 65-72 hours after transfection. A transfection efficiency of 20-40% was routinely obtained.
- Mutations of residues Lys20-Glu21-Gly22 (Mutant 1) completely abolish the binding of the Mab InnhCG64 recognizing the hCG- specific epitope cluster 06 and lead to partial binding of the 03/5 Mab 3E2.
- Mutant 2 (Pro24-Val25) fails to bind both 03 specific Mabs (InnLH1 and InnhCG111) and also reduces binding of 3E2 to 25-50%.
- the two 03 Mabs have separate but overlapping binding sites on ⁇ -hCG, because a single point mutation Pro24 ⁇ His (Mutant 5) completely abolishes binding of Mab InnLH1 but allows partial binding of InnhCG111 (63%), whereas the mutation Val25 ⁇ Tyr (Mutant 6) prevents binding of InnhCG111 and reduces the binding of InnLH1 to 63%. Combining all five point mutations of the N-terminal hairpin loop (Mutant 4) is required to reduce the binding of 3E2 to 13% compared to that of OT3A.
- the design of the mutants was guided by three main principles. Residues selected for mutation should contribute to epitope formation, they should be common to ⁇ -hCG and the cross-reacting luteinizing hormone, and their modification should not significantly influence the overall folding of the molecule at distant sites.
- the successful deletion mutants clearly achieved the primary objective of preserving the functional structure of the ⁇ 1 -specific epitope but considerable progress has also been made towards the secondary aim of identifying the location of the epitopes themselves. We have shown that, even with a molecule like hCG which has complex non-contiguous B-cell epitopes it is possible, to make radical changes in structure which remove unwanted epitopes yet maintain other desirable epitopes.
- the mutant can either be linked to a carrier such as tetanus toxoid, or engineered as a fusion protein with an appropriately immunogenic partner.
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- Chemical & Material Sciences (AREA)
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- Medicinal Chemistry (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Reproductive Health (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU65250/96A AU701702B2 (en) | 1995-07-19 | 1996-07-19 | Modified human chorionic (beta-hCG) proteins and their medical use |
NZ313232A NZ313232A (en) | 1995-07-19 | 1996-07-19 | Modified <beta>-human choronic gonadotropin (<beta>-hcg) and its use in diagnosing and treating kaposi sarcoma |
EP96924986A EP0839197A2 (en) | 1995-07-19 | 1996-07-19 | MODIFIED HUMAN CHORIONIC ($g(b)-hCG) PROTEINS AND THEIR MEDICAL USE |
US08/983,397 US6469139B1 (en) | 1995-07-19 | 1996-07-19 | Modified human chorionic gonadotropin (β-hCG) proteins and their medical use |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9514816.9 | 1995-07-19 | ||
GBGB9514816.9A GB9514816D0 (en) | 1995-07-19 | 1995-07-19 | Substances and their medical use |
Publications (2)
Publication Number | Publication Date |
---|---|
WO1997004098A2 true WO1997004098A2 (en) | 1997-02-06 |
WO1997004098A3 WO1997004098A3 (en) | 1997-02-27 |
Family
ID=10777945
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB1996/001717 WO1997004098A2 (en) | 1995-07-19 | 1996-07-19 | MODIFIED HUMAN CHORIONIC (β-hCG) PROTEINS AND THEIR MEDICAL USE |
Country Status (6)
Country | Link |
---|---|
US (1) | US6469139B1 (en) |
EP (1) | EP0839197A2 (en) |
AU (1) | AU701702B2 (en) |
GB (1) | GB9514816D0 (en) |
NZ (1) | NZ313232A (en) |
WO (1) | WO1997004098A2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6469139B1 (en) | 1995-07-19 | 2002-10-22 | University College London | Modified human chorionic gonadotropin (β-hCG) proteins and their medical use |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
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US20040209999A1 (en) * | 2002-08-16 | 2004-10-21 | Bohling James Charles | Method of manufacturing polypeptides, including T-20 and T-1249, at commercial scale, and polypeptide compositions related thereto |
CA2540838C (en) | 2003-10-03 | 2014-12-16 | Thorn Bioscience, Llc | Process for the synchronization of ovulation for timed breeding without heat detection |
CA2615460A1 (en) * | 2005-08-08 | 2007-02-15 | Onconon, Llc | Antibody compositions, methods for treating neoplastic disease and methods for regulating fertility |
EP2422794A3 (en) | 2006-03-07 | 2013-03-13 | Geeta Shroff | Compositions comprising human embryonic stem cells and their derivatives, methods of use, and methods of preparation |
BRPI1014362A2 (en) | 2009-04-23 | 2016-04-05 | Pennatek Llc | method and composition to synchronize insemination time |
JP2016508030A (en) | 2012-11-28 | 2016-03-17 | ジェイビーエス ユナイテッド アニマル ヘルス セカンド エルエルシー | Methods and compositions for synchronizing the timing of insemination in heifers |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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FR2319379A1 (en) * | 1975-07-29 | 1977-02-25 | All India Inst Medical Scie | Contraceptive vaccine for women - comprising a conjugate of tetanus anatoxin andhuman chlorionic gonadotrophin (3-sub-unit with no strong reaction with anti-LH serium) |
Family Cites Families (8)
Publication number | Priority date | Publication date | Assignee | Title |
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US4302386A (en) | 1978-08-25 | 1981-11-24 | The Ohio State University | Antigenic modification of polypeptides |
US4310455A (en) | 1979-04-17 | 1982-01-12 | Research Corporation | Antigen for early pregnancy test and contraceptive vaccine |
US4803164A (en) * | 1981-08-31 | 1989-02-07 | Genentech, Inc. | Preparation of hepatitis b surface antigen in yeast |
US4966888A (en) | 1985-07-08 | 1990-10-30 | Cornell Research Foundation, Inc. | hCG-hLH receptor and hCG-hLH receptor-hCG complex as antigens, antibodies thereto and contraceptive vaccine |
JP3330373B2 (en) | 1990-05-08 | 2002-09-30 | ユニバーシティー オブ メディスン アンド デンティストリー オブ ニュー ジャージー | Glycoprotein hormone analogs with altered immunological properties, performance and / or receptor specificity |
JPH08502062A (en) | 1992-09-30 | 1996-03-05 | ジ・オハイオ・ステイト・ユニバーシティ・リサーチ・ファウンデーション | Vaccines and antigen conjugates |
US5688506A (en) | 1994-01-27 | 1997-11-18 | Aphton Corp. | Immunogens against gonadotropin releasing hormone |
GB9514816D0 (en) | 1995-07-19 | 1995-09-20 | Ucli Ltd | Substances and their medical use |
-
1995
- 1995-07-19 GB GBGB9514816.9A patent/GB9514816D0/en active Pending
-
1996
- 1996-07-19 NZ NZ313232A patent/NZ313232A/en unknown
- 1996-07-19 AU AU65250/96A patent/AU701702B2/en not_active Ceased
- 1996-07-19 US US08/983,397 patent/US6469139B1/en not_active Expired - Fee Related
- 1996-07-19 WO PCT/GB1996/001717 patent/WO1997004098A2/en not_active Application Discontinuation
- 1996-07-19 EP EP96924986A patent/EP0839197A2/en not_active Withdrawn
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2319379A1 (en) * | 1975-07-29 | 1977-02-25 | All India Inst Medical Scie | Contraceptive vaccine for women - comprising a conjugate of tetanus anatoxin andhuman chlorionic gonadotrophin (3-sub-unit with no strong reaction with anti-LH serium) |
Non-Patent Citations (7)
Title |
---|
9TH INTERNATIONALCONGRESS OF IMMUNOLOGY, vol. 0, no. 0, 23 July 1995 - 29 July 1989, SAN FRANCISCO, CALIFORNIA, USA., page 191 XP002021739 A.M. JACKSON ET AL: "Construction of hCG epitope-loss mutants" * |
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 70, no. 2, 1976, ORLANDO, FL US, pages 525-532, XP000615372 O.P. BAHL ET AL: "Immunological properties of the beta-subunit of human chorionic gonadotropin" * |
ENDOCRINOLOGY, vol. 107, no. 5, October 1980, pages 1556-1563, XP000615394 R.D. GHAI ET AL: "Immunological properties of the beta-subunit of human Chorionic Gonadotropin. I. Effect of chemical and enzymatic modifications" * |
FASEB JOURNAL FOR EXPERIMENTAL BIOLOGY, vol. 7, 1993, BETHESDA, MD US, pages 1381-1385, XP002021737 S. DIRNHOFER ET AL: "Functional and immunological relevance of the COOH-terminal extension of human Chorionic Gonadotropin beta : Implications for the WHO birth control vaccine" cited in the application * |
JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 265, no. 15, 25 May 1990, MD US, pages 8511-8518, XP002021738 W.R. MOYLE ET AL: "Localization of residues that confer Antibody binding specificity using human Chorionic Gonadotropin/Luteinizing Hormone beta subunit chimeras and mutants" * |
JOURNAL OF ENDOCRINOLOGY , vol. 141, April 1994, pages 153-162, XP000612146 S. DIRNHOFER ET AL: "The molecular basis for epitopes on the free beta-subunit of human Chorionic Gonadotropin(hCG), its carboxyl-terminal peptide and the hCGbeta-core fragment" cited in the application * |
JOURNAL OF REPRODUCTIVE IMMUNOLOGY , vol. 31, no. 1-2, 1996, pages 21-36, XP000615397 A.M. JACKSON ET AL: "Identification and selective destruction of shared epitopes in human chorionic gonadotropin beta subunit" * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6469139B1 (en) | 1995-07-19 | 2002-10-22 | University College London | Modified human chorionic gonadotropin (β-hCG) proteins and their medical use |
Also Published As
Publication number | Publication date |
---|---|
EP0839197A2 (en) | 1998-05-06 |
GB9514816D0 (en) | 1995-09-20 |
AU6525096A (en) | 1997-02-18 |
NZ313232A (en) | 1998-10-28 |
WO1997004098A3 (en) | 1997-02-27 |
US6469139B1 (en) | 2002-10-22 |
AU701702B2 (en) | 1999-02-04 |
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