CA2090486A1 - Human zona pellucida protein zp3 - Google Patents
Human zona pellucida protein zp3Info
- Publication number
- CA2090486A1 CA2090486A1 CA002090486A CA2090486A CA2090486A1 CA 2090486 A1 CA2090486 A1 CA 2090486A1 CA 002090486 A CA002090486 A CA 002090486A CA 2090486 A CA2090486 A CA 2090486A CA 2090486 A1 CA2090486 A1 CA 2090486A1
- Authority
- CA
- Canada
- Prior art keywords
- polypeptide
- human
- antibodies
- exon
- nucleic acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 108090000623 proteins and genes Proteins 0.000 title claims description 14
- 102000004169 proteins and genes Human genes 0.000 title claims description 4
- 210000004340 zona pellucida Anatomy 0.000 title description 9
- 101000976442 Homo sapiens Zona pellucida sperm-binding protein 3 Proteins 0.000 claims abstract description 89
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 65
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 62
- 229920001184 polypeptide Polymers 0.000 claims abstract description 59
- 102000055956 human ZP3 Human genes 0.000 claims abstract description 20
- 230000000694 effects Effects 0.000 claims abstract description 6
- 229960005486 vaccine Drugs 0.000 claims abstract description 4
- 239000012634 fragment Substances 0.000 claims description 23
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- 238000012258 culturing Methods 0.000 claims 1
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- 108010038807 Oligopeptides Proteins 0.000 description 2
- 102000015636 Oligopeptides Human genes 0.000 description 2
- 102000008937 Zona Pellucida Glycoproteins Human genes 0.000 description 2
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- 150000007513 acids Chemical class 0.000 description 2
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- 108010043837 egg surface sperm receptor Proteins 0.000 description 2
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- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
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- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
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- 101000976425 Mus musculus Zona pellucida sperm-binding protein 3 Proteins 0.000 description 1
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- ROHDXJUFQVRDAV-UWVGGRQHSA-N Phe-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 ROHDXJUFQVRDAV-UWVGGRQHSA-N 0.000 description 1
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- 241000282898 Sus scrofa Species 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000003302 anti-idiotype Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 229960001714 calcium phosphate Drugs 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
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- 208000000509 infertility Diseases 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 231100000535 infertility Toxicity 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
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- 210000000287 oocyte Anatomy 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
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- 210000004681 ovum Anatomy 0.000 description 1
- 108010051242 phenylalanylserine Proteins 0.000 description 1
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- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- 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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- 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
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
Abstract
The invention comprises a novel polypeptide or a functional derivative thereof having human ZP3 activity or human ZP3 antigenicity. It also comprises epitopes of said polypeptide, antibodies to the polypeptides or the epitopes, as well as vaccines for contraception and methods for expressing the polypeptides in a suitable host.
Description
2 0 9 0 4 g ~ PCI/EP9l/0153X
HUMAN ZONA PELLUCIDA PROTEIN ZP3.
The invention relates to a polypeptide or a functional derivative thereof having human 7.P3 activity or having, at least in part, human ZP3 antigenicity.
During the process of fertilization, the first interaction between mammalian gametes is mediated by binding of sperm cells to a species specific receptor on the zona pellucida (ZP) that surrounds the female gamete. The ZP is an extracellular matrix which comprises three glycoproteins, designated ZP1, ZP2 and ZP3, of which ZP3 has been identified as the sperm receptor (reviewed in Wassarman, Development 108, 1-17; 1990). . ..
Numerous in vitro and in vivo studies using both porcine and murine ZP
proteins have indicated that ZP3 is an important candidate target antigen in experimental strategies aimed at the development of immuno-coneraception (Henderson et al., ~. Reprod. Fert. 83, 325-343; 1988 and refs. therein). The recent cloning and characterization of the murine ZP3 cDNA by screening of a mouse ovary cDNA expression library with anti-mouse ZP3 antibodies represents an important step towards this end (Ringuet~e et al., Developmental Biology 127, 287-29~; 1988). Subsequently, ZP3 cDNA
probes were used for isolation of the corresponding genomic ZP3 DNA
(Chamberlin and Dean, Devèlopmental Biology 131, 207-214, 1988; Kinloch et al., Proc. Natl. Acad. Sci. US~ 85, 6409~6413,1989).
The potential of ZP3 for contraception was emphasized by studies that revealed a long term infertility following vaccination of female mice with an oligopeptide derived from the murine ZP3 amino acid sequence (Millar et al., Science 246, 935-938; 1989). ;:~
For immunocontraception in man, mouse polypeptides are not suitable since - -the ZP3 sperm receptor is species specific. Moreover, polypeptides from non-human origin may lead to unwanted immune responses in man.
Therefore, development of a safe and efficient contraceptive vaccine based on (part of) the human sperm receptor requires information on and availability of the human ZP3 polypeptide.
W0 92/03548 2 0 9 0 9 8;6 i PCI /EP91/0153R
The isolation of sufficient amounts of human ZP3 polypeptide from female gametes is of course not possible. However, we have succeeded in identifying a human gene and elucidating its sequence. This opens the possibility to produce ZP3 polypeptides and/or ZP3 polypeptide fragments either by -~ -recombinant DNA technology or solid phase polypeptide synthesis.
The invention therefore comprises a novel DNA-molecule coding for at least a part of human ZP3 polypeptide. Said DNA-molecule comprises at least a part of the sequence shown in Fig. 2.
The invention also comprises a polypeptide coded for by at least part of the above-mentioned DNA-molecule. Said polypeptide may comprise at least a part of the amino acid sequence shown in Fig. 2.
Of course, functional derivatives as well as fragments of the polypeptides .;
according to the invention are also included in the present invention.
Functional derivatives are meant to include polypeptides in which one or more of the arnino acids have been replaced with a chemically comparable one.
The crux is of course that they should still show human ZP3 antigenicit~.
This means that upon administration (possibly with an adjuvant) they should give rise to antibodies which recognize human ZP3.
The polypeptides according to the invention can be produced either synthetically or by recombinant DNA technology. Methods for producing -svnthetic polypeptides are well known in the art and do not need any further elaboration.
Production of polypeptides by recombinant DNA techrliques is a general method which is known, but which has a lot of possibilities all leading to ;
somewhat different results. The polypeptide to be expressed is coded for by a DNA sequence or more accurately by a nucleic acid sequence.
This nucleic acid sequence must be transcribed (optionally) and translated to the wanted polypeptide. In order to reach that goal the nucleic acid sequence is normally cloned into a vector with which a host cell is transformed. The vector can be either self replicating or it may integrate into the DNA of the host.
.
,.
- WC) 92/03548 2 0 ~ 0 4 8 6 Pcr/Epsl/ols38 , Different host cells lead to different polypeptides~ Prokaryotes do notpossess the organelles necessary for glycosylation~ The polypeptides produced by them will be without carbohydrate side chains~ Eukar,yotes do have the glycosylation machinery, but yeast cells will give a different glycosylation pattern than mamrnalian cells.
Preferred for the polypeptides according to the invention is an expression -system which gives the most "natural" glycosylation pattern. Towards this end ~ -marnmalian cells are most preferred.
We have also identified epitopes on the human ZP3 polypeptide which may have contraceptive potential. These epitopes comprise at least a part of one of the following sequences:
-ThrLeuMetValMetValSerLys-, - --SerArgArgGlnProHisValMetSerGln-, -GluValGly~euHisGluCysGlyAsnSerMetGlnValThrAspAspAlaLeu~
-PheSerLeuArgLeuMetGluGluAsnTrpAsnAlaGluLysArgSerProThrPhe-, , .
-CysGlyThrProSerHisSerArgArgGlnProHisValMetSerGlnTrpSer-, or -SerGlnTrpSerThrSerAlaSerArgAsnArgArgHisValThrGlUGlu AlaAspValCysValGlyAlaThrAspLeuProGlyGlnGluTrp-. .
Small polypeptides like these are more easily produced synthetically. They can also be linked together with the same or different "epitopes" to form a larger more antiger,lic polypeptide.
One aim of all the polypeptides according to the invention is to produce a contraceptive vaccine using them. The vaccination may be either a passive or an active immunization.
HUMAN ZONA PELLUCIDA PROTEIN ZP3.
The invention relates to a polypeptide or a functional derivative thereof having human 7.P3 activity or having, at least in part, human ZP3 antigenicity.
During the process of fertilization, the first interaction between mammalian gametes is mediated by binding of sperm cells to a species specific receptor on the zona pellucida (ZP) that surrounds the female gamete. The ZP is an extracellular matrix which comprises three glycoproteins, designated ZP1, ZP2 and ZP3, of which ZP3 has been identified as the sperm receptor (reviewed in Wassarman, Development 108, 1-17; 1990). . ..
Numerous in vitro and in vivo studies using both porcine and murine ZP
proteins have indicated that ZP3 is an important candidate target antigen in experimental strategies aimed at the development of immuno-coneraception (Henderson et al., ~. Reprod. Fert. 83, 325-343; 1988 and refs. therein). The recent cloning and characterization of the murine ZP3 cDNA by screening of a mouse ovary cDNA expression library with anti-mouse ZP3 antibodies represents an important step towards this end (Ringuet~e et al., Developmental Biology 127, 287-29~; 1988). Subsequently, ZP3 cDNA
probes were used for isolation of the corresponding genomic ZP3 DNA
(Chamberlin and Dean, Devèlopmental Biology 131, 207-214, 1988; Kinloch et al., Proc. Natl. Acad. Sci. US~ 85, 6409~6413,1989).
The potential of ZP3 for contraception was emphasized by studies that revealed a long term infertility following vaccination of female mice with an oligopeptide derived from the murine ZP3 amino acid sequence (Millar et al., Science 246, 935-938; 1989). ;:~
For immunocontraception in man, mouse polypeptides are not suitable since - -the ZP3 sperm receptor is species specific. Moreover, polypeptides from non-human origin may lead to unwanted immune responses in man.
Therefore, development of a safe and efficient contraceptive vaccine based on (part of) the human sperm receptor requires information on and availability of the human ZP3 polypeptide.
W0 92/03548 2 0 9 0 9 8;6 i PCI /EP91/0153R
The isolation of sufficient amounts of human ZP3 polypeptide from female gametes is of course not possible. However, we have succeeded in identifying a human gene and elucidating its sequence. This opens the possibility to produce ZP3 polypeptides and/or ZP3 polypeptide fragments either by -~ -recombinant DNA technology or solid phase polypeptide synthesis.
The invention therefore comprises a novel DNA-molecule coding for at least a part of human ZP3 polypeptide. Said DNA-molecule comprises at least a part of the sequence shown in Fig. 2.
The invention also comprises a polypeptide coded for by at least part of the above-mentioned DNA-molecule. Said polypeptide may comprise at least a part of the amino acid sequence shown in Fig. 2.
Of course, functional derivatives as well as fragments of the polypeptides .;
according to the invention are also included in the present invention.
Functional derivatives are meant to include polypeptides in which one or more of the arnino acids have been replaced with a chemically comparable one.
The crux is of course that they should still show human ZP3 antigenicit~.
This means that upon administration (possibly with an adjuvant) they should give rise to antibodies which recognize human ZP3.
The polypeptides according to the invention can be produced either synthetically or by recombinant DNA technology. Methods for producing -svnthetic polypeptides are well known in the art and do not need any further elaboration.
Production of polypeptides by recombinant DNA techrliques is a general method which is known, but which has a lot of possibilities all leading to ;
somewhat different results. The polypeptide to be expressed is coded for by a DNA sequence or more accurately by a nucleic acid sequence.
This nucleic acid sequence must be transcribed (optionally) and translated to the wanted polypeptide. In order to reach that goal the nucleic acid sequence is normally cloned into a vector with which a host cell is transformed. The vector can be either self replicating or it may integrate into the DNA of the host.
.
,.
- WC) 92/03548 2 0 ~ 0 4 8 6 Pcr/Epsl/ols38 , Different host cells lead to different polypeptides~ Prokaryotes do notpossess the organelles necessary for glycosylation~ The polypeptides produced by them will be without carbohydrate side chains~ Eukar,yotes do have the glycosylation machinery, but yeast cells will give a different glycosylation pattern than mamrnalian cells.
Preferred for the polypeptides according to the invention is an expression -system which gives the most "natural" glycosylation pattern. Towards this end ~ -marnmalian cells are most preferred.
We have also identified epitopes on the human ZP3 polypeptide which may have contraceptive potential. These epitopes comprise at least a part of one of the following sequences:
-ThrLeuMetValMetValSerLys-, - --SerArgArgGlnProHisValMetSerGln-, -GluValGly~euHisGluCysGlyAsnSerMetGlnValThrAspAspAlaLeu~
-PheSerLeuArgLeuMetGluGluAsnTrpAsnAlaGluLysArgSerProThrPhe-, , .
-CysGlyThrProSerHisSerArgArgGlnProHisValMetSerGlnTrpSer-, or -SerGlnTrpSerThrSerAlaSerArgAsnArgArgHisValThrGlUGlu AlaAspValCysValGlyAlaThrAspLeuProGlyGlnGluTrp-. .
Small polypeptides like these are more easily produced synthetically. They can also be linked together with the same or different "epitopes" to form a larger more antiger,lic polypeptide.
One aim of all the polypeptides according to the invention is to produce a contraceptive vaccine using them. The vaccination may be either a passive or an active immunization.
3~48 2 0 9 0 4 8 6 pcr/Epsl /01 ~3X - ~
. , .
. \ . . ~ , For an active immunization a polypeptide according to the invention is adnunistered to a female (possibly ~,vith an adjuvant). The administration will giYe rise to an immune response by the female. Antibodies will be produced -which recognize human ZP3 on the ovum. These antibodies will specifically bind to the sperm receptor binding site so that sperrna~ozoa cannot bind, or ~ -otherwise the antibodies will prevent this binding through steric hindrance.
Passive irnmunization basically works the same way. Instead of the antigen or its rnirnicry the antibodies against it are directly adrninistered. Thus, antibodies have to be raised against the polypeptides according to the invention.
This is achieved through an active immunization scheme of a suitable mammal. The B-lymphocytes of the marmnal are harvested after a suitable period of time and immortalized through fusion or transformation These methods are well known in the art. Antibodies can be isolated from the culture of the immortalized Iymphocytes.
There is, however, a problem with antibodies of animal origin. I~pon repeated administration they will give rise to an anti-antibody response in the immunized woman. It is therefore preferred to use humanized antibodies or small parts of antibodies which will not lead to an immune response.
Methods for humanizing antibodies, such as CDR-grafling~ are known ~Jones et al., Nature 321, 522-525, 1986). Methods for producing fragments of antibodies which are still specific for the antigen of the original antibod are also known (Udaka et al., Molec. lmmunol. 27, 25-35~ 1990). Ano~her possibility to avoid antigenic response to antibodies to polypeptides according to the invention is the use of human antibodies or fragmenrs or derivatives thereof.
Human antibodies can be produced by in vltro stimulation of isolated B-Iymphocytes, or they can be isolated from (immortalized) B-lymphocytes which have been harvested from a female immunized w~th at least one polypeptide according to the invention. Another object of the invention is the use of ZP3 polypeptide and antibodies directed to it in diagnostic test kits.
Some antibodies to human ZP3 have now been obtained. It lies within the skill of the art to produce anti-idiotype antibodies which recoenize the antigen binding site of the antibody and therefore are an "internal image" of the antigen.
. ,::
~ WO 92/03548 2 0 9 ~ 4 8 6 pcr/Epsl/ol53~ .
..
These anti idio~ype antibodies will be also very useful for vaccine and diagnostic purposes.
In the following experimental part the isolation of the human ~P3 DNA-sequence and the human ZP3 amino acid sequence, as well as a method of expressing a recombinant polypeptide showing zn antigenicity, as well as a ~ .procedure tO make a synthetic polypeptide showing such antigenici2y are :
shown~ These examples are merely meant to illustra1e the invention and should not be constructed as a limita2ion of its scope.
` ' ' WO 9V03548 2 0 9 0!4 8 6 pcr/Ep9l/ol53~
EXAMPLES.
' MATERIALS AND METHODS.
A~l recombinant DNA procedures were performed essentially according lo procedures known as such (Sambrook et al., Molecular Cloning, a laboratory manual, 2nd edition, CSH-laboratory press, 1989).
Restriction enz~nes and DNA modi~ing enzyrnes were used as recornrnended by the suppliers.
A human ovary gtlO cDNA library was purchased from Stratagene. `
Oligonucleotide synthesis.
Oligonucleotides were prepared on an Applied Biosvstems 381A DNA ;
synthesizer and directly used for cloning purposes.
Peptide svnthesis.
Oligopeptides were produced by solid phase peptide svnthesis using procedures described by Fields and Noble (Int. J. Pept. Prot. Res. ~5, 160-214;
1990).The following ZP3 peptides were synthesized:(amino acids are given `:
in 3 letter code) - ZP3(93-110):
GluValGlyLeuHisGluCysGlyAsnSerMetGlnValThrAspAspAlaLeu ~ `: - ZP3(172-190):
PheSer~euArgLeuMetGluGluAsnTrpAsnAlaGluLysArgSerProThrPhe ' ' - ZP3(327-344):
CvsGlyThrProSerHisSerArgArgGlnProHisValMetSerGlnTrpSer - ZP3(341-360):
SerGlnTrpSerThrSerAlaSerArgAsnArgArgHisValThrGluGluA~aAspVal - ZP3(362-3~2): ;;
CysValGlyAlaThrAspLeuProGlyGlnGluTrp :
An~:EQon.
Polyclonal antisera were raised with various antigens:
- Total human zona-pellucida. Salt stored human zonae pellucidae were heat solubilized and mixed with freunds adjuvants for immunization of rabbits. Antiseraum titers were assaved on ELISA plates coated ~ith porcine ZP-proteins.
. . .
",` ', ` ~ WO 92/03~48 2 0 9 0:~ 8 6 pcrr/Epsl/ols3x .
nc; al~P3 fusion protein. The fusion protein was partially purified from sonicated bacteria based on its insolubility and separated by SDS
polyacrylarnide gelelectrophoresis (SDS-P~GE). By electro-blotting the proteins were transferred to a nitrocellulose membrane. l he region carrving the hybrid protein was excised and dissolved in DMSO. This was mixed in a 1:1 ratio with Freunds adjuvants and used for immul~ization of rabbits.
- CHO produced ZP3. A similar procedure was followed for the ZP3 protein produced by Chinese harnster ovary (CHO) cells. Concentrated culture medium was separated by SDS-PAGE. The 40-60 kiloDalton region 'r ~ .
was excised and used for immur~ization of rnice.
- ()ligopeptides. Synthesized peptides were physicallv linked to l;eyhole limpet hemocyanin (KLH) and injected in rabbits. As a control rabbits were immunized with KLH only. Sera were screened on ELISA microtitre plates coa~ed with peptide.
Human egg fluorescence assav.
Salt stored unfertilized human eggs (after 48 hour incubation with sperm in an IVF program) were incubated with antisera (1:50 diluted in buffer A
[PBS + 5~o BSA]). After three washes eggs were incubated with a second antibody (swine anti rabbit or mouse conjugated to FITC, 1:100 diluled in buffer A) and incubated for 1 hour at 37 C. Following three additional wash steps fluorescence was measured with a Nikon microscope and exposure analyser. Non-stained (negative) control egPs are darli. with a iong exposure time, and positivelv stained zonae pellucidae have shorter exposure-times.
Human sperm-zona bindin~ assay. -Human eggs (see above), were incubated in rabbit serum, washed 3 times with buffer (BWW + 3% BSA), incubated in buffer (control) or antibodies -(undiluted, 37C,lhour), washed 3 times and introduced in droplets of human capacitated spermatozoa (107 sperm/ml, 37 C~ 16 hour). Loosely adherent spermatozoa were removed from eggs by repeated pipetting.
Bound spermatozoa were fixed and stained with BWW containing 1~c glutaraldehyde and Hoechst (H33258, 20 g/ml) and the number of bound spermatozoa were counted with a fluorescence microscope.
. ">,., .~ .. ~ . .. ..... . . . . . .
.... ... . . . ~ - ......................... .. .. . : ~
- . - - . , ~ ........................... . . .:
. :: , , - : :: :
WO 92/03548 2 0 9 0 ~ 8 6' ` pcr/Ep9l/ols3y .
RESULTS~
Synthesis and co~truction pf a mpuse ZP3 Eobe~
A 135 bp mouse ZP3 probe was constructed by assembling 8 synthetic oligonucleotides (27-51 mers). This fragment, comprising part of exon 5 and 6 of the murine gene (position 771 to 909 in Ringuette et al., Developmental : ~
Biology 127, 287-295; 1988) was provided with unique 5' BamHI and 3' ~ -HindIII restriction sites and subcloned in pGEM3 (Promega). Subsequently its nucleotide seguence was verified.
: .
ClQning and characterization of the human ZP3 ~ene.
A human genomic EMBL3 library was screened with a 32P-labeled ZP3 probe (135 bp fragment described above). Three overlapping EMBL clones ;
showed a strong hybridization and were characterized in more detail. A
limited physical map of these clones is schematically depicted in Fig. 1.
Restriction fragments from clones 11 and D1 were subcloned in pGEM
vectors and M13mpl8/19 and used for genomic characterization:of the -human gene. This involved localization of the exons bv Southern blot .:',` ,~!;.` .
analysis with 32P-labeled oligonucleotides from the rnouse ZP3 sequence followed by sequence analysis. As is demonstrated in Fig. 1, the human gene consists of 8 exons spread over approximately 20 }ib of genomic DNA. All identified exons are flanked by consensus splice donor and splice acceptor -signals (not shown). From sequence analysis of all exons the complete coding sequence for the ZP3 gene could be deduced (presented in Fig. 2). -The human ZP3 gene encodes a polypeptide of 372 amino acids with a calculated molecular weight of 41437 Dalton.
ClQnin~ of human ZP3 cl:)N~
A human ovary cDNA library tgt10) was screened with 32P-labeled fragments from human ZP3 exon 1, 5 and 7. This yielded several partial cDNA's that contained only the 3' half of the ZP3 coding seguence t exon 5 to8).
' ~
"'''''' .
. - ' WO 92/03548 2 0 9 0 4 8 6 ; PCl /EP9 1 /01 53g Sequence analysis of 3 independent cDNA clones confirmed the previously determined genomic ZP3 sequence except for one residue in exon 7 (see Fig.
2)~ At nucleotide position 1064 a G and C residue was found in genomic and cDNA respectively. In the encoded amino acid sequence this yields an arginine or a threonine residue respectively at position 345. This sequence difference probably represents a polvmorphism in the human ZP3 gene and polypeptide.
Expression of recombinant ZP3 in CHO cells.
For expression in CHO cells the size of the ZP3 gene has been reduced to allow insertion in a mammalian expression vector. Various ZP3 DNA
fragments have been assembled to a 'minigene~ as is illustrated in Fig. 3.
Exon 1 and 2 were joined by PCR-technology (Horton et al., Gene 77, 61-68, 1989; Yon and Fried, Nucl. Acids Res. 17, 4895, 1989)) and provided with 5' EcoRI and 3' XbaI sites respectively. Subsequently, this DNA
fragment was ligated to a genornic XbaI-SalI fragment carrying exon 3, 4 and part of exon 5 and the partial cDNA harboring exon 5 to 8 on a SalI-HindIII fragment. The resulting 2.7 kb 'minigene' contains a truncated intron between exon 2 and 3 and the natural introns between ZP3 exons 3 and 4 and exon 4 and 5. The integrity of the polypeptide encoding nucleotide information of this ZP3 construct was completely verified by sequence ~ `
analysis.
The ZP3 `minigene' was subsequently inserted in a mammalian expression vector in which the ZP3 gene is driven by the strong SV40 early promoter. In addition, this vector harbored ~-GIobin splicing and SV40 poly-adenylation signals for correct RNA processing of the expressed gene and the selectable marker gene aminoglycosylphosphotransferase (Colbere-Garapin et al., J.
Mol. Biol. 150, 1~14, 1981~ which allows for isolation of stable transformants by selection for G418 resistance. CHO cells were transfected with the ZP3 expression construct using the calcium-phosphate precipitation technique (Graham and van der Eb, Virology 52, 456-467, 1973; Wigler et al., Proc.
Natl. Acad. Sci USA 76,1373-1376, 1979). Mass populations of G418 resistant transformants ~representing 300-5~0 independent clones) were analysed for expression of the recombinant ZP3 gene.
- ., - ,. ~ . - ,.
. ~ , . , ~ - - -, -- - . . . , -WO 92/03~48 2 0 9 0 4 8 6 PCT/EP9l/01538-;.
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As is demonstrated in Fig~ 4, Northern blot analysis of total RNA from pooled transformants with a 32P-labeled ZP3 probe reveals a relatively high level of expression of the transfected ZP3 gene (as compared to ~he highly -expressed actin gene, not shown). Moreover, it was demonstrated that the minigene RNA is correctly processed to a mRNA which is slightlv larger ~ -then the natural transcript found in human oYary RNA (see Fig. 4). This size difference is most likely due to flanking 5' and 3' sequences present in the expression vector. .
Correct splicing and processing of the recombinant gene was further confirmed by isolation of a partial cDNA carlying exon 1 to 5 from G418 resistant CHO transformants. Sequence data of this cDNA were similar to the transfected DNA and support the conclusion that the introns are correctly spliced from the ZP3 rninigene transcript.
Culture media of growing mass populations were examined for the presence -of recombinant ZP3 polypeptide by Western blot analysis. With a polyclonal antiserum directed against deglycosylated porcine ZP3 (Henderson et al., Gamete Research 18, 251-265, 1987) recombinant ZP3 polypeptide could be detected in culture medium. This signal was absent in medium from non-transfected CHO cells.
Expression of recombinant ZP3 in E. coli.
For expression in E.coli first a full lenght ZP3 cDNA was constructed using a cDNA fragment covering exon 1 to 5 isolated by PCR from CHO
transformants and the partial cDNA clone ~harboring exon 5 to 8) isolated ;, from a human ovary cDNA library. Four 2;P3 cDNA fragments were cloned in frame to the E.coli LacZ gene in pEX-plasmids (Biores) allowing the production of a B-galactosidase-ZP3 fusion polypeptide as reported by Stanley and Luzio, EMBO J. 3, 1429-1434, 1984). The expression of fusion polypeptides in E.coli was examined by SDS polyacrylamide ~ -gelelectrophoresis (SDS-PAGE) and western blot analysis. The results of these experiments are presented in Fig. 5. Following heat shock induction, all four LacZ-ZP3 constructs yielded fusion polypeptides, although to a ;
much lesser extend then the LacZ gene. The molecular weight of the hybrid polypeptides, as observed on coomassie stained gels (see Fig. 5A), were in agreement with the expressed ZP3 parts. 'rhis was further confirmed by -western blot analysis with anti-B-galactosidase antibodies (not shown) and a rabbit antiserum raised against ZP3 amino acids 341 to 360 coupled to KLH - :
~: .
- , - . .. . ~ . . . . . . . .. . .. . . ..
WO 92/03548 2 0 9 0 4 8 6 PCl/EP9l/0l53 , (Fig. 5B), Only fusion polypeptides carrying this region, i.e. ZP3-A and ZP3-I:), were recognized by the an~iserum. An antiserum against ~LH yielded a result sirn~lar to the background staining in Fig. SB (not shown). In addition, ~Gal-ZP3 fusion polypeptides were recognized by antisera raised against total human zonae pellucidae (not shown).
Binding of ZP3 antibodies to human eggs.
A human egg fluorescence assay was developed to characterize the binding of the various ZP antibodies to human zonae pellucidae. The results of three i i~
different experiments are depicted in Fig. 6. Serum against KLH and a normal mouse serum failed to give fluorescent staining whereas with all ;
antisera against ZP components strong to intermediate levels of fluorescence were observed. From the presented data it can be inferred that antisera against ZP3(93-110), ZP3(172-190), ZP3(327-344), ZP3(341-360) and ZP3(362-372) recognize human oocytes. ~-.
Effect of ZP antibodies on sperm-egg recognition. ~-The contraceptive potential of the ZP3 antibodies was analYsed in a human sperm-zona bindin" assay. The results of this assay is presented in Fig. 7.
Antibodies against total human zonae pellucidae showed 75 /c inhibition of sperm-zona binding. An antiserum against KLH does not interfere with sperm-egg binding. In contrast, a m~xture of ZP3-peptide antibodies (peptides: ZP3(93-110), ZP3(172-190), ZP3(341-360) and ZP3(362-372~j showed a relative inhibition of sperm binding of 35%. Antibodies a~ainst recombinant ZP3 from CHO cells reduced sperm binding with 55~G.
WO ~2/03548 2 0 9 0 ~ 8 ~` pcr/Ep9l/ol53p Legends to ~he Figures.
Figure 1.
Restriction maps of EMBL clones I1, Dl and El (A) and genomic organization of the human ZP3 gene. Exons are indicated by blacl~ boxes. ' -Restriction sites: S, SalI; B, BarnHI.
Figure 2. f Nucleotide and arnino acid sequence of human ZP3. At position 1064 a G or ~ `
C residue was found in genomic and cDNA respectively. This apparent polyrnorphism yields a ~hr or Alg amino acid residue. Arrows indicate exon junctions. Exon number is indicated in circies. The po]vadenv]ation signa] is underlined.
Figure 3. :Size reduction of ZP3 gene for insertion in a mammalian expression vector.
A 2.7 kb ~P3 'rninigene' was assembled from the following three fragments~
A, exon 1 and exon 2 joined in frame to each other by PCR-techno]ogv yielding a EcoRI-XbaI fragment; B, a genomic XbaI-Sa]I fragment with exon 3, d, and part of exon 5; C, a SalI-HindlII ZP3 cDNA fragment with exon 5 tO
8 (provided with artificial 3' BamHI and HindIII siles). The three fra~ments were cloned in a pGEM vector (Promega) opened with EcoRl and HindII~
and subsequently as a 2.7 kb EcoRI-BamHI fragment placed behind the SV40 promoter in a mammalian expression vector.
,: .
Figure 4.
Detection of ZP3 mRNA in total RNA (15 g) from C~O mass populations transfected with the :ZP3 DNA and poly(A)+ RNA (j g) from a human ovarv. Lane l, CHO; lane 2, CHO ZP3 transformants; lane 3. human ovary. ~ `
Total ZP3 cDNA in pGEM3 was used as a probe. `
Figure S.
Production of ~Gal-ZP3 fusion polypeptides in E.coli strain POP~136 carrving pEX-plasmids with human ZP3 cDNA inserts.
.
:` :
WO 92/03548 2 ~ 9 0 ~ 8 6 PCr/EP91/0153X
, .. , ,: .
.. . .
A~ Coomassie staining of polyacrylamide gel (lO~o) and B. Western blot : -analysis with anti-ZP3 341-360 antiserum (1:150 dilution). From left to right the follo~ving samples are loaded on the gels (-, before induction; + 2 hours after induction): control pEX; pEX-ZP3A (KpnI-BamHI fragment; total cDNA); pEX-ZP3B (KpnI-SalI fragment; cDNA truncated at exon 5 encoding amino acid 1-241); pEX ZP3C (Kpr~-SphI fragment; cDNA
truncated at end of exon 1 encoding arnino acid 1-102); pEX-ZP3D (SmaI- i-BarnHI fragment, 3' half of exon 5 + exon 6,7 and 8 encoding arruno acid 257-372). Molecular weight (M) markers are at the extreme left (indicated in kiloDalton). Bacteria were grown at 30 C till OD600 = 0.~-1 and '' ',!,~
subsequently induced for two hours at 42 C. Aliquots were taken, centrifuged and total cells Iysates were analysed.
Figure 6.
Binding of different antibodies (see materials and methods) to human eggs. - ~-Amount of second antibody labeled with FITC was measured with an ~ -exposure analyser and expressed as percentage of conlrol (100C/G = dark, O~c ;
= fluorescent eggs). 1: control; 2: antiserum against human zonae - pellucidae; 3: antiserum against KLH; 4-8: antisera agains~ ZP3(93-110), ~.
ZP3(172-l90), ZP3S327 344), ZP3(341-350) and ZP3(362-372); 9: antiserum against ~Ga]-ZP3; lO:mixture of sera of 3 mice immunized with recombinant CHO produced ZP3; 11: mixture of sera of 3 control mice. Average exposure time as percentage of control and standard error of the mean are -given. . . ..
:. .
Figure 7. .
Inhibition of sperm-zona binding by anti-ZP3 antibodies (see materials and methods). l: control; 2: antiserum against KLH; 3: antiserum against human zonae pellucidae 4: rnixture of antis~ra against ZP3(93-110), ZP3(172-190), ZP3(341-360) and ZP3~362-372); 5: mixture of sera of 3 mice immunized with recombinant CHO produced ZP3. Average number of boupd spermatozoa on total zonae pellucidae and standard error of the mean are given.
~' ,. .. . . _ . .. .. . ~
. , .
. \ . . ~ , For an active immunization a polypeptide according to the invention is adnunistered to a female (possibly ~,vith an adjuvant). The administration will giYe rise to an immune response by the female. Antibodies will be produced -which recognize human ZP3 on the ovum. These antibodies will specifically bind to the sperm receptor binding site so that sperrna~ozoa cannot bind, or ~ -otherwise the antibodies will prevent this binding through steric hindrance.
Passive irnmunization basically works the same way. Instead of the antigen or its rnirnicry the antibodies against it are directly adrninistered. Thus, antibodies have to be raised against the polypeptides according to the invention.
This is achieved through an active immunization scheme of a suitable mammal. The B-lymphocytes of the marmnal are harvested after a suitable period of time and immortalized through fusion or transformation These methods are well known in the art. Antibodies can be isolated from the culture of the immortalized Iymphocytes.
There is, however, a problem with antibodies of animal origin. I~pon repeated administration they will give rise to an anti-antibody response in the immunized woman. It is therefore preferred to use humanized antibodies or small parts of antibodies which will not lead to an immune response.
Methods for humanizing antibodies, such as CDR-grafling~ are known ~Jones et al., Nature 321, 522-525, 1986). Methods for producing fragments of antibodies which are still specific for the antigen of the original antibod are also known (Udaka et al., Molec. lmmunol. 27, 25-35~ 1990). Ano~her possibility to avoid antigenic response to antibodies to polypeptides according to the invention is the use of human antibodies or fragmenrs or derivatives thereof.
Human antibodies can be produced by in vltro stimulation of isolated B-Iymphocytes, or they can be isolated from (immortalized) B-lymphocytes which have been harvested from a female immunized w~th at least one polypeptide according to the invention. Another object of the invention is the use of ZP3 polypeptide and antibodies directed to it in diagnostic test kits.
Some antibodies to human ZP3 have now been obtained. It lies within the skill of the art to produce anti-idiotype antibodies which recoenize the antigen binding site of the antibody and therefore are an "internal image" of the antigen.
. ,::
~ WO 92/03548 2 0 9 ~ 4 8 6 pcr/Epsl/ol53~ .
..
These anti idio~ype antibodies will be also very useful for vaccine and diagnostic purposes.
In the following experimental part the isolation of the human ~P3 DNA-sequence and the human ZP3 amino acid sequence, as well as a method of expressing a recombinant polypeptide showing zn antigenicity, as well as a ~ .procedure tO make a synthetic polypeptide showing such antigenici2y are :
shown~ These examples are merely meant to illustra1e the invention and should not be constructed as a limita2ion of its scope.
` ' ' WO 9V03548 2 0 9 0!4 8 6 pcr/Ep9l/ol53~
EXAMPLES.
' MATERIALS AND METHODS.
A~l recombinant DNA procedures were performed essentially according lo procedures known as such (Sambrook et al., Molecular Cloning, a laboratory manual, 2nd edition, CSH-laboratory press, 1989).
Restriction enz~nes and DNA modi~ing enzyrnes were used as recornrnended by the suppliers.
A human ovary gtlO cDNA library was purchased from Stratagene. `
Oligonucleotide synthesis.
Oligonucleotides were prepared on an Applied Biosvstems 381A DNA ;
synthesizer and directly used for cloning purposes.
Peptide svnthesis.
Oligopeptides were produced by solid phase peptide svnthesis using procedures described by Fields and Noble (Int. J. Pept. Prot. Res. ~5, 160-214;
1990).The following ZP3 peptides were synthesized:(amino acids are given `:
in 3 letter code) - ZP3(93-110):
GluValGlyLeuHisGluCysGlyAsnSerMetGlnValThrAspAspAlaLeu ~ `: - ZP3(172-190):
PheSer~euArgLeuMetGluGluAsnTrpAsnAlaGluLysArgSerProThrPhe ' ' - ZP3(327-344):
CvsGlyThrProSerHisSerArgArgGlnProHisValMetSerGlnTrpSer - ZP3(341-360):
SerGlnTrpSerThrSerAlaSerArgAsnArgArgHisValThrGluGluA~aAspVal - ZP3(362-3~2): ;;
CysValGlyAlaThrAspLeuProGlyGlnGluTrp :
An~:EQon.
Polyclonal antisera were raised with various antigens:
- Total human zona-pellucida. Salt stored human zonae pellucidae were heat solubilized and mixed with freunds adjuvants for immunization of rabbits. Antiseraum titers were assaved on ELISA plates coated ~ith porcine ZP-proteins.
. . .
",` ', ` ~ WO 92/03~48 2 0 9 0:~ 8 6 pcrr/Epsl/ols3x .
nc; al~P3 fusion protein. The fusion protein was partially purified from sonicated bacteria based on its insolubility and separated by SDS
polyacrylarnide gelelectrophoresis (SDS-P~GE). By electro-blotting the proteins were transferred to a nitrocellulose membrane. l he region carrving the hybrid protein was excised and dissolved in DMSO. This was mixed in a 1:1 ratio with Freunds adjuvants and used for immul~ization of rabbits.
- CHO produced ZP3. A similar procedure was followed for the ZP3 protein produced by Chinese harnster ovary (CHO) cells. Concentrated culture medium was separated by SDS-PAGE. The 40-60 kiloDalton region 'r ~ .
was excised and used for immur~ization of rnice.
- ()ligopeptides. Synthesized peptides were physicallv linked to l;eyhole limpet hemocyanin (KLH) and injected in rabbits. As a control rabbits were immunized with KLH only. Sera were screened on ELISA microtitre plates coa~ed with peptide.
Human egg fluorescence assav.
Salt stored unfertilized human eggs (after 48 hour incubation with sperm in an IVF program) were incubated with antisera (1:50 diluted in buffer A
[PBS + 5~o BSA]). After three washes eggs were incubated with a second antibody (swine anti rabbit or mouse conjugated to FITC, 1:100 diluled in buffer A) and incubated for 1 hour at 37 C. Following three additional wash steps fluorescence was measured with a Nikon microscope and exposure analyser. Non-stained (negative) control egPs are darli. with a iong exposure time, and positivelv stained zonae pellucidae have shorter exposure-times.
Human sperm-zona bindin~ assay. -Human eggs (see above), were incubated in rabbit serum, washed 3 times with buffer (BWW + 3% BSA), incubated in buffer (control) or antibodies -(undiluted, 37C,lhour), washed 3 times and introduced in droplets of human capacitated spermatozoa (107 sperm/ml, 37 C~ 16 hour). Loosely adherent spermatozoa were removed from eggs by repeated pipetting.
Bound spermatozoa were fixed and stained with BWW containing 1~c glutaraldehyde and Hoechst (H33258, 20 g/ml) and the number of bound spermatozoa were counted with a fluorescence microscope.
. ">,., .~ .. ~ . .. ..... . . . . . .
.... ... . . . ~ - ......................... .. .. . : ~
- . - - . , ~ ........................... . . .:
. :: , , - : :: :
WO 92/03548 2 0 9 0 ~ 8 6' ` pcr/Ep9l/ols3y .
RESULTS~
Synthesis and co~truction pf a mpuse ZP3 Eobe~
A 135 bp mouse ZP3 probe was constructed by assembling 8 synthetic oligonucleotides (27-51 mers). This fragment, comprising part of exon 5 and 6 of the murine gene (position 771 to 909 in Ringuette et al., Developmental : ~
Biology 127, 287-295; 1988) was provided with unique 5' BamHI and 3' ~ -HindIII restriction sites and subcloned in pGEM3 (Promega). Subsequently its nucleotide seguence was verified.
: .
ClQning and characterization of the human ZP3 ~ene.
A human genomic EMBL3 library was screened with a 32P-labeled ZP3 probe (135 bp fragment described above). Three overlapping EMBL clones ;
showed a strong hybridization and were characterized in more detail. A
limited physical map of these clones is schematically depicted in Fig. 1.
Restriction fragments from clones 11 and D1 were subcloned in pGEM
vectors and M13mpl8/19 and used for genomic characterization:of the -human gene. This involved localization of the exons bv Southern blot .:',` ,~!;.` .
analysis with 32P-labeled oligonucleotides from the rnouse ZP3 sequence followed by sequence analysis. As is demonstrated in Fig. 1, the human gene consists of 8 exons spread over approximately 20 }ib of genomic DNA. All identified exons are flanked by consensus splice donor and splice acceptor -signals (not shown). From sequence analysis of all exons the complete coding sequence for the ZP3 gene could be deduced (presented in Fig. 2). -The human ZP3 gene encodes a polypeptide of 372 amino acids with a calculated molecular weight of 41437 Dalton.
ClQnin~ of human ZP3 cl:)N~
A human ovary cDNA library tgt10) was screened with 32P-labeled fragments from human ZP3 exon 1, 5 and 7. This yielded several partial cDNA's that contained only the 3' half of the ZP3 coding seguence t exon 5 to8).
' ~
"'''''' .
. - ' WO 92/03548 2 0 9 0 4 8 6 ; PCl /EP9 1 /01 53g Sequence analysis of 3 independent cDNA clones confirmed the previously determined genomic ZP3 sequence except for one residue in exon 7 (see Fig.
2)~ At nucleotide position 1064 a G and C residue was found in genomic and cDNA respectively. In the encoded amino acid sequence this yields an arginine or a threonine residue respectively at position 345. This sequence difference probably represents a polvmorphism in the human ZP3 gene and polypeptide.
Expression of recombinant ZP3 in CHO cells.
For expression in CHO cells the size of the ZP3 gene has been reduced to allow insertion in a mammalian expression vector. Various ZP3 DNA
fragments have been assembled to a 'minigene~ as is illustrated in Fig. 3.
Exon 1 and 2 were joined by PCR-technology (Horton et al., Gene 77, 61-68, 1989; Yon and Fried, Nucl. Acids Res. 17, 4895, 1989)) and provided with 5' EcoRI and 3' XbaI sites respectively. Subsequently, this DNA
fragment was ligated to a genornic XbaI-SalI fragment carrying exon 3, 4 and part of exon 5 and the partial cDNA harboring exon 5 to 8 on a SalI-HindIII fragment. The resulting 2.7 kb 'minigene' contains a truncated intron between exon 2 and 3 and the natural introns between ZP3 exons 3 and 4 and exon 4 and 5. The integrity of the polypeptide encoding nucleotide information of this ZP3 construct was completely verified by sequence ~ `
analysis.
The ZP3 `minigene' was subsequently inserted in a mammalian expression vector in which the ZP3 gene is driven by the strong SV40 early promoter. In addition, this vector harbored ~-GIobin splicing and SV40 poly-adenylation signals for correct RNA processing of the expressed gene and the selectable marker gene aminoglycosylphosphotransferase (Colbere-Garapin et al., J.
Mol. Biol. 150, 1~14, 1981~ which allows for isolation of stable transformants by selection for G418 resistance. CHO cells were transfected with the ZP3 expression construct using the calcium-phosphate precipitation technique (Graham and van der Eb, Virology 52, 456-467, 1973; Wigler et al., Proc.
Natl. Acad. Sci USA 76,1373-1376, 1979). Mass populations of G418 resistant transformants ~representing 300-5~0 independent clones) were analysed for expression of the recombinant ZP3 gene.
- ., - ,. ~ . - ,.
. ~ , . , ~ - - -, -- - . . . , -WO 92/03~48 2 0 9 0 4 8 6 PCT/EP9l/01538-;.
~t ~
As is demonstrated in Fig~ 4, Northern blot analysis of total RNA from pooled transformants with a 32P-labeled ZP3 probe reveals a relatively high level of expression of the transfected ZP3 gene (as compared to ~he highly -expressed actin gene, not shown). Moreover, it was demonstrated that the minigene RNA is correctly processed to a mRNA which is slightlv larger ~ -then the natural transcript found in human oYary RNA (see Fig. 4). This size difference is most likely due to flanking 5' and 3' sequences present in the expression vector. .
Correct splicing and processing of the recombinant gene was further confirmed by isolation of a partial cDNA carlying exon 1 to 5 from G418 resistant CHO transformants. Sequence data of this cDNA were similar to the transfected DNA and support the conclusion that the introns are correctly spliced from the ZP3 rninigene transcript.
Culture media of growing mass populations were examined for the presence -of recombinant ZP3 polypeptide by Western blot analysis. With a polyclonal antiserum directed against deglycosylated porcine ZP3 (Henderson et al., Gamete Research 18, 251-265, 1987) recombinant ZP3 polypeptide could be detected in culture medium. This signal was absent in medium from non-transfected CHO cells.
Expression of recombinant ZP3 in E. coli.
For expression in E.coli first a full lenght ZP3 cDNA was constructed using a cDNA fragment covering exon 1 to 5 isolated by PCR from CHO
transformants and the partial cDNA clone ~harboring exon 5 to 8) isolated ;, from a human ovary cDNA library. Four 2;P3 cDNA fragments were cloned in frame to the E.coli LacZ gene in pEX-plasmids (Biores) allowing the production of a B-galactosidase-ZP3 fusion polypeptide as reported by Stanley and Luzio, EMBO J. 3, 1429-1434, 1984). The expression of fusion polypeptides in E.coli was examined by SDS polyacrylamide ~ -gelelectrophoresis (SDS-PAGE) and western blot analysis. The results of these experiments are presented in Fig. 5. Following heat shock induction, all four LacZ-ZP3 constructs yielded fusion polypeptides, although to a ;
much lesser extend then the LacZ gene. The molecular weight of the hybrid polypeptides, as observed on coomassie stained gels (see Fig. 5A), were in agreement with the expressed ZP3 parts. 'rhis was further confirmed by -western blot analysis with anti-B-galactosidase antibodies (not shown) and a rabbit antiserum raised against ZP3 amino acids 341 to 360 coupled to KLH - :
~: .
- , - . .. . ~ . . . . . . . .. . .. . . ..
WO 92/03548 2 0 9 0 4 8 6 PCl/EP9l/0l53 , (Fig. 5B), Only fusion polypeptides carrying this region, i.e. ZP3-A and ZP3-I:), were recognized by the an~iserum. An antiserum against ~LH yielded a result sirn~lar to the background staining in Fig. SB (not shown). In addition, ~Gal-ZP3 fusion polypeptides were recognized by antisera raised against total human zonae pellucidae (not shown).
Binding of ZP3 antibodies to human eggs.
A human egg fluorescence assay was developed to characterize the binding of the various ZP antibodies to human zonae pellucidae. The results of three i i~
different experiments are depicted in Fig. 6. Serum against KLH and a normal mouse serum failed to give fluorescent staining whereas with all ;
antisera against ZP components strong to intermediate levels of fluorescence were observed. From the presented data it can be inferred that antisera against ZP3(93-110), ZP3(172-190), ZP3(327-344), ZP3(341-360) and ZP3(362-372) recognize human oocytes. ~-.
Effect of ZP antibodies on sperm-egg recognition. ~-The contraceptive potential of the ZP3 antibodies was analYsed in a human sperm-zona bindin" assay. The results of this assay is presented in Fig. 7.
Antibodies against total human zonae pellucidae showed 75 /c inhibition of sperm-zona binding. An antiserum against KLH does not interfere with sperm-egg binding. In contrast, a m~xture of ZP3-peptide antibodies (peptides: ZP3(93-110), ZP3(172-190), ZP3(341-360) and ZP3(362-372~j showed a relative inhibition of sperm binding of 35%. Antibodies a~ainst recombinant ZP3 from CHO cells reduced sperm binding with 55~G.
WO ~2/03548 2 0 9 0 ~ 8 ~` pcr/Ep9l/ol53p Legends to ~he Figures.
Figure 1.
Restriction maps of EMBL clones I1, Dl and El (A) and genomic organization of the human ZP3 gene. Exons are indicated by blacl~ boxes. ' -Restriction sites: S, SalI; B, BarnHI.
Figure 2. f Nucleotide and arnino acid sequence of human ZP3. At position 1064 a G or ~ `
C residue was found in genomic and cDNA respectively. This apparent polyrnorphism yields a ~hr or Alg amino acid residue. Arrows indicate exon junctions. Exon number is indicated in circies. The po]vadenv]ation signa] is underlined.
Figure 3. :Size reduction of ZP3 gene for insertion in a mammalian expression vector.
A 2.7 kb ~P3 'rninigene' was assembled from the following three fragments~
A, exon 1 and exon 2 joined in frame to each other by PCR-techno]ogv yielding a EcoRI-XbaI fragment; B, a genomic XbaI-Sa]I fragment with exon 3, d, and part of exon 5; C, a SalI-HindlII ZP3 cDNA fragment with exon 5 tO
8 (provided with artificial 3' BamHI and HindIII siles). The three fra~ments were cloned in a pGEM vector (Promega) opened with EcoRl and HindII~
and subsequently as a 2.7 kb EcoRI-BamHI fragment placed behind the SV40 promoter in a mammalian expression vector.
,: .
Figure 4.
Detection of ZP3 mRNA in total RNA (15 g) from C~O mass populations transfected with the :ZP3 DNA and poly(A)+ RNA (j g) from a human ovarv. Lane l, CHO; lane 2, CHO ZP3 transformants; lane 3. human ovary. ~ `
Total ZP3 cDNA in pGEM3 was used as a probe. `
Figure S.
Production of ~Gal-ZP3 fusion polypeptides in E.coli strain POP~136 carrving pEX-plasmids with human ZP3 cDNA inserts.
.
:` :
WO 92/03548 2 ~ 9 0 ~ 8 6 PCr/EP91/0153X
, .. , ,: .
.. . .
A~ Coomassie staining of polyacrylamide gel (lO~o) and B. Western blot : -analysis with anti-ZP3 341-360 antiserum (1:150 dilution). From left to right the follo~ving samples are loaded on the gels (-, before induction; + 2 hours after induction): control pEX; pEX-ZP3A (KpnI-BamHI fragment; total cDNA); pEX-ZP3B (KpnI-SalI fragment; cDNA truncated at exon 5 encoding amino acid 1-241); pEX ZP3C (Kpr~-SphI fragment; cDNA
truncated at end of exon 1 encoding arnino acid 1-102); pEX-ZP3D (SmaI- i-BarnHI fragment, 3' half of exon 5 + exon 6,7 and 8 encoding arruno acid 257-372). Molecular weight (M) markers are at the extreme left (indicated in kiloDalton). Bacteria were grown at 30 C till OD600 = 0.~-1 and '' ',!,~
subsequently induced for two hours at 42 C. Aliquots were taken, centrifuged and total cells Iysates were analysed.
Figure 6.
Binding of different antibodies (see materials and methods) to human eggs. - ~-Amount of second antibody labeled with FITC was measured with an ~ -exposure analyser and expressed as percentage of conlrol (100C/G = dark, O~c ;
= fluorescent eggs). 1: control; 2: antiserum against human zonae - pellucidae; 3: antiserum against KLH; 4-8: antisera agains~ ZP3(93-110), ~.
ZP3(172-l90), ZP3S327 344), ZP3(341-350) and ZP3(362-372); 9: antiserum against ~Ga]-ZP3; lO:mixture of sera of 3 mice immunized with recombinant CHO produced ZP3; 11: mixture of sera of 3 control mice. Average exposure time as percentage of control and standard error of the mean are -given. . . ..
:. .
Figure 7. .
Inhibition of sperm-zona binding by anti-ZP3 antibodies (see materials and methods). l: control; 2: antiserum against KLH; 3: antiserum against human zonae pellucidae 4: rnixture of antis~ra against ZP3(93-110), ZP3(172-190), ZP3(341-360) and ZP3~362-372); 5: mixture of sera of 3 mice immunized with recombinant CHO produced ZP3. Average number of boupd spermatozoa on total zonae pellucidae and standard error of the mean are given.
~' ,. .. . . _ . .. .. . ~
Claims (13)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. Polypeptide having human ZP3 activity comprising the following sequence:
2. Polypeptide according to claim 1, characterized in that it is at least partially glycosylated.
3. Polypeptide fragment of a protein having human ZP3 activity, characterized that it comprises one or more amino acid sequences selected from the group consisting of:
, , , , , , and subsequences thereof having human ZP3 activity.
, , , , , , and subsequences thereof having human ZP3 activity.
4. Nucleic acid sequence coding for a polypeptide according to any one of claims 1 to 3.
5. Nucleic acid sequence according to claim 4, characterized that it comprises the following sequence
6. A vector comprising a nucleic acid according to claims 4 or 5.
7. A host cell comprising a vector according to claim 6.
8. An immunogenic compound comprising a polypeptide according to any one of claims 1 to 3 linked to a carrier.
9. A pharmaceutical composition comprising an immunogenic compound according to claim 8.
10. An immunocontraceptive vaccine comprising an immunogenic compound according to claim 8 and an adjuvant.
11. An immunocontraceptive vaccine comprising an antibody which recognizes a polypeptide according to any one of the claims 1 to 3.
12. A method for producing a polypeptide according to any one of the claims 1 to 3, characterized in that a host cell is transformed with a vector comprising a nucleic acid sequence coding for such a polypeptide and culturing said host cell in a suitable medium and isolating the polypeptide from the culture.
13. A method for producing an antibody to a polypeptide according to any one of the claims 1 to 3, characterized in that a suitable animal is injected with said polypeptide and an adjuvant, and that the B-lymphocytes of said animal are harvested after a suitable period of time and that antibody producing cells are selected and immortalized, after which they are cultured and the antibodies are isolated from the culture.
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CA002090486A Abandoned CA2090486A1 (en) | 1990-08-27 | 1991-08-13 | Human zona pellucida protein zp3 |
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JP (1) | JPH06500690A (en) |
KR (1) | KR930702522A (en) |
CN (1) | CN1060499A (en) |
AU (1) | AU8328591A (en) |
CA (1) | CA2090486A1 (en) |
FI (1) | FI930897A (en) |
HU (1) | HUT64394A (en) |
IE (1) | IE912872A1 (en) |
NZ (1) | NZ239518A (en) |
PT (1) | PT98780A (en) |
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ZA (1) | ZA916432B (en) |
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US5641487A (en) * | 1989-06-12 | 1997-06-24 | The Government Of The United States Of America As Represented By The Secretary Department Of Health And Human Services | Contraceptive vaccine based on alloimmunization with zona pellucida polypeptides |
AU9094191A (en) * | 1990-11-21 | 1992-06-25 | Washington University | Recombinant avirulent salmonella antifertility vaccines |
WO1994010304A1 (en) * | 1992-11-02 | 1994-05-11 | Akzo Nobel N.V. | Marmoset zona pellucida protein zp3 |
WO1994011019A1 (en) * | 1992-11-09 | 1994-05-26 | Zonagen, Inc. | Materials and methods for immunocontraception |
US6001599A (en) * | 1992-11-09 | 1999-12-14 | Zonagen, Inc. | DNAs encoding mammalian ZPBs |
BR9508748A (en) * | 1994-08-22 | 1997-08-12 | Akzo Nv | Peptide use of the same antibodies cell lines hybridoma cell line pharmaceutical composition contraceptive vaccine process to detect autoimmune antibodies in a patient's serum test kit and diagnostic reagent |
DE19622289A1 (en) * | 1996-05-23 | 1997-11-27 | Schering Ag | Zona Pellucida Proteins for contraception |
ATE284443T1 (en) * | 1998-02-19 | 2004-12-15 | Eastern Virginia Med School | RECOMBINANT ACTIVE HUMAN ZONA PELLUCIDA PROTEIN 3 (HZP3) |
US7037663B2 (en) * | 1998-02-19 | 2006-05-02 | Eastern Virginia Medical School | Human zona pellucida protein 3 and uses thereof |
US7148021B2 (en) | 2001-08-02 | 2006-12-12 | Eastern Virginia Meical School | Human zona pellucida proteins and methods of their use in diagnosing male infertility |
CN101906163B (en) * | 2009-06-05 | 2012-06-27 | 上海交通大学医学院 | Immunocontraceptive synthetic peptide and immunocontraceptive chimeric peptide and application thereof |
EP3847187A1 (en) * | 2018-09-03 | 2021-07-14 | Laboratoire HRA-Pharma | Zp3 fragments in immunotherapy of ovarian cancer |
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US4996297A (en) * | 1987-10-07 | 1991-02-26 | Zonagen, Inc. | Recombinantly expressed rabbit zona pellucida polypeptides |
AU636895B2 (en) * | 1989-06-12 | 1993-05-13 | United States of America, as represented by the Secretary, U.S. Department of Commerce, The | Contraceptive vaccine based on cloned zona pellucida gene |
-
1991
- 1991-08-13 IE IE287291A patent/IE912872A1/en unknown
- 1991-08-13 CA CA002090486A patent/CA2090486A1/en not_active Abandoned
- 1991-08-13 KR KR1019930700637A patent/KR930702522A/en not_active Application Discontinuation
- 1991-08-13 AU AU83285/91A patent/AU8328591A/en not_active Abandoned
- 1991-08-13 JP JP3513881A patent/JPH06500690A/en active Pending
- 1991-08-13 HU HU9300537A patent/HUT64394A/en active IP Right Revival
- 1991-08-13 WO PCT/EP1991/001538 patent/WO1992003548A1/en active Application Filing
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- 1991-08-26 CN CN91109289A patent/CN1060499A/en active Pending
- 1991-08-26 PT PT98780A patent/PT98780A/en not_active Application Discontinuation
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ZA916432B (en) | 1992-06-24 |
JPH06500690A (en) | 1994-01-27 |
IE912872A1 (en) | 1992-03-11 |
NZ239518A (en) | 1994-03-25 |
HU9300537D0 (en) | 1993-05-28 |
AU8328591A (en) | 1992-03-17 |
FI930897A (en) | 1993-04-05 |
KR930702522A (en) | 1993-09-09 |
PT98780A (en) | 1992-07-31 |
CN1060499A (en) | 1992-04-22 |
WO1992003548A1 (en) | 1992-03-05 |
FI930897A0 (en) | 1993-02-26 |
HUT64394A (en) | 1993-12-28 |
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