+

WO1999003490A1 - Proteines et peptides pour vaccins contraceptifs et diagnostics de fertilite - Google Patents

Proteines et peptides pour vaccins contraceptifs et diagnostics de fertilite Download PDF

Info

Publication number
WO1999003490A1
WO1999003490A1 PCT/US1998/015094 US9815094W WO9903490A1 WO 1999003490 A1 WO1999003490 A1 WO 1999003490A1 US 9815094 W US9815094 W US 9815094W WO 9903490 A1 WO9903490 A1 WO 9903490A1
Authority
WO
WIPO (PCT)
Prior art keywords
peptide
sequence
arg
asp
ala
Prior art date
Application number
PCT/US1998/015094
Other languages
English (en)
Inventor
Erwin Goldberg
Original Assignee
Northwestern University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Northwestern University filed Critical Northwestern University
Priority to AU85049/98A priority Critical patent/AU8504998A/en
Publication of WO1999003490A1 publication Critical patent/WO1999003490A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin
    • 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

Definitions

  • NICHD Network-to-Network Interface
  • This invention relates to novel proteins and peptides and their use in contraceptive vaccines and to assess infertility.
  • the invention also relates to DNA molecules coding for the proteins and peptides and host cells containing the DNA molecules linked to expression control sequences for producing the proteins and peptides.
  • the invention relates to a topical contraceptive containing antibodies specific for the proteins of the invention.
  • Mammalian spermatozoa are highly specialized both in structure and function. These cells are the product of a developmental program that involves the expression of genes unique to the testes and of testis-specific variants of common somatic genes. Why testis and sperm should need specialized isoforms of common proteins or genes that are expressed only during spermatogenesis remains to be established.
  • Idiopathic infertility is characterized clinically as the inability to achieve a pregnancy by cohabiting couples with no apparent anatomical or functional reproductive pathology. In about 10% of such cases, the cause is attributed to immunological phenomena, including circulating antisperm antibodies in one or both partners. Presumably, such antibodies target to spermatozoa and, as a consequence, conception is blocked or fails. Additionally, there is indirect evidence of an association between infertility and antisperm antibodies in both male and female patients. With respect to the subject of immunologic infertility, see Witkin et al., Am. J. Obstet. Gynecol., 158, 59-62 (1988); Clarke et al., Fertil.
  • the invention provides purified proteins and peptides whose sequences comprise the sequence of an epitope of one of the proteins.
  • the proteins and peptides are described in detail below.
  • the proteins are unique to sperm and testis, and the proteins and peptides can be used in vaccines for contraception in mammals. Accordingly, the invention further provides: (1) immunogens comprising a peptide linked to a carrier, the peptide being capable of producing an antibody that reacts specifically with one of the proteins of the invention and having a sequence comprising a sequence which forms a B-cell epitope of the protein; and (2) vaccines comprising the peptides and immunogens in a delivery system.
  • proteins and peptides can be used in diagnostic assays for assessing infertility.
  • the assays and kits for performing the assays are also part of the invention.
  • the invention further provides DNA molecules coding for the proteins and peptides, and host cells containing the DNA molecules linked to expression control sequences, for producing the proteins and peptides.
  • the immunogens of the invention can be used to produce antibodies that bind specifically to the proteins of the invention, and these antibodies are incorporated into pharmaceutical formulations suitable for intravaginal administration for contraception in mammals.
  • the invention provides a purified protein which is a testis-specific isoform of calpastatin.
  • Testis-specific is used herein to mean that the isoform is found in the testes and sperm, but is not found in other tissues.
  • somatic isoforms are those found in one or more, generally several, types of tissues.
  • the somatic isoforms may be found in testes and sperm but, if so, will also be found in at least one other type of tissue.
  • Clone Y-19 coding for a human testis-specific isoform of calpastatin, was identified by screening a human testis cDNA library with sera from infertile patients positive for antisperm antibodies as described in Liang et al., Reprod. Fertil. Dev., >, 297-305 (1994) .
  • the complete sequence of this human testis-specific isoform of calpastatin is given in Chart A below.
  • Calpastatin is the peptide inhibitor of calpain, a cysteine protease. Calpain has been localized to the sperm head and appears to be involved in the acrosome reaction. See, Schollmeyer, Biol. Reprod., 34, 721-731 (1986) . Although not wishing to be bound by any particular theory, it is believed that infertility in individuals having antibodies directed to testis-specific calpastatin occurs as follows. The acrosome reaction, which must occur in order for the sperm to penetrate the zona pellucida of the egg, is triggered by an influx of Ca +2 . Wasserman, Annu. Rev. Cell Biol., 3, 109-142 (1987) .
  • the Y-19 protein is a human protein.
  • Corresponding proteins in other mammals would be expected to be at least 70% homologous to these human proteins.
  • the corresponding proteins in other mammals can be obtained by the method described in Example 1 or by using the sequence given in Chart A to design DNA probes which can be used to screen testis gene libraries, preferably cDNA libraries, of other mammals. Methods of making gene (e.g., cDNA) libraries, designing probes for screening them, identifying and isolating a desired clone, producing protein from the clone, etc., are well known in the art.
  • Testis cDNA libraries can also be purchased from ClonTech Laboratories, Inc., 1020 E. Meadow Circle, Palo Alto, CA 94303-4230.
  • homologous means that a sequence is at least 70% identical to the specified sequence, preferably 80% identical, most preferably 90% identical. Methods of determining the homology of proteins are well known in the art.
  • the proteins of the invention can be used in contraceptive vaccines in mammals.
  • a protein from the same species of mammal that is to be immunized is used in the vaccine.
  • proteins from other species, especially closely-related species can be used.
  • Immunogenic portions of the proteins can also be used in the vaccines. Immunogenic portions of the proteins must include at least a B-cell epitope from the sequences found on the testis-specific isoform, but not found on the somatic isoforms.
  • testis-specific calpastatin or an immunogenic portion thereof, since cross-reaction with the somatic isoforms may occur if the complete protein or an immunogenic portion containing an immunogenic somatic sequence is used in the vaccine. This may cause deleterious side effects and should be avoided except when the vaccine is to be used for contraception in pest species (e.g., rodents).
  • peptides derived from the proteins of the invention are used in the vaccines.
  • the peptides must comprise at least a B-cell epitope from the sequences found on the testis-specific isoform but not found on the somatic isoforms.
  • the peptide may include other sequences besides those which form the B- cell epitope, but these sequences must be chosen so that the antibody produced as a result of immunization with the vaccine containing the peptide will react specifically with the protein found in testis and sperm.
  • the human testis-specific isoform of calpastatin has the following sequence at its N-terminal:
  • Gly Ala Arg Glu Ala Val Arg lie Phe Gin Asp Gin Ala 30 35 40
  • This sequence of 41 amino acids is unique to the testis- specific isoform of calpastatin.
  • Peptides having this sequence, or a portion of it that includes the sequence from amino acid 26 through amino acid 41, or multiple copies of such sequences can be used to elicit antibodies that react with the testis-specific isoform of calpastatin, but do not react with somatic isoforms of calpastatin.
  • Amino acids 26-41 in the above sequence have been identified as a B-cell epitope (see Example 1) .
  • the peptides comprising a B-cell epitope of one of the proteins of the invention are preferably used in the vaccines in the form of an immunogen comprising the peptide linked to a carrier.
  • Suitable carriers are compounds capable of stimulating the production of antibodies to haptens coupled to them in a host animal . Many such carriers are well-known.
  • the carrier may be a high molecular weight compound.
  • Suitable high molecular weight compounds include proteins, polypeptides, carbohydrates, polysaccharides, lipopolysaccharides, nucleic acids, and the like of sufficient size and immunogenicity.
  • Preferred high molecular weight compounds are proteins and polypeptides.
  • Suitable immunogenic carrier proteins and polypeptides will generally have molecular weights between 4,000 and 10,000,000, and preferably greater than 15,000.
  • suitable carriers include proteins such as albumins (e.g., bovine serum albumin, ovalbumin, human serum albumin), immunoglobulins, thyroglobulins (e.g., bovine thyroglobulin) , hemocyanins (e.g..).
  • toxins e.g., diptheria toxoid, tetanus toxoid
  • polypeptides such as polylysine or polyalaninelysine.
  • diptheria toxoid and tetanus toxoid are preferred.
  • the peptide may be coupled to the carrier with conjugating reagents such as glutaraldehyde, a water soluble carbodiimide such as 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydro- chloride (ECDI), N-N-carbonyldiimidazole, 1— hydroxybenzotriazole monohydrate, N-hydroxysuccinimide, 6- maleimidocaproyl-N-hydroxysuccinimide, n-trifluoroacetylimidazole cyanogen bromide, 3- (2' — benzothiazolyl-dithio) propionate succinimide ester hydrazides or affinity labeling methods. See also Pierce Handbook and General Catalog (1989) for a list of possible coupling agents.
  • conjugating reagents such as glutaraldehyde, a water soluble carbodiimide such as 1- (3-dimethylaminopropyl) -3-ethy
  • the number of peptides attached to the high molecular weight carrier is called the "epitopic density.”
  • the epitopic density can range from 1 to the number of available coupling groups on the carrier molecule.
  • the epitopic density on a particular carrier will depend upon the molecular weight of the carrier and the density and availability of coupling sites.
  • the carrier may also be a peptide which has a sequence comprising the sequence of a T-cell epitope of one of the proteins of the invention or of another protein.
  • Methods of identifying T-cell epitopes are known. See, O'Hern and Goldberg, in Techniques In Protein Chemistry IV, pages 481- 490 (1993) ; O'Hern and Goldberg, Proceed. Intern. Symp. Control Rel. Bioact. Mater., 20, 394-395 (1993) .
  • the three criteria for selection of a T-cell epitope are: a size of 8-12 amino acids; hypervariability; and one or more representations of the tetrapeptide motif previously reported to be associated with T-cell epitopes. O'Hern and Goldberg, in Techniques In Protein Chemistry IV, pages 481- 490 (1993); O'Hern and Goldberg, Proceed. Intern. Symp. Control Rel. Bioact. Mater., 20, 394-395 (1993) .
  • the carrier is a peptide which has a sequence comprising the sequence of a promiscuous T-cell epitope.
  • a promiscuous T-cell epitope is a T-cell epitope that is recognized by individuals of several different major histocompatability (MHC) types. Promiscuous T-cell epitopes are known. See, Ho et al., Eur. J. Immunol., 20,
  • a preferred promiscuous T-cell epitope has the following sequence: Val Asp Asp Ala Leu lie Asn Ser Thr Lys lie Tyr Ser Tyr
  • a peptide carrier which has a sequence comprising the sequence of a T-cell epitope may include other sequences linked to the N-terminal or C-terminal of the T-cell epitope.
  • additional amino acids may be provided to link the B-cell epitope on the peptide to the T-cell epitope on the carrier. These linking amino acids should form a four-residue ⁇ -turn based on examination of 33 patterns in native proteins that code for corners. Efimov, FEBS Lett., 166, 33 (1984); Kaumaya et al., Biochemistry, 29, 13-23 (1990) .
  • Peptides comprising a B-cell epitope may be coupled to a peptide carrier comprising a T-cell epitope in the same manner as described above for high molecular weight proteins and polypeptides to form the immunogen.
  • immunogens are preferably synthesized as a single peptide in the ways described below for the synthesis of peptides .
  • the vaccines contain one or more of the proteins (or an immunogenic portion thereof) , peptides and immunogens of the invention in a delivery system. Suitable delivery systems are well known. For instance, the delivery system may simply be a solvent (such as saline and buffers) or other liquid (such as an oil) . However, the delivery system preferably enhances the immune response.
  • Such delivery systems include aluminum salts, water-oil emulsions (such as incomplete Freund's adjuvant), saponins, liposomes, immune stimulating complex, lipopolysaccharides, mycobacterial adjuvants (such as Freund's complete adjuvant) , Squalene-Arlacel A containing the synthetic muramyl dipeptide N-acetyl-nor-muramyl-L-alanyl-D- isoglutamine (CGP11637; Ciba-Geigy Pharmaceuticals, Basel, Switzerland) , live vectors, antigen immunotargeting materials, and polymers (e.g., biodegradable microspheres, such as polylactide-polyglycolide microspheres, and block copolymers for sustained release) .
  • biodegradable microspheres such as polylactide-polyglycolide microspheres, and block copolymers for sustained release
  • the vaccines may be administered in any conventional manner, including orally, intradermally, subcutaneously, intramuscularly, etc. to male or female mammals to inhibit fertilization of eggs by sperm. Suitable routes of administration and effective amounts (effective dosages and number of doses) necessary to inhibit conception can be determined empirically as is known in the art.
  • inhibitor is meant at least a 50% reduction in the number of female mammals becoming pregnant as a result of the administration of the vaccine. Preferably at least a 75%, most preferably at least a 90%, reduction is achieved.
  • the proteins and peptides comprising a B-cell epitope can also be used in assays to assess infertility.
  • the peptides may be used as such or may be linked to a carrier.
  • the carriers e.g., large molecular weight and T-cell epitope carriers
  • methods of linking the peptides to the carriers are the same as described above for the immunogens.
  • the protein, peptide or peptide linked to a carrier is contacted with a body fluid of a patient under conditions that permit antibodies in the body fluid to bind to it.
  • the assays are immunoassays that allow for the determination of whether the body fluid of a patient contains antibodies that bind to the protein, peptide or peptide linked to a carrier.
  • Suitable immunoassays and reagents for use therein are well known in the art, and those skilled in the art will be able to determine operative and optimal assay conditions using only ordinary skill in the art.
  • the protein, peptide or peptide linked to a carrier will be immobilized on a solid surface.
  • Suitable solid surfaces are well-known and include glass, polystyrene, polypropylene, polyethylene, nylon, paper, fiberglass, polyacrylamide and agaroses.
  • the immobilized material is contacted with the body fluid so that antibodies present in the body fluid can bind to the protein, peptide or peptide linked to a carrier.
  • a labeled secondary antibody or other material which binds specifically to the antibody in the body fluid is added as a means to detect and quantitate the antibody bound to the protein, peptide or peptide linked to a carrier.
  • Suitable labels are well known in the art. They include enzymes, fluorophores, radionucleotides, bioluminescent labels, chemiluminescent labels, and particulate labels. The binding and detection of these labels can be accomplished using standard techniques well known to those skilled in the art.
  • the body fluid may be any body fluid that contains antibodies. Suitable body fluids include serum, plasma, cervical mucus and seminal plasma.
  • the assays may be used to assess infertility in patients unable to conceive. If the patient has antibodies specific for one of the proteins of the invention, then this may be the cause, or one of the causes, of the infertility. The assays may also be used to evaluate whether administration of the vaccines of the invention has been effective in immunizing recipients of the vaccines.
  • the invention also comprises a kit.
  • the kit is a packaged combination of one or more containers holding reagents useful in performing the immunoassays. Suitable containers for the reagents include bottles, vials, test tubes, microtiter plates, a solid phase (see listing above) held in a molded plastic device, and other containers known in the art.
  • the kit will contain at least one container holding a protein, peptide comprising a B-cell epitope or such a peptide linked to a carrier.
  • the kit may also comprise a container of a labeled component useful for detecting or quantitating the antibodies in the body fluids that bind to the protein, peptide or peptide linked to a carrier.
  • the kit may also contain other materials which are known in the art and which may be desirable from a commercial and user standpoint, such as buffers, enzyme substrates, diluents, standards, etc.
  • the kit may include containers, such as test tubes and microtiter plates, for performing the immunoassay.
  • the peptides of the invention may be made in a variety of ways. For instance, solid phase synthesis techniques may be used. Suitable techniques are well known in the art, and include those described in Merrifield, in Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds. 1973); Merrifield, J. Am. Chem. Soc , 85, 2149 (1963); Davis et al . , Biochem. Int'l, 10, 394-414 (1985); Stewart and Young, Solid Phase Peptide Synthesis (1969); U.S. Patents Nos.
  • Solid phase synthesis is the preferred method of making the peptides of the invention.
  • the peptides may also be produced by culturing a host cell comprising a DNA molecule coding for the peptide operatively linked to expression control sequences under conditions permitting expression of the peptide.
  • the proteins of the invention may also be produced in this manner.
  • the proteins and peptides can be produced in transformed host cells using recombinant DNA techniques. Such techniques and suitable host cells and other reagents for use therein are well known in the art. For instance, the selection of a particular host cell is dependent upon a number of factors recognized by the art. These include, for example, compatibility with the chosen expression vector, use and toxicity of the protein or peptide encoded by the expression vector, rate of transformation, expression characteristics, bio-safety, and costs.
  • useful host cells include bacteria, yeast and other fungi, animal cell lines, animal cells in an intact animal, or other host cells known in the art.
  • the host cells may be transformed with a vector comprising DNA encoding the peptide or protein.
  • the coding sequence must be operatively linked to a promoter.
  • the promoter used in the vector may be any sequence which shows transcriptional activity in the host cell and may be derived from genes encoding homologous or heterologous proteins and either extracellular or intracellular proteins, such as amylase, glycoamylases, proteases, lipases, cellulases, and glycolytic enzymes.
  • the promoter need not be identical to any naturally-occurring promoter. It may be composed of portions of various promoters or may be partially or totally synthetic. Guidance for the design of promoters is provided by studies of promoter structure such as that of
  • the location of the promoter relative to the transcription start may be optimized. See Roberts, et al., Proc. Natl Acad. Sci . USA, 1_6' 760-4 (1979) .
  • the promoter may be inducible or constitutive, and is preferably a strong promoter.
  • strong it is meant that the promoter provides for a high rate of transcription in the host cell.
  • the coding sequences must be operatively linked to transcription termination sequences, as well as to the promoter.
  • the coding sequence may also be operatively linked to expression control sequences other than the promoters and transcription termination sequences. These additional expression control sequences include activators, enhancers, operators, stop signals, cap signals, polyadenylation signals, ribosome binding sites, and other signals involved with the control of transcription and translation.
  • the site at which the ribosome binds to the messenger includes a sequence of 3-9 purines.
  • the consensus sequence of this stretch is 5'-AGGAGG-3 ' , and it is frequently referred to as the Shine-Dalgarno sequence.
  • the sequence of the ribosome binding site may be modified to alter expression. See Hui and DeBoer, Proc. Natl. Acad. Sci. USA, 8T4, 4762-66 (1987) . Comparative studies of ribosomal binding sites, such as the study of Scherer, et al.. Nucleic Acids Res., Si, 3895-3907 (1987), may provide guidance as to suitable base changes.
  • the ribosome binding site lies 3-12 bases upstream of the start (AUG) codon.
  • a ribosome binding site and spacer that provide for efficient translation in the prokaryotic host cell should be provided.
  • a preferred ribosome binding site and spacer sequence for optimal translation in E. coli are described in Springer and Sugar, Proc. Nat'l Acad. Sci. USA, 84 ⁇ , 8961-65 (1987) and von Bodman et al . , Proc. Nat'l Acad. Sci. USA, 83, 9443-47 (1986) .
  • the sequence of this ribosome binding site and spacer is: AGGAGAACAA CAACC [SEQ ID NO: 11] .
  • the consensus sequence for the translation start sequence of eukaryotes has been defined by Kozak (Cell, 44, 283-292 (1986)) to be: C (A/G) CCAUGG. Deviations from this sequence, particularly at the -3 position (A or G) , have a large effect on translation of a particular mRNA. Virtually all highly expressed mammalian genes use this sequence. Highly expressed yeast mRNAs, on the other hand, differ from this sequence and instead use the sequence (A/Y)A(A/U)AAUGUCU (Cigan and Donahue, Gene, 5_9, 1-18 (1987) ) . These sequences may be altered empirically to determine the optimal sequence for use in a particular host cell.
  • DNA molecules encoding for the protein or peptide could be excised from genes or cDNA clones by methods well known in the art.
  • the DNA molecules encoding a protein or peptide of the invention are preferably chemically synthesized. Methods of chemically synthesizing DNA are well known in the art. Chemical synthesis is preferable for several reasons.
  • codons preferred by the host in which the DNA sequence will be expressed may be used to optimize expression. Not all of the codons need to be altered to obtain improved expression, but greater than 50%, most preferably at least about 80%, of the codons should be changed to host- preferred codons.
  • the codon preferences of many host cells, including E. coli, yeast, and other prokaryotes and eukaryotes, are known. See Maximizing Gene Expression, pages 225-85 (Reznikoff & Gold, eds., 1986). The codon preferences of other host cells can be deduced by methods known in the art.
  • chemically synthesized DNA also allows for the selection of codons with a view to providing unique or nearly unique restriction sites at convenient points in the sequence. The use of these sites provides a convenient means of constructing the synthetic coding sequences. In addition, if secondary structures formed by the messenger RNA transcript interfere with transcription or translation, they may be eliminated by altering the codon selections.
  • Chemical synthesis also allows for the use of optimized expression control sequences with the DNA sequence coding for a protein or peptide. In this manner, optimal expression of the protein or peptide can be obtained. For instance, as noted above, promoters can be chemically synthesized and their location relative to the transcription start optimized. Similarly an optimized ribosome binding site and spacer can be chemically synthesized and used with coding sequences that are to be expressed in prokaryotes.
  • DNA coding for a signal or signal-leader sequence may be located upstream of the DNA sequence encoding the protein or peptide.
  • a signal or signal-leader sequence is an amino acid sequence at the amino terminus of a protein which allows the protein to which it is attached to be secreted from the cell in which it is produced. Suitable signal and signal-leader sequences are well known. Although secreted proteins are often easier to purify, secretion is generally not preferred since expression levels are much lower than those that can be obtained in the absence of secretion.
  • the vector used to transform the host cells may have one or more replication systems which allow it to replicate in the host cells. In particular, when the host is a yeast, the vector should contain the yeast 2u replication genes REP 1-3 and origin of replication. Many bacterial replicons are known.
  • an integrating vector may be used which allows the integration into the host cell's chromosome of the sequence coding for the protein or peptide.
  • the copy number of the coding sequence in the host cells would be lower than when self-replicating vectors are used, transformants having sequences integrated into their chromosomes are generally quite stable.
  • the vector When the vector is a self-replicating vector, it is preferably a high copy number plasmid so that high levels of expression are obtained.
  • a "high copy number plasmid" is one which is present at about 100 copies or more per cell. Many suitable high copy number plasmids are known.
  • the vector desirably also has unique restriction sites for the insertion of DNA sequences and a sequence coding for a selectable or identifiable phenotypic trait which is manifested when the vector is present in the host cell ("a selection marker") . If a vector does not have unique restriction sites, it may be modified to introduce or eliminate restriction sites to make it more suitable for further manipulations.
  • the vector comprising the sequence coding for the protein or peptide is prepared, it is used to transform the host cells. Methods of transforming host cells are well known in the art, and any of these methods may be used. Transformed host cells are selected in known ways and then cultured to produce the protein or peptide.
  • the methods of culture are those well known in the art for the chosen host cell, but the use of enriched media (rather than minimal media) is preferred since higher yields are obtained.
  • the expressed protein or peptide may be recovered using methods of recovering and purifying proteins from cell cultures which are well known in the art.
  • the immunogens of the invention can also be used to produce antibodies which can, in turn, be used as topical contraceptives. Methods of making antibodies are well known in the art (see, e.g., the discussion of vaccines above) .
  • Antibody is used herein to mean any class and subclass of antibody, any form of antibody, fragments of antibodies, and any material having the ability to bind specifically to an antigen, and includes antisera, purified polyclonal antibodies, monoclonal antibodies, IgG antibodies, IgM antibodies. Fab, Fab', F(ab') 2 , and engineered antibodies such as single-chain antibodies.
  • Effective dosages of the antibody or antibodies may be determined empirically, and making such determinations is within the skill of the art. It is understood by those skilled in the art that the dosage amount will vary with the type of antibody (ies) , the affinity of the particular antibody (ies) , the form of the topical contraceptive formulation, the species of the mammal, and like factors well known in the medical and veterinary arts. Mammals treatable according to the invention include dogs, cats, other domestic animals and humans.
  • Suitable topical contraceptive formulations comprise an effective amount of the antibody (ies) and a carrier suitable for intravaginal use.
  • “Acceptable” means that the carrier ingredients are compatible with the antibody (ies) and each other and are not injurious to the patient.
  • Suitable carriers and carrier ingredients are well known in the art.
  • the antibody (ies) are combined with the carrier ingredients to produce topical contraceptive dosage forms by conventional methods known to those of skill in the art.
  • the amount of the antibody (ies) which will be combined with carrier ingredients to produce a single doage form will vary depending upon all of the factors described above.
  • the topical contraceptive formulations may be in the form of suppositories, pessaries, tampons, creams, gels, pastes, foams or sprays .
  • a computer-generated hydropathy plot of the first 41 residues of somatic calpastatin versus the first 41 residues of testis-specific calpastatin was performed. This hydropathy plot was generated using algorithms described in Hopp and Woods, Proc. Natl. Acad. Sci. USA, 78, 3824-28 (1981) and Kyte and Doolittle, J. Mol. Biol., 157, 105 (1982) . Residues 26-41 of testis-specific calpastatin are both hydrophilic and unique to the testis isoform. Therefore, this segment was chosen as a testis- specific B-cell epitope. This segment has the sequence: Glu Arg Arg Gly Ala Arg Glu Ala Val Arg lie Phe Gin Asp
  • Example 1 (with terminal Ala omitted) linked to a carrier comprising a universal T-cell epitope derived from tetanus toxin.
  • the T-cell epitope had the following sequence:
  • Female cynomologous macaques can be immunized with either lOO ⁇ g or 300 ⁇ g of the peptide immunogen [SEQ ID NO: 1]
  • the immunogen can be administered intramuscularly in Squalene-Arlacel A containing the synthetic muramyl dipeptide N-acetyl-nor- muramyl-L-alanyl-D-isoglutamine (CGP11637; Ciba-Geigy Pharmaceuticals, Basel, Switzerland) or in Montanide ISA 51
  • a single booster injection consisting of the same dose in the same delivery system can be administered intramuscularly ten days after the initial injection.
  • ELISA titers can be determined on microtiter plates coated with the testis-specific calpastatin B-cell epitope peptide (SEQ ID NO: 2; see Example 1) conjugated to bovine serum albumin (BSA) .
  • BSA bovine serum albumin
  • the B-cell epitope peptide can be synthesized with a non-natural cysteine at the amino terminus and conjugated to BSA as described in O'Hern et al., Biol. Reprod., 52, 331-339 (1995).
  • the ELISA can be performed as described in Laerimore et al . , J. Virol., 69, 6077-6089 (1995) .
  • the microtiter plate can be coated with peptide-conjugated BSA or BSA alone. After standard washing and blocking procedures, goat anti-human IgG conjugated to horseradish peroxidase can be added to detect bound antibody.
  • a macaque testis cDNA library (obtained from Dr. John Herr, University of Virginia) was screened with the human testis-specific calpastatin cDNA as probe, and B-cell epitopes identified.
  • a B-cell epitope of macaque testis-specific calpastatin was identified and has the following sequence:
  • Example 2 The antiserum produced in Example 2 was tested in a number of assays. First, an ELISA was performed to determine the antibody titer against two test antigens. To do so, microtiter plates were coated with the test antigens and incubated with dilutions of the antiserum, after which a standard ELISA was performed as described in O'Hern et al., Biol . Reprod. , 52, 331-339 (1995). Antibody titers were calculated in arbitrary units based on a control plate. The results are presented in the following table.
  • *T-CAST-GST is a fusion protein of the full-length testis-specific calpastatin (SEQ ID NO:l) with glutathione S-transferase.
  • the two entries are the results obtained using two different preparations of the protein.
  • +T-CAST is T-CAST-GST from which the GST has been proteolytically removed.
  • a Western blot was performed using anti-T- CAST:TT.
  • a 10% w/v extract of human liver (autopsy specimen) was prepared by homogenizing the tissue in 0.1 M sodium phosphate buffer, pH 7.0. Human sperm were concentrated by gentle centrifugation at 1000 x g, washing with buffer, and disruption by three cycles of freezing and thawing. About 25 ⁇ g of each of these preparations were loaded on SDS-PAGE (10% gels, reducing conditions) . The blots were probed with pre-immune serum and immune serum at 1:10,000 dilution.
  • a dark clear band was detected at about 60 KDa, the expected size of testis-specific calpastatin, in the sperm extract by anti-T-CAST:TT, but not with the pre-immune serum and not in the liver extract.
  • SIT complement-mediated sperm cytotoxic or sperm immobilization test
  • Freshly ejaculated and liquified sperm samples were diluted (1:2) with Biggers, Whitten and Whittingham (BWW) medium (see Methods In Mammalian Embryology, J.D. Daniel, Freeman, San Francisco, CA) and spun for 5 minutes at 450 x g.
  • the pellet was then washed twice in BWW containing 10 mg of human serum albumin (HSA) per ml at 250 x g for 4 minutes.
  • the resulting pellet was then layered with 2 ml of the wash medium for 1 hour at 37°C.
  • the swim- up cells were then diluted to a concentration of 20 million per ml, and 1 ⁇ l of this preparation was used for the SIT.
  • Five ⁇ l of the anti-T-CAST:TT were used for each reaction. It was diluted to 1:1 with BWW containing 30 mg HSA/ml and decomplemented for 30 minutes at 56°C.
  • Guinea pig serum absorbed twice with human spermatozoa, diluted 1:1 with sperm wash medium (see above) and used at 2 ⁇ l/reaction to give about 10-15 CH50 units/ reaction in the final mix.
  • 10 ⁇ l of the 1:1 diluted and decomplemented anti-T-CAST:TT plus 1 ⁇ l of the 20 million/ml swim-up sperm containing approximately 95% actively motile sperm plus 2 ⁇ l of the 1:1 diluted guinea pig complement were mixed and incubated at 37°C for 60 minutes. The percentage of motile sperm in the control and in the test vials were then counted.
  • the sperm immobilization or cytotoxic value (SIV)
  • testis-specific calpastatin is a cell-surface antigen which is accessible to cytotoxic antibodies and that it is of sufficient abundance to bind antibodies close enough to one another to initiate complement complexes.
  • GCAATTGCCT TCAATTTTTA GAGGAAAAGC TTTATATTTG TGTTATTACT TCTTCATCTT 1776

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne de nouvelles protéines et les peptides dérivés de ces protéines. Ces protéines sont spécifiques du sperme et des testicules, et ces protéines et peptides peuvent être utilisés dans des vaccins contraceptifs pour mammifères. Les protéines et les peptides conviennent également à des méthodes diagnostiques permettant d'évaluer l'infertilité. L'invention concerne également des molécules d'ADN codantes pour ces protéines et peptides, ainsi que des cellules hôtes renfermant les molécules d'ADN liées à des séquences de régulation de l'expression et servant à la production de ces protéines et peptides. Enfin, l'invention a pour objet des formulations pharmaceutiques comprenant un ou plusieurs anticorps, spécifiques d'une protéine de l'invention, utilisés comme contraceptif chez les mammifères.
PCT/US1998/015094 1997-07-21 1998-07-21 Proteines et peptides pour vaccins contraceptifs et diagnostics de fertilite WO1999003490A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU85049/98A AU8504998A (en) 1997-07-21 1998-07-21 Proteins and peptides for contraceptive vaccines and fertility diagnosis

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US5333797P 1997-07-21 1997-07-21
US60/053,337 1997-07-21
US11914998A 1998-07-20 1998-07-20
US09/119,149 1998-07-20

Publications (1)

Publication Number Publication Date
WO1999003490A1 true WO1999003490A1 (fr) 1999-01-28

Family

ID=26731737

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/015094 WO1999003490A1 (fr) 1997-07-21 1998-07-21 Proteines et peptides pour vaccins contraceptifs et diagnostics de fertilite

Country Status (2)

Country Link
AU (1) AU8504998A (fr)
WO (1) WO1999003490A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997026001A1 (fr) * 1996-01-16 1997-07-24 Northwestern University Proteines et peptides pour vaccins contraceptifs et diagnostic de la sterilite

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997026001A1 (fr) * 1996-01-16 1997-07-24 Northwestern University Proteines et peptides pour vaccins contraceptifs et diagnostic de la sterilite

Also Published As

Publication number Publication date
AU8504998A (en) 1999-02-10

Similar Documents

Publication Publication Date Title
JPH03502571A (ja) 不妊と避妊のための透明帯抗原と抗体の調製法及び使用
Adiga et al. Prospects of riboflavin carrier protein (RCP) as an antifertility vaccine in male and female mammals
US5693496A (en) DNA encoding the mouse and human PH30 beta chain protein
US7211255B2 (en) Contraceptives based on SP22 and SP22 antibodies
WO2009062034A1 (fr) Procédés, compositions, et séquences de peptides de liaison zp pour l'immunocontraception de chiens et d'autres animaux
US6258364B1 (en) Purified sperm surface antigen, monoclonal antibody therefor and application therefor
NAZ Fertilization‐related sperm antigens and their immunocontraceptive potentials
WO1997026001A1 (fr) Proteines et peptides pour vaccins contraceptifs et diagnostic de la sterilite
Afzalpurkar et al. Induction of native protein reactive antibodies by immunization with peptides containing linear B-cell epitopes defined by anti-porcine ZP3β monoclonal antibodies
Sivapurapu et al. Efficacy of antibodies against a chimeric synthetic peptide encompassing epitopes of bonnet monkey (Macaca radiata) zona pellucida‐1 and zona pellucida‐3 glycoproteins to inhibit in vitro human sperm‐egg binding
WO1999003490A1 (fr) Proteines et peptides pour vaccins contraceptifs et diagnostics de fertilite
Sivapurapu et al. Efficacy of antibodies against Escherichia coli expressed chimeric recombinant protein encompassing multiple epitopes of zona pellucida glycoproteins to inhibit in vitro human sperm–egg binding
HUT77262A (hu) Új immunokontraceptív peptidek
Naz et al. Antibodies to human sperm YLP12 peptide that is involved in egg binding inhibit human sperm capacitation/acrosome reaction
EP2711019B1 (fr) Constructions de phages, séquences et compositions antigéniques pour l'immunocontraception d'animaux
Yurewicz et al. Generation and characterization of site-directed antisera against an amino-terminal segment of a 55 kDa sperm adhesive glycoprotein from zona pellucida of pig oocytes
US6455041B1 (en) Immunogenic epitopes of the human zona pellucida protein (ZP1)
US20060121051A1 (en) Heat shock fusion-based vaccine system
WO2000056144A1 (fr) Antigenes de surface de sperme humain
Naz et al. Monoclonal antibody against human sperm-specific YLP 12 peptide sequence involved in oocyte binding
JP2003521902A (ja) 卵特異的表面タンパク質
US6190670B1 (en) Use of mCRP to enhance immune responses
Apostolidis et al. The peptide loop consisting of amino acids 139–157 of human granzyme B (fragmentin 2) contains an immunodominant epitope recognized by the mouse
WO1997039020A2 (fr) Sequences antigeniques d'une proteine seminale et methodes immunocontraceptives
AU2011232618B2 (en) Phage constructs, sequences and antigenic compositions for immunocontraception of animals

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 1999507837

Format of ref document f/p: F

NENP Non-entry into the national phase

Ref country code: CA

122 Ep: pct application non-entry in european phase
点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载