WO1997026001A1 - Proteines et peptides pour vaccins contraceptifs et diagnostic de la sterilite - Google Patents

Proteines et peptides pour vaccins contraceptifs et diagnostic de la sterilite Download PDF

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WO1997026001A1
WO1997026001A1 PCT/US1997/000908 US9700908W WO9726001A1 WO 1997026001 A1 WO1997026001 A1 WO 1997026001A1 US 9700908 W US9700908 W US 9700908W WO 9726001 A1 WO9726001 A1 WO 9726001A1
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lys
glu
ser
peptide
val
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PCT/US1997/000908
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Erwin Goldberg
Patricia O'hern Weinberg
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Northwestern University
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Priority to AU17053/97A priority Critical patent/AU1705397A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—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
    • 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.
  • 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. Gvnecol.. 158. 59-62 (1988); Clarke et al., Fertil. Steril..
  • the invention provides purified proteins and peptides whose sequences comprise the sequence of an epitope of one of these 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
  • vaccines comprising the proteins (or immunogenic portions thereof) , 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 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.
  • Figure 1 Diagram comparing the sequences of somatic and testis-specific isoforms of calpastatin.
  • Figure 2 Computer-generated hydropathy plot comparing the first forty-one amino acids of somatic (solid bars) and testis-specific (open bars) isoforms of calpastatin.
  • Figure 3 Western blot of human tissue extracts (lane 1 - testis, lane 2 - sperm, lane 3 - liver) probed with affinity-purified rabbit antiserum to a peptide having the sequence of a B-cell epitope found only on the testis- specific isoform of calpastatin.
  • Figure 4 Graph of ELISA results. In particular, absorbance at 405 nm is plotted versus weeks post primary immunization of macaques with a peptide having the sequence of a B-cell epitope found only on testis-specific isoform of calpastatin linked to a universal T-cell epitope by a four-amino acid linker.
  • Figure 5 Diagram of the technique of epitope mapping by nested deletions for clone C-2 and photograph of Coomasie blue-stained PAGE gel after separation of the resultant truncated proteins.
  • Figure 6 Western blots of truncated proteins produced by nested deletions performed to identify B-cell epitopes on the protein produced by clone C-2.
  • Figure 7 Diagram illustrating epitope identification for clone C-2.
  • Figure 8 Computer-generated plot of the occurrence of the amino acid valine along the length of the clone L-7 protein.
  • Figure 9 Western blots of truncated proteins produced by nested deletions performed to identify B-cell epitopes on the protein produced by clone L-7.
  • Figure 10 Diagram illustrating epitope identification for clone L-7.
  • 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 (see Example 1 below) .
  • the complete sequence of this human testis-specific isoform of calpastatin is given in Chart A below.
  • Affinity-purified antiserum specific for this testis- specific isoform of calpastatin was used to localize the isoform on human sperm by immuno-fluorescence. Diffuse, granular fluorescence was observed throughout the acrosome, and intense fluorescence was observed in the equatorial segment of the sperm (see Example 4) .
  • 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.. 2, 109-142 (1987) .
  • Calpain then, in the presence of the Ca +2 would hydrolyze calpastatin, thereby releasing protease inhibition and permitting proteolytic activity in membrane fusion phenomena. Goll et al., Bioessays. 14. 549-556 (1992) . Perturbation of this sequence of events by antibodies directed to testis- specific calpastatin would compromise fertilization and concomitantly cause infertility. Preliminary studies have demonstrated loss of calpastatin immunoreactivity from acrosome-reacted sperm, a result predicted from this theory. Also, the immunofluorescence studies described above show that testis-specific calpastatin is found on the surface of sperm and would, therefore, be accessible to antibodies.
  • the invention further provides a protein which is the protein produced by clone C-2.
  • Clone C-2 is a human cDNA clone that was identified by screening a human testis cDNA library with sera from infertile patients positive for antisperm antibodies (see Example 1 below) .
  • the C-2 protein is found in testis and sperm, but it is not found in other tissues.
  • the complete amino acid sequence of the C-2 protein is set forth in Chart B below.
  • the invention also provides a protein which is the protein produced by clone L-7.
  • Clone L-7 is a human cDNA clone that was identified by screening a human testis cDNA library with sera from infertile patients positive for antisperm antibodies (see Example 1 below) .
  • the L-7 protein is found in testis and sperm, but it is not found in other tissues. Affinity-purified antiserum specific for the L-7 protein was used to localize the L-7 protein on human sperm by immunofluorescence. Fluorescence was observed throughout the acrosome.
  • the complete amino acid sequence of the L-7 protein is set forth in Chart C below.
  • the Y-19, C-2 and L-7 proteins are human proteins. 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 sequences given in Charts A, B and C 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. See, e.g.. Ausubel et al.. Current Protocols In Molecular Biology. Volumes 1 and 2 (John Wiley and Sons, New York 1989) and Sambrook et al.. Molecular Cloning; A Laboratory Manual (Cold Spring Harbor
  • Testis cDNA libraries can also be purchased from ClonTech Laboratories, Inc., 1020 E. Meadow Circle, Palo Alto, CA 94303-4230.
  • the proteins of the invention can be used in contraceptive vaccines in mammals. Preferably a protein from the same species of mammal that is to be immunized is used in the vaccine. However, given the expected close homology of the proteins from different mammalian species, it is expected that 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. In choosing an immunogenic portion of testis-specific calpastatin, a portion must be chosen which includes sequences found on the testis-specific isoform but not found on the somatic isoforms.
  • testis-specific calpastatin or an immunogenic portion thereof, since somatic isoforms exist, and cross-reaction with these 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 of the protein.
  • a peptide derived from testis- specific calpastatin must include 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.
  • 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 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.
  • the protein coded for by clone C-2 contains the following sequence:
  • Pro Glu Pro Lys lie lie Pro Ser Glu Glu Asp Pro Thr Phe 15
  • Peptides having this sequence, or a portion of it that includes the sequence from amino acid 4 through amino acid 17, can be used to elicit antibodies that react specifically with the C-2 protein.
  • Amino acids 4-17 in the above sequence have been identified as a B-cell epitope.
  • the protein coded for by clone L-7 contains the following sequence:
  • SEQ ID NO:11 and SEQ ID NO:12 have been identified as B-cell epitopes, and peptides having these sequences can be used to elicit antibodies that react specifically with the protein.
  • 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) , he ocyanins (e.g.. Keyhole Limpet he ocyanin) , toxins (e.g.. diptheria toxoid, tetanus toxoid) and polypeptides such as polylysine or polyalaninelysine. Preferred are diptheria toxoid and tetanus toxoid.
  • the peptide may be coupled to the carrier with conjugating reagents such as glutaraldehyde, a water soluble carbodiimide such as l-(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.
  • conjugating reagents such as glutaraldehyde, a water soluble carbodiimide such as l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro ⁇ chloride (ECDI
  • 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 ⁇ Svm . 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 ⁇ Svmp. 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. 477-483 (1990); Kaumaya, et al., J. Molec. Reco ⁇ .. 6_, 81-94 (1993) .
  • a preferred promiscuous T-cell epitope has the following sequence:
  • 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 j8-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. See Goldberg, in Gamete Interaction: Prospects For Immunocontraception. pages 63- 73 (1990); Alexander et al., Reprod. Ferti1. Dev.. 6_, 273- 80 (1994); O'Hern et al., Biol. Reprod.. 52. 331-339 (1995) .
  • 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 used as such or may be linked to a carrier.
  • the carriers e.g. f 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. 3,941,763, 4,782,136, 4,990,596; Finn et al., in The Proteins , 3rd ed.
  • 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.
  • 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 In the vector, 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. 84. 4762-66 (1987) . Comparative studies of ribosomal binding sites, such as the study of Scherer, et al.. Nucleic Acids Res.. 8., 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 Sligar, Proc. Nat'l Acad. Sci. UH , M 8961-65 (1987) and von Bod an 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:28] .
  • 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. 59. 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. First, chemical synthesis is desirable because 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.
  • 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, opti al 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.
  • 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. Although 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.
  • a human testis cDNA library was screened with sera from infertile patients positive for antisperm antibodies. This screening was performed as described in Liang et al. , Reprod. Ferti1. Dev.. 6_, 297-305 (1994) . It is interesting to note that these patients, although infertile, were otherwise healthy. A total of 43 unique cDNA inserts were detected by the screening, of which four were testis-specific by Northern blot analysis (performed as described in Liang et al., Reprod. Fertil. Dev.. 6_, 297-305 (1994) ; see below) . One of the four clones turned out to encode a truncated mRNA for a somatic peptide and was not evaluated further. The remaining three clones were designated Y-19, C-2 and L-7.
  • Figure 1 shows the relationship between the published sequence of DNA coding for somatic calpastatin (solid) and the testis-specific region of clone Y-19 (diagonal stripes) .
  • Clone Y-19 appears to be a product of alternative splicing whereby DNA coding for somatic calpastatin domains L and 1 has been deleted and replaced with DNA coding for a unique, testis-specific L domain of approximately 65 amino acids (stripes) .
  • the rest of the cDNA sequence of clone Y-19 is virtually identical to the published sequence of somatic calpastatin.
  • DNA coding for testis-specific calpastatin contains 2 unique restriction sites (arrows) .
  • a lkb fragment of clone Y-19 was used to probe a Northern blot of human poly A+ RNA from eight different human tissues (leukocytes, colon, small intestine, ovary, testis, prostate, thymus and spleen; Multiple Tissue Northern blots purchased from Clonetech, Palo Alto, CA) . Two mRNAs of 4.3 and 2.8kb were detected by the probe in all tissues. A third mRNA of 1.9kb was detected only in testis.
  • Serum YM The serum that identified clone Y-19 (serum YM) agglutinates human sperm in a head-to-head orientation and completely inhibits cervical mucus penetration.
  • Figure 2 shows a computer-generated hydropathy plot of the first 41 residues of somatic calpastatin (solid lines) versus the first 41 residues of testis-specific calpastatin (open bars) .
  • 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) .
  • Only 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:
  • testis-specific calpastatin has a hydrophobic tail. This hydrophobic tail could serve as a membrane anchor for the protein.
  • a peptide immunogen was prepared containing the testis-specific calpastatin B-cell epitope identified in
  • Example 3 linked to a carrier comprising a universal T-cell epitope derived from tetanus toxoid.
  • the T-cell epitope had the following sequence: Val Asp Asp Ala Leu lie Asn Ser Thr Lys lie Tyr Ser Tyr
  • This immunogen [SEQ ID NO:7] was synthesized at the Salk Institute (under Contract NOl-HD-0-2906 with the NIH) and made available by the Contraceptive Development Branch, Center for Population Research, NICHD (Bethesda, MD) .
  • the affinity-purified antiserum was used to probe a Western blot of human tissue extracts.
  • the tissue extracts were made and the Western blots were performed as described in Diekman and Goldberg, Biol. Reprod.. 50. 1087-1093 (1994) .
  • the antiserum recognized a single protein of approximately 65Kd in human testis extracts (lane 1) and a slightly larger protein of approximately 68Kd in human sperm extracts (lane 2) .
  • There was no reactivity with human liver extracts (lane 3) although liver is known to be rich in the somatic isoforms of calpastatin.
  • the affinity-purified antiserum was also used to localize testis-specific calpastatin on human sperm by immunofluorescence, performed as described in Wright et al., Biol. Reprod.. 42. 693-701 (1990). Diffuse, granular fluorescence was observed throughout the acrosome, and intense fluorescence was observed in the equatorial segment of the sperm.
  • mice Female cynomologous macaques (three per group) were immunized with either lOO ⁇ g or 300 ⁇ g of the peptide immunogen [SEQ ID NO:7] prepared in Example 4.
  • the immunogen was administered intramuscularly in Sgualene- Arlacel A containing the synthetic muramyl dipeptide N- acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP11637; Ciba- Geigy Pharmaceuticals, Basel, Switzerland) .
  • a single booster injection consisting of the same dose in the same delivery system was administered intramuscularly ten days after the initial injection.
  • ELISA titers were determined on microtiter plates coated with the testis-specific calpastatin B-cell epitope peptide (SEQ ID NO:2; see Example 3) conjugated to bovine serum albumin (BSA) .
  • BSA bovine serum albumin
  • the B-cell epitope peptide was 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 was performed as described in Laerimore et al., J. Virol.. 69. 6077-6089 (1995) .
  • the microtiter plate was coated with peptide-conjugated BSA or BSA alone.
  • the cDNA insert of clone C-2 was used to probe a Northern blot of human poly A+ RNA from eight different human tissues as described above in Example 2. A single mRNA of 2.lkb was detected in testis only.
  • clone C-2 cDNA encodes a unique and previously undescribed protein.
  • the mRNA is approximately 2.1 kb. It has an open reading frame (ORF) of 1.4 kb translating to a peptide of 65-70 Kd. There are no significant sequence motifs or unusual properties.
  • the original antiserum that detected clone C-2 (number 629) is 100% effective in blocking fertilization in vitro of human ova by human sperm (see table below) .
  • Serum 629 which has been absorbed with sperm no longer blocks binding of sperm to zona (see table below) .
  • the peptide coded for by a 900 bp fragment from the 3' end of the C-2 cDNA was expressed as a glutathione-s- transferase (GST) fusion protein using cloning methods well known in the art. See, e.g.. Smith and Johnson, Gene. 67. 31-40 (1988); Johnson et al. , Nature. 338. 585-587 (1989); Kemp et al.. Gene. 94. 223-28 (1990); Kaelin Jr. et al., Cell. 64. 521-532 (1991); Chittenden Jr. et al. , Cell. 65. 1073-1082 (1991); Kaelin Jr. et al.. Cell. 7_0, 351-364 (1992) .
  • the clone encoding this fusion protein was designated clone GST-C2.
  • Each of the truncated GST-C2 fusion proteins was partially purified and used as the target for Western blots (all as described in Example 6) probed with the original patient 629 serum.
  • the results are shown in Figure 6.
  • the full-length fusion protein and the first 4 deletions were strongly positive for the antibody.
  • Time points 5-10 were negative, as was GST alone. Therefore, the C2 epitope recognized by the original human serum resides within time point 4.
  • Each of the 10 nested deletions was sequenced using an oligo primer specific for the pGEX vector (see Pharmacia Biotech GST Gene Fusion Manual) .
  • the results are shown in Figure 7.
  • the first 3 time points showed deletion of the 3' untranslated region (UTR) .
  • Time point 4 from which the 9 carboxy terminal amino acids were deleted, was still antibody positive.
  • Time point 5 with deletion of an additional 26 amino acids, was antibody negative. Therefore, the relevant B-cell epitope (cross-hatched box) resides within the region of amino acids 426-454.
  • the sequence of amino acids 426-454 is as follows:
  • EXAMPLE 8 Preparation of C-2 Immunogen An immunogen comprising the B-cell epitopes identified in Example 7 was prepared as described in Example 4. The sequence of this immunogen is:
  • the cDNA insert of clone L-7 was used to probe a Northern blot of human poly A+ RNA from eight different human tissues as described above in Example 2. A single mRNA of 2.5kb was detected in testis only.
  • the sequence of the cDNA insert of clone L-7 was determined as described in Liang et al. , Reprod. Ferti1. Dev. f 6_, 297-305 (1994) .
  • the DNA sequence of the insert and the corresponding amino acid sequence are set forth in Chart C below. Homology searches of the GenEMBL databases found that the sequence of the cDNA insert of clone L-7 was not represented. Thus, clone L-7 cDNA encodes an unique and previously undescribed protein.
  • This protein is relatively large (66 kD) and consists of several domains of as yet unknown functional significance.
  • the protein contains an endoplasmic reticulum signal sequence and appears to be anchored in the sperm plasma membrane at its amino terminus, but with surface accessible epitopes.
  • This plot was generated using PC/Gene software from Intelligenetics, Inc., 700 E. El Ca ino Rd. , Mountainview, CA 94047.
  • This computer analysis revealed the following features.
  • Residues 88-328 contain very little valine and 9 potential protein kinase C (PKC) phosphorylation sites (P) .
  • Residues 329 to 493 contains many valines and no PKC phosphorylation sites.
  • Residues 329-493 also contain 11 repeats of a 15 amino acid motif (see below) .
  • the consensus sequence of the motif is KgqEaQVKKsesgVp [SEQ ID NO:16].
  • Residues 494-568 contain few valines and 3 potential PKC phosphorylation sites. From the computer analysis and the protein's sequence, the following domain organization of the L-7 protein is proposed:
  • Domain I contains a consensus endoplasmic reticulum localization signal (p>0.85)
  • Domain IV again has a high isoelectric point and contains 2 bipartite nuclear translocation signals (see Robbins et al., Cell. 64. 615-623 (1991)).
  • L-7 was expressed and purified as a GST fusion protein as described in Example 6 above. This clone was designated GST-L7. Sera from three infertile patients (numbers 44, 65 and 66) recognized the fusion protein on Western blots (performed as described in Example 6) .
  • Epitope 1 is amino acids 500-517
  • epitope 2 is amino acids 389-408. These epitopes have the following sequences: Lys Gly Gin Glu Ala Gin Val Lys Lys Arg Glu Ser Val Val
  • Immunogens comprising the two B-cell epitopes identified in Example 10 were prepared as described in Example 4. The sequences of these two immunogens are:
  • Example 14 was used to immunize rabbits as described in Example 4.
  • the rabbit antiserum was affinity purified, and the affinity-purified rabbit antiserum was used to probe a Western blot of human tissue extracts, all as described in Example 4.
  • the affinity-purified antiserum recognized a single protein of approximately 58 Kd in human testis extracts and a protein of approximately 68 Kd in human sperm extracts. There was no reactivity with human liver extracts.
  • a B-cell epitope of macaque testis-specific calpastatin was identified and has the following sequence:
  • This B-cell epitope is 85% homologous to the B-cell epitope identified above for human testis-specific calpastatin [SEQ ID NO:2] .
  • the B-cell epitope of the macaque protein corresponding to the human protein produced by clone C-2 has a sequence identical to that of the B-cell epitope of the C-2 protein [SEQ ID NO:8]. Thus, in this case, there was 100% homology between the sequences.
  • Peptides having the sequences of the B-cell epitopes identified in Examples 3, 7 and 10 can be synthesized and coupled to diptheria toxin to produce immunogens that can -34- be used to immunize mammals, all as described in O'Hern et al., Biol. Reprod.. 5_2_, 331-339 (1995).
  • EXAMPLE 16 Sequencing Of Clones Y-19.
  • C-2 and L-7 DNA fragments of clones Y-19, C-2 and L-7 were subcloned into the pBluescriptll SK+ phagemid (Stratagene, Palo Alto, CA) and sequenced by a modification of the method of Kraft et al., Biotechniques. 6_, 544-547 (1988) as described in O'Hern et al., Biol. Reprod.. 52. 331-339 (1995) .
  • the DNA sequences and deduced amino acid sequences are presented in Charts A (Y-19) , B (C-2) and C (L-7) .
  • GAG AAG GCC AAA GAA GAA GAC CGT GAA AAG CTT GGT 657 Glu Lys Ala Lys Glu Glu Asp Arg Glu Lys Leu Gly 185 190
  • GGA GGT AAA GCG AAG GAT TCA GCA AAG ACA ACA GAG 1341 Gly Gly Lys Ala Lys Asp Ser Ala Lys Thr Thr Glu
  • Glu Leu Ser Ser lie Lys Asn Leu Gin His Asn lie 105 110 CAT CTG AAG GAG CTC TTT CTC ATG GGG AAC CCA TGT 432 His Leu Lys Glu Leu Phe Leu Met Gly Asn Pro Cys 115 120 125
  • TGG TAC ACA GAC ATC AAT GCT ACT CTT TCC TCT TTA 720
  • Arg Arg Pro Glu Pro Lys lie lie Pro Ser Glu Glu 440 445 GAC CCA ACC TTT GAA GAC AAC CCT GAA GTG CCT CCG 1440 Asp Pro Thr Phe Glu Asp Asn Pro Glu Val Pro Pro 450 455 460
  • GTA CTA AAA GGA CAG GAA GCC CAA GAA AAG AAG GAG 1694 Val Leu Lys Gly Gin Glu Ala Gin Glu Lys Lys Glu
  • GGT GAA AAA TCA AAA GGC TCG AAA AGG CGA AGG CAA 1874 Gly Glu Lys Ser Lys Gly Ser Lys Arg Arg Arg Gin
  • GAG AAG GCC AAA GAA GAA GAC CGT GAA AAG CTT GGT 657
  • GGA GGT AAA GCG AAG GAT TCA GCA AAG ACA ACA GAG 1341 Gly Gly Lys Ala Lys Asp Ser Ala Lys Thr Thr Glu
  • GAG AGC AAA GAC CAC CTA CAG GCA CCA GAC ATA GAG 756
  • GCT GTC ATC CTG ACT CTA CTG GGA CTT GCC
  • GCT ATT TTG TTA ACA AGA TGG GCA CGA CGT
  • CAC CTT CAG CAC CCG CGG TCA CCC ATG GCA CCC ATA 758 His Leu Gin His Pro Arg Ser Pro Met Ala Pro He 165 170 175
  • GCA AGG TCT CAG ATA GCC
  • GAG AAG AAA ACA AGG AAG 974 Ala Arg Ser Gin He Ala Glu Lys Lys Thr Arg Lys 240 245
  • GTA CCA AAA GGA CAA GAA GGC CAA GTA GAG AAG ACT 1514
  • GGT GAA AAA TCA AAA GGC TCG AAA AGG CGA AGG CAA 1874 Gly Glu Lys Ser Lys Gly Ser Lys Arg Arg Arg Gin 540 545 ATA CAG GAA GGA AGT ACA ACA AAA AAG TGG AAG AGT 1910 He Gin Glu Gly Ser Thr Thr Lys Lys Trp Lys Ser 550 555 560

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Abstract

L'invention porte sur de nouvelles protéines et des peptides dérivés de ces dernières. Lesdites protéines sont spécifiques du sperme et des testicules et ces protéines et peptides peuvent servir à produire des vaccins contraceptifs pour mammifères et à effectuer des tests de diagnostic de la stérilité. L'invention porte également sur des molécules d'ADN, codant pour ces protéines et peptides, et des cellules hôtes contenant ces molécules d'ADN liées à des séquences de régulation d'expression, et servant à l'obtention de ces protéines et peptides.
PCT/US1997/000908 1996-01-16 1997-01-16 Proteines et peptides pour vaccins contraceptifs et diagnostic de la sterilite WO1997026001A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999003490A1 (fr) * 1997-07-21 1999-01-28 Northwestern University Proteines et peptides pour vaccins contraceptifs et diagnostics de fertilite
WO2000026360A1 (fr) * 1998-11-03 2000-05-11 Adherex Technologies Inc. Composes et methodes permettant de moduler des fonctions induites par les claudines
US6723700B1 (en) 1998-11-03 2004-04-20 Adherex Technologies, Inc. Compounds and methods for modulating claudin-mediated functions
WO2004055185A1 (fr) * 2002-12-18 2004-07-01 Takeda Pharmaceutical Company Limited Nouvelle proteine et adn associe
EP2277905A3 (fr) * 2002-03-13 2011-05-25 Ganymed Pharmaceuticals AG Produits géniques d'expression différentielle dans les tumeurs et leur utilisation

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BIOCHEMISTRY AND MOLECULAR BIOLOGY INTERNATIONAL, May 1994, Vol. 33, No. 2, WANG et al., "Calpastatin in Human Testis", pages 245-252. *
JOURNAL OF MOLECULAR RECOGNITION, 1993, Vol. 6, KAUMAYA et al., "Peptide Vaccines Incorporating a 'Promiscuous' T-cell Epitope Bypass Certain Haplotype Restricted Immune Responses and Provide Broad Spectrum Immunogenicity", pages 81-94. *
REPRODUCTION FERTILITY AND DEVELOPMENT, 1994, Vol. 6, LIANG et al., "Human Testis cDNAs Identified by Sera from Infertile Patients: a Molecular Biological Approach to Immunocontraceptive Development", pages 297-305. *
TECHNIQUES IN PROTEIN CHEMISTRY, 1993, Vol. IV, O'HEARN et al., "The Use of Molecular Modelling to Delineate B-cell and T-cell Epitopes of Human Sperm-specific LDH-C4", pages 481-490. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999003490A1 (fr) * 1997-07-21 1999-01-28 Northwestern University Proteines et peptides pour vaccins contraceptifs et diagnostics de fertilite
WO2000026360A1 (fr) * 1998-11-03 2000-05-11 Adherex Technologies Inc. Composes et methodes permettant de moduler des fonctions induites par les claudines
US6723700B1 (en) 1998-11-03 2004-04-20 Adherex Technologies, Inc. Compounds and methods for modulating claudin-mediated functions
US6756356B2 (en) 1998-11-03 2004-06-29 Adherex Technologies, Inc. Compounds and methods for modulating claudin-mediated functions
US6830894B1 (en) 1998-11-03 2004-12-14 Adherex Technologies, Inc. Compounds and methods for modulating claudin-mediated functions
EP2277905A3 (fr) * 2002-03-13 2011-05-25 Ganymed Pharmaceuticals AG Produits géniques d'expression différentielle dans les tumeurs et leur utilisation
WO2004055185A1 (fr) * 2002-12-18 2004-07-01 Takeda Pharmaceutical Company Limited Nouvelle proteine et adn associe

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