EP1007667A1 - Nucleic acids encoding sperm antigens and reprosa-i polypeptides - Google Patents

Nucleic acids encoding sperm antigens and reprosa-i polypeptides

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Publication number
EP1007667A1
EP1007667A1 EP98908674A EP98908674A EP1007667A1 EP 1007667 A1 EP1007667 A1 EP 1007667A1 EP 98908674 A EP98908674 A EP 98908674A EP 98908674 A EP98908674 A EP 98908674A EP 1007667 A1 EP1007667 A1 EP 1007667A1
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EP
European Patent Office
Prior art keywords
polypeptide
reprosa
seq
human
antibody
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.)
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Application number
EP98908674A
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German (de)
French (fr)
Inventor
Cynthia K. French
Lorna I. Neilson
Patrick A. Schneider
Karen K. Yamamoto
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Reprogen Inc
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Reprogen Inc
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Publication date
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Publication of EP1007667A1 publication Critical patent/EP1007667A1/en
Withdrawn legal-status Critical Current

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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
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • an anti-sperm immune response in a patient is correlated with infertility.
  • anti-sperm antibodies are detected more frequently in the sera, seminal fluid, and/or cervical or vaginal fluid of couples experiencing unexplained infertility than in couples whose infertility is due to tubal factors.
  • vasectomized men experiencing reduced fertility upon vasectomy reversal have been found to exhibit anti-sperm antibodies (Bronson et al., 1984, Fertil. Steril. 42:171-183; Lehman et al., 1989, Am. J. Reprod. Immunol. 19:43-52; Naz et al., 1990, Human Reproduction 5:511-518).
  • Contraceptive vaccines have long been viewed as a highly desirable form of contraception, because they are relatively inexpensive, easy to administer, and capable of providing prolonged protection against pregnancy (Aitken et al., 1993, British Med. Bull. 49:88-99).
  • the primary targets for development of a contraceptive vaccine are (i) human chorionic gonadotrophin, (ii) the zona pellucida, and (iii) sperm.
  • a vaccine directed against sperm, particularly a sperm-surface antigen would have the advantage of having fewer effects on the hormonal milieu of the patient and fewer endocrinological side effects.
  • few suitable sperm antigens have been identified.
  • sources of sperm proteins, especially sperm surface proteins that can serve as antigenic targets, and for the creation of vaccines and other medicaments directed against sperm antigens.
  • immunological infertility is often a consequence of the presence of anti-sperm antibodies in a patient.
  • immunological infertility is a diagnosis of exclusion, due to the lack of simple and inexpensive assays for detecting naturally occurring anti-sperm antibodies.
  • assays that can be used in the diagnosis of immunological infertility. The present invention addresses these and other needs.
  • the invention provides a substantially pure ReproSA- 1 polypeptide, or fragment of a ReproSA- 1 polypeptide.
  • the polypeptide has a sequence substantially homologous to, or the same as, SEQ ID NO:2, or is a polypeptide having between about eight and about 509 contiguous residues of a polypeptide having the amino acid sequence of SEQ ID NO:2.
  • substantially pure polypeptides that are specifically immunoreactive with an antibody raised against a polypeptide having the amino acid sequence of SEQ ID NO:2.
  • any of the aforementioned polypeptides may be coupled to a solid support, for example, for use in assays for anti-ReproSA-1 antibodies.
  • the invention provides an isolated polynucleotide comprising a sequence encoding a ReproSA-1 polypeptide or fragment thereof, such as a peptide having between about eight and about 509 contiguous residues of a polypeptide having the amino acid sequence of SEQ ID NO:2.
  • the ReproSA-1 coding sequences may be operably linked to a promoter.
  • a recombinant cell, or cell line comprising a polynucleotide encoding ReproSA- 1 or a fragment thereof.
  • the cell line is capable of producing a human ReproSA- 1 polypeptide or a fragment thereof.
  • the invention provides an isolated or recombinant oligonucieotide or polynucleotide having a sequence of at least 10 contiguous nucleotides of SEQ ID NO: 1. In one embodiment, the oligonucieotide or polynucleotide has at least 12 contiguous nucleotides of SEQ ID NO:l.
  • the invention provides a substantially purified anti- ReproSA-1 antibody, or fragment thereof, that specifically binds to human ReproSA- 1.
  • the antibody may be polyclonal, monoclonal, human or humanized.
  • the antibody binds ReproSA- 1 with an affinity of at least about 10 8 M "1 .
  • a cell or hybridoma that secretes the antibody.
  • the invention also provides a medicament for contraception containing anti-ReproSA-1 antibodies in a pharmacologically acceptable carrier.
  • the invention provides vaccines comprising ReproSA-1 polypeptides which are capable of inducing an immune response in a human or non-human subject.
  • the vaccines may include a ReproSA- 1 polypeptide (comprising at least about eight consecutive residues of SEQ ID NO:2) or a polynucleotide encoding ReproSA- 1, in a pharmacologically acceptable carrier.
  • the vaccine includes an adjuvant.
  • the anti-ReproSA-1 antibodies elicited by the vaccine may be found in mucosal secretions, e.g., mucosal secretions of the reproductive tract, e.g., the vaginal mucosa.
  • the invention provides methods of immunizing a human to achieve a contraceptive effect by administering the vaccines.
  • the invention provides methods for: diagnosing immunological infertility in a human patient, for example, by detecting anti-ReproSA-1 antibodies in a biological sample from a patient; detecting an abnormal ReproSA- 1 protein in sperm of a patient by contacting the sperm of the patient with an antibody that specifically binds to an epitope of the ReproSA- 1 polypeptide having the amino acid sequence of SEQ ID NO:2 and determining the binding of the antibody to the sperm of the patient, where failure of the sperm to bind indicates the presence of abnormal ReproSA- 1 protein; and detecting a mutation in a ReproSA- 1 gene in a patient by comparing the sequence of a ReproSA- 1 gene of the patient with the sequence of the ReproSA- 1 gene having the sequence of SEQ ID NO:l, wherein a difference in the two sequences indicates a mutation in the ReproSA- 1 gene of the patient.
  • kits for carrying out the methods listed above are useful for determining the presence in a biological sample of antibodies to human ReproSA- 1 polypeptide and contains (i) an antibody specifically reactive with ReproSA- 1 and (ii) a polypeptide bound to a solid support, wherein the polypeptide comprises at least about eight consecutive amino acids of SEQ ID NO:2.
  • FIGURES Figure 1 shows the nucleotide sequence of the ReproSA- 1 cDNA [SEQ ID NO: 1]
  • Figure 2 shows a diagram illustrating the 1.8 and 2.8 KB ReproSA- 1 transcripts as well as four ReproSA- 1 clones.
  • Figure 3 shows a multiple-tissue Northern Blot probed with a labeled EcoRI/XhoI fragment of the ReproSA- 1 cDNA.
  • RNA was from human spleen (lane 1), thymus (lane 2), prostrate (lane 3), testis (lane 4), ovary (lane 5), small intestine (lane 6), colon (lane 7) and peripheral blood lymphocytes (lane 8).
  • Molecular weight is indicated in kilobases.
  • Figure 4 shows a Southern blot of EcoRI digested genomic DNA collected from human, monkey, rat, mouse, dog, cow, rabbit, chicken and yeast probed with a labeled probe from the ReproSA- 1 cDNA clone insert. Molecular weight is indicated in kilobase pairs.
  • Figure 5 shows the expression and purification of a poly(His) tagged ReproSA-1 polypeptide (encoded by Clone ReproSA-1.0).
  • Figure 6 shows Western blots of normal human sperm extracts probed with anti-ReproSA-1 antisera.
  • Lane 1 anti-(ReproSA-l recombinant polypeptide) antibody [rabbit #1]
  • lane 2 anti-(ReproSA-l recombinant polypeptide) antibody [rabbit #2]
  • lane 3 pre-immune serum from rabbit #1 used in lane 1
  • lane 4 pre-immune serum from rabbit #2 used in lane 2
  • lane 5 affinity purified anti-(Re ⁇ roSA armadillo repeat peptide) antibody [pool of two rabbits]
  • lane 6 affinity purified anti-(ReproSA C-terminus peptide) antibody [pool of two rabbits]
  • lane 7 unpurified anti-(ReproSA armadillo repeat peptide) serum [one rabbit]
  • lane 8 unpurified anti-(ReproSA C-terminus peptide) serum [one rabbit]
  • Lane 9 pooled pre-imm
  • the present invention is related to the identification and isolation of a human gene that encodes the sperm protein ReproSA- 1.
  • the gene is conserved in non-human primates and rodents, and its expression is testis-specific.
  • the gene and its RNA and protein products, as well as related nucleic acids, peptides and polypeptides are useful for contraception in humans and other animals, for example by vaccination, as well as for diagnosis and treatment of infertility.
  • this invention provides substantially pure or isolated ReproSA- 1 polypeptides, i.e., that are related to and/or derived from human ReproSA- 1 polypeptides.
  • ReproSA- 1 polypeptides comprise an amino acid sequence substantially identical to the amino acid sequence shown in Figure 1 [SEQ ID NO:2] or are immunologically related to the ReproSA-1 polypeptide of SEQ ID NO:2.
  • the polypeptide of the invention has between about eight and about 509 contiguous residues of a polypeptide having the amino acid sequence of SEQ ID NO:2.
  • ReproSA-1 polypeptides are components of the sperm (e.g., sperm tail) in humans and other species (e.g., non-human primates and rodents).
  • the full-length ReproSA- 1 polypeptides of the invention contain eight contiguous armadillo ("arm") repeats (Peifer et al., 1994, Cell 76:789).
  • a number of proteins with diverse cellular functions also contain armadillo repeats including pendulin, Rchl, importin, SRP-1, and armadillo (Peifer et al., supra).
  • ReproSA-1 shows additional homology to the PF16 flagellar protein of Chlamydomonas reinhardt, a protein believed to be involved in protein-protein interactions important for central microtubule stability and flagellar motility (Smith et al., 1996, J Cell Biology 132:359-370).
  • armadillo motifs of the ReproSA- 1 protein are involved in protein-protein interactions with other sperm proteins (e.g., flagellar proteins).
  • the armadillo repeat regions (i.e., motifs and sequences) of the ReproSA- 1 proteins, genes, and RNA transcripts provide a target for disruption of ReproSA- 1 activity or expression, and/or disruption or diminution of sperm motility or viability.
  • ReproSA- 1 activity includes the ability of the protein to assemble in the sperm, to interact with other sperm proteins, or any other activity or function of naturally occurring ReproSA- 1 polypeptides or nucleic acids.
  • Table 1 infra, shows an alignment of the ReproSA- 1 armadillo repeats with the "universal" consensus (Peifer et al., supra) and the PF16 armadillo repeat consensus sequence. Residues showing identity with the consensus are shown in bold (all polypeptide sequences described herein are presented in the conventional amino-terminus- > carboxy-terminus orientation).
  • ReproSA-1 (65) GRLANYNDD AEAWKCDI LPQLVYS AE QNRFYKKAAA FV [SEQ ID (107) LRAVGKHSPQ AQAIVDCGA DTLVIC ED FDPGVKEAAA A
  • LGQIGRHTPE HARAVAVTNT PVLLSLYMS TESSEDLQVK SK Although described in terms of the amino acid sequence shown in Figure 1, it will be apparent to those of skill that the polypeptides of the present invention include those peptides having the listed amino acid sequence or immunologically active (i.e., specifically immunoreactive or immunogenic) fragments thereof, as well as those polypeptides having amino acid sequences that are substantially homologous to the listed sequence.
  • the terms “substantially homologous” and “substantially identical refer to two amino acid sequences which, when optimally aligned, have at least 75% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and still more preferably at least about 95% sequence identity.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • substantially pure or isolated when referring to proteins and polypeptides, denote those polypeptides that are separated from proteins or other contaminants with which they are naturally associated.
  • a protein or polypeptide is considered substantially pure when that protein makes up greater than about 50% of the total protein content of the composition containing that protein, and typically, greater than about 60% of the total protein content. More typically, a substantially pure or isolated protein or polypeptide will make up from about 75 to about 90% of the total protein. Preferably, the protein will make up greater than about 90%, and more preferably, greater than about 95% of the total protein in the composition.
  • polypeptides of the invention may also be characterized by their ability to bind (i) antibodies that are specifically immunoreactive with a polypeptide having the sequence shown in Figure 1, or (ii) antibodies that are specifically immunoreactive with a substantially pure polypeptide having at least about eight, and preferably at least about 10, more preferably at least about 11 or about 12 contiguous amino acids as shown in Figure 1.
  • the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, Harlow and Lane
  • Harlow and Lane (hereinafter referred to as "Harlow") is incorporated by reference in its entirety and for all purposes.
  • Specific immunoreactivity is usually characterized by a specific binding affinity of an antibody for a protein (e.g. , ReproSA-1) of at least 10 7 , 10 8 , 10 9 , or 10 10 M "1 .
  • polypeptides of the present invention may generally be prepared using recombinant or synthetic methods that are well known in the art. Recombinant techniques are generally described in Sambrook et al., 1989, MOLECULAR CLONING: A LABORATORY MANUAL, (2nd ed.) Vols. 1-3, Cold Spring Harbor Laboratory, (hereinafter referred to as "Sambrook") and in Ausubel et al, Eds., 1995, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley and Sons, Inc., New York, (hereinafter referred to as "Ausubel”), both of which are incorporated by reference in their entirety and for all purposes.
  • polypeptides of the present invention may be expressed by a suitable host cell that has been transfected with a nucleic acid of the invention, as described in greater detail below.
  • Methods for synthesizing polypeptides are generally described in Merrifield, 1963, Amer. Chem. Soc. 85:2149-2456, Atherton et al, 1989, SOLID PHASE PEPTIDE SYNTHESIS: A PRACTICAL APPROACH, IRL Press, and Merrifield, 1986, Science 232:341-347.
  • polypeptides of the present invention can be carried out by methods that are generally well known in the art.
  • the polypeptides may be purified using readily available chromatographic methods, e.g., ion exchange, hydrophobic interaction, HPLC or affinity chromatography, to achieve the desired purity.
  • ReproSA- 1 polypeptide is purified using affinity chromatography.
  • antibodies raised against the ReproSA- 1 polypeptide described in Figure 1 may be coupled to a suitable solid support and contacted with a mixture of proteins containing the ReproSA- 1 polypeptide under conditions conducive to the association of this polypeptide with the antibody.
  • ReproSA- 1 protein is obtained from a human sperm homogenate, which can be prepared using methods well known in the art (e.g., Paradisi et al, 1995, J Repro. Imm., 28:61-73).
  • the solid support is washed to remove unbound material and/or nonspecifically bound proteins.
  • the desired polypeptide may then be eluted from the solid support in substantially pure form by, e.g., a. change in salt, pH or buffer concentration.
  • the present invention also provides fusion proteins which contain these polypeptides or fragments. Fusion proteins may be useful in providing for enhanced expression of the ReproSA- 1 polypeptide constructs, or in producing ReproSA- 1 polypeptides having other desirable properties, e.g., labeling groups, e.g., enzymatic reporter groups, binding groups, antibody epitopes, etc.
  • the term "fusion protein" as used herein generally refers to a composite protein, i.e., a single contiguous amino acid sequence, made up of two distinct, heterologous polypeptides which are not normally fused together in a single amino acid sequence.
  • a fusion protein may include a single amino acid sequence that contains two entirely distinct amino acid sequences or two similar or identical polypeptide sequences, provided that these sequences are not normally found together in a single amino acid sequence.
  • Fusion proteins may generally be prepared using either recombinant nucleic acid methods, i.e., as a result of transcription and translation of a gene fusion, which fusion comprises a segment encoding a polypeptide of the invention and a segment encoding a heterologous protein, or by chemical synthesis methods well known in the art.
  • the ReproSA- 1 polypeptide in is expressed in a cell as a fusion protein having a label or an "epitope tag" to aid in purification.
  • Fusion protein systems can also be used to facilitate efficient production and isolation of ReproSA- 1 proteins or peptides.
  • the non- ReproSA- 1 sequence portion of the fusion protein comprises a short peptide that can be specifically bound to an immobilized molecule such that the fusion protein can be separated from unbound components (such as unrelated proteins in a cell lysate).
  • a peptide sequence that is bound by a specific antibody.
  • a peptide comprising polyhistidine tracts e.g. (His) 6 or histidine-tryptophan sequences that can be bound by a resin containing nickel or copper ions (i.e., metal-chelate affinity chromatography) .
  • the ReproSA- 1 polypeptide is a variant of that of SEQ ID NO:2, in which insertions, deletions and substitutions of amino acids are made.
  • conservative amino acid substitutions may be made, i.e., substitution of selected amino acids with different amino acids having similar structural characteristics, e.g., net charge, hydrophobicity and the like. Examples of such conservative substitutions include, e.g., Ala:Val:Leu:Ile:Met, Asp:Glu, Lys:Arg, Asn:Gln, Phe:Tyr and Ser:Thr. Glycosylation modifications, either changed, increased amounts or decreased amounts, as well as other sequence modifications may also be made.
  • a ReproSA- 1 polypeptide may be substituted with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) to generate more stable peptides.
  • D-amino acid of the same type e.g., D-lysine in place of L-lysine
  • modification of the amino or carboxy terminals may also be used to confer stabilizing properties upon the polypeptides of the invention, e.g., amidation of the carboxy-terminus or acylation of the amino-terminus.
  • polypeptides one of skill in the art, upon reading the instant specification, will appreciate that structural analogs and derivatives of the above-described polypeptides, e.g., peptidomimetics, and the like. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compounds are termed “peptide mimetics” or “peptidomimetics” (Fauchere, 1986, Adv. DrugRes. 15:29; Veber and Freidinger, 1985, Trends Neurosci. p. 392; and Evans et al., 1987, J Med. Chem. 30:1229), and are usually developed with the aid of computerized molecular modeling.
  • Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent therapeutic effect.
  • detectable groups may comprise a detectable protein group, e.g., an assayable enzyme or antibody epitope as described above in the discussion of fusion proteins.
  • the detectable group may be selected from a variety of other detectable groups or labels, such as radiolabels (e.g., 125 1, 32 P or 35 S) or a chemiluminescent or fluorescent group.
  • the detectable group may be a substrate, cofactor, inhibitor or affinity ligand.
  • polypeptides of the invention find use as vaccines, immunogens, as antigens in diagnostic kits, and for other uses that will be apparent to those of skill.
  • the present invention provides antibodies that are specifically immunoreactive with human ReproSA- 1. Accordingly, the antibodies of the invention will specifically recognize and bind polypeptides which have an amino acid sequence that is substantially identical to the amino acid sequence shown in Figure 1 , or an immunogenic fragment thereof.
  • immunogen and “immunogenic” are used herein according to their ordinary meaning, i.e, an immunogen is a molecule, such as a protein, that can elicit an adaptive immune response upon injection into a person or an animal.
  • immunogenecity can be determined by injecting a polypeptide and adjuvant into an animal (e.g., a rabbit) and assaying for the appearance of antibodies directed against the injected polypeptide (see, e.g., Harlow, supra, chapter 5).
  • the antibodies of the invention usually exhibit a specific binding affinity for ReproSA- 1 of at least about 10 7 , 10 8 , 10 9 , or l0 I0 M-'.
  • an appropriate target immune system typically a mouse or rabbit, but also including goats, sheep, cows, guinea pigs, monkeys and rats.
  • the substantially purified antigen is presented to the immune system in a fashion determined by methods appropriate for the animal. These and other parameters are well known to immunologists. Typically, injections are given in the footpads, intramuscularly, intradermally, perilymph nodally or intraperitoneally.
  • the immunoglobulins produced by the host can be precipitated, isolated and purified by routine methods, including affinity purification. Substantially monospecific antibody populations can be produced by chromatographic purification of polyclonal sera.
  • monoclonal antibodies For monoclonal antibodies, appropriate animals will be selected and the desired immunization protocol followed. Monoclonal antibodies with affinities of 10 8 liters/mole, preferably 10 9 to 10 10 or stronger, will be produced by the methods described infra.
  • the production of non-human monoclonal antibodies, e.g., murine, lagomorpha, equine is well known and can be accomplished by, for example, immunizing an animal with a preparation containing ReproSA- 1 or fragments thereof.
  • Antibody-producing cells obtained from the immunized animals are immortalized and screened, or screened first for the production of antibody which binds to the ReproSA-1 polypeptide and then immortalized.
  • the spleens of the animals are excised and individual spleen cells are fused, typically, to immortalized myeloma cells under appropriate selection conditions. Thereafter, the cells are clonally separated and the supernatants of each clone (e.g., hybridoma) are tested for the production of an appropriate antibody specific for the desired region of the antigen.
  • Techniques for producing antibodies are well known in the art. See, e.g. , Goding et al., MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.) Acad. Press, N.Y., (which is incorporated in its entirety and for all purposes) and Harlow, supra.
  • human antibodies against a ReproSA- 1 polypeptide are provided. Some human antibodies are selected by competitive binding experiments, or otherwise, to have the same epitope specificity as a particular mouse antibody.
  • Human antibodies to a ReproSA- 1 polypeptide can be produced by screening a DNA library from human B cells according to the general protocol outlined by Huse et al., supra. Antibodies binding to the ReproSA- 1 polypeptide are selected. Sequences encoding such antibodies (or a binding fragments) are then cloned and amplified. The protocol described by Huse is rendered more efficient in combination with phage-display technology.
  • libraries of phage are produced in which members display different antibodies on their outer surfaces.
  • Antibodies are usually displayed as Fv or Fab fragments. Phage displaying antibodies with a desired specificity are selected by affinity enrichment to an ReproSA- 1 polypeptide.
  • human antibodies having the binding specificity of a selected murine antibody can be produced. See Winter, WO 92/20791.
  • either the heavy or light chain variable region of the selected murine antibody is used as a starting material. If, for example, a light chain variable region is selected as the starting material, a phage library is constructed in which members displays the same light chain variable region (i.e., the murine starting material) and a different heavy chain variable region. The heavy chain variable regions are obtained from a library of rearranged human heavy chain variable regions.
  • a phage showing strong specific binding for the ReproSA- 1 polypeptide e.g., at least 10 8 and preferably at least 10 9 M "1 ) is selected.
  • the human heavy chain variable region from this phage then serves as a starting material for constructing a further phage library.
  • each phage displays the same heavy chain variable region (i.e., the region identified from the first display library) and a different light chain variable region.
  • the light chain variable regions are obtained from a library of rearranged human variable light chain regions. Again, phage showing strong specific binding are selected. These phage display the variable regions of completely human anti- ReproSA- 1 antibodies. These antibodies usually have the same or similar epitope specificity as the murine starting material.
  • Human monoclonal antibodies against a known antigen can also be made using transgenic animals having elements of a human immune system (see, e.g., U.S. Patent Nos. 5,569,825; 5,545,806; 5,693,762; 5,693,761; and 5,7124,350, all of which are inco ⁇ orated by reference in their entirety for all purposes) or using human peripheral blood cells (Casali et al., Science 234:476-479, 1986).
  • the antibodies of the invention may be chimeric, human-like or humanized, in order to reduce their potential antigenicity, without reducing their affinity for their target.
  • Chimeric, human-like and humanized antibodies have been described in the art. See, e.g., Queen, et al., 1989, Proc. Nat'lAcad. Sci. USA 86:10029; Verhoeyan et al., 1988, Science 239:1534-1536 (1988) and U.S. Patent Nos. 5,585,089 and 5,530,101, all of which are incorporated by reference in their entirety and for all purposes.
  • Humanized immunoglobulins have variable framework regions substantially from a human immunoglobulin (termed an acceptor immunoglobulin) and complementarity determining regions substantially from a non-human (e.g., mouse) immunoglobulin (referred to as the donor immunoglobulin).
  • the constant region(s), if present, are also substantially from a human immunoglobulin.
  • the humanized antibodies of the present invention offer several advantages over the mouse donor antibody: (1) The human immune system should not recognize the framework or constant region of the humanized antibody as foreign, and therefore the antibody response against such an injected antibody should be less than against a totally foreign mouse antibody or a partially foreign chimeric antibody. (2) Because the effector portion of the humanized antibody is human, it may interact better with other parts of the human immune system. (3) Injected mouse antibodies have been reported to have a half-life in the human circulation much shorter than the half-life of normal human antibodies (Shaw et al., J Immunol. 138:4534-4538 (1987)). Injected humanized antibodies have a half-life essentially equivalent to naturally occurring human antibodies, allowing smaller and less frequent doses.
  • the cloning and sequencing of cDNA encoding the ReproSA- 1 polypeptide heavy and light chain variable regions may be carried out using routine procedures. See, e.g., Jones & Bendig, Bio/Technology 9:88-89 (1991) and Bio/Technology 9:579 (1991); Sambrook, supra, and Harlow, supra.
  • the substitution of mouse CDRs into a human variable domain framework is most likely to result in retention of their correct spatial orientation if the human variable domain framework adopts the same or similar conformation to the mouse variable framework from which the CDRs originated. This is achieved by obtaining the human variable domains from human antibodies whose framework sequences exhibit a high degree of sequence identity with the murine variable framework domains from which the CDRs were derived.
  • the heavy and light chain variable framework regions can be derived from the same or different human antibody sequences.
  • the human antibody sequences can be the sequences of naturally occurring human antibodies or can be consensus sequences of several human antibodies. See Kettleborough et al., Protein Engineering 4:773 (1991); Kolbinger et al., Protein Engineering 6:971 (1993).
  • Suitable human antibody sequences are identified by computer comparisons of the amino acid sequences of the mouse variable regions with the sequences of known human antibodies. The comparison is performed separately for heavy and light chains but the principles are similar for each.
  • the humanized antibodies of the invention comprise variable framework regions substantially from a human immunoglobulin and complementarity determining regions substantially from a mouse anti-ReproSA-1 immunoglobulin. Once the complementarity determining regions of the murine antibody are identified and appropriate human acceptor immunoglobulins, the next step is to determine which, if any, residues from these components should be substituted to optimize the properties of the resulting humanized antibody.
  • substitution of human amino acid residues with murine should be minimized, because introduction of murine residues increases the risk of the antibody eliciting a HAMA response in humans.
  • Amino acids are selected for substitution based on their possible influence on CDR conformation and/or binding to antigen. Investigation of such possible influences is by modeling, examination of the characteristics of the amino acids at particular locations, or empirical observation of the effects of substitution or mutagenesis of particular amino acids.
  • the human framework amino acid should usually be substituted by the equivalent mouse amino acid if it is reasonably expected that the amino acid noncovalently binds antigen directly, is adjacent to a CDR region, is part of a CDR region under the alternative definition proposed by
  • substitution of all or most of the amino acids fulfilling the above criteria is desirable. Occasionally, however, there is some ambiguity about whether a particular amino acid meets the above criteria, and alternative variant immunoglobulins are produced, one of which has that particular substitution, the other of which does not.
  • the CDR regions in humanized antibodies are substantially identical, and more usually, identical to the corresponding CDR regions in the anti-ReproSA-1 antibody. Although not usually desirable, it is sometimes possible to make one or more conservative amino acid substitutions of CDR residues without appreciably affecting the binding affinity of the resulting humanized immunoglobulin. Occasionally, substitution of CDR regions can result in enhanced binding affinity.
  • the framework regions of humanized immunoglobulins are usually substantially identical, and more usually, identical to the framework regions of the human antibodies from which they were derived.
  • many of the amino acids in the framework region make little or no direct contribution to the specificity or affinity of an antibody.
  • many individual conservative substitutions of framework residues can be tolerated without appreciable change of the specificity or affinity of the resulting humanized immunoglobulin.
  • substitutions are undesirable.
  • a variety of methods are available for producing such immunoglobulins. Because of the degeneracy of the code, a variety of nucleic acid sequences will encode each immunoglobulin amino acid sequence.
  • the desired nucleic acid sequences can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of an earlier prepared variant of the desired polynucleotide.
  • Oligonucleotide-mediated mutagenesis is a preferred method for preparing substitution, deletion and insertion variants of target polypeptide DNA. See Adelman et al., DNA 2:183 (1983). Briefly, the target polypeptide DNA is altered by hybridizing an oligonucieotide encoding the desired mutation to a single-stranded DNA template. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template that incorporates the oligonucieotide primer, and encodes the selected alteration in the target polypeptide DNA.
  • variable segments of humanized antibodies produced as described supra are typically linked to at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • Human constant region DNA sequences can be isolated in accordance with well-known procedures from a variety of human cells, but preferably immortalized B-cells (see Kabat et al., supra, and WO87/02671) (each of which is incorporated by reference in its entirety for all purposes).
  • the antibody will contain both light chain and heavy chain constant regions.
  • the heavy chain constant region usually includes CHI, hinge, CH2, CH3, and CH4 regions.
  • Recombinant expression of antibodies can be carried out by inserting nucleic acids encoding humanized light and heavy chain variable regions, optionally linked to constant regions, into expression vectors.
  • the light and heavy chains can be cloned in the same or different expression vectors.
  • the DNA segments encoding immunoglobulin chains are operably linked to control sequences in the expression vector(s) that ensure the expression of immunoglobulin polypeptides.
  • control sequences include a signal sequence, a promoter, an enhancer, and a transcription termination sequence.
  • Expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Immunoglobulin light and heavy chains are expressed using standard methods.
  • vectors containing the polynucleotide sequences encoding the heavy and light chain encoding sequences and expression control sequences can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts. (See generally Sambrook, supra).
  • the vectors are co-transfected to obtain expression and assembly of intact immunoglobulins.
  • cell lines expressing immunoglobulin products are cell selected. Cell lines capable of stable expression are preferred (i.e., undiminished levels of expression after fifty passages of the cell line).
  • the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms of the present invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see generally Scopes, Protein Purification (Springer- Verlag, N.Y., 1982).
  • Substantially pure immunoglobulins of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity most preferred, for pharmaceutical uses.
  • fragments of the intact antibodies described above are provided. Typically, these fragments compete with the intact antibody from which they were derived for specific binding to the ReproSA- 1 polypeptide, and bind with an affinity of at least 10 7 , 10 8, 10 9 M "1 , or 10 10 M "1 .
  • Antibody fragments include separate heavy chains, light chains Fab, Fab' F(ab') 2 , Fabc, and Fv. Fragments can be produced by enzymic or chemical separation of intact immunoglobulins.
  • a F(ab') 2 fragment can be obtained from an IgG molecule by proteolytic digestion with pepsin at pH 3.0-3.5 using standard methods such as those described in Harlow, supra.
  • Fab fragments may be obtained from F(ab') 2 fragments by limited reduction, or from whole antibody by digestion with papain in the presence of reducing agents. Fragments can also be produced by recombinant DNA techniques. Segments of nucleic acids encoding selected fragments are produced by digestion of full-length coding sequences with restriction enzymes, or by de novo synthesis. Often fragments are expressed in the form of phage-coat fusion proteins.
  • the antibodies of the present invention can be used with or without modification.
  • the antibodies will be labeled by joining, either covalently or non- covalently, a substance which provides for a detectable signal.
  • labels include those that are well known in the art, e.g., radioactive, fluorescent, or bioactive (e.g., enzymatic) labels.
  • the antibodies of the invention may be particularly useful in, e.g., diagnostic applications, for detecting the presence of sperm containing wild-type (i.e., normal) ReproSA- 1 protein.
  • Sperm antibodies assays are well known, see, e.g., Rajah SV, et al., 1992, Fertility and Sterility 57:1300.
  • preferred antibodies are those that interfere with sperm mobility. This will be a property of many or most anti-ReproSA-1 antibodies.
  • An antibody (or antisera) can be tested for its ability to immobilize sperm using methods well known in the art (e.g. sperm agglutination). See, e.g., Harlow, supra, and Isojima, 1992, Am. J. Obstret Gynecol 112: 199; Fichorova, 1991 , Journal of Reproductive Immunology 20:1; World Health Organization - WHO Laboratory Manual for the examination of human semen and semen-cervical mucus interaction, 2nd ed.
  • Anti-idiotypic antibodies which can be isolated by the above procedures.
  • Anti-idiotypic antibodies may be prepared by, for example, immunization of an animal with the primary antibody (i.e., anti-ReproSA-1 antibodies or ReproSA- 1 -binding fragments thereof).
  • anti-ReproSA-1 antibodies anti-idiotype antibodies whose binding to the primary antibody is inhibited by a ReproSA- 1 polypeptide or fragments thereof are selected.
  • the anti-idiotypic immunoglobulin may represent the "internal image" of an epitope and thus may substitute for the ReproSA- 1 polypeptide in assays or may be used to bind (i.e., inactivate) anti-ReproSA-1 antibodies in a patient.
  • Many of the immunoglobulins described above can undergo non-critical amino-acid substitutions, additions or deletions in both the variable and constant regions without loss of binding specificity or effector functions, or intolerable reduction of binding affinity (i.e., below about 10 7 M "1 ).
  • immunoglobulins incorporating such alterations exhibit substantial sequence identity to a reference immunoglobulin from which they were derived.
  • a mutated immunoglobulin can be selected having the same specificity and increased affinity compared with a reference immunoglobulin from which it was derived.
  • Phage-display technology offers powerful techniques for selecting such immunoglobulins. See, e.g., Dower et al., WO 91/17271 McCafferty et al, WO 92/01047; and Huse, WO 92/06204.
  • the invention also provides hybrid antibodies that share the specificity of antibodies against a ReproSA- 1 polypeptide but are also capable of specific binding to a second moiety.
  • one heavy and light chain pair is usually from an anti- complex antibody and the other pair from an antibody raised against another epitope. This results in the property of multi-functional valency, i.e., ability to bind at least two different epitopes simultaneously, where at least one epitope is the epitope to which the anti-complex antibody binds.
  • Such hybrids can be formed by fusion of hybridomas producing the respective component antibodies, or by recombinant techniques.
  • Immunoglobulins can also be fused to functional regions from other genes (e.g., enzymes) to produce fusion proteins (e.g., immunotoxins) having novel properties.
  • the antibodies of the invention may be of any isotype, e.g., IgM, IgD, IgG, IgA, and IgE, with IgG, IgA and IgM most referred.
  • Secreted IgG, IgD, and IgE isotypes are typically found in monomeric form.
  • Secreted IgM isotype is found in pentameric form; secreted IgA can be found in both monomeric and dimeric form. See generally, Paul, ed., 1989, FUNDAMENTAL IMMUNOLOGY 2nd ed. Raven Press, N.Y., Ch. 7 (incorporated by reference in its entirety for all purposes).
  • Humanized antibodies may comprise sequences from more than one class or isotype.
  • a composition comprising an antibody is substantially pure when at least about 80%, more often at least about 90%, even more often at least about 95%, most often at least about 99% or more of the immunoglobulin molecules present in a preparation specifically bind a ReproSA- 1 polypeptide.
  • the antibodies of the inventions are useful in a number of applications, e.g. , as probes in immunoassays in diagnostic or therapeutic applications, or in contraceptive compositions. These applications are discussed in greater detail, infra. IV. Nucleic Acids and Cell Lines
  • the present invention provides nucleic acids which encode the polypeptides of the invention, as well as expression vectors that include these nucleic acids, and cell lines and organisms that are capable of expressing these nucleic acids.
  • the nucleic acid composition has the sequence of the ReproSA-
  • nucleic acids will comprise a segment having more than about 20 contiguous nucleotides from the nucleotide sequences shown in Figure 1. Also provided are substantially similar nucleic acid sequences and allelic variations of the above described nucleic acids. Also included are chemically modified and substituted nucleic acids, e.g., those which incorporate modified nucleotide bases or which incorporate a labeling group.
  • Substantial identity in the nucleic acid context, means that the segments, or their complementary strands, when compared, are the same when properly aligned, with the appropriate nucleotide insertions or deletions, in at least about 70%, more typically, at least about 80%), usually, at least about 90%), and more usually, at least about 95% to 98% of the nucleotides.
  • Algorithms suitable for calculating the degree of identity include that of Smith and Waterman, supra, Needleman and Wunsch, supra, Lipman, supra, or by computerized implementations of these algorithms (e.g., FASTN in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, WI).
  • Another example of algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al, J. Mol. Biol.
  • HSPs high scoring sequence pairs
  • Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (ee, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • stringent conditions will be those in which the salt concentration is at least about 0.02 molar at pH 7 and the temperature is at least about 60 °C.
  • the combination of parameters is more important than the absolute measure of any one.
  • nucleic acid compositions of the invention encode a polypeptide that is specifically immunoreactive with an antibody that specifically binds the polypeptide shown in Figure 1 [SEQ ID NO:2].
  • nucleic acids and “polynucleotides” are used interchangeably and include RNA, cDNA, genomic DNA, synthetic forms and mixed polymers, both sense and antisense strands.
  • the nucleic acids described herein include self replicating plasmids and infectious polymers of DNA or RNA.
  • the nucleic acids of the present invention may be present in whole cells, cell lysates or in partially pure or substantially pure or isolated form.
  • the terms “substantially pure” or “isolated” generally refer to the nucleic acid separated from contaminants with which it is generally associated, e.g., lipids, proteins and other nucleic acids.
  • the substantially pure or isolated nucleic acids of the present invention will be greater than about 50% pure. Typically, these nucleic acids will be more than about 60% pure, more typically, from about 75% to about 90% pure and preferably from about 95% to about 98%o pure.
  • the nucleic acids of the present invention will be used in expression vectors for the preparation of the polypeptides of the present invention, namely those polypeptides which are derived from or related to ReproSA- 1.
  • expression vector generally refers to nucleotide sequences that are capable of affecting expression of a structural gene in hosts compatible with such sequences. These expression vectors typically include at least suitable promoter sequences and optionally, transcription termination signals. Commonly, expression vectors will contain selection markers, e.g., tetracycline or neomycin, to permit detection of those cells transformed with the desired DNA sequences (see, e.g.,
  • DNA encoding the ReproSA- 1 polypeptides of the present invention will typically be incorporated into DNA constructs capable of introduction into and expression in an in vitro cell culture. Often, the nucleic acids of the present invention may be used to produce a suitable recombinant host cell. (As used herein, a cell or cell line that contains a nucleic acid sequence made using recombinant techniques can be called a "recombinant cell” or "recombinant cell line”). Specifically, DNA constructs will be suitable for replication and expression in a prokaryotic host, such as bacteria, e.g., E. coli, or may be introduced into a cultured mammalian, plant, insect, e.g., Sf9, yeast, fungi or other eukaryotic cell line.
  • a prokaryotic host such as bacteria, e.g., E. coli
  • Sf9 e.g., Sf9, yeast, fungi or other eukaryotic cell line.
  • E. coli is one prokaryotic host useful particularly for cloning the polynucleotides of the present invention.
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species.
  • bacilli such as Bacillus subtilus
  • enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species.
  • any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.
  • the promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation.
  • Other microbes, such as yeast, may also be used for expression.
  • Saccharomyces is a preferred host, with suitable vectors having expression control sequences, such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired.
  • promoters including 3-phosphoglycerate kinase or other glycolytic enzymes
  • origin of replication including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired.
  • mammalian tissue cell culture may also be used to express and produce the polypeptides of the present invention (see Winnacker, From Genes to Clones (VCH Publishers, N.Y., N.Y., 1987).
  • Eukaryotic cells are actually preferred, because a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed.
  • Preferred suitable host cells for expressing nucleic acids encoding the immunoglobulins of the invention include: monkey kidney CV1 line transformed by S V40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293)
  • monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); and, TRI cells (Mather, et al., Annals N.Y. Acad. Sci. 383:44-46 (1982)); baculovirus cells.
  • DNA constructs prepared for introduction into a particular host will typically include a replication system recognized by the host, the intended DNA segment encoding the desired polypeptide, and transcriptional and translational initiation and termination regulatory sequences operably linked to the polypeptide encoding segment.
  • a DNA segment is operably linked when it is placed into a functional relationship with another DNA segment.
  • a promoter or enhancer is operably linked to a coding sequence if it stimulates the transcription of the sequence.
  • DNA for a signal sequence is operably linked to DNA encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide.
  • DNA sequences that are operably linked are contiguous, and in the case of a signal sequence both contiguous and in reading phase.
  • enhancers need not be contiguous with the coding sequences whose transcription they control. Linking is accomplished by ligation at convenient restriction sites or at adapters or linkers inserted in lieu thereof.
  • the selection of an appropriate promoter sequence will generally depend upon the host cell selected for the expression of the DNA segment. Examples of suitable promoter sequences include prokaryotic, and eukaryotic promoters well known in the art. See, e.g., Sambrook, supra.
  • the transcriptional regulatory sequences will typically include a heterologous enhancer or promoter (i.e., not naturally operably linked to a human ReproSA- 1 gene) which is recognized by the host.
  • a heterologous enhancer or promoter i.e., not naturally operably linked to a human ReproSA- 1 gene
  • the selection of an appropriate promoter will depend upon the host, but promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters are known and available. See Sambrook, supra.
  • the control sequences will include a promoter and preferably an enhancer derived from immunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence, and may include splice donor and acceptor sequences.
  • expression vectors which include the replication system and transcriptional and translational regulatory sequences together with the insertion site for the polypeptide encoding segment may be employed. Examples of workable combinations of cell lines and expression vectors are described in, e.g., Sambrook, supra, Metzger et al., 1988, Nature 334:31-36, and Gossen et al, 1994, Curr. Opin. Biotech 5:516.
  • suitable expression vectors may be expressed in, e.g., insect cells, e.g., Sf9 cells), mammalian cells, (e.g., CHO cells), and bacterial cells (e.g., E. coli).
  • the cDNA encoding the polypeptides of the invention may be cloned into an appropriate baculovirus expression vector, e.g., pBacPAK8 vector (Clontech, Palo Alto,
  • the recombinant baculovirus may then be used to transfect a suitable insect host cell, e.g., Spodoptera frugiperda (Sf9) cells, which may then express the polypeptide.
  • a suitable insect host cell e.g., Spodoptera frugiperda (Sf9) cells
  • Sf9 cells Spodoptera frugiperda (Sf9) cells
  • the DNA is isolated from a genomic or cDNA library using labeled oligonucieotide probes specific for sequences in the desired DNA. Restriction endonuclease digestion of genomic DNA or cDNA containing the appropriate genes can be used to isolate the DNA encoding the polypeptides of the invention.
  • a panel of restriction endonucleases can be constructed to give cleavage of the DNA in desired regions, i.e., to obtain segments which encode biologically active polypeptides or fragments of the invention.
  • DNA encoding the polypeptides of the invention is identified by its ability to hybridize with a nucleic acid probe in, for example, a Southern blot format. These regions are then isolated using standard methods. See, e.g., Sambrook, supra.
  • the nucleic acids of the invention are also useful for isolating and expressing full-length ReproSA- 1 genes from humans and non-human animals, using well known methods, e.g., Sambrook, supra, and Ausubel, supra.
  • a nucleic acid probe comprising all or some of the ReproSA- 1 cDNA sequence (typically a restriction fragment from the 5* end of the cDNA clone) is labeled and used to screen a genomic or cDNA library by hybridizing at high stringency.
  • the cDNA library may be oligo-dT or random primed.
  • the cDNA or genomic clones that hybridize are isolated and analyzed by restricting mapping, Southern hybridization, and DNA sequencing using methods that are well known in the art.
  • a polynucleotide encoding a full-length ReproSA- 1 polypeptide is cloned into an expression vector or synthesizes using sequence obtained from multiple clones, and expressed, as is described supra.
  • the invention provides antisense, triplex, and ribozyme reagents that target ReproSA- 1 nucleic acids.
  • the present invention provides antisense oligo- and polynucleotides (e.g., DNA, RNA, PNA or the like) that can be used to inhibit expression of the ReproSA- 1 gene, e.g., for contraceptive purposes.
  • Antisense methods are generally well known in the art and may be carried out using modified ReproSA- 1 polynucleotides (see, e.g., PCT publication WO 94/12633, and Nielsen et al., 1991, Science 254:1497; OLIGONUCLEOTIDES AND ANALOGUES, A PRACTICAL APPROACH, edited by F. Eckstein, IRL Press at Oxford University Press (1991); ANTISENSE RESEARCH AND APPLICATIONS (1993, CRC Press) all inco ⁇ orated by reference.
  • modified ReproSA- 1 polynucleotides see, e.g., PCT publication WO 94/12633, and Nielsen et al., 1991, Science 254:1497; OLIGONUCLEOTIDES AND ANALOGUES, A PRACTICAL APPROACH, edited by F. Eckstein, IRL Press at Oxford University Press (1991); ANTISENSE RESEARCH AND APPLICATIONS (1993, CRC Press) all
  • the invention provides oligo- and polynucleotides that bind to double-stranded or duplex ReproSA- 1 nucleic acids (e.g., in a folded region of the ReproSA- 1 RNA or in the ReproSA- 1 gene), forming a triple helix-containing, or "triplex" nucleic acid.
  • Triple helix formation results in inhibition of ReproSA- 1 expression by, for example, preventing transcription of the ReproSA-1 gene, (see, e.g., Gee et al., in Huber and Carr, 1994, MOLECULAR AND IMMUNOLOGIC APPROACHES, Futura Publishing Co, Mt Kisco NY and Rininsland et al., 1997, Proc.
  • the invention provides ribozymes useful for inhibition of ReproSA- 1 expression or activity.
  • the ribozymes of the invention bind and specifically cleave and inactivate ReproSA- 1 mRNA.
  • Useful ribozymes can comprise 5'- and 3'-terminal sequences complementary to the ReproSA- 1 mRNA and can be engineered by one of skill on the basis of the ReproSA- 1 mRNA sequence disclosed herein. See, e.g., PCT publications WO 94/02595 and WO 93/23569).
  • compositions of the invention have a number of uses including, but not limited to, use in contraceptive vaccines, as topical contraceptive agents (e.g., spermicidal agents), and for diagnosis and treatment of human diseases such as infertility.
  • contraceptive vaccines as topical contraceptive agents (e.g., spermicidal agents)
  • topical contraceptive agents e.g., spermicidal agents
  • human diseases such as infertility.
  • the invention provides methods of contraception by immunization, i.e., administering an effective amount of a vaccine comprising at least one ReproSA- 1 polypeptide or polynucleotide.
  • An effective amount of a contraceptive vaccine is an amount sufficient to induce an immune response, e.g. , production in a subject (e.g, a human or non-human animal) of antibodies that bind to a sperm ReproSA- 1 polypeptide (i.e., an anti-sperm immune response).
  • administering results in an elevation of anti-ReproSA-1 antibodies to atiter of at least about three times the antibody titer prior to vaccination, more preferably the elevation is at least about 50-fold or at least about 100-fold.
  • the isotype of the antibodies is IgA.
  • the anti-ReproSA-1 antibodies are found in mucosal secretions, e.g., mucosal secretions of the reproductive tract, e.g., the vaginal mucosa. Appearance of anti-ReproSA-1 antibodies can be correlated with reduced fertility, i.e., a contraceptive effect.
  • a contraceptive vaccine results in inhibition of fertilization by at least about fifty percent, more often by at least about 75% relative to untreated controls.
  • Protocols for determining the efficacy of a contraceptive are well known, and can be carried out in humans or in non-human animals. Contraceptive vaccines are described generally in Aitken et ⁇ l, 1993, Brit. Med. Bull. 49:88- 99. Although it is expected that the primary use of the vaccines of the invention will be in humans, they may also be used as contraceptives in nonhuman animals that produce sperm having a ReproSA- 1 protein.
  • the vaccines of the invention may be polypeptide or nucleic acid based.
  • vaccines are produced using recombinantly expressed peptides or polypeptides, or from synthetic peptides or mimetics.
  • the immunogens of the invention will include a subsequence of a ReproSA- 1 polypeptide.
  • the vaccine will comprise peptides or polypeptides derived from the ReproSA- 1 sequence shown in Figure 1.
  • immunogenic ReproSA- 1 peptides will range in size from about 8 amino acids in length to about 509 amino acids in length. More typically, the active fragments will be from about 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acids in length to about 50 amino acids in length.
  • ReproSA- 1 polypeptide sequences for use in vaccines include MSTESSEDLQVKSKKAIKNILQKCTY [SEQ ID NO:5], LPHDSKARRLFVTSGGLKKVQEIKAEPG [SEQ ID NO:6],
  • a vaccine will include from about 1 to about 50 micrograms of antigen or antigenic protein or peptide. More preferably, the amount of protein is from about 15 to about 45 micrograms. Typically, the vaccine is formulated so that a dose includes about 0.5 milliliters.
  • the vaccine may be administered by any route known in the art including oral, parenteral (e.g., subcutaneous or intramuscular), intrarectal, or vaginal.
  • the treatment may consist of a single dose of vaccine or a plurality of doses over a period of time.
  • the antigen may be combined or mixed with various solutions and omer compounds as is known in the art, as well as with other antigens.
  • it may be administered in any pharmacologically acceptable carrier including water, saline or buffered vehicles, with or without various adjuvants or immunodiluting agents.
  • adjuvants or agents examples include aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X, Corynebacterium parvum (Propionibacterium acnes), Bordetella pertussis, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants.
  • aluminum hydroxide aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide
  • Such adjuvants are available commercially from various sources, for example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) or Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Michigan).
  • suitable adjuvants are Amphigen (oil-in-water), Alhydrogel (aluminum hydroxide), or a mixture of Amphigen and Alhydrogel.
  • compositions may be sterilized by conventional, well known sterilization techniques.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH-adjusting and buffering agents, tonicity adjusting agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.
  • auxiliary substances as required to approximate physiological conditions such as pH-adjusting and buffering agents, tonicity adjusting agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.
  • Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Sciences, 17th Ed., Mack Publishing Company, Easton, Pennsylvania (1985), which is inco ⁇ orated herein by reference.
  • the proportion of antigen and adjuvant can be varied over a broad range so long as both are present in effective amounts.
  • aluminum hydroxide can be present in an amount of about 0.5% of the vaccine mixture (Al 2 O 3 basis).
  • the amount of the antigen can range from about 0.1 ⁇ g to about 100 ⁇ g protein per patient.
  • a preferable range is from about 1 ⁇ g to about 50 ⁇ g per dose.
  • a more preferred range is about 15 ⁇ g to about 45 ⁇ g.
  • a suitable dose size is about 0.5 ml. Accordingly, a dose for intramuscular injection, for example, would comprise 0.5 ml containing 45 ⁇ g of antigen in admixture with 0.5% aluminum hydroxide.
  • the vaccine may be inco ⁇ orated into a sterile container which is then sealed and stored at a low temperature, for example 4°C, or it may be freeze-dried. Lyophilization permits long-term storage in a stabilized form.
  • a sterile container which is then sealed and stored at a low temperature, for example 4°C, or it may be freeze-dried. Lyophilization permits long-term storage in a stabilized form.
  • cyclization or circularization of a peptide can increase the peptide's antigenic and immunogenic potency. See U.S. Pat. No. 5,001,049 which is inco ⁇ orated by reference herein.
  • an antigen can be conjugated to a suitable carrier, usually a protein molecule. This procedure has several facets.
  • an antigen such as a peptide
  • the carrier may possess properties which facilitate transport, binding, abso ⁇ tion or transfer of the antigen.
  • the conjugation between a peptide and a carrier can be accomplished using one of the methods known in the art. Specifically, the conjugation can use bifunctional cross-linkers as binding agents as detailed, for example, by Means and Feeney, Bioconjugate Chem. 1:2-12.
  • the epitopes are typically segments of amino acids which are a small portion of the whole protein.
  • Such derivatives may include peptide fragments, amino acid substitutions, amino acid deletions and amino acid additions within the ReproSA- 1 amino acid sequence.
  • Epitopes may be conformational (i.e. , discontinuous). That is, they may be formed from amino acids encoded by noncontiguous parts of a primary sequence (e.g., SEQ ID NO:2) that have been brought together by protein folding. It is understood by those of skill that conformational epitopes can be formed using recombinant techniques.
  • a polynucleotide with sequences that encode the amino acid residues that form two parts of a conformational epitope, and other sequences that allow the first part of a conformational epitope and the second part of a conformational epitope to associate in the native conformation of the ReproSA- 1 protein can be constructed and used, for example, as a vaccine or for purification of anti-ReproSA-1 antibody.
  • composition of the vaccine and/or the method of administration are chosen so as to generate persisting high levels of anti-ReproSA-1 IgA (e.g., soluble IgA).
  • anti-ReproSA-1 IgA e.g., soluble IgA
  • the nucleic acids of the present invention may be used as vaccines for the prevention of conception (i. e. , contraceptive vaccines).
  • plasmid DNA comprising the nucleic acids of the present invention may be directly administered to a patient, e.g., by injection into muscle. Expression of this "naked" DNA will have effects similar to the injection of the actual polypeptides. Specifically, the patient's immune response to the presence of the proteins expressed from the DNA, will result in the production of antibodies to that protein. See Sedegah et al., Proc. Nat'l Acad. Sci.
  • the isolated nucleic acid sequence coding for ReproSA- 1 or its homologous polypeptides can also be used to transform viruses which transfect host cells in the susceptible organism.
  • Live attenuated viruses such as vaccinia or adenovirus, are convenient alternatives to conventional vaccines because they are inexpensive to produce and are easily transported and administered.
  • Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Patent No. 4,722,848, inco ⁇ orated herein by reference and Ulaeto and Hruby, 1994, Curr. Opin. Biotech. 5:501 inco ⁇ orated herein by reference.
  • Suitable viruses for use in the present invention include, but are not limited to, pox viruses, such as, canarypox and cowpox viruses, and vaccinia viruses, alpha viruses, adenoviruses, and other animal viruses.
  • the recombinant viruses can be produced by methods well known in the art: for example, using homologous recombination or ligating two plasmids together.
  • a recombinant canarypox or cowpox virus can be made, for example, by inserting the gene encoding an immunologically active segment of ReproSA- 1 into a plasmid so that it is flanked with viral sequences on both sides. The gene is then inserted into the virus genome through homologous recombination.
  • the recombinant virus of the present invention can be used to induce anti- ReproSA-1 polypeptide antibodies in humans and other mammals.
  • the recombinant virus can be used to produce the ReproSA- 1 polypeptides by infecting host cells which in turn express the polypeptide.
  • compositions of the invention also find use as spermicidal agents for use in contraception.
  • ReproSA- 1 binding agents e.g., anti-ReproSA-1 antibodies (i.e., monoclonal antibodies and ReproSA- 1 -binding fragments thereof) are used to bind to sperm, resulting in immobilization and/or agglutination and thus preventing fertilization.
  • the antibody composition is applied vaginally in a suitable carrier, such as a pharmaceutically acceptable carrier, e.g., a gel.
  • the composition can be applied to a contraceptive device, such as a condom or diaphragm.
  • ReproSA- 1 polypeptides are useful as topical contraceptives, e.g., via protein- protein interactions with endogenous (sperm) ReproSA- 1 protein, e.g., dominant-negative effects.
  • ReproSA-1 polypeptides are useful as agents for enhancing fertilization, e.g. by binding to anti-ReproSA-1 antibodies present during a potential fertilization event.
  • the peptides bind anti-ReproSA-1 antibodies present and block the binding of the antibodies to sperm, enabling the sperm to participate in the fertilization event.
  • an effective amount of ReproSA- 1 polypeptide is administered to a site, either in vitro or in vivo, at which fertilization is attempted.
  • an effective amount of ReproSA- 1 polypeptide is defined as the amount of ReproSA- 1 polypeptide required to bind to more than about 50% of the total anti-ReproSA-1 antibodies present during a potential fertilization event, preferably, more than about 75% of the ReproSA- 1 antibodies present, more preferably greater than about 90% or about 95% of the anti-ReproSA-1 antibodies present.
  • the anti-ReproSA-1 antibody-binding polypeptide composition is applied vaginally in a suitable carrier, e.g., a pharmaceutically acceptable solution or gel.
  • the composition is contacted with, e.g., added to, a sperm preparation prior to performing in vitro fertilization.
  • compositions of the invention are used diagnostically in a number of contexts.
  • they are used to determine the presence of abnormal ReproSA- 1 genes, especially in men, e.g., men suffering from infertility.
  • they are useful in diagnostic assays to detect serum, seminal, mucosal and/or follicular antibodies to ReproSA- 1 proteins (and thus to sperm) in order to diagnose immunological infertility, e.g., in patients having difficulty conceiving.
  • compositions of the invention are used in an assay to quantitate titers of levels of antibodies to the antigen in contraceptively vaccinated individuals.
  • the polypeptides of the invention are useful for assaying the presence of anti-ReproSA-1 antibodies in a biological sample (e.g., serum, cervical or vaginal fluid, or semen). The presence of such antibodies will aid in diagnosing immunological infertility. Immunological infertility has heretofore been a diagnosis of exclusion, and thus surrounded by uncertainty. The present invention allows a more certain diagnosis to be made.
  • a male or female patient is diagnosed as expressing anti-ReproSA-1 antibodies
  • appropriate therapeutic measures may be taken, such as administration of appropriate pharmaceutical agents, e.g., immunosuppressive agents to increase the chance of conception.
  • suitable assays are known to those of skill for assaying the presence of antibodies to a known antigen (e.g., ReproSA- 1), some of which are described in Harlow, supra.
  • the polypeptide of the invention is coated on a solid support, such as a microtiter plate.
  • a biological sample from a patient e.g., including but not limited to serum or other blood fraction, semen, saliva, or cervical secretions is applied to the solid support that has been coated with polypeptide and anti-ReproSA-1 antibodies, if present, are allowed to bind the polypeptide.
  • Such antibodies can be detected by well known means, for example using anti-human immunoglobulin, e.g., IgG, conjugated to a detectable label.
  • novel polynucleotide and antibody reagents of the invention are also used for determining the presence of mutant or alternate allelic forms of the ReproSA- 1 genes or proteins, especially in men. Mutant or abnormal ReproSA- 1 genes or proteins may result in infertility in men carrying the genes or expressing the proteins.
  • a gene in a patient is compared to the sequence of the ReproSA- 1 gene provided in SEQ ID NO: 1. A difference in the sequences is indicative of a mutation (including alternative alleles) in the patient's gene.
  • Methods for analysis and comparison of nucleic acid sequences are well known. Preferred methods include use of the polymerase chain reaction (PCR).
  • primers designed using the sequence provided in Figure 1 [SEQ ID NO:l] can be used to amplify (e.g., by PCR) the ReproSA-1 gene from a patient, or a fragment thereof.
  • the sequence of the ReproSA- 1 gene from the patient can then be determined e.g., by sequencing and compared to SEQ ID NO:l.
  • differences between a patient's gene and SEQ ID NO:l can be determined by hybridization, for example, using a high density oligonucieotide array (e.g., Pease et al, 1994, Proc. Natl. Acad. Sci. USA 91 :5022-5026) or often means known in the art.
  • a silent mutation in a nucleic acid sequence is one that does not change the identity of an amino acid in an encoded polypeptide.
  • missense mutations, deletions, insertions, and frameshifts that result in a deviation in an encoded ReproSA- 1 amino acid sequence from that of SEQ ID NO:2 are of particular interest.
  • compositions of the invention have numerous additional uses not listed above.
  • the antibodies of the invention may be used to trap sperm, for example, to aid in counting sperm or for collecting samples in forensic investigations.
  • kits for use by physicians, clinical laboratories or patients to carry out the assays and diagnoses described above will contain one or more of the following in a container: oligonucieotide primers or probes corresponding to the ReproSA-1 cDNA sequence, anti-ReproSA-1 antibodies, ReproSA-1 polypeptides or fragments, optionally coated in a solid surface (such as a slide, multiple well plate, or test tube), a ReproSA- 1 polynucleotide (e.g., for use as positive controls in assays), and tubes. Instructions for carrying out the methods of the invention, and calibration curves may also be included.
  • the present invention provides methods for identifying and cloning proteins that interact with the ReproSA- 1 polypeptide or a region thereof (e.g., an armadillo repeat region).
  • these interacting proteins are identified using a two hybrid screen system according to methods well known in the art (see, e.g.., Chien et al. , 1991, Proc. Natl. Acad. Sci. USA 88:9578, Ausubel et al, supra, at Ch. 20; Fields and Song, 1989, Nature 340:245; U.S. Patent Nos: 5,283,173 and 5,468,614, which are inco ⁇ orated herein by reference in their entirety and for all pu ⁇ oses).
  • the two- hybrid screen identifies protein-protein interactions in vivo through reconstitution of a transcriptional activator, e.g., the yeast Gal4 transcription protein (see, Fields and Song, 1989, Nature 340:245).
  • the method is based on the properties of the yeast Gal4 protein, which consists of separable domains responsible for DNA-binding and transcriptional activation.
  • Polynucleotides, usually expression vectors, encoding two hybrid proteins are constructed.
  • One polynucleotide comprises the yeast Gal4 DNA-binding domain fused to a polypeptide sequence of a protein to be tested for a ReproSA- 1 interaction.
  • the yeast Gal4 DNA-binding domain is fused to cDNAs from a human cell, thus creating a library of human proteins fused to the Gal4 DNA binding domain for screening for
  • the other polynucleotide comprises the Gal4 activation domain fused to an ReproSA- 1 polypeptide sequence.
  • the constructs are introduced into a yeast host cell. Upon expression, intermolecular binding between the ReproSA- 1 polypeptide and the test protein can reconstitute the Gal4 DNA-binding domain with the Gal4 activation domain. This leads to the transcriptional activation of a reporter gene (e.g., lacZ, HIS3) operably linked to a Gal4 binding site. By selecting for, or by assaying the reporter, gene colonies of cells that contain a ReproSA-1 interacting protein can be identified.
  • a reporter gene e.g., lacZ, HIS3
  • the invention provides a method for detecting an interaction between a first polypeptide and a second polypeptide, where either the first polypeptide or the second polypeptide is a ReproSA- 1 protein or fragment, and the other polypeptide is a polypeptide sequence to be tested for interaction with the ReproSA- 1 protein or fragment.
  • the method involves providing a host cell and two chimeric genes that are capable of being expressed in the host cell.
  • the host cell which may be yeast, bacterial, or of another type, contains a detectable gene that expresses a detectable protein, when the detectable gene is activated by an amino acid sequence that includes a transcriptional activation domain.
  • the first chimeric gene has a DNA sequence that encodes a first hybrid protein.
  • the first hybrid protein contains a DNA-binding domain that recognizes a binding site on the detectable gene in the host cell, and the first polypeptide.
  • the second chimeric gene has a DNA sequence that encodes a second hybrid protein, which includes the transcriptional activation domain and the second polypeptide.
  • the interaction between the first polypeptide and the second polypeptide in the host cell causes the transcriptional activation domain to activate transcription of the detectable gene when the first chimeric gene and the second chimeric gene are introduced into the host cell and the host cell is subjected to conditions under which the first hybrid protein and the second hybrid protein are expressed.
  • the interaction is then detected by determining whether the detectable gene has been expressed to a degree greater than expression in the absence of an interaction between the first polypeptide and the second polypeptide.
  • the poly(A) + RNA ranged in size from 0.2 to 10 Kb as determined by denaturing gel electrophoresis (Technical Data Sheet and communication for Clontech product: Human Testis Poly(A) + RNA).
  • oligo-dT primer containing an internal, protected Xhol site was annealed in the presence of a nucleotide mixture containing 5 -methyl dCTP, and extended by MMTV reverse transcriptase (Stratagene ZAP Express cDNA Synthesis Technical Manual revision #065001, 1996).
  • Second strand synthesis of the testis cDNA/RNA hybrid was completed by the addition of RNase H, DNA polymerase I, and dNTPs to the first strand synthesis reaction.
  • Pfu DNA polymerase was used to blunt-end the double stranded testis cDNA followed by the ligation of EcoRI adapters.
  • the cDNA was kinased and digested with Xhol and EcoRI before size fractionation on Sephacryl S-500 columns. The size fractionated cDNA was recovered and then quantified on ethidium bromide containing plates against a set of serially-diluted DNA standards.
  • the cDNA contained in the first two column fractionations was directionally ligated, in the sense orientation, to J ⁇ oT/EcoRT-digested ZAP Express phage vector arms. Initially, approximately 26 ng (per fraction) of the cDNA was ligated and packaged into bacteriophage particles. Subsequently, the approximately 100 ng remaining cDNA in fractions 1 and 2 was packaged into bacteriophage particles using several reaction of the lambda phage packaging extract (Stratagene).
  • the primary human testis library was titered by infection of the XL1 Blue MRF' host strain.
  • the ratio of recombinanfcnonrecombinant phage was determined by plating infected XL1 Blue MRF' in the presence of IPTG and XGal.
  • the number of blue (non-recombinant) or white (recombinant) plaques were quantified using a Manostat colony counter.
  • Ninety-eight and one-half (98.5) percent of the phage in the human testis cDNA library were recombinant and the primary phage library base consisted of 9.97 x 10 6 independent clones.
  • the human testes cDNA library was amplified once and re-titered as above. The titer of the amplified library was 6 x 10 8 pfu/ml.
  • the average size of the cDNA inserts was determined by PCR amplification. Twenty well-isolated phage plaques were cored and the cDNA inserts were amplified using T7/T3 specific oligonucleotides which hybridize to sites flanking the cDNA insertion site contained within the lambda phage vector. The PCR products were analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining. Seventy percent of the cored plaques contained inserts varying in size from 0.5 to 2.5 Kb with an average size of 1.5 Kb.
  • patient serum with high levels of anti-sperm antibodies was used as a probe to screen the testes specific cDNA expression library described in Example I.
  • Serum was obtained from a male patient diagnosed as immunologically infertile.
  • the patient's semen contained antibodies to sperm as detected by a direct anti- sperm Ab test (See Munroe et al, 1990, Fertility & Sterility, 54:114; Bronson, 1984, Ann. NY Acad. Sci. 1984:436 ; Clarke, 1985, Am. J. Reprod. Immunol. Microbiol 7:143 Bronson, 1982, Am. J. Reprod. Immunol. 2:222).
  • Antibody test results were: IgG 50% positive and 52% positive; IgA 45% positive and 41% positive. A score greater than 20% is considered positive.
  • the patient's serum was able to agglutinate sperm in an agglutination assay (Hirano et al., 1996, Arch Androl 37:163-170). Magnified examination of sperm agglutination studies with the infertile patient serum used to isolate ReproSA- 1 showed that the site of contact was at the central part of the sperm tail.
  • Antibodies that react with expression library host strains were immunoadsorbed from patient serum by using BNN97 and Y1090 E. coli lysate-conjugated Sepharose beads (5prime-3prime, West Chester, PA) following the manufacturer's protocols. Briefly, 2 ml of host stain lysate-conjugated Sepharose beads were washed twice with sterile Tris buffered saline (TBS). The beads were resuspended in 4 mis of serum diluted 1/2 in TBS. Following a 16 hour incubation at 4° C, the sepharose beads were collected by centrifugation at 1,000 x g for 2 min.
  • the supernatant was removed and the beads were washed with 4 mis of sterile TBS. After centrifugation at 1,000 x g for 2 min, the supernatants were collected, pooled and used to screen the testes-specific cDNA expression library.
  • testes-specific cDNA library Screening the testes-specific cDNA library.
  • phage particles were incubated with XI- 1 blue MRF' host cells and plated at density of 50,000 phage per 150 mm dish using standard protocols (Stratagene). After incubating for 5 hours at 42° C, the phage plaques were overlaid with nitrocellulose membranes (Protran; Schleicher & Schuell) that had been soaked in a 10 mM isopropyl-1-thio- ⁇ -D-galactopyranoside (IPTG) solution. Following a 4 hour incubation at 37 °C, the membranes were removed and washed three times for 15 min in TBS with 0.05% Tween 20 (TTBS).
  • TBS Tween 20
  • the membranes were incubated with blocking solution (1 % Bovine serum albumin [fraction V] in TBS) for 1 hour at room temperature prior to a 1 hour incubation with preadsorbed patient serum diluted 1/10 (final dilution of 1/40) in blocking solution.
  • the membranes were washed three time for 15 min with
  • TTBS prior to the addition of alkaline phosphatase-conjugated goat anti-human Ig(G,A,M) (Pierce) diluted 1/25,000 in blocking solution.
  • enzyme substrate Western Blue; Promega
  • stop solution Tris-HCl pH 2.9; 1 mM EDTA
  • phage plaques were selected and transferred to 500 ul of SM buffer (100 mM NaCl, 8 mM MgSO 4 , 50 mM Tris-HCl pH7.5, 0.01 % gelatin) containing 20 ul of chloroform.
  • SM buffer 100 mM NaCl, 8 mM MgSO 4 , 50 mM Tris-HCl pH7.5, 0.01 % gelatin
  • the selected phage were eluted from the agar and plated at a density of approximately 1,000 phage per 100 mm dish and screened as described above.
  • ReproSA- 1.0 represented a single clone the screening process was repeated a third time as described above.
  • Plasmid containing the cDNA insert for ReproSA- 1 was excised from the phage clone using the manufacturer's protocols (Stratagene). The size of the ReproSA- 1.0 insert was determined by releasing the cDNA fragment from the rescued pBK-CMV/ReproSA-1 plasmid with the restriction enzymes, EcoRI and Xhol. The released insert was size fractionated by agarose-gel electrophoresis and the apparent length of the insert was determined by comparing its migration position with a DNA standard (1 kb ladder; Gibco BRL). The insert migrated at approximately 1.5 Kb.
  • the nucleotide sequence of ReproSA- 1.0 was determined using a modified protocol of the dideoxy chain termination method of Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74:5463-5467 and USB Sequences 2.0 (USB, Cleveland, Ohio). See Figure 1.
  • the amino acid sequence was predicted using the Intelligenetic TRANSLATE program and sequence homologies were determined with BLAST data base search algorithms.
  • the deduced amino acid sequence (in the expected frame for a fusion protein) showed similarity to the PF16 flagellar protein of Chlamydomonas reinhardt (Smith et al, 1996, J. Cell. Biology 132:359-370) indicating that ReproSA- 1 is a sperm-tail protein.
  • 5 'RACE Frohman et al., 1988 Proc. Acad. Sci USA 85;8998 .
  • total RNA isolated from testis was converted to cDNA using a ReproSA- 1 specific primer and Superscript reverse transcriptase (Life Technologies).
  • the resulting ReproSA- 1 cDNA was incubated with dCTP and terminal transferase to add CTP molecules to the 3' end, which was used as a binding site for the arbitrary primer that contained a complementary stretch of GTPs.
  • the resulting double-stranded ReproSA- 1 DNA (flanked with the arbitrary primer and the sequence-specific primer) was PCR amplified.
  • the PCR amplicon was used as template to generate a probe that contained 5' ReproSA- 1 sequences.
  • the testis library was screened with the 5'ReproSA-l probe, and full-length clones were isolated and sequenced. These clones are identified as clones C, E and X in Figure 2. These clones were sequenced by standard methods.
  • the ReproSA- 1 expression distribution was detected by Northern blot analysis using poly(A) + RNA collected from human spleen, thymus, prostate, testis, ovary, small intestine, colon, and peripheral blood leukocytes (MTN human blot II; Clontech) and human heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas (MTN human blot I; Clontech) and the manufacturer' suggested protocols (Clontech).
  • MTN human blot II peripheral blood leukocytes
  • MTN human blot I MTN human blot I
  • Clontech Clontech
  • the immobilized poly(A) + RNA samples were incubated with a random prime labeled probe that represented the ReproSA- 1.0 insert.
  • the probe was generated by using and EcoRI/XhoI released fragment of ReproSA- 1.0 as template for a random prime labeled reaction as described in the manufactures manual (Megaprime kit; Amersham).
  • the Northern blot membranes were prehybrized with 6 mis of ExpressHyb solution (Clontech) followed by a 1 hour incubation at 68 °C with approximately 0.5 ng of radiolabeled probe (approx. 2x10 s cpm) in 5 mis of ExpresHyb solution.
  • RNA species and tissue distribution were determined by autoradiography. Expression was only detected in testes, as a pair of mRNA transcripts of approximately 1.8 and 2.8 Kb ( Figure 3).
  • Example IV EXPRESSION OF REPRO-SA-1 POLYPEPTIDES
  • the ReproSA- 1 cDNA insert was PCR amplified using a T3 primer and prlAXhoHis (5'GTGGTGGTGCTCGAGTGGTTGATAGCTGTCC [SEQ ID NO:23]), according to standard protocols.
  • the PCR amplicons were separated by agarose-gel electrophoresis and purified with glass beads (BIO 101).
  • the purified fragments were prepared for ligation into a pET21b expression plasmid (Novagen, Madison, WI) and transfected into DH5 ⁇ E. coli.
  • Colonies were screened with a 32 P labeled ReproSA- 1 probe and by sequencing.
  • a plasmid encoding a full-length ReproSA- 1.0 insert in the correct orientation was purified and transfected into BL-21 E. coli bacteria for expression of recombinant protein.
  • the pET21b expression system produces a fusion protein with T7-Tag® and His- Tag® (Novagen, Madison, WI).
  • the bacteria transfected with pET21/ReproSA-l was grown in 30 mis of LB media with 50 ⁇ g/ml ampicilin to an OD 600 of 0.6 prior to induction of protein expression with IPTG (final concentration of 1 mM).
  • the bacterial cells were harvested by centrifugation and washed twice with PBS.
  • the bacterial pellet was resuspended in GMAC- 5 (20 mM Tris-HCL pH 7.4, 0.5 M NaCl, 6 M guanidine-HCl, 1 mM PMSF, 1 mM Imidazole) and sonicated on ice for 50 pulses until the OD 600 dropped to approximately one tenth of the original O.D.
  • Example V POLYCLONAL ANTI-REPRO-SA-1 SERA PRODUCTION Anti-ReproSA-1 anti-sera were made using three different immunogens. Peptides were synthesized corresponding to the C-terminal end of the protein
  • Antisera from immunized rabbits were tested for the presence of anti- ReproSA-1 antibodies using standard protocols (i.e., enzyme-linked immunosorbent assays using the ReproSA- 1 peptide or recombinant polypeptide immunogen immobilized onto microtiter plates; see Harlow, supra).
  • the anti-ReproSA-1 antibodies were affinity purified using appropriate peptide conjugated to a matrix support (see, CURRENT PROTOCOLS IN IMMUNOLOGY, supra, pp.9.4.9-9.4.10).
  • Example VI CHROMOSOME LOCATION OF REPRO-SA-1 Fluorescence in situ hybridization (FISH) was performed using a labeled ReproSA- 1 probe. Specific hybridization identified chromosome arm 1 Op 11.2 as the locus of the ReproSA- 1 gene.
  • Example VII RETROSPECTIVE SCREENING OF FERTILE AND INFERTILE PATIENT SERA Enzyme-linked immunosorbent assays (ELISA) are performed on a series of sera from (i) patients that have been clinically defined as infertile and (ii) a matched control (fertile) group, using standard methods. See, Enzyme-Linked Immunosorbent Assays., in Current Protocols, pages.2.1.3-2.1.5). The purified recombinant ReproSA-1 is immobilized on microtiter plates by diluting the protein to a concentration of 5 ⁇ g/ml in carbonate binding buffer and incubating the plates overnight. The patient sera is diluted in sample buffer and incubated with the immobilized ReproSA- 1 protein.
  • Bound anti-ReproSA-1 antibodies are detected with horseradish peroxidase conjugated anti-human immunoglobulin and the appropriate substrate. The signals generated in each well are analyzed to determine levels of anti-ReproSA-1 antibodies found in patient infertile serum relative to normal control (fertile) serum.
  • ELISA results are confirmed by using the purified recombinant ReproSA- 1 protein in an immunoblot analysis (see, Immunoblotting and Immunodetection, in CURRENT PROTOCOLS IN IMMUNOLOGY, pages.8.10.1-8.10.7). Briefly, 10 ⁇ g ofReproSA-1 protein is size fractionated on SDS-PAGE (10%) and transferred to a nitrocellulose membrane. The membrane is incubated with patient (infertile) sera or normal control (fertile) sera diluted in sample buffer. Bound anti-ReproSA-1 antibodies are detected with alkaline phosphatase conjugated anti-human immunoglobulin and the appropriate substrate.
  • Example VIII ISOLATION OF HUMAN ANTI-REPRO-A-1 IMMUNOGLOBULIN SEQUENCES Human antibodies are isolated or engineered from the antibody repertoire of infertile patients. These human antibodies are used to formulate spermicidal contraceptives.
  • PBLs peripheral blood lymphocytes
  • PBLs are isolated from a patient expressing anti-ReproSA-1 antibodies. Eight ml of blood are collected in Vacutainer CPT tubes (Becton-Dickinson) and the PBLs are isolated as per the manufacturer's recommendations. The PBLs are presorbed against non- reproductive tissue or cells (i.e. muscle, heart, skin, liver, kidney). The unabsorbed PBLs are collected and used to construct a sfFv library in the filamentous phage (fUSE5) (Scott, J & G. Smith, Science 249:386,1990).
  • Poly(A) + RNA is isolated from peripheral blood lymphocytes obtained in step A. cDNA complementary to the entire repertoire of the poly(A) + RNA is synthesized. The polymerase chain reaction is used to amplify the variable region heavy chain (V ⁇ -constant region heavy chain (CHI) and the kappa and lambda light chain genes, with additional complementary coding sequences for a peptide linker at the 3' end of the heavy chain genes and the 5' end of the light chain genes. PCR is then used to amplify the V H -linker- variable region light chain (V ) scFv cDNAs.
  • V ⁇ -constant region heavy chain CHI
  • V scFv cDNAs V H -linker- variable region light chain
  • the scFv cDNAs are ligated into the filamentous phage fUSE5 such that they are expressed as a fusion protein. Each resulting fusion phage will display 4 or 5 copies of a scFv molecule fused to the minor envelope protein p3. See, e.g., Mole et al. , Journal of Clinical Microbiology, 32:2212 ( 1994). C. Panning.
  • Recombinantly expressed ReproSA- 1 peptides are coated onto polystyrene dishes. Phage from the scFv library are precipitated in 4% (wt/vol) PEG/0.5 M NaCl and resuspended in water. Approximately 10 11 transforming units are added to the protein or peptide in 2 ml of binding buffer. Unbound phage is washed away with PBS, and bound phage eluted in elution buffer. The eluted phage are amplified as described in and the panning process will be repeated three times. See, e.g., Scott et al., Science 249:386 (1990) and Cai et al., Proc. Natl. Acad. Sci., USA 92; 6537 (1995), both of which are inco ⁇ orated in their entirety for all pu ⁇ oses.
  • the eluted phage are mixed with E. Coli K91 Kan cells at low phage to cell ratios, and plated on 2x TY/tetracycline plates. Individual colonies are inoculated into 2x TY/tetracycline medium and grown. Positives are verified by ELISA assay to ReproSA- 1 in which bound phage are detected using a conjugated anti-M13 polyclonal antibody. Those phage that bind with highest affinity are selected and the immunoglobulin gene sequences being expressed are characterized and used to design high affinity ReproSA- 1 -binding molecules. See, e.g., Scott et al., supra, and Cai et al., supra.

Abstract

Purified or recombinant peptides and polypeptides having a sequence of a human sperm protein are disclosed. Also provided are polynucleotides encoding the polypeptide, and antibodies that specifically bind the polypeptide. The novel reagents of the invention are used for diagnosis and treatment of human diseases such as immunological infertility, and in contraceptive formulations and vaccines.

Description

NUCLEIC ACIDS ENCODING SPERM ANTIGENS AND REPROSA-I POLYPEPTIDES
BACKGROUND OF THE INVENTION
The presence of an anti-sperm immune response in a patient is correlated with infertility. For example, anti-sperm antibodies are detected more frequently in the sera, seminal fluid, and/or cervical or vaginal fluid of couples experiencing unexplained infertility than in couples whose infertility is due to tubal factors. In addition, vasectomized men experiencing reduced fertility upon vasectomy reversal have been found to exhibit anti-sperm antibodies (Bronson et al., 1984, Fertil. Steril. 42:171-183; Lehman et al., 1989, Am. J. Reprod. Immunol. 19:43-52; Naz et al., 1990, Human Reproduction 5:511-518). Evidence that anti-sperm antibodies are the causative factor of infertility is indicated by cases of autoimmunization against sperm antigens in men resulting in aspermatogenic orchitis, and immunization of fertile women with sperm eliciting anti-sperm antibodies and resulting infertility (Mancini et al., 1965, J Clin. Endocrinol. Metab. 25:859-875; Baskin et al, 1932, Am. J. Obstet. Gynecol. 24:892-897; Escuder et al, 1936, Am. Fac. Med. Montevideo. 21:889-937). Also, treatment of immunoinfertile couples with immunosuppressive agents which lower antibody titers has resulted in successful conception (Hendry et al, 1979, Lancet ii:498-501 ; Shulman et al, 1986, Am. J. Reprod. Immunol.
Microbiol. 10:86-89). Additionally, evidence from human in vitro fertilization-embryo transfer programs indicates that the presence of anti-sperm antibodies can block fertilization, and that absorption of the anti-sperm antibodies caused a disappearance of the block, resulting in normal fertilization (Bronson et ah, 1984, Eert/7. Steril. 42:171-183; Tusuk et al., 1986, Fertil. Steril. 46:92-96). These data suggest that anti-sperm antibodies reduce fertility by causing sperm immobilization or by blocking sperm/ovum interactions/fusion. This naturally occurring immune response can be used to develop methods for diagnosis of infertility, and anti-fertility (i.e., contraceptive) vaccines or spermicidal contraceptives.
Contraceptive vaccines have long been viewed as a highly desirable form of contraception, because they are relatively inexpensive, easy to administer, and capable of providing prolonged protection against pregnancy (Aitken et al., 1993, British Med. Bull. 49:88-99). The primary targets for development of a contraceptive vaccine are (i) human chorionic gonadotrophin, (ii) the zona pellucida, and (iii) sperm. A vaccine directed against sperm, particularly a sperm-surface antigen, would have the advantage of having fewer effects on the hormonal milieu of the patient and fewer endocrinological side effects. However, few suitable sperm antigens have been identified. Thus a need exists for sources of sperm proteins, especially sperm surface proteins, that can serve as antigenic targets, and for the creation of vaccines and other medicaments directed against sperm antigens.
As noted above, immunological infertility is often a consequence of the presence of anti-sperm antibodies in a patient. However, at present, immunological infertility is a diagnosis of exclusion, due to the lack of simple and inexpensive assays for detecting naturally occurring anti-sperm antibodies. Thus, a need also exists for assays that can be used in the diagnosis of immunological infertility. The present invention addresses these and other needs.
SUMMARY OF THE INVENTION It is a general object of the present invention to provide isolated, recombinant, or substantially purified nucleic acids, polypeptides and antibodies related to the ReproSA- 1 polypeptide, as well as methods of using these nucleic acids, polypeptides and antibodies in screening, diagnostic and therapeutic applications.
In a first aspect, the invention provides a substantially pure ReproSA- 1 polypeptide, or fragment of a ReproSA- 1 polypeptide. In a preferred embodiment the polypeptide has a sequence substantially homologous to, or the same as, SEQ ID NO:2, or is a polypeptide having between about eight and about 509 contiguous residues of a polypeptide having the amino acid sequence of SEQ ID NO:2. Also provided are substantially pure polypeptides that are specifically immunoreactive with an antibody raised against a polypeptide having the amino acid sequence of SEQ ID NO:2. According to the invention, any of the aforementioned polypeptides may be coupled to a solid support, for example, for use in assays for anti-ReproSA-1 antibodies.
In a second aspect, the invention provides an isolated polynucleotide comprising a sequence encoding a ReproSA-1 polypeptide or fragment thereof, such as a peptide having between about eight and about 509 contiguous residues of a polypeptide having the amino acid sequence of SEQ ID NO:2. The ReproSA-1 coding sequences may be operably linked to a promoter. Also provided is a recombinant cell, or cell line, comprising a polynucleotide encoding ReproSA- 1 or a fragment thereof. In a preferred embodiment, the cell line is capable of producing a human ReproSA- 1 polypeptide or a fragment thereof.
In a third aspect, the invention provides an isolated or recombinant oligonucieotide or polynucleotide having a sequence of at least 10 contiguous nucleotides of SEQ ID NO: 1. In one embodiment, the oligonucieotide or polynucleotide has at least 12 contiguous nucleotides of SEQ ID NO:l.
In a fourth aspect, the invention provides a substantially purified anti- ReproSA-1 antibody, or fragment thereof, that specifically binds to human ReproSA- 1. The antibody may be polyclonal, monoclonal, human or humanized. In a preferred embodiment, the antibody binds ReproSA- 1 with an affinity of at least about 108 M"1. Also provided is a cell or hybridoma that secretes the antibody. The invention also provides a medicament for contraception containing anti-ReproSA-1 antibodies in a pharmacologically acceptable carrier.
In a fifth aspect, the invention provides vaccines comprising ReproSA-1 polypeptides which are capable of inducing an immune response in a human or non-human subject. The vaccines may include a ReproSA- 1 polypeptide (comprising at least about eight consecutive residues of SEQ ID NO:2) or a polynucleotide encoding ReproSA- 1, in a pharmacologically acceptable carrier. In a preferred embodiment the vaccine includes an adjuvant. The anti-ReproSA-1 antibodies elicited by the vaccine may be found in mucosal secretions, e.g., mucosal secretions of the reproductive tract, e.g., the vaginal mucosa. In related aspects, the invention provides methods of immunizing a human to achieve a contraceptive effect by administering the vaccines.
In other aspects, the invention provides methods for: diagnosing immunological infertility in a human patient, for example, by detecting anti-ReproSA-1 antibodies in a biological sample from a patient; detecting an abnormal ReproSA- 1 protein in sperm of a patient by contacting the sperm of the patient with an antibody that specifically binds to an epitope of the ReproSA- 1 polypeptide having the amino acid sequence of SEQ ID NO:2 and determining the binding of the antibody to the sperm of the patient, where failure of the sperm to bind indicates the presence of abnormal ReproSA- 1 protein; and detecting a mutation in a ReproSA- 1 gene in a patient by comparing the sequence of a ReproSA- 1 gene of the patient with the sequence of the ReproSA- 1 gene having the sequence of SEQ ID NO:l, wherein a difference in the two sequences indicates a mutation in the ReproSA- 1 gene of the patient.
The invention also provides kits for carrying out the methods listed above. For example, one kit of the invention is useful for determining the presence in a biological sample of antibodies to human ReproSA- 1 polypeptide and contains (i) an antibody specifically reactive with ReproSA- 1 and (ii) a polypeptide bound to a solid support, wherein the polypeptide comprises at least about eight consecutive amino acids of SEQ ID NO:2.
BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the nucleotide sequence of the ReproSA- 1 cDNA [SEQ ID
NO:l] and the amino acid sequence of the ReproSA-1 polypeptide [SEQ ID NO:2]. Figure 2 shows a diagram illustrating the 1.8 and 2.8 KB ReproSA- 1 transcripts as well as four ReproSA- 1 clones.
Figure 3 shows a multiple-tissue Northern Blot probed with a labeled EcoRI/XhoI fragment of the ReproSA- 1 cDNA. RNA was from human spleen (lane 1), thymus (lane 2), prostrate (lane 3), testis (lane 4), ovary (lane 5), small intestine (lane 6), colon (lane 7) and peripheral blood lymphocytes (lane 8). Molecular weight is indicated in kilobases.
Figure 4 shows a Southern blot of EcoRI digested genomic DNA collected from human, monkey, rat, mouse, dog, cow, rabbit, chicken and yeast probed with a labeled probe from the ReproSA- 1 cDNA clone insert. Molecular weight is indicated in kilobase pairs.
Figure 5 shows the expression and purification of a poly(His) tagged ReproSA-1 polypeptide (encoded by Clone ReproSA-1.0). A. SDS-polyacrylamide gel electrophoresis B. Western Blot probed with anti-T7-Tag® antibody (Novagen, Madison,
WT).
Figure 6 shows Western blots of normal human sperm extracts probed with anti-ReproSA-1 antisera. Lane 1: anti-(ReproSA-l recombinant polypeptide) antibody [rabbit #1]; lane 2: anti-(ReproSA-l recombinant polypeptide) antibody [rabbit #2]; lane 3: pre-immune serum from rabbit #1 used in lane 1 ; lane 4; pre-immune serum from rabbit #2 used in lane 2; lane 5: affinity purified anti-(ReρroSA armadillo repeat peptide) antibody [pool of two rabbits]; lane 6 :affinity purified anti-(ReproSA C-terminus peptide) antibody [pool of two rabbits]; lane 7: unpurified anti-(ReproSA armadillo repeat peptide) serum [one rabbit]; lane 8: unpurified anti-(ReproSA C-terminus peptide) serum [one rabbit]; Lane 9: pooled pre-immune sera from ReproSA C-terminal peptide- and ReproRA armadillo repeat peptide immunized rabbits; lane 10: secondary antibody only. Binding to the approximately 60 kDa ReproSA-1 protein is seen in lanes 1, 2, 5, and 8.
DETAILED DESCRIPTION OF THE INVENTION
I. General
The present invention is related to the identification and isolation of a human gene that encodes the sperm protein ReproSA- 1. The gene is conserved in non-human primates and rodents, and its expression is testis-specific. The gene and its RNA and protein products, as well as related nucleic acids, peptides and polypeptides are useful for contraception in humans and other animals, for example by vaccination, as well as for diagnosis and treatment of infertility.
II. Polypeptides
In one aspect, this invention provides substantially pure or isolated ReproSA- 1 polypeptides, i.e., that are related to and/or derived from human ReproSA- 1 polypeptides. ReproSA- 1 polypeptides comprise an amino acid sequence substantially identical to the amino acid sequence shown in Figure 1 [SEQ ID NO:2] or are immunologically related to the ReproSA-1 polypeptide of SEQ ID NO:2. In one embodiment, the polypeptide of the invention has between about eight and about 509 contiguous residues of a polypeptide having the amino acid sequence of SEQ ID NO:2. ReproSA-1 polypeptides are components of the sperm (e.g., sperm tail) in humans and other species (e.g., non-human primates and rodents).
In one notable aspect, the full-length ReproSA- 1 polypeptides of the invention contain eight contiguous armadillo ("arm") repeats (Peifer et al., 1994, Cell 76:789). A number of proteins with diverse cellular functions also contain armadillo repeats including pendulin, Rchl, importin, SRP-1, and armadillo (Peifer et al., supra). ReproSA-1 shows additional homology to the PF16 flagellar protein of Chlamydomonas reinhardt, a protein believed to be involved in protein-protein interactions important for central microtubule stability and flagellar motility (Smith et al., 1996, J Cell Biology 132:359-370). It is believed that armadillo motifs of the ReproSA- 1 protein are involved in protein-protein interactions with other sperm proteins (e.g., flagellar proteins). Thus, the armadillo repeat regions (i.e., motifs and sequences) of the ReproSA- 1 proteins, genes, and RNA transcripts provide a target for disruption of ReproSA- 1 activity or expression, and/or disruption or diminution of sperm motility or viability. Such disruption or diminution may be achieved using antibodies that binds one or more of the ReproSA- 1 armadillo repeat regions (or a subsequence thereof); using peptide or nucleic acid vaccines comprising a sequence from an armadillo repeat region; using antisense or ribozyme molecules targeted to nucleic acids comprising these regions; or the like. ReproSA- 1 activity includes the ability of the protein to assemble in the sperm, to interact with other sperm proteins, or any other activity or function of naturally occurring ReproSA- 1 polypeptides or nucleic acids. Table 1, infra, shows an alignment of the ReproSA- 1 armadillo repeats with the "universal" consensus (Peifer et al., supra) and the PF16 armadillo repeat consensus sequence. Residues showing identity with the consensus are shown in bold (all polypeptide sequences described herein are presented in the conventional amino-terminus- > carboxy-terminus orientation).
TABLE 1 ALIGNMENT OF ARMADILLO REPEATS UNIVERSAL CONSENSUS L+NLSX+XXX N+XALLXXGG LPALV+LLXS X+EXXLXX-A AX [SEQ ID NO: 13] A II I I
W V V
PF16 CONSENSUS LGXIAKHSPD LAQAWDAGA LPXLVXCLSE XDXXXKXXAA XA
[SEQ ID NO: 14] S Ξ I V
ReproSA-1 (65) GRLANYNDD AEAWKCDI LPQLVYS AE QNRFYKKAAA FV [SEQ ID (107) LRAVGKHSPQ AQAIVDCGA DTLVIC ED FDPGVKEAAA A
NOS: 15-22] (149) RYIARHNAE SQAWDAGA VPL VLCIQE PEIALKRIAA SA
(191) SDIAKHSPE AQTWDAGA VAH AQMILN PDAKLKHQIL SA
(233) LSQVSKHSVD LAEMWEAEI FPWLTCLKD KDEYVKKNAS TL
(275) IREIAKHTPE LSQLWNAGG VAAVIDCIGS CKGNTRLPGI MM (317) LGYVAAHSEN AMAVIISKG VPQ SVC SE EPEDHIKAAA AW
(360) LGQIGRHTPE HARAVAVTNT PVLLSLYMS TESSEDLQVK SK Although described in terms of the amino acid sequence shown in Figure 1, it will be apparent to those of skill that the polypeptides of the present invention include those peptides having the listed amino acid sequence or immunologically active (i.e., specifically immunoreactive or immunogenic) fragments thereof, as well as those polypeptides having amino acid sequences that are substantially homologous to the listed sequence. When referring to polypeptides the terms "substantially homologous" and "substantially identical refer to two amino acid sequences which, when optimally aligned, have at least 75% sequence identity, preferably at least about 85% sequence identity, more preferably at least about 90% sequence identity, and still more preferably at least about 95% sequence identity. Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (USA) 85:2444, or by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, WI) or by visual inspection (see generally Ausubel et al, supra). For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
The terms "substantially pure" or "isolated," when referring to proteins and polypeptides, denote those polypeptides that are separated from proteins or other contaminants with which they are naturally associated. A protein or polypeptide is considered substantially pure when that protein makes up greater than about 50% of the total protein content of the composition containing that protein, and typically, greater than about 60% of the total protein content. More typically, a substantially pure or isolated protein or polypeptide will make up from about 75 to about 90% of the total protein. Preferably, the protein will make up greater than about 90%, and more preferably, greater than about 95% of the total protein in the composition. The polypeptides of the invention may also be characterized by their ability to bind (i) antibodies that are specifically immunoreactive with a polypeptide having the sequence shown in Figure 1, or (ii) antibodies that are specifically immunoreactive with a substantially pure polypeptide having at least about eight, and preferably at least about 10, more preferably at least about 11 or about 12 contiguous amino acids as shown in Figure 1. The phrase "specifically immunoreactive," or "specifically binds" when referring to the interaction between an antibody and a protein or polypeptide, refers to an antibody that specifically recognizes and binds with relatively high affinity to the protein of interest, e.g., ReproSA- 1, such that this binding is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, Harlow and Lane
(1988) ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. Harlow and Lane (hereinafter referred to as "Harlow") is incorporated by reference in its entirety and for all purposes. Specific immunoreactivity is usually characterized by a specific binding affinity of an antibody for a protein (e.g. , ReproSA-1) of at least 107, 108, 109, or 1010 M"1.
The polypeptides of the present invention may generally be prepared using recombinant or synthetic methods that are well known in the art. Recombinant techniques are generally described in Sambrook et al., 1989, MOLECULAR CLONING: A LABORATORY MANUAL, (2nd ed.) Vols. 1-3, Cold Spring Harbor Laboratory, (hereinafter referred to as "Sambrook") and in Ausubel et al, Eds., 1995, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley and Sons, Inc., New York, (hereinafter referred to as "Ausubel"), both of which are incorporated by reference in their entirety and for all purposes. The term recombinant is intended to encompass the expression of heterologous genetic material which is introduced into a host cell through transfection techniques. In one aspect, the polypeptides of the present invention may be expressed by a suitable host cell that has been transfected with a nucleic acid of the invention, as described in greater detail below. Methods for synthesizing polypeptides are generally described in Merrifield, 1963, Amer. Chem. Soc. 85:2149-2456, Atherton et al, 1989, SOLID PHASE PEPTIDE SYNTHESIS: A PRACTICAL APPROACH, IRL Press, and Merrifield, 1986, Science 232:341-347.
Isolation and purification of the polypeptides of the present invention can be carried out by methods that are generally well known in the art. For example, the polypeptides may be purified using readily available chromatographic methods, e.g., ion exchange, hydrophobic interaction, HPLC or affinity chromatography, to achieve the desired purity. In a preferred embodiment, ReproSA- 1 polypeptide is purified using affinity chromatography. For example, antibodies raised against the ReproSA- 1 polypeptide described in Figure 1 may be coupled to a suitable solid support and contacted with a mixture of proteins containing the ReproSA- 1 polypeptide under conditions conducive to the association of this polypeptide with the antibody. In one embodiment, ReproSA- 1 protein is obtained from a human sperm homogenate, which can be prepared using methods well known in the art (e.g., Paradisi et al, 1995, J Repro. Imm., 28:61-73). Once the ReproSA-1 polypeptide is bound to the immobilized antibody, the solid support is washed to remove unbound material and/or nonspecifically bound proteins. The desired polypeptide may then be eluted from the solid support in substantially pure form by, e.g., a. change in salt, pH or buffer concentration.
In addition to those polypeptides and fragments described above, the present invention also provides fusion proteins which contain these polypeptides or fragments. Fusion proteins may be useful in providing for enhanced expression of the ReproSA- 1 polypeptide constructs, or in producing ReproSA- 1 polypeptides having other desirable properties, e.g., labeling groups, e.g., enzymatic reporter groups, binding groups, antibody epitopes, etc. The term "fusion protein" as used herein, generally refers to a composite protein, i.e., a single contiguous amino acid sequence, made up of two distinct, heterologous polypeptides which are not normally fused together in a single amino acid sequence. Thus, a fusion protein may include a single amino acid sequence that contains two entirely distinct amino acid sequences or two similar or identical polypeptide sequences, provided that these sequences are not normally found together in a single amino acid sequence. Fusion proteins may generally be prepared using either recombinant nucleic acid methods, i.e., as a result of transcription and translation of a gene fusion, which fusion comprises a segment encoding a polypeptide of the invention and a segment encoding a heterologous protein, or by chemical synthesis methods well known in the art. In one embodiment, the ReproSA- 1 polypeptide in is expressed in a cell as a fusion protein having a label or an "epitope tag" to aid in purification. Fusion protein systems can also be used to facilitate efficient production and isolation of ReproSA- 1 proteins or peptides. For example, in some embodiments, the non- ReproSA- 1 sequence portion of the fusion protein comprises a short peptide that can be specifically bound to an immobilized molecule such that the fusion protein can be separated from unbound components (such as unrelated proteins in a cell lysate). One example is a peptide sequence that is bound by a specific antibody. Another example is a peptide comprising polyhistidine tracts e.g. (His)6 or histidine-tryptophan sequences that can be bound by a resin containing nickel or copper ions (i.e., metal-chelate affinity chromatography) .
In some embodiments the ReproSA- 1 polypeptide is a variant of that of SEQ ID NO:2, in which insertions, deletions and substitutions of amino acids are made. For example, in some aspects, conservative amino acid substitutions may be made, i.e., substitution of selected amino acids with different amino acids having similar structural characteristics, e.g., net charge, hydrophobicity and the like. Examples of such conservative substitutions include, e.g., Ala:Val:Leu:Ile:Met, Asp:Glu, Lys:Arg, Asn:Gln, Phe:Tyr and Ser:Thr. Glycosylation modifications, either changed, increased amounts or decreased amounts, as well as other sequence modifications may also be made. In addition, in some embodiments it may be desirable to substitute one or more amino acids of a ReproSA- 1 polypeptide with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) to generate more stable peptides. Similarly, modification of the amino or carboxy terminals may also be used to confer stabilizing properties upon the polypeptides of the invention, e.g., amidation of the carboxy-terminus or acylation of the amino-terminus. Furthermore, although primarily described in terms of "proteins" or
"polypeptides" one of skill in the art, upon reading the instant specification, will appreciate that structural analogs and derivatives of the above-described polypeptides, e.g., peptidomimetics, and the like. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compounds are termed "peptide mimetics" or "peptidomimetics" (Fauchere, 1986, Adv. DrugRes. 15:29; Veber and Freidinger, 1985, Trends Neurosci. p. 392; and Evans et al., 1987, J Med. Chem. 30:1229), and are usually developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent therapeutic effect. Generally, peptidomimetics are structurally similar to a naturally occurring polypeptide but have one or more peptide linkages optionally replaced by a linkage selected from the group consisting of: -CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH- (cis and trans), -COCH2-, -
CH(OH)CH2-, and -CH2SO-, by methods known in the art and further described in the following references: Spatola, A.F., 1983, in CHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS, PEPTIDES, AND PROTEINS, B. Weinstein, eds., Marcel Dekker, New York, p. 267; Spatola, A.F., Vega Data (March 1983), 1:3, "Peptide Backbone Modifications"; Morley, J.S., 1980, Trends Pharm. Sci. p. 463-468. Peptide mimetics may have significant advantages over polypeptide embodiments, including, for example, more economical production and greater chemical stability. Structural mimetics can also be used as immunogens to elicit anti-telomerase or anti-ReproSA-1 protein antibodies.
For many applications, it will also be desirable to provide the polypeptides of the invention as labeled entities, i.e., covalently attached or linked to a detectable group, to facilitate identification, detection and quantification of the polypeptide in a given circumstance. These detectable groups may comprise a detectable protein group, e.g., an assayable enzyme or antibody epitope as described above in the discussion of fusion proteins. Alternatively, the detectable group may be selected from a variety of other detectable groups or labels, such as radiolabels (e.g., 1251, 32P or 35S) or a chemiluminescent or fluorescent group. Similarly, the detectable group may be a substrate, cofactor, inhibitor or affinity ligand.
As is described in more detail infra, the polypeptides of the invention find use as vaccines, immunogens, as antigens in diagnostic kits, and for other uses that will be apparent to those of skill.
III. Antibodies
In a second aspect, the present invention provides antibodies that are specifically immunoreactive with human ReproSA- 1. Accordingly, the antibodies of the invention will specifically recognize and bind polypeptides which have an amino acid sequence that is substantially identical to the amino acid sequence shown in Figure 1 , or an immunogenic fragment thereof. The terms "immunogen" and "immunogenic" are used herein according to their ordinary meaning, i.e, an immunogen is a molecule, such as a protein, that can elicit an adaptive immune response upon injection into a person or an animal. Thus, immunogenecity can be determined by injecting a polypeptide and adjuvant into an animal (e.g., a rabbit) and assaying for the appearance of antibodies directed against the injected polypeptide (see, e.g., Harlow, supra, chapter 5). The antibodies of the invention usually exhibit a specific binding affinity for ReproSA- 1 of at least about 107, 108, 109, or l0I0 M-'.
For production of polyclonal antibodies, an appropriate target immune system is selected, typically a mouse or rabbit, but also including goats, sheep, cows, guinea pigs, monkeys and rats. The substantially purified antigen is presented to the immune system in a fashion determined by methods appropriate for the animal. These and other parameters are well known to immunologists. Typically, injections are given in the footpads, intramuscularly, intradermally, perilymph nodally or intraperitoneally. The immunoglobulins produced by the host can be precipitated, isolated and purified by routine methods, including affinity purification. Substantially monospecific antibody populations can be produced by chromatographic purification of polyclonal sera.
For monoclonal antibodies, appropriate animals will be selected and the desired immunization protocol followed. Monoclonal antibodies with affinities of 108 liters/mole, preferably 109 to 1010 or stronger, will be produced by the methods described infra. The production of non-human monoclonal antibodies, e.g., murine, lagomorpha, equine is well known and can be accomplished by, for example, immunizing an animal with a preparation containing ReproSA- 1 or fragments thereof. Antibody-producing cells obtained from the immunized animals are immortalized and screened, or screened first for the production of antibody which binds to the ReproSA-1 polypeptide and then immortalized. In one method, after the appropriate period of time, the spleens of the animals are excised and individual spleen cells are fused, typically, to immortalized myeloma cells under appropriate selection conditions. Thereafter, the cells are clonally separated and the supernatants of each clone (e.g., hybridoma) are tested for the production of an appropriate antibody specific for the desired region of the antigen. Techniques for producing antibodies are well known in the art. See, e.g. , Goding et al., MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.) Acad. Press, N.Y., (which is incorporated in its entirety and for all purposes) and Harlow, supra. Other suitable techniques involve the in vitro exposure of lymphocytes to the antigenic polypeptides or alternatively, to selection of libraries of antibodies in phage or similar vectors (Huse et al., 1989, Science 246:1275-1281 which is incorporated by reference).
In another aspect of the invention, human antibodies against a ReproSA- 1 polypeptide are provided. Some human antibodies are selected by competitive binding experiments, or otherwise, to have the same epitope specificity as a particular mouse antibody. Human antibodies to a ReproSA- 1 polypeptide can be produced by screening a DNA library from human B cells according to the general protocol outlined by Huse et al., supra. Antibodies binding to the ReproSA- 1 polypeptide are selected. Sequences encoding such antibodies (or a binding fragments) are then cloned and amplified. The protocol described by Huse is rendered more efficient in combination with phage-display technology. See, e.g., Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047 (each of which is incorporated by reference in its entirety for all purposes). In these methods, libraries of phage are produced in which members display different antibodies on their outer surfaces. Antibodies are usually displayed as Fv or Fab fragments. Phage displaying antibodies with a desired specificity are selected by affinity enrichment to an ReproSA- 1 polypeptide.
In a variation of the phage-display method, human antibodies having the binding specificity of a selected murine antibody can be produced. See Winter, WO 92/20791. In this method, either the heavy or light chain variable region of the selected murine antibody is used as a starting material. If, for example, a light chain variable region is selected as the starting material, a phage library is constructed in which members displays the same light chain variable region (i.e., the murine starting material) and a different heavy chain variable region. The heavy chain variable regions are obtained from a library of rearranged human heavy chain variable regions. A phage showing strong specific binding for the ReproSA- 1 polypeptide (e.g., at least 108 and preferably at least 109 M"1) is selected. The human heavy chain variable region from this phage then serves as a starting material for constructing a further phage library. In this library, each phage displays the same heavy chain variable region (i.e., the region identified from the first display library) and a different light chain variable region. The light chain variable regions are obtained from a library of rearranged human variable light chain regions. Again, phage showing strong specific binding are selected. These phage display the variable regions of completely human anti- ReproSA- 1 antibodies. These antibodies usually have the same or similar epitope specificity as the murine starting material.
Human monoclonal antibodies against a known antigen can also be made using transgenic animals having elements of a human immune system (see, e.g., U.S. Patent Nos. 5,569,825; 5,545,806; 5,693,762; 5,693,761; and 5,7124,350, all of which are incoφorated by reference in their entirety for all purposes) or using human peripheral blood cells (Casali et al., Science 234:476-479, 1986).
Additionally, the antibodies of the invention may be chimeric, human-like or humanized, in order to reduce their potential antigenicity, without reducing their affinity for their target. Chimeric, human-like and humanized antibodies have been described in the art. See, e.g., Queen, et al., 1989, Proc. Nat'lAcad. Sci. USA 86:10029; Verhoeyan et al., 1988, Science 239:1534-1536 (1988) and U.S. Patent Nos. 5,585,089 and 5,530,101, all of which are incorporated by reference in their entirety and for all purposes. Humanized immunoglobulins have variable framework regions substantially from a human immunoglobulin (termed an acceptor immunoglobulin) and complementarity determining regions substantially from a non-human (e.g., mouse) immunoglobulin (referred to as the donor immunoglobulin). The constant region(s), if present, are also substantially from a human immunoglobulin. By incorporating as little foreign sequence as possible in the hybrid antibody, the antigenicity is reduced. Preparation of these hybrid antibodies may be carried out by methods well known in the art. The humanized antibodies of the present invention offer several advantages over the mouse donor antibody: (1) The human immune system should not recognize the framework or constant region of the humanized antibody as foreign, and therefore the antibody response against such an injected antibody should be less than against a totally foreign mouse antibody or a partially foreign chimeric antibody. (2) Because the effector portion of the humanized antibody is human, it may interact better with other parts of the human immune system. (3) Injected mouse antibodies have been reported to have a half-life in the human circulation much shorter than the half-life of normal human antibodies (Shaw et al., J Immunol. 138:4534-4538 (1987)). Injected humanized antibodies have a half-life essentially equivalent to naturally occurring human antibodies, allowing smaller and less frequent doses. The cloning and sequencing of cDNA encoding the ReproSA- 1 polypeptide heavy and light chain variable regions may be carried out using routine procedures. See, e.g., Jones & Bendig, Bio/Technology 9:88-89 (1991) and Bio/Technology 9:579 (1991); Sambrook, supra, and Harlow, supra. The substitution of mouse CDRs into a human variable domain framework is most likely to result in retention of their correct spatial orientation if the human variable domain framework adopts the same or similar conformation to the mouse variable framework from which the CDRs originated. This is achieved by obtaining the human variable domains from human antibodies whose framework sequences exhibit a high degree of sequence identity with the murine variable framework domains from which the CDRs were derived. The heavy and light chain variable framework regions can be derived from the same or different human antibody sequences. The human antibody sequences can be the sequences of naturally occurring human antibodies or can be consensus sequences of several human antibodies. See Kettleborough et al., Protein Engineering 4:773 (1991); Kolbinger et al., Protein Engineering 6:971 (1993).
Suitable human antibody sequences are identified by computer comparisons of the amino acid sequences of the mouse variable regions with the sequences of known human antibodies. The comparison is performed separately for heavy and light chains but the principles are similar for each. As noted supra, the humanized antibodies of the invention comprise variable framework regions substantially from a human immunoglobulin and complementarity determining regions substantially from a mouse anti-ReproSA-1 immunoglobulin. Once the complementarity determining regions of the murine antibody are identified and appropriate human acceptor immunoglobulins, the next step is to determine which, if any, residues from these components should be substituted to optimize the properties of the resulting humanized antibody. In general, substitution of human amino acid residues with murine should be minimized, because introduction of murine residues increases the risk of the antibody eliciting a HAMA response in humans. Amino acids are selected for substitution based on their possible influence on CDR conformation and/or binding to antigen. Investigation of such possible influences is by modeling, examination of the characteristics of the amino acids at particular locations, or empirical observation of the effects of substitution or mutagenesis of particular amino acids. When an amino acid differs between a murine anti-peptide/MHC antibody variable framework region and an equivalent human variable framework region, the human framework amino acid should usually be substituted by the equivalent mouse amino acid if it is reasonably expected that the amino acid noncovalently binds antigen directly, is adjacent to a CDR region, is part of a CDR region under the alternative definition proposed by
Chothia et al., supra, or otherwise interacts with a CDR region, or participates in the VL-VH interface.
Other candidates for substitution are acceptor human framework amino acids that are unusual for a human immunoglobulin at that position. These amino acids can be substituted with amino acids from the equivalent position of more typical human immunoglobulins. Alternatively, amino acids from equivalent positions in anti- peptide/MHC antibody can be introduced into the human framework regions when such amino acids are typical of human immunoglobulin at the equivalent positions.
In general, substitution of all or most of the amino acids fulfilling the above criteria is desirable. Occasionally, however, there is some ambiguity about whether a particular amino acid meets the above criteria, and alternative variant immunoglobulins are produced, one of which has that particular substitution, the other of which does not. Usually the CDR regions in humanized antibodies are substantially identical, and more usually, identical to the corresponding CDR regions in the anti-ReproSA-1 antibody. Although not usually desirable, it is sometimes possible to make one or more conservative amino acid substitutions of CDR residues without appreciably affecting the binding affinity of the resulting humanized immunoglobulin. Occasionally, substitution of CDR regions can result in enhanced binding affinity.
Other than for the specific amino acid substitutions discussed above, the framework regions of humanized immunoglobulins are usually substantially identical, and more usually, identical to the framework regions of the human antibodies from which they were derived. Of course, many of the amino acids in the framework region make little or no direct contribution to the specificity or affinity of an antibody. Thus, many individual conservative substitutions of framework residues can be tolerated without appreciable change of the specificity or affinity of the resulting humanized immunoglobulin. However, in general, such substitutions are undesirable. Having conceptually selected the CDR and framework components of humanized immunoglobulins, a variety of methods are available for producing such immunoglobulins. Because of the degeneracy of the code, a variety of nucleic acid sequences will encode each immunoglobulin amino acid sequence. The desired nucleic acid sequences can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of an earlier prepared variant of the desired polynucleotide. Oligonucleotide-mediated mutagenesis is a preferred method for preparing substitution, deletion and insertion variants of target polypeptide DNA. See Adelman et al., DNA 2:183 (1983). Briefly, the target polypeptide DNA is altered by hybridizing an oligonucieotide encoding the desired mutation to a single-stranded DNA template. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template that incorporates the oligonucieotide primer, and encodes the selected alteration in the target polypeptide DNA.
The variable segments of humanized antibodies produced as described supra are typically linked to at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Human constant region DNA sequences can be isolated in accordance with well-known procedures from a variety of human cells, but preferably immortalized B-cells (see Kabat et al., supra, and WO87/02671) (each of which is incorporated by reference in its entirety for all purposes). Ordinarily, the antibody will contain both light chain and heavy chain constant regions. The heavy chain constant region usually includes CHI, hinge, CH2, CH3, and CH4 regions.
Recombinant expression of antibodies can be carried out by inserting nucleic acids encoding humanized light and heavy chain variable regions, optionally linked to constant regions, into expression vectors. The light and heavy chains can be cloned in the same or different expression vectors. The DNA segments encoding immunoglobulin chains are operably linked to control sequences in the expression vector(s) that ensure the expression of immunoglobulin polypeptides. Such control sequences include a signal sequence, a promoter, an enhancer, and a transcription termination sequence. Expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Immunoglobulin light and heavy chains are expressed using standard methods. For example, vectors containing the polynucleotide sequences encoding the heavy and light chain encoding sequences and expression control sequences can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts. (See generally Sambrook, supra). When heavy and light chains are cloned on separate expression vectors, the vectors are co-transfected to obtain expression and assembly of intact immunoglobulins. After introduction of recombinant DNA, cell lines expressing immunoglobulin products are cell selected. Cell lines capable of stable expression are preferred (i.e., undiminished levels of expression after fifty passages of the cell line).
Once expressed, the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms of the present invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see generally Scopes, Protein Purification (Springer- Verlag, N.Y., 1982). Substantially pure immunoglobulins of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity most preferred, for pharmaceutical uses.
The recombinant techniques described above can also be used for expression of native sequences encoding human or murine antibodies. This approach is particularly advantageous for expression of human antibodies that are isolated as unstable cell lines. In another embodiment of the invention, fragments of the intact antibodies described above are provided. Typically, these fragments compete with the intact antibody from which they were derived for specific binding to the ReproSA- 1 polypeptide, and bind with an affinity of at least 107, 108, 109 M"1, or 1010 M"1. Antibody fragments include separate heavy chains, light chains Fab, Fab' F(ab')2, Fabc, and Fv. Fragments can be produced by enzymic or chemical separation of intact immunoglobulins. For example, a F(ab')2 fragment can be obtained from an IgG molecule by proteolytic digestion with pepsin at pH 3.0-3.5 using standard methods such as those described in Harlow, supra. Fab fragments may be obtained from F(ab')2 fragments by limited reduction, or from whole antibody by digestion with papain in the presence of reducing agents. Fragments can also be produced by recombinant DNA techniques. Segments of nucleic acids encoding selected fragments are produced by digestion of full-length coding sequences with restriction enzymes, or by de novo synthesis. Often fragments are expressed in the form of phage-coat fusion proteins. The antibodies of the present invention can be used with or without modification. Frequently, the antibodies will be labeled by joining, either covalently or non- covalently, a substance which provides for a detectable signal. Such labels include those that are well known in the art, e.g., radioactive, fluorescent, or bioactive (e.g., enzymatic) labels. As labeled binding entities, the antibodies of the invention may be particularly useful in, e.g., diagnostic applications, for detecting the presence of sperm containing wild-type (i.e., normal) ReproSA- 1 protein. Sperm antibodies assays are well known, see, e.g., Rajah SV, et al., 1992, Fertility and Sterility 57:1300.
In some cases, for example when antibodies are used in a spermicidal medicament (described infra), preferred antibodies are those that interfere with sperm mobility. This will be a property of many or most anti-ReproSA-1 antibodies. An antibody (or antisera) can be tested for its ability to immobilize sperm using methods well known in the art (e.g. sperm agglutination). See, e.g., Harlow, supra, and Isojima, 1992, Am. J. Obstret Gynecol 112: 199; Fichorova, 1991 , Journal of Reproductive Immunology 20:1; World Health Organization - WHO Laboratory Manual for the examination of human semen and semen-cervical mucus interaction, 2nd ed. (1987) The Press Syndicate of the University, Cambridge UK, 3-26, all of which are incorporated herein by reference in their entirety and for all purposes. Such immobilizing antibodies can be referred to as "spermicidal" antibodies. Also useful are anti-idiotype antibodies which can be isolated by the above procedures. Anti-idiotypic antibodies may be prepared by, for example, immunization of an animal with the primary antibody (i.e., anti-ReproSA-1 antibodies or ReproSA- 1 -binding fragments thereof). For anti-ReproSA-1 antibodies, anti-idiotype antibodies whose binding to the primary antibody is inhibited by a ReproSA- 1 polypeptide or fragments thereof are selected. Because both the anti-idiotypic antibody and the ReproSA- 1 polypeptide or fragments thereof bind the primary immunoglobulin, the anti-idiotypic immunoglobulin may represent the "internal image" of an epitope and thus may substitute for the ReproSA- 1 polypeptide in assays or may be used to bind (i.e., inactivate) anti-ReproSA-1 antibodies in a patient. Many of the immunoglobulins described above can undergo non-critical amino-acid substitutions, additions or deletions in both the variable and constant regions without loss of binding specificity or effector functions, or intolerable reduction of binding affinity (i.e., below about 107 M"1). Usually, immunoglobulins incorporating such alterations exhibit substantial sequence identity to a reference immunoglobulin from which they were derived. Occasionally, a mutated immunoglobulin can be selected having the same specificity and increased affinity compared with a reference immunoglobulin from which it was derived. Phage-display technology offers powerful techniques for selecting such immunoglobulins. See, e.g., Dower et al., WO 91/17271 McCafferty et al, WO 92/01047; and Huse, WO 92/06204.
The invention also provides hybrid antibodies that share the specificity of antibodies against a ReproSA- 1 polypeptide but are also capable of specific binding to a second moiety. In hybrid antibodies, one heavy and light chain pair is usually from an anti- complex antibody and the other pair from an antibody raised against another epitope. This results in the property of multi-functional valency, i.e., ability to bind at least two different epitopes simultaneously, where at least one epitope is the epitope to which the anti-complex antibody binds. Such hybrids can be formed by fusion of hybridomas producing the respective component antibodies, or by recombinant techniques.
Immunoglobulins can also be fused to functional regions from other genes (e.g., enzymes) to produce fusion proteins (e.g., immunotoxins) having novel properties.
The antibodies of the invention may be of any isotype, e.g., IgM, IgD, IgG, IgA, and IgE, with IgG, IgA and IgM most referred. Secreted IgG, IgD, and IgE isotypes are typically found in monomeric form. Secreted IgM isotype is found in pentameric form; secreted IgA can be found in both monomeric and dimeric form. See generally, Paul, ed., 1989, FUNDAMENTAL IMMUNOLOGY 2nd ed. Raven Press, N.Y., Ch. 7 (incorporated by reference in its entirety for all purposes). Humanized antibodies may comprise sequences from more than one class or isotype. Methods for purification of antibodies are well known. As used herein, a composition comprising an antibody (e.g. an anti-ReproSA-1 antibody) is substantially pure when at least about 80%, more often at least about 90%, even more often at least about 95%, most often at least about 99% or more of the immunoglobulin molecules present in a preparation specifically bind a ReproSA- 1 polypeptide. The antibodies of the inventions are useful in a number of applications, e.g. , as probes in immunoassays in diagnostic or therapeutic applications, or in contraceptive compositions. These applications are discussed in greater detail, infra. IV. Nucleic Acids and Cell Lines
In another aspect, the present invention provides nucleic acids which encode the polypeptides of the invention, as well as expression vectors that include these nucleic acids, and cell lines and organisms that are capable of expressing these nucleic acids. In one aspect, the nucleic acid composition has the sequence of the ReproSA-
1 cDNA shown in Figure 1 , or a fragment thereof. Such fragments are useful, for example, as probes, primers, antisense reagents, and the like, and will generally comprise a segment of from about 8 nucleotides to about 500 nucleotide bases or basepairs, more often between about 10 and about 250 nucleotides, still more often between about 12 and about 150 nucleotides, even more often between about 15 and about 50 nucleotides. In one embodiment, the nucleic acids will comprise a segment having more than about 20 contiguous nucleotides from the nucleotide sequences shown in Figure 1. Also provided are substantially similar nucleic acid sequences and allelic variations of the above described nucleic acids. Also included are chemically modified and substituted nucleic acids, e.g., those which incorporate modified nucleotide bases or which incorporate a labeling group.
Substantial identity, in the nucleic acid context, means that the segments, or their complementary strands, when compared, are the same when properly aligned, with the appropriate nucleotide insertions or deletions, in at least about 70%, more typically, at least about 80%), usually, at least about 90%), and more usually, at least about 95% to 98% of the nucleotides. Algorithms suitable for calculating the degree of identity include that of Smith and Waterman, supra, Needleman and Wunsch, supra, Lipman, supra, or by computerized implementations of these algorithms (e.g., FASTN in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, WI). Another example of algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al, J. Mol. Biol.
215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences ( ee, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
Alternatively, substantial identity exists when the segments will hybridize under stringent hybridization conditions to a strand, or its complement, typically using a sequence of at least about 50 contiguous nucleotides derived from the nucleotide sequences shown in Figure 1. Stringent hybridization exists when hybridization occurs under hybridization and wash conditions that are selected to be about 5°C, more often about 10°C lower than the thermal melting point (TM) for the specific sequence at a defined ionic strength and pH. The TM is the temperature (under defined ionic strength and pH) at which
50% of the target sequence hybridizes to a perfectly matched probe. Typically, stringent conditions will be those in which the salt concentration is at least about 0.02 molar at pH 7 and the temperature is at least about 60 °C. As other factors may significantly affect the stringency of hybridization, including, among others, base composition and size of the complementary strands, the presence of organic solvents and the extent of base mismatching, the combination of parameters is more important than the absolute measure of any one. Hybridization conditions and parameters, and formulae for calculating TM are found in Sambrook, supra.
In preferred embodiments, the nucleic acid compositions of the invention encode a polypeptide that is specifically immunoreactive with an antibody that specifically binds the polypeptide shown in Figure 1 [SEQ ID NO:2].
As used herein, the terms "nucleic acids" and "polynucleotides" are used interchangeably and include RNA, cDNA, genomic DNA, synthetic forms and mixed polymers, both sense and antisense strands. The nucleic acids described herein include self replicating plasmids and infectious polymers of DNA or RNA. The nucleic acids of the present invention may be present in whole cells, cell lysates or in partially pure or substantially pure or isolated form. When referring to nucleic acids, the terms "substantially pure" or "isolated" generally refer to the nucleic acid separated from contaminants with which it is generally associated, e.g., lipids, proteins and other nucleic acids. The substantially pure or isolated nucleic acids of the present invention will be greater than about 50% pure. Typically, these nucleic acids will be more than about 60% pure, more typically, from about 75% to about 90% pure and preferably from about 95% to about 98%o pure.
Typically, the nucleic acids of the present invention will be used in expression vectors for the preparation of the polypeptides of the present invention, namely those polypeptides which are derived from or related to ReproSA- 1. The phrase "expression vector" generally refers to nucleotide sequences that are capable of affecting expression of a structural gene in hosts compatible with such sequences. These expression vectors typically include at least suitable promoter sequences and optionally, transcription termination signals. Commonly, expression vectors will contain selection markers, e.g., tetracycline or neomycin, to permit detection of those cells transformed with the desired DNA sequences (see, e.g.,
U.S. Patent 4,704,362). Additional factors necessary or helpful in effecting expression may also be used as described herein. DNA encoding the ReproSA- 1 polypeptides of the present invention will typically be incorporated into DNA constructs capable of introduction into and expression in an in vitro cell culture. Often, the nucleic acids of the present invention may be used to produce a suitable recombinant host cell. (As used herein, a cell or cell line that contains a nucleic acid sequence made using recombinant techniques can be called a "recombinant cell" or "recombinant cell line"). Specifically, DNA constructs will be suitable for replication and expression in a prokaryotic host, such as bacteria, e.g., E. coli, or may be introduced into a cultured mammalian, plant, insect, e.g., Sf9, yeast, fungi or other eukaryotic cell line.
E. coli is one prokaryotic host useful particularly for cloning the polynucleotides of the present invention. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. The promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation. Other microbes, such as yeast, may also be used for expression.
Saccharomyces is a preferred host, with suitable vectors having expression control sequences, such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired.
In addition to microorganisms, mammalian tissue cell culture may also be used to express and produce the polypeptides of the present invention (see Winnacker, From Genes to Clones (VCH Publishers, N.Y., N.Y., 1987). Eukaryotic cells are actually preferred, because a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed. Preferred suitable host cells for expressing nucleic acids encoding the immunoglobulins of the invention include: monkey kidney CV1 line transformed by S V40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293)
(Graham et al, J Gen. Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary-cells-DHFR (CHO, Urlaub and Chasin, Proc. Natl Acad. Sci. (USA) 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); and, TRI cells (Mather, et al., Annals N.Y. Acad. Sci. 383:44-46 (1982)); baculovirus cells.
DNA constructs prepared for introduction into a particular host, e.g., insect or bacteria, will typically include a replication system recognized by the host, the intended DNA segment encoding the desired polypeptide, and transcriptional and translational initiation and termination regulatory sequences operably linked to the polypeptide encoding segment. A DNA segment is operably linked when it is placed into a functional relationship with another DNA segment. For example, a promoter or enhancer is operably linked to a coding sequence if it stimulates the transcription of the sequence. DNA for a signal sequence is operably linked to DNA encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide. Generally, DNA sequences that are operably linked are contiguous, and in the case of a signal sequence both contiguous and in reading phase. However, enhancers need not be contiguous with the coding sequences whose transcription they control. Linking is accomplished by ligation at convenient restriction sites or at adapters or linkers inserted in lieu thereof. The selection of an appropriate promoter sequence will generally depend upon the host cell selected for the expression of the DNA segment. Examples of suitable promoter sequences include prokaryotic, and eukaryotic promoters well known in the art. See, e.g., Sambrook, supra. The transcriptional regulatory sequences will typically include a heterologous enhancer or promoter (i.e., not naturally operably linked to a human ReproSA- 1 gene) which is recognized by the host. The selection of an appropriate promoter will depend upon the host, but promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters are known and available. See Sambrook, supra. For eukaryotic cells, the control sequences will include a promoter and preferably an enhancer derived from immunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence, and may include splice donor and acceptor sequences.
Conveniently available expression vectors which include the replication system and transcriptional and translational regulatory sequences together with the insertion site for the polypeptide encoding segment may be employed. Examples of workable combinations of cell lines and expression vectors are described in, e.g., Sambrook, supra, Metzger et al., 1988, Nature 334:31-36, and Gossen et al, 1994, Curr. Opin. Biotech 5:516. For example, suitable expression vectors may be expressed in, e.g., insect cells, e.g., Sf9 cells), mammalian cells, (e.g., CHO cells), and bacterial cells (e.g., E. coli).
Where an insect cell line is selected as the host cell of choice to express the polypeptide, the cDNA encoding the polypeptides of the invention may be cloned into an appropriate baculovirus expression vector, e.g., pBacPAK8 vector (Clontech, Palo Alto,
CA). The recombinant baculovirus may then be used to transfect a suitable insect host cell, e.g., Spodoptera frugiperda (Sf9) cells, which may then express the polypeptide. See, e.g., Morrison et al., Cell 58:649-657 (1989), M.D. Summers and G.E. Smith, A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Station, College Station, Texas (1987).
There are various methods of isolating the nucleic acids which encode the polypeptides of the present invention. Typically, the DNA is isolated from a genomic or cDNA library using labeled oligonucieotide probes specific for sequences in the desired DNA. Restriction endonuclease digestion of genomic DNA or cDNA containing the appropriate genes can be used to isolate the DNA encoding the polypeptides of the invention.
From the nucleotide sequence given in Figure 1, a panel of restriction endonucleases can be constructed to give cleavage of the DNA in desired regions, i.e., to obtain segments which encode biologically active polypeptides or fragments of the invention. Following restriction endonuclease digestion, DNA encoding the polypeptides of the invention is identified by its ability to hybridize with a nucleic acid probe in, for example, a Southern blot format. These regions are then isolated using standard methods. See, e.g., Sambrook, supra.
The nucleic acids of the invention are also useful for isolating and expressing full-length ReproSA- 1 genes from humans and non-human animals, using well known methods, e.g., Sambrook, supra, and Ausubel, supra. For example, a nucleic acid probe comprising all or some of the ReproSA- 1 cDNA sequence (typically a restriction fragment from the 5* end of the cDNA clone) is labeled and used to screen a genomic or cDNA library by hybridizing at high stringency. The cDNA library may be oligo-dT or random primed. The cDNA or genomic clones that hybridize are isolated and analyzed by restricting mapping, Southern hybridization, and DNA sequencing using methods that are well known in the art. A polynucleotide encoding a full-length ReproSA- 1 polypeptide is cloned into an expression vector or synthesizes using sequence obtained from multiple clones, and expressed, as is described supra. In other embodiments, the invention provides antisense, triplex, and ribozyme reagents that target ReproSA- 1 nucleic acids. Thus, the present invention provides antisense oligo- and polynucleotides (e.g., DNA, RNA, PNA or the like) that can be used to inhibit expression of the ReproSA- 1 gene, e.g., for contraceptive purposes. Antisense methods are generally well known in the art and may be carried out using modified ReproSA- 1 polynucleotides (see, e.g., PCT publication WO 94/12633, and Nielsen et al., 1991, Science 254:1497; OLIGONUCLEOTIDES AND ANALOGUES, A PRACTICAL APPROACH, edited by F. Eckstein, IRL Press at Oxford University Press (1991); ANTISENSE RESEARCH AND APPLICATIONS (1993, CRC Press) all incoφorated by reference. In an other embodiment, the invention provides oligo- and polynucleotides that bind to double-stranded or duplex ReproSA- 1 nucleic acids (e.g., in a folded region of the ReproSA- 1 RNA or in the ReproSA- 1 gene), forming a triple helix-containing, or "triplex" nucleic acid. Triple helix formation results in inhibition of ReproSA- 1 expression by, for example, preventing transcription of the ReproSA-1 gene, (see, e.g., Gee et al., in Huber and Carr, 1994, MOLECULAR AND IMMUNOLOGIC APPROACHES, Futura Publishing Co, Mt Kisco NY and Rininsland et al., 1997, Proc. Natl. Acad. Sci. USA 94:5854; Ferrin and Camerini-Otero, 1991, Science 354:1494; Ramdas et al, 1989, J. Biol. Chem. 264:17395; Strobel et al., 1991, Science; each of which is incoφorated herein by reference).
In an other embodiment, the invention provides ribozymes useful for inhibition of ReproSA- 1 expression or activity. The ribozymes of the invention bind and specifically cleave and inactivate ReproSA- 1 mRNA. Useful ribozymes can comprise 5'- and 3'-terminal sequences complementary to the ReproSA- 1 mRNA and can be engineered by one of skill on the basis of the ReproSA- 1 mRNA sequence disclosed herein. See, e.g., PCT publications WO 94/02595 and WO 93/23569).
V. Methods of Use
The compositions of the invention have a number of uses including, but not limited to, use in contraceptive vaccines, as topical contraceptive agents (e.g., spermicidal agents), and for diagnosis and treatment of human diseases such as infertility. A. Vaccines
In one aspect, the invention provides methods of contraception by immunization, i.e., administering an effective amount of a vaccine comprising at least one ReproSA- 1 polypeptide or polynucleotide. An effective amount of a contraceptive vaccine is an amount sufficient to induce an immune response, e.g. , production in a subject (e.g, a human or non-human animal) of antibodies that bind to a sperm ReproSA- 1 polypeptide (i.e., an anti-sperm immune response). Typically, administration of a ReproSA- 1 vaccine results in an elevation of anti-ReproSA-1 antibodies to atiter of at least about three times the antibody titer prior to vaccination, more preferably the elevation is at least about 50-fold or at least about 100-fold. In a preferred embodiment the isotype of the antibodies is IgA. In another preferred embodiment, the anti-ReproSA-1 antibodies are found in mucosal secretions, e.g., mucosal secretions of the reproductive tract, e.g., the vaginal mucosa. Appearance of anti-ReproSA-1 antibodies can be correlated with reduced fertility, i.e., a contraceptive effect. Usually, administration of a effective amount of a contraceptive vaccine results in inhibition of fertilization by at least about fifty percent, more often by at least about 75% relative to untreated controls. Protocols for determining the efficacy of a contraceptive are well known, and can be carried out in humans or in non-human animals. Contraceptive vaccines are described generally in Aitken et αl, 1993, Brit. Med. Bull. 49:88- 99. Although it is expected that the primary use of the vaccines of the invention will be in humans, they may also be used as contraceptives in nonhuman animals that produce sperm having a ReproSA- 1 protein.
The vaccines of the invention may be polypeptide or nucleic acid based. Thus, in one aspect, vaccines are produced using recombinantly expressed peptides or polypeptides, or from synthetic peptides or mimetics. Usually, the immunogens of the invention will include a subsequence of a ReproSA- 1 polypeptide. In one embodiment, the vaccine will comprise peptides or polypeptides derived from the ReproSA- 1 sequence shown in Figure 1. Typically, immunogenic ReproSA- 1 peptides will range in size from about 8 amino acids in length to about 509 amino acids in length. More typically, the active fragments will be from about 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acids in length to about 50 amino acids in length. Particularly preferred ReproSA- 1 polypeptide sequences for use in vaccines include MSTESSEDLQVKSKKAIKNILQKCTY [SEQ ID NO:5], LPHDSKARRLFVTSGGLKKVQEIKAEPG [SEQ ID NO:6],
SKGVPQLSVCLSEEPEDHIKAA [SEQ ID NO:7], YPEEIVRYYSPGYSDTLLQRVDS [SEQ ID NO:8],MSQRQVLQVFEQYQKARTQFVQMVAELATRP [SEQ ID NO:9], YNDDLAEAVVKCDILPQLVYSLAEQNRFYKKAAAFVLRAVGKHSPQLA [SEQ ID NO: 10], LLVLCIQEPEIALKRIAAS ALSDIAKHSPELAQTVVDAGAVAHLAQ
MILNPDAKLKHQILSALSQVSKHSVDLAEMVVEAEIF [SEQ ID NO: 11], TCLKDKDEYVKKNASTLIREIAKHTPELSQLV [SEQ ID NO: 12], and immunogenic fragments thereof (e.g., immunogenic fragments comprising at least 8, 9, 10, 11, or 12 contiguous amino acids). Typically, a vaccine will include from about 1 to about 50 micrograms of antigen or antigenic protein or peptide. More preferably, the amount of protein is from about 15 to about 45 micrograms. Typically, the vaccine is formulated so that a dose includes about 0.5 milliliters. The vaccine may be administered by any route known in the art including oral, parenteral (e.g., subcutaneous or intramuscular), intrarectal, or vaginal. The treatment may consist of a single dose of vaccine or a plurality of doses over a period of time.
The antigen may be combined or mixed with various solutions and omer compounds as is known in the art, as well as with other antigens. For example, it may be administered in any pharmacologically acceptable carrier including water, saline or buffered vehicles, with or without various adjuvants or immunodiluting agents. Examples of such adjuvants or agents include aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X, Corynebacterium parvum (Propionibacterium acnes), Bordetella pertussis, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants. Such adjuvants are available commercially from various sources, for example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) or Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Michigan). Other suitable adjuvants are Amphigen (oil-in-water), Alhydrogel (aluminum hydroxide), or a mixture of Amphigen and Alhydrogel.
These solutions are sterile and generally free of particulate matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH-adjusting and buffering agents, tonicity adjusting agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Sciences, 17th Ed., Mack Publishing Company, Easton, Pennsylvania (1985), which is incoφorated herein by reference.
The proportion of antigen and adjuvant can be varied over a broad range so long as both are present in effective amounts. For example, aluminum hydroxide can be present in an amount of about 0.5% of the vaccine mixture (Al2O3 basis). On a per-dose basis, the amount of the antigen can range from about 0.1 μg to about 100 μg protein per patient. A preferable range is from about 1 μg to about 50 μg per dose. A more preferred range is about 15 μg to about 45 μg. A suitable dose size is about 0.5 ml. Accordingly, a dose for intramuscular injection, for example, would comprise 0.5 ml containing 45 μg of antigen in admixture with 0.5% aluminum hydroxide. After formulation, the vaccine may be incoφorated into a sterile container which is then sealed and stored at a low temperature, for example 4°C, or it may be freeze-dried. Lyophilization permits long-term storage in a stabilized form. There are a number of strategies for amplifying an antigen's effectiveness, particularly as related to the art of vaccines. For example, cyclization or circularization of a peptide can increase the peptide's antigenic and immunogenic potency. See U.S. Pat. No. 5,001,049 which is incoφorated by reference herein. More conventionally, an antigen can be conjugated to a suitable carrier, usually a protein molecule. This procedure has several facets. It can allow multiple copies of an antigen, such as a peptide, to be conjugated to a single larger carrier molecule. Additionally, the carrier may possess properties which facilitate transport, binding, absoφtion or transfer of the antigen. The conjugation between a peptide and a carrier can be accomplished using one of the methods known in the art. Specifically, the conjugation can use bifunctional cross-linkers as binding agents as detailed, for example, by Means and Feeney, Bioconjugate Chem. 1:2-12.
Those of skill will readily recognize that it is only necessary to expose a patient to appropriate epitopes in order to elicit effective contraception. The epitopes are typically segments of amino acids which are a small portion of the whole protein. Using recombinant genetics, it is routine to alter a natural protein's primary structure to create derivatives embracing epitopes that are identical to or substantially the same as (immunologically equivalent to) the naturally occurring epitopes. Such derivatives may include peptide fragments, amino acid substitutions, amino acid deletions and amino acid additions within the ReproSA- 1 amino acid sequence. For example, it is known in the protein art that certain amino acid residues can be substituted with amino acids of similar size and polarity without an undue effect upon the biological activity of the protein. Thus, modifications should generally preserve conformation to produce a protective immune. Epitopes may be conformational (i.e. , discontinuous). That is, they may be formed from amino acids encoded by noncontiguous parts of a primary sequence (e.g., SEQ ID NO:2) that have been brought together by protein folding. It is understood by those of skill that conformational epitopes can be formed using recombinant techniques. For example, a polynucleotide with sequences that encode the amino acid residues that form two parts of a conformational epitope, and other sequences that allow the first part of a conformational epitope and the second part of a conformational epitope to associate in the native conformation of the ReproSA- 1 protein can be constructed and used, for example, as a vaccine or for purification of anti-ReproSA-1 antibody.
It will often be desirable to design a contraceptive vaccine that results in high levels of mucosal immunity so that sperm introduced into the mucosa of the female reproductive tract will be effectively immobilized. In some embodiments the composition of the vaccine and/or the method of administration (e.g., parenteral, oral, intravaginal, intrarectal immunization or a combination) are chosen so as to generate persisting high levels of anti-ReproSA-1 IgA (e.g., soluble IgA). Methods are known in the art for designing vaccines that induce secretory immunity and are described in, e.g. , Local Immunity in reproductive tissues, Griffin and Johnson, eds. Oxford Univ. Press, Oxford 1993, which is incoφorated herein by reference, e.g., at pages 488-89 and chapters 3, 4, and 28, which are incoφorated in their entirety and for all puφoses.
The nucleic acids of the present invention may be used as vaccines for the prevention of conception (i. e. , contraceptive vaccines). For example, plasmid DNA comprising the nucleic acids of the present invention may be directly administered to a patient, e.g., by injection into muscle. Expression of this "naked" DNA will have effects similar to the injection of the actual polypeptides. Specifically, the patient's immune response to the presence of the proteins expressed from the DNA, will result in the production of antibodies to that protein. See Sedegah et al., Proc. Nat'l Acad. Sci. (1994) 91:9866-9870 and Ulmer et α/., 1993, Science, 259:1745; Montgomery et al, 1994, Curr. Opin. Biotech. 5:505, all of which are incoφorated in their entirety and for all puφoses.
The isolated nucleic acid sequence coding for ReproSA- 1 or its homologous polypeptides can also be used to transform viruses which transfect host cells in the susceptible organism. Live attenuated viruses, such as vaccinia or adenovirus, are convenient alternatives to conventional vaccines because they are inexpensive to produce and are easily transported and administered. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Patent No. 4,722,848, incoφorated herein by reference and Ulaeto and Hruby, 1994, Curr. Opin. Biotech. 5:501 incoφorated herein by reference.
Suitable viruses for use in the present invention include, but are not limited to, pox viruses, such as, canarypox and cowpox viruses, and vaccinia viruses, alpha viruses, adenoviruses, and other animal viruses. The recombinant viruses can be produced by methods well known in the art: for example, using homologous recombination or ligating two plasmids together. A recombinant canarypox or cowpox virus can be made, for example, by inserting the gene encoding an immunologically active segment of ReproSA- 1 into a plasmid so that it is flanked with viral sequences on both sides. The gene is then inserted into the virus genome through homologous recombination.
The recombinant virus of the present invention can be used to induce anti- ReproSA-1 polypeptide antibodies in humans and other mammals. In addition, the recombinant virus can be used to produce the ReproSA- 1 polypeptides by infecting host cells which in turn express the polypeptide.
B) Topical Contraceptive Agents
The compositions of the invention also find use as spermicidal agents for use in contraception. ReproSA- 1 binding agents, e.g., anti-ReproSA-1 antibodies (i.e., monoclonal antibodies and ReproSA- 1 -binding fragments thereof) are used to bind to sperm, resulting in immobilization and/or agglutination and thus preventing fertilization. In one embodiment, the antibody composition is applied vaginally in a suitable carrier, such as a pharmaceutically acceptable carrier, e.g., a gel. In an alternative embodiment, the composition can be applied to a contraceptive device, such as a condom or diaphragm. Similarly, ReproSA- 1 polypeptides are useful as topical contraceptives, e.g., via protein- protein interactions with endogenous (sperm) ReproSA- 1 protein, e.g., dominant-negative effects.
C) Fertilization Enhancing Agents
ReproSA-1 polypeptides are useful as agents for enhancing fertilization, e.g. by binding to anti-ReproSA-1 antibodies present during a potential fertilization event. The peptides bind anti-ReproSA-1 antibodies present and block the binding of the antibodies to sperm, enabling the sperm to participate in the fertilization event. Thus, in one embodiment of the invention, an effective amount of ReproSA- 1 polypeptide is administered to a site, either in vitro or in vivo, at which fertilization is attempted. An effective amount of ReproSA- 1 polypeptide is defined as the amount of ReproSA- 1 polypeptide required to bind to more than about 50% of the total anti-ReproSA-1 antibodies present during a potential fertilization event, preferably, more than about 75% of the ReproSA- 1 antibodies present, more preferably greater than about 90% or about 95% of the anti-ReproSA-1 antibodies present. In one embodiment, the anti-ReproSA-1 antibody-binding polypeptide composition is applied vaginally in a suitable carrier, e.g., a pharmaceutically acceptable solution or gel. In an alternative embodiment, the composition is contacted with, e.g., added to, a sperm preparation prior to performing in vitro fertilization.
D) Diagnostic Methods and Kits
The compositions of the invention (e.g., antibodies, polynucleotides and polypeptides) are used diagnostically in a number of contexts. In one embodiment, for example, they are used to determine the presence of abnormal ReproSA- 1 genes, especially in men, e.g., men suffering from infertility. In a second embodiment, they are useful in diagnostic assays to detect serum, seminal, mucosal and/or follicular antibodies to ReproSA- 1 proteins (and thus to sperm) in order to diagnose immunological infertility, e.g., in patients having difficulty conceiving. In a third embodiment, the compositions of the invention are used in an assay to quantitate titers of levels of antibodies to the antigen in contraceptively vaccinated individuals. The polypeptides of the invention, especially monoclonal antibodies, are useful for assaying the presence of anti-ReproSA-1 antibodies in a biological sample (e.g., serum, cervical or vaginal fluid, or semen). The presence of such antibodies will aid in diagnosing immunological infertility. Immunological infertility has heretofore been a diagnosis of exclusion, and thus surrounded by uncertainty. The present invention allows a more certain diagnosis to be made. Once a male or female patient is diagnosed as expressing anti-ReproSA-1 antibodies, appropriate therapeutic measures may be taken, such as administration of appropriate pharmaceutical agents, e.g., immunosuppressive agents to increase the chance of conception. A number of suitable assays are known to those of skill for assaying the presence of antibodies to a known antigen (e.g., ReproSA- 1), some of which are described in Harlow, supra. In one suitable assay, the polypeptide of the invention is coated on a solid support, such as a microtiter plate. A biological sample from a patient, e.g., including but not limited to serum or other blood fraction, semen, saliva, or cervical secretions is applied to the solid support that has been coated with polypeptide and anti-ReproSA-1 antibodies, if present, are allowed to bind the polypeptide. Such antibodies can be detected by well known means, for example using anti-human immunoglobulin, e.g., IgG, conjugated to a detectable label. These and similar methods will also be useful for quantitating titers of anti-ReproSA- 1 antibodies in contraceptively vaccinated individuals. The novel polynucleotide and antibody reagents of the invention are also used for determining the presence of mutant or alternate allelic forms of the ReproSA- 1 genes or proteins, especially in men. Mutant or abnormal ReproSA- 1 genes or proteins may result in infertility in men carrying the genes or expressing the proteins. According to this invention, a gene in a patient is compared to the sequence of the ReproSA- 1 gene provided in SEQ ID NO: 1. A difference in the sequences is indicative of a mutation (including alternative alleles) in the patient's gene. Methods for analysis and comparison of nucleic acid sequences are well known. Preferred methods include use of the polymerase chain reaction (PCR). For example, primers designed using the sequence provided in Figure 1 [SEQ ID NO:l] can be used to amplify (e.g., by PCR) the ReproSA-1 gene from a patient, or a fragment thereof. The sequence of the ReproSA- 1 gene from the patient can then be determined e.g., by sequencing and compared to SEQ ID NO:l. Alternatively, differences between a patient's gene and SEQ ID NO:l can be determined by hybridization, for example, using a high density oligonucieotide array (e.g., Pease et al, 1994, Proc. Natl. Acad. Sci. USA 91 :5022-5026) or often means known in the art. It will be recognized that the mutations of most interest are those other than "silent mutations." A silent mutation in a nucleic acid sequence is one that does not change the identity of an amino acid in an encoded polypeptide. In contrast, missense mutations, deletions, insertions, and frameshifts that result in a deviation in an encoded ReproSA- 1 amino acid sequence from that of SEQ ID NO:2 are of particular interest.
Those of skill will recognize that the compositions of the invention have numerous additional uses not listed above. For example, the antibodies of the invention may be used to trap sperm, for example, to aid in counting sperm or for collecting samples in forensic investigations.
The invention also includes kits for use by physicians, clinical laboratories or patients to carry out the assays and diagnoses described above. Typically the kit will contain one or more of the following in a container: oligonucieotide primers or probes corresponding to the ReproSA-1 cDNA sequence, anti-ReproSA-1 antibodies, ReproSA-1 polypeptides or fragments, optionally coated in a solid surface (such as a slide, multiple well plate, or test tube), a ReproSA- 1 polynucleotide (e.g., for use as positive controls in assays), and tubes. Instructions for carrying out the methods of the invention, and calibration curves may also be included.
E) Identification of ReproSA- 1 Associated Proteins
In a further aspect, the present invention provides methods for identifying and cloning proteins that interact with the ReproSA- 1 polypeptide or a region thereof (e.g., an armadillo repeat region). In one embodiment, these interacting proteins are identified using a two hybrid screen system according to methods well known in the art (see, e.g.., Chien et al. , 1991, Proc. Natl. Acad. Sci. USA 88:9578, Ausubel et al, supra, at Ch. 20; Fields and Song, 1989, Nature 340:245; U.S. Patent Nos: 5,283,173 and 5,468,614, which are incoφorated herein by reference in their entirety and for all puφoses). Briefly, the two- hybrid screen identifies protein-protein interactions in vivo through reconstitution of a transcriptional activator, e.g., the yeast Gal4 transcription protein (see, Fields and Song, 1989, Nature 340:245). The method is based on the properties of the yeast Gal4 protein, which consists of separable domains responsible for DNA-binding and transcriptional activation. Polynucleotides, usually expression vectors, encoding two hybrid proteins are constructed. One polynucleotide comprises the yeast Gal4 DNA-binding domain fused to a polypeptide sequence of a protein to be tested for a ReproSA- 1 interaction. Alternatively the yeast Gal4 DNA-binding domain is fused to cDNAs from a human cell, thus creating a library of human proteins fused to the Gal4 DNA binding domain for screening for
ReproSA- 1 associated proteins. The other polynucleotide comprises the Gal4 activation domain fused to an ReproSA- 1 polypeptide sequence. The constructs are introduced into a yeast host cell. Upon expression, intermolecular binding between the ReproSA- 1 polypeptide and the test protein can reconstitute the Gal4 DNA-binding domain with the Gal4 activation domain. This leads to the transcriptional activation of a reporter gene (e.g., lacZ, HIS3) operably linked to a Gal4 binding site. By selecting for, or by assaying the reporter, gene colonies of cells that contain a ReproSA-1 interacting protein can be identified.
Thus, in one embodiment, the invention provides a method for detecting an interaction between a first polypeptide and a second polypeptide, where either the first polypeptide or the second polypeptide is a ReproSA- 1 protein or fragment, and the other polypeptide is a polypeptide sequence to be tested for interaction with the ReproSA- 1 protein or fragment. The method involves providing a host cell and two chimeric genes that are capable of being expressed in the host cell. The host cell, which may be yeast, bacterial, or of another type, contains a detectable gene that expresses a detectable protein, when the detectable gene is activated by an amino acid sequence that includes a transcriptional activation domain. The first chimeric gene has a DNA sequence that encodes a first hybrid protein. The first hybrid protein contains a DNA-binding domain that recognizes a binding site on the detectable gene in the host cell, and the first polypeptide. The second chimeric gene has a DNA sequence that encodes a second hybrid protein, which includes the transcriptional activation domain and the second polypeptide. The interaction between the first polypeptide and the second polypeptide in the host cell causes the transcriptional activation domain to activate transcription of the detectable gene when the first chimeric gene and the second chimeric gene are introduced into the host cell and the host cell is subjected to conditions under which the first hybrid protein and the second hybrid protein are expressed. The interaction is then detected by determining whether the detectable gene has been expressed to a degree greater than expression in the absence of an interaction between the first polypeptide and the second polypeptide.
Those of skill will appreciate that there are numerous variations of the 2- hybrid screen, e.g., the LexA system (Bartel et al, 1993, in Cellular Interactions in Development: A Practical Approach Ed. Hartley, D.A. Oxford Univ. Press pp. 153-79). Additional variations include a three-hybrid system (see, e.g., Zhang et al, 1996, Anal. Biochem. 242:68; Licitra et al, 1996, Proc. Natl. Acad. Sci. USA 93:12817 and the E. co/7/BCCP interactive screening system (Germino et al. , 1993, Proc. Natl. Acad. Sci.. 90:933; Guarente, 1993, Proc. Natl. Acad. Sci. 90:1639).
EXAMPLES The following examples are for illustration only and are not intended to limit the invention in any way.
Example I CLONING REPRO-S A- l cDNA
Testis poly(A)+ RNA isolated from a pool of four Caucasian male testes, aged 22-31 years (Clonetech, Inc., Palo Alto, CA) was used as a template for first strand cDNA synthesis. The poly(A)+ RNA ranged in size from 0.2 to 10 Kb as determined by denaturing gel electrophoresis (Technical Data Sheet and communication for Clontech product: Human Testis Poly(A)+ RNA). An oligo-dT primer containing an internal, protected Xhol site was annealed in the presence of a nucleotide mixture containing 5 -methyl dCTP, and extended by MMTV reverse transcriptase (Stratagene ZAP Express cDNA Synthesis Technical Manual revision #065001, 1996).
Second strand synthesis of the testis cDNA/RNA hybrid was completed by the addition of RNase H, DNA polymerase I, and dNTPs to the first strand synthesis reaction. Pfu DNA polymerase was used to blunt-end the double stranded testis cDNA followed by the ligation of EcoRI adapters. The cDNA was kinased and digested with Xhol and EcoRI before size fractionation on Sephacryl S-500 columns. The size fractionated cDNA was recovered and then quantified on ethidium bromide containing plates against a set of serially-diluted DNA standards. The cDNA contained in the first two column fractionations was directionally ligated, in the sense orientation, to J^oT/EcoRT-digested ZAP Express phage vector arms. Initially, approximately 26 ng (per fraction) of the cDNA was ligated and packaged into bacteriophage particles. Subsequently, the approximately 100 ng remaining cDNA in fractions 1 and 2 was packaged into bacteriophage particles using several reaction of the lambda phage packaging extract (Stratagene).
After packaging, the primary human testis library was titered by infection of the XL1 Blue MRF' host strain. The ratio of recombinanfcnonrecombinant phage was determined by plating infected XL1 Blue MRF' in the presence of IPTG and XGal. The number of blue (non-recombinant) or white (recombinant) plaques were quantified using a Manostat colony counter. Ninety-eight and one-half (98.5) percent of the phage in the human testis cDNA library were recombinant and the primary phage library base consisted of 9.97 x 106 independent clones. The human testes cDNA library was amplified once and re-titered as above. The titer of the amplified library was 6 x 108 pfu/ml.
The average size of the cDNA inserts was determined by PCR amplification. Twenty well-isolated phage plaques were cored and the cDNA inserts were amplified using T7/T3 specific oligonucleotides which hybridize to sites flanking the cDNA insertion site contained within the lambda phage vector. The PCR products were analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining. Seventy percent of the cored plaques contained inserts varying in size from 0.5 to 2.5 Kb with an average size of 1.5 Kb.
Example II LIBRARY SCREENING
To identify sperm-specific proteins that contain epitopes involved in immune- mediated infertility, patient serum with high levels of anti-sperm antibodies (greater than 35% reactivity by IBT) was used as a probe to screen the testes specific cDNA expression library described in Example I.
i) Source of patient serum
Serum was obtained from a male patient diagnosed as immunologically infertile. The patient's semen contained antibodies to sperm as detected by a direct anti- sperm Ab test (See Munroe et al, 1990, Fertility & Sterility, 54:114; Bronson, 1984, Ann. NY Acad. Sci. 1984:436 ; Clarke, 1985, Am. J. Reprod. Immunol. Microbiol 7:143 Bronson, 1982, Am. J. Reprod. Immunol. 2:222). Antibody test results (two tests) were: IgG 50% positive and 52% positive; IgA 45% positive and 41% positive. A score greater than 20% is considered positive. The patient's serum was able to agglutinate sperm in an agglutination assay (Hirano et al., 1996, Arch Androl 37:163-170). Magnified examination of sperm agglutination studies with the infertile patient serum used to isolate ReproSA- 1 showed that the site of contact was at the central part of the sperm tail.
ii) Preadsoφtion of Serum.
Antibodies that react with expression library host strains were immunoadsorbed from patient serum by using BNN97 and Y1090 E. coli lysate-conjugated Sepharose beads (5prime-3prime, West Chester, PA) following the manufacturer's protocols. Briefly, 2 ml of host stain lysate-conjugated Sepharose beads were washed twice with sterile Tris buffered saline (TBS). The beads were resuspended in 4 mis of serum diluted 1/2 in TBS. Following a 16 hour incubation at 4° C, the sepharose beads were collected by centrifugation at 1,000 x g for 2 min. The supernatant was removed and the beads were washed with 4 mis of sterile TBS. After centrifugation at 1,000 x g for 2 min, the supernatants were collected, pooled and used to screen the testes-specific cDNA expression library.
iii) Screening the testes-specific cDNA library.
Approximately 106 infectious phage particles were incubated with XI- 1 blue MRF' host cells and plated at density of 50,000 phage per 150 mm dish using standard protocols (Stratagene). After incubating for 5 hours at 42° C, the phage plaques were overlaid with nitrocellulose membranes (Protran; Schleicher & Schuell) that had been soaked in a 10 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) solution. Following a 4 hour incubation at 37 °C, the membranes were removed and washed three times for 15 min in TBS with 0.05% Tween20 (TTBS). The membranes were incubated with blocking solution (1 % Bovine serum albumin [fraction V] in TBS) for 1 hour at room temperature prior to a 1 hour incubation with preadsorbed patient serum diluted 1/10 (final dilution of 1/40) in blocking solution. The membranes were washed three time for 15 min with
TTBS prior to the addition of alkaline phosphatase-conjugated goat anti-human Ig(G,A,M) (Pierce) diluted 1/25,000 in blocking solution. After a 1 hour incubation at room temperature the membranes were washed three times with TTBS as described above and once with TBS. The membranes were incubated with enzyme substrate (Western Blue; Promega) for approximately 30 min and the enzymatic reaction was terminated by briefly incubating the membranes with stop solution (Tris-HCl pH 2.9; 1 mM EDTA). Several immunoreactive phage plaques were selected and transferred to 500 ul of SM buffer (100 mM NaCl, 8 mM MgSO4, 50 mM Tris-HCl pH7.5, 0.01 % gelatin) containing 20 ul of chloroform. The selected phage were eluted from the agar and plated at a density of approximately 1,000 phage per 100 mm dish and screened as described above. To insure that the selected phage plaque, named ReproSA- 1.0, represented a single clone the screening process was repeated a third time as described above.
iv) Excision of ReproSA- 1.0 phagemid.
Plasmid containing the cDNA insert for ReproSA- 1 was excised from the phage clone using the manufacturer's protocols (Stratagene). The size of the ReproSA- 1.0 insert was determined by releasing the cDNA fragment from the rescued pBK-CMV/ReproSA-1 plasmid with the restriction enzymes, EcoRI and Xhol. The released insert was size fractionated by agarose-gel electrophoresis and the apparent length of the insert was determined by comparing its migration position with a DNA standard (1 kb ladder; Gibco BRL). The insert migrated at approximately 1.5 Kb.
Example III FURTHER CHARACTERIZATION OF REPRO-SA-1 GENES AND POLYPEPTIDES A. Sequence analysis of ReproSA-1.0
The nucleotide sequence of ReproSA- 1.0 was determined using a modified protocol of the dideoxy chain termination method of Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74:5463-5467 and USB Sequences 2.0 (USB, Cleveland, Ohio). See Figure 1. The amino acid sequence was predicted using the Intelligenetic TRANSLATE program and sequence homologies were determined with BLAST data base search algorithms. The deduced amino acid sequence (in the expected frame for a fusion protein) showed similarity to the PF16 flagellar protein of Chlamydomonas reinhardt (Smith et al, 1996, J. Cell. Biology 132:359-370) indicating that ReproSA- 1 is a sperm-tail protein. B. Identification of additional clones
To obtain additional 5' sequence of the ReproSA- 1 protein coding sequence, 5 'RACE (Frohman et al., 1988 Proc. Acad. Sci USA 85;8998 ) was carried out, Briefly, total RNA isolated from testis was converted to cDNA using a ReproSA- 1 specific primer and Superscript reverse transcriptase (Life Technologies). The resulting ReproSA- 1 cDNA was incubated with dCTP and terminal transferase to add CTP molecules to the 3' end, which was used as a binding site for the arbitrary primer that contained a complementary stretch of GTPs. Once the second strand cDNA was produced, the resulting double-stranded ReproSA- 1 DNA (flanked with the arbitrary primer and the sequence-specific primer) was PCR amplified. The PCR amplicon was used as template to generate a probe that contained 5' ReproSA- 1 sequences. The testis library was screened with the 5'ReproSA-l probe, and full-length clones were isolated and sequenced. These clones are identified as clones C, E and X in Figure 2. These clones were sequenced by standard methods. Conceptual translation analysis of the cDNA clones identified an open reading frame of 509 amino acids, with the first ATG Met start codon at nucleotide 125 and a translation stop codon TAA at 1652, encoding a protein with a calculated molecular weight of about 55,600 kDa. Two transcriptional stop are present at 1731 and 2357 (Figure 2). The presence of alternative polyadenylation sites is consistent with the appearance of two transcripts in Northern Blots (see Figure 3) and is not unusual for testis-specific transcripts.
B. Tissue expression analysis.
The ReproSA- 1 expression distribution was detected by Northern blot analysis using poly(A)+ RNA collected from human spleen, thymus, prostate, testis, ovary, small intestine, colon, and peripheral blood leukocytes (MTN human blot II; Clontech) and human heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas (MTN human blot I; Clontech) and the manufacturer' suggested protocols (Clontech). The immobilized poly(A)+ RNA samples were incubated with a random prime labeled probe that represented the ReproSA- 1.0 insert. The probe was generated by using and EcoRI/XhoI released fragment of ReproSA- 1.0 as template for a random prime labeled reaction as described in the manufactures manual (Megaprime kit; Amersham). The Northern blot membranes were prehybrized with 6 mis of ExpressHyb solution (Clontech) followed by a 1 hour incubation at 68 °C with approximately 0.5 ng of radiolabeled probe (approx. 2x10s cpm) in 5 mis of ExpresHyb solution. Unbound probe was removed by washing the membranes three times with 2 X SSC, 0.05% SDS at room temperature for 10 min for each wash followed by twice with 0.1 X SSC, 0.1 % SDS at 50°C for 15 min for each wash. The apparent length of RNA species and tissue distribution was determined by autoradiography. Expression was only detected in testes, as a pair of mRNA transcripts of approximately 1.8 and 2.8 Kb (Figure 3).
C. Homologue analysis. To determine the level of nucleotide conservation of ReproSA- 1 in different species, a Southern blot analysis using EcoRI digested genomic DNA collected from human, monkey, rat, mouse, dog, cow, rabbit, chicken and yeast was performed as described in the manufacture's manual (ZooBlot; Clontech). Briefly, the immobilized EcoRI digested genomic DNA samples were prehybridized with 6 mis of ExpressHyb (Clontech) and incubated for 1 hour at 68 °C with 1.0 ng (approx. 4x105 cpm in 5 mis) of a random prime labeled probe that represented the ReproSA- 1.0 insert. Unbound probe was removed by washing the membranes once with 2 X SSC, 0.05% SDS at room temperature for 30 min followed by one wash with 0.1 X SSC, 0.1% SDS at 50 °C for 30 min. Identification of homologues in different species was determine by autoradiography. (See Figure 4) The sequence is highly conserved between human and non-human primates (Monkey), and a homologue can be detected in rodents (Rat).
Example IV EXPRESSION OF REPRO-SA-1 POLYPEPTIDES The ReproSA- 1 cDNA insert was PCR amplified using a T3 primer and prlAXhoHis (5'GTGGTGGTGCTCGAGTGGTTGATAGCTGTCC [SEQ ID NO:23]), according to standard protocols. The PCR amplicons were separated by agarose-gel electrophoresis and purified with glass beads (BIO 101). The purified fragments were prepared for ligation into a pET21b expression plasmid (Novagen, Madison, WI) and transfected into DH5α E. coli.
Colonies were screened with a 32P labeled ReproSA- 1 probe and by sequencing. A plasmid encoding a full-length ReproSA- 1.0 insert in the correct orientation was purified and transfected into BL-21 E. coli bacteria for expression of recombinant protein. The pET21b expression system produces a fusion protein with T7-Tag® and His- Tag® (Novagen, Madison, WI).
The bacteria transfected with pET21/ReproSA-l was grown in 30 mis of LB media with 50μg/ml ampicilin to an OD600 of 0.6 prior to induction of protein expression with IPTG (final concentration of 1 mM). The bacterial cells were harvested by centrifugation and washed twice with PBS. The bacterial pellet was resuspended in GMAC- 5 (20 mM Tris-HCL pH 7.4, 0.5 M NaCl, 6 M guanidine-HCl, 1 mM PMSF, 1 mM Imidazole) and sonicated on ice for 50 pulses until the OD600 dropped to approximately one tenth of the original O.D. The insoluble material was removed by centrifugation and the lysate applied to His-Bind Resin (Novagen, Madison, WI). The recombinant ReproSA- 1 protein was purified from the bacterial lysate according to the manufacture's protocols (Novagen, Madison, WI). Figure 5 shows SDS-PAGE of purified protein of approximately 25 kDa. The purified protein was used to generate polyclonal anti-ReproSA-1 antibodies (infra).
Example V POLYCLONAL ANTI-REPRO-SA-1 SERA PRODUCTION Anti-ReproSA-1 anti-sera were made using three different immunogens. Peptides were synthesized corresponding to the C-terminal end of the protein
[KVLPHDSKARRLFVT] and a ReproSA-1 armadillo repeat [YMSTESSEDLQVKSK]. These two peptides, along with the recombinant polypeptide described in Example IV, were used to generate anti-ReproSA-1 sera in rabbits using standard techniques (see, Coligan et al., eds., CURRENT PROTOCOLS IN IMMUNOLOGY pp.2.4.1-2.4.4. [Production of Polyclonal Antisera] John Wiley and Sons, Inc., New York. )
Antisera from immunized rabbits were tested for the presence of anti- ReproSA-1 antibodies using standard protocols (i.e., enzyme-linked immunosorbent assays using the ReproSA- 1 peptide or recombinant polypeptide immunogen immobilized onto microtiter plates; see Harlow, supra). For the antisera made to the C-terminal and armadillo repeat peptides, the anti-ReproSA-1 antibodies were affinity purified using appropriate peptide conjugated to a matrix support (see, CURRENT PROTOCOLS IN IMMUNOLOGY, supra, pp.9.4.9-9.4.10). To characterize the anti-ReproSA-1 antibodies, Western analysis was carried out (see, e.g., Harlow, supra) using human sperm total protein extracts. Sperm protein extracts were prepared from normal donor semen by incubation at 100° C for 10 minutes in one volume of SDS-loading buffer (100 mM Tris-CL pH 6.8, 200 mM dithiothreitol, 4% SDS, 0.2%) bromophenol blue, 20% glycerol). The human sperm proteins extracts were separated by reducing SDS-PAGE, transferred to nitrocellulose and blocked. Filters were incubated with each of the three anti-Repro-SA-1 sera according to standard Western blot protocols, using appropriate controls. Anti-ReproSA-1 antibodies made against each of the three classes of immunogen identified a 60 kDa protein by Western blot analysis (see Figure 6).
Example VI CHROMOSOME LOCATION OF REPRO-SA-1 Fluorescence in situ hybridization (FISH) was performed using a labeled ReproSA- 1 probe. Specific hybridization identified chromosome arm 1 Op 11.2 as the locus of the ReproSA- 1 gene.
Example VII RETROSPECTIVE SCREENING OF FERTILE AND INFERTILE PATIENT SERA Enzyme-linked immunosorbent assays (ELISA) are performed on a series of sera from (i) patients that have been clinically defined as infertile and (ii) a matched control (fertile) group, using standard methods. See, Enzyme-Linked Immunosorbent Assays., in Current Protocols, pages.2.1.3-2.1.5). The purified recombinant ReproSA-1 is immobilized on microtiter plates by diluting the protein to a concentration of 5 μg/ml in carbonate binding buffer and incubating the plates overnight. The patient sera is diluted in sample buffer and incubated with the immobilized ReproSA- 1 protein. Bound anti-ReproSA-1 antibodies are detected with horseradish peroxidase conjugated anti-human immunoglobulin and the appropriate substrate. The signals generated in each well are analyzed to determine levels of anti-ReproSA-1 antibodies found in patient infertile serum relative to normal control (fertile) serum.
ELISA results are confirmed by using the purified recombinant ReproSA- 1 protein in an immunoblot analysis (see, Immunoblotting and Immunodetection, in CURRENT PROTOCOLS IN IMMUNOLOGY, pages.8.10.1-8.10.7). Briefly, 10 μg ofReproSA-1 protein is size fractionated on SDS-PAGE (10%) and transferred to a nitrocellulose membrane. The membrane is incubated with patient (infertile) sera or normal control (fertile) sera diluted in sample buffer. Bound anti-ReproSA-1 antibodies are detected with alkaline phosphatase conjugated anti-human immunoglobulin and the appropriate substrate.
Example VIII ISOLATION OF HUMAN ANTI-REPRO-A-1 IMMUNOGLOBULIN SEQUENCES Human antibodies are isolated or engineered from the antibody repertoire of infertile patients. These human antibodies are used to formulate spermicidal contraceptives.
A. Isolation of human antibodies from peripheral blood lymphocytes (PBLs)
PBLs are isolated from a patient expressing anti-ReproSA-1 antibodies. Eight ml of blood are collected in Vacutainer CPT tubes (Becton-Dickinson) and the PBLs are isolated as per the manufacturer's recommendations. The PBLs are presorbed against non- reproductive tissue or cells (i.e. muscle, heart, skin, liver, kidney). The unabsorbed PBLs are collected and used to construct a sfFv library in the filamentous phage (fUSE5) (Scott, J & G. Smith, Science 249:386,1990).
B. Construction of the library.
Poly(A)+RNA is isolated from peripheral blood lymphocytes obtained in step A. cDNA complementary to the entire repertoire of the poly(A)+ RNA is synthesized. The polymerase chain reaction is used to amplify the variable region heavy chain (V^-constant region heavy chain (CHI) and the kappa and lambda light chain genes, with additional complementary coding sequences for a peptide linker at the 3' end of the heavy chain genes and the 5' end of the light chain genes. PCR is then used to amplify the VH-linker- variable region light chain (V ) scFv cDNAs. The scFv cDNAs are ligated into the filamentous phage fUSE5 such that they are expressed as a fusion protein. Each resulting fusion phage will display 4 or 5 copies of a scFv molecule fused to the minor envelope protein p3. See, e.g., Mole et al. , Journal of Clinical Microbiology, 32:2212 ( 1994). C. Panning.
Recombinantly expressed ReproSA- 1 peptides are coated onto polystyrene dishes. Phage from the scFv library are precipitated in 4% (wt/vol) PEG/0.5 M NaCl and resuspended in water. Approximately 1011 transforming units are added to the protein or peptide in 2 ml of binding buffer. Unbound phage is washed away with PBS, and bound phage eluted in elution buffer. The eluted phage are amplified as described in and the panning process will be repeated three times. See, e.g., Scott et al., Science 249:386 (1990) and Cai et al., Proc. Natl. Acad. Sci., USA 92; 6537 (1995), both of which are incoφorated in their entirety for all puφoses.
D. Cloning.
The eluted phage are mixed with E. Coli K91 Kan cells at low phage to cell ratios, and plated on 2x TY/tetracycline plates. Individual colonies are inoculated into 2x TY/tetracycline medium and grown. Positives are verified by ELISA assay to ReproSA- 1 in which bound phage are detected using a conjugated anti-M13 polyclonal antibody. Those phage that bind with highest affinity are selected and the immunoglobulin gene sequences being expressed are characterized and used to design high affinity ReproSA- 1 -binding molecules. See, e.g., Scott et al., supra, and Cai et al., supra.
All references cited herein are incoφorated herein by reference in their entirety and for all puφoses to the same extent as if each individual publication or patent application was specifically and individually indicated to be incoφorated by reference in its entirety for all puφoses.
Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

WHAT IS CLAIMED IS:
1. A substantially pure or recombinant ReproSA- 1 polypeptide, or fragment thereof.
2. The ReproSA- 1 polypeptide of claim 1 wherein the polypeptide comprises an amino acid sequence substantially identical to SEQ ID NO:2.
3. The polypeptide of claim 1 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:2.
4. A substantially pure polypeptide specifically immunoreactive with an antibody raised against a polypeptide having the amino acid sequence of SEQ ID NO:2.
5. The polypeptide of claim 4, wherein said polypeptide is coupled to a solid support.
6. A substantially pure polypeptide consisting essentially of between about eight and about 509 contiguous residues of a polypeptide having the amino acid sequence of SEQ ID NO:2.
7. The polypeptide of claim 6, comprising at least eight contiguous amino acids of a polypeptide selected from the group consisting of MSTESSEDLQVKSKKAIKNILQKCTY [SEQ ID NO:5]; LPHDSKARRLFVTSGGLKKVQEIKAEPG [SEQ ID NO:6];
SKGVPQLSVCLSEEPEDHIKAA [SEQ ID NO:7];
YPEEIVRYYSPGYSDTLLQRVDS [SEQ ID NO:8];
MSQRQVLQVFEQYQKARTQFVQMVAELATRP [SEQ ID NO:9];
YNDDLAEAWKCDILPQLVYSLAEQNRFYKKAAAFVLRAVGKHSPQLA [SEQ ID NO: 10];
LLVLCIQEPEIALKRIAASALSDIAKHSPELAQTVVDAGAVAHLAQ MILNPDAKLKHQILSALSQVSKHSVDLAEMVVEAEIF [SEQ ID NO:l 1]; and TCLKDKDEYVKKNASTLIREIAKHTPELSQLV [SEQ ID NO: 12].
8. The polypeptide of claim 7 wherein the polypeptide is selected from the group consisting of MSTESSEDLQVKSKKAIKNILQKCTY [SEQ ID NO:5] ;
LPHDSKARRLFVTSGGLKKVQEIKAEPG [SEQ ID NO:6];
SKGVPQLSVCLSEEPEDHIKAA [SEQ ID NO:7]; and
YPEEIVRYYSPGYSDTLLQRVDS [SEQ ID NO:8];
MSQRQVLQVFEQYQKARTQFVQMVAELATRP [SEQ ID NO:9]; YNDDLAEAWKCDILPQLVYSLAEQNRFYKKAAAFVLRAVGKHSPQLA [SEQ ID
NO: 10];
LLVLCIQEPEIALKRIAASALSDIAKHSPELAQTVVDAGAVAHLAQ
MILNPDAKLKHQILSALSQVSKHSVDLAEMVVEAEIF [SEQ ID NO:l 1]; and
TCLKDKDEYVKKNASTLIREIAKHTPELSQLV [SEQ ID NO:12].
9. An isolated or recombinant polynucleotide comprising a sequence encoding the polypeptide of claim 1.
10. An isolated or recombinant polynucleotide comprising a sequence encoding a polypeptide, said polypeptide comprising between about eight and about 509 contiguous residues of SEQ ID NO:2.
11. The polynucleotide of claim 10, wherein the sequence encoding the polypeptide is operably linked to a promoter.
12. A recombinant cell line comprising a polynucleotide of claim 11 , wherein the cell line is capable of expressing a human ReproSA- 1 polypeptide or a fragment thereof, and wherein the promoter is a heterologous promoter.
13. An isolated polynucleotide comprising a sequence encoding the polypeptide of claim 3.
14. The polynucleotide of claim 13, wherein the sequence encoding the polypeptide of claim 3 is operably linked to a promoter.
15. A cell comprising a polynucleotide of 13, wherein the cell is capable of expressing a human ReproSA- 1 polypeptide or a fragment thereof, and wherein the promoter is a heterologous promoter.
16. An isolated or recombinant polynucleotide comprising a sequence of at least 10 contiguous nucleotides of SEQ ID NO:l.
17. The polynucleotide of claim 16 comprising at least 12 contiguous nucleotides of SEQ ID NO:l.
18. A substantially purified antibody, or fragment thereof, wherein the antibody or antibody fragment specifically binds to human ReproSA- 1.
19. A monoclonal antibody or fragment thereof, wherein the antibody or antibody fragment is specifically immunoreactive with a ReproSA- 1 polypeptide.
20. The monoclonal antibody or fragment thereof of claim 19, wherein the
ReproSA- 1 polypeptide is human.
21. The antibody of claim 19, wherein the antibody binds with an affinity ofat least about 108 M-'.
22. The monoclonal antibody of claim 19, wherein the monoclonal antibody is a human monoclonal antibody.
23. A cell capable of secreting the antibody of claim 21.
24. A hybridoma capable of secreting the antibody of claim 22.
25. A contraceptive vaccine comprising a polypeptide comprising at least eight consecutive residues of SEQ ID NO:2 in a pharmacologically acceptable carrier.
26. The vaccine of claim 25, further comprising an adjuvant.
27. A method of immunizing a human to achieve to produce an anti-sperm immune response, comprising administering to a human the vaccine of claim 25.
28. A vaccine comprising the polynucleotide of claim 10.
29. A method of immunizing a human to achieve an anti-sperm immune response, comprising administering to a human the vaccine of claim 28.
30. A method for diagnosing immunological infertility in a human patient comprising detecting anti-ReproSA-1 antibodies in a biological sample from a patient.
31. The method of claim 30, wherein the biological sample is serum, semen, saliva, a secretion of cervical mucosa or a secretion of vaginal mucosa.
32. A method for detecting a mutation in a ReproSA-1 gene in a patient comprising comparing the sequence of a ReproSA- 1 gene of the patient with the sequence of the ReproSA- 1 gene having the sequence of SEQ ID NO:l, wherein a difference in the two sequences indicates a mutation in the ReproSA- 1 gene of the patient.
33. The method of claim 32 wherein a mutation other than a silent mutation is identified.
34. A medicament for contraception comprising anti-ReproSA- 1 antibodies in a pharmacologically acceptable carrier.
35. A kit for determining the presence in a body sample of antibodies to human ReproSA- 1 polypeptide comprising (i) an antibody specifically reactive with ReproSA- 1 and (ii) a polypeptide bound to a solid support, wherein the polypeptide comprises at least eight consecutive amino acids of SEQ ID NO:2.
36. A method for detecting an interaction between a first polypeptide and a second polypeptide, wherein eimer the first polypeptide or the second polypeptide is a ReproSA- 1 protein, or fragment thereof, said method comprising:
(a) providing a host cell containing a detectable gene, wherein the detectable gene expresses a detectable protein when the detectable gene is activated by an amino acid sequence including a transcriptional activation domain; (b) providing a first chimeric gene that is capable of being expressed in the host cell, the first chimeric gene comprising a DNA sequence that encodes a first hybrid protein comprising a DNA-binding domain that recognizes a binding site on the detectable gene in the host cell, and the first polypeptide;
(c) providing a second chimeric gene that is capable of being expressed in the host cell, the second chimeric gene comprising a DNA sequence that encodes a second hybrid protein, the second hybrid protein comprising the transcriptional activation domain and the second polypeptide; wherein one of the first polypeptide and the second polypeptide is a ReproSA- 1 protein, or fragment thereof, and the other of the first polypeptide and the second polypeptide is a polypeptide sequence to be tested for interaction with the ReproSA- 1 protein or fragment; and wherein interaction between the first polypeptide and the second polypeptide in the host cell causes the transcriptional activation domain to activate transcription of the detectable gene; (d) introducing the first chimeric gene and the second chimeric gene into the host cell;
(e) subjecting the host cell to conditions under which the first hybrid protein and the second hybrid protein are expressed
(f) determining whether the detectable gene has been expressed to a degree greater than expression in the absence of an interaction between the first polypeptide and the second polypeptide.
EP98908674A 1997-02-18 1998-02-17 Nucleic acids encoding sperm antigens and reprosa-i polypeptides Withdrawn EP1007667A1 (en)

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