EP0877757A1 - Methode zur auswertung und beeinflussung der männlichen fertilität - Google Patents

Methode zur auswertung und beeinflussung der männlichen fertilität

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Publication number
EP0877757A1
EP0877757A1 EP97904191A EP97904191A EP0877757A1 EP 0877757 A1 EP0877757 A1 EP 0877757A1 EP 97904191 A EP97904191 A EP 97904191A EP 97904191 A EP97904191 A EP 97904191A EP 0877757 A1 EP0877757 A1 EP 0877757A1
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EP
European Patent Office
Prior art keywords
sperm
fertility
antibody
animal
protein
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EP97904191A
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English (en)
French (fr)
Inventor
Gary Klinefelter
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US Environmental Protection Agency
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US Environmental Protection Agency
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Publication of EP0877757A1 publication Critical patent/EP0877757A1/de
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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/52—Sperm; Prostate; Seminal fluid; Leydig cells of testes
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to a sperm protein which can be used for evaluating and/or inhibiting male fertility, as well as antibodies to the sperm protein.
  • the present application is a continuation in part of application Serial No. 08/593,677, filed January 29, 1996, the entire contents of which are hereby incorporated by reference.
  • New contraceptives should be superior to existing products, e.g., oral contraceptives used by millions of women over the last 30 years are not only safe and effective, but even protect women against some cancers. However, other methods of contraception are still needed by many segments of the world's population, as many women do not have reliable access to oral contraceptives, or
  • ISA/EP may suffer adverse reactions to the hormones used in oral contraceptives.
  • biochemical assays of semen have not resulted in simple procedures which may be performed in either the physician's office or a dedicated semen evaluation lab. Most biochemical markers have failed to demonstrate correlations with sperm number, motility, or fertility. Activity of fu arase, an enzyme present in semen, has been found to correlate to both sperm count and percentage motility, Crabbe, J. Reprod. Fert . 51 : 73-76 (1977) . Crabbe measured fumarase activity by spectrophotometric measurements. Unfortunately, spectrophotometric assays are not generally suitable for office assays because of the cost of these specialized devices as well as the training required for accurate and reproducible operation.
  • Dorian in U.S. Patent No. 5,434,057, expanded on Crabbe's method by providing devices for assessing sperm " ⁇ number and motility in semen samples comprising a solid support having a carrier matrix containing a fumarase substrate and malate dehydrogenase.
  • the sample is applied to the carrier matrix and a visual signal is detected from the solid support resulting from metabolism of the fumarase substrate by fumarase in the sample. While this assay detects motile sperm in a semen sample, there is no method for inhibiting fertility nor of selecting out the most fertile sperm in a sample.
  • Feuchter et al . in U.S. Patent No. 5,250,417, disclose a method for detecting the ability of sperm to undergo the acrosome reaction to permit determination of the fertility of male mammals.
  • the acrosome reaction is a process by which sperm release hydrolytic enzymes that degrade the zona pellucida, which must be penetrated to enable the sperm and ovum to come into contact, fuse, and complete the fertilization process.
  • MHS-10 that cross-reacts with the entire acrosomal region. It is associated with the outer aspect of the inner acrosomal membrane and the inner aspect of the outer acrosomal membrane of mature human sperm. It has been reproduced recombinantly in an Escherichia coli expression system.
  • PH-20 a guinea pig sperm protein of 64 kD, is present on both the plasma membrane and inner acrosomal membrane of sperm. It is essential for adhesion of sperm to the zona ⁇ pellucida, the initial step in the fertilization process. Active immunization with PH-20 causes infertility in both male and female guinea pigs for a period ranging from six to fifteen months.
  • the protein SP-22 based upon initial electrophoretic runs, was originally thought to be a 16-kD protein, with a pi of 5.5, and was so identified in parent application Serial No. 08/593,677. However, after comparing several types of molecular weight standards, the molecular weight of this protein was found consistently to have an apparent molecular weight of 22 kD in 11% acrylamide gels. For purposes of the present invention, the protein will be identified as SP-22, although in the parent application USSN 08/593,677, the protein was identified as SP-16.
  • SP-22 has a partial amino acid sequence of Thr-Ser-Gly- Pro-Leu-Ala-Lys (SEQ ID NO:l) .
  • the present invention provides a convenient, reliable test for evaluating fertility, based upon changes to SP-22, a specific protein predictive of male fertility, as well as methods of calibrating changes in SP-22 in screening assays so as better to predict the likelihood of the sperm being fertile. Additionally, the present invention provides a method for blocking the expression of SP-22 so as to render the sperm infertile.
  • the present invention provides a reliable test for evaluating fertility following exposure to reproductive toxicants, in candidate sires for artificial insemination, and for men considering assisted reproductive technology. Moreover, as indicated above, the invention offers a means to render men infertile through immunocontraceptive technology.
  • Figure 1 shows the results of a regression analysis correlating the amount of SP-22 to fertility.
  • Figure 2 illustrates using an antibody to SP-22 to distinguish fertile sperm from infertile sperm.
  • Figure 3A and 3B illustrate immunolocalization of SP- 22 in a detergent extract of sperm using an antibody to SP 22.
  • Figure 4 illustrates immunolocalization of SP-22 on sperm using an antibody to SP-22.
  • SP-22 was purified by reverse-phase HPLC, 2D gel electrophoresis, and subsequent electroelution. Briefly, 50- 75 rats were killed and a detergent extract was prepared from the sperm recovered from cauda epididymal sperm. Next, 2 mg of concentrated, desalted extract was subjected to reversed-phase high pressure liquid chromatography (HPLC) . Aliquots of fractions were analyzed by mini-2D SDS gel electrophoresis, and the remaining volume of 5 fractions containing SP-22 were pooled and the protein (approximately 100 micrograms) was resolved by mini-2-D SDS gel electrophoresis.
  • HPLC reversed-phase high pressure liquid chromatography
  • the Coomassie-stained SP-22 then was punched from the gels, electroeluted and either used for amino acid sequencing or mixed with Freund's adjuvant for polyclonal antibody generation.
  • SP-22 as digested with TRCK- trypsin in 4 M urea, pH 8.0, and peptides were generated with a Microbore C8 HPLC column with 0.1% TFA/acetonitrile. Sequence analysis then was performed on an ABI 473 A sequenator. Since a characteristic fragment of SP-22 is known, i.e., Thr-Ser-Gly-Pro-Leu-Ala-Lys (SEQ ID N0:1) , it is possible to prepare functional derivatives of SP-22 as well.
  • ком ⁇ онент is meant a fragment,variant, analog, or chemical derivative of SP-22, which terms are defined below.
  • a “functional derivative” retains at least a portion of the amino acid sequence of SP-22, which permits its utility in accordance with the present invention, namely, determining or affecting male fertility.
  • a “fragment” of SP-22 refers to any subset of the SP-22 molecule, that is, a shorter peptide. Fragments of interest are those which can be used to determine or affect male fertility.
  • a “variant” of SP-22 refers to a molecule which is ⁇ substantially similar to either the entire SP-22 protein or a fragment thereof. Variant peptides may be conveniently prepared by direct chemical synthesis of the variant peptide, using methods well known in the art.
  • amino acid sequence variants of SP-22 can be prepared by mutations in the DNAs which encode the synthesized SP-22.
  • Such variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence. Any combination of deletion, insertion, and substitution may also be made to arrive at the final construct, provided that the final construct possesses the desired activity.
  • the mutations that will be made in the DNA encoding the variant peptide must not alter the reading frame and preferably will not create complementary regions that could produce secondary mRNA structure (cf . European Patent Publication No. EP 75,444) .
  • these variants ordinarily are prepared by site-directed mutagenesis, as exemplified by
  • the variants typically exhibit the same qualitative biological activity as the nonvariant peptide.
  • an "analog" of SP-22 refers to a molecule which is substantially similar to either the entire molecule or a fragment thereof .
  • the analog may be prepared by chemical synthesis.
  • a "chemical derivative" of SP-22 contains additional chemical moieties not normally part of the SP-22 amino acid sequence. Covalent modifications of the amino acid sequence are included within the scope of this invention. Such modifications may be introduced into the SP-22 by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Amino terminal residues can be reacted with succinic or other carboxyric acid anhydrides.
  • Suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4- pentanedione; and transaminase-catalyzed reaction with glyoxylate .
  • Carboxyl side groups such as aspartyl or glutamyl are selectively modified by reaction with carbodiimides (R'N-C-N-R*) such as l-cyclohexyl-3- [2-morpholinyl- (4-ethyl)] carbodiimide or 1- ethyl-3- (4-azonia-4, 4 dimethylpentyl) carbodiimide . Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions .
  • muteins or “variants” refers to analogs of SP-22 in which one or more of the amino acid residues of the natural SP-22, preferably 1-10, and more preferably 1-5, residues, or even only a single residue, are replace by different amino acid residues or are deleted, or one or more amino acid residues, such as 1-10, 1-5, or only one residue are added to the natural sequence of SP-22.
  • muteins are prepared by known synthesis techniques and/or site-directed mutagenesis techniques, or by any other known technique suitable therefor. The substitutions are preferably conservative, see, e.g., Schulz, G.E. et al .
  • substitutions are defined to be exchanges between two of groups (I)-(V) above which are limited to supergroup (A) , comprising (I) , (II) and (III) above, or to supergroup (B) , comprising (IV) and (V) above.
  • Substitutions are not limited to the genetically encoded, or even the naturally occurring amino acids.
  • the desired amino acid may ⁇ be used directly.
  • a genetically encoded amino acid may be modified by reacting it with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.
  • SP-22 Antibody Screening with SP-22 Antibody It is apparent from the above description of SP-22 — antibodies that a wide variety of diagnostic tests is possible using the antibodies of the invention. In attempting to diagnose causes of infertility, an immunoassay to detect decreased levels of SP-22 on sperm is a useful adjunct to known hormone assays. Further uses for the antibodies include testing livestock for candidates for artificial insemination: the higher the levels of SP-22 in the potential donor, the more likely artificial insemination is to be successful. Isolation of SP-22 allows production of antisera containing antibody to SP-22 for possible crossreaction with other species, including human SP-22. This antibody enables preparation of an enzyme-linked immunosorbent assay (ELISA) .
  • ELISA enzyme-linked immunosorbent assay
  • polystyrene microwells are precoated with extract of a particular epididymal sperm (rat) or ejaculate (horse, bull, human) sample containing an unknown amount of SP-22.
  • SP-22 antibody is added, followed by the addition of avidin- biotin-peroxidase complex.
  • a precipitate is formed when a substrate such as DAB is oxidized by peroxidase in the presence of hydrogen peroxide.
  • a standard curve for SP-22 is generated using increasing known amounts of SP-22. The amount of SP-22 in a sample is then determined by the optical density of the colored precipitate in the sample and the linear regression obtained from the set of SP-22 standards .
  • the amount of SP-22 present on the surface of sperm in a sample can be determined using quantitative fluorescence spectroscopy or fluorescent light microscopy. For this, sperm will be incubated with SP-22 antibody and then with labelled Rhodamine or FITC-conjugated second antibody. It is first necessary to determine the relationship between fluorescence of a sample in a fluorometer or a microscopic image, and the optical density of SP-22 separated by 2D gel electrophoresis. Once this is established, fluorescence can be related to fertility.
  • SP-22 for assisted reproductive technologies such as intra uterine transfer or IVF following dissociation of SP-22 expressing sperm from the SP-22 antibody.
  • sperm binding SP-22 antibody are evaluated by quantitative indirect fluorescence microscopy. For this, Rhodamine or FITC immunolabeling is performed on an aliquot of sperm equivalent to that used for in utero insemination, and the number of sperm that fluoresce is determined along with the relative degree of fluorescence of individual sperm in a sample. The resulting fluorescence histograms are related to fertility assessed by artificial (in utero) insemination.
  • amino acid substitutions according to the present invention are known in the art and would be expected to maintain the biological and structural properties of the polypeptide after such amino acid substitutions. Most deletions, insertions and substitutions according to the present invention are those which do not produce radical changes in the characteristics of the protein or peptide molecules. One skilled in the art will appreciate that the effect of substitutions can be evaluated by routine screening assays, either immunoassays or bioassays.
  • a mutant typically is made by site-specific mutagenesis of the peptide molecule-encoding nucleic acid, expression of the mutant nucleic acid in recombinant cell culture, and, optionally, purification from the cell culture, or a biological sample containing SP-22, for example, by immunoaffinity chromatography using a specific antibody on a column to absorb the mutant by binding to at least one epitope.
  • Antibodies to SP-22 are made by site-specific mutagenesis of the peptide molecule-encoding nucleic acid, expression of the mutant nucleic acid in recombinant cell culture, and, optionally, purification from the cell culture, or a biological sample containing SP-22, for example, by immunoaffinity chromatography using a specific antibody on a column to absorb the mutant by binding to at least one epitope.
  • Antibodies to SP-22 can be prepared by any conventional mans, e.g., by coincubating purified SP-22 with hybrid mouse cells in vi tro to produce monoclonal antibodies, or by generating polyclonal antibodies in mice or rabbits in vivo . The antibody produced by the hybrid cultures is collected by chromatography for subsequent use. Preparation of Antibodies to SP-22
  • Antibodies (both polyclonal and monoclonal) to SP-22 may be prepared for diagnostic and therapeutic uses including but not limited to fertility control (contraception) and fertility assessment (screening) .
  • "Antibody” in this context refers to a synthetic protein which binds SP-22 and negates its biological function.
  • Antibodies to SP-22 are prepared by either polyclonal or monoclonal techniques:
  • polyclonal Antibody Production For polyclonal antibody product, adult mice or rabbits are immunized with 25 or 100 mg of SP-22 suspended in Freund's complete adjuvant. This preparation is injected subcutaneously and is followed by booster injections of SP-22 mixed with incomplete adjuvant. Sera obtained after the final booster injection are checked for titer, affinity, and specificity.
  • Figure 3A shows, in the upper photograph, a silver- stained, two-dimensional gel profile of detergent-extracted cauda epididymal rat sperm.
  • SP-22 is indicated by a circle.
  • the acidic lower molecular weight protein appears to be a product of SP-22 degradation.
  • Figure 3B is an immunoblot of a sample that is identical to the one used in Figure 3A using mouse anti-rat SP-22 antiserum. Both authentic SP-22 and its degradation products are localized with antiserum.
  • antiserum to SP-22 was produced as follows.
  • a detergent extract of cauda epididymal sperm was chromatographed by reversed-phase HPLC and fractions enriched in SP-22 were run an analytical 2D gels.
  • Coomassie-stained SP-22 punches were subsequently subjected to electroelution and the electroeluted material was desalted, concentrated, and assayed for protein.
  • 25 micrograms was mixed with Freund's complete adjuvant and injected subcutaneously into each of six mice. Four other mice received only adjuvant .
  • each mouse was boosted with 12.5 micrograms of SP-22 mixed with Freund's incomplete adjuvant. After another ten days, a final similar boost was given.
  • mice were euthanized ten days after the final boost, and the serum was collected.
  • mice are immunized with SP-22 adjuvant emulsion described above. Each mouse first receives 0.2 mL of this emulsion intraperitoneally, and then is reinjected in similar fashion with 0.1 mL six weeks later.
  • Mouse serum is obtained ten days after the second injection and then tested for anti-FRP activity via ELISA.
  • the mouse exhibiting the highest absolute anti- FRP activity is chosen for cell fusion.
  • Spleen cell suspension containing B-lymphocytes and macrophages is prepared by perfusion of the spleen. The cell suspension is washed and collected by centrifugation. Myeloma cells are also washed in this manner. Live cells are counted and the cells placed into a 37"C water bath. One L of 50% polyethylene glycol in DMEM is added slowly.
  • the cells are incubated in the PEG for one to 1.5 minutes at 37 " C, after which the PEG is diluted by the slow addition of media.
  • the cells are pelleted and 35 to 40 L of DMEM containing 10% fetal bovine serum is added.
  • the cells are then dispensed into tissue culture plates and incubated overnight in a 37 ' C, 5% C0 2 , humidified incubator.
  • HAT medium hypoxanthine, thymidine, and aminopterin
  • SP-22 is identified by its biological functions and activities set forth herein, as well as by its size of approximately 22 kD and isoelectric point of 5.25. However, changes in form and the substitution of fragments or equivalents are contemplated as circumstances may suggest or render expedient. For instance, it may be necessary to generate polyclonal antibodies to peptide fragments of SP-22 if sufficient amounts of purified SP-22 cannot be obtained relatively easily.
  • the present invention embraces epitopes which are substantially homologous with such antibodies.
  • substantially homologous when used in connection with amino acid sequences, refers to sequences which are substantially identical to or similar in sequence with each other, giving rise to a homology of conformation and thus to retention, to a useful degree, of one or more biological (including immunological) activities. The term is not intended to imply a common evolution of the sequences.
  • Substantially homologous peptide epitopes may be identified by a variety of techniques. It is known in the art that one may synthesize all possible single substitution mutants of a know peptide epitope. Geysen et al . , Proc . Na t . Acad. Sci . (USA) 81 : 3998-4002, 1984. While the effects of different substitutions are not always additive, it is reasonable to expect that two favorable or neutral single substitutions at different residue positions in the epitope- can safely be combined in most cases.
  • SP-22 may be produced by methods other than recovery from male animals.
  • a cDNA probe is prepared against a partial sequence of SP-22 and used to identify the SP-22 genome in cells from any mammalian species. The identified genome is then inserted into a plasmid which is then employed to produce recombinant SP-22 in proliferating bacteria or other hosts according to methods known in the art. This will be useful in the methodologies, e.g., immunocontraception, _ addition to sperm, described herein. However, not only will these utilities require large amounts of SP-22, they will also require large quantities of the SP-22 antibody. This is accomplished in batch hybridoma cell culture using proven methods .
  • Antibodies to SP-22 can be used for contraception as well as for assaying fertility.
  • a reversible contraceptive vaccine is provided by administering to an animal subject SP-22 as described above in an amount effective to reduce the fertility of that subject via generation of antibodies to SP-22. Partial reduction in fertility, i.e., effects which are reflected as a reduction in fertility in a population of subjects, are intended as within the scope of the present invention.
  • Any animal which expresses sperm surface SP-22 may be treated by the immunocontraceptive method of the present invention, including both birds and mammals.
  • Exemplary mammals include mice, rabbits, dogs, cats, cows, pigs, sheep, horses, and humans. Mammalian subjects are preferred.
  • the vaccine can be administered to either females or males by any suitable means, including by intramuscular injection, by intravenous injection, or by intraperitoneal injection.
  • the antibody can be administered topically, as by vaginal foam or by any convenient topical method in an appropriate carrier, e.g., by nasal spray.
  • protection is intended to include prevention or suppression of production of fertile sperm.
  • administration may be systemic or topical.
  • administration of such a composition may be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, vaginal or buccal routes.
  • parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, vaginal or buccal routes.
  • parenteral administration can be by bolus injection or by gradual release over time.
  • the suitable dose of a composition according to the present invention will depend upon the age, health and weight of the recipient. However, the most preferred dosage can be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation. This typically involves adjustment of a standard dose, e.g., reduction of the dose if the patient has a low body weight.
  • a drug Prior to use in humans, a drug is first evaluated for safety and efficacy in laboratory animals. In human clinical trials, one begins with a dose expected to be safe in humans, based on the preclinical data for the drug in question, and on customary doses for analogous drugs, if any. If this dose is effective, the dosage may be decreased to determine the minimum effective dose, if desired. If this dose is ineffective, it will be cautiously increased, with the patients monitored for signs of side effects. See, e.g., Berkow et al . , eds., The Merck Manual , 15th edi tion, Merck and Co., Rahway, N.J., 1987; Goodman et al .
  • the appropriate dosage form depends on the composition administered, i.e., the carriers used for the antibody, as well as the mode of administration. Modes of administration include tablets, capsules, lozenges, dental pastes, suppositories, inhalants, solutions, ointments, and parenteral depots. See, e.g., Berker, supra , Goodman, supra , Avery, supra and Ebadi, supra, which are entirely incorporated herein by reference, including all references cited therein.
  • a pharmaceutical vaccine composition may contain suitable pharmaceutically acceptable carriers, such as excipients, carriers, and/or auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • suitable pharmaceutically acceptable carriers such as excipients, carriers, and/or auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • the amount of antigen administered depends upon factors such as route of administration, species, and the use of booster administrations. In general, a dose of about 0.1 to about 100 micrograms per kg of body weight may be used.
  • the antigen to SP-22 may be prepared as both human and veterinary vaccine formulations.
  • Vaccine formulations of the present invention comprise the antigen in a pharmaceutically acceptable carrier.
  • the antigen is included in the carrier in an amount which is effective to reduce the fertility of the subject being treated.
  • Pharmaceutically acceptable carriers are preferably liquid, particularly aqueous carriers, such as sodium phosphate buffered saline.
  • the vaccine formulations may be stored in a sterile glass container sealed with a rubber stopper through which liquids may be injected and formulations withdrawn by syringe.
  • Vaccine formulations of the present invention may optionally contain one or more adjuvants. Any suitable adjuvant can be used, such as aluminum hydroxide, aluminum phosphate, plant and animal oils, and the like, with the amount of adjuvant depending on the nature of the particular adjuvant employed.
  • the vaccine formulations may also contain at least one stabilizer, such as carbohydrates such as sorbitol, mannitol, starch, sucrose, dextrin, and glucose, as well as proteins such as albumin or casein, and buffers such as alkaline metal phosphates and the like.
  • stabilizer such as carbohydrates such as sorbitol, mannitol, starch, sucrose, dextrin, and glucose, as well as proteins such as albumin or casein, and buffers such as alkaline metal phosphates and the like.
  • compositions according to the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active ingredients into preparations for pharmaceutical use.
  • the preparations contain from about 0.1 to about 99 percent, preferably from about 25 to 85 percent of active ingredient, together with the excipient.
  • the excipient may be any pharmaceutically acceptable excipient or carrier which can be used with the antigen or antibody. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol and sorbitol, cellulose preparations and derivatives and/or calcium phosphates.
  • binders such as starch, gelatin, gums, methyl cellulose, hydroxyproplymethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone.
  • Lubricants such as silica, talc, stearic acid, or salts thereof, and/or polyethylene glycol can also be used.
  • suppositories for vaginal application, suppositories, lotions, creams, sprays, or foams may be used to incorporate the active ingredient.
  • Suitable suppository bases are, for example, natural or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols or higher alkanols.
  • Foam formulations may include oily suspensions or aqueous solutions of the active ingredient with suitable foaming agents.
  • Other topical carriers for vaginal application include pharmaceutically acceptable liquids in which the active ingredient is suspended or dissolved.
  • the active ingredient is incorporated into a pharmaceutically acceptable liquid that can be sprayed into the nose.
  • SP-22 can also be used to identify male animals who are good candidates for supplying sperm for artificial insemination. Since many livestock animals are reproduced by artificial insemination or embryo transfer, it is important to be able to identify males who are fertile as well as possessing desirable characteristics to pass on to the next generation. Techniques for reproducing animals by embryo transfer are described in U.S. Patents 3,854,479; 4, 816, 257f and 4,326,505, the entire contents of each of which are hereby incorporated by reference. By determining the amount of SP-22 in the sperm of a subject animal, the fertility of the animal can be predicted.
  • sperm proteins are affected by toxicants and pollutants.
  • the changes in SP-22 level are calibrated to predict the likelihood of the sperm having been rendered infertile because of exposure to the toxicant.
  • sperm are subjected to analysis for SP-22.
  • Adult (90 to 120 day old) male Sprague-Dawley rats are housed two to three per cage with laboratory-grade pine shavings as bedding. The rats are maintained under controlled temperature (22 °C) and humidity (40-50%) conditions, and are given Purina laboratory rat chow and tap water ad libi tum. Males are maintained in a 14-hour light, 10-hour dark schedule. Each male is numbered and randomly assigned to a treatment group. The test toxicant is administered either as a single intraperitoneal injection or as four daily injections.
  • the rats are killed in the caudal epididymides of each male is placed in a 35-mm culture dish containing 2 mL of Medium 199.
  • Detergent extracts representing 10-40 x 10 6 mL sperm, depending on treatment, are prepared and aliquots equivalent to 30 micrograms are electrophoresed in a mini, two-dimensional electrophoresis system (BioRad) for quantitative analysis of SP-22.
  • sperm are transferred to a microcentrifuge tube and washed twice by centrifugation (3000 x g, five minutes) in Dulbecco's — phosphate buffered saline, pH 7.2, with freshly added 0.2 M phenylmethylsulfonyl fluoride (PMSF) . After the final wash the sperm are extracted for one hour at room temperature with 1 mL of 40 mM n-octyl- -glucopyranoside in 10 mM Tris, pH 7.2, containing freshly added PMSF. Following a final centrifugation at 3000 x g, the supernatant is removed and frozen at -70 ° C.
  • PMSF phenylmethylsulfonyl fluoride
  • each extract is concentrated in 1 M Tris buffer by two centrifugations (3000 x g for 45 minutes at 4 ' C) in Centricon-10 units (Amicon) .
  • Protein concentration is determined using a Pierce protein assay kit . Sample volumes containing 30 ⁇ g protein are lyophilized, and protein is solubilized for 30 minutes at room temperature in 45 ⁇ L of sample buffer consisting of 5.7 g urea, 4 mL 10% NP-40, 0.5 mL ampholytes (70% 3-10, 30% 5-7) and 0.1 g dithiothreitol per 10 mL.
  • Isoelectric focusing (750 V, 3.5 hours) is carried out in gels consisting of 6.24 g urea, 1.5 g acrylamide (30% acrylamide, 1.2% bisacrylamide) , 2.25 mL 10% NP-40, and 0.65 mL ampholytes (60% 3-10, 40% 5-7) per 10 mL.
  • Molecular weight separation is carried out in 11% acrylamide gels (200 V, 45 minutes) . Gels are soaked in 50% methanol and silver stained.
  • a Kepler 2D gel analysis system (Large Scale Biology Corp., Rockville, MD) was used for background correction, spot matching, and spot area quantitation.
  • EDS comprises the fertilizing ability of sperm by affecting epididymal function directly. Fertilizing ability, sperm motility, serum testosterone, and tissue testosterone were evaluated. In addition, sperm proteins were extracted and analyzed by quantitative two dimensional gel electrophoresis. An 18 kD protein was well correlated with fertility. However, the authors felt that changes in this protein were not specific either to EDS itself or the dose that was tested.
  • the animals exposed to the known antiandrogen hydroxyflutamide were castrated and implanted with testosterone implants just prior to the first injection.
  • the vehicle controls for all treatments except hydroxyflutamide received daily injections of 30% DMSO in water.
  • the vehicle controls for the hydroxyflutamide animals were castrated, implanted with testosterone implants, and given daily injections of 15% ethanol.
  • the caput/corpus was frozen on dry ice for subsequent steroid extraction and testosterone assay.
  • Sperm were released from the epididymal tubule into insemination medium and held in a C0 2 incubator at 34 * C for no more than 15 minutes until insemination.
  • F SP.22 is the fertility at protein concentration SP-22
  • F 0 is the fertility at 0 protein concentration.
  • a and B are constants (A is the initial increase in fertility and B is the rate of exponential decay of the increase in ⁇ fertility) .
  • the dotted lines represent the 95% confidence limits around the fitted line.
  • an antibody to SP-22 can be used to evaluate the fertility of sperm in an epididymal sperm sample or an ejaculate. Since the antibody to SP-22 recognizes a single protein on immunoblots of gels of both human and stallion sperm extracts (not shown) , this antibody will most likely be applicable to evaluation of animals in which maximum fertility is important, e.g., cattle, horses, dogs, and humans, among other animals. Additional endpoints may be included to predict fertility.
  • Antibodies against SP-22 can be prepared by any conventional means, and can be either polyclonal or monoclonal. They may be raised in rabbits, mice, or other animals or tissue culture cells derived therefrom, or can be products or cells of human origin. They may also be produced by recombinant DNA technology either in a form identical to that of the native antibody or as chimeric molecules, constructed by recombination of antibody molecules of human or animal origin or in other forms chosen to make the antibodies most suitable for use in therapy.
  • either purified SP-22 or a peptide identical to the known sequence or a fragment thereof, e.g., to the N-terminal protein sequence, may be used to immunize animals.
  • a further possibility is to fuse one of the possible nucleotide sequences coding a fragment of SP-22 to the gene coding for Protein A, to express the antibody.
  • the antibody is then purified by affinity chromatography on a Sepharose column and used to immunize animals.
  • the monoclonal antibodies according to the present invention are prepared using conventional hybridoma techniques (Kohler et al . , (1975) Na ture 256 : 495; Kohler et al (1976) Eur. J. Immunol . 6 : 511) . After immunization, spleen cells alone or together with lymph node cells of the immunized animals are isolated and fused with a suitable myeloma cell line. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium and then cloned. The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding SP-22.
  • the desired clones are grown in bulk, either in suspension culture or in ascitic fluid, by injecting the cells into the peritoneum of suitable host mice.
  • the monoclonal antibodies produced by the hybridomas are then isolated and purified.
  • the monoclonal antibodies may also be immunized and used for the purification of SP- 22 in affinity purification procedures using an immunoadsorbent column.
  • antibody is meant to include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, anti- idiotypic antibodies to antibodies that can be labeled in soluble or bound form, as well as active fractions thereof provided by any known technique, such as, but not limited to, enzymatic cleavage, peptide synthesis, and recombinant techniques.
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen.
  • a monoclonal antibody contains a substantially homogeneous population of antibodies specific to antigens, which population contains substantially similar epitope binding sites.
  • Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having the variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Chimeric antibodies are primarily used to reduce immunogenicity in application and to increase yields in production, for example, where murine monoclonal antibodies have high yields from hybridomas but higher immunogenicity in humans, such that human murine chimeric monoclonal antibodies are used.
  • An anti-idiotypic antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody.
  • An anti-idiotypic antibody can be prepared by immunizing an animal of the same species and genetic type (e.g., a mouse strain) as the source of the monoclonal antibody with the monoclonal antibody to which an anti-idiotypic antibody is being prepared. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants, i.e., the antiidiotypic activity. See, for example, U.S. Patent No. 4,699,880, the entire contents of which are hereby incorporated by reference.
  • the anti-idiotypic antibody may also be used as an immunogen to produce an immune response in yet another animal, producing a so-called anti-anti-idiotypic antibody.
  • the anti- anti- idiotypic antibody may be epitopically identical to the original monoclonal antibody which induces the anti-idiotypic antibody.
  • SP-22, and related proteins of the present invention may be used to induce anti-idiotypic antibodies in suitable animals, such as BALB/c mice.
  • Spleen cells from such immunized mice are used to produce anti-idiotypic hybridomas secreting antiidiotypic monoclonal antibodies.
  • the anti-idiotypic monoclonal antibodies can be coupled to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunize additional BALB/c mice.
  • KLH keyhole limpet hemocyanin
  • Sera from these mice will contain anti-anti idiotypic antibodies that have the binding properties of the original monoclonal antibodies specific for SP- 22 or epitopes thereof.
  • antibody is also meant to include both intact molecules as well as active fractions thereof, such as, for example, those which are capable of binding antigen.
  • Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non specific tissue binding than an intact antibody, cf. Wahl et al . , J. Nucl . Med. 24 : 316-325, 1983.
  • compositions according to the present invention are prepared for administration by mixing the SP-22 antibody or its derivatives with physiologically acceptable carriers, stabilizers, and excipients, and prepared in dosage form, e.g., by lyophilization in dosage vials, foam, generating compositions, creams, jellies, lotions, suppositories, etc. ⁇
  • the amount of antibody to be administered will depend on the route of administration, the size of the patient, etc.
  • NAME U.S. ENVRONMENTAL PROTECTION AGENCY
  • B STREET: 401 M Street S.W.

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US6965016B2 (en) 1996-01-29 2005-11-15 The United States Of America As Represented By The U.S. Environmental Protection Agency Method for evaluating and affecting male fertility
US7211255B2 (en) 1996-01-29 2007-05-01 United States Of America The U.S. Environmental Protection Agency Contraceptives based on SP22 and SP22 antibodies
US6197940B1 (en) 1996-01-29 2001-03-06 U.S. Environmental Protection Agency Method for evaluating and affecting male fertility
EP1071712B1 (de) * 1998-04-23 2006-05-17 United States Environmental Protection Agency Sp22, ein mit fruchtbarkeit assoziiertes protein aus spermien, und ein verfahren zum abschätzen und beeinflussen der männlichen fruchtbarkeit durch dessen gebrauch
CN1300777A (zh) * 1999-12-22 2001-06-27 上海博德基因开发有限公司 一种新的多肽－人精蛋白45和编码这种多肽的多核苷酸
AU2003239901A1 (en) 2002-05-28 2003-12-12 Kathy M. Ensrud Crisp polypeptides as contraceptives and inhibitors of sperm capacitation
WO2007095530A2 (en) 2006-02-13 2007-08-23 U.S. Environmental Protection Agency Diagnostic kits to detect sp22 and sp22 antibodies

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