CA2098188A1 - Sperm cell surface protein ph-20 use in a contraceptive vaccine - Google Patents

Abstract

The disclosure relates to isolated DNA encoding all or a portion of a surface protein present in sperm of a mammal. This surface protein of sperm is essential for fertilization in the mammal.
Preferably, the sperm surface protein is the PH-20 protein.

Description

~)92/lOS69 2 0 g 8 1 ~ ~3 PCr/llS91/09098 SPERM CELL SURFACE PROTEIN.PH-20. USE IN A CONTACEPTIVE VACCINE

sac~rou~d Immunization of male and female animals with extracts of whole sperm cells is known to cause infertility (Tung, X., et al., of Re~roductive Immunol., l: 145-158 (1979) and Menge, A., et al., ~i~l~_o$ ReDroduction, Z0: 931-937 (1979)~. Also, men and women who spontaneously produce antisperm antibodies are infertile, but otherwise healthy (Bronson, R. et al., Fert. and Steril., 42: 171-183 (1984)). Although the ~ritical sperm antigens are unknown, these observations have led to the proposal that sperm proteins might be useful in the development of a contraceptive vaccine.
In mammalian species, sperm proteins have been proposed to have a role in sperm adhesion to the zona pellucida of the egg. In the mouse, it has been shown that a sperm surface galactosyl transferase is an adhesion protein that functions in acrosome-intact sperm binding to the zona (Shur, B.E., Galactosyl transferase as a recoqnition molecule durina fertilization and develo~ment, In: "The Molecular Biology of Fertilization," Eds. Schatten, H., and Schatten, G., Academic Press, pps. 37-71 (1989)). on rat sperm, there is a galactose receptor, (RTG-r), related to the hepatic asialoglycoprotein receptor, which could function through its lectin properties in 6perm binding to zona oligosaccharides (Abdullah, M., and Xierszenbaum, A.L., J. Cell Biol., 108: 367-375 (1989)). A boar sperm plasma membrane protein (AP2), distinct from galactosyl transferase, and a rabbit sperm protein have also been reported to have a role in ~perm-zona adhesion (Peterson, R.N. and Hunt, W.P., Gam. Res., 23: 103-118 (1989) and O'Rand, M.G., et al., Dev. 8iol., 129: 231-240 (1988)~.

WO92/10569 ~ 0~ gl ~ 8 PCT/~S91/090 ~ he guinea pig sperm surface protein PH-20 has been shown to have a required function in sperm adhesion to the extracellular coat ~zona pellucida) of the egg, a necessary initial step in fertilization. In male and female guinea pigs immunized with PH-20, 100%
effective contraception was obtained. Antisera from immunized females had high titers, specifically recognized PH-20 in sperm extracts and blocked sperm adhesion to egg zona pellucida, in_vitro. The contraceptive effect was long-lasting and reversible;
immunized females mated at intervals of 6-15 months after immunization progressively regained fertility.
Other sperm proteins tested as contraceptive immunogens include the sperm enzymes hyaluronidase, acrosin and lactate dehydrogenase C-4. Immunization of female animals with these enzymes had either no effect on fertility or partial effects on fertility, which were not large enough to make these proteins suitable as contraceptive agents. The high contraceptive e$fectiveness of PH-20, in the guinea pig, seems to depend on several of its specific properties, including its presence on the sperm surface, its strong immunogenicity and its essential role in fertilization.
Mammalian sperm-zona adhesion is in most cases species specific. Sperm from other mammalian species are like guinea pig sperm in that they can bind to the zona pellucida either before or after the acrosome reaction. The identification and isolation of sperm surface proteins essential for fertilization in species other than guinea pig would be useful for developing vaccines for effective immunization and providing long W O 92/10569 PC~r/US91/09098

2~9~

lasting contraception in those species. The lack of biochemical identification, isolation and cloning of candidate adhesion proteins of sperm has hindered scientists in developing effective contraceptive vaccines for humans as well as other mammalian species.

ummary ~f t~ Snv~ntion The present invention relates to isolated DNA
encoding all or a portion of a surface protein present in the ~perm of a mammal. This surface protein of sperm is essential for fertilization in the mammal.
Preferably the surface protein is the protein PH-20.
Such DNA sequences can be inserted, in expressible form, into a DNA expression vector to create a DNA
expression construct. Such a construct can be used to produce PH-20 protein for use in contraceptive immunization.
Current methods of contraception include physical and chemical methods such as surgical sterilization and drug treatments which alter the production of female hormones and interrupt the reproductive cycle. Each of these types of methods present their own distinct disadvantages. Sterilization, requiring surgery, causes permanent contraception and cannot, in general, be changed once performed. Barrier methods have lower theoretical effectiveness, low effectiveness in practice and are unacceptable to many potential users.
The chemical methods provide temporary contraception ~nd have been reported to cause an increased risk in cancer for women in certain age brackets. They must be taken repeatedly to ensure effectiveness and have 2 ~

~ctual or perceived side effects that make them unacceptable to many women. Chemical methods are not available for men and are not available for other mammals.
The present invention provides an alternative approach to contraception as a contraceptive vaccine that is longer lasting than the oral contraceptive pill yet is not a pexmanent form of contraception such as occurs with surgical sterilization. Hence, the present invention is as effective or more effective than other methods of contraception, is more convenient and utilizes the widely accepted medical practice of vaccination. In addition, it is more suitable than various other alternatives in that it is long lasting but not permanent.

Brief De~r~ptio~ of t~lprawiD~s Figure 1 is a diagram representing a partial restriction map of DNA encoding the guinea pig PH-20 protein, and the relative positions of 5 cDNA clones.
Figure 2 is a diagram representing the guinea pig cDNA sequence encoding the PH-20 protein, and the deduced amino acid sequence of the guinea pig PH-20 protein presented in single letter code.
Figure 3 is a diagram representing the murine DNA
sequence encoding the PH-20 protein.
Figure 4 is a diagram representing the human DNA
~equence encoding one form of the human PH-20 protein, and the deduced amino acid sequence presented in three letter code.

~'O92/10569 2 0 9 81 8 8 PCT/US91/09098 Figure S is a diagram representing the human DNA
seguence encoding a portion of a second form of the human PH-20 protein, and the deduced amino acid seguence presented in three letter code.

~etail-d D-s~riDtio~ of t~a Invention The PH-20 gene encodes a protein which is present on the ~urface of sperm cells and is essential for fertilization. The present invention is based, in part, on the isolation and cloning of DNA encoding the mammalian PH-20 protein and the discovery that the DNA
encoding PH-20 in one mammalian species is cross-reactive (i.e., hybridizable) with genomic DNA from all other mammals tested. The existence of these homologues in other mammalian species was an unexpected finding since mammalian sperm zona pellucida adhesion is, in most cases, species specific.

SDerm Surface ~roteins Sperm surface proteins which are useful in the present invention include surface proteins which are essential for fertilization. A sperm surface protein is defined as essential for fertilization if a monoclonal antibody to the protein or a polyclonal antibody raised against the purified protein, when bound to sperm, inhibits in vitro or in vivo fertilization or any step in in vitro fertilization.
The process of fertilization is defined as the binding or fusion of two gametes (sperm and egg) followed by the fusion of their nuclei to form the genome of a new organism. The surface protein can be located in the WO92/10569 0 9 S 1 8 8 PCT/US91/ogog~- ~

plasma membrane of sperm and/or the inner acrosomal membrane. It can be a protein or glycoprotein. The isolated surface protein used for immunization can comprise the entire surface protein or some portion of the protein (external to the cell) which is immunogenic. A preferred sperm surface protein is the PH-20 surface protein.

~rQductiQn a~d Puri~icatio~ pf Immunoaen A preferred method for producing sperm surface proteins for use as a contraceptive immunogen is by recombinant DNA technology. To produce the protein using this technology it is necessary to isolate and clone DNA encoding the protein, or an immunogenic portion thereof. Those skilled in the art are familiar with a variety of approaches which can be used in an effort to clone a gene of interest. However, having nothing more than the isolated protein of interest, ~uccess in such an effort can not be predicted with a reasonable degree of certainty.
In Example l which follows, Applicants' report the isolation and cloning of DNA encoding the guinea pig PH-20 gene. The method used to isolate DNA encoding the 3' portion of the PH-20 gene involved the screening of a cDNA expression library with polyclonal sera reactive with the PH-20 protein. Anchored PCR was used to isolate the 5' portion of the gene.

~ W092/10569 2 ~ 9 ~ 1 8 8 PCT/US9l/09098 Example 2 reports the ~urprising finding that a broad spectrum of mammalian genomic DNA contains DNA
sequenc~s which hybridize to guinea pig PH-20 eequences under the hybridization conditions described. In fact, cross-reacting sequences were identified in each of the mammalian ~amples analyzed.
The information presented in Exampes 1 and 2, enable one skilled in the art to i~olate and clone the ;PH-20 gene from any mammalian species. For example, a cDNA library is prepared from testis or spermatogenic cells isolated from a mammal of interest (e.g., feline, equine, canine, bovine, etc. ? . .T~is can be a time consuming process, but it is technicaily straightforward. One skilled in the art would approach this task with a high degree of certainty with regard to success.
Such a cDNA library is then creened using, for example, labeled guinea pig PH-20 DNA probes. DNA
encoding all or a portion of PH-20 is characterized by the ability to hybridize to such a probe seguence under hybridization conditions such as those described in Example 2. Nethods of labeling and screening by hybridization are very well known in the art. Positive clones are analyzed, and a full length gene is constructed by conventional methods. In light of Applicants' teaching that each of the 7 mammals analyzed contained cross-hybridizing ~equences, one skilled in the art would expect all mammals to contain W O 9ttlO569 PC~r/US91/09098 2o~l88 cross-hybridizing species. It is this methodology which enabled Applicants to isolate and clone the murine ~nd human PH-20 genes, as described in gre~ter detail below.
The cloned gene, or portions thereof which encode an immunogenic region of the PH-20 protein, can be expressed by inserting the coding region into an expression vector to produce an expression construct.
Many such expression vectors are known to those skilled in the art. These vectors contain a pro~oter for the gene of interest as well as additional ~ranscriptional and translational signals. Expression vectors ~or both eukaryotic host cells and prokaryotic host cells are widely available. The DNA expression construct is used to transform an appropriate host cell.
Eukaryotic, in particular mammalian, host cells are preferred for the expression of the sperm surface protein. It has ~een found, for example, that eukaryotic proteins frequently exhibit folding problems when expressed in prokaryotic cells. In addition, production of authentic, biologically active eukaryotic proteins from cloned DNA frequently requires post-translational modification such as disulfide bond formation, glycosylation, phosphorylation or specific proteolytic cleavage processes that are not performed in bacterial cells. This is especially true with membrane proteins. The sperm surface protein is produced using the transcriptional and translational components of the host cell. After an appropriate growth and expression period, the host cell culture is lysed and the sperm surface protein is purified from WO92/10569 2 0 9 ~ 1 8 8 PCT/US91/09098 the lysate. Lysis buffers typically include non-ionic detergent, chelating agents, protease inhibitors, etc.
From the solubilized cell extract, the sperm surface protein can be purified and isolated by physical and biochemical methods such as ultracentrifugation, column chromatography, high performance liguid chromatography, electrophoresis, etc. Alternatively, the sperm ~urface protein can be isolated by affinity chromatography using monoclonal or polyclonal antibodies (see Primakoff et al., ~iol. of Repxod. 38: 921-934 (1988)). Such methods for purifying proteins are well known to those skilled in the art.
As mentioned above, antigenic portions of the sperm surface protein are useful as immunogen, in addition to the full length protein. Antigenic fragments can be produced, for example, by proteolytic digestion of the full length protein, followed by isolation of the desired fragment. Alternatively, chemical synthesis can be used to generate the desired frag~ent starting with monomer amino acid residues.

Contraceptive ~accine once the sperm surface protein has been produced ~nd purified, ~ vaccine can be produced by combining the sperm surface protein with a suitable carrier for administration to a subject for immunization. A
vaccine can contain one or more sperm surface proteins.
Sperm ~urface proteins of the present invention can be combined with ~djuvants which contain non-specific stimulators of the immune system. Proper use of W092/l0569 2 U9 ~1 88 PCT/US9l/~9~

adjuvants can induce a strong antibody response to foreign antigens (i.e., sperm surface proteins). The action of adjuvants i8 not fully understood, but most adjuvants incorporate two components. one is a ~ubstance designed to form a deposit which protects the antigen from catabolism. Two methods of forming a deposit are to use mineral oils or aluminum hydroxide precipitates. With mineral oils, such as ~reund's adjuvant, the im~unogen is prepared in a water-in-oil emulsion. ~or aluminum hydroxide, the immunogen is either adsorbed to preformed precipitants or is trapped during precipitation. ~lternative delivery systems include liposomes or synthetic surfactants. Liposomes are only effective when the immunogen is incorporated 15 into the outer lipid layer; entrapped molecules are not seen by the immune system.
The second component required for an effective adjuvant is a substance that will stimulate the immune 6ystem nonspecifically. These substances stimulate the production of a large set of soluble peptide factors known as lymphokines. In turn, lymphokines stimulate the activity of antigen-processing cells directly and cause a local inflammatory reaction at the site of injection. A component of lipopolysaccharide known as lipid A is commonly used. Lipid A is available in a number of synthetic and natural forms that are much less toxic than lipopolysaccharides, but still retain most of the desirable adjuvant properties of the lipopolysaccharide molecules. Lipid A compounds are often delivered using liposomes. The two bacteria that are commonly used in adjuvants as non-specific ~092/10569 2 0 9 ~ 1 8 8 PCT/US91/09098 stimulants are Bordatellq pertussis and Mycobacterium ~uberculosis. When used as whole bacteria, they must be heat-killed prior to use. The immunomodulatory ~ediators Of ~ Ç=Y~i~ include a lipopolysaccharide component ~nd the pertussis toxin. The pertussis toxin has been purified and is available commercially.
tuberculosis is commonly found in complete Freund's adjuvant. The most active component of M. tuberculosis has been localized to muramyl dipeptide which is available in a number of forms.

Immunizations (Inoculation and Booster &kots) The subject to be immunized can be any mammal which possesses a competent immune system. Examples of subject ma~mals include humans and domestic animals (e.g., dogs, cats, cows, horses, etc.), as well as animals intended for experimental or other purposes (e.g., ~ice, rats, rabbits, etc.).
Two different criteria are important to consider in determining the proper dose for the initial immunization. First, the optimum dose to achieve the strongest response and second, the minimum dose likely to induce the production of useful polyclonal antibodies. Much of the injected material will be catabolized and cleared before reaching the appropriate target immune cell. The efficiency of this process will vary with host factors, the route of injection, the use of adjuvants, and the intrinsic nature of the surface protein injected. Thus, the effective dose delivered to the immune system may bear little relationship to the introduced dose and consequently W O 92/10569 PC~r/US91/0909~ 2 0 ~

dose requirements must be determined empirically.
These determinations can be readily made by one skilled in the art. Secondary injections and later boost can be given with amounts similar to or less than the primary injection.
The r~ute of injection is guided by three practical decisions: 1) what volume must be delivered;
2) what buffers and other components will be injected with the immunogen; and 3) how quickly should the immunogen be released into the lymphatics or circulation. For example, with rabbits, large volu~e injections normally are given at multiple ~ubcutaneous sites. ~or mice, large volumes are only possible with intraperitoneal injections. If adjuvants or particulate matter are included in the injection, the immunogen should not be delivered intravenously. If a ~low release of the inoculant is desired, the injections should be done either intramuscularly or intradermally. For immediate release, use intravenous in;ections.
Primary antibody responses often are very weak, particularly for readily catabolized, soluble antigens.
Hence, secondary or booster injections are required after the initial i~munization. A delay iS needed before reintroducing the protein into a primed subject.
A minimum of 2 or 3 weeks is recommended but greater intervals are possible. The antibody responses to secondary and subsequent injections is much stronger.
~igher titers of antibody are reached, but more i~portantly, the nature and quantity of the antibodies present in serum changes. These changes yield high-WO92/10569 2 0 9 31 8 ~ PCT/US91/09098 affinity antibodies. The intervals between secondary, tertiary and subsequent injections may also be varied, but usually need to be extended to allow the circulating level of antibody to drop enough to prevent r~pid clear~nce of newly injected antigen.
Subsequent booster injections will be required to increase reduced circulating ~ntibody for continued contraception. The actual intervals for these injections will differ form pecies to species.
However, the intervals can be determined by one skilled in the art by monitoring serum levels of sperm surface protein antibodies.
In another embodiment, subjects can be administered with alloantisera, or monoclsnal antibodies, directed to a sperm surface protein to achieve contraception. The alloantiserum is raised in another individual of the same species, isolated from the serum of the individual and prepared in a suitable carrier for injection into the recipient subject.
Those skilled in the art are familiar with methods for preparing and formulating monoclonal antibodies for administration.
The present invention is further explained in the following exemplification.

'~ O ~ X ~

XAMP~E8 B~ample 1: I~ol~ion o~_pNA Enco~in~ Gu~ ic ~-20 ,LibrarY ConstryctiQn--~nq ~creen~
A population of guinea pig ~esticular cells, enriched for sper~atoqenic cells on a Percoll gradient was u~ed for the isolation of ~permatogenic cell total RNA. The pelleted cell6 were lysed with detergent in the presence of ~anadyl-ribonucleoside complexes (VRC) in 0.5-1.0 ~1 of solution containing lOmM Tris (p~
8.6), 0.5% NP-40, 0.14 M NaCl, 1.5 mM MgCl2 and lOmM
VRC. After pelleting cellular debris, O.5 volu~e of 2X
Proteinase K buffer (2X=0.2M Tris (pH 7.5), 25 m~ EDTA
tpH 8.0), 0.3 M NaCl, and 2.0% SDS) and 200 ~g/ml Proteinase ~ was added to the supernant. PolyA+ RNA
was purified from the total RNA by oligo-dT cellulose chrom~tography. cDNA was synthesized using standard methods. Size selected cDNA (0.5-7kb) was ligated with lambda gtll arms and packaged into lambda coat proteins, utilizing ~its and protocols from ~mersham Corporation.
The unamplified library was plated at 20,000 plaques/150 mm plate for Ecreening. A single ; nitrocellulose filter from each plate was immunoblotted with rabbit anti-PH-20 polyclonal antiserum, raised against affinity-purified PH-20 protein (Primakoff et a~, Biol, Reprod. 38:921-934 tl988)), and diluted 1/500 in TBST (lOmM Tris (pH 8.0), 0.15 M NaCl, 0.05%
Tween-20) containing 2 mg/ml E. coli protein. The E.
coli protein was prepared by pelleting an overnight W092tlO569 ~ PCT/US91/09098 culture of Y1090 cells, resuspending the cells in a minimal volume of T~ST and freezing in liquid nitrogen.
The thawed cells were sonicated and the protein concentration determined using the BCA reagent (Pierce Chemical). Six positive plaques were detected with an anti-rabbit IgG alkaline phosphatase-conjugated 6e~0nd ~ntibody (Promega Biotec). Size of the fusion protein ~ade by plaque-purifled positive clones was determined to ~ary between 118-157 kD as determined by the analysis of E. coli extracts containing the fusion protein on 5DS-PAGE. Inserts from the six positive clones were subcloned into pUCl9 and sequenced at least partially.
Two of the inserts were confirmed to code for the PH-20 protein by locating the sequences of two PH-20 tryptic peptides in their derived amino acid 6equence.
Both of these inserts (gpPH-20-1, nucleotide (nt) 1016-2152 and gpPH-20-2, nt 1010-2125, Figure 1 and 2) contained a long (-925 nt) open readinq frame, a stop ~odon, a 3' untranslated region and a polyA tail. Thus these two inserts were concluded to represent the 3' end of a cDNA for PH-20. The other four antibody-positive lambda clones were unrelated to PH-20.
The 5' portion of the PH-20 cDNA was cloned utilizing anchored PCR following the protocol of Frohman et al. (Proc. Natl~cad. Sci. USA ~5: 8998-9002 (1988)). PolyA+ RNA $rom spermatogenic cells (2~g in 10 ~1 dH20) was heated to 65C for 3 min and then reverse transcribed by adding 4 ~1 10 X RTC buffer (lX
buffer is 50 mM Tris (pH 8.3), 50 mM KCl, 4 ~M

WO92/10569 PCT/US91/0~09~
2Y81 &~3 dithiothreitol, 10 mM MgCl2)), and 4 ~l 10 ~M stoc~ of each dN~P (1 ~M final), 2 ~l of 80 mM sodium pyrophosphate ~4 mM final), 1 ~l (40 units) of RNasin tPromega Biotec~, 40 pmol PH-20 specific primer (PH-20-RT), 18 units AMV reverse transcriptase tLife Sciences)and 40 ~Ci 32P-dCTP in 40 ~l total volume. After 1 hour of incubation at 42C, an additional 1 ~l of reverse transcriptase was ædded and incubation continued for a second hour. The PH-20-RT primer was a 17 nucleotide (nt) oligomer (nt 1242-1258, ~igure 2), ~250 bases downstream from the 5' end of the insert gpP~-20-1 (Figure 1).
The single strand cDNA was separated from excess PH-20-RT by column chromatography, tailed with polyA
and diluted to l.0 ml. Second strand synthesis and PCR
amplification were performed with a GeneAmp kit (Perkin Elmer Cetus) in a 100 ~l reaction containing 10 ~l of the reverse transcription product, 20 pmol (dT) 17 adapter, 50 pmol adapter and 50 pmol PH-20-ANP primer.
The PH-20-~MP primer was a 17 nt oligomer (nt 1202-1218, Figure 2) located upstream from the PH-20-RT
primer. The PCR product was purified from unincorporated primers and free nucleotides by spin column chromatography (columns from Boehringer-Mannheim). It was subsequently digested with HgiA I
and Sal I, gel purifi~d and ligated into p81ueseript digested with ~ct I and Sal I. The major PCR product was 1.2 kb, and Southern Blot analysis confirmed that this bAnd hybridized with the labeled insert gpPH-20-1.
The major PCR products from three separate reactions ~092/10569 PCT/US91/09098 2 ~ 8 ~

were cloned and one insert from each of the three reactions was ~equenced (gpPH-20-3, nt 1-1175, gpPH-20-4, nt 24-1175 and gpPH-20-5, nt 2~5-1175).
The complete cDNA sequence and the deduced amino acid sequence were obtained from the five cDNA in~erts (Figure 2) that were sequenced in their entirety on both ~trands. The cDNA sequence contains a 354 nt S' untranslated region, a 1590 nt open reading frame, and a 208 nt 3' untranslated region. The derived amino acid sequence contains all the tryptic peptide sequences obtained from purified PH-20, confirminq that the cDNAs are authentic PH-20 clones. Hybridization experiments indicated that guinea pig genomic DNA
contained a single gene for PH-20. Computer searches revealed no significant homology of the guinea pig PH-20 amino acid sequence with other known 6equences.

xampl6 2: PR-20 ~omologues ~n Othe~ Mamm~ p-Ci~3 To determine if there is a homologue of the PH-20 gene in the genomic DNA of other species, cross ~pecies Southern blots were performed. Genomic DNA was isolated from guinea pig, rat, rabbit, mouse, and hamster spleens by detergent lysis-Proteinase R
digestion. Other DNA samples (i.e., human, monkey and chicken) were provided by other investigators at the University of Connecticut Health Center. DNA from salmon sperm and bovine thymus were purchased from SigDa and reconstituted at 1 mg/ml in TE (10 mM Tris (pH 8.0), 1 mM EDTA (pH 8.0)). All species DNA's (10~g) were cut with restriction enzymes and ~eparated on a 1% agarose gel. The Southern transfer was carried W092/iO569 PCT/US91/0909P

~!3~

out by capillary transfer onto nylon membrane. The membranes were prehybridized in a solution consisting of 6XSSC, lX Denhardt's, 250 mg/ml 8almon ~perm DNA, 1%
SDS, ~nd 50 mM NaP0~ ~pH 7.4), for 1-2 hours at 65C.
The ~embranes were hybridized overnight at 55C in prehybridization buffer plus 2 x lo6 cpm/ml probe.
Probes were prepared by the random hexamer method. The blot was washed 3 X 5 min in 2XSSC + 1.0% SDS at room temperature, 2 X 30 min in 2XSSC + 0.1% SDS at room at 50C, and 2 X 30 min in lXSSC + 0.1% SDS at 60C. The blot was wrapped in plastic wrap and exposed to film with an intensifying screen at -70C.
The blots were probed with a mix of labeled gpPH-20-3 and gpPH-20-2. The Southern blots exhibited a weakly hybridizing band at -10 kb for chicken DNA and strongly hybridizing bands for mouse, rat, hamster, rabbit and human DNA. In addition, hybridization was observed with bovine and monkey DNA.

B~mDle ~ ol~tion of ~Nb Enoodin~ Mou~e P~o2 0 PolyA+ RNA was isolated from murine round spermatids and used to produce a cDNA library in lambda J using conventional ~ethods. The library was screened using a labeled full length guinea pig PH-20 cDNA
probe. The probe was produced by first isolating guinea pig PolyA+ RNA. An oligo-dT primer was hybridized to the poly(A) tract and reverse transcriptase was used to generate a first cDNA ~trand.
Two oligonucleotides, a first being complementary to a portion of the guinea pig PH-20 5' untranslated region ~ ~92/10569 PCT/US91/09098 2093 ~ ~

and a second being complementary to the 3' untranslated region, were added to the reaction mixture and a full length double stranded DNA sequence containing the entire coding region was generated by polymerase chain S reaction. The product of this reaction was a double ~tranded DNA fragment of between 1.5 1.6 kb. The rag~ent was cloned and the cloned fragment was analyzed to confirm that it did, in fact, encode the guinea pig PH-20 protein. Labeled probe was generated - 10 from this clone by conventional methods.
The murine cDNA library was screened using the guinea pig probe described above. Two positive clone~
were identified. The two clones represent about 1500 base pairs of DNA. Neither of the clones contained sequences from the 5' portion of the cDNA. Anchored PCR using a set of primers complementary to the 5' end of one of the positive clones was used to clone the 5' portion of the murine gene. The DNA sequence is set forth in Figure 3.

Bxample ~: Isolation o~ DNA Encodin~ ~u~n P~-20 DNA encoding human PH-20 was isolated and cloned by screening a human testis library in lambda gtll.
The library was plated at a density of about 3,000 plaques per 90mm plate. Phage plaques were transferred to duplicate filters and screened with a mix of two radioactively labeled DNA probes, a mouse PH-20 cDNA
and a guinea pig PH-20 cDNA. More specifically, the guinea pig probe was the labeled full length guinea pig PH-20 probe described above and the murine clone was one of the two murine clones which lacked sequences WO92/10569 PCT/US91/09Og 2~9~

from the 5' end of the murine cDNA.
Positive plagues that hybridized with the mix of two probes were picked and purified. The cDNA inserts were subcloned and the DNA sequence determined using ~tandard techniques. Two cDNA clones were obtained.
Each ~f the two encode a different form of human PH-20.
one human clone is desiqnated H18 lFigure 4) and one is designated H16 (Figure 5).
H18 is a full-length clone which contains an open reading frame of 510 amino acids and short 5' and 3' untranslated regions. The protein encoded in the open reading frame of H18 is 59~ identical and 74S similar (includes conservative substitutions) to guinea pig PH-20.
X16 is a partial length clone that encodes the carboxyl terminal half of human PH-20. Nucleotide 1 in H16 corresponds with nucleotide 814 in H18. The seguence of H16 from nucleotide 1-781 is identical to the sequence of H18 from nucleotide 814-1594; the sequence of H16 beginning at nucleotide 782 and continuing to nucleotide 1675 is different from the sequence of H18 beginning at nucleotide 1595 and continuing to nucleotide 1696. In terms of the encoded PH-20 protein, the partial protein encoded by H16 is identical to the protein encoded by H18 between amino acids 236-496 (amino acid numbering based on H18 ~equence). H16 then encodes amino acids 497-511 and H18 encodes amino acids 497-510 and the sequences are different at each residue.

2()9~1~8 ession and Purification of Human PH-20 The full-length clone for PH-20 (Hl8) was subcloned into two E. coli expression ve~tors, pMAL-p and pMAL-c (New England Biolabs, Beverly, MA). In both S ve~tors, PH-20 is made as a fusion protsin, the N-terminal fusion partner being the maltose binding (MBP) protein of E. Ç~li. In pNAL-p, the encoded ~BP (which is normally a periplasmic protein) has its usual signal sequence which results in the MBP-PH-20 fusion being targeted to the periplasm. For fusion proteins that can be successfully exported to the periplasm, this location has the advantage that disulfide bonds fo~m (twelve cysteines are present in human PH-20) yielding a potentially more immunogenic protein. In pMAL-c, the signal sequence for M3P is not present, and the fusion protein is found in the cytoplasm and does not form disulfides. Human PH-20 is produced from both pMAL-p and pMAL-c. However, in pMAL-p carrying strains, the amount of hPH 20 made is low, whereas in pMAL-c carrying strains, the amount of PH-20 made is high (the fusio~ protein is the major band in an E. coli extract on a Coomassie blue-stained SDS-PAGE gel). To purify the human PH-20 fusion protein, the NBP-PH-20 fusion protein is bound to an amylose resin (to which MBP
binds) and eluted with maltose.

WO92/10569 ~J~ 8 1 8 g PCT/US91/0909 Equiva~e~t~
Those Rkilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiment of the invention described ~pecifically herein. Such equivalents are intended to be compassed in the scope of the following claims.

Claims (23)

?? 92/10569 PCT/US91/09098

1. Isolated DNA encoding all or a portion of a surface protein present in the sperm of a mammal, the surface protein being essential for fertilization in the mammal.

2. Isolated DNA of Claim 1, wherein the surface protein is PH-20.

3. Isolated DNA encoding all or a portion of a mammalian PH-20 sperm surface protein.

4. Isolated DNA of Claim 3 wherein the isolated DNA
encodes a human PH-20 sperm surface protein.

5. Isolated DNA of Claim 4, wherein the DNA encoding all or a portion of the human PH-20 protein is characterized by the ability to hybridize to a DNA
sequence selected from the DNA sequences of Figure 4 and Figure 5.

6. A DNA expression construct comprising DNA encoding all or a portion of mammalian PH-20 protein in expressible form.

7. A DNA expression construct of Claim 6, wherein the encoded PH-20 protein is human PH-20 protein.

8. A DNA expression construct of Claim 7, wherein the DNA encoding all or a portion of human PH-20 protein is characterized by the ability to hybridize to n DNA sequence selected from the DNA
sequences of Figure 4 and Figure 5.

9. A method for producing recombinant PH-20 protein comprising transforming a cell with a DNA
expression construct encoding PH-20 protein in expressible form.

10. A method of Claim 9, wherein the PH-20 is human PH-20 protein.

11. A method of Claim 10, wherein the DNA encoding all or a portion of human PH-20 protein is characterized by the ability to hybridize to a DNA
sequence selected from the DNA sequences of Figure 4 and Figure 5.

12. A PH-20 protein produced by transforming a cell with a DNA expression construct containing DNA
encoding a mammalian PH-20 protein in expressible form.

13. The mammalian PH-20 protein of Claim 12, wherein the mammalian PH-20 protein is human PH-20 protein.

14. The human PH-20 protein of Claim 12, wherein the DNA encoding the human PH-20 protein is characterized by the ability to hybridize to a DNA
sequence selected from the sequences of Figure 4 and Figure 5.

15. Essentially pure mammalian DNA encoding PH-20, or a portion thereof, the isolated DNA being characterized by the ability to hybridize to a DNA
sequence selected from the group consisting of the DNA sequences of Figure 2, Figure 3, Figure 4 and Figure 5.

16. A method of contraception for a non-Cavia porcelles mammal comprising the active immunization of said mammal with a composition comprising an isolated surface protein present in sperm of the same species of said mammal wherein aid surface protein is essential for fertilization in said species.

17. A method of Claim 16 wherein said surface protein is essential for sperm-egg adhesion.

18. A method of Claim 16 wherein the mammal is a human, canine, feline and bovine.

19. A vaccine for inducing contraception in a non-Cavia porcelles mammal comprising a composition including an isolated surface protein present in sperm of the same species of said mammal to be vaccinated wherein said surface protein is essential for fertilization in said species.

20. A vaccine of Claim 19 wherein the mammal is selected from the group consisting of human, canine, feline and bovine.

21. A vaccine of Claim 19 wherein said isolated surface protein is combined with an adjuvant.

22. A method of contraception for a non-Cavia porcelles mammal comprising passive immunization of said mammal with alloantisera directed against a surface protein present in sperm of the same species of said mammal wherein said surface protein is essential for fertilization in said species.

23. A method of Claim 22 wherein the mammal is selected from the group consisting of human, canine, feline and bovine.