IE914128A1 - Gestational agents for controlling cell proliferation - Google Patents

Abstract

The present invention relates to substantially purified agents normally expressed during mammalian pregnancy that may be used to control the proliferation of cells, and, in particular, provides for proliferative agents as well as antiproliferative agents. The antiproliferative agents may be used to limit undesirable proliferation of cells, for example, in the treatment of cancer. The proliferative agents may be utilized to increase cell proliferation and may be used, for example, in the treatment of infertility.

Description

GESTATIONAL AGENTS FOR CONTROLLING CELL PROLIFERATION 1. INTRODUCTION The present invention relates to 5 substantially purified agents normally expressed during mammalian pregnancy that may be used to control the proliferation of cells, and, in particular, provides for proliferative agents as well as antiproliferative agents. The antiproliferative 10 agents may be used to limit undesirable proliferation of cells, for example, in the treatment of cancer.

The proliferative agents may be utilized to increase cell proliferation and may be used, for example, in the treatment of infertility. 2. BACKGROUND OF THE INVENTION Human gestation is divided into two developmental stages: the embryonic period, which extends from conception to the end of the eighth week, followed by the fetal period, which lasts until parturition. The embryonic period is characterized by the genesis of virtually all essential structures. During the fetal period, these structures grow and become more elaborate (see, for review, Moore, 1977, . . in The Developing Human, second edition, W. B. Saunders Company, Philadelphia). Therefore, it is during the embryonic period when the developing conceptus is in greatest flux, passing through numerous changes to recreate the human blueprint and to establish the maternal connection necessary for its survival.

Very early in the embryonic period, the fertilized egg, or zygote, undergoes a number of cell 35 divisions to form a ball of about 15 small cells, called the morula. The morula enters the uterus and develops an inner cavity, thereby becoming a blastocyst consisting of (1) an inner cell mass which gives rise to the embryo; (2) a blastocyst cavity; and (3) an outer layer of cells, called the trophoblast.

About five or six days after conception, the blastocyst attaches to the endometrial epithelium of the uterus and the trophoblastic cells invade the uterine wall.

With time, the actively erosive trophoblast invades the endometrial stroma, and the blastocyst is gradually engulfed by the endometrium (Id., p. 33).

The trophoblast differentiates into two types: cytotrophoblast and syncytiotrophoblast. The syncytiotrophoblast is adjacent to the developing embryo and becomes a multinucleated protoplasmic mass in which no cell boundaries are discernible (Id., p. 34). Isolated spaces, called lacunae, appear in the syncytiotrophoblast at about day 9 and become filled with a fluid consisting of maternal blood and secretions. This fluid, or embryolymph, provides nutrition to the developing embryo and marks the beginning of the uteroplacental circulation. Eventually, the endometrium forms the maternal part, and the trophoblast forms the fetal part, of the placenta (Id., p. 36).

Once the primitive placental circulation is established, the embryo begins to develop at astonishing speed. By 20 days, the brain and spinal cord have begun to form. At about 22 days, the embryonic heart begins to beat. At 27 days arm and leg buds have appeared. By 30 days, the eyes and nose are forming. By 40 days, the arms are bent at the elbow, early fingers and ears are apparent, and the embryo is only one centimeter long.

V.

This period of rapid development is accomplished by a carefully regulated program of cell division and differentiation which is, at this time, incompletely understood. An interest in the signals involved in initiating or terminating embryonic development has prompted analysis of hormones and cytokines associated with placental, embryonic, or fetal tissue. A brief list of some results of such studies follows.

Two we11-documented protein products of the placenta are (1) human chorionic gonadotrophin (hCG) and (2) human chorionic somatomamotropin (hCS), also known as human placental lactogen (hPL) (Id., p. 105). hCG is produced by the trophoblast, and acts to prevent degeneration of the corpus luteum, an ovarian structure that produces progesterone. During early pregnancy, circulating hCG levels increase linearly until eight weeks of gestation, reaching a plateau at between nine and ten weeks of gestation,and thereafter declining until term. This secretory pattern is considered to be an important indicator of normal trophoblast development. In fact, if circulating hCG levels continue to increase beyond ten weeks of gestation, trophoblastic neoplasia, such as a hydatiform mole, should be suspected (Delf, 1957, Obstet. Gynecol. 9:1). On the other hand, a premature plateau and decrease in hCG level generally indicate early pregnancy failure (Aspillaga et al., 1982, Am.

J. Obstet. Gynecol. 147:903).

Yoshida, Japanese Patent No. 59078694, May 7, 1984, reports the identification of a cobaltactivated substance in fetal or placental tissue which inhibits the action of a carcinogenic protein-forming enzyme.

Japanese Patent No. 2215730, August 28, 1990, reports the isolation of the cytokine transforming growth factor-beta (TGF-beta) from human placenta.

Massague (1983, J. Biol. Chem. 258:1361413620) reports the binding of epidermal growth-factor like transforming growth factor to epidermal growth factor receptors in human placenta membranes.

Roberts et al. (1985, Proc. Natl. Acad. Sci. IQ U.S.A. 82:119-123) reports that TGF-beta may be isolated from human placenta, among other tissues. It was observed that the response of cells to TGF-beta appeared to be bifunctional, in that TGF-beta was able to stimulate reversible transformation of murine fibroblasts, but was also able to inhibit anchoragedependent growth of normal rat kidney fibroblasts and of human tumor cells by increasing cell cycle time.

Letnansky (1987, Immunology 175:681 reports the isolation and characterization of a bovine placenta protein which specifically inhibits the proliferation of tumor cells. This protein, termed decidua inhibitory factor (DIF), was estimated to have a molecular weight of about 60 kD by SDS-PAGE.

Barnea et al. (1989, Placenta 10:331-3441 report that human embryonal extracts modulate placental function in the first trimester. They observed that extracts of specific tissues were capable of decreasing or, alternatively, increasing hCG secretion. In particular, water extract of 3θ embryonal lung was found to produce a twofold decrease in hCG production by placental explants; a protein having a molecular weight less than 8000 daltons appeared to be the active agent, but was not purified.

Plowman et al. (1990, Mol. Cell. Biol. :1969-1981) report the isolation of a gene encoding I amphiregulin (AR), a cytokine that is evolutionarily related to epidermal growth factor and to transforming growth factor alpha (TGF-alpha). AR was observed to be an 84 amino acid protein capable of acting as a bifunctional growth modulator, being able to promote the growth of normal epithelial cells and also to inhibit the growth of certain carcinoma cell lines. Human placenta and ovaries were found to express significant amounts of AR-encoding RNA. The unglycosylated AR precursor minus the signal peptide was predicted to have a molecular weight of about 25,942 D.

Further toward understanding the control of development, and in addition to the abovementioned hormones and cytokines, several lines of evidence suggest that there is an interdependence in mammals between the trophoblast and the embryo. Fetal death in both sheep and rats results in a decline in placental lactogen secretion (Ramsay et al., 1985, Biol. Neonate 47:42; Albrecht et al., 1984, Endocrinol. 107:766; Taylor et al., 1984, Res. Vet. Sci. 35:22; Robertson et al., 1984, Endocrinol. 114:22). Barnea et al. (supra) investigated the possible role of embryonic visceral organs as trophoblastic function regulators by examining the effects of dilute organ extracts upon in vitro secretion of hCG, and found an hCG-suppressive effect in certain fractions. It therefore appears that the developing embryo is not entirely subject to control 30 by its environment; rather, the embryo itself appears to play an active role in the physiology of pregnancy. 3. SUMMARY OF THE INVENTION The present invention provides for gestational agents that may be used to control cell proliferation. It is based, at least in part, on the discovery that several agents produced by the developing embryo appear to play an important role in achieving a system of checks and balances which regulate proliferation and differentiation of cells during pregnancy.

In particular embodiments, the present invention provides for a substantially purified protein having a molecular weight of less than about ,000 daltons and preferably about 6,500-7,000 daltons that is expressed in mammalian embryos and that has an antiproliferative effect on certain cancer cells and on certain embryonal cells. This protein, termed JDK protein, was observed to exert an antiproliferative effect on all cancer cell lines tested, and was found to prevent tumor formation and to improve the survival of nude mice inoculated with fibrosarcoma cells. The antiproliferative gestational protein of the invention may be utilized in the prevention and treatment of cancer. It may also be used as a contraceptive to prevent pregnancy.

In alternative embodiments, the present invention provides for a substantially purified protein having a molecular weight of less than about 3,000 daltons which is expressed in mammalian embryonal tissue and has a proliferative effect on morular cells. This gestational proliferative agent of the invention, termed GPA-1 has further been observed to increase the secretion of human chorionic gonadotrophin by placental explants, to promote the development of a morula into a blastocyst and to facilitate implantation. In further embodiments, the present invention provides for a second gestational proliferative agent, termed GPA-2, that has a molecular weight greater than 20,000 daltons. These proliferative agents may be used in the treatment of infertility and/or hypoproliferative disorders. 4. DESCRIPTION OF THE FIGURES 5 FIGURE 1. Results of HPLC analysis of the <8,000 MW fraction from spinal cord showing three dominant bands.

FIGURE 2. Results of PAGE analysis of the <10,000 MW fraction from spinal cord.

FIGURE 3. Superfusion apparatus. 1. GROWTH CHAMBERS: They contained the tissue being cultured. 2. TUBING SYSTEM: Silicone permitted the gas to get in the tubing system. 3. HEAT/GAS EXCHANGER: It maintains the thermal environment in and around the growth chambers. 4. GAS TRANSFER: Its achieved by flooding the system with the desired gas.

. INCOMING GAS: (5% C02, 95% air). 6. ENTRANCE OF WARM WATER: (37° C) . 7. EXIT OF WATER: After heating the exchanger. 8. ON: Starts the pump 9. MINIMUM: 0.01 ml/min.

. MAXIMUM: 3 ml/min. ii. DIRECTION: The direction of the lighted arrow indicates direction of fluid through the pump tubing. 12. FLOW RATE: From 00>99 represents a percentage of normal pumping rate. 13. AT THIS POINT, another peristaltic pump is added in order to add GnRH, or some other factors. 14. DIVISION of the principal tube.

. MEDIUM RESERVOIR 16. PERISTALTIC PUMP.

FIGURE 4. A. Suppressive effect of the <8,000 MW fraction of extract on secretion of hCG by placental explants. Efferent sample fractions were collected every 2.4 minutes and were sequentially numbered.

B. Suppressive effect of <8,000 MW fraction of extract on secretion of hCG by placental explants.

C. Sample identical to that of (B) lost the ability to suppress hCG secretion after heat inactivation.

FIGURE 5. Ability of various dilutions of total extract to suppress hCG secretion by placental explants. The following dilutions of extract in 0.05 M Tris-HCl PMSF-DTT (pH 7.4) were used: 1/20, 1/200, and 1/2000. Buffer alone was used as a control.

FIGURE 6. Suppressive activity of the <8,000 MW fraction of extract on the proliferation of cancer cell lines. The number of cells in buffer-treated cultures serves as a control. 20 FIGURE 7. Percent suppression of MCF-7 proliferation as a function of increasing dosage of <8,000 MW fraction protein.

FIGURE 8. Suppressive effect of embryo extract on mouse embryonal development in vitro. A. Percentage of untreated control embryos attaining the morular stage.

B. Percentage of untreated control embryos exhibiting hatching/adhesion.

FIGURE 9. The effect of various molecular weight fractions of extract on nude mice survival following inoculation with fibrosarcoma. Fraction 1 corresponds to MW <3,000; Fraction 2 corresponds to MW <30,000 MW; Fraction 3 corresponds to MW <8,000; Fraction 4 corresponds to MW <10,000; Fraction 5 corresponds to MW <100,000; and Fraction 6 corresponds to buffer-treated control.

FIGURE 10. A <3,000 MW fraction of embryo extract 5 increased secretion of hCG by placental explants.

FIGURE 11. Results of HPLC analysis of the <3,000 D molecular weight fraction associated with proliferative activity.

. DETAILED DESCRIPTION OF THE INVENTION The present invention provides for gestational agents that may be used to control cell proliferation. It is based, at least in part, on the theory that pregnancy operates, figuratively speaking, like a reversible cancer. The products of conception, similar to cancer, are invasive, penetrate the circulation and metastasize, express similar surface antigens, and secrete certain cancer-related compounds, such as alphafetoprotein and carcinoembryonic antigen. Furthermore, the conceptus, like a tumor, is not rejected by the body, but rather harnesses maternal resources to secure its well-being. Unlike cancer, however, the invasiveness and tolerance associated with pregnancy are reversible at almost any time.

It is an object of the present invention to isolate agents that operate to control the development of the embryo such that proliferation, invasiveness, 30 and differentiation may occur without substantially injuring the maternal host. It has been discovered that several agents produced by the embryo appear to play an important role in its development. The various embodiments of the invention relate to such agents, and their use, for example, in the treatment of cancer and infertility, or as a contraceptive.

For purposes of clarity of description and not by way of limitation, the detailed description of the invention is divided into the following subsections: (i) preparation of gestational antiproliferative agent; (ii) preparation of gestational IO proliferative agents; (iii) antibodies of the invention; (iv) the use of gestational agents in cancer therapy; (v) the use of gestational agents in controlling fertility; and (vi) additional utilities of the invention. .1. PREPARATION OF GESTATIONAL ANTIPROLIFERATIVE 20 AGENT_ The present invention provides for a gestational antiproliferative agent that is comprised in an extract of mammalian embryonal tissue and that has a molecular weight less than about 10,000 daltons.

In particular, the agent has a molecular weight between about 6,500 and 7,000 daltons, and is termed JDK.

The gestational antiproliferative agent JDK may be prepared from any suitable mammalian embryo.

As the size of the embryo will affect the ease with which individual organs are identified and isolated, it is desirable that the embryo be obtained from a relatively large mammal, such as, for example, but not by way of limitation, a human, non-human primate, horse, cow, sheep, or pig, etc. If obtained from a human, the embryo may be obtained, after proper consent, from the products of an elective or spontaneous abortion.

It is desirable further that the embryo be of a developmental stage in which rapid development is occurring or has just begun to taper off. For example, in humans, it may be desirable to utilize an embryo which is between six and nine weeks of gestation. For non-human animals, embryos at a stage comparable to weeks six through nine of human gestation may preferably be used.

Any organ of the embryo may be utilized. In preferred embodiments of the invention the brain and spinal cord, (i.e. neural elements) or adrenal gland are preferably used. Visceral organs, such as lung, liver, or kidney may also desirably be used.

The gestational antiproliferative agent, JDK, of the invention may be prepared as follows according to a specific, nonlimiting embodiment of the present invention.

Embryonal tissue may be minced and placed in cold Tris-HCl at pH 7.4 containing 10 mM dithiothreitol (DTT) and 2 mM phenyl-methyl-sulfonyl-fluoride (PMSF), then sonicated on ice for about 60 seconds and then centrifuged at about 2400 rpm for about ten minutes to remove cellular debris. An initial amount of embryonal tissue weighing about 50 mg may, for example, be processed in about 1 ml of buffer and may yield about 1 ml of supernatant. The supernatant from the centrifuged material may then be separated into 30 fractions based on molecular weight, using, for example, the Filtron System Omega Cell (Biolab), 30 mm diameter, under nitrogen, and the fractions containing molecular weights less than about 10,000 daltons may desirably be obtained. This fraction may, preferably, be further purified by polyacrylamide gel I electrophoresis (PAGE). That portion of the resulting gel corresponding to molecular weights between about 3,000 and 8,000 daltons, and preferably between about 6,500 and 7,000 daltons may then be separated from the rest of the gel and eluted to produce the gestational antiproliferative agent JDK. Alternatively, the fraction may be further purified by HPLC as described in Section 6.1.3., infra, in which case three major bands may be expected to be produced, as set forth in IQ Figure 1.

Accordingly, the present invention provides for a substantially purified protein having a molecular weight less than about 10,000 daltons, and preferably between about 6,500 and 7,000 daltons, that is normally expressed in mammalian embryonal tissue and that has an antiproliferative effect on certain cancer cells and certain embryonal cells. The fact that it is normally expressed in a mammalian embryo is distinct from the substantially purified form of the protein recited, as the protein does not normally, (i.e. naturally) occur in substantially purified form.

The present invention further provides for a substantially purified protein, as described supra. that corresponds to the protein comprised in peak I, peak II, or peak III of the chromatogram depicted in Figure 1.

The gestational proliferative agent, or JDK, of the invention may also be prepared by any other method known in the art, including, but not limited to, chemical synthesis or recombinant DNA technology.

Furthermore, the present invention provides for the preparation and use of fragments or derivatives of JDK, including fragments produced chemically, by recombinant DNA, or by protease activity, and 35 derivatives obtained by, for example, glycosylation, phosphorylation, dephosphorylation or chemical conjugation of any compound to JDK or a fragment thereof. The present invention also provides for a protein related to JDK but normally expressed in a non-human embryo that is at least about 70 percent homologous to JDK.

At least a portion of JDK may be sequenced, and the amino acid sequence so obtained may be used to deduce oligonucleotide probes that may be used jo directly to screen recombinant DNA libraries, or in polymerase chain reaction, in order to obtain JDKencoding nucleic acid sequences. These sequences may then be inserted into an appropriate expression vector system and then may be expressed in quantity using standard techniques. Alternatively, JDK may be produced by chemical synthesis or by any other method known in the art. .2. PREPARATION OF GESTATIONAL PROLIFERATIVE AGENTS The present invention provides for a gestational proliferative agent termed GPA-1 that is comprised in an embryonal tissue extract and that has a molecular weight less than about 3,000 daltons and preferably about 1,500-2,000 D. The source of suitable embryonal tissue is as described in Section 5.1., supra. except that embryos at a developmental stage comparable to six weeks of human gestation may also be used.

GPA-1 may be prepared as follows according 3® to a specific, nonlimiting embodiment of the present invention. Embryonal tissue may be minced and placed in cold Tris-HCl at pH 7.4 containing 10 mM dithiothreitol (DTT) and 2 mM phenyl-methyl-sulfonylfluoride (PMSF), then sonicated on ice for about 60 seconds, and then centrifuged at about 2400 rpm for about ten minutes to remove cellular debris. An initial amount of embryonal tissue weighing about 50 mg may, for example, be processed in about 1 ml of buffer and may yield about 1 ml of supernatant. The supernatant from the centrifuged material may then be separated into fractions based on molecular weight, using, for example, the Filtron System Omega Cell (Biolab), 30 mm diameter, under nitrogen and the fraction containing 10 molecular weights less than 3,000 D may desirably be obtained. This fraction may, preferably, be further purified by polyacrylamide gel electrophoresis. That portion of the gel corresponding to a molecular weight less than about 3,000 D may then be separated from the rest of the gel and eluted to form the gestational proliferative agent GPA-1. See Section 8, infra.

Accordingly, the present invention provides for a substantially purified protein, GPA-1, having a molecular weight of less than about 3,000 daltons that is expressed in mammalian embryonal tissue and that has a proliferative effect on morular cells. In preferred embodiments, this protein has a molecular weight greater than 1,400 D, and in most preferred embodiments, the molecular weight is about 2,000 D.

GPA-1 also is capable of increasing the secretion of hCG by placental explants, of promoting the development of a morula into a blastocyst, and of facilitating implantation.

The present invention further provides for a second gestational proliferative agent termed GPA-2 that has a molecular weight above 20,000. .3 ANTIBODIES OF THE INVENTION According to the invention, gestational proliferative agents (i.e. GPA-1 and GPA-2) and gestational antiproliferative agent (i.e. JDK) or active fragments or derivatives thereof, may be used as immunogens to generate antibodies.

To improve the likelihood of producing an immune response, the amino acid sequence of the gestational agent may be analyzed in order to identify 10 portions of the molecule which may be associated with increased immunogenicity. For example, the amino acid sequence may be subjected to computer analysis to identify surface epitopes. Alternatively, the deduced amino acid sequences of gestational agent from different species could be compared, and relatively non-homologous regions identified; these nonhomologous regions would be more likely to be immunogenic across various species.

For preparation of monoclonal antibodies directed toward the antigens of the invention, any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used. For example, the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-4971. as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, in “Monoclonal Antibodies and Cancer 30 Therapy, Alan R. Liss, Inc. pp. 77-96) and the like are within the scope of the present invention.

The monoclonal antibodies may be human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72-79; Olsson et al., 1982, Meth. Enzymol. 92:3-16). Chimeric antibody molecules may be prepared containing a mouse antigenbinding domain with human constant regions (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851, Takeda et al., 1985, Nature 314:452).

Various procedures known in the art may be 10 used for the production of polyclonal antibodies to epitopes of the antigens of the invention. For the production of antibody, various host animals can be immunized by injection with antigen, or fragment or derivative thereof, including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and, Corvnebacterium parvum.

Antibody molecules may be purified by known techniques, e.g.. immunoabsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography), or a combination thereof, etc.

Antibody fragments which contain the idiotype of the molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab')2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragment, and the 2 Fab or Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent. .4. THE USE OF GESTATIONAL AGENTS IN CANCER THERAPY The present invention provides for a method of inhibiting the proliferation of cancer cells comprising exposing said cells to an effective concentration of gestational antiproliferative agent (i.e. JDK). In preferred embodiments of the invention, the gestational antiproliferative agent is a substantially purified protein as described in Section 5.1., supra. or its analog.

The present invention provides for a method of treating a patient suffering from cancer comprising administering to the patient an effective amount of gestational antiproliferative agent JDK in a suitable 20 pharmaceutical carrier. In preferred embodiments of the invention, the gestational antiproliferative agent is a substantially purified protein as described in Section 5.1., supra♦ The present invention may be used to inhibit proliferation of a wide variety of cancer cells and to treat a wide variety of cancers. As exemplified in Section 7, infra. JDK was observed to inhibit the proliferation of all cancer cell lines tested, suggesting that it has a broad spectrum of activity both in vitro and in vivo. Accordingly, the present invention may be used to inhibit proliferation of cancer cells arising from any tissue, including, but not limited to breast, bone marrow, lymphoreticular system, ovary, kidney, lung, brain, intestine, stomach, esophagus, pancreas, spinal cord, mucosa, germ cell, bone, muscle, skin (e.g. melanoma) etc.

V The present invention may be used to treat cancer arising from any tissue in a patient, including, but not limited to, cancer of the breast, ovary, kidney,lung,brain, intestine, bone marrow, lymphoreticular system, stomach, esophagus, pancreas, spinal cord, mucosa, germ cell, bone, muscle, skin (e.g. melanoma), choriocarcinoma, etc.

An effective concentration of JDK may be defined as any concentration that is capable of inhibiting the proliferation of MCF-7 cells,under conditions as set forth in Section 7, infra, by at least 10 percent relative to untreated control. An effective amount of JDK to be used in patient treatment may be defined as that amount that produces an effective concentration of JDK in the tissue of interest.

In methods of treatment according to the invention, JDK may be administered by any route known in the art, including, but not limited to, intravenous, subcutaneous, intramuscular, by injection into a tumor or by other local injection, intranasal, intraocular, intraperitoneal, oral, etc. Further, JDK may be coupled to another molecule, such as an antibody, or to a magnetic bead in order to target delivery. JDK may be administered via a solid or semisolid implant, such as a sustained-release implant. JDK may be incorporated into microcapsules, microspheres, or liposomes. Further, JDK may be coupled to another molecule, such as an antibody, or 30 to a magnetic bead in order to target delivery. If orally administered, it may be desirable to provide a means to protect JDK from denaturation in the stomach.

A suitable pharmaceutical carrier, according to the invention, is any vehicle which is relatively nontoxic to humans and which preserves the activity of JDK. Suitable carriers include, but are not limited to, water, saline, phosphate buffered saline, dextrose, etc. .5. THE USE OF GESTATIONAL AGENTS IN CONTROLLING FERTILITY According to the invention, gestational antiproliferative agent (i.e. JDK) may be used as a contraceptive, and gestational proliferative agents (i.e. GPA-l and/or GPA-2) may be used to enhance fertility and to protect early pregnancy.

The present invention provides for a method of contraception comprising administering to a female subject an effective amount of gestational antiproliferative agent JDK in a suitable 15 pharmaceutical carrier. In preferred embodiments of the invention, the gestational antiproliferative agent JDK is a substantially purified protein.

The present invention further provides for a 2Q method of improving the fertility of a subject comprising administering to the subject an effective amount of gestational proliferative agent such as GPA1 and/or GPA-2 in a suitable pharmaceutical carrier.

In preferred embodiments of the invention, the gestational proliferative agent(s) is (are) a substantially purified protein(s).

A subject is any human or non-human mammalian subject. The fertility promoting aspect of the invention may be particularly useful in improving the fertility of livestock.

An effective amount of gestational antiproliferative agent is construed, in embodiments relating to contraception, to refer to an amount that will result in a concentration in at least a portion of the reproductive organs of the subject that is the same as a concentration that will inhibit morular development of mouse embryos, under conditions as described in Section 7.1.3., by at least about 50 percent relative to untreated controls.

An effective amount of gestational 5 proliferative agent is construed, in embodiments relating to fertility, to refer to an amount that will result in a concentration in at least a portion of the reproductive organs of the subject that is the same as a concentration that will increase the number of 1Q morula that progress to form blastocysts by at least 50 percent relative to untreated controls under conditions as described in Section 8, infra.

The fertility-controlling agents may be administered by any method known in the art, as described in Section 5.4., supra.

Suitable pharmaceutical carriers that may be used according to the invention are relatively nontoxic to the host and preserve the activity of the agent, and include those set forth in Section 5.4., supra.

In further embodiments of the invention antibody directed toward gestational proliferative agent (i.e. GPA-1 and/or GPA-2) may be used in methods of contraception. The present invention provides for a method of contraception comprising administering to a female subject an effective amount of antibody that binds to a gestational proliferative agent. In alternative embodiments, the present invention provides for a method of contraception comprising 3° immunizing a female subject with an immunogenic preparation comprising a gestational proliferative agent. Such an immunogenic composition may comprise an adjuvant, such as Freund’s complete adjuvant, and may desirably comprise gestational proliferative agent coupled to an immunogenic compound or a gestational proliferative agent obtained from a species that is different from the subject to be immunized. Of note, such immunization may result in irreversible contraception. .6. ADDITIONAL UTILITIES OF THE INVENTION In additional embodiments of the invention, gestational antiproliferative agent may be used in the treatment of disorders of increased cell proliferation 10 including, but not limited to, premalignant conditions, keloids, Von Recklinghausen disease, familial polyposis, benign neoplasms (e.g. breast adenomas), autoimmune diseases such as rheumatoid arthritis, etc.

In still further embodiments of the invention, a gestational proliferative agent (i.e. GPA-1 and/or GPA-2) may be used in the treatment of disorders of decreased cell proliferation, such as burnt-out myeloproliferative disorders, pernicious anemia, Sjogren’s syndrome, etc. Gestational proliferative agent(s) may also be used to treat conditions in which increased cell proliferation is desirable, for example, to replace cells damaged by infarct, infection, injury, or exposure to a toxic agent (e.g. post-myocardial infarction myocardium, nervous system tissue that has been damaged or lost by infarction, infection, injury or exposure to toxic agent bone marrow that has been damaged by disease or by exposure to a toxic agent, skin that has been 3® damaged or lost, e.g. in burn patients). In a specific, nonlimiting embodiment of the invention, gestational proliferative agent(s) may be used to promote liver regeneration. In another specific, nonlimiting embodiment of the invention, gestational proliferative agent(s) may be used to facilitate the incorporation of grafted tissue into a subject. In other specific, nonlimiting embodiments of the invention, gestational proliferative agent(s) may be used to promote wound healing and/or hair growth.

The gestational agents of the invention may be utilized in vivo or in vitro. For example and not by way of limitation, GPA-1 and/or GPA-2 may be used to promote the growth of cells or tissue in culture. 6. EXAMPLE: PREPARATION OF GESTATIONAL ANTIPROLIFERATIVE AGENT JDK 6.1. MATERIALS AND METHODS 6.1.1. EXTRACTION Human embryos of up to 9 weeks, which were 15 the products of terminated pregnancy and obtained with permission, were isolated from the other products of conception aseptically, and gestational ages were confirmed by last menstrual period and crown-rump measurement of the specimen, with an experimental error of + 3 days. Embryonal organs were dissected from the embryo and identified using a dissecting microscope, and were placed on ice. All subsequent steps were carried out at 4 °C.

Visceral organs comprising lung, liver, kidney and adrenal, and/or neural organs such as spinal cord and brain, were extracted by sonication on ice followed by centrifugation (2,400 rpm x 10 min.) in 0.5 M Tris-HCl buffer at pH 7.4, dithiotrietol (DTT) 10 mM and pheny1-roethyl-sulphony1-fluoride 30 (PMSF) 2 mM. Extraction with ethanol resulted in an approximately 3-fold lower activity product. 6.1.2. PREPARATION OF DIFFERENT MOLECULAR WEIGHT FRACTIONS Using the Filtron System Omega Cell (Biolab Labs, 30 mm diameter), under nitrogen, the extract obtained according to (A) above from embryonic spinal cord was filtered through different molecular weight (M.W.) filters using the PBS buffer with DTT and PMSF. Each 1 ml of extract was filtered with 30 ml of buffer. The collected extract was lyophylized and the sediment was suspended in distilled water and stored at -20°C.

The following fractions were prepared (the symbol < being used, as conventional, to indicate fractions having less than the indicated M.W.): <100,000, <30,000, <10,000, <8,000 and <3,000. All fractions were tested for the ability to modulate HCG production of placental explants by using the superfusion model described by Barnea and Kaplan (1989, J. Clin. Endocrinol. Metab. 69:215-217) (see 2q Section 7, infra). Only fractions below about 10,000 were found to be biologically active. 6.1.3. HIGH PRESSURE LIQUID CHROMATOGRAPHY ANALYSIS High pressure liquid chromatography (HPLC) analysis was performed on the <8,000 MW fraction of spinal cord extract using a Hitachi HPLC apparatus and a 25 cm RP-18 column at 25°C. The sample was applied in water and eluted using 40 percent H20, 60 percent CH3OH at a flow rate of 1 ml/min.

Approximately 50 mg of tissue was sonicated in 0.5 ml of 0.2 M Tris buffer containing 10'3 M PMSF and 2 mM DTT pH 7.4, centrifuged at 20,000 rpm and the supernatant was filtered through a 0.2 μπ» filter. A sample (335 μΐ of 16 mg protein/ml) was applied on a HPLC column (Superdex 75HR 10/30, LKB, Pharmacia) which was previously equilibrated with the same buffer. Samples of 0.5 ml were collected at a flow rate of 0.5ml/min and frozen at -70°C until used. The molecular weights of the various fractions were estimated from the retention time of proteins with known molecular weights. 6.1.4. POLYACRYLAMIDE GEL ELECTROPHORESIS ANALYSIS Polyacrylamide gel electrophoresis (PAGE) 10 was performed on the <10,000 MW fraction using a 12.5 percent gel. The resulting gel was then stained with Coomassie blue using standard techniques. 6.2. RESULTS AND DISCUSSION 15 Extracts of various embryonic organs, prepared as described above, were subjected to fractionation by molecular weight. Only fractions having a molecular weight less than 10,000 daltons were found to be capable of altering HCG production of placental explants.

When the <8,000 MW fraction was subjected to HPLC analysis, three peaks were visualized as shown in Figure 1. Similarly, when the <10,000 MW fraction from spinal cord was subjected to PAGE, three dominant 25 bands appeared at molecular weights of approximately 3,000 to 8,000 daltons, as shown in Figure 2. Further HPLC analysis indicated that gestational antiproliferative agent, called JDK,·* appears to have a molecular weight of about 6.43 D. 7. EXAMPLE: JDK HAD A SUPPRESSIVE EFFECT ON HCG SECRETION BY PLACENTAL EXPLANTS, AN ANTIPROLIFERATIVE EFFECT ON CANCER CELLS AND A SUPPRESSIVE EFFECT ON EMBRYONIC DEVELOPMENT 7.1 MATERIALS AND METHODS 5 7.1.1. ANALYSIS OF EXTRACT FRACTIONATED BY MOLECULAR WEIGHT FOR ABILITY TO SUPPRESS HCG SECRETION BY PLACENTAL EXPLANTS In order to determine suppressive activity, a super fusion apparatus (Accusyst, Endotronics, St.Paul, MN) (Figure 3) with a multichannel peristaltic pump and fraction collector (model 272, ISCO, Durham,NC) was used to study the short term dynamics of human chorionic gonadotropin (hCG) secretion, as described in Barnea and Kaplan (1989, J. Clin, ib Endocrinol. 69:215-217). Placental explants (200-300 mg wet weight) were placed into the culture chambers and a 18 mM HEPES/DMEM solution was washed through it in an atmosphere of 5% CO2 and 95% air at 37°C. Sample corresponding to a particular molecular weight fraction was applied by administering a one minute pulse such that all fractions could be analyzed in parallel. Experiments were conducted for a 120-minute period. A 1 ml sample from the effluent of the apparatus was collected every 2.4 minutes for hCG measurements by radioimmunoassay. In such experiments, one channel served as control and four served as experimental channels. At given intervals a 1-minute pulse of an aqueous solution of the tested fraction was given through a peristaltic pump equipped with a digital flowmeter (Ismatech DD, Chicago, IL) . The fractions that caused a significant change in hCG secretion following administration of the pulses were considered as active samples. 7.1.2. TESTING OF JDK ACTIVITY ON CANCER CELL LINES Cell lines for testing, obtained from the American Type Culture Collection, Rockville, Maryland, were MCF-7, a breast cell line (accession number: HTB22) and HBL-40, a breast cell line exposed to SV40 virus, (accession number: HTB124). Other cell lines used included CRL, a kidney cell line, and GNL, an ovarian cell line.

Cells were cultured in RPMI-1640 with 10-15% fetal calf serum at a concentration of 10,000 cells per milliliter in a volume of 0.5 ml, and kept at 37°C in a 95 percent air/5 percent C02 atmosphere. 10-50 microliter aliquots of extract from <8,000 and <10,000 fractions, corresponding, respectively, to protein concentrations of 0.2-1.0 microgram per ml were added to identical cultures in order to evaluate the doseresponse. Two control cultures were used for each cell line. The first was an untreated culture. The 2Q second control was a culture to which 50 microliters of Tris-HCl were added. Extract was added to cultures only at the beginning of the experiment. After 3-5 days, the number of cells was counted using an automatic cell counter. 7.1.3. ANALYSIS OF THE EFFECT OF JDK ON EMBRYONAL DEVELOPMENT IN VITRO ICR-strain mice were obtained from Charles River Laboratory. Females (between six and seven weeks old) were mated with ten to twelve-week old vV males. About 72 hours later, the females were sacrificed and morula-stage embryos were removed by flushing out the fallopian tubes with HAM-F1. The embryos were then placed in EBSS medium containing 10 percent newborn cord serum maintained in a humidified, 95 percent air/5 percent CO2 atmosphere. For trophoblast development studies, the culture dishes were pre-coated with a 1 mm thickness of collagen. 7.1.4 ANALYSIS OF THE EFFECT OF EXTRACT FRACTIONS ON THE SURVIVAL OF NUDE MICE 5 INOCULATED WITH FIBROSARCOMA_ Adult mice (Balb/c) were treated by sublethal irradiation with x-rays to destroy their ability to form an immunological response.

Experiments were carried out in which mice were 1θ subcutaneously inoculated with 100,000 cells of a fibrosarcoma cell line. Five mice were used for each experiment, and five mice that were not inoculated served as controls. In addition, inoculated mice were treated by injecting 50 μΐ of about 1 Mg/ml of protein-like material from different molecular weight fractions obtained as described supra immediately after tumor inoculation. Subsequently, the animals were kept in a germ free environment, fed ad libitum. and were examined weekly. After 3 weeks the mice were . . sacrificed and examined for the presence of tumors. 7.2. RESULTS AND DISCUSSION 7.2.1. EXTRACT FRACTIONS CONTAINING MOLECULES HAVING A MOLECULAR WEIGHT LESS THAN 25 10,000 DALTONS SUPPRESSED HCG SECRETION BY PLACENTAL EXPLANTS When placental explants were exposed to fractions of embryo extract corresponding to molecular weights <100,000, <30,000, <10,000, <8,000, and <3,000 daltons, only those fractions representing molecular weights less than 10,000 daltons were observed to suppress hCG secretion. The <10,000 fraction appeared to exhibit lower suppressive activity than the <8,000 fraction, whereas the <3,000 fraction exhibited a 35 stimulatory activity.

Figure 4A shows the ability of extract to suppress hCG secretion. As Figures 4B and 4C illustrate, the ability of short pulses of the <8,000 MW fraction to inhibit hCG secretion (Figure 4B) is eliminated by heat inactivation at 57°C (Figure 4C), indicating that JDK is heat labile.

Figure 5 illustrates that even a 1/2000 dilution of extract, corresponding to a protein concentration of about 100 ng/ml, was observed to achieve about 50 percent of the suppressive effect associated with control. 7.2.2. JDK SUPPRESSED THE GROWTH OF CANCER CELL LINES IN VITRO 15 Fifty microliter aliquots of <8,000 MW and <10,000 molecular weight fractions of embryo extract were added to cell cultures of MCF-7, CRL, HBL-40, and GNL as described above and the number of cells in each treated culture was counted at the end of 3-5 days and 2Q compared to the number of cells in untreated control cultures (expressed as 100%) and cultures to which buffer had been added (buffer control).

As shown in Table I, infra, the cell count in MCF-7 cultures treated with the <8,000 MW fraction was 11 percent of control, representing an 89 percent inhibition of growth. In another experiment, after only three days of culture in the presence of a 6.428 KD molecular weight fraction of extract, cellular proliferation was inhibited by a statistically significant 10 percent (p <0.05). Similarly treated cultures of (i) CRL attained a cell count of 63 percent of control, representing 37 percent inhibition; (ii) HBL-40 attained a cell count of 3 percent of control, representing 97 percent inhibition; and (iii) GNL reached a cell count of 23 percent of control, representing 77 percent inhibition. Interestingly, the <10,000 MW fraction showed significant inhibition in only MCF-7 cultures. This may reflect the presence of some compound(s) that may limit the effect of the active fraction on the other cell lines.

TABLE I Percentage Of Untreated Control Untreated Buffer Cell Line Control <8.000 MW <10.000 MW Control MCF-7 100 11 50 97 CRL 100 63 100 100 HBL-40 100 3 103 105 GNL 100 23 104 98 2o The results of another, similar experiment are presented in Figure 6. In this experiment, extract had a more pronounced effect on CRL cells than in the experiment depicted in Table I; this may be a function of heterogeneity in the extract itself or of the proliferative state of the cells prior to each experiment.

Table II, infra, and Figure 7 illustrate the dose dependency of the antiproliferative effect of JDK in the <8,000 MW fraction on MCF-7 cells in culture.

Cell count reduction became detectable when 10 μΐ of extract was used, corresponding to a protein concentration of 0.2 pg/ml. Maximal decrease was obtained with 50 μΐ of extract corresponding to 1.0 gg/ml of protein.

TABLE II Treatment Untreated control Buffer control Percent Count Reduction Ml <8,000 extract (0.2 25 Ml <8,000 extract (0.5 50 Ml <8,000 extract (1.0 0 3 pg/ml protein) 36 pg/ml protein) 52 pg/ml protein) 89 7.2.3. SUPPRESSIVE EFFECT OF JDK IN EXTRACT ON EMBRYONAL DEVELOPMENT IN VITRO MOUSE As shown in Figure 8, mouse embryos treated . with JDK in <8,000 MW fraction of extract did not develop normally, as compared to untreated control embryos. The number of extract-treated embryos which reached morular stage was observed to be only about twelve percent of untreated controls (Figure 8A) and the number of extract-treated embryos which exhibited hatching/adhesion (measured by light microscopy) was only about seven percent of untreated controls (Figure 8B). This is consistent with the antiproliferative activity of JDK and supports its use as a 25 contraceptive agent. 7.2.4. JDK-TREATED NUDE MICE INOCULATED WITH FIBROSARCOMA SHOW IMPROVED SURVIVAL AND FEWER TUMORS Thirty nude mice were inoculated with fibrosarcoma cells and divided into six groups of five mice each. Five uninoculated mice served as negative controls. One group of inoculated mice was treated only with Tris-HCl buffer. The remaining five groups were treated with 50 μΐ of about 1 pg/ml of one of the following extract fractions: <3,000 MW; <8,000 MW; <8,000 MW; <10,000 MW; and <30,000 MW. As shown in Table III, all five mice in the group treated with the <8,000 MW fraction were tumor-free. The only other group that contained tumor-free animals was the group receiving the <8,000 molecular weight fraction (2 out of 5) . Of the remaining groups. Tumors were found to occur at the site of inoculation.

Treatment None (control) PBS Buffer <3,000 <8,000 <8,000 <10,000 <30,000 TABLE III Tumor Free /5 0/5 0/5 /5 2/5 0/5 0/5 In a similar experiment, the survival of mice inoculated with fibrosarcoma and then either untreated (control) or treated with various molecular weight fractions of extract was evaluated over a three week period. Survival was measured as the number of mice surviving after the three week period. The tumor-cell inoculated mice receiving fraction 3, corresponding to <8,000 MW fraction of extract, exhibited a survival equivalent to those of mice that had not been inoculated with tumor.

·· V - 32 8. EXAMPLE: PREPARATION OF ACTIVE GESTATIONAL PROLIFERATIVE AGENTS 8.1. MATERIALS AND METHODS 8.1.1. PREPARATION OF GESTATIONAL PROLIFERATIVE AGENT-CONTAINING EXTRACTS The <3,000 molecular weight fraction was obtained by mincing embryonal spinal cord in cold Tris-HCl at pH 7.4 containing 10 mM DTT and 2 mM PMSF, sonicating the minced tissue on ice for about l minute, and then centrifuging at about 2400 rpm for 10 minutes. The supernatant from the centrifuged material was then subjected to sizing using a Filtron System Omega Cell (Biolab) under nitrogen, and the fractions containing molecular weights less than 3,000 D were collected. 8.1.2. PREPARATION OF MOUSE EMBRYOS ICR-strain mice were obtained from Charles River Laboratory. Females (between six and seven weeks old) were mated with ten to twelve-week old males. About 72 hours later, the females were sacrificed and morula-stage embryos were removed by flushing out the fallopian tubes with HAM-F1. The embryos were then placed in EBSS medium containing 10 percent newborn cord serum maintained in a humidified, 95 percent AIR/5 percent CO2 atmosphere. For trophoblast development studies, the culture dishes were pre-coated with a 1 mm thickness of collagen. 8.1.3. ANALYSIS OF EFFECT OF EXTRACT ON HCG SECRETION BY PLACENTAL EXPLANTS The effect of the 3,000 MW extract on hCG secretion by placental explants was evaluated using a superfusion apparatus as set forth in Section 7.1.1., supra. 8.1.4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ANALYSIS HPLC analysis was performed as set forth in 10 Section 6.1.3., supra. See Figure 11. 8.2. RESULTS AND DISCUSSION 8.2.1. GPA-1 CAUSES INCREASED SECRETION OF HCG BY PLACENTAL EXPLANTS It was observed that the <3,000 MW fraction, containing a protein termed GPA-1, of extract was able to increase hCG secretion by placental explants. Normally, hCG is secreted by placental extracts in a spontaneous pulsatile fashion with a relatively stable frequency. When a one minute pulse of the <3,000 MW fraction was administered to explants via the superfusion apparatus, a major increase in hCG secretion was observed, as shown in Figure 10. This was evidenced by a four-fold increase in the area under the curve noted in the channel of the superfusion apparatus treated with the <3,000 MW fraction compared to the control channel. In contrast, no effect on hCG secretion was observed in the channels treated with the <100,000 MW, <50,000 MW, and <30,000 MW fractions. Of note, treatment of explants with <3,000 MW fraction (GPA-1) which had been heat inactivated at 57°C for 30 minutes had no effect on hCG secretion. In addition, extraction of the fraction by charcoal/dextran (as is used to extract steroids) also eliminated its activity. 39322 I 8.2.2. GPA-1 PROMOTED MORUIAR DEVELOPMENT The number of morula treated with the <3,000 MW fraction (GPA) that matured to form blastocysts was about three-fold greater than the number of blastocysts formed by morulas treated with buffer only. This observation occurred when morulas were treated with 10 microliters of GPA-1, which has a protein content of about 0.2 Mg/ml. Exposure of the embryos to 50 microliters of GPA-1 containing fraction was found to increase the survival of the blastocysts by a factor of two relative to control, as measured over a 48 hour period.

Heat inactivated GPA-1 failed to affect morular development. 8.2.3. GPA-1 PROMOTED TROPHOBLAST DEVELOPMENT When trophoblast development was evaluated using collagen-coated culture dishes, it was observed that the same level of development achieved by normal controls within 24 hours had been achieved by GPA-1containing fraction-treated embryos within only 14 hours. These developmental changes included adhesion of the blastocyst to the collagen surface and penetration of the collagen by the differentiating trophoblastic cells. The process accelerated in vitro by GPA corresponds to the in vivo implantation process, and indicates that GPA-1 may be used to facilitate implantation, and, hence, increase fertility, in human beings.

Various publications are cited herein which are hereby incorporated by reference in their entirety.

Claims (35)

WHAT IS CLAIMED IS:

1. A substantially purified protein having a molecular weight of less than about 10,000 daltons that is normally expressed in mammalian embryonal 5 tissue and that has an antiproliferative effect on certain cancer cells.

2. A substantially purified protein having a molecular weight of less than about 10,000 daltons 10 that is normally expressed in mammalian embryonal tissue and that has an antiproliferative effect on certain embryonal cells.

3. The substantially purified protein of 15 claim 1 having a molecular weight of between about 6,300 and 6,600 daltons.

4. The substantially purified protein of claim 2 having a molecular weight of between about 20 6,300 and 6,600 daltons.

5. The substantially purified protein of claim 1 that corresponds to the protein comprised in peak I of the chromatogram depicted in Figure 1.

6. The substantially purified protein of claim 1 that corresponds to the protein comprised in peak II of the chromatogram depicted in Figure l. 30

7. The substantially purified protein of claim 1 that corresponds to the protein comprised in peak III of the chromatogram depicted in Figure 1. - 36

8. A substantially purified protein having the characteristics of a protein prepared by a process comprising: (a) extracting visceral organs or 5 neural organs or a combination of visceral and neural organs collected from a mammalian embryo in Tris-HCl at pH 7.4 containing 10 mM dithiothreitol and 2 mM 10 phenyl-methyl-sulfonyl fluoride by sonication on ice followed by centrifugation for 10 minutes at 2400 rpm and collecting the supernatant; and 15 (b) selecting those molecules from the supernatant that have a molecular weight less than 10,000 daltons into a fraction; (c) subjecting the fraction to high 20 pressure liquid chromatography using an RP-18 column and using, as, eluting buffer, a mixture of 40 percent water and 60 percent methanol; and (d) collecting the material from the HPLC 25 column corresponding to Peak I of Figure 1.

9. A substantially purified protein having the characteristics of a protein prepared by a process comprising: (a) extracting visceral organs or neural organs or a combination of visceral and neural organs collected from a mammalian embryo in phosphate buffered saline at pH 7.4 containing 10 mM dithiothreitol and 2 mM phenyl-methylsulfonyl fluoride by sonication on ice followed by centrifugation for 10 minutes at 2400 rpm and collecting the supernatant; and (b) selecting those molecules from the supernatant that have a molecular weight less than 10,000 daltons into a fraction; (c) subjecting the fraction to high pressure liquid chromatography using an RP-18 column and using, as, eluting buffer, a mixture of 40 percent water and 60 percent methanol; and (d) collecting the material from the HPLC column corresponding to Peak II of Figure 1.

10. A substantially purified protein having 20 the characteristics of a protein prepared by a process comprising: (a) extracting visceral organs or neural organs or a combination of visceral and neural organs collected from a 25 mammalian embryo in phosphate buffered saline at pH 7.4 containing 10 mM dithiothreitol and 2 mM phenyl-methylsulfonyl fluoride by sonication on ice followed by centrifugation for 10 minutes at 2400 rpm and collecting the supernatant; and (b) selecting those molecules from the supernatant that have a molecular weight less than 10,000 daltons into a fraction; (c) subjecting the fraction to high pressure liquid chromatography using an RP-18 column and using, as, eluting buffer, a mixture of 40 percent water 5 and 60 percent methanol; and (d) collecting the material from the HPLC column corresponding to Peak III of Figure 1. 10 11. A method for inhibiting the proliferation of cancer cells comprising exposing said cells to an effective concentration of the substantially purified protein of claim 1.

11. 15 12. A method for inhibiting the proliferation of cancer cells comprising exposing said cells to an effective concentration of the substantially purified protein of claim 3. 20 13. A method for inhibiting the proliferation of cancer cells comprising exposing said cells to an effective concentration of the substantially purified protein of claim 5. 25 14. A method for inhibiting the proliferation of cancer cells comprising exposing said cells to an effective concentration of the substantially purified protein of claim 6. 3® 15. A method for inhibiting the proliferation of cancer cells comprising exposing said cells to an effective concentration of the substantially purified protein of claim 7.

12. 16. A method of treating a patient suffering from cancer comprising administering to the patient an effective amount of the substantially purified protein of claim 1 in a suitable 5 pharmaceutical carrier.

13. 17. A method of treating a patient suffering from cancer comprising administering to the patient an effective amount of the substantially 10 purified protein of claim 3 in a suitable pharmaceutical carrier.

14. 18. A method of treating a patient suffering from cancer comprising administering to the 15 patient an effective amount of the substantially purified protein of claim 5 in a suitable pharmaceutical carrier.

15. 19. A method of treating a patient

16. 20 suffering from cancer comprising administering to the patient an effective amount of the substantially purified protein of claim 6 in a suitable pharmaceutical carrier. 25 20. A method of treating a patient suffering from cancer comprising administering to the patient an effective amount of the substantially purified protein of claim 7 in a suitable pharmaceutical carrier.

17. 21. The method of claim 16 in which the patient is suffering from breast cancer.

18. 22. The method of claim 16 in which the patient is suffering from ovarian cancer.

19. 23. The method of claim 16 in which the patient is suffering from renal cancer.

20. 24. A method for inhibiting the 5 proliferation of cancer cells comprising exposing said cells to an extract prepared by a method comprising: (a) sonicating, on ice, a mixture of (i) visceral organs or neural organs or a mixture of visceral and neural organs 10 collected from a mammalian embryo and (ii) a physiologically compatible solution; (b) centrifuging the product of sonication at about 2400 rpm for about ten minutes 15 and collecting the supernatant; and (c) selecting that portion of the supernatant that has a molecular weight of less than about 10,000 daltons, comprised in a suitable pharmaceutical 20 carrier.

21. 25. A method of contraception comprising administering to a female subject an effective amount of the substantially purified protein of claim 1 in a 25 suitable pharmaceutical carrier.

22. 26. A method of contraception comprising administering to a female subject an effective amount of the substantially purified protein of claim 3 in a 30 suitable pharmaceutical carrier.

23. 27. A method of contraception comprising administering to a female subject an effective amount of the substantially purified protein of claim 5 in a 35 . . . suitable pharmaceutical carrier.

24. 28. A method of contraception comprising administering to a female subject an effective amount of the substantially purified protein of claim 6 in a suitable pharmaceutical carrier.

25. 29. A method of contraception comprising administering to a female subject an effective amount of the substantially purified protein of claim 7 in a suitable pharmaceutical carrier.

26. 30. A substantially purified protein having a molecular weight of less than about 3,000 daltons that is expressed in a mammalian embryonal tissue and that has a proliferative effect on morular cells.

27. 31. A substantially purified protein having a molecular weight of less than about 3,000 daltons that is expressed in a mammalian embryonal tissue and that promotes the development of the morular into a 20 blastocyst.

28. 32. A substantially purified protein having a molecular weight of less than about 3,000 daltons that is expressed in a mammalian embryonal tissue and 25 that increases the secretion of human chorionic gonadotrophin by a placental explant.

29. 33. A method of improving the fertility of a subject comprising administering to the subject an 3® effective amount of the substantially purified protein of claim 30 in a suitable pharmaceutical carrier.

30. 34. The substantially purified protein of claim 1 that is normally expressed in a human embryo. - 42

31. 35. A substantially purified protein that is normally expressed in a non-human embryo that is at least about 70 percent homologous to the substantially purified protein of claim 34.

32. 36. A substantially purified protein substantially as hereinbefore described by way of Example.

33. 37. A substantially purified protein of claim 1 for use in inhibiting the proliferation of cancer cells.

34. 38. A substantially purified protein as claimed in claim 1 for use in treating a patient suffering from cancer.

35. 39. A substantially purified protein as claimed in claim 1 for use in a method of contraception.