WO1988008719A1

WO1988008719A1 - Reproductive hormone-specific immunocontraceptives and methods of using them

Info

Publication number: WO1988008719A1
Application number: PCT/US1988/001541
Authority: WO
Inventors: Stephen Grimes; Eliezer Benjamini; Robert J. Scibienski
Original assignee: Aphton Corporation
Priority date: 1987-05-12
Filing date: 1988-05-04
Publication date: 1988-11-17
Also published as: AU1962788A

Abstract

This invention relates to immunogens comprising anti-idiotypic antibodies formed against anti-reproductive hormone antibodies, which anti-idiotypic antibodies express internal images of reproductive hormone antigenic determinants and to a method for contraception comprising the immunization of a female or male mammal against one or more reproductive hormones using a composition comprising an effective amount of such immunogens. This invention also relates to a method for contraception comprising the passive immunization of a female or male mammal against one or more reproductive hormones using a composition comprising an effective amount of antibodies against reproductive hormones.

Description

REPRODUCTIVE HORMONE-SPECIFIC IMMUNOCONTRACEPTIVES AND METHODS OF USING THEM

TECHNICAL FIELD OF THE INVENTION

This invention relates to the control of mammalian fertility. More specifically/ this invention relates to immunoglobin immunogens that induce immunity directed against reproductive hormones which are essential to the maintenance o fertility as well as to a method of contraception comprising the active or passive immunization of female and male mammals using such immunogens. According to this invention, antibodie that express antigenic internal images of epitopes otherwise unique to reproductive hormones are administered to mammals in order to actively immunize a mammal against reproductive hormones and thereby prevent conception. Alternatively, contraceptive antibodies can be generated and used to passivel immunize the mammal.

BACKGROUND OF THE INVENTION

Conventional methods of contraception include the use of mechanical or chemical barriers to fertilization, administration of hormones, the use of mechanical means to prevent implanation of a fertilized ovum, sterilization, and natural family planning (the "rhythm method") . Typically, these methods have serious drawbacks, including practical inconvenience, incomplete effectiveness and various undesirabl side effects. Mechanical means of contraception can be inconvenient, may cause infection and, as with the chemical barrier and natural family planning methods, are often not sufficiently effective. Sterilization is usually not readily reversible. The oral administration of hormones, commonly referred to as "the pill", has been linked to many physiological problems, including neoplasia, hypertension, venous thromboembolism, stroke and myocardial infarction.

In view of the disadvantages of such methods, much effort has been directed at developing improved contraceptive technologies (see, for example, Fourteenth Annual Report. Special Programme of Research, Development and Research Training in Human Reproduction. World Health Organizations. Geneva» December 1985). An important approach emerging from these endeavors concerns the utilization of the body's immune system to prevent conception. Vaccines eliciting immune responses against a variety of antigenic targets unique to th reproductive system, including both structural and hormonal antigens, have been shown to affect contraception in males an females in numerous mammalian species [D.B. Crighton, Immunological Aspects of the Reproduction in Mammals (1984), D.J. Anderson and N.J. Alexander, "A new look at anti-fertili vaccines," Fertility and Sterility, 40, p. 557 (1983); W.R. Jones, Immunoloσical Fertility Regulation (1982)].

Mammalian fertility is regulated by a limited number of reproductive hormones [see M. Johnson and B. Everitt, Essential Reproduction (1984)]. Each of these hormones acts a concentration-dependant fashion; deviations from the hormon levels required for the maintenance of the fertile state can result in infertility. Antibodies directed against reproductive hormones are capable of neutralizing the hormone biological activities and/or accelerating the elimination of the hormone by opsonization. The net effect is a functional depletion of a specific hormonal activity, resulting in a los of fertility.

Consequently, a major approach towards immunocontraception involves the induction of humoral immunit against reproductive hormones. Antibody responses directed against the principal hormones responsible for the regulation of mammalian fertility, including luteinizing hormone releasi hormone (LHRH) , luteinizing hormone (LH) , follicle stimulatin hormone (FSH) , chorionic gonadotropin (CG) , and the sex steroids (progestagens, androgens, and estrogens), can result in infertility. Methods of contraception have been developed that rely upon the generation of immunity against reproductiv hormones (See United States Patent 4,526,716). However, these methods have disadvantages which have hindered or prevented their practical application. Immunization against the sex steriods has not yet been proven to be readily applicable as a practical means of contraception. A problem associated with this approach is tha antibody mediated neutralization of the sex steroids can cause an increase in gonadotropin (FSH and LH) levels which, in turn enhance sex steroid synthesis. The increased steroid output necessitates further elevations in contraceptive titers. This effect, which presumably results from the disruption of the inhibition of gonadotropin release normally mediated by the se steroids, is unpredictable. Thus, the sex steroids are not, b themselves, presently considered to be acceptable immunocσntraceptive targets.

On the other hand, LHRH and the gonadotropins are suitable targets for immunocontraception. However, there are serious drawbacks associated with the vaccines currently - utilized to elicit responses against these reproductive hormones. A major disadvantage associated with anti- gonadotropin vaccines is their lack of specificity. LH, FSH, and human CG exhibit extensive irumunological cross reactivity, which further extends to a non-gonadotropin, thyroid, stimulating hormone. Commonly employed gonadotropin vaccines consist of homologous hormones (or their major subunits) obtained from mammals of a different species than that to be immunized. Such preparation elicit antibody responses, comprised of both specific and cross reactive antibodies, whi can mediate undesirable side effects. For example, immunization of mice with human CG evokes antibodies that rea with both human CG and LH. A similar response would be most undesirable in human applications, wherein the goal of induci an anti-CG response is to provide contraception without affecting a woman's normal (non-pregnant) reproductive physiology.

In one approach, designed to overcome this problem, -noncrossreactive epitopes expressed by the target gonadotropi are identified, isolated or synthesized, then chemically coupled to an immunogenic carrier to yield an immunogen. Suc "subunit" vaccines can elicit specific humoral responses that mediate contraception (see United States Patent 4,302,386). However, the "subunit" approach also suffers from drawbacks which impede its practicality. First, the isolation and identification of noncrossreactive epitopes is time consuming and laborious. Second, it may be impossible to identify conformational epitopes by this approach. Conformational epitopes consist of molecular subunits that are sequentially separate but brought into close proximity as a molecule assum its native conformation; such epitopes are lost upon denaturation of secondary, tertiary, or quaternary structure. Most conformational epitopes could not be readily detected or duplicated by current subunit technology. Third, the gonadotropins are glycoproteins; epitopes crucial to the immunoneutralization of these hormones can consist partly or entirely of carbohydrates. Immunocontraceptive vaccines containing carbohydrate based epitopes cannot be synthesized o the requisite scale by current technology; therefore, they can not be incorporated into "subunit" immunocontraceptives.

To elicit immune responses against haptenic substance such as LHRH and the hormonal epitopes reproduced in the subunit approach, it is necessary to render the haptens immunogenic by chemically coupling them to appropriate carrier antigens. The preparation of such immunogens on the scale required for mass innoculation programs can be extremely expensive and technically infeasible.

SUMMARY OF THE INVENTION

The present invention provides a method of contraception comprising eliciting an active immune response i mammals with anti-idiotypic antibodies that possess "internal images' of antigenic determinants expressed by reproductive hormones. Alternatively, contraceptive antibodies can be generated and then used to passively immunize a mammal against the reproductive hormones. Thus, the method of the present invention involves either actively eliciting contraceptive antibodies specific for one or more reproductive hormones wit a vaccine comprising an effective amount of anti-idiotypic antibodies to anti-hormone antibodies, or by passively administering such contraceptive antibodies to the mammal.

The present invention also relates to anti-hormone idiotypic antibodies which are useful in the foregoing method of contraception, particularly when used to prepare a contraceptive anti-idiotypic vaccine for administration to mammals. The anti-hormone idiotypic antibodies may also be utilized as immunocontraceptives when passively administered mammals.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts the results of an experiment in whi female mice were immunized with three injections of LHRH-diptheria toxoid conjugate.

Figure 2 depicts the results of an inhibition study o the binding of anti-LHRH serum antibodies and normal mouse ser to LHRH-BSA by Free LHRH.

Figure 3 depicts the results of a comparision study o the duration of infertility in female mice following the passive immunization with four different anti-LHRH monoclonal antibodies. Figure 4 depicts the results of an experiment concerning the percent inhibition of binding of a monoclonal mouse anti-idiotypic antibody to rabbit anti-LHRH by free LHRH

Figure 5 depicts the results of an assay for the cross-reactivity of anti-hCG monoclonal antibodies with other hormones. In this assay the anti-hCG antibodies were each screened by an ELISA against other human hormones known to share cross-reactive epitopes with hCG. The other hormone antigens were as follows: luteinizing hormone (LH) , follicle stimulating hormone (FSH) , and thyroid stimulating hormone (TSH) .

Figure 6 depicts the results of a typical assay for the biological activity of anti-hCG monoclonal antibodies whi measured the in vivo neutralization of hCG. The assay measur the percent inhibition of hCG-induced rat uterine weight gain by anti-hCG mAb mixtures. The mixtures tested were: mAb mix = a mixture of mAbs designated 342-1, 342-2, 342-3 and 349-6; mAb mix 2 = a mixture of mAbs designated 367-2 and 366-1; and mAb mix 3 = mAb mix 1 + mAb mix 2.

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention herein described be more fully understood, the following detailed description is set forth. In the description, the following terms are employed: REPRODUCTIVE HORMONES — As used herein, this term encompasses all hormones directly or indirectly involved in t regulation of mammalian reproductive physiology. It includes all forms of these- hormones in males and females. It also includes hormones present during pregnancy and hormones prese in the non-pregnant state of a female.

The term "reproductive hormones" specifically includes, but is not limited to, the following hormones: luteinizing hormone releasing hormone (LHRH; also known as gonadotropin releasing hormone, GnRH) ; luteinizing hormone (LH) ; follicle stimulating hormone (FSH) ; chorionic gonadotropin (CG) ; inhibin; and the sex steroids, including a forms of progestagens, androgens, and estrogens.

As used herein, "reproductive hormones" encompasses all forms of each reproductive hormone that are capable of eliciting contraceptive antibodies to the hormone. This includes whole hormones, parts or subunits of hormones, enzymatically digested or denatured forms of hormones, agonis and antagonist analogs of reproductive hormones, molecules lacking hormonal activity but which immunologically cross-rea with reproductive hormones, or any combinations thereof. In addition, "reproductive hormones" includes any hormones or hormone subunits which are not inherently immunogenic but whi can be utilized as immunogens when they are chemically linked to immunogenic carrier molecules. - 10 -

The term "reproductive hormone", as used in this application, includes any epitopes expressed on said hormones that are: unique to each hormone within a species; shared by two or more different hormones within a species; shared by analogous hormones from different species; shared by two or more different hormones from different species.

ANTI-IDIOTYPE — The discovery of idiotypes by J. Oudin and M. Michel, "Une nouvelle form d'allotypic des globulines du serum de lapin, apparrement liee a la fonctio et a la specificite anticorps," Cornpt. Rend. Acad. Sci (Paris 257, p. 805 (1963), elucidated the potential of the variable region of an antibody to act as an antigenic determinant or epitope. Anti-antibodies directed to this region of the immunoglobulin molecule may be highly specific, i.e., unreactive with other antibodies. The variable-region antigenic determinants expressed by an antibody that induce anti-antibody formation are referred to as the idiotypic determinants or idiotopes [see A. Nisonoff, Introduction To Molecular Immunology (1984)]. Antibodies specific for the antibody's various variable-region antigenic determinants or idiotopes are referred to an anti-idiotypes ("anti-id").

Anti-idiotypic antibodies can express an "internal image" of the antigen to which the original antibody was formed. These anti-id antibodies combine with the antibody' binding site, by virtue of the anti-id antibodies' possessio 11 -

of the internal image of the target antigen epitope or a par thereof [see E.S. Golub, The Cellular Basis of the Immune Response, pp. 178-179 (1979)].

INTERNAL IMAGE — An epitope, expressed by antibody that immunochemically resembles an epitope expressed by antigen. The immunochemical similarity between the internal image and the antigen's epitope may be partial or complete [ I.M. Roitt et al., Immunology, p. 10.4 (1985)].

MONOCLONAL ANTIBODIES — Monoclonal antibodies are obtained by fusing an antibody-producing cell, which typical secretes a single species of antibody molecule, with a myelo tumor cell. A cell line is then propagated from the fused cell. The fused cell line, referred to as a hybrido a, has characteristic immortality of a tumor cell line, and secrete the desired antibody in large amounts. Hybridomas, which ca be made according to the methods of G. Kohler and C. Milstei ["Continuous Cultures of Fused Cells Secreting Antibodies of Predefined Specificities," Nature. 256, p. 495, (1975)], are major source of homogeneous antibodies. Unlimited amounts, o monoclonal antibodies formed in this manner can be prepared against the desired antigens or haptens [see C. Milstein "Monoclonal Antibodies", Scientific American, 243 , p. 66, (1980); A. Nisonoff, Molecular Immunology (2d Edition), pp. 169-81, (1984)]. - 12 -

ACTIVE IMMUNIZATION — The process of eliciting an immune response to an antigen or hapten by administration to the host of an immunogenic form of the antigen.

PASSIVE IMMUNIZATION — The process of supplementin body's immune system by injecting preformed antibodies speci for a given antigen. This is opposed to active immunization wherein the immune system is induced to produce antibodies against an antigen in response to innoculation with that antigen.

One aspect of this invention relates to a method fo contraception comprising the step of administering to a mamm a composition comprising an effective amount of anti-idiotyp antibodies to anti-reproductive hormone antibodies. Some of the anti-idiotypic antibodies are antibodies which possess internal images of specific antigenic determinants found on reproductive hormones. The anti-idiotypic antibodies may be obtained by the production of antibodies against a reproduct hormone's antigenic determinants (epitopes) followed by the of these antibodies to elicit anti-antibodies. Some of thes anti-antibodies possess antigenic determinants which immunochemically cross-react with similar antigenic determinants on the reproductive hormone. The portion of th anti-antibodies which are capable of binding to the anti- reproductive hormones antibodies' antigen binding sites poss internal images of the reproductive hormone determinants against which the anti-reproductive hormone antibodies are directed.

Advantageously, the method of this invention allows for the production of antibodies by male and female mammals against anti-id^' antibodies expressing internal images of the antigenic determinants of reproductive hormones. These anti antibodies are used in place of the actual antigen as a means to elicit reproductive hormone-specific antibodies and there prevent conception.

The present invention provides distinct advantages over previous methods of eliciting active immune responses against reproductive hormones.

Because an internal image represents a single, distinct epitope expressed by an antigen, internal image vaccines allow for the precise regulation of the heterogeneit and epitopic specificity of anti-reproductive hormone immune responses. As a result the cross-reactivity problems associated with anti-gonadotropin vaccines can be avoided by internal images of epitopes unique to the targeted gonadotrop

Internal images provide advantages over subunit or peptide vaccines because they mimic structures that the latte formulations cannot. For example, conformational epitopes formed by the secondary, tertiary or quaternary structures assumed by complex protein antigens and determinants containi carbohydrate can be targeted by internal image vaccines, but - 14 -

not by peptide or subunit vaccines. Furthermore, when generated in a foreign species, internal images are inherentl immunogenic and need not be coupled to carrier molecules to elicit i munity.

In our especially preferred embodiment, monoclonal internal image vaccines are used to produce, inexpensively an reliably, the large quantities required for vaccination programs.

According to one embodiment of this invention, to obtain internal images of epitopes expressed by a selected reproductive hormone, the appropriate reproductive hormone preparation is used to immunize mice whose anti-reproductive hormone antibody levels are assayed by standard serological procedures. The reproductive hormone preparation used to induce immunity can consist of intact hormone chemically modified hormone, multi- or mono-epitope hormone fragments, o compounds consisting of sequential repeats of hormone epitopes. These forms may be obtained and derived from naturally occurring compounds and/or man-made by biochemical recombinant DNA procedures. Such forms can be selectively altered to enhance their immunogenicity, such as by their chemical conjugation to immunogenic carrier molecules. When anti—reproductive hormone antibody titers have reached acceptable levels, the anti-reproductive hormone antibodies a tested for their effect in the target species. Alternatively - 15 -

the anti-serum may be tested for its capacity to specificall neutralize the biological activity of the hormone in vitro. Cells from mice which are producing contraceptive anti-hormo antibodies are then used to produce hybridomas. Reproductiv hormone-specific monoclonal antibodies are selected by means standard assays that detect anti-hormone antibodies, and monoclonal cell lines secreting anti- reproductive hormone antibodies are established. The monoclonal anti-reproductiv hormone immunoglobulins are subsequently screened for contraceptive potential on the basis of their ability to blo in vivo fertilization (and/or in vitro hormone biological activity) . Mouse monoclonal antibodies which are contracept in the targeted species are then used to elicit anti-antibod which express internal images of reproductive hormone antige determinants. When mice are to be immunized with anti-reproductive hormone monoclonal antibodies, the monoclo antibodies should be coupled to an immunogenic "carrier" protein to enhance immunogenicity; this is not required when immunizing other species with mouse immunoglobulins. Animal which have been immunized with anti-reproductive hormone and which show a positive titer may subsequently be used for the production of serum anti-idiotypic antibodies by further immunization and bleeding. Mice and rats may also serve as antibody producing cell donors for the production of monoclo anti-idiotypic antibodies derived by standard hybridoma techniques. - 16 -

In addition to anti-reproductive hormone antibodies, any molecule, compound or structure that specifically binds t a reproductive hormone can be used to generate antibodies tha bear internal images of the hormone. Thus, internal images c be generated by immunizing an animal with fragments of anti-reproductive hormone antibodies if the fragments are capable of specifically binding to the hormone. For example, antibody Fab fragments, which are formed by enzymatic cleavag of antibodies by standard techniques and which retain the binding specificity of the native antibody, could be used to generate internal image bearing antibodies. Similarly, any other hormone binding compounds, including compounds that are polypeptides, proteins, glycoproteins, lipoproteins, carbohydrates, lipopolysaccharides, fatty acids, lipids, or nucleic acids, can be used to generate antibodies that expres internal images of the hormone. Such compounds can be in unmodified or chemically modified forms. (In some instances may^" be necessary to render a hormone-binding compound immunogenic by coupling it to a carrier molecule by standard techniques.)

The choice of species in which to raise the anti-idiotypic antibodies is dictated by the intended use of the antibodies. If one desires to produce monoclonal anti-idiotypic antibodies expressing internal images, mice an rats are the preferred species. If it is intended that sera 17 -

serve as the source of anti-idiotypic antibodies, larger animals (e.g., horses or swine) are preferable since they yi greater quantities of sera. To prepare vaccines for use in humans, dogs, cats or other large mammals, large species, su as burrows, horses or goats are preferred. It is preferable generate the internal images in species other than the one t be immunized, so that there is no need to couple the interna image antibodies to an immunogenic carrier in order to gener an immune response.

The method of this invention may be used to treat a mammal, including, but not limited to, dogs, rabbits, rats, mice, pigs, horses, cattle, or primates, including humans. Anti-idiotypic antibodies to anti-reproductive hormone antibodies that are effective in preventing contraception in particular species of mammal may not be contraceptive in all mammals. This is due to antigenic differences in the reproductive hormones of different species. In view of the foregoing, it will be understood that in practicing the meth of the present invention, the initial immunogen, reproductiv hormone, must be capable of eliciting contraceptive antibodi that can react with the specified reproductive hormone of th species targeted for fertility control. The reproductive hormone may be obtained from the target species or from an appropriate alternative species. The reproductive hormone, any appropriate epitopes thereof, may also be obtained synthetically or by the techniques and methodologies of geneti engineering.

The sex and species of animal to be immunized are als factors in selecting the reproductive hormone to be targeted b a vaccine. Immunity against a particular hormone must confer infertility upon the host. For example, specific immunity against human CG can prevent pregnancy in a woman; however, it would not affect fertility in a man or in a non-primate species, neither of which relies upon human CG for fertility. Therefore, immunocontraceptives that elicit human CG-specific antibodies can be used for fertility control in women, but not in men or in non-primate species.

It should be noted that the contraceptive hormone-specific monoclonal antibodies, which are utilized herein to induce anti-idiotypic antibodies, can also be utilized as mediators of contraception when injected in a nonimmunogenic form into the appropriate host (i.e., passive immunization). This use is subject to conditions stated above plus the additional restriction that the monoclonal antibody donor and the recipient must be of the same species to avoid the induction of anti-antibodies that neutralize the injected hormone-specific antibodies. This additional restriction regarding the administration of foreign antibodies would not apply if the passively administered monoclonal anti-hormone antibodies are administered under conditions that prevent the host from generating an immune response against the foreign anti-hormone antibodies.

The contraceptive capacity of the anti-idiotypic antibodies obtained, as described above, may be tested in the target species as follows: the animals are immunized with purified or partially purified preparations of anti-idiotypic antibodies expressing internal images of one or more reproductive hormone antigenic determinants and the resulting anti-reproductive hormone antibodies are tested for their ability to inhibit in vitro biological activity of the hormon and in vivo fertilization in the target species. Anti-idiotypic antibodies which elicit anti-reproductive hormone responses in the target mammal effectively prevent conception and/or implantation when they are used to prepare a contraceptive vaccine. Each dose of the vaccine should contai an amount of anti-idiotypic antibodies that will elicit the production of a quantity of anti-reproductive hormone antibodies sufficient to elicit a contraceptive effect in the target species.

The means by which anti-reproductive hormone antibodies prevent conception has not been thoroughly established. Without being bound by theory, we believe that the contraceptive effect of our vaccine is due to the steric hindrance resulting from the attachment of antibody to reproductive hormones rendering the hormones incapable of effectively binding to their specific receptors. Anti-hormone antibodies can also reduce the concentration of free hormone i the body through opsonization. Specifically, by adhering to the hormone, these antibodies make the hormone a better target for the body's immune system, which then efficiently destroys it through phagocytosis. In addition, anti-human CG antibodies, for example, may bind to human CG which is expressed on the trophectoderm's surface, and thereby prevent implantation. When anti-reproductive hormone antibody levels recede following the cessation of immunization procedures, fertility is regained.

A vaccine of this invention may be either monovalent or multivalent. That is, it may comprise monoclonal antibodie expressing a single internal image, or it may comprise different types of anti-reproductive hormone monoclonal antibodies, each type of antibody having a different antigenic specificity. Alternatively monovalent and multivalent vaccine of this invention may comprise serum antibodies that express internal images. Multivalent vaccines may contain anti-idiotypic antibodies expressing internal images of two or more epitopes found on a single reproductive hormone. Multivalent vaccines may also contain internal images of epitopes found on different hormones, in order to elicit antibodies against more than one hormone. As a matter of practical convenience, the contraceptive vaccines described herein may also be combined with, and thus jointly administered with, other vaccines in such cases wherein the administered components are compatible and have no negative consequences on-the immune responses against each antigenic component of the mixture.

Administration of these anti-idiotypic antibodies, or pharmaceutically acceptable derivatives thereof, may be via an of the conventionally accepted modes of administration of agents which are intended to induce immunce responses. These include various parenteral routes, including, but not limited to, subcutaneous, intramuscular, intraperitoneal, and intravenous administrations. When applicable, non-parenteral routes of immunization may also be employed.

The compositions used in these applications may also be in a variety of forms. The anti-idiotypic antibodies, whic will generally be administered using the foregoing methods, also will preferably include conventional pharmaceutically acceptable carriers typically used in vaccine formulations, such as sterile water or sterile saline solution. Vaccine compositions comprising the anti-antibodies may also include other medicinal agents, adjuvants, excipients, etc. Preferably, the compositions of this invention are in the form of a unit dose. The amount of anti-idiotypic antibodies administered as a vaccination at one time, or over a period of time, will depend on the subject being treated, the manner and form of administration, and the judgment of the treating physician or veterinarian. However, a contraceptively effective dose may be in the range of from about 1 ng/kg to about 1 mg/kg, preferably about 10 ug/kg to about 100 ug/kg, i being recognized that lower and higher doses may also be useful. Generally, an immunization series can consist of one such dose of vaccine followed at 14 day intervals by three boosts of similar dose, and will provide a contraceptive effec for a period of about 6-12 months.

In addition, it should be recognized that the invention described herein can be applied to any condition wherein the presence of reproductive hormone-specific antibodies is deemed to be suitable. For example, the elicitation of antibodies specific for LHRH, or specific for FSH plus LH, may be a useful means of controlling prostate cancer in males. Antibodies expressing internal images of these hormones can, by means of the methods described herein, be utilized to evoke hormone-specific antibodies that aid in the control of prostate cancer. Alternatively, such control could be effected by the passive transfer of monoclonal antibodies specific for these hormones. Similarly as a secon example, the induction of humoral immunity against LHRH or against FSH plus LH in breast cancer patients by means of the methods of this invention may be a useful approach towards controlling estrogen-dependent breast cancers. As a third example, antibodies against LHRH have been shown to result in increased weight gain in beef cattle. The invention described herein could be utilized to evoke LHRH-specific antibodies in beef cattle, by means of active immunization with LHRH interna image bearing antibodies or by passive immunization with monoclonal anti-LHRH antibodies and thereby result in increase weight gain.

In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner.

EXAMPLE

In this example we demonstrate a means of producing vaccines capable of eliciting a contraceptive antibody respons against LHRH. In broad outline, mice are immunized against LHRH coupled to an immunogenic carrier. After it is verified that the mice are producing antibody to LHRH, a cell fusion is performed to produce hybridomas secreting anti-LHRH antibody. The monoclonal antibody is then shown to be contraceptive in vivo. Following this, the monoclonal anti-LHRH antibodies are coupled to an immunogenic carrier and injected into mice to induce anti-idiotypic antibodies. A second cell fusion is the performed to produce monoclonal anti-idiotypic antibodies, fro which are selected those monoclonal antibodies that express immunogenic internal images of LHRH. Finally, the LHRH—internal image antibodies are utilized as immunogens to induce contraceptive LHRH-specific antibodies in rabbits.

A. Production of Contraceptive

Monoclonal Anti-LHRH Antibodies

We first prepared immunogens consisting of LHRH coupled to succinylated human gamma globulin (sHGG) or to diptheria toxoid (DT), as follows. To succinylate HGG, 1.2 g of succinic anhydride (Aldrich Chemical) was slowly added to 1.0 g of HGG (Sigma Chemical) dissolved in 10.0 ml of 0.15 M phosphate buffer, pH=7.2. The pH was maintained at 7.0 with the addition of 5.0 M NaOH. The reaction was carried out at room temperature with stirring for 1 hour. The sHGG was next precipitated by adjusting the pH to 4.0 with the addition of 9.0 M acetic acid. We collected the precipitate by centrifugation at 12,000 xg for 30 minutes, then resuspended in 50 ml saline, to which 5.0 M NaOH was added to adjust the to 7.2. We then dialyzed the sHGG against saline at 4°C.

To couple LHRH to the sHGG, 30 mg of [glu¹]-LHRH (Vega Biochemicals) was added to 150 mg of sHGG in 20 ml saline. 0.1 M NaOH was used to adjust the pH to 7.5. 192 mg of l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI) (Pierce Chemical) was then added with the pH maintained at 7.5. The mixture was stirred for 8 hours at room temperature, then dialyzed at 4°C against saline. The LHRH-sHGG conjugate was stored at -20°C. LHRH-DT was prepared in an identical manner except that DT was not succinylated prior to conjugatio to [glu¹]-LHRH.

Anti-LHRH antibody responses were induced in mice as follows. Four-month old female CAF../J mice were injected intraperitoneally with 100 ug of LHRH-sHGG (in 0.1 ml) emulsified 1:1 in 0.1 ml of FCA (H37 Ra) (DIFCO), the standard oil-based adjuvant used in immunological studies in mice. Fou subsequent booster injections were given at approximately weekly intervals. Each boost, consisting of 100 ug LHRH-sHGG in 0.1 ml saline, was administered intraperitoneally. Epinephrine (0.1 ml) (1:5000) was injected intraperitoneally with each boost. Mice were also immunized with LHRH-DT via an identical protocol. We thus elicited the production of murine antibodies specific for LHRH.

In order to ascertain whether the mice were producing LHRH-specific antibodies, we conducted an Enzyme-Linked Immunosorbent Assay ("ELISA"). In this assay, the antibody is labelled by a covalently attached enzyme (instead of the radiolabel used in a radioimmunoassay) . The enzyme attached t the antibody is one that can react with a colorless substrate to give a colored product. The amount of product released in fixed period of time depends on the concentration of enzyme, and this in turn is a measure of the amount of antibody present. Spectrophotometric equipment reads the optical densities (O.D.), which correlates with the amount of bound antibody.

We established our assay, as follows: we first synthesized a target antigen for the ELISA by conjugating LHRH to bovine serum albumin (BSA) . 5.52 mg n-maleimido-benzoyl-N- hydroxysuccinimide ester (Pierce Chemical) was added to 1.0 ml N,N-dimethylformamide (DMF) (Pierce Chemical). To a second 1. ml DMF was added 50 mg [glu ]-LHRH. The two DMF solutions were mixed and stirred 16 hours at room temperature to form MBS-LHRH. 50 g BSA was dissolved in 10.0 ml of 0.2 M sodium bicarbonate buffer (pH=9.0). To this was added 4 mg 2-iminothiolane HC1 (Pierce Chemical), following which 1.0 ml of the MBS-LHRH solution was added. The mixture was stirred a room temperature for 8 hours, then dialyzed against saline. Final LHRH-BSA concentration was determined by absorption (A_2g ) using a Gilford Spectrophotometer #260. To conduct the ELISA, we coated Immulon II U-Plate (made by Dynatech) wells with 25 ul LHRH-BSA at 2-10 ug/ l in glycine coating buffer (0.1M, pH=9.5) and maintained the coated wells overnigh at 4°C. The wells were rinsed four times with a wash solution consisting of saline to which we had added 0.5% Tween-20 and 0.02% NaN, . We then added 25 ul of primary antibodies (tenfold serial dilutions of anti-LHRH-sHHG sera obtained from mice immunized with LHRH-sHGG, running from 10^~ through 10 —8; or cell culture supernatants when testing for monoclonal anti-LHRH antibodies) per well and incubated for on hour at 22°C. All anti-sera dilutions were made in diluting buffer, consisting of FTA Hemagglutination Buffer + 0.05%

Tween-20 + 0.02% NaN_ . To verify antibody specificity, binding could be inhibited by first adding to each well 25 ul of [glu ]-LHRH (usually at 1.0 mg/ml) in diluting buffer

(before the addition of primary antibody), ELISA O.D. values from these wells were compared with O.D. values from wells to which 25 ul of diluting buffer alone had been added. We then rinsed the wells again. We followed these with 25 ul of secondary antibodies per well, consisting of either biotinylated rabbit anti-mouse Ig (1:1000 in diluting buffer) or (for isotyping) rabbit anti-mouse Ig (polyspecific or monospecific isotyping reagents, 1:1000) and incubated for one hour at 22°C. We again rinsed the wells and then added to eac well 25 ul of either avidin-alkaline phosphatase conjugate

(1:1000) or, for isotyping, goat anti-rabbit Ig-alkaline phosphatase conjugate (1:2000) and incubated for one hour at

22°C. We again rinsed the wells, and we then added to each well, 25 ul of 1 mg/ml p-nitrophenyl-phosphate (in 10% diethanolamine, 0.5mM MgCl₂, 0.02% NaN, , pH=9.8) and allowed the color to develop for 5-30 minutes. We determined the optical densities with a Dynatech Microelisa Reader. We coated other protein antigens, such as hen egg lysosome, bovine serum albumin, and chicken ovalbumin, as negative controls for antibody specificities onto plates and analyzed them in a manner similar to that described above.

We performed ELISAs on 1:10 serial dilutions of serum obtained via tail vein bleedings of immunized mice using our dilution solution. Figure 1 depicts the results of a typical experiment, in which three injections of a LHRH-diptheria toxoid conjugate were sufficient to elicit strong serum antibody response against LHRH. Our immunization protocols generally elicited anti-LHRH serum antibody titers of 10 when measured by ELISA. The specificity of the response was ascertained by inhibition with free LHRH. As shown in Figure 2, binding was reduced to background levels by increasing concentrations of free LHRH. Identical concentrations of inhibitor had no effect upon the binding of carrier (diptheria toxoid) specific antibody to the carrier (data not shown). Thus, cells obtained from the LHRH-immune mice could be used as fusion partners to obtain hybrids secreting LHRH-specific antibodies.

We then performed cell fusions according to the methods of B.B. Mishell and S.M. Shiigi [Selected Methods in Cellular Immunology. W.H. Freeman and Co., San Francisco (1980)]. We created hybridomas producing monoclonal anti-LHRH antibodies by fusing spleen cells from LHRH-sBSA immunized mic with P3 tumor cells using polyethylene glycol. We selected ou hybrids by feeding on Hypoxanthine-Aminopterin-Thymidine [HAT] supplemented media and then screening for specific antibody protection using the ELISA protocol defined above. Two fusion (F18 and F35) each yielded several distinct hybridoma lines. We considered cell lines to be established when their cloning efficiencies reached 100%. Samples of established cell lines were frozen and stored uner liquid N„ .

We generated working quantities of monoclonal antibodies as ascites tumors in the peritoneal cavities of mic and collected the ascites fluid, according to the methods of Mishell, supra. We injected CAF. mice with 0.5 ml of Pristane, intraperitoneally. Three days later, we injected 2 10 hybrid cells, suspended in 0.5 ml saline, into the mide intraperitoneally. After collecting the ascites fluid from th peritoneal cavity of mice, we centrifuged the fluid (400 x g for 10 minutes) to remove the cells.

Referring now to Table I, we have provided therein th LHRH-specific monoclonal antibodies that we obtained as described above, along with their isotypes. We determined the isotypes by ELISA as described above. TABLE I

LHRH-specific Monoclonal Antibodies

Cell line/Antibody Isotvpe

18 - 1 IgG - 1

18 - 2 IgG - 1

18 - 3 IgG - 2b

35 - 1 IgG - 1

35 - 2 IgG - 1

35 - 5 IgG - 1

35 - 6 IgG - 1

35 - 7 IgG - 1

35 - 8 IgG - 1

We next assessed the contraceptive potential of the monoclonal anti-LHRH antibodies by passively immunizing fertil mice with the monoclonals, and mating the antibody recipients with fertile breeder animals. Figure 3 presents the results o a typical experiment, in which fertile female CAF,/J mice were each injected intravenously with 1.0 mg of individual monoclonals. A second injection was administered 7 days later at which time the mice were cohabitated with fertile male

CAF./J mice. Sham immunized control mice, which had received

1.0 mg of monoclonal antibody specific for an unrelated protei antigen, were included in each cage. The monoclonal antibodie were prepared for injection by running a 40% saturated ammoniu sulphate precipitation of immunoglobulins from each ascites fluid, followed by dialysis against water and then lyophilization. The duration of infertility was taken as the time period from the date of the second antibody injection to the estimated day of conception. (Conception day = day litter delivered less 19 days.) As shown in Figure 3, each monoclona antibody preparation was capable of inducing a reversible stat of infertility in mice. The duration of the period of infertility varied between monoclonal antibodies. Because our monoclonal antibodies were shown to be contraceptive in vivo, we subsequently utilized them to induce anti-idiotypic antibodies in mice.

B. Production of Monoclonal Anti-idiotypic

Antibodies Expressing Internal Images of LHRH

The next stage of our work involved the use of our contraceptive monoclonals as immunogens to elicit murine anti-idiotypic antibody responses. We subsequently derived therefrom, monoclonal anti-idiotypic antibodies which express immunogenic internal images of LHRH.

We induced anti-idiotype responses by immunizing mice with monoclonal anti-LHRH antibodies conjugated to immunogenic carriers. We prepared our immunogens by chemically coupling our monoclonal antibodies to keyhole limpet hemocyanin ("KLH") an immunogenic protein, using equal amounts of antibodies and KLH.

We isolated KLH, which we had purchased as an ammoniu sulphate haemolymph precipitate, by dialyzing the precipitate slurry against 0.5M NaCl followed by gel filtration over a Sephacryl 400 column (50 x 1.5 cm, 15 ml/hour, 1M NaCl) . We pooled KLH-containing fractions and determined the protein concentrations using standard spectrophotometric techniques with a Gilford Spectrophotometer 260 (A_2aQ measurements) . We then concentrated the combined material to 5.0 mg/ml on an Amicon concentrator.

In order to purify our monoclonal antibodies, we precipitated each with ammonium sulphate (40%) , dialyzed the precipitates against H_0, and then lyophilized the dialyzed solutions. We dissolved 10.0 mg of each lyophilized antibody in 2.5 ml of 0.5 M NaCl containing 5.0 mg KLH. After centrifuging the mixtures to remove small amounts of insoluble material (10 minutes at 2000 x g) , we added 50 mg of l-ethyl-3-(3-dimethylaminoproρyl) carbodiimide hydrochloride t each mixture while stirring. We allowed the reactions to proceed for 16 hours at room temperature and then dialyzed the mixtures against 0.5 M NaCl. The dialyzed mixtures were store frozen at -20° and thawed just prior to use.

We immunized CAF, mice with 0.1 ml injections (containing 100 ug of each conjugate per injection) by a protocol identical to that described for immunization against LHRH-sBSA. Mice received either antigen mixtures containing anti-LHRH antibody-KLH conjugates with each injection, or a sequential series of injections wherein each injection consisted of a different anti-LHRH antibody-KLH conjugate. The precise epitopic mimicry of an internal image is determined by the antigenic specificity of the antibody used t elicit the anti-idiotypic antibody response. Due to the small size of the LHRH peptide (ten amino acids), the epitopic fine-specificities of our monoclonal anti-LHRH antibodies most likely overlapped. However, the degree of similarity between the fine-specificities of the various monoclonal antibodies wa unknown; it could range from partial to complete identity. Therefore, to maximize the probability of inducing LHRH internal images, we elicited murine anti-idiotypic antibody responses by two approaches.

In the first approach, it was assumed that none of anti-LHRH monoclonal antibodies possessed identical epitopic fine-specificities; consequently, none of the anti-LHRH monoclonal antibodies would possess antigen binding sites that were biochemically, and thus immunochemically, identical. Therefore, as each of the monoclonal anti-LHRH antibodies woul elicit unique internal images (expressed by anti-idiotypic antibodies), a mixture containing approximately equal quantities of each KLH-monoclonal antibody was used to evoke anti-idiotypic antibody responses in mice. Under this protocol, there was an equal opportunity for each monoclonal anti-LHRH antibody paratope (binding site) to elicit anti- idiotypic antibodies. After an initial (primary) immunization, subsequent reexposure to most antigens, including immunoglobulins and KLH, induces an enhanced (secondary) immune response. In the secon approach, it was assumed that a minimum of two monoclonal anti LHRH antibody species had identical fine specificities. By sequentially immunizing with the KLH-monoclonal anti-LHRH antibody conjugates, secondary (enhanced) responses would be evoked upon the injection of a monoclonal anti-LHRH antibody conjugate that possessed a paratope that was biochemically and immunochemically identical to that of another conjugate agains which the host had been previously immunized. Idiotopes not shared by the different monoclonal anti-LHRH antibodies would elicit primary (smaller) responses. Thus, to enhance the production of internal image-bearing anti-idiotypic antibodies against shared antigen binding sites (relative to the production of anti-idiotypic antibodies specific for non-share determinants), we immunized mice with the monoclonal anti-LHRH antibody conjugates by means of a sequential immunization protocol in which a different conjugate was administered with each injection.

(We later determined that each of these two approache successfully elicited potential internal image bearing anti- idiotypic antibodies.)

To obtain monoclonal anti-idiotypic antibodies that express internal images of LHRH, we performed cell fusions in which spleen cells obtained from mice immunized with anti-LHRH antibody-KLH conjugates were fused with P3 cells by the method already described herein.

As a first step in screening for monoclonal antibodie expressing internal images of LHRH, we assayed the cell cultur supernatants for the presence of antibodies that bound to rabbit anti-LHRH antibodies. We selected anti-idiotypic antibodies whose binding was inhibited by LHRH when the rabbit anti-LHRH antibody binding sites were occupied by LHRH. To generate the rabbit anti-LHRH, a rabbit was injected subcutaneously with 1.0 mg LHRH-sHHG in 0.2 ml FCA. The rabbi was boosted subcutaneously at three week intervals with 1.0 mg of LHRH-sHHG in alum. Blood samples were periodically obtaine from the central ear artery, and sera samples were assayed for the presence of anti-LHRH antibody by ELISA (as previously set forth herein) .

To detect monoclonal anti-idiotypic antibodies expressing LHRH internal images, we utilized the previously described ELISA with the following modifications: Rabbit sera containing anti-LHRH antibodies was coated onto Immulon U Plat wells by diluting the sera 1:500 in glycine coating buffer, then adding 25 ul per well and incubating overnight at 4°C. After the coated wells were rinsed, we added 25 ul of 1.0 mg/m [glu ]-LHRH in diluting buffer to half of the wells; to the remaining wells we added 25 ul of diluting buffer. After incubating the plates for 30 minutes at room temperature, we added 25 ul samples of hybrid culture supernatant to the wells. Each hybrid sample was added to a well with LHRH and t a well with only coating buffer. After a 1 hour incubation at room temperature, the wells were rinsed. Following this, we followed the basic ELISA protocol to detect presence of mouse antibody. Potential LHRH internal image anti-idiotypic antibodies were then selected on the basis of their capacity t bind to the rabbit anti-LHRH antibodies only in the absence of LHRH. Generally, over 25 potential internal image antibodies per fusion were identified by this procedure.

The specificity of the binding by the anti-idiotypic antibodies was ascertained by inhibition with various quantities of free LHRH. The results of a typical ELISA for one monoclonal anti-idiotypic antibody are presented in Figure 4, wherein the anti-idiotypic antibody to rabbit anti-LHRH antibody binding was progressively inhibited by increasing quantities of LHRH.

We developed cell lines secreting potential internal image anti-idiotypic antibody, as identified by the ELISA, int hybrid cell lines secreting monoclonal antibodies, using the methods previously described herein. Ascites fluid containing monoclonal antibody was produced for each anti-idiotypic hybri cell line, also as already described herein. Each potential LHRH-internal image monoclonal antibod was next subjected to a selection procedure designed to distinguish those antibodies that expressed internal images of LHRH. In this procedure, we assessed the capacity of each monoclonal anti-idiotypic antibody to induce anti-LHRH antibodies when used as an immunogen in mice and rabbits.

Anti-idiotypic monoclonal antibodies were prepared as immunogens by individually precipitating each monoclonal antibody from ascites fluid with 40% saturation with ammonium sulphate, followed by dialysis against saline. Antibody ' concentrations were determined by spectrophotometry at _₈₀ and then adjusted to 1.0 mg/ml in saline.

To immunize rabbits with anti-idiotypic antibodies, w prepared mixtures of monoclonal anti-idiotypic antibodies containing up to 10 individual antibodies, in which each monoclonal antibody was present in a dose (per injection) of either 10 ug or 500 ug. Rabbits received four injections, the first two administered in 0.2 ml FCA, the last two in 0.2 ml alum. Injections were given subcutaneously at 14 day intervals, and blood samples were obtained 10 to 14 days following each injection. Rabbit anti-LHRH responses were assayed by ELISA as previously described herein.

To immunize mice with monoclonal anti-idiotypic antibodies, we first chemically conjugated each monoclonal antibody to KLH as previously described herein. Mice were immunized with either individual conjugates or with mixtures of up to 4 conjugates, at doses of either 5 ug or 100 ug of each conjugate per injection. The mice received 4 intraperitoneal injections at 14 day intervals; the first two injections were administered in 0.2 ml FCA (H37 Ra), and the second two injections were given in 0.2 ml alum. Blood samples were obtained by tail vein bleedings 10 to 14 days after each injection, and the serum was assayed for the presence of anti-LHRH antibodies by means of the ELISA herein described.

After screening anti-idiotypes for their expression of immunogenic LHRH internal images, and identification of monoclonal antibodies that express LHRH internal images, their immunocontraceptive capacities are assessed by immunization in numerous species, including (but not limited to) mice, rabbits, dogs, cats and cattle.

There are two principal stages in the development of internal image immunogens. The first stage involves the production of the appropriate antigen-specific antibodies, or idiotypes (ids) . This is followed by the second stage, wherei the ids are utilized to generate anti-idiotypic antibodies (anti-ids) which must be tested for internal-image expression on the basis of their capacity to induce antigen-specific immunity in animals.

A critical factor in the development of any internal image vaccine relates to the number of ids used to induce anti-ids. Because the potential number of anti-ids derived against each id is quite substantial (conceivably in the hundreds), it is vital that the correct ids be selected prior to the induction of the anti-ids. If the wrong ids were used, that is, ids directed against irrelevant epitopes (on the antigen) or ids directed against only a portion of important epitopes, it would not be possible to obtain the best internal images. Therefore, it is crucial that ids with the correct specificities be selected before they are used to induce anti-ids. If this were not done, it would be necessary to screen such large numbers of anti-ids that it would be extremely difficult to obtain the desired internal image(s).

The correct ids must be selected on the basis of both their specificity and their ability to mediate the desired biological effect. In general, it is desirable that the ids b specific for the targeted antigen, with minimal cross-reactivity against non-related antigens. Numerous ^* standard immunological assays can be employed to test for this. To demonstrate that the ids mediate a specific biological effect, one must use an independent functional test that distinguishes effective ids from those lacking in biological activity. It is essential that this test be conducted at the stage prior to the induction of anti-ids in order to limit the number of ids used to make the anti-ids. The normal functions of the target antigen and the biological effect resulting from the neutralization of the antigen by specific antibodies dictate the specific test to be employed.

The importance of this selection at an early stage (i.e., when the ids are formed) is illustrated by the following: to generate internal images of hCG, we first had t obtain the appropriate ids. After generating numerous monoclonal anti-hCG antibodies (ids), each was screened by ELISA against other human hormones known to share cross-reactive epitopes with hCG. These other hormones included luteinizing hormone (LH) , follicle stimulating hormo (FSH) , and thyroid stimulating hormone (TSH) . The anti-hCG monoclonal antibodies which cross-reacted with one or more of the other hormones were discarded. Only antibodies that specifically reacted with hCG were retained. An example of such a selection is illustrated in Figure 5. As shown in Figure 5 monoclonal antibodies 369-4 and 369-5 which reacted with hCG only were subsequently used for the induction of anti-ids; monoclonal antibodies 369-1, 369-2 and 369-3 which cross reacted with other relevant human hormones and were discarded. We thus were able to select antibodies that were specific for our target antigen.

To test our anti-hCG monoclonal antibodies for biological activity, we employed a previously established ass in which the increase in uterine weight induced in young fema rats by a set quantity of hCG can be counteracted by antibodi capable of neutralizing the biological activity of hCG. Using this assay we demonstrated that specific combinations of certain anti-hCG monoclonal antibodies were effective in neutralizing the biological activity of hCG (see Figure 6). These antibodies were subsequently used for the induction of anti-ids. Antibodies which had no biological effect were discarded.

In both phases of the hCG-specific id selection process (specificity testing and biological activity testing), numerous monoclonal antibodies (ids) were found to be unsuitable for the induction of internal images (anti-ids). Had we not employed these selection procedures, it would have been necessary to induce anti-ids against not only the few relevant ids but also against the many irrelevant ids. Screening this enormous number of anti-ids for internal image expression would have been very difficult and time-consuming. The selection of the proper ids is vital to the successful generation of internal images of hCG.

To ensure that the best epitopes are represented in any internal image vaccine, it is necessary that may idiotypic antibodies obtained from numerous cell fusions be subjected to the selection process. Only by screening large numbers of idiotypic antibodies for antigenic specificity and biological activity can it be assured that the most desirable are selecte to be used as immunogens to generate anti-idiotypic antibodies. It would be totally infeasible to bypass this important step and screen instead at the anti-idiotypic antibody level, due to the enormous number of anti-idiotypic antibodies that would need to be tested in immunization and biological activity experiments. The selection of the proper ids is vital to the successful generation of the internal imag vaccines.

While we have presented a number of embodiments of this invention, it is apparent that our basic construction can be altered to provide other embodiments which utilize the processes and compositions of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the claims appended hereto rather than by the specific embodiments which have been presented by way of example.

Claims

1. A vaccine comprising an effective amount of anti idiotypic antibodies to anti-reproductive hormone antibodies, which anti-idiotypic antibodies express internal images of reproductive hormone antigenic determinants.

2. A vaccine for use in passive immunization agains reproductive hormones comprising antibodies formed to.the . anti-idiotypic antibodies of claim 1.

3. A method for preparing a contraceptive vaccine comprising anti-idiotypic antibodies that express internal images of a specific reproductive hormone which comprises:

a) producing antibodies that react with the specific reproductive hormone; b) screening the antibodies so produced by reacting a quantity of the antibodies with the specific reproductive hormone and with other molecules that share cross-reactive epitopes with the specific hormone; c) selecting the antibodies that react with th specific hormone and that do not cross-react with the other molecules and discarding the antibodies that cross-react with one or more of the other hormones; d) testing a quantity of the selected- antibodies of step (c) for biological activity by assaying the antibodies to determine if the antibodies can counteract the biological activity of a measured quantity of the specific hormone; e) selecting the antibodies that neutralize th biological activity of the specific hormone and disregarding the antibodies that do not counteract the biological activity of the specific hormone; f) using the selected antibodies of step (e) o fragments thereof to produce anti-idiotypic antibodies that express internal images of the specific hormone and that react with the idiotypes on the selected antibodies; and g) using the anti-idiotypic antibodies so produced to produce contraceptive vaccines.

4. The contraceptive vaccine produced by the metho of claim 3.

5. The method of claim 3, wherein the antibodies that react with the specific reproductive hormone are monoclonal antibodies.

6. The method of claim 3, wherein the anti-idiotyp antibodies are monoclonal antibodies.

7. The method of to claim 3, wherein the antibodies that react with the specific reproductive hormone are obtained by immunization with an immunogenic compound that comprises on or more epitopes that cross react with epitopes expressed by the hormone.

8. The method of claim 3, wherein the specific reproductive hormone is selected from the group consisting of LHRH, LH, FSH, CG, estrogen, androgen and progestin.

9. A method for preparing a contraceptive vaccine comprising anti-idiotypic antibodies that express internal images of specific reproductive hormones which comprises:

a) selecting compounds that bind with the specific reproductive hormone; b) screening the selected compounds by reactin a sample of each compound with the specific reproductive hormone and with other hormones known to share cross-reactive epitopes with the specific hormone; c) selecting the compounds that react with the specific hormone and that do not cross-react with the other hormones and discarding the compounds that cross-react with on or more of the other hormones; d) testing the selected compounds of step (c) for biological activity by assaying the compounds to determine if the compounds can counteract the biological activity of a measured quantity of the specific hormone; e) selecting the compounds that neutralize the biological activity of the specific hormone and disregarding the compounds that do not counteract the biological activity o the specific hormone; f) using the selected compounds of step (e) to produce antibodies that express internal images of the specifi hormone and that react with the epitopes on the selected compounds; and g) using the internal image antibodies so produced to produce contraceptive vaccines.

10. The contraceptive vaccine produced by the method of claim 9.

11. A method for contraception comprising the step o administering to a male or female mammal a composition comprising an effective amount of the vaccine according to claim 1, 2, 4 or 10.

12. The method according to claim 11 wherein the composition is administered at a dosage of from about 1 ng/kg of body weight to about 100 mg/kg of body weight.

13. The vaccine of claim 1, wherein the reproductiv hormone is LHRH.

14. The vaccine of claim 1, wherein the reproductiv hormone is LH.

15. The vaccine of claim 1, wherein the reproductiv hormone is FSH.

16. The vaccine of claim 1, wherein the reproductiv hormone is an androgen.

17. The vaccine of claim 1, wherein the reproductiv hormone is an estrogen.

18. The vaccine of claim 1, wherein the reproductiv hormone is CG .

19. The vaccine of claim 1, wherein the reproductiv hormone is progestin.

20. A method for treating prostrate cancer comprisi administering to a male mammal an effective amount of the vaccine of claim 13, 14, 15 or 16.

21. The method according to claim 20 wherein the composition is administered at a dosage of from about 1 ng/kg of body weight to about 100 mg/kg of body weight.

22. A method for treating breast cancer comprising administering to female mammal a composition comprising an effective amount of the vaccine of claim 13, 14, 15 or 17.

23. The method according to claim 22 wherein the composition is administered at a dosage of from about 1 ng/kg of body weight to about 100 mg/kg of body weight.

24. A method for increasing weight gain in mammals comprising administering an effective amount of the vaccine of claim 13, 14, 15 or 16.

25. The method according to claim 24 wherein the composition is administered at a dosage of from about 1 ng/kg of body weight to about 100 mg/kg of body weight.

26. The use of a pharmaceutically effective amount o anti-idiotypic antibodies expressing internal images of a reproductive hormone for the production of a pharmaceutically acceptable composition for use in contraception, a treatment o prostate cancer, treatment of breast cancer or a method for increasing weight gain in mammals.