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WO1988008719A1 - Immunocontraceptifs specifiques d'hormones de reproduction et leurs procedes d'utilisation - Google Patents

Immunocontraceptifs specifiques d'hormones de reproduction et leurs procedes d'utilisation Download PDF

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Publication number
WO1988008719A1
WO1988008719A1 PCT/US1988/001541 US8801541W WO8808719A1 WO 1988008719 A1 WO1988008719 A1 WO 1988008719A1 US 8801541 W US8801541 W US 8801541W WO 8808719 A1 WO8808719 A1 WO 8808719A1
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WIPO (PCT)
Prior art keywords
antibodies
hormone
specific
vaccine
reproductive
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PCT/US1988/001541
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English (en)
Inventor
Stephen Grimes
Eliezer Benjamini
Robert J. Scibienski
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Aphton Corporation
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Publication of WO1988008719A1 publication Critical patent/WO1988008719A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens

Definitions

  • 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.
  • 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.
  • contraceptive antibodies can be generated and used to passivel immunize the mammal.
  • 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") .
  • 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.
  • 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.
  • LHRH and the gonadotropins are suitable targets for immunocontraception.
  • 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.
  • 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.
  • -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).
  • 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.
  • 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.
  • 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.
  • contraceptive antibodies can be generated and then used to passively immunize a mammal against the reproductive hormones.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • LHRH luteinizing hormone releasing hormone
  • GnRH gonadotropin releasing hormone
  • FSH follicle stimulating hormone
  • CG chorionic gonadotropin
  • inhibin inhibin
  • sex steroids including a forms of progestagens, androgens, and estrogens.
  • 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.
  • 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 -
  • reproductive hormone 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, "Unech 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.
  • 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 -
  • 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.
  • 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.
  • 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.
  • 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.
  • cross-reactivity problems associated with anti-gonadotropin vaccines can be avoided by internal images of epitopes unique to the targeted gonadotrop
  • monoclonal internal image vaccines are used to produce, inexpensively an reliably, the large quantities required for vaccination programs.
  • 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.
  • mice 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 -
  • 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.
  • 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.
  • 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.
  • 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.)
  • mice an rats are the preferred species. If it is intended that sera 17 -
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the invention described herein can be applied to any condition wherein the presence of reproductive hormone-specific antibodies is deemed to be suitable.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ELISA Enzyme-Linked Immunosorbent 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.
  • FIG. 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.
  • 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).
  • cells obtained from the LHRH-immune mice could be used as fusion partners to obtain hybrids secreting LHRH-specific antibodies.
  • FIG. 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
  • each monoclonal 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.
  • KLH keyhole limpet hemocyanin
  • 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) .
  • KLH-containing fractions and determined the protein concentrations using standard spectrophotometric techniques with a Gilford Spectrophotometer 260 (A 2aQ measurements) .
  • 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.
  • 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.
  • mice Idiotopes not shared by the different monoclonal anti-LHRH antibodies would elicit primary (smaller) responses.
  • 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.
  • 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.
  • 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.
  • the first stage involves the production of the appropriate antigen-specific antibodies, or idiotypes (ids) .
  • ids antigen-specific antibodies
  • anti-ids antigen-specific antibodies
  • 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.
  • FIG. 5 An example of such a selection is illustrated 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.

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Abstract

Immunogènes comprenant des anticorps anti-idiotypiques dressés contre les anticorps d'hormones d'anti-reproduction, lesdits anticorps anti-idiotypiques exprimant des images internes de déterminants antigéniques d'hormones de reproduction, et procédé de contraception comprenant l'immunisation d'un mammifère femelle ou mâle contre une ou plusieurs hormones de reproduction, utilisant une composition comprenant une quantité efficace desdits immunogènes. L'invention se rapporte également à un procédé de contraception comprenant l'immunisation passive d'un mammifère femelle ou mâle contre une ou plusieurs hormones de reproduction utilisant une composition comprenant une quantité effective d'anticorps dressés contre des hormones de reproduction.
PCT/US1988/001541 1987-05-12 1988-05-04 Immunocontraceptifs specifiques d'hormones de reproduction et leurs procedes d'utilisation WO1988008719A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993020199A1 (fr) * 1992-03-30 1993-10-14 Akzo Nobel N.V. Recepteur de gonadotrophine humaine (recepteur hfs)

Citations (5)

* Cited by examiner, † Cited by third party
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