GnRH/REDUCED PSEUDOMONAS EXOTOXIN CONJUGATES

Abstract: Conjugates of GnRH are constructed from GnRH or an analog thereof, a reduced Pseudomonas exotoxin, or a variant thereof, and a unique linking group. The conjugates are administered to male and female animals to sterilize said animals or to reduce tumors that require sex steroids for growth. The instant conjugates are therefore useful as sterilizing agents and anticancer agents.

Description: TITLE OF THE INVENTION GnRH/REDUCED PSEUDOMONAS EXOTOXIN CONJUGATES CROSS REFERENCE TO RELATED APPLICATIONS This application is based on, and claims priority from, provisional application No. 60/005,899 filed October 27, 1995. BACKGROUND OF THE INVENTION Considerable interest exists with respect to the subject of sterilization of animals. This is especially true of those concerned with veterinary medicine and animal husbandry, particularly as they relate to the subject of sterilization of domestic animals such as dogs, cats, cattle, sheep, horses, pigs, and the like. Sterilization may be used to control undesirable gonadal steroid hormone driven behavior such as aggression in males and estrus behavior in females, to improve carcass quality in food animals such as swine and cattle, and to eliminate boar taint in the carcasses of male pigs. Various methods have been developed over the years to accomplish sterilization. For example, with respect to male cattle, the most widely used procedure for eliminating problems of sexual or aggressive behavior is sterilization through surgical castration. This is done in various ways, e.g., crushing the spermatic cord, retaining the testes in the inguinal ring, or use of a rubber band, placed around the neck of the scrotum, to cause sloughing off of the scrotum and testes. However, most of these "mechanical" castration methods have proven to be undesirable in one respect or another; for example they (1) are traumatic, (2) may introduce the danger of anesthesia and specialized instruments, (3) are apt to produce infection, and (4) require trained personnel. Moreover, all such mechanical castration methods result in complete abolition of the testes and this of course implies complete removal of the anabolic effects of any steroids which are produced by the testes and which act as stimuli to growth and protein deposition. These drawbacks have caused consideration of various alternative sterilization techniques such as the use of chemical sterilization agents. However, the use of chemical sterilization agents has its own set of advantages and disadvantages. On the positive side, chemical sterilization eliminates the stress and danger associated with mechanical castration. Chemical sterilization also has the added advantage of allowing for retention of certain anabolic effects resulting from a continued presence of low levels of circulating testosterone. This is especially valuable in the case of animals raised for human consumption since circulating testosterone promotes growth, efficiency of feed conversion and protein deposition. Unfortunately, there are several disadvantages associated with chemical sterilization. For example, chemical sterilization is often temporary rather than permanent; it also sometimes produces extremely severe, and even fatal, side effects. In WO 9009799 to Nett certain GnRH analogs are coupled to a variety of toxins through an optional linking group consisting of 2- iminothiolane, SPDP (N-succinimidyl-3-(2-ρyridyldithio/propionate), bis-diazabenzidine and glutaraldehyde. The compounds are disclosed as sterilizing agents which purportedly act by selectively killing gonadotrophs.. In addition, Myers et al., Biochemical Journal 227: 1 pg 343 (1985) discloses a conjugate of a GnRH analog and the diphtheria A chain and pokeweed antiviral toxins coupled through SPDP. W093/15751 also discloses chimeric molecules of GnRH, or analogs thereof, and cytotoxins. The chimera of this disclosure is a molecule in which GnRH peptides are directly linked to a modified Pseudomonas exotoxin molecule. At each site of peptide binding to the toxin molecule, there is only one GnRH peptide bound. Administration of such chimeric molecules is reported to result in the destruction of GnRH receptor bearing cells in the pituitary gland, with concomitant reduction in the secretion of sex hormones. The ultimate result of this process is chemosterilization and reduction of steroid stimulated tumor proliferation. UK Application No. 2,282,812 teaches GnRH attached to a cyclic scaffold containing multiple lysine units, termed a MAP (multiple antigen peptide) or lysine tree, and the scaffold is in turn coupled to a cytotoxin such as Pseudomonas exotoxin. The use of the multi-lysine scaffold permits attaching more than one GnRH per cytotoxin linking site; however, the MAP approach is not necessarily an advantage because it remains unproven whether additional GnRH molecules bound to carrier beyond a given threshold results in a more effective vaccine; and furthermore, MAP conjugates generally have the attribute of high insolubility in hydrophobic and hydrophilic solvents rendering them more difficult to formulate. Another approach in animal sterilization involves the use of GnRH vaccines, i.e., immunosterilization. Typically a GnRH molecule, which is only weakly immunogenic, is coupled to an immunogenic macromolecule, such as a protein, in order to enhance the immunogenicity of GnRH; alternatively, a fusion protein containing a GnRH and an immunogenic peptide may be constructed for the same purpose. Animals administered the immunoconjugate or fusion protein develop antibodies against GnRH, which down regulate the action of GnRH resulting in the drastic reduction of sex hormones and the atrophy of hormone dependent organs. A number of GnRH immunoconjugates or fusion proteins suitable for use as vaccines have been described. A commercial GnRH vaccine is currently being marketed in Australia by Arthur Webster & Co Pty Ltd under the name of Vaxstrate® for use in cattle (see e.g., R. M. Hoskinson et at, Aust. J. BioltechnoL. 1990, 4: 166). This vaccine, which is reported to consist of GnRH conjugated to ovalbumin, is poorly immunogenic [the package insert recommends two 3 mg doses per cow and implies less than 100% efficacy]. This vaccine formulated in mineral oil and DEAE-dextran produces severe injection site reactions and lesions. US 4,975,420 discloses immunosterilants comprising a GnRH analog in which the amino acid 1, 6 or 10 has been replaced by cysteine, coupled to a carrier protein. WO88/05308 discloses immunoneutering compositions containing penta-, hexa-, or heptapeptide fragments of native GnRH conjugated with an immunogenic protein WO93/08290 describes fusion proteins comprising GnRH and a leukotoxin polypeptide. The leukotoxin serves as a carrier protein to increase the immunogenicity of the antigen. EP 578,293 discloses fusion proteins comprising a part of an E.coli P-fimbrial filament and GnRH. This carrier system is said to be capable of eliciting a greatly improved immune response against GnRH, and when used in a vaccine, avoids the need for aggressive adjuvants such as complete/incomplete Freunds adjuvant (CFA/IFA). W092/ 19746 teaches recombinant polypeptides comprising GnRH, at least one T-cell epitope and a purification site. WO90/02187 discloses fusion proteins comprising hepatitis B surface antigen and GnRH. The constructs are said to be sufficiently immunogenic to render unnecessary the use of adjuvants and multiple injections. US 5 ,324,512 teaches GnRH linked through an N-terminal glutamine to a carrier protein. The conjugates are claimed to be useful as antifertility vaccines and in the treatment of prostate cancer. WO94/25060 discloses immunogenic peptide containing GnRH, a T-cell epitope and, optionally, an invasin domain. The peptides are useful as antifertility vaccine and for treating androgen-dependent carcinoma. UK 2,228,262 discloses conjugates in which [D-Lys6]GnRH (i.e. amino acid 6 (glycine) of native GnRH has been replaced by D-Lys) is linked to a carrier protein through the ε-amino group of the D-Lys. The conjugates may be used to control fertility or for therapy of prostate cancer. U.S. patent 4.545.985 teaches that Pseudomonas exotoxin A can be chemically conjugated to an antibody or to epidermal growth factor. While this patent further teaches that these conjugates can be used to kill human tumor cells, these chemically linked toxins have been shown to have undesirable, nonspecific levels of activity. Allured, et al.. "PNAS USA" 8^:1320-1324 (1986). This article teaches the three dimensional structure of the Pseudomonas exotoxin A protein. Hwang. et al.. "Cell" 48:129-136 (1987). This article teaches that the Pseudomonas exotoxin A protein can be divided into three distinct functional domains responsible for: binding to mammalian cells, translocating the toxin protein across lysosomal membranes, and ADP ribosylating elongation factor 2 inside mammalian cells. This article further teaches that these functional domains correspond to distinct regions of the Pseudomonas exotoxin A protein. European patent application 0 261 671. published 30 March 1988, teaches that a portion of the Pseudomonas exotoxin A protein can be produced which lacks the cellular binding function of the whole Pseudomonas exotoxin A protein but possesses the translocating and ADP ribosylating functions of the whole Pseudomonas exotoxin A protein (MW 66,000). The portion of the Pseudomonas exotoxin A protein that retains the translocating and ADP ribosylating functions of the whole Pseudomonas exotoxin A protein is called PE-40 (MW 40,000). PE-40 consists of amino acid residues 252-613 of the whole Pseudomonas exotoxin A protein as defined in Gray, et al., PNAS USA 81:2645-2649 1984. This patent application further teaches that PE-40 can be linked to transforming growth factor-alpha to form a hybrid fusion protein produced in bacteria using recombinant DNA techniques. Chaudharv. et al.. "PNAS USA" 84:4538-4542 (1987). This article teaches that hybrid fusion proteins formed between PE-40 and transforming growth factor-alpha and produced in bacteria using recombinant DNA techniques will bind to and kill human tumor cells possessing epidermal growth factor receptors. Bailon. "Biotechnology", pp. 1326-1329 Nov. (1988). This article teaches that hybrid fusion proteins formed between PE-40 and interleukin 2 and produced in bacteria using recombinant DNA techniques will bind to and kill human cell lines possessing interleukin 2 receptors. Edwards, et al.. "Mol. Cell. Biol." 9: 2860-2867 (1989) describe the preparation of the modified TGF- alpha - PE40 hybrid molecules that have been found to have utility in treating bladder tumor cells. Heimbrook. et al.. "Proc. Natl. Acad. Sci. USA" £7: 4697- 4701 (1990) describe the in vivo efficacy of modified TGF-alpha - PE40 in significantly prolonging the survival of mice containing human tumor cell xenografts. The instant invention utilizes various constructs of GnRH, a linking group and a modified Pseudomonas exotoxin (PE). However, unlike the prior art constucts are difficult to analyze for amino acid content because there is no way of knowing how many of the GnRH peptides are bonded with the toxin, the instant linking groups, upon degradation in analytical assays, release a specific marker in the form of an unnatural amino acid. The amount of this marker allows the facile calculation of the exact ratio of peptide groups to toxins. SUMMARY OF THE INVENTION This invention is concerned with an agent which is capable of eliciting anti-GnRH antibodies in a host given the agent. More particularly, the agent of the invention comprises a GnRH moiety and a modified Pseudomonas exotoxin. The conjugation of these two moieties is accomplished with specific and improved linking groups that also aid in the analysis of the GnRH to Pseudomonas exotoxin ratio. These new, chimeric conjugates are useful as vaccines for use in immunosterilizing animals, fertility control, and for treatment of steroid hormone stimulated tumors. DESCRIPTION OF THE INVENTION The conjugates of the present invention are described in the following structural representation: Q-Ser-Tyr- W-X 1 - Arg- Y-Z Ll rPE (I) where: S is sulfur, rPE is a reduced Pseudomonas exotoxin linked to Ll via the thiol -S- group; χl is Leu or Nle (norleucine); Y is Pro or 4-hydroxy-Pro; and Z is Gly-NH2, D-Ala-NH2, NH-Et, NH-Pr or Aza-Gly-NH2; and Q is PyroGlu-His-Trp, N-acetyM-Cl-Phe^-Trp or 3-indolylpropionyl ; where PyroGlu is , and N-Acetyl-4-Cl-Phel> 2 -Tφ is and W is the D or L amino acid of the following structure: O (X 1 )-C-CH-(CH 2 )r-B-(CH 2 )m-N-(amino acid)n-(L 1 ); NH R 1 (Tyr) where: r is 1 or 2; m is an integer of 1 to 4; n is 0 or 1 ; B is CH2, O, S or N; and R 1 is hydrogen, Cl-C6alkyl or C3-C8cycloalkyl; (amino acid)n is a naturally occuring L-amino acid or its D stereoisomer; and (X 1 ), (Tyr) and (Ll) indicate the sites of attachment of χl, Tyr and Ll to W; Ll is independently ,W) -C fi χ 2 -γ-»S> . wr 9c_ (S) or where X 2 is C1-C5 alkylene, phenyl, or C5-C6 cycloalkylene; and (W) and (S) indicate the sites of attachment of W and S to Ll . Illustrative compounds of the instant invention are depicted in the following structure: PyroGlu-His-Tφ-Ser-Tyr-W-Leu-Arg-Y-Z 1 2 3 4 5 1 6 7 8 9 10 Ll I S I rPE (II) where the decapeptide is GnRH with the normal 6-position amino acid (W=Gly) deleted and replaced by W=D- or L-Lys or D-Orn. The use of D- or L-Lys"-GnRH is a preferred; however, it will be recognized that variations of Lys"-GnRH that will still elicit anti-GnRH antibodies in the host will be useful in this invention. All that is required is that the 6- position amino acid possess an amino group for binding to the linking group and that the remainder of the peptide retains its immunogenic properties. Z in the above formula is Gly-NH2, ethylamide, or Arg-Gly- NH2; Y is Pro or 4-hydroxy-Pro; Ll is independently: where X 2 is C1-C5 alkylene, phenyl or C5-C6 cycloalkylene. "rPE" is the reduced Pseudomonas exotoxin NLysPE38QQR, or a variant thereof. It will be appreciated by those skilled in the art that 4- hydroxy-Pro can exist as D and L isomers and as cis and trans isomers. All such isomers, and the racemic mixture of the D and L isomers are intended to be included in this invention. Unless otherwise specified, the following terms have the meaning defined below. The term "GnRH" is intended to encompass the native GnRH and analogs or derivatives thereof that are capable of eliciting anti- GnRH antibodies when administered to a host in accordance with the present invention, and are within the scope of the formula: Q-Ser-Tyr-W-Xl-Arg-Y-Z in which Q, W, χl , Y and Z are as defined above. When a particular GnRH molecule is meant, its amino acid sequence will be specified. Native GnRH, also known as luteinizing hormone releasing hormone (LHRH), is a decapeptide having the amino acid sequence pGlu-His-Tφ-Ser-Tyr-Gly-Leu- Arg-Pro-Gly-NH2 in which pGlu is pyroglutamate. The term "reduced Pseudomonas exotoxin (rPE)" used in the present invention is a form of Pseudomonas exotoxin wherein the disulfide bond between Cys265 n( j c ys 287 nas Deen reduced. The reduction of disulfide bond is achieved using conventional reagents, for example, dithiothreitol, in the presence of a chelating agent, e.g., the disodium salt of ethylenediaminetetraacetic acid, to remove metals that can oxidize thiols. The term "Pseudomonas exotoxin" includes the native Pseudomonas exotoxin as well as modified forms thereof. Pseudomonas exotoxin is a protein composed of 613 amino acids arranged into 3 major, and one minor domain. The preferred Pseudomonas exotoxins are variants thereof having decreased toxicity, for example, segments of Pseudomonas exotoxin wherein the binding or the ADP ribosylating activity has been attenuated or inactivated through deletion or partial deletion, insertion or substitution of amino acids in the binding or ribosylating domain, or where the PE holotoxin has been inactivated, for example by photoinactivation. The efficacy of PE immunoconjugates is independent of the toxin activity of the PE. One example of a Pseudomonas exotoxin with decreased toxin activity has had amino acids 1-252 deleted, which comprise most or all of the binding region and retaining amino acids 253-613 which contain the cell translocation region and the toxin region. This Pseudomonas exotoxin fragment has been identified as PE-40 - See Hwang et al., infra, Kondo et al J. Biol Chem 263 pg 9470-9475 (1988), Chaudhary et_al, PNAS-USA, 87 pg 308-312 (1990) and US Patent 4892827 to Pastan et aL The Pseudomonas exotoxin fragment PE-40 has been further modified by removing additional amino acids 365-380 to provide PE-38. PE-40 and PE-38 may be further modified by adding lysine containing oligopeptide fragments to their N-termini. Addition of the 9 amino acid peptide (M)ANLAEEAFK (the "Lys" peptide, starting with methionine) to the N-terminus of PE-40 and PE-38 produces Pseudomonas exotoxins identified as Lys PE-40 and Lys PE-38, respectively; addition of the 10 amino acid peptide (M)LQGTKLMAEE (the "NLys" peptide) produces Pseudomonas exotoxins identified as NLys PE-40 and NLys PE-38, respectively. Replacing PE-38 lysines at 590 and 606 with glutamine, and lysine 613 with arginine generates the Pseudomonas exotoxin identified as PE-38QQR. Lys PE-38QQR and NLys PE-38QQR have, at their N- termini, the "Lys" and "NLys" peptides, respectively. The various Pseudomonas exotoxin fragments are prepared using the techniques of biotechnology and recombinant DNA, and are described in Debinski and Pastan, Bioconjug. Chem.. 1994, 5(l):40-46, and references cited therein. The amino acid sequence of NLys PE-38QQR is shown below [SEQ ID No.: 1]; the underlined 4 amino acids represent the N-terminal amino acids of PE-38: MetAlaGluGlv.... MetLeuGlnGlyThrLysLeuMetAlaGluGluGlyGlySer euAlaAla euT rAlaHisGlnAlaCysHisLeuProLeuGluThrPheThrArgHis ArgGlnProArgGlyTrpGluGlnLeuGluGlnCysGlyTyrProValGln ArgLeuVa1Ala euTyrLeuAlaAlaArgLeuSerTrpAsnGlnValAsp GlnValIleArgAsnAlaLeuAlaSerProGlySerGlyGlyAspLeuGly GluAlalleArgGluGlnProGluGlnAlaArgLeuAlaLeuThrLeuAla AlaAlaGluSerGluArgPheValArgGlnGlyT rGlyAsnAspGluAla GlyAlaAlaAsnGlyProAlaAspSerGlyAspAlaLeuLeuGluArgAsn TyrProThrGlyAlaGluPheLeuGlyAspGlyGlyAspValSerPheSer T rArgGlyThrGlriAsriTrpThrValGluArgLeuLeuGlnAlaHisArg GlnLeuGluGluArgGlyTyrValPheValGlyTyrHisGlyThrPheLeu GluAlaAlaGlnSerlleValPheGlyGlyValArgAlaArgSerGlnAsp LeuAspAlalleTrpArgGlyPheTyrlleAlaGlyAspProAlaLeuAla TyrGlyTyrAlaGlnAspGlnGluProAspAlaArgGlyArglleArgAsn GlyAlaLeuLenArgValTyrValProArgSerSerLeuProGlyPheTyr ArgThrSerLeuThrLeuAlaAlaProGluAlaAlaGlyGluValGluArg euIleGlyHisProLeuProLeuArgLeuAspAlalleThrGlyProGlu GluGluGlyGlyArg euGluThrlleLeuGlyTrpPro euAlaGluArg ThrValVallleProSerAlalleProThrAspProArgAsnValGlyGly Asp euAspProSerSerlleProAspGlnGluGlnAlalleSerAlaLeu ProAspTyrAlaSerGlnProGlyGlnProProArgGluAspLeuArg The preferred rPE for the present invention is reduced NLys PE-38QQR. REACTION SCHEME 1 N ε H 2 MPS PyroGlu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-Gly-NH 2 p j ^p pitr^ 1 PyroGlu-His-Tφ-Ser-Tyr-D-Lys-Leu-Arg-Pro-Gly-NH 2 2 SS 1 | DTT NLysPE38QQR Cys 265 Cys 287 EDTA pH 8.0 3 phosphate buffer NLysPE38QQR REACTION SCHEME I (CONT'D) NLysPE38QQR O Br NH, SH NLysPE38QQR Cys 265 . Cys 287 [DLys 6 ] GnRH- Pro 9 NHEt A SH NH[DLys 6 ] GnRH"Pro 9 NHEt NLysPE38QQR Cys 265 , Cys 287 NH[DLys 6 ] GnRH- Pro 9 NHEt i S o In Reaction Scheme 1 the first step is the reaction of a GnRH with Ll , which for puφoses of illustration is shown as D-Lys"-GnRH and the maleimidoyl alkanoyl group, respectively. The D-Lys"-GnRH is prepared using known peptide synthesis techniques, preferably the solid phase peptide synthesis. "Et" is ethyl and "NεH2" is the epsilon amino group of lysine. The reaction for the preparation of the Ll-D-Lys"-GnRH is carried out using an active ester of the maleimidoyl alkanoyl group. Preferred esters are the esters made from maleimidoyl alkanoic acid and N-hydroxy succinimide, pentafluorophenol or p-nitro phenol. The ester with N-hydroxy succinimide is most preferred. The reaction is carried out in a polar solvent with a base selected from either (a) a non- nucleophilic organic base such as N,N-diisopropyl ethylamine (DIEA) or (b) a weak inorganic base such as sodium or potassium carbonate. The polar solvent can be N,N-dimethylformamide, water, acetonitrile or mixture thereof. N,N-Dimethylformamide is preferred. The reaction is carried out at from 0 to 25 °C, preferably at room temperature and is generally complete in from 10 to 90 minutes. The work-up of the reaction is to initially neutralize the base present with an acid such as trifluoroacetic acid, and the pH of the mixture is brought to about 2-4. The product is than isolated using techniques known to those skilled in the art. The second reaction is for the reduction of the disulfide bond of the PE, and is carried out in an aqueous buffer which provides for a pH of about 8. In the reaction scheme, the reduction of the disulfide bond is illustrated with the non-limiting example NLys PE38QQR. Included in the reaction mixture preferably is dithiothreitol and/or the disodium salt of ethylenediamine-tetraacetic acid. These reagents are generally added in considerable excess to reduce the Cys 265,287 disulfide bond of NLys PE38QQR. Excess reagents prevent these reactive groups from forming disulfides with like groups. For the same reason, the reaction is carried with the strict exclusion of oxygen, generally by using a nitrogen atmosphere. The reaction is carried out at from 0 to 25 °C, preferably room temperature, and is generally complete in from 5 to 18 hours. Before the reduced Pseudomonas exotoxin can be conjugated with the Ll-D-Lys 6 -GnRH, the dithiothreitol (DTT), the ethylenediaminetetraacetic acid disodium salt must be removed. Thus the reaction mixture is purified of such reagents prior to the next step. The most convenient method for doing so is to dialyze the reaction mixture. The dialysis solution free of extraneous reagents is used in the next step without further treatment. The final step is the coupling of the Ll D-Lys^GnRH with the reduced Pseudomonas exotoxin which is carried out under nitrogen at a pH of from 8 to 10 with a excess of the Ll D-Lys^GnRH. Generally from 2 to 20 equivalents of the GnRH reagent are used for each equivalent of the thiol substituent on the Pseudomonas exotoxin. The reaction is generally very fast and is complete in just 1-5 minutes although further aging of up to 2 hours has not been found to be detrimental. The coupled D-Lys"GnRH Ll -Pseudomonas exotoxin is isolated using techniques known to those skilled in the art. It has been found that dialysis of the reaction mixture is a convenient method for the removal of unwanted products. Since the conjugated product will generally be administered by injection, the resultant dialysis solution may be sterile filtered and used directly for percutaneous administration. It will be understood that other linkers Ll may be used in the preparation of the conjugates. For example, Scheme 1 further illustrates the use of an alkanoyl linker. Thus, the GnRH moiety is reacted with bromoacetic anhydride to provide 6, which in turn is reacted with reduced NLys PE38QQR to provide the conjugate 7. The intermediate compounds 2 and 6 are realized in the following structural formula: pGlu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Y-Z, I where Ll is where X is Cl-C5alkylene, phenyl or C5-C6cycloalkylene; and halo is iodo, bromo or chloro; and The linking groups, Ll, are bonded to the available primary amines of the D-Lys"-GnRH and the reduced PE, preferably NLys PE 38QQR. More than one thiol is available on the reduced PE; therefore, more than one L group will be reacted therewith. The most preferred GnRH derivative is D-Lys"-GnRH; thus, the most preferred intermediates of the instant invention are: also written as Pgl-H-W-S-Y-DK-L-R-P-NHCH 2 CH 3 I NHCOCH 2 Br (or Cl) The instant conjugates which connect a GnRH analog with a reduced PE through a unique linking group offer significant advantages in the preparation and analysis of the toxin conjugates. A further complication arises when, as often occurs, the resultant conjugate is actually a mixture of conjugates with differing numbers of GnRH analogs coupled to the PE. The analysis of the GnRH-PE conjugates is usually done through amino acid analysis; however, since the GnRH and and the Pseudomonas exotoxin all break down to normal amino acids, the determination of the number of GnRH moieties bonded to the PE is a very long and tedious process. The instant linking group, however, under amino acid analysis breaks down to beta-alanine, an unnatural amino acid. Thus during amino acid analysis, the ratio of lysine to beta-alanine is determined which reveals the extent of the conjugation. This is a much more direct and accurate method of determining the degree of conjugation which greatly facilitates the use of the toxin conjugates as chemical sterilization agents. Breakdown of conjugates prepared with bromo or (chloro) acetylated peptide, affords a novel amino acid: S-carboxyl-methyl- cysteine (SCMC). This moiety will only appear if a successful conjugation reaction occurred. Conjugates of the present invention have great utility in human medicine as well as in veterinary medicine. This follows from the fact that there are several important biological reasons for employing castration and antifertility drugs in humans. For example, breast and prostate cancers are but two examples of sex steroid-dependent tumors which respond to such hormonal manipulation. At present, the only reliable way to inhibit steroid-dependent tumor growth is through administration of counter-regulatory hormones (e.g., DES in prostate cancer), sex-steroid hormone binding inhibitors (e.g., tamoxifen in breast cancer) or surgical castration. Thus the potential medical uses of such chemical castration compounds are vast and varied. For example, prostate cancer remains an important cause of cancer deaths and represents the second leading cancer of males. The present palliative treatment for advanced prostate cancer cases involves reduction of serum testosterone/DHT levels through use of surgical castration. It should also be noted that for puφoses of disease and/or fertility control, especially in humans, it may be desirable to use applicant's compounds to ablate pituitary gonadotrophs in conjunction with other modes of treatment. For example, it is anticipated that chronic administration of progestins and estrogens to females and androgens to males might be necessary to prevent loss of secondary sex characteristics, behavior and osteoporosis. However, through judicious use of the herein disclosed compounds, especially in combination with appropriately administered sex steroids, desirable antifertility effects can be achieved. Another area of application in human medicine is treatment of endometriosis. This condition, which produces painful growth of endometrial tissue in the female peritoneum and pelvis also responds to inhibition of sex steroid synthesis. Those skilled in this art will also appreciate that the herein disclosed compounds could be used to partially reduce sex-steroid secretions, and thus reduce or eliminate certain hormone related behavior problems while retaining improved growth stimulation. The dose/time adjustments associated with the use of these compounds can vary considerably and will depend on a variety of factors such as the species of animal to be treated, the particular GnRH and/or carrier used, the adjuvant, the age of the animal, and the desired outcome of vaccination. In general, the conjugates are administered by subcutaneous or intramuscular injection into a mammal at a rate of 1 μg to 1000 μg of conjugate per dose . A single dose of the conjugates of the present invention may be all that is required to achieve sterilization, but multiple dises spaced at one to six week intervals are alternative sterilization schemes. Furthermore, as sterilization agents, the compounds of this invention can be used before or after puberty; thus they can delay sterilization, which is especially useful in those areas of animal husbandry where the anabolic benefits associated with the flexibility of timing of non-surgical sterilization can contribute positively to feed efficiency, meat production and/or quality. In swine, the conjugates can be used to maximize the boar¬ like growth efficiency and carcass quality while eliminating the offensive odor and taste of boar meat. This can be accomplished with one or two intramuscular or subcutaneous injections administered at various times during the grow out period. An example of convenient and efficacious schedule consists of an initial vaccination at the time of housing in the grower/finisher facility (9-16 weeks of age) with a booster late in the grow out (between 18 and 22 weeks of age). Each vaccination may be at a dose of about 1 μg to about 1000 μg of the conjugate, preferably about 10 μg to about 100 μg is used. For a single dose regimen, the amount of the conjugate is generally at a higher level than that used for two or more doses. Feedlot cattle could be treated in a manner similar to that used for swine, with vaccinations at the time of entry into the feedlot and at another time which would be determined by the effect desired, i.e., prevention of pregnancy in the females or growth maximization in the males. Females need to complete the vaccination prior to entering mixed sex housing to prevent pregnancy; however, in housing segregated by sex, vaccination could occur at time of arrival and then 4-12 weeks later to prevent estrus. Bulls should be vaccinated late enough to maximize feed efficiency, but early enough to prevent aggression and to provide marbling of meat. In companion animals such as dogs and cats, the GnRH vaccine could be administered by subcutaneous or intramuscular injection at times when the standard vaccinations are given (between 6 and 21 weeks of age with a booster at 6 months and annual boosters thereafter). For neutering adult animals such as dogs, cats and horses, two doses administered at 2-8 week intervals followed by and annual boosters should be sufficient to produce neutering. The actual dose and formulation remain to be determined and may vary with the particular conjugate used. However, a dose of 1 to 2000 ug, preferably about 500 ug of an conjugate of the present invention formulated on alum and administered in a volume of 1-3 mis may be sufficiently potent when administered as described above. In man, conjugates of the present invention can be used to treat sex steroid responsive tumors. Two doses at 1 to 1000 ug per dose of the vaccine can be administered at 2 to 8 week intervals with boosters at 6 to 12 months until the tumor is eliminated or ceases to be responsive to hormonal therapy. The conjugates of the present invention can be used for the above-mentioned application without the use of an aggressive adjuvant such as Complete Freund's Adjuvant, which cause injection site lesions and downgrading of feed animal carcasses. Suitable adjuvants are any of those substances recognized by the art as enhancing the immunological response of a mammal to an immunogen without causing an unacceptable adverse reaction, and include aluminum compounds or water in oil emulsions such as Incomplete Freund's Adjuvant (IFA). In a preferred embodiment, the immunoconjugates of the present invention is administered in an oil-in-water emulsion containing a metabolizable oil, a non-ionic surfactant such as a polyoxypropylene (POP)-polyoxyethylene (POE) block polymer, an emulsifier, and optionally an immune response enhancer of formula 1 wherein R 1 is H,. C2-8 alkenyl, Cl-8 alkyl, benzyl, phenyl or COR 4 , wherein R 4 is H, Cl-8 alkyl, C2-8 alkenyl, benzyl or phenyl wherein the phenyl moiety may have up to three substituents selected from the group consisting of hydroxy, carboxy of 1- 4 carbon atoms, halo, Cl-4 alkoxy, Cl-4 alky, and C2- 4alkenyl, SO3M or PO3M, wherein M is H or sodium or potassium; R is H or OR 1 ; R3 is ORl or R3 and R4 together form an oxo; R 4 , R5, R6, and R? are independently H or methyl; with the proviso that when R3 and R4 together form an oxo, R5, R6, R7 and R2 are each H; and that when R 2 is H, R 4 , R^, R6 and R? are each hydrogen, and R^ is ORl. In the vaccine composition, the metabolizable oil may be an oil of 6 to 30 carbon atoms including alkanes, alkenes, alkynes, and their corresponding acids and alcohols, the ethers and esters therof, and mixtures thereof. The oil may be any vegetable oil, fish oil, animal oil or synthetically prepared oil which can be metabolized in the body of the subject to which the adjuvant is administered, and which is not toxic to the organism. Examples of vegetable oil include peanut oil, soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil, and corn oil. Animal oils are usually solids at physiological temperature; however, fatty acids are obtainable from animal fats by partial or complete triglyceride saponifiction which provides the free fatty acids. Most fish contain metabolizable oils which may be readily recovered. For example, cod liver oil, shark liver oils, and whale oil such as spermaceti exemplify several of the fish oils. A number of branched chain oils are synthesized biochemically in 5-carbon isoprene units and are generally refened as teφenoids. Shark liver oil contains a branched, unsaturated teφenoids known as squalene. Squalane, the saturated analog of squalene is a particularly preferred oil for the present invention. The oil component of the adjuvant compositions and vaccines of the invention will usually be present in an amount between 1 % and 10%, but preferably in an amount between 2.5 and 5%. The term " poly oxypropylene-polyoxy ethylene block polymer" refers to a polymer made by the sequential addition of propylene oxide and then ethylene oxide to a low molecular weight, reactive compound, usually propylene glycol. These block polymers can be prepared by the methods set out in U.S. Pat. No. 2,674,619 issued to Lunsted, and are commercially available from BASF-Wyandotte under the trademark Pluronic®. The characteristics of these block polymers are determined by the molecular weight of the POP nucleus and of the percentage POE in the product. The POP section imparts hydrophobic characteristics to the block polymer, while the POE section imparts hydrophilic characteristics. Pluronic® block polymers are designated by a letter prefix followed by a two or a three digit number. The letter prefixes (L, P, or F) refer to the physical form of each polymer, (liquid, paste, or flakeable solid). The first one or two digits is a code for the average molecular weight of the POP base, while the last digit indicates the amount of POE. For example, Pluronic® L101 is a liquid having a polyoxypropylene base of average molecular weight 3,250, with 10% polyoxyethylene present at the ends of the molecule. The preferred block polymers are those which are liquid over a temperature range between about 15°-40° C. In addition, polymer mixtures of liquid and paste, liquid, paste and flakeable solid or liquid and flakeable solid mixtures which are liquid within the specified temperature range may have utility in this invention. Preferred block polymers are those having a POP base ranging in molecular weight between about 2250 and 4300 and POE in an amount between about 1 and 30%. More preferred are those polymers wherein POP has a molecular weight falling between 3250 and 4000 and the POE component comprises 10-20%. The Pluronic® block polymers L101, L121 and L122 fall within this definition. Most preferred are the block polymers wherein POP has a molecular weight of 4000 and POE in an amount of 10% or POP has a molecular weight of 3250 and POE in an amount of 10% e.g. Pluronic® block polymers L121 and L101 respectively. The block polymer is preferably used in an amount between 0.001 and 10%, most preferably in an amount between 0.001 and 5%. The term "emulsifier" refers to non-toxic surface active agents capable of stabilizing the emulsion. There are a substantial number of emulsifying and suspending agents generally used in the pharmaceutical sciences. These include naturally derived materials such as gums, vegetable protein, alginates, cellulose derivatives, phospholipids (whether natural or synthetic), and the like. Certain polymers having a hydrophilic substituent on the polymer backbone have emulsifying activity, for example, povidone, polyvinyl alcohol, and glycol ether-based compounds. Compounds derived from long chain fatty acids are a third substantial group of emulsifying and suspending agents usable in this invention. Though any of the foregoing emulsifiers can be used so long as they are non-toxic, glycol ether-based emulsifiers are preferced. Preferred emulsifiers are non-ionic. These include polyethylene glycols (especially PEG 200, 300, 400, 600 and 900), Span®, Arlacel®, Tween®, Myrj®, Brij® (all available from ICI America Inc., Wilmington, Del.), polyoxyethylene, polyol fatty acid esters, polyoxyethylene ether, polyoxypropylene fatty ethers, bee's wax derivatives containing polyoxyethylene, polyoxyethylene lanolin derivatives, polyoxyethylene fatty glycerides, glycerol fatty acid esters or other polyoxyethylene acid alcohol or ether derivatives of long-chain fatty acids of 12-21 carbon atoms. The presently preferred emulsifier is Tween® 80 (otherwise known as polysorbate 80 or polyoxyethylene 20 sorbitan monooleate), although it should be understood that any of the above-mentioned emulsifiers would be suitable after lack of toxicity is demonstrated. The emulsifier is usually used in an amount of about 0.05 to about 5.0%, preferably about 0.2 to 1%. The aqueous portion of the adjuvant compositions of the invention is preferably buffered isoosmotic saline. It is preferred to formulate these solutions so that the tonicity is essentially the same as normal physiological fluids in order to prevent post-administration swelling or rapid absoφtion of the composition due to differential ion concentrations between the composition and physiological fluids. It is also prefened to buffer the saline in order to maintain a pH compatible with normal physiological conditions. Also, in certain instances, it may be necessary to maintain the pH at a particular level in order to insure the stability of certain composition components, such as the glycopeptides. Any physiologically acceptable buffer may be used herein, but it has been found that it is most convenient to use a phosphate buffer. Any other acceptable buffer such as acetate, Tris, bicarbonate, carbonate, and the like can be used as a substitute for a phosphate buffer. It is prefened to use phosphate buffered saline, or saline buffered with a mixture of phosphate and acetate. The immune response enhancers of formula 1, are either compounds well known in the art (e.g. dehydroepiandrosterone) or they may be prepared according to the disclosures of US Patent 5,277,907 of R. M. Loria, or WO95/10527 of Neurocrine Biosciences. In a prefened embodiment of the vaccine composition, an immunopotentiating amount of the immune response enhancer is included. More preferably, the immune response enhancer is a compound of formula I wherein Rl, R 2 , R5, R6, and R^ are each H, and R3 and R 4 together form an oxo group, this compound being dehydroepiandrosterone or DHEA. In a prefened embodiment of the vaccine composition, the oil-in-water emulsion comprises squalane, Tween® 80 and Pluronic® L121. More prefened, the vaccine includes DHEA as the immune response enhancer. The adjuvant composition is prepared by emulsification using a mixer to form a homogenous emulsion. Typically, the adjuvant composition, including the immune response enhancer, is microfluidized prior to adding the GnRH-conjugate. The emulsion is cycled through the microfluidizer about 2-20 times, the GnRH-conjugate is then added to the adjuvant composition and the mixture is again cycled through the microfluidizer, generally 2-10 times until substantially all of the volume of the oily particles in the emulsion is present in particles having a diameter of about 0.03 μm and 0.5 μm, preferably between 0.05 and 0.2 μm. In the following experimental procedures all reagents were used as received from the supplier. In the case of solvents, HPLC-grade was used where available. HPLC (binary gradient) was performed on a Waters 600E system with Waters 484 tunable U.V. detector (Aufs=0.1 analytical or 2.0 preparative scale) and recorded on a Waters 746 Data Module. A Waters WISP™712 autosampler (200 μL sample loop) was used for analytical samples. A Rheodyne 7125 manual injection port (5000 μL sample loop) was used for preparative samples. A = H2θ, 0.1% TFA; B=CH3CN, 0.1% TFA. Mass spectra were taken on a Finnegan MAT 90, spectrophotometer (positive ion, NBA matrix). Abbreviations: Standard amino acid abbreviations are used. RT, room temperature; DCC, 1,3-dicyclohexylcarbodiimide; HOBT, 1- hydroxy-benzotriazole; TFA, trifluoroacetic acid; DIEA, N,N- diisopropyl-ethylamine; MPS, β-maleimidopropionic acid N- hydroxysuccinimide ester; GnRH, gonadotropin releasing hormone; PBS, phosphate buffered saline; DTT, dithiothreitol; EDTA-2Na, ethylenediaminetetraacetic acid disodium salt; NBA, 3-nitro-benzyl alcohol. PREPARATION OF STARTING MATERIALS 1. DLys^-GnRH, 1 :PyroGlu-His-Tφ-Ser-Tyr-D-Lys-Leu-Argι Pro-Gly-NH9: The peptide was synthesized on Rink amide MBHA resin (0.25 mmol, Nova Biochem) by solid phase peptide synthesis (SPPS) using an ABI model 431 A synthesizer and single couplings (DCC/HOBT). The peptide was cleaved (2 h, RT) from the resin using reagent R (1 mL/100 mg resin, TFA thioanisole/ethanedithiol/anisole, 90:5:3:2). The peptide was precipitated from the concentrated cleavage mixture with diethyl ether and purified by preparative reverse phase HPLC (Waters PrepPak®25 x 10™ Cl8; 10 mL/min; 10-20% B, 0-20 min.; then 20-35% B, 20-40 min.; λ=230 nm). 6-D-Lys-GnRH: FAB-MS (positive ion, NBA matrix) Calc. M+l 1254.44; Found M+l = 1254.4 2. (Nε-maleimidopropanoyl)-DLys6-GnRH,2:PyroGlu-His- Tφ-Ser-Tyr-fN -maleimidopropanoyP-D-Lys-Leu-Arg-Pro-Gly-NH?: DLys 6 -GnRH (10 mmol, 12.5 mg) was dissolved in N,N- dimethylformamide (0.5 mL/mg) and DIEA (50 mmol, 9 μL) added. The mixture was stined briefly (RT) and β-maleimidopropionic acid N- hydroxysuccinimide ester (MPS; 20 mmol, 5.2 mg) was introduced in one portion. After 30 min reaction time, 10 μL TFA was added to the reaction mixture and the solvent removed in vacuo. The peptide was purified by reverse phase HPLC (Waters PrepPak® 25 x 10™ Delta- Pak™ Cl8; 10 mL/min; 10-25% B, 0-30 min.; then 25% B, 30-35 min; λ=230 nm). FAB-MS (positive ion, NBA matrix) Calc. M+l : 1405.56; Found M+l=1405.6. (N £ -BromoacetylV6-D-Lvs-GnRH Bromoacetic anhydride was formed by allowing 3.35 mg of bromoacetic acid, 1.5 ml of dichloromethane, 4.9 mg dicyclohexyl¬ carbodiimide to react for three hours. The DLys^-GnRH peptide (12.3 mg) produced above was dissolved in 1 ml of dry degassed DMF and 20 microiiters of diisopropylethylamine (Hunig's base) were added. The bromoacetic anhydride solution (filtered to remove dicyclohexylcarbodiimide) was added to the peptide solution. After 30 minutes of reaction 100 microiiters of water containing 0.1% trifluoroacetic acid (TFA) was added to quench the reaction. The reaction mixture was concentrated in vacuo and the residue was taken up in 2.5 ml 10% aqueous acetonitrile containing 0.1% TFA. The reaction product was isolated and purified on a Waters Preparatory chromatography column to yield 3.2 mg. The mass spectrum showed a molecular ion peak by FAB, M+H = 1346.6. 4. N-Ac-l,2-Di-p-Chloro-Phe-DLys6-GnRH, Ac-4-Cl-Phe-4- Cl-Phe-Tφ-Ser-Tyr-DLvs-Leu-Arg-Pro-Glv-NH?: The peptide is synthesized on Rink amide MBHA resin (0.25 mmol, Nova Biochem) by solid phase peptide synthesis (Fmoc chemistry) using an ABI model 431 A synthesizer and double couplings (DCC/HOBT) for 4-Cl-Phe and single couplings for the remaining residues. The amino terminus is capped by treatment with acetic anhydride (5-10 mL) until the resin beads give a negative Kaiser test for the presence of an amine (0.5-8 h). The peptide is cleaved (2 h-4 h, RT) from the resin using reagent R (0.5 mL-3 mL/100 mg resin, TFA thioanisole/ethanedithiol/anisole, 90:5:3:2). The peptide is precipitated from the concentrated cleavage mixture with diethyl ether and purified by preparative reverse phase HPLC and characterized by FAB-MS. 5. DLy s6-D Ala 10-GnRH, H-Pgl-His-Tφ-Ser-Tyr-DLy s-Leu- Arg-Pro-DAla-NH?: The peptide was synthesized on Rink amide MB HA resin (0.25 mmol, Nova Biochem) by solid phase peptide synthesis (Fmoc chemistry) using an ABI model 431 A synthesizer and single couplings (DCC HOBT). The peptide was cleaved (3 h, RT) from the resin using reagent R (2.0 mL/100 mg resin, TFA/thioanisole/ethanedithiol anisole, 90:5:3:2). The peptide was precipitated from the concentrated cleavage mixture with diethyl ether and purified by preparative reverse phase HPLC. Characterization by FAB-MS of 6-D-Lys-10-D-Ala-GnRH (positive ion, NBA matrix) Calc (m+l) = 1268.5; Found (m+l) = 1267.5. 6. DLys6-Pro9-NHEt-GnRH, H-Pgl-His-Tφ-Ser-Tyr-DLys- Leu-Arg-Pro-NHEt: The peptide is synthesized on Oxime or Merrifield resin by solid phase peptide synthesis (Boc chemistry) using an ABI model 431 A synthesizer and single couplings (DCC/HOBT). The peptide is cleaved (2 h-72 h, RT) from the resin with anhydrous ethyl amine. The crude protected peptide is precipitated with diethyl ether, collected by suction filtration, and dried overnight (over P2O5). The protecting groups are removed from the dry peptide by treatment with anhydrous HF (0°C, 0.5- 2 h, 5-30 mL) in the presence of anisole (0.2-2 mL) and dimethyl phosphite (0.1-1 mL). The excess HF is removed in vacuo and the residue triturated with diethyl ether. The peptide is purified by preparative reverse phase HPLC and characterized by FAB-MS. 7. DOrn6-GnRH, H-Pgl-His-Tφ-Ser-Tyr-DOrn-Leu-Arg-Pro^ Glv-NH?: The peptide was synthesized on Rink amide MBHA resin (0.25 mmol, Nova Biochem) by solid phase peptide synthesis (Fmoc chemistry) using an ABI model 431 A synthesizer and single couplings (DCC/HOBT). The peptide was cleaved (3 h, RT) from the resin using reagent R (2.0 mL/100 mg resin, TFA thioanisole/ethanedithiol anisole, 90:5:3:2). The peptide was precipitated from the concentrated cleavage mixture with diethyl ether and purified by preparative reverse phase HPLC. Characterization by FAB-MS of 6-D-Orn-GnRH (positive ion, NBA matrix) Calc (m+l) 1239.4; Found (m+l) 1239.5. 8. 3-Indolylpropionyl-DLys6-GnRH,3-Indolylpropionyl-Ser- Tyr-DLvs-Leu-Arg-Pro-Glv-NH9: The peptide was synthesized on Rink amide MB HA resin (0.25 mmol, Nova Biochem) by solid phase peptide synthesis (Fmoc chemistry) using an ABI model 431 A synthesizer and single couplings (DCC/HOBT). The peptide was cleaved (3 h, RT) from the resin using reagent R (2.0 mL/100 mg resin, TFA thioanisole/ethanedithiol/anisole, 90:5:3:2). The peptide was precipitated from the concentrated cleavage mixture with diethyl ether and purified by preparative reverse phase HPLC. Characterization by FAB-MS of 3-indolylpropionyl-6-D-Lys- GnRH (positive ion, NBA matrix) Calc (m+l) 990.2; Found (m+l) 990.7. 9. 3-Indolylpropionyl-DLys6-Pro9-NHEt-GnRH, 3- Indolylpropionyl-Ser-Tyr-DLvs-Leu-Arg-Pro-NHEt: The peptide was synthesized on Oxime or Merrifield resin by solid phase peptide synthesis (Boc chemistry) using an ABI model 431 A synthesizer and single couplings (DCC/HOBT). The 3- indolylpropionyl moiety was incoφorated as 3-indolepropionic acid. The peptide was cleaved (2 h-72 h, RT) from the resin with anhydrous ethyl amine. The crude protected peptide was precipitated with diethyl ether, collected by suction filtration, and dried overnight (over P2O5). The protecting groups are removed from the dry peptide by treatment with anhydrous HF (0°C, 0.5-2 h, 5-30 mL) in the presence of anisole (0.2-2 mL) and dimethyl phosphite (0.1-1 mL). The excess HF was removed in vacuo and the residue triturated with diethyl ether. The peptide was purified by preparative reverse phase HPLC. Characterization by FAB¬ MS of DLys6-Pro9-NHEt-GnRH (positive ion, NBA matrix) showed M + 1 =1223.9. IO Preparation of NLvs PE38QOR Plasmid PJH4 (Ref. Hwang. J. "Cell" (1987, 48; 129-136) contains the coding sequence for PEl-613- Oligonucleotide directed mutagenesis as described in 15.51-15.73, Molecular Cloning. 2nd ed (1989) edited by Sambrook, Fritch & Maniatis (Cold Spring Harbor Press) has been used as a covenient way to make deletions/mutations in the PE molecule. An Ndel Hind III double digest is canied out on PJH4 resulting in linearization of the construct and clipping of a 12 bp segment which includes the ATG start codon of the PE coding sequence. Two complementary oligonucleotides are synthesized, annealed and ligated into the Ndel/Hind III splice site. The oligomers have the following nucleotide sequence: 1-5' TAT GCT GCA GGG TAC CAA GCT TAT GGC CGA AGA 3 ' and II - 5' AGC TTC TTC GGC CAT AAG CTT GGT ACC CTG CAG CA3'. The modified PE insert has a sequence of MLQGTKLMAEE constructed at the N-terminus. This plasmid is designated PJH42. The plasmid PJH42 is partially cut with Ava I. The linear form of DNA is isolated, completely digested with Hind III, and the resulting 5.1 Kb fragment isolated. SI nuclease treatment is carried out to create blunt ends for ligation and the plasmid is recircularized and designated PJH43. This results in a PE with deletion of amino acids 4- 252. A 553 bp Sal I/Bam HI fragment of plasmid PJH43 is cloned into Ml 3 mpl9. An oligonucleotide, 50 nucleotides in length with the structure 5' GGC GTC GCC GCT GTC CGC CGG GCC GTT GGC CGC GCC GGC CTC GTC GTT GC3', is synthesized and annealed to the single stranded M13 vector to facilitate (loop out) mutagenesis generating a deletion of amino acids 365-380 of the PE insert, resulting in the sequence: . . . .AGAANGPADSGDALL. . . . TT 364381 A 505 bp Sal I Bam HI fragment is excised from the replicative form of the mutant DNA in Ml 3 and ligated with a 3.7 Kb Sal 1 Bam HI fragment of the plasmid PJH43. This new plasmid is designated PJH44. A Bam Hl EcoR 1 fragment of 460 nucleotides is excised from PJH44 and cloned into Ml 3 mpi 9. This fragment contains the nucleotide sequence for three lysines that are mutated at the carboxy end of the coding sequence: lysines 590, 606 are mutated to glutamines and lysine 613 is mutated to an arginine. Oligo directed mutations are then caπied out successively at each of the lysines with the following oligomers: Lysines 590-5' GCT GAT CGC CTG TTC TTG GTC GGG GAT GCT GGA C 3' Lysines 606-5' GTC CTC GCG CGG CGG TTG GCC GGG CTG GCT G 3' Lysines 613-5' CGG TCG CGG CAG TTA ACG CAG GTC CTC GCG CGG 3' The Bam HI EcoR 1 fragment is excised from the replicative form of the mutant DNA in Ml 3 and ligated with a 3.4 Kb Bam Hl EcoR 1 fragment of the plasmid PJH44. The linearized plasmid is then recircularized, designated PJH45 and used for expression of the modified PE, identified as Lys PE38M, from a commercially available strain of E, coli. HB 101, available from Bethesda Research Laboratories. Lys PE38M is described in published WO 93/15751 (Merck & Co., Inc.). IL Reduced NLvs PE38QOR A solution of the toxin, NLys PE 38QQR, 10 ml containing 12.8 mg toxin, was adjusted to pH 8 by the addition of 175 mg of pH 8 buffer salts. EDTA disodium salt-dihydrate (372.24 mg) and 154.25 mg dithiothreitol were added and the mixture was shaken under dry nitrogen overnite at room temperature to complete the reduction of the cysteine 265, 287 disulfide bond in the toxin. The solution was transfened to a dialysis bag and dialyzed versus the buffer: 0.1 M phosphate (sparged with nitrogen) at room temperature for about eight hours. The reduced toxin solution was then dialyzed versus 0.01 M phosphate buffer overnite at room temperature with nitrogen sparging. At the end of the dialysis procedure the reaction contents from the dialysis bag were transfened to a sterile plastic centrifugation tube which was sampled for thiol content and the purified reduced toxin dithiol was retained for subsequent reaction. The following examples are illustrative of carrying out the invention as contemplated by the inventors and should not be construed as being limits on die scope or spirit of the invention described herein. EXAMPLE 1 Procedure for the Preparation of a DLys 6 -GnRH/NLys-PE38QQR Conjugate NLysPE38QQ pH8 phosphate buffer To 5 ml of the reduced NLys PE38QQR prepared above (0.286 micromol SH titre) was added 1.2 mg of the N- maleimidopropanoyl-6-D-Lys-GnRH derivative prepared and described above in Preparation 2. The reaction mixture was shaken overnite at room temperature under dry nitrogen. The reaction contents were transfened to a dialysis bag and the reaction solution was dialyzed versus 0.01 M pH7 phosphate buffer overnite at 4 degrees C. Then the contents were dialyzed versus phosphate buffered saline 6/N overnite. The compound was confirmed by chromatography. EXAMPLE 2 CONJUGATION Dlys 6 GnRH + LysPE38(C 265 , C 287 )M ε . SH SH pH8 phosphate » buffer LysPE 38 M-(Cys LysPE38M (Cys 265 ' 287 ) capped with DLys(N 6 -dithiopyridyl propanoyl) 6 -GnRH To 5 ml of a solution of the reduced toxin dithiol from Example 4 was added 1.24 mg of the pyidinedithiol and the reaction mixture was shaken overnite at room temperature. The reaction was dialyzed versus 1) 0.01 M pH phosphate, and then versus 2) sterile water, each dialysis conducted sequentially overnite at room temperature. The compound was confirmed by chromatography. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Tolman, Richard L. and Lombardo, Victoria K. (ii) TITLE OF INVENTION: GnRH/Reduced Pseudomonas Exotoxin Conjugates (iii) NUMBER OF SEQUENCES: 1 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Mollie M. Yang (B) STREET: P.O. Box 2000 (C) CITY: Rahway (D) STATE: New Jersey (E) COUNTRY: USA (F) ZIP: 07065 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: Power Macintosh 7500/100 (C) OPERATING SYSTEM: MS-DOS 7.1 (D) SOFTWARE: MS Word 5.1a (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Mollie M. Yang (B) REGISTRATION NUMBER: 32,718 (C) REFERENCE/DOCKET NUMBER: 18957 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (908) 594-6343 (B) TELEFAX: (908) 594-4720 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 355 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: Met Ala Asn Leu Ala Glu Glu Ala Phe Lys Gly Gly Ser Leu Ala Ala 1 5 10 15 Leu Thr Ala His Gin Ala Cys His Leu Pro Leu Glu Thr Phe Thr Arg 20 25 30 His Arg Gin Pro Arg Gly Trp Glu Gin Leu Glu Gin Cys Gly Tyr Pro 35 40 45 Val Gin Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn 50 55 60 Gin Val Asp Gin Val lie Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly 65 70 75 80 Gly Asp Leu Gly Glu Ala lie Arg Glu Gin Pro Glu Gin Ala Arg Leu 85 90 95 Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gin Gly 100 105 110 Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Gly Pro Ala Asp Ser Gly 115 120 125 Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly 130 135 140 Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gin Asn Trp Thr 145 150 155 160 Val Glu Arg Leu Leu Gin Ala His Arg Gin Leu Glu Glu Arg Gly Tyr 165 170 175 Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gin Ser He 180 185 190 Val Phe Gly Gly Val Arg Ala Arg Ser Gin Asp Leu Asp Ala He Trp 195 200 205 Arg Gly Phe Tyr He Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala 210 215 220 Gin Asp Gin Glu Pro Asp Ala Arg Gly Arg He Arg Asn Gly Ala Leu 225 230 235 240 Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe Tyr Arg Thr 245 250 255 Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Leu 260 265 270 He Gly His Pro Leu Pro Leu Arg Leu Asp Ala He Thr Gly Pro Glu 275 280 285 Glu Glu Gly Gly Arg Leu Glu Thr He Leu Gly Trp Pro Leu Ala Glu 290 295 300 Arg Thr Val Val He Pro Ser Ala He Pro Thr Asp Pro Arg Asn Val 305 310 315 320 Gly Gly Asp Leu Asp Pro Ser Ser He Pro Asp Gin Glu Gin Ala He 325 330 335 Ser Ala Leu Pro Asp Tyr Ala Ser Gin Pro Gly Gin Pro Pro Arg Glu 340 345 350 Asp Leu Arg 355

Claims: WHAT IS CLAIMED IS: 1. A conjugate having the formula: Q-Ser-Tyr- W-X 1 - Arg- Y-Z I Ll I rPE (I) where: S is sulfur, rPE is a reduced Pseudomonas exotoxin linked to Ll via the thiol -S- group; χl is Leu or Nle (norleucine); Y is Pro or 4-hydroxy-Pro; Z is Gly-NH2, D-Ala-NH2, NH-Et, NH-Pr or Aza-Gly-NH2; and Q is PyroGlu-His-Trp, N-acetyl-4-Cl-Phe 1 ' 2 -Tφ or 3-indolylpropionyl; where PyroGlu is , and N-Acety -Cl-Phe^-Trp Cl and W is the D or L amino acid of the following structure: O (X 1 )-b'-CH-(CH 2 )r-B-(CH 2 )m-N-(amino acid)n-(L 1 ); NH R 1 (Tyr) where: r is 1 or 2; m is an integer of 1 to 4; n is 0 or 1 ; B is CH2, O, S or N; and R 1 is hydrogen, C 1 -Cόalkyl or C3-C8cycloalkyl; (amino acid) n is a naturally occuring L-amino acid or its D stereoisomer; and (Xl), (Tyr) and (Ll) indicate the sites of attachment of χl, Tyr and Ll to W; Ll is where X 2 is C1-C5 alkylene, phenyl, or C5-C6 cycloalkylene; and (W) and (S) indicate the sites of attachment of W and S. 2. A conjugate of Claim 1 having the formula: PyroGlu-His-Trp-Ser-Tyr-W-Leu-Arg-Y-Z S rPE where W is D- or L-Lys, and Y, Z, Ll, and rPE are as defined in Claim 1. 3. The conjugate of Claim 2 where the Pseudomonas exotoxin has been modified such that the binding domain has been partly or completely deleted. 4. The conjugate of Claim 3 where the Pseudomonas exotoxin has been modified to delete amino acids 1-252 and retain amino acid 253-613. 5. The conjugate of Claim 1 of the structure: wherein rPE is NLysPE38QQR. 6. The conjugate of Claim 1 of the structure: wherein rPE is NLysPE38QQR. 7. A process for the preparation of a compound of Claim 1 which comprises treating a GnRH derivative having the formula: Q-Ser-Tyr-W-X 1 -Arg-Y-Z where Q, W, χl, Y and Z are defined in Claim 1, and Ll is where X 2 is as defined in Claim 1, and halo is Br, Cl, or I, with a reduced Pseudomonas exotoxin as defined in Claim 1 having at least one free thiol group. 8. The process of Claim 7 where the GnRH derivative has the formula: PyroGlu-His-Trp-Ser-Tyr- W-Leu- Arg- Y-Z I Ll wherein W is D- or L-Lys, and Y, Z and Ll are as defined in Claim 7. 9. The process of Claim 7 where the GnRH derivative is D-Lys 6 -GnRH. 10. The process of Claim 7 where the reduced Pseudomonas exotoxin is reduced NLysPE38QQR. 11. A method for the sterilization of animals which comprises administering to such animals an effective amount of a conjugate of Claim 1. 12. A composition useful for the sterilization of animals which comprises an inert canier and an effective amount of a conjugate of Claim 1.

Inventors: LOMBARDO VICTORIA K, TOLMAN RICHARD L, LOMBARDO, VICTORIA, K., TOLMAN, RICHARD, L.
Applicants: LOMBARDO VICTORIA K, MERCK & CO INC, TOLMAN RICHARD L, LOMBARDO, VICTORIA, K., MERCK & CO., INC., TOLMAN, RICHARD, L.
Application year: 1996
Main classification: A61K38
IPC codes: A61K31, A61K38, A61K39, A61K47, A61P15, A61P35, C07K1, C07K7, C07K14, C07K17, C07K19