EP0871481A1 - L'exotoxine de pseudomonas: un vecteur immunogene pour vaccins de synthese conjugues - Google Patents

L'exotoxine de pseudomonas: un vecteur immunogene pour vaccins de synthese conjugues

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Publication number
EP0871481A1
EP0871481A1 EP96940252A EP96940252A EP0871481A1 EP 0871481 A1 EP0871481 A1 EP 0871481A1 EP 96940252 A EP96940252 A EP 96940252A EP 96940252 A EP96940252 A EP 96940252A EP 0871481 A1 EP0871481 A1 EP 0871481A1
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EP
European Patent Office
Prior art keywords
gnrh
gly
leu
peptide
arg
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96940252A
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German (de)
English (en)
Inventor
Gerard J. Hickey
Kenneth L. Mohn
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merck and Co Inc
Original Assignee
Merck and Co Inc
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Filing date
Publication date
Priority claimed from GBGB9602878.2A external-priority patent/GB9602878D0/en
Application filed by Merck and Co Inc filed Critical Merck and Co Inc
Publication of EP0871481A1 publication Critical patent/EP0871481A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/18—Feminine contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides
    • 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

Definitions

  • the present invention relates to a method for eliciting antibodies against gonadotropin releasing hormone (GnRH) in an animal comprising adminstering to said animal an immunogenic carrier system which comprises GnRH and Pseudomonas exotoxin or a variant thereof.
  • GnRH gonadotropin releasing hormone
  • the method of the present invention represents an effective and preferred method for sterilizing animals. The method may also be used to arrest development of steroid hormone stimulated tumors.
  • Sterilization may be used to control undesirable gonadal steroid hormone driven behavior such as aggression in males and estrus behavior in females, to improve feed efficiency and carcass quality in food animals such as swine and cattle, and to eliminate boar taint in the carcasses of male pigs.
  • the GnRH-cytotoxin approach is illustrated by the disclosure of WO90/09799, which teaches certain sterilizing agents comprising GnRH analogs coupled to a variety of toxins through an optional linking group consisting of 2-iminothiolane, SPDP (N- succinimidyl-3-(2-pyridyldithio)propionate), bis-diazabenzidine and glutaraldehyde.
  • 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 Pseudomonas exotoxin molecule.
  • 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.
  • a MAP multiple antigen peptide
  • 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 MAP conjugates generally have the attribute of high insolubility in hydrophobic and hydrophilic solvents rendering them more difficult to formulate.
  • GnRH coupled to Pseudomonas exotoxin has been previously reported, these conjugates were used to deliver the toxin into the cytosol of cells expressing GnRH receptors.
  • the use of a GnRH- Pseudomonas exotoxin as a vaccine to raise anti-GnRH antibodies was not contemplated.
  • GnRH vaccines i.e., immunosterilization.
  • 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 androgen dependent organs.
  • a number of GnRH immunoconjugates or fusion proteins suitable for use as vaccines have been described.
  • 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).
  • CFA/IFA complete/incomplete Freunds adjuvant
  • 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 vaccine 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-Lys 6 ]GnRH
  • the conjugates may be used to control fertility or for therapy of prostate cancer.
  • the vaccines as described generally require large amounts of the vaccine concomitant with severe adjuvants to obtain any antibody response.
  • the adjuvants most commonly used are complete or incomplete Freund's adjuvant (CFA or IFA), which causes a variable amount of chronic inflammatory reaction at the site of injection, and are generally not acceptable to regulatory authorities. These reported vaccines do not elicit adequate levels of anti-GnRH antibodies, and are incapable of providing 100% effectiveness in all animals of a given group.
  • GnRH As previously discussed, Pseudomonas exotoxin has been coupled to GnRH, and the resulting construct used for the destruction of gonadotrophs; the GnRH of the construct acts to deliver the toxin into cells bearing GnRH receptors, and once inside the cell, the toxin is released and exerts its cytotoxic activity to effect cell killing.
  • the strategy of using a receptor binding ligand to deliver Pseudomonas toxin into the target cells has been well documented with a number of ligands other than GnRH.
  • 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.
  • Bailon. Biotechnology, pp. 1326-1329 Nov. (1988) teach 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.
  • the use of Pseudomonas exotoxin to increase immunogenicity of a hapten is described in W092/12173, which teaches fusion proteins of Pseudomonas exotoxin and specific regions of human P-glycoproteins; these fusion proteins are used to raise antibodies against P-glycoprotein.
  • Conjugate vaccines composed of Staphylococcus aureus capsular polysaccharide and recombinant protein derived from Pseudomonas exotoxin A is reported in Fattom, A. et al, Inf. Immun.. 1993, 61(3): 1023-1032.
  • European patent application 0 261 671 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 253- 613 of the whole Pseudomonas exotoxin A protein as defined in Gray, et al., PNAS USA £1:2645-2649 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.
  • the present invention relates to the use of Pseudomonas exotoxin (PE) as an immunogenic carrier protein for GnRH.
  • the GnRH- PE immunogenic carrier system may be GnRH-PE conjugates produced by chemical means, or GnRH-PE chimeric hybrid proteins produced using recombinant DNA technology.
  • the GnRH-PE immunogenic carrier system may be used in mammalian or avian species for conditions in which it is desirable to reduce or eliminate reproduction, reproductive hormone driven behavior, physiology or anatomy, or to treat sex steroid responsive tumors.
  • the present invention in one aspect provides a method for eliciting antibodies in a synthetic conjugate vaccine, and in particular anti-GnRH antibodies in an animal which comprises preferably administering to said animal an immunogenic carrier system comprising a GnRH associated with Pseudomonas exotoxin or a variant thereof, in an amount effective to elicit anti-GnRH antibodies. More particularly, there is provided a method for sterilizing an animal which comprises administering to said animal an immunogenic carrier system comprising a GnRH associated with Pseudomonas exotoxin or a variant thereof, in an amount effective to achieve sterilization.
  • the immunogenic carrier system comprising a GnRH associated with Pseudomonas exotoxin may be a GnRH-Pseudomonas exotoxin conjugate wherein said GnRH is attached to the Pseudomonas exotoxin by chemical means via a linker, or it may be a GnRH- Pseudomonas exotoxin hybrid protein.
  • GnRH TFA trifluoroacetic acid
  • 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.
  • GnRH molecule 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 [SEQUENCE ID NO.: 1]:
  • GnRH is pyroglutamate.
  • Many analogs or derivatives of native GnRH have been reported, and they may be obtained by addition, deletion, replacement or other alterations of the native GnRH.
  • Non- limiting examples of GnRH analogs or derivatives that may be suitable for use in the present inventions include those disclosed in UK Patent 2,228,262 (e.g. [D-Lys6]GnRH); US Patent 4,975,420 (e.g. [D- Cys6]GnRH); US Patent 4,608,251 (e.g. N-terminus modified nonapeptide or decapeptide); European Patent Application 464,124 (e.g two GnRH in tandem); European Patent Application 293,530 (e.g. C- terminus extended GnRH); PCT Application 88/05308 (e.g. truncated fragments of GnRH); and US Patent 5,324, 12 (pGlu of native GnRH replaced by Gin).
  • the native GnRH is preferably modified to include an amino acid that provides a functional group through which the GnRH can be linked to the Pseudomonas exotoxin; such an amino acid may be located at the N- or C-terminus, or it may replace amino acid 6 (Gly) of the native GnRH.
  • the GnRH includes a free amino or sulfhydryl group. Free amino group may be obtained by, for example, replacing the N- terminal pGlu with Gin, or by replacing Gly6 of the native GnRH with Lys.
  • Free sulfhydryl group may be obtained by replacing one of the amino acids, for example, the amino acid at position 1, 6 or 10, with cysteine; or alternatively, a free amino group may be thiolated using homocysteine thiolactone or mercaptopropanoic acid to provide the free sulfhydryl group.
  • a free amino group may be thiolated using homocysteine thiolactone or mercaptopropanoic acid to provide the free sulfhydryl group.
  • GnRH may be represented by the sequence [SEQUENCE ID NO.: 2]:
  • B is a thiol containing linker of the formula HS-(CH2)n-Co-Q-; p is O or 1; n is 1 to 10;
  • Q is pGlu or Gin
  • W is a D- or L- amino acid selected from glycine, alanine, cysteine, homocysteine, ornithine, and lysine;
  • T Leu or Nle
  • U is Pro or 4-hydroxy-Pro
  • V is Gly-NH 2 , D-Ala-NH 2 , NH-Et, NH-Pr or Arg-Gly-NH ; with the proviso that at least one of Q or W has a free amino or sulfhydryl group.
  • GnRH More preferred GnRH are of the formula [SEQUENCE ID NO.: 3]:
  • GnRH is preferably the native GnRH or an analog or derivative thereof that can be manufactured using recombinant DNA techniques, for example, two native GnRH molecules in tandem, or a GnRH analog comprising only natural amino acids.
  • Pseudomonas exotoxin is a protein (mw 66,000) 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 partially 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.
  • PE-GnRH conjugate or hybrid protein may be inactivated, e.g. by photoinactivation, and still retain its immuogenic properties .
  • 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 e l 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 aJ.
  • 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 oliogopeptide fragments to their N-termini.
  • the immunogenic carrier system comprising a Pseudomonas exotoxin and a GnRH may be prepared by chemically coupling a GnRH to the PE carrier protein, or by using recombinant DNA techniques to produce GnRH-PE hybrid proteins. Each of these methods will be discussed hereinbelow.
  • a cross linking reagent such as SPDP (N-succinimidyl 3- (2-pyridyldithio)propionate), glutaraldehyde, iminothiolane, N-acetyl- homocysteine thiolactone, bromoalkanoic anhydrides, maleimido- benzoyl-N-hydroxy-succinimide ester, 3-maleimidopropionic acid N- hydroxy succinimide ester, and the like.
  • SPDP N-succinimidyl 3- (2-pyridyldithio)propionate
  • glutaraldehyde iminothiolane
  • N-acetyl- homocysteine thiolactone N-acetyl- homocysteine thiolactone
  • bromoalkanoic anhydrides bromoalkanoic anhydrides
  • maleimido- benzoyl-N-hydroxy-succinimide ester 3-
  • nucleophilic and electrophilic groups are provided on the reacting partners.
  • the preferred cross linking agent for the present invention providing an electrophylic partner for the coupling reaction are active esters of maleimidoylalkanoic acids, and bromoalkanoic anhydrides.
  • Preferred cross linking partners providing a nucleophile for the coupling reaction are N-acetyl homocysteine thiolactone and imino thiolactone.
  • the present invention also comprehends a system wherein the GnRH component is first attached to an oligopeptidyl scaffold, and the GnRH-scaffold assembly is linked to the PE.
  • the scaffold can be designed to contain more than one GnRH linking site, each binding site on the PE can carry more than one GnRH.
  • the afore-mentioned cross linking reagents are also applicable for linking GnRH to the scaffold, and the scaffold to the PE. In either type of conjugation, i.e. with or without the scaffold, any unconjugated haptens should be removed after conjugation reaction.
  • GnRH-Pseudomonas conjugate within the limits of the linker definition as described above thus encompasses any GnRH- PE conjugate with or without the scaffold.
  • the GnRH-scaffold PE conjugate may be represented by the formula:
  • A is independently an amino acid selected from Gly, Ser, Thr, ⁇ -
  • L i is a linker optionally attached to an internal marker
  • L 2 is independently a linker
  • X is a GnRH
  • Yl and Y2 are independently Lys or Orn; m is 0 to 3; n is 1 to 10; p is 0 to 1 ; q is 1 or 2; r is 1 to 10.
  • the scaffold is a linear oligopeptide having up to a total of twenty seven amino acids in the sequence with at least two of those being independently ornithine or lysine.
  • the other amino acids are selected from amino acids of small size with at least one of the non-Lys non-Orn amino acid being a hydrophilic amino acid.
  • suitable small amino acids are alanine, ⁇ -alanine, glycine, serine, and threonine; serine and threonine are in addition hydrophilic.
  • the hydrophilic amino acid is preferably serine or threonine, and up to 5 hydrophilic amino acid residues may be incorporated into the scaffold.
  • the scaffold oligopeptide has from 5 to 10 amino acids, one or two of which are Ser or Thr.
  • the lysine/ornithine residues are separated from each other by at least one amino acid, preferably the spacer between lysine/ornithine residues is about 3 to 8 amino acids.
  • Examples of scaffold are Lys-An-Lys wherein n is 4 to 7, more preferably 5 or 6; one of A is Ser or Thr, and the others are selected from Gly, Ala and ⁇ Ala.
  • Lys-Gly-Gly-Ser-Gly-Gly-Lys More specific examples include [SEQUENCE ID NO: 5] Lys-Gly-Gly-Ser-Gly-Gly-Lys and [SEQUENCE ID NO: 6] Lys-Gly-Gly-Thr-Gly- ⁇ Ala-Gly-Lys.
  • the term "internal marker” means an unnatural amino acid that has been incorporated to facilitate characterization of the conjugate and the calculation of the ratio of GnRH to carrier protein. Examples include ⁇ -Ala and norleucine.
  • the "linker” for chemical conjugation of GnRH and Pseudomonas exotoxin may be derived from any heterobifunctional cross-linking agents carrying functionalities that are reactive with amino group and sulfhydryl group; for examples, SPDP (N-succinimidyl 3-(2- pyridyldithio)propionate), glutaraldehyde, iminothiolane, bromoalkanoic anhydrides, maleimido-benzoyl-N-succinimide ester, 3- maleimidopropionic acid N-hydroxysuccinimide ester, and the like may be used.
  • Preferred linkers have the formula (where the terminal N and S are derived from the peptides that are linked):
  • R is C 1-C5 alkylene, phenyl or C5-C5 cycloalkylene; and s is 1 or 2.
  • R a is a linker group, such as
  • the first step is the reaction of DLys"-GnRH with a heterobifunctional linker such as maleimidopropanoyl N- hydroxysuccinimide (MPS), bromoacetic anhydride, or SPDP.
  • a heterobifunctional linker such as maleimidopropanoyl N- hydroxysuccinimide (MPS), bromoacetic anhydride, or SPDP.
  • MPS maleimidopropanoyl N- hydroxysuccinimide
  • SPDP bromoacetic anhydride
  • 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.
  • Pseudomonas exotoxin having one or more free sulfhydryl functionality is carried out under nitrogen at a pH of from 8 to 10 with a excess of the linker-DLy s"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 DLys ⁇ -GnRH conjugate 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.
  • the scaffold bearing a protected intemal marker ⁇ -alanine is reacted with bromoacetic anhydride to effect bromoacetylation of the terminal amino groups (N ⁇ ) of the Y 1 and Y2 residues of the scaffold.
  • the reaction is carried out under nitrogen in an inert organic solvent such as methylene chloride, dimethylformamide or a combination thereof, typically at ambient temperature.
  • the bromoacetylated scaffold is reacted with a GnRH having a free sulfhydryl group, for example [DCys6]GnRH or HSCH2CH2CO-[Glnl]GnRH, resulting in scaffold supported GnRH.
  • the reaction is carried out in aqueous acetonitrile at room temperature, and the pH of the reaction mixture is preferably maintained at about 8, and generally between 7.5 and 8.5.
  • the Fmoc protecting group is removed using e.g., piperidine, and the deprotected peptide is reacted with a maleimidyl alkanoic acid activated ester such as MPS, at room temperature in an inert organic solvent such as dimethylformamide, and in the presence of a base such as diisopropylethylamine, to provide maleimidated scaffold supported GnRH.
  • the Pseudomonas exotoxin carrier protein having a free thiol is reacted with the scaffold-supported GnRH product shown in Scheme 1 to provide the desired conjugate.
  • the free thiol may be associated with a free cysteine, or generated by reducing disulfide bond(s) in the carrier protein using, for example, dithiothreitol, or introduced onto the carrier protein by reacting the carrier protein with a thiolating agent, such as N-alkanoylhomocysteine thiolactone. More than one free thiol may be present on the carrier protein, and therefore more than one scaffold-supported GnRH may be loaded onto the carrier protein.
  • the immunogenic carrier system of the present invention may also be hybrid proteins of GnRH and Pseudomonas exotoxin A .
  • Such hybrid proteins contain contiguous sequences of the constituent proteins or peptides.
  • the hybrid proteins are preferably manufactured through expression of recombinant DNA sequences.
  • the DNAs used in the practice of the invention may be natural or synthetic.
  • the recombinant DNA segments containing the nucleotide sequences coding for the embodiments of the present invention can be prepared by the following general processes:
  • a desired DNA sequence is cut out from a plasmid in which it has been cloned, or the sequence can be chemically synthesized;
  • any promoter is usable as long as the promoter is suitable for expression in the host used for the gene expression.
  • the promoters can be prepared enzymatically from the corresponding genes, or can be chemically synthesized.
  • Ligations of vector and insert DNAs are performed with T4 DNA ligase in 66 mM Tris-HCl, 5 mM MgC12, ImM DTE, ImM ATP, pH 7.5 at 15°C for up to 24 hours. In general 1 to 200 ng of vector and 3- 5x excess of insert DNA are preferred.
  • Selection of E. coli containing recombinant plasmids involve streaking the bacteria onto appropriate antibiotic containing LB agar plates or culturing in shaker flasks in LB liquid (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, pH 7.4) containing the appropriate antibiotic for selection when required.
  • Choice of antibiotic for selection is determined by the resistance markers present on a given plasmid or vector.
  • vectors are selected by ampicillin.
  • Harvesting involves recovery of E. coli cells by centrifugation. For protein production, cells are harvested 3 hours after induction though other times of harvesting could be chosen.
  • any vector such as a plasmid
  • a host cell of choice is E. coli HB101.
  • E. coli strain DH5a(BRL) can be used for routine cloning.
  • the hybrid proteins can be separated and purified by appropriate combinations of well-known separating and purifying methods. These methods include methods utilizing a solubility of differential such as salt precipitation and solvent precipitation, methods mainly utilizing a difference in molecular weight such as dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide gel electrophoresis, methods utilizing a difference in electric charge such as ion-exchange column chromatography, methods utilizing specific affinity such as affinity chromatography, methods utilizing a difference in hydrophobicity such as reverse-phase high pressure liquid chromatography, methods utilizing a difference in isoelectric point, such as isoelectricfocusing electrophoresis, and methods using denaturation and reduction and renaturation and oxidation.
  • differential such as salt precipitation and solvent precipitation
  • methods mainly utilizing a difference in molecular weight such as dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide gel electrophoresis
  • methods utilizing a difference in electric charge such as ion
  • Immunogenic carrier system of the present invention have 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. 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 effects in certain disease states.
  • the immunogenic carrier system of the present invention can also be used in veterinary medicine or animal husbandry for conditions in which it is desirable to reduce or eliminate reproduction and/or reproductive hormone driven behavior, physiology or anatomy. Sterilization of animals has primarily been achieved by surgical removal of the gonads. Surgery necessarily involves some degree of pain, trauma and stress for the animal with the potential for infection and death. In food animals, neutering has been used as a means of controlling undesirable behaviors or meat characteristics but it has resulted in substantial production losses. In the case of swine, intact males have a much higher feed efficiency (approximately 18%) than castrates. However, androstenone is deposited in the fat of the intact male giving the meat an undesirable smell and odor.
  • another aspect of the present invention provides a method of sterilizing animals comprising administering to said animal an immunogenic carrier system comprising a GnRH associated with Pseudomonas exotoxin or a variant thereo, in an amount effective to achieve sterilization.
  • Vaccination using the immunogenic carrier system of the present invention eliminates the need for surgical neutering. Therefore, it eliminates the pain, stress, trauma, infection, death, production loss and animal welfare issues associated with surgical neutering.
  • Desirable sequellae of vaccination include transient sterilization, controlling undesirable gonadal steroid hormone driven behavior such as aggression in males and estrus behavior in females, improving feed efficiency and carcass quality in food animals such as swine and cattle and a method for eliminating boar taint in the carcasses of male pigs.
  • 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.
  • the immunogenic carrier system are administered by subcutaneous or intramuscular injection into a mammal at a rate of 1 ⁇ g to 1000 ⁇ g of conjugate per dose .
  • a single dose of the immunogenic carrier system of the present invention may be all that is required to achieve sterilization, but multiple doses spaced at one to six week intervals are altemative sterilization schemes.
  • 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.
  • the immunogenic carrier system 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 ⁇ g to about 1000 ⁇ g of the immunogenic carrier system, preferably about 10 ⁇ g to about 100 ⁇ g is used.
  • the amount of the immunogenic carrier system 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.
  • 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).
  • two doses administered at 2-8 week intervals followed by and annual boosters should be sufficient to produce neutering.
  • the actual dose and formulation remains to be determined and may vary with the particular immunogenic carrier system used. However, a dose of 1 to 2000 ug, preferably about 500 ug of an immunogenic carrier system 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.
  • immunogenic carrier system 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 immunogenic carrier system of the present invention can be used for the above-mentioned application without the use of aggressive adjuvants such as Complete Freund's Adjuvant, which cause injection site lesions and downgrading of meat 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 such as Al(OH)3, AIPO4, Al2(S ⁇ 4)3, water-in-oil emulsions such as Incomplete Freund's Adjuvant (IFA), Bayol F® or Marcol F®, vitamin-E acetate solubilisate, saponins, muramyl dipeptides in an appropriate solvent such as squalane or squalene.
  • IFA Incomplete Freund's Adjuvant
  • Bayol F® Bayol F® or Marcol F®
  • vitamin-E acetate solubilisate saponins
  • the immune carrier system 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
  • a non-ionic surfactant such as a polyoxypropylene (POP)-polyoxyethylene (POE) block polymer
  • POP polyoxypropylene
  • POE polyoxyethylene
  • 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-
  • R3 is ORl or R3 and R4 together form an oxo;
  • R 4 , R5, R6, and R 7 are independently H or methyl; with the proviso that when R3 and R4 together form an oxo, R ⁇ , R6, R7 and R2 are each H; and that when R2 is H, R 4 , R ⁇ , R6 and R 7 are each hydrogen, and R3 is ORl.
  • 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 com 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 and shark liver oils, exemplify several of the fish oils. Whale oils such as spermaceti are also acceptable. A number of branched chain oils are synthesized biochemically in 5-carbon isoprene units and are generally referred as te ⁇ enoids. Shark liver oil contains a branched, unsaturated te ⁇ enoids 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%.
  • polyoxypropylene-polyoxyethylene 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.
  • 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.
  • 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%.
  • emulsifier refers to non-toxic surface active agents capable of stabilizing the emulsion.
  • 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.
  • emulsifiers can be used so long as they are non-toxic, glycol ether-based emulsifiers are preferred.
  • 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 0.5%, preferably about 0.2 to 1%.
  • the aqueous portion of the adjuvant compositions of the invention is preferably buffered isoosmotic saline. It is prefened 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 abso ⁇ tion of the composition due to differential ion concentrations between the composition and physiological fluids. It is also preferred 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 preferred 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.
  • an immunopotentiating amount of the immune response enhancer is included.
  • the immune response enhancer is a compound of formula a wherein R 1, R2, R5, R6, and R 7 are each H, and R3 and R 4 together form an oxo group, this compound being dehydroepiandrosterone or DHEA.
  • the immunogenic carrier system may be any GnRH-PE conjugates or GnRH- PE hybrid
  • the immunogenic carrier system of the present invention may also be hybrid proteins of GnRH and Pseudomonas exotoxin A .
  • Such hybrid proteins contain contiguous sequences of the constituent proteins/peptides.
  • the hybrid proteins are preferably manufactured through expression of recombinant DNA sequences.
  • the DNAs used in the practice of the invention may be natural or synthetic.
  • the recombinant DNA segments containing the nucleotide sequences coding for the embodiments of the present invention can be prepared by the following general processes:
  • a desired DNA sequence is cut out from a plasmid in which it has been cloned, or the sequence can be chemically synthesized;
  • a second DNA sequence, the targeted DNA sequence is cleaved at a specific location; and (c) The desired DNA sequence is then brought into alignment with the cut in the targeted DNA sequence and the two sequences are connected together through standard ligation procedures.
  • the resulting recombinant gene is ligated down stream from a suitable promoter in an expression vector.
  • any promoter is usable as long as the promoter is suitable for expression in the host used for the gene expression.
  • the promoters can be prepared enzymatically from the corresponding genes, or can be chemically synthesized. Conditions for usage of all restriction enzymes are in accordance with those of the manufacturer, including instructions as to buffers and temperatures. The enzymes may be obtained from New England Biolabs, Bethesda Research Laboratories, Boehringer Mannheim and Promega. Ligations of vector and insert DNAs are performed with T4
  • DNA ligase in 66 mM Tris-HCl, 5 mM MgC12, ImM DTE, ImM ATP, pH 7.5 at 15 °C for up to 24 hours. In general 1 to 200 ng of vector and 3- 5x excess of insert DNA are preferred.
  • E. coli containing recombinant plasmids involve streaking the bacteria onto appropriate antibiotic containing LB agar plates or culturing in shaker flasks in LB liquid (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, pH 7.4) containing the appropriate antibiotic for selection when required.
  • Choice of antibiotic for selection is determined by the resistance markers present on a given plasmid or vector.
  • vectors are selected by ampicillin.
  • Harvesting involves recovery of E. coli cells by centrifugation. For protein production, cells are harvested 3 hours after induction though other times of harvesting could be chosen.
  • any vector such as a plasmid
  • a host cell of choice is E. coli HB 101.
  • E. coli strain DH5a(BRL) can be used for routine cloning.
  • the hybrid proteins can be separated and purified by appropriate combinations of well-known separating and purifying methods. These methods include methods utilizing a solubility of differential such as salt precipitation and solvent precipitation, methods mainly utilizing a difference in molecular weight such as dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide gel electrophoresis, methods utilizing a difference in electric charge such as ion-exchange column chromatography, methods utilizing specific affinity such as affinity chromatography, methods utilizing a difference in hydrophobicity such as reverse-phase high pressure liquid chromatography, methods utilizing a difference in isoelectric point, such as isoelectricfocusing electrophoresis, and methods using denaturation and reduction and renaturation and oxidation.
  • differential such as salt precipitation and solvent precipitation
  • methods mainly utilizing a difference in molecular weight such as dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide gel electrophoresis
  • methods utilizing a difference in electric charge such as ion
  • GnRH-PE conjugates are used, and these may include the scaffold supported GnRH-PE conjugates, as well as other known GnRH-PE conjugates.
  • the oil-in-water emulsion comprises squalane, Tween 80 and Pluronic L121. More preferred, the vaccine includes DHEA as the immune response enchancer. Even more preferably, the Pseudomonas exotoxin is NLys PE38QQR.
  • the oil-in-water emulsion adjuvant composition is prepared by emulsification using a mixer to form a homogenous emulsion.
  • the adjuvant composition is microfluidized prior to adding the GnRH-conjugate; the emulsion is cycled through the microfluidizer about 2-20 times.
  • the GnRH-conjugate is then mixed with the adjuvant composition, and the mixture may be again cycled through the microfluidizer.
  • the immune response enhancer e.g. DHEA, if included in the vaccine composition, may be added to the adjuvant composition prior to microfluidization, or it may be added after the GnRH-conjugate has been mixed with the adjuvant composition.
  • the oily particles in the emulsion preferably have diameters of about 0.03 ⁇ m and 0.5 ⁇ m, more preferably between 0.05 and 0.2 ⁇ m.
  • the peptide was synthesized on Rink amide MBHA resin (0.25 mmol, Amino Tech) by solid phase peptide synthesis (SPPS) using an ABI model 431 A synthesizer and single couplings (DCC/HOBT).
  • SPPS solid phase peptide synthesis
  • 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 prepared, up to the Gin 1 residue, on an ABI 431 A peptide synthesizer (Fmoc chemsitry) using unloaded Rink Amide MB HA resin (NovaBiochem, 0.25 mmol). The initial Gly residue was double coupled onto the resin. All additional amino acids, except for
  • Arg were single coupled. A standard double coupling protocol was used for Arg 8 .
  • the N-terminal Fmoc group was removed from the peptide and the resin dried (N2).
  • the resin was transfened to a manual peptide synthesis vessel with fritted bottom (N2 used for agitation).
  • the resin was suspended in DMF (5 mL) and 3-mercaptopropionic acid (1 mmol) was manually coupled to the amino terminus using PyBOP/HOBt activation (1 mmol PyBOP, 0.1 mmol HOBt) and N2 agitation until a Kaiser test of the resin showed that the reaction was complete (16-48 hr).
  • the peptide was cleaved from the resin with 20 mL of degassed "reagent R" (90:5:3:2 TFA/thioanisole/ethanedithiol/anisole) for 3 hr at RT. The mixture was filtered and the filtrate concentrated in vacuo. The remaining residue was triturated with ether and the precipitate collected and dried (272.1 mg).
  • the peptide was prepared, up to the Gin 1 residue, on an ABI 431 A peptide synthesizer (Fmoc chemsitry) using unloaded Rink Amide MBHA resin (NovaBiochem, 0.25 mmol). The initial Gly residue was double coupled onto the resin. All additional amino acids, except for Arg, were single coupled. A standard double coupling protocol was used for Arg 8 . The N-terminal Fmoc group was removed from the peptide (regular piperidine cycle on ABI) and the resin dried (N2). The resin was transfened to a manual peptide synthesis vessel with fritted bottom (N2 used for agitation).
  • the resin was suspended in DMF (5 mL) and 3- mercaptopropionic acid ( 1 mmol) was manually coupled to the amino terminus using PyBOP/HOBt activation (1 mmol PyBOP, 0.1 mmol HOBt) and N2 agitation until a Kaiser test of the resin showed that the reaction was complete (16-48 hr).
  • the peptide was cleaved from the resin with 20 mL of degassed "reagent R" (90:5:3:2 TFA/thioanisole/ethanedithiol/anisole) for 3 hr at RT. The mixture was filtered and the filtrate concentrated in vacuo. The remaining residue was triturated with ether and the precipitate collected and dried (232.0 mg).
  • the peptide was synthesized on or Menifield resin by solid phase peptide synthesis (Boc chemistry) using an ABI model 431 A synthesizer and single couplings (DCC/HOBT).
  • the peptide was cleaved (24 h-72 h, RT) from the resin with anhydrous ethyl amine.
  • the cmde protected peptide was precipitated with diethyl ether, collected by suction filtration, and dried overnight (over P2O5).
  • the protecting groups were 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 peptide was synthesized on Rink amide MBHA resin (0.25 mmol, Amino Tech) 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/ethanedifhiol/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.
  • the peptide is synthesized on Oxime or Merrif ⁇ eld resin by solid phase peptide synthesis (Boc chemistry) using an ABI model 431 A synthesizer and single couplings (DCC/HOBT).
  • the 3-indolylpropionyl moiety is inco ⁇ orated as N-formyl-3-indolepropionic acid.
  • the peptide is cleaved (2 h-72 h, RT) from the resin with anhydrous ethyl amine.
  • the cmde 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.
  • 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 carried out on PJH4 resulting in linearization of the constmct and clipping of a 12 bp segment which includes the ATG start codon of the PE coding sequence.
  • oligonucleotides 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 allow blunt end 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 mpi 9.
  • oligonucleotide 50 nucleotides in length with the stmcture 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:
  • 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 M13 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 carried 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
  • the Bam HI EcoR 1 fragment is excised from the replicative form of the mutant DNA in M13 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 NLys PE38QQR, from a commercially available strain of E. coli. HB 101, available from Bethesda Research Laboratories.
  • NLys PE38QQR is also known as Lys PE38M as described in published WO 93/15751 (Merck & Co., Inc.).
  • the reduced toxin solution was then dialyzed versus 0.01 M phosphate buffer ovemite 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.
  • Pseudomonas Exotoxin A (10 mg, 0.152 mmol) (Sigma Chemical Co. ) was dissolved in 10 mL of water and to this was added 43 mg of 8-azidoadenosine (0.14 mmol). This did not completely dissolve and the small amount of precipitate was centrifuged and the supematant was charged to a Pyrex photoreactor, cooled with ice water and irradiated with a 450 W Hanovia lamp for 8 min. The resultant solution was dialyzed for 17.5 hr against 4 L of PBS and then analyzed by TSK 2000 HPSEC which showed an absence of low molecular weight material (i.e. unreacted 8-azidoadenosine). The solution was also assayed for ADP ribosylating activity and found to have only 4% of the original level.
  • DLys 6 -GnRH (10 mmol, 12.5 mg) was dissolved in N,N- dimethylformamide (0.5 mL/mg) and DIEA (50 mmol, 9 ⁇ L) added. The mixture was stirred briefly (RT) and ⁇ -maleimidopropionic acid N- hydroxysuccinimide ester (MPS; 20 mmol, 5.2 mg) was introduced in one poition. After 30 min reaction time, 10 ⁇ L TFA was added to the reaction mixture and the solvent removed in vacuo.
  • MPS ⁇ -maleimidopropionic acid N- hydroxysuccinimide ester
  • FAB-MS positive ion, NBA matrix
  • NLys-PE38QQR (0.22 ⁇ mol, 30. mL, 2.8 mg/mL) in PBS.
  • the pH of the solution was adjusted to 10.8 by the addition of 350 ⁇ L of 1.0 M, pH 11.0 borate buffer.
  • Dithiothreitol (11.0 ⁇ mol, 1.7 mg) and EDTA-2Na (22.1 ⁇ mol, 8.2 mg) were added and the protein mixture vortexed until all solids were in solution.
  • N-Acetylhomocysteine thiolactone (22.1 ⁇ mol, 3.5 mg) was introduced in one portion and the solution degassed and purged with nitrogen (degas/purge repeated 5X).
  • the mixture was aged in an nitrogen box at RT for 6.5 h, then charged to Spectropor 2 dialysis tubing and dialyzed (RT) as follows: vs. 1) 4L degassed, nitrogen sparged, 0.1 M, pH 8.0 phosphate buffer which contained 10 mg EDTA-2Na, and 0.25 mg DTT (16 h); 2) 4L degassed, nitrogen sparged, 0.01 M, pH 8.0 phosphate buffer which contained 10 mg EDTA-2Na, and 0.25 mg DTT (6 h). The thiolated exotoxin was then transfened to a sterile 15 mL polyethylene centrifuge tube (3.90 mL).
  • Bromoacetic anhydride was prepared by reaction of bromoacetic acid (31.5 mg, 0.226 mmol, 3.96 eq.) and DCC (23.4 mg, 0.113 mmol, 1.98 eq.) in dry (Aldrich Sure-Seal) dichloromethane (2 mL) at room temperature for 1 h. This mixture was filtered (sintered funnel, N 2 pressure) directly into a solution of the linear scaffold peptide from step 1 (50.0 mg, 0.057 mmol) that was dissolved in dry (4 A sieves) degassed DMF (5 mL). The reaction was allowed to proceed at room temperature under N 2 .
  • [DCys 6 ]-GnRH (68.1 mg, 0.055 mmol, 2.04 eq.) was dissolved in water (2 mL) and a few drops of acetonitrile were added to clear the solution.
  • the solution of [ ⁇ Cys 6 ]- GnRH was added dropwise (slow addition over 35 to 40 minutes) to the bis-bromoacetylated scaffold while maintaining the pH of the reaction mixture between pH 7.8 to 8.1 with the addition of dilute ammonium hydroxide.
  • the final pH of the reaction mixture was 8.0.
  • the reaction mixture was stirred at room temperature for 10 min then sonicated for 20 min after which time the mixture became cloudy. Stirring was again maintained at room temperature for a total of 2 h reaction time.
  • reaction mixture was quenched by the addition of TFA (10 ⁇ L) and concentrated in vacuo. The remaining residue was taken up into 10% aq. CH 3 CN (0.1% TFA) and purified by RP-HPLC (DeltaPak C l8 , RCM 25X10, 10 ml/min, 20 to 40% CH 3 CN, 30 min). The fractions containing the desired material were combined and lyophilized ovemight, providing the maleimidated scaffold supported [DCys6]-GnRH as a white powder (6.9 mg, .0021 mmol, 62%).
  • the tube was transferred into an N 2 -filled box and the cap replaced by a bber septum.
  • the tube was evacuated briefly and purged with N 2 (repeated 5X).
  • N-acetylhomocysteine thiolactone 100 mg, 0.629 mmol was added in one portion and the mixture vortexed until all solids were in solution and the tube re-evacuated and purged with N 2 (repeated 5X).
  • the tube was capped and allowed to age in the N 2 -filled box ovemight (20 h) at room temperature.
  • the reaction mixture was transferred to a dialysis bag and dialyzed against (a) 4L, 0.1 M pH 8.0 phosphate buffer with N 2 sparging (19 h); (b) 4L, 0.01 M pH 8.0 phosphate buffer containing 100 mg EDTA with N 2 sparging (8 h); and (c) 4L, 0.01 M pH 8.0 phosphate buffer containing 100 mg EDTA with N 2 sparging (20 h).
  • the maleimidated peptide of step 4 (5.0 mg 1.49 ⁇ mol) was dissolved in water (0.1 % TFA) and placed in a sterile 50 mL plastic centrifuge tube and lyophilized ovemight.
  • the thiolated protein of step 5 (0.083 ⁇ mol SH/mL protein, 16.9 mL, 1.40 ⁇ mol) was added to the lyophilized peptide and the tube capped and vortexed briefly.
  • the tube was sealed with parafilm and placed on a Clay-Adams nutator and tumbled ovemight at 4 °C (17 h).
  • the reaction mixture was transfened to a dialysis bag (Spectropor 2) and dialyzed (4 °C) vs.
  • TP40 a chimeric protein containing transforming growth factor alpha at the N-terminus and a derivative of a 40 kDa segment (PE40deltacys) of Pseudomonas exotoxin) and NLys PE38QQR.
  • Example 1 The conjugate of Example 1 (0.5 mL) was added to 9.5 mL of phosphate buffered saline (PBS) containing 4.16 mg of 8- azidoadenosine affording a solution which is .29 mM in conjugate and 1.35 mM in azidoadenosine.
  • PBS phosphate buffered saline
  • This solution was charged to a Pyrex photoreactor with a cooling jacket through which ice water was pumped. It was then inadiated using a 450 W Hanovia lamp at a distance of 6 inches for 6 min. The solution was then dialyzed vs. 4 L of PBS 16 hr affording solution which had no low molecular weight materials as indicated by TSK 2000 HPSEC.
  • the ADP ribosylating activity of this material was only 20% of the original as indicated by a wheat germ assay.
  • step 4 (1.88 mg, 450 nanomoles) was dissolved in 100 ⁇ L of water and 95 ⁇ L of this was added to 4 mL of the thiolated, photoinactivated PE holotoxin prepared in step 2. This was degassed and aged for 16 hr. At this time TSK 2000 HPSEC shows considerable low m.w. material remains. The solution was then dialyzed for 7 hr against 4L of PBS and then for 178 hr against a fresh 4 L of PBS at 4 ⁇ C. An HPSEC assay shows no small molecules remain. An amino acid analysis indicates that there are 90 ⁇ g of scaffold / mL ( ⁇ -alanine content) and 517 ⁇ g of PE toxin / mL.
  • the vaccines were prepared by combining the peptide solutions with 0.9% sodium chloride injection USP (Baxter, Lot C255075) and Incomplete Freund's Adjuvant (Sigma, F 5506, Lot 062H- 8802) in the proportions listed below in a 20 cc, glass, luer-lok syringe (Popper & Sons). Homogenization was achieved by passing the mixture between two 20 cc glass syringes through a 20 gauge, double hubbed, homogenization needle (Popper & Sons) until stiff.
  • an alum adjuvanted vaccine was also prepared by mixing 6 ml of immunogconjugate of Example 3 (0.117 mg/ml in saline), 1 ml saline and 7 ml Alhydrogel 1.3% (Superfos Biosector a/s, Batch 1983) to yield antigen final concentration of 50 ug/ml (vaccine No. 5).
  • PBS-BSA was prepared by dissolving one packet of BupH modified Dulbecco's phosphate buffered saline mix (Pierce, No. 28374, Lot 920521084) in 400 ml of deionized water and adding 2 gm of bovine albumin fraction V (Gibco No. 810-1018IL, Lot 76P9623) and 5 ml of a 1% w/v thimerosal solution (Sigma, T-5125, Lot 23H0526). Once the BSA was dissolved, deionized water was added to a final volume of 500 ml.
  • Dextran-coated charcoal suspension was prepared by washing 2.5 g of charcoal (Sigma, C-5385, Lot 102H0336 )with deionized water multiple times to remove the fines.
  • PBS was prepared by dissolving two packet of BupH Modified Dulbecco's Phosphate Buffered Saline mix (Pierce, No. 28374, Lot 920521084) in 1000 ml of deionized water. 0.25g of dextran, 70,000 mw (Sigma, D1390, Lot 122H0349) was dissolved in 500 ml of PBS. The washed charcoal was added to this solution. 10 ml of 1% w/v thimerosal solution (Sigma, T-5125, Lot 23H0526) plus an additional 500 ml of PBS were added and the charcoal suspension was stirred for 3 days at 4 C.
  • the antibody titer results were as follows:
  • Values are expresses as the average percentage of input 125 I-labeled peptide which was bound at a 1/1000 dilution of semm except for pretreatment which is neat semm.
  • Semm samples were submitted to the USDA lab at Athens, GA under the supervision of Dr. George Rampacek for semm LH quantitation by radioimmunoassay.
  • the assay is a standard radioimmunoassay using 125 I labelled porcine LH and anti-bovine LH antisera which recognize porcine LH. This assay is described by R. Kraeling et al in J. Anim. Sci. (1982) 54: 1212. The results below are the means for each group. Week 6 is the pooled mean for the three bleedings. The results are presented in ng of LH/ml. 0.15 ng LH/ml of semm is the lower limit of detection for the assay so all values which fell below the level of detection were assigned the value of 0.15 ng/ml.
  • Squalane 500 mg
  • Pluronic 121 250 mg; a block copolymer of polyethylene oxide and polypropylene oxide (BASF co ⁇ .) and Tween 80 (20 mg) are weighed into a 15 mL Dounce tissue homogenizer tube. This is then covered with 9.25 mL of PBS (pH 7.4) and the resultant mixture homogenized with about ten strokes . The solution is then transfened to a vial and a small magnetic stir bar added. About 3 mL of this mixture is transfened into cylinder of an Avestin Emulsiflex ® microfluidizer.
  • Emulsiflex is positioned so that it is submerged below the surface of the remaining liquid in the vial
  • the vial is cooled in an ice bath, and twenty passes of the Emulsiflex are effected while the liquid is magnetically stirred. In this manner 9.5 mL of STP are obtained.
  • the same product can be obtained by adding the requisite amount of the conjugate (in PBS) to the squalane:Tween 80: Pluronic 121 mixture in the Dounce homogenizer in Procedure 1, and subjecting the four part mixture to 20 passes in the microfluidizer.
  • MOLECULE TYPE peptide
  • SEQUENCE DESCRIPTION SEQ ID NO: :
  • Trp Arg Gly Phe Tyr lie Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr 210 215 220

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Abstract

L'exotoxine de Pseudomonas et certaines de ses variantes sont des protéines vecteurs immunogènes puissantes de la GnRH. Les vaccins contenant une GnRH en association avec l'exotoxine de Pseudomonas permettent d'obtenir chez des animaux des titres élevés d'anticorps anti-GnRH. Il en résulte que de tels vaccins conviennent particulièrement au contrôle de la fertilité, à l'atténuation des comportements indésirables dépendants des hormones de reproduction, et au traitement des tumeurs réagissant aux stéroïdes sexuels.
EP96940252A 1995-10-27 1996-10-23 L'exotoxine de pseudomonas: un vecteur immunogene pour vaccins de synthese conjugues Withdrawn EP0871481A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US801895P 1995-10-27 1995-10-27
US8018P 1995-10-27
GBGB9602878.2A GB9602878D0 (en) 1996-02-13 1996-02-13 Pseudomonas exotoxin as immunogenic carrier in synthetic conjugate vaccines
GB9602878 1996-02-13
PCT/US1996/017008 WO1997015325A1 (fr) 1995-10-27 1996-10-23 L'exotoxine de pseudomonas: un vecteur immunogene pour vaccins de synthese conjugues

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JP (1) JPH11515013A (fr)
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US5688506A (en) * 1994-01-27 1997-11-18 Aphton Corp. Immunogens against gonadotropin releasing hormone
US6838553B1 (en) * 1999-10-05 2005-01-04 Academia Sinica Peptide repeat immunogens
EP1303533A2 (fr) 2000-05-05 2003-04-23 Aphton Corporation Peptides chimeres a efficacite immunogene, leur preparation et utilisation
CN1683416A (zh) * 2004-04-13 2005-10-19 李相哲 高效低毒的系列功能蛋白
EP2332970B1 (fr) 2005-07-29 2015-12-23 The Government of the United States of America, as represented by the Secretary of Health and Human Services Exotoxines de Pseudomonas mutées a antigenicité reduite
AU2012216642B2 (en) * 2005-07-29 2014-06-12 THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY OF HEALTH AND HUMAN SERVICES NATIONAL INSTITUTES OF HEALTH Mutated pseudomonas exotoxins with reduced antigenicity
CN101343328B (zh) * 2008-08-25 2011-06-01 北京博翱泰生物技术有限公司 靶特异性双突变体融合蛋白质
AU2020265451A1 (en) * 2019-05-02 2021-12-23 The University Of Sydney Peptide derivatives and conjugates thereof for treating cancer

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AU5186090A (en) * 1989-02-23 1990-09-26 Colorado State University Research Foundation Gnrh analogs for destroying gonadotrophs

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See references of WO9715325A1 *

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