WO1993015751A1 - TOXINES CHIMERIQUES SE FIXANT AU RECEPTEUR DE GnRH - Google Patents

TOXINES CHIMERIQUES SE FIXANT AU RECEPTEUR DE GnRH Download PDF

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Publication number
WO1993015751A1
WO1993015751A1 PCT/US1993/001263 US9301263W WO9315751A1 WO 1993015751 A1 WO1993015751 A1 WO 1993015751A1 US 9301263 W US9301263 W US 9301263W WO 9315751 A1 WO9315751 A1 WO 9315751A1
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Prior art keywords
toxin
gnrh
pseudomonas exotoxin
arg
leu
Prior art date
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PCT/US1993/001263
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English (en)
Inventor
Victoria K. Lombardo
Richard L. Tolman
Stephen Marburg
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Merck & Co., Inc.
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Publication date
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Publication of WO1993015751A1 publication Critical patent/WO1993015751A1/fr

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    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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
  • optional linking group consisting of 2-iminothiolane, SPDP (N-succinimidyl-3-(2-pyridyldithio/ propionate), bis-diazabenzidine and glutaraldehyde.
  • SPDP N-succinimidyl-3-(2-pyridyldithio/ propionate
  • bis-diazabenzidine glutaraldehyde.
  • the compounds are disclosed as sterilizing agents.
  • conjugation can occur to one or through multiple attachments.
  • determination of the number of attachments of ligands is difficult or impossible.
  • An unverified assumption of these prior works is that all toxin conjugates, regardless of their stiochiometry, are efficacious or equally efficacious.
  • a toxin can be coupled to a GnRH analog through a linking group
  • the instant linking groups are unique and have been found to offer unique advantages in the efficacy of the final toxin conjugate, in the process for the manufacture thereof and in the analysis of conjugated peptide and toxin.
  • various constructs of bacterial or plant toxins have been prepared in attempts to specifically target the toxin to certain cells or organs.
  • 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.
  • antibodies can be conjugated to the A chain or the B chain of ricin which is a toxin obtained from plants.
  • Patent 4,664,911 further teaches that these conjugates can be used to kill human tumor cells.
  • U.S. patent 4,675,382 teaches that hormones such as melanocyte stimulating hormone (MSH) can be linked to a portion of the diphtheria toxin protein via peptide bonds.
  • MSH melanocyte stimulating hormone
  • Patent 4,675,382 further teaches that the genes which encode these proteins can be joined together to direct the synthesis of a hybrid fusion protein using recombinant DNA techniques.
  • This fusion protein has the ability to bind to cells that possess MSH receptors. Murphy, et al., PNAS USA 83:8258-8262
  • alpha-melanocyte-stimulating hormone will bind to and kill human melanoma cells.
  • 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.
  • 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.
  • Pseudomonas exotoxin A protein that retains the translocating and ADP ribosylating functions of the whole Pseudomonas exotoxin A protein is called Pseudomonas exotoxin - 40 or PE-40
  • 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. Kelley. et al., PNAS USA.85: 3980-3984
  • diphtheria toxin protein joined to interleukin 2 functions in mice to suppress cell mediated immunity.
  • Targeting A term for the selective delivery of chemotherapeutic agents to specific cell populations is "targeting".
  • Drug targeting to specific cells can be accomplished in several ways. One method relies on the presence of specific receptor molecules found on the surface of cells. Other molecules, referred to as “targeting agents”, can recognize and bind to these cell surface receptors. These “targeting agents” include, e.g., antibodies, growth factors, or hormones. “Targeting agents” which recognize and bind to specific cell surface receptors are said to target the cells which possess those receptors. For example, pituitary cells that release lutenizing hormone (LH) possess a protein on their surfaces that recognizes and binds with GnRH. GnRH is therefore, a "targeting agent” for these cells.
  • LH lutenizing hormone
  • “Targeting agents” by themselves do not kill cells.
  • Other molecules including cellular poisons or toxins can be linked to "targeting agents” to create hybrid molecules that possess both cell targeting and cellular toxin domains. These hybrid molecules function as cell selective poisons by virtue of their abilities to target selective cells and then kill those cells via their toxin component.
  • Some of the most potent cellular poisons used in constructing these hybrid molecules are bacterial toxins that inhibit protein synthesis in mammalian cells. Pseudomonas exotoxin A is one of these bacterial toxins, and has been used to construct hybrid "targeting - toxin" molecules (U.S. Patent 4,545,985).
  • Pseudomonas exotoxin A intoxicates mammalian cells by first binding to the cell's surface, then entering the cell cytoplasm and inactivating elongation factox 2 which is a cellular protein required for protein synthesis. Pseudomonas exotoxin A has been used to construct anticancer hybrid molecules using monoclonal antibodies and protein hormones.
  • hybrid molecules containing Pseudomonas exotoxin A exhibit toxicity towards normal cells. At least part of the toxicity associated with hybrid molecules containing Pseudomonas exotoxin A is due to the ability of Pseudomonas exotoxin A by itself to bind to and enter many types of mammalian cells.
  • hybrid molecules formed between Pseudomonas exotoxin A and specific "targeting agents” can bind to many normal cells in addition to the cells recognized by the "targeting agent".
  • One method of dealing with this problem is to modify Pseudomonas exotoxin A so that it is no longer capable of binding to normal cells. This can be accomplished by removing that portion of the Pseudomonas exotoxin A molecule which is responsible for its cellular binding activity.
  • a truncated form of the Pseudomonas exotoxin A molecule has been prepared which retains the ability to inactivate elongation factor 2 but no longer is capable of binding to mammalian cells.
  • This modified Pseudomonas exotoxin A molecule is called Pseudomonas exotoxin - 40 or PE 40 (Hwang, et al., Cell 48:129-136 1987).
  • the instant invention utilizes various constructs of GnRH, a linking group and a toxin.
  • the instant invention describes the preparation of site-specific toxin constructs using the
  • This invention is concerned with an agent which is selectively toxic to the LH releasing cells of the pituitary. This selective toxicity is
  • conjugates based on Pseudomonas exotoxin A are preferred.
  • chimeric toxic agents are best described in the following structural representation:
  • Q is PyroGlu-His-Trp, N-acetyl-4-Cl-Phe 1,2 -Trp or 3-indolylpropionyl
  • W is the D or L amino acid with a pendant linking functionality such as
  • r is 1 or 2;
  • n 1 to 4.
  • n 0 or 1
  • B is CH 2 , O, S or N
  • R 1 is hydrogen, C 1 -C 6 alkyl or
  • X is Leu or me
  • Y is Pro or 4- -hydroxy-Pro
  • Z is Gly, NH 2 , D-Ala-NH 2 , NH-Et, NH-Pr or
  • L 1 , L 2 and T are as defined below.
  • Preferred compounds of the instant invention are realized in the following structure:
  • D-Lys 6 -GnRH is a preferred targeting agent with which to bind the toxic construct to the LH releasing pituitary cells, it will be recognized that variations of D-Lys 6 -GnRH that will still bind to the GnRH receptor of the pituitary will be useful in this invention. All that is required is that the 6- ⁇ osition amino acid possess an amino group for binding to the linking group and that the remainder of the peptide bind to the GnRH receptor on the pituitary gland cells.
  • Z in the above formula is Gly-NH 2 ,
  • Y is Pro or 4-hydroxy-Pro
  • L 1 and L 2 are independently
  • X is C 1 -C 5 alkylene, phenyl or C 5 -C 6
  • R is C 1 -C 3 alkanoyl
  • n 1 or 2;
  • T is a toxin group; provided that the carbonyl ends of each of L 1 and L 2 are bonded to either the GnRH derivative or to the toxin.
  • the toxin can be any toxin that is capable of destroying the LH releasing cells of the
  • the toxins can be plant derived toxins, bacteria derived toxins or chemical toxins.
  • plant derived toxins are ricin, modeccin, abrin, pokeweed antiviral protein, ⁇ -amanatin, gelonin ribosome inhibiting protein, (RIP) or RIP derived from wheat, corn, rye, flax and the like.
  • bacteria derived toxins are diphtheria toxin, Pseudomonas exotoxin, shiga toxin and the like.
  • Examples of chemical toxins are melphalan, metbotrexate, nitrogen mustard, doxorubicin, daunomycin and the like.
  • the preferred toxins are those derived from Pseudomonas exotoxin.
  • the most preferred toxins are those segments of Pseudomonas exotoxin wherein the binding domain has been deleted or partially deleted so that the toxin retains its potential for cell toxicity but that the toxin lacks the ability to bind to animal cells, except when coupled with the GnRH targeting agent.
  • Pseudomonas toxin has had amino acids 1-252 deleted, which comprises 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., supra, Kondo et al J. Biol Chem 263 pg 9470-9475 (1988), Chaudharg et al, DNAS-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 and further providing the point
  • Lys is the amino acid that is bonded to the L 1 or L 2 at least one Lys is preferred to be retained in the Pseudomonas exotoxin peptide fragments.
  • PE-40 has been designated PE-38M and it is shown in Table 1 where the numbers above the peptide sequence refer to the Pseudomonas exotoxin sequence.
  • the various Pseudomonas exotoxin fragments are prepared using the techniques of biotechnology and recombinant DNA. However, once the Pseudomonas exotoxin has been prepared, it is bonded to the linking groups L 1 and L 2 and the D-Lys 6 -GnRH using synthetic organic chemical techniques.
  • Figure 1 is a mass spectrometric analysis of the toxin conjugate of DLs 6 -GnRH coupled to PE38 with f rom 1 to 5 GnRH moieties per toxin .
  • the s ix is a mass spectrometric analysis of the toxin conjugate of DLs 6 -GnRH coupled to PE38 with f rom 1 to 5 GnRH moieties per toxin .
  • Time-of-fli-ht (TOF) technique or a matr ix cons i sted matrix description TOF mass spectrometer is a mass specromative analysis of the PE38 starting material of approximately 38K molecular weight. The two smaller peaks represent doubly or tripled charged molecules.
  • Figures 3, 4 and 5 are SDS-PAGE Gel electrophoreis of the conjugates of PE38 D-Lys 6 GnRH ( Figures 3 and 4) or PE38M and D-Lys 6 -GnRH ( Figure 5)
  • the preparation of the instant toxin conjugates is shown in Reaction Scheme 1 using D-Lys 6 GnRH and PE-38M for exemplification.
  • the GnRH is first modified with one of the L 1 and L 2 linking groups and the modified Pseudomonas exotoxin peptide is modified with the other of the L 1 and L 2 linking groups.
  • the L 1 and L 2 linking groups are attached to all free or unprotected primary amines on the GnRH derivative and the Pseudomonas exotoxin.
  • the linking group is bonded to the amine at the end of the alkyl chain of the Lys. Since the N-terminus of the D-Lys 6 GnRH is a pyroglutamyl, no reaction can occur since no free primary amine is present. In the case of the Pseudomonas exotoxin, reaction can occur at any Lys present.
  • the PE-38M modified protein has only one Lys but other modified Pseudomonas exotoxins may have more than one.
  • the N-terminus of the Pseudomonas exotoxin is a free amine which is available for reaction with L 1 and L 2 .
  • the first step is the reaction of D-Lys 6 -GnRH with L 1 which for purposes of illustration is shown as the maleimidoyl alkanoyl group.
  • L 1 which for purposes of illustration is shown as the maleimidoyl alkanoyl group.
  • the D-Lys 6 -GnRH is jprepared using known peptide synthesis techniques, preferably the solid phase peptide synthesis.
  • the reaction for the preparation of the Li ⁇ D-Lys 6 -GnRH is carried out using an active ester of the maleimidoyl alkanoyl group.
  • 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 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
  • 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 Pseudomonas exotoxin is reacted with the other linking group L 2 at any and all of the unprotected primary amines.
  • the other linking group L 2 for purposes of illustration the
  • PE-38M Pseudomonas exotoxin which has two primary amines available for reaction, the N-terminus amine and the epsilon amine or the Lys, shown as N ⁇ .
  • L 1 -D-Lys 6 GnRH will react with each equivalent of the PE-38M.
  • the reaction is carried out in an aqueous buffer which provides for a pH of greater than 10. Borate buffer solution with a pH of about 11 is preferred. Included in the reaction mixture
  • reaction preferably is dithiothreitol and/or the disodium salt of ethylenediaminetetraacetic acid.
  • these reagents are generally added in considerable excess in order to prevent the reactive mercapto group from forming a disulfide bond with like groups.
  • 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.
  • L 2 -Pseudomonas exotoxin can be conjugated with the L 1 -D-Lys 6 -GnRH, the dithiothrietol, the ethylenediaminetetraacetic acid disodium salt and any
  • 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 L 1
  • 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 6 GnRH L 1 -L 2 -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.
  • the intermediate compounds 2 and 3 are important aspects of the instant invention and represent novel compounds-.
  • the novel intermediates are realized in the following structural formulae: p-Glu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Y-Z
  • X is C 1 -C 5 alkylene , phenyl or C 5 -C 6 cycloalkylene ; R i s C 1 -C 3 alkanoyl ;
  • T is a toxin .
  • the linking groups, L are bonded to the available primary amines of the D-Lys 6 -GnRH and the toxin, preferably Pseudomonas exotoxin A or fragment thereof. Where, as in the Pseudomonas exotoxin, more than one primary amine is available, more than one L group will be reacted therewith.
  • L While either value of L may be bound to either the D-Lys 6 -GnRH or the toxin, it is preferred to bond the maleimido alkanoyl group to the GnRH and the N-alkanoyl homocysteinyl group to the toxin.
  • the most preferred toxin is PE-38M and the most preferred GnRH derivative is D-Lys 6 -rGnRH, thus, the most preferred intermediates of the instant invention are:
  • the instant site-specific toxins which connect a GnRH analog with a toxin through a unique linking group offer significant advantages in the preparation and analysis of the toxin conjugates.
  • the toxin moieties often have more than one amine function which can be coupled to the GnRH analog through various linking groups (See Nett et al).
  • toxin conjugates are usually done through amino acid analysis, however, since the GnRH and toxin all break down to normal amino acids, the determination of the number of GnRH moieties bonded to the toxin is a very long and tedious process.
  • the ratio of lysine to beta-alanine is determined which reveals the extent of the
  • conjugates of the instant invention are particularly effective in causing the toxic compound T to be specifically targeted to the gonadotropin-secreting cells of the anterior pituitary gland. Indeed, they are the only cells to which the gonadotropin-releasing hormone portion of the conjugate will bind. Hence, these toxic compounds, bound to an analog of
  • gonadotropin-releasing hormone can be employed to permanently destroy a subpopulation of the anterior pituitary cells and thereby eliminate the gland's ability to secrete gonado.tropins. This in turn causes the animal's gonads to atrophy and lose their ability to function for reproductive purposes. That is to say that, without functioning gonadotrophs, an animal is not able to secrete luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and thus is rendered sterile.
  • LH luteinizing hormone
  • FSH follicle-stimulating hormone
  • Applicants have postulated that the compounds of this patent disclosure inhibit synthesis of LH, and presumably other proteins made by gonadotrophs, because they tend to inhibit all protein synthesis once these compounds gain entry into a cell. It should also again be noted that applicants' compounds allow "chemical castration" to be employed in place of surgical castration.
  • 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.
  • progestins and estrogens to females and androgens to males might be necessary to prevent loss of secondary sex characteristics, behavior and osteoporosis.
  • dose/time adjustments associated with the use of these compounds can vary considerably; however, these compounds are preferably administered by injection into a mammal in concentrations of from about 0.1 to about 10 milligrams per kilogram of the mammal's body weight. Sterilization may be
  • the compounds of this patent disclosure can be used before or after puberty. They too are especially useful in those areas of animal husbandry where the anabolic benefits of non-surgical sterilization techniques can be used.
  • the compounds of this invention are administered to male cattle between the ages of about 8 weeks and 20 weeks at least once and in a concentration of from about 0.1 to about 10 milligrams per kilogram of the animal's body weight.
  • the toxic moieties T of the herein disclosed compounds are obtainable from both natural and synthetic sources.
  • pokeweed antiviral protein can be isolated from leaves of pokeweed plants and purified by gel filtration chromatography.
  • the peptide was synthesized on Rink amide MBHA resin (0.25 mmol, Amino Tech) by solid phase peptide synthesis (SPPS) using an ABI model 431A synthesizer and single couplings (DCC/HOBT).
  • SPPS solid phase peptide synthesis
  • DCC/HOBT ABI model 431A synthesizer and single couplings
  • 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).
  • 6-D-Lys-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 portion. 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
  • Lys-PE38M (.221 ⁇ mol, 3.0 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 ethylenediaminetetraacetic acid disodium salt (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 por.tion and the solution degassed and purged with N 2 (degas/purge repeated 5X). The mixture was aged in an N 2 box at RT for 6.5 h, then charged to Spectropor 2 dialysis tubing and dialyzed (RT) as follows: vs. 1) 4L degassed,
  • the conjugate was centrifuged to pellet any unsuspended material and passed through a sterile filter (Millipore 0.22 ⁇ m, Millex ® -GV). This provided 4, which had HPLC characteristics that were distinct from unconjugated
  • Plasmid PJH4 (Ref. Hwang. J. Cell (1987, 48; 129-136) contains the coding sequence for PE 1 _ 613 . Oligonucleotide directed mutagenesis as described in 15.51-15.73, Molecular Cloning, 2nd ed (1989) edited by Sambrook, Fritch &. Maniatis (Cold Spring Hafbor 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 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.
  • S1 nuclease treatment is carried out to allow blunt end ligation of the sticky ends and the plasmid is recircularized and designated PJH43. This results in a PE with deletion of
  • a 553 bp Sal I/Bam H1 fragment of plasmid PJH43 is cloned into M13 mp19.
  • An oligonucleotide 50 nucleotides in length with the structure 5' GGC GTC GCC GCT GTC CGC CGG G.CC 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
  • a 505 bp Sal I Bam H1 fragment is excised from the replicative form of the mutant DNA in M13 and ligated with a 3.7 Kb Sal 1 Bam H1 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 mpl9. 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
  • 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 H1 EcoR 1 fragment is excised from the replicative form of the mutant DNA in M13 and ligated with a 3.4 Kb Bam.H1/EcoR 1 fragment of the plasmid PJH44.
  • the linearized plasmid is then recircularized, designated PJH45 and used for
  • the peptide is synthesized on Rink amide MBHA resin (0.25 mmol, Amino Tech) by solid phase peptide synthesis (Fmoc chemistry) using an ABI model 431A 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.
  • 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 431A 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
  • the peptide is synthesized on Oxime or Merrifield resin by solid phase peptide synthesis (Boc chemistry) using an ABI model 431A 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
  • 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.
  • 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 431A 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-0rn-GnRH (positive ion, NBA matrix) Calc (m+1) 1239.4; Found (m+1) 1239.5.
  • 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 431A 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.
  • the peptide is synthesized on Oxime or
  • 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. SEQUENCE LISTING

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  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Endocrinology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
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  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
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Abstract

Des analogues de GnRH sont fonctionnalisés avec des groupes de liaison uniques pour qu'ils puissent se coupler à une molécule destructrice de cellules. Cette toxine chimérique comprend un analogue de GnRH, un groupe de liaison et un constituant toxine qui sont administrés à des animaux mâles et femelles dans l'organisme desquels la toxine chimérique est transportée vers les organes contenant des cellules avec récepteur de GnRH, telles que les glandes pituitaires, afin de réduire les sécretions de stéroïdes sexuels, ce qui a pour conséquence d'entraîner la stérilité ou la réduction des tumeurs nécessitant des stéroïdes sexuels pour croître. Les composés décrits ici sont utilisés comme agents de stérilisation et comme agents antitumoraux. Des dérivés de GnRH modifiés au moyen des groupes de liaison décrits ici présentent un avantage par rapport aux chimères de la technique actuelle, préparées par conjugaison, en ce que, lors de l'analyse des acides aminés du conjugué, le dérivé de GnRH modifié libère un acide aminé non naturel qui est facilement quantifié, révélant ainsi le degré de conjugaison entre l'analogue de GnRH et la toxine.
PCT/US1993/001263 1992-02-14 1993-02-12 TOXINES CHIMERIQUES SE FIXANT AU RECEPTEUR DE GnRH WO1993015751A1 (fr)

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US836,031 1992-02-14
US918693A 1993-01-26 1993-01-26
US009,186 1993-01-26

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997015316A1 (fr) * 1995-10-27 1997-05-01 Merck & Co., Inc. Conjugues de gonadoliberine
WO1997015317A1 (fr) * 1995-10-27 1997-05-01 Merck & Co., Inc. CONJUGUES GnRH/EXOTOXINE DE PSEUDOMONAS REDUITE
WO1997046259A2 (fr) * 1996-06-04 1997-12-11 Yissum Research Development Company Of The Hebrew University Of Jerusalem Toxines chimeres pour therapie ciblee
WO1998051349A1 (fr) * 1997-05-09 1998-11-19 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Conjugue renfermant un antagoniste de l'acide folique et un excipient
EP0975354A1 (fr) * 1997-03-27 2000-02-02 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Compositions de ligands/peptides lytiques et leurs procedes d'utilisation
US6635740B1 (en) 1997-03-27 2003-10-21 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Ligand/lytic peptide compositions and methods of use
US6680058B1 (en) 1997-09-03 2004-01-20 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Compositions and methods for contraception in or sterilization of mammals
EP1610808A2 (fr) * 2003-03-26 2006-01-04 University of Texas Medical School Fixation par liaison covalente de ligands a des proteines nucleophiles dirigees par une liaison non covalente
EP1878438A2 (fr) * 1997-03-27 2008-01-16 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Compositions de peptide ligand/lytique et procédés d'utilisation
US7704506B2 (en) 1995-12-18 2010-04-27 Yissum Research Development Company Of The Hebrew University Of Jerusalem Fcε-PE chimeric protein for targeted treatment of allergy responses a method for its production and pharmaceutical compositions containing the same
US10111966B2 (en) 2016-06-17 2018-10-30 Magenta Therapeutics, Inc. Methods for the depletion of CD117+ cells
CN109248324A (zh) * 2018-10-16 2019-01-22 承德医学院 GnRH类似物－抗肿瘤药物偶联物、其制备方法及用途
US10434185B2 (en) 2017-01-20 2019-10-08 Magenta Therapeutics, Inc. Compositions and methods for the depletion of CD137+ cells
WO2020220085A1 (fr) * 2019-05-02 2020-11-05 The University Of Sydney Dérivés peptidiques et leurs conjugués pour le traitement du cancer

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4855285A (en) * 1985-12-04 1989-08-08 The Ohio State University Research Foundation Antigenic modification of polypeptides
GB2228262A (en) * 1989-01-25 1990-08-22 Nat Inst Immunology Antigenic derivative of GnRH
WO1990009799A1 (fr) * 1989-02-23 1990-09-07 Colorado State University Research Foundation ANALOGUES DE GnRH DETRUISANT LES GONADOTROPES

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4855285A (en) * 1985-12-04 1989-08-08 The Ohio State University Research Foundation Antigenic modification of polypeptides
GB2228262A (en) * 1989-01-25 1990-08-22 Nat Inst Immunology Antigenic derivative of GnRH
WO1990009799A1 (fr) * 1989-02-23 1990-09-07 Colorado State University Research Foundation ANALOGUES DE GnRH DETRUISANT LES GONADOTROPES

Cited By (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997015317A1 (fr) * 1995-10-27 1997-05-01 Merck & Co., Inc. CONJUGUES GnRH/EXOTOXINE DE PSEUDOMONAS REDUITE
WO1997015316A1 (fr) * 1995-10-27 1997-05-01 Merck & Co., Inc. Conjugues de gonadoliberine
US7740853B2 (en) 1995-12-18 2010-06-22 Yissum Research Development Company Of The Hebrew University Of Jerusalem Fcepsilon-pe chimeric protein for targeted treatment of allergy responses a method for its production and pharmaceutical compositions containing the same
US7704506B2 (en) 1995-12-18 2010-04-27 Yissum Research Development Company Of The Hebrew University Of Jerusalem Fcε-PE chimeric protein for targeted treatment of allergy responses a method for its production and pharmaceutical compositions containing the same
US6933271B2 (en) 1996-06-04 2005-08-23 Yissum Research Development Company Of The Hebrew University Of Jerusalem Chimeric toxins for targeted therapy
WO1997046259A2 (fr) * 1996-06-04 1997-12-11 Yissum Research Development Company Of The Hebrew University Of Jerusalem Toxines chimeres pour therapie ciblee
WO1997046259A3 (fr) * 1996-06-04 1998-03-12 Yissum Res Dev Co Toxines chimeres pour therapie ciblee
EP0975354A1 (fr) * 1997-03-27 2000-02-02 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Compositions de ligands/peptides lytiques et leurs procedes d'utilisation
EP1878438A2 (fr) * 1997-03-27 2008-01-16 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Compositions de peptide ligand/lytique et procédés d'utilisation
EP0975354A4 (fr) * 1997-03-27 2003-07-16 Univ Louisiana State Compositions de ligands/peptides lytiques et leurs procedes d'utilisation
US6635740B1 (en) 1997-03-27 2003-10-21 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Ligand/lytic peptide compositions and methods of use
US7566777B2 (en) 1997-03-27 2009-07-28 Board Of Supervisors Of Louisana State University And Agricultural And Mechanical College Genes encoding hormone and lytic peptides
EP0988048A4 (fr) * 1997-03-27 2003-05-28 Demeter Biotech Ltd Compositions de peptides lytiques/ligands et leurs procedes d'utilisation
EP0988048A1 (fr) * 1997-03-27 2000-03-29 Demeter Biotechnologies, Ltd. Compositions de peptides lytiques/ligands et leurs procedes d'utilisation
EP1878438A3 (fr) * 1997-03-27 2008-05-21 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Compositions de peptide ligand/lytique et procédés d'utilisation
US6720304B1 (en) 1997-05-09 2004-04-13 Deutsches Krebsforschungszentrum Stiftung Des Offentlichen Rechts Conjugate comprising a folic acid antagonist and a carrier
EP0879604A1 (fr) * 1997-05-09 1998-11-25 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Conjugué comprenant un antagoniste de l'acide folique et un porteur
WO1998051349A1 (fr) * 1997-05-09 1998-11-19 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Conjugue renfermant un antagoniste de l'acide folique et un excipient
US6680058B1 (en) 1997-09-03 2004-01-20 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Compositions and methods for contraception in or sterilization of mammals
EP1610808A2 (fr) * 2003-03-26 2006-01-04 University of Texas Medical School Fixation par liaison covalente de ligands a des proteines nucleophiles dirigees par une liaison non covalente
EP1610808A4 (fr) * 2003-03-26 2011-04-06 Sudhir Paul Fixation par liaison covalente de ligands a des proteines nucleophiles dirigees par une liaison non covalente
US10111966B2 (en) 2016-06-17 2018-10-30 Magenta Therapeutics, Inc. Methods for the depletion of CD117+ cells
US10434185B2 (en) 2017-01-20 2019-10-08 Magenta Therapeutics, Inc. Compositions and methods for the depletion of CD137+ cells
US10576161B2 (en) 2017-01-20 2020-03-03 Magenta Therapeutics, Inc. Compositions and methods for the depletion of CD137+ cells
CN109248324A (zh) * 2018-10-16 2019-01-22 承德医学院 GnRH类似物－抗肿瘤药物偶联物、其制备方法及用途
CN109248324B (zh) * 2018-10-16 2021-11-30 承德医学院 GnRH类似物－抗肿瘤药物偶联物、其制备方法及用途
WO2020220085A1 (fr) * 2019-05-02 2020-11-05 The University Of Sydney Dérivés peptidiques et leurs conjugués pour le traitement du cancer

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