GB2282812A - Cytotoxic/receptor ligand conjugates linked via lysine radicals - Google Patents

Abstract

Cytotoxic receptor ligand scaffolds, CRLS, are prepared by convergent synthesis and coupling of soluble precursors which may be modified by addition of solubilizing groups and hydrophilic groups enhancing the efficiency of coupling. Linear and cyclic peptide receptor ligands are conveniently incorporated, as are non-peptidyl ligands. The receptor ligand scaffold are represented by the general formula: (Lys)n(Unh)m(Sol)m-(X)n-Z, wherein: Lys represents lysine; Unh represents sterically unhindered groups; Sol represents hydrophilic, solubilizing groups; n is 3 to 10; m is 0 to 5; X represents a receptor ligand; and Z is a cytotoxin.g

Description

TITLE OF THE INVENTION CYTOTOXIC RECEPTOR LIGAND SCAFFOLDS BACKGROUND OF THE INVENTION This patent disclosure relates to scaffolds of receptor ligands linked to cytotoxic agents. In one embodiment of the invention the receptor ligand is a gonadotropin releasing hormone, GnRH, or an analog thereof, and the toxin is pseudomonas exotoxin. This embodiment of the invention is an effective chemosterilant and it may also be used to arrest development of steroid hormone stimulated tumors.

In the art of receptor ligand binding, it has become accepted practice to produce chimeric molecules of a receptor binding ligand and a toxin. In this manner, the chimeric molecules are targeted to specific receptor bearing cells. Upon endocytosis of the receptor bound, chimeric toxin-liland complex. the toxin functionality kills the cell. In this fashion, physiological actions mediated through cells bearing the targeted receptor are down regulated or abolished.

To this end, WO 93/113777 disclosed chimeric molecules of GnRH or analogs thereof and cytotoxins. The chimera of that disclosure was a molecule in which GnRH peptides were directly linked to a pseudomonas exotoxin molecule. At each site of peptide binding to the toxin molecule, there was only one GnRH peptide bound.

Administration of such chimeric molecules was reported to result in the destruction of GnRH receptor bearing cells in the pituitary gland, with concomitant reduction in the secretion of sex hormones. The ultimate result of this process is chemosterilization and reduction of steroid stimulated tumor proliferation. Similar compounds and concepts are embodied in WO 92/22322 and WO 90/09799.

Considerable interest exists with respect to the subject of sterilization of animals. This is especially true of those concerned with veterinary medicine and animal husbandry, particularly as they relate to the subject of sterilization of domestic animals such as dogs, cats, cattle, sheep, horses, pigs, and the like. Various methods have been developed over the years to accomplish sterilization. For example, with respect to male cattle, the most widely used procedure for eliminating problems of sexual or aggressive behavior is sterilization through surgical castration.

This is done in various ways, e.g., crushing the spermatic cord, retaining the testes in the inguinal ring, or use of a rubber band, placed around the neck of the scrotum, to cause sloughing off of the scrotum and testes. However, most of these "mechanical" castration methods have proven to be undesirable in one respect or another; for example they (1) are traumatic, (2) introduce the danger of anesthesia, (3) are apt to produce infection. and (4) require trained personnel. Moreover, all such mechanical castration methods result in complete abolition of the testes and this of course implies complete removal of the anabolic effects of any steroids which are produced by the testes and which act as stimuli to growth and protein deposition.

These drawbacks have caused consideration of various alternative sterilization techniques such as the use of chemical sterilization agents. However, the use of chemical sterilization agents has its own set of advantages and disadvantages. On the positive side, chemical sterilization eliminates the stress and danger associated with mechanical castration. Chemical sterilization also has the added advantage of allowing for retention of certain anabolic effects resulting from a continued presence of low levels of circulating testosterone.

This is especially valuable in the case of animals raised for human consumption since circulating testosterone promotes growth, efficiency of feed conversion and protein deposition. Unfortunately, there are several disadvantages associated with chemical sterilization. For example chemical sterilization is often temporary rather than permanent; it also sometimes produces extremely severe, and even fatal.

side effects.

In WO 9009799 to Nett certain GnRH analogs are coupled to a variety of toxins through an optional linking group consisting of 2iminothio- lane, SPDP (N-succinimi dyl-3-(2-pyri dy ldithio/ propionate) bis-diazabenzidine and glutaraldehyde. The compounds are disclosed as sterilizing agents.

Myers et al Biochemical Journal 227:1 pg 343 (1985) discloses a conjugate of a GnRH analog and the diphtheria A chain and pokeweed antiviral toxins coupled through SPDP.

Various constructs of bacterial or plant toxins have been prepared in attempts to specifically target the toxin to certain cells or organs.

U.S. patent 4.545985 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.

U.S. patent 5*036*047 teaches that free LHRH (alternate nomenclature for GnRH) can be administered along with an immunogenic conjugate of LHRH and a specific nonapeptide or decapeptide. The administration is reported to induce reversible sterilization.

U.S. patent 4.664.911 teaches that 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. Patent 4,675.3X' 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 (1986) teach that a hybrid fusion protein produced in bacteria using recombinant DNA technology and consisting of a portion of the diphtheria toxin protein joined to alpha-melanocyte-stimulating hormone will bind to and kill human melanoma cells.

Allured et al., PNAS USA Sss:1320-1324 (1986) teach the three dimensional structure of the Pseudomonas exotoxin A protein.

Hxan(T et al., Cell 48:129-136 (1987) teach that the Pseudomonas exotoxin A protein can be divided into three distinct functional domains responsible for: binding to mammalian cells, translocating the toxin protein across lysosomal membranes, and ADP ribosylating elongation factor 2 inside mammalian cells. This article further teaches that these functional domains correspond to distinct regions of the Pseudomonas exotoxin A protein.

Chaudhary. et al., PNAS USA 84:4538-4543 (1987) teach that hybrid fusion proteins formed between PE-40 and transforming growth factor-alpha and produced in bacteria using recombinant DNA techniques will bind to and kill human tumor cells possessing epidermal growth factor receptors.

European patent application Q -'kl 671 published 30 March 1988* teaches that a portion of the Pseudomonas exotoxin A protein can be produced which lacks the cellular binding function of the whole Pseudomonas exotoxin A protein but possesses the trans locating and ADP ribosylating functions of the whole Pseudomonas exotoxin A protein. The portion of the Pseudomonas exotoxin A protein that retains the trans locating and ADP ribosylating functions of the whole Pseudomonas exotoxin A protein is called seudomonas 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.

Kellev et al., PNAS USA 85:3980-3984 (1988) teach that a hybrid fusion protein produced in bacteria using recombinant DNA technology and consisting of a portion of the diphtheria toxin protein joined to interleukin 2 functions in mice to suppress cell mediated immunity.

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.

Edwards, et al., Mol. Cell. Biol. 9: 2860-2867 (1989) describe the preparation of the modified TGF-alpha - PE40 hybrid molecules that have been found to have utility in treating bladder tumor cells.

Heimbrook, et al., Proc. Natl. Acad. Sci. USA ras7: 46974701 (1990) describe the in vivo efficacy of modified TGF-alpha PE40 in significantly prolonging the survival of mice containing human tumor cell xenografts.

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.

"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. However, a problem encountered in the past has been that upon conjugation to a toxin, the targeting agent loses a great deal of its binding affinity, and hence much of its specificity. The danger of this is that toxins are more likely to be directed to unwanted targets when the ligand has reduced specificity.

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 - toy in" molecules (U.S. Patent 4545,985).

Pseudomonas exotoxin A intoxicates mammalian cells by first binding to the cell's surface, then entering the cell cytoplasm and inactivating elongation factor 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. However one problem with these hybrid molecules is that they 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. Therefore, 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 - An or PEqo (H wang et al..

Cell 4 9-136 1987). The use of PE-40 helps reduce the non specificty of ligand-toxin conjugates. However. simply using PE-40 does not alleviate the problem of reduced ligand binding affinity.

A further limitation of the known receptor ligand-toxin chimeras is that the GnRH peptides were directly linked to a pseudomonas exotoxin molecule. Therefore, at each site of peptide binding to the toxin molecule. there was only one GnRH peptide bound.

However, ligand binding affinity and internalization by GnRH receptor bearing cells using such chimera has been found to be suboptimal.

In the art of vaccine production it has become common to produce conjugates of poorly immunogenic epitopes and strongly immunogenic carriers for the purpose of enhancing the immunogenicity of the poorly immunogenic epitopes. For a recent review of conjugate vaccines, see Dintzis, R. Z., Pediatric Res., 32:376-356 (1992). Thus, capsular bacterial polysaccharides are conjugated to proteins, see for example US Patent 4,695,624, and peptidyl epitopes have likewise been conjugated, see for example, EP 0 467 700.

One disadvantage in these techniques is that a large percentage of the mass of such conjugates is represented by the carrier which, while enhancing the immunogenicity of the polysaccharide or peptidyl epitope to which it is conjugated, generally does not itself generate an immune response protective against the pathogen of concern.

One solution to this problem is the multiple antigen peptide.

or MAP, advanced by James Tam [Vaccines 89, Cold Spring Harbor, p21-25; Tam, J.P., P.N.A.S., 85:5409 (1988); see also Prosnett, et al., J.

Biol. Chem., 263:1719 (1988)]. The prototype MAP described by Tam consisted of a small peptidyl core matrix bearing seven radially branching Iysl residues to which eight synthetic peptide antigens were attached to form an outer surface layer of acetylated synthetic peptides.

According to that prototype both the peptidyl core and the outer layer of peptides were synthesized as a single operation on a polymeric support by conventional step-wise, solid-phase (Merrifield) methods.

Tam reported immune responses to fourteen different MAP constructs in rabbits and mice, and in eleven out of the fourteen constructs, the antibodies were specific for the native proteins from which the peptide epitopes were derived. While appearing to be effective, this method was limited in that desirable cyclic peptides could not be incorporated into the MAP.

Since Tam's description of the MAP, several other workers have reported on the use of the MAP concept. Thus, EP 0 339 695 [published 11/2/89; see also Drijfhout, J. W., and Bloemhoff, W., Int. J.

Peptide Protein Res., 37:27-32, (1991)], describes the separate preparation of the lysine core, the peptide antigens, and their subsequent coupling through disulfide bond formation. In addition, the incorporation of a solubilizing group. such as glutamic acid, between the lysine core and the antigenic peptide, was also described. This work added some flexibility and solubility to the MAP concept, but was limited in that coupling of peptidyl antigens through disulfides results in the loss of antigen upon oxidation, resulting in diminished epitope presentation. Furthermore, the coupling efficiency of this system is subject to steric hindrance such that cyclic peptides are difficult to couple.

The concept of the MAP immunogen was extended to the development of anti-HIV-l antigens by Nardelli, et al., [J. Immunoi., 148:914-920 (1992); see also European Patent Application 89301288.0].

In that work. synthetic linear peptides from the V3 region of the gp 120 of the IIIB, RF and MN isolates of HIV-I were used as the peptidyl antigens. Isolate specific antibody responses were raised by these immunogens in rabbits and mice. In EP A 0 328,403 dendritic multiple antigen peptides using linear HIV peptides were prepared.

In WO 9222583. a concept similar to the MAP was used to produce tri- and tetra-valent mono-specific antigen-binding proteins consisting of 3 or 4 Fab fragments bound to a connecting structure for treating cancer.

In WO 9218528 (published Oct. 29. 1992). a branched peptide was described wherein a lysine core was linked to peptide epitopes with the interposition of a spacer comprising 1-20 glycine residues. In PCT/US90/02039, a MAP in which malaria antigens were bound to a core molecule was described.

In W093/03766 (published 4 March 1993), a MAP including a peptide from the V3 loop of gpl20. from cysteine 303 to cysteine 338 of HIV-l, was disclosed. The only type of circularized peptide envisioned, however. were disulfide bonded peptides (see W093/03766, page 9, lines 29-33). Such peptides are notoriously labile, and in the physiological mileu. are likely to spend a large percentage of time in a linear conformation.

While the MAP concept has been applied to linkage of linear antigens or disulfide-bonded cycles it has not been applied to stably cyclic antigens. According to Satterthwait, A.C. et al. [Phil.

Trans. R. Soc. Lond. B323:565-572 (1989); Bulletin of the World Health Organization, 68 (Suppl.):17-25 (1990)], small conformationally constrained cyclic peptides were reported to be useful anti-malaria immunogens. Lacroix, et al. [V Conf. Int. SIDA, Ed. Morrisset R, Abstract W.B.P.l 10, Montreal (June 1989)], compared the performance of cyclic and linear peptides covering an immunodominant region of HIV-I and HIV-2 transmembrane glycoproteins in an ELISA. They concluded that the cyclic peptides were superior antigens than the linear counterparts, and that they can form the basis of highly sensitive and specific tests for HIV-antibody detection.

The instant inventors have made the unexpected discovery that in at least certain contexts. chimera in which GnRH peptides are directly linked to a pseudomonas exotoxin molecule are inadequate to achieve efficient endocytosis and cell destruction of receptor bearing cells. The instant invention now provides a scaffold for receptor ligands which overcomes this problem and which improves on the MAP concept used to produce vaccines and which induces efficient chimeric ligand/toxin-receptor complex endocytosis and cellular destruction. A further unexpected discovery is that the binding affinity of the conjugated targeting agent may be preserved at close to the affinity of the free ligand when conjugated onto the receptor ligand scaffold of the instant invention.

SUMMARY OF THE INVENTION Cytotoxic receptor ligand scaffolds (CRLS) are prepared by convergent synthesis and coupling of soluble precursors which may be modified by addition of solubilizing groups and groups enhancing the efficiency of coupling. Linear and cyclic peptide receptor ligands and cytotoxic moieties are conveniently incorporated, as are non-peptidyl ligands. The receptor ligand scaffold may be represented by the general formula: (Lys)a(Unh)b(Sol)b-(X)a-Z wherein: Lys represents lysine; Unh represents sterically unhindered groups; Sol represents hydrophilic, solubilizing groups; a is 3 to 10; b is 0 to 5; X represents a receptor ligand; and Z is a cytotoxin.

In a preferred embodiment of the invention, the CRLS has the structure:

wherein: S is a sulfur atom, forming a thioether bond: X is a receptor ligand with each X being linked through the oc- or amino group of lysine, with each X being the same or different: Y is a linker; Z is a cytotoxic moiety linked to the ligand scaffold; p is 0 to 5; mis 3 to 10; and n is 1 to 10.

Where X is a gonadotropin releasing hormone (GnRH. also known as luteinizing hormone releaesing hormone or LHRH), or an analog thereof, the scaffold binds with high affinity to pituitary cells bearing GnRH receptors. When Z is pseudomonas exotoxin, internalization of the scaffold is efficient and results in destruction of the GnRH receptor bearing cells, causing reduction or elimination of steroid hormone secretion. Consequently, this embodiment of the invention is an effective chemosterilant and is also useful to arrest proliferation of steroid hormone stimulated tumor growth.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1: A binding curve showing displacement of 125I labeled buserelin from pituitary membranes by unlabeled buserelin, 6-D-Lys-GnRH, 6-D Lys-GnRH chimera of the prior art (WO 93/15751), of Lys-PE38M, and of natural GnRH (-[logIC50] = 9, S.S, 6.9, none, and 7.8 respectively).

Fig. 2: A binding curve showing diplacement of 125I labeled buserelin from pituitary membranes by unlabeled buserelin, 6-D Cys-GnRH-CRLS of this invention with conjugated toxin and 6-D-Cys GnRH receptor ligand scaffold before conjugation with toxin ( [lo1C501 = 9.1.8.15 and 8.25 respectively).

Fig.3: An SDS-PAGE analysis of toxin and CRLS with conjugated 6-D Cys-GnRIl: LANE SAMPLE Blank 2 BIO-RAD Molecular Weight Standards 3 CRLS-6-D-Cys-GnRH, 3 g 4 Unconjugated Toxin, Mr ~ 33,000, 3 g 5 Blank 6 BIO-RAD Molecular Weight Standards 7 Unrelated protein size marker, Mr = 50,000 8 CRLS-6-D-Cys-GnRH 3 ltg 9 BIO-RAD Molecular Weight Standards 10 CRLS-6-D-Cys-GnRH, 3 g 11 Unconjugated Toxin.Mr ~ 33,000, 3 g 12 BlO-RAD Molecular Weight Standards DETAILED DESCRIPTiON OF THE INVENTION Cytotoxic receptor ligand scaffolds, hereinafter also referred to as CRLS, of this invention are made by: a) Synthesizing a multi-lysine core optionally incorporating solubilizing or sterically unhindered groups and purifying this component; b) Synthesizing a receptor ligand optionally incorporating a terminal group of low steric hindrance and purifying this component: and c) Coupling the purified core component of step (a) with the purified receptor ligand of step (b). and purifying the receptor ligand scaffold.

RLS thus formed: and d) Coupling the RLS of step (c) with a cytotoxic functionality to form a cytotoxic receptor ligand scaffold. CRLS.

The order of the foregoing steps need not be precisely as described above. For example. the multi-lysine core may be linked to the cytotoxic functionality before coupling the receptor ligands.

The multi-lysine core may consist of a matrix of a few or many lysines. Practically. however. a tri- or tetralysine core is generally sufficient. easily and reproducibly synthesized. and capable of being conjugated to four receptor ligand molecules. The lysine core is prepared by standard solid phase peptide synthetic methods. For example. a resin functionalized with a 13-Ala is reacted with the free carboxy group of lysine. To this group. two more Lines may be reacted. one with the alpha amino group and the other with the epsilon amino. The result is a trilysine, dendritic structure with four amino groups available for linkage to receptor ligands.This embodiment extends the MAP vaccine concepts of Tam et al. (see background of the invention above) to the production of a CRLS.

In a preferred embodiment of this invention a linear lysine oligomer is cyclized to form an annular structure. According to this embodiment. several lines are linked through standard solid phase chemistry and peptide bonds (using either the alpha or epsilon amino group of each lysine to extend the chain) to form, for example, a tetralysine oligopeptide. At the carboxy terminus, a cysteine is added to provide a sulkydryl for ring closure. The amino terminus is bromoacetylated. Once the oligopeptide is liberated from the solid phase synthesis resin, the peptide is cyclized to form a circular peptide or annulus through a thioether linkage.

Steric hindrance may be reduced by coupling a small, nonsteric group to the free amino group of each of the lysines. Thus, for example, each lysine may be acylated to a glycine. In this way, once the annulus is fully loaded with receptor ligands, the ligands will still be able to fully interact with the target receptor. Thus, groups of low steric hindrance, such as glycine or a similar compound of formula: NH2-(CH2)x-COOH.

wherein x is 1-10, and is preferably glycine (x = 1), are appended on the peptide at the site of conjugation.

We have discovered that it is desirable to purify the lysine core before reacting the core with receptor ligands. Starting with a purified core eliminates incompletely synthesized scaffold cores. To enhance solubility, it is advantageous to add hydrophylic groups. such as senne. onto the nascent lysine core, for example. between each lysine and the sterically unhindered group such as glycine, before it is cleaved from the resin. It is important not to utilize groups having functionalities which potentially can react with free amino groups (such as carboxyllic acid groups on glutamic or aspartic acid). While many such solubilizing groups may be added, a point is reached where such groups net little increase in the solubility of the core while adding mass to the core.In our experience, addition of one to five serines, is sufficient to render CRLS soluble even when hydrophobic receptor ligands are linked to the receptor ligand scaffold. Naturally, the more hydrophobic the ligands, the more solubilizing groups will be needed.

Thus, in a preferred embodiment of this invention the CRLS core has the general formula: (Lys)a(Unh)b(Sol)b-(X)a-Z wherein: Lys represents lysine: Unh represents sterically unhindered groups: Sol represents hydrophilic solubilizing groups: X represents a receptor ligand: Z is a cytotoxin; bisOtoS: and a is 3 to 10.

In a branched, or dendritic form, the RLS core has the structure, (showing four free amino groups):

Onto each of the four free amino groups from one to five solubilizing groups such as serines may be added before cleaving the core from the resin. In addition unhindered groups, such as glycine or a lower alkyl of one to four carbons, with a terminal carboxyl or amino functionality, may be added. The cytotoxin is acylated onto the free carboxy terminus of this dendritic RLS.

In a preferred embodiment of the invention, the RLS core has the structure:

wherein: S is a sulfur atom, forming a thioether bond; X is a receptor ligand with each X being linked through the or- or amino group of lysine. with each X being the same or different; Y is a linker; Z is a cytotoxic moiety linked to the ligand scaffold: p is 0 to 5; m is 7 to 10: and n is 1 to 10.

This annular structure is preferred because it provides a very compact and easily synthesized core for the scaffold which. once the receptor ligand has been linked exhibits the ligands surrounding the annuls thus essentially obscuring the presence of the core as completely as possible.

Once the core has been synthesized as described above, it is purified according to standard peptide purification procedures known in the art. A particularly preferred method is to purify the core by chromatographic methods including reverse-phase high performance liquid chromatography (RPHPLC), or by affinity chromatography using the receptor ligand specific affinity media such as an anti-GnRH antibody column, as shown in the examples below.

Any of a number of different receptor ligands may be linked to the RLS core. For example, ligands specific to the alpha. beta.

or another subtype of the adrenergic receptor family could be used.

Receptor ligands for the angiotensin II receptor could be used. Any of the ligands described in the background section, such as the melanotropin stimulating hormone, epidermal growth factor, interleukins, tissue growth factors and antibodies, could be used as the ligands according to this invention.

Linkage of receptor ligands to the receptor ligand scaffold core may be accomplished in a variety of different ways. In one method. the free amino groups of the core are bromoacylated, or maleimidated. The core may then be reacted with receptor ligands bearing free sulfhydryl groups, as in 6-D-Cys-GnRH, with receptor ligands which have been thiolated, for example by provision of a cysteine anywhere in the ligand molecule that does not interfere with receptor binding affinity, or reaction with a reagent such as Nacetyhomocycteine thiolactone to thiolate free amines on the receptor ligand. Alternatively free amino groups of the core may be thiolated, for example by reaction with N-acety homocysteine thiolactone. and the receptor ligands may be bromoacylated or maleimidated.Essentially anv method where nucleophillic and electrophillic groups are provided on the reacting partners is sufficient to achieve linkage of receptor ligands to the cytotoxic receptor ligand scaffold. As a result of these diverse linking strategies linkages including but not restricted to the following are produced: (a) Direct coupling through an amide linkage to yield peptide bonds, as in coupling of active esters with free amines.

(b) Coupling through a linker as in the use of a reagent such as succinic anhydride to produce a linker of formula -NHCO(CH2)nCO-, wherein this "n" may between one and five.

(c) Coupling through a linker, as in the use of a bromoacylating agent and a thiol linkage, to produce, for example, a linker such as -NH CO(CH2)n-S- between the receptor ligand and the CRLS. As noted above however, any of a variety of linkages may be used to produce the conjugate CRLS of this invention and this discussion is meant to be mearly illustrative, as are the examples which follow.

The toxin may be any of the known toxins such as diphtheria toxin, pokeweed toxin, resin pseudomonas exotoxin, and preferably, PE-40, or a variant thereof, such as PE38m, see below..

As a particular example of how to make and use the CRLS of this invention, gonadotropin releasing hormone receptor ligands are described and their activity within the CRLS context is demonstrated.

As described in the background section of this patent disclosure, a number of publications have disclosed attempts to provide GnRH chimeric toxins for the purpose of destroying GnRH receptor expressing cells in the pituitary. The disclosure of WO 93/11 3777, which disclosed chimeric molecules of GnRH or analogs thereof and cytotoxins in which GnRH receptor binding ligands were directly linked to a toxin functionality, and of WO 92/22322 and WO 90/09799. are hereby incoroporated by rereference for what they teach in regard to GnRH and its analogs.

Naturally occurring GnRH has the formula: Pyr-His-Trp-Ser-Tyr-Gly-Leu-Ar-Pro-Gly-NHn, [SEQ ID:I:] (Pyr is pyroglutamic acid). a decapeptide (Mr) which stimulates the synthesis and secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH) by the anterior pituitary. LH and FSH stimulate the production of sex hormones by the gonads. This compound. and analogs thereof in which linkage to the CRLS is enabled by inclusion of easily derivatized amino acids at the naturally occurring Gly at the six position may be used in the CRLS of this invention.

The GnRH ligand may be defined with reference to the following structure [Seq. lD:2:]:

wherein: Q is PyroGlu-His-Trp. N-acetyl-4-Cl-Phe12-Trp or 3indolylpropionyl; CRLS is the cytotoxic receptor ligand scaffold to which the GnRH peptide is linked; W is a D or L amino acid with a pendant linking functionality such as cysteine or lysine; X is Leu or Nle: Y is Pro or 4-hydroxy-Pro: and V is Gly, NH2, D-Ala-NH2. NH-Et. NH-Pr or Arg-Gly-NH2.

L1 and L2 are portions of the linker as described above.

Preferred compounds of the instant invention are realized in the following structure [Seq. ID:3:] PyroGlu-His-Trp-Ser-Tyr-W-Leu-Arg-Y-V 1 2 3 4 5 |6 7 8 9 10 L1 L2 RLS where the decapeptide is GnRH with the normal 6-position amino acid (W=Gly) deleted and replaced by W=D-Lys. D-Om or D-Cys.

D-Lvs6-GnRH or D-Cys6-GnRH are preferred targeting agents with which to bind the CRLS to the LH releasing pituitary cells.

It will be recognized that variations of these GnRH analogs that bind to the GnRH receptor of the pituitary will be useful in this invention. All that is required is that the 6-position amino acid possess a group for binding to the linking group and that the remainder of the peptide bind to the GnRH receptor on the pituitary gland cells.

L1 and L2 may include any of the linkages described above. In addition L1 and L may comprise, independently. the groups

wherein: X is Cl-Cs alkylene, phenyl or C5-C6 cycloalkylene; R is C1-C3 alkanoyl; and n is 1 or 2.

It will be appreciated by those skilled in the art that 4 hydroxy-Pro can exist as D and L isomers and as cis and trans isomers.

All such isomers and the racemic mixture of the D and L isomers are intended to be included in this invention.

The cytotoxin can be any toxin that is capable of destroying the LH releasing cells of the pituitary referred to as gonadotrophs. The toxins can be plant derived toxins bacteria derived toxins or chemical toxins. Examples of plant derived toxins are ricin, modeccin, abrin, pokeweed antiviral protein, a-amanatin, gelonin ribosome inhibiting protein, (RIP) or RIP derived from wheat. corn, rye, flax and the like.

Examples of bacteria derived toxins are diphtheria toxin, Pseudomonas exotoxin, shiga toxin and the like. Examples of chemical toxins are melphalan, methotrexate, 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.

One example of such a 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 Twang 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 mutations of deleting one or more of the Lys amino acids. At least one Lys is preferred to be retained in the Pseudomonas exotoxin peptide fragment which can be bromoacetylated.

One such modified PE-40 has been designated PE-3AM which is shown below 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 RLS at the position Z by providing a free amino and/or on the core's carboxy terminus to which a bromoacetylated exotoxin is acylated (See Ex.).

The amino acid sequence of PE-38M is (SEQ ID:4:]: 253 MetLeuGlnGlyThrLysLeuMetAlaGluGluGlyGlySerLeuAlaAla LeuThrAlaHisGlnAlaCysHisLeuProLeuGluThrPheThrArgHis ArgGlnProArgGlyTrpGluGlnLeuGluGlnCysGlyTyrProValGln ArgGlnProArgGlyTrpGluGlnLeuGluGlnCysGlyTyrProValGln ArgLeuValAlaLeuTyrLeuAlaAlaArgLeuSerTrpAsnGlnValAsp GlnValIleArgAsnAlaLeuAlaSerProGlySerGlyGlyAspLeuGly GluAlaIleArgGluGlnProGluGlnAlaArgLeuAlaLeuThrLeuAla AlaAlaGluSerGluArgPheValArgGlnGlyThrGlyAsnAspGluAla GlyAlaALaAsnGlyProAlaAspSerGLyAspAlaLeuLeuGluArgAsn TyrProThrGlyAlaGluPheLeuGlyAspGlyGlyAspValSerPheSer ThrArgGlyThrGlnAsnTrpThrValGluArgLeuLeuGlnAlaHisArg GlnLeuGluGluArgGlyTyrValPheValGlyTyrHisGlyThrPheLeu GluAlaAlaGlnSerIleValPheGlyGlyValArgAlaArgSerGlnAsp LeuAspAlaIleTrpArgGlyPheTyrIleAlaGlyAspProAlaLeuAla TyrGlyTyrAlaGlnAspGlnGluProAspAlaArgGlyArgIleArgAsn GlyAlaLeuLeuArgValTyrValProArgSerSerLeuProGlyPheTyr ArgThrSerLeuThrLeuAlaAlaProGluAlaAlaGlyGluValGluArg LeuIleGlyHisProLeuProLeuArgLeuAspAlaIleThrGlyProGlu GluGluGlyGlyArgLeuGluThrIleLeuGlyTrpProLeutlaGlun.ro ThrValValIleProSerAlaIleProThrAspProArgAsnValGlyGly AspLeuAspProSerSerIleProAspGlnGluGlnAlaIleSerAlaLeu ProAspTyrAlaSerGlnProGlyGlnProProArgGluAspLeuArg S3 The instant inventors have found that CRLS's of this invention, incorporating GnRH analogs, are particularly effective in causing the toxic compound Z 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 analogs of gonadotropin-releasing hormone can be employed to permanently destroy a subpopulation of the anterior pituitary cells and thereby eliminate the glandes ability to secrete gonadotropins. 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.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.

In a particular embodiment of this invention the CRLS is[no Seq. ID:: provided as compound is branched, see CFR 1.821(a)]:

wherein: X is 6-D-Cys GnRH; and Z is pseudomonas exotoxin PE38M. However as disclosed above, any GnRM peptide and any cytotoxin could be used.

The use of these compounds has great utility in human medicine as well as in veterinary medicine. This follows from the fact that there are several important biological reasons for employing castration and antifertility drugs in humans. For example, breast and prostate cancers are but two examples of sex steroid-dependent tumors which respond to such hormonal manipulation. At present the only reliable way to inhibit steroid-dependent tumor growth is through administration of counter-regulatory hormones (e.g. DES in prostate cancer), sex-steroid hormone binding inhibitors (e.g. tamoxifen in breast cancer) or surgical castration. Thus the potential medical uses of such chemical castration compounds are vast and varied. For example, prostate cancer remains an important cause of cancer deaths and represents the second leading cancer of males.The present palliative treatment for advanced prostate cancer cases involves reduction of serum testosterone/DHT levels through use of surgical castration. It should also be noted that for purposes of disease and/or fertility control especially in humans, it may be desirable to use applicants compounds to ablate pituitary gonadotrophs in conjunction with other modes of treatment. For example, it is anticipated that chronic administration of progestins and estrogens to females and androgens to males might be necessary to prevent loss of secondary sex characteristics, behavior and osteoporosis. However, through judicious use of the herein disclosed compounds, especially in combination with appropriately administered sex steroids, desirable antifertility effects can be achieved.Another area of application in human medicine is treatment of endometriosis.

This condition, which produces painful growth of endometrial tissue in the female peritoneum and pelvis also responds to inhibition of sex steroid synthesis. Those skilled in this art will also appreciate that the herein disclosed compounds could be used to partially reduce sexsteroid secretions, and thus reduce or eliminate certain hormone related behavior problems while retaining improved growth stimulation.

The dose/time adjustments associated with the use of these compounds can vary considerably. 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 accomplished with as few as one injection: but multiple treatments (e.g., based upon concentrations of from about 0.03 milligrams once every 4 days to about 1 milligram per kilogram of body weight for 20 days) are alternative sterilization schemes.

Furthermore. as sterilization agents, 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 contribute to meat production and/or quality. In one preferred embodiment of this invention 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 Z of the herein disclosed compounds are obtainable from both natural and synthetic sources. For example, pokeweed antiviral protein can be isolated from leaves of pokeweed plants and purified by gel filtration chromatography. It can then, by way of example, be conjugated to a RLS linked to D-Lys6-desGlyl0- Pro9GnRH-ethylamide via the amino group on the lysine through a sulfhydryl group introduced into the pokeweed antiviral protein at the bromoacetylated RLS carboxy terminus. In any event, one of the chief advantages of the CRLS compounds of this invention is their ability to produce permanent sterilization without strong toxic side effects. Hence these compounds may be used on mammals such as human beings, domestic animals, pets or wild animals.Moreover, they can be administered as a single injection which can induce permanent and irreversible sterility in both male and female mammals. However, an alternative approach to achieve sterilization is through multiple injections at lower dosages than those employed in a single treatment or by slow release implants (i.e., biodegradable formulations).

The following examples are provided to further define but not to limit this invention.

EXAMPLE 1 PREPARATION OF AN ANNULAR ANTIGEN SCAFFOLD CORE:

wherein: S is a sulfur atom, contributed by the cysteine shown, forming a thioether bond; X is -NH2; Z is ss-alanine-OH.

1. Svnthesis of the linear peptide: Lvs-Lvs-Lvs-Lvs-Cvs [Seq.ID:5:]: Boc-ssAla-PAM Resin (Peninsula Labs, Lot 027925, 0.15 meq/g, 1 g) was washed three times with DCM, deprotected with 5057c TFA/DCM for five minutes, and again for twenty minutes. The resin was washed again with DCM twice. twice with 5%DIEA. and then five more times with DCM.

Onto this resin, the active ester of N-Fmoc-S-Trityl-L-Cys [Bachem lot 2J541, 0.26g, prepared in NMP with BOP (0.19g) and NMM (0.45g, 0.49 mL)] was coupled to a negative ninhydrin. The amino terminus was then deprotected with 20% Piperidine in NMP followed by five NMP washes. Next, N-e-Boc-N-a-Fmoc-L-Lys (Bachem lot 2J724, 0.21 it) was prepared in NMP with BOP (0.19g) and NMM (0.45g, 0.49 mL) and was acylated onto the peptide to a negative ninhydrin. An additional three cycles of lysine addition, following almost exactly the above procedure for addition of the first lysine, was then conducted, and the amino terminus deprotected by treatment with 20% piperidine in NMP for twenty minutes, followed by five NMP washes.

2. Cvclization of the linear peptide to form a thioether linked annular antigen scaffold core: The deprotected amino terminus was bromoacetylated by addition of bromoacetic acid (Aldrich. AW03119ET, 0.45mmoles, 63 mg) plus dicyclohexylcarbodiimide (DCC, 0.225 moles. 46 mg) in DCM. This bromoacetylation was allowed to run for 1.5 hours and then repeated.

by addition of the same reagents, to ensure complete bromoacetylation.

for another 20 minutes.

The resin was washed five times with DCM. The Boc protection was removed using 50% TFA for 20 minutes, in the presence of 0.5 mL triethyl silane as a scavanger. Four DCM washes and one diethyl ether wash followed.

The peptide was cleaved from the resin in dry hydrofluoric acid (HF), without any scavanger, for one hour at 0 C. The HF was then evaporated. The peptide was separated from the resin by dissolution in TFA, filtering off the resin, and removing the TFA in a rotary evaporator. Cyclization of the peptide was allowed to occur by dissolving the peptide in 800 mL of double deionized water, and adding solid NaHCO3 until the pH reached 8. The solution was stirred. and cyclization was allowed to proceed over approximately AS hours. The liquid was then removed by rotary evaporation.

The peptide was dissolved in 8 mL of double deionized water and the cyclized AASC was recovered by loading onto a preparative Vydac C18 HPLC column at 3 mL/minute, and then eluting at 10 mL/minute using a gradient from 0-25% CH3CN over ten minutes. Sample elution was monitored at 215 nm, with full scale deflection set at 2. Two peaks were collected and rechromatographed (2.5% CH3CN-12.5% CH3CN over 60 minutes) after drying in a rotary evaporator. A peak eluting at 23.4 minutes was collected and lyophilized to yield 17.6 mg of peptide.

The mass was analyzed by FAB-MS, and the predicted mass of 746 for the AASC was confirmed.

EXAMPLE 2 PREPARATION OF BROMOACETYLATED CORE: Bromoacetic anhydride (BrAc)2O was prepared by dissolving 15.2 mg of bromoacetic acid (BrAcOH) in 2 mL of dry methylene chloride. Dicyclohexyl carbodiimide (DCC. 8 equivalents.

I I. mg) was added and the mixture stirred at room temperature for one hour in the dark. At the same time. tetralnsine cyclic core. TLCC (5.1 mg 6.8 mol) was dissolved in dry, degassed DhlF. The (BrAc)2O was filtered through glass wool to remove dicyclohexylurea DCU and added to the solution of TLCC.After one hour. a 2 L aliquot was removed for Kaiser analysis which was negative. A second 2 L aliquot was removed. diluted with 100uL water and l0pL was injected into an analytical, reverse phase HPLC (RPHPLC, 1.5 mL/min, Vydac C18, 10-60%CH2CN over 20 minutes. I=215nm, 0.1 abs. units full scalej. Four peaks were observed.

An additional equivalent of (BrAc)rO (7.6 mg BrAcOH.

5.6 mg DCC) was prepared in 0.5 mL methylene chloride. This was filtered, added to the reaction mix, and the bromoacetylation was allowed to proceed overnight. When RPHPLC analysis showed no change after overnight reaction, the reaction was concentrated in vacuo and an additional two equivalents of (BrAc)2O in 500 L methylene chloride was prepared. The concentrated reaction was taken up in 1 mL of 1:1 DMF/methylene chloride, and the filtered (BrAc)rO was added.

The reaction was stirred at room temperature for 5 hours. RPHPLC analysis revealed 3 major product peaks.

The reaction was concentrated in vacuo and products were isolated in a single RPHPLC run on a RCM25X10 Cl 8 delta pak column with a gradient from 20-26go. over 30 minutes at 10 mL/min. Four pooled fractions were collected, peaking at the indicated times: All minutes; B-15 minutes; C-l8 minutes; D-21 minutes. The C fraction was identified as having the correct mass of 5818 for tetrabromoacetylated product by electrospray mass spectrometry.

EXAMPLE 3 PREPARATION OF GnRH ANALOGS: General: All reagents were used as received by the supplier. In the case of solvents HPLC-grade was used where available. HPLC (binary gradient) was performed on a Waters 600E system with Waters AS tunable U.V. detector (Aufs=0.l analytical or 2.0 preparative scale) and recorded on a Waters 746 Data Module. A Waters WISPTM712 autosampler (2000 mL sample loop) was used for analytical samples. A Rheodyne 7125 manual injection port (5000 mL sample loop) was used for preparative samples. A = H2O, 0.1% TFA; B=CH3CN. 0.15c TFA.

Mass spectra were taken on a Finnegan MAT 90. spectrophotometer (positive ion, NBA matrix).

Abbreviations: Standard amino acid abbreviations are used. RT, room temperature; DCC, 1,3-dicyclo-hexylcarbodiimide; HOBT, 1hydroxybenzotriazole; TFA, trifluoroacetic acid; DIEA, N,Ndiisopropylethyl-amine; MPS, ss-maleimidopropionic acid N-hydroxysuccinimide ester; GnRH, gonadotropin releasing hormone; PBS, phosphate buffered saline; DTT, dithiothreitol; EDTA-2Na.

ethylenediaminetetraacetic acid disodium salt; NBA, 3-nitro-benzyl alcohol.

6-D-Lys-GnRH, 1 :PyroGlu-His-Trp-Ser-Tyr-D-Lys-Leu-Arg- Pro-Gly-NH2[Seq.ID:6:]: 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).

The peptide was cleaved (2 h, RT) from the resin using reagent R (1 mL/100 mg resin, TFA/thioanisole/ethanedithiol/anisole, 90:5:3:2). The peptide was precipitated from the concentrated cleavage mixture with diethyl ether and purified by preparative reverse phase HPLC (Waters PrepPak#25 x 10TM C18: 10 mL/min: 10-20% B 0-20 min.: then 2035% B, 20-40 min.: g='30 nm).

6-D-Lys-GnRH: FAB-MS (positive ion NBA matrix) Calc. M+1 1254.44: Found M+1 = 1254.4 (Ne-maleimidopropanoyl)-6-D-Lys-GnRH, 2:PyroGlu-His- Trp-Ser-Tyr-(Ne-maleimidopropanoyl)-D-Lys-Leu-Arg- Pro-Gly-NH2 [Seq. ID:7:]: 6-D-Lys-GnRH (10 mmol. 12.5 mg) was dissolved in NN- dimethylformamide (0.5 mL/mg) and DIEA (50 mmol 9 mL) added.

The mixture was stirred briefly (RT) and ss-maleimidopropionic acid Nhydroxysuccinimide ester (MPS; 20 mmol, 5.2 mg) was introduced in one portion. After 30 min reaction time 10 mL TFA was added to the reaction mixture and the solvent removed in vacuo. The peptide was purified by reverse phase HPLC (Waters PrepPak# 25 x 10TM Delta PakTM C18: 10 mL/min; 10-25% B. 0-30 min.: then 25% B 30-35 min: g=230 nm).

(Ne-maleimidopropanoyl)-6-D-Lys-GnRH,FAB-MS (positive ion NBA matrix) Calc. M+1: 1405.56; Found M+1=1405.6 (Ne-Succinyl)-6-D-Lys-GnRH,2:PyroGlu-His- Trp-Ser-Tyr-(Ne-succinyl-D-Lys-Leu-Arg- Pro-Glv-NH9 ESEO.ID:12:1: 6-D-Lys-GnRH (7.98 !lmol, 10 mg) was dissolved in N,N- dimethylformamide (1 mL) and DIEA (8 mol, 8 L) added, followed by succinic anhydride (1.6 mg, 16 mol, 2 eq). The mixture was stirred for about 12 hours at room temperature. The solvent was removed in vacuo. The residue was dissolved in 0.1% TFA (1.4 mL) and purified by reverse phase HPLC (Vydac RCM8x 10 Delta-PakTM C I g; 3 mL/min; 15-20% acetonitrile over30 min.). Combined product peaks were lyophilized (9.7 mg).An portion of the sample was analyzed by FAB-MS and the predicted mass for the succinylated product was confirmed.

(N,-succinyl)-6-D-Lys-GnRH,FAB-MS (positive ion, NBA matrix) Calc. M+l:; Found M+l= Preparation of other GnRH Analogs N-Ac-1,2-Di-p-Chloro-Phe-6-DLys-GnRH, Ac-4-Cl-Phe A-Cl-Phe-Trp-Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH2 [Seq ID:8:]: The peptide is synthesized on Rink amide WIBHA 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.

6-DLys-10-DAla-GnRH, H-Pgl-His-Trp-Ser-Tyr-DLys-Leu Arg-Pro-DAla-NH2 [Seq. ID:9:]: 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/thio ani sole/ethanedithiol/ani sole. 90:5:3:2). The peptide was precipitated from the concentrated cleavage mixture with diethyl ether and purified by preparative reverse phase HPLC. Characterization by FAB-MS of 6 D-Lys-l0-D-Ala-GnRH (positive ion, NBA matrix) Calc (m+l) = 1268.5; Found (m+l) = 1267.5.

6-DLys-9-Pro-NHEt-GnRH, H-Pgl-His-Trp-Ser-Tyr-DLys- Leu-Arg-Pro-NHEt [Seq. ID:10:]: 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 (2h-72 h, RT) from the resin with anhydrous ethyl amine. The crude protected peptide is precipitated with diethyl ether, collected by suction filtration. and dried overnight (over P,O5). 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.

6-DOrn-GnRH, H-Pgl-His-Trp-Ser-Tyr-DOrn-Leu-Arg- Pro-Gly-NHo [Seq. ID:l 1:1: 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 43IA 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, TFAAhio anisole/ethanedithiol/anisole, 90:5:3:2). The peptide was precipitated from the concentrated cleavage mixture with diethyl ether and purified by preparative reverse phase HPLC. Characterization by FAB-MS of 6 D-Orn-GnRH (positive ion, NBA matrix) Calc (m+l) 1239.4; Found (m+l) 1239.5.

3-Indolylpropionyl-6-DLys-GnRH,3-Indolylpropionyl- Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH2 [Seq. ID: 12:]: 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/thio anisolekthanedithiol/anisole 90:5:3:2). The peptide was precipitated from the concentrated cleavage mixture with diethyl ether and purified by preparative reverse phase HPLC. Characterization by FAB-MS of 3- indolxrlpropionyl-6-D-Lys-GnRH (positive ion, NBA matrix) Calc (m+l) 990.2; Found (m+l) 990.7.

3-Indolylpropionyl-6-DLys-9-Pro-NHEt-GnRH, 3-lndolyl- propionyl-Ser-Tyr-DLys-Leu-Arg-Pro-NHEt [Seq. ID: 13: ]: The peptide is synthesized on Oxime or Merrifield resin by solid phase peptide synthesis (Boc chemistry) using an ABI model at 1 A synthesizer and single couplings (DCC/HOBT). The 3-indolylpropionyl moiety is incorporated as N-formyl-3-indolepropionic acid. The peptide is cleaved (2 h-72 h RT) from the resin with anhydrous ethyl amine. The crude protected peptide is precipitated with diethyl ether, collected by suction filtration, and dried overnight (over P2O5). The protecting groups are removed from the dry peptide by treatment with anhydrous HF (0 C, 0.5-2 h, 5-30 mL) in the presence of anisole (0.2-2 mL) and dimethyl phosphite (0.1-1 mL). The excess HF is removed in vacuo and the residue triturated with diethyl ether. The peptide is purified by preparative reverse phase HPLC and characterized by FAB-MS.

6-D-Cys-GnRH [SEQ.ID:14:]: This peptide is prepared by solid phase synthesis as with any of the foregoing compounds with the addition of D-cysteine at the 6 position as follows: Rink amide resin (591 mg) was coupled sequentially with Gly, Pro, Arg, Leu, D-Cys. Tyr, Ser, Trp, His, Pyroglutamic acid. The Arg and Gly residues were double-coupled and the other residues were single-coupled. The resin was washed with methanol three times and dried under nitrogen (1.069 g resin = 0.539 t weight gain).

The peptide was cleaved for three hours, the resin was filtered off. and the peptide was dried under vacuum. The residue was triturated with diethyl ether and the precipitated peptide was collected by suction filtration and lyophilized (431.7 mg).

A portion of the peptide (195.5 mg) was dissolved in 0.1% TFA. 10% acetonitrile (3 mL), filtered. and purified by RPHPLC (1545% acetonitrile over 30 minutes using two 25Xl0 RCM delta pak C18 columns in tandem. A repeat purification of a second portion of peptide was conducted (190.5 mg). Peak fractions were collected. combined and lyophilized, and an aliquot was analyzed by FAB-MS and amino acid analysis. The predicted mass (1229) and amino acid composition were confirmed.

6-D-Glu-GnRH [SEQ.ID:15:]: This peptide is prepared by solid phase synthesis as with any of the foregoing compounds with the addition of D-glutamic acid at the 6 position. as follows: Rink amide MBHA resin (694 mg, 0.36 mmol/g) in an ABI 431A synthesizer was coupled sequentially with Gly, Pro, Arg, Leu, D Glu, Tyr, Ser, Trp, His, Pyroglutamic acid. The Arg and Gly residues were double-coupled and the other residues were single-coupled. The resin was washed with DCM three times and dried under nitrogen (1.314 g resin).

The peptide was cleaved for three hours, the resin was filtered off, and the peptide was dried under vacuum. The residue was triturated with diethyl ether and the precipitated peptide was collected by suction filtration and lyophilized (200.S mg).

The peptide was dissolved in 0.1% TFA, and purified by RPHPLC (10-60% acetonitrile over 30 minutes using two 25X10 RCM delta pak Cl8 columns in tandem, 13 mL/min) in two portions. Peak fractions were collected, combined and lyophilized (108.3 mg), and an aliquot was analyzed by FAB-MS and amino acid analysis. The predicted mass (1253) and amino acid composition were confirmed.

EXAMPLE 4 Preparation of PE38M Plasmid PJH4 (Ref. Hwang. J. Cell (1987, 48; 129-136) contains the coding sequence for PE1613. 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 NDE1/Hind III double digest is carried out on PJHA 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 AGA3 [Seq. ID:16:]: and II - 5' AGC TTC TTC GGC CAT AAG CTT GGT ACC CTG CAG CA3' [Seq. ID:17:]:. The modified PE insert has a sequence of MetLruGlnGlyThrLysLeuMetAlaGluGlu [SEQ.ID:18:] 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 amino acids 4-252 A 553 bp Sal I/Bam HI fragment of plasmid PJH43 is cloned into M13 mpl9.An oligonucleotide 50 nucleotides in length with the structure 5' GGC GTC GCC GCT GTC CGC CGG GCC GTT GGC CGC GCC GGC CTC GTC GTT GC3' [Seq ID:19:]:. is synthesized and annealed to the single stranded Ml3 vector to facilitate (loop out) mutagenesis generating a deletion of amino acids 365-380 of the PE insert.

A 505 bp Sal I Bam Hl fragment is excised from the replicative form of the mutant DNA in M13 and ligated with a 3.7 Kb Sal 1 Bam Hl fragment of the plasmid PJH43. This new plasmid is designated PJH44.

A Bam Hl/EcoR 1 fragment of 460 nucleotides is excised from PJHAA and cloned into Ml3 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 successively at each of the lvsines with the following olioomers: Lysines 590-5' GCT GAT CGC CTG TTC TTG GTC GGG GAT GCT GGA C3'[Seq.ID:20:]: Lysines 606-5 GTC CTC GCG CGG CGC TTG GCC GGG CTG GCT G3' [Seq.ID:21:]: Lysines 613-5' CGG TCG CGG CAG TTA ACG CAG GTC CTC GCG CGG 3' [Seq.ID:22:]: The Bam Hl 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 PE3Am from a commercially available strain of E. coli HB 101. available from Bethesda Research Laboratories.

EXAMPLE 5 Preparation of GnRH Receptor Lizand Scaffold: Step 1: Preparation of Bromoacetvlated Core:

Summary: Step 1 converts X from -NH2 to bromoacetyl; Z = ss-Ala-OH Step 2 converts X to GnRM linked scaffold; Z = JA-Ala OM.

Step 3 converts Z to ss-Ala-NH-(CH2)2NH2 Step 4 converts Z to lS-Ala-NH-(CH2)2-NH-PE38M.

Bromoacetic anhydride,(BrAc)2O was prepared by dissolving 58.6 mg (0.42 mmol) of bromoacetic acid (BrAcOH) in 3 mL of dry methylene chloride. Dicyclohexyl carbodiimide (DCC, 43.5 mg, 0.21 mmol) was added and the mixture stirred at room temperature for one hour in the dark. At the same time, annular antigen scaffold core, AASC, (26.2 mg, 35.1 pLmol) was dissolved in dry, degassed DMF (3 mL). The (BrAc)2O was filtered to remove dicyclohexylurea DCU, and added to the solution of AASC, and the reaction was stirred for one hour at room temperature. Kaiser analysis on an aliquot was negative.

The reaction was concentrated in vacuo and products were isolated in a single RPHPLC run on a delta prep 3000 using two RCM25X10 C18 delta pak columns with a gradient from 20-27% acetonitrile over 30 minutes at 15 mL/min. Three fractions were collected, peaking at the indicated times: A- 17 minutes; B- 19 minutes; C- 20 minutes. The B fraction was identified as having the correct mass of 1229 for tetraacetylated product by fast atom bombardment mass spectrometry (FAB-MS).

Step 2: Linkage of GnRH to the Bromoacetvlated Core: Bromoacetylated core from step 1 (11.3 mg, 9.2 Limol, FW =1929) was dissolved in degassed nitrogen sparged pH 8 phosphate buffer (0.1 M 2 mL), and stirred under an atmosphere of nitrogen. A 2 L aliquot was removed and diluted with 100uL of 0.1 '7e aqeous TFA and saved as a reference. To the bromoacetylated core was added D Cys6-GnRH(45.2 mg.36.8 mol) and an additional 2 mL of buffer, followed by 500 L acetonitrile). The reaction was stirred overnight at room temperature. Analytical RPHPLC showed that there was no residual starting material.The reaction was therefore fractionated by preparative RPHPLC (RCM 25x10. 10 mL/minute. delta pak C18. 2030%CH3CN over 30 minutes, monitored at 230 nm with 1.25 absorbance units full scale). Product (12.3 mg) was collected and lyophilized. This material gave the correct mass of 5817 for a core having four D-Cys6-GnRH peptides by FAB-MS.

Step 3. Provision of free amine at the core C-terminus: The peptidylated core from step 2 (12.3 mg, 2.1 mol) was dissolved in dry, degassed DMF. To the solution of peptidylated core was added diaminoethane-BOC (1.7 nig, A.2 pmol) followed by DIEA (0.8 L. A.2 Rmol) followed by bezotriazolyl-N-oxy- tris(dimethylamino)-phosphonium hexafluorophosphate, B OP (1.9 mg, 4.2 mol), and 1-hydroxybenzotriazole hydrate, HOBt, (0.6 mg, 4.2 Rmol). The reaction was allowed to stir at room temperature and then at 4 C for 48 hours.The sample was concentrated in vacuo, and the residue was taken up in aqueous 5% CH3CN, filtered (0.45 m nylon filter) and fractionated by RPHPLC (RCM 25x10, 20-35'Xc CH3CN.

over 30 minutes. 10 mL/min. monitored at 230 nm. 1.5 absorbance unit full scale, delta pak Cl 8, 300 angstrom packing). The main peak was collected and lyophilized. By FAB-MS, the product had the correct mass of 5959.

The t-Boc product (7.3 mg, 1.22 !lmol) was resuspended in methylene chloride (3 mL). Anhydrous anisole (1 mL) was added and the solution was stirred at 0 C. TFA (3 mL) was added and the solution stirred for forty minutes at 0 C and then allowed to reach ambient temperature over twenty minutes. The product was concentrated in vacuo. followed by lyophilization to remove all of the anisole, yielding 8.3 mg of product. The residue was dissolved in 0.1% TFA (3 mL). A 2 pL sample was analyzed in a Kaiser test which showed a faint blue color, confirming the presence of a free amino group appended to the receptor ligand scaffold at the position Z. The product was lyophilized overnight (7.3 mg yield), which gave the predicted mass by electrospray mass spectrometry.

Step 4: Linkage of Pseudomonas Exotoxin: A. Bromoacetylation of the peptidylated core: The free amino group was bromoacetylyated by addition of bromoacetic anhydride, prepared as follows: DCC (10 mg) and bromoacetate (13.5 mg) were dissolved in dry methylene chloride (AX0 pL). The anhydride was allowed to form for one hour at room temperature. The anhydride was filtered and added (12.2 RL, 0.1 M) to the dissolved peptide (1.22 pmol in 2.5 mL DMF). The solution was stirred for one hour at room temperature in the dark. The reaction was then concentrated under vacuum to remove all the DMF.The sample was redissolved in 20% acetonitrile in 0.1% TFA, and separated on RPHPLC (Delta Pak C1S, RCM 25x10, 10 mL/min., 20-35So CH3CN over 30 minutes, monitored at 230 nm, 1.5 absorbance units full scale).

Three product peaks (A, B, C) were collected and lyophilized. The A peak was starting material. The B peak (3.7 mg) was the correct product by electrospray mass spectrometry (5980). The C peak was an unidentified contaminant.

B. Thiolation of Exotoxin: A solution of NLPE38QQR [Seq. ID:4:] (8.96 mg. 7.0 mL was adjusted to a pH = 11 by addition of pH 11 borate buffer salts (292 mg). To this was added EDTA (50 m) and DTT (10 mg). The solution was covered with an atmosphere of nitrogen and vortexed until all solids were dissolved. N-acetyl homocysteine thiolactone (N-Ac HCTL. 50 mg) was added and the atmosphere of nitrogen replaced.

The reaction was allowed to proceed overnight at romm temperature and then dialized against 0.1 M pH 8.0 phosphate buffer (4 L) at 25 C for eight hours. The dialysis was then continued in fresh buffer 0. i IvI pH 8.0 phosphate buffer (4 L) with added DTT (0.25 mg) for about 16 hours. All dialysis was conducted under an atmosphere of nitrogen.

The thiolated protein was transferred to a sterile plastic (15 mL) centrifuge tube under nitrogen. An Ellman's test on 300 uL of thiolated protein gave 0.085 mol SH/mL (7.45 mL of product = 0.633 mol SH total).

C. Conjugation of bromoacetvlated peptide core and thiolated Toxin: Thiolated exotoxin (0.502 umol 5.90 mL) and bromoacetylated GnRH scaffold (3.0 mg. 0.502 umol) ere mixed and the reaction was allowed to proceed under an atmosphere of nitrogen.

The reaction was tumbled for six hours at room temperature. The conjugate was dialized against pH 7.0 phosphate buffer (0.01 M. 4 L; at A-C for 15 hours and then against 0.9 % NaCI (4 L) at A-C for 9 hours.

This was repeated with fresh NaCI for 15 hours.

The dialized sample was recovered and the protein concentration was obtained by BIO-RAD protein assay (325 ug/mL) , and an aliquot was measured by SDS-PAGE (see figure 3). From this analysis it is clear that some contaminating pseudomonas exotoxin (Mr=39.000) remains in the product (Mr=50.000). The residual contaminating free exotoxin may be removed by passage of the product over an anti-GnRH affinity column.

EXAMPLE 5 PITUITARY GnRH RECEPTOR BINIDING ASSAY Annular ligand-toxin scaffolds of this invention were tested for their ability to bind to pituitary membranes as follows: A. Preparation of pituitary membranes 1. Frozen rat pituitary glands were homogenized in 10 volume of ice cold buffer (Na2HCO3, 1 mM; dithiothreitol. 2.5 mM).

2. Centrifuged at 750 x g for 10 min.

3. Centrifuge the supernatant at 20,000 x g for 15 min.

4. Wash the pellet with Tris.HCl (20 mM, pH 7.4). Centrifuge at 20,000 x g for 15 min. Repeat washing one more time.

5. Resuspend the pellet in Tris.HCl buffer (140 mg fresh weight/ml).

B. Binding Assav 1. Membranes were incubated in a ice bath in 0.25 ml of medium (pH 7.5) containing 50 mM Tris.HCl. 1 mM dithiothritol. 0.1% BSA.

I-125 Buserelin (20,000 dpm) and desired concentration of test compound.

2. After 90-120 min. incubation, 5 ml of ice cold PBS (pH 7.5) were added to each tube and the content of tubes were rapidly filtered through a glass fiber filter which was been pre-soaked in 1% BSA in PBS. Wash the membrane on filter two times with 5 ml of cold PBS.

3. Determine the radioactivity of bound In 125 Buserelin in a gama counter. The IC50 of compounds for the inhibition of the I-125 Buserelin binding were estimated from competition curves.

C. Results: In figures 1 and 2, binding curves of compounds of the instant invention, as compared with those of the prior art (see WO 93/15751 ) are shown.

The -[logIC50] of the scaffold of the instant invention, (6-D-Cys)4 (Lys)4-Cys-iS-Ala-PE38, was measured according to this assay and found to be 8.2. Because the product is approximately 50% contaminated with underivatized exotoxin, the actual -[loglC50] is actually higher by about 50%, than reported here (see Example Aj.

Chimaeric conjugates of the prior art (see WO 93/15751, 6-D-Lys GnRH-PE38 conjugate) exhihit a -[logIC50]= 6.9. The -[logIC50 for buserelin. 5-oxoPro-His-Trp-Ser-Tyr-D-Ser(tBu)-Leu-Arg- Pro-NH-C2H5 [Seq. ID:23:] is 9.1.

Thus while conjugation of GnRH analogs to exotoxin in the past greatly reduced the binding affinity (by a factor of about 100 fold), the present invention provides a scaffold of GnRH receptor ligands which unexpectedly retain their high-affinity receptor binding upon conjugation to toxin.

Claims (15)

WHAT IS CLAIMED IS:

1. A cytotoxic receptor ligand scaffold, CRLS, of formula: (Lys)n(Unh)m(Sol)m-(X)n-Z, wherein: Lys represents lysine; Unh represents sterically unhindered groups; Sol represents hydrophilic, solubilizing groups; n is 3 to 10; m is 0 to 5; X represents a receptor ligand; and Z is a cytotoxin.

2. The CRLS of Claim 1 having the formula:

wherein: S is a sulfur atom, forming a thioether bond; X is a receptor ligand with each X being linked through the ot- or amino group of lysine, with each X being the same or different; Y is a linker; Z is a cytotoxic moiety linked to the ligand scaffold; p is 0 to 5; m is 3 to 10: and n is 1 to 10.

3. The CRLS of Claim 2 capable of destroying the LH releasing cells of the pituitary gland.

4. The CRLS of Claim 3 where the toxin is a plant derived toxin a bacteria derived toxin or a chemical toxin.

5. The CRLS of Claim 4 where the plant derived toxin is ncin modeccin, abrin. poke weed antiviral protein. a-amantin gelonin ribosome inhibiting protein (RIP) or RIP derived from wheat.

corn, rye or flax.

6. The CRLS of Claim 4 where the chemical toxin is melphalan methotrexate. nitrogen mustard. doxorubicin or daunomycin.

7. The CRLS of Claim 1 where the bacteria derived toxin is diphtheria toxin. Thseudomonas exotoxin or shiga toxin.

8. The CRLS of Claim 7 where the bacteria derived toxin is Pseudomonas exotoxin or a segment thereof.

9. The CRLS of Claim 8 where the Pseudomonas exotoxin has been modified such that the binding domain has been partly or completely deleted.

10. The CRLS of Claim 9 where the Pseudomonas exotoxin has been modified to delete amino acids 1-252 and retain amino acid 253-613.

11. The CRLS of Claim 4 wherein the receptor ligand is a GnRH peptide.

12. The CRLS of Claim 11 wherein the GnRH peptide is: Q-Ser-Tyr-W-X-Arg-Y-V | L1 L2 CRLS wherein: Q is PyroClu-His-Trp N-acetyl-4-Cl-Phe1,2-Trp or 3indolylpropionyl; CRLS is the cytotoxic receptor ligand scaffold to which the GnRH peptide is linked; W is the D or L amino acid with a pendant linking functionality such as cysteine, or

where r is 1 or 2; m is 1 to 4; n is 0 or 1; B is CH2, O, S or N; and R1 is H, C1-C6alkyl or C3-CScycloalkyl; X is Leu or Nle; Y is Pro or 4-hydroxy-Pro; V is Gly, NH2, D-Ala-NH2, NH-Et, NH-Pr or Arg-Gly-NH2; and L1 and L2 are independently:

wherein: X is C1-C5 alkylene phenyl or C5-C6 cycloalkylene: R is Cl-Cn alkanoyl: and n is 1 or 2.

13. The CRLS of Claim 12 wherein the GnRH peptide has the structure: PyroGlu-His-Trp-Ser-Tyr-W-Leu-Arg-Y-V 1 2 3 4 5 1 6 7 8 9 10 L1 L2 RLS where the decapeptide is GnRH with the normal 6-position amino acid (W=Gly) deleted and replaced by W=D-Lys, D-Cys or D-Orn.

14. A cytotoxic receptor ligand scaffold having the formula:

wherein: X is 6-D-Cys; and Z is pseudomonas exotoxin, PE38M.

15. A method for preparing a cytotoxic receptor ligand scaffolds CRLS, which comprises: a) Synthesizing a multi-lysine core optionally incorporatint solubilizing or sterical unhindered groups and purifying this component: b) Synthesizing a receptor ligand optionally incorporating a terminal group of low steric hindrance and purifying this component; and c) Coupling the purified core component of step (a) with the purified receptor ligand of step (b), and purifying the receptor ligand scaffold thus formed: and d) Coupling the RLS of step (c) with a cytotoxic functionality to form a cytotoxic receptor ligand scaffold CRLS.

1 6. A method for the sterilization of animals which comprises administerinr to such animals an effective amount of a toxin conjugate of Claim 1.

1 7. A composition useful for the sterilization of animals which comprises an inert carrier and an effective amount of a toxin conjugate of Claim 1.