CA2467769A1 - Methods and formulations for testosterone suppression - Google Patents

Abstract

Compositions comprising immunogenic GnRH multimer/leukotoxin chimeras in combination with aluminum-based adjuvants and Montanide.TM., are described.
The compositions elicit anti-GnRH antibodies and are useful for suppressing testosterone levels, e.g., in males with prostate cancer.

Description

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TECHNICAL FIELD
The present invention pertains generally to GnRH f~mulatio~s. In ~ticul, the invention relates to vaccine compositions comprising immunogenic GnRH
multimer/leukotoxin chimeras in combination with particular adjuvants, that elicit anti-GnRH
antibodies. The vaccines are useful for suppressing testosterone levels, e.g., in males with prostate cancer.
BACKGROUND
Prostate cancer has become the most common cancer in men, excluding basal cell carcinoma of the skin. Approximately 200,000 new cases of prostate cancer are diagnosed per year in the United States and it is estimated that one in five men will eventually develop prostate cancer. Although slow growing, prostate cancer not only results in significant morbidity, but it is also the second leading cause of cancer deaths in men with approximately 31,000 deaths occurring per year in the United States. Adequate therapy is therefore important for such patients.
At the time of initial diagnosis, most prostate cancers have spread beyond the ability of surgery or radiation to achieve a cure (Kirby, R.S. (1996) BJCP 50:88).
Palliative therapy is thus critical in the optimum management of patients with prostate cancer.
Unlike many other types of canatr, palliative therapy can ~sult in pilong~ hods of survival that typically last for years. It is therefore necessary to continue identifying new palliative therapies that can be used long teen to extend both the quantity and quality of life of prostate cancer patients.
Sexual maturation and function of the gonads and gonadal accessory tissues such as the prostate gland are under the control of Gonadotropin releasing hormone (GnRH) (formerly designated LHRH). GnRH is produced in the hypothalamus and GnRH
initiates the sex hormone pathway. In particular, GnRH causes the release of the gonad stimulating hormones, luteinizing hormone (LH) and follicle stimulating hormone (FSH) from the pituitary gland. ~'Eiese, in turn, control the production of the sex hormones, testosterone in men and estrogen and progesterone in women, which act on tissues such as the prostate gland, breast, ovaries and endometrium.
Prostate tumors typically develop slowly and are initially testosterone-dependent.
Therefore, the current standard of therapy involves surgical castration, or medical castration using GnRH analogs or antiandrogens. GnRH analogs, when administered continuously at high doses, suppress pituitary LH and FSH release, and thereby reduce gonadal testosterone production.
Studies using experimental GnRH vaccines over more than 20 years have established that immunization can stimulate formation of antibodies of sufficient titer and binding affinity to inhibit gonadal steroid production in animals (Fraser et al., J.
Endocrinol. (1974) 63:399-406), including primates (Giri et al., Exper. and Molec. Path. (1991) 54:255-264).
Effects include atrophy of the prostate gland in normal animals (Jayashankar et al., Prostate (1991) 14:3-11 ), as well as growth inhibition of the Dunning hormone-dependent tumor in male rats (Fuerst et al., Prostate (1997) 32:77-84) and estrogen-dependent 7,12 dimetylbenz (a) antracene-induced mammary tumors in rats (Ravdin et al., Life Sci. (1988) 43:117-123).
GnRH chimeric molecules that include GnRH multimers fused to a leukotoxin carrier have also been shown to stimulate an immune response (see, e.g., U.S. Patent Nos.
5,837,268 and 6,521,746).
In the year 2000, the British Cancer Research Campaign reported preliminary results of a trial of an anti-GnRH vaccine called D 17DT (Simms et al., British Journal of Cancer (2000) 83:443-446). This molecule consists of the GnRH decapeptide linked to diptheria Eax~id. Tvuelpatients vxrith lacally advaneaei prostate-canter were treated with three injections of either 30 ug or 100 pg doses of the vaccine over a six-week period. Patients were followed for estimation of serum testosterone, PSA and anti-GnRH antibody titer. In five patients, a significant reduction in testosterone and PSA was observed.
Castrate levels of testosterone of less than 50 ng/rnl wachieved i~r feur ~t~~rr~tta d w~laintailbr up to nine months. The patients with the best antibody responses had the best response in terms of testosterone suppression.
Nevertheless, there is a continuing need for the development of effective GnRH
vaccines to reduce serum testosterone levels. The slow, often asymptomatic early phase of t~d'~~~er~ea~ts ~~t at t?t~ °~ dia~tiosis, many patie~tts already have cancerous cells that have metastasized to other sites. Medical castration with GnRH analogs is commonly used at this stage. However, such therapy is costly, subject to poor patient compliance and has undesirable side-effects. For example, GnRH analogs are known to initially cause a "testosterone flare" which is a rapid increase in testosterone clinically characterized by impQt~nce, hot flashes and log of libido. GnRH analogs are often adminislc~ed in conjunction with antiandrogens, some of which also have side-effects. Thus, the discovery of new formulations for the treatment of prostate cancer would be extremely desirable.
Montanide ISA 51T"'' (SEPPIC, Paris, France) is an oiUwater vaccine adjuvant that can be mixed one to one (v/v) with aqueous solutions. It has been successfully used as an adjuvant in both animals and humans. In particular, Montanide ISA SITM has been used as an adjuvant to raise antibodies against Plasmodium B epitopes in mice (Del Guercio et al., Vaccine (1997) 15:441-448) and Plasmodium jalciparum pre-erythrocytic antigens in monkeys (Perlaza et al., Infect. Immun. (1998) 66:3423-3428). Additionally, a hydrophobized GM3 ganglioside/Neisseria meningitides outer membrane protein complex vaccine that incorporated Montanide ISA 51 TM has been shown to induce tumor protection in B16 marine melanoma (Alonso et al., Int. J. Oncol. (1999) 15:59-66).
In humans, a vaccine containing Montanide ISA 51T'''' and HIV-1-specific peptides has been used to induce delayed-type hypersensitivity in asymptomatic HIV-1-positive individuals (Turner et al., AIDS (1994) 8:1429-1435). HIV Tat Toxoid emulsified in Montanide ISA 51~ has been shown to be safe and immunogenic in immunocompromised HIV-1-infected patients (Gringeri et al., J. Hum. Virol. (1998) 1:293-298).
Montanide ISA
51TM has also been incorporated into a human papilloma virus (HPV)-based vaccine that used HPV-16 peptides for safe and effective vaccination of patients with advanced cervical carcinoma (Pinto et al., AIDS (I999) 13:2003-2012}. HIV-specific immunity has been shown in asymptomatic HIV-infected patients following vaccination with HIV
synthetic envelope peptides emulsified in Montanide ISA 51TM (Pinto et al., AIDS (1999) 13:2003-2012). Rosenberg et al., Nature Med. (1998) 4:321-326 showed that it is possible to immunize patients with metastatic cancer using a modified self peptide in Montanide ISA
51TT' to generate lymphocyte precursors against growing tumor and to mediate tumor regression. U.S. Patent No. 6,303,123 describes GnRH-diptheria toxin conjugates emulsified 1!3~ I~A'~3°cor~i~tng 1.$o aluminum monostearate.

However, the use of Montanide ISA 51 T"' in combination with GnRH multimeric compounds has not heretofore been described.
SUMMARY OF THE INVENTION
The present invention is based on the discovery of novel formulations comprising GnRIi multimsr/leukotoxin chimeras. The formulations display surprisingly enhanced immunogenicity as compared to previous GnRH multimer/leukotoxin compositions and are useful for suppressing serum testosterone levels in male mammals, such as in humans.
Accordingly, in one embodiment, the invention is directed to a composition comprising an immunogenic GnRH multimer/leukotoxin chimera, an aluminum-based adjuvant, a MontanideTM adjuvant and a pharmaceutically acceptable excipient, wherein the GnRH multimer present in the chimera has the general formula [GnRH-X-GnRH]~, wherein:
GnRH is a GnRH polypeptide;
X is a peptide linkage or an amino acid spacer group; and n is an integer greater than or equal to 1.
The GnRH polypeptides of [GnRH-X-GnRH) can be the same or different.
In certain embodiments, the leukotoxin polypeptide lacks cytotoxic activity.
The leukotoxin polypeptide can be LKT 111. Moreover, the GnRH multimer/leukotoxin chimera may comprise the contiguous sequence of amino acids depicted in Figures 2A-2F (SEQ
>D N0:6), or an immunogenic amino acid sequence with 75% s~uence identity thereto.
In certain embodiments the Montanide is Montanide ISA S1TM.
In additional :.em>xadiments, the aluminum-based adjuvxnt is an ~il(OH)3 gel.
In yet further embodiments, the invention is directed to a composition comprising:
(a) a GnRH multimer/leukotoxin chimera comprising the contiguous sequence of amino acids depicted in Figures 2A-2F (SEQ ID N0:6), or an immunogenic amino acid sequence with 75% sequence identity thereto; (b) Al(OH)3 gel; (c) Montanide ISA S1T""; and (d) a pharmaceutically acceptable excipient.
In further embodiments, the GnRH multimer/leukotoxin chimera comprises the contiguous amino acid sequence of Figures 2A-2F (SEQ ID N~7:6).

In additional embodiments, the GnRH multimer/leukotoxin chimera is present in an amount of about 20 pg per mL to about 2 mg per mL, such as in an amount of 100 wg per mL
to 500 wg per mL.
In further embodiments, the Al(OH)3 gel is present in an amount of about 0.1%
to about 1% (w/v), such as in an amount of 0.2% to 0.5% (w/v).
In yet additional embodiments, the Montanide ISA S1T"' is present in an amount of about .25 mL to about .75 mL per mL of the composition, such as in an amount of 0.5 mL
per mL of the composition.
In another embodiment, the invention is directed to a composition comprising:
(a) 100 pg per mL to 500 wg per mL of a GnRH multimer/leukotoxin chimera comprising the contiguous sequence of amino acids depicted in Figures 2A-2F (SEQ ID N0:6);
(b) 0.2% to 0.5% (wlv) Al(OH)3 gel;(c) 0.5 mL per mL of composition Montanide ISA 51~; and (d) a pharmaceutically acceptable excipient.
In further embodiments, the invention is directed to a method of producing an immune response in a mammalian subject comprising administering to the subject a therapeutically effective amount of a first composition, wherein the first composition is any one of the compositions above.
In certain embodiments, the method further comprises boosting the subject with one or more additional doses of a second composition, such as with 4-10 additional doses of the second composition. The second composition can be the same as the first composition or different than the first composition.
~ ;addi~ ex~~dimtl~ immure response .t~usea a reduction in serum testosterone levels.
In yet further embodiments, cyclophosphamide is administered prior to, concurrent with, or subsequent to administration of the first composition.
In additional embodiments, the mammal is a male human.
In further embodiments, the invention is directed to a method of reducing serum testosterone levels in a male human, said method comprising: (a) administering to the male human a therapeutically effective amount of a first composition, wherein the first composition comprises (i) 100 ug per mL to 500 p,g per mL of a GnRH
multimer/leukotoxin ~~e~a '~m~g ~1~~ ti~iius ~~u~~' c~ ~~lr~~ ~~~ ~~'e~ ~ ~. . ~ 2A-' (~

ID N0:6), (ii) 0.2% to 0.5% (w/v) Al(OH)3 gel, (iii) 0.5 mL per mL of composition Montanide ISA 51TM, and (iv) a pharmaceutically acceptable excipient; (b) boosting the male human with 4-10 additional doses of the first composition, to result in a reduction of serum testosterone levels.
In certain embodiments, cyclophosphamide is administered prior to administration of the first composition.
These and other embodiments of the subject invention will readily occur to those of skill in the art in view of the disclosure herein.
BRIEF DESCRIPTION OF THE FIGURES
Figures lA (SEQ ID NOS:1 and 2) and 1B (SEQ ID NOS:3 and 4) show the nucleotide sequences and amino acid sequences of the GnRH constructs used in the chimeric leukotoxin-GnRH polypeptide gene fusions. Figure lA depicts GnRH-1 which includes a single copy of a GnRH decapeptide; Figure 1B depicts GnRH-2 which includes four copies of a GnRH decapeptide when n=1, and eight copies of GnRH when n=2, etc.
Figures 2A-2F (SEQ ID NOS:S and 6) show the nucleotide sequence and predicted amino acid sequence of the LKT-GnRH chimeric protein termed "IPS-21" herein.
The chimeric protein includes GnRH multimers consisting of two repeats of the GnRH
sequence shown in Figure 1 B on each of the 5' and 3' ends of the LKT 111 polypeptide.
Thus, the LKT 111 nucleotide and amino acid sequences are found at positions 334-1779 and 112-592, respectively, of Figure 2A-2F.
DETAILED DESCRIPTION OF THE INVENTION
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA
technology, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual; DNA Cloning, Vols. I and II (D.N. Glover ed.);
Oligonucleotide Synthesis (M.J. Gait ed.); Nucleic Acid Hybridization (B.D.
Hames & S.J.
l~~s Vii;.); . Peal, A practical Guide to Molecular Cloning; the series, Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); and Handbook of Experimental Immunology, Vols. I-IV (D.M. Weir and C.C. Bla;ckwell eds., Blackwell Scientific Publications).
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entireties.
The following amino acid abbreviations are used throughout the text:
Alanine: Ala (A) Arginine: Arg (R) Asparagine: Asn (N) Aspartic acid: Asp (D) Cysteine: Cys (C) Glutamine: Gln (Q) Glutamic acid: Glu Glycine: Gly (G) (E) Histidine: His (H) Isoleucine: Ile (I) Leucine: Leu (L) Lysine: Lys (K) Methionine: Met (M) Phenylalanine: Phe (F) Proline: Pro (P) Serine: Ser (S) Threonine: Thr (T) Tryptophan: Trp (W) Tyrosine: Tyr (Y) Valine: Val (V) I. DEFINITIONS
In d:a~cribing the pit ireret'ntioi~, ttia~frsllic2is twill be tiriplo~ed, arid uce intended to be defined as indicated below.
It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a GnRH multimer" includes a mixture of two or more su~th multimers, and the like.
The term "Gonadotropin releasing hormone" or "GnRH" refers to a decapeptide secreted by the hypothalamus which controls release of both luteinizing hormone (LH) and follicle stimulating hormone (FSH) in vertebrates (Fink, G., British Medical Bulletin (1979) 35:15-160). The amino acid sequence of GnRH is highly conserved among vertebrates, and especially in mammals. In this regard, GnRH derived from most mammals including human, bovine, porcine and ovine GnRH has the amino acid sequence pyroGiu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NHZ {SEQ )D N0:7) (Murad et al., Hormones and Hormone Antagonists, in The Pharmacological Basis of Therapeutics, Sixth Edition (1980) and Seeburg et al., Nature (1984) 311:666-668).
As used herein a "GnRH polypeptide" includes a molecule derived from a native GnRH sequence, as well as recombinantly produced or chemically synthesized GnRH
polypeptides having amino acid sequences which are substantially homologous to native GnRH and which remain immunogenic and retain the ability to stimulate an immune response and preferably to suppress testosterone secretion, as described below. Thus, the term encompasses derivatives and analogues of GnRH including any single or multiple amino acid additions, substitutions and/or deletions occurring internally or at the amino- or carboxy-termini of the peptide. Accordingly, under the invention, a "GnRH
polypeptide"
includes molecules having the native sequence as well as analogs of GnRH.
Representative GnRH analogs include an analog with an N-terminal Gln or Glu residue rather than a pyroGlu residue, an analog having Asp at amino acid position 2 instead of His; a GnRH analog with an N-terminal addition such as Cys-Gly-GnRH (see, e.g., Prendiville et al., J. Animal Sci. (1995) 73:3030-3037); a carboxyl-containing GnRH analog (see, e.g., Jago et al., J. Animal Sci. (1997) 75:2609-2619; Brown et al., J.
Reproduc. Fertil.
( 1994) 101:1 S-21 ); the GnRH analog (D-Trp6-Pro9-ethyl amide)GnRH (see, e.g., Tilbrook et al., Hormones and Behavior (1993) ~:S-28) or (D-Trp6)GnRH (see, e.g., Chaffaux et al., R~c.'~cei.~ ~e tl~edecl~te vet~rinatre (185) 161:133-145); GnRH analogs with the first, sixth and/or tenth normally occurring amino acids replaced by Cys and/or wherein the N-terminus is acetylated and/or the C-terminus is amidated (see, e.g., U.S. Patent Nos.
4,608,251 and 4,975,420); the GnRH analog pyroGlu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Gly-Y-Z (SEQ
ID
N0:8) wherein X is Gly or a D-amino acid, Y is one or more amino acid residues which may be the same or different, preferably 1-3 Gly residues, and Z is Cys or Tyr (see, UK Patent Publication No. GB 2196969); GnRH analog described in U.S. Patent No.

5,688,506, including the GnRH analogue Cys-Pro-Pro-Pro-Pro-Ser-Ser-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly (SEQ ID N0:9), pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Ser-Ser-Pro-Pro-Pro-Pro-Cys (SEQ ID NO:10), pyroGlu-Hid-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Arg-Pro-Pro-Pro-Pro-Cys (SEQ ID NO;11); the GnRH analogue known as Deslorelin, commercially available from Apeptech (Australia), and OvuplantT'~; and molecules with other amino acid additions, substitutions and/or deletions which retain the ability to elicit formation of antibodies that cross-react with naturally occurring GnltH and preferably suppress testosterone secretion.
Thus, the term "GnRH polypeptide" includes a GnRH molecule differing from the reference sequence by having one or more amino acid substitutions, deletions and/or additions and which has at least about 50% amino acid identity to the reference molecule, more preferably about 75-85% identity and most preferably about 90-95%
identity or mop, to the relevant portion of the native polypeptide sequence in question. The amino acid sequence will have not more than about 1-5 amino acid substitutions, or not more than about 1-3 amino acid substitutions. Particularly preferred substitutions will generally be conservative in nature, i.e., those substitutions that take place within a family of amino acids.
In this regard, amino acids are generally divided into four families: (1) acidic -- aspartate and glutamate; (2) basic -- lysine, arginine, histidine; (3) non-polar --alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar -glycine, asparagine, glutamine, cystine, serine threonine, tyrosine.
Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonably predictable that an isolated replacement of leucine with isoleucine or valine, or vice versa; an aspartate with a glutamate or vice versa; a threonine with a serine or vice versa; or a similar conservative replacement of an amino acid with a structurally related amino ,acid, will not have a major effect on the activity. Proteins having substantially the same amino acid sequence as the reference molecule, but possessing minor amino acid substitutions that retain the desired activity, are therefore within the definition of a Gnltl-I
polypeptide.
A "GnRH polypeptide" also includes peptide fragments of the reference GnRH
molecule, so long as the molecule retains the desired activity. Epitopes of GnRH are also captured by the definition. For purposes of the present invention, a GnRH
polypeptide may be derived from any of the various known GnRH sequences, described above, including without limitation, GnRH polypeptides derived from human, bovine, porcine, ovine, canine, feline, cervine subjects, rodents such as hamsters, guinea pigs, gerbils, ground hogs, gophers, lagomorphs, rabbits, ferrets, squirrels, reptilian and avian subjects.
A "GnRH peptide" is a GnRH polypeptide, as described herein, which includes less than the full-length of the reference GnRH molecule in question and which includes at least one epitope as defined below. Thus, a vaccine composition comprising a GnRH
peptide would include a portion of the full-length molecule but not the entire GnRH
molecule in question. Particular GnRH peptides for use herein include, for example, GnRH
peptides with 5, 6 or 7 amino acids, particularly those peptides which include the amino terminus or the carboxy terminus, such as GnRH peptides including amino acids 1-S, 1-6, 1-7, 2-8, 3-8, 3-10, 4-10 and 5-10 of the native sequence (see, e.g., International Publication No.
WO 88/05308).
By "GnRH multimer" is meant a molecule having more than one copy of a selected GnRH polypeptide, GnRH peptide or epitope, or multiple tandem repeats of a selected GnRH
polypeptide, GnRH peptide or epitope. The GnRH multimer may correspond to a molecule with repeating units of the general formula (GnRH-X-GnRH)y wherein GnRH is a GnRH
polypeptide, peptide or epitope, X is one or more molecules selected from the group consisting of a peptide linkage, an amino acid spacer group and [GnRH]", where n is an integer greater than or equal to 1, y is an integer greater than or equal to 1, and further wherein "GnRH" may comprise any GnRH polypeptide, peptide or epitope. Y may therefore define 1-40 or more repeating units, more preferably, 1-30 repeating units and most preferably, 1-20 repeating units. Further, the selected GnRH sequences may all be the same, or may correspond to different derivatives, analog, variants or epitopes of GnRH, so long as t~cy re,taixt the ,ability to ,elxcat :an immune responses Additionally, if the GnRH unit: are linked either chemically or recombinantly to a carrier, GnRH molecules may be linked to either the 5'-end, the 3'-end, or may flank the carrier in question. Further, the GnRH
multimer may be located at sites internal to the earner. Particularly contemplated herein are multimers of GnRH including repeating sequences of GnRH polypeptides such as multimers including 2, 4, 8, 16, 32 copies, etc. of one or more GnRH polypeptides, optionally including spacer sequences, such as those described in U.S. Patent Nos. 5,837,268 and 6,521,746, incorporated herein by reference in their entirties. A representative multimer of GnRH is shown in Figure 1 B herein. Such multimers are described more fully below.

The term "epitope" refers to the site on an antigen or hapten to which a specific antibody molecule binds. Since GnRH is a very small molecule, the identification of epitopes thereof which are able to elicit an antibody response is readily accomplished using techniques well known in the art. Regions of a given polypeptide that include an epitope can be identified using any number of epitope mapping technique, well known in the art. fee, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E.
Morns, Ed., 1996) Humans Press, Totowa, New Jersey. For example, linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., U.S. Patent No. 4,708,871; Geysen et al.
(1984) Proc. Natl.
Acad. Sci. USA 81:3998-4002; Geysen et al. (1985) Proc. Natl. Acad. Sci. USA
82:178-182; Geysen et al. (1986) Molec. Immunol. 23:709-715, all incorporated herein by reference in their entireties. Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra.
Antigenic regions of proteins can also be identified using standard antigenicity and hydropathy plots, such as those calculated using, e.g., the Omiga version 1.0 software program available from the Oxford Molecular Group. This computer program employs the Hopp/Woods method, Hopp et al., Proc. Natl. Acad. Sci USA (1981) 78:3824-3828 for determining antigenicity profiles, and the Kyte-Doolittle technique, Kyte et al., ,7. Mol. Biol.
(198x) 157:105-132 for hydropathy plots.
As used herein the term "T-cell epitope" refers to a feature of a peptide structure which is capable of inducing T-cell immunity towards the peptide structure or an associated hapten. In this regard, it is accepted in the art that T-cell epitopes comprise linear peptide determinants that assume extended conformations within the peptide-binding cleft of MHC
molecules, (Unanue et al., Science (1987) 236:551-557). Conversion of polypeptides to MHC class II-associated linear peptide determinants (generally between S - 14 amino acids in length) is termed "antigen processing" which is carried out by antigen presenting cells (APCs). More particularly, a T-cell epitope is defined by local features of a short peptide ~tn~, ~tc~ a~ pr'~m~y amino acid sequence properties involving charge and hydrophobicity, and certain types of secondary structure, such as helicity, that do not depend on the folding of the entire polypeptide. Further, it is believed that short peptides capable of recognition by helper T-cells are generally amphipathic structures comprising a hydrophobic side (for interaction with the MHC molecule) and a hydrophilic side (for interacting with the T-cell receptor), (Margalit et al., Computer Prediction of T cell Epitopes, New Generation Vaccines Marcel-Dekker, Inc, ed. G.C. Woodrow et al., (1990) pp. 109-116) and further that the amphipathic structures have an a-helical configuration (see, e.g., Spouge et al., J.
Immunol. (1987) 138:204-212; Berkower et al., J. Immunol. (1986) 136:2498-2503).
Hence, segments of proteins which include T-cell epitopes can be readily predicted using numerous computer programs. (See e.g., Margalit et al., Computer Prediction of T cell Epitopes, New Generation Vaccines Marcel-Dekker, Inc, ed. G.C. Woodrow et al., (1990) pp. 109-116). Such programs generally compare the amino acid sequence of a peptide to sequences known to induce a T-cell response, and search for patterns of amino acids which are believed to be required for a T-cell epitope.
An "immunogenic protein" or "immunogenic amino acid sequence" is a protein or amino acid sequence, respectively, which elicits an immunological response in a subject to which it is administered. Under the invention, an "immunogenic" GnRH multimer refers to a GnRH molecule which, when fused to a carrier molecule, such as a leukotoxin polypeptide as described herein, and introduced into a host subject, stimulates an immunological response and preferably suppresses testosterone secretion.
An "immunological response" to an antigen or vaccine is the development in the host of a cellular and/or antibody-mediated immune r~~ponse to the composition or vaccine of interest. Usually, such a response includes but is not limited to one or more of the following effects; the production of antibodies, B cells, helper T cells, suppressor T
cells, and/or cytotoxic T cells and/or y8 T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. An immunological response can be detected using any of several immunoassays well known in the art.
The term "leukotoxin polypeptide" or "LKT polypeptide" intends a polypeptide which serves as a carrier for a GnRH multimer. Generally, an LKT polypeptide will include at least one T-cell epitope. Moreover, an LKT polypeptide is derived from a protein llridg t~ fih'~ ~~ly o~molecules characterized by the carboxy-terminus consensus amino acid sequence Gly-Gly-X-Gly-X-Asp (Highlander et al., DNA (1989) 8:15-28), where X is Lys, Asp, Val or Asn. Such proteins include, among others, leukotoxins derived from P.
haemolytica and Actinobacillus pleuropneumoniae, as well as E. coli alpha hemolysin (Strathdee et al., Infect. Immun. (1987) 55:3233-3236; Lo, Can. J. Vet. Res.
(1990) 54:533-535; Welch, Mol. Microbiol. (1991) 5:521-528). This family of toxins is known as the "RTX" family of toxins (Lo, Can. J. Vet. Res. (1990) 54:533-535). In addition, the term "leukotoxin polypeptide" refers to a leukotoxin polypeptide which is chemically synthesized, isolated from an organism expressing the same, or recombinantly produced.
Furthermore, the term intends a protein having an amino acid sequence substantially homologous to a contiguous amino acid sequence found in the particular native leukotoxin molecule. Thus, the term includes both full-length and partial sequences, as well as analogues. Although native full-length leukotoxins display cytotoxic activity, the term "leukotoxin" also intends molecules which remain useful carriers for GnRH yet lack the cytotoxic character of native leukotoxins. The nucleotide sequences and corresponding amino acid sequences for several leukotoxins are known. See, e.g., U.S. Patent Nos. 4,957,739 and 5,055,400; Lo et al., Infect.
Immun. (1985) 50:667-67; Lo et al., Infect. Immun. (1987) 55:1987-1996;
Strathdee et al., Infect. Immun. (1987) 55:3233-3236; Highlander et al., DNA (1989) 8:15-28;
Welch, Mol.
Microbiol. (1991) 5:521-528. In the chimeras produced according to the present invention, a selected leukotoxin polypeptide sequence imparts enhanced immunogenicity to one or more fused GnRH multimers by providing, among other things, T-cell epitopes comprising small peptide segments in the range of five to fourteen amino acids in length which are capable of complexing.with MHC clues II molecules: for presentation to, and activation of, T-helper cells. As discussed further below, these T-cell epitopes occur throughout the leukotoxin molecule and are thought to be concentrated in the N-terminus portions of leukotoxin, i.e., between amino acid residues 1 to 199.
The term "GnRH multimer/leukotoxin chimera" intends a molecule comprising a GnRH multimer, as defined above, and a leukotoxin polypeptide, as defined above. The GnRH multimer can be present at the 5' end, the 3' end, or both the 5' and 3' ends of the leukotoxin polypeptide. Moreover, the term is not limited by the method of production of the chimera. Thus, the chimera can be produced recombinantly by a fusion between the use encol#Ing the iJr~rnu~f~m~r anti the leukvtoxin polypep~.d~; or the GnRH

multimer and the leukotoxin polypeptide can be chemically fused after individual production of the multimer and the leukotoxin. Alternatively, the molecule can be chemically synthesized.
As used herein, a leukotoxin polypeptide "which lacks cytotoxic activity"
refers to a leukotoxin polypeptide as described above which lacks significant cytotoxicity as compared to a native, full-length leukotoxin (such as the full-length P, haemolytica leukotoxin described in U.S. Patent Nos. 5,055,400 and 4,957,739) yet still retains immunogenicity and at least one T-cell epitope. Leukotoxin polypeptides can be tested for cytotoxic activity using any of several known assays such as the lactate dehydrogenase release assay, described by Korzeniewski et al., Journal of Immunological Methods 64:313-320, wherein cytotoxicity is measured by the release of lactate dehydrogenase from bovine neutrophils. A
leukotoxin molecule is identified as cytotoxic if it causes a statistically significant release of lactate dehydrogenase when compared to a control non-cytotoxic molecule.
The provision of LKT-GnRH chimeras comprising leukotoxin polypeptides which lack cytotoxic activity provides several important benefits. A leukotoxin polypeptide which lacks cytotoxic activity is desirable since the injection of an active toxin into a subject can result in localized cell death (PMNs and macrophages) and, in tum, cause a severe inflammatory response and abscess at the injection site. Cytotoxic activity resulting in the killing of macrophages may lead to reduced antigen presentation and hence a suboptimal immune response. The removal of the cytotoxic portion as found in the non-cytotoxic LKT
polypeptides used in producing the fusion proteins of the invention also results in a truncated LKT gene which is capable of being exprat much higher levels than full-length LKT.
Further, the use of non-cytotoxic LKT polypeptides in the fusions constructed herein which retain sufficient T-cell antigenicity reduces the overall amount of leukotoxin-GnRH antigen which needs to be administered to a host subject to yield a sufficient B-cell response to the selected GnRH polypeptides. A particular example of an immunogenic leukotoxin polypeptide which lacks cytotoxic activity is LKT 111. The nucleotide sequence of this gene and the corresponding amino acid sequence are disclosed in U.S. Patent Nos.
5,837,268 and 6,521,746, incorporated herein by reference in their entireties. The LKT 111 nucleotide and amino acid sequences are found at positions 334-1779 and 112-592, respectively, of Figures 2A-~F' herein. '~'~ie gene includes an internal deletion approximately 1300 by in length. The LKT 111 polypeptide has an estimated molecular weight of 52 kDa, but retains portions of the leukotoxin molecule containing T-cell epitopes which are necessary for sufficient T-cell immunogenicity, and portions of the leukotoxin C-terminus containing convenient restriction sites for use in producing fusion proteins for use in the present invention.
An "immunogenic composition" is a composition that comprises at least one immunogenic polypeptide (e.g., an immunogenic GnRH multimer/leukotoxin chimera).
Hy "immunological adjuvant" is meant an agent which acts in a nonspecific manner to increase an immune response to a particular antigen, thus reducing the quantity of antigen necessary in any given vaccine, and/or the frequency of injection necessary in order to generate an adequate immune response to the antigen of interest. See, e.g., A.C. Allison J. Reticuloendothel. Soc. (1979) 26:619-630.
"Substantially purified" generally refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance comprises the majority percent of the sample in which it resides. Typically in a sample a substantially purified component comprises SO%, preferably 80%-85%, more preferably 90-95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.
By "isolated" is meant, when referring to a polypeptide, that the indicated molecule is separate and discrete from the whole organism with which the molecule is found in nature or is present in the substantial absence of other biological macro-molecules of the same type.
Tl~ ~"~ _ "with tit to a polynucl~tide~ is~ nucleic ~aeid mol~aut~ e3~~vd, i~r' whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome.
The term "derived from" as it is used herein, denotes an actual or theoretical source or origin of the subject molecule or immunogen. For example, an immunogen that is "derived from" a prarticular GnRH molecule will bear close sequ~rice similarity with a relevant portion of the reference molecule. Thus, an immunogen that is "derived from" a particular GnRH
molecule may include all of the wild-type GnRH sequence, or may be altered by insertion, ~el'ron .or s~b~titt~tion o-amltio acid residues, so long as the derived sequence provides for an immunogen that corresponds to the targeted GnRH molecule. Immunogens derived from a denoted molecule will contain at least one epitope specific to the denoted molecule.
By a "reduction in serum testosterone" is meant a statistically significant reduction in serum testosterone levels as measured using a standard assay, as described herein, as compared with the serum testosterone levels expected in a typical uncastrated, untreated male, of the same age and species. For example, testosterone can be measured using ELISAs and RIAs well known in the art. Particularly convenient measures may be made using commercially available test kits, e.g., The ICN Biomedicals, Inc. (Costa Mesa, CA) double antibody RIA kit. Another test uses the Coat-A-Count Total Testosterone KitTM
(Diagnostic Products Corporation, Los Angeles, CA). This kit is a solid-phase RIA designed for the quantitative measurement of testosterone in serum, based on testosterone-specific antibody immobilized to the wall of a polypropylene tube. See, also Schanbacher et al., J. Andrology (1982) 3:45-51, for a description of a direct RIA for determining testosterone levels. In preferred embodiments, testosterone levels are reduced to castration levels (e.g., less than 50 1 S ng/dL and preferably less than 30 ng/dL).
By a "substantially reduced" level of testosterone is meant that the level of testosterone is at least about 50% less than expected in a typical uncastrated, untreated male, of the same age, preferably at least about 75% less, and more preferably at least about 80% to 90% or less.
"Homology" refers to the percent identity between two polynucleotide or two polypeptide moieties. Two nucleic acid, or two polypeptide sequences are "substantially h~l~~lpgpus" to e,~eh other when the aequ$nces axhibit at least about 5041n , preferably at least about 75%, more preferably at least about 80%-85%, preferably at least about 90%, and most preferably at least about 95%-98% sequence identity over a defined length of the molecules. As used herein, substantially homologous also refers to sequences showing complete identity to the specified sequence.
In general, "identity" refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity can be determined by a direct comparison of the sequence information between two molecules (the reference sequence and a sequence with unknown i~dtfit~ tip the reference sequence) by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the reference sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M.O. Dayhoff ed., 5 Suppl. 3:353-358, National biomedical Research Foundation, Washington, DC, which adapts the local homology algorithm of Smith and Waterman Advances in Appl. Math. 2:482-489, 1981 for peptide analysis. Programs for determining nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, W17 for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent identity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.
Another method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the "Match" value reflects "sequence identity." Other suitable programs for calculating the percent identity or similarity betwe~tn sZquences are gencrally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters:
genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10;
Matrix =
BLOSUM62; Descriptions '= 50 sequences; sort by = HIGH SCORE; Databases =
non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + Swiss protein + Spupdate + PIR. Details of these programs are readily available.
Alternatively, homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion tivltl'i ~inTe-stranded-specific nuclease(s), and size determination of the digested fragments.

DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art.
See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.
"Recombinant" as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation is not associated with all or a portion of the polynucleotide with which it is associated in nature. The term "recombinant" as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide.
In general, the gene of interest is cloned and then expressed in transformed organisms, as described further below. The host organism expresses the foreign gene to produce the protein under expression conditions.
By "mammalian subject" is meant any mammal including, without limitation, mammals such as rodents, cattle, pigs, sheep, goats, horses and humans; and domestic 1 S animals such as dogs and cats. The term does not denote a particular age.
Thus, both adult and newborn animals are intended to be covered.
II. MODES OF CARRYING OUT THE INVENTION
Before describing the present invention in detail, it is to be understood that this invention is not limited to particular formulations or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.
Although a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein.
The present invention is based on the discovery of novel immunogenic compositions that induce anti-GnRH antibodies and that have a measurable effect on circulating serum testosterone levels. Hence, the present compositions are useful for any condition in which decreased testosterone levels are desired, such as in the treatment of prostate cancer or to regulate fertility. The compositions herein include GnRH multimers provided as fusions with leukotoxin polypeptides in combination with aluminum-based adjuvants and one or more Montanide adjuvants (Seppic, Paris, France), a group of oiUsurfactant-based adjuvants in which different surfactants are combined with either a non-metabolizable mineral oil, a metabolizable oil, or a mixture of the two. Particularly prefer ed is the use of a Montanide adjuvant suitable for use in humans, such as Montanide ISA 51~ or Montanide ISA 720TM.
Montanide ISA S1TM includes approximately 8-12% by weight mannide oleate and 88-92%
by weight Drakeol 6VR mineral oil. The aluminum-bawd adjuvants pr~aent in the compositions are generally either aluminum phosphate or aluminum hydroxide (alum), preferably in the form of an aluminum gel, such as an aluminum hydroxide gel (alhydrogel).
The leukotoxin polypeptide acts as a carrier protein which presents selected GnRH multimers to a subject's immune system in a highly immunogenic form. Although GnRH is generally recognized as "self ' and hence nonimmunogenic, the compositions described herein are able to confer superior immunogenicity to the GnRH/leukotoxin chimeras. The Montanide-containing formulations are surprisingly superior to formulations of GnRH/leukotoxin chimeras that do not include a Montanide adjuvant.
Preferably, the immune response generated to the compositions of the present invention is sufficient to substantially reduce serum testosterone levels. In particularly preferred embodiments, serum testosterone levels are reduced to less than 100 ng/dL, preferably to castration levels of less than 50 ng/dL, and even more preferably to less than 30 ng/dL, measured using a commercially available RIA assay, such as The ICN
Biomedicals, Inc. (Costa Mesa, CA) double antibody RIA kit or the Coat-A-Count Total Testosterone KitTM (Diagnostic Products Corporation, Los Angeles, CA). Moreover, these levels are pr~f~tly acbisareti within x:20 weep or 1~, preferably within 6415 w~slcs or 1, more preferably within 6-12 weeks or less from the beginning of treatment. In preferred embodiments, reduced levels of testosterone are maintained for a minimum of at least 30 days, preferably at least 60 days and even more preferably at least 90 days after initiation of treatment. Thus, the formulations provide a reliable and effective alternative to invasive sterilization procedures currently practiced in treating such diseases as prostate cancer, such as surgical castration, and the like.

The leukotoxin chimeras include a leukotoxin polypeptide fused to more than one GnRH polypeptide. Particular embodiments of the present invention include chimeras comprising a leukotoxin polypeptide fused to one or more GnRH multimers, wherein the multimers have at least one repeating GnRH decapeptide sequence, or at least one repeating unit of a sequence corresponding to at least one epitope of a selected GnRH
molecule.
Further, the selected GnRH peptide sequences may all be the same, or may correspond to different derivatives, analogues, variants or epitopes of GnRH so long as they retain the ability to elicit an immune response and preferably reduce testosterone levels. A
representative nucleotide sequence of a GnRH decapeptide is depicted in Figure 1 A. The depicted GnRH sequence is modified by the substitution of a glutamine residue at the N-terminal in place of pyroglutamic acid which is found in the native sequence.
This particular substitution renders a molecule that retains the native glutamic acid structure but also preserves the uncharged structure of pyroglutamate. Accordingly, the resulting peptide does not require cyclization of the glutamic acid residue and may be produced in the absence of conditions necessary to effect cyclization.
In order to further an understanding of the invention, a more detailed discussion is provided below regarding GnRH multimers and chimeras with leukotoxin polypeptides, as well as the subject compositions and methods.
GnRH Multimers and GnRH Leukotoxin Chimeras As explained above, the formulations of the present invention include GnRH
ncttrltim~=fui ~ lotoxin polypeptides. The GnRH multimer will have more than one copy of a selected GnRH polypeptide, peptide or epitope, as described above, or multiple tandem repeats of a selected GnRH polypeptide, peptide or epitope. Thus, the GnRH
multimers may comprise either multiple or tandem repeats of selected GnRH
sequences, multiple or tandem repeats of selected GnRH epitopes, or any conceivable combination ther~f. GnRH epi~pe~ may be identified using techniques as described in detail above.
For example, the GnRH multimer may correspond to a molecule with repeating units of the general formula (GnRH-X-GnRH)y wherein GnRH is a GnRH polypeptide, X is selected from the group consisting of a peptide linkage, an amino acid spacer group, and ",'tt~ ~t~-'! "1't'~ 1, y is an integer greater i~an or equal to 1, and further wherein "GnRH" may comprise any GnRH polypeptide. Thus, the GnRH
multimer may contain from 2-64 or more GnRH polypeptides, mare preferably 2-32 or 2-16 GnRH polypeptid~. Further, the selected GnRH sequences may all be the same, or may correspond to different derivatives, analogues, variants or epitopes of GnRH
so long as they retain the ability to elicit an immune response.
Spacer sequences may be present between the GnRH moieties. For example, Ser-Gly-Ser trimers and Gly-Ser dimers are present in the GnRH multimers exemplified herein which provide spacers between repeating sequences of the GnRH immunogens. See, e.g., Figure 1B. The strategic placement of various spacer sequences between selected GnRH
polypeptides can be used to confer increased immunogenicity on the subject constructs.
Accordingly, under the invention, a selected spacer sequence may encode a wide variety of moieties such as a single amino acid linker or a sequence of two to several amino acids.
Selected spacer groups may preferably provide enzyme cleavage sites so that the expressed multimer can be processed by proteolytic enzymes in vivo (by APCs, or the like) to yield a number of peptides, each of which contain at least one T-cell epitope derived from the carrier portion, and which are preferably fused to a substantially complete GnRH
polypeptide sequence.
The spacer groups may be constructed so that the junction region between selected GnRH moieties comprises a clearly foreign sequence to the immunized subject, thereby conferring enhanced immunogenicity upon the associated GnRFi immunogens.
Additionally, spacer sequences may be constructed so as to provide T-cell antigenicity, such as those ~quenwhich encode amphipathic and/or a-hclical peptide uenc'~ which a~ geiWlly recognized in the art as providing immunogenic helper T-cell epitopes. The choice of particular T-cell epitopes to be provided by such spacer sequences rnay vary depending on the particular vertebrate species to be vaccinated. Although particular GnRH
portions are exemplified which include spacer sequences, it is also an object of the invention to provide one or more GnRH multimers comprising directly adjacent GnRH sequences (without intervening spacer sequences).
The GnRH multimeric sequence thus produced renders a highly immunogenic GnRH
antigen for coupling to the leukotoxin carriers used in the compositions of the invention.

Leukotoxin polypeptides have been found to impart excellent immunogenicity to GnRH when in association with the molecule. These carriers serve to non-specifically stimulate T-helper cell activity and to help direct an immunogen of interest to antigen presenting cells (APCs) for processing and presentation at the cell surface in association with mple~ule~ of the major histocompatibility complex (MHC). Accordingly, the present invention utilizes GnRH multimerlleukotoxin chimeras to provide highly immunogenic compositions.
For example, leukotoxin polypeptides, as defined above, such as a Pasteurella haemolytica leukotoxin (LKT) polypeptide can be fused to the GnRH multimer of interest.
The nucleotide sequences and corresponding amino acid sequences for several leukotoxin carriers are known. See, e.g., U.S. Patent Nos. 5,422,110, 5,708,155, 5,723,129 and 6,521,746, all of which are incorporated herein by reference in their entirties. Particular examples of immunogenic leukotoxin polypeptides for use with the GnRH
multimers include LKT 342, LKT 352, LKT 111, LKT 326 and LKT 101 which are described in the patents cited above. Particularly preferred are LKT 111 and LKT 114. The gene encoding includes an internal deletion of approximately 1300 by in length from the native leuktoxin gene. The LKT 111 polypeptide has an estimated molecular weight of 52 kDa (as compared to the approximately 100 kDa full-length leukotoxin), but retains portions of the full-length leukotoxin N-terminus containing T-cell epitopes which are necessary for sufficient T-cell immunogenicity, and portions of the full-length leukotoxin C-terminus containing convenient restriction sites for use in producing the fusion proteins of the present invention. LKT 114 d~'tffeEcs from tT 111 by virtue of an additional amino acid deletion from the internal portion of the molecule. See, e.g., U.S. Patent Nos. 5,837,268 and 6,521,746, for descriptions of these molecules. The LKT 111 nucleotide and amino acid sequences are found at positions 334-1779 and 112-592, respectively, of Figures 2A-2F.
Additionally, the GnRH immunogens may be linked to either the 5'-end, the 3'-end, or may flank the carrier in question. Further, the GnRH multimer may be located at sites internal to the carrier. A particularly preferred GnRH leukotoxin chimera for use with the present invention is represented by the GnRH leukotoxin chimera shown in Figures 2A-2F
herein. This chimera contains 8 tandem repeats of the GnRH sequence fused to both the S' arid ~' en~~ ol! a tl~~ 111 leuk~toxt~ polypeptide. Each alternating GnRH
sequence has a change in the fourth base in the sequence from cytosine to guanosine. This results in a single amino acid change in the second amino acid of the GnRH molecule from His to Asp. See, Figures lA and 1B.
The GnRH multimers and chimeras can be produced using a number of techniques well known in the art. In particular, GnRH polypeptides can be isolated directly from native sources, using standard purification techniques. Alternatively, the polypeptides, multimers and chimeras can be recombinantly produced using nucleic acid expression systems, well known in the art and described in, e.g., Sambrook et al., supra. Methods of recombinantly producing GnRH multimers and GnRH leukotoxin chimeras are well known and described in e.g., U.S. Patent Nos. 5,837,268 and 6,521,746, incorporated herein by reference in their entirties. GnRH polypeptides can also be synthesized using chemical polymer syntheses such as solid phase peptide synthesis. Such methods are known to those skilled in the art.
See, e.g., J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, IL (1984) and G. Barany and R. B. Merrifield, The Peptides:
Analysis, Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 2, Academic Press, New York, (1980), pp. 3-254, for solid phase peptide synthesis techniques.
Once obtained, the GnRH leukotoxin chimeras may be formulated into compositions, such as vaccine compositions as described further below, in order to elicit antibody production.
GnRH Compositions Qz~ce the ,~bov~GnRH lee>leotoxin chimeras are produced, they are formulated into compositions for delivery to a mammalian subject in order to stimulate an immune response.
The polypeptides may be formulated into compositions in either neutral or salt forms.
Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the active polypeptides) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

Also present in the compositions of the present invention is an aluminum-based adjuvant, such as aluminum phosphate or aluminum hydroxide. Preferably, the adjuvant is an aluminum-based gel, such as an alhydrogel. Such aluminum adjuvants are well known in the art. The compositions of the present invention also include a Montanide adjuvant, preferably suitabhfor use in humans, such as Montanide ISA 51~ or Montanide ISA 720TM, available from Seppic (Paris, France). The compositions also generally include a pharmaceutically acceptable vehicle or excipient. Suitable vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and additional adjuvants. The compositions of the present invention can also include ancillary substances, such as pharmacological agents, cytokines, or other biological response modifiers.
The compositions of the present invention are normally prepared as injectables.
Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 18th edition, 1990. The composition to be administered will contain a quantity of the GnRH muhimerlleukotoxin chimera adequate to achieve the desired state in the subject being treated, the exact amount being readily determined by one skilled in the art, wherein the amount depend on the animal to be tre,~ted, the capacity of the animal's immune system to synthesize antibodies, and the degree of immunoneutralization of GnRH desired.
For formulations to be administered to humans, the amount of GnRH
multimer/leukotoxin chimera present in the compositions will generally be approximately 20 p.g to about 2 mg, more generally about 50 pg to about 1 mg, more preferably 100 wg to 400 N.g, such as 150... 175... 180... 190... 200... 220... 250... 300... 350...
400... 450... 500... 1000...
2000 pg, or any amount within these stated ranges, per mL of injected solution.
The ratio of GnRH multimer to leukotoxin carrier present in the vaccine formulation will vary based on the particular leuktoxin carrier and GnRH multimer selected to construct such molecules. One preferred vaccine composition contains a GnRH
multimerlleukotoxin chimera having about 1 to 90% GnRH, preferably about 3 to 80% and most preferably about t'o ~~'o r ~lypd#de per fusion molecule. Increases in the percentage of ~nRH

present in the LKT-GnRH fusions reduce the amount of total antigen which must be administered to a subject in order to elicit a sufficient inununological response to GnRH, The amount of aluminum-based adjuvant present, such as alhydrogel (an Al(OH)3 gel), will vary depending on the amount of aluminum prc~scnt in the alhydrogel. Typically, the amount of alhydrogel present will be on the order of about 0.1 % to about 1 % (w/v), preferably about 0.2% to about .7S%, such as 0.2... 0.25... 0.3... 0.35...
0.4... O.S... 0.6... 0.7...
0.8... 0.9... 1 %, or any amount within these stated ranges. The content of aluminum in the alhydrogel will typically range from about .S mg to about 10 mg, preferably .7S mg to S mg, more preferably 1 mg to 3 mg, such as 0.5... .75... 1Ø.. 1.25... 1.5...
2Ø.. 2.5... 3Ø.. 3.5...
4Ø.. 5Ø.. 6Ø.. 7Ø.. 8Ø.. 9Ø.. 10 mg of aluminum, or any amount within these stated ranges.
The amount of Montanide present per mL of liquid will generally be about .2S
mL to 7S mL, such as .25... .3... .35... .4... .45... .5... .?S mL of Montanide per mL of formulation.
The formulations are generally prepared by mixing the GnRH multimer/leukotoxin chimera, 1 S aluminum-based adjuvant and appropriate excipients, adding water to the desired volume, adding Montanide and homogenizing the composition, using methods well known in the art.
The subject is administered a primary immunization, in at least one dose, and optionally, two or more doses. The primary administrations) is followed with one or more boosts with the same or different GnRH compositions, in order to substantially reduce the circulating level of testosterone. Particularly useful, is the administration of multiple doses of the compositions of the present invention over a sustained period of time.
For example, subj:eets: can be administered from 2-20 doses, such as 4-10, doses, over a period of months or years. Typically, doses will initially be administered more frequently, such as every one to three weeks, e.g., every 10 days to two weeks. After several treatments, e.g., 4-6 2S treatments, dosing can be spread further apart, such as monthly. Thus, one particular dosing regimen would be initial delivery (day 1 ) with boosting at days 1 S, 29, 43 and S7 (i.e., every two weeks), followed by boosts on or near a monthly basis, such as at 90, 120, 1 SO days, and the like.
Any suitable pharmaceutical delivery means may be employed to deliver the compositions to the vertebrate subject. For example, conventional needle syringes, spring or eased =g~ ij ~tj~(~'.. less. 1,,'to moot; f,'';~ to Laurens;

3,853,125 to Clark et al.; 4,596,556 to Morrow et al.; and 5,062,830 to Dunlap), liquid jet injectors (U.S. Patent Nos. 2,754,818 to Scherer; 3,330,276 to Gordon; and 4,518,385 to Lindmayer et al.), and particle injectors (LJ.S. Patent Nos. 5,149,655 to McCabe et al. and 5,204,253 to Sanford et al.) are all appropriate for delivery of the compositions.
Preferably, the composition is administered intramuscularly, subcutaneously, intravenously, subdermally, intradennally, transdennally or transmucosally to the subject. If a jet injector is used, a single jet of the liquid vaccine composition is ejected under high pressure and velocity, e.g., 1200-1400 PSI, thereby creating an opening in the skin and penetrating to depths suitable for immunization.
Prior, concurrent with, or subsequent to vaccination with the GnRH
formulations of the present invention, the subject can be given an agent to potentiate the immune response.
This is especially useful in patients that are immunocompromised. Such agents are known in the art and include, any agent with T-cell suppressing activity including, without limitation, various cytokines, as well any of the various chemotherapeutics such as cisplatin and 1 S particularly, chemotherapeutics useful in the treatment of prostate cancer, such as mitoxantrone and the like. Particularly useful is cyclophosphamide. If administered concurrently, these agents can be provided in the same or in a different composition. If administered prior to the vaccine administration, generally the agent is delivered 1 month to 1 day prior to vaccine delivery, such as 14 days to 1 day, preferably 7 days to 1 day, such as S days to 3 days prior, to vaccine delivery. If administered subsequent to vaccine delivery, the agent will typically be administered 1 day to 1 month following delivery, preferably 1 c~;y to 14 days subsequent to delivery, even more preferably 1 day to 7 days;
following delivery, such as 3 days to 5 days after delivery. Moreover, the agent can be administered only once, e.g., prior to, concurrent with, or subsequent to the first vaccination, or may be administered multiple times. The amount to be provided is readily determined by one of skill in the art. For example, if cyclophosmamide is used, generally 100 rng/m2 to approximately 500 mg/m2, preferably 150 mg/mz to about 400 mg/m2, even more preferably 200 mg/m2 to 300 mg/mZ.

III. Experimental Below are examples of specific embodiments for carrying out the present invention.
The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.
Example 1 v ~ ~ - ~~, ~~ ~~ a ~;;
y~~"0~;_ With Alhydroael and Montanide to Reduce Testosterone levels In order to study the safety and immunogenicity of a vaccine formulation composed of a GnRH multimer/leukotoxin chimera (IPS-21 ) formulated with alhydrogel and fiuther emulsified with Montanide ISA 51~, the following study was performed on patients with adenocarcinoma of the prostate.
~~. , ~~~ ,~~~~ ~. fY r ~'l~'~~
1. Patient Population. Patients with the following criteria were included in this study:
(1) Male between the ages of 18 to 77 years old inclusive (2) Had biopsy-proven adenocarcinoma of the prostate (3) Was a candidate for non-urgent chemical or surgical castration (Hormone Sensitive Prostate Cancer) (4) Had a serum testosterone level >200 ng/dL
(5) Was able and willing to follow instructions and able to make all required study visits (6) Was able and willing to give written consent to participate in this study (7) Had life expectancy of at least 6 months (8) Had an ECOG status of 0 or 1.
Patients with the following criteria were excluded from this study (1) Known allergy or hypersensitivity to test article ingredients (2) Any prior history of prescription drug hormonal therapy within the past 3 months (3) Hormone-refractory prostate cancer (4) Chemotherapeutic agents for any malignancy within 4 weeks prior to study entry (5) Requirement for urgent castration including known pathological fractures or spinal cord involvement, urethral or ureteral obstruction, or disseminated intravascular coagulation (DIC) (6) Clinically significant cardiovascular, pulmonary, renal, endocrine, hepatic, respiratory, neumlogic, psychiatric, immunologic, gastrointestinal, hematologic, metabolic or any other condition or laboratory abnormality that, in the opinion of the investigator deemed the patient unsuitable for participation in the study (7) ECOG performance status greater than 1 (8) Another primary cancer (excluding basal cell skin cancer) during the past 5 years or currently (9)Received treatment with any other investigational drug within the preceding one (1) month (10)Any recent therapy (<6 months) with immunosuppressive agents (11) Any condition that might render the patient immunocompromised, e.g., autoimmune disorders, including systemic lupus erythematosus (SLE) or rheumatoid arthritis Potential study participants underw~t a scrc3ening evaluation consisting of a medical history, general physical examination, and laboratory tests to assess general medical condition and prostate cancer status. Patients were assigned to a treatment regimen if they met all of the inclusion criteria and none of the exclusion criteria. Within two weeks of the first vaccine administration, patients were evaluated for GnRH antibody levels, as well as serum PSA and serum testosterone levels.
II. GnRN Multimers. The GnRH multimers used in the vaccine consisted of a carrier protein from Pasturella hemolytica, with eight copies of GnRH at the 5' and 3' end of the leukotoxin ''"' 1"~oWh~ an aspartic acid residue in place of histidine at the second position from the amino terminus of the GnRH peptide. This formulation was called the "IPS-21 antigen." This antigen was produced recombinantly in E. coli. as described in U.S. Patent No.6.521.746, incorporated herein by reference in its entirety.
The nucleotide and amino acid sequence of the IPS-21 antigen is shown in Figures 2A-2F herein.
S
Ill. Vaccine Composition. This vaccine was prepared from stock solutions provides as (1) 3 mL vial containing 1 mL of the antigen with alhydrogel (this mixture was termed "NOR-1"), (2) a 5 mL ampoule of Montanide ISA S 1TM containing 3 mL of the adjuvant, and (3) an empty sterile 10 mL vial for mixing. The individual components are as follows in the tables below.
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To prepare the vaccine, the Montanide ISA 51~ and antigen solutions were mixed in the clinic prior to injection. 1.0 mL of Montanide ISA 51~'' was withdrawn from the Montanide ISA 51 T"' vial and put into the third sterile vial. 1.0 mL of NOR-1 was also withdrawn and put into the third vial now containing the Montanide ISA S lr"".
This process was done with a 3 mL syringe (rubber free, latex free piston) with a size 18-type needle. The contents of the vial were then homogenized by withdrawing back and forth with the syringe into a vial 10 times. A final 1.0 mL of the emulsion, comprising the ingredients first referenced above, was withdrawn for IM injection in the gluteal region using a size 20 gauge 1 %z type needle.
It will be appreciated that, if desired, controlled and alternative dilution schemes will provide vaccine compositions containing unit doses of antigen different from the 200 pg dose exemplified above.
Each patient also received cyclophosphamide 3 days before the first immunization.
The cyclophosphamide was given at a dose of 200 mg per meters as an intravenous bolus.
For this propose, SOOmg of cyclophosphamide was provided in a vial with 25 mL
of sterile water for injection USP, and 10 mL was injected per meter squared of body surface. In the alternative, an equivalent dose of cyclophosphamide can be given orally.
IY. Serum Testosterone. Serum testosterone levels were evaluated as follows.
The ICN
Biomedicals, Inc. (Costa Mesa, CA) double antibody RIA kit was used to measure total testosterone. In the assay, a limited amount of specific rabbit polyclonal antibody is reacted with :t~estostelabeled with IZSI. Upon ~eidition of:an incriamount of non-labeled testosterone in the patient sample, a correspond decreased fraction of radiolabel led testosterone is bound to the antibody. After separation of the bound from the free radiolabel led testosterone using a precipitating antibody, the amount of radioactivity remaining is measured and the testosterone concentration is determined from a standard curve.
Y. Sere~m PSA. Serum PSA levels were evaluated as follows. The Abbott Axsym methodology (Abbott Park, IL) was used to measure total PSA. The method is based on Microparticle Enzyme Immunoassay (MEIA) technology. Sample, anti-PSA coated mir~pa~ialW t~t~ifi ie~togh~ in one well oi"'~he reaction vessel.
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During the incubation of this reaction mixture, the PSA in the specimen binds to the anti-PSA
coated microparticles, forming an antibody-antigen complex. An aliquot of the reaction mixture is transferred to the matrix cell. The microparticles bind irreversibly to the glass fiber matrix. The matrix cell is washed to remove unbound materials. The anti-PSA
alkaline phosphatase conjugate is dispensed onto the matrix cell and binds to the antibody-antigen complex. The matrix cell is washed to remove unbound materials. The substrate, methylumbelliferyl phosphate, is added to the matrix cell and the fluorescent product is measured by the MEIA optical assembly.
VI. GnRH Antibody Titers. A radioimmunoassay (RIA) was used to detect antibodies against GnRH in serumlplasma samples. Radiolabeled GnRH ('ZSI-GnRH) was added to the samples and following a 48 hour incubation period, the antibody-bound l2sl -GnRH was separated from the free'ZSI -GnRH by a charcoal/centrifugation step. The resultant charcoal pellet, which contains the free (unbound) lzsl _GnRH, was counted in a gamma counter.
Samples that contain antibodies to GnRH have lower counts than samples that do not contain GnRH
antibodies.
VII. Administration Schedule. Subjects were divided into three groups, A, B
and C. All subjects received one dose of cyclophosphamide intravenously or orally (200 mg/m2) and then received the first of 4 doses of the vaccine composition 3 days later, administered by the intramuscular route. The vaccine was given intramuscularly on the following schedule:
Group A: Days 1, 15, 29 ark 43 in 12 patients. Patients were followed up on a monthly basis for one year. Patients that developed anti-GnRH antibodies were boosted 4 times during follow-up. Testosterone levels in Group A patients were assessed at Day 90 and the decision whether or not to continue patients in the study was made at that time. Group B patients had their testosterone assessed at Day 180 and were then randomized to either 3 or 6 month boosters (group C).
VIII. General Monitoring Parameters. Within two weeks prior to the first vaccine administration, GnRH antibody levels, as well as serum PSA and serum testosterone levels were evaluated in X11 subjects. While being monitored for efficacy during the first 3 months of treatment, all patients had blood drawn for serum testosterone, PSA
concentrations and for GnRH antibody levels. In addition, the injection site was examined for local reaction for the first three months. Depending on the individual response to treatment, these efficacy determinations are conducted monthly thereafter for 9 more months unless the patient reverts to standard therapy.
IX. Patient Classification from 8 Weeks to 5 Months (Group A). Based on the results of samples taken 8-20 weeks from initiation of treatment, a decision was made to either revert to standard therapy for prostate cancer, to provide a booster vaccination or to continue monitoring the response to therapy. In Groups B and C the decision is made at Day 180 because evidence of immunogenicity was demonstrated in a majority of patients at Day 90 in Group A.
i) Testosterone NOT suppressed to castrate levels at 8-20 weeks If serum testosterone has not reached castration levels (<50 ng/dL) at 8-20 weeks, the patient was considered to be a nonresponder and booster ineli ig ble.
n) ~~'' '~ 'but suppression is not maintained to 3 months If serum testosterone reached castration levels (<SO ng/dL), by the time of the week 8 assessment and the testosterone levels were noted to rise above castration levels in samples taken after 8 weeks up to 3 months following initiation of therapy, the patient was considered to be a responder and booster ell 'able.
ifi) Test~sterane suppre_estad to castrate Levels at 8 we.~s and sugnrassion is maintained to 3 months Patients were classified as responders if they achieved suppression of testosterone concentrations to castrate levels (<SO ng/dL), within 8 weeks of initiation of treatment and maintained suppression until a minimum of 3 months after initiation of treatment. In responders, a single booster dose was administered at months 3, 6, 9 and 1 Z.
X. Monitoring after 6 Months of Treatment Initiation: In patients who are responders, i.e., who have achieved suppression of testosterone concentrations to castration levels (<50 .~k 3:x levels were assayed monthly for 12 months. In responders, patients were randomized to boosters on one of two schedules: at months 6, 9 and 12 or months 6 & 12 of the study. In Groups B and C, all patients receive monthly boosters Month 3. The decision on further boosters will be made at Month 6.
XI. Safety Analysis. The following key safety parameters were evaluated:
1. Adverse events assessments 2. Prostate cancer status 3. Clinical laboratory evaluations 4. Physical examination, vital signs 5. Local injection site reactions.
RESULTS
There were 12 patients entered in the study. All of these subjects were in Group A.
All were males aged 48-77, with a median age of 72. Patients were administered the vaccine according to tlm schedule detailed above. Results for all patients to date are shown in Table 1.

As can be seen in the above table, positive antibody titers were observed in subjects. Testosterone declined sufficiently in 2 of those 6 patients to warrant entry into the booster phase of the study. Castrate testosterone and normalized PSA was subsequently observed in both subjects. Patient RCH left the study as per protocol due to insufficient testoste~orte d~line by Day 90 and was retun~ied to "watchful waiting".
Because positive data was demonstrated in patients after he left the study, he was approached to re-enter the study and did so at Day 210 receiving a booster at that time and continues to receive boosters every 3 months.
A further analysis of the data was conducted on the data looking at the anti-GnRH
antibody assay results in greater detail. In previous studies of a composition as used herein but without the Montanide adjuvant in animals, antibody titers were assessed in terms of binding at the 1:100 dilution across time. Binding at 10% or greater is required for a positive result at a given dilution. Therefore, this analysis was applied to the human data with the following results shown in Table 2. Positive results are indicated.
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Table 2: Percentage Binding at 1:100 Across Time It was concluded from this analysis that 7/12 had positive titers and that a further 3 or 4 patients had rising titers. It thus appeared that the study cut off at Day 90 may have been too early to allow the antibodies to exert their effect. Furthermore, it appears that the fifth dose (Day 90 booster) was important in the testosterone castration process.
Patients AAM
and D1ZB had this booster and shortly thereafter experienced dramatic drops in both testosterone and PSA. Patient AAM did not receive that booster and once he did receive a booster, his testosterone and PSA declined more slowly. Interestingly in patient AAM, despite not having a booster for 168 days, his testosterone level did not begin to revert back to the prestudy level.
As explained above, the safety of the vaccine was also monitored. Results are shown in Table 3. No serious adverse effects were seen in this study.
Table 3: Incidence of Acute Adverse Effects (AE) by Maximum Grade and Visit 9100-OOOIp PATENT
To summarize, the vaccine was well tolerated. Positive anti-GnRH titers were demonstrated in a majority of the patients. The duration of effect of the vaccine may be up to 6 months allowing for boosters every six months in responders.
Further studies are conducted in which the 5'h dose is moved back to Day 57.
Then all patients receive boosters at Days 90, 120 and 150. The decision to continue the subjects is based on castrate testosterone at Day 180. The subjects are then randomized to receive boosters every 3 or 6 months.
In Groups B and C, the decision is made at Day 180 because evidence of immunogenicity was demonstrated in a majority of patients at Day 90 in Group A. The treatment regimen is as follows:
Group B: Days 1, 15, 29, 43 and 57, 90, 120 and 150 in 6 patients. Then boosters at Months 6, 9, 12 Group C: Days 1, 15, 29, 43 and 57, 90, 120 and 150 in 6 patients. Then boosters at Months 6 & 12 Thus, novel GnRH formulations and methods for decreasing serum testosterone levels are disclosed. Although preferred embodiments of the subject invention have been described in some detail, it is understood that obvious variations can be made without departing from the spirit and the scope of the invention as described herein.

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: YM Biosciences, Inc.
(B) STREET: 5045 Orbitor Drive (C) CITY: Mississauga (D) STATE: Ontario (E) COUNTRY: Canada (F) POSTAL CODE (ZIP): L4W 4Y4 (ii) TITLE OF INVENTION: Methods and Formulations for Testosterone Suppression (iii) NUMBER OF SEQUENCES: 11 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (v) CURRENT APPLICATION DATA:
APPLICATION NUMBER: CA 2,467,769 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..30 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Gln His Trp Ser Tyr Gly Leu Arg Pro Gly (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Gln His Trp Ser Tyr Gly Leu Arg Pro Gly (2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 147 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..147 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

GlnHisTrpSer TyrGly LeuArgProGly SerGly SerG1nAspTrp SerTyrGlyLeu ArgPro GlyGlySerSer GlnHis TrpSerTyrGly LeuArgProGly SerGly SerGlnAspTrp SerTyr GlyLeuArgPro Gly (2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Gln His Trp Ser Tyr Gly Leu Arg Pro Gly Ser Gly Ser Gln Asp Trp Ser Tyr Gly Leu Arg Pro Gly Gly Ser Ser Gln His Trp Ser Tyr Gly Leu Arg Pro Gly Ser Gly Ser Gln Asp Trp Ser Tyr Gly Leu Arg Pro Gly (2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2102 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..2102 (xi)SEQUENCE ID
DESCRIPTION: N0:
SEQ 5:

MetAla ThrValIle AspArg SerGlnHisTrp SerTyr GlyLeuArg ProGly SerGlySer GlnAsp TrpSerTyrGly LeuArg ProGlyGly SerSer GlnHisTrp SerTyr GlyLeuArgPro GlySer GlySerGln AspTrp SerTyrGly LeuArg ProGlyGlySer GlnHis TrpSerTyr GlyLeu ArgProGly SerGly SerGlnAspTrp SerTyr GlyLeuArg ProGly GlySerSer GlnHis TrpSerTyrGly LeuArg ProGlySer GlySer GlnAspTrp SerTyr Gly~euArgPro GlyGly SerSerPhe ProLys ThrGlyAla LysLys IleIleLeuTyr IlePro GlnAsnTyr GlnTyr AspThrGlu GlnGly AsnGlyLeuGln AspLeu ValLysAla AlaGlu GluLeuGly IleGlu ValGlnArgGlu GluArg AsnAsnIle AlaThr AlaGlnThr SerLeu GlyThrIleGln ThrAla IleGlyLeu ThrGlu ArgGlyIle ValLeu SerAlaProGln IleAsp LysLeuLeu GlnLys ThrLysAla GlyGln AlaLeuGlySer AlaGlu SerIleVal AAT AAT ACT TCT
AAA
GCC

GlnAsnAla AsnLysAla LysThrVal LeuSer GlyIleGln SerIle LeuGlySer ValLeuAla GlyMetAsp LeuAsp GluAlaLeu GlnAsn AsnSerAsn GlnHisAla LeuAlaLys AlaGly LeuGluLeu ThrAsn SerLeuIle GluAsnIle AlaAsnSer ValLys ThrLeuAsp GluPhe GlyGluGln IleSerGln PheGlySer LysLeu GlnAsnIle LysGly LeuGlyThr LeuGlyAsp LysLeuLys AsnIle GlyGlyLeu AspLys AlaGlyLeu GlyLeuAsp ValIleSer GlyLeu LeuSerGly AlaThr AlaAlaLeu ValLeuAla AspLysAsn AlaSer ThrAlaLys LysVal GlyAlaGly PheGluLeu AlaAsnGln ValVal GlyAsnIle ThrLys AlaValSer SerTyrIle LeuAlaGln ArgVal AlaAlaGly LeuSer SerThrGly ProValAla AlaLeuIle AlaSer ThrValSer LeuAla IleSerPro LeuAlaPhe AlaGlyIle AlaAsp LysPheAsn HisAla LysSerLeu GluSerTyr AlaGluArg PheLys LysLeuGly TyrAsp GlyAspAsn LeuLeuAla GluTyrGln ArgGly ThrGlyThr IleAsp AlaSerVal ThrAlaIle AsnThrAla LeuAla AlaIleAla GlyGly ValSerAla AlaAlaAla AspLeuThr PheGlu LysValLys HisAsn Leu Val Ile Thr Asn Ser Lys Lys Glu Lys Val Thr Ile Gln Asn Trp Phe Arg Glu Ala Asp Phe Ala Lys Glu Val Pro Asn Tyr Lys Ala Thr Lys Asp Glu Lys Ile Glu Glu Ile Ile Gly Gln Asn Gly Glu Arg Ile AAG GTT AAA ATT
GGT

ThrSer LysGlnVal AspAspLeu IleAlaLys GlyAsnGly LysIle ThrGln AspGluLeu SerLysVal ValAspAsn TyrGluLeu LeuLys HisSer LysAsnVal ThrAsnSer LeuAspLys LeuIleSer SerVal SerAla PheThrSer SerAsnAsp SerArgAsn ValLeuVal AlaPro ThrSer MetLeuAsp GlnSerLeu SerSerLeu GlnPheAla ArgGly SerGln HisTrpSer TyrGlyLeu ArgProGly SerGlySer GlnAsp TrpSer TyrGlyLeu ArgProGly GlySerSer GlnHisTrp SerTyr GlyLeu ArgProGly SerGlySer GlnAspTrp SerTyrGly LeuArg ProGly GlySerGln HisTrpSer TyrGlyLeu ArgProGly SerGly SerGln AspTrpSer TyrGlyLeu ArgProGly GlySerSer GlnHis TrpSer TyrGlyLeu ArgProGly SerGlySer GlnAspTrp SerTyr GlyLeu ArgProGly GlySer* LeuAlaSer His (2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 700 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 6:
Met Ala Thr Val Ile Asp Arg Ser Gln His Trp Ser Tyr Gly Leu Arg Pro Gly Ser Gly Ser Gln Asp Trp Ser Tyr Gly Leu Arg Pro Gly Gly Ser Ser Gln His Trp Ser Tyr Gly Leu Arg Pro Gly Ser Gly Ser Gln Asp Trp Ser Tyr Gly Leu Arg Pro Gly Gly Ser Gln His Trp Ser Tyr Gly Leu Arg Pro Gly Ser Gly Ser Gln Asp Trp Ser Tyr Gly Leu Arg Pro Gly Gly Ser Ser Gln His Trp Ser Tyr Gly Leu Arg Pro Gly Ser Gly Ser Gln Asp Trp Ser Tyr Gly Leu Arg Pro Gly Gly Ser Ser Phe Pro Lys Thr Gly Ala Lys Lys Ile Ile Leu Tyr Ile Pro Gln Asn Tyr Gln Tyr Asp Thr Glu Gln Gly Asn Gly Leu Gln Asp Leu Val Lys Ala Ala Glu Glu Leu Gly Ile Glu Val Gln Arg Glu Glu Arg Asn Asn Ile Ala Thr Ala Gln Thr Ser Leu Gly Thr Ile Gln Thr Ala Ile Gly Leu Thr Glu Arg Gly Ile Val Leu Ser Ala Pro Gln Ile Asp Lys Leu Leu Gln Lys Thr Lys Ala Gly Gln Ala Leu Gly Ser Ala Glu Ser Ile Val Gln Asn Ala Asn Lys Ala Lys Thr Val Leu Ser Gly Ile Gln Sex Ile Leu Gly Ser Val Leu Ala Gly Met Asp Leu Asp Glu Ala Leu Gln Asn Asn Ser Asn Gln His Ala Leu Ala Lys Ala Gly Leu Glu Leu Thr Asn Ser Leu Ile Glu Asn Ile Ala Asn Ser Val Lys Thr Leu Asp Glu Phe Gly Glu Gln Ile Ser Gln Phe Gly Ser Lys Leu Gln Asn Ile Lys Gly Leu Gly Thr Leu Gly Asp Lys Leu Lys Asn Ile Gly Gly Leu Asp Lys Ala Gly Leu Gly Leu Asp Val Ile Ser Gly Leu Leu Ser Gly Ala Thr Ala Ala Leu Val Leu Ala Asp Lys Asn Ala Ser Thr Ala Lys Lys Val Gly Ala Gly Phe Glu Leu Ala Asn Gln Val Val Gly Asn Ile Thr Lys Ala Val Ser Ser Tyr Ile Leu Ala Gln Arg Val Ala Ala Gly Leu Ser Ser Thr Gly Pro Val Ala Ala Leu Ile Ala Ser Thr Val Ser Leu Ala Ile Ser Pro Leu Ala Phe Ala Gly Ile Ala Asp Lys Phe Asn His Ala Lys Ser Leu Glu Ser Tyr Ala Glu Arg Phe Lys Lys Leu Gly Tyr Asp Gly Asp Asn Leu Leu Ala Glu Tyr Gln Arg Gly Thr Gly Thr Ile Asp Ala Ser Val Thr Ala Ile Asn Thr Ala Leu Ala Ala Ile Ala Gly Gly Val Ser Ala Ala Ala Ala Asp Leu Thr Phe Glu Lys Val Lys His Asn Leu Val Ile Thr Asn Ser Lys Lys Glu Lys Val Thr Ile Gln Asn Trp Phe Arg Glu Ala Asp Phe Ala Lys Glu Val Pro Asn Tyr Lys Ala Thr Lys Asp Glu Lys Ile Glu Glu Ile Ile Gly Gln Asn Gly Glu Arg Ile Thr Ser Lys Gln Val Asp Asp Leu Ile Ala Lys Gly Asn Gly Lys Ile Thr Gln Asp Glu Leu Ser Lys Val Val Asp Asn Tyr Glu Leu Leu Lys His Ser Lys Asn Val Thr Asn Ser Leu Asp Lys Leu Ile Ser Ser Val Ser Ala Phe Thr Ser Ser Asn Asp Ser Arg Asn Val Leu Val Ala Pro Thr Ser Met Leu Asp Gln Ser Leu Ser Ser Leu Gln Phe Ala Arg Gly Ser Gln His Trp Ser Tyr Gly Leu Arg Pro Gly Ser Gly Ser Gln Asp Trp Ser Tyr Gly Leu Arg Pro Gly Gly Ser Ser Gln His Trp Ser Tyr Gly Leu Arg Pro Gly Ser Gly Ser Gln Asp Trp Ser Tyr Gly Leu Arg Pro Gly Gly Ser Gln His Trp Ser Tyr Gly Leu Arg Pro Gly Ser Gly Ser Gln Asp Trp Ser Tyr Gly Leu Arg Pro Gly Gly Ser Ser Gln His Trp Ser Tyr Gly Leu Arg Pro Gly Ser Gly Ser Gln Asp Trp Ser Tyr Gly Leu Arg Pro Gly Gly Ser * Leu Ala Ser His (2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:1 (D) OTHER INFORMATION:/note= "The amino acid at this position is pyroGlu."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
Xaa His Trp Ser Tyr Gly Leu Arg Pro Gly (2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:1 (D) OTHER INFORMATION:/note= "The amino acid at this position is pyroGlu."
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:6 (D) OTHER INFORMATION:/note= "The amino acid at this position is either Gly or a D-amino acid."
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:11 (D) OTHER INFORMATION:/note= "The amino acid at this position is one or more amino acid residue."

(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:12 (D) OTHER INFORMATION:/note= "The amino acid at this position is either Cys or Tyr."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Xaa His Trp Ser Tyr Xaa Leu Arg Pro Gly Xaa Xaa (2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Cys Pro Pro Pro Pro Ser Ser Glu His Trp Ser Tyr Gly Leu Arg Pro Gly (2) INFORMATION FOR SEQ ID N0: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:1 (D) OTHER INFORMATION:/note= "The amino acid at this position is pyroGlu."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Xaa His Trp Ser Tyr Gly Leu Arg Pro Gly Ser Ser Pro Pro Pro Pro Cys (2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:1 (D) OTHER INFORMATION:/note= "The amino acid at this position is pyroGlu."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
Xaa His Trp Ser Tyr Gly Leu Arg Pro Gly Arg Pro Pro Pro Pro Cys

Claims (28)

1. A composition comprising an immunogenic GnRH multimer/leukotoxin chimera, an aluminum-based adjuvant, a Montanide.TM. adjuvant and a pharmaceutically acceptable excipient, wherein the GnRH multimer present in the chimera has the general formula [GnRH-X-GnRH]n, wherein:
GnRH is a GnRH polypeptide;
X is a peptide linkage or an amino acid spacer group; and n is an integer greater than or equal to 1.

2. The composition of claim 1, wherein the GnRH polypeptides of [GnRH-X-GnRH]
are the same.

3. The composition of claim 1, wherein the GnRH polypeptides of [GnRH-X-GnRH]
are different.

4. The composition of claim 1, wherein the leukotoxin polypeptide lacks cytotoxic activity.

5. The composition of claim 4, wherein the leukotoxin polypeptide is LKT 111.

6. The composition of claim 5 wherein the GnRH multimer/leukotoxin chimera comprises the contiguous sequence of amino acids depicted in Figures 2A-2F
(SEQ ID
NO:6), or an immunogenic amino acid sequence with 75% sequence identity thereto.

7. The composition of claim 6, wherein the GnRH multimer/leukotoxin chimera comprises the contiguous amino acid sequence of Figures 2A-2F (SEQ ID NO:6).

8. The composition of claim 1, wherein the Montanide is Montanide ISA 51.TM..

9. The composition of claim 1, wherein the aluminum-based adjuvant is an Al(OH)3 gel.

10. A composition comprising:

(a) a GnRH multimer/leukotoxin chimera comprising the contiguous sequence of amino acids depicted in Figures 2A-2F (SEQ ID NO:6), or an immunogenic amino acid sequence with 75% sequence identity thereto;
(b) Al(OH)3 gel;
(c) Montanide ISA 51.TM.; and (d) a pharmaceutically acceptable excipient.

11. The composition of claim 10, wherein the GnRH multimer/leukotoxin chimera comprises the contiguous amino acid sequence of Figures 2A-2F (SEQ ID NO:6).

12. The composition of claim 10, wherein the GnRH multimer/leukotoxin chimera is present in an amount of about 20 µg per mL to about 2 mg per mL.

13. The composition of claim 12, wherein the GnRH multimer/leukotoxin chimera is present in an amount of 100 µg per mL to 500 µg per mL.

14. The composition of claim 10, wherein the Al(OH)3 gel is present in an amount of about 0.1% to about 1% (w/v).

15. The composition of claim 14, wherein the Al(OH)3 gel is present in an amount of 0.2% to 0.5% (w/v).

16. The composition of claim 10, wherein the Montanide ISA 51.TM. is present in an amount of about .25 mL to about .75 mL per mL of the composition.

17. The composition of claim 16, wherein the Montanide ISA 51.TM. is present in an amount of 0.5 mL per mL of the composition.

18. A composition comprising:

(a) 100 µg per mL to 500 µg per mL of a GnRH multimer/leukotoxin chimera comprising the contiguous sequence of amino acids depicted in Figures 2A-2F
(SEQ ID
NO:6);

(b) 0.2% to 0.5% (w/v) Al(OH)3 gel;

(c) 0.5 mL per mL of composition Montanide ISA 51.TM.; and (d) a pharmaceutically acceptable excipient.

19. A method of producing an immune response in a mammalian subject comprising administering to the subject a therapeutically effective amount of a first composition, wherein the first composition is the composition of any one of claims 1-18.

20. The method of claim 19, further comprising boosting the subject with one or more additional doses of a second composition.

21. The method of claim 20, wherein the subject is boosted with 4-10 additional doses of the second composition.

22. The method of claim 21, wherein the second composition is the same as the first composition.

23. The method of claim 22, wherein the immune response causes a reduction in serum testosterone levels:

24. The method of claim 23, wherein cyclophosphamide is administered prior to, concurrent with, or subsequent to administration of the first composition.

25. The method of claim 24, wherein cyclophosphamide is administered prior to administration of the first composition.

26. The method of claim 19, wherein the mammal is a male human.

27. A method of reducing serum testosterone levels in a male human, said method comprising:
(a) administering to the male human a therapeutically effective amount of a first composition, wherein the first composition comprises (i) 100 µg per mL to 500 µg per mL of a GnRH multimer/leukotoxin chimera comprising the contiguous sequence of amino acids depicted in Figures 2A-2F (SEQ ID NO:6), (ii) 0.2% to 0.5% (w/v) Al(OH)3 gel, (iii) 0.5 mL per mL of composition Montanide ISA 51.TM., and (iv) a pharmaceutically acceptable excipient;

(b) boosting the male human with 4-10 additional doses of the first composition, to result in a reduction of serum testosterone levels.

28. The method of claim 27, wherein cyclophosphamide is administered prior to administration of the first composition.