[Gln']-luteinizing hormone releasing hormone conjugate of tetanus vaccine and its uses

The present prevention provides an effective, fast acting method of vaccination useful in suppressing gonadotropic hormone release. The vaccine utilizes LHRH conjugated at its amino terminus to a protein carrier and can be mixed with either adjuvants or detergents in order to provide an effect vaccine.

BACKGROUND OF THE INVENTION 
The possibility of developing an antifertility vaccine for male mammals 
based on active immunization against Luteinizing Hormone Releasing Hormone 
(LHRH) is currently under investigation in several laboratories. These 
studies are based on the findings that antibodies against LHRH prevent 
gonadotropin release. Prevention of gonadotropin release causes regression 
of Leydig cells and suppression of testosterone production and 
spermatogenesis with subsequent infertility. See e.g. Fraser et al., 
"Effect of Active Immunization to Luteinizing Hormone Releasing Hormone on 
Serum and Pituitary Gonadotropins, Tests and Accessory Sex Organs in Male 
Rat", J. Endocrinol., 63:399-405 (1974); Fraser et al., "LHRH Antibodies: 
Their Use in the Study of Hypothalamic LHRH and Testicular LHRH-like 
Material, and Possible Contraceptive Applications", In: Sandlet, ed 
Progress Toward a Male Contraceptive. New York, John Wiley, (1982) pp. 
41-78. 
Production of anti-LHRH antibodies would also be useful to treat certain 
forms of cancer such as prostate cancer where such therapy could replace 
orchiectomy or LHRH analogue treatment. 
Recurrent or metastatic cancer of the prostate is a major cause of cancer 
mortality in the United States with more than 25,000 deaths occurring 
annually because of this malignancy. Silverberg and Lubera, "Cancer 
Statistics", CA 38:1-19 (1988). It is the most common malignancy in men 
older than 70. Carcinoma within the prostate is a common finding at 
autopsy, being found in 20-40% of men between 70-79 years of age. 
Unfortunately, approximately one third of all prostate cancers are 
diagnosed only after the patient has clinically apparent disseminated 
disease with bone or visceral metastases outside the pelvis. 
For patients with disseminated disease at the time of diagnosis the 
classical treatment is surgical orchiectomy. Huggins and Hodges, "Studies 
of Prostatic Cancer. Effect of Castration, Estrogen, and Androgen 
Injection of Serum Phosphatases in Metastatic Carcinoma of the Prostate", 
Cancer Res., 1:293-297 ( 1941 ). This procedure decreases serum 
testosterone levels by 95% and causes objective tumor regression in 
approximately 40% of cases and disease stabilization in an additional 40% 
of cases. Alternative strategies to surgical orchiectomy include treatment 
with diethylstilbestrol (DES) or with LHRH analogues. These strategies 
also seem to work by decreasing serum testosterone levels, and have 
similar response rates to those seen with orchiectomy. Results of first 
line single agent hormonal manipulations are favorable but average 
response durations are less than one year and actuarial survival less than 
2 years. Seftel et al., "Hormonal Therapy for Advanced Prostatic 
Carcinoma", J. Surg. Onc. Suppl., 1: 14-20 ( 1989 ). 
Recent results have suggested that the addition of an anti-androgen to 
initial orchiectomy or an LHRH antagonist may improve response rates. 
Using combined androgen blockade in patients with stage D2 prostate 
cancer, Labtie initially reported a 90% survival rate at 2 years. When 
this therapy was tested in a Canadian double-blinded randomized trial, the 
results still statistically significantly favored combined therapy 
although the improvement in survival was less striking (52% survival at 18 
months with simple orchiectomy versus 66% survival with combined therapy). 
Beland et al., "Total Androgen Blockade for Metastatic Cancer of the 
Prostate", Am. J. Clin. Onc., 11 (Suppl. 2):S18714 S190 (1988) This modest 
but statistically significant improvement in survival provided by combined 
therapy was also seen in an American randomized study. Thus the optimal 
therapy for cancer of the prostate at this time seems to be a combination 
of orchiectomy and therapy that blocks peripheral androgen action. 
Current therapies for the suppression of androgen production are not 
universally acceptable to all patients. Orchiectomy is psychologically 
unacceptable to many patients. The user of LHRH analogues is dependent on 
repetitive application of the analogue and involves long term 
inconvenience and expense. Use of estrogens such as DES is generally 
considered inferior to the other two treatment modalities due to 
cardiovascular complications and side effects such as gynecomastia. 
Immunization against LHRH has been proposed as an alternative 
anti-neoplastic strategy for sex steroid dependent tumors. Raydin and 
Jordan, "Active Immunization to Luteinizing Hormone to Inhibit the 
Induction of Mammary Tumors in the Rat", Life Sci., 43:117-123 (1988). 
This strategy has been shown to be effective in animal models, and depends 
on the immunoneutralization of LHRH after active immunization of the host. 
This blockade of LHRH causes decreased production of gonadotropins and 
then secondarily decreased sex steroid production by the ovaries or 
testes. It would be of great significance in the treatment of this disease 
to have a LHRH vaccine capable of rapidly stimulating production of high 
titers of anti-LHRH antibodies. 
The availability of synthetic LHRH has provided anti-LHRH antisera, needed 
for development of radioimmunoassays and immunocytochemistry. 
Unfortunately, the use of synthetic LHRH in humans to induce production of 
anti-LHRH antibodies is precluded by the necessity of using adjuvants not 
suitable for use in humans in order to achieve an effective titer within 
an acceptable time period. In the majority of the reported experiments, 
Freund's complete adjuvant (FCA) was used as an immune enhancer to obtain 
anti-LHRH antisera. Chappel et al., "Active Immunization of Male Rhesus 
Monkeys Against Luteinizing Hormone Releasing Hormone", Biol. Reprod., 
22:333-342 (1974); Fraser et al., "Preparation of Antisera to Luteinizing 
Hormone Releasing Factor", J. Endocrinol , 61:756-769 (1974); and Awoniyi 
et al., "Changes in Testicular Morphology in Boars Actively Immunized 
Against Gonadotropin Hormone-Releasing Hormone", J. Androl., 9:160-171 
(1988). 
In developing vaccines for man, only materials permitted for use in humans 
can be utilized. This criterion eliminates FCA, the most potent immune 
enhancer. Thus other methods of inducing highly effective anti-LHRH 
antibody in the shortest possible time are necessary to enable anti-LHRH 
production in man. Steblay, "Glomerulonephritis Induced in Sheep By 
Injections of Heterologous Glomerular Basement Membrane and Freund's 
Complete Adjuvant", Exp. Med., 116:253-172 (1962); Thau et al., "Effects 
of Immunization with the 8-Subunit of Ovine Luteinizing Hormone on Corpus 
Luteum Function in the Rhesus Monkey", Fertil. Steril., 31:200-204 (1979); 
and Dalsgaard, "Adjuvants", Vet. Immunol. Immunopathol., 17:145-152 
(1987). 
The studies described below were designed to examine the ability of LHRH 
conjugated to tetanus toxoid (TT) either at the N-terminal, C-terminal, or 
mid-section of the LHRH to induce biologically effective antibodies in the 
shortest possible time. 
It has now been shown that conjugating a protein carrier to LHRH enhances 
immunogenicity in a site specific manner. Carriers such as TT and purified 
protein derivative (PPD, derived from tuberculin) which are suitable for 
use in man and have been shown to stimulate the immune response are useful 
as the protein carrier. Fraser et al., (1982); Shastri et al., (1981); 
Talwar, "Immunobiology of Gonadotropin-Releasing Hormone", J. Steroid 
Biochem., 23: 795-800 ( 1985 ); and Ellouz et al., "Minimal Structural 
Requirements for Adjuvant Activity of Bacterial Peptidoglycan Derivative", 
Biochem. and Biophys. Res. Comm.,59:1317-1325 (1974).

DETAILED DESCRIPTION OF THE INVENTION 
The present invention shows that the conjugation site of a hapten to the 
LHRH molecule plays an important role in stimulating the immune system. 
Previous studies have shown that antibodies can be produced against 
unconjugated LHRH emulsified in FCA. For instance, using unconjugated LHRH 
in FCA, complete suppression of gonadal function was achieved in 100% of 
rats in 32 weeks with 8 injections. Several investigators reported that 
only a small percentage of animals immunized against unconjugated LHRH 
developed sufficient antibodies to suppress the biological actions of 
LHRH. Pique et al., (1978 ); Catelli et al., (1985); Hodges et al , (1979 
); and Jeffcoat et al., "Anti-LHRH Sera in the Investigation of 
Reproduction", In: Edwards and Johnson, eds. Physiological Effects of 
Immunity Against Hormones", Cambridge: Cambridge University Press, pp. 
121-136 (1976). 
A more uniform production of anti-LHRH antibodies could be achieved by 
conjugating the decapeptide LHRH to a carrier protein. Several proteins, 
such as bovine serum albumin (BSA), human serum albumin (HSA) 
thyroalobulin and tetanus toxoid (TT) have been tested as carrier 
molecules. Fraser et al., (1974); Nett et al., "A Radioimmunoassay for 
Gonadotropin Releasing Hormone (Gn-RH) In Serum", J. Clin. Endocrinol. 
Metab., 36:880-885 (1973); Copeland et al., "Luteinizing Hormone-Releasing 
Hormone: Sequential Versus Conformational Specificity of Antiluteinizing 
Hormone-Releasing Hormone Sera", Endocrinology, 104:1504-1512 (1979); 
Arimura et al., "The Antigenic Determinant of the LH-Releasing Hormone for 
Three Different Antisera", Acta Endocrinol Metab., 36:880-885 (1975); 
Copeland et al., (1979); Bercu et al., "Permanent Impairment of Testicular 
Development After Transient Immunological Blockade of Endogenous 
Lutenizing Hormone Releasing Hormone in the Neonatal Rat", Endocrinology, 
101:1871-1879 (1977), and Sharpe et al., "The Influence of Sexual 
Maturation and Immunization Against LHRH on Testicular Sensitivity to 
Gonadotropin Stimulation in vitro", Int. J. Androl., 1:501-508 (1978). 
However, no systematic studies have been done to compare the effects of 
the conjugation sites of any hapten with the LHRH molecule on 
immunological and, consequently, biological responses. Thus a vaccine must 
be 100% percent effective and preferably work quickly, particularly in the 
case of carcinomas where a delay can be critical to the outcome. Although 
it has previously been shown that LHRH.sub.10 -TT suppresses gonadotropic 
hormones, the effect is not 100% complete until five months after therapy 
is initiated. This delay is far too long to allow the use of LHRH.sub.10 
-TT in inducing infertility or in treating carcinomas. Ladd et al., 
"Active Immunization Against Gonadotropin-Releasing Hormone Combined With 
Androgen Supplementation is a Promising Antifertility Vaccine for Males", 
Am. J. Rep. Immunol. and Microbiol., 17:121-127 (1988). 
It has now been found .that the conjugation site of the LHRH molecule is 
critical to the immunological activity of LHRH. Mammals have now been 
found to respond to active immunization against LHRH conjugated to a TT at 
the N-terminus (LHRH.sub.1 -TT) by developing anti-LHRH antibodies capable 
of suppressing pituitary gonadotropin secretion significantly faster and 
more uniformly than animals immunized against LHRH.sub.6 -TT or 
LHRH.sub.10 -TT. LHRH.sub.1 -TT is the preferred embodiment of the present 
invention. Further, other protein carriers such as PPD (purified protein 
derivative obtained from tuberculin) and BSA have been found to form 
suitable conjugates. Thus, the invention is not limited to TT as a protein 
carrier; any immunogenic protein carrier suitable for use in humans is 
within the scope of the present invention. Unlike previously reported 
work, immunity to LHRH was accomplished by utilizing only materials 
permitted of use in humans. 
LHRH.sub.1 -TT can be produced by the methods outlined below or by any 
suitable method known in the art of protein conjugation. 
LHRH.sub.1 -TT is administered to subjects including but not limited to 
animals including man. In particular, subjects may include but are not 
limited to males with prostate or testicular carcinoma, benign prostatic 
hyperplasia and healthy males for the purpose of inducing infertility. 
Further, female subjects are included. In females, suppression of LHRH 
causes decreased estrogen and progesterone levels. Such treatment is 
suitable for a variety of disorders including but not limited to 
endometriosis, benign uterine tumors, recurrent functional ovarian cysts 
and severe premenstrual syndrome. 
LHRH.sub.1 -TT is administered to a subject by any suitable method of 
injection including but not limited to parenteral, subcutaneous, 
intramuscular and intraperitoneal. 
LHRH.sub.1 -TT is suspended in a sterile, physiologically acceptable 
vehicle prior to administration to a subject. Such vehicles include but 
are not limited to physiological saline, or an isotonic buffering 
compound. The vehicle may also include adjuvants including but not limited 
to murabutide, Freund's incomplete adjuvant, and Freund's complete 
adjuvant. In humans, adjuvants such as Freund's are not acceptable since 
they cause adverse reactions; for that reason only adjuvants suitable for 
use in humans are indicated when the subject is a human. For instance, the 
composition can be mixed with immunostimulating complexes as described by 
Takahashi et al., "Induction of CDS+Cytotoxic T Cells by Immunization With 
Purified HIV-1 Envelope Protein and ISCOMS", Nature 344:873-875 (1990 ). 
As with all immunogenic compositions for eliciting antibodies, the 
immunogenically effective amounts of the composition of the invention must 
be determined empirically. Factors to be considered include the choice of 
adjuvant or carrier, the immunogenic molecule to which LHRH is coupled, 
the route of administration and the number of immunizing doses to be 
administered. Such factors are known in the vaccine art and it is well 
within the skill of immunologists to make such determinations without 
undue experimentation. Edelman, "Vaccine Adjuvants", Rev. Inf. Dis., 
2:370-383 (1980). It has now been found that adjuvant is not necessary to 
induce an immune response utilizing the peptide of the present invention. 
As is described in detail below, when LHRH.sub.1 -TT is mixed with an 
isotonic solution containing detergents such as Tween 80 (also known as 
Polysorbate 80) and Pluronic L121, the vaccine is at least equally 
effective to vaccines containing commonly used adjuvants. This obviates 
the expense of these adjuvants and avoids any possible side effects 
inherent in their use. Although both TWEEN 80 (Polyoxyethylene (20) 
sorbitan monopoleate) and PLURONIC L121, a liquid block copolymer of 
propylene oxide and ethylene oxide, the liquid block copolymer having a 
molecular weight of approximately 3.5.times.10.sup.3 and 15 weight percent 
ethylene oxide belong to a large group of related compositions, few of 
these related compositions are suitable for use in vaccines. The preferred 
composition is a mixture of Tween 80 and Pluronic L121. It is further 
preferred that they be combined in a range of proportion of 1 to 40, more 
preferably 1 to 25. Several other TWEENS or Pluronics are suitable for use 
in the present invention, but are thought to be less preferred. See e.g. 
Murray et al., "Mineral Oil Adjuvants; Biological and Chemical Studies", 
Anals of Allergy, 30:146-151 (1972). 
The present invention is useful for inducing infertility in males and as an 
effective therapy for prostate cancer patients. In the case of prostate 
cancer treatment, the present invention can be used alone or in 
combination with other standard treatments such as orchiectomy or 
anti-androgen treatment. In addition, during treatment of men according to 
the present invention, androgen replacement therapy is recommended for 
maintenance of libido. A particularly suitable androgen supplementation is 
described in copending U.S. patent application No. 07/335,039, filed Apr., 
7, 1989 to Bardin et al. which is incorporated herein by reference. U.S. 
patent application No. 07/335,039 describes compositions for androgen 
supplementation which are testosterone derivatives having a non-hydrogen 
substituent in the 6.alpha. or 7.alpha. position. 
It has now been found that antibody production may not depend on the dose 
of an antigen (LHRH.sub.10 -TT), provided a minimal (threshold) dose has 
been surpassed. For instance, when LHRH.sub.1 -TT was used as an antigen, 
no differences in time course or magnitude of immune response was 
observed. Ineffective doses of LHRH.sub.1 -TT were not tested in this 
experiment, and therefore the threshold dose of this conjugate remains 
unknown. However, a complete suppression of testicular steroidogenesis, as 
evidenced by a marked decrease in serum T levels paralleled by the 
decrease in gonadal size (as estimated by palpation) was achieved 12 weeks 
faster than previously found. This decrease in time of suppression means 
that LHRH levels can now be suppressed within a therapeutically meaningful 
time frame, 4-8 weeks. Although in these experiments, mating to ensure 
infertility was not performed, in several of our previous experiments rats 
with testis size below 30% of normal and serum T levels below 0.03 nmol/L 
were infertile. Ladd et al., "Active Immunization Against 
Gonadotropin-Releasing Hormone Combined With Androgen Supplementation is a 
Promising Antifertility Vaccine for Males", Am. J. Reprod. Immunol., 
17:121-127 (1988), and Ladd et al., "Effects of Long-Term Immunization 
Against LHRH and Androgen Treatment on Gonadal Function", J. Reprod. 
Immunol., 15:85-101 (1989). 
The following experiments are provided to illustrate but not limit the 
present invention. 
EXAMPLE 1 
Synthesis of LHRH Conjugates 
Synthetic LHRH with a free carboxyl terminal (LHRH.sub.10 -Gly-OH, code 
#139-183-20), [D-Lys.sup.6 ]-LHRH, code #139-185-10, and [Gln.sup.1 
]-LHRH, code #139-133-30 were generously provided by the Salk Institute 
(La Jolla, CA). Tetanus toxoid (TT) was obtained from the Wyeth Laboratory 
(Marietta, PA). 
For conjugation, a solution consisting of 20 mg LHRH.sub.10 -Gly-OH, 20 mg 
purified TT, and 100 mg 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in a 
total volume of 20 mL water was incubated at room temperature for a 24 
hour, followed by an 18 hour incubation at 4.degree. C. The reaction 
mixture was dialyzed against distilled water for 2 days and lyophilized. 
The lyophilized LHRH.sub.10 -TT conjugate was used as an antigen. A 
similar procedure was used to conjugate TT at the N-terminal of [Gln.sup.1 
]-LHRH and is described in Example 10. 
LHRH.sub.6 -TT conjugate was prepared by reacting TT (25 mg in 0.9 mL of 
0.1 M phosphate buffer) and 15 mg succinimidyl-4-(N-maleimidomethyl ) 
cyclohexane-1-carboxylate in 1.1 mL dimethyl formamide at room temperature 
for 5 hours. The product was purified on a Sephadex G-25 column. The 
thiolated derivative of [D-Lys.sup.6 ]-LHRH was prepared by reacting 12.3 
mg [D-Lys.sup.6 ]-LHRH in 1.2 mL of 0.1 M NaC1, 0.1 M phosphate buffer, pH 
7.5, and 12.9 mg succinimidyl-3-(2-pyridyldithio) propionate in 1.2 mL 
dimethyl formamide. The reaction was carried out at room temperature for 
3.5 hour. At the end of the reaction, 6.6 mg dithiothreitol was added, and 
the product was purified by Sephadex G-10 chromatography. The [D-Lys.sup.6 
]-LHRH-TT (LHRH.sub.6 -TT) conjugate was prepared by reacting TT 
derivative and thiolated [D-Lys.sup.6 ]-LHRH at room temperature for 24 h. 
The reaction product was dialyzed, lyophilized, and used as immunogen. 
N-acetyl normuramyl-L-alanyl-D-isoglutamine (MDP A-5, CIBA-GEIGY Limited, 
Basel, Switzerland) emulsified in a mixture of TWEEN 80 (0.2%) (Sigma 
Chemical) and PLURONIC L121 (2.5%) (BASF) in 0.9% NaCl (TP) was used as an 
adjuvant in the experiments provided below unless indicated otherwise. 
EXAMPLE 2 
Animals Used In Antisera Production 
Sexually mature male rats (Sprague-Dawley, body weight 350-400 g) and 
rabbits (New Zealand White, body weight 3.3-3.6 kg, Dutchland, Denver, PA) 
were kept under standard conditions. The animals were immunized by 
subcutaneous injection at designated time intervals without anesthesia. 
Blood was collected from the middle caudal artery under ether-nembutal 
anesthesia (rats) and from the central ear artery (rabbits). The sera were 
separated and stored frozen until analyzed. 
EXAMPLE 3 
Radio immunoassays 
Serum T was measured by radioimmunoassay (RIA) using a Coat-a-Count T Kit 
obtained from Diagnostic Products (Los Angeles, CA). Detection limits were 
from 0.01 to 0.03 pmol/L. Inter- and intra-assay coefficients of variation 
were 11.9 and 14.7%, respectively. 
Serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) 
levels were measured with RIA kits obtained from the National Hormone and 
Pituitary Program (Baltimore, MD). Rabbit FSH levels were expressed as 
ng/mL of AFP-538C. The limit of detection was 52 pg/mL. Rabbit LH levels 
were expressed as ng/mL of AFP-7818C. The limit of detection was 13 pg/mL. 
Rat LH levels were expressed as ng/mL of AFP-5666C. The limit of detection 
was 10 pg/mL. Rat FSH levels were expressed as ng/mL of AFT-4621B. All 
samples from each experiment were analyzed in duplicate in a single assay. 
RIA data were analyzed by a PDP-11/2 minicomputer. 
EXAMPLE 4 
LHRH Specific Antibody Titers 
In the following experiments, LHRH antibody titers were determined by the 
following procedure. Antisera were diluted 1:100 in 1% bovine serum 
albumin (BSA) Sigma, St. Louis, MO). 100 .mu.L of diluted antiserum were 
incubated overnight at room temperature in 1% BSA-precoated polystyrene 
tubes (100 .mu.L) with 100 .mu.L of [.sup.125 I]-LHRH (approximately 
15,000 cpm). Antibody-bound [.sup.125 I]-LHRH was precipitated with 400 
.mu.L polyethylene glycol 8,000 (25% solution ) and 200 .mu.L of bovine 
.gamma.-globulin (5 mg/mL). Antibody titers were expressed as nmoles of 
[.sup.125 I]-LHRH bound per liter of sera. Intra- and inter-assay 
coefficients of variation were 7.6 and 11.8%, respectively. 
EXAMPLE 5 
Tissue Preparation 
At the end of each experiment, testes, epididymides, prostates, and seminal 
vesicles were removed and placed immediately into Bouin's solution for 
fixation. Following fixation, tissues were embedded in paraplast and cut 
into 5-.mu.m slices. These were stained with periodic 
acid-Schiff/hematoxylin. The glass-mounted sections of the testes were 
evaluated under a light microscope as described by Clermont and 
Morgentaler, "Quantitative Study of Spermatogenesis in the 
Hypophysectomized Rat", Endocrinology, 57:369-386 (1955 ). 
EXAMPLE 6 
Effects of increasing doses of LHRH.sub.10 -TT Vaccine 
Six groups of rats (12/group)were immunized against LHRH using 2.5, 5, 10 
.mu.g of LHRH.sub.10 -TT conjugate per rat. Rats were immunized by 
subcutaneous injection at weeks 0, 3, 6, and 10. Blood was collected every 
4 weeks and analyzed for antibody titers (AT) and serum testosterone (T) 
levels. At the time of blood collection, testes were palpated to estimate 
their size as a semi-quantitative criterion for success of the effect of 
the immunization. 
Rats immunized with either 2.5 or 5 .mu.g of LHRH.sub.10 -TT developed 
barely measurable AT levels of 0.5 nmol/L in 5 and 7 of 12 rats in each 
group, respectively, by week 16. Ten .mu.g of LHRH.sub.10 -TT per rat 
induced AT of 0.9.+-.0.2 nmol/L (mean.+-.SE) at week 8. By week 16, AT 
levels reached 2 nmol/L in this group. Rats immunized with any of the 
three higher doses (50, 175, or 612 .mu.g/rat) of LHRH.sub.10 -TT started 
developing antibodies faster than rats immunized with the dose of 10 .mu.g 
of LHRH. Four weeks after the primary immunization, AT levels in the 
former groups ranged from 1 to 2 nmol/L. However, by week 8 there was no 
significant difference in antibody titers between the four groups of 
animals immunized with 50 or more .mu.g/rat of LHRH.sub.10 -TT. This 
remained true until the end of the experiment, at week 20 (FIG. 1B). 
Rats immunized with either 2.5 or 5 .mu.g of LHRH.sub.10 -TT had serum T 
levels similar to nonimmunized controls. Serum T levels in animals 
immunized with any of the higher doses of LHRH.sub.10 -TT (from 10 to 612 
.mu.g/rat) gradually declined and reached castration levels (&lt;0.03 nmol/L) 
by week 20 (FIG. 1A). At this time the weights of testes and accessory sex 
organs in all groups of rats immunized with 10 .mu.g/rat of LHRH.sub.10 
-TT or more were significantly reduced as compared with nonimmunized 
controls or rats immunized with 2.5 or 5 .mu.g of LHRH. The weights of the 
prostates and seminal vesicles were comparable with those of castrated 
rats. The reduction in weight of the testes and accessory sex organs was 
not dependent on the dose of LHRH.sub.10 -TT injected (FIG. 2). Black bars 
inside of nonimmunized control (0) bars represent organ weights of 
castrated male rats of comparable age and body weight. Organ weights of 
all groups of immunized rats injected with 10, 50, 175 or 612 .mu.g 
LHRH.sub.10 -TT are significantly lower than in nonimmunized controls and 
rats injected with 2 5 or 5 .mu.g LHRH.sub.10 -TT (P&lt;0.0001). 
Histological evaluations of cross-sections of the testes showed severe 
impairment of spermatogenesis in rats immunized with 10 .mu.g LHRH.sub.10 
-TT or more, as manifest by reduced diameters of seminiferous tubules 
(approximately one-fourth of those of nonimmunized controls) and atrophied 
Leydig and Sertoli cells. Doses of 2.5 and 5 .mu.g/rat of LHRH.sub.10 -TT 
had no effect on testicular functions. FIG. 3(A) shows a cross section of 
a rat testis from a nonimmunized control. FIG. 3(B) shows a cross section 
of a rat testis from a rat immunized with 10 .mu.g/rat of LHRH.sub.10 -TT. 
Note the significantly reduced diameter of the seminiferous tubules and 
atrophied Leydig and Sertoli cells. Spermatogenesis did not progress 
beyond the spermatogonial stage in the immunized rat. 
This Example shows that vaccination with LHRH.sub.10 -TT suppresses serum T 
levels in animals but not before 20 weeks of treatment. 
EXAMPLE 7 
Effects of increasing doses of LHRH.sub.1 -TT Vaccine 
Three groups of rats (12/group) were immunized against LHRH using 50, 175, 
or 612 .mu.g of LHRH.sub.1 -TT conjugate per rat. The adjuvant (MDP A-5), 
emulsifier (TP), immunization, and blood collection schedule were the same 
as in Example 6. 
Animals immunized with LHRH.sub.1 -TT developed AT levels ranging from 2.0 
to 2.4 nmol/L by week 4, and responded to the following booster injections 
by rapidly increasing AT. By week 8, unlike the animals in Example 6, AT 
levels exceeded 3.5 nmol/L (FIG. 4) in all three groups of immunized 
animals, regardless of the dose of LHRH.sub.1 -TT utilized in this 
experiment. 
Biological effects of active immunization against three doses of LHRH.sub.1 
-TT used in this experiment were expressed as a reduction of serum T 
levels (FIG. 4) due to significantly suppressed levels of serum 
gonadotropins (FIG. 5). In FIG. 5, the black bars inside of the 
nonimmunized control bars (0) represent serum LH and FSH levels in 
hypophysectomized rats. In this experiment serum T levels were suppressed 
below the limits of RIA sensitivity (&lt;0.03 nmol/L) by week 8 after the 
primary immunization in all three groups of immunized animals. By week 8, 
testes were reduced to less than 30% of normal size, as estimated by 
palpation in all groups of immunized rats. 
This Example shows that vaccination with LHRH.sub.1 -TT suppresses serum T 
levels to castration levels by 8 weeks of treatment. This is 12 weeks 
sooner than vaccination with LHRH.sub.10 -TT. (Example 6). LHRH.sub.1 -TT 
thus works quickly and efficiently to suppress serum T levels. 
EXAMPLE 8 
Effects of LHRH conjugation site in rats 
Rats (12/group) were immunized using subcutaneous injections of either 
LHRH.sub.1 -TT or LHRH.sub.10 -TT (50 .mu.g/rat) emulsified in TP; MDP A-5 
(250 .mu.g/rat) was used as an adjuvant. Nonimmunized (control) rats 
received injections of MDP A-5 in TP only. Booster injections were given 
3, 6, and 9 weeks after the primary immunization. The experiment was 
terminated 14 weeks after the primary immunization. 
Both immunological and biological effects were monitored. The results 
presented in FIG. 6 show that four weeks after primary immunization, rats 
immunized against LHRH.sub.1 -TT developed AT levels of 2.1.+-.0.1 nmol/L 
(mean.+-.SE, n=12). By week 8, AT reached 4 nmol/L and remained above this 
level until the end of the experiment. Rats immunized against LHRH10-TT 
developed AT levels of approximately 2 nmol/L only by week 16, and the 
levels of AT in this group never exceeded 3 nmol/L. 
The biological effects were evidenced in serum T levels and testes size 
reduction. Serum T levels in 11 of 12 rats immunized against LHRH.sub.1 
-TT were suppressed to castration level by week 4. By week 8, all 12 rats 
in this group had nondetectable serum T levels and had testes reduced down 
to approximately 30 percent of normal size. Although serum T levels were 
significantly reduced (2.3.+-.0.5 nmol/L) as compared with 
pre-immunization level (20.8.+-.2.4 nmol/L) in the LHRH.sub.10 
-TT-immunized group, none of the rats had levels below 0.03 nmol/L even 
sixteen weeks after the primary immunization. At this time, 5 of 12 rats 
had reduced testicular size, but not below 50% of normal and serum T 
levels ranging from 0.3 to 0.6 nmol/L. 
This Example shows that LHRH.sub.1 -TT reduces serum T levels to castration 
levels in almost 100% of subjects by 4 weeks and 100% of animals by 8 
weeks, whereas LHRH.sub.10 -TT does not reduce serum T levels to 
castration levels in the subjects even sixteen weeks after immunization. 
LHRH.sub.1 -TT is thus more effective and acts more rapidly than 
LHRH.sub.10 -TT indicating that LHRH.sub.1 -TT is more suited to treatment 
of patients requiring reduction of gonadotropic hormone levels. 
EXAMPLE 9 
Effects of the LHRH conjugation site in rabbits 
Rabbits (6/group) were immunized by subcutaneous injection of 500 .mu.g of 
conjugate (LHRH.sub.1 -TT, LHRH.sub.6 -TT, or LHRH.sub.10 -TT) emulsified 
in TP MDP A-5 was used as an adjuvant in a dose of 200 .mu.g/rabbit. 
Nonimmunized controls were injected with MDP A-5 emulsified in TP only. 
Booster injections were given 3, 6, and 9 weeks after primary immunization 
at week 0. Blood was collected from the central ear artery every other 
week. 
Immune responses to active immunization against various LHRH conjugates in 
rabbits were similar to those in rats: rabbits immunized against 
LHRH.sub.1 -TT had AT levels of 3.1.+-.0.5 nmol/L 4 weeks after the 
primary immunization and responded to the following booster injections. At 
week 9, AT was 5.3.+-.0.7 nmol/L and remained close to 4 nmol/L until the 
end of the experiment (FIG. 7). Rabbits immunized against HRH.sub.6 -TT 
had their maximum antibody titers (2.8.+-.0.6 nmol/L) at week 10, 1 week 
after the last booster injection, followed by a rapid decline in antibody 
titer. Similar results (maximum AT of 2.4.+-.0.5 nmol/L at week 10) were 
obtained in LHRH.sub.10 -TT-immunized rabbits. 
At the end of the experiment (16 weeks after the primary immunization) an 
LHRH-challenge test was performed in order to evaluate whether circulating 
LHRH antibodies are capable of inhibiting LHRH-induced LH and FSH release. 
Rabbits were injected subcutaneously with 30 .mu.g of LHRH (LH-FSH-RH, 
chloride form, batch #2, National Hormone and Pituitary Program) dissolved 
in 1 mL 0.9% NaC1. Blood from the central ear artery was collected at 0, 
30, 60 and 120 minutes after the injection of LHRH. Sera were separated 
and stored at -20.degree. C. for further serum LH and FSH analysis. 
The LHRH-challenge test performed 16 weeks after the primary immunization 
revealed a lack of LH and FSH response to exogenous LHRH in LHRH.sub.1 -TT 
immunized rabbits (FIG. 8). In FIG. 8, lines represent mean values for 
each group of rabbits. Closed and open circles and triangles represent 
individual serum LH and FSH levels. In rabbits immunized with either 
LHRH.sub.6 -TT or LHRH.sub.10 -TT, changes in serum gonadotropins 
following the injection of LHRH were dependent on AT. Animals with AT 
levels above 1.0 nmol/L showed significantly suppressed pituitary LH and 
FSH response after injection of LHRH. However, only two rabbits immunized 
against LHRH.sub.10 -TT and two rabbits immunized against LHRH.sub.6 -TT 
had AT of .gtoreq.1 nmol/L; therefore, the mean increase of serum 
gonadotropins after the injection of LHRH in these two groups was not 
significantly different from controls. 
The biological effects of immunization using various LHRH conjugates are 
clearly demonstrated in the suppression of serum T levels While in the 
LHRH.sub.1 -TT-immunized group 5 of 6 rabbits had serum T at castration 
level at week 7, LHRH.sub.6 -TT- or LHRH.sub.10 -TT-immunized rabbits had 
serum T levels comparable with nonimmunized controls throughout the 
experiment (FIG. 7). Testicular weights in rabbits immunized against 
LHRH.sub.1 -TT were significantly reduced as compared with nonimmunized 
controls (FIG. 9). Histological evaluation of testicular cross-sections 
revealed arrest of spermatogenesis in this but not in two other groups of 
immunized rabbits (FIG. 3B). FIG. 3(C) shows a cross section of rabbit 
testis from a nonimmunized control rabbit. FIG. 3(D) shows a cross section 
of rabbit testis from a rabbit immunized with LHRH.sub.1 -TT. Note the 
significantly reduced diameter of the seminiferous tubules and the 
atrophied Leydig and Sertoli cells in the immunized rabbit. 
Spermatogenesis did not progress beyond the spermatogonial stage in the 
immunized rabbit. 
The results of this experiment demonstrate that LHRH.sub.1 -TT acts more 
rapidly and efficiently than either LHRH.sub.6 -TT or LHRH.sub.10 -TT to 
reduce serum T levels to clinically significant levels. 
EXAMPLE 10 
Clinical Trials 
Animal studies of the LHRH.sub.1 -TT vaccine have used 0.25 mg murabutide 
per injection in rats and rabbits. Human studies using murabutide in 
combination with tetanus vaccine gave best results using 0.05 mg/kg of 
approximately 3.1 mg per dose. The dose of 1.0 mg is selected for the 
first clinical study of the LHRH.sub.1 -TT vaccine. 
The following are the drug substances used in manufacturing of the vaccine 
for human use and their release specifications: 
(i)[Gln.sup.1 ]-LHRH is obtained from the Clayton Foundation Laboratories 
for Peptide Biology, the Salk Institute, through the Contraceptive 
Development Branch of the National Institute for Child Health and Human 
Development. It is identified by high performance liquid chromatography 
with Bio-Sil T5K-250 column with 50 mM sodium sulfate in 20 mM phosphate 
buffer at pH 6.8 as the mobile phase. The compound should be a single peak 
by HPLC analysis with no more than 5% contamination of native LHRH. 
Tetanus toxoid is obtained from Wyeth Laboratories, Radnor, Pennsylvania. 
1-Ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride is purchased 
from Sigma Chemical Company, St. Louis, Missouri. NaCl is provided by 
Mallinkrodt. Sodium phosphate monobasic is provided by Mallinkrodt. TWEEN 
80 is purchased from Sigma Chemical Company, St. Louis, MI. PLURONIC L121 
is purchased from BASF Wyandotte Corporation, Wyandotte, Michigan. 
Thimerasol is purchased from Roger Chemical Co., New Jersey. 
The drug product is dispensed in two vials. One vial contains the 
[Gln.sup.1 ]-LHRH-TT conjugate and the other vial contains a lyophilized 
powder of the adjuvant murabutide. A single dose of vaccine consists of 
0.5 mL vaccine solution plus 1 mg murabutide. 
The contents of the vaccine vial per 0.5 mL solution are as follows: 
______________________________________ 
(i) [Gln.sup.1 ]-LHRH-TT 0.5 mg 
(ii) Sodium chloride 4.4 mg 
(iii) Sodium phosphate, monobasic 
0.7 mg 
(iv) TWEEN 80 1.0 mg 
(v) PLURONIC L121 12.5 mg 
(vi) Thimerosal 0.05 mg 
______________________________________ 
Each vaccine vial contains 0.7 mL, of which 0.5 mL are used per injection. 
The above list of contents is that contained in 0.5 mL. 
The content of the murabutide vial is as follows: 
______________________________________ 
(i) Murabutide lyophilized powder 
5 mg 
______________________________________ 
Prior to administration, 0.25 mL of vaccine solution is removed aseptically 
from the vaccine vial with a 1 mL tuberculine syringe and added to the 
vial containing 5 mg murabutide powder. The murabutide vial is then gently 
agitated to dissolve the powder, creating a solution containing 1 mg 
murabutide per 0.05 mL. Then 0.05 mL of this solution is transferred from 
the murabutide vial into the 1 mL syringe. 0.45 mL of vaccine solution 
from the vaccine vial is added to the syringe. The total contents of the 
syringe, consisting of 0.5 mL vaccine solution and 1 mg murabutide, is 
administered intramuscularly by injection to each patient. 
Preparation of [Gln.sup.1 ]-LHRH-TT conjugate is as follows. A solution 
consisting of 150 mg [Gln.sup.1 ]-LHRH, 150 mg TT and 750 mg 
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride in 60 mL of 
water is reacted at room temperature for 24 hours. The reaction mixture is 
dialyzed against 5 mM phosphate buffer at pH 7.4 for two days. The 
conjugate is filtered through a sterile filter unit (Falcon filter unit, 
0.22 micron). The amount of [Gln.sup.1 ]-LHRH-TT conjugate in the solution 
is determined by Lowry protein assay. The amount of LHRH in the conjugate 
is determined by radioimmunoassay. 
Lowry protein assay: 
(i) Reagents: 
Reagent A: 2% Na.sub.2 CO.sub.3 in 0.1 N NaOH 
Reagent B1: 2% NaK tartrate 
Reagent B2: 1% CuSO.sub.4 .cndot.5H.sub.2 O 
Reagent C: Mixture of Reagents A:Bi:B.sub.2 (100:1:1) 
Reagent E: 1 N phenol reagent (Fisher Scientific) 
(ii) Procedure: 
The protein standard is prepared from 1 mg/mL bovine serum albumin (BSA) 
stock solution to give concentrations of 25, 50, 75 and 100 .mu.g/500 
.mu.L. Water is used as the blank tube. To each 500 .mu.L sample tube, add 
2.5 mL reagent C, vortex, and let stand for 10 minutes. Add 0.25 mL 
Reagent E, vortex and let stand for 30 minutes. Determine absorbancy at 
540 nm. A standard curve is plotted using .mu.g BSA concentration vs. 540 
nm absorbance. 
(iii) Concentration of [Gln.sup.1 ]-LHRH-TT in the solution is determined 
based on the standard curve of BSA. Radioimmunoassay: 
(i) Reagents: 
[.sup.125 I]-LHRH: purchased from New England Nuclear, Boston, MA. 
Antiserum: prepared by immunization of rabbits against [Gln.sup.1 
]-LHRH-TT. 
[Gln.sup.1 ]-LHRH: supplied by Salk Institute. 
Phosphate buffer - saline (PBS): 0.15 M NaCl, 0.01 M phosphate buffer at pH 
7.5. 
Bovine .gamma.-globulin: 5 mg/mL. 
Polyethyleneglycol: 25% solution. 
(ii) Procedure: 
Samples for standard curve are prepared by diluting [Gln.sup.1 ]-LHRH to 
concentrations of 0.05, 0.1, 0.2, 0.4, 0.6, 1.0, and 2.5 ng/mL. The 
[Gln.sup.1 ]-LHRH-TT conjugate is similarly diluted to 2, 5, 10, 30, 100 
and 500 ng/mL. All samples are prepared in duplicate. 0.5 mL aliquots of 
the diluted standard, blank, and test samples are distributed to test 
tubes and 0 1 mL of a solution of [.sup.125 I]-LHRH containing about 
50,000 cpm is added to each. 0.2 mL of the diluted antiserum is added to 
each except for one pair of blank tubes. 0.2 mL of 0.1 M EDTA in PBS is 
added to the blank pair. 
The solutions are mixed thoroughly with a vortex mixer and incubated in a 
water bath at 37.degree. C. for two hours and then overnight at 4.degree. 
C. At the end of the incubation period, 0.2 mL of a solution containing 5 
mg of bovine .gamma.-globulin per mL and one mL of 25% polyethylene glycol 
in PBS are added and the solution thoroughly mixed with a vortex mixer. 
The mixture is then centrifuged at 2000.times.g for 40 min. The 
supernatant is decanted and precipitate counted in a gamma counter. 
Counts are corrected by subtracting the counts found in the blank samples. 
The ratio of counts of test samples over counts without test sample 
(B/B.sub.o) is plotted against a logarithmic scale of concentration of 
test samples. A regression line is drawn. 
(iii.) Calculation of the amount of [Gln.sup.1 ]-LHRH present in the 
[Gln.sup.1 ]-LHRH-TT conjugate: 
The aim of the assay is to determine the amount of [Gln.sup.1 ]-LHRH-TT 
conjugate which is assayable by radioimmunoassay (RIA). [Gln]-LHRH is used 
as standard in the assay system. The corresponding molar amount of 
assayable [Gln.sup.1 ]-LHRH on [Gln.sup.1 ]-LHRH-TT is measured and 
expressed as the molar ratio of [Gln.sup.1 ]-LHRH to [Gln.sup.1 ]-LHRH-TT. 
The amount of [Gln.sup.1 ]-LHRH is an underestimate of its actual amount 
in [Gln.sup.1 ]-LHRH-TT because the conjugated [Gln.sup.1 ]-LHRH (MW 
1.2kD) in the interior part of the tetanus toxoid (MW 150kD) is not 
accessible to the antibody in this RIA system. 
The concentration of [Gln.sup.1 ]-LHRH and [Gln.sup.1 -LHRH-TT 
corresponding to 50% inhibition of binding of [.sup.125 I]-LHRH is 
determined from the B/B.sub.o vs logarithm of concentration curve. The 
molecular weight of [Gln.sup.1 ]-LHRH and [Gln.sup.1 ]-LHRH-TT are 
1.2.times.10 and 1.5.times.10 Daltons respectively. The molar ratio of 
[Gln.sup.1 ]-LHRH/ [Gln.sup.1 ]-LHRH-TT is obtained by: 
##EQU1## 
(iv.) Control specification of [Gln.sup.1 ]-LHRH-TT: The molar ratio of 
[Gln.sup.1 ]-LHRH: [Gln.sup.1 ]-LHRH-TT should be more than 1. 
High performance liquid chromatography (HPLC): 
(i.) Column reagents and procedures: 
column: Bio-Sil TSK-250 (300.times.7.5 mm), purchased from Bio Rad 
Mobil phase: 50 mM sodium sulfate, 20 mM phosphate buffer (pH 6.8) 
Flow rate: 1.0 mL per minute 
Chart speed: 5 min/cm 
Detector: 254 nm 
Loop size: 20 .mu.L. 
(ii.) Control specification of [Gln.sup.1 ]-LHRH-TT: 
No unreacted [Gln.sup.1 ]-LHRH is present in the product 
One major broad peak with shoulder. 
Subjects are men with metastatic prostate cancer (Stage D2) who have 
undergone orchiectomy. History, physical exam including vital signs and 
performance status and laboratory tests of hematology, chemistry, and 
endocrine function are performed prior to immunization. They are repeated 
on the day of each of the three immunizations, every four weeks thereafter 
for the first six months of the study, and then every three months for up 
to two years. In subjects with measurable disease, X-rays or scans are 
done prior to immunization, on the day of the third immunization, and 
every four weeks thereafter. Pathology reports are reviewed on all 
subjects. Subjects requiring radiation or chemotherapy in the first eight 
weeks of the study, and those developing signs of immediate 
hypersensitivity or signs of systemic, renal, hematologic, endocrinologic, 
or neurologic adverse effects receive no further immunizations, but are 
followed for 6 months. Subjects with a history of autoimmune disease or 
hypersensitivity to tetanus toxoid, with brain metastases documented 
within 6 months prior to enrollment, or who are undergoing radiation or 
chemotherapy are not enrolled in the study. 
Blood samples are drawn on the day of the initial immunization and at 2, 4, 
8, 12, 16, 20 and 24 weeks, and every 3 months thereafter (for up to 2 
years) for use in assays to detect antibody titers to LHRH. Each sample 
for this is 10 mL of blood collected in an non-anticoagulant treated tube. 
This sample is allowed to clot over approximately one hour at room 
temperature and the serum collected after centrifugation. This sample is 
frozen at -20.degree. C. 
Blood samples are drawn on the day of the initial immunization and at 2, 4, 
8, 12, 16, 20 and 24 weeks and every 3 months thereafter (for up to two 
years) for use in radioimmunoassays to detect serum LH, FSH, growth 
hormone, prolactin, and testosterone levels. Each sample is 10 mL of blood 
collected in an non-anticoagulant treated tube. This sample is allowed to 
clot over approximately one hour at room temperature and the serum 
collected after centrifugation. This sample is either immediately assayed 
as described in Example 3 or frozen at -20.degree. C. and subsequently 
assayed. 
If the tumor is measurable then evaluation is done at 4 week intervals. 
These examinations allow a quantitative assessment of the disease extent. 
Criteria for response are the standard SWOG criteria. 
EXAMPLE 11 
Comparison of Adjuvant-Effects of Murabutide, MDP A-5 and a Mixture of 
Tween 80 and Pluronic (TP) 
In order to determine whether MDP A-5 or Murabatide were more effective at 
inducing antigenicity, rats were injected with MDP A-5 in TP (250 
.mu.g/rat) or Murabutide in TP (250 .mu.g/rat) as described in Example 6. 
The results obtained are shown in Table 1. Antibody titers were determined 
as described in Example 3. 
TABLE 1 
______________________________________ 
Group Batch of LHRH.sub.1 -TT 
Conjugate Adjuvant 
No. Conjugate (dose) (dose) 
______________________________________ 
1 x-19 50 .mu.g/rat 
MDP in TP 
(250 .mu.g/rat) 
2 x-19 50 .mu.g/rat 
Murabutide 
in TP 
(250 .mu.g/rat) 
______________________________________ 
Antibody Titers 
(nmol/L, mean .+-. SE, N = 6 to 7) at week: 
3 5 7 
______________________________________ 
Group 1 2.0 .+-. 0.3 3.7 .+-. 0.5 
3.4 .+-. 0.5 
Group 2 1.5 .+-. 0.3 3.2 .+-. 0.4 
3.3 .+-. 0.2 
Serum testosterone 
(nmol/L, mean .+-. SE, N = 6 to 7) 
Group 1 1.7 .+-. 0.6 0.03 .+-. 0 
0.03 .+-. 0 
Group 2 4.4 .+-. 1.5 0.03 .+-. 0 
0.03 .+-. 0 
______________________________________ 
Testes Weights (g) (Mean .+-. SE, N = 6) 
Group 1: 0.74 .+-. 0.12 
Group 2: 0.50 .+-. 0.04 
(Control: 1.7 .+-. 0.03) 
______________________________________ 
No significant differences were observed in antibody titers, serum 
testosterone levels or sex organ weights (at week 7) between MDP A-5 and 
Murabutide. 
In order to determine whether MDP A-5 or TP were contributing to 
antigenicity, rats were injected as above with one of the following: TP 
(no adjuvant); MDP A-5 (250 .mu.g/rat) in TP; and MDP A-5 in SPAN 80 
(Sorbitan monoleate, (Ruger Chemical Co., Irvington NJ) and TWEEN 80. The 
TP and MDP/A-5 injections were prepared as described in Example 6; MDP A-5 
in SPAN 80 and TWEEN 80 were prepared by mixing all the components in an 
electric blender until an emulsion was achieved. The final injections 
contained 12 mg TWEEN 80, 48 mg SPAN 80, 0.34 ml H.sub.2 O, 0.66 ml peanut 
oil and 175 .mu.g LHRH.sub.1 -TT. Antibody titers were determined as 
described in Example 3. The results obtained are shown in Table 2. 
TABLE 2 
______________________________________ 
Group Batch of LHRH.sub.1 -TT 
Conjugate Adjuvant 
No. Conjugate (dose) (dose) 
______________________________________ 
1 YT-1X-140 175 .mu.g/rat 
TP (no 
adjuvant) 
2 YT-1X-140 175 .mu.g/rat 
MDP A-5 
(250 .mu.g/rat) 
in TP 
3 YT-1X-140 175 .mu.g/rat 
MDP A-5 
(250 .mu.g/rat) 
SPAN 80 and 
TWEEN 80 
______________________________________ 
Antibody Titers (nmol/L) 
(Mean .+-. SE, N = 10 to 12) at weeks: 
Group 4 8 12 16 
______________________________________ 
1 2.0 .+-. 0.1 
3.8 .+-. 0.2 
4.0 .+-. 0.3 
3.0 .+-. 0.3 
2 1.5 .+-. 0.1 
4.5 .+-. 0.2 
4.6 .+-. 0.2 
4.2 .+-. 0.3 
3 1.1 .+-. 0.1 
2.7 .+-. 0.3 
3.0 .+-. 0.3 
2.0 .+-. 0.4 
______________________________________ 
Serum Testosterone 
(nmol/L, Mean .+-. SE, N = 10 to 12) 
Week 
Group 4 8 12 16 
______________________________________ 
1 3.4 .+-. 1.5 
0.03 .+-. 0 0.3 0.03 
2 5.3 .+-. 1.6 
0.1 .+-. 0.05 
0.03 0.03 
3 7.4 .+-. 1.7 
4.0 .+-. 1.4 
1.3 .+-. 0.6 
7.0 .+-. 2.6 
______________________________________ 
The results of this study demonstrate that, although animals in Group 2 
(MDP A-5 used for all injections) have developed slightly higher antibody 
titers then those in Group 1 (LHRH.sub.1 -TT emulsified in TP without 
addition of adjurant), serum testosterone levels were sufficiently 
suppressed in both groups by week 8. In contrast, animals in Group 3 
(LHRH.sub.1 -TT emulsified in SPAN 80+TWEEN 80 with MDP A-5) developed 
significantly lower antibody titers and serum testosterone levels were not 
suppressed even by week 16. 
This suggests that the presence of TP in immunogen is more important than 
the presence of MDP A-5. 
In order to determine the importance of TP, rats were immunized with either 
MDP A-5 in TP or MDP A-5 in SPAN 80 plus alum. Antibody titers were 
obtained and the results were compared. The MDP A-5 TP injections were 
prepared as described above. The MDP A-5 alum injections were prepared as 
follows. The LHRH.sub.1 -TT was dissolved in phosphate buffered saline 
(PBS, 10 mM NaPO.sub.4, 150 mM NaCl pH 7.2) to obtain a final 
concentration of 0.5 mg/ml. To the LHRH.sub.1 -TT/PBS solution was added 
0.4 mL 10% AlK (SO.sub.4).sub.2 .times.10H.sub.2 O per mg of protein. A 
7.5% NaHCO.sub.3 solution was added to obtain maximum precipitation and 
the product was mixed well and centrifuged at 2,000 rpm for 10 minutes. 
After centrifugation the supernatant was discarded, the pellet was washed 
twice and resuspended in the vehicle to the desired concentration. Animals 
were injected and bled as described in Example 6 and antibody titers were 
determined as described in Example 3. The results obtained are shown in 
Table 3. 
TABLE 3 
______________________________________ 
Group Batch of LHRH.sub.10 -TT 
Conjugate Adjuvant 
No. Conjugate (dose) (dose) 
______________________________________ 
1 YT-1X-122 175 .mu.g/rat 
MDP A-5 
(250 .mu.g) in TP 
2 YT-1X-122 175 .mu.g/rat 
MDP A-5 
(250 .mu.g) in 
Span 80 + Alum 
______________________________________ 
Antibody Titers 
(nmol/L, mean .+-. SE, N = 11 to 12) at weeks: 
Group 4 8 12 20 
______________________________________ 
1 1.1 .+-. 0.3 
1.0 .+-. 0.1 
1.6 .+-. 0.2 
2.9 .+-. 0.2 
2 1.6 .+-. 0.4 
1.8 .+-. 0.2 
1.7 .+-. 0.2 
2.7 .+-. 0.2 
______________________________________ 
Serum Testosterone 
(nmol/L, mean .+-. SE, N = 10 to 12) at week: 
Group 4 8 12 20 
______________________________________ 
1 7.3 .+-. 0.5 
8.5 .+-. 1.4 
3.4 .+-. 0.8 
0.8 .+-. 0.3 
2 8.1 .+-. 1.1 
1.8 .+-. 0.5 
3.3 .+-. 1.0 
3.8 .+-. 1.0 
______________________________________ 
Number of rats with serum testosterone levels below 0.03 nmol/L 
by week 20: 
Group 1: 6 out of 12 
Group 2: 1 out of 12 
______________________________________ 
The results of this experiment, using LHRH.sub.10 -TT, clearly demonstrate 
the importance of TP in enhancing an immune response to LHRH. 
EXAMPLE 12 
Induction of Anti-LHRH Antibodies in Female Cats 
Four sexually mature female cats were immunized against LHRH.sub.1 -TT, 
using 500 .mu.g/injection, as described in Example 6, and four cats were 
used as nontreated controls. The animals were checked daily for signs of 
estrus. Antibody titers were determined as described in Example 3. All 
immunized cats developed measurable antibody titers by week 4. By week 8, 
antibody titers exceeded 2 nmol/L (FIG. 11). None of the immunized cats 
displayed any signs of estrus during the 16 weeks of the experimental 
period. Non-immunized cats displayed signs of estrus from one to four 
times (FIG. 12). In FIG. 12 dotted bars indicate time cats were tested for 
signs of estrus and black bars indicate the times cats displayed signs of 
estrus. In FIGS. 11 and 12 the numbers 645, 372, 646 and 228 indicate cats 
immunized with LHRH.sub.1 -TT. In FIG. 12, the numbers 164, 605, 648 and 
300 indicate non-immunized controls. The animals were killed sixteen weeks 
after the initial immunization. Ovaries in the immunized cats were 
approximately one-tenth the size of those in non-immunized controls. 
EXAMPLE 13 
Active Immunization Against LHRH Prevents the Growth of Androgen-Dependent 
Prostatic Carcinoma 
In order to determine whether active immunization against LHRH can be used 
as treatment for androgen-dependent prostatic carcinoma the following 
experiment was performed. 
Fifty male rats of the Copenhagen Fisher F.sub.1 strain were implanted 
subcutaneously with Dunning R-3327 prostate carcinoma. This carcinoma cell 
line was obtained from Dr. Karr at the University of Miami, School of 
Medicine. Rats were outfitted with self-piercing numbered ear-tags and 
randomly assigned to one of the following groups: 
Group 1--non-immunized controls 
Group 2--non-immunized, treated with LHRH-34 (250 .mu.g/kg/day) for 120 
days 
Group 3--immunized against LHRH.sub.1 -TT 
Group 4--immunized against LHRH.sub.1 -TT and treated with LHRH-34 (an LHRH 
antagonist: [Ac-D2Nal.sup.1, 4ClDPhe.sup.2, D3Pal.sup.3, Arg.sup.5, 
DGlu.sup.6 (Anisole Adduct), DAla.sup.10 ]-LHRH (obtained 6 from Dr. 
Sundaram) like Group 2 for the first 21 days. 
Tumor sizes were measured weekly. Immunization and/or treatment with 
LHRH-34 was initiated when tumors were at least 3cm.sup.2 in size (19 
weeks after insertion of the tumor cells.) Each of the above listed group 
of animals consisted of 4 rats with tumors from 3 to 4 cm.sup.2, 4 rats 
with tumors from 4 to 8 cm.sup.2 and 4 to 5 rats with tumors larger than 8 
cm.sup.2 ]. The results obtained from rats immunized with LHRH.sub.1 -TT 
and treated with LHRH-34 are shown in closed triangles. Note that the 
antibody titers in nonimmunized rats are within the shaded area and were 
0.4 nmol/L. 
Animals in Group 1 (non-immunized controls) were injected daily with 
sterile saline. Rats in Group 2 were injected daily with LHRH-34 (250 
.mu.g/kg/day) dissolved in 5% mannitol in bacteriostatic water (0.5 
ml/rat). Rats from Groups 3 and 4 were immunized against LHRH.sub.1 -TT 
(50 .mu.g/rat/injection) emulsified in a mixture of Tween 80 and Pluronic 
in 0.9% NaCl as described in Example 12. Immunogen injections were given 
at weeks 19, 21 and 23 after the implantation of Dunning prostate cancer 
cells. Animals in Group 4 were immunized as in Group 3 and concomitantly 
treated with LHRH-34 as in Group 2 for the first 3 weeks after 
immunization. 
Blood from the central caudal artery was collected approximately every 3 
weeks from the beginning of LHRH immunization/treatment. The experiment 
was terminated approximately 4 months after initiation of the treatment. 
Testes, epididymides, prostates and seminal vesicles were dissected and 
weighed. 
The results obtained showed that animals immunized against LHRH.sub.1 -TT 
(Groups 3 and 4) developed anti-LHRH anti-body titers above 3 nmol/L by 
week 22, only 3 weeks after the immunization (FIG. 13). FIG. 13 is a graph 
depicting the anti-LHRH antibody titers in the rats described above. 
Arrows represent the time of immunization against LHRH. The results 
obtained from the control rats are shown by the open circles. The results 
obtained from the rats treated with LHRH-34 are shown in closed circles. 
The results obtained from rats immunized with LHRH-TT are shown in open 
triangles. Animals from Group 4 (immunized against LHRH.sub.1 -T and 
concomitantly treated with LHRH-34) had anti-LHRH antibody titers lower 
than those without LHRH-34 treatment even after the LHRH-34 injections 
were discontinued. However, in both groups of immunized animals the amount 
of anti-LHRH antibodies was sufficient to suppress T to castration levels 
(below 0.03 nmol/L) by week 24. In animals treated with LHRH-34 alone 
serum T levels were suppressed by the time of the first blood collection 
at week 20 (FIG. 14). FIG. 14 is a graph depicting serum testosterone 
levels in the rats of the present example. The different groups are 
depicted as described above for FIG. 13. Note that immediately after 
treatment at nineteen weeks serum T levels dropped below 0.03 nmol/L in 
animals treated with LHRH-34 whereas in animals immunized with LHRH.sub.1 
-TT serum T levels dropped to castration level by week 24, five weeks 
after initial immunization. All animals regardless of the treatment 
regimen, had serum T levels below 0.03 nmol/L through the end of the 
experiment at week 35. Testes and accessory sex organ weights in all 
groups of treated animals were significantly lower than those in 
non-immunized controls (FIG. 15). FIG. 15 is a series of bar graphs 
depicting organ weights of the various treatment groups following sixteen 
weeks of androgen suppression treatment. The control group is represented 
by the open bar, the LHRH treated group is represented by the slanted 
lines, the LHRH.sub.1 -TT immunized group is represented by the vertical 
lines and the LHRH-34 treated, LHRH.sub.1 -TT immunized group is 
represented by the cross-hatched bars. Note that the organ weights did not 
differ significantly between groups. 
The effects of treatment with LHRH-34 and/or active immunization against 
LHRH on the growth of the tumors depended on tumor size. Androgen 
depletion through immunization against LHRH, daily injections with 
LHRH-34, or a combination of the two, resulted in significant suppression 
of tumor growth if treatment was initiated before tumors were larger than 
8 cm.sup.2. Once the tumors were larger than this size, however, neither 
immunization nor LHRH-34 treatment caused suppression of tumor growth 
(FIG. 16). FIG. 16 depicts the change in tumor size relative to initial 
tumor size for the various groups of rats. The difference is expressed as 
the difference in tumor area (mean+SE, N=3 to 4) at the beginning and end 
of treatment. The various groups are depicted as described above for FIG. 
13. 
The results presented above demonstrate that active immunization against 
LHRH can be successfully used as convenient and cost-effective treatment 
to suppress tumor growth in androgen-dependent prostatic carcinoma.