Immunotherapy of prostate cancer in a murine model using a novel GnRH based vaccine candidate

Available online at www.sciencedirect.com

Vaccine 25 (2007) 8460–8468

Immunotherapy of prostate cancer in a murine model using a novel GnRH based vaccine candidate Jes´us A. Junco a,∗ , Peter Peschke b , Ivan Zuna b , Volker Ehemann d , Franklin Fuentes a , Eddy Bover a , Eulogio Pimentel a , Roberto Basulto a , Osvaldo Reyes c , Lesvia Calzada a , Mar´ıa D. Castro a , Niurka Arteaga a , Yovisleidis L´opez a , Hilda Garay c , H´ector Hern´andez a , Ricardo Bringas c , Gerardo E. Guill´en c b

a Department of Cancer, Center for Genetic Engineering and Biotechnology of Camaguey, CP 70100, Apdo 387, Camaguey, Cuba Clinical Cooperation Unit Nuclear Medicine, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany c Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana, Cuba d Institute for Pathology, University of Heidelberg, Im Neuenheimer Feld 400, D-69120 Heidelberg, Germany

Received 30 April 2007; received in revised form 14 September 2007; accepted 15 September 2007 Available online 4 October 2007

Abstract Previous studies with gonadotrophin releasing hormone (GnRH/LHRH) vaccines have shown the usefulness of immunization against this hormone in prostate cancer. To this end, we have generated a completely synthetic peptide modified at position 6 and attached to the 830–844 tetanic toxoid (TT) helper T cell sequence. Through this work we have demonstrated that the GnRHm1-TT molecule was highly immunogenic when it is formulated as an oil-based emulsion adjuvated with Montanide ISA 51. That results correlated directly with testosterone reduction and tumor growth inhibition of the Dunning R3327-H androgen responsive prostate tumor model in rats. GnRHm1-TT, proved to be safe and useful for future clinical trials. © 2007 Elsevier Ltd. All rights reserved. Keywords: Hormone-sensitive prostate cancer; Immunotherapy; Cancer vaccines

1. Introduction In the 1940s Huggins and Hodges established that androgen deprivation in patients suffering from prostate cancer resulted in a favorable response in most patients [1]. From that moment on androgen deprivation has become the standard treatment for those with locally advanced or metastatic disease. Androgen deprivation can be achieved either medically or surgically and surgical castration is still considered the gold standard endocrine treatment for prostate cancer [2]. Medical castration can be achieved in many ways. Though initially Dietil-stilbestrol (DES) was used, this had many cardiovascular side effects. Therefore alterna∗

Corresponding author. Tel.: +53 32 261014; fax: +53 7 2714764. E-mail address: [email protected] (J.A. Junco).

0264-410X/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2007.09.033

tive drugs such as antiandrogens and gonadotropin-releasing hormone (GnRH/LHRH) analogs were introduced in the early 1980s to achieve reversible pharmacological castration. However, these drugs are very expensive and must be used for a lifetime [3]. An alternative approach to the use of GnRH analogs is the immunoneutralization of GnRH using a new GnRH like peptide that could serve as vaccines when attached to more immunogenic molecules like tetanic toxoid (TT) epitopes [4–7]. Similar approaches had been used in rodents and primates and reported to produce atrophy of the prostate [8–12]. Since the GnRH is a completely conserved structure in rats and monkeys, it serves as homologous model for humans [13–15]. In the 1990s clinical trials with the construction of LHRH vaccines were started in order to produce an androgen deprivation in patients with advanced prostate cancer [4,16,17] and in

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post-menopausal women to search gonadotropins inhibition. The efficacy of neutralizing the GnRH/LHRH function through the action of anti GnRH specific antibodies has been demonstrated actively by vaccination or passively by infusion of purified anti GnRH antibodies [18–20]. However, other researchers have mentioned that used LHRH peptide coupled to tetanus or diphtheria toxoid (DT) molecules as carriers [4,16,17] have been reported to cause carrier induced antihaptenic immunosuppression [21–23]. Furthermore, the conjugation process used for the peptide-carrier fusion, resulted in losses during conjugation and impractical commercial goals [13], and the passive immunization has turned out to be an expensive product with limited results [17]. Recently, a research group used multiple T helper epitopes chemically bound to GnRH to improve the immunogenicity and the castration levels in the potential recipients [24]. In this report, we have designed a vaccine candidate called GnRHm1-TT [25], based on a completely synthetic immunogen. Its active compound is a GnRH decapeptide, modified at position 6, in which the local glycine has been substituted by a proline attached to a 15 amino-acid tetanic toxoid T helper epitope through the GnRH carboxyl terminal. The peptide was finally formulated as a white, semiviscous water in oil preparation. In healthy animals, this vaccine was shown to be very immunogenic, resulting in high anti-GnRH antibodies titers, reduction in testosterone and significant decrease of the prostate and testes weight. In healthy adult male and female dogs, the histology showed degenerative changes in testes and ovarian cellular morphology, that produced a decrease in the number and survival time of spermatozoa in male, and atretic follicles in females [26]. In the present report, we emphasize the immunogenic capacity of GnRHm1-TT and the correlation with reduction in testosterone and tumor burden in the Dunning R3327-H androgen responsive prostate tumor model. Moreover it was demonstrated that the vaccine candidate was safe when formulated as an oil-based emulsion with Montanide ISA 51 adjuvant, which makes it a candidate for use in future clinical trials.

2. Materials and methods 2.1. GnRH peptides The mammalian GnRH-I [pGlu-His-Trp-Ser-Tyr-GlyLeu-Arg-Pro-GlyNH2 ] and the modified variant GnRHm1TT [pGlu-His-Trp-Ser-Tyr-Pro-Leu-Arg-Pro-GlyNH2 ] were prepared by conventional solid phase F-moc methodology and purified by preparative C18 reverse phase HPLC at The Center for Genetic Engineering and Biotechnology, Havana, Cuba, as previously detailed [27]. The construction of a GnRHm1-TT synthetic peptide meant the link of a GnRH molecule modified at position 6 in which the glycine was changed to a more rigid proline and joined through a couple

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Fig. 1. Primary Structure of the native LHRH/GnRH molecule and the GnRHm1-TT peptide. The GnRHm1-TT molecule is the result of the solid phase synthesis and chemical binding of the GnRHm1 molecule with a TT epitope. The synthetic process was carried out coupling the GnRHm1 peptide to the 17 amino acid sequence 830–844 of TT trough their carboxi-terminal using a glycine-glycine (GG) spacer branch.

of glycine residues to a T helper (Th) epitope corresponding to 830–844 region of Tetanic Toxoid (TT). This peptide fragment has been reported to be a promiscuous Th epitope that stimulate a broad range of histocompatibility backgrounds. As result of the chemical synthesis, a high purity 27 aminoacid peptide was obtained (Fig. 1). Mass spectrophotometry was performed for verification. 2.2. Animals, tumor system and experimental design Fresh pieces (2 mm × 2 mm × 2 mm) of tumor tissue from the R3327-H subline of the Dunning prostate tumor system were transplanted subcutaneusly (s.c). into the distal right thigh of anesthetized male young adult (180 g) Copenhagen rats (Charles River, Germany) 4 months before the beginning of treatments (immunization and castration). The R3327H subline, was kindly supplied to us by J.T. Isaacs, Johns Hopkins University, Baltimore MD, USA. It is a highly differentiated, hormone dependent carcinoma of the prostate with a mean volume doubling time (VDT) of about 17 days [28]. Therapeutic intervention started when the tumors had reached a diameter of about 10 mm. The three study arms consist of; (A) 9 immunized animals with the GnRH immunogen (GnRHm1-TT); (B) animals which received a placebo preparation containing water in oil emulsion (n = 7); and (C) 10 animals surgically castrated according to the therapeutic “gold standard” for hormone dependent prostate tumors. For volumetric determination of growth delay, tumors were measured routinely once a week. The tumor volume was calculated with the formula 4/3πr3 , r being the mean of two orthogonal radii. Animals were maintained with a commercial diet and fresh water, ad libitum. Then they were housed, two per cage, under humidity-and temperature-controlled conditions and a light/dark cycle of 12-h intervals. All the guidelines established for laboratory animals by the German Government were considered. 2.3. Animals immunization The immunogen was prepared by emulsifying the aqueous phase containing the lyophilized peptide with the oily adjuvant Montanide ISA 51 (v/v) (Seppic, France). Male Copenhagen rats (250 g) were immunized with GnRHm1-TT (750 ␮g/rat). All the injections, six in total, were administered as a bolus s.c. (500 ␮L/rat) within an interval of 15 days.

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2.4. Serum collection Blood samples from all animals were collected via tail vein puncture before the start of the experiment and every 15 days prior to the immunization procedure. During blood collection, animals were kept under a short-time anesthesia using a mixture of halothane, oxygen and nitrous oxide. The blood samples were kept for 2 h at 4 ◦ C followed by centrifugation at 4000 × g, 20 min (4 ◦ C). The serum was collected and frozen at −20 ◦ C until use. 2.5. Anti-GnRH antibody titer screening Concentration of circulating antibodies was determined by an enzyme-linked-immunoassay. Microtiter plates were coated with natural GnRH peptide (5 ␮g/mL) in 100 mM Na2 CO3 (pH 9.6) and incubated overnight at 4 ◦ C. After several washes with PBS (pH 7.4), the plates were saturated for 1 h with 2% BSA in PBS. Animal serum was diluted (range: 1/60 until 1/2000) in PBS containing 1% BSA, Tween 20 (0.01%, w/v) and incubated for 3 h at 37 ◦ C. The plates were washed several times with PBS and allowed to react with peroxidase-conjugated goat antirat antibody (Sigma Biochemical, USA) for 1 h at 37 ◦ C. After another wash, the plates were allowed to react with Ortophenilen-diamine (OPD) containing hydrogen peroxidase as substrate. The antibody titers were calculated as the maximum dilution at which the sample value was two times higher than the absorbency of the cut off value. 2.6. Testosterone determination Testosterone levels were determined using the commercial TESTO CT2 kit (CisBio, International, France). The sensitivity of the method defined as being the detectable concentration equivalent to twice the standard deviation of the zero-binding value, is approximately 0.1 nmol/L. The specificity of the test kit for testosterone is superior to 99%. To carry out the determinations, 25 ␮L of serum from each sample was plated directly in the pre-coated tubes. Duplicates from all samples were incubated for 1 h at 37 ◦ C. Finally the tubes were washed with distilled water and read in gamma counter. The results were recorded as nmol/L.

the Student’s t-test at a level of significance P < 0.05. For animal survival analysis the log rank statistical test method was used. A Student’s t-test was used for antibody and testosterone analysis. Biostatistical analysis was performed using a statistical program package (SAS Institute, Inc., SAS/STATTM Userˇıs Guide, Release 6.03 Edition. Cary, NC: SAS Institute Inc., 1988. 1028 pp).

3. Results 3.1. Immunization with GnRHm1-TT produced neutralizing anti natural GnRH antibodies that drop testosterone in pre-implanted Copenhagen rats To evaluate the ability of GnRHm1-TT vaccine candidate to produce effective anti-GnRH response in a tumoral model, mature male Copenhagen rats were implanted with the Dunning R3327-H tumor fragments 4 months before beginning treatments (Immunization and castration). Solid tumors were obtained in all the 26 implanted rats used in the experiment. Based on our established vaccination protocol, we undertook a total of 6 immunizations in a fortnightly schedule in the pre-implanted Copenhagen rats. Eight out of nine rats generated a variable neutralizing anti-GnRH antibodies, as shown by an indirect anti GnRH ELISA (Fig. 2). Antibody levels started to increase at day 45 from the beginning of immunization. Five out of eight animals developed high anti GnRH seroconversion after the four immunization and among the remaining three animals, two of them developed moderate anti GnRH seroconversion at day 60 post immunization. One rat did not raise significant anti- GnRH antibodies. This animal was considered a non responder (Fig. 2).

2.7. Statistical analysis The treatment response was judged on the basis of tumor growth delay (GD). For that purpose the time T5 in days, the tumors needed to reach five times their pre-treatment volume (V0 ), was calculated. Non-parametric statistics (median, 25%-, 75%-quantils, Mann–Whitney) were used [29]. A bestfit regression (linear or quadratic) was determined from the median GD’s of the control and the two treatment groups. To enable a parametric evaluation of the data (analysis of variance, and Student’s t and F tests) we used mean and standard deviation, instead of nonparametric statistics for the characterization of T5 . The treatment groups were compared using

Fig. 2. Immunogenicity profile of male rats immunized with GnRHm1-TT vaccine candidate. Rats were immunized subcutaneous at 0, 15, 30, 45, 60 and 75 days (labelled by arrows) with 750 ␮g of GnRHm1-TT peptide emulsified with Montanide ISA 51. Each animal is represented by a symbol. Animals were bled at days 0, 45, 60, 75 and 90 after immunization and tested by indirect ELISA for anti GnRH antibody seroconversion. GnRHm1TT produced anti-GnRH antibody titres that correlated with the androgen withdrawal observed in the major vaccine responders.

J.A. Junco et al. / Vaccine 25 (2007) 8460–8468 Table 1 Sequential testosterone measurements of male Copenhagen rats at days 0, 60, 75 and 90 after the experiment beginning (a) immunized group (n = 9), (b) castrated group (n = 10), (c) placebo group (n = 7) (a) N0. of the immunized rata

Blood extraction Day 0

Day 60

Day 75 2.40 0.07 0.23 0.10 0.08 0.73 –c 0.11 –c

1.70 0.01 0.14 0.07 0.00 0.00 –c 0.18 –c

Day 75

Day 90

(b) N0. of the castrated rata

Blood extraction Day 0

Day 60

Sequential testosterone levels determination (nmol/L)b 35 4.23 0.00 0.00 38 3.92 0.00 0.01 39 2.95 0.06 0.00 40 3.87 3.40 2.32 41 4.12 0.95 0.00 44 3.12 0.00 0.07 45 2.99 0.00 0.00 48 3.45 0.00 0.06 50 2.03 0.00 –c 52 2.54 0.00 0.00

0.00 0.04 0.01 1.73 0.00 0.03 0.00 0.00 –c 0.00

(c) N0. of the placebo rata

Blood extraction Day 0

Day 60

Sequential testosterone levels determination 31 6.75 6.76 33 4.43 7.21 34 2.43 2.32 47 2.28 2.22 53 4.34 3.47 54 4.10 3.36 57 2.85 –c

Day 75

Day 90

(nmol/L)b 6.89 5.01 –c 2.12 –c 3.78 –c

fast and powerful testosterone fall. Similarly, in two animals of this group that just resembled moderate anti GnRH seroconversion and titers, testosterone dropped until castration level (Table 2). No anti-GnRH serum antibodies were observed in either the placebo, castrated or immunized rats before the start of the experiment.

Day 90

(nmol/L)b

Sequential testosterone levels determination 32 2.63 0.68 36 4.84 0.19 43 3.78 0.95 46 3.89 1.15 49 3.33 0.00 51 4.21 0.79 55 4.87 0.84 56 2.63 0.73 60 2.63 1.90

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–c 6.94 –c 3.22 –c 3.69 –c

a Rats were randomized using the statistical program package (SAS Institute Inc., SAS/STATTM . b Testosterone was determined using the radioimmunoassay kit TESTOCT2 manufactured by CIS-BIO International, France. c Means that the determination was not carried out since the rat died.

Castration levels in the immunized animals were detected just after the four immunization. The individual Testosterone sequential determination practiced to the immunized, castrated and placebo group, are summarized in Table 1(a)–(c) respectively. As expected, the anti GnRH seroconversion in this cohort of vaccinated animals was correlated with significant testosterone decrease in six of the eight immunoresponding rats (Table 2). So, rats 46, 49 and 51, that developed the major anti GnRH seroconversion and titers showed the

3.2. Therapeutic immunization with GnRHm1-TT vaccine induced tumor growth retardation in pre-implanted Copenhagen rats Given the ability of the tumor pre-implanted Copenhagen rats immunized with GnRHm1-TT to develop anti-GnRH antibodies and in turn, reduce testosterone, the study was performed in order to evaluate the extent of tumor inhibition. In these experiments, therapy was administered to rats carrying subcutaneous Dunning R3327-H tumors that reached a volume of around 10 mm. Placebo, castrated and immunized groups were evaluated. By the end of the day 60-observation period, tumor increased growth sharply in six out of seven animals of the Placebo group (Fig. 3a). However, in the majority of immunized rats (6/9) and castrated (6/10), a tumor growth retardation was observed (Fig. 3b and c). Around day 105 after treatment when the placebo rats died or been sacrificed due to the tumor size, the majority of immunized and castrated animals were alive and a significant tumoral growth inhibition was noted (P = 0.014). Taking together the testosterone and anti-GnRH seroconversion data in correlation with the tumor growth burden (Table 3), the treated rats were classified as: good, moderate and non responders. So, the 75% of the immunized rats were considered moderate or good responders. However, in one immunized rat (rat 51), regardless of the high levels of anti GnRH antibodies and Testosterone depletion, a vigorous tumor growth was observed. Furthermore, one rat (rat 60), did not generate anti GnRH antibodies and was unable to drop Testosterone and hence tumor grew vigorously. Finally, one animal within this group with a potent anti GnRH seroconversion, testosterone reduction and evident tumor retardation at day 60 (rat 55), was not considered in the statistical analysis since it died accidentally (Fig. 3b). In the castrated group, similar to the immunized one, a clear tumor inhibition was found in 6 out of 10 animals (Fig. 3c). The tumor growth comparison between castrated and placebo groups revealed a statistical significance of (P = 0.023). However, as expected, no statistical differences were found between Immunized and castrated groups. (P > 0.05). 3.3. GnRHm1-TT vaccine increase the survival of immunized Copenhagen rats preimplanted with Dunning R3327-H tumor To determine the effects of GnRHm1-TT vaccination on long term survival, animals were followed over a period of 300 days. The Kaplan–Meier survival plot showed that at

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Table 2 GnRHm1-TT effect on antibody production and survival in immunized Copenhagen rats Number of immunized animals

Time of anti GnRH seroconversion (in days)a

Anti GnRH titres after the last immunizationb

Last testosterone determination (nmol/L)

Survival (in days)

32 36 43 46 49 51 55 56 60

60 45 45 45 45 45 45 60 –

1/200 1/400 1/100 1/800 1/800 1/1600 1/200 1/100 –

1.70 0.01 0.14 0.07 0.00 0.00 0.84 0.18 1.90

150 150 265 195 265 95 60 265 85

a b

Number of days after the first immunization at which the individual animals exceeded the seroconversion cut off line. Maximal dilution at which the anti-GnRH antibodies levels were over the cut off line (0.38 OD), when measured at 492 nm.

Table 3 Effectiveness of GnRHm1-TT vaccine candidate in pre-implanted Copenhagen rats compared with controls Type of treatment

Placebo Immunized Castrated a

Animal response per group Testosterone respondersa

(%)

Testosterone (nmol/L) mean per group

Tumor growth retardation

Mean survival (days)

0/7 6/8 9/10

0 75 90

4.18 0.52 0.10

0/7 6/8 7/10

77 183 174

Number of animals with testosterone under castration levels.

day 105, 100% of the placebo animals were dead or had to be sacrificed due to their tumor size. The survival analysis of each group of animals using the log rank statistical test to compare the cumulative survival probability, showed that the immunized and castrated controls survived twice as long as the placebo (Fig. 4).

4. Discussion GnRHm1-TT, the putative vaccine described in this study is an effective and practical synthetic peptide based compound useful for treatment of androgen-responsive prostate cancer in which a simple replacement of the glycine aminoacid at position 6 of the GnRH molecule by the more rigid proline have been changed and instead of a conjugation step for the carrier attachment, the high immunogenic 830–844 Tetanic Toxoid T helper region has been included in the same process of peptide synthesis [25]. The mode of action for this immunotherapeutic vaccine is the androgen deprivation of the androgen- dependent prostatic neoplasms. The vaccine consists of 750 ␮g of the active peptide diluted in water for injection and emulsified with Montanide ISA 51 oil adjuvant. This preparation has been used successfully to immunize prepuberal pigs, adult dogs and rats, where it corroborated the advantage of using this single step method in comparison with the conjugation process practiced in the earlier studies [4,16,30]. The competence of GnRHm1-TT in causing anti-GnRH seroconversion and testosterone reduction in the models tested, have been mainly produced by the change at position 6 of GnRH molecule

of a glycine by the more rigid proline aminoacid and on the other hand taking advantage of the use of the universal 830–844 Tetanus Toxoid epitope, which we used in a similar attempt to those practiced by Etlinger et al. to bypass the carrier-induced suppression caused by the whole molecule. GnRHm1-TT adjuved in Montanide ISA 51 has shown to be effective in generating high anti GnRH antibody levels, including the tumor implanted Copenhagen rats (Fig. 2, Table 2). These results are in agreement with those reports of good immune humoral response when using peptide and proteins antigens administered in the same adjuvant [31–34]. The therapeutic immunization practiced using these vaccine candidate, revealed that (88.8%) of the animals generated neutralizing anti-GnRH antibodies when analyzed by ELISA. These results were comparable or slightly higher than those obtained with the veterinary vaccine VAXTRATE used for the immunization of healthy pigs [35], as well as those obtained with the use of the D17DT candidate from Apthon Corporation in pre-clinical experiments and in clinical settings [17]. In the study carried out, a direct correlation between antiGnRH titers and castration levels was observed. So, the three animals with the higher antibodies titers corresponded to those with the fast and major testosterone castration. Moreover, three rats with moderate anti GnRH titers, showed a good testosterone depletion, showing that once the titers reach a significant level, testosterone drops until castration. What is more, according to the relation between the anti GnRH seroconversion and castration, it appears that once the seroconversion overcomes 0.3 OD, with a dilution serum of 1/50, it result enough to drop testosterone until castration

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Fig. 3. Follow up of tumor growth rates for Dunning R3327-H prostate tumor fragments implanted in Copenhagen rats. Placebo, Castrated and Immunized male Copenhagen rats were implanted subcutaneously in the distal right thigh with 2 × 2 × 2 tumor fragments of Dunning R3327-H as described in Section 2. Placebo group (n = 7; part a) was injected with 500 ␮L of water in oil emulsion without the peptide. Immunized rats (n = 9; part b), were injected fortnightly with 750 ug of GnRHm1-TT peptide mixed with Montanide ISA 51. Castrated rats (n = 10; part c) were surgically orchiectomized at the beginning of the experiment. Tumor growth was measured at weekly interval and showed a tumor growth retardation in the immunized and castrated animals in comparison to the intact placebo.

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Fig. 4. Kaplan–Meier representation of the cumulative survival probability of the different experimental groups. The Immunized and castrated rats showed a significant tumor growth retardation (P = 0.014 and P = 0.023) respectively; compared to the placebo group.

regardless the final serum titer. On other hand, the animals that never responded to immunization, were the ones that did not deplete testosterone and were the first to die in the group. These results, with the use of a single T helper epitope in a vaccine preparation, although employing a different immunoassay system to the serum screening, were similar to those described by Talwar et al. in their GnRH vaccines experiences and the work published by Finstad et al. using the multi-T helper epitopes vaccine candidate UBITh® [13,16,24]. The Dunning R3327-H tumor model for androgenresponsive prostate cancer in Copenhagen rats has permitted the efficacy of the GnRHm1-TT vaccine candidate for hormone-deprivation therapy to be tested and validated. So, in the experiment described above, the tumor implantation in Copenhagen rats, resulted in the development of a solid tumor which reached a mean volume of 10 mm in around 4 months. The histology, biochemical and enzymatic profile of these tumoral models are similar to those of the well-differentiated human prostatic cancer, which coincides with other reports [24,36,37]. To evaluate the mean tumor growth in each group, the experiment was extended to around 300 days and the difference at day 105 was correlated. The mean tumor volume of the immunized and castrated animals was significantly different to placebo (P = 0.014 and P = 0.023), respectively. In the case of the experimental GnRHm1-TT group, it was 75% effective in arresting the growth of established tumors in the therapeutic model used once the testosterone levels were suppressed to castrating levels by the anti- GnRH antibodies. However, in one case despite the good correlation between anti GnRH titers and testosterone suppression, a fast tumorgrowing rate was seen. This behavior was similar to those found in three castrated animals, in which, irrespective of the surgical castration practiced and the resulting testosterone depression until zero, (0),

the tumor became apparently hormone-insensitive and grew independent of castration. Similar trends following castration have been reported by Isaacs et al. [38], who measured the effects of surgical castration on the growth of Dunning H tumors and observed delayed growth of tumor implants in castrated animals when compared to placebo’s animals; where tumors continued to grow in the androgen-independent environment with a marked delay. These observations suggested the presence of a local LHRH loop within the rat prostate and the Dunning H tumors, in addition to the LHRH loop of the pituitary-gonadal axis [37,39]. In the case of the castrated animals used in the experiment, it is interesting to note that regardless of the surgical procedure practiced to all rats of this group, one failed to lessen testosterone to castration levels. Although there is no obvious explanation for this phenomenon; this animal, was one of the non responders. Finally, to evaluate the animals group survival, we used the log rank statistical test to compare the cumulative survival probability of each group by Kaplan-Meier. These data showed that the immunized and castrated rats, survived around 2 times longer than the placebo group P < 0.01. These pre-clinical studies all together, have important implications for the clinical situation where conventional androgen suppression therapy serve as a palliative care to suppress the prostate neoplasm rather than destroying the tumor. Hormone deprivation action of GnRHm1-TT emulsified in Montanide ISA 51 inhibited testosterone synthesis and may have other applications. The most prominent is related with the immunotherapy of androgen-sensitive prostate cancer and other hormone sensitive-diseases as breast cancer. At the same time, these applications can be used for reversible treatment of benign prostate hyperplasia (BPH), contraception, and in the swine industry to avoid the boar taint caused by androgens [37,40–43]. Previous clinical trials using an LHRH decapeptide with a lysine instead of a glycine at position 6 to bind a DT molecule to GnRH, have demonstrated to be safe and clinically beneficial in patients with advanced prostate cancer [44]. However, a drawback of these vaccines was the relatively low number of responders apparently caused by the use of a mild adjuvant like alumina. [22,23]. Other studies with a GnRH vaccine were carried out by Simms et al. [17]. They used the vaccine candidate D17DT of the Aphton Corporation, which coupled GnRH to the whole DT molecule through their amino terminal. However in these studies major problems arose and were related to the high number of non responders apparently due to the epitopic suppression caused by previous exposition to DT. In conclusion these studies highlight the efficacy of GnRHm1-TT in inhibiting testosterone synthesis. As such this method would important implications not only in the treatment of androgen-sensitive prostate cancer but also in other hormone sensitive diseases such as breast cancer. Additionally, our novel approach could have applications as in the

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treatment of BPH [40], contraception [16] and in the swine industry [40]. Though there have been other clinical trials with GnRH immunogenes in the past [16,17,39] these earlier trials demonstrated various limitations including a low number of responders triggered by either the use a mild adjuvant [22,23] or because of epitopic suppression [17]. Our approach not only overcomes these limitations but is also well tolerated and this offers a new and effective alternative for the treatment of hormone sensitive prostate cancer.

Acknowledgements We would like to thank German Academic Exchange Service (DAAD) by the funds provided to permit the development of this work in Germany and the excellent attention of Ms. Elke Massa in this purpose. We thank similarly to DKFZ, Heidelberg, Germany, and especially to Dr. Peter Pescke belonging to the Department of Radiation Oncology for permit us to carry out these work. Moreover we would like to thank Professor Eric Hahn for his valuable suggestions and technician Alexandra Tietz for the help in tumor implantation. Twan Lammers by his important recommendations and Gabriele Becker for their unconditional support and help. We thanks English language specialist, Luis Daniel Gonz´alez and Orestes Padr´on Yordy, for their careful English revision and professor Fouad Habib by his meticulous arrangements and important suggestions to the final version of this paper.

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