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Synthetic luteinizing hormone releasing hormone (LHRH) vaccine for effective androgen deprivation and its application to Connie L. Finstad , Chang Yi Wang , Jacek Kowalski , Meilun Zhang , Ming Lie Li , Xuan Mao Li , Wei Guo Xia , Maarten C. Bosland , Krishna K. Murthy , Alan M. Walfield , Wayne C. Koff , Timothy J. Zamb a United Biomedical Inc., 25 Davids Drive, Hauppauge, NY 11788, USA b New York University School of Medicine, Tuxedo, NY 10987, USA c Southwest Foundation for Biomedical Research, San Antonio, TX 78227, USA Received 9 June 2003; accepted 17 August 2003 Abstract
We have designed a peptide-based immunotherapeutic vaccine for treatment of androgen-responsive prostate cancer. The vaccine targets the luteinizing hormone-releasing hormone (LHRH) decapeptide that results in an androgen-deprivation immunotherapy. The design ele-ments of the peptide immunogens are the LHRH peptide or B cell epitope synthetically linked to different promiscuous helper T cell (Th)sequences, the UBITh® epitopes, derived from four natural pathogens for effective immunogenicity in outbred populations, and in somecases, also linked to an adjuvanting peptide from Yersinia invasin (Inv) protein. The UBITh® LHRH immunogens are adsorbed on Alhydro-gel or formulated as several different oil-based emulsions and tested in rodents, dogs, and a non-human primate, baboons. The immunogensgenerate an anti-LHRH antibody response specific to the LHRH decapeptide element in contrast to LHRH conjugate-carrier protein vaccineswhere only a small portion of the antibody response is directed to the target epitope and epitopic suppression is noted. Individual UBITh®peptide domains, but not the LHRH and Inv peptide domains, are stimulatory in lymphocyte cultures. The UBITh® LHRH immunogens in aclinically applicable formulation, controlled the growth of Dunning R3327-H androgen-responsive prostate tumor cells in rats. The resultsdemonstrate universal responsiveness and long duration of androgen deprivation from three diverse species, and thus vaccine efficacy.
2003 Elsevier Ltd. All rights reserved.
Keywords: Androgen-deprivation immunotherapy; Prostate cancer; LHRH vaccine; Baboons 1. Introduction
and follicle stimulating hormone (FSH). LH and FSHbind to receptors in the testis and ovary and regulate go- A treatment for prostate cancer is described based on a nadal function by promoting sex steroid production and synthetic peptide vaccine that effects hormone-deprivation gametogenesis. In males, LHRH induces maturation of the therapy. The active ingredient of the vaccine is a mixture testosterone-secreting interstitial cells of the testis. Testos- of entirely synthetic peptide immunogens that direct an terone is converted to dihydrotestosterone, the form of the immune response against luteinizing hormone-releasing hormone that interacts with androgen receptors on prostatic hormone (LHRH). LHRH, also known as gonadotropin epithelial cells to control their proliferation and apoptosis.
releasing hormone (GnRH), is synthesized in the hypotha- Therapies with LHRH agonists interfere with the action lamus and transported by the hypothalamic-hypophyseal of LHRH and block the effects of this hormonal cascade portal system to the anterior pituitary where it acts to ef- The growth of hormone-dependent tumors arising in fect secretion of gonadotropins, luteinizing hormone (LH) the prostate gland can also be controlled by removal ofthe growth-promoting hormone(s) by blocking theLHRH pathway with immunotherapy ∗ Corresponding author. Tel.: +1-631-273-2828; fax: +1-631-273-1717.
The early landmark studies of Huggins and Hodges E-mail address: (C.L. Finstad).
1 established the hormonal dependence of prostate cancer and Present Address: Wyeth Vaccines, Pearl River, NY 10965, USA.
2 Present Address: International AIDS Vaccine Initiative, New York, provided the basis for the use of androgen deprivation in its treatment. Reduction of plasma testosterone to castrate 0264-410X/$ – see front matter 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.vaccine.2003.08.044 C.L. Finstad et al. / Vaccine 22 (2004) 1300–1313 levels, either through surgical castration (orchiectomy) or solid-phase synthesis with F-moc chemistry using termi- use of oral or injectable estrogens, became the standard of nus and side chain-protected amino acids Ap- therapy for disseminated prostate cancer for the next 40 plied Biosystems Peptide Synthesizers, Models 430A or years. In the early 1980’s, LHRH analogues were added as 431. After complete elongation of a desired peptide, the an alternative to achieve reversible pharmacologic castra- co-polymeric styrene beads (Rink Amide MBHA resin, Cat.
tion Several randomized trials have demonstrated ther- No. 01-64-0037; Calbiochem-Novabiochem, San Diego, apeutic equivalence of low dose estrogens, LHRH agonists CA, USA) were treated according to standard procedures (e.g. leuprolide, buserelin, goserelin), and orchiectomy in with 90% trifluoroacetic acid (TFA) to cleave the peptide rate of response, failure-free survival and overall survival from the resin and de-block the functional groups on the By mid 1990’s, an immunological approach, LHRH amino acid side chains. Each free peptide was washed, vaccines, had been designed and tested in men to achieve lyophilized and dissolved in distilled water to the desired androgen deprivation as a treatment of prostate cancer concentration. Peptides were purified (>90%) by reverse and in post-menopausal women to test the ability to inhibit phase HPLC with gradient elution consisting of an ace- tonitrile water mixture with added TFA. Synthetic peptides The efficacy of neutralizing LHRH/GnRH action through were characterized for the correct composition by amino the involvement of hormone-specific antibodies has been acid analysis or by N-terminal sequence analysis using demonstrated in a wide range of animal species including hu- Edman degradation chemistry and matrix-assisted laser mans. Such studies have involved either passive immuniza- desorption time-of-flight mass spectrometry using an Ettan tion by infusion of anti-LHRH antibodies vaccination MALDI-ToF mass spectrometer (Amersham Bioscience, with LHRH peptide coupled to tetanus or diphtheria toxoid Piscataway, NJ, USA). Each purified peptide was stored (DT) molecules as carriers LHRH in multiple (lyophilized or in solution) at −20 ◦C until needed.
antigen peptide (MAP) constructs These approachesare impractical for widespread commercial application since passive immunity is inefficient and expensive and the useof peptide–toxoid conjugates and MAP constructs produce For Sprague–Dawley rat studies, individual peptide im- variable results. In addition, the peptide–toxoid conjugates munogens (25–400 ␮g per 0.5 ml dose) and mixtures of pu- and MAP immunogens are difficult to manufacture, and rified peptides (100 ␮g total peptide per 0.5 ml dose) in equal the use of toxoids can lead to carrier-induced anti-haptenic molar ratios were adsorbed to aluminum hydroxide gel immunosuppression Recombinant LHRH toxoid (gift of Dr. E.B. Lindblad, Superfos Biosector a/s, Vedbaek, fusion molecules have the same disadvantages. These lim- Denmark) in 0.72% NaCl and 0.066 M sodium phosphate itations are overcome by the UBITh® synthetic peptide buffer, pH 7.0 by mixing overnight at 4 ◦C.
For Copenhagen rat studies, UBITh® LHRH immuno- ically defined, homogenous peptide structure that can be gen Mix #1 containing equal molar ratios of peptides p607, reproducibly synthesized and characterized and is readily p667, p669, p500 (100 ␮g total peptide per 0.5 ml dose) was adaptable to large scale manufacturing processes.
adsorbed on aluminum hydroxide gel (Alhydrogel 85; EM The basis of our strategy is to induce an “anti-self” immu- Sergeant Pulp and Chemical Co. Inc., Clifton, NJ, USA) in nity to LHRH by altering the target molecule on a synthetic 0.72% NaCl and 0.066 M sodium phosphate buffer, pH 7.0 peptide immunogen. Immunization with LHRH peptide im- munogens produces the desired hormone neutralization ef- For dog studies, UBITh® LHRH immunogen Mix #1 fect or “immunological castration” by eliciting anti-LHRH (400 ␮g total peptide per 0.5 ml dose) was either adsorbed antibodies to the decapeptide (pEHWSYGLRPG-NH2). We on Alhydrogel or formulated in an oil-in-water emulsion of have developed entirely synthetic LHRH immunogens, in 30% Emulsigen (MVP Laboratories Inc., Ralston, NE, USA) contrast to other groups, who have linked the LHRH de- containing 5.93 mg/ml dimethyldioctadecyl ammonium bro- capeptide to carrier proteins report describes mide A; Eastman Kodak, Rochester, NY, USA), the design of our synthetic LHRH peptide immunogens and 0.68% Tween, 4.2% ethanol, 0.68% NaCl. Placebo control the development of vaccines to effect androgen deprivation formulations contained Alhydrogel alone or the emulsion in rodents, dogs, and baboons, and as an immunotherapy for delivery system without immunogen Mix #1.
androgen-responsive prostate cancer.
For baboon protocol #1, UBITh® LHRH immunogen Mix #2 containing equal molar ratios of peptides p607E, p667,p500 (400 ␮g total peptide in 0.15 M NaCl per 0.5 ml dose) 2. Materials and methods
in solution were filtered aseptically and combined withtwo different water-in-oil emulsions, Montanide® ISA51 (50:50 (v/v)) or Montanide® ISA720 (70:30 (v/v)), orwith a water-in-oil-in-water emulsion, Montanide® ISA206 Peptide antigens for immunoassays and peptide im- (Seppic Inc., Fairfield, NJ, USA), each containing 100 ␮g munogens for vaccines were synthesized using automated monophosphoryl lipid A 0.8% squalene. For baboon C.L. Finstad et al. / Vaccine 22 (2004) 1300–1313 protocol #2, UBITh® LHRH immunogen Mix #2 (25, 100, kins Oncology Center, Baltimore, MD) to test the efficacy or 400 ␮g total peptide in 0.15 M NaCl per 0.5 ml dose) was of UBITh® LHRH immunogen Mix #1 at 100 ␮g total pep- combined with Montanide® ISA51 containing 0.9% DDA.
tide per dose formulated in Alhydrogel to control the growthof the Dunning H androgen-responsive prostate tumor The original R3327 tumor, discovered by Dunning Groups of male Sprague–Dawley rats (8–12 weeks of was a spontaneously occurring prostatic adenocarcinoma in age; Taconic, Germantown, NY, USA) were immunized in an old inbred male Copenhagen rat. A number of so-called the hind leg by intramuscular route at 0, 3, 6 weeks with Dunning R3327 prostate tumor lines were derived, includ- individual peptide immunogens (100–400 ␮g per dose) or ing the Dunning H tumor line, which is predominantly peptide mixtures (25–100 ␮g per dose) adsorbed on alu- androgen-dependent and rarely metastatic, and which grows minum hydroxide. Testes and prostate glands were removed with a doubling time of 21 ± 6 days when implanted under and weighed at autopsy at the end of the study. The mean the skin of intact male Copenhagen rats Dunning average weight in vaccine-treated animals was compared H subline is a heterogeneous tumor composed of clones with that of placebo control animals to evaluate organ of both androgen-dependent and androgen-independent tu- mor cells and will respond to castration of the host by Groups of sexually mature intact male dogs (randomly di- cessation of growth eventually followed by re-growth as vided between mongrel and pure bred animals 8–10 months hormone-independent tumor The androgen-sensitive of age; Pfizer Animal Health, Lincoln, NE, USA) were im- Dunning H tumor used in this study was received as a munized in the hind leg by intramuscular injection at 0, 3, frozen piece of tumor tissue which was passaged in intact 6 weeks with 400 ␮g per dose of UBITh® LHRH immuno- male Copenhagen rats. Tumor pieces (approximately 1 mm3 gen Mix #1 in Alhydrogel or in Emulsigen + DDA. Placebo each) were implanted subcutaneously into three groups of control intact male dogs received the adjuvant in the delivery eight rats each. The sizes of tumors growing in the rats system without peptide immunogens and control dogs were were measured in three dimensions using calipers and tu- surgically castrated without receiving any vaccine treatment.
mor volume was approximated by the formula: length (cm) Testes and prostate glands were removed and weighed at × width (cm) × height (cm) × 0.5236 and expressed as autopsy (end of the study). Organ atrophy in dogs was de- fined as the percentage change in organ weight per totalbody weight. All organ weight data are expressed as a per- centage of the total body weight to account for size differ-ences in animals comprising both the treated and control 2.5.1. Solid-phase enzyme-linked immunoassay (ELISA) for detection of antibodies to synthetic peptides Adult male baboons (Papio cynocephalus; 6–14 years of LHRH decapeptide, individual T helper (Th) peptide age; Southwest Foundation for Biomedical Research, San domains (UBITh® peptides), or adjuvanting peptide from Antonio, TX, USA) in protocol #1 were immunized at 0, Yersinia invasin protein (Inv peptide) were coated on 96-well 3, 6, 16 weeks with 400 ␮g per dose of UBITh® LHRH microtitre plates at 5 ␮g/ml and dried overnight. Serum immunogen Mix #2 in three oil-based emulsions each con- samples were serially diluted 10-fold with a starting serum taining adjuvant or with Mix #2 immunogens containing dilution of 1:100. Briefly, 100 ␮l samples of diluted sera adjuvant alone. Baboons in protocol #2 were immunized at were incubated in the wells for 60–90 min at 37 ◦C, washed 0, 4, 34 weeks with 25, 100, or 400 ␮g per dose of Mix with phosphate buffered saline and incubated for 60 min at #2 immunogens formulated in Montanide® ISA51 contain- 37 ◦C with horseradish peroxidase (HRP)-conjugated goat ing DDA. Testes length and width were approximated by or rabbit species-specific immunoglobulin G. HRP-goat caliper at 2–4 week intervals and calculated as the mean anti-rat IgG (Cappel, ICN Biomedicals Inc., Costa Mesa, CA, USA), HRP-rabbit anti-dog IgG (Jackson Im- Toxicity (i.e. pain, redness and swelling) at the intramus- munoResearch Laboratories, West Grove, PA, USA), and cular injection site (upper hind leg for rodents, dogs and HRP-goat anti-human IgG (Fc) for detection of baboon baboons) was not observed with UBITh® LHRH immuno- IgG (Anogen, Division of Yes Biotech Laboratories Ltd., gens in Alhydrogel. Transient redness was noted in some Mississauga, Ontario, Canada) were purchased as indi- baboons receiving the more potent water-in-oil emulsions.
cated. The plates were washed again and incubated witheither O-phenylenediamine (1.67 mg/ml; Sigma, St. Louis, 2.4. Dunning H rat prostate tumor model MO, USA) or 3, 3 , 5, 5 -tetramethyl benzidine (0.2 mg/ml;Sigma) plus hydrogen peroxide as substrate for 15 min at Groups of male Copenhagen (COP/N) rats (Frederick 37 ◦C. The enzyme-substrate reaction was stopped by the Cancer Research and Developmental Center, Frederick, MD, addition of stop solution (1.0 M sulphuric acid). ELISA USA) were implanted subcutaneously with 1 mm3 pieces of titres were quantitated using an automated plate reader at Dunning R3327-H tumor (gift of Dr. J.T. Isaacs, Johns Hop- 492 nm (Dynex Technologies, Chantilly, VA, USA).
C.L. Finstad et al. / Vaccine 22 (2004) 1300–1313 2.5.2. Radioimmunoassay (RIA) for detection of Functional domains of UBITh® LHRH peptide immunogens The anti-LHRH titre was determined by incubation of 125I labeled LHRH (DuPont Co., Wilmington, DE, USA) with serum samples from rats and dogs at 1:100 dilution and ba- boons samples at 1:50 dilution. Briefly, serum samples were diluted with 1% bovine serum albumin, mixed (1:1) with radiolabeled-LHRH, and incubated overnight at room tem- perature. Bovine gamma-globulin (0.5%) and polyethylene a Peptides are synthesized with a UBITh® sequence covalently linked to glycol (25%) were added, mixed and the tubes centrifuged the N-terminus of LHRH decapeptide through a glycine–glycine (GG) spacer, at 4000 rpm for 25 min at 4 ◦C. Supernatant was aspirated from each tube and the radiolabeled-immune complex (pel- b UBITh® epitopes are T helper (Th) cell sequences found in several let) was counted (Packard Cobra II-Auto Gamma Model pediatric vaccines: hepatitis B virus surface antigen (HBsAg) measlesvirus fusion protein (MVF) toxin (TT) pertussis toxin 5010, Meriden, CT). The results were adjusted to mean ex- perimental values and calculated as nmol/l based on stan- c In some cases the N-terminus of the adjuvanting domain from Yersinia dard curve measurements and serum dilution factors.
invasin (Inv) protein added to the N-termini of the UBITh® LHRHimmunogens through a GG spacer.
2.5.3. Radioimmunoassay (RIA) for detection of serumtestosterone a distinct helper T cell epitope, was produced by United Testosterone levels were determined by a standard RIA Biomedical Inc. The Th epitopes (designated as UBITh®) kit using 125I labeled testosterone (Cat. No. TKTT-500; are covalently linked to the anti-LHRH antibody-eliciting Diagnostic Products Corp., Los Angeles, CA, USA) using epitope (B cell epitope) by continuous solid-phase synthe- serum samples from rats, dogs and baboons at 1:100 dilu- sis. The UBITh® domains are promiscuous Th epitopes that tion. Testosterone levels were calculated as nmol/l based on stimulate helper T cells of a broad range of histocompatibil- ity backgrounds. The UBITh® epitopes used here range from 2.6. Lymphocyte proliferation analysis 15 to 24 residues in length and they correspond to Th se-quences found in several pediatric vaccines including hepati- Peripheral blood mononuclear cells from baboons were tis B virus surface antigen (HBsAg19–33) virus isolated by Ficoll-hypaque gradient centrifugation and fusion protein (MVF288–302) toxin (TT830–844) washed three times with sodium phosphate buffered saline.
or pertussis toxin (PT18–41) A further modifi- Cells (2 × 105 per well) were cultured in 96-well plates con- cation to some of the UBITh® LHRH peptides is the ad- taining RPMI-1640 medium (GIBCO Laboratories, Grand dition to the amino-terminus of a domain from Yersinia Island, NY, USA), supplemented with 10% heat-inactivated invasin protein (Inv718–732) has demonstrated fetal bovine serum. The cells were cultured alone or with significant adjuvanting activity The individual func- individual peptide immunogens (p607E, p667, p500), in- tional domains of the UBITh® immunogens are separated by dividual peptide domains (LHRH decapeptide; UBITh®4, glycine–glycine (GG) spacers. the indi- UBITh®5, UBITh®6 peptides; Inv peptide) or an unrelated vidual peptide segments comprising UBITh® LHRH peptide peptide (p1412). On day 6, 1 ␮Ci of 3H thymidine (3H-TdR; New England Nuclear, Boston, MA, USA) was added toeach of three replicate wells. After 18 h of incubation, cells 3.2. Immunization with UBITh® LHRH immunogens were harvested with a multiple well harvesting apparatus, and 3T-TdR incorporation was determined (Packard Tri-Carb Liquid Scintillation Analyzer, Model 1900TR, Meri- den, CT, USA). The stimulation index (SI) was calculated individual UBITh® LHRH immunogens to test their by dividing the mean counts per minutes 3H-TdR incor- effectiveness to obtain castrate levels of testosterone. His- porated into cells stimulated with 5 ␮g/ml of a peptide or torically, this animal strain has been used to evaluate other peptide domain by the mean counts per minutes 3H-TdR testosterone-deprivation therapies These rats are out- incorporated into cells cultured with medium alone.
bred, express different histocompatibility antigens and arerepresentative of a genetically heterogeneous population.
3. Results
3.1. Design of LHRH synthetic peptide immunogens Four different UBITh® LHRH immunogens (p588, p583, p550D, p500) without the Yersinia invasin (Inv) adjuvanting A diverse array of chimeric LHRH peptide immunogens, peptide and the same four immunogens with the Inv pep- each carrying both the target LHRH decapeptide site and tide sequence attached (p607, p667, p669, p668) were tested C.L. Finstad et al. / Vaccine 22 (2004) 1300–1313 Table 2Efficacy study of individual UBITh® LHRH immunogens in male rats Animals response per group ( n = 5) a Individual UBITh® LHRH immunogens (100 ␮g per dose) were adsorbed on Alhydrogel and administered at 0, 3, 6 weeks by intramuscular route.
b Number of animals with anti-LHRH antibody levels above 0.50 nmol/l per group of five animals at 10 weeks following three immunizations.
c Number of animals with testosterone levels below 0.05 nmol/l per group of five animals at 10 weeks following three immunizations.
d Some immunogens were synthesized with glycine–glycine (GG) linkers separating the functional domains as indicated.
e Peptide p607E differs from p607 by a single residue substitution of glutamic acid (E) for aspartic acid (D) in the UBITh®4 peptide segment for in groups of five rats to assess the immunopotency of each of testosterone and atropied testes at all three dose levels UBITh® LHRH immunogen adsorbed to aluminum hydrox- and was identified as the preferred mixture. Although nei- ide (Superfos) and administered at 100 ␮g per dose. The an- ther p550D nor p669 immunogens had striking anti-LHRH imals were immunized at 0, 3, 6 weeks and serum was col- titres, when Mix C was compared to Mix #1; it is appar- lected at 0, 3, 5, 8, 10 weeks for evaluation of anti-LHRH ent that p669, with the Inv peptide attached, was preferred antibody and serum testosterone levels. Six UBITh® LHRH to immunogen p550D without Inv. Individual peptide im- immunogens, without or with the Inv peptide, were se- munogens were ranked for potency based on the number of lected for additional studies based on immunopotency results animals immunocastrated in protocol #1 (as well as shown in our UBITh® LHRH immunogens (p607, from additional dose escalation studies of individual LHRH p588, p667 and p500) showed strong immunopotency and immunogens (data not shown). The immunogen with the two immunogens (p550D, p669) showed anti-LHRH activ- strongest anti-LHRH antibody response was p607. The or- ity in only one of five rats. Two immunogens had minimal der of anti-LHRH immunopotency for individual immuno- or baseline anti-LHRH activity (p583 and p668) and were Two groups of 12 rats were also immunized at 0, 3, 6 Briefly, groups of five rats were immunized with four weeks with UBITh® LHRH Mix #2 consisting of three im- UBITh® LHRH immunogen mixtures (Mix A, Mix B, Mix munogens (p607E, p667, p500) combined in equimolar ra- C, and Mix #1). Four mixtures of four immunogens, com- tio at 25 and 100 ␮g per dose and adsorbed on aluminum bined in equimolar ratio, were adsorbed on aluminum hy- hydroxide (Alhydrogel). Serum was collected at 0, 3, 5, 8, droxide (Superfos) and tested at 25, 50 and 100 ␮g per dose 11, 16, 20 weeks for evaluation of anti-LHRH antibody and (in order to identify the most immunopotent com- serum testosterone levels. At 11 weeks, all rats had castrate bination. The animals were immunized at 0, 3, 6 weeks and levels of testosterone at both dose levels. At 20 weeks, 11 of serum was collected at 0, 3, 5, 8, 10 weeks for evaluation 12 rats at 100 ␮g dose and 9 of 12 rats at 25 ␮g dose level re- of anti-LHRH antibody and serum testosterone levels. Mix mained castrated. The p669 immunogen was removed from A contained four UBITh® LHRH immunogens without the UBITh® LHRH Mix #1 because of poor peptide stabil- Inv peptide (p588 + p583 + p550D + p500). The composi- ity. The Mix #2 immunogens demonstrated improved im- tion of the other three mixtures substituted the correspond- munopotency and were used in vaccines for the non-human ing peptide covalently linked to the Inv peptide as follows: Mix B substituted p607 for p588; Mix C also substitutedp607 and p667 for p583; Mix #1 also substituted p607, p667 3.3. Immunization with UBITh® LHRH immunogens and p669 for p550D; and p500 was present in all four mix- tures. A two-fold dose escalation permitted the selection ofthe immunogen mixture with the strongest immunopotency.
The Dunning R3327-H rat tumor model was used to test Specifically, p607 + p667 + p669 + p500 mixture (Mix the immunotherapy for efficacy by showing the effect of #1) demonstrated that five of five rats had castrate levels the LHRH vaccine on the growth of an androgen-dependent C.L. Finstad et al. / Vaccine 22 (2004) 1300–1313 Table 3Efficacy study of UBITh® LHRH peptide immunogen mixtures in male rats Number of animals per total animals in group a Mixtures of UBITh® LHRH immunogens (25–100 ␮g per dose as indicated) were adsorbed on Alhydrogel and administered at 0, 3, 6 weeks by b Number of animals with anti-LHRH antibody levels above 0.50 nmol/l per total number of animals tested per group (n = 5) at 10 weeks following c Number of animals with testosterone levels below 0.05 nmol/l per total number of animals tested per group at 10 weeks following three immunizations.
d Number of animals with atrophied testes at 10 weeks per total number of animals evaluated per group. Atrophied testis weights were less than 25% of the mean weight of the control group. All animals immunized with Mix #2 had atrophied testes at 10 weeks but were followed for an additional 10weeks to monitor duration of response.
prostate tumor. Copenhagen male rats were each implanted and continued to grow in two of the remaining five animals.
subcutaneously with 1 mm3 fragments of Dunning H tumor Testosterone levels in the other three animals remained less at week 0 and they were divided into three groups of eight than 0.5 nmol/l and tumors of palpable size were not noted Copenhagen male rats in Group 1 implanted with Dun- Copenhagen male rats in Group 3 were implanted subcu- ning H tumors were immunized with 100 ␮g total peptide taneously with Dunning H tumor pieces (1 mm3 size) and the per dose of UBITh® LHRH immunogen Mix #1 adsorbed tumors were permitted to grow until they reached a volume on Alhydrogel at 0, 3, 6 weeks and then boosted at 26 and of approximately 0.4 cm3 at which time the animals were 58 weeks post-tumor implantation when testosterone levels immunized with 100 ␮g total peptide per dose of UBITh® became detectable in serum. Rats in Group 2 implanted with LHRH immunogen Mix #1 adsorbed on Alhydrogel at 26, Dunning H tumors were also immunized at 0, 3, 6, 26, 58 29, 32 weeks post-tumor implantation, and later boosted at weeks with Alhydrogel alone. Blood was collected by tail 58 and 82 weeks. Blood was collected for determinations vein cannulation under light anesthesia at 2 week intervals of anti-LHRH and testosterone levels every 2 weeks. Note for assays. Tumor growth was followed with tumor measure- that tumors started to appear by approximately 20 weeks ments every 2 weeks. Androgen ablation was monitored for and by 30 weeks remained at approximately 0.4 cm3 (mean serum testosterone and palpation for testes re-growth, and tumor volume) throughout the study period in the eight an- anti-LHRH antibody titres were determined by RIA (data imals receiving Mix #1 in Alhydrogel in Group 3 in com- not shown). Tumors did not appear in any of the eight an- parison to the rapidly growing tumors in the eight Group imals receiving the LHRH vaccine (Group 1) when com- 2 control animals receiving the Alhydrogel alone as illus- pared to the eight control animals (Group 2) as illustrated trated in panel). At the end of the study, testos- in panel). The mean tumor size represents the terone levels were permitted to rebound and, as expected, mean of the volumes of all nodules in all animals in each the androgen-sensitive Dunning H tumors started to grow group. At the end of the study (120+ weeks), approximately again in 4 of 5 of the remaining animals. Tumor re-growth 20 weeks after the testosterone levels started to increase in was not noted in one animal that appeared to remain cas- the Group 1 rats, small tumors of palpable size appeared trated by the prior LHRH immunization.
C.L. Finstad et al. / Vaccine 22 (2004) 1300–1313 Mean Tumor Volume 0.0
Weeks Post Tumor Inoculation
Weeks Post Tumor Inoculation
Fig. 1. Proof of concept using Dunning R3327-H rat tumor model. Three groups of Copenhagen male rats (n = 8) were implanted subcutaneously witha small fragment of Dunning H tumor (as described in at week 0. In the Prophylaxis Model (left panel), Group 1 (solid circles) rats wereimmunized with UBITh® LHRH immunogen Mix #1 beginning at 0, 3, 6, 26, 58 weeks after tumor implant and compared with control Group 2 (opencircle symbol) rats immunized with Alhydrogel alone at the same schedule. In the Therapeutic Model (right panel), Group 3 (solid circles) rats wereimmunized with UBITh® Mix #1 beginning at 26, 29, 32, 58, 82 weeks after tumor implant and compared with control Group 2 rats. Tumor growthwas measured every 2 weeks and mean tumor volume was calculated. The priming and booster immunizations are indicated by arrows.
3.4. Efficacy studies of UBITh® LHRH immunogens in eight dogs in Groups 2 at week 20 Intact control dogs (Group 3) and surgically castrated dogs (Group 4) didnot have detectable anti-LHRH activity. All animals in the Intact young adult mongrel and pure-bred male dogs were placebo control Group 3 had detectable testosterone levels immunized with 400 ␮g of UBITh® LHRH immunogen Mix throughout the study period while the surgically castrated #1 adsorbed on Alhydrogel at 0, 3, 6 weeks (Group 1) or dogs of Group 4 had castrate levels as expected (data not formulated in Emulsigen containing DDA at 0, 3, 6 weeks (Group 2). Placebo control intact dogs were immunized with At week 20, the animals were sacrificed and their rel- either Alhydrogel alone or Emulsigen/DDA delivery system evant reproductive organs (i.e. testes and prostate glands) alone (Group 3). Castrated control dogs did not receive any were dissected and weighed. The mean organ weights of vaccines (Group 4). Blood samples were collected from all the vaccine-treated dogs were compared to the mean or- animals at 0, 3, 6, 8, 10, 12, 16, 20 weeks for determination of gan weights of the placebo control dogs. Data from the anti-LHRH titres and testosterone levels in serum. By week non-responsive animal in Group 1 were not included for 6, significant levels of anti-LHRH antibodies were observed the mean weight calculation. To correct for differences in in six of seven animals in Group 1 (data not shown) and the sizes of the animals, organ weights were expressed as in all eight animals in Group 2 (The potent LHRH a fraction of the total body weight of the originating ani- antibody responses caused a concomitant decrease in serum mal. The mean organ weights for the placebo control Group testosterone in the treated animals. By week 6, serum testos- 3 was set at 100%. The mean weights of the testes and terone was at castration levels (<0.05 nmol/l) in six of seven prostate glands for Group 1 were 43.7 and 30.3%, respec- animals immunized with UBITh® LHRH immunogens ad- tively, and for Group 2 they were 21.8 and 22.5%, respec- sorbed to Alhydrogel (Group 1) and in all eight dogs re- tively. The increased organ size in Group 1 is related to the ceiving the Emulsigen/DDA formulation (Group 2) and they rebound of testosterone in most animals at 20 weeks when remained at this level through at least week 12 after which compared to Group 2 where all animals maintained castrate time testosterone levels started to rebound in some animals levels of testosterone. The consistent efficacy in the dogs in Group 1. The failure to castrate one of seven dogs in of the Group 2 oil-in-water formulation over the Group 1 Group 1 is attributable to the lack of a sustained LHRH an- Alhydrogel formulation suggested that a more immunopo- tibody response in this animal. Antibody responses in dogs tent vaccine delivery system would be necessary for the parallel those observed in rats, where LHRH-specific an- UBITh® LHRH peptide immunogens to attain efficacy in tibody titres of 0.5 nmol/l must be maintained for several higher animals. The following non-human primate studies weeks to achieve castration. Castrate levels of testosterone were evaluated with water-in-oil emulsion-based delivery were noted in one of seven dogs in Group 1 and in all C.L. Finstad et al. / Vaccine 22 (2004) 1300–1313 ol/L) 1.5
Anti-LHRH (Log
RIA Anti-LH 0.0
(nMol/L) 30
one (nMol/L)
Weeks Post Immunization
Fig. 2. Efficacy study in intact male dogs. Dogs were immunized at 0, 3, 6 weeks (indicated by arrows) with 400 ␮g total peptide per dose ofUBITh® LHRH immunogen Mix #1 formulated in Emulsigen containing DDA (Group 2). Animals were bleed at 0, 3, 6, 8, 10, 12, 16, and20 weeks and tested for anti-LHRH antibody levels (upper panel) and Testes size (cm
testosterone levels (lower panel). Each solid black symbol represents anindividual dog; the anti-LHRH titres were sufficient to effect androgen deprivation in all dogs at 20 weeks (end of study).
3.5. Efficacy studies of UBITh® LHRH immunogens in Weeks Post Immunization
Fig. 3. Efficacy study in adult male baboons. In protocol #1, individual Three adult male baboons (Papio cynocephalus), 6–14 baboons were immunized at 0, 3, 6, 16 weeks (indicated by arrows) withUBITh® LHRH immunogen Mix #2 at 400 ␮g formulated in Montanide® years of age, were immunized with UBITh® LHRH im- ISA 720 (black circles, baboon #7446), in Montanide® ISA 206 (black munogen Mix #2 (p607E + p667 + p500) at 400 ␮g to- triangles, baboon #7663) or in Montanide® ISA 51 (black squares, baboon tal peptide per dose formulated in either of three different #8010), each containing monophospholipid A. Baboons were bleed at water-in-oil emulsion vaccine delivery systems containing week 0, bimonthly from 4 to 36 weeks and then at 4–6 week intervals monophosphoryl lipid A as adjuvant (The im- until 52 weeks and tested for anti-LHRH levels by radioimmunoassay(upper panel), serum testosterone levels (middle panel), and testes size munization schedule was 0, 3, 6, 16 weeks. High titred anti-LHRH antibody was noted to support immunocastra-tion by 6 weeks in the three baboons (#7446, #7663, #8010)receiving emulsion-based formulations. Duration of andro-gen deprivation was observed for at least 40 to >52 weeksdepending on the formulated emulsion used (One C.L. Finstad et al. / Vaccine 22 (2004) 1300–1313 Table 4Site-directed immunoreactivity of immune baboon serum samples to LHRH immunogen Mix #2 a Solid-phase enzyme-linked immunoassay (ELISA) tests of individual peptide domains are described in samples at week 10 are taken from baboon protocol #1 and tested at 10-fold serial dilutions from starting dilution 1:100.
b Results are expressed as log10 ELISA titre of anti-peptide antibody to each domain at 10 weeks. Pre-bleed ELISA titre results at week 0 are between 0.12 and 0.26 for all samples tested. Anti-LHRH antibody titres of >1:103–1:104 are detected. Anti-UBITh® antibody titres are all negligible. Anti-Invpeptide reactivity was negative or weakly positive after three immunizations but returned to background levels (titre <1:102) within 1 month after thethird immunization.
c Serum testosterone deprivation, measured by radioimmunoassay, is noted at 8 weeks in all three baboons (#7446, #7663, #8010) immunized with Mix #2 immunogens formulated in Montanide® oil-based emulsions containing adjuvant but not in the baboon (#7052) immunized with Mix #2 immunogensformulated in squalene with adjuvant.
baboon (#7052), immunized with 400 ␮g per dose of Mix #2 serum testosterone when tested 2 weeks after the boost. Two immunogens in a squalene-based adjuvant suspension, de- baboons showed rebound of testosterone (#8477, #9186) veloped low anti-LHRH antibody levels and maintained nor- whereas four baboons maintained castrate levels of testos- mal levels of testosterone. In the three baboons with castrate terone at 95 weeks (end of study). Organ re-growth occurred levels of testosterone, three clinical endpoints were mon- approximately 1 month after the testosterone rebound in two itored: reversibility, duration of androgen deprivation and animals. The testes remained atrophied in the other four ba- re-growth of testes. Androgen rebound was noted in one of boons and were observed to be 24–36% of the original organ three baboons at 40 weeks (#7663) and detection of serum sizes as calculated by cross-sectional area.
testosterone preceded observable re-growth of testes by ap-proximately 10 weeks. Castrate levels of testosterone were 3.6. Proof of concept for UBITh® LHRH maintained in the other two baboons at 52 weeks (end of The LHRH decapeptide, UBITh® peptides, and Inv pep- tide domains from the three UBITh® LHRH immunogens Six adult male baboons were immunized with UBITh® in Mix #2 were synthesized as unlinked peptide segments, LHRH immunogen Mix #2 at 25 ␮g (#8477, #8616), 100 ␮g purified and then coated onto separate microtitre plate wells.
(#8617, #8995) or 400 ␮g (#8518, #9186) per 0.5 ml dose of Results by ELISA using serum samples at week 10 from the total peptide formulated in Montanide ISA 51 emulsion con- four baboons in protocol #1 demonstrated that the LHRH taining DDA. The immunization schedule of this dose esca- decapeptide was highly immunogenic and the T cell helper lation study was 0, 4, 34 weeks and blood was collected at 2 peptides or UBITh® epitopes were not immunoreactive with week intervals to week 52 and then monthly until week 95 the baboon antibodies in the three baboons immunized with (end of study). The baboons were followed for anti-LHRH water-in-oil emulsions (The Inv peptide showed a antibody levels, duration of androgen deprivation and testes transient low-titred reactivity in serum samples from some involution and re-growth. Results from representative ba- animals immediately following the boost injection at 16 wpi boons at 100 ␮g (#8517) and 400 ␮g (#8518) dose levels in protocol #1 but returned to baseline within 4 weeks of the through week 95 are shown in After the two prim- boost injection. Results from baboon protocol #2 also re- ing doses, the level of anti-LHRH antibody was at least vealed predominant anti-LHRH peptide reactivity for all six 0.5 nmol/l in all six baboons at 6 weeks and castrate levels animals immunized with UBITh® LHRH immunogen Mix of testosterone were noted in all animals. In four of six ba- #2 emulsions; the UBITh® peptides and Inv peptide were boons (#8517, #8518, #8615, #8995), testosterone rebound not immunoreactive by ELISA tests when animals were im- was observed around 22–24 weeks and testes re-growth was munized two times at 0 and 4 weeks (data not shown).
noted at 26–28 weeks. In the other two baboons (#8477, To demonstrate specific T helper cell immunogenicity for #9186), testosterone rebound was noted following decreases the UBITh® epitope, mononuclear blood cells collected and in anti-LHRH levels below 0.5 nmol/l.
purified at 0, 4, 6, 12 and 14 weeks from all six animals in ba- At 34 weeks post-initial immunization, the six baboons boon protocol #2 were co-cultured with individual peptides received booster injections of the UBITh® LHRH immuno- (data not shown) or peptide domains of the three UBITh® gen Mix #2 in Montanide® ISA 51 containing DDA at the LHRH immunogens in Mix #2 to access lymphocyte stim- respective dose levels. All six baboons had castrate levels of ulation in vitro. Lymphocyte proliferation studies demon- C.L. Finstad et al. / Vaccine 22 (2004) 1300–1313 (nMol/L)
RIA Anti-LHRH 0.0
Testosterone (nMol/L)
Testes Size
Weeks Post Immunization
Weeks Post Immunization
Fig. 4. Efficacy study in adult male baboons. In protocol #2, individual baboons were immunized at 0, 4, 34 weeks (indicated by arrows) with UBITh®LHRH immunogen Mix #2 at 100 ␮g per dose (black circles, baboon #8995, left panels) or at 400 ␮g per dose (black triangles, baboon #8518, rightpanels) formulated in Montanide® ISA 51 containing DDA. Animals were bleed at week 0, bimonthly from weeks 4 to 52 weeks and then monthly to95 weeks and evaluated for anti-LHRH by radioimmunoassay (upper panels), serum testosterone levels (middle panels) and testes size (lower panels).
strated that specific UBITh® T cell helper peptides were baboons (#8995, #8518). The LHRH decapeptide, Inv pep- each strongly stimulatory in this assay system in baboons tide, and an unrelated control peptide did not stimulate any receiving immunogens at all three dose levels. of the baboon lymphocyte cultures regardless of the LHRH ulates the stimulation index when each peptide domain is immunogen dose used for the immunizations. In contrast, the incubated with lymphocyte cultures from two representative three UBITh® peptide segments were stimulatory in vitro C.L. Finstad et al. / Vaccine 22 (2004) 1300–1313 Table 5Site-directed lymphocyte stimulation of immune baboon samples by individual peptide domains a Serum samples from baboons immunized at 0, 4 weeks by intramuscular route are indicated by asterisk (*).
b Lymphocyte stimulation assay is described in Stimulation index is calculated from mean count values of triplicate cell cultures in the presence of synthetic peptide domains and divided by the mean count values of cultures without peptides added. Positive stimulation index is >3.0(boldface results).
c Serum testosterone deprivation, measured by radioimmunoassay, is noted between weeks 6 and 12 in baboons (#8995, #8518) immunized with Mix #2 immunogens formulated in Montanide® oil-based emulsions containing DDA.
Fig. 5. Prototype UBITh® LHRH synthetic peptide immunogen. The LHRH decapeptide is synthesized with a helper T cell (Th) domain (UBITh® epitope)covalently linked to the amino terminus of LHRH. These UBITh® epitopes mimic epitopes found in several pediatric vaccines. A further modification tosome UBITh® LHRH immunogens is the addition to the amino-terminus of a domain from Yersinia invasin protein. High levels of anti-LHRH antibodieswere detected in immune sera from samples collected at week 10 but no appreciable levels of anti-UBITh® antibodies Conversely, UBITh®peptides stimulated proliferation of cultured T lymphocytes from samples collected at 4, 6, 12, 14 weeks after immunizations at 0 and 4 weeks but notthe LHRH decapeptide antibody target or the immunostimulatory Inv peptide and lymphocyte cultures from each baboon displayed dif- gens either adsorbed on aluminum hydroxide (Alhydrogel) ferent patterns of response. Cultures from all six baboons or formulated with adjuvants as water-in-oil emulsions. The were strongly stimulated by UBITh®6 (TT830–844) peptide; studies in rodents and dogs used four different UBITh® they were also moderately or weakly stimulated by either LHRH immunogens (Mix #1) and studies in adult male ba- UBITh®4 (HBsAg19–33) or UBITh®5 (MVF288–302) pep- boons used an improved mixture of three UBITh® LHRH tides or both peptides. Review of the clinical history of the immunogens (Mix #2). The design elements of each im- baboons at Southwest Foundation indicated that these ani- munogen include the LHRH decapeptide as the B cell tar- mals had not received prior TT vaccines. get epitope, a UBITh® epitope for T cell help, and in some cally summarizes these observations and the proof of con- cases, an adjuvanting Inv peptide from Yersinia invasin pro- cept design for UBITh® LHRH immunogens.
tein. The LHRH immunogens are formulated as mixtures inorder to maximize the immune response in individual ani-mals. These studies demonstrated efficacy, universal respon- 4. Discussion
siveness, and long duration of castrate levels of testosteroneas well as proof of concept for the immunogen design.
An effective and practical synthetic peptide-based im- Individual UBITh® LHRH immunogens, each carrying a munotherapeutic vaccine for treatment of androgen-respon- distinct T helper cell epitope from protein antigens found sive prostate cancer has been designed and tested in rats, in pediatric vaccines, have been identified and shown to dogs and baboons. The mode of action for the immunother- be effective in generating anti-LHRH antibody levels suffi- apeutic is androgen deprivation of the androgen-dependent ciently high (>0.5 nmol/l) to effect androgen deprivation in prostate cancer The LHRH/GnRH immunotherapeutic rats, dogs and baboons as illustrated in this report. Efficacy comprises a mixture of UBITh® LHRH peptide immuno- was readily noted in rodents with our individual UBITh® C.L. Finstad et al. / Vaccine 22 (2004) 1300–1313 LHRH peptide immunogens adsorbed on aluminum hydrox- years by anti-LHRH antibody that effectively suppressed testosterone synthesis. When anti-LHRH antibody levels peptides in a safe and effective delivery system for human were sufficiently diminished and a testosterone rebound was application has been challenging. Results presented in the noted, this was followed by re-growth of quiescent Dunning dog protocol illustrate that mineral salt-based formulations H tumor cell populations. At the end of the study, small (e.g. Alhydrogel) were transiently effective but not durable tumors became evident in two of five animals remaining enough to sustain an anti-LHRH response whereas, addition in the early LHRH-immunized Group 1 and continued tu- of an adjuvant (e.g. DDA) to oil-based emulsions were 100% mor cell growth was notable in the established tumors in effective in dogs for at least 20 weeks (end of study). In four of five animals remaining in the late LHRH-immunized baboon protocol #1, emulsion-based formulations enhanced Group 3. These observations confirm viability of the tu- the effectiveness of the anti-LHRH response (In mor implants after remaining quiescent in vivo for over 2 baboon protocol #2, the effective UBITh® LHRH immuno- years. The results of these rodent studies also have impor- gen dose was decreased by including the DDA adjuvant in tant implications for the clinical situation where conven- the emulsion-based formulations. These studies demonstrate tional androgen-deprivation therapy suppresses prostate can- that emulsions can effectively support long-term duration of cer growth rather than destroying tumor cells.
a specific anti-LHRH peptide response and also specifically The hormone deprivation action of the UBITh® LHRH stimulate responsiveness to UBITh® helper T cell epitopes immunogen vaccines in blocking testosterone synthesis (These results are proof of efficacy for the product has several applications. In a clinical setting, LHRH im- munotherapy is a treatment option for androgen-responsive The Dunning R3327-H tumor model for androgen-respon- prostate cancer or for other hormone-responsive benign sive prostate cancer in Copenhagen rats has permit- tumors (e.g. endometriosis, uterine fibroids, etc.) presently ted the efficacy of the UBITh® LHRH immunogens for treated by LHRH agonists In companion animals, hormone-deprivation therapy to be tested and validated. The LHRH immunotherapy can be used for immunocontracep- immunotherapy was 100% effective in preventing develop- tion reversible behavior modification or for treatment ment of the androgen-dependent Dunning H tumors in the of benign prostatic hyperplasia (BPH) or androgen- Prophylaxis Model and in arresting the growth of established dependent prostatic intraepithelial neoplasia (PIN) in elderly tumors of palpable size in the Therapeutic Model as long sexually intact dogs whose owners do not elect to surgi- as testosterone levels were suppressed to castrate levels by cally castrate The prevalence of BPH or PIN in dogs anti-LHRH antibody at serum levels that were sufficient to is apparently influenced by age and presence of testicular neutralize circulating LHRH. The Dunning H rat tumor ex- androgens as in humans. In the swine industry the periment extended over 2 years in duration. It is of particular LHRH vaccine can be used for removal of androstenones relevance to clinical applications that the Dunning H tumors or boar taint, known to cause off-flavor of the pork from did not grow in the androgen-deficient environment evoked boars, as well as for growth promotion.
by the immunotherapy. This is in contrast to early observa- The LHRH secreted from the hypothalamus stimulates the tions of Isaacs et al. measured the effects of surgical anterior pituitary to secrete LH and FSH that is required for castration on the growth of Dunning H tumors and observed normal spermatogenesis. Prolonged treatment with LHRH delayed growth of tumor implants in castrated animals when agonists desensitizes the LHRH receptors and results in sup- compared to control animals; tumors continued to grow in pression of gonadotropins and spermatogenesis In the androgen-independent environment with a time delay.
our two baboon protocols, castrate levels of testosterone These results suggested the presence of a local LHRH loop were maintained for at least 1 year in six of nine baboons.
within the rat prostate and Dunning H tumors, in addition The reason for continued androgen deprivation in these ani- to the LHRH loop of the pituitary–gonadal axis. Additional mals is not known but could be due to suppression of LHRH studies support a local LHRH loop in the prostate. Reddy receptors or loss of Leydig cells, as reported after extended et al. direct inhibitory action of LHRH on the use of LHRH agonists in non-human primates after accessory organs in the rat. Srkalovic et al. 2 years of LHRH agonist therapy in humans or contin- strated LHRH receptors and their modulation by LHRH an- ued high levels of effective anti-LHRH antibody. However, tagonists on Dunning H tumor cells. Fuerst et al. androgen rebound was observed in three of nine animals de- strated that active immunization with an LHRH-diphtheria spite the presence of elevated anti-LHRH antibody in serum.
toxoid conjugated vaccine leads to down-regulation of go- In protocol #1, hormonal rebound was noted after 34 weeks nadotropins and testosterone and atrophy of the testes and of castration in baboon #7663 and in protocol #2, after the 34 prostate glands. Growth inhibition of the androgen-sensitive week booster immunization, rebound was noted after another Dunning R3327-PAP tumor by this LHRH–DT conjugated 36 weeks in baboon #8477 and after only 16 weeks in ba- vaccine was shown to be caused by suppression of cell boon #9186. Several reasons may account for the hormonal division rather than increase in tumor cell death rebound noted, including immunogen dose, emulsion for- In our study, control of tumor growth of the androgen- mulation, reduced anti-LHRH antibody level and/or younger dependent tumor cell population was controlled for over 2 age. Experiments in baboons (Papio hamadryas), ranging in C.L. Finstad et al. / Vaccine 22 (2004) 1300–1313 age from 6 to 15 years, showed that animal to animal vari- Chang for anti-LHRH antibody and testosterone assay de- ation in response to hormonal stimuli differed in relation to terminations; Lori Horton for her assistance with the Dun- the basal LH concentrations and to the pituitary–gonadal re- ning H tumor rat experiments; Maria Salas for lymphocyte sponse to administration of LHRH agonists rather than to proliferation assays in baboons; Claire Chen for data pre- the age of the animal, although degenerative changes were sentation, and Rosanne W. Boyle for advice on compliance with regulatory requirements for preparation of clinical trial The LHRH/GnRH immunotherapy for androgen-respon- grade material. We also thank Dr. Pele C.-S. Chong for sug- sive prostate cancer has a definable endpoint (testosterone gestions and comments. This work was supported in part level) to evaluate vaccine efficacy, which is distinct from by the National Cancer Institute Grant CA-83450 to United the markers to assess cancer progression (e.g. PSA level). In Biomedical Inc., and by NIH Center Grants CA-16087 and one candidate LHRH/GnRH vaccine, the LHRH decapep- tide was modified by replacement of glycine at position 6with d-lysine linked through a spacer to diphtheria toxoidand adsorbed on Alhydrogel The immunotherapy was References
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