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Receptors for Luteinizing Hormone Releasing Hormone Expressedon Human Renal Cell Carcinomas Can Be Used for TargetedChemotherapy with Cytotoxic Luteinizing HormoneReleasing Hormone AnaloguesGunhild Keller,1,2,3 Andrew V. Schally,1,2 Timo Gaiser,4 Attila Nagy,1,2 Benjamin Baker,1,2

Gabor Halmos,1,2 andJo« rg B. Engel1,2

Abstract Purpose:To determine the expression of luteinizing hormone releasing hormone (LHRH) recep-tors in specimens and cell lines of human renal cell carcinoma (RCC) and to evaluate the antitumorefficacy of targeted therapy with a cytotoxic analogue of LHRH, AN-207, invivo. AN-207, consist-ing of [D-Lys6] LHRH linked to a cytotoxic radical, 2-pyrrolinodoxorubicin (AN-201), binds withhigh affinity to LHRH receptors and can be targeted to tumors expressing these receptors.Experimental Design: The expression of LHRH receptors was investigated in 28 surgicallyremoved specimens of human renal cell carcinoma (RCC) by immunohistochemistry and in threehuman RCC cell lines A-498, ACHN, and 786-0 by radioreceptor assays,Western immunoblot-ting, and reverse transcription-PCR analysis. Antitumor efficacy of AN-207 was examined inexperimental models of these cell lines.Results: Positive staining for LHRH receptors was found in all (28 of 28) of the examined humanRCC specimens. mRNA for LHRH receptor, receptor protein, and LHRH binding sites weredetected in all three cell lines. AN-207 significantly (P < 0.05) inhibited the growth of A-498,ACHN, and 786-0 xenografts in vivo producing a 67.8% to 73.8% decrease in tumor volume anda 62.2% to 77.3% reduction in tumor weight. Nontargeted cytotoxic radical AN-201had no sig-nificant antitumor effects. Blockade of LHRH receptors by an excess of LHRH agonist Decapeptylsuppressed tumor inhibitory effects of AN-207.Conclusions:Our findings indicate that LHRHreceptors are expressed inhuman RCC specimensand can be used for targeted chemotherapy with cytotoxic LHRH analogues.

In 2004, about 36,000 patients in the United States will bediagnosed with renal cancer, with an estimated 12,000 to13,000 deaths (1). Renal cell carcinoma (RCC) accounts forf85% of all kidney tumors and its incidence has beenincreasing by 2% to 4% per year since the 1970s (2, 3). Thestandard treatment for patients diagnosed with localized RCC issurgical removal (4). However, 30% to 50% of all patients will

show recurrence of disease and one third of the cases initiallypresent with evidence of systemic spread (5). With a 5-yearsurvival rate of <10%, these patients face a dismal prognosisand currently no standard treatment modality is effective in thetherapy of metastatic RCC (3). Systemic chemotherapy resultsin partial and complete response rates only in 4.7% and 1.3%of the cases, respectively (6). Immunotherapy with IFNs,interleukin-2, and lymphokine-activated killer cells achievesresponse rates of 10% to 20%, some of which were lasting(7, 8). However, these therapeutic approaches are accompaniedby severe side effects and consequently by a low quality of life.These clinical observations underline the importance ofdeveloping a new, more effective treatment for RCC.

The elucidation of molecular characteristics of cancer cellsprompted the development of a new class of drugs known astargeted therapeutics. The targets for antibodies or conjugates ofligands linked to toxins, radionuclides, or chemotherapeuticagents include surface antigens, growth factor signaling path-ways, and peptide hormone receptors (9 – 16). Targetedchemotherapy can be based on peptide hormones, such asluteinizing hormone releasing hormone (LHRH), somatostatin,or bombesin (13, 15). A direct delivery of the cytotoxic agent tothe tumor cells is expected to increase therapeutic efficacy andreduce the systemic toxicity. Recently, we developed a targetedcytotoxic conjugate AN-207, which consists of the LHRH

Cancer Therapy: Preclinical

Authors’Affiliations: 1Endocrine, Polypeptide and Cancer Institute, VeteransAffairs Medical Center ; 2Section of Experimental Medicine, Department ofMedicine, Tulane University School of Medicine, New Orleans, Louisiana;3Supported by a fellowship within the post doctorate program of the GermanAcademic Exchange Service; and 4Department of Pathology and BiomedicalResearch, Klinikum Kassel, GermanyReceived 12/1/04; revised 4/25/05; accepted 5/11/05.Grant support: Medical research service of the Veterans Affairs Department andZENTARIS Gmbh, Frankfurt am Main, Germany toTulane University (A.V. Schally).The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.Note:This work is dedicated to the late Ana-Maria Comaru-Schally, M.D., F.A.C.P.,who died recently from thyroid cancer, for her intellectual, spiritual, and personalcontribution to this project and for the inspiration she provided.Requests for reprints: Andrew V. Schally, Veterans Affairs Medical Center, 1601Perdido Street, New Orleans, LA 70112-1262. Phone: 504-589-5230; Fax: 504-566-1625; E-mail: [email protected].

F2005 American Association for Cancer Research.

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agonist [D-Lys6] LHRH linked to a highly potent derivative ofdoxorubicin 2-pyrrolino-DOX (AN-201; refs. 17–19). AN-207effectively inhibits the growth of experimental breast, ovarian,endometrial, and prostate cancers, which are known to expressspecific LHRH receptors (13, 15). In nonmalignant humantissue, receptors for LHRH seem expressed mainly in organsregulated by the pituitary-gonadal axis but not in tissues such aslymph nodes or hematopoietic stem cells (20). However,binding sites for LHRH were also detected in human tumorspecimens and cancer cell lines originating from liver, larynx,pancreas, colon, skin, and brain (21–27). In addition, specificbinding for a LHRH agonist, [125I] buserelin, was found in fourof six human RCC specimens (28).

These findings prompted us to investigate the expression ofLHRH receptors in human RCC specimens as well as in threehuman RCC cell lines and to evaluate the efficacy of cytotoxicLHRH analogue AN-207 in models of human RCC in vivo .

Materials andMethods

Immunohistochemistry. Tissues of 37 human RCC specimens derivedfrom primary tumors and metastases as well as three normal humankidneys were fixed for 16 to 20 hours in 4% neutral buffered formalinand then embedded in paraffin. Two- to 4-Am-thin sections of selectedtissue blocks were cut, mounted on siliconized glass slides (SigmaChemical Co., St. Louis, MO), and dried at 60jC for 30 minutes and at38jC overnight. Sections were deparaffinized by incubation in a xylenebath for 5 minutes twice, in acetone for 5 minutes twice, and finally indistilled water for 5 minutes. Heat pretreatment of the sections was donein 10 mmol/L citrate buffer (pH 6.0), in a steamer for 30 minutesfollowed by washing in distilled water. Endogenous peroxidase wasblocked by incubation in a freshly prepared solution of 0.3% H2O2 inmethanol for 20 minutes at room temperature followed by washing indistilled water for 5 minutes. Except for counterstaining with hematox-ylin and mounting, the following steps were done overnight using theTecan-Immunostainer Genesis RSP 200 (Software: Gemini 3.40),according to the manufacturer’s EnVision Plus–staining procedure(DAKO, Carpinteria, CA): The slides were rinsed twice in PBS/0.05%Tween (pH 7.4, PBS buffer) for 7 minutes and incubated with anantibody for LHRH receptors (clone A9E4, Novocastra, New Castle,United Kingdom) for 4 hours (1:10 dilution in antibody diluent;DAKO). The reaction was stopped with 100 AL PBS buffer per slide. Afterwashing in 1,400 AL PBS buffer for 7 minutes, the slides were incubatedwith 120 AL EnVision horseradish peroxidase antimouse antibody(DAKO) for 30 minutes. After washing as before, the staining reactionwas done with 120 AL per slide 3,3V-diaminobenzidine solution (DAKO;1:50 dilution in substrate buffer) for 10 minutes. The reaction wasstopped with 100 AL PBS buffer for 20 minutes followed by washingwith 1400 AL PBS buffer for 7 minutes. The slides were then washedthrice with PBS buffer every 2 hours. Finally, the slides were rinsed inwater, counterstained with Harris’ hematoxylin, and covered with a glassslide. The slides were examined by light microscopy and the expressionof LHRH receptors was estimated on a four-point scale as absent (�),weak expression (+), distinct expression (++), and strong expression (+++).

Peptides and cytotoxic agents. Cytotoxic LHRH analogue AN-207was synthesized in our laboratory by coupling one molecule of 2-pyrrolino-DOX-14-O-hemiglutarate to the q-amino-group of carrierpeptide [D-Lys6] LHRH (19). Cytotoxic radical AN-201 was prepared asdescribed (18). For the i.v. injection, the compounds were dissolved in20 AL of 0.01 N aqueous acetic acid and diluted with 5% (w/v) aqueousD-mannitol solution (Sigma Chemical).

Cell lines. Human RCC cell lines A-498, ACHN, and 786-0 wereobtained from American Type Culture Collection (Manassas, VA). Cellswere maintained in RPMI 1640 supplemented with 10% heat-

inactivated FCS (Sigma Chemical), 50 AL penicillin G, 50 ALgentamicin, and 100 AL streptomycin (all from Life Technologies,Grand Island, NY).

Animals. Five- to 6-week-old male athymic nude mice (Ncr nu/nu)were obtained from the National Cancer Institute (Bethesda, MD). Theanimals were housed in sterile cages under laminar flow hoods in atemperature-controlled room with a 12-hour light/12-hour darkschedule and were fed autoclaved chow and water ad libitum .

Experimental protocol. Tumors resulting 6 to 10 weeks after s.c. cellinjection in donor animals were aseptically dissected and mechanicallyminced. Pieces of tumor tissue, about 3 mm3, were transplanted s.c.

into the experimental animals by a trocar needle. Tumor volume(length � width � height � 0.5236) and body weight were measuredweekly. At the end of each experiment, mice were sacrificed underanesthesia and necropsy was done. The right kidneys were fixed in 4%neutral buffered formalin. Tumors were excised, weighed, snap frozen,and stored at �70jC.

All experiments were in accordance with the institutional guidelines

for the welfare of experimental animals and the NIH Guide for Care and

Use of Laboratory animals.Experiment 1. Four weeks after transplantation of A-498 tumors,

mice were assigned to three groups of nine animals each, with anaverage tumor size of f50 to 60 mm3. Mice received the followingtreatment as single i.v. injections into the jugular vein on day 1: group 1,control, vehicle solution (5% mannitol); group 2, cytotoxic analogueAN-207 at a dose of 200 nmol/kg; group 3, cytotoxic radical AN-201 ata dose of 200 nmol/kg. The experiment was terminated on day 29.

Experiment 2. Nude mice bearing ACHN tumors were divided into

three groups of nine animals each, with an average tumor volume of

f50 mm3. Mice received the following treatment on days 1 and 29 of

the experiment: group 1, control, vehicle solution; group 2, AN-207 at

200 nmol/kg; group 3, AN-201 at 200 nmol/kg. The experiment was

terminated on day 57.Experiment 3. Animals with 786-0 tumors were assigned to six

groups when tumors had reached a size of 40 to 50 mm3. The mice

received the following treatment on days 1 and 15: group 1, control,

vehicle solution (n = 10 mice); group 2, AN-207 at 150 nmol/kg (n =

10 mice); group 3, AN-201 at 150 nmol/kg (n = 10 mice); group 4, the

carrier [D-Lys6] LHRH at 150 nmol/kg (n = 5 mice); group 5,

unconjugated mixture of the cytotoxic radical AN-201 and the carrier

[D-Lys6] LHRH at 150 nmol/kg (n = 5 mice); group 6, 200 Ag of the

LHRH agonist Decapeptyl ([D-Trp6] LHRH) i.v. 15 minutes before the

i.v. injection of cytotoxic analogue AN-207 at 150 nmol/kg (n = 6

mice). The experiment was terminated on day 29.Evaluation of systemic and local renal toxicity. General toxicity was

evaluated on the bases of total leukocyte count (WBC) and bodyweight.WBC was determined with the Unopette microcollection kit (BectonDickinson, Franklin Lakes, NJ) before, and 7 days after the drugadministration as well as at the end of the experiments. Bodyweight wasmeasured weekly.

Toxic effects to renal tissue were examined by serum creatinine, urineanalyses, and light microscopy of kidney tissue. For serum creatinineanalyses, nude mice were assigned to three groups and received thefollowing treatment: group 1, control, vehicle solution (n = 4 mice);group 2, AN-207 at a dose of 200 nmol/kg (n = 5 mice); group 3, AN-201at a dose of 200 nmol/kg (n = 5 mice). Eight days after the injection bloodwas collected during necropsy and serum creatinine levels were measuredby Antech Diagnostics (Lake Success, NY). Urine was collected from mice7 days after the application of AN-207 and AN-201 as well as fromcontrol animals. Urine analyses were carried out by Antech Diagnostics.Kidneys from mice treated with single or repeated injections of AN-207and AN-201 and from control animals were excised and fixed in 10%buffered formalin. The specimens were embedded in paraplast (OxfordLabware, St. Louis, MO), sliced, and stained with H&E. All slides wereexamined carefully by light microscopy for any signs of cellular damage.

RNA extraction and reverse transcription-PCR. Tumor tissue fromhuman RCC cell lines: RNA was isolated from approximately 100 mg


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of tumor tissue according to the protocol for TRI-Reagent (Sigma-Aldrich, St. Louis, MO), following the manufacturer’s instructions. Thereverse transcription reaction was done with the iScript cDNAsynthesis kit from Bio-Rad (Hercules, CA) according to the manu-facturer’s instructions. Five hundred nanograms of RNA weretranscribed into cDNA in a final volume of 10 AL. All PCR reactionswere carried out in an Applied Biosystems PCR system 2700(Norwalk, CT). For the amplification of the cDNA, gene-specificprimers for the LHRH receptor were used as described (29). Tenmicroliters of each PCR product were loaded on a 1.8% agarose geland subjected to electrophoresis, staining with ethidium bromide, andanalysis using Kodak 1D 3.6 (Rochester, NY) imaging analysissoftware. A total RNA negative control, which received only water inthe reverse transcription reaction, was used in each PCR to rule out

genomic DNA contamination. DU-145 human prostate cancer cellline was used as a positive control for LHRH receptors in the PCRreaction.

Mouse kidney. For the detection of the LHRH receptor in mousekidney tissue, normal mouse kidney was homogenized and RNAisolated in Tri reagent according to the manufacturer’s protocol. Fivemicrograms of total RNA were used in the Superscript III cDNAsynthesis system (Invitrogen, Carlsbad, CA) in a volume of 20 AL. ThecDNA was amplified using the Amplitaq system (Applied Biosystems)in a reaction volume of 25 AL for 40 cycles using the primer sequencespecific for mouse LHRH receptor: sense 5V-TGATGGTGGTGATTAGCC-3V, antisense 5V-CACATTGCGAGAAGACTG-3V.

Western immunoblot analysis. For the immunodetection of LHRHreceptors, an extraction of membrane protein from A-498, ACHN, and

Table1. LHRH receptor expression in surgically removed human RCC determined by immunohistochemistry

Expression level

Primary tumorsWell-differentiated clear-cell RCC +Well-differentiated clear-cell RCC with cystic parts ++Moderately differentiated clear-cell RCC +Moderately differentiated clear-cell RCC +Moderately differentiated chromophilic papillary RCC ++/+++Moderately differentiated chromophilic papillary RCC +Infiltrating, moderately differentiated clear-cell RCC +Moderately differentiated clear-cell RCC ++Moderately differentiated clear-cell RCC ++Moderately differentiated clear-cell RCC +Moderately differentiated chromophilic papillary RCC with some sarcomatoid parts ++Poorly differentiated clear-cell RCC with sarcomatoid parts ++Moderately differentiated clear cell RCC +++Moderately differentiated chromophilic papillary RCC ++Moderately differentiated clear-cell RCC ++Well-differentiated chromophilic RCC ++Moderately differentiated clear-cell RCC +/++Moderately differentiated clear-cell RCC ++Well-differentiated clear-cell RCC +/++Moderately differentiated chromophilic RCC ++Moderately differentiated clear-cell RCC ++Poorly differentiated chromophilic RCC +/++Moderately differentiated clear-cell RCC �/+Well-differentiated clear-cell RCC +/++Well-differentiated clear-cell RCC �/+

Metastases of renal cell carcinomaCervical lymph node metastasis of a papillary RCC ++Bone metastasis in femur of a clear-cell RCC ++Bone metastasis in humerus of a clear-cell RCC ++Lung metastasis of a clear-cell RCC ++Liver metastasis of a clear-cell RCC ++Bone metastasis of a clear-cell RCC ++Lymph node metastasis of a papillary RCC ++Lung metastasis of a clear-cell RCC ++/+++Metastasis in the adrenal gland of a clear-cell RCC ++/+++Metastasis in the opposite kidney of a clear-cell RCC ++Lung metastasis of a clear-cell RCC ++Metastasis in the retperitoneum of a clear-cell RCC +

NOTE: �, no expression; �/+, tumor with parts of negative and low receptor expression; +, low expression; +/++, tumor with parts of low and distinct receptor expres-sion; ++, distinct receptor expression; ++/+++, tumor with parts of distinct and high receptor expression; +++, high receptor expression.

LHRHReceptorsTargeting in Renal Cell Carcinoma




786-0 samples as well as from normal mouse kidneys was done aspreviously reported (30). The presence of LHRH receptor protein wasthen shown by Western blotting using a goat polyclonal antihumanLHRH receptor antibody (Santa Cruz Biotechnology, Santa Cruz, CA),as described (30).

Luteinizing hormone releasing hormone receptor binding studies.

Receptors for LHRH on A-498, ACHN, and 786-0 tumors from theexperimental groups were characterized by the ligand competitionassay. Preparation of tumor membrane fractions and receptor bindingstudies of LHRH were done as described (31, 32). The LIGAND-PCcomputerized curve-fitting program of Munson and Rodbard (33) wasused to determine the type of receptor binding, dissociation constant(Kd) and maximal binding capacity of the receptors (Bmax).

Statistical analysis. Data are expressed as means F SE. Differencesbetween mean values were evaluated by two-tailed Student’s t test.P < 0.05 was considered significant.

Results

Immunohistochemistry of human renal cell carcinoma speci-mens. A total of 37 human RCC specimens were obtainedfrom primary tumors and metastases and evaluated for LHRH

receptor expression by immunohistochemistry. Positive stain-ing for LHRH receptors was found in all samples examined,independently of the histologic subtype. In only two of theprimary RCCs, areas with no LHRH receptor expression weredetected. In six primary RCCs, LHRH receptor expression waslow, whereas the majority of the primary RCC specimens(17 of 25) showed moderate or high LHRH receptor expression.Eleven of 12 samples of RCC metastases were moderately orstrongly stained, whereas weak receptor expression was foundin only one sample. In surrounding nonmalignant tissue,binding sites for LHRH were detected in cells of the renaltubular system (13 of 17 samples) with varying intensity. Othercells (e.g., cells of glomeruli, blood vessels, or connective tissue)showed no or only marginal LHRH receptor expression. Threenormal, human kidneys examined showed a moderate LHRHreceptor expression in the tubular system, which was compa-rable with the findings in the surrounding tissue of the tumors.(Table 1; Fig. 1).

Inhibition of tumor growth in experimental renal cellcarcinoma models. In experiment 1, a single administration

Fig. 1. Expression of LHRH receptors insurgically removed human RCCspecimens:The tissue was stained byimmunohistochemistry with mouse LHRHreceptor monoclonal antibody (cloneA9E4,Novocastra). A , positive control, humanpituitary gland (anterior lobe). Intensestaining of LHRH receptor ^ positive cellsand a capillary with unstained endothelialcells in the center. B, negative control,human colon; no specific staining.C, intermediate (2+) expression of LHRHreceptor in a lung metastasis of a humanRCC. D, strong (3+) expression of LHRHreceptors in tubuli of normal human kidney.E , weak (1+) expression of LHRH receptorsin a human RCC. F, intermediate (2+)expression of LHRH receptors in a humanRCC. G, strong (3+) expression of LHRHreceptors in a human RCC. H, bonemetastasis of a human RCC with a strongstaining for LHRH receptors on cellmembranes. Magnification: 25� (B), 100�(C, E, G), 400� (A , D, F, H).





of AN-207 at 200 nmol/kg significantly (P < 0.001) inhibitedthe growth of human A-498 RCC, resulting in a 71.1%reduction of tumor volume. In this group, tumor weight wassignificantly (P < 0.01) decreased by 77.3% and tumor-doubling time was significantly (P < 0.01) prolonged com-pared with controls. Equimolar doses of the cytotoxic radicalAN-201 had no significant effects on any tumor growthcharacteristics (Fig. 2; Table 2).

In experiment 2, two injections of AN-207 at 200 nmol/kgon days 1 and 29 completely arrested the growth of humanACHN RCC tumors, until the end of the experiment on day

57 when final tumor volume was 73.8% (P < 0.001) smallerthan in the control group. Tumor weights in the group treatedwith AN-207 were 75.5% lower than in the control animals(P < 0.01) and tumor-doubling time was also significantlyextended (P < 0.05). The cytotoxic radical AN-201 atequimolar doses had no significant effects on tumor growth.(Fig. 3; Table 2).

In experiment 3, the administration of AN-207 at a dose of150 nmol/kg on days 1 and 15 significantly inhibited thegrowth of human 786-0 RCC xenografts by 67.8% comparedwith the control group (P < 0.001). AN-207 significantlydecreased tumor weight by 62.2% (P < 0.001) and significantly(P < 0.001) prolonged tumor-doubling time. Equimolar dosesof AN-201, the carrier [D-Lys6] LHRH or the mixture of AN-201and [D-Lys6] LHRH had no significant effects on any variablesof tumor growth. The effect of AN-207 could be blocked byinjecting 200 Ag of the LHRH agonist Decapeptyl, 15 minutesbefore the administration of AN-207 (Fig. 4; Table 2).

Toxicity. In all treatment groups, a slight loss of body weightwas observed 8 days after treatment, which ranged from 0.3%to 6.9% in the AN-207 groups and from 1.9% to 11.6% in theAN-201 groups (Table 3). In experiment 1, body weights in theAN-201 group were significantly (P < 0.05) reduced from day8 until the end of the experiment. No significant loss of bodyweight was observed in any group in experiments 2 and 3(Table 3).

AN-207 did not significantly decrease the WBC in any of thethree experiments. AN-201 significantly (P < 0.001) suppressedWBC on day 8 in all three experiments compared with controls.In experiments 2 and 3, the number of leucocytes in the bloodof mice treated with AN-201 was within reference ranges beforethe second injection. However, the second administration of

Fig. 2. Effects of targeted cytotoxic LHRHanalogue AN-207 andits radical AN-201on the growth of A-498 human RCC xenografts.Arrows , treatment. *,P < 0.05versus controls; **,P <0.001versus controls.

Table 2. Effects of therapy with cytotoxic LHRH analogue (AN-207), the cytotoxic radical AN-201, the carrier[D-Lys6] LHRH, an unconjugated mixture of AN-201and [D-Lys6] LHRH, and AN-207 after blockade of the receptorswith Decapeptyl on the growth of A-498, ACHN, and 786-0 human RCC xenografted in nude mice

Treatment Tumor volume (mm3) Tumor-doubling time (d) Tumor weight (mg), % inhibition

Initial Final (% inhibition)

Experiment1 (A-498)Control 59.7 F12.1 361.6 F 54.0 12.7 F 1.1 405.4 F 74.5AN-207 53.4 F 5.2 104.6 F 15.1* (71.1) 34.3 F 2.9c 91.9 F 15.2c (77.3)AN-201 58.3 F 10.9 338.5 F 60.9 (�6.5) 14.5 F 2.4 381.6 F 70.9 (5.9)

Experiment 2 (ACHN)Control 50.8 F 6.7 207.0 F 45.7 47.3 F 10.1 263.2 F 73.0AN-207 47.7 F 6.9 54.3 F 12.8c (73.8) 1,055.7 F 315.2b 64.6 F16.7c (75.5)AN-201 53.3 F 6.7 153.5 F 31.3 (25.8) 73.4 F 20.3 173.8 F 32.6 (34.0)

Experiment 3 (786-0)Control 46.1 F6.1 342.4 F 44.5 10.5 F 0.6 389.6 F 46.2AN-207 46.5 F 7.7 110.6 F10.6* (67.8) 21.3 F 2.4c 147.1 F13.4*(62.2)AN-201 45.5 F 6.2 318.8 F 51.6 (6.9) 10.9 F 0.7 363.4 F 52.4(6.7)Carrier 49.6 F 7.7 388.7 F 63.8 (�13.5) 10.1 F 1.2 421.7 F 73.2 (�8.3)Mixture 45.9 F 6.9 298.6 F 56.2 (12.8) 13.0 F 2.6 378.9 F 89.3 (2.7)Blockade 50.0 F 11.6 244.4 F 61.8 (28.6) 15.5 F2.9 316.5 F 70.1 (18.8)

*P < 0.001versus controls.cP < 0.05 versus controls.bP < 0.01versus controls.





the cytotoxic radical significantly (P < 0.05) decreased the WBCin both studies until the experiments were terminated.

In experiment 2, one mouse died on day 32 in the AN-201group and one on day 45 in the AN-207 group. In experiment3, one mouse died in the AN-201 group on day 11 (Table 3).

Eight days after the injection of AN-207 and AN-201 at 200nmol/kg serum creatinine levels did not differ from the valuesin the control animals. Mean serum creatinine levels were 0.20mg/d in the control and the AN-201 group and 0.22 mg/d inmice treated with AN-207 (Table 3).

Urine analyses did not show any differences between controlanimals and mice after treatment with AN-207 or AN-201. Noleucocytes, RBC, or casts were detectable. Total protein waswithin the reference range for nude mice (Table 3).

Microscopic examinations of mouse kidneys showed minorchanges after treatment with AN-207 or AN-201. Few apoptoticand necrotic cells were detected in the tubular system ofkidneys of both groups. No mononuclear cell infiltration wasfound in periglomerular or peritubular areas (Table 3).

Expression of mRNA for luteinizing hormone releasinghormone receptors in tumors of human renal cell carcinoma celllines. mRNA for the human LHRH receptor was found inhuman A-498, ACHN, and 786-0 RCC xenografts by reversetranscription-PCR analyses. The PCR products were of theexpected size of 319 bp. No PCR products were amplified fromthe negative controls, ruling out the possibility of genomiccontamination (Fig. 5A).

Expression of luteinizing hormone releasing hormone receptorprotein in tumors of human renal cell carcinoma cell lines. Thepresence of LHRH receptor protein in untreated A-498, ACHN,and 786-0 tumor tissues was evaluated by Western blottingusing specific antibodies. A specific band at Mr = 64,000 wasfound in all investigated tumor samples (Fig. 5B).

Expression of mRNA for luteinizing hormone releasing hormonereceptors and luteinizing hormone releasing hormone receptorprotein in nonmalignant renal tissue of nude mice. mRNA forthe LHRH receptors and the LHRH receptor protein in mousekidneys were detectable at low levels in two of the threesamples (Fig. 5C-D).

Luteinizing hormone releasing hormone receptor bindingstudies. In membranes of A-498, ACHN, and 786-0 RCCtumors from the control group, receptor analyses revealed a

single class of specific, high-affinity binding sites for LHRH(Table 4). Mean Kd values were 8.51 nmol/L for A-498 tumors,4.35 nmol/L for ACHN RCC, and 10.6 nmol/L for 786-0tumors. Mean Bmax values were 439.8 fmol/mg membraneprotein in A498 tumors, 654.2 fmol/mg membrane protein forACHN tumors, and 437.2 fmol/mg membrane protein for 786-0 RCC. Treatment with AN-201 and AN-207 given as single ortwo consecutive injections did not affect the affinity or theconcentration of LHRH receptors compared with LHRHreceptor characteristics in the controls in all three cell lines(Table 4).

Discussion

Patients with advanced RCC face a dismal prognosis and noeffective therapy currently exists for the management ofmetastatic disease (3). A new class of drugs known as targetedtherapeutics, directed against specific proteins in cancer cells,such as surface antigens, growth factors, or hormone receptors,is being developed (9–15). Recently, Bevacizumab, an anti-body targeted to vascular endothelial growth factor, showedsome promising results in patients with advanced RCC in aphase II clinical study (34).

Chemotherapy targeted to hormone receptors is a novelapproach designed to deliver chemotherapeutic agents directlyto cancer cells to increase antitumor activity and decrease theside effects. Because the receptors for peptide hormones werefound on a wide variety of cancers, several cytotoxic hormoneanalogues were synthesized in our laboratory includingcytotoxic LHRH analogue AN-207. AN-207 consists of [D-Lys6]LHRH covalently linked to superactive derivative of doxorubi-cin, 2-pyrrolino-DOX (AN-201) and it shows enhancedantitumor activity in various LHRH receptor-positive experi-mental models of human cancers (13, 15). Previously, specificbinding for LHRH agonist buserelin was detected in four of sixhuman RCC (28). This finding prompted us to evaluate LHRHreceptors in samples of surgically removed human RCCspecimens derived from primary tumors or metastatic lesions.

Fig. 3. Effects of targeted cytotoxic LHRHanalogueAN-207 and its radical AN-201on the growth of ACHN human RCC xenografts. Arrows , treatment. *, P < 0.05versus controls.

Fig. 4. Effects of targeted cytotoxic LHRH analogue AN-207, the cytotoxic radicalAN-201, the carrier [D-Lys6] LHRH, an unconjugated mixture of AN-201and[D-Lys6] LHRH, and AN-207 after blockade of the LHRHreceptors with Decapeptylon the growth of 786-0 human RCC. Arrows , treatment. *, P < 0.001versuscontrols.





Immunohistochemical analysis showed positive staining forLHRH receptors in all 25 tested specimens of primary RCCindependently of histologic subtype and in all 12 samples ofRCC metastases. These findings indicate that a very highpercentage of primary RCC express receptors for LHRH andthat there is no loss of LHRH receptor expression inmetastatic disease. This suggests that treatment with targetedLHRH analogue AN-207 might be a promising therapy foradvanced stages of RCC. In surrounding, nonmalignanttissue, LHRH receptor expression was only detected in cellsof the renal tubular system. In line with our findings inhuman RCC specimens, specific, high-affinity LHRH receptorsand the corresponding mRNA were also found in threehuman RCC cell lines, A-498, ACHN, and 786-0, by reversetranscription-PCR, Western blot analysis, and radioligandbinding assays. Xenografts of these three RCC cell lines innude mice were used to evaluate the efficacy and toxicity ofAN-207 in vivo . These studies were designed to provide arationale for a possible use of AN-207 in targeted chemo-therapy of RCC.

In all three RCC models, targeted therapy with cytotoxicLHRH analogue AN-207 was highly efficacious and a complete

arrest of tumor growth occurred in experiment 2 with ACHNRCC. To show that the growth-inhibiting effects of AN-207 aredue to targeting to LHRH receptors, in experiment 3 we alsoincluded groups that received an unconjugated mixture ofAN-201 and the carrier peptide, [D-Lys6] LHRH, or the carrieralone. Because a significant tumor inhibitory activity wasobserved only in the group injected with AN-207, the highefficacy of AN-207 cannot be attributed to any of itscomponents but rather to the ability of [D-Lys6] LHRH todeliver AN-201 to malignant cells. The targeting concept isfurther supported by the fact that a blockade of LHRH receptorsby Decapeptyl reduces the antitumor activity of the targetedcytotoxic analogue AN-207.

The binding characteristics and the concentration of LHRHreceptors on tumor cell membranes were not changed bya single administration nor two consecutive injections ofAN-207. This suggests that multiple dosing schedules ofAN-207 are possible.

The toxicity of targeted cytotoxic LHRH analogue AN-207and its cytotoxic radical AN-201 was compared with respect tosystemic and local effects. The overall mortality in this studywas very low with only one death after treatment with AN-207

Table 3. Systemic and local renal toxicity of cytotoxic LHRH analogue AN-207 and the cytotoxic radical AN-201inmice bearing A-498, ACHN, and 786-0 human RCC tumors

Variables of toxic side effects AN-207 AN-201

No. deaths/treated animals 1/28 2/28Decrease inWBC 8 days after first injection compared with controls 2.6-20.3% 39.7-69.6%Decrease inWBC 8 days after second injection compared with controls 7.4-22.7% 48.3-64.8%Decrease inWBC at the end of the experiments compared with controls 4.6-15.4% 44.6-50.1%Loss of bodyweight on days 8 compared with controls 0.3-6.9% 1.9-11.6%Microscopic examinations of mouse kidneys Few apoptotic and necrotic cells Few apoptotic and necrotic cellsUrine analyses Normal NormalSerum creatinine 0.22 mg/dl 0.20 mg/dl*

*The same as control.

Fig. 5. A , expression of mRNA for LHRH receptors in A-498, ACHN, and 786-0 human RCC xenografts as revealed by reverse transcription-PCR. Lane M, 100-bp ladder;lanes1-3,786-0; lanes 4-6, ACHN; lanes 7-9, A498; lane�, RNA negative control; lane +, positive control (DU-145 prostate cancer).B, expression of LHRHreceptor protein inA-498, ACHN, and 786-0 human RCC xenografts as revealed byWestern blot analysis. LaneM, molecular weight marker; lane1, ES2 ovarian cancer (positive control);lanes 2-4, 786-0; lanes 5-7, A-498; lanes8-10, ACHN.C, expression of mRNA for LHRH receptors in normal mouse kidneys as revealed by reverse transcription-PCR. LaneM,100-bp ladder; lanes1-3, normal mouse kidney; lane +, positive control; lane�, RNA-negative control. D, expression of LHRH receptor protein in normal mouse kidneys asrevealed byWestern blot analysis. laneM, molecular weight marker; lane +, ES2 ovarian cancer ( positive control); lanes 1and 2, normal kidneys.





and two after AN-201. In all treatment groups, except for thecarrier group, a slight loss of body weight was observed 8 daysafter the therapy. However, a significant weight loss wasobserved only in experiment 1 in animals treated with AN-201. The myelotoxicity is usually the most serious side effectand the dose-limiting factor of chemotherapy. In the presentstudy, AN-207 did not significantly lower the number ofleucocytes at any time, whereas in groups receiving AN-201, asignificant decrease of WBC was observed in all the experi-ments. This indicates that targeting of the cytotoxic radicalAN-201 to LHRH receptors in the form of AN-207 can reduceits systemic toxicity. As normal kidneys were previously foundto express low levels of mRNA (35) and 13 of 17 specimens inthis study showed LHRH receptor expression in normaltubular cells, we also addressed the possibility of local toxicityby AN-207 in nonmalignant renal cells. Therefore, mouse

kidneys were examined for LHRH receptor mRNA expressionand nonmalignant renal tissue was histologically evaluatedafter therapy with AN-207 and AN-201. As in the case ofhuman kidneys, some of the mouse kidneys were positive forLHRH receptors. Microscopic examination of kidneys aftersingle or repeated injections of AN-207 and AN-201 showedonly a few apoptotic and necrotic cells in both groups,indicating that the targeted therapy causes no specific toxicityto renal tissue. In addition, serum creatinine levels and urineanalyses 8 days after the injection of AN-207 or AN-201showed no differences compared with the control group,showing that renal function is not greatly affected by targetedtherapy. A possible, specific toxicity of AN-207 to the LH-producing cells of the anterior pituitary has been investigatedin previous studies in rats (36). The results of these experi-ments indicated that AN-207 causes no permanent damage tothe pituitary function at its maximum tolerated dose. The lowlocal toxicity of the cytotoxic radical AN-201 in AN-207 inLHRH receptor – positive nonmalignant tissue may beexplained by the fact that AN-201 mainly kills rapidlyproliferating cells.

The current study shows that LHRH receptors are expressedin a very high percentage (28 of 28) of human RCCspecimens. In addition, our work shows that cytotoxic LHRHanalogue AN-207 can be successfully targeted in vivo tohuman RCC, resulting in a high antitumor activity. Thus, areduction of systemic side effects compared with the cytotoxicradical AN-201 can be achieved by targeting to tumoralLHRH receptors. In conclusion, our findings suggest thattargeted therapy with cytotoxic LHRH analogues could be apromising new therapeutic modality for the treatment ofadvanced RCC.

Acknowledgments

We thank Dr. Gabriela Westphal, Patricia Armatis, David Chism, Miriam Ducruet,and Harold Valerio for excellent experimental assistance and Dudley R. Callais forhelp in preparation of the article.

Table 4. Binding characteristics of LHRH receptors inA-498, ACHN, and 786-0 human RCC xenograftedinto nude mice

Cell line Treatment Kd (nmol/L) Bmax (fmol/mg protein)

A-498 Control 8.51 F0.38 439.8 F 21.9AN-207 9.11 F0.83 398.4 F 55.8AN-201 8.08 F 0.77 414.2 F 32.7

ACHN Control 4.35 F 0.33 654.2 F 35.4AN-207 4.11 F0.74 599.1 F51.6AN-201 4.73 F 0.58 670.2 F 58.8

786-0 Control 10.6 F 0.25 437.2 F 39.9AN-207 11.4 F 0.76 404.6 F 47.1AN-201 9.93 F 0.58 415.5 F 28.3

NOTE: Binding characteristics were obtained from ligand competition assays(each done in duplicate or triplicate tubes) based on binding of radiolabeled[D-Trp6] LHRH to tumor membrane homogenates. All values represent meanF SE.

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2005;11:5549-5557. Clin Cancer Res Gunhild Keller, Andrew V. Schally, Timo Gaiser, et al. Hormone Releasing Hormone Analoguesfor Targeted Chemotherapy with Cytotoxic LuteinizingExpressed on Human Renal Cell Carcinomas Can Be Used Receptors for Luteinizing Hormone Releasing Hormone

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