Somatostatin Receptor Subtype Specificity and in Vivo Binding of a Novel Tumor Tracer, 99Mtc-P8291

[CANCER RESEARCH 58, 1850-1859. May 1. 1998] Somatostatin Receptor Subtype Specificity and in Vivo Binding of a Novel Tumor Tracer, 99mTc-P8291

Irene Virgolini,2 Maria Leimer, Hirsch Handmaker, Secondo Lastoria, Claudia Bischof, Pietro Muto, Thomas Pangerl, Doris Gludovacz, Markus Peck-Radosavljevic, John Lister-James, Gerhard Hamilton, Klaus Kaserer, Peter Valent, and Richard Dean Departments of Nuclear Medicine /I. V.. M. L. C. B.. T. P.. D. G.Â¡, Gastroenterologe Â¡M.P-R.Â¡. Surgery Â¡G.H.Â¡.Pathology Â¡K.K.Â¡,and Internal Medicine I. Division of Hematolog\ [P. V.}, University of Vienna, A-1090 Vienna, Austria; Arizona Institute of Nuclear Medicine, Phoenix. Arizona 85016 [H. H.J; Department of Nuclear Medicine, National Cancer Institute, 80131 Naples, Italy Â¡S.L, P. M.]: and Diatide, Inc.. Londonderry, New Hampshire 03053 Â¡J.L-J., R. D.Â¡

ABSTRACT strated (1, 2, 5). In fact, such tumors frequently coexpress VIP and SST/OCT binding sites. Recent data suggest that somatostatin receptors (SSTRs) are expressed An interesting phenomenon is that VIP and OCT can cross-compete on various tumor cells. High-level expression of SSTR on the tumor cell for binding to tumor cell membrane receptors (2). The molecular basis surface provides the basis for the successful clinical use of radiolabeled of this phenomenon could not readily be explained thus far. However, ligands for the in vivo localization of tumor sites. We have characterized the in vitro binding properties of the novel SSTR ligand "mTc-P829 using the molecular cloning of SSTR and VIPR has recently provided new primary human tumors (carcinoids, breast cancers, intestinal adenocar- insights into the biology and interactions of VIP and SST. To date, cinomas, pheochromocytomas, small cell and non-small cell lung cancer, five different human SSTRs (6-13) and two different VIPRs (14-19) and melanomas; n = 28), various tumor cell lines, and COS7 cells trans- have been characterized in detail and have been cloned. Using trans fected with the human SSTR (hSSTR) subtypes 1, 2, 3, 4, and 5. """Tc- fected peptide receptors, hSSTRS has recently been identified as a P829 bound to primary tumor cells and tumor cell lines with high affinity potential common acceptor site for both SST/OCT and VIP (20). and high capacity. The dissociation constants (A,,I ranged between 1 and Several efforts have been undertaken to identify hSSTR subtypes 20 IIM. '"""Tc-1'829 also bound with high affinity to the transfected expressed in primary human cancers (for a review, see Ref. 21). A hSSTR2 (Ka, 2.5 nM), hSSTRS (Ad, 2 nM), and hSSTR3 (KÂ¿,1.5 nM). Binding of "'""Tt-PH29 to hSSTR3 was found to be displaceable by unla- number of observations suggest that hSSTR2 is expressed in many different tumors, including neuroendocrine tumors and breast cancer beled P829/([ReO]-P829), SST-14, and vasoactive intestinal peptide (VIP; IC50, 2 nM) and, less effectively, by Tyr'-octreotide (IC50, 20 nM). In (22). However, other hSSTR subtypes have also been detected (23, contrast, the binding of 99mTc-P829 to hSSTR2 and hSSTRS could be 24). We recently have demonstrated expression of hSSTRS mRNA in displaced by P829/([ReO]-P829) and Tyr'-octreotide but not by VIP. a variety of human tumors, including breast cancers, melanomas, and 99l"Tc-P829 scintigraphy revealed in vivo binding to primary or metastatic neuroendocrine tumors (21, 25). Because this receptor (hSSTR3) was tumor sites in seven of eight patients with breast cancer and six of six found to bind both VIP and OCT (20), it was hypothesized that this patients with melanoma. In summary, our data show that 99n>Tc-P829 site is responsible for the observed cross-competition of these peptides binds with high affinity to many different types of primary and cloned in primary human tumors. tumor cells. Furthermore, our data identify hSS TK2. the VIP acceptor Despite the clinical usefulness of "'In-OCT (3) and 123I-VIP (4), hSSTR3, and hSSTRS as the respective target receptors. Because these several attempts have been made to label hSSTR ligands with 99mTc receptors are frequently expressed at high levels on primary tumor cells, because of its optimal decay properties and cost effectiveness (26- 99mTc-P829 appears to be a promising novel peptide tracer for tumor 28). Recently, P829, a peptide containing a sequence that mimics the imaging. binding domain of SST, has been identified as a suitable hSSTR ligand that can be labeled with 99mTc (29). However, the spectrum of human tumors that can be visualized by 99mTc-P829 has not been INTRODUCTION defined yet. Also, the binding behavior of this novel hSSTR ligand The high-level expression of peptide receptors on various tumor onto various subtypes of hSSTR is not known. The aims of the present cells as compared with normal tissue or blood (1,2) provides the study were to evaluate the binding characteristics and hSSTR subtype molecular basis for the successful use of radiolabeled SST* analogues specificity of 99mTc-P829, as well as the binding affinity of this novel (such as OCT) and VIP as tumor tracers in nuclear medicine (3, 4). tracer for primary human tumors. Thus, using primary tumor cells or cell lines, specific binding of both VIP and OCT to the cell surface of various tumors has been demon- MATERIALS AND METHODS

Received 9/19/97; accepted 3/3/98. Synthesis and Labeling of P829. The peptide P829 was synthesized using The costs of publication of this article were defrayed in part by the payment of page solid-phase peptide synthesis techniques and /v'-(9-fluorenyl)methoxycarbonyl charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. chemistry and was purified by preparative high-performance liquid chroma- ' These studies have been supported in part by the Austrian National Bank (JubilÃ¤ums tography as described (29). [ReO]-P829 was prepared by ligand exchange fonds Projects 5460 and 6512) and by a Foundation of the Mayor of the City of Vienna. using tetrabutyl ammonium oxorhenium tetrabromide, which was prepared Parts of the studies were sponsored by Diatide. Inc. 2 To whom requests for reprints should be addressed, at Department of Nuclear according to Cotton and Lippard (30). For practical use, we formulated an Medicine. University of Vienna, WÃ¤hringer GÃ¼rtel18-20. A-1090 Vienna. Austria. instant kit containing 50 /ig of the P829 peptide. This P829 kit was reconsti Phone/Fax: 43-1-40400-7835; E-mail: [email protected]. tuted with 500 MBq-3 GBq "'"Tc-pertechnetate (CIS Bio-International, Paris, 3 The abbreviations used are: SST, somatostatin; SSTR, SST receptor; hSSTR. human SSTR; VIP, vasoactive intestinal peptide; VIPR, VIP receptor; 123I-VIP, '"l-labeled VIP; France) in a final volume of 1-5 ml. The reconstituted product was heated for IC50, concentration of unlabeled ligand necessary to induce 50% inhibition of labeled 15 min and then kept at room temperature for 45 min. An aliquot of this ligand binding; Kd, dissociation constant (concentration of labeled ligand necessary to preparation was used for the in vitro series of experiments. The radiochemical produce half-maximal binding); LNN, lymph node mÃ©tastases;NSCLC, non-small cell purity of the 99mTc-P829 product was determined by instant TLC (ITLC-SG; lung cancer; OCT, octreotide; "'In-OCT. '"In-labeled OCT: P829. novel SST-14 ana Gelman Sciences, Ann Arbor, MI) developed in saturated saline [99mTc-P829 logue; |ReO]-P829, oxorhenium complex of P829; PMNC, peripheral blood mononuclear and 99mTc-microcolloid, relative fraction (rf) 0-0.75] and ITLC-SG developed cell; SPECT, single-photon emission computed tomography; SSM, superficial spreading melanoma. in pyridine:acetic acid:water (5:3:1.5) for determination of "Tc-microcolloid 1850

Table 1 Binding Â¡if9v'"Tc-PH29. Approximately 500 MBq WmTc-P829 were injected i.V.. and planar and SPECT images were acquired within 24 h after injection. 99mTc-P829 identified primary tumors and mÃ©tastasesas indicated in the Table. NP, not performed.

Detection of tumor lesions by scintigraphy with

Patient No./initials Site of disease" WmTc-P829 "in-OCT I-VIP BreastcancerI/EE2/SB3/RS4/JA5/HB6/BW''7/LT*8/CCfr9/SM'IO/NE'1 (right)Primarybreast cancer (right)Primarybreast cancer (left)NHLbreast cancer (left)Primary(MALToma) of the breast (left)Supraclavicularianductal breast cancer (left)Multiple LNN positive>l() tissueRecurrentLNN in bone, liver, soft positiveNegative1/1 (left)LNN. lobular breast cancer thoraxPrimaryleft lower positiveNPNPNPNPPositivePositive1/1 tumorPrimaryductal tumorPrimarylobular I/RE'12/TS''Melanomas1/LR'2/CF3/SA*4/VS"-'5/MR'"6/BMC'"''7/TJc8/KL''-'CarcinoidsI/RE*"2/TK"'rSCLCI/RM'NSCLC15/SP1" tumorPrimaryductal tumorPrimaryductal

choroid)PrimarySSM (right toe)LNN SSM (left (leftgroin)LNN positive1/1 (leftaxilla)LNN positive>!(> groin)LNN(left and right positive1/1 (rightaxilla)s.c. positiveI/I metastasiss.c. positive3/3 mÃ©tastasesLNN. positive1/1 rightaxillaBone positive5/5 mÃ©tastasesLNN, positive4/4 groins.c.left, right positive2/2 mÃ©tastasesLung positiveI/I positiveNPNPNPNPNPNPNP'smetastasisPrimary NMs.c. mÃ©tastasesLNN mÃ©tastasesLiver

metastasisLNN (abdomen)PrimarymÃ©tastases

tumorPrimary

tumorPositivePositivePositivePositiveNegative1/1 NHL,non-Hodgkin' melanoma:alreadylymphoma; MALToma. mucosa-associated lymphoid tissue tumor; NM. nodular small cell lung cancer;NPNPNPNPNPNPNPNPNPNPPositiveNPPositiveNPNPNPNPNPNPNPNPNPNPNPNPNPNPPositivePositivePositivePositiveNPNPNP.not performed. was'Primary tumor scintigraphy.,resected at the lime of From these patientsPrimary tissue specimens were obtained for in v/fm receptor analysis (seealso Table 2).NPNPNPNPNPNPNPNPNPPositivePositivePositivePositivePositivePositivePositivePositivePositivePositivePositivePositivePositivePositivePositivePositivePositivePositivePositivePositivePositivePositivePositivePositiveSCLC.

(rf 0-0.25). The radiochemical purity of 99mTc-P829 was >90% in all exper Cell lines were cultured in RPMI 1640 (A431. HT29, T47D. BT20, ZR75-1. MCF7. and KU8I2) or in Iscove's modified Dulbecco's medium iments. Preparation of '"In-OCT. ' ''In-OCT was prepared by reacting 10 Â¿igof (HMC-1) supplemented with 10% PCS, i.-glutamine. and antibiotics in 5% diethylenetriaminepentaacetic acid-OCT for 10 min at 22Â°Cwith 100-200 CO,/95% O2 air at 37Â°C.Cells were fed two to four times per week. Adherent MBq of '"In-Cl, according to the manufacturer's description (Mallinckrodt cells were passaged with trypsin (Worthington. Freehold, NJ) after confluency Medical. St. Louis. MO). This product was routinely applied for scintigraphy. was reached. Before being used in binding experiments, cells were washed in and an aliquot of this preparation was used for in vitro studies. 50 mM Tris-HCI buffer (pH 7.5) and then resuspended in 50 mM Tris-HCI Preparation of Primary Tumor Cells. Written informed consent was assay buffer (4Â°C;pH 7.5), containing 5 mM MgCl, and 0.1% BSA. About obtained from all patients undergoing surgery. Tumor tissue specimens (0.5-1 5 X IO7 cells suspended in 5 ml were used for one series of experiments. ml) were collected at surgery and immediately placed into liquid nitrogen. The Normal PMNCs and blood platelets were prepared according to published diagnoses were established by histological examination and immunohisto- techniques (32). chemistry according to WHO criteria. In particular, tissue was analyzed in Transfection. Plasmids containing the receptor cDNAs for the human SST patients with carcinoids (n = 2), pheochromocytomas (n = 4), colonie ade- receptors (hSSTRl through hSSTR4) were kindly provided by Dr. G. Bell nocarcinomas (n = 3), ductal breast cancer (n = 4), lung cancer (n = 2). and melanomas (n = 12). Tissue was stored at â€”70Â°Cuntilit was used for in vitro (Howard Hughes Medical Institute. Chicago, IL). The plasmid carrying hSSTR5 was kindly provided by Dr. A. M. O'Carroll (NIH, Bethesda, MD). studies. Tumor cell membrane fractions were prepared according to established Correctness of the receptor clones was checked by agarose gel analysis of techniques (2). Briefly, tissue was thawed, cut into pieces, put into 50 mM Tris-HCI buffer (pH 7.5) and 5 mM MgCl-,. and homogenized by means of a restriction fragments. Furthermore, a complete sequence analysis was per glass homogenizer. The cell homogenate was centrifuged at 5000 x g for 10 formed for each clone. Isolation of plasmid DNA was carried out with the min at 4Â°C. washed, and resuspended in assay buffer containing 50 mM Qiagen plasmid purification kit (Qiagen. Hilden. Germany). Tris-HCI (pH 7.5) and 5 mM MgCl, at a concentration of 100-1000 /xg COS7 cells were grown in RPMI 1640 containing 10% fetal bovine serum and antibiotics (Life Technologies, Inc., Vienna, Austria). Transient transfec- protein/ml, and measured by the method of Bradford (31). Preparation of Tumor Cell Lines and Blood Cells. The epidermoid tion of COS7 cells with different receptor cDNAs was performed using the yV-[l-(2.3-dioleoyloxyl)propyl]-A',A'./V-trimethylammoniunimethyl sulfate carcinoma cell line A431; the colonie adenocarcinoma cell line HT29; the transfection reagent (Boehringer Mannheim) according to the manufacturer's melanoma cell line 518A2; and the breast cancer cell lines T47D. BT20. ZR75-1. and MCF7 were purchased from American Type Culture Collection description. Twenty-four h after transfection. the medium was exchanged, and (Rockville. MD). The basophil cell line KU812 was kindly provided by Dr. K. after 48 h in culture, the cells were harvested, an aliquot was recovered for Kishi (Nijgata University, Nijgata. Japan), and the mast cell line HMC-1 by RNA extraction and Northern blot analysis, and the remaining cells were used Dr. J. H. Butterfield (Mayo Clinic, Rochester, MN). immediately for binding studies. 1851

Table 2 â„¢"'Tc-P829 binding to primary tumors in vitro and mRNA expression Tumor tissue was removed during surgery, and tumor membrane fraciions were prepared as described in the texl. All concentrations listed are in nM. The ÃŸmaxrefers lo Ihe lolal number of specific binding siles esiimaied from ihe binding of 99mTc-P829 in Ihe concenlralion range of [99mTc-P829] listed, and Ihe KÂ¿value is the dissocialion conslanl for Ihis inleraclion. The IC50s indicale the concentralion of unlabeled ligand necessary lo reduce Ihe specific binding of 99mTc-P829 (al the concentration of [99n'Tc-P829] used in the assay) by half. NP. not performed. Northern blot score: + + +, very strong expression; + +, strong expression; +, weak expression; -, no expression.

experimenlsPalient Saturalion values)["mTc- experimenls (IC50 expression2++NPNP+NPNP-(+)NPNPNPâ€”++NP+

[ReO]- Tyr1- No./inilialsCarcinoidsI/RF2/TKPheochromocylomas3/GO4/TO5/BJ6/TMColonieadenocarcinomas7/HW8/SK9/HADuclaldiagnosisLiver P829]32.50.90.932.511.72.75.032.58.78.71.01.0582691.70.10.2NP10.8NP12.50.83551NP0.15P829/OCT<30P829 SST-14 1NP3

melasiasis"'Lymph XIO121.7 3003 NP ++ melasi."'Normalnode X10"Negative1.8 NegativeNegative3 +Negalive +NPNP+NPNP++NPNPNPNPNP+(+)NP+NP+ mucosaPrimaryjejunal Negative30Negative NPNP XIO123.6 lumorPrimary Negative3NP NP35 XIO121.2 lumorPrimary .0-2600.3-130.16-354.3-2720.9^Â» NP4 NP X10"1.5 tumorPrimary 5NP1.2 NP3.3 X10"3.0 tumorPrimary 330 3 NPNPNPNP

XIO129 lumorPrimary NegativeIONP +++ XIO12Negali lumorNormal 10.9^111.6-711.6-713.6-2638.5-2690.3-130.9-410.01-19.41.7-75.40.18-71.5NP0.1-67NP'2.1-96.51.1-73(Fig.NPNegative NP +NP+ mucosaPrimarycolonie ve3 NP3 Negative NP10 XIO123 lumorPrimary NP3 NP NPNP ++NP+ cancer10/SMll/NE12/RE13/TSLungbreast lumor"Primary XIO121.8 NP<60 NP NPNP lumor"'Primary XIO121.2 NP<50 NP +NP500NP ++++ ++ lumor"Primary X10"4.2 NP5 NP ++NP+NPNPNPNP+ M)SCLC"NSCLClumor" (Fig. XIO119 NP<3 500 ++NP++NPNPNPNP+(Fig.1C)NP- cancer14/RM15/SPMelanomas16/SE17/TJ18/NM19/LR20/HH21/ZJ22/RH23/VS24/PL25/KL26/PX27/MR28/BMCPalienlXIO12''6.2 <11 NP NPNPNP XIO121.2 IB)Primary(Fig. NPNP 2 +NPNPNPNP+NPNP+NP+ (Fig.ID)NPNPNPNP+ XIO123 (SSM)Primarylumor NP8 NP NPNegalive (NNM)"Primarylumor XIO12NP1 814 NP NPNP (SSM)Primarylumor 2.5NP NP NPNP (SSM)''lymphlumor XIO12NPNegative1 NP1 NP NPNPNP melasiasislymphnode NPNegative NP ++NPNP++NPNP+ +NPNPNPNP+++NP melasiasislymphnode NP0.8Negative NPNP melastasislymphnode XIO12NPNegative3 Negative4NP NPNPNP melastasis''s.c.node 4B +5B)0.05-630.02-60NP0.1-23NP(siles/mg)3.0 5Negative NP ++NP++ ++NP+++NP5NP+NPNPNPNPNP++NP+NP+++ metastasis"'*s.c. Negative1Negative NP0.4 metastasis"'s.c. XIO12NP6 Negative8.62.3 -Negalive +++ metastasis"'s.c. NegativeNPNegative â€”NP +++ ++ melasiasis''s.c. XIO12NP151Negative15411.5155Negative2510152.41.65152NP1NPNegative1NPNegative1NP4.5NPDisplacementNPNP NP NPNegalive +NP3+ ++NP4++ melasiasisP8294.3-2720.17-100.17-104.3-2721 63 NegativeVIP NPSSTR-mRNA " In these patients, In-OCT scintigraphy visualized the tumor site in vivo prior to its surgical removal. b In these patients, 123I-VIP scintigraphy visualized the tumor site in vivo prior to its surgical removal. * In this patient, In-OCT was used as a radioligand. *' In these patients, 99mTc-P829 scintigraphy visualized the melanoma tissues (see also Fig. 4).

RNA Extraction and Northern Blot Analysis. Snap-frozen tumor speci Radioligand Binding Assay. To evaluate expression of P829-binding sites mens were put in liquid nitrogen and homogenized in Trizol Reagent (Life on tumor cells, saturation and displacement studies were performed. In prin Technologies) using a glass homogenizer. Total RNA from the homogenate ciple, the assay conditions were the same as those reported previously for other was extracted following the Trizol extraction protocol, a modification of the peptide ligands (2). All saturation studies were performed under steady-state RNA extraction described by Chomczynski and Sacchi (33). RNA of trans- conditions at 4Â°C.In saturation experiments, the intact cells or the membrane fected and control COS? cells was extracted using the same technique. Integ fractions were incubated with increasing concentrations of "mTc-P829 (typi rity of RNA was confirmed by agarose gel electrophoresis and staining with cally 0.01-100 nM. unless otherwise specified) in the absence (total binding) ethidium bromide. Northern blot analysis of receptor subtype expression was and the presence of the unlabeled peptide P829 ( 1 /AM,nonspecific binding). carried out as described (34). Northern transfer of 10-20 (Â¿goftotal RNA was Alternatively, SST-14 or [ReO]-P829 was used to assess the nonspecific performed using S&S Nytran membrane (Schleicher & Schuell, Vienna, Aus binding. In displacement experiments, the intact cells or the membrane frac tria) by capillary blotting overnight. RNA was fixed to the membranes by UV tions were incubated at 4Â°Cwith 99mTc-P829 in the absence (total binding, cross-linking. Specific probes for Northern hybridization were generated by 0.1-10 nM if not otherwise indicated) and in the presence of increasing restriction cutting of the plasmids carrying the probes with the appropriate concentrations (0.001-1000 nM) of the unlabeled ligands P829. [ReO]-P829, restriction enzymes. After separation with an agarose gel. the probes were Tyr'-OCT, SST-14, and VIP. purified with the Qiaex gel purification kit (Qiagen) and labeled using the After incubation of intact cells for 45 min, the reaction mixture was diluted Redivue random prime labeling kit (Amersham, Buckinghamshire, United 1:10 with assay buffer (4Â°C)and rapidly centrifuged (5000 X g for 10 min at Kingdom) and [32P]dCTP (Amersham). Hybridization was carried out as 4Â°C)to separate membrane-bound from free ligand. The resulting pellet was described (33. 34). Briefly, the membranes were prehybridized at 42Â°Cin a washed twice with buffer and counted in a gamma counter for 1 min. Filters hybridization solution containing 50% formamide, 5X Denhardt's solution, were presoaked with 0.1% BSA. 5X SSC (sodium chloride and sodium citrate), 0.2% SDS, and 100 Â¿ig/ml Specific binding was determined as the difference between total and non salmon sperm DNA. After 4 h of hybridization, the labeled probe was added specific binding. Binding data were analyzed according to Scatchard (35). In in fresh hybridization buffer, and hybridization was carried out overnight. selected binding experiments, ' " In-OCT was used as a ligand for comparative Thereafter, blots were rinsed twice at room temperature with 2X SSC buffer binding studies. containing 0.1 % SDS and then twice at 42Â°Cwith 0.2 x SSC buffer containing In Vivo Studies: Patients and Gamma Camera Imaging. The adminis 0.1% SDS and exposed to an X-ray film (Hyperfilm; Amersham). tration of 99"'Tc-P829 to patients was approved by the local Institutional 1852

2 3 4 99mTc-P829 (nmol/L) 11Tc-P829(nmol/L)

*- CM CO ce o: rr cc tr co co co co co D,_Â£Â£COcoCMcoco.CcoOÃco(0.C^J.coco.CIO(TCOco-C CO CO CO CO CO je .c

18S 18S 28S 28S â€¢Â«I

ÃŸactinâ€¢ ÃŸactinâ€¢

Fig. I. Specific binding of "mTc-P829 to a primary human breast cancer (A; Table 2. patient 13| and a NSCLC (B: Table 2, patient 15) The tumors were obtained intraoperatively, and tumor membrane fractions were used for the binding studies. These were incubated with increasing concentrations of "mTc-P829. The specific binding (shown) was calculated by subtracting the amount of "mTc-P829 bound in the presence of an excess of unlabeled P829 or [ReO]-P829 (1000 nM; nonspecific binding) from that bound in its absence (total binding). Saturation curves and Scatchard analysis (A and B. insets) indicated a saturable number of specific binding sites for Wn)Tc-P829 for both tumors. A single high-affinity binding class was demonstrable for the breast tumor displaying a Bma^ of 4.2 X IO11 sites (i.e., 720 fmol)/mg protein and a KÂ¿of 2.4 nw. Scatchard plots derived for the saturation data of NSCLC identified two classes of specific high-affinity 99nlTc-P829 binding sites with a Bmaxl of 6 X 10" (i.e.. 1 pmol) and a ÃŸmâ€ž2of5.4 X IO'2 (i.e., 8 pmol) sites/trig protein and a Kd, of 0.25 and a KÂ¿2of5 nM, respectively. mRNA analysis by Northern blotting identified in the breast tumor the presence of hSSTRJ. hSSTR2, hSSTR4. and hSSTRS (O and in NSCLC, the presence of hSSTR2, hSSTR3, and hSSTR4 (D).

Review Board and was performed according to the Declaration of Helsinki. All X-40 (breast cancer; SMV, Twinsbury, OH) gamma camera equipped with a patients gave written informed consent to participate in the study. Eight high-resolution collimator. The pulse-height analyzer window centered around the patients with breast tumors and six patients with melanomas were studied as "Tc peak (140 keV) with a window width of Â±20%.The planar images were part of Phase Ila/IIb clinical investigations to evaluate 99mTc-P829 binding to acquired with a matrix resolution of 128 X 128 pixels, 1000 kilocounts for the tumor tissues in vivo (Table 1). Four patients had ductal breast adenocarcino- whole body, 300 kilocounts for the head and extremities, and 500 kilocounts for mas and one a non-Hodgkin's lymphoma (mucosa-associated lymphoid tissue the chest and abdomen. SPECT was performed at 2 h postinjection using a matrix type). In three patients, the primary breast tumor had already been resected at of 128 X 128 pixels, 64 steps (30 s/step). and a 360Â°rotation. SPECT images were the time of scintigraphy. These three patients presented with LNN and/or bone reconstructed in conventional axial, sagittal, and coronal projections, using either and soft tissue mÃ©tastases.Among the six melanoma patients, two patients had a Shepp Logan Hanning reconstruction filter (Orbiter II) or a Butterworth filter a primary SSM, and in five of the six patients, multiple sites of mÃ©tastaseswere (SMV-X-40). Scans were viewed by two independent nuclear medicine physi present at the time of scintigraphy (Table 1). Scintigraphy with "mTc-P829 cians. Using standard nuclear medicine techniques, foci of increased labeled (370-550 MBq) was performed within 1 month following conventional stag peptide accumulation were considered as true positive sites of disease when ing procedures (biopsy, computed tomography and/or magnetic resonance corroborated by computed tomography and/or magnetic resonance imaging find imaging, mammography, and bone scintigraphy). ings or confirmed by histology after surgical biopsy. One group of the patients who had the "Tc-P829 scan was also imaged by "'in-OCT. In a group of patients in whom tumor tissue was analyzed for expression of "mTc-P829 binding sites, I23I-VIP and/or "'In-OCT scintigra RESULTS phy were performed prior to surgery according to Refs. 3 and 4, whereas In Vitro Binding of WmTc-P829 to Primary Human Tumors and "TC-P829 scintigraphy was not available. A summary of patients who MÃ©tastases.A variety of primary tumors were examined for expres underwent scintigraphy is shown in Table 1. sion of 99mTc-P829 binding sites. For this purpose, tumor membrane Whole-body and planar scanning, as well as SPECT studies, were performed within 24 h after injection of 99mTc-P829. Planar and SPECT images were preparations were used. The primary tumors, as well as their mÃ©tas tases, bound 99mTc-P829 in a saturable manner, indicating the pres- acquired either with an Orbiter II (melanoma; Siemens, Erlangen, Germany) or 1853

Table 3 99"'Tc-PX29 binding to tumor cell lines und SSTR mRNA expression

Intact tumor cells or normal peripheral blood cells were used for the experiments listed. All values are listed in niu. The ÃŸmasnumber of binding sites estimated from the specific binding of WmTc-P829 determined in the concentration range of |''9l"Tc-P829] listed, and the K0 value is the dissociation constant for this interaction. The IC50s indicate the concentration of unlabeled ligand necessary to reduce the specific binding of "mTc-P829 (at the concentration of |'Wl"Tc-P829] used in the assay) by half. NP. not performed. All experiments were repeated three to six times. Northern blot score: , very strong expression: + + . strong expression: +. weak expression: â€”.no expression. studyBmÂ¡lx (IC5()s)V1-51-510-201-510-15Negative1-510-200.55-151-55-101-510-151-510-303-53-5""'TC-P8296.7-10.56.7-10.55-10.56.7-10.51.1-27.29.9-11.31.1-86.4-7.80.2-13.5-6.73.5-6.7P829/study (mRNA)VIP subtype expression

(sites/cell)0.6-1 [ReO|-P829Negative10-151-15Negative3-3010-300.3-310-301-53-103-10SST-14Negative10-15NP'Negative3-301-10NP10-30NPNP100Tyr'-OCTNegative10-15NPNegative50-30010-10010-5030NPNegative100SSTR3NP 1 2 5(+) T47DBT20ZR75-IMCF7HT29KU8I2HMCIA431518A2*PlateletsPMNCs"mTc-P8290.8-740.8-740.8-741X10s0.3-1 +10-15 (+) ++ XIO50.2-1 +Negative ( + ) + +( XIO62-5 XIO51 +NP â€” (+ ) + )+NP XIO6Negative0.8-1.5 .9-740.1-21980.6-6000.1-12741.6-72170.1-750.17-560.4-56Saturation NP3-30 NP NP NP( XIO50.3-3 +3-30 (+ ) + + + >(+)NP XIO61-10 XIO53-30 NP10-30 NP NP NPNP XIO60.5-1 XIO60.9-1.8 NPNP NP NP NPNP XIO70.6-1.2 XIO60.5-1. NP1-10 NP NP NP+ 2 XIO73 XIO124.2 +3-10 (+ ) ( + ) + + + ++NP XIO133-6 XIO34-6 NP1-10 NP NP NP( X IO3Displacement - ( + ) +4 + ) " ÃŸnlaxand KÂ¿are given for the two high-affinity binding classes, where two values appear. For the experiments with 518A2 melanoma cells, membrane fractions were used, and the sites/mg protein are listed. ' NP. not performed. enee of specific 99"'Tc-P829 binding sites. Table 2 shows the total Binding of WmTc-P829 to Transfected hSSTR Subtypes. To number of binding sites (Bma%s)andthe binding affinities (A"ds)forthe examine the hSSTR subtype specificity of 99mTc-P829binding, COS? various tumors and/or mÃ©tastases.Mosttumor cell preparations ana cells transfected with cDNAs encoding for hSSTR 1 through hSSTRS lyzed were found to contain two classes of high-affinity binding sites. were used (Fig. 2, A-E). mRNA expression of hSSTR subtypes was In the case of small tumor samples, preventing extensive binding checked in each series of experiments by Northern blot analysis. The analysis, high-affinity binding of 99mTc-P829was also obtained but binding of 99mTc-P829and unlabeled P829/[ReO]-P829 to hSSTRl could not be subdivided into two classes of binding sites. Typical through hSSTRS was assessed by saturation and displacement studies binding isotherms for the specific binding of 99mTc-P829to a primary in parallel. ductal breast adenocarcinoma (Kd, 2.4 nM) and a NSCLC (Kal and In saturation studies, 99mTc-P829binding to hSSTR2 (Fig. 2ÃŸ), Ka2,0.25 and 5 nM, respectively) are shown in Fig. 1. As opposed to hSSTR3 (Fig. 2O, and hSSTRS (Fig. 2E) was saturable, demonstrat primary tumor tissues, no significant in vitro binding of 99l"Tc-P829 ing an interaction of the labeled ligand with a high-affinity binding to normal intestinal mucosa (n = 2) was detectable. domain on the transfected hSSTR subtype. The respective binding data are depicted in Table 4, indicating A'jSin the lower nanomolar In two patients with liver or lymph node mÃ©tastasesspreadfrom carcinoid tumors, the mÃ©tastaseswerefound to bind 99n'Tc-P829 in range for these ligand-receptor interactions. Noteworthy is the inter vitro, whereas the normal jejunal mucosa did not bind 99"'Tc-P829 action of ""TC-P829 with hSSTRS (Fig. 2C), which also binds (Table 2). In four patients with pheochromocytomas (A"d,1-15 nM),in labeled (I23I-VIP) and unlabeled VIP (20). In contrast, 99mTc-P829 three patients with colonie adenocarcinomas (Kd 2-15 nM), in four did not bind to hSSTRl (Fig. 2/1) and hSSTR4 (Fig. 2D). patients with ductal breast cancers (Fig. \A; Kd, 2.4-15 nM), and in As opposed to 99mTc-P829, "'in-OCT bound to hSSTRl and one patient with NSCLC (Fig. IÃŸ;Kd5 nM),99mTc-P829bound to the hSSTRS with a similar binding affinity in terms of Kds (1 nM), primary tumor tissues with high affinity. In four of four patients with whereas the binding affinity for hSSTR3 was significantly lower (Kd, melanomas, 99mTc-P829bound to primary tumors; in three of four 5-50 versus 1-10 nM).As with 99mTc-P829,no substantial binding to melanoma patients, 99mTc-P829also bound to lymph node mÃ©tasta hSSTRl and hSSTR4 was observed for '"In-OCT. ses; and in four of five patients, "mTc-P829 also bound to s.c. Ability of Unlabeled Peptides to Compete for the Binding of melanoma mÃ©tastases. "mTc-P829. Displacement studies were performed using tumor Binding of WnlTc-P829to Tumor Cell Lines. Surfaceexpression membrane fractions and intact tumor cell lines, as well as COS7 cells of 99n'Tc-P829 binding sites was analyzed by using a number of transfected with hSSTRl through hSSTRS. different cell lines (intact cells; Table 3). The breast cancer cell lines The binding of 99mTc-P829 to primary tumor cell membranes T47D, BT20, and ZR75-1 bound "mTc-P829 in a specific manner, (Table 2) was competed for by SST-14 and/or Tyr3-OCT, as well as whereas the MCF7 breast cancer cell line did not bind 99"'Tc-P829. In by P829/[ReO]-P829 (Fig. 3, A and B). In addition, using membrane addition, positive binding results were obtained for the HT29 adeno fractions of carcinoid (n = 1), pheochromocytomas (n = 3), and carcinoma cell line, 518A2 melanoma cell line, KU812 basophil cell colonie cancer (n = 1), VIP was found to be a competitor for line, HMC-1 mast cell line, and A431 epithelial cell line (Table 3). For 99mTc-P829binding, with IC5()sin the nanomolar range (Table 2). most cell lines, the binding could be subdivided into two high-affinity Specifically bound 99lnTc-P829was displaced by unlabeled P829 or binding classes. Significantly higher numbers of binding sites were [ReO]-P829 from the cell surface of various tumor cell lines (Table 3). observed for tumor cells as compared with normal peripheral blood For most cell lines, SST-14 and Tyr3-OCT were effective in compet cells (platelets and PMNCs; P < 0.001). A summary of the binding ing for 99"'Tc-P829 binding sites, with IC50sin the lower nanomolar results is shown in Table 3. range. Furthermore, unlabeled VIP was a significant competitor (Kd, 1854

2.5 -,

O m o> CM CO Q_ 0.5-

T1Tc-P829(nmol/L) T1Tc-P829(nmol/L)

O ZI O I m O 2-

20 40 60 80 100 120 T1Tc-P829 (nmol/L) 99mTc-P829 (nmol/L)

6-1 - W (/> C/5 CO W O O O O O 0 0 0 0 0 8 co 18S 28S

râ€” rÂ». r-. W W W co CO O o o o o o m 2 o o o o o

18S 28S

02468 99mTc-P829 (nmol/L)

Fig. 2. Specific binding of 99mTc-P829 to COS? cells transfected with hSSTRl (A), hSSTR2 (B), hSSTR3 (O. hSSTR4 (D). and hSSTRS (E). COS? cells were transiently transfected with hSSTR I through hSSTRS. Intact transfected cells were incubated with increasing concentrations of l)I)mTc-P829. The specific binding (â€¢)was calculated by subtracting the amount of "mTc-P829 bound in the presence of an excess of unlabeled P829 or [ReO]-P829 (1000 nM; nonspecific binding, T) from that bound in its absence (total binding. A). Saturation curves and Scatchard analysis (B, C, and E, inserta) indicated a saturable number of specific binding sites for 'w"Tc-P829 binding to hSSTR2. hSSTR3, and hSSTR5. However, no specific binding of 99mTc-P829 could be observed for hSSTRl and hSSTR4. as indicated by unsaturable total binding (Aj and nonspecific binding (T). The respective binding data are listed in Table 3, which shows a Kd value of 2.5 nM for hSSTR2 (2 fmol/106 cells; i.e.. 1500 sites/cell, n = 6), 1.5 nM for hSSTR3 (6 fmol/106 cells; i.e.. 3600 sites/cell), and 2 nM for hSSTRS (6 fmol/106 cells; i.e.. 3600 sites/cell). F. mRNA analysis by Northern blotting for control (i.e., nontransfected COS? cells; bottom) and COS? cells transfccled with the respective hSSTR subtypes 1-5 (top) used for these studies. 1855

Table 4 Binding of WmTc-P829 to SSTR subtype expressed on COS? cells COS7 cells were transiently transfected with hSSTRl through hSSTRS (see also Figs. 3 and 4). Intact transfected cells were incubated either with Wr"Tc-P829 or ' "in-OCT (data in brackets). The results listed indicate the median (range) of at least six independent experiments. The ÃŸmaxrefers to the total number of binding sites estimated from the specific binding of |'WmTc-P829] determined in the concentration range of [9MlIÃŒTc-P829]listed;and the Kd. is the dissociation constant of this interaction. The IC50s indicate the concentration of unlabeled ligand necessary to reduce the specific binding of WmTc-P829 (at the concentration of |yt mTc-P829] used in the assay) by half. All values are listed in nM.

study(sites/cell)NegativeNegative1.500(IC50s)KJNegativeNegative2.5(0.25-5)1.5(0.1-2)1.5(1-10)15 study

SSTRlSSTR2SSTR3SSTR4SSTR5"mTc-P829['"in-OCT]0.09-58410.09-6.90.1-5709[0.01-13.80.01-20000[0.03-960.4-2598[0.04-960.15-37[0.007-96Saturation (1,200-10.000)720(240-1.200)3.600(600-10,000)1.200(1.000-4.000)NegativeNegative3.600(0.3-3)1 (0.3-1)1 (0.3-3)1 (0.3-3)1 (300Negative]

<1.200-6.000)6.200(600-12.000)Displacement(0.1-10)0.5 (0.3-30)1 (0.3-30)1 (3-30)5(1-30)VIPNegativeNegative]>500>IOOO]2 (0.5-10)99mTc-P829["'in-OCT]0.7-1320.03-1.30.7-9.60.14-7.60.7-5.60.15-7.80.19-1100.2-5.72.2-4.40.1-5.7|ReO]-P829NegativeNegative1(

1-30 nM) for the sites recognized by 99mTc-P829 in most tumor cell DISCUSSION lines (Table 3). 99nTc-P829 binding to HSSTR2, HSSTR3. and hSSTRS (transfÃ©rants) The observation that a variety of tumors express significantly was displaced by unlabeled P829/[ReO]-P829, by Tyr'-OCT, and by higher amounts of SST/VIP binding sites as compared with normal SST-14, with IC5()sin the nanomolar range, as indicated in Table 4. No peripheral blood cells and tissues led to the clinical use of radiolabeled substantial difference was observed for the displacement of the specific SST/OCT analogues (3) as well as of VIP (4) for the in vivo local binding of "mTc-P829 and ' " In-OCT bound to HSSTR2 and hSSTR3 ization of tumors expressing peptide binding sites. In the present by unlabeled peptides. The rank order of potency for both ligands at study, the binding behavior and receptor subtype specificity of the hSSTR2 sites was SST-14 = P829 = Tyr'-OCT Â» VIP. The rank order novel SST analogue 99n'Tc-P829 has been evaluated. The results of of potency for 99mTc-P829 binding to hSSTRS sites was SST- our study show that 99mTc-P829 binds to many different types of 14 > P829 > VIP > Tyr'-OCT, and that for "'In-OCT was SST- tumor cells, including breast cancers, intestinal adenocarcinomas, and 14 > P829 > Tyr'-OCT = VIP. The rank order of magnitude for both melanomas. Furthermore, our study identities hSSTR2, hSSTR3, and ligands for hSSTRS sites was P829 = SST-14 = Tyr'-OCT Â» VIP. hSSTRS as "'"Tc-P829-binding sites. Because these receptors are expressed at high levels in many different tumors, 99mTc-P829 ap Detection of hSSTR mRNA by Northern Blotting. The presence of hSSTR subtype-specific mRNA in primary tumors (Table 2) and pears as a promising novel tumor-imaging agent. Indeed, our initial imaging studies demonstrate the in vivo binding capacity of ""Tc- tumor cell lines (Table 3) was analyzed by Northern blotting. As can be seen in Fig. 1, C and D, as well as in Tables 2 and 3. primary P829 in melanoma and breast cancer patients. In the first phase of our study, we analyzed the binding of 99mTc- tumors and immortalized tumor cell lines all expressed HSSTR3. Also in most primary tumors and tumor cells, expression of P829 to a variety of primary human tumors. Similarly to OCT and Tyr'-OCT (36), this novel SST-analogue bound to virtually all tumor hSSTR2 mRNA as well as hSSTR4 and hSSTRS mRNA was detectable. However, most of the tumors did not express hSSTRl. cell membrane fractions, with high affinity and high capacity. The number of 99mTc-P829 binding sites expressed on these tumors was in Depending on the tumor type, expression of hSSTR subtype mRNA differed with respect to the intensity of Northern blot the range reported earlier for other SSTR ligands (36). Furthermore, the binding of 99mTc-P829 to the membrane fractions could be dis signals (see Tables 2 and 3). In Vivo Binding of 99mTc-P829 to Primary Tumors or Tumor placed by unlabeled P829, [ReO]-P829, SST-14, and Tyr'-OCT. This suggests that 99mTc-P829 acts as a SSTR ligand in many human MÃ©tastases.To test the in vivo applicability of the novel tumor tracer 99mTc-P829, a cohort of patients suffering either from breast tumors tumors. Similar results were obtained with a number of tumor cell (n = 8) or melanomas (n = 6) was subjected to 99mTc-P829 scintig- lines showing high level expression of 99"'Tc-P829 binding sites on intact tumor cells. In contrast, the number of 99mTc-P829 binding sites raphy. In four melanoma patients, we were able to perform compar ative in vitro-in vivo experiments using 99"'Tc-P829. detectable on normal human peripheral blood cells (PMNCs and 99mTc-P829 was well tolerated by all patients. No adverse reactions platelets), as well as normal tissues (see Table 3), was in a lower range. All of these observations suggest that 99mTc-P829 is a prom were observed after peptide injection or during the study period. 99mTc-P829 visualized the primary breast tumor in four of five pa ising novel imaging agent. Indeed, using 99mTc-P829 as an imaging tients (Fig. 4A). 99mTc-P829 also indicated multiple sites of mÃ©tasta agent, primary tumors (breast cancers and melanomas) and their ses in three of three patients (Table 4: Fig. 4ÃŸ).Furthermore, 99mTc- mÃ©tastasescould be detected with high specificity. Whether all pri mary tumors expressing P829 binding sites can be imaged by 99mTc- P829 scintigraphy indicated two primary melanomas (two of two) and multiple sites of melanoma lymph node mÃ©tastases,bone, and s.c. P829 remains to be determined in a larger clinical study. mÃ©tastasesin five of five patients (Table 1). Lymphonodal packages, A remarkable finding of this study was that almost all tumor cell clinically not evident and unknown prior to scintigraphy were iden lines expressed two classes of high-affinity 99n'Tc-P829 binding sites. tified in three of these patients (Fig. 4B). This observation confirms our previous data obtained with other In four melanoma patients in whom the primary tumor, LNN radiolabeled SST-14 analogues (Tyr'-OCT and SST-14; Ref. 2). With mÃ©tastases,or s.c. mÃ©tastaseswere visualized by 99mTc-P829 scintig regard to primary tumor cells, it was not possible to analyze the raphy (Table 1), in vitro receptor binding experiments revealed ex presence of the two classes of binding sites due to the low amount of pression of 99mTc-P829 binding sites. A summary of in vivo receptor available primary tumor tissue. However, in all donors in whom the binding results is provided in Table 1. two binding classes could be analyzed, we were indeed able to detect 1856

100

n o> CM 00 50 - O. 6

0.001 0.01 0.1 1 10 100 1000 O.OOt 0.01 0.1 1 10 100 1000 unlabeled ligand (nmol/L) unlabeled ligand (nmol/L)

0.001 0.01 0.1 1 10 100 1000 0.001 O.Ot 0.1 t 10 100 1000 unlabeled ligand (nmol/L) unlabeled ligand (nmol/L)

0.001 0.01 0,1 1 10 100 1000 unlabeled ligand (nmol/L)

Fig. 3. Ability of unlabeled peptides to compete with WnTc-P829 for binding to a primary NSCLC (A: Table 2. patient IS) and a melanoma lymph node metastasis (ÃŸ.-Table 2. patient 23). as well as to COS? cells Iransfected with hSSTR2 (C). hSSTR3 (DÃ¬.orhSSTRS (Ã‰).Eachassay tube contained '""Tc-P829 (concentrations as indicated below), unlabeled peptides (O.OOI-1000 nM). and primary tumor membrane fractions or intaci COS7 cells transiently translectcd with hSSTR2. hSSTR3. or hSSTRS. Experiments with primary tumor cells were performed in triplicate (n = 1); experiments with Iransfected COS7 cells were done in duplicate or triplicate (Â«â€”6). A, in NSCLC. the 100% control value for the binding of WmTc-P829 (0.2 nM) in the absence of unlabeled agonists was 250 fmol/mg protein. The IC5(1swere I nM for P829/(ReO]-P829 (â€¢)and 2 nM for SST-14 (A). A. in a melanoma lymph node metastasis, the 100% control value for the binding of l|'""Tc-P829 (3 nM) in the absence of unlabeled agonists was 2 pmol/mg protein. The ICM,s were 4 nM for P829/(ReO|-P829 (â€¢)and 5 nw for Tyr'-OCT (â€¢).C. for COS? cells transfected with hSSTR2. the 100% control value for the binding of Wl"Tc-P829 (0.7-9.6 nM) in the absence of unlabeled agonists was 0.3-1.6 fmol/10'1 cells. The IC50s were 1 nM for P829/[ReO]-P829 (â€¢).1 nM for SST-14 (A). 3 nM for Tyr'-OCT (â€¢),and >500 nw for VIP (T). D. for COS? cells transfected with hSSTR3. the 100% control value for the binding of '""TC-P829 (0.7-5.6 nM) in the absence of unlabeled agonists was 0.7-5.5 fmol/10" cells. The IC,,,s were 1 nM for P829/[ReO]-P829 (â€¢).1 nM for SST-14 (A). 30 nM for Tyr'-OCT (â€¢),and 2 nM for VIP (V). Â£.for COS? cells transfected with hSSTRS. the 100% control value for the binding of '''""Tc-P829 (2.2-4.4 nM) in the absence of unlabeled agonists was 3.4-4.5 fmol/106 cells. The lC,,,s were 1 nM for P829/|ReO|-P829 {â€¢).1 nM for SST-14 (A). 3.5 nM for Tyr'-OCT (â€¢).and 400 nM for VIP (T).

1857

Fig. 4. 99mTc-P829 scintigraphy in a breast cancer patient (Table 1. patient I ) and in a melanoma patient (B; Table 1. patient 4). ')9mTc-P829 (500 MBq) significantly concentrated in the 2-cm primary ductal breast cancer (A, right lateral Â¡mageinprone position; matrix 128 X 128, 1 min postinjection), as well as in multiple sites of melanoma lymph node mÃ©tastases (B. right axilla; C, pelvis; matrix 128 X 128. 2 h postinjection). The tumor sites were still visible at 24 h postinjection in both patients. the two binding classes. The molecular basis of the heterogeneity of intestinal adenocarcinomas, melanomas, and most neuroendocrine the binding sites remains unknown. One attractive hypothesis is that tumors using Northern blotting (28). This observation was confirmed different types of hSSTR exhibit diverging binding affinity for"mTc- in the present study (Fig. 1, C and D). In particular, all investigated P829. In this regard, it is also noteworthy that all of the tumor tissues tumors and cell lines expressed significant amounts of hSSTR3 analyzed expressed multiple types of hSSTR at the mRNA level (see mRNA and hSSTR2 mRNA, whereas the other hSSTR subtypes were below). only weakly expressed or absent. The observation that 99mTc-P829 Previous data have shown that VIP and SST cross-compete for binds to hSSTRS is of considerable importance both in terms of its binding to tumor cell membrane receptors (2). This cross-competition potential use as a radioligand and its use as a potential radiopharma- has been observed with primary tumor cells as well as with different ceutical. tumor cell lines (2). In the present study, we asked whether the hSSTR In conclusion, our data show that 99n'Tc-P829 binds to a number of ligand P829 and VIP cross-compete for binding to primary or immor different human tumors, possibly via hSSTR2, hSSTR5, and the VIP talized tumor cells. Indeed, cross-competition between these ligands acceptor hSSTR3. Because these receptors are frequently expressed in was demonstrable, suggesting that 99nlTc-P829 identifies the common human tumors and 99mTc labeling is simple and relatively inexpensive receptor for SST and VIP (20). The next question was whether any of and exhibits desirable decay characteristics, 99mTc-P829 may be a the known hSSTR subytpes or VIPR subtypes mediates this cross- most useful tumor-imaging agent in nuclear medicine. competition. For this purpose, cDNAs encoding for hSSTR 1 through hSSTRS were transfected into COS7 cells to determine the binding specificity of the radioligand 99mTc-P829. In these experiments, the ACKNOWLEDGMENTS hSSTR subtypes 2, 3, and 5 were identified as P829 binding sites. The binding of 99mTc-P829 to hSSTR2 was displaced by Tyr3-OCT and We are grateful to Prof. B. Niederle (Department of Surgery. University of SST-14 but not by VIP. In contrast, binding of 99mTc-P829 to Vienna) for tissue supply, as well as to Dr. W. Acampa for help with the 99mTc-P829 scintigraphy. We are grateful to the technicians Martha Seif, hSSTR3 was displaced by Tyr3-OCT and SST-14, as well as by VIP. Elisabeth Hangelmann, and Qiong Yang for laboratory assistance. These data confirm that hSSTR3 is the common receptor for SST-14 and VIP and that 99mTc-P829 is a hSSTR3 ligand. One interesting aspect of our data is that 99mTc-P829 and 123I-VIP bind with higher REFERENCES affinity to hSSTR3 as compared with Tyr'-OCT. The reason for this 1. Reubi, J. C.. Maurer. K., von Werder. K.. Torhorst, J., Klijn, G. M., and Lamberts. phenomenon is not known. One possibility could be that the differ S. W. J. Somatostatin receptors in human endocrine tumors. Cancer Res., 47: ences are due to the different chemical structure of the peptides. In this 551-558, 1987. 2. Virgolini. !.. Yang. Q.. Li, S.. Angelberger, P., Neuhold. N.. Niederle, B.. respect, it is noteworthy that OCT, in contrast to VIP, binds to Scheithauer. W., and Valent, P. Cross-competition between vasoactive intestinal hSSTR2 and hSSTR5, but not to hSSTRl or hSSTR4. The inability of peptide and somatostatin for binding to tumor cell membrane receptors. Cancer Res.. hSSTRl and hSSTR4 to bind SST analogues such as OCT is consist 54: 690-700. 1994. 3. Krenning. E. P.. Kwekkeboom. D. J.. Bakker, W. H.. Breeman. W. A. P.. Kooij. ent with previous reports (for review, see Ref. 37). The alternative P. P. M.. Oei, H. Y.. van Hagen, M.. Poslerna. P. T. E., de Jong. M.. Reubi. J. C.. explanation, i.e., an ineffective transfection, seems rather unlikely, Visser. T. J.. Reijy. A. E. M.. Hofland. L. J.. KÃ¶per.J. W., and Lamberts, S. W. J. Somatostatin receptor scintigraphy with '"In-DTPA-D-Phe1 and '23I-Tyr'-oct- because Northern blotting confirmed the expression of these receptors reotide; the Rotterdam experience with more than 1000 patients. Eur. J. NucÃ.Med.. at the RNA level (hSSTRl through hSSTRS). 20: 716-731. 1993. A number of studies have recently been conducted to characterize 4. Virgolini. I.. Raderer. M.. Kurtaran. A.. Angelberger, P., Banyai, S., Yang, Q., Li, S.. hSSTR expressed on primary tumors. Using different techniques, Banyai. J.. Pidlich. J., Scheithauer. W., and Valent. P. Vasoactive intestinal peptide (VIP) receptor imaging for the localization of intestinal adenocarcinomas and endo hSSTR2 through hSSTRS were found to be expressed in primary crine tumors. N. Engl. J. Med.. 331: 1116-1121, 1994. human tumors (for a review, see Ref. 24). We have recently shown 5. Reubi. J-C. In vitru identification of vasoactive peptide receptors in human tumors; implications for tumor imaging. J. NucÃ.Med.. .io: 1846-I853, 1995. that among these receptors, hSSTR3 is frequently and strongly ex 6. Yamada, Y., Post, S. R., Wang, K., Tager, H. S., Bell, G. I., and Scino, S. Cloning pressed in a large variety of human tumors, including breast cancers, and functional characterization of a family of human and mouse somatostatin recep- 1858

lors expressed in brain, gastrointestinal tract and kidney. Proc. Nati. Acad. Sci. USA. 22. Reubi. J.. Schaer. J. C.. Waser, B.. and Mengod. G. Expression and localization of 89: 251-255. 1992. somatostatin receptor SSTR1. SSTR2. and SSTR3 mRNAs in primary human tumors 7. Yamada, Y., Reisine, S., Law, S. F., Ihara, Y.. Kubota. A.. Kagimoto, S.. Scino. M.. using in \tiu hybridization. Cancer Res., 54: 3455-3459. 1994. Seino, Y.. Bell, I., and Seino. S. Somatostatin receptors, an expanding gene family: 23. Buscail. L.. Saint-Laurent. N.. Chastre. E.. Vaillant, J. C.. Gespach. C.. Capella. G., cloning and functional characterization of human SSTR3. a protein coupled to Kalthoff, H., Luis. F., Vaysse, N., and Susini. C. Loss of sst2 somatostatin receptor adenylyl cyclase. Mol. Endocrinol.. 6.- 2136-2142, 1992. gene expression in human pancreatic and colorectal cancer. Cancer Res.. 56: 1823- 8. Yamada, Y.. Kagimoto. S.. Kubota. A.. Yasuda. K.. Masuda. K.. Someya. Y.. Ihara. 1827, 1996. Y.. Li. Q.. Imura, S., Seino, S., and Seino, Y. Cloning, functional expression and 24. Vikic-Topic. S., Raisch. K. P.. Kvols. L.. and Vuk-Pavlovic. S. Expression of pharmacological characterization of a fourth (hSSTR4) and fifth (hSSTR5) human somatostatin receptor subtype. Biochem. Biophys. Res. Commun., 195: 844-852, somatostatin receptor subtypes in breast carcinoma, carcinoid tumors, und renal cell carcinoma. J. Clin. Endocrinol. Metab.. 80: 2974-2979. 1995. 1993. 9. Yasuda, K., Res-Domiano, S.. Breder. C. A.. Law, S. F., Saper, C., Reisine, T., and 25. Virgolini, I.. Pangerl. T.. Bischof. C.. Leimer. M.. Yang. Q.. Peck-Radosavljevic. M., Bell, G. I. Cloning of a novel somatostatin receptor, SST3, coupled to adenylate Kaserer. K.. Niederle. B.. Angelberger. P.. Gangl. A., and Valent. P. Somatostatin cyclase. J. Biol. Chem.. 267; 20422-20428, 1992. (SSTI. and vasoactive intestinal peptide (VIP) receptor (R) subtype gene expression. 10. Demchyschyn. L. L.. Srikant. C. B.. Sunahara. R. K.. Kent. G.. Seeman. P.. van Toi. Eur. J. NucÃ.Med.. 23: 1101. 1996. H. H.. Panctta, R.. Palei. Y. C., and Ni/nik. H. B. Cloning and expression of a human 26. Maina. T.. Stolz. B.. Albert, R.. Bruns. C.. Koch. P.. and Maecke. H. Synthesis, somatostatin-14-selective receptor variant (somatostatin receptor 4) located on chro radiochemistry and biological evaluation of a new somatostatin analogue (SDZ mosome 20. Mol. Pharmacol.. 43: 894-901. 1993. 219-387) labeled with technetium-99m. Eur. J. NucÃ.Med., 21: 437-444. 1994. 11. Bell. G. I.. Yasada, K.. and Kong, H. Molecular biology of somatostatin receptors. 27. Krois, D., Riedel, C., Angelberger, P., Kalchbergcr. P.. Virgolini. I., and Lehner. H. Ciba Found. Symp.. 190: 65-88. 1995. Synthesis of Â¿V-a-ÃÃ³-hydrazinonicotinoylt-octreotide: a precursor of a 'iynlTc-com- 12. Comess. J. D., Demchyschyn. L. L.. and Seeman. P. A human somatostatin receptor plex. Liebigs Ann. Chem.. 1463-1469. 1996. (SSTR3). located on chromosome 22. displays preferential affinity for somalosla- 28. Mather. S. J.. and Ellison. D. Technetium-99m labeled hybrid receptor binding tin-14 like peptides. FEBS Lett.. 321: 279-284, 1993. peptides. J. NucÃ.Biol. Med.. 38: 480-481, 1994. 13. Rohrer. L.. Raulf. F.. Bruns, C., Buelter. R.. Hofstaedter. F.. and Schule. R. Cloning 29. Vallabhajosula. S.. Moyer. B. R.. Lister-James. J.. McBride. B. J., Lipszyc. H.. Lee. and characterization of a fourth human somatostatin receptor. Proc. Nati. Acad. Sci. H.. Bastidas. D.. and Dean. R. T. Preclinical evaluation of technetium-99m-lahcled USA. 90: 4196-4200. 1993. somatostatin receptor-binding peplides. J. NucÃ.Med.. 37: 1016-1022. 1996. 14. Robberecht, P.. de Neef. P., Gourlet. P.. Cauvin. A., Coy, D. H., and Christophe. 30. Cotton, F. A., and Lippard, S. J. Chemical and structural studies of the rhcnium(V) J. Pharmacological characterization of the novel helodermin/VIP receptor present in human SUP-T1 lymphoma cell membranes. Regul. Pepi.. 26: 117-126. 1989. oxyhalide complexes. Complexes from rhenium(III) bromide. Inorg. Chem.. 5: 9-16, 15. Ishihara. T.. Shigemoto. R.. Mori. K.. Takahashi, K.. and Nagata. S. Functional 1996. expression and tissue distribution of a novel receptor for vasoactive intestinal 31. Bradford. M. M. A rapid and sensitive method for the quantitation of microgram polypeptide. Neuron. S: 811-819. 1992. quantities of protein utilizing the principal of protein-dye binding. Anal. Biochcm.. 16. Lutz, E. M.. Sheward, W. J., West, K. M., Fink, G.. and Marmar. A. J. The VIP2 72: 248-254. 1976. receptor: molecular characterization of a cDNA encoding a novel receptor for 32. Virgolini, L, Sillaber, C.. Majdic, O., Sinzinger, H.. Bettelheim. K., and Valent. P. vasoactive intestinal peptide. FEBS Lett.. 334: 3-8. 1993. Characterization of prostaglandin (PG) binding sites expressed on human basophils. 17. Svoboda. M., Tastenoy. M.. van Rampelsbergh. J.. de Neef. P.. Waelbroeck. M.. and J. Biol. Chem.. 267: 12700-12708. 1992. Robberecht, O. Molecular cloning and functional characterization of a human VIP 33. Chomczynski. P.. and Sacchi, N. Single-step method of RNA isolation by acid receptor from sup-Tl lymphoblasts. Biochem. Biophys. Res. Commun.. 205: 1617- guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem.. 762: 156- 1624. 1994. 159. 1987. 18. Usdin, T. B., Bonner. T. I., and Mezey. E. Two receptors for vasoaclive intestinal 34. Sambrook. J.. Fritsch. E. F.. and Maniatis. T. Molecular Cloning: A Laboratory polypeptide with similar specificity and complementary distributions. Endocrinology. Manual. Ed. 2, p. 740. Cold Spring Harbor. NY: Cold Spring Harbor Laboratory. 135: 2662-2680, 1994. 1989. 19. Couvineau, A., Rouyer-Fessard, C.. Darmoul, D., Maoret, J. J., Carrero, I., Ogier- 35. Scatchard, G. The attractions of proteins for small molecules and ions. Ann. NY Denis. E., and Laburthe, M. Human intestinal VIP receptor: cloning and functional Acad. Sci.. 51: 660-672, 1949. expression of two cDNA encoding proteins with different N-terminal domains. Biochem. Biophys. Res. Commun.. 200: 769-776. 1994. 36. Virgolini, L, Angelberger, P., Li, S., Yang. Q.. Kurlaran. A.. Raderer. M. Neuhold. N.. 20. Virgolini. I.. Kurtaran. A.. Lcimer. M.. Kaserer. K.. Peck-Radosavljevic. M.. Kaserer. K.. Leimer. M.. Peck-Radosavljevic. M.. Scheilhauer. W.. Niederle. B.. Eichler. H. G.. and Valent. P. In vitro and in vivo studies on three radiolaheled Angelberger. P.. HÃ¼bsch.P.. Dvorak. M.. Valent. P.. and Niederle. B. Visualization somatostatin analogues: 121l-octreolide (OCT). '"I-Tyr'-OCT and '"in-DTPA-D- of a VIPoma by vasoactive intestinal peptide receptor scintigraphy. J. NucÃ.Med.. in press, 1997. Phe 1-OCT. Eur. J. NucÃ.Med.. 23: 1388-1399. 19%. 21. Virgolini, I., Pangerl. T.. Bischof. C.. Smith-Jones. P.. and Pcck-Radosavljcvic. M. 37. Lamberts, S. W. J.. Bakker. W. H.. Reubi. J. C.. and Krenning. E. P. Somatostatin- Somatostatin receptor subtype expression in human tissues: a prediction for diagnosis receptor imaging in the localization of endocrine tumors. N. Engl. J. Med.. 323: and treatment of cancer? Eur. J. Clin. Invest., 27: 645-647, 1997. 1246-1249. 1990.

1859

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1998 American Association for Cancer Research. Somatostatin Receptor Subtype Specificity and in Vivo Binding of a Novel Tumor Tracer, 99mTc-P829

Irene Virgolini, Maria Leimer, Hirsch Handmaker, et al.

Cancer Res 1998;58:1850-1859.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/58/9/1850

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/58/9/1850. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.