immunoscintigraphic detection of the ed-b domain of ... · advances in brief immunoscintigraphic...

Advances in Brief

Immunoscintigraphic Detection of the ED-B Domain of Fibronectin,a Marker of Angiogenesis, in Patients with Cancer1

Monica Santimaria, Giovanni Moscatelli,Giuseppe L. Viale, Leonardo Giovannoni,Giovanni Neri, Francesca Viti,Alessandra Leprini, Laura Borsi,Patrizia Castellani, Luciano Zardi,2 Dario Neri,and Pietro RivaServizio di Medicina Nucleare, Ospedale M. Bufalini, 47023 Cesena,Italy [M. S., G. M., P. R.]; Division of Neurosurgery Di.S.C.A.T.Department of Surgery, University of Genoa, Medical School, 16132Genoa, Italy [G. L. V.]; Philogen S.r.l., 53100 Siena, Italy [L. G.,G. N., F. V., A. L.]; Istituto Nazionale per la Ricerca sul Cancro,16132 Genova, Italy [L. B., P. C., L. Z.]; Institute of PharmaceuticalSciences, Swiss Federal Institute of Technology Zurich, CH-8057Zurich, Switzerland [D. N.]; and Istituto Oncologico Romagnolo,Forlı, Italy [P. R.]

AbstractPurpose: ED-B fibronectin is expressed only during

angiogenic processes and in tissues undergoing growthand/or extensive remodeling. We demonstrated previouslythe possibility to target and selectively deliver therapeuticsubstances to tumor vasculature in experimental animalmodels using a human recombinant antibody fragment, L19,specific for the ED-B domain of fibronectin. Here we eval-uate the possibility of targeting primary tumors and meta-static lesions in cancer patients through immunoscintigra-phy using 123I-labeled dimeric L19 [L19(scFv)2].

Experimental Design: Twenty patients (34–79 years ofage) with lung, colorectal, or brain cancer, whose tumorshad been confirmed by imaging techniques and/or histolog-ically, were admitted to the immunoscintigraphic investiga-tion.

Results: The dimeric L19 antibody selectively localizedin tumor lesions in aggressive types of lung cancer andcolorectal cancer. Because ED-B fibronectin is expressedonly during angiogenic processes and in tissues undergoinggrowth and/or extensive remodeling, L19(scFv)2 is able todistinguish between quiescent and actively growing lesions.No side effects were observed.

Conclusions: The ability of L19(scFv)2 to target tumorsin patients provides the foundations for new therapeuticapplications, in which the L19 antibody is engineered toselectively deliver bioactive molecules to primary tumors aswell as to metastases.

IntroductionAngiogenesis, i.e., the proliferation of new blood vessels

from pre-existing ones, is a characteristic feature of aggressivesolid tumors and other relevant disorders, such as age-relatedmacular degeneration, diabetic retinopathy, and rheumatoid ar-thritis (1–3). The switch of solid tumors from a poorly vascu-larized state to a condition in which exuberant angiogenesisprovides tumor cells with oxygen and nutrients often corre-sponds to the onset of a more aggressive phenotype (4).

The noninvasive imaging of angiogenesis and tissue re-modeling processes in vivo would prove to be dramaticallyadvantageous over current methods, because it would yieldinformation about both the location and the growth dynamics oflesions. Because neovasculature and tissue remodeling are re-quired for the growth of all aggressive solid tumors, the sameimaging approach could be used for different types of cancer.Furthermore, the proven ability of a molecule (e.g., a humanmonoclonal antibody fragment) to selectively localize in newblood vessels or actively remodeling tissues would herald anumber of therapeutic strategies in which bioactive compoundsare selectively targeted to angiogenic sites (5, 6).

Imaging of angiogenesis in animal models and in patientswith cancer has been attempted, using computed tomography,MRI, ultrasound, and scintigraphic techniques, to assesschanges in vascular permeability and tumor blood flow duringantiangiogenic therapy (7). However, a quantitative distinctionbetween vascularity and angiogenesis would be expedient, be-cause a number of benign tumors are known to be highlyvascular despite a low rate of new blood vessel formation (8).Imaging of angiogenesis using molecular targeting agents (e.g.,antibody fragments) may provide the means to achieve this goal.

Specific ligands of the integrin �v�3 have been used forimaging of angiogenesis in preclinical models (9, 10), but re-sults from an immunoscintigraphic clinical trial with the human-ized anti�v�3 antibody Vitaxin has thus far been disappointing(11). We focused our attention on the ED-B3 of fibronectin, amarker of angiogenesis and tissue remodeling (8, 12–15). Thissequence of 91 amino acids, identical in mouse, rat, rabbit, dog,and humans, can be inserted into the fibronectin molecule by amechanism of alternative splicing at the level of the primaryfibronectin transcript. Fibronectin containing ED-B (B-FN) ac-

Received 5/9/02; revised 7/29/02; accepted 8/21/02.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 Supported in part by the Italian Association for Cancer Research(AIRC), and the European Union Project QLK3-CT-2001-01495.2 To whom requests for reprints should be addressed, at Laboratorio diBiologia Cellulare, Istituto Nazionale per la Ricerca sul Cancro, LargoRosanna Benzi 10, 16132 Genova, Italy. Phone: 39-010-5737371; Fax:39-010-352855; E-mail: [email protected].

3 The abbreviations used are: ED-B, extra-domain B; 123I, iodine-123;SPECT, single-photon emission computerized tomography; CT, com-puterized tomography; MRI, magnetic resonance imaging.

571Vol. 9, 571–579, February 2003 Clinical Cancer Research

Research. on May 18, 2020. © 2003 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

http://clincancerres.aacrjournals.org/

cumulates around neovascular structures in aggressive tumorsand other tissues undergoing angiogenesis and remodeling, suchas neoplasia, some ocular structures in pathological conditions,and fetal tissues, but is otherwise undetectable in normal adulttissues with the exception of the female reproductive system,where tissue remodeling and angiogenesis are recurrent physi-ological processes.

To date, the production of monoclonal antibodies directlyrecognizing the ED-B domain in B-FN has not been possibleusing hybridoma technology because of tolerance. We haveovercome this problem, using large synthetic antibody reper-toires (16–19), in combination with phage display (20, 21) oriterative colony filter screening (22). Antibodies specific toB-FN selectively target the neovasculature in vivo, as shown intumor-bearing mice (23–26) and in rabbit models of ocularangiogenesis (27), thus underpinning the possibility to selec-tively deliver therapeutic molecules to new blood vessels. ThescFv(L19) antibody fragment, with picomolar affinity for theED-B domain (21), was chemically coupled to a photosensitizer,and was shown to mediate the selective and complete occlusionof ocular neovasculature in a rabbit model after irradiation withnear infrared light (27). Nilsson et al. (28) reported that thefusion protein composed of L19 and the extracellular domain oftissue factor mediates the selective thrombosis of new bloodvessels in different types of murine tumor models. Furthermore,scFv(L19) has been shown recently to dramatically increase thetherapeutic index of cytokines when used to deliver these to thetumor neovasculature (29, 30).

In this article we report on the immunoscintigraphic find-ings obtained using the noncovalent homodimeric form of L19scFv [L19(scFv)2] labeled with the �-emitter 123I in 20 patientswith cancer.

Patients and MethodsAntibody Preparation, Immunohistochemistry, and

Antibody Radioiodination. The scFv(L19) antibody frag-ment was produced in the supernatant of Escherichia coli asdescribed (24), according to the guidelines for preparation ofrecombinant DNA products in Phase I trials in patients withCancer of the Cancer Research Campaign (31–33). In short, theantibody was affinity purified from the bacterial supernatantona ED-B-Agarose resin (24), followed by dialysis in 10 mM

HEPES (pH 7.0) at 1 mg/ml concentration; the resulting proteinsolution mainly contained the noncovalent homodimer of thescFv that was separated from the monomer by cation exchangechromatography on a Resource S column (Amersham Pharma-cia, Uppsala, Sweden). The L19(scFv)2 gave a single band inSDS-PAGE loading up to 20 �g/lane, and was a pure nonco-valent homodimer, as judged by size-exclusion chromato-graphy. Purity, immunoreactivity, identity, and absence ofendotoxins were additionally tested by chromatography, matrix-assisted desorption ionization-time of flight, ELISA, BIAcore,and Limulus amoebocyte lysate, as well as in tumor-targetingexperiments in tumor-bearing mice and in a rabbit-based pyro-gen test. Absence of acute toxicity at doses �10-fold higherthan the ones used in patients was assessed in mice, rats, andguinea pigs, under good laboratory practice conditions. Thefinal purified antibody bulk was pooled, sterile filtered, and

frozen in aliquots in GMP conditions by Bioreliance (Stirling,Scotland) at A280 � 1.14 in saline solutions.

Immunohistochemical studies were performed as described(8, 20), using 5-�m cryostat sections of freshly frozen tumorspecimens, including samples from 2 patients with newly diag-nosed lesions, who had been imaged previously with 123I-L19(scFv)2.

Immediately before the scintigraphic studies, theL19(scFv)2 antibody was labeled with 123I (Sorin Amersham,Saluggia, Italy), using the chloramine-T method (31). The ra-dioiodinated antibody was purified from the reaction mixture bygel filtration (PD-10 columns equilibrated with saline solution;Amersham Pharmacia Biotech) and 0.22-�m sterile filtered.

The 123I incorporation ranged between 70% and 90%, asjudged by radioactive counting of the PD-10 eluate, thin layerchromatography, and high-performance liquid chromatographygel-filtration. In all of the cases tested, the immunoreactivitywas �85%, as measured by affinity chromatography on anED-B resin, as described (27), with a single exception where itwas �50%.

Immunoscintigraphic Procedures. Twenty patients(34–79 years of age) with brain, lung, or colorectal cancer [2brain tumors: 1 pylocitic astrocytoma, 1 glioblastoma multi-forme; 16 lung cancers: 7 squamous cell carcinomas, 4 smallcell carcinomas (2 of which with liver metastases), 1 large cellanaplastic carcinoma, 3 adenocarcinomas (1 of which bromchi-oloalveolar), 1 sarcoma; and 2 colorectal carcinomas with livermetastases], whose tumor had been confirmed by imaging tech-niques and/or histologically, were admitted to the immunoscin-tigraphic investigation after giving their informed consent. Onemg of 123I- L19(scFv)2 (5–14 mCi) in 10 ml of 0.9% sodiumchloride was administered i.v. over 2 min, followed by flushingwith 0.9% sodium chloride. The planar images were obtained 4and 24 h after the i.v. infusion of the radiolabeled monoclonalantibody. A computer-assisted, large field of view GE gammacamera, equipped with a low energy and high-resolution colli-mator, was used. Brain, chest, abdomen, and pelvis, in anteriorand posterior view, were imaged by collecting 300 k counts.SPECT examination was carried out by using the same gammacamera. If appropriate, images at other time points were col-lected. In hospitalized patients the thyroid uptake of possiblefree 123I was prevented by administration of potassium perchlo-rate (Pertiroid; PIAM, Genoa, Italy), 400 mg three times a daystarting on day �4 and continuing till day 1. The study wasperformed according to good clinical practice standards, incompliance with the Italian Decreto Legislativo 26 May 2000, n.187, regulating clinical investigations, which follows the Euro-pean Community guideline 97/43/EURATOM. Immunoscinti-graphic studies with scFv(L19) received authorization number:800/II/I.27.15/1172 of the Italian Ministry of Health. The doseto the target tumor and to major organs was calculated accordingto the methodology described previously (34).

ResultsExpression of B-FN in Different Tumor Types. Exten-

sive immunohistochemical analyses of B-FN expression in tu-mors have been described elsewhere, and practically all of thesolid tumors show the presence of ED-B (8, 13–15, 35, 36). The

572 Tumor Imaging by Recombinant Antibodies



tumor types that, based on immunohistochemical findings, nor-mally show a particularly abundant presence of ED-B are lungcancers, high-grade astrocytomas (37), and liver metastases. Bycontrast, ED-B was constantly undetectable in normal lung,brain, and liver specimens.

Fig. 1 shows the immunohistochemical findings for someof the tumor types that were investigated in this study. A strikingcontrast in ED-B staining can be observed between aggressivebrain tumors (e.g., glioblastoma; Fig. 1a), where practically allof the blood vessels reacted positively with the anti-ED-Bantibody, and low-grade astrocytomas (e.g., pilocytic astrocy-toma; Fig. 1b) where ED-B was undetectable, regardless of thevascular density (8). Fig. 1d shows a section from a specimen ofa lung squamous carcinoma stained using the L19(scFv)2; astrong and diffuse staining of the tumor stroma is clearly visible.

Fig. 1e shows a section of a large cell anaplastic lung carcinomastained with the L19(scFv)2: it is easier to observe ED-B-positive vascular structures here, because the connective com-ponents of the tumor stroma in this tumor type are less abundantcompared with squamous lung cancer. By contrast, no ED-Bstaining could be detected in normal lung specimens (Fig. 1f).We also investigated liver metastases of colorectal cancer, andfound strong positive staining using the recombinant antibody toED-B (Fig. 1c). These immunohistochemical findings promptedus to initiate our immunoscintigraphic trial in patients with lungcancer, cerebral glioma, or liver metastasis.

Antibody Radiolabeling and Fractional Blood Clear-ance. L19(scFv)2 was radiolabeled with 123I and given i.v. to20 patients with brain, lung, or colorectal cancer. The injecteddose of 123I-labeled L19(scFv)2 ranged from 5 to 14 mCi

Fig. 1 Immunohistochemical findings in sections of human tumor specimens, stained (in red) with an anti-ED-B antibody and counterstained withhematoxylin as described in “Materials and Methods.” a, glioblastoma; b, pylocytic astrocytoma: the clusters of cells are vascular structures that reactwith antifactor VIII antibodies (data not shown) but are completely negative for ED-B; c, liver metastasis from a colon carcinoma; d, squamous lungcarcinoma, with a diffuse staining of the tumor stroma; e, large cell anaplastic lung carcinoma (vascular structures are visible and strongly positivefor ED-B); f, section of normal lung, showing no reaction with the anti-ED-B antibody.

573Clinical Cancer Research



(185–518 MBq) corresponding to 1–1.5 mg of protein with asingle exception where the injected dose was 0.4 mg.

A biphasic clearance profile of radioiodinated L19(scFv)2

from blood was observed. Fitting of the curve with a biexpo-nential function yielded half-lives of 35 min (� phase, account-ing for 86% of the injected dose) and 5.2 h (� phase, accountingfor 14% of injected dose). Antibody clearance was mediatedprincipally by the kidneys, as determined by counting of urinesamples. Begent et al. (31) reported previously that the clear-ance of a radiolabeled scFv anticarcinoembryonic antigen in-jected in patients was kidney mediated. However, Begent et al.(31) used a monomeric scFv (27 kDa), whereas here we used thehomodimeric L19(scFv)2 of �57 kDa. Size exclusion chroma-tography analysis of blood samples at different time pointsshowed that �80% of radioactivity in blood was associated withradiolabeled L19 (scFv)2 (85% at 3.5 h; 84% at 6.5 h; and 82%at 22 h), and immunoreactivity was �50% at 3 h after injection.

Immunoscintigraphic Study. A total of 20 cancer pa-tients were injected with 123I-radiolabeled L19(scFv)2. All ofthe patients tolerated the scFv injection well without showingside effects. No early or late allergic reaction was observed. Thehematological parameters were not affected, and no adverseeffects according to common toxicity criteria were seen (38).Sixteen of 20 patients showed different levels of antibody ac-cumulation either in the primary tumors or metastases. The fourcompletely negative scans were from patients with a lung sar-coma, a bronchioloalveolar carcinoma, a squamous cell lungcarcinoma, and a low-grade pylocitic astrocytoma. The negativeresults of the low-grade astrocytoma were expected, becausethis type of tumor does not express ED-B. We have no expla-nation at the moment for the 3 other negative patients, other thanthe possibility that the tumors were in a quiescent phase.

Fig. 2 shows representative anterior planar scans, at 18 h,of the thorax of patients injected with 123I-labeled L19(scFv)2.

Fig. 2a shows the scan of a patient with liver metastases ofcolorectal cancer that typically exhibited a strong and selectiveantibody uptake in the liver lesions. Fig. 2b shows the scan ofthe thorax of a small cell lung carcinoma patient with a miliaryinvolvement of both lungs, revealing a diffuse accumulation ofthe radiolabeled L19(scFv)2 in both lungs.

Brain Tumors. The SPECT analysis of Fig. 3a shows astrong and selective accumulation of 123I-labeled L19(scFv)2 inthe tumor mass of a patient with recurrent glioblastoma, grow-ing within the postoperative cavity and in the adjacent tissue(Fig. 3b). By contrast, Fig. 3c shows no selective accumulationin the brain of a patient with a benign brain tumor (pylocyticastrocytoma), which could be removed only partially by brainsurgery (Fig. 3d). The differences in antibody uptake correlatedwith the different levels of antigen expression always found inthese pathologies, as exemplified in Fig. 1, a and b, and with thedifferences in the integrity of the blood-brain barrier in thesetumor types (39).

Lung Cancer. Fig. 4 shows SPECT images obtained 6 hafter injection of radioiodinated L19(scFv)2 in a patient with anewly diagnosed large cell anaplastic lung carcinoma who hadnot received chemotherapy previously. A selective antibodyaccumulation in the tumor lesion was clearly detectable. Thetumor size was estimated to be 5 7 5.5 cm (on the basis ofSPECT images), and was consistent with the tumor diameterestimate obtained by CT. Immunohistochemistry of the tumor,after surgical removal, confirmed a strong and diffuse ED-Bexpression in the tumor stroma, which is rich in vascular struc-tures (Fig. 1e).

Liver Metastases. Fig. 5 shows an array of SPECTscans from patients who had undergone surgery for removalof the primary tumor and who later presented with bulky livermetastases. Fig. 5a shows a selective antibody accumulationin a large (8 cm) liver metastasis of a colon carcinoma 21 h

Fig. 2 Anterior planar images of the thorax, recorded 18 h after injection of 123I-L19(scFv)2, in patients with (a) colorectal cancer with livermetastases and (b) small cell lung carcinoma with a miliary involvement of both lungs.




after injection. The lesion was clearly detectable already 6 hafter injection, with tumor:background ratios increasing at21 h. Tumor:normal tissue and tumor:liver ratios were 3.7and 1.6 at 6 h, and 9.2 and 4.6 at 21 h, respectively. At thistime point, the antibody dose delivered to the tumor wasgreater than for all of the other organs, including kidneys.Fig. 5b shows the immunoscintigraphic detection of a livermetastasis from a small cell lung carcinoma, obtained 6 hafter injection. Fig. 5, c and d, show SPECT images of largeliver metastases in the same patient, recorded 6 h afterinjection. A strong and selective antibody accumulation isvisible in the peripheral part of the lesion but not in thenecrotic center of the tumor mass, which yielded a charac-teristic doughnut-shape staining pattern. At this time point,tumor:normal tissue (soft tissue of the shoulder) and tumor:nontumoral part of the liver ratios were 4.8 and 1.9, respec-tively, calculated as reported previously (34).

DiscussionThe tumor targeting performance of L19(scFv)2, a human

antibody fragment with identical affinity for the ED-B domainof fibronectin from different animal species (21), was assessed

previously by quantitative biodistribution analysis in a numberof murine tumor models (24–26). The results presented in thisarticle show the ability of L19(scFv)2, with its low molecularweight and its rapid renal clearance, to efficiently localize inaggressive primary tumors as well as metastases in patients withcancer. Accumulation of L19(scFv)2 in tumor lesions contrastswith the pharmacokinetic behavior of most chemotherapeuticdrugs, which typically exhibit tumor:normal organ ratios as lowas 1:10–1:20 at different time points after i.v. injection (40).Moreover, L19(scFv)2 demonstrated an impressive ability todeliver bioactive agents (procoagulant factors, cytokines, cyto-toxic agents, radionuclides, and photosensitizers) to tumorsin syngeneic animal models, with dramatic therapeutic results(27–30).

This study, carried out on 20 patients, the majority ofwhom had colorectal or lung cancer, clearly demonstrates thatthe antibody L19(scFv)2 selectively localized in patient tumors.This observation offers a number of important diagnostic andtherapeutic prospects. Because the antigen recognized by theL19 recombinant antibody is always associated with angiogen-esis and tissue remodeling, it is possible to obtain informationon the growth potential of the lesion with a noninvasive proce-

Fig. 3 Localization of 123I-L19(scFv)2 in brain tumors. SPECT gamma camera transaxial section (a) and MRI (b), from a patient with a recurrentglioblastoma lesion growing around the postoperatory cavity. SPECT transaxial scans (c) and CT (d) from a patient with a low-grade pilocyticastrocytoma, which could be removed only subtotally by surgery. Residual tumor tissue adjacent to the brain stem is indicated by the arrow (d). CThad to be performed for this patient, because a metallic implant prevented MRI.




dure, and relatively simple and commonly used tools. At pres-ent, this same kind of information is available only with sophis-ticated and expensive means such as positron emissiontomography. Thus, immunoscintigraphy using L19(scFv)2 canprovide important diagnostic information in the follow-up oflow-grade (ED-B-negative) astrocytomas, which may switch toanaplastic astrocytomas that express large amounts of ED-B.The ability of 123I-labeled L19(scFv)2 to image high-grade, butnot low-grade, astrocytomas indicates an avenue for the nonin-vasive discrimination between these two classes of brain tumors.Furthermore, immunoscintigraphy with L19(scFv)2 may proveuseful in differentiating between postsurgery reactions, such asfibrosis, and tumor recurrence in a number of cancers, includinglung cancer, where differential diagnosis is often impossibleusing classic radiodiagnostic procedures. We observed throughimmunohistochemistry4 that lymph nodes infiltrated by neoplas-tic cells show the presence of ED-B, whereas the noninfiltratedlymph nodes do not. Therefore, immunoscintigraphy using ra-diolabeled L19(scFv)2 would allow the noninvasive distinctionbetween the two. Furthermore, the use of L19(scFv)2 couldintegrate approaches used for the identification of “sentinellymph nodes” in breast cancer patients.

A particularly high accumulation of radiolabeledL19(scFv)2 was observed in hepatic metastatic lesions. If

similar results were to be found in hepatocarcinomas, thisotherwise untreatable cancer might well benefit from diag-nostic and/or therapeutic approaches based on the use ofradiolabeled L19(scFv)2. At the moment we have no evi-dence that immunoscintigraphy using L19(scFv)2 can iden-tify micrometastases that are undetectable with current stand-ard radiological procedures: the smallest lesion detected thusfar is a subcentimetric liver metastasis (4 – 6 mm). However,the results of this study seem to suggest that L19(scFv)2

would be able to detect the more aggressive micrometastases,regardless of their size.

In view of the large number of ongoing clinical trialsinvestigating antiangiogenic substances and in view of the an-tiangiogenic activity of most cytotoxic anticancer drugs (41,42), the repeated imaging with 123I-labeled L19(scFv)2 mightallow the better follow-up of patient response to treatment. Theintroduction of novel gamma cameras has lead to noteworthyimprovements in immunoscintigraphic detection as well as theuse of different radioisotopes (43–45). The use of positronemission tomography with suitable nuclides may enhance res-olution and sensitivity, allowing a better three-dimensional lo-calization of the tumor and more reliable quantitations. Further-more, because ED-B is a pan-tumoral marker, new clinical trialson selected tumor types are necessary and are about to bestarted. The clinical studies presented in this article provide astrong rationale and a convincing incentive to rapidly introduceL19(scFv)2-based therapeutic fusion proteins into cancer clini-cal trials.

4 P. Castellani, E. Balza, L. Borsi, B. Carnemolla, D. Neri, and L. Zardi,unpublished observations.

Fig. 4 Localization of 123I-L19(scFv)2 in a pa-tient with lung adenocarcinoma. SPECT images,obtained 6 h after injection, showing thetransaxial, sagittal, and coronal projection of thethorax area of a patient with adenocarcinoma ofthe lung, were matched to the CT scan of thethorax of the same patient.




AcknowledgmentsWe thank Thomas Wiley for manuscript revision.

References1. Folkman, J. Angiogenesis in cancer, vascular, rheumathoid and otherdisease. Nat. Med., 1: 27–31, 1995.2. Carmeliet, P. Mechanisms of angiogenesis and arteriogenesis, Nat.Med., 6: 389–395, 2000.

3. Ferrara, N., and Alitalo, K. Clinical applications of angiogenicgrowth factors and their inhibitors. Nat. Med., 5: 1359 –1364,1999.4. Hanahan, D., and Folkman, J., Patterns and emerging mechanisms ofthe angiogenic switch during tumorigenesis. Cell, 86: 353–564, 1998.5. Thorpe, P. E., and Ran, S., Tumor Infarction by Targeting TissueFactor to Tumor Vasculature. Cancer J., 66: 237–244, 2000.6. Halin, C., Zardi, L., and Neri, D. Antibody-based targeting of an-giogenesis. News Physiol. Sci., 16: 191–194, 2001.

Fig. 5 Localization of 123I-L19(scFv)2 in bulky liver metastases from patients with colorectal cancer (a) or lung cancer (b–d). a, CT scan and SPECTimages, obtained 21 h after antibody injection, showing intersecting transaxial, sagittal, and coronal projection of the abdomen. The liver metastasisis indicated with arrowheads in the CT scan. B, CT scan and SPECT images (6 h) of a liver metastasis in a patient with small cell lung carcinoma.c and d, CT scan and SPECT images, obtained 6 h after antibody injection, showing intersecting transaxial, sagittal, and coronal projection of theabdomen, from a patient with small cell lung carcinoma with multiple liver metastases.




7. Weber, W. A., Haubner, R., Vabuliene, E., Kuhnast, B., Wester,H. J., and Schwaiger, M. Tumor angiogenesis targeting using imagingagents. Q. J. Nucl. Med., 45: 179–182, 2001.

8. Castellani, P., Viale, G., Dorcaratto, A., Nicolo, G., Kaczmarek, J.,Querze, G., and Zardi. L The fibronectin isoform containing the ED-Boncofetal domain: a marker of angiogenesis. Int. J. Cancer, 59: 612–618, 1994.

9. Haubner, R., Wester, H. J., Weber, W. A., Mang, C., Ziegler, S. I.,Goodman, S. L., Senekowitsch-Schmidtke, R., Kessler, H., andSchwaiger, M. Noninvasive imaging of �(v) �3 integrin expressionusing 18F-labeled RGD-containing glycopeptide and positron emissiontomography. Cancer Res., 61: 1781–1785, 2001.

10. Sipkins, D. A., Cheresh, D. A., Kazemi, M. R., Nevin, L. M.,Bednarski, M. D., and Li, K. C. Detection of tumor angiogenesis in vivoby �V�3-targeted magnetic resonance imaging. Nat. Med., 4: 623–626,1998.

11. Posey, J. A., Khazaeli, M. B., DelGrosso, A., Saleh, M. N., Lin,C. Y., Huse, W., and LoBuglio, A. F A pilot trial of Vitaxin, ahumanized anti-vitronectin receptor (anti � v � 3) antibody in patientswith metastatic cancer. Cancer Biother. Radiopharm., 16: 125–132,2001.

12. Halin, C., and Neri, D. Antibody-based targeting of angiogenesis.Crit. Rev. Ther. Drug Carrier Syst., 18: 299–339, 2001.

13. Kaczmarek, J., Castellani, P., Nicolo’, G., Spina, B., Alemanni, G.,and Zardi, L. Distribution of oncofetal fibronectin isoforms in normal,hyperplastic and neoplastic human breast tissues. Int. J. Cancer, 58:11–16, 1994.

14. Scarpino, S., Stoppacciaro, A., Pellegrini, C., Marzullo, A., Zardi,L., Tartaglia, F., Viale, G., and Ruco. L. P Expression of EDA/EDBisoforms of fibronectin in papillary carcinoma of the thyroid. J. Pathol.,188: 163–167, 1999.

15. Karelina, T. V., and Eisen, A. Z. Interstitial collagenase and theED-B oncofetal domain of fibronectin are markers of angiogenesis inhuman skin tumors. Cancer. Detect. Prev., 22: 438–444, 1998.

16. Viti, F., Nilsson, F., Demartis, S., Huber, A., and Neri, D. Designand use of phage display libraries for the selection of antibodies andenzymes. Methods Enzymol., 326: 480–505, 2000.

17. Rader, C., and Barbas, C. F., III. Phage display of combinatorialantibody libraries. Curr. Opin. Biotechnol., 8: 503–508, 1997.

18. Winter, G., Griffiths, A. D., Hawkins, R. E., and Hoogenboom,H. R. Making antibodies by phage display technology. Annu. Rev.Immunol., 12: 433–455, 1994.

19. Hoogenboom, H. R., Henderikx, P., and de Haard, H. Creating andengineering human antibodies for immunotherapy. Adv. Drug Deliv.Res., 31: 5–31, 1998.

20. Carnemolla, B., Neri, D., Castellani, P., Leprini, A., Neri, G., Pini,A., Winter, G., and Zardi, L. Phage antibodies with pan-species recog-nition of the oncofoetal angiogenesis marker fibronectin ED-B domain.Int. J. Cancer, 68: 397–405, 1996.

21. Pini, A., Viti, F., Santucci, A., Carnemolla, B., Zardi, L., Neri, P.,and Neri, D. Design and use of a phage display library. Human anti-bodies with subnanomolar affinity against a marker of angiogenesiseluted from a two-dimensional gel. J. Biol. Chem., 273: 21769–21776,1998.

22. Giovannoni, L., Viti, F., Zardi, L., and Neri, D. Isolation of anti-angiogenesis antibodies from a large combinatorial repertoire by colonyfilter screening. Nucleic Acids Res., 29: e27, 2001.

23. Neri, D., Carnemolla, B., Nissim, A., Leprini, A., Querze, G.,Balza, E., Pini, A., Tarli, L., Halin, C., Neri, P., Zardi, L., and Winter,G. Targeting by affinity-matured recombinant antibody fragments of anangiogenesis associated fibronectin isoform. Nat. Biotechnol., 15:1271–1275, 1997.

24. Viti, F., Tarli, L., Giovannoni, L., Zardi, L., and Neri, D. Increasedbinding affinity and valence of recombinant antibody fragments lead toimproved targeting of tumoral angiogenesis. Cancer Res., 59: 347–352,1999.

25. Tarli, L., Balza, E., Viti, F., Borsi, L., Castellani, P., Berndorff,D., Dinkelborg, L., Neri, D., and Zardi. L A high-affinity humanantibody that targets tumoral blood vessels. Blood, 94: 192–198,1999.

26. Demartis, S., Tarli, L., Borsi, L., Zardi, L., and Neri, D. In vivotargeting of tumour neo-vasculature by a radiohalogenated human an-tibody fragment specific for the ED-B domain of fibronectin. Eur.J. Nucl. Med., 28: 534–539, 2001.

27. Birchler, M., Viti, F., Zardi, L., Spiess, B., and Neri, D. Selectivetargeting and photocoagulation of ocular angiogenesis mediated by aphage-derived recombinant antibody. Nat. Biotechnol., 17: 984–988,1999.

28. Nilsson, F., Koshmehl, H., Zardi, L., and Neri, D. Targeted deliveryof tissue factor to the ED-B domain of fibronectin, a marker of angio-genesis, mediates the infarction of solid tumours in mice. Cancer Res.,61: 711–716, 2001.

29. Carnemolla, B., Borsi, L., Balza, E., Castellani, P., Meazza, R.,Berndt, A., Ferrini, S., Kosmehl, H., Neri, D., and Zardi, L. Enhance-ment of the anti-tumor properties of interleukin-2 by its targeted deliv-ery to the tumor blood vessel extracellular matrix. Blood, 99: 1659–1665, 2002.

30. Halin, C., Rondini, S., Nilsson, F., Berndt, A., Kosmehl, H., Zardi,L., and Neri. D. Enhancement of the anti-tumor activity of interleu-kin-12 by targeted delivery to neo-vasculature. Nat. Biotechnol., 20:264–269, 2002.

31. Begent, R. H., Verhaar, M. J., Chester, K. A., Casey, J. L.,Green, A. J., Napier, M. P., Hope-Stone, L. D., Cushen, N., Keep,P. A., Johnson, C. J., Hawkins, R. E., Hilson, A. J., and Robson, L.Clinical evidence of efficient tumor targeting based on single-chainFv antibody selected from a combinatorial library. Nat. Med., 2:979 –984, 1996.

32. Begent, R. H., Chester, K. A., Connors, T., Crowther, D., Fox, B.,Griffiths, E., Hince, T. A., Ledermann, J. A., McVie, J. G., Minor, P.,and et al. Cancer Res. Campaign operation manual for control recom-mendations for products derived from recombinant DNA technologyprepared for investigational administration to patients with cancer inphase I trials. Eur. J. Cancer, 29A: 1907–1910, 1993.

33. Mayer, A., Chester, K. A., Bhatia, J., Pedley, R. B., Read, D. A.,Boxer, G. M., and Begent, R. H. Exemplifying guidelines for prepara-tion of recombinant DNA products in phase I trials in cancer: prepara-tion of a genetically engineered anti-CEA single chain Fv antibody. Eur.J. Cancer, 34: 968–976, 1998.

34. Riva, P., Franceschi, G., Frattarelli, M., Lazzari, S., Riva, N.,Giuliani, G., Casi, M., Sarti, G., Guiducci, G., Giorgetti, G., Gentile, R.,Santimaria, M., Jermann, E., and Maeke, H. R. Loco-regional radioim-munotherapy of high-grade malignant gliomas using specific mono-clonal antibodies labeled with 90Y: a phase I study. Clin. Cancer Res.,5(10 Suppl): 3275s–3280s, 1999.

35. Kosmehl, H., Berndt, A., Strassburger, S., Borsi, L., Rousselle, P.,Mandel, U., Hyckel, P., Zardi, L., and Katenkamp, D. D. Distribution oflaminin and fibronectin isoforms in oral mucosa and oral squamous cellcarcinoma. Br. J. Cancer., 81: 1071–1079, 1999.

36. Berndt, A., Borsi, L., Luo, X., Zardi, L., Katenkamp, D., andKosmehl, H. Evidence of ED-B fibronectin synthesis in human tissuesby non-radioactive RNA in situ hybridization. Investigations on carci-noma (oral squamous cell and breast carcinoma), chronic inflammation(rheumatoid synovitis) and fibromatosis (Morbus Dupuytren). Histo-chem. Cell Biol., 109: 249–55, 1998.

37. Castellani, P., Borsi, L., Carnemolla, B., Biro, A., Dorcaratto, A.,Viale, G. L., Neri, D., and Zardi, L. Differentiation between high- andlow-grade astrocytoma using a human recombinant antibody to the extradomain-B of fibronectin. Am. J. Pathol., 161: 1695–1700, 2002.

38. Operation manual for control of production, preclinical toxicologyand phase I trials of antitumour antibodies and drug antibodies conju-gates. Prepared by a Joint Committee of the Cancer Research CampaignNational Institute for Biological Standards and Control. Br. J. Cancer,54: 557–668, 1986.




39. Del Sole, A., Falini, A., Ravasi, L., Ottobrini, L., De Marchis, D.,Bombardieri, E., and Lucignani, G. Anatomical and biochemical inves-tigation of primary brain tumours. Eur. J. Nucl. Med., 28: 1851–1872,2001.40. Bosslet, K., Straub, R., Blumrich, M., Czech, J., Gerken, M.,Sperker, B., Kroemer, H. K., Gesson, J. P., Koch, M., and Monneret, C.Elucidation of the mechanism enabling tumor selective prodrug mono-therapy. Cancer Res., 58: 1195–201, 1998.41. Browder, T., Butterfield, C. E., Kraling, B. M., Shi, B., Marshall,B., O’Reilly, M. S., and Folkman, J. Antiangiogenic scheduling ofchemotherapy improves efficacy against experimental drug-resistantcancer. Cancer Res., 60: 1878–1886, 2000.42. Klement, G., Huang, P., Mayer, B., Green, S. K., Man, S., Bohlen,P., Hicklin, D., and Kerbel, R. S. Differences in therapeutic indexes ofcombination metronomic chemotherapy and an anti-VEGFR-2 antibody

in multidrug-resistant human breast cancer xenografts. Clin. CancerRes., 8: 221–232, 2002.43. Beekman, F. J., Kamphuis, C., King, M. A., van Rijk, P. P., andViergever, M. A. Improvement of image resolution and quantitativeaccuracy in clinical Single Photon Emission Computed Tomography.Comput. Med. Imaging Graph., 25: 135–146, 2001.44. Liberatore, M., Neri, D., Neri, G., Pini, A., Iurilli, A. P., Ponzo, F.,Spampinato, G., Padula, F., Pala, A., and Colella, A. C. Efficientone-step direct labelling of recombinant antibodies with technetium-99m. Eur. J. Nucl. Med., 22: 1326–1329, 1995.45. Verhaar, M. J., Keep, P. A., Hawkins, R. E., Robson, L.,Casey, J. L., Pedley, B., Boden, J. A., Begent, R. H., and Chester,K. A. Technetium-99m radiolabeling using a phage-derived single-chain Fv with a C-terminal cysteine. J. Nucl. Med., 37: 868 – 872,1996.




2003;9:571-579. Clin Cancer Res Monica Santimaria, Giovanni Moscatelli, Giuseppe L. Viale, et al. CancerFibronectin, a Marker of Angiogenesis, in Patients with Immunoscintigraphic Detection of the ED-B Domain of

Updated version

http://clincancerres.aacrjournals.org/content/9/2/571

Access the most recent version of this article at:

Cited articles

http://clincancerres.aacrjournals.org/content/9/2/571.full#ref-list-1

This article cites 43 articles, 10 of which you can access for free at:

Citing articles

http://clincancerres.aacrjournals.org/content/9/2/571.full#related-urls

This article has been cited by 43 HighWire-hosted articles. Access the articles at:

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

SubscriptionsReprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://clincancerres.aacrjournals.org/content/9/2/571To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/content/9/2/571.full#ref-list-1

http://clincancerres.aacrjournals.org/content/9/2/571.full#related-urls

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



immunoscintigraphic detection of the ed-b domain of ... · advances in brief immunoscintigraphic...

Documents