Epidermal Growth Factor Receptor-targeted Therapy with C225and Cisplatin in Patients with Head and Neck Cancer1

Dong M. Shin,2 Nicholas J. Donato,Roman Perez-Soler,3 Hyung Ju C. Shin,Ji Y. Wu, Peter Zhang, Kristie Lawhorn,Fadlo R. Khuri, Bonnie S. Glisson, Jeffrey Myers,Gary Clayman, David Pfister, John Falcey,Harlan Waksal, John Mendelsohn, andWaun Ki HongDepartments of Thoracic/Head and Neck Medical Oncology[D. M. S., R. P-S., P. Z., K. L., F. R. K., B. S. G., W. K. H.],Bioimmunotherapy [N. J. D., J. Y. W.], Pathology [H. J. C. S.],Head and Neck Surgery [J. My., G. C.], and ExperimentalTherapeutics [J. Me.], The University of Texas M. D. AndersonCancer Center, Houston, Texas 77030; Department of MedicalOncology, Memorial Sloan-Kettering Cancer Center, New York,New York 10021 [D. P.]; and ImClone Systems, Inc., Somerville,New Jersey 08876 [J. F., H. W.]

ABSTRACTC225, a human-mouse chimerized monoclonal antibody

directed against the epidermal growth factor receptor(EGFr), has a synergistic effect with cisplatin in xenograftmodels. To determine the tumor EGFr saturation dose withC225 and the fate of infused C225, we conducted a Phase Ibstudy with C225 in combination with cisplatin in patientswith recurrent squamous cell carcinoma of the head andneck. Using tumor samples, we assessed tumor EGFr satu-ration by antibody using immunohistochemistry studies, theEGFr tyrosine kinase assay, and detection of the EGFr/C225complex formation by immunoblot. Potential candidateswere screened for EGFr expression in their tumors, and 12patients who had high levels of EGFr expression and tumorseasily accessible for repeated biopsies (pretherapy, 24 h afterfirst C225 infusion, 24 h before third C225 infusion) wereentered at three different dose levels of C225 with a fixeddose of cisplatin. The median value of tumor EGFr satura-

tion increased to 95% at the higher dose levels. EGFr tyro-sine kinase activity was significantly reduced after C225infusion, and EGFr/C225 complexes were also detected athigher doses of C225. The loading dose of C225 at 400mg/m2 with a maintenance dose at 250 mg/m2 achieved ahigh percentage of saturation of EGFr in tumor tissue, andthese doses were recommended for Phases II or III clinicaltrials. Six (67%) of nine evaluable patients achieved majorresponses, including two (22%) complete responses. Mild tomoderate degrees of allergic reaction and folliculitis-likeskin reactions were demonstrated. We conclude that infusedC225 binds and significantly saturates tumor EGFr, whichmay render a high degree of antitumor activity, and pro-vides a novel mechanism for targeting cancer therapy forpatients who have EGFr expression in their tumors.

INTRODUCTIONEGFr4 is a 170,000-kDa transmembrane glycoprotein

found primarily on cells of epithelial origin (1–3). EGFr repre-sents one of the most important growth-regulatory signal-trans-duction molecules and exerts this function mainly through itsintrinsic tyrosine kinase activity, which can be activated uponligand binding (4–7). EGFr frequently is overexpressed inbreast cancer, ovarian cancer, prostate cancer, bladder cancer,glioblastoma, non-small cell lung cancer, and head and neckcancer and has also been found to play a significant role in theprogression of several human malignancies. EGFr positivitymay also be an indicator of poor prognosis (8–12).

Earlier studies suggested that tumors with high EGFr ex-pression appear to be more susceptible to chemotherapeuticagents or radiotherapy (13–15). When ovarian cancer cells bear-ing high levels of EGFr on the cell surface were pretreated withEGF, the sensitivity of these cells to cisplatin was substantiallyincreased (13). Similarly, when squamous carcinoma cells werepretreated with EGF, their sensitivity to radiation was enhancedin relation to the number of EGFrs on their surfaces (15). Theseobservations indicate that EGFr expression and its signal trans-duction pathways may be crucial determinants of sensitivity tochemotherapeutic agents or irradiation, and alterations in recep-tor expression or function may influence response to thesetherapies.

Because of the relationship between overexpression ofEGFr and aggressive behavior of tumor cells, monoclonal anti-bodies directed against this receptor might prove to be effectivetherapeutic agents. The anti-EGFr monoclonal antibody 225 wasgenerated and has been shown to have antitumor activityin vitroand in xenograft models (16–18). This antibody has been chi-merized with human IgG1 (C225) in its constant region to

Received 10/6/00; revised 1/3/01; accepted 1/26/01.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisementin accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 This work was partly supported by grants from ImClone Systems, Inc.;NCI [Grants CA69025 and CA75603 (to D. M. S.), Grant CA73018 (toN. J. D.)]; and by the M. D. Anderson Cancer Center Core Grant (GrantCA 16672). J. Me. is on the Board of Directors of ImClone Systems,Incorporated, and also holds stock options. W. K. H. is an AmericanCancer Society Clinical Research Professor.2 To whom requests for reprints should be addressed, at Department ofMedicine, Division of Hematology-Oncology, University of PittsburghCancer Institute, 200 Lothrop Street, N-755 UPMC Montefiore, Pitts-burgh, PA 15213-2582.3 Present address: Kaplan Cancer Center, New York University, NewYork, NY 10016. 4 The abbreviation used is: EGFr, epidermal growth factor receptor.

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increase clinical utility by lowering the potential for generationof human antimouse antibodies in recipients (19). C225 wasable to inhibit the growth of cultured EGFr-expressing tumorcell lines and to repress thein vivogrowth of these tumors whengrown as xenografts in nude mice (18–21). A therapeutic strat-egy combining C225 with chemotherapeutic agents such asdoxorubicin or cisplatin was found to be markedly synergistic inwell-established human xenograft models, and complete regres-sion of tumor growth in these mice was noted (22, 23). Com-bination therapy of cisplatin and C225 may block activation ofreceptor tyrosine kinase and induce EGFr down-regulation (23).

We conducted a Phase Ib study of C225 in combinationwith cisplatin in patients with recurrent squamous cell carci-noma of the head and neck to determine the optimal biologicaldose of C225 (i.e.,tumor EGFr-saturating dose) and to establisha safety profile of C225 in a different range of dose levels incombination with cisplatin.

MATERIALS AND METHODSStudy Design

A Phase Ib clinical and biological translational study usingC225 and cisplatin was designed for patients with recurrent ormetastatic squamous cell carcinoma of the head and neck. To beeligible for the study, patients had to have a histologicallyproven squamous cell carcinoma of the head and neck that wasconsidered incurable with standard therapy, have a good per-formance status (Zubrod scale# 2), have disease accessible torepeated biopsies, have measurable disease, have adequate ma-jor organ function, and sign a written informed consent. EGFr intumor cells should have been overexpressed in a prescreeningtest of all eligible patients.

Monoclonal antibody C225, manufactured and suppliedby ImClone System Incorporated (Somerville, NJ), was ad-ministered by i.v. infusion with a loading dose and mainte-nance dose weekly for 6 weeks, with a fixed dose of cisplatin(100 mg/m2) every 3 weeks. The cycle was repeated every 6weeks. Because of potential allergic reaction to C225, allpatients received premedication with 20 mg of dexametha-sone i.v. and 50 mg of diphenhydramine i.v. 30 min beforethe C225 infusion. At the time of initial C225 infusion,emergency kits including i.v. epinephrine, solumedrol, anddiphenhydramine were placed at each patient’s bedside totreat possible anaphylactic reaction. On the basis of a previ-ous pharmacokinetic study (24), the starting dose of C225was 100 mg/m2 for the loading dose and 100 mg/m2 weeklyfor the maintenance doses. Subsequent dose escalations innewly enrolled patients were 500 mg/m2 (loading) plus 250mg/m2 weekly maintenance and 400 mg/m2 (loading) plus250 mg/m2 weekly maintenance. Repeated doses of C225were allowed every 6 weeks according to the tumor responsestatus and the patient’s tolerance of the treatment.

Responses were assessed by computed tomography ormagnetic resonance imaging scans of the head and neck beforetreatment began and after each 6-week course of treatment.Designation of complete or partial response, no change or stabledisease, or progressive disease was based on standard WorldHealth Organization criteria (25).

EGFr Saturation StudiesTumor Specimens. Before therapy began, fresh tumor

specimens were obtained by punch biopsy by a cytopathologist(H. J. S.); two specimens were obtained at each time point fromeach patient and distributed for immunohistochemistry, immu-noprecipitation, and immunoblot analyses for tumor EGFr sat-uration and C225/EGFr complex studies. Posttherapy, freshtumor specimens were also obtained by punch biopsy 24 h afterthe first infusion (posttherapy 1) and 24 h before the thirdinfusion (posttherapy 2) of C225; two specimens again wereobtained from each patient. All tumor specimens at each timepoint were stained with H&E to evaluate viability of tumor cellsand processed accordingly for further studies.

Immunohistochemistry and Image Analysis. Tumorbiopsy specimens obtained from patients at each time point wereembedded in OCT (Miles Lab, Naperville, IL), snap-frozen inliquid nitrogen, and stored at270°C until used. Four-mm sec-tions were cut and mounted on saline-coated slides (HCS, Inc.,Glen Head, NY). Briefly, the sections were fixed in acetone for5 min and quickly transferred into PBS. Endogenous peroxidaseactivity was quenched by incubating the slides with 3% H202 inmethanol for 30 min, followed by three rinses with PBS. Theslides were then incubated for 30 min in 1% normal horse serumto reduce any nonspecific staining, allowed to react with 20mg/ml M225 (murine monoclonal antibody; ImClone System)overnight at 4°C, and washed with PBS three times for 15 mineach. Slides were then incubated with a secondary antibody(biotinylated horse antimouse IgG, diluted 1:200; Vector Lab-oratory, Burlingame, CA) for 1 h at room temperature, followedby incubation with avidin-biotin complex, and finally incubatedwith the peroxidase substrate, 0.1% diaminobenzidine in thepresence of H202. Stained sections were mounted with Eukitmounting medium. The degree of EGFr staining was assessedquantitatively in each sample by image analysis (26); baselinesamples (pretherapy) were compared with posttreatment sam-ples (24 h after first infusion of C225 and/or 24 h before thirdinfusion of C225).

Measurement of EGFr Tyrosine Kinase Activity. Inearly studies (Access No. 1–7), the EGFr level was determinedafter EGFr/C225 immune complexes were precleared with an-tihuman IgG and associated EGFr. Supernatants derived fromantihuman IgG immunoprecipitates were normalized in volume,and equal aliquots were placed into two tubes. Anti-EGFr anti-body (A108; 2.5mg; Rhone-Poulenc Rorer) was added to onealiquot, and the other received BSA or an isotype-specificirrelevant antibody (ZME-018). Samples were incubated for 90min, and 40ml of Pansorbin were added to facilitate EGFrimmunoprecipitation (as described below). The pellets werewashed extensively in lysis buffer and resuspended in 20ml of20 mM HEPES buffer (pH 7.4) containing 0.4 mM Na3VO4. Toinitiate EGFr phosphorylation, 20ml of HEPES buffer contain-ing 20 mM MnCl2 and 10mCi of [32P]ATP were added to eachtube, and samples were incubated at 30°C for 15 min. SDSsample buffer (53) was added to each tube, samples wereheated, and proteins were resolved by SDS-PAGE. The gel wasfixed, dried, and exposed to X-ray film to detect32P-labeledEGFr. Counts were quantitated by Phosphorimager, and sam-ples receiving anti-EGFr were compared with those receiving

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irrelevant antibody to determine the level of specificversusnonspecific32P incorporation. This technique was used previ-ously to detect EGFr levels in clinical specimens and to estimatethe level of EGFr saturation with an anti-EGFr monoclonalantibody (27).

Detection of EGFr/C225 Complexes. Tumor specimenswere obtained prior to treatment and at indicated intervals afterC225 infusion from patients whose disease was accessible torepeated biopsies. The frozen tumor tissues (;2.5 mm3 orlarger) were stored at280°C until all specimens (pre- andposttherapy) from individual patients had been collected. Frozensample weights were recorded (5–125 mg), and tissues werehomogenized on ice (Polytron) in lysis buffer [20 mM sodiumphosphate (pH 7.4), 5 mM EDTA, 150 mM NaCl, 50 mM NaF, 1mM Na3 VO4, 2 mM phenylmethylsulfonyl fluoride, 10mg/mlaprotinin, 10mg/ml leupeptin, and 1% (v/v) Triton X-100] at afixed tissue-to-volume ratio (20 mg of tissue:1 ml of lysisbuffer). All procedures were carried out on ice unless otherwisespecified. Homogenates were centrifuged (18,0003 g, 40 min,4°C), and supernatants were retained. Equal volumes of super-natant from individual patient samples were incubated 60–90min with 10mg/ml affinity-purified goat antihuman IgG (SigmaChemical Co.), and immune complexes were precipitated with50 ml of Pansorbin (Calbiochem) for 30 min. Supernatants weretransferred to a fresh test tube, and immunoprecipitation withgoat antihuman IgG was repeated. The two resultant pelletswere combined and washed extensively with lysis buffer byresuspension and brief centrifugation before heating in SDSsample buffer. Proteins were resolved by SDS-PAGE, trans-ferred to nitrocellulose membranes, and immunoblotted withanti-EGFr (Transduction Laboratory) followed by detectionwith secondary antibody (Bio-Rad) and ECL reagents (Amer-sham). In some experiments, A431 cell lysate (1mg) was loadedonto gels with immunoprecipitates as a positive control.

RESULTSPatient Characteristics. Twenty-two potential patients

were prescreened for tumor EGFr expression. In 20 (91%) of 22patients, high [$21in the scale from 0 (no expression) to 31(highest expression)] amounts of EGFr expression were seen.Among 20 candidates, 12 patients were enrolled on the protocol,

and the remaining 8 patients were excluded because their tumorswere not accessible for repeated biopsies. The characteristics ofthe 12 enrolled patients are summarized in Table 1. The medianage was 55 years (range, 36–78 years). Seven of the 12 patientswere men, and 5 were women. Six patients had dermal metas-tases, five had locoregional recurrence, and one had distantmetastasis. Six patients had recurrent disease after surgery andpostoperative radiation therapy, and six patients received sys-temic chemotherapy when disease recurred after surgery and/orradiation therapy (one patient received paclitaxel plus gemcit-abine, one received 5-fluorouracil plus cisplatin, one received5-fluorouracil plus cisplatin plus paclitaxel, one received 5-fluorouracil plus cisplatin and IFN-a plus 13-cis-retinoic acid,one receivedp53 gene therapy, and one received bryostatin).

Five patients in the first cohort were treated with C225 at100 mg/m2 as a loading dose with maintenance doses at 100mg/m2 weekly and cisplatin at 100 mg/m2 every 3 weeks. Fourpatients in the second cohort received C225 at 500 mg/m2 as aloading dose with maintenance doses at 250 mg/m2 weekly withthe same dose of cisplatin every 3 weeks. Three patients in thethird cohort received C225 at 400 mg/m2 as a loading dose withmaintenance doses at 250 mg/m2 weekly with the same dose ofcisplatin every 3 weeks.

Immunohistochemistry Studies. Immunohistochemis-try studies with image analysis were performed in tumor spec-imens using pre- and posttherapy biopsied tissue (24 h after thefirst infusion of C225 and 24 h before the third infusion ofC225) in eight cases (Acc 1, 2, 3, 4, 5, 7, 8, and 9) using frozentissue sections as described in “Materials and Methods.” For theother four cases (Acc 6, 10, 11, and 12), we were not able toperform the assay because no tissue was available or tumortissue became completely necrotic after therapy. The results arelisted in Table 2. At a C225 loading dose of 100 mg/m2 andmaintenance doses of 100 mg/m2, the median value of tumorEGFr saturation was 33% (range, 12–76%) 24 h after the firstdose of C225, suggesting a modest degree of tumor EGFrsaturation. At a C225 loading dose of 500 mg/m2 with a main-tenance dose of 250 mg/m2, tumor EGFr saturation in two cases(Acc 7 and 8) after the first dose of C225 was 76 and 39%,respectively. EGFr saturation before the third dose of C225 inAcc 7 decreased to 36% from 76% possibly because of inter-

Table 1 Patient characteristics and C225 doses

Acc no.C225 dose (mg/m2;

loading/weekly) Age (yr)/Sex Primary site Recurrent site Previous therapy

1 100/100 63/F Base of tongue Dermal Sa 1 RT2 100/100 36/M Oral tongue Locoregional S1 RT 1 gene therapy3 100/100 63/M Alveolar ridge Locoregional S1 RT4 100/100 72/M Tonsil Dermal S1 RT5 100/100 46/F Soft palate Locoregional S1 RT 1 5-fluorouracil1 cisplatin; IFN-a 1 13-cRA6 500/250 74/M Base of tongue Dermal S1 RT; paclitaxel1 gemcitabine7 500/250 50/M Base of tongue Dermal S1 RT8 500/250 55/F Larynx Dermal S1 RT; 5-fluorouracil1 cisplatin

10 500/250 78/F Mucosal lip Locoregional S1 RT9 400/250 55/M Hypopharynx Dermal S1 RT

11 400/250 68/M Retromolar trigone Distant S1 bryostatin12 400/250 39/F Oral tongue Locoregional S1 RT; 5-fluorouracil1 cisplatin1 paclitaxela S, surgery; RT, radiation therapy; 13-cRA, 13-cis-retinoic acid.

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nalization of the receptor or reduction in EGFr levels. However,EGFr saturation before the third dose of C225 in Acc 8 in-creased to 95%. In this case, complete tumor regression wasnoted, as shown in Fig. 1.

At a C225 loading dose of 400 mg/m2 with a maintenancedose of 250 mg/m2, tissue was available in one patient (Acc 9)for the EGFr assay; tumor EGFr saturation was 57% within 24 hafter the first dose and 79% within 24 h before the third dose ofC225.

Detection of EGFr/C225 Complexes by Immunopre-cipitation. Additional evidence of an association betweenC225 and EGFr in tumor specimens was provided by independ-ent biochemical analysis. Tumor samples taken before and afterC225 infusion were collected and initially analyzed for residualEGFr tyrosine kinase activity after immunodepletion of C225/EGFr complexes with antihuman IgG. EGFr was collected inimmunoprecipitates with A108 monoclonal antibody, whichrecognizes an epitope on EGFr distinct from that recognized byC225. As shown in Fig. 2, tumor specimens were available foranalysis by this technique in 4 of 12 cases, and in each case,EGFr tyrosine kinase activity was detected in pretherapy tumorspecimens. After the initial C225 infusion, EGFr tyrosine kinaseactivity was reduced by 50% or more in two cases (Acc 1 and7) and was not detected in specimens derived from patients withlow receptor kinase activity (Acc 3 and 5). EGFr tyrosine kinaseactivity was further reduced in specimens taken after the thirdinfusion of C225 (Fig. 2,Acc #7 post-therapy 2). In one case,EGFr tyrosine kinase activity was completely suppressed (Fig.2, Acc #1 post-therapy 2). These results provide further evi-dence that C225 binds EGFr in tumor tissue and forms EGFr/C225 complexes. Tumors achieving a high level of saturationwith C225 correlated with reduction in EGFr tyrosine kinaseactivity even in patients who received lower doses of C225.

More direct evidence of EGFr/C225 complex formationwas provided in five of six patients by examining antihumanIgG immune complexes for the presence of EGFr by directimmunoblotting. As shown in Table 2 and Fig. 3, tumor-derivedEGFr was immunocomplexed with antihuman IgG in patientsreceiving C225. EGFr was not detected in antihuman IgG im-mune complexes in patients prior to C225 infusion, except in

one specimen (Acc 1). In samples for which both EGFr tyrosinekinase and EGFr/C225 complex assays were performed (Acc 1,5, and 7), the decreased EGFr tyrosine kinase activity fromantihuman IgG-immunodepleted supernatants (Fig. 2) corre-lated with the appearance of EGFr/C225 complexes in theantihuman IgG precipitate.

Clinical Responses and Toxic Effects. Although theprimary objectives of this study were to assess tumor EGFrsaturation and to determine the optimal biological dose andsafety profile of C225 when combined with cisplatin, we alsoassessed the antitumor activity of C225 in patients who hadrecurrent squamous cell carcinoma of the head and neck.Among 12 patients entered in this protocol, 9 had diseaseresponse that could be evaluated, whereas 3 did not (2 at a C225loading dose of 100 mg/m2, 1 at a C225 loading dose of 500mg/m2). One patient terminated therapy because of an allergicreaction during the loading dose of C225, one patient stoppedtreatment because of severe peripheral neuropathy, and onepatient withdrew from the trial after three doses of C225.Among the nine patients, disease response was complete in twoand partial in four. There was one case each of mixed response,stable disease, and progressive disease. The overall responserate was 67% (six of nine patients). Among the six respondingcases, three had progressive disease during cisplatin-containingchemotherapy before enrollment in this protocol. Measurablediseases of the responders were located inside of the previousradiation port in four patients and outside of radiation port in theremaining two patients.

The toxic effects of C225 combined with cisplatin arelisted in Table 3. No bone marrow suppression was observed inrelation to C225 infusion. Four patients had grade 3 or grade 4neutropenia, and one had neutropenic fever, which may havebeen related to cisplatin. Among nonhematological side effects,two patients had grade 3 fatigue, one had grade 3 peripheralneuropathy, and two had grade 3 orthostatic hypotension; allmay have been related to cisplatin infusion. Allergic reactiondeveloped in two patients: one grade 2 reaction, and one grade3. Both patients experienced shortness of breath and chesttightness right after initiation of the loading dose of C225: oneat 100 mg/m2, and one at 500 mg/m2. This was severe enough

Table 2 Tumor EGFr saturation studies

Acc no.

EGFr saturation status (%)

EGFr saturation by IHCa Tyrosine kinase activity unbound EGFr/C225 complex

Pre tx 24 h post 1st tx 24 h pre 3rd tx Pre tx 24 h post 1st tx 24 h pre 3rd tx Pre tx 24 h post 1st tx 24 h pre 3rd tx

1 0 12 59b 100 60 0 1c 111 112 0 76 32b ND ND ND ND ND ND3 0 53 10b 100 0 0 ND ND ND4 0 33 45 ND ND ND ND ND ND5 0 26 25 100 20 0 2 111 17 0 76 36 100 70 60 2 11 18 0 39 95 ND ND ND 2d 2d 2d

9 0 57 79 ND ND ND 2 111 112 0 ND ND ND ND ND 2 111 2a IHC, immunohistochemistry; tx, therapy; ND, not done or could not be evaluated.b Biopsies obtained 48 h after third dose of C225.c 1, 11, 111, weak, moderate, and strong band of EGFr/C225 complex, respectively;2, no EGFr/C225 complex formation.d Tissue samples from Acc no. 8 may have undergone tumor necrosis and/or proteolysis.

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Fig. 1 Tumor EGFr saturation with C225 de-tected by immunohistochemistry studies on tumorspecimens from a patient with responding disease.Pretherapy photographic illustration of tumorshowing multiple dermal metastases (A) in theanterior neck and upper chest were analyzed forEGFr expression (B) by immunohistochemistry(3200) and H&E staining (3200;C) of the adja-cent section. After one course of C225 with cis-platin, multiple dermal metastases have com-pletely regressed (D), and EGFr expression wasmarked down-regulated (E).F, H&E staining ofthe adjacent tissue section. Of note, 95% EGFrsaturation with C225 in tumor tissue was observedby image analysis after therapy.

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Fig. 1 D–F Continued.

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for the second patient to receive epinephrine and steroids torecover from the allergic reaction, and the patient was taken offthe study after recovering from the reaction. The allergic reac-tion was modest in the first patient, and the therapy was con-tinued with additional premedication (20 mg of dexamethasoneand 25 mg of diphenhydramine as i.v. infusion) and slowerinfusion of C225. Grade 3 folliculitis-like skin rashes developedin two patients (one had received a loading dose of 100 mg/m2

C225 with a maintenance dose of 100 mg/m2; one had receiveda loading dose of 500 mg/m2 C225 with a maintenance dose of250 mg/m2). Both patients received more than six doses ofC225, and the rashes appeared to gradually worsen with thecumulative amount of C225. However, topical and oral antibi-otics helped to improve the folliculitis-like skin rashes in bothpatients.

DISCUSSIONMonoclonal antibody C225 against the human EGFr po-

tentially blocks activation of receptor tyrosine kinase (16, 17,28). Preclinical studies indicate that EGFr activation can beblocked by C225, which subsequently inhibits the growth ofmalignant tumor cells (18–20). Furthermore, C225-mediatedcell growth inhibition involves down-regulation of EGFr ex-pression (21, 29). Preclinical studies also suggest that blockade

of EGFrs allows augmentation of antitumor activities whencombined with doxorubicin or cisplatin in A431 cell xenografts(23, 29, 30). In particular, C225 therapy in combination with themaximum tolerated dose of cisplatin (23, 29, 30) produced curesin tumor-bearing mice whose follow-up was observed for.6months.

The current study describes the use of a dose escalation ofC225 in combination with cisplatin in patients with head andneck cancer, a highly desirable tumor target for this combinedtherapy because a majority of squamous cell carcinomas havehigh EGFr expression. (26, 31) One important aspect of thisstudy was to understand the fate of the infused C225. Therefore,emphasis was placed on assessing the binding of infused C225to tumor EGFrs (i.e.,EGFr/C225 complex formation). Threeindependent approaches were used to investigate this process. Inthe first approach, immunohistochemical methods were used toevaluate the tumor EGFr status before and after C225 infusion.Infused C225 may compete with receptor ligands (i.e., EGF ortumor growth factor-a) for EGFrs on the cell surface (29). Thetumor EGFr saturation status was assessed by measuring differ-ential staining for EGFr expression by immunohistochemistryand quantitated by image analysis on tumor tissue obtainedbefore and after C225 infusion. Tumor EGFr saturation withC225 was relatively low at lower C225 doses compared withhigher amounts of C225. These findings are supported by our

Fig. 2 Effect of C225 infusion on EGFr tyrosine kinase activity in headand neck tumor specimens derived from patients treated with a combi-nation of C225 and cisplatin. Tumor specimens obtained from fourpatients (Acc# 1,3, 5, and7) prior to (pre-therapy) and at two intervalsafter C225/cisplatin (post-therapy 1and2; see “Materials and Methods”for details) were homogenized, and human IgG complexes were re-moved by immunoprecipitation. The supernatant fraction was dividedinto two equal fractions, with one fraction receiving anti-EGFr. Sampleswere treated with Pansorbin and washed extensively, and EGFr tyrosinekinase activity was measured by phosphorylation in the presence of[32P]ATP. Radiolabeled EGFr was resolved by SDS-PAGE, detected byautoradiography, and quantitated by Phosphorimager. Samples that didnot receive anti-EGFr (2) were used as controls to determine the levelof background phosphorylation. Quantitation estimated that the percent-age of change in tyrosine kinase activity (based on pretherapy controls)was 40 and 100% reduced in the posttherapy 1 and 2 samples of Acc 1,respectively, whereas 80 and 95% reduction was estimated in Acc 7specimens. The kinase activity could not be detected in either post-therapy specimen from Acc 3 and 5.Arrow depicts migration of the170-kDa EGFr band.

Fig. 3 Detection of EGFr/C225 complexes by immunoblot in tumortissues derived from patients with head and neck cancer treated withC225/cisplatin. Tumor specimens were obtained from six patients be-fore and after therapy and were subjected to immunoprecipitation withantihuman IgG. Immune complexes were examined for the presence ofEGFr by immunoblotting. The specificity of the antibody for EGFr wasconfirmed by immunoblotting A431 cell lysates with prepared patientsample immunoprecipitates (Acc #9). EGFr was absent or minimallydetected in pretherapy specimens of Acc 8 (no detectable EGFr band ineither pre- or posttherapy samples because of a high degree of necrosisin this tissue). In five of the six cases (Acc #1,5, 7, 9, and12), infusionwith C225 (post-therapy 1) resulted in a marked increase in EGFr/C225complexes in antihuman IgG immunoprecipitates. Subsequent treatmentwith C225 resulted in a reduction of the level of EGFr recovered in thesecomplexes.Arrow depicts migration of the 170-kDa EGFr band.

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previousin vitro observation that inhibition of A431 cell pro-liferation by monoclonal antibody was more prominent in low-cell density cultures than in high-density cultures, possiblybecause of lower concentrations of antibody-competing auto-crine factors. As had been predicted but not previously tested,higher amounts of C225 infusion in patients resulted in higherlevels of tumor EGFr saturation, in agreement with preclinicalstudies (28).

To confirm the immunohistochemical results and to exam-ine the association and saturation of tumor EGFr with C225invivo, two additional biochemical assays were used. The firstassay estimated receptor saturation by gauging tyrosine kinaseactivity associated with murine monoclonal anti-EGFr antibodyin patients with cancer (27). In the present study, tissue homo-genates were immunoprecipitated with anti-EGFr (A108)invitro after immunodepletion of EGFr/C225 complexes withhuman-specific anti-IgG. Signals were recorded by measuringthe tyrosine kinase activity of the EGFr in the immune complex,and we were able to demonstrate EGFr autophosphorylation inthe majority of the specimens obtained (some of the samplesfailed to provide a signal because of insufficient tissue or tissuenecrosis). By comparing the autokinase activities of pre- andpost-C225 specimens from the same patient (with pretherapyunbound kinase activity defined as 100% loss of EGFr kinaseactivity), we used measurements for C225-treated specimens asan estimate of tumor EGFr saturation. From this analysis, itappears that lower receptor expression in individual tumorscorrelates with more effective C225 saturation. However, otherfactors that are unrelated to C225 saturation may also contributeto this apparent loss of kinase activity in these fractions. Thestate of EGFr tyrosine phosphorylation, apoptosis stimulated bychemotherapy, limited proteolysis of the receptors, or partialinhibition of enzymatic activity can also lead to an apparentreduction in EGFr kinase activity following treatment withC225 and cisplatin. Therefore, different or perhaps more con-vincing ways of measuring EGFr/C225 association in clinicalsamples were investigated.

To maximize evaluation of clinical tissues for EGFr/C225association, the primary antihuman IgG immunoprecipitate wassubjected to EGFr immunoblotting and compared (in somespecimens) with the results obtained for kinase activity assess-ments of EGFr recovery. As shown in Fig. 3, EGFr was notimmunoprecipitable with antihuman antibody without priorC225 infusion. In specimens analyzed by both the kinase activ-ity assay and EGFr immunoblotting, loss of kinase activity fromthe supernatant fraction (after immunodepletion of antihumanimmune complexes) correlated with an increase in EGFr in theantihuman IgG/C225 immunoprecipitate (see Fig. 3). In samplestaken after initial C225 therapy, an increase in EGFr recoverywas noted in five of six patient samples. (Acc 8 may haveundergone tumor necrosis and proteolysis.) Further treatmentwith C225 and cisplatin (posttherapy 2) resulted in reducedrecovery of EGFr in this fraction. This reduced recovery ofEGFr/C225 complexes may be attributable to several factors,including the possibility for C225-induced down-regulation ofEGFrs. However, tumor apoptosis and necrosis brought aboutthrough the combination of C225 with cisplatin therapy cannotbe ruled out as a contributor to this apparent loss of detectableC225/EGFr complexes. Although these possibilities cannot bedifferentiated by the current study, the results provided byC225/EGFr complex studies and immunohistochemical studiessuggest that treatment with C225/cisplatin results in a high-affinity interaction between EGFr and C225 that affects EGFrexpression or recovery in patients with head and neck cancerand potentiates tumor regression (19, 29).

Although the primary objectives of this study were assess-ment of tumor EGFr saturation and determination of the bio-logical dose of C225, we evaluated antitumor activity in theparticipating patients. All patients had failed primary therapy,including surgical resection and/or postoperative radiotherapy.Six of the 12 patients had been treated with systemic chemo-therapy or gene therapy after disease recurrence. Among ninepatients who could be assessed for disease response, six (67%)had major responses, including two complete responses. Amongthese six cases, three patients had received prior cisplatin-containing combination chemotherapy. Two of these three pa-tients had resistance to cisplatin; they experienced disease pro-gression while receiving cisplatin-based chemotherapy just priorto enrollment on this protocol. Such antitumor activity is highlyencouraging. In second-line therapy with single-agent cisplatinfor recurrent head and neck cancer, one may expect to see aresponse rate,10% (32, 33). Therefore, C225 therapy in com-bination with cisplatin appears to have a synergistic effect inrecurrent head and neck cancer as predicted in xenograft mod-els. The detailed mechanism of this synergistic effect must bemore fully addressed in future studies.

Combined C225 and cisplatin therapy does not appear toinduce overlapping toxic effects. Bone marrow suppression,emesis, and peripheral neuropathy in association with orthos-tatic hypotension have been associated with cisplatin (32, 33),whereas folliculitis-like skin rashes and allergic reaction may beassociated with C225 infusion as described previously (24, 34).This study particularly emphasizes that biological end points(i.e., EGFr tumor saturation, tumor EGFr tyrosine kinase activ-ity, and degree of C225/EGFr complex formation) are far morerelevant than the traditional end point (i.e., determination of

Table 3 Maximum toxic effects (n5 12)

Toxic effects

Number of patients

CTCa

grade 2CTC

grade 3CTC

grade 4

HematologicNeutropenia 2 3 1Neutropenic fever 1 0 0Thrombocytopenia 1 0 0Anemia 3 1 0

NonhematologicFatigue 3 2 0Nausea/vomiting 4 0 0Peripheral neuropathy 1 1 0Creatinine elevation 0 0 0Orthostatic hypotension 0 2 0Allergic reactionb 1c 1d 0Skin reactionb (folliculitis-like reaction) 0 2e 0

a CTC, National Cancer Institute Common Toxicity Criteria.b Related to C225 infusion.c,d Dose of C225:c 100 mg/m2; d 500 mg/m2.e One patient had received a loading dose of 100 mg/m2; one had

received 500 mg/m2.

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maximum tolerated dose or dose-limiting toxic effects) in PhaseI studies when biologicals are given alone or in combinationwith chemotherapeutic agents.

In summary, a C225 loading dose of 400 mg/m2 with amaintenance C225 dose of 250 mg/m2 achieved nearly com-plete saturation of EGFr in tumor tissue, and the combinationof C225 with cisplatin achieved a high percentage of tumorresponses. There is also a suggestion of synergism betweenC225 and cisplatin in recurrent head and neck cancer. On thebasis of this EGFr saturation study, a Phase II study withlarge sample size is being conducted using 400 mg/m2 C225for the loading dose and 250 mg/m2 for the maintenance dosein combination with cisplatin for refractory patients withrecurrent head and neck cancer. A Phase III study is alsobeing conducted through the Eastern Cooperative OncologyGroup comparing C225 plus cisplatin with placebo pluscisplatin in recurrent head and neck cancer. A multi-institu-tional randomized Phase III trial is also underway to deter-mine the role of C225 in combination of radiation therapyversusradiation therapy alone in locally advanced head andneck cancer. The doses of C225 in this study are 400 mg/m2

for loading and 250 mg/m2 weekly for maintenance duringthe radiation therapy. These studies should ultimately assistin determining the potential impact of targeted EGFr biolog-ical therapy in combination with cytotoxic chemotherapeuticagents or with radiation therapy for the treatment of EGFr-expressing epithelial cancers.

ACKNOWLEDGMENTSWe thank the patients who participated in this study and their

families. We also thank Vanessa Valiare for preparation of the manu-script and Julia Starr for editorial comments on the manuscript.

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2001;7:1204-1213. Clin Cancer Res   Dong M. Shin, Nicholas J. Donato, Roman Perez-Soler, et al.   and Cisplatin in Patients with Head and Neck CancerEpidermal Growth Factor Receptor-targeted Therapy with C225

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