Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
[CANCER RESEARCH57. 2181—2186, June I, 1997J Enhanced Antitumor Activity of Combination Radioimmunotherapy Monoclonal Antibody A33) with Chemotherapy Jörg Tschmelitsch, Els Barendswaard, Clarence Williams, (1311-labeled (Fluorouracil)' Jr., Tzy-Jyun Yao, Alfred M. Cohen, Lloyd J. Old, and Sydney Welt2 New York Branch, Ludwig institute for Cancer Research IE. B., C. W., L J. 0., S. W.j, Department of Surgery If. T., A. M. CI, and Department of Epidemiology IT-f. Y.J, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 analysis showed significant survival benefit for adjuvant 5-FU + LV ABSTRACT Monoclonal antibody (mAb) A33 reacts with an antigen expressed by >95% of colon cancer and normal colon epithelial cells. An earlier Phase I trial of ‘3'I-labeled mAb A33 (‘3'I-mAb A33) demonstrated bone mar row suppression as the dose-limiting toxicity, and although modest anti tumor effects were seen, no normal colon toxicity was observed. In this study, a nude mouse model was used to test whether combinations of low-dose ‘311.mAb A33 (0.1 mCI) and chemotherapy [5-fluorouracil (5FU) or 5-FU + leucovorin, doxorubicin, or carmustine] enhance the antitumor effects, compared to ‘311-mAb A33 alone or either drug regi men alone. 5-FU was administered either at 30 mg/kg/day for 5 days or at 75 mg/kg/day on days 1 and 5. In assessing the reduction in tumor volumes over the first 28 days of the experiment, 5-FU treatment (with or without leucovorin) in combination with ‘3m1-mAb A33 showed a statistically significant additive antitumor effect compared to ‘311-mAb A33 alone or to chemotherapy alone. When long-term survival was used as an end point, 38% of the mice treated with 5-FU and ‘311.mAb A33 were disease free at 276 days comparedto none from any other group,suggestinga synergistic effect. These data indicate that Phase II clinical trials combin. Ing radlolabeled antibody therapy with 5-FIJ-based treatments are war ranted. @ INTRODUCTION Cancer of the colon and rectum is the second most common cancer in theUnitedStates(1).Surgeryis thecornerstoneof treatment,but surgeryalonehas a highrecurrencerate evenin the treatmentof primary, localized disease, Only lately have adjuvant chemotherapy regimens for colon cancer, or adjuvant chemoradiotherapy regimens for rectal cancer, shown an impact on survival (2—6), 5-FU,3 the mainstay ot' treatment, blocks the activity of thymidylate synthetase and/or is activated to fluoro-UTP and incorporated into the RNA, However, the efficacy rate of 5-FU is limited (7—9).Recently, many attempts have been made to increase the antitumor activity of t'Iuoropyrimidine using biochemical modulators. LV is one such mod ulator, and its mode of action is considered to be an increase in thymidylate synthetase inhibition through formation of a ternary com plex of 5, 10-methylene tetrahydrot'olate from LV, thymidylate syn thetase, and t1uoro@dUMP (10—12), Whether the strategy of 5-FU modulation by LV is clinically significantremainsunclear,SomePhaseIIIclinicaltrialshaveshown that 5-FU ±LV provides a significantly higher response rate than 5-FU alone ( 13—16),and a recently published multicenter, pooled Received 7/29/96; accepted 4/7/97. The costs of publication of this article were defrayed in part by the payment of page charges. This @rtjcI@ must therefore be hereby marked atl@'eriiseit,ei,t ifl accordance with 18 U.S.C. Section 1734 solely to indicate this fact. â€T̃his study was supported in part by funds provided by Prof. F. Bodner, CA-05748 from the National Cancer Institute. whom requrstu for reprints should Research, Memorial Sloan-Kettering 10021-6007. 3 The abbreviations used are: 5-FU, be addressed, at Ludwig Institute for treatment after colon resection ( 17). However, other studies have not shown a significant benefit (18, 19). Even under these improved conditions, the response rate is at best only 35%. Therefore, new therapeutic approaches are needed to treat this type of cancer. Another approach to enhance the efficacy of 5-FU is to take advantage of the additive (20, 21) or even synergistic effect of the combination of 5-FU and radiation (22—25), which is believed to account in part for better control of local recurrences of rectal cancer. In primary or advanced colon cancer and advanced rectal cancer, external beam radiation therapy is not applicable for technical reasons and because of adjacent radiosensitive organs. Radiolabeled mAbs targeting colon cancer might be able to deliver the needed radiation dose precisely and without major toxicity. A Phase I/I! ‘3'I-mAbA33 therapy trial demonstrated bone marrow as the dose-limiting organ toxicity (26). Due to the localization of radiolabeled antibody to normal colon, gastrointestinal toxicity was examined closely and was found to be minimal. Antitumor effects were observed in 5 of 23 assessable patients. despite the fact that only a single dose could be administered due to development of an antimouse immunoglobulin response (26). To determine toxicity, treatment schedule, and thera peutic efficacy of a combined approach, different combinations of low-dose (0, 1 mCi) ‘‘I-mAbA33 and chemotherapeutic agents were evaluated in a nude-mouse, human colon cancer-xenograt't model. Because the radiation dose delivered to solid tumors by radiolabeled antibodies is low in clinical studies (27), we selected a subtherapeutic dose of ‘311-mAb A33 for this study to search for enhanced antitumor effects at low radiation dosages, MATERIALS AND METHODS Cell Lines. Humancolon carcinomacell line SWI222 was obtainedfrom the cell bank of the Ludwig Institute for Cancer Research at Memorial Sloan-Kettering Cancer Center and maintained in Eagle's MEM containing 1% nonessential amino acids and supplemented with 10% FCS, 2 mM glutamine, 100 units/mI penicillin. and 100 @g/mlstreptomycin. Cells were cultured in a 37°Cincubator containing 5% [email protected] cells were harvested using 0. 1% trypsin and 0.02 EDTA (Life Technologies, Inc., Grand Island, NY). Human Tumor Xenografts, Four- to 6-week old female Swiss (nu/nu) athymic mice (20—25g body weight) were obtained from the Memorial Sloan-Kettering Cancer Center nude mouse facility and injected i.m. with 10 x io@cells in 250 @i1 PBS into the left thigh muscle. After I week, mice bearing tumors weighing approximately 100—5(X) tug were selected. Mice were anesthetized with 0.5 ml Avertin (2,2,2-tribromoethanol, 97%; PlaIts and Bauer, Waterbury, CT) by i.p. injection. Labeling of mAim, lodination of mAbs was carried out using the chlor amine T method. mAb A33 and mAb FB5, an lgG2a against human neovas cular endothelium used as an isotype control, were mixed with the requisite quantity Chairman, Second Department of Surgery, University of Innubruck, Innsbruck, Austria, and Grant 2 To and Biostatistics of ‘@‘i (1 mg/20 mCi). Two hundred @Ll of a freshly prepared solution of 2 mg/mI chioramine T (in 150 mM sterile phosphate buffer at p1-17.4) were added to the mixture. After I mm, the reaction was quenched by the addition Cancer Cancer Center, 1275 York Avenue, New York, NY of 200 @ilof a freshlypreparedsolutionof 10 mg/mlsodiummetabisulfite. Labeled protein was separated from the free iodine on a Sephadex 025 column 5-fluorouracil; LV, leucovorin; mAb, monoclonal antibody; ‘3'l-mAbA33, ‘3'I-labeled mAb A33; ‘3'J-mAb FBS, ‘3'I-labeled mAb PBS; MTD,maximumtolerateddose;AUC,areaunderthe fittedgrowthcurve;% 101g.% of injecteddose/g;MTS,meantumorsi@c. (Pharmacia),saturatedwith 0.5% BSA/PBS.Antibodyfractionswith peak radioactivity were pooled, and aliquots of the ptoled fractions were tested for the percentage of precipitable protein-bound ‘@‘i by the trichioroacetic acid 2181 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1997 American Association for Cancer Research. COMBINATION RADIOIMMUNOTHERAPYAND CHEMOTHERAPY precipitation method. The amount of trichloroacetic acid-precipitable Table 1 MeancurveTreatment and SE of the area under the fitted logarithmic tumor gr protein bound ‘@‘I was 97—99%. Immunoreactivity for mAb A33 was determined as described previously (28) by adding 0.1 @Ci of ‘311-mAb A33 to 15 X 106 antigen-positive cell pellets. To determine background cpm, cell pellets were pretreated with >100 X excess unlabeled Immunoreactivity from day 0 to day 28owth day)SEExperiment 20.12.55-FUFB5 —1.61.95-Ri bolus mAb A33 prior to addition of ‘311-mAbA33. was calculated by subtracting background cpm from AUC (ln (cm3) X I 13'I-mAb —48.36.0@3'I-m@b A33 ‘@‘I —73.411.35-FU bolus + ‘3'I-mAbA33 mAb A33 cpm bound to the cell pellet, after washing twice in PBS and dividing the remaining fraction by the total cpm added. Mean immunoreac —7.82.05-FU bolus + ‘3'I-mAbFB5 daily tivity was 34%. A single dose of 0.1 mCi of ‘3'I-mAbA33 or ‘3'I-mAbFB5 5-nj daily+ ‘3'I-mAb A33 —77.7 5-FU daily + ‘311-mAbFBS 2.1Control —3.42.5 in 0.2 ml of PBS was injected iv. into the retro-orbital plexus. Formulation and Administration of Drugs. 5-FU (50 mg/mI;SoloPak —4.8 25.53.9Experiment 2 Laboratories, Franklin Park, IL) was obtained as a pharmaceutical preparation —38.34.6s-at ‘31I@bA33 and diluted to a concentration —12.83.95-nj + LV —53.84.05-FU + LV + ‘3'I-mAb A33 of 5 mg/ml or 10 mg/mI in sterile 0.9% saline. It was injected i.p. either at 30 mg/kg/day for 5 days or at 75 mg/kg/day on days 1 and 5. The injected volume ranged from 0.12 to 0.19 ml. 9.0 16.73.4Experiment + LV + ‘31I-mAbFB5 — 3 —37.93.85-FU t311-mAbA33 LV (Burroughs Wellcome Co., Research Triangle Park, NC) was dissolved —74.44.7Doxorubicin + LV + ‘311-mAbA33 in 0.9% sterile saline at a concentration of 10 mg/mI. Mice treated daily received 90 mg/kg LV i.p. 2 h prior to 5-FU. The injected volume was 0.18 ml/20 g body weight. Doxorubicin (Sigma Chemical Company, St. Louis, MO) was given i.p. as a single dose of 10 mg/kg. Carmustine (Bristol Myers Squibb, Princeton, NJ) was injected i.p. as a single dose of 30 mg/kg. Both drugs were dissolved in —39.96.1Doxorubicin + 1311-mAbA33 1.05.3Carmustine + ‘ 3'I-mAbFB5 — —27.04.8Carmustine + ‘3'I-mAbA33 15.64.2‘3'I-m@b + ‘311-mAb FB5 20.22.5Control FB5 26.45.3 PBS sufficientto yield a volume thatpermittedadministrationin a dose of 0.1 mi/lO g body weight and injected on day 1. The doses used were selected from the literature and first tested in tumor bearing animals in preliminary toxicity studies to determine the MTh. Tumor Therapy. Mice were divided into groups of six to eight animals. Toxicity was determined by measuring change in body weight. All animals were weighed at the start of the experiments and at 8-day intervals. Tumor size @ was determined by digital caliper measurement. Two perpendicular diameters were used to calculate tumor volumes (29). Measurements were taken imme diately before therapy and at 4-day intervals until day 68 or until tumor volume reached 2 cm3 (2 g). In the first experiment, one group of mice each received 0. 1 mCi of either ‘311-mAbA33 or control ‘3'l-mAbFB5, 5-Ri daily X 5, or 5-FU on days 1 and 5. Additional groups were treated either with a combination regimen of 5-nj @ @ daily X 5 + ‘311-mAb A33, 5-Ri on days I and 5 + ‘3'I-mAb A33, 5-Ri daily X 5 + ‘3'I-mAb FB5, or 5-Ri on days 1 and 5 + ‘311-mAb FB5. Growth controls consisted of mice receiving no treatment. In the second experiment, the effectiveness of 5-FU + LV + ‘ ‘I-mAb A33 was compared with that of ‘‘I-mAbA33 alone and with 5-FU + LV. Again, an untreated control group was included. In the third experiment, the effect of 5-FU + LV on tumor growth was compared to doxorubicin or carmustine, each with or without ‘311-mAb A33. Statistical Methods. Within each treatment group, the growth curves of tumors were fitted with a linear regression model, up to second order, of time (day) after logarithmic transformation. The model can be displayed as: log(tumor volume) = A + B X day + C X day2 The regression coefficients A, B, and C were estimated using a generalized estimation equation (30). In comparing tumor growth between groups, the AUC (measuring tumor volume accumulated over time) from day 0 to day 28 was the endpoint of comparisons, with AUC defined as: B AUC = 28A + 282_ C + 2 28@— 3 where A, B, and C are the estimated regression coefficients. A smaller AUC compared to the control group AUC indicates a treatment effect on tumor growth inhibition. We assumed that the effect of a regimen composed of two agents is equivalent to the sum of the separate effects of the two agents plus the synergistic effect, if it exists. As an analogue to the test of interaction in the two-way ANOVA, the synergy between agents A and B was tested by applying a Z test on the difference between (a) AUC of treatment A and B + AUC of control and (b) AUC of treatment A + AUC of treatment B. If the tests of synergy were not significant, the tests of main effects were pooled over different treatment combinations using the Z test. For observations beyond 28 days, the proportion of long-term survival between groups was compared using Fisher's exact test. Three separate experiments were per formed. Only the treatment groups within the same experiment were compared to each other. For the comparisons within an experiment, Type I errors were adjusted using Bonferroni's method. RESULTS A dose-response curve for ‘‘I-mAbA33 was established previ ously4; the lowest dose of ‘311-m.Ab A33 giving an antitumor effect was 0.1 mCi. This dose was tested again and confirmed to be the lowest dose producing the smallest measurable tumor-suppressive effect. It was therefore selected for our studies (see below). The mean AUCs from day 0 to day 28 for each group are shown in Table I. Many of the AUCs are negative due to logarithmic transfor mation of small values (< 1). In a preliminary experiment, 5-Ri was tested at doses ranging from 15 to 30 mg/kg daily X 5 or at 40—75 mg/kg on days I and 5 (data not shown), but even the maximum dose given by daily injection or on days 1 and 5 achieved only a minimum antitumor effect (Fig. 1). In an earlier study, unlabeled mAb A33 had shown no demonstrable effect on the growth of established tumors in this model.4 Likewise, the isotype-matched control ‘311-m.Ab FB5 showed no significant effect on tumor growth when compared to untreated controls (Fig. 2). The combination of 5-FU given daily X 5 or on days 1 and 5 + ‘311-mAb FB5 at 0. 1 mCi showed no increased tumor-suppressive effects compared to 5-FU alone (Fig. 3). The combination of 5-FU and ‘311-mAb A33 resulted in significantly higher antitumor activity (Fig. 4). In the groups of mice that were treated either with 5-FU daily X 5 + ‘31I-mAbA33orwith5-FUondays 1 and5 + ‘311-mAb A33, one xenograft in both groups escaped the therapy relatively early. Three of the eight mice in each group developed minimal tumor growth 54—60days after treatment. The other four animals in each group had no evidence of tumor growth until 80—84 days after treatment, when one of the four mice in each group had a recurrence. The remaining three animals (38%) in both groups had no recurrence as late as 276 days following treatment. Thus, statistical analysis showed that long-term, disease-free survival for both of these corn 4 E. Barendswaard, S. Welt, F. Daghighian, A. Scott, M. Graham, and unpublished observations. 2182 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1997 American Association for Cancer Research. L. J. Old, COMBINATIONRADIOIMMUNOTHERAPY AND CHEMOTHERAPY agents did not improve the antiturnor compared to radiolabeled antibody alone Two deaths unrelated to tumor growth one animal of eight (12.5%) in the 2.5 2.0 Control 1.5 activity of 13‘I-mAbA33 (data not shown). occurred during treatment: group treated with 5-Hi daily X 5 + ‘31I-mAb PBS and one ofsix (16.5%) in the group treated 1.0 with doxorubicin + ‘311-mAb FB5. Minor weight loss was observed in several animals treated with ‘31I-mAbPBS and with doxorubicin. No signs of toxicity were seen in animals treated with 5-Ri and LV 0.5 0.0 as single agents and with any combinationplus ‘311-mAb A33. 048121620242832364044485256606468 DISCUSSION ci;-. 2.5 2 2M E 5-Ri 30 mg/kg daily x 5 11.5 > Adjuvant chemotherapy, radiation, and immunotherapy have shown some usefulness in the treatment of primary colorectal carci norna and its more advanced stages (3 1). However, agents with better antitumor activity need to be identified if significant progress is to be made. Recent studies have shown a significant survival benefit (6, 32) for patients treated with chemotherapy and external beam radiation after curative surgery for rectal cancer. The underlying cause for this phenomenon is thought to be radiosensitization. In vitro and in vivo studies of the combined effects of 5-Hi and radiation (2 1, 33—37) have suggested that any increase of effect over radiation alone is not due to inhibition of repair of sublethal or potentially lethal damage. The mode of interaction of the drug with radiation remains unclear. In addition, the sequencing of 5-Hi and radiation treatments may be a crucial parameter. It has been suggested that synergism could be achieved only by 5-Hi infusions, with brief injection schedules yield ing additive effects (35). Other investigators (21, 38) found only additive antitumor effects without evidence for sequence dependence. Nevertheless, clinical trials have suggested a greater than expected 1.0 5- 0 E — 0.0 048121620242832364044485256606468 2.5 @ @@..##F:_F(J 75 mg/kg on days 1 and 5 @ 0.0! t t I I 048121620242832364044485256606468 I local control rate with 5-Hi chemoradiation for both squamous cell I days after start of treatment Fig. I. Effect of 5-FU on tumor growth. Control, n = 8; MTS, 0.375 cm3. 5-Ri daily, n = 6; MTS, 0.344 cm3. 5-FU bolus, n 6; MTS, 0.369 cm3. carcinomas and adenocarcinomas (39). In colon cancer, external beam radiation has its limits for technical reasons and because of toxicity to adjacent organs. Lacking these drawbacks, radiolabeled antibodies might be an alternative to deliver 2.5 bination therapies was significantly different from that of 5-Hi alone or ‘311-rnAb A33 alone (Fisher's exact test, P < 0.05). A synergistic antitumor effect is suggested when the groups are compared for @ Control survival over the entire observation period (276 days). @ A similar set of experiments comparing the combination of 5-Hi + LV and the addition of ‘311-mAbA33 showed that chemotherapy and radioimmunotherapy have significantly more antitumor effects (Fig. 5), whereas the addition of ‘31I-mAbFBS to 5-PU + LV did not increase antitumor activity significanfly (Fig. 5). To determine whether combination therapy had additive or synergistic effect on reduction of tumor size, differences in tumor volume were analyzed. When the tumor weight of mice reached 2 g, the @ animals @ 2.0 1,5 were sacrificed. Therefore, the analysis was carried ,— 0.5 ‘ E -@ > 048121620242832364044485256606468 2.5 out only 2 0 until tumor volumes reached 2 cm3. No statistical differences were observed in the analysis evaluating synergistic antitumor effects (by 1.5 1311-mAbFBS tumor volume) for the combination of 5-Ri given daily X 5 or on days 1 and 5 + ‘311-mAbA33 and either treatment alone for days 1—28.However, the two different treatment modalities had additive antitumor activity. To determine whether the enhanced tumor-suppressive effect of 5-Hi + ‘311-mAb A33 chemotherapeutic with or without LV might be common 1.0 0.5 0.0 048121620242832364044485256606468 to days after start of treatment agents in general or specific to certain drugs, we tested combinations of doxorubicin + ‘311-mAbA33 and carmustine + ‘311-mAb A33. The addition of these two chemotherapeutic Fig.2. Effectof control‘311-mAb J2@5 on tumorgrowth.Control,n = 8; MTS,0.375 cm3.‘311-mAb FB5.n = 6; MTS.0.345cm3. 2183 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1997 American Association for Cancer Research. COMBINATION RADIOIMMUNOTHERAPYAND CHEMOTHERAPY radiation therapy in colon cancer. The realistic evaluation of radiola 2.5 beled antibodies as radiotherapeutic agents, however, depends on 2.0 accurate dosimetry calculations. This is key information for radioim munotherapy trials in solid tumors that will require continuous mon itoring of isotope levels in essential tissues for the length of time the isotope is retained. Positron emission tomography analysis offers an approach to obtaining this information. Another important consideration is rnicrodosimetry, which takes into account the architecture of the tumor and the distribution of 1.5 1311-mAbA33 1.0 0.5 0.0 .@ (%J c'J %@ Q@o r'@ m //@ %@ @O ‘0 antibody. Our earlier studies with mAb A33 have shown that local ization was specific when @mTc@albunün was compared to radiola 2.5 2.0 2.5 5-FU30 mg/kg daily xS 1.5 2.0 1.0 1.5 5-Ri 30 mg/kg daily x S 0.5 1.0 0.0 0.5 @ @ 0 4—I--I e'J ‘.4 -@ @O m 0 @e ‘0 C'J i―. ‘@ ‘0 ‘0 N. 01 0.0 5-FU 30 mg/kg daily x 5 048121620242832364044485256606468 ‘@—2.5 E + w E @ 25 @ 2.0 @ 1.5 + 1311-mAb @ 0.5 FB5 c@-.. A33 @/I! 1 ..—.—. S 0.0 1.0 @ 1.5 1.0 5-Ri 30 mg/kg daily x 5 @ 1311-mAb I ‘@ 2.0 0 -GO-C (‘.4 ‘-I ‘4 10 (,@ ‘@ 0 C―J ‘0 N. ‘0 10 01 N. 0.5 ,@0.0 E 2.5 0 4 8121620242832364044485256606468 E 2.0 1.5 0 > @ — @ @ 2.5 1.0 2.0 0.5 5-FU75mg/kgondays1and5 . 0.0 1.5 days I and S 0 c@4 — ‘4 c@J ‘0 0 (@J — ‘a r'j ‘0 rn . . . I, -a 0 ‘0 (Si N- @0 0 ‘0 C@J N- ‘4 ‘0 01 F, ‘0 N. 1.0 0.5 2.5 0.0 2.0 048121620242832364044485256606468 1.5 1.0 @ @ 2.5 0.5 2.0 0.0 1.5 5-nj75 mg/kgon 1.0 days 1 and 5 + 1311-mAbFB5 ‘0 Q, ‘0 N. days after start of treatment Fig. 4. Effect of addition of specific 31I-mAb A33 radioimmunotherapy to chemo therapy on tumor growth. ‘31I-mAb A33, n = 8; MTS, 0.329 cm3. 5-RI daily, n = 6; MTS, 0.344 cm3. 5-FU daily + ‘311-mAbA33, n = 8; MTS, 0.392 cm3. 5-Ri bolus, n = 6; MTS, 0.369 cm3. 5-PU bolus 4- ‘311-mAb A33, n = 8; MTS, 0.376 cm3. 0.5 0.0 0 4 8 121620242832364044485256606468 days after start of treatment Fig. 3. Effect of nonspecific radioimmunotherapy beled mAb A33 as a marker for tumor blood pool uptake, and when in combination with chemotherapy on tumor growth. 5-FU daily, n = 6; MTS, 0.344 cm3. 5-FU daily + ‘31I-mAb FB5, n = 8; MTS, 0.342 cm3. 5-FU bolus, n FB5, n = 8; MTS, 0.349 cm3. 6; MTS, 0.369 cm3. 5-FU bolus + ‘311-mAb a ‘31I-labeledisotype-matched control antibody was compared to ‘25I@@@b A33 (28). Using autoradiographs, we were able to show that isotope accumulation in tumors corresponded to the antibody binding 2184 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1997 American Association for Cancer Research. COMBINATION RADIOIMMUNOTHERAPYAND CHEMOTHERAPY in other therapeutic systems, it was shown that human xenografts in mice seem to retain histology and chemosensitivity of the original tumor (40) and that radiosensitivity of individual tumor cells corre lates well with the original tumor (41). For the studies presented here, we selected a ‘311-mAbA33 dose that was 20% of the MTD. On the basis of the % ID/g tumor observed in this animal model, peak ‘@I uptake corresponds to 0.035 mCi/g. In our clinical studies with ‘311-mAb A33, uptake (% Dig) was meas ured by biopsy on day 7 or 8 after antibody administration. In dosimetry studies, patients with the highest uptake of 1311-mAb A33 would achieve isotope concentrations of 0.015—0.045 mCi/g if treated at the MTh (75 mCi/rn2). Thus, the effects observed in the present 2.5 2.0 5-FU+ leucovorin 1.5 1.0 0.5 0.0 048121620242832364044485256606468 ‘@— E 2 2.5 study may be correlated directly to the subgroup of patients with the 2.0 highest radiolocalization indices for ‘311-mAb A33. Our results show an additive effect of combination chemotherapy (5-Hi or 5-Hi + LV) plus low-dose radioimmunotherapy. When survival was used as an end point, a synergistic effect was found, in contrast to earlier findings in a mouse radioimmunotherapy model using an anti-CEA antibody (42). No differences were observed in antitumor 11.5 > 1.0 + leucovorin 5- + 1311-mAb FB5 0 E 0.5 — 0.0 effects when the two 5-Hi treatment schedules were compared, and 048121620242832364044485256606468 2.5 2.0 1.5 S-nj + teucovorin 4 1311-mAb A33 1.0 0.5 0.0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 days after start of treatment Fig. 5. Effect of addition of LV to combination chemoradioimmunotherapy. 5-FUlLy, n = 8; MTS, 0.350 cm3. 5-FUlLy + ‘311-mAbFBS, n = 8; MTS, 0.325 cm3. 5-Ri! LV + ‘31mAb A33, n = 8; MTS, 0.387 cm3. specifically to the tumor cells while the surrounding stromal cells and vasculature did not concentrate the isotope. Thus, dosimetry calcula tions have provided only estimates of radiation doses delivered to tumor cells. Single-cell dosimetry with long-range @3-emitterssuch as ‘@‘i provide an even greater challenge. In general, our present knowl edge is based on external and biopsy-based measurements; however, tumor tissue doses reported to be absorbed are only a fraction of what is needed to eradicate tumors (27). Human cancer xenograft models in nude mice have proved to be valuable in testing the therapeutic potential of new reagents such as radiolabeled antibodies. However, when evaluating the results of these models, an important consideration is the limitation in predicting efficacy in human studies. One major consideration is that the anti body uptake by the tumor can be 3000-fold higher in the mouse model than in patient studies (30% versus 0.01% DIg tumor). This is partially compensated for by the higher total dose used in humans compared to mice. Because the MTD of 131!in humans is almost 300 times that in mice, this still leaves a 10-fold higher tumor tissue dose in mice than in humans at the MTD. Thus, it is important to select doses for experiments with animal models that are a fraction of the MTD. In general, animal models may in some cases exaggerate the antitumor effect of experimental therapy regimens, as it does with radiolabeled antibodies. However, when this issue has been examined both demonstrated enhanced activity when combined with specific radioimmunotherapy. In the clinic, the dose-limiting toxicity for bolus and continuous-infusion 5-Ri is different, and the relative lack of hematological toxicity of the continuous-infusion schedule makes this an attractive partner for ‘311-labeled antibody-based therapies. Due to the low nontumor radiation dose and specific uptake of radiolabeled antibody, we could not detect any signs of toxicity in our animals. In patients treated with 5-Hi/mAb A33 chemoradioimmunotherapy, we might expect enhanced antitumor effects with little change in toxicity, due to the low nonspecific radiation doses delivered to critical tissues manifesting 5-Ri toxicity. The enhanced antitumor effects were spe cific for 5-Hi-based treatments as the addition of other chemothera peutic agents (doxorubicin and carmustine or even LV) to ‘311-mAb A33 had no beneficial effect. In theoty, mAbs can deliver radiation therapy continuously over pro longed periods. Hyperfractionated radiation dosing, in which the rationale is based on growth and on radiation repair patterns of tumor cells, has shown advantages over single doses (43—45).After a single antibody administration, the actual dose rate to the tumor does not simulate hyperfractionation protocols due to the half-life effect of the isotope used (e.g., ‘@‘I) and the continuous clearance of isotope from the tumor. With the generation and clinical use of humanized antibodies, therapeutic regimens may be developed that will approximate hyperfractionation because the humanized antibody can be given repeatedly. The question of whether the response to continuous, exponentially decreasing irradiation, which is characteristic of radioimmunotherapy, is biologically equivalent to conventionally fractionated, high-dose rate irradiation remains to be solved. Very few studies have addressed this question (46—53).Our results here have clearly demonstrated an improvement in antitumor effects by chemoradioimmunotherapy in an animal model. This finding should be tested in clinical studies in subsets of patients with advanced disease, who have not yet been treated with 5-Hi. We have observed that in a group of heavily pretreated patients, who received chemotherapy after a Phase I radio immunotherapy trial with ‘251-mAbA33, a significant number had major responses (54). These unexpected findings need to be explored further and verified in well-designed clinical studies. Preclinical stud ies to identify other chemotherapeutic agents with additive or syner gistic activity when combined with radiolabeled antibodies should continue, especially with the newer agents identified recently as having activity in colon cancer. 2185 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1997 American Association for Cancer Research. COMBINATION RADIOIMMUNOTHERAPYAND CHEMOTHERAPY 29. Houghton, P. J., Houghton, J. A., Myers, L, Chesire, P., Howbert, J. J., and Grindey, REFERENCES G. B. Evaluationof N-(5-indanylsulfonyl)-N-(4-chlorophenyl)-urea againstxenograftsof I . Schneebaum, S., and Arnold, M. W. Adjuvant treatment for rectal cancer: current status. Oncology (Basel), 7: 83—90,1993. 2. Gastrointestinal Tumor Study Group. Prolongation of the disease free interval in surgically treated rectal carcinoma. N. EngI. J. Med., 312: 1465—1472, 1985. 3. Douglass, H. 0., Jr., Moertel, C. G., Mayer, R. J., Thomas, P. R., Lindblad, A. S., Mittleman, A., Stablein, D. M., and Bruckner, H. W. Survival after postoperative combination treatment of rectal cancer. N. EngI. J. Med.. 315: 1294—1295, 1986. 4. Fisher, B., Wolmark, N., Rockette, H., Redmond, C., Deutsch, M., Wickerham, D. L., Fisher, E. R., Caplan, R., Jones, J., and Lamer, H. Postoperative adjuvant chemo therapy or radiation therapy for rectal cancer: results from NSABP protocol R-Ol. J. NatI. Cancer Inst., 80: 21—28,1988. 5. Gastrointestinal Tumor Study Group. Radiation therapy and fluorouracil with or without semustine for the treatment of patients with surgical adjuvant adenocarci noma of the rectum. J. Clin. Oncol., 10: 549—557,1992. 6. Krook, J. E., Moertel, C. G., Gunderson, L. L., Wieand, H. S., Collins, R. T., Beart, U. R. W., Kubista, T. P., Poon, M. A., Meyers, W. C., Mailliard, J. A., Twito, D. I., Morton, R. F., Veeder, M. H., Witzig, T. E., Cbs, S., and Vidyarthi, S. C. Effective surgical adjuvant therapy for high-risk rectal carcinoma. N. Engl. J. Med., 324: 709—715, 1991. 7. Comis, R. L., and Carter, S. K. Integration of chemotherapy into combined modality treatment of solid tumors. Cancer Treat. Rev., I: 221—238,1974. 8. Ramming. K. P., and Haskell, C. M. Colorectal malignancies. In: C. M. Haskell (ed), Cancer Treatment, Ed. 2., pp. 295—334. Philadelphia: W. B. Saunders, 1985. 9. Cohen, A. M., Shank, B., and Friedman, M. A. Colorectal cancer. In: V. T. De Vita, Jr., S. Hellman, and S. A. Rosenberg (eds.), Cancer: Principles and Practice of Oncology, Vol. 1, Ed. 3, pp. 895—964. Philadelphia: J. B. Lippincott, 1989. 10. Bleyer, W. A. New vistas for leucovonn in cancer chemotherapy. Cancer (Phila.), 63: 995—1007, 1989. I 1. Rustum, Y. M. Toxicity and antitumor activity of 5-fluorouracil in combination with leucovorin. Cancer(Phila.). 63: 1013-1017,1989. 12. Houghton, J. A., Williams, L. G., Loftin, S. K., Cheshire, P. J., Morton, C. L., Houghton, P. J., Dayan. A., and Jolivet, J. Factors that influence the therapeutic activityof 5-fluorouracil(6RS)leucovorincombinationsin colonadenocarcinoma xenografts. Cancer Chemother. Pharmacol., 30: 423—432,1992. I3. Advanced Colorectal Cancer Mets-analysis Project. Modulation of fluorouracil by rates. J. Clin. Oncol., 10: 896—903,1992. 14. Laufman, L. R., Brenckman, W. D., Stydnicki, K. A., Morgan, E. D., Collier, M., Knick, V. B., Mullin, R., and Ferone, R. Clinical experience with leucovorin and 5-fluorouracil. Cancer (Phila.), 63: 1031—1035,1989. 15. Arbuck, S. G. Overview of clinical trials using 5-fluorouracil and leucovorin for the treatmentof colorectalcancer.Cancer(Phila.),63: 1036—1044, 1989. 16. Mini, E., Trave, F., Rustum, Y. M., and Bertino, J. R. Enhancement of the antitumor effects of 5-fluorouracil by folinic acid. Pharmacol. Ther., 47: 1—19, 1990. 17. Intemational Multicenter Pooled Analysis of Colon Cancer Trials (IMPACT) Inves tigators. Efficacy of adjuvant fluorouracil and folinic acid in colon cancer. Lancet, 345: 939—944,1995. 18. Laufman, L. R., Bukowski, R. M., Collier, M. A., Sullivan, B. A., McKinnis, R. A., Clendennin, N. J., Guaspari, A., and Brenckman, W. D., Jr. A randomized, double plus placebo versus fluorouracil plus oral leucovorin in patients with metastatic colorectal cancer. J. Clin. Oncol., II: 1888—1893,1993. 19. Leichman, C. G., Fleming. T. R.. Muggia, F. M., Tangen, C. M., Ardalan, B., Doroshow, J. H., Meyers, F. J., Holcombe, R. F., Weiss, G. R., and Mangalik, A. Phase II study of fluorouracil and its modulation in advanced colorectal cancer: a Southwest Oncology Group Study. J. Clin. Oncol., 13: 1303—1 3 11, 1995. 20. Fu, K. F., Rayner, P. A., and Lam, K. N. Modification of effects of continuous low dose rate irradiation by concurrent chemotherapy infusion. Int. J. Radiat. Oncol. Biol. Phys.,10: 1473—1478, 1984. 21 . Weinberg, M. J., and Rauth, A. M. 5-Fluorouracil infusions and fractionated doses of radiation: studies with a murine squamous cell carcinoma. mt. J. Radiat. Oncol. BioI. Phys.,13: 1691—1699, 1987. 22. Byfield, J. E., Chan, P. Y. M., and Seagren, S. L. Radiosensitization 18: 74, 1977. 23. Byfield, J. E., Barone, R. M., Seagren, S. L., Frankel, S., Quinol, L., and Mendelsohn, the survival of mammalian cells in vitro and on early recovery between fractionated X-ray doses. Br. J. Radiol., 34: 458—465,1966. 35. Byfield, J. E., Calabro-Jones, P., Klisak, I., and Kullianian, F. Pharmacologic require mentsfor obtainingsensitizationof humantumorcellsin vitroto combined5-flu orouracil or Ftorafur and X-rays. tnt. J. Radiat. Oncol. Biol. Phys., 8: 1923-1933, 1982. 36. Nakajima, V., Miyamoto, Y., Tanabe, M., Watanabe, I., and Terasima, T. Enhance ment of mammalian cell killing by 5-fluorouracil in combination with X-rays. Cancer Res., 39: 3763—3767,1979. 37. Rich, T. A., Kavanaugh, B., Williams, M., Bock, S., Murray. D., Meyn, R., and Brock, W. A. Effects of continuous post-irradiation low dose 5-fluorouracil on log and plateau phase CHO cells. Proceedings, Chemical Modifiers ofCancer Treatment, Clearwater, FL, Abstr. 6—21,1985. 38. Stephens, J. C., Peacock, S. H., and Steel, G. G. Cell survival in Bl6 melanoma after treatment with combination of cytotoxic agents: lack of potentiation. Br. J. Cancer, 36: 84—93,1977. 39. Schein, P. S., Gore, M. E., and Buckman, R. A. Radiation therapy and 5-fluorouracil. Drugs Exp. Clin. Rca., 8: 587—592,1982. 40. Winograd, B., Boven, E., Lobezzo, M. W., and Pinedo, H. M. Human tumor xenografts in the nude mouse and their value as test models in anti-cancer drug development. In Vivo, 1: 1—14, 1987. growth delay and survival data. in Vivo,4: 253—258,1990. 42. Chalandon, Y., Mach, J-P., Pèlegrin, A., Folli, S., and Buchegger, F. Combined radioimmunotherapy and chemotherapy of human colon carcinoma grafted in nude mice: advantages and limitations. Anticancer Res., 12: 1131—1140,1992. 43. Fowler, J. F. Briefsummary ofradiobiological principles in fractionated radiotherapy. Semin. Radiat. Oncol., 2: 16—21,1992. 44. Fowler, J. F. Intercomparisons of new and old schedules in fractionated radiotherapy. Semin. Radiat. Oncol., 2: 67—72,1992. 45. Thames, H. D., Jr. Withers, H. R., and Fletcher, G. H. Changes in early and late radiation responses with altered dose fractionation: implications for dose survival relationship. Int. J. Radiat. Oncol. Biol. Phys., 8: 219—226, 1992. 46. Knox, S. J., Goris, M. L., and Wessels, B. W. Overview of animal studies comparing radioimmunotherapy with dose equivalent external beam irradiation. Radiother. On col., 23: 111—117, 1992. 47. Buchsbaum, D. J., Ten Haken, R. K., Heidorn, D. B., Lawrence, T. S., Glatfelter, A. A., Terry, V. H., Guilbault, D. M., Steplewski, Z., and Lichter, A. S. A comparison of ‘311-labeledmonoclonal antibody 17—IAtreatment to external beam radiation on the growth of LSI74T human colon cancer xenografts. mt. J. Radiat. Oncol. Biol. Phys., 18: 1033-1041, 1990. 48. Buras, R. R., Wong, J. Y. C., and Kuhn, J. A. Comparison of radioimmunotherapy and external beam radiotherapy in colon cancer. Int. J. Radiat. Oncol. Biol. Phys., 16: 12—16,1991. 49. Knox, S. J., Levy, R., Miller, R. A., Uhland, W., Schiele, J., Ruehl, W., Finston, R., monoclonal antibodies. Cancer Res., 50: 4935—4940, 1990. 50. Neacy, W. P., Wessels, B. W., Bradley, E. W., Kovandi, S., Justice, T., Danskin, S., and Sands, H. Comparison of radioimmunotherapy and 4 MV external beam J. Biweekly infused 5-fluorouracil and X-rays in advanced gastro-intestinal cancer. Proc. Am. Soc. Clin. Oncol., 18: 322—324,1978. 24. Calabro-Jones, P. M., Byfield, J. E., Ward, J. F., and Sharp, T. R. Time dose relationships for 5-fluorouracil cytotoxicity against human epithelial cells in vitro. radiotherapy of tumor xenografts in athymic mice. J. Nucl. Med., 27: 902—903, 1986. 51 . Palme, D. F., Berkopec, J. M., Elson, M. K., Bono, M. D., Wessels, B. W., Lange, P. H., andVessella, R. L. Immunotherapyof renalcell carcinomaxenograftsby 1-131 monoclonal antibody A6H compared to single fraction external beam irradiation. Cancer Res., 42: 4413—4415,1982. F., and Valeriote, F. Combined effect of X-radiation and 5-fluorouracil on survival of transplanted leukemia cells. J. NatI. Cancer Inst., 47: 865—870, 1971. 26. Welt, S., Divgi, C. R., Kemeny, N., Finn, R. D., Scott, A. M., Graham, M., St. Germain, J., Carswell Richards, E., Larson, S. M., Oettgen, H. F., and Old, L. J. Phase I/Il study of iodine 131-labeled monoclonal antibody A33 in patients with advanced colon cancer. J. Clin. Oncol., 12: 1561—1571, 1994. J. Nucl. Med., 28: 651, 1987. 52. Vessella, R. L., Palme, D. F., Berkopec, J. M., Elson, M. K., Truillo, G., Wessels, B. W., andLange,P. H. A comparisonof escalatingsingle andmultiplefractionlow dose rate radioimmunotherapy with monoclonal antibody A6H 1-131 conjugates to high dose rate external beam therapy on human renal cell carcinoma xenografts. J. Nucl. Med., 29: 875, 1988. 53. Wessels, B. W., Vessella, R. L., Palme, D. F., Berkopec, J. M., Smith, G. K., and Bradley, E. W. Radiobiological comparison of extemal beam irradiation and radio 27. Goldenberg, D. M. (ed). Cancer Therapy with Radiolabeled Antibodies. Boca Raton: CRC Press, 1995. 28. Welt, S., Divgi, C. R., Real, F. X., Yeh, S. D., Gain Lancet, 343: 1177—1 183, 1994. 32. Gastrointestinal Tumor Study Group. Survival after postoperative combination treat ment of rectal cancer. N. Engl. J. Med., 315: 1294—1295,1986. 33. Bagshaw, M. A. Possible role of potentiators in radiation therapy. Am. J. Roentgenol., 85: 822—833,1961. 34. Berry, R. J. Effects of some metabolic inhibitors on X-ray dose response curves for Day-Lollim, P., and Goris, M. L. Determinants of the antitumor effect of radiolabeled by 5-Ri: molecular origins and clinical scheduling implications. Proc. Am. Assoc. Cancer Res., 25. Vieni, T., Eggerding, Wine, J., and German Cancer Aid 17—IAStudy Group. Randomised trial of mono clonal antibody for adjuvant therapy of resected Dukes C colorectal carcinoma. 41. Schwachofer, J. H. M., Hoogenhout, J., Kal, B., Koedam, J., and Van Wezel, H. P. N. Radiosensitivity of different human tumor lines grown as xenografts determined from leucovorin in patients with advanced colorectal cancer: evidence in terms of response blind trial of fluorouracil pediatric rhabdomyosarcoma. Cancer Chemother. Pharmacol., 24: 84—88,1989. 30. Liang, K., and Zeger, S. L. Longitudinal data analysis using generalized linear models. Biometrika, 73: 13—22,1986. 31. RiethmUller, G., Schneider-GEdicke, E., Schlimok, G., Schmiegel, W., Raab, R., Hdffken, K., Gruber, R., Pichlmaier, H., Hirche, H., Pichimayr, R., Buggisch, P., Chesa, P., Finstad, C. L., Sakamoto, J., Cohen, A., Sigurdson, E. R., Kemeny, N., Carswell, E. A., Oettgen, H. F., and Old, L. L. Quantitative analysis of antibody localization in human metastatic colon cancer: a phase I study with monoclonal antibody A33. J. Clin. Oncol., 8: 1894—1906,1990. immunotherapy in renal cell carcinoma xenografts. mt. J. Radiat. Oncol. Biol. Phys., 17: 1257—1263, 1989. 54. Welt, S., Scott, A. M., Divgi, C. R., Kemeny, N. E., Finn, R. D., Daghighian, F., St. Germain, 1., Richards, E. C., Larson, S. M., and Old, L J. Phase 1/11study of iodine 125-labeled monoclonal antibody A33 in patients with advanced colon cancer. J. Clin. Oncol., 14: 1787—1797, 1996. 2186 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1997 American Association for Cancer Research. Enhanced Antitumor Activity of Combination Radioimmunotherapy ( 131I-labeled Monoclonal Antibody A33) with Chemotherapy (Fluorouracil) Jörg Tschmelitsch, Els Barendswaard, Clarence Williams, Jr., et al. Cancer Res 1997;57:2181-2186. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/57/11/2181 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1997 American Association for Cancer Research.