(CANCER RESEARCH 50, 159-163. January I. 1990]

Enhancement of Monoclonal Antibody Uptake in Human Colon Tumor Xenograftsfollowing Irradiation1

H. Kalofonos, G. Rowlinson, and A. A. Epenetos2

Imperial Cancer Research Fund Oncology Group, Royal Postgraduate Medical School, Hammersmith Hospital, London W120HS, England

ABSTRACT

Indium-Ill-labeled Al Al tumor-associated monoclonal antibody

raised against an antigen of colon adenocarcinoma was used to evaluatethe effect of ionizing radiation on antibody uptake by the LoVo adenocarcinoma cell line grown as a xenograft in nude mice. Tumors wereexposed to single doses of external X-irradiation of between 400 and

1600 cGy followed, 24 h later, by administration of specific or nonspecificantibody. Animals were sacrificed 3 days after antibody administration.At doses higher than 400 cGy, tumor uptake with both specific andnonspecific antibody was significantly increased. No difference in changesin tumor volume was observed between the groups receiving irradiationand the controls. Specific antibody uptake by tumors was always significantly higher than nonspecific having an approximate 4-fold binding

advantage. Vascular permeability and the vascular volume of irradiatedand control tumors was measured 24 and 72 h after irradiation, usingiodine-125-labeled nonspecific antibody and labelling of the red bloodcells in vivo with "TcO.t. At doses higher than 400 cGy, vascular

permeability in the tumor 24 h after irradiation was significantly increased (P < 0.05), while the vascular volume decreased (P < 0.001)compared to control values. However at 72 h after irradiation there wasno difference between treated and control groups.

The results obtained in this study suggest a potential value of externalirradiation to increase monoclonal antibody uptake by tumors governedmainly by the increased vascular permeability of the tumor vasculaturesoon after the irradiation exposure.

INTRODUCTION

The usefulness of radiolabeled monoclonal antibodies fordiagnosis (1) and therapy (2) of human tumors has been recognized, but a significant limitation for their application in vivois the low absolute amount reaching the target (3). The localization of monoclonal antibodies on the solid tumors is assumedto be governed by passive diffusion and binding on tumor cells.This depends on many factors such as antibody affinity, bindingof the injected antibody by circulating antigen, density of theantigen expressed on tumor cells, vascular content of the tumorand accessibility of the antibody to solid tumors (4-6).

It has been previously shown that a single dose of externalirradiation can increase monoclonal antibody uptake by tumors such as colorectal carcinoma (7), melanoma (8), andhepatoma (9), grown in mice and in an anecdotal hepatomacase reported by Order et al. (10). Ionizing radiation has beenshown to change the vascular volume and the vascular permeability to macromolecules in tumors. Paradoxically increasedand decreased vascular volume have been observed dependingupon the study and the time of observation after the irradiation(11, 12). The exposure of several different tissues to ionizingirradiation leads to a disruption of the vascular endothelium(13-15) and a quantitative increase in vascular permeability(15-18).

The purpose of this study was to conduct a comprehensiveevaluation of the effect of a single dose of external irradiation

Received 3/20/89; revised 7/31/89; accepted 10/2/89.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by the Imperial Cancer Research Fund.2To whom requests for reprints should be addressed, at ICRF Oncology

Group, Hammersmith Hospital, DuCane Road, London W120HS. England.

on the uptake of specific and nonspecific monoclonal antibodyby a human colon adenocarcinoma xenograft in nude mice. Afurther aim was to assess and correlate the vascular volume andvascular permeability in this tumor model following irradiation.

MATERIALS AND METHODS

Animals and Tumors

Twelve-week-old male athymic nu/nu (nude) mice, from a colony ofmixed genetic background, were bred in the ICRF Animal BreedingUnit (South Mimms, Hertfordshire, UK). The mice were housed insterile cages and maintained on irradiated diet and acidified water (pH2.8). Xenografts were established s.c. by the inoculation under the skinof the flank of a single cell suspension of 5.106 cells in 0.1-ml medium

of a human tumor cell line of a colon carcinoma designated LoVo (19).Tumor cells were grown in large tissue culture flasks in RPMI 1640medium supplemented with 10% FCS.3 Cells were harvested by incu

bation with 0.02% EDTA for 5 min and washed with serum freemedium. Dimensions of tumors were measured before the irradiationas well as at the day of dissection to determine whether irradiation hada detectable effect on tumor volume. Tumor volume was determinedfrom orthogonal measurements of three diameters using the formula:

y = (4ir/3) x (dl x d2 x d3)/8

where V is the tumor volume, dl, d2, and d3 are the three differentdiameters. Mice were randomly allocated to treatment groups of fiveto six. The mean tumor volume in each group of mice was not significantly different from the others.

Monoclonal Antibodies

AUA1. This is a murine IgG, raised in the conventional fashion byimmunising BALB/c mice with the colon adenocarcinoma cell lineLoVo (20). It recognizes a M, 35,000 antigen located on basolateralaspect of the cell membrane of a wide variety of neoplastic cells and alimited set of normal epithelial tissues (21). AUA1 is an epithelialspecific antibody and reacts with the majority of human gastrointestinal, ovarian and breast carcinomas, as well as proliferating epithelialcells in tissues such as normal colon.

HMFG1. This is a murine IgG, and defines a tumor-associatedantigen on a high molecular weight glycoprotein which is stronglyexpressed in lactating breast as well as in a range of neoplasms such asbreast cancer, ovarian, and nonsmall cell lung cancer (22). This antibodydoes not react with LoVo adenocarcinoma and was used as a negativecontrol.

Radiolabeling of Antibodies

Monoclonal antibodies were radiolabeled with '"In (0.04 M, carrier

free, INS. 1; Amersham International, Amersham, UK) using DTPA,the cyclic anhydride (Sigma), as described by Hnatowich et al. (23). Inthe vascular studies the HMFG1 was labeled with '"I (IBS.3, Amer

sham International, Amersham, UK), in order to determine the permeability of tumor vasculature to IgG. lodination was carried out iniodogen coated tubes using an established technique (24). lodinatedantibody was purified before each experiment by passage through acolumn with Sephadex G50 (Pharmacia, Sweden) to remove the free

' The abbreviations used are: PCS, fetal calf serum; FACS. fluorescence-

activated cell sorting; FPLC, fast protein liquid chromatography: Ht. haematocrit;PBS, phosphate buffered saline: SDS-PAGE, sodium dodecyl sulfate-polyacryl-amide gel electrophoresis; VV, vascular volume; VP, vascular permeability.

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ANTIBODY UPTAKE BY TUMORS AFTER IRRADIATION

iodine. Radiolabeled antibodies were 0.22 /mi millipore filtered priorto administration to mice.

Immunoreactivity

A radiobinding assay under conditions of antigen excess was performed as described by I .indino et al. (25). The immunoreactive fractionof the radiolabeled antibody was determined by extrapolation in adouble-inverse linear plot corresponding to infinite antigen excess.

Affinity was determined from Scatchard plot of the binding of labeledantibody to whole LoVo cells. Nine successive 1:3 dilutions of antibody,starting at 30 ^g/ml in 100 /il, in PBS buffer with 2% PCS and 0.02%sodium azide were mixed with 100 n\ 2 x IO6 LoVo cells/ml in LP-3

tubes and allowed to incubate for l h at room temperature with periodicmixing. Standards of the radiolabeled antibody were left for comparison. The cells were then washed three times with PBS containing 2%PCS, and bound radioactivity was counted following centrifugation, bysubtracting the nonspecifically bound radioactivity which was determined separately in tubes containing excess unlabeled antibody.

Flow cytometric analysis of cell surface phenotypes was carried outusing indirect fluorescence. LoVo cells (1 x IO6 in 100 pi) were

incubated for l h at room temperature with 100 n\ of the variousmonoclonal antibodies (10 ¿ig/ml).The cells were then washed threetimes with PBS containing 2% PCS, after which they were mixed withfluorescein isothiocyanate-conjugated rabbit antimouse IgG (Dako,Denmark) followed by 30 min incubation at 4°C.The cells were then

washed three times and resuspended in 0.5 ml buffer and subjected tocell sorting. Flow microfluorometry was performed using fluorescenceactivated cell sorting (Epics profile, Hialeah, PL).

In Vitro and in Vivo Stability

In vitro and in vivo stability were tested by performing serial SDS-PAGE and autoradiography. SDS-PAGE was performed using 8%gradient acrylamide gels, with a 4% stacking gel under nonreducingconditions before and after the radiolabeling of monoclonal antibodiesto reaffirm purity and to determine the molecular weight. In addition,aliquots of the radiolabeled antibody were subjected to FPLC analysisusing a Superóse6 column (Pharmacia).

The characteristics of antibody circulating in the blood were alsoevaluated as a measure of the in vivo stability. The circulating radioactivity which was not associated with the antibody was assessed bycolumn chromatography (Sephadex G50).

Immunohistological Staining

A two-step indirect immunoperoxidase reaction was performed onfresh frozen tissue sections. Negative and positive control antibodieswere included in all tissue sections. The concentration of the antibodiesused was

External Beam Irradiation

Tumors were exposed to a single dose of X-rays ranging from 400to 1600 cGy using a beam of radiation from a 240- KeV machine (half-value layer =1.1 mm of Cu from a PANTAK, MRC Cyclotron Unit,Hammersmith Hospital). All mice were irradiated under anesthesiawith fentanyl (0.79 mg/kg) and fluanisone (25 mg/kg; Hypnorm, CrownChemical Co. Ltd., Lamberhurst, UK) and midazolam (12.5 mg/kg;Hypnovel, Roche, Welwyn Garden City, UK), in a specially constructedjig with 0.3-cm-thick lead to shield the normal tissues. Tumors wereexposed to radiation via 20- x 15-mm holes in the lead screen. Groupsof at least five mice were exposed to a range of single doses (400, 800,1200, 1600 cGy). Control animals were prepared and treated in thesame way as those to be irradiated, except for the radiation exposureitself. To ensure uniform doses throughout the tumor volume, the micewere turned 180°half-way through each irradiation to obtain optimum

uniformity of dose distribution. The exposure dose rate, as determinedby inserting thermoluminescent dosimeter ribbons into tumors of micefor dosimetrie purposes was 91.6 Gy/min.

Localization Study

with Indium-Ill. Each mouse received a dose of between 15 and 20nC\, depending on the specific activity. Three days later, tumor dimensions were remeasured, mice were killed by exsanguination and immediately dissected. Blood, visceral organs, muscle samples, bone andtumor were obtained, washed in PBS containing 0.5% heparin, blotteddry, weighed, and counted in a gamma-counter (Canberra Packard5550) against standards of the injectate. Data were analyzed as percentage of injected dose per gram in tissue relative to that in blood tominimize the possible variation of isotope dose among the animals, dueto the i.p. route of administration, which can result in variable absorption of antibody.

Vascular Studies

Vascular volume and vascular permeability were determined in thetumor, muscle, liver, spleen, and kidney of irradiated and control mice24 and 72 h after irradiation or control procedures using the methodof Song and Levitt (12) as modified by Sands et al. (6). The radiationdose to the tumor was 400, 800, 1200, or 1600 cGy in one fraction.Erythrocytes were radiolabeled with ""Te in vivo after the administra

tion of 1.2 /<nin 100 //I of stannous fluoride (Amersham International,Amersham, UK) into the tail vein. Thirty min later a mixture of 25 f/Ciof 99mTcO4and 5 ¿iCiof '"I-HMFG1 was administered i.v. in order to

determine the total plasma in the tissues. One h after injection ofradioactivity the animals were sacrificed by cervical dislocation and theblood and the indicated tissues removed weighed and counted in agamma-counter against standards of the injectâtes.After sufficient timehad passed for the total decay of the "Te (half-life 6 h), samples wererecounted to establish their content of 125I.The VV and VP were

calculated as follows:

W = ("Tc/g tissue)/C""Tc/ml blood)

VP = [(125I/gtissue)/('"I/ml blood)] - [VV (1 - Ht/100)]

The vascular permeability was determined by measuring the amount ofiodine-125-labeled nonspecific antibody which extravasated out of tumor or normal tissue vasculature in l h and was expressed as nl/g/h.Vascular volume was expressed as n\/g tissue.

Statistics

Irradiated and control data were compared using the two-tailedStudent's t test. P values of less than 0.05 were considered significant.

Data are reported as the mean ±SD.

RESULTS

Radiolabeling. Indium-111-labeled antibodies had a labelingefficiency between 80 and 85% and specific activity between 1.5and 2 mCi/mg, resulting in approximately one molecule ofradiolabel being attached to one molecule of antibody as previously described (26).

In addition, HMFG1 was labeled with iodine-125 for thevascular studies. Labeling efficiencies of 90-95% were achievedwith a specific activity in the 2-3 mCi/mg range.

Immunoreactivity. The immunoreactive fraction of the in-dium-111-labeled AUA1 was approximately 65%. A Scatchardplot of indium-111-AU AI binding to viable LoVo cells is shownin Fig. 1. The abscissa shows the concentration of specifically

0.5-

0.4

u. 0-31m

0.2

0.1-

0.00.0 0.1 0.2

B ug/ml

Twenty-four h after irradiation of the tumor, all mice were injected Fig. , Scatchard plot analysis of the "'ln-AUAI monoclonal antibody bind-

i.p. with 10 ¿igin 100 «Iof specific or nonspecific antibody radiolabeled ing to whole LoVocells.

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ANTIBODY UPTAKE BY TUMORS AFTER IRRADIATION

bound antibody and the ordinate is the ratio of the concentrations of specifically bound over reactive free antibody. Fromthe intercept value on the abscissa the number of moleculesbound per cell was 4 x IO5 and from the slope of the line theassociation constant was Ka = 2.2 x IO8(M"').

Flow microfluorometric analysis of LoVo cells showed that97% of the LoVo cells were AU AI positive.

In Vitro and in Vivo Stability. The stability of the radiolabeledantibody in vitro and in vivo was satisfactory. SDS-PAGE andautoradiographs of serum samples demonstrated that the circulating radioactivity was on a M, \ 50,000 molecule, consistentwith mouse immunoglobulin. Radiochemical purity of the "' In-

labeled antibodies averaged 98 ±1% as determined by FPLCanalysis. The amount of free radioiodine in the '"I-labeledantibody preparation was less than 2%. The amount of '"In in

the blood which was not associated with the AU AI monoclonalantibody 3 days after antibody administration was 3%. Similarresults were found with the HMFG1 monoclonal antibody.

Immunohistochemical Staining. By immunoperoxidase staining of frozen sections from LoVo tumors it was confirmed thatnearly all the tumor cells were expressing on the cell surfacethe antigen recognized by AUA1. The specificity of binding wasconfirmed by the lack of staining when the control antibodyHMFG1 was used (Fig. 2).

Tumor Development and Localization Study. The xenograftedtumors in the mice were allowed to grow for 4-6 weeks. Themean tumor volume on the day of irradiation was 0.20 ±0.06cm3 for the control group and 0.15 ±0.07, 0.28 ±0.09, and

Fig. 2. Top, indirect immunoperoxidase staining of a LoVo xenograft tumorsection with AUAI monoclonal antibody (10 ¿<g/ml).Note dark staining of thecells indicating positive reaction with the antibody. Bottom, indirect ¡mmunoper-oxidase staining of LoVo xenograft tumor section with irrelevant antibodyHMFGI (10 ,.1; ml). Note lack of staining indicating negative reaction with thisantibody.

0.21 ±0.05 cm3 for groups irradiated with 800, 1200, and 1600

cGy, respectively. The mean tumor volume on the day ofdissection was 0.27 ±0.10 cm1 for the control group and 0.26±0.09, 0.39 ±0.11, and 0.25 ±0.07 cm3 for the groups

irradiated with 800, 1200, and 1600 cGy, respectively. Themean tumour weight on the day of dissection was 0.16 ±0.03g for the control group and 0.14 ±0.05, 0.25 ±0.07, and 0.16±0.04 g for the groups irradiated with 800, 1200, and 1600cGy, respectively. Therefore, irradiation did not cause tumorregression at any dose at 3 days postirradiation.

The tissue distribution of '"In-labeled antibodies in mice is

shown in Tables 1 and 2. Tissue/blood ratios have been usedto standardize the data from different animals which may haveabsorbed different amounts of radiolabeled antibody from i.p.injection. Progressive increase in antibody uptake was readilyapparent in the tumors with both specific and nonspecificantibody in the irradiated groups, expressed as percentage ofthe injected dose per gram of tissue relative to blood (Fig. 3).Three days after injection of radiolabeled AUA1, the controlanimals had blood activity levels of 9.9 ±2.9% of administereddose per gram, and no treatment group had blood levels significantly different from this. The increase in antibody uptake bytumors compared to control was significant at doses greaterthan 400 cGy, with a mean enhancement of 90%. Antibodyuptake by normal organs did not differ significantly after anydose of radiation to the tumor for either antibody. Specificantibody uptake by tumors was always significantly higher thannonspecific, having about a 4-fold binding advantage in vivo.

Vascular Studies. One hour after the i.v. administration of99mTcO4,99% of the radiolabel in blood was bound to the red

cells. Vascular volume in tumors 24 h after irradiation wasfound to be significantly decreased following exposure to doseshigher than 400 cGy (P < 0.001) as shown in Fig. 4. Vascularpermeability in tumors was significantly higher (P < 0.05) inall irradiated groups after exposure to doses higher than 400cGy compared to nonirradiated values (Fig. 4). Vascularchanges at 72 h were not significant compared to control values.Vascular changes in the muscle, liver, spleen, and kidney inirradiated groups at 24 and 72 h were not significant comparedto control values.

DISCUSSION

The results of this study show that exposure of tumors to asingle dose of radiation can lead to vascular changes and increased monoclonal antibody uptake.

In the present study we used nude mice xenografted with ahuman colon carcinoma cell line (LoVo), because this modelhas the advantage of testing the ability of radiolabeled AU AImonoclonal antibody to reach and bind to a human tumor ///vivo (27). The specificity of binding of AU AI monoclonalantibody was tested first in tissue sections of LoVo xenografttumours as well as with flow cytometry, and then in vivo whereit was confirmed by the low degree of accumulation of nonspecific antibody in the tumor. Recent studies have demonstrateda large difference in percentage of binding of monoclonal antibodies against colon carcinoma (28, 29) due to heterogeneityin antigenic expression in tumors. In our study it was observedthat nearly all tumor cells expressed the antigen recognized bythe AU AI monoclonal antibody.

Antibody molecules are delivered and transported to thetumor cells through the vascular and interstitial compartmentof the tumor. Tumor vasculature and interstitial compartmenthave significant differences in structure and function from thenormal tissues (30). Tumor capillaries are devoid of smooth

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Table

ANTIBODY UPTAKE BY TUMORS AFTER IRRADIATION

Percentage of administered dose/g of normal tissue relative to Mood, three days postinjection of A VA I monoclonal antibody at 0 and 400, 800, 1200, 1600cGy radiation doses

RadiationdoseUrineBloodTumorStomachIntestineKidneysSpleenLungLiverSkinHeartBone00.47

±0.321.01.05

±0.140.26±0.140.28±0.161.52

±0.470.50+0.160.49±0.080.66±0.200.14

±0.030.22+0.060.23±0.090.251.170.130.201.200.410.350.540.140.170.24too±0.071.0±0.16±0.02±0.04±

0.35±0.08±0.06±0.08±0.04±

0.03±0.088000.29

±0.101.01.64

±0.340.12+0.030.23+0.091.62

±0.590.42±0.110.46±0.080.61

±0.140.13±0.020.17±0.020.23

±0.0612000.4

+0.181.02.28

±0.80.21±0.100.36±0.161.52

+0.610.68±0.210.61±0.230.73

+0.210.15±0.040.22

+0.050.31±0.1216000.63

+0.431.02.19

±0.570.20+0.100.29

0.091.810.440.840.350.520.160.770.290.18±0.060.28

+0.090.27+ 0.13

Table 2 Percentage of administered dose/g of tissue relative to Mood, 3 days postinjection ofHMFGI monoclonal antibody al Oand 400, 800, 1200, 1600 cCyradiation doses

RadiationdoseUrineBloodTumorStomachIntestineKidneysSpleenLungsLiverSkinHeartBone00.29

+0.141.00.24

0.050.110.030.220.041.020.230.540.140.460.100.560.100.140.040.260.090.21

0.064000.30

±0.131.00.31

±0.050.12+0.020.22±0.031.01

+0.150.43+0.030.42±0.040.57±0.060.14+0.030.20±0.060.18+ 0.028000.31

±0.161.00.44

0.100.120.030.190.011.060.310.560.230.480.110.650.090.130.030.500.200.22

0.0912000.38

±0.171.00.51

±0.120.14+0.040.21

+0.041.08+0.300.57

±0.210.47±0.120.61

±0.110.15±0.030.32

+0.090.27+ 0.0716000.44

+0.211.00.50

+0.090.13±0.040.22

±0.041.42±0.430.66

+0.270.52±0.140.59

+0.120.14±0.040.24

±0.060.24+ 0.08

2-

0 4 8 12Radiation dose (Gy)

16

Fig. 3. Tumorblood ratios plotted against radiation dose ¡nmice injected 24h postirradiation and dissected 72 h postinjection of AUAl or HMFG1 antibody.Each point represents the mean and the standard deviation of 12 mice.

•¿�§.30nS"'ermeability

(u'olume(ul/g)-•rooo33D

vp0WIltT^viÌ_LTiii10

4 8 1216Radat<>nDose (Gy)

Fig. 4. VP and VV of irradiated tumors at 24 h after irradiation. Each pointrepresents the mean and the standard deviation of 18 mice.

muscle cells, being composed only of endothelial cells surrounded by a basement membrane (31). The architecture of thetumor vascular network, the VV and VP to macromolecules, aswell as the tumor interstitial compartment are factors whichaffect the monoclonal antibody uptake by the tumors (9, 30,31). The enhancement of monoclonal antibody binding by thetumor observed in this study suggests that irradiation probablyaffects the mechanisms of antibody localization.

This study indicates that the pooling of blood in the tumorvascular compartment was not the explanation for the enhancedantibody binding. This improvement was probably the result ofincreased antibody accessibility to the tumor extravascular compartment. It is of interest that the vascular volume of the tumors

was decreased following the irradiation. The decrease of tumorVV early after irradiation has been reported in the past by manyinvestigators (12, 32-34). Contradictory results have also beenpublished where the VV was unchanged (10, 16, 35), or increased (11). There are several possible mechanisms which mayexplain the decrease of the VV. Histological changes of theendothelial cells were observed in different tissues as early as24 h after irradiation, such as swelling and disruptions of theendothelial cells and occlusion of capillary lumen (15, 36).Other mechanisms include the increased transport of proteinsthrough vasculature to the interstitial fluid with subsequentincrease of the tissue oncotic pressure (16, 36), the release ofvasoactive agents from dying cells (37) and the decreased production of prostacyclin (38) by the irradiated vascular endothe-lium. Prostacyclin is a prostaglandin produced by endotheliumand acts as a potent inhibitor of platelet aggregation and as avasodilator.

We observed that the vascular permeability of tumors increased following external beam irradiation. This observationis consistent with the findings of other groups (16, 17, 18, 39).Evans et al. (18) reported a quantitative measurement ofchanges in VP of several normal tissues. At 1 day postirradia-tion of thorax with 1800 and 2500 cGy, the VP of these normaltissues increased, but by day 4 declined to control levels. However later on, they observed a second increase between 14 and38 days, which was supressed with the administration of dexa-methazone (40).

It has also been reported by different investigators (13, 41)that the increased vascular permeability early after irradiationwas, at least in part, due to vascular response to histamine.They could show a pronounced suppression of the increase inVP after irradiation with the use of the antihistamines or withother antiinflammatory compounds. Song et al. (13) reportedthat antiinflammatory compounds such as indomethacin, pro-methazine HC1 and epsilon-amino-w-caproic acid suppress theincrease in VP of guinea pig skin following irradiation.

The measurement of the tumor volume before irradiation as162

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ANTIBODY UPTAKE BY TUMORS AFTER IRRADIATION

well as prior to dissection 4 days later, showed that at dosesgiven and in a short period of time, irradiation did not have ameasurable effect on tumor size in our tumor model. Thisindicates that change in tumor size is not the explanation forthe higher antibody uptake in this study, although Wong et al.(7), studying nude mice with xenografts of colorectal carcinoma,found a significant decrease in tumor mass 2 days after irradiation with 200 and 2000 cGy. Our results are consistent withStickney et al. (9), who studied human melanoma xenografts inmice, and 7 days after a single exposure of 1000 cGy found thatthe differences between tumor volumes in groups receivingirradiation and the controls were not significant and tumorscontinued to increase.

In conclusion, the results of the present study show thatsingle doses of external beam irradiation can increase monoclonal antibody localization in the tumor despite the decrease invascular volume. The increase in antibody uptake varied directlywith the dose of irradiation, as did the vascular permeability.Whether the increased vascular permeability plays the mostimportant role in the enhancement of antibody deposition intumors is unknown but it is likely to be one of the centralparameters. Further studies are required to determine the optimal time for antibody administration following irradiation,the optimal radiation dose and fractionation as well as the roleof the various physiological parameters and how they affectantibody uptake by the tumors, in order to devise techniquesfor enhanced tumor targeting.

ACKNOWLEDGMENTS

The authors would like to thank A. Silvester and the MRC CyclotronUnit, Hammersmith Hospital, for assistance with dosimetry and forthe use of the X-ray facility; and S. Pervez, Royal Postgraduate MedicalSchool, Department of Histopathology, for the immunohistochemicalstaining of tissue sections.

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1990;50:159-163. Cancer Res   H. Kalofonos, G. Rowlinson and A. A. Epenetos  Tumor Xenografts following IrradiationEnhancement of Monoclonal Antibody Uptake in Human Colon

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