micropharmacology of monoclonal antibodies in solid …[cancer research 52, 5144-5153. october 1....

[CANCER RESEARCH 52, 5144-5153. October 1. 1992)

Micropharmacology of Monoclonal Antibodies in Solid Tumors: DirectExperimental Evidence for a Binding Site BarrierMalik Juweid, Ronald Neumann, Chaing Paik, Miguel J. Perez-Bacete, Jun Sato, William van Osdol, andJohn N. Weinstein1

Nuclear Medicine Department, Clinical Center Â¡M.J., R. N., C. P., M. J. P-B.J, National Cancer Institute Â¡J.S., W. v. O., J. N. W.}, NIH, Bethesda, Maryland20892, and Takeda, Inc., Osaka, Japan fJ. S.J

ABSTRACT

Monoclonal antibodies (MAbs) often distribute nonuniformly in tumors. In part, that observation reflects intrinsic heterogeneity within thetumor; in part, it reflects poor penetration through tumor substance.Several years ago, we proposed the "binding site barrier" hypothesis

(J. N. Weinstein, R. R. Eger, D. G. Covell, C. D. V. Black, J. Mulshine,J. A. Carrasquillo, S. M. Larson, and A. M. Keenan, Ann. NY Acad.Sci., 507:199-210, 1987; K. Fujimori, D. C. Covell, J. E. Fletcher, andJ. N. Weinstein, Cancer Res., 49: 5656-5663, 1989), the idea that

antibodies (and other ligands) could be prevented from penetrating tumors by the very fact of their successful binding to target antigen.Calculations suggested that this might be a significant factor in thetherapy of even microscopic nodules. The higher the affinity and thehigher the antigen density, the greater the barrier. Here, we providedirect experimental evidence of such a barrier to the percolation of D3M Ab through intradermally implanted line 10 carcinoma of guinea pigs.After affinity purification using glutaraldehyde-fixed line 10 cells, the

D3 had an average immunoreactivity of 88%, a binding constant of1.6 Â±0.3 (SEM) X IO10 M ', and saturation binding of 355,000 Â±15,000

molecules/cell. Using a combination of double-label autoradiographyand double-chromagen immunohistochemistry, we determined simulta

neously the distribution of (a) i.v. injected D3 MAb; (b) coinjectedisotype-matched control IgG (BL3); (e) D3 antigen; (a) blood vessels.

The previously developed mathematical models aided in the design ofthese experiments. Double immunochemical staining of the tumorsshowed antigen-rich patches 100-800 urn across, surrounded by blood

vessels. At a low MAb dose (30 Â¿eg),binding to antigen severely hindered penetration into antigenic patches as small as 200 urn. even at 72h. Explanation of this finding by a physical barrier was ruled out by theobservation that BL3 distributed uniformly in the same patches. At ahigher dose (1000 Â¿ig),the binding site barrier could be partially overcome. The same general principles of micropharmacology may apply tobiological ligands other than antibodies, including those secreted bygenetically modified cells.

INTRODUCTIONWhen MAbs2 are administered i.v. for diagnosis or treatment

of a cancer, they tend to distribute heterogeneously within thetumor substance. This nonuniformity often reflects the intrinsicheterogeneity of the mass with respect to factors such as antigendensity, vascularization, capillary permeability, degree of necrosis, and interstitial pressure (1-11); but it also may reflect inefficient penetration from the blood vessels into tumor nodules(11-17). Modeling studies led us a few years ago to formulatethe hypothesis that antibody molecules (and other ligands) canbe effectively prevented from penetrating the substance of atumor by the very fact of their successful binding to antigen(14, 15, 18-25). Formation of antigen-antibody complexes per

Received 1/6/92; accepted 7/27/92.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 in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1To whom requests for reprints should be addressed, at 9000 Rockville Pike.National Cancer Institute/NIH, Building 10, Room 4B-54, Bethesda, MD 20892.

2 The abbreviations used are: MAb. monoclonal antibody; PBS, phosphate-bufTcred saline; BSA, bovine serum albumin.

se does not present a mechanical barrier to the uncomplexedMAb. However, binding to antigens in the immediate proximityof the blood vessels might significantly decrease the number offree, diffusible molecules available for penetration deeper intothe tumor substance. The term "binding site barrier" was intro

duced (21-23) to describe this phenomenon. It was predicted

that greater antigen density, higher MAb affinity, and fasterMAb internalization and metabolism by cells would increasethe "barrier" effect, all else being equal.

Experimental studies from a number of laboratories haveshown heterogeneous distribution of antibodies in tumors, andsome of these studies have shown preferential localization ofantibody near blood vessels in the stroma. To demonstrate abinding site barrier convincingly, however, one must considerthe distributions of four different entities: antibody; antigen;control antibody; and blood vessels. To rule out the possibilitythat the nonuniform distribution of antibody simply reflects theheterogeneity of antigen expression, the antigen distributionmust be determined. To rule out a physical barrier (e.g., tightjunctions) to the progress of macromolecules through the tumor, it must be shown that a matched, nonbindable immuno-globulin distributes uniformly. To circumvent the problems associated with the use of immunohistochemical techniques todemonstrate the distribution of nonbindable antibody, Â¡intonidiographic techniques should be used. In addition, it is important to know the location of blood vessels as sources of theantibody.

Here, we report direct experimental evidence for the bindingsite barrier hypothesis. For these studies, we used a syngeneictumor system, line 10 (LIO) guinea pig cholangiocarcinoma inSewall-Wright strain 2 guinea pigs. The antibody was D3, amurine monoclonal IgG] directed against a heterodimeric A/r290,000 antigen on the cell membranes of L10 cells (26, 27). Anisotype-matched monoclonal murine antibody, BL3, directedagainst an idiotope on the surface of a particular human B-celllymphoma, served as the control (13). We used our previouslydeveloped mathematical models to aid in designing experiments that combined double-chromagen immunohistochemistry with double-label autoradiography to determine simultaneously the distribution of D3 antibody, D3 antigen, BL3antibody, and blood vessels.

MATERIALS AND METHODS

Tumor Model. L10 carcinoma (26) of Sewall-Wright strain 2 guineapigs is a chemically induced, transplantable tumor thought to be of bileduct origin (26, 28). LIO cells were stored in M 199 medium with 15%glycerol in liquid nitrogen. To produce L10 ascites, ~5 x 106cells were

injected i.p. into strain 2 guinea pigs, and cells were obtained from theascites in approximately 10-14 days. The fresh ascites cells were thenused to produce tumors in other guinea pigs by injecting 2-3 x IO6cells/300-500-g animal intradermally in the back of the neck. Seven tofourteen days later a ~ 1-cm intradermal tumor was present in all of theanimals.

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MICROPHARMACOLOGY OF MONOCLONAL ANTIBODIES IN SOLID TUMORS

Antibody Labeling and Quality Assessment. The purity of D3 and our previous studies, and from preliminary' single-label studies in thisBL3 before radiolabeling was assessed by size-exclusion high-performance liquid chromatography and isoelectric focusing. High-performance liquid chromatography showed both D3 and BL3 as single peaksat the typical retention time for IgG molecules (data not shown). BothD3 and BL3 were labeled with radioiodine using the chloramine-Tmethod as described previously (29). Each milligram of protein wasreacted with 12.5 Mgof Chloramine-T (Fisher Scientific, Fairlawn, NJ)plus an amount of either Na125I or Na'"I determined by the specific

activity required. After 2 min, the reaction was stopped by the additionof 44 Mg of sodium thiosulfate (Fisher Scientific). The radiolabeledprotein was then separated from unbound iodide by size exclusionchromatography on Sephadex G-25 (Pharmacia, Inc., Piscataway, NJ).For coinjection studies, D3 was labeled with "'I, and BL3 was labeledwith ' 25I.For experiments not involving coinjection, D3 was sometimeslabeled with I25I. The immunoreactivity of labeled D3 antibody before

purification ranged from 51 to 84%. For in vivo studies, however,radiolabeled D3 antibody was affinity purified using a method describedelsewhere by Juweid et al. (30). Briefly, line 10 cells were fixed with0.25% glutaraldehyde for 30 min at room temperature in 25 ml PBS atpH 7.4. Fifty ml of a PBS-1% BSA buffer containing labeled antibody(140 Mg,420 MCi)were added to 3 x IO8 packed, fixed cells in a 50-ml

centrifuge tube. After incubation for 1 h, the cells were washed threetimes with a total volume of 150 ml PBS-1% BSA at pH 7.4. The cellswere then washed three times with 10 ml of glycine buffer (pH 3) todetach bound antibody. The pH was immediately neutralized by passage over a Sephadex G25 column preequilibrated with PBS-1% BSA atpH 7.4. The fixed cells remained stable in their binding characteristicsover at least 2 months at 4Â°C,and they could be reused repeatedly. This

purification technique resulted in an increase in immunoreactivity tovalues between 81 and 93%. The immunoreactivity of BL3 to LIO cellswas, as expected, practically zero (0.05 Â±0.55).

Size exclusion high-performance liquid chromatography profilesafter radiolabeling and affinity purification revealed no significantaggregation of the antibody. After i.v. injection (vide infra), therewas no unusual accumulation in the liver that might indicate majoraggregation.

Antibody Affinity. The affinity of D3 was assessed in a cell bindingassay. Briefly, different dilutions of monoclonal D3 (10-0.005 fig/ml)were added to 2 x 10s cells in PBS-1% BSA at pH 7.4 in a final volumeof 1 ml. After incubation for l h at 4Â°C,the cells were washed two times

with PBS-1% BSA, and the pellets were counted in a gamma counter.Binding was analyzed by simultaneously fitting the expressions

B = B0-F/(F+ \/KJ + a-F

where B (molecules/cell) is the observed cell-associated D3 antibody,fins(molecules/cell) is the observed cell-associated D3 in the presence ofexcess unlabeled D3 antibody, fi0 (molecules/cell) is the specific bindingat saturation, /â€¢'(M) is the final free concentration, A., is the associationconstant (M~'), and a (molecules-cell"1 -M~') is the constant for non

specific binding. These two simultaneous equations were fitted to thedata by nonlinear least-squares regression with Gaussian kernel weighting, using MLAB (Civilized Software, Bethesda, MD) as describedpreviously (31).

Simulation of D3 Distribution in a Line 10 Tumor Nodule. To aid indesigning the double-label experiments on antibody localization, we didmathematical simulations using the PERC program package as previously described (20-22). PERC splices together information on (a) theglobal pharmacokinetics, (b) transcapillary transport by diffusionand/or convection, (c) "percolation" through the interstitial space of a

tumor, (</)binding to antigen, (e) metabolism and/or dehalogenation,and (/) lymphatic clearance. The particular tumor nodule geometrysimulated was spherical (23). Although the antigen-rich patches ofLIO tumors were not generally spherical, this mathematical formulation was expected to give reasonable order-of-magnitude approximations to be used in designing in vivo experiments. Values for the variousinput parameters (see Fig. 3) were obtained from the literature, from

tumor/antibody system. Parameters used in the simulations (keyedto symbols and definitions in Table 2 of Ref. 23) are: radius of antigenicpatch (R) = 150 Mm;effective interstitial diffusion coefficient of MAb(D) = 1.3 x 10~8cm2/s; transcapillary transport coefficient (K) = 3.9 x10~7 liters/min/g; interstitial volume efflux (lymphatic) coefficient(L) = 2.83 x 10~6 liter/min/g; ratio of interstitial volume to plasma

volume (<t>)= 0.17; mean density of interstitial tissue (p) = 1.0 g/cm3;pseudomonovalent rate constant for binding of MAb to Ab (kt) = 1.6 xIO5 M~' s~'; pseudomonovalent rate constant for release of MAb fromMAb-Ag complex (kr) = 10~5 s~' (the last two values corresponding tothe measured K, = kf/k, = 1.6 x 10'Â°M~'); valence of MAb-Ag binding

(n) = 2; initial MAb plasma concentration (cpo) = 10 HM; antigenconcentration in interstitial space [Ag] = l MM;first- and second-rateconstants for plasma clearance of MAb (X, and X2)= 1.9 x 10~4and 7.4x 10~6 s"1; fractions cleared from plasma with first- and second-rate

constants (a, and a2) = 0.27 and 0.73 (23).Animal Studies. Four different experiments were performed. In the

first, a low dose of ' 25I-labeled D3 (30 Mg,200 MCi)was injected i.v. (via

the penile vein) in six guinea pigs. The animals were sacrificed 24 h afterinjection. In the second experiment, 125I-labeled BL3 (30 Mg,200 MCi)

was injected i.v. into two2 guinea pigs, and the animals were sacrificed24 h later. In the third and fourth experiments, '"I-labeled D3 andI25l-labeled BL3 were coinjected in order to determine the distribution

of both antibodies in the same histolÃ³gica! sections. It was, therefore,necessary to choose doses of radioactivity such that (a) autoradiogramsdone soon after sacrifice would show principally the mI-D3; (Â¿>)auto-radiograms done several ml half-lives later would show principally the125I(BL3); (c) there would be sufficient radioactivity to give good au-

toradiographic images for each isotope. The mathematical simulationsdescribed above, in conjunction with preliminary experimental results,were used to optimize the experimental design. A preliminary studyusing radioactive gelatin standards showed that the sensitivity of theautoradiographic film (Kodak, SB5) for '-"I was about 5 times that forI25I (on a microcurie-by-microcurie basis). In addition, the tumor uptake of D3 at salient time points was 3-10 times greater than that ofBL3.

In the third experiment, D3 was labeled with '-"I, and BL3 waslabeled with I25I to monitor the distribution of irrelevant, isotype-matched IgG after the decay of the '"I. Four guinea pigs received

coinjections of a low protein dose (30 Mg,33 MCi)of D3 and an equalprotein dose of I2Ã•IBL3 with a larger amount of radioactivity (30 Mg,

200 MCi).The 1:6 ratio of radioactivity permitted us to obtain autoradiograms showing principally I25Icounts within about 2 months (given

the film sensitivity, pharmacokinetics, microscopic localization, etc.).The animals were killed 6 h (n = 2) and 24 h (n = 2) postinjection.

In the fourth experiment, the antibodies were again coinjected. Ahigh protein dose (1 mg of each antibody) was administered i.v. in sixanimals, and a lower protein dose (55 Mg)was injected in another sixanimals. The amounts of radioactivity were the same for the two groups(70 MCiof '"I-D3 and 200 MCiof I25I-BL3), and unlabeled antibody

was added to increase total protein. The animals in each group weresacrificed 6 h (n = 3) and 72 h (n = 3) after injection.

In all experiments, the tumors were excised, weighed, counted in awell-type gamma counter, and immediately frozen. Later, serial thicksections (15-20 Mm)were prepared for immunohistochemistry, am oradiography, and hematoxylin-eosin staining. Blood samples wereweighed and counted. Radioactivity, expressed as cpm/g of tissue, wascompared with that of a standard of the injected dose, and the percentage of injected dose per gram of tissue was calculated.

Immunostaining. Immunohistochemical techniques were used onadjacent tissue sections to compare localization of antigen with localization of injected D3. In all cases, an avidin-biotin immunoperoxidasetechnique, as previously described by Hsu et al. (32), was used. Thetissue sections were fixed with acetone for 10 min, and endogenousperoxidase was blocked by incubating the slides in 1% Ilo. in distilledwater for 10 min. The tissue sections were then exposed to 20% normalhorse serum. D3 antigen was stained by incubating the sections overnight in a humidity chamber at 4Â°Cwith the primary antibody (D3 in a

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dilution of 1:50, i.e., 20 Mg/ml), then for 45 min at room temperaturewith the second antibody (biotinylated horse anti-mouse IgG), and finally for 45 min at room temperature with avidin-biotin-peroxidase

complex (Vectastain Kit; Vector Laboratories, Burlingame, CA). Thereaction was developed using 0.5 mg/ml 3,3-diaminobenzidine tetrahy-drochloride (Sigma, St Louis, MO) and 3 /Â¿I/mlH2O2 in 50 miviTris-buffered saline (pH 7.6). After each step the slides were washed threetimes in Tris-buffered saline.

Sections were stained for Factor VIII in the same way but with aprimary polyclonal rabbit antibody against Factor VIII (at 1:300 dilution) replacing D3 in the first step and with goat anti-rabbit IgG as thesecond antibody.

To determine the in vivo localization of D3, the above steps wereperformed without adding D3 as the primary antibody. To enhance thesensitivity of the immunostaining for in vivo localization of D3, especially when a low dose of D3 was injected, two different enhancementmethods were used: (a) cobalt with benzidine or (b) the avidin-biotin-peroxidase complex incubation step repeated 2 to 4 times. Both approaches enhanced the staining without significantly increasing thebackground.

Autoradiography. Frozen thick sections (15-20 urn) correspondingto the largest cross-sectional area of the tumor were cut at -20Â°C in a

cryomicrotome. They were then placed in light-proof cassettes at roomtemperature in direct contact with SB5 Kodak film. To determine thedistribution of '"I-labeled D3 or '"I-labeled BL3, the films were ex

posed for various time periods from 2 to 14 days, as appropriate. Incoinjection studies, the films were initially exposed for 24-48 h toobtain the distribution of "'I-labeled specific antibody (D3). After allowing '"I to decay (approximately 8 half-lives, 64 days), we exposedadditional films for 48-96 h to determine the distribution of 125I-BL3.

The late exposures were approximately twice as long as the initial onesin order to correct for the decay of 12SI.

RESULTS

Binding Studies. Fig. 1 shows the binding of I25I-D3 to LIOcells in vitro. From this plot an antibody affinity ATa(= 1/Kd) of1.6 Â±0.3 (SEM) x IO10 M"' and a maximal antibody binding

(B0) of 355,000 Â±15,000 (SEM) molecules/cell were calculated. Nonspecific binding of D3 in the assay (i.e., binding notblocked by excess unlabeled D3) was 15,600 Â±2,200 (SEM)molecules-cell"1 -HM"'. There was no measurable specific bind

ing of BL3.Mathematical Simulation of D3 Distribution in Antigen-rich

Patches. To aid in designing the experiments, we used ourpreviously described quantitative model for microscopic tumornodules (22). Input parameter values were obtained from the in

600,000

480.000 â€¢¿�

360.000 -

240.000

120.000 1311D3 * Excess unlabeled D3_ , _ - -"

0 2.2 4.4 6.6

03 Antibody Concentration (nM)

Fig. 1. Binding of I25l-labe!cd D3 to LIO cells in vitro.

11.0

vitro binding studies, preliminary pharmacokinetic experiments, and literature values. Of particular interest here was thedistribution of antibody in antigen-rich areas. Although perfectly spherical nodules were a rarity in this tumor, a sphericalnodule geometry was used to simplify calculations. An averagenodule diameter of 300 ^m was chosen. In line 10 tumors, theantigen-rich patches ranged in size from about 100 to about 800/xm in their smallest dimension. As shown by Factor VIII staining, the vessels were located almost entirely outside of theseareas. The results of this simulation are shown in Fig. 2 for twodoses of antibody, 30 and 1000 ng. This modeling predicts thatantibody at the lower dose will penetrate about 25 urn in 6 h and45 Mmin 72 h; it predicts that antibody at the higher dose willpenetrate about 95 urn in 6 h and 150 ^m in 72 h. The cutoffsare quite sharp because they represent moving boundaries ofantigen binding site saturation. Since the IgG is bivalent, saturation is seen at 500 nM, given 1 Ã•IMantigen (normalized perunit interstitial space). These results are qualitatively and, to acertain degree, even quantitatively in agreement with our measurements from the in vivo studies. We emphasize, however,that these data should be interpreted as an aid to experimentaldesign and as a demonstration of feasibility; the quantitativedata are not sufficient to justify fitting to the model.

Radioactivity in Tumors. Table 1 summarizes the radioactivity in tumors after injection of labeled antibody. Since therewere only 3 animals/group, the quantitative results should beinterpreted with caution, but the following observations weremade. I25I-D3 and U1I-D3 counts (expressed as percentages of

injected counts/g tumor) were higher 24 h after injection than at6 or 72 h. I31I-D3 accumulation appeared slightly lower when

a higher dose of unlabeled D3 antibody was coinjected, probably because the unlabeled antibody competed to some extentwith labeled antibody (20-fold higher dose of unlabeled antibody than of labeled antibody). As would be expected, the lowertumor uptake of nonspecific BL3 counts appeared to be independent of the dose injected. The apparently lower D3 counts intumor at 72 h than at 24 h may have resulted from dehaloge-nation. D3:BL3 specificity ratios for the low antibody dose(30-50 Mg)were 2.6,8.5, and 7.7 at 6,24, and 72 h, respectively.For the higher antibody dose (1000 Mg)the ratios were 1.6 and4.5 at 6 and 72 h, respectively. As expected, the plasma clearances of specific and nonspecific IgGs were very similar.

Microscopic Distribution of D3 and BL3 in the Tumors. Themain objective of this study was to test the binding site barrierhypothesis with respect to penetration of antibodies into antigen-rich patches of a tumor. It seemed reasonable to assumethat this effect would be most clearly demonstrated in animalsinjected with a low dose of D3 and killed at a reasonably earlytime point after injection. We therefore focused on tumors ofanimals injected with a low dose (30-50 Â¿ig)and killed at 6 h.The results are shown in Fig. 3.

The D3 antigen was heterogeneously distributed (Fig. 3, Aand A'). Some areas that clearly consisted of tumor cells asshown by hematoxylin-eosin staining (see Fig. 6, C and D) wereapparently antigen-negative. This was a common finding in allof the tumors. Antigen-positive and antigen-negative areas varied in size, shape, and intensity of staining from one tumor toanother as well as within a tumor. Such antigenic heterogeneityis the norm for human carcinomas as well.

Fig. 3Ã„and ff shows immunostaining for low-dose D3 antibody in tumor sections adjacent to those in Fig. 3A and A. Theantibody distribution was highly heterogeneous. D3 was confined to the periphery of antigen-rich patches and did not

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Low Dose High Dose

6 hours 6 hours

- te

72 hours 72 hours

S

Fig. 2. Simulation of D3 antibody distribution in a 300-nm diameter antigen-rich spherical patch 6 and 72 h after bolus i.v. injection. .1. 30-^g dose at 6 li; B, 30-jigdose at 72 h; C, 1000-^g dose at 6 h; /'. 1000-^g dose at 72 h. The binding site barrier is predicted to limit penetration at low doses, but saturation of antigenic sitesis predicted to overcome the barrier at high dose. These simulations, used to aid in the design of experiments, were done as explained in "Materials and Methods" and

by van Osdol et al. (23). The two horizontal axes represent distances (in urn) from the center (0)of the hypothetical patch. Antibody diffuses with radial symmetry fromthe edges toward the center.

penetrate deeply into them. This observation was confirmed inthe same way for all tumors of animals injected with a low doseof antibody and killed 6 h after injection. When injected alone,the nonbindable IgG (BL3) showed no discernible staining (notshown), in part because the levels of nonspecific antibody uptake were too low, but principally because BL3 washed out ofthe section during the immunostaining procedure.

Autoradiography for injected D3 (Fig. 3, C and C) correlatedvery well with the immunostained consecutive sections in Fig. 3(B and ff). D3 counts localized in the periphery of antigen-positive areas (including the "fetus-shaped" antigen-positive

patch; Fig. 3, arrows). Antigen-negative areas showed only minimal (nonspecific) localization of radiolabel.

As noted above, the in vivo localization of BL3 could not bedetermined reliably using our immunohistochemical lech

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Table 1 Accumulation of D3 and BL3 antibodies in line 10 tumor(% dose/g)

Data represent mean Â±SEM of 2-3 animals/group.

I25ID3 125IBL3

TimeExperiment

124hExperiment

224hExperiment

36h24

hExperiment

46h

72 hLow

dose1.77

Â±0.021.2

Â±0.234.5 Â±1.51.43

Â±0.040.92 Â±0.03High

dose Lowdose0.19

Â±0.030.45

Â±0.070.53 Â±0.080.88

Â±0.09 0.56 Â±0.0040.80 Â±0.01 0.12 + 0.004High

dose0.54

Â±0.050.14 Â±0.001




Ag

BD3Ab B'

Â«X

â€¢¿�â€¢â€¢â€¢

D3Ab

m D1 125|-BL3 Ab

Fig. 3. Immunohistochemical and autoradiographic studies of serial sections cut from an intradermal LIO tumor in a guinea pig that received an injection of 30 Mgeach ("low" dose) of i:"I-D3-specific antibody and 125I-BL3 control IgG. The animal was killed 6 h postinjection. A, A', antigen staining at low and high magnification,respectively; B. B, antibody staining at the same low and high magnifications; C, C, immediate autoradiography (predominantly 13II-D3 distribution) at low and highmagnification, respectively; D, D1,late autoradiography (I25I-BL3 distribution). Arrows, "fetus-shaped" antigenic patch (drawn schematically in insets, A-C). Bars, 1

mm. D, enlargement of the central region of D; the apparent difference in darkness is due to photographic technique. D3 antibody is localized in the periphery ofantigenic patches, whereas BL3 is uniformly distributed throughout the tumor.

niques, since free antibody might be washed out during theimmunohistochemical procedure. However, autoradiogramsobtained after the decay of 13II (Fig. 3, D and D1)showed the in

vivo distribution of radiolabeled BL3. This distribution wasgenerally quite homogeneous and did not show the distinctivepatchy features seen for I3II-D3 in the immediately exposed

autoradiograms. The homogeneous distribution in both antigen-positive and antigen-negative areas indicates that physicalbarriers were not preventing BL3 from entering the centers ofantigen-positive patches.

Immediate autoradiograms obtained from animals injectedwith the nonbindable IgG (125I-BL3) alone also showed a

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125I-BL3 125I-BL3

Fig. 4. Immediate autoradiograms (,4 and B) of tumors from animals that received an injection of 30 Â»ig(200 jiCi) of 125I-BL3 (no '"I) and were killed 24 h afterinjection. The distribution is relatively uniform in the absence of any "'I-D3 and at an early time after injection, confirming that the I25I-BL3 distributes

homogeneously.

125I-D3B

125|-D3

Fig. 5. Autoradiograms of tumors from animals that received an injection of 30 /ig of 12*I-D3 (no "M) and were killed 24 h after injection. A. immediateautoradiogram; B, autoradiogram of the same tissue section 2 months later. The patterns are essentially identical, ruling out the possibility that "'I had migratedbetween the times of immediate and late autoradiograms in Fig. 3.

homogeneous distribution of antibody (Fig. 4, A and B). Thisfinding indicates that the distribution pattern of I25I-BL3 seenin the autoradiograms exposed 2-3 months after the experiment is a true reflection of nonspecific antibody distribution,not the result of migration in the histolÃ³gica! sections duringstorage or of dehalogenation with subsequent migration of iodine. When cut sections from animals that received injectionsof 125I-D3 alone were autoradiographed immediately (Fig. 5A)

and again after 6 months (Fig. 5Ã„), the paired exposuresshowed exactly the same heterogeneous patterns, further indicating that there was very little, if any, dehalogenation of iodi-nated antibodies.

Fig. 6 (Cand D) shows hematoxylin and eosin staining at twodifferent magnifications. The antigen-rich patches seen withinuminosi ai Ming do not idled obvious patterns of cellularity inthe tumor.

The pattern of tumor vessels seen in Fig. 6 (A and B) wasconsistent with the distribution pattern of D3. That is, almostall of the vessels were located outside of the antigen-positiveareas. There were very few blood vessels within or traversing the

antigen-positive areas; antibody apparently percolated fromvessels in the antigen-negative areas into the edges of antigen-

rich patches.Despite the limited numbers of animals in groups studied at

later time points and/or receiving injections of higher doses ofD3, certain general trends can be identified. At a low dose ofantibody there appeared to be slightly better penetration intothe antigen-positive areas at 72 h, as compared with 6 h; theantibody penetrated a few cell layers deeper into these areas(not shown). However, it was far from uniform, and large numbers of antigen-positive cells remained unstained.

At a higher dose of antibody there was a clearly better penetration of the antigen-positive areas at 6 h. Penetration seemedto be complete at 72 h, with essentially no difference betweenstaining for D3 antigen and staining for D3 antibody (Fig. 7,A and B). The remaining heterogeneity in D3 antibody distribution within the tumors was, in these samples, attributable to heterogeneous tumor antigen distribution. Early autoradiograms of adjacent sections showed |;"I-D3 distribution

(Fig. 1C) correlated well with the immunostaining of antibody5149




BV

B BV

H&E

H&E

Fig. 6. A and B, vascular pattern, at low and high magnifications, respectively,as indicated by immunohistochemical staining with factor Vili. C and />, hema-toxylin and eosin staining, at low and high magnifications, respectively, of thetumor in Fig. 3; Bars, 1 mm.

in Fig. IB; late autoradiograms (Fig. ID) showed homogeneousdistribution of 125I-BL3, as expected. The effect of dose on

antibody penetration will be described in more detail elsewhere.3

DISCUSSION

The main aim of this study was to test the hypothesis of abinding site barrier, the idea that bindable immunoglobulinmolecules (and other biological ligands) are sometimes retardedin their penetration of tumor by the very fact of their successfulbinding (14, 15, 18-24). For this purpose, we used the LIOguinea pig carcinoma and D3 antibody. The cells bound355,000 Â±15,000 D3 antibody molecules/cell at saturation,with an apparent affinity of 1.6 Â±0.3 x 10'Â°M~'. The immu-

noreactivity ranged from 51 to 84% in various batches. However, we developed an affinity purification protocol that increased the immunoreactivity of labeled D3 to values between81 and 93%, an important step for this type of study.

The tumor-antibody model used here has advantages overmouse xenograft systems. In particular, the combination ofsyngeneic solid tumor and xenogeneic antibody is analogous tothe situation in which murine antibodies are used in humans.Although not important for the present studies, the immunesystem of guinea pigs is more like that of a human than is themouse's.

Immunostaining of the tumors showed heterogeneous antigen distribution, as is also the norm for human carcinomas(3, 33, 34). The antigen tended to be expressed in irregularlyshaped patches characteristically 100-800 urn across (Fig. 3,A and A'). The first question, then, was whether D3 antibody

would distribute freely in these patches. As shown by the staining in Fig. 3 (B and ff), it did not. D3 apparently had access toonly the outer cell layers within the first 6 h after i.v. injection.

Heterogeneous localization of MAb has been seen in otherstudies, in vivo (13, 16, 35), in muItÂ¡cellularspheroids (36-39),and in cells grown in hollow fibers (40). However, the findingthat antibody has not reached all of the antigen does not sufficeto demonstrate that binding sites are the cause. Lack of penetration could be explained at the microscopic level by mechanical barriers, e.g., by a network of intercellular tight junctions orby elements of basement membrane. Pervez et al. (16) haveconsidered such a "mechanical" barrier. It is also worth noting

that localization near the edges of a bulky tumor could beexplained by elevated interstitial pressure in the center of thetumor and by the consequent outwardly directed volume flows(7, 11, 17). Such a macroscopic pattern of nonuniformity wasnot prominent in our experiments. Nonspecific antibody distributed relatively homogeneously.

To test the possibility of such nonspecific barrier effects, wecoinjected an irrelevant, isotype-matched immunoglobulin(BL3) along with the D3. One would expect a mechanical orhydraulic barrier to exclude the BL3 as well as the D3, whereasa binding site barrier would leave the BL3 to distribute more orless uniformly in the tumor. For this comparison, it was notclear that we could rely on standard immunohistochemicaltechniques, since the BL3 might redistribute or be washed outof the tissue section during sequential incubations. Hence, weturned to double-label autoradiography with I31I-D3 and I25I-BL3. Fig. 3C (low magnification) and Fig. 3C' (high magnifi

cation) show autoradiography of a tissue section cut adjacent to

1M. Juweid et al., manuscript in preparation.

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Ag

BfP D3Ab

A

13i|-D3Ab

that of the histochemistry in Fig. 3/4 and A. Note the same"fetus-shaped" antigen-rich regions (arrows). The autoradio-

gram matches the histochemistry in showing very little penetration by D3 antibody. In contrast, autoradiograms taken after2 months (Fig. 3, D and />) show quite uniform distribution ofBL3. Given these observations, the localization of bindable im-munoglobulin in the periphery of antigenic patches can reliablybe ascribed to antigen binding. Metabolic breakdown of theantibody or release of label after binding will, of course, increasethe effect.

Ong and Mattes (18) examined nonbindable IgG in a modeltumor system by immunohistochemistry. Perhaps because ofwashout during the staining procedure (as well as low IgGconcentrations in the tumor), they reported the staining as onlyweakly detectable and distributed only in the connective tissues.Because gradients of nonbindable IgG in antigen-positive areascould not be ruled out, it was not possible to determine fromthose experiments whether the barrier was mechanical or due tobinding. The combination of immunohistochemical and auto-radiographic techniques used here circumvents the problems ofwashout and low sensitivity.

To understand more fully our experimental findings, it wasimportant to define the vascular architecture and therefore toidentify where antibodies might leak from vessels and diffuseinto antigen-positive areas. If, e.g., the vessels tended to runthrough the centers of antigen-rich patches, one might haveexpected D3 to be held up in small regions surrounding thosecentral vessels. Dvorak and Â«workers (8) have described theexistence of two tumor vascular patterns in LIO carcinoma: (a)branching vessels penetrating the nodule and (b) a vascularplexus surrounding the nodule. Their study did not include anattempt to correlate vascular patterns with D3 antigen distribution. In the present work, we saw predominantly the latterpattern with respect to antigen-rich patches. Fig. 6 (A and B)shows the brown-stained antigenic patches and redder factorVIII staining for vessels and other stromal elements. Hence,antibodies would be expected to leak from vessels outside ofantigen-positive patches and to be held up in the periphery ofthe patches, as was in fact observed.

These simultaneous determinations of antigen distribution,specific antibody distribution, nonspecific antibody distribution, and vascular pattern provide direct experimental evidencefor a binding site barrier. Further correlative evidence was provided by experiments in which substantially larger amounts ofantibody were injected. We expected from preliminary calculations (see Fig. 2, C and D) that the high dose (1000 tig) mightsuffice to saturate the binding sites in the outer cell layers ofantigen-rich patches. In this case, specific antibody would beexpected to penetrate deeper (21,41). Time after injection wasalso expected to play an important role. With increased time,more antibody molecules are delivered to the tumor, and, in theabsence of rapid metabolism, they would tend to penetratedeeper after the superficial layers of antigen had been largelysaturated. Fig. 7, A and B, shows immunohistochemical patterns of antigen and D3 antibody, respectively, for the high doseat 72 h. Although the antigen-rich regions happen to be smallerand different in shape from those of the tumor in Fig. 3, it is

Fig. 7. Immunohistuchcmical and autoradiographic studies ol"serial sections

cut from an intradermal LIO tumor in a guinea pig that received an injection of1000Mgeach ("high"dose)of '"I-D3 specificantihody and '"I-BI.3control IgG.

The animal was killed 72 h postinjcction. A, antigen staining at low magnification;

B, antibody staining of adjacent section at the same magnification: C, immediateautoradiography (predominantly ' "I l>Ã®distribution); I), late autoradiography(125I-BL3 distribution). Bars. 1 mm. The antigen and antibody are distributedalmost identically. D3 antibody is uniformly distributed in antigenic patches,whereas BL3 is uniformly distributed throughout the entire tumor.

5151




apparent that penetration has been much more effective; antigen and antibody distributions are indistinguishable. The corresponding autoradiograms in Fig. 7 (C and D) confirm thatU1I-D3 antibody appears to distribute quite uniformly in antigen-rich regions, whereas the I25I-BL3 distributes uniformly

throughout the entire tumor, as expected. More complete results for the effects of time and dose will be presented elsewhere.1 In brief, the low dose (30-50 Mg)showed poor penetra

tion at times up to 72 h, whereas the high dose (1000 Mg)showed relatively poor penetration at 6 h but quite uniformdistribution by 72 h.

The lower dose (30-50 Mg)that we injected into guinea pigscorresponded on a weight basis to about 5-9 mg/70-kg human.However, our mathematical simulations suggest that completepenetration of a 300-nm nodule with characteristics similar to

those of LIO could not be accomplished without administeringmuch higher doses. Even higher doses would, of course, berequired to penetrate still larger nodules. The dose of antibodycan be raised in order to overcome the binding site barrier, buta higher dose is also expected to decrease the selectivity ratiowithin a tumor and to decrease the ratio of uptake in tumor tothat in distant organs (15, 41). The high dose in these experiments (1000 Mg)corresponded to about 175 mg in a human.

Heterogeneous antibody distribution, which was dramaticallydemonstrated by immunohistochemistry, appeared somewhatless sharp in autoradiograms. The difference can be explainedby the path length of '-"I /3-particles. Similar "fuzzing" effects

are expected for the pattern of radiation dose rate in vivo. Inradioimmunotherapy with /3-particles the distribution of radiation dose to tumor cells will be more homogeneous than theactual distribution of the radioiodinated antibody molecules.Nevertheless, the heterogeneity could be considerable, as indicated by the microdosimetric calculations of Fujimori et al. (22)and Hui et al. (42). Dehalogenation would also complicate thissituation; the longer the time required for antibody to penetrate,the larger the fraction of radioactive iodine released from antibody. Cellular internalization and metabolism of antibody areexpected to have similar, and sometimes very important, consequences. The binding site barrier effect is expected to be muchmore important when short-range Â«-particles (which typicallyhave path lengths shorter than 100 Mm), rather than ÃŸemitters,are used for radioimmunotherapy. It may be even more prominent with drugs and toxin conjugates if they must reach eachindividual cell to be effective. At the opposite extreme, poorpenetration may be a distinct advantage if the aim is to destroytumor blood vessels with a short-range radioisotope.

In conclusion, this study provides the first direct experimental evidence in support of the binding site barrier hypothesis.Elsewhere (19,43), we have discussed how this principle shouldbe taken into account when designing new immunoglobulin-likemolecules and other biological ligands, including lymphokinesand cytokines to be secreted locally within the body by genetically modified cells.

ACKNOWLEDGMENTS

We are grateful to Drs. T. Saga, T. Heya, and M. S. Abu-Asab forthoughtful comments on the manuscript. We thank the National Cancer Institute for allocation of computing time and staff support at theAdvanced Scientific Computing Laboratory of the Frederick ResearchFacility.

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1992;52:5144-5153. Cancer Res Malik Juweid, Ronald Neumann, Chaing Paik, et al. Direct Experimental Evidence for a Binding Site BarrierMicropharmacology of Monoclonal Antibodies in Solid Tumors:

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