0883~2897/91$3.00 + 0.00
Nucl. Med. Biol. Vol. 18, No. 7, pp. 753-764, 1991 Int. J. Radial. Appl. Instrum. Part B Printed in Great Britain
Perpmon Press plc
The Effect of Antigen Concentration, Antibody Valency and Size, and Tumor Architecture on Antibody Binding in Multicell Spheroids* VIRGINIA K. LANGMUIR’tS, JUDETH K. McGANN’S, FRANZ BUCHEGGER’ and ROBERT M. SUTHERLAND’S ‘University of Rochester Cancer Center, Rochester, NY 14642, U.S.A. and *Institute of Biochemistry, University of Lausanne, Switzerland (Received
28 November
1990)
Intact IgG, and F(ab’), anti-carcinoembryonic antigen antibodies penetrate human colon adenocarcinoma multicell spheroids much more slowly than Fab fragments and the molecular weight and the binding site valency appear to be the most important factor in determining the rate of penetration. The rate is also influenced considerably by the number of antigen binding sites per cell, with a high antigen concentration slowing penetration appreciably. The tumor cell architecture appears to have a minor effect on antibody penetration when compared to antibody size or antigen concentration.
Introduction The success of radiolabeled antibody therapy is dependent on the dose distribution obtained in the tumor. If regions of viable tumor cells receive a low radiation dose, these cells may survive to repopulate the tumor and produce treatment failure. The evenness of dose distribution is dependent on three factors, the distribution of radiolabeled Ab in the tumor and the energy and penetration distance
of the radionuclide
emissions.
This paper
will address the first factor, antibody distribution in tumor. In vivo studies of radiolabeled Ab uptake have generally shown a heterogeneous pattern of Ab distribution (Del Vecchio et al., 1989; Griffith ef al., 1988). There are many factors which influence this pattern in tissue. These can be divided into vascular, tumor, and antibody factors. Vascular factors include tumor vessel blood flow and permeability. There has been *Research supported by the Wilmot Foundation and the H. Gardner and S. Edelman Foundation. We gratefully acknowledge NeoRx Corporation for supplying the NRLU-10 antibody. tTo whom requests for reprints should be addressed. $Present address: SRI International, 333 Ravenswood Ave, Menlo Park, CA 94025, U.S.A. Abbreviations used: Ab, antibody; RIT, radioimmunotherapy; CEA, carcinoembryonic antigen; DMEM, Dulbecco’s modified essential medium.
some indirect support of the effect of blood flow in patients with metastatic colon cancer who were infused with ‘3’I-1083-17-1A F(ab’), (Gatenby et al., 1988). Tumor oxygen levels were measured with an electrode and those with a mean ~0, < 16 mm Hg did not image successfully despite the presence of antigen positive cells on subsequent immunohistochemistry. Although other factors such as tissue pH may have been important, Ab binding appeared to correlate with oxygen delivery and presumably therefore blood flow. As tumors increase in size, the proportion of poorly vascularized tumor increases resulting in decreased Ab uptake per gram of tissue (Philben et al., 1986). It has been suggested by Jain and Baxter, (1988) that high interstitial pressure and low microvascular pressure may lead to inhibition of movement of Ab into the tumor. This problem is enhanced by the fact that there are no functioning lymphatics in tumors to aid in decreasing the interstitial pressure. Before Ab can bind to a tumor cell, it must first pass through the vascular endothelium and any factor that will enhance this passage should improve Ab binding. Studies of radiolabeled Ab uptake in a renal cell carcinoma line grown in nude mice have demonstrated remarkably high percentage injected dose per gram of tumor and this has been related to vascular permeability (Chiou et al., 1988). Sands et al. (1985) have devised a technique for assessing vascular permeability and vascular volume simultaneously which 153
VIRGINIA K. LANGMUIR et al.
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should be useful for assessing the role of these vascular factors in Ab binding in vivo. Antibody factors that influence antibody distribution include molecular weight, antibody concentration and antibody affinity. The molecular weights of intact IgG, F(ab’), , and Fab are approx. 1.5 x 10s, 1.0 x 10’ and 5.0 x 104, respectively. It has been shown in the multicell tumor spheroid mode1 using human colon adenocarcinoma and anti-CEA that at Ab concentrations of 1-5 pg/mL after 4 h incubation, the binding of IgG was only to a depth of 1-3 cell layers but that of F(ab’)z and Fab was up to 6-8 cells (Sutherland et af., 1987). Presumably the smaller size of the fragments allowed better diffusion. Buchegger et al. (1983) and Pervez et al. (1988) have shown similar results in human tumor xenografts. These findings are supported by modeling analysis (Fujimori et al., 1990). It has been shown in the multicell spheroid model that increasing the antibody concentration does improve the rate of penetration presumably because of the more rapid saturation of binding sites (Langmuir et al., 1990). It has been suggested that decreased Ab affinity could lead to a more even distribution of Ab in the tumor as Ab would be less likely to be bound to the first available binding site encountered (Fujimori et al., 1990; Weinstein et al., 1986). Other modeling analysis has suggested that high affinity Ab provides a substantial advantage in tumor/non-tumor ratios, particularly for decreased molecular size, but the microscopic distribution of Ab in the tumor was not addressed (Thomas et al., 1989). Tumor factors affecting Ab penetration include number of available antigen binding sites per cell, distribution of antigen-positive cells in the tumor, composition of the tumor interstitium, and cell-cell interactions. A large number of binding sites per cell should lead to a less even distribution of Ab because it would take longer to saturate the most accessible binding sites and move into those that are less accessible. The tumor architecture may influence Ab distribution independent of these other factors by producing a mechanical effect on Ab penetration. Tightly packed tumor cells with strong cell-cell interactions such as tight junctions may inhibit Ab penetration whereas a large interstitial volume may enhance penetration. The possibility of this effect of tumor architecture has not been studied. The purpose of this paper is to address the roles of tumor architecture, antigen concentration and Ab size on Ab penetration. To study these factors directly and independently of the effects of the vasculature on Ab penetration, multicell tumor spheroids were used.
Materials and Methods Cell lines Two human colon carcinoma cell lines were used for these experiments, LS174T and HT29.
LS174T was obtained from Jeffrey Schlom (National Institutes of Health, Bethesda, Md) and was established in 1974 from a patient with a moderately well differentiated adenocarcinoma of the colon (Tom et al., 1976). HT29 was obtained from J-P. Mach (University of Lausanne, Lausanne, Switzerland) and was established from a patient with adenocarcinoma of the colon (Fogh and Trempe, 1975). Monolayer cells were grown in DMEM (Gibco, Grand Island, N.Y.) with added penicillin/streptomycin, L-glutamine, and fetal calf serum, 10% for LS174T and 5% for HT29. They were maintained at 37°C in a humidified 5% CO,/air atmosphere. To grow multicell spheroids, 5 x 10’ cells were plated in complete medium over 2% agar (Bactoagar, Difco, Detroit, Mich.). After 4-5 days, when the cell aggregates had reached approx. 80 pm in diameter, they were transferred to spinner flasks, gassed with 3% CO,/air and maintained at 37°C on a magnetic stir plate. Medium was replenished every 2-3 days and spheroid diameter was measured under an inverted microscope using an ocular micrometer. The geometric mean diameter was calculated from two perpendicular diameter measurements and the mean from 30 spheroids was taken. Spheroids of 600-800 pm diameter were used for most experiments. Antibodies CE25-B7 (B7), an IgG, murine monoclonal Ab to CEA, was used for these experiments. The intact Ab and its F(ab’h and Fab fragments were obtained courtesy of J-P. Mach and F. Buchegger (University of Lausanne, Switzerland) (Buchegger et al., 1988). The affinity of intact B7 and its Fab fragment have been calculated by Buchegger to be 1.6 x lOi and 3.1 x lOa, respectively in PBS. PX63, an IgG, nonbinding Ab was used as a control. NRLU-10, a murine monoclonal IgG2b Ab to a pancarcinoma antigen (Okabe et al., 1984), was obtained from NeoRx Corporation (Seattle, Wash.). Antibody labeling Abs were labeled using a modification of the chloramine-T method (Hunter and Greenwood, 1962). I251(New England Nuclear, Billerica, Mass.) was added to Ab at 4°C at a ratio of 1OpCi “I/pg Ab. Chloramine-T was then added and after 4min the reaction was stopped with sodium metabisulfite. Carrier potassium iodide was then added followed by bovine serum albumin (final concentration of BSA = 1 pg/mL). The labeled Ab was then passed through a Sephadex PD-10 column (Pharmacia, Uppsala, Sweden) to separate the bound from the free iodine. The percent of activity that was protein bound was then estimated by performing trichloroacetic acid precipitation. This was usually greater than 96% and specific activity was between 4 and 8 pCi/pg. For the experiments, labeled Ab was diluted with complete medium to the desired concentration.
Antibody penetration in multicell spheroids Cells antibody binding assay
Exponentially growing monolayer cells were harvested with 0.01% trypsin in 0.02% EDTA and placed on a shaker in complete medium overnight. If the trypsinization had stripped CEA from the cell surface, this would give time for re-expression. The cells were then harvested, counted and labeled with 51Cr (New England Nuclear, Billerica, Mass.) as follows: 2-4 x 10’ cells were washed in 1% BSAjPBS twice and resuspended in a total volume of O.S1.OmL. 0.5 mCi “Cr (1 mCi/mL) was then added and the mixture incubated for 4 min at room temperature. The cells were then washed three times in 1% BSA/PBS. The cells were diluted, recounted using a hemocytometer, and counted in a y counter to determine cpm/cell. The cells were resuspended at a concentration of 1-2 x 10’ cells/ml. 0.2 mL of the cell suspension was added to 0.2 mL labeled Ab at varying concentrations. This mixture was then incubated at 4°C with continuous shaking. After 4 h, three 0.1 mL aliquots of the mixture were then spun through oil (see below) and the pellets counted in a dual channel well-type y counter (Packard, Downers Grove, Ill.). To determine the total number of binding sites per cell, the ratio of bound Ab to free Ab was calculated and plotted against bound Ab. The total number of binding sites per cell was determined from the x-intercept of the linear portion of this plot. Spheroid antibody binding assay
All experiments were performed at 4°C to avoid the confounding effects of spheroid growth and metabolism. Replicate samples of 6 spheroids were measured for spheroid diameter, placed in 5 mL snap-cap tubes (Falcon, Lincoln Park, N.J.), 2 mL of complete medium at 4°C was added, and then 1 mL of labeled Ab to give a final Ab concentration of 1 pg/mL. The tubes were then placed horizontally on a rotary shaker at 60rpm in a 4°C room. At intervals from 15 min to 48 h, duplicate tubes were removed. To assess Ab binding, the spheroids were washed four times in 1 mL medium for 5 min each. The spheroids were then counted in a well-type y counter. Ab binding was expressed as fmoles Ab bound per mm) spheroid volume. The spheroids were then fixed in neutral buffered formalin, embedded in paraffin, and sectioned at 5 ,um thickness for autoradiography. To evaluate Ab diffusion as well as binding, duplicate tubes were removed at intervals and the spheroids immediately spun through oil (see below). The supematant was removed and the tube was counted in a y counter both before and after removing the spheroids. The spheroids were then placed in embedding compound (Tissue Tek OCT Compound, Miles Laboratories Inc., Elkhart, Ind.) and flash frozen in 2-methyl butane (Kodak, Rochester, N.Y.) cooled in liquid nitrogen. 5 and 10 pm frozen sections were then made
755
on a cryostat and placed on gelatin-subbed glass slides for autoradiography. After 24 or 48 h incubation, duplicate samples of spheroids were washed, counted in the gamma counter, and resuspended in complete medium at 4°C for evaluation of the rate of Ab fall-off. Some spheroids were put back into snap-cap tubes on the rotary shaker and some were placed over 2% agar and kept at 4°C to evaluate the effect of shaking on Ab fall-off. After periods between 1 and 72 h, the tubes of spheroids were removed and washed as described above, counted in a y counter and fixed for autoradiography. Oil centrifuge assay
A 4:1 mixture of dibutylphthalate:corn oil was made with a resultant specific gravity of 1.02. 0.8 mL aliquots of this mixture were placed in 1 mL microcentrifuge tubes. For cell suspensions, 0.1 mL aliquots (3/sample) were layered over the oil and centrifuged for 1.5 min to pellet the cells but not the medium. The whole tube was counted in a dual channel y counter (12’1and Vr). The tip of the tube containing the cell pellet was then cut off and counted in the y counter. In the case of spheroids, they were placed on top of the oil using a Pasteur pipette, transferring as little medium as possible. The tube was then spun very briefly (10 s) so that the spheroids were at the bottom of the tube but not distorted. The medium was aspirated, the tube counted in a y counter, the spheroids removed, and the tube recounted. Autoradiography
Paraffin sections were deparaffinized prior to autoradiography. Frozen sections were dehydrated and fixed in acetone. Although this may have removed some of the radiolabel, adequate histology was not possible without this step. For liquid emulsiori autoradiography, NTB-3 emulsion (Kodak, Rochester, N.Y.) was diluted 1: 1 with distilled water and heated to 4244°C. The slides were dipped into the emulsion three times slowly, placed at a 60 angle, and allowed to dry for 1 h. The slides were then placed in slide boxes and kept at 4°C. Slides were developed in D 19 developer (Kodak, Rochester, N.Y.) at intervals from 10 days to 3 months later depending on the activity present in the sample. The slides were then stained with hematoxylin and eosin. For film autoradiography, deparaffinized or dehydrated slides were attached to cardboard and ‘HUltrofilm (LKB, Uppsala, Sweden) was placed over the slides and secured with tape. This was then placed in a cassette, wrapped in foil, and kept at 4°C with compression to ensure intimate contact between the sections and the film. At intervals from 1 h to 10 days, film was removed and developed in GBX developer (Kodak, Rochester, N.Y.).
VIRGINIA K. LANGMUIR et al.
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Results Cell antibody binding Antibody binding after 4 h incubation is shown in Fig. 1 for both LS174T and HT29 cells. From the binding data at several Ab concentrations, the number of antigenic binding sites per cell was estimated by linear regression of a plot of bound Ab vs bound Ah/free Ab to be 6.3 x 10’ for LSl74T and 1.7 x 10’ for HT29 (correlation coefficient = 0.98 and 0.88, respectively). INCUBATION
Spheroid antibody binding Binding of Ab to washed spheroids over time is shown in Fig. 2. Each point is the mean of two samples of 6 spheroids each. Ab concentration was 1 pg/mL, which is equivalent to nM concentrations of 6.7, 10, and 20 for IgG, F(ab’),, and Fab, respectively. Therefore, the fragments were present in equal binding site concentrations, but the IgG was at a slightly lower binding site concentration. The estimated number of Ab molecules bound per cell in LS174T and HT29 spheroids at 24 h were 1.8 f 0.3 and 0.6 f 0.2 for intact B7 and 7.9 It 0.8 an 3.7 k 0.3 for B7 Fab (all x 105). This was determined from the Ab binding data and the cell yield per spheroid after dissociation with trypsin, and counting of cells. The proportion of total Ab in a spheroid that was actually bound was estimated by comparing the radioactivity in washed spheroids to that in spheroids that were spun through oil. By 24 h, almost 100% of
TIME
(HRS)
250 > 0 0
= HT29 = LS174T
-0
(B) 0
12
24 INCUBATION
600.. “I
0 0
36 TIME
40
(HRS)
= HT29 = LS174T
soo-
(C) J
0
12
24 INCUBATION
(A) 0.1
1
0.1
I 1.0 ANTIBODY
10 CONC.
100
100
10
$ z
1
(HRS)
Fig. 2. Antibody binding per mm3 spheroid volume for (A) intact B7 (circles) and control PX63 (triangles), (B) B7 F(ab’)z and (C) B7 Fab.
the Ab (intact and fragments) in LS174T spheroids was bound whereas an average of 75% of the Ab was bound in HT29. This is likely explained by the larger interstitial space in HT29 (Fig. 3). The rate of loss of bound Ab was similar for both lines, about one-third in 48 h. There was no significant difference between the intact Ab and the fragments.
K G 4
TIME
4%
(NM)
1000 Y d 0 In 0
36
Autoradiography
d E 01 0.1
10
1.0 ANTIBODY
CONC.
100
(NM)
Fig. 1. Binding of B7 antibody, its fragments, and nonspecific Ab (PX63) to (A) LS174T and (B) HT29 cells.
Intact B7. Penetration of intact B7 was poor in LSl74T spheroids. At 7 h, there was primarily surface binding in LSl74T but by 24 h, although most binding was still near the spheroid surface, there was some penetration about 6-7 cell layers deep, and by 48 h, 10-14 cell layers (Fig. 4). In HT29, penetration was
Fig. 3. Transmission electron micrographs of (A) HT29 (4000x) and (B) LS174T (2000x) spheroids. Nc the Iloosely bound cells in HT29 with a large extracellular space and the more tightly bound LS 174T CC with an acinar-like structure at the left.
Fig . 4. Liquid emulsion autoradiographs of LS174T spheroids after 7 h (A) and 48 h (B) incubation with ‘251, -B7. Spheroids were washed prior to autoradiography so the grains represent bound antit >ody. Bar = 12/rm (A), 19pm (B).
758
Fig. 5. Liquid emulsion autoradiographs of HT29 spheroids after 24 h incubation in ‘*‘I-B7 (A) and 1251-NRLU-10 (B). Bar = 19 pm (A), 12 pm (B).
759
Fig. 6(A) and (B). Caption on p. 761.
760
Fig. 6(C) and (D) Fig. 6. Contact film autoradiographs of LS174T spheroids after incubation in ‘251-B7F(ab’), for 1 and 24 h (A and B) and in ‘251-B7Fab for 1 and 24 h (C and D). Autoradiography was done after washing and fixation of spheroids. Bar = 360 pm (A and B), 280 pm (C and D).
761
__ Fig. 7. Liquid emulsion autoradiograph
of HT29 spheroid after 24 h incubation in ‘WB7 F(ab’), Bar = 12 pm.
762
Antibody penetration in multicell spheroids slightly more rapid with grains visible within the necrotic center by 24 h. The grains seen in the necrotic center may be due to specific binding to CEA that has been shed by dying cells in the necrotic center. lt could also be due to non-specific binding to necrotic cells. NRLU-10 was used in HT29 to compare the relative importance of spheroid architecture and the number of binding sites per cell. The estimated number of binding sites per cell in HT29 spheroids for NRLU-10 is approx. 4 times that for intact B7 (data not shown). It was found that penetration of NRLU-10 was very slow with few grains past the first three cell layers by 24 h (Fig. 5). The affinity of NRLU-10 is only slightly less than that of B7 (5 x IO9 vs 1.6 x 10” M-‘) so differences in affinity were unlikely to be the source of these differences in penetration. 87 F&b’),. Penetration of F(ab’), was similar to intact Ab in both lines with some suggestion that, as with intact Ab, penetration was faster in HT29 [Figs 6(A), (B) and 71. B7 Fub. Penetration of Fab into both HT29 and LS174T spheroids was much faster than with intact Ab or F(ab’),. Penetration appeared to occur at similar rates in the two lines with penetration to several cell layers apparent by 1-4 h [Fig. 6(C) and (D)].
Discussion Fab binding/cell was higher in spheroids than predicted from the number of binding sites/cell in monolayer cells, presumably because of increased antigen expression due to some degree of differentiation in the spheroids. Sutherland et al. (1987) have shown that CEA expression is increased when CO1 12 (a human colon carcinoma cell line) is grown as spheroids. Hollow fiber culture of LSl74T cells also allows differentiation to occur and CEA expression has been shown to be increased (Rutzy et al., 1979). Binding of intact B7 in spheroids is less than Fab and autoradiography shows this to be due to reduced penetration. Direct antibody application to frozen sections of spheroids shows that the expression of CEA is heterogeneous with some cells binding Ab avidly and others not at all. The glandular structures sometimes seen in the LS174T spheroids stained positively for CEA. HT29 spheroids appear to have a larger extracellular space than LSl74T spheroids with looser cell-cell contacts (Fig. 3), supported by the finding that only 75% of the Ab in unwashed spheroids at 24 h is actually bound. This may explain the slightly faster diffusion of intact B7 and F(ab’)> into HT29 spheroids. The fact that there were fewer binding sites per cell in HT29 was probably most important in determining the rate of intact Ab and F(ab’), penetration because antigenic sites at a particular depth would be saturated more quickly. This is supported by the finding that incubation of HT29 spheroids with NRLU-IO led to much slower Ab
163
penetration. In contrast, the rate of penetration of Fab did not appear to be significantly different between the two lines. Fab may diffuse in SO easily that binding sites per cell and histologic architecture may become less important. The lower affinity that is usually found with Fab fragments may also contribute to the ease of diffusion into spheroids but studies in LS174T spheroids using a different Ab showed similar results to those presented here and the affinity of the intact Ab was less than twice that of the Fab (Langmuir et at., 1990). It is not clear why the binding of intact B7 appeared to plateau by 2448 h when it was clear from the autoradiography that not all antigenic binding sites were occupied. It may be that there were areas in the spheroid that were completely inaccessible to the intact Ab. It could also be that, for some reason, the labeled Ab was unavailable for binding after 2448 at 4°C perhaps because of dehalogenation or reduced immunoreactivity. However, Fab binding was continuing to increase at 2448 h. These experiments confirm the findings by Sutherland et al. (1987) that Ab size influences the ease of penetration into tumors. F(ab’), and intact IgG behave in a similar fashion, penetrating fairly slowly. Although F(ab’), may penetrate somewhat faster, it is still much slower than Fab. The number of binding sites per cell is a second factor of importance in Ab penetration in that a higher number of sites slows penetration. There are two factors that are likely to be most important in producing the more rapid penetration of Fab relative to intact IgG and F(ab’), The first is the smaller molecular size of Fab. The second factor is the valency. In the case of the univalent Fab, if one binding site dissociates, the molecule is free to diffuse further into the tumor. For IgG and F(ab’),, if both binding sites had attached to antigen, dissociation of one site would not allow further diffusion into the tumor. The effect of Ab concentration has been presented in another paper (Langmuir er al., 1990) and increasing Ab concentration did lead to more rapid saturation of binding sites with subsequent faster penetration, but the marked difference between intact Ab and Fab was still apparent. The distribution of binding sites may influence the differences between intact Ab and Fab. Immunohistochemistry in spheroid sections has revealed antigen expression within acinar structures that bind intact Ab only after prolonged incubation of the intact spheroid. Presumably there is a physical barrier to Ab penetration because of intimate cellcell contacts. In this situation, Ab size may be important as we have seen early binding within these structures with Fab. Kwok et al. (1988) demonstrated similar results to ours in human melanoma spheroids with maximal uptake of intact Ab by 24 h. The rate of penetration of Ab into spheroids appeared to be slightly more rapid in their studies which may have been due to
764
VIRGINIAK. LANGMUIR et al.
fewer binding sites per cell, lower Ab affinity, temperature differences, and spheroid growth during the incubation period. It was not due to a higher Ab concentration as they used a concentration of 0.2 pg/mL compared with 1 pg/mL in our experiments.
References Buchegger F., Vacca A., Carrel S., Schreyer M. and Mach J.-P. (1988) Radioimmunotherapy of human colon carcinoma by “‘I-lab&d monoclonal anti-CEA antibodies in a nude mouse model. Int. J. Cuncer 41, 127. Buchegger F., Haskell C. M., Schreyer M., Scazziga B. R., Randin S., Carrel S. and Mach J.-P. (1983) Radiolabeled fragments of monoclonal antibodies against carcinoembryonic antigen for localization of human colon carcinoma grafted into nude mice. J. Exp. Med. 158,413. Cdiou R. K., Vessella R. L., Limas C., Shafer R. B., Elson M. K., Arfman E. W. and Lange P. H. (1988) Monoclonal antibody-targeted radiotherapy of renal cell carcinoma using a nude mouse model. Cancer 61, 1766. Del Vecchio S., Reynolds J. C., Carrasquillo J. A., Blasberg R. G., Newmann R. D., Lotze M. T., Bryant G. J, Farkas R. J. and Larson S. M. (1989) Local distribution and concentration of intravenously ‘injected “‘I-9.2.27 monoclonal antibody in human malignant melanoma. Cancer Res. 49, 2783. Fogh J. and Trempe G. (1975) New human tumor cell lines. In Human Tumor Cells In Vitro (Edited bv Fonh J.). Chap. 5. Plenum Press, New York: ’ ” Fujimori K., Cove11 D. G., Fletcher J. E. and Weinstein J. N. (1990) A modeling analysis of the global and microscopic distribution of IgG, F(ab’),, and Fab in tumors. Cancer Rex 49, 5656. Gatenby R. A., Moldofsky P. J. and Weiner L. M. (1988) Metastatic colon cancer: correlation of oxygen levels with I-131 F(ab’), uptake. Radiology 166, 757. Griffith M. H., Yorke E. D., Wessels B. W., DeNardo G. L. and Neacy W. P. (1988) Direct dose confirmation of quantitative autoradiography with micro-TLD measurements for radioimmunotherapy. J. Nucl. Med. 29, 1795. Hunter W. M. and Greenwood F. C. (1962) Preparation of iodine-13 1 labeled human growth hormone of high specific activity. Nature 194, 495. Jain R. K. and Baxter L. T. (1988) Mechanisms of heterogeneous distribution of monoclonal antibodies in tumors: significance of elevated interstitial pressure. Cancer Rex 48,701l.
Kwok C. S., Cole S. E. and Liao S.-K. (1988) Uptake kinetics of monoclonal antibodies by malignant melanoma multicell spheroids. Cancer Res. 48. 1856. Langmuir V. K., Atche; R. W., Hines J. J. and’Brechbie1 M. W. (1990) ‘251-NRLU-10 kinetic studies and 2’2BiNRLU-i0 toxicity in LS174T multicell spheroids. J. Nucl. Med. 31, 1527. Okabe T., Kaizu T., Fujisawa M., Watanabe J., Kojima K., Yamashita T. and Takaku F. (1984) Monoclonal antibodies to surface antigens of small cell carcinoma of the lungs. Cancer Res. 44, 5273. Pervez S., Epenetos A. A., Mooi W. J., Evans D. J., Rowlinson G.. Dhokia B. and Krausz T. 11988) Localization of monoclonal antibody AUAl aid-& F(ab’), fragments in human tumour xenografts: an autoradiographic and immunohistochemical study. In!. J. Cancer 3, (Suppl.) 23. Philben V. J., Jakowatz J. G., Beatty B. G., Vlahos W. G., Paxton R. J., Williams L. E., Shively J. E. and Beatty J. D. (1986) The effect of tumor CEA content and tumor size on tissue uptake of indium-labeled anti-CEA monoclonal antibody. Cancer 57, 571. Rutzky L. P., Tomita J. T., Calenoff M. A. and Kahan B. D. (1979) Human colon adenocarcinoma cells. III. In vitro organoid expression and carcinoembryonic antigenkinetics in hollow fiber culture. J. N&l. Cancer Inst. 63, 893. Sands H., Shah S. A. and Gallagher B. M. (1985) Vascular volume and permeability of human and murine tumors grown in athymic mice. Cancer Letf. 27, 15. Sutherland R. M., Buchegger F., Schreyer M. et al. (1987) Penetration and binding of radiolabeled anti-carcinoembryonic antigen monoclonal antibodies and their antigen binding fragments in human colon multicellular spheroids. Cancer Rex 47, 1627. Thomas G. D., Chappell M. J., Dykes P. W., Ramsden D. B., Godfrey K. R., Ellis J. R. M. and Bradwell A. R. (1989) Effect of dose, molecular size, affinity, and protein binding on tumor uptake of antibody or ligand: a biomathematical model. Cancer Res. 49, 3290. Tom B. H., Rutzky L. P., Jakstys M. M., Oyasu R., Kaye C. I. and Kahan B. D. (1976) Human colonic adenocarcinema cells. I. Establishment and description of a new line. In Vitro 12, 180. Weinstein J. N., Cove11D. G., Barbet J., Eger R. R., Holton 0. D., Talley M. J., Parker R. J. and Black C. D. V. (1986) Local and cellular factors in the pharmacology of monoclonal antibodies. In Membrane Mediated Cy~otoxicily (Edited by Bonavida B. and Collier R. J.), p. 279. Liss, New York.
Nucl. Med. Biol. Vol. 18, No. 7, pp. 753-764, 1991 Int. J. Radial. Appl. Instrum. Part B Printed in Great Britain
Perpmon Press plc
The Effect of Antigen Concentration, Antibody Valency and Size, and Tumor Architecture on Antibody Binding in Multicell Spheroids* VIRGINIA K. LANGMUIR’tS, JUDETH K. McGANN’S, FRANZ BUCHEGGER’ and ROBERT M. SUTHERLAND’S ‘University of Rochester Cancer Center, Rochester, NY 14642, U.S.A. and *Institute of Biochemistry, University of Lausanne, Switzerland (Received
28 November
1990)
Intact IgG, and F(ab’), anti-carcinoembryonic antigen antibodies penetrate human colon adenocarcinoma multicell spheroids much more slowly than Fab fragments and the molecular weight and the binding site valency appear to be the most important factor in determining the rate of penetration. The rate is also influenced considerably by the number of antigen binding sites per cell, with a high antigen concentration slowing penetration appreciably. The tumor cell architecture appears to have a minor effect on antibody penetration when compared to antibody size or antigen concentration.
Introduction The success of radiolabeled antibody therapy is dependent on the dose distribution obtained in the tumor. If regions of viable tumor cells receive a low radiation dose, these cells may survive to repopulate the tumor and produce treatment failure. The evenness of dose distribution is dependent on three factors, the distribution of radiolabeled Ab in the tumor and the energy and penetration distance
of the radionuclide
emissions.
This paper
will address the first factor, antibody distribution in tumor. In vivo studies of radiolabeled Ab uptake have generally shown a heterogeneous pattern of Ab distribution (Del Vecchio et al., 1989; Griffith ef al., 1988). There are many factors which influence this pattern in tissue. These can be divided into vascular, tumor, and antibody factors. Vascular factors include tumor vessel blood flow and permeability. There has been *Research supported by the Wilmot Foundation and the H. Gardner and S. Edelman Foundation. We gratefully acknowledge NeoRx Corporation for supplying the NRLU-10 antibody. tTo whom requests for reprints should be addressed. $Present address: SRI International, 333 Ravenswood Ave, Menlo Park, CA 94025, U.S.A. Abbreviations used: Ab, antibody; RIT, radioimmunotherapy; CEA, carcinoembryonic antigen; DMEM, Dulbecco’s modified essential medium.
some indirect support of the effect of blood flow in patients with metastatic colon cancer who were infused with ‘3’I-1083-17-1A F(ab’), (Gatenby et al., 1988). Tumor oxygen levels were measured with an electrode and those with a mean ~0, < 16 mm Hg did not image successfully despite the presence of antigen positive cells on subsequent immunohistochemistry. Although other factors such as tissue pH may have been important, Ab binding appeared to correlate with oxygen delivery and presumably therefore blood flow. As tumors increase in size, the proportion of poorly vascularized tumor increases resulting in decreased Ab uptake per gram of tissue (Philben et al., 1986). It has been suggested by Jain and Baxter, (1988) that high interstitial pressure and low microvascular pressure may lead to inhibition of movement of Ab into the tumor. This problem is enhanced by the fact that there are no functioning lymphatics in tumors to aid in decreasing the interstitial pressure. Before Ab can bind to a tumor cell, it must first pass through the vascular endothelium and any factor that will enhance this passage should improve Ab binding. Studies of radiolabeled Ab uptake in a renal cell carcinoma line grown in nude mice have demonstrated remarkably high percentage injected dose per gram of tumor and this has been related to vascular permeability (Chiou et al., 1988). Sands et al. (1985) have devised a technique for assessing vascular permeability and vascular volume simultaneously which 153
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should be useful for assessing the role of these vascular factors in Ab binding in vivo. Antibody factors that influence antibody distribution include molecular weight, antibody concentration and antibody affinity. The molecular weights of intact IgG, F(ab’), , and Fab are approx. 1.5 x 10s, 1.0 x 10’ and 5.0 x 104, respectively. It has been shown in the multicell tumor spheroid mode1 using human colon adenocarcinoma and anti-CEA that at Ab concentrations of 1-5 pg/mL after 4 h incubation, the binding of IgG was only to a depth of 1-3 cell layers but that of F(ab’)z and Fab was up to 6-8 cells (Sutherland et af., 1987). Presumably the smaller size of the fragments allowed better diffusion. Buchegger et al. (1983) and Pervez et al. (1988) have shown similar results in human tumor xenografts. These findings are supported by modeling analysis (Fujimori et al., 1990). It has been shown in the multicell spheroid model that increasing the antibody concentration does improve the rate of penetration presumably because of the more rapid saturation of binding sites (Langmuir et al., 1990). It has been suggested that decreased Ab affinity could lead to a more even distribution of Ab in the tumor as Ab would be less likely to be bound to the first available binding site encountered (Fujimori et al., 1990; Weinstein et al., 1986). Other modeling analysis has suggested that high affinity Ab provides a substantial advantage in tumor/non-tumor ratios, particularly for decreased molecular size, but the microscopic distribution of Ab in the tumor was not addressed (Thomas et al., 1989). Tumor factors affecting Ab penetration include number of available antigen binding sites per cell, distribution of antigen-positive cells in the tumor, composition of the tumor interstitium, and cell-cell interactions. A large number of binding sites per cell should lead to a less even distribution of Ab because it would take longer to saturate the most accessible binding sites and move into those that are less accessible. The tumor architecture may influence Ab distribution independent of these other factors by producing a mechanical effect on Ab penetration. Tightly packed tumor cells with strong cell-cell interactions such as tight junctions may inhibit Ab penetration whereas a large interstitial volume may enhance penetration. The possibility of this effect of tumor architecture has not been studied. The purpose of this paper is to address the roles of tumor architecture, antigen concentration and Ab size on Ab penetration. To study these factors directly and independently of the effects of the vasculature on Ab penetration, multicell tumor spheroids were used.
Materials and Methods Cell lines Two human colon carcinoma cell lines were used for these experiments, LS174T and HT29.
LS174T was obtained from Jeffrey Schlom (National Institutes of Health, Bethesda, Md) and was established in 1974 from a patient with a moderately well differentiated adenocarcinoma of the colon (Tom et al., 1976). HT29 was obtained from J-P. Mach (University of Lausanne, Lausanne, Switzerland) and was established from a patient with adenocarcinoma of the colon (Fogh and Trempe, 1975). Monolayer cells were grown in DMEM (Gibco, Grand Island, N.Y.) with added penicillin/streptomycin, L-glutamine, and fetal calf serum, 10% for LS174T and 5% for HT29. They were maintained at 37°C in a humidified 5% CO,/air atmosphere. To grow multicell spheroids, 5 x 10’ cells were plated in complete medium over 2% agar (Bactoagar, Difco, Detroit, Mich.). After 4-5 days, when the cell aggregates had reached approx. 80 pm in diameter, they were transferred to spinner flasks, gassed with 3% CO,/air and maintained at 37°C on a magnetic stir plate. Medium was replenished every 2-3 days and spheroid diameter was measured under an inverted microscope using an ocular micrometer. The geometric mean diameter was calculated from two perpendicular diameter measurements and the mean from 30 spheroids was taken. Spheroids of 600-800 pm diameter were used for most experiments. Antibodies CE25-B7 (B7), an IgG, murine monoclonal Ab to CEA, was used for these experiments. The intact Ab and its F(ab’h and Fab fragments were obtained courtesy of J-P. Mach and F. Buchegger (University of Lausanne, Switzerland) (Buchegger et al., 1988). The affinity of intact B7 and its Fab fragment have been calculated by Buchegger to be 1.6 x lOi and 3.1 x lOa, respectively in PBS. PX63, an IgG, nonbinding Ab was used as a control. NRLU-10, a murine monoclonal IgG2b Ab to a pancarcinoma antigen (Okabe et al., 1984), was obtained from NeoRx Corporation (Seattle, Wash.). Antibody labeling Abs were labeled using a modification of the chloramine-T method (Hunter and Greenwood, 1962). I251(New England Nuclear, Billerica, Mass.) was added to Ab at 4°C at a ratio of 1OpCi “I/pg Ab. Chloramine-T was then added and after 4min the reaction was stopped with sodium metabisulfite. Carrier potassium iodide was then added followed by bovine serum albumin (final concentration of BSA = 1 pg/mL). The labeled Ab was then passed through a Sephadex PD-10 column (Pharmacia, Uppsala, Sweden) to separate the bound from the free iodine. The percent of activity that was protein bound was then estimated by performing trichloroacetic acid precipitation. This was usually greater than 96% and specific activity was between 4 and 8 pCi/pg. For the experiments, labeled Ab was diluted with complete medium to the desired concentration.
Antibody penetration in multicell spheroids Cells antibody binding assay
Exponentially growing monolayer cells were harvested with 0.01% trypsin in 0.02% EDTA and placed on a shaker in complete medium overnight. If the trypsinization had stripped CEA from the cell surface, this would give time for re-expression. The cells were then harvested, counted and labeled with 51Cr (New England Nuclear, Billerica, Mass.) as follows: 2-4 x 10’ cells were washed in 1% BSAjPBS twice and resuspended in a total volume of O.S1.OmL. 0.5 mCi “Cr (1 mCi/mL) was then added and the mixture incubated for 4 min at room temperature. The cells were then washed three times in 1% BSA/PBS. The cells were diluted, recounted using a hemocytometer, and counted in a y counter to determine cpm/cell. The cells were resuspended at a concentration of 1-2 x 10’ cells/ml. 0.2 mL of the cell suspension was added to 0.2 mL labeled Ab at varying concentrations. This mixture was then incubated at 4°C with continuous shaking. After 4 h, three 0.1 mL aliquots of the mixture were then spun through oil (see below) and the pellets counted in a dual channel well-type y counter (Packard, Downers Grove, Ill.). To determine the total number of binding sites per cell, the ratio of bound Ab to free Ab was calculated and plotted against bound Ab. The total number of binding sites per cell was determined from the x-intercept of the linear portion of this plot. Spheroid antibody binding assay
All experiments were performed at 4°C to avoid the confounding effects of spheroid growth and metabolism. Replicate samples of 6 spheroids were measured for spheroid diameter, placed in 5 mL snap-cap tubes (Falcon, Lincoln Park, N.J.), 2 mL of complete medium at 4°C was added, and then 1 mL of labeled Ab to give a final Ab concentration of 1 pg/mL. The tubes were then placed horizontally on a rotary shaker at 60rpm in a 4°C room. At intervals from 15 min to 48 h, duplicate tubes were removed. To assess Ab binding, the spheroids were washed four times in 1 mL medium for 5 min each. The spheroids were then counted in a well-type y counter. Ab binding was expressed as fmoles Ab bound per mm) spheroid volume. The spheroids were then fixed in neutral buffered formalin, embedded in paraffin, and sectioned at 5 ,um thickness for autoradiography. To evaluate Ab diffusion as well as binding, duplicate tubes were removed at intervals and the spheroids immediately spun through oil (see below). The supematant was removed and the tube was counted in a y counter both before and after removing the spheroids. The spheroids were then placed in embedding compound (Tissue Tek OCT Compound, Miles Laboratories Inc., Elkhart, Ind.) and flash frozen in 2-methyl butane (Kodak, Rochester, N.Y.) cooled in liquid nitrogen. 5 and 10 pm frozen sections were then made
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on a cryostat and placed on gelatin-subbed glass slides for autoradiography. After 24 or 48 h incubation, duplicate samples of spheroids were washed, counted in the gamma counter, and resuspended in complete medium at 4°C for evaluation of the rate of Ab fall-off. Some spheroids were put back into snap-cap tubes on the rotary shaker and some were placed over 2% agar and kept at 4°C to evaluate the effect of shaking on Ab fall-off. After periods between 1 and 72 h, the tubes of spheroids were removed and washed as described above, counted in a y counter and fixed for autoradiography. Oil centrifuge assay
A 4:1 mixture of dibutylphthalate:corn oil was made with a resultant specific gravity of 1.02. 0.8 mL aliquots of this mixture were placed in 1 mL microcentrifuge tubes. For cell suspensions, 0.1 mL aliquots (3/sample) were layered over the oil and centrifuged for 1.5 min to pellet the cells but not the medium. The whole tube was counted in a dual channel y counter (12’1and Vr). The tip of the tube containing the cell pellet was then cut off and counted in the y counter. In the case of spheroids, they were placed on top of the oil using a Pasteur pipette, transferring as little medium as possible. The tube was then spun very briefly (10 s) so that the spheroids were at the bottom of the tube but not distorted. The medium was aspirated, the tube counted in a y counter, the spheroids removed, and the tube recounted. Autoradiography
Paraffin sections were deparaffinized prior to autoradiography. Frozen sections were dehydrated and fixed in acetone. Although this may have removed some of the radiolabel, adequate histology was not possible without this step. For liquid emulsiori autoradiography, NTB-3 emulsion (Kodak, Rochester, N.Y.) was diluted 1: 1 with distilled water and heated to 4244°C. The slides were dipped into the emulsion three times slowly, placed at a 60 angle, and allowed to dry for 1 h. The slides were then placed in slide boxes and kept at 4°C. Slides were developed in D 19 developer (Kodak, Rochester, N.Y.) at intervals from 10 days to 3 months later depending on the activity present in the sample. The slides were then stained with hematoxylin and eosin. For film autoradiography, deparaffinized or dehydrated slides were attached to cardboard and ‘HUltrofilm (LKB, Uppsala, Sweden) was placed over the slides and secured with tape. This was then placed in a cassette, wrapped in foil, and kept at 4°C with compression to ensure intimate contact between the sections and the film. At intervals from 1 h to 10 days, film was removed and developed in GBX developer (Kodak, Rochester, N.Y.).
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Results Cell antibody binding Antibody binding after 4 h incubation is shown in Fig. 1 for both LS174T and HT29 cells. From the binding data at several Ab concentrations, the number of antigenic binding sites per cell was estimated by linear regression of a plot of bound Ab vs bound Ah/free Ab to be 6.3 x 10’ for LSl74T and 1.7 x 10’ for HT29 (correlation coefficient = 0.98 and 0.88, respectively). INCUBATION
Spheroid antibody binding Binding of Ab to washed spheroids over time is shown in Fig. 2. Each point is the mean of two samples of 6 spheroids each. Ab concentration was 1 pg/mL, which is equivalent to nM concentrations of 6.7, 10, and 20 for IgG, F(ab’),, and Fab, respectively. Therefore, the fragments were present in equal binding site concentrations, but the IgG was at a slightly lower binding site concentration. The estimated number of Ab molecules bound per cell in LS174T and HT29 spheroids at 24 h were 1.8 f 0.3 and 0.6 f 0.2 for intact B7 and 7.9 It 0.8 an 3.7 k 0.3 for B7 Fab (all x 105). This was determined from the Ab binding data and the cell yield per spheroid after dissociation with trypsin, and counting of cells. The proportion of total Ab in a spheroid that was actually bound was estimated by comparing the radioactivity in washed spheroids to that in spheroids that were spun through oil. By 24 h, almost 100% of
TIME
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600.. “I
0 0
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soo-
(C) J
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(A) 0.1
1
0.1
I 1.0 ANTIBODY
10 CONC.
100
100
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$ z
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Fig. 2. Antibody binding per mm3 spheroid volume for (A) intact B7 (circles) and control PX63 (triangles), (B) B7 F(ab’)z and (C) B7 Fab.
the Ab (intact and fragments) in LS174T spheroids was bound whereas an average of 75% of the Ab was bound in HT29. This is likely explained by the larger interstitial space in HT29 (Fig. 3). The rate of loss of bound Ab was similar for both lines, about one-third in 48 h. There was no significant difference between the intact Ab and the fragments.
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TIME
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36
Autoradiography
d E 01 0.1
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Fig. 1. Binding of B7 antibody, its fragments, and nonspecific Ab (PX63) to (A) LS174T and (B) HT29 cells.
Intact B7. Penetration of intact B7 was poor in LSl74T spheroids. At 7 h, there was primarily surface binding in LSl74T but by 24 h, although most binding was still near the spheroid surface, there was some penetration about 6-7 cell layers deep, and by 48 h, 10-14 cell layers (Fig. 4). In HT29, penetration was
Fig. 3. Transmission electron micrographs of (A) HT29 (4000x) and (B) LS174T (2000x) spheroids. Nc the Iloosely bound cells in HT29 with a large extracellular space and the more tightly bound LS 174T CC with an acinar-like structure at the left.
Fig . 4. Liquid emulsion autoradiographs of LS174T spheroids after 7 h (A) and 48 h (B) incubation with ‘251, -B7. Spheroids were washed prior to autoradiography so the grains represent bound antit >ody. Bar = 12/rm (A), 19pm (B).
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Fig. 5. Liquid emulsion autoradiographs of HT29 spheroids after 24 h incubation in ‘*‘I-B7 (A) and 1251-NRLU-10 (B). Bar = 19 pm (A), 12 pm (B).
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Fig. 6(A) and (B). Caption on p. 761.
760
Fig. 6(C) and (D) Fig. 6. Contact film autoradiographs of LS174T spheroids after incubation in ‘251-B7F(ab’), for 1 and 24 h (A and B) and in ‘251-B7Fab for 1 and 24 h (C and D). Autoradiography was done after washing and fixation of spheroids. Bar = 360 pm (A and B), 280 pm (C and D).
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__ Fig. 7. Liquid emulsion autoradiograph
of HT29 spheroid after 24 h incubation in ‘WB7 F(ab’), Bar = 12 pm.
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Antibody penetration in multicell spheroids slightly more rapid with grains visible within the necrotic center by 24 h. The grains seen in the necrotic center may be due to specific binding to CEA that has been shed by dying cells in the necrotic center. lt could also be due to non-specific binding to necrotic cells. NRLU-10 was used in HT29 to compare the relative importance of spheroid architecture and the number of binding sites per cell. The estimated number of binding sites per cell in HT29 spheroids for NRLU-10 is approx. 4 times that for intact B7 (data not shown). It was found that penetration of NRLU-10 was very slow with few grains past the first three cell layers by 24 h (Fig. 5). The affinity of NRLU-10 is only slightly less than that of B7 (5 x IO9 vs 1.6 x 10” M-‘) so differences in affinity were unlikely to be the source of these differences in penetration. 87 F&b’),. Penetration of F(ab’), was similar to intact Ab in both lines with some suggestion that, as with intact Ab, penetration was faster in HT29 [Figs 6(A), (B) and 71. B7 Fub. Penetration of Fab into both HT29 and LS174T spheroids was much faster than with intact Ab or F(ab’),. Penetration appeared to occur at similar rates in the two lines with penetration to several cell layers apparent by 1-4 h [Fig. 6(C) and (D)].
Discussion Fab binding/cell was higher in spheroids than predicted from the number of binding sites/cell in monolayer cells, presumably because of increased antigen expression due to some degree of differentiation in the spheroids. Sutherland et al. (1987) have shown that CEA expression is increased when CO1 12 (a human colon carcinoma cell line) is grown as spheroids. Hollow fiber culture of LSl74T cells also allows differentiation to occur and CEA expression has been shown to be increased (Rutzy et al., 1979). Binding of intact B7 in spheroids is less than Fab and autoradiography shows this to be due to reduced penetration. Direct antibody application to frozen sections of spheroids shows that the expression of CEA is heterogeneous with some cells binding Ab avidly and others not at all. The glandular structures sometimes seen in the LS174T spheroids stained positively for CEA. HT29 spheroids appear to have a larger extracellular space than LSl74T spheroids with looser cell-cell contacts (Fig. 3), supported by the finding that only 75% of the Ab in unwashed spheroids at 24 h is actually bound. This may explain the slightly faster diffusion of intact B7 and F(ab’)> into HT29 spheroids. The fact that there were fewer binding sites per cell in HT29 was probably most important in determining the rate of intact Ab and F(ab’), penetration because antigenic sites at a particular depth would be saturated more quickly. This is supported by the finding that incubation of HT29 spheroids with NRLU-IO led to much slower Ab
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penetration. In contrast, the rate of penetration of Fab did not appear to be significantly different between the two lines. Fab may diffuse in SO easily that binding sites per cell and histologic architecture may become less important. The lower affinity that is usually found with Fab fragments may also contribute to the ease of diffusion into spheroids but studies in LS174T spheroids using a different Ab showed similar results to those presented here and the affinity of the intact Ab was less than twice that of the Fab (Langmuir et at., 1990). It is not clear why the binding of intact B7 appeared to plateau by 2448 h when it was clear from the autoradiography that not all antigenic binding sites were occupied. It may be that there were areas in the spheroid that were completely inaccessible to the intact Ab. It could also be that, for some reason, the labeled Ab was unavailable for binding after 2448 at 4°C perhaps because of dehalogenation or reduced immunoreactivity. However, Fab binding was continuing to increase at 2448 h. These experiments confirm the findings by Sutherland et al. (1987) that Ab size influences the ease of penetration into tumors. F(ab’), and intact IgG behave in a similar fashion, penetrating fairly slowly. Although F(ab’), may penetrate somewhat faster, it is still much slower than Fab. The number of binding sites per cell is a second factor of importance in Ab penetration in that a higher number of sites slows penetration. There are two factors that are likely to be most important in producing the more rapid penetration of Fab relative to intact IgG and F(ab’), The first is the smaller molecular size of Fab. The second factor is the valency. In the case of the univalent Fab, if one binding site dissociates, the molecule is free to diffuse further into the tumor. For IgG and F(ab’),, if both binding sites had attached to antigen, dissociation of one site would not allow further diffusion into the tumor. The effect of Ab concentration has been presented in another paper (Langmuir er al., 1990) and increasing Ab concentration did lead to more rapid saturation of binding sites with subsequent faster penetration, but the marked difference between intact Ab and Fab was still apparent. The distribution of binding sites may influence the differences between intact Ab and Fab. Immunohistochemistry in spheroid sections has revealed antigen expression within acinar structures that bind intact Ab only after prolonged incubation of the intact spheroid. Presumably there is a physical barrier to Ab penetration because of intimate cellcell contacts. In this situation, Ab size may be important as we have seen early binding within these structures with Fab. Kwok et al. (1988) demonstrated similar results to ours in human melanoma spheroids with maximal uptake of intact Ab by 24 h. The rate of penetration of Ab into spheroids appeared to be slightly more rapid in their studies which may have been due to
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VIRGINIAK. LANGMUIR et al.
fewer binding sites per cell, lower Ab affinity, temperature differences, and spheroid growth during the incubation period. It was not due to a higher Ab concentration as they used a concentration of 0.2 pg/mL compared with 1 pg/mL in our experiments.
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